307

Degradation of poly-L-lactide Part 1 in vitro andin vivo physiological temperature degradation

N A Weir1 F J Buchanan1 J F Orr1 and G R Dickson21School of Mechanical and Manufacturing Engineering Queenrsquos University Belfast Belfast UK2Department of Trauma and Orthopaedic Surgery Queenrsquos University Belfast Belfast UK

Abstract Poly-L-lactide (PLLA) is one of the most significant members of a group of polymersregarded as bioresorbable The degradation of PLLA proceeds through hydrolysis of the ester linkagein the polymerrsquos backbone and is influenced by the polymerrsquos initial molecular weight and degree ofcrystallinity To evaluate its degradation PLLA pellets were processed by compression moulding intotensile test specimens and by extrusion into 2 mm diameter lengths of rod prior to being sterilizedby ethylene oxide gas (EtO) and degraded in both in vitro and in vivo environments On retrieval atpredetermined time intervals procedures were used to evaluate the materialrsquos molecular weightcrystallinity mechanical strength and thermal properties Additionally the in vivo host tissuersquos bio-logical response was analysed The results from this study suggest that in both the in vitro and invivo environments degradation proceeded at the same rate and followed the general sequence ofaliphatic polyester degradation ruling out enzymes contributing and accelerating the degradationrate in vivo Additionally the absence of cells marking an inflammatory response suggests that thePLLA rods investigated in vivo were biocompatible throughout the 44 weeks duration of the studybefore any mass loss was observed

Keywords bioresorbable poly-L-lactide degradation molecular weight crystallinity

NOTATION 1 INTRODUCTION

Over the past 40 years polymer scientists workingDSC differential-scanning calorimetryclosely with those in the medical device and clinical fieldsEtO ethylene oxide gashave made tremendous advances in understanding andGPC gel-permeation chromatographyapplying bioresorbable polymers to more sophisticatedLM light microscopyapplications Currently applications for these promisingMn number average molecular weightbiomaterials cover a broad range of clinical and scientificm0 initial massdisciplines including sutures and fracture-fixation devicesm

tmass at time t

to support tissue regeneration [1] as drug deliveryPDLA poly-D-lactidesystems in the pharmaceutical industry [2] and withPDLLA poly-DL-lactiderecent scientific advances as scaffolds in the field ofPGA polyglycolidetissue engineering [3]PLA polylactide

Bioresorbable polymers belonging to the aliphaticPLLA poly-L-lactidepolyester family currently represent the most attractivePI polydispersity indexgroup of polymers that meet the various medical andSEM scanning electron microscopyphysical demands for safe clinical applications ThisTEM transmission electron microscopyis mainly due to their high level of biocompatibilityTg glass transition temperatureacceptable degradation rates and versatility regardingTm melting temperaturephysical and chemical properties [4] Undoubtedly twoof the most significant members of the aliphatic poly-DHmelt enthalpy of fusionester family are the poly(a-hydroxy acids) polyglycolide

The MS was received on 2 February 2004 and was accepted after revision (PGA) and polylactide (PLA) [5] High molecularfor publication on 17 June 2004 weight PGA and PLA were first introduced in the 1950s Corresponding author School of Mechanical and Manufacturing

[6 ] and [7] however they were initially discarded andEngineering Queenrsquos University Belfast Ashby Building StranmillisRoad Belfast BT9 5AH UK email fbuchananqubacuk research was abandoned into the polymerization of other

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308 N A WEIR F J BUCHANAN J F ORR AND G R DICKSON

a-hydroxy acids because of their poor thermal and hydro- molecular weight mechanical strength crystallinity andmass change at predetermined time intervals throughoutlytic stabilities which did not allow them to be used as

regular plastics for long-term industrial use [8] Never- degradation and is a follow up to a previous study thatexamined the processing annealing and sterilization oftheless this very instability has proven to be of immense

importance enabling PGA PLA and other aliphatic PLLA [25] Additionally this study examines the in vivohost tissuersquos biological response to determine the bio-polyesters to be developed as synthetic bioresorbable

polymers for medical use compatibility of the processed material Test techniquesutilized include tensile and shear testing for mechanicalPLA was first investigated for medical use by Kulkarni

et al [9] who reasoned that it would be useful for strength differential-scanning calorimetry (DSC) to deter-mine thermal properties and crystallinity gel-permeationsurgical implants since hydrolysis would yield lactic acid

a normal intermediate of carbohydrate metabolism and a chromatography (GPC) to measure molecular weightand conventional histological analysis techniques forproduct of muscle contraction [10] Due to the asymmetry

of the PLA molecule it exists as two optical isomers examining any inflammatory response to the implantsin vivo The aim of the study is to develop a completepoly-D-lactide (PDLA) and poly-L-lactide (PLLA)

with a synthetic blend of D-lactide and L-lactide yielding understanding of the processed PLLArsquos degradationprofile and to provide the necessary control data forpoly-DL-lactide (PDLLA) [11] PDLLA is an amorphous

polymer while PLLA the naturally occurring isomer investigation of methods to accelerate the degradationof PLLA relative to its physiological degradation rateis semicrystalline and the most common bioresorbable

polymer used for orthopaedic devices [12] due to itsrelatively high tensile strength and low elongation PLLA

2 MATERIALS AND METHODSlike all aliphatic polyesters degrades in vivo throughsimple hydrolysis of the hydrolytically unstable ester

21 Materialslinkage in the polymerrsquos backbone with the degradationproducts ultimately metabolized to carbon dioxide and The polymer studied in this investigation poly-L-lactidewater and eliminated from the body [13] (PLLA) ResomerA L (batch number 26033) was sup-

For semicrystalline aliphatic polyesters like PLLA plied in a sealed moisture-proof container by Boehringerthere is a general concensus that degradation proceeds Ingelheim (Ingelheim Germany) in pellet formvia random bulk hydrolysis in two distinct stages [14]The first stage is characterized by the preferential attack

22 Methodsof the ester linkages in the more accessible amorphousregions [15] with the second stage characterized by the 221 Processingonset of mass loss and attack of the less accessible

The PLLA was processed by compression mouldingcrystalline regions [16ndash18] In conjunction with thisinto plates 08 mm thick and by extrusion into a 2 mmtwo-stage mechanism work by Li et al [19] examiningdiameter rod using techniques detailed previously bythe degradation of PDLLA showed conclusively thatWeir et al [25] ASTM D638-99 type-V tensile samplesdegradation occurs more rapidly in the centre than atwere then cut from the compression-moulded plates andthe surface This is known as heterogeneous degradation30 mm lengths cut from the extruded rod The tensileand is widely accepted as occurring in both lactide andand extruded rod samples were then annealed at 120 degCglycolide polymers [20] and [21] This heterogeneous orfor a period of four hours in a preheated air-circulatingautocatalytic degradation mechanism results from theoven (see Fig 1) prior to being sterilized using ethylenehydrolytic cleavage of the ester bonds forming new acidicoxide gas (EtO) by Griffith Microscience (Derbyshirecarboxyl end groups As degradation proceeds the solubleUK) on their standard EtO cycle for medical polymersoligomers produced close to the surface can escape whileie lsquoCycle 33rsquo [25]those in the centre cannot diffuse out of the polymer

This results in a higher internal acidity with the carboxyl222 In vitro degradation

end groups catalysing the ester hydrolysis reactionand a differentiation between the surface and interior The initial mass m0 of each of the tensile and extruded

rod samples were recorded Individual specimens weredegradation rates The degradation rate of PLLA andaliphatic polyesters in general is strongly related to their then placed in 28 ml screw-top glass bottles fully

immersed in a pH 74 phosphate-buffered solution inmaterial properties with crystallinity molecular weightand distribution orientation unreacted monomer and accordance with ISO 158141999 and placed in an air-

circulating oven maintaining the temperature at a con-the presence of impurities all playing significant roles[1] This is illustrated with reports on the time for the stant 37 degC The pH of the solutions was periodically

monitored throughout the duration of each of the studiescomplete resorption of PLLA ranging from 80 weeks[22] to 57 years [23] and [24] although in each case the ratio of the phosphate-buffered

solution in millimetres to the polymerrsquos mass in gramsThis study investigates the in vitro and in vivodegradation of PLLA by evaluating its properties of was greater than 301

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309DEGRADATION OF POLY-L-LACTIDE PART 1

Eight PLLA extruded rod samples were removed at strengths of the retrieved extruded rod samples weremeasured as an indication of mechanical strength Theeach follow-up time (five mechanical test samples and

three mass-change samples) while six PLLA tensile shear test employed was adapted from BS 2782 Part 3Method 340B 1978 Determination of Shear Strength ofsamples were removed at each follow-up time (three

mechanical test samples and three mass change samples) Sheet Material and was similar to the method employedby Suuronen et al [26 ] The retrieved PLLA rod samplesFollow-up times for the in vitro studies are given in

Table 1 were slotted into a hole aligned between two halves ofa shear bracket and sheared simultaneously at both ends

223 In vivo degradation under a constant strain rate of 5 mmmin Shear strengthin MPa was calculated from equation (1)The Sprague Dawley rat was chosen as the animal model

with the 30 mm lengths of 2 mm diameter extruded shear strength (MPa)=F2A=F2pr2 (1)PLLA rod solely investigated The samples were pre-

where F= load at maximum (N) and r=average radiuspared similarly to the rods for the in vitro studies how-of sample (mm) The overall shear strength was dividedever before sterilization the ends of each of the rods wereby two in equation (1) to account for the double shearingblunted by grinding on a Struers (Roslashdovre Denmark)action taking placerotating grinding machine using Struers silicon carbide

In accordance with ISO 158141999 the retrievedpaper (grit 1200) and the initial mass (m0) of each ofmaterial underwent mechanical testing while lsquowetrsquo withthe samples was recorded Three PLLA rod samples weretesting conducted within three hours of retrieval fromimplanted per rat one each for mechanical testing massboth the in vitro buffered solution and in vivo animalloss and histological analysis to examine the host tissuersquosmodelresponse A total of 12 male-weight-matched (350 g)

Sprague Dawley rats were used for the in vivo studies Mass change On retrieval the in vitro and in vivowith the material implanted subcutaneously in the ratsrsquo samples were dried immediately with a paper towel todorsum with the three samples placed about the dorsum remove any surface moisture before being weighed usingmidline an electronic balance (Mettler Toledo Fisher Scientific

At predetermined time intervals (Table 1) the PLLA UK) to determine the percentage swelling of the polymerrod samples intended for mechanical strength and mass- and water uptake The samples were then dried in achange analysis were surgically removed from the ratsrsquo vacuum oven (Townson+Mercer Altrincham UK) atdorsal subcutaneous tissue and separated from any approximately 30 degC for 48 hours at a vacuum of 068 baradhering tissue The rod samples intended for histological and reweighed to obtain their mass at time t(m

t) The

analysis were also removed together with surrounding overall percentage mass change after drying was thenadherent tissue and placed in a fixative solution and calculated from equation (2)stored in a refrigerator at 4 degC to preserve the tissue untilit was ready to be processed for light microscopy (LM) percentage mass change=

mtminusm0

m0times100 per cent

scanning electron microscopy (SEM) and transmissionelectron microscopy (TEM) Three rats were sacrificed

(2)at each time interval by administering an increasing doseof CO2 gas All experimental procedures were carried Molecular weight and thermal properties Followingout under approved Home Office Project and Personal mass change measurements the dried PLLA samplesLicence cover were reused for gel-permeation chromatography (GPC)

analysis to determine their weight and number average224 Characterization of retrieved in vitro and in vivomolecular weights (Mr) throughout degradation and alsomaterialfor differential-scanning calorimetry (DSC) to determine

Mechanical properties The mechanical properties of their thermal properties and percentage crystallinitythe PLLA tensile samples were determined using aJJ Lloyd EZ 50 tensile testing machine (Hampshire Molecular weight The GPC analysis was conducted

by Rapra Technology Ltd (Shropshire UK) SamplesUK) equipped with a 1 kN load cell and tested at aconstant strain rate of 10 mmmin Youngrsquos modulus were prepared by adding 10 ml of choloform solvent to

20 mg of sample taken through a cross-section of thetensile strength and extension at break were calculatedfrom each of the load versus extension curves The shear material A Pl gel-mixed bed column with a refractive

Table 1 In vitro and in vivo follow-up times

Follow up Weeks

In vitro 4 10 20 26 32 38 44 50 57 65In vivo 10 26 38 44

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310 N A WEIR F J BUCHANAN J F ORR AND G R DICKSON

index response detector was used The GPC system was at a heating rate of 10 degCmin providing measurementsof glass transition temperature Tg melting point Tm andcalibrated with polystyrene and all results were expressed

as lsquopolystyrene equivalentrsquo molecular weights It should enthalpy of fusion DHmelt in Jg The DSC results werederived from this single heating cycle to provide a truebe noted that this is a relative technique rather than an

absolute technique for determining molecular weight indication of changes in the polymersrsquo thermal propertiesand morphology as a direct result of degradation TheMolecular weight is considered to be the most

important and sensitive parameter for modelling bio- enthalpy of fusion DHmelt was then used to calculatethe polymersrsquo percentage crystallinities relative to theresorbable polymer degradation [27ndash29] In particular

with Mn directly related to the scission of the polymers enthalpy of fusion of a 100 per cent crystalline sampleof PLLA reported to be 93 Jg [32]chains a number of relationships have been derived

relating the changes in Mn with time to the hydrolysispercentage crystallinity=(DHmelt 93)times100 per cent

rate of the unstable ester linkages Anderson [30] andChu [17] reported a statistical method for relating (7)molecular weight to hydrolysis rate assuming that theextent of degradation was not large they reported the Investigation of the biological host tissuersquos response Thefollowing kinetic relationship based on the polymers Mn retrieved PLLA extruded rod samples from the in vivo

animal model were prepared and investigated using LM1Mnt=1Mn

0+kt (3)

TEM and SEM to observe structure ultrastructure andwhere Mn

t=Mn at time t Mn0=Mn at t=0 k=rate surface topography of the tissue-implant environment

constant and t=time If the theory holds true a linear Conventional techniques were used in the preparationrelationship should exist between 1Mn versus time up of each of the samples [33] and [34] Specimens foruntil the point of mass loss LM were fixed in 10 per cent buffered formaldehyde

However a disadvantage of this statistical approach while those for SEM and TEM were preserved in 3 peris that it does not account for the possibility of auto- cent glutaraldehyde in 01 M sodium cacodylate buffercatalysis accelerating the polymerrsquos degradation rate Pitt pH 72ndash74 The local host biological tissuersquos responseand Gu [31] derived a relationship based on the kinetics to the implant was analysed after 36 and 44 weeksof the ester-hydrolysis reaction accounting for auto-catalysis by the generated carboxylic acid end groupsdescribed by the rate equation 3 RESULTS

d(E)dt=minusd(COOH)dt=minusk(COOH)(H2O)(E)31 Visual examination

(4)Initially at 0 weeks the annealed PLLA tensile and

where (COOH) (H2O) and (E) represent the con- extruded rod specimens were opaque and off-whitecentrations of carboxyl end groups water and esters in colour (Fig 1) At 32 weeks small areas of therespectively tensile specimens became more intensely white and as

On further analysis of equation (4) and assuming that degradation time increased more white areas becamethe ester and water concentrations remain constant and visible (Fig 2) Both the in vitro and in vivo extrudedthe concentration of acid end groups is equal to 1Mn rod specimens remained opaque and off-white in colourit can be shown that throughout the duration of the study (Fig 1) with no

whiter areas becoming visible The sizes and shapes ofMnt=Mn

0eminuskt (5)

both the tensile and extruded rod specimens did notIf this relationship holds true a linear relationship change during ageingshould exist between the ln Mn versus time up until thepoint of mass loss

ln Mnt=minuskt+ ln Mn

0(6)

An advantage of using GPC to measure molecularweight is that it also provides information on thesamplesrsquo molecular weight distribution through con-sideration of the whole GPC chromatogram providingfurther insights into the complex nature of bioresorbablepolymer degradation

Thermal properties The thermal properties of the driedretrieved PLLA samples were analysed using a PerkinElmer DSC 6 (Beaconsfield Buckinghamshire UK) test-

Fig 1 PLLA tensile and extruded rod test samplesing machine over a temperature range of 40 degC to 200 degC

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311DEGRADATION OF POLY-L-LACTIDE PART 1

Table 2 In vitro and in vivo molecular weight results versus time results (PI=polydispersity index)

Tensile samples Extruded rod samples

Degradation In vitro In vitro In vivotime(weeks) Mw Mn PI Mw Mn PI Mw Mn PI

0 424 000 158 500 267 339 000 155 000 218 399 000 155 000 2574 339 000 143 000 237 312 500 129 000 242

10 309 000 120 000 258 249 000 85 250 29 226 500 87 750 25620 199 000 72 500 274 176 000 82 900 21226 199 000 93 850 211 149 000 70 150 213 153 500 70 050 22332 159 000 65 800 24 127 000 60 800 20838 133 500 53 050 251 89 100 40 850 218 105 500 54 250 19444 74 900 22 500 333 76 400 37 850 202 69 800 32 600 215

Table 3 Thermal properties of extruded PLLA rod in vitro

Degradation time(weeks) crystallinity Tm degC Tg onset degC

0 407 1842 68910 455 1846 70726 449 1848 67232 408 1838 65538 534 1836 63644 536 1824 587

Considering the data presented in Table 2 in con-junction with equations (3) and (6) Fig 4 presentsplots of 1Mn and ln Mn versus time modelling theuncatalysed and autocatalysed degradation models withlinear trendlines fitted and R2 correlation coefficientsdisplayed In both cases a higher correlation coefficientwas achieved for the ln Mn versus time relationshipFig 2 Degraded PLLA tensile samples at 37 degC in vitrodescribing the autocatalytic degradation mechanism

The GPC chromatograms for the PLLA tensile32 Molecular weight versus timesamples remained monomodal throughout successive

The molecular weights of the PLLA tensile and extruded weeks of degradation (Fig 5) and shifted towards lowerrod samples in vitro and in vivo decreased with time molecular weights as degradation time increased A(Table 2) After 44 weeks the Mw of the extruded rod similar trend was observed for the extruded rod samplessamples decreased by approximately 80 per cent while in vitro and in vivothe Mw for the tensile samples in vitro and extruded rodin vivo decreased by approximately 82 per cent A similardecreasing trend was also observed for the extruded rod 33 Mass change versus timeand tensile samplesrsquo Mn in vitro and in vivo (Fig 3)

Before drying a similar pattern for the percentage massHowever no obvious pattern in polydispersity indexchange of both the tensile and extruded rod samples(PI ) (Table 3) the ratio of MwMn with time could bein vitro was observed (Fig 6) After four weeks the massderived from the molecular weight dataof both sets of samples had increased by approximately06 per cent this increase remained relatively constantup until about week 44 when the polymerrsquos massincreased to approximately 1 per cent Further increasesin mass before drying were observed throughout sub-sequent weeks up to approximately 25 per cent for boththe tensile and extruded rod samples at 65 weeks Afterdrying a similar pattern was again observed for bothsets of samples with minimal mass loss observed at 57weeks for both the tensile and extruded rod samplesHowever at 65 weeks the tensile samplesrsquo mass had

Fig 3 Comparison between in vitro and in vivo samplesrsquo Mn decreased by approximately 12 per cent with a 09 per

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312 N A WEIR F J BUCHANAN J F ORR AND G R DICKSON

Fig 4 Uncatalysed and autocatalysed models for tensile and extruded rod samples

cent mass loss observed for the extruded rod samples Asimilar pattern was also observed for the extruded rodsamples mass change in vivo however the increases inmass before drying were not as large peaking at approxi-mately 08 per cent after 44 weeks with no significantmass loss observed after drying

34 DSC analysis versus time

For both the tensile and extruded rod samples in vitroa general trend was observed of increasing crystallinityand decreasing Tg onset temperature with degradationtime (Tables 3 and 4) Additionally a slight but signifi-Fig 5 PLLA tensile samples molecular weight distributionscant decrease in both the tensile and extruded rodat 0 10 32 and 44 weeks in vitrosamples melting point Tm was also observed after 44weeks The results of the thermal analysis conducted on

Table 4 Thermal properties of PLLA tensile samples in vitro

Degradation time(weeks) crystallinity Tm degC Tg onset degC

0 448 1821 6694 455 1814 688

10 416 1816 67720 421 1821 66826 470 1814 65732 519 1803 61938 548 1810 63244 587 1791 573

Fig 6 In vitro mass change analysis

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313DEGRADATION OF POLY-L-LACTIDE PART 1

the extruded rod in vivo also followed this general trend time with degradation proceeding further the mainmelting peak began to shift to lower temperatures untilwith the in vitro and in vivo extruded rod results proving

to be very similar eventually the two peaks merged and the smaller peakappeared as a shoulder on the larger peak (Fig 7(f ))The DSC thermograms for the tensile samples at 0 4

10 20 38 and 44 weeks are shown in Fig 7 At 0 weeks(Fig 7(a)) a small endothermic peak commencing atapproximately 67 degC was observed relating to stress 35 Mechanical strength versus timerelaxation at the polymerrsquos Tg [35] As the temperature

The mechanical strengths of both the tensile andincreased further a small endothermic dip was observed

extruded rod samples deteriorated with time with thejust before melting commenced followed by the main

tensile strength of the compression-moulded samplesmelting peak It is suggested that the dip before melting

reduced to approximately zero in 44 weeks (Table 5)was caused by some crystallization of the polymer

After 44 weeks the samples were very brittle and couldAlthough the polymer was annealed prior to degradation

not be gripped in the tensile test grips without fracturingwith the aim of limiting crystallization throughout the

A similar trend was observed initially for the loss ofstudy close to the polymerrsquos melting point the chain

shear strength for the extruded rods in vitro and in vivomobility would have increased allowing some of the

(Fig 8) However after 44 weeks in vitro the rodamorphous segments to order themselves into a more

samples had lost approximately 52 per cent of theircrystalline structure

original strength compared to only 263 per cent for theAt ten weeks (Fig 7(c)) as degradation increased

samples in vivopresumably in the amorphous regions the initial dipbefore melting observed at 0 and 4 weeks was reducedwith less amorphous regions remaining capable of

36 Biological host tissuersquos responsecrystallization

At 20 weeks (Fig 7(d)) the endothermic dip before The combination of LM TEM and SEM proved usefulin determining the relationship between the PLLA rodmelting had disappeared and a small peak appeared to

form in its place It is speculated that this new peak implants and surrounding biological tissue After 36weeks the PLLA implant appeared to stimulate the pro-represented the melting of new crystallites formed by the

crystallization of internal degradation by-products The duction of a fibrous tissue capsule (Figs 9(a) and (b))in which type-1 collagen fibre production was extensivereduction of the amorphous regions and crystallization

of the degradation by-products resulted in the polymerrsquos (Fig 9(c)) The TEM image of the fibrous capsulesurrounding the PLLA implant showed the presence ofoverall crystallinity increasing throughout degradation

(Tables 3 and 4)At weeks 38 and 44 (Figs 7(e) and (f )) the newly Table 5 Deterioration in PLLArsquos tensile properties

formed peak appeared to have grown and moved to a throughout degradationhigher temperature evidence that the newly formed

Degradation Youngrsquos Tensile Extensioncrystallitesrsquo size may have been increasing At the sametime modulus strength at break(weeks) (MPa) (MPa) (mm)

0 6684 643 164 6184 538 16

10 6252 603 1520 4330 237 0826 5287 369 1032 2265 99 0438 2842 82 0344 ndash 10 04

Fig 7 PLLA tensile samplesrsquo DSC thermograms at 0 4 10 Fig 8 Shear strength comparison of PLLA rod versus timein vitro and in vivo20 38 and 44 weeks

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314 N A WEIR F J BUCHANAN J F ORR AND G R DICKSON

the absence of cells marking an inflammatory responseat 36 and 44 weeks would suggest that the PLLA rodinvestigated was biocompatible throughout the 44 weeksduration of the study

4 DISCUSSION

41 Degradation mechanisms

The results presented show that the in vitro and in vivodegradation of PLLA commences almost immediatelywith the in vitro tensile and extruded rod samples losingapproximately 20 per cent of their initial molecularweight at four weeks the first time point analysed

Autocatalysis The higher R2 correlation coefficients(Fig 4) achieved for the ln Mn versus time relationshipshows a closer approximation of the autocatalysed model(equation (6)) to the experimental data compared tothe uncatalysed model with degradation accelerated bythe newly formed carboxylic acid end groups generatedby the continual ester hydrolysis reaction However itcannot be concluded that the mechanism is exclusivelyautocatalytic Investigating the in vitro degradation ofSR-PLLA at 37 degC Pohjonen and Tormala [36 ] observeda similar trend They reported correlation coefficients of0989 for the autocatalysed model and 0910 for theuncatalysed model confirming the findings of the presentstudy and at least according to theory suggesting anautocatalytic degradation mechanism In contrast in acomparative study investigating the molecular weightversus time data available in literature for semicrystallinealiphatic polyesters such as PLLA and amorphous poly-mers such as PDLLA Anderson [30] reported that noclear distinction could be derived between the uncatalysed1Mn and autocatalysed ln Mn plots versus time for semi-crystalline polymers However for amorphous polymersthe results were reported to be much more consistentwith an autocatalytic mechanism with higher correlationcoefficients achieved for plots of ln Mn versus timeAlthough the correlation coefficients for each study werenot given making comparisons to the present studydifficult As a result of these studies Anderson [30] con-cluded that the hydrolytic degradation of semicrystallinepolyesters may not proceed exclusively by non-catalyticor autocatalytic mechanisms speculating that both maycontribute to the rate of chain scission

Relationship between molecular weight distribution anddegradation Considering the GPC curves for the tensileFig 9 Images of PLLA biological tissue after 36 weeks

implantation samples (Fig 5) it is interesting to note that they remainedmonomodal throughout successive weeks of degradationIn contrast many researchers have reported that asfibroblasts (Fig 9(c)) in an extracellular matrix (inset)

composed extensively of type-1 collagen fibre bundles the degradation of PLA and PGA aliphatic polyestersproceeds the initially monomodal GPC curve becomesrunning in different orientations While tissue disturbance

during surgery produces an initial inflammatory response bimodal and even multimodal in nature [14] and [37]

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315DEGRADATION OF POLY-L-LACTIDE PART 1

The bimodal nature of these GPC curves was originally butions were the result of preferential degradation of theamorphous regions a view supported by Fischer et alassigned to the difference in degradation rates in the[32] It would be anticipated that due to the semicrystallineamorphous and crystalline regions [14] [32] and [37]nature of the compression-moulded and extruded PLLAHowever with molecular weight usually determined byinvestigated in the present study [25] the GPC curvestaking samples from the bulk of the polymer comprisingof both would become bimodal and even multimodalthe interior of a lower molecular weight than the surfacein nature due to the preferential degradation of the[37] Li et al [19] were the first to assign this bimodalamorphous regions However Fig 5 shows that this isbehaviour to the autocatalytic effect and faster internalnot the case with the molecular weight distributionsdegradation It is currently understood that for PLA andremaining monomodal throughout the 44 weeks durationPGA polymers and their copolymers this bimodal natureof the study contradicting the findings of Li et al [20]can be accounted for by three different mechanismsand Pistner et al [39] It must also be assumed that withrelated to the polymers morphology [14] First by fasterthe samples intended for molecular weight analysis takeninternal degradation however this mechanism is mostthrough a cross-section of the material the suspectedcommonly observed for initially amorphous polymersautocatalytic mechanism did not result in a large enoughwhich are not believed to be capable of crystallizationsurface-interior differentiation to yield curves containingeven throughout degradation [37] for example a 5050two distinct molecular weight speciescopolymer of PLA and PDLA [19] Second for semi-

crystalline polymers the bimodal nature has been attri-buted to selective degradation of the amorphous regions Bulk degradation The time delay before mass losswith the surface-interior differentiation reported not to observed in this study (Fig 6) is in agreement withbe large enough to yield bimodal GPC chromatograms the reported general sequence of aliphatic polyester[14] supporting Andersonrsquos theory [30] that the hydro- degradation which suggests molecular weight loss islytic degradation of semicrystalline polyesters does not observed first before loss of mechanical strength andproceed exclusively by non-catalytic or autocatalytic before any physical mass loss is observed [8] This ismechanisms Finally the bimodal nature of the GPC accounted for by the fact that water diffusion into thechromatograms has been attributed to the crystallization polymer is faster than the hydrolytic degradation ofof low molecular weight degradation by-products in the polymerrsquos ester linkage suggesting that ester-bondinitially amorphous polymers for example amorphous cleavage is the rate-limiting step in the degradation ofPLLA and a 7525 PLAPGA copolymer [38] which are aliphatic polyesters [40] This results in degradation pro-capable of crystallizing throughout degradation Once ceeding in the bulk of the polymer resulting in a time-

lag before any mass loss is observed as the polymerrsquosthe low molecular by-products crystallize they becomemolecular weight has to be reduced to a critical valueresistant to degradation and appear as a low molecularbefore soluble oligomers can be released In contrast forweight peak on the GPC curvebioresorbable polymers regarded as surface erodingThe monomodal nature of the GPC curves obtainedsuch as those belonging to the polyanhydride and poly-for the semicrystalline PLLA prepared by annealingorthoester families [41] mass loss is observed almostand investigated in this study appears to contradictimmediately as the chain scission of their more reactivethe findings of other researchers investigating similarunstable linkages in comparison to the ester linkage insemicrystalline PLLA Li et al [20] investigating thealiphatic polyesters is faster than the diffusion of waterdegradation of semicrystalline PLLA prepared by anneal-molecules into the polymer [40]ing at 130 degC for two hours with an initial crystallinity

of 72 per cent deduced from XRD measurementsobserved that the initial monomodal molecular weight Polymer morphology and degradation The results of thedistribution became multimodal after 18 weeks After 50 DSC analysis (Fig 7) appear to provide evidence thatweeks Li et al [20] observed that the GPC curve became the low molecular weight degradation by-products arebimodal with the peak corresponding to high molecular capable of crystallizing due to their greater mobilityweight being more prominant for the surface than for and contribute to the samplesrsquo increasing crystallinitythe centre suggesting autocatalysis At 90 weeks the This is evident by the emergence of a small peak formingGPC chromatogram then became almost monomodal and eventually merging with the main melting peak Theand was composed of a single low molecular weight crystallization of these internal degradation by-productspeak Pistner et al [39] observed a similar profile for the resulted in the polymer maintaining its structural integrityGPC chromatograms of semicrystalline PLLA with an throughout the duration of the study In contrast hollowinitial crystallinity of 73 per cent measured by DSC with structures have been reported for intrinsically amorphousa low molecular weight shoulder observed after eight polymers since their degradation products are notweeks becoming more important as degradation time believed to be capable of crystallizing for example inproceeded Both Li et al [20] and Pistner et al [39] the case of a 5050 copolymer of PLLAPDLLA [19]

The decreasing peak melting temperature observed mostconcluded that the multimodal molecular weight distri-

H01004 copy IMechE 2004 Proc Instn Mech Engrs Vol 218 Part H J Engineering in Medicine

316 N A WEIR F J BUCHANAN J F ORR AND G R DICKSON

significantly at 44 weeks (Tables 3 and 4) and deter- In vitro and in vivo degradation rates Literature regard-ing the role of enzymes on the degradation of aliphaticmined from a single heating cycle is consistent with the

hypothesis that the initially crystalline regions are resistant polyesters is often contradictory Many authors havereported that enzymes may be involved in the latter stagesto degradation resulting in a two-stage degradation mech-

anism with the amorphous regions being preferentially of degradation when the polymer has fragmented and themolecular weight is sufficiently small [47ndash49] Howeverattacked [14] However once the amorphous regions have

been exhausted the less accessible crystalline regions the role of enzymes during hydrolysis of the polymerbulk remains unclear In comparative in vitro and in vivoare then solely attacked and disrupted resulting in a

decreased size of the initially present crystallites and studies Vasenius et al [50] have reported significantlyfaster degradation of PGA rods in vivo with Matsusuehence a reduced melting point [42] Although the melt-

ing point of bioresorbable polymers is also known to be et al [51] also reporting faster in vivo degradationof PLLA In each case the faster in vivo degradation ratedependent on molecular weight the extent of this is most

readily determined by considering a reheat DSC run was attributed in some part to the action of enzymesHowever comparative studies by Hooper et al [52] andSince the fusion of the first run destroys the polymerrsquos

initial crystalline structure crystallization on cooling Pitt et al [53] have reported no significant differences inthe degradation rates of poly(a-hydroxy acids) in vivoinvolves the degraded chains only [36 ] confirming that

in the present study the decrease in melting temperature with Cam et al [54] reporting their degradation tobe practically independent of enzymes The similaritydetermined from a single heating cycle was most likely a

result of a reduction in crystallite size and not decreasing between the results of the molecular weight (Fig 3) andDSC analysis for the PLLA rods investigated in vitromolecular weight It is speculated that the decreasing Tg

observed as degradation time increased (Tables 3 and 4) and in vivo in the present study suggests that thedegradation of PLLA is independent of enzymes and inis related to the reduction in molecular weight of the

polymerrsquos chains in the amorphous regions with a similar agreement with Timmins and Lenz [55] who reportedthat enzymes capable of catalysing hydrolysis are them-trend also observed by Li [14] Duek et al [43] Joukainen

et al [44] and Kellomaki et al [45] Interestingly a selves macromolecules unable to penetrate into the poly-mer bulk Therefore any enzyme-contributed reactionsmall amount of water within a polymer is also known

to have a marked plasticizing effect causing a reduction would be heterogeneous and confined to the surface ofthe polymer with a reduction in mass observed but littlein the polymerrsquos Tg A study by Siemann [46 ] investi-

gating the influence of water on the glass transition of change in the polymers overall molecular weight [4]The significant loss of molecular weight (Table 2) andpoly(dl-lactic acid) by DSC reported a 12 K decrease

in Tg after samples were exposed to water for six hours negligible mass loss (Fig 6) observed for the PLLA rodinvestigated in vivo in the present study would suggestprior to testing However in a further study investi-

gating samples exposed to water and then dried to a that degradation proceeded predominantly in the bulkof the polymer by non-enzymatic hydrolysis similar toconstant mass before testing the Tg remained the same

as the untreated samples In the present study the the mechanism observed in vitro However this doesnot rule out the influence of enzymes at later stages ofsamples were dried to constant mass before DSC testing

was conducted ruling out water acting as a plasticizer the degradation process particularly when mass lossbecomes significantand confirming a reduction in molecular weight as the

most probable cause for the decreasing trend in TgHowever this underlines the problem that for accurateTg measurements representative of the polymerrsquos con-

42 Biological responsedition in service test regimes need to be developed thatcan accurately monitor the polymerrsquos Tg while the The production of a fibrous capsule around bio-

resorbable implants has been observed previously [56ndash58]samples remain lsquowetrsquoand is regarded as part of the bodyrsquos natural responseto implants made of diverse materials [49] SurgicalMechanical strength Since degradation predominantly

occurred in the amorphous regions disrupting the tie intervention such as the implantation procedure under-taken in this study would initiate inflammation as achains holding the crystallites together coupled with the

decreasing molecular weight and increasing crystallinity response to injury However the absence of inflam-matory cells at 36 and 44 weeks suggests that PLLAit is not surprising that the mechanical properties of the

PLLA investigated decreased so rapidly However con- is biocompatible throughout the early stages of itsdegradation It is understood that the onset of mass losssidering the similarities between the molecular weight

loss and the results derived from the DSC analysis for particularly in fast degrading aliphatic polyesters suchas PGA can result in an inflammatory reaction due tothe extruded PLLA rod in vitro and in vivo it is difficult

to speculate at this stage why the samples in vitro the sudden release of acidic degradation by-productscausing a large change in pH of the surrounding mediaappeared to lose their strength more rapidly than those

in vivo [59] In the present investigation the PLLA degradation

H01004 copy IMechE 2004Proc Instn Mech Engrs Vol 218 Part H J Engineering in Medicine

317DEGRADATION OF POLY-L-LACTIDE PART 1

scaffolds and cells In Synthetic Biodegradable Polymerstudy in vivo was terminated before any mass loss wasScaffolds (Eds A Atala and D J Mooney) 1997 pp 1ndash14observed although it is speculated that any inflam-(Birkhauser Boston MA USA)matory response observed as a direct result of the onset

4 Li S and Vert M Biodegradation of aliphatic poly-of polymer mass loss would be mild In comparison toesters In Degradable Polymers Principles amp Applicationsfast degrading PGA implants it is anticipated that the(Eds G Scott and D Gilead) 1995 pp 43ndash87 (Chapman

release of acidic degradation products from the slower amp Hall London)degrading PLLA would be less intense This would 5 Chu C C Biodegradable polymeric biomaterials anresult in the surrounding tissue being more capable of overview In The Biomedical Engineering Handbookeliminating any such debris more efficiently reducing the (Ed J D Bronzino) 1995 pp 611ndash626 (CRC Press Boca

Raton FL USA)risk of a severe inflammatory reaction developing that6 Higgins N A Condensation of Polymers of Hydroxyaceticwould require further surgical intervention

Acid US Patent 2 676 945 19547 Schneider A K Polymers of High Melting Lactide US

Patent 2 703 316 19555 CONCLUSIONS8 Middleton J C and Tipton A J Synthetic biodegradable

polymers as orthopedic devices Biomaterials 2000 21The results of the analytical characterization studies 2335ndash2346conducted on the retrieved PLLA samples in vitro and 9 Kulkarni R K Pani K C Neuman C and Leonard Fin vivo provides strong evidence to support the findings Polylactic acid for surgical implants Arch Surg 1966of other researchers investigating similar bioresorbable 93 839ndash843polymers Additionally the results from the in vivo 10 Hofmann G O Biodegradable implants in orthopaedic

surgerymdasha review on the state-of-the art Clin Materstudies would suggest that throughout the first stage of1992 10 75ndash80degradation before mass loss is observed PLLA is bio-

11 Ciccone W J Motz C Bentley C and Tasto J Pcompatible and degrades at the same rate in vitro andBioabsorbable implants in orthopaedics new developmentsin vivo However the results of the present studies doand clinical applications J Am Acad Orthop Surg 2001appear to indicate that for semicrystalline polymers like 9 280ndash288

the PLLA investigated no clear differentiation between 12 Barber F A Resorbable materials for arthroscopicsurface and interior degradation could be observed that fixation a product guide Orthopedic Special Edn 2002would clearly point to an autocatalytic degradation 8 29ndash37mechanism As a result it is speculated that as poly- 13 Hayashi T Biodegradable polymers for biomedical uses

Prog Polym Sci 1994 19 663ndash702mer crystallinity increases the importance of the auto-14 Li S Hydrolytic degradation characteristics of aliphaticcatalysis degradation mechanism may become less

polyesters derived from lactic and glycolic acids J BiomedsignificantMater Res (Appl Biomater) 1999 48 342ndash353

15 Vert M Li S and Garreau H New insights on thedegradation of bioresorbable polymeric devices based onACKNOWLEDGEMENTSlactic and glycolic acids Clin Mater 1992 10 3ndash8

16 Ali S Doherty P J and Williams D F MechanismsThe authors would like to thank Mr David Farrar of polymer degradation in implantable devices 2at Smith amp Nephew Group Research Centre (York Poly(DL-lactic acid) J Biomed Mater Res 1993 27UK) Boehringer Ingelheim (Ingelheim Germany) for 1409ndash1418

17 Chu C C Degradation and biocompatibility of syn-supplying the PLLA Griffith Microscience (Derbyshirethetic absorbable suture materials general biodegradationUK) for the ethylene oxide sterilization and Rapraphenomena and some factors affecting biodegradationTechnology Limited (Shropshire UK) for the molecularIn Biomedical Applications of Synthetic Biodegradableweight characterization Finally the EPSRC (SwindonPolymers (Ed J O Hollinger) 1995 pp 103ndash128 (CRCUK) for financial assistancePress Boca Raton FL USA)

18 Mainil-Varlet P Curtis R and Gogolewski S Effect ofin vivo and in vitro degradation on molecular and mech-

REFERENCES anical properties of various low-molecular-weight poly-lactides J Biomed Mater Res 1997 36 360ndash380

1 Tormala P Pohjonen T and Rokkanen P Bioabsorbable 19 Li S M Garreau H and Vert M Structure-propertypolymers materials technology and surgical applications relationships in the case of the degradation of massiveProc Instn Mech Engrs Part H J Engineering in Medicine aliphatic poly-(a-hydroxy acids) in aqueous media Part 11998 212 101ndash112 poly(DL-lactic acid) J Mater Sci Mater Med 1990

2 Chasin M Biodegradable polymers for controlled drug 1 123ndash130delivery In Biomedical Applications of Synthetic Bio- 20 Li S Garreau H and Vert M Structure-propertydegradable Polymers (Ed J O Hollinger) 1995 pp 1ndash15 relationships in the case of the degradation of massive(CRC Press Boca Raton FL USA) poly(a-hydroxy acids) in aqueous media Part 3 influence

3 Chaignaud B E Langer R and Vacanti J P The history of the morphology of poly(L-lactic acid) J Mater SciMater Med 1990 1 198ndash206of tissue engineering using synthetic biodegradable polymer

H01004 copy IMechE 2004 Proc Instn Mech Engrs Vol 218 Part H J Engineering in Medicine

308 N A WEIR F J BUCHANAN J F ORR AND G R DICKSON

a-hydroxy acids because of their poor thermal and hydro- molecular weight mechanical strength crystallinity andmass change at predetermined time intervals throughoutlytic stabilities which did not allow them to be used as

regular plastics for long-term industrial use [8] Never- degradation and is a follow up to a previous study thatexamined the processing annealing and sterilization oftheless this very instability has proven to be of immense

importance enabling PGA PLA and other aliphatic PLLA [25] Additionally this study examines the in vivohost tissuersquos biological response to determine the bio-polyesters to be developed as synthetic bioresorbable

polymers for medical use compatibility of the processed material Test techniquesutilized include tensile and shear testing for mechanicalPLA was first investigated for medical use by Kulkarni

et al [9] who reasoned that it would be useful for strength differential-scanning calorimetry (DSC) to deter-mine thermal properties and crystallinity gel-permeationsurgical implants since hydrolysis would yield lactic acid

a normal intermediate of carbohydrate metabolism and a chromatography (GPC) to measure molecular weightand conventional histological analysis techniques forproduct of muscle contraction [10] Due to the asymmetry

of the PLA molecule it exists as two optical isomers examining any inflammatory response to the implantsin vivo The aim of the study is to develop a completepoly-D-lactide (PDLA) and poly-L-lactide (PLLA)

with a synthetic blend of D-lactide and L-lactide yielding understanding of the processed PLLArsquos degradationprofile and to provide the necessary control data forpoly-DL-lactide (PDLLA) [11] PDLLA is an amorphous

polymer while PLLA the naturally occurring isomer investigation of methods to accelerate the degradationof PLLA relative to its physiological degradation rateis semicrystalline and the most common bioresorbable

polymer used for orthopaedic devices [12] due to itsrelatively high tensile strength and low elongation PLLA

2 MATERIALS AND METHODSlike all aliphatic polyesters degrades in vivo throughsimple hydrolysis of the hydrolytically unstable ester

21 Materialslinkage in the polymerrsquos backbone with the degradationproducts ultimately metabolized to carbon dioxide and The polymer studied in this investigation poly-L-lactidewater and eliminated from the body [13] (PLLA) ResomerA L (batch number 26033) was sup-

For semicrystalline aliphatic polyesters like PLLA plied in a sealed moisture-proof container by Boehringerthere is a general concensus that degradation proceeds Ingelheim (Ingelheim Germany) in pellet formvia random bulk hydrolysis in two distinct stages [14]The first stage is characterized by the preferential attack

22 Methodsof the ester linkages in the more accessible amorphousregions [15] with the second stage characterized by the 221 Processingonset of mass loss and attack of the less accessible

The PLLA was processed by compression mouldingcrystalline regions [16ndash18] In conjunction with thisinto plates 08 mm thick and by extrusion into a 2 mmtwo-stage mechanism work by Li et al [19] examiningdiameter rod using techniques detailed previously bythe degradation of PDLLA showed conclusively thatWeir et al [25] ASTM D638-99 type-V tensile samplesdegradation occurs more rapidly in the centre than atwere then cut from the compression-moulded plates andthe surface This is known as heterogeneous degradation30 mm lengths cut from the extruded rod The tensileand is widely accepted as occurring in both lactide andand extruded rod samples were then annealed at 120 degCglycolide polymers [20] and [21] This heterogeneous orfor a period of four hours in a preheated air-circulatingautocatalytic degradation mechanism results from theoven (see Fig 1) prior to being sterilized using ethylenehydrolytic cleavage of the ester bonds forming new acidicoxide gas (EtO) by Griffith Microscience (Derbyshirecarboxyl end groups As degradation proceeds the solubleUK) on their standard EtO cycle for medical polymersoligomers produced close to the surface can escape whileie lsquoCycle 33rsquo [25]those in the centre cannot diffuse out of the polymer

This results in a higher internal acidity with the carboxyl222 In vitro degradation

end groups catalysing the ester hydrolysis reactionand a differentiation between the surface and interior The initial mass m0 of each of the tensile and extruded

rod samples were recorded Individual specimens weredegradation rates The degradation rate of PLLA andaliphatic polyesters in general is strongly related to their then placed in 28 ml screw-top glass bottles fully

immersed in a pH 74 phosphate-buffered solution inmaterial properties with crystallinity molecular weightand distribution orientation unreacted monomer and accordance with ISO 158141999 and placed in an air-

circulating oven maintaining the temperature at a con-the presence of impurities all playing significant roles[1] This is illustrated with reports on the time for the stant 37 degC The pH of the solutions was periodically

monitored throughout the duration of each of the studiescomplete resorption of PLLA ranging from 80 weeks[22] to 57 years [23] and [24] although in each case the ratio of the phosphate-buffered

solution in millimetres to the polymerrsquos mass in gramsThis study investigates the in vitro and in vivodegradation of PLLA by evaluating its properties of was greater than 301

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309DEGRADATION OF POLY-L-LACTIDE PART 1

Eight PLLA extruded rod samples were removed at strengths of the retrieved extruded rod samples weremeasured as an indication of mechanical strength Theeach follow-up time (five mechanical test samples and

three mass-change samples) while six PLLA tensile shear test employed was adapted from BS 2782 Part 3Method 340B 1978 Determination of Shear Strength ofsamples were removed at each follow-up time (three

mechanical test samples and three mass change samples) Sheet Material and was similar to the method employedby Suuronen et al [26 ] The retrieved PLLA rod samplesFollow-up times for the in vitro studies are given in

Table 1 were slotted into a hole aligned between two halves ofa shear bracket and sheared simultaneously at both ends

223 In vivo degradation under a constant strain rate of 5 mmmin Shear strengthin MPa was calculated from equation (1)The Sprague Dawley rat was chosen as the animal model

with the 30 mm lengths of 2 mm diameter extruded shear strength (MPa)=F2A=F2pr2 (1)PLLA rod solely investigated The samples were pre-

where F= load at maximum (N) and r=average radiuspared similarly to the rods for the in vitro studies how-of sample (mm) The overall shear strength was dividedever before sterilization the ends of each of the rods wereby two in equation (1) to account for the double shearingblunted by grinding on a Struers (Roslashdovre Denmark)action taking placerotating grinding machine using Struers silicon carbide

In accordance with ISO 158141999 the retrievedpaper (grit 1200) and the initial mass (m0) of each ofmaterial underwent mechanical testing while lsquowetrsquo withthe samples was recorded Three PLLA rod samples weretesting conducted within three hours of retrieval fromimplanted per rat one each for mechanical testing massboth the in vitro buffered solution and in vivo animalloss and histological analysis to examine the host tissuersquosmodelresponse A total of 12 male-weight-matched (350 g)

Sprague Dawley rats were used for the in vivo studies Mass change On retrieval the in vitro and in vivowith the material implanted subcutaneously in the ratsrsquo samples were dried immediately with a paper towel todorsum with the three samples placed about the dorsum remove any surface moisture before being weighed usingmidline an electronic balance (Mettler Toledo Fisher Scientific

At predetermined time intervals (Table 1) the PLLA UK) to determine the percentage swelling of the polymerrod samples intended for mechanical strength and mass- and water uptake The samples were then dried in achange analysis were surgically removed from the ratsrsquo vacuum oven (Townson+Mercer Altrincham UK) atdorsal subcutaneous tissue and separated from any approximately 30 degC for 48 hours at a vacuum of 068 baradhering tissue The rod samples intended for histological and reweighed to obtain their mass at time t(m

t) The

analysis were also removed together with surrounding overall percentage mass change after drying was thenadherent tissue and placed in a fixative solution and calculated from equation (2)stored in a refrigerator at 4 degC to preserve the tissue untilit was ready to be processed for light microscopy (LM) percentage mass change=

mtminusm0

m0times100 per cent

scanning electron microscopy (SEM) and transmissionelectron microscopy (TEM) Three rats were sacrificed

(2)at each time interval by administering an increasing doseof CO2 gas All experimental procedures were carried Molecular weight and thermal properties Followingout under approved Home Office Project and Personal mass change measurements the dried PLLA samplesLicence cover were reused for gel-permeation chromatography (GPC)

analysis to determine their weight and number average224 Characterization of retrieved in vitro and in vivomolecular weights (Mr) throughout degradation and alsomaterialfor differential-scanning calorimetry (DSC) to determine

Mechanical properties The mechanical properties of their thermal properties and percentage crystallinitythe PLLA tensile samples were determined using aJJ Lloyd EZ 50 tensile testing machine (Hampshire Molecular weight The GPC analysis was conducted

by Rapra Technology Ltd (Shropshire UK) SamplesUK) equipped with a 1 kN load cell and tested at aconstant strain rate of 10 mmmin Youngrsquos modulus were prepared by adding 10 ml of choloform solvent to

20 mg of sample taken through a cross-section of thetensile strength and extension at break were calculatedfrom each of the load versus extension curves The shear material A Pl gel-mixed bed column with a refractive

Table 1 In vitro and in vivo follow-up times

Follow up Weeks

In vitro 4 10 20 26 32 38 44 50 57 65In vivo 10 26 38 44

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310 N A WEIR F J BUCHANAN J F ORR AND G R DICKSON

index response detector was used The GPC system was at a heating rate of 10 degCmin providing measurementsof glass transition temperature Tg melting point Tm andcalibrated with polystyrene and all results were expressed

as lsquopolystyrene equivalentrsquo molecular weights It should enthalpy of fusion DHmelt in Jg The DSC results werederived from this single heating cycle to provide a truebe noted that this is a relative technique rather than an

absolute technique for determining molecular weight indication of changes in the polymersrsquo thermal propertiesand morphology as a direct result of degradation TheMolecular weight is considered to be the most

important and sensitive parameter for modelling bio- enthalpy of fusion DHmelt was then used to calculatethe polymersrsquo percentage crystallinities relative to theresorbable polymer degradation [27ndash29] In particular

with Mn directly related to the scission of the polymers enthalpy of fusion of a 100 per cent crystalline sampleof PLLA reported to be 93 Jg [32]chains a number of relationships have been derived

relating the changes in Mn with time to the hydrolysispercentage crystallinity=(DHmelt 93)times100 per cent

rate of the unstable ester linkages Anderson [30] andChu [17] reported a statistical method for relating (7)molecular weight to hydrolysis rate assuming that theextent of degradation was not large they reported the Investigation of the biological host tissuersquos response Thefollowing kinetic relationship based on the polymers Mn retrieved PLLA extruded rod samples from the in vivo

animal model were prepared and investigated using LM1Mnt=1Mn

0+kt (3)

TEM and SEM to observe structure ultrastructure andwhere Mn

t=Mn at time t Mn0=Mn at t=0 k=rate surface topography of the tissue-implant environment

constant and t=time If the theory holds true a linear Conventional techniques were used in the preparationrelationship should exist between 1Mn versus time up of each of the samples [33] and [34] Specimens foruntil the point of mass loss LM were fixed in 10 per cent buffered formaldehyde

However a disadvantage of this statistical approach while those for SEM and TEM were preserved in 3 peris that it does not account for the possibility of auto- cent glutaraldehyde in 01 M sodium cacodylate buffercatalysis accelerating the polymerrsquos degradation rate Pitt pH 72ndash74 The local host biological tissuersquos responseand Gu [31] derived a relationship based on the kinetics to the implant was analysed after 36 and 44 weeksof the ester-hydrolysis reaction accounting for auto-catalysis by the generated carboxylic acid end groupsdescribed by the rate equation 3 RESULTS

d(E)dt=minusd(COOH)dt=minusk(COOH)(H2O)(E)31 Visual examination

(4)Initially at 0 weeks the annealed PLLA tensile and

where (COOH) (H2O) and (E) represent the con- extruded rod specimens were opaque and off-whitecentrations of carboxyl end groups water and esters in colour (Fig 1) At 32 weeks small areas of therespectively tensile specimens became more intensely white and as

On further analysis of equation (4) and assuming that degradation time increased more white areas becamethe ester and water concentrations remain constant and visible (Fig 2) Both the in vitro and in vivo extrudedthe concentration of acid end groups is equal to 1Mn rod specimens remained opaque and off-white in colourit can be shown that throughout the duration of the study (Fig 1) with no

whiter areas becoming visible The sizes and shapes ofMnt=Mn

0eminuskt (5)

both the tensile and extruded rod specimens did notIf this relationship holds true a linear relationship change during ageingshould exist between the ln Mn versus time up until thepoint of mass loss

ln Mnt=minuskt+ ln Mn

0(6)

An advantage of using GPC to measure molecularweight is that it also provides information on thesamplesrsquo molecular weight distribution through con-sideration of the whole GPC chromatogram providingfurther insights into the complex nature of bioresorbablepolymer degradation

Thermal properties The thermal properties of the driedretrieved PLLA samples were analysed using a PerkinElmer DSC 6 (Beaconsfield Buckinghamshire UK) test-

Fig 1 PLLA tensile and extruded rod test samplesing machine over a temperature range of 40 degC to 200 degC

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311DEGRADATION OF POLY-L-LACTIDE PART 1

Table 2 In vitro and in vivo molecular weight results versus time results (PI=polydispersity index)

Tensile samples Extruded rod samples

Degradation In vitro In vitro In vivotime(weeks) Mw Mn PI Mw Mn PI Mw Mn PI

0 424 000 158 500 267 339 000 155 000 218 399 000 155 000 2574 339 000 143 000 237 312 500 129 000 242

10 309 000 120 000 258 249 000 85 250 29 226 500 87 750 25620 199 000 72 500 274 176 000 82 900 21226 199 000 93 850 211 149 000 70 150 213 153 500 70 050 22332 159 000 65 800 24 127 000 60 800 20838 133 500 53 050 251 89 100 40 850 218 105 500 54 250 19444 74 900 22 500 333 76 400 37 850 202 69 800 32 600 215

Table 3 Thermal properties of extruded PLLA rod in vitro

Degradation time(weeks) crystallinity Tm degC Tg onset degC

0 407 1842 68910 455 1846 70726 449 1848 67232 408 1838 65538 534 1836 63644 536 1824 587

Considering the data presented in Table 2 in con-junction with equations (3) and (6) Fig 4 presentsplots of 1Mn and ln Mn versus time modelling theuncatalysed and autocatalysed degradation models withlinear trendlines fitted and R2 correlation coefficientsdisplayed In both cases a higher correlation coefficientwas achieved for the ln Mn versus time relationshipFig 2 Degraded PLLA tensile samples at 37 degC in vitrodescribing the autocatalytic degradation mechanism

The GPC chromatograms for the PLLA tensile32 Molecular weight versus timesamples remained monomodal throughout successive

The molecular weights of the PLLA tensile and extruded weeks of degradation (Fig 5) and shifted towards lowerrod samples in vitro and in vivo decreased with time molecular weights as degradation time increased A(Table 2) After 44 weeks the Mw of the extruded rod similar trend was observed for the extruded rod samplessamples decreased by approximately 80 per cent while in vitro and in vivothe Mw for the tensile samples in vitro and extruded rodin vivo decreased by approximately 82 per cent A similardecreasing trend was also observed for the extruded rod 33 Mass change versus timeand tensile samplesrsquo Mn in vitro and in vivo (Fig 3)

Before drying a similar pattern for the percentage massHowever no obvious pattern in polydispersity indexchange of both the tensile and extruded rod samples(PI ) (Table 3) the ratio of MwMn with time could bein vitro was observed (Fig 6) After four weeks the massderived from the molecular weight dataof both sets of samples had increased by approximately06 per cent this increase remained relatively constantup until about week 44 when the polymerrsquos massincreased to approximately 1 per cent Further increasesin mass before drying were observed throughout sub-sequent weeks up to approximately 25 per cent for boththe tensile and extruded rod samples at 65 weeks Afterdrying a similar pattern was again observed for bothsets of samples with minimal mass loss observed at 57weeks for both the tensile and extruded rod samplesHowever at 65 weeks the tensile samplesrsquo mass had

Fig 3 Comparison between in vitro and in vivo samplesrsquo Mn decreased by approximately 12 per cent with a 09 per

H01004 copy IMechE 2004 Proc Instn Mech Engrs Vol 218 Part H J Engineering in Medicine

312 N A WEIR F J BUCHANAN J F ORR AND G R DICKSON

Fig 4 Uncatalysed and autocatalysed models for tensile and extruded rod samples

cent mass loss observed for the extruded rod samples Asimilar pattern was also observed for the extruded rodsamples mass change in vivo however the increases inmass before drying were not as large peaking at approxi-mately 08 per cent after 44 weeks with no significantmass loss observed after drying

34 DSC analysis versus time

For both the tensile and extruded rod samples in vitroa general trend was observed of increasing crystallinityand decreasing Tg onset temperature with degradationtime (Tables 3 and 4) Additionally a slight but signifi-Fig 5 PLLA tensile samples molecular weight distributionscant decrease in both the tensile and extruded rodat 0 10 32 and 44 weeks in vitrosamples melting point Tm was also observed after 44weeks The results of the thermal analysis conducted on

Table 4 Thermal properties of PLLA tensile samples in vitro

Degradation time(weeks) crystallinity Tm degC Tg onset degC

0 448 1821 6694 455 1814 688

10 416 1816 67720 421 1821 66826 470 1814 65732 519 1803 61938 548 1810 63244 587 1791 573

Fig 6 In vitro mass change analysis

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313DEGRADATION OF POLY-L-LACTIDE PART 1

the extruded rod in vivo also followed this general trend time with degradation proceeding further the mainmelting peak began to shift to lower temperatures untilwith the in vitro and in vivo extruded rod results proving

to be very similar eventually the two peaks merged and the smaller peakappeared as a shoulder on the larger peak (Fig 7(f ))The DSC thermograms for the tensile samples at 0 4

10 20 38 and 44 weeks are shown in Fig 7 At 0 weeks(Fig 7(a)) a small endothermic peak commencing atapproximately 67 degC was observed relating to stress 35 Mechanical strength versus timerelaxation at the polymerrsquos Tg [35] As the temperature

The mechanical strengths of both the tensile andincreased further a small endothermic dip was observed

extruded rod samples deteriorated with time with thejust before melting commenced followed by the main

tensile strength of the compression-moulded samplesmelting peak It is suggested that the dip before melting

reduced to approximately zero in 44 weeks (Table 5)was caused by some crystallization of the polymer

After 44 weeks the samples were very brittle and couldAlthough the polymer was annealed prior to degradation

not be gripped in the tensile test grips without fracturingwith the aim of limiting crystallization throughout the

A similar trend was observed initially for the loss ofstudy close to the polymerrsquos melting point the chain

shear strength for the extruded rods in vitro and in vivomobility would have increased allowing some of the

(Fig 8) However after 44 weeks in vitro the rodamorphous segments to order themselves into a more

samples had lost approximately 52 per cent of theircrystalline structure

original strength compared to only 263 per cent for theAt ten weeks (Fig 7(c)) as degradation increased

samples in vivopresumably in the amorphous regions the initial dipbefore melting observed at 0 and 4 weeks was reducedwith less amorphous regions remaining capable of

36 Biological host tissuersquos responsecrystallization

At 20 weeks (Fig 7(d)) the endothermic dip before The combination of LM TEM and SEM proved usefulin determining the relationship between the PLLA rodmelting had disappeared and a small peak appeared to

form in its place It is speculated that this new peak implants and surrounding biological tissue After 36weeks the PLLA implant appeared to stimulate the pro-represented the melting of new crystallites formed by the

crystallization of internal degradation by-products The duction of a fibrous tissue capsule (Figs 9(a) and (b))in which type-1 collagen fibre production was extensivereduction of the amorphous regions and crystallization

of the degradation by-products resulted in the polymerrsquos (Fig 9(c)) The TEM image of the fibrous capsulesurrounding the PLLA implant showed the presence ofoverall crystallinity increasing throughout degradation

(Tables 3 and 4)At weeks 38 and 44 (Figs 7(e) and (f )) the newly Table 5 Deterioration in PLLArsquos tensile properties

formed peak appeared to have grown and moved to a throughout degradationhigher temperature evidence that the newly formed

Degradation Youngrsquos Tensile Extensioncrystallitesrsquo size may have been increasing At the sametime modulus strength at break(weeks) (MPa) (MPa) (mm)

0 6684 643 164 6184 538 16

10 6252 603 1520 4330 237 0826 5287 369 1032 2265 99 0438 2842 82 0344 ndash 10 04

Fig 7 PLLA tensile samplesrsquo DSC thermograms at 0 4 10 Fig 8 Shear strength comparison of PLLA rod versus timein vitro and in vivo20 38 and 44 weeks

H01004 copy IMechE 2004 Proc Instn Mech Engrs Vol 218 Part H J Engineering in Medicine

314 N A WEIR F J BUCHANAN J F ORR AND G R DICKSON

the absence of cells marking an inflammatory responseat 36 and 44 weeks would suggest that the PLLA rodinvestigated was biocompatible throughout the 44 weeksduration of the study

4 DISCUSSION

41 Degradation mechanisms

The results presented show that the in vitro and in vivodegradation of PLLA commences almost immediatelywith the in vitro tensile and extruded rod samples losingapproximately 20 per cent of their initial molecularweight at four weeks the first time point analysed

Autocatalysis The higher R2 correlation coefficients(Fig 4) achieved for the ln Mn versus time relationshipshows a closer approximation of the autocatalysed model(equation (6)) to the experimental data compared tothe uncatalysed model with degradation accelerated bythe newly formed carboxylic acid end groups generatedby the continual ester hydrolysis reaction However itcannot be concluded that the mechanism is exclusivelyautocatalytic Investigating the in vitro degradation ofSR-PLLA at 37 degC Pohjonen and Tormala [36 ] observeda similar trend They reported correlation coefficients of0989 for the autocatalysed model and 0910 for theuncatalysed model confirming the findings of the presentstudy and at least according to theory suggesting anautocatalytic degradation mechanism In contrast in acomparative study investigating the molecular weightversus time data available in literature for semicrystallinealiphatic polyesters such as PLLA and amorphous poly-mers such as PDLLA Anderson [30] reported that noclear distinction could be derived between the uncatalysed1Mn and autocatalysed ln Mn plots versus time for semi-crystalline polymers However for amorphous polymersthe results were reported to be much more consistentwith an autocatalytic mechanism with higher correlationcoefficients achieved for plots of ln Mn versus timeAlthough the correlation coefficients for each study werenot given making comparisons to the present studydifficult As a result of these studies Anderson [30] con-cluded that the hydrolytic degradation of semicrystallinepolyesters may not proceed exclusively by non-catalyticor autocatalytic mechanisms speculating that both maycontribute to the rate of chain scission

Relationship between molecular weight distribution anddegradation Considering the GPC curves for the tensileFig 9 Images of PLLA biological tissue after 36 weeks

implantation samples (Fig 5) it is interesting to note that they remainedmonomodal throughout successive weeks of degradationIn contrast many researchers have reported that asfibroblasts (Fig 9(c)) in an extracellular matrix (inset)

composed extensively of type-1 collagen fibre bundles the degradation of PLA and PGA aliphatic polyestersproceeds the initially monomodal GPC curve becomesrunning in different orientations While tissue disturbance

during surgery produces an initial inflammatory response bimodal and even multimodal in nature [14] and [37]

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315DEGRADATION OF POLY-L-LACTIDE PART 1

The bimodal nature of these GPC curves was originally butions were the result of preferential degradation of theamorphous regions a view supported by Fischer et alassigned to the difference in degradation rates in the[32] It would be anticipated that due to the semicrystallineamorphous and crystalline regions [14] [32] and [37]nature of the compression-moulded and extruded PLLAHowever with molecular weight usually determined byinvestigated in the present study [25] the GPC curvestaking samples from the bulk of the polymer comprisingof both would become bimodal and even multimodalthe interior of a lower molecular weight than the surfacein nature due to the preferential degradation of the[37] Li et al [19] were the first to assign this bimodalamorphous regions However Fig 5 shows that this isbehaviour to the autocatalytic effect and faster internalnot the case with the molecular weight distributionsdegradation It is currently understood that for PLA andremaining monomodal throughout the 44 weeks durationPGA polymers and their copolymers this bimodal natureof the study contradicting the findings of Li et al [20]can be accounted for by three different mechanismsand Pistner et al [39] It must also be assumed that withrelated to the polymers morphology [14] First by fasterthe samples intended for molecular weight analysis takeninternal degradation however this mechanism is mostthrough a cross-section of the material the suspectedcommonly observed for initially amorphous polymersautocatalytic mechanism did not result in a large enoughwhich are not believed to be capable of crystallizationsurface-interior differentiation to yield curves containingeven throughout degradation [37] for example a 5050two distinct molecular weight speciescopolymer of PLA and PDLA [19] Second for semi-

crystalline polymers the bimodal nature has been attri-buted to selective degradation of the amorphous regions Bulk degradation The time delay before mass losswith the surface-interior differentiation reported not to observed in this study (Fig 6) is in agreement withbe large enough to yield bimodal GPC chromatograms the reported general sequence of aliphatic polyester[14] supporting Andersonrsquos theory [30] that the hydro- degradation which suggests molecular weight loss islytic degradation of semicrystalline polyesters does not observed first before loss of mechanical strength andproceed exclusively by non-catalytic or autocatalytic before any physical mass loss is observed [8] This ismechanisms Finally the bimodal nature of the GPC accounted for by the fact that water diffusion into thechromatograms has been attributed to the crystallization polymer is faster than the hydrolytic degradation ofof low molecular weight degradation by-products in the polymerrsquos ester linkage suggesting that ester-bondinitially amorphous polymers for example amorphous cleavage is the rate-limiting step in the degradation ofPLLA and a 7525 PLAPGA copolymer [38] which are aliphatic polyesters [40] This results in degradation pro-capable of crystallizing throughout degradation Once ceeding in the bulk of the polymer resulting in a time-

lag before any mass loss is observed as the polymerrsquosthe low molecular by-products crystallize they becomemolecular weight has to be reduced to a critical valueresistant to degradation and appear as a low molecularbefore soluble oligomers can be released In contrast forweight peak on the GPC curvebioresorbable polymers regarded as surface erodingThe monomodal nature of the GPC curves obtainedsuch as those belonging to the polyanhydride and poly-for the semicrystalline PLLA prepared by annealingorthoester families [41] mass loss is observed almostand investigated in this study appears to contradictimmediately as the chain scission of their more reactivethe findings of other researchers investigating similarunstable linkages in comparison to the ester linkage insemicrystalline PLLA Li et al [20] investigating thealiphatic polyesters is faster than the diffusion of waterdegradation of semicrystalline PLLA prepared by anneal-molecules into the polymer [40]ing at 130 degC for two hours with an initial crystallinity

of 72 per cent deduced from XRD measurementsobserved that the initial monomodal molecular weight Polymer morphology and degradation The results of thedistribution became multimodal after 18 weeks After 50 DSC analysis (Fig 7) appear to provide evidence thatweeks Li et al [20] observed that the GPC curve became the low molecular weight degradation by-products arebimodal with the peak corresponding to high molecular capable of crystallizing due to their greater mobilityweight being more prominant for the surface than for and contribute to the samplesrsquo increasing crystallinitythe centre suggesting autocatalysis At 90 weeks the This is evident by the emergence of a small peak formingGPC chromatogram then became almost monomodal and eventually merging with the main melting peak Theand was composed of a single low molecular weight crystallization of these internal degradation by-productspeak Pistner et al [39] observed a similar profile for the resulted in the polymer maintaining its structural integrityGPC chromatograms of semicrystalline PLLA with an throughout the duration of the study In contrast hollowinitial crystallinity of 73 per cent measured by DSC with structures have been reported for intrinsically amorphousa low molecular weight shoulder observed after eight polymers since their degradation products are notweeks becoming more important as degradation time believed to be capable of crystallizing for example inproceeded Both Li et al [20] and Pistner et al [39] the case of a 5050 copolymer of PLLAPDLLA [19]

The decreasing peak melting temperature observed mostconcluded that the multimodal molecular weight distri-

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316 N A WEIR F J BUCHANAN J F ORR AND G R DICKSON

significantly at 44 weeks (Tables 3 and 4) and deter- In vitro and in vivo degradation rates Literature regard-ing the role of enzymes on the degradation of aliphaticmined from a single heating cycle is consistent with the

hypothesis that the initially crystalline regions are resistant polyesters is often contradictory Many authors havereported that enzymes may be involved in the latter stagesto degradation resulting in a two-stage degradation mech-

anism with the amorphous regions being preferentially of degradation when the polymer has fragmented and themolecular weight is sufficiently small [47ndash49] Howeverattacked [14] However once the amorphous regions have

been exhausted the less accessible crystalline regions the role of enzymes during hydrolysis of the polymerbulk remains unclear In comparative in vitro and in vivoare then solely attacked and disrupted resulting in a

decreased size of the initially present crystallites and studies Vasenius et al [50] have reported significantlyfaster degradation of PGA rods in vivo with Matsusuehence a reduced melting point [42] Although the melt-

ing point of bioresorbable polymers is also known to be et al [51] also reporting faster in vivo degradationof PLLA In each case the faster in vivo degradation ratedependent on molecular weight the extent of this is most

readily determined by considering a reheat DSC run was attributed in some part to the action of enzymesHowever comparative studies by Hooper et al [52] andSince the fusion of the first run destroys the polymerrsquos

initial crystalline structure crystallization on cooling Pitt et al [53] have reported no significant differences inthe degradation rates of poly(a-hydroxy acids) in vivoinvolves the degraded chains only [36 ] confirming that

in the present study the decrease in melting temperature with Cam et al [54] reporting their degradation tobe practically independent of enzymes The similaritydetermined from a single heating cycle was most likely a

result of a reduction in crystallite size and not decreasing between the results of the molecular weight (Fig 3) andDSC analysis for the PLLA rods investigated in vitromolecular weight It is speculated that the decreasing Tg

observed as degradation time increased (Tables 3 and 4) and in vivo in the present study suggests that thedegradation of PLLA is independent of enzymes and inis related to the reduction in molecular weight of the

polymerrsquos chains in the amorphous regions with a similar agreement with Timmins and Lenz [55] who reportedthat enzymes capable of catalysing hydrolysis are them-trend also observed by Li [14] Duek et al [43] Joukainen

et al [44] and Kellomaki et al [45] Interestingly a selves macromolecules unable to penetrate into the poly-mer bulk Therefore any enzyme-contributed reactionsmall amount of water within a polymer is also known

to have a marked plasticizing effect causing a reduction would be heterogeneous and confined to the surface ofthe polymer with a reduction in mass observed but littlein the polymerrsquos Tg A study by Siemann [46 ] investi-

gating the influence of water on the glass transition of change in the polymers overall molecular weight [4]The significant loss of molecular weight (Table 2) andpoly(dl-lactic acid) by DSC reported a 12 K decrease

in Tg after samples were exposed to water for six hours negligible mass loss (Fig 6) observed for the PLLA rodinvestigated in vivo in the present study would suggestprior to testing However in a further study investi-

gating samples exposed to water and then dried to a that degradation proceeded predominantly in the bulkof the polymer by non-enzymatic hydrolysis similar toconstant mass before testing the Tg remained the same

as the untreated samples In the present study the the mechanism observed in vitro However this doesnot rule out the influence of enzymes at later stages ofsamples were dried to constant mass before DSC testing

was conducted ruling out water acting as a plasticizer the degradation process particularly when mass lossbecomes significantand confirming a reduction in molecular weight as the

most probable cause for the decreasing trend in TgHowever this underlines the problem that for accurateTg measurements representative of the polymerrsquos con-

42 Biological responsedition in service test regimes need to be developed thatcan accurately monitor the polymerrsquos Tg while the The production of a fibrous capsule around bio-

resorbable implants has been observed previously [56ndash58]samples remain lsquowetrsquoand is regarded as part of the bodyrsquos natural responseto implants made of diverse materials [49] SurgicalMechanical strength Since degradation predominantly

occurred in the amorphous regions disrupting the tie intervention such as the implantation procedure under-taken in this study would initiate inflammation as achains holding the crystallites together coupled with the

decreasing molecular weight and increasing crystallinity response to injury However the absence of inflam-matory cells at 36 and 44 weeks suggests that PLLAit is not surprising that the mechanical properties of the

PLLA investigated decreased so rapidly However con- is biocompatible throughout the early stages of itsdegradation It is understood that the onset of mass losssidering the similarities between the molecular weight

loss and the results derived from the DSC analysis for particularly in fast degrading aliphatic polyesters suchas PGA can result in an inflammatory reaction due tothe extruded PLLA rod in vitro and in vivo it is difficult

to speculate at this stage why the samples in vitro the sudden release of acidic degradation by-productscausing a large change in pH of the surrounding mediaappeared to lose their strength more rapidly than those

in vivo [59] In the present investigation the PLLA degradation

H01004 copy IMechE 2004Proc Instn Mech Engrs Vol 218 Part H J Engineering in Medicine

317DEGRADATION OF POLY-L-LACTIDE PART 1

scaffolds and cells In Synthetic Biodegradable Polymerstudy in vivo was terminated before any mass loss wasScaffolds (Eds A Atala and D J Mooney) 1997 pp 1ndash14observed although it is speculated that any inflam-(Birkhauser Boston MA USA)matory response observed as a direct result of the onset

4 Li S and Vert M Biodegradation of aliphatic poly-of polymer mass loss would be mild In comparison toesters In Degradable Polymers Principles amp Applicationsfast degrading PGA implants it is anticipated that the(Eds G Scott and D Gilead) 1995 pp 43ndash87 (Chapman

release of acidic degradation products from the slower amp Hall London)degrading PLLA would be less intense This would 5 Chu C C Biodegradable polymeric biomaterials anresult in the surrounding tissue being more capable of overview In The Biomedical Engineering Handbookeliminating any such debris more efficiently reducing the (Ed J D Bronzino) 1995 pp 611ndash626 (CRC Press Boca

Raton FL USA)risk of a severe inflammatory reaction developing that6 Higgins N A Condensation of Polymers of Hydroxyaceticwould require further surgical intervention

Acid US Patent 2 676 945 19547 Schneider A K Polymers of High Melting Lactide US

Patent 2 703 316 19555 CONCLUSIONS8 Middleton J C and Tipton A J Synthetic biodegradable

polymers as orthopedic devices Biomaterials 2000 21The results of the analytical characterization studies 2335ndash2346conducted on the retrieved PLLA samples in vitro and 9 Kulkarni R K Pani K C Neuman C and Leonard Fin vivo provides strong evidence to support the findings Polylactic acid for surgical implants Arch Surg 1966of other researchers investigating similar bioresorbable 93 839ndash843polymers Additionally the results from the in vivo 10 Hofmann G O Biodegradable implants in orthopaedic

surgerymdasha review on the state-of-the art Clin Materstudies would suggest that throughout the first stage of1992 10 75ndash80degradation before mass loss is observed PLLA is bio-

11 Ciccone W J Motz C Bentley C and Tasto J Pcompatible and degrades at the same rate in vitro andBioabsorbable implants in orthopaedics new developmentsin vivo However the results of the present studies doand clinical applications J Am Acad Orthop Surg 2001appear to indicate that for semicrystalline polymers like 9 280ndash288

the PLLA investigated no clear differentiation between 12 Barber F A Resorbable materials for arthroscopicsurface and interior degradation could be observed that fixation a product guide Orthopedic Special Edn 2002would clearly point to an autocatalytic degradation 8 29ndash37mechanism As a result it is speculated that as poly- 13 Hayashi T Biodegradable polymers for biomedical uses

Prog Polym Sci 1994 19 663ndash702mer crystallinity increases the importance of the auto-14 Li S Hydrolytic degradation characteristics of aliphaticcatalysis degradation mechanism may become less

polyesters derived from lactic and glycolic acids J BiomedsignificantMater Res (Appl Biomater) 1999 48 342ndash353

15 Vert M Li S and Garreau H New insights on thedegradation of bioresorbable polymeric devices based onACKNOWLEDGEMENTSlactic and glycolic acids Clin Mater 1992 10 3ndash8

16 Ali S Doherty P J and Williams D F MechanismsThe authors would like to thank Mr David Farrar of polymer degradation in implantable devices 2at Smith amp Nephew Group Research Centre (York Poly(DL-lactic acid) J Biomed Mater Res 1993 27UK) Boehringer Ingelheim (Ingelheim Germany) for 1409ndash1418

17 Chu C C Degradation and biocompatibility of syn-supplying the PLLA Griffith Microscience (Derbyshirethetic absorbable suture materials general biodegradationUK) for the ethylene oxide sterilization and Rapraphenomena and some factors affecting biodegradationTechnology Limited (Shropshire UK) for the molecularIn Biomedical Applications of Synthetic Biodegradableweight characterization Finally the EPSRC (SwindonPolymers (Ed J O Hollinger) 1995 pp 103ndash128 (CRCUK) for financial assistancePress Boca Raton FL USA)

18 Mainil-Varlet P Curtis R and Gogolewski S Effect ofin vivo and in vitro degradation on molecular and mech-

REFERENCES anical properties of various low-molecular-weight poly-lactides J Biomed Mater Res 1997 36 360ndash380

1 Tormala P Pohjonen T and Rokkanen P Bioabsorbable 19 Li S M Garreau H and Vert M Structure-propertypolymers materials technology and surgical applications relationships in the case of the degradation of massiveProc Instn Mech Engrs Part H J Engineering in Medicine aliphatic poly-(a-hydroxy acids) in aqueous media Part 11998 212 101ndash112 poly(DL-lactic acid) J Mater Sci Mater Med 1990

2 Chasin M Biodegradable polymers for controlled drug 1 123ndash130delivery In Biomedical Applications of Synthetic Bio- 20 Li S Garreau H and Vert M Structure-propertydegradable Polymers (Ed J O Hollinger) 1995 pp 1ndash15 relationships in the case of the degradation of massive(CRC Press Boca Raton FL USA) poly(a-hydroxy acids) in aqueous media Part 3 influence

3 Chaignaud B E Langer R and Vacanti J P The history of the morphology of poly(L-lactic acid) J Mater SciMater Med 1990 1 198ndash206of tissue engineering using synthetic biodegradable polymer

H01004 copy IMechE 2004 Proc Instn Mech Engrs Vol 218 Part H J Engineering in Medicine

309DEGRADATION OF POLY-L-LACTIDE PART 1

Eight PLLA extruded rod samples were removed at strengths of the retrieved extruded rod samples weremeasured as an indication of mechanical strength Theeach follow-up time (five mechanical test samples and

three mass-change samples) while six PLLA tensile shear test employed was adapted from BS 2782 Part 3Method 340B 1978 Determination of Shear Strength ofsamples were removed at each follow-up time (three

mechanical test samples and three mass change samples) Sheet Material and was similar to the method employedby Suuronen et al [26 ] The retrieved PLLA rod samplesFollow-up times for the in vitro studies are given in

Table 1 were slotted into a hole aligned between two halves ofa shear bracket and sheared simultaneously at both ends

223 In vivo degradation under a constant strain rate of 5 mmmin Shear strengthin MPa was calculated from equation (1)The Sprague Dawley rat was chosen as the animal model

with the 30 mm lengths of 2 mm diameter extruded shear strength (MPa)=F2A=F2pr2 (1)PLLA rod solely investigated The samples were pre-

where F= load at maximum (N) and r=average radiuspared similarly to the rods for the in vitro studies how-of sample (mm) The overall shear strength was dividedever before sterilization the ends of each of the rods wereby two in equation (1) to account for the double shearingblunted by grinding on a Struers (Roslashdovre Denmark)action taking placerotating grinding machine using Struers silicon carbide

In accordance with ISO 158141999 the retrievedpaper (grit 1200) and the initial mass (m0) of each ofmaterial underwent mechanical testing while lsquowetrsquo withthe samples was recorded Three PLLA rod samples weretesting conducted within three hours of retrieval fromimplanted per rat one each for mechanical testing massboth the in vitro buffered solution and in vivo animalloss and histological analysis to examine the host tissuersquosmodelresponse A total of 12 male-weight-matched (350 g)

Sprague Dawley rats were used for the in vivo studies Mass change On retrieval the in vitro and in vivowith the material implanted subcutaneously in the ratsrsquo samples were dried immediately with a paper towel todorsum with the three samples placed about the dorsum remove any surface moisture before being weighed usingmidline an electronic balance (Mettler Toledo Fisher Scientific

At predetermined time intervals (Table 1) the PLLA UK) to determine the percentage swelling of the polymerrod samples intended for mechanical strength and mass- and water uptake The samples were then dried in achange analysis were surgically removed from the ratsrsquo vacuum oven (Townson+Mercer Altrincham UK) atdorsal subcutaneous tissue and separated from any approximately 30 degC for 48 hours at a vacuum of 068 baradhering tissue The rod samples intended for histological and reweighed to obtain their mass at time t(m

t) The

analysis were also removed together with surrounding overall percentage mass change after drying was thenadherent tissue and placed in a fixative solution and calculated from equation (2)stored in a refrigerator at 4 degC to preserve the tissue untilit was ready to be processed for light microscopy (LM) percentage mass change=

mtminusm0

m0times100 per cent

scanning electron microscopy (SEM) and transmissionelectron microscopy (TEM) Three rats were sacrificed

(2)at each time interval by administering an increasing doseof CO2 gas All experimental procedures were carried Molecular weight and thermal properties Followingout under approved Home Office Project and Personal mass change measurements the dried PLLA samplesLicence cover were reused for gel-permeation chromatography (GPC)

analysis to determine their weight and number average224 Characterization of retrieved in vitro and in vivomolecular weights (Mr) throughout degradation and alsomaterialfor differential-scanning calorimetry (DSC) to determine

Mechanical properties The mechanical properties of their thermal properties and percentage crystallinitythe PLLA tensile samples were determined using aJJ Lloyd EZ 50 tensile testing machine (Hampshire Molecular weight The GPC analysis was conducted

by Rapra Technology Ltd (Shropshire UK) SamplesUK) equipped with a 1 kN load cell and tested at aconstant strain rate of 10 mmmin Youngrsquos modulus were prepared by adding 10 ml of choloform solvent to

20 mg of sample taken through a cross-section of thetensile strength and extension at break were calculatedfrom each of the load versus extension curves The shear material A Pl gel-mixed bed column with a refractive

Table 1 In vitro and in vivo follow-up times

Follow up Weeks

In vitro 4 10 20 26 32 38 44 50 57 65In vivo 10 26 38 44

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310 N A WEIR F J BUCHANAN J F ORR AND G R DICKSON

index response detector was used The GPC system was at a heating rate of 10 degCmin providing measurementsof glass transition temperature Tg melting point Tm andcalibrated with polystyrene and all results were expressed

as lsquopolystyrene equivalentrsquo molecular weights It should enthalpy of fusion DHmelt in Jg The DSC results werederived from this single heating cycle to provide a truebe noted that this is a relative technique rather than an

absolute technique for determining molecular weight indication of changes in the polymersrsquo thermal propertiesand morphology as a direct result of degradation TheMolecular weight is considered to be the most

important and sensitive parameter for modelling bio- enthalpy of fusion DHmelt was then used to calculatethe polymersrsquo percentage crystallinities relative to theresorbable polymer degradation [27ndash29] In particular

with Mn directly related to the scission of the polymers enthalpy of fusion of a 100 per cent crystalline sampleof PLLA reported to be 93 Jg [32]chains a number of relationships have been derived

relating the changes in Mn with time to the hydrolysispercentage crystallinity=(DHmelt 93)times100 per cent

rate of the unstable ester linkages Anderson [30] andChu [17] reported a statistical method for relating (7)molecular weight to hydrolysis rate assuming that theextent of degradation was not large they reported the Investigation of the biological host tissuersquos response Thefollowing kinetic relationship based on the polymers Mn retrieved PLLA extruded rod samples from the in vivo

animal model were prepared and investigated using LM1Mnt=1Mn

0+kt (3)

TEM and SEM to observe structure ultrastructure andwhere Mn

t=Mn at time t Mn0=Mn at t=0 k=rate surface topography of the tissue-implant environment

constant and t=time If the theory holds true a linear Conventional techniques were used in the preparationrelationship should exist between 1Mn versus time up of each of the samples [33] and [34] Specimens foruntil the point of mass loss LM were fixed in 10 per cent buffered formaldehyde

However a disadvantage of this statistical approach while those for SEM and TEM were preserved in 3 peris that it does not account for the possibility of auto- cent glutaraldehyde in 01 M sodium cacodylate buffercatalysis accelerating the polymerrsquos degradation rate Pitt pH 72ndash74 The local host biological tissuersquos responseand Gu [31] derived a relationship based on the kinetics to the implant was analysed after 36 and 44 weeksof the ester-hydrolysis reaction accounting for auto-catalysis by the generated carboxylic acid end groupsdescribed by the rate equation 3 RESULTS

d(E)dt=minusd(COOH)dt=minusk(COOH)(H2O)(E)31 Visual examination

(4)Initially at 0 weeks the annealed PLLA tensile and

where (COOH) (H2O) and (E) represent the con- extruded rod specimens were opaque and off-whitecentrations of carboxyl end groups water and esters in colour (Fig 1) At 32 weeks small areas of therespectively tensile specimens became more intensely white and as

On further analysis of equation (4) and assuming that degradation time increased more white areas becamethe ester and water concentrations remain constant and visible (Fig 2) Both the in vitro and in vivo extrudedthe concentration of acid end groups is equal to 1Mn rod specimens remained opaque and off-white in colourit can be shown that throughout the duration of the study (Fig 1) with no

whiter areas becoming visible The sizes and shapes ofMnt=Mn

0eminuskt (5)

both the tensile and extruded rod specimens did notIf this relationship holds true a linear relationship change during ageingshould exist between the ln Mn versus time up until thepoint of mass loss

ln Mnt=minuskt+ ln Mn

0(6)

An advantage of using GPC to measure molecularweight is that it also provides information on thesamplesrsquo molecular weight distribution through con-sideration of the whole GPC chromatogram providingfurther insights into the complex nature of bioresorbablepolymer degradation

Thermal properties The thermal properties of the driedretrieved PLLA samples were analysed using a PerkinElmer DSC 6 (Beaconsfield Buckinghamshire UK) test-

Fig 1 PLLA tensile and extruded rod test samplesing machine over a temperature range of 40 degC to 200 degC

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311DEGRADATION OF POLY-L-LACTIDE PART 1

Table 2 In vitro and in vivo molecular weight results versus time results (PI=polydispersity index)

Tensile samples Extruded rod samples

Degradation In vitro In vitro In vivotime(weeks) Mw Mn PI Mw Mn PI Mw Mn PI

0 424 000 158 500 267 339 000 155 000 218 399 000 155 000 2574 339 000 143 000 237 312 500 129 000 242

10 309 000 120 000 258 249 000 85 250 29 226 500 87 750 25620 199 000 72 500 274 176 000 82 900 21226 199 000 93 850 211 149 000 70 150 213 153 500 70 050 22332 159 000 65 800 24 127 000 60 800 20838 133 500 53 050 251 89 100 40 850 218 105 500 54 250 19444 74 900 22 500 333 76 400 37 850 202 69 800 32 600 215

Table 3 Thermal properties of extruded PLLA rod in vitro

Degradation time(weeks) crystallinity Tm degC Tg onset degC

0 407 1842 68910 455 1846 70726 449 1848 67232 408 1838 65538 534 1836 63644 536 1824 587

Considering the data presented in Table 2 in con-junction with equations (3) and (6) Fig 4 presentsplots of 1Mn and ln Mn versus time modelling theuncatalysed and autocatalysed degradation models withlinear trendlines fitted and R2 correlation coefficientsdisplayed In both cases a higher correlation coefficientwas achieved for the ln Mn versus time relationshipFig 2 Degraded PLLA tensile samples at 37 degC in vitrodescribing the autocatalytic degradation mechanism

The GPC chromatograms for the PLLA tensile32 Molecular weight versus timesamples remained monomodal throughout successive

The molecular weights of the PLLA tensile and extruded weeks of degradation (Fig 5) and shifted towards lowerrod samples in vitro and in vivo decreased with time molecular weights as degradation time increased A(Table 2) After 44 weeks the Mw of the extruded rod similar trend was observed for the extruded rod samplessamples decreased by approximately 80 per cent while in vitro and in vivothe Mw for the tensile samples in vitro and extruded rodin vivo decreased by approximately 82 per cent A similardecreasing trend was also observed for the extruded rod 33 Mass change versus timeand tensile samplesrsquo Mn in vitro and in vivo (Fig 3)

Before drying a similar pattern for the percentage massHowever no obvious pattern in polydispersity indexchange of both the tensile and extruded rod samples(PI ) (Table 3) the ratio of MwMn with time could bein vitro was observed (Fig 6) After four weeks the massderived from the molecular weight dataof both sets of samples had increased by approximately06 per cent this increase remained relatively constantup until about week 44 when the polymerrsquos massincreased to approximately 1 per cent Further increasesin mass before drying were observed throughout sub-sequent weeks up to approximately 25 per cent for boththe tensile and extruded rod samples at 65 weeks Afterdrying a similar pattern was again observed for bothsets of samples with minimal mass loss observed at 57weeks for both the tensile and extruded rod samplesHowever at 65 weeks the tensile samplesrsquo mass had

Fig 3 Comparison between in vitro and in vivo samplesrsquo Mn decreased by approximately 12 per cent with a 09 per

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312 N A WEIR F J BUCHANAN J F ORR AND G R DICKSON

Fig 4 Uncatalysed and autocatalysed models for tensile and extruded rod samples

cent mass loss observed for the extruded rod samples Asimilar pattern was also observed for the extruded rodsamples mass change in vivo however the increases inmass before drying were not as large peaking at approxi-mately 08 per cent after 44 weeks with no significantmass loss observed after drying

34 DSC analysis versus time

For both the tensile and extruded rod samples in vitroa general trend was observed of increasing crystallinityand decreasing Tg onset temperature with degradationtime (Tables 3 and 4) Additionally a slight but signifi-Fig 5 PLLA tensile samples molecular weight distributionscant decrease in both the tensile and extruded rodat 0 10 32 and 44 weeks in vitrosamples melting point Tm was also observed after 44weeks The results of the thermal analysis conducted on

Table 4 Thermal properties of PLLA tensile samples in vitro

Degradation time(weeks) crystallinity Tm degC Tg onset degC

0 448 1821 6694 455 1814 688

10 416 1816 67720 421 1821 66826 470 1814 65732 519 1803 61938 548 1810 63244 587 1791 573

Fig 6 In vitro mass change analysis

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313DEGRADATION OF POLY-L-LACTIDE PART 1

the extruded rod in vivo also followed this general trend time with degradation proceeding further the mainmelting peak began to shift to lower temperatures untilwith the in vitro and in vivo extruded rod results proving

to be very similar eventually the two peaks merged and the smaller peakappeared as a shoulder on the larger peak (Fig 7(f ))The DSC thermograms for the tensile samples at 0 4

10 20 38 and 44 weeks are shown in Fig 7 At 0 weeks(Fig 7(a)) a small endothermic peak commencing atapproximately 67 degC was observed relating to stress 35 Mechanical strength versus timerelaxation at the polymerrsquos Tg [35] As the temperature

The mechanical strengths of both the tensile andincreased further a small endothermic dip was observed

extruded rod samples deteriorated with time with thejust before melting commenced followed by the main

tensile strength of the compression-moulded samplesmelting peak It is suggested that the dip before melting

reduced to approximately zero in 44 weeks (Table 5)was caused by some crystallization of the polymer

After 44 weeks the samples were very brittle and couldAlthough the polymer was annealed prior to degradation

not be gripped in the tensile test grips without fracturingwith the aim of limiting crystallization throughout the

A similar trend was observed initially for the loss ofstudy close to the polymerrsquos melting point the chain

shear strength for the extruded rods in vitro and in vivomobility would have increased allowing some of the

(Fig 8) However after 44 weeks in vitro the rodamorphous segments to order themselves into a more

samples had lost approximately 52 per cent of theircrystalline structure

original strength compared to only 263 per cent for theAt ten weeks (Fig 7(c)) as degradation increased

samples in vivopresumably in the amorphous regions the initial dipbefore melting observed at 0 and 4 weeks was reducedwith less amorphous regions remaining capable of

36 Biological host tissuersquos responsecrystallization

At 20 weeks (Fig 7(d)) the endothermic dip before The combination of LM TEM and SEM proved usefulin determining the relationship between the PLLA rodmelting had disappeared and a small peak appeared to

form in its place It is speculated that this new peak implants and surrounding biological tissue After 36weeks the PLLA implant appeared to stimulate the pro-represented the melting of new crystallites formed by the

crystallization of internal degradation by-products The duction of a fibrous tissue capsule (Figs 9(a) and (b))in which type-1 collagen fibre production was extensivereduction of the amorphous regions and crystallization

of the degradation by-products resulted in the polymerrsquos (Fig 9(c)) The TEM image of the fibrous capsulesurrounding the PLLA implant showed the presence ofoverall crystallinity increasing throughout degradation

(Tables 3 and 4)At weeks 38 and 44 (Figs 7(e) and (f )) the newly Table 5 Deterioration in PLLArsquos tensile properties

formed peak appeared to have grown and moved to a throughout degradationhigher temperature evidence that the newly formed

Degradation Youngrsquos Tensile Extensioncrystallitesrsquo size may have been increasing At the sametime modulus strength at break(weeks) (MPa) (MPa) (mm)

0 6684 643 164 6184 538 16

10 6252 603 1520 4330 237 0826 5287 369 1032 2265 99 0438 2842 82 0344 ndash 10 04

Fig 7 PLLA tensile samplesrsquo DSC thermograms at 0 4 10 Fig 8 Shear strength comparison of PLLA rod versus timein vitro and in vivo20 38 and 44 weeks

H01004 copy IMechE 2004 Proc Instn Mech Engrs Vol 218 Part H J Engineering in Medicine

314 N A WEIR F J BUCHANAN J F ORR AND G R DICKSON

the absence of cells marking an inflammatory responseat 36 and 44 weeks would suggest that the PLLA rodinvestigated was biocompatible throughout the 44 weeksduration of the study

4 DISCUSSION

41 Degradation mechanisms

The results presented show that the in vitro and in vivodegradation of PLLA commences almost immediatelywith the in vitro tensile and extruded rod samples losingapproximately 20 per cent of their initial molecularweight at four weeks the first time point analysed

Autocatalysis The higher R2 correlation coefficients(Fig 4) achieved for the ln Mn versus time relationshipshows a closer approximation of the autocatalysed model(equation (6)) to the experimental data compared tothe uncatalysed model with degradation accelerated bythe newly formed carboxylic acid end groups generatedby the continual ester hydrolysis reaction However itcannot be concluded that the mechanism is exclusivelyautocatalytic Investigating the in vitro degradation ofSR-PLLA at 37 degC Pohjonen and Tormala [36 ] observeda similar trend They reported correlation coefficients of0989 for the autocatalysed model and 0910 for theuncatalysed model confirming the findings of the presentstudy and at least according to theory suggesting anautocatalytic degradation mechanism In contrast in acomparative study investigating the molecular weightversus time data available in literature for semicrystallinealiphatic polyesters such as PLLA and amorphous poly-mers such as PDLLA Anderson [30] reported that noclear distinction could be derived between the uncatalysed1Mn and autocatalysed ln Mn plots versus time for semi-crystalline polymers However for amorphous polymersthe results were reported to be much more consistentwith an autocatalytic mechanism with higher correlationcoefficients achieved for plots of ln Mn versus timeAlthough the correlation coefficients for each study werenot given making comparisons to the present studydifficult As a result of these studies Anderson [30] con-cluded that the hydrolytic degradation of semicrystallinepolyesters may not proceed exclusively by non-catalyticor autocatalytic mechanisms speculating that both maycontribute to the rate of chain scission

Relationship between molecular weight distribution anddegradation Considering the GPC curves for the tensileFig 9 Images of PLLA biological tissue after 36 weeks

implantation samples (Fig 5) it is interesting to note that they remainedmonomodal throughout successive weeks of degradationIn contrast many researchers have reported that asfibroblasts (Fig 9(c)) in an extracellular matrix (inset)

composed extensively of type-1 collagen fibre bundles the degradation of PLA and PGA aliphatic polyestersproceeds the initially monomodal GPC curve becomesrunning in different orientations While tissue disturbance

during surgery produces an initial inflammatory response bimodal and even multimodal in nature [14] and [37]

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315DEGRADATION OF POLY-L-LACTIDE PART 1

The bimodal nature of these GPC curves was originally butions were the result of preferential degradation of theamorphous regions a view supported by Fischer et alassigned to the difference in degradation rates in the[32] It would be anticipated that due to the semicrystallineamorphous and crystalline regions [14] [32] and [37]nature of the compression-moulded and extruded PLLAHowever with molecular weight usually determined byinvestigated in the present study [25] the GPC curvestaking samples from the bulk of the polymer comprisingof both would become bimodal and even multimodalthe interior of a lower molecular weight than the surfacein nature due to the preferential degradation of the[37] Li et al [19] were the first to assign this bimodalamorphous regions However Fig 5 shows that this isbehaviour to the autocatalytic effect and faster internalnot the case with the molecular weight distributionsdegradation It is currently understood that for PLA andremaining monomodal throughout the 44 weeks durationPGA polymers and their copolymers this bimodal natureof the study contradicting the findings of Li et al [20]can be accounted for by three different mechanismsand Pistner et al [39] It must also be assumed that withrelated to the polymers morphology [14] First by fasterthe samples intended for molecular weight analysis takeninternal degradation however this mechanism is mostthrough a cross-section of the material the suspectedcommonly observed for initially amorphous polymersautocatalytic mechanism did not result in a large enoughwhich are not believed to be capable of crystallizationsurface-interior differentiation to yield curves containingeven throughout degradation [37] for example a 5050two distinct molecular weight speciescopolymer of PLA and PDLA [19] Second for semi-

crystalline polymers the bimodal nature has been attri-buted to selective degradation of the amorphous regions Bulk degradation The time delay before mass losswith the surface-interior differentiation reported not to observed in this study (Fig 6) is in agreement withbe large enough to yield bimodal GPC chromatograms the reported general sequence of aliphatic polyester[14] supporting Andersonrsquos theory [30] that the hydro- degradation which suggests molecular weight loss islytic degradation of semicrystalline polyesters does not observed first before loss of mechanical strength andproceed exclusively by non-catalytic or autocatalytic before any physical mass loss is observed [8] This ismechanisms Finally the bimodal nature of the GPC accounted for by the fact that water diffusion into thechromatograms has been attributed to the crystallization polymer is faster than the hydrolytic degradation ofof low molecular weight degradation by-products in the polymerrsquos ester linkage suggesting that ester-bondinitially amorphous polymers for example amorphous cleavage is the rate-limiting step in the degradation ofPLLA and a 7525 PLAPGA copolymer [38] which are aliphatic polyesters [40] This results in degradation pro-capable of crystallizing throughout degradation Once ceeding in the bulk of the polymer resulting in a time-

lag before any mass loss is observed as the polymerrsquosthe low molecular by-products crystallize they becomemolecular weight has to be reduced to a critical valueresistant to degradation and appear as a low molecularbefore soluble oligomers can be released In contrast forweight peak on the GPC curvebioresorbable polymers regarded as surface erodingThe monomodal nature of the GPC curves obtainedsuch as those belonging to the polyanhydride and poly-for the semicrystalline PLLA prepared by annealingorthoester families [41] mass loss is observed almostand investigated in this study appears to contradictimmediately as the chain scission of their more reactivethe findings of other researchers investigating similarunstable linkages in comparison to the ester linkage insemicrystalline PLLA Li et al [20] investigating thealiphatic polyesters is faster than the diffusion of waterdegradation of semicrystalline PLLA prepared by anneal-molecules into the polymer [40]ing at 130 degC for two hours with an initial crystallinity

of 72 per cent deduced from XRD measurementsobserved that the initial monomodal molecular weight Polymer morphology and degradation The results of thedistribution became multimodal after 18 weeks After 50 DSC analysis (Fig 7) appear to provide evidence thatweeks Li et al [20] observed that the GPC curve became the low molecular weight degradation by-products arebimodal with the peak corresponding to high molecular capable of crystallizing due to their greater mobilityweight being more prominant for the surface than for and contribute to the samplesrsquo increasing crystallinitythe centre suggesting autocatalysis At 90 weeks the This is evident by the emergence of a small peak formingGPC chromatogram then became almost monomodal and eventually merging with the main melting peak Theand was composed of a single low molecular weight crystallization of these internal degradation by-productspeak Pistner et al [39] observed a similar profile for the resulted in the polymer maintaining its structural integrityGPC chromatograms of semicrystalline PLLA with an throughout the duration of the study In contrast hollowinitial crystallinity of 73 per cent measured by DSC with structures have been reported for intrinsically amorphousa low molecular weight shoulder observed after eight polymers since their degradation products are notweeks becoming more important as degradation time believed to be capable of crystallizing for example inproceeded Both Li et al [20] and Pistner et al [39] the case of a 5050 copolymer of PLLAPDLLA [19]

The decreasing peak melting temperature observed mostconcluded that the multimodal molecular weight distri-

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316 N A WEIR F J BUCHANAN J F ORR AND G R DICKSON

significantly at 44 weeks (Tables 3 and 4) and deter- In vitro and in vivo degradation rates Literature regard-ing the role of enzymes on the degradation of aliphaticmined from a single heating cycle is consistent with the

hypothesis that the initially crystalline regions are resistant polyesters is often contradictory Many authors havereported that enzymes may be involved in the latter stagesto degradation resulting in a two-stage degradation mech-

anism with the amorphous regions being preferentially of degradation when the polymer has fragmented and themolecular weight is sufficiently small [47ndash49] Howeverattacked [14] However once the amorphous regions have

been exhausted the less accessible crystalline regions the role of enzymes during hydrolysis of the polymerbulk remains unclear In comparative in vitro and in vivoare then solely attacked and disrupted resulting in a

decreased size of the initially present crystallites and studies Vasenius et al [50] have reported significantlyfaster degradation of PGA rods in vivo with Matsusuehence a reduced melting point [42] Although the melt-

ing point of bioresorbable polymers is also known to be et al [51] also reporting faster in vivo degradationof PLLA In each case the faster in vivo degradation ratedependent on molecular weight the extent of this is most

readily determined by considering a reheat DSC run was attributed in some part to the action of enzymesHowever comparative studies by Hooper et al [52] andSince the fusion of the first run destroys the polymerrsquos

initial crystalline structure crystallization on cooling Pitt et al [53] have reported no significant differences inthe degradation rates of poly(a-hydroxy acids) in vivoinvolves the degraded chains only [36 ] confirming that

in the present study the decrease in melting temperature with Cam et al [54] reporting their degradation tobe practically independent of enzymes The similaritydetermined from a single heating cycle was most likely a

result of a reduction in crystallite size and not decreasing between the results of the molecular weight (Fig 3) andDSC analysis for the PLLA rods investigated in vitromolecular weight It is speculated that the decreasing Tg

observed as degradation time increased (Tables 3 and 4) and in vivo in the present study suggests that thedegradation of PLLA is independent of enzymes and inis related to the reduction in molecular weight of the

polymerrsquos chains in the amorphous regions with a similar agreement with Timmins and Lenz [55] who reportedthat enzymes capable of catalysing hydrolysis are them-trend also observed by Li [14] Duek et al [43] Joukainen

et al [44] and Kellomaki et al [45] Interestingly a selves macromolecules unable to penetrate into the poly-mer bulk Therefore any enzyme-contributed reactionsmall amount of water within a polymer is also known

to have a marked plasticizing effect causing a reduction would be heterogeneous and confined to the surface ofthe polymer with a reduction in mass observed but littlein the polymerrsquos Tg A study by Siemann [46 ] investi-

gating the influence of water on the glass transition of change in the polymers overall molecular weight [4]The significant loss of molecular weight (Table 2) andpoly(dl-lactic acid) by DSC reported a 12 K decrease

in Tg after samples were exposed to water for six hours negligible mass loss (Fig 6) observed for the PLLA rodinvestigated in vivo in the present study would suggestprior to testing However in a further study investi-

gating samples exposed to water and then dried to a that degradation proceeded predominantly in the bulkof the polymer by non-enzymatic hydrolysis similar toconstant mass before testing the Tg remained the same

as the untreated samples In the present study the the mechanism observed in vitro However this doesnot rule out the influence of enzymes at later stages ofsamples were dried to constant mass before DSC testing

was conducted ruling out water acting as a plasticizer the degradation process particularly when mass lossbecomes significantand confirming a reduction in molecular weight as the

most probable cause for the decreasing trend in TgHowever this underlines the problem that for accurateTg measurements representative of the polymerrsquos con-

42 Biological responsedition in service test regimes need to be developed thatcan accurately monitor the polymerrsquos Tg while the The production of a fibrous capsule around bio-

resorbable implants has been observed previously [56ndash58]samples remain lsquowetrsquoand is regarded as part of the bodyrsquos natural responseto implants made of diverse materials [49] SurgicalMechanical strength Since degradation predominantly

occurred in the amorphous regions disrupting the tie intervention such as the implantation procedure under-taken in this study would initiate inflammation as achains holding the crystallites together coupled with the

decreasing molecular weight and increasing crystallinity response to injury However the absence of inflam-matory cells at 36 and 44 weeks suggests that PLLAit is not surprising that the mechanical properties of the

PLLA investigated decreased so rapidly However con- is biocompatible throughout the early stages of itsdegradation It is understood that the onset of mass losssidering the similarities between the molecular weight

loss and the results derived from the DSC analysis for particularly in fast degrading aliphatic polyesters suchas PGA can result in an inflammatory reaction due tothe extruded PLLA rod in vitro and in vivo it is difficult

to speculate at this stage why the samples in vitro the sudden release of acidic degradation by-productscausing a large change in pH of the surrounding mediaappeared to lose their strength more rapidly than those

in vivo [59] In the present investigation the PLLA degradation

H01004 copy IMechE 2004Proc Instn Mech Engrs Vol 218 Part H J Engineering in Medicine

317DEGRADATION OF POLY-L-LACTIDE PART 1

scaffolds and cells In Synthetic Biodegradable Polymerstudy in vivo was terminated before any mass loss wasScaffolds (Eds A Atala and D J Mooney) 1997 pp 1ndash14observed although it is speculated that any inflam-(Birkhauser Boston MA USA)matory response observed as a direct result of the onset

4 Li S and Vert M Biodegradation of aliphatic poly-of polymer mass loss would be mild In comparison toesters In Degradable Polymers Principles amp Applicationsfast degrading PGA implants it is anticipated that the(Eds G Scott and D Gilead) 1995 pp 43ndash87 (Chapman

release of acidic degradation products from the slower amp Hall London)degrading PLLA would be less intense This would 5 Chu C C Biodegradable polymeric biomaterials anresult in the surrounding tissue being more capable of overview In The Biomedical Engineering Handbookeliminating any such debris more efficiently reducing the (Ed J D Bronzino) 1995 pp 611ndash626 (CRC Press Boca

Raton FL USA)risk of a severe inflammatory reaction developing that6 Higgins N A Condensation of Polymers of Hydroxyaceticwould require further surgical intervention

Acid US Patent 2 676 945 19547 Schneider A K Polymers of High Melting Lactide US

Patent 2 703 316 19555 CONCLUSIONS8 Middleton J C and Tipton A J Synthetic biodegradable

polymers as orthopedic devices Biomaterials 2000 21The results of the analytical characterization studies 2335ndash2346conducted on the retrieved PLLA samples in vitro and 9 Kulkarni R K Pani K C Neuman C and Leonard Fin vivo provides strong evidence to support the findings Polylactic acid for surgical implants Arch Surg 1966of other researchers investigating similar bioresorbable 93 839ndash843polymers Additionally the results from the in vivo 10 Hofmann G O Biodegradable implants in orthopaedic

surgerymdasha review on the state-of-the art Clin Materstudies would suggest that throughout the first stage of1992 10 75ndash80degradation before mass loss is observed PLLA is bio-

11 Ciccone W J Motz C Bentley C and Tasto J Pcompatible and degrades at the same rate in vitro andBioabsorbable implants in orthopaedics new developmentsin vivo However the results of the present studies doand clinical applications J Am Acad Orthop Surg 2001appear to indicate that for semicrystalline polymers like 9 280ndash288

the PLLA investigated no clear differentiation between 12 Barber F A Resorbable materials for arthroscopicsurface and interior degradation could be observed that fixation a product guide Orthopedic Special Edn 2002would clearly point to an autocatalytic degradation 8 29ndash37mechanism As a result it is speculated that as poly- 13 Hayashi T Biodegradable polymers for biomedical uses

Prog Polym Sci 1994 19 663ndash702mer crystallinity increases the importance of the auto-14 Li S Hydrolytic degradation characteristics of aliphaticcatalysis degradation mechanism may become less

polyesters derived from lactic and glycolic acids J BiomedsignificantMater Res (Appl Biomater) 1999 48 342ndash353

15 Vert M Li S and Garreau H New insights on thedegradation of bioresorbable polymeric devices based onACKNOWLEDGEMENTSlactic and glycolic acids Clin Mater 1992 10 3ndash8

16 Ali S Doherty P J and Williams D F MechanismsThe authors would like to thank Mr David Farrar of polymer degradation in implantable devices 2at Smith amp Nephew Group Research Centre (York Poly(DL-lactic acid) J Biomed Mater Res 1993 27UK) Boehringer Ingelheim (Ingelheim Germany) for 1409ndash1418

17 Chu C C Degradation and biocompatibility of syn-supplying the PLLA Griffith Microscience (Derbyshirethetic absorbable suture materials general biodegradationUK) for the ethylene oxide sterilization and Rapraphenomena and some factors affecting biodegradationTechnology Limited (Shropshire UK) for the molecularIn Biomedical Applications of Synthetic Biodegradableweight characterization Finally the EPSRC (SwindonPolymers (Ed J O Hollinger) 1995 pp 103ndash128 (CRCUK) for financial assistancePress Boca Raton FL USA)

18 Mainil-Varlet P Curtis R and Gogolewski S Effect ofin vivo and in vitro degradation on molecular and mech-

REFERENCES anical properties of various low-molecular-weight poly-lactides J Biomed Mater Res 1997 36 360ndash380

1 Tormala P Pohjonen T and Rokkanen P Bioabsorbable 19 Li S M Garreau H and Vert M Structure-propertypolymers materials technology and surgical applications relationships in the case of the degradation of massiveProc Instn Mech Engrs Part H J Engineering in Medicine aliphatic poly-(a-hydroxy acids) in aqueous media Part 11998 212 101ndash112 poly(DL-lactic acid) J Mater Sci Mater Med 1990

2 Chasin M Biodegradable polymers for controlled drug 1 123ndash130delivery In Biomedical Applications of Synthetic Bio- 20 Li S Garreau H and Vert M Structure-propertydegradable Polymers (Ed J O Hollinger) 1995 pp 1ndash15 relationships in the case of the degradation of massive(CRC Press Boca Raton FL USA) poly(a-hydroxy acids) in aqueous media Part 3 influence

3 Chaignaud B E Langer R and Vacanti J P The history of the morphology of poly(L-lactic acid) J Mater SciMater Med 1990 1 198ndash206of tissue engineering using synthetic biodegradable polymer

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310 N A WEIR F J BUCHANAN J F ORR AND G R DICKSON

index response detector was used The GPC system was at a heating rate of 10 degCmin providing measurementsof glass transition temperature Tg melting point Tm andcalibrated with polystyrene and all results were expressed

as lsquopolystyrene equivalentrsquo molecular weights It should enthalpy of fusion DHmelt in Jg The DSC results werederived from this single heating cycle to provide a truebe noted that this is a relative technique rather than an

absolute technique for determining molecular weight indication of changes in the polymersrsquo thermal propertiesand morphology as a direct result of degradation TheMolecular weight is considered to be the most

important and sensitive parameter for modelling bio- enthalpy of fusion DHmelt was then used to calculatethe polymersrsquo percentage crystallinities relative to theresorbable polymer degradation [27ndash29] In particular

with Mn directly related to the scission of the polymers enthalpy of fusion of a 100 per cent crystalline sampleof PLLA reported to be 93 Jg [32]chains a number of relationships have been derived

relating the changes in Mn with time to the hydrolysispercentage crystallinity=(DHmelt 93)times100 per cent

rate of the unstable ester linkages Anderson [30] andChu [17] reported a statistical method for relating (7)molecular weight to hydrolysis rate assuming that theextent of degradation was not large they reported the Investigation of the biological host tissuersquos response Thefollowing kinetic relationship based on the polymers Mn retrieved PLLA extruded rod samples from the in vivo

animal model were prepared and investigated using LM1Mnt=1Mn

0+kt (3)

TEM and SEM to observe structure ultrastructure andwhere Mn

t=Mn at time t Mn0=Mn at t=0 k=rate surface topography of the tissue-implant environment

constant and t=time If the theory holds true a linear Conventional techniques were used in the preparationrelationship should exist between 1Mn versus time up of each of the samples [33] and [34] Specimens foruntil the point of mass loss LM were fixed in 10 per cent buffered formaldehyde

However a disadvantage of this statistical approach while those for SEM and TEM were preserved in 3 peris that it does not account for the possibility of auto- cent glutaraldehyde in 01 M sodium cacodylate buffercatalysis accelerating the polymerrsquos degradation rate Pitt pH 72ndash74 The local host biological tissuersquos responseand Gu [31] derived a relationship based on the kinetics to the implant was analysed after 36 and 44 weeksof the ester-hydrolysis reaction accounting for auto-catalysis by the generated carboxylic acid end groupsdescribed by the rate equation 3 RESULTS

d(E)dt=minusd(COOH)dt=minusk(COOH)(H2O)(E)31 Visual examination

(4)Initially at 0 weeks the annealed PLLA tensile and

where (COOH) (H2O) and (E) represent the con- extruded rod specimens were opaque and off-whitecentrations of carboxyl end groups water and esters in colour (Fig 1) At 32 weeks small areas of therespectively tensile specimens became more intensely white and as

On further analysis of equation (4) and assuming that degradation time increased more white areas becamethe ester and water concentrations remain constant and visible (Fig 2) Both the in vitro and in vivo extrudedthe concentration of acid end groups is equal to 1Mn rod specimens remained opaque and off-white in colourit can be shown that throughout the duration of the study (Fig 1) with no

whiter areas becoming visible The sizes and shapes ofMnt=Mn

0eminuskt (5)

both the tensile and extruded rod specimens did notIf this relationship holds true a linear relationship change during ageingshould exist between the ln Mn versus time up until thepoint of mass loss

ln Mnt=minuskt+ ln Mn

0(6)

An advantage of using GPC to measure molecularweight is that it also provides information on thesamplesrsquo molecular weight distribution through con-sideration of the whole GPC chromatogram providingfurther insights into the complex nature of bioresorbablepolymer degradation

Thermal properties The thermal properties of the driedretrieved PLLA samples were analysed using a PerkinElmer DSC 6 (Beaconsfield Buckinghamshire UK) test-

Fig 1 PLLA tensile and extruded rod test samplesing machine over a temperature range of 40 degC to 200 degC

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311DEGRADATION OF POLY-L-LACTIDE PART 1

Table 2 In vitro and in vivo molecular weight results versus time results (PI=polydispersity index)

Tensile samples Extruded rod samples

Degradation In vitro In vitro In vivotime(weeks) Mw Mn PI Mw Mn PI Mw Mn PI

0 424 000 158 500 267 339 000 155 000 218 399 000 155 000 2574 339 000 143 000 237 312 500 129 000 242

10 309 000 120 000 258 249 000 85 250 29 226 500 87 750 25620 199 000 72 500 274 176 000 82 900 21226 199 000 93 850 211 149 000 70 150 213 153 500 70 050 22332 159 000 65 800 24 127 000 60 800 20838 133 500 53 050 251 89 100 40 850 218 105 500 54 250 19444 74 900 22 500 333 76 400 37 850 202 69 800 32 600 215

Table 3 Thermal properties of extruded PLLA rod in vitro

Degradation time(weeks) crystallinity Tm degC Tg onset degC

0 407 1842 68910 455 1846 70726 449 1848 67232 408 1838 65538 534 1836 63644 536 1824 587

Considering the data presented in Table 2 in con-junction with equations (3) and (6) Fig 4 presentsplots of 1Mn and ln Mn versus time modelling theuncatalysed and autocatalysed degradation models withlinear trendlines fitted and R2 correlation coefficientsdisplayed In both cases a higher correlation coefficientwas achieved for the ln Mn versus time relationshipFig 2 Degraded PLLA tensile samples at 37 degC in vitrodescribing the autocatalytic degradation mechanism

The GPC chromatograms for the PLLA tensile32 Molecular weight versus timesamples remained monomodal throughout successive

The molecular weights of the PLLA tensile and extruded weeks of degradation (Fig 5) and shifted towards lowerrod samples in vitro and in vivo decreased with time molecular weights as degradation time increased A(Table 2) After 44 weeks the Mw of the extruded rod similar trend was observed for the extruded rod samplessamples decreased by approximately 80 per cent while in vitro and in vivothe Mw for the tensile samples in vitro and extruded rodin vivo decreased by approximately 82 per cent A similardecreasing trend was also observed for the extruded rod 33 Mass change versus timeand tensile samplesrsquo Mn in vitro and in vivo (Fig 3)

Before drying a similar pattern for the percentage massHowever no obvious pattern in polydispersity indexchange of both the tensile and extruded rod samples(PI ) (Table 3) the ratio of MwMn with time could bein vitro was observed (Fig 6) After four weeks the massderived from the molecular weight dataof both sets of samples had increased by approximately06 per cent this increase remained relatively constantup until about week 44 when the polymerrsquos massincreased to approximately 1 per cent Further increasesin mass before drying were observed throughout sub-sequent weeks up to approximately 25 per cent for boththe tensile and extruded rod samples at 65 weeks Afterdrying a similar pattern was again observed for bothsets of samples with minimal mass loss observed at 57weeks for both the tensile and extruded rod samplesHowever at 65 weeks the tensile samplesrsquo mass had

Fig 3 Comparison between in vitro and in vivo samplesrsquo Mn decreased by approximately 12 per cent with a 09 per

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312 N A WEIR F J BUCHANAN J F ORR AND G R DICKSON

Fig 4 Uncatalysed and autocatalysed models for tensile and extruded rod samples

cent mass loss observed for the extruded rod samples Asimilar pattern was also observed for the extruded rodsamples mass change in vivo however the increases inmass before drying were not as large peaking at approxi-mately 08 per cent after 44 weeks with no significantmass loss observed after drying

34 DSC analysis versus time

For both the tensile and extruded rod samples in vitroa general trend was observed of increasing crystallinityand decreasing Tg onset temperature with degradationtime (Tables 3 and 4) Additionally a slight but signifi-Fig 5 PLLA tensile samples molecular weight distributionscant decrease in both the tensile and extruded rodat 0 10 32 and 44 weeks in vitrosamples melting point Tm was also observed after 44weeks The results of the thermal analysis conducted on

Table 4 Thermal properties of PLLA tensile samples in vitro

Degradation time(weeks) crystallinity Tm degC Tg onset degC

0 448 1821 6694 455 1814 688

10 416 1816 67720 421 1821 66826 470 1814 65732 519 1803 61938 548 1810 63244 587 1791 573

Fig 6 In vitro mass change analysis

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313DEGRADATION OF POLY-L-LACTIDE PART 1

the extruded rod in vivo also followed this general trend time with degradation proceeding further the mainmelting peak began to shift to lower temperatures untilwith the in vitro and in vivo extruded rod results proving

to be very similar eventually the two peaks merged and the smaller peakappeared as a shoulder on the larger peak (Fig 7(f ))The DSC thermograms for the tensile samples at 0 4

10 20 38 and 44 weeks are shown in Fig 7 At 0 weeks(Fig 7(a)) a small endothermic peak commencing atapproximately 67 degC was observed relating to stress 35 Mechanical strength versus timerelaxation at the polymerrsquos Tg [35] As the temperature

The mechanical strengths of both the tensile andincreased further a small endothermic dip was observed

extruded rod samples deteriorated with time with thejust before melting commenced followed by the main

tensile strength of the compression-moulded samplesmelting peak It is suggested that the dip before melting

reduced to approximately zero in 44 weeks (Table 5)was caused by some crystallization of the polymer

After 44 weeks the samples were very brittle and couldAlthough the polymer was annealed prior to degradation

not be gripped in the tensile test grips without fracturingwith the aim of limiting crystallization throughout the

A similar trend was observed initially for the loss ofstudy close to the polymerrsquos melting point the chain

shear strength for the extruded rods in vitro and in vivomobility would have increased allowing some of the

(Fig 8) However after 44 weeks in vitro the rodamorphous segments to order themselves into a more

samples had lost approximately 52 per cent of theircrystalline structure

original strength compared to only 263 per cent for theAt ten weeks (Fig 7(c)) as degradation increased

samples in vivopresumably in the amorphous regions the initial dipbefore melting observed at 0 and 4 weeks was reducedwith less amorphous regions remaining capable of

36 Biological host tissuersquos responsecrystallization

At 20 weeks (Fig 7(d)) the endothermic dip before The combination of LM TEM and SEM proved usefulin determining the relationship between the PLLA rodmelting had disappeared and a small peak appeared to

form in its place It is speculated that this new peak implants and surrounding biological tissue After 36weeks the PLLA implant appeared to stimulate the pro-represented the melting of new crystallites formed by the

crystallization of internal degradation by-products The duction of a fibrous tissue capsule (Figs 9(a) and (b))in which type-1 collagen fibre production was extensivereduction of the amorphous regions and crystallization

of the degradation by-products resulted in the polymerrsquos (Fig 9(c)) The TEM image of the fibrous capsulesurrounding the PLLA implant showed the presence ofoverall crystallinity increasing throughout degradation

(Tables 3 and 4)At weeks 38 and 44 (Figs 7(e) and (f )) the newly Table 5 Deterioration in PLLArsquos tensile properties

formed peak appeared to have grown and moved to a throughout degradationhigher temperature evidence that the newly formed

Degradation Youngrsquos Tensile Extensioncrystallitesrsquo size may have been increasing At the sametime modulus strength at break(weeks) (MPa) (MPa) (mm)

0 6684 643 164 6184 538 16

10 6252 603 1520 4330 237 0826 5287 369 1032 2265 99 0438 2842 82 0344 ndash 10 04

Fig 7 PLLA tensile samplesrsquo DSC thermograms at 0 4 10 Fig 8 Shear strength comparison of PLLA rod versus timein vitro and in vivo20 38 and 44 weeks

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314 N A WEIR F J BUCHANAN J F ORR AND G R DICKSON

the absence of cells marking an inflammatory responseat 36 and 44 weeks would suggest that the PLLA rodinvestigated was biocompatible throughout the 44 weeksduration of the study

4 DISCUSSION

41 Degradation mechanisms

The results presented show that the in vitro and in vivodegradation of PLLA commences almost immediatelywith the in vitro tensile and extruded rod samples losingapproximately 20 per cent of their initial molecularweight at four weeks the first time point analysed

Autocatalysis The higher R2 correlation coefficients(Fig 4) achieved for the ln Mn versus time relationshipshows a closer approximation of the autocatalysed model(equation (6)) to the experimental data compared tothe uncatalysed model with degradation accelerated bythe newly formed carboxylic acid end groups generatedby the continual ester hydrolysis reaction However itcannot be concluded that the mechanism is exclusivelyautocatalytic Investigating the in vitro degradation ofSR-PLLA at 37 degC Pohjonen and Tormala [36 ] observeda similar trend They reported correlation coefficients of0989 for the autocatalysed model and 0910 for theuncatalysed model confirming the findings of the presentstudy and at least according to theory suggesting anautocatalytic degradation mechanism In contrast in acomparative study investigating the molecular weightversus time data available in literature for semicrystallinealiphatic polyesters such as PLLA and amorphous poly-mers such as PDLLA Anderson [30] reported that noclear distinction could be derived between the uncatalysed1Mn and autocatalysed ln Mn plots versus time for semi-crystalline polymers However for amorphous polymersthe results were reported to be much more consistentwith an autocatalytic mechanism with higher correlationcoefficients achieved for plots of ln Mn versus timeAlthough the correlation coefficients for each study werenot given making comparisons to the present studydifficult As a result of these studies Anderson [30] con-cluded that the hydrolytic degradation of semicrystallinepolyesters may not proceed exclusively by non-catalyticor autocatalytic mechanisms speculating that both maycontribute to the rate of chain scission

Relationship between molecular weight distribution anddegradation Considering the GPC curves for the tensileFig 9 Images of PLLA biological tissue after 36 weeks

implantation samples (Fig 5) it is interesting to note that they remainedmonomodal throughout successive weeks of degradationIn contrast many researchers have reported that asfibroblasts (Fig 9(c)) in an extracellular matrix (inset)

composed extensively of type-1 collagen fibre bundles the degradation of PLA and PGA aliphatic polyestersproceeds the initially monomodal GPC curve becomesrunning in different orientations While tissue disturbance

during surgery produces an initial inflammatory response bimodal and even multimodal in nature [14] and [37]

H01004 copy IMechE 2004Proc Instn Mech Engrs Vol 218 Part H J Engineering in Medicine

315DEGRADATION OF POLY-L-LACTIDE PART 1

The bimodal nature of these GPC curves was originally butions were the result of preferential degradation of theamorphous regions a view supported by Fischer et alassigned to the difference in degradation rates in the[32] It would be anticipated that due to the semicrystallineamorphous and crystalline regions [14] [32] and [37]nature of the compression-moulded and extruded PLLAHowever with molecular weight usually determined byinvestigated in the present study [25] the GPC curvestaking samples from the bulk of the polymer comprisingof both would become bimodal and even multimodalthe interior of a lower molecular weight than the surfacein nature due to the preferential degradation of the[37] Li et al [19] were the first to assign this bimodalamorphous regions However Fig 5 shows that this isbehaviour to the autocatalytic effect and faster internalnot the case with the molecular weight distributionsdegradation It is currently understood that for PLA andremaining monomodal throughout the 44 weeks durationPGA polymers and their copolymers this bimodal natureof the study contradicting the findings of Li et al [20]can be accounted for by three different mechanismsand Pistner et al [39] It must also be assumed that withrelated to the polymers morphology [14] First by fasterthe samples intended for molecular weight analysis takeninternal degradation however this mechanism is mostthrough a cross-section of the material the suspectedcommonly observed for initially amorphous polymersautocatalytic mechanism did not result in a large enoughwhich are not believed to be capable of crystallizationsurface-interior differentiation to yield curves containingeven throughout degradation [37] for example a 5050two distinct molecular weight speciescopolymer of PLA and PDLA [19] Second for semi-

crystalline polymers the bimodal nature has been attri-buted to selective degradation of the amorphous regions Bulk degradation The time delay before mass losswith the surface-interior differentiation reported not to observed in this study (Fig 6) is in agreement withbe large enough to yield bimodal GPC chromatograms the reported general sequence of aliphatic polyester[14] supporting Andersonrsquos theory [30] that the hydro- degradation which suggests molecular weight loss islytic degradation of semicrystalline polyesters does not observed first before loss of mechanical strength andproceed exclusively by non-catalytic or autocatalytic before any physical mass loss is observed [8] This ismechanisms Finally the bimodal nature of the GPC accounted for by the fact that water diffusion into thechromatograms has been attributed to the crystallization polymer is faster than the hydrolytic degradation ofof low molecular weight degradation by-products in the polymerrsquos ester linkage suggesting that ester-bondinitially amorphous polymers for example amorphous cleavage is the rate-limiting step in the degradation ofPLLA and a 7525 PLAPGA copolymer [38] which are aliphatic polyesters [40] This results in degradation pro-capable of crystallizing throughout degradation Once ceeding in the bulk of the polymer resulting in a time-

lag before any mass loss is observed as the polymerrsquosthe low molecular by-products crystallize they becomemolecular weight has to be reduced to a critical valueresistant to degradation and appear as a low molecularbefore soluble oligomers can be released In contrast forweight peak on the GPC curvebioresorbable polymers regarded as surface erodingThe monomodal nature of the GPC curves obtainedsuch as those belonging to the polyanhydride and poly-for the semicrystalline PLLA prepared by annealingorthoester families [41] mass loss is observed almostand investigated in this study appears to contradictimmediately as the chain scission of their more reactivethe findings of other researchers investigating similarunstable linkages in comparison to the ester linkage insemicrystalline PLLA Li et al [20] investigating thealiphatic polyesters is faster than the diffusion of waterdegradation of semicrystalline PLLA prepared by anneal-molecules into the polymer [40]ing at 130 degC for two hours with an initial crystallinity

of 72 per cent deduced from XRD measurementsobserved that the initial monomodal molecular weight Polymer morphology and degradation The results of thedistribution became multimodal after 18 weeks After 50 DSC analysis (Fig 7) appear to provide evidence thatweeks Li et al [20] observed that the GPC curve became the low molecular weight degradation by-products arebimodal with the peak corresponding to high molecular capable of crystallizing due to their greater mobilityweight being more prominant for the surface than for and contribute to the samplesrsquo increasing crystallinitythe centre suggesting autocatalysis At 90 weeks the This is evident by the emergence of a small peak formingGPC chromatogram then became almost monomodal and eventually merging with the main melting peak Theand was composed of a single low molecular weight crystallization of these internal degradation by-productspeak Pistner et al [39] observed a similar profile for the resulted in the polymer maintaining its structural integrityGPC chromatograms of semicrystalline PLLA with an throughout the duration of the study In contrast hollowinitial crystallinity of 73 per cent measured by DSC with structures have been reported for intrinsically amorphousa low molecular weight shoulder observed after eight polymers since their degradation products are notweeks becoming more important as degradation time believed to be capable of crystallizing for example inproceeded Both Li et al [20] and Pistner et al [39] the case of a 5050 copolymer of PLLAPDLLA [19]

The decreasing peak melting temperature observed mostconcluded that the multimodal molecular weight distri-

H01004 copy IMechE 2004 Proc Instn Mech Engrs Vol 218 Part H J Engineering in Medicine

316 N A WEIR F J BUCHANAN J F ORR AND G R DICKSON

significantly at 44 weeks (Tables 3 and 4) and deter- In vitro and in vivo degradation rates Literature regard-ing the role of enzymes on the degradation of aliphaticmined from a single heating cycle is consistent with the

hypothesis that the initially crystalline regions are resistant polyesters is often contradictory Many authors havereported that enzymes may be involved in the latter stagesto degradation resulting in a two-stage degradation mech-

anism with the amorphous regions being preferentially of degradation when the polymer has fragmented and themolecular weight is sufficiently small [47ndash49] Howeverattacked [14] However once the amorphous regions have

been exhausted the less accessible crystalline regions the role of enzymes during hydrolysis of the polymerbulk remains unclear In comparative in vitro and in vivoare then solely attacked and disrupted resulting in a

decreased size of the initially present crystallites and studies Vasenius et al [50] have reported significantlyfaster degradation of PGA rods in vivo with Matsusuehence a reduced melting point [42] Although the melt-

ing point of bioresorbable polymers is also known to be et al [51] also reporting faster in vivo degradationof PLLA In each case the faster in vivo degradation ratedependent on molecular weight the extent of this is most

readily determined by considering a reheat DSC run was attributed in some part to the action of enzymesHowever comparative studies by Hooper et al [52] andSince the fusion of the first run destroys the polymerrsquos

initial crystalline structure crystallization on cooling Pitt et al [53] have reported no significant differences inthe degradation rates of poly(a-hydroxy acids) in vivoinvolves the degraded chains only [36 ] confirming that

in the present study the decrease in melting temperature with Cam et al [54] reporting their degradation tobe practically independent of enzymes The similaritydetermined from a single heating cycle was most likely a

result of a reduction in crystallite size and not decreasing between the results of the molecular weight (Fig 3) andDSC analysis for the PLLA rods investigated in vitromolecular weight It is speculated that the decreasing Tg

observed as degradation time increased (Tables 3 and 4) and in vivo in the present study suggests that thedegradation of PLLA is independent of enzymes and inis related to the reduction in molecular weight of the

polymerrsquos chains in the amorphous regions with a similar agreement with Timmins and Lenz [55] who reportedthat enzymes capable of catalysing hydrolysis are them-trend also observed by Li [14] Duek et al [43] Joukainen

et al [44] and Kellomaki et al [45] Interestingly a selves macromolecules unable to penetrate into the poly-mer bulk Therefore any enzyme-contributed reactionsmall amount of water within a polymer is also known

to have a marked plasticizing effect causing a reduction would be heterogeneous and confined to the surface ofthe polymer with a reduction in mass observed but littlein the polymerrsquos Tg A study by Siemann [46 ] investi-

gating the influence of water on the glass transition of change in the polymers overall molecular weight [4]The significant loss of molecular weight (Table 2) andpoly(dl-lactic acid) by DSC reported a 12 K decrease

in Tg after samples were exposed to water for six hours negligible mass loss (Fig 6) observed for the PLLA rodinvestigated in vivo in the present study would suggestprior to testing However in a further study investi-

gating samples exposed to water and then dried to a that degradation proceeded predominantly in the bulkof the polymer by non-enzymatic hydrolysis similar toconstant mass before testing the Tg remained the same

as the untreated samples In the present study the the mechanism observed in vitro However this doesnot rule out the influence of enzymes at later stages ofsamples were dried to constant mass before DSC testing

was conducted ruling out water acting as a plasticizer the degradation process particularly when mass lossbecomes significantand confirming a reduction in molecular weight as the

most probable cause for the decreasing trend in TgHowever this underlines the problem that for accurateTg measurements representative of the polymerrsquos con-

42 Biological responsedition in service test regimes need to be developed thatcan accurately monitor the polymerrsquos Tg while the The production of a fibrous capsule around bio-

resorbable implants has been observed previously [56ndash58]samples remain lsquowetrsquoand is regarded as part of the bodyrsquos natural responseto implants made of diverse materials [49] SurgicalMechanical strength Since degradation predominantly

occurred in the amorphous regions disrupting the tie intervention such as the implantation procedure under-taken in this study would initiate inflammation as achains holding the crystallites together coupled with the

decreasing molecular weight and increasing crystallinity response to injury However the absence of inflam-matory cells at 36 and 44 weeks suggests that PLLAit is not surprising that the mechanical properties of the

PLLA investigated decreased so rapidly However con- is biocompatible throughout the early stages of itsdegradation It is understood that the onset of mass losssidering the similarities between the molecular weight

loss and the results derived from the DSC analysis for particularly in fast degrading aliphatic polyesters suchas PGA can result in an inflammatory reaction due tothe extruded PLLA rod in vitro and in vivo it is difficult

to speculate at this stage why the samples in vitro the sudden release of acidic degradation by-productscausing a large change in pH of the surrounding mediaappeared to lose their strength more rapidly than those

in vivo [59] In the present investigation the PLLA degradation

H01004 copy IMechE 2004Proc Instn Mech Engrs Vol 218 Part H J Engineering in Medicine

317DEGRADATION OF POLY-L-LACTIDE PART 1

scaffolds and cells In Synthetic Biodegradable Polymerstudy in vivo was terminated before any mass loss wasScaffolds (Eds A Atala and D J Mooney) 1997 pp 1ndash14observed although it is speculated that any inflam-(Birkhauser Boston MA USA)matory response observed as a direct result of the onset

4 Li S and Vert M Biodegradation of aliphatic poly-of polymer mass loss would be mild In comparison toesters In Degradable Polymers Principles amp Applicationsfast degrading PGA implants it is anticipated that the(Eds G Scott and D Gilead) 1995 pp 43ndash87 (Chapman

release of acidic degradation products from the slower amp Hall London)degrading PLLA would be less intense This would 5 Chu C C Biodegradable polymeric biomaterials anresult in the surrounding tissue being more capable of overview In The Biomedical Engineering Handbookeliminating any such debris more efficiently reducing the (Ed J D Bronzino) 1995 pp 611ndash626 (CRC Press Boca

Raton FL USA)risk of a severe inflammatory reaction developing that6 Higgins N A Condensation of Polymers of Hydroxyaceticwould require further surgical intervention

Acid US Patent 2 676 945 19547 Schneider A K Polymers of High Melting Lactide US

Patent 2 703 316 19555 CONCLUSIONS8 Middleton J C and Tipton A J Synthetic biodegradable

polymers as orthopedic devices Biomaterials 2000 21The results of the analytical characterization studies 2335ndash2346conducted on the retrieved PLLA samples in vitro and 9 Kulkarni R K Pani K C Neuman C and Leonard Fin vivo provides strong evidence to support the findings Polylactic acid for surgical implants Arch Surg 1966of other researchers investigating similar bioresorbable 93 839ndash843polymers Additionally the results from the in vivo 10 Hofmann G O Biodegradable implants in orthopaedic

surgerymdasha review on the state-of-the art Clin Materstudies would suggest that throughout the first stage of1992 10 75ndash80degradation before mass loss is observed PLLA is bio-

11 Ciccone W J Motz C Bentley C and Tasto J Pcompatible and degrades at the same rate in vitro andBioabsorbable implants in orthopaedics new developmentsin vivo However the results of the present studies doand clinical applications J Am Acad Orthop Surg 2001appear to indicate that for semicrystalline polymers like 9 280ndash288

the PLLA investigated no clear differentiation between 12 Barber F A Resorbable materials for arthroscopicsurface and interior degradation could be observed that fixation a product guide Orthopedic Special Edn 2002would clearly point to an autocatalytic degradation 8 29ndash37mechanism As a result it is speculated that as poly- 13 Hayashi T Biodegradable polymers for biomedical uses

Prog Polym Sci 1994 19 663ndash702mer crystallinity increases the importance of the auto-14 Li S Hydrolytic degradation characteristics of aliphaticcatalysis degradation mechanism may become less

polyesters derived from lactic and glycolic acids J BiomedsignificantMater Res (Appl Biomater) 1999 48 342ndash353

15 Vert M Li S and Garreau H New insights on thedegradation of bioresorbable polymeric devices based onACKNOWLEDGEMENTSlactic and glycolic acids Clin Mater 1992 10 3ndash8

16 Ali S Doherty P J and Williams D F MechanismsThe authors would like to thank Mr David Farrar of polymer degradation in implantable devices 2at Smith amp Nephew Group Research Centre (York Poly(DL-lactic acid) J Biomed Mater Res 1993 27UK) Boehringer Ingelheim (Ingelheim Germany) for 1409ndash1418

17 Chu C C Degradation and biocompatibility of syn-supplying the PLLA Griffith Microscience (Derbyshirethetic absorbable suture materials general biodegradationUK) for the ethylene oxide sterilization and Rapraphenomena and some factors affecting biodegradationTechnology Limited (Shropshire UK) for the molecularIn Biomedical Applications of Synthetic Biodegradableweight characterization Finally the EPSRC (SwindonPolymers (Ed J O Hollinger) 1995 pp 103ndash128 (CRCUK) for financial assistancePress Boca Raton FL USA)

18 Mainil-Varlet P Curtis R and Gogolewski S Effect ofin vivo and in vitro degradation on molecular and mech-

REFERENCES anical properties of various low-molecular-weight poly-lactides J Biomed Mater Res 1997 36 360ndash380

1 Tormala P Pohjonen T and Rokkanen P Bioabsorbable 19 Li S M Garreau H and Vert M Structure-propertypolymers materials technology and surgical applications relationships in the case of the degradation of massiveProc Instn Mech Engrs Part H J Engineering in Medicine aliphatic poly-(a-hydroxy acids) in aqueous media Part 11998 212 101ndash112 poly(DL-lactic acid) J Mater Sci Mater Med 1990

2 Chasin M Biodegradable polymers for controlled drug 1 123ndash130delivery In Biomedical Applications of Synthetic Bio- 20 Li S Garreau H and Vert M Structure-propertydegradable Polymers (Ed J O Hollinger) 1995 pp 1ndash15 relationships in the case of the degradation of massive(CRC Press Boca Raton FL USA) poly(a-hydroxy acids) in aqueous media Part 3 influence

3 Chaignaud B E Langer R and Vacanti J P The history of the morphology of poly(L-lactic acid) J Mater SciMater Med 1990 1 198ndash206of tissue engineering using synthetic biodegradable polymer

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311DEGRADATION OF POLY-L-LACTIDE PART 1

Table 2 In vitro and in vivo molecular weight results versus time results (PI=polydispersity index)

Tensile samples Extruded rod samples

Degradation In vitro In vitro In vivotime(weeks) Mw Mn PI Mw Mn PI Mw Mn PI

0 424 000 158 500 267 339 000 155 000 218 399 000 155 000 2574 339 000 143 000 237 312 500 129 000 242

10 309 000 120 000 258 249 000 85 250 29 226 500 87 750 25620 199 000 72 500 274 176 000 82 900 21226 199 000 93 850 211 149 000 70 150 213 153 500 70 050 22332 159 000 65 800 24 127 000 60 800 20838 133 500 53 050 251 89 100 40 850 218 105 500 54 250 19444 74 900 22 500 333 76 400 37 850 202 69 800 32 600 215

Table 3 Thermal properties of extruded PLLA rod in vitro

Degradation time(weeks) crystallinity Tm degC Tg onset degC

0 407 1842 68910 455 1846 70726 449 1848 67232 408 1838 65538 534 1836 63644 536 1824 587

Considering the data presented in Table 2 in con-junction with equations (3) and (6) Fig 4 presentsplots of 1Mn and ln Mn versus time modelling theuncatalysed and autocatalysed degradation models withlinear trendlines fitted and R2 correlation coefficientsdisplayed In both cases a higher correlation coefficientwas achieved for the ln Mn versus time relationshipFig 2 Degraded PLLA tensile samples at 37 degC in vitrodescribing the autocatalytic degradation mechanism

The GPC chromatograms for the PLLA tensile32 Molecular weight versus timesamples remained monomodal throughout successive

The molecular weights of the PLLA tensile and extruded weeks of degradation (Fig 5) and shifted towards lowerrod samples in vitro and in vivo decreased with time molecular weights as degradation time increased A(Table 2) After 44 weeks the Mw of the extruded rod similar trend was observed for the extruded rod samplessamples decreased by approximately 80 per cent while in vitro and in vivothe Mw for the tensile samples in vitro and extruded rodin vivo decreased by approximately 82 per cent A similardecreasing trend was also observed for the extruded rod 33 Mass change versus timeand tensile samplesrsquo Mn in vitro and in vivo (Fig 3)

Before drying a similar pattern for the percentage massHowever no obvious pattern in polydispersity indexchange of both the tensile and extruded rod samples(PI ) (Table 3) the ratio of MwMn with time could bein vitro was observed (Fig 6) After four weeks the massderived from the molecular weight dataof both sets of samples had increased by approximately06 per cent this increase remained relatively constantup until about week 44 when the polymerrsquos massincreased to approximately 1 per cent Further increasesin mass before drying were observed throughout sub-sequent weeks up to approximately 25 per cent for boththe tensile and extruded rod samples at 65 weeks Afterdrying a similar pattern was again observed for bothsets of samples with minimal mass loss observed at 57weeks for both the tensile and extruded rod samplesHowever at 65 weeks the tensile samplesrsquo mass had

Fig 3 Comparison between in vitro and in vivo samplesrsquo Mn decreased by approximately 12 per cent with a 09 per

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312 N A WEIR F J BUCHANAN J F ORR AND G R DICKSON

Fig 4 Uncatalysed and autocatalysed models for tensile and extruded rod samples

cent mass loss observed for the extruded rod samples Asimilar pattern was also observed for the extruded rodsamples mass change in vivo however the increases inmass before drying were not as large peaking at approxi-mately 08 per cent after 44 weeks with no significantmass loss observed after drying

34 DSC analysis versus time

For both the tensile and extruded rod samples in vitroa general trend was observed of increasing crystallinityand decreasing Tg onset temperature with degradationtime (Tables 3 and 4) Additionally a slight but signifi-Fig 5 PLLA tensile samples molecular weight distributionscant decrease in both the tensile and extruded rodat 0 10 32 and 44 weeks in vitrosamples melting point Tm was also observed after 44weeks The results of the thermal analysis conducted on

Table 4 Thermal properties of PLLA tensile samples in vitro

Degradation time(weeks) crystallinity Tm degC Tg onset degC

0 448 1821 6694 455 1814 688

10 416 1816 67720 421 1821 66826 470 1814 65732 519 1803 61938 548 1810 63244 587 1791 573

Fig 6 In vitro mass change analysis

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313DEGRADATION OF POLY-L-LACTIDE PART 1

the extruded rod in vivo also followed this general trend time with degradation proceeding further the mainmelting peak began to shift to lower temperatures untilwith the in vitro and in vivo extruded rod results proving

to be very similar eventually the two peaks merged and the smaller peakappeared as a shoulder on the larger peak (Fig 7(f ))The DSC thermograms for the tensile samples at 0 4

10 20 38 and 44 weeks are shown in Fig 7 At 0 weeks(Fig 7(a)) a small endothermic peak commencing atapproximately 67 degC was observed relating to stress 35 Mechanical strength versus timerelaxation at the polymerrsquos Tg [35] As the temperature

The mechanical strengths of both the tensile andincreased further a small endothermic dip was observed

extruded rod samples deteriorated with time with thejust before melting commenced followed by the main

tensile strength of the compression-moulded samplesmelting peak It is suggested that the dip before melting

reduced to approximately zero in 44 weeks (Table 5)was caused by some crystallization of the polymer

After 44 weeks the samples were very brittle and couldAlthough the polymer was annealed prior to degradation

not be gripped in the tensile test grips without fracturingwith the aim of limiting crystallization throughout the

A similar trend was observed initially for the loss ofstudy close to the polymerrsquos melting point the chain

shear strength for the extruded rods in vitro and in vivomobility would have increased allowing some of the

(Fig 8) However after 44 weeks in vitro the rodamorphous segments to order themselves into a more

samples had lost approximately 52 per cent of theircrystalline structure

original strength compared to only 263 per cent for theAt ten weeks (Fig 7(c)) as degradation increased

samples in vivopresumably in the amorphous regions the initial dipbefore melting observed at 0 and 4 weeks was reducedwith less amorphous regions remaining capable of

36 Biological host tissuersquos responsecrystallization

At 20 weeks (Fig 7(d)) the endothermic dip before The combination of LM TEM and SEM proved usefulin determining the relationship between the PLLA rodmelting had disappeared and a small peak appeared to

form in its place It is speculated that this new peak implants and surrounding biological tissue After 36weeks the PLLA implant appeared to stimulate the pro-represented the melting of new crystallites formed by the

crystallization of internal degradation by-products The duction of a fibrous tissue capsule (Figs 9(a) and (b))in which type-1 collagen fibre production was extensivereduction of the amorphous regions and crystallization

of the degradation by-products resulted in the polymerrsquos (Fig 9(c)) The TEM image of the fibrous capsulesurrounding the PLLA implant showed the presence ofoverall crystallinity increasing throughout degradation

(Tables 3 and 4)At weeks 38 and 44 (Figs 7(e) and (f )) the newly Table 5 Deterioration in PLLArsquos tensile properties

formed peak appeared to have grown and moved to a throughout degradationhigher temperature evidence that the newly formed

Degradation Youngrsquos Tensile Extensioncrystallitesrsquo size may have been increasing At the sametime modulus strength at break(weeks) (MPa) (MPa) (mm)

0 6684 643 164 6184 538 16

10 6252 603 1520 4330 237 0826 5287 369 1032 2265 99 0438 2842 82 0344 ndash 10 04

Fig 7 PLLA tensile samplesrsquo DSC thermograms at 0 4 10 Fig 8 Shear strength comparison of PLLA rod versus timein vitro and in vivo20 38 and 44 weeks

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314 N A WEIR F J BUCHANAN J F ORR AND G R DICKSON

the absence of cells marking an inflammatory responseat 36 and 44 weeks would suggest that the PLLA rodinvestigated was biocompatible throughout the 44 weeksduration of the study

4 DISCUSSION

41 Degradation mechanisms

The results presented show that the in vitro and in vivodegradation of PLLA commences almost immediatelywith the in vitro tensile and extruded rod samples losingapproximately 20 per cent of their initial molecularweight at four weeks the first time point analysed

Autocatalysis The higher R2 correlation coefficients(Fig 4) achieved for the ln Mn versus time relationshipshows a closer approximation of the autocatalysed model(equation (6)) to the experimental data compared tothe uncatalysed model with degradation accelerated bythe newly formed carboxylic acid end groups generatedby the continual ester hydrolysis reaction However itcannot be concluded that the mechanism is exclusivelyautocatalytic Investigating the in vitro degradation ofSR-PLLA at 37 degC Pohjonen and Tormala [36 ] observeda similar trend They reported correlation coefficients of0989 for the autocatalysed model and 0910 for theuncatalysed model confirming the findings of the presentstudy and at least according to theory suggesting anautocatalytic degradation mechanism In contrast in acomparative study investigating the molecular weightversus time data available in literature for semicrystallinealiphatic polyesters such as PLLA and amorphous poly-mers such as PDLLA Anderson [30] reported that noclear distinction could be derived between the uncatalysed1Mn and autocatalysed ln Mn plots versus time for semi-crystalline polymers However for amorphous polymersthe results were reported to be much more consistentwith an autocatalytic mechanism with higher correlationcoefficients achieved for plots of ln Mn versus timeAlthough the correlation coefficients for each study werenot given making comparisons to the present studydifficult As a result of these studies Anderson [30] con-cluded that the hydrolytic degradation of semicrystallinepolyesters may not proceed exclusively by non-catalyticor autocatalytic mechanisms speculating that both maycontribute to the rate of chain scission

Relationship between molecular weight distribution anddegradation Considering the GPC curves for the tensileFig 9 Images of PLLA biological tissue after 36 weeks

implantation samples (Fig 5) it is interesting to note that they remainedmonomodal throughout successive weeks of degradationIn contrast many researchers have reported that asfibroblasts (Fig 9(c)) in an extracellular matrix (inset)

composed extensively of type-1 collagen fibre bundles the degradation of PLA and PGA aliphatic polyestersproceeds the initially monomodal GPC curve becomesrunning in different orientations While tissue disturbance

during surgery produces an initial inflammatory response bimodal and even multimodal in nature [14] and [37]

H01004 copy IMechE 2004Proc Instn Mech Engrs Vol 218 Part H J Engineering in Medicine

315DEGRADATION OF POLY-L-LACTIDE PART 1

The bimodal nature of these GPC curves was originally butions were the result of preferential degradation of theamorphous regions a view supported by Fischer et alassigned to the difference in degradation rates in the[32] It would be anticipated that due to the semicrystallineamorphous and crystalline regions [14] [32] and [37]nature of the compression-moulded and extruded PLLAHowever with molecular weight usually determined byinvestigated in the present study [25] the GPC curvestaking samples from the bulk of the polymer comprisingof both would become bimodal and even multimodalthe interior of a lower molecular weight than the surfacein nature due to the preferential degradation of the[37] Li et al [19] were the first to assign this bimodalamorphous regions However Fig 5 shows that this isbehaviour to the autocatalytic effect and faster internalnot the case with the molecular weight distributionsdegradation It is currently understood that for PLA andremaining monomodal throughout the 44 weeks durationPGA polymers and their copolymers this bimodal natureof the study contradicting the findings of Li et al [20]can be accounted for by three different mechanismsand Pistner et al [39] It must also be assumed that withrelated to the polymers morphology [14] First by fasterthe samples intended for molecular weight analysis takeninternal degradation however this mechanism is mostthrough a cross-section of the material the suspectedcommonly observed for initially amorphous polymersautocatalytic mechanism did not result in a large enoughwhich are not believed to be capable of crystallizationsurface-interior differentiation to yield curves containingeven throughout degradation [37] for example a 5050two distinct molecular weight speciescopolymer of PLA and PDLA [19] Second for semi-

crystalline polymers the bimodal nature has been attri-buted to selective degradation of the amorphous regions Bulk degradation The time delay before mass losswith the surface-interior differentiation reported not to observed in this study (Fig 6) is in agreement withbe large enough to yield bimodal GPC chromatograms the reported general sequence of aliphatic polyester[14] supporting Andersonrsquos theory [30] that the hydro- degradation which suggests molecular weight loss islytic degradation of semicrystalline polyesters does not observed first before loss of mechanical strength andproceed exclusively by non-catalytic or autocatalytic before any physical mass loss is observed [8] This ismechanisms Finally the bimodal nature of the GPC accounted for by the fact that water diffusion into thechromatograms has been attributed to the crystallization polymer is faster than the hydrolytic degradation ofof low molecular weight degradation by-products in the polymerrsquos ester linkage suggesting that ester-bondinitially amorphous polymers for example amorphous cleavage is the rate-limiting step in the degradation ofPLLA and a 7525 PLAPGA copolymer [38] which are aliphatic polyesters [40] This results in degradation pro-capable of crystallizing throughout degradation Once ceeding in the bulk of the polymer resulting in a time-

lag before any mass loss is observed as the polymerrsquosthe low molecular by-products crystallize they becomemolecular weight has to be reduced to a critical valueresistant to degradation and appear as a low molecularbefore soluble oligomers can be released In contrast forweight peak on the GPC curvebioresorbable polymers regarded as surface erodingThe monomodal nature of the GPC curves obtainedsuch as those belonging to the polyanhydride and poly-for the semicrystalline PLLA prepared by annealingorthoester families [41] mass loss is observed almostand investigated in this study appears to contradictimmediately as the chain scission of their more reactivethe findings of other researchers investigating similarunstable linkages in comparison to the ester linkage insemicrystalline PLLA Li et al [20] investigating thealiphatic polyesters is faster than the diffusion of waterdegradation of semicrystalline PLLA prepared by anneal-molecules into the polymer [40]ing at 130 degC for two hours with an initial crystallinity

of 72 per cent deduced from XRD measurementsobserved that the initial monomodal molecular weight Polymer morphology and degradation The results of thedistribution became multimodal after 18 weeks After 50 DSC analysis (Fig 7) appear to provide evidence thatweeks Li et al [20] observed that the GPC curve became the low molecular weight degradation by-products arebimodal with the peak corresponding to high molecular capable of crystallizing due to their greater mobilityweight being more prominant for the surface than for and contribute to the samplesrsquo increasing crystallinitythe centre suggesting autocatalysis At 90 weeks the This is evident by the emergence of a small peak formingGPC chromatogram then became almost monomodal and eventually merging with the main melting peak Theand was composed of a single low molecular weight crystallization of these internal degradation by-productspeak Pistner et al [39] observed a similar profile for the resulted in the polymer maintaining its structural integrityGPC chromatograms of semicrystalline PLLA with an throughout the duration of the study In contrast hollowinitial crystallinity of 73 per cent measured by DSC with structures have been reported for intrinsically amorphousa low molecular weight shoulder observed after eight polymers since their degradation products are notweeks becoming more important as degradation time believed to be capable of crystallizing for example inproceeded Both Li et al [20] and Pistner et al [39] the case of a 5050 copolymer of PLLAPDLLA [19]

The decreasing peak melting temperature observed mostconcluded that the multimodal molecular weight distri-

H01004 copy IMechE 2004 Proc Instn Mech Engrs Vol 218 Part H J Engineering in Medicine

316 N A WEIR F J BUCHANAN J F ORR AND G R DICKSON

significantly at 44 weeks (Tables 3 and 4) and deter- In vitro and in vivo degradation rates Literature regard-ing the role of enzymes on the degradation of aliphaticmined from a single heating cycle is consistent with the

hypothesis that the initially crystalline regions are resistant polyesters is often contradictory Many authors havereported that enzymes may be involved in the latter stagesto degradation resulting in a two-stage degradation mech-

anism with the amorphous regions being preferentially of degradation when the polymer has fragmented and themolecular weight is sufficiently small [47ndash49] Howeverattacked [14] However once the amorphous regions have

been exhausted the less accessible crystalline regions the role of enzymes during hydrolysis of the polymerbulk remains unclear In comparative in vitro and in vivoare then solely attacked and disrupted resulting in a

decreased size of the initially present crystallites and studies Vasenius et al [50] have reported significantlyfaster degradation of PGA rods in vivo with Matsusuehence a reduced melting point [42] Although the melt-

ing point of bioresorbable polymers is also known to be et al [51] also reporting faster in vivo degradationof PLLA In each case the faster in vivo degradation ratedependent on molecular weight the extent of this is most

readily determined by considering a reheat DSC run was attributed in some part to the action of enzymesHowever comparative studies by Hooper et al [52] andSince the fusion of the first run destroys the polymerrsquos

initial crystalline structure crystallization on cooling Pitt et al [53] have reported no significant differences inthe degradation rates of poly(a-hydroxy acids) in vivoinvolves the degraded chains only [36 ] confirming that

in the present study the decrease in melting temperature with Cam et al [54] reporting their degradation tobe practically independent of enzymes The similaritydetermined from a single heating cycle was most likely a

result of a reduction in crystallite size and not decreasing between the results of the molecular weight (Fig 3) andDSC analysis for the PLLA rods investigated in vitromolecular weight It is speculated that the decreasing Tg

observed as degradation time increased (Tables 3 and 4) and in vivo in the present study suggests that thedegradation of PLLA is independent of enzymes and inis related to the reduction in molecular weight of the

polymerrsquos chains in the amorphous regions with a similar agreement with Timmins and Lenz [55] who reportedthat enzymes capable of catalysing hydrolysis are them-trend also observed by Li [14] Duek et al [43] Joukainen

et al [44] and Kellomaki et al [45] Interestingly a selves macromolecules unable to penetrate into the poly-mer bulk Therefore any enzyme-contributed reactionsmall amount of water within a polymer is also known

to have a marked plasticizing effect causing a reduction would be heterogeneous and confined to the surface ofthe polymer with a reduction in mass observed but littlein the polymerrsquos Tg A study by Siemann [46 ] investi-

gating the influence of water on the glass transition of change in the polymers overall molecular weight [4]The significant loss of molecular weight (Table 2) andpoly(dl-lactic acid) by DSC reported a 12 K decrease

in Tg after samples were exposed to water for six hours negligible mass loss (Fig 6) observed for the PLLA rodinvestigated in vivo in the present study would suggestprior to testing However in a further study investi-

gating samples exposed to water and then dried to a that degradation proceeded predominantly in the bulkof the polymer by non-enzymatic hydrolysis similar toconstant mass before testing the Tg remained the same

as the untreated samples In the present study the the mechanism observed in vitro However this doesnot rule out the influence of enzymes at later stages ofsamples were dried to constant mass before DSC testing

was conducted ruling out water acting as a plasticizer the degradation process particularly when mass lossbecomes significantand confirming a reduction in molecular weight as the

most probable cause for the decreasing trend in TgHowever this underlines the problem that for accurateTg measurements representative of the polymerrsquos con-

42 Biological responsedition in service test regimes need to be developed thatcan accurately monitor the polymerrsquos Tg while the The production of a fibrous capsule around bio-

resorbable implants has been observed previously [56ndash58]samples remain lsquowetrsquoand is regarded as part of the bodyrsquos natural responseto implants made of diverse materials [49] SurgicalMechanical strength Since degradation predominantly

occurred in the amorphous regions disrupting the tie intervention such as the implantation procedure under-taken in this study would initiate inflammation as achains holding the crystallites together coupled with the

decreasing molecular weight and increasing crystallinity response to injury However the absence of inflam-matory cells at 36 and 44 weeks suggests that PLLAit is not surprising that the mechanical properties of the

PLLA investigated decreased so rapidly However con- is biocompatible throughout the early stages of itsdegradation It is understood that the onset of mass losssidering the similarities between the molecular weight

loss and the results derived from the DSC analysis for particularly in fast degrading aliphatic polyesters suchas PGA can result in an inflammatory reaction due tothe extruded PLLA rod in vitro and in vivo it is difficult

to speculate at this stage why the samples in vitro the sudden release of acidic degradation by-productscausing a large change in pH of the surrounding mediaappeared to lose their strength more rapidly than those

in vivo [59] In the present investigation the PLLA degradation

H01004 copy IMechE 2004Proc Instn Mech Engrs Vol 218 Part H J Engineering in Medicine

317DEGRADATION OF POLY-L-LACTIDE PART 1

scaffolds and cells In Synthetic Biodegradable Polymerstudy in vivo was terminated before any mass loss wasScaffolds (Eds A Atala and D J Mooney) 1997 pp 1ndash14observed although it is speculated that any inflam-(Birkhauser Boston MA USA)matory response observed as a direct result of the onset

4 Li S and Vert M Biodegradation of aliphatic poly-of polymer mass loss would be mild In comparison toesters In Degradable Polymers Principles amp Applicationsfast degrading PGA implants it is anticipated that the(Eds G Scott and D Gilead) 1995 pp 43ndash87 (Chapman

release of acidic degradation products from the slower amp Hall London)degrading PLLA would be less intense This would 5 Chu C C Biodegradable polymeric biomaterials anresult in the surrounding tissue being more capable of overview In The Biomedical Engineering Handbookeliminating any such debris more efficiently reducing the (Ed J D Bronzino) 1995 pp 611ndash626 (CRC Press Boca

Raton FL USA)risk of a severe inflammatory reaction developing that6 Higgins N A Condensation of Polymers of Hydroxyaceticwould require further surgical intervention

Acid US Patent 2 676 945 19547 Schneider A K Polymers of High Melting Lactide US

Patent 2 703 316 19555 CONCLUSIONS8 Middleton J C and Tipton A J Synthetic biodegradable

polymers as orthopedic devices Biomaterials 2000 21The results of the analytical characterization studies 2335ndash2346conducted on the retrieved PLLA samples in vitro and 9 Kulkarni R K Pani K C Neuman C and Leonard Fin vivo provides strong evidence to support the findings Polylactic acid for surgical implants Arch Surg 1966of other researchers investigating similar bioresorbable 93 839ndash843polymers Additionally the results from the in vivo 10 Hofmann G O Biodegradable implants in orthopaedic

surgerymdasha review on the state-of-the art Clin Materstudies would suggest that throughout the first stage of1992 10 75ndash80degradation before mass loss is observed PLLA is bio-

11 Ciccone W J Motz C Bentley C and Tasto J Pcompatible and degrades at the same rate in vitro andBioabsorbable implants in orthopaedics new developmentsin vivo However the results of the present studies doand clinical applications J Am Acad Orthop Surg 2001appear to indicate that for semicrystalline polymers like 9 280ndash288

the PLLA investigated no clear differentiation between 12 Barber F A Resorbable materials for arthroscopicsurface and interior degradation could be observed that fixation a product guide Orthopedic Special Edn 2002would clearly point to an autocatalytic degradation 8 29ndash37mechanism As a result it is speculated that as poly- 13 Hayashi T Biodegradable polymers for biomedical uses

Prog Polym Sci 1994 19 663ndash702mer crystallinity increases the importance of the auto-14 Li S Hydrolytic degradation characteristics of aliphaticcatalysis degradation mechanism may become less

polyesters derived from lactic and glycolic acids J BiomedsignificantMater Res (Appl Biomater) 1999 48 342ndash353

15 Vert M Li S and Garreau H New insights on thedegradation of bioresorbable polymeric devices based onACKNOWLEDGEMENTSlactic and glycolic acids Clin Mater 1992 10 3ndash8

16 Ali S Doherty P J and Williams D F MechanismsThe authors would like to thank Mr David Farrar of polymer degradation in implantable devices 2at Smith amp Nephew Group Research Centre (York Poly(DL-lactic acid) J Biomed Mater Res 1993 27UK) Boehringer Ingelheim (Ingelheim Germany) for 1409ndash1418

17 Chu C C Degradation and biocompatibility of syn-supplying the PLLA Griffith Microscience (Derbyshirethetic absorbable suture materials general biodegradationUK) for the ethylene oxide sterilization and Rapraphenomena and some factors affecting biodegradationTechnology Limited (Shropshire UK) for the molecularIn Biomedical Applications of Synthetic Biodegradableweight characterization Finally the EPSRC (SwindonPolymers (Ed J O Hollinger) 1995 pp 103ndash128 (CRCUK) for financial assistancePress Boca Raton FL USA)

18 Mainil-Varlet P Curtis R and Gogolewski S Effect ofin vivo and in vitro degradation on molecular and mech-

REFERENCES anical properties of various low-molecular-weight poly-lactides J Biomed Mater Res 1997 36 360ndash380

1 Tormala P Pohjonen T and Rokkanen P Bioabsorbable 19 Li S M Garreau H and Vert M Structure-propertypolymers materials technology and surgical applications relationships in the case of the degradation of massiveProc Instn Mech Engrs Part H J Engineering in Medicine aliphatic poly-(a-hydroxy acids) in aqueous media Part 11998 212 101ndash112 poly(DL-lactic acid) J Mater Sci Mater Med 1990

2 Chasin M Biodegradable polymers for controlled drug 1 123ndash130delivery In Biomedical Applications of Synthetic Bio- 20 Li S Garreau H and Vert M Structure-propertydegradable Polymers (Ed J O Hollinger) 1995 pp 1ndash15 relationships in the case of the degradation of massive(CRC Press Boca Raton FL USA) poly(a-hydroxy acids) in aqueous media Part 3 influence

3 Chaignaud B E Langer R and Vacanti J P The history of the morphology of poly(L-lactic acid) J Mater SciMater Med 1990 1 198ndash206of tissue engineering using synthetic biodegradable polymer

H01004 copy IMechE 2004 Proc Instn Mech Engrs Vol 218 Part H J Engineering in Medicine

312 N A WEIR F J BUCHANAN J F ORR AND G R DICKSON

Fig 4 Uncatalysed and autocatalysed models for tensile and extruded rod samples

cent mass loss observed for the extruded rod samples Asimilar pattern was also observed for the extruded rodsamples mass change in vivo however the increases inmass before drying were not as large peaking at approxi-mately 08 per cent after 44 weeks with no significantmass loss observed after drying

34 DSC analysis versus time

For both the tensile and extruded rod samples in vitroa general trend was observed of increasing crystallinityand decreasing Tg onset temperature with degradationtime (Tables 3 and 4) Additionally a slight but signifi-Fig 5 PLLA tensile samples molecular weight distributionscant decrease in both the tensile and extruded rodat 0 10 32 and 44 weeks in vitrosamples melting point Tm was also observed after 44weeks The results of the thermal analysis conducted on

Table 4 Thermal properties of PLLA tensile samples in vitro

Degradation time(weeks) crystallinity Tm degC Tg onset degC

0 448 1821 6694 455 1814 688

10 416 1816 67720 421 1821 66826 470 1814 65732 519 1803 61938 548 1810 63244 587 1791 573

Fig 6 In vitro mass change analysis

H01004 copy IMechE 2004Proc Instn Mech Engrs Vol 218 Part H J Engineering in Medicine

313DEGRADATION OF POLY-L-LACTIDE PART 1

the extruded rod in vivo also followed this general trend time with degradation proceeding further the mainmelting peak began to shift to lower temperatures untilwith the in vitro and in vivo extruded rod results proving

to be very similar eventually the two peaks merged and the smaller peakappeared as a shoulder on the larger peak (Fig 7(f ))The DSC thermograms for the tensile samples at 0 4

10 20 38 and 44 weeks are shown in Fig 7 At 0 weeks(Fig 7(a)) a small endothermic peak commencing atapproximately 67 degC was observed relating to stress 35 Mechanical strength versus timerelaxation at the polymerrsquos Tg [35] As the temperature

The mechanical strengths of both the tensile andincreased further a small endothermic dip was observed

extruded rod samples deteriorated with time with thejust before melting commenced followed by the main

tensile strength of the compression-moulded samplesmelting peak It is suggested that the dip before melting

reduced to approximately zero in 44 weeks (Table 5)was caused by some crystallization of the polymer

After 44 weeks the samples were very brittle and couldAlthough the polymer was annealed prior to degradation

not be gripped in the tensile test grips without fracturingwith the aim of limiting crystallization throughout the

A similar trend was observed initially for the loss ofstudy close to the polymerrsquos melting point the chain

shear strength for the extruded rods in vitro and in vivomobility would have increased allowing some of the

(Fig 8) However after 44 weeks in vitro the rodamorphous segments to order themselves into a more

samples had lost approximately 52 per cent of theircrystalline structure

original strength compared to only 263 per cent for theAt ten weeks (Fig 7(c)) as degradation increased

samples in vivopresumably in the amorphous regions the initial dipbefore melting observed at 0 and 4 weeks was reducedwith less amorphous regions remaining capable of

36 Biological host tissuersquos responsecrystallization

At 20 weeks (Fig 7(d)) the endothermic dip before The combination of LM TEM and SEM proved usefulin determining the relationship between the PLLA rodmelting had disappeared and a small peak appeared to

form in its place It is speculated that this new peak implants and surrounding biological tissue After 36weeks the PLLA implant appeared to stimulate the pro-represented the melting of new crystallites formed by the

crystallization of internal degradation by-products The duction of a fibrous tissue capsule (Figs 9(a) and (b))in which type-1 collagen fibre production was extensivereduction of the amorphous regions and crystallization

of the degradation by-products resulted in the polymerrsquos (Fig 9(c)) The TEM image of the fibrous capsulesurrounding the PLLA implant showed the presence ofoverall crystallinity increasing throughout degradation

(Tables 3 and 4)At weeks 38 and 44 (Figs 7(e) and (f )) the newly Table 5 Deterioration in PLLArsquos tensile properties

formed peak appeared to have grown and moved to a throughout degradationhigher temperature evidence that the newly formed

Degradation Youngrsquos Tensile Extensioncrystallitesrsquo size may have been increasing At the sametime modulus strength at break(weeks) (MPa) (MPa) (mm)

0 6684 643 164 6184 538 16

10 6252 603 1520 4330 237 0826 5287 369 1032 2265 99 0438 2842 82 0344 ndash 10 04

Fig 7 PLLA tensile samplesrsquo DSC thermograms at 0 4 10 Fig 8 Shear strength comparison of PLLA rod versus timein vitro and in vivo20 38 and 44 weeks

H01004 copy IMechE 2004 Proc Instn Mech Engrs Vol 218 Part H J Engineering in Medicine

314 N A WEIR F J BUCHANAN J F ORR AND G R DICKSON

the absence of cells marking an inflammatory responseat 36 and 44 weeks would suggest that the PLLA rodinvestigated was biocompatible throughout the 44 weeksduration of the study

4 DISCUSSION

41 Degradation mechanisms

The results presented show that the in vitro and in vivodegradation of PLLA commences almost immediatelywith the in vitro tensile and extruded rod samples losingapproximately 20 per cent of their initial molecularweight at four weeks the first time point analysed

Autocatalysis The higher R2 correlation coefficients(Fig 4) achieved for the ln Mn versus time relationshipshows a closer approximation of the autocatalysed model(equation (6)) to the experimental data compared tothe uncatalysed model with degradation accelerated bythe newly formed carboxylic acid end groups generatedby the continual ester hydrolysis reaction However itcannot be concluded that the mechanism is exclusivelyautocatalytic Investigating the in vitro degradation ofSR-PLLA at 37 degC Pohjonen and Tormala [36 ] observeda similar trend They reported correlation coefficients of0989 for the autocatalysed model and 0910 for theuncatalysed model confirming the findings of the presentstudy and at least according to theory suggesting anautocatalytic degradation mechanism In contrast in acomparative study investigating the molecular weightversus time data available in literature for semicrystallinealiphatic polyesters such as PLLA and amorphous poly-mers such as PDLLA Anderson [30] reported that noclear distinction could be derived between the uncatalysed1Mn and autocatalysed ln Mn plots versus time for semi-crystalline polymers However for amorphous polymersthe results were reported to be much more consistentwith an autocatalytic mechanism with higher correlationcoefficients achieved for plots of ln Mn versus timeAlthough the correlation coefficients for each study werenot given making comparisons to the present studydifficult As a result of these studies Anderson [30] con-cluded that the hydrolytic degradation of semicrystallinepolyesters may not proceed exclusively by non-catalyticor autocatalytic mechanisms speculating that both maycontribute to the rate of chain scission

Relationship between molecular weight distribution anddegradation Considering the GPC curves for the tensileFig 9 Images of PLLA biological tissue after 36 weeks

implantation samples (Fig 5) it is interesting to note that they remainedmonomodal throughout successive weeks of degradationIn contrast many researchers have reported that asfibroblasts (Fig 9(c)) in an extracellular matrix (inset)

composed extensively of type-1 collagen fibre bundles the degradation of PLA and PGA aliphatic polyestersproceeds the initially monomodal GPC curve becomesrunning in different orientations While tissue disturbance

during surgery produces an initial inflammatory response bimodal and even multimodal in nature [14] and [37]

H01004 copy IMechE 2004Proc Instn Mech Engrs Vol 218 Part H J Engineering in Medicine

315DEGRADATION OF POLY-L-LACTIDE PART 1

The bimodal nature of these GPC curves was originally butions were the result of preferential degradation of theamorphous regions a view supported by Fischer et alassigned to the difference in degradation rates in the[32] It would be anticipated that due to the semicrystallineamorphous and crystalline regions [14] [32] and [37]nature of the compression-moulded and extruded PLLAHowever with molecular weight usually determined byinvestigated in the present study [25] the GPC curvestaking samples from the bulk of the polymer comprisingof both would become bimodal and even multimodalthe interior of a lower molecular weight than the surfacein nature due to the preferential degradation of the[37] Li et al [19] were the first to assign this bimodalamorphous regions However Fig 5 shows that this isbehaviour to the autocatalytic effect and faster internalnot the case with the molecular weight distributionsdegradation It is currently understood that for PLA andremaining monomodal throughout the 44 weeks durationPGA polymers and their copolymers this bimodal natureof the study contradicting the findings of Li et al [20]can be accounted for by three different mechanismsand Pistner et al [39] It must also be assumed that withrelated to the polymers morphology [14] First by fasterthe samples intended for molecular weight analysis takeninternal degradation however this mechanism is mostthrough a cross-section of the material the suspectedcommonly observed for initially amorphous polymersautocatalytic mechanism did not result in a large enoughwhich are not believed to be capable of crystallizationsurface-interior differentiation to yield curves containingeven throughout degradation [37] for example a 5050two distinct molecular weight speciescopolymer of PLA and PDLA [19] Second for semi-

crystalline polymers the bimodal nature has been attri-buted to selective degradation of the amorphous regions Bulk degradation The time delay before mass losswith the surface-interior differentiation reported not to observed in this study (Fig 6) is in agreement withbe large enough to yield bimodal GPC chromatograms the reported general sequence of aliphatic polyester[14] supporting Andersonrsquos theory [30] that the hydro- degradation which suggests molecular weight loss islytic degradation of semicrystalline polyesters does not observed first before loss of mechanical strength andproceed exclusively by non-catalytic or autocatalytic before any physical mass loss is observed [8] This ismechanisms Finally the bimodal nature of the GPC accounted for by the fact that water diffusion into thechromatograms has been attributed to the crystallization polymer is faster than the hydrolytic degradation ofof low molecular weight degradation by-products in the polymerrsquos ester linkage suggesting that ester-bondinitially amorphous polymers for example amorphous cleavage is the rate-limiting step in the degradation ofPLLA and a 7525 PLAPGA copolymer [38] which are aliphatic polyesters [40] This results in degradation pro-capable of crystallizing throughout degradation Once ceeding in the bulk of the polymer resulting in a time-

lag before any mass loss is observed as the polymerrsquosthe low molecular by-products crystallize they becomemolecular weight has to be reduced to a critical valueresistant to degradation and appear as a low molecularbefore soluble oligomers can be released In contrast forweight peak on the GPC curvebioresorbable polymers regarded as surface erodingThe monomodal nature of the GPC curves obtainedsuch as those belonging to the polyanhydride and poly-for the semicrystalline PLLA prepared by annealingorthoester families [41] mass loss is observed almostand investigated in this study appears to contradictimmediately as the chain scission of their more reactivethe findings of other researchers investigating similarunstable linkages in comparison to the ester linkage insemicrystalline PLLA Li et al [20] investigating thealiphatic polyesters is faster than the diffusion of waterdegradation of semicrystalline PLLA prepared by anneal-molecules into the polymer [40]ing at 130 degC for two hours with an initial crystallinity

of 72 per cent deduced from XRD measurementsobserved that the initial monomodal molecular weight Polymer morphology and degradation The results of thedistribution became multimodal after 18 weeks After 50 DSC analysis (Fig 7) appear to provide evidence thatweeks Li et al [20] observed that the GPC curve became the low molecular weight degradation by-products arebimodal with the peak corresponding to high molecular capable of crystallizing due to their greater mobilityweight being more prominant for the surface than for and contribute to the samplesrsquo increasing crystallinitythe centre suggesting autocatalysis At 90 weeks the This is evident by the emergence of a small peak formingGPC chromatogram then became almost monomodal and eventually merging with the main melting peak Theand was composed of a single low molecular weight crystallization of these internal degradation by-productspeak Pistner et al [39] observed a similar profile for the resulted in the polymer maintaining its structural integrityGPC chromatograms of semicrystalline PLLA with an throughout the duration of the study In contrast hollowinitial crystallinity of 73 per cent measured by DSC with structures have been reported for intrinsically amorphousa low molecular weight shoulder observed after eight polymers since their degradation products are notweeks becoming more important as degradation time believed to be capable of crystallizing for example inproceeded Both Li et al [20] and Pistner et al [39] the case of a 5050 copolymer of PLLAPDLLA [19]

The decreasing peak melting temperature observed mostconcluded that the multimodal molecular weight distri-

H01004 copy IMechE 2004 Proc Instn Mech Engrs Vol 218 Part H J Engineering in Medicine

316 N A WEIR F J BUCHANAN J F ORR AND G R DICKSON

significantly at 44 weeks (Tables 3 and 4) and deter- In vitro and in vivo degradation rates Literature regard-ing the role of enzymes on the degradation of aliphaticmined from a single heating cycle is consistent with the

hypothesis that the initially crystalline regions are resistant polyesters is often contradictory Many authors havereported that enzymes may be involved in the latter stagesto degradation resulting in a two-stage degradation mech-

anism with the amorphous regions being preferentially of degradation when the polymer has fragmented and themolecular weight is sufficiently small [47ndash49] Howeverattacked [14] However once the amorphous regions have

been exhausted the less accessible crystalline regions the role of enzymes during hydrolysis of the polymerbulk remains unclear In comparative in vitro and in vivoare then solely attacked and disrupted resulting in a

decreased size of the initially present crystallites and studies Vasenius et al [50] have reported significantlyfaster degradation of PGA rods in vivo with Matsusuehence a reduced melting point [42] Although the melt-

ing point of bioresorbable polymers is also known to be et al [51] also reporting faster in vivo degradationof PLLA In each case the faster in vivo degradation ratedependent on molecular weight the extent of this is most

readily determined by considering a reheat DSC run was attributed in some part to the action of enzymesHowever comparative studies by Hooper et al [52] andSince the fusion of the first run destroys the polymerrsquos

initial crystalline structure crystallization on cooling Pitt et al [53] have reported no significant differences inthe degradation rates of poly(a-hydroxy acids) in vivoinvolves the degraded chains only [36 ] confirming that

in the present study the decrease in melting temperature with Cam et al [54] reporting their degradation tobe practically independent of enzymes The similaritydetermined from a single heating cycle was most likely a

result of a reduction in crystallite size and not decreasing between the results of the molecular weight (Fig 3) andDSC analysis for the PLLA rods investigated in vitromolecular weight It is speculated that the decreasing Tg

observed as degradation time increased (Tables 3 and 4) and in vivo in the present study suggests that thedegradation of PLLA is independent of enzymes and inis related to the reduction in molecular weight of the

polymerrsquos chains in the amorphous regions with a similar agreement with Timmins and Lenz [55] who reportedthat enzymes capable of catalysing hydrolysis are them-trend also observed by Li [14] Duek et al [43] Joukainen

et al [44] and Kellomaki et al [45] Interestingly a selves macromolecules unable to penetrate into the poly-mer bulk Therefore any enzyme-contributed reactionsmall amount of water within a polymer is also known

to have a marked plasticizing effect causing a reduction would be heterogeneous and confined to the surface ofthe polymer with a reduction in mass observed but littlein the polymerrsquos Tg A study by Siemann [46 ] investi-

gating the influence of water on the glass transition of change in the polymers overall molecular weight [4]The significant loss of molecular weight (Table 2) andpoly(dl-lactic acid) by DSC reported a 12 K decrease

in Tg after samples were exposed to water for six hours negligible mass loss (Fig 6) observed for the PLLA rodinvestigated in vivo in the present study would suggestprior to testing However in a further study investi-

gating samples exposed to water and then dried to a that degradation proceeded predominantly in the bulkof the polymer by non-enzymatic hydrolysis similar toconstant mass before testing the Tg remained the same

as the untreated samples In the present study the the mechanism observed in vitro However this doesnot rule out the influence of enzymes at later stages ofsamples were dried to constant mass before DSC testing

was conducted ruling out water acting as a plasticizer the degradation process particularly when mass lossbecomes significantand confirming a reduction in molecular weight as the

most probable cause for the decreasing trend in TgHowever this underlines the problem that for accurateTg measurements representative of the polymerrsquos con-

42 Biological responsedition in service test regimes need to be developed thatcan accurately monitor the polymerrsquos Tg while the The production of a fibrous capsule around bio-

resorbable implants has been observed previously [56ndash58]samples remain lsquowetrsquoand is regarded as part of the bodyrsquos natural responseto implants made of diverse materials [49] SurgicalMechanical strength Since degradation predominantly

occurred in the amorphous regions disrupting the tie intervention such as the implantation procedure under-taken in this study would initiate inflammation as achains holding the crystallites together coupled with the

decreasing molecular weight and increasing crystallinity response to injury However the absence of inflam-matory cells at 36 and 44 weeks suggests that PLLAit is not surprising that the mechanical properties of the

PLLA investigated decreased so rapidly However con- is biocompatible throughout the early stages of itsdegradation It is understood that the onset of mass losssidering the similarities between the molecular weight

loss and the results derived from the DSC analysis for particularly in fast degrading aliphatic polyesters suchas PGA can result in an inflammatory reaction due tothe extruded PLLA rod in vitro and in vivo it is difficult

to speculate at this stage why the samples in vitro the sudden release of acidic degradation by-productscausing a large change in pH of the surrounding mediaappeared to lose their strength more rapidly than those

in vivo [59] In the present investigation the PLLA degradation

H01004 copy IMechE 2004Proc Instn Mech Engrs Vol 218 Part H J Engineering in Medicine

317DEGRADATION OF POLY-L-LACTIDE PART 1

scaffolds and cells In Synthetic Biodegradable Polymerstudy in vivo was terminated before any mass loss wasScaffolds (Eds A Atala and D J Mooney) 1997 pp 1ndash14observed although it is speculated that any inflam-(Birkhauser Boston MA USA)matory response observed as a direct result of the onset

4 Li S and Vert M Biodegradation of aliphatic poly-of polymer mass loss would be mild In comparison toesters In Degradable Polymers Principles amp Applicationsfast degrading PGA implants it is anticipated that the(Eds G Scott and D Gilead) 1995 pp 43ndash87 (Chapman

release of acidic degradation products from the slower amp Hall London)degrading PLLA would be less intense This would 5 Chu C C Biodegradable polymeric biomaterials anresult in the surrounding tissue being more capable of overview In The Biomedical Engineering Handbookeliminating any such debris more efficiently reducing the (Ed J D Bronzino) 1995 pp 611ndash626 (CRC Press Boca

Raton FL USA)risk of a severe inflammatory reaction developing that6 Higgins N A Condensation of Polymers of Hydroxyaceticwould require further surgical intervention

Acid US Patent 2 676 945 19547 Schneider A K Polymers of High Melting Lactide US

Patent 2 703 316 19555 CONCLUSIONS8 Middleton J C and Tipton A J Synthetic biodegradable

polymers as orthopedic devices Biomaterials 2000 21The results of the analytical characterization studies 2335ndash2346conducted on the retrieved PLLA samples in vitro and 9 Kulkarni R K Pani K C Neuman C and Leonard Fin vivo provides strong evidence to support the findings Polylactic acid for surgical implants Arch Surg 1966of other researchers investigating similar bioresorbable 93 839ndash843polymers Additionally the results from the in vivo 10 Hofmann G O Biodegradable implants in orthopaedic

surgerymdasha review on the state-of-the art Clin Materstudies would suggest that throughout the first stage of1992 10 75ndash80degradation before mass loss is observed PLLA is bio-

11 Ciccone W J Motz C Bentley C and Tasto J Pcompatible and degrades at the same rate in vitro andBioabsorbable implants in orthopaedics new developmentsin vivo However the results of the present studies doand clinical applications J Am Acad Orthop Surg 2001appear to indicate that for semicrystalline polymers like 9 280ndash288

the PLLA investigated no clear differentiation between 12 Barber F A Resorbable materials for arthroscopicsurface and interior degradation could be observed that fixation a product guide Orthopedic Special Edn 2002would clearly point to an autocatalytic degradation 8 29ndash37mechanism As a result it is speculated that as poly- 13 Hayashi T Biodegradable polymers for biomedical uses

Prog Polym Sci 1994 19 663ndash702mer crystallinity increases the importance of the auto-14 Li S Hydrolytic degradation characteristics of aliphaticcatalysis degradation mechanism may become less

polyesters derived from lactic and glycolic acids J BiomedsignificantMater Res (Appl Biomater) 1999 48 342ndash353

15 Vert M Li S and Garreau H New insights on thedegradation of bioresorbable polymeric devices based onACKNOWLEDGEMENTSlactic and glycolic acids Clin Mater 1992 10 3ndash8

16 Ali S Doherty P J and Williams D F MechanismsThe authors would like to thank Mr David Farrar of polymer degradation in implantable devices 2at Smith amp Nephew Group Research Centre (York Poly(DL-lactic acid) J Biomed Mater Res 1993 27UK) Boehringer Ingelheim (Ingelheim Germany) for 1409ndash1418

17 Chu C C Degradation and biocompatibility of syn-supplying the PLLA Griffith Microscience (Derbyshirethetic absorbable suture materials general biodegradationUK) for the ethylene oxide sterilization and Rapraphenomena and some factors affecting biodegradationTechnology Limited (Shropshire UK) for the molecularIn Biomedical Applications of Synthetic Biodegradableweight characterization Finally the EPSRC (SwindonPolymers (Ed J O Hollinger) 1995 pp 103ndash128 (CRCUK) for financial assistancePress Boca Raton FL USA)

18 Mainil-Varlet P Curtis R and Gogolewski S Effect ofin vivo and in vitro degradation on molecular and mech-

REFERENCES anical properties of various low-molecular-weight poly-lactides J Biomed Mater Res 1997 36 360ndash380

1 Tormala P Pohjonen T and Rokkanen P Bioabsorbable 19 Li S M Garreau H and Vert M Structure-propertypolymers materials technology and surgical applications relationships in the case of the degradation of massiveProc Instn Mech Engrs Part H J Engineering in Medicine aliphatic poly-(a-hydroxy acids) in aqueous media Part 11998 212 101ndash112 poly(DL-lactic acid) J Mater Sci Mater Med 1990

2 Chasin M Biodegradable polymers for controlled drug 1 123ndash130delivery In Biomedical Applications of Synthetic Bio- 20 Li S Garreau H and Vert M Structure-propertydegradable Polymers (Ed J O Hollinger) 1995 pp 1ndash15 relationships in the case of the degradation of massive(CRC Press Boca Raton FL USA) poly(a-hydroxy acids) in aqueous media Part 3 influence

3 Chaignaud B E Langer R and Vacanti J P The history of the morphology of poly(L-lactic acid) J Mater SciMater Med 1990 1 198ndash206of tissue engineering using synthetic biodegradable polymer

H01004 copy IMechE 2004 Proc Instn Mech Engrs Vol 218 Part H J Engineering in Medicine

313DEGRADATION OF POLY-L-LACTIDE PART 1

the extruded rod in vivo also followed this general trend time with degradation proceeding further the mainmelting peak began to shift to lower temperatures untilwith the in vitro and in vivo extruded rod results proving

to be very similar eventually the two peaks merged and the smaller peakappeared as a shoulder on the larger peak (Fig 7(f ))The DSC thermograms for the tensile samples at 0 4

10 20 38 and 44 weeks are shown in Fig 7 At 0 weeks(Fig 7(a)) a small endothermic peak commencing atapproximately 67 degC was observed relating to stress 35 Mechanical strength versus timerelaxation at the polymerrsquos Tg [35] As the temperature

The mechanical strengths of both the tensile andincreased further a small endothermic dip was observed

extruded rod samples deteriorated with time with thejust before melting commenced followed by the main

tensile strength of the compression-moulded samplesmelting peak It is suggested that the dip before melting

reduced to approximately zero in 44 weeks (Table 5)was caused by some crystallization of the polymer

After 44 weeks the samples were very brittle and couldAlthough the polymer was annealed prior to degradation

not be gripped in the tensile test grips without fracturingwith the aim of limiting crystallization throughout the

A similar trend was observed initially for the loss ofstudy close to the polymerrsquos melting point the chain

shear strength for the extruded rods in vitro and in vivomobility would have increased allowing some of the

(Fig 8) However after 44 weeks in vitro the rodamorphous segments to order themselves into a more

samples had lost approximately 52 per cent of theircrystalline structure

original strength compared to only 263 per cent for theAt ten weeks (Fig 7(c)) as degradation increased

samples in vivopresumably in the amorphous regions the initial dipbefore melting observed at 0 and 4 weeks was reducedwith less amorphous regions remaining capable of

36 Biological host tissuersquos responsecrystallization

At 20 weeks (Fig 7(d)) the endothermic dip before The combination of LM TEM and SEM proved usefulin determining the relationship between the PLLA rodmelting had disappeared and a small peak appeared to

form in its place It is speculated that this new peak implants and surrounding biological tissue After 36weeks the PLLA implant appeared to stimulate the pro-represented the melting of new crystallites formed by the

crystallization of internal degradation by-products The duction of a fibrous tissue capsule (Figs 9(a) and (b))in which type-1 collagen fibre production was extensivereduction of the amorphous regions and crystallization

of the degradation by-products resulted in the polymerrsquos (Fig 9(c)) The TEM image of the fibrous capsulesurrounding the PLLA implant showed the presence ofoverall crystallinity increasing throughout degradation

(Tables 3 and 4)At weeks 38 and 44 (Figs 7(e) and (f )) the newly Table 5 Deterioration in PLLArsquos tensile properties

formed peak appeared to have grown and moved to a throughout degradationhigher temperature evidence that the newly formed

Degradation Youngrsquos Tensile Extensioncrystallitesrsquo size may have been increasing At the sametime modulus strength at break(weeks) (MPa) (MPa) (mm)

0 6684 643 164 6184 538 16

10 6252 603 1520 4330 237 0826 5287 369 1032 2265 99 0438 2842 82 0344 ndash 10 04

Fig 7 PLLA tensile samplesrsquo DSC thermograms at 0 4 10 Fig 8 Shear strength comparison of PLLA rod versus timein vitro and in vivo20 38 and 44 weeks

H01004 copy IMechE 2004 Proc Instn Mech Engrs Vol 218 Part H J Engineering in Medicine

314 N A WEIR F J BUCHANAN J F ORR AND G R DICKSON

the absence of cells marking an inflammatory responseat 36 and 44 weeks would suggest that the PLLA rodinvestigated was biocompatible throughout the 44 weeksduration of the study

4 DISCUSSION

41 Degradation mechanisms

The results presented show that the in vitro and in vivodegradation of PLLA commences almost immediatelywith the in vitro tensile and extruded rod samples losingapproximately 20 per cent of their initial molecularweight at four weeks the first time point analysed

Autocatalysis The higher R2 correlation coefficients(Fig 4) achieved for the ln Mn versus time relationshipshows a closer approximation of the autocatalysed model(equation (6)) to the experimental data compared tothe uncatalysed model with degradation accelerated bythe newly formed carboxylic acid end groups generatedby the continual ester hydrolysis reaction However itcannot be concluded that the mechanism is exclusivelyautocatalytic Investigating the in vitro degradation ofSR-PLLA at 37 degC Pohjonen and Tormala [36 ] observeda similar trend They reported correlation coefficients of0989 for the autocatalysed model and 0910 for theuncatalysed model confirming the findings of the presentstudy and at least according to theory suggesting anautocatalytic degradation mechanism In contrast in acomparative study investigating the molecular weightversus time data available in literature for semicrystallinealiphatic polyesters such as PLLA and amorphous poly-mers such as PDLLA Anderson [30] reported that noclear distinction could be derived between the uncatalysed1Mn and autocatalysed ln Mn plots versus time for semi-crystalline polymers However for amorphous polymersthe results were reported to be much more consistentwith an autocatalytic mechanism with higher correlationcoefficients achieved for plots of ln Mn versus timeAlthough the correlation coefficients for each study werenot given making comparisons to the present studydifficult As a result of these studies Anderson [30] con-cluded that the hydrolytic degradation of semicrystallinepolyesters may not proceed exclusively by non-catalyticor autocatalytic mechanisms speculating that both maycontribute to the rate of chain scission

Relationship between molecular weight distribution anddegradation Considering the GPC curves for the tensileFig 9 Images of PLLA biological tissue after 36 weeks

implantation samples (Fig 5) it is interesting to note that they remainedmonomodal throughout successive weeks of degradationIn contrast many researchers have reported that asfibroblasts (Fig 9(c)) in an extracellular matrix (inset)

composed extensively of type-1 collagen fibre bundles the degradation of PLA and PGA aliphatic polyestersproceeds the initially monomodal GPC curve becomesrunning in different orientations While tissue disturbance

during surgery produces an initial inflammatory response bimodal and even multimodal in nature [14] and [37]

H01004 copy IMechE 2004Proc Instn Mech Engrs Vol 218 Part H J Engineering in Medicine

315DEGRADATION OF POLY-L-LACTIDE PART 1

The bimodal nature of these GPC curves was originally butions were the result of preferential degradation of theamorphous regions a view supported by Fischer et alassigned to the difference in degradation rates in the[32] It would be anticipated that due to the semicrystallineamorphous and crystalline regions [14] [32] and [37]nature of the compression-moulded and extruded PLLAHowever with molecular weight usually determined byinvestigated in the present study [25] the GPC curvestaking samples from the bulk of the polymer comprisingof both would become bimodal and even multimodalthe interior of a lower molecular weight than the surfacein nature due to the preferential degradation of the[37] Li et al [19] were the first to assign this bimodalamorphous regions However Fig 5 shows that this isbehaviour to the autocatalytic effect and faster internalnot the case with the molecular weight distributionsdegradation It is currently understood that for PLA andremaining monomodal throughout the 44 weeks durationPGA polymers and their copolymers this bimodal natureof the study contradicting the findings of Li et al [20]can be accounted for by three different mechanismsand Pistner et al [39] It must also be assumed that withrelated to the polymers morphology [14] First by fasterthe samples intended for molecular weight analysis takeninternal degradation however this mechanism is mostthrough a cross-section of the material the suspectedcommonly observed for initially amorphous polymersautocatalytic mechanism did not result in a large enoughwhich are not believed to be capable of crystallizationsurface-interior differentiation to yield curves containingeven throughout degradation [37] for example a 5050two distinct molecular weight speciescopolymer of PLA and PDLA [19] Second for semi-

crystalline polymers the bimodal nature has been attri-buted to selective degradation of the amorphous regions Bulk degradation The time delay before mass losswith the surface-interior differentiation reported not to observed in this study (Fig 6) is in agreement withbe large enough to yield bimodal GPC chromatograms the reported general sequence of aliphatic polyester[14] supporting Andersonrsquos theory [30] that the hydro- degradation which suggests molecular weight loss islytic degradation of semicrystalline polyesters does not observed first before loss of mechanical strength andproceed exclusively by non-catalytic or autocatalytic before any physical mass loss is observed [8] This ismechanisms Finally the bimodal nature of the GPC accounted for by the fact that water diffusion into thechromatograms has been attributed to the crystallization polymer is faster than the hydrolytic degradation ofof low molecular weight degradation by-products in the polymerrsquos ester linkage suggesting that ester-bondinitially amorphous polymers for example amorphous cleavage is the rate-limiting step in the degradation ofPLLA and a 7525 PLAPGA copolymer [38] which are aliphatic polyesters [40] This results in degradation pro-capable of crystallizing throughout degradation Once ceeding in the bulk of the polymer resulting in a time-

lag before any mass loss is observed as the polymerrsquosthe low molecular by-products crystallize they becomemolecular weight has to be reduced to a critical valueresistant to degradation and appear as a low molecularbefore soluble oligomers can be released In contrast forweight peak on the GPC curvebioresorbable polymers regarded as surface erodingThe monomodal nature of the GPC curves obtainedsuch as those belonging to the polyanhydride and poly-for the semicrystalline PLLA prepared by annealingorthoester families [41] mass loss is observed almostand investigated in this study appears to contradictimmediately as the chain scission of their more reactivethe findings of other researchers investigating similarunstable linkages in comparison to the ester linkage insemicrystalline PLLA Li et al [20] investigating thealiphatic polyesters is faster than the diffusion of waterdegradation of semicrystalline PLLA prepared by anneal-molecules into the polymer [40]ing at 130 degC for two hours with an initial crystallinity

of 72 per cent deduced from XRD measurementsobserved that the initial monomodal molecular weight Polymer morphology and degradation The results of thedistribution became multimodal after 18 weeks After 50 DSC analysis (Fig 7) appear to provide evidence thatweeks Li et al [20] observed that the GPC curve became the low molecular weight degradation by-products arebimodal with the peak corresponding to high molecular capable of crystallizing due to their greater mobilityweight being more prominant for the surface than for and contribute to the samplesrsquo increasing crystallinitythe centre suggesting autocatalysis At 90 weeks the This is evident by the emergence of a small peak formingGPC chromatogram then became almost monomodal and eventually merging with the main melting peak Theand was composed of a single low molecular weight crystallization of these internal degradation by-productspeak Pistner et al [39] observed a similar profile for the resulted in the polymer maintaining its structural integrityGPC chromatograms of semicrystalline PLLA with an throughout the duration of the study In contrast hollowinitial crystallinity of 73 per cent measured by DSC with structures have been reported for intrinsically amorphousa low molecular weight shoulder observed after eight polymers since their degradation products are notweeks becoming more important as degradation time believed to be capable of crystallizing for example inproceeded Both Li et al [20] and Pistner et al [39] the case of a 5050 copolymer of PLLAPDLLA [19]

The decreasing peak melting temperature observed mostconcluded that the multimodal molecular weight distri-

H01004 copy IMechE 2004 Proc Instn Mech Engrs Vol 218 Part H J Engineering in Medicine

316 N A WEIR F J BUCHANAN J F ORR AND G R DICKSON

significantly at 44 weeks (Tables 3 and 4) and deter- In vitro and in vivo degradation rates Literature regard-ing the role of enzymes on the degradation of aliphaticmined from a single heating cycle is consistent with the

hypothesis that the initially crystalline regions are resistant polyesters is often contradictory Many authors havereported that enzymes may be involved in the latter stagesto degradation resulting in a two-stage degradation mech-

anism with the amorphous regions being preferentially of degradation when the polymer has fragmented and themolecular weight is sufficiently small [47ndash49] Howeverattacked [14] However once the amorphous regions have

been exhausted the less accessible crystalline regions the role of enzymes during hydrolysis of the polymerbulk remains unclear In comparative in vitro and in vivoare then solely attacked and disrupted resulting in a

decreased size of the initially present crystallites and studies Vasenius et al [50] have reported significantlyfaster degradation of PGA rods in vivo with Matsusuehence a reduced melting point [42] Although the melt-

ing point of bioresorbable polymers is also known to be et al [51] also reporting faster in vivo degradationof PLLA In each case the faster in vivo degradation ratedependent on molecular weight the extent of this is most

readily determined by considering a reheat DSC run was attributed in some part to the action of enzymesHowever comparative studies by Hooper et al [52] andSince the fusion of the first run destroys the polymerrsquos

initial crystalline structure crystallization on cooling Pitt et al [53] have reported no significant differences inthe degradation rates of poly(a-hydroxy acids) in vivoinvolves the degraded chains only [36 ] confirming that

in the present study the decrease in melting temperature with Cam et al [54] reporting their degradation tobe practically independent of enzymes The similaritydetermined from a single heating cycle was most likely a

result of a reduction in crystallite size and not decreasing between the results of the molecular weight (Fig 3) andDSC analysis for the PLLA rods investigated in vitromolecular weight It is speculated that the decreasing Tg

observed as degradation time increased (Tables 3 and 4) and in vivo in the present study suggests that thedegradation of PLLA is independent of enzymes and inis related to the reduction in molecular weight of the

polymerrsquos chains in the amorphous regions with a similar agreement with Timmins and Lenz [55] who reportedthat enzymes capable of catalysing hydrolysis are them-trend also observed by Li [14] Duek et al [43] Joukainen

et al [44] and Kellomaki et al [45] Interestingly a selves macromolecules unable to penetrate into the poly-mer bulk Therefore any enzyme-contributed reactionsmall amount of water within a polymer is also known

to have a marked plasticizing effect causing a reduction would be heterogeneous and confined to the surface ofthe polymer with a reduction in mass observed but littlein the polymerrsquos Tg A study by Siemann [46 ] investi-

gating the influence of water on the glass transition of change in the polymers overall molecular weight [4]The significant loss of molecular weight (Table 2) andpoly(dl-lactic acid) by DSC reported a 12 K decrease

in Tg after samples were exposed to water for six hours negligible mass loss (Fig 6) observed for the PLLA rodinvestigated in vivo in the present study would suggestprior to testing However in a further study investi-

gating samples exposed to water and then dried to a that degradation proceeded predominantly in the bulkof the polymer by non-enzymatic hydrolysis similar toconstant mass before testing the Tg remained the same

as the untreated samples In the present study the the mechanism observed in vitro However this doesnot rule out the influence of enzymes at later stages ofsamples were dried to constant mass before DSC testing

was conducted ruling out water acting as a plasticizer the degradation process particularly when mass lossbecomes significantand confirming a reduction in molecular weight as the

most probable cause for the decreasing trend in TgHowever this underlines the problem that for accurateTg measurements representative of the polymerrsquos con-

42 Biological responsedition in service test regimes need to be developed thatcan accurately monitor the polymerrsquos Tg while the The production of a fibrous capsule around bio-

resorbable implants has been observed previously [56ndash58]samples remain lsquowetrsquoand is regarded as part of the bodyrsquos natural responseto implants made of diverse materials [49] SurgicalMechanical strength Since degradation predominantly

occurred in the amorphous regions disrupting the tie intervention such as the implantation procedure under-taken in this study would initiate inflammation as achains holding the crystallites together coupled with the

decreasing molecular weight and increasing crystallinity response to injury However the absence of inflam-matory cells at 36 and 44 weeks suggests that PLLAit is not surprising that the mechanical properties of the

PLLA investigated decreased so rapidly However con- is biocompatible throughout the early stages of itsdegradation It is understood that the onset of mass losssidering the similarities between the molecular weight

loss and the results derived from the DSC analysis for particularly in fast degrading aliphatic polyesters suchas PGA can result in an inflammatory reaction due tothe extruded PLLA rod in vitro and in vivo it is difficult

to speculate at this stage why the samples in vitro the sudden release of acidic degradation by-productscausing a large change in pH of the surrounding mediaappeared to lose their strength more rapidly than those

in vivo [59] In the present investigation the PLLA degradation

H01004 copy IMechE 2004Proc Instn Mech Engrs Vol 218 Part H J Engineering in Medicine

317DEGRADATION OF POLY-L-LACTIDE PART 1

scaffolds and cells In Synthetic Biodegradable Polymerstudy in vivo was terminated before any mass loss wasScaffolds (Eds A Atala and D J Mooney) 1997 pp 1ndash14observed although it is speculated that any inflam-(Birkhauser Boston MA USA)matory response observed as a direct result of the onset

4 Li S and Vert M Biodegradation of aliphatic poly-of polymer mass loss would be mild In comparison toesters In Degradable Polymers Principles amp Applicationsfast degrading PGA implants it is anticipated that the(Eds G Scott and D Gilead) 1995 pp 43ndash87 (Chapman

release of acidic degradation products from the slower amp Hall London)degrading PLLA would be less intense This would 5 Chu C C Biodegradable polymeric biomaterials anresult in the surrounding tissue being more capable of overview In The Biomedical Engineering Handbookeliminating any such debris more efficiently reducing the (Ed J D Bronzino) 1995 pp 611ndash626 (CRC Press Boca

Raton FL USA)risk of a severe inflammatory reaction developing that6 Higgins N A Condensation of Polymers of Hydroxyaceticwould require further surgical intervention

Acid US Patent 2 676 945 19547 Schneider A K Polymers of High Melting Lactide US

Patent 2 703 316 19555 CONCLUSIONS8 Middleton J C and Tipton A J Synthetic biodegradable

polymers as orthopedic devices Biomaterials 2000 21The results of the analytical characterization studies 2335ndash2346conducted on the retrieved PLLA samples in vitro and 9 Kulkarni R K Pani K C Neuman C and Leonard Fin vivo provides strong evidence to support the findings Polylactic acid for surgical implants Arch Surg 1966of other researchers investigating similar bioresorbable 93 839ndash843polymers Additionally the results from the in vivo 10 Hofmann G O Biodegradable implants in orthopaedic

surgerymdasha review on the state-of-the art Clin Materstudies would suggest that throughout the first stage of1992 10 75ndash80degradation before mass loss is observed PLLA is bio-

11 Ciccone W J Motz C Bentley C and Tasto J Pcompatible and degrades at the same rate in vitro andBioabsorbable implants in orthopaedics new developmentsin vivo However the results of the present studies doand clinical applications J Am Acad Orthop Surg 2001appear to indicate that for semicrystalline polymers like 9 280ndash288

the PLLA investigated no clear differentiation between 12 Barber F A Resorbable materials for arthroscopicsurface and interior degradation could be observed that fixation a product guide Orthopedic Special Edn 2002would clearly point to an autocatalytic degradation 8 29ndash37mechanism As a result it is speculated that as poly- 13 Hayashi T Biodegradable polymers for biomedical uses

Prog Polym Sci 1994 19 663ndash702mer crystallinity increases the importance of the auto-14 Li S Hydrolytic degradation characteristics of aliphaticcatalysis degradation mechanism may become less

polyesters derived from lactic and glycolic acids J BiomedsignificantMater Res (Appl Biomater) 1999 48 342ndash353

15 Vert M Li S and Garreau H New insights on thedegradation of bioresorbable polymeric devices based onACKNOWLEDGEMENTSlactic and glycolic acids Clin Mater 1992 10 3ndash8

16 Ali S Doherty P J and Williams D F MechanismsThe authors would like to thank Mr David Farrar of polymer degradation in implantable devices 2at Smith amp Nephew Group Research Centre (York Poly(DL-lactic acid) J Biomed Mater Res 1993 27UK) Boehringer Ingelheim (Ingelheim Germany) for 1409ndash1418

17 Chu C C Degradation and biocompatibility of syn-supplying the PLLA Griffith Microscience (Derbyshirethetic absorbable suture materials general biodegradationUK) for the ethylene oxide sterilization and Rapraphenomena and some factors affecting biodegradationTechnology Limited (Shropshire UK) for the molecularIn Biomedical Applications of Synthetic Biodegradableweight characterization Finally the EPSRC (SwindonPolymers (Ed J O Hollinger) 1995 pp 103ndash128 (CRCUK) for financial assistancePress Boca Raton FL USA)

18 Mainil-Varlet P Curtis R and Gogolewski S Effect ofin vivo and in vitro degradation on molecular and mech-

REFERENCES anical properties of various low-molecular-weight poly-lactides J Biomed Mater Res 1997 36 360ndash380

1 Tormala P Pohjonen T and Rokkanen P Bioabsorbable 19 Li S M Garreau H and Vert M Structure-propertypolymers materials technology and surgical applications relationships in the case of the degradation of massiveProc Instn Mech Engrs Part H J Engineering in Medicine aliphatic poly-(a-hydroxy acids) in aqueous media Part 11998 212 101ndash112 poly(DL-lactic acid) J Mater Sci Mater Med 1990

2 Chasin M Biodegradable polymers for controlled drug 1 123ndash130delivery In Biomedical Applications of Synthetic Bio- 20 Li S Garreau H and Vert M Structure-propertydegradable Polymers (Ed J O Hollinger) 1995 pp 1ndash15 relationships in the case of the degradation of massive(CRC Press Boca Raton FL USA) poly(a-hydroxy acids) in aqueous media Part 3 influence

3 Chaignaud B E Langer R and Vacanti J P The history of the morphology of poly(L-lactic acid) J Mater SciMater Med 1990 1 198ndash206of tissue engineering using synthetic biodegradable polymer

H01004 copy IMechE 2004 Proc Instn Mech Engrs Vol 218 Part H J Engineering in Medicine

314 N A WEIR F J BUCHANAN J F ORR AND G R DICKSON

the absence of cells marking an inflammatory responseat 36 and 44 weeks would suggest that the PLLA rodinvestigated was biocompatible throughout the 44 weeksduration of the study

4 DISCUSSION

41 Degradation mechanisms

The results presented show that the in vitro and in vivodegradation of PLLA commences almost immediatelywith the in vitro tensile and extruded rod samples losingapproximately 20 per cent of their initial molecularweight at four weeks the first time point analysed

Autocatalysis The higher R2 correlation coefficients(Fig 4) achieved for the ln Mn versus time relationshipshows a closer approximation of the autocatalysed model(equation (6)) to the experimental data compared tothe uncatalysed model with degradation accelerated bythe newly formed carboxylic acid end groups generatedby the continual ester hydrolysis reaction However itcannot be concluded that the mechanism is exclusivelyautocatalytic Investigating the in vitro degradation ofSR-PLLA at 37 degC Pohjonen and Tormala [36 ] observeda similar trend They reported correlation coefficients of0989 for the autocatalysed model and 0910 for theuncatalysed model confirming the findings of the presentstudy and at least according to theory suggesting anautocatalytic degradation mechanism In contrast in acomparative study investigating the molecular weightversus time data available in literature for semicrystallinealiphatic polyesters such as PLLA and amorphous poly-mers such as PDLLA Anderson [30] reported that noclear distinction could be derived between the uncatalysed1Mn and autocatalysed ln Mn plots versus time for semi-crystalline polymers However for amorphous polymersthe results were reported to be much more consistentwith an autocatalytic mechanism with higher correlationcoefficients achieved for plots of ln Mn versus timeAlthough the correlation coefficients for each study werenot given making comparisons to the present studydifficult As a result of these studies Anderson [30] con-cluded that the hydrolytic degradation of semicrystallinepolyesters may not proceed exclusively by non-catalyticor autocatalytic mechanisms speculating that both maycontribute to the rate of chain scission

Relationship between molecular weight distribution anddegradation Considering the GPC curves for the tensileFig 9 Images of PLLA biological tissue after 36 weeks

implantation samples (Fig 5) it is interesting to note that they remainedmonomodal throughout successive weeks of degradationIn contrast many researchers have reported that asfibroblasts (Fig 9(c)) in an extracellular matrix (inset)

composed extensively of type-1 collagen fibre bundles the degradation of PLA and PGA aliphatic polyestersproceeds the initially monomodal GPC curve becomesrunning in different orientations While tissue disturbance

during surgery produces an initial inflammatory response bimodal and even multimodal in nature [14] and [37]

H01004 copy IMechE 2004Proc Instn Mech Engrs Vol 218 Part H J Engineering in Medicine

315DEGRADATION OF POLY-L-LACTIDE PART 1

The bimodal nature of these GPC curves was originally butions were the result of preferential degradation of theamorphous regions a view supported by Fischer et alassigned to the difference in degradation rates in the[32] It would be anticipated that due to the semicrystallineamorphous and crystalline regions [14] [32] and [37]nature of the compression-moulded and extruded PLLAHowever with molecular weight usually determined byinvestigated in the present study [25] the GPC curvestaking samples from the bulk of the polymer comprisingof both would become bimodal and even multimodalthe interior of a lower molecular weight than the surfacein nature due to the preferential degradation of the[37] Li et al [19] were the first to assign this bimodalamorphous regions However Fig 5 shows that this isbehaviour to the autocatalytic effect and faster internalnot the case with the molecular weight distributionsdegradation It is currently understood that for PLA andremaining monomodal throughout the 44 weeks durationPGA polymers and their copolymers this bimodal natureof the study contradicting the findings of Li et al [20]can be accounted for by three different mechanismsand Pistner et al [39] It must also be assumed that withrelated to the polymers morphology [14] First by fasterthe samples intended for molecular weight analysis takeninternal degradation however this mechanism is mostthrough a cross-section of the material the suspectedcommonly observed for initially amorphous polymersautocatalytic mechanism did not result in a large enoughwhich are not believed to be capable of crystallizationsurface-interior differentiation to yield curves containingeven throughout degradation [37] for example a 5050two distinct molecular weight speciescopolymer of PLA and PDLA [19] Second for semi-

crystalline polymers the bimodal nature has been attri-buted to selective degradation of the amorphous regions Bulk degradation The time delay before mass losswith the surface-interior differentiation reported not to observed in this study (Fig 6) is in agreement withbe large enough to yield bimodal GPC chromatograms the reported general sequence of aliphatic polyester[14] supporting Andersonrsquos theory [30] that the hydro- degradation which suggests molecular weight loss islytic degradation of semicrystalline polyesters does not observed first before loss of mechanical strength andproceed exclusively by non-catalytic or autocatalytic before any physical mass loss is observed [8] This ismechanisms Finally the bimodal nature of the GPC accounted for by the fact that water diffusion into thechromatograms has been attributed to the crystallization polymer is faster than the hydrolytic degradation ofof low molecular weight degradation by-products in the polymerrsquos ester linkage suggesting that ester-bondinitially amorphous polymers for example amorphous cleavage is the rate-limiting step in the degradation ofPLLA and a 7525 PLAPGA copolymer [38] which are aliphatic polyesters [40] This results in degradation pro-capable of crystallizing throughout degradation Once ceeding in the bulk of the polymer resulting in a time-

lag before any mass loss is observed as the polymerrsquosthe low molecular by-products crystallize they becomemolecular weight has to be reduced to a critical valueresistant to degradation and appear as a low molecularbefore soluble oligomers can be released In contrast forweight peak on the GPC curvebioresorbable polymers regarded as surface erodingThe monomodal nature of the GPC curves obtainedsuch as those belonging to the polyanhydride and poly-for the semicrystalline PLLA prepared by annealingorthoester families [41] mass loss is observed almostand investigated in this study appears to contradictimmediately as the chain scission of their more reactivethe findings of other researchers investigating similarunstable linkages in comparison to the ester linkage insemicrystalline PLLA Li et al [20] investigating thealiphatic polyesters is faster than the diffusion of waterdegradation of semicrystalline PLLA prepared by anneal-molecules into the polymer [40]ing at 130 degC for two hours with an initial crystallinity

of 72 per cent deduced from XRD measurementsobserved that the initial monomodal molecular weight Polymer morphology and degradation The results of thedistribution became multimodal after 18 weeks After 50 DSC analysis (Fig 7) appear to provide evidence thatweeks Li et al [20] observed that the GPC curve became the low molecular weight degradation by-products arebimodal with the peak corresponding to high molecular capable of crystallizing due to their greater mobilityweight being more prominant for the surface than for and contribute to the samplesrsquo increasing crystallinitythe centre suggesting autocatalysis At 90 weeks the This is evident by the emergence of a small peak formingGPC chromatogram then became almost monomodal and eventually merging with the main melting peak Theand was composed of a single low molecular weight crystallization of these internal degradation by-productspeak Pistner et al [39] observed a similar profile for the resulted in the polymer maintaining its structural integrityGPC chromatograms of semicrystalline PLLA with an throughout the duration of the study In contrast hollowinitial crystallinity of 73 per cent measured by DSC with structures have been reported for intrinsically amorphousa low molecular weight shoulder observed after eight polymers since their degradation products are notweeks becoming more important as degradation time believed to be capable of crystallizing for example inproceeded Both Li et al [20] and Pistner et al [39] the case of a 5050 copolymer of PLLAPDLLA [19]

The decreasing peak melting temperature observed mostconcluded that the multimodal molecular weight distri-

H01004 copy IMechE 2004 Proc Instn Mech Engrs Vol 218 Part H J Engineering in Medicine

316 N A WEIR F J BUCHANAN J F ORR AND G R DICKSON

significantly at 44 weeks (Tables 3 and 4) and deter- In vitro and in vivo degradation rates Literature regard-ing the role of enzymes on the degradation of aliphaticmined from a single heating cycle is consistent with the

hypothesis that the initially crystalline regions are resistant polyesters is often contradictory Many authors havereported that enzymes may be involved in the latter stagesto degradation resulting in a two-stage degradation mech-

anism with the amorphous regions being preferentially of degradation when the polymer has fragmented and themolecular weight is sufficiently small [47ndash49] Howeverattacked [14] However once the amorphous regions have

been exhausted the less accessible crystalline regions the role of enzymes during hydrolysis of the polymerbulk remains unclear In comparative in vitro and in vivoare then solely attacked and disrupted resulting in a

decreased size of the initially present crystallites and studies Vasenius et al [50] have reported significantlyfaster degradation of PGA rods in vivo with Matsusuehence a reduced melting point [42] Although the melt-

ing point of bioresorbable polymers is also known to be et al [51] also reporting faster in vivo degradationof PLLA In each case the faster in vivo degradation ratedependent on molecular weight the extent of this is most

readily determined by considering a reheat DSC run was attributed in some part to the action of enzymesHowever comparative studies by Hooper et al [52] andSince the fusion of the first run destroys the polymerrsquos

initial crystalline structure crystallization on cooling Pitt et al [53] have reported no significant differences inthe degradation rates of poly(a-hydroxy acids) in vivoinvolves the degraded chains only [36 ] confirming that

in the present study the decrease in melting temperature with Cam et al [54] reporting their degradation tobe practically independent of enzymes The similaritydetermined from a single heating cycle was most likely a

result of a reduction in crystallite size and not decreasing between the results of the molecular weight (Fig 3) andDSC analysis for the PLLA rods investigated in vitromolecular weight It is speculated that the decreasing Tg

observed as degradation time increased (Tables 3 and 4) and in vivo in the present study suggests that thedegradation of PLLA is independent of enzymes and inis related to the reduction in molecular weight of the

polymerrsquos chains in the amorphous regions with a similar agreement with Timmins and Lenz [55] who reportedthat enzymes capable of catalysing hydrolysis are them-trend also observed by Li [14] Duek et al [43] Joukainen

et al [44] and Kellomaki et al [45] Interestingly a selves macromolecules unable to penetrate into the poly-mer bulk Therefore any enzyme-contributed reactionsmall amount of water within a polymer is also known

to have a marked plasticizing effect causing a reduction would be heterogeneous and confined to the surface ofthe polymer with a reduction in mass observed but littlein the polymerrsquos Tg A study by Siemann [46 ] investi-

gating the influence of water on the glass transition of change in the polymers overall molecular weight [4]The significant loss of molecular weight (Table 2) andpoly(dl-lactic acid) by DSC reported a 12 K decrease

in Tg after samples were exposed to water for six hours negligible mass loss (Fig 6) observed for the PLLA rodinvestigated in vivo in the present study would suggestprior to testing However in a further study investi-

gating samples exposed to water and then dried to a that degradation proceeded predominantly in the bulkof the polymer by non-enzymatic hydrolysis similar toconstant mass before testing the Tg remained the same

as the untreated samples In the present study the the mechanism observed in vitro However this doesnot rule out the influence of enzymes at later stages ofsamples were dried to constant mass before DSC testing

was conducted ruling out water acting as a plasticizer the degradation process particularly when mass lossbecomes significantand confirming a reduction in molecular weight as the

most probable cause for the decreasing trend in TgHowever this underlines the problem that for accurateTg measurements representative of the polymerrsquos con-

42 Biological responsedition in service test regimes need to be developed thatcan accurately monitor the polymerrsquos Tg while the The production of a fibrous capsule around bio-

resorbable implants has been observed previously [56ndash58]samples remain lsquowetrsquoand is regarded as part of the bodyrsquos natural responseto implants made of diverse materials [49] SurgicalMechanical strength Since degradation predominantly

occurred in the amorphous regions disrupting the tie intervention such as the implantation procedure under-taken in this study would initiate inflammation as achains holding the crystallites together coupled with the

decreasing molecular weight and increasing crystallinity response to injury However the absence of inflam-matory cells at 36 and 44 weeks suggests that PLLAit is not surprising that the mechanical properties of the

PLLA investigated decreased so rapidly However con- is biocompatible throughout the early stages of itsdegradation It is understood that the onset of mass losssidering the similarities between the molecular weight

loss and the results derived from the DSC analysis for particularly in fast degrading aliphatic polyesters suchas PGA can result in an inflammatory reaction due tothe extruded PLLA rod in vitro and in vivo it is difficult

to speculate at this stage why the samples in vitro the sudden release of acidic degradation by-productscausing a large change in pH of the surrounding mediaappeared to lose their strength more rapidly than those

in vivo [59] In the present investigation the PLLA degradation

H01004 copy IMechE 2004Proc Instn Mech Engrs Vol 218 Part H J Engineering in Medicine

317DEGRADATION OF POLY-L-LACTIDE PART 1

scaffolds and cells In Synthetic Biodegradable Polymerstudy in vivo was terminated before any mass loss wasScaffolds (Eds A Atala and D J Mooney) 1997 pp 1ndash14observed although it is speculated that any inflam-(Birkhauser Boston MA USA)matory response observed as a direct result of the onset

4 Li S and Vert M Biodegradation of aliphatic poly-of polymer mass loss would be mild In comparison toesters In Degradable Polymers Principles amp Applicationsfast degrading PGA implants it is anticipated that the(Eds G Scott and D Gilead) 1995 pp 43ndash87 (Chapman

release of acidic degradation products from the slower amp Hall London)degrading PLLA would be less intense This would 5 Chu C C Biodegradable polymeric biomaterials anresult in the surrounding tissue being more capable of overview In The Biomedical Engineering Handbookeliminating any such debris more efficiently reducing the (Ed J D Bronzino) 1995 pp 611ndash626 (CRC Press Boca

Raton FL USA)risk of a severe inflammatory reaction developing that6 Higgins N A Condensation of Polymers of Hydroxyaceticwould require further surgical intervention

Acid US Patent 2 676 945 19547 Schneider A K Polymers of High Melting Lactide US

Patent 2 703 316 19555 CONCLUSIONS8 Middleton J C and Tipton A J Synthetic biodegradable

polymers as orthopedic devices Biomaterials 2000 21The results of the analytical characterization studies 2335ndash2346conducted on the retrieved PLLA samples in vitro and 9 Kulkarni R K Pani K C Neuman C and Leonard Fin vivo provides strong evidence to support the findings Polylactic acid for surgical implants Arch Surg 1966of other researchers investigating similar bioresorbable 93 839ndash843polymers Additionally the results from the in vivo 10 Hofmann G O Biodegradable implants in orthopaedic

surgerymdasha review on the state-of-the art Clin Materstudies would suggest that throughout the first stage of1992 10 75ndash80degradation before mass loss is observed PLLA is bio-

11 Ciccone W J Motz C Bentley C and Tasto J Pcompatible and degrades at the same rate in vitro andBioabsorbable implants in orthopaedics new developmentsin vivo However the results of the present studies doand clinical applications J Am Acad Orthop Surg 2001appear to indicate that for semicrystalline polymers like 9 280ndash288

the PLLA investigated no clear differentiation between 12 Barber F A Resorbable materials for arthroscopicsurface and interior degradation could be observed that fixation a product guide Orthopedic Special Edn 2002would clearly point to an autocatalytic degradation 8 29ndash37mechanism As a result it is speculated that as poly- 13 Hayashi T Biodegradable polymers for biomedical uses

Prog Polym Sci 1994 19 663ndash702mer crystallinity increases the importance of the auto-14 Li S Hydrolytic degradation characteristics of aliphaticcatalysis degradation mechanism may become less

polyesters derived from lactic and glycolic acids J BiomedsignificantMater Res (Appl Biomater) 1999 48 342ndash353

15 Vert M Li S and Garreau H New insights on thedegradation of bioresorbable polymeric devices based onACKNOWLEDGEMENTSlactic and glycolic acids Clin Mater 1992 10 3ndash8

16 Ali S Doherty P J and Williams D F MechanismsThe authors would like to thank Mr David Farrar of polymer degradation in implantable devices 2at Smith amp Nephew Group Research Centre (York Poly(DL-lactic acid) J Biomed Mater Res 1993 27UK) Boehringer Ingelheim (Ingelheim Germany) for 1409ndash1418

17 Chu C C Degradation and biocompatibility of syn-supplying the PLLA Griffith Microscience (Derbyshirethetic absorbable suture materials general biodegradationUK) for the ethylene oxide sterilization and Rapraphenomena and some factors affecting biodegradationTechnology Limited (Shropshire UK) for the molecularIn Biomedical Applications of Synthetic Biodegradableweight characterization Finally the EPSRC (SwindonPolymers (Ed J O Hollinger) 1995 pp 103ndash128 (CRCUK) for financial assistancePress Boca Raton FL USA)

18 Mainil-Varlet P Curtis R and Gogolewski S Effect ofin vivo and in vitro degradation on molecular and mech-

REFERENCES anical properties of various low-molecular-weight poly-lactides J Biomed Mater Res 1997 36 360ndash380

1 Tormala P Pohjonen T and Rokkanen P Bioabsorbable 19 Li S M Garreau H and Vert M Structure-propertypolymers materials technology and surgical applications relationships in the case of the degradation of massiveProc Instn Mech Engrs Part H J Engineering in Medicine aliphatic poly-(a-hydroxy acids) in aqueous media Part 11998 212 101ndash112 poly(DL-lactic acid) J Mater Sci Mater Med 1990

2 Chasin M Biodegradable polymers for controlled drug 1 123ndash130delivery In Biomedical Applications of Synthetic Bio- 20 Li S Garreau H and Vert M Structure-propertydegradable Polymers (Ed J O Hollinger) 1995 pp 1ndash15 relationships in the case of the degradation of massive(CRC Press Boca Raton FL USA) poly(a-hydroxy acids) in aqueous media Part 3 influence

3 Chaignaud B E Langer R and Vacanti J P The history of the morphology of poly(L-lactic acid) J Mater SciMater Med 1990 1 198ndash206of tissue engineering using synthetic biodegradable polymer

H01004 copy IMechE 2004 Proc Instn Mech Engrs Vol 218 Part H J Engineering in Medicine

315DEGRADATION OF POLY-L-LACTIDE PART 1

The bimodal nature of these GPC curves was originally butions were the result of preferential degradation of theamorphous regions a view supported by Fischer et alassigned to the difference in degradation rates in the[32] It would be anticipated that due to the semicrystallineamorphous and crystalline regions [14] [32] and [37]nature of the compression-moulded and extruded PLLAHowever with molecular weight usually determined byinvestigated in the present study [25] the GPC curvestaking samples from the bulk of the polymer comprisingof both would become bimodal and even multimodalthe interior of a lower molecular weight than the surfacein nature due to the preferential degradation of the[37] Li et al [19] were the first to assign this bimodalamorphous regions However Fig 5 shows that this isbehaviour to the autocatalytic effect and faster internalnot the case with the molecular weight distributionsdegradation It is currently understood that for PLA andremaining monomodal throughout the 44 weeks durationPGA polymers and their copolymers this bimodal natureof the study contradicting the findings of Li et al [20]can be accounted for by three different mechanismsand Pistner et al [39] It must also be assumed that withrelated to the polymers morphology [14] First by fasterthe samples intended for molecular weight analysis takeninternal degradation however this mechanism is mostthrough a cross-section of the material the suspectedcommonly observed for initially amorphous polymersautocatalytic mechanism did not result in a large enoughwhich are not believed to be capable of crystallizationsurface-interior differentiation to yield curves containingeven throughout degradation [37] for example a 5050two distinct molecular weight speciescopolymer of PLA and PDLA [19] Second for semi-

crystalline polymers the bimodal nature has been attri-buted to selective degradation of the amorphous regions Bulk degradation The time delay before mass losswith the surface-interior differentiation reported not to observed in this study (Fig 6) is in agreement withbe large enough to yield bimodal GPC chromatograms the reported general sequence of aliphatic polyester[14] supporting Andersonrsquos theory [30] that the hydro- degradation which suggests molecular weight loss islytic degradation of semicrystalline polyesters does not observed first before loss of mechanical strength andproceed exclusively by non-catalytic or autocatalytic before any physical mass loss is observed [8] This ismechanisms Finally the bimodal nature of the GPC accounted for by the fact that water diffusion into thechromatograms has been attributed to the crystallization polymer is faster than the hydrolytic degradation ofof low molecular weight degradation by-products in the polymerrsquos ester linkage suggesting that ester-bondinitially amorphous polymers for example amorphous cleavage is the rate-limiting step in the degradation ofPLLA and a 7525 PLAPGA copolymer [38] which are aliphatic polyesters [40] This results in degradation pro-capable of crystallizing throughout degradation Once ceeding in the bulk of the polymer resulting in a time-

lag before any mass loss is observed as the polymerrsquosthe low molecular by-products crystallize they becomemolecular weight has to be reduced to a critical valueresistant to degradation and appear as a low molecularbefore soluble oligomers can be released In contrast forweight peak on the GPC curvebioresorbable polymers regarded as surface erodingThe monomodal nature of the GPC curves obtainedsuch as those belonging to the polyanhydride and poly-for the semicrystalline PLLA prepared by annealingorthoester families [41] mass loss is observed almostand investigated in this study appears to contradictimmediately as the chain scission of their more reactivethe findings of other researchers investigating similarunstable linkages in comparison to the ester linkage insemicrystalline PLLA Li et al [20] investigating thealiphatic polyesters is faster than the diffusion of waterdegradation of semicrystalline PLLA prepared by anneal-molecules into the polymer [40]ing at 130 degC for two hours with an initial crystallinity

of 72 per cent deduced from XRD measurementsobserved that the initial monomodal molecular weight Polymer morphology and degradation The results of thedistribution became multimodal after 18 weeks After 50 DSC analysis (Fig 7) appear to provide evidence thatweeks Li et al [20] observed that the GPC curve became the low molecular weight degradation by-products arebimodal with the peak corresponding to high molecular capable of crystallizing due to their greater mobilityweight being more prominant for the surface than for and contribute to the samplesrsquo increasing crystallinitythe centre suggesting autocatalysis At 90 weeks the This is evident by the emergence of a small peak formingGPC chromatogram then became almost monomodal and eventually merging with the main melting peak Theand was composed of a single low molecular weight crystallization of these internal degradation by-productspeak Pistner et al [39] observed a similar profile for the resulted in the polymer maintaining its structural integrityGPC chromatograms of semicrystalline PLLA with an throughout the duration of the study In contrast hollowinitial crystallinity of 73 per cent measured by DSC with structures have been reported for intrinsically amorphousa low molecular weight shoulder observed after eight polymers since their degradation products are notweeks becoming more important as degradation time believed to be capable of crystallizing for example inproceeded Both Li et al [20] and Pistner et al [39] the case of a 5050 copolymer of PLLAPDLLA [19]

The decreasing peak melting temperature observed mostconcluded that the multimodal molecular weight distri-

H01004 copy IMechE 2004 Proc Instn Mech Engrs Vol 218 Part H J Engineering in Medicine

316 N A WEIR F J BUCHANAN J F ORR AND G R DICKSON

significantly at 44 weeks (Tables 3 and 4) and deter- In vitro and in vivo degradation rates Literature regard-ing the role of enzymes on the degradation of aliphaticmined from a single heating cycle is consistent with the

hypothesis that the initially crystalline regions are resistant polyesters is often contradictory Many authors havereported that enzymes may be involved in the latter stagesto degradation resulting in a two-stage degradation mech-

anism with the amorphous regions being preferentially of degradation when the polymer has fragmented and themolecular weight is sufficiently small [47ndash49] Howeverattacked [14] However once the amorphous regions have

been exhausted the less accessible crystalline regions the role of enzymes during hydrolysis of the polymerbulk remains unclear In comparative in vitro and in vivoare then solely attacked and disrupted resulting in a

decreased size of the initially present crystallites and studies Vasenius et al [50] have reported significantlyfaster degradation of PGA rods in vivo with Matsusuehence a reduced melting point [42] Although the melt-

ing point of bioresorbable polymers is also known to be et al [51] also reporting faster in vivo degradationof PLLA In each case the faster in vivo degradation ratedependent on molecular weight the extent of this is most

readily determined by considering a reheat DSC run was attributed in some part to the action of enzymesHowever comparative studies by Hooper et al [52] andSince the fusion of the first run destroys the polymerrsquos

initial crystalline structure crystallization on cooling Pitt et al [53] have reported no significant differences inthe degradation rates of poly(a-hydroxy acids) in vivoinvolves the degraded chains only [36 ] confirming that

in the present study the decrease in melting temperature with Cam et al [54] reporting their degradation tobe practically independent of enzymes The similaritydetermined from a single heating cycle was most likely a

result of a reduction in crystallite size and not decreasing between the results of the molecular weight (Fig 3) andDSC analysis for the PLLA rods investigated in vitromolecular weight It is speculated that the decreasing Tg

observed as degradation time increased (Tables 3 and 4) and in vivo in the present study suggests that thedegradation of PLLA is independent of enzymes and inis related to the reduction in molecular weight of the

polymerrsquos chains in the amorphous regions with a similar agreement with Timmins and Lenz [55] who reportedthat enzymes capable of catalysing hydrolysis are them-trend also observed by Li [14] Duek et al [43] Joukainen

et al [44] and Kellomaki et al [45] Interestingly a selves macromolecules unable to penetrate into the poly-mer bulk Therefore any enzyme-contributed reactionsmall amount of water within a polymer is also known

to have a marked plasticizing effect causing a reduction would be heterogeneous and confined to the surface ofthe polymer with a reduction in mass observed but littlein the polymerrsquos Tg A study by Siemann [46 ] investi-

gating the influence of water on the glass transition of change in the polymers overall molecular weight [4]The significant loss of molecular weight (Table 2) andpoly(dl-lactic acid) by DSC reported a 12 K decrease

in Tg after samples were exposed to water for six hours negligible mass loss (Fig 6) observed for the PLLA rodinvestigated in vivo in the present study would suggestprior to testing However in a further study investi-

gating samples exposed to water and then dried to a that degradation proceeded predominantly in the bulkof the polymer by non-enzymatic hydrolysis similar toconstant mass before testing the Tg remained the same

as the untreated samples In the present study the the mechanism observed in vitro However this doesnot rule out the influence of enzymes at later stages ofsamples were dried to constant mass before DSC testing

was conducted ruling out water acting as a plasticizer the degradation process particularly when mass lossbecomes significantand confirming a reduction in molecular weight as the

most probable cause for the decreasing trend in TgHowever this underlines the problem that for accurateTg measurements representative of the polymerrsquos con-

42 Biological responsedition in service test regimes need to be developed thatcan accurately monitor the polymerrsquos Tg while the The production of a fibrous capsule around bio-

resorbable implants has been observed previously [56ndash58]samples remain lsquowetrsquoand is regarded as part of the bodyrsquos natural responseto implants made of diverse materials [49] SurgicalMechanical strength Since degradation predominantly

occurred in the amorphous regions disrupting the tie intervention such as the implantation procedure under-taken in this study would initiate inflammation as achains holding the crystallites together coupled with the

decreasing molecular weight and increasing crystallinity response to injury However the absence of inflam-matory cells at 36 and 44 weeks suggests that PLLAit is not surprising that the mechanical properties of the

PLLA investigated decreased so rapidly However con- is biocompatible throughout the early stages of itsdegradation It is understood that the onset of mass losssidering the similarities between the molecular weight

loss and the results derived from the DSC analysis for particularly in fast degrading aliphatic polyesters suchas PGA can result in an inflammatory reaction due tothe extruded PLLA rod in vitro and in vivo it is difficult

to speculate at this stage why the samples in vitro the sudden release of acidic degradation by-productscausing a large change in pH of the surrounding mediaappeared to lose their strength more rapidly than those

in vivo [59] In the present investigation the PLLA degradation

H01004 copy IMechE 2004Proc Instn Mech Engrs Vol 218 Part H J Engineering in Medicine

317DEGRADATION OF POLY-L-LACTIDE PART 1

scaffolds and cells In Synthetic Biodegradable Polymerstudy in vivo was terminated before any mass loss wasScaffolds (Eds A Atala and D J Mooney) 1997 pp 1ndash14observed although it is speculated that any inflam-(Birkhauser Boston MA USA)matory response observed as a direct result of the onset

4 Li S and Vert M Biodegradation of aliphatic poly-of polymer mass loss would be mild In comparison toesters In Degradable Polymers Principles amp Applicationsfast degrading PGA implants it is anticipated that the(Eds G Scott and D Gilead) 1995 pp 43ndash87 (Chapman

release of acidic degradation products from the slower amp Hall London)degrading PLLA would be less intense This would 5 Chu C C Biodegradable polymeric biomaterials anresult in the surrounding tissue being more capable of overview In The Biomedical Engineering Handbookeliminating any such debris more efficiently reducing the (Ed J D Bronzino) 1995 pp 611ndash626 (CRC Press Boca

Raton FL USA)risk of a severe inflammatory reaction developing that6 Higgins N A Condensation of Polymers of Hydroxyaceticwould require further surgical intervention

Acid US Patent 2 676 945 19547 Schneider A K Polymers of High Melting Lactide US

Patent 2 703 316 19555 CONCLUSIONS8 Middleton J C and Tipton A J Synthetic biodegradable

polymers as orthopedic devices Biomaterials 2000 21The results of the analytical characterization studies 2335ndash2346conducted on the retrieved PLLA samples in vitro and 9 Kulkarni R K Pani K C Neuman C and Leonard Fin vivo provides strong evidence to support the findings Polylactic acid for surgical implants Arch Surg 1966of other researchers investigating similar bioresorbable 93 839ndash843polymers Additionally the results from the in vivo 10 Hofmann G O Biodegradable implants in orthopaedic

surgerymdasha review on the state-of-the art Clin Materstudies would suggest that throughout the first stage of1992 10 75ndash80degradation before mass loss is observed PLLA is bio-

11 Ciccone W J Motz C Bentley C and Tasto J Pcompatible and degrades at the same rate in vitro andBioabsorbable implants in orthopaedics new developmentsin vivo However the results of the present studies doand clinical applications J Am Acad Orthop Surg 2001appear to indicate that for semicrystalline polymers like 9 280ndash288

the PLLA investigated no clear differentiation between 12 Barber F A Resorbable materials for arthroscopicsurface and interior degradation could be observed that fixation a product guide Orthopedic Special Edn 2002would clearly point to an autocatalytic degradation 8 29ndash37mechanism As a result it is speculated that as poly- 13 Hayashi T Biodegradable polymers for biomedical uses

Prog Polym Sci 1994 19 663ndash702mer crystallinity increases the importance of the auto-14 Li S Hydrolytic degradation characteristics of aliphaticcatalysis degradation mechanism may become less

polyesters derived from lactic and glycolic acids J BiomedsignificantMater Res (Appl Biomater) 1999 48 342ndash353

15 Vert M Li S and Garreau H New insights on thedegradation of bioresorbable polymeric devices based onACKNOWLEDGEMENTSlactic and glycolic acids Clin Mater 1992 10 3ndash8

16 Ali S Doherty P J and Williams D F MechanismsThe authors would like to thank Mr David Farrar of polymer degradation in implantable devices 2at Smith amp Nephew Group Research Centre (York Poly(DL-lactic acid) J Biomed Mater Res 1993 27UK) Boehringer Ingelheim (Ingelheim Germany) for 1409ndash1418

17 Chu C C Degradation and biocompatibility of syn-supplying the PLLA Griffith Microscience (Derbyshirethetic absorbable suture materials general biodegradationUK) for the ethylene oxide sterilization and Rapraphenomena and some factors affecting biodegradationTechnology Limited (Shropshire UK) for the molecularIn Biomedical Applications of Synthetic Biodegradableweight characterization Finally the EPSRC (SwindonPolymers (Ed J O Hollinger) 1995 pp 103ndash128 (CRCUK) for financial assistancePress Boca Raton FL USA)

18 Mainil-Varlet P Curtis R and Gogolewski S Effect ofin vivo and in vitro degradation on molecular and mech-

REFERENCES anical properties of various low-molecular-weight poly-lactides J Biomed Mater Res 1997 36 360ndash380

1 Tormala P Pohjonen T and Rokkanen P Bioabsorbable 19 Li S M Garreau H and Vert M Structure-propertypolymers materials technology and surgical applications relationships in the case of the degradation of massiveProc Instn Mech Engrs Part H J Engineering in Medicine aliphatic poly-(a-hydroxy acids) in aqueous media Part 11998 212 101ndash112 poly(DL-lactic acid) J Mater Sci Mater Med 1990

2 Chasin M Biodegradable polymers for controlled drug 1 123ndash130delivery In Biomedical Applications of Synthetic Bio- 20 Li S Garreau H and Vert M Structure-propertydegradable Polymers (Ed J O Hollinger) 1995 pp 1ndash15 relationships in the case of the degradation of massive(CRC Press Boca Raton FL USA) poly(a-hydroxy acids) in aqueous media Part 3 influence

3 Chaignaud B E Langer R and Vacanti J P The history of the morphology of poly(L-lactic acid) J Mater SciMater Med 1990 1 198ndash206of tissue engineering using synthetic biodegradable polymer

H01004 copy IMechE 2004 Proc Instn Mech Engrs Vol 218 Part H J Engineering in Medicine

316 N A WEIR F J BUCHANAN J F ORR AND G R DICKSON

significantly at 44 weeks (Tables 3 and 4) and deter- In vitro and in vivo degradation rates Literature regard-ing the role of enzymes on the degradation of aliphaticmined from a single heating cycle is consistent with the

hypothesis that the initially crystalline regions are resistant polyesters is often contradictory Many authors havereported that enzymes may be involved in the latter stagesto degradation resulting in a two-stage degradation mech-

anism with the amorphous regions being preferentially of degradation when the polymer has fragmented and themolecular weight is sufficiently small [47ndash49] Howeverattacked [14] However once the amorphous regions have

been exhausted the less accessible crystalline regions the role of enzymes during hydrolysis of the polymerbulk remains unclear In comparative in vitro and in vivoare then solely attacked and disrupted resulting in a

decreased size of the initially present crystallites and studies Vasenius et al [50] have reported significantlyfaster degradation of PGA rods in vivo with Matsusuehence a reduced melting point [42] Although the melt-

ing point of bioresorbable polymers is also known to be et al [51] also reporting faster in vivo degradationof PLLA In each case the faster in vivo degradation ratedependent on molecular weight the extent of this is most

readily determined by considering a reheat DSC run was attributed in some part to the action of enzymesHowever comparative studies by Hooper et al [52] andSince the fusion of the first run destroys the polymerrsquos

initial crystalline structure crystallization on cooling Pitt et al [53] have reported no significant differences inthe degradation rates of poly(a-hydroxy acids) in vivoinvolves the degraded chains only [36 ] confirming that

in the present study the decrease in melting temperature with Cam et al [54] reporting their degradation tobe practically independent of enzymes The similaritydetermined from a single heating cycle was most likely a

result of a reduction in crystallite size and not decreasing between the results of the molecular weight (Fig 3) andDSC analysis for the PLLA rods investigated in vitromolecular weight It is speculated that the decreasing Tg

observed as degradation time increased (Tables 3 and 4) and in vivo in the present study suggests that thedegradation of PLLA is independent of enzymes and inis related to the reduction in molecular weight of the

polymerrsquos chains in the amorphous regions with a similar agreement with Timmins and Lenz [55] who reportedthat enzymes capable of catalysing hydrolysis are them-trend also observed by Li [14] Duek et al [43] Joukainen

et al [44] and Kellomaki et al [45] Interestingly a selves macromolecules unable to penetrate into the poly-mer bulk Therefore any enzyme-contributed reactionsmall amount of water within a polymer is also known

to have a marked plasticizing effect causing a reduction would be heterogeneous and confined to the surface ofthe polymer with a reduction in mass observed but littlein the polymerrsquos Tg A study by Siemann [46 ] investi-

gating the influence of water on the glass transition of change in the polymers overall molecular weight [4]The significant loss of molecular weight (Table 2) andpoly(dl-lactic acid) by DSC reported a 12 K decrease

in Tg after samples were exposed to water for six hours negligible mass loss (Fig 6) observed for the PLLA rodinvestigated in vivo in the present study would suggestprior to testing However in a further study investi-

gating samples exposed to water and then dried to a that degradation proceeded predominantly in the bulkof the polymer by non-enzymatic hydrolysis similar toconstant mass before testing the Tg remained the same

as the untreated samples In the present study the the mechanism observed in vitro However this doesnot rule out the influence of enzymes at later stages ofsamples were dried to constant mass before DSC testing

was conducted ruling out water acting as a plasticizer the degradation process particularly when mass lossbecomes significantand confirming a reduction in molecular weight as the

most probable cause for the decreasing trend in TgHowever this underlines the problem that for accurateTg measurements representative of the polymerrsquos con-

42 Biological responsedition in service test regimes need to be developed thatcan accurately monitor the polymerrsquos Tg while the The production of a fibrous capsule around bio-

resorbable implants has been observed previously [56ndash58]samples remain lsquowetrsquoand is regarded as part of the bodyrsquos natural responseto implants made of diverse materials [49] SurgicalMechanical strength Since degradation predominantly

occurred in the amorphous regions disrupting the tie intervention such as the implantation procedure under-taken in this study would initiate inflammation as achains holding the crystallites together coupled with the

decreasing molecular weight and increasing crystallinity response to injury However the absence of inflam-matory cells at 36 and 44 weeks suggests that PLLAit is not surprising that the mechanical properties of the

PLLA investigated decreased so rapidly However con- is biocompatible throughout the early stages of itsdegradation It is understood that the onset of mass losssidering the similarities between the molecular weight

loss and the results derived from the DSC analysis for particularly in fast degrading aliphatic polyesters suchas PGA can result in an inflammatory reaction due tothe extruded PLLA rod in vitro and in vivo it is difficult

to speculate at this stage why the samples in vitro the sudden release of acidic degradation by-productscausing a large change in pH of the surrounding mediaappeared to lose their strength more rapidly than those

in vivo [59] In the present investigation the PLLA degradation

H01004 copy IMechE 2004Proc Instn Mech Engrs Vol 218 Part H J Engineering in Medicine

317DEGRADATION OF POLY-L-LACTIDE PART 1

scaffolds and cells In Synthetic Biodegradable Polymerstudy in vivo was terminated before any mass loss wasScaffolds (Eds A Atala and D J Mooney) 1997 pp 1ndash14observed although it is speculated that any inflam-(Birkhauser Boston MA USA)matory response observed as a direct result of the onset

4 Li S and Vert M Biodegradation of aliphatic poly-of polymer mass loss would be mild In comparison toesters In Degradable Polymers Principles amp Applicationsfast degrading PGA implants it is anticipated that the(Eds G Scott and D Gilead) 1995 pp 43ndash87 (Chapman

release of acidic degradation products from the slower amp Hall London)degrading PLLA would be less intense This would 5 Chu C C Biodegradable polymeric biomaterials anresult in the surrounding tissue being more capable of overview In The Biomedical Engineering Handbookeliminating any such debris more efficiently reducing the (Ed J D Bronzino) 1995 pp 611ndash626 (CRC Press Boca

Raton FL USA)risk of a severe inflammatory reaction developing that6 Higgins N A Condensation of Polymers of Hydroxyaceticwould require further surgical intervention

Acid US Patent 2 676 945 19547 Schneider A K Polymers of High Melting Lactide US

Patent 2 703 316 19555 CONCLUSIONS8 Middleton J C and Tipton A J Synthetic biodegradable

polymers as orthopedic devices Biomaterials 2000 21The results of the analytical characterization studies 2335ndash2346conducted on the retrieved PLLA samples in vitro and 9 Kulkarni R K Pani K C Neuman C and Leonard Fin vivo provides strong evidence to support the findings Polylactic acid for surgical implants Arch Surg 1966of other researchers investigating similar bioresorbable 93 839ndash843polymers Additionally the results from the in vivo 10 Hofmann G O Biodegradable implants in orthopaedic

surgerymdasha review on the state-of-the art Clin Materstudies would suggest that throughout the first stage of1992 10 75ndash80degradation before mass loss is observed PLLA is bio-

11 Ciccone W J Motz C Bentley C and Tasto J Pcompatible and degrades at the same rate in vitro andBioabsorbable implants in orthopaedics new developmentsin vivo However the results of the present studies doand clinical applications J Am Acad Orthop Surg 2001appear to indicate that for semicrystalline polymers like 9 280ndash288

the PLLA investigated no clear differentiation between 12 Barber F A Resorbable materials for arthroscopicsurface and interior degradation could be observed that fixation a product guide Orthopedic Special Edn 2002would clearly point to an autocatalytic degradation 8 29ndash37mechanism As a result it is speculated that as poly- 13 Hayashi T Biodegradable polymers for biomedical uses

Prog Polym Sci 1994 19 663ndash702mer crystallinity increases the importance of the auto-14 Li S Hydrolytic degradation characteristics of aliphaticcatalysis degradation mechanism may become less

polyesters derived from lactic and glycolic acids J BiomedsignificantMater Res (Appl Biomater) 1999 48 342ndash353

15 Vert M Li S and Garreau H New insights on thedegradation of bioresorbable polymeric devices based onACKNOWLEDGEMENTSlactic and glycolic acids Clin Mater 1992 10 3ndash8

16 Ali S Doherty P J and Williams D F MechanismsThe authors would like to thank Mr David Farrar of polymer degradation in implantable devices 2at Smith amp Nephew Group Research Centre (York Poly(DL-lactic acid) J Biomed Mater Res 1993 27UK) Boehringer Ingelheim (Ingelheim Germany) for 1409ndash1418

17 Chu C C Degradation and biocompatibility of syn-supplying the PLLA Griffith Microscience (Derbyshirethetic absorbable suture materials general biodegradationUK) for the ethylene oxide sterilization and Rapraphenomena and some factors affecting biodegradationTechnology Limited (Shropshire UK) for the molecularIn Biomedical Applications of Synthetic Biodegradableweight characterization Finally the EPSRC (SwindonPolymers (Ed J O Hollinger) 1995 pp 103ndash128 (CRCUK) for financial assistancePress Boca Raton FL USA)

18 Mainil-Varlet P Curtis R and Gogolewski S Effect ofin vivo and in vitro degradation on molecular and mech-

REFERENCES anical properties of various low-molecular-weight poly-lactides J Biomed Mater Res 1997 36 360ndash380

1 Tormala P Pohjonen T and Rokkanen P Bioabsorbable 19 Li S M Garreau H and Vert M Structure-propertypolymers materials technology and surgical applications relationships in the case of the degradation of massiveProc Instn Mech Engrs Part H J Engineering in Medicine aliphatic poly-(a-hydroxy acids) in aqueous media Part 11998 212 101ndash112 poly(DL-lactic acid) J Mater Sci Mater Med 1990

2 Chasin M Biodegradable polymers for controlled drug 1 123ndash130delivery In Biomedical Applications of Synthetic Bio- 20 Li S Garreau H and Vert M Structure-propertydegradable Polymers (Ed J O Hollinger) 1995 pp 1ndash15 relationships in the case of the degradation of massive(CRC Press Boca Raton FL USA) poly(a-hydroxy acids) in aqueous media Part 3 influence

3 Chaignaud B E Langer R and Vacanti J P The history of the morphology of poly(L-lactic acid) J Mater SciMater Med 1990 1 198ndash206of tissue engineering using synthetic biodegradable polymer

H01004 copy IMechE 2004 Proc Instn Mech Engrs Vol 218 Part H J Engineering in Medicine

317DEGRADATION OF POLY-L-LACTIDE PART 1

scaffolds and cells In Synthetic Biodegradable Polymerstudy in vivo was terminated before any mass loss wasScaffolds (Eds A Atala and D J Mooney) 1997 pp 1ndash14observed although it is speculated that any inflam-(Birkhauser Boston MA USA)matory response observed as a direct result of the onset

4 Li S and Vert M Biodegradation of aliphatic poly-of polymer mass loss would be mild In comparison toesters In Degradable Polymers Principles amp Applicationsfast degrading PGA implants it is anticipated that the(Eds G Scott and D Gilead) 1995 pp 43ndash87 (Chapman

release of acidic degradation products from the slower amp Hall London)degrading PLLA would be less intense This would 5 Chu C C Biodegradable polymeric biomaterials anresult in the surrounding tissue being more capable of overview In The Biomedical Engineering Handbookeliminating any such debris more efficiently reducing the (Ed J D Bronzino) 1995 pp 611ndash626 (CRC Press Boca

Raton FL USA)risk of a severe inflammatory reaction developing that6 Higgins N A Condensation of Polymers of Hydroxyaceticwould require further surgical intervention

Acid US Patent 2 676 945 19547 Schneider A K Polymers of High Melting Lactide US

Patent 2 703 316 19555 CONCLUSIONS8 Middleton J C and Tipton A J Synthetic biodegradable

polymers as orthopedic devices Biomaterials 2000 21The results of the analytical characterization studies 2335ndash2346conducted on the retrieved PLLA samples in vitro and 9 Kulkarni R K Pani K C Neuman C and Leonard Fin vivo provides strong evidence to support the findings Polylactic acid for surgical implants Arch Surg 1966of other researchers investigating similar bioresorbable 93 839ndash843polymers Additionally the results from the in vivo 10 Hofmann G O Biodegradable implants in orthopaedic

surgerymdasha review on the state-of-the art Clin Materstudies would suggest that throughout the first stage of1992 10 75ndash80degradation before mass loss is observed PLLA is bio-

11 Ciccone W J Motz C Bentley C and Tasto J Pcompatible and degrades at the same rate in vitro andBioabsorbable implants in orthopaedics new developmentsin vivo However the results of the present studies doand clinical applications J Am Acad Orthop Surg 2001appear to indicate that for semicrystalline polymers like 9 280ndash288

the PLLA investigated no clear differentiation between 12 Barber F A Resorbable materials for arthroscopicsurface and interior degradation could be observed that fixation a product guide Orthopedic Special Edn 2002would clearly point to an autocatalytic degradation 8 29ndash37mechanism As a result it is speculated that as poly- 13 Hayashi T Biodegradable polymers for biomedical uses

Prog Polym Sci 1994 19 663ndash702mer crystallinity increases the importance of the auto-14 Li S Hydrolytic degradation characteristics of aliphaticcatalysis degradation mechanism may become less

polyesters derived from lactic and glycolic acids J BiomedsignificantMater Res (Appl Biomater) 1999 48 342ndash353

15 Vert M Li S and Garreau H New insights on thedegradation of bioresorbable polymeric devices based onACKNOWLEDGEMENTSlactic and glycolic acids Clin Mater 1992 10 3ndash8

16 Ali S Doherty P J and Williams D F MechanismsThe authors would like to thank Mr David Farrar of polymer degradation in implantable devices 2at Smith amp Nephew Group Research Centre (York Poly(DL-lactic acid) J Biomed Mater Res 1993 27UK) Boehringer Ingelheim (Ingelheim Germany) for 1409ndash1418

17 Chu C C Degradation and biocompatibility of syn-supplying the PLLA Griffith Microscience (Derbyshirethetic absorbable suture materials general biodegradationUK) for the ethylene oxide sterilization and Rapraphenomena and some factors affecting biodegradationTechnology Limited (Shropshire UK) for the molecularIn Biomedical Applications of Synthetic Biodegradableweight characterization Finally the EPSRC (SwindonPolymers (Ed J O Hollinger) 1995 pp 103ndash128 (CRCUK) for financial assistancePress Boca Raton FL USA)

18 Mainil-Varlet P Curtis R and Gogolewski S Effect ofin vivo and in vitro degradation on molecular and mech-

REFERENCES anical properties of various low-molecular-weight poly-lactides J Biomed Mater Res 1997 36 360ndash380

1 Tormala P Pohjonen T and Rokkanen P Bioabsorbable 19 Li S M Garreau H and Vert M Structure-propertypolymers materials technology and surgical applications relationships in the case of the degradation of massiveProc Instn Mech Engrs Part H J Engineering in Medicine aliphatic poly-(a-hydroxy acids) in aqueous media Part 11998 212 101ndash112 poly(DL-lactic acid) J Mater Sci Mater Med 1990

2 Chasin M Biodegradable polymers for controlled drug 1 123ndash130delivery In Biomedical Applications of Synthetic Bio- 20 Li S Garreau H and Vert M Structure-propertydegradable Polymers (Ed J O Hollinger) 1995 pp 1ndash15 relationships in the case of the degradation of massive(CRC Press Boca Raton FL USA) poly(a-hydroxy acids) in aqueous media Part 3 influence

3 Chaignaud B E Langer R and Vacanti J P The history of the morphology of poly(L-lactic acid) J Mater SciMater Med 1990 1 198ndash206of tissue engineering using synthetic biodegradable polymer

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318 N A WEIR F J BUCHANAN J F ORR AND G R DICKSON

21 Hurrell S and Cameron R E Polyglycolide degradation 37 Vert M Li S M Spenlehauer G and Guerin PBioresorbability and biocompatibility of aliphatic poly-and drug release Part I changes in morphology during

degradation J Mater Sci Mater Med 2001 12 811ndash816 esters J Mater Sci Mater Med 1992 3 432ndash44638 Li S Garreau H and Vert M Structure-property22 Bergsma J E Rozema F R Bos R R M Boering G

de Bruijn W C and Pennings A J In vivo degradation relationships in the case of the degradation of massivealiphatic poly-(a-hydroxy acids) in aqueous media Part 2and biocompatibility study of in vitro pre-degraded

as-polymerized polylactide particles Biomaterials 1995 degradation of lactideglycolide copolymers PLA375GA25and PLA75GA25 J Mater Sci Mater Med 1990 116 267ndash274

23 Bergsma J E de Bruijn W C Rozema F R 131ndash13939 Pistner H Bendix D R Muhling J and Reuther JBos R R M and Boering G Late degradation tissue

response to poly(L-lactide) bone plates and screws Poly(L-lactide) a long-term degradation study in vivoPart III Analytical characterization Biomaterials 1993Biomaterials 1995 16 25ndash31

24 Gunatillake P A and Adhikari R Biodegradable synthetic 14 291ndash29840 Von Burkersroda F Schedl L and Gopferich A Whypolymers for tissue engineering Eur Cell Mater 2003

5 1ndash16 degradable polymers undergo surface erosion or bulkerosion Biomaterials 2002 23 4221ndash423125 Weir N A Buchanan F J Orr J F Farrar D F and

Boyd A Processing annealing and sterilisation of poly-L- 41 Gopferich A and Tessmar J Polyanhydride degradationand erosion Advanced Drug Delivery Reviews (ADDR)lactide Biomaterials 2004 25 3939ndash3949

26 Suuronen R Pohjonen T Taurio R Tormala P 2002 54 911ndash93142 Von Recum H A Cleek R L Eskin S G andWessman L et al Strength retention of self-reinforced

poly-L-lactide screws and plates an in vivo and in vitro Mikos A G Degradation of polydispersed poly(L-lacticacid) to modulate lactic acid release Biomaterials 1995study J Mater Sci Mater Med 1992 3 426ndash431

27 Farrar D F and Gillson R K Hydrolytic degradation of 16 441ndash44743 Duek E Zavaglia C and Belangero W In vitro studypolyglyconate B the relationship between degradation

time strength and molecular weight Biomaterials 2002 of poly( lactic acid) pin degradation Polymer 1999 406465ndash647323 3905ndash3912

28 Gopferich A Mechanisms of polymer degradation and 44 Joukainen A Pihlajamaki H Makela A EAshammakhi N et al Strength retention of self-reinforcederosion Biomaterials 1996 17 103ndash114

29 Reed A M and Gilding D K Biodegradable polymers drawn poly-LDL-lactide 7030 (SR-PLA70) rods andfixation properties of distal femoral osteotomies with thesefor use in surgery poly(glycolic)poly( lactic acid) homo

and copolymers 2 In vitro degradation Polymer 1981 rods An experimental study on rats J Biomater SciPolymer Edn 2000 11 1411ndash142822 494ndash498

30 Anderson J M Perspectives on the in vivo responses of 45 Kellomaki M Paasimaa S and Tormala P Pliable poly-lactide plates for guided bone regeneration manufacturingbiodegradable polymers In Biomedical Applications of

Synthetic Biodegradable Polymers (Ed J O Hollinger) and in vitro Proc Instn Mech Engrs Part H J Engineeringin Medicine 2000 214 615ndash6291995 pp 223ndash233 (CRC Press Boca Raton FL USA)

31 Pitt C G and Gu Z-W Modification of the rates of chain 46 Siemann U The influence of water on the glass transitionof poly(dl-lactic acid) Thermochimica Acta 1985 85cleavage of poly(e-caprolactone) and related polyesters in

the solid state J Control Release 1987 4 283ndash292 513ndash51647 An Y H Woolf S K and Freidman R J Pre-clinical32 Fischer E W Sterzel H J and Wegner G Investi-

gation of the structure of solution grown crystals of lactide in vivo evaluation of orthopaedic bioabsorbable devicesBiomaterials 2000 21 2635ndash2652copolymers by means of chemical reactions Kolloid-Z u

Z Polymere 1973 251 980ndash990 48 Woodward S C Brewer P S Moatamed FSchindler A and Pitt C G The intracellular degradation33 Dickson G R Chemical fixation and the preparation of

calcified tissues for transmission electron microscopy In of poly(e-caprolactone) J Biomed Mater Res 1985 19437ndash444Methods of Calcified Tissue Preparation (Ed G R Dickson)

1984 pp 79ndash145 (Elsevier Science Amsterdam Oxford 49 Pietrzak W S Sarver D R and Verstynen M LBioabsorbable polymer science for the practicing surgeonNew York)

34 Boyde A Methodology of calcified tissue specimen J Craniofac Surg 1997 8 87ndash9150 Vasenius J Vainionpaa S Vihtonen K Makela Apreparation for scanning electron microscopy In Methods

of Calcified Tissue Preparation (Ed G R Dickson) 1984 Rokkanen P et al Comparison of in vitro hydrolysis sub-cutaneous and intramedullary implantation to evaluate thepp 251ndash306 (Elsevier Science Amsterdam Oxford New

York) strength retention of absorbable osteosynthesis implantsBiomaterials 1990 11 501ndash50435 Hutchinson J M Studying the glass transition by DSC

and TMDSC J Therm Analysis Calorimetry 2003 72 51 Matsusue Y Yamamuro T Oka M Shikinami Y et alIn vitro and in vivo studies on bioabsorbable ultra-high-619ndash629

36 Pohjonen T and Tormala P Hydrolytic degradation of strength poly(L-lactide) rods J Biomed Mater Res 199226 1553ndash1567ultra-high-strength self-reinforced poly-L-lactide A tem-

perature dependence study In Biodegradable Implants in 52 Hooper K A Macon N D and Kohn J Com-parative histological evaluation of new tyrosine-derivedFracture Fixation (Ed P C Leung) 1994 pp 75ndash88

(Department of Orthopaedics and Traumatology Chinese polymers and poly(L-lactic acid) as a function of polymerdegradation J Biomed Mater Res 1998 41 443ndash454University of Hong Kong and World Scientific)

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319DEGRADATION OF POLY-L-LACTIDE PART 1

53 Pitt C G Chasalow F I Hibionada Y M Klimas D M experience Injury Int J Care Injured 2002 33S-B4ndashB16and Schindler A Aliphatic polyesters I The degradation

of poly(e-caprolactone) In-Vivo J Appl Polym Sci 1981 57 Suuronen R Pohjonen T Hietanen J and Lindqvist CA 5-year in vitro and in vivo study of the biodegradation26 3779ndash3787

54 Cam D Hyon S-H and Ikada Y Degradation of of polylactide plates J Oral Maxillofacial Surg 199856 604ndash614high molecular weight poly(L-lactide) in alkaline medium

Biomaterials 1995 16 833ndash843 58 Lowry K J Hanson K R Bear L Peng Y BCalaluce R Evans M L et al Polycaprolactoneglass55 Timmins M R and Lenz R W Enzymatic biodegradation

of polymers the polymer chemistsrsquo perspective Trends in bioabsorbable implant in a rabbit humerus fracture modelJ Biomed Mater Res 1997 36 536ndash541Polymer Science (TRIP) 1994 2(1) 15ndash19

56 Gutwald R Schon R Gellrich N-C Schramm A 59 Athanasiou K A Agrawal C M Barber A andBurkhart S Orthopaedic applications for PLA-PGA bio-Schmelzeisen R and Pistner H Bioresorbable implants in

maxillo-facial osteosynthesis experimental and clinical degradable polymers Arthroscopy 1998 7 726ndash737

H01004 copy IMechE 2004 Proc Instn Mech Engrs Vol 218 Part H J Engineering in Medicine