designing of hydroxyapatite-gelatin correlation with bio compatibility aspects

8/2/2019 Designing of Hydroxyapatite-Gelatin Correlation With Bio Compatibility Aspects

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1. Introduction

Bone is a natural composite in the body, boneserves a number of functions, such as providing thecells found in the marrow that differentiate into

blood cells, and also acting as a calcium reservoir.Nevertheless, its primary purpose is to providemechanical support for soft tissues and serves as ananchor for the muscles that generate motion [1].The incidence of fractures increases rapidly withage. This is partly due to extraosseous factors suchas the impaired reflex of the elderly, their reducedproprioceptive efficiency, reduced cushioning byfat, weakened musculature and by osseous factorssuch as the structural changes in the shape and size

of the bone and by deterioration of the condition ofthe bone material itself [2].

Bone graft materials are quickly becoming a vitaltool in reconstructive orthopedic surgery anddemonstrate considerable variability in theirappearance. Functions of bone graft materials and

bone healing provide a structural substrate for theseprocesses, and serve as a vehicle for direct antibi-otic delivery. The three primary types of bone graftmaterials are allografts, autografts, and syntheticbone grafts substitutes [3]. During the last 5 yearsbone cement materials have grown in popularityand are very promising osteoconductive substitutesfor bone graft, they are prepared like acryliccements.Hydroxyapatite (HA), which has molecular stoi-

chiometric formula Ca10(PO4)6(OH)2, has beenextensively investigated due to its excellent bio-

201

*Corresponding author, e-mail: [email protected] BME-PT and GTE

Designing of hydroxyapatite-gelatin based porous matrix as

bone substitute: Correlation with biocompatibility aspects

H. Bundela, A. K. Bajpai*

Bose Memorial Research Laboratory, Department of Chemistry, Government Model Science College. Jabalpur (M.P),

India

Received 10 December 2007; accepted in revised form 3 February 2008

Abstract. In the present study polyacrylamide (PAm)-gelatin-hydroxyapatite (HA) composites have been synthesized bysuspension polymerization method. The prepared composites were characterized by Fourier transform spectroscopy (FTIR)which revealed the presence of functional groups in the composite. The X-ray diffraction (XRD) studies indicated that HApowder was present in nano size. Thermogravimetric analysis (TGA) revealed that composite is more thermally stable thanthe polymer matrix alone. The morphology of composite studied by optical microscopy (OPM) and scanning electronmicroscopy (SEM) suggested that pore size was between 320 m. The composites showed adequately good mechanicalproperties as evident from the varying compressive strength and modulus in the range 31.578.16 MPa and 745388 MPa,respectively. The water sorption behavior was found to be dependent on the chemical composition of the matrix and thesorption data were used to calculate network parameters. The porosity of composite varied between 4 to 30.66%.The in

vitro blood compatibility indicated that the adsorption of bovine serum albumin (BSA) varied from 0.11 to 0.24 mgg1, thepercentage haemolysis was between 2.4 to 6.9% and the weight of blood clot formed on the composite surfaces were foundin the range 11 to 52 mg.

Keywords:polymer composites, hydroxyapatite, polyacrylamide, gelatin, blood compatibility

eXPRESS Polymer Letters Vol.2, No.3 (2008) 201213

Available online at www.expresspolymlett.com

DOI: 10.3144/expresspolymlett.2008.25


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compatibility, bioactivity and osteoconductivity aswell as its similarities to the main mineral compo-nent of bone. However, the poor compressivestrength and fatigue failure limits its applicabilityto the low or non load-bearing sites in human body

[4, 5]. Additionally, it has been reported that HA inthe form of powders, used for the treatment of bonedefects, has problem associated with migration toplaces other than implanted areas. It is known thatvarious biocomposites existing in nature, such asshells and pearls, are all organic/inorganic compos-ites with good mechanical properties, which mayprovide a route to resolve the above problems.Extensive research has been carried out in thisregard and composite materials based on HA and a

variety of polymers have been worked out [6].Among them, hydroxyapatite is frequently used inorthopedic, dental and maxillofacial applications,meaning that it supports bone growth and osteoin-tegration [79]. The development of bonelike com-posites with enhanced biocompatibility calls for abiomimetic approach using natural bone as a guide.Natural bone is a composite of collagen, a protein-based hydrogel template, and carbonated apatitecrystals with varying compositions and microstruc-

tures. The unusual combination of a hard inorganicmaterial and an underlying elastic hydrogel net-work gives bone, unique mechanical properties,such as low stiffness, resistance to tensile and com-pressive forces, and high fracture toughness [10].Gelatin is a natural biopolymer obtained as ahydrolysis product of collagen, which is a fibrousprotein, found abundantly in the animal kingdom inthe form of hides, skins, bones and connective tis-sues. This natural water-soluble biopolymer has toits credit a large number of applications in pharma-ceuticals, medicine, food and other allied fields[11]. However the hydrogels based on Polyacry-lamide (PAm) have been widely used in drugrelease system, membranes for dialysis, oxidationdevices, fixation of chemical enzymes etc. [12, 13].Authors are interested in a bottom-up approach tothe design and synthesis of artificial bone. Thisentails the design of simple model system withwell-designed chemical, physical, and biologicalproperties, followed by an iterative increase in

complexity of the system to realize a higher orderapproximation of natural bone . In the present studycrosslinked PAm-gelatin-HA composites havebeen synthesized using sedimentation approach.

Furthermore structural characterization, bloodcompatibility tests were also carried out. Theseresults provide a framework for generating syn-thetic composites with defined organic/inorganicinterfaces similar to natural bone.

2. Materials and methods

Calcium hydroxide Ca(OH)2 was purchased formE. Merck and used as received. Orto-phosphoricacid (H3PO4, 8893%), was purchased from Qali-gens Fine Chemicals. Acrylamide (E. Merck, India)was freed from inhibitor after recrystallizing ittwice from methanol and drying over anhydroussilica for a week. Gelatin purified was received

from E. Merck, India. MBA (N,N-methylene bis-Acrylamide) were purchased from central drughouse Mumbai (India). KPS (Potassium persul-phate), obtained from Loba chemicals India, wasemployed as a polymerization initiator.

2.1. Synthesis of HA

HA was synthesized by the slow addition of 0.6 MH3PO4 to an aqueous suspension of 1.0 M Ca(OH)2

under constant heating at 150C in N2 atmosphereas per the method reported in literature [14]. Inorder to obtain uniform size crystals of HA the pHof the reaction mixture was strictly controlled i.e.during addition of solutions the pH was maintainedin the range of 1112 while after washing off thepowder the pH was below 9.

2.2. Preparation of PAm-gelatin-HA

composites

PAm-gelatin-HA composites were prepared by freeradical polymerization of acrylamide in the imme-diate presence of a crosslinker (MBA) and HAtaken in pre-calculated amounts. In a typical exper-iment, 0.5 g of gelatin was dissolved into 15 ml ofdistilled water followed by the addition of28.13 mM of Am, 3.0 g of HAp, 0.129 mM ofMBA and 0.073 mM of KPS as free radical initia-tor. The whole reaction mixture was homogenizedby manual mixing and poured into rectangular

glass moulds of definite size (25 mm 25 mm 10 mm). The mould containing the reaction mix-ture was kept at 70C for 4 h, then the upper poly-mer layer was separated from the lower slab which

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Bundela and Bajpai eXPRESS Polymer Letters Vol.2, No.3 (2008) 201213


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was a white solid composite of PAm, gelatin andHA. The composite was then purified by immers-ing it in distilled water so as to allow the compositeto swell till equilibrium. In this way the unreactedmonomer and other reagents were leached out from

the composite thus purifying the prepared PAm-gelatin-HA composite. The swollen soft compositewas cut into desired shape, dried at 70C for 24 hand then kept in air-tight containers.

2.3. Characterization

2.3.1. FTIR-studies

IR studies of the powdered specimens wererecorded on a FTIR-8400S, Shimadzu spectropho-

tometer. Prior to analysis KBr pellets were pre-pared by mixing 1:10 of sample: KBr (wt/wt)followed by uniaxial pressing the powders undervacuum. Spectra were obtained between 4400450 cm1 at 2 cm1 resolution.

2.3.2. XRD-studies

The XRD apparatus (Philips PW 1820) powder dif-fractometer, was used to investigate the crys-

tallinity and phase content of PAm-gelatin-HAcomposites. The diffraction data were collectedfrom 2 to 60, 2 values with a step size of 0.02and counting time of 2 sstep1 at i.e. 1.54 .

2.3.3. TGA

To evaluate thermal stability of the PAm-gelatin-HA composites, TGA was performed on MET-TLER TA 3000 instrument in the temperaturerange of 50800C, in nitrogen atmosphere at a

heating rate of 10Cmin1. The sample weightswere in the range of 1520 mg.

2.3.4. Microscopy study

An approximate idea of the morphology of com-posites was deduced by using Optical microscope,MIOTIC DIGITAL MICROSCOPE DMWB-series.In order to study the morphology of the preparedcomposites SEM was carried out on STEREO

SCAN, 430, Lecica SEM, USA.

2.3.5. Mechanical testing

Compression tests (dry test) were performed usingan Universal Testing Machine (Instron series IX)possessing a load cell of 5 kN, at room temperature.The gauge length and diameter of all specimens

were 6 mm and 2 mm respectively. Tests were con-ducted with a constant strain rate of 1 mmmin1,and up to failure or until 60% reduction in speci-men height. The modulus (E) was determined bylinear regression from the slopes in the initial elas-tic portion of the stress- strain diagram. A mini-mum number of 10 specimens were tested, andthenEwas averaged from the 5 measurements.

2.3.6. Swelling studiesThe extent of swelling was determined by a con-ventional gravimetric procedure as reported in liter-ature [15]. In a typical experiment, preweighedpieces of PAm-gelatin-HA composites wereallowed to swell in distilled water for a predeter-mined time period (up to equilibrium swelling),thereafter the pieces were taken out from the waterand gently pressed in-between the two filter papersto remove excess of water and finally weighed

using a sensitive balance. The swelling ratio wasdetermined by the Equation (1):

(1)

2.3.7. Porosity determination

The apparent porosity of a porous scaffold can

influence its mechanical strength, permeability, andpresence of structural defects [16].The porosity wasdetermined by the method reported in literature[17]. In brief, the known volume and weight of thesamples noted as V0 and W0 respectively. After thatsamples were immersed into the dehydrated alco-hol for 48 h till absorbing dehydrated alcohol satu-rated the samples. The weight gained by the sampleis measured as W1. Finally the porosity (P) of theopen pores in the composites were evaluated using

formula given in Equation (2):

(2)0

01

V

WWP

=

)(

)(

geldryofweight

gelswollenofweight

)(ratiowelling

d

w

r

S

S

SS

=

=

203



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where is the density of the dehydrated alcohol.

2.3.8. Blood compatibility

A biomaterial is a substance used in medical

devices for contact with the living body for theintended method of application and for the intendedtime period. To acquire biocompatibility, the mate-rials used in medical applications must meet certainregulatory requirements. The surface of biomateri-als is believed to play an important role in deter-mining biocompatibility. For materials that comeinto contact with blood, the formation of clot is themost undesirable but frequently occurring eventthat restricts the clinical acceptance of a material to

be used as biomaterial. Therefore, certain test pro-cedures have been developed and they need to beemployed to judge the haemofriendly nature ofmaterials.

2.3.8.1. Clot formation tests

The anti-thrombogenic potential of the compositesurface was judged by the blood-clot formationtest, as described elsewhere [18]. In brief, the spec-

imens were equilibrated with saline water (0.9% w/vNaCl) at 37C for 24 h in a constant temperaturebath. To these swollen samples was added 0.5 ml ofACD blood followed by the addition of 0.03 ml ofCaCl2 solution (4 moll1) to start the thrombus for-mation. Adding 4.0 ml of deionized water stoppedthe reaction and the thrombus formed was sepa-rated by soaking in water for 10 min at room tem-perature and then fixed in 36% formaldehydesolution (2.0 ml) for another 10 min. The fixed clotwas placed in water for 10 min and after drying itsweight was recorded. The same procedure wasrepeated for glass surface, blood bags and for thecomposites of varying compositions and respectiveweights of thrombus formed was recorded.

2.3.8.2. % Haemolysis tests

Haemolysis experiments were performed on thesurfaces of prepared PAm-gelatin-HA compositesas described elsewhere [19]. In a typical experi-

ment, dry composite pieces (4 cm2) were equili-brated in normal saline water (0.9% w/v NaCl) at37C for 24 h and human ACD blood (0.25 ml) wasadded into the composites after 20 min 2.0 ml of

saline water was added into the specimens to stophaemolysis and the samples were incubated for60 min at 37C. Positive and negative controlswere obtained by adding 0.25 ml of human ACD(acid citrate dextrose) blood and 0.9% NaCl respec-

tively to 2.0 ml of doubly distilled water. Incubatedsamples were centrifuged for 45 min, the super-natant was taken and its absorbance at 545 nm wasrecorded using a spectrophotometer. The percent-age of haemolysis was calculated using the follow-ing relationship, given in Equation (3):

(3)

whereA is absorbance. The absorbance of positiveand negative controls was found to be 1.73 and0.048, respectively.

2.3.8.3. Protein (BSA) adsorption

Adsorption of BSA onto the composite materialswas performed by the batch contact processreported elsewhere [20]. For protein adsorptionexperiment, protein (BSA) solutions were prepared

in 0.5 M PBS (Phosphate buffer saline) at physio-logical pH 7.4. A fresh solution of BSA was alwaysprepared prior to adsorption experiments. The com-posite of definite weights were equilibrated withPBS for 24 h. The adsorption was then carried outby gently shaking a BSA solution of known con-centration containing preweighed and fully swollencomposites. By taking fully swollen samples, thepossibility of soaking of BSA solution within thecomposite becomes minimum. Shaking was per-formed so gently that no froth was produced, other-wise BSA would adsorb in air-water interface.After a definite time period, the samples wereremoved and the adsorbed protein was assayed forthe remaining concentration of BSA by recordingthe absorbance at of protein solution at 272 nm on aUV spectrophotometer (Systronics, model no. 2201,India).

2.3.8.4. Platelet adhesion test

A piece of composite of definite composition wasincubated in platelet rich plasma (PRP) at 37C for6 min. The blood was obtained from a healthydonor and PRP was separated by automated aphaere-

controlcontrol

controlsampletest

AA

AA

)()(

)([%]Haemolysis

+

=

204



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sis. After incubation, the material was fixed in 2.5%gluteraldehyde aqueous solution for 10 min andkept in a 70% ethanol solution in order to avoiddehumidification and microorganism proliferation.The amount and morphology of adhered platelets

were analyzed by SEM [21].

3. Results and discussion

3.1. FTIR-studies

The FTIR spectra of native HA sintered at 70C isshown in Figure 1a which clearly shows peaks at602, 962, 1035 cm1, corresponding to PO43 ion[22] and a small and sharp band observed at3572 cm1, corresponds to the stretching mode of

OH group, which is characteristic of hydrated cal-cium phosphate such as HA [23]. A weak peakobserved at 876 cm1 and strong peak at 1450 cm1

corresponds to the stretching vibration of CO32

ions. These observations confirm that HA crystalswere prepared, partially substituted by CO32

groups. Therefore, HA crystals are CO32 contain-ing HA [24]. Also the broad bands at about 3200and 2800 cm1, correspond to the absorbed hydrateand the sharp medium and short peaks between

35703670 cm1

belong to the stretching vibrationsof lattice OH ions of hydroxyapatite [25]. It has

been reported that the FTIR spectra of PAm wherepeaks at about 3298, 3184 and 1660 cm1 showedthe presence of primary amide (NH stretchingvibration) group and peaks near about 1595 and1429 cm1 corresponding to C=O asymmetric

stretching vibration [26]. Gelatin being a protein,has been reported to contain the characteristicamide absorption bands at about 1690 and1530 cm1 [27]. The FTIR spectra of PAm-gelatin-HA composite is shown in Figure 1b, which con-tains all the characteristic absorption peaks of HA,PAm and gelatin. As there are no considerableshifts of peaks of any group in the composite spec-trum, it is confirmed that PAm-gelatin-HA com-posite is only a mixture and no chemical reaction

has taken place between the individual compo-nents.

3.2. XRD studies

The XRD pattern of the synthesized native HA isshown in Figure 2a which exhibits sharp apatite

205


Figure 1. (a) FTIR spectra of native HA sintered at 70Cfor 24 h; (b) FTIR spectra of PAm-gelatin-HAcomposite

Figure 2. (a) XRD pattern of native HA sintered at 70Cfor 24 h; (b) XRD pattern of native PAm-gela-tin-HA composite with given composition,[AM] = 28.13 mM, [gelatin] = 0.5 g, [HAp] =3.0 g, [MBA] = 0.129 mM


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peaks due to crystal growth alternatively calciumcarbonate peak at 2 value of about 29 presenttogether with apatite phase. However , the -trical-cium phosphate (-Ca3P2O7) phase was notdetected at any temperature. Also, the CaO peaks at

37.469 and 54.029 were not detected. The accom-panying two peaks at about 32.22 and 32.23 ofequal intensities were also detected which clearlyconfirm the presence of well crystallized HA phase[28]. The mean grain size was calculated usingDebye-Scherrer formula [29, 30] as shown in Equa-tion (4):

(4)

where d is mean grain size, k is the shape factor(0.9), is broadening of the diffraction angle and is diffraction wavelength (1.54 ).The estimatedaverage grain size of HA was found to be 6.53 nmand the width was found to be 1.278 nm. Figure 2bshows the XRD spectra of PAm-gelatin-HA com-posite with slight broadening of the apatite peaksshowing the decrease in crystallinity of HAbecause of incorporation of polymer. The spectrumalso shows a XRD peak at about 31.99 (2) indi-

cating well crystalline nature of hydroxyapatiteeven in composite state.

3.3. TGA analysis

In order to evaluate thermal stability and under-stand the phase transformation in the prepared sam-ples, the TGA studies were performed in the range50 to 800C and the results are shown in Figure 3.It is clear from the thermogram of native HA (Fig-ure 3a) that the weight of the sample decreasesquickly with increasing temperature and about total66% weight loss is observed. The obtained weightloss is possibly due to evaporation of water. Theobtained TGA results were further confirmed by asimple experiment that involved heating the com-posite of known initial weight in an oven in thetemperature range 50 to 800C. After heating wasover the weight of composite was recorded and aweight loss of about 66% was noticed. It is revealedby the data that the weight loss that occurred at

about 580 and 620C could be attributed to thedecomposition of trace CO32 ions [31]. Theexothermic dissociation of CO32 is reported tooccur at the temperature range between 500 to

890C in nitrogen atmosphere. Figure 3b corre-sponds to the thermogram of PAm-gelatin-HAcomposite where initial weight loss from 50 to200C may be due to the evaporation of surfaceadsorbed water molecules and the quick weightloss from about 300 to 600C may be due to theendothermic dissociation of CO32 ions [32]. Fur-

thermore, the TGA of natural compact bone shownin Figure 3c, may also be compared with that ofprepared composite and a fair resemblance may benoticed.

3.4. Microscopy study

In order to study the morphology of the preparedcomposites and adhesion of platelets OPM studyhas been performed as shown in Figure 4a and 4b,respectively. The comparison of both the imagesindicates a change in surface of the compositematerial after the addition of platelets.Since the morphology of a biomaterial contributessignificantly to its biocompatibility and consideringthis important aspect, the morphology of the sur-face has been examined by recording SEM of thePAm-gelatin-HA composite. The SEM images ofthe composites are shown in Figure 5, whichclearly shows that the composite surface is highlyporous in nature. The size of the pores varies in the

range 3 to 20 m as indicated by arrows in the samefigure.

=

cos

kd

206


Figure 3. (a) TGA curve of native HA; (b) TGA curve ofPAm-gelatin-HA composite with given compo-sition [Am] = 28.13, [gelatin] = 0.5 g mM,

[HAp] = 3.0 g, [MBA] = 0.129 mM; (c) TGAcurve of natural compact bone


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3.5. Mechanical testing

To examine the mechanical properties of the PAm-gelatin-HA composites (having composition[Am] = 28.13, [gelatin] = 0.5 g mM, [HAp] =4.0 g, [MBA] = 0.129 mM) compression tests ofthe specimens were conducted, in dry condition.The modulus (E) was found to be 745 388 MPafor the control set of composites and compressivestrength was found to be 31.57 8.16 MPa. Theenhanced modulus provides increased fracturetoughness to the samples. These results show thatPAm-gelatin-HA composites possess quite goodmechanical properties for being used in tissue engi-neering (TE). The values show good agreementwith those obtained for normal human articular car-

tilage, which has been reported to have a compres-sive strength ranging from 1.9 to 14.4 MPa [33,34]. The compressive strength and Youngs modu-lus of trabecular bone were reported rangingbetween 210 MPa and 50100 MPa, respectively[35, 36].

3.6. Swelling behavior of composite

One of the prime factors to contribute to biocom-patible nature of synthetic biomaterials is theamount of water content which imparts severalunique physiochemical properties to the material. Apolymer matrix imbibing an adequate of water,shows living tissue like membrane, physiologicalstability, low interfacial tension, permeability tobiomolecules etc. Thus realizing the unusual signif-icance of water sorption capacity of a material, thePAm-gelatin-HAp composites have been investi-gated for water sorption capacity and the influenceof chemical composition of the composites on their

water intake has been investigated as discussedbelow.

3.6.1. Effect of Am

The influence of Am content of the composite onits swelling behavior has been studied by varyingits concentration in the range 14.06 to 56.27 mM.The observed results are shown in Figure 6a whichclearly indicate that the swelling ratio constantlyincreases with increasing amount of PAm.Theobserved results are quite expected as PAm is ahighly hydrated polymer and its increasing contentin the composite makes it more hydrophilic in

207


Figure 5. SEM micrographs of PAm-gelatin-HA compos-

ite, pores have been indicated with arrows

Figure 4. Optical micrographs of PAm-gelatin-HA com-posite. (a) composite without platelet; (b) com-posite with platelet (indicated with arrows)


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nature which eventually results in an enhancedswelling.

3.6.2. Effect of gelatin

The effect of gelatin on the swelling ratio of thehydrogel was investigated by varying the concen-tration of gelatin in the range 0.5 to 2.0 g in the feedmixture of the composite. The results are shown inFigure 6b which clearly reveal that the swellingratio constantly increases with increasing concen-tration of gelatin up to 2.0 g. It is to be mentionedhere that beyond this concentration the feedingmixture becomes too viscous to form the compos-ite. The observed results may be explained by thefact that, since gelatin itself has a natural tendency

to form reversible gel, its increasing pressure inPAm-gelatin-HAp mixture lowers the weight frac-tions of HA and PAm in the feed mixture. In thisway a lower degree of crystallinity results in an

enhanced swelling. Gelatin is a hydrophilic poly-mer and its increasing concentration in the feedmixture increases water sorption capacity of com-posite.

3.6.3. Effect of hydroxyapatite

Impregnation of hydroxyapatite into the polymermatrix brings about a significant change in watersorption behavior and mechanical properties of thematrix. In order to obtain the effect of HA on theswelling ratio of the composite, the concentrationof the HA was varied in the range 1.0 to 4.0 g in thefeed mixture. The results are shown in Figure 6c,which clearly reveal that the swelling ratio con-stantly decreases with increasing HA content in the

composite. The results are quite expected and maybe explained by the fact that due to relatively lowerhydrophilicity of the apatite in comparison to PAm-gelatin matrix, its increasing fraction in the com-

208


Figure 6. (a) Variation of swelling ratio with varying amounts of Am i.e. 14.06 to 56.27 mM; (b) variation of swellingratio with varying amounts of gelatin i.e. 0.5 to 2.0 g; (c) variation of swelling ratio with varying amounts ofHA i.e. 1.0 to 4.0 g; (d) variation of swelling ratio with varying amounts of MBA i.e. 0.064 to 0.259 mM


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posite results in a lower water sorption by the com-posite. Alternatively, the increasing polymer-HAinteraction with increasing concentration of HAresults in a slower relaxation of polymer chains,which also decreases the swelling ratio.

3.6.4. Effect of crosslinker

One of the effective ways to modify the water sorp-tion behavior of a polymer matrix is to employvarying amounts of crosslinking agent at the timeof polymerization reaction. The addition of acrosslinker not only enhances the degree ofcrosslinking but also increases the glass transitiontemperature (Tg) of the polymer. In the presentstudy the effect of crosslinker content of the com-posite on its swelling ratio has been studied byvarying its concentration in the range of 0.064 to0.259 mM. The results are shown in Figure 6dreveal the fact that with the increasing content ofMBA in the feeding mixture the crosslink densityof the network increases [37] which results inhigher crosslink density of the network. This obvi-ously slows down the diffusion of water moleculesinto the composite and eventually results in a fall inwater intake [38].

3.7. Network parameters

One of the most important structural parameterscharacterizing a crosslinked density is the averagemolecular mass between crosslinks (Mc), which isdirectly related to the crosslink density. The magni-tude ofMc significantly affects the physical andmechanical properties of crosslinked polymers, andits determination has great practical significance.Eqilibrium swelling is widely used to determine

Mc. Early research by Flory and Rehner laid thefoundation for the analysis of equilibrium swelling.According to the theory of Flory and Rehner, for aperfect network (Equation (5)):

(5)

where in Equation (5)Mc is the number averagemolar mass of the chain between crosslinks, v1 isthe molar volume [mlmol1], dp is the polymerdensity [gml1], Vs is the volume fraction of poly-mer in the swollen gel and is the Flory and Hug-gins interaction parameter between solvent andpolymer. The swelling ratio is equal to 1/Vs. Herethe crosslink density q is defined as the mole frac-tion of crosslinked units, as shown by Equation (6):

(6)

whereM0 is the molar mass of the repeating unit.Some authors defined a crosslink density, Ve, as thenumber of elastically effective chains totallyincluded in a perfect network per unit volume, sim-ply related to q since, as given by Equation (7),

(7)

where NA is Avogadros number. The density ofpolymer dp was determined to be 0.65 gcm3.Other parameters such as v1 and were taken fromthe literature . Using Equations (5)(7) the valuesofMc, q, and Ve have been calculated for the vary-ing compositions of PAm-gelatin-HA composites[39]. The values are summarized in Table 1.

c

Ape

M

NdV =

cM

Mq 0=

2

3/1

1)1ln(

2

sss

ss

pcVVV

VVdvM

++

=

209


Table 1. Data showing the structural parameters of PAm-gelatin-HA composites of varying compositions

MBA (crosslinker)

[mM]

Am

[mM]

Gelatin

[g]

HAp

[g]

Swelling

ratioMc q102 Ve1020

0.064 28.13 0.5 3.0 2.43 189.40 037.52 2.380.129 28.13 0.5 3.0 2.13 083.90 084.71 5.380.259 28.13 0.5 3.0 1.86 055.14 128.00 8.17

0.129 14.06 0.5 3.0 2.08 730.00 009.73 6.170.129 28.13 0.5 3.0 2.13 083.90 084.71 5.380.129 56.27 0.5 3.0 2.45 200.00 035.54 2.25

0.129 28.13 0.5 3.0 2.13 083.90 084.71 5.38

0.129 28.13 1.0 3.0 2.21 123.00 057.78 3.660.129 28.13 2.0 3.0 2.70 047.05 151.07 9.58

0.129 28.13 0.5 1.0 3.00 394.40 020.34 1.290.129 28.13 0.5 2.0 2.22 142.00 050.05 3.170.129 28.13 0.5 4.0 1.67 051.77 137.29 8.71


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3.8. Percent porosity

Porosity characterization is based on the presenceof open pores, which are related to properties suchas permeability, and surface area of the porousstructure. The measured porosity of the PAm-gela-

tin-HA composites is given in Table 2, whichreveals the fact that as the amount of crosslinker isincreased in feed mixture porosity becomes low,the results are quite obvious since increased num-ber of crosslinks make polymer network more com-pact which results in the lower mesh size anddecreased porosity. It was found that the addition ofHA results in more dense and thicker pore wallswith lower porosity [40] as shown in Table 2. How-ever the decrease in porosity observed as the

amount of polymer like PAm is increased in feedmixture is mainly due to the increase in theHAP/polymer ratio in the dispersed phase which inturns improves the sintering and porosity reduction[41]. As we increase the amount of gelatin in feed

mixture porosity increases, this may be explainedon the basis of the fact that gelatin is a hydrophilicpolymer and during the formation of matrix it letsthe polymer network to swell in greater amount,which in turns results in greater pore size within the

network and after the drying of the matrix, it yieldsgreater porosity.

3.9. Blood compatibility

Blood compatibility of a material is intimatelyrelated to various intrinsic factors such as organiza-tion of water molecules in the polymer matrix,chemical architecture and topology of the surfaceetc. In the present study also various in vitro tests

were applied to observe the blood compatibility ofthe prepared PAm-gelatin-HA composite. Theresults are shown in Table 3, which may be dis-cussed as below:

210


Table 2. Data showing the percent porosity of PAm-gelatin-HA composites of different compositions

Table 3. Blood compatibility parameters of PAm-gelatin-HA composites of different compositions

(MBA)

[mM]

Am

[mM]

Hap

[g]

Gelatin

[g]

Swelling

ratio

BSA

adsorption

[mgg1]

Percentage

haemolysis

Blood clot

formation

[mg]

0.064 28.13 3.0 0.5 2.43 0.021 2.4 11.00.129 28.13 3.0 0.5 2.13 0.020 3.4 32.00.259 28.13 3.0 0.5 1.86 0.023 6.9 52.0

0.129 14.06 3.0 0.5 2.08 0.022 4.5 49.00.129 28.13 3.0 0.5 2.13 0.020 3.4 32.00.129 56.27 3.0 0.5 2.45 0.018 2.0 21.0

0.129 28.13 1.0 0.5 3.00 0.018 2.9 25.00.129 28.13 3.0 0.5 2.13 0.020 3.4 32.00.129 28.13 4.0 0.5 1.67 0.024 3.7 43.0

0.129 28.13 1.0 0.5 2.13 0.020 4.2 32.00.129 28.13 3.0 1.0 2.21 0.015 5.0 29.00.129 28.13 4.0 2.0 2.70 0.011 3.4 23.0

Glass surface 44.0Polyethylene bag 30.4 35.0

S. No (MBA) [mM] Am [mM] HAp [g] Gelatin [g] % Porosity

01. 0.064 28.13 3.0 0.5 13.0002. 0.129 28.13 3.0 0.5 11.2903. 0.259 28.13 3.0 0.5 04.00

04. 0.129 14.06 3.0 0.5 30.6605. 0.129 28.13 3.0 0.5 11.2906. 0.129 56.27 3.0 0.5 04.0307. 0.129 28.13 1.0 0.5 11.2908. 0.129 28.13 3.0 0.5 11.2909. 0.129 28.13 4.0 0.5 05.6010. 0.129 28.13 1.0 0.5 11.2911. 0.129 28.13 3.0 1.0 15.1812. 0.129 28.13 4.0 2.0 18.56


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When the concentration of crosslinker is raised inthe range 0.064 to 0.259 mM, the amount of bloodclot formed increases with increasing MBA contentin the composite. The results summarized in Table 3also reveal that both the percent haemolysis and

protein adsorption also increase with increasingMBA content. The results obtained are consistentwith each other and suggest that the compositeshows decreasing blood compatibility with increas-ing number of crosslinks. The results may beexplained by the fact that since MBA is a hydropho-bic crosslinker, its increasing content results ingreater protein-surface interaction and, therefore,shows more clot formation and percent haemolysis.The results reveal the fact that the weight of blood

clot constantly decreases with increasing amount ofPAm and gelatin in the feed mixture. The resultsmay be explained on the basis of the fact that bothPAm and gelatin are hydrophilic polymers andtherefore, are not expected to provoke any damageto blood cells or any change in the structure of theplasma proteins. It has also been realized that withincreasing concentrations of PAm and gelatin, thecomposite acquires more smoothness of their sur-faces and this consequently results in an improve-

ment in antithrombogenic property of material.The prepared composites were tested for haemolyticactivity and the results obtained are quite satisfac-tory. Percent haemolysis is maximum (100%) for adistilled waterwater-added blood sample (positivecontrol). The results obtained clearly indicate that,with increasing PAm and gelatin content, the extentof haemolysis steadily decreases. The observedresults may be attributed to the reason that, with theincrease in PAm and gelatin weight fractions in thecomposite, the surface composition favorablychanges, which improves the blood compatiblequality of the material.One of the essential components of the compositeis HA and its concentration in the composite isexpected to influence blood compatibility of thematrix. In order to examine this the concentrationof HA powder was varied in the range 1.0 to 4.0 gand blood compatibility parameters were evalu-ated. The data summarized in Table 3 reveal thatwith increasing HA content, the blood compatibil-

ity of the composite shows a decrease, i.e. all thethree parameters increase. The reason for theobserved more thrombogenicity is that the ionicgroups of HAp may react with the blood compo-

nents and produce greater blood-surface interac-tions. This is likely to cause thrombogenic behaviorof the composite.The above discussion clearly suggests that a lesscrosslinked composite with more PAm-gelatin and

low HAp content may prove to be more biocompat-ible.

4. Conclusions

Nanosized hydroxyapatite powder is obtained bythe hydrothermal method and the free radical poly-merization of acrylamide in presence of HA andgelatin forms a nanocomposite material. The HA-composites when examined by FTIR spectroscopy

clearly show the presence of HA and polymer com-ponents in the composite. The XRD studies alsoconfirm the nanosized mean grain size of the HApowder in native state as well as in composites.Thermal properties of the material assigned byTGA analysis, meet the thermal requirements ofthese types of materials. The optical microscopyand SEM studies reveal the morphologicalanisotropy of the material.Swelling studies show the effect of composition on

the swelling. It is found that as the PAm and gelatincontents increases, the swelling ratio also increases.The extent of swelling decreases with increasingamounts of HA and crosslinker.The PAm-gelatin-HA composite also display a fairlevel of blood compatibility as confirmed by invitro experiments of blood clot formation, proteinadsorption and haemolysis. It is clear by the data, aswe increase the amount of crosslinker in the feedmixture in the range 0.0640.259 mM, the amountof clot formation increases. Similar type of resultsare observed when the amount of HA increases inthe feed mixture of the composite. In case of poly-mers as the amounts of PAm and gelatin increase incomposite mixture degree of blood clot formationdecreases due to the increased swelling of compos-ite. Percentage haemolysis tests for haemolyticactivity of composites indicate that as the contentof crosslinker and HA increases in feed mixturepercent of haemolysis also increases. The observedresults are consistent with blood clot formation test

results and conclude that a material surface show-ing reluctance to BSA adsorption proves to be morebiocompatible. BSA absorption results also show

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the similar type of trends as those of percentagehaemolysis and clot formation results.

Acknowledgements

The authors gratefully acknowledge the authorities ofBRNS (Board of Research in Nuclear Sciences), BARC(Bhabha Atomic Research Center) Mumbai, India , for pro-viding financial assistance in the form of a project (Sanc-tion no. 2005/35/26-BRNS/RTAC) and performing TGA,XRD and other analysis of prepared composite materials.

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designing of hydroxyapatite-gelatin correlation with bio compatibility aspects

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