New material for tissue engineering DegraPol®

2

Index 1. Introduction....................................................................................................................................3 2. DegraPol®.......................................................................................................................................4

2.1 Polyurethanes..........................................................................................................................5 2.2 Synthesis of hard segment......................................................................................................7

2.2.1 Characteristics of PHB................................................................................................8 2.2.2 Characteristics of ethylenglycole..............................................................................10

2.3 Synthesis of soft segment.....................................................................................................13 2.3.1 Characteristics of poly ε-caprolactone…………………..………………………….13 2.3.2 Characteristics of copolymer ε-caprolactone-diglycolid.……………………..…....14

2.4 Synthesis of DegraPol®......................................................................................………..…15 3. Scaffold........................................................................................................................................17 4. Experimental plan........................................................................................................................19 5. Current application.......................................................................................................................22 6. Possibile development..................................................................................................................24 References........................................................................................................................................25

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1. Introduction The research programme at Ab Medica is trying to define a new kind of polyurethane material

addressing the needs of tissue engineering.

Tissue engineering is today aimed at creating materials which promote, possibly in a controlled

way, cell adehesion. In particular, the current trend is aimed at creating "bioartificial hybrids

organs" in which the artificial component has a three-dimensional structure capable of promoting

the growth and diversification of cells. The artificial support should also preferably reorganize and

be absorbed simultaneously to cell growth and then gradually be replaced by new tissue. From this

point of view, the bioartificial material cannot be considered as a standing substitute of a tissue, but

as a temporary structure which promotes regeneration.

A biomaterial is a material designed to interface with biological systems for assessing, giving

support or replacing any tissue, organ or function of the body.

Biomaterials can be used in both permanent installations or prostheses and devices in contact with

the human body for a limited period of time.

In particular the basic requirements a biomaterial must have in order to b used for tissue engineering

applications are:.

appropriate mechanical, physical and chemical properties;

reproducibility of preparation with an acceptable degree of purity;

ease of sterilization and lack of inflammatory reactions during the initial contact with the

biological environment and lack of side effects after implantation.

Moreover, the structure of a biomaterial should be as homogeneous as possible to allow a uniform

distribution of cells and the extracellular matrix produced by them.

These features are dependent on both the nature of polymers used and the processes of the material.

The research work was initially focused on the detection of formulations more suitable for

biomaterials with appropriate physical and mechanical properties, then processes to obtain a three-dimensional structure capable of promoting the growth and diversification of cells and finally the creation of scaffolds obtained with the elettrospinning technique and phase separation method. The first material which has been studied is Degrapol®, a biocompatible and biodegradable

polyestereurethane, in which it is possible to modulate the chemical and physical properties

depending on the chemical composition and processes.

4

2. DegraPol®

DegraPol® is a polyester-urethane, it consists of two blocks of polymers which impart very different

physical and mechanical properties to the final product. It consists of polyhydroxybutyrate-diol

(Hard Segment) and polycaprolactole-dyglicol-diol (Soft Segment). Both are biodegradable

polymers and their degradation products are not toxic. Using various ratios of hard and soft

segments it’s possibile to modulate the mechanical properties of the final product.

Unlike traditional materials, DegraPol® shows a broad range of elastic modulus, making it a

potential new material for the regeneration of many types of biological tissues. Figure 1 shows the

comparison of Young’s modulus of DegraPol® with both traditionally used polymers

(Polyhydroxybutyrate (PHB), poly-lactic acid) and body tissues to regenerate. It is visible how

DegraPol® has Young’s modulus comparable with many types of cellular tissues, nerves, blood

vessels, while traditional materials applications are limited.

Figure 1. Form elastic of biological tissue and biopolymers

The biocompatibility of DegraPol® has been demonstrated both in vitro and in vivo in previous

studies.[1] The cell interaction, in particular the inflammatory response and cytotoxicity of

degradation products are the most important factors which determine the biocompatibility. For this

purpose, fibroblasts cultures and murine macrophages have been used. In vitro fibroblasts maintain

2

4

6

8

10

12

Literatures:

F.H. Silver, Biological Materials: Structure, Mechanical Properties, and Modeling of Soft Tissues, 1987, New York University Press.

J. Black, G. Hasting, Handbook of Biomaterial Properties, 1998, Chapman & Hall.

J. Jurvelin, I. Kivirant, A.M. Tammi, H.J. Helminen, J. Biomechanics, 1990, 23(12), 1239.

F.A. Grieshaber, U. Faust, Biomed. Technik, 1992, 37, 278.

J. Kohn, R. Langer in Ratner et. al "Biomaterial Science" AP (1996). Bioresorbable and bioerodable materials

Estimated properties for tissue and bioresorbable polymers

Values for synthetic polymersEstimation for tissue

Bone

Po

lyo

rth

oe

ste

rs

Hydro

gels

Po

lyim

ino

ca

rbo

na

tes

PL

A (

D,

L,

DL

)

PH

B

De

gra

Po

l®N

erv

es

Aort

a

Ve

ins

Me

nis

cu

s

Skin

Cart

ilage

Tra

b. bone

log

Te

nsile

Mo

du

ls [

Pa

]

5

their phenotype and produce large amounts of proteins of the extracellular matrix; macrophages are

not activated when grown on polymer, retain their capacity to respond to bacterial toxins and do not

seem to present signs of cell damage, or apoptosis. Tests in vivo confirm the good compatibility

found in vitro.

After two months from the implant of the DegraPol® film in rats, it has been possible to observe

how the fibrotic capsule formed presents a very small thickness and no significant differences in the

specific guideline of fibroblasts were found.

The degradation, in physiological conditions, occurs by means of hydrolysis of ester links. The

timing of degradation depends on the material hydrophobicity. The use of polyglycolic acid, as

copolymer of the soft segment, speeds the process of bioerosion up since the glycolic acid is

characterized by greater hydrophily (the alifatic chain is shorter than the polycaprolactone, 1 to 6-

CH2), making the copolymer more degradable for hydrolysis.

Using different percentages of polycaprolactone and poly-glycolic acid it is possible to vary the

time of degradation from several weeks to several years, without interfering with the mechanical

properties of DegraPol®. This feature allows to choose DegraPol® according to the time for the

regeneration of tissues. In vivo studies[2] demonstrate the biodegradability of DegraPol®, pointing

out that after 1 years from subcutaneous implant in rats, the molecular weight of polymer is reduced

by 50%.

2.1 Polyurethanes The polyurethanes are an important subclass in the family of thermoplastic elastomers. They

contain an urethanic link1 (similar to the carbamate group) and are made of blocks of "soft" (or

flexible) and "hard" (rigid) segments.

The soft segments generally have a low glass transition temperature (Tg) and consist of polyethers,

polyester or polyols. The hard segments normally have a high Tg and are made of diisocyanates

linked to chain extension. As chain extenders diols or diamines with low molecular weight are

commonly used and polyurethanes and polyurethane-urea are obtained respectively.

The polyurethanes may have, depending on their composition, significantly different physical

properties: this is due to the possibility of changing the chemical nature and the molecular weight of

1 Characteristic group RH-NO-CO-R

6

the various components, and the reaction proportion. New rigid and very brittle materials are

realized as well as other soft, amorphous and viscous.

Commonly, these polymers show phase separation between the two segments: the structure is made

up of hard domains dispersed in a soft matrix. It is believed that this feature is responsible for the

peculiar physical and mechanical properties that contribute to make these materials particularly

biocompatible.

Figure 2. Outline of structural organization of polyurethanes

Depending on the type of monomers selected it is also possibile to obtain polymers with degradable

chemical bonds in physiological environment. The modulability of mechanical properties and

biodegradability make polyurethanes excellent candidates for applications in soft tissue

engineering.

Basic requirement for such applications is to obtain non-toxic degradation products. The polyols

used in the synthesis of polyurethanes generally have a molecular weight between 400 and 6000

and functionality (number of hydroxy group reactive per molecule) between 2 and 8. They can be

polyethers (polyether-polyols) polyester (polyols-polyester) and polycarbonates; they are available

in different lengths of chain and in atomic arrangement from linear to variously branched.

Degradable polyurethanes are often obtained by using hydrolyzeble polymers as soft segments as

polylattidi and especially polyester. They are insoluble in water but degradable for hydrolytic

attack to ester link . In vivo this type of degradation can occur in present of special enzymes such as

the 'α-chimotripsina. The mechanisms and timing of degradation dipend on various parameters such

as molecular weight (the greater the length of soft segment, the higher the degree of hydrolysis) the

7

degree of crystallinity, the glass transition temperature and the hydrophily of monomers.

Degrapol® is made of poly-hydroxybutyrate-diol (Hard Segment) and polycaprolactole-diglycole-

diol (Soft Segment).

2.2 Synthesis of hard-segment

The hard-segment is obtained from poly-hydroxybutyrate (PHB), which can be extracted and

isolated from the chemical degradation of a number of microorganisms.

The PHB undergoes a first transformation with ethylene glycol to reduce the molecular weight, to

cutting the long chains of polymer gaining as a result short-chain diols of the hydroxybutyric acid.

O

H

O

O

H HO

OH

+

H

O

O

O

O

O

O

H

m

n

x

x > 1000

1<m<50

1<n<50

Scheme 1. Synthesis of PHB-diol

The initiator is a metal complex, and if the polymer is designed for applications in vivo, preferably

the octanoate tin as initiator is used, since it has the approval of the FDA (Food and Drug

Administration) as a food preservative.

The reaction is considered completed when the average molecular weight (Mw) of polymer reaches

the value of 2600. The chlorobenzene is removed by extraction with dichloromethane. While

methanol, a non-solvent of the polymer, is used for the precipitation.

The polymer obtained, called PHB-diol, appears as a white powder and has the same mechanical

characteristics of PHB, but differs for the molecular weight and the thermal properties.

8

Mw T melting point [°C]

PHB 500000 190 PHB-diol 2600 145

Table 1: Comparison PHB and PHB-diol

The reaction of trans-esterification and depolymerization is conducted in a batch reactor at 140°C,

using chlorobenzene as a solvent, in which the PHB and ethylenglycol are dissolved, according to

the following reaction:

H

O

O

O

O

O

O

H

m

nO

O

O

O

+

H

O

O

O

O

O

O

x+y

n

O

O

O

O

H

dibutyltin laureate

H

O

O

O

O

O

O

n

O

O

O

O

O

y

O

H

1<x+y<501<m<501<n<50

Scheme 2. Synthesis of the hard segment from monomers units

9

2.2.1 Characteristics of PHB

The PHB belongs to polyhydroxyalkanoate family.

The polyhydroxyalkanoate (PIA) are macromolecules synthesized by more than 90 kinds of bacteria

gram + and gram-. In appropriate culture conditions and in particular shortage of some nutrient (eg

N, P, S), polyhydroxyalkanoate accumulate into the bacterium in the form of granules (φ = 0.5 µm)

to a concentration that can reach 90% of dry weight of bacterial mass. The main types of

polyhydroxyalkanoate identified by now are polyester linear head-tail, made up of monomers

belonging to the group of β/3 (R-) hydroxyacids (in much lesser extent also γ, δ, ε (R-)

hydroxyacids). The synthetic scheme of polymerization is shown in Diagram 3.

HO

R

OH

O

n

O

R

*

O

n

+ (n-1) H2O

Scheme 3. Synthetic scheme of polymerization

The lateral group R in position β (3) is an alkyl with C ÷ 13 = 1, which can be linear or branched,

saturated or unsaturated, epoxidised and with halogenated or aromatic substituents.

The exact composition of polyhydroxyalkanoate depends on the type of bacterium from which they

are synthesized and on the culture. For the polyhydroxyalkanoate synthesis, the different types of

bacteria use monomers from different metabolic pathways.

The extreme variability of the chemical nature of the chains side lays the basis of a wide range of

properties of polyhydroxyalkanoate (ranging from typical thermoplastic polymers, such as the

polyhydroxibutyrate, to tires as polyhydroxyottanoate) and of possibility of secondary chemical

interventions (eg cross-linkage ).

The molecular mass of polymers synthesized by bacteria varies from 5,104 to 1,106 Da.

The main polyhydroxyalkanoate are listed in Table 2.

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n*=1 n*=2 n*=3 n*=4 R=H Poly (3-

hydroxypropionate) Poly (4-

hydroxybutirate) Poly (5-

hydroxyvalerate)

R=CH3 Poly (3-hydroxybutirate) Poly (4-hydroxyvalerate)

Poly (5-hydroxyexanoate)

R=C2H5 Poly (3-hydroxyvalerate) R=C3H7 Poly (3-

hydroxyexanoate)

R=C5H11 Poly (3-hydroxyoctanoate)

R=C6H13 Poly (6-hydroxydodecanoate)

R=C9H19 Poly (3 hydroxydodecanoate)

*n=number of CH2 in the linear chain

Table 2. Main polyhydroxyalkanoate

The polyhydroxibutyrate was identified in 1926 by the Institute Pasteur Lemoigne as constituent of

the microorganism Bacillus Megaterium. The production Zeneca/Monsanto (trademark Biopol) is

based on the fermentation of a mutant of Ralstonia on glucose, with the addition of propionate in

shortage of phosphorus.

The strong interest in technology for this polymer is linked to the increasing use of natural polymers

in replacements for traditional polymers derived from oil, and then by non-renewable sources, and

the need to reduce the amount of waste by using biodegradable polymers.

The data reported in Table 3 are related to commercial product Biopol (Zeneca/Monsanto).

This is a thermoplastic polymer, isotactic, orthorhombic, high crystallinity, optically active, with a

molecular mass of about 0.5 106 Da (polydispersion expressed as Mw/Mn = 1.8).

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Properties PHB Polypropilen Tg (°C) +15 -10 Tf (°C) 190 176

Cristallinity (%) 80 70 Resistance (Mpa) 40 38

Elongation at break (%) 8 40 Tensile (GPA) 3.5 -

Resistance to bending Izod-n (J/m) 60 100 Dielectric constant at 1 MHz 3 -

Resistivity (ohm•cm) ≥1016 ≥1016

Higher temperature od use(°C) 130 135 Chemical Resistance

Acid Alkali Alcool

Oils and fats

Low Low

Discreet Good

Excellent Excellent Excellent

Good UV Resistance Discreet Low

Table 3: comparison PHB e Polypropylen

The polyhydroxibutyrate is, for many reason, similar to polypropylene, but unlike this has a glass

transition temperature (Tg) too high and a resistance too low. In addition, the melting temperature is

very close to that of degradation, which makes it problematic, if not impossible, to process with

conventional techniques in use for thermoplastic polymers.

The polyhydroxibutyrate results to be a highly biodegradable polymer. In physiological conditions

ester link are split with an hydrolytic mechanism. The degree of biodegradation of these polymers is

determined primarily by molecular weight, exposed surface, crystallinity and in the case of

copolymers, by the chemical composition and distribution of monomer units. The bioresorption and

the ultimate degradation products may involve macrophages, neutrophils and lymphocytes,

different classes of white blood cells activated by the immune system in the presence of an antigen.

The final degradation product of PHB is the (R)-3-hydrxybutiryc acid, a constituent of human

blood, which is bioassimilable without any problem.

Many micro-organisms (bacteria, fungi) in soil, in urban and industrial discharges, in estuaries of

the rivers, can degrade the polyhydroxibutyrate and its copolymers out of the cells.

For this purpose, they are provided with appropriate enzymes secretion (depolymerasis) that adhere

to particles of polymer and catalyze scrapping to simple water-soluble molecules. In turn, these

molecules are used by microorganisms themselves in their metabolism. The final products of

biochemical demolition were found to be H2O and CO2 in aerobic environment and CO2 and CH4 in

12

anaerobic environment.

2.2.2 Characteristics of ethylenglycol

The ethylenglycol, IUPAC name 1,2-ethandiol, is

the most common diol. At room temperature looks

like a transparent liquid, miscible with water,

appearance and syrupy liquid with a sweet taste.

Figure 2. Chemical structure of ethylenglycol

Produced in small quantities during World War I as coolant and as an ingredient in explosives,

ethylenglycol is produced on a large scale since 1937, when its precursor, ethylene oxide, becomes

cheaply available. It is produced from ethylene, via the intermediate ethylene oxide. Ethylene oxide

reacts with water to produce ethylene glycol accordino to the following chemical equation:

C C

H

HH

H

+ O2

O

HO

OHH2O

Scheme 4. Synthesis of ethylenglycol

This reaction can be catalyzed by either acids or bases, or can occur at neutral pH at elevated

temperatures. The highest yields of ethylene glycol occur at acidic or neutral pH with a large excess

of water. Under these conditions, ethylene glycol yields of 90% can be achieved. The main

byproducts are the ethylene glycol oligomers: diethylene glycol, triethylene glycol and tetraethylene

glycol.

The presence of hydroxylic groups makes it very responsive and is widely used in polymerization

reactions for polyester.

13

Table 4: Ethylenglycol properties

2.3 Synthesis of Soft-segment

The soft segment is synthetized from polymerization process which involves the opening of the ring

of the ε-caprolactone, which is linked to diglycol units through glycol which plays the role of

initiator, with the task of controlling the molecular weight.

The synthesis takes place in the liquid phase at 135°C until it stabilizes the molecular weight. The

reaction is considered completed when the volume of retention, obtained by GPC, is constant over

the time. The value of molecular weight is determined by the operator and from the mass it’s

possible to determine the amount of monomers to be used.

Properties Molecular formula C2H6O2

Molar mass 62,07 Aspect Colorless liquid

CAS number 107-21-1 Chimical-fisic properties

Density (g/cm3, in c.s.) 1,11

Solubility in water 1000 g/l a 20°C Melting point (K) 260 (-13°C) Boiling point (K) 470,6 (197,6°C)

ΔebH0 (kJ·mol-1) 49,66

Termochemical properties

ΔfH0 (kJ·mol-1) -460

S0m(J·K-1mol-1) 163,2

C0p,m(J·K-1mol-1) 148,6

Hazards Flash point (K) 384 (111°C) Explosion limits 3,2 - 53% vol.

Autoignition temperature (K) 683 (410°C)

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2.3.1 Characteristics of poly ε-caprolactone

The use of poly(ε-caprolactone) (PCL) for biomedical applications has become a real possibility

after its appearance on the market as degradable packaging material.

Later it was shown that the PCL may be degraded in physiological conditions through the same

hydrolityc mechanism of the other hydroxyacids. The bioerosion of the PCL is significantly slower

because of of more distinctly hydrophobic repeated units. The PCL is commonly considered a non-

toxic and biocompatible material.

The long aliphatic main chain of PCL gives to the polymeric material some properties uncommon

to other aliphatic polyesters such as an extremely low glass transition temperature (Tg ≈ -60°C), a

moderate melting temperature (Tf ≈ 60°C ), a high solubility in organic solvents, a significant

thermal stability (Td > 350°C) and the ability to form single-phase mixtures with many polymeric

materials. The high permeability of PCL matrices, which are always in rubbery state at

physiological temperature, has allowed to use it in systems controlled release of drugs.

2.3.2 Characteristics of copolymer ε-caprolactone-diglycolid

Polycaprolactone degradation is very long and therefor, in order to increase the degree of

degradation, a copolymer of caprolactone and diglycolid is used.

During polymerization the diglycolid forms the poly glycolic acid according to the following

reaction:

OH

HO

O

O

O

O

O

*

O

O

*

n

Glycolic Acid Poly (glycolic acid)

Scheme 5. Polymerization of glycolic acid

15

Variables ratios of caprolactone and glycolid are used, depending on the time of degradation

desired. The reaction of copolymerization occurs as follows:

O

O

O

O

O

O

+ +HO

OH

O

O

O

O

O

H H

xy

Scheme 6. Copolymerization reaction

x, y depend on the percentage of monomers used.

The polymer obtained contains fractions of glycolic acid, characterized by higher hydrophylia

(aliphatic chain is shorter than the polycaprolactone, 1 to 6-CH2). It is precisely this property that

makes the copolymer more degradable for hydrolysis, it speeds up the process of bioerosion.

The presence of diglycolid besides change the timing of degradation, change the mechanical

properties: ε-caprolactone has a long-term degradation, low σ and high percentage of elongation

while the glycolid degrades easily, has a high and low percentage σ elongation. The percentages of

diglycolid normally used are 15, 30 and 40%.

2.4 Synthesis of DegraPol®

Both hard and soft segments undergo a subsequent polymerization in presence of a urethanic

linkant. The point of junction is created through the use of TMDI (2-4-4 trimetilexametilene

diisocyanate), which is a toxic component and is used in stechiometric quantities and controlled by

FTIR to assess their presence in solving reaction.

Hard segment (HO-R1-OH) and soft segment (HO-R2-OH) are dissolved in dioxane at 80°C, in ratio

that vary according to the application.

Before adding the TMDI it is necessary to eliminate the water present, due to both the high

hygroscopy of PHB-diol and the humidity presents in the air. The TMDI could react with water and

16

form insoluble ureae as follows:

R

N

C

O+ H2O

R

HN

C

O

OH

R NH2 CO2+

Isocyanide Carbamic Acid Amine

1)

2)R

N

C

O+ R NH2

R

HN

HN

O

R

Isocyanide Amine Urea disubstitued

Scheme 7.

The removal of water from the reaction takes place by distillation of the solvent: dioxane and water

temperatures have very close boiling point (101°C and 100°C), this way it's possible to eliminate

50% H2O. Subsequently, the reaction mixture is anhydrificated in Soxhlet reflux on molecular

sieves.

The reaction between the diisocyanate and diol is a simple addition reaction with movement of a

hydrogen atom. The product of the reaction is a urethane, as follows:

R

N

C

O+ R' OH R

HN O

O

R' 1

Isocyanide Urethane (carbamate)Hydroxyl

Scheme 8.

The addition reaction is exothermic with H -105 kJ /-NCO and Energy activation 42 kJ / piers. The

diols used in the reaction have only primary hydroxyl group that react quickly with the urethanic

group.

In case of DegraPol® the reaction takes place by means of addition to the main chain. The

orientation of the groups R1 and R2 is random because it is not currently possible to establish a

regularity in the structure.

17

R1HO OH + R2HO OH +

N

C

O

N

C

O

R1

HO OHN

O

NH

O

R2

O

OH

Scheme 9. Synthesis of DegraPol®

The reaction is considered completed when all TMDI is consumed and the molecular weight of

polymer reaches the value of 100,000. The product, recovered by extraction with methanol,

contains traces of catalyst (Dilaurate of dibutyltin) and impurities found in solvents and in the

reaction.

A good purification of the polymer obtained is necessary, since the presence, even minimal, of

impurity limits its use in the biomedical field.

A solution of DegraPol® in chloroform is percolate through a bed of silica gel (silicon dioxide

hydrate), polymer of the orthosilicic acid. The gel has an amorphous structure, similar to that of

glass, which do not identify structural elements repetitive and ordered as crystal in the state. The

silica can be stratified into thin layers: up to 100 cm2 per gram of silica. The surface of the gel,

strongly polar for the presence of numerous groups OH-free, holds the polar compounds and

impurities solid, purifying the polymer which remains dissolved in chloroform.

18

3. Scaffold

A scaffold is a three-dimensional porous support made of biocompatible and bioerodible material

on which initial adhesion of cells and subsequent cell regrowth up to formation of tissue can take

place and having a degradation speed similar to cell regrowth.

The main features of these support are:

high porosity and three-dimensionality with the presence of a network of interconnected

pores to allow cell growth, transport of nutrients and elimination of waste substances

high biocompatibility so as not to generate any form of rejection by the host cells;

controlled biodegradability;

bioreabsorption with a degree of reabsorption that allows cell growth in vitro and /or in vivo;

surface chemically suitable for adhesion, proliferation and differentiation of cells;

mechanical properties similar to those of tissues that must play on the scaffold

reproducibility in various shapes and sizes

The cells to reproduce need a suitable environment, so when they are seeded on a polymer scaffold,

it is necessary that the substrate has the right characteristics of good biocompatibility, low

cytotoxicity, good biodegradability. Moreover, toh ave a correct cell growth, it is necessary that

there is similarity between the mechanical properties of regenerated tissues and those of the scaffold

on which these cells are seeded.

The mechanical properties essential to bear in mind is Young’s modulus, since for high values of

this quantity corrispond to hard and brittle materials that do not work as support for cell regrowth.

The first fundamental principle is that the Young's moduli of materials, used as support for

regrowth, have comparable values to the relative moduli from regenerate tissue; if this does not

apply, a wrong cell regrowth will accour with, possibly, death of cells. In fact, the tissue necrosis is

due to stress which is created at the interface between support and tissue, due to the movement that

generates between scaffold and tissue, caused by their difference in Young's modulus. Another

possible reason is the absence of effective transfer of stress through the interface support/tissue.

The second basic principle is the analysis of viscoelastic behaviour of support materials, because it

is necessary to analyse how the mechanical properties change in time (variable data) affecting the

process of tissue regrowth.

19

The surface topography of a material is usually classified according to the roughness, texture and

porosity; each of these aspects is important in monitoring the adhesion and cell functionality.

Indeed in tissues and organs, cells are organized in well-defined space relatively to specific

functional requirements. Indeed, during the development of a tissue or an organ, dividing cells

recognize gradients of biochemical signals that guide their polarization and final position. Many of

these signals are produced in relation to the topography of the environment surrounding the cells.

The techniques used to obtain scaffold in our laboratory are:

• the separation phase, it exploits the principles of thermodynamics to create two phases to

different concentrations within the polymer solution. Phase poor of polymer is removed,

leaving a highly porous polymer network. This technique allows to create microporose

sponges but show little interconnections;

• fibre production, through elettrospinning, involves the formation of polymeric networks

with large areas of cell attack that can ensure a rapid diffusion of nutrients.

While the technique of phase separation allows to obtain scaffold with controlled porosity, using

solutions with different concentrations of Degrapol®, the electrospinning allows to control the

topography of the scaffold, choosing appropriately the plot, and the alternation of layers of

Degrapol®.

20

4. Experimental Plan

The strength of the product is the ability to modify the property in a manner that is fully

independent on the application.

The properties of Degrapol® are according to:

operating conditions of the production process (temperature)

the chemical composition (ratio of copolymers)

productive processes

Production process Mechanical properties Composition

Mechanical properties

Degradation time Manufacturing process

Mechanical properties

Surface properties

The production process has been optimized at ETH Zurich; Ab Medica’s task is to make it

reproducible, controlling operating conditions. For example, the temperature must remain constant

throughout the process, it is noted that a temperature lower than 90°C results in a polymer low

molecular weight, and then with poor mechanical properties that do not make it suitable for

elettrospinning. The polymerizations must be conducted in a controlled environment to reduce

contamination and the thermal fluctuations, also the molecular weight of polymer needs to be

monitored in order to establish the reaction time needed to obtain Degrapol® molecular weights

known.

The chemical composition determines both the mechanical properties and the time degradation

because the individual blocks, as seen previously, have different mechanical properties. A different

ratio of hard and soft segments makes the material harder or softer and therefore the choice of

different percentage mixture is made according to the type of tissue to regenerate: for example, to

regenerate bones it is necessary to use a very strong material, while for the skin a soft material is

preferable. The different contents of poly glycolic acid adjust times of degradation. One of our

objectives is to identify the chemical and physical characteristics, suitable for different

compositions and establish standard formulations for each regenerating tissue.

As seen above, the polymer support must ensure the growth and cell differentiation. These features

are conferred by sponging techniques previously described. For each application should be

21

established:

the ratio empty / full such as to ensure adequate mechanical properties

the percentage of interconnected pores

the surface topography

To evaluate the properties of surface morphology of pores, their size and the presence of

interconnections, analytical technique of Microscopy scanning electron (SEM) has been used.

For the tensile tests (Young modulus and rupture elongation) and creep tests (visco-elastic

behaviour), a isotonic position transducer must be used (model 7006 of Ugo Basile Biological

Research Apparatus), designed to measure size and polymeric changes in muscle fiber.

All the mechanical tests should be performed on both scaffold and compact film of the same

material, in order to assess the influence of the microproduction on such properties. It is also

necessary to know the dimensions, the techniques to carry out the test and then regulations of

reference for the mechanical tests used in engineering tissue.

Determination of biotoxicity, biocompatibility and degradation time is carried out by institutes of

cell biology.

The biocompatibility is guaranteed by the use of biocompatible materials, while biotoxicity depends

on the presence of traces of catalysts and solvents, toxic substances that should be removed before

using the material for cell cultures. The timing of degradation varies from a few weeks to few years,

generally it assesses the changing of weight of the support over time under certain conditions that

are similar to those physiological.

It will be necessary to make a market investigation on the scaffold, to meet the needs of potential

buyers of the product and thus establish an experimental plan that takes this information into

account.

The diagram (Figure 4) shows the interconnection between the various disciplines involved in

tissue engineering. It is essential to cooperation with institutes involved in cell growth and

characterization of biomaterials in order to design the material depending on the application.

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23

5. Current applications Studies in the literature of both in vitro and in vivo cultures of DegraPol® have demonstrated the

real possibility of using it as a scaffold, in fact apart from being a good support for cell growth, it

makes it possible to preserve the specific properties of cells.

The first study of compatibility between the material and biological tissue covered the culture of

osteoblasts, cells isolated from bone tibia rats [3]. After 8 days from seeding on highly porous

DegraPol® (pore size of between 100-150 µm), the osteoblasts showed both a multi-lamellar

structure and migration inside the pores of polymer, with high degree of cell growth. It was also

observed that the amount of collagen type I and osteocalcium produced, remains constant over the

time in cultures of osteoblasts, indicating that the osteoblasts maintain their phenotype.

These data are confirmed by another study[4]. In vitro the growth of osteoblasts of rats and a variety

of human cells (HF01, MC3T3) on sponges of DegraPol® (pore size of between 100-400 µm)

confirmed the previous data. The possibility of using the sponges of DegraPol® as carrier for BMP

(bone morphogenetic protein) was also assessed. The BMP are biologically active proteins that

induce the formation of bone in vivo. Discs of DegraPol® with BMP adsorbed were implanted under

the skin on the back of rats and after 2 weeks from the implant the histological activity showed

both the activity of the alkaline phosphatase enzyme (ALP), which leads to target substances for the

renewal of bone cells, and the presence of calcium, confirmed by the X-ray analysis, attesting its

calcification.

Another study of in vivo cell regeneration[5] refers to the possibility of using DegraPol®, as a guide

for the regrowth of nerves, nerve growth channel (NGC). The NGC are polymeric tubular structures

in which the ends of nerves are inserted and sutured and which are able to issue, within their lumen,

tropics factors that improve the regeneration device. Tubular structures of 3 different DegraPol®,

containing various percentages of PHB-diol were planted on 26 rats; after 4 weeks in 23 rats,

regardless of the type of DegraPol® used, it is noted that into the channel epineural tissue

surrounded by myelinic axons and Schwann cells is present. This study also shows how a low-

PHB-diol induces a faster degradation.

There have been inflammatory phenomena that affect only small fragments of polymers because of

the high air interface. These small fragments are caused by the splitting of soft segment, which are

however phagocytized by macrophages without interfering with the cell regeneration.

There were also studied of chondrocytes’s culture derived from human tracheal cartilage on porous

24

and three-dimensional structures of DegraPol®[6]. After 8 weeks from seeding it was observed that

large numbers of type II collagen is produced by chondrocytes while collagen type I was absent.

This indicates a reduced loss of cell phenotype because collagen type II, interconnected with the

type of collagen IX, is a typical component of cartilage.

Furthermore, SEM observations demonstrated that the cells assume a spherical configuration

similar to that of native cartilaginous tissue cells. It therefore has a cell-specific growth, reflecting

in terms of size and shape the phenotype cell. These data are confirmed by other studies in vitro

about the regeneration of chondrocytes isolated from xiphoid of rats and from costal area of

cattles.[7] [8]

Another recent study regards the culture of cells in the smooth muscle tissue on the DegraPol®

scaffold (pore size of between 100-300 µm)[9]. After 2 days from seeding, cells have adhered well to

the surface of DegraPol® and retain their characteristic fusiform shape confirming that the porosity

of the scaffold allows cell penetration and adherence.

After 8 days apoptosis (the programmed death of cells and the loss of morphology) is observed.

This event is due to the static conditions of the culture rather then the support; indeed this type of

cell is much more sensitive to culture conditions compared to chondrocytes primary used in

previous examples.

All studies confirm that the scaffolds of DegraPol® ensure both the growth and the maintenance of

cellular phenotype of many biological tissues and that it can be used for many applications.

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6. Possible development

In the body, the interactions between cells and the surrounding environment are based on the

recognition of certain biological molecular structures through specific membrane receptors. Thus, a

convenient method to confer biological activity to a polymer surface is represented by blending

natural or covalently molecular species which promote the phenomena of adehesion and

proliferation. It's possible to functionalize diols with proteins, making the cell recognition faster

and more effective. This will reduce the time of adehesion on the scaffold and regeneration time of

the tissues.

This technique is necessary to assess compatibility with diols used to produce DegraPol® and the

feasibility of the process to industrial level.

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References

[1] Saad B., Keiser O.M., Welti M., Uhlschmid G.K., Neuenschwander P., Suter U.W.,

Multiblock copolyesters as biomaterials: in vitro biocompatibility testing. Journal of

Materials Science: Materials in Medicine. 1997; 8: 497-505B.

[2] Saad , T.D. Hirt, M. Welti, G.K. Uhlschmid, P. Neuenschwander, U.W. Suter,

Development of degradable polyesterurethanes for medical applications:In vitro and in vivo

evalutions. Journal of Biomedical Materials Research. 1997; 36: 65-74

[3] B. Saad, S. Matter, G. Ciardelli, G. K: Uhlschmid, M. Welti, P. Neuenschwander, U.

W.Suter, Interaction of osteoblasts and macrophages with biodegradable and higly porous

polyesterurethane foam and its degration products. Journal of biomedical Materials

Reserch.1996; 32:355-366

[4] B. Saad, Y. Kuboki, M. Welti, G.K. Uhlschmid, P. Neuenschwander, U. W.Suter,

DegraPol-Foam: a degradable and highly porous polyesterurethane foam as a new

substrate for bone formation. Artificial Organs. 2000; 24: 936-945

[5] M. Borkenhage, R.C. Stoll, P. Neuenschwander,U.W: Suter, P. Aebischer, In vivo

performance of a new biodegradable polyester urethane system used as a nerve guidance

channel. Biomaterials.1998; 19:2155-2165

[6] Yang L., Korom S., Weltia M., Hoerstrup S.P., Zund G., Jung F.J., Neuenschwander P.,

Weder W., Tissue engineered cartilage generated from human trachea using DegraPol®

scaffold. European Journal of Cardio-thoracic Surgery. 2003; 24: 201-207

[7] B. Saad, M. Moro, A. Tun-Kyi, M. Welti, P. Schmutz, G.K. Uhlschmid, P.

Neuenschwander, U. W.Suter, Condrocyte-biocompatibility of DegraPol-Foam: in vitro

evaluations. Journal of Biomaterials Sci. Polymer. 1999; 10:1107-1119

27

[8] B. Saad, M. Moro, A. Tun-Kyi, M. Welti, P. Schmutz, G.K. Uhlschmid, P.

Neuenschwander, U. W.Suter, Hyghly porous and biodegrable DegraPol-foam as

sugstrate for the formation of neo-cartilage: in vitro evalution. 9th simposium-Materials

in clinical Application. 1999

[9] Danielsson C., Ruault S., Simonet M., Neuenschwander P., Frey P., Polyesterurethane foam

scaffold for smooth muscle cell tissue engineering. Biomaterials. 2006; 27: 1410-15