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Polymer International

Polym Int 56 :145–157 (2007)

Polymer International Polym Int 56 :145–157 (2007) Review Biodegradable polymers applied in tissue engineering
Polymer International Polym Int 56 :145–157 (2007) Review Biodegradable polymers applied in tissue engineering

Review Biodegradable polymers applied in tissue engineering research: a review

Monique Martina 1 and Dietmar W Hutmacher 2

1 Department of Orthopedic Surgery, Yong Loo Lin School of Medici ne, National University of Singapore, 10 Kent Ridge Crescent, Singapore 119260 2 Division of Bioengineering, Faculty of E ngineering, Department of Orthopaedic Surgery, Yong Loo Lin School of Medicine, National University of Singapore, 10 Kent Ridge Crescent, Singapore 119260

Abstract: Typical applications and research areas of po lymeric biomaterials include tissue replacement, tissue augmentation, tissue support, and drug delivery. In many cases the body needs only the temporary presence of a device/biomaterial, in which instance biodegradable an d certain partially biodegradable polymeric materials are better alternatives than biostable ones. Recent treatme nt concepts based on scaffold-based tissue engineering principles differ from standard tissue replacement and drug therapies as the engineered tissue aims not only to repair but also regenerate the target tissue. Cells have b een cultured outside the body for many years; however, it has only recently become possible for scientists and engineers to grow complex three-dimensional tissue grafts to meet clinical needs. New generations of scaffolds based on synthetic and natural polymers are being developed and evaluated at a rapid pace, aimed at mimicking the structural characteristics of natural extracellular matrix. This review focuses on scaffolds made of more recently developed synthetic polymers for tissue engineering applications. Currently, the design and fabrication of biodegradable synthetic scaffolds is driven by four material categories:

(i) common clinically established polym ers, including polyglycolide, polylac tides, polycaprolactone; (ii) novel di- and tri-block polymers; (iii) newl y synthesized or studied polymeric biomaterials, such as polyorthoester, polyanhydrides, polyhydroxyalkanoate, polypyrroles, po ly(ether ester amide)s, elastic shape-memory polymers; and (iv) biomimetic materials, supramolecular polymers formed by self-assembly , and matrices presenting distinctive or a variety of biochemical cues. This paper aims to review the latest developments from a scaffold material perspective, mainly pertaining to categories (ii) and (iii) listed above. 2006 Society of Chemical Industry

Keywords: scaffolds; biodegradable polymers; tissue engineering; matrices

INTRODUCTION A great number of current tissue engineering strate- gies are based on the development of a cell–scaffold construct whose role is to repair and regenerate tissue defects (Fig. 1). During the first phase of tissue engi- neering in the 1990s research utilized either US Food and Drug Administration (FDA)/CE mark approved devices or used so-called conventional scaffold fabrica- tion technologies in combination with FDA/CE mark approved biomaterials of synthetic and natural origin. This work has been reviewed in detail elsewhere. 13 Currently, the design and fabrication of syn- thetic scaffolds is driven by four material categories:

(i) biodegradable and bioresorbable polymers, which have been effectively used for clinically established products, including polyglycolide (PGA), polylactides (PLA), poly-L-lactic acid (PLLA), poly-D,L-lactic acid (PDLA), polycaprolactone (PCL); (ii) novel di- and tri-block polymers which predominantly incor- porate PGA, PLA, and other resorbable polymers in different chain arrangements which confer both

degradation and mechanical property customization; (iii) polymers that are regulatory approved for spe- cific applications and/or are in clinical trials, e.g. polyorthoester (POE), polyanhydrides, polyhydrox- yalkanoate (PHA), and newly synthesized polymeric biomaterials, such as polypyrroles (PPy), poly(ether ester amide)s (PEEA), and elastic shape-memory poly- mers; and (iv) biomimetic materials, supramolecular polymers formed by self-assembly, and matrices pre- senting distinctive or a variety of biochemical cues. The aim of this review is to capture the latest develop- ments from a scaffold material point of view covering the aforementioned categories (ii) and (iii).

DI- AND TRI-BLOCK POLYMERS BASED ON ALIPHATIC POLYESTERS In attempts to tune aliphatic polymer properties to wider applications for scaffold-based tissue engi- neering, di- and tri-block polymers are seen as promising alternatives. By modifying the backbone

M Martina, DW Hutmacher

M Martina, DW Hutmacher Figure 1. Schematic of scaffold-ba sed tissue engineering. of the polymers, some

Figure 1. Schematic of scaffold-based tissue engineering.

of the polymers, some of the characteristics, such as degradation properties, mechanical properties, and even biocompatibility, of the polymers can be changed to a significant extent. 46 Li et al . 4 developed a series of di- and tri-block polymers based on PCL, PLA, and poly(ethylene gly- col) (PEG)/poly(ethylene oxide) (PEO). They used a tri-block made of PLA/PEO/PLA, with PEO as the hydrophilic and swollen component; and PLA chains as the nanometric nodes in the gel network. These physically crosslinked hydrogels possessed interesting degradation properties. Incorporation of PLA blocks at the end of the PEO segments decreased the degra- dation rate when compared to pure PEO. Weight losses of 49% after 10 days and 77% after 30 days were observed. A fast initial loss was observed due to the release of PEO-rich segments. Enzymatic degra- dation promoted a much faster degradation rate which reached 59% weight loss after 80 h of immersion, com- pared to 5% under hydrolytic degradation conditions. The author of this review and co-workers prepared a series of scaffolds from these materials using a rapid prototyping system (Fig. 2) and the scaffolds were studied in vitro and in vivo. 6 PCL/PGA di-block systems 5 have certain elastic properties. Scaffolds with pore sizes of 250 ± 50 µm and a porosity of 93% were produced using a sol- vent casting and particulate leaching method. Due

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to its elasticity (elongation up to 250% and recovery up to 98% after applied strain of 120%) Lee et al . 5 predicted the potential of this di-block in muscle tis- sue engineering. Preliminary in vitro studies using rat smooth muscle cells (SMC) displayed growth and tis- sue formation on the PCL/PGA scaffold. A summary of the properties of this system is given in Table 1. PCL-based copolymers for soft tissue engineering have been investigated by Cohn’s group, 7 who synthesized tri-blocks based on PCL/PEO/PCL (soft segment with hexamethylene diisocyanate chain extension as the hard segment). Incorporation of PEO enhanced water permeability. These tri-blocks have a water uptake of up to 94%, which also influences the mechanical properties and biodegradability. The length of the blocks affect polymer properties: for example, longer PEO segments lead to a decrease in the degree of crystallinity, an increase in water uptake, and an increase in degradation rate. Possible applications include soft tissue replacement and drug delivery systems. The cell biocompatibility of this system was studied by the authors of this review (Fig. 3(a)–(c)). One of the current challenges facing polymer usage in cell therapy is the ability to add bioac- tive molecules to enhance cytocompatibility. Con- ventional biodegradable polymers lack this charac- teristic. A strategy to overcome this limitation is

Polym Int 56 :145–157 (2007) DOI: 10.1002/pi

Reference

26–28

24

23

23

22

5

Biocompatibility

in vitro

Rat osteoblast

Rabbit BMSC

Rat SMC

Rat SMC

Rat SMC

Rat SMC

Subcutaneous implantation using rat model, no tissue necrosis, vascularized, no giant cells Subcutaneous and cranial implantation using rabbit model, 5–15 layers of fibrous capsule, and inflammatory cells are seen

Subcutaneous implantation using mouse model, appropriate SM tissue formation after 3 weeks

Biocompatibility

in vivo

Soft tissue engineering

Soft tissue engineering

Soft tissue engineering

Smooth muscle tissue engineering

Application

engineering

engineering

Bone tissue

Bone tissue

90–100% mass loss

in 60 days ( in vivo)

weeks (in PBS, C)

loss in 8 weeks (in

loss in 8 weeks (in

13.37–20.7% mass

67.7% mass loss in

25.4–47.3% mass

13.6–27.9 ± 1.8%

days (PBS, C). Approx.

50% mass loss in 6 weeks (PBS,

Degradation

mass loss in

PBS, 37 C)

PBS, 37 C)

rate

C)

60

37

12
37

37

354.1–1024.2 MPa compression modulus

0.97–1.64 MPa tensile strength (3D porous scaffold) 0.59–1.68 MPa tensile strength (3D porous scaffold) 13 ± 4 MPa tensile strength (nanofibre)

>250% elongation at break

Mechanical

properties

Abbreviation

LDI-glucose

PEEUU

Table 1. Summary of polymer properties

PGCL

PEUU

PEUU

PPF

Lysine diisocyanate-glucose

Poly(propylene fumarate)

Poly(ester urethane)urea

Poly(ester urethane)urea

co-

urethane)urea

caprolactone)

Poly(ether ester

Poly(glycolide-

Polymer

Polym Int 56 :145–157 (2007) DOI: 10.1002/pi

Biodegradable polymers in tissue engineering

147

Reference

16,17,19

33,34

10

12

Fibroblast human cell lines and primary rabbit fibroblast

Osteoblast cell lines

Biocompatibility

Fibroblast cell lines

in vitro

Rabbit tibial model, minimal inflammatory response Subcutaneous implantation using rat model, sterile inflammation with normal foreign body reaction Subcutaneous implantation using rat model, similar inflammatory response to PLGA Subcutaneous implantation using rat model, tissue ingrowth similar to collagen. Rabbit model

Biocompatibility

in vivo

Microvasculature, soft

Application

Anterior cruciate

Cardiovascular

engineering

Bone tissue

ligament

tissue

17 ± 6% mass loss in

Less than 5% mass loss in 12 weeks (in PBS, 37 C)

weeks (in PBS, C)

Totally absorbed

days ( in vitro).

No mass loss after

Degradation

Approx. 6% after

implantation

rate

weeks’

(in vivo)

30

60

37

52

200 MPa compression modulus, and remains after 2 weeks

57 ± 8 MPa ultimate tensile strength

0.282 ± 0.025 MPa tensile Young’s modulus

1–10 MPa Young’s modulus

Mechanical

properties

Poly(DTE carbonate)

Poly(TMC/CL)

Abbreviation

PPHOS/HA

PGS

tyrosyl-tyrosine ethyl ester carbonate)

Poly(ethyl glycinate/ p-methyl phenoxy) phosphazene/ hydroxyapatite Poly(trimethylene

Poly(glycerol sebacate)

Table 1. Continued

carbonate/ε- caprolactone)

Poly(desamino

Polymer

M Martina, DW Hutmacher

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Polym Int 56 :145–157 (2007) DOI: 10.1002/pi

Biodegradable polymers in tissue engineering

Biodegradable polymers in tissue engineering Figure 2. Polyester–polyether block copolymers composed of PCL or PLA and

Figure 2. Polyester–polyether block copolymers composed of PCL or PLA and poly(ethylene glycol) (PEG) have attracted much attention as they offer the possibility of varying the ratio of hydrophobic/hydrophilic constituents to modulate the degradability and hydrophilicity of the polymer matrix and surface. Scaffolds were fabricated using a rapid prototyping (RP) machine built in-house at the National University of Singapore. System details are reported elsewhere. 7 Briefly, in contrast to traditional RP systems such as fused deposition modelling, three-dimensional printing, stereolithography, and selective laser sintering, which mainly focus on a single mode of material processing, this system can accommodate a much larger variety of synthetic and/or natural biomaterials. A lay-down pattern of 0/90 was used to form the honeycomb patterns of square with a single fill gap (FG) of 1 mm. Porous sheets measuring 40 × 40 × 3 mm were fabricated using the system, with a 0.5 mm diameter nozzle. PCL–PEG diblock copolymer was synthesised by Li et al . 4 The extrusion temperature was set at 110 C for PCL–PEG. PCL–PEG was dispensed with an air pressure of 3.5 bar and a speed of 30 mm min 1 in the x / y axis. Scanning electron micrographs (right) of melt extruded PCL–PEG scaffolds display typical honeycomb morphology (A). Alternate layers of filaments were positioned at right angles to one another creating pore sizes of 600 µm. Confocal laser microscopy of scaffold/cell constructs was used to study cell attachment and proliferation.

to use polymers containing functional side groups. Guan and co-workers 8 attempted to synthesize an ABA tri-block polymer that had this particular property. Fabrication from ABA-type tri-block poly- mer PLGBG–PEG–PLGBG consisting of PEG and poly[(lactic acid)- co-(glycolic acid)- alt-(γ -benzyl-L- glutamic acid)] that had undergone catalytic hydro- genation resulted in PLGG–PEG–PLGG, which contained carboxyl pendant groups. These carboxyl pendant groups may be useful to facilitate the attach- ment of oligosaccharides, drug molecules, or short peptides. Due to its amphiphilic nature, the polymer formed micelles and may find useful applications in drug delivery systems. Another new tri-block system are thermogelling copolymers 9 made of PEG/PCL/PEG. At a copolymer concentration of less than 1% and at a temperature of 20 C, micelle aggregates of 10 and 23 nm coex- isted, but at 25–45 C micelle aggregates of 23 nm were dominant. Increasing the copolymer concentra- tion also increased the micelle aggregation size. Upon varying the PCL block’s length, sol–gel temperature decreased due to the hydrophobic interaction that drove gel formation. This system is appropriate for in situ gel forming whereby entrapment and depot formation can be envisaged with minimally invasive therapy.

NEWLY SYNTHESIZED OR STUDIED POLYMERIC BIOMATERIALS Polycarbonate Polycarbonate in its pure form is an amorphous polymer that possesses low moisture absorption, and is not susceptible to microbial attack, which implies non-biodegradability of the polymer. How- ever, the mechanical properties of the polymer attract researchers keen to develop polycarbonate-based material that can withstand mechanical loading while tissue is being regenerated. Since resorbability is the main problem, Bourke et al . 10 investigated a member

Polym Int 56 :145–157 (2007) DOI: 10.1002/pi

of the tyrosine-derived polycarbonates that was not only resorbable, but also possessed high strength. The structure of this material is shown in Fig. 4(a). How- ever, in that study, the degradation rate was shown to be very slow, with no mass loss observed after 30 weeks of incubation in PBS, at 37 C.

mate-

rial by melt extrusion at 60–90 C, which was above the glass transition temperature. In the second phase, they tried the melt-spinning technique at 181–183 C. The fabricated fibres were aligned to mimic the structure of ACL (anterior cruciate ligament). The ultimate tensile strength of this material was found to be comparable with natural ACL (57 MPa). Sub- cutaneous implantation using a rat model revealed tissue ingrowth 4 weeks post-operative with mainly fibroblast-like structures observed surrounding the fibres. Interestingly, although the structure was intact 8 weeks post-implantation, strength retention dropped to 40%; this was in contrast to in vitro studies which showed almost 90% strength retention, which implied that the in vivo environment induced molecular relax- ation, causing reduction in strength. Further studies based on polycarbonate’s good biocompatibility and ease of biochemical modification towards cell adhesivity 11 have been undertaken. Using the polycondensation technique, copolymers of poly ethylglycol/poly(desamino tyrosyl-tyrosine ethyl ester carbonate) (PEG/poly(DTE carbonate)) were obtained to study keratinocyte migration. It was concluded that PEG provided a better attachment and migration substrate for fibroblasts and keratinocytes, whereas poly(DTE carbonate) acted to maintain the structural integrity of the graft.

In the first phase, Bourke et al . fabricated this

Polyphosphazene The search for polymeric materials that are versatile for various hard and soft tissue engineering appli- cations has generated interest in another group of polyphosphazene-based materials. 1214 The unique properties arise from the unusual flexible backbone allowing torsional and angular freedom within the

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M Martina, DW Hutmacher

M Martina, DW Hutmacher (a) (b) (c) Figure 3. (a) Soft-PCL scaffold fabricated via a rapid

(a)

M Martina, DW Hutmacher (a) (b) (c) Figure 3. (a) Soft-PCL scaffold fabricated via a rapid

(b)

M Martina, DW Hutmacher (a) (b) (c) Figure 3. (a) Soft-PCL scaffold fabricated via a rapid

(c)

Figure 3. (a) Soft-PCL scaffold fabricated via a rapid prototyping technique (unpublished data). (i) High flexibility of scaffold is demonstrated; (ii, iii) swelling ability is demonstrated (soft segments in polymer chain allow scaffold to swell up to 1.5 times). (b) Light micrographs of soft-PCL (P) alginate/thrombin (M) construct seeded with human adipose derived precursor cells (hADAS). Formulations of cell aggregates and extracellular material (white arrows) illustrate cell proliferation upon culturing: (i) day 1, (ii) day 7, (iii) day 22, (iv) day 42. Bar represents 100 µm. (c) Environmental scanning electron micrograph (left) of scaffold/cell construct after 3 weeks of culturing exhibited intact cell/alginate/thrombin structure (M) inside the soft-PCL pore morphology. Confocal laser micrograph (right) of soft-PCL (P) alginate/thrombin construct after 3 weeks of culturing. Cell viability is shown by life/death assay (FDA; green fluorescence in confocal laser micrograph) inside the alginate/thrombin matrix. (Cells are indicated by white arrows).

P–N skeletal system (Fig. 5(a)). The most com- mon fabrication technique is thermal ring-opening polymerization of dichlorophosphazene. The current

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technology enables fabrication of this polymer derived from polydichlorophosphazene using two different substituents. These substituents induce highly tuned

Polym Int 56 :145–157 (2007) DOI: 10.1002/pi

(a) Biodegradable polymers in tissue engineering (b) (c) (d) Figure 4. Structures of (a) poly(desamino

(a)

Biodegradable polymers in tissue engineering

(a) Biodegradable polymers in tissue engineering (b) (c) (d) Figure 4. Structures of (a) poly(desamino tyrosyl-tyrosine

(b)

(a) Biodegradable polymers in tissue engineering (b) (c) (d) Figure 4. Structures of (a) poly(desamino tyrosyl-tyrosine

(c)

(a) Biodegradable polymers in tissue engineering (b) (c) (d) Figure 4. Structures of (a) poly(desamino tyrosyl-tyrosine

(d)

Figure 4. Structures of (a) poly(desamino tyrosyl-tyrosine ethyl ester carbonate) (poly(DTE carbonate)), modified from of Bourke et al .; 10

(b)

PPF/PPF-DA crosslinked, modified from of Horch et al .; 25 (c) poly DTH-adipate for R = hexyl and y = 4, modified from of Schachter and Kohn; 42

(d)

soft segment and hard segment of poly(ether ester amide), modified from Deschamps et al . 44

(a)

(b)

amide), modified from Deschamps et al . 4 4 (a) (b) (c) Figure 5. Preparation schemes
amide), modified from Deschamps et al . 4 4 (a) (b) (c) Figure 5. Preparation schemes
(c)
(c)

Figure 5. Preparation schemes of (a) polydichlorophosphazene, modified from Ambrosio et al .; 12 (b) PEUU, modified from Stankus et al .; 24

(c) poly(glycerol sebacate), modified from Wang et al . 33

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properties such as crystallinity, degradability, and hydrophilicity/hydrophobicity which provide the ver- satility of the polymers. Ambrosio et al . 12 explored amino acid ester polyphosphazene in bone tissue engineering. They chose poly[(ethylglycinate phosphazene)- co-( p-me- thylphenoxy phosphazene)] (PPHOS) in combination with hydroxyapatite (HA) to develop a composite. Degradation was achieved by incorporating side chains that sensitized the polymer backbone to hydrolysis; in this case, the amino acid side chains. Phenoxy side chains inhibited degradation, and in this study, PPHOS/HA remained stable mechanically and main- tained a compressive modulus of around 200 MPa, after 12 weeks’ degradation in vitro. PLAGA/HA con- structs that were used as a control showed almost insignificant compressive modulus. Degradation prod- ucts could easily be detoxified by the body as the degradation by-products are amino acids, phosphates, and ammonia. PPHOS possesses a combination of surface- and bulk-eroding properties. Other types of polyphosphazene, those with polyethyloxybenzoate and polypropyloxybenzoate, were combined with HA resulting in composites suitable for bone grafts. 15 Syntheses of these polyphos- phazene/HA composites were achieved through acid–base reactions to form HA precursors, which increased the pH of the solution and in the presence of polyphosphazenes resulted in carboxyl formation in the surface layer. These carboxyl groups then reacted with calcium ions to form calcium crosslinks on the surface, which played a role in nucleation and deposi- tion of HA. In another study, a PPHOS/PLAGA blend exhibited a higher pH in the degradation solution compared to PLAGA. 13 The buffering phenomenon was attributed to phosphates which were shown to be present in the solution using 31 P NMR. Cell adhesion experiments using MC3T3-E1 cells revealed PPHOS/HA to possess a similar cell attachment and proliferation when compared to tissue culture plastic (TCPS). Nair and colleagues 14 investigated the effect of sol- vent, needle diameter, solution concentration, and applied voltage on the fabrication of polyphosphazene nanofibre scaffolds. Using chloroform as the solvent produced the most uniform fibres. Decreasing the nee- dle diameter resulted in decreased fibre diameter, but too fine a needle produced fibres with beads. Increas- ing the solution concentration generally resulted in larger fibre diameters. The applied voltage affected the shape of the nanofibres generated: 27–30 kV resulted in a curly or distorted shape, 33 kV resulted in cylindri- cal and rod-like structures, while 36 kV gave even finer and more distinct rod-like structures. Investigation of the polyphosphazene nanofibres extended to cell compatibility studies. In vitro cell adhesion of bovine coronary artery endothelial cells showed adhesion 24 h post-seeding. The materila also exhibited cytocom- patibility to osteoblast cell lines, MC3T3-E1, which adhered and proliferated for 7 days. This presents an

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opportunity for this material to be utilized in appli- cations such as coatings to enhance tissue integration and wound dressings.

Trimethylene carbonate-based materials The elastomeric properties of poly(trimethylene car- bonate) (poly(TMC)) make it a potential can- didate for scaffold-based soft tissue engineering applications. 1619 Interestingly, this polymer exhibits slow degradation in vitro, but rapid degradation in vivo. 16 Poly(TMC) showed negligible mass loss after 2 years in physiological solution, in which degra- dation mainly occurred due to hydrolysis of ester bonds. In contrast, in vivo implantation of polymer discs into Wistar rats resulted in a high degradation rate based on a cell-triggered surface erosion mecha- nism. After 52 weeks, implanted poly(TMC) was 1% of its initial mass. Incorporation of other monomers such as D,L- lactide acid (DLLA) or ε-caprolactone (CL) mod- ulated the degradation properties in vitro as well as in vivo. In vivo degradation characteristics of these copolymers coincided well with in vitro degradation behaviour, in which hydrolysis was mainly responsi- ble for both conditions. TMC/DLLA decreased to 1% of the initial mass after 52 weeks, with rapid loss observed after week 12, while TMC/CL displayed a linear and continuous reduction throughout a year of investigation, with mass loss less than 7% of the initial mass. The difference in degradation behaviour of the two copolymers was caused by autocatalytic activity of DLLA, which did not occur in TMC/CL. Furthermore, the in vivo tissue response of these copolymers is similar to sterile inflammatory reactions followed by normal foreign body reactions, which are commonly seen after implantation of biodegradable polymers. Due to its flexibility, poly(TMC/DLLA) was tai- lored towards heart tissue engineering applications. 17 Poly(TMC/DLLA), although glassy at room temper- ature, was rubbery at body temperature which made it difficult to achieve a porous structure. A combination of coprecipitation, compression moulding, and salt leaching techniques were used to produce a porous stable scaffold with a pore size of around 100 µm, a porosity of 85–95%, and a compressive modulus up to 430 kPa. In vitro degradation studies in physiolog- ical solution showed that porous scaffolds shrunk by up to 65% after the third week. Preliminary investi- gation of cell compatibility using rat cardiomyocytes showed average cell attachment and proliferation on the copolymer surface compared to normal TCPS plates. Poly(TMC/CL) was explored for nerve grafting. 16 Human dermal fibroblasts (HDFs) were subjected to extracts of the polymer. Results showed that HDFs subjected to the extraction vehicle maintained high cell viability and cell metabolism activity. Schwann cells were plated onto copolymer discs and demonstrated good proliferation, with a higher proliferation rate after

Polym Int 56 :145–157 (2007) DOI: 10.1002/pi

6 days compared with those cells plated onto PLGA. Implanted tubular poly(TMC/CL) scaffolds were still smooth and flexible at 60 days post-implantation, and adhered well to the surrounding tissue. In another in vivo study, a lesion in the spinal cord of female Wistar rats exhibited extensive growth of Schwann cells and extracellular matrix after implantation of poly(TMC/CL) to the lesion. 20 Grijpma et al . 19 also explored the possibility of a photocrosslinkable polymer via UV functionalization of poly(TMC/CL) or poly(TMC/DLLA) with fumaric acid monoethyl ester (FAME).

Polyurethanes Polyurethanes are another area of investigation, especially for soft tissue engineering applications, in contrast to aliphatic linear polyesters that are better suited to hard tissue engineering due to their high glass transition temperature and high modulus. Polyurethanes exhibit a wide range of properties through variability of the hard segment (diisocyanate), the soft segment (polyethers or polyesters), the chain extenders, and the ratios in which they are reacted. Previously, polyurethanes had a limited usage due to the toxicity of their degradation product (2,4- diaminotoluene). Hence, the challenge presented was to develop polyurethanes with non-toxic degradation products. Non-toxic polyurethanes with diisocyanate replace- ments which could give rise to non-toxic degrada- tion products have been developed by at least two groups. 2124 Guan et al . 23 synthesized the polymers made from PCL and 1,4-diisocyanatobutane (BDI) with putrescine as chain extender (poly(etherurethane urea), PEUU) (Fig. 5(b)). BDI was used because, upon degradation, it would release putrescine, a polyamine that is essential for cell growth and pro- liferation. Zhang et al . 22 synthesized polyurethane from highly pure lysine diisocyanate (hard segment) and polymerized it with glucose, which resulted in major degradation products lysine and glucose (LDI- glucose). Scaffold fabrication routes for these polyurethanes include thermally induced phase separation, electro- spinning, and water foaming. These create differ- ent porosities, surface-to-volume ratios, and three- dimensional structures, with concomitant changes in mechanical properties which are wide-ranging and can be varied to suit potential applications in the biomed- ical field, such as engineering blood vessels and bone (Table 1). The degradation mechanisms of the polymers are important and need to be investigated further. Non-toxic degradation products are necessary and, moreover, mechanical properties are also influenced by degradation mechanisms. LDI-glucose polymer, for example, is degraded by hydrolysis of urethane bonds to liberate lysine, glucose, ethanol, and CO 2 . Ethanol could inhibit cell–cell adhesion, but a study reported that concentrations less than 30 mM (0.5%

Polym Int 56 :145–157 (2007) DOI: 10.1002/pi

Biodegradable polymers in tissue engineering

v/v) are harmless to the cell. 21 Moreover, in contrast to PLA and PLGA degradation mechanisms, the study showed no significant increase in pH of the solution. PEUU degradation products were also shown to be non-toxic to endothelial cells. The polymer showed a linear degradation with no signs of autocatalytic effects when compared to PLA or PLGA degradation behaviour. 23 Scaffolds were prepared by thermally induced phase separation and a subsequent solvent extraction technique. The results were porous structures (porosity >80%) with pore diameters ranging from 12 to 232 µm. The pore shape was dependent upon the quenching temperature and polymer concentration. The tensile strength of these polymers during degradation decreased by about 12% after 1 week and 31% after 2 weeks from the initial value of 0.57–1.69 MPa. Stankus et al . 24 were able to machine these polyurethanes via electrospinning to produce scaffolds. The tensile strength of the nanofibres produced ranged from 2 to 13 MPa and breaking strains from 160 to 280%. Incorporation of type I bovine collagen reduced the tensile strength, but also reduced water contact angles. This represents an increase in hydrophilicity and, in turn, enhanced attachment of rat smooth muscle cells. In vitro and in vivo observations demonstrated that these polymers had no harmful effects on cell viability, growth, and proliferation. Subcutaneous implantation using rat models revealed that LDI-glucose polymer did not enhance capsule formation, accumulation of foreign body giant cells, or tissue necrosis. 22 The versatility of these polyurethanes was demon- strated through attempts to enhance cell and tissue compatibility via: addition of soft segments with PEG to the backbone in order to enhance hydrophilicity; mixing with collagen type I for better cell attachment; and addition of ascorbic acid to the polymer mixture to enhance osteoblast lineage progression. All of these proved to be feasible. Polyurethane-based materials are consequently considered promising candidates for biomaterial-based applications.

Polyfumarate Polyfumarate-based materials have been developed mainly for bone tissue engineering. 2528 The main advantages of these materials are their injectable and in situ crosslinkable properties. 29,30 With N -vinylpyrrolidone (N-VP) as crosslinker, Payne et al . 28 showed during an in vitro study that fully crosslinked poly(propylene fumarate) (PPF) had potential as a substrate for supporting rat osteoblasts. Cell proliferation, ALP activity, and osteocalcin and calcium production of cells on fully crosslinked PPF did not exhibit significant differences with those cells grown on TCPS. Poly(caprolactone fumarate) (PCLF) and poly(ethylene glycol fumarate) (PEGF) were also investigated as injectable, self-crosslinkable polymers which circumvented the requirement for crosslinking agents that may be toxic. The polymers

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were shown to harden and self-crosslink when under physiological conditions, and tissue compatibility studies using rat models demonstrated no inflamma- tory reactions. 29,30 Bone and soft tissue responses have been inves- tigated in New Zealand white rabbit models. 27 A PEG block was added in order to make a hydrogel (oligo(PEG fumarate), OPF). Tissue response and in vitro degradation behaviour of four groups were investigated. The groups included longer PEG blocks,

higher crosslinking density, and the addition of cell- adhesive peptides to the hydrogels. Hydrogels with higher crosslinking were observed to have the least mass loss after 12 weeks in vitro. As the degradation product was fumaric acid, a drop in pH was expected.

A pH drop of not more than 0.5 was reported (which

was considered to be harmless to the cells). In contrast,

in vivo results suggested that hydrogels with longer PEG block resulted in more tissue infiltration, and hence more active degradation. Fibrous capsule for- mation with 5–15 cell layers including inflammatory cells was observed for all groups. Degradation rate and degree of swelling were also dependent on the degree of crosslinking per macromer chain. Less crosslinking gave rise to an increase in water uptake and increased swelling. As degradation occurred mainly due to the cleavage of ester bonds within the crosslinked network by hydrolysis, a greater number of chains were accessible to water and so the polymer degraded faster. The higher the water uptake, the faster the degradation. One type of OPF investigated even appeared soft and jelly-like after 12 weeks under physiological conditions. As the applications of polyfumarate-based materials

were first investigated for bone substitutes, mechanical properties are an ultimate concern. Cortical bone has a compressive modulus in the range 17–20 GPa, com- pressive strength of 106–144 MPa, flexural modulus of 15.5 GPa, and flexural strength of about 180 MPa. This presents a major challenge for the tissue engineer using polyfumarate-based materials. This unique char- acteristic of bone is a result of its composite make-up comprising the interaction of inorganic material, i.e. HA, with organic material such as collagen fibres. Researchers have considered ways to optimize the mechanical properties of poyfumarate-based polymers using the principle of bone architecture. 25,26 Horch et al . 25 developed PPF/poly(propylene fumarate diacrylate) (PPF/PPF-DA) (Fig. 4(b)) with

a surface modification using carboxylate alumoxane

nanoparticles. The polymer composites were gener- ated by mixing in a chloroform solvent, and the solvent was then removed by rotary evaporation and high- vacuum drying. Crosslinking was achieved by UV radiation. Interactions of the inorganic and organic matrix were achieved by covalent bonding, and the presence of organophilic chains in the organic matrix enhanced dispersion. Covalent bonding alone was not enough; dispersion played a key role in the mechanical

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properties of the composite. Without proper disper- sion, the inorganic particles tended to aggregate and create crack propagation sites, which, in turn, made compressive fracture and flexural fracture strength worse than that of PPF/PPF-DA. Flexural testing was done on samples fabricated by injecting a nanocomposite mixture into a mould, and crosslinking the mixture using UV radiation. The samples that showed enhanced dispersion and covalent bonding with PPF/PPF-DA matrix exhibited the best flexural modulus of more than threefold higher (5.4 GPa) compared with blank PPF/PPF-DA, which has a flexural strength of 1.5 GPa. Compressive strength, however, was not significantly affected by these modifications. Incorporation of β-tricalcium phosphate (β-TCP) was also attempted to enhance the mechanical properties of polyfumarate-based polymers. 26 The polymer was produced by radical polymerization using benzoyl peroxide and dimethyltoluidine as initiator and accelerator. N-VP was used as crosslinking reagent. Scaffolds were then fabricated by mixing PPF with β-TCP and leachable porogen NaCl. Groups with β-TCP concentrations of 0.5gg 1 of PPF exhibited bending strengths of up to 16 MPa, compressive strengths of up to 79 MPa, moduli in bending of up to 1270 MPa, and moduli in compression of up to 1020 MPa. An approximately twofold increase in bending and compressive strength and bending and compression modulus for a twofold increase in β-TCP concentration was observed. Mechanical testing showed compression and bending modulus of elasticity to be of the same order of magnitude as that of trabecular bone.

Polyorthoester Polyorthoester is a hydrophobic polymer fabricated by polycondensation of diketene acetals and diols. This fabrication creates ortho-ester bonds that are stable at neutral pH, but hydrolyse rapidly at phagosomal pH, i.e. pH 5.5. Today, it is mainly used in drug delivery systems. 31,32

Poly(glycerol sebacate) The search for soft and mechanically stable elastomeric materials to be implanted in dynamic environments led to the investigation of polymers that are anal- ogous with vulcanized rubber, having a crosslinked three-dimensional network in combination with ran- dom coil characteristics. Polycondensation of glycerol and sebacic acid renders a polymer having hydro- gen bonding interactions through hydroxyl proline hydroxyl groups. 33,34 With building blocks made of glycerol (a basic building block of lipids) and sebacic acid (a natural metabolic intermediate in ω-oxidation of medium- to long-chain fatty acids), such materi- als are expected to be biocompatible and non-toxic (Fig. 5(c)). The hydrophilic characteristics of the material are a result of the hydroxyl groups attached to its

Polym Int 56 :145–157 (2007) DOI: 10.1002/pi

backbone. The material is insoluble but swells in

water by approximately 2%. It is totally amorphous

at 37 C like vulcanized rubber, a thermoset polymer.

However, the uncrosslinked polymer can be melted to

a liquid form which is soluble in common organic

solvents. It is tougher than hydrogels with tensile strength of less than 0.5 MPa and tensile strain more than 300%. 33 In vivo and in vitro investigations showed an acceptable biocompatibility. In vitro studies using NIH 3T3 fibroblast cell lines grown in PGS- coated Petri dishes exhibited higher cell growth compared with PLGA-coated Petri dishes. An in vivo study with Sprague-Dawley rats showed less fibrous capsule formation compared to PLGA control after subcutaneous implanation. While simple foams were fabricated using a salt leaching technique, lithography was used to fabricate

capillary networks. 34 The surface of the wafer-like scaffold was coated by a pentapeptide derived from fibronectin to improve cell attachment. Adherence of the HUVEC to the PGS was observed and proliferation occurred for at least 10 days. After 14 days, the surface of the network was nearly confluent and cultures were kept for up to 4 weeks. PGS has another potential application as nerve guides. 35 In vitro studies demonstrated PGS as non-toxic to Schwann cells and supported better proliferation compared to PLLA. In vivo studies revealed less inflammatory response possibly due to its degradation profile via surface erosion.

Elastic shape-memory polymers Lendlein and Langer 36,37 investigated polymers that could change shape by increases in temperature. This thermally induced shape-memory effect required two components: a switching segment, having transition temperature to fix temporary shape, and a segment for crosslinking to determine the permanent shape. The switching segment used was oligo(ε-caprolactone)diol or dimethacrylates, while crosslinking segments were either n-butylacrylate or oligo( p-dioxanone)diol. The mechanism of changing the shape from permanent to temporary relies on the transition temperature of the material. A polymer in a permanent

shape is heated above T trans while applying an external stress. The temporary shape is obtained by reducing the temperature to below T trans . Releasing the external stress and heating up the material will reform the temporary shape back to the permanent shape. Using this mechanism, prior to surgery, implants can be compressed to a smaller and more compact temporary shape, inserted by minimally invasive surgery, and then using body heat, it will expand back

to

the permanent shape. Another possible application

is

as sutures in endoscopic surgery, whereby the suture

knot can be applied loosely in its temporary shape, followed by an increase in the temperature, which would tighten the knot as it goes back to its permanent shape.

Polym Int 56 :145–157 (2007) DOI: 10.1002/pi

Biodegradable polymers in tissue engineering

Another development by Lendlein and co-workers 38

as regards elastic shape-memory polymers was light-

induced shape-memory polymers. With a similar mechanism to that of a heat-sensitive shape-memory polymer, this too consisted of two segments: molecular

switches that were photoresponsive, and netpoints for covalent crosslinking that determined the permanent shape, using cinnamic acid as a molecular switch. When the polymer was stretched, the coiled segments

of amorphous polymer chain were elongated. Upon

exposure to UV radiation of wavelength >260 nm, the elongated segments were partially fixed due to the formation of new photoresponsive crosslinks. If the loading was removed and the polymer exposed to UV radiation of wavelength <260 nm, the crosslinks were cleaved and the polymer returned to its original shape. The discovery of this material eliminated temperature constraints associated with thermally induced shape- memory polymers for medical applications.

Polypyrrole Electronic interaction is one of the factors that may influence neuronal tissue regeneration and growth.

A class of polymers that have also been explored in

the field of nerve tissue engineering are conducting polymers. Polypyrrole (PPy) is an electrodeposited polymer that can be doped to modify its physical,

chemical, and electrical properties, and has conse-

quently emerged as a potential candidate for scaffolds

in neuronal tissue regeneration. 39 Using PPy doped

with polystyrene-sulfonate (PSS) and sodium dode- cylbenzene sulfonate (NaDBS) at different deposition temperatures and solvents, George et al . 39 investi- gated the influence of these parameters on neural tissue growth in vivo. Immunofluorescence analysis of the neural tissue displayed a more complete bridging when compared to a Teflon implant, which is cur- rently a gold standard for neural implants. The data presented implied that PPy implants generally exhib- ited greater tissue integration and less inflammation. The feasibility of incorporation of neuronal growth factors (NGFs) was also shown, with even more neu- ral tissue formation observed for NGF-incorporated implants. Moreover, the conductivity of PPy can be exploited for the fabrication of bioelectrical circuits that integrate electrical and neural signals. Cui et al . investigated the use of PPy as a neural probe. 40,41 These neural probes facilitated the functional stimulation and recording from the peripheral or central nervous system. The conductivity of PPy might induce selective neurons to attach to the electrode and achieve neuronal tissue regeneration. However, problems were encountered, including loss of the ability to record neural activity with time. Modifications by patterning peptide or peptide/protein polymer blends on the surface were explored to enhance interaction and anchorage between electrode and neurons.

155

M Martina, DW Hutmacher

Polyarylates Schachter and Kohn introduced an interesting concept of using a group of polyarylates for drug delivery vehicles as an alternative to conventional PLA, PGA, or PLGA systems. 42 The polyarylates (Fig. 4(c)) used were tyrosine-derived polymers which possessed sites for interaction with peptides. Some possible interactions included hydrogen bonding and hydrophobic interactions. Addition of PLGA system as a ‘delayed excipient’ induced a decrease of internal pH during its degradation, and weakened the sensitive interaction between peptide and polymer. Using Integrillin as the drug model, the glass transition temperature of the system varied in the range 36–40 C as the amount of loaded drug varied. The delayed time of the Integrillin release increased as the initial molecular weight of the PLGA increased. While degradation of PLGA affected the release of the peptide, degradation of the polyarylates did not have such an effect. Preliminary in vitro studies showed the polymer to be versatile towards drug release kinetics by tuning in the pendant chain and the backbone unit.

Poly(ether ester amide) Another candidate for drug delivery systems arises from the family of poly(ether ester amide)s (PEEAs). They are mainly fabricated by polycondensation of PEG and diester-diamide to create an amphiphilic system. 43,44 Diester-diamide acts as a hydrophobic block for creating reversible physical crosslinks that account for stronger mechanical properties in the swollen state. In this case, PEG is the ‘soft’ segment and diester-diamide acts as the ‘hard’ segment (Fig. 4(d)). The structure gives versatility allowing the tailoring of hydrogels to suit different drug release profiles. Bezemer et al . 43 fabricated the polymer by a two-step mechanism. The first step involved transesterification of diamide-dimethyl ester monomers with PEG, and the second step was polycondensation at 220 C. The resulting polymers had an intrinsic viscosity ranging from 0.58 to 0.78 dL g 1 , and they were not completely amorphous; both depended on the length of the polymer blocks. It was shown that the soft segment length in a microsphere influenced the degree of swelling, in vitro degradation, and release rate. 43 Cytocompatibility was investigated using HUVEC. 44 These surfaces did not perform as pos- itively as TCPS, possibly due to the PEO con- tent. Deschamps et al . 44 implanted a low-content- PEG PEEA subcutaneously in rats. Mass loss was 7–12 wt% at 14 weeks post-implantation and a slow decrease in intrinsic viscosity led to bulk hydrolysis degradation. Tissue responses were found to be sim- ilar to tissue reactions observed upon implantation of other biodegradable polymers. Fibrous capsules were observed, with macrophages and blood vessel infiltra- tion accompanied by cracks on the polymer surface.

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A porous scaffold was also fabricated from this mate-

rial using compression moulding followed by a salt leaching technique. Another amphiphilic drug delivery system uses a PCL-based polymer construct containing hydrophilic PEEA. 45 The possibility of using PCL macromers of different molecular weights changed the PEEA properties including crystallinity and the hydrophilic- ity/hydrophobicity, and this would affect the drug release rate. In vitro drug release studies using three different drugs of different natures revealed that PEEA successfully enhanced the completion of drug release. An acidic drug was released rapidly after 2 h incuba- tion, while a drug of a basic nature exhibited longer controlled release.

Poly(amido amine) The use of hydrogels as matrices for tissue engineering applications and the search for versatile, easily fab- ricated, biodegradable, and biocompatible matrices continue. One novel hydrogel is made of poly(amido amine), a polycationic polymer in nature. It con- tains ter-amino and amido groups regularly arranged along the polymer chain, obtained by Michael-type polyaddition of primary or secondary amines to bis- acrylamides. The method of fabrication made it possible to have side substituents with biomimick- ing properties, such as carboxyl, ter-amino, hydroxyl, allyl, or bioactive molecules like proteins and peptides. In vitro assays performed by Ferruti et al . 46 inves- tigated cytotoxicity, compatibility, and proliferation of the materials and the cells in contact with them. Biological evaluation results exhibited non-toxicity, comparable cell proliferation (more than 70%) to nor- mal TCPS plates, and normal cell morphology.

Furthermore, degradation products were non-toxic and the rate of degradation could be fine tuned depending on the structure and degree of crosslinking, which, in turn, affected the degradation via a hydrolysis mechanism. For example, hybrids of PAA and BSA (bovine serum albumin) degraded fully up to 8 months

in simulated body fluids. 46 It was suspected that this

was because of the protective characteristics of BSA. Apart from providing a protective layer, BSA made the hybrid substrate more supportive to cell adhesion and proliferation. An attempt to make it more bioactive was achieved by incorporating agmatine, a decarboxylated product of arginine, derived from RGD sequences. 47 Cell adhesion and proliferation assays exhibited up to 80% capacity compared to normal polystyrene culture plates.

CONCLUSIONS Biodegradable synthetic polymers, from a material standpoint, are a key area of interest for the devel- opment of new scaffold-based tissue engineering strategies. A critical issue in scaffold-based tissue engineering is the assembly of cells and extracel- lular material into a three-dimensional architecture

Polym Int 56 :145–157 (2007) DOI: 10.1002/pi

that allows for both structure and functionality that mimics the native tissue that is being replaced and/or repaired. As the scaffolds for tissue engi- neering will be implanted in the human body, the scaffold materials should be non-antigenic, non- carcinogenic, non-toxic, non-teratogenic, and possess high cell/tissue biocompatibility so that they will not trigger pathological reactions after implantation. In addition to materials issues, the macro- and microstructural properties of the scaffold are also very important. In general, the scaffolds require individual external shape and well-defined internal structure with interconnected porosity to host most cell types. From a biological point of view the designed matrix should serve various functions, including (1) as an immobi- lization site for transplanted cells, (2) formation of a protective space to prevent unwanted tissue growth into the wound bed and allow healing with dif- ferentiated tissue, (3) directing migration or growth of cells via surface properties of the scaffold, and (4) directing migration or growth of cells via release of soluble molecules such as growth factors, hormones, and/or cytokines. Some technology platforms of FDA- approved polymeric degradable scaffold systems have already found promising clinical applications. New polymers are under development and their applica- tions need to be a part of the systemic approach to tissue engineering; namely the need to coordi- nate interaction between the parameters of scaffolds, cells, bioreactors, and biomolecular factors as well as the controlling features of the host response to re- implanted constructs, including phenomena of angio- genesis, inflammation, and immune response. Based on these results, novel therapeutic options in the area of polymeric scaffold-based tissue replacement can be expected in the 21st century.

ACKNOWLEDGEMENTS The work reported in this review was supported in part by the Biomedical Research Council (BMRC) grants R-397-000-005-305 (to DWH). The authors thank Maria Woodruff for editing the manuscript.

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