Novel Agmatine-Containing Poly(amidoamine) Hydrogels as Scaffolds

Their toxicity was found in several tests to be constantly lower by 2 or 3 orders ... for cell culturing, even if their cell adhesion properties were ...
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Biomacromolecules 2005, 6, 2229-2235

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Novel Agmatine-Containing Poly(amidoamine) Hydrogels as Scaffolds for Tissue Engineering Paolo Ferruti,* Sabrina Bianchi, and Elisabetta Ranucci Dipartimento di Chimica Organica e Industriale, Universita` di Milano, via Venezian 21, 20133 Milano, Italy

Federica Chiellini and Anna Maria Piras Dipartimento di Chimica e Chimica Industriale, Universita` di Pisa, via Risorgimento 35, 56125 Pisa, Italy Received March 19, 2005; Revised Manuscript Received April 15, 2005

Novel biocompatible and biodegradable amphoteric poly(amidoamine) (PAA) hydrogels were designed for applications as scaffolds for tissue engineering. These hydrogels (PAA-AG1 and PAA-AG2) were obtained by polyaddition of 2,2-bisacrylamidoacetic acid with 2-methylpiperazine and 4-aminobutyl guanidine, a bioactive molecule with a known ability to induce adhesion to cell membranes. They contain carboxylic functions in their main chain and interchain connections deriving from two different cross-linking agents: for PAA-AG1, a multifunctional primary amine, that is, 1,10-decanediamine; for PAA-AG2, a purposely synthesized PAA (PAA-NH2) containing pendant NH2. Both PAA-AG1 and PAA-AG2 proved noncytotoxic and adhesive to cell membranes, as ascertained by means of cytotoxicity and proliferation tests carried out on fibroblast cell lines. Good apparent mechanical strength was also observed in the case of PAA-AG2, cross-linked with the PAA-NH2. Both PAA-AG1 and PAA-AG2 underwent degradation tests under controlled conditions simulating the biological environments, that is, Dulbecco medium at pH 7.4 and 37 °C. They completely dissolved within 10 and about 40 days, respectively. In both cases, the degradation products were completely noncytotoxic. All the results of this paper point to the conclusion that agmatine-based PAA hydrogels are excellent substrates for cell proliferation. Introduction The basic requirements for hydrogels in view of applications as scaffolds for tissue engineering are biocompatibility, biodegradability, high water content, and cell membrane adhesion. Poly(amidoamine)s (PAAs) are a family of synthetic polymers containing ter-amino and amido groups regularly arranged along their polymer chain.1,2 They are obtained by Michael-type polyaddition of primary or secondary amines to bis-acrylamides (Scheme 1). PAAs containing as side substituents other chemical functions, such as additional ter-amino groups, carboxyl groups, hydroxyl groups, and allyl groups, can be easily obtained by using suitably functionalized monomers. Peptides and proteins can also participate in the polyaddition reaction through their terminal amino groups as well as -lysine amino groups, if present.3 PAAs are, normally, biodegradable, with a degradation rate depending on their specific structure4 and, what is most relevant for biomedical applications, most of them are almost nontoxic, despite their polycationic nature. Their toxicity was found in several tests to be constantly lower by 2 or 3 orders of magnitude than that of poly-L-lysine.5,6 Amphoteric PAAs, carrying carboxyl groups as side substituents, are even less toxic and may be approximately as biocompatible as dextran. The same PAAs when injected in animals are endowed with * Author to whom correspondence should be addressed. Tel: +39-0250314128; fax: +39-02-50314129; e-mail: [email protected].

“stealth” properties and undergo passive concentration in solid tumors by the EPR (enhanced permeation and retention) effect.6 Cross-linked PAAs can be obtained by using multifunctional amines as cross-linking agents.1,2 For instance, diaminoalkanes contain four mobile hydrogens and behave as tetrafunctional monomers. Cross-linked PAAs are typical hydrogels, absorbing large amounts of water if their cross-linking degree is not too high. As such, they have, in principle, a potential as scaffolds for tissue engineering.7 According to recent studies, PAA-based hydrogels have a definite potential as biodegradable and biocompatible substrates for cell culturing, even if their cell adhesion properties were modest.8 Recently, it has been demonstrated that some oligopeptides are capable of reproducing the receptorial sites of proteins playing a fundamental role in cell adhesion, such as fibronectin, laminin, and vitronectin.9 Among these, the tripeptide arginin-glycin-aspartic acid (RGD) is presently the most popular. Grafted on a material’s surface, it is capable of promoting a strong cell adhesion even at very low surface density.10 The structure of RGD sequence in proteins is shown in Scheme 3. The peculiar biological properties of RGD seem to be mainly related to the presence of the guanidine side group deriving from arginine. The decarboxylation product of arginine, that is, 4-aminobutyl guanidine or agmatine (Scheme 3), apparently shares some of the biological properties of

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Scheme 1. Synthesis of PAAs

Scheme 2. Scheme of a Cross-Linked PAA Segment

Scheme 3. Structure of RGD Tripeptide Sequence in Proteins

Scheme 4. Structure of Agmatine

RGD. For instance, PEG hydrogels surface-modified with the introduction of agmatine residues are capable, in contrast with plain PEG hydrogels, to promote cell adhesion.11As regards soluble polymers, it may be mentioned that in a series of vinyl cationic polymers studied as transfection agents, the only one showing significant activity contained agmatine residues as side substituents.12 The above considerations prompted us to investigate if the introduction of side guanidine groups in PAA hydrogels would improve cell adhesion preserving their already advantageous biological properties. The aim of this paper is to report on the synthesis of amphoteric agmatine-based PAA hydrogels and on their preliminary evaluation as scaffolds for tissue engineering applications by cytotoxicity and cell proliferation tests carried out on fibroblast lines as well as by degradation tests performed under conditions mimicking the physiological environment. Experimental Part Instruments. Size exclusion chromatography (SEC) traces of the linear PAA-based cross-linker were obtained making

use of TSK-gel G5000 PW, TSK-gel G4000 PW, and TSKgel G3000 PW columns produced by TosoHaas. The three columns were connected in series and the mobile phase was Tris buffer pH 8,10; flow rate 1 mL/min (Waters model HPLC pump 515); the UV detector was a Waters model 486, operating at 230 nm; the refractive detector was a Waters model 2410. The samples were prepared in Tris buffer with a 1% concentration in polymer (10 mg of polymer in 1 mL of buffer). The 1H and 13C spectra were acquired on a Bruker Avance 400 spectrometer, operating at 400.133 MHz (1H) and at 100.00 MHz (13C). For cytotoxicity experiments, cells were grown at 37 °C in 5% CO2 enriched atmosphere in a Hera Cell incubator (Heraeus Instruments). Cell manipulation was carried out under a Hera Safe HS12 laminar flow (Heraeus Instruments). Quantitative evaluation of cell proliferation and viability was carried out using a Benchmark Microplate Reader (Biorad). Materials. Triethylamine (100%), 1,10-diaminodecane (97%), and LiOH‚H2O (99%) were purchased from Fluka and were used as received. 2-Methylpiperazine (2MP) (98%), Fluka, was purified by crystallization from n-heptane, and purity was checked by acidimetric titration. Agmatine sulfate (97%) was purchased from Aldrich and was used as received. 2,2-Bis(acrylamido) acetic acid (BAC) was synthesized as previously described.13 Cell line BALB/3T3 Clone A31 mouse embryo fibroblasts (CCL163) were obtained from American Type Culture Collection (ATCC) and propagated as indicated by the supplier. Dulbecco’s Modified Eagles Medium (DMEM), 0.01 M pH 7.4 phosphate buffer saline without Ca2+ and Mg2+ (PBS), fetal bovine serum (FBS), trypsine/EDTA, glutamine, and antibiotics (penicillin/streptomycin) were purchased from GIBCO Brl. Cell proliferation reagent WST-1 was purchased from Roche Diagnostic. Toluidine blue was purchased from Sigma. Tissue culture grade disposable plastics were obtained from Corning Costar. Methods. Synthesis of PAA-AG1. BAC (0.70 g, 3.5 mmol) and LiOH‚H2O (0.15 g, 3.5 mmol) were dissolved in water (0.6 mL). Agmatine sulfate (0.29 g, 1.2 mmol) and LiOH‚H2O (0.05 g, 1.2 mmol) were then added to the mixture and stirred until a homogeneous solution was obtained. 2MP (0.13 g, 1.2 mmol) and a methanol solution of 1,10-diaminodecane (0.09 g, 0.5 mmol in 0.6 mL) was prepared and added to the BAC/agmatine reactive mixture. The polymerizing solution was flushed with nitrogen and then was injected into the mold, consisting of two interfaced

Agmatine-Containing Poly(amidoamine) Hydrogels

silanized, nonadhesive glass plates carrying a 1-mm-thick silicone spacer, and was maintained at room temperature for 5 days. A transparent and soft hydrogel was obtained. The PAA hydrogel obtained by this procedure was purified from low molecular weight impurities by first extracting with excess ethanol and then by phosphate buffer pH 7.4. Treating directly ethanol-swollen hydrogel samples with aqueous media caused an osmotic shock that, in some cases, leads to serious damages. The adopted procedure was, therefore, to expose the ethanol-swollen samples to a gradient buffer concentration by soaking first in ethanol and then in buffer/ ethanol mixtures with increasing buffer concentrations, until pure buffer is used. The extraction time was at least 1 h each step. Synthesis of PAA-NH2 Cross-Linker. The PAA-based cross-linker was obtained according to an experimental procedure already reported.14 In brief, BAC (0.50 g, 2.4 mmol) and triethylamine (0.24 g, 2.4 mmol) were dissolved in water (1.5 mL). Acetic acid (0.15 g, 2.6 mmol) was added, and the solution was maintained under stirring for 15 min at room temperature. After this time, EDA (0.15 g, 2.4 mmol) was added and the reaction mixture was allowed to stand under inert atmosphere, with occasional stirring for 2 days. The aqueous solution of PAA-NH2 was treated with triethylamine (0.25 g, 2.4 mol) just before use. 1H NMR (D O, δ, ppm) ) 2.75 (m, 4H, CH CH -NH ); 2 2 2 2 2.80 (m, 4H, CH2CO); 3.3 (m, 4H, CO-C-CH2-N); 5.55 (s, 2H, -NH-); 13C NMR (D O, δ, ppm) ) 27.5-28 (-NCH CH -NH); 2 2 2 30-48 (NH-C-CH2-); 39.64 (NH CH2-); 170 (-CON-); 172.5 (-COOH-). Number average molecular weight ) 10 300; polydispersity ) 2.3. Synthesis of PAA-AG2 Hydrogel. BAC (0.99 g, 4.8 mmol) and LiOH‚H2O (0.21 g, 4.8 mmol) were dissolved in water (1.6 mL). Agmatine sulfate (0.28 g, 1.2 mmol) and LiOH‚H2O (0.05 g, 1.2 mmol) were then added to the mixture and stirred until a homogeneous solution was obtained. 2MP (0.13 g, 1.2 mmol) and then PAA-NH2 (2.21 g) were added to the BAC/2MP reactive mixture. The reactive solution obtained was injected into the glass mold and was maintained at room temperature for 1 day. A transparent soft and pliable hydrogel was obtained. The PAA hydrogel obtained by this procedure was purified as described in the case of PAA-AG1. Swelling Tests. The tests were carried out on PAA hydrogel sheets with dimension 10 × 20 × 1 mm. The specimens were dried at 20 °C and 0.1 Torr. Their weight, in the dry state, was 21.2 ( 0.6 mg on average. Each specimen, of initial mass Mo, was placed inside a 10-mL test tube containing 5 mL water maintained at 37 °C. At regular intervals, the specimen was taken out of the test tube, any visible surface moisture was wiped off, and then it was weighed. After this, the specimen was returned to the test tube and the uptake of water was measured until the maximum mass was obtained. The percent amount of water absorbed was calculated using the following formulas:

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abs% )

Mat × 100 Ma0

(1)

des% )

Mdt × 100 Md∞

(2)

where Mat is the water mass absorbed at time t, Mao is the mass of sample at time zero, Mdt is the water mass desorbed at time t, and Md∞ is the mass of the sample at equilibrium. Degradation Experiments. The degradation tests were carried out on PAA hydrogel sheets with the same shape and dimension as in the swelling tests. Each specimen, of initial mass Mo, was placed inside a 10-mL test tube containing 5 mL Dulbecco Modified Eagles medium at pH 7.0 and was maintained at 37 °C. At regular intervals, the specimen was taken out of the test tube, any visible surface moisture was wiped off, and then the specimen was weighed. Biological Tests Cell Line. Cell adhesion and proliferation assays were carried out using the 3T3/BALB-C Clone A31. Cells were grown in DMEM containing 10% FBS, 4 mM of glutamine, and 100 U/mL:100µg/mL penicillin:streptomycin (complete DMEM). Subculturing. A 25-mL flask containing exponentially growing 3T3 cells was observed under an inverted microscope for cell confluence. The complete DMEM media was then removed, and cells were rinsed for a few minutes with PBS. The buffer solution was removed, and cells were incubated with 0.5 mL of trypsin/EDTA solution at 37 °C in 5% CO2 incubator for 5 min or until the monolayer started to detach from the flask. Cells were suspended in 5 mL of complete DMEM media and then were centrifuged at 700g for 5 min. The pellet was suspended in an appropriate volume of DMEM and plated at a split ratio of 1:6 or 1:10 in a 75 cm2 flask. Direct Contact Assays. For the determination of cytotoxicity by direct contact assays, cells were seeded at appropriate density and the hydrogels were placed in direct contact with cell monolayer. The culture medium was then removed, thus removing also the cells that did not adhere, and replaced with fresh complete DMEM. Cells were allowed to proliferate for 72 h, and then evaluation of cell viability was carried out by means of a WST-1 assay as well as by morphological investigation on fixed and stained cells. Cell Adhesion Assay. To evaluate the ability of PAAAG hydrogels to sustain cell adhesion and proliferation, cells were seeded at appropriate density directly onto hydrogel samples. The culture medium was then removed, thus removing also the cells that did not adhere, and replaced with fresh complete DMEM. Cells were allowed to proliferate for up to 10 days, and then evaluation of cell viability was carried out by means of a WST-1 assay as well as by morphological investigation on fixed and stained cells. Cytotoxicity Evaluation of Degradation Products. Hydrogels were allowed to incubate in DMEM at 37 °C, 5% CO2 for 38 days. At the end of the degradation experiments, cells were exposed to DMEM containing degradation

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Scheme 5. Synthesis of PAA-AG1

products undiluted or diluted 1:2 and 1:4 in complete DMEM, for 24 h, and then were incubated with WST-1 cell proliferation reagent for the quantitative evaluation of cell proliferation. Cell Proliferation Assay. Quantitative proliferation was assayed by using the Cell Proliferation Reagent WST-1 and by following the protocol indicated by the manufacturer. Briefly, cells were allowed to proliferate either in direct contact with hydrogels or exposed to DMEM containing degradation products and then were incubated for 4 h with an appropriate volume of WST-1 tetrazolium salts. Formazan production was detected at 450 nm, with 620 nm as reference wavelength using an ELISA microplate reader (Biorad.). Cell Fixing and Staining. Cells grown onto hydrogels were washed three times with PBS, incubated for 1 h at room temperature in a 3.8% solution of paraformaldehyde in 0.01 M pH 7.4 PBS, and finally carefully rinsed with PBS. For postfixation staining, cells were incubated with Toluidine blue solution in PBS, rinsed with PBS, and stored at 4 °C. Microscopy. For routine culturing and qualitative evaluation of morphology, cells were analyzed under an inverted microscope Nikon Eclipse TE2000-U. Results and Discussion Synthesis. Previous findings have shown that, on the whole, amphoteric PAAs deriving from 2,2-bisacrylamidoacetic acid (BAC) are most promising with regard to lack of toxicity, “stealth” properties, and transfection ability.14 Therefore, it was decided to investigate the potential as scaffolds for tissue engineering of hydrogels obtained by the polyaddition of agmatine to BAC. Agmatine contains a primary amino group and a guanidine group carrying five potentially mobile hydrogens that could participate in the

polyaddition reaction. It is, therefore, a potential cross-linking agent in PAA synthesis like primary diamines as, for instance, 1,2-diaminoethane (ethylenediamine, EDA) and 1,10-diaminodecane, the latter adopted to this purpose in PAAAG1. However, in the case of EDA, where a large difference exists in the basic properties of the two different amino groups (pKa values 10.71 and 7.56, respectively), partial protonation can be employed as a tool for protecting one of the amino groups during polyaddition. Starting from monoprotonated EDA and BAC, linear PAAs with pendant primary amino groups were in fact obtained.15 A large difference in basic properties exists also between the amine and the guanidine groups of agmatine. The latter has a pKa of 12.5, much higher that that of any aliphatic amine, and remains protonated under the conditions employed in PAA synthesis. Agmatine is usually sold as sulfate. By adding a single mole of a strong base (lithium hydroxide was found particularly convenient for the ease of dosage and of elimination from the end product), only the amine group loses its proton, and monoprotonated agmatine in which the proton is localized on the guanidine group is obtained. A protonated guanidine group does not participate in the polyaddition reaction and in the absence of cross-linking agents linear soluble agmatine-based PAAs are formed (data not shown). Cross-linked agmatine-containing PAAs can only be prepared in the presence of cross-linking agents, the general method for traditional PAAs. This paper reports on synthesis, properties, and performance as scaffolds of amphoteric agmatine-containing PAA hydrogels obtained by adding monoprotonated agmatine to polymerizing mixtures containing BAC, 2-methylpiperazine, and polyaminic cross-linkers. The structure of the agmatine units of these PAAs (Schemes 5 and 7) is similar to that of RGD tripeptide (Scheme 3). In other words, agmatine-

Agmatine-Containing Poly(amidoamine) Hydrogels Scheme 6. Structure of the PAA-NH2 Cross-Linker

containing PAAs might be regarded as a synthetic mimic of RGD and can be reasonably expected to share some of its biological properties. In a first instance, 1,10-diaminodecane was used as crosslinking agent, thus obtaining PAA-AG1 hydrogel, whose structure is reported in Scheme 5. To obtain a hydrogel with better mechanical properties, a new hydrogel (PAA-AG2) was prepared. In this hydrogel, the cross-linker consisted of a purposely synthesized PAA containing pendant NH2 groups, PAA-NH2 (see Scheme 6). The structure of PAAAG2 is schematically represented in Scheme 7. In both preparations, the monomer ratio was adjusted so as to balance the number of aminic and acrylamido functions. In the polymerization recipes of both PAA-AG1 and PAAAG2, 30% of the aminic hydrogens belonged to the crosslinking agent. Agmatine and 2MP were present in equimolar amounts. Both PAA hydrogels were prepared between two glass plates as 1-mm-thick sheets. They were transparent,

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soft, and elastic. The mechanical strength of PAA-AG2 was obviously superior than that of PAA-AG1. Both hydrogels could easily be cut into small parallelepipeds (10 × 20 × 1 mm in dimensions) for further evaluations, as measurement of water uptake, toxicological, and long-term degradation tests. The swelling tests demonstrated that both PAA hydrogels had a high water absorption capability, 1100% for PAA-AG1 and 700% for PAA-AG2. Biological Evaluation. Cell adhesion tests carried out on agmatine-containing PAA hydrogels were performed following the indications of the standard ISO-10993 (EN 30993), Part 10995: test for cytotoxicity, in vitro methods. Both PAA-AG1 and PAA-AG2 proved to be noncytotoxic and bioactive in terms of allowing cell adhesion and further proliferation. Quantitative results relative to PAA-AG2 are shown in Figure 1. Similar data were obtained in the case of PAA-AG1. The cytobiocompatibility of agmatine-containing PAA hydrogels in direct contact assays was also qualitatively evaluated by optical microscopy. The morphology of cells grown onto the surface of both hydrogels were comparable with that of cells grown on the tissue culture polystyrene plates used as control. In all cases, cell confluence was reached after 10 days from the beginning of the experiment. The results relative to PAA-AG2 and PAA-AG1 are reported in Figures 2 and 3, respectively. As expected, no toxic substances were apparently released from the hydrogels during incubation.

Scheme 7. Schematic Representation of PAA-AG2 Synthesized by Using PAA-NH2 as Cross-Linkera

a

The solid line stands for PAA-NH2 residue.

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Figure 1. Quantitative results of cell adhesion and proliferation assay of PAA-AG2 evaluated by means of WST-1 tetrazolium salt. Data presented were obtained on nine replicates for each time point. The exact figures are (a) 6 days in culture: cell viability 28,3 ( 0,8%; (b) 10 days in culture: cell viability 85,9 ( 3,5%.

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Figure 3. Morphology of mouse embryo fibroblasts grown on PAAAG1 hydrogels after 10 days of culture.

Figure 4. Results of the degradation tests of PAA-AG1 (pink line) and PAA-AG2 (blue line) in Dulbecco medium at 37 °C and pH 7.0.

Figure 2. Morphology of mouse embryo fibroblasts grown on PAAAG2 hydrogels after (A) 6 days and (B) 10 days of culture.

It may be finally observed that both hydrogel samples did not significantly alter the pH of the culture medium, which remained in the range 7.6-8.1 during all the evaluation time. Degradation. The degradation of PAA-AG1 and PAAAG2 samples was carried out under conditions mimicking the physiological environment, that is, Dulbecco medium at pH 7.0 and 37 °C. The percent weight decrease of hydrogel samples with time under the reported conditions is shown in Figure 4. It was found that PAA-AG1 dissolved completely within 10 days, but the dissolution of PAA-AG2 went

to completion after about 40 days. The longer dissolution time of PAA-AG2 might be ascribed to its lower water absorption capability. The cytotoxicity of the degradation products of both hydrogels was evaluated. Degradation was carried out for 38 days under sterile conditions in Dulbecco medium at pH 7.0 and 37 °C. This reaction time was arbitrarily chosen. It was preferred to perform the experiment at the same reaction time for both hydrogels, even if some undissolved material was still present in the case of PAA-AG2. Cell viability was then assessed using mouse embryo fibroblast cell lines. The degradation products of both hydrogels proved devoid of significant toxicity even if undiluted. The results relative to PAA-AG2 are reported in Figure 5. The results of PAAAG1 were practically superimposable. Conclusions This paper reports on the preliminary evaluation of novel biodegradable and biocompatible agmatine-containing PAA hydrogels as scaffolds for tissue engineering. The following conclusions can be drawn: (1) Amphoteric PAA hydrogels, that is, containing tertamino and carboxyl groups in their repeating units, can be

Agmatine-Containing Poly(amidoamine) Hydrogels

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containing membrane-active oligopeptide moieties, including RGD, will be the object of forthcoming papers. Acknowledgment. This work was funded by the Italian Ministry of University and Research (MIUR), Project COFIN 2004. The authors wish to thank Dr. Cristina Bartoli and Mr Matteo Gazzarri for their contribution in carrying out biological experiments. References and Notes

Figure 5. Quantitative results of cytotoxicity evaluation of the degradation products of PAA-AG2. Cell viability was assessed after exposing 3T3 mouse embryo fibroblasts for 72 h to a growth medium containing the degradation products.

synthesized by using agmatine as comonomer and multifunctional amines as cross-linkers. Their agmatine-containing repeating unit can be considered as a mimic of RGD peptide. (2) The apparent mechanical strength is higher when a second PAA carrying primary amino groups is used as crosslinking agent. (3) Degradation tests carried out under conditions mimicking the physiological environment demonstrate that the degradation rate can be tuned by properly selecting the aminic cross-linker. (4) The degradation products of both hydrogels are nontoxic. (5) Cell adhesion and proliferation on fibroblast cell lines is excellent irrespectively of the nature of the cross-linker. (6) As a final conclusion, agmatine-containing amphoteric PAA hydrogels definitely warrant attention as biomimetic materials for tissue engineering applications. A comparison between their performances and those of PAA hydrogels

(1) Ferruti, P. Ion-Chelating Polymers (Medical Applications). In Polymeric Materials Encyclopedia; Salamone, J. C., Ed.; CRC Press Inc.: Boca Raton, FL, 1996; Vol. 5, pp 3334EnDash3359. (2) Ferruti, P.; Marchisio, M. A.; Duncan, R. Macromol. Rapid Commun. 2002, 23, 332-355. (3) Ranucci, E.; Bignotti, F.; Paderno, P. L.; Ferruti, P. Polymer 1995, 36, 2989-2994. (4) Ferruti, P.; Ranucci, E.; Sartore, L.; Bignotti, F.; Marchisio, M. A.; Bianciardi, P.; Veronese, F. M. Biomaterials 1994, 15, 1235-1241. (5) Ranucci, E.; Spagnoli, G.; Ferruti, P.; Sgouras, D.; Duncan, R. J. Biomater. Sci, Polym. Ed. 1991, 2, 303-315. (6) Richardson, S.; Ferruti, P.; Duncan, R. J. Drug Targeting 1999, 6, 391-404. (7) Lanza, R. P.; Langer, R.; Chick, W. L. Principles of tissue engineering; Academic Press: 2001. (8) Ferruti, P.; Bianchi, S.; Ranucci, E.; Chiellini, F. Macromol. Biosci. 2005, in press. (9) Massia, S. P.; Hubbel, J. A. J. Cell Biol. 1991, 114, 1089. (10) Tanahashi, K.; Jo, S.; Mikos, A. G. Biomacromolecules 2002, 3, 1030-1037. (11) Raasch, W.; Schafer, U.; Chun, J.; Dominiak, P. Br. J. Pharmacol. 2001, 133, 755-780. (12) Dubruel, P.; Dekie, L.; Schacht, E. Biomacromolecules 2003, 4, 1168-1176. (13) Ferruti, P.; Ranucci, E.; Trotta, F.; Gianasi, E.; Evagorou, G. E.; Wasil, M.; Wilson, G.; Duncan, R. Macromol. Chem. Phys. 1999, 200, 1644-1654. (14) Ferruti, P.; Manzoni, S.; Richardson, S. C. W.; Duncan, R.; Pattrick, N. G.; Mendichi, R.; Casolaro, M. Macromolecules 2000, 33, 77937800. (15) Malgesini, B.; Verpilio, I.; Duncan, R.; Ferruti, P. Macromol. Biosci. 2003, 3, 59-66.

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