Biomacromolecules Electrostatic Self-Assembly on 3-Dimensional

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Biomacromolecules 2004, 5, 1933-1939

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Biomacromolecules Electrostatic Self-Assembly on 3-Dimensional Tissue Engineering Scaffold Huiguang Zhu,† Jian Ji,* and Jiacong Shen Department of Polymer Science and Engineering, Zhejiang University, Hangzhou, 310027 China Received April 23, 2004; Revised Manuscript Received June 21, 2004

A poly(ethylenimine) (PEI) was employed to obtain a stable positively charged surface on a poly( D,Llactide) (PDL-LA) tissue engineering scaffold. An extracellular matrix (ECM)-like biomacromolecule, gelatin, was selected as polyelectrolyte and deposit alternately with PEI on the activated PDL-LA scaffold via ESA technique. The ζ-potential result showed alternating charge of polyelectrolytes (PEI/gelatin) layering on PDL-LA microspheres. Quartz crystal microbalance (QCM) measurement further verified the gradual deposition of PEI/gelatin on the PDL-LA thin film. The combination of PEI aminolysis and the layer-bylayer technique was then explored to construct gelatin coating onto the 3-D porous PDL-LA scaffold. Scanning electronic microscopy showed that there is no notable difference between modified and unmodified PLA scaffolds, with regard to the porosity, pore diameter, and scaffold integration. The dual-tunnel confocal laser scanning microscopy indicated uniform gelatin distribution on the inner surface of the 3-D porous scaffold. The gradual build-up of protein layer on scaffold was investigated by radioiodination technique. Chondrocyte was chosen to test the cell behavior on modified and unmodified PDL-LA scaffolds. The results of the cell viability, total intracellular protein content, and cell morphology on the PEI/gelatin multilayers modified PDL-LA scaffold showed to promote chondrocyte growth. Comparing conventional coating methods, polyelectrolyte multilayers are easy and stable to prepare. It may be a promising choice for the surface modification of complex biomedical devices. These very flexible systems allow broad medical applications for drug delivery and tissue engineering. Introduction Tissue engineering has developed rapidly to a major field in biotechnology in recent years. New developments in biomaterial, innovative cell culture technique, and newly discovered growth factors open novel avenues to engineering vital transplantable tissues and organs for research and clinical application.1 Specially designed three-dimensional biomaterials provide one of the fundamental tools to shape and guide the tissue development in vitro and in vivo. An ideal tissue scaffold should (1) be biocompatible, i.e., not provoke any tissue response to the implant, but at the same time have the right surface chemistry which will promote cell attachment and function, (2) be biodegradable into nontoxic products, leaving a living tissue, (3) have the mechanical strength needed for the creation of macroporous scaffold that will retain its structure after implantation, and (4) be easily processed to have a variety of configurations.2 However, few scaffolds can fit all of these qualifications. The synthetic biodegradable polyester, poly(lactide) (PLA), has long been used in the biomedical field as sutures, drug delivery carriers, implants, and recently as scaffold for tissue engineering. However, the high hydrophobicity of PLA * To whom correspondence should be addressed. Tel: +86-57187951108. Fax: +86-571-87951948. E-mail: [email protected]. † Currently working as postdoc at Institute for Micromanufacturing (IfM) at Louisiana Tech University, Louisiana, U.S.A.

material has made it difficult to get a stable biomimetic coating of hydrophilic natural biomacromolecules, and the lack of functional groups to covalent cell-recognition molecules to PLA molecules has limited its further application in tissue engineering and other biomedical areas. Though different strategies have been proposed,3-7 very few examples have achieved uniformly surface modification for the complex 3D tissue-engineering scaffold. In nature, the biological world is built up via precise selfassembly of biomacromolecules. It has served as a great inspiration for investigators to explore an engineered scaffold via macromolecules self-assembly.8-13 Layer-by-layer electrostatic self-assembly (ESA) of polyelectrolytes constitutes a novel and promising technique to modify surfaces in a controlled way.14-22 Also there have been lots of attempts to build biomimetic films based on ESA in recent years, for example, with alginate/polylysine,23 chitosan/dextran sulfate,24 hyaluronic acid/poly(lysine),25 poly(lysine)/poly(glutamic acid),26 and collagen.27 The principle of alternate adsorption was invented in the pioneering works of Iler17 and Decher et al.20-22 introduced a related method for film assembly by means of alternate adsorption of linear polycations and polyanions or bipolar amphiphiles. One of the most important properties of such assembly comes from the fact that they exhibit an excess of alternatively positive and negative charges. Not only does this constitute the motor of their buildup but also it allows, by simple contact, the films to absorb a great variety of compounds such as dyes,

10.1021/bm049753u CCC: $27.50 © 2004 American Chemical Society Published on Web 08/03/2004

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particles, or proteins. More recently, the activity of a protein embedded in a film and of a peptide coupled to one of the polyelectrolytes has been evidenced.15,28,29 This remarkable property opens up the possibility to construct multilayer incorporating specific ligands that keep their biological activity and promote specific cell function. Our previous study30,31 indicated that the natural biomacromolecules, for example, gelatin and chitosan, could be deposited on the surface of poly(lactide) (PLA) film via aminolysis and layer-by-layer electrostatic self-assembly, which obviously promote the cytocompatibility of the chondrocyte and endothelial cells. The current research will explore to construct extracellular matrix-like multilayers on 3-D macroporous PLA scaffold via ESA technique. Chondrocytes behavior was evaluated to assess the potential of this novel surface treatment for cartilage implant and tissue engineering scaffold for cartilage reconstruction. Experimental Section Chemicals. Poly(D,L-lactide) (PDL-LA, abbreviated as PLA in this paper) was prepared by ring-opening polymerization of D,L-lactide using stannous octoate as a catalyst32 in our laboratory, molecular weight was 200 000 obtained from GPC analysis, MWD)1.75. Poly(ethylenimine) (PEI) was purchased from Aldrich (Mw ) 25 000). The biomacromolecule used in this study was gelatin (Fluka, Gelatin from Porcine Skin with medium gel strength). Sodium chloride was ground and sieved to provide particles of relatively uniform diameter. Methylene chloride was used to dissolve the PLA matrix for the preparation of the scaffold. All of the chemicals were used without further purification. ζ-Potential Measurement. PLA microspheres were prepared by an emulsification technique33 for ζ-potential measurement. The microelectrophoretic mobility of unmodified and coated PLA microspheres was measured with a ζ-potential analyzer (Zeta Plus, Brookhaven Instruments Corporation) by taking the average of five measurements at the stationary level. The mobilities (µ) were converted to the electrophoretic potentials (ζ) using the Smoluchowski relation ζ ) µη/, where η and  are the viscosity and permittivity of the solution, respectively. All measurements were performed on PLA microspheres re-dispersed in DIwater. Quartz Crystal Microbalance (QCM) Measurements. A QCM (QCM100 quartz crystal microbalance, QCM25 Crystal Oscillator, SRS) was used to investigate the gradual deposition of the polyelectrolyte layers on the PLA film. The QCM crystal was first cleaned by using cleaning solution containing 1 part of 45% KOH, 39 parts of ethanol, and 60 parts of DI water. The crystal was immersed in a low concentration of PLA/acetone solution and then dried by nitrogen to get a thin film of PLA on the crystal. The crystal with the thin PLA film was activated by 1 mg/mL PEI solution for 3 h, rinsing with DI water, and nitrogen-dried. The activated substrate was then immersed in a 0.5 mg/mL gelatin, pH7.4, for 20 min. After a 30 s washing of the substrate with deionized water, the substrate was dipped into a 1 mg/mL PEI solution, pH 7.4, for 20 min. Following the

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same washing procedure, the substrate was exposed to gelatin solution for 20 min and rinsed with water again. The further growth of PEI/gelatin bilayers was accomplished by the repetition of the same cycle of immersion into the solution of PEI, rinsing, immersion into the gelatin solution, and rinsing. The cycle was repeated n times to obtain a film of desirable thickness. After each step of the polyelectrolyte adsorption, the crystals were washed thoroughly with DI water and nitrogen-dried, and the QCM frequency change in air (f) was measured. The ∆f (frequency difference before and after adsorption) was used to determine the mass adsorbed after each immersion step according to the Sauerbrey equation: ∆f ) -Cf∆m where ∆f is the observed frequency changes in Hz, ∆m is the change in mass per unit area, in g/cm2, and Cf is the sensitivity factor for the crystal. The thickness of each layer can therefore calculated from the change in mass based on known crystal surface area. Multilayer Preparation on PLA Porous Scaffold and Scaffold Characterization. The PLA scaffolds were prepared using a particulate-leaching technique as described by Mikos et al.34 PLA/ethylene chloride solution was mixed with sodium chloride particles with 100-150 µm and then pour into a spherical mould to allow the evaporation of the solvent. After the ethylene chloride was dried completely, the PLA/ salt composite was put into water to allow salt leaching out of the scaffold. The 90% porosity scaffold was prepared to obtain uniformly interconnected-pore structure. To obtain uniform surface modification of PEI on the PLA scaffold, the scaffold was preimmersed into ethanol solution under vacuum to exclude the air in the scaffold, and then the ethanol was replaced by water, and after that, the water was replaced by PEI solution. The scaffold was activated by treating with PEI at the concentration of 1 mg/mL in DI water for 3 h, followed by washing repeatedly with water. The multilayer preparation on PLA porous scaffold was achieved as following. The dip started with 0.5 mg/mL gelatin solution for 40min, followed by rinse in water bath for 10 min, a 40 min dip in 1 mg/mL PEI, another rinse in water bath for 10 min again. After final assembly cycle, the scaffold with gelatin outlayer was immersed in 5 mg/mL solution of glutaraldehyde for 20 min to further fix the protein/polyion architecture for tissue engineering application. After rinsing with water, the scaffold was dried under vacuum for at least 48 h. Six bilayers of PEI/gelatin were deposited by alternative dip as above. Scaffold analysis was performed with scanning electronic microscopy (SEM), confocal laser scanning microscopy (CLSM), and radiolabeling technique. The dried scaffolds were sliced to show the inner part and sputtered with gold for the SEM investigation. Gelatin and PEI were conjugated with fluorescein isothiocyanate (FITC) and tetramethylrhodamine-isothiocyanate (TRITC) under sodium bicarbonate buffers and then purified, respectively. The conjugated PEI and gelatin were alternate deposited on PLA scaffold for confocal study. Before confocal investigation, the scaffold was sliced to show the inner part, though the confocal

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investigation can itself give deep scanning within the sample. The Iodogen method35,36 was used to iodinate the gelatin for the radiolabeling test of the PEI/gelatin assembly on the PLA scaffold. A detailed description of the radiolabeling of gelatin can be found in our previously work.30 The PLA scaffolds used for radio-iodination investigation were cut into 2 × 4 × 4 mm3, and the assembly process of PEI/gelatin was the same as what is described above, except that a radio-labeled gelatin solution was added. After each bilayer assembly, the scaffold was put into RIA tube and then count the aliquots for radioactivity in the γ counter. Cell Culture and Investigation. Chondrocytes from rabbit ear were isolated according to the method of Klagsbrum.37 The chondrocytes were grown in Ham’s F12 (Gibco.) supplemented with 10% fetal bovine serum (FBS, Sijiqin Biotech. Co., China, Lot number: 020613.2), 1.176 µg/mL NaHCO3, 0.3 µg/mL L-glutamic acid, 0.05 µg/mL vitamin C, 80 U/mL penicillin, and 100 µg/mL streptomycin. The scaffolds were sterilized in 75% ethanol and swollen in PBS and then placed into 24-well tissue culture polystyrene (TCPS) plates (NUCLON, Cat. No.167008), following the chondrocytes (300 × 104 cells/ml) injection and then vortex. The samples were kept at 37 °C in a humidified, 5% CO2 atmosphere. Following incubation, the scaffolds were washed twice with PBS to remove nonattached cells. Because of the difficulties in finding another standard porous scaffold as the control sample, the result from unmodified PLA scaffold was set as 100% control in our study to compare the result from modified PLA scaffold. For the cell assay on scaffolds in our study, the scaffolds with cells were first sliced into small pieces. These pieces were then put into the eppendorf tube with 1 mL of 1% Triton X-100 solution and ultrasonicated for 30 min to break cells. The solutions were collected for cell assays. The lysates were stored in eppendorf under -20 °C until use. For the total intracellular protein content test, BCA protein assay reagents38 were mixed with the lysate and the absorbency was measured by using microplate reader (BIO-RAD, model 550) at wavelength 570 nm. At least three similar scaffolds for unmodified and modified PLA scaffolds were taken for the cell tests. Cell activity was determined by an MTT assay,39 which is based on the mitochondrial conversion of the tetrazolium salt, 3-(4,5-dimethylthiazol-2-yl) 2,5-diphenyl tetrazolium bromide (MTT). Briefly, after the chondrocytes were incubated on different scaffolds in due time, the MTT solution (5 mg/ml in PBS) was added to each sample and incubated at 37 °C for 4 h for MTT formazan formation. The dissolution of the MTT formazan results in a purple solution, which can be measured by using microplate reader (BIORAD, model 550) at wavelength 490 nm. After seeding for 7 days, the scaffolds with cells were fixed in a 2.5% glutaraldehyde solution for at least 15 min and washed with PBS three times. The samples were then dehydrated using different graded ethanol and dried by critical-point drier for the SEM study. The cells on PLA scaffolds were also stained with fluorescein diacetate (FDA, Sigma) for confocal laser scanning microscopy (CLSM) (Bio-Rad, Radiance 2100; ZEISS AXIOVERT 200) investigation. FDA is an indicator of

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Figure 1. ζ-Potential of microcrystal as a function of polyelectrolyte layer number. The unmodified PLA microspheres do not has any charge on the surface. PEI layer shows +20 mV of ζ-potential and gelatin layer shows -19 mV of ζ-potential.

membrane integrity and cytoplasmic esterase activity.40 Enzymatic hydrolysis of the fluorogenic ester substrate of FDA results in the intracellular accumulation of the green fluorescent product fluorescein in cells with intact plasma membranes. Stock solutions were prepared by dissolving 5.0 mg/mL FDA in acetone. The working solution was freshly prepared by adding 5. 0µL of FDA stock solution to 5.0 mL PBS. 20 µL of an FDA solution was added in each well and incubated for 5 min. The substrates were then washed twice with PBS and placed on a glass slide for confocal examination. The 488 nm wavelength of the laser was used to excite the dye. Results and Discussion ζ-Potential Measurement. ζ-Potential measurements were utilized to follow the adsorption of the layers on the PLA microspheres. The microelectrophoretic mobility of the unmodified PLA microsphere, PEI-activated PLA microspheres, and subsequently polyelectrolytes (PEI/gelatin) of alternating charge, was measurement. Figure 1 shows the ζ-potential as a function of the polyelectrolyte coating layer number. The unmodified PLA microspheres do not have any charge on the surface as shown from Figure 1. It switches to positive charge after the PEI-activation exhibited a value of +20 mV. Furthermore, PEI/gelatin alternate surface yielded a ζ-potential of +20 mV and -19 mV, respectively. These data confirm the charging of the PLA microspheres surface through the adsorption of the polyelectrolytes. Quartz Crystal Microbalance (QCM) Measurements. The adsorbed mass of polyelectrolytes on the QCM crystal can be measured via the QCM frequency shift according to the Sauerbrey relation. According to the Sauerbrey equation, the coated PLA thin film was about 4.3 nm, and the activated PEI layer on PLA film was calculated as 1.2 nm. The following gradual deposition of PEI/gelatin alternate layer was calculated (film density was assumed 1 g/cm3 in this study) as near 0.8 nm and 2.85 nm respectively (Figure 2). The thickness of gelatin layer obtained from QCM measurement is in close agreement with the data we got from the radiolabeling method (2.24 nm).30

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Figure 2. QCM monitoring of the growth of PEI/gelatin 4bilayers on PLA-coated QCM electrode. The coated PLA film is about 4.3 nm. The increasing of the multiplayer is approximately 0.80 nm for PEI layer and 2.85 nm for gelatin layer.

Multilayer Assembly on PLA Porous Scaffold. The above results and our previous study30 indicated PEI aminolysis reaction can activate the PLA film to obtain stable positively charged surface that can be further used to deposit polyelectrolytes by ESA technique. This stable activated surface was obtained by the chemical reaction between PEI and PLA molecules based on the aminolysis of ester group.30 Since the aminolysis reaction is independent of the substrate shape, we can also construct positive charge surface on PLA macroporous scaffold via aminolysis. Furthermore, the layerby-layer technique is valid whatever the shape of the solid as reported by many literatures. The combination of PEI aminolysis and the layer-by-layer technique was then explored to construct extracellular matrix-like biomacromolecule coating onto 3-D porous scaffold. SEM Investigation. The PLA scaffold was prepared by salt-leaching method in our study with 90% porosity for tissue engineering application. It was therefore activited by PEI solution and deposited with PEI/gelatin multilayers. The unmodified PLA scaffold and PEI/gelatin assembly modified PLA scaffolds were investigated by SEM. It was found in Figure 3 that, from the microscale for tissue engineering application, there is no notable difference among these three PLA scaffolds considering about the porosity, pore diameter and scaffold integration. Confocal Investigation of the Scaffold. As a nanoscale surface modification and low concentration of the biomacromolecules used, it allows easy penetration of the polyelectrolytes into the scaffold and get uniform modification of the inner surface of the scaffold that will not affect the microscale scaffold structure. Comparing with the unmodified PLA scaffold imaged by rhodamine 6G (physicaladsorption dye; Figure 4a), it has uniform distribution of the rhodamine-labeled PEI all over the inner surface of PLA scaffold (Figure 4b). From the result of Figure 4c (investigated by dual-tunnel CLSM, in which the upper left green one is fluorescein isothiocyanate (FITC) labeled gelatin, the upper right red one is rhodamine labeled PEI, and the bottom one is the overlap image of the upper two.), we can see the scaffold modified by the ESA method presents a uniform

Figure 3. SEM photos of different PLA scaffolds with pore size 100150 µm and porosity 90%. [(a) Unmodified PLA, (b) six bilayers of PEI/gelatin assembly PLA scaffold].

PEI and gelatin distribution all over the inner surface of the scaffold. Radioiodination Investigation. The radio-labeling technique can also be used to investigate the protein assembly process on PLA scaffold via Iodogen method35,36 with high precision. The PLA scaffolds used for radio-iodination investigation were cut into 2 × 4 × 4 mm3, and the assembly process of PEI/gelatin was the same as what is described in the Experimental Section, except that a radio-labeled gelatin solution was added. After each bilayer assembly, the scaffold was put into RIA tube to test the radio-activity. Figure 5 showed the linear increasing of gelatin content with assembly cycle on PLA scaffold. An exception can be found at the first two bilayers, which showed much more gelatin adsorption than those of the later bilayers. The above results demonstrated that it could construct protein surface on biodegradable PLA 3-D tissue engineering scaffold via biomacromolecules electrostatic self-assembly. From the above result, we can conclude that the ESA technique was successfully applied to modifying PLA tissue engineering scaffold with biomacromolecules in our study. Chondrocyte was therefore selected as model system for testing the cell behavior on modified PLA scaffolds. Cell Behavior. Cell Viability Test. The chondrocytes viability in different PLA scaffold was measured by MTT method.39 Figure 6 showed the cell viability of chondrocytes

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Figure 5. Radio-labeling test of the gelatin assembly on PLA scaffold. (Scaffold volume: 2 × 4 × 4 mm3).

Figure 6. Chondrocytes viability in unmodified and 6bilayers PEI/ gelatin assembly modified PLA scaffold. Results represent mean(SD of triplicates from six separate experiments (P < 0.05).

Figure 4. Confocal laser scanning microscopy (CLSM) images of the cross-section part of different scaffold. ((a) Unmodified PLA scaffold stained by Rhodamine 6G, (b) PEI-activated PLA scaffold, PEI conjugated with tetramethylrhodamine-isothiocyanate (TRITC), (c) 4bilayers of PEI/gelatin modified PLA scaffold. The upper left one showed the gelatin conjugated with fluorescein isothiocyanate (FITC), the upper right one showed the PEI conjugated with TRITC, while the bottom image is the overlap of two upper images.)

in different scaffolds over a period of 7 days. It was found that the cell viability on PEI/gelatin assembly modified PLA scaffold is much higher than that on the unmodified PLA scaffold. The results of chondrocytes activity in PEI/gelatin assembly modified PLA scaffold is 171.1%, comparing with the control PLA scaffold as 100%. Total Intracellular Protein Content Measurement. The total intracellular protein content of the chondrocyte shows

Figure 7. Total protein content of chondrocyte in unmodified PLA scaffold and 6bilayers PEI/gelatin assembly modified PLA scaffold after 7 days. Results represent mean ( SD of triplicates from three separate experiments (P < 0.05).

the protein-synthesizing activity within the cells, which can reflect, to some extent, the proliferation ability of the chondrocyte in scaffold. Figure 8 showed the total protein content result of chondrocytes on unmodified PLA scaffold, PEI/gelatin assembly modified PLA scaffold after 7 days. It can be seen that the PEI/gelatin assembly modified PLA scaffold (897.0 µg/mL) showed much higher total protein content than that of unmodified PLA scaffold (568.3 µg/ mL), which indicated that the ECM-like surface on PLA

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Figure 8. SEM of the chondrocytes in different PLA scaffolds after 7days. Samples were prepared by critical-point drying. Arrows show the chondrocyte on the surface of scaffold. ((a) Unmodified PLA scaffold, (b) six bilayers PEI/gelatin assembly modified PLA scaffold.)

scaffold in our study could promote the proliferation of the chondrocytes and provide an ideal microenvironment for cells. SEM Investigation. Figure 8 showed that the chondrocytes attaching to different matrixes. After 7 days, it is very interesting to see that the chondrocytes cultured on the PEI/ gelatin assembly modified PLA film had undergone some degree of proliferation and full covered the inner surface of the modified PLA scaffold (Figure 8b). However, there are few chondrocytes on unmodified PLA scaffold, most of them maintained round morphology and not spreading completely (Figure 8a). Confocal Investigation. The living cells in different PLA scaffolds were stained by fluorescein diacetate (FDA) and investigated under confocal microscopy. The result showed that the chondrocytes with high density attaching at the inner surface of the PEI/gelatin assembly modified PLA scaffold (Figure 9b) are well spreading in the pore (Figure 9c). As a control, few chondrocytes penetrated into PLA scaffold, and most of them are clotted and not at a normal spreading status (Figure 9a). We proved in our investigation that, as illustrated in other related papers, it was possible to replace the polyelectrolyte with other proteins or polysaccharides such as collagen, alginate, or chitosan. More importantly, it has the potential

Figure 9. Chondrocytes in different PLA scaffold seeding after 7days and stained by fluorescein diacetate (FDA). [(a) PLA virgin scaffold, (b) six bilayers PEI/gelatin assembly modified PLA scaffold, and (c) enlarged image from (b) showed cells attached on the inner surface of one scaffold pore.]

to substitute the PLA substrate with other matrixes such as poly (glycolic acid) (PGA), poly(caprolactone) (PCL), poly(urethane), or poly(ortho ester). These very flexible systems allow broad medical applications for drug delivery and tissue engineering. Conclusion In summary, ζ-potential and QCM measurements verified the buildup of PEI/gelatin layers on PLA microsphere and

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film. Since both the aminolysis reaction and layer-by-layer assembly are independent of the substrate shape, the combination of the PEI aminolysis and the layer-by-layer technique was explored to construct extracellular matrix-like biomacromolecule coating onto 3-D porous scaffold. SEM, confocal microscopy, and radiolabeling technique investigation all indicated that gelatin was successfully anchored as an outlayer on the surface of 3-D PLA scaffold via a layerby-layer electrostatic self-assembly technique. The cytocompatible surface was proved to promote chondrocyte growth. The buildup is easy and the procedure can be adapted to the matrix with complex 3-D architecture. It has a high potential to replace the polyelectrolyte with other proteins or polysaccharides such as collagen, alginate, or chitosan to construct extracellular matrix-like surface. It also has high potential to substitute the PLA substrate with other matrixes such as poly(glycolic acid) (PGA), poly(caprolactone) (PCL), poly(urethane), or poly(ortho ester) containing an ester group on molecules, which offers a possible way to aminolysis these materials to get charged surface and further modified by layer-by-layer technique. The deposition of biomacromolecules represents a new, alternative solution for combining the good mechanical property of synthetic biomaterials with good cytocompatibility of natural biomacromolecules, thus opens new way to design tissue engineering scaffold with specific biological properties. Acknowledgment. This research was financially supported by Major State Basic Research Foundation of China (Grant No. G1999054305) and the Natural Science Foundation of China (NSFC-20174035). References and Notes (1) Sittinger, M.; Perka, C.; Schultz, O.; Haupl, T.; Burmester, G. R. Z Rheumatol. 1999, 58(3), 130-135. (2) Cima, L. G.; Vacanti, J. P.; Vacanti, C.; Ingber, D.; Moony, D.; Langer, R. J. Biomech. Eng. 1991, 113 (2), 143-151. (3) Quirk, R. A.; Chan, W. C.; Davies, M. C. Biomaterials 2001, 22 (8), 865-872. (4) Otsuka, H.; Nagasaki, Y.; Kataoka, K. Biomacromolecules 2000, 1 (1), 39-48. (5) Barrera, D. A.; Zylstra, E.; Lansbury, Jr., P. T.; Langer, R. J. Am. Chem. Soc. 1993, 115 (23), 11010-11011. (6) Quirk, R. A.; Davies, M. C.; Tendler, S. J. B.; Shakesheff, K. M.; Macromolecules 2000, 33 (2), 258-260. (7) Quirk, R. A.; Davies, M. C.; Tendler, S. J. B.; Chan, W. C.; Shakesheff, K. M. Langmuir 2001, 17 (9), 2817-2820. (8) Whitesides, G. M.; Ostuni, E.; Takayama, S.; Jiang, X.; Ingber, D. E. Annu. ReV. Biomed. Eng. 2001, 3, 335-373. (9) Holland, N. B.; Qiu, Y. X.; Ruegsegger, M.; Marchant, R. E. Nature 1998, 392 (6678), 799-801.

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