Bioactive Thin Film of Acidic Fibroblast Growth Factor Fabricated by

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Bioconjugate Chem. 2005, 16, 1316−1322

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Bioactive Thin Film of Acidic Fibroblast Growth Factor Fabricated by Layer-by-Layer Assembly Zhengwei Mao, Lie Ma, Jie Zhou, Changyou Gao,* and Jiacong Shen Department of Polymer Science and Engineering, Zhejiang University, Hangzhou 310027, China. Received October 10, 2004; Revised Manuscript Received June 24, 2005

A new class of bioactive thin films using growth factors as building blocks has been fabricated via layer-by-layer assembly (LBL) technique. Acid fibroblast growth factor (aFGF) in the presence of heparin was used as negatively charged polyelectrolytes, while poly(ethyleneimine) (PEI) was chosen as a positively charged counterpart. The self-deposition process and surface morphology of the resultant multilayers were monitored and detected by UV-vis absorbance spectra, advanced contact angle measurements, and scanning force microscopy (SFM) observations. Cell culture was performed to assess the efficiency of the growth factors. The fibroblasts proliferated faster on the surface assembled with five bilayers of (aFGF/heparin)/PEI with apparent higher cytoviability than on those surfaces modified by one bilayer of (aFGF/heparin)/PEI, five bilayers of aFGF/PEI, or five bilayers of heparin/ PEI, and tissue culture polystyrene. Enhanced secretion of collagen type I and interleukin 6 (IL-6) by the fibroblasts seeded on the five bilayers of (aFGF/heparin)/PEI was also verified by immunohistochemical examination. The bioactivity of the (aFGF/heparin)/PEI multilayers could be largely preserved when stored at -20 °C.

INTRODUCTION

The field of tissue engineering exploits living cells in a variety of ways to restore, maintain, or enhance tissues and organs (1). New developments in biomaterials, innovative cell culture technique, and newly discovered growth factors open novel avenues to engineering living tissues in vitro for fundamental research and clinical applications (2). Many kinds of polymers, e.g. poly(lactide) (PLA)1 and poly(lactide-co-glycotide) (PLGA), have been widely used in medical fields because of manipulable properties such as biodegradability and good mechanical properties (3). Yet their drawbacks such as poor cytocompatibility lead to inefficiency in constructing a bioactive interface with living cells. Layer-by-layer assembly (LBL) technique, which is based on the alternating physiosorption of oppositely charged polyelectrolytes, has been extended from fundamental studies to practical applications, in particular surface modification of biomaterials. Sequential adsorptions of anionic and cationic polyelectrolytes allow the construction of multilayer films on most types of substratum. The method has many important advantages over other techniques; for example, the assembly is based * E-mail: [email protected], Tel: +86-571-87951108, Fax: +86-571-87951948. 1Abbreviations: LBL, layer-by-layer; aFGF, acid fibroblast growth factor; PEI, poly(ethyleneimine); SFM, scanning force microscopy; PLA, poly(lactide); PLGA, poly(lactide-co-glycotide); ECM, extracellular matrix; FGFs, fibroblast growth factor family; bFGF, basic fibroblast growth factor; HMDA, hexamethylenediamine; EDAC, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide; NHS, N-hydroxysuccinimide; FCS, fetal calf serum; DMEM, Dulbecco eagle’s minimum essential medium; TCPS, tissue culture polystyrene; MTT, methylthiazoletetrazolium; CLSM, confocal laser scanning microscopy; FDA, fluorescein diacetate; IL, interleukin; PDGF, platelet-derived growth factor; NGF, tumor necrosis factor; TGF, transforming growth factor.

on spontaneous adsorptions and can proceed under mild conditions without involvement of harmful organic solvent, and the assembled molecular films exhibit a much larger thermal and mechanical stability and can be prepared up to hundreds of layers (4). Moreover, the method is valid whatever the shape and the inner structure of substratum. By this technique, many charged biopolymers including a lot of extracellular matrix (ECM) and extracellular matrix-like molecules have been incorporated for the purpose of ameliorating biocompatibility of polymers (5-7). Growth factors, which are generally soluble signaling proteins, play an important role in tissue regeneration and differentiation. Acidic fibroblast growth factor (aFGF) is a paradigm of a group of nine closely related, multifunctional proteins known as the fibroblast growth factor family (FGFs). FGFs induce mitosis, migration, and differentiation in most mesoderm and neuroectodermderived cells, influence angiogenesis, and regulate various biological responses (8-10). For instance, T. Imaizumi et al. find that 1 ng/mL of human recombinant basic fibroblast growth factor (bFGF, another paradigm of FGFs) in collagen gel can accelerate the proliferation of fibroblasts and decrease the contraction of collagen fibers induced by the fibroblasts (11). Owing to their powerful bioactivity, the FGFs are being tested to treat surgical, burn, and periodontal tissue wounds, gastric ulcers, segmental bony defects, and ligament and spinal cord injury (12). Toshi Fujisato et al. observed that the composite of chondrocytes and the collagen scaffold impregnated with bFGF is very promising for cartilage reconstruction (13). Ichiro Ono reports that administration of bFGF at the time of wound closure significantly increases the breaking strength after a full thickness incisional wound operation (14). But unfortunately, successful use of growth factors for human tissue regeneration has been notoriously difficult, since they typically have half-lives only on the order of

10.1021/bc049755b CCC: $30.25 © 2005 American Chemical Society Published on Web 08/25/2005

Technical Notes

minutes (15-17) and may cause uncontrolled cell growth when overdosed, as that which occurs in carcinoma (18, 19). To prolong the lives of growth factors and decrease their side effects, alot of controlled release systems for growth factors including microspheres, hydrogels, and matrixes have been widely explored in tissue engineering. Katsuya Kawai et al. reported that bFGF incorporated in gelatin microspheres is gradually released in artificial dermis, which exhibits a remarkably enhanced angiogenic effect to that of free bFGF (20). Xanthe M. Lam et al. encapsulated insulin-like growth factor-I in PLGA microspheres. The growth factor maintains its bioactivity and can be sustained release for up to 14 days in vivo (21). The heparin family is nearly ubiquitous in animal tissues as heparan sulfate proteoglycans on cell surfaces and in ECM. They possess a linear anionic polysaccharide chain and are typically heterogeneously sulfated on alternating L-iduronic acid and D-glucosamino sugars. Recently, Ram Sasisekharan et al. reported that the activation and protection of FGFs requires heparin-like molecules (22). Heparin and aFGF can form a complex in solution even at ultralow concentrations (∼pmol/L). Both the life and bioactivity of aFGF are improved in the complex. J. Feijen et al. immobilized heparin on collagen matrixes for the loading of bFGF. The incorporated bFGF exhibited sustained release over a prolonged period of time (23). Yoshihiro Ito et al. built micropattern-immobilized heparin for guiding cell growth. After treatment in FGF solution, they found that mouse fibroblasts have enhanced bioactivity on the heparin-immobilized regions (24). Herein, we intend to extend the LBL technique to build a new class of bioactive thin films by using growth factors as building blocks. aFGF with the protection of heparin is used as a negatively charged polyelectrolyte, while poly(ethyleneimine) (PEI) is chosen as the positively charged counterpart. Culture of fibroblasts in vitro demonstrates that the activity of aFGF in multilayers is largely preserved in the presence of heparin. This would provide a new strategy to immobilize cell growth factors in a more controllable manner, for example, the amount, stability, and durability of the growth factors. Because the charge interaction is nonspecific and not restricted to the macroscopic shape of the substratum, this method can also be extended to tissue regeneration scaffolds with porous and/or irregular inner structure. EXPERIMENTAL SECTION

Materials. Acidic fibroblast growth factor (aFGF, Mw )16 000, 3 mg/mL, in phosphate buffer solution) was kindly donated by Wanxing Bio-Pharmaceutical Co., Ltd. China. Poly(ethyleneimine) (PEI, Mw ) 750 000, 50 wt %) and heparin (sodium salt) were purchased from Sigma-Aldrich. All the chemicals were used as received. Triple distilled water was used throughout the experiments. aFGF (0.2 mg/mL) and PEI (1 mg/mL) were labeled with 1 mg/mL fluorescein isothiocyanate (FITC, Sigma) at 4 °C for 48 h, followed by dialysis with water for 4 weeks. To label the heparin molecules with FITC, hexamethylenediamine (HMDA) was first coupled to introduce active amino groups. For this to occur, 1 mg/mL heparin was reacted with 1 mg/mL HMDA (heparin: HMDA ) 1:100 mol) in 20 mM/10 mM 1-ethyl-3-(3dimethylaminopropyl)carbodiimide (EDAC) and N-hydroxysuccinimide (NHS) solution for 24 h, followed by dialysis with water for 1 week to obtain aminolyzed

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heparin, which was then labeled with FITC similarly to that of PEI. Multilayer Preparation. Multilayer thin films were deposited on a tissue culture polystyrene (TCPS) (Nunc, Denmark) sheet. The sheet was incubated in PEI solution (1 mg/mL with 0.15 M NaCl) for 20 min to adsorb a layer of PEI and then rinsed with phosphate buffer (PBS) three times. In the next step, the surface charge of the sheet was reversed by the adsorption of a layer of aFGF/ heparin mixture (aFGF 0.2 mg/mL and heparin 0.4 mg/ mL in PBS), followed by rinsing with PBS three times. Repeating this cycle produced self-organized multilayer films. Characterization of the Multilayers. UV-vis spectra were measured on a UV-visible spectrophotometer (CARY 100 BIO, America). Water contact angle was measured at room temperature using a sessile drop technique on a DSA10-MK2 contact angle measuring system from Kru¨ss. Advancing contact angle was measured to reveal the surface wettability. Before the measurement, all the samples were dried at 25 °C under reduced pressure overnight and then were equilibrated in ambient air for 1 day. The scanning force microscopy (SFM) image was obtained on a scanning probe microscope (SPA400, Seiko) in a dynamic mode. Human Fibroblasts Culture. The human fibroblasts were isolated from foreskins (25) and routinely cultured on TCPS. The cells of 5-10 passages were then seeded on the TCPS wells assembled previously with (aFGF/ heparin)/PEI multilayers, heparin/PEI multilayers, and aFGF/PEI multilayers as well as on the blank TCPS. The fibroblasts were incubated in a culture medium consisting of 10% (v/v) fetal calf serum (FCS, Sijiqing Biotech.Co., China) and 90% (v/v) DMEM (Gibcobrl Co.) supplemented with 100 units/mL of penicillin and 100 µg/mL of streptomycin in humidified air containing 5% CO2 at 37 °C. The culture medium was changed every 2 days. The cell numbers were averaged from three parallel measurements by trypsinization of the fibroblasts and counting the cell number under a hemocytometer at every 2 days, respectively. The cell viability was measured using methylthiazoletetrazolium (MTT) method according to refs (26, 27). The absorbance that has a proportional relationship with the number of living cells and cell viability is recorded at a wavelength of 570 nm. To observe the cell morphology under confocal laser scanning microscopy (CLSM, Bio-Rad 2100), the fibroblasts were stained with 5 µg/mL fluorescein diacetate (FDA, Aldrich) solution in the incubator for 15 min after being cultured for 8 days. Each value was averaged from three parallel experiments and expressed as mean(standard deviation. Measurement of Secreted Collagen Type I. After being cultured for 10 days, the fibroblasts were fixed with 4% paraformaldehyde/PBS solution for 30 min at room temperature, followed by five washings with PBS. Then they were challenged with rabbit polyclonal antibodies against human type I collagen (The immunoreagents were purchased from Zhongshan Golden Bridge Co., Ltd., Beijing, China) overnight at 4 °C. After washing with PBS, poly-HRP anti-mouse/rabbit IgG detection system was used to stain collagen type I (brown color). After rinsing with PBS, the specimens were observed under a microscope. Negative controls of the same specimen were incubated with rabbit normal serum or directly reacted with poly-HRP anti-mouse/rabbit IgG detection system in the absence of primary antibody, and then they were processed as outlined above (28). No positive immunoreactivity was found in these negative controls.

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Technical Notes

Figure 1. (a) UV-vis spectra of FITC-aFGF to show the layer growth process of (aFGF/heparin)/PEI multilayers. (b) Absorbance maximum at 490 nm to show the linear increase of layer thickness. The multilayers were deposited directly onto TCPS surfaces. For detail, see the text.

Measurement of Released Interleukin 6. Secretion of the interleukin 6 (IL-6) was measured in the supernatant of the cultured fibroblasts by IL-6 enzyme linked immunosorbent assay (ELISA, The Fourth Military Medical University, Xi’an, China). Spectrophotometric readings were performed at 405 nm. For detail measuring process, see refs 29 and 30. Final results were obtained by comparison with a standard curve constructed using dilutions of standard human IL-6. Each value was averaged from three parallel experiments. The Stability of the Multilayers. Multilayer thin films deposited on the TCPS sheet were incubated in PBS at 37 °C. The normalized maximum absorbance at 490 nm was plotted as a function of incubation time to approximately depict the reservation of the building blocks. Each value was averaged from three parallel experiments. Evaluation of Multilayer Activity after Storage. Five bilayers of (aFGF/heparin)/PEI were built on TCPS and then were incubated in culture medium at 37 °C for 24 h or 48 h, or stored at -20 °C for 3 months. Fibroblast cultures were similarly performed and evaluated as described above. Each value was averaged from three parallel experiments. RESULTS AND DISCUSSION

Monitoring the LBL Deposition Process. In each LBL assembly, one of the normal aFGF, heparin, or PEI was substituted by a FITC-labeled counterpart for the purpose of monitoring layer growth by UV-vis spectroscopy. Total of three kinds of multilayer films were assembled with only one labeling component in each film. The spectra of all the multilayers exhibit a maximum absorbance at 490 nm arising from the fluorescein groups (Figure 1a). The maximum absorbance of all the labeled components increased linearly along with the increase of layer number (Figure 1b), demonstrating the homogeneous growth of the layer thickness or the total amount of the building blocks. Interestingly, the slopes of each component are different, with PEI being the sharpest and aFGF being the flattest. This would mean that the ratio of heparin to aFGF in the multilayers increased along with the layer number, e.g. more heparin will be incorporated into the multilayers compared with aFGF. However, this would not mean that the relative amount of PEI in each layer is larger than that of the negative components, since it equilibrates with both negative building blocks. Theoretically, the total absorbance of the

Figure 2. Advancing contact angle as a function of the layer number of (aFGF/heparin)/PEI multilayers. Odd numbers represent films with PEI as the outermost layer, whereas even number films have aFGF/heparin as the outermost layer.

negative layer can be derived from the summary of the corresponding value of heparin and aFGF, since the multilayers are assembled under identical conditions. The summarized values (dashed line in Figure 1b) indeed exhibit the same slope as that of the polycation, demonstrating the steady increase of both positive and negative layer thickness. The reason the relative mass of heparin increased faster than aFGF is not very clear at present. One possible reason is that the stronger charge property of heparin arises from its sulfate groups. The surface wettability of the sequentially layered PEI and aFGF/heparin as a function of layer number (Figure 2) also confirmed the occurrence of LBL assembly. The measurement of contact angle has been widely employed to diagnose the surface chemistry of synthesized surfaces such as a self-assembled monolayer. The surface wettability measured by an advancing contact angle is controlled primarily by the outermost layer if the layer is uniform (31, 32). Thus, it is possible to monitor the deposition process by determining how surface wettability changes in a LBL manner. In the present case, samples with an odd number of layers have PEI as the outermost layer, whereas samples with an even number of layers have aFGF/heparin as the outermost layer. The advancing contact angle of the raw TCPS sheet was determined to be 69.1°. Figure 2 shows that the contact angles of the (aFGF/heparin)/PEI multilayers displayed

Technical Notes

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Figure 3. SFM images of (a) the TCPS surface, (b) deposited by one bilayer of (aFGF/heparin)/PEI and (c) by five bilayers of (aFGF/ heparin)/PEI.

Figure 4. (a) Fibroblast proliferation and (b) cell viability seeded on multilayers (PEI being the outmost layers) or TCPS control.

a distinct oscillation as the outermost layer changed from PEI to aFGF/heparin, demonstrating the increased hydrophilicity of samples having aFGF/heparin as the outermost layer than that with PEI. However, the same outermost layer did not present the same contact angle. There was some fluctuation in a range of about 3-7°. This might be caused by the layer interpenetration between neighboring PEI and aFGF/heparin layers where the segments of the underlying layer are able to influence the surface hydrophilicity (32). It is worth noting that a more hydrophobic surface was found when heparin was partially substituted by aFGF, since a contact angle of 42° was measured for (aFGF/heparin)/PEI multilayers (the outmost layer is aFGF/heparin) and 28° for PEI/ heparin multilayers (the outmost layer is heparin), respectively (33). This should be caused by the fact that aFGF is comparatively more hydrophobic than heparin. It is known that the surface morphology of biomaterials also has a big influence on cell attachment, proliferation, and function in addition to the surface chemistry. Figure 3 shows that there exist minimal fluctuations on both the TCPS and the multilayer surface, which are in the range of nanometers as revealed by the roughness (RMS) measurements. The RMS increased from 1.38 nm (TCPS) to 1.90 nm (one bilayer) and to 4.52 nm (five bilayers). Some aggregates appeared on the first bilayer (Figure 3b), while some holes in a range of nanometers could be visualized after deposition of five bilayers (Figure 3c). This would mean that defects were formed either during layer assembly or drying process, or both. Yet compared with the fluctuation of the original TCPS, the alteration of the surface morphology after multilayer deposition is

rather minimal. It is worth noting that these images measured in the dry state could only reflect the surface morphology to some extent, since multilayer swelling cannot be avoided in hydrated state, e.g. in the cell culture medium. Cell Response to the (aFGF/Heparin)/PEI Multilayers. The aFGF built in the multilayers provides the possibility of promoting the mitosis of cells and inducing a series of signal pathways. Exemplified here with human fibroblasts, the cell culture results indeed showed a more positive cell response to the aFGF-assembled films in the presence of heparin (Figure 4). Both the cell proliferation ratio and the viability were substantially improved on the surface assembled with five bilayers of (aFGF/heparin)/PEI (with PEI being the outmost layer if not otherwise indicated), especially at longer culture time, while the multilayers composed of five bilayers of heparin/PEI exhibited somewhat improvement of cell proliferation and viability. On the other hand, during all the culture time no significant difference was found between the surfaces modified with five bilayers of aFGF/ PEI, one bilayer (aFGF/heparin)/PEI, and an unmodified one, i.e., TCPS control. The results that no difference was found between the aFGF/PEI and TCPS control would mean that the aFGF had lost its effect upon being assembled into the multilayers, which most possibly caused by the denaturation of aFGF or tight bonding (see below). Therefore, a strategy to ensure its bioactivity should be adopted during the LBL assembly process. We used here the heparin as a co-building block with aFGF. In the presence of heparin (22-24), the aFGF could actually act on the

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Technical Notes

Figure 5. The morphology of fibroblasts observed under CLSM after being cultured for 8 days and stained with FDA. (a) Deposited by five bilayers of (aFGF/heparin)/PEI, (b) deposited by five bilayers of heparin/PEI, and (c) TCPS control.

Figure 6. The immunohistorical stain of collagen type I secreted by fibroblasts after cultured for 8 days. (a) Deposited by five bilayers of (aFGF/heparin)/PEI, (b) deposited by five bilayers of heparin/PEI, and (c) TCPS control.

fibroblasts as shown in Figure 4. It is worth noting that PEI has similar ability to support cell attachment and growth as TCPS since no difference was observed for PEIcovered TCPS and TCPS control in our results (data not shown). At the first 2 days, the fibroblasts cultured on five bilayers of (aFGF/heparin)/PEI did not show apparent difference with others in terms of cell number and viability. This may attribute to the facts that it takes 12 to 20 h for the fibroblasts to attach and spread on the culture substrate, and the main function of aFGF is inducing cell mitosis rather than cell attachment and spread. At longer culture time, the aFGF can then act on the fibroblasts to yield a higher proliferation ratio and viability than others. The fibroblasts cultured on five bilayers of (aFGF/heparin)/PEI for 10 days eventually reached 2.5 times the cell number and 3.1 times the MTT absorbance than that of TCPS control, respectively. However, aFGF in only one bilayer of (aFGF/heparin)/ PEI had not shown an effect on acceleration of fibroblast growth. This is attributed to the minimal amount of aFGF that existed on the material surface, leading to no obvious effect on the cells. The fibroblasts cultured on the heparin/PEI multilayers also presented a higher proliferation ratio and viability. Heparin is also a kind of active factor which can stimulate cell proliferation at low concentration (9). Observed under CLSM after culturing for 8 days, all the fibroblasts were spread well with their typical shapes. The cells on the five bilayers of (aFGF/heparin)/PEI displayed the highest density, and most of them reached confluence (Figure 5). Hence, one can conclude that the fibroblasts presented a larger number, higher viability, and normal morphology on the surface modified with five bilayers of (aFGF/heparin)/PEI than those on the heparin/PEI multilayers and TCPS control. A regulated synthesis of interstitial collagens, the most abundant being type I collagen, is important during

development and wound healing but also in a number of pathological conditions. Studies on these diseases as well as various in vitro models have shown that collagen synthesis and deposition are influenced by cytokines, growth factors, and mechanical tension (34, 35). aFGF is one of the important regulatory factors for collagen synthesis in cultured fibroblasts. In the present case, human collagen type I (brown color, Figure 6) secreted by fibroblasts on all the substrates existed in the extracelluar matrix, in the cytoplasm, and in the nucleus. However, fibroblasts on the five bilayers of (aFGF/ heparin)/PEI obviously secreted the greatest extent of collagen type I (Figure 6a), proving that aFGF built in the multilayers in the presence of heparin can stimulate the synthesis of collagen type I in fibroblasts. aFGF not only induces fibroblast proliferation and influences collagen synthesis, but also acts as a multifunctional regulator of other water soluble factors such as interleukins (ILs), platelet-derived growth factor (PDGF), tumor necrosis factors (TNFs), and transforming growth factors (TGFs), etc. (36). Interleukin 6 (IL-6) which can be produced by human fibroblasts is a molecule that has both proinflammatory and antiinflammatory function, a modulator of bone resorption, a promoter of hematopoiesis, and an inducer of plasma cell development (37). Figure 7 shows that at first 4 days, fibroblasts on the surface covered by five bilayers of (aFGF/heparin)/ PEI produced 2.9 times IL-6 of others. This value decreased to 1.3 at a still longer culture time. This is attributed to the limited life of aFGF which is only a few days in vitro/in vivo even stabilized by heparin. Although heparin can accelerate cell proliferation, very similar IL-6 secretion level was determined on both surfaces of heparin/PEI multilayers and unmodified TCPS, implying that heparin cannot stimulate the synthesis of IL-6 in human fibroblasts. The (Bio)stability of the Multilayers. The stability of the multilayers in the culture conditions takes an

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Technical Notes

Figure 7. The IL-6 secretion of fibroblasts cultured on TCPS assembled with five bilayers of (aFGF/heparin)/PEI, five bilayers of heparin/PEI, or TCPS control.

46% aFGF and 28% heparin had been released from the (aFGF/heparin)/PEI multilayers after incubated for 48 h, respectively. By contrast, heparin in heparin/PEI multilayers was rather stable. This proved that the structure of the (aFGF/heparin)/PEI multilayers was largely destroyed in a few days. As PEI/heparin multilayers were stable over several weeks (33), the damage of the (aFGF/heparin)/PEI multilayers must be caused by the introduction of aFGF. Therefore, this instability should be reasonably caused by the weak binding strength of PEI and aFGF/heparin complex. Consequently, the multilayers may swell in the buffer to enable the release of the building blocks, noting that hydrolysis of the biomacromolecules might also have partial contribution. Hence, suitable storage is of practical importance since the materials may not be used immediately in many cases. Several storage conditions were determined with respect to bioactivity reservation. Figure 9 shows that stored at -20 °C even for 3 months the fibroblasts on the multilayers still behaved like those on the freshly made substrate, although a limited decrease in proliferation ratio and viability was detected. This result indicates that -20 °C is a suitable storage condition to keep the activity of the thin films. However, when the multilayers were incubated in vitro for 24 h, the activity largely decreased. A still longer incubation, e.g. for 48 h, had deteriorated the bioactivity dramatically, whose value was even lower than freshly made heparin/PEI multilayers. Besides the partial dissociation of the bioactive components from the multilayers, the denaturing of the native factors may also be one important reason. CONCLUSIONS

Figure 8. The normalized Abs as a function of incubation time in PBS at 37 °C.

important role in retaining their function. The multilayers were incubated in PBS at 37 °C to mimic the culture conditions. Figure 8 shows that the absorbance of the multilayers was reduced gradually as a function of incubation time, revealing that aFGF and heparin were released into the solution. For example, approximately

We show here that the bioactive aFGF has been successfully deposited onto the TCPS sheet surface in the presence of heparin via a layer-by-layer manner. The aFGF built in the multilayers obviously enhances fibroblast proliferation and viability and regulates the secretion of collagen type I and IL-6. The bioactivity of the multilayers can be largely preserved when stored at -20 °C even for 3 months, while being diminished in vitro after a few days. In conclusion, herein a practical and simple technique has been provided to immobilize bioactive growth factors on a material surface with preserved bioactivity.

Figure 9. (a) Fibroblast proliferation and (b) cell viability seeded on five bilayers of (aFGF/heparin)/PEI, which were stored at different conditions previously.

1322 Bioconjugate Chem., Vol. 16, No. 5, 2005 ACKNOWLEDGMENT

We thank Prof. H. Mo¨hwald, Prof. M. Grunze, and W. J. Tong for their stimulating discussion. This work was financially supported by the Science and Technology Program of Zhejiang Province (2004C21022), the Natural Science Foundation of China (20434030, 90206006), and the National Science Fund for Distinguished Young Scholars (50425311). LITERATURE CITED (1) Griffith, L. G., and Naughton, G. (2002) Tissue engineeringcurrent challenges and expanding opportunities. Science 295, 1009-1014. (2) Sittinger, M., Perka, C., Schutz, O., Haupl, T., and Burmester, G. R. (1999) Joint cartilage regeneration by tissue engineering. Z. Rheumatol. 8, 130-135. (3) Seal, B. L., Otero, T. C., and Panitch, A. (2001) Polymeric biomaterials for tissue and organ regeneration. Mater. Sci. Eng. R. 34, 147-230. (4) Chen W., McCarthy T. J. (1997) Layer-by-layer deposition: A tool for polymer surface modification.Macromolecules 30, 78-86. (5) Zhu, Y. B., Gao, C. Y. He, T., Liu, X. Y., and Shen, J. C. (2003) Layer-by-layer assembly to modify poly(L-lactic acid) surface toward improving its cytocompatibility to human endothelial cells. Biomacromolecules 4, 446-452. (6) Elbert, D. L., Herbert, C. B., and Hubbell, J. A. (1999) Thin polymer layers formed by polyelectrolyte multilayer techniques on biological surfaces. Langmuir 15, 5355-5362. (7) Grant, G. G. S., Koktysh, D. S., Yun, B., Matts, R. L., and Kotov, N. A. (2001) Layer-by-layer assembly of collagen thin films: controlled thickness and biocompatibility. Biomed. Microdevices 3, 301-310. (8) Bartletl, P. F., Brooker, G. J., and Faux, C. H. (1998) Regulation of neural stem cell differentiation in the forebrain. Immunol. Cell. Biol. 76, 414-418. (9) Rcsengart, T. K., Budenbender, K. T., and Duenas, M. (1997) Therapeutic angiogenesis: a comparative study of the angiogenic potential of acidic fibroblast growth factor and hepain. J. Vasc. Surg. 26, 302-312. (10) Szabo, S., and Sandor, Z. (1996) Basic fibroblast growth factor and PDGF in GI disease. Gastroenterol. Clin. N. 10, 97-112. (11) Imaizumi, T., Jean-Louis, F., Dubertret, M. L., Bailly, C., and Cicurel, L. (1996) Effect of human basic fibroblast growth factor on fibroblast proliferation, cell volume, collagen lattice contraction: in comparison with acidic type. J. Dermatol. Sci. 11, 134-141. (12) Yoneda, A., Asada, M., Oda, Y., Suzuki, M., and Imamura, T. (2000) Engineering of an FGF-proteoglycan fusion protein with heparin-independent, mitogenic activity. Nature Biotechnol. 18, 641-644. (13) Fujisato, T., Sajiki, T., Qiang, L., and Ikada, Y. (1996) Effect of basic fibroblast growth factor on cartilage regeneration in chondrocyte-seeded collagen sponge scaffold. Biomaterials 17, 155-162. (14) Ono, I. (2002) The effect of basic fibroblast growth factor (bFGF) on the breaking strength of acute incisional wounds. J. Dermatol. Sci. 29, 104-113. (15) Kim, D. C., Sugiyama, Y., Satoh, H., Fuwa, T., Iga, T., and Hanano, M. (1988) Kinetic analysis of in vivo receptordependent binding of human epidermal growth factor by rat tissues. J. Pharm. Sci. 77, 200-207. (16) Konturek, S. J., Pawlik, W., Mysh, W., Gustaw, P., Sendur, R., Mikos, E., and Bielanski, W. (1990) Comparison of organ uptake and disappearance half time of human epidermal growth factor and insulin. Regul. Pept. 30, 137-148. (17) Edelman, E. R., Nugent, M. A., and Karnovsky, M. J. (1993) Perivascular and intravenous administration of basic fibroblast growth factor: vascular and solid organ deposition. Proc. Natl. Acad. Sci. U.S.A. 90, 1513-1517. (18) Aaronson, S. A. (1991) Growth factors and cancer. Science 1146-1153. (19) Kliln, J. G., Berns, P. M., Schmitz, P. I., and Foekens, J. A. (1992) The clinical significance of epidermal growth factor

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