J. Biomater. Sci. Polymer Edn, Vol. 19, No. 5, pp. 677– 691 (2008) Koninklijke Brill NV, Leiden, 2008.
Also available online - www.brill.nl/jbs
Electrospun chitosan-P(LLA-CL) nanofibers for biomimetic extracellular matrix FENG CHEN 1 , XIAOQIANG LI 2 , XIUMEI MO 1,2,∗ , CHUANGLONG HE 1 , HONGSHENG WANG 1 and YOSHITO IKADA 3 1 Institute
of Biology Science and Biotechnology, Donghua University, 2999 North Renmin Road, Songjiang District, Shanghai 201620, P.R. China 2 College of Material Science and Engineering, Donghua University, Shanghai 201620, P.R. China 3 Department of Indoor Environmental Medicine, Nara Medical University, Nara, Japan Received 6 April 2001; accepted 20 September 2007 Abstract—Chitosan-poly(L-lactic acid-co-ε-caprolactone)(50:50) (P(LLA-CL)) (CS/P(LLA-CL)) blends were electrospun into nanofibers using 1,1,1,3,3,3-hexafluoro-2-propanol (HFIP) and trifluoroacetic acid (TFA) as solvents. Chitosan, which is difficult to electrospin into nanofibers, could be easily electrospun into nanofibers with addition of a small portion of P(LLA-CL). The fiber diameter depended on both the polymer concentration and the blend ratio of chitosan to P(LLA-CL). The average fiber diameter increased with increasing polymer concentration and decreasing the blend ratio of chitosan to P(LLA-CL). X-ray diffractometry (XRD) and Fourier-transform infrared (FT-IR) spectra were measured to characterize blended nanofibers. The porosity of CS/P(LLA-CL) nanofiber mats increased with increasing the weight ratio of chitosan to P(LLA-CL), while both the tensile strength and the ultimate strain increased with increasing P(LLA-CL) ratio. Fibroblast cell growth on nanofiber mats were investigated with MTT assay and scanning electron microscope (SEM) observation. The highest cell proliferation was observed on the nanofiber mats when the weight ratio of chitosan to P(LLA-CL) was 1:2. As SEM images shown, fibroblast cells showed a polygonal shape on blend nanofiber mats and migrated into the nanofiber mats. Key words: Electrospinning; chitosan; poly(L-lactic acid-co-ε-caprolactone); biomimetic extracellular matrix; tissue engineering.
INTRODUCTION
Native extracellular matrix (ECM) is a chemically and physically cross-linked complex network of three important classes of biomacromolecules secreted locally by cells. These are fibrous proteins, proteoglycans and non-fibrous proteins. In a ∗ To
whom correspondence should be addressed.
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typical connective tissue, structural fibers such as collagen fibers and elastin fibers have diameters ranging from several ten to several hundred nanometers. Nanoscale fibers entangle with each other to form a non-woven mesh that provides tensile strength and elasticity for the tissues. It does much more than just provide a physical support for cells, it provides a substrate with specific ligands for cell adhesion and migration, and regulates cellular proliferation and function by providing various growth factors [1]. Biomimetic ECM has been widely used in tissue engineering, wound healing, drug delivery system, gene therapy and so on. The key factors of biomimetic ECM are to create a three-dimensional scaffold with nanoscale fibers which have a suitable degradation rate and mechanical and chemical properties. Three methods have been used to produce such nanoscale polymeric fibers, including electrospinning, self-assembly and phase separation. Among them electrospinning is the most simple and efficient technique [1]. Both synthetic and naturally polymers have been used for fabrication of nanofibrous scaffolds by the electrospinning method, such as poly(ε-caprolactone) (PCL) [2], poly(DL-lactide) (PDLLA), poly(L-lactide) (PLLA) [3], polyglycolide (PGA) [4], collagen [5, 6], elastin [7], hyaluronic acid [8], fibrinogen [9], gelatin [10], chitosan [11], P(LLA-CL) [12] and collagen-elastin [13]. It has been found that nanofibers made from natural polymers are more positive than these from synthetic polymers in the interaction between cells and biomimetic ECM, whereas the latter have more favorable mechanical characteristics than the former. It is reasonable to expect that an ideal biomimetic ECM should mimic both the mechanical characteristic and the chemical composition of the native ECM. Therefore, many kinds of composite nanofibers have been fabricated by electrospinning, such as collagen-blended P(LLA-CL) [14], polyaniline-contained gelatin [15] and chitosan/PEO [16]. P(LLA-CL) is a co-polymer of LLA and CL. It has been investigated as biomaterial for surgery and drug-delivery systems due to its good biodegradability [17]. Chitosan is biologically renewable, biodegradable, non-antigenic and biocompatible [18], and has been used in wound healing [19], drug-delivery carrier [20] and tissue-engineering applications [21 – 23]. However, no use has been reported of chitosan and P(LLA-CL) blend nanofibers. In this study, the blend of CS/P(LLA-CL) is used to fabricate electrospun nanofiber mats. The morphology, X-ray diffractometry (XRD) and Fouriertransform infrared (FT-IR) spectra, porosity and mechanical performance of blended nanofibers, as well as fibroblast cell behavior on the mats were investigated. MATERIALS AND METHODS
Materials A co-polymer of P(LLA-CL)(50:50), which has a composition of 50 mol% L -lactide, was used. Squid pen chitosan, with a deacetylation degree of 60%,
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was purchased from Fine Chemical Sales Carbohydrate Chemistry Team Industrial Research (New Zealand). 1,1,1,3,3,3-Hexafluoro-2-propanol (HFIP) was obtained from Daikin (Japan). Trifluoroacetic acid (TFA) was purchased from Shanghai Runjie Chemical Reagent (China). Mouse fibroblasts were obtained from institute of biochemistry and cell biology (Chinese Academy of Sciences, China). Unless specifically explained, all culture media and reagents were purchased from Gibco Life Technologies (USA). Preparation of solution for electrospinning Chitosan was dissolved in HFIP and TFA (9:1, v/v), while P(LLA-CL) was dissolved in pure HFIP. After the two solutions were prepared, they were mixed together at different blend ratios and stirred for 30 min for electrospinning. Nanofiber fabrication The prepared polymer solutions were fed into a plastic syringe with a needle (inner diameter 0.21 mm). A syringe pump (789100C, Cole-Palmer, Germany) was used to feed the solutions to the needle at a feed rate of 1.0 ml/h. An electrospinning voltage of 20 kV was applied to the needle using a high-voltage power supplier (BGG6-358, BMEI, China). A grounded collection plate of aluminum foil was located at a fixed distance, 13 mm from the needle tip. Morphology Morphology of the nanofibers was observed using a scanning electron microscope (SEM, JSM-5600, Jeol, Japan) at an accelerated voltage of 15 kV. The diameter range of the fabricated nanofibers was measured based on SEM images using an image visualization software ImageJ developed at Upper Austria University of Applied Sciences. XRD pattern The samples were tested using an X-ray diffraction instrument (D/MAX-2550PC, Rigaku, Japan) under the condition of Cu Kα1, 40 kV and 300 mA. In this work, chitosan, P(LLA-CL) as-received and electrospun CS/P(LLA-CL) fibers with different ratios chitosan to P(LLA-CL) were studied using XRD. FT-IR spectra Chitosan raw materials, chitosan nanofibers, P(LLA-CL) nanofibers and CS/P(LLA-CL)(1:1) nanofibers were prepared for the FT-IR test on an AVATAR 380 FT-IR instrument (Therm Electron, USA).
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Porosity of nanofibrous mats Porous blended CS/P(LLA-CL) nanofiber mats were fabricated at a concentration of 8 wt% and different blend ratios. The thickness of the nanofiber mats was changed by the collection time and measured with a micrometer. The apparent density and porosity of nanofiber mats were calculated according to the following equations [24]: Apparent density (g/cm3 ) =
Mat mass (mg)
, Mat thickness (µm) × Mat area (cm2 ) Apparent density (g/cm3 ) × 100%. Porosity (%) = 1 − Bulk density of raw P(LLA-CL)(g/cm3 ) Mechanical measurements Mechanical measurements were achieved by applying tensile test loads to specimens prepared from the electrospun fine non-woven nanofiber mats at a concentration of 8 wt% and different blend ratios of chitosan to P(LLA-CL) (4:1, 3:2, 2:3, 1:4, 0:1). In this study, five specimens were prepared according to the method described by Huang et al. for each proportion [10]. First, a white paper was cut into templates with width × gauge length = 10 mm × 50 mm and double-sided tapes were glued onto the top and bottom areas of one side. Secondly, the aluminum foil was carefully peeled off and single side tapes were applied onto the gripping areas as end-tabs. The resulting specimens had a planar dimension of width × gauge length = 10 mm × 30 mm. Mechanical properties were tested by a materials testing machine (H5K-S, Hounsfield, UK) at 20◦ C, relative humidity of 65% and a elongation speed of 10 mm/min. Viability and morphology study of fibroblasts on nanofiber mats Fibroblast cells were cultured in DMEM medium with 10% fetal calf serum, 100 units/ml penicillin and 100 units/ml streptomycin in humidified incubator with a 5% CO2 at 37◦ C, and the medium was replaced every 3 days. The coverslips (14 mm in diameter) were put onto the aluminum foil to collect the electrospun nanofiber mats fabricated with 8 wt% solution. The nanofiber mats were vacuum dried in a vacuum oven for a week to release the residual solvents. Then the mats were fixed in 24-well plates with stainless rings and sterilized with 75% alcohol solution, which was replaced with phosphate-buffered saline (PBS) solution after 2 h for washing. For the cell viability test, fibroblasts were seeded onto the coverslips and nanofiber mats with different ratios of chitosan to P(LLA-CL) at a density of 1.5 × 104 cells/cm2 . At 24, 48 and 96 h after cell seeding, unattached cells were washed out with PBS solution and attached cells amounts were evaluated by MTT kit (C0009, Beyotime Institute of Biotechnology, China) and Enzyme-labeled Instrument (MK3, Thermo, USA).
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Fibroblasts were seeded onto the coverslips and nanofiber mats at a density of 5.0 × 104 cells/cm2 for cell morphology by SEM (S-2700, Hitachi, Japan). The cells cultured on the coverslips and the mats were washed with PBS and then fixed with 4% glutaraldehyde for 45 min at 4◦ C. Thereafter, the samples were dehydrated in 50, 75 and 100% alcohol solutions, and dried under vacuum. The samples were sputter coated with gold and observed with SEM at a voltage of 15 kV. Statistical analysis Statistical analysis was performed using Origin 7.5 (OriginLab, USA). Statistical comparisons were made by analysis of one-way ANOVA. In all evaluations, P < 0.05 was considered as statistically significant.
RESULTS AND DISCUSSION
Morphology and diameter change of CS/P(LLA-CL) nanofibers The dependence of the electrospun CS/P(LLA-CL) nanofiber diameter on the blend ratio is shown in Fig. 1A–F, where the SEM micrographs of CS/P(LLACL) nanofibers electrospun from 6 wt% solutions at blend ratios of chitosan to P(LLA-CL) from 10:0 to 0:10 are given. 6 wt% chitosan solution in HFIP/TFA (9:1, v/v) was difficult to be spun into fibers with only beans formed as in Fig. 1A, while ultrafine fibers were spun out when chitosan solution was mixed with a small portion of P(LLA-CL) at CS/P(LLA-CL) = 8:2 (Fig. 1B). Fibers could be more easily fabricated from mixed solution with increasing blend ratio of P(LLA-CL) to chitosan. Figure 2 shows the relationship between the average diameter of nanofibers and the blend ratio of chitosan to P(LLA-CL). It can be seen that the fiber diameter varied with the blend ratio. The average diameter decreased from 124 to 426 nm with decreasing the blend ratio of chitosan to P(LLA-CL) from 8:2 to 0:10. A similar result was reported in the electrospinning of chitin/silk fibroin blend nanofibers. In this case, the average diameter of blended nanofibers decreased gradually from 1260 to 130 nm with decreasing the chitin content in the blends [25]. The gradual decrease in the fiber diameter with addition of chitosan in the blends can be explained by the conductivity increase of electrospinning solutions. The addition of salts (e.g., NaCl and KH2 PO4 ) to polymer solutions has found to affect the fiber diameter. The presence of charged ions in solution caused a substantial decrease in fiber diameter [3]. Chitosan has higher polarity compared to P(LLA-CL), and the formation of salts between TFA molecules and the amino groups of chitosan is thought to occur in the process of dissolution of chitosan in TFA [26]. The complex solution may have higher conductivity than the P(LLA-CL) solution, and the conductivity may increase with the increasing chitosan content. Thus, the diameter of blended fibers decreased with increasing chitosan to P(LLACL) blend ratio.
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Figure 1. SEM images of electrospun CS/P(LLA-CL) nanofibers (A–F, 6 wt%; G, 4 wt%; H, 8 wt%). (A) CS/P(LLA-CL) = 1:0, (B) CS/P(LLA-CL) = 8:2, (C) CS/P(LLA-CL) = 6:4, (D) CS/ P(LLA-CL) = 4:6, (E) CS/P(LLA-CL) = 2:8, (F) CS/P(LLA-CL) = 0:1, (G) CS/P(LLA-CL) = 1:1, (H) CS/P(LLA-CL) = 1:1.
The effect of CS/P(LLA-CL) solution concentration on the fiber diameter was investigated by changing the concentration from 4 to 8 wt% at the same blend ratio of chitosan to P(LLA-CL) (1:1). Figure 1G and H shows SEM micrographs of the nanofibers electrospun from 4 and 8 wt% solution. Nanofiber diameter was calculated from 100 nanofibers in the SEM micrographs. As seen from Fig. 3, with the decrease of CS/P(LLA-CL) concentration from 8 to 4 wt%, the average fiber diameters reduced from 370 to 223 nm with the same electrospinning parameters. The electrospinning jet with lower polymer concentration certainly gives thinner fiber after solvents evaporated during electrospinning. A similar result
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Figure 2. Dependence of the diameter of electrospun CS/P(LLA-CL) fibers on electrospinning of blends of different ratios.
Figure 3. Dependence of the diameter of electrospun CS/P(LLA-CL) fibers on the concentration of electrospinning solution.
was observed in the electrospinning of P(LLA-CL) [12], gelatin [27] and many other electrospinning systems. XRD study Figure 4 shows XRD patterns of chitosan, P(LLA-CL) and electrospun CS/P(LLACL) nanofibers. Chitosan has a low intensity peak at 2θ of 49.9◦ before electrospinning, but the crystalline peak disappeared after electrospun into nanofibers. The
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Figure 4. XRD pattern of (a) chitosan as received, (b) P(LLA-CL) as received, (c) chitosan fibers, (d) blended fibers (1:1) and (e) P(LLA-CL) fibers.
reason may be that the chitosan chains can not crystalize during electrospinning. P(LLA-CL) has sharp peaks at 2θ of 16.7 and 18.9◦ . Electrospun P(LLA-CL) nanofibers show a higher intensity peak at 2θ of 16.7◦ and a lower intensity at 2θ of 18.9◦ , not so much different from other P(LLA-CL) materials. That means that P(LLA-CL) molecular chains can crystallize during electrospinning at room temperature, which can be explained from the mobility of the molecular chains of P(LLA-CL) at electrospinning temperature. P(LLA-CL) has a rubbery state at room temperature so the molecular chains have sufficient mobility to crystallize during electronspinning and form the crystal structure. However, with chitosan blended to P(LLA-CL) most of the regular arrangements of P(LLA-CL) were destroyed; as a result, the XRD pattern of electrospun CS/P(LLA-CL) fibers showed an amorphous structure. FT-IR spectra The FT-IR spectra presented in Fig. 5A demonstrate that chitosan has characteristic absorption bands at 1650, 1320 and 1250 cm−1 . The peak at 1650 cm−1 represents amide I absorption, while the peaks at 1320 and 1250 cm−1 represent amide III absorptions [28]. The FT-IR spectra of electrospun chitosan in Fig. 5B show an amide II characteristic absorption band at 1530 cm−1 and amide I at 1675 cm−1 . The amide I band moves to 1675 cm−1 , and an amide II peak appears. This may be caused by TFA and the peak at 1675 cm−1 corresponds to the stretching of the protonated amino groups (–NH3+ ). Three absorption peaks around 840–720 cm−1 indicate the presence of TFA in the chitosan nanofibers as amine salts [29]. The additional TFA alerts some of amide III into amide II. Electrospun P(LLA-CL) has a characteristic peak of –COOC– at 1735 cm−1 and –CH2− – at 1450 cm−1 , as
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Figure 5. FT-IR spectra of (A) chitosan raw material, (B) chitosan nanofibers, (C) P(LLA-CL) nanofibers and (D) CS/P(LLA-CL) = 1:1 nanofibers.
shown in Fig. 5C. The FT-IR spectra corresponding to CS/P(LLA-CL) nanofibers are shown in Fig. 5D. The spectra show both characteristic peaks of electrospun chitosan and P(LLA-CL), this is evidence to illuminate that there may be no reaction between chitosan and P(LLA-CL). Porosity of CS/P(LLA-CL) nanofiber mats Table 1 and Fig. 6 show the apparent density and porosity of CS/P(LLA-CL) nanofibers with various blend ratios of chitosan to P(LLA-CL), respectively. The apparent density of all blended nanofibers was in the range of 0.14–0.42 g/cm3 and decreased with increasing the weight ratio of chitosan to P(LLA-CL). In contrast, as shown in Fig. 6, the porosity of CS/P(LLA-CL) nanofibers increased with the increasing weight ratio of chitosan to P(LLA-CL). The porosity increase of nanofiber mats by adding chitosan in the blend can also be explained as the increasing of solution conductivity. If the conductivity of the solution increased, more electronic charges would be carried by the electrospinning jet, causing the stronger repulsive forces among fibers during depositing to the collector. Another effect of the increased charges may be that it results in a greater deposition area of electrospun fibers [30]. Thus, it may be in favor of increasing the diffusion rate of the residual solvents before nanofiber collecting. The
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Apparent density (g/cm3 )
0:10 2:8 4:6 6:4 8:2
0.41 ± 0.01 0.34 ± 0.04 0.33 ± 0.03 0.19 ± 0.02 0.16 ± 0.02
Data are representative of three independent experiments and all data are given as means ± SD (n = 3).
Figure 6. The porosity of chitosan/P(LLA-CL) blended nanofibers with various blend ratios. Data are representative of three independent experiments and all data points are plotted as means ± SD (n = 3).
evidence is the presence of merged nanofibers in Fig. 1E and F, while they cannot be observed in Fig. 1B–D, G and H. The nanofiber mats formed layer by layer by depositing of electrospun nanofibers could have higher porosity than those formed by merged nanofibers. Thus, the resultant CS/P(LLA-CL) nanofiber mats have a looser structure than P(LLA-CL). The porosity of the nanofiber mats increased with increasing blend ratio of chitosan to P(LLA-CL). To allow for transport of cells and metabolites, the mat must have a large pore volume fraction as well as an interconnected pore network. High porosity usually favors cell adhesion to the mat, promotes ECM regeneration and minimizes diffusion constraints during in vitro culture. Thus, porous material is an essential premise for delivering nutrition and eliminating metabolic waste for tissue engineering to mimic the in vivo condition of tissue generate in vitro. CS/P(LLA-CL) nanofibers show high porosity and adequate property for cell in-growth.
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Mechanical properties of CS/P(LLA-CL) nanofibers Figure 7 shows the typical tensile stress–strain curve of CS/P(LLA-CL) nanofiber mats. The stiffness of the blended nanofiber decreases with increasing P(LLA-CL) content. The tensile strength and ultimate strain obtained from five independent tests are summarized in Table 2. Both strength and strain increased with increasing the blend ratio of P(LLA-CL) to chitosan. This may be because that dry chitosan nanofibers have higher stiffness and lower strength than P(LLA-CL) fibers. The addition of chitosan, thus, reduced the tensile strength of blended nanofibers. The porosity and fibers diameter which changed in blend system were also affect mechanical properties of blend nanofibers. The mechanical properties of nanofibers are important for their successful application in tissue engineering. The mechanical properties of blended nanofibers increased with the increasing of P(LLA-CL) content. Therefore, we could adjust
Figure 7. Mechanical properties of chitosan/P(LLA-CL) nanofibers with various blend ratios. Table 2. Mechanical properties of chitosan/P(LLA-CL) nanofibers with different blend ratios CS/P(LLA-CL) (w/w)
Tensile stress (MPa)
Ultimate strain (%)
0:10 2:8 4:6 6:4 8:2
4.7 ± 0.62 3.6 ± 0.63 2.7 ± 0.33 2.8 ± 0.53 1.4 ± 0.50
121.6 ± 27.67 102.5 ± 17.33 49.5 ± 11.00 18.5 ± 5.36 22.5 ± 5.44
Data are representatives of five independent experiments and all data are used as means ± SD (n = 5).
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the mechanical properties to meet the requirement in practice through changing the blend ratio of chitosan to P(LLA-CL). For instance, one of the main failure modes of synthetic vascular grafts is intimal hyperplasia, caused by the shear stress disturbances due to compliance mismatch at the end-to-end anastomosis between an artery and a rigid graft [31]. Therefore, it was possible to find a blend ratio of nanofibers with suitable mechanical property and porosity to meet the requirement of blood vessel scaffold through this adjustment. Viability and morphology of fibroblasts Figure 8 shows the viability of fibroblasts on blend nanofibers and coverslip. At 24, 48 and 96 h, cell proliferation on all blend nanofiber mats was faster compared with that on the coverslip. At 24 and 48 h there was no obvious difference among the blend nanofiber mats. However, at 96 h, among the CS/P(LLA-CL) nanofibers, the one with the CS to P(LLA-CL) weight ratio of 1:2 was the most excellent and showed significant difference compared with the control. Meanwhile, there was no significant difference among blend nanofiber mats with other blend ratio at 96 h. The proliferation of fibroblast was not promoted by addition more chitosan. It may be because the fiber dimension, porosity and mechanical properties of nanofiber mats are very important for cell growing and migration. In this blend system, they were all affected by blended ratio. The addition of chitosan decreased the presence of merge fibers which have been found in electrospun P(LLA-CL) nanofibers (Fig. 1F), and increased the porosity of blended nanofiber mats. Thus, compared with P(LLA-CL), the blended nanofiber mats can provide larger potential space for cell migration. In our test, among the blend nanofibers, the one with the CS
Figure 8. Viability of fibroblasts cultured on P(LLA-CL), CS/P(LLA-CL) nanofibers and coverslips (control). Data are representative of three independent experiments and all data are plotted as means ± SD (n = 3). *P < 0.05, # P < 0.01.
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Figure 9. SEM images of fibroblasts cultured on the nanofibers and coverslips. (A) P(LLA-CL), (B–D) CS/P(LLA-CL) nanofibers with various blend ratios (CS/P = 1:2, 1:1 and 2:1), (E) coverslip and (F) higher magnification image showing fibroblasts growing along the CS/P(LLA-CL) nanofibers with a blend ratio of 1:2.
to P(LLA-CL) weight ratio of 1:2 might supply the most suitable fiber dimension, porosity and mechanical properties conditions in culturing of fibroblast. Figure 9 shows SEM micrographs of fibroblasts on P(LLA-CL) (Fig. 9A), coverslips (Fig. 9E) and CS/P(LLA-CL) (Fig. 9B–D) mats after seeding for 24 h. As SEM images shown, cells on coverslip could not spread well, but they showed polygonal shape on blend nanofiber mats and migrated into the nanofiber mats. Figure 9F shows higher magnification image of fibroblasts growing along the CS/P(LLA-CL) nanofibers with the blend ratio of 1:2. As Fig. 9 shows, the blended nanofiber mats can maintain their fibrous structure well in medium. Thus, they can supply steady physical and chemical support for cell growing. Chitosan is a hydrophilic material and has structural characteristics similar to glycosaminoglycans and distinctive biological properties. It has been reported that chitosan is able to promote cell attachment and maintain the characteristic morphology and viability of various cells such as human osteoblasts and chondrocytes [16]. The presence of chitosan improves the interface between the nanofiber mats and cells. Thus, the fibroblasts can show better morphology cultured on CS/ P(LLA-CL) nanofibers than P(LLA-CL) nanofibers and coverslips.
CONCLUSIONS
Blended CS/P(LLA-CL) was electropun into nanofibers for the first time. The average diameter of electrospun blended nanofibers was affected by the solution concentration and the blend ratio of CS/P(LLA-CL). The diameter decreased with
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decreasing concentration of electrospinning solution and with increasing chitosan content at the same concentration. The XRD pattern and FT-IR spectra indicated that the blend system was a simple mixture where both chitosan and P(LLACL) kept the molecular structure intact. The porosity of CS/P(LLA-CL) blended nanofibers increased with increasing the weight ratio of chitosan to P(LLA-CL). The tensile strength and ultimate strain increased with increasing P(LLA-CL) content in the blend. After the cell viability and morphology studied on P(LLA-CL) and chitosan blended nanofiber mats, it was found that the nanofibers mat with weight ratio of 1:2 is the most appropriate for fibroblasts proliferation. The cells cultured on blended nanofiber mats showed better spreading phenotype than those on the P(LLA-CL) nanofiber mat or on the coverslip, and could migrate along the blended nanofibers. All these results suggest that electrospun CS/P(LLA-CL) nanofibers have potential as tissue engineering scaffolds to biomimic the extracellular matrix. Acknowledgements This research was supported by National Nature Science Foundation of China (grant 30570503) and Shanghai Sci. & Tech. Committee China (grants 05DJ14006 and 05PJ14013).
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