Characterization of the Surface Biocompatibility of the Electrospun

Lihong Huang , Xiuli Zhuang , Jun Hu , Le Lang , Peibiao Zhang , Yu Wang .... Felipe Xavier Costa , Melinda Larsen , Alexander Khmaladze , James Castr...
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Biomacromolecules 2005, 6, 2583-2589

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Characterization of the Surface Biocompatibility of the Electrospun PCL-Collagen Nanofibers Using Fibroblasts Y. Z. Zhang,*,†,‡ J. Venugopal,†,§ Z.-M. Huang,| C. T. Lim,†,‡,§ and S. Ramakrishna†,‡,§ Division of Bioengineering, Department of Mechanical Engineering, and NUS Nanoscience and Nanotechnology Initiative, National University of Singapore, Singapore 117576, and School of Aeronautics, Astronautics & Mechanics, Tongji University, 1239 Siping Road, Shanghai 200092, People’s Republic of China Received May 6, 2005; Revised Manuscript Received June 23, 2005

The effect of nanofiber surface coatings on the cell’s proliferation behavior was studied. Individually collagencoated poly(-caprolactone) (PCL) nanofibers (i.e., Collagen-r-PCL in the form of a core-shell structure) were prepared by a coaxial electrospinning technique. A roughly collagen-coated PCL nanofibrous matrix was also prepared by soaking the PCL matrix in a 10 mg/mL collagen solution overnight. These two types of coated nanofibers were then used to investigate differences in biological responses in terms of proliferation and cell morphology of human dermal fibroblasts (HDF). It was found that coatings of collagen on PCL nanofibrous matrix definitely favored cells proliferation, and the efficiency is coating means dependent. As compared to PCL, the HDF density on the Collagen-r-PCL nanofiber membrane almost increased linearly by 19.5% (2 days), 22.9% (4 days), and 31.8% (6 days). In contrast, the roughly collagen-coated PCL increased only by 5.5% (2 days), 11.0% (4 days), and 21.0% (6 days). SEM observation indicated that the Collagen-r-PCL nanofibers encouraged cell migration inside the scaffolds. These findings suggest that the Collagen-r-PCL nanofibers can be used as novel functional biomimetic nanofibers toward achieving excellent integration between cells and scaffolds for tissue engineering applications. 1. Introduction The regeneration of lost or damaged tissue requires that reparative cells assemble three-dimensionally around and inside the supporting scaffold via a series of biological activities such as adhering, migrating, growing, and differentiating to attain a proper integration between cells and scaffold for synthesizing a new tissue. The majority of human tissues and organs such as bone, tendon, and skin are deposited on hierarchically organized fibrous structures with the fibril/fiber size realigning from nanometer to millimeter scale. As such, nanofibers have now been extensively used to mimic these natural tissue matrixes. Using nanofibers, the engineering of a number of tissues including cartilage,1 bone,2 arterial blood vessel,3 heart,4 and nerve5,6 has been attempted. Various fabrication methods have been used, and these include electrospinning,7,8 phase separation,9 and self-assembly.10 Among these, electrospinning is a technique that uses high voltage electrical field to generate nano/microscale fibers and is by far the most prevalent method used. This is because electrospinning possesses quite a few appreciable merits - simple, straightforward, versatile, cost-effective, and scaleable. * To whom correspondence should be addressed. Tel.: +65-6874 6567. Fax: +65-6874 6567. E-mail: [email protected]. † Division of Bioengineering, National University of Singapore. ‡ Department of Mechanical Engineering, National University of Singapore. § NUS Nanoscience and Nanotechnology Initiative, National University of Singapore. | Tongji University.

Representative biodegradable polymers including synthetic ones such as poly(lactic acid) (PLA),11,12 poly(glycolic acid) (PGA),13 poly(lactic-co-glycolic acid) (PLGA),1,14 poly(caprolactone) (PCL),2,15,16 or natural ones such as collagen,17 gelatin,18 silk,19,20 and chitosan21,22 have been electrospun into nanofibers. Many studies have shown that the nanofibrous scaffolds do affect cellular responsiveness very favorably (see a recent review article23). Nanofibers are dimensionally small but mechanically weak. Their similarity in physical structure to native ECM partially contributes to the favorable interaction between cells and the nanofibrous scaffolds. Effective use of polymer nanofibers for engineering tissues relies not only on the buildup elements of fibers being nanoscale, which can mimic the physical structure of the native extracellular matrix, but also on the biochemistry characteristics of the materials used. This biochemical characteristic can be controlled via the functionalization of the nanofibers. Functionalizing the nanofibers can be achieved through three methods: (1) mixing bioactive agents with the biodegradable polymer solutions to prepare bioactive composite nanofibers by means of electrospinning;24,25 (2) conducting surface modification on the whole nanofibrous scaffolds26-28 after electrospinning; and (3) employing an advanced coaxial electrospinning technique, which was illustrated very recently29-31 but has not been attempted for such functionalization application yet. Regarding the coaxial electrospinning, it is essentially a modification or extension of the traditional electrospinning technique with a major difference being the compound spinneret used. From this

10.1021/bm050314k CCC: $30.25 © 2005 American Chemical Society Published on Web 07/27/2005

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Figure 1. Coaxial electrospinning (a), and cross-sectional view of resultant bi-component composite fiber (b).

compound spinneret (Figure 1), two (or more) components can be fed through different coaxial capillary channels and are integrated into a core-shell structured composite fiber to fulfill different application purposes. The primary objective of this study was to investigate the efficacy of using surface functionalized nanofibers in regulating cell-scaffold interactions by using the human dermal fibroblasts as the sample cells for skin tissue engineering. A coaxial electrospinning technique was employed to produce the individually surface-coated nanofibers, while a rough surface coating was accomplished by simply soaking a base nanofibrous scaffold in a coating medium. For this study, PCL and collagen were selected as the scaffold material and coating reagent, respectively. The effect of coating on the influence of cell proliferation and morphology was then reported in the line of forming dermal substitution for skin regeneration. 2. Materials and Methods 2.1. Materials. Polymers of granular PCL (Mn 80 000, Aldrich) and Type I Collagen from calf skin in foam form, and solvents of 1,1,1,3,3,3-hexafluoro-2-propanol (HFP) and 2,2,2-trifluoroethanol (TFE) (purity g99.0%, Fluka, Buchs, Switzerland), were purchased from Sigma-Aldrich, Inc. (St. Louis, MO). Methanol and chloroform were obtained from Merck, Germany. These polymers and solvents for electrospinning were used as received without further purification. 2.2. Surface Coating of Electrospun PCL Nanofibers. Two techniques were employed to produce different coatings on the PCL nanofibers prior to cell culture, that is, coaxial electrospinning and post coating on the electrospun nanofibers. The coaxial electrospinning was achieved using a setup reported earlier.31 In brief, the compound spinneret used consisted of a syringe PP Luer fitting (OD 2.5 mm, ID 1.5 mm) attached to a 10 mL standard syringe, and an 18G blunted stainless steel needle (OD 1.2 mm, ID 0.84 mm) inside the fitting. The inner and outer solutions used were PCL/TFE (100 mg/mL) and collagen/TFE (72 mg/mL), respectively. By adjusting the processing parameters such as applied voltage, gap distance, and flow rate, a continuous and stable core-shell jet can be formed and maintained. Electrospun composite fibers from our homemade coaxial device were denoted as Collagen-r-PCL, which means the collagen was the shell material, and PCL was the wrapped core component. The electrospinning parameters for obtaining the Collagen-r-PCL nanofibers were as follows: 12 cm

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gap distance, 15.8 kV applied voltage, 0.7 mL/h inner flow rate, 21.5 °C ambient temperature, and 75% ambient humidity. All of the electrospun fibrous membranes were stored in desiccators for a couple of days before subsequent use. For the post electrospinning coating, the PCL nanofibers were first prepared using a traditional electrospinning process. To electrospin PCL nanofibers, PCL solution (7.5% w/v in methanol and chloroform mixed at a ratio of 1:3) was placed in a 5 mL syringe, which was connected to a 30 cm long Teflon tube with an internal diameter of 1.0 mm. The positive electrode of the high voltage power supply is clamped directly to a blunted needle (0.84 mm ID) connected to the exit of the Teflon tube. The negative electrode was connected to a metallic lab rack with its plateau wrapped with aluminum foil, which was located at a distance of 13 cm from the needle tip. On the aluminum foil, 15 mm diameter coverslips were placed for the collection of nanofibers. Electrospinning solution was delivered at 0.75 mL/h via a syringe pump (KD100, KD Scientific Inc.). A voltage of 12.5 kV was applied using a high voltage power supply (RR50-1.25R/230/DDPM, Gamma High Voltage Research, USA) during the electrospinning process. After electrospinning, the PCL nanofibrous membranes on the coverslips were then subjected to a rough collagen coating by immersing the PCL nanofibrous scaffolds into a collagen solution (10 mg/mL in 0.05 M acetic acid) overnight. Afterward, the constructs were washed three times with PBS and kept air-dried. For control purpose, collagen nanofibers from collagen/ HFP (75 mg/mL) were also prepared through this normal electrospinning using the same processing parameters as that of PCL. 2.3. Electron Microscopy. The electrospun nanofibrous membranes were sputter coated (JEOL JFC-1200 Fine Coater) with gold up to 90 s, and their morphologies were observed by a scanning electron microscope (JEOL JSM5600, Japan) at an accelerating voltage of 15 kV. Diameters of the electrospun fibers were analyzed on the basis of the obtained SEM images. The core-shell structure of the coaxially electrospun Collagen-r-PCL composite nanofibers was examined using a JEOL JEM-2010F FasTEM field emission transmission electron microscope. The samples for TEM were prepared by directly depositing the as-spun nanofibers onto a copper grid, which was coated in advance with a supportive Formvar film followed by a carbon coating. The samples were kept in a vacuum oven for 48 h for drying at room temperature before the TEM imaging. The TEM machine was operated at 100 kV. 2.4. Fibroblast Culture. The fibroblasts used for the present cultures were a gift from the National University Hospital, Singapore. The donor human normal skin was obtained by surgical removal under local anesthesia after informed consent. Epidermis and subdermal fat were removed from sterile biopsies of the normal skin. The specimens were minced into pieces of 1-2 mm3 in sterile tissue culture dishes and gently overlaid with DMEM supplemented with 10% FBS with antibiotics. Explants were incubated at 37 °C in a humidified CO2 incubator for 10 days and fed every 3 days, and fibroblasts were harvested

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Electrospun PCL-Collagen Nanofibers

Figure 2. SEM images of electrospun PCL (a) and collagen (b) nanofibers.

from primary cultures by trypsin-EDTA treatment and replated. The human dermal fibroblast cultures at 2-4 passages were used for this study. 2.5. Cell Proliferation. The nanofibrous scaffolds of PCL, collagen, and Collagen-r-PCL collected on circular glass coverslips (φ15 mm) were placed in 24 well cell culture plates for sterilizing and prewetting by decreasing concentration of ethanol for 1-2 days. Thereafter, the scaffolds were soaked in phosphate buffered saline (PBS, pH ) 7.4) and in cell culture medium overnight prior to cell seeding to facilitate protein absorption and cell attachment onto the nanofibers. The human dermal fibroblasts were seeded (1 × 104 cells/ cm2) on control tissue culture plate (TCP), PCL, collagen, collagen-coated PCL, and Collagen-r-PCL nanofibrous matrixes on 24-well tissue culture plates. The cell proliferation was monitored after 2, 4, and 6 days by MTS assay (3-(4,5dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt). To monitor cell adhesion and proliferation on different substrates, the number of cells was determined by using the colorimetric MTS assay (CellTiter 96 Aqueous Assay). The mechanism behind this assay is that metabolically active cells react with tetrozolium salt in the MTS reagent to produce soluble formazan dye that can be observed at 490 nm. The cellular constructs were rinsed with PBS followed by incubation with 20% MTS reagent in serum free medium for 3 h. Thereafter, aliquots were pipetted into 96-well plates, and the samples were read using the spectrophotometric plate reader (FLUOstar OPTIMA, BMG Lab Technologies, Germany) at 490 nm. 2.6. Cell Morphology. After 6 days of cell proliferation, the HDF grown on different scaffolds was washed with PBS to remove nonadherent cells, fixed in 4% glutaraldehyde for 1 h at room temperature, dehydrated through a series of graded alcohol series, and finally critical point dried with hexamethyldisilazan overnight to maintain the normal cell morphology. The dried cellular constructs were sputter coated with gold and observed under the SEM machine at an accelerating voltage of 15 kV. 2.7. Statistical Analysis. All quantitative results were obtained from triplicate samples. Data were expressed as the mean ( SD. Statistical analysis was carried out using an unpaired student’s t-test. A value of P < 0.05 was considered to be statistically significant.

Figure 3. TEM image of an individual Collagen-r-PCL composite nanofiber (a) with collagen as the shell material, and PCL the support; for comparison purposes, (b) is the TEM image of a pure PCL nanofiber.

3. Results 3.1. Morphology of the Nanofibers. With our optimized electrospinning parameters, beads free and relatively uniform ultrafine fibers of PCL and collagen were prepared as shown in Figure 2. The SEM images show that the electrospun nanofibers possess a common feature of random array and very porous structure. The average diameters measured for PCL and collagen nanofibers are in the ranges of 318 ( 131 and 216 ( 72 nm, respectively. For the coaxially electrospun Collagen-r-PCL nanofibers, TEM image (Figure 3a) clearly indicates the formation of core-shell structure, with the dark component inside the composite being a structural support of PCL. Such a structure resembles a “coating” of collagen onto individual PCL nanofibers. For comparison purposes, Figure 3b gives the TEM image of the mono-PCL nanofibers produced using a traditional electrospinning setup. The inset in Figure 3a is the SEM microphotograph of Collagen-r-PCL composite nanofibers, which had morphology similar to that of the PCL and collagen nanofibers with an average diameter of 385 ( 82 nm, and a coating thickness of 64 ( 26 nm. 3.2. Cell Proliferation. Proliferation data of the dermal fibroblasts after 2, 4, and 6 days of in vitro culturing were plotted in Figure 4. The mono-PCL nanofibers showed a significant level (P < 0.05) of slow increase in cell proliferation throughout the designated three time intervals as compared to the other material groups. To study the coating efficiency on cell proliferation, we may limit our comparison to the nanofibers of the mono-PCL, collagencoated PCL, and Collagen-r-PCL. It was found that coatings on the PCL nanofibers by both of the coating techniques

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Figure 4. Cell proliferation of human dermal fibroblasts by MTS assay.

gave rise to very distinct and accelerated differences; the significance level increases to P < 0.001 at day 6 from P < 0.05 at day 2. Furthermore, we identified that the HDF proliferation was exactly as expected to be coating-dependent; Collagen-r-PCL is significantly more favorable (P < 0.001) for cells proliferation than that of the roughly collagen-coated PCL. As compared to the mono-PCL, the HDF density on the Collagen-r-PCL almost linearly increased by 19.5% (2 days), 22.9% (4 days), and 31.8% (6 days). In contrast, the roughly collagen-coated PCL increased only by 5.5% (2 days), 11.0% (4 days), and 21.0% (6 days). Comparing the Collagen-r-PCL and the collagen nanofibers, although proliferation data of the collagen-r-PCL are not as significant as those of the collagen, the t-test indicates that there is no statistically significant difference in cell proliferation between the two nanofibrous matrixes. 3.3. Cellular Morphology. Morphology observations of the HDF after 6 days of in vitro culture on the different

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scaffolds were examined by SEM and presented in Figures 5 and 6. Although the dermal fibroblasts were attached and spread well and normal cell morphology was seen with all of the culturing substrates, the cell-scaffold interactions indeed varied for different scaffolds. This was first reflected by a difference in reaching confluence of the HDF cells. Confluence phenomenon was observed in the substrates of the collagen (Figures 5d and 6d) and TCP control (similar images, not shown), whereas this did not happen for the others. Visually, varied extents of cell spreading around the nanofibrous scaffolds of the PCL, collagen-coated PCL, and Collagen-r-PCL already indicate the differences in cell proliferation. The PCL nanofibers subjected to coatings (Figures 5b and c) were attached with many more cells than those of the pristine PCL. This is consistent with the proliferation analysis done in section 3.2. A close view of cell-scaffold interaction in terms of cell migration in-depth level is presented in Figure 6. Cell ingrowth in electrospun collagen-based nanofibers has been reported by us and others.17,18 In the present situation, because HDFs on the collagen fibrous scaffold were already in confluence, we can only see the layered cells. For the cases of PCL involved nanofibers, interestingly, we found that cells penetrating beneath the Collagen-r-PCL composite nanofibers can be clearly seen around the scaffolds (Figure 6a). However, there is no such finding either in the pristine PCL or in the collagen-coated PCL nanofibrous scaffold, implying that a direct coating of collagen onto PCL would only be helpful in improving the cell proliferation behavior facially. Its action in favoring the cell-scaffold integration forming a 3-D ingrowth in the scaffold is not apparent. 4. Discussion 4.1. Core-Shell Structured Composite Nanofiber. As shown in Figure 3, although the inset SEM image indicated

Figure 5. Cell morphology of HDF at low magnification on different fibrous scaffolds: (a) pure PCL; (b) PCL with surface roughly collagen coated; (c) individually collagen-coated PCL (Col-r-PCL); and (d) pure collagen nanofibers.

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Figure 6. A comparison of cells ingrowth behavior on different nanofibrous scaffolds: (a) individually collagen-coated PCL (Col-r-PCL); (b) pure PCL; (c) PCL with surface roughly collagen coated; and (d) pure collagen nanofibers.

no difference between a nanofiber mat obtained by coaxial electrospinning and the one by single dope electrospinning, the TEM examination showed a clear core-shell structure. The sharp boundary has been explained as the characteristic time of the bending instability during electrospinning that was much shorter than that of diffusion spread.30 Coaxial electrospinning is able to create interesting compound nanofiber structures, but the complex electrohydrodynamics involved in the coaxial electrospinning are yet to be illuminated.32 Detailed knowledge of how the processing parameters such as flow rate, electrical field, viscosity, etc., affect the fiber morphology of the resultant composite fiber needs further study. Here, on the basis of our results, we only highlight the capability of coaxial electrospinning in producing novel and functionalized nanofibers. Design and functionalization of nanofibers are important concerns in the effective applications of these nanostructured materials. For example, surface-coated polymer nanofibers can be used to develop highly sensitive bio- or chemical sensors.33 Hollow nanofibers can find applications in highly efficient filtrations,34,35 whereas nanofibers containing drugs can perform controlled releases.36,37 With respect to nanofibers for tissue engineering applications, our interest is to improve the cell-scaffold interaction or biocompatibility of nanofibrous scaffolds with living cells upon culturing. Surface modification techniques to improve the hydrophilicity and biochemistry affinity have been used.26-28 A common scheme in this regard is the inert polymer that is first subjected to pretreatment via techniques such as argon plasma pretreatment, or UV irradiation to generate reactive species (e.g., hydroxyl, peroxide, and hydroperoxide) before proceeding to subsequent graft copolymerization and immobilization of biomacromolecules. However, because the nanofibers are so delicate as compared to its bulk form, their nanofibrous form can be severely

destroyed and mechanical properties compromised tremendously after experiencing those harsh pretreatment conditions.26,27 Furthermore, as the plasma effect only happens to a depth of several hundred angstroms, a full depth surface modification of the scaffold structure may be difficult to attain. There is also another simple coating technique to functionalize the surface of nanofibrous matrixes. This is usually done through precoating of protein substances such as collagen by simply immersing the nanofibrous scaffolds into the protein-rich media for a certain period of time prior to cell seeding so as to facilitate cell attachment.1,2 Because the majority of polyester-type biodegradable polymers, for example, PLA and PCL, are hydrophobic and taking into account the fact that nanofibrous structures contribute to increasing hydrophobicity,38,39 the coating is likely to occur only on the outer shallow surface of the whole nanofibrous structure rather than on individual fiber level. This may result in a poor coating effect. Comparatively, core-shell structured nanofibers can be conveniently prepared by coaxial electrospinning, with the advantages of being able to control the shell thickness and manipulate the overall mechanical strength and degradation properties of the resulting composite nanofibers without offsetting biocompatibility. Such coreshell structured composite nanofibers can also be functionalized for potential uses in controlled releases and development of highly sensitive sensors, highly conductive nanocables, reactive compound nanofibers, and super strong engineering composites, etc. 4.2. Cell-Scaffold Interaction. Numerous studies have demonstrated that cell-scaffold interaction in the first place is dependent on the chemical characteristics of scaffolds. For the polymeric scaffolds, it is well known that polymers from both synthetic and natural origins possess their intrinsic pros and cons if used separately for making biodegradable porous scaffolds. For instance, synthetic polymers have advantages

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in proccessability, good mechanical properties, and manipulating degradation rate, but they lack cell recognition signals and hinder successful cell seeding because of their hydrophobic trait. On the other hand, natural biodegradable polymers such as collagen, as a protein substance, are hydrophilic and good for specific cell interaction. However, scaffolds fabricated purely from collagen exhibit poor mechanical strength and are not easy to handle. As such, hybridization40,41 of the two has been frequently used to develop a novel artificial biodegradable material for scaffold applications. In a previous work, we used a mixture of PCL and gelatin (1:1) to prepare biomimetic gelatin/PCL composite nanofibers through electrospinning, and we achieved encouraging results in improving the cell-scaffold interactions.18 In this study, we have made the hybridization of synthetic and natural polymers in the form of a core-shell structure. With synthetic polymer (better mechanical performance) as the core and structural component and natural biomacromolecules presented on the sheath for functional purposes, such nanofibers are advantageous for those where biocompatibility and mechanical properties are equally important. The experimental results have shown that, although synthetic biodegradable PCL supports cell growth, to proliferate more and encourage cell ingrowth for better integration between cells and the scaffold, the biologically inert PCL nanofibers need effective surface coatings of bioactive molecules. In other words, materials complex at nano- and macro-level gave rise to apparent difference in cell response propensity. Our investigation also implied a direct coating by simply soaking the nanofibrous membrane in the coating medium had a poor coating effect. Because the fiber diameters for the PCL and collagen-r-PCL are comparable as reported in section 3.1, the favorable response of HDF on the collagenr-PCL nanofibrous membranes can be explained as a result of the effective presence of collagen biomacromolecules on the PCL nanofibers, instead of the fiber size effect. It is necessary to discuss the cell inward growth here. Nanofibrous scaffolds are very porous, but the “pores” (it is not appropriate to use the term “pore size” to quantify the porous feature of a nanofibrous structure) formed in the electrospun fibrous structure are much smaller than the normal cell size of a few to tens of micrometers, which would inhibit cell migration. This inhibition phenomenon is similar to that of filtration by nanoscale fibers in the filter structure,42,43 resulting in efficient exclusion of microsized dust particles. As such, the phenomenon of cell ingrowth into a nanofiber structure has been doubted by many researchers. Results from our present work and earlier reports17,18 indicated the capability of cells infiltration. We speculate that two reasons are responsible for this phenomenon: (1) Good hydrophilicity and the presence of biological signals from biomimetic nanofibers provide a nano/micro environment like a surrounding of real 3-D natural ECM does,44 which would facilitate transportation of nutrient and metabolic waste, and hence regulate cell migration to a deeper level of the nanofibrous scaffolds. (2) Both the loosely interlaced fibrous structure and the weak nanoscale fibers provide least obstruction and matched mechanical properties

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for cell movements. Unlike “dead” microsized dust particles, the living cells entering into the matrix through amoeboid movement to migrate through the pores can push the surrounding fibers aside to expand the hole as the small and weak nanofibers offer little resistance to the cell movement. This dynamic architecture of the fibers allows the cells to adjust according to the pore size and grow into the nanofiber matrixes.18,45 Therefore, proper match of the mechanical properties and the loosely laced fibro-porous nanofibrous structure could favor the cells modulating themselves and the artificial ECM toward favorable interactions for synthesizing a “real” tissue. With a recent successful measurement of single nanoscale fiber,15 how strong nanofibers affect cell movement is possibly a topic to be studied soon. Cell-scaffold interaction involves a series of proper biological responses of cells to the passive substratum, including adhesion, cell morphology, growth, differentiation, and migration. Scaffolds encouraging and sustaining such biological activities upon culturing can be deemed biologically active or “bioactive”. At this point, Collagen-r-PCL composite nanofibers can be potentially effective in creating bioactive scaffolds because they mimic the natural ECM to a certain extent in terms of the physical structure and biochemical characteristics. By selecting proper core and shell materials, an optimized balance between structural strength and biological compatibility can be achieved for the resulting core-shell structured nanofibers. 5. Conclusions This study demonstrated the feasibility and efficacy of using core-shell composite nanofibers for improving cellscaffold interactions in tissue engineering applications. Our preliminary experimental results indicated individual nanofiber coated with collagen tends to resemble the natural ECM rather than the rough collagen coating or the pristine PCL nanofibers. Therefore, cells have more propensities to interact well with the former than the latter, suggesting a greater ability in constructing complex sets of cellular interactions as biomimetic nanofibers in engineering tissues. Acknowledgment. We thank Dr. X. J. Xu for her assistance in using the TEM. Z.-M.H. acknowledges the financial support of the NanoSciTech Promote Center, the Shanghai Science & Tech. Committee (0352nm091). This study was supported by a research grant from the National University of Singapore. References and Notes (1) Li, W.-J.; Laurencin, C. T.; Caterson, E. J.; Tuan, R. S.; Ko, F. K. J. Biomed. Mater. Res. 2002, 60, 613-621. (2) Yoshimoto, H.; Shin, Y. M.; Terai, H.; Vacanti, J. P. Biomaterials 2003, 24, 2077-2082. (3) Xu, C. Y.; Inai, R.; Kotaki, M.; Ramakrishna, S. Biomaterials 2004, 25, 877-886. (4) Zong, X.; Bien, H.; Chung, C.; Yin, L.; Kim, K.; Fang, D.; Chu, B.; Hsiao, B.; Entcheva, E. Polym. Prepr. 2003, 44, 96-97. (5) Yang, F.; Murugan, R.; Ramakrishna, S.; Wang, X.; Ma, Y.-X.; Wang, S. Biomaterials 2004, 25, 1891-1900. (6) Silva, G. A.; Czeisler, C.; Niece, K. L.; Beniash, E.; Harrington, D. A.; Kessler, J. A.; Stupp, S. I. Science 2004, 303, 1352-1355. (7) Huang, Z.-M.; Zhang, Y.-Z.; Kotaki, M.; Ramakrishna, S. Compos. Sci. Technol. 2003, 63, 2223-2253.

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