KEY WORDS Collagen-blended PLGA nanofibres, electrospinning

COLLAGEN-BLENDED BIODEGRADABLE POLYMER NANOFIBERS: POTENTIAL SUBSTRATES FOR WOUND HEALING IN SKIN TISSUE ENGINEERING [K. Ma] 1, [T. Yong] 2, [K.C. Chan] 3-5, [S. Ramakrishna] 2-4 [Graduate Program in Bioengineering, GPBE] 1, [Department of Mechanical Engineering] 2, [Division of bioengineering] 3, [Nanoscience and Nanotechnology Initiative, NUS] 4, [Department of Orthopedic Surgery, NUH] 5 National University of Singapore, Nanoscience & Nanotechnology Initiative, Blk E3 # 05-14 Biochemistry Laboratory 2, Engineering Drive 3, 117576 Singapore ABSTRACT Due to the limited area of normal skin, immune rejection and high expenses, no wound dressings could completely satisfy clinical demands for extensive burns and large open wounds. An alternative to skin engraftment is to the development of engineering skin constructs to facilitate wound healing, which include a scaffold, support cells, growth factors and extracellular matrix (ECM). In this study we fabricated and optimized collagen-blended PLGA nanofibers by electrospinning techniques. Both Scanning Electron Microscopy (SEM) and Transmission Electron Microscopy (TEM) micrographs verify the nanometer scale and smooth morphology of the blended nanofibers, revealed that 8% is the best concentration for preparing the polymer solution. Attenuated Total Reflectance - Fourier Transform Infrared (ATR-FTIR) demonstrates that blended nanofibers are a mixture of PLGA and collagen. Mechanical tests of their tensile properties show the ultimate strain value and tensile modulus of blended nanofibers are comparable to those of human skin. Contact Angle Assessment shows their decreased hydrophobicity compared with pure PLGA nanofibers and suggests the improved capability for cell attachment. Therefore, this new type of blended nanofiber substrate holds a great potential to facilitate wound healing in skin tissue engineering by being loaded with necessary growth factors and ECM molecules on it. KEY WORDS Collagen-blended PLGA nanofibres, electrospinning 1. INTRODUCTION Electrospun polymer nanofibers (NFs) have elicited great interests in skin tissue engineering because the nanometer scale architecture of the NFs mimics the natural extracellular matrix (ECM), which is composed of a three-dimensional network of nanoscaled fibrous proteins embedded in a glycosaminoglycan (GAG) hydrogel[1]. In addition to their special morphology, polymer NFs have additional desirable features as skin grafts, such as a high surface area-to-volume ratio (5-100 m²/g), which is efficient for fluid absorption and dermal delivery during the process of wound healing[2]. Another advantage lies in g0500566@nus.edu.sg their high porosity but small pore size. Non-woven nanofibrous scaffolds for wound dressing usually have pore sizes ranging from 500nm to 1µm, small enough for cell migration as well as protection of the wound from bacterial penetration [2]. Numerous scaffolds have been developed from naturally derived biodegradable polymers for culturing human fibroblasts and keratinocytes [3, 4]. Of these, collagen -based porous scaffolds are the most popular for skin tissue engineering [5, 6]. Collagen can be used alone or in combination with other ECM components such as glycosaminoglycan (GAG) and growth factors to improve cell attachment and proliferation. The problems that compromise the effects of collagen-based scaffolds are their rapid absorption rate and weal mechanical strength. Synthetic biodegradable polymers such as poly (L-lactide) (PLA), poly (glycolide) (PGA), poly (caprolactone) (PCL) and poly (DL-lactide-co-glycolide) (PLGA) have also been used as matrices for skin regeneration [7, 8, 9, 10]. The biggest disadvantage of synthetic materials is the lack of cell recognition signals [11]. However, it can be overcome by surface immobilization or simply by physically blending the two materials, which may improve the biocompatibility of the synthetic NFs while preserving their mechanical strength simultaneously. The objective of our present study was to fabricate collagen-blended biodegradable polymer NFs for skin tissue engineering. We chose a random copolymer Poly (lactide-co-glycolide) (PLGA), because its degradation rate can be controlled readily by adjusting the ratio of the two components. In previous experiments performed in our laboratory, we found that PLGA (a feed ratio of lactide and glycolide: 75:25) NF scaffolds, when seeding dermal fibroblasts on them, degraded within 2-3 weeks, which is within the time frame for skin to regenerate. The collagen-blended PLGA NFs were fabricated by electrospinning the solution of a collagen-polymer mixture in 1, 1, 1, 3, 3, 3-hexafluoro-2-propanol (HFP). The blended nanofiber substrate, serves as a biomimetic synthetic ECM both in fiber diameter and constituent components and thus promises to be an alternative skin graft for wound healing. 555a-011 262 Abstract Submission

2. MATERIALS AND METHODS Materials Poly (DL-lactide-co-glycolide) (PLGA 75:25) copolymer was purchased from Sigma-Aldrich, at a molecular weight of 90,000-126,000. 1, 1, 1, 3, 3, 3-hexafluoro-2-propanol (HFP) was purchased from Sigma-Aldrich (St. Louis, MO). Koken Type I Collagen powder (Medical Grade) was purchased from INNOMED PTE LD (Singapore). Fabrication of collagen-blended PLGA NFs PLGA and collagen powders were mixed at a ratio (w:w 1:1) overnight at room temperature in different concentrations of HFP (w:v 4.5%, 5%, 6%, 8%). The mixture was then placed in a plastic syringe with a needle tip diameter of 0.21 mm. With the aid of a syringe pump, the solution was dispersed at a feed rate of 1.2 ml/h at the humidity of 55% - 60% and temperature of 20-24 0 C. Electrospinning voltage was applied to the needle at 18-kV DC voltage, using a high-voltage power supply (Gamma High Voltage Research, Ormond Beach, FL).The electric field generated by the surface charge caused the solution drop at the tip of the needle to distort into a Taylor cone. Once the electric potential at the surface charge exceeded a critical value, the electrostatic forces would overcome the solution surface tension and a thin jet of solution would erupt from the surface of the cone. The schematic setup and the optimized parameters for fabrication of collagen-blended PLGA NFs are shown in Fig. 1. The resultant NFs were collected on an aluminum collector located 12 cm from the needle tip. After solvent evaporation, the PLGA collagen-coated aluminum films were placed in a vacuum dryer overnight to remove any remaining solvent. collagen-blended PLGA NFs were obtained with a JSM-5800LV scanning electron microscope (JEOL, Tokyo, Japan). Transmission Electron Microscopy (TEM) micrographs of the nanofibers were obtained with a JEM-2010F FasTEM field emission electron microscope (JEOL) operated at 100 kev. Electrospun NFs for the TEM observation were prepared by directly depositing the electrospun NFs onto copper grids that had been coated with a supportive Formvar film (Structure Probe, SPI Supplies Division, West Chester, PA). The diameter range of the fabricated NFs was measured on the basis of SEM images, using image analysis software (ImageJ; National Institutes of Health, Bethesda, MD). Attenuated Total Reflectance-Fourier Transform Infrared (ATR-FTIR) spectra were obtained with an AVATAR 380 FTIR machine (Thermo Electron, Waltham, MA). Tensile tests were carried out with a 5848 microtester (Instron, Norwood, MA) at a stroke rate of 10 mm/min and with a 40-mm gauge length. NFs were rectangular (10 x 60 mm) in shape. This tensile test was performed in the same manner as standard mechanical tests for fabric materials. Hydrophilicity was determined by Contact Angle Assessment. The water contact angles for samples of pure PLGA NFs and collagen-blended NFs are measured by the sessile drop method. Drops of distilled water were deposited onto the surface of the samples and the direct microscopic measurement of the contact angles was done with the computer software. The water droplet was 0.5 ml to prevent a gravitational distortion of the spherical profile. The result of each sample was obtained by averaging 5 tests. Statistical analysis Values (at least triplicate) were averaged and expressed as means ± standard deviation (SD). Each experiment was repeated two or three times. Statistical differences were determined by Student two-tailed t test. Differences were considered statistically significant at p< 0.05. 3. RESULTS Fig. 1 Schematic setup and optimized operation parameters (voltage, feed rate, and collector distance) for fabrication of the random collagen-blended PLGA nanofibers by electrospinning techniques. Modified with approval from [1]. Characterization of collagen-blended PLGA NFs Scanning electron microscopy (SEM) micrographs of Scanning Electron Microscopy (SEM) SEM micrographs of collagen-blended PLGA NFs prepared with various concentrations in HFP (w:v 4.5%, 5%, 6%, 8%) are shown in Figure 2. Fig. 2A shows that NFs with a ratio of 4.5% are characterized with many bubbles and beads, which were once observed to exert adverse effects on cell attachment and migration (data not shown). The bubbles disappear while the pores still exist when the ratio is increased up to 5% (Fig. 2B). When the concentration of polymer solution reaches 6%, neither bubbles nor beads are observed in Fig. 2C, but their diameters are not uniform. Fig. 2D reveals that 8% is the optimal concentration for electrospinning the copolymer solution since NFs are well characterized by non-bubbles, non-beads, uniform and interconnected structures under 263

this concentration. The diameters of the blended NFs were 383 ± 132 nm, directly measured from Fig.2D. It shows a well-controlled diameter distribution of the blended NFs. Transmission Electron Microscopy (TEM) TEM images of collagen-blended NFs are shown in Fig.3, which verified the nanometer scale and smooth morphology of the blended NFs. However, in order to identify the distribution of PLGA and collagen in NFs, the following measurement is introduced. A B Mechanical Tests of tensile properties of NFs Fig. 5 shows the typical stress strain curves of collagen-blended PLGA NFs and pure PLGA NFs under tensile loading. Mechanical evaluation of both NFs shows a linear segment up to the proportionality limit followed by a non-linear curve. Incorporation of collagen in NFs leads to a significant decrease in tensile strength and ultimate strain (p < 0.05). Tensile properties of the NFs obtained from two independent experiments as well as the human skin [12] are also summarized in Table 1. The tensile modulus of the collagen blended NF overlaps well with that of the human skin. Moreover, compared with PLGA NFs, blended NFs have a more desirable ultimate strain value, which is within the value range of human skin. C D Even though the Ultimate Tensile Stress (MPa) value of blended NFs is low compared to that of human skin, it doesn t weaken its potential as skin grafts, since they are seldom under a high tensile strength when immobilized on the wound sites. On the other hand, its desirable tensile modulus value similar to that of human skin provides it with good resilience and compliance to movement as skin grafts. Fig. 2 SEM images (x1600) of collagen-blended PLGA NFs prepared in different concentrations (w:v) in HFP (A: 4.5%, B: 5%, C: 6%, D: 8%) E F G Fig. 3 TEM images of collagen-blended PLGA NFs (E), Pure PLGA NFs (F) and Pure collagen NFs (G). Attenuated Total Reflectance-Fourier Transform Infrared (ATR-FTIR) The chemical composition of collagen-blended PLGA NFs was verified by ATR-FTIR spectrometry. Fig. 4 shows the infrared spectra of pure collagen NFs (Fig. 4A), pure PLGA NFs (Fig. 4B), and collagen-blended PLGA NFs (Fig. 4C). Spectra of blended NFs revealed peaks characteristic of type I collagen at wave numbers of 3310 cm -1 (N-H stretch), 1649 cm -1 (amide I bond) and 1550 cm -1 (amide II bond) and a PLGA peak at 1759 cm -1 (ester C=O bond), verifying that blended NFs are a mixture of PLGA and collagen. Since ATR-FTIR can probe 100nm into the surface of materials, therefore, in order to further understand its further surface composition, the XPS (X-ray Photoelectron Spectroscopy should be employed. Fig. 4 ATR-FTIR spectra of (A) Pure collagen NFs. (B) Pure PLGA NFs. (C) Collagen-blended PLGA NFs. 264

Tensi l e St r ess ( MPa) St r ess- st r ai n cur ve f or t he el ect r ospun nanof i ber s 6 5 4 3 2 1 0-0.5 0 0.5 1 1.5 2 2.5 Tensile Strain (%) Col l agen- bl ended PLGA PLGA Fig. 5 Typical stress-strain curves for elctrospun pure PLGA NFs (above) and collagen-blended PLGA NFs (below). Measurement of Wettability through Contact Angle Assessment The mean water in air contact angle observed for blended NFs was 66.4 ± 3.3, which is significantly lower than pure PLGA NFs 126.7 ± 4.5. The decreased contact angle value in collagen-blended PLGA NFs substrate indicates its decreased hydrophobicity and enhanced capability for cell attachment. Tensile (MPa) Ultimate Stress (MPa) Ultimate (%) modulus Tensile Strain PLGA Collagen- blended PLGA Human Skin 185.64 ± 14.12 13.10 ± 6.38 15-150 4.86 ± 0.31 0.74 ± 0.14 5-30 179 ± 20 97 ± 15 35-115 Table 1 Tensile Properties of Electrospun NFs and Human Skin 4. DISCUSSION In this study, the biodegradable PLGA and collagen were mixed at a ratio (w:w 1:1) with different concentrations in HFP (w:v 4.5%, 5%, 6%, 8%). At low concentrations (4.5%, 5%), defects in the form of beads and droplets can be observed because the process under these conditions was characteristic of electrospaying rather than electro spinning [13]. Increasing concentration up to 8% yielded uniform fibers with non-droplet, non-beads, inter -connected favorable nanofiberous structures. When further the concentration reaches 10%, 12%, we found that the NFs still showed smooth and uniform morphology similar to those at 8% except the diameter increased up to 397 ± 145 nm, 427 ± 199nm respectively, still within nanoscale (pictures not shown). In consideration of the high cost of PLGA and collagen, we choose 8% as the best concentration for electrospinning the solution. Herein we applied the efficient, rapid processing techniques - electrospinning to fabricate NFs. Besides, proteins can be introduced onto a substrate surface by other methods including physical coating [20] and chemical grafting [21]. However, one of the problems these surface modification techniques face is the slow mass transfer of proteins into the three-dimensional porous materials. For example, it took more than 3 days to effectively graft gelatin molecules onto poly (ethylene terephthalate) (PET) NF surfaces [21]. Compared with the surface modification of polymer NFs, direct electrospinning of a blended collagen polymer mixture is much simpler because it avoids the slow mass transfer process and also uses lesser amounts of chemical reagents during protein immobilization. The blending method has an additional potential to readily refine the composition of NFs by adding new components such as growth factors, proteoglycan, and glycoproteins to the NFs according to different cell types. Moreover, the existence of collagen molecules on the surface and inside the NFs provides sustained cell recognition signals with polymer degradation, which is important for cell function development. Also, blended NFs may have improved mechanical strength compared with pure non-cross-linked collagen NFs, which would combine the advantages of both synthetic and natural materials [1]. Bone Marrow-derived stem cells were reported to facilitate skin regeneration in both acute and chronic wounds and deep burns [14-16]. Therefore, the in vitro biocompatibility studies are needed to observe the cell -scaffold interactions. Other ECM components such as fibronectin, hyaluronic acid, glycosaminoglycans (GAGs) [17,18] or other ligands or growth factors like epidermal growth factor (EGF) and keratinocyte growth factor (KGF) plateletderived growth factor (PDGF) can be loaded on the NF scaffold to improve the treatment for non-healing epidermal wounds and/or acute large-defect epidermal wounds [19]. Finally, the animal test is also required to evaluate the efficacy of fabricated NF scaffolds as skin grafts in wound healing. 5. CONCLUSION The electrospinning technique was employed to fabricate collagen-blended PLGA (75:25) NFs. 8% has been chosen as the optimized concentration for preparing the polymer solution in HFP (w:v) because NFs electrospun under this parameter shows smooth surface morphology and a well-controlled diameter distribution. The existence of collagen in the blended NFs was verified by ATR-FTIR. In addition, the blended NFs showed their desirable mechanical properties and improved biocompatibility due to enhanced hydrophilicity relative to pure polymer NFs and we can also refine the composition of NFs readily by loading necessary ingredients according to different cell types. All of these advantages strongly indicate a great potential of the collagen-blended NFs for skin tissue 265

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