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Keywords:

  • electrospinning;
  • gelatin;
  • PLA;
  • skin;
  • tissue engineering

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. References

Repair or replacement of damaged tissues using tissue engineering technology is considered to be a fine solution for enhanced treatment of different diseases such as skin diseases. Although the nanofibers made of synthetic degradable polymers, such as polylactic acid (PLA), have been widely used in the medical field, they do not favour cellular adhesion and proliferation. To enhance cell adherence on scaffold and improve biocompatibility, the surface of PLA scaffold was modified by gelatin in our experiments. For electrospinning, PLA and gelatin were dissolved in hexafluoroisopropanol (HFIP) solvent at varying compositions (PLA:gelatin at 3:7 and 7:3). The properties of the blending nanofiber scaffold were investigated by Fourier transform infrared (FT-IR) spectroscopy and scanning electron microscopy (SEM). Modified PLA/gelatin 7/3 scaffold is more suitable for fibroblasts attachment and viability than the PLA or gelatin nanofiber alone. Thus fibroblast cultured on PLA/gelatin scaffold could be an alternative way to improve skin wound healing.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. References

As the largest organ in the body, the skin is vital for survival through maintaining homeostasis, performing immune surveillance and protecting body tissues from different types of damage, including those resulting from infections and burns (Atiyeh et al., 2005). Treatment of burns with cultured skin substitutes is now routine, but has been limited by poor engraftment and scarring. Tissue engineering implantation with suitable cells, such as fibroblasts and keratinocytes, are desirable to promote tissue regeneration (Wu and Ding, 2005; Tangsadthakun et al., 2007; Heunis and Dicks, 2010). It is a simple and effective technique widely used for generating biological tissue to repair, replace or maintain the function of defective or damaged tissues (Liu et al., 2010). In this regard, scaffolding materials can be used for cell attachment, proliferation, differentiation and new tissue generation. An ideal scaffold should have optimal mechanical strength, non-toxic degradation products, biodegradability and, most importantly, excellent biocompatibility (Mei et al., 2005, 2010; Xie et al., 2009). One of the best methods for ideal scaffold synthesis is electrospinning (Meng et al., 2010; Wang et al., 2012). This is a low-cost, novel and simple technique (Xu et al., 2011) for generating extremely long fibers with diameters on both the micro- and nanoscale (Yim and Leong, 2005; Baharvand et al., 2006; Hashemi et al., 2009). Various synthetic polymers have been utilised, among which polylactic acid (PLA) has many advantages and considerable potential.

Many methods on surface modification and hydrophilicity of scaffolds have been employed to improve cellular adhesion (Zhang et al., 2005; Kim et al., 2008; Meng et al., 2010; Wang et al., 2013). Modification of synthetic materials with natural products is one of the most effective methods (Chen, 2005; Mei et al., 2005; Ghasemi-Mobarakeh et al., 2009; Xu et al., 2011; Browning et al., 2011; Xing et al., 2013). Gelatin promotes cell differentiation, proliferation and adhesion; it also has good biocompatibility and biodegradation. Gelatin is used because it mimics natural extracellular matrix (ECM) architecture and is cheap (Ma et al., 2003; Tangsadthakun et al., 2007; Kanokpanont et al., 2007; Gui-Bo et al., 2010; Meng et al., 2010; Wang et al., 2012). In this study, pure PLA, pure gelatin and PLA/gelatin were directly dissolved in hexafluoroisopropanol (HFIP) before nanofibers were synthesised by electrospinning. The method optimises the hydrophilicity and cellular affinity using the gelatin/PLA blending scaffold coated with matrigel. Fibroblast cultured scaffolds were implanted in the wound area.

Materials and methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. References

Materials

PLA, gelatin and HFIP were purchased from Sigma. The human embryonic fibroblasts (STO) were obtained from Iran Pasteur Institute, Tehran. The culture media was Dulbecco's Modified Eagle's Medium (DMEM, Gibco, Carlsbad, CA, USA) supplemented with 10% (V/V) heat-inactivated fetal bovine serum (FBS, Gibco), 100 U/mL penicillin and 100 mg/mL streptomycin (Gibco). Other reagents used included EGF, FGFb were purchased from Gibco; 0.25% trypsinase and 3-(4,5-dimethylthiazol-2-yl)-diphenyltetrazolium bromide (MTT) were purchased from Sigma Company.

Electrospinning and preparation of the scaffold

Electrospinning conditions of the pure PLA (MW = 5,000) and gelatin (type A), and also he PLA/gelatin blending nanofibers, were as previously reported (Kim et al., 2008; Yu et al., 2008; Meng et al., 2010; Xu et al., 2011; Browning et al., 2011; Wang et al., 2012). Different concentrations of PLA and gelatin solutions (2.5 and 8%, w/v respectively) with good spinnability were electrospun (dissolved in HFIP). To prepare blending polymers, the PLA and gelatin solutions were mixed in two ratios (7:3 and 3:7). A fluid jet was ejected (10–16 kV) from a 22 ga needle at a flow rate of 0.5 mL h−1 towards a grounded collector with 50 mm diameter at the rate of 1,000 rpm. The samples were placed in a vacuum drying oven overnight to evaporate remaining solvent.

Material characterisations

For analysis of the morphology of the fibers, the samples were sputter-coated with gold and examined by scanning electron microscopy (SEM, XL-30, Philips, Netherland) at 20 kV. The diameter of the nanofibers was measured from 20 different, arbitrarily selected samples by analysing SEM images with a measurement software program and averaged. Cell-containing scaffolds were taken out from the culture plate and rinsed with PBS gently before the specimens were fixed with 2.5% glutaraldehyde in PBS for 1–2 h, followed by washing with PBS, and subsequent dehydration sequentially in 30, 50, 70, 80, 90 and 100% ethanol at 37°C for 10–15 min.

Fourier transform infrared (FT-IR) spectroscopy

FT-IR spectra were obtained in the spectral region of 4,000–400 cm−1; curves were obtained through a thermal analysis instrument at a heating rate of 10°C/min, scan range of 40–400°C, and a nitrogen gas flow-rate of 120 mL/min.

Swelling ratio determination

The swelling ratio (Sr) of the electrospun nanofibers was examined by immersing the samples within a phosphate buffer saline (PBS) pH 7.4 at a temperature of 37°C. The swelling ratio was determined by the following formula: Sr = (W2 − W1) × 100 (where W1 = dry weight and W2 = wet weight).

In vitro degradation and pH

In vitro biodegradation test of the scaffolds with different ratios was carried out by calculating weight. Three rectangular (15 × 15 mm2) scaffolds of each type were immersed in phosphate buffered saline (PBS, pH 7.4) at 37°C for 12 weeks. After 2, 4, 6, 8, 10 and 12 weeks, samples were removed from the medium, rinsed with distilled water, dried, weighed and the pH of the PBS solution was measured by pH meter.

Cell culture on the scaffolds

Human embryonic fibroblasts (STO) were cultured in DMEMu, supplemented with 10% FBS and 50 U/mL penicillin and 50 U/mL streptomycin, with the medium being changed every 2 days. STO fibroblasts were plated on the prepared nanofiber specimens (15 mm in diameter, sterilised with UV and coated with 1% matrigel) with a final seeding density of 5 × 04 cells/scaffold (pure gelatin, PLA:gelatin at 3:7, PLA:gelatin at 7:3 and pure PLA) in 24-well plates and incubated in DMEM supplemented with 10 ng/mL FGFb (basic fibroblast growth factor) and 10 ng/mL EGF (epidermal growth factor) under standard condition.

Cell viability and proliferation assay

The relative cell viability of cultured fibroblasts on the scaffolds was evaluated with 3-(4,5-dimethylthiazol-2-yl)-diphenyltetrazolium bromide (MTT) at 1, 3, 5 and 7 days. Briefly, 30 µL of MTT solution (5 mg/mL) was freshly added to each well and incubated at 37°C for 4 h for the MTT formazan formation. After removing the medium, the intracellular formozan was dissolved in 300 mL dimethyl sulfoxide (DMSO). The absorbance at 570 nm was measured with a microplate reader (Expert 96, Asys Hitch, Ec Austria).

Attachment and spreading of human fibroblasts on scaffold

DAPI staining was used to assess cell adhesion and spreading. Scaffold matrices were seeded with human fibroblasts and cultured in DMEM supplement with FGF and EGF for 1, 3 and 5 days. After fixation, cell attachment and proliferation was quantitatively measured after DAPI staining.

In vivo and histological analysis

Twelve male Wistar rats (8 weeks old, 200–250 g), obtained from the Pasteur Institute of Iran chosen for in vivo experiments, were divided randomly into two groups. All rats were anesthetised with an intraperitoneal injection of a mixture of ketamine, xylazine and acepromazine (25:5:1 mg/mL at 1 mL/kg body weight). The dorsal skin (1 cm in diameter) was burned (first degree burn) and subcutaneous pockets were made in the backs of animals. In the treatment group, the wounds on the rat's body were filled with fibroblast cultured scaffolds (PLA/gelatin 7:3). Rats without cell implantation were used as controls. All rats were followed for 3 weeks after transplantation. The affected sites were removed from euthanised animals (associated with the surrounding skin) and fixed immediately in 10% buffered formalin, dehydrated, and embedded in paraffin blocks. Seven micrometre paraffin sections were stained with hematoxylin–eosin (H&E) and Masson's trichrome stain for histological observations using standard protocols. Histological analysis was carried out using a light microscope (Olympus, 1 × 51, USA) and image tool software (version 3). Cell density, morphology and collagen deposition were determined on stained sections. For determining cell density, a random sample of recovered wound images (1.0 mm2) was taken in high magnification (400 and 1,000), and nuclei of the cells were counted by image tool software. Collagen deposition was quantified by the image tool software. The results were the average of at least six specimens.

Statistical analysis

Statistical analysis was performed by one-way ANOVA analysis followed by the paired t-test. Statistical calculations used the SPSS system for windows, version 16, Statistical Package for Social Sciences (SPSS). P < 0.05 was considered to be statistically significant. Each value was averaged from three parallel measurements.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. References

Morphology of electrospun nanofiber and cell seeding

SEM micrographs of the gelatin and PLA (Figures 1a and 1b) show continuous fibrous structure with an average diameter of 150 and 650 nm, respectively. Significant changes in the average fiber diameter of PLA fibers that were electrospun with different amounts of gelatin are shown in Figure 1c and d. The composite scaffolds composed of PLA nanofibers and gelatin (PLA/gelatin 3:7, 7:3 w/w) had average fiber diameters of 250 and 350 nm, respectively (Figures 1c and 1d). Gelatin was distributed uniformly in the PLA fibrous structure, and appeared to adhere strongly to PLA nanofibers. Fiber diameter decreased significantly with increase in the amount of gelatin (Figure 1). These findings could be attributed to the changes in conductivity and viscosity influenced by the gelatin content. These findings are similar to the results from other studies in which gelatin was added to various polymers to decrease fiber diameter (Ma et al., 2003; Meng et al., 2010; Xu et al., 2011).

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Figure 1. (a) SEM micrographs with and without cells. (b) Diameters of the nanofibers. The fiber size is reported as the average. (a) The fibers of pure gelatin scaffold (b) The fibers of pure PLA scaffold (c) The fibers of PLA/gelatin (3:7) scaffold (d) The fibers of PLA/gelatin (7:3) scaffold (e and f) plated fibroblast cells in PLA/gelatin (7:3) scaffold with matrigel covering.

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The behaviour of cells on biomaterial depends on the scaffold's surface characteristics. Cell morphology and behavior on the PLA/gelatin scaffold during the culture by SEM shows that they appear to have spread themselves favourably over the scaffold surface (Figures 1e and 1f). After 5 days, cells had proliferated significantly on PLA/gelatin and covered almost all the surface. Our observations show that a large surface area benefits the excellent in-growth of cells; hence the cells were well distributed and firmly attached on the surface of the PLA/gelatin scaffold.

We chose PLA/gelatin (7:3) scaffold because it has superior properties for cellular adhesion and proliferation; these results indicate that gelatin modification dramatically enhances the cellular affinity and compatibility of PLA scaffold.

Cell adhesion and proliferation test

STO cells (Figure 2) were seeded on scaffolds and cultured in DMEM medium for 5 days. Cell number increased with time on each scaffold and a great number of fibroblasts could be grown on the surfaces of scaffolds. In cell adhesion on day 1 after seeding, there was no significant difference between gelatin, PLA/gelatin (3:7 and 7:3), and PLA scaffolds (Figure 3). Scaffolds with 30% gelatin showed a significantly higher (P < 0.05) relative cellular proliferation compared to PLA and gelatin scaffolds at day 3 and 5 (Figure 3). These results demonstrated that the composition of scaffolds had a positive effect on the cell growth, and the gelatin could promote cellular adhesion and proliferation.

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Figure 2. Optical microscopy images of STO fibroblast cells, 40× (Bar = 50 µm).

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Figure 3. Fluorescent microscopic observation of fibroblast cells cultured on scaffolds for (A) 1 day (B) 3 days (C) 5 days. The cells are stained by DAPI. And Cell density within the section samples, 40× (Bar indicates = 100 µm, *P < 0.05 and Values are mean (n = 3)).

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Swelling test

The swelling ratios of various scaffolds are shown in Figure 4. In descending order, gelatin showed the best swelling (456%), whereas the swelling ratio of PLA/gelatin scaffold (3:7) was a little less than gelatin at 388%. PLA/gelatin scaffold (7:3) was the next one at 358% swelling and the fourth is related to PLA at 276%. PLA is a hydrophobic material, so PBS solution could not easily penetrate into the interior pores of the scaffold. Swelling of PLA/gelatin could be attributed to both PLA and the hydrophilicity property of gelatin, which decreases when PLA increases.

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Figure 4. Equilibrium swelling ratio of PLA/gelatin scaffolds with different blending compositions. Values are mean (n = 3).

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In vitro biodegradability and pH change of PBS solution

Figure 5 compares the biodegradation degree of gelatin, PLA scaffold and the PLA/gelatin scaffolds. These results show that the presence of PLA can significantly improve the biostability of gelatin. Among the scaffolds, gelatin had the highest weight reduction with the total weight loss after 8 weeks, whereas PLA remained relatively constant over 12 weeks. Therefore composition of gelatin with polymers such as PLA could prolong biodegradability. A dimensional decrease of gelatin scaffolds was seen prior to the stage where it gradually became brittle. PLA/gelatin (3:7) and pure gelatin scaffolds had dramatic weight loss after 10 and 8 weeks, respectively (Figure 5).

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Figure 5. Time history of relative weight of scaffold samples after incubation for 12 weeks. Values are mean (n = 3).

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The pH value decreased gradually during immersion in PBS, which could be attributed to the formation of acidic degradation products from the scaffolds. The pH change was substantially synchronised with weight loss. No significant decrease in pH was detected during the whole degradation period, and the lowest pH was accompanied with the highest percentage weight loss (Figure 6).

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Figure 6. Time history of pH value of the PLA/gelatin scaffold sample.

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FT-IR spectrophotometric analysis

For PLA fibers, the spectrum shows the characteristic absorption band at 1,754 cm−1 which represents C[DOUBLE BOND]O carbonyl group, and the C[BOND]O[DOUBLE BOND]C stretching at 1,090–1,187 cm−1 characteristics of ester bonds. Another moderate intensity peak is at 1,456 cm−1, which might be related to bending vibration of CH3. For gelatin, the two main characteristic peaks were at 1,532 and 1,645 cm−1, which respectively represent amide I and amide ΙΙ functional groups of gelatin. Another broad peak with moderate intensity at 3,000–3,600 cm−1 relates to N[BOND]H and OH[BOND]H stretching vibration and intermolecular hydrogen bonding. For PLA/gelatin blend fibers with two PLA to gelatin ratio (spectrum C and D), in addition to PLA characteristics peaks, two amide І and amide ІІ peaks are also visible. In PLA/gelatin (3:7) (spectrum C) two amides peak has more intensity than PLA/gelatin with ratio (7:3) (spectrum D). These results confirm other studies. The peak place of these spectra (C and D) is not changed dramatically. These show that PLA/gelatin blend fibers were mixed physically and no chemical bonds were formed between the two polymers (Figure 7).

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Figure 7. FT-IR spectra of PLA/gelatim scaffolds with different blending compositions: (A) pure PLA, (B) Pure gelatin, (C) PLA/gelatin (3:7) and (D) PLA/gelatin (7:3). Values are mean (n = 3).

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Cell viability

The viability of STO cells seeded was followed for 7 days. After 1 day there was no significant difference (Figure 8). However there was a remarkable increase in cell viability on the PLA/gelatin (7:3) scaffolds after 7 days of culture, which showed considerably more cell adherence on the PLA/gelatin scaffold than the others after 3 and 5 days. The PLA modification with gelatin could significantly improve the viability and attachment of fibroblasts.

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Figure 8. MTT assay. Formosan absorbance expressed as a measure of cell viability from the fibroblast cells cultured on nanofibrous scaffold for a 7-day period (*P < 0.05 and values are mean (n = 3)).

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Histology

The histological results of the PLA/gelatin scaffold embedded in the rat skin as a wound healing study are shown in Figure 9. Macroscopic observations had indicated a faster healing of wound in treatment group (wounds dressed with fibroblasts cultured scaffolds). In the treated group, the epidermal and dermal layers seemed to be in a normal condition after 3 weeks. The epidermal layer was thick, compact and dark. A natural level of collagen production and fibroblast proliferation could also be seen in the dermal layer (Figure 9). However, we saw re-epithelialisation, acanthotic epidermis, granulation tissue, mild infiltration of acute inflammatory cells, chronic inflammatory cells, reticular collagen deposition, dermal appendages and decreased thickness of dermis in control samples (Figure 9). We also detected swelling and destruction of epidermis, surface necrotic, massive neutrophilic infiltration, dermal destruction and edema in wheal areas after 3 weeks. The difference between healing rates in treatment and control groups were completely clear after 21 days and recovery was significantly better in the treated group than controls. Comparison of fibroblast cell number, and percentage of collagen synthesis in control and treated samples, also represents a significant increase in treated sample after 14 and 21 days (Figures 10 and 11).

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Figure 9. The histological response to the PLA/gelatin scaffolds after being embedded in rat skin for 21 days. Stains: hematoxylin and eosin (H&E), Masson's trichrome (MT), 100×, Bar indicates 500 µm.

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Figure 10. Cell density within the section samples. Representative H&E-stained sections of 21-day samples are shown. Gradual increase in cell density is shown.

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Figure 11. Percent collagen synthesis within the control and treatment samples. Representative Masson's trichrome-stained sections of 14 and 21-day samples are shown.

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After implantation in the skin, the scaffolds were gradually degraded and finally replaced by the regenerating tissue. These results demonstrate that the cell seeding scaffolds are efficient and helpful in wound recovery, a process which fibroblasts can accelerate.

Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. References

Natural ECM has an important function in growth, proliferation and behaviour of cells. A synthetic three-dimensional system to replace natural ECM is one of the most important goals in tissue engineering. Structural proteins such as collagen and gelatin are crucial for the mechanical support of tissues, and provide attachment sites for cell receptors (Gillette et al., 2011). Having a similar chemical structure to collagen (Wu and Ding, 2005; Canbolat et al., 2011) also, splitting and elongation of the jet became easy as gelatin content increased (Gui-Bo et al., 2010; You-Zhu, 2010; Gillette et al., 2011; Rossen, 2011). (Missing material – please amend and rephrase.) Thus, we used gelatin to generate suitable scaffolds.

In the electrospinning method, the characteristics of the solvent (e.g. volatility and polarity) have important effects on the morphology and diameter of the fibers. The use of polar solvents with low surface tension, such as HFIP, generally produce ultra-thin fibers with a smaller average diameter (Xie et al., 2009). Therefore, we preferred HFIP as the solvent for the PLA and gelatin nanofiber matrix.

Cytocompatibility, with a potential application as wound dressing materials in tissue engineering, is necessary in cell–scaffold interactions; cell adhesion, proliferation and spreading on scaffolds contribute crucially to tissue regeneration (Kanokpanont et al., 2007; Tangsadthakun et al., 2007; Park and Daley, 2009; Domingos et al., 2013). To achieve this goal, we used gelatin associated PLA covered with matrigel as a natural polymer to improve the hydrophilicity of scaffolds. Gelatin probably provides much more amino groups for cell adhesion and proliferation (Mei et al., 2005; Chen et al., 2005; Willerth, 2009; Xie et al., 2009; Gui-Bo et al., 2010; You-Zhu, 2010; Xu et al., 2011).

Degradation of scaffold plays a crucial role in tissue regeneration (Wu and Ding, 2005; Li et al., 2008). Its rate affects cell vitality, growth and even host response (Moisenovich et al., 2012). It is accepted that the ideal in vivo degradation rate is equal to or slightly less than the rate of tissue formation (Wu and Ding, 2005).

Another important feature is survival and proliferation of cells cultured on the scaffolds. Our results show that the PLA modification with gelatin could significantly improve the viability of fibroblasts. In general, three-dimensional nanofibrous scaffolds have a high surface area to volume ratio and provide a more suitable substrate for cell attachment, growth, proliferation and immigration (Xu et al., 2011; Browning et al., 2011).

Our results (Lai, 2013) show that modified scaffold with fibroblasts can be used for clinical treatment. After implantation in the skin, the scaffolds are gradually degraded and finally replaced by the regenerating tissue. These findings demonstrate that the fibroblast seeded scaffolds are very efficient in wound recovery.

In conclusion, we studied the structural properties and cytocompatibility of the gelatin-modified PLA scaffold in a rat wound healing model. PLA/gelatin nanofiber structures were obtained by electrospinning and the average fiber diameter was 350 nm, found to be more desirable to mimic the natural extracellular matrix. Importantly, the cell adhesion and proliferation on the PLA/gelatin scaffolds were considerably improved after modification. The results suggest that the cell culture using PLA-based scaffolds containing 30% gelatin enhanced fibroblast adhesion and proliferation compared to PLA scaffold alone. Histopathological analyses also confirmed the formation of new tissue, dermis and epidermis, after 21 days in fibroblast cultured PLA/gelatin scaffold. The research has developed a simple but efficient modification, enhancing the potential application of PLA scaffold in the field of tissue engineering and wound healing.

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. References
  • Atiyeh BS, Hayek SN, Gunn SW (2005) New technologies for burn wound closure and healing-review of the literature. Burns J Int Soc Burn Injuries 31: 94456.
  • Baharvand H, Hashemi SM, Kazemi Ashtiani S, Farrokhi A (2006) Differentiation of human embryonic stem cells into hepatocytes in 2D and 3D culture systems in vitro. Int J Dev Biol 50: 64552.
  • Canbolat MF, Tang C, Bernacki SH, Pourdeyhimi B, Khan S (2011) Mammalian cell viability in electrospun composite nanofiber structures. Macromol Biosci 11: 134656.
  • Domingos M, Intranuovo F, Gloria A, Gristina R, Ambrosio L, Bartolo PJ, Favia P (2013) Improved osteoblast cell affinity on plasma-modified 3-D extruded PCL scaffolds. Acta Biomater. 9: 59976005.
  • Ghasemi-Mobarakeh L, Morshed M, Karbalaie K, Fesharaki MA, Nematallahi M, Nasr-Esfahani MH, Baharvand H (2009) The thickness of electrospun poly (epsilon-caprolactone) nanofibrous scaffolds influences cell proliferation. Int J Artif Organs 32: 1508.
  • Gillette BM, Rossen NS, Das N, Leong D, Wang M, Dugar A, Sia SK (2011) Engineering extracellular matrix structure in 3D multiphase tissues. Biomaterials 32: 806776.
  • Gui-Bo Y, You-Zhu Z, Shu-Dong W, De-Bing S, Zhi-Hui D Wei-Guo F (2010) Study of the electrospun PLA/silk fibroin-gelatin composite nanofibrous scaffold for tissue engineering. J Biomed Mater Res A 93: 15863.
  • Hashemi SM, Soleimani M, Zargarian SS, Haddadi-Asl V, Ahmadbeigi N, Soudi S, Gheisari Y, Hajarizadeh A, Mohammadi Y (2009) In vitro differentiation of human cord blood-derived unrestricted somatic stem cells into hepatocyte-like cells on poly(epsilon-caprolactone) nanofiber scaffolds. Cells Tissues Organs 190: 13549.
  • Heunis TD, Dicks LM (2010) Nanofibers offer alternative ways to the treatment of skin infections. J Biomed Biotechnol doi: 10.1155/2010/510682
  • Kim HW, Yu HS, Lee HH (2008) Nanofibrous matrices of poly(lactic acid) and gelatin polymeric blends for the improvement of cellular responses. J Biomed Mater Res A 87: 2532.
  • Lai JY (2013) Corneal stromal cell growth on gelatin/chondroitin sulfate scaffolds modified at different NHS/EDC molar ratios. Int J Mol Sci 14: 203655.
  • Li W, Guo Y, Wang H, Shi D, Liang C, Ye Z, Qing F, Gong J (2008) Electrospun nanofibers immobilized with collagen for neural stem cells culture. J Mater Sci Mater Med 19: 84754.
  • Liu T, Zhang S, Chen X, Li G, Wang Y (2010) Hepatic differentiation of mouse embryonic stem cells in three-dimensional polymer scaffolds. Tissue Eng Part A 16: 111522.
  • Ma L, Gao C, Mao Z, Zhou J, Shen J, Hu X, Han C (2003) Collagen/chitosan porous scaffolds with improved biostability for skin tissue engineering. Biomaterials 24: 483341.
  • Mei N, Chen G, Zhou P, Chen X, Shao ZZ, Pan LF, Wu CG (2005) Biocompatibility of Poly(epsilon-caprolactone) scaffold modified by chitosan – the fibroblasts proliferation in vitro. J Biomater Appl 19: 32339.
  • Meng ZX, Wang YS, Ma C, Zheng W, Li L, Zheng YF (2010) Electrospinning of PLGA/gelatin randomly-oriented and aligned nanofibers as potential scaffold in tissue engineering. Mater Sci Eng C 30: 120410.
  • Moisenovich MM, Pustovalova O, Shackelford J, Vasiljeva TV, Druzhinina TV, Kamenchuk YA, Guzeev VV, Sokolova OS, Bogush VG, Debabov VG, Kirpichnikov MP, Agapov II (2012) Tissue regeneration in vivo within recombinant spidroin 1 scaffolds. Biomaterials 33: 388798.
  • Park IH, Daley GQ (2009) Human iPS cell derivation/reprogramming. Curr Protoc Stem Cell Biol Unit 4A.1. doi: 10.1002/9780470151808.sc04a01s8
  • Tangsadthakun C, Kanokpanont S, Sanchavanakit N, Pichyangkura R, Banaprasert T, Tabata Y, Damrongsakkul S (2007) The influence of molecular weight of chitosan on the physical and biological properties of collagen/chitosan scaffolds. J Biomater Sci Polym Ed 18: 14763.
  • Wu L, Ding J (2005) Effects of porosity and pore size on in vitro degradation of three-dimensional porous poly(D,L-lactide-co-glycolide) scaffolds for tissue engineering. J Biomed Mater Res A 75: 76777.
  • Xie J, Willerth SM, Li X, Macewan MR, Rader A, Sakiyama-Elbert SE, Xia Y (2009) The differentiation of embryonic stem cells seeded on electrospun nanofibers into neural lineages. Biomaterials 30: 35462.
  • Xing ZC, Han SJ, Shin YS, Koo TH, Moon S, Jeong Y, Kang IK (2013) Enhanced osteoblast responses to poly(methyl methacrylate)/hydroxyapatite electrospun nanocomposites for bone tissue engineering. J Biomater Sci Polym Ed 24: 6176.
  • Xu X, Browning VL, Odorico JS (2011) Activin, BMP and FGF pathways cooperate to promote endoderm and pancreatic lineage cell differentiation from human embryonic stem cells. Mech Dev 128: 41227.
  • Yim EK, Leong KW (2005) Proliferation and differentiation of human embryonic germ cell derivatives in bioactive polymeric fibrous scaffold. J Biomater Sci Polym Ed 16: 1193217.
  • Zhang S, Gelain F, Zhao X (2005) Designer self-assembling peptide nanofiber scaffolds for 3D tissue cell cultures. Semin Cancer Biol 15: 41320.