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- Materials and methods
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.
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- Materials and methods
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.