Composite Scaffold Of Gelatin And PLLA For Corneal Tissue Engineering

Tissue engineering is an interdisciplinary feld that applies the principles ofengineering and the life sciences toward the development of biological substitutes thatrestore, maintain or improve tissue function. Corneal repair and regeneration hasfocused on the construction of tissue engineered substitutes, which is challenged by itshigher transparency, mechanical properties and organizations. Therefore, how todevelop an ideal scaffold suitable for cell growth and tissue regeneration is one of theproblems that are most urgent to be solved in this field.Electrospinning provides a simple and cost-effective means to produce scaffoldswith3-diamentional inter-connected pore structure, fiber diameters in the sub-micronrange. Electrospun fibers have been investigated as promising tissue engineeringscaffolds since they mimic the nanoscale properties of native extracellular matrix. Whileinterest in electrospinning has recently escalated in tissue enginnering, the strategy ofelectrospinning is undoubtedly one of the best methods for the research in mechanismof interactions between scaffold and corneal cells. Compared to gel scaffold, which wascommon in corneal tissue engineering, research in the application of electrospun fibrousmembranes for corneal tissue engineering is still rare reports. In this dissertation, we apply the strategies of electrospinning and principles oftissue engineering to study the preparation of suitable scaffolds and the construction oftissue substitutes. Based on development tendency of tissue regeneration and restoration,we designed a novel electrospinning process; according to the ideas and strategies ofcells in vitro culture and tissue engineering, we successfully prepared a series of randomand aligned gelatin-PLLA fibrous scaffolds, achieved controlled biomechanicalproperties such as geometric configuration, pore structure, mechanical property anddegradation characteristics, and so on, and preliminary evaluated the correspondingcells compatibility and corneal damaged repair performance.Firstly, to study what role does the electrospinning process play in the fibermorphology, and whether it is possible to achieve control of biomechanical propertiesby tuning material composition, we selected a traditional electrospinning technologywith a plate as collector to prepare a series of gelatin-PLLA composite fibrousmembranes. Results show that the best fiber morphology can be acquired underappropriate electrospinning process parameters, and the water content, porosity,transmittance as well as degradation safety improve with the addition of PLLA. In short,we can achieve control of biomechanical properties through tuning materialcomposition and electrospinning process parameters.Secondly, to achieve controlled alignment of electrospun fibers, we designed novelelectrospinning processes by using a parallel plate and a rotating drum as the collector,and conducted a detailed study on their corresponding role in the fiber alignment andmorphology. Interactions between fiber chemical composition, scaffold physicalstructure and their corresponding biomechanical properties were also researched in thissection. Results show that controllable fiber alignment can be achieved through tuningelectrospinning process parameters, the tensile properties and transmittance of scaffoldcan be improved through highly ordered fibers, physical structure and biomechanicalproperties of scaffold can be influenced significantly by fiber alignment, while patterns and rate of scaffold degradation are mainly determined by fiber chemical composition.All these experimental results show that we can control the alignment of gelatin-PLLAfibers through improved electrospinning process.Thirdly, to design functional structured scaffold, we established mature technologyof corneal cells separation and cultivation combined with tissue engineering and cellular,molecular biology technology. The effect of fiber alignment and chemical compositionon corneal cells compatibility was conducted, and the guided mechanism of materialsand structures of scaffold on corneal cells was speculated. The effect of fber alignmenton cells behavior was evaluated by cell morphology, specifc protein expression, adhesion, and proliferation. Different corneal cells respond uniquely to fber alignment ofscaffold, keratocytes interacting more favorably on alignment scaffold and cornealepithelial cells more favorably on randomly oriented scaffold. These results confrm thatfber alignment of scaffold should be beneft for cell proliferation if its contact guidancecoincided with the cell shape and cytoskeletal tension. The study also found that moregelatin in fibers can induce higher proliferative activity, faster growth rate and betterfunctionality of corneal cells can be guided by. These results confrm that cellproliferation and adhesion ability can be regulated by fiber chemical composition whichhas impact on surface chemical properties, porosity and degradation rate of electrospungelatin-PLLA scaffold. In short, we achieve comprehensive control of the surfaceproperties, structure parameters and mechanical strength as well as degradeperformance of electrospun gelatin-PLLA scaffold through turning fiber alignment andchemical composition, which regulate the corresponding biological function.Fourthly, according to the guidance of fiber alignment on biological behavior ofkeratocytes and the strategy of tissue engineering for tissue repair, we select theuniaxially aligned fibrous scaffold of gelatin-PLLA as tissue engineered scaffold toachieve rabbit corneal stromal damage repair in-situ. Research in tissue repair processshows that physical structure and biomechanical properties of scaffold effect the hestogenesis. Uniaxially aligned fibrous scaffold of gelatin-PLLA, with excellentcorneal tissue compatibility, can successfully activate the native keratocytes, providephysical support, induce migration and maintain growth polarity of these cells.Meanwhile, the synchronous biodegradability of scaffold can provide physical space forregeneration and structure reconstruction of freshman collagen fibers and corneal tissue.Finally, repair is end with the replacement of cambium. The establishment of this repairmodel will promote the clinical application of corneal stromal damage repair in-situ.At last, according to the classic tissue engineering technology and the guidedmechanism of materials and structures of scaffold on corneal epithelial cells, we selectthe random fibrous scaffold of gelatin-PLLA as tissue engineered scaffold to achievereconstruction of corneal epithelial substitute in vitro and future investigated itspotential in corneal epithelial damage repair. Results show that proliferative activity and3-diamentional growth trend of corneal epithelial cells are maintained on the scaffold ofR-G8P2. Meanwhile, damaged corneal epithelium can be repaired by transplantation ofthis transparent and flexile cell-scaffold composite. All of these results demonstrated apossibility of corneal repair and regeneration by tissue engineering with electrospuncomposite fibrous scaffold.In conclusion, in this dissertation, we focus on the design of electrospungelatin-PLLA scaffold with fine properties by electrospinning and the strategy of tissueengineering, and realize different modulation strategy of biological properties of tissueengineered scaffold, such as the electrospinning process, fiber chemical compositionand fiber alignment. All these results provide references for the controlled preparationof such kind of fibrous scaffold for tissue engineering. We believe that these findingswill also provide new strategies for the preparation of complicated structured scaffoldfor more tissue and organ regeneration.