| IntroductionThe cornea is a transparent,avascular tissue on the surface of the human eye.Its integrity is very important for normal vision.It includes layered epithelial cells that maintain the integrity and smoothness of the corneal surface while providing a barrier against external stress.Many factors,such as mechanical,thermal,chemical damage and microbial infections,can cause opacity of the transparent cornea,resulting in vision loss,and even blindness,known as corneal disease.Corneal disease has a high prevalence rate and is blindingly strong.It is the second blindest eye disease in the world,second only to cataracts.Currently,there are about 20 million people in the world who suffer from corneal disease blindness,and our country has existing corneal blindness.There are more than 3 million patients.Allogeneic corneal transplantation is currently the most effective method for patients with corneal diseases.However,because of the lack of donors,immune rejection or biocompatibility,and infection,it is difficult to obtain a good therapeutic effect and meet increasing medical needs.Stem cells in the marginal region present an ability to replace and regenerate the corneal epithelium and participate in the dynamic balance process of the corneal surface.The corneal limbal stem cells have self-regenerating and multi-directional differentiation capabilities,and amniotic membrane-based transplantation has become an effective means of regenerating epithelial cells.However,this method requires a long incubation time.At the same time,we do not know whether the amniotic membrane transplantation provided by the donor will increase the safety of infection,inflammation,and other biological research.In order to solve the above problems,allogenic corneas are urgently needed for artificial corneas.With the rise of cell therapy and tissue engineering,clinical medicine is entering a new stage of "regenerative medicine".Biomaterials,seed cells and cytokines are the three key elements in constructing tissue engineering.In the process of tissue repair,seed cells occupy a very important component.They can accumulate at the site of injury,proliferate,and secrete the extracellular matrix needed for repair.Currently,common seed cells for corneal tissue engineering include limbal stem cells(LESCs),mesenchymal stem cells(MSCs),embryonic stem cells(ESCs)and induced pluripotent stem cells(iPSs).These cells have their own advantages,but also have certain drawbacks,such as limbal stem cells to obtain access is not easy,easily lead to secondary damage.In recent years,induced pluripotent stem cell-derived mesenchymal stem cells(iPSC-MSCs)have shown the same characteristics as stem cells,can be differentiated into various types of tissue cells,and are readily available in vitro.We regard it as a type of Exogenous seed cells were selected.However,no literature has reported the feasibility and effectiveness of transplantation of exogenous iPSC-MSCs to promote corneal repair in vivo.Stromal cell-derived factor(SDF-1)acts to recruit CXCR4-positive cells in various organ repairs.Previous studies have shown that SDF-1 alpha complex thermo-sensitive chitosan/gelatin scaffolds can promote stem cell recruitment and reduce inflammatory cell infiltration.Studies have also confirmed that corneal fibroblasts express SDF-1 and its receptor CXCR4;and the SDF-1/CXCR4 axis is crucial for maintaining the characteristics of LESCs in limbal stem cells.These studies suggest that SDF-1 may have the potential to recruit stem cells to promote corneal repair,but its specific role in corneal injury repair has not been studied.The use of scaffold materials in combination with stem cells or growth factors/cytokines,etc.,can promote the regeneration of damaged tissues and bring new opportunities for improving the quality of repair of corneal and other tissues.In this study,we investigated whether the composite scaffold could further promote the repair of damaged corneas by using composite SDF-1 and temperature-sensitive chitosan/gelatin scaffolds.Our research will bring a new way to repair the damaged cornea.Our study was divided into three parts:(1)Preparation and Characterization of Thermo-sensitive Chitosan/Gelatin Hydrogel;(2)Construction of a Thermo-sensitive Chitosan/Gelatin Sheath with Interstitial Cell Derived Factor(SDF-1)Tissue engineering scaffolds are used to repair corneal epithelium and evaluate the role of endogenous SDF-1 chemotaxis cells in repairing corneal injury;(3)Exogenous seed cells(iPSC-MSCs)combined with thermosensitive chitosan/Gelatin scaffolds to construct tissue-engineered corneas to evaluate the promotion of corneal stromal injury repairChapter1 Preparation and Characterization of Thermosensitive Chitosan/Gelatin HydrogelPurpose:Chitosan is derived from natural chitin and has many excellent biological properties.The purpose of this section is to study the preparation and characterization of temperature-sensitive chitosan/gelatin scaffolds.Methods and results:In in vitro tests,we prepared preparations of temperature-sensitive chitosan/gelatin scaffolds and their characterization.The results showed that the temperature-sensitive chitosan/gelatin scaffold has temperature sensitivity,excellent light transmission,tissue compatibility,and its surface has a pore size,capable of sustained release of SDF-1 alpha.Conclusion:Temperature-sensitive chitosan/gelatin scaffold has excellent properties and can release cytokines,which may be an ideal material for corneal epithelial and stromal damage repair.Chapter 2 Stromal cell-derived factor(SDF-1)combined with thermo-sensitive chitosan/gelatin scaffold to construct tissue engineering scaffold for repair of corneal epithelial damagePurpose:The purpose of this study was to construct a bioactive,thermo-sensitive chitosan/gelatin scaffold that selectively promotes endogenous cell migration for damage to the corneal epithelium by compounding exogenous mesenchymal cell-derived factors.repair.Methods and results;In vitro,we evaluated the proliferation,metastasis and related gene expression of rat limbal stem cells(LESCs)and corneal mesenchymal stem cells(MSCs)by exogenous mesenchymal cell-derived factor(SDF-1).The effect of the scaffold constructed to promote regeneration of corneal epithelium was determined in a rat model of corneal severe alkali burn.In vitro results showed that LESCs and MSCs correlate corneal repair-related gene expression and cell migration to exogenous SDF-1.In the rat model of alkali burn injury,exogenous SDF-1 can recruit more limbal stem cells to repair and repair at the site of injury 7 days after surgery,expressing more growth factors and reducing the infiltration of inflammatory cells,thereby accelerating the corneal epithelium.repair.After 13 days,the SDF-1 treated group showed more transparent corneas and fewer corneal neovascularizations than the control group with simple material,while the regenerated epithelium was arranged closer to the normal cornea.Conclusion:This study clarified that synergistic effects of exogenous SDF-1 and thermosensitive chitosan/gelatin scaffolds can be achieved by increasing the accumulation of endogenous limbal stem cells,enhancing local endogenous SDF-1 and epidermal growth factor,and the liver.Cell growth factors,etc.,reduce the accumulation of inflammatory cells to increase the efficiency of corneal epithelial regeneration.Therefore,exogenous SDF-1 composite thermosensitive chitosan/gelatin scaffold provides a practical method for corneal epithelial tissue engineering.Chapter 3 Exogenous Seed Cells(iPSC-MSCs)Combined with Temperature-Sensitive Chitosan/Gelatin Stent to Construct Tissue-Engineered Cornea to Assess the Role of Promoting Corneal Lamina RepairPurpose:To investigate whether the iPSC-MSCs composite heat-sensitive chitosan/gelatin scaffold can promote corneal stromal repair.Methods and results:In vitro,we assessed the characteristics of stem cells identified from iPSC-MSCs and successfully extracted exosomes secreted by iPSC-MSCs and identified exosomes.The characteristics of exosome proliferation and migration of corneal epithelial cells and corneal mesenchymal stem cells were studied.In the in vivo test,we selected the rat superficial layer injury model as an animal model.The iPSC-MSCs and their exosomes were planted on a temperature-sensitive chitosan/gelatin scaffold and transplanted into rats to evaluate their repair effects.The results showed that iPSC-MSCs showed general characteristics of stem cells.The exosomes were successfully extracted and characterized in vitro.Cells grown in vitro proliferate well on thermosensitive chitosan/gelatin scaffolds without cytotoxicity.At 3 and 7 days postoperatively in vivo,it promoted the growth of more fibroblast-like cells and reduced the infiltration of inflammatory cells.In addition,the iPSC-MSCs group and the exosomes added with its origin performed close,the repaired cornea was more transparent,and the collagen structure was closer to the normal cornea.Conclusion:Transplanted iPSC-MSCs promote the repair of damaged corneal tissue by releasing exosomes and related growth factors.Therefore,iPSC-MSCs composite temperature-sensitive chitosan/gelatin scaffold is a corneal tissue engineering scaffold with certain clinical application value. |
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