Preparation Of Compound Modified Collagen Films And Injection Of Mesenchymal Stem Cells For Corneal Repair

The cornea is the outermost transparent tissue of the eye,which is susceptible to damage and further develops into corneal diseases,which have become the second largest blinding corneal diseases in China.At present,corneal transplantation is the gold standard for treating corneal blindness.However,the scarcity of corneal donors in worldwide is the biggest obstacle affecting surgical treatment.The problem of immune rejection after transplantation cannot be ignored.Therefore,there is an urgent need to find alternative materials for corneal regeneration and repair and to solve the problem of postoperative immune rejection.As the main component of natural corneal extracellular matrix,collagen is widely used in corneal regeneration and repair.However,the poor mechanical properties of collagen and hard to suture limited its development in biomedicine.Cellulose nanocrystals(CNCs)have excellent mechanical properties and good biocompatibility.By combining collagen and cellulose nanocrystals,collagen-based composite materials with good mechanical properties and biocompatibility can be obtained.Mesenchymal stem cells are a class of adult stem cells with multi-directional differentiation potential,can promote tissue repair and anti-inflammatory,have received great attention in regenerative medicine,and have been extensively studied in the treatment of corneal injury.Therefore,this study intends to use composite modified collagen films and mesenchymal stem cells for regeneration and repair after corneal injury.In this study,collagen was used as a basic material.The collagen-based cellulose nanocrystals composite films were obtained by adding aqueous dispersions of cellulose nanocrystals with different contents to the collagen solution.The mass ratios were 0,1,3,5,7 and 10 wt%.The physical properties and in vitro cell experimental of collagen-based composite films with different cellulose nanocrystals contents were studied.The results shown that the addition of cellulose nanocrystals enhanced the mechanical strength of collagen.Compared with pure collagen film,the tensile strength and elastic modulus of the collagen-based composite film contain 10 wt% CNCs increased by 3.5-and 2.7-fold,respectively;the collagen content rate and light transmission of collagen/cellulose nanocrystals composites films did not affect,but the degradation of collagen/cellulose nanocrystals composite films were accelerated,and the degradation of collagen-based composite film with 7 wt% CNCs was similar to that of pure collagen film.In vitro cell experiment shown that rabbit corneal epithelial cells and keratocytes could well cell morphology and proliferated on the collagen-based composite films,the collagen-based cellulose nanocrystals composites films have good biocompatibility.In vitro wound healing experiments and the condition of TGF-?1 introduced keratocytes differentiated into myofibroblasts,which shown that collagen-based composite film with 7 wt% CNCs can accelerate wound healing and inhibit the differentiation of keratocytes into myofibroblasts.Therefore,collagen-based composite film containing 7 wt% CNCs has good performance in all aspects,can meet the needs of corneal repair,and has potential application value in corneal regeneration and repair.In addition,this study extracted rabbit bone marrow mesenchymal stem cells(BMSCs)by whole bone marrow adherent method and through in vitro identified.Then,co-culture of BMSCs and keratocytes to study the differentiation of BMSCs into corneal epithelial-like cells and keratocytes differentiated into myofibroblasts by TGF-?1.Next,to transplant 7 wt% CNC and inject BMSCs for the repair of corneal mechanical injury.The results showed that the cells extracted in this study were identified as BMSCs.BMSCs differentiated into corneal epitheliallike cells in the co-cultured environment of BMSCs and keratocytes.Under TGF-?1 induction,the differentiation of keratocytes into myofibroblasts was effectively inhibited in co-cultured with BMSCs.In vivo animal experiments showed that BMSCs can inhibit the fibrosis of the corneal matrix to a certain extent,the transplantation of COL-GA accelerated and maintained the complete epithelialization process in a short time,and the combined use of transplanted COL-GA and injected BMSCs always maintained complete re-epithelialization.In summary,the study is based on these problems of the poor mechanical properties of collagen materials and inflammation and cornea haze after corneal injury.By introducing different content of cellulose nanocrystals into collagen-based cellulose nanocrystalline composite films with good mechanical properties,the research of physical properties and biological properties of collagen-based cellulose nanocrystalline composite films.Rabbit bone marrow mesenchymal stem cells were extracted and identified,and the potential with multidirectional differentiation and anti-fibrosis of bone marrow mesenchymal stem cells was studied.Finally,glutaraldehyde-fumigated pure collagen membrane and mesenchymal stem cell injection are used for corneal injury repair.These provide theories references for the study of corneal regeneration and repair,and has important reference value for the development of corneal repair and regeneration alternative materials and the development of corneal stem cell injection therapy.