| Background:Keratoprosthesis(KPro)has been widely used in clinical practice and is the last choice for restoring vision in patients with corneal diseases.The KPro usually consists of a central optical region and a stent,and its long-term presence depends on the stable binding of the stent to the surrounding corneal tissue.In recent years of our clinical work,we have innovatively used aurionic cartilage auxiliary stents to strengthen and fix the stability of the surrounding tissues and the KPro,which achieved good clinical results.However,the disadvantage of this procedure is that it causes damage to the ear.Therefore,there is an urgent need to find an alternative to the ear cartilage to avoid this damage.Purpose:The KPro in the current market has certain shortcomings,and this study intends to combine the advantages and disadvantages of a variety of artificial corneas to develop a new type of KPro,and detect its tissue compatibility and presence.Synthetic materials similar to ear cartilage are critical to solving the above challenges.In this study,human umbilical cord mesenchymal stem cells(hUC-MSCs)are induced into chondrocytes in a three-dimensional porous polyethylene glycol acrylate scaffold(P scaffold)and a three-dimensional porous gelatin scaffold(G scaffold),and the cell-laden material is implanted into the cornea in order to check its biocompatibility and whether increase the thickness and strength of the recipient cornea.Method:1.Implant the self-developed and designed PMMA KPro into the rabbit eye to observe the histocompatibility,presence and corneal sealing of the new artificial cornea.2 Identification of hUC-MSCs cell phenotypes using flow cytometry,induction differentiation in the direction of cartilage formation and identification.3.Make a rabbit corneal laminae pocket model,inject hUC-MSCs cells and hUC-MSCs cells induced into cartilage into the sac,take anterior section photographs at different time points after implantation,and observe and record corneal transparency changes,thickness changes,neovascularization,etc.HE stainning of corneal tissue was performed to detect the survival differentiation of hUC-MSCs in the cornea.4.Prepare P/G and measure the physical parameters.To detect whether hUC-MSCs are capable of differentiation into chondrocytes in the P/G scaffold.5.Implant the P/G scaffold into the corneal capsule,and record the changes in corneal transparency,thickness,and neovascularization through anterior segment photography and observation.HE stainning of corneal tissue to detect the biocompatibility of P/G scaffold within the cornea.6.Induce hUC-MSCs into chondrocytes in P scaffold,P scaffold and cells are jointly implanted in rabbit corneal plate sacs,and anterior segment photography and anterior OCT scan are performed at different time points after implantation to observe and record corneal transparency changes,P scaffold changes,neovascularization,etc.HE stainning,histochemical stainning,immunohistochemical stainning,and immunofluorescence stainning were also carried out to explore the pathophysiological changes of hUC-MSCs and P scaffold in the cornea,and to observe the correlation.Scanning electron microscopy(SEM)observed the cell-P scaffold-cornea,and evaluated the effect of P scaffold combined with hUC-MSCs in enhancing the tissue compatibility of artificial corneal scaffolds.Result:1.The PMMA KPro implanted rabbit eye with independent design and development has dissolution around the KPro without cartilage reinforcement,and there is no dissolution around the KPro with cartilage reinforcement,and the in vitro time is longer than 5 months.2.hUC-MSCs are morphologically similar to fibroblasts and can differentiate in vitro in the direction of cartilage.Alisinlan stained positive,immunofluorescence stained SOX9,COMP,Agreecan stain positive.3.Simple injection of hUC-MSCs cells can be stored in the cornea in a small amount,but due to the lack of carrier,cells are easy to lose,can not form an effective cell concentration,can only form a small volume of clump-like changes in the cornea,can not significantly increase the overall thickness of the cornea.4.P scaffold is thin,the micropores in the material are rich,the diameter is about 36.4±15.6μm,suitable for cell reproduction and growth,hUC-MSCs can be successfully induced into chondrocytes in the P scaffold,the cell volume is large,the nucleus is large,the cytoplasm is rich,and the cell stretch is closely attached to the material.Immunofluorescence stainning COMP,Agreecan stainning marker positive.Alisinland stain positive.5.P scaffold can be stably present after implantation in the corneal capsule,and can slightly increase the thickness of the cornea,promote the infiltration of inflammatory cells,promote the repair of corneal damage,and does not lead to serious inflammatory reactions of the cornea and changes such as perforation of corneal ulcers.It was confirmed that it was biocompatible.6.Micropores in G scaffolds are abundant,but no growth and differentiation of hUC-MSCs into microwells was observed,and no induction and induction of hUC-MSCs showed a tendency to differentiate into interstitial cells.Moreover,the G scaffolds is completely degraded 8w after implantation in the cornea,and the considered material should not be used as a carrier to be implanted with hUC-MSCs in the cornea.7.The unincipered group and the induction group of hUC-MSCs-P scaffoldwere implanted into the rabbit corneal plate sac respectively,and the in vivo observation found that the uninvited hUC-MSCs and P scaffold s were translucent after implantation,and the hUCMSCs and P scaffold s were located in the middle of the cornea.Anterior segment photography shows that the P scaffoldimplanted in the corneal plate layer is stable,the neovascularization in the stromal layer is generated,and the neovascular area covers the implantation range of the material.HE stainning showed intact corneal epithelium,massive cell infiltration of the stromal layer,more neovascularization,a fishnet-like material,and cells scattered in the scaffold.hUC-MSCs do not induce corneal thickness in the group to be lower than the cornea thickness in the hUC-MSCs induced group.The thickness of the cornea in the induction group increased significantly,and the anterior segment photographs showed a significantly thickened pale yellowish thick tissue.Anterior OCT measurement showed that the normal rabbit cornea thickness was 350-400 microns,and the cornea thickened after implantation of P scaffold for 8w,and the material showed medium and high echo.There are more cells in the scaffold,and the cartilage markers COMP and Agreecan are positive for immunofluorescence expression.Conclusion:1.The PMMA KPro of the self-developed and designed is in good condition,and the KPro of the cartilage reinforcement group is firm,and there is no leakage and dissolution.2.P scaffold is beneficial for the growth of hUC-MSCs and induces differentiation into chondroenchys.3.P-material implanted in the cornea can support the growth of host cells and achieve a good state of biocompatibility.4.After induction of differentiation in the direction of cartilage,hUC-MSCs in the P scaffoldcan be implanted in the rabbit corneal plate layer,which can significantly increase the cornea thickness and support and strengthen the receptor cornea. |
