Feasibility Study On Construction Of Tissue Engineering Rabbit Corneal Endothelium By Femtosecond Laser Pulse Technique

Purpose:Femtosecond laser-Acellular Porcine Cornea Matrix（FS-APCM）was used as a scaffold to utilize human umbilical mesenchymal stem cells（HUMSCs）.The rabbit cultured rabbit corneal endothelial cells were promoted to be seed cells,and artificial corneal endothelial grafts were constructed in vitro to investigate the feasibility of tissue engineering corneal endothelium construction.Method:The intact cornea of adult New Zealand white rabbits was taken out under aseptic conditions,and the post-elastic layer with endothelial cells was completely separated.After in vitro digestion and purification,rabbit corneal endothelial cells（RCECs）were obtained and cultured.P3-P7 generation HUMSCs were selected,subcultured and resuspended at 5×10⁵/cm2,and 10%,30%,50%,70%,100%different concentrations of HUMSCs conditioned medium（human umbilical mesenchymal stem cells condition medium,HUMSCs-CM）cultured to observe the effects of different concentrations of HUMSCs-CM on the proliferation of RCECs.By comparing traditional manual cutting technology with femtosecond laser pulse technology to find suitable conditions for optimizing the stent,ultra-thin porcine corneal stroma scaffold with a post-elastic layer（thickness of about 100μm and diameter of about 8.5 mm）was obtained by femtosecond laser pulse technique.After deactivating at 4°C for 24 h in 0.5%SDS solution,FS-APCM was obtained.The RCECs cultured in 70%concentration of HUMSCs-CM were used for Dil fluorescent labeling.The suspension of RCECs was carefully added to the pretreated FS-APCM scaffold by suspension culture.Under inverted microscope,2 h after inoculation,respectively.3d,5d,1w were observed to observe the morphological changes of endothelial cells after in vitro reconstruction.Result:1.The primary culture of RCECs in vitro has good activity.RCECs grow well in vitro,and can form a tight monolayer at the bottom of the dish in 6 days.The cell size is uniform and the morphology is basically the same,mostly similar to the cobble-stone endothelium morphology.2.70%HUMSCs-CM can effectively promote the proliferation of RCECs in vitro.RCECs morphology and MTT assay showed that at the same time（24h）,different concentration gradients（10%CEC-CM,30%HUMSCs-CM,50%HUMSCs-CM,70%HUMSCs-CM,100%HUMSCs-CM）Under the action,it can promote the proliferation of RCECs,and 70%of HUMSCs-CM has the most significant effect,and P1 to P4 have consistent results.The results of plate cloning experiments showed that 70%of HUMSCs-CM could effectively promote the proliferation of RCECs in vitro.3.Compared with the traditional manual cutting stent,the femtosecond laser-assisted ultra-thin decellularization scaffold with a post-elastic layer（thickness about 100μm,diameter about 8.5mm）has good repeatability and operability.Histological examination found that the traditional manual circumcision cut porcine corneal group（T-PC group）had the thickest corneal thickness,femtosecond laser-assisted porcine corneal deep-layer matrix slice group（FS-PC）tissue section was very thin,similar A thin sheet.The water content test results showed that the NPC group had the highest water content after swelling,followed by T-PC,and the FS-PC group had the lowest water content.HE staining,DAPI staining and DNA content detection showed that there was no cell residue in the decellularized 0.5%SDS acellular porcine corneal stroma.4.In vitro simulation of artificial corneal endothelial grafts confirmed that endothelial cells survived well.The RCECs seed cells were inoculated into the FS-APCM scaffold and cultured for 1 w.The anisin red staining and Dil fluorescence tracking showed that the RCECs could form a continuous single cell layer on the FS-APCM.Compact,cell count density is over 2000/mm^2.conclusion:1.70%human umbilical cord blood mesenchymal stem cell condition medium（HUMSCs-CM）can effectively stimulate the proliferation of rabbit corneal endothelial cells in vitro,providing a simple and efficient method for in vitro culture of corneal endothelial cells.2.Femtosecond laser-assisted ultra-thin acellular porcine corneal stroma（FS-APCM）can accurately cut the corneal stroma and provide a better choice for tissue engineering scaffold material optimization.