| According to WHO estimates,4.9 million people worldwide have bilateral corneal blindness,and the number of unilateral blindness is about 23 million.For severe corneal injury,corneal transplantation is the most effective treatment.However,the number of corneas donated worldwide is limited.One way to solve this shortage is to use tissue-engineered corneal grafts with full or partial thickness.Bone marrow mesenchymal stem cells(BMSCs)are the first choice of seed cells because of their wide sources,convenient use and low immunogenicity.At present,amniotic membrane with good biocompatibility is often used as scaffold material to load stem cells in the treatment of corneal injury.At present,it has been found that BMSCs can differentiate into corneal epithelial cells by indirect contact coculture in vitro.However,in the treatment of corneal injury with BMSCs,it is difficult for BMSCs to survive in the injured site due to the influence of microenvironment at the early stage of injury;amniotic membrane as scaffold material faces the problems of limited material source and high cost.In order to solve the above problems,we studied the effect of plasma fibrin membrane as scaffold loaded with BMSCs and corneal epithelial cells as tissue engineering corneal grafts.The research work mainly includes the following contents:1 Isolation,culture and identification of rat BMSCs and corneal epithelial cellsBMSCs were extracted by whole bone marrow adherent method,and cultured in vitro.They were spindle shaped,arranged in a vortex shape and directional.Flow cytometry showed that the extracted BMSCs were positive for CD90 and negative for CD34,which was consistent with the literature reports.The corneal epithelial cells were isolated and extracted by enzyme digestion method.The cells adhered to the wall and grew in a polygonal shape.Immunofluorescence results showed that the extracted cells expressed the specific protein CK12 of corneal epithelial cells2 Induced differentiation of corneal epithelial cells to contact co-cultured BMSCsBMSCs were labeled with CFSE,and then BMSCs were co cultured with corneal epithelial cells at the ratio of 1:3,1:1 and 3:1 for 7 days.Then,BMSCs were selected by flow cytometry,and the mRNA levels of p63 and CK12 were detected by QRT PCR.The results showed that the BMSCs that did not express p63 and CK12 genes expressed these two kinds of corneal epithelial cell related genes after contact co culture with corneal epithelial cells,and the expression levels of these two kinds of corneal epithelial cell related genes increased with the increase of the proportion of corneal epithelial cells in the co culture system.The results showed that BMSCs were induced to differentiate into corneal epithelial cells by direct contact coculture,and the proportion of corneal epithelial cells in co culture system affected the differentiation degree of BMSCs.3 Preparation and performance testing of plasma fibrin membrane3.1 Preparation The whole blood was obtained from the jugular sinus.After centrifugation,the upper plasma was taken out.After adding Ca2+,the plasma gel was obtained after coagulation.After removing the excess fluid in the gel,the plasma fibrin membrane was obtained.3.2 The structure of the membrane was observed by SEM Scanning electron microscopy showed that the micropores on the surface of plasma fibrin membrane were evenly distributed,and the pore size was mostly 25?30?M.the porous structure was conducive to cell attachment and nutrient transport.3.3 The viscoelasticity of the membrane was measured by rheometer The results show that the elastic modulus G 'of freeze-dried rehydrated membrane is greater than that of viscosity modulus G ",and G' and G" of freeze-dried rehydrated membrane are greater than that of fresh membrane.The results showed that the plasma fibrin membrane which was rehydrated after lyophilization had better viscoelasticity than the fresh membrane,and was not easy to deform,that is,the stiffness was higher.It was more convenient to use the plasma fibrin membrane for transplantation after loading cells.3.4 The cell loading on plasma fibrin membrane was observed by scanning electron microscopeBMSCs and corneal epithelial cells were seeded on the lyophilized and rehydrated plasma fibrin membrane.After 24 hours of culture,the dried membrane of loaded cells was obtained by supercritical drying technology.Scanning electron microscopy showed that the two kinds of cells existed on the surface of the membrane,which indicated that the plasma fibrin membrane could be used as a scaffold for loading cells4 Treatment of corneal alkali burn in rats with plasma fibrin membrane loaded with BMSCs and corneal epithelial cells4.1 The effect of alkali burn on corneal wound healing in ratsThe healing of corneal epithelial injury was detected by sodium fluorescein staining on 3 and 7 days after alkali burn.The results showed that with the increase of the days after injury,the rate of corneal epithelial defect in the injury group,blank membrane group and loading cell membrane group decreased to varying degrees;at 3 and 7 days,the defect rate of corneal epithelial cells in the injury group>blank membrane treatment group>membrane loaded group,and there were significant differences between each group(P<0.0001).On the 7th and 14th day after alkali burn,he staining was used to observe the corneal injury.It was found that with the increase of days,the epithelium and stroma structure of the three groups of rats were restored to varying degrees.The corneal injury site structure of the blank group and the load cell membrane group recovered better,and the structure of the corneal injury site was the best,and it was close to the normal corneal structure on the fourteenth day.These results indicate that the self-made loading cell membrane can effectively promote the healing of corneal injury.4.2 The effect on inflammatory reaction after alkali burn in ratsThe expression of matrix metalloproteinase-9(MMP-9),tissue inhibitor of metalloproteinases-1(TIMP-1),interleukin-6(IL-6)and tumor necrosis factor-?(TNF-?)were detected by immunofluorescence staining on the 7th and 14th day after corneal alkali burn.The results showed that on the 7th and 14th day,the expression of these four inflammatory related factors in the cornea of the cell loaded membrane treatment group and the blank membrane treatment group decreased to varying degrees compared with the injury group,and the expression of these inflammatory related factors in the cell loaded membrane treatment group was the least.This indicates that fibrin membrane can reduce the inflammatory reaction of rat cornea after alkali burn,and the inhibition effect of cell loading on the membrane is better.4.3 The effect on corneal fibrosis after alkali burn in ratsThe expression of alpha smooth muscle actin(?-SMA)was detected by immunofluorescence staining on the 7th and 14th day after corneal alkali burn.The results showed that the expression of ?-SMA in the cells loaded membrane treatment group and blank membrane treatment group was significantly decreased compared with the injury group on the 7th and 14th days,and the expression of ?-SMA in the membrane treatment group loaded with cells was the least.This indicates that fibrin membrane can inhibit corneal fibrosis after alkali burn in rats,and the inhibition effect is stronger after loading cells on the membrane.4.4 Differentiation and implantation of BMSCs in plasma fibrin membrane loaded with BMSCs and corneal epithelial cells in alkali burned greater corneaBMSCs were labeled with 5-bromo-1-(2-deoxy-?-D-ribofuranosyl)uracil 5-bromouracil deoxyriboside(5-BrdU)to detect the implantation of BMSCs in injured corneal tissue.The results showed that BMSCs were not detected in the corneal injury site on the 7th day,but a small amount of BMSCs were detected in the corneal injury site on the 14th day,and the expression of CK12 in BMSCs was detected by immunofluorescence staining.These results indicate that BMSCs can be implanted into the injured site and differentiate into corneal epithelial cells in the early repair stage(7-21 days)under the microenvironment composed of plasma fibrin membrane scaffold and loaded corneal epithelial cells.In conclusion,BMSCs and corneal epithelial cells were loaded on the self-made plasma fibrin membrane scaffold to prepare the tissue observation cell membrane.The transplantation of BMSCs and corneal epithelial cells in rats with alkali burn of cornea showed that it can effectively reduce the inflammatory reaction of corneal injury site,inhibit the degree of fibrosis of corneal injury site,and accelerate the healing of corneal wound.It was also found that some BMSCs were successfully implanted into the injured cornea and differentiated into corneal epithelial cells.These results suggest that the tissue-engineered cell membrane is worthy of further study,and may provide a new therapeutic scheme for corneal injury and a new combination for the construction of tissue-engineered cornea. |
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