| Objective: In this study,human corneal stroma lens from SMILE was acellular and cross-linked with ultraviolet A riboflavin to construct tissue engineered human anterior lamellar cornea in vitro.The tissue engineered human corneal stroma lens was transplanted into the anterior lamellar cornea of New Zealand white rabbits to explore its biological characteristics,function and the impact of graft rejection after transplantation,and finally transplanted in patients with corneal stroma defect or corneal microperforation,Observe the clinical effect.The human corneal stroma lens from ultraviolet A riboflavin crosslinked SMILE was used to construct tissue engineering corneal stroma and carry out the experimental study of anterior lamellar keratoplasty,providing a new source of tissue engineering corneal stroma scaffold materials,and providing a new safe and effective treatment method for patients with corneal defects,corneal ulcer and corneal microperforation.Methods:1.Construction of tissue engineering human corneal anterior lamella in vitro: SMILE surgery takes out human corneal stroma lens and puts it into sterile EP tube containing PBS,and transfers it to the laboratory for the next operation.Place human corneal stroma lens in 0.1% sodium dodecyl sulfate(SDS)of acellular solution for acellular preparation,and then immerse human corneal stroma lens in 0.1% riboflavin dextran solution for 90 seconds,using a wavelength of 370 nm,Irradiance 30 m W/cm2UV-A irradiates the cornea for 90 seconds.The changes of tissue structure were observed by HE staining,DPI staining and Masson staining,the ultrastructure was detected by transmission electron microscopy,the acellular efficiency was directly reflected by the determination of DNA concentration,the biomechanical measurement of the cornea in vitro was performed by the in situ mechanical test system,and the human corneal epithelial cells were inoculated on the surface of the acellular human corneal stroma lens crosslinked by ultraviolet A riboflavin for the culture of human corneal epithelial cells.The growth and structure of cells on the surface of tissue-engineered human corneal stroma lens were observed by HE staining,and the epithelial cell specific marker CKl2 was detected by immunofluorescence;2.The experimental study of tissue engineering human corneal stroma lens for anterior lamellar keratoplasty on New Zealand white rabbits: the acellular human corneal stroma lens crosslinked by ultraviolet A riboflavin was used for anterior lamellar keratoplasty in New Zealand white rabbits,and a trephine with a diameter of 5mm was used to make the anterior lamellar keratoplasty bed with about 1/3 rabbit corneal depth,and then a trephine with a diameter of 5.25 mm was used to drill the tissue engineering corneal stroma lens as a graft,Use 10-0 nylon thread and12-needle intermittent suture to fix the graft on the implant bed.After the operation,dexamethasone was injected under the bulbar conjunctiva.After the operation,the operation eyes were marked 4 times a day.The anterior segment photography and fluorescein sodium staining were performed on the 7th,15 th,30th,60 th and 90 th days after the operation,and the anterior segment optical coherence tomography,corneal topography and corneal confocal microscopy were performed on the 30 th and 90 th days after the operation.The specimens were taken for histological examination on the 90 th day after the operation.3.Observation on the clinical treatment effect of human anterior lamellar keratoplasty with tissue-engineered human corneal stroma lens: select the patients with corneal stroma defect or corneal perforation to be included in the group,sign the informed consent of the operation,and agree to participate in the clinical research and treatment.The acellular human corneal stroma lens crosslinked by ultraviolet A riboflavin was given to the selected recipient patients for lamellar keratoplasty.The corneal lesions were removed by trephine combined with scalpel sectioning,and the bottom and periphery of the implant bed were trimmed.The single-layer or double-layer grafts were prepared according to the size of the lesion area and the thickness of the defect.The grafts were fixed on the implant bed by 10-0 nylon suture with 8 stitches intermittently,and the suture was removed intermittently 4-6 weeks after the routine operation,If the suture is loose,remove it at any time.After the operation,slit lamp microscope and fluorescein sodium staining were performed every day.At the 7th,15 th,30th,60 th and 90 th days after the operation,anterior segment photography was performed to observe the transparency of the graft,whether the graft had autolysis,whether there was drainage,and whether the corneal epithelium was repaired;Observe the inflammatory reaction of conjunctiva,cornea and anterior chamber,the depth of anterior chamber and whether there is suture reaction;On the 30 th and 90 th days after operation,optical coherence tomography(OCT)of the anterior segment was performed to observe the morphology of the corneal cross section,and confocal laser corneal microscopy was used to detect the status of corneal epithelial and stromal cells,and whether there were inflammatory cell infiltration.Results: 1.HE staining and DAPI staining showed that there were corneal stroma cells in the normal human corneal stroma lens taken from SMILE surgery.The acellular human corneal stroma lens crosslinked by ultraviolet A riboflavin had no residual cell components.Masson staining showed that the structure of collagen fibers remained intact after the acellular and ultraviolet A riboflavin crosslinked,and there was no tissue damage such as fibrosis,The transmission electron microscope shows that the collagen fibers of normal human corneal stroma lens are arranged regularly and orderly,the structure of corneal stroma cells is complete,and the collagen fibers of the acellular human corneal stroma lens crosslinked by ultraviolet A riboflavin are arranged regularly and tightly,and the collagen gap is not damaged,and the residual voids and vacuoles after the separation of cells are visible,DNA extraction experiment showed that the DNA concentration of acellular human corneal stromal lens group was significantly lower than that of normal human corneal stromal lens group(P<0.05).The biomechanics of the isolated cornea measured by the in-situ mechanical experimental system showed that the human corneal stroma lens group crosslinked by ultraviolet A riboflavin was superior to the normal human corneal stroma lens group in terms of stress and elastic modulus,with significant statistical difference(P<0.05).Under the light microscope,human corneal epithelial cells cultured in vitro have high transparency,plump morphology and stable epithelioid cell morphology.After passage at the ratio of l/2,the cells grew rapidly and were passaged once every 3 days on average.Human corneal epithelial cells can adhere and grow on the acellular human corneal stromal lens cross-linked by ultraviolet A riboflavin.The corneal epithelial cell marker CK12 can be detected by immunofluorescence detection.2.During the observation period of 90 days after operation,8 rabbits survived,of which 1 rabbit’s experimental eye had suture loosening and graft detachment after operation,1 rabbit’s experimental eye had corneal rejection,and the other 6 rabbit’s experimental eyes had no adverse reactions.The time of complete epithelization of the graft was about 15 days,and the graft was basically transparent 90 days after operation.The results of optical coherence tomography of the anterior segment showed that the implant showed high signal at 90 days after surgery,but no obvious edema.At 90 days after operation,the corneal topography showed no keratoconus and other corneal deformation in the eyes.90 days after surgery,confocal laser corneal microscopy showed that the basal cells of the corneal graft epithelium were arranged regularly and tightly,with clear cell boundaries and a layered structure.The density of epithelial cells is close to that of the contralateral healthy eye.There is nerve growth in the graft.The corneal stromal cells of the graft are similar to those of the normal cornea,and no inflammatory cell infiltration is found.HE staining showed that the graft grew and fused well on the recipient’s cornea 90 days after the operation.The distribution of corneal stromal cells in the graft was consistent with that of the surrounding recipient’s cornea stromal cells.There was no infiltration of lymphocytes and neutrophils.Collagen was arranged neatly and tightly.The corneal epithelial cells on the surface of the graft grew closely,with normal corneal epithelial characteristics.At 90 days after operation,the expression of CK12 in the surface epithelial cells of tissue engineered human anterior lamellar cornea was detected by immunohistochemistry.CD4 and CD8 were not expressed in the corneal stroma of the experimental group and the control group by immunohistochemistry,and the inflammatory cytokine IL-1 was detected by ELISA β、IL-12、IFN-γ,There was no difference in the expression of inflammatory cytokines between the experimental group and the control group(P>0.05).3.During the observation period of 90 days after operation,the tissue engineering corneal stroma grafts of all cases were successfully sutured,the corneal perforation was closed,the grafts and the recipient cornea fused well,no aqueous leakage,graft fusion,displacement,suture loosening,graft and bed neovascularization,corneal infection and immune rejection were found.Epithelialization of cornea was completed about 1 week after operation.90 days after operation,the results of optical coherence tomography of anterior segment showed that the corneal ulcer was completely healed and the graft grew and fused well.Confocal laser corneal microscope showed that the epithelial cells of the graft were arranged regularly and tightly,with clear cell boundaries and stratified structure.Normal stromal cells could be seen in the stromal layer,and the graft had nerve growth,without inflammatory cell infiltration and connective tissue wrapping.Conclusion: The tissue engineered human anterior lamellar cornea,which is made of human corneal stroma lens from SMILE after acellular treatment and cross-linked with ultraviolet A riboflavin,has a similar phenotype and structure to normal human corneal tissue,and has better biomechanical properties.The tissue engineered human corneal stroma lens can be used in the anterior lamellar cornea transplantation of New Zealand white rabbits to restore the integrity of the cornea,and has good biological functional characteristics,The patients with corneal defect,corneal ulcer and corneal microperforation have been successfully treated in clinical practice.This is a safe and effective alternative treatment method for patients who need to rescue vision but cannot wait for corneal donor materials. |
PDF Full Text Request