ABSTRACT
AIM:To observe the sustained-release effect of compound betamethasone by subconjunctival injection on immunological rejection after ostrich-rabbit lamellar keratoplasty.METHODS:Sixteen healthy New Zealand white rabbits with 6wk old received corneal lamellar keratoplasty,and the corneal graft was ostrich acellular corneal stroma.After surgery all subjects were divided into two groups,Group A (experimental group) were administrated with subconjunctival injection of compound betamethasone injection (once every 7d),and Group B (control group) were administrated with subconjunctival injection of dexamethasone sodium phosphate (once every 7d).At 1,2wk,1,2mo after the surgery,rabbit corneas were taken for paraffin sections,and were observed with H-E staining,in the meantime changes of CD4+ and CD8+ T lymphocytes were observed by immunofluorescence.RESULTS:Two months after surgery,in Group A corneal grafts remained transparenct,and showed little neovascularization;HE staining and indirect immunofluorescence showed that only a few neutrophil infiltration,no CD4+ and CD8+T lymphocytes.In Group B,the inflammatory reaction was observable at different time points,the corneal graft was turbid;and the tissue sections and indirect immunofluorescence staining showed that neutrophil infiltration was predominant,and CD4+,CD8+T lymphocytes were also seen.CONCLUSION:Compound betamethasone is able to inhibit the ostrich-rabbit corneal transplantation immune rejection,prolong the survival time of the grafts.The present study lay the foundation for further research and clinical application.
ABSTRACT
AIM:To observe the transplantation of acellular porcine corneal stroma on the treatment of superficial keratitis by drug-resistant fungal.METHODS:We performed a retrospective analysis of 16 cases of fungal keratitis received the transplantation of acellular porcine corneal matrix from June 2015 to March 2016 with a follow-up of 6mo.We analyzed on items as postoperative visual acuity, corneal graft status, postoperative recurrence and postoperative complications.RESULTS:We observed a healing time of corneal epithelium in 7 to 10d postoperatively generally and the absence of corneal edema in 1mo, while the cornea gradually returned transparent in the 16 cases.Two cases required medication for an epithelial recovery and 3 cases received intervention for decreasing intraocular pressure to a certain level.During the follow-up we observed no cases of cornea degeneration, recurrence of infection or rejection.The vision acuity showed 1.27±0.22, 1.11±0.13, 0.79±0.22 in 1, 3 and 6mo after operation respectively.There was no statistical difference between vision in 1mo and the vision before surgery (P=0.06);while we found a statistical difference when comparing the vision of 3 and 6mo with vision before surgery (P=0.01,0.001).The vision in 6mo increased with a statistic difference to the vision at 1 and 3mo (P<0.001) while no statistic difference was observed between 1 and 3mo(P=0.11).CONCLUSION:Transplantation of acellular porcine corneal matrix is a safe and efficient treatment for fungal keratitis.
ABSTRACT
Objective To select the largest non-toxic leaching solution concentration through the experimental observation of the cytotoxicity of the ostrich acellular corneal stromal leaching solution to the Chinese hamster lung fibroblasts cells(CHL) for the further chromosome distortion experiment.Methods The leaching solution made from the ostrich acellular corneal stromal material was diluted with concentrate of 1 ∶ 2,1 ∶ 4 and the original concentration were used to culture with the CHL cells,the negative and positive control were also set up at the same time,to evaluate the impact on cell growth after 24 hour by MTT colorimetric method.Results The leaching solution diluted with 1∶4 was non-toxic,and could promote the growth of the cells.Conclusion Combined with the results of classification and cell morphological features,this cytotoxicity test can be used to screen the best benchmark non-toxic concentrations for the chromosome aberration test of the CHL cells.
ABSTRACT
Background Corneal transplantation faces a great challenge because of the shortage of corneal donors and difficulty of human corneal endothelial cells (HCECs) regeneration in vitro.So the study on tissue engineering cornea is still a main topic.Previous research showed that mouse embryonic stem cell conditioned medium (ESC-CM) improved the proliferative capacity of HCECs in vitro,and acellular porcine corneal stroma (APCS) was a good saffold material.However,whether HECEs cultured by mouse ESC-CM can form cell sheet in vitro were rarely studied.Objective This study was to investigate the potential that HCECs cultured by mouse ESC-CM form a monolayer cell sheet.Methods The supernatant of ESC-CM was collected after mouse ES-E14 cells were cultured,and the cultured medium was centrifuged and mixed with 75％ human corneal endothelium medium (CEM)at a proportion of 1 ∶ 3 to prepare the 25％ ESC-CM system.Primary cultures of HCECs were established from explants of corneal limbal with Descemet's membrane,and the cells were identified by using reverse-transcription PCR to determine the expressions of collagen Ⅷ (Col Ⅷ) mRNA and neuron-specific enolase (NSE) mRNA in the cells.APCS was prepared by decellularization with phospholipase A2 and bicarbonate solution,and the second generation of HCECs were inoculated on the sterilized APCS at a 800/mm2 density.The morphology of the cells was observed by hematoxylin-eosin staining under the phase-contrast microscope.The expressions of zona occludens protein-1 (ZO-1)and Na+-K+-ATPase in the cell sheet were detected by immunofluorescence staining.Results The second generation of HCECs cultured with 25％ ESC-CM in vitro showed the hexagon in shape with positive expressions for Col Ⅷ mRNA and NSE mRNA.Decellularization APCS was transparent,and no corneal cells were seen,the structures of corneal collagenous fibres were regular.HCECs attached closely to APCS and formed monolayer sheet 7 days after culture on the APCS with the cell density of (2 694±143)/mm2.ZO-1 and Na+-K+-ATPase were positively expressed on the HCECs sheet.Conclusions Twenty-five percent ESC-CM promotes the proliferation and maintains the normal morphology of HCECs.APCS provide a good scoffold and microenvironment for the formation of HCEC sheet.The HCEC sheet Possesses the pump function of HCECs and is a good corneal donor for transplantation.
ABSTRACT
Background Ostrich acellular corneal stroma possesses a similar constitution to human corneal stroma,so it is expected to become one of ideal biological corneal carriers.Objective This study was to investigate the immunogenicity of acellular stroma carrier of ostrich cornea and offer the information for the development of industrialization and clinical use of acellular stroma carrier of ostrich cornea.Methods Twenty fresh ostrich eyeballs and 20 porcine eyeballs were collected.Acellular corneal stroma carriers of ostriches and pigs were prepared using low temperature freezing joint enzyme digestion method and desiccant dehydration method and sterilized by cobalt-60 irradiation.The corneal stroma carriers were preserved using drying and dehydration method.Forty-five male BALB/c mice were randomly divided into the sham operation group,ostrich acellular corneal stroma group and porcine acellular corneal stroma group.Acellular corneal stroma carriers of ostriches and pigs(wet weight after rewatering was 10 mg/piece) were subcutaneously implanted to the back of BALB/c mice,respectively.Wound healing and inflammatory response on the operative site were observed,and phenotype and activating rate of CD4+,CD8+ and CD25+in peripheral blood of mice were dynamically detected 7,14 and 28 days after ectopically implantation of heterogeneous corneal stroma by immunofluorescence labeling and flow cytometry analysis.Results No swelling and exudation were seen in the skin of operative site of the mice with a good healing of wound after surgery.There were no significant differences in the activating rates of CD4+,CD8+ and CD25+ cells in the peripheral blood of mice among the sham operation group,the porcine acellular corneal stroma group and ostrich acellular corneal stroma group in the three time points after surgery(CD4+:F=0.74,P=0.50;F=0.39,P=0.05;F=3.46,P=0.58.CD8+:F=1.75,P=0.21 ;F=1.14,P=0.35;F=0.78,P=0.48.CD25+:F=0.52,P=0.61 ;F=3.53,P=0.62;F=2.42,P=0.13).Conclusions The ostrich acellular heterogeneous corneal stroma carrier possesses low immunogenicity.It is inferred that ostrich acellular corneal stroma carrier can be used in heterogeneous corneal transplantation.
ABSTRACT
AIM: To explore the feasibility of culturing human umbilical vein endothelial cells ( HUVEC ) on acellular corneal stroma and performing the posterior lamellar endothelial keratoplasty ( PLEK ) treating corneal endothelial decompensation. METHODS: Thirty New-Zealand rabbits were divided into three groups randomly, 10 rabbits for experimental group, 10 for stroma group and 10 for control group. Corneal endothelial cells were removed to establish animal model of corneal endothelial failure. PLEK was performed on the rabbits of experimental group and stroma group, and nothing was transplantated onto the rabbits of control group with the deep layer excised only. Postoperative observation was taken for 3mo. The degree of corneal edema and central corneal thickness were recorded for statistical analysis. RESULTS:Corneas in experimental group were relieved in edema obviously compared with that in stroma group and the control group, and showed increased transparency 7d after the operation. The average density of endothelial cells was 2 026. 4±129. 3cells/mm2 , and average central corneal thickness was 505. 2±25. 4μm in experimental group, while 1 535. 6±114. 5μm in stroma group and 1 493. 5±70. 2μm in control group 3mo after operation. CONCLUSION:We achieved preliminary success in our study that culturing HUVEC on acellular corneal stroma and performing PLEK for corneal endothelial decompensation. HUVEC transplanted could survive in vivo, and have normal biological function of keeping cornea transparent. This study provides a new idea and a new way clinically for the treatment of corneal endothelial diseases.