| Background:Limbal stem cells deficiency can lead to blindness.Stem cells therapy have given these patients a great hope to recovery.Recently,autologous limbal tissue or limbal stem cells transplantation gained a great effect in the clinic.However,when bilateral limbal stem cells deficiency was too severe to isolate and culture,we needed to seek for other suitable stem cells for treatment.Bone mesenchymal stem cells were widely studied because of myriad of advantages.It has been reported that bone mesenchymal stem cells was successfully induced into corneal-like stem cells in vitro,but few studies have examined the differential orientation of bone mesenchymal stem cells after transplantation in vivo.Objective: To observe the differentiation of uninduced rabbit BMSCs transplanted onto the ocular surface of totally deficient limbal stem cells rabbit model.Methods: This experimental research consisted of 24 Japanese clean white rabbits about 3-5 months as models.Right eye limbal stem cells deficient models were prepared with annulus limbal dissection and central corneal epithelium scraping.Ocular surfaces were observed after 4 weeks then HE staining,PAS staining and DAPI staining helped us identify whether models were established successfully or not.From our model selection procedure 18 successful models(36 eyes)were taken into account and grouped randomly.The control group consisted of 18 normal left eyes,total limbal stem cells deficient model group consisted of 6 right eyes and model transplanted with amniotic membrane group(6 right eyes)and the experimental group consisted of model transplanted in amniotic membrane with BMSCs group(6 right eyes).The 3rd passage BMSCs,with denuded amniotic membrane as a carrier,were planted on models while growing to cover more than 90% of amniotic membrane surface.12 weeks later,the picture of ocular surfaces were obtained by digital camera where corneal structures were displayed by HE staining and the expression of CECs’ differentiation marker CK3+12 was tested by immunofluoresence.Results: After 4 weeks of establishing the models,ocular surface scores of21 rabbits were 7 or up to 7.HE staining,PAS staining,DAPI staining showed that limbal and corneal epithelial cells layer were removed,plenty of inflammatory cells infiltrated and neovascularization formed in the superficial basal layer,goblet cells formed,indicating that models were built successfully.After 12 weeks of transplantation,compared with the normal left cornea,we observed the opacity of the model(right cornea)with a large number of neovascularization,amniotic membrane group right eye cornea was slightly turbid with minimal neovascularization,amniotic membrane with BMSCs group cornea became transparent without any neovascularization.HE staining showed that corneal structure was complete in amniotic membrane with BMSCs group.Immunofluorescence showed that the specific marker CK3+12 was expressed in amniotic membrane with BMSCs group but not in amniotic membrane group.Conclusion: Rabbit BMSCs transplantation onto the totally deficient rabbit limbal stem cells model in vivo can differentiate into corneal epithelial cells or corneal like epithelial cells,which can help restore the ocular surface structure and function. |
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