AIM:To evaluate the clinical efficacy of small-diameter acellular porcine corneal stroma(SAPS)for the treatment of peripheral corneal ulceration(PCU).METHODS:This retrospective clinical study included 18 patients(18 e...AIM:To evaluate the clinical efficacy of small-diameter acellular porcine corneal stroma(SAPS)for the treatment of peripheral corneal ulceration(PCU).METHODS:This retrospective clinical study included 18 patients(18 eyes)with PCU between April 2018 and December 2020.All patients had PCU and underwent lamellar keratoplasty with SAPS.Observation indicators included preoperative and postoperative best-corrected visual acuity(BCVA)and transparency of SAPS.The infection control rate in the surgical eye-lesion area was also calculated.RESULTS:Eighteen patients underwent lamellar keratoplasty with SAPS to treat PCU.None of the patients experienced rejection after 6mo(18/18)and 12mo(16/16)of follow-up.The BCVA(0.47±0.30)at the 6mo followup after operation was significantly improved compared with the baseline(0.99±0.80),and the difference was statistically significant(Z=-3.415,P<0.05).The BCVA at the 12mo follow-up after operation was not statistically significant compared to the 6mo(Z=0,P=1).With time,the SAPS graft gradually became transparent.At the 6mo(18/18)and 12mo(16/16)follow-up,none of the patients had recurrent corneal infection.CONCLUSION:SAPS is clinically effective in the treatment of PCU,improving the patient’s BCVA and reducing the incidence of rejection after keratoplasty.展开更多
AIM: To establish an untransfected human corneal stromal (HCS) cell line and characterize its biocompatibility to acellular porcine corneal stoma (aPCS). METHODS: Primary culture was initiated with a pure population o...AIM: To establish an untransfected human corneal stromal (HCS) cell line and characterize its biocompatibility to acellular porcine corneal stoma (aPCS). METHODS: Primary culture was initiated with a pure population of HCS cells in DMEM/F12 media (pH 7.2) containing 20% fetal bovine serum and various necessary growth factors. The established cell line was characterized by growth property, chromosome analysis, tumorigenicity assay, expression of marker proteins and functional proteins. Furthermore, the biocompatibility of HCS cells with aPCS was examined through histological and immunocytochemistry analyses and with light, electron microscopies. RESULTS: HCS cells proliferated to confluence 2 weeks later in primary culture and have been subcultured to passage 140 so far. A continuous untransfected HCS cell line with a population doubling time of 41.44 hours at passage 80 has been determined. Results of chromosome analysis, morphology, combined with the results of expression of marker protein and functional proteins suggested that the cells retained HCS cell properties. Furthermore, HCS cells have no tumorigenicity, and with excellent biocompatibility to aPCS. CONCLUSION: An untransfected and non-tumorigenic HCS cell line has been established, and the cells maintained positive expression of marker proteins and functional proteins. The cell line, with excellent biocompatibility to aPCS, might be used for in vitroreconstruction of tissue-engineered HCS.展开更多
AIM： To assess acellular ostrich corneal matrix used as a scaffold to reconstruct a damaged cornea. METHODS： A hypertonic saline solution combined with a digestion method was used to decellularize the ostrich cornea...AIM： To assess acellular ostrich corneal matrix used as a scaffold to reconstruct a damaged cornea. METHODS： A hypertonic saline solution combined with a digestion method was used to decellularize the ostrich cornea. The microstructure of the acellular corneal matrix was observed by transmission electron microscopy （TEM） and hematoxylin and eosin （H＆E） staining. The mechanical properties were detected by a rheometer and a tension machine. The acellular corneal matrix was also transplanted into a rabbit cornea and cytokeratin 3 was used to check the immune phenotype, RESULTS： The microstructure and mechanical properties of the ostrich cornea were well preserved after the decellularization process, in vitro, the methyl thiazolyl tetrazoUum results revealed that extracts of the acellular ostrich corneas （AOCs） had no inhibitory effects on the proliferation of the corneal epithelial or endothelial cells or on the keratocytes, The rabbit lamellar keratoplasty showed that the transplanted AOCs were transparent and completely incorporated into the host cornea while corneal turbidity and graft dissolution occurred in the acellular porcine cornea （APC） transplantation, The phenotype of the reconstructed cornea was similar to a normal rabbit cornea with a high expression of cytokeratin 3 in the superficial epithelial cell layer, CONCLUSION： We first used AOCs as scaffolds to reconstruct damaged corneas. Compared with porcine corneas, the anatomical structures of ostrich corneas are closer to those of human corneas. In accordance with the principle that structure determines function, a xenograft lamellar keratoplasty also confirmed that the AOC transplantation generated a superior outcome compared to that of the APC graft.展开更多
Background Prostaglandin E2 （PGE2） is a key modulator of dendritic cells （DCs） function, and cornea-derived transforming growth factor beta 2 （TGF-β2） promotes the generation of phenotypically and functionally ...Background Prostaglandin E2 （PGE2） is a key modulator of dendritic cells （DCs） function, and cornea-derived transforming growth factor beta 2 （TGF-β2） promotes the generation of phenotypically and functionally immature DCs. Therefore, this study was carried out to investigate whether PGE2 is involved in the suppressive effect on DCs maturation mediated by corneal stroma cells （CSCs） and whether PGE2 and TGF-β2 have additive effects in this immunosuppressive mechanism. Methods Bone marrow-derived DCs （BM-DCs）, splenic T cells and CSCs culture supernatant were obtained from mice via various protocols. After that, the level of PGE2 in CSCs culture supernatant was analyzed by enzyme-linked immunosorbent assay. Then, immature BM-DCs pretreated by E-prostanoid 2 receptor antagonist AH6809 or dimethyl sulfoxide were induced to mature in the presence of lipopolysaccharide, with or without CSCs culture supernatant. In parallel experiments, neutralizing TGF-β2 antibody or normal goat IgG was added into the supernatant. Next, the cellular surface markers for DCs maturation, including CD80, CD86, and major histocompatibility complex class Ⅱ （MHC Ⅱ）, were analyzed by flow cytometry; the capability of stimulating the proliferation of T lymphocytes was evaluated by allogeneic mixed lymphocyte reactions and the function of endocytosis was assessed by fluorescein isothiocyanate-dextran uptake. Results Higher concentration of PGE2 was detected in CSCs culture supernatant than in the fresh medium. In addition, compared with control group, after treated with the supernatant in the mature stage, BM-DCs displayed lower expression of CD80, CD86 and MHC Ⅱ, lower T cell stimulatory capacity and higher endocytosis function. However, after the application of AH6809, BM-DCs partially regained T cell stimulatory capacity and expression of CD86 and MHC Ⅱ, but partially lost endocytic activity. Moreover, after the application of AH6809 and neutralizing TGF-β2 antibody, the result of statistical analysis indicated that there was a statistical difference of interaction in the expression of MHC Ⅱ and T cell stimulatory capacity. Conclusions PGE2 contributes to the suppressive effect on BM-DCs maturation mediated by CSCs in vitro, and PGE2 and TGF-β2 have additive effects on the immunosuppression of BM-DCs.展开更多
Cellular therapy of the corneal stroma,with either ocular or extraocular stem cells,has been gaining a lot of interest over the last decade.Multiple publications from different research groups are showing its potentia...Cellular therapy of the corneal stroma,with either ocular or extraocular stem cells,has been gaining a lot of interest over the last decade.Multiple publications from different research groups are showing its potential benefits in relation to its capacity to improve or alleviate corneal scars,improve corneal transparency in metabolic diseases by enhancing the catabolism of the accumulated molecules,generate new organized collagen within the host stroma,and its immunosuppressive and immunomodulatory properties.Autologous extraocular stem cells do not require a healthy contralateral eye and they do not involve any ophthalmic procedures for their isolation.Mesenchymal stem cells have been the most widely assayed and have the best potential to differentiate into functional adult keratocytes in vivo and in vitro.While embryonic stem cells have been partially abandoned due to ethical implications,the discovery of the induced pluripotent stem cells(iPSC)has opened a new and very promising field for future research as they are pluripotent cells with the capacity to theoretically differentiate into any cell type,with the special advantage that they are obtained from adult differentiated cells.Cellular delivery into the corneal stroma has been experimentally assayed in vivo in multiple ways:systemic versus local injections with or without a carrier.Encouraging preliminary human clinical data is already available although still very limited,and further research is necessary in order to consolidate the clinical applications of this novel therapeutic line.展开更多
基金Key R&D Plan of Shaanxi Province(No.2021SF-331).
文摘AIM:To evaluate the clinical efficacy of small-diameter acellular porcine corneal stroma(SAPS)for the treatment of peripheral corneal ulceration(PCU).METHODS:This retrospective clinical study included 18 patients(18 eyes)with PCU between April 2018 and December 2020.All patients had PCU and underwent lamellar keratoplasty with SAPS.Observation indicators included preoperative and postoperative best-corrected visual acuity(BCVA)and transparency of SAPS.The infection control rate in the surgical eye-lesion area was also calculated.RESULTS:Eighteen patients underwent lamellar keratoplasty with SAPS to treat PCU.None of the patients experienced rejection after 6mo(18/18)and 12mo(16/16)of follow-up.The BCVA(0.47±0.30)at the 6mo followup after operation was significantly improved compared with the baseline(0.99±0.80),and the difference was statistically significant(Z=-3.415,P<0.05).The BCVA at the 12mo follow-up after operation was not statistically significant compared to the 6mo(Z=0,P=1).With time,the SAPS graft gradually became transparent.At the 6mo(18/18)and 12mo(16/16)follow-up,none of the patients had recurrent corneal infection.CONCLUSION:SAPS is clinically effective in the treatment of PCU,improving the patient’s BCVA and reducing the incidence of rejection after keratoplasty.
基金National High Technology Research and Development Program("863" Program) of China(No.2006AA02A132)
文摘AIM: To establish an untransfected human corneal stromal (HCS) cell line and characterize its biocompatibility to acellular porcine corneal stoma (aPCS). METHODS: Primary culture was initiated with a pure population of HCS cells in DMEM/F12 media (pH 7.2) containing 20% fetal bovine serum and various necessary growth factors. The established cell line was characterized by growth property, chromosome analysis, tumorigenicity assay, expression of marker proteins and functional proteins. Furthermore, the biocompatibility of HCS cells with aPCS was examined through histological and immunocytochemistry analyses and with light, electron microscopies. RESULTS: HCS cells proliferated to confluence 2 weeks later in primary culture and have been subcultured to passage 140 so far. A continuous untransfected HCS cell line with a population doubling time of 41.44 hours at passage 80 has been determined. Results of chromosome analysis, morphology, combined with the results of expression of marker protein and functional proteins suggested that the cells retained HCS cell properties. Furthermore, HCS cells have no tumorigenicity, and with excellent biocompatibility to aPCS. CONCLUSION: An untransfected and non-tumorigenic HCS cell line has been established, and the cells maintained positive expression of marker proteins and functional proteins. The cell line, with excellent biocompatibility to aPCS, might be used for in vitroreconstruction of tissue-engineered HCS.
基金Supported by National Natural Science Foundation of China(No.31200724)Key Innovation Project of Shaanxi Science and Technology Plan(No. 2012KTCQ03-11)+1 种基金Shenzhen Peacock Plan(No. KQCX20130628155525051)Projects of Basic Research of Shenzhen(No.JCYJ20120614193611639,No.JCYJ 20140509172959988)
文摘AIM： To assess acellular ostrich corneal matrix used as a scaffold to reconstruct a damaged cornea. METHODS： A hypertonic saline solution combined with a digestion method was used to decellularize the ostrich cornea. The microstructure of the acellular corneal matrix was observed by transmission electron microscopy （TEM） and hematoxylin and eosin （H＆E） staining. The mechanical properties were detected by a rheometer and a tension machine. The acellular corneal matrix was also transplanted into a rabbit cornea and cytokeratin 3 was used to check the immune phenotype, RESULTS： The microstructure and mechanical properties of the ostrich cornea were well preserved after the decellularization process, in vitro, the methyl thiazolyl tetrazoUum results revealed that extracts of the acellular ostrich corneas （AOCs） had no inhibitory effects on the proliferation of the corneal epithelial or endothelial cells or on the keratocytes, The rabbit lamellar keratoplasty showed that the transplanted AOCs were transparent and completely incorporated into the host cornea while corneal turbidity and graft dissolution occurred in the acellular porcine cornea （APC） transplantation, The phenotype of the reconstructed cornea was similar to a normal rabbit cornea with a high expression of cytokeratin 3 in the superficial epithelial cell layer, CONCLUSION： We first used AOCs as scaffolds to reconstruct damaged corneas. Compared with porcine corneas, the anatomical structures of ostrich corneas are closer to those of human corneas. In accordance with the principle that structure determines function, a xenograft lamellar keratoplasty also confirmed that the AOC transplantation generated a superior outcome compared to that of the APC graft.
文摘Background Prostaglandin E2 （PGE2） is a key modulator of dendritic cells （DCs） function, and cornea-derived transforming growth factor beta 2 （TGF-β2） promotes the generation of phenotypically and functionally immature DCs. Therefore, this study was carried out to investigate whether PGE2 is involved in the suppressive effect on DCs maturation mediated by corneal stroma cells （CSCs） and whether PGE2 and TGF-β2 have additive effects in this immunosuppressive mechanism. Methods Bone marrow-derived DCs （BM-DCs）, splenic T cells and CSCs culture supernatant were obtained from mice via various protocols. After that, the level of PGE2 in CSCs culture supernatant was analyzed by enzyme-linked immunosorbent assay. Then, immature BM-DCs pretreated by E-prostanoid 2 receptor antagonist AH6809 or dimethyl sulfoxide were induced to mature in the presence of lipopolysaccharide, with or without CSCs culture supernatant. In parallel experiments, neutralizing TGF-β2 antibody or normal goat IgG was added into the supernatant. Next, the cellular surface markers for DCs maturation, including CD80, CD86, and major histocompatibility complex class Ⅱ （MHC Ⅱ）, were analyzed by flow cytometry; the capability of stimulating the proliferation of T lymphocytes was evaluated by allogeneic mixed lymphocyte reactions and the function of endocytosis was assessed by fluorescein isothiocyanate-dextran uptake. Results Higher concentration of PGE2 was detected in CSCs culture supernatant than in the fresh medium. In addition, compared with control group, after treated with the supernatant in the mature stage, BM-DCs displayed lower expression of CD80, CD86 and MHC Ⅱ, lower T cell stimulatory capacity and higher endocytosis function. However, after the application of AH6809, BM-DCs partially regained T cell stimulatory capacity and expression of CD86 and MHC Ⅱ, but partially lost endocytic activity. Moreover, after the application of AH6809 and neutralizing TGF-β2 antibody, the result of statistical analysis indicated that there was a statistical difference of interaction in the expression of MHC Ⅱ and T cell stimulatory capacity. Conclusions PGE2 contributes to the suppressive effect on BM-DCs maturation mediated by CSCs in vitro, and PGE2 and TGF-β2 have additive effects on the immunosuppression of BM-DCs.
文摘Cellular therapy of the corneal stroma,with either ocular or extraocular stem cells,has been gaining a lot of interest over the last decade.Multiple publications from different research groups are showing its potential benefits in relation to its capacity to improve or alleviate corneal scars,improve corneal transparency in metabolic diseases by enhancing the catabolism of the accumulated molecules,generate new organized collagen within the host stroma,and its immunosuppressive and immunomodulatory properties.Autologous extraocular stem cells do not require a healthy contralateral eye and they do not involve any ophthalmic procedures for their isolation.Mesenchymal stem cells have been the most widely assayed and have the best potential to differentiate into functional adult keratocytes in vivo and in vitro.While embryonic stem cells have been partially abandoned due to ethical implications,the discovery of the induced pluripotent stem cells(iPSC)has opened a new and very promising field for future research as they are pluripotent cells with the capacity to theoretically differentiate into any cell type,with the special advantage that they are obtained from adult differentiated cells.Cellular delivery into the corneal stroma has been experimentally assayed in vivo in multiple ways:systemic versus local injections with or without a carrier.Encouraging preliminary human clinical data is already available although still very limited,and further research is necessary in order to consolidate the clinical applications of this novel therapeutic line.
