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@#AIM: To investigate the feasibility of constructing corneal stromal scaffolds and the optimal preservation conditions of corneal stromal lenses obtained from the small incision lenticule extraction(Smile)surgery.<p>METHODS: Constructing a bilayer lens by adhering together two corneal stromal lenses with human fibrin sealant(FS). Human corneal fibroblasts were isolated and cultured from Smile derived corneal stromal lenses <i>in vitro</i>, and the toxicity of FS on human corneal fibroblasts was detected by MTT method. The bilayer lenses were then placed in anhydrous glycerin, sodium hyaluronate eye drops, a simulated wet room environment and fetal bovine serum groups respectively, and stored at 4℃ for 14d. The transparency, hardness and stability of the scaffolds were then compared. Afterwards, the bilayer lens scaffolds were stored in anhydrous glycerin at room temperature, 4℃ and -20℃. After 14d of preservation, the diverse effects of temperature on the transparency and hardness of the scaffolds were compared.<p>RESULTS: MTT results showed that the cells of the experimental group and the control group had similar proliferation trend within 0-72h. The cytotoxicity rating of the experimental group was 0 at 36-48h and 1 at 24h and 60-72h. The relative survival rate of the cells within 0-72h was over 90%. FS-bonded bilayer lens scaffold had a smooth surface, close bonding, good transparency and suitable hardness. After 14d of storage at 4℃, none of the nine bilayer lens scaffolds in the anhydrous glycerol group showed signs of cracking cracking after rehydration, and their transparency was good. In the sodium hyaluronate group, three of the nine scaffolds cracked and the remaining six were still intact. In the simulated wet room environment group, none of the 9 scaffolds cracked, but there were different degrees of shrinkage, their surface was rough and transparency was lower. In the fetal bovine serum group, all the 9 stents were cracked, and the single corneal stromal lens was soft and edema was serious. Out of the 15 bilayer lens scaffolds preserved in anhydrous glycerol at room temperature, 2 remained colourless and transparent, 5 slightly yellowed but still remained transparent, 8 yellowed substantially with a significant reduction in transparency. Out of the 15 bilayer lens scaffolds preserved in anhydrous glycerol at 4℃, 5 remained colourless and transparent, and 10 slightly yellowed while remaining transparent. Of the 15 bilayer lens scaffolds preserved in anhydrous glycerol at -20℃, none of the scaffolds yellowed, therefore, remaining colourless and transparent.<p>CONCLUSION: FS is a safe and non-toxic bio-gel. It can be used to glue Smile-derived corneal stromal lenses to construct corneal stromal scaffolds with good stability, high transparency and suitable hardness. Anhydrous glycerol at -20℃ is the best preservation condition for corneal stromal lens scaffolds.
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BACKGROUND:Previous studies from Shaanxi Institute of Ophthalmology have shown that ostrich cornea has the advantages to be developed into the alternatives of human corneal material. OBJECTIVE:To determine the potential toxic effects of ostrich corneal stromal scaffold on cel s. METHODS:Cel culture methods were used to culture L-929 cel s in the extracts of ostrich acel ular corneal stroma which was dried and dehydrated. 3-(4,5)-Dimethylthiahiazo(-z-y1)-3,5-di-phenytetrazoliumromide assay was used to evaluate the growth and proliferation of cel s after cultured for 1, 2 and 3 days. RESULTS AND CONCLUSION:After the cel s were cultured in the extracts of ostrich acel ular corneal stroma subjected to dryness and dehydration for 1, 3 and 5 days, and the toxicity level of cultured cel s was graded as level 1. The cytotoxicity test was conducted according to the“National Standard of the People's Republic of China GB/T16886.5-2003”. After cultured in the extracts of ostrich acel ular corneal stroma, a smal number of cel s were round in shape and loosely adherent without intracytoplasmic granules, and cel lysis could be observed occasional y. The results of 3-(4,5)-dimethylthiahiazo(-z-y1)-3,5-di-phenytetrazoliumromide assay showed that the ostrich acel ular corneal stromal scaffold which was dried and dehydrated had level 1 of cytotoxicity and could be considered as a qualified material.
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Great progress has been made in tissue engineering cornea construction (i.c.constructing human corneal equivalence by using tissue engineering technique) during the past 20 years.However,a kind of tissue engineering cornea which can be applied to corneal transplantation as human cornea equivalent is yet to be availablc.Scaffold is an indispensable part of tissue engineering cornea.Searching for some kinds of scaffolds with good biocompability,some extent of biodegradation and euough biomechanics property are the issue needing to be resolved immediately in the tissue engineering cornea filed.This article reviewed the development of tissue engineering cornea scaffolds,represented the merits and defects of different scaffolds in order to optimize the project of choosing scaffolds and furthermore lay the foundation for constructing a kind of tissue engineering cornea which may be applied to corneal transplantation as human cornea equivalent in the future.
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Background Corneal blindness caused by ocular surface disease is one of the main reasons for the global blinding corneal diseases.With the development and progress of tissue engineering technology,tissueengineered cornea offers a new approach to the treatment of ocular surface disease.Objective This study was to obscrve the growth and differentiation of human umbilical cord mesenchymal stem cclls (UC-MSCs) on thc corneal stroma of receipts and investigate the feasibility of human UC-MSCs differentiated into corneal epithelium-like cells and the reparation of injury cornea.Methods Human UC-MSCs were isolated from human umbilical cord using collagenase Ⅳ digestion and passaged in DMEM/F12 containing fetal bovine serum in vitro.The immunophenotype of cultured human UC-MSCs was evaluated by flow cytometry.The differentiated osteoblasts from the human UC-MSCs by directional induce was identified.Twenty-four New Zealand albino rabbits were randomly divided into 2 groups.The human UC-MSCs were cultured on porcine corneal matrix without corneal epithelium for 4 days and then transplanted onto the 12 left eyes of 12 New Zealand albino rabbits,and porcine corneal matrix without corneal epithelium was transplanted onto the left eyes of other 12 New Zealand albino rabbits as control group.The rabbits received keratoplasty were examined using in vivo confocal microscope through focusing(CMTF).The eyeballs were taken off after 2,4 and 8 weeks,the growth and differentiation,expression of cytokeratin 3 (CK3),CK12 and ATP-binding cassette superfamily G memben 2 (ABCG2)of human UC-MSCs were observed by histopathology and immunofluorescence staining.This use of the experimental animals complied with ARVO Statement.Results Digestive human UCMSCs formed round in shape and was large in size.The attached cells displayed long-fusiform shape like fibroblasts.The cultured human UC-MSCs phenotype was CD105+/CD29+/CD44+/CD34-/CD45-and could be induced toward osteoblast differentiation under the appropriate experimental conditions.Human UC-MSCs grew well on the porcine corneal matrix.The corneal grafts survived wcll without rejection till the experiment end in experimental eyes,but the rejection of corneal graft occurred in control eyes.Confocal microscope could observe corneal epithelium-like cells.The corneal epithelium cells showed the positive response for CK3 and CK12 and absent response for ABCG2.Conclusions Human UC-MSCs with porcine corneal matrix can survive,proliferate and differentiate into corneal epithelium-like cells after transplanting onto the corneal stroma of rabbits.This result suggests that human UC-MSCs is able to repair and reconstruct the injured corneal surfaces.
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Background The tissue-engineered cornea is becoming the hot spot in the ophthalmologic field,while the research of corneal substitute is in the ascendant,because it is more similar to the corneal morpha and easy to survive in vivo.Objective This study was to investigate the biocompatibility of recombinant human type-Ⅲ collagen/poly9 ( 3-( methacryloylamino ) propyl dimethyl ( 3-sulfopropyl ) ammonium hydroxide ) ( PMPDSAH ) interpenetrating polymer network (IPN) (RHC-Ⅲ/PMPDSAH IPN) hydrogel as a tissue-engineered cornea in rabbit eye and its feasibility as the corneal substitute.Methods One hundred and eight rabbits were randomly divided into experimental group( 90 rabbits) and normal control group ( 3 rabbits),and 15 rabbits ( 30 eyes ) used as the donor corneas.RHC-Ⅲ/PMPDSAH IPN,NGF PMPDSAH IPN and corneal grafts were lamellarly transplanted into the right eyes in RHC-Ⅲ/PMPDSAH IPN group,NGF PMPDSAH IPN group and allograft group respectively.The corneal transparency and neovascularization were examined and scored under the slim lamp and compared among three groups using Kraskal-Wallis H test.The corneal epithelization time was observed and compared among these three groups using one way analysis of variance and LSD-t test.The histological examination of corneas was performed at the 3rd day,1st and 2nd week,1 st,3rd and 6th month after the surgery.The immunohistochemistry was used to detect the expression of K3 in cornea at the 6th month.Results The grafts were well attached in RHC-Ⅲ/PMPDSAH IPN group,NGF PMPDSAH IPN group and allograft group,and no rejection reaction was found throughout 6-month following up.Compared with normal control group,no significant differences were found in the scores of corneal opacification and neovescularization in these three groups (x2 =4.34,P =0.23 ;x2 =2.60,P =0.46 ) at the 6th month.NGF PMPDSAH IPN group achieved reepithelialization in (4.97±0.63) days and was obviously shorted than that in RHC-Ⅲ/PMPDSAH IPN group and allograft group ( t =11.97,P =0.00; t =5.80,P =0.00).The re-epithelialization time in RHC-Ⅲ/PMPDSAH IPN was (6.86±0.71) days,and that of allograft group was (5.87±0.43 ) days,showing a significant difference ( t =6.32,P =0.00).Hematoxylin-eosin staining results demonstrated that implanted materials integrated into the host corneal tissue well and support corneal epithelialization.Part of the material degraded at the 2nd week and degraded completely 1 month later.Regular alignment and distribution of collagen fibers were seen in the regenerated cornea and were similar to those of the normal stroma in 6 months.Immunohistochemistry showed the positive expression of keratin-3 in corneal epithelial cells.Conclusions RHC-Ⅲ/PMPDSAH IPN has a good biocompatibility without toxicity to corneal tissue.Furthermore,NGF can promote the corneal wound-healing and re-epithelialization.The material can be used as safe and reliable corneal substitute after improving the mechanical strength.