Research progress of ocular surface reconstruction with ex vivo cultured tissue engineered cell sheet / 中华实验眼科杂志

Limbal stem cells (LSCs),the source of corneal epithelial cells,play an important role in the ocular surface. In recent years, with the development of somatic stem cell application and tissue engineering, biomaterials and cell culture technology,progress has been made on the basic researches and clinical applications of ocular surface reconstruction with ex vivo cultured limbal epithelial and oral mucosal epithelial cell sheet transplantation. However, there are several issues, including the successful clinical outcomes for ocular surface reconstruction,and the in vivo tracking of donor stem cells,remained indefinitive. This article introduced and compared recent advancements of tissue engineering techniques ex vivo cultured autologous or allogeneic limbal,oral mucosal epithelial cells in ocular surface reconstruction,so as to provide a useful direction for the future research of ocular surface reconstruction.

Feasibility of adipose tissue-derived stem cell sheet fabricated ex vivo on temperature-responsive scaffolds as seed cells for ocular surface reconstruction / 中华实验眼科杂志

Background Development of corneal tissue engineering creates a new therapeutic method for severe corneal diseases.However,ideal seed cells and scaffold for corneal surface reconstruction have not yet been investigated well.Adipose-derived stem cells (ADSCs) are varified to have a self-renewal ability and epithelioid features,and temperature-responsive scaffolds (TRSs) can offer technical support for stem cell sheet.Objective This study was to investigate the characteristics of ADSCs cultured on TRSs and compare these features to typical oral mucosal epithelial cells (OMECs),and therefore to explore the feasibility of reconstruction of ocular surface with ADSCs as seed cells.Methods Self-made TRSs were prepared by adding isopropyl alcohol dissolved poly-Nisopropylacrylamide (PNIPAAm) to each polystyrene tissue culture dish and then irradiating using an election beam.Subcutaneious fatty tissue of rabbit neck was obtained to culture ADSCs,and 4 pieces of oral cavity mucosal tissue were digested and cultured to obtain OMECs.Then the ADSCs and OMECs were incubated on TRSs,and cell morphology,growth rate,detached duration and survival counts were compared between ADSCs and OMECs.The ADSCs sheet and OMECs sheet were stained with hematoxylin and eosin for morphological examination.Immunochemistry was used to observe the expressions of stem-cell biomakers and epithelioid-cell biomakers in the cells.The ultrastructure of cell surface was observed under the scanning electron microscope.Results Self-made TRSs were similar to ordinary culture dish in the transparancy and smoothness.The water contact angle of 4 in 5 samples were ＞10° with the effective rate upto 80％.A DSCs showed the elongated fusiform in shape,while OMECs showed a cobblestone appearance.The growth cycle,detached duration and cell number of ADSCs were 12-14 days,(46.0 ±9.6) minutes and (7.9 ±1.1)×105/sheet,and those of OMECs were 14-16 days,(91.9 ±10.9) minutes and (45.8 ±26.5)×105/sheet,respectively,showing statistically significant differences in the detached duration and cell counts between ADSCs and OMECs (P=0.002,0.028).Hematoxylin and eosin staining showed that ADSCs sheet comprised only 1-3 layer cells,while OMECs showed 4-5 layer cells.ATP-binding cassette superfamily G member 2 (ABCG2),p63 and cytokeratin 12 (CK12) were positively expressed in both ADSCs sheet and OMECs sheet.Closely packed cells and typical eithelial microvilli in the cell surface were exhibited in both ADSCs sheet and OMECs sheet under the scanning electron microscope.Conclusions Self-made TRSs can be used as scaffold of ADSCs.The ADSCs sheet on the TRSs appears to have a good cell vitality and therefore is a new seed source of ocular surface reconstruction.

Efficacy assessment of drug-loaded amniotic membrane transplantation for infectious corneal ulceration / 中华实验眼科杂志

Background Treatment of corneal ulceration by transplanting drug-loaded amniotic membrane has been used widespreadly abroad,however,seldom study is found in China up to now.Objective This study was to explore the sustained release property and the efficacy of the drug-loaded amniotic membrane.Methods The bacteriostatic area of amniotic membrane fragments immersed with different concentrations (5,20,30 g/L) of levofloxacin for different time points (5,15,30,60 minutes) was evaluated by in vitro test.Bacterial corneal ulceration models were established in 20 rabbits by injected 0.7 MCF staphylococcus aureus suspension (0.1 ml) into the central corneal stroma to form the cloudy area of 4.0-6.0 mm.Then the rabbits were randomized into two groups.Regular amniotic membrane transplantation was performed laterally and 0.5％ levofloxacin drops was topically administered after operation in the amnion+levofloxacin drops group,and drug-loading amnion transplantation was used in the drug-loading amnion group.Aqueous humor of 0.1 ml was collected in 30 minutes,1 hour,2,3.5,5.5 hours after levofloxacin was administered and 1 day,3,7,10,14,21 days after operation for the detect of levofloxacin level with high-performance liquid chromatography.The corneal symptom was scored based on McNeill's criteria in 1 week,2 weeks and 4 weeks and the ulceration area was assessed under the slit lamp in the first week.The pathological examination was carried out in the fourth week after surgery.Results The mean bacteriostatic area was bigger with the increase of levofloxacin concentration,and bacteriostatic area in amnion immersed for 15 minutes was bigger than that of 5 minutes (P＜0.01).The levofloxacin concentration of aqueous humor after transplantation was decreased by extending the time,and that in 30 minutes and 5.5 hours after operation was (0.873±0.264) mg/L and (0.106±0.027) mg/L,respectively,in the amnion+levofloxacin drops group,and that in day 1,3,7 after surgery in the drug-loading amnion group was higher than at 30 minutes in the amnion+levofloxacin drops group,showing all significant differences (all P =0.00).In the first,second and fourth week after operation,the corneal symptom score was 1.7±0.6,1.3±0.5,0.2±0.4 in the drug-loading amnion group and 2.2±0.8,2.0±0.6,1.5±0.8 in the amnion+ levofloxacin drops group,with the significant differences among the different groups and time points (Fgroup =9.49,P =0.01 ;Ftime =22.96,P=0.01).The corneal ulceration area was (1.6±0.6) mm2 in the drug-loading amnion group and (3.2±0.8) mm2 in the amnion+levofloxacin drops group 1 week after operation,showing a significant difference between them (t =3.98,P =0.00).Histopathological revealed that the various layers of cornea tissue appeared irregular arrangement in the amnion + levofloxacin drops group 4 weeks after operation with 1-2 layers of new squamous epithelium.Disorder hypothallus structure,more inflammatory cells and residual vascular cavity were visible.However,new squamous epithelium of 4-5 layers was seen in the drug-loading amnion group,and inflammatory cells and residual vascular cavity were less than the amnion+levofloxacin drops group 4 weeks after operation.Conclusions Levofloxacin-loaded amniotic membrane can sustained release levofloxacin and maintain an effective drug concentration in aqueous humor,which improves the treating efficacy for staphylococcus aureus corneal ulceration.

Mucous membrane grafting for the post-Steven–Johnson syndrome symblepharon: A case report.

An 18-year-old woman was referred with late sequelae of chloroquine-induced Steven–Johnson syndrome. At the time of presentation, the symblepharon was involving the upper lids to almost the whole of the cornea, and part of the lower bulbar conjunctiva with the lower lid bilaterally. Other ocular examinations were not possible due to the symblepharon. B-scan ultrasonography revealed acoustically clear vitreous, normal chorioretinal thickness, and normal optic nerve head, with an attached retina. Conjunctivo-corneal adhesion released by superficial lamellar dissection of the cornea. Ocular surface reconstruction was carried out with a buccal mucous membrane. A bandage contact lens was placed over the cornea followed by the symblepharon ring to prevent further adhesion. The mucosal graft was well taken up along with corneal re-epithelization. Best corrected visual acuity of 20/120 in both sides after 1 month and 20/80 after 3 months was achieved and maintained till the 2.5-year follow-up.

A Case of Ocular Surface Reconstruction Using Nasal and Oral Mucosa Autograft

PURPOSE: To report a case of ocular surface reconstruction using nasal and oral mucosa auto grafts on a man whose ocular surface was deformed by a chemical burn. CASE SUMMARY: A 48-year-old man was injured when his right eye was exposed to alkali solution. He underwent fifteen operations, including amniotic membrane auto grafts and fornix reconstructions. However, his right eye still suffered from symble pharon, drying, corneal neovascularization, and erosion. We performed ocular surface reconstruction with free autologous nasal and oral mucosal transplantation and amniotic membrane graft. By one month after surgery, his eye showed no evidence of symble pharon or fibrovascularization recurrence on the cornea or conjunctiva. By six months after surgery, his ocular surface had stabilized. CONCLUSIONS: Ocular surface reconstruction with free autologous nasal and oral mucosal transplantation is a useful method on eyes that are injured by chemical burns.

Microsurgicai techniques in amniotic membrane patching for mild to moderate ocular surface burns / 中华显微外科杂志

Objective To evaluate the use of amniotic membrane patches for mild or moderate eye burns at the stage of acute burns and the involved microsurgical techniques.Methods Thirty-four eyes with corneal burns of Ⅱ to Ⅲ degree in which may have partial limbal necrosis were accepted amniotic membrane patching (21 eyes) or pharmaceutical treatment (13 eyes).Four amniotic patches were procured and examined by transmitting electronic microscope when the exposed corneal surface became re-epithelialization after amniotic membrane inching.Results Amniotic patches became partially melting or prolapsed 5-10 (11± 2) days after surgery.The uncovered corneal surface showed quickly re-epithehalized.There were a few thin fibrovascular membrane invaded onto corneal surface in the patients with more than a half limbal necrosis.Postoperative visual acuity increased 1 to 6 (3.3 ± 1.2) lines.The electronic findings showed that most of the infiltrated polymorphonuclear neutrophils in the anmiotic patches became apoptosis.Corneal melting presented in four of 13 eyes who received only drug treatment and lamellar keratoplasty was then performed.The other eyes manifested pseudo-pterygium or symblepharon at different extent.Conclusion Amniotic,patches may reduce the inflammation of burned cornea and corneal neovascularization,accelerate re-epithelialization of corneal surface,even improve the rehabilitation of burned limbal stem cells.

New Strategy of Ocular Surface Disease: Ocular Surface Reconstruction Using Amniotic Membrane and Limbal Stem Cell Transplantation

Amniotic membrane is the innermost layer of the placenta and consists of a thick basement membrane and an avascular stromal matrix. Amniotic membrane transplantation facilitates rapid healing with recovery of a normal epithelial phenotype in the epithelium, and reduces inflammation, vascularization, and scarring in the stroma. Amniotic membrane has two major role, basement membrane and cocktail of cytokines. Amniotic membrane as a native matrix can be used as a graft to restore conjunctival surfaces and corneal surfaces suffering from intractable corneal disease or limbal stem cell deficiency. To restore limbal stem cell deficiency, the source of stem cell has been introduced the autograft transplantation, allograft transplantation, ex vivo expansion and in vivo expansion.

Microsurgical techniques in amniotic membrane transplantation for conjunctival surface reconstruction / 中华显微外科杂志

Objective To investigate the long-term results of amniotic membrane transplantation for conjunctival surface reconstruction and analyze the involved microsurgical techniques and associated factors. Methods Fifty-one consecutive cases (55 eyes) with symblepharon at degree due to eye burns or Stevens-Johnson syndrome were accepted amniotic membrane transplantation after lysis of symblepharon. Results Observation time varied from 36 to 44 months (mean value, 38?4 months). Fifty-six point four percent(31/55) eyes got enough conjunctival fornix and resolution of eye movement restrict. About sixteen percent of them (9/55) recurred less symblepharon. Fifteen-five eyes of them(27.3%) recurred moderate symblepharon. The results of AMT for those patients with symblepharon at different degree and between those patients who were performed in different time after eye burns had significant difference statistically ( ? 2 test, P

The Effect of Limbal Transplantation & Cyclosporine A for Chemically Damaged Rabbit Cornea

The authors have evaluated the effect of topically applied 1% cyclosporine A for four weeks after limbal allograft transplantation for ocular surface stability in severely ocular surface damaged rabbits. A total eleven severely damaged rabbits were subdivided into three groups of four limbal autograft (AUTO), three limbal allograft (ALLO), four limbal allograft with topical application of 1% cyclosporine A five times daily for four weeks (ALLO-C). We examined ocular surface smoothness, clarity, corneal vascularization before surgery and three months after surgery, and classified three grade (success, partial success, failure) according to result. In AUTO, three eyes were success, one eye was failure. In ALLO, one eye showed inferior graft detachment and one eye was success, two eyes were failire. In ALLO-C, no graft detachment detected and one eye was success, two eyes were partial success, one eye was failure. We knew that. ALLO-C show more effective ocular surface stability than ALLO. These result suggest ALLO-C can be used as alternative treatment of AUTO in ocular surface reconstruction. But, rnore study for duration and concentration of cyclosporin A will be need.

The current status and future in limbal stem cells research / 眼科

The discovery of limbal stem cells is the key advancement in ophthalmology in the 20th century.Many clinical researches have proved that limbal stem cells transplantation is an effective method to treat severe ocular surface diseases.However, some important issues associated with the identification,isolation,standardized culture in vitro and differentiation after transplantation are still not clear and need to be paid more attention.Strengthening the underlaying research in this field will play important role in treating corneal blindness.