组织工程口腔黏膜上皮治疗角膜缘干细胞缺陷症的实验研究

目的探讨体外培养的自体组织工程口腔黏膜上皮重建兔角膜上皮的可行性。方法制作兔角膜缘干细胞缺陷模型32只,实验组Ⅰ～Ⅲ以自体口腔黏膜上皮细胞为种子细胞制作组织工程上皮,移植到实验组模型兔角膜表面,分别观察2周、1个月、3个月,对照组移植空白载体膜,观察3个月。术后裂隙灯显微镜下观察,以角膜新生血管、混浊度及上皮染色评分评价移植效果。用组织病理、免疫组织化学和印迹细胞技术评价角膜上皮重建的可能性。结果模型兔角膜混浊,有大量新生血管和杯状细胞。实验组移植后角膜透明,印迹细胞检查PAS(-)。实验组与对照组术后角膜总评分差异有统计学意义(P=0.000),p63表达阳性,角膜上皮的组织特点及角蛋白的表达与正常角膜上皮相似。结论组织工程口腔黏膜上皮在角膜基质微环境的诱导下可分化为角膜样上皮细胞,有重建角膜上皮的作用。     

Midterm outcomes of penetrating keratoplasty after cultivated oral mucosal epithelial transplantation in chemical burn

Purpose

To evaluate the midterm outcomes of penetrating keratoplasty (PKP) after cultivated oral mucosal epithelial transplantation (COMET) in patients with bilateral total limbal stem cell deficiency (LSCD) due to chemical burn.

Analysis of angiogenesis induced by cultured corneal and oral mucosal epithelial cell sheets in vitro

The aim of this study was to compare angiogenesis-induction capabilities of cultured corneal epithelial cells (CCE) and cultured oral mucosal epithelial cells (COE) in vitro, and identify candidate factors that induce corneal neovascularization after transplantation of COE sheets. Rabbit corneal and oral mucosal epithelial cells were co-cultured with mitomycin C-treated NIH/3T3 cells on culture plates and inserts. After CCE and COE were multilayered, culture medium was replaced by basal medium and incubated. Angiogenic potential was examined by invasion, migration and tube formation assays with human umbilical vein endothelial cells (HUVECs). Protein secretion of fibroblast growth factor 2 (FGF2), vascular endothelial growth factor (VEGF), angiopoietin-1 and transforming growth factor beta1 was assessed in conditioned medium by ELISA. Gene expression of FGF2 and VEGF was also quantified by real-time RT-PCR and neutralizing antibodies against FGF2 and VEGF were employed for blocking assays. COE induced significantly greater invasion, migration and tube formation of HUVECs, when compared to CCE. CCE secreted a significantly lower amount of FGF2 than COE, while amounts of VEGF were approximately equal in both culture media. Similarly, significantly higher expression of FGF2 mRNA was observed with COE, while no significant difference in VEGF mRNA expression was observed between COE and CCE. Only anti-FGF2 neutralizing antibody significantly suppressed HUVEC invasion and migration induced by COE, without suppression in CCE. In conclusion, angiogenic potential of COE is greater than that of CCE and FGF2 is a candidate involved in the induction of corneal neovascularization after COE sheet transplantation.     

Transplantable cultivated mucosal epithelial sheet for ocular surface reconstruction

Ocular surface reconstruction by tissue engineering using somatic stem cells is a second-generation therapeutic modality. In view of future treatment of bilaterally affected, severe ocular surface disorders, two types of transplantable cultivated mucosal epithelial sheets can be used for reconstruction. One is an allogeneic corneal epithelial stem cell sheet, and the other is an autologous oral mucosal epithelial cell sheet. We first investigated the feasibility of amniotic membrane as an epithelial carrier, and found that denuded amniotic membrane was the most appropriate substrate for this purpose. Thus, cultivated corneal epithelial stem cell sheets were created by co-culturing with 3T3 fibroblast and air-lifting on amniotic membrane. These epithelial sheets demonstrated positive keratin 3 and 12 specific to in vivo corneal epithelium, light junction related proteins and proliferative activity. The transplanted allogeneic human corneal epithelial sheets existed successfully on the corneal surface, and were quite effective in achieving ocular surface stability in severe ocular surface disorders. A few cases, however, developed immunological reactions or opportunistic infections, etc. Secondly, we established transplantable autologous cultivated oral mucosal epithelial sheets in rabbits. The in vitro oral mucosal epithelial sheets showed histological characteristics similar to those of in vivo corneal epithelial sheets; for example, positive keratin 3 expression. Based on the fact that, the transplanted autologous oral mucosal epithelial sheets resembled corneal epithelium and that we achieved the recovery of corneal transparency in rabbits, we propose that cultivated oral mucosal epithelium may become the substitute for corneal epithelium in ocular surface reconstruction.     

自体口腔黏膜上皮细胞体外培养异体角膜移植治疗眼表重度烧伤

目的观察自体口腔粘膜上皮细胞体外培养联合异体角膜基质治疗重度眼表烧伤临床效果。方法随机将重度眼表烧伤患者分为两组,移植组行自体口腔粘膜上皮细胞联合异体角膜移植术,对照组为异体角膜移植术,分别在术后1 d、7 d、30 d、90 d、120 d观察角膜荧光染色、植片水肿及角膜新生血管情况。结果在各观察点角膜荧光染色得分对照组均高于移植组;在第7天、30天时其水肿程度好于对照组;术后30 d、90 d、120 d新生血管数量和范围对照组均高于移植组。结论对于严重的双眼化学烧伤者,自体口腔粘膜上皮细胞联合异体角膜板层移植具有较好疗效,为挽救眼球,争取二次复明手术提供了一个新的可行性方法。     

Oral epithelial cells transplanted on to corneal surface tend to adapt to the ocular phenotype

To understand the response of oral epithelial cells, transplanted on corneal surface to the ocular cues in vivo. The corneal button obtained after penetrating keratoplasty (PK) of an eye of a patient with total limbal stem cell deficiency (LSCD), previously treated with cultured oral mucosal epithelial transplantation (COMET) was examined by immunohistochemistry for the expression of keratins, p63, p75, PAX6, Ki-67, CD31, and CD34. COMET followed by optical-PK has improved visual acuity to 20/40 and rendered a stable ocular surface. The excised corneal tissue showed the presence of stratified epithelium with vasculatures. The epithelial cells of the corneal button expressed K3, K19, Ki-67, p63, p75 and the cornea-specific PAX6 and K12. This study confirms that the oral cells, transplanted to corneal surface, survive and stably reconstruct the ocular surface. They maintain their stemness at the ectopic site and acquire some of the corneal epithelial-like characters.     

兔骨髓间充质干细胞及人羊膜上皮细胞移植治疗兔角膜缘干细胞缺损的研究

背景角膜缘干细胞缺乏可引起致盲性眼病,但传统的治疗方法疗效欠佳。最近的研究表明,骨髓间充质干细胞（BMSCs）和人羊膜上皮细胞（AECs）有分化成多种细胞的能力,但其对角膜缘于细胞缺乏的疗效仍有待研究和评价。目的观察和对比兔BMSCs和人AECs移植治疗兔角膜缘干细胞缺损的治疗效果。方法将18只新西兰白兔采用随机数字表法分为羊膜基质（AS）移植组、兔BMSCs移植组和人AECs移植组,每组6只。将浸有NaOH溶液的滤纸贴附于角膜表面建立碱烧伤角膜缘干细胞缺损的动物模型。抽取兔髂窝处骨髓并收集人胎盘组织分别分离、制备兔BMSCs和人AECs,通过密度梯度离心加贴壁培养法及胰蛋白酶多次分步消化法获取兔BMSCs和人AECs,采用逆转录聚合酶链反应（RT—PCR）法对培养细胞进行鉴定,再接种于人去上皮AS上,按照动物的分组分别将上述材料缝合至动物模型的角膜表面。术后28d,对各组动物的角膜新生血管（CNV）评分以及角膜混浊度评分进行对比,对角膜组织行组织病理学检查并针对角膜上皮细胞特异性标记物细胞角蛋白3（CK3）行免疫组织化学染色。结果第3代兔BMSCs接种于AS载体上12h后贴附生长,第1代人AECs接种于AS载体上48h后呈单层膜状生长,传代细胞经RT—PCR鉴定符合目标细胞的特征。兔BMSCs移植组术后28d,免疫组织化学染色结果显示,兔BMSCs移植组和人AECs移植组的角膜表面细胞CK3均呈阳性表达,而AS组CK3表达阴性。与AS移植组比较,兔BMSCs移植组和人AECs移植组CNV评分以及角膜混浊度评分明显降低,差异均有统计学意义（BMSCs：Z=-2．983,P=0．003;Z=-2．844,P=0．004;AECs：Z=-2．817,P=0．005;Z=-2．041,P=0．041）。组织病理学检查显示,AS组兔角膜组织有较多的炎性细胞生长,胶原纤维排列紊乱。兔BMSCs组术后角膜表层形成了角膜上皮样的复层结构,无明显杯状细胞,角膜基质内无新生血管生长,炎性细胞减少,胶原纤维排列更加规则,且兔BMSCs移植组角膜透明度明显优于人AECs移植组,差异有统计学意义（Z=-2．091,P=0．037）,而2组间CNV评分的差异无统计学意义（Z=-0．267,P=0．789）。结论移植至兔角膜缘干细胞缺损角膜表面的兔BMSCs和人AECs均能分化为角膜上皮细胞样细胞,可抑制CNV,减轻角膜混浊。在改善角膜透明度方面,兔BMSCs优于人AECs。     

Efficacy and outcome of simple limbal epithelial transplantation for limbal stem cell deficiency verified by epithelial phenotypes integrated with clinical evaluation

PurposeTo evaluate the efficacy and outcome of simple limbal epithelial transplantation (SLET) for limbal stem cell deficiency (LSCD) using epithelial phenotype detection integrated with clinical manifestation.MethodsThis prospective multicenter study included patients with LSCD who underwent autologous SLET (autoSLET) and living-related allogenic SLET (Lr-alloSLET). All patients were assessed by slit-lamp biomicroscopy, in vivo confocal microscopy (IVCM), and impression cytology with immunofluorescence staining (ICIF) before and after surgery. The criteria for success were the presence of a clinically non-conjunctivalized cornea and corneal epithelium detected by IVCM or ICIF. Otherwise, the case would be considered a failure. Visual improvement and risk factors for SLET failure were analyzed.ResultsA total of 28 eyes of 26 patients (11 autoSLET and 17 Lr-alloSLET) were included. The median age was 53 years (range, 35–63), and the follow-up time was 29.5 months (range, 17.5–39.8). The overall survival rate was 89.3% at 2 years and 75.6% at 3 years with no difference between autoSLET and Lr-alloSLET (p = 0.24). Seven eyes subsequently underwent penetrating keratoplasty. Immunohistochemistry analysis showed that all corneal buttons had corneal epithelium and limbal stem cell markers. Visual improvement was achieved in both SLET groups (p < 0.001). Failed SLET developed between 5 and 32 months postoperatively. However, absolute risk factors for SLET failure were unidentified.ConclusionThe efficacy of autoSLET and Lr-alloSLET for LSCD was excellent. Limbal explants can regenerate and restore the corneal surface while maintaining the characteristics of limbal stem cells as shown by epithelial phenotype detection and immunohistochemistry integrated with clinical evaluation.     

Adult human buccal epithelial stem cells: identification, ex-vivo expansion, and transplantation for corneal surface reconstruction

Purpose

To identify adult human buccal epithelial stem cells (SCs) on the basis of two parameters (high p63 expression and greater nucleus/cytoplasmic (N/C) ratio) and to evaluate clinical efficacy of ex-vivoexpanded autologous epithelium in bilateral limbal SC-deficient (LSCD) patients.

Methods

The epithelial cells were isolated from buccal biopsy and cultured on human amnion in culture inserts with 3T3 feeder layer. The SCs were identified on the basis of two-parameter analysis using confocal microscopy, surface markers, and colony-forming efficiency (CFE). The cultured epithelium was transplanted in 10 LSCD patients followed by penetrating keratoplasty in 4 patients. The clinical outcome was followed up to 3 years.

Results

A distinct population (3.0±1.7%) of small cells expressing high levels of p63 with greater N/C ratio was observed in buccal epithelium. The N/C ratio was found to be more appropriate than cell diameter for two-parameter analysis. These cells located in the basal layer were negative for connexin-43 and positive for melanoma-associated chondroitin sulfate proteoglycan, containing holoclones with 0.2% CFE, thus representing the SC population. After transplantation of cultured epithelium with increased (sixfold) SC content, anatomical and visual improvement was observed at 13–34 months in 3/10 LSCD patients.

Conclusions

The two-parameter SC marker is useful to identify and quantify buccal epithelial SCs. The transplantation of bioengineered SC-rich buccal epithelium is a strategy for corneal surface reconstruction in bilateral LSCD. However, further studies are required to optimize the culture conditions and to look for other sources of adult SCs for better visual outcome.     

A single cell atlas of human cornea that defines its development,limbal progenitor cells and their interactions with the immune cells

PurposeSingle cell (sc) analyses of key embryonic, fetal and adult stages were performed to generate a comprehensive single cell atlas of all the corneal and adjacent conjunctival cell types from development to adulthood.MethodsFour human adult and seventeen embryonic and fetal corneas from 10 to 21 post conception week (PCW) specimens were dissociated to single cells and subjected to scRNA- and/or ATAC-Seq using the 10x Genomics platform. These were embedded using Uniform Manifold Approximation and Projection (UMAP) and clustered using Seurat graph-based clustering. Cluster identification was performed based on marker gene expression, bioinformatic data mining and immunofluorescence (IF) analysis. RNA interference, IF, colony forming efficiency and clonal assays were performed on cultured limbal epithelial cells (LECs).ResultsscRNA-Seq analysis of 21,343 cells from four adult human corneas and adjacent conjunctivas revealed the presence of 21 cell clusters, representing the progenitor and differentiated cells in all layers of cornea and conjunctiva as well as immune cells, melanocytes, fibroblasts, and blood/lymphatic vessels. A small cell cluster with high expression of limbal progenitor cell (LPC) markers was identified and shown via pseudotime analysis to give rise to five other cell types representing all the subtypes of differentiated limbal and corneal epithelial cells. A novel putative LPCs surface marker, GPHA2, expressed on the surface of 0.41% ± 0.21 of the cultured LECs, was identified, based on predominant expression in the limbal crypts of adult and developing cornea and RNAi validation in cultured LECs. Combining scRNA- and ATAC-Seq analyses, we identified multiple upstream regulators for LPCs and demonstrated a close interaction between the immune cells and limbal progenitor cells. RNA-Seq analysis indicated the loss of GPHA2 expression and acquisition of proliferative limbal basal epithelial cell markers during ex vivo LEC expansion, independently of the culture method used. Extending the single cell analyses to keratoconus, we were able to reveal activation of collagenase in the corneal stroma and a reduced pool of limbal suprabasal cells as two key changes underlying the disease phenotype. Single cell RNA-Seq of 89,897 cells obtained from embryonic and fetal cornea indicated that during development, the conjunctival epithelium is the first to be specified from the ocular surface epithelium, followed by the corneal epithelium and the establishment of LPCs, which predate the formation of limbal niche by a few weeks.ConclusionsOur scRNA-and ATAC-Seq data of developing and adult cornea in steady state and disease conditions provide a unique resource for defining genes/pathways that can lead to improvement in ex vivo LPCs expansion, stem cell differentiation methods and better understanding and treatment of ocular surface disorders.     

白细胞介素-6对糖尿病小鼠角膜缘干细胞活化的促进作用和角膜上皮愈合的加速作用

背景 白细胞介素(IL)-6既介导炎症反应过程又在损伤修复中发挥重要作用,但其具体机制及其在糖尿病角膜组织损伤修复中的作用值得探讨. 目的 探讨IL-6在正常和糖尿病小鼠角膜缘干细胞活化和角膜上皮修复过程中的作用及其机制.方法 正常6～8周龄C57B L/6小鼠52只,采用随机数字表法随机分为正常对照鼠32只和糖尿病模型鼠20只,采用50 mg/kg链脲佐菌素连续腹腔内注射5d的方法诱导建立小鼠糖尿病模型.正常对照小鼠和糖尿病模型小鼠均行角膜上皮刮除术,然后分别于刮除后即刻和48 h结膜下注射IL-6或等容量PBS,采用荧光素染色法评价随时间延长的角膜上皮的愈合情况.体外培养小鼠角膜上皮干/祖细胞(TKE2细胞系),采用结晶紫染色法评估不同质量浓度IL-6处理后细胞克隆率(CFE),并与空白对照组进行比较;采用免疫荧光检测法和Western blot法检测细胞和小鼠再生上皮中干细胞标志物△NP63、Ki67的表达以及关键转录因子STAT3磷酸化水平;采用实时荧光定量PCR法和ELISA法检测小鼠角膜再生上皮中IL-6的mRNA及蛋白水平.结果 角膜荧光素染色检查显示,正常对照小鼠和糖尿病模型小鼠PBS注射组与IL-6注射组注射后24、48和72 h残留角膜上皮缺损面积占原始缺损面积的百分比随角膜上皮损伤后时间延长均明显缩小,同时间点IL-6注射组角膜上皮缺损面积均明显小于PBS注射组,组间总体差异均有统计学意义(正常对照组:F分组=19.982,P＜0.01;F时间=589.350,P＜0.01;糖尿病组:F分组=25.411,P＜0.01;F时间=334.807,P＜0.01).空白对照组及10、20、50、100 ng/ml IL-6处理组CFE分别为(13.23±1.12)％、(15.87±1.30)％、(21.69±1.62)％、(25.33±1.28)％和(18.67±1.54)％,随着IL-6质量浓度的增加CFE逐渐增加,总体比较差异有统计学意义(F=35.547,P＜0.01).50 ng/ml IL-6处理5、10、15、30和60 min细胞中△NP63、Ki67和p-STAT3蛋白的相对表达量随着处理时间的延长均逐渐增加,各时间点细胞中△NP63、Ki67和p-STAT3蛋白的相对表达量均明显高于空白对照组,差异均有统计学意义(均P＜0.05).糖尿病组和正常对照组小鼠角膜上皮刮除后24 h的再生上皮中IL-6 mRNA相对表达量分别为0.45±0.21和1.00±0.16,糖尿病组小鼠角膜再生上皮中IL-6 mRNA相对表达量较正常对照组小鼠下降56％,差异有统计学意义(t=3.42,P=0.03),糖尿病小鼠角膜上皮刮除后48 h的再生上皮中IL-6蛋白质量浓度为(257±12)ng/μl,明显低于正常对照组小鼠的(323±17) ng/μl,差异有统计学意义(t=5.60,P＜0.01). 结论 IL-6能够通过激活STAT3信号通路促进正常和糖尿病小鼠角膜缘干细胞的活化和增生,进而促进角膜上皮修复,而封闭内源性IL-6则会延迟小鼠角膜上皮的修复.     

Functional expression of transient receptor potential vanilloid 3 (TRPV3) in corneal epithelial cells: Involvement in thermosensation and wound healing

Transient receptor potential vanilloid 3 (TRPV3), a member of the calcium-permeable thermosensitive TRP (thermoTRP) subfamily of receptors, is an important cutaneous sensor that detects thermal and chemical stimuli. TRPV3 is activated by innocuous warm temperature stimuli (>33° C) and a variety of physiologically active substances. While the corneal epithelium is known to respond to such stimuli, it is unknown whether TRPV3 is involved in this phenomenon. We show here that TRPV3 mRNA and protein are abundantly expressed in the epithelial cells of human and mouse cornea. Carvacrol, an agonist of TRPV3, elevated cytosolic Ca²⁺ concentration in both primary mouse corneal epithelial cells and cultured human corneal epithelial cells (HCE-T cells). The response to carvacrol was inhibited by ruthenium red, a TRPV channel antagonist. Moreover, repetitive agonist stimulation sensitized the response with gradually increasing amplitude, suggesting that the TRPV3 in the cornea has similar physiological and pharmacological characteristics to that in skin keratinocytes. Finally, a wound healing assay revealed that appropriate calcium ion influx via activated TRPV3 in corneal epithelial cells accelerated their proliferation. Thus, functional TRPV3 is present in corneal epithelial cells and may play a role not only in thermosensation, but also in the regulation of cell proliferation.     

Evidence for heterogeneity in a small squamous cell type (‘light’ cells) in the rabbit corneal epithelium—a scanning electron microscope study

The rabbit corneal epithelial surface, as viewed by scanning electron microscopy (SEM), is composed of a mosaic of polygonal cells with different appearances (light, medium and dark) with the lighter cells having a characteristically high density of microplicae. From the central zone of the corneal epithelial surface of 16 female New Zealand White rabbits (2 kg), the lighter-appearing cells had an average area of 108±47 μm² (n=567, ±SD). A subgroup of atypical lighter cells had an average area of 294±67 μm² (n=53) compared to typical light cells of 88±12 μm (n=514). These atypical lighter cells had fused microplicae at their periphery (instead of a uniform arrangement of densely packed microplicae), tended to be rounder in shape (as opposed to being angular), could show signs of desquamation and were not decorated with the epithelial craters found on almost all (92%) other light cells. All of these features are suggestive of these atypical lighter cells being the terminal phenotype of the light cells just prior to desquamation from the ocular surface. The observation of the desquamation of lighter cells supports a hypothesis that they constitute a distinct sub-population of cells at the corneal epithelial surface.     

Epidermal growth factor receptor transactivation by the cannabinoid receptor (CB1) and transient receptor potential vanilloid 1 (TRPV1) induces differential responses in corneal epithelial cells

Corneal epithelial injury induces release of endogenous metabolites that are cannabinoid receptor 1 (CB1) and transient receptor potential vanilloid 1 (TRPV1) agonists. We determined the functional contributions by CB1 and TRPV1 activation to eliciting responses underlying wound healing in human corneal epithelial cells (HCEC). Both the selective CB1 and TRPV1 agonists (i.e., WIN55,212-2 [WIN] and capsaicin [CAP], respectively) induced EGFR phosphorylation whereas either inhibition of its tyrosine kinase activity with AG1478 or functional blockage eliminated this response. Furthermore, EGFR transactivation was abolished by inhibitors of proteolytic release of heparin bound EGF (HB-EGF). CB1-induced Ca²⁺ transients were reduced during exposure to either the CB1 antagonist, AM251 or AG1478. Both CAP and WIN induced transient increases in Erk1/2, p38, JNK1/2 MAPK and Akt/PI-3K phosphorylation status resulting in cell proliferation and migration increases which mirrored those elicited by EGF. Neither EGF nor WIN induced any increases in IL-6 and IL-8 release. On the other hand, CAP-induced 3- and 6-fold increases, which were fully attenuated during exposure to CPZ, but AG1478 only suppressed them by 21%. The mixed CB1 and TRPV1 antagonist, AM251, enhanced the CAP-induced rise in IL-8 release to a higher level than that elicited by CAP alone. In conclusion, CB1 and TRPV1 activation induces increases in HCEC proliferation and migration through EGFR transactivation leading to global MAPK and Akt/PI-3K pathway stimulation. On the other hand, the TRPV1-mediated increases in IL-6 and IL-8 release are elicited through both EGFR dependent and EGFR-independent signaling pathways.     

A multicenter report of the use of plasma rich in growth factors (PRGF) for the treatment of patients with ocular surface diseases in North America

PurposeTo investigate the efficacy and safety of plasma rich in growth factors (PRGF) eyedrops in the management of patients with ocular surface diseases in North America.MethodsMulticenter interventional case series of patients using PRGF eyedrops for the first time. A cohort of patients was analyzed for corneal staining score at initial visit and at 3 months of therapy with PRGF. Another cohort responded to a 10-item questionnaire that evaluated patients' satisfaction and safety, which included the symptom assessment questionnaire in dry eye (SANDE) score, after 6 months of PRGF treatment.ResultsA total of 153 patients were analyzed. Of these, 102 were reviewed for corneal epitheliopathy and 99 patients responded to the questionnaire. The mean (±SD) age of the population was 63.7 ± 17 years and 72.5% were female. The clinical indications for PRGF usage were dry eye (60%), neurotrophic keratopathy (15%), dormant corneal ulcers (12%), limbal stem cell deficiency (10%), and cicatrizing conjunctivitis (4%). At the final visit, 74.3% of patients showed an improvement of their corneal staining. Those who had punctate epithelial erosions or epithelial defects were reduced from 76.5% to 47% and 23.5% to 7.8% respectively (p < 0.0001). Symptoms, measured via SANDE score, significantly decreased from a median of 90 to 34.6 out of 100 points on follow-up (p < 0.0001). Only one patient (0.98%) complained of ocular burning sensation as a side effect.ConclusionsThis multicentric study demonstrates the safety and efficacy of the use of PRGF for treating signs and symptoms in patients with significant ocular surface diseases.     

Long-term outcomes of cultivated cell sheet transplantation for treating total limbal stem cell deficiency

PurposeThis study was conducted to determine the long-term outcomes of cultivated cell sheet transplantation (CCST), and to clarify risk factors that affected the outcomes.MethodsWe retrospectively analyzed the medical charts and photographs of 246 consecutive surgeries (162 eyes from 139 patients) that used CCST for treating total limbal stem cell deficiency. Deficiency types included Stevens–Johnson syndrome (SJS) or toxic epidermal necrolysis (SJS/TEN; n = 80), ocular surface burns (Burn; n = 75), ocular cicatricial pemphigoid (OCP) or pseudo-OCP (n = 58), and others (n = 33). The methods of CCST included 103 cultivated limbal epithelial transplantations (28 autologous, 62 allogeneic, and 13 living-related relatives), and 143 cultivated oral mucosal epithelial transplantations. We analyzed the rate of successful ocular surface reconstruction, clear corneas, and best-corrected visual acuity equal to or better than 20/200.ResultsWith a mean observation period of 357 weeks, successful ocular surface reconstruction and clear corneas at last visit were achieved in 65.1% of the patients. Corrected visual acuity equal to or better than 20/200 was achieved in 19.0% of the patients. Eyes with SJS/TEN exhibited worse outcomes compared with the Burn group. Postoperative complications included corneal ulcerations or perforations (18.3%), glaucoma (13.8%), and infections (8.5%). Multivariate analyses using the logistic mixed-effects model indicated that the presence of preoperative corneal epithelial defects was a significant risk factor for postoperative failure.ConclusionsIn this series, the success rate of CCST after a mean observation period of 357 weeks was 65.1%, and preoperative epithelial defect was the most relevant risk factor.