Allo-limbal transplantation in patients with limbal stem cell deficiency

AIM: To report the outcome of a series of patients with stem cell deficiency who underwent allo-limbal transplantation and to describe a technique for this procedure. METHODS: Six consecutive patients underwent allo-limbal stem cell transplantation. The primary diagnosis included alkali burn (n = 2), trachoma (n = 1), chronic rosacea blepharitis and kerato-conjunctivitis (n = 1), aniridia (n = 1), and Stevens-Johnson syndrome (n = 1). The limbal rim consisted of peripheral cornea and perilimbal sclera. FK-506 was used postoperatively for immunosuppression. RESULTS: The length of follow up ranged from 3 to 24 months (mean follow up 11.8 (SD 9.3) months). The outcome was considered satisfactory in five of six cases. The corneal surface was completely epithelialised within 2 weeks, and there was a substantial improvement in vision and symptoms. One patient had recurrent epithelial defects related to eyelid abnormalities. No side effects associated with systemic immunosuppression were noted. CONCLUSION: Allo-limbal transplantation, with systemic immunosuppression with FK-506 is useful in reconstruction of the ocular surface with improvement in vision in patients with severe stem cell deficiency.     

Midterm outcomes of penetrating keratoplasty following allogeneic cultivated limbal epithelial transplantation in patients with bilateral limbal stem cell deficiency

AIM: To evaluate the midterm outcomes of penetrating keratoplasty (PK) following allogeneic cultivated limbal epithelial transplantation (CLET) for bilateral total limbal stem cell deficiency (LSCD). METHODS: Ten patients (10 eyes) with bilateral LSCD were enrolled in this prospective noncomparative case series study. Each participant underwent PK approximately 6mo after a CLET. Topical tacrolimus, topical and systemic steroids, and oral ciclosporin were administered postoperatively. Best-corrected visual acuity (BCVA), intraocular pressure (IOP), ocular surface grading scores (OSS), corneal graft epithelial rehabilitation, persistent epithelial defect (PED), immunological rejection, and graft survival rate were assessed. RESULTS: The time interval between PK and allogeneic CLET was 6.90±1.29 (6-10)mo. BCVA improved from 2.46±0.32 logMAR preoperatively to 0.77±0.55 logMAR post-PK (P<0.001). Kaplan-Meier analysis of mean graft survival revealed graft survival rates of 100% at 12 and 24mo and 80.0% at 36mo. PEDs appeared in 5 eyes at different periods post-PK, and graft rejection occurred in 4 eyes. The total OSS decreased from 12.4±4.4 before allogeneic CLET to 1.4±1.51 after PK. CONCLUSION: A sequential therapy design of PK following allogeneic CLET can maintain a stable ocular surface with improved BCVA despite the relatively high graft rejection rate.     

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。     

Post-chemotherapy bilateral limbal stem cell deficiency

We report an extremely rare case of bilateral total limbal stem cell deficiency (LSCD) induced by long-term systemic chemotherapy for squamous cell carcinoma of the lung, and the outcomes of bilateral keratolimbal allograft transplantation (KLAT) in such a case. A 34-year-old male patient had underlying disease of squamous cell carcinoma in the right upper lung with extensive mediastinum and lymph node metastasis and received a series of systemic chemotherapy, including a combination of vinorelbine and cisplatin and a combination of gemcitabine, cisplatin, and docetaxel. According to clinical manifestations of ocular surface disorder, pathology findings, and immunostaining of cytokeratin 13 (CK13), LSCD secondary to systemic chemotherapy was diagnosed. KLAT was performed in both eyes. Although graft rejection developed in the right eye, complete re-epithelialization with favorable visual acuity occurred in the left eye 3 years after limbal transplantation.     

Short-term outcome of Boston Type 1 keratoprosthesis for bilateral limbal stem cell deficiency

This study reports the short-term functional and anatomical outcome of Boston Type 1 keratoprosthesis (Boston Kpro) implantation for bilateral limbal stem cell deficiency (LCSD). Retrospective analysis was done on eight eyes of eight patients who underwent Boston Kpro implantation between July 2009 and October 2009. The best corrected visual acuity (BCVA) and slit-lamp biomicroscopy findings were assessed at 1, 3 and 6 months postoperatively. All eight eyes retained the prosthesis. BCVA of 20/40 or better was achieved in 8, 6, and 5 eyes at 1, 3, and 6 months, respectively, postoperatively. One patient each developed epithelial defect, sterile stromal melt and fungal keratitis in the late postoperative period associated with antecedent loss of the soft contact lens from the eye. Boston Kpro has good short-term visual and anatomical outcome in patients with bilateral LSCD, provided compliance with postoperative care can be ensured.     

Quantification of corneal neovascularization after ex vivo limbal epithelial stem cell therapy

AIM: To assess cultured limbal epithelial stem cell transplantation in patients with limbal stem cell deficiency by analyzing and quantifying corneal neovascularization.METHODS: This retrospective, interventional case series included eight eyes with total limbal stem cell deficiency. Ex vivo limbal epithelial stem cells were cultured on human amniotic membrane using an animal-free culture method. The clinical parameters of limbal stem cell deficiency, impression cytology, and quantification of corneal neovascularization were evaluated before and after cultured limbal stem cell transplantation. The area of corneal neovascularization, vessel caliber (VC), and invasive area (IA) were analyzed before and after stem cell transplantation by image analysis software. Best-corrected visual acuity (BCVA), epithelial transparency, and impression cytology were also measured.RESULTS: One year after surgery, successful cases showed a reduction (improvement) of all three parameters of corneal neovascularization [neovascular area (NA), VC, IA], while failed cases did not. NA decreased a mean of 32.31% (P=0.035), invasion area 29.37% (P=0.018) and VC 14.29% (P=0.072). BCVA improved in all eyes (mean follow-up, 76±21mo). Epithelial transparency improved significantly from 2.00±0.93 to 0.88±1.25 (P=0.014). Impression cytology showed that three cases failed after limbal epithelial stem cell therapy before 1y of follow-up.CONCLUSION: This method of analyzing and monitoring surface vessels is useful for evaluating the epithelial status during follow-up, as successful cases showed a bigger reduction in corneal neovascularization parameters than failed cases. Using this method, successful cases could be differentiated from failed cases.     

分期实施异体角膜缘干细胞移植与穿透性角膜移植术治疗严重化学伤

有1位于1996年实施右眼穿透性角膜移植术失败的双眼严重眼表化学伤的患者, 62岁,右眼视力手动,左眼视力光感。于2006-09-16/2007-02-07分别对右眼实施了异体角膜缘干细胞移植术和穿透性角膜移植术,经过术后5m的药物治疗观察,最终获得了右眼最佳矫正视力为6/30,角膜移植片透明,眼表得以重建,并未发现明显排斥迹象的良好效果。     

Dyskeratosis congenita and limbal stem cell deficiency

In light of the latest developments in the field of molecular hematology, we herein discuss the reported cases that have presented dyskeratosis congenita as one of the inherited stem cell diseases causing limbal stem cell deficiency.     

利用组织工程方法治疗角膜缘干细胞缺失的相关研究进展

角膜缘干细胞在角膜上皮更新和创伤愈合中起重要作用,角膜缘干细胞缺失可导致各种严重眼表面疾患。因为治疗上存在自体移植的健眼损伤问题,同种异体移植的排斥反应以及供体不足等问题,近年来随着对干细胞研究的深入以及组织工程学的发展,有利于克服和解决上述问题,为角膜缘干细胞缺乏的治疗带来了曙光。本文通过近年来组织工程治疗角膜缘干细胞缺失的相关研究的回顾,归纳了种子细胞和支架材料选择两方面的研究进展,以探索该领域存在的问题和未来的研究方向。（国际眼科纵览, 2017,  41:   346-351）     

Transplantation of tissue-engineered human corneal epithelium in limbal stem cell deficiency rabbit models

AIM: To evaluate the biological functions of tissue-engineered human corneal epithelium (TE-HCEP) by corneal transplantation in limbal stem cell deficiency (LSCD) rabbit models. METHODS: TE-HCEPs were reconstructed with DiI-labeled untransfected HCEP cells and denuded amniotic membrane (dAM) in air-liquid interface culture, and their morphology and structure were characterized by hematoxylin-eosin (HE) staining of paraffin-sections, immunohistochemistry and electron microscopy. LSCD models were established by mechanical and alcohol treatment of the left eyes of New Zealand white rabbits, and their eyes were transplanted with TE-HCEPs with dAM surface outside by lamellar keratoplasty (LKP). Corneal transparency, neovascularization, thickness, and epithelial integrality of both traumatic and post transplantation eyes were checked once a week by slit-lamp corneal microscopy, a corneal pachymeter, and periodic acid-Schiff (PAS) staining. At day 120 post surgery, the rabbits in each group were sacrificed and their corneas were examined by DiI label observation, HE staining, immunohistochemistry and electron microscopy. RESULTS: After cultured for 5 days on dAM, HCEP cells, maintaining keratin 3 expression, reconstructed a 6-7 layer TE-HCEP with normal morphology and structure. The traumatic rabbit corneas, entirely opaque, conjunctivalized and with invaded blood vessels, were used as LSCD models for TE-HCEP transplantation. After transplantation, obvious edema was not found in TE-HCEP-transplanted corneas which became more and more transparent, the invaded blood vessels reduced gradually throughout the monitoring period. The corneas decreased to normal thickness on day 25, while those of dAM eyes were over 575μm in thickness during the monitoring period. A 4-5 layer of epithelium consisting of TE-HCEP originated cells attached tightly to the anterior surface of stroma was reconstructed 120 days after TE-HCEP transplantation, which was similar to the normal control eye in morphology and structure. In contrast, intense corneal edema, turbid, invaded blood vessels were found in dAM eyes, and no multilayer epithelium was found but only a few scattered conjunctiva-like cells appeared. CONCLUSION: The TE-HCEP, with similar morphology and structure to those of innate HCEP, could reconstruct a multilayer corneal epithelium with normal functions in restoring corneal transparency and thickness of LSCD rabbits after transplantation. It may be a promising HCEP equivalent for clinical therapy of corneal epithelial disorders.     

角膜上皮移植术治疗角膜缘干细胞功能障碍临床探究

目的 探讨多种角膜上皮移植术式治疗角膜缘干细胞功能衰竭的手术技术和成败因素。方法根据角膜缘受损程度，用全角膜上皮移植、指环形上皮移植、次全角膜上皮移植和蕈状瓣移植4种不同术式治疗38例（42眼）角膜缘干细胞功能衰竭；术后环孢素和／或糖皮质激素口服2～9个月，联合局部滴眼至少12个月防范移植排斥；总结临床规律和成败因素。结果术后平均随访14．5个月（3～25个月），35眼（83．3％）视力≥0．05，其中31眼裸眼视力≥0．3；失败5例中，4例为Ⅳ度眼表烧伤，并发眼睑缺损和睑球粘连；另一例因术中植床穿破，术后长期双前房导致植片水肿混浊。结论同种异体角膜上皮移植术通过角膜缘干细胞移植，使患眼回归正常的生理状态而恢复视力或重新获得稳定的眼表；为尔后的增视性角膜和／或晶状体手术提供了稳定的解剖学基础。     

Ex vivo expansion of limbal epithelial stem cells: amniotic membrane serving as a stem cell niche

Identification, maintenance, and expansion of stem cells for subsequent transplantation has become a new strategy for treating many diseases in most medical subspecialties. The stem cells of the corneal epithelium are located in the limbal basal layer and are the ultimate source for constant corneal epithelial renewal. Like those in other tissues, limbal stem cells are supported by a unique stromal microenvironment called the stem cell niche, which consists of certain extracellular matrix components, cell membrane-associated molecules, and cytokine dialogues. Destructive loss of limbal stem cells or dysfunction of their stromal environment renders many corneas with a clinical entity called limbal stem cell deficiency, which is characterized by variable extents of conjunctival ingrowth depending on the severity of limbal damage. A new strategy of treating limbal stem cell deficiency is to transplant a bio-engineered graft by expanding limbal epithelial stem cells ex vivo on amniotic membrane. This review summarizes the published literature data collectively explaining how amniotic membrane is an ideal biological substrate that can help maintain and support the expansion of limbal epithelial stem cells.     

角膜缘干细胞研究新进展

位于角膜最外层的角膜上皮细胞,正常损耗或创伤后,由角膜缘干细胞不断自我更新补充。当损伤等致角膜缘干细胞缺乏时,则会导致角膜溃疡、新生血管、混浊等角膜病变而影响视力。目前治疗这些角膜疾病的重要方式之一为移植体外培养的角膜缘干细胞。本文将从角膜缘干细胞的定位、体外培养、新细胞来源等方面进行综述。     

角膜缘干细胞移植治疗翼状胬肉的远期疗效观察

目的:探讨角膜缘干细胞移植治疗复发性翼状胬肉的远期疗效。方法:复发性翼状胬肉110例患者中,有19例(A组)接受翼状胬肉切除+丝裂霉素手术,91例(B组)接受了翼状胬肉切除+角膜缘干细胞移植术。结果:实施翼状胬肉切除术+丝裂霉素的19例患者中,术后6例翼状胬肉复发;接受翼状胬肉切除+角膜缘干细胞移植的91例患者中,术后有2例翼状胬肉复发。结论:经观察复发性翼状胬肉远期疗效证实,翼状胬肉切除+角膜缘干细胞移植术是治疗复发性翼状胬肉的理想方法。年轻患者较年老患者手术复发率高。     

翼状胬肉扩大切除联合自体角膜缘干细胞移植术的临床疗效观察

目的:探讨翼状胬肉扩大切除联合自体带结膜瓣的角膜缘干细胞移植术的手术方法及其临床疗效。方法:选择达到或超过瞳孔缘的35例36眼翼状胬肉患者,其中有4眼为复发性,其余均为原发性,于显微镜下做扩大的翼状胬肉切除,包括变性的球筋膜,联合自体带结膜瓣的角膜缘干细胞移植术,术后随访6～24.4mo,中位数为14.6mo,观察术眼手术前后视力变化及术后复发情况。结果:术前视力为指数/眼前～0.6,中位数为0.25,术后1mo视力为指数/眼前～1.2,中位数为0.7(秩和检验u=5.325,P<0.01)。无1例术后复发,复发率为0。结论:翼状胬肉扩大切除联合自体带结膜瓣的角膜缘干细胞移植术,术后复发率低,视力提高显著,是治疗翼状胬肉的有效方法,其术后的长期疗效仍需进一步观察。     

兔角膜缘上皮细胞培养后自体移植修复

目的：运用培养角膜缘上皮细胞联合人羊膜行自体移植的方法，观察植片修复兔眼角膜上皮的疗效。方法：选用健康新西兰白兔20只，制成右眼角膜缘干细胞缺乏的兔眼模型，其中12只兔行角膜缘上皮细胞培养联合羊膜自体移植，另外8只兔只进行单纯羊膜移植。术后每周对眼表情况进行评分，术后1mo眼角膜进行苏木素-伊红(HE)染色和透射电镜观察。结果：移植了含有自体角膜上皮细胞的兔眼，术后早期都形成了角膜上皮化并明显抑制了新生血管的再生，HE染色和电镜观察表明培养并移植的角膜上皮与正常的角膜上皮无明显差异；而只接受羊膜移植的兔眼，术后又出现角膜混浊和明显的新生血管，表明角膜表面被结膜上皮覆盖。结论：该方法术后早期可以恢复角膜上皮化，重建正常眼表，疗效明显优于单纯羊膜移植。     

自体角膜缘干细胞移植治疗翼状胬肉取不同经线宽度植片的疗效比较

目的：比较自体角膜缘干细胞移植治疗翼状胬肉取不同经线宽度结膜植片的疗效。方法：对182例252眼初发翼状胬肉行翼状胬肉切除联合角膜缘干细胞移植,按术中结膜植片的经线宽度分为两组：小植片组86例110眼,行干细胞移植时取结膜植片经线宽度为3 mm;大植片组96例142眼,取结膜植片经线宽度为5mm,术后1、6、12、24mo随访,观察两组的复发率统计分析其差异。结果：术后早期（1～12mo）小植片组与大植片组复发率比较无统计学意义（P＞0．05）,术后远期（24mo）复发率差异有统计学意义（P＜0．05）。结论：翼状胬肉切除联合自体角膜缘干细胞移植治疗翼状胬肉,较大经线宽度的结膜植片可以降低远期的复发率。     

Corneal surface reconstruction using adult mesenchymal stem cells in experimental limbal stem cell deficiency in rabbits

Purpose: To investigate the ability of mesenchymal stem cells (MSC) to transdifferentiate to corneal epithelial cells in experimental limbal stem cell deficiency in rabbits. Methods: Total limbal stem cell deficiency was produced in 21 right eyes of 21 New Zealand rabbits; 6 eyes served as controls (group 1, G1). After removal of the conjunctival overgrowth, five eyes received amniotic membrane transplantation (AMT; G2). In four eyes, autologous limbal stem cell transplantation from the healthy eye was performed with AMT (G3). In another six eyes, enriched autologous MSC were injected under the amniotic membrane (AM) (G4). Within 280 days, corneoscleral discs were analysed for goblet cells, cytokeratin (CK) 3/12, connexin 43, β₁‐integrin, CK 19, α‐enolase, p63 and ATP‐binding cassette transporter subtype G‐2 (ABCG‐2) distribution patterns. Results: Cultivated MSC were positive for CK 3/12 and α‐enolase, but negative for ABCG‐2, p63 and connexin 43. On rabbit corneas, CK 3/12 was expressed in all corneal regions in all groups, but with significantly different intensities. Among all other parameters, expression levels of ABCG‐2, β₁‐integrin and connexin 43 were significantly different between the transplanted groups and the control group. After a mean follow‐up time of 172 (47–280) days, goblet cells were rarely present in the central cornea (G1‐4). Conclusion: CK 3/12 is not highly specific for differentiated corneal epithelium. Further, goblet cells are not a reliable marker for conjunctivalization in rabbits. Expression of ABCG‐2, β₁‐integrin and connexin 43 after mesenchymal stem cell transplantation may indicate their ability to maintain their stem cell character or to transdifferentiate to epithelial progenitor cells.     

翼状胬肉切除联合自体角膜缘干细胞移植术临床疗效观察

目的观察翼状胬肉切除联合自体角膜缘干细胞移植术治疗翼状胬肉的临床疗效。方法选择2013年2月-2014年2月眼科收治的翼状胬肉患者82例（86眼）,随机分为观察组43例（45眼）和对照组39例（41眼）。观察组行翼状胬肉切除联合自体角膜缘干细胞移植术,对照组行单纯翼状胬肉切除术。结果观察组患者角膜上皮化愈合时间短于对照组,术后眼内异物感、畏光、流泪等不适症状持续时间短于对照组,差异有统计学意义（P〈0.05）。观察组患者复发率低于对照组,差异有统计学意义（P〈0.05）。结论翼状胬肉切除联合自体角膜缘干细胞移植术取材容易,简单易行,可明显降低翼状胬肉术后的复发率,是一种治疗翼状胬肉行之有效的好方法。在翼状胬肉切除治疗中有效促进了眼表结构和功能的修复,对降低翼状胬肉复发率有重要意义。