Corneal stromal mesenchymal stem cells: reconstructing a bioactive cornea and repairing the corneal limbus and stromal microenvironment

Corneal stroma-derived mesenchymal stem cells(CS-MSCs) are mainly distributed in the anterior part of the corneal stroma near the corneal limbal stem cells(LSCs). CS-MSCs are stem cells with self-renewal and multidirectional differentiation potential. A large amount of data confirmed that CS-MSCs can be induced to differentiate into functional keratocytes in vitro, which is the motive force for maintaining corneal transparency and producing a normal corneal stroma. CS-MSCs are also an important component of the limbal microenvironment. Furthermore, they are of great significance in the reconstruction of ocular surface tissue and tissue engineering for active biocornea construction. In this paper, the localization and biological characteristics of CS-MSCs, the use of CS-MSCs to reconstruct a tissue-engineered active biocornea, and the repair of the limbal and matrix microenvironment by CS-MSCs are reviewed, and their application prospects are discussed.

The Limbal Niche and Its Role in Maintaining Corneal Regeneration

In recent years, stem cells have been a focal point in research designed to evaluate the efficacy of ophthalmologic therapies, specifically those for corneal conditions. The corneal epithelium is one of the few regions of the body that maintains itself using a residual stem cell population within the adjacent limbus. Stem cell movement has additionally captivated the minds of researchers due to its potential application in different body regions. The cornea is a viable model for varying methods to track stem cell migratory patterns, such as lineage tracing and live imaging from the limbus. These developments have the potential to pave the way for future therapies designed to ensure the continuous regeneration of the corneal epithelium following injury via the limbal stem cell niche. This literature review aims to analyze the various methods of imaging used to understand the limbal stem cell niche and possible future directions that might be useful to consider for the better treatment and prevention of disorders of the cornea and corneal epithelium. .

角巩膜缘与角膜切口超声乳化术治疗老年白内障疗效对比

目的 对比角巩膜缘与角膜切口超声乳化术治疗老年白内障的临床疗效。方法 随机将80例老年白内障患者分为2组。2组均行超声乳化术,对照组采用角膜切口,观察组采用角巩膜缘切口。比较2组上皮修复时间、切口修复时间、裸眼视力、最佳矫正视力(BCVA)、角膜散光度、手术源性角膜散光度、泪囊破裂时间(BUT)、角膜荧光素染色评分(FLCs)、泪液分泌试验(SIt)水平以及术后并发症发生率。结果 观察组上皮修复、切口修复时间均早于对照组(P<0.05);观察组术后1周、2周、4周时裸眼视力、BCVA水平以及BUT、Sit水平均高于对照组(P<0.05);观察组术后1周、2周、4周时角膜散光度、手术源性角膜散光度以及FLCs水平水平均低于对照组(P<0.05);2组术后并发症发生率(18%与12%)差异无统计学意义(P>0.05)。结论 角巩膜缘切口超声乳化术治疗老年白内障临床疗效较好,患者术后角膜散光度较低,且视力、泪膜功能稳定性恢复较好。

An updated narrative review of treatment for limbal epithelial stem cell deficiency

Background and Objective:Nearly 30 years have passed since limbal stem cell deficiency(LSCD)was first identified by pioneers and given clinical attention.LSCD remains a difficult disease to treat.It can potentially lead to blinding.At present,understanding of limbal stem cells(LSCs)has deepened and various treatment options for LSCD have been devised.The objective of this review is to summarize basic knowledge of LSCD and current treatment strategies.Methods:PubMed search was performed to find studies published in English on LSCs and LSCD including original reports and reviews.Literatures published from 1989 to 2022 were reviewed.Key Content and Findings:LSCs are enigmatic stem cells for which no specific marker has been discovered yet.Although LSCD is not difficult to diagnose,it is still challenging to treat.An important advancement in the treatment of LSCD is the provision of guidelines for selecting systematic surgical treatment according to the patient’s condition.It is also encouraging that stem cell technologies are being actively investigated for their potential usefulness in the treatment of LSCD.Conclusions:Although various treatment options for LSCD have been developed,it should be kept in mind that the best chance of treatment for LSCD is in the early stage of the disease.Every effort should be made to preserve as many LSCs as possible in the early treatment of LSCD.

An interferometric ex vivo study of corneal biomechanics under physiologically representative loading,highlighting the role of the limbus in pressure compensation

Background:The mechanical properties of the cornea are complex and regionally variable.This paper uses an original method to investigate the biomechanics of the cornea in response to hydrostatic loading over the typical physiological range of intraocular pressure(IOP)fluctuations thereby increasing understanding of clinically relevant corneal biomechanical properties and their contributions to the refractive properties of the cornea.Methods:Displacement speckle pattern interferometry(DSPl)was used to measure the total surface displacement of 40 porcine and 6 human corneal-scleral specimens in response to pressure variations up to 1 mmHg from a baseline of 16.5 mmHg.All specimens were mounted in a modified artificial anterior chamber(AAC)and loaded hydrostatically.Areas of high strain in response to loading were identified by comparing the displacements across different regions.Results:The nature of the response of the corneal surface to loading demonstrated high regional topographic variation.Mechanical properties were shown to be asymmetrical,and deformation of the limbal and pre-limbal regions dominated these responses respectively with over 90%(nasal-temporal)and 60%(superior-inferior)of the total maximum displacement occurring in these regions indicating high strain.In contrast,the curvature of the central cornea remained relatively unchanged merely translating in position.Conclusions:The limbal and pre-limbal regions of the cornea appear to be fundamental to the absorption of small pressure fluctuations facilitating the curvature of the central cornea to remain relatively unchanged.The differential mechanical properties of this region could have important implications for the application of corneal surgery and corneal crosslinking,warranting further investigation.

An interferometric ex vivo study of corneal biomechanics under physiologically representative loading,highlighting the role of the limbus in pressure compensation

Background:The mechanical properties of the cornea are complex and regionally variable.This paper uses an original method to investigate the biomechanics of the cornea in response to hydrostatic loading over the typical physiological range of intra-ocular pressure(IOP)fluctuations thereby increasing understanding of clinically relevant corneal biomechanical properties and their contributions to the refractive properties of the cornea.Methods:Displacement speckle pattern interferometry(DSPI)was used to measure the total surface displacement of 40 porcine and 6 human corneal-scleral specimens in response to pressure variations up to 1 mmHg from a baseline of 16.5 mmHg.All specimens were mounted in a modified artificial anterior chamber(AAC)and loaded hydrostatically.Areas of high strain in response to loading were identified by comparing the displacements across different regions.Results:The nature of the response of the corneal surface to loading demonstrated high regional topographic variation.Mechanical properties were shown to be asymmetrical,and deformation of the limbal and pre-limbal regions dominated these responses respectively with over 90%(N-T)and 60%(S-I)of the total maximum displacement occurring in these regions indicating high-strain.In contrast,the curvature of the central cornea remained relatively unchanged merely translating in position.Conclusions:The limbal and pre-limbal regions of the cornea appear to be fundamental to the absorption of small pressure fluctuations facilitating the curvature of the central cornea to remain relatively unchanged.The differential mechanical properties of this region could have important implications for the application of corneal surgery and corneal crosslinking,warranting further investigation.

自体角膜缘干细胞移植与常规翼状胬肉切除术疗效比较

目的通过翼状胬肉单纯切除术及翼状胬肉切除加自体角膜缘干细胞移植术2种手术方式治疗翼状胬肉,比较其治愈及复发情况及角膜上皮修复时间,评价其疗效。方法将86例(88眼)翼状胬肉患者随机分为翼状胬肉单纯切除术组(A组)43例44眼及翼状胬肉切除加自体角膜缘干细胞移植术组(B组)43例44眼,组间比较采用χ2检验比较其疗效。结果A组10例10眼复发,B组仅有1例1眼复发,2组比较差异有统计学意义(P<0.05);A组角膜上皮修复时间平均为(5.15±0.92)d,B组角膜上皮修复时间平均为(3.36±0.82)d,2组比较差异有统计学意义(P<0.05)。结论翼状胬肉切除加自体角膜缘干细胞移植术可以降低复发率,缩短角膜上皮修复时间,是治疗翼状胬肉较为安全有效的方法。

正常角膜缘组织中与血管生成相关的生长因子及其受体的表达

目的探查正常人角膜缘组织里与血管生成相关的生长因子及其受体的分布。方法用冰冻切片和链亲合素过氧化物酶染色系统，选择一组兔抗人多克隆抗体，即：血管内皮生长因子（ＶＥＧＦ）、碱性成纤维细胞生长因子（ｂＦＧＦ）、血小板源性生长因子（ＰＤＧＦ－Ａ）和转化生长因子－β １（ＴＧＦ－β １）及其受体对１１例角膜移植供体材料的角膜缘组织进行了检查。结果ＶＥＧＦ，ｂＦＧＦ，ＰＤＧＦ－Ａ和ＴＧＦ－β １ ４种生长因子仅在１或２例标本中不表达外，在大部分标本中表达阳性。４种生长因子的受体在角膜缘上皮、基底膜、上皮下组织和角巩膜组织内阳性表达分别是：ＶＥＧＦ ９例、７例、１０例和８例；ｂＦＧＦ ８例、９例、９例和８例；ＰＤＧＦ ５例、４例、１０例和７例；ＴＧＦ－β １３例、０例、１１例和６例。结论在正常角膜缘组织中，存在ＶＥＧＦ，ｂＦＧＦ，ＰＤＧＦ－Ａ和ＴＧＦ－β １及其受体，可能在维持正常的角膜功能和调节角膜缘组织的生长和增殖中起着重要作用。

HLA匹配角膜缘干细胞联合羊膜移植修复翼状胬肉

背景:HLA匹配的角膜缘干细胞联合羊膜移植治疗翼状胬肉是眼科医生治疗的新理念,该方法治疗后并发症少、康复快、复发率低,尤其是能减少移植排斥反应等已被广泛接受。目的:探讨HLA匹配的角膜缘干细胞联合羊膜移植治疗翼状胬肉疗效。方法:选取2013年3月1日至2014年4月30日行HLA匹配的角膜缘干细胞联合羊膜移植治疗的翼状胬肉患者47例(47眼)作为试验组;回顾性分析2010年3月1日至2012年12月1日采用单纯翼状胬肉切除方法治疗的翼状胬肉患者40例(47眼)作为对照组,对两组患者治愈和复发情况进行比较。结果与结论:试验组47例,全部顺利完成治疗,无并发症,其中1例切口感染,特殊对症治疗后均已好转出院,全部患者均无移植排斥反应。试验组治愈45眼,复发2眼,复发率为4.3%。对照组47眼中治愈32眼,复发15眼,复发率31.9%。结果表明HLA匹配的角膜缘干细胞联合羊膜移植治疗翼状胬肉并发症少、复发率低,是治疗翼状胬肉一种行之有效的方法。

组织块培养角膜缘上皮细胞的生物学特征观察

目的 观察以人羊膜为底物组织块培养角膜缘上皮细胞的生物学特征。方法 取含有上皮细胞大小约 2mm×2mm、厚约 2 0 0 μm兔角膜缘组织 ,剪切成 4个小的组织块 ,用消化液消化至组织块变疏松后放在去除上皮细胞的羊膜上培养 ,每 2～ 3d用倒置显微镜观察 ,定期取羊膜进行细胞组织学、免疫组织化学及电镜检查。结果 角膜缘组织块在羊膜上培养 8d ,羊膜上培养的角膜缘上皮细胞出现汇合并部分成层 ,培养 16d ,细胞形成 3～ 4层 ,并很好地附着于下面的羊膜上 ,羊膜上培养的细胞经广谱角蛋白 (pan keratin ,PK)抗体和抗增殖细胞核抗原 (proliferatingcellnuclearanti gan ,PCNA)单克隆抗体免疫组织化学染色呈阳性反应。结论 羊膜作为底物组织块培养的角膜缘上皮细胞短期内能形成多层且具有较强增殖能力 。

ROCK抑制剂Y-27632在角膜保存时对角膜缘干细胞活性的促进作用

背景 角膜缘干细胞（LSCs）对维持角膜上皮的稳定和角膜组织透明性有重要作用.Rho关联卷曲螺旋蛋白激酶（ROCK）抑制剂Y-27632能够促进人胚胎干细胞、角质上皮细胞等的增生,减少细胞凋亡.目的 探讨Y-27632对离体兔角膜保存和体外LSCs扩增能力的影响. 方法 在细胞培养基MEM中加入质量分数12.5％硫酸软骨素、质量分数10.0％低分子右旋糖酐、20.0 mg/L地塞米松、100 mg/L妥布霉素注射液、9.5 g/L Hepes,使用时添加0.375 mg/L L-谷氨酰胺,制备成角膜活性保存液.将新西兰大白兔的角膜组织片分别置于含或不含Y-27632的角膜活性保存液中保存4、7、14d,采用质量分数0.25％锥虫蓝和质量分数0.2％茜素红染色法观察角膜内皮细胞密度及形态,采用Giemsa染色法并使用Image J图像分析软件计数克隆球数量和LSCs活性,计算LSCs存活率和克隆形成效率. 结果 角膜片保存4d时,Y-27632保存液组和单纯角膜中期保存液组角膜内皮细胞形态无明显差别,角膜片保存7d时,Y-27632保存液组角膜内皮细胞形态仍保持规则的六边形,而单纯角膜中期保存液组角膜内皮细胞的细胞膜轻微皱缩,少数细胞体积变大.角膜片保存14 d时单纯角膜中期保存液组角膜内皮细胞可见较多的茜素红斑.单纯角膜中期保存液组角膜内皮细胞计数为（2 262±75）/mm2,Y-27632保存液组角膜内皮细胞计数为（2 425 ±95）/mm2,差异有统计学意义（P＜0.001）.新鲜角膜片和角膜片保存4d时,Y-27632保存液组和单纯角膜中期保存液组LSCs的克隆球均较大,其内细胞数较多,而保存7d和14 d时,与Y-27632保存液组比较,单纯角膜中期保存液组LSCs克隆球直径明显缩小.新鲜分离的角膜片和角膜片保存4d时,Y-27632保存液组与单纯角膜中期保存液组LSCs的克隆形成率和角膜上皮细胞存活率的差异均无统计学意义（均P＞0.05）;角膜片保存7d和14d时,角膜缘上皮细胞的活性率分别为（73.00±2.12）％和（56.00±0.71）％,明显高于单纯角膜中期培养液组的（66.00±4.00）％和（49.00±0.71）％,差异均有统计学意义（t=3.098,P=0.018;t=9.798,P=0.000）;角膜片保存7d和14 d时,Y-27632保存液组与单纯角膜中期保存液组LSCs的克隆形成效率分别为（11.05±0.21）％和（3.10±1.97）％,明显高于单纯角膜中期保存液组中的（2.05±1.20）％和（0.40±0.14）％,差异均有统计学意义（t=18.107,P=0.000;t=3.184,P=0.017）.结论角膜保存液中添加Y-27632可明显提高离体角膜保存的效果,同时可维持LSCs的活性及克隆形成能力.Y-27632可作为角膜保存液的有效添加成分。

制作角膜缘干细胞完全缺乏动物模型的新方法

目的提出一种新的角膜缘干细胞完全缺乏模型制作方法，为干细胞缺乏疾病的治疗性研究提供可靠的模型。方法利用9mm环钻划界，板层切除角膜组织，通过临床评分、印迹细胞学、组织病理及免疫组织化学验证模型的成功。结果板层切除术后30～45d，模型眼角膜混浊、上皮缺损、大量新生血管长入，印迹细胞学可见PAS（＋）的杯状细胞，组织病理学检查上皮符合结膜细胞表型，免疫荧光染色可见结膜杯状细胞特异性标志MUC5AC阳性表达。结论环钻划界加板层切除法为一种可靠、有效的角膜缘干细胞完全缺乏模型制作方法。

角膜缘干细胞自体移植联用羊膜治疗复发性翼状胬肉

目的:观察自体角膜缘干细胞移植联合羊膜移植治疗复发性翼状胬肉的临床疗效。方法:自体角膜缘干细胞移植联合羊膜移植治疗复发性翼状努肉30例,术后随访1年,观察胬肉复发及手术并发症情况。结果:无复发及睑球粘连等并发症发生。结论:自体角膜缘干细胞移植联合羊膜移植治疗复发性翼状胬肉是安全有效的治疗方法。

翼状胬肉模型的建立

目的人为地造成局部角膜缘干细胞缺损以及慢性结膜炎症的眼表模型,探讨翼状胬肉的形成原因。方法40只新西兰大白兔随机分为A、B、C、D 4组,设右眼为实验眼,左眼为对照眼。采用手术切除1个象限角巩膜缘组织的方法产生角膜缘干细胞缺损模型,眼部以稀盐酸(A、B组)或滑石粉(C、D组)定期点眼产生慢性角结膜炎模型。实验眼在同一眼上观察同时产生角膜缘干细胞缺损和慢性角结膜炎模型后的形态学变化,对照眼分别采用两种模型之一的相应形态学改变。于2、4、8、12周每只眼记录角膜上皮缺损、球结膜充血和结膜新生物情况,评分,并随机每组取材2只作病理。结果切除角膜缘干细胞的兔眼,可以在短期内在切除角膜缘干细胞的方向上出现类胬肉增殖组织样改变。类胬肉增殖组织的形成、程度和是否有局部的炎症反应以及角膜上皮完整性的破坏呈正相关性。结论局限性角膜缘干细胞的缺损和长期慢性炎症刺激的同时作用可能是翼状胬肉形成的主要原因。

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。

组织工程技术构建角膜组织研究进展

同种异体角膜移植是目前治疗角膜盲最常用、最有效的治疗方法,但由于供体来源匮乏限制了此技术的应用。组织工程技术是应用细胞生物学和工程学原理,研究开发制作用于修复、改善损伤组织结构和功能替代物的一门技术。本文就组织工程技术构建角膜组织的研究进展进行综述。

培养的角膜缘上皮细胞增殖能力的动态研究

目的 探讨组织块培养角膜缘上皮细胞在不同时间以及第 2、3代培养的角膜缘上皮细胞早期 (6～ 8d)的增殖能力。方法 应用组织块培养法培养角膜缘上皮细胞 ,在d8、12、16、2 0、2 4用流式细胞仪检测其细胞周期 ,同时对组织块培养 16d的角膜缘上皮细胞进行传代 ,用同样方法检测第 2、3代细胞出现汇合时 (6～ 8d)的细胞周期。结果 组织块培养角膜缘上皮细胞增殖指数 (ProliferativeIndex ,PI)在高水平上呈下降趋势 (分别为 31 2 %、2 0 1%、17 6 %、15 6 %、13 4 % ) ;2、3代培养细胞早期的增殖指数较高 (分别为 30 3%、2 6 % )。结论 组织块培养的角膜缘上皮细胞增殖能力在 8～ 2 4d内在高水平上呈下降趋势 ,第 2、3代培养的细胞在培养早期的增殖能力也较高 。

糖尿病泪液功能和眼表面变化

目的:探讨糖尿病患者泪液功能和眼表面失调及其影响因素。方法:研究60例120眼非胰岛素依赖型患者,根据病程将其分为A,B,C3个组:A组患者病程<5a,B组患者病程5～9a,C组患者病程≥10a;40例80眼无糖尿病同年龄段者设为对照组。所有研究者均观察角膜荧光素染色、泪膜破裂时间及Schirmer试验;对糖尿病组及对照组各20眼行结膜印记细胞学检查。结果:糖尿病组泪膜破裂时间较对照组短(u=13.54,P<0.01),角膜荧光素染色阳性细胞检出率高于对照组(χ2=52.21,P<0.01),泪液分泌试验较对照组少(t=2.05,P<0.05)。B,C组泪膜破裂时间较A组短,泪液分泌亦减少。结膜印记细胞学检查糖尿病组结膜上皮细胞异常较多,杯状细胞数较少。结论:糖尿病患者是干眼症的易患人群;糖尿病患者的眼表疾病以泪液的质和量的失调,鳞状上皮化生、杯状细胞减少为特征。泪膜破裂时间、Schirmer试验、角膜荧光素染色检查可作为糖尿病患者的眼科常规检查。

人角膜上皮细胞的体外培养方法探讨

目的：探寻角膜上皮移植细胞的体外培养方法。方法：用改良的细胞悬液培养法与组织块培养法，描绘两种方法获取细胞的生长曲线，并计算细胞倍增时间；用＾３Ｈ胸腺嘧啶核苷（＾３Ｈ－ＴｄＲ）掺入与液体闪烁技术观察细胞ＤＮＡ合成能力。结果：改良的细胞悬液法与组织块培养法培养的细胞平均倍增时间分别为５４．１５±４．２８小时和６７．６８±１．９６小时（Ｐ〈０．０１）；应用前者培养的细胞ＤＮＡ合成也明显活跃（Ｐ〈０．

以羊膜为载体体外培养大鼠角膜缘干细胞的实验研究

目的比较角膜缘干细胞(LSCs)在不同羊膜上的生长情况,寻求LSCs体外培养的最佳载体。方法取健康剖宫产孕妇胎盘的羊膜组织,分别制备保留上皮的新鲜羊膜、去除上皮的新鲜羊膜和冷冻羊膜。消化法分离大鼠LSCs后将其按相同浓度分别种植于3种事先准备好的羊膜载体和无载体孔中,分为4组。实验1组:保留上皮的新鲜羊膜组;实验2组:去除上皮的新鲜羊膜组;实验3组:去除上皮的冷冻羊膜组;对照组:无羊膜载体组。4组细胞分别作细胞凋亡检测,p63、PCNA免疫荧光检测,检测阳性细胞计数。结果培养第3天,3个实验组培养的细胞排列紧密,融合形成单层,其中可见大小不等的团块状集落,细胞大小基本一致,多呈圆球形或卵圆形。对照组培养的细胞排列相对稀疏,基本融合,形成单层,可见散在的、大小不等的桑葚样细胞集落,集落细胞的生长特征及形态特点与实验组相似。实验组的细胞凋亡率均低于对照组,p63阳性率和PCNA阳性率均高于对照组(P<0.01),尤以实验1组变化最为明显(P<0.01)。结论羊膜载体有利于体外培养大鼠LSCs的扩增,保留上皮的新鲜羊膜能更好地维持体外培养LSCs的特性,是体外角膜缘干细胞培养的理想载体。