Chondromodulin-I expression in rat articular cartilage

The localization and expression of chondromodulin-I (ChM-I), an angiogenesis inhibitor, in the rat articular cartilage during maturation from 2 to 10 weeks of age were examined by immunohistochemistry, Western blot analysis and ribonuclease protection assay, and the results were compared with those in the epiphyseal cartilage. ChM-I was found to be diffusely immunostained in the inter-territorial space of the cartilage matrix from the intermediate to the deep layers at the immature stage. As the articular cartilage matured, the immunoreactivity was localized around the hypertrophic chondrocytes in the deep layer and the immunoreactivity became weak after maturation. In contrast, the ChM-I immunoreactivity was intense in the epiphyseal cartilage at all ages examined. ChM-I was detected by Western blotting as a broad band or occasionally as a cluster of multiple bands (approximately 25 kDa) in both the articular and the epiphyseal cartilage. The intensity of the bands decreased gradually with age in the articular cartilage, but was unchanged in the epiphyseal cartilage at all ages. Ribonuclease protection assay revealed that ChM-I mRNA also decreased gradually with age in the articular cartilage in parallel with the maturation of the articular cartilage, while no decrease in ChM-I mRNA was found in the epiphyseal cartilage. The expression of ChM-I mRNA in the articular cartilage was less than that in the epiphyseal cartilage at all ages. The decrease in amount of ChM-I in the mature articular cartilage suggests that ChM-I plays a more important role in the maintenance of avascularity in the immature articular cartilage than in the mature one. The avascular condition may be preserved by angiogenic inhibitors or mechanisms other than ChM-I in the mature articular cartilage.     

小鼠角膜的发生与细胞凋亡

目的 通过观察小鼠角膜的发生过程,探讨角膜细胞的增殖与凋亡对角膜结构修复与塑形的作用。方法 各日龄共计120只小鼠,用HE染色或4’,6-二脒基-2-苯基吲哚（DAPI）染色对小鼠角膜的一般结构进行观察;用5′-溴脱氧尿嘧啶核苷（BrdU）技术标记角膜增殖细胞和免疫荧光法标记干细胞和凋亡细胞。结果 胚胎发育及出生后早期,角膜以实质层的发育为主。出生14d（P14）左右,角膜上皮细胞层开始增殖分化为两层细胞,同时内皮细胞也开始分化。至P30时,我们可以辨别出角膜的6层结构。BrdU阳性细胞主要存在实质层中的成纤维细胞,出生以后也可见于角膜上皮细胞层和内皮细胞层。随着角膜发育,P10左右,其他层的BrdU阳性细胞都消失,仅存在于角膜上皮细胞层。增殖细胞核抗原（PCNA）阳性细胞在发育早期散在分布于角膜的各层,P14以后PCNA阳性细胞均匀的分布于角膜上皮细胞的基底层,并维持在稳定状态。在角膜发育早期,在各层可见许多细胞凋亡。结论 角膜的发育与其感光功能形成的过程相一致,角膜干细胞的增殖与其修复有关;有大量的凋亡细胞参与角膜结构的塑形。     

活体及体外条件下对角膜上皮干细胞的定位

背景：眼表存在两种形式的上皮干细胞即角膜上皮干细胞和结膜上皮干细胞,角膜上皮干细胞在角膜上皮细胞更新和角膜透明的维持方面起着重要作用。 目的：采用活体激光扫描角膜共焦显微镜和免疫荧光染色技术相结合的方法,从活体和体外层面上对角膜上皮干细胞进行定位研究。 方法：收集2009年9月至2012年9月来河南省眼科研究所就诊的单侧角膜缘干细胞缺乏患者,使用活体激光扫描角膜共焦显微镜检查患者双眼,健侧眼为对照。扫描方位依次为中央角膜及上、下、左、右方的角膜缘,记录扫描图像并分析。眼球材料来自于河南省眼库,切取角膜中央和角膜缘组织,组织包埋剂包被、冰冻切片,切片厚度5-7 μm;免疫荧光染色技术检测p63、ABCG2、K3和Connexin 43在角膜中央及角膜缘上皮层的表达。 结果与结论：共有24例患者确诊为单侧角膜缘干细胞缺乏,活体激光扫描角膜共焦显微镜下患侧眼角膜病变区可见结膜细胞及杯状细胞;角膜缘区域Vogt栅栏状结构消失,色素细胞消失,取而代之的是大量纤维瘢痕化组织。免疫荧光染色示表达ABCG2和p63的细胞主要在角膜缘上皮基底层,尤其在近结膜侧的角膜缘及角膜缘中间部表达相对较高,而中央角膜上皮层细胞不表达;K3及Connexin43在角膜缘上皮基底细胞层不表达,中央角膜上皮全层表达。通过活体激光扫描角膜共焦显微镜观察及干细胞标记物检测显示角膜上皮干细胞主要存在于角膜缘外2/3区域的Vogt栅栏基底部及钉突结构中。中国组织工程研究杂志出版内容重点：干细胞;骨髓干细胞;造血干细胞;脂肪干细胞;肿瘤干细胞;胚胎干细胞;脐带脐血干细胞;干细胞诱导;干细胞分化;组织工程全文链接：     

Experimental and morphological as well as physical and chemical interaction of laser radiation of a close infrared range with the fibrous eye membrane

Experimental-and-morphological as well as physical-and-chemical changes occurring in the eye fibrous coat tissues, i.e. the cornea and sclera, at non-ablation impact of fiber laser radiation (Er-glass) with a wavelength of 1.56 mcm were analyzed. Necrobiotic or necrotic changes occur in the tissues with regard for laser radiation capacity and impact duration; subsequently, the regenerative process involving a retraction of the sclera and a flattening of the cornea takes place in the end. When the cornea is heated by fiber laser radiation with a minimally possible intensity (up to 0.3 Wt), a temporary weakening is observed in the corneal rigidity. Should it be accompanied by a simultaneous occurrence of retraction forces coming from the sclera (at its coagulation), it can be used to change, in the clinical controllable mode, the corneal curvature radius.     

Survival of cultured allogeneic limbal epithelial cells following corneal repair

In this study a technique for determining donor cell fate following corneal grafting was evaluated. Patients treated for limbal deficiency with allogeneic cultured corneal epithelial cells were studied to determine the fate of the grafted cells. The technique was evaluated initially through the use of donor eyes and then applied to the clinical analysis of 7 patients who had received a cultured corneal epithelial allograft. Cells removed from the cornea and any retrieved tissue were analyzed via polymerase chain reaction (PCR) genotyping to determine the origin of the cells populating the patients' healed cornea. A mixture of genotypes was detected in a cornea retrieved from a patient following a fully penetrating keratoplasty who had received a mixture of allogeneic tissue. Donor cells were no longer detected on the corneal surface of all 7 cases beyond 28 weeks postgraft. At these later time points, only patient genotype could be detected. These results demonstrate that PCR genotyping can be used to determine the origin of cells populating the surface of the cornea following the grafting of cultured allogeneic cells and demonstrates that transplanted cultured limbal epithelial cells do not persist on the surface of the host cornea for more than 28 weeks.     

K14 + Compound niches are present on the mouse cornea early after birth and expand after debridement wounds

Background: We previously identified compound niches (CNs) at the limbal:corneal border of the mouse cornea that contain corneal epithelial progenitor cells, express Keratin 8 (K8), and goblet cell mucin Muc5AC. During re‐epithelialization after 2.5 mm epithelial debridement wounds, CNs migrate onto the cornea and expand in number mimicking conjunctivalization. When CNs form during development and whether they express corneal epithelial progenitor cell enriched K14 was not known. Results: To provide insight into corneal epithelial homeostasis, we quantify changes in expression of simple (K8, K18, K19) and stratified squamous epithelial keratins (K5, K12, K14, and K15) during postnatal development and in response to 2.5 mm wounds using quantitative polymerase chain reaction (Q‐PCR), confocal imaging and immunoblots. K14 + CNs are present 7 days after birth. By 21 days, when the eyelids are open, K8, K19, and Muc5AC are also expressed in CNs. By 28 days after wounding, the corneal epithelium shows enhanced mRNA and protein expression for K14 and retains mRNA and protein for corneal epithelial specific K12. Conclusions: The keratin phenotype observed in corneal epithelial cells before eyelid opening is similar to that seen during wound healing. Data show K14 + corneal epithelial progenitor cells expand in number after 2.5 mm wounds. Developmental Dynamics 245:132–143, 2016. © 2015 The Authors. Developmental Dynamics published by Wiley Periodicals, Inc.     

Involvement of macrophage chemotactic protein-1 and interleukin-1beta during inflammatory but not basic fibroblast growth factor-dependent neovascularization in the mouse cornea

Corneal neovascularization develops in several pathologic conditions, but its underlying mechanisms remain elusive. We used a mouse inflammatory corneal model (corneas cauterized with silver nitrate) and assessed the role of monocyte/macrophage-attracting factors, macrophage chemotactic protein-1 (MCP-1), and a proinflammatory cytokine, IL-1beta, on macrophage recruitment and neovascularization. Both MCP-1, IL-1beta protein, and mRNA levels increased markedly 12 hours after the chemical cauterization. In situ hybridization showed that MCP-1 was located in corneal epithelial cells, and IL-1beta was located in corneal epithelial cells and infiltrating inflammatory cells. In addition, double staining of corneas with antibodies specific for monocytes/macrophages and IL-1beta revealed that IL-1beta was found in infiltrating monocytes/macrophages at Day 2 after cauterization. Both IL-1beta and MCP-1 induced neovascularization in a rat cornea model, and the cauterization-induced corneal neovascularization was partially inhibited by subconjunctival injection of anti-IL-1beta or anti-MCP-1. Coadministration of two antibodies inhibited corneal neovascularization slightly more than that by the administration of each. In contrast, administration of the anti-MCP-1 or anti-IL-1beta showed minimal inhibition of basic fibroblast growth factor-driven corneal neovascularization by mouse cornea assay. Cauterized corneas treated with anti-MCP-1 antibody had significantly fewer monocytes/macrophages than control. These results indicate the existence of distinct monocyte/macrophage-involved angiogenic pathways in mouse cornea, in which MCP-1 released from corneal epithelial cells attracts monocytes/macrophages into the cornea, where they release IL-1beta leading to inflammatory neovascularization. In addition, the IL-1beta and MCP-1 released from the corneal epithelial cells may directly induce corneal neovascularization.     

Comparative Analysis of Human-Derived Feeder Layers with 3T3 Fibroblasts for the Ex Vivo Expansion of Human Limbal and Oral Epithelium

Corneal transplantation with cultivated limbal or oral epithelium is a feasible treatment option for limbal stem cell deficiency (LSCD). Currently utilized co-culture of stem cells with murine 3T3 feeder layer renders the epithelial constructs as xenografts. To overcome the potential risks involved with xenotransplantation, we investigated the use of human-derived feeder layers for the ex vivo expansion of epithelial (stem) cells. Human limbal and oral epithelium was co-cultured with mouse 3T3 fibroblasts, human dermal fibroblasts (DF), human mesenchymal stem cells (MSC), and with no feeder cells (NF). Cell morphology was monitored with phase-contrast microscopy, and stem cell characteristics were assessed by immunohistochemistry, real-time PCR for p63 and ABCG2, (stem cell markers), and by colony-forming efficiency (CFE) assay. Immunohistochemical analysis detected positive staining for CK3 (cornea specific marker) and Iβ1 and p63 (putative stem cell markers) in all culture conditions. The level of Iβ1 and p63 was significantly higher in both limbal and oral cells cultured on the 3T3 feeder, as compared to the MSC or NF group (p<0.01). This level was comparable to the cells cultured on DF. Expression of p63 and ABCG2 in limbal and oral epithelial cells in the 3T3 and DF groups was significantly higher than that in the MSC or NF group (p<0.01). No statistical difference was detected between 3T3 and DF groups. The CFE of both limbal and oral cells co-cultured on 3T3 fibroblasts was comparable to cells grown on DF, and was significantly higher than that of cells co-cultured with MSC or NF (p<0.01). Epithelial cells grown on a DF feeder layer maintained a stem cell-like phenotype, comparable to cells grown on a 3T3 feeder layer. In conclusion, DF provides a promising substitute for 3T3 feeder cells during cultivation of xenobiotic-free corneal equivalents.     

A high-density PEG interfacial layer alters the response to an EGF tethered polydimethylsiloxane surface

Previously, epidermal growth factor (EGF)-modified surfaces have shown promise in supporting cellular growth and adhesion on synthetic polymeric substrates. Surfaces prepared using a novel modification technique were investigated in the current work for their ability to support corneal epithelialization, important to the integration of a synthetic artificial cornea. EGF could be tethered to PDMS surfaces via a high-density, hetero-bifunctional PEG-NSC linking layer with a tunable surface concentration of up to 300 ng/cm(2). Only a small fraction of the EGF on these surfaces could be removed with SDS rinsing, indicative of covalent tethering. Studies with human corneal epithelial cells suggest a relatively linear increase in the number of corneal epithelial cells with increasing EGF concentration at all times. However, confluence was not achieved at any time point. It is believed that the presence of the non-adsorbent PEG layer, useful for preventing non-specific adsorption of proteins, may limit the cellular response by minimizing the adsorption of adhesion molecules. The effects of the EGF alone are clearly not sufficient to result in epithelialization of an artificial cornea surface. Altering both the adhesion and growth of corneal epithelial cells in a controlled manner may be necessary for epithelialization of an artificial cornea.     

A high-density PEG interfacial layer alters the response to an EGF tethered polydimethylsiloxane surface

Previously, epidermal growth factor (EGF)-modified surfaces have shown promise in supporting cellular growth and adhesion on synthetic polymeric substrates. Surfaces prepared using a novel modification technique were investigated in the current work for their ability to support corneal epithelialization, important to the integration of a synthetic artificial cornea. EGF could be tethered to PDMS surfaces via a high-density, hetero-bifunctional PEG-NSC linking layer with a tunable surface concentration of up to 300 ng/cm². Only a small fraction of the EGF on these surfaces could be removed with SDS rinsing, indicative of covalent tethering. Studies with human corneal epithelial cells suggest a relatively linear increase in the number of corneal epithelial cells with increasing EGF concentration at all times. However, confluence was not achieved at any time point. It is believed that the presence of the non-adsorbent PEG layer, useful for preventing non-specific adsorption of proteins, may limit the cellular response by minimizing the adsorption of adhesion molecules. The effects of the EGF alone are clearly not sufficient to result in epithelialization of an artificial cornea surface. Altering both the adhesion and growth of corneal epithelial cells in a controlled manner may be necessary for epithelialization of an artificial cornea.     

Effects of insulin-like growth factor 2 and its receptor expressions on corneal repair

Limbal stem cell (LSC) on the basal layer of cornea plays an important role in the epithelial repair after corneal injury as it can proliferate, differentiate and migrate into injury sites under the direction of cytokines. This study explored the signaling pathway and cellular mechanism between corneal epithelial cells LSC, on a mouse model with mechanic corneal injury. Ipsilateral corneal mechanic injury model was prepared on mice using the contralateral eye as the control. Tissues from both central and peripheral regions of cornea were collected, cultured and quantified for expression of various cytokines including epidermal growth factor (EGF), fibroblast growth factor-β (FGF-β), heparin-like growth factor (HGF), keratinocyte growth factor (KGF), transforming growth factor-β1 (TGF-β1), IGF-1 and IGF-2. The effects of these factors on the differentiation of LSC and fibroblasts were also studied. Most of those cytokines had elevated gene expressions after the corneal injury. Among those IGF-2 had significantly increased expression, along with the high expression of IGF-2 receptor in corneal peripheral cells. IGF-2 also induced the differentiation of LSC into keratin-12-positive cells. Further studies showed the prominent expression of α-actin in injured tissues, suggesting the potential transformation of fibroblasts into myofibroblasts. Both IGF-2 and its receptor had elevated expressions after corneal injury. They may facilitate the transformation of LSC into epithelial cells, in addition to the role in transformation from fibroblasts to myofibroblasts.     

体外培养人角膜缘上皮细胞抑制激活态角膜基质细胞的生长

背景：角膜受到损伤后,角膜基质细胞激活转变为成纤维细胞,引起角膜基质瘢痕化,导致视力下降甚至丧失。 目的：观察角膜不同部位上皮细胞与角膜基质细胞的相互作用,探索角膜缘上皮细胞群能否抑制激活态角膜基质细胞的生长。 方法：采用酶消化及机械外力相结合的方法获取人角膜中央、角膜旁中央及角膜缘处角膜上皮细胞与浅层角膜基质细胞,进行体外培养。相差显微镜下观察细胞形态及生长变化。待培养角膜上皮细胞与基质细胞发生接触抑制时,记作“0 周”,采用免疫荧光染色技术检测培养细胞中PCNA及p63蛋白的表达。 结果与结论：培养的角膜上皮细胞与成纤维细胞发生接触抑制时,两种细胞间有明显分界线。角膜缘组上皮细胞中PCNA及p63蛋白均有较高的表达;角膜旁中央组PCNA有较高的表达,p63蛋白阴性表达;角膜中央组PCNA表达较低,p63蛋白阴性表达;从鉴定结果中可以得出只有角膜缘组中存在一定比例的角膜缘上皮干细胞。角膜缘组上皮细胞逐渐包围并化解成纤维细胞,在相互作用4周后,成纤维细胞聚集成死细胞团,缺乏角膜缘干细胞的中央组及旁中央组中成纤维细胞生长面积增加,上皮细胞生长受到抑制甚至死亡。说明体外培养的角膜缘上皮细胞群可以抑制激活态角膜基质细胞的生长。     

Maintenance of the corneal epithelium is carried out by germinative cells of its basal stratum and not by presumed stem cells of the limbus

The purpose of this investigation was to analyze the proliferative behavior of rabbit corneal epithelium and establish if any particular region was preferentially involved in epithelial maintenance. [³H]-thymidine was injected intravitreally into both normal eyes and eyes with partially scraped corneal epithelium. Semithin sections of the anterior segment were evaluated by quantitative autoradiography. Segments with active replication (on) and those with no cell division (off) were intermingled in all regions of the tissue, suggesting that the renewal of the epithelial surface of the cornea followed an on/off alternating pattern. In the limbus, heavy labeling of the outermost layers was observed, coupled with a few or no labeled nuclei in the basal stratum. This suggests that this region is a site of rapid cell differentiation and does not contain many slow-cycling cells. The conspicuous and protracted labeling of the basal layer of the corneal epithelium suggests that its cells undergo repeated cycles of replication before being sent to the suprabasal strata. This replication model is prone to generate label-retaining cells. Thus, if these are adult stem cells, one must conclude that they reside in the corneal basal layer and not the limbal basal layer. One may also infer that the basal cells of the cornea and not of the limbus are the ones with the main burden of renewing the corneal epithelium. No particular role in this process could be assigned to the cells of the basal layer of the limbal epithelium.     

Reconstruction of damaged corneas by transplantation of autologous limbal epithelial cells

BACKGROUND: Stevens-Johnson syndrome, ocular pemphigoid, and thermal or chemical burns can cause scarring and opacification of the cornea and loss of vision. Transplantation of epithelial cells from the limbus of the contralateral cornea can restore useful vision. However, this procedure requires a large limbal graft from the healthy eye and is not possible in patients who have bilateral lesions. METHODS: We took specimens of limbal epithelial cells from the healthy contralateral eyes of six patients with severe unilateral corneal disease. The epithelial cells were cultured and expanded on amniotic membrane. The amniotic membrane, together with the sheet of limbal epithelial cells, was transplanted to the denuded corneal surface of the damaged eye after superficial keratectomy to remove fibrovascular ingrowth. The mean (+/-SD) follow-up period was 15+/-2 months. RESULTS: Complete reepithelialization of the corneal surface occurred within two to four days of transplantation in all six eyes receiving transplants. By one month, the ocular surface was covered with corneal epithelium, and the clarity of the cornea was improved. In five of the six eyes receiving transplants (83 percent), the mean visual acuity improved from 20/112 to 20/45. In one patient with a chemical burn who had total opacification of the cornea, the acuity improved from the ability to count fingers at 40 cm to 20/200. No patient had recurrent neovascularization or inflammation in the transplanted area during the follow-up period. CONCLUSIONS: Transplantation of autologous limbal epithelial cells cultured on amniotic membrane is a simple and effective method of reconstructing the corneal surface and restoring useful vision in patients with unilateral deficiency of limbal epithelial cells.     

Decellularized human cornea for reconstructing the corneal epithelium and anterior stroma

In this project, we strived to develop a decellularized human cornea to use as a scaffold for reconstructing the corneal epithelium and anterior stroma. Human cadaver corneas were decellularized by five different methods, including detergent- and nondetergent-based approaches. The success of each method on the removal of cells from the cornea was investigated. The structural integrity of decellularized corneas was compared with the native cornea by electron microscopy. The integrity of the basement membrane of the epithelium was analyzed by histology and by the expression of collagen type IV, laminin, and fibronectin. Finally, the ability of the decellularized corneas to support the growth of human corneal epithelial cells and fibroblasts was assessed in vitro. Corneas processed using Triton X-100, liquid nitrogen, and poly(ethylene glycol) resulted in incomplete removal of cellular material. Corneas processed with the use of sodium dodecyl sulfate (SDS) or with sodium chloride (NaCl) plus nucleases successfully removed all cellular material; however, only the NaCl plus nuclease treatment kept the epithelial basement membrane completely intact. Corneas processed with NaCl plus nuclease supported both fibroblast and epithelial cell growth in vitro, while corneas treated with SDS supported the growth of only fibroblasts and not epithelial cells. Decellularized human corneas provide a scaffold that can support the growth of corneal epithelial cells and stromal fibroblasts. This approach may be useful for reconstructing the anterior cornea and limbus using autologous cells.     

Cytocompatibility of Three Corneal Cell Types with Amniotic Membrane

Rabbit limbal corneal epithelial cells, corneal endothelial cells and keratocytes were cultured on amniotic membrane. Phase contrast microscope examination was performed daily. Histological and scan electron microscopic examinations were carried out to observe the growth, arrangement and adhesion of cultivated cells. Results showed that three corneal cell types seeded on amniotic membrane grew well and had normal cell morphology. Cultured cells attached firmly on the surface of amniotic membrane. Corneal epithelial cells showed singular layer or stratification. Cell boundaries were formed and tightly opposed. Corneal endothelial cells showed cobblestone or polygonal morphologic characteristics that appeared uniform in size. The cellular arrangement was compact. Keratocytes elongated and showed triangle or dendritic morphology with many intercellular joints which could form networks. In conclusion, amniotic membrane has good scaffold property, diffusion effect and compatibility with corneal cells. The basement membrane side of amniotic membrane facilitated the growth of corneal epithelial cells and endothelial cells and cell junctions were tightly developed. The spongy layer of amniotic membrane facilitated the growth of keratocytes and intercellular joints were rich. Amniotic membrane is an ideal biomaterial for layering tissue engineered cornea.