The Corneal Epithelium as A Source of Mammalian Somatic Mitoses

Dividing cells that occur abundantly in the lowermost layers of the corneal epithelium of various laboratory mammals provide a reliable and readily procurable source of mitotic figures for studies of chromosome number and form. Preparations may be secured quickly by fixing and staining entire eyes in an orcein-alcohol-acetic-acid mixture, or by fixing in acetic-alcohol and then staining with orcein or with leucobasic f uchsin as used in the Feulgen technic. For cytological examination of the epithelial cells and for determination of mitotic rates, the cornea is dissected from the eye and mounted as a flat preparation. Squashes of the epithelial cells are useful if mitotic counts are not required.     

胎盘脱细胞基质作为组织修复材料的初步研究

目的 制备胎盘脱细胞基质并评价其生物相容性，探讨其作为组织修复材料的可行性。方法 利用分娩废弃物胎盘组织，进行病毒灭活、脱细胞处理、冷冻干燥得到胎盘脱细胞海绵状基质材料，HE染色观察脱细胞效果及扫描电镜观察材料微观结构。同时，选取健康雄性SD大鼠39只，体质量120～150 g，随机分为实验1组、实验2组及对照组。对构建的基质材料进行大鼠皮下植入实验，实验1组植入基质材料，实验2组植入基质材料及脐带间充质干细胞，对照组为假手术组。于手术后第3、5、7天进行动物血常规检测，分析淋巴细胞、粒细胞等炎性细胞数量；于手术后第1、2、4、8、9周取材料植入处及周围组织样本进行HE染色分析。结果 构建的胎盘脱细胞基质肉眼观呈乳白色海绵状，HE染色未见细胞残留，电镜观察材料内部空隙比较明显，材料交联度较好，总孔隙率为（77.54±2.53）%。皮下植入之后，切口处愈合良好，血常规结果未见明显的炎性细胞增多；术后7 d植入材料切片HE染色即可见血管形成，且脐带间充质干细胞的加入能够加快材料与机体的融合，促进细胞的深入生长及血管化。结论 胎盘脱细胞海绵基质材料具有良好的生物相容性，可作为组织工程材料的理想来源。     

Glycerolized Reticular Dermis as a New Human Acellular Dermal Matrix: An Exploratory Study

Human Acellular Dermal Matrices (HADM) are employed in various reconstructive surgery procedures as scaffolds for autologous tissue regeneration. The aim of this project was to develop a new type of HADM for clinical use, composed of glycerolized reticular dermis decellularized through incubation and tilting in Dulbecco’s Modified Eagle’s Medium (DMEM). This manufacturing method was compared with a decellularization procedure already described in the literature, based on the use of sodium hydroxide (NaOH), on samples from 28 donors. Cell viability was assessed using an MTT assay and microbiological monitoring was performed on all samples processed after each step. Two surgeons evaluated the biomechanical characteristics of grafts of increasing thickness. The effects of the different decellularization protocols were assessed by means of histological examination and immunohistochemistry, and residual DNA after decellularization was quantified using a real-time TaqMan MGB probe. Finally, we compared the results of DMEM based decellularization protocol on reticular dermis derived samples with the results of the same protocol applied on papillary dermis derived grafts. Our experimental results indicated that the use of glycerolized reticular dermis after 5 weeks of treatment with DMEM results in an HADM with good handling and biocompatibility properties.     

Changes of Cytochemical Markers in the Conjunctival and Corneal Epithelium After Corneal Debridement

1. The aim of this study was to determine the epithelial changes of the conjunctiva and cornea up to 7 days after corneal debridement and the changes highlighted included (1) proliferation, (2) production of growth factor, (3) changes in calcium binding protein marker, (4) production of cytokine, and (5) maturity of the regeneration corneal epithelium.2. The cytochemical changes of the corneal and conjunctival epithelia of rabbit were analyzed up to 7 days after debridement.3. An increase in proliferating cell nuclear antigen (PCNA) was observed in the limbal epithelia 12 hr after lesion and reached a peak by 48 hr.4. Some proliferating limbal cells also contained epidermal growth factor (EGF) beginning 24 hr after injury. The early limbal cell proliferation and the EGF production and their persistence until 7 days after lesion were likely involved with the process of regeneration.5. Other positive markers appeared after lesion included tumor necrosis factor (TNF) and calcium binding proteins S100A and S100B, which appeared mainly within the first 48 hr after lesion and then started to decline. The short appearance and the relatively small quantity of TNF indicated that this cytokine was probably not very important in the repair process and its appearance might be related to the injury induced. The presence of S100A and S100B could be associated with both cell death after injury and the proliferation of new epithelium.6. The cornea epithelium was still immature 7 days after lesion in that it still contained cytokeratin.7. In conclusion, the critical hours of peak conjunctival and corneal changes after corneal debridement were in the first 2 days.     

Immunoregulation Effects of Bone Marrow-Derived Mesenchymal Stem Cells in Xenogeneic Acellular Nerve Grafts Transplant

This study evaluated whether bone marrow-derived mesenchymal stem cells (BM-MSCs) combined with xenogeneic acellular nerve grafts (xANGs) would reduce the inflammation reaction of xANGs transplantation. BM-MSCs were extracted, separated, purified, and cultured from the bone marrow of rats. Then BM-MSCs were seeded into 5 mm xANGs as experimental group, while xANGs group was chosen as control. Subcutaneous implantation and nerve grafts transplantation were done in this study. Walking-track tests, electrophysiological tests, H&E staining, and immunostaining of CD4, CD8, and CD68 of subcutaneous implantations, cytokine concentrations of IL-2, IL-10, IFN-γ and TNF-α in lymphocytes supernatants and serum of the two groups were evaluated. Walking-track tests and electrophysiological tests suggested the group of BM-MSCs with xANGs obtained better results than xANGs group (P < 0.05). H&E staining and immunostaining of CD4, CD8, and CD68 of subcutaneous implantations showed there were less inflammatory cells in the group of BM-MSCs when compared with the xANGs group. The cytokine concentrations of IL-2, IFN-γ, and TNF-α in BM-MSCs group were lower than xANGs group in lymphocytes supernatants and serum (P < 0.05). However, IL-10 concentrations in BM-MSCs group were higher than xANGs group (P < 0.05). xANG with BM-MSCs showed better nerve repair function when compared with xANG group. Furthermore, xANG with BM-MSCs showed less inflammatory reaction which might indicate the reason of its better nerve regeneration.     

下颌下腺脱细胞基质材料的生物相容性及毒理学研究

为探讨下颌下腺脱细胞基质支架材料的生物相容性,应用3%TritonX-100对SD大鼠的下颌下腺组织进行脱细胞处理,制备脱细胞基质支架材料,将该材料的浸提液注入小鼠体内进行全身急性毒性试验,观察小鼠全身反应.将该材料植入Wistar鼠肌内进行体内植入试验,不同时间观察支架材料与组织反应.用传代培养的第2代下颌下腺细胞与支架材料体外复合培养,第7 d时进行MTT检测,观察支架材料对细胞增殖的影响.全身急性毒性试验结果显示,实验组与对照组无显著性差别(P＞0.05),体内植入试验2、4、8 W时光镜下表现与对照组基本相似,MTT检测结果,细胞相对增长率为91.66%,支架材料的毒性为0级.结果可见,经3%TritonX-100脱细胞处理后所制备的下颌下腺生物衍生支架材料具有良好的生物相容性,对机体无毒害作用.     

A Decellularization Methodology for the Production of a Natural Acellular Intestinal Matrix

Successful tissue engineering involves the combination of scaffolds with appropriate cells in vitro or in vivo. Scaffolds may be synthetic, naturally-derived or derived from tissues/organs. The latter are obtained using a technique called decellularization. Decellularization may involve a combination of physical, chemical, and enzymatic methods. The goal of this technique is to remove all cellular traces whilst maintaining the macro- and micro-architecture of the original tissue.Intestinal tissue engineering has thus far used relatively simple scaffolds that do not replicate the complex architecture of the native organ. The focus of this paper is to describe an efficient decellularization technique for rat small intestine. The isolation of the small intestine so as to ensure the maintenance of a vascular connection is described. The combination of chemical and enzymatic solutions to remove the cells whilst preserving the villus-crypt axis in the luminal aspect of the scaffold is also set out. Finally, assessment of produced scaffolds for appropriate characteristics is discussed.     

Extracellular Matrix Aggregates from Differentiating Embryoid Bodies as a Scaffold to Support ESC Proliferation and Differentiation

Embryonic stem cells (ESCs) have emerged as potential cell sources for tissue engineering and regeneration owing to its virtually unlimited replicative capacity and the potential to differentiate into a variety of cell types. Current differentiation strategies primarily involve various growth factor/inducer/repressor concoctions with less emphasis on the substrate. Developing biomaterials to promote stem cell proliferation and differentiation could aid in the realization of this goal. Extracellular matrix (ECM) components are important physiological regulators, and can provide cues to direct ESC expansion and differentiation. ECM undergoes constant remodeling with surrounding cells to accommodate specific developmental event. In this study, using ESC derived aggregates called embryoid bodies (EB) as a model, we characterized the biological nature of ECM in EB after exposure to different treatments: spontaneously differentiated and retinoic acid treated (denoted as SPT and RA, respectively). Next, we extracted this treatment-specific ECM by detergent decellularization methods (Triton X-100, DOC and SDS are compared). The resulting EB ECM scaffolds were seeded with undifferentiated ESCs using a novel cell seeding strategy, and the behavior of ESCs was studied. Our results showed that the optimized protocol efficiently removes cells while retaining crucial ECM and biochemical components. Decellularized ECM from SPT EB gave rise to a more favorable microenvironment for promoting ESC attachment, proliferation, and early differentiation, compared to native EB and decellularized ECM from RA EB. These findings suggest that various treatment conditions allow the formulation of unique ESC-ECM derived scaffolds to enhance ESC bioactivities, including proliferation and differentiation for tissue regeneration applications.     

Loss of Notch1 Disrupts the Barrier Repair in the Corneal Epithelium

The corneal epithelium is the outermost layer of the cornea that directly faces the outside environment, hence it plays a critical barrier function. Previously, conditional loss of Notch1 on the ocular surface was found to cause inflammation and keratinization of the corneal epithelium. This was in part attributed to impaired vitamin A metabolism, loss of the meibomian glands and recurrent eyelid trauma. We hypothesized that Notch1 plays an essential role in the corneal epithelial barrier function and is a contributing factor in the pathologic changes in these mice. Notch1 was conditionally deleted in adult Notch1^flox/flox, K14-Cre-ERT^+/- mice using hydroxy-tamoxifen. The results indicated that conditional deletion of Notch1 on the ocular surface leads to progressive impairment of the epithelial barrier function before the onset of corneal opacification and keratinization. Loss of the barrier was demonstrated both by an increase in in vivo corneal fluorescein staining and by enhanced penetration of a small molecule through the epithelium. Corneal epithelial wounding resulted in significant delay in recovery of the barrier function in conditional Notch1^-/- mice compared to wild type. Mice with conditional deletion of Notch1 did not demonstrate any evidence of dry eyes based on aqueous tear production and had normal conjunctival goblet cells. In a calcium switch experiment in vitro, Notch1^-/- cells demonstrated delayed membrane localization of the tight junction protein ZO-1 consistent with a defect in the epithelial tight junction formation. These findings highlight the role of Notch1 in epithelial differentiation and suggest that intrinsic defects in the corneal epithelial barrier recovery after wounding is an important contributing factor to the development of inflammatory keratinization in Notch1^-/- mice.     

Generation of Corneal Epithelial Cells from Induced Pluripotent Stem Cells Derived from Human Dermal Fibroblast and Corneal Limbal Epithelium

Induced pluripotent stem (iPS) cells can be established from somatic cells. However, there is currently no established strategy to generate corneal epithelial cells from iPS cells. In this study, we investigated whether corneal epithelial cells could be differentiated from iPS cells. We tested 2 distinct sources: human adult dermal fibroblast (HDF)-derived iPS cells (253G1) and human adult corneal limbal epithelial cells (HLEC)-derived iPS cells (L1B41). We first established iPS cells from HLEC by introducing the Yamanaka 4 factors. Corneal epithelial cells were successfully induced from the iPS cells by the stromal cell-derived inducing activity (SDIA) differentiation method, as Pax6⁺/K12⁺ corneal epithelial colonies were observed after prolonged differentiation culture (12 weeks or later) in both the L1B41 and 253G1 iPS cells following retinal pigment epithelial and lens cell induction. Interestingly, the corneal epithelial differentiation efficiency was higher in L1B41 than in 253G1. DNA methylation analysis revealed that a small proportion of differentially methylated regions still existed between L1B41 and 253G1 iPS cells even though no significant difference in methylation status was detected in the specific corneal epithelium-related genes such as K12, K3, and Pax6. The present study is the first to demonstrate a strategy for corneal epithelial cell differentiation from human iPS cells, and further suggests that the epigenomic status is associated with the propensity of iPS cells to differentiate into corneal epithelial cells.     

Time Dependency in the Regenerative Response to Injury of the Rat Corneal Epithelium

In the present study a central corneal epithelial defect (diameter 3.5 mm) was made in both eyes at 12:00 h in one group of rats and at 24:00 h in another group to see if the regenerative proliferation is influenced by circadian rhythms. The labeling index and the mitotic rate were registered at 4-h intervals in the perilimbal conjunctiva, the limbal area, and different parts of the cornea from the following morning until noon the day after that. The most pronounced regenerative proliferation was seen in the midperipheral and peripheral cornea. The regenerative response occurred in both groups 24-28 h after the injury, but was highly influenced by the normal circadian rhythms, especially with regard to the mitotic rate. The results support the theory that even regeneration is influenced by a circadian proliferative factor.     

Type I Collagen as an Extracellular Matrix for the In Vitro Growth of Human Small Intestinal Epithelium

Background

We previously reported in vitro maintenance and proliferation of human small intestinal epithelium using Matrigel, a proprietary basement membrane product. There are concerns over the applicability of Matrigel-based methods for future human therapies. We investigated type I collagen as an alternative for the culture of human intestinal epithelial cells.

Methods

Human small intestine was procured from fresh surgical pathology specimens. Small intestinal crypts were isolated using EDTA chelation. Intestinal subepithelial myofibroblasts were isolated from a pediatric sample and expanded in vitro. After suspension in Matrigel or type I collagen gel, crypts were co-cultured above a confluent layer of myofibroblasts. Crypts were also grown in monoculture with exposure to myofibroblast conditioned media; these were subsequently sub-cultured in vitro and expanded with a 1∶2 split ratio. Cultures were assessed with light microscopy, RT-PCR, histology, and immunohistochemistry.

Results

Collagen supported viable human epithelium in vitro for at least one month in primary culture. Sub-cultured epithelium expanded through 12 passages over 60 days. Histologic sections revealed polarized columnar cells, with apical brush borders and basolaterally located nuclei. Collagen-based cultures gave rise to monolayer epithelial sheets at the gel-liquid interface, which were not observed with Matrigel. Immunohistochemical staining identified markers of differentiated intestinal epithelium and myofibroblasts. RT-PCR demonstrated expression of α-smooth muscle actin and vimentin in myofibroblasts and E-Cadherin, CDX2, villin 1, intestinal alkaline phosphatase, chromogranin A, lysozyme, and Lgr5 in epithelial cells. These markers were maintained through several passages.

Conclusion

Type I collagen gel supports long-term in vitro maintenance and expansion of fully elaborated human intestinal epithelium. Collagen-based methods yield familiar enteroid structures as well as a new pattern of sheet-like growth, and they eliminate the need for Matrigel for in vitro human intestinal epithelial growth. Future research is required to further develop this cell culture system for tissue engineering applications.     

The Effect of <Emphasis Type="Italic">para</Emphasis>-Aminobenzoic Acid on Apoptosis Processes in the Adult Rat Conjunctiva and Corneal Epithelium in vivo after Hypobaric Hypoxia

The initial stages of apoptotic damage to the conjunctiva and corneal epithelium, in the renewing cell populations of the eye has been studied in adult rats exposed to acute hypobaric hypoxia in vivo, and the effect of para-aminobenzoic acid (PABA) administered as the Aktipol pharmaceutical preparation (0.007% PABA) has been assessed in this experimental setup. Apoptotic damage to cell DNA was aggravated after two instances of hypoxia, and this change was accompanied by an increase in the intensity of the regenerative process inferred from the expression levels of phosphorylated histone pH3, a mitotic cell marker, and phosphorylated histone γ-H2AX, a DNA repair marker. Parabulbar injection of Aktipol 24 h before hypoxia has been shown to block development of the apoptotic process and to maintain a physiologically normal level of apoptosis, and thus the preparation exerted a protective effect in hypoxia.     

兔泪腺摘除后的泪液分泌及其角膜上皮的形态学改变

目的观察摘除泪腺后,兔泪液S1T值、角膜荧光素染色和虎红染色的变化及其角膜上皮超微结构的变化,评估兔干眼症模型的建立。方法摘除兔泪腺和Harder氏腺,比较泪腺摘除前后S1T、角膜荧光素染色和虎红染色分值的变化,并在透射电镜下观察角膜上皮形态学的变化。结果泪腺摘除后平均S1T值(15.88±6.29mm/5min)低于摘除前的平均S1T值(20.25±5.52mm/5min),差别有统计学意义(P=0.0062)。平均角膜荧光素染色和虎红评分(分别为8.22±1.99和7.67±0.87)明显高于摘除前(分别为0.22±0.44和0.67±0.5),差别有统计学意义(两组均P<0.0001)。以上3个检查指标的变化说明摘除泪腺后出现水液性泪液分泌缺乏,导致角膜上皮点状剥脱,干燥及坏死。在形态学上泪腺摘除前后的角膜上皮也发生了明显的改变,透射电镜检查发现液化的表层上皮细胞,表层细胞膜破裂。以上的改变均符合干眼症的特征。结论摘除兔泪腺和Harder氏腺后兔泪液分泌减少,角膜上皮坏死,能有效形成泪液缺乏型干眼模型。     

Diurnal Variation of Tight Junction Integrity Associates Inversely with Matrix Metalloproteinase Expression in Xenopus laevis Corneal Epithelium: Implications for Circadian Regulation of Homeostatic Surface Cell Desquamation

Background and Objectives

The corneal epithelium provides a protective barrier against pathogen entrance and abrasive forces, largely due to the intercellular junctional complexes between neighboring cells. After a prescribed duration at the corneal surface, tight junctions between squamous surface cells must be disrupted to enable them to desquamate as a component of the tissue homeostatic renewal. We hypothesize that matrix metalloproteinase (MMPs) are secreted by corneal epithelial cells and cleave intercellular junctional proteins extracellularly at the epithelial surface. The purpose of this study was to examine the expression of specific MMPs and tight junction proteins during both the light and dark phases of the circadian cycle, and to assess their temporal and spatial relationships in the Xenopus laevis corneal epithelium.

Methodology/Principal Findings

Expression of MMP-2, tissue inhibitor of MMP-2 (TIMP-2), membrane type 1-MMP (MT1-MMP) and the tight junction proteins occludin and claudin-4 were examined by confocal double-label immunohistochemistry on corneas obtained from Xenopus frogs at different circadian times. Occludin and claudin-4 expression was generally uniformly intact on the surface corneal epithelial cell lateral membranes during the daytime, but was frequently disrupted in small clusters of cells at night. Concomitantly, MMP-2 expression was often elevated in a mosaic pattern at nighttime and associated with clusters of desquamating surface cells. The MMP-2 binding partners, TIMP-2 and MT1-MMP were also localized to surface corneal epithelial cells during both the light and dark phases, with TIMP-2 tending to be elevated during the daytime.

Conclusions/Significance

MMP-2 protein expression is elevated in a mosaic pattern in surface corneal epithelial cells during the nighttime in Xenopus laevis, and may play a role in homeostatic surface cell desquamation by disrupting intercellular junctional proteins. The sequence of MMP secretion and activation, tight junction protein cleavage, and subsequent surface cell desquamation and renewal may be orchestrated by nocturnal circadian signals.     

Actin is a Constituent of the Nuclear Matrix and Chromosome Scaffold in Physarum poly cephalum

Nuclear matrices and chromosome scaffolds were obtained by digesting and extracting, respectively with DNase Ⅰ and 2 mol/L NaC1, the nuclei and chromosomes isolated from the plasmedia of Physarum polycephalum Schw. The results of the indirect immunofluorescence of tacit antiactin antibody as immunomarker indicated that the nuclear matrices and chromosome scaffolds both had positive reaction with the antibody. The results of the indirect immunodotting experiment further verified the presence of actin antibody in their constituent. Protein A-colloidal gold immunoelectron microscopy technique revealed that gold particles were distributed in the interphase nuclei and metaphase chromosomes. The above results showed that actin is a constituent of the nuclear matrix and chromosome scaffold of P. polycephalum.     

Human Umbilical Mesenchymal Stem Cells-Seeded Bladder Acellular Matrix Grafts for Reconstruction of Bladder Defects in a Canine Model

Background

The goal of this study was to explore the feasibility of utilizing human umbilical mesenchymal stem cells (HUMSCs)-seeded Bladder acellular matrix graft (BAMG) for bladder reconstruction in a canine model.

Methodology/Principal Findings

HUMSCs were isolated from newborn umbilical cords and identified by flow cytometry. Partial cystectomy was performed in the experimental and control group. Bladder defects were repaired with HUMSCs-BAMG in the experimental group and repaired with unseeded-BAMG in control group. The implanted grafts were harvested after surgery. H&E and immunohistochemistry staining were performed to evaluate the regeneration of the bladder defect. Primary cultured HUMSCs displayed typical fibroblast morphology with spindle-shaped. Flow cytometry indicated that these cells were positive for CD105 (97.3%) and CD44 (99%), but negative for CD34 (2.8%), CD31 (2.1%), and CD45 (1.7%). Immunohistochemistry staining showed that a multilayered urothelium and well-developed smooth muscle were observed at 12 weeks in experiment group. In contrast, multilayered urothelial tissues were also observed at 12 weeks in group B, but well-developed smooth muscle bundles were observed.

Conclusions/Significance

Our preliminary results demonstrate that UMSC-seeded BAMGs are superior to unseeded BAMGs to promote the regeneration of bladder defects. Our findings indicated that HUMSCs may be a potential cell source for bladder tissue engineering.