A dual-layer silk fibroin scaffold for reconstructing the human corneal limbus

Membranes prepared from Bombyx mori silk fibroin have shown potential as a substrate for human limbal epithelial (L-EC) and stromal cell cultivation. Here we present fibroin as a dual-layer construct containing both an epithelium and underlying stroma for corneolimbal reconstruction. We have compared the growth and phenotype of L-EC on non-porous versus porous fibroin membranes. Furthermore, we have compared the growth of limbal mesenchymal stromal cells (L-MSC) in either serum-supplemented medium or the MesenCult-XF(?) culture system within fibroin fibrous mats. The co-culture of L-EC and L-MSC in fibroin dual-layer constructs was also examined. L-EC on porous membranes displayed a squamous monolayer; in contrast, L-EC on non-porous fibroin appeared cuboidal and stratified. Both constructs maintained evidence of corneal phenotype (cytokeratin 3/12) and distribution of ΔNp63(+) progenitor cells. L-MSC cultivated within fibroin fibrous mats in serum-supplemented medium contained less than 64% of cells expressing the characteristic MSC phenotype of CD73(+)CD90(+)CD105(+) after two weeks, compared with over 81% in MesenCult-XF(?) medium. Dual-layer fibroin scaffolds consisting of L-EC and L-MSC maintained a similar phenotype as on the separate layers. These results support the feasibility of a 3D engineered limbus constructed from B.?mori silk fibroin, and warrant further studies into the potential benefits it offers to corneolimbal tissue regeneration.     

Multipotent stem cells in human corneal stroma

Keratocytes of the corneal stroma secrete a specialized extracellular matrix essential for vision. These quiescent cells exhibit limited capacity for self-renewal and after cell division become fibroblastic, secreting nontransparent tissue. This study sought to identify progenitor cells for human keratocytes. Near the corneal limbus, stromal cells expressed ABCG2, a protein present in many adult stem cells. The ABCG2-expressing cell population was isolated as a side population (SP) by cell sorting after exposure to Hoechst 33342 dye. The SP cells exhibited clonal growth and continued to express ABCG2 and also PAX6, product of a homeobox gene not expressed in adult keratocytes. Cloned SP cells cultured in medium with fibroblast growth factor-2 lost ABCG2 and PAX6 expression and upregulated several molecular markers of keratocytes, including keratocan, aldehyde dehydrogenase 3A1, and keratan sulfate. Cloned corneal SP cells under chondrogenic conditions produced matrix staining with toluidine blue and expressed cartilage-specific markers: collagen II, cartilage oligomatrix protein, and aggrecan. Exposure of cloned SP cells to neurogenic culture medium upregulated mRNA and protein for glial fibrillary acidic protein, neurofilament protein, and beta-tubulin II. These results demonstrate the presence of a population of cells in the human corneal stroma expressing stem cell markers and exhibiting multipotent differentiation potential. These appear to be the first human cells identified with keratocyte progenitor potential. Further analysis of these cells will aid elucidation of molecular mechanisms of corneal development, differentiation, and wound healing. These cells may be a resource for bioengineering of corneal stroma and for cell-based therapeutics.     

生物工程角膜与人供体角膜治疗真菌性角膜溃疡的比较

文题释义： 生物工程角膜：又称脱细胞猪角膜基质,是取材于猪眼角膜经病毒灭活与脱细胞等工艺制备而成,由前弹力层和部分基质层构成的纤维支架组织。所有的脱细胞猪角膜基质均通过细胞毒性测试和组织相容性测试。 板层角膜移植：是一种部分厚度的角膜移植,通常是切除病变的角膜组织,将相应厚度的植片移植到角膜植床上。 背景：寻找替代角膜供体的材料是临床上治疗真菌性角膜溃疡的研究热点。 目的：观察生物工程角膜和人供体角膜治疗真菌性角膜溃疡的临床疗效。 方法：选择2016年10月至2017年2月北部战区总医院收治的真菌性角膜溃疡患者44例(44眼),随机分2组：生物工程角膜组(n=22)采用脱细胞猪角膜基质材料进行板层角膜移植治疗,人供体角膜组(n=22)采用人供体角膜材料进行板层角膜移植治疗。随访12个月,观察两组感染控制率、视力、角膜植片透明度、角膜上皮化时间、并发症等情况。试验已通过北部战区总医院医学伦理委员会批准[K(2018)05号]。 结果与结论：①两组感染控制率比较差异无显著性意义(91%,91%,P > 0.05);②两组术后视力随时间延长逐渐改善,人供体角膜组术后12个月的矫正视力视力优于生物工程角膜组(P < 0.05);③人供体角膜组术后1,3,6个月的角膜植片透明度优于生物工程角膜组(P < 0.05),两组术后12个月的角膜植片透明度无差异(P > 0.05);④两组角膜植片上皮愈合时间比较差异无显著性意义[(6.6±2.0),(6.7±1.9) d,P > 0.05];⑤两组角膜上皮愈合延迟、角膜植片排斥反应、新血管长入、原病复发等并发症发生率比较差异无显著性意义,生物工程角膜组角膜植片溶解发生率高于人供体角膜组(32%,8%,P < 0.05);⑥结果表明,采用生物工程角膜行板层角膜移植治疗真菌性角膜溃疡的临床效果显著、安全性高、预后较好。因此在无人供体角膜的情况下,生物角膜可作为板层角膜移植的材料用于治疗真菌性角膜溃。 ORCID: 0000-0002-1297-4684(刘志玲) 中国组织工程研究杂志出版内容重点：生物材料;骨生物材料; 口腔生物材料; 纳米材料; 缓释材料; 材料相容性;组织工程     

Acanthamoeba adherence to soft contact lens and human corneal stroma

In the present study, the adhesibility of Acanthamoeba castellani trophozoites and cysts to hydrogel contact lenses and to human cornea was investigated. Segments of unworn contact lenses were prepared (120 segments in total). In addition, 8 corneal buttons were obtained from 8 enucleated eyes. And each cornea cut into 8 radial segments. To these lens and corneal segments, 1.2 x 106/ml and 1.3 x 106/ml of cysts and trophozoites respectively were added under varying conditions. The adhesion was checked at 0, 3 and 24 hours after the exposure. Adhesion analysis showed that the trophozoites adhered equally well to lens or cornea. There is an increase in the number of trophozoites adhering to contact lenses as well as cornea. This difference is more significant for contact lenses. Washing of contact lenses significantly decreased the adherence of the trophozoites after 3 hours of incubation. When the comparison of adhesion was done between the unwashed worn and unwashed unworn contact lenses, it was observed that there was a significant difference in adherence to new lenses where the adherence was much lower. The study shows that washing of contact lenses does decrease the chances of colonization by Acantamoeba catellani and also that older lenses have higher chances of getting colonized probably due to the occurrence of scratches et. on the surface which may help in colonization.     

脱细胞猪角膜基质的基底膜在修复兔角膜损伤中的作用

目的 探讨脱细胞角膜基质支架材料表面保存基底膜样结构对修复角膜损伤的必要性. 方法 将采用反复冻融及酶消化法制备的脱细胞猪角膜基质,移植到兔角膜损伤模型上,通过大体及组织学分别观察材料表面基底膜结构的有无对修复兔角膜基质损伤的影响. 结果 脱细胞猪角膜基质材料表面有基底膜的实验组（n=8）兔角膜损伤全部修复,无穿孔,支架材料与受体角膜整合.材料表面无基底膜的对照组（n=8）中,有6例植入的支架材料溶解,兔角膜穿孔.两组结果比较差异有显著统计学意义. 结论 脱细胞角膜基质支架材料表面具有基底膜样结构,有利于形成正常角膜上皮屏障,从而防止了生物材料的过快降解,有利于材料与宿主基质的整合和改建.     

A novel population of repair cells identified in the stroma of the human cornea

The transmembrane protein CD133 is expressed on somatic stem cells of various adult human tissues. To investigate whether human corneal stroma also contains CD133-expressing cells and to analyze their functional features, stromal cells were isolated by collagenase digestion, immunophenotyped, and transferred to different culture systems to determine their stem cell properties as well as their differentiation potentials. For comparison, the embryonic keratocyte cell line EK1.Br, the dermal stromal cell line NHDF, and stromal cells of diseased corneas were studied. On average, 5.3% of the normal stromal cells expressed the stem cell marker CD133 and 3.6% co-expressed CD34. Expression of CD133 but not CD34 was also demonstrated for EK1.Br cells, whereas NHDF cells were negative for both markers. Further analysis of CD133(+) normal corneal cells revealed that a significant proportion displayed a monocytic phenotype with co-expression of CD45 and CD14. In diseased corneas, up to 26.8% of the stromal cells showed expression of CD133, and virtually all CD133(+) cells co-expressed CD14 but not CD45. Moreover, using a standard clonogenic assay, normal stromal cells had the capacity to form colonies of the macrophage lineage. These colonies could be further differentiated into lumican-expressing keratocytes. Our data suggest that the human corneal stroma harbors CD133(+) monocytic progenitor cells, which possess the potential to differentiate into the fibrocytic lineage. Thus, CD133(+) /CD45(+) /CD14(+) cells might represent stromal repair cells that differentiate into keratocytes via a CD133(+)/CD45()/CD14(+) intermediate stage. The findings from our study may shed new light on regenerative processes of the human corneal stroma.     

Using genipin-crosslinked acellular porcine corneal stroma for cosmetic corneal lens implants

Acellular porcine corneal stroma (APCS) has been proven to maintain the matrix microenvironment and is therefore an ideal biomaterial for the repair and reconstruction of corneal stroma. This study aims to develop a method to prepare cosmetic corneal lens implants for leukoma using genipin-crosslinked APCS (Gc-APCS). The Gc-APCS was prepared from APCS immersed in 1.0% genipin aqueous solution (pH 5.5) for 4?h at 37?°C, followed by lyophilization at -10?°C. The color of the Gc-APCS gradually deepened to dark-blue. The degree of crosslinking was 45.7?±?4.6%, measured by the decrease of basic and hydroxy amino acids. The porous structure and ultrastructure of collagenous lamellae were maintained, and the porosity and BET SSA were 72.7?±?4.6% and 23.01?±?3.45?m(2)/g, respectively. The Gc-APCS rehydrated to the physiological water content within 5?min and was highly resistant to collagenase digestion. There were no significant differences in the areal modulus and curvature variation between Gc-APCS and nature porcine cornea. The dark-blue pigments were stable to pH, light and implantation in?vivo. Gc-APCS extracts had no inhibitory effects on the proliferation of keratocytes. Corneal neovascularization, graft degradation and corneal rejection were not observed within 6 months.     

以猪角膜脱细胞基质为载体培养人脐静脉内皮细胞构建实验性角膜后板层

 目的： 探讨以异种后弹力膜/基质为载体诱导人脐静脉内皮细胞向角膜内皮细胞分化的可行性及移植术后在活体的生理功能。方法：体外分离培养脐静脉内皮细胞作为诱导移植的种子细胞;取当地质检合格的猪眼球以直径6.2 mm、厚100 μm、冷冻脱水进行角膜深板层载体的制备;接种经CM-DiI荧光标记的第2~3代人脐静脉内皮细胞于载体的后弹力层面,体外培养7~10 d行细胞形态、密度、组织学和扫描电镜观察,待细胞与后弹力层面融合形成单层后,用于兔角膜移植。受眼：正常健康新西兰大白兔24只（24眼）,实验组（人脐静脉内皮细胞移植组）12眼,对照组（单纯猪角膜深板层移植组）12眼,全角膜范围去除术眼内皮细胞,实施移植手术。结果：人脐静脉内皮细胞在猪后弹力膜/基质载体上贴附生长,形成紧密连接的单层,形态近似六角形,呈铺路石状分布,具有正常兔角膜内皮细胞的超微结构。术后8周实验组角膜基本透明,周边角膜略有水肿。对照组单纯猪角膜深板层移植后,移植角膜明显水肿、混浊。结论：以异种角膜后弹力膜/基质为载体培养的人脐静脉内皮细胞,行异种异体移植后,能够在活体上成活,并能维持角膜透明,具有正常角膜内皮细胞的生物学功能。深板层角膜内皮移植术是体外培养血管内皮细胞移植的一种有效术式。     

Bioactive self-assembled peptide nanofibers for corneal stroma regeneration

Defects in the corneal stroma caused by trauma or diseases such as macular corneal dystrophy and keratoconus can be detrimental for vision. Development of therapeutic methods to enhance corneal regeneration is essential for treatment of these defects. This paper describes a bioactive peptide nanofiber scaffold system for corneal tissue regeneration. These nanofibers are formed by self-assembling peptide amphiphile molecules containing laminin and fibronectin inspired sequences. Human corneal keratocyte cells cultured on laminin-mimetic peptide nanofibers retained their characteristic morphology, and their proliferation was enhanced compared with cells cultured on fibronectin-mimetic nanofibers. When these nanofibers were used for damaged rabbit corneas, laminin-mimetic peptide nanofibers increased keratocyte migration and supported stroma regeneration. These results suggest that laminin-mimetic peptide nanofibers provide a promising injectable, synthetic scaffold system for cornea stroma regeneration.     

Intermediate filament expression by normal and diseased human corneal epithelium

Cicatricial conjunctivitis may be a sequel to systemic disorders (eg, Stevens-Johnson syndrome, cicatricial pemphigoid) or local disorders such as chemical burns. The cicatrisation is often associated with corneal epithelial changes that cause visual loss. These have been attributed to encroachment of the conjunctival epithelium over the cornea. However, the epithelial anomalies are poorly understood. We investigated the corneal epithelial changes in cicatricial conjunctivitis with an immunohistochemical study of intermediate filaments in normal and pathological specimens. Our results show that the normal corneal epithelium is immunoreactive for cytokeratin 3 (CK 3) but not cytokeratin 19 (CK 19), whereas normal conjunctival epithelium is CK 3 negative and CK 19 positive. Conjunctiva artificially transposed over the cornea (after therapeutic conjunctival flap reconstruction) retained the normal pattern of conjunctival cytokeratin expression (CK 3 negative, CK 19 positive). Conversely, the entire corneal epithelium exhibited the normal cytokeratin pattern (CK 3 positive, CK 19 negative) in 82% of Stevens-Johnson, 80% of cicatricial pemphigoid, and 69% of chemical burns specimens. The findings suggest that conjunctival encroachment is not responsible for the changes at the corneal surface in cicatricial conjunctivitis and that the abnormal corneal epithelium is derived from native corneal cells in these diseases.     

Construction of the recellularized corneal stroma using porous acellular corneal scaffold

Acellular porcine cornea stroma (APCS) prepared using pancreatic phospholipase A(2) was proven to be promising corneal scaffold. However, its dense ultrastructure provides insufficient space that prevents the seeded cells from organizing into a functional tissue. In this report, freezing dry APCS (FD-APCS) biomaterials containing pores with different sizes were fabricated at different pre-freezing temperatures of -10, -80 or -198°C, and the percentage of large pores (equivalent circle diameter ≥10 μm) was 93.55%, 69.36%, 35.79%, while the small pores (<10 μm) were account for 6.45%, 30.64%, 64.21%, respectively. Both porosity and specific surface area increased in FD-APCS fabricated with decreased pre-freezing temperature, and they were dramatically higher than those in APCS. The three FD-APCS groups displayed higher permeability than APCS, and the -10°C FD-APCS possessed the highest permeability. The keratocytes seeded in the FD-APCS construct survived well in vitro, and maximal cell proliferation was observed in the -10°C FD-APCS. The light transmittance of the FD-APCS-transplanted cornea after interlamellar keratoplasty in rabbit eyes displayed no significant difference from the APCS-transplanted or native cornea. This study indicated that the porous FD-APCS prepared using pancreatic phospholipase A(2) is capable of serving as potential scaffold for constructing tissue-engineered cornea with biological properties.     

The keratocyte or fibroblast of the cornea: morphological and biochemical characteristics in normal stroma and a few cases of corneal dystrophies]

Keratocytes are differentiated mesenchymal fibroblast which are specialized in the production and maintenance of the transparent corneal stroma. These elongated cells with large nuclei and scant cytoplasm produce the collages, proteoglycans, and glycoproteins which make up the stroma, as well as the enzymes involved in post-translation modifications and catabolism of these macromolecules. The characteristic morphological features and specific metabolic activity of keratocytes undergo significant changes during tissue repair and in several corneal diseases. Alterations in keratocyte biosynthetic activity, regardless of their cause, ultimately lead to fibrillar alterations responsible for a loss of corneal transparency.     

ATP-induced [Ca2+]i changes in the human corneal epithelium

ATP, when leaked from damaged cells, is capable of eliciting responses in neighboring cells. A better understanding of the mechanism of this response is essential for designing therapeutic strategies for disease, there have been only a limited number of studies on the effect of ATP on the human cornea. We examined ATP-induced intracellular Ca2+ ([Ca2+]i) changes in the human corneal epithelia, cultured to near confluence. Cells were loaded with the Ca2+ indicators, Indo-1 or Fluo-4, and [Ca2+]i was monitored. ATP was found to induce an increase in [Ca2+]i, which was initiated from the perinuclear region and the nuclear envelope per se, and then propagated gradually towards the periphery. Intranuclear Ca2+ was momentarily increased. UTP elicited an identical response, but adenosine and alpha, beta-methylene ATP had no effect. Pretreatment with U73122 or thapsigargin inhibited the ATP-induced increase in [Ca2+]i. When a cell was topically stimulated with ATP, the [Ca2+]i increase spread beyond the cell boundary. The intercellular communications that accompanied the [Ca2+]i changes were inhibited by octanol. We conclude that extracellular ATP in the human cornea caused the mobilization of Ca2+ from intracellular Ca2+ stores (e.g. the endoplasmic reticulum and nuclear envelope) via P2Y purinoceptors of the epithelial cell. The response to ATP appears to spread to neighboring regions through gap junctions in the epithelium.     

Electrical profiles in the corneal epithelium

1. The potentials and resistances associated with the cell membranes of the rabbit corneal epithelium were studied with 3 M-KCl-filled micro-electrodes.2. In the isolated cornea, the transepithelial potential was identical in polarity and magnitude to the simultaneously measured total corneal potential. In contrast to previous findings, the stromal potential was positive to the tear side. Negative stromal potentials apparently derive from inadequate electrodes or method of penetration, and were not found to be a function of filling solution. Transepithelial potential was also identical to over-all corneal potential in the living rabbit eye.3. In the isolated preparation, the average potential profile occurred in three distinct steps across the epithelium. By means of iontophoretic dye injection it was shown that these steps occurred across the outer membrane of the squamous cell, the transition region between the wing and basal cell, and across the inner membrane of the basal cell.4. The transverse membrane resistance of the outer epithelial membrane accounted for 60% of total corneal resistance. As a result, short-circuit current, which depolarizes the cornea, led to a hyperpolarization of the outer membrane, while affecting deeper membrane potentials little or not at all.5. The spontaneous potential of the outer membrane varied inversely with corneal potential in both normal and chloride-free Ringer, while the potential of the inner membrane of the basal cell was relatively constant, approaching the theoretical Nernst potential for potassium. The potential of the outer membrane was at chloride equilibrium and was sensitive to extracellular shunts. A Thevenin equivalent drawn for the epithelium suggested that half of the outer membrane potential could be attributed to loop currents. The potential step between wing and basal cells could be accounted for in terms of loop currents driven by the corneal potential through the epithelium.6. The potential profile of the frog corneal epithelium was similar to that of the rabbit. However, the major resistance in the frog cornea was associated with the basal cell membrane rather than with the squamous cell outer membrane. Quasi-instantaneous rectification was found for both epithelia. In the rabbit chloride rectified inwardly.     

Collagen-immobilized poly(vinyl alcohol) as an artificial cornea scaffold that supports a stratified corneal epithelium

The cornea is a transparent tissue of the eye, which is responsible for the refraction of incoming light. Both biological corneal equivalents and synthetic keratoprostheses have been developed to replace donor tissue as a means to restore vision. However, both designs have drawbacks in terms of stability and biocompatibility. Clinically available synthetic devices do not support an intact epithelium, which poses a risk of microbial infection or protrusion of the prosthesis. In the present study, type I collagen was immobilized onto poly(vinyl alcohol) (PVA-COL) as a possible artificial cornea scaffold that can sustain a functional corneal epithelium. Human and rabbit corneal epithelial cells were air-lift cultured with 3T3 feeder fibroblasts to form a stratified epithelial layer on PVA-COL. The epithelial sheet expressed keratin 3/12 differentiation markers, the tight junction protein occludin, and had characteristic microvilli structures on transmission electron microscopy. Functionally, the stratified epithelium contained normal glycogen levels, and an apical tight-junction network was observed to exclude the diffusion of horseradish peroxidase. Furthermore, the epithelium-PVA-COL composite was suturable in the rabbit cornea, suggesting the possibility of using PVA-COL as a biocompatible material for keratoprosthesis.     

Concise review: Stem cells in the corneal stroma

The cornea is a tough transparent tissue admitting and focusing light in the eye. More than 90% of the cornea is stroma, a highly organized, transparent connective tissue maintained by keratocytes, quiescent mesenchymal cells of neural crest origin. A small population of cells in the mammalian stroma displays properties of mesenchymal stem cells, including clonal growth, multipotent differentiation, and expression of an array of stem cell-specific markers. Unlike keratocytes, the corneal stromal stem cells (CSSCs) undergo extensive expansion in vitro without loss of the ability to adopt a keratocyte phenotype. Several lines of evidence suggest CSSCs to be of neural crest lineage and not from bone marrow. CSSCs are localized in the anterior peripheral (limbal) stroma near to stem cells of the corneal epithelium. CSSCs may function to support potency of the epithelial stem cells in their unique limbal niche. On the other hand, little information is available documenting a role for CSSCs in vivo in stromal wound healing or regeneration. In vitro CSSCs reproduce the highly organized connective tissue of the stroma, demonstrating a potential use of these cells in tissue bioengineering. Direct introduction of CSSCs into the corneal stroma generated transparent tissue in a mouse model of corneal opacity. Human CSSCs injected into mice corneas did not elicit immune rejection over an extended period of time. The CSSCs therefore appear offer an opportunity to develop cell- and tissue-based therapies for irreversible corneal blindness, conditions affecting more than 10 million individuals worldwide.