Cultured autologous oral mucosal epithelial cell sheet (CAOMECS) transplantation for the treatment of corneal limbal epithelial stem cell deficiency.

PURPOSE: Total bilateral corneal limbal epithelial stem cell deficiency (LSCD) cannot be treated with the surgical transplantation of autologous limbus or cultured autologous limbal epithelium. Transplantation of allogenic limbal epithelium is possible but requires immunosuppressive treatments. Cultured autologous oral mucosal epithelial cell sheet (CAOMECS) is a transparent, resistant, viable, and rapidly bioadhesive cell sheet, cultured with the UpCell-Insert technology (CellSeed, Inc., Tokyo, Japan), which allows for grafting onto the patient's corneal stroma without suturing. It has therefore been proposed as an alternative treatment for LSCD. METHODS: The objectives were to assess the safety and efficacy of CAOMECS, using a prospective Gehan's design. Safety was measured in terms of ocular adverse events during the study period, and efficacy was measured using a composite criterion based on epithelial defect, punctate epithelial keratopathy, conjunctival epithelium on the cornea, number of vascular pediculi, and vessel activity. RESULTS: CAOMECS was found to be safe and effective. In total, 26 eyes of 25 patients received a graft. Two patients experienced serious adverse events classified as not product related. Twenty-five patients were included in the efficacy analysis, as one patient was lost to follow-up. The treatment was found to be effective in 16 of 25 patients at 360 days after grafting. Of the 23 patients who completed follow-up at 360 days, 22 had no ulcers, and 19 showed a decrease in the severity of the punctate epithelial keratopathy. CONCLUSIONS: CAOMECS is a well-tolerated and safe tissue-engineered product. These results suggest its efficacy for reconstructing the ocular surface in patients with total bilateral corneal LSCD.

Keratin 13 immunostaining in corneal impression cytology for the diagnosis of limbal stem cell deficiency.

PURPOSE: The aim of this study was to develop a validated, reliable, and minimally invasive technique for diagnosing limbal stem cell deficiency (LSCD) by immunocytochemical detection of conjunctival and corneal keratins on epithelial cells collected by impression cytology (IC). METHODS: After validation of labeling techniques on a cohort of 10 healthy control patients, keratins K12, K13, and K19 were labeled on corneal IC of 10 eyes suspected of LSCD. Positive scores for the conjunctival markers K13/K19, coupled with the rarity of the corneal marker K12, were diagnostic proof of LSCD. RESULTS: IC is a reliable and noninvasive technique for collecting epithelial cells. The labeling validation phase has permitted K3 labeling to be eliminated due to lack of corneal specificity. Among patients with LSCD, nine samples were diagnosed with LSCD (K13+/K19+), which was severe (K12-) in eight cases and mild (K12+) in one case. One sample could not be analyzed due to lack of cells. CONCLUSIONS: K13 has shown to be a new marker of conjunctival differentiation. The immunocytochemical search for the K13/K19 couple by corneal IC provides a simple and reliable method for diagnosing LSCD, whereas the level of K12 could provide a score of disease severity. On the other hand, the authors question the corneal specificity of K3 as conventionally established.

Use of magnetically oriented orthogonal collagen scaffolds for hemi-corneal reconstruction and regeneration.

We recently showed that the highly organized architecture of the corneal stroma could be reproduced using scaffolds consisting of orthogonally aligned multilayers of collagen fibrils prepared using a high magnetic field. Here we show that such scaffolds permit the reconstruction in vitro of human hemi-corneas (stroma + epithelium), using primary human keratocytes and limbal stem cell derived human keratinocytes. On the surface of these hemi-corneas, a well-differentiated epithelium was formed, as determined both histologically and ultrastructurally and by the expression of characteristic markers. Within the stroma, the keratocytes aligned with the directions of the fibrils in the scaffold and synthesized a new extracellular matrix with typical collagen markers and small, uniform diameter fibrils. Finally, in vivo experiments using a rabbit model showed that these orthogonally oriented multi-layer scaffolds could be used to repair the anterior region of the stroma, leading to re-epithelialization and recovery of both transparency and ultrastructural organization.

Reconstruction of a full-thickness collagen-based human oral mucosal equivalent.

Tissue engineered human oral mucosa has the potential to be applied to the closure of surgical wounds after tissue deficits due to facial trauma, malignant lesion surgery or preposthetic procedure. It can also be used to elucidate the biology and pathology of oral mucosa and as a model alternative to animals for safety testing of oral care products. Using the technology previously developed in our laboratory for the production of a skin equivalent, we were able to reconstruct a nonkeratinized full-thickness human oral mucosal equivalent closely mimicking human native oral mucosa. The successive coculture of human lamina propria fibroblasts and human oral epithelial cells isolated from the nonkeratinized region of oral cavity in a porous collagen-glycosaminoglycan (GAG)-chitosan scaffold gave rise to a lamina propria equivalent (LPE) and then to an oral mucosa equivalent (OME). The results of the histology, immunohistology and transmission electron microscopy of this OME demonstrated the presence of a nonkeratinized pluristratified and differentiated epithelium as in native nonkeratinized human oral mucosa expressing both K13 and K3/76. This epithelium was firmly anchored to the LPE by a continuous and ultrastructurally well-organized basement membrane. In the LPE, fibroblasts synthesized new extracellular matrix where the average collagen fibre diameter was 28.4 nm, close to that of native oral mucosa. The proliferative capacity of the basal cells was demonstrated by the expression of Ki67.

Characterisation of human fibroblasts as keratinocyte feeder layer using p63 isoforms status.

Large-scale culture of primary keratinocytes allows the production of large epidermal sheet surfaces for the treatment of extensive skin burns. This method is dependent upon the capacity to establish cultures of proliferating keratinocytes in conditions compatible with their clonal expansion while maintaining their capacity to differentiate into the typical squamous pattern of human epidermis. Feeder layers are critical in this process because the fibroblasts that compose this layer serve as a source of adhesion, growth and differentiation factors. In this report, we have characterise the expression patterns of p63 isoforms in primary keratinocytes cultured on two different feeder layer systems, murine 3T3 and human fibroblasts. We show that with the latter, keratinocytes express a higher ratio of Delta N to TAp63 isoform, in relation with higher clonogenic potential. These results indicate that human fibroblasts represent an adequate feeder layer system to support the culture of primary human keratinocytes.

Development of a reconstructed cornea from collagen-chondroitin sulfate foams and human cell cultures.

PURPOSE: To develop an artificial cornea, the ability to coculture the different cell types present in the cornea is essential. Here the goal was to develop a full-thickness artificial cornea using an optimized collagen-chondroitin sulfate foam, with a thickness close to that of human cornea, by coculturing human corneal epithelial and stromal cells and transfected human endothelial cells. METHODS: Corneal extracellular matrix was simulated by a porous collagen/glycosaminoglycan-based scaffold seeded with stromal keratocytes and then, successively, epithelial and endothelial cells. Scaffolds were characterized for bulk porosity and pore size distribution. The performance of the three-dimensional construct was studied by histology, immunofluorescence, and immunohistochemistry. RESULTS: The scaffold had 85% porosity and an average pore size of 62.1 microm. Keratocytes populated the scaffold and produced a newly synthesized extracellular matrix as characterized by immunohistochemistry. Even though the keratocytes lost their CD34 phenotype marker, the absence of smooth muscle actin fibers showed that these cells had not differentiated into myofibroblasts. The epithelial cells formed a stratified epithelium and began basement membrane deposition. An endothelial cell monolayer beneath the foam was also apparent. CONCLUSIONS: These results demonstrate that collagen-chondroitin sulfate scaffolds are good substrates for artificial cornea construction with good resilience, long-term culture capability, and handling properties.

Variations in the characteristics of keratocytes in culture in relation to their location in human cornea.

To reconstruct artificial stroma close to corneal stroma, it is necessary to use keratocytes with high proliferative potential that maintain the keratocyte phenotype as characterised by CD34. To select such cells, we tested the proliferative potential and characterised the keratocytes isolated from 4 different areas of the human cornea: superior perilimbal, inferior perilimbal, superior central and inferior central. Keratocytes isolated from these different areas had significantly different growth rates (p<0.05), as measured by population doublings: superior perilimbal (42.59+/-11.78) > inferior perilimbal (38.23+/-12.67) > superior central (35.69+/-8.07) > inferior central (25.35+/-7.63). Their clonogenic potential evolved in the same order. Moreover, CD34 labelling gave higher levels in the central areas in relation to the perilimbal areas. We found the best location for isolating keratocytes for stromal reconstruction. The superior perilimbal area had the greatest capacity for proliferation, as well as the best clonogenic potential and the average CD34 level (70%) remained high.

Reconstructed corneas: effect of three-dimensional culture, epithelium, and tetracycline hydrochloride on newly synthesized extracellular matrix.

PURPOSE: To evaluate the influence of the 3-dimensional collagen-glycosaminogycan-chitosan (CGC 3D) scaffold, epithelialization, and the addition of tetracycline hydrochloride on the ultrastructural organization, measured by the diameter and spacing of newly synthesized collagen I fibrils. METHODS: Little is known about the role of interactions between epithelial cells and fibroblasts in controlling the extracellular matrix of the cornea. We developed a hemicornea from a CGC 3D matrix cocultured with keratocytes and human epithelial cells. The keratocytes colonized this substrate, proliferated, and synthesized the extracellular matrix, reproducing a living stroma equivalent. RESULTS: Without a 3D scaffold, the collagen fibrils produced had an average diameter that was 42.7 nm and sigma = 16.9 nm. In the CGC 3D scaffold, the fibrils had an average diameter of 33.4 nm, with little dispersion (sigma = 6.7 nm), suggesting a greater regulation. The epithelium permitted a significant reduction in fibril diameter and interfibrillar spacing. Tetracycline hydrochloride had no effect on spacing but did have a significant effect on fibril diameter. We found positive interactions between the epithelium and tetracycline hydrochloride on the regulation of collagen fibrils but not on spacing. The presence of epithelium led to the increased formation of collagens I and V in the subepithelial area of the newly formed matrix. Type VI collagen was localized around the keratocytes throughout the matrix. CONCLUSIONS: Epithelialization and the 3D scaffold had a great influence on the diameter of collagen I fibrils.

Orthogonal scaffold of magnetically aligned collagen lamellae for corneal stroma reconstruction.

The creation of 3D scaffolds that mimic the structure of physiological tissue required for normal cell function is a major bioengineering challenge. For corneal stroma reconstruction this necessitates the creation of a stroma-like scaffold consisting of a stack of orthogonally disposed sheets of aligned collagen fibrils. This study demonstrates that such a scaffold can be built up using magnetic alignment. By allowing neutralized acid-soluble type I collagen to gel in a horizontal magnetic field (7 T) and by combining a series of gelation-rotation-gelation cycles, a scaffold of orthogonal lamellae composed of aligned collagen fibrils has been formed. Although initially dilute, the gels can be concentrated without noticeable loss in orientation. The gels are translucent but their transparency can be greatly improved by the addition of proteoglycans to the gel-forming solution. Keratocytes align by contact guidance along the direction of collagen fibrils and respect the orthogonal design of the collagen template as they penetrate into the bulk of the 3D matrix. The scaffold is a significant step towards the creation of a corneal substitute with properties resembling those of native corneal stroma.

Supplementation with a complex of active nutrients improved dermal and epidermal characteristics in skin equivalents generated from fibroblasts from young or aged donors.

Cultured skin equivalent (SE, Mimeskin) was generated by co-culturing skin fibroblasts and keratinocytes on a collagen-glycosaminoglycan-chitosan dermal substrate. In order to examine donor age effect, fibroblasts from 19- (young) or 49- (aged) year-old females were used. Culture medium was supplemented with nutrients complex containing soy extract, tomato extract, grape seed extract, white tea extract, sodium ascorbate, tocopherol acetate, zinc gluconate and BioMarine complex. Epidermal and dermal structure and composition were examined after 42 and 60 days of culture. In untreated samples, SE generated from young fibroblasts was superior to SE from aged fibroblasts in all characteristics. Those include number and regularity of keratinocyte layers, number of keratinocytes expressing proliferation marker Ki67, content of collagen type I, fibrillin-1, elastin, and SE lifespan. Effects of nutritional supplementation were observed in SE from both young and aged fibroblasts, however, those effects were more pronounced in SE from aged fibroblasts. In epidermis, the treatment increased number of keratinocyte layers and delayed epidermal senescence. The number of cells expressing Ki67 was nine folds higher than those of controls, and was similar to that of young cell SE. In dermis, the treatment increased mRNA synthesis of collagen I, fibrillin-1 and elastin. In conclusion, skin cell donor age had major important effect on formation of reconstructed SE. Imperfections in epidermal and dermal structure and composition as well as life span in SE from aged cells can be improved by supplementation with active nutrients.

Development of an optimised culture medium for keratocytes in monolayer.

UNLABELLED: Our objective was to formulate a medium for monolayer culture optimising both keratocyte growth and preservation of the keratocyte phenotype. METHODS: An experimental matrix selected 14 media to test, using 7 components. Selection criteria were growth rates over 5 passages and expression of the CD34 marker. RESULTS: Acetylcholine, insulin and vitamin C had no effect on growth and differentiation. The DMEM + Ham F12 1 : 1 based medium was selected for its initial effect on growth. At concentrations of 5 ng/ml, b-FGF improved the percentage of CD34+ cells without reducing growth rates. New-born calf serum (NCS) had a greater effect on growth than foetal calf serum (FCS). We showed three major interactions: between b-FGF and IGF-1, FCS and IGF-1 and NCS and b-FGF. CONCLUSION: We selected the following medium, which provides optimal growth and preservation of the CD34+ phenotype: DMEM/HAM-F12 + 10% NCS + 5 ng/ml b-FGF + antibiotics.