Decellularized human corneal stromal cell sheet as a novel matrix for ocular surface reconstruction.

The shortage of donor corneas as well as the limitations of tissue substitutes leads to the necessity to develop alternative materials for ocular surface reconstruction. Corneal surface substitutes must fulfill specific requirements such as high transparency, low immunogenicity, and mechanical stability combined with elasticity. This in vitro study evaluates a decellularized matrix secreted from human corneal fibroblasts (HCF) as an alternative material for ocular surface reconstruction. HCF from human donors were cultivated with the supplementation of vitamin C to form a stable and thick matrix. Furthermore, due to enhanced cultivation time, a three-dimensional like multilayered construct which partly mimics the complex structure of the corneal stroma could be generated. The formed human cell-based matrices (so-called cell sheets [CS]) were subsequently decellularized. The complete cell removal, collagen content, ultrastructure, and cell toxicity of the decellularized CS (DCS) as well as biomechanical properties were analyzed. Surgical feasibility was tested on enucleated porcine eyes. After decellularization and sterilization, a transparent, thick, cell free, and sterile tissue substitute resulted, which allowed expansion of limbal epithelial stem cells with no signs of cytotoxicity, and good surgical feasibility. DCS seem to be a promising new corneal tissue substitute derived from human cells without the limitation of donor material; however, future in vivo studies are necessary to further elucidate its potential for ocular surface reconstruction.

Stiffening of rabbit corneas by the bacteriochlorophyll derivative WST11 using near infrared light.

PURPOSE: We evaluated the efficacy and safety of photochemical corneal stiffening by palladium bacteriochlorin 13'-(2-sulfoethyl)amide dipotassium salt (WST11) and near infrared (NIR) illumination, using ex vivo and in vivo rabbit eye models. METHODS: Corneas of post mortem rabbits and living rabbits were pretreated topically with 2.5 mg/mL WST11 in saline or in 20% dextran T-500 (WST-D), washed and illuminated with an NIR diode laser (755 nm, 10 mW/cm(2). Studies with corneas of untreated fellow eyes served as controls. Tensile strength measurements, histopathology, electron spin resonance, and optical spectroscopy and fluorescence microscopy were used to assess treatment effects. Comparative studies were performed with standard riboflavin/ultraviolet-A light (UVA) treatment. RESULTS: WST11/NIR treatment significantly increased corneal stiffness following ex vivo or in vivo treatment, compared to untreated contralateral eyes. The incremental ultimate stress and Young's modulus of treated corneas increased by 45, 113, 115%, and 10, 79, and 174% following 10, 20, and 30 minutes of incubation with WST11, respectively. WST-D/NIR had a similar stiffening effect, but markedly reduced post-treatment edema and shorter time of epithelial healing. WST11/NIR and WST-D/NIR generate hydroxyl and superoxide radicals, but no singlet oxygen in the cornea. Histology demonstrated a reduction in the keratocyte population in the anterior half of the corneal stroma, without damage to the endothelium. CONCLUSIONS: Treatment of rabbit corneas, with either WST11/NIR or WST-D/NIR, increases their biomechanical strength through a mechanism that does not involve singlet oxygen. The WST-D/NIR treatment showed less adverse effects, demonstrating a new potential for clinical use in keratoconus and corneal ectasia after refractive surgery.

Stromal niche controls the plasticity of limbal and corneal epithelial differentiation in a rabbit model of recombined tissue.

PURPOSE: The adult rabbit limbal basal epithelium contains corneal epithelial stem cells, which have been characterized by a negative expression of keratin-3 (K3) and a lower expression of connexin 43 (Cx43). This study was conducted to determine whether the limbal stroma dictates the plasticity of limbal and corneal epithelial differentiation. METHODS: Viable epithelial sheets of the central cornea and the pigmented limbus were isolated from Dutch belted rabbits by incubation of 50 mg/mL of dispase II in supplemental hormonal epithelial medium (SHEM) for 18 hours at 4 degrees C. The cleavage plane was studied by immunostaining with antibodies against K3, Cx43, integrin beta1, and collagen IV. Viability of single cells derived from these sheets was assessed by a live-dead assay. Such limbal (L) and corneal (K) epithelial sheets were recombined with either limbal (Ls) or corneal (Ks) stroma, and cultured in SHEM for 10 days before lifting to the air-fluid interface for 1 week. The resultant epithelial phenotype was determined by histology and immunostaining to K3 and Cx43, and apoptosis was investigated by Hoechst and TUNEL nuclear staining. RESULTS: Viability of isolated limbal and corneal epithelial sheets was determined to be 91.1% +/- 2.9%. The basal epithelium of isolated limbal epithelial sheets was positive for integrin beta1, negative for K3, but weakly positive for Cx43, and still retained patches of collagen IV. All recombinants showed stratified epithelia, with intraepithelial cysts with desquamated debris noted only in K/Ks, and epithelial outgrowth onto the insert filter from L/Ls. As expected, expression of K3 was negative in the basal layer of L/Ls, but positive in that of K/Ks. Expression of K3 was sporadically positive in the basal layer of L/Ks but largely negative in that of K/Ls. Expression of Cx43 was uniformly expressed in the basal layer of the K/Ks, but weak in that of L/Ls, K/Ls, and L/Ks. A higher apoptosis index was only noted in intraepithelial cysts of K/Ks. CONCLUSIONS: These results strongly indicate that the limbal stroma modulates epithelial differentiation, proliferation and apoptosis in the direction favoring stemness, whereas the corneal stroma promotes differentiation. Further investigation into elements constituting such a niche should help unveil the secrecy whereby stemness is controlled.

The influence of corneal stromal matrix proteins on the migration of human corneal fibroblasts.

Motivated by the alterations seen in the corneal matrix composition after photorefractive keratectomy and the migration of corneal keratocytes seen following this procedure, the locomotor response of corneal stromal fibroblasts to various extracellular matrix proteins was determined. In addition, the involvement of integrin mediated attachment to the matrix proteins was investigated. Quantitative invasion assays were performed using collagen gels, supplemented with either fibronectin, tenascin, collagen type V, collagen type VI, chondroitin sulfate or keratan sulfate. The ultrastructure of the gels was visualized by scanning electron microscopy and related to the migration results. The extent of alpha(1)beta(1), alpha(2)beta(1), alpha(3)beta(1)and alpha(5)beta(1)integrin mediated attachment to the matrix proteins was evaluated using blocking antibodies. Fibronectin increased corneal fibroblast migration significantly, and served as an excellent substrate for cellular attachment, mediated by the alpha(5)beta(1)integrin. Addition of tenascin to the fibronectin-containing gels disrupted these effects, while attachment to this matrix also involved the integrins alpha(2)beta(1)and alpha(3)beta(1). Chondroitin sulfate and collagen types V and VI primarily altered the structure of the collagen matrix, resulting in an inhibition of migration by the collagens and an increase by chondroitin sulfate. They all served as poor substrates for attachment. Thus, the migratory activity of corneal fibroblasts in vitro is influenced by the composition of the surrounding extracellular matrix, either by integrin mediated cell-matrix interactions or through matrix-matrix interactions. This study provides evidence that the provisional matrix deposited in a corneal stromal wound may facilitate the entry of migrating corneal fibroblasts.

Factors affecting corneal strip stroma-to-stroma adhesion.

BACKGROUND: Successful laser in situ keratomileusis depends on strong adhesion between the dissected anterior corneal flap and the underlying stromal bed, without suturing. This study attempts to generate new information about the mechanism of this adhesion. METHODS: Strips of fresh bovine corneal stroma were dissected and split apart. The split strips were then made to adhere to each other with varying amounts of combined heat and drying. A relationship was developed between heat applied and hydration of strips. After each heat and drying treatment, the strips were pulled apart and the force needed to accomplish this task was measured. RESULTS: There was a tight linear correlation between increase in temperature and decrease in water content (R2 = 0.7414) and between decrease in water content and increase in the adhesive force (R2 = 0.3355). CONCLUSION: This model suggests that drying increases stromal-stromal adhesion. We speculate this is due to the increased concentration of surface molecules, which have high ionic charge densities and ionic bonding. A higher concentration of these molecules produces stroma-to-stroma adhesion via ionic bonding.

pH, ionized calcium and osmotic artifacts can affect determination of rabbit corneal deturgescence in vitro.

Freshly isolated stroma-endothelial preparations were perfused with a glucose, adenosine and glutathione-supplemented Ringer solution at 37 degrees C at a hydrostatic pressure of 20 cm H2O. The composition (tCO2, pH, Ca2+ and osmolality) of the stromal-side bathing solution was analyzed during a 90-min equilibration period during which this solution was covered or deliberately left open to the atmosphere. If exposed to the atmosphere, even for a few minutes, substantial changes in pH, osmolality and ionized calcium occurred such that, when silicone oil was subsequently applied to the stromal surface, significant increases in both the rate and amplitude of deturgescence were observed. The rates and amplitude were proportional to the corneal thickness prior to silicone oil application. The results further indicate that the physicochemical state of the stroma is an important determinant in the corneal deturgescence mechanism.