Isolation and Propagation of Human Corneal Stromal Keratocytes for Tissue Engineering and Cell Therapy.

The human corneal stroma contains corneal stromal keratocytes (CSKs) that synthesize and deposit collagens and keratan sulfate proteoglycans into the stromal matrix to maintain the corneal structural integrity and transparency. In adult corneas, CSKs are quiescent and arrested in the G0 phase of the cell cycle. Following injury, some CSKs undergo apoptosis, whereas the surviving cells are activated to become stromal fibroblasts (SFs) and myofibroblasts (MyoFBs), as a natural mechanism of wound healing. The SFs and MyoFBs secrete abnormal extracellular matrix proteins, leading to corneal fibrosis and scar formation (corneal opacification). The issue is compounded by the fact that CSK transformation into SFs or MyoFBs is irreversible in vivo, which leads to chronic opacification. In this scenario, corneal transplantation is the only recourse. The application of cell therapy by replenishing CSKs, propagated in vitro, in the injured corneas has been demonstrated to be efficacious in resolving early-onset corneal opacification. However, expanding CSKs is challenging and has been the limiting factor for the application in corneal tissue engineering and cell therapy. The supplementation of serum in the culture medium promotes cell division but inevitably converts the CSKs into SFs. Similar to the in vivo conditions, the transformation is irreversible, even when the SF culture is switched to a serum-free medium. In the current article, we present a detailed protocol on the isolation and propagation of bona fide human CSKs and the morphological and genotypic differences from SFs.

Morphofunctional Properties of Corneal Stromal Cells.

Human corneal stromal cells were isolated by enzymatic digestion from a new source, lenticules obtained during laser vision correction by the ReLEx SMILe method. The resulting culture was mainly presented by fibroblast-like cells with a phenotype CD90-/CD73+/CD105+/keratocan-/lumican-/ALDH1A1+ that differentiate into keratocytes in a specialized medium. The concentration of fetal calf serum-derived growth factors affects the rate of proliferation, production of erythropoietin and brain neurotrophic factor by corneal fibroblasts, and to a lesser extent, their migration activity and production of extracellular matrix components. Thus, the high functional potential of fibroblast-like cells isolated from stromal lenticles can be used to develop cell technologies in ophthalmology.

Pathophysiology and inflammatory biomarkers of sulfur mustard-induced corneal injury in rabbits.

Sulfur mustard (SM) is a cytotoxic, vesicating, chemical warfare agent, first used in 1917; corneas are particularly vulnerable to SM exposure. They may develop inflammation, ulceration, neovascularization (NV), impaired vision, and partial/complete blindness depending upon the concentration of SM, exposure duration, and bio-physiological conditions of the eyes. Comprehensive in vivo studies have established ocular structural alterations, opacity, NV, and inflammation upon short durations (<4 min) of SM exposure. In this study, detailed analyses of histopathological alterations in corneal structure, keratocytes, inflammatory cells, blood vessels, and expressions of cyclooxygenase (COX)-2, matrix metalloproteinase (MMP)-9, vascular endothelial growth factor (VEGF), and cytokines were performed in New Zealand white rabbits, in a time-dependent manner till 28 days, post longer durations (5 and 7 min) of ocular SM exposure to establish quantifiable endpoints of injury and healing. Results indicated that SM exposure led to duration-dependent increases in corneal thickness, opacity, ulceration, epithelial-stromal separation, and epithelial degradation. Significant increases in NV, keratocyte death, blood vessels, and inflammatory markers (COX-2, MMP-9, VEGF, and interleukin-8) were also observed for both exposure durations compared to the controls. Collectively, these findings would benefit in temporal delineation of mechanisms underlying SM-induced corneal toxicity and provide models for testing therapeutic interventions.

Human platelet lysate as a replacement for fetal bovine serum in human corneal stromal keratocyte and fibroblast culture.

The isolation and propagation of primary human corneal stromal keratocytes (CSK) are crucial for cellular research and corneal tissue engineering. However, this delicate cell type easily transforms into stromal fibroblasts (SF) and scar inducing myofibroblasts (Myo-SF). Current protocols mainly rely on xenogeneic fetal bovine serum (FBS). Human platelet lysate (hPL) could be a viable, potentially autologous, alternative. We found high cell survival with both supplements in CSK and SF. Cell numbers and Ki67+ ratios increased with higher fractions of hPL and FBS in CSK and SF. We detected a loss in CSK marker expression (Col8A2, ALDH3A1 and LUM) with increasing fractions of FBS and hPL in CSK and SF. The expression of the Myo-SF marker SMA increased with higher amounts of FBS but decreased with incremental hPL substitution in both cell types, implying an antifibrotic effect of hPL. Immunohistochemistry confirmed the RT-PCR findings. bFGF and HGF were only found in hPL and could be responsible for suppressing the Myo-SF conversion. Considering all findings, we propose 0.5% hPL as a suitable substitution in CSK culture, as this xeno-free component efficiently preserved CSK characteristics, with non-inferiority in terms of cell viability, cell number and proliferation in comparison to the established 0.5% FBS protocol.

Tissue engineering of corneal stroma via melt electrowriting.

The cornea serves as the main refractive component of the eye with the corneal stroma constituting the thickest component in a stratified layered system of epithelia, stroma, and endothelium. Current treatment options for patients suffering from corneal diseases are limited to transplantation of a human donor cornea (keratoplasty) or to implantation of an artificial cornea (keratoprosthesis). Nevertheless, donor shortage and failure of artificial corneas to integrate with local tissue constitute important problems that have not been yet circumvented. Recent advances in biofabrication have made great progress toward the manufacture of tailored biomaterial templates with the potential of guiding partially or totally the regeneration process of the native cornea. However, the role of the corneal stroma on current tissue engineering strategies is often neglected. Here, we achieved a tissue-engineered corneal stroma substitute culturing primary keratocytes on scaffolds prepared via melt electrowriting (MEW). Scaffolds were designed to contain highly organized micrometric fibers to ensure transparency and encourage primary human keratocytes to self-orchestrate their own extracellular matrix deposition and remodeling. Results demonstrated reliable cell attachment and growth over a period of 5 weeks and confirmed the formation of a dense and highly organized de novo tissue containing collagen I, V, and VI as well as Keratocan, which resembled very closely the native corneal stoma. In summary, MEW brings us closer to the biofabrication of a viable corneal stroma substitute.

Safety of grafting acellular human corneal lenticule seeded with Wharton's Jelly-Derived Mesenchymal Stem Cells in an experimental animal model.

The present study was conducted to evaluate safety of grafting acellular human corneal lenticule seeded with Wharton's Jelly-derived Mesenchymal Stem Cells (WJSC) in an experimental animal model. Human corneal lenticules were decellularized with a rate of about 97% with an acceptable lack of cytotoxicity and relatively intact ultrastructure of the lenticules. 12 rabbits underwent unilateral stromal pocketing with implantation of decellularized lenticules. Implantation was performed for 6 rabbits along with graft recellularization with WJSCs. Rabbits were euthanized after 1 month (n = 6) and 3 months (n = 6) to evaluate progression of graft bio-integration. No clinical rejection sign was detected during the study. Histopathological analysis showed that, grafts were integrated well with the least distortion of surrounding collagen bundles. After 3 months, labeled WJCS was detected representing viability of stem cells in the host. Increased expression of keratocyte-specific markers showed the potential of recruiting WJSCs as keratocyte progenitor cells to reinforce corneal ultrastructure.

Keratocyte mechanobiology.

In vivo, corneal keratocytes reside within a complex 3D extracellular matrix (ECM) consisting of highly aligned collagen lamellae, growth factors, and other extracellular matrix components, and are subjected to various mechanical stimuli during developmental morphogenesis, fluctuations in intraocular pressure, and wound healing. The process by which keratocytes convert changes in mechanical stimuli (e.g. local topography, applied force, ECM stiffness) into biochemical signaling is known as mechanotransduction. Activation of the various mechanotransductive pathways can produce changes in cell migration, proliferation, and differentiation. Here we review how corneal keratocytes respond to and integrate different biochemical and biophysical factors. We first highlight how growth factors and other cytokines regulate the activity of Rho GTPases, cytoskeletal remodeling, and ultimately the mechanical phenotype of keratocytes. We then discuss how changes in the mechanical properties of the ECM have been shown to regulate keratocyte behavior in sophisticated 2D and 3D experimental models of the corneal microenvironment. Finally, we discuss how ECM topography and protein composition can modulate cell phenotypes, and review the different methods of fabricating in vitro mimics of corneal ECM topography, novel approaches for examining topographical effects in vivo, and the impact of different ECM glycoproteins and proteoglycans on keratocyte behavior.

Isolation and Culture of Corneal Stromal Stem Cells.

An increasing body of evidence authenticates the benefit of corneal stroma-derived stem cells (CSSCs) in tissue engineering and regeneration oriented research, and potentially in the development of clinically relevant cellular therapies. Postmortem corneal tissue obtained from otherwise discarded material after keratoplasties is oftentimes the source of the cells for ex vivo research. Relatively easy to isolate and cultivate as well as inexpensive to culture, CSSCs now represent a well-described cell type with attributes of mesenchymal stem cells (MSCs). These include differentiation- and immunosuppressive potential, as well as a favorable capacity to expand in vitro. Here, we in detail describe two straightforward methods to isolate and establish CSSC cultures ex vivo.

HC-HA/PTX3 Purified From Human Amniotic Membrane Reverts Human Corneal Fibroblasts and Myofibroblasts to Keratocytes by Activating BMP Signaling.

Purpose: Fibrosis or scarring is a pathological outcome of wound healing and is characterized by terminally differentiated myofibroblasts. Heavy chain-hyaluronic acid/pentraxin 3 (HC-HA/PTX3) is a unique matrix component purified from amniotic membrane that exerts an anti-inflammatory effect. Herein, we investigate whether HC-HA/PTX3 can also exert an antiscarring effect. Methods: Human corneal fibroblasts and myofibroblasts were seeded on plastic, immobilized HA or HC-HA/PTX3 or on plastic with or without soluble HA and HC-HA/PTX3 in DMEM+10% FBS, with or without AMD3100 or SB431542 in DMEM+ITS with or without transforming growth factor-ß1 (TGF-ß1). Transcript expression of keratocyte and signaling markers was determined by RT-qPCR. Immunostaining was performed to monitor cytolocalization of signaling markers and α-SMA. Western blotting was used to measure relative protein level. Results: Human corneal fibroblasts and myofibroblasts cultured in or on HC-HA/PTX3, but not HA, were refrained from cytoplasmic expression of αSMA and nuclear translocation of pSMAD2/3 when challenged with exogenous TGF-ß1. Such an antiscarring action by suppressing canonical TGF-ß1 signaling was surprisingly accompanied by phenotypic reversal to keratocan-expressing keratocytes through activation of BMP signaling. Further investigation disclosed that such phenotypic reversal was initiated by cell aggregation mediated by SDF1-CXCR4 signaling highlighted by nuclear translocation of CXCR4 and upregulation of CXCR4 transcript and protein followed by activation of canonical BMP signaling. Conclusions: These findings collectively provide mechanistic understanding explaining how amniotic membrane transplantation exerts an antiscarring action. In addition, HC-HA/PTX3 and derivatives may be developed into a new biologic to treat corneal blindness caused by stromal scar or opacity in the future.

Influence of micropatterned substrates on keratocyte phenotype.

Substrate topographic patterning is a powerful tool that can be used to manipulate cell shape and orientation. To gain a better understanding of the relationship between surface topography and keratocyte behavior, surface patterns consisting of linear aligned or orthogonally aligned microchannels were used. Photolithography and polymer molding techniques were used to fabricate micropatterns on the surface of polydimethylsiloxane (PDMS). Cells on linear aligned substrates were elongated and aligned in the channel direction, while cells on orthogonal substrates had a more spread morphology. Both linear and orthogonal topographies induced chromatin condensation and resulted in higher expressions of keratocyte specific genes and sulfated glycosaminoglycans (sGAG), compared with non-patterned substrates. However, despite differences in cell morphology and focal adhesions, many genes associated with a native keratocyte phenotype, such as keratocan and ALDH3A1, remain unchanged on the different patterned substrates. This information could be used to optimize substrates for keratocyte culture and to develop scaffolds for corneal regeneration.

Corneal Stroma Cell Density Evolution in Keratoconus Corneas Following the Implantation of Adipose Mesenchymal Stem Cells and Corneal Laminas: An In Vivo Confocal Microscopy Study.

Purpose: To report the corneal stroma cell density evolution identified by in vivo corneal confocal microscopy in humans using injected autologous adipose-derived adult stem cells (ADASCs) and corneal decellularized laminas in corneas with advanced keratoconus. Methods: Interventional prospective, consecutive, randomized, comparative series of cases. A total of 14 keratoconic patients were randomly distributed into three groups for three types of surgical interventions: group 1 (G-1), autologous ADASC implantation (n = 5); group 2 (G-2), decellularized human corneal stroma (n = 5); and group 3 (G-3), autologous ADASCs + decellularized human corneal stroma (n = 4). Results: A gradual and significant increase (P < 0.001) was observed in the cellularity in the anterior and posterior stroma of patients in G-1, G-2, and G-3 a year after the surgery in comparison with the preoperative density level. The same result was observed at the mid-corneal stroma in G-1 and at the anterior and posterior surfaces and within the laminas in G-2 and G-3. The cell density of patients receiving ADASC recellularized laminas (G-3) was statistically significantly higher (P = 0.011) at the anterior surface and within the lamina (P = 0.029) and at the posterior surface than in those implanted only with decellularized laminas (G-2). Conclusions: A significant increase in cell density occurred up to 1 postoperative year at the corneal stroma following the implantation of ADASCs alone, as well as in those cases implanted with decellularized and recellularized laminas at the different levels of the analysis. However, this increase was significantly higher in the ADASC recellularized laminas.

Exosomal miR-19a from adipose-derived stem cells suppresses differentiation of corneal keratocytes into myofibroblasts.

In this study, we investigated the effects of exosomal microRNAs (miRNAs) from adipose-derived stem cells (ADSCs) on the differentiation of rabbit corneal keratocytes. Keratocytes grown in 10% FBS differentiated into myofibroblasts by increasing HIPK2 kinase levels and activity. HIPK2 enhanced p53 and Smad3 pathways in FBS-induced keratocytes. Keratocytes grown in 10% FBS also showed increased levels of pro-fibrotic proteins, including collagen III, MMP9, fibronectin, and α-SMA. These effects were reversed by knocking down HIPK2. Moreover, ADSCs and exosomes derived from ADSCs (ADSCs-Exo) suppressed FBS-induced differentiation of keratocytes into myofibroblasts by inhibiting HIPK2. Quantitative RT-PCR analysis showed that ADSCs-Exos were significantly enriched in miRNA-19a as compared to ADSCs. Targetscan and dual luciferase reporter assays confirmed that the HIPK2 3'UTR is a direct binding target of miR-19a. Keratocytes treated with 10% FBS and ADSCs-Exo-miR-19a-agomir or ADSCs-Exo-NC-antagomir showed significantly lower levels of HIPK2, phospho-Smad3, phospho-p53, collagen III, MMP9, fibronectin and α-SMA than those treated with 10% FBS plus ADSCs-Exo-NC-agomir or ADSCs-Exo-miR-19a-antagomir. Thus, exosomal miR-19a derived from the ADSCs suppresses FBS-induced differentiation of rabbit corneal keratocytes into myofibroblasts by inhibiting HIPK2 expression. This suggests their potential use in the treatment of corneal fibrosis.

Requirement for the collagen receptor Endo180 in collagen gel contraction mediated by corneal fibroblasts.

The interaction of keratocytes with extracellular matrix components plays an important role in the maintenance of corneal transparency and shape as well as in the healing of corneal wounds. In particular, the interaction of these cells with collagen and cell-mediated collagen contraction contribute to wound closure. Endo180 is a receptor for collagen that mediates its cellular internalization. We have now examined the role of Endo180 in collagen contraction mediated by corneal fibroblasts (activated keratocytes). Antibodies to Endo180 inhibited the contractile activity of mouse corneal fibroblasts embedded in a three-dimensional collagen gel and cultured in the presence of serum, with this effect being both concentration and time dependent and essentially complete at an antibody concentration of 0.2 µg/ml. Whereas corneal fibroblasts cultured in a collagen gel manifested a flattened morphology with prominent stress fibers under control conditions, they showed a spindlelike shape with few stress fibers in the presence of antibodies to Endo180. Antibodies to Endo180 had no effect on the expression of α-smooth muscle actin or the extent of collagen degradation in collagen gel cultures of corneal fibroblasts. Immunohistofluorescence analysis did not detect the expression of Endo180 in the unwounded mouse cornea. However, Endo180 expression was detected in keratocytes migrating into the wound area at 3 days after a corneal incisional injury. Together, our results suggest that Endo180 is required for the contraction of collagen matrix mediated by corneal fibroblasts and that its expression in these cells may contribute to the healing of corneal stromal wounds.

Effects of Soft Toric, Rigid Gas-Permeable, and Mini-Scleral Lenses on Corneal Microstructure Using Confocal Microscopy.

OBJECTIVE: To determine effects of wearing soft toric silicone hydrogel, rigid gas-permeable (RGP), and mini-scleral lenses on corneal microstructure using confocal microscopy. METHOD: A prospective cohort study was conducted on 33 neophyte patients fitted with contact lenses (avg. age: 26±7 years) in the tertiary eye center. Patients were instructed to wear soft toric silicone hydrogel, RGP, or mini-scleral lenses based on clinical diagnoses. Inclusion criteria were age greater than 18 years and best-corrected visual acuity ≥3/10. Patients with a history of eye-involving systemic diseases were excluded. Baseline examinations included log of minimal angle of resolution visual acuity (Early Treatment Diabetic Retinopathy Study chart), refraction, slit-lamp, and fundoscopy. Confocal microscopy was used to measure subbasal nerve (SBN) density (mm/mm), keratocyte cell density (cells/mm), basal epithelial cell density (cells/mm), and endothelial cell density (cells/mm). Data were gathered on the first and follow-up visits. The follow-up visit happened after 6 months when the subjects had stopped wearing contact lenses for 12 hr. Comparative analysis was conducted within each group using the paired t test. RESULTS: The changes in visual acuity, SBN, and keratocyte cell density were insignificant after 6 months of wearing lenses in all three groups. The basal epithelial cell density significantly decreased (P<0.05) in RGP and mini-scleral groups. In addition, the endothelial cell density decreased significantly (P<0.05) in the RGP group. No significant changes were detected in soft toric silicone hydrogel lens wearers. CONCLUSIONS: Soft toric silicone hydrogel lenses seemed to have the least impact on the corneal cellular microstructure for a wear period of 6 months, controlling confounding factors of prior cross-sectional investigations. The coarse (three layers) versus fine (five layers) division of stroma, the repeatability and reproducibility of stromal layers' demarcation, and the cohort size and its diversity in terms of initial corneal diagnoses (particularly in the mini-scleral wearing group) can potentially influence the outcomes, and their impact remains to be further investigated.

The Effect of Cytokine-Stimulation and Pharmacologic Intervention on PGE2 Production in Primary Human Conjunctival and Corneal Cells.

We studied the production of PGE2 by human conjunctival and corneal cells in response to inflammation, and reduction of inflammation with non-steroidal anti-inflammatory drugs. Primary cultures of human conjunctival epithelial cells, fibroblasts, corneal epithelial cells, and keratocytes were incubated with IL-4 and TNF-α. PGE2 and COX-2 levels were analyzed. Effects of anti-inflammatory and anti-immune drugs on PGE2 production were also investigated. IL-4 and TNF-α induced the generation of PGE2 and COX-2 in conjunctival and corneal cells. Epithelial PGE2 production was significantly lower than in keratocytes and fibroblasts, which was down-regulated by aspirin. IL-4 and TNF-α enhanced the inflammatory response via prostaglandin production which contributed to ocular surface inflammation. Prostaglandin production was higher in stromal cells than epithelial cells. These results suggest that the epithelial barrier disruption may contribute to ocular allergic inflammation by the PGE2 production from stromal cells. Moreover, NSAIDs were effective in suppressing PGE2 production in our experiment.

A high-throughput microfluidic method for fabricating aligned collagen fibrils to study Keratocyte behavior.

In vivo, keratocytes are surrounded by aligned type I collagen fibrils that are organized into lamellae. A growing body of literature suggests that the unique topography of the corneal stroma is an important regulator of keratocyte behavior. In this study we describe a microfluidic method to deposit aligned fibrils of type I collagen onto glass coverslips. This high-throughput method allowed for the simultaneous coating of up to eight substrates with aligned collagen fibrils. When these substrates were integrated into a PDMS microwell culture system they provided a platform for high-resolution imaging of keratocyte behavior. Through the use of wide-field fluorescence and differential interference contrast microscopy, we observed that the density of collagen fibrils deposited was dependent upon both the perfusion shear rate of collagen and the time of perfusion. In contrast, a similar degree of fibril alignment was observed over a range of shear rates. When primary normal rabbit keratocytes (NRK) were seeded on substrates with a high density of aligned collagen fibrils and cultured in the presence of platelet derived growth factor (PDGF) the keratocytes displayed an elongated cell body that was co-aligned with the underlying collagen fibrils. In contrast, when NRK were cultured on substrates with a low density of aligned collagen fibrils, the cells showed no preferential orientation. These results suggest that this simple and inexpensive method can provide a general platform to study how simultaneous exposure to topographical and soluble cues influence cell behavior.

Cell loaded 3D bioprinted GelMA hydrogels for corneal stroma engineering.

Tissue engineering aims to replace missing or damaged tissues and restore their functions. Three-dimensional (3D) printing has been gaining more attention because it enables the researchers to design and produce cell loaded constructs with predetermined shapes, sizes, and interior architecture. In the present study, a 3D bioprinted corneal stroma equivalent was designed to substitute for the native tissue. Reproducible outer and inner organization of the stroma was obtained by optimizing printing conditions such as the nozzle speed in the x-y direction and the spindle speed. 3D printed GelMA hydrogels were highly stable in PBS during three weeks of incubation (8% weight loss). Live-Dead cell viability assay showed 98% cell viability on day 21 indicating that printing conditions were suitable for keratocyte printing. Mechanical properties of the cell loaded 3D printed hydrogels increased 2-fold during this incubation period and approached those of the native cornea (ca. 20 kPa vs. 27 kPa, respectively). Expression of collagens types I and V, and proteoglycan (decorin) in keratocytes indicates maintenance of the phenotype in the hydrogels. Transparency of cell-loaded and cell-free hydrogels was over 80% (at 700 nm) during the three week culture period and comparable to that of the native cornea (85%) at the same wavelength. Thus, GelMA hydrogels bioprinted with keratocytes mimic the biological and physical properties of the corneal stroma with their excellent transparency, adequate mechanical strength, and high cell viability.

Fidelity of long-term cryopreserved adipose-derived stem cells for differentiation into cells of ocular and other lineages.

Adipose-Derived Stem Cells (ADSCs) have an important contribution in regenerative medicine ranging from testing stem cell therapy for disease treatment in pre-clinical models to clinical trials. For immediate use of stem cells for therapy, there is a requirement of the high dose of stem cells at different time points which can be met by cryopreservation. In this study, we evaluated the characteristics of long-term cryopreserved ADSCs and their regenerative potential after an average of twelve-year cryopreservation. Revived ADSCs were examined for cell viability and proliferation by trypan blue, Calcein/Hoechst and MTT assay. Expression of stem cell markers was examined by flow cytometry, immunostaining and qPCR. Colony forming efficiency and spheroid formation ability were also assessed. Multilineage differentiation potential was evaluated by induction into osteocytes, adipocytes, neural cells, corneal keratocytes and trabecular meshwork (TM) cells. Post-thaw, ADSCs maintained expression of stem cell markers CD90, CD73, CD105, CD166, NOTCH1, STRO-1, ABCG2, OCT4, KLF4. ADSCs retained colony and spheroid forming potential. These cells were able to differentiate into osteocytes, confirmed by Alizarin Red S staining and elevated expression of osteocalcin and osteopontin; into adipocytes by Oil Red O staining and elevated expression of PPARγ2. ADSCs could differentiate into neural cells, stained positive to ß-III tubulin, neurofilament, GFAP as well as elevated expression of nestin and neurofilament mRNAs. ADSCs could also give rise to corneal keratocytes expressing keratocan, keratan sulfate, ALDH and collagen V, and to TM cells expressing CHI3L1 and AQP1. Differentiated TM cells responded to dexamethasone treatment with increased Myocilin expression, which could be used as in vitro glaucoma model for further studies. Conditioned medium from ADSCs was found to impart a regenerative effect on primary TM cells. In conclusion, ADSCs maintained their stemness and multipotency after long-term cryopreservation with variability between different donors. This study can have great repercussions in regenerative medicine and pave the way for future clinical trials using cryopreserved ADSCs.

Methacrylated gelatin hydrogels as corneal stroma substitutes: in vivo study.

Methacrylated gelatin (GelMA) hydrogels were prepared to serve as corneal stroma equivalents. They were highly transparent (ca. 95% at 700 nm), mechanically strong and withstood handling and had high human corneal keratocyte viability (98%) after 21 days of culture period. In order to test the in vivo performance of the cell free GelMA hydrogels a pilot in vivo study was carried out using eyes of two white New Zealand rabbits. Hydrogel was implanted in a mid-stromal pocket created and without suture fixation, and observed for 8 weeks under a slit lamp. No edema, ulcer formation, inflammation or infection was observed in both the control (sham) and hydrogel implanted corneas. Corneal vascularization on week 3 was treated with one dose of anti-VEGF application. Hematoxylin and Eosin staining showed that the hydrogel was integrated with the host tissue with only a minimal foreign body reaction. Results demonstrated some degradation in the construct within 8 weeks as evidenced by the decrease of the diameter of the hydrogel from 4 mm to 2.6 mm. High transparency, adequate mechanical strength, biocompatibility and well integration with the host tissue, indicates that this hydrogel is a viable alternative to the current methods for the treatment of corneal blindness and deserves testing on larger number of rabbits and more extensively using microscopy, histology and immune histochemistry.

Potential for combined delivery of riboflavin and all-trans retinoic acid, from silk fibroin for corneal bioengineering.

Millions of people worldwide suffer from vision impairing conditions resulting from corneal injury or disease. Silk fibroin (SF) is an emerging biopolymer that has been used for several applications including the fabrication of bioengineered corneas and ocular prostheses. To improve the cell response to SF, riboflavin (RF) and all-trans retinoic acid (RA) were coupled onto SF matrices. RF is a photo-initiator that has previously been combined with ultraviolet light to crosslink corneal collagen while RA has been used to regulate the phenotype of corneal stromal cells and their extracellular matrix deposition. Different concentrations of RF and RA were respectively photo-crosslinked and covalently bound through carbodiimide coupling onto 2% SF matrices. The effect of incorporating these molecules on the physical, chemical and mechanical properties of the matrices was evaluated. The biological response of human corneal stromal cells to the matrices was examined using cellular adhesion assays, proliferation assays, cytoskeleton staining, gene expression analysis and immunocytochemical staining. RF and RA both led to changes in the surface nanostructure and hydrophilicity while just RF increased the material stiffness. Cells cultured on the matrices containing both biomolecules displayed improved cellular proliferation, increased GAG deposition and increased expression of keratocyte genes that are normally associated with healthy corneal stromal tissue. These in vitro studies serve as a starting point for the optimization of loading bioactive molecules on SF based matrices for formulating clinically relevant ocular implants.