Mithun Santra

Epigenetics regulates gene activity without altering the underlying DNA sequences. Numerous studies have highlighted the importance of epigenetics in diverse physiological processes, including cell growth, differentiation, and tissue development. Increasingly, epigenetic modifications are recognized for their involvement in various diseases, notably corneal disorders. Corneal fibrosis, a common consequence of ocular injury or infection, significantly contributes to visual impairment and blindness worldwide. Recent evidence indicates that epigenetic changes regulate key processes in corneal pathogenesis, such as inflammation, wound healing, extracellular matrix remodeling, fibrosis, and neovascularization. These findings underscore the potential of developing novel therapeutic strategies that specifically target epigenetic mechanisms to treat or mitigate corneal pathology. Nevertheless, bringing epigenetic therapies into clinical practice remains challenging given the complexity of epigenetic regulation. Future research leveraging multi-omics technologies and specific gene manipulation will be essential to elucidate the mechanisms underlying epigenetic regulation in corneal diseases and to identify specific therapeutic targets. Such advancements will drive the development of effective, clinically relevant treatments for corneal fibrosis and related disorders.

Corneal fibrosis, clinically referred to as corneal scarring, disrupts the normal architecture and transparency of the cornea and remains a major cause of visual impairment worldwide. Although corneal transplantation can restore vision, its effectiveness is constrained by limited accessibility, donor tissue shortages, and the risk of allograft rejection. Treatments with human corneal stromal stem cells (hCSSCs) have demonstrated scarless healing in preclinical models of acute corneal injury. Here, we report that hCSSCs also modulated pre-existing corneal opacities. We established a reproducible in vivo model of chronic corneal opacity. Given that scar severity varies among corneas even after identical injuries, we developed a non-invasive, image-based method to quantify opacity volume longitudinally in individual corneas. Using this approach, we evaluated the scar-reducing potential of three hCSSC batches previously shown to inhibit acute scarring. Following cell treatment, the pre-existing opacity volumes gradually decreased. In vitro, hCSSCs exposed to pro-inflammatory stimulus exhibited increased metalloproteinase (MMP) activity relative to tissue inhibitor of metalloproteinase (TIMP), as indicated by an elevated MMP2/TIMP2 ratio. This shift may promote matrix remodeling and scar resolution. Overall, our findings provide proof-of-concept for hCSSC-based therapy as a strategy to reduce established corneal scarring and restore corneal transparency.

Purpose: Corneal neovascularization (CNV) threatens corneal transparency and is a leading cause of vision loss. This study evaluates the anti-angiogenic effects of extracellular vesicles (EVs) derived from human corneal stromal stem cells (CSSCs). Methods: Cultured human umbilical vein endothelial cells (HUVECs) were treated with CSSC-EVs to examine the influence on cell growth and modulation of angiogenesis using xCELLigence and tube formation assays. In vivo , a mouse model of CNV was induced by silver nitrate (AgNO₃) cauterization, and the injured corneas were treated with CSSC-EV eye drops twice daily for four days. On day 10, new vessel formation on mouse corneas was graded, and the expression of angiogenic markers was assessed using immunostaining and qPCR. Results were analyzed using one-way ANOVA. Results: HUVEC cultures treated with CSSC-EVs showed reduced growth with a significant increase in cell doubling time. On Geltrex-coated culture surface, HUVECs incubated with CSSC-EVs generated reduced numbers of vascular tube structures, with significantly reduced junctions and nodes. In vivo , CNV developed in AgNO₃-cauterized mouse corneas by day 10 post-injury. Eye drop treatment with CSSC-EVs attenuated the formation of new vessels expressing vascular marker CD31 and lymphatic marker LYVE1. Compared to injured controls treated with phosphate-buffered saline (PBS) eye drops, the expression of angiogenic markers, including ANGPT1 and 2, CD31, VEGFA, VEGFR1, and R2, was significantly downregulated in EV-treated corneas ( P < 0.05). Conclusion: The anti-angiogenic effect of CSSC-EVs demonstrated their potential to inhibit CNV. Overall, the topical application of CSSC-EVs was safe and effective for addressing CNV-related pathologies.

Biomaterials that integrate multiple functionalities, mimic the extracellular matrix (ECM) microenvironment to support cellular growth, and adhere robustly to damaged tissues are highly needed to advance tissue engineering. Protein‐based biomaterials are promising due to their inherent biocompatibility, biomimicry, biodegradation, and cell‐supportive properties. Herein, by leveraging the unique ability of dithiolanes to generate on‐demand in situ thiols, a new class of dithiolane‐modified, protein‐based biomaterial that combines unique, seemingly opposing functions for tissue engineering is developed. Dithiolane‐modified gelatin, a model protein used herein, enabled photoinitiator‐free photo‐crosslinking to form multi‐functional gelatin‐dithiolane (GelDT) hydrogels, which displayed exceptional long‐term stability in cell culture media (>28 days) to support the growth of both surface‐seeded and encapsulated cells. GelDT hydrogels allowed pre‐gelation tuning of biomechanical properties and biodegradation via introducing physical crosslinks, and post‐gelation tuning of matrix stress‐relaxation rate, via responding to exogenous thiols, independently of other parameters. Furthermore, GelDT enabled covalent immobilization of bio‐active molecules, glutathione‐responsive drug release, supported efficient 3D bioprinting due to its shear‐thinning ability, and demonstrated robust tissue adhesion in various contexts (bare skin, ex‐vivo, in‐vivo) due to covalent disulfide coupling with endogenous tissue thiols. Together, this study presents a novel multi‐responsive and multi‐functional protein‐based biomaterial, anticipated to advance tissue engineering and regeneration.

The challenge of treating corneal scarring through keratoplasties lies in the limited availability of donor tissue. Various studies have shown the therapeutic use of cultivated corneal stromal stem cells (CSSCs) to mitigate tissue inflammation and suppress fibrosis and scar tissue formation in preclinical corneal wound models. To develop CSSC therapy for clinical trials on patients with corneal scarring, it is necessary to generate clinical-grade CSSCs in compliant to Good Manufacturing Practice (GMP) regulations. This chapter elucidates human CSSC isolation, culture, and cryopreservation under GMP-compliant conditions. It underscores quality assessment encompassing morphological traits, expression of stemness markers, anti-inflammatory activity, and keratocyte differentiation potency.

The cornea is a transparent and vitally multifaceted component of the eye, playing a pivotal role in vision and ocular health. It has primary refractive and protective functions. Typical corneal dysfunctions include opacities and deformities that result from injuries, infections, or other medical conditions. These can significantly impair vision. The conventional challenges in managing corneal ailments include the limited regenerative capacity (except corneal epithelium), immune response after donor tissue transplantation, a risk of long-term graft rejection, and the global shortage of transplantable donor materials. This review delves into the intricate composition of the cornea, the landscape of corneal regeneration, and the multifaceted repercussions of scar-related pathologies. It will elucidate the etiology and types of dysfunctions, assess current treatments and their limitations, and explore the potential of regenerative therapy that has emerged in both in vivo and clinical trials. This review will shed light on existing gaps in corneal disorder management and discuss the feasibility and challenges of advancing regenerative therapies for corneal stromal scarring.

Background: Mesenchymal stem cells in the adult corneal stroma (named corneal stromal stem cells, CSSCs) inhibit corneal inflammation and scarring and restore corneal clarity in pre-clinical corneal injury models. This cell therapy could alleviate the heavy reliance on donor materials for corneal transplantation to treat corneal opacities. Herein, we established Good Manufacturing Practice (GMP) protocols for CSSC isolation, propagation, and cryostorage, and developed in vitro quality control (QC) metric for in vivo anti-scarring potency of CSSCs in treating corneal opacities. Methods: A total of 24 donor corneal rims with informed consent were used—18 were processed for the GMP optimization of CSSC culture and QC assay development, while CSSCs from the remaining 6 were raised under GMP-optimized conditions and used for QC validation. The cell viability, growth, substrate adhesion, stem cell phenotypes, and differentiation into stromal keratocytes were assayed by monitoring the electric impedance changes using xCELLigence real-time cell analyzer, quantitative PCR, and immunofluorescence. CSSC’s conditioned media were tested for the anti-inflammatory activity using an osteoclastogenesis assay with mouse macrophage RAW264.7 cells. In vivo scar inhibitory outcomes were verified using a mouse model of anterior stromal injury caused by mechanical ablation using an Algerbrush burring. Results: By comparatively assessing various GMP-compliant reagents with the corresponding non-GMP research-grade chemicals used in the laboratory-based protocols, we finalized GMP protocols covering donor limbal stromal tissue processing, enzymatic digestion, primary CSSC culture, and cryopreservation. In establishing the in vitro QC metric, two parameters—stemness stability of ABCG2 and nestin and anti-inflammatory ability (rate of inflammation)—were factored into a novel formula to calculate a Scarring Index (SI) for each CSSC batch. Correlating with the in vivo scar inhibitory outcomes, the CSSC batches with SI < 10 had a predicted 50% scar reduction potency, whereas cells with SI > 10 were ineffective to inhibit scarring. Conclusions: We established a full GMP-compliant protocol for donor CSSC cultivation, which is essential toward clinical-grade cell manufacturing. A novel in vitro QC–in vivo potency correlation was developed to predict the anti-scarring efficacy of donor CSSCs in treating corneal opacities. This method is applicable to other cell-based therapies and pharmacological treatments.

Corneal scarring, a significant cause of global blindness, results from various insults, including trauma, infections, and genetic disorders. The conventional treatment to replace scarred corneal tissues includes partial or full-thickness corneal transplantation using healthy donor corneas. However, only 1 in 70 individuals with treatable corneal scarring can undergo surgery, due to the limited supply of transplantable donor tissue. Our research focuses on cell-based strategies, specifically ex vivo-expanded corneal stromal stem cells (CSSCs), to address corneal scarring. Preclinical studies have demonstrated the efficacy of CSSC treatment in reducing corneal inflammation and fibrosis, inhibiting scar formation, and regenerating native stromal tissue. Mechanisms include CSSC differentiation into stromal keratocytes and the expression of regenerative cytokines. Here, we present a good manufacturing practice (GMP)compliant protocol to isolate and expand human CSSCs. This method paves the way to produce clinical-grade CSSCs for transplantation and clinical trials.

A transparent cornea is paramount for vision. Corneal opacity is one of the leading causes of blindness. Although conventional corneal transplantation has been successful in recovering patients’ vision, the outcomes are challenged by a global lack of donor tissue availability. Bioengineered corneal tissues are gaining momentum as a new source for corneal wound healing and scar management. Extracellular matrix (ECM)-scaffold-based engineering offers a new perspective on corneal regenerative medicine. Ultrathin stromal laminar tissues obtained from lenticule-based refractive correction procedures, such as SMall Incision Lenticule Extraction (SMILE), are an accessible and novel source of collagen-rich ECM scaffolds with high mechanical strength, biocompatibility, and transparency. After customization (including decellularization), these lenticules can serve as an acellular scaffold niche to repopulate cells, including stromal keratocytes and stem cells, with functional phenotypes. The intrastromal transplantation of these cell/tissue composites can regenerate native-like corneal stromal tissue and restore corneal transparency. This review highlights the current status of ECM-scaffold-based engineering with cells, along with the development of drug and growth factor delivery systems, and elucidates the potential uses of stromal lenticule scaffolds in regenerative therapeutics.

Corneal blindness due to scarring is conventionally treated by corneal transplantation, but the shortage of donor materials has been a major issue affecting the global success of treatment. Pre-clinical and clinical studies have shown that cell-based therapies using either corneal stromal stem cells (CSSC) or corneal stromal keratocytes (CSK) suppress corneal scarring at lower levels. Further treatments or strategies are required to improve the treatment efficacy. This study examined a combined cell-based treatment using CSSC and CSK in a mouse model of anterior stromal injury. We hypothesize that the immuno-regulatory nature of CSSC is effective to control tissue inflammation and delay the onset of fibrosis, and a subsequent intrastromal CSK treatment deposited collagens and stromal specific proteoglycans to recover a native stromal matrix. Using optimized cell doses, our results showed that the effect of CSSC treatment for suppressing corneal opacities was augmented by an additional intrastromal CSK injection, resulting in better corneal clarity. These in vivo effects were substantiated by a further downregulated expression of stromal fibrosis genes and the restoration of stromal fibrillar organization and regularity. Hence, a combined treatment of CSSC and CSK could achieve a higher clinical efficacy and restore corneal transparency, when compared to a single CSSC treatment.