Biological materials derived from extracellular matrix (ECM) proteins have garnered interest as their composition is very similar to that of native tissue. Herein, we report the use of human cornea derived decellularized ECM (dECM) microparticles dispersed in human fibrin sealant as an accessible therapeutic alternative for corneal anterior stromal reconstruction. dECM microparticles had good particle size distribution (≤10 µm) and retained the majority of corneal ECM components found in native tissue. Fibrin–dECM hydrogels exhibited compressive modulus of 70.83 ± 9.17 kPa matching that of native tissue, maximum burst pressure of 34.3 ± 3.7 kPa, and demonstrated a short crosslinking time of ~17 min. The fibrin–dECM hydrogels were found to be biodegradable, cytocompatible, non-mutagenic, non-sensitive, non-irritant, and supported the growth and maintained the phenotype of encapsulated human corneal stem cells (hCSCs) in vitro. In a rabbit model of anterior lamellar keratectomy, fibrin–dECM bio-adhesives promoted corneal re-epithelialization within 14 days, induced stromal tissue repair, and displayed integration with corneal tissues in vivo. Overall, our results suggest that the incorporation of cornea tissue-derived ECM microparticles in fibrin hydrogels is non-toxic, safe, and shows tremendous promise as a minimally invasive therapeutic approach for the treatment of superficial corneal epithelial wounds and anterior stromal injuries.

Purpose: Failure of rapid re-epithelialization within 10–14 days after corneal injury, even with standard supportive treatment, is referred to as persistent corneal epithelial (CE) defect (PED). Though an array of genes regulates reepithelization, their mechanisms are poorly understood. We sought to understand the network of genes driving the re-epithelialization in PED. Method: After obtaining informed consent, patients underwent an ophthalmic examination. Epithelial scrapes and tears samples of six PED patients and six individuals (control) undergoing photorefractive keratectomy (PRK) were collected. RNA isolation and quantification were performed using either the epithelial scrape taken from PED patients or from HCLE cells treated with control tears or tears of PED patients. Quantitative real-time polymerase chain reaction (qRT-PCR) was performed to detect the expression of a few important genes in CE homeostasis, inflammation, and cell–cell communication, viz., Kruppel-like factor 4 (KLF4), GPX4, IL6, TNFα, STING, IL8, desmoglein, and E-cadherin, among others. Their expressions were normalized with their respective housekeeping genes and fold changes were recorded. KLF4 localization and MMPs activity was carried out via immunofluorescence and zymography, respectively. Results: KLF4, a transcription factor important for CE homeostasis, was upregulated in tears-treated HCLE cells and downregulated in PED patients compared to the healthy PRK group. Cell–cell communication genes were also upregulated in tears-treated cells, whereas they were downregulated in the PED tissue group. Genes involved in proinflammation (IL6, 282-fold; TNFα, 43-fold; IL8, 4.2-fold) were highly upregulated in both conditions. MMP9 activity increased upon tears treatment. Conclusions: This study suggests that tears create an acute proinflammatory milieu driving the PED disease pathology, whereas the PED patients scrapes are an indicator of the chronic stage of the disease. Interferons, pro-inflammatory genes, and their pathways are involved in PED, which can be a potential target for inducing epithelialization of the cornea.

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Three-dimensional bioprinting (3DBP) is an additive manufacturing technique that has emerged as a promising strategy for the fabrication of scaffolds, which can suc¬cessfully recapitulate the architectural, biochemical, and physical cues of target tis¬sues. More importantly, 3DBP offers fine spatiotemporal control and high submicron scale resolution, which can be leveraged for the incorporation and directional gra¬dient release of single or multiple biomimetic cues, including cell-derived exosomes (EXOs). EXOs are extracellular vesicles that originate from the endosomal compart¬ment of various cell types, with sizes ranging from 30 to120 nm. They act as cell medi¬ators and contain discrete cell constituents, including growth factors, cytokines, lipid moieties, nucleic acids, metabolites, and cell surface markers, depending on the cell type. Essentially, owing to their therapeutic potential, EXOs derived from mesenchy¬mal stem cells (MSCs) have been recently investigated in several clinical trials for the treatment of various conditions, including cancer, diabetes, dry eyes, periodontitis, and acute ischemic stroke. The 3DBP strategy of EXOs is especially useful in tissue engineering and regenerative medicine applications, as tissues can be biofabricat¬ed to closely mimic the complex microarchitecture and developmental profiles of native heterogeneous tissues for restoring biological functions. Moreover, EXOs can be manipulated to carry exogenous cargo such as genes or proteins of therapeutic interest, confer multifunctional attributes, and further enhance their tissue regenera¬tive potential. However, significant challenges, including the selection of appropriate bioink, pattern resolution, engineering-defined exosomal gradient, spatial presen¬tation and modulation of EXO release kinetics, as well as EXO stability and storage conditions, must be addressed for the successful translation of therapeutic grade EXOs to clinical settings. In this review, we highlight the recent advances and offer future perspectives on the bioprinting of EXOs as regenerative biotherapeutics for the fabrication of complex heterogeneous tissues that are suitable for clinical trans¬plantation.