fabrication-surgical-application-enriched-photo-crosslinked

Fabrication and Surgical Application of Enriched Photo-crosslinked Gelatin&#8211;Riboflavin Hydrogels for Corneal Wound Repair in Rabbits

This article presents a step&#8209;by&#8209;step protocol to fabricate, supplement, and surgically apply an in situ blue&#8209;light&#8211;crosslinkable ...

University of the Basque Country UPV/EHU

IIS Biobizkaia

The aim of this study is to assess if an adhesive biopolymer, sodium hyaluronate (NaHA), has synergistic effects with s-PRGF (a serum derived from plasma rich in growth factors and a blood derivative that has already shown efficacy in corneal epithelial wound healing), to reduce time of healing or posology. In vitro proliferation and migration studies, both in human corneal epithelial (HCE) cells and in rabbit primary corneal epithelial (RPCE) cultures, were carried out. In addition, we performed studies of corneal wound healing in vivo in rabbits treated with s-PRGF, NaHA, or the combination of both. We performed immunohistochemistry techniques (CK3, CK15, Ki67, ß4 integrin, ZO-1, α-SMA) in rabbit corneas 7 and 30 days after a surgically induced epithelial defect. In vitro results show that the combination of NaHA and s-PRGF offers the worst proliferation rates in both HCE and RPCE cells. Addition of NaHA to s-PRGF diminishes the re-epithelializing capability of s-PRGF. In vivo, all treatments, given twice a day, showed equivalent efficacy in corneal epithelial healing. We conclude that the combined use of s-PRGF and HaNA as an adhesive biopolymer does not improve the efficacy of s-PRGF alone in the wound healing of corneal epithelial defects.

Hyaluronic Acid Combined with Serum Rich in Growth Factors in Corneal Epithelial Defects.

Transplantation of human cultured limbal epithelial stem/progenitor cells (LESCs) has demonstrated to restore the integrity and functionality of the corneal surface in about 76% of patients with limbal stem cell deficiency. However, there are different protocols for the expansion of LESCs, and many of them use xenogeneic products, being a risk for the patients' health. We compared the culture of limbal explants on the denuded amniotic membrane in the culture medium-supplemental hormone epithelial medium (SHEM)-supplemented with FBS or two differently produced human sera. Cell morphology, cell size, cell growth rate, and the expression level of differentiation and putative stem cell markers were examined. Several bioactive molecules were quantified in the human sera. In a novel approach, we performed a multivariate statistical analysis of data to investigate the culture factors, such as differently expressed molecules of human sera that specifically influence the cell phenotype. Our results showed that limbal cells cultured with human sera grew faster and contained similar amounts of small-sized cells, higher expression of the protein p63α, and lower of cytokeratin K12 than FBS cultures, thus, maintaining the stem/progenitor phenotype of LESCs. Furthermore, the multivariate analysis provided much data to better understand the obtaining of different cell phenotypes as a consequence of the use of different culture methodologies or different culture components.

Expansion of Human Limbal Epithelial Stem/Progenitor Cells Using Different Human Sera: A Multivariate Statistical Analysis.

The vision impairments suffered by millions of people worldwide and the shortage of corneal donors show the need of substitutes that mimic native tissue to promote cell growth and subsequent tissue regeneration. The current study focused on the in vitro assessment of protein-based biomaterials that could be a potential source for corneal scaffolds. Collagen, soy protein isolate (SPI), and gelatin films cross-linked with lactose or citric acid were prepared and physicochemical, transmittance, and degradation measurements were carried out. In vitro cytotoxicity, cell adhesion, and migration studies were performed with human corneal epithelial (HCE) cells and 3T3 fibroblasts for the films' cytocompatibility assessment. Transmittance values met the cornea's needs, and the degradation profile revealed a progressive biomaterials' decomposition in enzymatic and hydrolytic assays. Cell viability at 72 h was above 70% when exposed to SPI and gelatin films. Live/dead assays and scanning electron microscopy (SEM) analysis demonstrated the adhesion of both cell types to the films, with a similar arrangement to that observed in controls. Besides, both cell lines were able to proliferate and migrate over the films. Without ruling out any material, the appropriate optical and biological properties shown by lactose-crosslinked gelatin film highlight its potential for corneal bioengineering.

Cytocompatibility and Suitability of Protein-Based Biomaterials as Potential Candidates for Corneal Tissue Engineering.

The transplantation of expansions of limbal epithelial stem cells (LESC) remains one of the most efficient therapies for the treatment of limbal stem cell deficiency (LSCD) to date. However, the available donor corneas are scarce, and the corneas conserved for long time, under hypothermic conditions (after 7 days) or in culture (more than 28 days), are usually discarded due to poor viability of the endothelial cells. To establish an objective criterion for the utilisation or discarding of corneas as a source of LESC, we characterized, by immunohistochemistry analysis, donor corneas conserved in different conditions and for different periods of time. We also studied the potency of LESCs isolated from these corneas and maintained in culture up to 3 cell passages. We hoped that the study of markers of LESCs present in both the corneoscleral histological sections and the cell cultures would show the adequacy of the methods used for cell isolation and how fit the LESC enrichment of the obtained cell populations to be expanded was. Thus, the expressions of markers of the cells residing in the human limbal and corneal epithelium (cytokeratin CK15 and CK12, vimentin, Collagen VII, p63α, ABCG2, Ki67, Integrin β4, ZO1, and melan A) were analysed in sections of corneoscleral tissues conserved in hypothermic conditions for 2-9 days with post-mortem time (pmt) < 8 h or for 1 day with pmt > 16 h, and in sclerocorneal rims maintained in an organ culture medium for 29 days. Cell populations isolated from donor corneoscleral tissues were also assessed based on these markers to verify the adequacy of isolation methods and the potential of expanding LESCs from these tissues. Positivity for several putative stem cell markers such as CK15 and p63α was detected in all corneoscleral tissues, although a decrease was recorded in the ones conserved for longer times. The barrier function and the ability to adhere to the extracellular matrix were maintained in all the analysed tissues. In limbal epithelial cell cultures, a simultaneous decrease in the melan A melanocyte marker and the putative stem cell markers was detected, suggesting a close relationship between the melanocytes and the limbal stem cells of the niche. Holoclones stained with putative stem cell markers were obtained from long-term, hypothermic, stored sclerocorneal rims. The results showed that the remaining sclerocorneal rims after corneal transplantation, which were conserved under hypothermic conditions for up to 7 days and would have been discarded at a first glance, still maintained their potential as a source of LESC cultures.

Characterisation of corneas following different time and storage methods for their use as a source of stem-like limbal epithelial cells.

Extrusion bioprinting based on the development of novel bioinks offers the possibility of manufacturing clinically useful tools for wound management. In this study, we show the rheological properties and printability outcomes of two advanced dressings based on platelet-rich plasma (PRP) and platelet-poor plasma (PPP) blended with alginate and loaded with dermal fibroblasts. Measurements taken at 1 h, 4 days, and 18 days showed that both the PRP- and PPP-based dressings retain plasma and platelet proteins, which led to the upregulation of angiogenic and immunomodulatory proteins by embedded fibroblasts (e.g., an up to 69-fold increase in vascular endothelial growth factor (VEGF), an up to 188-fold increase in monocyte chemotactic protein 1 (MCP-1), and an up to 456-fold increase in hepatocyte growth factor (HGF) 18 days after printing). Conditioned media harvested from both PRP and PPP constructs stimulated the proliferation of human umbilical vein endothelial cells (HUVECs), whereas only those from PRP dressings stimulated HUVEC migration, which correlated with the VEGF/MCP-1 and VEGF/HGF ratios. Similarly, the advanced dressings increased the level of interleukin-8 and led to a four-fold change in the level of extracellular matrix protein 1. These findings suggest that careful selection of plasma formulations to fabricate wound dressings can enable regulation of the molecular composition of the microenvironment, as well as paracrine interactions, thereby improving the clinical potential of dressings and providing the possibility to tailor each composition to specific wound types and healing stages.

Plasma-Based Bioinks for Extrusion Bioprinting of Advanced Dressings.

Bilastine is a non-sedating, highly selective H1-antihistamine with proven efficacy and safety in treating allergic rhinoconjunctivitis and urticaria in adults and children. Allergic conjunctivitis, a common ocular condition, negatively impacts quality of life. Topical eye drops are the standard treatment, though ocular bioavailability is often low. Incorporating biopolymers such as hyaluronic acid (HA) into topical formulations enhances adhesive properties, prolongs retention on the ocular surface, and ultimately improves drug bioavailability. This study evaluated the new multidose preservative-free bilastine 0.6% solution with sodium HA against eight commercially available antiallergic eye drops. Using an ex vivo bovine cornea model, bilastine 0.6% demonstrated the highest bioadhesion strength (0.025 mJ), indicating superior retention on the ocular surface. It also showed strong protective effects against in vitro dehydration, mainly due to the presence of HA, and did not exhibit cytotoxicity in human primary conjunctival cells. In wound healing assays, preservative-free ketotifen 0.025%, bilastine 0.6%, and azelastine 0.05% promoted corneal wound repair at 72 h, outperforming preserved formulations. Overall, preservative-free bilastine 0.6% with HA enhances corneal hydration, retention, and re-epithelialization in vitro, suggesting potential benefits for the management of allergic conjunctivitis and offering promising advancements in treating this widespread condition.

The new bilastine eye drop formulation protects against conjunctival dehydration and promotes corneal wound healing in a comparative in vitro study.

Corneal persistent epithelial defects are common ophthalmic injuries that can cause significant visual and structural damage. While diagnosis is straightforward, treatment remains challenging. Noninvasive therapies like eye drops are preferred, but severe neurotrophic keratopathy may require surgical interventions. This study explores gelatin-based hydrogels as noninvasive alternatives for corneal repair. Four photo-cross-linkable hydrogels with gelatin and riboflavin phosphate (RFP) were evaluated: a control and variants incorporating 2.5% dextran (D), 0.4% hyaluronic acid (HA), or 1% methylcellulose (MC). In vitro assessments included physicochemical properties, biocompatibility, and release kinetics alongside ex vivo wound healing assays. The gelatin-RFP hydrogel maintained corneal transparency, while additives reduced it. Dextran slowed compound release, and HA and MC reduced the release rate of larger molecules. All hydrogels showed excellent biocompatibility, and ex vivo models confirmed re-epithelialization, though slower than controls. The unmodified gelatin-RFP hydrogel demonstrated the best potential for corneal tissue engineering, supporting its future clinical translation.

In Vitro Evaluation of Gelatin-Based Hydrogels as Potential Fillers for Corneal Wounds.

The objective of this study was to develop a reliable, cost-effective, and rapid in vitro model employing real-time PCR to assess inflammatory responses in hydrogel-based systems, and to comparatively evaluate the anti-inflammatory efficacy of human serum (HS), serum derived from plasma rich in growth factors (sPRGF), and human amniotic membrane extracts (HAMe) incorporated into gelatin-based hydrogels. An in vitro model of corneal inflammation was established by quantifying IL-1β expression via qPCR in TNFα-stimulated SV-40 immortalised human corneal epithelial (HCE) cells. Hydrogels functionalised with HS, sPRGF, or HAMe sourced from proximal, medial, distal, or pooled amniotic regions were evaluated for their anti-inflammatory potential. In vivo validation was conducted in a rabbit anterior stromal keratectomy model, assessing epithelial wound closure and clinical signs of irritation following application of unmodified hydrogels or hydrogels functionalised with autologous serum (AS) or HAMe. In vitro, hydrogels incorporating HS, followed by sPRGF and pooled HAMe, significantly attenuated IL-1β expression, whereas unmodified hydrogels exacerbated the inflammatory response; region-specific HAMe hydrogels demonstrated inconsistent effects. In vivo, AS-functionalised hydrogels facilitated complete re-epithelialisation by day 7 and achieved the lowest irritation scores, indicating both therapeutic efficacy and high tolerability. All hydrogel formulations were found to be biocompatible throughout the study period. These findings underscore the significant anti-inflammatory and regenerative potential of gelatin-based hydrogels functionalised with blood-derived products and support their development as bioactive platforms for ocular surface therapy. Furthermore, the in vitro model provides a robust preclinical screening tool, contributing to the refinement and reduction of animal use in biomedical research.

An in vitro pre-screening model to evaluate the corneal anti-inflammatory effect of human blood-derived products and amniotic membrane extracts incorporated into gelatine-based hydrogels.

Persistent epithelial defects (PEDs) and chronic corneal ulcers are lesions resistant to treatment for over 2 weeks, risking inadequate healing, reduced sensitivity, and corneal lysis or perforation. This study evaluates the regenerative potential of functionalized gelatin-based hydrogels for treating rabbit corneal wounds as a non-surgical alternative.

Article	Year
Hyaluronic Acid Combined with Serum Rich in Growth Factors in Corneal Epithelial Defects. International journal of molecular sciences\| PubMed ID: 30987108	2019
Expansion of Human Limbal Epithelial Stem/Progenitor Cells Using Different Human Sera: A Multivariate Statistical Analysis. International journal of molecular sciences\| PubMed ID: 32854428	2020
Cytocompatibility and Suitability of Protein-Based Biomaterials as Potential Candidates for Corneal Tissue Engineering. International journal of molecular sciences\| PubMed ID: 33807473	2021
Characterisation of corneas following different time and storage methods for their use as a source of stem-like limbal epithelial cells. Experimental eye research\| PubMed ID: 34389315	2021
Plasma-Based Bioinks for Extrusion Bioprinting of Advanced Dressings. Biomedicines\| PubMed ID: 34440227	2021
The new bilastine eye drop formulation protects against conjunctival dehydration and promotes corneal wound healing in a comparative in vitro study. Scientific reports\| PubMed ID: 40055363	2025
In Vitro Evaluation of Gelatin-Based Hydrogels as Potential Fillers for Corneal Wounds. Biomacromolecules\| PubMed ID: 40079491	2025
An in vitro pre-screening model to evaluate the corneal anti-inflammatory effect of human blood-derived products and amniotic membrane extracts incorporated into gelatine-based hydrogels. Experimental eye research\| PubMed ID: 40889613	2025
Comparison of the regenerative potential of different functionalized gelatin-based hydrogels as fillers of rabbit corneal wounds. Frontiers in medicine\| PubMed ID: 41070062	2025

Noelia Andollo

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