Collagen Gels Crosslinked by Photoactivation of Riboflavin for the Repair and Regeneration of Corneal Defects.

Bioengineered corneal tissue is a promising therapeutic modality for the treatment of corneal blindness as a substitute for cadaveric graft tissue. In this study, we fabricated a collagen gel using ultraviolet-A (UV-A) light and riboflavin as a photosensitizer (PhotoCol-RB) as an in situ-forming matrix to fill corneal wounds and create a cohesive interface between the crosslinked gel and adjacent collagen. The PhotoCol-RB gels supported corneal epithelialization and exhibited higher transparency compared to physically crosslinked collagen. We showed that different riboflavin concentrations yielded gels with different mechanical and biological properties. In vitro experiments using human corneal epithelial cells (hCECs) showed that hCECs are able to proliferate on the gel and express corneal cell markers such as cytokeratin 12 (CK12) and tight junctions (ZO-1). Using an ex vivo burst assay, we also showed that the PhotoCol-RB gels are able to seal corneal perforations. Ex vivo organ culture of the gels filling lamellar keratectomy wounds showed that the epithelium that regenerated over the PhotoCol-RB gels formed a multilayer compared to just a double layer for those that grew over physically cross-linked collagen. These gels can be formed either in situ directly on the wound site to conform to the geometry of a defect, or can be preformed and then applied to the corneal wound. Our results indicate that PhotoCol-RB gels merit further investigation as a way to stabilize and repair deep and perforating corneal wounds.

Clinical outcomes of intravitreal treatment for ocular toxoplasmosis: systematic review and meta-analysis.

BACKGROUND: Ocular toxoplasmosis is the leading cause of infectious posterior uveitis worldwide, accounting for 30-50% of all cases in immunocompetent patients. Conventional treatment is associated with adverse effects and does not prevent recurrence. Intravitreal drug administration can improve disease outcomes and reduce side effects. Herein, we conducted a systematic review and meta-analysis on the efficacy of intravitreal injections for treating ocular toxoplasmosis. METHODS: The systematic search was conducted using PubMed, SciELO, and Google Scholar with the descriptors "ocular toxoplasmosis" AND "intravitreal". We analyzed studies that met the inclusion criteria, i.e., experimental cases in patients treated intravitreally for ocular toxoplasmosis. Considering the systematic review, we focused on the number of intravitreal injections, the therapeutic drug class, and the presence of preexisting conditions. To assess the efficacy of intravitreal injections, a meta-analysis was performed using visual acuity, side effects, disease recurrence, and inflammatory responses as variables. RESULTS: Intravitreal injection-induced side effects were rarely observed (0.49% [0.00, 1.51%] ). The use of antiparasitic and anti-inflammatory drugs afforded improved visual acuity (99.81% [98.60, 100.00%]) and marked effectiveness in treating ocular toxoplasmosis. CONCLUSIONS: Intravitreal injections may facilitate the successful treatment of ocular toxoplasmosis. However, clinicians should carefully evaluate the presence of preexisting conditions for ocular toxoplasmosis or previous diseases, as these can impact the decision to administer intravitreal injections.

In Situ-forming Collagen Hydrogels Crosslinked by Multifunctional Polyethylene Glycol as a Matrix Therapy for Corneal Defects: 2-Month Follow-up In Vivo.

PURPOSE: We recently showed that in situ-forming collagen gels crosslinked through multifunctional polyethylene glycol (PEG) supported corneal epithelialization 7 days after treatment of lamellar keratectomy wounds. In this study, we aimed to evaluate the longer-term regenerative effects of this gel in animals. METHOD: Corneal wound healing was assessed 60 days after lamellar keratectomy and gel treatment using slitlamp examination, optical coherence tomography (OCT), pachymetry, corneal topography, an ocular response analyzer, and tonometry. The corneas were evaluated for the presence of beta-tubulin, cytokeratin 3, zonula occludens-1, and alpha smooth muscle actin (SMA) markers. Gene expression of aldehyde dehydrogenase 3A1 (ALDH3A1), cluster of differentiation 31, CD163, alpha-SMA, hepatocyte growth factor, and fibroblast growth factor 2 (FGF-2) and protein expression of CD44 and collagen VI were evaluated. RESULTS: Intraocular pressure, corneal thickness, and hysteresis for the corneas treated with collagen-PEG gels did not significantly change compared with the saline group. However, placido disk topography revealed greater regularity of the central cornea in the gel-treated group compared to the saline group. The gel-treated group exhibited a lower degree of epithelial hyperplasia than the saline group. Immunohistochemical and gene expression analysis showed that the gel-treated corneas exhibited lower alpha-SMA expression compared with the saline group. CD163 and CD44 were found to be elevated in the saline-treated group compared with normal corneas. CONCLUSIONS: The in situ-forming collagen-PEG gel promoted epithelialization that improved central corneal topography, epithelial layer morphology, and reduced expression of fibrotic and inflammatory biomarkers after 60 days compared to the saline group.

Clinical outcomes of intravitreal treatment for ocular toxoplasmosis: systematic review and meta-analysis

ABSTRACT Background: Ocular toxoplasmosis is the leading cause of infectious posterior uveitis worldwide, accounting for 30-50% of all cases in immunocompetent patients. Conventional treatment is associated with adverse effects and does not prevent recurrence. Intravitreal drug administration can improve disease outcomes and reduce side effects. Herein, we conducted a systematic review and meta-analysis on the efficacy of intravitreal injections for treating ocular toxoplasmosis. Methods: The systematic search was conducted using PubMed, SciELO, and Google Scholar with the descriptors "ocular toxoplasmosis" AND "intravitreal". We analyzed studies that met the inclusion criteria, i.e., experimental cases in patients treated intravitreally for ocular toxoplasmosis. Considering the systematic review, we focused on the number of intravitreal injections, the therapeutic drug class, and the presence of preexisting conditions. To assess the efficacy of intravitreal injections, a meta-analysis was performed using visual acuity, side effects, disease recurrence, and inflammatory responses as variables. Results: Intravitreal injection-induced side effects were rarely observed (0.49% [0.00, 1.51%] ). The use of antiparasitic and anti-inflammatory drugs afforded improved visual acuity (99.81% [98.60, 100.00%]) and marked effectiveness in treating ocular toxoplasmosis. Conclusions: Intravitreal injections may facilitate the successful treatment of ocular toxoplasmosis. However, clinicians should carefully evaluate the presence of preexisting conditions for ocular toxoplasmosis or previous diseases, as these can impact the decision to administer intravitreal injections.

In Situ-Forming Collagen-Hyaluronate Semi-Interpenetrating Network Hydrogel Enhances Corneal Defect Repair.

Purpose: Millions worldwide suffer vision impairment or blindness from corneal injury, and there remains an urgent need for a more effective and accessible way to treat corneal defects. We have designed and characterized an in situ-forming semi-interpenetrating polymer network (SIPN) hydrogel using biomaterials widely used in ophthalmology and medicine. Methods: The SIPN was formed by cross-linking collagen type I with bifunctional polyethylene glycol using N-hydroxysuccinimide ester chemistry in the presence of linear hyaluronic acid (HA). Gelation time and the mechanical, optical, swelling, and degradation properties of the SIPN were assessed. Cytocompatibility with human corneal epithelial cells and corneal stromal stem cells (CSSCs) was determined in vitro, as was the spatial distribution of encapsulated CSSCs within the SIPN. In vivo wound healing was evaluated by multimodal imaging in an anterior lamellar keratectomy injury model in rabbits, followed by immunohistochemical analysis of treated and untreated tissues. Results: The collagen-hyaluronate SIPN formed in situ without an external energy source and demonstrated mechanical and optical properties similar to the cornea. It was biocompatible with human corneal cells, enhancing CSSC viability when compared with collagen gel controls and preventing encapsulated CSSC sedimentation. In vivo application of the SIPN significantly reduced stromal defect size compared with controls after 7 days and promoted multilayered epithelial regeneration. Conclusions: This in situ-forming SIPN hydrogel may be a promising alternative to keratoplasty and represents a step toward expanding treatment options for patients suffering from corneal injury. Translational Relevance: We detail the synthesis and initial characterization of an SIPN hydrogel as a potential alternative to lamellar keratoplasty and a tunable platform for further development in corneal tissue engineering and therapeutic cell delivery.

Supramolecular host-guest hyaluronic acid hydrogels enhance corneal wound healing through dynamic spatiotemporal effects.

Severe corneal wounds can lead to ulceration and scarring if not promptly and adequately treated. Hyaluronic acid (HA) has been investigated for the treatment of corneal wounds due to its remarkable biocompatibility, transparency and mucoadhesive properties. However, linear HA has low retention time on the cornea while many chemical moieties used to crosslink HA can cause toxicity, which limits their clinical ocular applications. Here, we used supramolecular non-covalent host-guest interactions between HA-cyclodextrin and HA-adamantane to form shear-thinning HA hydrogels and evaluated their impact on corneal wound healing. Supramolecular HA hydrogels facilitated adhesion and spreading of encapsulated human corneal epithelial cells ex vivo and improved corneal wound healing in vivo as an in situ-formed, acellular therapeutic membrane. The HA hydrogels were absorbed within the corneal stroma over time, modulated mesenchymal cornea stromal cell secretome production, reduced cellularity and inflammation of the anterior stroma, and significantly mitigated corneal edema compared to treatment with linear HA and untreated control eyes. Taken together, our results demonstrate supramolecular HA hydrogels as a promising and versatile biomaterial platform for corneal wound healing.

Use of a slow-release intravitreal clindamycin implant for the management of ocular toxoplasmosis.

PURPOSE: To report the first patient with ocular toxoplasmosis treated with a slow-release biodegradable intravitreal clindamycin implant. OBSERVATIONS: A 39-year-old human immunodeficiency virus (HIV)-positive woman with recurrent toxoplasmic retinochoroiditis and vitritis for whom oral medication was medically contraindicated was treated with an intravitreal slow-release clindamycin implant and three monthly intravitreal injections of clindamycin and dexamethasone. Serial ophthalmologic examinations demonstrated gradual, complete resolution of posterior uveitis and healing of the retinochoroidal lesion with cicatricial changes, as well as gradual improvement of cells in the anterior chamber. There was no significant change in electroretinography waves after treatment with the implant. The presence of the implant, or part of it, was detectable in the vitreous cavity for 4 months. To date, the patient has been monitored for 30 months, and there has been no reactivation of ocular toxoplasmosis. CONCLUSION: The slow-release clindamycin implant was safe for intravitreal use in this patient and may have contributed to the long-term control of toxoplasmosis chorioretinitis.

Effect of mesenchymal stromal cells encapsulated within polyethylene glycol-collagen hydrogels formed in situ on alkali-burned corneas in an ex vivo organ culture model.

BACKGROUND AIMS: Corneal inflammation after alkali burns often results in vision loss due to corneal opacification and neovascularization. Mesenchymal stem cells (MSCs) and their secreted factors (secretome) have been studied for their anti-inflammatory and anti-angiogenic properties with encouraging results. However, topical instillation of MSCs or their secretome is often accompanied by issues related to delivery or rapid washout. Polyethylene glycol (PEG) and collagen are well-known biomaterials used extensively in scaffolds for tissue engineering. To effectively suppress alkaline burn-induced corneal injury, the authors proposed encapsulating MSCs within collagen gels cross-linked with multi-functional PEG-succinimidyl esters as a means to deliver the secretome of immobilized MSCs. METHODS: Human MSCs were added to a neutralized collagen solution and mixed with a solution of four-arm PEG-N-hydroxysuccinimide. An ex vivo organ culture was conducted using rabbit corneas injured by alkali burn. MSCs were encapsulated within PEG-collagen hydrogels and injected onto the wounded cornea immediately following alkali burn and washing. Photographs of the ocular surface were taken over a period of 7 days after the alkali burn and processed for immunohistochemical evaluation. Samples were split into three groups: injury without treatment, MSCs alone, and MSCs encapsulated within PEG-collagen hydrogels. RESULTS: All corneas in ex vivo organ culture lost their transparency immediately after alkali burn, and only the groups treated with MSCs and MSCs encapsulated within PEG-collagen hydrogels recovered some transparency after 7 days. Immunohistochemical analysis revealed increased expression of vimentin in the anterior corneal stroma of the group without treatment indicative of fibrotic healing, whereas less stromal vimentin was detected in the group containing MSCs encapsulated within the PEG-collagen hydrogels. CONCLUSIONS: PEG-collagen hydrogels enable the encapsulation of viable MSCs capable of releasing secreted factors onto the ocular surface. Encapsulating MSCs within PEG-collagen hydrogels may be a promising method for delivering their therapeutic benefits in cases of ocular inflammatory diseases, such as alkali burn injuries.

In situ-forming collagen hydrogel crosslinked via multi-functional PEG as a matrix therapy for corneal defects.

Visually significant corneal injuries and subsequent scarring collectively represent a major global human health challenge, affecting millions of people worldwide. Unfortunately, less than 2% of patients who could benefit from a sight-restoring corneal transplant have access to cadaveric donor corneal tissue. Thus, there is a critical need for new ways to repair corneal defects that drive proper epithelialization and stromal remodeling of the wounded area without the need for cadeveric donor corneas. Emerging therapies to replace the need for donor corneas include pre-formed biosynthetic buttons and in situ-forming matrices that strive to achieve the transparency, biocompatibility, patient comfort, and biointegration that is possible with native tissue. Herein, we report on the development of an in situ-forming hydrogel of collagen type I crosslinked via multi-functional polyethylene glycol (PEG)-N-hydroxysuccinimide (NHS) and characterize its biophysical properties and regenerative capacity both in vitro and in vivo. The hydrogels form under ambient conditions within minutes upon mixing without the need for an external catalyst or trigger such as light or heat, and their transparency, degradability, and stiffness are modulated as a function of number of PEG arms and concentration of PEG. In addition, in situ-forming PEG-collagen hydrogels support the migration and proliferation of corneal epithelial and stromal cells on their surface. In vivo studies in which the hydrogels were formed in situ over stromal keratectomy wounds without sutures showed that they supported multi-layered surface epithelialization. Overall, the in situ forming PEG-collagen hydrogels exhibited physical and biological properties desirable for a corneal stromal defect wound repair matrix that could be applied without the need for sutures or an external trigger such as a catalyst or light energy.

Characterizing the impact of 2D and 3D culture conditions on the therapeutic effects of human mesenchymal stem cell secretome on corneal wound healing in vitro and ex vivo.

The therapeutic effects of secreted factors (secretome) produced by bone marrow-derived human mesenchymal stem cells (MSCs) were evaluated as a function of their growth in 2D culture conditions and on 3D electrospun fiber scaffolds. Electrospun fiber scaffolds composed of polycaprolactone and gelatin were fabricated to provide a 3D microenvironment for MSCs, and their mechanical properties were optimized to be similar to corneal tissue. The secretome produced by the MSCs cultured on 3D fiber matrices versus 2D culture dishes were analyzed using a Luminex immunoassay, and the secretome of MSCs cultured on the 3D versus 2D substrates showed substantial compositional differences. Concentrations of factors such as HGF and ICAM-1 were increased over 5 times in 3D cultures compared to 2D cultures. In vitro proliferation and scratch-based wound healing assays were performed to compare the effects of the secretome on corneal fibroblast cells (CFCs) when delivered synchronously from co-cultured MSCs through a trans-well co-culture system versus asynchronously after harvesting the factors separately and adding them to the media. Cell viability of CFCs was sustained for 6 days when co-cultured with MSCs seeded on the fibers but decreased with time under other conditions. Scratch assays showed 95% closure at 48 h when CFCs were co-cultured with MSCs seeded on fibers, while the control group only exhibited 50% closure at 48 h. Electrospun fibers seeded with MSCs were then applied to a rabbit corneal organ culture system, and MSCs seeded on fibers promoted faster epithelialization and less scarring. Corneas were fixed and stained for alpha smooth muscle actin (α-SMA), and then analyzed by confocal microscopy. Immunostaining showed that expression of α-SMA was lower in corneas treated with MSCs seeded on fibers, suggesting suppression of myofibroblastic transformation. MSCs cultured on electrospun fibers facilitate wound healing in CFCs and on explanted corneas through differential secretome profiles compared to MSCs cultured on 2D substrates. Future work is merited to further understand the nature and basis of these differences and their effects in animal models. STATEMENT OF SIGNIFICANCE: Previous studies have shown that the secretome of bone marrow-derived mesenchymal stem cells (MSC) is promotes corneal wound healing by facilitating improved wound closure rates and reduction of scarring and neovascularization. The present research is significant because it provides evidence for the modulation of the secretome as a function of the MSC culture environment. This leads to differential expression of therapeutic factors secreted, which can impact corneal epithelial and stromal healing after severe injury. In addition, this article shows that co-continuous delivery of the MSC secretome improves cell migration and proliferation over aliquoted delivery, and that MSCs grown on three-dimensional electrospun fiber constructs may provide a favorable microenvironment for cultured MSCs and as a carrier to deliver their secreted factors to the ocular surface.