Hossein Aghamollaei

This review examines the potential of Wharton’s jelly–derived mesenchymal stem cells (WJ-MSCs) as regenerative therapies for corneal ectasia, with an emphasis on keratoconus. Current standards, including corneal cross-linking, intrastromal corneal ring segments, and keratoplasty, stabilize or reshape the cornea but do not restore native extracellular matrix (ECM) architecture or correct underlying cellular dysfunction. WJ-MSCs offer a scalable, ethically accessible cell source with high proliferative capacity, low immunogenicity, and robust paracrine activity. Preclinical studies suggest that WJ-MSCs may acquire corneal epithelial- and keratocyte-like characteristics under in vitro induction conditions and may promote epithelial healing, reduce inflammation and haze, and improve stromal organization and biomechanics, collectively supporting their potential role in corneal regeneration. Early clinical experiences with MSC-based therapies in ocular surface disease indicate favorable safety and preliminary evidence of efficacy, while a phase 1b clinical trial of locally delivered allogeneic bone marrow–derived MSCs supports the feasibility and short-term safety of ocular MSC administration, providing indirect translational support for future WJ-MSC studies. Mechanistically, WJ-MSCs act via coordinated paracrine signaling and extracellular vesicles, immunomodulation (TGF-β, IL-10, IDO, HLA-G), anti-fibrotic effects, ECM remodeling, and potential neurotrophic support. Cell-free derivatives (secretome/EVs) may replicate therapeutic benefits with simplified manufacturing and repeat dosing. Key challenges include optimizing dose, route, and timing; enhancing intrastromal persistence and potency (e.g., via biomaterials and preconditioning); and standardizing Good Manufacturing Practice (GMP) production. Rigorous, multicenter randomized trials with harmonized imaging, biomechanical, and patient-reported outcomes are needed to confirm durability and clinical benefit, including in combination strategies with cross-linking. The regulatory pathway also requires early engagement with relevant regulatory agencies, science-based chemistry, manufacturing, and controls (CMC), standardized product characterization, validated potency assays, ethical umbilical-cord sourcing, and appropriate preclinical toxicology before clinical implementation. With disciplined clinical development, WJ-MSC-based therapies, including cells or cell-free products, could evolve into disease-modifying options that complement existing care and, in selected cases, reduce reliance on keratoplasty.

Persistent corneal epithelial defects (PEDs) and microbial keratitis are major contributors to vision loss. Current treatments, including topical eye drops, are constrained by low bioavailability and inadequate patient adherence. This study introduces a multifunctional, biocompatible contact lens composed of gelatin (G), amniotic membrane (AM), and the antimicrobial peptide CM11 (P). It is specifically developed to promote corneal healing and provide sustained drug delivery. Three hydrogel scaffolds (G, G-AM, and G-AM-P) were fabricated and systematically characterized, with emphasis on peptide release dynamics, antibacterial performance, and cytocompatibility, as these parameters are central to their therapeutic functionality. In vivo efficacy and safety were evaluated in a rabbit model using slit-lamp examination, anterior segment optical coherence tomography, and histopathological analysis. G-AM-P demonstrated acceptable optical clarity, significant porosity (~ 94%), moderate hydrophilicity (contact angle ≈ 70°), and an elastic modulus of ~ 0.55 MPa. Also, a biphasic release characterized by an initial rapid phase followed by plateau for up to 72 h. G-AM-P inhibited Staphylococcus aureus with a 15 mm zone of inhibition. This scaffold showed no cytotoxicity on the human corneal epithelial cells with viability exceeding 80%, after 72 h. In rabbits, G-AM-P exhibited ocular tolerance, facilitated re-epithelialization, and did not provoke inflammation, neovascularization, or stromal haze. This study demonstrates, the development of a protein-releasing G-AM-P therapeutic contact lens that integrates optical transparency, mechanical robustness, biocompatibility, and targeted antibacterial activity against Gram-positive pathogens. These findings highlight its potential as a promising platform for corneal epithelial repair with in vitro antibacterial activity, supporting further investigation toward clinical translation.

Introduction: This investigation was designed to assess the therapeutic efficacy of a novel anti- GRP78 nanobody in the treatment of dry eye syndrome (DES) in a murine model. The focus was on the nanobody's anti-inflammatory properties and its ability to increase tear production. materials and methods: 2.1. Development of anti-GRP78 nanobody Anti-GRP78 nanobody was previously developed by Aghamollaei et al. (13). The construct was cloned into the pET28a(+) and transformed into E. coli BL21. Briefly, the protein expression was induced by isopropyl-β-D-thiogalactopyranoside (IPTG); then, the purification was performed by Ni-NTA affinity chromatography due to the presence of HisTag in c-terminus of protein, followed by a dialysis (<10 KDa), as previous studies (13,14). The purity and integrity of the nanobody were confirmed by SDS-PAGE. The final concentration of purified nanobody was determined by the Bradford assay, and aliquots were stored at -80°C until further use. 2.2. MTT Cytotoxicity Assay Prior to in vivo administration, the cytotoxicity of the anti-GRP78 nanobody was evaluated using the MTT assay. Briefly, human dermal fibroblast (HDF) cells were seeded in a 96-well plate at a density of 10,000 cells per well. Cells were treated with varying concentrations of the nanobody (5-400 µg/ml) for 24 and 48 hours. Following incubation, 20 µl of MTT reagent (5 mg/ml) was added to each well and incubated for an additional 4 hours at 37°C. The resulting formazan crystals were dissolved in dimethyl sulfoxide (DMSO), and absorbance was measured at 570 and 630 nm using a microplate reader. The highest non-toxic concentration was selected for in vivo application. 2.3. Animal Model and Ethics Statement All experimental procedures were conducted in compliance with institutional guidelines for the care and use of laboratory animals and were approved by the Institutional Animal Care and Use Committee (approval code: IR.BMSU.AEC.1402.055). Female BALB/c mice (6-8 weeks old) were used for the study and housed under standard laboratory conditions with a 12-hour light/dark cycle, controlled temperature, and free access to food and water. 2.4. Induction of Dry Eye Syndrome ES was induced in mice following the method described in previous studies, using 1% atropine sulfate eye drops (Sigma-Aldrich, USA). Atropine was administered three times daily into the lower conjunctival sac of each eye for 10 consecutive days to reduce tear secretion and mimic the clinical manifestations of DES (15). The induction of DES was confirmed by fluorescein staining of the cornea and the assessment of tear volume using the Schirmer tear test. 2.5. Experimental Groups and Interventions The mice were randomly assigned into five groups (n=6 per group): non-induced DES group, control group (received no treatment after DES induction), standard treatment group (received betamethasone eye drops (0.1%)), non-induced DES group treated with nanobody, and DES-induced nanobody-treated group. In the groups that required treatment, one drop of the designated drug was administered 15 minutes after each application of atropine sulfate. The Schirmer’s test was performed twice, with a 4-hour interval, on the first, third, fifth, and seventh days following the initial administration of the atropine sulfate solution. 2.6. Histopathological Analysis Following the treatment period, mice were euthanized, and their eyes were enucleated for histopathological examination. Corneal and conjunctival tissues were fixed in 10% formalin, embedded in paraffin, and sectioned at 5-μm thickness. Tissue sections were stained with hematoxylin and eosin (H&E) to evaluate inflammatory cell infiltration and structural changes. Fluorescein staining was also performed to assess epithelial integrity and damage. 2.7. Quantification of Inflammatory Cytokines Levels of mTNF-α, mIL-1α, mIL-1β, and mIL-6 in corneal tissues of models were quantified using commercially available ELISA kits (Karmania ParsGene, Iran) according to the manufacturer’s instructions. Absorbance was read at 450 nm using a microplate reader, and cytokine concentrations were determined based on standard curves. Also, total RNA was extracted from corneal tissues using an RNA extraction kit (Norex, Iran), and cDNA was synthesized using a reverse transcription kit (Jena Bioscience, USA). Quantitative real-time PCR (qPCR) was performed using SYBR Green Master Mix and specific primers for mTNF-α, mIL-1α, mIL-1β, mIL-6, and GAPDH as an internal control. Reactions were run on a real-time PCR system (Applied Biosystems, USA) under the following conditions: 95°C for 10 minutes, followed by 40 cycles of 95°C for 15 seconds and 60°C for 1 minute. Relative gene expression levels were calculated using the 2^-ΔΔCt method. Primers used in this study was listed in Table1. 2.8. Statistical Analysis Data were expressed as mean ± standard deviation (SD) and analyzed using GraphPad Prism software (version 10.4). Statistical comparisons between groups were performed using two-way analysis of variance (ANOVA) followed by Tukey’s post-hoc test. A p-value of <0.05 was considered statistically significant. Methods: Topical atropine sulfate was implemented to induce DES in BALB/c rodents. Subsequently, mice were administered betamethasone (as a positive control), the purified anti-GRP78 nanobody, or a vehicle. The Schirmer tear test, ELISA, and qRT-PCR were implemented to quantify corneal inflammatory cytokines (TNF-α, IL-1α, IL-1β, IL-6), as well as a histopathological examination. Results: Treatment with the anti-GRP78 nanobody meaningfully improved tear production and reduced levels of key inflammatory cytokines in corneal tissues. Histopathological analysis demonstrated noticeably reduced corneal epithelial damage and inflammatory cell infiltration, with efficacy comparable to betamethasone....

Cell-based therapies offer an alternative to corneal transplantation for the management of corneal diseases. However, these approaches require a deeper understanding of the principles of cell therapy, and the ability to predict and diagnose outcomes pre- and post-operatively is highly desirable. Recently, the development of innovative techniques that leverage predefined data from multiple cohorts with corneal diseases has received considerable attention. Approaches using artificial intelligence (AI) can address major concerns in corneal cell therapy, including the identification of novel biomarkers, improvements in cell delivery processes, and the acceleration of personalized treatments. This review summarizes real-world examples of AI applications from preclinical through clinical studies, with a focus on corneal cell-based therapies.

Cancer is a global health problem despite the most developed therapeutic modalities. The delivery of specific therapeutic agents to a target increases the effectiveness of cancer treatment by reducing side effects and post‐treatment issues.Our aim in this study was to design a recombinant protein consisting of nanobody molecules and exotoxin that targets the surface GRP78 receptor on tumor cells. Bioinformatics methods make drug design and recombinant protein evaluation much easier before the laboratory steps.Two constructs were designed from a single‐variable domain on heavy chain nanobody domains and PE toxin domains II, Ib, and III. The physicochemical properties, secondary structure, and solubility of the chimeric protein were analyzed using different software. Prostate cancer DU‐145 and breast cancer MDA‐MB‐468 cell lines were used as GRP78‐positive and negative controls, respectively. Accordingly, the cytotoxicity, binding affinity, cell internalization, and apoptosis were evaluated using MTT, enzyme‐linked immunosorbent assay, and western blot. The results showed that in the DU‐145 cell line, the cytotoxicity of two recombinant immunotoxins is dose and time‐dependent. In MDA‐MB‐468 and HEK‐293 cells, such an event does not occur. It is possible that two constructs designed for immunotoxins can attach to GRP78‐positive cancer cells and then eradicate cancer cells by internalization and apoptosis. As our in vitro results were in line with in silico data confirming the Bioinformatics predictions, it can be concluded that the designed recombinant immunotoxins may exhibit therapeutic potential against GRP78‐positive tumor cells.

Purpose: Sulfur mustard (SM) is a potent blistering agent. This alkylating chemical agent has extremely toxic effects on the eye. MMP-2 and MMP-9 are the two most important matrix metalloproteinase enzymes involved in the pathology of chemical eye injuries. Curcumin is regarded as a natural anti-inflammatory agent. This study aims to compare the anti-inflammatory effects of curcumin versus doxycycline on chemically induced corneal injuries. Methods: The HCE-2 cell line was used as a model for corneal cells. The effective concentrations of 2-chloroethyl ethyl sulfide (CEES) – as an analog of SM – doxycycline, and curcumin were determined using the MTT assay. The gene expression of MMP-2, MMP-9, and tissue inhibitors of metalloproteinase (TIMP-1) was evaluated by the real-time PCR method. Also, the activity of MMP-2 and MMP-9 enzymes was determined by zymography. Results: The expression of the MMP-2 and MMP-9 genes increased 5- and 3.3-fold after exposure to CEES, respectively. Following the treatment with curcumin and doxycycline, MMP- 2 expression decreased significantly. Also, after treatment with curcumin and doxycycline, the MMP-9 expression decreased 2.5- and 1.6-fold, respectively. The reduction in activity was 32% for MMP-2 and 56% for MMP-9 after treatment with curcumin. The corresponding values were 12% and 40% following doxycycline treatment. There was no significant difference between the effects of curcumin and doxycycline on reducing MMP-2 expression, but the difference was statistically significant in the case of MMP-9. Conclusion: Doxycycline and curcumin can inhibit MMP expression and activity in chemically exposed corneal cells. Curcumin has a greater ability than doxycycline to inhibit MMP-2 and MMP- 9 enzymes; however, the difference is statistically significant only in the case of MMP-9. After further validation, these substances can be introduced as anti- inflammatory agents to treat corneal chemical burns.

Background: Dry eye disease (DED) is a multifactorial condition characterized by ocular surface inflammation, tear film instability, and corneal epithelial damage. Current treatments often provide temporary relief without addressing the underlying inflammatory mechanisms. Objectives: This study examined the therapeutic potential of crocin and nobiletin, two naturally derived compounds with well-known antioxidant and anti-inflammatory properties, in a mouse model of DED induced by lacrimal gland excision (LGE). Methods: Thirty female Balb/c mice were divided into five groups (n = 6 each): Control (sham surgery), untreated DED, nobiletin-treated DED (32.75 µM), crocin-treated DED (34 µM), and 1% betamethasone-treated DED. Treatments were administered three times daily for 28 days. Ocular tissues were evaluated using Hematoxylin and Eosin (H&E) staining and fluorescein staining. Conjunctival inflammatory cytokines, including interleukin-6 (IL-6), interleukin-1 beta (IL-1β), and tumor necrosis factor-alpha (TNF-α), were measured by enzyme-linked immunosorbent assay (ELISA). Results: Histological analysis showed that the crocin and nobiletin treatment groups exhibited reduced epithelial disruption, keratinization, and inflammatory cell infiltration compared to the untreated DED group. The ELISA assay revealed that both compounds efficiently inhibited the production of the pro-inflammatory cytokines IL-6, TNF-α, and IL-1β, which are key mediators of DED pathogenesis. Fluorescein staining further confirmed the protective impact of crocin and nobiletin on corneal epithelial integrity. Moreover, the anti-inflammatory and epithelial-preserving effects of these compounds were comparable to those of the corticosteroid betamethasone. Conclusions: Overall, these findings suggest that crocin and nobiletin have therapeutic potential for DED management by modulating inflammatory responses and enhancing ocular surface healing. These naturally derived compounds offer promising avenues for the development of safer and more effective treatments for this challenging condition. However, further investigations, including clinical trials, are essential to elucidate the underlying mechanisms of action and optimize therapeutic approaches.