Focus on cell therapy to treat corneal endothelial diseases.

The cornea is a multi-layered structure which allows fine refraction and provides both resistance to external insults and adequate transparency. The corneal endothelium ensures stromal hydration, failure of which, such as in Fuchs endothelial corneal dystrophy, after trauma or in aging, may lead to loss of corneal transparency and induce blindness. Currently, no efficient therapeutic alternatives exist except for corneal grafting. Thus corneal tissue engineering represents a valuable alternative approach, which may overcome cornea donor shortage. Several studies describe protocols to isolate, differentiate, and cultivate corneal endothelial cells (CEnCs) in vitro. Two main in vitro strategies can be described: expansion of eye-native cell populations, such as CEnCs, or the production and expansion of CEnCs from non-eye native cell populations, such as induced Pluripotent Stem Cells (iPSCs). The challenge with these cells is to obtain a monolayer of CEnCs on a biocompatible carrier, with a specific morphology (flat hexagonal cells), and with specific functions such as programmed cell cycle arrest. Another issue for this cell culture methodology is to define the adapted protocol (media, trophic factors, timeframe) that can mimic physiological development. Additionally, contamination by other cell types still represents a huge problem. Thus, purification methods, such as Fluorescence Activated Cell Sorting (FACS), Magnetic Ativated Cell Sorting (MACS) or Sedimentation Field Flow Fractionation (SdFFF) are useful. Animal models are also crucial to provide a translational approach for these therapies, integrating macro- and microenvironment influences, systemic hormonal or immune responses, and exogenous interactions. Non-eye native cell graft protocols are constantly improving both in efficacy and safety, with the aim of being the most suitable candidate for corneal therapies in future routine practice. The aim of this work is to review these different aspects with a special focus on issues facing CEnC culture in vitro, and to highlight animal graft models adapted to screen the efficacy of these different protocols.

Combined intra-tendinous injection of Platelet Rich Plasma and bevacizumab accelerates and improves healing compared to Platelet Rich Plasma in tendinosis: comprehensive assessment on a rat model.

PURPOSE: the aim of our study was to assess the potential of combined intratendinous injection of an anti-angiogenic drug: bevacizumab (AA) and Platelet Rich Plasma (PRP) to treat tendinopathy in a murine model of patellar and Achilles tendinopathy, and to evaluate its local toxicity. MATERIAL AND METHOD: twenty rats (80 patellar and Achilles tendons) were used for the study. We induced tendinosis (T+) in 80 tendons (patellar=40 and Achilles=40) by injecting under ultrasonography (US) guidance Collagenase 1® (day 0 = D0). Clinical examination was performed at D3, immediately followed by either PRP and AA (AAPRPT+, n=40) or PRP (PRPT+ n=40, control) US-guided intratendinous injection. Follow-up at D6, D18 and D25 using clinical, US and histology, and comparison between the 2 groups were performed. To study AA+PRP toxicity, we looked for necrosis or rupture on the 40 AAPRPT+. RESULTS: all AAPRPT+ showed better joint mobilization compared to PRPT+ at D6 (p=0.03), D18 (p=0.04) and D25 (p=0.02). Similar results were found regarding US and histology, with smaller collagen fiber diameters (D6, p≤0.017, D25, p≤0.015), less disorganization and fewer neovessels (D25, p=0.004) in AAPRPT+ compared to PRPT+. No AA+PRP local toxicity was discovered in histology assessment. CONCLUSION: our study suggests that combined injection of AA and PRP in tendinosis accelerates and improves tendon's healing compared PRP used alone, with no local toxicity.

Acceleration of tendon healing using US guided intratendinous injection of bevacizumab: first pre-clinical study on a murine model.

PURPOSE: Tendinopathy shows early disorganized collagen fibers with neo-angiogenesis on histology. Peri-tendinous injection of corticosteroid is the commonly accepted strategy despite the abscence of inflammation in tendinosis. The aim of our study was to assess the potential of intratendinous injection of an anti-angiogenic drug (bevacizumab, AA) to treat tendinopathy in a murine model of patellar and Achilles tendinopathy, and to evaluate its local toxicity. MATERIALS AND METHOD: Forty rats (160 patellar and Achilles tendons) were used for this study. We induced tendinosis (T+) in 80 tendons by injecting under ultrasonography (US) guidance Collagenase 1(®) (day 0 = D0, patellar = 40 and Achilles = 40). Clinical examination and tendon US were performed at D3, immediately followed by either AA (AAT+, n = 40) or physiological serum (PST+, n = 40, control) US-guided intratendinous injection. Follow-up at D6 and D13 using clinical, US and histology, and comparison between the 2 groups were performed. To study AA toxicity we compared the 80 remaining normal tendons (T-) after injecting AA in 40 (AAT-). RESULTS: All AAT+ showed a better joint mobilization compared to PST+ at D6 (p = 0.004) with thinner US tendon diameters (p<0.004), and less disorganized collagen fibers and neovessels on histology (p<0.05). There was no difference at D13 regarding clinical status, US tendon diameter and histology (p>0.05). Comparison between AAT- and T- showed no AA toxicity on tendon (p = 0.18). CONCLUSION: Our study suggests that high dose mono-injection of AA in tendinosis, early after the beginning of the disease, accelerates tendon's healing, with no local toxicity.

Effects of subconjunctival bevacizumab on corneal neovascularization: results of a prospective study.

PURPOSE: To evaluate the effect of subconjunctival bevacizumab injections in patients with corneal neovascularization resulting from different ocular surface disorders. METHODS: Prospective case series. Twelve eyes of 11 patients with corneal neovascularization caused by different ocular surface disorders, such as healed corneal ulcers, long-standing chronic inflammatory diseases, and corneal ischemia secondary to burn, were included. All eyes received a single subconjunctival injection of 2.5 mg (0.1 mL) of bevacizumab. Morphological changes in neovascularization were evaluated during 3 months using slit-lamp biomicroscopy, corneal digital photography, and computer-assisted semiautomatic analysis of corneal neovascularization area. RESULTS: Recession of corneal vessels was observed in all eyes at 1 week postinjection. The surface of the neovascular tree continued to decrease noticeably for 1 month and then increased again for the remainder of the follow-up period. The corneal neovascularization area amounted to 11.25 ± 4.49% of the corneal surface preinjection, compared with 8.44 ± 3.37% postinjection (P = 0.02), reflecting a mean decrease in corneal neovascularization of 25%. No local or systemic adverse events possibly related to subconjunctival bevacizumab injection were observed. CONCLUSIONS: Short-term results suggest that subconjunctival bevacizumab can be used safely and effectively for corneal neovascularization resulting from different ocular surface disorders, providing an additional strategy to improve success of corneal grafts.

Intravitreal ranibizumab and bevacizumab in combination with full-fluence verteporfin therapy and dexamethasone for exudative age-related macular degeneration.

PURPOSE: To evaluate the efficacy and safety of triple therapy with intravitreal anti-vascular-endothelial-growth-factor (VEGF) antibody, dexamethasone and verteporfin photodynamic therapy (PDT) for exudative age-related macular degeneration (AMD). METHODS: Retrospective, comparative, interventional study. Records of treatment-naïve patients who received intravitreal bevacizumab or ranibizumab in monotherapy or in combination with dexamethasone and full-fluence verteporfin PDT in triple therapy were reviewed. logMAR visual acuity, foveal thickness (FT) on optical coherence tomography, intraocular pressure and endophthalmitis occurrence were recorded. RESULTS: Sixty-one eyes were included in the triple-therapy group, 40 eyes were included in the monotherapy group. The mean follow-up was 14.1 ± 3.4 months in the triple-therapy group and 16.3 ± 4.1 months in the monotherapy group. The triple-therapy group enjoyed a lower total number of treatments (1.92 ± 0.44 vs. 3.12 ± 0.37, p < 0.001) and a longer time before first retreatment (5.4 ± 3.3 vs. 3.6 ± 2.5 months, p = 0.001). A significant improvement of visual acuity and FT was present in both groups during the 12 months following first treatment. No adverse effects were observed. CONCLUSION: The combination of intravitreal bevacizumab or ranibizumabwith dexamethasone and full-fluence PDT for exudative AMD provided visual and anatomic improvement and a good safety profile. Triple therapy may reduce the number of retreatments when compared to anti-VEGF alone.