Potential Effect of Plasma Rich in Growth Factors-Endoret in Stromal Wound Healing in Additive Surgery.

PURPOSE: To compare the clinical and histological outcomes after intrastromal corneal ring segment (ICRS) implantation with and without plasma rich in growth factors (PRGF) in an experimental animal model. MATERIALS AND METHODS: First, the toxicity of PRGF was tested in hen's keratocyte cultures. Then, an animal model with 18 hens was randomly divided into 2 groups. In the first group, one ICRS was implanted in each eye (ICRS group). In the second group, the ICRS was firstly immersed 30 min in PRGF-Endoret solution, then implanted and, finally, PRGF-Endoret was inoculated into the channel (PRGF-ICRS group). Animals of each group were also separated into 3 groups regarding the time they were sacrificed, and corneal tissue was fixed for histological analysis at 2, 7 and 30 days. Cell death was detected by terminal uridine nick end labelling (TUNEL) assay. Proliferation was labelled by 5-bromo-2-deoxyuridine (BrdU) incorporation and myofibroblast differentiation by alpha-smooth muscle actin (αSMA) immunodetection. Clinical examination, analyzing epithelial wound closure, deposits and stromal haze, was carried out at the different study times. RESULTS: No toxic effect was observed by PRGF in hen stromal cell cultures. Clinically, in PRGF-ICRS corneas at 7 days, there were more deposits with higher intensity than in ICRS group. Histologically, at day 2 there was less epithelial damage over the segment in the PRGF-ICRS group, corneal oedema around the segment disappeared earlier and, at day 7, there was also double the number of cells around the segment than in the ICRS group displaying different morphologies. The number of TUNEL-positive cells was statistically higher in the PRGF-ICRS group at 7 and 30 days, and the number of BrdU-positive cells was statistically higher at all analyzed times. However, there were no differences in the number of αSMA-positive cells at 30 days between both groups. CONCLUSIONS: The ICRS immersion in PRGF-Endoret prior and after to its corneal implantation, in an experimental animal model, enhances clinical deposits and histological cell turnover without increasing myofibroblast differentiation reducing stromal wound-healing time after surgery.

Human corneal fibroblast migration and extracellular matrix synthesis during stromal repair: Role played by platelet-derived growth factor-BB, basic fibroblast growth factor, and transforming growth factor-ß1.

The development of treatments that modulate corneal wound healing to avoid fibrosis during tissue repair is important for the restoration of corneal transparency after an injury. To date, few studies have studied the influence of growth factors (GFs) on human corneal fibroblast (HCF) expression of extracellular matrix (ECM) proteins such as collagen types I and III, proteoglycans such as perlecan, or proteins implicated in cellular migration such as α5ß1-integrin and syndecan-4. Using in vitro HCFs, a mechanical wound model was developed to study the influence of the GFs basic fibroblast GF (bFGF), platelet-derived GF (PDGF-BB) and transforming GF-ß1 (TGFß1) on ECM protein production and cellular migration. Our results show that mechanical wounding provokes the autocrine release of bFGF and TGFß1 at different time points during the wound closure. The HCF response to PDGF-BB was a rapid closure due to fast cellular migration associated with a high focal adhesion replacement and a high expression of collagen and proteoglycans, producing nonfibrotic healing. bFGF stimulated nonfibrotic ECM production and limited the migration process. Finally, TGFß1 induced expression of the fibrotic markers collagen type III and α5ß1 integrin, and it inhibited cellular migration due to the formation of focal adhesions with a low turnover rate. The novel in vitro HCF mechanical wound model can be used to understand the role played by GFs in human corneal repair. The model can also be used to test the effects of different treatments aimed at improving the healing process. Copyright © 2016 John Wiley & Sons, Ltd.

Effects of TGFß1, PDGF-BB, and bFGF, on human corneal fibroblasts proliferation and differentiation during stromal repair.

In an effort to improve the regenerative nature of corneal repair, this study reports the use of an in vitro human corneal fibroblasts (HCFs) wound model after treatment with three of the main growth factors (GFs) involved in corneal healing: transforming growth factor beta 1 (TGFß1), platelet-derived growth factor BB-isoform (PDGF-BB), and basic fibroblast growth factor (bFGF) in order to delve in cell proliferation and differentiation processes. HCFs were mechanically wounded. The individual effect of TGFß1, PDGF-BB, and bFGF on cell proliferation and differentiation during the repair process was studied at different time points until wound closure. Wound dimensions and morphological changes were evaluated by microscopy. Cell proliferation and myofibroblast differentiation were analyzed by immunofluorescence cytochemistry. Changes in cell morphology were apparent at Day 4. PDGF-BB- and bFGF-treated cells had fibroblast-like morphology. TGFß1 stimulated proliferation in the wound edge and surrounding area, induced myofibroblast differentiation and inhibited cellular migration. PDGF-BB induced rapid wound closure due to proliferation, high motility, and late myofibroblast differentiation. The time course of closure induced by bFGF was similar to that for PDGF-BB, but was mostly due to proliferation in the wound area, and inhibited myofibroblast differentiation. Each of the GFs induced increases in responses promoting stromal repair differently. This study provides insight regarding how to optimize the outcome of stromal repair following corneal injury.

Clinical, Refractive and Histological Reversibility of Corneal Additive Surgery in Deep Stroma in an Animal Model.

PURPOSE: The aim was to evaluate the reversibility of the clinical and histological changes induced in the corneas of an animal model after removing an intracorneal ring segment (ICRS). METHODS: Surgery for this study was performed in 38 eyes of an experimental animal model (Gallus domesticus) for ICRS surgery (Ferrara technique). The animals without complications were randomized to two groups; in all of them, 1 segment was implanted in each eye and later removed at different times (1 and 3 months after implantation). In each group, after explantation, corneas were processed at different times for histological analysis with hematoxylin and eosin (H&E) stain and electronic microscopy. The refractive state of the eyes was also measured. RESULTS: In corneas without complications (88.23%), explantation was performed correctly. During the first few days, around the area where the ICRS was implanted we observed deposits of cells and a moderate degree of corneal opacity (haze). These signs decreased progressively without disappearing completely. Histologically, at 7 days, we observed hyperplasia and abnormal arrangement of collagen fibers. Later, these findings also decreased in both groups, albeit at a faster rate in group 1. Minimal changes were observed in electron microscopy up to the end of the study in both groups. Preoperative refractive state was achieved at 1 month after explantation in both groups. CONCLUSIONS: ICRS can safely be explanted from the cornea. Refractive reversibility was achieved at 1 month after explantation. However, the clinical and histological findings after ICRS explantation depend on the time from implantation to explantation.

Tissue reaction after intrastromal corneal ring implantation in an experimental animal model.

PURPOSE: To evaluate corneal wound healing in the hen animal model after additive surgery with an intracorneal ring segment (ICRS). METHODS: We implanted one ICRS in each eye of 76 hens. In control group 1 (n = 22 hens), the stromal channel was prepared but no ICRS was inserted. In control group 2 (n = 2 hens), no surgery was performed. Animals were randomly separated into groups and euthanized after clinical follow-up of 4 and 12 hours, 1, 2, 3, and 7 days, and 1, 2, 3, 4, and 6 months. Corneas were stained with hematoxylin-eosin. Apoptosis was measured by terminal uridine nick end-labeling assays. Cell proliferation and myofibroblast-like differentiation were assayed by BrdU and α-smooth muscle actin immunofluorescence microscopy. Stromal matrix changes were documented by electron microscopy. RESULTS: Epithelial and stromal cell apoptosis around the ICRS-implanted and control group 1 eyes peaked at 12 hours, but continued for 72 hours. In ICRS-implanted eyes, epithelial and stromal proliferation was present at 12 and 24 hours, respectively, and peaked at 7 days and 72 hours, respectively. Some proliferation in the ICRS-implanted group continued through the 6-month follow-up, and myofibroblast-like cells differentiated one to three months after ICRS implantation. The segments rotated within the stroma as the limbal inferior angle approached the epithelium. CONCLUSIONS: Wound healing after ICRS implantation in hen corneas was similar to that of other corneal surgical wounds in stages. However, there were some specific features related to the small size of the epithelial wound and the device permanently implanted inside the cornea.

Does mitomycin C cause toxicity in the cornea after photorefractive keratectomy? A comparative wound-healing study in a refractive surgery animal model.

PURPOSE: In this study, we investigated the wound-healing process after photorefractive keratectomy with mitomycin C (MMC) in hen corneas. In addition, we evaluated the synergistic effect of ethanol and MMC. METHODS: Forty-eight adult hens were divided into 3 groups: A: ethanol-assisted debridement plus MMC; B: mechanical debridement plus MMC; and C: mechanical debridement (MMC-untreated control). Photorefractive keratectomy was performed, and the animals were followed up for up to 60 days. Epithelial healing was measured with fluorescein. Apoptosis was evaluated by terminal deoxynucleotidyl transferase dUTP nick end-labeling assay and proliferation was measured by BrdU incorporation. Both myofibroblast differentiation and collagen deposition were evaluated by immunofluorescence and histology. RESULTS: Epithelial wound closure was similar in all 3 groups (P > 0.05). Significant reduction in haze was observed in groups A and B compared with C (P < 0.01), but there was no difference between groups A and B (P > 0.05). Furthermore, there was no difference in the number of apoptotic cells between groups. Proliferation was delayed in both groups A and B compared with C (P < 0.01), but groups A and B did not differ significantly (P > 0.05). Myofibroblasts, cellular density, and collagen deposition were lower in both groups A and B compared with C (P < 0.01), but they were not significantly different from each other (P > 0.05). CONCLUSIONS: Topical application of MMC in hen corneas reproduces the wound healing observed in humans by reducing haze, keratocyte proliferation, myofibroblast differentiation, and new collagen deposition. Synergistic cytotoxic effects of ethanol and MMC were not observed.

Efficacy and safety of short-duration topical treatment with azithromycin oil-based eyedrops in an experimental model of corneal refractive surgery.

PURPOSE: To assess the tolerance and side effects of azithromycin eyedrops at the ocular surface after corneal refractive surgery in an experimental animal model. METHODS: The effect of azithromycin eyedrops was evaluated in hen corneas that underwent laser-assisted in situ keratomileusis (LASIK) or photorefractive keratectomy (PRK) surgery in 1 eye, using the fellow eye (not manipulated) as a control. Animals were treated bid 3 days prior to surgery and 3 days after surgery with T1225 1.5% azithromycin eyedrops or saline eyedrops (balanced salt solution), or were left untreated as a control. Clinical course and cell biology (apoptosis, proliferation, and differentiation) measurements were assessed. RESULTS: Infections were present in the following proportions of corneas operated on by LASIK: 0% treated with azithromycin, 60% treated with BSS, and 30% untreated. No corneal abscess or keratitis were present in any PRK or unmanipulated corneas. Conjunctival edema and redness were less prevalent in LASIK-operated eyes treated with azithromycin than in BSS-treated or untreated eyes and were not observed in any PRK or unmanipulated corneas. In PRK-operated eyes treated with azithromycin, a decrease was observed in the apoptosis and an increase in the stromal proliferation. There were no differences in these parameters for LASIK and unmanipulated eyes. CONCLUSIONS: Topical administration of T1225 oil-based azithromycin eyedrops was well tolerated in both unmanipulated hen corneas and those treated with corneal refractive surgery (PRK and LASIK). T1225 demonstrated a potent antibiotic effect after LASIK treatment.