Elsevier

Experimental Eye Research

Volume 115, October 2013, Pages 153-161
Experimental Eye Research

Plasma rich in growth factors (PRGF-Endoret) stimulates corneal wound healing and reduces haze formation after PRK surgery

https://doi.org/10.1016/j.exer.2013.07.007Get rights and content

Highlights

  • Plasma rich in growth factors enhances corneal epithelial cell (HCE) proliferation.

  • Corneal wound healing was accelerated after PRGF-Endoret treatment.

  • PRGF-Endoret treatment reduces corneal haze formation after Photorefractive keratectomy.

Abstract

This study evaluated the efficacy of Plasma rich in growth factors (PRGF-Endoret) on the corneal wound healing process after Photorefractive keratectomy (PRK). To address this, blood from three healthy donors was collected, centrifuged and, the whole plasma column (WP) and the plasma fraction with the highest platelet concentration (F3) were collected. The effects of F3 and WP on the proliferation and migration of human corneal epithelial cells (HCE) were analyzed. PRK was performed on C57BL/6 mice. Animals were divided in three treatment groups: Control, F3, and WP. Corneal wound healing and haze formation were evaluated macroscopically. Eyes were collected at 1, 2, 3, and 7 days after surgery, and were processed for histological studies. Immunofluorescence was used to assess cellular proliferation, apoptosis and myofibroblast transformation in the mouse cornea. Results showed a significant increased on proliferation and wound healing after F3 and WP treatment when compared with control group. In vivo studies showed significant reduction on haze formation in mice treated with both PRGF-Endoret formulations (F3 and WP). Histological studies showed an increase of epithelial cell proliferation in corneas of control group, promoting an epithelial hyperplasia. The number of SMA-positive cells (corresponding to myofibroblast differentiation) was significantly lower in the PRGF-Endoret group than in the control group, correlating with the higher transparence results observed macroscopically in both PRGF-Endoret groups. According to this, it can be concluded that PRGF-Endoret accelerates corneal tissue regeneration after PRK, reducing haze formation.

Introduction

The cornea is the transparent tissue of the eye that consists mainly in three layers: an outer layer containing an epithelium, a middle stromal layer composed by specialized cells called keratocytes, and an inner layer of endothelial cells. The homeostatic mechanisms regulating the normal physiological renewal of the corneal tissue involves proliferation, migration and differentiation of cells (Dua et al., 1994).

After corneal injury, epithelial cells from the basal epithelial layer and from limbus proliferate and migrate to cover the wound bed before differentiating into new multilayered epithelium (Dua et al., 1994). In contrast, keratocytes underlying the damage area undergo apoptosis (Wilson et al., 1996), and the quiescent keratocytes adjacent to the injury change into activated cells, enter into the cell cycle and subsequently migrate to the site of injury (Del Pero et al., 1990, Fini and Stramer, 2005). These new cells have the characteristics of fibroblasts, and some of them develop actin-myosin contractile elements, consistent with myofibroblasts (Garana et al., 1992, Zieske et al., 2001). During corneal repair, the transformed myofibroblasts are responsible for wound contraction and for extracellular matrix (ECM) deposition and organization (Jester et al., 1999b). After complete tissue repair, the new ECM formed takes over the mechanical load and myofibroblast cells disappear by apoptosis or by differentiation to keratocytes (Maltseva et al., 2001, Tomasek et al., 2002). Abnormal tissue regeneration process can lead to a corneal ulceration and scarring (Dupps and Wilson, 2006).

This biological process of wound healing is mediated by many proteins, including several growth factors like, epithelial growth factor (EGF), fibroblast growth factor (FGF), and platelet-derived growth factors (PDGF), among others (Imanishi et al., 2000, Klenkler and Sheardown, 2004, Wilson et al., 1994). These growth factors may infiltrate from the tear film to the superficial tissues of the eye (Wilson et al., 2001).

Artificial tears are the most widely used approaches for the conventional management of ocular surface damage with good outcome regarding lubrication and relieve the symptoms of dry eye. However, they lack the biological components of healthy tears. Furthermore, they often contain preservatives, stabilizers, and other additives that potentially may induce toxic or allergic reactions (Noecker, 2001, Tripathi and Tripathi, 1989).

One interesting alternative to artificial tears is the topical use of blood derivates. In particular, the use of plasma rich in growth factors may represent a potential therapeutic approach for ocular surface disorders as a tear substitute. Apart from its lubricating properties, the technology of plasma rich in growth factors (PRGF-Endoret) contains numerous of morphogens including EGF, transforming growth factor-beta (TGF-β), PDGF and nerve growth factor (NGF) among others (Blair and Flaumenhaft, 2009, Nurden et al., 2008). This biological approach consists on the elaboration and use of a platelet enriched plasma obtained from patient's own blood. After activation with calcium chloride, this approach allows the in-situ formation of a biodegradable fibrin scaffold and the release of a pool of biologically active proteins that influence and promote a range of biological processes including cell recruitment, growth and differentiation (Anitua et al., 2008, Anitua et al., 2009).

Recently, we have observed that PRGF-Endoret significantly enhances proliferation and migration of both keratocytes and conjunctival fibroblasts. In addition, it prevents and inhibits TGF-β1-induced myofibroblast differentiation (Anitua et al., 2011). These interesting results suggest that PRGF-Endoret could have a promising role in myofibroblast modulation in the stroma of the ocular surface tissues.

In this study, concentrations of several factors considered to be important in corneal wound healing were measured in two different PRGF-Endoret-derived formulations, fraction 3 (F3: platelet enriched fraction) or whole plasma (WP) column (with lower amount of platelets than F3) obtained from PRGF-Endoret technology. The regenerative effects of these preparations on proliferation and migration of immortalized human corneal epithelial cells and on mice cornea after Photorefractive keratectomy surgery were evaluated.

Section snippets

PRGF-Endoret preparations

The study was performed following the principles of the Declaration of Helsinki. Blood from three healthy young male donors was collected after informed consent into 9-mL tubes with 3.8% (wt/v) sodium citrate. Samples were centrifuged at 580 g for 8 min at room temperature in a PRGF-Endoret System centrifuge (BTI Biotechnology Institute, S.L., Miñano, Álava, Spain). Half of the tubes were used to separate the whole plasma column (WP) over the buffy coat and the other half to take the milliliter

Results

Platelet enrichment of the PRGF-Endoret preparations were 2.6-fold for WP (481 × 106 platelets/ml) and 3.6-fold for F3 (663 × 106 platelets/ml) over the baseline concentration in whole blood. None of the preparations contained detectable concentrations of leukocytes. Table 1 shows the concentrations of several of the most important growth factors for each sample (WP and F3). No statistical differences were observed between both formulations.

Discussion

The cornea reacts to damage by releasing numerous substances, including cytokines, growth factors, proteases and neuropeptides with the aim of restoring anatomical integrity (re-epithelisation, stromal repair, and re-innervation). In chronological order, re-epithelisation is the first process that occurs during corneal tissue regeneration, through the stimulation of the proliferation, migration and differentiation of the adjacent epithelium. This process is initiated and controlled by the

Conclusions

In summary, our results indicate that different formulations of PRGF-Endoret enhance proliferation and wound healing of epithelial corneal cells, and enhance wound healing after excimer laser photoablation, reducing corneal haze formation. Although further studies are needed to determine the exact mechanisms underlying the effects of this autologous technology, results from this study suggest that the different PRGF-Endoret formulations (WP and F3) could improve the wound healing in ocular

Acknowledgments

This work is funded by Customized Eye Care. CEYEC (n° CEN-20091021) project, which has been supported by the Centre for Industrial Technological Development (CDTI) in the fifth edition of the CENIT program. The aim of this program is to promote the public-private stable cooperation in research, development and innovation (R + D + i), which is part of the Spanish government initiative INGENIO 2010.

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