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