Elsevier

Experimental Eye Research

Volume 119, February 2014, Pages 27-34
Experimental Eye Research

Effects of heat-treatment on plasma rich in growth factors-derived autologous eye drop

https://doi.org/10.1016/j.exer.2013.12.005Get rights and content

Highlights

  • Heat-treatment of PRGF eye drop preserves the content of most of the proteins.

  • Heat-inactivation reduces drastically the content of IgE and complement activity.

  • Heat-inactivated PRGF induces higher biological effects.

Abstract

We have developed and characterized a new type of plasma rich in growth factors (PRGF) derived eye-drop therapy for patients suffering from autoimmune diseases. To determine the concentration of several growth factors, proteins, immunoglobulins and complement activity of the heat-inactivated eye-drop and to study its biological effects on cell proliferation and migration of different ocular surface cells, blood from healthy donors was collected, centrifuged and PRGF was prepared avoiding the buffy coat. The half volume of the obtained plasma supernatant from each donor was heat-inactivated at 56 °C for 1 h (heat-inactivated PRGF). The concentration of several proteins involved on corneal wound healing, immunoglubolins G, M and E and functional integrity of the complement system assayed by CH50 test were determined. The proliferative and migratory potential of inactivated and non-inactivated PRGF eye drops were assayed on corneal epithelial cells (HCE), keratocytes (HK) and conjunctival fibroblasts (HConF). Heat-inactivated PRGF preserves the content of most of the proteins and morphogens involved in its wound healing effects while reduces drastically the content of IgE and complement activity. Heat-inactivated PRGF eye drops increased proliferation and migration potential of ocular surface cells with regard to PRGF showing significant differences on proliferation and migration rate of HCE and HConF respectively. In summary, heat-inactivation of PRGF eye drops completely reduced complement activity and deceased significantly the presence of IgE, maintaining the biological activity of PRGF on ocular surface cells.

Introduction

It is widely known that different ocular surface structures including cornea, conjunctiva, meibomian glands, globet cells and lacrimal glands and mucosal immunity system contribute to the tear film composition and to the maintenance of normal cornea integrity. These components are interrelated to each other by neural, hormonal and chemical feedback mechanisms (Mathers, 2000, Pflugfelder et al., 1986, Stern et al., 1998). Alteration of any of these components may modify this complex system, affecting the composition of the tear film and the homeostasis of the corneal tissues, increasing the risk to suffer a corneal injury (Muller et al., 2003, Nasu et al., 1984, Pflugfelder et al., 2002).

Several systemic immune-mediated disorders affecting the eye such as allergies, vasculitis, Primary or secondary Sjögren’s syndrome, or graft versus host disease are characterized by a deregulated tear film composition and dry eye syndrome (Fox and Kang, 1992, Read, 2004, Tabbara et al., 2009). The soluble components of immune system, including immunoglobulins and the complement system have a direct role with the pathogenesis of these ocular diseases (Daha et al., 2011, Patriarca et al., 2006, Read, 2004, Stern et al., 2013). In addition, inflammation and ocular tissue injury are also common signs of these types of ocular diseases.

Local medications with preservatives, commonly used to treat ocular inflammation and to reduce corneal injuries, are not well tolerated by patients suffering from systemic immune disorders. Combination of lubricants and steroids are also usually required, although they show poor tolerance and unsatisfactory response (Abelson et al., 2003, Balaram et al., 2005, Stern et al., 2013).

Recently, the use of an autologous eye-drop based on platelet-rich plasma (PRP) has been proposed for treatment of several acute and chronic eye surface disorders including corneal epithelial defects and dry eye syndrome (Alio et al., 2007a, Alio et al., 2007b, Alio et al., 2007c, Alio et al., 2012, Lopez-Plandolit et al., 2010, Lopez-Plandolit et al., 2011, Pezzotta et al., 2012). Plasma rich in growth factors (PRGF) is a type of platelet enriched plasma obtained from patient's own blood, which avoid the leukocytes collection, and which after activation with calcium chloride allows the release of a pool of biologically active proteins that influence and promote a range of biological process including proliferation, migration and differentiation, and protect against microbial contamination (Anitua et al., 2011, Anitua et al., 2012, Freire et al., 2012).

Clinical results from patients with dry-eye syndrome from different etiologies treated with PRP eye-drops show that around 80% of the patients have a very significant or moderate improvement, while around 20% show minimal or no changes in the disease status (Alio et al., 2007b, Lopez-Plandolit et al., 2011, Pezzotta et al., 2012). However, in patients with systemic autoimmune syndromes results are worse. This may due to the concentration of immunologic components in the eye drops obtained from the own patient's blood. These immunologic components including immunoglobulins and complement (Jensen et al., 1999, Nguyen et al., 2007, Patriarca et al., 2006) may hamper correct wound healing of the cornea.

In this study we propose a new type of plasma rich in growth factors (PRGF)-derived eye-drop therapy for patients suffering from autoimmune diseases. The new formulation has been obtained after heating the eye-drops at 56 °C. The hypothesis is that the heat-inactivated PRGF eye-drops will have a reduced amount of immunoglobulins or complement. It has been reported that heating serum or plasma at 56 °C reduces the complement system activity and destroy microorganism and viruses (Triglia and Linscott, 1980, Ward, 1979). However, it is also known that heat inactivation has a wide spectrum of denaturing effects on serum and plasma protein and factors (Ayache et al., 2006). Assuming this, a new heat-inactivated PRGF eye-drop formulation has been obtained and characterized. The concentration of several growth factors, proteins, immunoglobulins and complement activity of the eye-drop has been determined. In addition, the effects of the heat-inactivated eye-drop on cell proliferation and migration of different ocular surface cells have been evaluated.

Section snippets

Preparation of plasma rich in growth factors (PRGF) eye drops

Blood from four healthy donors was collected after informed consent into 9-mL tubes with 3.8% (wt/v) sodium citrate. The study was performed following the principles of the Declaration of Helsinki. Samples were centrifuged at 580 g for 8 min at room temperature in an Endoret System centrifuge (BTI Biotechnology Institute, S.L., Miñano, Álava, Spain). Whole column of plasma rich in growth factors was collected after centrifugation avoiding the buffy coat that contains the leukocytes using

Results

The mean platelet enrichment of the PRGF preparations was 2.45-fold over the baseline concentration of platelets in whole blood. None of the preparations contained detectable levels of leucocytes (data not shown).

Discussion

This study describes de development and characterization of a new type of plasma rich in growth factors (PRGF) eye-drop therapy directed towards the treatment of ocular surface pathologies in patients suffering from autoimmune diseases. The new formulation has been obtained after heating the eye-drops at 56 °C. The composition of the non-inactivated and heat-inactivated PRGF samples has been evaluated and compared as well as their biological potential determined in three different cells of the

Conclusions

In summary, this study describes the development and characterization of a new type of heat-inactivated autologous PRGF eye-drops. The latter has preserves the content of most of the proteins and morphogens involved in its wound healing effects while reduces drastically the content of IgE and complement activity. Although the present work represents only a preliminary study and additional in vivo studies are needed to effectively evaluate the potential of the new formulation, this is the first

Acknowledgments

This work is funded by Customized Eye Care. CEYEC (no. 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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