Optimization of a Plasma Rich in Growth Factors Membrane for the Treatment of Inflammatory Ocular Diseases

Abstract

The main purpose of the present study is to develop an immunosafe fibrin membrane obtained by plasma rich in growth factors technology (is-mPRGF) with improved mechanical properties that could be applied in patients with inflammatory ocular diseases. Blood was drawn from three healthy donors and centrifuged, and the collected PRGF was activated and distributed into two groups: (i) mPRGF: a PRGF membrane maintained at 37 °C for 30 min; (ii) IS5+30: mPRGF incubated at 37 °C for 5 min and then incubated at 56 °C for 30 min. The content of both membranes was analyzed for several growth factors such as IgE and the complement activation, as well as biological activity on different ocular surface cells. Furthermore, the physical and mechanical characterizations were also evaluated. IS5+30 completely reduced the complement activity and decreased the IgE while preserving the concentration of the main growth factors. IS5+30 induced similar biological activity regarding mPRGF on the different ocular surface cells analyzed. Furthermore, no significant differences in release kinetics or fibrin degradation were observed between both membranes. Summarizing, IS5+30 totally reduces complement activity while preserving the concentration of most growth factors and their biological activity. Furthermore, the physical and mechanical properties of the fibrin membrane are preserved after heat inactivation.

Keywords: PRP; autoimmune diseases; complement system; fibrin membrane; heat inactivation; ocular surface diseases; plasma rich in growth factors; platelet rich plasma.

Figure 1

IgE and complement activity levels in non-heat treated (mPRGF) and heat-treated (IS5+30) membranes. IgE levels were reduced by 20% in the heat-treated membrane (IS5+30) regarding mPRGF. No significant differences were observed in the IgE levels between the mPRGF and IS5+30. Heat treatment completely reduces complement activity in the mPRGF incubated at 37 °C for 5 min and at 56 °C for 30 min (IS5+30) compared to the mPRGF. * p < 0.05.

Figure 2

Percentage content of the different growth factors in the IS5+30 membrane with respect to the mPRGF evaluated in the study. Significant reduction (* p < 0.05) of TSP-1, ANG-1, and END levels was observed after heat treatment of the PRGF membrane (IS5+30) regarding the non-heated membrane (mPRGF).

Figure 3

The release kinetics of PDGF-AB, EGF, TGF-β1, FGFb, VEGF, and END were analyzed in both PRGF membranes (mPRGF and IS5+30) for 1 h and 1, 3, and 7 days. Except for TGF-β1 levels at 1d, no significant differences were found between the IS5+30 and mPRGF at any study time points in any of the growth factors evaluated. * p < 0.05 significant differences between mPRGF and IS5+30.

Figure 4

Proliferation percentage of different ocular surface cells (HCE, HConF, and HK) after treatment with the supernatant obtained from each of the PRGF membranes (mPRGF and IS5+30) at each time point of the kinetic release assay (1 h, and 1, 3, and 7 days) compared to the supernatant obtained after complete membrane squeezing of both PRGF membranes (control of mPRGF and IS5+30). No significant differences were observed among the different samples at any study time and in any of the cell types analyzed.