Ozone dosing alters the biological potential and therapeutic outcomes of plasma rich in growth factors.

BACKGROUND AND OBJECTIVE: Until now, ozone has been used in a rather empirical way. This in-vitro study investigates, for the first time, whether different ozone treatments of plasma rich in growth factors (PRGF) alter the biological properties and outcomes of this autologous platelet-rich plasma. MATERIAL AND METHODS: Human plasma rich in growth factors was treated with ozone using one of the following protocols: a continuous-flow method; or a syringe method in which constant volumes of ozone and PRGF were mixed. In both cases, ozone was added before, during and after the addition of calcium chloride. Three ozone concentrations, of the therapeutic range 20, 40 and 80 µg/mL, were tested. Fibrin clot properties, growth factor content and the proliferative effect on primary osteoblasts and gingival fibroblasts were evaluated. RESULTS: Ozone treatment of PRGF using the continuous flow protocol impaired formation of the fibrin scaffold, drastically reduced the levels of growth factors and significantly decreased the proliferative potential of PRGF on primary osteoblasts and gingival fibroblasts. In contrast, treatment of PRGF with ozone using the syringe method, before, during and after the coagulation process, did not alter the biological outcomes of the autologous therapy. CONCLUSION: These findings suggest that ozone dose and the way that ozone combines with PRGF may alter the biological potential and therapeutic outcomes of PRGF.

Morphogen and proinflammatory cytokine release kinetics from PRGF-Endoret fibrin scaffolds: evaluation of the effect of leukocyte inclusion.

The potential influence of leukocyte incorporation in the kinetic release of growth factors from platelet-rich plasma (PRP) may explain the conflicting efficiency of leukocyte platelet-rich plasma (L-PRP) scaffolds in tissue regeneration. To assess this hypothesis, leukocyte-free (PRGF-Endoret) and L-PRP fibrin scaffolds were prepared, and both morphogen and proinflammatory cytokine release kinetics were analyzed. Clots were incubated with culture medium to monitor protein release over 8 days. Furthermore, the different fibrin scaffolds were morphologically characterized. Results show that leukocyte-free fibrin matrices were homogenous while leukocyte-containing ones were heterogeneous, loose and cellular. Leukocyte incorporation produced a significant increase in the contents of proinflammatory cytokines interleukin (IL)-1ß and IL-16 but not in the platelet-derived growth factors release (<1.5-fold). Surprisingly, the availability of vascular endothelial growth factor suffered an important decrease after 3 days of incubation in the case of L-PRP matrices. While the release of proinflammatory cytokines was almost absent or very low from PRGF-Endoret, the inclusion of leukocytes induced a major increase in these cytokines, which was characterized by the presence of a latent period. The PRGF-Endoret matrices were stable during the 8 days of incubation. The inclusion of leukocytes alters the growth factors release profile and also increased the dose of proinflammatory cytokines.

Plasma rich in growth factors (PRGF-Endoret) stimulates tendon and synovial fibroblasts migration and improves the biological properties of hyaluronic acid.

PURPOSE: Cell migration plays an essential role in development, wound healing, and tissue regeneration. Plasma rich in growth factors (PRGF-Endoret) technology offers a potential source of growth factors involved in tissue regeneration. Here, we evaluate the potential of PRGF-Endoret over tendon cells and synovial fibroblasts migration and study whether the combination of this autologous technology with hyaluronic acid (HA) improves the effect and potential of the biomaterials over the motility of both types of fibroblasts. METHODS: Migration of primary tendon cells and synovial fibroblasts after culturing with either PRGF or PPGF (plasma poor in growth factors) at different doses was evaluated. Furthermore, the migratory capacity induced by the combination of PPGF and PRGF with HA was tested. RESULTS: PPGF stimulated migration of both types of cells but this effect was significantly higher when PRGF was used. Tendon cells showed an increase of 212% in migratory ability when HA was combined with PPGF and of 335% in the case of HA + PRGF treatment compared with HA alone. CONCLUSIONS: PRGF-Endoret stimulates migration of tendon cells and synovial fibroblasts and improves the biological properties of HA.

Fibroblastic response to treatment with different preparations rich in growth factors.

OBJECTIVES: Preparations rich in growth factors (PRGF) release them plus bioactive proteins at localized sites, with the aim of triggering healing and regenerative processes. The prevailing paradigm suggests that their influence on proliferation, angiogenesis and the extracellular matrix synthesis is minimal. However, variations in their composition and impact on different cell phenotypes have not been examined. MATERIALS AND METHODS: Sixteen fibroblast cultures obtained from three different anatomical sites (skin, synovium and tendon) of 16 donors were exposed to the molecular pool released from PRGF scaffolds, with increasing amounts of platelets. We evaluated cell proliferation, secretion of angiogenic growth factors (VEGF and HGF), synthesis of type I collagen and hyaluronic acid (HA), considering platelet dose and anatomical origin of the cells. Activity of transforming growth factor-beta (TGF-beta) in type I procollagen and HA synthesis was examined by adding exogenous TGF-beta to plasma preparations. RESULTS: All plasma preparations induced a significant proliferative response compared to non-stimulated cells (P < 0.05). Maximum proliferation rate was obtained with PRGF with 2-fold or 4-fold platelet concentration. Exposure to PRGF stimulated VEGF synthesis exclusively in tendon cells (P < 0.05), which also exhibited a different pattern of HGF production (P < 0.05). PRGF enhanced HA synthesis (P < 0.05), but did not alter collagen I production. Platelet-secreted TGF-beta may be involved in HA, but not in type I procollagen synthesis. CONCLUSIONS: Optimizing composition and use of platelet-rich products is crucial to enhancing the therapeutic potential of this technology. Our data show that the biological effects of PRGF may depend on concentration of platelets and on the anatomical source of the cells.

Platelet-released growth factors enhance the secretion of hyaluronic acid and induce hepatocyte growth factor production by synovial fibroblasts from arthritic patients.

OBJECTIVES: Autologous platelet-secreted growth factors (GFs) may have therapeutic effects in osteoarthritis (OA) capsular joints via multiple mechanisms. Our aim was to examine the effect of a platelet-derived preparation rich in growth factors (PRGFs) in OA synovial cell biology. METHODS: Synovial cells were isolated from 10 osteoarthritic patients and cultured in serum-free media (basal conditions) and exposed to either a platelet-poor preparation or PRGF for 72 h. Cells activated with interleukin-1beta (IL-1beta) for 48 h were also exposed to PRGF. Changes in several events relevant to joint homeostasis including (i) hyaluronic acid (HA) secretion, (ii) the balance between metalloproteinase-1, -3 and -13 (MMP-1, MMP-3 and MMP-13) and tissue inhibitor-1 (TIMP-1) and (iii) the secretion of transforming growth factor-beta1(TGF-beta1), vascular endothelial growth factor (VEGF) and hepatocyte growth factor (HGF), were all assessed. RESULTS: PRGF significantly enhanced HA secretion compared with platelet-poor preparations, P < 0.05; at the same time release of TIMP-1, MMP-1, MMP-3 and MMP-13 were not affected. An increased HGF production was observed (P < 0.05) but VEGF and TGF-beta1 levels remained unchanged. PRGF significantly enhanced the secretion of HA induced by IL-1beta activation, P < 0.05, but it did not modify the IL-1beta-induced rise in MMP-1, MMP-3 and VEGF. In contrast, PRGF-induced HGF production was abolished by the presence of IL-1beta during PRGF treatment, P < 0.05. CONCLUSIONS: Intra-articular administration of PRGF might be beneficial in restoring HA concentration and switching angiogenesis to a more balanced status but does not halt the effects of IL-1beta on synovial cells.

Structural and functional preservation of specific sequences of DNA and mRNA in apoptotic bodies from ES cells.

Retinoic acid-induced apoptosis of embryonic stem (ES) cells is an experimental system which resembles the physiological programmed cell death that occurs during differentiation in embryonic development. Our aim was to analyze the involvement of epigenetic modifications such as DNA methylation and chromatin structure in the apoptotic process and to investigate the metabolic activity of apoptotic bodies. We found a relationship between DNA methylation and apoptosis, shown by a dose-dependent induction of apoptosis after treatment with the inhibitor of DNA methylation 5-aza-2'-deoxycytidine. Interestingly, we found a slight demethylation of specific sequences of the U2afl-rs1 imprinted gene in those RA treated cells which were specifically undergoing apoptosis. In addition, apoptotic bodies exhibited an unexpected open chromatin conformation accessible to the endonuclease DNase-I. Furthermore, we observed a structural and functional preservation of specific DNA sequences and mRNA. These results suggest that biological activities, such as transcription or protein synthesis, could be maintained even towards the end of the apoptotic process.