Mar Zalduendo

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.

Reciprocal Actions of Platelet-secreted Tgf-β1 on the Production of Vegf and Hgf by Human Tendon Cells

Background: Autologous platelet-rich matrices can be an aid in surgery by promoting and accelerating tissue healing because of the release of growth factors including transforming growth factor (TGF)-&bgr;1 and platelet-derived growth factor (PDGF) from platelet &agr;-granules. Methods: PDGF and TGF-&bgr;1 were quantified in supernatants collected from platelet-rich matrices prepared in vitro (n = 45 donors) and they correlated with the number of platelets and showed a constant ratio (p < 0.05). Tendon cells in culture were exposed to the supernatants (n = 4 donors) from either platelet-rich or platelet-poor matrices, differing in their content of platelet-secreted molecules. These treatments were modified by either neutralizing or adding PDGF or TGF-&bgr;1. Effects were compared in terms of proliferation, procollagen I, vascular endothelial growth factor (VEGF), and hepatocyte growth factor (HGF) production. Results: PDGF was a partial contributor to cell proliferation, whereas exogenous TGF-&bgr;1 acted as a negative modulator (p < 0.05). The production of type I collagen was similar regardless of differences in the concentration of TGF-&bgr;1. Moreover, addition of exogenous TGF-&bgr;1 promoted a significant increase in collagen synthesis only in the absence of other platelet-released substances (p < 0.05). Exogenous TGF-&bgr;1 increased the synthesis of VEGF and simultaneously abolished the production of HGF. Furthermore, antibody-mediated neutralization of TGF-&bgr;1 induced a decrease in VEGF synthesis and concomitantly a substantial production of HGF (p < 0.05). Conclusion: The balance between TGF-&bgr;1 and the pools of platelet-secreted molecules may have important therapeutic implications in the control of angiogenesis and fibrosis.

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)-1β 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.

Leukocyte Inclusion within a Platelet Rich Plasma-Derived Fibrin Scaffold Stimulates a More Pro-Inflammatory Environment and Alters Fibrin Properties

One of the main differences among platelet-rich plasma (PRP) products is the inclusion of leukocytes that may affect the biological efficacy of these autologous preparations. The purpose of this study was to evaluate whether the addition of leukocytes modified the morphological, biomechanical and biological properties of PRP under normal and inflammatory conditions. The release of pro-inflammatory cytokines from plasma rich in growth factors (PRGF) and leukocyte-platelet rich plasma (L-PRP) scaffolds was determined by enzyme-linked immunosorbent assay (ELISA) and was significantly increased under an inflammatory condition when leukocytes were included in the PRP. Fibroblasts and osteoblasts treated with L-PRP, under an inflammatory situation, underwent a greater activation of NFĸB pathway, proliferated significantly less and secreted a higher concentration of pro-inflammatory cytokines. These cellular events were assessed through Western blot and fluorimetric and ELISA methods, respectively. Therefore, the inclusion of leukocytes induced significantly higher pro-inflammatory conditions.

Relationship between Investigative Biomarkers and Radiographic Grading in Patients with Knee Osteoarthritis

Objective. To examine new investigative biomarkers and their relevance for radiographic severity in knee osteoarthritis. Methods. The group comprised 63 patients with 73 knees examined. Patients were divided according to radiographic severity to allow for comparison of biomarker levels. Hyaluronic acid (HA), matrix metalloproteases (MMP-1, MMP-3 and MMP-13), tissue inhibitors of metalloproteases (TIMP-1 and TIMP-2), platelet-derived growth factor (PDGF-AB), transformed growth factor (TGF-β), vascular endothelial growth factor (VEGF), hepatocyte growth factor (HGF) and insulin-like growth factor (IGF-I) were measured on synovial fluid and in plasma releasate at a single time point. Principal component analysis (PCA) followed by analysis of covariance were applied to evaluate data. Results. Four different groups of biomarker were identified in plasma releasates. The first (platelet number, PDGF-AB and TGF-β) and second groups (HA and IGF-I) were related to radiographic severity, P = .005 and P = .022, respectively. The third (MMP-1 and TIMP-2) and fourth groups (MMP-3 and TIMP-1) represented the catabolic balance, but were not associated to radiographic grading. Three different clusters of biomarkers were found in synovial fluid but did not show any significant association to radiographic grading. Conclusions. New imaging approaches to assess structural deterioration and correlation with biomarker levels are warranted to advance in OA research.

Implementation of a more physiological plasma rich in growth factor (PRGF) protocol: Anticoagulant removal and reduction in activator concentration

Abstract Plasma rich in growth factors (PRGF) is a biological therapy that uses patient’s own growth factors for promoting tissue regeneration. Given the current European regulatory framework in which anticoagulant solution in blood extraction tubes could be considered as a medicinal product, a new PRGF protocol has been developed. The actual protocol (PRGF-A) and the new one (PRGF-B) have been performed and compared under Good Laboratory Practices. PRGF-A protocol uses extraction tubes with 0.9 mL of trisodium citrate as anticoagulant and 50 μL of calcium chloride/mL PRGF to activate it. The PRGF-B reduces the amount of sodium citrate and calcium chloride to 0.4 mL and to 20 μL, respectively. Basic hematological parameters, platelet function, the scaffold obtaining process, growth factors content, and the biological effect were compared between both PRGF obtaining protocols. Results: PRGF-B protocol led to a statistically significant higher enrichment and recovery of platelets regarding to the PRGF-A. Hypotonic stress response by platelets was significantly better in the new protocol. A statistically significant decrease in the basal platelet activation status of PRGF-B compared to PRGF-A was also observed. The duration of the lag phase in the platelet aggregation assay was statistically lower for the PRGF-B protocol. Both the clotting and the clot retraction time were significantly reduced in the B protocol. A higher growth factor concentration was detected in the plasma obtained using the PRGF-B protocol. The new PRGF obtaining protocol, with a reduction in the amount of anticoagulant and activator, has even improved the actual one.

PRGF exerts a cytoprotective role in zoledronic acid-treated oral cells.

ObjectivesBisphosphonates-related osteonecrosis of the jaw (BRONJ) is a common problem in patients undergoing long-term administration of highly potent nitrogen-containing bisphosphonates (N-BPs). This pathology occurs via bone and soft tissue mechanism. Zoledronic acid (ZA) is the most potent intravenous N-BP used to prevent bone loss in patients with bone dysfunction. The objective of this in vitro study was to evaluate the role of different ZA concentrations on the cells from human oral cavity, as well as the potential of plasma rich in growth factors (PRGF) to overcome the negative effects of this BP.Material and methodsPrimary human gingival fibroblasts and primary human alveolar osteoblasts were used. Cell proliferation was evaluated by means of a fluorescence-based method. A colorimetric assay to detect DNA fragmentation undergoing apoptosis was used to determine cell death, and the expression of both NF-κB and pNF-κB were quantified by Western blot analysis.ResultsZA had a cytotoxic effect on both human gingival fibroblasts and human alveolar osteoblasts. This BP inhibits cell proliferation, stimulates apoptosis, and induces inflammation. However, the addition of PRGF suppresses all these negative effects of the ZA.ConclusionsPRGF shows a cytoprotective role against the negative effects of ZA on primary oral cells.Clinical relevanceAt present, there is no definitive treatment for bisphosphonates-related osteonecrosis of the jaw (BRONJ), being mainly palliatives. Our results revealed that PRGF has a cytoprotective role in cells exposed to zoledronic acid, thus providing a reliable adjunctive therapy for the treatment of BRONJ pathology.

Progress in the Use of Autologous Regenerative Platelet-based Therapies in Implant Dentistry.

The field of medicine is rapidly moving towards the development of personalized treatments and non-invasive tools to achieve a more predictable and optimal tissue regeneration. In this sense, the goal of periodontal healing is to arrest disease progression and functionally regenerate all the tissues that comprise the periodontium. The latter implies a well-orchestrated interaction among oral cells, growth factors and extracellular matrix. Although several procedures are performed in an attempt to regenerate lost periodontal tissue, outcomes are not always predictable. Growth factors represent a class of biologically active polypeptides that have a critical role in the healing process. Their use provides a new paradigm to understand the regenerative medicine. The use of platelet- rich plasma (PRP) products as a local source and delivery system of autologous growth factors has emerged recently. Among them, PRGF stands for its remarkable stimulatory effect on oral tissue regeneration, making it a very safe and successful tool with a great value in Dentistry.

Effects of anti-aggregant, anti-inflammatory and anti-coagulant drug consumption on the preparation and therapeutic potential of plasma rich in growth factors (PRGF)

Abstract The prevalence and incidence of trauma-related injuries, coronary heart disease and other chronic diseases increase dramatically with age. This population sector is therefore a regular consumer of different types of drugs that may affect platelet aggregation and the coagulation cascade. We have evaluated whether the consumption of acetylsalicylic acid, acenocoumarol, glucosamine sulfate and chondroitin sulfate, and therefore their presence in blood, could interfere with the preparation and biological outcomes of plasma rich in growth factors (PRGF). Clotting time, clot retraction and platelet activation of PRGF was evaluated. PRGF growth factor content and the release of different biomolecules by tendon fibroblasts were also quantified, as well as cell proliferation and cell migration. The preparation and biological potential of PRGF is not affected by the intake of the evaluated drugs, and solely its angiogenic potential and its capacity to induce HA and fibronectin synthesis, is reduced in patients taking anti-coagulants.

Personalized plasma-based medicine to treat age-related diseases

As social and health needs are changing, new challenges to develop innovative alternatives arise to address unmet medical needs. Personalized medicine is emerging as a promising and appealing therapeutic option. The use of patients own plasma and platelets as therapeutics is providing new avenues in the treatment of acute and chronic tissue injuries by promoting tissue repair and regeneration. Plasma and platelet-based therapies mimic the physiological repair process by releasing autologous growth factors and creating a natural, biodegradable and transient scaffold that acts as transient matrix. This review summarizes the recent advances and challenges in the field of personalized plasma-based medicine and its potential to treat age-related diseases.