María Troya

Plasma Rich In Growth Factors Promote Gingival Tissue Regeneration by Stimulating Fibroblast Proliferation and Migration and by Blocking Transforming Growth Factor-β1-Induced Myodifferentiation

BACKGROUND Periodontitis involves inflammation and infection of the ligaments and bones that support the teeth. Gingival fibroblasts are the most abundant cells in periodontal tissue, and they play a role in maintaining the structural integrity of the tissue. Plasma rich in growth factors contain a pool of proteins and growth factors that promote wound healing and tissue regeneration. In the present study, we evaluate the potential of different formulations obtained with this approach to stimulate several biologic processes involved in wound healing, including fibroblast proliferation, migration, adhesion, and the autocrine release of some angiogenic factors and extracellular matrix components. Furthermore, the ability of this technology to prevent and inhibit transforming growth factor β1-induced myodifferentiation was also determined. METHODS Cell proliferation was evaluated through a colorimetric assay, cell migration was performed on culture inserts, and cell adhesion was studied through a fluorescence-based method. Enzyme-linked immunosorbent assay was used to determine some of the biomolecules released by gingival fibroblasts. Smooth muscle actin expression was assessed through immunofluorescence microscopy. RESULTS Results showed that plasma rich in growth factors significantly increased gingival fibroblast proliferation, migration, and cell adhesion on type I collagen matrix. In addition, it stimulated the autocrine expression of vascular endothelial growth factor, hepatocyte growth factor, and hyaluronic acid. The myofibroblast phenotype, which is characterized by expressing α-smooth muscle actin, was inhibited and reverted by treating with this technology. CONCLUSION These findings suggest that plasma rich in growth factors is capable of promoting regeneration of gingival connective tissue by stimulating some of the main processes involved in wound regeneration.

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.

An autologous platelet-rich plasma stimulates periodontal ligament regeneration.

BACKGROUND Regeneration of periodontal tissues is one of the most important goals for the treatment of periodontal disease. The technology of plasma rich in growth factors provides a biologic approach for the stimulation and acceleration of tissue healing. The purpose of this study is to evaluate the biologic effects of this technology on primary human periodontal ligament fibroblasts. METHODS The authors studied the response of periodontal ligament cells to this pool of growth factors on cell proliferation, cell migration, secretion of several biomolecules, cell adhesion, and expression of α2 integrin. Cell proliferation and adhesion were evaluated by means of a fluorescence-based method. Cell migration was performed on culture inserts. The release of different biomolecules by periodontal ligament fibroblasts was quantified through enzyme-linked immunosorbent assay. The α2 integrin expression was assessed through Western blot. RESULTS This autologous technology significantly stimulated cell proliferation, migration, adhesion, and synthesis of many growth factors from cells including vascular endothelial growth factor, thrombospondin 1, connective tissue growth factor, hepatocyte growth factor, and procollagen type I. The α2 integrin expression was lower in plasma rich in growth factor-treated cells compared to non-stimulated cells, although no statistically significant differences were observed. CONCLUSION This plasma rich in growth factors exerts positive effects on periodontal ligament fibroblasts, which could be positive for periodontal regeneration.

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.

The effect of different drugs on the preparation and biological outcomes of plasma rich in growth factors.

Chronic diseases are the major contributors to the global burden of disease and involve prodigious consumption of various drugs that usually affect platelet function. The autologous technology of plasma rich in growth factors (PRGF) provides a biological approach using autologous platelets as a reservoir and local delivery of proteins to promote tissue healing. The purpose of this study was to evaluate the effect of the consumption of acetylsalicylic acid, acenocoumarol and glucosamine sulfate on the preparation as well as on the biological properties of the PRGF technology. Clotting time and platelet activation of PRGF was evaluated. The latter was performed by flow cytometry. PRGF growth factor content and the release of various biomolecules by gingival fibroblasts were quantified by enzyme-linked immunosorbent assay. Cell proliferation was evaluated by means of a fluorescence-based method and cell migration was performed on culture inserts. None of the parameters evaluated was modified by the consumption of any of the three drugs tested; only the plasma of patients who had consumed acetylsalicylic acid and acenocoumarol expressed greater gingival fibroblast migration compared to plasma control. The intake of acetylsalicylic acid, acenocoumarol and glucosamine sulfate does not alter the preparation and biological properties of the autologous technology of PRGF.

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.

A novel personalized 3D injectable protein scaffold for regenerative medicine

Biomaterials should be designed to closely resemble the characteristics and functions of the native extracellular matrix to provide mechanical support and signals to direct biological events. Here we have developed a novel injectable plasma rich in growth factors (PRGF-Endoret)-based formulation that combines a thermal-denaturation step of plasma with an autologous fibrin crosslinking. Rheological and mechanical properties were evaluated. Additionally, the microstructure and biological capacity of the biomaterial was also characterized. This novel formulation exhibited ideal mechanical properties and a gel-like behavior with the ability to progressively release its growth factor load over time. The results also suggested that the novel injectable formulation is non-cytotoxic, biocompatible and suitable for cell ingrowth as it is deduced from the fibroblast proliferation within the scaffold. Finally, stimulation of both cell proliferation and matrix proteins synthesis demonstrated the regenerative potential of this autologous protein based injectable scaffold.Graphical abstract

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.