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Platelet-Poor Plasma as a Supplement for Fibroblasts Cultured in Platelet-Rich Fibrin.

ABSTRACT: The aim of this study was to evaluate the proliferation and adhesion of mesenchymal cells (3T3/NIH) in Dulbecco's Modified Eagle Medium(DMEM) supplemented with Platelet-Poor Plasma (PPP) in aPlatelet-Rich Fibrin (PRF) scaffold. Human blood was obtained and processed in a centrifuge considering the equation G=1.12xRx(RPM/1000)2 to obtain PRF and PPP.Cell adhesion and maintenance analyses were performed by MTTassays in a 96 well plate withsupplemented DMEM: PPP (90:10) for 24 hours. Besides, the PRF was deposited in a 48 well plate and 10x104 cells were seeded above each PRF (n=3) with 800µl of DMEM: PPP (90:10) and cultured for 7 days. Histological analysis and the immunohistochemical staining for Vimentin were performed. Results were analyzed by one-way ANOVA in Stata12®. A significant decrease (p<0.05) of cells adhesion in relationship to FBSwas observed. However, a similar ability of cell-maintenance for PPP 10% was observed (P>0.05). Fibroblasts culture for 7 days in PRF supplemented with PPP 10% was possible, showing positive staining for Vimentin. Therefore, PPP cell supplementation decreased the initial adhesion of cells but was able to maintain the proliferation of adhered cells and able to support their viability in PRF.It seems that this method has many clinical advantagessince it provides an autologous and natural scaffold with their respective supplement for cell culture by only one process, without using xenogeneic compounds. This could improve the potential of clinical translational therapies based on the use of PRF cultured cells, promoting the regenerative potential for future use in medicine and dentistry.

SUBMITTER: Chisini LA

PROVIDER: S-EPMC5548224 | biostudies-other | 2017 Jun

REPOSITORIES: biostudies-other

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Proteomic analysis of platelet-rich and platelet-poor plasma.

Project description:BackgroundAutologous blood products, such as platelet-rich plasma (PRP) are commercial products broadly used to accelerate healing of tissues after injuries. However, their content is not standardized and significantly varies in composition, which may lead to differences in clinical efficacy. Also, the underlying molecular mechanisms for therapeutic effects are not well understood.PurposeA proteomic study was performed to compare the composition of low leukocyte PRP, platelet poor plasma (PPP), and blood plasma. Pathway analysis of the proteomic data was performed to evaluate differences between plasma formulations at the molecular level. Low abundance regulatory proteins in plasma were identified and quantified as well as cellular pathways regulated by those proteins.MethodsQuantitative proteomic analysis, using multiplexed isotopically labeled tags (TMT labeling) and label-free tandem mass spectrometry, was performed on plasma, low leukocyte PRP, and PPP. Plasma formulations were derived from two blood donors (one donor per experiment). Pathway analysis of the proteomic data identified the major differences between formulations.ResultsNearly 600 proteins were detected in three types of blood plasma formulations in two experiments. Identified proteins showed more than 50% overlap between plasma formulations. Detected proteins represented more than 100 canonical pathways, as was identified by pathway analysis. The major pathways and regulatory molecules were linked to inflammation.ConclusionThree types of plasma formulations were compared in two proteomic experiments. The most represented pathways, such as Acute Phase Response, Coagulation, or System of the Complement, had many proteins in common in both experiments. In both experiments plasma sample sets had the same direction of biochemical pathway changes: up- or down-regulation. The most represented biochemical pathways are linked to inflammation.

| S-EPMC7770407 | biostudies-literature

Platelet-Rich Fibrin Increases BMP2 Expression in Oral Fibroblasts via Activation of TGF-β Signaling.

Project description:Solid platelet-rich fibrin (PRF), consisting of coagulated plasma from fractionated blood, has been proposed to be a suitable carrier for recombinant bone morphogenetic protein 2 (BMP2) to target mesenchymal cells during bone regeneration. However, whether solid PRF can increase the expression of BMPs in mesenchymal cells remains unknown. Proteomics analysis confirmed the presence of TGF-β1 but not BMP2 in PRF lysates. According to the existing knowledge of recombinant TGF-β1, we hypothesized that PRF can increase BMP2 expression in mesenchymal cells. To test this hypothesis, we blocked TGF-β receptor 1 kinase with SB431542 in gingival fibroblasts exposed to PRF lysates. RT-PCR and immunoassays confirmed that solid PRF lysates caused a robust SB431542-dependent increase in BMP2 expression in gingival fibroblasts. Additionally, fractions of liquid PRF, namely platelet-poor plasma (PPP) and the buffy coat (BC) layer, but not heat-denatured PPP (Alb-gel), greatly induced the expression of BMP2 in gingival fibroblasts. Even though PRF has no detectable BMPs, PRF lysates similar to recombinant TGF-β1 had the capacity to provoke canonical BMP signaling, as indicated by the nuclear translocation of Smad1/5 and the increase in its phosphorylation. Taken together, our data suggest that PRF can activate TGF-β receptor 1 kinase and consequently induce the production of BMP2 in cells of the mesenchymal lineage.

| S-EPMC8347014 | biostudies-literature

Initial Healing Effects of Platelet-Rich Plasma (PRP) Gel and Platelet-Rich Fibrin (PRF) in the Deep Corneal Wound in Rabbits.

Project description:Platelet concentrates (PCs), including platelet-rich plasma (PRP) gel and platelet-rich fibrin (PRF), are autologous blood-derived biomaterials containing numerous growth factors. This study aimed to evaluate the initial healing effects of PRP gel and PRF on deep corneal wounds. Thirty-three eyes from New Zealand white rabbits were divided into four groups: group 1, lamellar keratectomy (LK); group 2, LK + commercial porcine small intestinal submucosal membrane (SIS); group 3, LK + SIS + PRP gel; and group 4, LK + SIS + PRF. Postoperative clinical and histological findings were observed for eight weeks. Group 1 showed no neovascularization during the observation period, and incompletely recovered with a thin cornea. Group 2 showed active healing through neovascularization, and a thick cornea was regenerated through the sufficient generation of myofibroblasts. Although group 3 showed a healing effect similar to that of group 2, angiogenesis and subsequent vessel regression were promoted, and corneal opacity improved more rapidly. In group 4, angiogenesis was promoted during initial healing; however, the incidence of complications, such as inflammation, was high, and myofibroblasts were hardly generated in the corneal stroma, which adversely affected remodeling. In conclusion, while PRP gel is a safe surgical material for promoting remodeling through vascular healing and myofibroblast production in deep corneal wounds, the use of PRF is not recommended.

| S-EPMC9405118 | biostudies-literature

Platelet-rich fibrin suppresses in vitro osteoclastogenesis.

Project description:BACKGROUND:Platelet-rich fibrin (PRF) membranes can preserve alveolar ridge dimension after tooth extraction. Thus, it can be presumed that PRF suppresses the catabolic events that are caused by osteoclastic bone resorption. METHODS:To address this possibility, we investigated the impact of soluble extracts of PRF membranes on in vitro osteoclastogenesis in murine bone marrow cultures. Osteoclastogenesis was induced by exposing murine bone marrow cultures to receptor activator of nuclear factor kappa B ligand (RANKL), macrophage colony-stimulating factor (M-CSF) and transforming growth factor-beta 1 (TGF-?1) in the presence or absence of PRF. Osteoclastogenesis was evaluated based on histochemical, gene expression, and resorption analysis. Viability was confirmed by formation of formazan crystals, live-dead staining and caspase-3 activity assay. RESULTS:We report here that in vitro osteoclastogenesis is greatly suppressed by soluble extracts of PRF membranes as indicated by tartrate-resistant acid phosphatase (TRAP) staining and pit formation. In support of the histochemical observations, soluble extracts of PRF membranes decreased expression levels of the osteoclast marker genes TRAP, Cathepsin K, dendritic cell-specific transmembrane protein (DCSTAMP), nuclear factor of activated T-cells (NFATc1), and osteoclast-associated receptor (OSCAR). PRF membranes, however, cannot reverse the process once osteoclastogenesis has evolved. CONCLUSION:These in vitro findings indicate that PRF membranes can inhibit the formation of osteoclasts from hematopoietic progenitors in bone marrow cultures. Overall, our results imply that the favorable effects of PRF membranes in alveolar ridge preservation may be attributed, at least in part, by the inhibition of osteoclastogenesis.

| S-EPMC7155126 | biostudies-literature

Evaluation of blood clot, platelet-rich plasma, and platelet-rich fibrin-mediated regenerative endodontic procedures in teeth with periapical pathology: a CBCT study.

Project description:ObjectivesThis study compared the clinical and radiological outcomes of regenerative endodontic procedures (REPs) using blood clots (BCs), platelet-rich plasma (PRP), and platelet-rich fibrin (PRF) through intraoral periapical radiography (IOPAR) and cone-beam computed tomography (CBCT).Materials and methodsForty-five single-rooted necrotic teeth with periapical pathology were randomly allocated to receive BC, PRP, or PRF as an individual scaffold. Outcomes were evaluated in 35 teeth in 23 patients with a follow-up period of 12-24 months through qualitative IOPAR scoring and quantitative CBCT measurements. Healing of periapical lesions and in immature teeth, changes in the apical foramen diameter (AFD), root wall thickness (RWT), and root length (RL) were assessed. A p value less than 0.05 was considered to indicate statistical significance.ResultsAll teeth were asymptomatic except 1 in the PRP group. Periapical lesion healing was seen in all except 2 teeth in the BC group and 3 in the PRP group. Both IOPAR and CBCT revealed no significant differences in bone healing or changes in AFD, RWT, and RL among the 3 groups. A positive pulp sensibility response to the cold test was seen in 2 teeth in the BC group, but none to the electric pulp test. Intracanal calcification (ICC) was evident in more teeth in the BC group than in the PRP and PRF groups, and was also significantly higher in immature teeth.ConclusionsOur results revealed that BC, PRP, and PRF have similar potential as scaffolds in REPs, and ICC may be a concern for long-term outcomes.

| S-EPMC9715374 | biostudies-literature

Comparative effectiveness of hyaluronic acid, platelet-rich plasma, and platelet-rich fibrin in treating temporomandibular disorders: a systematic review and network meta-analysis.

Project description:ObjectiveThis study aims to compare the efficacy of intra-articular injections of hyaluronic acid (HA), platelet-rich plasma (PRP), and platelet-rich fibrin (PRF) for treating temporomandibular disorders (TMDs) and summarize their mechanisms of action.MethodsRandomized controlled trials (RCTs) published until November 13, 2021, were identified using electronic and manual searches. Each study was evaluated for the risk of bias using the Cochrane risk of bias tool. The studies found via searches were categorized by follow-up time (1, 3, or 6 months). Evidence quality was graded according to the GRADE system.ResultsTwelve RCTs were included that involved 421 patients with TMD. The network meta-analysis showed that all treatment groups improved compared to the placebo groups in terms of pain and maximal mouth opening (MMO). For pain evaluated via the visual analog scale, PRF exhibited better analgesic effects than PRP or HA after 1 and 3 months. PRP appeared to be more effective than PRF was after 6 months but there were no statistically significant differences between the two. For MMO, the effect of PRP was superior to those of PRF and HA after 1 month. However, after 3 and 6 months, PRF provided more encouraging results in improving MMO.ConclusionPRP and PRF exhibited similar short-term efficacy in treating TMD, while PRF was more advantageous in terms of long-term efficacy. Therefore, PRF was recommended for treating TMD.

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Autologous platelet-rich plasma: a biological supplement to enhance adipose-derived mesenchymal stem cell expansion.

Project description:Currently the use of non-autologous cell culture media (e.g., animal-derived or allogeneic serum) for clinical applications of mesenchymal stem cells (MSCs) is criticized by regulatory agencies. Autologous platelet-rich plasma (PRP) is proposed as a safer alternative medium supplement for adipose-derived mesenchymal stem cells (AT-MSC) culture. To study its efficiency on cell proliferation, AT-MSCs were cultured for 10 days in media supplemented with different concentrations of autologous non-activated PRP (nPRP) or thrombin-activated PRP (tPRP) (1-60%). AT-MSC proliferation, cell phenotype, multipotency capacity, and chromosome stability were assessed and compared to AT-MSCs expanded in a classical medium supplemented with 10% of fetal bovine serum (FBS). Culture media supplemented with nPRP showed dose-dependent higher AT-MSC proliferation than did FBS or tPRP. Twenty percent nPRP was the most effective concentration to promote cell proliferation. This condition increased 13.9 times greater AT-MSC number in comparison to culture with FBS, without changing the AT-MSC phenotype, differentiation capacity, and chromosome status. We concluded that 20% autologous nPRP is a safe, efficient, and cost-effective supplement for AT-MSC expansion. It should be considered as an alternative to FBS or other nonautologous blood derivatives. It could serve as a potent substitute for the validation of future clinical protocols as it respects good manufacturing practices and regulatory agencies' standards.

| S-EPMC4346379 | biostudies-literature

Angiogenic Properties of 'Leukocyte- and Platelet-Rich Fibrin'.

Project description:Leukocyte- and Platelet-Rich Fibrin (L-PRF) is an autologous platelet concentrate, consisting of a fibrin matrix enriched with platelets, leukocytes and a plethora of cytokines and growth factors. Since L-PRF is produced bedside from whole blood without the use of an anti-coagulant, it is becoming a popular adjuvant in regenerative medicine. While other types of platelet concentrates have been described to stimulate blood vessel formation, little is known about the angiogenic capacities of L-PRF. Therefore, this study aimed to fully characterize the angiogenic potential of L-PRF. With an antibody array, the growth factors released by L-PRF were determined and high levels of CXC chemokine receptor 2 (CXCR-2) ligands and epidermal growth factor (EGF) were found. L-PRF induced in vitro key steps of the angiogenic process: endothelial proliferation, migration and tube formation. In addition, we could clearly demonstrate that L-PRF is able to induce blood vessel formation in vivo, the chorioallantoic membrane assay. In conclusion, we could demonstrate the angiogenic capacity of L-PRF both in vitro and in vivo, underlying the clinical potential of this easy-to-use platelet concentrate.

| S-EPMC6168453 | biostudies-literature

Autologous platelet-rich fibrin stimulates canine periodontal regeneration.

Project description:Platelet-rich fibrin (PRF) provides a scaffold for cell migration and growth factors for promoting wound healing and tissue regeneration. Here, we report using PRF in periodontal healing after open flap debridement (OFD) in canine periodontitis. A split-mouth design was performed in twenty dogs. Forty periodontitis surgical sites were randomly categorized into 2 groups; OFD alone and OFD with PRF treatment. Clinical parameters of periodontal pocket depth, gingival index, and the cemento-enamel junction-alveolar bone levels/root length ratio were improved in the OFD + PRF group. The OFD + PRF group also demonstrated a dramatically decreased inflammatory score compared with the OFD group. Collagen accumulation was improved in the OFD + PRF group at later time points compared with baseline. PRF application also significantly reduced inflammatory cytokine expression (TNFA and IL1B), and promoted the expression of collagen production-related genes (COL1A1, COL3A1, and TIMP1) and growth factors (PDGFB, TGFB1, and VEGFA). These findings suggest that PRF combined with OFD provides a new strategy to enhance the overall improvement of canine periodontitis treatment outcomes, especially in terms of inflammation and soft tissue healing. Therefore, PRF use in treating periodontitis could play an important role as a regenerative material to improve canine periodontitis treatment.

| S-EPMC7002419 | biostudies-literature

Influence of Platelet-Rich and Platelet-Poor Plasma on Endogenous Mechanisms of Skeletal Muscle Repair/Regeneration.

Project description:The morpho-functional recovery of injured skeletal muscle still represents an unmet need. None of the therapeutic options so far adopted have proved to be resolutive. A current scientific challenge remains the identification of effective strategies improving the endogenous skeletal muscle regenerative program. Indeed, skeletal muscle tissue possesses an intrinsic remarkable regenerative capacity in response to injury, mainly thanks to the activity of a population of resident muscle progenitors called satellite cells, largely influenced by the dynamic interplay established with different molecular and cellular components of the surrounding niche/microenvironment. Other myogenic non-satellite cells, residing within muscle or recruited via circulation may contribute to post-natal muscle regeneration. Unfortunately, in the case of extended damage the tissue repair may become aberrant, giving rise to a maladaptive fibrotic scar or adipose tissue infiltration, mainly due to dysregulated activity of different muscle interstitial cells. In this context, plasma preparations, including Platelet-Rich Plasma (PRP) and more recently Platelet-Poor Plasma (PPP), have shown advantages and promising therapeutic perspectives. This review focuses on the contribution of these blood-derived products on repair/regeneration of damaged skeletal muscle, paying particular attention to the potential cellular targets and molecular mechanisms through which these products may exert their beneficial effects.

| S-EPMC6387315 | biostudies-literature

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