Platelet-rich Fibrin Clot Research Articles

Comparison of platelet rich fibrin and collagen as osteoblast‐seeded scaffolds for bone tissue engineering applications

The loss of jaw bone caused by different kinds of pathologies leads to dysfunction and reduced quality of life in affected patients. Thus, the pivotal goal in bone tissue engineering is to reconstruct these defects. The essential precondition for new tissue generation is an extracellular matrix which acts as a scaffold so that cells can migrate, differentiate, and proliferate. Fibrin, a biopolymer responsible for blood clot formation, has been shown to be suitable for tissue engineering applications. The aim of the present study is a comparison of platelet rich fibrin (PRF) with the commonly used collagen membrane BioGide(®) as a scaffold for human osteoblast cell seeding for bone tissue engineering. Human osteoblasts were cultured with eluates from PRF (n=7) and BioGide(®) (n=8) membranes incubated in serum-free cell culture medium. Vitality of these cells was assessed by fluorescein diacetate and propidium iodide staining, biocompatibility with the lactate dehydrogenase test and proliferation levels with the MTT ([3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium-bromide]), and BrdU (5-bromo-2-deoxyuridine) tests. In addition, human osteoblasts were seeded on both membrane systems and cell growth was compared by the water soluble tetrazolium (WST-1) (4-[3-(4-iodophenyl)-2-(4-nitrophenyl)-2H-5-tetrazolio]-1,3-benzene disulfonate) test and scanning electron microscopy (SEM). Osteoblastic differentiation was assessed by alkaline phosphatase activity measured by ELISA in the supernatant of osteoblasts cultivated on PRF membranes (n=10), PRF clots (n=10), and BioGide(®) membranes (n=10). Lactate dehydrogenase test values were higher for PRF compared to BioGide(®) . The BrdU test showed superior cell growth after cultivation in eluate from PRF than in eluate from BioGide(®) . The WST-1 assay demonstrated superior cell proliferation on PRF than on BioGide(®) . SEM revealed osteoblast colonization of both membranes. Cultivation of osteoblasts on PRF membranes and PRF clots showed significantly higher alkaline phosphatase activity than on BioGide(®) membranes. Metabolic activity and proliferation of human osteoblast cells in vitro were supported to a significant higher extent by eluates from PRF membranes. Both membranes are suitable as scaffolds for cultivation of human osteoblast cells in vitro; proliferation was significant higher on PRF membranes and on PRF clot than on BioGide(®) membranes.

Dissolution of pre-existing platelet thrombus by synergistic administration of low concentrations of bifunctional antibodies against β3 integrin.

Most antithrombotic approaches target prevention rather than the more clinically relevant issue of resolution of an existing thrombus. In this study, we describe a novel and effective therapeutic strategy for ex vivo clearance of pre-existing platelet thrombus by the combination of two bifunctional platelet GPIIIa49-66 ligands that target different parts of the arterial thrombus. We produced an additional GPIIIa49-66 agent (named APAC), which homes to activated platelets. Like our previously described SLK (which targets newly deposited fibrin strands surrounding the platelet thrombus), APAC destroys platelet aggregates ex vivo in an identical fashion with 85% destruction of platelet aggregates at 2 hours. The combined application of APAC and SLK demonstrated a ∼2 fold greater platelet thrombus dissolution than either agent alone at a low concentration (0.025 µM). Platelet-rich clot lysis experiments demonstrated the time required for 50% platelet-rich fibrin clot lysis (T50%) by APAC (95±6.1 min) or SLK (145±7.1 min) was much longer than that by combined APAC+SLK (65±7.6 min) at the final concentration of 0.025 µM (APAC+SLK vs APAC, p<0.05; APAC+SLK vs SLK, p<0.01). Thus these low concentrations of a combination of both agents are likely to be more effective and less toxic when used therapeutically in vivo.

Human blood-derived fibrin releasates: Composition and use for the culture of cell lines and human primary cells

We have evaluated the capacity of two human blood fractions to substitute for FBS as growth medium supplement for human and animal cell cultures. Non-anticoagulated blood from volunteer donors (N = 13) was centrifuged to isolate a supernatant serum (SS) and a platelet-rich fibrin (PRF) clot which was squeezed to extract the releasate (PRFR). Both materials were characterized for the content in PDGF-AB, TGF-β1, VEGF, bFGF, EGF, IGF, total protein, albumin, IgG, IgM IgA, fibrinogen, cholesterol, triglycerides, various chemistry analytes and hemoglobin. Cell growth promoting activity of pooled SS and PRFR at 1, 5, and 10% in growth medium was evaluated over 7 days using human (HEK293, MG-63) and animal (SIRC, 3T3) cell lines and two human primary cultures (gingival fibroblasts and periodontal ligaments). Viable cell count was compared to that in cultures in FBS free-medium and 10% FBS supplement. SS and PRFR at 1–10% stimulated cell growth significantly more than FBS-free medium and in a way similar to 10% FBS in all cultures apart from 3T3. These two human blood-derived fibrin releasates are equally efficient to substitute for FBS as supplement for cell cultures and could be useful for specialized applications in regenerative medicine, dentistry and oral implantology, or cell therapy.

Platelet-rich Fibrin Increases Proliferation and Differentiation of Human Dental Pulp Cells

Introduction Platelet-rich fibrin (PRF) by Choukroun's technique is derived from an autogenous preparation of concentrated platelets without any manipulation. When delicately pressed between 2 gauzes, the PRF clot becomes a strong membrane with high potential in clinical application. However, the effect of PRF on dental pulp cells (DPCs) remains to be elucidated. This study was to determine the biological effects of PRF on DPCs. Methods PRF samples were obtained from 6 healthy volunteers. Human DPCs were derived from healthy individuals undergoing extraction for third molars. Cell proliferation resulting from PRF was evaluated by colorimetric assay. Western blot was used to evaluate the expression of osteoprotegerin (OPG). Alkaline phosphatase (ALP) activity was examined by substrate assay. Results PRF did not interfere with cell viability of DPCs ( P > .05). DPCs were observed to attach at the edges of PRF by phase-contrast microscopy. PRF was found to increase DPC proliferation during 5-day incubation period ( P < .05). PRF was found to increase OPG expression in a time-dependent manner ( P < .05). ALP activity was also significantly up-regulated by PRF ( P < .05). Conclusions PRF was demonstrated to stimulate cell proliferation and differentiation of DPCs by up-regulating OPG and ALP expression. These findings might serve as a basis for preclinical studies that address the role of PRF in reparative dentin formation.

Platelet‐rich fibrin modulates the expression of extracellular signal‐regulated protein kinase and osteoprotegerin in human osteoblasts

Platelet-rich fibrin (PRF) by Choukroun's technique is produced in a totally natural manner, without using anticoagulant during blood harvest nor bovine thrombin and calcium chloride for platelet activation and fibrin polymerization. When delicately pressed between two gauzes, the PRF clot becomes a strong membrane with high potential in clinical application and tissue engineering. In this study, blood collection was carried out from healthy volunteers. Osteoblast cell line U2OS was used to evaluate the cell proliferation resulting from PRF by using colorimetric assay. Western blot was employed to evaluate the expression of phosphorylated extracellular signal-regulated protein kinase (p-ERK), receptor activator of nuclear factor-kappa B ligand (RANKL), and osteoprotegerin (OPG) in U2OS cells. PRF was found to increase osteoblast proliferation during 5 day incubation period (p < 0.05). PRF was found to increase ERK phosphorylation in U2OS cells (p < 0.05). OPG was significantly elevated by the stimulation with PRF (p < 0.05). However, there was no significant change in RANKL expression (p > 0.05). Taken together, PRF can stimulate osteoblasts proliferation. The activation of p-ERK and OPG expression by PRF suggests a potential role for new bone formation. The application of PRF may provide the benefit for the bone regeneration.

Three‐Dimensional Architecture and Cell Composition of a Choukroun's Platelet‐Rich Fibrin Clot and Membrane

Platelet-rich fibrin (PRF; Choukroun's technique) is a second-generation platelet concentrate for surgical use. This easy protocol allows the production of leukocyte and platelet-rich fibrin clots and membranes starting from 10-ml blood samples. The purposes of this study were to determine the cell composition and three-dimensional organization of this autologous biomaterial and to evaluate the influence of different collection tubes (dry glass or glass-coated plastic tubes) and compression procedures (forcible or soft) on the final PRF-membrane architecture. After centrifugation, blood analyses were performed on the residual waste plasmatic layers after collecting PRF clots. The PRF clots and membranes were processed for examination by light microscopy and scanning electron microscopy. Approximately 97% of the platelets and >50% of the leukocytes were concentrated in the PRF clot and showed a specific three-dimensional distribution, depending on the centrifugation forces. Platelets and fibrin formed large clusters of coagulation in the first millimeters of the membrane beyond the red blood cell base. The fibrin network was very mature and dense. Moreover, there was no significant difference in the PRF architecture between groups using the different tested collection tubes and compression techniques, even if these two parameters could have influenced the growth factor content and biologic matrix properties. The PRF protocol concentrated most platelets and leukocytes from a blood harvest into a single autologous fibrin biomaterial. This protocol offers reproducible results as long as the main production principles are respected.

Sinus Floor Augmentation With Simultaneous Implant Placement Using Choukroun's Platelet‐Rich Fibrin as the Sole Grafting Material: A Radiologic and Histologic Study at 6 Months

Sinus augmentation with simultaneous implant placement without bone graft material is a hotly debated technique. This technique could be improved and secured by the use of an autologous leukocyte- and platelet-rich fibrin (PRF) (Choukroun's technique) concentrate. The objectives of this study were to assess the relevance of PRF clots and membranes as the sole filling material during a lateral sinus lift with immediate implantation using radiologic and histologic analyses in a case series. Twenty-five sinus elevations with simultaneous implantation were performed on 20 patients with Choukroun's PRF as the sole filling biomaterial. For each patient, a presurgical exam and a 6-month post-surgical radiologic exam were performed with a panoramic x-ray and three-dimensional volumetric computed radiography (VCR) to evaluate the subsinus residual bone height and the final bone gain around the implants. In nine patients, 6 months after the sinus lift, bone biopsies were collected on the buccal wall of the alveolar ridge at the level of the osteotomy window, and evaluated by histomorphometry. In this study, 41 implants from three different systems with different screw designs (Biomet 3I Nanotite, MIS Seven, Intra-Lock Ossean) were placed. All implants were inserted in residual bone height between 1.5 and 6 mm (mean +/- SD: 2.9 +/- 0.9 mm). The final bone gain was always very significant (between 7 and 13 mm [mean +/- SD: 10.1 +/- 0.9 mm]). No implant was lost. After radiologic analyses, the position of the final sinus floor was always in the continuation of the end of the implant. All biopsies showed well organized and vital bone. From a radiologic and histologic point of view at 6 months after surgery, the use of PRF as the sole filling material during a simultaneous sinus lift and implantation stabilized a high volume of natural regenerated bone in the subsinus cavity up to the tip of the implants. Choukroun's PRF is a simple and inexpensive biomaterial, and its systematic use during a sinus lift seems a relevant option, particularly for the protection of the Schneiderian membrane.

Stability of an autologous platelet clot in the pericardial sac: An experimental and clinical study

Autologous platelet clots serve as slow-release delivery systems for platelet-derived growth factors and cytokines. Their application to the pericardial sac might facilitate salvage and repair of ischemically injured myocardium. However, little is known about platelet clot stability in the pericardial sac. We investigated the stability of platelet clots in vitro and after administration to the pericardial sac in pigs and patients. In 5 Yorkshire-Landrace pigs and 10 patients, in vitro manufactured autologous platelet gel (Medtronic Magellan Platelet Separator) and platelet-rich fibrin (Vivolution Vivostat System) were administered to the pericardial sac for 30 minutes. Two antifibrinolytics (tranexamic acid and aprotinin) were tested for their capacity to stabilize autologous platelet gel. In vitro clots, incubated at 37 degrees C for 48 hours, served as controls. Clot weight was measured before and after administration. In vitro, autologous platelet gel clots of either formula liquefied almost entirely within 60 minutes whereas platelet-rich fibrin clots remained intact. In the pig, platelet clot weight decreased to 16.7% +/- 7.8% (P < .05) and 66.4% +/- 3.2% (P < .05) of initial clot weight for autologous platelet gel and platelet-rich fibrin, respectively. Addition of antifibrinolytics to autologous platelet gel did not reduce clot degradation significantly. In patients, autologous platelet gel and platelet-rich fibrin clot weight remained 9.0% +/- 1.5% (P < .05) and 73.7% +/- 2.6% (P < .05) of initial clot weight, respectively. Autologous platelet gel is unstable both in vitro and in vivo, whereas platelet-rich fibrin remains intact in vitro and, compared with autologous platelet gel, is less subject to degradation in pigs and in patients.

Clinical Evaluation of a Modified Coronally Advanced Flap Alone or in Combination With a Platelet‐Rich Fibrin Membrane for the Treatment of Adjacent Multiple Gingival Recessions: A 6‐Month Study

The aim of this study was to determine whether the addition of an autologous platelet-rich fibrin clot (PRF) to a modified coronally advanced flap (MCAF) (test group) would improve the clinical outcome compared to an MCAF alone (control group) for the treatment of multiple gingival recessions. Twenty subjects, presenting three adjacent Miller Class I or II multiple gingival recessions of similar extent on both sides of the mouth, were enrolled in the study. The mean recession value at baseline was 2.9 +/- 1.1 mm for test sites and 2.5 +/- 0.9 mm for control sites. Each patient was treated on both sides by an MCAF technique; the combination treatment (with a PRF membrane) was applied on the test side. Probing depth (PD), recession width, clinical attachment level (CAL), keratinized gingival width, and gingival/mucosal thickness (GTH) were measured at baseline and at 6 months post-surgery. Gingival recession was measured at baseline and at 1, 3, and 6 months post-surgery. Mean root coverage after 1, 3, and 6 months was 81.0% +/- 16.6%, 76.1% +/- 17.7%, and 80.7% +/- 14.7%, respectively, at the test sites and 86.7% +/- 16.6%, 88.2% +/- 16.9%, and 91.5% +/- 11.4%, respectively, at the control sites. Differences between the two groups were statistically significant at 3 and 6 months. At 6 months, complete root coverage was obtained at 74.6% of the sites treated with the control procedure but at only 52.2% of the experimental sites. At 6 months, the increase in GTH was statistically significant when comparing the test sites (from 1.1 +/- 0.3 mm at baseline to 1.4 +/- 0.5 mm at 6 months) to the control sites (from 1.1 +/- 0.3 mm at baseline to 1.1 +/- 0.3 mm at 6 months). In the case of PD, there was no significant difference between the two groups at 6 months, but a significant CAL gain in favor of the control group was observed at that time. MCAF is a predictable treatment for multiple adjacent Miller Class I or II recession-type defects. The addition of a PRF membrane positioned under the MCAF provided inferior root coverage but an additional gain in GTH at 6 months compared to conventional therapy.

Platelet-rich fibrin (PRF): A second-generation platelet concentrate. Part III: Leucocyte activation: A new feature for platelet concentrates?

Platelet-rich fibrin (PRF) belongs to a new generation of platelet concentrates, with simplified processing and without biochemical blood handling. In this third article, we investigate the immune features of this biomaterial. During PRF processing, leucocytes could also secrete cytokines in reaction to the hemostatic and inflammatory phenomena artificially induced in the centrifuged tube. We therefore undertook to quantify 5 significant cell mediators within platelet poor plasma supernatant and PRF clot exudate serum: 3 proinflammatory cytokines (IL-1beta, IL-6, and TNF-alpha), an antiinflammatory cytokine (IL-4), and a key growth promoter of angiogenesis (VEGF). Our data are correlated with that obtained in plasma (nonactivated blood) and in sera (activated blood). These initial analyses revealed that PRF could be an immune regulation node with inflammation retrocontrol abilities. This concept could explain the reduction of postoperative infections when PRF is used as surgical additive.

Platelet-rich fibrin (PRF): A second-generation platelet concentrate. Part II: Platelet-related biologic features

Platelet-rich fibrin (PRF) belongs to a new generation of platelet concentrates, with simplified processing and without biochemical blood handling. In this second article, we investigate the platelet-associated features of this biomaterial. During PRF processing by centrifugation, platelets are activated and their massive degranulation implies a very significant cytokine release. Concentrated platelet-rich plasma platelet cytokines have already been quantified in many technologic configurations. To carry out a comparative study, we therefore undertook to quantify PDGF-BB, TGFbeta-1, and IGF-I within PPP (platelet-poor plasma) supernatant and PRF clot exudate serum. These initial analyses revealed that slow fibrin polymerization during PRF processing leads to the intrinsic incorporation of platelet cytokines and glycanic chains in the fibrin meshes. This result would imply that PRF, unlike the other platelet concentrates, would be able to progressively release cytokines during fibrin matrix remodeling; such a mechanism might explain the clinically observed healing properties of PRF.

Fresh frozen plasma in patients with disseminated intravascular coagulation or in patients with liver diseases

Disseminated intravascular coagulation (DIC) and liver diseases are complex clinical conditions. Both disorders frequently disturb the finely tuned coagulation and fibrinolysis equilibrium. In DIC, a wide range of underlying disorders can induce a systemic activation of the coagulation system with generation of soluble fibrin, possible deposition of platelet-rich fibrin clots in the microvasculature and subsequent micro- or macroembolism, impaired organ perfusion and organ failure. Such coagulation activation depletes platelets, coagulation factors, and inhibitors and clinically can result in severe, sometimes untreatable bleeding, especially when bone marrow or liver function is diminished or invasive procedures are performed. In addition, a secondary counterbalancing activation of the fibrinolytic system to dissolve microcirculatory clots adds to the bleeding tendency. In conjunction with other options based on prompt and rigorous treatment of the underlying cause of DIC, fresh frozen plasma plays an important role in therapeutic management when overt bleeding is present or anticipated in DIC patients with disturbed coagulation or when an invasive procedure is being planned. In liver disease, factor and inhibitor synthesis in both the coagulation and fibrinolytic system is impaired, both quantitatively and qualitatively. This destabilizes the balance between the two systems. In addition, the clearance of activated coagulation factors and fibrin(ogen) degradation products (FDP) from the systemic circulation is impaired. In patients with liver diseases and acute or imminent bleeding, or before invasive procedures, fresh frozen plasma (FFP) offers advantages over clotting factor concentrates. However, hypervolemia following the required doses of FFP might pose a problem in some liver disease patients. The complex pathophysiology both in DIC and in liver disease requires early diagnosis and adequate management including plasma and platelet substitution after treatment of the underlying disease. Due to the heterogeneity of DIC and liver disease, prospective randomized trials are difficult to perform. Therefore, treatment recommendations are mostly empirical and less evidence-based. Therapy must be accompanied by close and repeated clinical and laboratory monitoring.

Retention of Platelet Fibrin Stabilizing Factor during the Platelet Release Reaction and Clot Retraction

SummaryPlatelet FSF was found to be retained by rabbit, pig or human platelets during the release reaction induced by thrombin, collagen or plasmin and during a 2 hours incubation of platelet rich fibrin clots.