Thrombin-activated Platelet-rich Plasma Research Articles

Transplantation of Human Placenta Derived Mitochondria Promotes Cell Communication in Endometrium in a Murine Model of Disturbed Endometrium.

Herein, we investigated the regenerative potential of functional mitochondria to restore endometrial injury. The endometrium was disturbed with an intrauterine injection of 95% ethanol. Regeneration of the disturbed endometrium was achieved by transplantation of human placenta derived mitochondria followed by thrombin activated platelet rich plasma (hMTx). The transplantation method provided a biomimetic gel layer that stabilized and supported the functionality of the transplanted mitochondria to flourish regeneration of the disturbed endometrium. The presence of engrafted Rhodamine B labelled mitochondria was quantified at 12, 24, 48, and 72h after transplantation. Detection of human-specific mitochondria mRNA in recipient rat uterus showed significant up-regulation of MT ATP-8, MT COX-1, MT COX -3, MT COX -2, MT ATP-6 (p = 0.009) in the hMTx treated group compared to the disturbed endometrium group. The hMTx group demonstrated showed regeneration through increased expressions of α-SMA, CK-18, CK-19, Connexin-40, E Cadherin, Claudin-1, Zona Occludin as compared with disturbed endometrium group. Experimental hMTx endometrial cells had significantly higher values of activities of NADH, NADPH, Cytochrome B5, Cytochrome P450, Complex I, Complex II, Complex III, Complex IV compared with disturbed endometrium indicating the regeneration of damaged endometrial cells at 72h. Intrauterine hMTx was accounted to improve endometrial junction protein thus regeneration in the disturbed endometrium. Our Data provide the first evidence that hMTx promotes endometrial regeneration in the disturbed endometrium, paving the way for the development of a novel approach to human endometrial regeneration.

Pulsed Radiofrequency Neuromodulation Contributes to Activation of Platelet-Rich Plasma in In Vitro Conditions

Recent years have brought new developments in interventional chronic pain management, namely regenerative orthopedics utilizing platelet-rich plasma (PRP) as well as further evolution of pulsed radiofrequency neuromodulation (PRF). Both methods have been used separately. Here, we investigated whether PRF may potentiate the activation of platelets in PRP samples when both these techniques are combined together in in vitro conditions. Studies were performed on concentrated PRP samples (PRPs) obtained from acid citrate dextrose-treated blood taken from 11 healthy volunteers. PRPs were divided into four groups: 1) nonactivated PRP; 2) thrombin-activated PRP as a positive control for maximal platelets activation; 3) PRF-treated PRP exposed for 20 min to PRF energy generated by neurotherm radio frequency generator at 500 kHz, with a voltage of 40 V and maximal temperature of 42°C; and 4) a combination of groups 2 and 3. PRF-induced platelet activation measured by platelet factor 4 (PF4) and ATP release from PRPs was significantly higher compared to nonactivated PRPs, and similar to PF4 and ATP release from thrombin-activated PRPs. Thrombin activation did not potentiate PF4 release in PRF samples and even reduced ATP level. Additionally, PRF neither induced any platelet membrane damage measured by lactic dehydrogenase release from PRP nor modified any platelets viability or metabolism measured by MTT. We confirmed that PRF may activate PRP without additional platelet activators. So, a combination of both methods PRF and PRP application may provide a more effective opportunity for tissue regeneration in dentistry, surgery, dermatology, or in orthopedics.

Thrombin-activated platelet-rich plasma enhances osteogenic differentiation of human periodontal ligament stem cells by activating SIRT1-mediated autophagy

BackgroundPlatelet-rich plasma (PRP) has the potential to be used for bone regeneration. However, its effect on osteogenic differentiation of human periodontal ligament stem cells (hPDLSCs) and its effect on cell autophagy of hPDLSCs remain unknown. In this study, we investigated the effects of PRP on cell viability and osteogenic differentiation of hPDLSCs and the underlying molecular mechanisms.MethodshPDLSCs were isolated and identified by morphology and flow cytometry analysis. Next, thrombin-activated PRP was used to stimulate hPDLSCs. The MTT assay was used to analyze cell viability. Osteogenic differentiation was investigated using alkaline phosphatase (ALP) activity assay, alizarin red S (ARS) staining, and gene expression analysis of osteogenic markers. Expression of the autophagic proteins was determined using western blotting.ResultsThrombin-activated PRP significantly enhanced cell viability, ALP activity, osteogenic-related mRNA levels and alizarin red-mineralization activity in hPDLSCs in a dose-dependent manner. Furthermore, activated PRP dose-dependently increased LC3-II/I ratio and the expression of SIRT1 and Beclin-1. PRP treatment also enhanced the autophagic flux. It was also demonstrated that the inhibition of SIRT1 using sirtinol or suppression of autophagy by 3-methyladenine (3-MA) abrogated PRP-induced viability and osteogenic differentiation of hPDLSCs.ConclusionOur study suggested that thrombin-activated PRP accelerated the viability and osteogenic differentiation of hPDLSCs via SIRT1-mediated autophagy induction.

Activation of platelet-rich plasma by pulse electric fields: Voltage, pulse width and calcium concentration can be used to control and tune the release of growth factors, serotonin and hemoglobin.

Activated platelet-rich plasma (PRP) has been used in the clinical settings of wound healing and regenerative medicine, with activation typically induced by the addition of bovine thrombin. To eliminate issues with availability, cost and potential side effects associated with bovine thrombin, ex vivo PRP activation using pulse electric fields (PEF) has been proposed and demonstrated. The present study characterizes the effect of PEF voltage and pulse width, in combination with a range of calcium concentrations, on clot formation, growth factor release, and serotonin (5-HT) release from dense granules. The main findings are: 1) increasing calcium concentrations with most PEF conditions leads to increased levels of PDGF and 5-HT release; 2) whether EGF levels increase or decrease with increasing calcium concentration depends on the specific PEF parameters; 3) the pattern of PDGF and EGF levels in supernatants suggest that these molecules are localized differently within platelets; 4) significant levels of PDGF, EGF, and 5-HT can be released without inducing clot formation or hemoglobin release. In conclusion, voltage, pulse width and calcium concentration can be used to control and tune the release of growth factors, serotonin and hemoglobin from PEF-activated PRP. Because growth factor requirements vary for different types of wounds and for wounds at different stages of healing, the unique balance of factors in supernatants of PEF-activated PRP may provide more clinically advantageous than the current standard of bovine thrombin-activated PRP.

Platelet-rich plasma and stromal vascular fraction cells for the engineering of axially vascularized osteogenic grafts.

Avascular necrosis of bone (AVN) leads to sclerosis and collapse of bone and joints. We have previously shown that axially vascularized osteogenic constructs, engineered by combining human stromal vascular fraction (SVF) cells and a ceramic scaffold, can revitalize necrotic bone of clinically relevant size in a rat model of AVN. For a clinical translation, the fetal bovine serum (FBS) used to generate such grafts should be substituted by a nonxenogeneic culture supplement. Human thrombin-activated platelet-rich plasma (tPRP) was evaluated in this context. SVF cells were cultured inside porous hydroxyapatite scaffolds with a perfusion-based bioreactor system for 5 days. The culture medium was supplemented with either 10% FBS or 10% tPRP. The resulting constructs were inserted into devitalized bovine bone cylinders to mimic the treatment of a necrotic bone. A ligated vascular bundle was inserted into the constructs upon subcutaneous implantation in the groin of nude rats. After 1 and 8 weeks, constructs were harvested, and vascularization, host cell recruitment, and bone formation were analyzed. After 1 week in vivo, constructs were densely vascularized, with no difference between tPRP- and FBS-based ones. After 8 weeks, bone formation and vascularization was found in both tPRP- and FBS-precultured constructs. However, the amount of bone and the vessel density were respectively 2.2- and 1.8-fold higher in the tPRP group. Interestingly, the density of M2, proregenerative macrophages was also significantly higher (6.9-fold) following graft preparation with tPRP than with FBS. Our findings indicate that tPRP is a suitable substitute for FBS to generate vascularized, osteogenic grafts from SVF cells and could thus be implemented in protocols for clinical translation of this strategy towards the treatment of bone loss and AVN.

Effect of autologous PRP on wound healing in dental regenerative surgeries and its correlation with PDGF levels.

INTRODUCTION:Autologous platelet-rich plasma (PRP) is the fraction of blood plasma, with increased concentration of platelets, from baseline serum level. Growth factors (GFs) in PRP expedite the soft tissue and bony healing. However, estimation of their levels and role in healing had not been studied extensively. This study gives an insight to the quantification of platelet-derived GF-BB (PDGF-BB) present in PRP and its correlation with the clinical wound healing and bone regeneration.AIMS:This study aims to quantify PDGF-BB levels in PRP with its subsequent correlation with healing in dental regenerative surgeries.SETTINGS AND DESIGN:This was an experimental study including patients undergoing various dental regenerative surgeries.SUBJECTS AND METHODS:Autologous thrombin-activated PRP in the form of PRP gel was used in study group (n = 39) whereas no such intervention was given in control group (n = 30). PDGF-BB quantification was done in PRP samples using enzyme-linked immunosorbent assay. Clinicoradiological evaluation of healing was done in both the groups.STATISTICAL ANALYSIS USED:Descriptive analysis, independent Z-test, Correlation regression analysis, and ANOVA.RESULTS:Mean platelet concentration achieved in PRP was 5.79 times the baseline count. Mean PDGF-BB concentration in PRP was 31.92 ± 10.47 ng/ml which significantly correlated (P < 0.05) with the PRP platelet count. Study group showed significant healing clinically (P < 0.05). Significant bone fill observed in study group at 3 and 6 months when compared to the baseline as well as control group. Furthermore, bone fill at 6 months showed linear correlation with PGDF-BB levels (r = 0. 80).CONCLUSIONS:PRP led to enhanced bone regeneration and soft-tissue healing with former being directly related to higher concentration of PDGF-BB.

Abstract 10.55 PLATELET-RICH PLASMA VS. FETAL BOVINE SERUM IN ENGINEERING OF AXIALLY-VASCULARIZED OSTEOGENIC GRAFTS FROM HUMAN ADIPOSE-DERIVED CELLS TO TREAT AVASCULAR NECROSIS OF BONE

INTRODUCTION: Avascular necrosis of bone (AVN) leads to sclerosis and collapse of bone and joints. Engineered, axially vascularized, human adipose-derived stromal vascular fraction (SVF) cell based osteogenic constructs have been shown to revitalize necrotic bone of clinically-relevant size in a challenging rat model of AVN. To reduce regulatory difficulties towards clinical translation, fetal bovine serum (FBS) was substituted by thrombin-activated platelet-rich plasma (tPRP). METHODS: SVF cells from 5 lipoaspirate were isolated and cultured onto porous hydroxyapatite scaffolds within a perfusion-based bioreactor system for 5 days. The medium was supplemented either with 10% FBS or 10% tPRP. The resulting constructs were inserted into devitalized bone cylinders mimicking AVN-affected bone. A ligated vascular bundle was inserted upon subcutaneous implantation of constructs in nude rats. After 1 and 8 weeks, vascularization and bone formation were analyzed. RESULTS: After 1 week, neither maximal distance of vessels from the bundle nor vascular density was significantly different. However, FBS cultured grafts revealed significantly more human vessel (hCD34) and osteoclasts compared to tPRP cultured grafts. Overall macrophages and M2 macrophages were not different between FBS and tPRP cultured grafts. After 8 weeks in vivo, 3/5 samples from FBS culture and 1/5 sample from tPRP culture showed bone formation. Roughly 40% of the osteocytes were of human origin (36% from FBS and 42% from tPRP culture). DISCUSSION: Despite promising results in literature, replacement of FBS by tPRP resulted in a reduced capacity of bone formation in our series. Further studies are necessary to analyze and improve the low bone formation capacity, possibly caused by the heterogeneity of the SVF donors and tPRP samples.

Pooled thrombin-activated platelet-rich plasma: a substitute for fetal bovine serum in the engineering of osteogenic/vasculogenic grafts.

The use of fetal bovine serum (FBS) as a culture medium supplement in cell therapy and clinical tissue engineering is challenged by immunological concerns and the risk of disease transmission. Here we tested whether human, thrombin-activated, pooled, platelet-rich plasma (tPRP) can be substituted for FBS in the engineering of osteogenic and vasculogenic grafts, using cells from the stromal vascular fraction (SVF) of human adipose tissue. SVF cells were cultured under perfusion flow into porous hydroxyapatite scaffolds for 5 days, with the medium supplemented with either 10% tPRP or 10% FBS and implanted in an ectopic mouse model. Following in vitro culture, as compared to FBS, the use of tPRP did not modify the fraction of clonogenic cells or the different cell phenotypes, but increased by 1.9-fold the total number of cells. After 8 weeks in vivo, bone tissue was formed more reproducibly and in higher amounts (3.7-fold increase) in constructs cultured with tPRP. Staining for human-specific ALU sequences and for the human isoforms of CD31/CD34 revealed the human origin of the bone, the formation of blood vessels by human vascular progenitors and a higher density of human cells in implants cultured with tPRP. In summary, tPRP supports higher efficiency of bone formation by SVF cells than FBS, likely by enhancing cell expansion in vitro while maintaining vasculogenic properties. The use of tPRP may facilitate the clinical translation of osteogenic grafts with intrinsic capacity for vascularization, based on the use of adipose-derived cells. Copyright © 2015 John Wiley & Sons, Ltd.

Stimulation of the Superficial Zone Protein and Lubrication in the Articular Cartilage by Human Platelet-Rich Plasma

Background: Platelet-rich plasma (PRP) contains high concentrations of autologous growth factors that originate from platelets. Intra-articular injections of PRP have the potential to ameliorate the symptoms of osteoarthritis in the knee. Superficial zone protein (SZP) is a boundary lubricant in articular cartilage and plays an important role in reducing friction and wear and therefore is critical in cartilage homeostasis. Purpose: To determine if PRP influences the production of SZP from human joint-derived cells and to evaluate the lubricating properties of PRP on normal bovine articular cartilage. Study Design: Controlled laboratory study. Methods: Cells were isolated from articular cartilage, synovium, and the anterior cruciate ligament (ACL) from 12 patients undergoing ACL reconstruction. The concentrations of SZP in PRP and culture media were measured by enzyme-linked immunosorbent assay. Cellular proliferation was quantified by determination of cell numbers. The lubrication properties of PRP from healthy volunteers on bovine articular cartilage were investigated using a pin-on-disk tribometer. Results: In general, PRP stimulated proliferation in cells derived from articular cartilage, synovium, and ACL. It also significantly enhanced SZP secretion from synovium- and cartilage-derived cells. An unexpected finding was the presence of SZP in PRP (2.89 ± 1.23 μg/mL before activation and 3.02 ± 1.32 μg/mL after activation). In addition, under boundary mode conditions consisting of high loads and low sliding speeds, nonactivated and thrombin-activated PRP decreased the friction coefficient (μ = 0.012 and μ = 0.015, respectively) compared with saline (μ = 0.047, P < .004) and high molecular weight hyaluronan (μ = 0.080, P < .006). The friction coefficient of the cartilage with PRP was on par with that of synovial fluid. Conclusion: PRP significantly stimulates cell proliferation and SZP secretion by articular cartilage and synovium of the human knee joint. Furthermore, PRP contains endogenous SZP and, in a functional bioassay, lubricates bovine articular cartilage explants. Clinical Relevance: These findings provide evidence to explain the biochemical and biomechanical mechanisms underlying the efficacy of PRP treatment for osteoarthritis or damage in the knee joint.

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

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.

Platelet-Rich Plasma and Bone Defect Healing

On activation, platelets secrete an array of growth factors that contribute to bone regeneration. Combining platelet-rich plasma (PRP) with bone graft substitutes has the potential to reduce or replace the reliance on autografts. Lack of standardization and improper use may contribute to the conflicting outcomes reported within both preclinical and clinical investigations using PRP. This study investigates the effect of PRP dose on bone augmentation. Eighty critical-sized defects were created in the cancellous bone of the medial proximal tibia and the distal femur of 20 five-year-old female sheep. The defects were treated with three doses of an autologous thrombin-activated PRP combined with a biphasic calcium phosphate (BCP) or autograft and empty defects. Radiography, micro-computed tomography, histology, histomorphometry, and fluorochrome bone labels were examined at 4 weeks. The empty defects did not spontaneously heal. The highest dose of PRP treatment had a significantly greater micro-CT bone volume/total volume compared with the BCP alone (PRP: 30.6%±1.8%; BCP: 24.5%±0.1%). All doses of PRP treatment were significantly greater than the BCP alone for histomorphometric new bone area (PRP: 14.5%±1.3%; BCP: 9.7%±1.5%) and bone ingrowth depth (PRP: 2288±210 μm; BCP:1151±268 μm). From week 2 onward, PRP had a significant effect on the weekly bone ingrowth compared with BCP; however, autografts had the highest amount of weekly fluorescent bone labeling. PRP induces new bone formation with a dose-dependent response at 4 weeks when used with a BCP in sheep.

Platelet-mediated changes to neuronal glutamate receptor expression at sites of microthrombosis following experimental subarachnoid hemorrhage.

Glutamate is important in the pathogenesis of brain damage after cerebral ischemia and traumatic brain injury. Notably, brain extracellular and cerebrospinal fluid as well as blood glutamate concentrations increase after experimental and clinical trauma. While neurons are one potential source of glutamate, platelets also release glutamate as part of their recruitment and might mediate neuronal damage. This study investigates the hypothesis that platelet microthrombi release glutamate that mediates excitotoxic brain injury and neuron dysfunction after subarachnoid hemorrhage (SAH). The authors used two models, primary neuronal cultures exposed to activated platelets, as well as a whole-animal SAH preparation. Propidium iodide was used to evaluate neuronal viability, and surface glutamate receptor staining was used to evaluate the phenotype of platelet-exposed neurons. The authors demonstrate that thrombin-activated platelet-rich plasma releases glutamate, at concentrations that can exceed 300 μM. When applied to neuronal cultures, this activated plasma is neurotoxic, and the toxicity is attenuated in part by glutamate receptor antagonists. The authors also demonstrate that exposure to thrombin-activated platelets induces marked downregulation of the surface glutamate receptor glutamate receptor 2, a marker of excitotoxicity exposure and a possible mechanism of neuronal dysfunction. Linear regression demonstrated that 7 days after SAH in rats there was a strong correlation between proximity to microthrombi and reduction of surface glutamate receptors. The authors conclude that platelet-mediated microthrombosis contributes to neuronal glutamate receptor dysfunction and might mediate brain injury after SAH.

Synovial fluid growth factor and cytokine concentrations after intra-articular injection of a platelet-rich product in horses

Platelet-rich plasma (PRP) products may be useful for treatment of joint disease in horses, but may contain undesirable pro-inflammatory cytokines in addition to growth factors. This study investigated whether autologous PRP increases synovial fluid growth factor and cytokine concentrations when injected into normal equine metacarpophalangeal and metatarsophalangeal (fetlock) joints. Fetlock joints of seven healthy horses received one of four treatments: saline, resting PRP, CaCl2-activated PRP or thrombin-activated PRP. Synovial fluid was sampled prior to injection and at 6, 24, 48 and 96h post-injection. Platelet-derived growth factor (PDGF-BB), transforming growth factor β1 (TGFβ1), interleukin (IL)-6 and tumor necrosis factor α (TNFα) concentrations in synovial fluid and PRP were measured by ELISA. Synovial fluid PDGF-BB, TGFβ1, IL-6, TNFα and IL-1 concentrations were also measured in vitro after incubation for 6h with resting PRP only. Growth factor concentrations, but not cytokine concentrations, were significantly higher in activated PRP than in resting PRP samples. After intra-articular injection with resting or thrombin-activated PRP, synovial TGFβ1 increased significantly compared to baseline levels. TNFα and IL-6 were significantly increased in synovial fluid after thrombin-activated PRP injection. In vitro, growth factor concentrations increased significantly in synovial fluid after mixing with PRP, indicating that exogenous activation of PRP for intra-articular injection may be unnecessary, whereas cytokine levels did not. In conclusion, thrombin-activated PRP induced an inflammatory cytokine response in joints, whereas resting or CaCl2-activated PRP did not. Synovial growth factor levels were low overall; the reported benefits of intra-articular PRP may not be attributable to changes in local PDGF or TGFβ1 concentrations.

Abstract TP266: Platelet-Derived Glutamate Contributes to Neuronal Injury after Experimental Subarachnoid Hemorrhage

Background: Glutamate toxicity (excitotoxicity) is well-studied in the pathogenesis of brain damage after cerebral ischemia and traumatic brain injury. Patients with subarachnoid hemorrhage (SAH) also have increased intracerebral glutamate as detected by microdialysis. While neurons and glia are potential sources of glutamate, platelets also release glutamate as part of their recruitment and might mediate neuronal damage. Studies have shown that intraluminal platelets escape into brain parenchyma after SAH. Therefore, we studied the hypothesis that formation of platelet microthrombi after SAH, and their subsequent extravasation releases glutamate that mediates excitotoxic brain injury and neuron dysfunction after SAH. Methods: We used two models, primary neuronal cultures exposed to activated platelets, and a model of SAH created by injection of 300 μl of fresh, unheparinized arterial blood into the prechiasmatic cistern of Sprague-Dawley rats (300-350 g). Glutamate was measured using amperometric microelectrode arrays. Propidium iodide was used to evaluate neuronal viability in neuronal cultures, and surface glutamate receptor immunohistochemical staining was used to evaluate the phenotype of platelet-exposed cultured neurons and brain after SAH. Microthrombi were stained with anti-fibrinogen antibodies. Results: We first demonstrated that thrombin-activated platelet-rich plasma releases glutamate in concentrations that exceed 300 μmol/l. When applied to neuronal cultures, this activated plasma was neurotoxic, and neurotoxicity was attenuated by glutamate receptor antagonism. Exposure of cultured neurons to thrombin-activated platelets induced a marked downregulation of the surface glutamate receptor GluR2, a marker of excitotoxicity and a possible mechanism of neuron dysfunction. Microthrombi were detected in rat cerebral cortex 7 days after SAH and linear regression demonstrated a strong correlation between proximity to microthrombi and reduction of surface glutamate GluR2 receptors. Conclusions: These correlative data support the novel hypothesis that platelet-mediated microthrombosis contributes to neuronal glutamate receptor dysfunction and might therefore influence clinical outcome following SAH.

Mesenchymal stem cells and cardiac regeneration: a sophisticated approach depends on trophic effects—what's left over? Focus on “Activation of Toll-like receptor 3 amplifies mesenchymal stem cell trophic factors and enhances therapeutic potency”

application of stem cells for tissue, in particular cardiac, regeneration represents a promising field of investigation but has not achieved clinical utility ([Fig. 1][1]). From the latter point of view, Strauer and colleagues ([7][2]) in 2001 achieved an important milestone by their administration

Periodontal Tissue Engineering After Tooth Replantation

Blood-derived products, platelet-poor plasma (PPP) and platelet-rich plasma (PRP), constitute an approach in the enhancement of tissue healing. PRP has also been used as a scaffold for bone marrow stem cells in tissue engineering. This study evaluates the effect of PPP, calcium chloride-activated PRP (PRP/Ca), calcium chloride- and thrombin-activated PRP (PRP/Thr/Ca), and bone marrow mononuclear cells and PRP/Ca (BMMCs/PRP/Ca) on the healing of replanted dog teeth. After 30 minutes of extraction, teeth were replanted with 1) no material (control); 2) PPP; 3) PRP/Ca; 4) PRP/Thr/Ca; or 5) BMMCs/PRP/Ca. Histologic, histomorphometric, and immunohistochemical analysis was assessed 120 days after replantation. Data from histomorphometric analysis were analyzed statistically (analysis of variance, Tukey; P <0.05). Quantitative immunohistochemical analysis was analyzed by Kruskal-Wallis and Dunn post hoc test (P <0.05). Flow cytometry analysis showed 55.98% of CD34(+) and 32.67% of CD90/Thy-1 for BMMCs sample. BMMCs/PRP/Ca presented the largest areas of replacement resorption characterized by osseous ingrowth into cementum (P <0.05), with intense immunomarcation for tartrate-resistant acid phosphatase. The PRP/Ca group also showed areas of replacement resorption with significant immunomarcation for osteopontin. PRP/Thr/Ca presented no replacement resorption. PPP showed areas of inflammatory resorption, with immunomarcation for tartrate-resistant acid phosphatase. The results suggest that platelets activated with thrombin play an important role in the healing of tissues after tooth replantation. Additional studies are necessary to test other materials, because PRP/Ca did not present an appropriate scaffold for undifferentiated cells in the treatment of avulsed teeth.

Epidermal growth factor released from platelet-rich plasma promotes endothelial cell proliferationin vitro

The therapeutic benefits of platelet-rich plasma (PRP) for the promotion of healing and regeneration of periodontal tissues are thought to result from enrichment in growth factors released from platelets. The aim of this study was to evaluate the effects of specific growth factors released from PRP on endothelial cell proliferation. The levels of vascular endothelial growth factor (VEGF), platelet-derived growth factor BB (PDGF-BB), basic fibroblast growth factor (bFGF) and epidermal growth factor (EGF) in supernatants of calcium- and thrombin-activated PRP samples from five donors were quantified by enzyme-linked immunosorbent assay. Supernatants were treated with neutralizing antibodies specific to each growth factor, and the effects of these treatments on human umbilical vein endothelial cell (HUVEC) proliferation in vitro were determined. The effect of removing EGF from PRP supernatants with antibody-coated beads on HUVEC proliferation was also tested. Average concentrations of VEGF, PDGF-BB, bFGF and EGF in PRP supernatants were 189, 27,190, 39.5 and 513 pg/mL, respectively. The addition of EGF neutralizing antibodies to the PRP supernatants significantly reduced HUVEC proliferation (up to 40%), while such an inhibition was not observed following neutralization of the other growth factors. Removal of EGF from PRP supernatants by treatment with antibody-coated beads also resulted in a significant decrease in HUVEC proliferation. Recombinant EGF increased HUVEC proliferation in vitro in a dose-dependent manner. This study showed that PRP supernatants are highly mitogenic for endothelial cells and provided evidence that this effect may be due, at least in part, to the presence of EGF. In vivo experiments are needed to confirm the roles of specific growth factors released from PRP in the healing of oral surgical and/or periodontal wounds.

The Effect of Thrombin Activation of Platelet-Rich Plasma on Demineralized Bone Matrix Osteoinductivity

Demineralized bone matrix is an osteoinductive and osteoconductive material that is often used in orthopaedic surgery to induce bone formation. Autologous platelet-rich plasma, which contains proliferative and chemoattractant growth factors, has been used as a demineralized bone matrix adjuvant with mixed results. One variable during clinical use appears to be whether the platelet-rich plasma is activated with thrombin or is implanted in a liquid form with intact platelets. The objective of the present study was to determine if platelet-rich plasma can increase the osteoinductivity of demineralized bone matrix when used without thrombin activation. The bioactivity of the demineralized bone matrix was evaluated in vitro by determining alkaline phosphatase production by C2C12 myoblast cells. The effect of thrombin activation on platelet-rich plasma was studied in vitro by evaluating osteosarcoma and bone marrow stromal cells for cell number and transforming growth factor-beta1 activation. Demineralized bone matrices supplemented with platelet-rich plasma, with or without thrombin activation, were implanted intramuscularly in athymic rats and were examined at fourteen, twenty-eight, and fifty-six days. Histological samples were analyzed for osteogenesis and chondrogenesis. Osteogenesis was further characterized on the basis of alkaline phosphatase activity. In vitro, thrombin triggered an immediate release of growth factors from the platelet-rich plasma, and the platelet-rich plasma increased the number of both osteosarcoma and stromal cells in a dose-dependent manner. Thrombin activation of the platelet-rich plasma eliminated such stimulatory effects. In vivo, the platelet-rich plasma stimulated chondrogenesis on Day 14 and osteogenesis on Days 28 and 56, whereas thrombin-activated platelet-rich plasma acted as an inhibitor of such events. In addition, inflammatory cells were detected in demineralized bone matrix samples that were mixed with thrombin-activated platelet-rich plasma. These cells were not present in matrix mixed with platelet-rich plasma alone. Platelet-rich plasma significantly increased in vivo demineralized bone matrix osteoinductivity only when used without thrombin activation.

Role of Platelet-Rich Plasma in Acceleration of Bone Fracture Healing

Platelet-rich plasma (PRP) is a common therapy for acceleration of maxillofacial and spinal fusion bone-graft healing. This study analyzes the therapeutic role of PRP during long-bone fracture healing evaluated Lewis rats. Following creation of unilateral open femur fractures, either 500 microL thrombin-activated PRP (PRP treated group) or 500 microL saline (control group) were applied once to the fracture site. Fracture healing was analyzed after 1 and 4 weeks. Following 4 weeks of fracture healing, radiographic analysis demonstrated higher callus to cortex width ratio (P < 0.05) in the PRP group (PRP: 1.65 +/- 0.06; control: 1.48 +/- 0.05). Three-point load bearing showed increased bone strength following PRP treatment (PRP: 60.85 +/- 6.06 Newton, control: 47.66 +/- 5.49 Newton). Fracture histology showed enhanced bone formation in the PRP group. Immunohistochemistry and Western-blotting demonstrated healing-associated changes in transforming growth factor (TGF)-beta1 and bone morphogenetic protein (BMP)-2. Our results suggest that PRP accelerates bone fracture healing of rat femurs via modulation of TGF-beta1 and BMP-2 growth factor expression.

Effect of Platelet‐Rich Plasma on Cell Adhesion, Cell Migration, and Myofibroblastic Differentiation in Human Gingival Fibroblasts

Platelet-rich plasma is a blood-derived fraction that contains a high concentration of platelets and growth factors. It was proposed that the use of this platelet concentrate stimulates tissue repair. However, little is known about the biologic response of gingival fibroblasts to platelet's derived growth factors. In the present study, we evaluated whether platelet-rich plasma modulated cell adhesion, cell migration, and myofibroblastic differentiation in primary cultures of human gingival fibroblasts. We studied the response of primary cultures of gingival fibroblasts to thrombin-activated platelet-rich plasma fractions. Cell adhesion was evaluated through a colorimetric assay. Cell spreading, actin cytoskeleton remodeling, and focal adhesion distribution were assessed through light and immunofluorescence microscopy. Cell migration was analyzed using a bicameral cell culture system. Smooth muscle actin production was studied through Western blotting. Exposure of gingival fibroblasts to platelet-rich plasma stimulated adhesion and spreading of cells on fibronectin matrices, the development of actin-enriched cellular extensions, and formation of focal adhesions. Platelet-rich plasma also promoted cell migration and invasion through a reconstituted basement membrane matrix. Differentiation into the myofibroblastic phenotype, assessed through the production of smooth muscle actin, was also stimulated by platelet-rich plasma preparations. Platelet-rich plasma may modulate several cell responses potentially involved in wound healing such as cell adhesion, cell migration, and myofibroblastic differentiation.