Platelet-rich plasmas: growth factor content and roles in wound healing

Platelet-rich plasmas (PRPs) are used in a variety of clinical applications, based on the premise that higher growth factor content should promote better healing. In this study, we have determined the effects of calcium and thrombin on the release of EGF, TGF-alpha, IGF-1, Ang-2 and IL-1beta from PRPs, and assessed the mitogenic potential of PRP supernatants on osteoblast and endothelial cell division. ELISA assays indicate that (i) mean growth factor concentrations vary from traces (TGF-alpha) to 5.5 ng/mL (IGF-1), (ii) there are significant variations in growth factor concentrations between individuals, and (iii) calcium and thrombin regulate growth factor release, synthesis, and/or degradation in stereotyped patterns that are specific to each growth factor. PRP supernatants promote strong osteoblast and endothelial cell divisions, supporting the concept that PRPs may be beneficial in wound healing. Abbreviations: PRPs, platelet-rich plasmas; GFs, growth factors; EGF, epidermal growth factor; TGF-alpha, transforming growth factor-alpha; IGF-1, insulin-like growth factor-1; Ang-2, angiopoietin-2; IL-1beta, interleukin-1 beta; HUVECs, human umbilical vein endothelial cells; hFOB 1.19, human fetal osteoblasts; and FBS, fetal bovine serum.     

Platelet concentrates: effects of calcium and thrombin on endothelial cell proliferation and growth factor release

BACKGROUND: Clinical evidence suggests that platelet concentrate (PC) could have beneficial therapeutic effects on hard and soft tissue healing, due to the contents of growth factors (GFs) stored in the platelets. The objectives of this study were: 1) to determine the concentrations of platelet-derived growth factor-BB (PDGF-BB), transforming growth factor-beta1 (TGF-beta1), vascular endothelial growth factor (VEGF), and basic fibroblast growth factor (bFGF) released from PCs and whole blood (WB), before and after the addition of various concentrations of calcium and thrombin, and 2) to assess the physiological importance of the released GFs on angiogenesis. METHODS: WB and PCs were harvested and prepared from three healthy volunteers. Enzyme-linked immunosorbent assay tests, specific for PDGF-BB, TGF-beta1, VEGF, and bFGF, were performed on WB and PC supernatants, collected before and 30 minutes after the addition of various concentrations of calcium and thrombin. The supernatants were also added to human umbilical vein endothelial cell (HUVEC) cultures in order to measure their effects on endothelial cell proliferation. RESULTS: Growth factor concentrations detected in PC supernatants were significantly greater (280% to 800% increase) than concentrations present in WB supernatants. Calcium and thrombin induced immediate GF release from PCs in a dose-dependent fashion. Furthermore, PC supernatants led to greater HUVEC proliferation rates than WB supernatants. However, there was no correlation between the concentrations of specific GFs and HUVEC proliferation rates. CONCLUSION: These results suggest that PCs could stimulate blood vessel formation. They also reinforce the relevance for using PCs in regenerative therapies.     

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

Bertrand‐Duchesne M‐P, Grenier D, Gagnon G. Epidermal growth factor released from platelet‐rich plasma promotes endothelial cell proliferation in vitro. J Periodont Res 2009; doi: 10.1111/j.1600‐0765.2009.01205.x. © 2009 The Authors. Journal compilation © 2009 Blackwell Munksgaard Background and Objective: 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. Material and Methods: 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. Results: 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. Conclusion: 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.     

Differential growth factor retention by platelet-rich plasma composites.

PURPOSE: This study evaluates the temporal sequence and growth factor release from platelet-rich plasma (PRP) combined with different bone substitutes (BS), to identify an optimal substrate for extended growth factor retention. MATERIALS AND METHODS: PRP was clotted with bovine thrombin or thrombin receptor activator peptide-6 (TRAP). In addition, PRP was clotted using Allogro (Ceramed, Lakewood, CO), BioGlass (Mo-Sci, Rolla, MN), or BioOss (Osteohealth, Shirley, NY). The effects of media exchange and BS on platelet-derived growth factor (PDGF) and transforming growth factor-beta (TGF beta) release were quantified via enzyme-linked immunosorbent assay. RESULTS: At day 1, the thrombin group released 36% more PDGF than the TRAP group and 80% more than the BS groups. At 7 days, PDGF release was the greatest for the TRAP group. PDGF release was minimal for all groups at day 14, with BS groups retaining 60% more PDGF than thrombin clots. Similarly, the thrombin group released the greatest amount of TGF beta (81.4% of the total), whereas TRAP and BS groups released significantly less TGF beta at day 1. Compared with thrombin, TRAP retained 39.2% more TGF beta, whereas BS groups retained even greater levels (Allogro, 54.3%; BioOss, 45.8%; BioGlass, 67.0%). No significant difference in TGF beta release was observed among the substitutes after day 1. The BS groups continued to retain TGF beta after 14 days, whereas all TGF beta in the thrombin clots was depleted. CONCLUSIONS: PRP preparation with thrombin results in a large, immediate release of growth factors that could be lost into the interstitium in vivo. TRAP-BS may prove more efficacious than thrombin in sustaining growth factor levels critical for the cascade of events leading to bone formation.     

Efficacy of freeze-dried platelet-rich plasma in bone engineering

ObjectivePlatelet-rich plasma (PRP) is typically isolated and applied immediately after preparation, making it both a time- and labor-intensive addition to the operative procedure. Thus, it would be convenient if PRP could be preserved. We evaluated the efficacy of freeze-dried PRP (FD-PRP), as compared with freshly isolated PRP (f-PRP) for bone engineering.DesignFD-PRP was prepared by lyophilization of f-PRP and was subsequently preserved at −20 °C for one month. It was then rehydrated with an equal or 1/3 amount of distilled water (×1FD-PRP, ×3FD-PRP, respectively), and we assessed its gelation properties and the release of growth factors (PDGF-BB, TGF-β1, and VEGF). We also examined the bone forming ability with onlay-grafting on mice calvaria using β-TCP granules as a scaffold.ResultsFD-PRP showed comparable gelation as f-PRP. In terms of growth factor release, × 1FD-PRP released identical concentrations of PDGF-BB and TGF-β1 to f-PRP, while ×3FD-PRP released approximately 3-fold concentrations when compared with f-PRP. In vivo, ×1FD-PRP promoted identical levels of the bone formation as f-PRP, and ×3FD-PRP induced more abundant bone formation.ConclusionsThese results suggest that f-PRP can be stored without functional loss by freeze-drying and the concentration of PRP may improve its efficacy in bone engineering.     

Analysis of a rapid, simple, and inexpensive technique used to obtain platelet-rich plasma for use in clinical practice

The use of platelet-rich plasma (PRP) has become more generally accepted, and implant dentists are using PRP more frequently to promote the healing of oral surgical and/or periodontal wounds. Critical elements of PRP are thought to be growth factors contained within the concentrated platelets. These growth factors are known to promote soft-tissue healing, angiogenesis and osteogenesis. We present a rapid, simple, and inexpensive methodology for preparing PRP using the Cliniseal centrifuge method. This study demonstrates that platelets are concentrated approximately 6-fold without altering platelet morphology. Further we demonstrate that key growth factors, platelet-derived growth factor BB (PDGF-BB), transforming growth factor B (TGF-B1), vasculature endothelial growth factor (VEGF), and epidermal growth factor (EGF) are present in comparable or higher concentrations than those reported with the use of other techniques. Prolonged bench set time (>3 hours) after centrifugation resulted in decreased concentration of TGF-B1 but not decreased concentration of PDGF-BB, VEGF, or EGF. This study confirms the molecular aspects of PRP obtained using this inexpensive and efficient methodology.     

Impact of incubation method on the release of growth factors in non-Ca2+-activated PRP,Ca2+-activated PRP,PRF and A-PRF

The aim of this study was to investigate the influence of different incubation methods on the growth factor content of lysates of platelet-rich fibrin (PRF), advanced-platelet-rich fibrin (A-PRF) and platelet-rich plasma (PRP) products. A comparison of related studies suggests that the method of sample preparation has a significant influence on growth factor content. There are few reports on the comparison of non-Ca²⁺-activated PRP, Ca²⁺-activated PRP, A-PRF, and PRF, along with a lack of information on the release of PDGF-BB, TGF-β1, and VEGF among the different incubation methods.The lysate preparation was made of non-Ca²⁺-activated PRP, Ca²⁺-activated PRP, PRF, and A-PRF, using a room-temperature, 37 °C, or freeze–thaw–freeze incubation method. Afterwards the VEGF, PDGF-BB, and TGF-β1 content was investigated by running ELISA tests.Growth factor levels were significantly increased in the non-Ca²⁺-activated PRP with freeze–thaw–freeze incubation, and in the PRF preparation there was a significant disadvantage to using room temperature incubation for releasing growth factors.In conclusion, the freeze–thaw–freeze method is sufficient for releasing growth factors, and calcium activation is not necessary. Finally, the study demonstrates the possibility of preparing PRP products from platelet concentrates, so that preoperative blood sampling might not be required.     

富血小板血浆对人真皮细胞增殖及PDGF、TGF-β和VEGF表达的影响

目的:研究富血小板血浆(PRP)对人真皮成纤维细胞(hFbs)增殖、分化及胞质中PDGF、TGF-β和VEGF表达量的影响。方法:2次离心法制备PRP,成骨诱导条件下hFbs培养液中加入不同浓度PRP于第12天、21天钙-钴法染色,第21天茜素红染色;免疫细胞化学法检测加不同浓度PRP后第4天细胞PDGF、TGF-β和VEGF的表达;碱性磷酸酶染色(ALP)检测细胞活性;CCK-8法检测有和无成骨诱导条件下不同浓度PRP对细胞增殖的影响。结果:茜素红染色显示,2.8%PRP组钙结节形成最明显;钙-钴法染色显示,2.8%PRP组矿化作用最强;免疫细胞化学结果表明PDGF表达存在剂量依赖性,TGF-β和VEGF表达虽无剂量依赖性,但有适宜浓度的要求;ALP染色表明3%PRP组细胞增殖明显,活性最强。CCK-8检测表明各浓度PRP对hFbs均有促增殖作用,PRP加成骨诱导组比单纯PRP组表现出更明显的促增殖作用,其中4.8%PRP促增殖作用最强。结论:适宜浓度的PRP可促进hFbs增殖,但相关因子的表达存在适宜浓度的要求,PRP通过促进细胞增殖,增加相关因子表达促进创伤愈合。     

Platelet-rich plasma contains high levels of platelet-derived growth factor and transforming growth factor-beta and modulates the proliferation of periodontally related cells in vitro

BACKGROUND: Platelet-rich plasma (PRP) is a fraction of plasma, in which platelet-derived growth factor (PDGF) and transforming growth factor-beta (TGF-beta) are thought to be concentrated. It is plausible that topically-applied PRP up-regulates cellular activity and subsequently promotes periodontal regeneration in vivo. However, the concentrations of these growth factors in PRP have not been specifically determined and the biological effects of PRP at the cellular and molecular levels have not been determined. METHODS: PRP obtained from 20 healthy subjects was prepared from plasma by centrifugation. These PRP preparations were immediately subjected to an evaluation for PDGF-AB and TGF-beta1 using enzyme-linked immunosorbent assay (ELISA) kits. The biological effects of the PRP preparations were evaluated on osteoblastic, epithelial, fibroblastic, and periodontal ligament cells. Cellular mitogenic activity was evaluated by counting cell numbers or evaluating 5-bromodeoxyuridine (BrdU) incorporation. Expression of alkaline phosphatase (ALP) was immunocytochemically evaluated. RESULTS: In the PRP preparations, platelets were concentrated up to 70.9 x 10(4) cells/microl (283.4% of the unconcentrated plasma). The levels of PDGF-AB and TGF-beta1 were also concentrated up to 182.0 ng/ml (440.6%) and 140.9 ng/ml (346.6%), respectively. Scatter plots revealed significant correlations between platelet counts and levels of these growth factors. PRP stimulated osteoblastic DNA synthesis and cell division (138% of control), with simultaneous down-regulation of ALP, but suppressed epithelial cell division (80% of control). PRP also stimulated DNA synthesis in gingival fibroblasts and periodontal ligament cells. CONCLUSIONS: These data demonstrated that both PDGF-AB and TGF-beta1 were highly concentrated in the PRP preparations. It is suggested PRP modulates cell proliferation in a cell type-specific manner similar to what has been observed with TGF-beta1. Since synchronized behavior of related cell types is thought to be required for successful periodontal regeneration, it is further suggested these cell type-specific actions may be beneficial for periodontal regenerative therapy.     

Platelet rich plasma (PRP) as adjuvant tool for bone augmentation

New bone formation requires sufficient number of osteogenic progenitors capable of forming the bone desired. The site of engraftment must be filled with a matrix that facilitates attachment, migration and differentiation of osteoblastic progenitors. It is also necessary that the cells receive stimuli by growth factors that allow them to progress toward a bone phenotype. Another critical step in new tissue formation is the construction of new blood vessels--angiogenesis. Platelets contain growth factors that induce osteoinductive stimuli and accelerate angiogenesis. One strategy for harnessing this benefit is to apply platelet rich plasma (PRP) to bone graft site. The present article review platelets and growth factors physiology. We discuss the interaction between growth factors, thrombin and cells that form bone and blood vessels: osteoblasts, mesenchimal stem cells and endothelial cells. Methods and defaults of PRP preparation and safety issues are presented. The knowledge of platelet physiology and the mechanism by which growth factors effect cell proliferation and differentiation allow the dental surgeon to properly use this treatment modality and to achieve the ultimate goal of durable and effectively functioning bone.     

Platelet-rich plasma: growth factors and pro- and anti-inflammatory properties

BACKGROUND: Platelet-rich plasma (PRP) promotes regeneration of bone, presumably through the action of concentrated growth factors. However, it is not clear how PRP affects the inflammatory response. The purpose of this study was to analyze the growth factors in PRP and to study the effects of PRP on monocyte cytokine release and lipoxin A(4) (LXA(4)) generation. METHODS: PRP was prepared from healthy donors. Platelet-derived growth factor (PDGF)-AB, PDGF-BB, transforming growth factor-beta1, insulin-like growth factor-I, fibroblast growth factor-basic (FGF-b), epidermal growth factor (EGF), vascular endothelial growth factor, interleukin-12 (p40/70), and regulated on activation, normal T-cell expressed and secreted (RANTES) levels were evaluated by enzyme-linked immunosorbent assay and bead-based multiplexing. Peripheral blood monocytes were isolated and cultured with or without PRP. Cytokine, chemokine, and LXA(4) levels as well as monocyte chemotactic migration were analyzed. RESULTS: Growth factors were increased significantly in PRP compared to whole blood (WB) and platelet-poor plasma. Monocyte chemotactic protein-1 (MCP-1) was suppressed significantly by PRP, whereas RANTES was increased significantly in monocyte cultures. LXA(4) levels were significantly higher in PRP compared to WB. PRP stimulated monocyte chemotaxis in a dose-dependent fashion, whereas RANTES, in part, was responsible for PRP-mediated monocyte migration. CONCLUSIONS: PRP is a rich source of growth factors and promoted significant changes in monocyte-mediated proinflammatory cytokine/chemokine release. LXA(4) was increased in PRP, suggesting that PRP may suppress cytokine release, limit inflammation, and, thereby, promote tissue regeneration.     

VEGF在脱细胞骨基质复合富血小板血浆修复颅骨缺损中的表达

目的：研究血管内皮生长因子（VEGF）在脱细胞骨基质（acellular bone matrix,ABM）复合富血小板血浆（platelet-rich plasma,PRP）修复兔颅骨缺损时的表达及分布,探讨富血小板血浆促进成骨的机制。方法：雄性新西兰大白兔24只,体质量1.5～2.0kg。在兔颅骨两侧分别建立一个1cm×0.5cm全层骨缺损区,同时去除该区骨膜,注意勿伤及硬脑膜。随机选择一侧骨缺损作实验侧,植入复合PRP的ABM;另一侧为对照侧,仅植入ABM。术后第2、4、8、12周末分别处死6只兔取材。免疫组织化学法测定骨缺损修复区血管内皮细胞生长因子（Vascular endothelial growth factor,VEGF）的表达;应用Image-proplus 5.0图像分析软件测量VEGF表达强度的灰度值。采用SSPS16.0软件包进行t检验。结果：术后2周,实验组VEGF呈强阳性表达,随后急剧下降,以后趋于平缓。对照组在术后2、4周呈阳性表达,以后平缓下降。两组相比,在第2、4周时差异均有显著性（P〈0.05）。第8、12周时,2组表达差异无显著性。结论：VEGF在实验组早期阶段的强阳性表达,说明血管形成活跃。PRP促进骨修复的作用发生在植入后早期,启动了早期活跃的成骨。     

Cytokine regulation of syndecan-1 and -2 gene expression in human periodontal fibroblasts and osteoblasts

Cell-surface proteoglycans participate in several biological functions including interactions with a variety of growth factors and cytokines. Regulation of syndecan-1 and -2 gene expression was investigated in human periodontal ligament fibroblasts (PDLF), osteoblasts (OB) and gingival fibroblasts (GF), in response to platelet-derived growth factor (PDGF-BB), transforming growth factor (TGF-beta 1), and interleukin (IL-1 beta) by Northern blot analyses. We also compared the effect of PDGF-BB and TGF-beta 1, separately and in combination, in the prolonged presence of IL-1 beta on the expression of both syndecan genes. The results demonstrated that the three cell lines regulated the expression of syndecan-1 and -2 in response to growth factors and cytokines in different manners. These cell lines increased syndecan-1 mRNA levels in response to either PDGF-BB or TGF-beta 1 and decreased levels in response to IL-1 beta. The effect of IL-1 beta on syndecan-1 mRNA synthesis was partially reversed after adding PDGF-BB and TGF-beta 1, separately or in combination, in the presence of IL-1 beta. In contrast, syndecan-2 mRNA level was markedly upregulated in response to either TGF-beta 1 or IL-1 beta in OB when compared with the other two cell lines. However, the stimulatory effect of TGF-beta 1 on syndecan-2 mRNA production in OB was abolished in the prolonged presence of IL-1 beta. These findings lend support to the notion that syndecan-1 and syndecan-2 have distinct functions which correlate with their source and functions within the periodontium.     

The use of porous calcium phosphate scaffolds with transforming growth factor beta 1 as an onlay bone graft substitute

OBJECTIVES: Autogeneous bone grafting is regarded to be the golden standard for onlay grafts, but it requires a harvesting procedure and the remodeling pattern over time is unpredictable. New materials are constantly being sought to overcome these problems. An in vivo experiment was carried out to evaluate whether (1) porous calcium phosphate cement is a suitable biomaterial for onlay bone grafting, and (2) the addition of transforming growth factor beta 1 (TGF-beta1) accelerates de novo bone formation inside the cement porosity. MATERIAL AND METHODS: A carrier of porous calcium phosphate cement (Calcibon) was designed and 16 rats received one preshaped implant each. In 8 out of 16 implants 0.75 mug TGF-beta1 was applied. The animals were killed after 4 weeks and the characteristics of tissue ingrowth into the onlay graft were evaluated. RESULTS: Histologic and quantitative histomorphometrical measurements demonstrated osteoid-like tissue formation in both experimental groups. The addition of TGF-beta1 did not induce significantly more osteoid-like tissue formation. On the other hand, in TGF-beta-loaded implants, a higher number of pores contained an inflammatory infiltrate. CONCLUSION: This study indicated that porous calcium phosphate cement is a promising material for clinical situations where bone formation has to be supported.     

Platelet-derived growth factor enhancement of a mineral-collagen bone substitute

BACKGROUND: Anorganic bovine bone-collagen matrix is commercially available for bone regeneration procedures. Platelet-derived growth factor-BB (PDGF-BB) has been demonstrated to stimulate bone formation in vivo and in vitro. It was the aim of these studies to examine 1) the interaction of this mineral-collagen matrix with PDGF-BB and 2) determine if the adsorption of PDGF-BB to the mineral-collagen matrix stimulates osteoblastic cell proliferation above that of the untreated matrix. METHODS: Measurement of PDGF-BB adsorption and release was accomplished using 125I radiolabeled growth factor. The PDGF-BB was incubated with the anorganic bovine bone-collagen matrix and the amount which adsorbed was determined. In the release studies, radiolabeled PDGF-BB was adsorbed to the matrix material, then the samples were incubated in buffer for various time periods. The amount of PDGF-BB retained on the matrix was measured and the percent of growth factor released calculated. The biological activity was tested in an in vitro assay with primary culture neonatal rat osteoblastic cells. Osteoblastic cells were cultured on bone mineral-collagen matrix with known amounts of adsorbed PDGF-BB. Proliferation of the cells was assessed by 3H-thymidine incorporation and cell attachment measured by prelabeling cells with 3H-leucine. RESULTS: PDGF-BB adsorbed to the mineralized-collagen matrix material in a rapid, concentration-dependent fashion. The growth factor was slowly released from the matrix such that approximately 30% of the adsorbed protein was liberated over 10 days. PDGF-BB treated mineralized-collagen matrix displayed significantly (P < 0.05, ANOVA) enhanced proliferation of cultured osteoblastic cells compared to the mineralized-collagen matrix alone. CONCLUSIONS: These results suggest that PDGF-BB is rapidly adsorbed then slowly released from the anorganic bovine bone-collagen matrix. PDGF-BB adsorbed to this material is able to stimulate proliferation of the attached osteoblastic cells. These data suggest that it may be clinically feasible to adsorb PDGF to this bone-collagen matrix and that this combination of bone growth factor and mineral-collagen matrix has the potential for clinical applications.     

Growth factors and cytokines in autologous platelet concentrate and their correlation to periodontal regeneration outcomes

AIM: To determine the concentration of naturally available biologic mediators in autologous platelet concentrates and their correlation with periodontal regeneration outcomes. MATERIAL AND METHODS: In 25 patients with two intra-bony defects each, an autologous platelet concentrate (APC) was prepared by a laboratory thrombocyte apheresis technique pre-operatively. Both defects were treated using a bioresorbable guided tissue regeneration-membrane in combination with tricalciumphosphate (TCP). In the test defect, APC was additionally applied. In the APC, platelets were counted and the levels of growth factors and cytokines were determined by ELISA. Correlations between the platelet counts or the growth factor/cytokine levels and the potential clinical and radiographic regeneration outcomes due to APC were calculated after 3, 6, and 12 months. RESULTS: The APC contained 2.2 x 10(6) platelets/mul, which was 7.9 times more than in the venous blood. Transforming growth factor-beta1 (TGF-beta1), insulin-like growth factor-I (IGF-I), platelet-derived growth factor-AB (PDGF-AB), PDGF-BB, vascular endothelial growth factor (VEGF), and epidermal growth factor (EGF) were found in the APC, whereas interleukin-1beta (IL-1beta), IL-6, tumor necrosis factor alpha (TNFalpha), IL-4, and IL-10 were not detectable. The regression analysis showed a weak correlation between the platelet counts or the growth factor levels and the clinical and radiographic regeneration outcomes (r2相似文献   

Platelet-rich plasma-derived fibrin clot formation stimulates collagen synthesis in periodontal ligament and osteoblastic cells in vitro

BACKGROUND: Platelet-rich plasma (PRP) contains several growth factors, including platelet-derived growth factor (PDGF) and transforming growth factor-beta (TGF-beta), at high levels. We have demonstrated the PRP functions like TGF-beta to modulate cell proliferation in a cell-type specific manner. In addition, PRP forms gel-like materials in several, but not all, cell cultures tested. This study was designed to investigate PRP's action on extracellular matrix production in periodontal ligament (PDL) and osteoblastic MG63 cell cultures. METHODS: PRP was prepared from the plasma obtained from autologous blood of healthy volunteers and stored at -20 degrees C until used. Cells treated with PRP (0.5% to 2%) were immunocytochemically stained for type I collagen and fibrin and the viscosity of the culture media was visually evaluated. Fibrinogen in PRP was detected by immunodot-blotting while endogenous thrombin expression in cells was detected by a modified enzyme-linked immunosorbent assay. RESULTS: Gel-like material rapidly (< 30 minutes) formed in cultures of either PDL or osteoblastic MG63 cell cultures after addition of PRP (> or = 0.5%). PRP changed cell shape and up-regulated type I collagen at 24 hours. Fibrinogen was detected in the PRP preparations and insoluble fibrin networks were found in the newly formed gel-like material. PRP's action on collagen synthesis was mimicked by purified fibrinogen and blocked by thrombin inhibitor. Thrombin was expressed both in PDL and MG63 cells. CONCLUSIONS: These findings demonstrated that the gel-like material formed in cell cultures of either PDL or MG63 cells is fibrin clot that is capable of up-regulating collagen synthesis in the extracellular matrix. Our data suggest the possibility that fibrinogen, converted to fibrin, in combination with growth factors present in PRP might effectively promote wound healing at sites of injury in periodontal tissue.     

The in vitro effect of different PRP concentrations on osteoblasts and fibroblasts

OBJECTIVES: The aim of this study was to assess the biological rationale for the use of platelet-rich plasma (PRP) by evaluating the effect of different concentrations of PRP on osteoblasts (OB) and fibroblasts (FB) function in vitro. MATERIAL AND METHODS: PRP was obtained from volunteer donors using standard protocols. Primary human cultures of oral FBs and OBs were exposed to both activated and non-activated plasma as well as various concentrations of PRP (2.5 x, 3.5 x and max (4.2-5.5 x)). Cell proliferation was evaluated after 24 and 72 h using an MTT proliferation assay. Production of osteocalcin (OCN), osteoprotegerin (OPG) and transforming growth factor beta1 (TGF-beta1) was evaluated in OB after 24 and 72 h. Statistical analysis was performed using one-way ANOVA. RESULTS: PRP-stimulated cell proliferation in both OBs and FBs. The effect of different PRP concentrations on cell proliferation was most notable at 72 h. The maximum effect was achieved with a concentration of 2.5 x, with higher concentrations resulting in a reduction of cell proliferation. Upregulation of OCN levels and downregulation of OPG levels were noted with increasing PRP concentrations at both 24 and 72 h. TGF-beta1 levels were stimulated by increasing concentrations of PRP, with the increased levels being maintained at 72 h. CONCLUSIONS: PRP preparations exert a dose-specific effect on oral FBs and OBs. Optimal results were observed at a platelet concentration of 2.5 x, which was approximately half of the maximal concentrate that could be obtained. Increased concentrations resulted in a reduction in proliferation and a suboptimal effect on OB function. Hence, different PRP concentrations may have an impact on the results that can be obtained in vivo.     

Activation of platelet-rich plasma using thrombin receptor agonist peptide.

PURPOSE: This study proposes an alternative preparation method of platelet-rich plasma (PRP). Specifically, we compare the use of thrombin receptor agonist peptide-6 (TRAP) and bovine thrombin as a clotting agent in the preparation of PRP. MATERIALS AND METHODS: PRP was prepared by centrifugation and clotted with thrombin or TRAP. In vitro clotting times were monitored as a function of TRAP concentration, and clot retraction was determined by measuring clot diameter over time. Following the optimization of TRAP concentration, experiments were repeated with the addition of several commercially available bone substitutes. The release of PRP-relevant growth factors as a function of PRP preparation was also determined. RESULTS: The most rapid polymerization of PRP takes place with the addition of thrombin, followed by TRAP/Allogro (Ceramed, Lakewood, CO), TRAP/BioGlass (Mo-Sci, Rolla, MN), TRAP/BioOss (Osteohealth, Shirley, NY), and TRAP alone. Thrombin caused considerable clot retraction (43%), whereas TRAP alone resulted in only 15% retraction. TRAP/Allogro, TRAP/BioOss, and TRAP/BioGlass all exhibited minimal retraction (8%). CONCLUSIONS: The use of TRAP to activate clot formation in the preparation of PRP may be a safe alternative to bovine thrombin. It results in an excellent working time and significantly less clot retraction than the currently available methods of PRP production.