Comparison of Point-of-Care Methods for Preparation of Platelet Concentrate (Platelet-Rich Plasma)

Purpose: This study analyzed the concentrations of platelets and growth factors in platelet-rich plasma (PRP), which are likely to depend on the method used for its production. Materials and Methods: The cellular composition and growth factor content of platelet concentrates (platelet-rich plasma) produced by six different procedures were quantitatively analyzed and compared. Platelet and leukocyte counts were determined on an automatic cell counter, and analysis of growth factors was performed using enzyme-linked immunosorbent assay. Results: The principal differences between the analyzed PRP production methods (blood bank method of intermittent flow centrifuge system/platelet apheresis and by the five point-of-care methods) and the resulting platelet concentrates were evaluated with regard to resulting platelet, leukocyte, and growth factor levels. The platelet counts in both whole blood and PRP were generally higher in women than in men; no differences were observed with regard to age. Statistical analysis of platelet-derived growth factor AB (PDGF-AB) and transforming growth factor Β1 (TGF-Β1) showed no differences with regard to age or gender. Platelet counts and TGF-Β1 concentration correlated closely, as did platelet counts and PDGF-AB levels. There were only rare correlations between leukocyte counts and PDGF-AB levels, but comparison of leukocyte counts and PDGF-AB levels demonstrated certain parallel tendencies. Conclusions: TGF-Β1 levels derive in substantial part from platelets and emphasize the role of leukocytes, in addition to that of platelets, as a source of growth factors in PRP. All methods of producing PRP showed high variability in platelet counts and growth factor levels. The highest growth factor levels were found in the PRP prepared using the Platelet Concentrate Collection System manufactured by Biomet 3i.     

Antimicrobial Activity of Platelet‐Rich Plasma and Other Plasma Preparations Against Periodontal Pathogens

Background: In addition to releasing a pool of growth factors during activation, platelets have many features that indicate their role in the anti‐infective host defense. The antimicrobial activities of platelet‐rich plasma (PRP) and related plasma preparations against periodontal disease–associated bacteria were evaluated. Methods: Four distinct plasma fractions were extracted in the formulation used commonly in dentistry and were tested for their antibacterial properties against three periodontal bacteria: Porphyromonas gingivalis, Aggregatibacter actinomycetemcomitans, and Fusobacterium nucleatum. The minimum inhibitory concentration of each plasma preparation was determined, and in vitro time‐kill assays were used to detect their abilities to inhibit bacterial growth. Bacterial adhesion interference and the susceptibility of bacterial adherence by these plasma preparations were also conducted. Results: All plasma preparations can inhibit bacterial growth, with PRP showing the superior activity. Bacterial growth inhibition by PRP occurred in the first 24 hours after application in the time‐kill assay. PRP interfered with P. gingivalis and A. actinomycetemcomitans attachment and enhanced exfoliation of attached P. gingivalis but had no influences on F. nucleatum bacterial adherence. Conclusions: PRP expressed antibacterial properties, which may be attributed to platelets possessing additional antimicrobial molecules. The application of PRP on periodontal surgical sites is advisable because of its regenerative potential and its antibacterial effects.     

Influence of antiplatelet substances on platelet-rich plasma.

PURPOSE: Platelets containing a number of growth factors (platelet-derived growth factor [PDGF], transforming growth factor-beta [TGF-beta], etc) can be obtained in high concentrations through centrifugal separation and are used in clinical applications as platelet-rich plasma (PRP). However, only a few studies have been conducted on the growth factors present in PRP. In this study, we focused on the concentrations of growth factors in PRP and clarified the influence of using antiplatelet substances in the process of platelet concentration to improve the concentration rate of growth factors in PRP. MATERIALS AND METHODS: We made platelet pellets from whole blood obtained from humans with or without some antiplatelet substances (prostaglandin E1, aspirin, apyrase). Platelet pellets were resuspended in phosphate-buffered saline as platelet resuspensions. We measured PDGF and TGF-beta1 concentrations in the samples. In measurements, we had the samples treated to release growth factors from platelets to measure accurate concentrations. RESULTS: PDGF and TGF-beta1 were concentrated to a mean of over 400% in the samples with antiplatelet substances as compared with the samples without antiplatelet substances. CONCLUSIONS: The antiplatelet substances were effective for efficiently concentrating growth factors in platelets.     

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.     

Platelet‐Poor and Platelet‐Rich Plasma Stimulate Bone Lineage Differentiation in Periodontal Ligament Stem Cells

Background: Plasma‐derived fractions have been used as an autologous source of growth factors; however, limited knowledge concerning their biologic effects has hampered their clinical application. In this study, the authors analyze the content and specific effect of both platelet‐rich plasma (PRP) and platelet‐poor plasma (PPP) on osteoblastic differentiation using primary cultures of human periodontal ligament stem cells (HPLSCs). Methods: The authors evaluated the growth factor content of PRP and PPP using a proteome profiler array and enzyme‐linked immunosorbent assay. HPLSCs were characterized by flow cytometry and differentiation assays. The effect of PRP and PPP on HPLSC bone differentiation was analyzed by quantifying calcium deposition after 14 and 21 days of treatment. Results: Albeit at different concentrations, the two fractions had similar profiles of growth factors, the most representative being platelet‐derived growth factor (PDGF) isoforms (PDGF‐AA, ‐BB, and ‐AB), insulin‐like growth factor binding protein (IGFBP)‐2, and IGFBP‐6. Both formulations exerted a comparable stimulus on osteoblastic differentiation even at low doses (2.5%), increasing calcium deposits in HPLSCs. Conclusions: PRP and PPP showed a similar protein profile and exerted comparable effects on bone differentiation. Further studies are needed to characterize and compare the effects of PPP and PRP on bone healing in vivo.     

Flow cytometric and morphological characterization of platelet-rich plasma gel

BACKGROUND OF PROBLEMS: Platelet-rich plasma (PRP) gel is derived from an autogenous preparation of concentrated platelets and is widely used in implant dentistry as a vector for cell growth factors. However, limited data are available on its structure and composition. The present study was aimed at providing a flow cytometric and ultrastructural characterization of PRP gel. MATERIALS AND METHODS: Twenty PRP gel samples were obtained from healthy volunteers. These PRP gel specimens were prepared for transmission (TEM) and scanning electron microscopy (SEM) examination of their morphological ultrastructure. Flow cytometry with CD41-PE monoclonal antibody was used to detect platelet cells, as this antibody recognizes human-platelet-specific antigen CD41. RESULTS: Both SEM and TEM showed that PRP gel contains two components: a fibrillar material with striated band similar to fibrin filaments, and a cellular component that contains human platelet cells. Both techniques indicated that no morphological elements were bound between the cellular component and the fibrillar material. The cells were confirmed as platelet cells by flow cytometric study after incubation with specific monoclonal antibody CD41-PE. CONCLUSION: PRP gel contains a fibrillar and a cellular (largely human platelet cell) component. This unique structure may be capable of acting as a vehicle for carrying of cells that are essential for soft/hard tissue regeneration.     

An in vitro and in vivo evaluation of autologous platelet concentrate in oral reconstruction

A platelet concentrate, when combined with calcified thrombin, produces a platelet gel that has been used to achieve hemostasis and modulate bone growth and wound healing. The recovery of high concentrations of viable platelets and their resulting growth factor levels represents the most important factor in the clinical utility of a platelet concentrate because only functional platelets can release the growth factors that are necessary to induce tissue growth and bone regeneration. The SmartPReP system's efficiency in recovery of platelets from a sample of whole blood averaged 70.6%, almost twice that of various manual techniques using laboratory centrifuges. Platelet concentrates prepared by the SmartPReP system had a viability equal to platelet concentrates prepared for transfusion as measured by hypotonic stress, platelet aggregation, and p-selectin. A series of clinical case studies demonstrates the use of autologous platelet gel in oral surgery.     

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.     

Activation of human platelet-rich plasmas: effect on growth factors release, cell division and in vivo bone formation

OBJECTIVES: Aims of this controlled study were to determine the effects of activated human platelet-rich plasmas (PRPs) on early and mature bone formation in vivo, and to characterize the effect of PRP activation on growth factors release and endothelial cell division in vitro. MATERIAL AND METHODS: PRPs were prepared from four volunteers with the platelet concentrate collector system (PCCS) system and activated with three concentrations of calcium and thrombin. Platelet-derived growth factor (PDGF)-BB, vascular endothelial growth factor (VEGF), transforming growth factor beta (TGF-beta) and interleukin-1beta (IL-1beta) levels released in supernatants were measured by ELISA, at time 0, 1h, 24h and 6 days following PRP activation. Mitogenic potential of PRP supernatants were tested on endothelial cells in vitro, and the effects of activated human PRPs on bone formation in vivo were measured in athymic rats by micro-CT analyses. RESULTS: Activation of PRPs with calcium and thrombin triggered an immediate release of VEGF, PDGF-BB and TGF-beta and a delayed release of IL-1beta in PRP supernatants. Higher endothelial cell division was observed with supernatants from activated PRPs than from non-activated PRPs. Positive correlations were observed between VEGF levels and endothelial cell division and bone formation. A negative correlation was also found between PDGF-BB concentration and bone formation. However, early and mature bone formations with activated PRPs did not significantly differ from the ones obtained in the control group. CONCLUSIONS: Activation of PRPs with calcium and thrombin regulates growth factors release and endothelial cell division in vitro. However, activated PRPs does not improve the early or mature bone formations in vivo in this athymic rat model.     

Modulation of platelet activation and initial cytokine release by alloplastic bone substitute materials

Objectives: Platelet‐derived cytokines play a crucial role in tissue regeneration. In regenerative dental medicine, bone substitute materials (BSM) are widely used. However, initial interactions of BSM and platelets are still unknown. The aim of this study was to evaluate the potential of platelet activation and subsequent initial cytokine release by different commercial alloplastic BSM. Material and methods: Eight commercial BSM of different origins and chemical compositions (tricalcium phosphate, hydroxyapatite, bioactive glass: SiO₂ and mixtures) were incubated with a platelet concentrate (platelet‐rich plasma, PRP) of three healthy volunteers at room temperature for 15 min. Platelet count, aggregation, degranulation (activated surface receptor CD62p) and cytokine release (Platelet‐derived growth factor, Vascular endothelial growth factor) into the supernatant were quantified. Highly thrombogenic collagen served as a reference. Results: The investigated PRP samples revealed different activation patterns when incubated with different BSM. In general, SiO₂‐containing BSM resulted in high platelet activation and cytokine release. In detail, pure bioactive glass promoted platelet activation most significantly, followed by hybrid BSM containing lower ratios of SiO₂. Additionally, we found indications of cytokine retention by BSM of large specific surfaces. Conclusions: Platelet activation as well as consecutive storage and slow release of platelet‐derived cytokines are desirable attributes of modern BSM. Within the limits of the study, SiO₂‐containing BSM were identified as promising biomaterials. Further investigations on cytokine adsorption and cytokine release kinetics by the respective BSM have to be conducted. To cite this article:
Klein MO, Kämmerer PW, Scholz T, Moergel M, Kirchmaier CM, Al‐Nawas B. Modulation of platelet activation and initial cytokine release by alloplastic bone substitute materials.
Clin. Oral Impl. Res. 21 , 2010; 336–345
doi: 10.1111/j.1600‐0501.2009.01830.x     

Growth Factor Measurement and Histological Analysis in Platelet Rich Fibrin: A Pilot Study

Objective

The aim of this study was to compare growth factor amount contained in platelet rich fibrin (PRF) and compare with that in platelet rich plasma (PRP), and in whole blood. And also to investigate distribution of growth factors and cellular components in PRF.

Materials and Methods

PRF and PRP were obtained from the same sample of peripheral blood. Extraction of proteins were done with lysis buffer, accompanied by freeze and thaw procedures. Concentration of two representative growth factors in platelets: platelet derived growth factor (PDGF) and transforming growth factor beta (TGF-β), were measured with enzyme-linked immunosorbent assay (ELISA). PRF was cut into three parts: (top, middle and bottom), and growth factor concentration was measured respectively. Paraffin embedded section of PRF was observed with Giemsa stain. Immuno-histochemical analysis with anti-PDGF and anti-TGF-β antibodies was also conducted.

Results

The growth factor levels in PRF was higher than in peripheral blood and comparable to those in PRP. Growth factor levels in bottom part of PRF was much higher than in top and middle part. Microscopically, platelets and mono-nucleated cells were concentrated just above the yellow–red interface. Poly-nucleated cells were concentrated below the interface.

Conclusion

The growth factors were surely concentrated in PRF. This result can support basis of good clinical outcomes. For effective application of PRF, the knowledge that growth factors and cells are not equally distributed in PRF should be utilized.     

Porphyromonas gingivalis-induced Platelet Aggregation in Human Plasma

The periodontal pathogen Porphyromonas gingivalis has the ability to aggregate human platelets. In this study, the mechanism of P. gingivalis-induced platelet aggregation in platelet-rich plasma (PRP) was investigated. Proteinase inhibitors toward Arg- and Lys-specific gingipain (Rgp and Kgp) did not suppress P. gingivalis-induced platelet aggregation in PRP and it was found that proteolytic activity of gingipains did not contribute to P. gingivalis-induced platelet aggregation in plasma. The study using mutant strains revealed that P. gingivalis-induced platelet aggregation in PRP depended on Rgp-, Kgp- and hemagglutinin A (HagA) -encoding genes that intragenically coded for adhesin Hgp44. Hgp44 adhesin on the bacterial cell surface, which was processed by Rgp and Kgp proteinases, was essential for P. gingivalis-induced platelet aggregation in PRP P. gingivalis cell-reactive IgG in plasma, and FcyRIIa receptor and to a lesser extent GP Ibα receptor on platelets were found to be essential for P. gingivalis-induced platelet aggregation in PRP These results reveal a novel mechanism of platelet aggregation by P. gingivalis and contribute to elucidation of the common nature of the interaction between platelets and pathogenic bacteria.     

Periodontal tissue engineering after tooth replantation

Background: 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. Methods: 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). Results: 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. Conclusions: 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.     

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.     

Growth factor levels in the platelet-rich plasma produced by 2 different methods: curasan-type PRP kit versus PCCS PRP system

PURPOSE: Potential treatments using autologous thrombocyte growth factors are an important reason to improve methods for isolating platelet-rich plasma (PRP). Two methods for extracting PRP directly by the surgeon are currently available; this study was conducted to compare the growth factor levels in the resulting PRP. MATERIALS AND METHODS: Whole blood was drawn from 46 healthy donors (17 men, 29 women) aged 20 to 59 years (29.9 +/- 7.8). PRP was then separated from each sample by both the PCCS (3i) and Curasan (PRP Kit, Curasan) methods. RESULTS: The growth factor content differed significantly for TGF-beta1 (PCCS 467.1 ng/mL; Curasan 79.7 ng/mL) (sign test P < .0001) and PDGF-AB (PCCS 251.8 ng/mL; Curasan 314.1 ng/mL) (P < .0001); this was less significant for IGF-I (PCCS 91.0 ng//mL; Curasan 69.5 ng/mL) (P < .02). The higher platelet count in the PCCS PRP (PCCS 2,232,500/microL; Curasan 1,140,500/microL) seemed to correlate with a higher level of TGF-beta1 (Spearman's correlation coefficient, r(s) = 0.7), whereas the higher leukocyte count in the Curasan PRP (PCCS 15,300/microL; Curasan 33,150/pL) had only a minor correlation with higher levels of PDGF-AB (r(s) = 0.46). DISCUSSION: The PCCS end product has both a higher platelet count and a higher total content of the growth factors investigated. Nevertheless, the biologic effect of the evaluated growth factor levels remains unknown. The amount of PRP necessary to achieve the intended biologic effects still remains unclear. CONCLUSION: PRP contains growth factors in high concentrations. Precise predictions of growth factor levels based on the thrombocyte counts of whole blood or PRP appeared limited. There are different sources for growth factors (platelets, leukocytes, plasma).     

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.     

In vitro evidence that the biological effects of platelet-rich plasma on periodontal ligament cells is not mediated solely by constituent transforming-growth factor-beta or platelet-derived growth factor

BACKGROUND: The biological actions of platelet-rich plasma (PRP) are thought to be mediated primarily by constituent transforming-growth factor-beta1 (TGF-beta1) and platelet-derived growth factor-AB (PDGF-AB). However, we previously demonstrated that type I collagen expression in periodontal ligament (PDL) cells is acutely stimulated through fibrin clot formation produced by the fibrinogen within PRP, rather than by the known growth factors. To investigate the possible involvement of other unidentified components in PRP action, we have now compared the effects of PRP with those of known recombinant growth factors on cell proliferation, alkaline phosphatase (ALP) activity, and collagen synthesis in human PDL cell cultures. METHODS: PRP was prepared by an established two-step centrifugation protocol using blood obtained from adult human volunteers. Cells cultured in serum-reduced medium on native or collagen-coated plates were treated with PRP, TGF-beta1, or PDGF-AB. Cellular DNA synthesis was evaluated by bromodeoxyuridine incorporation. ALP activity was assessed using p-nitrophenylphosphate with formalin-fixed cells, and cellular DNA content was subsequently quantified using bis-benzimide. Collagen synthesis was evaluated using a specific dye-based assay kit. RESULTS: 1) As did both TGF-beta1 and PDGF-AB, PRP stimulated cell proliferation. 2) However, only the initial mitogenic action of PRP was attenuated in collagen-coated plates. 3) PRP, but neither growth factor, immediately induced fibrin clot formation and subsequently stimulated cellular adhesion and collagen synthesis. 4) These effects were significantly augmented on collagen-coated plates. 5) PRP enhanced ALP activity, but neither TGF-beta1 nor PDGF-AB replicated this effect. CONCLUSIONS: When evaluated versus the concentrations of growth factors known to be contained by our PRP preparations, these data support the concept that PRP-constituent TGF-beta1 acts as a significant growth factor on PDL cells. However, our findings also strongly suggest that the PRP-induced increase in ALP activity is mediated by an as-yet-unidentified component(s). In conjunction with previously demonstrated fibrinogen-mediated actions, our data provide evidence that PRP produces a number of potent effects on PDL cells that does not solely reflect simple combination of its major known growth factors.     

The contribution of platelet-derived growth factor, transforming growth factor-beta1, and insulin-like growth factor-I in platelet-rich plasma to the proliferation of osteoblast-like cells

The aim of this study was to investigate the effect of platelet-rich plasma (PRP) on the proliferation of osteoblast-like cells in vitro. PRP was prepared using a centrifuge; the number of platelets (n = 32) and the levels of platelet-derived growth factor-AB (PDGF-AB), transforming growth factor-beta1 (TGF-beta1), and insulin-like growth factor-I (IGF-I) were measured (n = 16). For the proliferation assay, SaOS-2 was cultured in the presence of platelet-poor plasma (PPP), whole blood, or PRP. The cell number was counted after 36 and 72 hours. To investigate the effect of each growth factor, the cells were cultured with PRP in the absence or presence of neutralizing antibodies, and counted as described. The mean platelet count of PRP was 1546.36 +/- 382.25 x 10(3)/microL, and the mean levels of PDGF-AB, TGF-beta1 and IGF-I were 0.271 +/- 0.043, 0.190 +/- 0.039, and 0.110 +/- 0.039 ng/1500 x 10(3) platelets, respectively. Cell proliferation was enhanced in all PRP groups in a dose-dependent manner, and all neutralizing antibodies significantly suppressed proliferation compared with the PRP group, lacking antibody, at 36 hours. However, at 72 hours, the neutralizing antibodies of PDGF and TGF-beta1, but not IGF-I, significantly suppressed proliferation. These results show the beneficial abilities of PRP in the proliferation of osteoblast-like cells from the standpoint of growth factors, including the contribution of each factor.     

The effect of platelet-rich plasma on new bone formation by augmentation with osseoconductive bone substitute material in beagle dogs

Animal experiments were carried out with osseoconductive bone substitute beta-tricalcium phosphate (beta-TCP), with the aim of assessing the effect of the growth factors synthesized by platelets on the speed of beta-TCP incorporation and on the quality of newly formed bone. The question arises whether the results attained with this synthetic material approach are comparable to those attained with autologous bone. Defects in the mandibles of beagle dogs were filled with beta-TCP or with the mixture of beta-TCP and platelet rich plasma (PRP) obtained from autologous blood. The quality of the newly formed bone and the effect of PRP were studied by histologic and histomorphometric methods. On the 6th week, bone formation seemed to be more effective when PRP was applied in comparison with beta-TCP alone, but the difference was not significant. On the 12th week bone formation was significantly greater. The results demonstrate that the use of PRP accelerates the remodelling of the synthetic bone-substitute material beta-TCP.     

The Harvest Smart PRePTM system versus the Friadent-Schütze platelet-rich plasma kit

An important reason to improve methods for isolating platelet-rich plasma (PRP) is the potential use of autogenous platelet growth factors. In addition to the Curasan PRP kit (Curasan, Kleinostheim, Germany) and the platelet concentrated collection system (PCCSTM) system, two new methods for the preparation of PRP by the surgeon are now available. This study compared the suitability of these new methods for the preparation of PRP. Whole blood was drawn from 54 healthy donors (33 men and 21 women) aged 23-79 years (38.0 +/- 17.7 years). PRP was prepared from each donor's blood using both the Smart PRePTM system (Harvest Technologies Corporation, Munich, Germany) and the Friadent-Schütze method (PRP kit; Friadent-Schütze, Vienna, Austria). The platelet count in donor whole blood was 276 810 +/- 59 440 /microl. Platelet counts differed significantly between the Smart PRP preparation (1227 890 +/- 312 440 platelets/microl) and the Friadent-Schütze PRP preparation (1440 500 +/- 501 700 platelets/microl) (sign test, P < 0.001). The Smart PRePTM system had a significantly higher collection efficiency (63.4 +/- 7.9%) than the Friadent-Schütze kit (49.6 +/- 13.6%) (sign test, P < 0.001). The leukocyte contents in the two platelet concentrates were similar (Smart PRePTM, 19 261 +/- 8082 platelets/microl; Friadent-Schütze, 21 691 +/- 16 430). Transforming growth factor (TGF)-beta1 and platelet-derived growth factor (PDGF)-AB were higher in the Friadent-Schütze PRP (TGF-beta1, 196.8 +/- 109.6 ng/ml; PDGF-AB, 251.6 +/- 115.4 ng/ml) than in the Smart PRePTM (TGF-beta1, 77.2 +/- 54.8 ng/ml; PDGF-AB, 208 +/- 85.2 ng/ml). The sign test indicated significant differences between the two methods in the concentrations of TGF-beta1 (P < 0.001) and PDGF-AB (P < 0.01). Insulin-like growth factor (IGF)-1 levels in the two PRP preparations were similar (Friadent-Schütze PRP, 72.8 +/- 22.3 ng/ml; Smart PRePTM, 91.4 +/- 21.3 ng/ml). The Smart PRePTM system was superior with respect to ease of handling and preparation time. It also had a significantly higher platelet collection efficiency than the Friadent-Schütze PRePTM kit. The Friadent-Schütze PRP kit offers a slight advantage in the resulting PRP platelet concentration. However, this is easily compensated for in the Smart PRePTM system by reducing the volume of the resulting PRP.