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-rich plasma: a promising innovation in dentistry

The goal of periodontal therapy is to protect and maintain the patient's natural dentition for his or her lifetime. More specifically, after periodontal regenerative surgery, the aim is to achieve complete wound healing and regeneration of the periodontal unit. A recent innovation in dentistry is the preparation and use of platelet-rich plasma (PRP), a concentrated suspension of the growth factors found in platelets. These growth factors are involved in wound healing and are postulated as promoters of tissue regeneration. This clinical update outlines the specific effects of these growth factors, both in vitro and in vivo, on periodontal wound healing. The review focuses on current animal and human trials using PRP to promote tissue regeneration and alveolar bone repair. The article goes on to describe the clinical benefits of PRP and the step-by-step preparation of PRP in the dental office.     

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.     

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.     

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.     

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   

Comparative release of growth factors from PRP,PRF, and advanced-PRF

Objectives

The use of platelet concentrates has gained increasing awareness in recent years for regenerative procedures in modern dentistry. The aim of the present study was to compare growth factor release over time from platelet-rich plasma (PRP), platelet-rich fibrin (PRF), and a modernized protocol for PRF, advanced-PRF (A-PRF).

Materials and methods

Eighteen blood samples were collected from six donors (3 samples each for PRP, PRF, and A-PRF). Following preparation, samples were incubated in a plate shaker and assessed for growth factor release at 15 min, 60 min, 8 h, 1 day, 3 days, and 10 days. Thereafter, growth factor release of PDGF-AA, PDGF-AB, PDGF-BB, TGFB1, VEGF, EGF, and IGF was quantified using ELISA.

Results

The highest reported growth factor released from platelet concentrates was PDGF-AA followed by PDGF-BB, TGFB1, VEGF, and PDGF-AB. In general, following 15–60 min incubation, PRP released significantly higher growth factors when compared to PRF and A-PRF. At later time points up to 10 days, it was routinely found that A-PRF released the highest total growth factors. Furthermore, A-PRF released significantly higher total protein accumulated over a 10-day period when compared to PRP or PRF.

Conclusion

The results from the present study indicate that the various platelet concentrates have quite different release kinetics. The advantage of PRP is the release of significantly higher proteins at earlier time points whereas PRF displayed a continual and steady release of growth factors over a 10-day period. Furthermore, in general, it was observed that the new formulation of PRF (A-PRF) released significantly higher total quantities of growth factors when compared to traditional PRF.

Clinical relevance

Based on these findings, PRP can be recommended for fast delivery of growth factors whereas A-PRF is better-suited for long-term release.

     

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-rich plasma: clinical applications in dentistry

BACKGROUND: Platelet-rich plasma, or PRP, has become a valuable adjunct in wound healing in dentistry. Postsurgically, blood clots initiate the healing and regeneration of hard and soft tissues. Clinicians and scientists are investigating the use of PRP in dentistry as a way to enhance the body's natural wound-healing mechanisms. TYPES OF ARTICLES REVIEWED: The authors reviewed scientific articles that discuss the basic knowledge of wound healing mechanisms and that directly studied the growth factors shown to be concentrated in PRP. They also reviewed articles written by clinicians and researchers in dentistry fields, including oral and maxillofacial surgery and periodontics to determine applications of PRP in the field of dentistry. RESULTS: All of the reviewed articles expressed promise in PRP use and in the growth factors expressed by the platelets concentrated in PRP-namely platelet-derived growth factor, or PDGF, and transforming growth factor-beta, or TGF-beta--as an adjunct to postsurgical wound healing. Both PDGF and TGF-beta have been shown in vivo to accelerate wound healing through different mechanisms. The development of an autologous PRP has been shown to be relatively easy, to be effective as a surgical adjunct, to retain high levels of the desired growth factors after preparation and to be clinically effective in accelerating postsurgical healing in both periodontal and oral surgery applications. CLINICAL IMPLICATIONS: PRP has proven to be effective at improving surgical results in a variety of procedures in the field of oral and maxillofacial surgery. PRP also shows promise in periodontal regenerative therapy and should continue to be studied by scientists and clinicians alike.     

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.     

A novel drilling procedure and subsequent bone autograft preparation: a technical note

PURPOSE: To describe a new drilling system that allows the surgeon to obtain autologous living bone that, when associated with a plasma rich in growth factors (PRGF), can be used in bone grafting. MATERIALS AND METHODS: Bone particles collected using both conventional and new drilling systems were analyzed by means of optic and electronic microscopy in 10 patients. Blood was collected from 43 volunteers and used to prepare PRGF. Quantitative aspects of the PRGF, including number of platelets and concentration of growth factors (insulin growth factor [IGF-I], transforming growth factor [TGF-beta1], platelet-derived growth factor [PDGF-AB], vascular endothelial growth factor [VEGF], hepatocyte growth factor [HGF], and epidermal growth factor [EGF]) were assessed. A demonstrative case study was presented. RESULTS: Microscopic examination showed that the bone structure and the presence of living cells in the bone chips were conserved in all samples obtained from drilling at low speed, whereas material obtained by conventional drilling did not maintain these qualities. Mean counts for TGF-beta1 (55.27 +/- 16.23 ng/mL), PDGF-AB (27.96 +/- 12.13 ng/mL), VEGF (421.09 +/- 399.0 pg/mL), EGF (455.49 +/- 210.04 pg/mL), and HGF (605.70 +/- 269.20 pg/mL) were significantly correlated with the number of platelets (590,000 +/- 197,000 platelets/microL; P < .05). DISCUSSION AND CONCLUSION: The new drilling procedure was developed based on biologic criteria. The method may reduce damage to the host tissue and can be used to obtain a mass of living bone for subsequent grafting in association with autologous growth factors. This new procedure may present new possibilities for enhanced bone healing and needs to be evaluated in a clinical trial.     

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.     

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.     

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富血小板血浆是自体全血经离心后获得的具有高浓度血小板的血浆,激活后释放大量生长因子,可加速组织损伤的愈合。本文对富血小板血浆的制备、作用机制及其在口腔医学中应用现状做一综述。     

Releasing growth factors from activated human platelets after chitosan stimulation: a possible bio-material for platelet-rich plasma preparation

INTRODUCTION: Thrombin is commonly used for activating the platelets and releasing the growth factors on the application of platelet-rich plasma (PRP). We have previously reported that chitosan can enhance rabbit platelet aggregation. In this study, the effects of chitosan on the subsequent growth factors release after human platelets activation were examined to evaluate the possibility of chitosan being used as a substitute for thrombin during PRP preparation. MATERIAL AND METHODS: Human platelet activation was determined by aggregation, adhesion and alpha-granule membrane glycoprotein expression. Platelet aggregation was measured by the turbidimetric method, the adhesion was directly examined on chitosan-coated glass plates under light microscope and scanning electron microscope (SEM), and the alpha-granule membrane glycoprotein was detected by fluorescent isothiocyanate (FITC)-conjugated anti-CD61 antibody through flow cytometry. The subsequent epidermal growth factor (EGF), platelet-derived growth factor (PDGF)-AB and transforming growth factor (TGF)-beta1 release from platelets were assayed by ELISA after mixing with chitosan. RESULTS: The enhancing effects on the platelet adhesion and the aggregation from chitosan were observed. Under both microscopes, the adhesive platelets on the chitosan-coated plates were not only greater in number but also earlier in activation than those on the control plates. With flow cytometry, increased glycoprotein IIIa expression in platelets was detected after chitosan treatment. Greater concentrations of growth factors were measured from PRP after chitosan treatment than after the solvent treatment. CONCLUSION: Because of the observations of growth factors releasing from activated human platelets after chitosan stimulation, we suggest that chitosan may be an appropriate substitute for thrombin in PRP preparation.     

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.     

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.     

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.     

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.