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.     

Use of platelet-rich plasma,platelet-rich growth factor with arthrocentesis or arthroscopy to treat temporomandibular joint osteoarthritis: Systematic review with meta-analyses

Background

The authors conducted a systematic review and meta-analysis to determine whether arthrocentesis or arthroscopy combined with platelet-rich plasma (PRP) or platelet-rich growth factor (PRGF) injection compared with no injection or saline injection (control group) or hyaluronic acid (HA) injection reduced pain and increased maximum mouth opening (MMO) in patients with temporomandibular joint (TMJ) osteoarthritis (OA).

Comparison of platelet-rich plasma (PRP), platelet-rich fibrin (PRF), and concentrated growth factor (CGF) in rabbit-skull defect healing

Objectives

The objective of this study was to evaluate the effect of platelet-rich plasma (PRP), platelet-rich fibrin (PRF), and concentrated growth factor (CGF) on bone healing.

Study design

Twelve rabbits were included in this randomized, blinded, prospective study. 15-mm × 10-mm-sized defects were created in the parietal bone, filled with PRP, PRF, CGF, and void. The bone mineral density and bone volume were analyzed with microscopic computed tomography (micro-CT) and histomorphometrics at the 6th and 12th week.

Results

In micro-CT analysis, bone mineral density and bone volume were greater in the experimental group than in controls at both 6th and 12th week, but not among the experimental groups. Similarly, histomorphometric examination revealed that more bone formation was seen in the experimental group.

Conclusion

The addition of PRP, PRF, and CGF had significantly increased bone formation at the 6th week. The effect of PRP, PRF, and CGF was similar and may be useful in the future to increase the success rate of bone grafting.     

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.     

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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。     

富血小板血浆/藻酸盐缓释载体复合物修复骨缺损的实验研究

目的 比较富血小板血浆（PRP）/藻酸盐微囊和PRP/藻酸盐微珠2种复合材料的生物学性能。方法 42只新西兰兔分为3组,每组14只,切除兔桡骨中下段1.5 cm的骨质造成极限性骨缺损模型,分别植入PRP/藻酸盐微珠复合物、PRP/藻酸盐微囊复合物、PRP凝胶于骨缺损处。另取4只桡骨同样部位和大小骨缺损旷置作为空白对照。术后观察其一般情况并于8、12周每组分别处死7只实验动物,取材行X线片检查及组织学切片,观察成骨效果。结果 术后8周和12周各组骨缺损区均有新骨形成,成骨量随时间的推移而增加。经影像学检查和组织学评估,各组的成骨能力依次为：PRP/藻酸盐微珠〉PRP/藻酸盐微囊〉PRP/凝胶,各组间的差异有统计学意义（P〈0.05）,其中PRP/藻酸盐微珠的成骨能力最强。结论 PRP/藻酸盐微珠对骨缺损愈合有明显促进作用,可以作为组织工程学中一种替代骨组织的生物活性材料。     

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.     

Efficacy of platelet-rich plasma in alveolar bone grafting.

PURPOSE: In this study, we performed alveolar bone grafting with autologous iliac cancellous bone incorporation with platelet-rich plasma (PRP) and evaluated its efficacy in osteoregeneration. MATERIALS AND METHODS: Seven alveolar cleft patients with adult dentition (average age, 16.1 years) underwent iliac bone grafting with PRP. Quantitative evaluation of regenerated bone was made with 3-dimensional computed tomography scans and compared with controls. RESULTS: The average of the volume ratio of regenerated bone to alveolar cleft in cases with PRP was higher than in controls (P <.05). There were no complications from the blood draw or PRP. CONCLUSION: PRP was a safe and cost-effective source for growth factors and was easy to extract. It could enhance the osteogenesis of alveolar bone grafting in cleft lip and palate patients and may useful for subsequent orthodontic therapy.     

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。     

富血小板血浆诱导骨髓基质细胞碱性磷酸酶活性的研究

目的：体外观察不同浓度富血小板血浆（PRP）对自体骨髓基质细胞碱性磷酸酶活性,对PRP诱导骨髓基质细胞向成骨细胞分化的生物学效应进行评价.方法：抽取狗静脉血及骨髓,采用改进Appel方法制备不同浓度的PRP;采用传代培养筛选法,获得足够数量稳定的骨髓基质细胞（BMSC）,酶动力学法检测碱性磷酸酶活性.结果：（1）原代培养的骨髓基质细胞24h开始贴壁,12d后细胞融汇成单层,细胞多呈成纤维梭形细胞形态,经诱导液传代培养后,细胞形态渐变为方形、多角形.（2）酶动力学法测定各实验组细胞第三、六、九、十二天检测碱性磷酸酶活性,经过配对t检验,各组第六、九、十二天的ALP活性水平均较第三天明显升高,低浓度PRP组较高浓度组碱性磷酸酶活性升高,差异具有统计学意义（P＜0.05）.结论：一定浓度的PRP能促进骨髓基质细胞碱性磷酸酶的活性,PRP能有效的促使BMSC向前期成骨细胞分化.     

缓释人富血小板血浆生长因子壳聚糖微球的制备

目的：制备载人富血小板血浆（hPRP）生长因子壳聚糖微球,观察其体外缓释效果。方法：选用离子凝胶法制备壳聚糖微球,利用微球吸附hPRP生长因子;以微球对hPRP中主要生长因子TGF-β1的吸附率作为评价指标,优化微球制备配方,并观察优化配方微球体外缓释TGF-β1效果;以hPRP中PDGF-AB为对象考察优化配方微球对其吸附率、载生长因子率,验证微球吸附效果。结果：当壳聚糖溶液浓度为2.5mg/ml、10mg/ml多聚磷酸钠溶液滴加量为2.5ml时可获得优化的壳聚糖微球制备配方。优化配方微球对hPRP中主要生长因子TGF-β1、PDGF-AB吸附率分别为78.02%、58.28%,载生长因子率分别为3.70ng/mg、1.31ng/mg;载hPRP生长因子的微球体外21天缓释TGF-β1累计35.80ng,占吸附总量的85.52%。结论：本实验制备的壳聚糖微球可初步实现吸附hPRP复合生长因子,并初步达到体外缓释效果。     

Growth factor levels in platelet-rich plasma and correlations with donor age, sex, and platelet count.

INTRODUCTION: Platelet-rich plasma contains autologous thrombocyte growth factors and might be promising for acceleration of dentoalveolar bone regeneration. In this study, it was analysed for platelet counts and growth factor concentrations. MATERIAL AND METHOD: Platelet-rich plasma was isolated by discontinuous cell separation from 158 healthy men and 55 women aged 17-62 years. One hundred and fifteen specimens (stratified for age and gender of the donor) were analysed for growth factor concentrations and platelet count. RESULTS: The platelet count in platelet-rich plasma (1,407,640+/-320,100/microl) was 5 times higher than in donor blood (266,040+/-60,530/microl). Platelet-derived growth factor AB (117+/-63 ng/ml), transforming growth factor (TGF) beta -1 (169+/-84 ng/ml), and insulin-like growth factor (IGF) I (84+/-23 ng/ml) were found in large amounts, while platelet-derived growth factor (PDGF) BB (10+/-8 ng/ml) and transforming growth factor beta -2 (0.4+/-0.3 ng/ml) were found in small amounts only. The growth factor content was not well correlated with the platelet count in whole blood nor with the platelet-rich plasma (r(p)=0.35). No influence of gender or age on platelet count or growth factor concentrations was discovered (except IGF-I). CONCLUSIONS: While there was substantial variation in the growth factor content of platelet-rich plasma, the factors influencing this are still worthy of further investigation. Furthermore, a technique whereby the growth factor content could be rapidly assessed in platelet-rich plasma may be of therapeutic benefit.     

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.     

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.     

A comparative study of 2 methods for obtaining platelet-rich plasma.

PURPOSE: Double and single centrifugation are the most commonly used techniques for obtaining platelet-rich plasma (PRP) in dentistry. In this study, we used and compared 2 methods for obtaining PRP: double centrifugation (ACE system; Surgical Supply and Surgical Science Systems, Brockton, MA) and single centrifugation (Nahita system; Nahita, Navarra, Spain). MATERIALS AND METHODS: Blood samples were obtained from 30 random patients. Each blood sample was treated using the ACE system and Nahita system methods, after which the obtained material was analyzed by flow cytometry for platelet counts and by transmission electron microscopy (TEM) for ultrastructural analysis of the PRP gel. RESULTS: Platelet count analysis of the PRP obtained from both methods revealed that the ACE and Nahita systems accomplished platelet concentrations of (336%) and (227%), respectively. The platelet counting results obtained from the ACE system samples were more dispersed than their Nahita system counterpart. The ultrastructural (ie, TEM) study showed considerable alterations of the platelet aggregates in the ACE's PRP, especially when the samples were not mixed in the final stage of the procedure, whereas the Nahita aggregates always had a normal physiological appearance. CONCLUSIONS: The ACE double-centrifugation method is able to achieve higher platelet concentrations than the single-centrifugation Nahita system, although the results obtained by ACE were more dispersed. Nevertheless, the ACE system provoked alterations in the PRP ultrastructure, and it was more sensitive to small errors during preparation.