人富血小板血浆及其细化成分对小鼠成骨细胞增殖的影响

目的:观察人富血小板血浆(PRP)及其细化成分对小鼠成骨细胞MC3T3-E1增殖的影响.方法:抽取年轻男性志愿者静脉血制备富血小板血浆,并提取洗涤血小板(WPLT)和乏血小板血浆(PPP)等细化成分,使用细胞定量测定试剂盒测定细胞增殖情况.结果:50 ml/L的PRP对MC3T3-E1细胞的增殖没有明显影响,而50 ml/L的WPLT显著促进了MC3T3-E1细胞的增殖,但是,这种促进增殖的作用效果会随着WPLT浓度的倍比增加而显著降低,并且当其浓度为150 ml/L时反而会显著抑制细胞的增殖.另外,50 ml/L的PPP对MC3T3-E1细胞的增殖有明显的抑制作用,而且倍比添加PPP会显著抑制50 ml/LWPLT促进的MC3T3-E1细胞增殖.结论:人WPLT促进小鼠成骨细胞MC3T3-E1增殖的作用具有浓度特异性,PPP对MC3T3-E1的增殖有显著的抑制作用.     

自体来源富血小板血浆对大鼠牙髓细胞增殖作用的研究

目的:初步探讨富血小板血浆及之中生长因子对大鼠牙髓细胞增殖的作用.方法:采用Landesberg 法制备PRP,ELISA测定PRP中两种主要生长因子:转化生长因子(TGF - β1)和血小板源性生长因子(PDGF -AB)的浓度;CCK-8法观察5％、10％PRP在48h时对牙髓细胞的增殖作用;在5％PRP中加入TGF-β1抗体和PDGF-AB抗体分别拮抗TGF-β1和PDGF-AB的作用并观察对细胞增殖的影响.结果:所制备的PRP中血小板含量为1790.58±388.08×109个/L,ELISA的方法测定PRP中TGF-β1及PDGF-AB的浓度分别为3.080 ng/ml和10.706 ng/ml.CCK-8法测定5％和10％PRP对牙髓细胞均有增殖作用(P＜0.05,与对照组相比);屏蔽生长因子可以明显改变5％PRP对大鼠牙髓细胞的增殖作用(P＜0.05);拮抗PDGF-AB组比拮抗TGF - β1组降低增殖作用明显(P＜0.05).结论:本实验制备的PRP含有高浓度的TGF - β1及PDGF-AB,不同浓度的PRP均能有效促进牙髓细胞增殖;屏蔽PDGF-AB明显降低5％PRP对大鼠牙髓细胞的增殖作用.     

富血小板血浆促进人牙髓细胞增殖的实验研究

目的探讨不同浓度富血小板血浆(Platelet rich plasma,PRP)对人牙髓细胞(human dental pulp cells,HDPCs)的增殖作用。方法两步离心法制备PRP,通过ELISA的方法测定PRP中两种主要生长因子血小板源性生长因子(PDGF-AB)和转化生长因子(TGF-β1)的浓度;用四唑盐比色法(MTT)观察5%、10%、20%PRP在2d、4d时对牙髓细胞的增殖作用,并探讨这种作用是否依赖于胎牛血清(FBS)的存在。结果所制备的PRP中血小板的浓度大于1000×109个/L,为全血中的4倍以上,经ELISA的方法测定PRP及贫血小板血浆(Platelet poor plasma,PPP)中PDGF-AB、TGF-β1的浓度分别增加4倍以上。MTT法测定不同浓度组的PRP对牙髓细胞均有增殖作用,以10%PRP增殖效应最明显,P值均<0.05;4d时PRP对细胞的增殖作用明显强于2d时,P值<0.05;10%PRP组较10%胎牛血清组增殖作用明显,P值<0.05。结论本实验制备的PRP含有较高浓度的PDGF-AB及TGF-β1,不同浓度的PRP均能有效促进牙髓细胞增殖,以10%PRP浓度增殖效应最明显;并且这种增殖作用并不依赖于胎牛血清的存在;随着时间延长,PRP对细胞增殖作用增加。     

富血小板血浆支架诱导牙髓再生的体内研究

目的：探讨不同浓度的富血小板血浆（PRP）支架在体内诱导牙髓组织再生的能力。方法将小型猪乳牙牙根段进行化学预备,采用二次离心法制备PRP,根据注入根管中的成分不同将研究分为4组：（1）阴性对照组,即全血组;（2）100％ PRP组;（3）50％ PRP组;（4）空白组,即空的牙根段;每组5个样本,分别植入裸鼠背部皮下,于术后5周处死动物,取出样本进行组织学观察。结果植入5周后,100％ PRP组根管内充满了炎性细胞,50％ PRP组根管内有少量牙髓样的组织生成。结论合适浓度的PRP作为生物支架在体内再生牙髓样的组织是可行的。     

富血小板血浆对根尖乳头干细胞增殖及矿化的影响

目的：探讨不同浓度富血小板血浆（Platelet—rich plasma,PRP）对根尖乳头干细胞（Stem cell from the apical papilla,SCAP）增殖及矿化的影响。方法：两步离心法制备PRP,通过ELISA的方法测定PRP中血小板源性生长凶子（PDGF—AB）和转化生长因子（TGF—B1）的浓度;CCK-8法、ALP活性检测法观察5％、10％、15％、20％PRP与对照组在1d、2d、3d、4d、5d、6d时对根尖乳头干细胞增殖及矿化作用的影响。结果：PRP中血小板浓度大于全血中血小板浓度,为全血中的5倍以上。CCK-8法测定不同浓度组的PRP对根尖乳头细胞增殖均有促进作用,以10％PRP增殖作用显着,P〈0．05。PRP组与对照组相比,3d时,ALP活性无显著性差异（P〉0．05）;6d时,差异有显著性（P〈0．05）。结论：PRP对根尖乳头干细胞的增殖及矿化均具有促进作用,以10％PRP增殖效应最为显著。     

低浓度洗涤血小板促进人牙髓细胞增殖的作用机制探讨

目的:探讨低浓度洗涤血小板促进人牙髓细胞增殖的作用机制。方法:使用从成年男性志愿者静脉采集、制备的洗涤血小板(washed platelet,WPLT)作用于人牙髓细胞,人牙髓细胞增殖和产生的前列腺素E2(PGE2)采用细胞和PGE2测定试剂盒测定;表达的细胞内环氧酶-2(COX-2)mRNA采用Realtime RT-PCR方法测定。结果:50ml/LWPLT诱导人牙髓细胞COX-2mRNA的表达在作用后3h达到最高峰,而诱导PGE2的产生快于IL-1β;50ml/LWPLT诱导的人牙髓细胞增殖可以被消炎痛和地塞米松抑制,而添加PGE2可以逆转这种抑制作用;WPLT诱导的PGE2量随着其浓度的成倍增加呈现出明显的增多趋势,而PGE2在适当浓度范围内明显促进了人牙髓细胞的增殖。结论:低浓度WPLT可能通过诱导产生的PGE2促进了人牙髓细胞的增殖。     

富血小板血浆对牙周膜成纤维细胞在病变牙根表面胶原形成的影响

目的：研究不同浓度的富血小板血浆（platelet-rich plasma,PRP）对牙周膜成纤维细胞（peridontal fibro—blasts,PDLFs）在脱矿的病变牙根表面形成胶原的影响。以进一步探讨富血小板血浆在促牙周组织再生中的作用。方法：采用组织块培养法培养原代人牙周膜成纤维细胞,取5～8代细胞用于实验。富血小板血浆的制备采用二步密度梯度离心法,获取PRP待用,取因重度牙周病拔除的病牙,制取牙根片,收集的根片经高压处理后留存待用。观察富血小板血浆对牙周膜成纤维细胞在脱矿的病变牙根表面胶原的形成,用天狼星红组织特染的方法进一步观察病变根片表面胶原形成的情况。结果：MTT结果和天狼星红特异性染色结果：实验组与对照组均有阳性染色,用Histogram功能来分析统计胶原所占面积的百分数,取其平均值,实验组与对照组差异有显著性（P〈0．05）。结论：浓度为20％PRP为促进PDLFs在脱矿处理的病变牙根表面附着的最佳浓度,并能促进其在处理的脱矿的病变牙根表面胶原的形成。推测PRP和根面脱矿处理在牙周再生中起重要作用。     

富血小板血浆和乏血小板血浆包被的屏障膜对成骨细胞附着的影响

目的：观察富血小板血浆（plateletrich plasma，PRP）和乏血小板血浆（platelet poor plasma，PPP）包被的屏障膜对人牙槽骨成骨细胞附着的影响。方法取第4代人牙槽骨成骨细胞用于实验。健康成人的全血经过两次离心得到PRP和PPP。将A膜（GoreTex-ePTFE^TM膜）、B膜（GoreTex-Resolut^TM膜）和C膜（Inion-GTR^TM膜）冲切成直径为3mm的圆片并固定于24孔培养板底，用盖玻片作为阳性对照，将包被液分为PRP、PPP、磷酸盐缓冲液（phosphate buffer solution，PBS）3个组，分别包被屏障膜或玻片（仅用磷酸盐）2h，将成骨细胞以5×10^7个／L每孔接种于屏障膜或玻片上并孵育24h使细胞附着。苏木素染色，光镜下观察并计数，扫描电镜观察成骨细胞附着膜上的形态。结果：PRP组包被的A、B、C膜上的细胞数分别为23、35和41；PPP组包被的A、B、C膜上的细胞数分别15、12和22；PBS包被的A、B、C膜上的细胞数分别为3、4和6。成骨细胞在PRP、PPP组的附着数量明显高于磷酸盐组（P〈0．05）；PRP组较PPP组的附着数量多（P〈0．05）；盖玻片上的附着数量显著多于3种屏障膜。3种屏障膜相比，B膜和C膜的成骨细胞附着量高于A膜（P〈0．05）。扫描电镜结果显示，PRP组屏障膜上成骨细胞呈梭形，贴壁好，膜表面可见血小板、交织成网状的纤维蛋白，细胞呈现复层生长；PPP组或磷酸盐组成骨细胞贴壁不完全，呈圆形。结论：PRP和PPP能促进成骨细胞在屏障膜上的附着数量；PRP能改善成骨细胞在屏障膜上的附着方式。     

富血小板血浆对牙周膜成纤维细胞的增殖、迁移和分化的影响

目的: 体外研究不同浓度的富血小板血浆(platelet-rich plasma, PRP)对牙周膜成纤维细胞(periodontal ligament fibroblasts,PDLFs)的增殖、迁移和分化的影响.方法:不同浓度PRP(10, 50, 100, 200, 300, 500 ml/L),加入到原代培养的人PDLFs,培养时间为3、 5、 7 d,MTT法测定细胞增殖效果,transwell系统测定细胞迁移效果,AKP试剂盒测定碱性磷酸酶活性.结果:PRP有明显促进增殖作用,以200 ml/L浓度PRP效果最好,更高浓度促进作用反而减低.细胞迁移效果中,各浓度组均比阴性对照组效果好,尤其是100 ml/L的效果最佳.对于碱性磷酸酶活性,也具有明显的促进作用,以300 ml/L的效果最佳.结论:PRP在一定浓度范围内对人PDLFs功能有明显促进效果,但是在更高浓度下,这种促进效果反而减低.     

富血小板血浆对牙髓成纤维细胞附着、增殖的影响

目的 :探讨富血小板血浆 (PRP)对牙髓成纤维细胞附着、增殖的影响。方法 :利用原代细胞体外培养技术、PRP提取技术及四唑盐比色法 (MTT)检测 5 %PRP对牙髓成纤维细胞附着的影响和不同浓度PRP(5 %、10 %、2 0 %、30 %、4 0 %、5 0 % )对牙髓成纤维细胞增殖的影响。结果 :在附着实验中 ,5 %PRP组所测得的平均光密度值 (OD值 )明显高于空白对照组 (P <0 .0 1)。在增殖实验中 ,各不同浓度PRP组所测得的平均OD值均明显高于对照组 (P <0 .0 1)。结论 :PRP可促进牙髓成纤维细胞的附着及增殖     

富血小板血浆和贫血小板血浆处理的生物降解膜对牙周韧带成纤维细胞影响的扫描电镜观察

目的 :探索富血小板血浆 (platelet -richplasma ,PRP)和贫血小板血浆 (platelet -poorplasma,PPP)处理后的生物降解膜对附着的人牙周韧带成纤维细胞形态的影响。方法 :体外培养人牙周韧带成纤维细胞 ,应用梯度密度离心 ,从人新鲜全血中获取PRP和PPP ,并分别预处理两种生物降解膜。体外细胞培养 2 4h ,扫描电镜观察PRP和PPP对两种生物降解膜的附着效果 ,以及附着于膜上人牙周韧带成纤维细胞的超微结构。结果 :PRP和PPP能有效地贴附于生物降解膜 ,在未处理和处理后的生物降解膜上均可见附着的呈梭形或长扁形的人牙周韧带成纤维细胞 ,但在处理膜上人牙周韧带成纤维细胞明显地被包裹于PRP和PPP中 ,仅见增大的胞体、胞浆突起的轮廓 ,细胞彼此重叠或融合成片状。结论 :有效地贴附于生物降解膜的PRP和PPP能将人牙周韧带成纤维细胞网织在一起 ,并促进细胞的增殖。提示经PRP和PPP处理的生物降解膜可能更利于引导牙周组织再生。     

Effect of autologous and allogenic platelet-rich plasma on human gingival fibroblast function

Oral Diseases (2012) 18 , 494–500 Objective: Platelet‐rich plasma (PRP) has been proposed as a method of delivering growth factors to enhance regeneration. The aim of this study was to investigate the use of autogenous and allogenic PRP and platelet‐poor plasma (PPP) on migration and proliferation of human gingival fibroblasts in vitro. Methods: Various concentrations of PRP, as well as PPP, were prepared from autologous and allogenic sources and applied to primary gingival fibroblasts. Migration was determined by assessing the fibroblast response to a concentration gradient. ³H‐thymidine incorporation and crystal violet colorimetric assays were utilized to assess DNA synthesis and proliferation. Results: Platelet‐rich plasma provides a significant migratory stimulus to gingival fibroblasts. Furthermore, the various concentrations of PRP (50%, 20% and 10%) do not promote DNA synthesis in the short term (24 h), but over the longer term (5 days) they stimulate an increase in cell proliferation. Compared with PPP, PRP was superior in terms of encouraging migration, but was inferior in terms of promoting DNA synthesis and cell proliferation. No difference was noted between the autologous and allogenic PRP preparations on cell function. Conclusion: Both PPP and PRP promote gingival fibroblast migration and proliferation in vitro, without differences between preparations obtained from autologous and allogenic sources.     

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.     

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.     

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.