富含血小板血浆双相接种法构建的组织工程骨修复山羊颅骨缺损

目的：观察以富含血小板血浆（platelet-rich plasma PRP）介导的双相接种法构建的组织工程骨在体内的成骨是否优于普通双相接种法和传统静滴接种法。方法：用传统静滴法（对照组）和富含血小板血浆（PRP组）、乏血小板血浆（PPP组）双相接种法构建组织工程骨,并用其修复山羊颅骨临界缺损,术后3月通过CT扫描、组织病理学观察三组动物颅骨缺损处成骨情况。结果：PRP组的成骨量显著较PPP组合和对照组（P〈0.05）,PPP组合和对照组无显著差异（P〉0.05）。结论：用PRP介导的双相接种法构建组织工程骨在体内早期成骨优势显著;PRP双相接种法是安全、简便、实用、高效的细胞接种方法。     

新型多孔双相磷酸钙支架与兔骨髓间充质干细胞相容性的体外实验研究

目的：研究新型多孔双相磷酸钙支架与兔骨髓间充质干细胞的体外相容性。方法：体外分离、扩增兔骨髓间充质干细胞。取传二代细胞,按照经典组织工程构建方法,分别接种双相磷酸钙（对照组）和双相磷酸钙复合纳米羟基磷灰石（实验组）支架。单纯细胞培养为空白对照组。体外成骨诱导培养14天。倒置相差显微镜、扫描电镜观察细胞生长情况;MTT法检测细胞增殖功能;碱性磷酸酶（ALP）检测细胞成骨分化功能。结果：细胞在新型支架上吸附、生长良好。各组MTT及ALP值随培养时间延长而增大。培养各时段实验组均高于其他两组,差别有统计学意义（P〈0.01）。结论：新型BCP支架与兔BMSC在体外具有良好的相容性,二者具有体外构建工程骨的潜力。     

新型β-磷酸三钙的制备与成骨能力的实验研究

目的采用有机泡沫浸渍法制备多孔β-磷酸三钙(β-TCP)生物陶瓷,探讨其作为骨组织工程支架材料的可能性。方法有机泡沫浸渍法制备β-TCP支架材料,接种人骨髓基质干细胞,体外成骨诱导培养。相同条件下培养的单层细胞作为对照组。采用扫描电镜和噻唑蓝比色法观察细胞在材料上的增殖能力,同时测定碱性磷酸酶活性和骨钙蛋白含量来观察细胞在材料上的成骨分化能力。将体外培养7 d的细胞材料复合物和单纯材料回植裸鼠皮下,分别于术后4、8、12周取材观察异位成骨情况。结果扫描电镜观察显示实验组细胞在材料上增殖良好,MTT结果与对照组无明尼差异,但碱性磷酸酶活性和骨钙蛋白含量均高于单层培养的细胞。细胞材料复合物在裸鼠皮下4周已经成骨,并随时间延长骨量增多。单纯材料组在各时间点均未成骨。结论有机泡沫浸渍法制备的多孔β-TCP生物陶瓷具有良好的支持成骨能力,是一种较理想的骨组织工程支架材料。     

关节软骨细胞三维支架双相接种体外生成软骨组织的观察

目的联合应用自制生物凝胶和三维支架材料,建立组织工程种子细胞的三维支架双相接种技术,并对其构建生成组织工程关节软骨的效果进行初步观察。方法酶消化法分离幼兔关节软骨细胞,于培养瓶中接种、培养,收集第1代软骨细胞,以2.5×107/ml加入液态的自制生物凝胶液中充分混均,制成细胞-凝胶混悬液,接种于CPPf/PLLA(caliumpolyphosphatefiber/poly-L-lacticacid)三维支架材料中,经固化后形成工程化组织块,体外连续培养4周。行大体、倒置显微镜以及组织学、Ⅰ型和Ⅱ型胶原免疫组化光镜观察,二甲基亚甲蓝显色法定量检测硫酸糖胺多糖含量。结果(1)细胞-凝胶混悬液接种后完全充满CPPf/PLLA支架的孔隙,固化后三者很好地形成一个工程化组织整体。构建的工程化组织块在培养过程中不仅能够保持其初始外形,而且能保持种子细胞稳定的三维均相分布,无细胞脱落现象。同时,硬度亦不断增加,有弹性,表面湿润、光滑。(2)随着培养时间的延长,软骨组织的形成是由外周向中心进行。培养2周后,逐渐形成富含Ⅱ型胶原和蛋白聚糖、具有典型软骨组织结构的成熟工程化软骨。同时,Ⅰ型胶原逐渐转为阴性,支架材料亦不断降解。4周时硫酸糖胺多糖含量平均为(15.70±2.00)mg/g(湿重),为天然关节软骨的30%以上。结论种子细胞的三维支架双相     

组织工程骨软骨复合物的构建与形态学观察

目的探讨采用组织工程技术构建骨软骨复合物的可行性。方法将骨髓基质细胞(BMSCs)成诱导软骨后接种于快速成形的三维支架材料聚乳酸／聚羟乙酸共聚物(PLGA)构建组织工程软骨，经成骨诱导的BMSCs接种于聚乳酸／聚羟乙酸共聚物／磷酸三钙(PLGA／TCP)构建组织工程骨，在体外分别培养2周后，将两种工程化组织及两者以无损伤线缝合形成的组织工程骨软复合体分别植入自体股部肌袋，术后8周取材，行组织学观察。结果术后组织学观察表明。组织工程软骨在体内可形成软骨组织组织工程骨在体内可形成骨组织，两者的复合体在体内可形成骨软骨复合物。结论以骨髓基质细胞为种子细胞、以快速成形的生物降解材料为支架体外构建的组织工程骨软骨复合物，可在体内形成骨软骨组织，有望用于骨软骨缺损的修复。     

人胚胎成纤维细胞作为组织工程皮肤种子细胞的可行性研究

目的：评估人胚胎成纤维细胞（human embryonic fibroblasts,HEF）作为组织工程皮肤种子细胞的可行性和优越性。方法：取人流产胚胎皮肤、正常儿童皮肤,相同条件下分离培养成纤维细胞,比较两组细胞镜下、超微结构以及增殖特性,异体淋巴细胞混合实验对比其抗原性。以第三代细胞复合鼠尾胶原构建三维培养,ELASA法分别测定两组三维构建培养液中IL-6,TGF-β1含量。结果：与普通成纤维细胞相比,胚胎成纤维细胞扩增后具有更好的细胞形态和功能,生长速度快,分裂指数高,几乎不刺激异体淋巴细胞增殖。在鼠尾胶原支架中成纤维细胞生长状态良好,并且具有一定的组织强度。胎儿成纤维细胞组培养液中的TGF-β1和IL-6在各个时相上分别显著低于和高于普通成纤维细胞组。结论：胚胎成纤维细胞是组织工程皮肤较理想的种子细胞。     

联合培养VECs与ADSCs与部分脱蛋白生物骨体外构建组织工程骨的实验研究

目的本实验旨在研究体外血管内皮细胞和脂肪来源的联合培养体系在部分脱蛋白生物骨支架材料上的黏附、增殖,以及二者体外培养构建组织工程骨时移植入体内的最佳时机,为体内构建组织工程骨提供理论依据。方法取血管内皮细胞、脂肪干细胞和1：1联合培养细胞分别接种部分脱蛋白生物骨片,MTT法检测吸光度,评价三种不同细胞组在PDPBB生长情况,分析联合培养细胞在支架材料上增殖情况,扫描电镜观察不同细胞组在PDPBB生长情况。结果各细胞组在PDPBB上吸光度逐渐增加,第10天均达到高峰,1：1混合细胞组最高,各细胞组之间差异有统计学意义（P〈0．01）;10天可见联合培养细胞细胞组大量细胞与PDPBB附着,呈巢状分布。结论1：1混合细胞在PDPBB支架材料上的增殖优于单种细胞,10天为最佳移植时机。     

富含血小板血浆对牵引成骨过程中新骨生成的影响

目的　探讨富含血小板血浆 (PRP)对成骨细胞以及牵引成骨过程中新骨生成的作用。方法　通过体外成骨细胞的培养及牵引成骨动物模型的制备 ,采用MTT法检测成骨细胞的增殖情况 ,采用组织化学、四环素标记等方法观察骨组织新骨生成情况。结果　PRP可以促进体外培养成骨细胞的增殖 ,促进牵引成骨过程新骨的生成。结论　PRP可以加快牵引成骨过程的新骨生成 ,临床应用可能缩短牵引成骨的治疗过程。     

组织工程骨的牛血清白蛋白残余量检测

目的检测体外构建的组织工程骨中的牛血清白蛋白(Bovine serum albumin,BSA)残余量,并探讨减少残余量的方法。方法体外分离培养hBMSCs,将第2代细胞消化、离心、洗涤3次,获取细胞洗涤液样品。取第2代h BMSCs接种于β-TCP支架材料,体外成骨诱导2周,构建组织工程骨。生理盐水浸洗3次,获取洗涤液样品。然后加入PBS,37℃振荡浸提24h,获取浸提液样品。同法获取未接种细胞的单纯支架材料的浸提液样品作为对照。采用酶联免疫法检测洗涤液与组织工程骨样品浸提液中BSA的残余量,观察洗涤次数与洗涤液中BSA含量的变化关系。结果随洗涤次数增多,洗涤液与浸提液中的BSA含量明显降低。酶联免疫法测定的组织工程骨与单纯支架材料浸提液中BSA残余量分别为(19.54±6.70)ng和(15.67±5.49)ng,单位重量的残余量分别为(0.656±0.213)ng/mg和(0.796±0.205)ng/mg,两组无显著差异(P>0.05)。结论酶联免疫法适用于组织工程骨中BSA残余量的检测。因为支架材料较细胞更易吸附BSA,现有条件下构建的组织工程骨BSA残余量仍然较高,需要进一步探索降低残余量的方法 。     

血小板裂解物支持人骨髓基质细胞的扩增

目的探讨血小板裂解物体外培养、扩增人骨髓基质干细胞的可行性。方法将富含血小板血浆以冻融裂解法处理,培养人骨髓基质干细胞;体系中加入不同浓度的裂解物,MTT法检测细胞体外增殖情况;流式细胞仪分析细胞表型特征,并应用细胞化学染色法观察细胞体外成骨和成脂肪能力。结果血小板裂解物培养的骨髓基质干细胞呈典型的成纤维细胞形态,均一呈现CD14、CD31、CD34、CD45及HLA-DR阴性和CD73、CD90、CD105、CD166阳性,具备体外成骨、成脂分化能力。此外,与经筛选的胎牛血清比较,低浓度血小板裂解物即具有支持骨髓基质干细胞扩增的能力。结论血小板裂解物可替代胎牛血清,用于骨髓基质干细胞的体外扩增。     

Platelet‐rich plasma for resistant oral erosions of pemphigus vulgaris: A pilot study

Oral erosions and ulcers of pemphigus vulgaris (PV) are a debilitating condition that is usually difficult to treat. The wound healing properties of platelet‐rich plasma (PRP) encouraged us to evaluate its usefulness in treatment of non‐healing oral PV lesions. Seven patients with chronic oral PV, resistant to conventional therapy, were treated with weekly to monthly injections of PRP of affected mucosal membranes. All recruits reported improvement in pain and mastication and 6 of 7 patients had an improvement in pemphigus disease area index scores with PRP treatment. PRP injections seems to accelerate the healing process and decrease the pain and eating discomfort associated with the oral erosions and ulcers induced by PV.     

富血小板血浆治疗腱病概况

腱病是临床常见的疾病，给社会造成巨大的经济负担。注射富血小板血浆(PRP)作为对抗腱病发生发展的手段之一，有望为腱病的治疗带来新的选择。但是腱病是一大类疾病的统称，其具体分型间存在明显异质性。与此同时市售PRP本身也具有许多种类，因此本文对PRP在腱病中应用的文章进行了文献综述，以期科学评判PRP对腱病的治疗效果。对于传统保守治疗无效的慢性腱病患者而言，使用多个浓缩步骤制备的多次超声引导的PRP注射仍然具有治疗潜力，其中疗效最为明确的是膑腱炎以及肱骨外上髁炎。     

Proliferation and differentiation of human tenocytes in response to platelet rich plasma: an in vitro and in vivo study

Platelet rich plasma (PRP) is the autologous plasma fraction with a platelet-rich cellular component which is enriched with a number of growth factors. Due to its availability and low cost, PRP has become an increasingly popular clinical tool as an alternative source of growth factors for various applications, for example, tendon regeneration but with limited success in clinical trials. The main objective of the current study was to determine whether activated PRP [i.e., platelet rich plasma-clot release (PRCR)] could be used to induce the proliferation and collagen synthesis in human tenocyte in vitro. The advantage of using PRCR is that the platelet-derived bioactive factors are more concentrated and could initiate a more rapid and accelerated healing response than PRP. Our results demonstrated that 10% PRCR treatment accelerated the extent of cell proliferation and collagen production by human tenocytes in vitro. The expression of specific tenocyte markers were similar to conventional fetal bovine serum (FBS)-treated tenocytes implanted in mice within 14 days of implantation in diffusion chambers. Moreover, relatively more collagen fibrils were evident in PRCR-treated tenocytes in vivo as compared to 10% FBS-treated cells. Overall, our feasibility study has indicated that PRCR can induce human tenocyte proliferation and collagen synthesis which could be implemented for future tendon regeneration in reconstructive surgeries.     

凝胶包埋兔脂肪基质细胞接种三维支架动态构建组织工程化骨

目的 探讨高效的细胞种植方法,提高工程化骨构建质量.方法 成骨诱导化脂肪基质细胞,制备含rhBMP-2的纳米化壳聚糖/胶原蛋白/β磷酸三钙(Cs-Col-β-TCP)支架,构建工程化骨.静态种植静态培养(A组),振荡种植静态培养(B组),凝胶+振荡种植静态培养(c组),凝胶+静态种植静态培养(D组).第1、4、 8、 12、16、20、24、28天,扫描电镜、共聚焦显微镜观察,检测细胞存活率、细胞增殖、碱性磷酸酶、DNA、骨钙素水平.结果 C组细胞分布均匀、呈三维内生长、细胞外基质丰富,细胞存活率、增殖活力、碱性磷酸酶、DNA、骨钙素水平均高于其他组(79.53±2.67、0.59±0.20、65.16±6.85、207.62±19.36、55.22±8.51,P<0.05).结论 凝胶联合振荡种植可提高细胞接种效率及增强诱导成骨活性.     

Basic studies on the clinical applications of platelet-rich plasma

Platelets, which contain many growth factors such as platelet-derived growth factor (PDGF) and transforming growth factor-beta (TGF-beta), are being used in clinical applications as platelet-rich plasma (PRP). Only a few studies, however, have been conducted on the growth factors present in PRP and on the clinical applications using the drug delivery system (DDS). For the purpose of clinical application, we first modified the PRP preparation method and assessed the amounts of growth factors contained in the human platelet concentrates. Furthermore, we assessed fibrin glue as a DDS of platelet concentrates. Platelet precipitations were made by twice centrifuging human whole blood. The precipitated platelet was resuspended to yield the platelet concentrates. The growth factor concentrations were measured. Fibrin glue sheets containing this platelet concentrate were implanted in rabbit pinna and samples were obtained for immunostaining (anti-PDGF antibody) to assess the use of PRP over time using the fibrin glue as the DDS. The mean concentration of growth factors present in the platelet concentrates was three times or greater than that of conventional PRP. Furthermore, the results indicated that when the platelet concentrate was used with fibrin glue as a carrier, the contents were released over a period of about 1 week. This raises the possibility that this system may be useful in clinical applications.     

Autologous platelet-rich plasma in cardiac surgery: effect on intraoperative and postoperative transfusion requirements

The Southern Arizona Red Cross Blood program, in conjunction with participating hospitals and cardiac surgeons, evaluated the effect of a program to harvest autologous platelet-rich plasma (PRP) from patients immediately prior to undergoing cardiopulmonary bypass surgery. The PRP was transfused back to the patient after heparin neutralization was achieved at the completion of cardiopulmonary bypass. The effect of this autologous PRP product on homologous plasma and platelet usage was examined. The study demonstrates a significant decrease in homologous plasma and platelet usage when autologous PRP is used in cardiac surgery.     

Platelet-rich plasma stimulates porcine articular chondrocyte proliferation and matrix biosynthesis

OBJECTIVE: Platelet-rich plasma (PRP) is a fraction of plasma that contains high levels of multiple growth factors. The purpose of this study was to examine the effects of PRP on cell proliferation and matrix synthesis by porcine chondrocytes cultured in alginate beads, conditions that promote the retention of the chondrocytic phenotype, in order to determine the plausibility of using this plasma-derived material for engineering cartilage. DESIGN: PRP and platelet-poor plasma (PPP) were prepared from adult porcine blood. Adult porcine chondrocytes were cultured in the presence of 10% PRP, 10% PPP or 10% fetal bovine serum (FBS) for 3 days. Cell proliferation, proteoglycan (PG) and collagen synthesis were quantified, and the structure of newly synthesized PG and collagen was characterized. RESULTS: Treatment with 10% PRP resulted in a small but significant increase in DNA content (+11%, vs FBS; P<0.01; vs PPP; P<0.001). PG and collagen syntheses by the PRP-treated chondrocytes were markedly higher than those by chondrocytes treated by FBS or PPP (PG; PRP: +115% vs FBS; +151% vs PPP, both P<0.0001, collagen; PRP: +163% vs FBS; +163% vs PPP, both P<0.0001). Biochemical analyses revealed that treatment with PRP growth factors did not markedly affect the types of PGs and collagens produced by porcine chondrocytes, suggesting that the cells remained phenotypically stable in the presence of PRP. CONCLUSION: PRP isolated from autologous blood may be useful as a source of anabolic growth factors for stimulating chondrocytes to engineer cartilage tissue.     

Comparison of Conventional and Platelet‐Rich Plasma-Assisted Fat Grafting: A Systematic Review and Meta-analysis

BackgroundAutologous fat grafting (FG) is a popular technique for soft-tissue augmentation, but the fat survival rate is unpredictable. Platelet-rich plasma (PRP) has emerged as an adjuvant to enhance fat graft survival.ObjectivesThis literature review and meta-analysis aimed to investigate the effect of PRP on the survival rate of fat grafting.MethodsA comprehensive systematic literature search was done to identify clinical studies on PRP and fat cotransplantation in PubMed, Cochrane Library, Web of Science, and EMBASE databases up to May 2020. The reference lists of selected articles were reviewed to identify any additional related articles. A meta-analysis was conducted to compare PRP + FG and conventional FG in terms of fat graft survival rate, patient satisfaction rate, and recovery time after surgery.ResultsEleven studies consisting of 1125 patients were analyzed. Patients were followed up from 3 to 24 months post-FG. The fat survival rate varied from 20.5% to 54.8% in FG alone and from 24.1% to 89.2% in the PRP + FG groups. The survival rate was significantly higher and recovery time was significantly lower in the PRP + FG group than in the FG alone group. However, there was no significant difference in the patient satisfaction rate between the groups.ConclusionsThis study demonstrates that PRP-enhanced fat transplantation has better efficacy than conventional fat grafting. Further studies are required to provide the optimum concentration of PRP and the long-term efficacy of the technique. There is not enough evidence to compare the rate of complications with PRP and fat cotransplantation and conventional fat grafting.     

The effect of platelet-rich plasma on the cellular response of rat bone marrow cells in vitro.

The aim of this study was to investigate the effect of platelet-rich plasma (PRP) on the proliferation and the differentiation of rat bone marrow cells (RBMCs). PRP, platelet-poor plasma (PPP), and bone marrow cells were derived from the rats (hearts and tibia) and the cells were cultured with or without PRP or PPP (0 [control]), 0.2 approximately 10 microL/mL). The proliferation of RBMCs was measured on days 2 and 4, and alkaline phosphatase (ALP) staining and activity measurement were evaluated to determine the effect of PRP on the differentiation on days 4 and 8. PRP enhanced the proliferation significantly compared to the control group (P < .05). These enhancements were greater than ones induced by the addition of PPP. ALP staining appeared to show that PRP decreased the number of ALP positive cells and ALP activity significantly (P < .05). Our results demonstrate that PRP stimulates the proliferation but suppresses the differentiation of RBMCs.