PRP对纳米HA/PLGA复合支架上鼠骨骼肌卫星细胞增殖和成骨活性的影响

目的:研究富血小板血浆(platelet-rich plasma,PRP)对纳米羟基磷灰石(nano-hydroxyapatite,nHA)/聚乳酸乙醇酸(poly lactic acid-co-glycolic acid,PLGA)(nHA/PLGA)支架上SD大鼠骨骼肌卫星细胞(muscle satellite cells,MSCs)黏附、增殖和成骨活性的影响。方法:将体外培养、扩增的鼠MSCs与同一供体来源的PRP混合,滴加到nHA/PLGA支架上,形成MSCs/PRP/nHA/PLGA复合物(实验组),继续在体外培养,以MSCs/nHA/PLGA复合物作为对照组。通过扫描电镜观察MSCs在支架上黏附、生长和增殖情况;水溶性四氮唑法(WST-1)法测定MSCs增殖情况;碘化丙啶(PI)与钙黄绿素-AM(Calcein-AM)检测MSCs的荧光活性;组织化学方法检测培养液中碱性磷酸酶(alkaline phosphatase,ALP)活性和骨钙素(osteocalcin,OC)含量;RT-PCR检测骨桥蛋白(osteopontin,OPN)mRNA的表达。结果:扫描电镜观察显示,实验组大量MSCs黏附在支架表面及其洞壁上,并大量增殖和分泌骨基质,而对照组复合材料表面细胞附着较少;WST-1测定实验组吸光度值明显大于对照组(P<0.05);PI荧光染色二组的死细胞数量均较少;实验组ALP活性和OC含量均较对照组增高明显(P<0.05);RT-PCR结果显示OPN在实验组中表达明显增强。结论:PRP促进了nHA/PLGA支架上鼠MSCs黏附、增殖和成骨分化。

Influence of Platelet-rich Plasma on the Proliferation and Osteogenetic Differentiation of Rat Skeletal Muscle Satellite Cells on nano-HA/PLGA Composite Scaffold

Objective： To investigate the influence of platelet-rich plasma （PRP） on the proliferation and osteogenetic differentiation of rat skeletal muscle satellite cells （MSCs） on nHA/PLGA composite scaffold. Methods： The mixture of PRP and MSCs was pipetted onto the surface of the scaffolds, MSCs/PRP/nHA/PLGA composite was obtained （experimental group）. MSCs/nHA/PLGA composite was used as control group. The cell adhesion, growth and proliferation of MSCs on scaffolds were observed by scanning electron microscopy. WST-I assay was used to evaluate the effects of PRP on proliferation of MSCs on scaffolds. The cell viability was stained with PI and Calcein-AM after 7 days. The alkaline phosphatase （ALP） activities and osteocalcin were also evaluated. RT-PCR was used to examine the expression of OPN. Results： Scanning electron microphotos showed more cells were adhered to the scaffolds of experimental group than those in control group, and some asteroid MSCs were bound by the fibrin network. WST-1 assay showed significant higher optical density values in the experimental group compared to the control group （P〈O.05）. There was a significant increase in ALP activity and osteocalcin in the experimental group compared to the control group （P〈0.05）. The expression of OPN was intensified in experimental group. Conclusion： PRP could obviously promote the rat skeletal muscle satellite cells adhesion, proliferation and osteogenetic differentiation on nHA/PLGA scaffold.