富血小板血浆对珊瑚支架上骨髓基质细胞增殖和成骨分化的影响

Influence of Platelet-rich Plasma on Proliferation and Differentiation of MSCs in Coral Scaffold in Vitro.

目的:评价富血小板血浆(platelet rich plasma,PRP)对珊瑚支架上骨髓基质细胞(marrow stromal cells,MSCs)增殖和成骨分化的影响。方法:将体外培养、扩增和诱导的兔MSCs悬液与同一供体来源的PRP混合,滴加到珊瑚圆片上,再滴加牛凝血酶溶液,形成珊瑚/MSCs/PRP复合物,继续在体外培养。以珊瑚/MSCs复合物和珊瑚/PRP复合物作对照。8 d和14 d,通过MTT法检测MSCs增殖情况;通过组织化学方法检测培养液中碱性磷酸酶活性和骨钙素含量;通过扫描电镜观察MSCs在珊瑚支架上附着、生长和增殖情况。结果:珊瑚/MSCs/PRP组的光密度值明显大于珊瑚/MSCs组(P<0.05);珊瑚/MSCs/PRP组培养液中的ALP活性和OC含量明显大于珊瑚/MSCs组(P<0.05);扫描电镜显示:珊瑚/MSCs/PRP组,大量MSCs附着于珊瑚表面和孔洞壁,可见孔洞内有血小板和纤维网格样结构存在;珊瑚/MSCs组,珊瑚表面和孔洞壁有少量MSCs附着;珊瑚/PRP组,珊瑚表面及其孔洞内有大量的血小板和纤维网格样结构存在。结论:PRP促进了珊瑚支架上MSCs的增殖、成骨分化和粘附。

Objectives： To investigate the influence of platelet rich plasma （PRP） on proliferation and differentiation of marrow stromal cells （MSCs） in coral scaffold in vitro. Methods： A suspension of 5 x 106 MSCs in 50 μl1 medium were dissolved in 50μof PRP. The mixture of MSCs and PRP was spread on a coral disc with diameter of 8 mm and thickness of 2 ram, so coral/MSCs/PRP composite was obtained. A suspension of MSCs in 50μl medium was spread on a coral disc to obtain coral/MSCs composite. 50μlof PRP was added on a coral disc to obtain coral/PRP composite. Coral/MSCs/PRP composite was cultured in a humidified incubator for 8 and 14 days. The medium was changed every other day. Coral/MSCs composite and coral/PRP composite were used as control. Then three types of composites were processed to evaluate ALP activity, osteocalcin and proliferation of MSCs in the constructs. The composites were also evaluated cell adhesion, growth and proliferation of MSCs on coral scaffold by scanning electron microscope. Results： The ALP activity and content of osteocalcin were significantly higher in the samples of coral/MSCs/PRP group compared to that of coral/MSC group at 8 and 14 days in vitro （P 〈 0.05 ）. A comparison of the results of MSCs proliferation showed that cell growth increases as a function of PRP in coral scaffolds. Scanning electron micrographs showed more cells adhered to the scaffolds of coral/MSCs/ PRP than that of coral/MSCs, and some asteroid MSCs were bound by the fibrin network. Conclusion： These in vitro study ？ results suggest that MSCs may be encouraged to proliferate and osteogenically differentiate by PRP in the coral scaffold, leading to increased osteogenesis in vitro.