The influence of bone formation by thrombomodulin in platelet rich plasma gel

富血小板血浆（PRP）已被证明在局部应用后可以促进骨移植物的成熟，并在体外对成骨细胞具有生物学效应。然而，由于PRP在大骨缺损中疗效不佳，临床上并未单独应用。本研究旨在通过研究PRP与成骨细胞联合应用，并评估其移植后的效果，开发一种临床替代自体骨的材料。采用两步离心法制备小鼠外周血PRP和贫血小板血浆（PPP）。MC3T3-E1细胞在移植前用荧光碳花青素标记。将标记细胞与PRP凝胶联合皮下移植到SCID小鼠背部，4周后进行放射学和免疫组织学评价。移植前采用碱性磷酸酶（ALP）染色和von Kossa染色评价PRP的作用，逆转录-聚合酶链反应分析骨相关标志物的表达。移植前，与PPP相比，PRP增强了Osterix和骨涎蛋白mrna的表达。此外，PRP提高ALP活性，诱导矿化结节的形成。移植后，标记细胞与PRP凝胶结合形成矿化组织，荧光显微镜观察到移植细胞在组织中表达骨钙素和I型胶原。在成骨细胞中发现了局灶黏附分子（FAK）的磷酸化。这些结果表明，应用PRP/成骨细胞复合物通过促进成骨细胞分化，对工程细胞移植到骨缺损中具有有益的作用。

Platelet-rich plasma (PRP) has been shown to enhance the maturation of bone grafts following local application, and to have biological effects on osteoblasts in vitro. However, PRP is not applied by itself clinically due its poor benefits in large bone defects. The present study was undertaken to develop a clinical alternative to autologous bone, by investigating the application of PRP in combination with osteoblastic cells and evaluating its effects after transplantation.PRP and platelet-poor plasma (PPP) were prepared from blood obtained from ddY mice by two centrifugation steps. MC3T3-E1 cells were labeled with fluorescent carbocyanine just before transplantation. The combination of labeled cells and PRP gel was subcutaneously transplanted into the back of SCID mice, and the transplants were evaluated radiographically and immunohistologically after 4 weeks. The effects of PRP were assessed by alkaline phosphatase (ALP) staining and von Kossa staining and the expressions of bone-related markers were analyzed by reverse transcription-polymerase chain reaction before transplantation.Before transplantation, PRP enhanced the expressions of Osterix and bone sialoprotein mRNAs compared with PPP. Furthermore, PRP elevated ALP activity and induced the formation of mineralized nodules. After transplantation, the combination of labeled cells and PRP gel formed mineralized tissue and the transplanted cells visualized in the tissue using fluorescence microscopy expressed osteocalcin and type I collagen. And Phosphorylation of focal adhesion molecule (FAK) was recognized in osteoblasts with PRP application.These results suggest that application of a PRP/osteoblasts complex has beneficial effects for transplanting engineered cells into bone defects through the promotion of osteoblastic differentiation.