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

富血小板血浆（PRP）在局部应用后可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在大面积骨缺损中的益处较差，因此临床上不单独应用PRP。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来开发一种临床上可替代自体骨的方法。在移植前用荧光碳菁标记MC 3 T3-E1细胞。将标记细胞与PRP凝胶组合物皮下移植到SCID小鼠背部，4周后对移植物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，并采用逆转录-聚合酶链反应（RT-PCR）检测骨相关标志物的表达，结果表明：与PPP相比，PRP可促进骨相关标志物Osterix和骨唾液酸蛋白mRNA的表达。此外，PRP升高ALP活性并诱导矿化结节的形成。移植后，标记的细胞和PRP凝胶的组合形成矿化组织，并且使用荧光显微镜在组织中可见的移植细胞表达骨钙素和I型胶原。结果表明，PRP/成骨细胞复合物可促进成骨细胞的分化，有利于工程化细胞的骨缺损修复。

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.