Yoshikawa T, Ohgushi H, Tamai S
Department of Orthopaedic Surgery, Nara Medical University, Japan.
J Biomed Mater Res. 1996 Nov;32(3):481-92. doi: 10.1002/(SICI)1097-4636(199611)32:3<481::AID-JBM23>3.0.CO;2-I.
We employed culture technology, which provides bone tissue in vitro, to expand and promote the osteogenic ability of marrow cells in porous hydroxyapatite (HA). Marrow cells were obtained from rat femur and cultured in a standard medium for 10 days, then trypsinized to make composites of HA and the cells. An additional 2-week culture (subculture) was done for the composite in a standard medium with or without the addition of dexamethasone (Dex). The 2-week subcultured composites were implanted into subcutaneous sites of syngeneic rats. These implants were harvested and prepared for the biochemical analysis of alkaline phosphatase activity and bone Gla protein content, as well as histological analysis of decalcified and undecalcified sections. In Dex-treated composites, high alkaline phosphatase activity could be detected 1 week after implantation and was maintained until 8 weeks after implantation. The bone Gla protein content could also be detected 1 week after implantation, followed by a steady increase with the passage of time until 8 weeks after implantation. The histological analysis showed active bone formation even 1 week after implantation. The bone formation was evidenced by active osteoblast lining and the appearance of calcein labeling following calcein injection 1 week after implantation. Thus, Dex-treated subcultured marrow cells in pore regions of HA showed a high osteogenic response immediately after transplantation. In contrast, Dex-untreated composite did not show bone formation and contained traces of these biochemical parameters. These results indicate that the inherent osteogenic ability of marrow stromal stem cells in pore regions of HA can be stimulated using tissue culture technology; and thus, formed osteogenic HA can show immediate osteoblastic activity in in vivo situations, suggesting the applicability of the HA in clinical situations.
我们采用能在体外提供骨组织的培养技术，来扩增并促进多孔羟基磷灰石（HA）中骨髓细胞的成骨能力。从大鼠股骨获取骨髓细胞，并在标准培养基中培养10天，然后用胰蛋白酶处理以制备HA与细胞的复合物。将该复合物在添加或不添加地塞米松（Dex）的标准培养基中再培养2周（传代培养）。将传代培养2周后的复合物植入同基因大鼠的皮下部位。收集这些植入物并进行碱性磷酸酶活性和骨钙素蛋白含量的生化分析，以及脱钙和未脱钙切片的组织学分析。在经Dex处理的复合物中，植入后1周可检测到高碱性磷酸酶活性，并一直维持到植入后8周。植入后1周也可检测到骨钙素蛋白含量，随后随时间稳步增加，直至植入后8周。组织学分析显示，即使在植入后1周也有活跃的骨形成。植入后1周注射钙黄绿素后，活跃的成骨细胞排列和钙黄绿素标记的出现证明了骨形成。因此，HA孔隙区域经Dex处理的传代培养骨髓细胞在移植后立即显示出高成骨反应。相比之下，未经Dex处理的复合物未显示骨形成，且这些生化参数含量微量。这些结果表明，利用组织培养技术可以刺激HA孔隙区域骨髓基质干细胞的固有成骨能力；因此，形成的成骨性HA在体内情况下可显示立即的成骨细胞活性，这表明HA在临床情况下具有适用性。
