In vivo osteogenic capability of cultured allogeneic bone in porous hydroxyapatite: Immunosuppressive and osteogenic potential of FK506 in vivo
In vivo osteogenic capability of cultured allogeneic bone in porous hydroxyapatite: Immunosuppressive and osteogenic potential of FK506 in vivo
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DOI：
10.1359/jbmr.2000.15.6.1147
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发表时间：
2000-06-01
影响因子：
6.2
通讯作者：
Ichijima, K
中科院分区：
文献类型：
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作者：
Yoshikawa, T;Nakajima, H;Ichijima, K
Fischer 或 ACI 大鼠骨髓细胞取自股骨干，并在补充有 15% 胎牛血清的 Eagle's 基本必需培养基 (EMEM) 中培养至汇合。胰蛋白酶消化后，在 P-甘油磷酸和 10 nM 地塞米松 (Dex) 存在下，将细胞在多孔羟基磷灰石 (EIA；Interpore 500) 块上传代培养。继代培养 2 周后，HA 孔表面形成含有成骨细胞的矿化骨基质。将 ACI 或 Fischer 培养的骨组织/HA 构建体皮下植入 Fischer 大鼠的背部，并给予大鼠免疫抑制剂 FK506 4 周。植入后4周和8周收获植入物。 4 周时，ACI 构建体（同种异体移植物）显示出高水平的成骨参数（碱性磷酸酶 [ALP] 活性和骨钙素含量），并且观察到骨形成与活性成骨细胞一起，没有明显的炎症细胞积累。 8周时，仍然观察到活跃的成骨细胞和进行性骨形成，同时成骨参数仍然很高，并且检测到骨钙素信使RNA（mRNA）。如果不施用 FK506，同种异体移植物既不显示骨形成，也不显示骨钙素 mRNA，并且仅存在痕量水平的成骨参数。在 Fischer 构建体（同种移植物）的情况下，检测到广泛的骨形成，并且在第 4 周和第 8 周，使用 FK506 的所有成骨参数均高于未使用 FK506 的情况。这些结果表明，培养的骨组织/HA构建体即使作为同种异体移植物也具有很高的成骨潜力，并且FK506不仅具有免疫抑制作用，而且还促进骨形成。
Fischer or ACI rat marrow cells were obtained from femoral shafts and were cultured to confluence in Eagle's minimal essential medium (EMEM) supplemented with 15% fetal bovine serum. After trypsinization, the cells were subcultured on porous hydroxyapatite (EIA; Interpore 500) blocks in the presence of P-glycerophosphate and 10 nM dexamethasone (Dex). After 2 weeks of subculture, a mineralized bone matrix with osteogenic cells developed on the HA pore surfaces. ACI or Fischer cultured bone tissue/HA constructs were implanted subcutaneously into the backs of Fischer rats and the immunosuppressant FK506 was given to the rats for 4 weeks. Implants were harvested 4 weeks and 8 weeks after insertion. At 4 weeks, the ACI constructs (allografts) showed high levels of osteogenic parameters (alkaline phosphatase [ALP] activity and osteocalcin content) and bone formation was observed together with active osteoblasts without obvious accumulation of inflammatory cells. At 8 weeks, active osteoblasts and progressive bone formation were still observed, while osteogenic parameters remained high and osteocalcin messenger RNA (mRNA) was detected. Without FK506 administration, the allografts showed neither bone formation nor osteocalcin mRNA and there were only trace levels of the osteogenic parameters. In the case of Fischer constructs (isografts), extensive bone formation was detected and all the osteogenic parameters were higher with FK506 than without FK506 at both 4 weeks and 8 weeks. These results indicate that cultured bone tissue/HA constructs possess a high osteogenic potential, even as allografts, and that FK506 not only has an immunosuppressive action, but also promotes bone formation.