Nakahara H, Goldberg V M, Caplan A I
Osaka University Medical School, Japan.
J Orthop Res. 1991 Jul;9(4):465-76. doi: 10.1002/jor.1100090402.
Periosteal cells were enzymatically liberated from human rib periostea obtained from autopsies of 37 donors with an age distribution ranging from 25 weeks of gestation to 88 years old. These cells were introduced into cell culture and subcultured when they reached confluence. After subculture, the adherent periosteal-derived cells showed a nondescript, fibroblast-like morphology in cell culture. The cells from various passages of each donor were tested for in vivo osteochondrogenic potential with three different assay methods in athymic mice: (a) inoculation assay--the cells were directly inoculated into a subcutaneous site, (b) porous ceramics assay--the cells were combined with porous calcium phosphate ceramics, and this composite graft was implanted into a subcutaneous site, and (c) diffusion chamber assay--the cells were loaded into diffusion chambers and cultured in the peritoneal cavity. Frozen-preserved and recultured periosteal-derived cells were also assayed in the same way. In cases of donors younger than 19 years old, cultured, periosteal-derived cells from up to several passages consistently formed bone and/or cartilage in each of the three assays. Frozen-preserved and recultured cells from these donors also formed bone and/or cartilage after introduction into the three in vivo assays. In cases of donors older than 22 years of age, cultured, periosteal-derived cells formed neither bone nor cartilage in vivo. Cultured muscle fibroblasts from some of the same donors did not form bone or cartilage when assayed in vivo under identical conditions. These results suggest that periosteal cells with osteochondrogenic potentials can be liberated from the periosteum of a rib of human donors up to a certain age. Importantly, this potential is retained after enzymatic liberation, cell culture, subculturing, and freeze preservation. The present results suggest that culture-expanded human periosteal-derived cells from young donors may be useful in the repair of skeletal defects to foster cell-mediated regeneration of skeletal tissues, and that this methodology can be used to elucidate cellular, molecular, and genetic disorders in various metabolic bone diseases and skeletal dysplasias.
从37名捐赠者的尸检获取的人肋骨骨膜中酶解分离出骨膜细胞，这些捐赠者的年龄分布从妊娠25周直至88岁。将这些细胞引入细胞培养，当细胞达到汇合状态时进行传代培养。传代培养后，贴壁的骨膜来源细胞在细胞培养中呈现出难以描述的、成纤维细胞样的形态。对每个捐赠者不同传代的细胞，在无胸腺小鼠中用三种不同的检测方法测试其体内成骨软骨生成潜能：(a)接种检测——将细胞直接接种到皮下部位；(b)多孔陶瓷检测——将细胞与多孔磷酸钙陶瓷混合，然后将这种复合移植物植入皮下部位；(c)扩散盒检测——将细胞装入扩散盒并在腹腔中培养。冷冻保存并重新培养的骨膜来源细胞也以同样的方式进行检测。在年龄小于19岁的捐赠者中，培养的、传代数次的骨膜来源细胞在三种检测方法中的每一种中都始终能形成骨和/或软骨。将这些捐赠者的冷冻保存并重新培养的细胞引入三种体内检测后也能形成骨和/或软骨。在年龄大于22岁的捐赠者中，培养的骨膜来源细胞在体内既不形成骨也不形成软骨。在相同条件下对来自一些相同捐赠者的培养肌肉成纤维细胞进行体内检测时，它们也不形成骨或软骨。这些结果表明，具有成骨软骨生成潜能的骨膜细胞可以从人类捐赠者的肋骨骨膜中分离出来，直至一定年龄。重要的是，这种潜能在酶解分离、细胞培养、传代培养和冷冻保存后仍能保留。目前的结果表明，来自年轻捐赠者的培养扩增的人骨膜来源细胞可能有助于修复骨骼缺陷，促进细胞介导的骨骼组织再生，并且这种方法可用于阐明各种代谢性骨病和骨骼发育异常中的细胞、分子和遗传紊乱。
