Gan Jean C, Ducheyne Paul, Vresilovic Edward J, Shapiro Irving M
Center for Bioactive Materials and Tissue Engineering, Department of Bioengineering, School of Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA.
Clin Orthop Relat Res. 2003 Jun(411):315-24. doi: 10.1097/01.blo.0000063797.98363.d3.
The main objective of the current investigation was to regenerate cells of the nucleus pulposus without loss of phenotype. Nucleus pulposus cells were isolated from intervertebral discs from adult rabbits, grown in monolayer culture, and then maintained as a micromass pellet in tube culture. The specimens were evaluated by transmission and light microscopy, reverse transcriptase polymerase chain reaction, and immunohistochemistry. Nucleus pulposus cells proliferated in monolayer culture. When almost confluent, the cells were transferred to a tube and sedimented to form a pellet. The cells reverted to a rounded configuration and formed cell nests surrounded by extensive extracellular matrix, similar to that seen in vivo. These cells did not proliferate. Similar to that observed in situ, cells in pellet culture also expressed aggrecan, CD44, collagen Type II, and collagen Type I, but not collagen Type X, and had low alkaline phosphatase activity. The results of the investigation indicated that nucleus pulposus cells grown in monolayer culture might revert to their original characteristics when transferred to an environment that allows three-dimensional growth, such as upon implantation, a one-step approach. The results also indicated that the two-stage culture procedure might provide an expedient technique to regenerate nucleus pulposus tissue for disc repair.
当前研究的主要目的是再生髓核细胞且不丧失其表型。从成年兔的椎间盘分离出髓核细胞，在单层培养中生长，然后在试管培养中作为微团沉淀维持培养。通过透射电镜和光学显微镜、逆转录聚合酶链反应及免疫组织化学对标本进行评估。髓核细胞在单层培养中增殖。当接近汇合时，将细胞转移至试管中沉淀形成微团。细胞恢复为圆形形态并形成被大量细胞外基质包围的细胞巢，类似于在体内观察到的情况。这些细胞不再增殖。与原位观察到的情况相似，微团培养中的细胞也表达聚集蛋白聚糖、CD44、Ⅱ型胶原和Ⅰ型胶原，但不表达Ⅹ型胶原，且碱性磷酸酶活性较低。研究结果表明，单层培养的髓核细胞在转移至允许三维生长的环境（如植入时，一种一步法）时可能恢复其原始特性。结果还表明，两阶段培养程序可能为再生用于椎间盘修复的髓核组织提供一种便捷技术。
