Omlor G W, Nerlich A G, Tirlapur U K, Urban J P, Guehring T
Department of Orthopaedic Surgery and Trauma Surgery, Heidelberg University Hospital, 69118, Heidelberg, Germany.
Arch Orthop Trauma Surg. 2014 Dec;134(12):1673-81. doi: 10.1007/s00402-014-2097-2. Epub 2014 Oct 28.
Embryonic notochordal disc nucleus cells (NC) have been identified to protect disc tissue against disc degeneration but in human beings NC phenotype gets lost with aging and the pathophysiological mechanisms are poorly understood. NC may stimulate other cells via soluble factors, and NC-conditioned medium can be used to stimulate matrix production of other disc cells and mesenchymal stem cells and thus may be of special interest for biological disc repair. As this stimulatory effect is associated with the NC phenotype, we investigated how cell morphology and gene-expression of the NC phenotype changes with time in 3D-cell culture.
NC and inner annulus chondrocyte-like cells (CLC) from immature pigtails (freshly isolated cells/tissue, 3D-alginate beads, 3D-clusters) were cultured for up to 16 days under normoxia and hypoxia. Protein-expression was analysed by immunohistology and gene-expression analysis was carried out on freshly isolated cells and cultured cells. Cell morphology and proliferation were analysed by two-photon-laser-microscopy.
Two-photon-laser-microscopy showed a homogenous and small CLC population in the inner annulus, which differed from the large vacuole-containing NC in the nucleus. Immunohistology found 93 % KRT8 positive cells in the nucleus and intracellular and pericellular Col2, IL6, and IL12 staining while CLC were KRT8 negative. Freshly isolated NC showed significantly higher KRT8 and CAIII but lower Col2 gene-expression than CLC. NC in 3D-cultures demonstrated significant size reduction and loss of vacuoles with culture time, all indicating a loss of the characteristic NC morphology. Hypoxia reduced the rate of decrease in NC size and vacuoles. Gene-expression of KRT8 and CAIII in NC fell significantly early in culture while Col2 did not decrease significantly within the culture period. In CLC, KRT8 and CAIII gene-expression was low and did not change noticeably in culture, whereas Col2 expression fell with time in culture.
3D-culture caused a rapid loss of NC phenotype towards a CLC phenotype with disappearance of vacuoles, reduced cell size, increased proliferation, and gene-expression changes. These findings may be related to NC nutritional demands and support the latest hypothesis of NC maturation into CLC opposing the idea that NC get lost in human discs by cell death or apoptosis to be replaced by CLC from the inner annulus.
胚胎脊索盘核细胞（NC）已被证实可保护椎间盘组织免受退变影响，但在人类中，NC表型会随着年龄增长而丧失，其病理生理机制仍知之甚少。NC可能通过可溶性因子刺激其他细胞，NC条件培养基可用于刺激其他椎间盘细胞和间充质干细胞的基质产生，因此可能对生物椎间盘修复具有特殊意义。由于这种刺激作用与NC表型相关，我们研究了在三维细胞培养中NC表型的细胞形态和基因表达如何随时间变化。
将来自未成熟猪尾的NC和内环软骨样细胞（CLC）（新鲜分离的细胞/组织、三维藻酸盐珠、三维聚集体）在常氧和低氧条件下培养长达16天。通过免疫组织学分析蛋白质表达，并对新鲜分离的细胞和培养细胞进行基因表达分析。通过双光子激光显微镜分析细胞形态和增殖情况。
双光子激光显微镜显示内环中有均匀且数量少的CLC群体，这与髓核中含有大液泡的NC不同。免疫组织学发现髓核中有93%的KRT8阳性细胞，细胞内和细胞周围有Ⅱ型胶原、白细胞介素6和白细胞介素12染色，而CLC为KRT8阴性。新鲜分离的NC显示KRT8和碳酸酐酶Ⅲ（CAIII）基因表达显著高于CLC，但Ⅱ型胶原基因表达低于CLC。三维培养中的NC随着培养时间的延长，体积显著减小，液泡消失，所有这些都表明其特征性的NC形态丧失。低氧降低了NC体积和液泡的减小速率。NC中KRT8和CAIII的基因表达在培养早期显著下降，而Ⅱ型胶原在培养期内没有显著下降。在CLC中KRT8和CAIII基因表达较低，在培养过程中没有明显变化，而Ⅱ型胶原表达随培养时间下降。
三维培养导致NC表型迅速向CLC表型转变，表现为液泡消失、细胞体积减小、增殖增加以及基因表达变化。这些发现可能与NC的营养需求有关，并支持NC成熟为CLC的最新假说，反对NC在人类椎间盘中因细胞死亡或凋亡而丧失并被内环中的CLC取代的观点。
