Metabolism of the extracellular matrix formed by intervertebral disc cells cultured in alginate
Metabolism of the extracellular matrix formed by intervertebral disc cells cultured in alginate
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DOI：
10.1097/00007632-199712150-00011
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发表时间：
1997-12-15
期刊：
影响因子：
3
通讯作者：
Thonar, EJMA
中科院分区：
文献类型：
--
作者：
Chiba, K;Andersson, GBJ;Thonar, EJMA
研究设计.将来自正常兔髓核（NP）和纤维环（AF）的细胞在藻酸盐珠中培养长达14天，以使它们改革由两个隔室组成的基质：细胞相关基质（CM）和进一步去除的基质（FRM）。在不同的时间点，使用组织学、生物化学和免疫学方法分析每个细胞群产生的CM和FRM。研究藻酸盐中正常兔NP和AF细胞的代谢，通过表征每个细胞群形成的CM和FRM，并确定可能揭示椎间盘退变平台机制的代谢特性。对椎间盘细胞的代谢知之甚少，部分原因是缺乏适合于体外研究这些细胞的微培养系统。在我们实验室最近的研究中，有人提出，在藻酸盐珠中培养的关节软骨细胞保持表型稳定，并改革类似于它们在体内填充的基质。这种培养系统似乎非常适合于研究椎间细胞，只有在有限的数量。将通过顺序酶消化从基质释放的兔NP和AF细胞包封在藻酸盐珠粒中（20，000个细胞/珠粒）并培养长达14天。在选定的时间点，用钙螯合剂溶解珠粒，并分离CM和FRM。测量S-35-硫酸盐掺入蛋白多糖的速率和各种细胞外基质分子（总硫酸化蛋白多糖、抗原性硫酸角质素、透明质酸、胶原和吡啶交联）的含量。NP和AF细胞在整个培养期间在藻酸盐凝胶中保持表型稳定，并重建了由CM和FRM隔室组成的基质。这两个细胞群在体外代谢活性方面表现出许多差异。髓核细胞合成较少的蛋白多糖和胶原分子，并且与AF细胞相比，将这些分子整合到CM中的效率较低。椎间盘细胞，特别是NP细胞，在形成CM（富含蛋白聚糖和胶原分子的保护壳）方面极其缓慢。这可能有助于解释为什么髓核的损伤通常伴随着体内椎间盘的进行性退变。
Study Design. Cells from normal rabbit nucleus polposus (NP) and anulus fibrosus (AF) were cultured in alginate beads for as long as 14 days to allow them to reform a matrix made up of two compartments: the cell-associated matrix (CM) and further removed matrix (FRM). At different time points, the CM and FRM made by each cell population were analyzed using histologic, biochemical, and immunologic assays.Objectives. To study the metabolism of normal rabbit NP and AF cells in alginate by characterizing the CM and FRM formed by each cell population, and to identify metabolic properties that may shed light on mechanisms at plat in disc degeneration.Summary of Background Data. Little is known about the metabolism of intervertebral disc cells, in part because of the lack of microculture systems appropriate for the study of these cells in vitro. In recent studies from our laboratories, it was suggested that articular chondrocytes cultured in alginate beads remain phenotypically stable and reform a matrix similar to the one they populate in vivo. This culture system appears ideally suited for the study of intervertebral cells available only in limited numbers.Methods. Rabbit NP and AF cells released from the matrix by sequential enzyme digestion were encapsulated in alginate beads (20,000 cells/bead) and cultured for as long as 14 days. At selected time points, beads were solubilized with calcium chelating agents, and the CM and FRM were isolated. The rate of S-35-sulfate incorporation into proteoglycans, and the contents of various extracellular matrix molecules (total sulfated proteoglycans, antigenic keratan sulfate, hyaluronan, collagen, and pyridinium crosslinks) were measured.Results. Both NP and AF cells remained phenotypically stable in the alginate gel throughout the culture period and reestablished a matrix composed of CM and FRM compartments. The two cell populations exhibited numerous differences in their metabolic activities in vitro. Nucleus pulposus cells synthesized fewer proteoglycan and collagen molecules and were less effective in incorporating these into the CM than AF cells.Conclusions. Intervertebral disc cells, especially NP cells, are extremely sluggish in reforming a CM, a protective shell rich in proteoglycans and collagen molecules. This may help explain why damage to the NP often is accompanied by progressive degeneration of the disc in vivo.