Iu Jonathan, Santerre J Paul, Kandel Rita A
1 Institute of Biomaterials and Biomedical Engineering, University of Toronto , Toronto, Canada .
Tissue Eng Part A. 2014 Dec;20(23-24):3261-9. doi: 10.1089/ten.TEA.2013.0777.
Since there is currently no optimal treatment for chronic neck or back pain that restores full spine functionality and disc height, recent research has focused on developing a regenerative medicine approach. This requires a better understanding of the phenotype nature of the different cell types present in disc tissues. In particular, there is very little known about the differentiated states of cells that co-exist within the annulus fibrosus (AF), despite the critical importance of this tissue in maintaining disc functionality. Maintenance of the differentiated states of these AF cells is imperative to the production of appropriate extracellular matrix (ECM) macromolecules and to the engineering of functional AF tissues. The objective of this study was to determine whether inner annulus fibrosus (IAF) cells when grown on polycarbonate urethane (PU) scaffolds in vitro will produce ECM molecules characteristic of IAF cells and different from outer annulus fibrosus (OAF) cells. OAF and IAF cells isolated from bovine coccygeal intervertebral disc were grown on nanofibrous PU for approximately 14 days. The effect of culture time on ECM gene expression, DNA content, and the synthesis and retention of proteoglycans and collagens were evaluated for both OAF and IAF cells. The ECM accumulated was also characterized by immunostaining and Western blot. The tensile strengths of the tissue/scaffolds were evaluated at 14 days. Both OAF and IAF cells both attached to PU and had similar DNA contents over time. IAF cells maintained relatively higher levels of COL2A1, ACAN and VCAN gene expression, and relatively lower levels of COL1A1 gene expression when compared with OAF cells, by 10 days of culture. IAF cells synthesized and retained similar amounts of total collagen and proteoglycans when compared with OAF cells. While both OAF and IAF cells accumulated type I collagen, only IAF cells accumulated type II collagen. Both cell types accumulated similar amounts of aggrecan but IAF cells accumulated higher amounts of versican as determined by immunostaining. In conclusion, nanofibrous PU scaffolds enabled the maintenance of most of the characteristic features of the IAF cell phenotype, and were different from those of OAF cells.
由于目前尚无能够恢复脊柱全部功能和椎间盘高度的慢性颈部或背部疼痛的最佳治疗方法，因此近期研究聚焦于开发再生医学方法。这需要更好地了解椎间盘组织中不同细胞类型的表型性质。特别是，尽管纤维环（AF）组织在维持椎间盘功能方面至关重要，但对于共存在纤维环内的细胞的分化状态却知之甚少。维持这些AF细胞的分化状态对于产生适当的细胞外基质（ECM）大分子以及构建功能性AF组织至关重要。本研究的目的是确定体外在聚碳酸酯聚氨酯（PU）支架上培养时，纤维环内层（IAF）细胞是否会产生具有IAF细胞特征且不同于纤维环外层（OAF）细胞的ECM分子。从牛尾椎椎间盘分离出的OAF和IAF细胞在纳米纤维PU上培养约14天。评估了培养时间对OAF和IAF细胞的ECM基因表达、DNA含量以及蛋白聚糖和胶原蛋白的合成与保留的影响。通过免疫染色和蛋白质印迹对积累的ECM进行了表征。在第14天评估了组织/支架的拉伸强度。随着时间的推移，OAF和IAF细胞均附着于PU且DNA含量相似。培养10天时，与OAF细胞相比，IAF细胞维持相对较高水平的COL2A1、ACAN和VCAN基因表达，以及相对较低水平的COL1A1基因表达。与OAF细胞相比，IAF细胞合成并保留了相似量的总胶原蛋白和蛋白聚糖。虽然OAF和IAF细胞均积累I型胶原蛋白，但只有IAF细胞积累II型胶原蛋白。通过免疫染色确定，两种细胞类型积累的聚集蛋白聚糖量相似，但IAF细胞积累的多功能蛋白聚糖量更高。总之，纳米纤维PU支架能够维持IAF细胞表型的大部分特征，且与OAF细胞的特征不同。
