Inner and Outer Annulus Fibrosus Cells Exhibit Differentiated Phenotypes and Yield Changes in Extracellular Matrix Protein Composition In Vitro on a Polycarbonate Urethane Scaffold

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.