Study Of The Rabbit Nucleus Pulposus -like Cells Cultured In Vitro On Thermo-sensitive Scaffold

The intervertebral discs (IVD) of the human spine serve to link adjacent vertebral bodies, allow movement and protect the spine from damage during extremes of physical load. Each IVD is comprised of a central, gelatinous nucleus pulposus (NP), constrained by a fibrous annulus fibrosus (AF) and joined to the adjacent vertebrae by cartilaginous end plates (EP). As individuals age the NP begins to degenerate, due to a alteration in ECM and apoptosis of NP cells. In many cases these symptoms are associated with pain and, while it is multifactorial, IVD degeneration is considered to be one of the predominant causes of low back pain (LBP). The treatment may include surgical intervention to either remove tissue or fuse the affected level. All of these treatments are aimed at relieving pain and restoring biomechanics, rather than at repairing the tissue, and in many cases they lead to further complications such as accelerated degeneration in adjacent levels due to alterations in the biomechanics of the spine. Recent developments in the field of tissue engineering suggest that a regenerative medicine approach may be ideal to repair the degenerate IVD and restore spinal biomechanics. Several tissue engineering approaches using different cell types and hydrogels/scaffolds have been proposed. Owing to the unsuitable nature of degenerate cells for tissue engineering attention has focused on the use of mesenchymal stem cells (MSCs). Additionally, while rigid scaffolds have been demonstrated to allow MSC differentiation to the chondrocyte-like cells of the IVD, hydrogels are being increasingly studied as they allow minimally invasive implantation without extensive damage to the IVD.Here, we have studied the temperature-sensitive hydrogel : chitosan- glycerophosphate (C/Gp), seeded with human induced-ADSCs (NP-like cells)and cultured for 3 weeks in standard medium. We have analysed the gene xpression profile of the induced-ADSCs cultured in C/Gp and compared it to that of NP-like cells before cultured in C/Gp. We also have analysed the gene and protein expression profile of ADSCs co-cultured 2weeks with NP cells and compared to which induced by compound growth factors.Gene expression analysis for NP-cell marker genes demonstrated both co-cultured and growth-factor induced ADSCs differented to a phenotype which showed similarities to both articular chondrocytes and NP cells. ADSCs also secreted both proteoglycans and collagens in a ratio, which more closely resembled that of NP cells than articular chondrocytes. We also compounded the scaffold and NP-like cells. Then, the viability of NP-like cells in the C/GP hydrogel was observed 2 days after compound culture and the growth condition of NP-like cells on the scaffold was observed by SEM 7 days after compound culture. Their expression of Col II and aggrecan mRNA were analyzed by RT-PCR 21 days after compound culture and showed that the secretion of Col II and aggrecan mRNA in NP-like cells cultured in C/GP hydrogen scaffold is more strengths of producing matrix than that of monolayer culture without de-differentiation. These results therefore suggest that ADSC-seeded C/Gp gels could be used clinically for the regeneration of the degenerate human IVD.