Bilaminar coculture of stem cells and instructive cells for tissue regeneration

Tissue engineering is a growing and dynamic field with the potential to provide patients with minimally-invasive treatments that repair or replace dysfunctional musculoskeletal tissues. For the intervertebral disc, the goal is to re-establish pain-free motion by restoring the disc's physical and biochemical properties. Unfortunately, many of the biological processes involved remain unclear. Mesenchymal stem cells (MSCs) are an attractive component of disc tissue engineering given their ability to differentiate down multiple lineages. However, MSCs require a coordinated set of environmental cues to appropriately differentiate to a chondrogenic or disc-like cell.;Our goal is to identify mechanisms involved in disc regeneration and MSC differentiation to optimize disc tissue engineering strategies. We explored the benefits of coculturing nucleus pulposus cells (NPC) and adult mesenchymal stem cells (MSC) using a 3D system that exploits embryonic processes such as tissue induction and condensation.;The following dissertation reports on a novel "bilaminar cell pellet" (BCP) approach that structures cell-cell signaling between MSCs and instructive cells, in this case non-degenerative NPCs, in a 3D culture system. A spherical bi-layer pellet is used where one cell type forms an inner sphere enclosed within a shell of the other cell type. This technology has been patented by UCSF as of May 2008 (see Appendix).;The BCP system has been tested extensively under multiple conditions. We first tested the BCP under normal in vitro culture conditions and showed an increase in matrix production over controls. The BCP was subsequently tested in a bioreactor supplemented with inflammatory cytokines to simulate the degenerative disc environment. Again, the BCP proved to be resilient and produced more new matrix than controls. Finally, the BCP was evaluated in an in vivo rat tail model. Our data show that the beneficial behaviors previously reported in vitro translate to a more effective cell-based treatment in vivo. Future studies will explore the function of BCPs in larger discs that more closely mimic the human situation.;Our data show that spatial organization plays an important role in matrix production, and that structured communication between MSC and NPC enhances the efficacy of stem-cell-based strategies for nucleus regeneration beyond using MSCs alone.