Behaviour of Dental Pulp Stem Cells in 3D Culture Systems

The dental pulp contains a progenitor stem cell population that is vital for the repair of the tissue. Such dental pulp stem cells (DPSCs) exist within a 3D environment, however many studies investigate the potential of these cells in monolayer cultures.  The ability to culture DPSCs in a 3D matrix may provide a better understanding of DPSC behaviour. Objective: Investigate the biological response of DPSCs within 3D collagen environments. Methods: DPPCs were isolated from third molar teeth, pulps dissected and cell suspensions obtained by collagenase/dispase digestion.  Individual clones were isolated by fibronectin adhesion and maintained in culture. Population doublings (PDs) were monitored, as was stem cell marker expression (CD105/CD90/CD73) by RT-PCR. Type 1 rat tail collagen was prepared in a 1:1 ratio with culture medium to a concentration of 1.025mg/ml and seeded with 8x10⁴ cells/ml of either high proliferating or low proliferating DPSCs and cultured for 12 days. Cell morphology within collagen gels was examined by scanning electron microscopy and shrinkage of gels measured at day 0, 2, 4, 6 and 12. To determine the relative amounts of pro- and active-MMP species produced by DPPCs in the collagen, gelatin zymography was utilized using conditioned media from the cultures. Results:   Both DPPC clones appeared to remain viable and were uniformly distributed within the collagen during the culture period, with maintenance of stem cell markers. Cultured gels containing the high proliferating DPSCs demonstrated significant shrinkage, however in cultures containing low proliferating DPCS gel shrinkage was less marked over the first 48 hours. Gelatin zymography demonstrated increasing expression of MMP2 and MMP9 with increasing culture time. Conclusions: This 3D culture system provides a promising environment for the culture of DPSCs in 3D, allowing for further studies to evaluate their responses to exogenous stimuli such as cytokines, growth factors, oxidative stress and bioactive matrices.