Dental Pulp Cell Behaviour in Biomimetic Environments

There is emerging recognition of the importance of a physiologically
relevant in vitro cell culture environment to promote maintenance of stem
cells for tissue engineering and regenerative medicine purposes. In vivo,
appropriate cellular cues are provided by local tissue extracellular matrix
(ECM) and these are not currently recapitulated well in vitro using
traditional cultureware. We therefore hypothesized that better replication
of the in vivo environment for cell culture and differentiation could be
achieved by culturing dental pulp cells with their associated ECM. Primary
dental pulp cells were subsequently seeded onto pulp-derived ECM coated
cultureware. While at up to 24-hours they exhibited the same level of
adherence as those cells seeded on tissue culture treated surfaces, by 4-
days cell numbers and proliferation rates were significantly decreased in
cells grown on pulp ECM compared with controls. Analysis of stem cell and
differentiation marker transcripts, as well as Oct 3/4 protein distribution,
supported the hypothesis that cells cultured on ECM better maintained a
stem cell phenotype compared with those cultured on standard tissue
culture treated surfaces. Subsequent differentiation analysis of cells
cultured on ECM demonstrated they exhibited enhanced mineralisation as
determined by alizarin red staining and mineralised marker expression.
Supplementation of a 3% alginate hydrogel with pulp ECM components and
dental pulp cells followed by differentiation induction in mineralisation
medium resulted in a time-dependent mineral deposition at the periphery
of the construct, as demonstrated histologically and using micro-computed
tomography analysis, which was reminiscent of tooth structure. In
conclusion, data indicates that culture of pulp cells in the presence of ECM
better replicates the in vivo environment maintaining a stem cell
phenotype suitable for downstream tissue engineering applications.