Early angiogenic change in dental pulp stromal cells cultured on biomimetic matrices

Revascularisation of the devitalised root canal is the Holy Grail of Endodontics and is being hotly pursued by many teams of clinicians but has yet to be achieved. The overall aim of this work was to attempt to induce early angiogenesis in human dental pulp stromal cells (DPSCS) in vitro and in vivo using a biomimetic approach based on combining scaffolds comprised of ECM components with DPSCs as a first step towards a tissue engineering strategy for dental pulp regeneration. After isolating DPSCs using collagenase digest, they were cultured on 1% hyaluronic acid (HyA) or Types I and III collagen matrices used either singly or in combination to determine the ability of these scaffolds to support/induce early angiogenic change in DPSCs both in vitro and in vivo. Angiogenic change was determined using a combined approach of DNA quantification, histology, immunohistochemistry to detect the angiogenic markers CD31 and CD34 and quantitative RT-PCR. DPSCs were shown to attach and proliferate on Type I and III collagen membranes in vitro but early angiogenic change in vitro was evidenced only when 1% HyA gel was used, including in the absence of the morphogen rhVEGF165 as shown immunohistochemically. PCR at two and five days post-seeding showed an up-regulation of CD31 and CD34 genes dependant on culture conditions, with CD31 being upregulated early and CD34 later in the culture period. A modified tooth slice model containing a combination of HyA/collagen scaffold/DPSC constructs within its lumen also showed positive early angiogenic change in vitro. SEM examination further confirmed that DPSCs could attach, colonise and proliferate to/on the combined scaffold. The same combined scaffold-tooth slice model ± DPSCs used in vivo in nude mice showed cellular ingress into the lumen with a soft tissue closely resembling dental pulp-like tissues in its appearance with new tubule-like material grown on from the dentinal tubules of the tooth slice. There was a defined demarcation line between this latter material and the dentinal tubules of the tooth slice and the new material closely resembled predentine or dentine-like matrix in appearance and stained strongly for CD31 and CD34 markers. It also had a layer of cells adjacent to and in intimate contact with its deposition front, whose cell processes transited the new tubule-like material and continued into the dentine tubules of the tooth slice for some distance. Interestingly, this neo-tissue was independent of the addition of DPSCs to the construct. The results suggest that biomimetic scaffolds based upon components of the pulp extracellular matrix may provide a useful platform for future engineering of a vascularised replacement dental pulp.