In vitro investigations of bioactive glass scaffolds for dental tissue engineering

Abstract

The regeneration of large bone and dental defects is hampered by the lack of remaining available tissue and the limited degree of vascularisation to support the healing process. Current treatment strategies using autologous tissue are inadequate for regenerative medicine. By contrast, the development of smart biomaterials that promote angiogenesis and bone deposition is gaining interest. This thesis describes the in vitro investigations of two bioactive glasses, namely strontium-containing bioactive glass (SrBG) and cobalt-containing bioactive glass (CoBG), for their potential use in the repair and regeneration of dental related structures. Strontium was incorporated into the bioactive glass (BG) structure by virtue of its dual role in promoting new bone formation and preventing osteoclast-mediated bone resorption. Cobalt is a known hypoxia-mimicking agent, and the incorporation of cobalt into the BG structure may also help create the hypoxic environment that benefits vascularisation during the regeneration of hard and soft tissue. Dental stem cells from permanent (dental pulp stem cells, DPSC) and deciduous teeth (stem cells from exfoliated deciduous tooth, SHED) were used in the current project since both appeared suitable for the regeneration of oral and dental related structures.

Strontium-containing BG (SrBG) with 0%, 10% and 100% substitution of strontium for calcium were studied. Their dissolution profiles in culture medium (DMEM and RPMI) were assessed using inductively coupled plasma-optical emission spectroscopy (ICP-OES), and the dissolution products were tested on human osteosarcoma cells (Saos-2) and mouse monocyte cells (RAW264.7). At the selected SrBG powder to liquid ratio (SrBG in DMEM was 6.0 mg/ml, and SrBG in RPMI was 1.5 mg/ml), the bioactive glasses were non-cytotoxic. In addition, when dental pulp stem cells from the permanent and deciduous teeth (DPSC and SHED, respectively) were seeded on the 10% SrBG discs, both cell types had a higher proliferation rate when compared to those seeded on tissue culture plastic (Thermanox) after 21 days of culture.

The use of DPSC and SHED as a source of stem cells for dental tissue engineering was investigated on cobalt-containing BG (CoBG). These glasses act as hypoxia-mimicking materials, a desirable property since hypoxia is known to enhance stem cell proliferation, stimulate VEGF secretion through the HIF-1 pathway, which help to improve vessel formation. First, the dissolution products from CoBG (0%, 1%, 2% and 4% cobalt for calcium substitution in mole percent) were incubated with the dental stem cells, and in vitro assays showed that the cobalt-containing BG-conditioned media had a dose-dependent effect on metabolic activity and proliferation. In addition, the CoBG succeeded in activating the hypoxia pathway, leading to VEGF secretion. The DPSC and SHED also secreted a substantial amount of collagen. Next, CoBG powder was incorporated into a biodegradable polymer, polycaprolactone (PCL), and three-dimensional cobalt-containing BG with PCL (CoBG/PCL) scaffolds were produced. The CoBG/PCL scaffolds also promoted the DPSC and human mesenchymal stem cells (hMSC) metabolic activity, and the cobalt released had no negative effect on cell survival. The CoBG/PCL scaffolds were also able to stimulate the hypoxia pathway and caused both DPSC and hMSC to secrete VEGF. Following the osteogenic induction of cells seeded on the CoBG/PCL scaffolds, there was lower ALP activity suggesting the cells remained undifferentiated when exposed to the hypoxia-mimicking CoBG.

Although the DPSC and SHED were seeded only on the 10SrBG discs (10% of strontium substitution with calcium in BG), the results presented in this thesis demonstrate the potential of SrBG and CoBG scaffolds for dental tissue repair and regeneration. The CoBG as dissolution products from the CoBG powder and the incorporation of CoBG powder into scaffolds demonstrated activation of HIF pathway, which has promising potential for dental tissue repair and regeneration.