This dissertation thesis was designed to contribute to a better understanding of isolation, proliferation and differentiation of dental stem cells (DSCs) derived from human teeth under specific treatment conditions for bone regeneration. The extracted impacted third molar teeth are discarded source of human waste, which harbor tissues that have potential stem cells. The dental tissues that can be obtained from the teeth are dental pulp tissue, dental follicle tissue and periodontal tissue. These tissues can be enzymatically digested to isolate cells; DSCs obtained from all three tissues proliferated and could be differentiated osteogenically in our studies. The advantage of these cells over the more standard, more clinically developed mesenchymal stem cell (MSC) source derived from bone marrow tissues (BM-MSCs) is that DSCs can be obtained from discarded teeth from routine dental procedure, with low tissue site morbidity and the cell numbers obtained are higher.

Furthermore, the combination of various growth factors or hormones in culture media results in osteogenic differentiation. Hence, in this thesis, the effect of growth factors and hormones were compared on osteogenic differentiation ability. Bone morphogenetic proteins (BMP) are growth factors that play a critical role in tooth morphogenesis and were originally identified as proteins that induce bone formation at extra- skeletal sites, BMP-2 induces alveolar bone formation and BMP-6 regulates osteogenic differentiation of cells. Therefore, these growth factors were tested in study I on periodontal ligament cells (PDLCs) osteogenic differnetiation ability. However, BMP-2 and BMP-6 did not suppport the osteogenic differentiation of PDLCs. Thereafter, in study II, hormones such as vitamin D3 metabolites (VD) and dexamethasone (DEX) were compared for their osteogenic induction abilty in dental pulp stem cells (DPSCs) and dental follicle stem cells (DFSCs). Vitamin D3 metabolites were found to support osteogenic differentiation of the DPSCs and DFSCs better than DEX. Therefore, VD was chosen as an osteogenic inducer for our subsequent studies. Apart from that, by considering the ease of accessibility of the tissue and its stem cell potential, DPSCs were chosen to be further studied for this thesis.

There are several studies showing the potential of DPSCs over BM-MSCs in comparison to their proliferation, multilineage differentiation ability and immunosuppressive response. However, the culture techniques used for isolation of cells seems to be a major concern in terms of clinically applicability of DPSCs. In most of the studies, the cells are cultured in medium containing fetal bovine serum (FBS) animal derived component, which initiates immunogenic or allergic responses in patients treated with those cells. Therefore, in study III we have shown that DPSCs isolated and cultured in allogenic human serum medium (HS-M) maintained their stemness as they expressed the OCT3/4, SOX-2 markers that are responsible for altering the progenitor status and NANOG that has been reported to be a key gene for maintaining pluripotency. Furthermore, the cells were also cultured in serum free/xeno free medium (SF/XF-M), DPSCs proliferated at a slower rate in comparison to the cells cultured in HS and FBS media, however, the expression of the mesenchymal stem cell markers was similar in DPSCs cultured in all the media. Moreover, the differentiation in osteogenic, adipogenic and chondrogenic lineages was more in HS-M cultured DPSCs. Also, as shown in study IV, the HS-M cultured cells were able to proliferate and differentiate into osteoblasts on a medical grade β-Tricalcium phosphate/Poly L-Lactic acid- caprolactone (β-TCP/P (LLA-CL)) 3D biomaterial scaffold.