Isolation of periodontal ligament stem cell and investigation of chemical and mechanical stimuli in oral tissue regeneration

[Truncated] Tissue regeneration is a complex process involving regenerative cells, as well as creating a conducive environment for facilitating the process. Oral tissue regeneration, especially the bony defects from acquired disease or trauma and congenital agenesis like in oral cleft are the most difficult clinical challenges dentofacial esthetics. Hence, the goals of this project are to: 1) identify a reliable and easily accessible source of local resident stem cells, and 2) investigate potential stimuli both chemical stimuli (Nerve growth factor beta NGF-β, Epidermal growth factor EGF, Bone morphogenetic protein-4 BMP-4) and mechanical stimuli (Orthodontic tooth movement OTM, Distraction osteogenesis DO) that can facilitate oral tissue regeneration.

Mesenchymal stem cells (MSC) have been an ideal source for regenerative therapy with its ease of handling and diverse multipotent lineage. Its relative ease of access when compared to other sources of stem cells has also given the use of MSCs a strong advantage. To date, bone marrow mesenchymal stem cell (BMMSC) remain the most common source for MSC. However, due to its co-morbidities risks, other easier alternatives are preferred. In contrast, stem cells derived from the oral region is an appealing alternative source of MSC, as these cells can be utilised without the associated morbidity or mortality. The periodontal ligament in the oral region is a source of mesenchymal stem cells and these cells are termed Periodontal ligament stem cells (PDLSCs). PDLSCs can be cultivated from vestigial wisdom teeth. To date three putative markers of the PDLSC stem cell have been identified, namely murine IgM monoclonal antibody (STRO1), melanoma cell adhesion molecule (CD146) and ATP binding cassette subfamily G member 2 (ABCG2). However, thus far only a combination of two markers have been used in the isolation process. The aim is to combine all 3 pre-existing stem cell markers simultaneously in the isolation process to further isolate PDLSC which were never attempted. Using immune-selection and flow cytometry techniques, STRO-1, CD 146 and ABCG2 markers can be utilised to positively identify the stem cell population in periodontal ligaments. Following successful isolation of these stem cell populations, these PDLSC populations were validated for their multipotent ability and clonogenesis by means of colony forming unit assays (CFU). Clonogenesis was consistently demonstrated with the differential seeding density showing the CFU amongst the PDLSC. Osteogenic, adipogenic and chondrogenic potentials were validated using both histological stain as well as gene marker expressions. The PDLSC were strongly stained with Alkaline phosphatase (ALP), Alizarin red , oil red stain, safranin O and alcian blue which represented their multi-potency. Moreover, key gene expressions of each lineage were confirmed such as osteocalcin (OC), low density lipoprotein (LDL) and sex determining region Y (SOX9). The result supports the aim by combining all three stem cell markers a consistent isolation of a stem cell population of periodontal ligament are achievable. Hence PDLSC can be an alternative source for oral tissue regeneration. To further assess if stimuli such as environmental cues play important roles in oral tissue regeneration, the use of chemical and mechanical stimulus were investigated. Specifically in this thesis, chemical stimuli employed the use of growth factors such as nerve growth factor beta (NGF-β), epidermal growth factor (EGF) and bone morphogenetic protein-4 (BMP-4). All the above mentioned growth factors were selected on the basis of their known involvement in tissue regeneration in other tissue regions. Mechanical stimuli like orthodontic tooth movement (OTM) and distraction osteogenesis (DO) are the common clinical parameters adopted in dentofacial orthopedics in orthodontics. These mechanical stimuli were simulated using the Flexcell FX4000 system to investigate the responses of the jaw structures to these commonly used mechanical stimuli in clinical practice of orthodontics.