A thesis submitted to the College of Dental Medicine of Nova Southeastern University of the degree of Master of Science in Dentistry.
Background: Therapeutic management of bone loss in the craniofacial region as a consequence of trauma, surgery or congenital malformations presents a clinical challenge. Mesenchymal stem cells (MSCs), due to their inherent plasticity, are potential candidates for cell based therapies for the repair and reconstruction of craniofacial bone tissue. Guided differentiation of stem cells to osteogenic precursors is marked by spatio-temporally regulation of gene expression profiles including that of transcription factors, cytokines, extracellular matrix proteins, enzymes and several signaling pathways. Cytokines, produced by both immune and non-immune cells can influence both immuno- modulatory responses in the host and also affect cell physiology. Understanding the cytokine expression profiles will be of great advantage in developing methods for effective bone regeneration with minimal immunological insults either on the graft or on the host. Objective: The objective of the present study is to investigate the gene expression profiles of the various cytokines of HGMSCs in normal and osteogenic conditions. Methodology: HGMSCs were isolated from gingival tissues by standard enzymatic methods. HGMSCs were guided to osteogenic precursor cells and the differentiation process was monitored by measuring stage specific expression of genes and proteins. Mineral nodule formation of osteogenic differentiation was analyzed by using Alizarin red and Von Kossa Staining methods. Gene expression profiles of various pro- and anti-inflammatory cytokine profiles of HGMSCs were investigated using quantitative real time PCR at 1, 2 and 3 weeks post-induction with osteogenic medium. Results: The osteogenic differentiation of HGMSCs was confirmed by alkaline phosphate enzyme activity assay, gene and protein expression studies of osteogenic markers. Mineral nodule formation was observed after 4 weeks ofosteogenic induction. The results of cytokine profile expressions revealed that there was a significant upregulation in the expression of TGF-β at all-time points. The gene expression of IL-10 was more or less consistent with an overall increase of 40% over that of controls at all time points studied. We observed a significant decrease in the mRNA expression of IL-6 and IL-1? with respect to their control group (P<0.05) and the expression of IL-8 was upregulated significantly. Conclusion: There is an overall enhancement in the expression of anti-inflammatory cytokines IL-10 and TGF-β during the osteogenic differentiation of HGMSCs that indicates a potential shift of cytokines to dampen immune responses. The reduction of IL-6 and IL-1β expression is an added advantage to reduce the acute phase and inflammatory responses, favoring HGMSCs to be cells of choice for repair and regeneration of craniofacial bones. A beneficial combination of the cytokines expressed by HGMSCs during osteogenic differentiation to reduce acute phase and long term immune responses will facilitate the achievement of our long term goal.
| Identifer | oai:union.ndltd.org:nova.edu/oai:nsuworks.nova.edu:hpd_cdm_stuetd-1034 |
| Date | 01 January 2015 |
| Creators | Almashat, Reem |
| Publisher | NSUWorks |
| Source Sets | Nova Southeastern University |
| Detected Language | English |
| Type | thesis |
| Format | application/pdf |
| Source | Student Theses, Dissertations and Capstones |
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