Date of Award

Summer 8-5-2014

Degree Type

Thesis

Degree Name

MSOB (Master of Science in Oral Biology)

Primary Advisor

Sunday O. Akintoye

Abstract

Mesenchymal stem cells (MSCs) are emerging donor grafts for bone regeneration in dentistry. MSCs are phenotypically and functionally skeletal site- specific based on extensive studies using human and rodent MSCs but there is paucity of information on canine MSCs (cMSCs) and their regenerative applications in veterinary dentistry. We hypothesized that cMSCs are functionally skeletal-site specific and that mandible cMSCs (M-cMSCs) are highly osteogenic relative to femur cMSCs (F-cMSCs). Trabecular bone samples were obtained from mandible and femur of 2 healthy beagle dogs (ages: 3 weeks, females). Primary M-cMSCs and F-cMSCs were established in culture. Using early passage cells, colony-forming units (CFU), cell proliferation and population doubling capacity were assessed. Using established induction culture conditions, in vitro osteogenesis, chondrogenesis, adipogenesis, and neurogenesis were also assessed. Western blotting and real time PCR were used to assess the following osteogenic markers: alkaline phosphatase (ALP), bone sialoprotein (BSP), osteocalcin (OCN) and osteopontin (OPN). Chondrogenesis was assessed using pellet culture method and histologic sections were stained with Alcian blue; adipogenically induced-cultures were stained with Oil Red O. Neural differentiation was evaluated using morphological analysis and immunostaining to nestin and βIII-tubulin antibodies. Furthermore, in vivo osteogenesis was assessed using the mouse model of in vivo bone regeneration. Transplants were harvested at 6, 8 and 12 weeks for histological analysis.The M-cMSCs demonstrated 1.5 to 2 fold increases in cell proliferation (p =0.006) and life span (five more passages of survival) relative to F-cMSCs. Similar pattern was displayed by M-cMSCs based on expression levels of BSP (14 days p=0.05), ALP (14 days p= 0.004) and OCN (14 days p= 0.03) but OPN levels were not significantly different. Adipogenesis based on number of stained lipid droplets per unit area in M-cMSCs was significant higher than F-cMSCs (p=0.007) and chondrogenic response was also significant higher in M-cMSCs compared with F-cMSCs (4 weeks p= 0.009). Canine MSCs induced substantial in vivo bone formation. The canine MSCs phenotypic and functional properties are site-dependent as the M-cMSCs were apparently more responsive to multi-lineage differentiation relative to F-cMSCs. While the sample size in this study is limited, our findings are still consistent with previous studies using human, mouse and rat MSCs for site-to-site comparative characterizations (Akintoye et al, 2006; Yoshimura et al, 2007; Aghaloo et al, 2010; Lee et al, 2011). Additionally, it is imperative to further confirm these in a larger sample size and in other dog breeds since dogs exhibit an extremely wide range of body physique. New information will advance our understanding of pre-clinical applications of orofacial MSCs as donor graft materials for oral bone regeneration.

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