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Faculty of Health Sciences, Degree of Master of Science in Medicine. 9208366a / The initiation of new bone formation within the porous spaces of hydroxyapatite (HA) implants involves the expression of osteogenic markers belonging to the TGF-β superfamily. To study the genetic expression of these osteogenic markers in relation to the type of HA implant used and implantation period, five different types of porous HA biomaterials were implanted in the rectus abdominis muscles of adult baboons Papio ursinus, and were harvested at two, three and 12 months. The total RNA of all harvested samples was extracted and analysed using the Northern blot technique. The results showed that Collagen type IV, GDF-10 and BMP-7 were expressed at the early time points at relatively high levels, and their expression levels were significantly reduced at 12 months. The expression of these makers was not affected by the type of porous HA implant used. The histological sections of these specimens at two and three months showed vascularised connective tissue within the porous spaces of the implants with no bone formation. However, at 12 months there were substantial amounts of bone formed in all the studied implants. The down-regulation of the expressed osteogenic markers at 12 months correlates to the amount of bone formed, suggesting some negative feedback mechanism which may be acting via inhibitory Smads proteins in relation to the amount of bone formed. Neither TGF-β1 nor BMP-3 messages were detected in any of the studied samples, It is possible that these bone markers are not expressed locally within the vicinity of the porous HA implants but are adsorbed to the HA implants from the circulatory system.

Cardiovascular disease is most deadly medical condition in the United States. Medial vascular calcification is a disease that often precedes other more serious cardiovascular diseases that have high mortality. In order to research new therapies for the treatment of medial vascular calcification, an in vitro cell culture model must be developed that mimics the process in vivo. This disease is shown to be an active, cell-mediated process where the vascular smooth muscle cells (VSMCs) in the arteries are differentiating into osteoblast-like cells and depositing hydroxyapatite mineral in the artery walls. By administering inorganic phosphate to cell culture medium, an osteogenic shift can initiated in VSMCs in vitro resulting in calcium deposition and an increase in bone related proteins. We propose to develop and characterize a model for vascular calcification and investigate the effects of magnesium supplementation on in vitro calcification and cellular phosphate uptake.

Exercise associated muscle induced bone strain has potential osteogenic effects that may increase skeletal density, bone cross-sectional area and structural strength. Whether the effects of exercise and the muscle-bone relation are similar in weight bearing and nonweight bearing bones remains to be determined. This study compared bone density, geometry and biomechanical properties, and bone and muscle cross-sectional areas of 13 elite adolescent male cross-country skiers with height, weight, age and maturity matched non-athletic controls. Total bone mineral density (BMDror), and trabecular bone mineral density and total bone cross-sectional area (CSAror) were measured at the distal 4% of the radius (DR) of the dominant (D) and non-dominant (ND) arms, and tibia (DT) using peripheral quantitative computed tomography (pQCT); BMDror, CSAror, cortical BMD, cortical thickness (CrtTH) and area (CSAcoRr), stress-strain index polar, x, y, polar moment of inertia, axial moment of inertia, and muscle cross-sectional area (mCSA) were measured at the 66% length of the proximal tibia (PT) and proximal radius (PR) of the D and ND arms. Whole body BMD, whole body bone area, and hip areal bone mineral density were measured using dual energy X-ray absorptiometry (DXA). Speed of sound along the bone was measured using quantitative ultrasound (QUS) at the 1/3 DR and PT. There were no differences between the skiers and controls for any of the descriptive measures, however, there was a trend (p=0.06) for skiers to have lower percent body fat than controls. There were no differences between skiers and controls for the bone outcome measures using pQCT, DXA or QUS, except for CrtTH at the PT which was significantly higher in skiers (5.42±0.25mm, p=0.03) than controls (5.18±0.28mm). Cross-country skiers had increased CrtTH at the PT suggesting little differential effect of mechanical loading on bone density, geometry or biomechanical properties associated with skiing. / Thesis / Master of Science in Kinesiology

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There are two classes of transposable elements: DNA transposons and retrotransposons. DNA transposons spread in the genome by “cut and paste” mechanism. In contrast, retrotransposons use copy and paste strategy involving RNA and retrotranscriptase mediated mechanism; these include long interspersed nuclear elements-1 (LINE-1, L1) and short interspersed nuclear elements (SINE). In mammals, in order to maintain genome integrity both types of transposons are tightly repressed. However, some copies of retrotransposons are still active in germ cells contributing to natural variation. Surprisingly, recent reports indicate that also somatic cells support L1 reactivation in early development, in particular in the brain leading to mosaicism. However, whether L1 retrotransposition is a part of other cell lineage developmental programs and its functional significance in the context of cell differentiation remain to be elucidated. To address this question, I investigated whether L1 retrotransposition was occurring during in vitro osteogenic and adipogenic differentiation of bone marrow derived human mesenchymal stem cells (hMSCs). Interestingly, clinical observations have revealed loss of bone density in HIV-infected individuals treated with nucleoside analogs that inhibit HIV retrotranscriptase, as well as the endogenous one encoded by L1s. This observation made us to hypothesize that transposable elements played a positive role in post-natal bone homeostasis. I found that while adipogenesis is “retrotransposition free”, osteogenic differentiation is a “retrotransposition-prone” process and its inhibition blocks its genetic program. Indeed, L1 DNA content does not change during adipogenic differentiation and that of retrotranscriptase does not have any effect on the acquisition of a terminally differentiated phenotype. In contrast, soon after MSCs commitment into pre-osteoblasts, L1 retrotransposable elements increase their expression and actively transpose. Inhibition of retrotransposition and knock down of L1 mRNA strongly impairs matrix deposition. Moreover, I forced L1 expression in in vitro adipogenesis, by directly delivering L1 mRNA to the cells. Interestingly, overexpression of L1 elements was detrimental for in vitro adipogenesis. Then, I performed loss of function experiments in osteogenesis by directly targeting and degrading the L1 endogenous transcript. This experiment confirmed the positive role of L1 reactivation in the osteogenic context, suggesting also a possible role for L1 RNA, distinct from retrotransposition.

In this dissertation, small and large NaCl particle-derived surfaces (Ra > 40 microns) were generated on 2D Zn materials, and the surfaces were carefully studied concerning topography, corrosion behavior, and bone cell compatibility. Increases in surface roughness accelerated the corrosion rate, and cell viability was maintained. This method was then extended to 3D porous scaffolds prepared by a hybrid AM/casting technique. The scaffolds displayed a near-net shape, an interconnected pore structure, increasing porosity paralleled to an increased corrosion rate, an ability to support cell growth, and powerful antibacterial properties. Lastly, nano/micro (Rz 0.02–1 microns) topographies were generated on 2D Zn materials, and the materials were comprehensively studied with special attention devoted to corrosion behavior, biocompatibility, osteogenic differentiation, immune cell response, hemocompatibility, and antibacterial performance. For the first time, the textured nonhemolytic surfaces on Zn were shown to direct cell fate, and the micro-textures promoted bone cell differentiation and directed immune cells away from an inflammatory phenotype.

Introduction: The objective of this study is to investigate the role of osteoblasts in the pathophysiology of premature suture fusion in infants. Methods: Bone and periosteal tissue from fused and patent cranial sutures and adjacent bone were harvested from infants undergoing surgery for craniosynostosis and used to develop primary osteoblast cell cultures. Dural tissue was obtained from neurosurgical procedures in order to generate an osteoblast-dural co-culture. Osteoblast proliferation, differentiation, mineralization, protein expression (Noggin, BMP3 and Runx2) and response to exogenous FGF2 stimulation were assessed. Results: Cell cultures demonstrated significant (p<0.05) regional variations in osteoblast proliferation, differentiation markers and in vitro bone nodule formation. The expression of anti-osteogenic molecules (Noggin and BMP3) was decreased in osteoblasts from fused suture regions. Conclusion: The creation of a pro-osteogenic environment through the decreased expression of anti-osteogenic signalling molecules and increased expression of osteogenic factors may be responsible for premature suture fusion in infants.

Introduction: The objective of this study is to investigate the role of osteoblasts in the pathophysiology of premature suture fusion in infants. Methods: Bone and periosteal tissue from fused and patent cranial sutures and adjacent bone were harvested from infants undergoing surgery for craniosynostosis and used to develop primary osteoblast cell cultures. Dural tissue was obtained from neurosurgical procedures in order to generate an osteoblast-dural co-culture. Osteoblast proliferation, differentiation, mineralization, protein expression (Noggin, BMP3 and Runx2) and response to exogenous FGF2 stimulation were assessed. Results: Cell cultures demonstrated significant (p<0.05) regional variations in osteoblast proliferation, differentiation markers and in vitro bone nodule formation. The expression of anti-osteogenic molecules (Noggin and BMP3) was decreased in osteoblasts from fused suture regions. Conclusion: The creation of a pro-osteogenic environment through the decreased expression of anti-osteogenic signalling molecules and increased expression of osteogenic factors may be responsible for premature suture fusion in infants.

Student Number : 9501020M - M Sc dissertation - School of Clinical Medicine - Faculty of Health Sciences / An exciting and novel concept of tissue engineering and morphogenesis is the generation of bone by the implantation of smart biomaterials that in their own right can induce a desired and specific morphogenetic response from the host tissues without the addition of exogenously applied bone morphogenetic and osteogenic proteins.