published_or_final_version / Orthopaedic Surgery / Master / Master of Philosophy

The effect of zinc deficiency on the growth promoting effect of circulating IGF-I and the direct growth effect of GH on long bone growth were investigated. Food intake was decreased by lack of zinc in the diet. Tissue zinc content and plasma alkaline phosphatase activity were reduced by zinc deficiency. Systemic administration of human IGF-I increased the body weight, tail length and tibia epiphyseal cartilage width of control animals. This somatogenic action was impaired by zinc deficiency, as evidenced by continued weight loss, no increase in tail length and decreased tibial epiphyseal cartilage width of zinc deficient animals. Unilateral arterial infusion of GH increased the tibial epiphyseal width of the treated limb but not of the non-treated limb in control rats. However, no difference was found between the infused and the non-infused limb of zinc deficient animals, suggesting the occurrence of GH resistance on long bone growth in zinc deficiency. We conclude that zinc deficiency inhibits the growth promoting action of circulating IGF-I and the direct growth effect of GH on long bone growth.

Exercise is known to have a long-term benefit on bone mass in children, but little is known about the underlying mechanisms. The purpose of this investigation was to determine the acute effect of exercise on bone metabolism in pre-pubertal children. Biochemical markers of bone formation were measured in 4 male and 4 female children, 8 to 11 years of age. Each subject performed 50 vertical jumps. Serum osteocalcin and C-telopeptide of type I collagen (CTx), were determined prior to exercise and at 24 and 72 hours post exercise as indicators of bone formation and bone resorption. Osteocalcin concentration was 8.20 ± 3.65 ng•mL"' before exercise, and was 7.1 ± 3.7 ng•mL-' and7.4 ± 3.7 ng•mL-' at 24 hours and 72 hours post exercise, respectively (P > 0.05). CTx concentrations were 11632 ± 4093 pmol•L-' before exercise, and was 9831 ± 3159 pmol•L-' at 24 hours and 9722 ± 2426 pmol•L7' at 72 hours post exercise (P > 0.05). In conclusion an acute bout of ballistic exercise appears to have no effect on bone metabolism in pre-pubertal children. / School of Physical Education

No description available.

Made available in DSpace on 2014-11-10T11:09:56Z (GMT). No. of bitstreams: 0 Previous issue date: 2011-09-19Bitstream added on 2014-11-10T11:57:36Z : No. of bitstreams: 1 000697140_20151015.pdf: 232164 bytes, checksum: 0bed7471a6786fe38e3164319d6deb3e (MD5) Bitstreams deleted on 2015-08-07T12:19:55Z: 000697140_20151015.pdf,. Added 1 bitstream(s) on 2015-08-07T12:20:58Z : No. of bitstreams: 1 000697140.pdf: 6167998 bytes, checksum: bf8732b598c57469944be2ab9eac1b0c (MD5) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / Os objetivos deste estudo foram avaliar a influência da nanotopografia de superfícies de titânio no processo de osteogênese por meio de avaliação histológica do contato osso-implante, em ratos, sob efeito da inalação forçada da fumaça de cigarro; Avaliar a osteogênese e a expressão de citocinas em cultura de células primárias sobre discos de titânio com nanotopografia; E avaliar o efeito morfológico e molecular da incorporação da laminina-1 (LN-1) em superfícies com nanotopografia, em coelhos. Implantes e discos de titânio foram especialmente produzidos e submetidos a diferentes métodos para a obtenção de superfícies: usinada, micro e nanotopografia. As superfícies foram devidamente caracterizadas quanto a sua topografia, morfologia e química. Os resultados mostraram que: a nanotopografia é capaz de aumentar o contato osso-implante (BIC) mesmo na presença da inalação intermitente da fumaça de cigarro, e foi capaz de reduzir, porem não totalmente, seus efeitos deletérios e uma menor formação óssea; A nanotopografia de titânio pode ter papel importante no processo de mineralização da matriz extracelular e na modulação da expressão de citocinas; E que a incorporação de LN-1 a superfície de titânio com nanotopografia demonstrou favorecer uma maior expressão de importantes genes envolvidos na cascata da osseointegração. / The aims of this study were to evaluate the influence of nanotopography of titanium surfaces in the process of osteogenesis by means of histological assessment of bone-implant contact, in rats, under the effect of forced inhalation of cigarette smoke; To assess the osteogenesis process and the expression of cytokines in primary cell culture on titanium disks with nanotopography; And evaluate morphological and molecular the effect of incorporation of laminin-1 (LN-1) surfaces with nanotopography, in rabbits. Implants and titanium disks were specially produced and submitted to different methods for obtaining surfaces with turned, micro and nanotopography. The surfaces have been properly characterized as its topography, morphology and chemistry. The results showed that: nanotopography is able to increase bone-implant contact (BIC) even in the presence of intermittent inhalation of cigarette smoke, and was able to reduce, although not entirely, their harmful effects and a lower bone formation; Nanotopography may play a important role in mineralization process and in cytokine expression.; And the incorporation of LN-1 on titanium surface with nanotopography favor a higher expression of important genes involved in the cascade of osseointegration.

Emerging evidence indicates that the dysregulation of protein ubiquitination plays a crucial role in aging-associated diseases. Smad-dependent canonical BMP signaling pathway is indispensable for osteoblastic bone formation, which could be disrupted by the ubiquitination and subsequent proteasomal degradation of Smad1/5, the key molecules for BMP signaling transduction. However, whether the dysregulation of Smad1/5 ubiquitination and disrupted BMP signaling pathway are responsible for the age-related bone formation reduction is still underexplored. Casein kinase-2 interacting protein-1 (CKIP-1), also known as Pleckstrin homology domain-containing family O member 1 (PLEKHO1), is a previously identified ubiquitination-related molecule that could specifically target the linker region between the WW domains of Smurf1 to promote the ubiquitination of Smad1/5. Here, we found an age-related increase in the expression of CKIP-1 in bone specimens from either fractured patients or aging rodents, which was associated with the age-related reduction in Smad-dependent BMP signaling and bone formation. By genetic approach, we demonstrated that loss of Ckip-1 in osteoblasts could promote the Smad-dependent BMP signaling and alleviated the age-related bone formation reduction. In addition, osteoblast-specific Smad1 overexpression had beneficial effect on bone formation during aging, which could be counteracted after overexpressing Ckip-1 within osteoblasts. By pharmacological approach, we showed that osteoblast-targeted CKIP-1 siRNA treatment could enhance Smad-dependent BMP signaling and promote bone formation in aging rodents. Taken together, it suggests that the increased CKIP-1 could suppress Smad-dependent BMP signaling to inhibit bone formation during aging, indicating the translational potential of targeting CKIP-1 in osteoblast as a novel bone anabolic strategy for reversing established osteoporosis during aging.

Osteoporosis is characterized by chronic bone loss and deterioration of microarchitecture that can leave patients more susceptible to costly and debilitating fractures. A variety of treatment options have been developed that target different cells and pathways to disrupt its progression. In addition to pharmaceutical options, regular exercise is recommended, as external, mechanical loading has long been recognized as a stimulus bone can use to regulate its size and shape to meet mechanical demands. While bone cell signaling is undoubtedly multifaceted, meaningful changes in bone mass ultimately result from the actions of bone-forming osteoblasts and bone-resorbing osteoclasts. To this end, therapies are most traditionally described through their impacts on these cells, and are broadly categorized as anabolic (activating osteoblasts and having bone-building effects, such as parathyroid hormone injections and sclerostin antibody treatment), or anti-resorptive (targeting osteoclasts and slowing resorption, such as bisphosphonates and denosumab). Bone formation and resorption are rooted in two overarching processes: coupled bone remodeling (resorption followed by formation in the same space) and uncoupled bone modeling (formation or resorption occurring independently). Hematopoietic-lineage cells have an inherent, established role in bone remodeling, as descendent osteoclasts perform the resorption to initiate remodeling, but have only more recently been implicated as potential orchestrators of anabolic bone modeling in their preosteoclastic states, suggesting the extent of their differentiation may be a mechanism steer the bone response between maintenance remodeling and adaptive modeling regimes. Understanding how pharmaceutical treatments and mechanical loading work through these regimes, augment intrinsic sensing mechanisms, or tilt local signals to favor one or the other may provide valuable insight into optimizing or combining current treatments, and potentially suggest new therapeutic avenues. We first establish a method for quantifying modeling and remodeling in vivo using image registration on weekly micro-computed tomography scans. This technique is implemented in a study to assess the independent and combined effects of daily mechanical loading and parathyroid hormone injections in mice. We found that both resulted in significant increases in bone formation through anabolic modeling and remodeling, and while the modeling effects were usually additive or independent, the remodeling response was synergistic. Additionally, while PTH tended to exert its influence indiscriminately, the loading response was more targeted and pronounced in ways that mirrored local mechanical strains. Interestingly, this held true for catabolic modeling as well, where we observed a previously unreported phenomenon of load-induced increases in catabolic modeling in areas of low strain on the endosteal surface of cortical bone. We then began targeted interventions into the hematopoietic lineage cells, starting at their most terminally differentiated state in bone, the osteoclast. Using an injectable osteoclast maturation inhibitor, osteoprotegerin (OPG), we observed how arresting this process influenced modeling and remodeling in response to loading in normal mice, and in mice genetically modified to reduce sclerostin expression. We observed the expected reductions in catabolic modeling regardless of genotype. We also found that in sclerostin-depleted mice treated with OPG, anabolic modeling was elevated, and there was no added benefit of mechanical loading to the response in trabecular and endosteal compartments, suggesting the controlled manipulation of these factors can fully recapitulate the intrinsic mechanosensing capabilities. Since the loading response is largely modeling-based, these findings support the hypothetical determinant of the modeling/remodeling response being the preosteoclast/osteoclast ratio in these areas. In contrast, however, on the periosteal surface a pronounced load-induced anabolic modeling response persisted in all treatment conditions, suggesting the unique cell populations in the periosteum may have more robust, more finely tuned, or differentially regulated mechanosensing mechanisms. Finally, to probe the hematopoietic lineage further upstream and address the other side of the preosteoclast/osteoclast hypothesis, we utilized a novel genetically modified mouse model that allows for inducible macrophage (preosteoclast) ablation. Modeling and remodeling dynamics in response to loading were quantified in mice with normal or depleted macrophage quantities with concurrent normal or genetically-reduced sclerostin expression. In agreement with our hypothesis linking these cells to the anabolic modeling response, macrophage ablation resulted in significantly less anabolic modeling on trabecular and endosteal surfaces, which was not recovered by mechanical loading in either wild type or sclerostin deficient mice. Again, however, the periosteal surface was unique. Macrophage ablation did not reduce anabolic modeling on the periosteal surface, and loading still significantly increased it, regardless of sclerostin expression. Thus, similar to our findings with osteoprotegerin, a unique contrast existed between macrophage/preosteoclast ablation drastically reducing anabolic modeling and nullifying any mechanoresponse on trabecular and endosteal surfaces, but not the periosteal surface. Taken together, these studies outline and implement a novel method to quantify modeling and remodeling in response to loading and clinically relevant treatments, with an emphasis on perturbations of the hematopoietic lineage. Concurrent stimuli are used to observe and quantify overlaps and augmentations in treatment efficacies, with a focus on mechanisms related to mechanoadaptation. Future work will focus on targeted approaches to identify unique mechanosensing factors driving the periosteal response, more sophisticated data analysis tools to observe to what extent localized bone metabolism can be predicted by strain and morphology, and the protein and cellular-level dynamics that underlie our findings. As an addendum, a novel bone morphological parameter is described. A trabecular-cortical interface surface area metric (iSAM) is quantified on a set of cadaver bone segments from clinical high-resolution peripheral quantitative computed-tomography scans (a clinical analog to micro-computed tomography). iSAM is shown to correlate with stiffness and ultimate force derived from mechanical testing of the same samples, and improve correlations gleaned from traditional morphometric parameters alone.

No description available.

The effect of restricting energy intake (ad libitim vs. 75% ad libitum) elevation of vitamin and mineral levels (150% NRC vs 100% NRC) and the effects of aging on bone growth, histological lesioning and biochemical composition of articular cartilage from growing boars was examined. Growth of the radius and tibia was characterized by radiography at eight equal time points from 110 to 330 d of age. Although ad libitum-fed boars had larger bones in most cases, calculation of weight corrected differences suggested that restriction of energy resulted in lover weight gain but that reduced energy intake slightly increased the rate of bone growth of the restrict-fed boars. Although elevation of vitamin and mineral levels resulted in a few scattered differences in bone size, no consistent trends were evident. Neither restriction of energy intake nor elevation of vitamins and minerals had any consistent effect on the reduction of histological lesion severity. Age, expressed as days on test, produced no definite trends toward increasing or decreasing frequency of histological lesions. The frequency and severity of gross lesions were unaffected by altered energy or vitamin-mineral levels, however, a sevenfold age-related increase in frequency of gross lesions was observed over time. Uronic acid content of the articular cartilage from the femur was greater for the restrict-fed boars, however, uronic acid content of cartilage from the humerus and radius tended to be greater for the ad libitum fed boars. Elevation of vitamins and minerals had no effect on uronic acid content of articular cartilage from the femur, humerus or radius. Uronic acid content of articular cartilage decreased consistently with age throughout the trial period. Galactose and hydroxyproline levels in cartilage samples from the femur, humerus and radius were generally unaffected by the imposed dietary treatments. Galactose levels were unaffected by age. Hydroxyproline content of cartilage from the radius and humerus increased with age, whereas, cartilage from the femur remained unchanged throughout the experimental period. No changes in percentage fat-free dry weight of articular cartilage samples from the femur, humerus, or radius due to treatment or age were evident. Articular cartilage from the radius had a higher percentage of fat-free dry weight than either the humerus or femur. Articular cartilage from the radius also contained less uronic acid than the femur or humerus. Galactose content of the radius was less than that of the humerus, however, galactose levels of the femur and radius did not differ. Hydroxyproline levels were similar among all areas sampled. In summary, skeletal growth rate was slightly greater for restrict-fed boars after weight correction, however, elevation of vitamins and minerals had no overall impact on any parameters measured. Age had a more pronounced, though inconsistent influence on lesion frequency, severity, and biochemical composition of the articular cartilage than the imposed dietary treatments. / Master of Science

Indiana University-Purdue University Indianapolis (IUPUI) / The Cre/loxP system is a tool for targeted recombination of DNA. For applying Cre recombinase-mediated genome modifications, there is a requirement for reliable, high-fidelity, and specific transgenic expression of the Cre recombinase. This study focuses on the reliability of different bone cell specific Cre models in the Cre/loxP system. In this study, DMP1-Cre transgenic mouse which has a transgene driven by DMP1 promotor that allows Cre-expression only in late stage osteoblasts and osteocytes was used. Ctsk-Cre mouse with a driven by Ctsk promoter was used so that only osteoclasts would undergo Cre-mediated recombination. E2A-Cre mouse where the Cre recombinase is driven by a global promoter E2A was also included in this study as a control line to test the Cre reporter line Ai9. Dmp1-Cre, Ctsk-Cre and E2A-Cre mice were crossed to the fluorescent Cre-reporter line—Ai9, which harbors a floxed stop codon, followed by the fluorophoremTomato, inserted into the Rosa26 locus. This construct is expected to give red fluorescence when it recombines with Cre-expressing mouse cells and no fluorescence in non-recombinant mouse cells. Double positive (Ai9+/Cre+) offspring selected by PCR were perfused, and 5mu-m thick section of bone and soft tissues were examined for red fluorescent expression. Cre positive cells were quantitated using ‘ImageJ’ software program. The DMP1-vi Cre mouse results showed significant expression in the targeted osteocytes and osteoblasts. In addition, skeletal muscle tissue also showed significant Cre- expression. Ctsk-Cre mice showed significant expression in targeted osteoclasts. But brain tissue was positive in Cre-expression. Bone-Cre mouse models are expected to express Cre recombinase only in their respective bone cells and they have been used for gene deletion studies in bone cells. However, this study has revealed that the bone cell specific Cre mouse models DMP1-Cre and Ctsk-Cre have unexpected expression in muscle and brain respectively. In order to use these models for targeted gene deletion in bone cells, further testing and studies have to be conducted.