Age-at-death estimation in older adults is complicated because current techniques fail to capture an older individual’s age-at-death in a narrow enough range. This study aimed to test the relationship between osteoporotic change and age-at-death in the form of intracortical porosity. It was hypothesized that as individuals age, osteoporotic processes increase resulting in a higher percentage of intracortical porosity that may correlate with particular age cohorts. Forty ribs were analyzed, ranging in age from 15 to 84. Ribs sections were digitized using a 40X objective on a Nikon E600 microscope equipped with a motorized stage and a Turboscan montaging system (Objective Imaging Inc., UK). Images were made binary using image ImageJ 2.0. Nine measurements and calculations as described by the Agnew and Stout (2012) method were used: total subperiosteal area, endosteal area, cortical area, percent cortical area (%C/T), porosity area, percent porosity area, absolute cortical area, percent absolute cortical area (%CA/T), and the difference between %C/T and %CA/T. The ranges and mean values for intracortical porosity across the sample were calculated. The effect of intracortical porosity on measures of cortical area were interpreted using a calculation of the difference between %C/T and %CA/T. A Two Sample Independent T-Test was performed to see if there was a significant difference between sexes. Regression analyses were conducted to assess correlations between the traits and age-at-death. No significant difference was found between sexes in regards to intracortical porosity or the difference between %C/T and %CA/T. The mean intracortical porosity was found to be 15.30. The mean value of the difference was found to be 6.95%. Measures of intracortical porosity varied from 0.74% - 31.67% while measures of the effect of intracortical porosity varied from 0.32% - 17.85%. No correlation was found between either trait and age-at-death. There are a number of reasons intracortical porosity might not be correlated with an individual’s age-at-death including (1) processes that influence intracortical porosity, (2) hormonal changes, (3) the current understanding of the relationship between aging and bone cell functions. One should still account for intracortical porosity when conducting histomorphometric analyses, as failure to consider it would result in miscalculations.

BACKGROUND: In applications on dental/orthopedic implants and bone regeneration, biomaterials contained magnesium have been widely used. However, the mechanism underlying the biologic effects is still largely unknown. In addition, previous reports of osteogenic effect of magnesium mainly relied on studies using ATCC osteosarcoma cell lines but not normal human osteoblasts. OBJECTIVE: This study was designed to test the effect of magnesium on osteogenic phenotypic behaviors of normal human osteoblasts. METHODS: Normal human osteoblasts derived from human alveolar bone were cultured in triplicate in growth media with varies concentrations of supplemental magnesium: 0.5mM, 1mM, 2mM, 4mM, 8mM and 16mM as the study groups and 0mM as a control group for the time intervals of 7 days, 10 days, 14 days and 21days. Cell proliferation was measured by crystal violet dye staining. Expression of osteocalcin was measured by Quantikine Elisa and mineralization of cultures was measured by Alizarin Red staining. The data were normalized per cell basis. Statistical analysis was done using ANOVA and Student’s t test. Results: Osteocalcin expression was upregulated in groups with supplemented magnesium at 0.5mM (1.16folds, p<0.01 ), 1.0mM (1.22folds, p<0.01 ), 2.0mM (1.37folds, p<0.01 ) at day 21 compared to control, while at 4mM ( p<0.01 ) and above showed down-regulation. Alizarin Red stained cultures showed higher degree of mineralization at 1mM ( p=0.0228 ) and 2mM ( p=0.0142) compared to control. Groups with 4mM and above showed less calcium deposition. Similar results have been gained also on day 10 and day 14 for both assays. CONCLUSION: Osteogenesis of normal human osteoblasts could be significantly upregulated by 2mM supplemental magnesium. These data are important for manufacturing magnesium-containing biomaterials for bone tissue regeneration and implants.

Electrical Muscle Stimulation (EMS) is used as a countermeasure in animal disuse studies that seek to determine which forms of exercise are most effective in mitigating the effects of disuse atrophy on bone and muscle. Although EMS has been used for many years in our lab and others, few researchers have been able to quantify the levels of strain on rat tibiae during EMS and far fewer have investigated the causal relationship between torque produced at the ankle and strain on the tibia. This thesis sought to investigate the relationship between ankle torque and tibial strain by using a combination of techniques, namely: (1) the addition of rosette strain gages, (2) improved synchronization between ankle torque and tibial strain recordings, and (3) spectral analysis between torque and strain waveforms. In previous work, few methods existed to align torque and strain recordings temporally, as those data were recorded on separate computers and synchronizing events were not captured. Attempting to create a torque-strain crossplot with unsynchronized data does not always yield valid results, so a method of reliably synchronizing those data is required. This thesis developed methods to capture simultaneous (synchronizing) events in both torque and strain recordings and then used those captured events to synchronize data between two computers. Following that synchronization, stiffness calculations were run on torque-strain crossplots to determine linear-model relationships between torque and strain for each method of synchronization. The results from those regressions were then used to determine if one or more synchronization techniques are superior to others, in terms of repeatability or precision. The results of these analyses have shown that using portions of the curves can dramatically increase computing speed while providing high levels of repeatability in synchronization measures. After synchronization techniques had been investigated, 3-element rosette data were used to calculate the principal strains on the surface of the tibiae, and the percentage of principal strains that are accounted for in the axial direction. Since the strain environment changes along the axis of the bone, the principal strain data were plotted versus the distance from proximal epiphysis to rosette gage, and statistical analysis was presented. After rosette data were analyzed, the torque and strain data pairs were fed into a signal processing suite for the purpose of transfer function calculation. Using the synchronization methods outlined above, two means of synchronization were compared in the transfer function program. Results for these analyses demonstrated that transfer functions are slightly dependent on synchronization methods, but that calculated gains do not differ between synchronization techniques. The specific shapes of the transfer functions highlight the relative attenuation/amplification of frequencies in torque and strain signals. Specifically, a range of frequencies, commonly called a band, between 24 and 32Hz is attenuated by the soft tissues and mechanical linkages in the lower leg of rats. This finding gives researchers looking to increase or decrease modeling stimulus to bone a new piece of information about the relative efficiency of EMS exercise. For example, EMS performed at 24-25Hz might produce less strain in the tibia than EMS at 22-23Hz, despite the 22-23Hz stimulation producing marginally less torque.

Bone resorption involves osteoclast-mediated acidification via a vacuolar type H+-ATPase (V-ATPase) found in lysosomes and at the ruffled border membrane. V-ATPases are proton pumps that include the a3 subunit, one of four isoforms (a1-a4) in mammals. The a3 isoform is enriched in osteoclasts where it is essential for bone resorption. Over 50% of humans with osteopetrosis have mutations in the a3 subunit and a3 mutations in mouse also result in osteopetrosis. A mouse founder with an osteopetrotic phenotype was identified in an N-ethyl-N-nitrosourea (ENU) mutagenesis screen. This mouse bears a dominant missense mutation in the Tcirg1 gene that encodes the a3 subunit resulting in the replacement of a highly conserved amino acid, arginine 740, with serine (R740S). The heterozygous mice (+/R740S) exhibit high bone density but otherwise have a normal appearance, size and weight. Osteoblast parameters are unaffected whereas osteoclast number and marker expression are increased along with a decreased number of apoptotic osteoclasts. V-ATPases from +/R740S osteoclast membranes have severely reduced proton transport along with wild type levels of ATP hydrolysis, indicating that the R740S mutation uncouples ATP hydrolysis from proton transport. The mutation however has no effect on ruffled border formation or polarization of +/R740S osteoclasts. Mice homozygous for R740S (R740S/R740S) have more severe osteopetrosis than +/R740S mice and die by postnatal day 14. Similarly to the mouse models that lack the a3 subunit (oc/oc and Tcirg1-/-) R740S/R740S osteoclasts do not polarize and lack ruffled border membranes. However R740S/R740S osteoclasts exhibit unique phenotypic traits, including increased apoptosis and defective early stage autophagy. Intracellular and extracellular acidification is absent in R740S/R740S osteoclasts, providing evidence for a requirement for lysosomal acidification for cytoplasmic distribution of key osteoclast enzymes such as TRAP and other important osteoclast phenotypic traits. This work provides evidence that the a3 subunit of V-ATPases and the proton pumping function of a3-containing V-ATPases play a major role in osteoclast survival, maturation and function.

Bone resorption involves osteoclast-mediated acidification via a vacuolar type H+-ATPase (V-ATPase) found in lysosomes and at the ruffled border membrane. V-ATPases are proton pumps that include the a3 subunit, one of four isoforms (a1-a4) in mammals. The a3 isoform is enriched in osteoclasts where it is essential for bone resorption. Over 50% of humans with osteopetrosis have mutations in the a3 subunit and a3 mutations in mouse also result in osteopetrosis. A mouse founder with an osteopetrotic phenotype was identified in an N-ethyl-N-nitrosourea (ENU) mutagenesis screen. This mouse bears a dominant missense mutation in the Tcirg1 gene that encodes the a3 subunit resulting in the replacement of a highly conserved amino acid, arginine 740, with serine (R740S). The heterozygous mice (+/R740S) exhibit high bone density but otherwise have a normal appearance, size and weight. Osteoblast parameters are unaffected whereas osteoclast number and marker expression are increased along with a decreased number of apoptotic osteoclasts. V-ATPases from +/R740S osteoclast membranes have severely reduced proton transport along with wild type levels of ATP hydrolysis, indicating that the R740S mutation uncouples ATP hydrolysis from proton transport. The mutation however has no effect on ruffled border formation or polarization of +/R740S osteoclasts. Mice homozygous for R740S (R740S/R740S) have more severe osteopetrosis than +/R740S mice and die by postnatal day 14. Similarly to the mouse models that lack the a3 subunit (oc/oc and Tcirg1-/-) R740S/R740S osteoclasts do not polarize and lack ruffled border membranes. However R740S/R740S osteoclasts exhibit unique phenotypic traits, including increased apoptosis and defective early stage autophagy. Intracellular and extracellular acidification is absent in R740S/R740S osteoclasts, providing evidence for a requirement for lysosomal acidification for cytoplasmic distribution of key osteoclast enzymes such as TRAP and other important osteoclast phenotypic traits. This work provides evidence that the a3 subunit of V-ATPases and the proton pumping function of a3-containing V-ATPases play a major role in osteoclast survival, maturation and function.

Piezocision is a new periodontal method for cutting bone more precisely than conventional methods, such as bur drilling, with 3D ultrasonic vibration power. We have conducted a study on piezocision effect on periodontal tissue in rodents. Our previous animal studies demonstrated that piezocision hastens tooth movement in rodents compared to conventional methods. The histological results showed that piezocision induces bone resorption and regeneration quickly. In addition, we observed the same effect of piezocision surgery on clinical tooth movement in collaboration with the orthodontists at BUSDM. We believe that piezocision can contribute significantly to dental therapies. However, more studies of piezocision effects are necessary for a thorough understanding. Periodontal tissue healing requires the participation of regulatory molecules, cells, and scaffold or matrix. We hypothesized that piezosurgery induces alveolar bone regeneration by uncovered procedures. In this study, we focused on the cells contributing to synthesis or repair of periodontal tissue, such as osteocytes, osteoclasts, osteoblasts, white blood cells, and periodontal ligament cells in order to close the gap between clinical knowledge and cellular mechanisms Histological analysis and MRI data indicates that piezocision surgery enhanced alveolar bone degradation in the post-operative early phase (from day1 to day7), and induced bone regeneration in the post-operative mid phase (from day14 to day28). The structure of alveolar bone was similar to controls in the late phase (day70). Serum ALP activity, a bone formation marker, was significantly increased by Piezocision surgery (p<0.05). Piezocision increasd serum CTX, a bone degradation biomarker, at post-operative 7day in the 30Hz Piezocision surgery (p<0.05). In addition, Serum PINP, a bone formation biomarker, was significantly decreased in the post-operative early phase. TUNEL assays revealed that osteocyte apoptosis was induced in alveolar bone by piezocision at post-operative 1day. Apoptosis in osteocytes induces osteoclast activity that leads to bone degradation. In previous studies piezocision induced TRAP activity in the post-operative early phase. Runx2 positive osteoblast progenitor cells were observed in the post-operative day7 and 14 as assessed by Immunofluorescence microscopy analysis. The Runx2 positive cells accumulated near the new bone formation area. The structure of collagen was observed by histological staining with Pico Sirius Red. Piezocision resulted in deteriorated collagen structure in the post-operative early phase that recovered in the post-operative mid phase. Since the collagen fibers filled in the gap between alveolar bone and roots, we stained the sections with Periostin, a specific PDL biomarker. Periostin was observed on the collagen fibers that filled in the gap between bone and root. previous studies revealed that the expression of Periostin induced TGF beta, a pivotal molecule for osteoblast differentiation. Taken together, these studies indicate that periodontal tissue responds to Piezosurgery by alveolar bone decalcification and regeneration. Further elucidation of the role of the each conducting cells (eg. osteocytes, osteoclast, osteoblast, periodontal ligament cells) after piezosurgery in the periodontal tissue may provide a new target for the treatment of periodontal disease by stimulating the return to tissue homeostasis.

La vascularisation joue un rôle important dans la biologie de l’os. Les souris génétiquement modifiées sont devenues le modèle de choix dans ce champ de recherche ; cependant les outils permettant son exploration structurelle (réseau capillaire) et fonctionnelle (perfusion) dans ce modèle animal, demeurent imparfaits. Notre travail a consisté en une adaptation chez la souris de la technique d’histomorphométrie quantitative vasculaire des os longs, fondée sur le remplissage du réseau par du sulfate de baryum, précédemment développée chez le rat. Nous avons, par ailleurs, mis au point une technique reproductible de mesure de la perfusion osseuse dans le tibia en utilisant le Laser Doppler. En termes d’exploration structurelle, et grâce à une imagerie par microtomographie 3D (Synchrotron ou Nano scanner) nous montrons la supériorité du sulfate de baryum sur le Microfil®, (produit associant silicone et plomb) et sa compatibilité avec les colorations histologiques usuelles et les marquages immuno histochimiques de la paroi vasculaire. Nous établissons la reproductibilité de la technique et nous proposons une nomenclature. En termes de perfusion, après avoir optimisé le protocole en limitant les facteurs de variabilité, nous montrons que le Laser Doppler permet de mesurer de façon reproductible une perfusion strictement osseuse dans le tibia de souris. Sous réserve de recourir { des groupes d’animaux de taille suffisante, (n=15), il devient possible de réaliser des comparaisons intergroupes. Ces deux approches combinées et intégrées, menées sur des souris C57BL/6 et 129sv/Cd1 nous ont permis de montrer (1) qu’il n’y a pas de corrélation entre paramètres de perfusion et de densité vasculaire, (2) que lors du vieillissement chez les mâles, la cinétique d’évolution de la vascularisation osseuse diffère selon le fond génétique, et que la perte osseuse n’est pas accompagnée d’une diminution de la perfusion dans ce modèle, (3) que l’ovariectomie (OVX) induit une diminution de la vascularisation et de la perfusion osseuses qui précède la perte osseuse. Enfin, notre étude des effets vasculaires osseux structurels et fonctionnels de la parathormone 1-84 a montré qu’ils dépendent, à l’instar de ses effets sur la masse osseuse, de son mode d’administration, intermittent ou continu et qu’ils ne sont pas impactés par l’OVX / Vascularisation plays a major role in bone biology. Genetically modified mice became the most favorite model in this research field; however, the tools allowing bone vessel analysis on both structural (capillary network) and functional aspects (perfusion) in this animal model remain unsatisfactory. Our work consisted in adapting in mice the quantitative histomorphometry technique allowing the analysis of long bone vascularization, based on the infusion of barium sulfate, which was previously developed in the rat. In addition, we set up a reproducible Laser Doppler-Based technique for measurement of mouse tibia perfusion. In terms of structural analyses, and thank to 3D micro-Tomography imaging (Synchrotron or Nano scanner), we show the superiority of barium sulfate on a lead/silicon-Based contrast product (Microfil®)and its compatibility with common staining used in histology and with immuno-Histochemsitry of the vessel wall. We establish the reproducibility of the technique and propose a nomenclature. In terms of perfusion, we show, after having optimized the protocol by limiting the factors of variability, that Laser Doppler allows to measure, in a reproducible way, perfusion signals specific to bone. As long as the animal group size is appropriate (n=15), it becomes possible to carry out intergroup comparisons. These combined and integrated approaches, carried out on C57BL/6 and 129sv/Cd1 mice allowed us to show that (1) there is no correlation between perfusion and vascular density parameters (2) during ageing in males, the kinetics of bone vascularisation evolution differ according to the genetic background and that bone loss was not associated with decrease in perfusion in this model (3) that ovariectomy (OVX) induces a decrease in both bone vessel density and perfusion which precedes bone loss. Finally, we studied the structural and functional vascular effects of 1-84 parathyroid hormone and show that, as for its effects on bone mass, they depend on its mode of administration, intermittent or continuous and that they are not affected by OVX

<p>Approximately 6.3 million bone fractures occur annually in the USA, resulting in considerable morbidity, deterioration in quality of life, loss of productivity and wages, and sometimes death (e.g. hip fractures). Although anabolic and antiresorptive agents have been introduced for treatment of osteoporosis, no systemically-administered drug has been developed to accelerate the fracture healing process. To address this need, we have undertaken to target a bone anabolic agent selectively to fracture surfaces in order to concentrate the drug’s healing power directly on the fracture site. We report here that conjugation of dasatinib to a bone fracture-homing oligopeptide via a releasable linker reduces fractured femur healing times in mice by ~60% without causing overt off-target toxicity or remodeling of nontraumatized bones. Thus, achievement of healthy bone density, normal bone volume, and healthy bone mechanical properties at the fracture site is realized after only 3-4 weeks in dasatinib-targeted mice, but requires ~8 weeks in PBS-treated controls. Moreover, optimizations have been implemented to the dosing regimen and releasing mechanisms of this targeted-dasatinib therapy, which has enabled us to cut the total doses by half, reduce the risk of premature release in circulation, and still improve upon the therapeutic efficacy. These efforts might reduce the burden associated with frequent doses on patients with broken bones and lower potential toxicity brought by drug degradation in the blood stream. In addition to dasatinib, a few other small molecules have also been targeted to fracture surfaces and identified as prospective therapeutic agents for the acceleration of fracture repair. In conclusion, in this dissertation, we have successfully targeted dasatinib to bone fracture surfaces, which can significantly accelerate the healing process at dasatinib concentrations that are known to be safe in oncological applications. A modular synthetic method has also been developed to allow for easy conversion of a bone-anabolic warhead into a fracture-targeted version for improved fracture repair.</p><p></p>