OBJECTIVE: To determine the effects of vitamin C and insulin on osteoblasts harvested from neonatal mouse calvaria. To determine the effects of experimental media (vitamin C and insulin and a combination) on the ex-vivo live bone organ culture model and explore the capacity of addition of osteoblasts to allow for bone formation within a critical defect. To use the chick chorioallantoic membrane (CAM) model to explore bone formation within critical bone defect. METHODS: Osteoblasts were harvested from neonatal mice were tested using four types of experimental media: control DMEM, media prepared with 150 μg/ml vitamin C, 10 nM media, or a combination of both vitamin C, insulin, and a combination of vitamin C and insulin media. Cell were cultured for 18 days at 37°C. Neutral red was done to identify cellular activity and silver nitrate to detect calcium deposits. Two types of scaffolds were inserted into the defect: collagen membrane scaffold and NuOss (xenograft) with collagen scaffold. After 30 days the samples were collected for histological analysis. Neonatal mouse calvaria were harvested and a 2mm critical defect made on each calvaria. Each calvaria received a scaffold of collagen or NuOss with or without osteoblasts with one of three experimental media within the CAM model. After 7 days, the amnion membrane of the egg was dropped and a window was made. The calvaria with the scaffold samples were placed on the amnion membrane. The eggs were incubated for 10 days then the experiment was terminated. Calvaria were collected and processed for histological evaluation. RESULTS: Neutral red and silver nitrate of 2D in-vitro cells revealed calcium deposits in culture well using vitamin C media, cell cultured with insulin media showed calcium deposits and cell morphological change, and cell cultured with a combination of vitamin C and insulin media showed the most calcium deposits and morphological changed. Ex-Vivo samples with collagen scaffold had bone thickening but not enough nutrients for bone regeneration, despite the addition of cells. The collagen scaffold is a more suitable material than xenograft due to particle size. The CAM model showed new bone formation and new vessels were most abundant in areas closest to lining cells in collagen samples. Samples with additional osteoblasts added showed greater results. NuOss scaffold samples did not show the same bone formation or vessel growth. CONCLUSIONS: The results indicate and confirm the basic principles of tissue engineering. In order to have bone regeneration more cells allow for better results. The quality of the scaffold is important and should have stability as well as enough space for cellular migration and recruitment for new blood vessel to support regeneration of bone to its original state.

Pregnancy induces a dramatic change in hemodynamics due to increased blood volume and metabolic demands. The adaptation of the heart leads to physiological cardiac hypertrophy remodeling in healthy individuals during pregnancy. Metabolic syndrome (MetS) is known to predispose individuals to adverse cardiovascular event. Cardiac remodeling during pregnancy in obese individuals with or without MetS remains unclear. This study first observed differences in cardiac remodeling in human patients with excess weight during pregnancy. The pathophysiology of cardiac remodeling with pregnancy was then studied in a diet-induced animal model that recapitulates features of human MetS. Female mice fed with high fat diet (HFD) (45%kcal) for 4 months had increased body weight, impaired glucose tolerance and dyslipidemia. Pregnant female mice were kept on this HFD and were compared to nonpregnant females and normal diet (10%kcal fat) controls. HFD induced early-stage MetS led to cardiac hypertrophy at term that had features of pathological hypertrophy (PH), including fibrosis and upregulation of fetal genes associated with PH. Hearts from pregnant animals on the HFD had a distinct gene expression profile that likely underlies their pathological remodeling. Post-partum mice with preexisting MetS are also more susceptible to future pathological stimuli, with exacerbated cardiac hypertrophy and impaired cardiac function. These results suggested that preexisting MetS could change physiological into pathological cardiac remodeling during pregnancy, and predispose the heart to future cardiovascular risks. / Biomedical Sciences

Ischemic heart disease (IHD) accounts for the majority of heart disease-related deaths worldwide. Ubiquitin (UB), found in all eukaryotic cells, is a highly conserved low molecular weight (~8.5 kDa) protein. A well-known intracellular function of UB is to regulate protein turnover via the UB-proteasome system. UB is a normal constituent of plasma, and elevated levels of UB are observed in the serum of patients under a variety of pathological conditions. Recent studies provide evidence for cardioprotective potential of exogenous UB in the remodeling process of the heart in IHD, including effects on cardiac myocyte apoptosis, inflammatory response, and reorganization of the vasculature and extracellular matrix. This review summarizes functions of UB with an emphasis on the role of exogenous UB in myocardial remodeling in IHD.

Purpose: Determination of genotypic/phenotypic features of GATAD2B-associated neurodevelopmental disorder(GAND). Methods: Fifty GAND subjects were evaluated to determine consistentgenotypic/phenotypic features. Immunoprecipitation assays utilizing in vitrotranscription–translation products were used to evaluate GATAD2B missensevariants’ ability to interact with binding partners within the nucleosomeremodeling and deacetylase (NuRD) complex. Results: Subjects had clinical findings that included macrocephaly,hypotonia, intellectual disability, neonatal feeding issues, polyhydramnios,apraxia of speech, epilepsy, and bicuspid aortic valves. Forty-one novelGATAD2B variants were identified withmultiple variant types (nonsense, truncating frameshift, splice-site variants,deletions, and missense). Seven subjects were identified with missense variantsthat localized within two conserved region domains (CR1 or CR2) of the GATAD2Bprotein. Immunoprecipitation assays revealed several of these missense variantsdisrupted GATAD2B interactions with its NuRD complex binding partners. Conclusions: A consistent GAND phenotype was caused by a range of geneticvariants in GATAD2B that includeloss-of-function and missense subtypes. Missense variants were present inconserved region domains that disrupted assembly of NuRD complex proteins.GAND’s clinical phenotype had substantial clinical overlap with other disordersassociated with the NuRD complex that involve CHD3 and CHD4, with clinicalfeatures of hypotonia, intellectual disability, cardiac defects, childhoodapraxia of speech, and macrocephaly.

Peripheral arterial occlusive disease (PAOD) is an ischemic disease characterized by narrowing of the peripheral arteries due to the accumulation of atherosclerotic plaque in the inner lining of the vessels, which disrupts blood flow to downstream tissues. Blood can be redirected into collateral vessels, natural bypasses around arterial occlusions, causing shear-induced outward remodeling of the vessels. The enlarged vessels facilitate transfer of increased blood flow to downstream tissues. The remodeling process, however, may impair vasodilation, which in turn may cause or contribute to intermittent claudication- transient pain brought on by locomotion. To stimulate the growth of collateral arteries, the femoral arteries of young, otherwise healthy mice were ligated distally to the profunda femoris, the stem to the gracilis collateral circuit. The diameter of the profunda femoris artery was measured at rest and following gracilis muscle contraction 7 and 28 days post-surgery using intravital microscopy. Enlarged resting diameter, consistent with collateral enlargement, and impaired vasodilation was observed at day 7, but not at day 28. To determine if impaired functional vasodilation is due to impaired endothelial- or smooth muscle-dependent responses during outward remodeling, cell-dependent vasodilators were applied to the hindlimb. Endothelial- and smooth muscle-dependent vasodilation was significantly impaired 7 days post-ligation, but not 28 days after. This data supports the hypothesis that smooth muscle dysfunction causes impaired functional vasodilation in the early stages of collateral enlargement.

ABSTRACT A Study of Osteocyte Apoptosis by Region and Quadrant in Murine Cortical Bone Jessica Kristen Chan Osteocytes undergo apoptosis to spatially and temporally initiate bone remodeling. This study investigates the distribution of apoptotic osteocytes within different quadrants and regions of cortical bone and compares the frequency of osteocyte apoptosis to regional factors associated with bone remodeling. Specifically, the quantity of apoptosis was compared to levels of the bone morphagenic protein antagonists noggin and gremlin. Samples of unloaded right tibial bone obtained from C57/Bl/6 mice underwent TUNEL staining for apoptotic osteocytes and were counterstained with methyl green to detect osteocyte viability. Cross sectional areas of bone were divided into four quadrants (cranial, caudal, medial, and lateral) and three regions (proximal, midshaft, and distal) for analysis. Densities and percentages of osteocytes were measured within each area. While the results show that there were no differences among quadrants, regional variations were found in osteocyte apoptosis. A significantly higher density of apoptotic osteocytes was found in the midshaft region which also displayed higher levels of BMP antagonists. Using regression analysis, a positive linear relationship between apoptotic osteocytes and gremlin was established while noggin showed a negative linear correlation for the percentage of apoptotic osteocytes. Further studies are needed to observe the distribution of apoptotic osteocytes within loaded bone to confirm the exact relationship between osteocyte apoptosis and bone remodeling. Keywords: osteocyte apoptosis, bone remodeling

The purpose of this study is to characterize the compact bone remodeling of the ovariectomized ewe and its effectiveness as an animal model for studies of postmenopausal osteoporosis. The use of animal models is a beneficial way to evaluate the effectiveness of medical devices and therapeutic agents for treating diseases. The ovine model is an attractive option because of its large size and similar bone remodeling to humans. However, this species, like most animals, does not undergo a natural menopause, making an ovariectomy necessary for studies of estrogen depletion. The OVX (ovariectomized) sheep has been widely accepted as a model for loss of BMD. However, even with its advantages seasonality has been shown to have an effect on the bone remodeling in the ovine model. Thus, this study focused on characterizing the seasonal and anatomical variation in the compact bone remodeling in the ovine model. In doing so, 28 skeletally mature Columbia-Rambouillet cross ewes underwent an ovariectomy. The animals were divided into groups of 7 based on each season: autumn, winter, spring, and summer. During its specified season each group of 7 underwent ovariectomy and then was sacrificed 12 months post-surgery. The radii and ulnae were harvested and then divided into 6 anatomical locations: craniolateral, cranial, craniomedial, craniolateral, caudal, and caudomedial. This allowed for analysis through the fabrication of microradiographs. Histomorphometric analysis involved measurements to quantify the bone volume to tissue volume ratio, the percent of tissue and material remodeling, the mean secondary osteonal radius, and number of tissue and material cement line interfaces. Densitometry analysis was then performed to determine the density of each sector relative to an aluminum step wedge, serving as the key. Using a 2-way repeated measures ANOVA statistical analysis was performed to evaluate for seasonal and anatomical differences as well as a possible interaction between season and anatomical region. The OVX data showed significant seasonal and anatomical differences as well as seasonality within anatomical section in the remodeling parameters. When this data was combined with earlier data from a control, sham-cohort from the same experiment a 3-way ANOVA was also performed to evaluate the effects of season, anatomical sector, and treatment on the remodeling parameters. Again, there was significant seasonal, anatomical, and treatment differences, as well as interactions of all three. The results of this study showed that varying levels of remodeling occurs in the adult OVX ovine model and differences between the control and OVX model can be attributed to seasonal and anatomical variations. Thus, it will be an important consideration when developing new test protocols for research incorporating the sheep model for studying osteoporosis.

The onset of osteoporosis caused by aging, disease states, and post-menopausal conditions significantly impacts patient quality of life, required healthcare funding, personal autonomy losses from increased fracture risk and the subsequent corrective surgery. Research has indicated that osteocyte apoptosis may be a key parameter in bone remodeling, raising the possibility of remodeling rate modulation for the mitigation of bone mass resorption. By developing therapies that target osteocyte apoptosis, it may be possible to prevent undesired bone remodeling activity while maintaining a healthy balance between damage formation in the form of microcracks induced by the strain environment and the removal of damaged bone facilitated by resorbing BMUs, resulting in the preservation of bone mass. This preservation in turn results in a decreased risk of bone fracture. The purpose of the current study was to develop a computational model to predict bone density changes from empirical osteocyte apoptosis data. This was achieved by developing a simulation that correlates osteocyte apoptosis with activation frequency and mechanical stimulus, two key bone remodeling parameters. The results of the simulation show that it is possible to use osteocyte apoptosis to predict remodeling and the resulting bone porosity changes. Instances of increased osteocyte apoptosis occurred simultaneously with increases in porosity, and under-loaded force conditions were more deleterious to bone porosity then overload associated with heavy exercise, which agrees with previous models.

京都大学 / 新制・課程博士 / 博士(医学) / 甲第24199号 / 医博第4893号 / 新制||医||1060(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 武藤 学, 教授 斎藤 通紀, 教授 浅野 雅秀 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

Indiana University-Purdue University Indianapolis (IUPUI) / Bisphosphonates, drugs which inhibit bone resorption and remodeling, are currently prescribed for the treatment of osteoporosis. Previous research suggests that decreased bone turnover may lead to accumulation of microdamage, possibly increasing the risk for fracture in some sites. The effects of bisphosphonate therapy on the mandibular condyle have not been quantitatively studied. The purpose of the proposed study was to histomorphometrically quantify the effects of etidronate (a bisphosphonate) on trabecular bone sites of the dog mandibular condyle and to compare this to another trabecular bone site (vertebrae) to determine whether the two sites were affected differently. Eleven mature female dogs were treated with high- (5 mg/kg/d) and low- (0.5 mg/kg/d) dose etidronate therapy for seven months. Fluorochrome labels were used to mark sites of bone mineralization for the calculation of static and dynamic histomorphometric parameters. High-dose therapy resulted in a complete inhibition of remodeling, as shown by the reduction of mineral apposition rate (MAR), bone formation rate (BFR), and mineralizing surface (MS/BS) to zero. Low-dose therapy also decreased BFR and MS/BS. Osteoid accumulation was only significant in the high-dose therapy group, but there was no evidence of osteomalacia (osteoid volume < 5%). Etidronate treatment had no significant effect on bone volume, trabecular number, trabecular thickness, or trabecular separation. Vertebral trabeculae ranged from 5.5 to 9.5 times greater in number than mandibular trabeculae, but were 45 to 60 percent thinner and closer together. The interaction between dosage and site was insignificant for all parameters studied. Further investigation is needed to determine whether these effects will prove to be harmful to the mandibular condyle, especially over a long period of time.