Indiana University-Purdue University Indianapolis (IUPUI) / The hormonal and cellular mechanisms controlling bone formation are not completely understood. The proline-rich tyrosine kinase 2 (Pyk2) is important for osteoblast (OB) activity and bone formation. However, female mice lacking Pyk2 (Pyk2-KO) exhibit elevated bone volume/total volume. Previously, our laboratory found ovariectomized Pyk2-KO mice supplemented with 17β-estradiol (E2) exhibited a greater increase in bone volume than WT mice treated with E2. The overall hypotheses of our studies are that Pyk2 regulates OB activity by modulating the E2-signaling cascade and that a Pyk2-inhibitor will promote OB activity and be suitable for bone regeneration applications. In Aim1, we determined the mechanism of action of Pyk2 and E2 in OBs. Pyk2-KO OBs showed significantly higher proliferation, matrix formation, and mineralization than WT OBs. In the presence of E2 or raloxifene, a selective estrogen receptor (ER) modulator, both matrix formation and mineralization were further increased in Pyk2-KO OBs, but not WT OBs. Consistent with a role of Pyk2 in E2 signaling, Pyk2-depletion led to the proteasome-mediated degradation of ERα, but not ERβ. Finally, we found Pyk2-depletion and E2 have an additive effect on ERK phosphorylation, known to increase cell differentiation and survival. In Aim2, we developed a Pyk2-inhibitor loaded hydrogel and evaluated its viscosity, gelation time, swelling, degradation, and release behavior. We found that a hydrogel composed of PEGDA1000 plus 10% gelatin exhibited viscosity and shear-thinning behavior suitable for use as an injectable-carrier. Importantly, the Pyk2-inhibitor-hydrogel was cytocompatible, retained its inhibitory activity against Pyk2 leading to an increase in OB activity. In conclusion, therapeutic strategies targeting Pyk2 may improve systemic bone formation, while Pyk2-inhibitor loaded hydrogels may be suitable for targeted bone regeneration in craniofacial and/or the other skeletal defects.

Indiana University-Purdue University Indianapolis (IUPUI) / The Keap1-Nrf2 pathway regulates a wide range of cytoprotective genes, and has been found to serve a protective and beneficial role in many body systems. There is limited information available, however, about its role in bone homeostasis. While Nrf2 activation has been suggested as an effective method of increasing bone mass and quality, there have been conflicting reports which associate Keap1 deficiency with detrimental phenotypes. As Keap1 deletion is a common method of Nrf2 activation, further study should address the impacts of various methods of regulating Nrf2 expression. Also, little research has been conducted on the specific pathways by which Nrf2 activation improves bone quality. In this study, the effects of alterations to Nrf2 activation levels were explored in two specific and varied scenarios. In the first experiment, moderate Nrf2 activation was achieved via partial deletion of its sequestering protein, Keap1, in an aging mouse model. The hypothesis tested here is that moderate Nrf2 activation improves bone quality by affecting bone metabolism and response to mechanical loading. The results of this first experiment suggest a subtle, sex-specific effect of moderate Nrf2 activation in aging mice which improves specific indices of bone quality to varying degrees, but does not affect loading-induced bone formation. It is likely that the overwhelming phenotypic impacts associated with aging or the systemic effects of global Keap1 deficiency may increase the difficulty in parsing out significant effects that can be attributed solely to Nrf2 activation. In the second experiment, a cell-specific knockout of Nrf2 in the osteocytes was achieved using a Cre/Lox breeding system. The hypothesis tested here is that osteocyte-specific deletion of Nrf2 impairs bone quality by affecting bone metabolism and response to mechanical loading. The results of this experiment suggest an important role of Nrf2 in osteocyte function which improves certain indices of bone quality, which impacts male and female bones in different 7 ways, but did not significantly impact loading-induced bone formation. Further studies should modify the method of Nrf2 activation in an effort to refine the animal model, allowing the effects of Nrf2 to be isolated from the potential systemic effects of Keap1 deletion. Future studies should also utilize other conditional knockout models to elucidate the effects of Nrf2 in other specific cell types.

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第21673号 / 医博第4479号 / 新制||医||1036(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 松田 道行, 教授 滝田 順子, 教授 戸口田 淳也 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

In the United States, osteoporosis results in about 1.5 million annual fractures, costing approximately $15 billion. Calcitonin is safe and effective in slowing osteoporotic bone loss, but its effect is transient. The current studies were designed to explore the dose-dependent effects of salmon calcitonin on bone turnover in ovariectomized rats and to determine if the decrease in therapeutic effectiveness of calcitonin demonstrated over time with higher doses is due to oversuppression of bone turnover. Doses of 5, 15, & 50 IU/kg BW/day of calcitonin were compared to placebo in 12-week-old ovariectomized and sham-ovariectomized Sprague-Dawley rats for 24 weeks. The spinal bone mineral content (BMC) as measured by DXA in ovariectomized subjects receiving 5 & 15 IU/kg of calcitonin was not significantly different from sham-ovariectomized subjects, while spinal BMC of subjects receiving 50 IU/kg was significantly lower than shamovariectomized subjects (p<0.05). Femoral BMC of ovariectomized subjects was significantly lower than sham-ovariectomized subjects (p<0.05), but no significant differences were noted between treatment groups. Scanning electron microscopy (SEM) demonstrated a decrease in number and density of trabeculae and in cortical thickness when comparing femurs from ovariectomized with sham-ovariectomized subjects. SEM of subjects receiving 50 IU/kg displayed greater bone loss than other groups. No significant differences were noted between groups for levels of urinary helical peptides or serum pyridinoline [ELISA], indicators of bone resorption. Urinary calcium excretion [capillary ion electrophoresis] was significantly higher in subjects receiving 50 IU/kg of calcitonin than other ovariectomized subjects (p<0.05). Serum levels of osteocalcin [RIA], an indicator of bone formation, were significantly higher in subjects receiving 5 IU/kg of calcitonin than control subjects and those receiving 50 IU/kg (p<0.05). Production of antibodies to calcitonin [ELISA] by subjects in this study did not correlate with changes in bone turnover or bone density. The results of this study do not provide evidence higher doses of calcitonin result in oversuppression of bone turnover. However, urinary calcium excretion affected bone resorption in a reverse dose-dependent manner, suggesting the calciuric effect may be responsible for less effective outcomes seen with higher doses of calcitonin.

No description available.

Imatinib mesylate has been associated with the changes in bone turnover. We report a case of the development of tissue calcification in a patient on long-term therapy with this drug. A 48-year-old male patient with gastrointestinal stromal tumor and liver metastasis complained of abdominal pain. His treatment included hepatic artery chemoembolization and partial hepatectomy in addition to chronic imatinib mesylate for 4 years. On physical examination, he had a peritoneal mass just beneath the laparotomy incision scar that, after resection, was found to be dystrophic bone formation. Based on the previous studies suggesting bone changes due to chronic therapy with imatinib mesylate, we believe that the patient's new bone formation was causally related to the use of this drug. To our knowledge, there are no similar reported cases in the literature.

[Truncated abstract] Intracellular communication between osteoclasts and osteoblasts is imperative to maintain bone integrity. A myriad of molecules are responsible for regulating osteoblast and osteoclast activity. In particular, it is well documented that osteoblast-derived factors are crucial in directly controlling osteoclast formation and function. Since bone formation is coupled to bone resorption, it would be expected that osteoclasts also have some role in regulating the growth and function of osteoblast cells. However, despite extensive research upon osteoclast and osteoblast biology, the mechanisms by which osteoclasts regulate osteoblast growth and function is not well understood. In an attempt to further elucidate the mechanisms by which osteoclasts and osteoblasts communicate, the technique of subtractive hybridisation was used to identify a novel osteoclastderived factor identical to that of mouse Seminal Vesicle Secretion VII (SVS VII). Previous characterisation of the gene in bone demonstrated that SVS VII was abundantly and specifically expressed by mature osteoclasts (Phan, 2004). Additional research hinted that SVS VII acted as a novel osteoclast-derived factor, that by paracrine mechanisms, targeted osteoblast function (Phan, 2004). However, it remained open as to whether the SVS VII molecule did uniquely target the osteoblast, and whether this interaction influenced bone formation in vivo. Therefore, this thesis endeavoured to functionally characterise the role of the SVS VII molecule in the bone environment. ... Further work is needed to identigy a clear consensus binding sequence, to determine the specificity of the interaction between SVS VII protein and each phage clone, and to isolate a specific binding partner for SVS VII. In conclusion, the studies of this thesis sought to characterise the significance of SVS VII expression by mature osteoclasts, relative to its effects on osteoblast behaviour, but failed to conclusively determine a role for SVS VII in bone. Given that the effects of SVS VII on in vitro osteoblast activity and function are minimal, it is doubtful that SVS VII primarily acts as a paracrine factor integral to osteoblast function. Therefore, these findings conflict with those presented previously (Phan, 2004). However, it was demonstrated that SVS VII treatment was associated with in vivo effect on the skeleton, suggesting that SVS VII may target other elements of the bone microenvironment. Via mechanisms not yet understood, which possibly involves additional factors of the bone 11 extracellular matrix, SVS VII may target a subset of osteoprogenitor cells within the bone environment and act to regulate their proliferation. Therefore, SVS VII may enhance osteogenic precursor cell number at sites of bone formation which would increase the pool of cells that can differentiate down the osteoblast linage and contribute to bone formation. In this regard, SVS VII might function in a manner homologous to the Ly-6 molecule Sca-1 and act as an important factor that maintains a balance between the bone formation and resorption process. Clearly, more work focusing on alternative facets of bone biology is needed to identify whether there is a significant role for SVS VII in skeletal tissue.

<p>In this thesis, occurrence of heterotopic ossification (HO) following total hip arthroplasty (THA) was studied. Preventive effects and complications with non-steroidal anti-inflammatory drugs (NSAIDs) were analyzed. Experimental investigations on bone formation were employed to gain insight to the mechanism of NSAIDs action on bone.</p><p>(I). Fifty-six patients with bilateral THAs were analyzed. We found a strong correlation between HO on the two sides. Incidence and grade of HO were higher in men than in women.</p><p>(II). Sixty-nine patients with bilateral THAs who had been treated with NSAIDs after one or both THAs were analyzed for HO. Widespread HO occurred in untreated THAs, but in none of the treated THAs.</p><p>(III). A consecutive series of THAs were analyzed for HO. No widespread HO occurred in patients treated with NSAIDs for 21 days. In contrast, widespread HO occurred in 23% of patients not treated.</p><p>(IV). A randomized, double-blind, prospective study on 144 patients was performed to determine the efficacy and minimum treatment time with Ibuprofen for prophylaxis of HO after THA. Treatment with Ibuprofen was effective for preventing HO and a treatment time of 8 days was sufficient.</p><p>(V). A ten-year follow-up examination was performed on the patients from study IV. Thirteen patients had been revised. All but one belonged to groups treated with Ibuprofen. However, the prosthetic survival time was not statistically different for patients treated with NSAIDs compared to the control group. Eighty-four more patients underwent radiographic examination10 years after THA. Nine loose prostheses were found. These were equally distributed between NSAIDs-treated and non-treated THAs. When combining complications (revisions and radiographic loosening) no significant effects could be verified.</p><p>(VI). Experimental induction of heterotopic new bone with demineralized allogeneic bone matrix (DABM) and with bone autografts, was used in rats to study effects of NSAIDs on new bone formation. Indomethacin inhibited net bone formation in DABMs and in orthotopic fractured bone. In contrast, a net mineral loss occurred in autografts, but neither mineral content nor ⁴⁵Ca incorporation was affected by Indomethacin treatment. The amount of bone formed per mg implanted DABM was linearly correlated to implant size.</p>

In this thesis, occurrence of heterotopic ossification (HO) following total hip arthroplasty (THA) was studied. Preventive effects and complications with non-steroidal anti-inflammatory drugs (NSAIDs) were analyzed. Experimental investigations on bone formation were employed to gain insight to the mechanism of NSAIDs action on bone. (I). Fifty-six patients with bilateral THAs were analyzed. We found a strong correlation between HO on the two sides. Incidence and grade of HO were higher in men than in women. (II). Sixty-nine patients with bilateral THAs who had been treated with NSAIDs after one or both THAs were analyzed for HO. Widespread HO occurred in untreated THAs, but in none of the treated THAs. (III). A consecutive series of THAs were analyzed for HO. No widespread HO occurred in patients treated with NSAIDs for 21 days. In contrast, widespread HO occurred in 23% of patients not treated. (IV). A randomized, double-blind, prospective study on 144 patients was performed to determine the efficacy and minimum treatment time with Ibuprofen for prophylaxis of HO after THA. Treatment with Ibuprofen was effective for preventing HO and a treatment time of 8 days was sufficient. (V). A ten-year follow-up examination was performed on the patients from study IV. Thirteen patients had been revised. All but one belonged to groups treated with Ibuprofen. However, the prosthetic survival time was not statistically different for patients treated with NSAIDs compared to the control group. Eighty-four more patients underwent radiographic examination10 years after THA. Nine loose prostheses were found. These were equally distributed between NSAIDs-treated and non-treated THAs. When combining complications (revisions and radiographic loosening) no significant effects could be verified. (VI). Experimental induction of heterotopic new bone with demineralized allogeneic bone matrix (DABM) and with bone autografts, was used in rats to study effects of NSAIDs on new bone formation. Indomethacin inhibited net bone formation in DABMs and in orthotopic fractured bone. In contrast, a net mineral loss occurred in autografts, but neither mineral content nor 45Ca incorporation was affected by Indomethacin treatment. The amount of bone formed per mg implanted DABM was linearly correlated to implant size.

Sammanfattning Syfte: Syftet med denna systematiska litteraturöversikt var att testa hypotesen att det inte är någon skillnad rörande lyckandefrekvensen gällande implantatbehandling vid sinuslyft med olika bentransplantatmaterial Material och metod: Studien baseras på sökningar i PubMed, Cochrane Library och Web of Science i kombination med en manuell granskning av relevanta publikationer. Inkluderade publikationer var prospektiva studier av ≥ 10 patienter och med en uppföljningstid på ≥ 1 år. Autologt bentransplantat från mandibel/maxilla, oorganiskt ben (Bio-Oss), en kombination av dessa två samt sinuslyft utan insättning av bentransplant utvärderades. Kvaliteten på varje publikation bedömas enligt kriterier baserade på en modifiering av STROBE-statment. Resultat: Sökningen resulterade i 818 titlar och 15 inkluderade publikationer relevanta enligt förutbestämda inklusionskriterier. En studie var en randomiserad kontrollerad studie (RCT) och en var en kontrollerad klinisk studie (CCT). De återstående 13 studierna var observationsstudier. Uppföljningen varierade mellan ett och nio år, och antalet patienter mellan 10 och 191. Sinuslyft utan insättning av bentransplantat hade en implantatöverlevnad på 97,7% -100% och studier rörande autologt bentransplantat 98,8%. Bio-Oss resulterade i 86,3% -98,1% överlevnad och en kombination av Bio-Oss och autologt ben resulterade i en överlevnad på 90,7%. Slutsats: Enligt denna undersökning fanns ingen skillnad i implantat överlevnad mellan de olika bentransplantaten. Beroende på studiernas karaktär behövs fler RCT och CCT studier, som analyserar implantatutfallet, involverar sinuslyft och olika bentransplantat rörande implantatöverlevnad och lyckandefrekvens. / AbstractAims: The objective was to test the hypothesis that there is no difference in implant treatment outcome using different bone graft material for sinus floor augmentation.Material and methods: This systematic review is based on searches in PubMed, the Cochrane Library and the Web of Science and a hand search of relevant publications. Autologous bone, anorganic bovine bone (Bio-Oss), a combination of these two and elevation of the Schneiderian membrane with no graft material was evaluated. The quality of each publication was assessed according to criteria based on the STROBE-statements.Results: The search provided 818 titles and 15 were found relevant according to the predetermined inclusion criteria. One study was a randomized controlled trial (RCT) and one was a controlled clinical trial (CCT). The remaining 13 studies were observation studies. The follow-up time varied between one and nine years and the number of patients between 10 and 191. Studies on elevation of the Schneiderian membrane with no bone graft material reported an implant survival rate between 97.7% and100% and studies on only autologous bone graft material 98.8%. Bio-Oss as bone graft material resulted in 86.3%-98.1% survival rate and a combination of Bio-Oss and autologous bone graft resulted in a survival rate of 90.7%.Conclusion: There was no difference in implant outcome of the different bone graft material. More studies designed as RCT and CCT, which analyze the implant outcome involving sinus floor augmentation and different bone graft material are needed to improve evidence on survival and success rate.