No description available.

No description available.

No description available.

No description available.

No description available.

Indiana University-Purdue University Indianapolis (IUPUI) / Background: Suture expansion is a very important clinical approach to correct maxillary width deficiency, but it has a high potential for treatment relapse. Accelerating bone formation and mineralization in the midpalatal suture during suture expansion is beneficial in preventing relapse of the arch width and reducing the retention period. Pyk2 is tyrosine kinase which has been shown to mediate signaling pathways that are involved in the process of bone remodeling. Pyk2 knock-out (KO) mice have augmented bone formation and increased bone mass, suggesting that therapeutic strategies that inhibit Pyk2 may be useful to enhance bone remodeling and prevent suture relapse during suture expansion. Objectives: To determine if Pyk2-deficiency affects midpalatal suture bone mass and bone remodeling with or without suture expansion in mice. Methods: Thirty-six Pyk2-KO and thirty-six wild type (WT) 6 week-old male mice were randomly assigned into three groups: receiving no expansion force (0 g), 10 g or 20 g force of rapid maxillary expansion for 14 days. Half of the mice in each group were used for histology analysis; the other half was assigned for fluorescence analysis. Suture width, maxilla width and bone volume/tissue volume around suture bone edges were measured using micro-CT. Histological analyses of osteoclasts (tartrate resistant acid phosphatase, TRAP), osteoblasts (alkaline phosphatase, ALP) and chondrocytes (alcian blue) were performed. Results: The BV/TV ratio was significantly higher in Pyk2-KO control mice compared to WT control mice. Suture expansion in WT and Pyk2-KO mice led to an increase in bone marrow spaces around the suture edge and significantly reduced BV/TV. Expansion also led to a significant increase in suture width, suture fibrous area, osteoclast number, cartilage area and hypertrophic chondrocyte number. However, BV/TV in Pyk2-KO mice was significantly higher than in WT mice at both the 10 g and 20 g force levels. In addition, Pyk2-KO exhibited reduced suture width, maxilla width, fibrous area and osteoclast number per bone surface (OC.S/BS) compared to WT mice under expansion forces. Cartilage area and hypertrophic chondrocyte number were increased by force but were independent of mouse genotypes. Conclusion: Pyk2-KO mice have higher BV/TV and narrower suture width compared to WT mice, which may be due to decreased osteoclast activity. The higher BV/TV of the midpalatal sutures of Pyk2-KO mice following suture expansion may suggest the presence of a more stable suture that has a reduced potential for relapse. Therapeutic strategies to inhibit Pyk2 during RME may be beneficial in increasing bone mass and preventing relapse of the suture.

Resident tissue macrophages are an integral component of many tissues and are important in development, homeostasis and repair. Macrophages are present at sites of both pathologic bone deposition and loss, and can produce osteo-active factors. These observations link macrophages to bone disease, however their contribution to bone dynamics is poorly understood. The molecular and cellular mechanisms driving osteoblast differentiation, matrix deposition and mineralization in vivo are incompletely understood and this deficiency is translated to limited ability to clinically manipulate bone formation. The emerging understanding of the bi-directional interactions between the osseus and immune systems (osteoimmunology) provides a novel avenue to identify mechanisms involved in the regulation of bone formation. In this study, the presence and distribution of macrophages on bone surfaces was systematically analysed and their functional contribution to the bone microenvironment was investigated. Using immunohistochemistry a discrete population of mature resident tissue macrophages was demonstrated throughout resting murine osteal tissues, termed OsteoMacs. Utilising MacGreen mice (csf1r promoter drives eGFP transgene expression in macrophages and other myeloid cells), it was demonstrated that OsteoMacs were intercalated amongst other bone lining cells in both the endosteum and periosteum. OsteoMacs were TRAPneg in situ and had limited osteoclastogenic ability in vitro therefore they are unlikely to serve as the immediate physiologic osteoclast precursors in vivo. Microarray gene expression profiling demonstrated that macrophage gene expression was regulated in response to a characteristic feature of the bone microenvironment, elevated extracellular calcium. Quantitative PCR validated upregulation of sphingosine kinase 1, interleukin 1 receptor antagonise, progressive ankylosis, vascular endothelial growth factor c and dipepetidase 2 mRNA in response to elevated extracellular calcium, suggesting the potential roles of these genes in this unique niche. GNF Symatlas microarray and quantitative PCR demonstrated the expression of macrophage-restricted genes throughout a 21-day primary osteoblast differentiation time course, suggesting co-isolation of OsteoMacs with primary osteoblasts. Flow cytometry analysis confirmed that over all 15.9% of the digested primary calvarial cell preparations were OsteoMacs. Immunocytochemistry demonstrated that OsteoMacs persisted and expanded in standard 21-day osteoblast differentiation assays. Contrary to previous studies, we demonstrated it was the OsteoMacs, and not osteoblasts, within calvarial preparations that selectively detected patho-physiological concentrations of the bacterial product lipopolysaccharide (LPS). A protocol was developed to deplete OsteoMacs from calvarial digests to determine if their presence within these cultures facilitates osteoblast differentiation or function. OsteoMac removal did not affect expression of the early osteoblast differentiation marker genes collagen type I or alkaline phosphatase. However, OsteoMac removal significantly decreased gene expression of the osteoblast mineralisation marker osteocalcin and mineralisation function, assessed by von Kossa staining. Microarray gene expression profiling demonstrated that osteoblast enrichment had a broad impact on transcription within the culture, identifying both candidate OsteoMac marker genes as well as osteoblast expressed genes that are regulated by OsteoMacs. Potential OsteoMac-enriched candidate genes insulin-like growth factor a, dipepetidase 2, glycoprotein NMB, and macrophage expressed gene 1 as well as osteoblast-specific genes bone sialoprotein and thrombospondin 1 were selected based on their potential involvement in osteoblast function. In a transwell co-culture system of enriched osteoblasts and macrophages, it was demonstrated that macrophages were required for osteoblast mineralisation in response to the physiologic remodelling stimulus, elevated extracellular calcium. A blocking soluble receptor strategy provided evidence that this is mediated in a BMP-2 and -4 independent manner. To investigate the relevance of OsteoMacs to bone formation in vivo, immunohistochemistry staining for the mature tissue macrophage marker F4/80 was performed in long bone sections from rapidly growing mice. OsteoMacs were closely associated with areas of bone formation in situ, forming a distinctive canopy structure over mature cuboidal osteoblasts (collagen type I+, osteocalcin+) on endosteal cortical surfaces. Using adapted histomorphometic analysis, we determined that 77 ± 2.1% (n = 7) of the endosteal mature osteoblast surface was covered by the F4/80+ OsteoMac canopy. This observation suggested that OsteoMacs are optimally located to regulate osteoblast function in vivo. In summary, we have demonstrated that OsteoMacs are an integral component of bone lining tissues and play a novel role in bone dynamics through regulating osteoblast function. These observations implicate OsteoMacs, in addition to osteoclasts and osteoblasts, as principal participants in bone dynamics. Further delineation of OsteoMac functions is likely to provide new avenues for treating bone disease and assisting bone repair.

Resident tissue macrophages are an integral component of many tissues and are important in development, homeostasis and repair. Macrophages are present at sites of both pathologic bone deposition and loss, and can produce osteo-active factors. These observations link macrophages to bone disease, however their contribution to bone dynamics is poorly understood. The molecular and cellular mechanisms driving osteoblast differentiation, matrix deposition and mineralization in vivo are incompletely understood and this deficiency is translated to limited ability to clinically manipulate bone formation. The emerging understanding of the bi-directional interactions between the osseus and immune systems (osteoimmunology) provides a novel avenue to identify mechanisms involved in the regulation of bone formation. In this study, the presence and distribution of macrophages on bone surfaces was systematically analysed and their functional contribution to the bone microenvironment was investigated. Using immunohistochemistry a discrete population of mature resident tissue macrophages was demonstrated throughout resting murine osteal tissues, termed OsteoMacs. Utilising MacGreen mice (csf1r promoter drives eGFP transgene expression in macrophages and other myeloid cells), it was demonstrated that OsteoMacs were intercalated amongst other bone lining cells in both the endosteum and periosteum. OsteoMacs were TRAPneg in situ and had limited osteoclastogenic ability in vitro therefore they are unlikely to serve as the immediate physiologic osteoclast precursors in vivo. Microarray gene expression profiling demonstrated that macrophage gene expression was regulated in response to a characteristic feature of the bone microenvironment, elevated extracellular calcium. Quantitative PCR validated upregulation of sphingosine kinase 1, interleukin 1 receptor antagonise, progressive ankylosis, vascular endothelial growth factor c and dipepetidase 2 mRNA in response to elevated extracellular calcium, suggesting the potential roles of these genes in this unique niche. GNF Symatlas microarray and quantitative PCR demonstrated the expression of macrophage-restricted genes throughout a 21-day primary osteoblast differentiation time course, suggesting co-isolation of OsteoMacs with primary osteoblasts. Flow cytometry analysis confirmed that over all 15.9% of the digested primary calvarial cell preparations were OsteoMacs. Immunocytochemistry demonstrated that OsteoMacs persisted and expanded in standard 21-day osteoblast differentiation assays. Contrary to previous studies, we demonstrated it was the OsteoMacs, and not osteoblasts, within calvarial preparations that selectively detected patho-physiological concentrations of the bacterial product lipopolysaccharide (LPS). A protocol was developed to deplete OsteoMacs from calvarial digests to determine if their presence within these cultures facilitates osteoblast differentiation or function. OsteoMac removal did not affect expression of the early osteoblast differentiation marker genes collagen type I or alkaline phosphatase. However, OsteoMac removal significantly decreased gene expression of the osteoblast mineralisation marker osteocalcin and mineralisation function, assessed by von Kossa staining. Microarray gene expression profiling demonstrated that osteoblast enrichment had a broad impact on transcription within the culture, identifying both candidate OsteoMac marker genes as well as osteoblast expressed genes that are regulated by OsteoMacs. Potential OsteoMac-enriched candidate genes insulin-like growth factor a, dipepetidase 2, glycoprotein NMB, and macrophage expressed gene 1 as well as osteoblast-specific genes bone sialoprotein and thrombospondin 1 were selected based on their potential involvement in osteoblast function. In a transwell co-culture system of enriched osteoblasts and macrophages, it was demonstrated that macrophages were required for osteoblast mineralisation in response to the physiologic remodelling stimulus, elevated extracellular calcium. A blocking soluble receptor strategy provided evidence that this is mediated in a BMP-2 and -4 independent manner. To investigate the relevance of OsteoMacs to bone formation in vivo, immunohistochemistry staining for the mature tissue macrophage marker F4/80 was performed in long bone sections from rapidly growing mice. OsteoMacs were closely associated with areas of bone formation in situ, forming a distinctive canopy structure over mature cuboidal osteoblasts (collagen type I+, osteocalcin+) on endosteal cortical surfaces. Using adapted histomorphometic analysis, we determined that 77 ± 2.1% (n = 7) of the endosteal mature osteoblast surface was covered by the F4/80+ OsteoMac canopy. This observation suggested that OsteoMacs are optimally located to regulate osteoblast function in vivo. In summary, we have demonstrated that OsteoMacs are an integral component of bone lining tissues and play a novel role in bone dynamics through regulating osteoblast function. These observations implicate OsteoMacs, in addition to osteoclasts and osteoblasts, as principal participants in bone dynamics. Further delineation of OsteoMac functions is likely to provide new avenues for treating bone disease and assisting bone repair.

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第22145号 / 医博第4536号 / 新制||医||1039(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 戸口田 淳也, 教授 安達 泰治, 教授 松田 秀一 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

As alterações de superfície dos implantes de titânio (Ti) influenciam funções celulares e formação de tecido adjacente aos mesmos, mas é controverso se tais alterações trariam vantagens quando esses implantes fossem colocados em tecido ósseo de baixa qualidade. O objetivo deste estudo foi avaliar a resposta do tecido ósseo de baixa qualidade (tipo IV) a implantes de Ti com diferentes tratamentos de superfície. Para isso, implantes de Ti com superfície nanotexturizada (Ti-Nano), microtexturizada (Ti-Porous) e sem tratamento de superfície (Ti-Usinado) foram implantados em tíbias de coelhos. Após duas e seis semanas, o tecido ósseo formado ao redor dos implantes foi avaliado em cortes histológicos por desgaste e histomorfometricamente foram avaliados: a porcentagem de contato ossoimplante (BIC), porcentagem de área de osso mineralizado formado entre as espiras do implante (BABT), porcentagem da área de osso formada na área espelho (BAMA). Vários outros parâmetros morfométricos foram avaliados a partir de imagens e reconstruções tridimensionais obtidos por microtomografia. Ao final de seis semanas, foi avaliado o torque de remoção. Os resultados foram submetidos à análise de variância (ANOVA) com dois fatores de variação (tratamento de superfície e tempo de implantação) seguido do teste de Tukey (p≤0,05). Apesar de algumas diferenças estatisticamente significantes para alguns dos parâmetros morfométricos, tais diferenças não são biologicamente importantes para que pudessem ser atribuídas aos tratamentos de superfície. Tanto que, histologicamente não se observou diferença nos padrões de formação óssea ou tipo de osso formado que pudesse ser associado aos tratamentos de superfície e o torque de remoção também não foi afetado pelos tratamentos. Em conclusão, esses resultados mostraram que os tratamentos de superfície, embora sejam na escala micrométrica ou nanométrica, não induzem repostas do tecido ósseo tipo IV diferentes daquelas induzidas por superfícies sem tratamento. / Some alterations in titanium (Ti) implant surfaces influence cell functions and tissue formation in areas adjacent to the implants, but it is controversial whether these changes would bring advantages when these implants were placed in bone of poor quality. The aim of this study was to evaluate the response of poor quality bone tissue (type IV) to Ti implants with different surface treatments. For this, Ti implants with nanotextured surface (Ti-Nano), microtextured (Ti-Porous) and without surface treatment (Ti-Machined) were implanted into tibias of rabbits. After two and six weeks, the bone tissue around the implants was assessed in undecalcified histological sections and histomorphometrical analysis to evaluate: the percentage of bone-implant contact (BIC), percentage of the area of mineralized bone formed between threads (BABT), the percentage area of bone formed in the mirror area (BAMA). Several other morphometric parameters were evaluated from images and three-dimensional reconstructions obtained by microtomography. At the end of six weeks the removal torque was evaluated. The results were subjected to analysis of variance (ANOVA) with two factors of variation (surface treatment and time) followed by Tukey test (p≤0.05). Despite some statistically significant differences for some of the morphometric parameters, such differences are not biologically important and they could not be attributed to the surface treatments. In keeping with this, histological analysis failed to show any difference in bone formation that could be associated to the surface treatments and removal torque was not affected by treatments either. In conclusion, these results showed that the surface treatments, irrespective of being on the micrometer or nanometer scale, do not induce responses of type IV bone tissue different from those induced by untreated surfaces.