Recombinant human bone morphogenetic proteins (rhBMPs) are increasingly used for reconstructing bony defects, fracture non-unions, and augmenting existing bone volumes. For the numerous patients taking bisphosphonates, the impact of prior bisphosphonate treatment on rhBMP bone induction is not well understood. Objective: to evaluate the effect of the prior treatment with zoledronate on rhBMP induced bone formation in mice. Methods: 42 mice were pre-treated with 0, 2, and 20 µg of zoledronate/mouse. The osteoinductive activity of a bioimplant, containing rhBMP-2, was assessed using the mouse muscle pouch assay, and analyzed with micro CT and histology. Results: micro CT demonstrated that BMP bioimplants placed in mice pretreated with 20 µg of zoledronate, formed bony ossicles of greater volume but reduced bone density compared to controls. Histologically, the heterotopic ossicles from the 20 µg group consisted of more immature bone than those from the other groups. Conclusion: bone induced by rhBMP-2 in mice pre-treated with a high concentration of zoledronate was immature as evidenced by radiographic and histologic appearance.

No description available.

The osteoclasts, the bone cells responsible for bone degradation, have a crucial role in the age-related bone loss and post-menopause osteoporosis. Rac1 and Rac2, members of the Rho-family of small GTPases, are known for having a key role in osteoclast formation and activity, which could be translated to bone quality. In this study, we characterize the roles of Rac1 and Rac2 on bone quality using an aged and osteoporotic mouse model. Bones from wild type, Rac1KO and Rac2KO mice were harvested for mechanical tests, bone densitometry, micro-computed tomography and histomorphometric analyses to evaluate bone mineralization and architecture. We observed that the deletion of Rac1 or Rac2 in pre-osteoclasts minimized bone loss in both age-related and post-menopause osteoporosis. These results highlight the importance of the two small GTPases in bone remodeling and identify Rac1 and Rac2 as potential targets for the development of new therapies for the treatment of osteoporosis.

The cement line is the first mineralized matrix deposited on an implant surface during contact osteogenesis forming the bone/implant interface. The hypothesis underlying the present project was that non-collagenous cement line proteins must be deposited into the submicron undercuts on substrate surfaces prior mineralization. In vitro osteogenic cultures were used to grow bone nodules on Thermanox® coverslips modified with calcium phosphate nanocrystals, creating an undercutted surface. Electron microscopy was used to observe cement line formation. BSP immunogold labelling was used to determine if the cement line organic matrix is deposited within undercuts prior mineralization. The results showed the deposited bone nodules, and on test coverslips the deposited cement line was thicker and evenly distributed than control. Furthermore, positive BSP labelling was found within the undercuts prior to cement line mineralization. Thus, it can be concluded that cement line proteins are deposited into submicron undercuts on substrate surfaces prior to mineralization.

The osteoclasts, the bone cells responsible for bone degradation, have a crucial role in the age-related bone loss and post-menopause osteoporosis. Rac1 and Rac2, members of the Rho-family of small GTPases, are known for having a key role in osteoclast formation and activity, which could be translated to bone quality. In this study, we characterize the roles of Rac1 and Rac2 on bone quality using an aged and osteoporotic mouse model. Bones from wild type, Rac1KO and Rac2KO mice were harvested for mechanical tests, bone densitometry, micro-computed tomography and histomorphometric analyses to evaluate bone mineralization and architecture. We observed that the deletion of Rac1 or Rac2 in pre-osteoclasts minimized bone loss in both age-related and post-menopause osteoporosis. These results highlight the importance of the two small GTPases in bone remodeling and identify Rac1 and Rac2 as potential targets for the development of new therapies for the treatment of osteoporosis.

The cement line is the first mineralized matrix deposited on an implant surface during contact osteogenesis forming the bone/implant interface. The hypothesis underlying the present project was that non-collagenous cement line proteins must be deposited into the submicron undercuts on substrate surfaces prior mineralization. In vitro osteogenic cultures were used to grow bone nodules on Thermanox® coverslips modified with calcium phosphate nanocrystals, creating an undercutted surface. Electron microscopy was used to observe cement line formation. BSP immunogold labelling was used to determine if the cement line organic matrix is deposited within undercuts prior mineralization. The results showed the deposited bone nodules, and on test coverslips the deposited cement line was thicker and evenly distributed than control. Furthermore, positive BSP labelling was found within the undercuts prior to cement line mineralization. Thus, it can be concluded that cement line proteins are deposited into submicron undercuts on substrate surfaces prior to mineralization.

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.

Diss. (sammanfattning) Stockholm : Karolinska institutet, 2007. / Härtill 5 uppsatser.

Thesis (Ph.D.)--Tufts University, 2004. / Adviser: David L. Kaplan. Submitted to the Dept. of Biotechnology Engineering. Includes bibliographical references (leaves 162-222). Access restricted to members of the Tufts University community. Also available via the World Wide Web;