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Bone is a dynamic organ system exhibiting continuous modeling (de novo formation), and remodeling (resorption and reformation of resorbed tissue). These processes are dependent on the metabolic activity of its constituent cells: osteoblasts form bone, osteocytes maintain bone, while osteoclasts resorb bone. Nearly all bone diseases, or pathologies in fracture healing of bone, manifest aberrations in bone matrix production and/or the mineralization of this matrix.
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Primary bone formation during embryogenesis. Shown is a midsaggital section of an E15 mouse embryo. Fluorescent detection of newly forming bone mineral (red color) overlaid onto a differential contrast image. Cyan-outlined box denotes the developing c lavicle (red color). Yellow-outlined box indicates a region of primary bone formation in the developing clavicle.
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Primary bone formation during rapid modeling of young, growing bone. Shown is an H&E stained tissue section from 21 day-old rat tibial periosteum. Osteogenic cell types are indicated. Scale bar is 25 mm.
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The cellular activities in bone are tightly regulated by cytokines and hormones both in normal and pathologic states. For example, parathyroid hormone (PTH) and its somatic homolog PTHrP control bone modeling and remodeling. A major research effort focuses on the regulation of osteoblast function by PTH. Our initial approaches have been to analyze the effects of PTH on an osteoblast's ability to synthesize specific macromolecules found in bone matrix, properly assemble them in the extracellular environment, and mineralize this matrix. Using several osteoblastic models (from cell lines to primary tissue), we have discovered that PTH, through the activation of protein kinase A (PKA), can dramatically effect an osteoblast's ability to produce select matrix macromolecules, alter its ability to assemble these macromolecules into an extracellular matrix, regulate its mineralization of this matrix, and even change its shape by stimulating microfilament polymerization and cell spreading.
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PTH peptide and cAMP analogs inhibit in vitro bone mineral deposition. Osteoblastic cells deposit bone mineral in culture as illustrated by positive alizarin red-staining (control). Short-term treatment of osteoblastic cultures with 0.1 nM PTH peptide or 0.1 mM 8-bromo-cAMP results in a complete suppression of bone mineral deposition.
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Microfilament assembly and cell spreading in osteoblastic cells stimulated by PKA activation. Osteoblastic cultures treated short-term with agents that stimulate PKA activity were stained with rhodamine- phalloidin. Microfilament assembly and cell spreading occurs immediately after treatment, and reverts to the control state only after several hours of recovery.
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Our overall objectives are to define the molecular mechanisms operating in primary bone formation. This research will yield increased knowledge of an osteoblast's functions in bone homeostasis. Ultimately, it will provide a better understanding of bone development and pathology, and may offer new theurapies to augment bone healing and treat bone disease.
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X-ray and H&E histology of fracture callus healing in a stabilized rat tibia fracture model.
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