Bone and mineral最新文献

The object of this study was to determine the tissue level mechanism in which mechanical loading had an influence on ovariectomy-induced cancellous bone loss. Two experiments were performed: (1) 230 g female rats were divided into basal, age-related controls and right hindlimb immobilized (RHLI) group; (2) 250 g female rats were divided into basal, age-related, ovariectomized (OVX) controls and OVX combined with RHLI. The RHLI model immobilized (IM) or underloaded (UL) the right hindlimb and loaded (L) the left hindlimb compared to the right hindlimb. Both experiments lasted 60 days. Histomorphometric data was gathered from the secondary spongiosa of double fluorescent labeled proximal tibial metaphysis (PTM). The study confirmed that IM or UL induces cancellous bone loss, by decreasing bone formation, and increasing bone resorption and OVX results in cancellous bone loss, a higher bone turnover with bone resorption exceeding bone formation in the PTM. The OVX'd and immobilized (OVX + UL) PTM showed further decreased cancellous bone mass (−48%), decreased number (−43%), and decreased tissue-level bone formation rate (−54%) from that of OVX rats. However, the OVX’d and loaded (OVX + L) PTM partially prevented the OVX-induced cancellous bone loss. The cancellous bone area (+46%), number (+29%), and ratio of node to free end (+70%) were increased and percent eroded perimeter (−44%) and bone resorption rates (−30%) were decreased from OVX rats, but cancellous bone area and number were still significantly lower than those in age-related controls. We found that IM or UL accentuated cancellous bone loss in OVX rats by inhibiting bone formation and that loading partially prevented cancellous bone loss in OVX and RHLI rats by inhibiting bone resorption.

An Hologic QDR 1000-W dual energy X-ray absorptiometer (DXA) was used to make a densitometric evaluation and to optimize ex vivo scanning and analysis conditions for excised sheep bones. Articulated lumbar spines and femora, removed at autopsy from eight 1–2-year-old cross-bred merino ewes, were studied using standard lumbar spine and hip protocols (version 4.20). Spine data were analysed with the ultra-high resolution (UHR) protocol (version 4.25). Preliminary hip measurements also were made using the lumbar spine (version 4.20) and scoliosis protocols (version 4.25). Bone mineral density (BMD) precision for the total hip (n = 5) was 0.4% using version 4.20, and 0.8% using the scoliosis protocol (version 4.25) but was 1.2–3.4% for femoral sub-regions. Precision for the lumbar spine was 0.8% (version 4.20). There was considerable variation in BMD and bone mineral content (BMC) at the same site between animals. Right and left hip BMD were highly correlated (r = 0.98, P < 0.00001). Measurement variables evaluated to determine the optimal conditions included: Hologic scanning protocols, depth of water immersion of bone and dimensions of the region of interest. Accuracy was determined by ashing. Use of the scoliosis protocol (version 4.25) gave the best results for proximal femur measurement. Measured BMC decreased by 4.4% as the water depth increased from 0 to 22.5 cm. At 5 cm water, BMC accuracy was 99% for the total hip, and BMD precision values (n = 3) were 0.3% (total hip) and 0.4% (femoral neck, trochanteric region and femoral shaft). The much-improved precision was attributed to use of a positioning device and to optimizing of the depth of water immersion. Rib segments (length 2.5 cm) were measured using the ultra-high resolution protocol (version 4.25). BMC accuracy determined using 6 ribs was best (100 ± 2%) at 5 cm water. BMD precision (n = 5) was best (0.3%) at 0 cm water depth and was 0.5% at 4.5 cm water depth. Rib sample BMC (n = 15, 4.5 cm water depth) and ash weight were highly correlated (r = 0.985, P < 0.001). It is concluded that highly precise, accurate densitometry can be performed on excised small and large sheep bones. This technique will support studies evaluating the sheep as an animal model of human osteoporosis.

An activity that recruits osteoclasts has been identified and partially characterized from bone matrix. Bone-derived osteoclast recruiting activity (BORA) was co-purified with osteogenin, a bone inductive protein. Osteogenin was extracted from bovine bone with 6 M urea and purified by chromatography on hydroxyapatite, heparin-Sepharose and Sephacryl S-200 gel filtration. The biologically active osteoclast formation-stimulating material was further purified by C₁₈ reverse phase HPLC. BORA is obviously distinct from osteogenin and transforming growth factor β (TGF-β), since further purified osteogenin and pure TGF-β did not stimulate the formation of osteoclast-like cells. BORA (0.1–10 μg/ml) stimulated the formation of tartrate-resistant acid phosphatase (TRAP)-positive multinucleated cells (MNC) in a dose-dependent manner. These multinucleated cells resorbed bone when cultured on bovine bone slices. The effect of BORA is primarily directed to differentiate osteoclast precursors, since it did not stimulate osteoclast function in in vitro resorption assay where disaggregated rat osteoclasts were cultured on bovine bone slices. However, after 24 h preincubation with 50 nM PTH in the mouse calvaria assay, BORA at 10 μg/ml significantly stimulated bone resorption.

We have generated and characterized an antibody that recognizes the C-terminal sequence of the propeptide of human bone GLA protein (BGP/osteocalcin) (amino acid −26 to −1, with +1 being the amino terminus of the mature protein). The range of sensitivity of the antibody, as determined by enzyme-linked immunosorbent assay (ELISA), was 0.5–250 ng/ml. The antibody effectively recognized pro-BGP in cell layer extracts of transformed cells (KT-005), but did not recognize mature, propetide-less BGP in the medium from the same cultures. Strong labelling was obtained using this antibody in immunoperoxidase staining or immunofluorescence of both transformed and normal human bone cells in vitro. Monensin significantly altered the intracellular pattern of labelling in immunofluorescence studies, indicating that the recognized antigen was associated with the cellular secretory pathway. We also obtained a specific and strong staining of cells in tissue sections of human fetal bone. Antibodies against the mature protein strongly stained the mineralization front, but did not stain cells to any appreciable level. Newly embedded osteocytes were the predominant cell type stained in such material, suggesting that they may represent the major source of BGP in the intact tissue. These observations indicate that BGP synthesis is a late event in osteoblastic development and that antibodies generated against the propeptide sequence are a potentially powerful tool in the analysis of bone tumors and evaluation of osteoblastic differentiation.

The ability of trabeculae to reform following localized ablation may provide further insight into the sequence of events in cancellous regeneration. Histological features of cancellous repair were examined in the iliac crest of aged female sheep at intervals after removal of a 1-cm diameter biopsy. Comparison was made with normal intramembranous trabecular formation in the foetal lamb. The first immature trabeculae to form in the defects within 3 weeks were exclusively intramembranous, not endochondral, and the systematic process was indistinguishable from that in the intact growing foetal lamb. In both the young and old skeleton, two features were prominent. First, the damaged endosteum of the sheep functioned like the intact periosteum of the lamb to produce orderly migrating arrays of discrete coarse collagenous fibres, 5–25 µm thick, which penetrated the surrounding soft tissues to form a polarised preliminary framework. Without this structure, primary trabecular development did not take place. Throughout subsequent bone apposition the preliminary framework, which bonded hard to soft tissues and new bone to old, remained largely unmineralised. Second, intratra-becular resorption channels divided the established, thickened primary bars into networks of mature secondary trabeculae. It is concluded that the two features are central and universal to trabecular proliferation and may provide a morphological basis for future trabecular restitution of the depleted elderly skeleton.