Absence of bone sialoprotein (BSP) impairs cortical defect repair in mouse long bone

Matrix proteins of the SIBLING family interact with bone cells and with bone mineral and are thus in a key position to regulate bone development, remodeling and repair. Within this family, bone sialoprotein (BSP) is highly expressed by osteoblasts, hypertrophic chondrocytes and osteoclasts. We recently reported that mice lacking BSP (BSP−/−) have very low trabecular bone turnover. In the present study, we set up an experimental model of bone repair by drilling a 1 mm diameter hole in the cortical bone of femurs in both BSP−/− and +/+ mice. A non-invasive MRI imaging and bone quantification procedure was designed to follow bone regeneration, and these data were extended by μCT imaging and histomorphometry on undecalcified sections for analysis at cellular level. These combined approaches revealed that the repair process as reflected in defect-refilling in the cortical area was significantly delayed in BSP−/− mice compared to +/+ mice. Concomitantly, histomorphometry showed that formation, mineralization and remodeling of repair (primary) bone in the medulla were delayed in BSP−/− mice, with lower osteoid and osteoclast surfaces at day 15. In conclusion, the absence of BSP delays bone repair at least in part by impairing both new bone formation and osteoclast activity.     

Osteoblast-like cell adhesion to bone sialoprotein peptides.

A number of studies have suggested that biomimetic peptides can be used in the design of a new generation of prosthetic implants to promote the successful biointegration of the implant materials. In the current study, the in vitro bioactivities of several peptides representing RGD (Arg-Gly-Asp)-containing sequences of bone sialoprotein (BSP) toward an osteoblast-like cell line (MC3T3-E1) were examined to provide insight into the molecular basis of BSP's interaction with bone cells. BSP residues 283-288, 281-290, 278-293 and 278-302 were coated on polystyrene surfaces in 96-well non-tissue (untreated) culture plates, and their osteoblast adhesive properties compared to intact BSP and fibronectin as positive controls. BSP peptides 278-302 and 278-293 were found to be the most potent in their adhesive activity, increasing the number of adherent cells to 350% of control levels at an added concentration of 1 microM. Since these two peptides were equivalent in potency, it is suggested that the region 294-302 beyond the RGD domain is not necessary for cell binding. In comparison, peptides 283-288 and 281-290 were only active at concentrations greater than 200 microM. 50-70% of the peptide-stimulated adhesion was inhibited by the pretreatment of cell suspensions with solution phase RGD, suggesting that a portion of the peptides' adhesive effects was specific and integrin-mediated, although other non-RGD flanking regions were probably also involved in the mechanism of adhesion. Importantly, a modified BSP peptide, in which an aspartic acid residue at position 288 of the RGD sequence was replaced by a glutamic acid residue to form RGE, was completely inactive as a cell adhesion stimulus at concentrations up to 200 microM. Thus, despite the potential role of non-RGD flanking regions, an intact RGD tripeptide was essential for all of the adhesive activity of the BSP peptides.     

Further characterization of interaction between bone sialoprotein (BSP) and collagen

Bone sialoprotein (BSP) has an affinity to collagen fibrils [25]. A role of carbohydrate chains in the affinity was examined by removing sialic acids of BSP. Neuraminidase treatment of the BSP increased the binding to collagen. Binding sites of BSP on collagen were examined by biochemical and electron-microscopic methods. Purified bovine BSP was labeled with biotin. Collagen chains or CNBr peptides were separated by electrophoresis and transfered to nitrocellulose membranes. The membranes were incubated with the biotin-labeled BSP, and the bound BSP was visualized with avidin conjugated with alkaline phosphatase. The labeled BSP was preferentially bound to the 2 chain, and peptides derived from 2 chain. In another experiment, the labeled BSP was incubated with reconstituted native collagen fibrils. The mixture was put on a copper grid, reacted with avidin conjugated with gold particles, and observed with an electron microscope. The gold particles were seen mainly within hole zones of the fibrils. BSP bound to the 2 chain within the hole zones may regulate the onset of calcification at hole zones and the cell binding to collagen fibrils.     

Casodex (a nonsteroidal antiandrogen) reduces cancellous,endosteal, and periosteal bone formation in estrogen-replete female rats

Testosterone has been implicated in the preservation of the skeleton in women and female rats. In this study we investigated the role of androgens in estrogen-replete female rats by giving Casodex (pure nonsteroidal anti-androgen) daily and analyzing the effects on the skeleton using static and dynamic histomorphometric parameters after 3 weeks. There was a significant reduction in the bone formation rate in both the cancellous bone of the tibial metaphysis and in the periosteal and corticoendosteal diaphyseal bone (90%, 30%, and 100% reduction, respectively, compared with control animals, which was unaccompanied by a change in the indices of bone resorption. Casodex had no effect on cancellous bone volume and cortical bone area but this can be accounted for by the short duration of the experiment. The serum levels of dehydroepiandrosteronesulfate and androstenedione were significantly reduced in the Casodex-treated rats compared with the control animals and there was no difference in the plasma levels of estrone, estradiol, or testosterone between the two groups. This study demonstrates that androgens play a physiological role in regulating osteoblast activity in female rats.     

Osteoclast development in the coculture system of periostless metatarsal bones and hemopoietic cells studied by in situ hybridization with a probe for Y chromosomes

In the coculture system of periostless metatarsal bones of 17-day-old fetal mice and osteoclast progenitors, osteoclasts will develop. Our goal in the present report was to provide further evidence that in the coculture system of fetal metatarsal bone rudiments with hemopoietic cells, the osteoclasts developing inside the bone rudiments are exclusively derived from the cells suspended in the plasma clot and not from endogenous precursor cells of the bone explants themselves, by using the technique of in situ hybridization with a probe for the mouse Y chromosome. Osteoclast formation in unstripped male metatarsal rudiments, occurring after 3–4 days of culture, was compared with osteoclast formation in cocultures of female metatarsal rudiments and male bone marrow cells, occurring after 5–6 days of culture. Osteoclasts were recognized by their tartrate-resistant acid phosphatase activity. In paraffin sections of cultured male metatarsals, the mean percentage of microscopically identifiable osteoclast nuclei, in which the Y chromosome could be detected, was 43.1±4.2% (n=12). For cocultures of female metatarsal bones and male bone marrow cells this mean percentage was 40.9±5.7% (n=17). Statistical comparison by means of the two sample t-test indicated no significant difference in the percentages of osteoclast nuclei containing the Y chromosome for both groups. We concluded that the osteoclasts do derive from cocultured cells and not from precursor cells in the bone explant itself. Therefore, the coculture system is a reliable in vitro system for studying osteoclast formation from progenitor/precursor cells.     

Characterization of human bone cells in culture

Summary Cultures of human bone cells were established, maintained, and characterized with respect to several metabolic parameters. These studies were undertaken with a view to using the bone culture system as a means of studying mechanisms of bone metabolism. The donor patients' ages ranged from 1 to 90 years and their disease states included congenital limb anomalies, exostosis, and osteo- and rheumatoid arthritis. Cultures were maintained up to 5 months. The osteoblast-like character of these cells was confirmed with the use of measurements applied to bone cells from other systems. Analyses showed that (a) the cells' appearance resembled that of cultured osteoblasts from other animal sources, b) intracellular cAMP was stimulated by human parathyroid hormone, c) osteocalcin was detected in the medium of all tested bone cell cultures and its production was found to be stimulated by 1,25-dihydroxycholecalciferol, and d) newly synthesized collagen was almost exclusively type I. In contrast, cultures of human fibrolasts, established in one instance from tissue specimens of the same donor patient, grew faster, reached a higher limiting density, and produced a greater proportion of type III collagen than the corresponding bone cells. Furthermore, fibroblasts did not accumulate osteocalcin in their culture medium. The conditions described in this report to maintain human bone cells in culture should provide a suitable test system to study the regulation of human bone metabolism.     

Morphologic features of bone in human osteopetrosis

Trabecular bone biopsies obtained from six patients with malignant osteopetrosis, one patient with benign osteopetrosis, and two controls were examined by light and electron microscopy. Osteopetrotic osteoclasts showed little to no signs of active involvement in bone resorption. Ruffled borders and clear zones were absent in most cells. In all patients there were large numbers of osteoclasts. Numbers of osteoblasts, bone lining cells, and bone marrow stromal cells were extremely low in all patients with malignant osteopetrosis. In six of the patients a mineralized layer of amorphous organic material lacking collagen fibrils was seen covering large areas of the bone or cartilage matrix. We suggest that this layer represents a pathological calcification on which subsequently organic material has accumulated. The abnormalities in osteopetrotic bone are discussed in the light of the pathogenesis of this disease.     

Insulinlike growth factor gene expression in human fracture callus

Summary The effects of insulinlike growth factors on bone and cartilage-derived cells in culture have been extensively investigated, but there is little information on their rolein vivo in bone, especially in fracture healing. This study investigated insulinlike growth factor (IGF) I and II mRNA expression in normally healing human fractures byin situ hybridization. Endothelial and mesenchymal cells at the granulation tissue stage expressed IGF-II mRNA. At the stage of bone and cartilage formation, osteoblasts and nonhypertrophic chondrocytes expressed mRNA for both IGF-I and II. Some osteoclasts were positive for IGF-II mRNA at the stage of bone remodeling. The greater time span of IGF-II expression relative to IGF-I reflects the predominance of IGF-II in human bone matrix. Taken together with the known effects of IGFs on bone and cartilage cellsin vitro, these findings support a role for IGFs in local cellular regulation in human fracture healing.     

Cultured embryonic bone shafts show osteogenic responses to mechanical loading

Summary Pairs of 17-day embryonic chick tibiotarsi were removed and maintained in organ culture. One of each pair was subjected to a single 20-minute period of intermittent loading at 0.4 Hz, producing peak longitudinal compressive strains of 650 microstrain (). In the 18-hour culture period following loading, alkaline phosphatase levels in the osteoblasts of the loaded tibiotarsi were maintained whereas in controls they declined. In situ hybridization using a collagen type I cRNA riboprobe showed a substantial increase in expression of mRNA for collagen type I in the periosteal tissue of bones that were cultured for 18 hours after loading compared with that in similarly cultured controls and bones cullured for 4 hours. These results demonstrate that appropriate loading of embryonic chick bones in organ culture elicits adaptive regulation of matrix synthesis as evidenced by increased expression of the gene for type I collagen and alkaline phosphatase activity. This model may be useful as it must contain all the obligatory steps between strain change in the matrix and modified osteogenic activity.     

Patterns and Localization of Gene Expression During Intramembranous Bone Regeneration in the Rat Femoral Marrow Ablation Model

Tissue formation and repair are dependent upon cascades of biological events, but the signals involved and the possible gene coexpression patterns during intramembranous bone repair are only poorly understood. We sought to place this mode of regeneration in context by profiling quantitative gene expression for a panel of 39 genes between days 1 and 14 following rat femoral marrow ablation. In situ hybridization was employed to localize a subset of genes. Additionally, principal components analysis was conducted to identify underlying factors suggestive of coexpression patterns. During inflammation (days 1–5), several genes, including cyclooxygenase-1 and -2, showed downregulation. Other proinflammatory cytokines, tumor necrosis factor-α and interleukin-1β, exhibited increasing levels around day 5. During repair (days 3–10), growth factors, receptors, and inhibitor genes for transforming growth factor- β; basic fibroblast growth factor; bone morphogenetic proteins 2, 4, and 7; vascular endothelial growth factor; and insulin-like growth factor-I were upregulated. In addition, the gene for core binding factor-α1 and markers of osteoblast function such as alkaline phosphatase, collagen type I, osteonectin, osteopontin, and osteocalcin had peak expression at day 5 or 7. The remodeling phase (days 10–14) was characterized by peaks for cytokines associated with osteoclastic activity including receptor activator of nuclear factor-κB, receptor activator of nuclear factor-κB ligand (RANKL), cathepsin K, tumor necrosis factor-α, interleukin-6, and cyclooxygenase-2. In situ hybridization showed that the most common sites of increased signal were within osteoblastic cells on trabecular and endosteal surfaces. Principal components analysis identified eight underlying factors that together explained over 80% of the variance in the data. Shinji Kuroda and Amarjit S. Virdi, contributed equally to this report.     

A possible role of bone morphogenetic protein (BMP) in the regulation of bone remodeling

Bone morphogenetic protein (BMP) from an osteosarcoma powder was found to be solubilized in weak acid (pH 2.6–3.0), but not in acidic solutions of a lower or higher pH. This indicates that the solubility of BMP strictly depends on the hydrogen ion concentration of the solution. Ectopic osteogenesis induced by the osteosarcoma powder was greatly enhanced by treating the powder with acid solutions below pH 3.0. The acid-treated preparations were neutralized before bioassay. Acid treatment resulted in almost a five-fold increase in the amount of new bone formation. This finding suggests that a high concentration of hydrogen ion is important in regulating the activity of BMP. Lyophilized cultured cells of the osteosarcoma preserved the osteogenic activity even after incubation in acidic and neutral buffers at 37°C for six days. This observation suggests that BMP is stable to endogenous enzymes such as lysosomal or proteolytic enzymes. Based on these results, the role possibly played by BMP in the regulation of bone remodeling is discussed.     

Expression of type I,type II,and type X collagen genes during altered endochondral ossification in the femoral epiphysis of osteosclerotic (oc/oc) mice

The osteosclerotic (oc/oc) mouse, a genetically distinct murine mutation that has a functional defect in its osteoclasts, also has rickets and shows an altered endochondral ossification in the epiphyseal growth plate. The disorder is morphologically characterized by an abnormal extension of hypertrophic cartilage at 10 days after birth, which is later (21 days after birth) incorporated into the metaphyseal woven bone without breakdown of the cartilage matrix following vascular invasion of chondrocyte lacunae. In situ hybridization revealed that the extending hypertrophic chondrocytes expressed type I and type II collagen mRNA, as well as that of type X collagen and that the osteoblasts in the metaphysis expressed type II and type X collagen mRNA, in addition to type I collagen mRNA. The topographic distribution of the signals suggests a possible co-expression of each collagen gene in the individual cells. Immunohistochemically, an overlapping deposition of type I, type II, and type X collagen was observed in both the extending cartilage and metaphyseal bony trabeculae. Such aberrant gene expression and synthesis of collagen indicate that pathologic ossification takes place in the epiphyseal/metaphyseal junction of oc/oc mouse femur in different way than in normal endochondral ossification. This abnormality is probably not due to a developmental disorder in the epiphyseal plate but to the failure in conversion of cartilage into bone, since the epiphyseal plate otherwise appeared normal, showing orderly stratified zones with a proper expression of cartilage-specific genes.     

Effect of recombinant human granulocyte colony-stimulating factor (rh G-CSF) on rat bone: Inhibition of bone formation at the endosteal surface of vertebra and tibia

The effect of recombinant human granulocyte colony-stimulating factor (rh G-CSF) on bone was evaluated by histomorphometry using Sprague-Dawley rats. rh G-CSF was injected at doses of 0, 50, 150, and 450 μg/kg for 6 weeks.In vivo double fluorochrome labeling was performed before sacrifice. No significant change in body weight was observed. Bone mineral density (BMD) of lumbar vertebrae and femora was significantly decreased in G-CSF-treated groups. In the lumbar vertebra, osteoid surface, osteoid thickness, trabecular thickness, and labeled surface in G-CSF-treated groups were also significantly lower. In addition, osteoclast number and osteoclast surface were significantly higher in the G-CSF-treated groups. The endocortical surface at the mid-tibia showed lower labeled surface and mineral apposition rate in G-CSF-treated groups, without significant changes at the periosteal surface. Furthermore, numerous granulocytes fully occupied the bone marrow area. We conclude that proliferating granulocytes in the bone marrow may inhibit bone-forming cells from contacting the bone surface, resulting in reduction of bone formation;and increased osteoclastic bone resorption induced by G-CSF treatment contributed to the reduction of BMD.     

Effects of ovariectomy on iliac trabecular bone in baboons (Papio anubis)

Summary Biopsies were collected from the left iliac crest of six adult female baboons, after which three of the animals were ovariectomized. Biopsies were collected from the right iliac crest six months later. Histomorphometric evaluation of the biopsies revealed consistent increases in fractional forming surface, appositional rate, and volume-based bone formation rate after ovariectomy. The data indicate that bone turnover is increased following ovariectomy in the baboon.     

Cbfal基因与成骨细胞标志物在HOS TE85中的表达

目的 观察核心编码因子（cbfal)基因在人的类成骨细胞株TE85的表达与骨钙素，I型胶原和碱性磷酸酶合成及分泌的关系。方法 原位杂交组织化学技术，放射免疫法，硝基苯酚法和^3H脯氨酸掺入法。结果 （1）细胞培养至第3天可见cbfal基因表达，第9天达高峰，12d时表达减弱；（2）骨钙素和I型胶原的表达在第12天时达高峰，碱性磷酸酶在第15天时达峰值。结论 cbfal基因表达与成骨细胞标志物表达之间的时差关系，提示cbfal基因在成骨细胞增殖和分化中有重要作用。     

Expression of collagen,osteocalcin, and bone alkaline phosphatase in a mineralizing rat osteoblastic cell culture

Summary Rat calvaria bone cells isolated by collagenase digestion form a bone-like matrix which mineralizes in vitro in the presence of -glycerophosphate, in less than 2 weeks. The purpose of this work was to investigate, in this mineralizing rat osteoblastic cell culture, the synthesis of collagen, osteocalcin, and bone alkaline phosphatase (ALP). The results obtained indicate (1) After 15 days in culture, the extracellular-matrix contains collagen type I, V, and to some extent type III. Metabolic labeling at day 14, during the phase of nodules mineralization as well as new nodules formation, shows that collagen types I and type V are synthesized; (2) During the phase of cell growth, no osteocalcin could be detected in the medium, however, at the point of nodule formation, the osteocalcin level reached values of 3.55±1.39 ng/ml, followed by a 30-fold increase after nodules became mineralized. At day 14, after metabolic labeling, de novo synthesized osteocalcin was chromatographed on an immunoadsorbing column. With urea-SDS PAGE the apparent molecular weight was determined to be 9,000 daltons. (3) Specific activity of ALP was found to be 10 nmol/min/mg of proteins at cell confluence. At day 15, when nodules are mineralized, this activity was increased by 40-fold. The Michaelis constant was 1.58 10^-3 M/L. ALP was inhibited by L-homoarginine and levamisole but not by L-phenylalanine. ALP was shown to be heat sensitive at 56°C with two slopes of inhibition. On SDS-PAGE, apparent molecular weight of ALP showed one band at 116,000 daltons (d) when extracted at cell confluence and two bands at 116,000 and 140,000 d when extracted at the 15th day of culture. ³²P-labeled subunit of the enzyme migrated as one band at 75,000 d. Sialic acid content was demonstrated by neuraminidase treatment either on the dimeric form or on the ³²P-labeled subunit. These data indicate that ALP expressed in this culture is bone specific. The results of the present study show that this mineralizing rat osteoblastic cell culture system synthesizes collagen type I, V, and traces of type III, osteocalcin, and bone ALP isoenzyme. Medium osteocalcin was detected during nodule formation and increased during mineralization. Increase in ALP activity as well as the presence of an additional form of ALP occurred in the mineralization phase. Therefore, this culture may be a useful model for studying the functions of bone-specific proteins during the process of mineralization.     

Bone Sialoprotein (BSP) is a Crucial Factor for the Expression of Osteoblastic Phenotypes of Bone Marrow Cells Cultured on Type I Collagen Matrix

In this study, we demonstrated that type I collagen matrix induced the expression of osteoblastic phenotypes of bone marrow cells, and that antibone sialoprotein (BSP) monoclonal antibody suppressed the expression of these phenotypes. On the other hand, BSP accelerated the expression of osteoblastic phenotypes of bone marrow cells. The adherent bone marrow cells were harvested from rat femur and cultured on type I collagen matrix gels in medium containing 15% fetal calf serum, neither β-glycerophosphate nor glucocorticoid. Cells showed osteoblastic phenotypes (high alkaline phosphatase activity, osteocalcin synthesis, and responsiveness against parathyroid hormone) on collagen matrix gels at week 3 after the inoculation, and simultaneously, BSP was detected in the conditioned medium by Western blotting using an anti-BSP monoclonal antibody. However, cells in the conventional culture dishes did not show osteoblastic phenotypes during the experimental period. To investigate the physiological function of BSP in osteoblastic differentiation, bone marrow cells were cultured on collagen matrix with an anti-BSP monoclonal antibody for 3 weeks. This treatment suppressed the expression of the osteoblastic phenotypes, and the effect of the antibody was abolished by the addition of bovine bone BSP. Furthermore, bovine bone BSP stimulated the expression of osteoblastic phenotypes of bone marrow cells. Our results indicate that BSP plays a crucial role in the expression of osteoblastic phenotypes of bone marrow cells. Received: 17 February 1999 / Accepted: 14 December 1999     

LRIG1下调原代星形细胞瘤细胞增殖的机制

目的观察LRIGl蛋白表达对表皮生长因子（EGF）促肿瘤细胞增殖作用的影响，探讨LRIGl抑制肿瘤细胞增殖的机制。方法原代培养19例星形细胞瘤细胞，用原位杂交检测LRIGl的表达，用噻唑蓝（MTT）法观察EGF对培养细胞的促增殖作用，并分析培养细胞LRIGl表达和EGF干预后的细胞生长率的关系。结果（75．3±11．6）％的培养细胞表达LRIGl蛋白；EGF促进培养细胞增殖（38．0±14．8）％，EGF促细胞增殖的细胞生长率与LRIGl表达程度呈负相关。结论LRIGl蛋白可能通过抑制EGF—EGFR信号抑制肿瘤细胞的增殖。     

<Emphasis Type="Italic">In Vivo</Emphasis> Mechanical Loading Modulates Insulin-Like Growth Factor Binding Protein-2 Gene Expression in Rat Osteocytes

Mechanical stimulation is essential for maintaining skeletal integrity. Mechanosensitive osteocytes are important during the osteogenic response. The growth hormone-insulin-like growth factor (GH-IGF) axis plays a key role during regulation of bone formation and remodeling. Insulin-like growth factor binding proteins (IGFBPs) are able to modulate IGF activity. The aim of this study was to characterize the role of IGFBP-2 in the translation of mechanical stimuli into bone formation locally in rat tibiae. Female Wistar rats were assigned to three groups (n = 5): load, sham, and control. The four-point bending model was used to induce a single period of mechanical loading on the tibial shaft. The effect on IGFBP-2 mRNA expression 6 hours after stimulation was determined with nonradioactive in situ hybridization on decalcified tibial sections. Endogenous IGFBP-2 mRNA was expressed in trabecular and cortical osteoblasts, some trabecular and subendocortical osteocytes, intracortical endothelial cells of blood vessels, and periosteum. Megakaryocytes, macrophages, and myeloid cells also expressed IGFBP-2 mRNA. Loading and sham loading did not affect IGFBP-2 mRNA expression in osteoblasts, bone marrow cells, and chondrocytes. An increase of IGFBP-2 mRNA-positive osteocytes was shown in loaded (1.68-fold) and sham-loaded (1.35-fold) endocortical tibial shaft. In conclusion, 6 hours after a single loading session, the number of IGFBP-2 mRNA-expressing osteocytes at the endosteal side of the shaft and inner lamellae was increased in squeezed and bended tibiae. Mechanical stimulation modulates IGFBP-2 mRNA expression in endocortical osteocytes. We suggest that IGFBP-2 plays a role in the lamellar bone formation process.