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Exogenous type I collagen facilitates osteogenic differentiation and acts as a substrate for mineralization of rat marrow mesenchymal stem cells in vitro.

We cultured rat mesenchymal stem cells (MSCs) in a medium containing beta-glycerophosphate, ascorbic acid, and dexamethasone to show in vitro osteogenic differentiation of the MSCs. The differentiation was enhanced by adding solubilized type I collagen to the medium as evidenced by higher alkaline phosphatase activity as well as more calcium deposition than that without collagen. The exogenous collagen integrated well with the mineralized bone matrix and maintained the native triple helical structure. These findings indicate that exogenously supplemented type I collagen acts as a component of the extracellular matrix of MSCs, and deposited type I collagen facilitates osteogenic differentiation followed by maturation of mineralized bone matrix.

Animals↗

Histologic changes in Paget's disease treated with calcitonin.

In recent years the use of calcitonin in the medical treatment of Paget's disease of bone has been gaining in popularity. Seventeen patients on long term calcitonin therapy showed objective morphologic evidence of improvement in the quality of bone matrix deposition, in the trabecular organization, and in the parameters used to evaluate the rates of bone turnover. The usefulness of bone biopsy and fine detail radiography of the biopsy core in evaluating the progress of disease and the effects of therapy is discussed. It is concluded that the longer the period of therapy and follow-up, the higher the proportion of patients showing histologic evidence of improvement.

Aged↗

Extracellular matrix in bone marrow cell cultures. Synthesis of a 45k non-collagenous protein and a 17k protein sensitive to bacterial collagenase.

Proteins produced by bone marrow constituents are of importance to hematopoiesis and osteogenesis. To elaborate the role of bone marrow in these functions the proteins synthesized by rat bone marrow cells in culture were evaluated. In addition to types I and III procollagen and fibronectin, two novel proteins were produced by the adherent stromal cells, a 45 kDa protein, and a 17 kDa protein which is sensitive to bacterial collagenase. The 45k protein is sensitive to pepsin digestion, contains no disulfide bonds, has no precursors or breakdown products on pulse-chase analysis and is maximally synthesized in young cultures (5 days old) with decreased synthesis as time in culture increases. The 17k protein is sensitive to digestion by bacterial collagenase and pepsin, appears without precursors or breakdown products on pulse-chase analysis and is maximally synthesized in 5 day old cultures. Synthesis decreases with longer times in culture. The 17k protein is not a degradation product of a collagen precursor and appears to be a novel protein in bone marrow cell cultures.

Animals↗

Management strategy for unicameral bone cyst.

The management of a unicameral bone cyst varies from percutaneous needle biopsy, aspiration, and local injection of steroid, autogenous bone marrow, or demineralized bone matrix to the more invasive surgical procedures of conventional curettage and grafting (with autogenous or allogenous bone) or subtotal resection with bone grafting. The best treatment for a unicameral bone cyst is yet to be identified. Better understanding of the pathology will change the concept of management. The aim of treatment is to prevent pathologic fracture, to promote cyst healing, and to avoid cyst recurrence and re-fracture. We retrospectively reviewed 17 cases of unicameral bone cysts (12 in the humerus, 3 in the femur, 2 in the fibula) managed by conservative observation, curettage and bone grafting with open reduction and internal fixation, or continuous decompression and drainage with a cannulated screw. We suggest percutaneous cannulated screw insertion to promote cyst healing and prevent pathologic fracture. We devised a protocol for the management of unicameral bone cysts.

Adolescent↗

Osteopontin antisense deoxyoligonucleotides inhibit bone resorption by mouse osteoclasts in vitro.

Osteopontin (OPN) is an acidic phosphoprotein synthesized by osteoblasts and osteoclastic cells that are localized in the mineralized phase of bone matrix. OPN is thought to bind to the vitronection receptor on the osteoclast membrane and regulates bone resorption by the osteoclast. In this study, we investigated whether or not OPN can relate to osteoclast differentiation and bone resorption in a co-culture system. When C57Black/6N mouse bone marrow cells suspended on ivory slices coated with collagen were inoculated onto a MC3T3-G2/PA6 cell layer, colonies containing TRAP(+) mononuclear and multinuclear cells were formed in the presence of 1 alpha, 25-dihydroxyvitamin D3 and dexamethasone. At the end of culture period the number of TRAP(+) osteoclast-like cells were counted and the resorption pits were evaluated by reflected light microscopy. The mRNA of OPN was detected by in situ hybridization. Osteoclast-like cells expressed OPN mRNA. The addition of an OPN antisense oligomer (5' AAT CAC TGC CAA TCT CAT 3') at the start of the co-culture period decreased the number of TRAP(+) cells present after 7 d (30.3 +/- 3.4 vs 56.9 +/- 12.4), and the ratio of mononuclear and multinucleated cells was changed (77.6:23.2 vs 60.8:39.3). The total area of pits per ivory slice was also decreased by adding the OPN antisense oligomer (246813 vs 303139 microns2). These results showed that OPN can be an important mechanism for regulating differentiation and bone resorption.

3T3 Cells↗

[Cytoskeleton of cultured osteoclasts on bone slices and glass].

The cytoskeletal system in a osteoclast modulates its cell shape and motility. During bone remodeling, osteoclastic adhesion and escapement from bone matrix depend upon the formation and disassembly of podosomes. Highly dynamic adhesion of osteoclasts to the bone matrix is referred to as podosomes which consists of actin cytoskeleton and its associated proteins. The cytoskeleton modulates not only in mechanical support of cell shape and motility but also serves signal transducing function in response to external signals. For the better understanding of osteoclast function, our intention here is to describe a dynamic cytoskeleton of osteoclasts including a specific adhesion termed podosome.

English Abstract↗

Porous hydroxylapatite as a bone graft substitute in mandibular contour augmentation: a histometric study.

The buccal contour of the mandible was augmented in 17 dogs with 5 X 7.5 X 20 mm blocks of porous hydroxylapatite (HA) on one side and two-layered split rib autografts on the other. Both specimens were retrieved at three, six, 12, 24, and 48 months. Undecalcified sections were prepared for microradiography, light and UV microscopy, and histometry. A transmitted light video image digitizing system was used to trace implant and graft perimeters and calculate cross sectional areas. This system was also used to measure graft density and calculate bone and soft tissue compositions. The HA matrix, bone and soft tissue compositions of implant specimens were measured with a backscattered scanning electron microscope imaging digitizing system. All grafts became increasingly resorbed with time whereas all implants remained intact. Mature osteotonic bone ingrowth was present in all implants except one which failed to unite with the mandibular cortex. The mean graft areas decreased from 30.8 mm2 at three months to 0.7 mm2 at 48 months, while the implant areas averaged 35.5 mm2 and remained stable. The graft specimens were composed of 46.6% bone and 53.4% soft tissue or fluid space. The implant specimens were composed of 34.5% HA matrix, 28.6% bone, and 33.9% soft tissue. The HA matrix had a surface area of 9.8 mm2/mm3 that was 61.9% covered with bone ingrowth and 38.1% covered with soft tissue or fluid space. In contrast to the rapid resorption of graft onlays, the porous HA matrix demonstrated a long-term permanence with maintenance of contour and osseous incorporation over the four-year duration of this study.

Animals↗

Tetracycline administration normalizes the structure and acid phosphatase activity of osteoclasts in streptozotocin-induced diabetic rats.

Diabetes induces osteopenia, which is characterized by a deficiency of osteoid and decreased activity of osteoblasts. We recently found that tetracyclines prevent the loss of osteoid and bone matrix and the degeneration of osteoblasts in diabetic rats by a mechanism independent of their antimicrobial efficacy. However, bone remodeling requires the activity of osteoclasts as well as osteoblasts. To determine the in vivo effects of tetracycline on osteoclasts in long bones, either a tetracycline (minocycline, TC) or its chemically modified non-antibiotic analogue (CMT), 4-de-dimethylaminotetracycline, was administrated daily to streptozotocin-induced diabetic rats by oral intubation. After 21 days, the rats were perfusion-fixed with a mixture of formaldehyde and glutaraldehyde, and the humeri were dissected and processed for ultracytochemical demonstration of acid trimetaphosphatase (ACPase) activity. In untreated non-diabetic (control) rats, the osteoclasts at the zone of provisional ossification exhibited abundant mitochondria and cisterns of rough endoplasmic reticulum (RER) throughout the cytoplasm, prominent stacks of Golgi membranes, and lysosomes in the perinuclear cytoplasm, and numerous various pale vacuoles in the cytoplasmic area adjacent to well-developed ruffled border. Intense ACPase activity was observed in the Golgi saccules, lysosomes, pale vacuoles, and the extracellular canals of ruffled border. The reaction products were also noted along the resorbing bone surfaces associated with the osteoclast ruffled border. The osteoclasts in the untreated diabetic rats showed a cytoplasmic organization similar to that of the non-diabetic control rats, but showed little or no ruffled border which was replaced by a broad clear zone in some of these cells. However, most of the osteoclasts on bone matrix in the diabetics were devoid of both a ruffled border and a clear zone. ACPase activity was detected in the osteoclast cytoplasm of diabetic rat, as in the controls, but to a much lesser extent along the broad clear zone facing the resorbing bone surfaces. The osteoclasts in TC-treated diabetic rats possessed both a clear zone and a small ruffled border. However, in some cases, they lacked both structures reminiscent of the untreated diabetic cells. The osteoclasts of CMT-treated diabetic rats exhibited structural and enzymatic features essentially identical to those of the non-diabetic control rats. These results suggest that the diabetes-induced osteopenia results, at least in part, from degeneration of osteoclasts (as well as atrophic osteoblasts) and that tetracyclines may be effective in preventing these abnormalities by a mechanism not dependent on the drugs' antimicrobial properties.

Acid Phosphatase↗

Early bone in-growth ability of alumina ceramic implants loaded with tissue-engineered bone.

To enhance early bonding of an alumina ceramic implant to bone, we evaluated a method of seeding the implant surface with bone marrow mesenchymal cells that differentiated to osteoblasts and bone matrix prior to implantation. The usefulness of the method was evaluated in Japanese white rabbits. In our study, an alumina ceramic test piece loaded with differentiated osteoblasts and bone matrix by a tissue engineering technique was implanted into rabbit bones. Three weeks after the procedure, evaluation of mechanical bonding and histological examination were performed. Histological examination of the noncell-loaded implant surfaces showed no bone infiltration into the implant gap. However, the cell-loaded implant surfaces exhibited new bone infiltration into the implant gap with mechanical bonding. In the mechanical test, the average failure load was 0.60 kgf for the noncell-loaded side and 1.49 kgf for the cell-loaded side. Preculturing mesenchymal cells on the surface of the alumina ceramic prior to implantation increased the debonding strength by two and half times. The present findings indicate early bonding between the implant and bone three weeks after the procedure.

Alkaline Phosphatase↗

Correlative morphometric and biochemical analysis of purified extracellular matrix vesicles from rat alveolar bone.

Extracellular matrix vesicles were isolated from rat alveolar bone and purified by either gel filtration or discontinuous sucrose density gradient. Morphometric evaluation of electron-micrographs of pellets of purified and nonpurified vesicle fractions was correlated with the activity of vesicular enzymes. A high correlation was found between the percentage of area occupied by vesicles with electron-dense content (electron-dense vesicle fractional area) and the enzymatic activity. Highest enzymatic specific activities and electron-dense vesicle fractional area were recorded in the "light" vesicle-enriched fraction obtained after equilibrium density centrifugation. These parameters revealed lowest values in the "heavy" vesicle-enriched fraction resolved by the same methods. The combined electron-dense and electron-lucent fractional area (vesicular fractional area) was similar in the different purified fractions. It is therefore suggested that the fraction obtained by gel filtration contains both "light" and "heavy" vesicles. Morphometric study is proposed as an additional criterion for the degree of purification of matrix vesicle preparations.

Acid Phosphatase↗

Short-term effects of fluoride and strontium on bone formation and resorption in the mouse.

The early effects of sodium fluoride (0.80 mg/kg/d) and strontium chloride (0.27%) given alone, or in combination in drinking water, on bone metabolism were examined in the mouse using dynamic histomorphometric methods. Four weeks of oral strontium supplementation increased the osteoid surface and reduced the number of acid phosphatase-stained osteoclasts. However the trabecular calcified bone volume was not augmented. By contrast, short-term treatment with fluoride produced a rapid stimulatory effect on bone formation at a dose that did not affect the bone mineralization rate. Four weeks of fluoride supplementation induced a rapid 21.1% increase in the osteoblastic surface and a 26.3% stimulation of the bone matrix apposition rate evaluated by the double tritiated proline labelling method, which resulted in a 29% increase in the amount of osteoid. This rapid stimulation of the bone formation rate without detectable change in osteoclastic bone resorption led to a 12% increase in the trabecular calcified bone density. This study shows that fluoride and strontium produce distinct early effects on bone formation and resorption in the mouse and that fluoride exerts a rapid stimulatory effect on the bone matrix synthesis rate through an augmentation of the number of bone-forming cells.

Acid Phosphatase↗

Histological and ultrastructural appearance of the hydroxyapatite-bone interface.

This article describes the histological and ultrastructural appearance of the interface created in the implantation bed, between bone tissue and implants made of dense sintered hydroxyapatite (HA). Biopsies from dog subjects included: a) loaded permucosal dental implants for tooth substitution, b) subperiosteally placed implants for alveolar bone correction, c) endosseously placed dental root implants to retain ridge form following extraction. The light and electron microscopical results show extensive bone apposition on the osseous sides of the implant surfaces. There is an intimate, direct bone contact without any visible interruption. The bone is of normal lamellar type and continuously connected with the trabecular bone. Bone has grown into the finest surface irregularities of the implant. Collagen fibers of the calcified bone matrix are observed within a distance less than 500 A from the implant surface. The thin (20-100 A) electron dense layer at the bone-implant interface resembled the lamina limitans of organic bone matrix, also seen at the inner walls of the osteocytes lacunes. Deposition of bone gives rise to a biologically stable bone-implant interface, without disturbance of the physiological bone turnover. This is seen as very favorable for desired long term fixation of implant to bone.

Animals↗

Comparison of the effects of adriamycin and methotrexate on orthotopic and induced heterotopic bone in rats.

The effect of the two antineoplastic drugs, Adriamycin and methotrexate, on orthotopic bone, and on the induction of experimental heterotopic bone in rats was analyzed. The drugs were administered as single injections: Adriamycin in s.c. doses of 0.5 and 2 mg/kg body weight and methotrexate i.v. 100 and 250 mg/kg body weight followed by leucovorin rescue after 2 h. A passing, but significant, decrease in body weights occurred in the methotrexate-treated animals, but not in those given Adriamycin. Analysis of the amount of heterotopic bone formed 4 weeks after induction by demineralized bone matrix revealed a 30-40% decrease in the groups treated with either of the antineoplastic agents, whereas orthotopic bone was unaffected. Six weeks after the treatment the net effect on the induced bone had decreased. The present study shows that the two antineoplastic drugs Adriamycin and methotrexate inhibit heterotopic new bone formation induced by demineralized bone matrix in rats to an equal extent, although their mode of action on the cellular level is entirely different, and that the inhibitory effect of a single treatment diminishes in the presence of a continuous inductive process.

Animals↗

Developmental expression of osteopontin (OPN) mRNA in rat tissues: evidence for a role for OPN in bone formation and resorption.

Osteopontin (OPN) is a 34-kDa, highly-phosphorylated glycoprotein with cell attachment properties that is a prominent constituent of the bone matrix. To aid in elucidating the function of this protein we have studied the cellular expression of OPN mRNA during the formation, growth and maturation of rat calvarial (membranous) and tibial (endochondral) bone. From Northern hybridization analysis OPN expression was demonstrated in the kidney and gravid uterus as well as in bone tissues. Compared to collagen, the expression of OPN was low in early bone formation but increased subsequently and reached peak levels in 14-day-old bone. However, both the collagen and OPN mRNAs decreased markedly thereafter and remained low in young adult bone. From in situ hybridization studies using a [35S]-labelled rat OPN cRNA probe, OPN mRNA was localized to osteoblastic cells in newly-forming calvariae, jaw bones, and in the metaphyseal and periosteal bone of the tibia. In contrast to bone sialoprotein (BSP), which is expressed almost exclusively by osteoblasts at sites of de novo bone formation, OPN transcripts were present in cells lining both endosteal and periosteal bone surfaces, and in osteocytes. Moreover, expression of OPN persisted during the subsequent growth and remodelling of both membranous and endochondral bone and was expressed at particularly high levels by bone cells and hypertrophic chondrocytes at sites of osteoclastic resorption. In the more mature bone of young adult rats OPN expression was significantly reduced but remained detectable in bone cells lining periosteal and endosteal surfaces and in the primary and secondary spongiosa of the tibia. These studies on the developmental expression of OPN support the concept of a multifunctional role for OPN in bone formation and remodelling. Thus, the expression of OPN by osteoblasts early in bone development is consistent with a role for this protein in the formation of bone matrix, whereas the peak expression of OPN later in bone development, together with high expression at sites of rapid remodelling, indicate that OPN deposited on the surface of mineralized connective tissues may provide a template for osteoclastic resorption.

Animals↗

Vitamin D control of osteoblast function and bone extracellular matrix mineralization.

Vitamin D is the major regulator of calcium homeostasis and protects the organism from calcium deficiency via effects on the intestine, kidney, parathyroid gland, and bone. Disturbances in the vitamin D endocrine system (e.g., vitamin D-dependent rickets type I and type II), result in profound effects on the mineralization of bone. Recent studies with vitamin D receptor knockout mice also show effects on bone. It is questioned whether vitamin D has a direct effect on bone formation and mineralization. In rickets and particular vitamin D receptor knockout mice, calcium supplementation restores bone mineralization. However, the vitamin D receptor is present in osteoblasts, and vitamin D affects the expression of various genes in osteoblasts. This review focuses on the role of vitamin D in the control of osteoblast function and discusses the current knowledge of the direct effects of vitamin D on mineralization. Moreover, the role of vitamin D metabolism and the mechanism of action of vitamin D and interaction with other hormones and factors are discussed.

Alkaline Phosphatase↗

A chemosterilized antigen-extracted autodigested alloimplant for bone banks.

Limited chemical extraction of hydrophobic glycopeptides and subtotal autodigestion of the donor's cells and plasma membranes in undemineralized cortical bone in vitro reduces the putative quantity of haptenic substances absorbed by the recipient. Iodoacetic acid and sodium azide or other sulfhydryl group enzyme inhibitors added to the buffer solutions during in vitro autodigestion and estraction of intracellular alloantigens protects the bone matrix morphogenetic property against enzymatic degradation. The delayed hypersensitivity reaction induced by aseptically collected freeze-dried bone and the destruction of the bone morphogenetic property caused by radiation-sterilization is avoidable by sequential chemodigestion and chemosterilization of bone that preserves the maximum morphogenetic potential while transferring a minimum quantity of alloantigen.

Animals↗

Reduced osteoblastic population and defective mineralization in osteopetrotic (op/op) mice.

Osteopetrotic (op/op) mice fail to exhibit bone remodeling because of a defective osteoclast formation due to a lack of macrophage colony-stimulating factor. In this study, we investigated the femora of op/op mice to clarify whether the osteoblastic population and bone mineralization are involved in osteoclasts or their bone resorption. The op/op mice extended the meshwork of trabecular bones from the chondro-osseous junction to the diaphyseal region. In the femoral metaphyses of op/op mice, intense alkaline phosphatase (ALPase)-positive osteoblasts were observed on the metaphyseal bone in close proximity to the erosion zone of the growth plates. Von Kossa's staining revealed scattered mineralized nodules and a fine meshwork of mineralized bone matrices while the wild-type littermates developed well-mineralized trabeculae parallel to the longitudinal axis. In contrast to the metaphysis, some op/op diaphyses showed flattened osteoblasts with weak ALPase-positivity, and the other diaphyses displayed bone surfaces without a covering by osteoblasts. It is likely, therefore, that the osteoblastic population and activity were lessened in the op/op diaphyses. Despite the osteopetrotic model, von Kossa's staining demonstrated patchy unmineralized areas in the op/op diaphyses, indicating that a lower population and/or the activity of osteoblasts resulted in defective mineralization in the bone. Transmission electron microscopy disclosed few osteoblasts on the diaphyseal bones, and instead, bone marrow cells and vascular endothelial cells were often attached to the unmineralized bone. Osteocytes were embedded in the unmineralized bone matrix. Thus, osteoclasts appear to be involved in the osteoblastic population and activity as well as subsequent bone mineralization.

Alkaline Phosphatase↗