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Reduced expression of thrombospondins and craniofacial dysmorphism in mice overexpressing Fra1.

UNLABELLED: Fra1 transgenic (Tg) mice develop osteosclerosis and exhibit altered expression of bone matrix proteins. We found that expression of Thbs1 and Thbs2 was reduced in Fra1 Tg osteoblasts. Fra1 Tg and non-osteosclerotic Thbs1-/-Thbs2-/- mice share an edge-to-edge bite. Therefore, reduced expression of thrombospondins may contribute to craniofacial dysmorphism independently of osteosclerosis. INTRODUCTION: Tg mice overexpressing Fra1, a component of the transcription factor activator protein-1 (AP-1), show progressive osteosclerosis caused by cell autonomous abnormalities in osteoblasts. The expression of several bone matrix proteins, including matrix gla protein, is dysregulated in Fra1 Tg osteoblasts. MATERIALS AND METHODS: In osteoblastogenic cultures, altered bone matrix production by Fra1 overexpression was monitored using Alizarin red staining, quantitative RT-PCR, and Western blotting. Responsiveness to ovariectomy was examined by bone histomorphometry. Craniofacial parameters were measured on radiographs and using CT. RESULTS: Thrombospondin-1 (Thbs1) and thrombospondin-2 (Thbs2) were reduced in Fra1 Tg osteoblasts differentiated in vitro and in bones from Fra1 Tg mice. Despite alterations in bone matrix proteins, ovariectomy induces high turnover bone loss in Fra1 Tg mice as in wildtype mice. Fra1 Tg mice, as well as Thbs1-/- Thbs2-/- mice, which do not show osteosclerosis, exhibit an edge-to-edge bite phenotype associated with craniofacial dysmorphism. CONCLUSIONS: These data suggest that reduced expression of thrombospondins in Fra1 Tg mice underlies craniofacial dysmorphism, independent of osteosclerosis.

Animals↗

Chronic metabolic acidosis alters osteoblast differentiation from human mesenchymal stem cells.

Bone histology of distal renal tubular acidosis patients showed decreased bone formation with impaired bone matrix mineralization that is not entirely explained by an alteration in the mineral balance. Data from in vitro studies suggests a direct inhibitory effect of metabolic acidosis on osteoblast function. We investigated the effects of chronic metabolic acidosis on osteoblast differentiation from mesenchymal stem cells (MSCs). Human MSCs were allowed to differentiate into osteoblasts in culture. Concentrated hydrochloric acid was added to the medium to lower the bicarbonate concentration and pH. The expression of various osteoblastic genes and proteins and bone matrix mineralization were examined. Chronic metabolic acidosis enhanced the messenger RNA (mRNA) and protein expression of early osteoblast transcription factor, runx-2, whereas inhibiting osterix and having no effect on ATF-4. The expression of type I collagen, the most abundant bone matrix protein, was increased following the same pattern of runx-2. Likewise, metabolic acidosis slightly enhanced the expression of mature osteoblastic gene, osteocalcin. Study on mineralization revealed suppressed alkaline phosphatase mRNA and enzyme activity. Despite the augmented collagen deposit in acidic culture, bone matrix mineralization was impaired. In conclusion, chronic metabolic acidosis alters osteoblast differentiation from MSCs through its diverse effect on osteoblastic genes and proteins resulting in an impairment of bone formation.

Acidosis, Renal Tubular↗

[Cells of bone tissues].

Bone cells consist of osteoblasts, osteocytes, and osteoclasts. Osteoblasts secrete collagen and orientate directions of collagen fibrils to form bone lamellae in the bone matrix. Osteocytes are differentiated in the layer of osteoblasts as functionally different cells from the osteoblasts and sink into the bone matrix to form a network of cell processes to monitor the stress added to the bone. Osteoclasts, multinuclear giant cells on the bone, resorb the bone matrix on the bone surface. These three kinds of bone cells functionally cooperate to each others.

English Abstract↗

The use of demineralized freeze-dried bone-glycoprotein matrix grafts in treating baboon periodontal infrabony defects.

In an effort to improve regenerative capacity and establish higher levels of predictability, the use of a new graft material was investigated in baboon infrabony periodontal defects. A graft matrix composed of human demineralized freeze-dried bone combined with human connective tissue glycoprotein was implanted in 20 defects in 9 baboons. Control, debrided-only sites in the same animal served as a comparison. Grafted sites achieved a 1.8-mm greater reduction in probing depth and attachment gain compared to controls. In addition, 25.3% greater defect fill was found in grafted sites. Histologic analyses provided evidence of bone induction by the graft matrix and regeneration of new cementum, periodontal ligament, and bone in the grafted sites.

Alveolar Bone Loss↗

Chemotaxis of muscle-derived mesenchymal cells to bone-inductive proteins of rat.

In this investigation we examined the chemotaxis of muscle-derived mesenchymal cells from neonatal rats to partially purified extracts of demineralized bone matrix with osteoinductive properties. Using the modified Boyden chamber assay and muscle-derived mesenchymal cells obtained from neonatal Long-Evans rats, we tested the chemotactic properties of the 4 M guanidine-HCl extract from demineralized rat bone matrix and fractions thereof that were derived from sequential Sepharose CL-6B, TSK-3000 and HPLC-C18 chromatography. We have identified that those fractions exhibiting chemotactic properties were also osteoinductive. Therefore, demineralized bone matrix serves as its own soluble signal and insoluble substratum in the inductive process leading to endochondral bone formation in vivo.

Animals↗

Mechanisms underlying the effects of phosphate and calcitonin on bone histology in postmenopausal osteoporosis.

The aim of this study was to evaluate the mechanism underlying the beneficial effect of phosphate combined with calcitonin on trabecular bone mass in postmenopausal osteoporosis. Histomorphometric parameters of trabecular bone formation and resorption were assessed blindly on sections from tetracycline-labeled iliac crest bone biopsies from 44 women with postmenopausal osteoporosis obtained before and after 6 months of treatment with phosphate (n = 9), calcitonin (n = 13), combined therapy (n = 13), or double placebos (n = 9). Treatment with phosphate (1.5 g/day) increased the osteoblastic surface in correlation with the fractional trabecular surface with double tetracycline labeling. The mean wall thickness of the basic structural units increased significantly only in the two groups of patients treated with phosphate. Thus, oral phosphate therapy stimulated bone formation by increasing both the bone-forming surfaces and bone matrix production. The mean interstitial bone thickness, which is inversely related to the depth of resorbing cavities, was increased in the two groups treated with calcitonin (50 IU X 5 days every third week), indicating that calcitonin therapy partially inhibited the resorbing activity of osteoclasts. The combination of calcitonin and phosphate produced a reduction in bone resorption associated with a stimulation of bone matrix production. This effect resulted in a 22.1% increase in the thickness of the trabeculae and a 31.1% increase in trabecular bone volume. The data show that calcitonin combined with phosphate increased the trabecular bone volume in postmenopausal osteoporosis through reduction of bone resorption associated with stimulation of bone formation along the trabecular bone surface.

Administration, Oral↗

In vivo magnetic resonance imaging explorative study of ectopic bone formation in the rat.

In animal studies of tissue engineering of bone, histology remains the standard for assessing bone formation. As longitudinal studies with this method are feasible only at the cost of large numbers of animals, we looked for an alternative. Therefore, demineralized bone matrix (DBM) and inactivated demineralized bone matrix (iDBM) implants were subcutaneously implanted in a rat. At 1, 3, 5, and 7 weeks postimplantation soft X-ray and magnetic resonance imaging (MRI) were done to monitor bone formation in the implants. At 7 weeks, the animal was killed and the implants were retrieved for histology. Our results showed that in vivo MRI is well suited to assess bone formation larger than 0.5 mm in diameter and to monitor the complete three-dimensional shape of the newly formed bone noninvasively and longitudinally. The MRI results matched well with the histology results obtained at 7 weeks. In contrast, X-ray imaging appeared inappropriate to monitor the bone formation process in DBM.

Animals↗

Proteoglycans of alveolar bone of diabetic and non-diabetic mice: a histochemical study.

The effect of diabetes mellitus on the interdental alveolar bone has been long debated. The present study reported the distribution of glycosaminoglycans (GAG) in normal and diabetic alveolar bone using histochemical techniques. Animals were rendered diabetic and killed at 2, 4, 6 and 9 weeks after injections. Tissues were stained with Alcian blue 8GX dye (pH 2.5) to demonstrate GAG and the intensity of the staining reactions compared with age-matched controls. During the experiment, weights of control animals did not change significantly; weights of diabetic animals were significantly less than initial weights from 0-6 weeks (p less than 0.001), but became nearly equal by 9 weeks. Staining intensity of diabetic bone demonstrated initial decrease (0-4 weeks) followed by a marked increase (4-9 weeks) suggesting an early decline in bone GAG levels followed by increased bone GAG levels as compared to age-matched control and initial levels. Bone GAG levels were significantly different between diabetics and age-matched controls at 2 (p less than 0.005) 4 (p less than 0.001), 6 (p less than 0.001) and 9 (p less than 0.001) weeks after streptozotocin injections. Digestion with chondroitinase AC, ABC and streptomyces hyaluronidase suggested significant differences between control and diabetic bone matrix in the levels of chondroitin 4 and 6 sulfates (p less than 0.05) and hyaluronic acid (p less than 0.001) but not dermatan sulfate. In control and diabetic bone, chondroitin sulfates were located within the bone matrix, dermatan sulfate within bone matrix and Sharpey fiber bundles.(ABSTRACT TRUNCATED AT 250 WORDS)

Alveolar Process↗

In vitro bone formation induced by immunosuppressive agent tacrolimus hydrate (FK506).

When rat bone marrow cells were cultured with an immunosuppressive agent, tacrolimus hydrate (FK506), as well as with beta-glycerophosphate and vitamin C, numerous cell clusters became positive for alkaline phosphatase activity. Scanning electron microscopy revealed mineralized bone matrix in the cell clusters, which was identical to that of living bone. High levels of alkaline phosphatase (ALP), indicating osteoblastic activity, and high levels of osteocalcin (Oc) and calcium were found in the mature bone matrix of the cultures. There was significantly increased expression of mRNAs for ALP and Oc. These results indicate that the cultures contained both bone matrix and high osteoblastic activity, suggesting that FK506 induces ossification.

Animals↗

[Bone replacement using demineralized allogeneic bone in animal experiments].

The implantation of bone matrix in muscle bag or trepanation defects of rats with greatest reproducibility will induce osteogenesis and defect healing. The phenomenon of osteo-induction was possible to obtain only in first steps in higher developed mammals. Investigations in nude rats have shown a clear difference in biologic reaction on the matrix of various species. The osteo-regeneration in replacement weak defect of ulnar bone in dogs showed a massive greater reactivity of autogenous spongiosa, whereas bank spongiosa and bone matrix produced not more new bone as the control. An increase of osteoinductive matrix potency is a supposition for practical use in case of severe bone defects.

Animals↗

Increased cartilage and bone formation in spontaneously hypercholesterolemic rats.

Spontaneously hypercholesterolemic (SHC) rats are known to exhibit accelerated bone resorption. We compared endochondral bone formation induced by implantation of demineralized bone matrix (DBM) to 4-week-old SHC rats with that of age-matched Sprague-Dawley (SD) rats. When DBM prepared from adult SD rats was implanted, the cartilageous area enlarged, and C-propeptide of type II procollagen content on day 7 was higher in SHC rats. Alkaline phosphatase activity and calcium content on day 12 and tartrate-resistant acid phosphatase activity on day 19 were higher in SHC rats. These results suggest active chondrogenesis, with a subsequent increase in osteogenesis, and stimulated osteoclastic bone resorption in SHC rats. When DBM from 10-week-old SHC rats was implanted into SD or SHC rats, the levels of bone forming parameters on day 12 were reduced to one-third, suggesting inhibiting factor(s) for bone induction in bone matrix of SHC rats. In contrast, when DBM from 6-month-old SHC rats was implanted, although bone forming parameters in SD rats were comparable to the case of implantation of DBM from SD rats, the accelerated bone formation detected in SHC rats was blocked, indicating resistance to systemic bone inducing factor(s) of SHC rats in aged bone matrix. These results suggest that age-related decrease in responses to some systemic bone inducing factor may lead to the bone loss with advancing age.

Acid Phosphatase↗

Validation of quantitative backscattered electron imaging for the measurement of mineral density distribution in human bone biopsies.

The measurement of bone mineral density (BMD) using X-rays is usually employed to monitor the mineral content in a given portion of bone. However, this method cannot differentiate between changes in bone volume or in degree of mineralization of the bone matrix. In contrast to BMD, bone mineral density distribution (BMDD), as measured on bone sections by quantitative backscattered electron imaging (qBEI), is able to distinguish differences in the degree of mineralization. For routine clinical research, we have validated the method of calibration and standardization of the backscattered electron (BE) signal. Carbon and aluminum were used as reference materials for BE gray levels and osteoid and apatite for calcium concentration. Experiments were performed to get knowledge about precision (intraassay variance-instrumental stability and interassay variance-reproducibility) and accuracy (standardization) of this method as well as the biological variance (intraindividual and interindividual) in human bone. On transiliac biopsies or necropsies from 20 individuals having had accidental death (13 females, 7 males, age 30-85 years) BMDD measurements were conducted. The patients' medical history as well as the histomorphology of these bones showed no evidence of metabolic bone disease. For instance, the standard deviations of the weighted mean calcium concentrations were <0.3%, <0.4%, <0.9%, and <2.6% of the mean for the intraassay, interassay, intraindividual, and interindividual variations, respectively. In addition, a mean BMDD histogram for transiliac bone specimens was calculated from the 20 aforementioned individuals. The method used allows detection of the degree of mineralization independently from the actual bone volume, a result that seems to be of special interest in the assessment of the effect of treatments for osteoporosis. The power of this technique is demonstrated by using bone from a patient with a metabolic bone disease. In this case of osteomalacia due to celiac disease, the mean calcium concentration in the bone matrix was reduced by 19.3% as compared with normal.

Adult↗

Novel explant model to study mechanotransduction and cell-cell communication.

To understand in situ behavior of osteocytes, we characterized a model of osteocytes in their native bone matrix and demonstrated real-time biologic activity of osteocytes while bending the bone matrix. Using 43 male Sprague-Dawley rats, dumbbell-shaped explants were harvested from stainless steel femoral implants after 6-12 weeks and incubated in culture medium or fixed. Sixteen specimens were used to determine bone volume density (BV/TV), volumetric bone mineral density (BMD) and histology for different implantation periods. Osteocyte viability was evaluated by L-lactate dehydrogenase (LDH) activity in 12 cultured explants. Confocal microscopy was used to assess tracer diffusion in three explants and changes in osteocyte pH of a mechanically loaded explant. From 6 to 12 weeks, explant BV/TV and volumetric BMD trended up 92.5% and 101%, respectively. They were significantly and highly correlated. Tissues were uniformly intramembranous and all bone cell types were present. Explants maintained LDH activity through culture day 8. Diffusion at 200 microM was limited to 1,209 Da. Explants appeared capable of reproducing complex bone biology. This model may be useful in understanding osteocyte mechanotransduction in the context of a physiologically relevant bone matrix.

Animals↗

Calcium, physical activity and bone health--building bones for a stronger future.

Adequate provision of nutrients composing the bone matrix and regulating bone metabolism should be provided from birth in order to achieve maximal bone mass, compatible with individual genetic background, and to prevent osteoporosis later in life. Low calcium intake (<250 mg day(-1)) in children is associated with both a reduced bone mineral content and hyperparathyroidism. Optimal calcium intake is, however, still a matter of controversy. The minimisation of fracture risk would be the ideal functional outcome on which to evaluate lifetime calcium intakes, but proxy indicators, such as bone mass measurements or maximal calcium retention, are used instead. Calcium recommendations in Europe and the United States are based on different concepts as to requirements, leading to somewhat different interpretations of dietary adequacy. Minerals and trace elements other than calcium are involved in skeletal growth, some of them as matrix constituents, such as magnesium and fluoride, others as components of enzymatic systems involved in matrix turnover, such as zinc, copper and manganese. Vitamins also play a role in calcium metabolism (e.g. vitamin D) or as co-factors of key enzymes for skeletal metabolism (e.g. vitamins C and K). Physical activity has different effects on bone depending on its intensity, frequency, duration and the age at which it is started. The anabolic effect on bone is greater in adolescence and as a result of weight-bearing exercise. Adequate intakes of calcium appear necessary for exercise to have its bone stimulating action.

Adolescent↗

[Bone remodeling: evaluation methods].

Bone tissue undergoes continual renewal in order to keep the mechanical competence of the bone matrix. This is performed by two cells: osteoclasts which resorb the calcified matrix and osteoblast which synthetize a new bone matrix. Biochemical markers of bone remodelling allow the evaluation of the activity of each type of bone cells. An unbalance in this process induces modifications of the amount of calcified tissue and of the bone microarchitecture. Several technics based on X-ray and ultrasound allow a non invasive evaluation of these modifications.

Biomarkers↗

Spaceflight results in formation of defective bone.

Growing rats were flown on 19 day spaceflights aboard Cosmos 782 and 936 biosatellites. Spaceflight resulted in a prominent skeletal defect at the periosteal surface of the tibia diaphysis. The defect, termed an arrest line, was approximately 3 micron across and separated the bone formed in space from that formed following spaceflight. The bone matrix at the arrest line region was abnormal in that collagen fibers were preferentially orientated parallel to the periosteal surface. In addition, the bone matrix was hypomineralized. The altered bone was inferior to normal bone in resistance to abrasion and may be partially responsible for the decrease in torsional strength observed after spaceflight.

Animals↗

Allogenic bone and cartilage morphogenesis. Rat BMP in vivo and in vitro.

An allogenic aggregate of bone morphogenetic protein (BMP) and insoluble non-collagenous proteins (NCP) as well as a crude GuHCl extract were isolated from rats diaphyseal bones. Intramuscular implantation of 5 mg and 10 mg rat BMP/NCP in rats formed new ossicles, whereas 20 mg GuHCl extract failed to induce heterotopic bone formation. When 6 samples of inactivated rat bone matrix gelatin (BMG) were reconstituted with 0.75 mg of either BMP/NCP or GuHCl extract all 3 matrices reconstituted with BMP/NCP but only 1 out of 3 samples reconstituted with GuHCl extract induced heterotopic bone formation. Inactivated BMG alone did not show any osteoinductive activity. The small amount of BMP/NCP necessary for osteoinduction when recombined with inactivated BMG suggests that growth factors in bone matrix without inherent bone-forming activity enhance BMP activity. In vitro, connective tissue outgrowths of neonatal rat muscle on a substratum of inactivated rat BMG differentiated into cartilage in response to 0.05 microgram/ml, 0.5 microgram/ml and 5.0 micrograms/ml allogenic BMP/NCP added to the medium during the incubation period of 2 weeks. On day 14 of cultivation S35-sulphate incorporation into glycosaminoglycans (GAG) and H3-thymidine incorporation into DNA were measured, and the results related to the DNA content and the weight of the incubated muscle tissue, respectively. All doses of BMP/NCP increased GAG synthesis statistically significantly (p less than 0.05 to p less than 0.001). In contrast to that, DNA synthesis rate was not influenced by BMP/NCP. This suggests that GAG synthesis was not caused by cell proliferation but by cell differentiation.

Abdominal Muscles↗