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PubMed1992

Lanthanum tracer and freeze-fracture studies suggest that compartmentalisation of early bone matrix may be related to initial mineralisation.

In adult bone the calcified matrix and enclosed osteocytes are separated from the extracellular space by a continuous layer of bone lining cells. It thus appears that bone matrix is compartmentalised and, as such, may constitute a 'milieu intérieur' which is different from the general extracellular space. Since adult bone matrix is compartmentalised and matrix vesicles also form a microcompartment, it is conceivable that compartmentalisation, in early osteogenesis, may be a requirement for the initial events of the mineralisation process. We have therefore conducted an ultrastructural, tracer, and freeze-fracture study to determine the stage in which bone matrix becomes compartmentalised and also to find out whether there are tight junctions between osteoblasts. The results show that in early nonmineralised stages and in incipient mineralisation, lanthanum penetrates all intercellular spaces and the newly forming bone matrix which is rich in matrix vesicles and collagen. With the progression of mineralisation, when all matrix vesicles appear mineralised and calcification is 'spreading' to the surrounding matrix, lanthanum is restricted to intercellular spaces and conspicuous macular tight junctions are present between osteoblasts. We suggest that matrix vesicles act as microcompartments for calcification when the early bone matrix is in continuity with the surrounding extracellular space. In later stages, when lanthanum fails to penetrate the matrix, matrix vesicles may no longer be necessary because the bone matrix itself is compartmentalised, thus allowing for localised changes in composition that might favour mineral deposition.

Animals↗

PubMed1991

Altered differentiation of limb bud cells by transforming growth factors-beta isolated from bone matrix and from platelets.

A crude extract of demineralized bone matrix caused an altered differentiation of limb bud cells which was seen within 5 days in culture. Using this bioassay system we purified two factors to homogeneity and found that according to their N-terminal sequences they corresponded to TGF-beta 1 and TGF-beta 2 isolated from platelets. Biochemical analyses and biological studies (molecular mass determination, inactivation by reducing agents and proteases, antibody neutralization, competitive binding to TGF-beta receptors and influence on protein expression) provided additional evidence that the two proteins isolated from demineralized bone matrix were apparently identical to TGF-beta 1 and TGF-beta 2. Proteoglycan content, alkaline phosphatase activity and response of the cells to PTH stimulated adenylate cyclase were quantitatively changed by the factors. Culturing limb bud cells on polycarbonate membranes resulted in a rapid and extensive growth and differentiation of the cells to palpable tissue pieces. Relative to controls distinct cell and tissue morphology was observed macroscopically and in histological sections of these tissue pieces.

Alkaline Phosphatase↗

PubMed1985

[Osteogenesis induced by the addition of demineralized bone matrix to plaster pellets with antibiotics. Animal experiment].

The authors have previously shown the role of antibiotic loaded plaster-of-Paris pellets in the treatment of bone loss. In the present paper, a study has been made of the effects of osteogenesis of added decalcified bone matrix in powder. The experiment was made by the implantation of pellets in the muscles of rabbits. It was shown that bone induction by pellets of bone matrix was slightly lower when Rifamycin was added and was not modified by the addition of Fucidin or Gentamycin. The exudation of antibiotics was not diminished by the addition of bone matrix to plaster-of-Paris pellets. The double action, on infection and on bone induction, of composite pellets of plaster-of-Paris with antibiotics and bone matrix may lead to their use in the treatment of septic bone cavities since the pellets are absorbable.

Animals↗

PubMed1995

Sulfate incorporation into organic bone matrix of the tibiotarsus of broiler chicks is reduced by excess dietary methionine.

Two experiments were conducted in broiler chicks to determine whether dietary imbalances of sulfur amino acids (SAA), vitamin A, or interactions between the two nutrients could influence organic bone matrix metabolism measured with L-[35S]-methionine. In the first experiment, in vivo incorporation of 35S into the tibiotarsal bone matrix of 2-wk-old birds was unaffected by vitamin A treatment of 10 and 100 times the requirement when compared with that of birds receiving recommended amounts of vitamin A. However, 35S incorporation was significantly reduced by increasing the SAA concentration of the diet to 1.5 times the requirement relative to lysine. In the second experiment, in vitro incorporation of 35S, derived from L-[35S]-methionine, into bone matrix was reduced in birds consuming a diet containing 1.5 times the methionine requirement relative to lysine (Diet HS) when compared with those receiving .75 (Diet LS), 1.0 (Diet NS), or 1.25 (Diet MS) times the requirement. Birds consuming Diet LS incorporated significantly more 35S into organic bone matrix than birds consuming the other three diets. Although the ratio of SAA to lysine was that recommended (.76:1), on a weight basis the concentration of SAA in diet NS was relatively high (11.48 g/kg diet) compared with the NRC (1984) recommendation of 9.3 g/kg diet. The results show that excess SAA can affect organic bone matrix metabolism and suggest that SAA may play a role in the etiology of tibial dyschondroplasia. They also indicate the importance of distinguishing between nutrient content of the diet expressed as a ratio and that expressed on a weight basis.

Animals↗

PubMed1992

[The effect of various sterilization procedures on the osteoinductive properties of demineralized bone matrix].

To minimize potential infection following the transplantation of allogeneic bone, extremely rigorous selection of donors and careful processing and storage of samples are required. Other major problems related to allogeneic transplants, such as reduced osteogenic properties and immunological reactions, led to the development of demineralized bone matrix (DBM). This osteoinductive bone extract is largely free of antigens and is easy to produce. However, to eliminate the potential risk of infection, DBM should be sterilized prior to implantation. The purpose of this study was to investigate the influence of different sterilization techniques on the osteoinductive properties of DBM. A series of 76 cortical defects (drill holes) 0.6 cm in diameter in the tibiae of 11 Merino sheep were filled with DBM in addition to autogeneic and allogeneic cancellous bone. Prior to implantation DBM was sterilized by autoclaving, gamma irradiation, or application of ethylene oxide or ethyl alcohol. A further 12 drill holes were left empty as controls. The formation of new bone was examined 3 and 6 weeks postoperatively, using histological, fluorescent-optical and microradiographical techniques. The amount of newly formed bone was also quantified. Apart from autoclaved DBM all matrix grafts showed excellent new bone formation following sterilization, by far exceeding the formation with allogeneic cancellous bone.

Animals↗

PubMed2004

Osteoblast-like cells complete osteoclastic bone resorption and form new mineralized bone matrix in vitro.

Bone remodeling involves old bone resorption by osteoclasts and new bone formation by osteoblasts. However, the precise cellular mechanisms underlying these consecutive events remain obscure. To address this question in vitro, we have established a cell culture model in which the resorption lacunae are first created by osteoclasts and osteoblast-like cells accomplish the subsequent bone formation. We isolated osteoclasts from rat bone marrow and cultured them on bovine bone slices for 48 hours to create resorption lacunae. After removing osteoclasts, confluent differentiated primary osteoblast cultures were trypsinized and the cells were replaced on the resorbed bone slices for up to 14 days. The cultures were then examined by confocal microscopy, field emission scanning electron microscopy (FESEM), and transmission electron microscopy (TEM). Our data suggest that after osteoclastic bone resorption, osteoblast-like cells, not macrophages, remove the remaining organic matrix in the lacuna. After cleaning the lacuna, osteoblast-like cells deposit new collagen fibrils at the bottom of the lacuna and calcify the newly formed matrix only, as visualized by labeled tetracycline accumulation merely in the lacuna during the osteoblast culture. Furthermore, an electron-dense layer rich in osteopontin separates the old and new matrices suggesting formation of the cement line. Since the morphology of the newly formed matrix is similar to the natural bone with respect to the cement line and osteoid formation as well as matrix mineralization, the present method provides for the first time a powerful in vitro method to study the cellular mechanisms leading to bone remodeling also in vivo.

Animals↗

PubMed1982

Experimental osteogenesis with demineralized allogeneic bone matrix in extraskeletal sites.

Male albino rats were used to study the osteogenic potential of demineralized allogeneic bone matrix (DABM) grafts, in different stages of demineralization, placed in the anterior abdominal wall. The clinical evaluation, radiologic examination, calcium determination, and histologic (light and electron microscopic) studies showed that completely demineralized allogeneic bone matrix (CDABM) grafts were not rejected and that they stimulated new bone formation at the transplant sites. On the other hand, partially demineralized allogeneic bone matrix (PDABM) grafts were frequently rejected and showed minimal bone induction and gradual demineralization.

Abdominal Muscles↗

PubMed1981

[Morphology of the induction process during following bone matrix implantation in mice].

A decalcified bone matrix was implanted intramuscularly to normal mice, to mice irradiated by 450 R, to mice with curretaged femur and beneath the renal capsule in normal mice. In the course of implantation the bone induction was noted in 80% of cases. In all the series of experiments, the implants had different degrees of maturity of induced bone tissue and different extent of repopulation of the induced bone by hemopoietic cells. Hemopoietic cells inhabited young osteogenic tissue with a well marked osteoblastic layer and formed bone marrow cavity. No hemopoiesis was observed in the case of induction of cavity-free bone tissue plates or mature bones with bone marrow cavity but without osteoblastic layer.

Animals↗

PubMed1990

Inductive influences of demineralized dentin and bone matrix on pulp cells: an approach of secondary dentinogenesis.

The effects of demineralized dentin and bone matrix on dental ectomesenchymal cells were evaluated after observation periods of two or three weeks. Autogenous dentin and bone matrix, obtained from the crowns of primary molars or maxillary cortical bone, respectively, were demineralized with 3% acetic acid and implanted into pulpal or papilla sites of erupting dog teeth: first molars, fourth premolars, and canines. Dentin histogenesis associated with odontoblastic arrangement was demonstrated in relation to all dentin implants in pulpal sites. Deposition of osteodentin, followed in some areas by tubular predentin formation, was observed in contact with bone implants in pulpal sites. In papilla sites, the dentin implantation exhibited bone-like matrix formation, while bone implants were encapsulated by connective tissue. The interactions of pulp cells with demineralized dentin matrix constitute a model for experimental induction of secondary dentinogenesis and odontoblast-like cell differentiation.

Animals↗

PubMed1994

Bone matrix RGD glycoproteins: immunolocalization and interaction with human primary osteoblastic bone cells in vitro.

The interaction of cells with extracellular matrix is essential for their anchorage, proliferation, migration, and differentiation. In bone matrix there are multiple glycoproteins that contain the integrin-binding RGD sequence: fibronectin (FN), thrombospondin (TSP), osteopontin (OPN), bone sialoprotein (BSP), type I collagen (COLL I), and vitronectin (VN). In this study, the localization of TSP, FN, VN, and several integrins within developing human long bone using immunohistochemical methods was examined, as was the effect of all bone RGD proteins on the adhesion of human osteoblastic cells. Thrombospondin, fibronectin, and vitronectin showed distinct localization patterns within bone tissue. TSP was found mainly in osteoid and the periosteum; VN appeared to be present mainly in mature bone matrix. FN was present in the periosteum as well as within both mature and immature bone matrix. Using a panel of antiintegrin antibodies we found that bone cells in vivo and in vitro express alpha 4, alpha v, alpha 5 beta 1, alpha v beta 3, and beta 3/beta 5 integrins, and these receptors are for the most part expressed on all bone cells at different stages of maturation with quantitative rather than qualitative variations, with the exception of alpha 4, which is expressed mainly by osteoblasts. Cell attachment assays were performed using primary human cells of the osteoblastic lineage under serum-free conditions. COLL I, TSP, VN, FN, OPN, and BSP promoted bone cell attachment in a dose-dependent manner and were equivalent in action when used in equimolar concentrations. In the presence of GRGDS peptide in the medium, the adhesion to BSP, OPN, and VN was almost completely blocked (10, 10, and 15% of control, respectively), and attachment to FN, COLL I, and TSP was only slightly decreased (80, 75, and 55%, respectively). These results suggest that human bone cells may use RGD-independent mechanisms for attachment to the latter glycoproteins.

Alkaline Phosphatase↗

PubMed1983

Defective binding of macrophages to bone in rodent osteomalacia and vitamin D deficiency. In vitro evidence for a cellular defect and altered saccharides in the bone matrix.

In the osteomalacic as well as normal skeleton, few osteoclasts are associated with osteoid-covered bone surfaces. The reason for this particular cellular deficit is not clear, but may relate to the inability of osteoclasts and/or osteoclast precursors (monocyte-macrophages) to attach to immature, unmineralized bone matrix, a step apparently essential for normal resorptive activity and osteoclast differentiation. In this study, we have examined cell-bone binding using macrophages (M phi) and bone isolated from vitamin D-deficient rats and hypophosphatemic, osteomalacic mice and from their normal counterparts. The data show that M phi-bone attachment is greatly reduced (P less than 0.001) in both vitamin D deficiency and hypophosphatemia, but that the mechanisms responsible for this reduction are apparently different in the two disorders. In hypophosphatemia, the reduction in binding appears solely attributable to the absence or inaccessibility of bone matrix oligosaccharides or glycoproteins essential to the attachment process. In vitamin D deficiency, on the other hand, not only is the bone matrix defective as a binding substrate, but the M phi, per se, is limited in its capacity to attach to normal, vitamin D-deficient, and hypophosphatemic bone.

Animals↗

PubMed2004

[Use of bone-matrix transplants in rhinoplastic operations].

Defects of the external nose and nasal septum were corrected using bone-matrix transplants (bone matrix-BM and embryonic matrix-EM) in 220 patients. EM transplants were more osteoinductive. Functional studies show that demineralized transplants, especially EM, activate both microcirculation in the implantation focus, adjacent tissue and metabolic processes which improve the results of the functional tests in rhinoplasty.

Bone Matrix↗

PubMedDate not supplied

Phospholipids of a bone matrix calcification nucleator.

Phospholipids of a bone matrix calcification nucleator are identified as mono and diphosphoinositides and phosphatidyl serine. The nucleator, a protein-phospholipid complex, was dissociated by acidified-solvent porous-glass column chromatography. Analysis was by gas-liquid chromatography.

Actinomycetaceae↗

PubMed1991

The induction of bone in osteogenic composites of bone matrix and porous hydroxyapatite replicas: an experimental study on the baboon (Papio ursinus).

This study evaluated the morphogenetic properties of osteogenic composites of bone matrix and porous coralline hydroxyapatite (HA) replicas after intramuscular implantation in adult baboons (Papio ursinus). Composite implants were prepared by inserting rods of nonresorbable HA replicas, 20 mm in length, and 5 or 7 mm in diameter, into the medullary canals of 90 diaphyseal baboon bone cylinders, 20 mm in height, sequentially extracted and chemosterilized to obtain autolysed antigen-extracted allogeneic (AAA) bone matrix, preserving the bone morphogenetic protein (BMP) activity. Composites were implanted in the rectus abdominis and in the dorsal musculature of 24 adult male baboons. Before implantation, 45 composites were coated with an allogeneic fibrin-fibronectin protein concentrate (AFFP) prepared from fresh-frozen baboon plasma. Histologic analysis of undecalcified and decalcified specimens procured at 3, 6, and 9 months showed bone differentiation by induction along the endosteal surfaces of the chemosterilized matrix and within the porous spaces of the HA substratum. Bone formation was often extensive, culminating in complete penetration of the porous spaces. Histomorphometry showed that bone in the HA substratum increased at each time period. The biochemical treatment of the composite implants with AFFP did increase significantly the amount of induced bone, although only in the HA substratum. Bone differentiation was confirmed by fluorescence microscopy of the mineralization fronts after intravital double tetracycline labeling. The differentiation of bone in composites of bone matrix and porous HA replicas in extraskeletal sites of adult baboons may help to design appropriate delivery systems for the controlled therapeutic initiation of bone formation for craniofacial and orthopedic applications in man.

Animals↗

PubMed1990

Stimulation of bone matrix apposition in vitro by local growth factors: a comparison between insulin-like growth factor I, platelet-derived growth factor, and transforming growth factor beta.

Many recent in vitro studies have shown effects of insulin-like growth factor I (IGF I), platelet-derived growth factor (PDGF), and transforming growth factor-beta (TGF beta) on the proliferation and differential functions of bone-forming osteoblasts; however, the question whether these factors might ultimately lead to a net increase or decrease in bone formation has been difficult to assess. In this study, we have used an autoradiographic method based on the incorporation of [3H]proline into freshly synthesized bone matrix to determine the overall effects of these factors on bone matrix apposition in 21-day-old fetal rat calvariae. IGF I, PDGF, and TGF beta increased bone matrix apposition in a dose-dependent manner up to 2-fold within 48 h. In addition, they partially or completely reversed the inhibition of bone matrix apposition observed with PTH. Exogenously added TGF beta was significantly more potent than equimolar concentrations of PDGF or IGF I in stimulating bone formation. Matrix apposition was greatest when IGF I, PDGF, and TGF beta were added simultaneously to the culture medium, indicating that these factors can enhance each other in stimulating bone formation. In conclusion, our results provide direct evidence that IGF I, PDGF, and TGF beta are capable of stimulating bone formation in vitro.

Animals↗

PubMed1977

Proteolipid and bone matrix calcification in vitro.

Proteolipid was demonstrated to contain the nucleator of bone matrix calcification, in vitro. Crude phospholipid extracted from bone matrix was fractionated by gel filtration. A single, protein-containing fraction induced apatite crystallization in a metastable calcium phosphate solution. The fraction was identified as proteolipid. The result supports the validity of a microbiologic analogue for vertebrate calcification.

Actinomyces↗

PubMed1995

Response to demineralized bone matrix implantation in foals and adult horses.

Equine demineralized bone matrix, particle size 2 to 4 mm, was implanted SC and IM in 4 foals and 4 adult horses. The implants were removed between 5 and 8 weeks after implantation. Bone formation was induced by SC and IM implantations in all animals. The implantation site had a marked effect on the amount of bone that developed, bone being formed earlier and in greater amounts when the matrix was implanted IM. The amount of bone formed increased with increasing time after matrix implantation at both sites. Demineralized bone matrix implantation also led to formation of small amounts of chondroid tissue; this tissue was more common in IM than SC matrix implants, and increased in amount with increasing time after implantation. Formation of this chondroid tissue did not precede the formation of bone, and there was no evidence that implantation of demineralized bone matrix in horses induced endochondral ossification. Age of the host did not appear to affect the response.

Age Factors↗

PubMed1956

Electron microscopy of cartilage and bone matrix at the distal epiphyseal line of the femur in the newborn infant.

An examination of the fine structure of cartilage and bone matrix at the distal epiphyseal line of the femur of a newborn infant has revealed the following information. Cartilage matrix is composed of a network of widely spaced fibers without obvious periodic banding. Calcification is first seen about the level of the third chondrocyte capsule distal to the furthest penetration of the capillaries. It starts as a haphazard deposition of crystals which have no obvious relationship to the location of the fibers. The process of calcification is completed before ossification commences but the central zone of matrix remains only partly mineralized. Bone matrix is formed over a bar of calcified cartilage. Fibers, recognizable as collagen, are deposited in a loose network in a narrow zone between the osteoblasts and cartilage. These fibers are 2 to 5 times as wide as the fibers in epiphyseal cartilage. Calcification then begins in the osteoid, crystals being first laid down irregularly on or close to the fibers. As they increase in number, the crystals tend to line up along the fibers and eventually are arranged so that the periodicity of the underlying collagen is emphasized. In such an area the fibers are more tightly packed than when uncalcified. There is no change observed in the calcified cartilage at this level. The extracellular matrices of this epiphyseal cartilage and bone can be distinguished from one another in the electron microscope.

Bone Matrix↗