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PubMed1990

[Vertebral trabecular bone in various age groups and in osteoporosis-- morphometry and bone matrix biochemistry].

Vertebral trabecular bone was analysed by morphometry and bone matrix biochemistry. Trabecular bone volume (TBV) and mean trabecular plate thickness (MTPT) decreased with age. TBV was significantly correlated with MTPT and mean trabecular plate density (MTPD). The individual structure of trabecular bone could be described by both MTPT and MTPD together, but changes of these parameters, that were pathognomonic for osteopenia, were not found. By measuring TBV 3 cases of severe osteopenia were identified (TBV less than 2s of controls); 2 of them showed matrix abnormalities so far not described. In one case (a 67 year old woman without risk factors for osteoporosis) an abnormal high content of type III collagen was found, in the other case (a 44 year old woman with acromegaly) bone matrix analysis atypically revealed a significant fraction of type II collagen. Further studies will be needed to assess the pathogenetic or diagnostic importance of these new findings.

Adult↗
PubMed2005

Role of bovine bone morphogenetic proteins in bone matrix protein and osteoblast-related gene expression during rat bone marrow stromal cell differentiation.

Bone morphogenetic proteins (BMPs) are known to promote osteogenesis, and clinical trials are currently underway evaluating the ability of certain BMPs to promote bone graft and fracture healing. To observe the mechanism of osteoinductive and bone formation, 100 microg of bovine BMP was tested during osteogenic differentiation of rat bone marrow stromal cells (MSCs) and C2C12 line culture for 14 and 28 days. We examined alkaline phosphatase (ALP) by assay, immunohistochemical studies for bone matrix proteins, and mRNA expression of bone matrix proteins and osteoblast-related analysis by reverse-transcription polymerase chain reaction. ALP activity in MSC cultures was elevated by bovine BMP by two to fivefold (P < 0.05-0.001). DNA and protein content increased over 14 days. BMP significantly increased the mRNA expression of type I collagen, ALP, osterix, osteocalcin, osteopontin, vascular endothelial growth factor (VEGF), platelet-derived growth factor (PDGF)-A, and parathyroid hormone receptor time dependently during the osteoblastic differentiation. There was no markedly enhanced mRNA expression of bone sialoprotein (BSP) and glyceraldehyde-3-phosphate dehydrogenase compared with that of control. Immunohistochemical results also showed BMP increased immunoreactive positivity of type I collagen, osteocalcin, osteonectin, osteopontin, and BSP during the C2C12 differentiation. These data indicated that BMP enhances our ability to stimulate the differentiation of osteoblast-like cells and increases osteoinductivity, bone matrix protein formation and mineralization, angiogenesis, and chondrogenesis during osteoblast progenitor cell differentiation in vitro and that the role of chondrogenic is weak.

Alkaline Phosphatase↗
PubMed1992

Osteoclast recruitment in response to human bone matrix is age related.

The effect of bone matrix age on the recruitment and differentiation of osteoclast precursors was studied using the chick chorioallantoic membrane (CAM) implant system. Devitalized mineralized bone particles (75-250 microns) were prepared from human femoral cortical bone obtained postmortem from 8 men (age range: 18-72 years). The particles were implanted onto the CAM and 8 days later implants were harvested and processed for light microscopic, morphometric or immunohistochemical analysis. Histomorphometric analysis was performed on samples representing each donor age. The analysis was grouped into three categories consisting of bone from young adults (18-20 years), adults (34-53 years) and aged individuals (67 years and older). Total osteoclast number, osteoclast number per bone particle, cell area, cell size, number of nuclei per cell profile, nucleocytoplasmic ratio, and the presence of a distinctive osteoclast antigen defined by monoclonal anti-body 121F were determined. Bone matrix from older individuals, and therefore the oldest age group (67 years and older), elicited significantly fewer multinucleated cells when compared to bone matrix from younger donors. The number of nuclei per cell profile was highest in the adult population (34-53 years), and there was a continuous increase in cell area with aging. As a consequence, the nucleocytoplasmic ratio decreased from the youngest to the oldest age group. These findings indicate that, relative to factors that affect the recruitment and differentiation of osteoclast precursor cells, bone matrix of older individuals is changed in quality and/or quantity compared to bone matrix from younger individuals. It is hypothesized that this decline in osteoclast formation in response to older bone matrix may contribute to the impaired bone remodeling associated with aging.

Adolescent↗
PubMed1986

The use of demineralized bone matrix in the repair of segmental defects. Augmentation with extracted matrix proteins and a comparison with autologous grafts.

A soluble protein component of bone, bone morphogenetic protein, and decalcified bone matrix have been shown to induce the formation of bone in extraosseous tissue. Clinical and animal studies investigating the use of these materials as bone grafts have shown radiographic and histological evidence of formation of bone, but the clinical usefulness of these grafts remains unknown. This study compared the healing processes when plasma-coated demineralized bone matrix and autologous cancellous bone were used to graft segmental defects of bone. A standard procedure was used to make a two-centimeter defect bilaterally in the ulna of forty-eight skeletally mature New Zealand White rabbits. In each rabbit, one ulnar defect was grafted with autologous citrated plasma-coated demineralized bone matrix while the other defect served as a control and was grafted with either autologous cancellous bone from the iliac crest, demineralized bone matrix, or demineralized bone matrix augmented with bone proteins that had been extracted with guanidinium hydrochloride. The ulnar defect was stabilized by the intact radius, and no supplemental device was necessary for fixation. To examine spontaneous healing in this model, one group of rabbits had a control defect that was not grafted. The grafts were periodically evaluated by radiographs, and twelve weeks after surgery the grafts were harvested and tested to failure in a standard torsion-test machine. The mechanical parameters were calculated, and histological examination of major fragments of the grafts was performed. The results of the radiographic and histological evaluation showed that all of the grafted ulnae healed, with fusion of the graft to the cut ends of the defect and reformation of approximately normal anatomy. No ungrafted ulnar defects healed. The results from the mechanical tests were evaluated by comparing the defect that was grafted with plasma-coated demineralized bone matrix with the control graft in each animal. These data showed that: twelve weeks after grafting, the normal ulnae were significantly stronger than the ulnae that had been grafted with plasma-coated demineralized bone matrix; the ulnae that had been grafted with plasma-coated demineralized bone matrix and those that had been grafted with autologous bone were equivalent in strength; and twelve weeks after grafting, grafts of demineralized bone matrix that were augmented with extracted bone proteins were significantly stronger than those that had not been so augmented.

Amino Acids↗
PubMed1993

Skeletal growth after oral administration of demineralized bone matrix.

Oral administration of bone extracts obtained from bovine demineralized bone matrix to rats has a direct effect on bone metabolism, affecting bone proportions and some markers of bone formation such as bone malate dehydrogenase, serum alkaline phosphatase and serum osteocalcin. Furthermore collagen deposition, bone protein synthesis and nucleic acids content were significantly increased by the treatment.

Administration, Oral↗
PubMed1996

Partial purification and characterization of bone morphogenetic protein from bone matrix of the premature moose (Alces alces): degradation of bone-inducing activity during storage.

In spite of the advances in recombinant techniques in the production of bone morphogenetic proteins (BMPs), the best clinical results so far have been obtained with human and animal source-extracted BMPs. Also, the poor availability of recombinant products gives rise to continued research with different extracted and purified proteins. In a search for a new source of bone-matrix-derived BMP with high osteoinductive activity, BMP was extracted from fresh bone matrix of the premature moose (Alces alces). Bone-inducing activity was investigated by implanting 0.5-20 mg of BMP into thigh muscle pouches of BALB mice. Radiologically detectable formation of new bone required 2.0 mg of partially purified BMP. Immediately after the extraction, an analytic chromatogram with known molecular weight (MW) markers showed three fractions with different MWs. After 15 months of storage at +1 degree C lyophilized and desiccated, BMP was fractionated by HPLC gel filtration and bioassayed. New bone formation was evaluated qualitatively by histology and quantitatively by radiomorphometry, the quantity of calcified tissue per milligram of implanted agent being determined. Fractions I and III, with high (100-700 kD) and low MW (15-25 kD), respectively, were apparently more effective inducers of new bone than the second-time-tested partially purified BMP complex, the activity of which had significantly (p < 0.05) decreased during 15 months of storage compared to initial results after extraction. However, the bone-inducing activity of fractions I and III corresponded closely to the initial activity of the BMP complex. Fraction II, with medium MW (25-55 kD), caused an apparent inflammatory reaction and no bone formation, and was though to be immunogeneic. Fraction III was considered to include the dominant BMP component with MW 18.5 and fraction I an association of BMP with other non-collagenous bone matrix proteins after one-step gel filtration. The results suggest that BMP from the premature moose has high bone-forming activity. With identification and removal of apparently immunogenic protein fractions, the inflammatory reaction and inhibitory effect on bone induction could be eliminated, and still higher bone-forming activity was attained. Acid protease enzymes were assumed to be responsible for the observed decline in the inductive activity of semi-purified BMP after 15 months of storage, as both osteoinductive fractions proved to be acidic in isoelectric focusing.

Animals↗
PubMed1994

Experimental study on demineralized bone matrix (DBM) and coral as bone graft substitutes in maxillofacial surgery.

Three different bone substitutes were implanted in a standardized nasal bone defect in 30 Wistar rats. The results were assessed at 2 months by macroscopic examination, contact radiography, and histologic analysis. Demineralized osseous implants sterilized by ethylene oxide induced bone formation in 90% of the the cases, as no heat-treated graft showed any bone formation. Coralline grafts were osteointegrated in 50% of the cases, but osteoconduction was not sufficient to achieve complete bone repair. This study implies that ethylene oxide sterilization does not impair biologic properties of demineralized grafts, but further studies on more evolved animal species are necessary before human implantation.

Animals↗
PubMed1992

Demineralized bone matrix polydioxanone composite as a substitute for bone graft: a comparative study in rats.

Demineralized bone matrix (DBM) has been successfully used as a substitute for bone grafting. Autogenous bone grafts (ABG) may cause donor site morbidity and undergo significant resorption. DBM may overcome these problems but is mechanically unstable when originally placed. We explored using a slowly resorbable template, polydioxanone (PDS), in combination with DBM and compared it to ABG in a rat 9 x 9 mm cranial defect model. After both 1 and 3 months, histologically and biochemically well-formed bone was present in ABG/PDS and DBM/PDS-treated defects, but not in control defects (PDS alone). Mechanical push-out tests using a servohydraulic testing frame were conducted. Maximum load before failure of DBM/PDS increased from 65% at 1 month to 100% of that of intact skull at 3 months. In contrast, ABG/PDS was 50% as strong as DBM/PDS and not significantly stronger than PDS alone. ABG/DBM did not significantly increase in strength from 1 to 3 months. We conclude that DBM/PDS is better than ABG/PDS in treating cranial defects in the rat model, and that an absorbable osteoinductive bone substitute with superior mechanical advantage is possible without the disadvantages of ABG.

Animals↗
PubMed1994

[Bridging long tibial shaft defects by partially demineralized bone matrix].

The problems arising from the transplantation of autogenic and allogenic bone have significantly limited the use of these methods. Hence, there is an ever increasing demand for suitable transplant materials that could be readily available to orthopaedic surgeons throughout the country. Although the advantages of demineralized bone matrix over allogenic cancellous bone have been shown in numerous experimental studies, its broad clinical application has so far been limited. The purpose of this study was to investigate the osteogenic properties of partially demineralized bone matrix in clinically relevant and realistic conditions. Tibial defects 5 cm in length in 24 merino sheep were bridged by way of medullary nailing and filled with various preparations of bone matrix. Cortical bone displaying poor vascularization and rotation instability of the osteosynthesis ensured extremely difficult testing conditions for the transplant. Postoperatively, the extent of new bone formation was evaluated by means of regular X-ray examinations over a period of 12-20 weeks. In addition, histological, fluorescent-optical and microradiographic examinations of the final specimen were carried out. Good new bone formation regularly followed the transplantation of partially demineralized bone matrix with a particle size of 750 microns. Complete bridging of the defect was achieved when small amounts of bone marrow were added. The use of bone matrix with a smaller or larger particle size did not influence the rate of new bone formation perceptibly.

Animals↗
PubMed1973

Coagulation of blood plasma of guinea pig by the bone matrix.

Optimal amounts of demineralized bone matrix possess the ability to coagulate platelet-free heparinized, citrated, and oxalated blood plasmas of guinea pigs. Clotting constituents become denatured in contact with the insoluble coagulant proteins. Quantities in excess of optimal modify plasma so that it does not gel when thrombin is added. The newly described coagulant effects are not restricted to the bone matrix, but are present also in the demineralized matrices of tooth and ivory, and in denatured tendon as well. They are regulated properties that were not demonstrated in mineralized bone or native tendon. The coagulant attributes of bone matrix are consistent with those of electropositive polymers of a specific sort.

Amines↗
PubMed1999

Effect of demineralized bone matrix on polymorphonuclear leukocyte degranulation.

The potential use of allogenic demineralized bone matrix to augment or treat bone defects or nonunions in animals and humans is currently being investigated. Demineralized bone matrix induces osteogenesis by a multistep cascade of endochondral ossification that is mediated by bone-induction factors. The migration and activation of polymorphonuclear leukocytes appear to be critical in the initiation of the cascade of osteogenesis induced by demineralized bone matrix. This study examined the effects of demineralized bone matrix on the degranulation of polymorphonuclear leukocytes. Demineralized bone matrix stimulated the release of polymorphonuclear leukocyte-specific, but not azurophilic, granules in a time and dose-dependent manner. The ability of the bone matrix to induce this degranulation was independent of its size and species. The mechanism by which this degranulation occurs is not completely understood; however, it is known that it does not occur by means of a receptor that requires guanidine triphosphate-dependent regulatory proteins as does polymorphonuclear-leukocyte degranulation induced by N-formyl peptide. The factor that stimulates degranulation is not type-I collagen but rather appears to be a cytokine that has a heparin-binding domain and a molar mass of 10-70 kDa. Loss of the ability of demineralized bone matrix to induce degranulation of polymorphonuclear leukocytes correlated positively with the loss of its ability to induce bone formation.

Animals↗
PubMed1985

Comparison of bone inductive proteins of rat and porcine bone matrix.

Subcutaneous implantation of demineralized bone matrix in allogenic rats induces a sequence of events resulting in de novo formation of cartilage, bone and bone marrow. In the present study endochondral bone formation by demineralized porcine matrix was studied and compared with the rat bone matrix. Endochondral bone formation was induced by 4M guanidine hydrochloride fraction IV (less than 50,000 daltons) of Sepharose CL-6B gel filtration but not by whole extract or by demineralized porcine bone matrix. Sephacryl S-200 gel filtration of the osteoinductive proteins of fraction IV showed the Porcine osteoinductive factor to be associated with protein fraction III (less than 20,000 daltons) whereas the rat with fraction II (between 20,000 and 30,000 daltons) of the chromatographic profile indicating an apparent difference in molecular weight of the osteoinductive factors between these two species.

Animals↗
PubMed1975

Transmembrane bone matrix gelatin-induced differentiation of bone.

In response to chemically-defined bone matrix gelatin (BMG) inside a diffusion chamber implanted in a muscle pouch, mesenchymal cells migrate directionally, aggregate and differentiate into new bone, on the outside of the chamber. BMG diffuses through double membranes 275 to 300 mum in thickness. The inner membrane of pore size is 0.025 mum and the outer membrane of pore size is 0.45 mum. The inner membrane is 1/20 the pore size and the combination is twice the thickness of membranes previously reported to transfer osteoinductive activity of living cells. Autoradiographs show 35S-cysteine-labelled BMG produces very high trans-membrane grain counts while 3H-proline labelled BMG produces very low transmembrane grain counts. Electron micrographs demonstrate that gelatin-derived, uranyl-acetate-stained fine granules interspersed with ruthenium red-staining coarse granules, diffuse through the membrane of 0.025 mum pore size from the inside out. Solitary pale-staining collagen fibrils, possibly formed in interstitial fluid by renaturation of BMG are found in the interior of the chamber and in the interior of the outer 0.45 mum but not the inner 0.025 mum pore size membrane. Densely-stained new bone collagen fiber bundles cover the outer membrane, fill the 0.45 mum subsurface pores for a depth of 0.20 to 30 mum, and thereby attach the new cartilage and bone deposits to the outer surface of the chamber. BMG powders solubilize rapidly in diffusion chambers and produce high yields of new bone. The relationship between denatured collagen and renatured gelatin fibrils in the process of transfer of the bone morphogen from BMG to mesenchymal cell receptors is an intriguing subject for further investigation.

Animals↗
PubMed1990

[Clinical study on the application of demineralized bone matrix (DBM) in surgical orthodontics].

Demineralized bone matrix has been tested in a clinical study concerning the filling of bone defects in the surgical stomatology. It is reported on the clinical results and the X-ray evaluation of the replacement of bones by demineralized bone matrix. Fifty two implantations in the maxilla and mandible of 51 patients were carried out to be able to evaluate the efficacy of demineralized bone matrix.

Adolescent↗
PubMed1991

Architecture of implanted bone matrix gelatin influences heterotopic calcification and new bone formation.

Heterotopic bone formation in skeletal muscle induced by compacted demineralized bone matrix gelatin (BMG) was studied histologically and biochemically. BMG was obtained by dehydrating diaphyseal shafts of femora and tibiae of 4-week-old male Sprague-Dawley rats, cutting the bone into chips, and demineralizing and extracting the chips with various solutions. The BMG was treated with 4 M guanidine-HCl, and compacted BMG was prepared by centrifugation. The compacted BMG was implanted into the rectus abdominis muscle of 5-week-old male Sprague-Dawley rats. The resulting specimens were examined histologically, and their alkaline phosphatase activity and the calcium content of the tissues were measured 3, 5, 7, 10, and 15 days after implantation. The BMG (separated BMG) with 75- to 500-microns particle sizes were implanted into control rats. The results showed that calcification, alkaline phosphatase activity, and bone formation were suppressed by implantation of the compacted BMG and that scarcely any vascularization occurred. Calcification, vascularization, and alkaline phosphatase activity were related and were indispensable for bone formation. In the control group, bone formation was observed at sites of high activity of alkaline phosphatase and well-developed vascularization. These results suggested that compacting of BMG suppressed vascularization, decreased calcification, and consequently reduced the induction of bone formation.

Alkaline Phosphatase↗
PubMed1989

Bone induction by fetal and adult human bone matrix in athymic rats.

In the rat the intramuscular implantation of demineralized rat bone matrix induces local bone formation. In adult primates, however, allogenous bone matrix induces little or no bone formation in extraskeletal sites. To assay inductive properties, human demineralized bone matrix from 6 adult donors and 4 fetuses was implanted intramuscularly in athymic rats for 6 weeks. Fetal and adult matrix implants yielded about the same amount of bone: about half of the bone yield from rat or rabbit matrix in the same model. We conclude that human bone matrix has inductive properties and that failures to induce bone formation in adult primates may be due to an inability by the recipients to respond to inductive stimuli of adult bone matrix.

Adolescent↗
PubMed1991

[Morpho-functional correlations of the structure of bone cells and adjoining bone matrix in the developing bone].

By means of scanning and transmissive electron microscopy methods structure of the developing bone has been studied. Interconnection of the cell structure and spatial organization of the adjoining matrix has been demonstrated. On the surface of the growing bone not only forming areas have been revealed, where under osteoblasts at various functional states, osteoid layer is determined, but also areas of resorption and completed osteogenesis. This demonstrates an interrupted character of osteogenesis at modelling. At the same time for the remodelling process presence of erosive lacunae is specific; they are filled with a newly deposited collagenous matrix. Therefore, it is possible to suppose that formation of the bone as an organ during the postnatal development includes in itself both mechanisms supporting its form at outgrowth of the osseous matrix volume (modeling) and its continuous rearrangement and adaptation to real conditions of functioning (remodelling).

Animals↗
PubMed1992

Compositional analysis of the collagenous bone matrix. A study on adult normal and osteopenic bone tissue.

The collagenous constituents of mature bone of 30 individuals 22-93 years of age were studied by post-mortem morphological and biochemical analysis. Morphometric evaluation of the second lumbar vertebral body revealed striking interindividual differences in bone mass, mean trabecular density and mean trabecular thickness. Collagen extracted from vertebral trabecular bone by limited pepsin digestion consisted mainly of collagen I (92%) and collagen V (8%). Immunohistochemistry revealed a distinct distribution of these two collagen types within the bone matrix. The degree of lysyl hydroxylation of the alpha 2(I) collagen chain correlated inversely with the trabecular bone volume (TBV) and with the mean trabecular plate density. This correlation was statistically significant for the entire study group as well as for the female and male subgroups. Within the female subgroup, the lysyl hydroxylation/TBV ratio was higher in postmenopausal than in premenopausal women and was highest in women with established osteoporosis. No significant correlation was found between the level of lysyl hydroxylation and the age of the patients. The alpha 1(I) collagen chain showed a nearly constant degree of lysyl hydroxylation in all 30 samples. The results provide convincing evidence that morphometric changes associated with osteopenia in adult bone are accompanied by an altered level of lysyl hydroxylation of the alpha 2(I)-chain of collagen I. The biochemical alterations observed may be responsible for the deposition of a deficient bone matrix in osteopenic conditions.

Adult↗