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PubMed1992

Factors influencing synthesis and mineralization of bone matrix from fetal bovine bone cells grown in vitro.

This study of the in vitro synthesis and mineralization of bovine bone demonstrates that sheets of mineralized matrix can be produced consistently within 18-24 days of cell isolation. Mineralization surpasses that achieved by other systems with other species: The deposition of mineral extends beyond nodules to form branching trabeculae and then solid wafers of bone. Comparison of the fetal age of the bone source, enzyme digestion methods, seeding density, culture surface, nutritive media, and concentration of fetal calf serum and other additives, including insulin and ascorbic acid, has yielded a set of optimal culture conditions. In the presence of ascorbic acid and beta-glycerol phosphate, insulin has a dose-dependent effect on the morphology of the mineralized bone matrix produced. Quantitative analysis shows that in these cultures calcium accumulates most rapidly between days 6 and 10 after the introduction of mineralization medium but that mineral accretion continues throughout 14-16 days of culture. Alkaline phosphatase levels rise up to 200-fold, concomitant with a rapid increase in the number of cells per culture during the early mineralization phases; both fall as mineralization proceeds. This system has been used to study the induction of mRNA of type I collagen, alkaline phosphatase, and several noncollagenous bone proteins during the course of mineralization. Because of the degree of mineralization achieved with this system, it has many potential applications.

Alkaline Phosphatase↗
PubMed1986

Pulverized bone matrix as an injectable bone graft in rabbit radius defects.

A segment of the rabbit radius diaphysis was excised, demineralized, and pulverized. The demineralized matrix particles were mixed with autologous bone marrow from the femoral canal and injected into the defect from which it had been excised. On the contralateral side, the demineralized bone was reimplanted without pulverization, but with bone marrow. The bone yield was measured by radiographic planimetry and Tc99m MDP scintimetry. The forearms of the rabbits were sectioned into transverse segments, including the middle of the radius defect. The ash weights and the Ca45 content of these segments were measured. After two weeks, the ash weight was greater on the pulverized than on the unpulverized side; but by four weeks, measurements showed no difference. In general, the injectable bone matrix preparation did not interfere with bone repair. In comparing the Tc99m and Ca45 methods, the latter provided high-precision data with respect to the biologic variation.

Animals↗
PubMed1991

Effect of diet on bone matrix constituents.

Bone formation occurs in an integrated, highly ordered manner, beginning in the embryonic period. Nutrients may affect bone formation by delaying cellular differentiation, altering responses to bone growth factors, affecting supply of needed nutrients, and/or affecting rates of synthesis of the matrix constituents. Several growth factors, both systemic and local, are being identified which affect bone formation. Matrix constituents include collagen and noncollagenous proteins, each of which are thought to have specific roles in bone formation, maintenance, or resorption. Among the nutrients which are known to affect bone formation at a cellular level are vitamins A, D, and K, ascorbic acid, zinc, magnesium, and calcium. Nutrients that are known to affect protein synthesis in general also affect bone formation. It is necessary for nutritionists to consider cellular as well as systemic effects of nutrients on bone formation.

Bone Development↗
PubMed1998

Concentration of insulin-like growth factor (IGF)-I and -II in iliac crest bone matrix from pre- and postmenopausal women: relationship to age, menopause, bone turnover, bone volume, and circulating IGFs.

Insulin-like growth factor-I (IGF-I) and -II are important local regulators of bone metabolism, but their role as determinants of human bone mass is still unclear. In the present study, we analyzed the concentration of IGF-I and -II in the bone matrix of 533 human biopsies from the iliac crest that were obtained during surgery for early breast cancer. There was an inverse association of bone matrix IGF-I concentration with age that was unaffected by menopause. Bone matrix IGF-I was positively associated with histomorphometric and biochemical parameters of bone formation and bone resorption and with cancellous bone volume. Based on the estimates of the linear regression analysis, women with a bone matrix IGF-I concentration 2 SD above the mean had a 20% higher bone volume than women with a bone matrix IGF-I concentration 2 SD below the mean. In contrast, serum IGF-I was neither correlated with bone turnover nor with bone volume and was only weakly associated with bone matrix IGF-I when adjusted for the serum concentration of IGF binding protein-3. Bone matrix IGF-II was positively associated with the osteoblast surface, but in contrast to IGF-I, tended to be positively associated with age and was unrelated to cancellous bone volume. In summary, our study suggests the following. 1) The concentration of IGF-I in cancellous bone undergoes age-related decreases that are similar to those of circulating IGF-I. 2) Menopause has no effect on this age-related decline. 3) Physiological differences in bone matrix IGF-I are associated with differences in iliac crest cancellous bone volume. 4) Bone matrix IGF-I is a better predictor of cancellous bone volume than circulating IGF-I. 5) The role of IGF-II in human bone tissue is clearly distinct from that of IGF-I.

Adult↗
PubMed1999

Bioassayed demineralized bone matrix and calcium sulfate: use in bone-grafting procedures.

BACKGROUND AND AIMS: A combination product of bioassayed, demineralized bone matrix (AlloGro, AlloSource, Denver CO) and calcium sulfate pellets (OsteoSet, Wright Medical Technology, Arlington TN) was utilized in a prospective clinical study in 50 patients in need of bone-grafting procedures. It was proposed that the osteoinductive activity of the demineralized bone matrix combined with the osteoconduction and rapid dissolution of the calcium sulfate pellets would complement each other in promoting bone formation. MATERIALS AND METHODS: The patients were evaluated clinically and radiographically at regular intervals post-operatively by an independent clinician. A total 10-point healing score was used to determine healing characteristics and progress. Fifty patients (24 males and 26 females) were treated for benign bone lesions (35), nonunion (11), osteomyelitis (3), and acute fracture (1). The average age was 33 years (range, 3-64 years). Lesions were located in the femur (16), tibia (15), humerus (7), and other sites (12). RESULTS: The average length of follow-up was 14 months (range, 6-32 months). Forty-nine of 50 patients healed their lesions (98%), requiring an average time to heal of 11.8 weeks (range, 3-48 weeks). There were no graft-related complications. CONCLUSIONS: The results of this preliminary clinical study suggest that a combination of bioassayed demineralized bone matrix and calcium sulfate is very effective in treating benign lesions of bone, as well as nonhealing fractures, which is comparable to grafting with autograft. Future studies have been undertaken utilizing this combination in all acute operative settings and fracture management situations.

Adolescent↗
PubMed2004

Alkali-urea extraction of demineralized bone matrix removes noggin, an inhibitor of bone morphogenetic proteins.

Demineralized bone matrix (DBM) and native bone morphogenetic protein (nBMP) are complex mixtures of non-collagenous bone proteins. These mixtures contain many of the BMPs that are available as recombinant molecules. Information regarding the presence in these materials of molecules that may affect the availability and activity of the BMPs is very limited. We have devised a simple chemical extraction of DBM using alkali-urea that produces a water soluble extractate that inhibits the osteogenic activity of DBM. We have demonstrated the presence of noggin, an extracellular BMP ligand antagonist, in this material. We conclude that differential chemical extraction may be a useful means of removing inhibitory molecules from DBM and nBMP.

Alkalies↗
PubMed1999

Demineralized intramembranous bone matrix augments the healing of endochondral bone graft.

The aim of this study was to examine the osteogenic potential of demineralized bone matrix prepared from intramembranous bone (DBMIM) and to examine its effects on the healing of endochondral autogenous bone grafts. Twenty-four defects in 24 New Zealand white rabbits were used as experimental groups. Twelve defects were grafted with endochondral bone, and the other 12 defects were grafted with endochondral bone with DBMIM (EC-DBMIM). One rabbit from each group was sacrificed on days 1, 2, 3, 4, 5, 6, and 7 postgrafting, and the remaining 5 rabbits from each group were sacrificed on day 14 postgrafting. Another 8 defects in 4 rabbits were used as control groups: 4 defects were left empty (passive control), and 4 defects were grafted with rabbit skin collagen (positive control). They were all sacrificed at 14 days after grafting. Serial sections were made across the whole defect. Quantitative analysis was performed on 100 sections of the 14-day experimental groups by image analysis. Four hundred fourteen percent more new bone was formed in defects grafted with composite EC-DBMIM than in those grafted with endochondral bone alone (P < 0.0001). No bone was formed in either passive or positive controls. Histologic examination of the bone grafts revealed intermediate-stage cartilage, and immunohistochemical examination revealed earlier vascularization in the composite EC-DBMIM groups. In conclusion, DBMIM has extremely high osteoinductive properties and greatly enhances the integration of endochondral bone with defects of intramembranous bone in origin.

Analysis of Variance↗
PubMed1992

Ultrastructural observation of calcification preceding new bone formation induced by demineralized bone matrix gelatin.

Demineralized bone matrix gelatin (BMG) was implanted into the skeletal muscle of Sprague-Dawley rats, and the resulting ultrastructural changes of the BMG were examined 3, 5, 7, 10, 15 and 20 days later. Most of the implanted BMG became calcified 7-20 days after implantation. Calcification ('acellular mineral deposition') was first observed as needle-shaped crystalline deposits in the BMG matrix on day 7 after implantation, which gradually increased in size and fused with one another in some deposits. They appeared to be divided into small partitions consisting of denatured collagen fibers unlike those of noncalcified BMG. Some deposits had electron-lucent areas in their center adjacent to well-calcified peripheral areas, and the central area contained many collagen-like fibers and spherical vacuoles. Osteoblast-like cells were not present around these calcified deposits 7 days after implantation. After that, new bone formation was often seen near the area of acellular mineral deposition, and the fused calcified deposits remained until day 15 after implantation. The noncalcified BMG was mainly absorbed by macrophages, and the sites of acellular mineral deposition were absorbed by multinucleated cells resembling osteoclasts which were considered to be activated by the implants. Acellular mineral deposition probably started as calcium and phosphate deposits on some materials in the BMG matrix, and by heterogenic nucleation without osteoblasts or matrix vesicles, inducing bone formation. Thus the BMG may be not only a carrier of bone morphogenetic protein, gradually supplying it to the surrounding tissue but also a storage site for minerals that are indispensable for bone formation.(ABSTRACT TRUNCATED AT 250 WORDS)

Alkaline Phosphatase↗
PubMed1986

Effect of acute increases in bone matrix degradation on circulating levels of bone-Gla protein.

Serum bone Gla-protein (BGP), also called osteocalcin, is a specific and sensitive measure of bone turnover in a variety of metabolic bone disorders. Although some BGP diffuses into the circulation after synthesis by osteoblasts, most is incorporated into bone matrix where it remains until bone is resorbed. Thus, serum BGP could reflect bone formation, bone resorption, or a combination of both. The relationship of serum BGP to the components of bone turnover was evaluated in 18 normal women (mean age 48 yr; range 30-70) who received a continuous 24-h intravenous infusion of the 1-34 synthetic fragment of bovine parathyroid hormone. Mean +/- SE for urinary hydroxyproline excretion, an index of bone resorption, increased (from 22.7 +/- 2.2 to 38.5 +/- 3.7 micrograms/100 ml glomerular filtrate [GF], p less than .001), whereas levels of serum alkaline phosphatase, an index of bone formation, were unchanged (from 20 +/- 1 to 20 +/- 1 U/liter, NS). Despite the increase in bone resorption, levels of serum BGP decreased (from 8.8 +/- 0.8 to 6.8 ng/dl, p less than .001). The data suggest that circulating levels of BGP are a measure of bone formation but, at least in subjects with normal renal function, not a measure of bone resorption. Presumably BGP in bone matrix is degraded during osteoclastic resorption into fragments that either are not recognized by an antiserum raised against the native molecule or are rapidly cleared from the circulation.

Adult↗
PubMed1992

In situ hybridization of bone matrix proteins in undecalcified adult rat bone sections.

We have developed a method for in situ hybridization of adult bone tissue utilizing undecalcified sections and have used it to histologically examine the mRNA expression of non-collagenous bone matrix proteins such as osteocalcin (bone Gla protein, BGP), matrix Gla protein (MGP), and osteopontin in adult rats. Expression was compared with that in bone tissues of newborn rats. In the adult bone tissue, osteocalcin mRNA was strongly expressed in periosteal and endosteal cuboidal osteoblasts but not in primary spongiosa near the growth plate. Osteopontin mRNA was strongly expressed in cells present on the bone resorption surface, osteocytes, and hypertrophic chondrocytes, but not in cuboidal osteoblasts on the formation surface. Osteopontin and osteocalcin mRNAs were expressed independently and the distribution of cells expressing osteopontin mRNA corresponded with acid phosphatase-positive mononuclear cells and osteoclasts. Expression of MGP mRNA was noted only in hypertrophic chondrocytes. In newborn rat bone tissues, expression of osteocalcin mRNA was much weaker than in adult rat bone tissues. These results clearly indicate the differential expression of mRNAs of non-collagenous bone matrix proteins in adult rat bone tissues.

Animals↗
PubMed2005

TGF-beta regulates the mechanical properties and composition of bone matrix.

The characteristic toughness and strength of bone result from the nature of bone matrix, the mineralized extracellular matrix produced by osteoblasts. The mechanical properties and composition of bone matrix, along with bone mass and architecture, are critical determinants of a bone's ability to resist fracture. Several regulators of bone mass and architecture have been identified, but factors that regulate the mechanical properties and composition of bone matrix are largely unknown. We used a combination of high-resolution approaches, including atomic-force microscopy, x-ray tomography, and Raman microspectroscopy, to assess the properties of bone matrix independently of bone mass and architecture. Properties were evaluated in genetically modified mice with differing levels of TGF-beta signaling. Bone matrix properties correlated with the level of TGF-beta signaling. Smad3+/- mice had increased bone mass and matrix properties, suggesting that the osteopenic Smad3-/- phenotype may be, in part, secondary to systemic effects of Smad3 deletion. Thus, a reduction in TGF-beta signaling, through its effector Smad3, enhanced the mechanical properties and mineral concentration of the bone matrix, as well as the bone mass, enabling the bone to better resist fracture. Our results provide evidence that bone matrix properties are controlled by growth factor signaling.

Animals↗
PubMed1995

Age-related reduction in bone matrix protein mRNA expression in rat bone tissues: application of histomorphometry to in situ hybridization.

Age-related changes in the biological activity of osteoblastic cells have been studied extensively using histomorphometry, especially in relation to osteoporosis. Nevertheless, the changes occurring in the biological activity of individual osteoblastic cells are not sufficiently clarified by this technique. In the present study, age-related changes in the expression of bone matrix protein mRNAs in individual osteoblastic cells were analyzed in vivo by in situ hybridization using undecalcified bone sections. In the femurs of 8-week-old male rats, strong expression of type I collagen and osteocalcin mRNAs was detected in cuboidal osteoblasts on the bone formation surface. Osteopontin mRNA was detected in some of the mononuclear cells and osteoclasts on the bone resorption surface, and also in osteocytes. In the femurs of 60-week-old and 100-week-old male rats, expression of these bone matrix protein mRNAs was markedly decreased. Histomorphometrical analysis of 8-week-old and 60-week-old rats indicated that both the activity and number of osteoblasts expressing type I collagen mRNA, as well as the number of osteoclasts, were reduced in these tissues in older animals. These results indicate age-related reductions in both biological activity and numbers of osteoblasts.

Aging↗
PubMed1989

Bone matrix stimulates osteoclastic differentiation in cultures of rabbit bone marrow cells.

Cells showing osteoclastic characteristics have not been identified outside bone. Because osteoclasts originate from an extraosseous source, this suggests that identifiable osteoclastic features do not develop until the precursors enter bone, where the local microenvironment may signal osteoclastic differentiation or maturation. We assessed the influence of bone matrix on osteoclastic differentiation by incubating bone marrow cells, after removal of pre-existing osteoclasts, on plastic coverslips or slices of devitalized cortical bone. We found that there was a threefold increase in the number of osteoclast-specific MAb-positive cells on the bone matrix compared with plastic coverslips. The number of MAb-positive cells correlated with the extent of excavation of the surface of the bone slices. Multinuclearity correlated with MAb-positive cell density, and for any given density the proportion of MAb-positive cells that were multinucleate was similar on plastic and bone. We conclude that, in the presence of 1,25-(OH)2 vitamin D3, bone matrix stimulates the generation of osteoclasts but has no demonstrable influence on the fusion of mononuclear osteoclastic precursors.

Animals↗
PubMed1992

Calcification preceding new bone formation induced by demineralized bone matrix gelatin.

Demineralized bone matrix gelatin (BMG) was implanted into the skeletal muscle of Sprague-Dawley (S.D.) rats, and histological changes were examined 3, 5, 7, 10 and 15 days later. Before bone formation, a specific calcification process was found in most of the BMG from day 5 and 7 after implantation. The heterotopic calcified sites were not always consistent with the sites of the alkaline phosphatase activity. It was considered that this calcification progresses without any cellular components, and we distinguished this type of calcification as "acellular mineral deposition" from the calcification which occurs in new bone formation. This "acellular mineral deposition" was first observed as small spherical calcified deposits in the BMG on day 7 after implantation; these deposits then gradually grew and fused with each other. Some multinucleated cells appeared near the site of calcification on day 7 after implantation, but osteoblasts or osteoblast-like cells were scarcely observed around the calcified deposits in BMG until day 7. Vascularization was often observed near the "acellular mineral deposition" and the new bone formation. Fourier transform infrared spectroscopy showed that the calcified deposits in BMG were composed of hydroxyapatite, carbonateapatite and other calcium phosphate components, and that the first two components became prominent with time. It is believed that the "acellular mineral deposition" is due to the deposition of calcium and phosphate into the BMG by a process of heterogenic nucleation that does not involve osteoblasts or matrix vesicles. Bone formation induced by the BMG occurred after the "acellular mineral deposition." The experimental calcification shown in this paper seems a useful model for the study of biocalcification.

Alkaline Phosphatase↗
PubMed2006

The effects of demineralized bone matrix proteins and osteogenic protein-1 on bone cells isolated in culture.

With the growing number of bone-related traumas and the limitations of traditional bone repair, alternative methods of bone management must be investigated. Demineralized bone matrix protein (DBX) has been used to reconstruct bone. DBX, a type of demineralized bone matrix, is a combination of several different proteins including osteogenic protein-1 (OP-1). Osteogenic protein-1 or Bone Morphogenic Protein-7 (BMP-7) was the first BMP approved for clinical use in the United States. Previous studies have shown that proliferation of osteoblasts (bone forming cells) was stimulated by OP-1. However, the effects of DBM and OP-1 at the cellular level have not been clearly defined. MG-63 osteosarcoma cells were utilized as a model and subsequently plated onto 24 well tissue culture plates at a density of 1x 10(5) ml/well. Cells were exposed to different concentrations of DBX demineralized bone matrix and OP-1 for periods of 24, 48, and 72 hours and compared with untreated controls. After each incubation period, cell morphology, cell damage, cell number, and protein concentrations were determined. Results indicate a significant increase in cell number at 72 hours in cells treated with 30% (5.66 x 10(5)) and 100% (6.3 x 10(5)) DBX treated groups when compared with the control (1.4 x 10(5)). OP-1 results do not indicate a significant increase in cell number at the 24 and 48 hour treatment phases when compared with the control (p > 0.05), however, results do show a statistically significant difference (approximately twofold, p < 0.05) between the control cells (1.9 x 10(4)) and those cells treated with low (3.9 x 10(4)) and high (4.1 x 10(4)) concentrations of OP-1 at the 72 hour time phase. The increases in cell number indicate that both DBX and OP-1 are effective in stimulating cell growth. When comparing the results of the DBX treatments with those of the OP-1 treatments, the cells treated with DBX showed a more substantial increase in bone cell proliferation after treatment than those cells treated with OP-1. This does suggest that DBX provides the most effective treatment for bone cell proliferation. Closer evaluation of the morphology especially the changes occurring at the nuclear level need to be addressed in future studies.

Bone Demineralization Technique↗
PubMed1996

Detection of vitronectin in mineralized bone matrix.

Adhesive glycoproteins in the bone matrix are of critical importance for cell anchorage, proliferation, migration, differentiation, and regulation of bone metabolism. The localization of the adhesive glycoprotein vitronectin (Vn) in murine bone tissue was evaluated by immunohistochemical staining. Vitronectin was present throughout the mineralized bone matrix of cancellous and cortical bone, whereas cartilage was devoid of Vn staining. To exclude the possibility that the positive Vn staining resulted from plasma Vn in blood vessels within the bone sections, adjacent tissue sections were stained with antibodies to fibrinogen, and abundant plasma protein. Fibrinogen immunoreactivity was confined to blood vessels in the bone marrow and Haversian system, whereas the mineralized bone matrix was devoid of staining. The presence of Vn in murine bones was confirmed by sequential extraction, followed by fractionation of the resulting polypeptides by gel electrophoresis and immunoblotting analysis. Hydroxyapatite affinity chromatography raises the possibility that mineral interactions, at least in part, mediate the incorporation of Vn into the bone matrix. These results indicate that Vn is a specific component of bone tissue and raise the possibility that Vn is involved in regulation of bone metabolism.

Animals↗
PubMed1998

Sinus floor elevation using anorganic bovine bone matrix (OsteoGraf/N) with and without autogenous bone: a clinical, histologic, radiographic, and histomorphometric analysis--Part 2 of an ongoing prospective study.

One of the goals of the sinus elevation procedure is the creation of vital bone to effect the osseointegration of dental implants placed in the posterior maxilla. With this goal in mind, in 1993 the Department of Implant Dentistry at New York University College of Dentistry began a long-term clinical, histologic, histomorphometric, and radiographic study of the sinus elevation procedure. The primary parameters included the effects of graft material selection, time allowed for graft maturation, and the effect of barrier membrane placement on the creation of vital bone in the sinus cavity. The effects of these and other parameters on implant survival rates were also to be evaluated. This paper reports the data collected on a subgroup of 113 sinus elevations that used anorganic bovine bone matrix (OsteoGraf/N) alone or in combination with autogenous bone and/or demineralized freeze-dried bone as a graft material. This is the second in a proposed series of papers that will result from this ongoing research project. The results of this study indicate that: OsteoGraf/N appears to be an effective graft material with a 98.2% implant survival rate to date: vital bone formation increased with time; vital bone formation increased moderately when demineralized freeze-dried bone allograft was added, and increased substantially when intraoral autogenous bone was added or when an expanded polytetrafluoroethylene membrane was used; and the increased height achieved by the procedure was stable over a 3-year period. Because of the high overall implant survival rate, it was not possible to determine the relationship between vital bone formation or membrane usage and implant survival.

Animals↗