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PubMed1998

[Defect reconstruction using demineralized bone matrix. Experimental studies on piglets].

The aim of this study was to evaluate the bone stimulation forced by Demineralized Bone Matrix (DBM)-Chips and-Gel in comparison to the bone-ingrowth into a porous hydroxylapatite ceramic (Endobon) in mini pigs. The following results were obtained: 1. DBM-Chips and DBM-Gel did not stimulate bone healing when filled into cancellous bone defects. The defect did not heal within 12 weeks. 2. Up to 35 days the least amount of new bone formation was observed within porous hydroxylapatite ceramic. Up to 12 weeks complete bone ingrowth in to the ceramic has been seen with close bonding between new formed bone and the ceramic trabeculae. 3. By continuous labelling with fluorochromes the new bone formation could be analysed by fluorescence microscopy and the dynamics could be related to time after implantation.

Animals↗
PubMed2005

[Repair of articular cartilage defects with "two-phase" tissue engineered cartilage constructed by autologous marrow mesenchymal stem cells and "two-phase" allogeneic bone matrix gelatin].

OBJECTIVE: To investigate the effect of "two-phase" tissue engineered cartilage constructed by autologous marrow mesenchymal stem cells(MSCs) and allogeneic bone matrix gelatin(BMG) in repairing articular cartilage defects. METHODS: Thirty-two New Zealand white rabbits were involved in the experiment. "Two-phase" allogeneic BMG scaffold (one side of porous cancellous bone and the other side of cortical bone; 3 mm both in diameter and in thickness) was prepared from iliac bone and limb bone of 5 rabbits by sequentially chemical method. The MSCs were separated from 18 New Zealand white rabbits and induced to express chondrocytic phenotype. The chondrocyte precursor cells were seeded onto "two-phase" allogeneic BMG to construct tissue engineering cartilage. Masson's trichrome staining, PAS staining and scanning electronic microscopic observation were carried out at 1, 3 and 5 weeks. The defects of full thickness articular cartilage (3 mm both in diameter and in depth) were made at both sides of femoral medial condyles in 27 rabbits (including 18 of separated MSCs and the remaining 9). The defects were repaired with the tissue engineered cartilage at the right side (group A, n = 18), with BMG at the left side (group B, n = 18), and without any implant at both sides in the remaining 9 rabbits as a control (group C, n = 18). After 1, 3 and 6 months, the 6 specimens of femoral condyles were harvested in 3 groups, respectively. Gross observation, Masson's trichrome and Alcian blue staining, modified Wakitani scoring and in situ hybridization of collagen type I were carried out to assess the repair efficacy of tissue engineered cartilage. RESULTS: The "two-phase" BMG consisted of the dense cortical part and the loose cancellous part. In cancellous part, the pore size ranged 100-800 microm, in which the chondrocyte precursor cells being induced from MSCs proliferated and formed the cell-rich cartilaginous part of tissue engineered cartilage. In cortical part, the pore size ranged 10-40 microm, on which the cells arranged in a layer and formed the hard part of subchondral bone. After 1 month of transplantation, the cartilage and subchondral bone were regenerated in group A; during observation, the regenerated cartilage gradually thinned, but defect was repaired and the structure of the articular surface and subchondral bone was in integrity. In groups B and C, defects were not repaired, the surrounding cartilage of defect was abrased. According to the modified Wakitani scoring, the indexes in group A were significantly higher than those in group B and C (P < 0.01) except the thickness of cartilage at 6 months. The positive cell rate of in situ hybridization for collagen type II in group A was also higher than those in groups B and C (P < 0.01). CONCLUSION: "Two-phase" allogeneic BMG is a prospective scaffold for tissue engineered cartilage, which combines with autologous chondrocyte precursor cells induced from MSCs to construct the tissue engineering cartilage. The tissue engineered cartilage can repair defects of articular cartilage and subchondral bone.

Animals↗
PubMedDate not supplied

Reconstruction of the anterior laryngeal wall with a composite graft of demineralized bovine bone matrix and autogenous perichondrium. An experimental study in adult rabbits.

In this study the feasibility to reconstruct the anterior laryngeal wall in adult rabbits with a composite graft of demineralized bovine bone matrix and autogenous perichondrium was investigated. It is demonstrated that demineralized bovine bone, which is a porous biomaterial, is completely transformed into autologous cartilage, once it is implanted in a vascularized perichondrial pocket. It appeared to be possible to reconstruct the anterior laryngeal wall in adult rabbits with this newly formed cartilage. As the biomaterial has been transformed into autologous tissue it is entirely incorporated at the recipient site giving rise to solid connections (cartilaginous and/or fibrous) with the surrounding tissues.

Animals↗
PubMed1983

Incorporation of radioactive precursors into glycosaminoglycans by rat muscle fibroblasts exposed to a solubilized rat bone matrix fraction.

Confluent cultures of rat muscle fibroblastic cells respond by increased glycosaminoglycan (GAG) synthesis when cultured in medium containing a solubilized bone matrix fraction (SBM) at a concentration of 100 micrograms/ml. The metabolism of the GAG associated with the cell pellet, the cell surface and the tissue culture medium fractions was studied, in the presence and absence of SBM, by measuring the incorporation of radioactivity from [3H]glucosamine and [35S]SO4 into the isolated GAG. Net synthesis of hyaluronic acid and of chondroitin sulfate in the medium fraction increased more rapidly in cultures containing SBM compared to controls, and the accumulation of labelled GAG in the medium of the treated cultures was approximately linear with respect to the length of incubation. The addition of SBM also resulted in increased incorporation of 3H and of 35S into the GAG of the cell surface and cell pellet fractions. In these fractions, stimulation of incorporation of radioactivity occurred in two waves: an early, relatively minor increase and a later relatively major increase. The relatively major stimulation of radioactivity into the GAG of the cell surface fraction occurred between 24 and 48 h and was independent of any apparent effect of serum.

Animals↗
PubMed1992

Comparison of bone matrix-derived bone morphogenetic proteins from various animals.

Bone matrix-derived bone morphogenetic protein (BMP) was extracted from bovine, porcine, rabbit, Sprague-Dawley rat, and Wistar rat bone and purified. The purified fractions all had similar molecular weights and induced new bone in 3 weeks when implanted into muscle pouches of Wistar rats. Bone matrix-derived BMP is believed to consist of subunits and that of different animal origin to contain the same fraction with BMP activity.

Alkaline Phosphatase↗
PubMed1991

Ultrastructure, tartrate-resistant acid phosphatase activity and calcitonin responsiveness of osteoclasts at sites of demineralized bone matrix implant-induced osteogenesis.

The morphology, ultrastructure, tartrate-resistance acid phosphatase reactivity, and calcitonin responsiveness of osteoclasts induced at sites of demineralized bone matrix (DBM) implant-induced osteogenesis in rats were determined. Osteoclasts at these ectopic sites had a morphologic and ultrastructural appearance similar to osteoclasts normally found in skeletal tissues. When observed by scanning electron microscopy, resorption surfaces on the implants had well-defined resorption pits (Howship's lacunae), indicative of active bone resorption. The osteoclasts stained intensely for tartrate-resistance acid phosphatase, an enzyme that is specific for osteoclasts. In response to human calcitonin, hypocalcemia occurred and osteoclasts lost their ruffled borders, indicating that these cells are responsive to exogenous hormonal stimulation. The osteoclasts induced by subcutaneous implantation of DBM had morphologic and functional characteristics similar to osteoclasts normally found in skeletal tissues.

Acid Phosphatase↗
PubMed1996

The effects of various fluoride concentrations on new bone formation in intramuscular implants of bone matrix in mice.

The efficacy of fluoride therapy of osteoporosis remains a controversial issue, specially in clinical trials. The effect of fluoride on heterotopic bone formation in mice was analyzed with respect to the dose of the drug administered. Thirty days after implantation of demineralized bone matrix, the ash weight and mineral content of implants from animals receiving the highest dose of fluoride was approximately 49% lower than that of the controls. Thirty days of fluoride administration had no effect on blood composition. On the other hand, small concentrations of fluoride ions increased the dry and ash weight of implants (36%) and promoted bone mineralization. The results derived from this study may be directly applicable to the treatment of osteoporosis with fluoride and support our clinical trials.

Animals↗
PubMedDate not supplied

Differentiation of cartilage from calvarial bone under the influence of bone matrix gelatin in vitro.

Fetal rat calvarium in an optimum (CMRL-1066) culture medium (with respect to amino acids. Tween 80, DNA precursors and hydrogen acceptors used in oxidative metabolism) produces new lamellar bone. The explanted osteoprogenitor cells survive, proliferate on living membrane bone surfaces and remodel membrane bone into lamellar bone. In the same culture medium, calvarial connective tissue and osteoprogenitor cells grow out of the membrane bone interstices onto a substratum consisting of bone matrix gelatin (BMG) and differentiate not into bone but into hyaline cartilage. Outgrowths from either bone or muscle onto a substratum of BMG prepared from bone autodigested in neutral buffer solutions, produce only fibrous connective tissue. In BGJ, a culture medium containing suboptimal ingredients for cell proliferation, calvarial bone produces neither lamellar bone nor new cartilage but is gradually resorbed and replaced by fibroblasts. In CMRL culture medium, outgrowths of mesenchymal-like cells from muscle onto a substratum of living membrane bone produce an epiphyseal plate-like columnar deposit of new hyaline cartilage. These observations suggest that the function of BMG is to evoke mesenchymal cell differentiation into prechondroblasts during the latent or migratory morphogenetic phase while the effect of the culture medium is to provide the bionutritional requirements for synthesis of hyaline cartilage matrix by chondrocytes during the patent phase of development.

Animals↗
PubMed1981

Density of a sample bovine cortical bone matrix and its solid constituent in various media.

The density of a bovine cortical bone matrix sample was found in water, several ethanol-water solutions, and in dried state. Previously the density of the same mineralized bone was found fresh and when desiccated. The volume in each state was estimated from the dimensional changes axially, tangentially, and radially. Confirmation was found by determining the density of dried specimens upon immersion in xylene. The amount of imbibed xylene provided an estimate of the free pore volume in the dried matrix. The volume fraction of the solid constituent, S, in the wet matrix was found to be 0.57, from which the density of S in various solutions was calculated. Density of wet matrix in 0.15 M saline: 1.180 g/cc; for dried matrix, 1.246 g/cc. Density of wet S in saline: 1.33 g/cc; for dried S, 1.42 g/cc, which matches published values for collagen molecules. Dimensional changes between wet and dried state of matrix match published values for artificially cross-linked rat tail tendon fibers. Axially: 1.04, by area: 2.27; by volume: 2.62. Estimate of intrafibrillar volume, assuming 80% of mineral is within fibrils: 0.73 cc/g dry collagen.

Animals↗
PubMed1985

The effect of human bone matrix extracts on the biosynthesis of macromolecules by human mononuclear cells in culture.

In chronic rheumatoid arthritis, mononuclear cells (MC) accumulate in the subchondral bone and form a prominent part of both destructive lesions and repair reactions. A fraction from human bone matrix extracts (BME) stimulated glycosaminoglycan (gag) and glycoprotein synthesis by fibroblastic cells but its effects on MC metabolism had not been studied. A method was established for the study of incorporation of radioactive precursors into total protein, IgG and gag synthesized and secreted by peripheral blood MC cultured in microwells in the presence or absence of Concanavalin A (ConA). Relatively low concentrations of BME suppressed spontaneous synthesis of radioactive IgG (protein A bound) and TCA precipitable protein but had little effect on gag synthesis. In general, stimulation of the cultures with ConA overcame the inhibitory effects on protein synthesis by the BME. A large stimulation of gag synthesis induced by ConA was not affected by BME. The interactions between the BME and the stimulatory effect of ConA on DNA synthesis were studied in detail and were found to be complex, not immunologically specific and appeared to be due to binding of lectin by the carbohydrate moieties of the glycoproteins in the BME. On the basis of a model of the lectin-BME interaction, the hypothesis is postulated that the carbohydrate moieties of subchondral bone glycoproteins may have the capacity to act as a solid state "trap" for certain circulating antigens which may then also interact with surface glycoproteins of the MC accumulating in the subchondral bone. The physiological role of gag synthesis by MC is not known.

Arthritis, Rheumatoid↗
PubMed2007

A comparison of commercially available demineralized bone matrix for spinal fusion.

In an effort to augment the available grafting material as well as to increase spinal fusion rates, the utilization of a demineralized bone matrix (DBM) as a graft extender or replacement is common. There are several commercially available DBM substances available for use in spinal surgery, each with different amounts of DBM containing osteoinductive proteins. Each product may have different osteoinductivity potential due to different methods of preparation, storage, and donor specifications. The purpose of this study is to prospectively compare the osteoinductive potential of three different commercially available DBM substances in an athymic rodent spinal fusion model and to discuss the reasons of the variability in osteoinductivity. A posterolateral fusion was performed in 72 mature athymic nude female rats. Three groups of 18 rats were implanted with 1 of 3 DBMs (Osteofil, Grafton, and Dynagraft). A fourth group was implanted with rodent autogenous iliac crest bone graft. The rats were sacrificed at 2, 4, 6, and 8 weeks. A dose of 0.3 cm(3) per side (0.6 cm(3)per animal) was used for each substance. Radiographs were taken at 2 weeks intervals until sacrifice. Fusion was determined by radiographs, manual palpation, and histological analysis. The Osteofil substance had the highest overall fusion rate (14/18), and the highest early 4 weeks fusion rate of (4/5). Grafton produced slightly lower fusion rates of (11/17) overall, and lower early 4 weeks fusion rate of (2/5). There was no statistically significant difference between the rate of fusion after implantation of Osteofil and Grafton. None of the sites implanted with Dynagraft fused at any time point (0/17), and there was a significantly lower fusion rate between the Dynagraft and the other two substances at the six-week-time point and for final fusion rate (P = 0.0001, Fischer's exact test). None of the autogenous iliac crest animals fused at any time point. Non-decalcified histology confirmed the presence of a pseudarthrosis or the presence of a solid fusion, and the results were highly correlated with the manual testing. Although all products claim to have significant osteoinductive capabilities, this study demonstrates that there are significant differences between some of the tested products.

Animals↗
PubMed1993

A proteoglycan form of macrophage colony-stimulating factor that binds to bone-derived collagens and can be extracted from bone matrix.

We previously found that the human osteoblastic cell line MG-63 produces two molecular types of macrophage colony-stimulating factor (M-CSF). One is an 85-kD M-CSF, and the other is a proteoglycan form of M-CSF (PG-M-CSF) that has a binding affinity to type V collagen. The latter type of M-CSF showed dose-dependent binding to wells coated by pepsin-extracted bone collagens, whereas the 85-kD M-CSF did not. Immunoblot analysis of urea-extracted bone M-CSF revealed the presence of PG-M-CSF. PG-M-CSF contained in bone matrix may have physiological importance in the bone metabolism.

Bone Matrix↗
PubMed1998

Gene expression of bone matrix proteins and endothelin receptors in endothelin-1-deficient mice revealed by in situ hybridization.

Endothelin-1 (ET-1) was first found as a vasoconstrictor protein excreted by vascular endothelial cells, but recently ET-1 has been considered to have widespread functions that include regulation of osteochondrogenic metabolism. We analyzed sections of head regions in ET-1 knockout mice that are known to have abnormalities in pharyngeal arch-derived tissues and found that there was severe hypoplasia in facial bones. The hypoplasia suggests that the matrix mineralization system of facial bones is disrupted in ET-1-/- homozygous mice. To elucidate whether osteogenic cells in facial bones are the targets for ET-1 and whether expression of bone matrix genes are modulated by ET-1, we examined gene expression of ET-1 receptors, ETA and ETB, and that of the bone matrix proteins, osteonectin (ON) and osteopontin (OP), both in the head regions of ET-1+/- heterozygous and ET-1-/- homozygous mice by means of in situ hybridization. Different patterns of expression between ETA and ETB mRNAs were observed in both groups. In 18.5 days post coitus fetuses, ETA mRNA was most strongly expressed in osteogenic cells along craniofacial bones, but ETB mRNA was most strongly expressed in trunks of trigeminal nerve. This finding suggests that ET-1 may modulate osteogenic cells through ETA receptor but not through ETB receptor. The expression patterns of ETA, OP, and ON mRNAs were distinct between the two groups. In the lower jaw of ET-1+/- heterozygous mice, the ETA, ON, and OP mRNA positive cells were scattered in the inner and outer regions of the thick bone matrix, but in ET-1-/- homozygous mice, cells containing those mRNAs were located close to each other at the surface of thin bone matrix. However, cellular expression of ON and OP mRNAs in osteogenic cells of ET-1-/- homozygous mice was not suppressed as compared with ET-1+/- heterozygous mice. We conclude that ET-1 may regulate proliferation and migration of osteogenic cells in the maxillofacial region, rather than modulating the expression level of ON and OP mRNAs.

Animals↗
PubMedDate not supplied

Equine demineralized bone matrix: relationship between particle size and osteoinduction.

The osteoinductive capability of four particle sizes of equine demineralized bone matrix (DBM) was evaluated. Matrix particles were implanted in brachiocephalicus muscle pouches in six horses and were harvested 8 weeks later. Matrix particle sizes of 2.0 mm3 to 4.0 mm3 and 5.0 mm3 to 10.0 mm3 were associated with osteoinductive activity and minimal signs of local inflammation. The two smaller particle sizes (0.425 mm3 to 0.850 mm3 and 0.850 mm3 to 2.0 mm3) were minimally osteoinductive and were associated with a greater local inflammatory response. Microscopic events associated with new bone production in the two largest particle sizes included the presence of new osteoprogenitor cells in matrix vascular spaces, multinucleated osteoclast-like giant cells, and the formation of cartilage, osteoid, and new mineralized bone. The two smaller particle sizes were associated with increased fibrous tissue ingrowth and the presence of increased numbers of inflammatory cells. The temporal events of osteoinduction in response to heterotopic implantation of allogeneic DBM were delayed in horses when compared with other species. Although the demineralization technique used was effective in previous investigations on other species, incomplete demineralization occurred in the two smaller particle sizes that was not detected until after DBM implant harvest. The demineralization process may need to be altered for equine bone and precise monitoring for complete demineralization before implantation is required. Equine DBM is osteoinductive in muscle pouch sites in horses but matrix particle size does affect osteoinductive capability.

Animals↗
PubMed2001

Demineralized bone matrix as an alternative for mastoid obliteration and posterior canal wall reconstruction: results in an animal model.

HYPOTHESIS: This study was conducted to evaluate the use of Grafton human demineralized bone matrix as a graft material for mastoid cavity obliteration and canal wall reconstruction in an animal model. BACKGROUND: Canal wall down procedures in the treatment of cholesteatoma may result in a problematic mastoid cavity. Elimination of the mastoid cavity by obliteration or canal wall reconstruction can prevent or correct this problem. Many techniques and implant materials have been used for this application, yet no single material has proven to be ideal. METHODS: Athymic rats received tympanic bulla obliteration and lateral bulla wall reconstruction utilizing the Grafton Putty and Flex formulations, respectively. Wound healing was monitored twice a week. Auditory brainstem evoked responses were obtained 8 weeks after implantation. Nine weeks after implantation, the animals were killed, and histologic sections were prepared. A histologic bone formation score (range 0-4) was determined for each implant. RESULTS: Wound healing occurred without complication. Auditory brainstem response thresholds (average 23.5) fell within the normal range for all ears tested. The average histologic bone formation score for all implants was 3.7. The average scores for obliteration implants and wall reconstruction implants were 3.5 and 3.9, respectively. All wall reconstruction implants underwent partial or total collapse into the bulla. CONCLUSIONS: The high level of bone formation obtained by the use of Grafton implants in this study makes this material a promising resource for use in mastoid obliteration. The use of the material for canal wall reconstruction will likely require a sturdier preparation to prevent collapse into the mastoid cavity.

Animals↗
PubMed2004

Differential adherence of osteoarthritis and rheumatoid arthritis synovial fibroblasts to cartilage and bone matrix proteins and its implication for osteoarthritis pathogenesis.

In osteoarthritis (OA), cartilage and bone fragments have been described within the synovial tissue which are surrounded by synovial cells (i.e. detritus synovitis). These cells appear to attach actively to the cartilage and bone fragments. In rheumatoid arthritis (RA), on the other hand, synovial fibroblasts (SF) have also been shown to be localized at sites of invasion into cartilage and bone and to degrade extracellular matrix (ECM) by secreting proteolytic enzymes. One prerequisite for exerting their aggressive properties is the attachment to cartilage and bone ECM. This attachment appears to be mediated by the expression of different adhesion molecules for which corresponding binding sites on ECM components are known. As it has not been addressed to which ECM proteins SF adhere and with which affinity this process takes place, we investigated the adherence of SF from patients with OA and RA to different cartilage and bone matrix proteins. Synovial tissue samples were obtained during synovectomy or arthroplastic surgery and used for isolating and culturing SF. Synovial cells attaching to cartilage/bone fragments were characterized using immunohistochemistry. The adherence of SF to ECM proteins was examined using an adhesion assay with the following proteins coated on 96-well plates: aggrecan (AGG), bone sialoprotein (BSP), cartilage oligomeric matrix protein (COMP), collagen type I, II and VI, proline arginine-rich, end leucine-rich repeat protein (PRELP), osteopontin (OPN) and recombinant chondroadherin (CHAD). Bovine serum albumin was used as negative control. In addition, adhering fibroblasts were photographed using a phase-contrast microscope. As compared with RA-SF, significantly higher numbers of OA-SF adhering to collagen type II, OPN and CHAD could be detected (P < 0.05). In contrast, RA-SF showed increased attachment to collagen type II, OPN and BSP. Adhesion to AGG, COMP and PRELP appeared not to be significantly increased and differed widely among the SF samples, and, apart from one exception (BSP), OA-SF adhered in higher numbers to the matrix proteins than did RA-SF. Using immunohistochemistry, synovial cells attached to cartilage/bone fragments could be shown to predominantly express CD68 (>/=50%). The CD68-negative population was of the fibroblast phenotype (AS02 positive). The study demonstrates that the binding pattern of OA-SF and RA-SF to ECM proteins differs considerably and therefore provides novel insights into the difficult pathophysiology of OA and RA. In general, it appeared that SF adhere primarily to ECM proteins that contain known binding sites for adhesion molecules (e.g. integrins: collagen/integrin alpha(2)beta(1)) and that higher numbers of OA-SF adhered to the cartilage and bone matrix proteins than did RA-SF.

Arthritis, Rheumatoid↗