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PubMed2003

Clinical and radiographic treatment evaluation of class III furcation defects using GTR with and without inorganic bone matrix.

OBJECTIVE: The aim of this study was to evaluate the effect of guided tissue regeneration (GTR) alone and in conjunction with a bovine inorganic bone matrix in furcation defects. MATERIAL AND METHODS: Twenty class III furcation defects were treated in 18 non-smoker patients, 35-75 years old. Horizontal (CAL-H) and vertical clinical attachment levels (CAL-V), probing depths (PD), gingival margin levels (GML), horizontal (BDL-H) and vertical bone defect levels (BDL-V), and alveolar crest levels (ACL) were performed at baseline and at 6-month re-entry procedures. Subtraction radiography was used to assess gain or loss in optical density (OD) and area of bone fill (A) (baseline/6 months). After flap elevation, the sites were randomly assigned to receive GTR + Bio-Oss (test) or GTR treatment alone (control). Results were evaluated using anova. RESULTS: Differences were statistically significant between baseline and re-entry for PD, ACL (p < 0.01) and GML (p < 0.05) for the control group, and for BDL-V (p < 0.01) for the test group. There was a gain in ACL for the test group and a reduction in ACL for the control group (p < 0.01). No differences were observed for OD and A. CONCLUSION: The results of this study indicate that class III furcation defects are not predictably resolved utilizing GTR or GTR in combination with an inorganic bone matrix.

Adult↗
PubMed1977

Chondrogenesis in outgrowths of muscle tissue onto modified bone matrix in tissue culture.

Connective tissue outgrowths of muscle occur in vitro onto a substratum of bone matrix gelatin (BMG) chemically modified by various inorganic and organic cations or anions. Outgrowths onto BMG denatured by lyotropic concentrations of Ca2+ or complexed by only 1 mmole/1 of Cu2+ differentiate into fibrous tissue only. Onto BMG complexed with either positively or negatively charged organic molecules, the product was also fibrous tissue. Outgrowths onto undenatured and partially recalcified MBG differentiated into cartilage. These observations substantiate the view that differentiation of cartilage in tissue culture occurs in response to the undenatured form of a bone matrix protein molecular and not an unspecific surface net electrochemical charge.

Bone Matrix↗
PubMed2005

Restoration of bone defect and enhancement of bone ingrowth using partially demineralized bone matrix and marrow stromal cells.

PURPOSE: This study aimed to investigate the capability of combining marrow stromal cells (MSC) and partially demineralized bone matrix (PDBM) to fill bone defect and enhance bone ingrowth using a canine non-weight-bearing gap model. METHODS: Custom-made implants with 3mm gap between the porous surface and the host bone were used. The implants were inserted into the distal femurs of 25 mongrel dogs and the gaps were randomly assigned to be filled with culture-expanded autologous MSC-loaded PDBM, autograft, fresh-frozen allograft, PDBM alone, or nothing as controls. Histomorphometry using backscattered scanning electron microscopic examination, and mechanical push-out test were performed at 6 months after surgery. RESULTS: Histomorphometry showed that amounts of bone regeneration in the gap and bone ingrowth into the porous-coated surface in the MSC-loaded PDBM-treated group were comparable to those of autograft-treated group and were significantly greater than those of allograft-treated, PDBM-treated, or non-grafted groups. Mechanical test showed the same differences. CONCLUSION: The results of this study showed that combining PDBM and autologous culture-expanded MSC restored bone stock and enhanced bone ingrowth into the porous-coated area in a canine non-weight-bearing gap model. This combination may provide an option for reconstructing bone defect when we perform a cementless revision arthroplasty.

Animals↗
PubMed1986

Characterization of mitogenic activities extracted from bovine bone matrix.

The mitogenic activity in the unfractionated mixture of proteins released from adult bovine bone matrix during demineralization with ethylenediaminetetraacetate (EDTA) has been examined. Bovine bone extract (BBE) from 1 to 25 micrograms protein per ml stimulated proliferation of chick embryo calvaria bone cells, newborn mouse bone cells, and osteoblastlike cell lines MMB-1 and ROS 17/2.8. BBE also stimulated DNA synthesis in cells from chick embryo cartilage, skin and skeletal muscle tissues and fibroblastlike BALB/c 3T3 and NRK cells. BBE contained beta transforming growth factor (TGF) activity (NRK cell colony formation in soft agar in the presence of epidermal growth factor EGF). The cell specificity results suggest that BBE contains more than one growth factor, including a beta TGF and a factor that is not specific for bone cells, and all of the bone derived growth factor activities that have been described previously, including SGF, are apparently present in BBE. Maximal stimulation of chick embryo calvarial cell DNA synthesis by BBE was equal to or exceeded maximal stimulation by nonosseous growth factors that have been reported to stimulate DNA synthesis in bone organ cultures (EGF, fibroblast growth factor, platelet-derived growth factor, insulinlike growth factor I, and multiplication stimulating activity). Combinations of BBE with maximally stimulatory concentrations of each growth factor stimulated DNA synthesis to a greater magnitude than did each growth factor alone. These results suggest that combinations of bone derived and systemic factors can coordinately stimulate bone cell proliferation.

Animals↗
PubMed1993

Induction of new bone by allogeneic demineralized bone matrix combined to bioactive glass composite in the rat.

Allogeneic diaphyseal demineralized bone matrix (DBM) cylinders containing bioactive glass rods were implanted for 4 and 8 weeks in the abdominal muscle wall of rats. DBM without glass served as control. The results suggest that new bone induction by DBM was accelerated by the presence of bioactive glass implants. However, the bone formation induced by DBM on the glass surface was relatively small. The biocompatibility of the glass was verified by the absence of adverse cellular reactions in the interface region between glass and bone. The method used provides a simple and fast means of exploring the characteristics of potential bone substitutes.

Animals↗
PubMed1991

Bone matrix constituents stimulate interleukin-1 release from human blood mononuclear cells.

To test the hypothesis that mononuclear cells are stimulated to release interleukin 1 (IL-1) by bone fragments released in the bone microenvironment during the remodeling cycle, we have investigated the effects of bone matrix and some of its constituents on IL-1 secretin from peripheral blood mononuclear cells (PBMC). Increases in IL-1 activity were observed when either PBMC or adherent monocytes, but not lymphocytes depleted of monocytes, were co-cultured with either human or rat bone particles but not with latex particles of similar size. Co-culture of PBMC with bone particles in a transwell system where the cells were physically separated from the bone particles, or with osteoblast- or osteoclast-covered bone particles, did not stimulate IL-1 release, indicating that a physical contact between PBMC and the bone surface is required for eliciting IL-1 release. This was confirmed by the finding of a lower stimulatory effect of bone particles pretreated with etidronate, a bisphosphonate which decreases the bone binding capacity of PBMC. Constituents of bone matrix, such as collagen fragments, hydroxyproline, and, to a lesser extent, transforming growth factor-beta, but not osteocalcin, alpha 2HS glycoprotein, fragments of either bone sialoprotein or osteopontin, and fibronectin, stimulated PBMC IL-1 release in a dose-dependent fashion. Collagen-stimulated IL-1 release was partially and specifically inhibited by a monoclonal antibody directed against the alpha 2 beta 1-integrin cell surface collagen receptor. These data demonstrate that products of bone resorption, known to be chemotactic for mononuclear cells, stimulate PBMC IL-1 activity. These findings may help explain previous documentation of increased IL-1 secretion by circulating monocytes obtained from patients with high turnover osteoporosis.

Animals↗
PubMed1989

[A method for the preparation of demineralized bone matrix].

A procedure for preparation of a demineralized bone matrix (DBM) implant is demonstrated. The main steps of this method are refrigeration, milling/sieving, defatting, demineralization, freeze-drying and sterilization. These steps are standardized but also viable. Therefore this method is on the one hand international comparable on the other hand it creates the possibility for further investigations to improve the osteoinductive capacity of DBM.

Bone Matrix↗
PubMedDate not supplied

Antibacterial and osteoinductive properties of demineralized bone matrix treated with silver.

The problems incurred by storage of demineralized bone allograft material and its potential use in contaminated operative sites make an antibacterial property desirable. Silver was considered for this role because of its wide spectrum of antibacterial susceptibility, low incidence of resistance, and its ability to persistently inhibit bacteria after binding to collagen matrices. Demineralized bone matrix prepared from rat diaphyseal bone segments was treated by exposure fo AgCl, AgNO3 and NaNO3 solutions prior to lyophilization. The resulting material was tested for bacterial inhibition after incubation in saline solutions for various times and showed inhibition persisting for at least four weeks (Ag-treated material only). Silver treating the matrix was found to partially inhibit the osteoinductive capacity at 10(-3) and 10(-2) M but not at 10(-5) M as measured by intramuscular implantation in the rat for six weeks. Control and NaNo3-treated specimens showed normal bone growth as measured by ashing and by 99mTc binding, and confirmed by radiologic densities. Histologic sections showed dense microdeposits on dense material predominately near the decalcified bone surfaces, but also within the matrix. The results suggest that pretreatment with silver at concentrations in the 10(-4) range would render the implant material antibacterial, protect its sterility, and leave the osteoinductive capacity intact.

Animals↗
PubMed1989

Structure and biology of cartilage and bone matrix noncollagenous macromolecules.

Over recent years a number of cartilage and bone matrix molecules have been identified and characterized. These include major constituents such as collagens and proteoglycans as well as a number of less-abundant matrix proteins. In several cases these proteins have been characterized by cloning and sequence analysis of the corresponding cDNA. Some properties of the macromolecules have been studied and an understanding of their functions in the structure, assembly, and breakdown of connective tissue matrix is emerging. It appears that some of these molecules have structural roles whereas others participate in the assembly of the tissue. In this paper we attempt to give a current picture of the organization and role of the noncollagenous matrix macromolecules in cartilage and bone.

Animals↗
PubMed1997

Attachment characteristics and involvement of integrins in adhesion of breast cancer cell lines to extracellular bone matrix components.

Evidence is mounting that changes in the ability of cancer cells to adhere to extracellular matrices play a decisive role in metastatic spread. The mechanism underlying the preference of breast cancer cells to metastasize to bone is, however, poorly understood. We investigated the expression and involvement of integrin adhesion receptors in the adhesion of breast cancer cells to bone matrix (constituents) in two in vitro attachment assays using RGD peptides and anti-integrin antibodies. Breast cancer cells adhered rapidly to extracellular bone matrix. Adhesion of most cells to vitronectin, fibronectin, thrombospondin, osteopontin, and the fairly bone-specific bone sialoprotein was inhibited by the 200 micrograms/ml GRGDS peptide. These data suggest that integrin adhesion receptors can modulate the attachment of breast cancer cells to bone matrix molecules. In accordance with these findings, we found that alpha 1-alpha 5(beta 1) and alpha v(beta 3) integrins were expressed by mammary carcinoma cells. Highly tumorigenic MDA-MB-231 cells, which form osteolytic metastases in vivo, expressed relatively high levels of alpha 2 beta 1, alpha 3 beta 1, alpha 5 beta 1, alpha v beta 3 integrins, when compared to MCF-7, T47D, and ZR75-1 breast cancer cells. Addition of function-blocking anti-alpha 2 beta 1, -alpha 3 beta 1, -alpha 5 beta 1, and -alpha v beta 3 antibodies significantly inhibited the adhesion of MDA-MB-231 breast cancer cells to bone matrices. In conclusion, our data suggest a possible role for beta 1 and beta 3 integrin subfamily members in the establishment of skeletal metastases in advanced breast cancer patients. Clearly, functional evidence is required to understand the mechanisms involved in the development of skeletal metastases in breast cancer patients.

Bone and Bones↗
PubMed1990

Heterogeneity of latent transforming growth factor-beta isolated from bone matrix proteins.

Transforming growth factor-beta (TGF beta) is a family of 25-kDa peptides that appear to be important regulators of cell proliferation, differentiation, and differentiated function in many tissues. TGF beta is present in platelets and serum and is released by cultured cells as several distinct large mol wt complexes of TGF beta with other proteins. These complexes are biologically inactive and are generically called latent TGF beta (L-TGF beta). Large quantities of TGF beta are present in bone matrix. This study was undertaken to determine whether the TGF beta in bone matrix was present as a free 25-kDa peptide or a large mol wt L-TGF beta complex. TGF beta activity was determined by inhibition of [3H]methylthymidine incorporation in mink lung epithelial cells. The specificity of inhibition was determined by treating fractions with a polyclonal rabbit antiporcine TGF beta-blocking antibody before assay. Latency was examined by assaying untreated and acid-treated fractions for TGF beta activity. Acid treatment of EDTA extracts of the bovine bone matrix proteins increased TGF beta activity from a mean of 0.8 pg/microgram protein to 56 pg/micrograms. Under native conditions L-TGF beta eluted from S400 between the 600-400 kDa mol wt standards. No activity eluted in the fractions with authentic 25-kDa TGF beta. Eighty-five percent of the L-TGF beta bound to lentil lectin, and this separated into four discrete L-TGF beta peaks (I-IV) at 0.22, 0.25, 0.35, and 0.42 M NaCl with Mono-Q anion exchange chromatography. Mono-Q pools II and III were reseparated by molecular size on Superose-12 under native and dissociative conditions. Under native conditions TGF beta activity was latent and eluted in the large mol wt fractions. No 25-kDa TGF beta was present. With dissociating conditions (4 M GuHCl) all TGF beta activity eluted in the small mol wt fractions identical to the elution position of authentic 25-kDa TGF beta. The active fractions from the dissociative separation of Mono-Q pool III were separated by C4 reverse phase HPLC. There were three discrete peaks of TGF beta activity corresponding to TGF beta 1, TGF beta 2, and an unidentified form of TGF. Maximum activation of L-TGF beta in each Mono-Q peak occurred at pH 3-3.5. There was partial activation at pH 4.5, but no additional activation at pH 1.5.(ABSTRACT TRUNCATED AT 400 WORDS)

Acetates↗
PubMed1991

Demineralized allogeneic bone matrix for cartilage repair.

We tested the chondrogenic potential of demineralized allogeneic bone matrix (DABM) in the repair of osteochondral defects. In 42 adult rabbits, a 5-mm2 or 15-mm2 defect was created bilaterally in the intercondylar groove of distal femur. DABM was inserted directly in 37 defects, whereas in 35 it was inserted after previous placement in muscle for 4, 16, or 19 days. Another 12 defects were left empty, serving as controls. Subgroups of animals were killed at 6, 12, 18, and 26 weeks. The distal femora were excised and prepared for histologic evaluation in hematoxylin-eosin and toluidine blue. Cartilage-like repair tissue was observed in the majority of defects. However, there was a great variability in the experimental groups without any clear relationship to type of DABM implant, defect size, or postoperative time. Even individual knees exhibited varying stages of cartilage differentiation. Overall, DABM placed in muscle for 19 days appeared to yield the best repair of the defects. The most consistent findings of the present study were bone formation in the marrow of distal femur and, notably, the absence of bone differentiation toward the joint surface. Hence, it seems that the synovial environment prevents bone formation otherwise induced by DABM in vascular tissue. Although tissue formed in articular defects supplemented with DABM is of cartilaginous differentiation, which is retained over time, it is of highly variable quality. Hence, the described approach has to be optimized before it can be applied for the purpose suggested.

Animals↗
PubMed1980

Induction and inhibition of bone matrix resorption.

A method is described by which the action of hormones on cartilage and bone matrix in mice is ascertained by measuring 35S release from bone explants. Parathyroid extract was found to increase the rate of 35S release in vitro as well as in samples from pretreated animals, whereas calcitonin and 25-hydroxycholecalciferol were effective only in vitro.

Animals↗
PubMed1991

Interaction of allogeneic demineralized bone matrix and porous hydroxyapatite bioceramics in lumbar interbody fusion in rabbits.

Bone repair by autograft is effective in clinical practice. However, serious problems arise when a considerable volume of transplant is needed, as with spinal fusion procedures. The use of bone substitutes combined with osteoinductive agents may contribute to the solution of such problems. In this study, the effectiveness of such a procedure was tested in an experimental model of interbody fusion in rabbits in which the incorporation of a porous hydroxyapatite block (HA) was enhanced by the addition of allogeneic demineralized bone matrix (DBM). The latter was used as a delivery system for the osteoinductive activity of the bone morphogenetic protein contained in the matrix. A group implanted with combined HA + DBM showed significantly earlier stabilization of the fusion when compared to groups implanted with DBM alone, HA alone, and bone autografts. On the other hand, the general results of the fusion with HA + DBM were superimposable on those of autografts. With further research, the combination of a bone substitute and an osteoinductive agent may constitute an alternative to the use of bone autografts.

Animals↗
PubMed1987

Purification and characterization of an acid phosphatase that displays phosphotyrosyl-protein phosphatase activity from bovine cortical bone matrix.

An acid phosphatase activity that displayed phosphotyrosyl-protein phosphatase has been purified from bovine cortical bone matrix to apparent homogeneity. The overall yield of the enzyme activity was greater than 25%, and overall purification was approximately 2000-fold with a specific activity of 8.15 mumol of p-nitrophenyl phosphate hydrolyzed per min/mg of protein at pH 5.5 and 37 degrees C. The purified enzyme was judged to be purified based on its appearance as a single protein band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (silver staining technique). The enzyme could be classified as a band 5-type tartrate-resistant acid phosphatase isoenzyme. The apparent molecular weight of this enzyme activity was determined to be 34,600 by gel filtration and 32,500 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis in the presence of reducing agent, indicating that the active enzyme is a single polypeptide chain. Kinetic evaluations revealed that the acid phosphatase activity appeared to catalyze its reaction by a pseudo Uni Bi hydrolytic two-step transfer reaction mechanism and was competitively inhibited by transition state analogs of Pi. The enzyme activity was also sensitive to reducing agents and several divalent metal ions. Substrate specificity evaluation showed that this purified bovine skeletal acid phosphatase was capable of hydrolyzing nucleotide tri- and diphosphates, phosphotyrosine, and phosphotyrosyl histones, but not nucleotide monophosphates, phosphoserine, phosphothreonine, phosphoseryl histones, or low molecular weight phosphoryl esters. Further examination of the phosphotyrosyl-protein phosphatase activity indicated that the optimal pH at a fixed substrate concentration (50 nM phosphohistones) for this activity was 7.0. Kinetic analysis of the phosphotyrosyl-protein phosphatase activity indicated that the purified enzyme had an apparent Vmax of approximately 60 nmol of [32P]phosphate hydrolyzed from [32P]phosphotyrosyl histones per min/mg of protein at pH 7.0 and an apparent Km for phosphotyrosyl proteins of approximately 450 nM phosphate group. In summary, the results of these studies represent the first purification of a skeletal acid phosphatase to apparent homogeneity. Our observation that this purified bovine bone matrix acid phosphatase was able to dephosphorylate phosphotyrosyl proteins at neutral pH is consistent with our suggestion that this enzyme may function as a phosphotyrosyl-protein phosphatase in vivo.

Acid Phosphatase↗
PubMed1990

Estrogen inhibition of periosteal bone formation in rat long bones: down-regulation of gene expression for bone matrix proteins.

Estrogen is important for both the sexual dimorphism of the skeleton during growth and the maintenance of bone balance in adults. This report describes the in vivo effects of estrogen on bone formation and gene expression in the tibial diaphysis of ovariectomized rats. Rats were ovariectomized at 8 weeks of age and were given diethylstilbestrol (DES) or placebo 1 week later as sc sustained release pellets. Histomorphometry revealed that that the periosteal bone formation and apposition rates were reduced at the tibial diaphysis 1 week after beginning estrogen treatment and further reduced after 2 weeks. Interestingly, DES treatment had no effect on endosteal bone formation, but suppressed endosteal bone resorption. Northern analysis of freshly isolated periosteal cells from tibiae and femora revealed that DES treatment resulted in dramatic decreases in steady state mRNA levels for the bone matrix proteins osteocalcin, prepro alpha 2(I) chain of type 1 collagen, osteonectin, and osteopontin as well as the osteoblast marker enzyme alkaline phosphatase. The results suggest that the inhibitory effects of estrogen on radial bone growth in rats are mediated, or at least accompanied, by the inhibition of the expression of bone matrix protein genes in periosteal cells.

Alkaline Phosphatase↗
PubMed1984

An osteo-inductive bone matrix extract stimulates the in vitro conversion of mesenchyme into chondrocytes.

Urea and guanidine extracts of demineralized beef and rabbit bone matrix were assayed both in vivo and in vitro. One month following intramuscular implantation into mouse thighs, these extracts induced ectopic cartilage and bone. Seven days following continuous in vitro exposure to the same extracts, stage 24 chick limb bud mesenchymal cells in cultures had differentiated into greater numbers of chondrocytes than controls. These results suggest the feasibility of using limb bud mesenchymal cell cultures as an in vitro assay for bone matrix derived, extractable bioactive factors which effect the conversion of mesenchymal cells into chondrocytes as a requisite step in in vivo osteogenesis.

Animals↗