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PubMed1991

Analysis of bone morphogenetic protein (BMP) derived from human and bovine bone matrix.

Recently, Bone Morphogenetic Protein (BMP) has attracted the attention of a number of investigators, but its elucidation remains incomplete. At present, the determination of its amino acid sequence, which is necessary for its synthesis, and screening for a carrier that allows BMP to be effective in small amounts are unsolved problems. Bone morphogenetic protein is studied here to clarify its clinical applications. BMP was extracted from human and bovine bone matrix with 4 M guanidine-HCl and purified by liquid chromatography. Acrylamide electrophoresis (SDS-PAGE) and isoelectric focusing (IEF) showed that the purified BMP was homogeneous. We used type I collagen as the carrier in the bioassay. This BMP induced new bone in situ three weeks after implantation in muscle pouches in Wistar rats. The molecular weights of human and bovine bone matrix-derived BMP are 17.0 and 18.0 kDa by SDS-PAGE, and pI values for both are 4.9 by IEF. Human and bovine bone matrix-derived BMP are peptides containing 165 and 163 amino acids, respectively, according to amino acid analysis. The NH2-terminal sequence of bovine bone matrix-derived BMP was obtained from the bovine band, electroblotted onto polyvinylidene difluoride membrane, that corresponded to the final purified fraction. The sequence differs from previously designated BMPs25 and other proteins reported to have similar activity, but the physicochemical characteristics are comparable to the native preparations.

Amino Acid Sequence↗
PubMed2005

Effects of moisture and temperature on the osteoinductivity of demineralized bone matrix.

Demineralized bone matrix (DBM) is a well established osteoinductive bone graft material. It has been mixed with a variety of carriers to adjust to different forms of handling for a variety of applications. The impact of the various carriers on osteoinductivity remains largely unknown. Using an in vitro cell culture assay and in vivo intramuscular implantations into nude rats, the effects of moisture from water-based carriers and the storage temperature on osteoinductivity were studied. In the dry state, DBM can preserve its osteoinductive activity when temperatures reached 65 degrees C, but in the presence of moisture, the activity decreases with incubation time. Nearly 90% of the DBM activity is lost when maintained for 5 weeks at 65 degrees C. This study further indicates that the structure and stability of the collagen network in DBM not only provide a scaffold for osteogenitor cell proliferation and differentiation, but also controls the release rate of osteoinductive growth factors.

Alkaline Phosphatase↗
PubMed1993

Spinal fusion induced by porous hydroxyapatite blocks (HA). Experimental comparative study with HA, demineralized bone matrix and autogenous bone marrow.

Numerous materials have been studied and recently used in clinical practice as synthetic bone biosubstitutes to integrate or even replace autogenous bone grafts. This study consisted of an experimental vertebral fusion model in rabbits using porous hydroxyapatite (HA), both alone and in various combinations with demineralized bone matrix (DBM), as a vehicle for the osteoinductive agent (Bone Morphogenetic Protein-BMP), and with autogenous bone marrow (BM), which supplies BMP-sensitive reticular cells. The animals were divided into two main groups on the basis of the physical form of the biomaterial implanted: HA blocks and HA granules, which in turn included several subgroups based on the composition of the implants (HA, HA+DBM, HA+BM, HA+DBM+BM). There were also control groups with no implant or with bone autografts (ACB). Two months after the operation, analysis of the results (radiograph, fusion stability test on dynamic radiographs, histological evaluation) demonstrated the following: clinical and radiographical results of the group implanted with HA blocks were superimposable on those with autogenous bone grafts, especially in the subgroup treated with DBM and BM, while in the group implanted with granules the results were poor, thus indicating the fundamental influence of the physical state of the biomaterial on bone ingrowth. This was then further confirmed by the histological study in the HA block group, whereas the HA granule group showed a certain fibrous reaction around the granules with poor porous bone ingrowth.

Animals↗
PubMed1987

Bone matrix and marrow versus cancellous bone in rabbit radial defects.

Implants of demineralized bone matrix induce new bone formation. In order to estimate the possible clinical usefulness of this phenomenon, autologous cancellous bone grafts were compared with composite grafts of bone matrix and marrow. Cancellous bone from the tuber ischii of the rabbit was transplanted to a preformed radial defect in the same animal. On the opposite side, a similar defect was filled with a mixture of either allogenous or autogenous bone-matrix particles and autogenous bone marrow. After 25 days, calcium 45 was injected intravenously. Three days later the animals were killed. Standardized segments of the rabbit's forearms, containing the middle of the defect, were cut out, ashed, and analyzed for 45Ca activity. No side difference in 45Ca deposition was found. The callus ash weight of the allogenous matrix-transplanted side was approximately 60% of that of the cancellous bone side. This side difference of ash weights corresponds to the estimated initial mineral content of the cancellous graft. Nontransplanted defects had very low ash weight and 45Ca activity. Thus, in the rabbit, composite grafts of bone matrix and marrow produce a bone yield comparable to that of cancellous bone.

Animals↗
PubMed1988

Irradiation-sterilization of rat bone matrix gelatin.

Bone matrix gelatin induces bone formation in muscle, and when implanted orthotopically it improves bone repair. Co-60 sterilization of bone gelatin impairs the protein-bound induction mechanisms. Gelatin samples nonirradiated or irradiated by 25 or 50 kGy were implanted into a pouch in the abdominal wall of Sprague-Dawley rats, as well as into a 7-mm calvarial defect. Evaluation was done by histologic studies, histomorphometry of orthotopic implants, and determination of alkaline phosphatase in ectopic implants. Gelatin irradiated with 50 kGy was absorbed in the muscle bed without evidence of any specific host reaction. Irradiation of 25 kGy led to histologically confirmed ectopic bone formation, but the wet weight of the explants was only half that of the nonirradiated control samples. Alkaline phosphatase activity was equal in both of these groups. With orthotopic implantation, neither a histologic nor a morphometric effect was seen with 25 kGy. Loss of osteoinduction with 25-kGy irradiation is apparently masked by osteoconductive mechanisms with orthotopic implantation.

Alkaline Phosphatase↗
PubMed1989

Bone formation and static changes in the thoracic spine at uni- or bilateral experimental spondylodesis with demineralized bone matrix (DBM).

Bone repair by autografting is an effective but disabling procedure, especially when considerable amounts of transplants are needed, as may be the case in spinal fusion operations. While looking for suitable alternatives, the trend is to study new agents stimulating bone formation (induction) as well as synthetic Bone Matrix (DBM), showing an activation of the osteoinductive properties of a Bone Morphogenetic Protein (BMP), has been successfully used in experimental posterolateral spondylodesis procedures, as it is highly effective together with autogenous Bone Marrow (BM). In the present experiment with thoracal spondylodesis on still growing rabbits, DBM, when supplied alone, confirmed its osteoinductive potential leading to clinical stability within three months in 67% of the cases and total radiological union within five months in all cases. Scoliosis with a rotational component developed following such spondylodesis procedures, but it could not be directly correlated to fusion modalities in the experiment. The pathway's analysis of the amplitude of the spinal canal at the fusion site, when compared to that of the adjacent segments, showed changes as initial increase of the amplitude in the fusion area during early fusion stages, followed by later narrowing. The relation to the amount of implanted DBM in the spondylodesis site and the development of spinal deformities could not be significantly correlated.

Animals↗
PubMed1989

Clinical induction of bone repair with demineralized bone matrix or a bone morphogenetic protein.

Treatment of orthopaedic lesions through induction of bone regeneration has produced promising results in laboratory animals and preliminary human trials. Both demineralized bone powder and bone morphogenetic protein (BMP) are being used clinically to treat bony defects without the need for an autogenous bone harvesting procedure. The historical perspective and laboratory rationale for osteoinduction is presented, as well as current and future clinical applications of demineralized bone powder and BMP.

Animals↗
PubMed1996

Demineralized bone matrix supplied by bone banks for a carrier of recombinant human bone morphogenetic protein (rhBMP-2): a substitute for autogeneic bone grafts.

Four commercially available preparations of demineralized freeze-dried human bone powders (DFDB) were investigated for endogenous bone morphogenetic protein (BMP) as observed by osteoinductive activity. Composites of DFDB without and with 1 microgram or 10 micrograms of recombinant human bone morphogenetic protein-2 (rhBMP-2) were implanted into the hindquarter muscles of Swiss-Webster mice. The four batches of DFDB without rhBMP-2 were placed also in hindquarter muscles of athymic mice. Three weeks after implantation, the area of induced bone and cartilage formation was measured by radiographic and histomorphometric methods. In normal mice, without rhBMP-2, DFDB implants induced development of dense fibrous connective tissue with minimal, if any, new bone. In athymic mice, DFDB induced development of small patches of appositional new bone. In contrast, in normal mice, composites of DFDB and rhBMP-2 induced development of large areas of heterotopic bone and bone marrow formation. The bone morphogenetic response occurred with a statistically significant difference (p < 0.0001) between implants of 1 microgram and the 10-microgram rhBMP-2 composites. Thus, DFDB from all four bone banks demonstrated comparable capacity to serve as a carrier for rhBMP-2.

Analysis of Variance↗
PubMed1991

Influence of parathyroidectomy, 1,25-dihydroxyvitamin D3 and high dietary calcium intake on demineralized bone matrix powder-induced bone formation in the rat.

Demineralized bone matrix induces ectopic endochondral bone formation. We used this model to study the effect of parathyroidectomy (PTX), 1,25-dihydroxyvitamin D3 treatment, and calcium enriched diet on bone formation in the rat. Hypocalcemia and hyperphosphatemia in PTX rats were corrected by 1,25-dihydroxyvitamin D3 treatment (2 x 12.5 ng/day) or by calcium enriched diet (3% calcium). Serum 1,25-dihydroxyvitamin D3 concentration was decreased in PTX rats and in intact rats with high dietary calcium intake. Calcium content of ectopic new bones (42 days after bone matrix implantation) was reduced in PTX rats compared with intact control rats. This could be prevented by 1,25-dihydroxyvitamin D3 treatment. In contrast, calcium enriched diet led to diminished mineralization of ectopic bones both in intact and PTX rats. We conclude that the effect of parathyroidectomy on bone formation may be mediated by 1,25-dihydroxyvitamin D3. 1,25-Dihydroxyvitamin D3 directly stimulates bone formation in this model and this effect is not simply the result of increasing serum calcium concentration.

Animals↗
PubMed2000

Ultrastructural identification of cells involved in the healing of intramembranous bone grafts in both the presence and absence of demineralised intramembranous bone matrix.

Alveolar bone defects are conditions that impede the progress of orthodontic treatment. This study compared the mechanics of the healing of autogenous intramembranous (IM) bone grafts and grafts comprising a mixture of IM and demineralised bone matrix of autogenous intramembranous origin (DBMIM), in an attempt to determine the reliability of each material. Thirty-two New Zealand white rabbits had a single defect created in their skull. Sixteen were grafted with IM bone alone (Group I: autogenous IM), and the other 16 had a combined graft of composite IM sandwiched between two layers of DBMIM (Group II: composite IM-DBMIM). A third group (Group III) of eight rabbits each had two defects created in their skull; one defect was left empty (A: passive control) and the other filled with rabbit-skin collagen (B: active control). In Groups I and II, inflammatory cells were found to be present on Days 1 and 2 of tissue retrieval. The appearance of the mesenchymal cells and preosteoblasts, osteoblasts and osteocytes was earlier (Day 3) in Group II than in Group I (Day 5). In both groups, preosteoblasts, osteoblasts and osteocytes were observed with no cartilage at the intermediate stage. In conclusion, autogenous IM bone grafts and IM bone grafts in the presence of DBMIM healed through an osteogenic ossification route.

Animals↗
PubMed1987

Nature of bone morphogenetic protein (BMP) from decalcified rabbit bone matrix.

Rabbit bone morphogenetic protein (BMP) from demineralized and defatted rabbit bone matrix was partially purified. BMP activity was examined by the implantation of fractionated materials into the thigh muscle pouch of the mouse. Rabbit BMP was solubilized by both 4M guanidine hydrochloride (GuHCl) and 6M urea solutions. Crude BMP had isoelectric point precipitation at pH 3 in 6M urea and showed bone morphogenesis. Fractions eluted with 0 and 0.2 N NaCl in DEAE CL-6B ion exchange chromatography showed bone morphogenesis in each individual pH of pH 4 to pH 7 but the fraction eluted with 1.0 N NaCl did not show any activity. Sephadex G-75 filtration separated the crude material into three peaks and the peak of about 23,000 showed bone morphogenesis. In sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis and isoelectric focusing, rabbit BMP was thought to be an acidic protein having a molecular weight of 24,000 with an isoelectric point around 4.85.

Animals↗
PubMed1997

A quantitative assessment of osteoinductivity of human demineralized bone matrix.

Demineralized bone matrix (DBM) is widely used in the repair of pathologies associated with skeletal defects and periodontal diseases. The present study was directed at establishing in vivo and in vitro models for a quantitative assessment of the osteoinductivity of DBM before clinical use. Athymic mice were used in an in vivo assay to overcome the species limitations (for human DBM) found in xenogeneic animal models. Calcium contents of explants, as an indicator of new bone formation, were assayed and expressed as a change in the weight percent calcium in the explant as compared to the weight percent of calcium in the implanted material. A total of 82 mice (2 implants per mouse) were used in this study. Significant amounts of new bone were induced in this animal model in response to implantation of DBM. Muscular implantation was found to be more osteoinductive (increases of 10.0 +/- 0.4 calcium weight percent of explant) than subcutaneous implantation (increases of 1.62 +/- 0.27 calcium weight percent of explant) and new bone formation in muscular implantation sites of athymic mice mimics endochondral bone formation. Between weeks 1 to 4, the weight of explanted materials did not significantly differ from the weight of the implanted material; however, by week 5 the explant weight began to increase. Calcium deposition over the 5 weeks of implantation increased in a nearly linear fashion. Consequently week 4 was chosen as the optimum time for explantation in the in vivo assay in that sufficient calcium levels had been achieved without a significant increase in explant dry weight. Aliquots of 10, 20, 30, and 40 mg per implantation site were used in dose response studies in the in vivo bioassay. Dose response curves with DBM exhibited maximal activity at the 20 mg DBM implant dose in the in vivo bioassay. An in vitro bioassay was also developed where human periosteal (HPO) cells were chosen because osteoprogenitor cells found in bone repair typically come from periosteal tissue. Alkaline phosphatase (ALP) activity in confluent cell cultures of HPO cells exposed to DBM, as an indicator of osteoblast induction, reached its highest level on day 5 of DBM treatment. Aliquots of 2, 5, 10, 20, 30, and 40 mg DBM per flask were chosen in dose response studies using the in vitro bioassay. These dose response studies with DBM revealed that quantities approximating 5 to 10 mg DBM in the in vitro model provided for maximal levels of ALP in cell extracts. A linear correlation (R2 = 0.7397) was demonstrated between the in vivo calcium remineralization assay and the in vitro ALP assay of osteoinductivity of DBM, suggesting that the in vitro assay can be used to quantitatively assess the osteoinductive potential of DBM where production and distribution of clinically usable DBM dictates rapid analysis.

Adult↗
PubMed2005

Mechanisms of action of demineralized bone matrix in the repair of cortical bone defects.

Demineralized bone matrix commonly is used to enhance and to facilitate bone grafting after skeletal injury or disease; however, the biologic bases for its bone-inducing abilities remain obscure. We have taken advantage of a mouse model of cortical bone defect healing to elucidate its mechanisms of action in vivo. Demineralized bone matrix combined with hyaluronan improved skeletal healing by inducing early deposition of an osteoid matrix. Demineralized bone matrix combined with hyaluronan might accelerate bone formation because it serves as a scaffold on which osteoprogenitor cells attach. We tested this possibility by comparing demineralized bone matrix combined with hyaluronan with heat-inactivated demineralized bone matrix combined with hyaluronan and found that the intact material was superior in terms of its ability to stimulate new bone formation. We also compared the bone inducing capacity of demineralized bone matrix combined with hyaluronan with a synthetic collagen sponge and found that not only the synthetic collagen scaffold delayed bone healing but also impaired bony bridging at later stages of repair. Another important property of demineralized bone matrix combined with hyaluronan was its ability to become actively degraded by osteoclasts during healing. Therefore, demineralized bone matrix combined with hyaluronan may not only attract osteoblasts and stimulate their differentiation, but also induce bone matrix resorption, which is a critically important regulator of bone formation and mineralization.

Animals↗
PubMed1989

Affinity of osteogenin, an extracellular bone matrix associated protein initiating bone differentiation, for concanavalin A.

Subcutaneous implantation of demineralized bone matrix results in bone differentiation. The bone inductive protein, osteogenin, was isolated recently by heparin affinity chromatography. The affinity of osteogenin for various lectins was examined to attain further purification and characterization. Osteogenin extracted from bovine bone matrix binds to concanavalin A (Con A) but not to wheat germ agglutinin or soybean lectin. The present data indicate that the bone inductive protein, osteogenin, is a glycoprotein. The use of a Con A Sepharose affinity column followed by preparative gel electrophoresis resulted in a greater than 250,000 fold purification of osteogenin.

Animals↗
PubMed1996

Morphological changes of autoclaved autogenic bone implantation and autoclaved autogenic bone supplemented with allogenic demineralized bone matrix in rat parietal bone.

The healing process of resected, autoclaved (121 degrees C, 20 minutes) and re-implanted bone in the rat parietal bone was compared with that of autoclaved bone that was supplemented with allogenic bone matrix (AAA-bone), using a scanning electron microscope and a light microscope. In the implant without AAA-bone, bone union and replacement of the autoclaved bone was seen at 2 weeks after implantation. There was no evidence of any inflammatory reaction around the autoclaved bone. The implant was gradually replaced by the new bone. In the implant with AAA-bone, the new bone formation around the implanted bone was more abundant than that of the implant without AAA-bone. An inflammatory reaction was also observed after 1 week. The replacement of the implant with AAA-bone was inferior to the nonsupplemented group. The reason for the poor replacement was the disturbance of the blood supply in the implant by abundant new bone formation. In these results, the autoclaved bone re-implantation was an excellent bone substitute with osteoconductive ability and biocompatibility. The implantation with AAA-bone was good for the new bone formation, but the position and the technique of supplement with AAA-bone have to be more deeply investigated.

Animals↗
PubMed1993

Interactions between the bone matrix proteins osteopontin and bone sialoprotein and the osteoclast integrin alpha v beta 3 potentiate bone resorption.

We have investigated the mechanism by which osteoclasts adhere to and resorb bone. We show that these cells express beta 1 and beta 3 integrins which are involved in attachment to purified bone matrix proteins. Binding to osteopontin and bone sialoprotein is mediated by alpha v beta 3, while a beta 1 integrin is responsible for attachment to fibronectin. Both the rapid attachment by osteoclasts to intact bone particles and their subsequent resorption are blocked by a monoclonal antibody directed to the alpha v beta 3 complex but not by an antibody against beta 1 integrins. Attachment of osteoclasts to bone is also inhibited with soluble osteopontin, Arg-Gly-Asp-containing peptides derived from both osteopontin and bone sialoprotein, or a monospecific polyclonal antibody against osteopontin. We conclude that both osteoclast adherence to bone and subsequent resorption of its matrix are dependent on interactions between the bone matrix proteins osteopontin and/or bone sialoprotein and the integrin alpha v beta 3. Moreover, collagen, which constitutes 90% of its organic matrix, is minimally involved in binding of chicken osteoclasts to bone.

Amino Acid Sequence↗
PubMed2006

BMP stimulation of alkaline phosphatase activity in pluripotent mouse C2C12 cells is inhibited by dermatopontin, one of the most abundant low molecular weight proteins in demineralized bone matrix.

Demineralized bone matrix (DBM) is a complex mixture of osteoinductive bone morphogenetic proteins (BMPs), as well as BMP-binding proteins that regulate BMP bioactivity and localization. Our aim was to use modern proteomic methods to identify additional BMP-binding proteins in DBM, with initial emphasis on the most abundant. Relatively large, water-soluble noncollagenous proteins (NCPs) were preferentially extracted from DBM with alkalinized urea. The insoluble residue, which contained the BMP activity, was extracted with GuHCl/CaCl2, dialyzed versus citrate, defatted, resuspended in GuHCl, dialyzed sequentially against Triton X-100 and water, pelleted, and lyophilized. The proteins in this pellet were fractionated by hydroxyapatite affinity chromatography. Proteins that copurified with BMP bioactivity were separated by SDS-PAGE. Distinct bands were excised, and the proteins in them were reduced and alkylated, digested with trypsin, eluted, and subjected to MALDI/ToF MS (matrix-assisted laser-desorption ionization time-of-flight mass spectrometry). Computer-assisted peptide fingerprint analysis of the MS profiles was used to identify C-terminal lysine-6-oxidase; dermatopontin (DPT); histones H2A2, H2A3, and H2B; and trace amounts of gamma-actin. DPT is a 22-kDa, tyrosine-rich acidic matrix protein not previously recognized to be among the most abundant small proteins to copurify with BMP bioactivity in DBM. We tested the effects of DPT on BMP-2 stimulation of alkaline phosphatase (ALP) activity in C2C12 cells. BMP-2 stimulated ALP activity in C2C12 cells by 6.2-fold above basal levels. DPT alone had no effect on ALP activity in C2C12 cells. When added with BMP-2, DPT blocked 40% of the stimulatory effect of BMP-2 on ALP activity in C2C12 cells. DPT is an abundant protein in DBM, and it can inhibit the stimulatory effects of BMP-2 on ALP activity in C2C12 cells.

Alkaline Phosphatase↗