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PubMed1981

Purification, composition, and 31P NMR spectroscopic properties of a noncollagenous phosphoprotein isolated from chicken bone matrix.

Fractionation of the EDTA-soluble, noncollagenous proteins of the organic matrix of chicken bone by Sephadex G-100 molecular sieving has revealed that the majority of the organic phosphorus is present in two fractions, from one of which a homogeneous phosphoprotein has been isolated. The purified phosphoprotein has an apparent molecular weight of 12,000 and contains both O-phosphoserine and O-phosphothreonine. 31P-NMR spectroscopy demonstrates that all of the organic phosphorus exists in the form of phosphomonoesters which have an average pK2 of 6.8. The phosphoprotein is highly acidic due to its high content of dicarboxylic acids in addition to the presence of organic phosphorus. The characteristic amino acid composition of the phosphoprotein establishes its noncollagenous nature and highlights the differences among bone, dentin, and enamel phosphoproteins. The absence of gamma-carboxyglutamic acid distinguishes it from osteocalcin, the noncollagenous gamma-carboxyglutamic acid-containing peptide of bone matrix.

Amino Acids↗
PubMed2000

Reconstruction of complex cranial wounds with demineralized bone matrix and bilayer artificial skin.

Complex wounds involving loss of both cranium and scalp are difficult to reconstruct. Current solutions include both autogenous and alloplastic materials. Tissue engineering provides interesting alternatives for reconstruction of missing bone and soft tissue. Demineralized bone matrix and Integra, artificial bilayer skin substitute, have been used successfully to reconstruct bone and skin as isolated defects, but never in combination. This study investigates the possibility of using demineralized bone matrix in a gel carrier and Integra for combined cranial and scalp defects. The study was divided into two parts. In the first, demineralized bone matrix in the forms of Grafton Flex (n = 12) and Grafton putty (n = 12) was used to reconstruct 15-mm critical size cranial defects in female adult New Zealand White rabbits. In the control group (n = 6), the defect was left empty. The second part of the study investigated the use of Integra and Grafton Flex to reconstruct a 15-mm cranial defect with an overlying full-thickness scalp defect (n = 6). The first study revealed bony healing of the critical-size cranial defect with demineralized bone matrix. The second study demonstrated successful reconstitution of scalp and cranium with both demineralized bone matrix and Integra. We conclude that complex cranial defects involving bone and soft tissue may be successfully reconstructed with their tissue-engineered substrates, demineralized bone matrix (Grafton) and Integra artificial skin.

Absorptiometry, Photon↗
PubMed1977

The effects of zinc deprivation on the host response to intramuscular bone matrix implants in the rat.

Sterile sequentially extracted insoluble bone matrix gelatin and freeze-dried allogeneic demineralized whole bone matrix was implanted into a lumbar muscle pouch of 12 Sprague-Dawley rats fed a diet containing 0.9 ppm zinc and into 6 rats fed a diet containing 50 ppm zinc. Histological sections and microradiography demonstrated that skeletal tissue induction occurred in all instances. In the zinc-supplemented hosts, four weeks postimplantation, trabecular bone and bone marrow grew on both sides of the matrix implant which had been almost completely resorbed by matrixclast activity. However, in the zinc-deficient hosts, the central cavity of the matrix was first filled with mesenchyme and then cartilage cells which grew in the form of thin wedges into the implanted matrix. Poor vascularization appears to be an important factor in cell proliferation and abnormal differentiation in zinc deficiency.

Animals↗
PubMed1992

Osteogenesis associated with bone gla protein gene expression in diffusion chambers by bone marrow cells with demineralized bone matrix.

Diffusion chambers with rat bone marrow cells and demineralized bone matrix (DBM) were implanted subcutaneously to syngeneic 8-week-old rats and were harvested every week 3-7 weeks after implantation, and histochemical examination, determination of alkaline phosphatase activity, total calcium and phosphorus, the bone-specific vitamin K-dependent gla-containing protein (BGP) content, and detection of BGP mRNA relative to mineralization were performed. Alkaline phosphatase in diffusion chamber implants reached the highest activity at 4 weeks and then decreased. Calcium and phosphorus deposits occurred at 4 weeks after implantation and were followed by marked increases until 7 weeks, which was comparable to the accumulation of BGP. The BGP gene within the diffusion chambers began to be expressed at 5 weeks, and its expression increased markedly at 7 weeks after implantation. At 4-5 weeks after implantation, new bone adjacent to the membrane filters and cartilage toward the center of the diffusion chamber were observed histochemically. Light microscopic and immunohistologic examinations of chambers with marrow cells and DBM revealed production of mineralized matrices, typical of bone characterized by the appearance of BGP and mineralized nodules. In contrast, bone marrow cells alone did not show extensive bone formation and yielded very low values for these biochemical parameters. The present experiments demonstrate the potential of bone marrow cells and DBM to produce not only cartilage formation but also membranous bone formation associated with increasing expression of BGP mRNA during the later stages of bone formation, as well as a marked accumulation of BGP.

Alkaline Phosphatase↗
PubMed1989

Osteogenesis in cranial defects and diffusion chambers. Comparison in rabbits of bone matrix, marrow, and collagen implants.

In rabbits, we compared calcification and bone formation by bone marrow, acid-demineralized bone matrix and glutaraldehyde-cross-linked Type I collagen implanted in intramuscular diffusion chambers or in trephine skull defects. The rabbits were killed 4 weeks postimplantation and calcification and osteogenesis were evaluated radiographically and histologically, and by calcium and alkaline phosphatase assays. Bone marrow produced bone and fibrous tissue within the chambers and had high alkaline phosphatase levels. Bone matrix in chambers with intact filters failed to induce bone formation within and outside the chambers, while glutaraldehyde-cross-linked collagen produced only scant calcific deposits following implantation in either diffusion chambers or skull defects. Central areas of skull defects implanted with bone marrow were partially repaired with new bone and had high calcium and alkaline phosphatase levels, but not as high as defects implanted with demineralized bone matrix.

Animals↗
PubMed1994

Osteoclast recruiting activity in bone matrix.

An activity that recruits osteoclasts has been identified and partially characterized from bone matrix. Bone-derived osteoclast recruiting activity (BORA) was co-purified with osteogenin, a bone inductive protein. Osteogenin was extracted from bovine bone with 6 M urea and purified by chromatography on hydroxyapatite, heparin-Sepharose and Sephacryl S-200 gel filtration. The biologically active osteoclast formation-stimulating material was further purified by C18 reverse phase HPLC. BORA is obviously distinct from osteogenin and transforming growth factor beta (TGF-beta), since further purified osteogenin and pure TGF-beta did not stimulate the formation of osteoclast-like cells. BORA (0.1-10 micrograms/ml) stimulated the formation of tartrate-resistant acid phosphatase (TRAP)-positive multinucleated cells (MNC) in a dose-dependent manner. These multinucleated cells resorbed bone when cultured on bovine bone slices. The effect of BORA is primarily directed to differentiate osteoclast precursors, since it did not stimulate osteoclast function in in vitro resorption assay where disaggregated rat osteoclasts were cultured on bovine bone slices. However, after 24 h preincubation with 50 nM PTH in the mouse calvaria assay, BORA at 10 micrograms/ml significantly stimulated bone resorption.

Animals↗
PubMed1981

Bridging of large chronic osteoperiosteal gaps by allogeneic decalcified bone matrix implants in rabbits.

A circumferential complete osteoperiosteal gap (1 to 1.5 cm) was produced in the diaphysis of ulna of rabbits, and the ipsilateral radius was kept intact. In no case did spontaneous bridging of the gap occur. By 6 weeks after the creation of the gap the bone ends became smooth and tapered towards the ipsilateral radius and the defect was observed to be filled by thin fibrous tissue. Established defects at least 6 weeks old were then bridged by inserting a snugly fitting allogeneic bone matrix implant. The bone matrix was prepared by demineralization of tubular bones from healthy rabbits, using 0.6 M HCl, preserved in 75% alcohol and used within 3 weeks. The fate of the implant was observed clinically, radiologically, histologically by tetracycline fluorescence for up to 12 weeks. Radiologic evidence of bone formation was present in a majority by 6 weeks, and complete bridging of the gap was seen in 75% of cases by 12 weeks after implantation. Most of the initial bone that formed was the woven bone, i.e., immature bone formed directly from the mesenchymal cells, or formed in membrane, though areas of enchondral ossification were also observed in some cases. By 12 weeks, the bone had remodeled almost to the texture of a mature tubular bone. Successful bridging of chronic massive bone defects by allogeneic bone-matrix was confirmed in 75% of cases.

Animals↗
PubMed1976

A comparison of bone matrix and tendon with particular reference to glycoprotein content.

Bone matrix and tendon are compared in terms of their carbohydrate and non-collagenous protein composition. The collagen content of both tissues was similar (90-91%), but bone matrix had at least three times as much sialic acid (0.28%) as tendon (0.08%). Smaller differences were found in the analysis of hexoses and hexosamines. After digestion with bacterial collagenase, about 9% of the total protein from both tissues was non-diffusible on dialysis, and this contained only 0.15% (bone) and 0.7% (tendon) of the original hydroxyproline; recovery of sialic acid was 86-87%. The collagenase-resistant soluble material amounted to about 9% (bone matrix) and 5% (tendon); the insoluble residues were 1 and 4% respectively. There were clear differences in the carbohydrate contents of the digests, but the amino acid compositions were similar. When the soluble digests were chromatographed on DEAE-cellulose, the elution profiles indicated the presence in each tissue of a variety of glycoproteins and a proteoglycan fraction, and showed clearly that an acidic glycoprotein corresponding to bone sialoprotein was not present in tendon.

Amino Acids↗
PubMed1988

Demonstration of reduced mitogenic and osteoinductive activities in demineralized allogeneic bone matrix from vitamin D-deficient rats.

Osteoinduction is the formation of ectopic bone that follows implantation of demineralized allogeneic bone matrix (DABM) and is believed to be secondary to the release of associated inductive factors from bone matrix. To clarify the role of vitamin D in osteoinduction, we implanted DABM from vitamin D-deficient rats (-D rats) into normal rats (+D rats). Because mitogens and osteocalcin might be involved in osteoinduction, these were measured. Mitogenic activity in extracts from mineralized allogeneic bone matrix (ABM) and DABM from both +D and -D rats was determined with an assay that utilizes monolayer cultures of embryonic chick calvarial cells. Osteocalcin in serum and DABM was measured by radioimmunoassay. DABM from -D rats did not promote osteoinduction as effectively as DABM from +D rats. Resorption of implant matrix from -D rats was diminished compared with resorption of matrix from +D rats (P less than 0.01), and the decrease was attributed to a corresponding decrease in the number of osteoclasts in the implants (P less than 0.02). Bone formation (P less than 0.01) and total implant mineralization (P less than 0.001) were significantly reduced in implants from -D rats, and the reductions corresponded with a decline in the number of osteoblasts (P less than 0.05). Mitogenic activity in DABM from +D rats was only slightly decreased as compared with activity in ABM, but DABM from -D rats contained significantly less activity (P less than 0.001). No mitogenic activity was identified in implants of DABM from either +D or -D rats 3 wk after implantation. Serum osteocalcin was significantly higher in -D as compared with +D animals. In contrast, the concentrations of osteocalcin in DABM from the two groups of animals were not significantly different from each other. These findings indicate that the diminished osteoinductive activity of DABM from -D rats results from deficiency of one or more mitogenic factors that are essential for inducing the proliferation and differentiation of bone cells at the implant site and that osteocalcin does not play a role in this regard.

Animals↗
PubMed2004

Hybrid constructs for craniofacial reconstruction: sustained gene delivery using demineralized bone matrix putty.

These experiments evaluate the efficacy of a demineralized bone matrix putty engineered as a hybrid construct for sustained, site-directed gene transfer using an adenoviral vector. In vitro experiments were performed to evaluate the optimal dosing for gene transfer to fetal calvarial osteoblasts and dural cells and for the sustainability of gene transfer from the hybrid constructs. In the dosing experiments, hybrid constructs were created by combining 0.5 mL of demineralized bone matrix putty (DBX; SYNTHES Maxillofacial, Monument, CO) with 1 x 10(8), 1 x 10(7), or 1 x 10(6) particle-forming units (PFU) of an adenoviral vector carrying the gene encoding green fluorescent protein (AdGFP). These constructs were then placed in direct contact, or in transwell coculture, with fetal murine calvarial osteoblasts or dural cells at a multiplicity of infection (MOI = viral particle/cell ratio) of 1000, 100, and 10. The sustainability of gene transfer was tested through transfer of the hybrid construct to wells containing untransfected cells every 24 hours for 30 days. In both experiments, gene transfer was determined through the visualization of GFP using fluorescence light microscopy 24 hours after the onset of transfection. Optimal dosing for gene transfer occurred at an MOI of 10 for calvarial osteoblasts and 100 for dural cells. At greater concentrations, toxicity was observed in the majority of samples. Gene transfer to fetal dural cells and calvarial osteoblasts was sustained throughout the 30-day period. These experiments suggest that adenoviral vectors could be successfully incorporated within demineralized bone matrix to provide effective, sustained, site-directed gene transfer.

Adenoviridae↗
PubMed1999

New formulations of demineralized bone matrix as a more effective graft alternative in experimental posterolateral lumbar spine arthrodesis.

STUDY DESIGN: A rabbit model of posterolateral intertransverse process spine arthrodesis was used. OBJECTIVE: To determine the efficacy of two new formulations of demineralized bone matrix. SUMMARY OF BACKGROUND DATA: The flowable gel form of Grafton (Osteotech, Eatontown, NJ) demineralized bone matrix has been shown to have osteoinductive properties in various models and currently is used clinically as bone graft material in posterolateral lumbar spine arthrodesis. Two new formulations of Grafton, one made of flexible sheets (Flex) and the other made in a malleable consistency (Putty), have improved handling characteristics compared with the gel form. METHODS: In this study, 108 New Zealand white rabbits underwent bilateral posterolateral intertransverse spine arthrodesis at L5-L6 using autogenous iliac crest bone graft alone (control), one of the new forms of demineralized bone matrix (DBM; made from rabbit bone) alone or in combination with autogenous iliac crest bone. Rabbits were killed 6 weeks after surgery. The lumbar spines were excised, and fusion success or failure was determined by manual palpation and radiography. Specimens also were processed for undecalcified histologic analysis. RESULTS: Manual palpation of the harvested lumbar spines revealed that the fusion rates of the Flex-DBM/Auto group (9/9, 100%) and Putty-DBM/Auto group (10/10, 100%) were superior (P < 0.01) to those of the Auto/control group (3/9, 33%). As a stand-alone graft substitute, Flex-DBM performed superiorly with a fusion rate of 11/11 (100%) compared with that of Putty-DBM (10/12, 83%) and Gel-DBM (7/12, 58%). The devitalized version of Flex-DBM had a fusion rate of 4/11 (36%), which was comparable with the devitalized Putty-DBM rate of 4/12 (33%). Both were superior (P < 0.05) to the devitalized Gel-DBM rate of 0/12 (0%). More mature fusions with greater amounts of trabecular bone were present radiographically and histologically in rabbits that received all forms of demineralized bone matrix than in those in which autograft was used. CONCLUSIONS: The new flexible sheet and malleable putty forms of demineralized bone matrix were effective as graft extender and graft enhancer in a model of posterolateral lumbar spine fusion. These newer formulations of Grafton appear to have a greater capacity to form bone than the gel form or autogenous bone graft alone in this model.

Animals↗
PubMed1992

Accelerated endochondral osteoinduction in the absence of bone matrix particles in a rat model system.

Ethanol-precipitated proteins obtained from demineralized rat bone powder (DBP) by 4M guanidine-HCl extraction have been shown to reproducibly induce ectopic endochondral bone formation when subcutaneously implanted in rats in the absence of bone matrix particles. Histologic and biochemical analysis revealed a temporal sequence of chondrocyte differentiation, calcified cartilage formation, neovascularization, osteoblast differentiation, bone formation, osteoclastic bone remodeling, and hematopoietic marrow development that is complete by 21 days. In contrast to previous reports, these results clearly show an osteoinductive response independent of the presence of insoluble extracellular bone matrix. Compared with conventional DBP implants, the guanidine-extractable protein (GE) produces an accelerated and more robust osteoinductive response. Histologically, the initial chondrogenic response at days 6 to 9 is greatly amplified. Alkaline phosphatase specific activity peaks at day 9, several days earlier than for DBP, and is sixfold higher. Calcium accumulation in GE implants at day 12 is fivefold greater than with DBP, and all mineral is localized within the matrix of newly calcified cartilage and new bone. Osteoclasts are up to ninefold more abundant in the rapidly remodeling GE ossicle, making space for hematopoietic marrow. Delivery of GE coprecipitated with inert bone matrix particles was also more effective than DBP, although the response was somewhat attenuated compared with GE alone. Bony filling of 4-mm defects in rat mandibular rami was elicited by 10 mg of GE and followed an endochondral process with increased neovascularization compared with DBP and unimplanted controls. This guanidine-extractable protein fraction should prove useful for inducing quantities of chondrocytes and osteoclasts for in vitro study, and for analysis of osteoinductive requirements.(ABSTRACT TRUNCATED AT 250 WORDS)

Alkaline Phosphatase↗
PubMed2006

Safety and efficacy of use of demineralised bone matrix in orthopaedic and trauma surgery.

Demineralised bone matrix (DBM) acts as an osteoconductive, and possibly as an osteoinductive, material. It is widely used in orthopaedic, neurosurgical, plastic and dental areas. More than 500,000 bone grafting procedures with DBM are performed annually in the US. It does not offer structural support, but it is well suited for filling bone defects and cavities. The osteoinductive nature of DBM is presumably attributed to the presence of matrix-associated bone morphogenetic proteins (BMPs) and growth factors, which are made available to the host environment by the demineralisation process. Clinical results have not been uniformly favourable; however, a variable clinical response is attributed partly to nonuniform processing methods found among numerous bone banks and commercial suppliers. DBMs remain reasonably safe and effective products. The ultimate safe bone-graft substitute, one that is osteoconductive, osteoinductive, osteogenic and mechanically strong, remains elusive.

Animals↗
PubMed1988

Osteoinduction by implants of demineralized allogeneic bone matrix is diminished in vitamin D-deficient rats.

Experimental heterotopic bone formation was produced by subcutaneous implants of demineralized allogeneic bone matrix (DABM) in vitamin D-deficient (-D) animals that were either not treated or given vitamin D3 (+D) or 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) to determine the role of vitamin D and its most active metabolite in osteoinduction and implant remodeling. Histologically, implants in both +D and -D groups caused a similar acute inflammatory response, formation of a fibrous capsule, and chondrogenesis by 1 to 2 weeks after implantation. However, by 3 weeks after implantation implants in the -D animals had formed less bone matrix, had developed a defect in matrix mineralization, had reduced bone forming and bone resorbing surfaces, and had altered bone architecture resulting from defective bone remodeling. The altered histology in -D animals was not corrected by 10 weeks after implantation. Treatment of vitamin D-deficient rats with 1,25(OH)2D3, 65 pmol/day for 3 weeks, corrected both the defect in mineralization and the abnormal histology. The results indicate that (1) vitamin D deficiency does not alter either the timing or the sequence of histologic events associated with osteoinduction but dramatically reduces the magnitude of the response, (2) vitamin D deficiency not only impairs mineralization but also reduces bone formation and resorption, and (3) 1,25(OH)2D3 mimics all of the actions of vitamin D with regard to correcting the abnormal osteoinductive response and bone histomorphometry.

Animals↗
PubMed1978

The osteoninductive property of decalcified bone matrix. An experimental study,.

Demineralised homologous bone-matrix implant was used to bridge a large circumferential osteoperiosteal gap in the diaphysis of the ulna of rabbits. Periodic observations of the graft were made clinically, radiologically, histologically and by tetracycline fluorescence up to forty-two weeks. By the twelfth week after operation 81 per cent of the animals revealed bone formation in the implant and complete bridging of the gap. The new bone was laid on the surface and in the substance of the matrix, suggesting that the inductive principle was acting locally. The bone, once formed, remodelled to the texture of a mature tubular bone and did not undergo absorption during a long follow-up period. Demineralise bone-matrix proved to be a highly osteoinductive and readily osteoconductive material. The graft did not evoke any appreciable local foreign-body or immunogenic reaction. The high degree of success in bridging massive bone defects justifies further serious studies and hopes for a useful substitute for massive autologous bone grafts.

Animals↗
PubMed1994

The purification and partial characterization of bone resorptive polypeptides from bovine bone matrix.

Matrix proteins were extracted from bovine cortical bone with EDTA/Tris-HCl under non-dissociative conditions at neutral pH. Four distinct bone resorptive proteins with molecular masses of 14, 25, 29 and 40 kDa were purified and partially characterized using an in vitro neonatal mouse calvarial assay and a growth factor assay using BALB/c/3T3 cells. The 14 kDa protein was purified by anion exchange chromatography (Mono Q) and gel filtration (Superdex 75HR) using FPLC (fast protein liquid chromatography); this factor stimulated the proliferation of MCF-7 human breast cancer cells, a bioassay which is specific for the insulin-like growth factors (IGFs). The 25, 29 and 40 kDa proteins were purified by sequential chromatography as follows: anion-exchange (Mono Q), heparin-Sepharose, hydroxyapatite, concanavalin A-Sepharose, phenyl-Superose, reversed phase high performance liquid chromatography (HPLC) and sodium dodecylsulfate polyacrylamide gelelectrophoresis (SDS-PAGE). The 25 kDa protein was identified as TGF-beta by its inhibitory effect on the proliferation of mink lung cells. The 40 kDa protein enhanced the formation of multinucleate tartrate-resistant acid phosphatase positive cells in a murine bone marrow differentiation assay, but was without effect in an isolated osteoclast assay and had no growth factor activity; this protein is likely to be a colony stimulating factor. The 29 kDa protein was also without growth factor activity; it was, however, able to stimulate bone resorption in the isolated osteoclast assay, suggesting a direct action in osteoclast function. The 29 and 40 kDa proteins may be osteoblast gene products that have been sequestrated by the bone matrix in a similar fashion to TGF-beta and the IGFs. This is the first report of proteins isolated from bone matrix which directly stimulate osteoclast differentiation and activity.

Animals↗
PubMed1993

Heat tolerance of activity toward ectopic bone formation by rabbit bone matrix protein.

The 4 M guanidine hydrochloride (GuHCl)-soluble bone matrix proteins which contained bone morphogenetic protein (BMP) were found capable of inducing ectopic bone and cartilage formation in mammals. This osteogenic capacity was resistant to high temperatures and mild heating actually increased the amount of bone induced, with a maximum occurring at 70 degrees C. The osteogenic potential was preserved after heating at 170 degrees C for 10 min or 140 degrees C for 30 min.

Animals↗
PubMed2001

Osteomalacia in hyp mice is associated with abnormal phex expression and with altered bone matrix protein expression and deposition.

To explore how the loss of Phex function contributes to the pathogenesis of osteomalacia, we examined the abnormalities of mineralization, Phex, and bone matrix protein expression occurring in Hyp mice in vivo and in ex vivo bone marrow cell cultures. The results in vivo show that mineralization was decreased significantly in Hyp mouse bone. Phex protein was identifiable in osteoblasts and osteocytes in wild-type mice, but not in Hyp mice. In Hyp mice, osteocalcin, bone sialoprotein, and vitronectin expression were down-regulated, whereas biglycan and fibrillin-1 expression were up-regulated in osteocytes and bone matrix relative to those in their wild-type counterparts. Parallel studies ex vivo demonstrated that cells derived from 18-day Hyp mouse bone marrow cell cultures had a 3'-Phex deletion, no Phex protein expression, decreased alkaline phosphatase activity, collagen deposition, and calcium accumulation, and reduced osteocalcin, bone sialoprotein, and vitronectin at both the protein and messenger RNA levels. Furthermore conditioned medium from Hyp mouse bone marrow cultures could induce analogous defects in bone marrow cell cultures of wild-type cells. These novel findings indicate that there is an intrinsic osteogenic cell differentiation defect in addition to the known hypomineralization of bone in Hyp mice, which may be inducible by an autocrine/paracrine secreted factor. These results suggest that alterations in the Phex gene may control bone matrix mineralization indirectly by regulating the synthesis and deposition of bone matrix proteins.

Animals↗