Age effects on bone induction by demineralized bone powder

It has been previously shown that osteoinduction by demineralized bone powder (DBP) in the rat decreases as the age of the recipient animal increases. In the present study, the effects of age on osteoinduction by DBP were evaluated in the rat by varying the age of the donor and the recipient animal. Cartilage and bone formation in subcutaneous pouches was assessed using a histologic grading technique in which a composite score was derived from analysis of multiple histologic sections from each specimen. The results confirm the previously reported decrease in osteoinduction in middle-aged adult animals compared with younger ones. However, DBP prepared from middle-aged adult rats was more inductive than that prepared from either prepubertal or young postpubertal animals. The latter result contradicts the widely held belief that demineralized bone matrix from younger animals is more inductive than that from older ones. This finding may help to further elucidate the mechanism of ectopic bone formation and lead to more inductive bone graft substitutes for human use.     

The bonding of glass ceramics to bone

Summary In this study the bonding behaviour of glass ceramics, containing apatite and wollastonite, to bone tissue is shown to vary depending on the amount of alumina they contain. We have experimented with three types of material: A·W-GC, AW-6 and AW-AL. Rectangular plates were implanted into the tibiae of rabbits. Ten weeks later a segment of bone around the plate was removed for examination, and the load of breaking by traction (failure load) was measured by an autograph. This was lowest for AW-AL and highest for A·W-GC (with AW-6 in between), and the figures differed significantly from each other (P<0.01). The interface was examined by a scanning electron micro-analyser and an energy dispersive X-ray micro-analyser (SEM-EPMA) and the reactive zone, the calcium-phosphorus rich layer, was assessed. Silicon and magnesium decreased, the calcium did not change, and the phosphorus increased. The reactive zone of A·W-GC was wider than that of AW-6. A Ca-P rich layer was not present between AW-AL and the bone. It is suggested that the strong bonding between glass-ceramics and bone was made through the formation of the Ca-P rich layer.

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Résumé Cette étude montre que la liaison entre les céramiques, contenant de l'apatite et de la wollastonite, et le tissue osseux, varient en fonction de la quantité d'alumine qu'elles contiennent. Nous avons expérimenté trois types de matériaux: AW-GC, AW-6 et AW-AL. Des plaques rectangulaires ont été insérées dans des tibias de lapin. Dix jours plus tard on a découpé, en vue d'examen, un segment d'os autour de la plaque. La charge de rupture par traction a été mesurée. Elle est la plus basse pour le AW-AL et la plus élevée pour le AW-GC (le AW-6 est entre deux) et les chiffres diffèrent significativement l'un de l'autre (P<0,01). L'interface a été examinée par micro-analyse électronique et radiologique et la zone réactive, la couche riche en calcium et phosphore a été étudiée. Le silicium et le magnésium avaient diminué, le calcium était resté stable et le phosphore avait augmenté. La zone réactive de l'AW-GC était plus large que celle de l'AW-6. Il n'existait pas de couche phopho-calcique entre l'AW-AL et l'os. Il semble que la forte liaison entre la céramique et l'os soit due à la formation d'une importante couche phopho-calcique.

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This study was supported by Japan Orthopaedics and Traumatology Foundation Inc, grant number 0020     

Comparison of TGF-beta/BMP pathways signaled by demineralized bone powder and BMP-2 in human dermal fibroblasts.

Demineralized bone induces chondrogenic differentiation of human dermal fibroblasts in vitro. Analyses of signaling gene expression showed that DBP and BMP-2 regulate common and distinct pathways. Although BMP-2 was originally isolated as a putative active factor in DBP, rhBMP-2 and DBP do not affect all the same genes or in the same ways. INTRODUCTION: Demineralized bone powder (DBP) induces chondrogenic differentiation of human dermal fibroblasts (hDFs) in 3D culture, but the initiating mechanisms have not been identified. We tested the hypotheses that DBP would affect expression of signaling genes and that DBP's effects would differ from the effects of bone morphogenetic proteins (BMPs). MATERIALS AND METHODS: A chondroinduction model was used in which hDFs were cultured with and without DBP in a porous collagen sponge. BMP-2 was delivered in a square of absorbable collagen felt inserted into a collagen sponge. Total RNA was isolated after 3 days of culture, a time that precedes expression of the chondrocyte phenotype. Gene expression was evaluated with two targeted macroarray screens. Effects of DBP and rhBMP-2 were compared by macroarray, RT-PCR, and Northern hybridization analysis of selected genes in the transforming growth factor (TGF)-beta/BMP signaling pathways. RESULTS: By macroarray analysis of 16 signal transduction pathways, the following pathways were modulated in hDFs by DBP: TGF-beta, insulin/LDL, hedgehog, PI3 kinase/AKT, NF-kappaB, androgen, retinoic acid, and NFAT. There was convergence and divergence in DBP and rhBMP-2 regulation of genes in the TGF-beta/BMP signaling pathway. Smad target genes were the predominant group of DBP- or rhBMP-2-regulated genes. Several genes (IGF-BP3, ID2, and ID3) showed similar responses (increased expression) to DBP and rhBMP-2. In contrast, many of the genes that were greatly upregulated by DBP (TGFBI/betaig-h3, Col3A1, TIMP1, p21/Waf1/Cip1) were barely affected by rhBMP-2. CONCLUSION: These findings indicate that multiple signaling pathways are regulated in fibroblasts by DBP, that one of the major pathways involves Smad target genes, and that DBP and rhBMP-2 elicit different gene expression responses in hDFs. Although BMP-2 was originally isolated as a putative inductive factor in DBP, rhBMP-2 and DBP do not affect all the same genes or in the same ways.     

Use of osteopromotive growth factors, demineralized bone matrix, and ceramics to enhance spinal fusion

Recently developed materials that can enhance fusion rates for posterolateral lumbar arthrodesis may be used alone or in combination with autogenous bone grafts. Novel osteopromotive growth factor preparations are currently under scrutiny; these include autogenous growth factor concentrate, bovine bone-derived osteoinductive protein, and recombinant human MP52. Demineralized bone matrix products may enhance or extend grafts. However, few studies, especially prospective randomized clinical trials, have assessed their efficacy, so it is difficult to compare formulations. Ceramics have been evaluated in animal studies and human clinical trials for a variety of applications in spinal surgery. These materials function best as bone graft extenders or as bioactive osteoinductive material carriers in posterolateral lumbar fusions. They have the advantage of variable porosity, low cost, and ease of manufacture. Hydroxyapatite/tricalcium phosphate ceramics have been shown to perform as well as autogenous bone grafts but with fewer complications.     

Induced healing of aneurysmal bone cysts by demineralized bone particles

Two cases of induced healing of aneurysmal bone cyst (ABC) following intralesional implantation of a bone paste made of autogeneic bone marrow and allogeneic bone powder are reported. The calcaneum in one case and the superior pubic ramus in the other were blown out by an ABC and would have required extensive surgery. Via a minimal exposure, the cyst was partially evacuated and filled with an admixture of a partially demineralized bone particles with bone marrow. Ossification of the peripheral shell was the first sign of healing and was observed within the first 3 postoperative months. Successful healing was observed in both cases. The rationale underlying this intralesional treatment was that the bone grafting material might reverse ABC expansion by promoting ossification through a bone induction mechanism. The concept of this treatment was to retain the ABC tissue, using its own intrinsic osteogenic potential to promote healing. By triggering intralesional new bone formation, the bone paste represented an effective means to reverse the expanding phase of ABC. The particulated bone allograft was easy to handle and to introduced in an irregular cavity. Moreover, as a complete cyst evacuation was not required, a minimal surgical approach could be used so that the risks and morbidity associated with an extensive approach were reduced. Its use is of particular interest in poorly accessible areas like the pelvis and spine.     

Induction of endochondral bone by demineralized bone matrix from diabetic rats

Summary In this investigation we examined the osteoinductive potential of demineralized bone matrix derived from chronically diabetic (streptozotocin-induced) rats. Long-Evans rats (28–31 days) were made diabetic with a single injection of streptozotocin (65 mg/kg) and provided food and waterad lib for 2 months. Diaphyseal shafts of femurs and tibias removed from the diabetic rats and their sibling controls were dehydrated, pulverized, sieved to 74–420 μm particles, and demineralized Matrix was then bioassayed for its ability to induce endochondral bone on day 11 following subcutaneous implantation over the thorax of Long-Evans rats. The resulting plaques of tissue were subjected to histological analysis, determination of alkaline phosphatase activity, and calcium content. Bone matrix derived from diabetic animals proved to be a significantly better inducer of endochondral bone than did control matrix.     

Cellular events associated with the induction of bone by demineralized bone

Implantation of demineralized bone (DB) in the form of powder or intact segments in extra skeletal sites stimulates new bone formation. Urist and co-workers presented substantial evidence that there is a noncollagenous protein that has the ability to induce bone formation. One aim of this study was to trace the process of bone formation when DB, in the form of perforated rectangular plates, is implanted subcutaneously in 2-month-old rats. A second objective was to determine whether cartilage cells play a role in the formation of bone in this model. Various DB plates with 0.25 mm diameter holes were implanted subcutaneously for 1-4 weeks in rats. One week after implantation, DB plates were covered by vascularized connective tissue that invaded the perforations. Aggregates of chondrocytes were observed within the holes and on periosteal surfaces in only a few specimens. Further cartilage proliferation was not observed, and by the 2nd week there was no evidence of endochondral bone formation. Where these cartilage-like cells were present, a thin layer of mineral was deposited around them; resorption and fibrous tissue infiltration followed. This aborted form of endochondral calcification was not followed spatially by bone formation. Patent vascularized channels were invaded by alkaline phosphatase-positive mononuclear cells and fibroblasts, and became enlarged by the enzymatic action of macrophages. The next step involved the calcification of DB plates adjacent to the wide spaces. Osteoclasts now appeared leading to the resorption of this recalcified matrix. The eroded and now enlarged lacunar surfaces were lined by newly formed bone and osteoblasts. This process continued so that, at the end of 4 weeks following implantation, the original DB plates were replaced by trabecular bone. Biochemical data on calcium and alkaline phosphatase levels in the implants paralleled the morphological observations.     

Bridging large defects in bone by demineralized bone matrix in the form of a powder. A radiographic, histological, and radioisotope-uptake study in rats

Demineralized bone powder was used as an osteoinductive substance to bridge very large defects (more than 50 per cent of the total length of the bone) in one radius of each of thirty-three rats. An identical defect was produced in the contralateral radius of each animal for use as a control. The defect on the control side was left unbridged or was bridged by large chips of autologous bone or an autologous inlay graft. All rats showed formation of new bone throughout the length of the radial defect only on the side in which the demineralized bone powder had been implanted. The control side, in which an autologous graft in the form of chips or inlay had been implanted, showed resorption of the graft. The maximum rate of formation of bone occurred fifteen to twenty-one days after implantation of the demineralized bone powder. At thirty-five days, the experimental defect was fully bridged, forming solid bone, in 71 per cent of the rats, and the remaining 29 per cent showed bridging of 95.8 per cent of the length of the defect, with union on one side. Analysis of the sequential radiographs, technetium-99m scans, and histological findings showed that the formation of bone and bridging of the defect were superior on the side in which the demineralized bone powder had been implanted compared with the side in which pieces of autologous bone or an autologous inlay graft had been used.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of hydrogen peroxide on osteoinduction by demineralized bone

The osteoinductive capacity of demineralized bone matrix (DBM) has led to wide use of this material for surgical reconstruction. Preparation of DBM often includes sterilization with ethylene oxide, disinfection with various chemical agents, or irradiation. Exposure to hydrogen peroxide (H2O2) is used for both sterilization and bleaching of bone, the latter primarily for cosmetic reasons. We investigated the effect of H2O2, on the osteoinductive capacity of DBM. Cortical bone implants prepared from rat femurs were placed into 3% H2O2 solution. Control specimens were not exposed to H2O2. Bones were then lipid-extracted, demineralized, sterilized with ethylene oxide, and freeze-dried in an identical manner. Allografts were implanted into rat hosts for 1 to 3 weeks. Osteoinduction proceeded rapidly in implants not exposed to H2O2, with chondrocytes and new bone appearing in the implant. After 3 weeks, perforations in the implant were largely replaced with new bone. In contrast, osteoinduction did not occur in implants treated with H2O2. Perforations in H2O2-treated implants were filled with vascularized fibrous tissue, but no cartilage or bone. These findings reveal that H2O2 used for disinfection or bleaching of DBM can abolish its osteoinductive capacity in rats.     

Treatment of aneurysmal bone cysts by introduction of demineralized bone and autogenous bone marrow

BACKGROUND: On the assumption that an aneurysmal bone cyst has an intrinsic potential to heal by ossification, a new, minimally invasive protocol was developed. Demineralized bone powder mixed with bone-marrow aspirate was introduced into the cyst to halt the expansion phase and to allow the cyst to ossify. We hypothesized that, in order to induce bone-healing, cells from the cyst are needed to respond to the inductive material but that curettage or extensive surgery is not necessary. The goals of the present study were to assess cyst-healing and to determine the prevalence of recurrence associated with this new procedure. METHODS: Thirteen biopsy-proven primary aneurysmal bone cysts were entered through a small incision, and a paste of demineralized bone and autologous bone marrow was introduced with an applicator. The study group included three male and ten female patients with a mean age of 16.6 years. The cyst was located in a long bone in six patients, the pelvis in five patients, and the scapular glenoid and the calcaneus in one patient each. Five patients had not received treatment previously, whereas one had had a preoperative embolization and seven had recurrent lesions that had been treated previously. RESULTS: After a mean duration of follow-up of 3.9 years, healing was achieved in eleven patients. CONCLUSIONS: This minimally invasive method is able to promote the self-healing of a primary aneurysmal bone cyst. As no curettage is required, the proposed treatment avoids extensive surgery and blood loss and is convenient for the treatment of poorly accessible lesions such as those occurring in the pelvis. LEVEL OF EVIDENCE: Therapeutic Level IV.     

Immune response to perforated and partially demineralized bone allografts

Immune responses have been shown to be involved in the pathogenesis of clinical complications of cortical bone allografts. In an attempt to reduce the immunogenicity of these allografts, we evaluated cortical bone allografts modified by laser perforation and partial demineralization transplanted orthotopically into sheep tibiae. The recipient animals were divided into three groups, of eight animals each, according to the type of cortical allograft that was transplanted: group 1, no treatment (control); group 2, demineralization only; and group 3, laser perforation and partial demineralization. All animals were tissue-typed by biochemical definition of MHC class I molecules, using unidimensional isoelectric focusing and Western blotting. Mismatches of donors and recipients were assessed by testing samples of each donor and recipient pair in parallel and by comparing their individual bands. Donor-specific alloantibodies were detected by a similar technique, using an enzyme-linked immunosorbent assay (ELISA) format. Negative controls were included in all tests. All grafts were poorly immunogenic, whether they were untreated, processed by partial demineralization, or processed by both laser perforation and partial demineralization. Only two recipient animals showed a transient, antibody-mediated donor-specific immune response. One of these animals had received a control allograft, whereas the other animal had received a laser-perforated and partially demineralized bone allograft. All of the grafts in this study, including control grafts, were stripped of soft tissues and their bone marrow was removed; cellular sources of alloantibody stimulation may have been eliminated by these processes. The results of this study suggest that immune responses to bone allografts may be reduced by removing the bone marrow and adjacent soft tissues. The processing of cortical bone allografts by laser perforation and partial demineralization appeared to have little effect on immune responses.     

Calcificationin vitro of demineralized bone matrix

Collagen was prepared from compact sheep bone by decalcification with EDTA and from rat tail tendons by acetic acid extraction and reconstitution with NaCl. The deposition of apatite in sheep bone collagen in a metastable calcification solution was studied chemically and by electron microscopy. The bone collagen was shown to be a good nucleation catalyst for mineral deposition, while rat tail collagen was a poor catalyst. Mineral deposition in bone collagen occured in two separate kinetic phases, a rapid phase of nucleation and crystal growth, giving rise to small calcified islands, and a second slow phase, ascribed to growth in regions not involving the catalytic sites. This second phase of mineral deposition is considered to be the result of impaired ion diffusion through the closely-aligned collagen fibrils, thus leaving large areas of the collagen free of mineral even though the buffer remains highly supersaturated. Electron micrographs suggested that the catalytic sites might be in some relationship to the 640 Å periodicity of collagen, but a role for non-collagenous material bound to the collagen has not been excluded.The poor catalytic activity of reconstituted collagen was not due to the presence of loosely-bound inhibitors, although inhibitors could be strongly bound to this type of collagen and be absent from bone collagen. The differences in catalytic activity may have a bearing on physiological calcification. A more general hypothesis for nucleation of a mineral phase in biological systems is required.This work was supported in part by the European Atomic Energy Community (EURA-TOM), Brussels, Belgium.     

Effect of demineralized bone matrix on polymorphonuclear leukocyte degranulation.

The potential use of allogenic demineralized bone matrix to augment or treat bone defects or nonunions in animals and humans is currently being investigated. Demineralized bone matrix induces osteogenesis by a multistep cascade of endochondral ossification that is mediated by bone-induction factors. The migration and activation of polymorphonuclear leukocytes appear to be critical in the initiation of the cascade of osteogenesis induced by demineralized bone matrix. This study examined the effects of demineralized bone matrix on the degranulation of polymorphonuclear leukocytes. Demineralized bone matrix stimulated the release of polymorphonuclear leukocyte-specific, but not azurophilic, granules in a time and dose-dependent manner. The ability of the bone matrix to induce this degranulation was independent of its size and species. The mechanism by which this degranulation occurs is not completely understood; however, it is known that it does not occur by means of a receptor that requires guanidine triphosphate-dependent regulatory proteins as does polymorphonuclear-leukocyte degranulation induced by N-formyl peptide. The factor that stimulates degranulation is not type-I collagen but rather appears to be a cytokine that has a heparin-binding domain and a molar mass of 10-70 kDa. Loss of the ability of demineralized bone matrix to induce degranulation of polymorphonuclear leukocytes correlated positively with the loss of its ability to induce bone formation.     

Bone induction by composite of bioerodible polyorthoester and demineralized bone matrix in rats.

A composite of a local, sustained, drug-release system, Alzamer bioerodible polyorthoester, and demineralized bone-matrix (DBM) particles implanted in the abdominal muscle of 89 Wistar rats induced cartilage and bone formation at the same rate as DBM when evaluated histologically and by 85Sr uptake. The composite implant was technically easier to use than DBM alone.     

生物活性玻璃陶瓷对成骨细胞影响的体外实验研究

目的：探讨生物活性玻璃作为成骨细胞培养支架的可行性，为骨组织工程寻找一种良好的细胞载体。方法：采用骨膜源性成骨细胞与生物活性玻璃陶瓷体外复合培养，通过光镜、电镜、上清液ALP测定及流式细胞仪观察及检测生物活性玻璃陶瓷对成骨细胞生物学性状的影响。结果：成骨细胞在生物活性玻璃陶瓷的表面及空隙内生长状态良好，上清液ALP测定值及细胞凋亡率(Ap)两组对照无显著性差异。结论：生物活性玻璃陶瓷具有良好的生物相容性、生物活性和成骨作用，可以成为骨组织工程中一种新型的细胞支架材料。     

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.     

Accelerated repair of cortical bone defects using a synthetic extracellular matrix to deliver human demineralized bone matrix.

Injectable hydrogel and porous sponge formulations of Carbylan-GSX, a crosslinked synthetic extracellular matrix (ECM), were used to deliver human demineralized bone matrix (DBM) in a rat femoral defect model. A cortical, full-thickness 5-mm defect was created in two femurs of each rat. Six rats were assigned to each of five experimental groups (thus, 12 defects per group). The defects were either untreated or filled with Carbylan-GSX hydrogel or sponges with or without 20% (w/v) DBM. Radiographs were obtained on day 1 and at weeks 2, 4, 6, and 8 postsurgery of each femur. Animals were sacrificed at week 8 postsurgery and each femur was fixed, embedded, sectioned, and processed for Masson's Trichrome staining. The bone defects were measured from radiographs and the fraction of bone healing was calculated. The average fractions of bone healing for each group were statistically different among all groups, and all treatment groups were significantly better than the control group. The Carbylan-GSX sponge with DBM was superior to the sponge without DBM and to the hydrogel with DBM. Histology showed that defects treated with the Carbylan-GSX sponge plus DBM were completely filled with newly generated bone tissue with a thickness comparable to native bone. Carbylan-GSX sponge was an optimal delivery vehicle for human DBM to accelerate bone healing.