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PubMed1996

Periodontal repair in dogs: evaluation of rhBMP-2 carriers.

This study evaluated candidate carriers for recombinant human bone morphogenetic protein-2 (rhBMP-2)-driven periodontal regeneration. Previous experiments indicated the ability of rhBMP-2 in a particulate delivery system to result in substantial regeneration of bone and periodontal regeneration. In the present study, canine demineralized bone matrix (DBM), bovine deorganified crystalline bone matrix (Bio-Oss), an absorbable collagen sponge (ACS) of type I bovine collagen, poly(D,L-lactide-co-glycolide) microparticles (PLGA), and polylactic acid granules (Drilac) were tested for their ability to support rhBMP-2 (0.2 mg/mL implant volume)-driven periodontal regeneration. The implants were tested in routine critical size canine supra-alveolar periodontal defects with transgingival tooth positioning. Contralateral defects in six beagle dogs were semirandomly assigned to receive: DBM/rhBMP-2, DBM (no rhBMP-2), Bio-Oss/rhBMP-2, ACS/rhBMP-2, PLGA/rhBMP-2, or Drilac/rhBMP-2. Animals were sacrificed 8 weeks postsurgery, and block sections of the defects were processed for light microscopy. Substantial bone regeneration was observed in all defects implanted with rhBMP-2. Other measures of periodontal healing, including cementum regeneration and presence of ankylosis, were more variable between the implants. DBM and Bio-Oss performed well as carriers for rhBMP-2-driven periodontal regeneration, although other impediments to their clinical use exist. This study indicates that qualities of the carrier system, including its space-maintaining capacity can affect the ability of rhBMP-2 to regenerate both alveolar bone and periodontal attachment.

Alveolar Bone Loss↗
PubMed1993

[Osteoinduction with the dog tibial defect model].

The objectives of this study were to investigate the osteoinductive effect of demineralized bone matrix powder and of cancellous bone in a comparative experiment in a standardized dog defect model. 16 adult shepard dogs were divided into two test subgroups. One group underwent application of demineralized bone matrix (DBM) into the defect. The second group had their defects treated with autogenous cancellous bone (ACG) only. The progress of bone formation was observed for twelve weeks. The excised tibiae were examined by correlating serial X-rays with histologic sections stained with Giemsa and fluorescent labels. In this experiment no osteoinductive effect of demineralized bone powder could be demonstrated. The amount and localisation of bone regeneration in the matrix group seemed to be spontaneous, corresponding to the radiological and histological parameters. In the group of cancellous bone grafts, the defect (length 30 mm) was bridged by incorporation of the graft and graft mediated bone formation within twelve weeks. Disturbing factors such as instability, infection, insufficient vascularity, and cellular immune response to the graft were excluded in this model. The failure of the osteoinductive implants in our experiment compared with the good results obtained in rodents limits their value also in clinical situations. As long as there is no explanation for this failure, this technique should not be used in a clinical situation.

Animals↗
PubMed2006

Plate fixation of ununited humeral shaft fractures: effect of type of bone graft on healing.

BACKGROUND: Delayed union or nonunion of a fracture of the humerus is an infrequent but debilitating complication. Open reduction and internal fixation combined with autologous bone-grafting can result in reliable healing of the fracture; however, there is morbidity associated with the bone-graft donor site. This study was designed to evaluate healing of ununited fractures of the humeral shaft treated by one surgeon at one institution with a strict and consistent surgical protocol but with the use of two different types of bone graft: autologous iliac crest bone graft and demineralized bone matrix. METHODS: A consecutive retrospective cohort series was analyzed. From 1992 to 1999, forty-five patients with an aseptic, atrophic delayed union or nonunion of a humeral shaft fracture were treated with open reduction and internal fixation with a plate and autologous iliac crest bone graft. The mean time from the fracture to the surgery was 14.0 months, and the mean duration of follow-up was 32.8 months. From 2000 to 2003, thirty-three patients with the same condition were treated with the same protocol with the exception that demineralized bone matrix was used instead of autologous iliac crest bone graft. The mean time from the fracture to the surgery in that group was 22.6 months, and the mean duration of follow-up was 20.4 months. All patients in both groups were assessed clinically and radiographically. RESULTS: Osseous union was noted clinically and radiographically following the index surgery in 100% of the forty-five patients treated with autologous bone graft and 97% (thirty-two) of the thirty-three patients treated with demineralized bone matrix. The mean time to union was 4.5 months in the group treated with autologous bone graft and 4.2 months in the group treated with demineralized bone matrix. The overall functional outcome did not differ between the groups; however, twenty (44%) of the autologous bone-graft recipients had donor site morbidity, including a prolonged pain in the majority and a superficial infection requiring irrigation and débridement in one patient. CONCLUSIONS: Healing of an ununited humeral shaft fracture can be achieved consistently with rigid plate fixation and lag-screw compression augmented with either autologous cancellous bone graft or commercially available demineralized bone matrix. The harvest of the autologous bone graft is frequently associated with complications. LEVEL OF EVIDENCE: Therapeutic Level III. See Instructions to Authors for a complete description of levels of evidence.

Adolescent↗
PubMed2001

An unexpected outcome during testing of commercially available demineralized bone graft materials: how safe are the nonallograft components?

STUDY DESIGN: Radiographic and histologic analyses of commercially available bone graft materials were performed. OBJECTIVE: To compare the osteoinductive efficacy of commercially available demineralized bone matrix material. SUMMARY OF BACKGROUND DATA: The relative in vivo bone formation and toxicology of the nonallograft components the make up various commercially available demineralized bone matrix products are not known. METHODS: An in vivo bone formation model was used in 30 athymic rats. Six different bone grafting materials were tested in subcutaneous and intermuscular locations. After 4 weeks, radiographic and histologic testing of bone formation was performed. RESULTS: Eight of nine rats implanted with Grafton demineralized bone matrix products died 1 to 4 days after implantation of the bone graft material. None of the remaining 10 animals implanted with the four other grafting materials died. The experiment was modified and completed with a lower dose of bone graft material. Pathologic analysis indicated that the cause of death was hemorrhagic necrosis of the kidneys, most likely caused by a toxic effect on the glomeruli and tubules. A possible causative factor may have been the glycerol in the graft material. CONCLUSIONS: Although the volume of Grafton product per kilogram of body weight used in this study was approximately eight times the maximum volume used in humans, the authors believe that this data must be reported because this product is used substantially in clinical settings. In addition, the osteoinductive performance and relative safety of the nonallograft components in all commercially available demineralized bone grafts are not known.

Animals↗
PubMed1997

Heritable diseases of the skeleton. Part II: Molecular insights into skeletal development-matrix components and their homeostasis.

A range of osteochondrodysplasias is caused by mutations in components of the extracellular matrix in cartilage and bone and in molecules that are important for posttranslational processing of such components. Mutations in the genes encoding the two polypeptide subunits of collagen I cause defects in the structure of bone matrix while mutations in genes encoding cartilage-specific collagens are responsible for several chondrodysplasias. Abnormalities in cartilage structure and function can also be due to mutations in structural noncollagenous components such as aggrecan and cartilage oligomeric matrix protein. Finally, several cartilage and bone disorders are due to abnormalities in sulfate transport and regulation of bone matrix homeostasis.

Animals↗
PubMed2004

The use of Novabone and Norian in cranioplasty: a comparative study.

Bone replacement products have enhanced the ease of reconstructing bone while improving morbidity related to bone harvest. Although these products are successfully used, studies of bone healing and biomechanical strength are lacking. We aimed to compare how Norian CRS (cranial replacement substance) and Novabone C/M heal in a cranial defect. Adult New Zealand rabbits underwent removal of a critical size cranial defect. The defect was filled with Novabone (n = 8), Novabone plus demineralized bone matrix (n = 8), or Norian (n = 8), or it was left empty (n = 8). Rabbits were euthanized at 8 weeks. Cranial specimens were harvested and soft radiographs, contact microradiographs, and biomechanical testing were done. Soft radiographs revealed opacification like adjacent bone with Novabone, which was augmented when Novabone was combined with demineralized bone matrix. Norian maintained an opaque appearance. The control group did not heal. Contact microradiographs demonstrated bone within the healing defect with Novabone, which was augmented by demineralized bone matrix. Norian was not replaced with bone but served as a scaffold for bone formation. Biomechanical indentation testing demonstrated that the stiffness of Norian was the highest. Novabone plus demineralized bone matrix had a higher stiffness than Novabone alone. All experimental groups had a statistically significant difference compared with Norian. None of the groups achieved the strength of unoperated native bone. Studying two popular products, we found evidence that Novabone was incorporated into cranial bone, regenerating the bone. Novabone healed at a faster rate, creating a stronger product, with demineralized bone matrix. The biomechanical strength of the healed defect was higher in the Norian group, because the bone cement remained solid and was not incorporated, unlike crania reconstructed with Novabone.

Animals↗
PubMed1998

Changes in the extracellular matrix on the surface of sintered bovine bone implanted in the femur of a rabbit: an immunohistochemical study.

The interface of implanted True Bone Ceramics (TBC; sintered bovine bone; Koken, Tokyo, Japan) was examined. In the primary experiment, TBC was implanted into the bone marrow of a rabbit's femur. The extracellular matrices (types I, II, and III collagens and fibronectin) of decalcified specimens collected 1-48 weeks postoperatively were immunohistochemically examined. Undecalcified sections collected 6 weeks postoperatively were used for line analyses of calcium and phosphorus, by a scanning electron microscope-electron probe microanalysis (SEM-EPMA) method. In a secondary experiment, TBC was implanted into an osteochondral defect of a femoral condyle, harvested 1-12 weeks postoperatively, and decalcified to examine the extracellular matrices at the interface. In the bone marrow in the early phase, TBC had absorbed quantities of fibronectin. Immature bone (containing both types I and III collagens) in direct apposition to the ceramic surface had matured (containing type I collagen alone) in the TBC pores. SEM-EPMA revealed the continuity of high levels of calcium and phosphorus at the TBC-bone interface. In the secondary experiment, enchondral ossification or fibrous tissue formation was observed near the articular surface. However, in the subchondral layer, direct bone formation was observed in the TBC pores. It was concluded that TBC has excellent bioactivity for inducing maturation of new bone matrix on porous surfaces.

Animals↗
PubMed1980

Changes in synthesis of types-I and -III collagen during matrix-induced endochondral bone differentiation in rat.

The changes in rates of hydroxyproline formation and biosynthesis of types-I and -III collagen during bone matrix-induced sequential differentiation of cartilage, bone and bone marrow in rat were investigated. Biosynthesis of types-I and -III collagen at different stages of this sequence was studied by labelling in vivo and in vitro with [2,3-3H]proline. Pepsin-solubilized collagens were separated by sodium dodecyl sulphate/polyacrylamide-slab-gel electrophoresis. The results revealed that maximal amounts of type-III collagen were synthesized on day 3 during mesenchymal-cell proliferation. Thereafter, there was a gradual decline in type-III collagen synthesis. On days 9--20 during bone formation predominantly type-I collagen was synthesized. Similar results were obtained by the use of labelling techniques both in vivo and in vitro.

Animals↗
PubMed1976

Phosphotungstic acid-iron-haematoxylin staining method for osteoid, boundary bone and bone components in paraffin sections.

A new staining technique which stains osteoid and bone tissue differentially and also demonstrates boundary bone, pathological osteoid and the changes in ageing, pathological and dead bone matrix in decalcified paraffin or low-viscosity-nitrocellulose bone sections was developed. This phosphotungstic acid-iron-haematoxylin (PTAIH) method is based on pretreating the sections with phosphotungstic acid followed by an iron alum mordant and staining in haematoxylin with subsequent timed differentiation, at certain stages of which the features listed above appear. Van Gieson's picrofuchsin is then used as a counterstain. After standard differentiation osteoid appears red in sharp contrast with the black bone, young and woven bone, old and lamellar bone, and allows one to demonstrate changes in stainability of diseased osteoid and bone matrix, and dead bone. With the differentiation done individually and interrupted at certain stages it is possible to distinguish between various bone components depending on the amount and quality of their in vivo mineralisation. Comparison with controls showed that in this respect the method is more sensitive than the curremt staining techniques of undecalcified bone sections since it demonstrates not only unmineralised and fully mineralised tissues but also shows the poorly calcified, demineralised and ill-calcified bone components. The advantages of the method compared with those using undecalcified sections are its simplicity, suitability for fixed and decalcified material in any unspecialised histological laboratory and the fact that osteoid and other bone components can be studied in sections of unlimited size and in undisturbed relationship to their surrounding soft tissues.

Aging↗
PubMed2005

[Levels of bone mineral matrix organization and the mechanisms determining parameters of its formation].

Authors suggest to regard bone mineral matrix as the four-level structure. The first level is represented by an internal structure of a mineral, the second--by mineral morphological structure, the third--by coplanar association of minerals, and the fourth--by macroassociation of minerals in a single complex inside each bone. The most probable mechanisms determining stability of reproduction of mineral matrix parameters on each of these levels are shown. As a result of their functioning, the variants of bone mineral matrix structures are formed that are the programmed reflection of specificity of the given site of organic structures.

Animals↗
PubMed1994

Biochemistry of bone induction and dystrophic calcification.

The propensity of crosslinked collagen matrices to calcify when implanted in animals and human beings is well documented. This article demonstrates that the covalent binding of the biphosphonate, 3-amino-1-hydroxy-propane-1-1-diphosphonic acid, to active bone matrix inhibits bone formation as well as dystrophic calcification. The inhibitory effects on calcium accumulation and alkaline phosphatase activity are greater than those achieved by inactivation of the matrix by protease digestion of the bone morphogenetic factors present in demineralized bone. Whereas bone formation is a cell-mediated effect, artificially crosslinked collagen matrices calcify extensively inside diffusion chambers, reflecting a non-cell-mediated nucleation process. The fundamental differences described should shed light on new modalities to modulate these processes in living systems.

Acid Phosphatase↗
PubMed1986

Partial isolation and characterization of a chemotactic factor from adult bovine bone for mesenchymal cells.

Demineralized adult bone matrix has the capacity to initiate de novo ectopic endochondral bone formation 2-3 weeks following intramuscular implantation into suitable hosts. An early step in this process is the migration of mesenchymal cells to the implant site; these cells later differentiate into cartilage and bone. Adult bone has been shown to contain a number of bioactive factors, such as chemotactic factors for various cell types, including osteoblasts. We have used embryonic chick limb bud mesenchymal cells to construct an in vitro assay for testing chemotactic activity derived from bone matrix extracts. With a modified Boyden chamber, water-soluble components from a 4 M guanidinium chloride extract of demineralized adult bovine bone matrix were found to stimulate the directional migration of these chick embryonic limb bud mesenchymal cells as well as embryonic muscle-derived fibroblasts and cells derived from embryonic skin. The chemotactic activity was destroyed by treatment with heat (100 degrees C) or trypsin. Partial purification by molecular sieve chromatography suggested that the chemotactic factor(s) has a molecular weight of between 50,000 and 90,000. This factor can be separated from bone matrix-derived chondrogenic stimulating activity by either ion exchange or molecular sieve chromatography. These observations confirm that bone matrix contains a chemoattractant for mesenchymal cells that may be important for in vivo recruitment of cells as part of the process of ectopic bone formation or in cases of bone repair.

Animals↗
PubMed2000

Possibilities of extraction and characterization of ancient plasma proteins in archaeological bones.

Due to the mineral matrix bone proteins are capable of surviving during centuries after inhumation, but cross-linking with other bone proteins as well as fragmentation and complex reactions with humic acids and microorganisms lead to considerable alterations in molecular weight and structure of these proteins. Our group concentrates on polymorphic plasma proteins which diffuse out of the capillary system into the bone matrix where they adsorb to the mineralic substrate. So far, only little is known about the degradation and alteration of these proteins in fossil bones. It has to be evaluated whether the aged proteins still contain some of the information which renders them a valuable tool for forensic questions and population genetics in recent populations. Therefore we tried by modification of already existing methods to expand plasma protein identification and subtyping into the new field of aged plasma proteins.

Adult↗
PubMed2002

Dose-dependent effects of combined IGF-I and TGF-beta1 application in a sheep cervical spine fusion model.

Combined IGF-I and TGF-beta1 application by a poly-(D,L-lactide) (PDLLA) coated interbody cage has proven to promote spine fusion. The purpose of this study was to determine whether there is a dose-dependent effect of combined IGF-I and TGF-beta1 application on intervertebral bone matrix formation in a sheep cervical spine fusion model. Thirty-two sheep underwent C3/4 discectomy and fusion. Stabilisation was performed using a titanium cage coated with a PDLLA carrier including no growth factors in group 1 ( n=8), 75 micro g IGF-I plus 15 micro g TGF-beta1 in group 2 ( n=8), 150 micro g IGF-I plus 30 micro g TGF-beta1 in group 3 ( n=8) and 300 micro g IGF-I plus 60 micro g TGF-beta1 in group 4 ( n=8). Blood samples, body weight and temperature were analysed. Radiographic scans were performed pre- and postoperatively and after 1, 2, 4, 8, and 12 weeks. At the same time points, disc space height and intervertebral angle were measured. After 12 weeks, the animals were killed and fusion sites were evaluated using quantitative computed tomographic (CT) scans to assess bone mineral density, bone mineral content and bony callus volume. Biomechanical testing was performed and range of motion, and neutral and elastic zones were determined. Histomorphological and histomorphometrical analysis were carried out and polychrome sequential labelling was used to determine the time frame of new bone formation. In comparison to the group without growth factors (group 1), the medium- and high-dose growth factor groups (groups 3 and 4) demonstrated a significantly higher bony callus volume on CT scans, a higher biomechanical stability, an advanced interbody bone matrix formation in histomorphometrical analysis, and an earlier bone matrix formation on fluorochrome sequence labelling. Additionally, the medium- and high-dose growth factor groups (groups 3 and 4) demonstrated a significantly higher bony callus volume, a higher biomechanical stability in rotation, and an advanced interbody bone matrix formation in comparison to the low-dose growth factor group (group 2). No significant difference could be determined between the medium- and the high-dose growth factor groups (groups 3 and 4, respectively). The local application of IGF-I and TGF-beta1 by a PDLLA-coated cage significantly improved results of interbody bone matrix formation in a dose-dependent manner. The best dose-response relationship was achieved with the medium growth factor dose (150 micro g IGF-I and 30 micro g TGF-beta1). With an increasing dose of these growth factors, no further stimulation of bone matrix formation was observed. Although these results are encouraging, safety issues of combined IGF-I and TGF-beta1 application for spinal fusion still have to be addressed.

Animals↗
PubMed1993

The effects of three different demineralization agents on osteopontin localization in adult rat bone using immunohistochemistry.

Immunohistochemical localization of osteopontin, a phosphorylated acidic glycoprotein, was compared in adult rat femur fixed in 4% paraformaldehyde at 4 degrees C for 48 h and demineralized at 4 degrees C in ethylenediaminetetraacetic acid (EDTA), modified Jenkin's solution, or 15% formic acid, until radiographs indicated demineralization was complete. Formic acid was also evaluated at room temperature. EDTA solution (15 days) resulted in intense staining of osteocytes, periosteal osteoclasts and osteoblastic cells in osteonal bone. Osteoblasts were negative in the periosteum. No megakaryocyte staining was present; however, occasional neutrophils in the bone marrow were non-specifically stained. Demineralization in modified Jenkin's solution (16 days) showed osteopontin localization in bone matrix, hypertrophic and articular chondrocytes, and osteocytes. In cortical bone, almost all cement lines demarcating osteons showed very dense labeling. In the bone marrow, occasional megakaryocytes were immunopositive and neutrophils were non-specifically stained. Jenkin's produced non-specific staining of skeletal muscle and connective tissue. Formic acid demineralization (14 days, 4 degrees C) resulted in osteopontin expression in osteoblasts, osteocytes, osteoclast precursors, bone matrix, osteoid, cement lines, and chondrocytes; osteoclasts, although present in very low numbers, were also positive. More labeled osteoblasts could be identified compared to Jenkin's demineralization. Also more intense non-specific staining of the bone marrow neutrophils was obtained than with Jenkin's. Harsh, rapid demineralization with formic acid (4 days, room temperature) produced a loss in antigenicity demonstrated by a reduction in staining intensity not experienced with the 4 degrees C protocol; however, osteopontin was still localized in bone matrix and hypertrophic zone chondrocytes.(ABSTRACT TRUNCATED AT 250 WORDS)

Acetates↗
PubMed1992

Osteogenin, a bone morphogenetic protein, adsorbed on porous hydroxyapatite substrata, induces rapid bone differentiation in calvarial defects of adult primates.

Osteogenin, a bone morphogenetic protein, in conjunction with insoluble collagenous bone matrix initiates local endochondral bone differentiation by induction in vivo. This study, by exploiting the affinity of native osteogenin for hydroxyapatite, was designed to construct a delivery system for the expression of the biologic activity of osteogenin in nonhealing calvarial defects of adult primates. After exposure of the calvaria, 64 cranial defects, 25 mm in diameter, were prepared in 16 adult male baboons (Papio ursinus). Defects were implanted with disks of porous nonresorbable and resorbable hydroxyapatite substrata obtained after hydrothermal conversion of calcium carbonate exoskeletons of corals. In each animal, one disk of each hydroxyapatite preparation was treated with osteogenin isolated and purified from baboon bone matrix after sequential chromatography on heparin-Sepharose, hydroxyapatite, and Sephacryl S-200 gel filtration columns. The remaining two defects were implanted with one disk of each hydroxyapatite preparation without osteogenin as control. Histomorphometry on decalcified sections prepared on days 30 and 90 showed superior osteogenesis in osteogenin-treated nonresorbable hydroxyapatite specimens as compared with controls. On day 90, substantial bone formation also had occurred in control nonresorbable hydroxyapatite specimens. On day 90, but not on day 30, significantly greater amounts of bone had formed in osteogenin-treated resorbable specimens as compared with resorbable controls. Overall, resorbable substrata performed poorly when compared with nonresorbable substrata, perhaps due to a premature dissolution of the implants. These results provide evidence that the biologic activity of osteogenin can be restored and delivered by a substratum other than the organic collagenous matrix, inducing rapid bone differentiation in calvarial defects of adult nonhuman primates. The adsorption strategy of osteogenin on porous inorganic nonimmunogenic substrata may help to design appropriate osteogenic delivery systems for craniofacial and orthopedic applications in humans.

Adsorption↗
PubMedDate not supplied

Ultrastructural characterization of otospongiotic lesions in re-embedded celloidin sections.

A technique of using re-embedded celloidin sections for ultrastructural analysis was used for the study of otospongiosis in human temporal bones. Celloidin sections stored in 80% alcohol with active lesions of cochlear otospongiosis were processed and re-embedded in epoxy resin. Semithin and thin sections were cut and analysed for a characterization of the ultrastructural cellular histopathology. The predominant cell types were found to be osteoblasts/osteocytes and macrophages. Lymphocytes were also noted but were rare. Several osteoblasts showed signs of active collagen and bone matrix production, indicative of ongoing new bone formation and repair. Macrophages often interacted physically to form cell clusters. The macrophages were frequently observed to endocytose the non-mineralized bone matrix as well as to degrade mononuclear cells presumed to represent osteoblasts. The observations may support the notion that increased osteolysis in active ostospongiosis is partly caused by a recruited osteoclast activity and partly by an impaired bone repair mechanism due to a macrophage digestion of osteoblast-deposited non-mineralized bone matrix. These two conditions may act in concert with cellular degradation of bone-producing cells.

Bone Resorption↗
PubMed1990

Dose-dependent reduction of bone inductive properties by ethylene oxide.

Sterilisation of demineralised bone matrix with ethylene oxide has been claimed to destroy the ability of bone matrix to induce new bone formation on intramuscular implantation. Other workers have routinely used ethylene oxide sterilised bone matrix for assays in rodents without detrimental effects. We studied the effects of various lengths of exposure to ethylene oxide gas, and found that bone induction properties are destroyed in a dose-dependent manner. After a short exposure, bone induction properties were moderately diminished. However, this short ethylene oxide treatment did not kill Bacillus subtilis spores. A sterilisation procedure that killed these spores rendered the implants incapable of bone-induction.

Animals↗