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PubMed2006

The effect of residual calcium in decalcified freeze-dried bone allograft in a critical-sized defect in the Rattus norvegicus calvarium.

Demineralized freeze-dried bone allograft (DFDBA), a widely used graft material in periodontal regenerative procedures, is processed with hydrochloric acid in the attempt to expose proteins located within the bone matrixes that are capable of inducing new bone formation. However, the degree of DFDBA demineralization varies between tissue banks, which may have an effect on clinical regeneration. This study uses the critical-sized defect (CSD) model to evaluate the wound-healing response to the residual calcium of donor bone. If the percentage of residual calcium in a graft were demonstrated to significantly enhance wound healing, then periodontal patients may benefit from further standardization of human-allograft processing. Sixty adult, male, Harlan Sprague-Dawley rats (Rattus norvegicus) were randomly and equally divided into 4 test groups (ie, DFDBA at 1%, 2%, and 3% to 6% residual calcium levels and FDBA at 23% residual calcium) and a control group (no allograft). An 8-mm-diameter craniotomy was made in the rat calvarium, and polytetrafluoroethylene membranes with pore sizes of 0.50 microm were placed intracranially and ectocranially. Treatment materials were carefully placed into the CSD with a new sterilized dental amalgam carrier. Tetracycline hydrochloride was injected intraperitoneally for labeling new bone growth, and animals were euthanized 12 weeks postsurgery. As a result, histomorphometric bone fill at 12 weeks showed a statistically significant increase in the 2% DFDBA group as compared to all other groups. The authors conclude that a 2% residual calcium level in human DFDBA appears to significantly (P < or = .05) enhance osseous wound healing in the rat calvarium.

Absorptiometry, Photon↗
PubMed1998

Localized ridge augmentation with chin grafts and resorbable pins: case reports.

Six block grafts harvested from the mandibular symphysis were used to augment partially atrophied ridges. Three maxillary defects and three mandibular defects were treated in five patients. Autologous bone grafts from the chin were stabilized in the recipient sites with resorbable pins and no membranes were used over the grafts. Healing proceeded without complications. At 3 to 4 months the external cortical surface of the grafts progressively resorbed and the profiles of the pins protruded from underneath the buccal tissue that covered the augmented areas. However, the pins never perforated the tissue and they were resorbed macroscopically within 4 to 6 months. At 6 months the areas treated showed successful ridge augmentation and when exposed for stage 2 surgery, remnants of the pin holes on the external surface of the repaired defects were detected. Radiographic evaluation of the block grafts was performed at 3 and 6 months and histologic specimens were obtained at 6 months; the specimens demonstrated incomplete pin resorption and encapsulation. A severe foreign-body reaction was detected in one case. The presence of an acellular bone matrix in certain sections and a normal bone pattern with a cellular component in others was a consistant finding. ITI endosseous Implants were placed with excellent primary stability in all treated cases.

Absorbable Implants↗
PubMed1992

The use of a new form of allograft bone in implantation or osseointegrated dental implants--a preliminary report.

The use of and early results with a new form of demineralized allograft bone tissue with the immediate placement of a dental implant are described. No occlusive membranes were utilized. GRAFTON Allogeneic Bone Matrix (ABM) is processed from human cortical bone into a thick gel consistency and packaged sterile in disposable syringes. The material was utilized in seven patients with seven implants. New bone formation was noted and, with the exception of two implants with a small amount of thread exposure, complete. It appears that treatment with the bone-grafting material produced clinical results similar to those reported with the use of barrier membranes and may provide an alternative to guided tissue regeneration. Further investigation appears warranted.

Bone Matrix↗
PubMed1989

Osteoclast features of cells that resorb demineralized and mineral-containing bone implants in rats.

The comparative temporal tissue response to demineralized bone matrix powder (DBP) and devitalized mineral containing bone powder (MCP) implanted subcutaneously in rats was studied. The tissue response to implanted DBP followed the well described endochondral osteogenic pathway and included the appearance of osteoblasts and osteoclasts. On the other hand, implanted MCP resulted in the appearance of a large population of giant cells that resorbed the implants. At later times (3-4 weeks), most of the cells in the MCP implants appeared as typical foreign body giant cells with extensive membrane foldings, usually away from bone surface. Some cells did, however, have the histological appearance of osteoclasts, although this could not be completely confirmed by transmission electron microscopy. Scanning electron microscopy of anorganic preparations of the MCP following implantation showed resorption pits covering most of the surface, providing additional evidence that the resorption of bone by osteoclasts and giant cells may involve similar mechanisms. The observations suggest that both osteoclasts and giant cells may be involved with the resorption of ectopic MCP.

Animals↗
PubMed1978

Systemic effects of the VX2 carcinoma on the osseous skeleton. A quantitative study of trabecular bone.

The skeleton of rabbits bearing the transplantable VX2 carcinoma has been studied by a combination of radiographic and histomorphometric techniques. It has previously been shown that this tumor produces and secretes large amounts of prostaglandin E2. In vivo experiments, as well as cell and organ culture studies, have led to the conclusion that the hyerpcalcemia observed in tumor-bearing rabbits is due to excessive secretion of prostaglandin E2 by the tumor and its subsequent action on bone throughout the organism. Our new findings reveal (1) no evidence of local invasion of bone by the VX2 tumor or osseous metastases; (2) radiographic evidence of generalized osteopenia; (3) histomorphometric documentation in trabecular bone of reduced volume density of bone matrix, consistent with increased resorption in trabecular bone at sites distant from tumor; and (4) a marked generalized reduction in surface density of osteoblastic layers, volume density of osteoid, osteoid seam thickness, and surface density of osteoid. These findings in trabecular bone of iliac crest and vertebra offer quantitative data in support of the conclusion that VX2 carcinoma affects skeletal metabolism systemically.

Animals↗
PubMed2004

[Effect of sodium fluoride on ectopic induction of bone tissue].

Fluoride is a very potent bone stimulating factor. The aim of the study was to assess the influence of sodium fluoride on bone formation and resorption in the process of experimental ectopic bone formation. Ectopic bone formation was induced 10 days after the start of treatment. Two groups of 15 rats each, study and control, were formed. The study group received 100 ppm sodium fluoride in water. Devitalised bone matrix grafts were implanted intramuscularly in two thorax regions. Eight weeks later the ossicles were removed and prepared without decalcification for histomorphometric analysis. Osteoid perimeter (O.Pm) and osteoblast number (N.Ob) were higher in the study as compared to the control group (42.24 +/- 4.14 vs. 12.18 +/- 2.89; p < 0.01 and 26.07 +/- 3.26 vs. 10.24 +/- 1.39; p < 0.01, respectively). Osteoclast number (N.Oc) was increased in the study group (4.31 +/- 0.40 vs. 2.53 +/- 0.31; p < 0.01). No significant differences in bone area (B.Ar/T.Ar) were found (19.36 +/- 2.26 vs. 18.67 +/- 3.71). It was concluded that sodium fluoride not only increases osteoblast activity but also stimulates bone resorption in bone matrix grafts.

Administration, Oral↗
PubMed1990

[Experimental studies on wound healing of the jaw bone fracture].

The author has studied on wound healing of the jaw bone fractures in 5 Malaysian monkeys aging around 2 years and weighing about 3 Kg. For the experimental studies, fractures of the jaw bone in terms of oblique osteotomies from angle to sigmoid notch of the mandible of the Malaysian monkeys were made by using #700 fissure bur and reduced and fixed them in terms of interosseous wiring. The experimental animals were sacrificed on the 1st, 2nd, 3rd, 4th and 5th week thereafter, and the fracture sites were obtained to make microscopic slides stained with Hematoxylin-Eosin, and Van Gieson method. The results obtained were as follows: 1. The blood of hematoma situated in fractured crevice are getting replaced with granulation tissue 1 week after fracture. 2. The granulation tissue occurred in fractured crevice are getting replaced with connective tissue as bone matrix 2 weeks after fracture. 3. Immature new bone formation with bony trabeculation formed 3 weeks after fracture. 4. The secondary bony callus with abundant osteoblasts and osteocytes in bone matrix formed 4 weeks after fracture. 5. The fractured wound completely healed with a new bone formation 5 weeks after fracture.

Animals↗
PubMed1985

Influence of indomethacin on experimental bone metabolism in rats.

The effect of indomethacin on heterotopic and orthotopic bone formation in rats was analyzed with respect to dose and time of initiation of drug administration. Three weeks after implantation of demineralized bone matrix, the ash weight of implants from animals receiving the highest doses of indomethacin was approximately 25% lower than that of controls. The ash weight of implants was only affected in rats receiving indomethacin from the week before implantation or from the time of implantation. In a separate study, no effect on the rate of resorption measured by elimination of 3H-proline and 45Ca or on the amount of ash could be detected. Orthotopic bone remained unaffected by indomethacin treatment. The study showed that in order to inhibit experimental new bone formation, indomethacin must be present before or at the time of implantation of demineralized bone matrix.

Animals↗
PubMed2004

Bone sialoprotein, matrix metalloproteinase 2, and alpha(v)beta3 integrin in osteotropic cancer cell invasion.

BACKGROUND: Bone sialoprotein (BSP) interacts separately with both matrix metalloproteinase 2 (MMP-2) and integrin alpha(v)beta3 and is overexpressed in many metastatic tumors. Its role in tumor biology, however, remains unclear. We investigated whether BSP enhances cancer cell invasiveness by forming a trimolecular complex with MMP-2 and cell-surface integrin alpha(v)beta3. METHODS: Invasiveness of breast, prostate, lung, and thyroid tumor cell lines was measured with a modified Boyden chamber assay. Binding and co-localization of BSP, MMP-2, and integrin alpha(v)beta3 were investigated with immunoprecipitation and in situ hybridization. All statistical tests were two-sided. RESULTS: Treatment with BSP increased invasiveness of many breast, prostate, lung, and thyroid cancer cells through Matrigel in a dose-dependent manner. BSP at 50 nM increased the invasiveness of SW-579 thyroid cancer cells (95.2 units, 95% confidence interval [CI] = 90.4 to 100 units) by approximately 10-fold compared with that of untreated control SW-579 cells (9.1 units, 95% CI = 5.7 to 12.5 units) (P<.001). Addition of an inactive mutated BSP, in which BSP's integrin-binding RGD tripeptide was altered, or addition of integrin alpha(v)beta3-blocking antibodies resulted in invasiveness equivalent to that of untreated cells. Inhibiting cellular MMP-2 activity with chemical inhibitors or a specific antibody also blocked BSP-enhanced invasiveness. Osteopontin and dentin matrix protein 1, proteins related to BSP that also bind integrin alpha(v)beta3 and form complexes with other MMPs (but not MMP-2), did not enhance invasiveness. Immunoprecipitation showed that a complex containing BSP, integrin alpha(v)beta3, and MMP-2 formed in vitro. Addition of BSP increased the amount of MMP-2 bound by cells in an integrin-dependent fashion. Co-expression of BSP, integrin alpha(v)beta3, and MMP-2 in papillary thyroid carcinoma cells was shown by in situ hybridization. CONCLUSION: Cancer cells appear to become more invasive when BSP forms a cell-surface trimolecular complex by linking MMP-2 to integrin alpha(v)beta3.

Bone Neoplasms↗
PubMed1989

Procollagenase associated with the noncalcified matrix of bone and its regulation by parathyroid hormone.

Collagenolytic enzyme activity associated with the noncalcified pool of collagen was studied using calvarial matrices from which the periosteal cell envelope had been removed. Aminophenylmercuric acetate (APMA) stimulated degradation of about 5% of the noncalcified collagen in matrices prepared from freshly dissected bone. Significantly more activity was detected if intact calvaria were cultured 24 h before removal of the cells, in which case 20-30% of the noncalcified collagen was degraded following treatment with APMA. Trypsin elicited a similar response. The collagen being degraded was representative of the entire pool of noncalcified collagen and was not underhydroxylated. Treatment of intact calvaria with parathyroid hormone (PTH) before removal of the cells increased the level of both active collagenase and procollagenase activity associated with the matrix. Enhanced 3H release was noted for PTH treated intact bone in the prior 24 h. Inactivation of endogenous procollagenase by phenanthroline had no effect on the ability of isolated calvarial cells to resorb the bone upon treatment with resorptive agents. The data show that PTH-stimulated collagenolysis of noncalcified collagen involves increased deposition of procollagenase onto the noncalcified matrix in addition to activation of the enzyme.

Animals↗
PubMed1988

Composition of the cement line and its possible mechanical role as a local interface in human compact bone.

Human compact bone may be viewed as a fiber reinforced composite material in which the secondary osteons act as the fiber reinforcements. The cement line, which is the interface between the 'fibers' (osteons) and extraosteonal bone matrix, may impart important mechanical properties to compact bone. The nature of these properties is not known partly because the composition of the cement line is unknown. This analysis examines the constituents of the osteon cement line using scanning electron microscopy and X-ray microprobe analysis to address its biomechanical functions as a local interface. The analysis suggests that the cement line is a region of reduced mineralization which may contain sulfated mucosubstances. This composition is consistent with the hypothesis that the cement line provides a relatively ductile interface with surrounding bone matrix, and that it provides the point specific stiffness differences, poor 'fiber'-matrix bonding and energy transfer qualities required to promote crack initiation but slow crack growth in compact bone.

Adult↗
PubMed1976

Changes in shape, ossification and quality of bones in children with spina bifida.

Changes in the cross-sectional shape, size, bone mass and amount of unmineralised osteoid tissue were studied in 17 dissected tibiae from spina-bifida babies who died with paralysis and foot deformities and in 14 tibiae from non-spina bifida controls of matching age. In addition, 12 tibiae from young experimental rats with myotomy of foot dorsiflexors and foot plantiflexors were double-labelled with bone-seeking markers and studied in order to find the role of experimental muscle imbalance in the dynamic remodelling of the developing long bones. It was found that in tibiae from spina-bifida children with paralysis the total area of cortical bone, its thickness, number of Haversian systems and number of large remodelling cavities are diminished. Significant changes in the cross-sectional shape of the midshaft of the tibia were found, ranging from the triangular shape seen in normal babies and in those with spina bifida and calcaneus-type foot deformity, to the circular shape of tibiae from babies with spina-bifida paralysis and no foot deformity or with spina bifida and equinovarus-type of deformity. Results of experimental myotomy on growing rats showed the direct influence of working muscles on the remodelling process of growing tibiae. On the side of myotomy the flat cortex resumed a bulging convex shape and the centre of gravity shifted towards the myotomised side. These principles cannot on their own explain the specific changes in the shape of human tibiae found during anatomical studies. There is, however, a common denominator in these apparently contradictory findings. This is the combined action of two factors previously reported: the combination of paralysis of the growing limb and mechanical intra-uterine pressure acting on it. The findings in the present study also indicate that they played a major role in the production of deformities. The total amount of osteoid tissue in spina-bifida paralysed bone is increased. This delay of mineralisation of newly laid-down bone matrix would lead to softening of the new bone matrix and osteoid-rich subepiphyseal and metaphyseal regions. This 'paralytic rickets', together with the diminished total bone mass found, could probably be the cause of the common spina-bifida fractures in these regions.

Animals↗
PubMed2000

Comparison of radiometric and histometric features of bone samples from ribs.

OBJECTIVE: To correlate quantitative data gathered from conventional radiograms as well as from quantitative computed tomography (QCT) images of bone samples with histometric coefficients of the same specimens. STUDY DESIGN: Bone samples were obtained from standardized sites of ribs during autopsy of 40 males aged 15-84 years. In the bone samples the following assessments were performed: densitometry of bone using QCT, estimation of radiostructural quantitative coefficients (coefficient of trabecular arrangement anisotropy, trabecular thickness coefficient); histometry (area of bone matrix, trabecular thickness coefficient); relative weights of dry, decalcified bone and bone in ash form; and concentration of calcium in ash. Radiometry and histometry were performed using image analysis methods with IBAS for Windows (Kontron, Eching b. München, Germany) equipment. Radiostructure of bone was evaluated with the use of our own algorithm; concentration of calcium was measured spectrophotometrically. Analysis of variance, cross correlation, regression analysis and others were the statistical tools applied. RESULTS: From the variables analyzed, the following indicates a significant correlation with the histometric area of bone matrix: radiologic trabecular anisotropy, QCT densitometry (especially of cancellous bone) and the ratio of weight of dry sample to fresh sample. The coefficient of radiologic trabecular thickness appeared not to be a valuable feature for estimation of bone histology. CONCLUSION: Quantitative estimation of radiostructure of bone carried out on conventional radiograms may be helpful in the detection of histologic properties of bone, an important factor in assessment of the fragility of bone.

Adolescent↗
PubMed2003

The effects of serine proteinase inhibitors on bone resorption in vitro.

The aims of this study were to identify the role and sites of action of serine proteinases (SPs) in bone resorption, a process which involves a cascade of events, the central step of which is the removal of bone matrix by osteoclasts (OCs). This resorbing activity, however, is also determined by recruitment of new OCs to future resorption sites and removal of the osteoid layer by osteoblasts (OBs), which enables OCs to gain access to the underlying mineralized bone. The resorption systems we have studied consisted of (i) neonatal calvarial explants, (ii) isolated OCs cultured on ivory slices, (iii) mouse OBs cultured on either radiolabelled type I collagen films or bone-like matrix, (iv) bone marrow cultures to assess OC formation and (v) 17-day-old fetal mouse metatarsal bone rudiments to assess OC migration and fusion. Two separate SP inhibitors, aprotinin and alpha(2)-antiplasmin dose-dependently inhibited (45)Ca release from neonatal calvarial explants: aprotinin (10(-6) M) was the most effective SP inhibitor, producing a maximum inhibitory effect of 55.9%. Neither of the SP inhibitors influenced either OC formation or OC resorptive activity. In contrast, each SP inhibitor dose-dependently inhibited OB-mediated degradation of both type I collagen fibrils and non-mineralized bone matrix. In 17-day-old metatarsal explants aprotinin produced a 55% reduction in the migration of OCs from the periosteum to the mineralized matrix after 3 days in culture but after 6 days in culture aprotinin was without effect on OC migration. Primary mouse osteoblasts expressed mRNA for urokinase type plasminogen activator (uPA), tIssue type plasminogen activator (tPA), the type I receptor for uPA, plasminogen activator inhibitor types I and II and the broad spectrum serine proteinase inhibitor, protease nexin I. In situ hybridization demonstrated expression of tPA and uPA in osteoclasts disaggregated from 6-day-old mouse long bones. We propose that the regulation of these various enzyme systems within bone tIssue determines the sites where bone resorption will be initiated.

Amyloid beta-Protein Precursor↗
PubMed2002

Recovery of proximal tibia bone mineral density and strength, but not cancellous bone architecture, after long-term bisphosphonate or selective estrogen receptor modulator therapy in aged rats.

Various bisphosphonates and the selective estrogen receptor modulator (SERM) raloxifene are approved treatments of postmenopausal osteoporosis. They increase bone mineral density (BMD), decrease bone turnover, and reduce vertebral fracture incidence through different cellular mechanisms. We investigated the bone cellular activities, architecture, mineral content/density, and strength of ovariectomized (ovx) rats on a long-term bisphosphonate or SERM treatment, at doses of either agent correcting bone strength. Eleven weeks postovariectomy, 6-month-old rats were treated with the SERM MDL 103,323 or with the bisphosphonate pamidronate for 5 months. Doses of pamidronate and MDL 103,323 were selected from previous studies showing correction of the ovx-induced decrease of ultimate strength of proximal tibia. Ultimate and yield strengths, BMD, and histomorphometric parameters were all quantified at the same site, i.e., the proximal tibia metaphysis. Long-term pamidronate decreases bone turnover and bone formation activity, leading to trabecular thinning. MDL 103,323 decreases bone turnover to a lesser extent, and slightly protects trabecular architecture by uncoupling bone resorption and formation activities. The yield strength is corrected by pamidronate, but not by MDL 103,323 treatment. However, neither compound restores the ovariectomy-induced cancellous bone loss. Total tissue area and cortical thickness are unchanged with pamidronate or MDL 103,323 treatment, indicating that cortical bone mass, thickness, and cross-sectional area are not modified. The discrepancy between proximal tibia BMD and mechanical resistance to fracture modifications, on the one hand, and cancellous bone volume, on the other hand, could be due to changes in the degree of mineralization of bone matrix and/or of the intrinsic properties of the bone matrix.

Aging↗
PubMed1992

Bone maturation and resorption by peritoneal macrophages in noninsulin-dependent streptozocin-induced diabetes in the rat.

The existence of osteopenia in noninsulin-dependent diabetes mellitus (NIDDM) is controversial. In this regard rats with a diabetic condition that simulates NIDDM have normal bone mineral content. However, normal total mineral content in bones does not preclude abnormalities in more delicate functions in bones of rats with NIDDM (specifically, bone maturation and response of bone matrix to resorptive activity). To evaluate these functions, bone maturation was studied in tibias from 14-week old male and female rats with streptozocin-induced NIDDM and controls. The ability of macrophages to resorb bone was studied in bone particles that were obtained from femors of male rats with NIDDM and controls. The diabetic rats had the overt NIDDM condition for 8 weeks at the time of the study. We found that bone maturation/mineralization remained intact in the diabetic rats and that response of the bone particles that were obtained from their femors to the resorptive activity of macrophages was similar to that of the bone particles that were obtained from controls. These data suggest that in the rat, a diabetic condition that simulates NIDDM does not alter the physiologic functions of the bone matrix.

Analysis of Variance↗
PubMed1983

Autoradiographic and liquid scintillation assessment of beta-amino-propionitrile-induced inhibition of collagen maturation using 3H-proline and 3H-lysine.

BAPN (0.1 mg/day) was injected into chick embryos for 4 days starting on the 7th day of incubation. On the 11th day, the embryos were administered either 3H-proline or 3H-lysine. 36 h later, the incorporation of each isotope by the periosteal osteogenic cells as well as into bone matrix was investigated by autoradiography. The incorporation of the two isotopes into whole bones was assessed by liquid scintillation counting. 3H-proline incorporation into the cellular or matrical compartments was unaffected by treatment. As compared to the controls, 3H-lysine label in BAPN-treated embryonic bones was significantly higher in the cellular compartment but was reduced over the bone matrix. The data provide the first direct morphological evidence that BAPN probably induces certain changes in the maturation of collagen involving lysyl residues which result in an inhibition of cross-linkage formation in collagen.

Aminopropionitrile↗
PubMed1986

In vitro degradation of bone particles by human monocytes is decreased with the depletion of the vitamin K-dependent bone protein from the matrix.

The bone vitamin K-dependent protein osteocalcin has been suggested to play a role in bone resorption. By administering sodium warfarin to rats, it is possible to inhibit the vitamin K-dependent addition of the their gamma-carboxyglutamic acid residues to osteocalcin. This results in reduced amounts of osteocalcin bone, probably because devoid of the calcium-binding Gla residues, the protein no longer accumulates in bone. Preparations of bone obtained from rats treated with sodium warfarin for 6 weeks contained only 0.2% of normal levels of osteocalcin and were 90% reduced in the concentration of Gla. This bone could not be degraded by human monocytes in vitro as well as control bone (only 54% of control; P less than 0.003). Defects in the movement of cells to the bone were documented by phase contrast microscopy. Only 60% as many monocytes attached to the osteocalcin-depleted bone as to control bone in an in vitro attachment assay. These effects do not appear to be related to direct cellular toxicity. The degradation of bone in this in vitro system appears to be dependent on the osteocalcin content in matrix. This may result from defective movement of cells to bone and/or attachment to the bone.

Animals↗