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PubMed1993

Defects of the rat premaxilla as a model of alveolar clefts for testing bone-inductive agents.

Critical-sized defects were made in the premaxillary bone of Wistar male rats using a surgical trephine and a low-speed dental engine as a model of the maxillary alveolar cleft for testing bone-inductive agents. The defects were treated with either 7 mg of demineralized bone matrix (DBM) or were left nongrafted. The nongrafted group healed mainly with fibrous connective tissue, with a small amount of bone formation at the periphery. There was no significant change in alkaline phosphatase activity and 45Ca incorporation. The DBM-grafted group produced new bone with osseous bridging in the defect by day 35. Alkaline phosphatase activity increased significantly from day 10, reaching a maximum on day 14, and 45Ca incorporation increased on day 14. These results indicate that this nonhealing bony wound of the premaxilla in rats may be useful as a model for studying the effect of bone-inductive agents on the healing of alveolar clefts.

Alkaline Phosphatase↗
PubMed2003

Failure to induce supracrestal bone growth between and around partially inserted titanium implants using bone morphogenetic protein (BMP): an experimental study in dogs.

The effect of bone morphogenetic protein on supracrestal bone growth around partially inserted implants in a dog model is described. The lower premolar teeth (P1, P2, P3 and P4) were extracted on both sides of the mandible in six dogs. At a surgical exposure 12 weeks later, two 10-mm turned titanium implants were partially inserted, approximately 15 mm apart, in the areas of the P1 and P3 in each side of the mandible, allowing five threads to protrude from the bone crest. A titanium mesh was fastened to the coronal aspect of the two fixtures and the space beneath the mesh was filled with bone morphogenetic protein (S300 BMP) in combination with an insoluble bone matrix carrier, or with the carrier alone. The mesh was covered with an ePTFE membrane. Thus, a space for potential bone formation was created between the two implants. The surgical flaps were coronally positioned and secured with vertical mattress sutures. After 16 weeks of healing, biopsy specimens were retrieved and examined histologically. Bone was not formed around the protruding implants or in the created space between the implants in any case. The carrier was incompletely resorbed. We conclude that supracrestal bone growth beyond the crestal limit with or without BMP in such a large space as in this experimental design may not be possible.

Absorbable Implants↗
PubMed1958

Autoradiographic studies of the utilization of Ca45 by the chick embryo.

Calcium-45 was injected into the dense albumen of fertile hen's eggs, to the extent of 25 microc. per egg. The eggs were incubated under standard conditions and three or more embryos removed daily and fixed in 10 per cent neutral formalin. Stripping-film autoradiograms were prepared from paraffin sections of the tibiofibulae. Exposure varied with the isotope concentration. The tissue sections with their autoradiograms in place were stained with dilute Giemsa, while other sections were stained with hematoxylin-azure-eosin and by von Kossa to demonstrate bone salt. At about 9 days, Ca(45) is found in the cartilage template both intra- and extracellularly. Between 9 and 11 days, a primary diaphyseal lamella is deposited which is largely acellular. The lamella is eroded by capillaries from the periosteum and a resorption center is established in the cartilage. New lamellae of bone are deposited centrifugally in an imbricated pattern. Bone matrix formation precedes calcification by about 1 to (1/2) days, and calcification in a particular lamella is not uniform. Endochondral bone formation is described, as well as calcification of the epiphyseal/diaphyseal cartilage. Calcium-45 occurs intracellularly in the osteocyte during bone formation.

Animals↗
PubMed1992

Fibroblast growth factor: effects on osteogenesis and chondrogenesis in the chick embryo.

In vivo effects of fibroblast growth factor (FGF) on osteogenesis were evaluated in the chick embryo. Autoradiographic studies of 3H-proline labeling over bone matrix indicated that 24 h after treatment on day 11, FGF stimulated osteogenic cell proliferation, while inhibiting the production of bone matrix collagen. However, 4 days after multiple doses of FGF, the large pool of newly formed osteogenic and chondrogenic cells expressed their function with the increased formation of matrix. The data provide in vivo evidence of the effects of exogenous FGF on osteogenesis and also point to a possible role for FGF both in embryonic osteogenesis and in fracture repair.

Animals↗
PubMed1997

Detection of osteopontin in calcified human aortic valves.

Cardiac valve calcification often results in obstruction of blood flow, which eventually leads to valve replacement. The molecular mechanisms resulting in valve calcification are unknown. Collagen and specific bone matrix proteins are thought to provide the framework for ectopic tissue calcification. This investigation was performed to determine whether the bone matrix protein osteopontin was present in calcified human aortic valves. Proteins extracted from human aortic valve tissue were subjected to polyacrylamide gel electrophoresis followed by Western blotting, using polyclonal antibodies directed against osteopontin. Fresh frozen tissue sections were also screened for osteopontin and macrophages using immunohistochemical techniques. Osteopontin was present in both heavily and minimally calcified aortic valves and absent in noncalcified purely regurgitant or normal aortic valves by both radioimmunoassay (n = 16) and immunohistochemical techniques (n = 8). Osteopontin colocalized with valvular calcific deposits, and macrophages were identified in the vicinity of osteopontin. These results, in addition to showing that osteopontin is present in calcified human aortic valves, suggest that osteopontin is a regulatory protein in pathological calcification. Identification of the cells producing osteopontin in abnormal cardiac valves and of proximate stimuli for its secretion may lead to novel therapeutic strategies to prevent and/or reverse calcific valve disease.

Adult↗
PubMed1993

[Effect of parathyroidectomy (PTX) on aluminum (Al) induced bone disease].

Al-treated or untreated decalcified sections of bone matrix were implanted subcutaneously into the abdominal wall of parathyroidectomized (PTX) or control rats for 21 or 28 days. The bone remodeling and Al, Ca content in the implants were measured by using histomorphometry and atomic absorption spectrum method. The results showed that bone area, osteoclast number, osteoblast number and bone mineralization rate of Al-treated or untreated bone in PTX rats and of Al-treated bone in control rats were significantly lower than those of untreated bone in control rats (P < 0.05). There was no marked difference between these parameters of Al-treated bone in control rats and untreated bone in PTX rats (P > 0.05), and these of Al-treated bone in PTX rats were the lowest (P > 0.05- < 0.05). Ca contents were consistent with the histological parameters. Bone Al removing rate between PTX and control rats was not markedly different. In conclusion, both PTX and Al impair bone formation and mineralization, bone biopsy and removal of Al from bone is necessary before PTX treatment in uremic patients with secondary hyperparathyroidism.

Aluminum↗
PubMed1979

Autoradiography of technetium-labelled diphosphonate in rat bone.

The passage of technetium-labelled methylene diphosphonate (99mTc-MDP) across rat bone was examined by autoradiography. The autoradiographs showed that shortly after an injection of the bone-seeking agent there was activity outside the bone, within the bone marrow and also adjacent to the highly vascular epiphysial plate; the distribution of the isotope in the incubated bone appeared to be non-uniform, a high concentration being seen adjacent to the epiphysial plate and also on the surfaces of the bone. The evidence suggested that a two-fold mechanism resulted in the uneven distribution of 99mTc-MDP. The first factor probably represented the regional distribution of blood flow with a transcapillary movement of the tracer from the capillary bed to the extravascular space; the subsequent incorporation of the tracer into bone appeared to depend on the nature of the bone matrix.

Animals↗
PubMed1998

In vitro osteoinduction of demineralized bone.

Among numerous available materials for osseous repair and reconstruction, those presenting osteoinductive characteristics and promoting bone regeneration are preferable. Fresh autologous bone is one of the most effective, but it has some disadvantages and risks. Demineralized bone matrix (DBM) is considered to be a valid alternative, because it seems to show osteogenic potential, ascribed to the presence of bone morphogenetic proteins. In addition it can be prepared without difficulty and preserved without losing osteoinductive properties. The aim of the study was to evaluate the osteoinductive ability of xenogenic DBM, by testing DBM powder obtained from rabbit long bones, in cell culture of murine fibroblasts, alone or associated with electromagnetic field (EMF), that are known to exhibit biologic effects on cells: in particular they are used in orthopedics to improve bone formation. At the end of experiment, alkaline phosphatase (ALP) activity, calcium levels and cell proliferation and morphology were evaluated. A statistically significant stimulation of ALP activity and cell proliferation and a morphological change of fibroblasts were found. The results obtained show how DBM and EMF have different effects on cells, and that together they have synergic action toward bone induction.

Animals↗
PubMedDate not supplied

Bone histomorphometry in 1,25(OH)2D3- and vitamin D3-treated aged laying hens.

Aged laying hens at oviposition were given four levels of dietary vitamin D3 and three levels of 1,25-dihydroxyvitamin D3 (1,25[OH]2D3) to determine the effects of the additives on the medullary and endosteal bone. Oxytetracycline hydrochloride labeling and histomorphometry of undecalcified sections were used in the determination of changes in medullary bone and endosteal surface of mid-diaphyseal tibiotarsal bones. Treatment with 1,25(OH)2D3 increased mineralized medullary bone area, endosteal surface length, amount of tetracycline labeling in medullary and endosteal bone, total number of osteoclasts on endosteal and medullary bone, and appositional rate of mineralizing bone. Vitamin D3 increased mineralized medullary bone area and total tetracycline labeling in endosteal bone but decreased endosteal mineral apposition rate. Medullary bone matrix with no mineral was observed only in the group receiving neither supplement. These areas were much larger than typical osteoid seams usually seen in trabecular bone. Both 1,25(OH)2D3 and vitamin D3 were effective in stimulating bone formation and retention of mineral within medullary bone matrix at oviposition.

Aging↗
PubMed2005

Current insights into the role of transforming growth factor-beta in bone resorption.

Transforming growth factor-beta (TGF-beta) elicits a variety of effects on cellular proliferation and differentiation. The major repository for TGF-beta is bone, where it possesses separate facilitative and suppressive actions on osteoclast differentiation and bone resorption. Without a direct enabling stimulus from TGF-beta monocytes cannot form osteoclasts but instead follow macrophage differentiation pathways. This facilitative action depends on an ability to promote a state in which precursors are resistant to anti-osteoclastic inflammatory signals. Following the initiation of resorption TGF-beta is released from bone matrix. This acts on osteoblasts to reduce the availability of the osteoclast differentiation factor, RANKL (receptor activator of NFkappaB ligand) and thereby indirectly limits further osteoclast formation. Thus TGF-beta has a fundamental role in the control of bone resorption having actions that first allow monocytes to develop into osteoclasts then subsequently limiting the extent and duration of resorption after its release from the bone matrix.

Animals↗
PubMed2002

Purified bovine BMP extract and collagen for spine arthrodesis: preclinical safety and efficacy.

STUDY DESIGN: Rabbit and nonhuman primate posterolateral intertransverse process spinal fusions were performed. OBJECTIVES: To determine the preclinical safety and efficacy of bBMPx product (a composite of collagen and bovine bone morphogenetic protein extract) mixed with demineralized bone matrix for posterolateral intertransverse process spinal fusions. SUMMARY OF BACKGROUND DATA: A dose response of bovine BMP extract with collagen in demineralized bone matrix has demonstrated spinal fusion to a rate of 100% in a rabbit spinal fusion model. This study furthers the research to demonstrate safety in the rabbit and efficacy of bovine BMP extract for spinal fusion applications in nonhuman primates. Additionally, preliminary human clinical data are presented. METHODS: For the safety portion of the study, 45 New Zealand white rabbits underwent posterolateral intertransverse process spinal fusion after laminectomy. Nine additional rabbits served as nontreated control subjects. Graft material (autograft or bBMPx product) was placed in the gutters in 30 animals, and no material was used in 15 animals. The animals were observed for abnormal physical activity, then killed at 8, 29, or 57 days. Histologic evaluation was performed on explanted spines. In the nonhuman primate efficacy studies, 54 rhesus macaques also underwent bilateral posterolateral intertransverse process fusion. bBMPx product with varying bovine BMP extract doses was implanted bilaterally. The animals were killed at 4, 8, 12, 18, or 24 weeks. Plain radiograph, computed tomography scanning, and histology were performed. RESULTS: The rabbit safety study demonstrated that any spinal cord compression or degeneration was caused by the inflammatory response after surgery and was equivalent in all groups. These issues resolved over time. Efficacy data demonstrated an autograft fusion rate of only 21% in the rhesus macaques. The bovine BMP extract demonstrated a dose response in which 3 mg per side gave twice the fusion rate as autograft. CONCLUSIONS: bBMPx product is safe and effective as an autograft substitute for posterolateral intertransverse process spinal fusion. Clinical studies are underway.

Animals↗
PubMed2001

Mechanisms governing bone metastasis in prostate cancer.

Why does prostate cancer metastasize to bone? Why is there increased turnover of the bone matrix in the presence of prostate cancer? A recent autopsy study supports a traditional hypothesis that gross, anatomic patterns of blood flow influence the distribution of metastatic deposits. On the other hand, recent developments in animal models of prostate cancer bone metastasis have renewed interest in the traditional 'seed and soil' hypothesis: several studies point to specific biological interactions between prostate cancer cells and the bone microenvironment that can explain the tendency of prostate cancer cells to colonize bone and grow into clinically relevant metastatic deposits. Some studies implicate mechanisms including chemoattraction and enhanced adherence to bone endothelium. Additional data suggest that prostate cancer cells are 'osteomimetic', that is, they take on the properties and behaviors of osteoblasts or osteoclasts upon arrival in bone. These activities lead to enhanced turnover of the bone matrix and may explain the propensity of prostate cancer to grow in bone. Finally, a series of studies have implicated other molecular markers as distinguishing characteristics of bone-metastatic prostate cancer tissue.

Animals↗
PubMed1984

Induction of heterotopic and orthotopic cartilage and bone formation in mice.

Heterotopic cartilage, bone and bone-marrow formation was achieved in mice by transplantation of a variety of xenogeneic established cell lines, by the transitional epithelium or by implants of demineralized bone matrix. The pattern and the sequence of events were always the same, regardless of the inducer used; viz., hyaline cartilage appeared 6-7 days after implantation, and endochondral bone formation followed. However, in cases of allogeneic implants of transitional epithelium into species other than the mouse, an intramembranous osteogenesis was the main mode of bone formation. When the yield of induced bone was high enough, a true myelopoiesis developed after three weeks. Heterotopically-induced bones had a relatively short life-span. Periosteal membranes of bones at the sites of sarcomes induced by M-MSV responded with rapid and extensive proliferation, with subsequent bone and, sometimes, hyaline cartilage deposition. This phenomenon was observed in long and cranial bones. However, bone induced heterotopically by demineralized bone matrix did not respond in such a way to the presence of M-MSV-induced sarcoma, suggesting that the connective tissue-encapsulated heterotopic bone was not a functioning periosteum. M-MSV-induced sarcoma also stimulates proliferation of elastic cartilage.

Animals↗
PubMed2000

Immunohistochemical expression of bone morphogenetic protein-2 in pilomatricoma.

BACKGROUND: The mechanism of occurrence of calcification and ossification in pilomatricoma remains unclear. OBJECTIVES: To elucidate the pathogenesis of calcification and ossification in pilomatricoma we examined the role of bone morphogenetic protein (BMP)-2, which plays important parts in inducing ectopic bone formation both in vivo and in vitro. METHODS: Twenty cases of pilomatricoma were studied immunohistochemically using anti-BMP-2 monoclonal antibody. RESULTS: In normal skin, including hair follicles, there was no BMP-2 expression. In all pilomatricomas, BMP-2 was found exclusively in the cytoplasm of shadow cells but not in basophilic cells. In two cases of bone formation seen in pilomatricoma, osteoblasts in the periosteal area showed a strong positive reaction, while bone trabeculum (bone matrix) showed no reaction. CONCLUSIONS: Our findings indicate that shadow cells positive for BMP-2 may play an important part in generating bone formation in pilomatricoma.

Adolescent↗
PubMed1996

Establishment and hormonal regulation of a conditionally transformed preosteocytic cell line from adult human bone.

Osteocytes are differentiated forms of osteoblasts that arise upon entrapment within the bone matrix. In this report, we describe the establishment and hormonal regulation of the first conditionally transformed human preosteocytic cell line. Primary adult bone cells were obtained from protease cell line. Primary adult bone cells were obtained from protease digestion of cancellous chips. The cells were infected with adenovirus-ori- SV40 tsA 209, which encodes for a temperature-sensitive large T-antigen. After immortalization, we isolated a clone designated HOB-01-C1. This cell line expressed the mutant T-antigen and proliferated at the permissive temperature (34 C) but stopped dividing at the nonpermissive temperature (39-40 C). Electron microscopy of cells incubated at 39 C demonstrated the presence of preosteocytic cellular processes, some of which appeared to form gap junctions or were rich in microfilaments. The clone expressed alpha 1 type (I) procollagen messenger RNA (mRNA) and secreted type I procollagen C peptide at both temperatures, and this expression was elevated 1.6-fold to 1.8-fold at 40 degrees C. The cells expressed very low basal levels of alkaline phosphatase activity (approximately 0.02 nmol/min.mg), which was increased 2- to 5-fold in a dose-dependent manner by 0.1-100 nM 1 alpha,25-dihydroxyvitamin D3 (vitamin D3) at both temperatures. Vitamin D3 also increased osteocalcin secretion in a dose-dependent manner when the clone was maintained at 34 C (approximately 6-fold), and this stimulation was enhanced > 5 fold at 40 C. In contrast to the low expression of alkaline phosphatase, the cells secreted high amounts of osteocalcin in response to vitamin D3 (approximately 15 ng/mg cell protein); this biochemical profile also resembled that of preosteocytes. Alizarin red-S histochemical staining demonstrated that these cells rapidly produced mineralized nodules at both temperatures. PTH (10 and 100 nM) had no effect on the intracellular accumulation of cAMP at 34 C but stimulated a 14- to 18-fold increase in the production of this second messenger at 40 C. In contrast, 100 nM prostaglandin E2 and 1 microM forskolin stimulated cAMP synthesis better at 34 C. Western blot analysis indicated that the cells expressed CD44, a putative osteocytic marker, at both temperatures. Finally, interleukin-1 beta and tumor necrosis factor-alpha (1-1000 pM) stimulated dose-dependent increases in the secretion of interleukin-6 and monocyte chemoattractant protein-1 at 34 C and 40C. We conclude that the HOB-01-C1 cell line has a preosteocytic phenotype. Moreover, these cells respond to calcitropic hormones and bone resorbing cytokines.

1-Methyl-3-isobutylxanthine↗
PubMed2003

Studies on the effects of biomedicinal agents on serum concentration of Ca2+, P and ALP activity in osteoporosis-induced rats.

The present study was carried out to investigate the effects of biomedicinal agents on Ca2+, P and alkaline phosphatase (ALP) levels in ovariectomized rats. Rats were ovariectomized bilaterally and were fed up with Ca2+ and P-free diet during 8(9,10) weeks to induce osteoporosis. Osteoporosis was determined by the extent of bone density and by lowering the concentrations of serum Ca2+, P and ALP activity every week. Rats in antler, safflower, ipriflavon, or co-administrated with estrogen groups were administrated with feed supplement for 5 weeks to elucidate the protective and therapeutic effects against osteoporosis. The bone tissue was examined with electron microscope to determine the effects of each treatment on osteoporosis. 1. The levels of serum Ca2+ and P in osteoporosis-induced rats, administrated with antler, ipriflavon and estrogen groups, were little higher than those of control rats. However, the levels of serum Ca and P in ovariectomized rats were significantly higher than those of control group (p<0.05). 2. The activities of serum ALP in osteoporosis-induced rats, administrated with antler extract, safflower, ipriflavon, or co-administrated with estrogen, were little increased in comparing with those of control group, but were significantly decreased in with combination of estrogen for 5 weeks. However, The connections were interrupted and the bone matrix was destroyed in the osteoporosis-induced rats. 3. The inter-trabecular connections were examined under electron microscope. The connections were well maintained and bone loss was without in the administration with antler, safflower, and ipriflavon with combination of estrogen for 5 weeks. However, The connections were interrupted and the bone matrix was destroyed in the osteoporosis-induced rats.

Alkaline Phosphatase↗
PubMed2001

Characterisation of the temporal sequence of osteoblast gene expression during estrogen-induced osteogenesis in female mice.

Osteoblast differentiation under in vitro conditions is associated with increased expression of non-collagenous bone proteins including osteocalcin, osteopontin, and osteonectin, the exact function of which remain poorly understood. To determine whether these proteins play an important role in the formation of mineralised bone matrix by osteoblasts in vivo, we analysed the time-course of their expression during estrogen-induced osteogenesis in female mice, and compared this with the formation of new cancellous bone. Female mice were sacrificed prior to or following treatment with 17beta-estradiol for up to 32 days (500 microg/animal/week). Total RNA was extracted from femurs, and changes in expression of genes for a range of osteoblast-derived proteins assessed by Northern blot analysis. In parallel experiments, the time course of cancellous bone formation was determined by measuring bone mineral density (BMD) of the distal femur. Estrogen led to a rapid increase in BMD, which reached significance by Day 16. This was preceded by three-fold increases in expression of alkaline phosphatase (ALP) and type I collagen (COL I) at Days 8 and 12 respectively. In contrast, osteocalcin, osteopontin, and osteonectin expression showed no change during this initial period, although modest increases were observed at later times (i.e., Days 20 and 24). Our results suggest that osteocalcin, osteopontin, and osteonectin are not involved in the initial phase of the osteogenic response to estrogen, suggesting that these non-collagenous bone proteins do not play a direct role in the formation of mineralised bone matrix by osteoblasts in vivo.

Alkaline Phosphatase↗