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Inductive specificity of mineralized bone matrix in ectopic osteoclast differentiation.

The present report describes the first in a series of studies designed to identify the factor or factors responsible for eliciting osteoclast differentiation. Particles of mineralized and demineralized bone, hydroxyapatite (HA), and eggshell were grafted onto the chorioallantoic membranes (CAMs) of chick embryos. After 3 of 6 days, portions of CAMs with associated grafts were harvested, processed for light and electron microscopy, and examined for the presence of multinucleated giant cells with the morphological characteristics of osteoclasts. Light microscopic examination revealed that, within only 3 days, many particles of mineralized materials had become surrounded or engulfed by multinucleated giant cells. Ultrastructurally, all such cells possessed a vacuolated and mitochondria-enriched cytoplasm, but they differed in the nature of the contacts formed at the cell-particle interface. With eggshell, the cells developed filopodia but lacked clear zones and ruffled membranes. With HA, clear zones were evident but cytoplasmic extensions and membrane ruffling were absent. Implants of mineralized bone, however, elicited the formation of giant cells with prominent clear zones and ruffling of the plasma membrane like that observed in bonafide osteoclasts. In contrast, grafts of demineralized bone did not evoke giant cell formation but rather recruited two cell types morphologically akin to either fibroblasts or macrophages. We conclude that the factor(s) responsible for osteoclast differentiation resides specifically within bone matrix and is intimately associated with the mineral phase. Further, in response to such a factor(s), osteoclast differentiation can occur ectopically, outside of the developing vertebrate body.

Allantois↗

The effect of food restriction and germ-free environment on age-related changes in bone matrix.

Studies were performed to determine the effects of food restriction or a germ-free environment on age-related changes in bone. Four groups of male Lobund-Wistar rats were examined at 6 months, 18 months, and 30 months of age. Conventional-free-fed rats were housed in routine laboratory cages and fed ad libitum. Conventional-restricted diet rats were fed 12 grams a day, which becomes restrictive at 8 weeks of age. Germ-free rats were maintained using gnotobiotic procedures and were free of pathogens. The germ-free rats were maintained on either the full-fed or restricted diet regimens. Serum bone Gla protein and matrix concentrations of calcium, magnesium, and hydroxyproline (reflecting collagen content) and bone Gla protein were not significantly different between the four treatment groups. All of these parameters except hydroxyproline, however, showed age-related declines in all four treatment groups. We conclude that prolongation of rat life span by dietary restriction and/or a germ-free environment did not alter the measured parameters in each age group, and did not alter or slow the age-related changes in the bone matrix.

Aging↗

Cartilage transformation in a composite graft of demineralized bovine bone matrix and ear perichondrium used in a child for the reconstruction of the nasal septum.

For the first time it was observed in a boy that a xenogenic implant of demineralized bovine bone matrix, enfolded in a pedicled flap of autogenic ear perichondrium, was transformed into autogenic cartilage. This new cartilage could be adequately used as a graft to bridge the cartilaginous defect of a nasal septum perforation. The defect was successfully closed by a technique employing four bipedicled mucosal advancement flaps of the septum and the nasal floor. The result of this pilot study, which is based on experiments in growing rabbits, appears to open the possibility to create de novo young and growing autogenic cartilage of substantial quantity in children, without the loss of the cartilage present.

Child↗

Expression of bone matrix protein mRNAs by primary and cloned cultures of the regenerative phenotype of human periodontal fibroblasts.

The successful regeneration of periodontal tissues is dependent, in part, on the ability of cells to reconstitute the mineralized tissues of cementum and bone. The aim of the present study was to characterize regeneration-associated cells in terms of their ability to express mineralized tissue macromolecules. Following guided tissue regeneration, cell cultures were established from regenerating tissue, periodontal ligament, and gingiva. Additionally, these cells were transfected, and single-cell-derived clones were established. Following treatment with platelet-derived growth factor-BB and insulin-derived growth factor-1, the presence of mRNA for alkaline phosphatase, osteocalcin, bone sialoprotein, osteopontin, and bone morphogenetic proteins-2 and -4 was assessed. The three cell types expressed similar mRNA levels for alkaline phosphatase, bone morphogenetic protein-2, and bone morphogenetic protein-4, whereas the expression of osteopontin, osteocalcin, and bone sialoprotein was greater in the periodontal ligament and regenerating tissue fibroblasts compared with the gingival fibroblasts. The two growth factors did not affect the expression of any of the genes. This study has identified markers that correlate with the known ability of periodontal ligament and regenerating tissue-derived fibroblasts to facilitate regeneration of the mineralized tissues of the periodontium.

Alkaline Phosphatase↗

Expression of bone matrix proteins mRNA during distraction osteogenesis.

Distraction osteogenesis is a recently advanced principle of bone lengthening in which a bone separated by osteotomy is subjected to slow progressive distraction using an external fixation device. Appropriate mechanical tension-stress is believed not to break the callus but rather to stimulate osteogenesis. To study the molecular features of this process, the expression and localization of the mRNAs encoding osteopontin (OPN), osteocalcin (OC), matrix Gla protein (MGP), osteonectin (ON), and collagen type I and I during distraction osteogenesis were examined by in situ hybridization and Northern blot analysis. The process can be divided into three distinct phases: the lag phase for 7 days between osteotomy and the beginning of distraction, the distraction phase for 21 days, and the consolidation phase for several weeks. The histologic and molecular events taking place during the lag phase were similar to those observed in fracture healing. The osteotomy site was surrounded by external callus consisting of hyaline cartilage. As distraction started at the rate of 0.25 mm/12 h, the cartilaginous callus was elongated, deformed, and eventually separated into proximal and distal segments. The chondrocytes were stretched along the tension vector and became fibroblast-like in shape. Although morphologically these cells were distinguishable from osteogenic cells, they expressed OPN, OC, and alkaline phosphatase mRNAs. As distraction advanced, the cartilaginous callus was progressively replaced by bony callus by endochondral ossification and thereafter new bone was formed directly by intramembranous ossification. OPN mRNA was detected in preosteoblasts and osteoblasts at the boundary between fibrous tissue and new bone. ON, MGP, and OC mRNAs appeared early in the differentiation stage. The variety of cell types expressing mRNA encoding bone matrix proteins in distraction osteogenesis was much greater than that detected in the embryonic bone formation and fracture healing process. Moreover, the levels of OPN, ON, MGP, and OC mRNA expression markedly increased during the distraction phase. These results suggested that mechanical tension-stress modulates cell shape and phenotype, and stimulates the expression of the mRNA for bone matrix proteins.

Animals↗

Biochemical and immuno- and lectin-histochemical studies of solubility and retention of bone matrix proteins during EDTA demineralization.

The present study utilized biochemical and immuno- and lectin-histochemical methods to demonstrate solubility and retention of mineral-binding non-collagenous proteins in rat midshaft subperiosteal bone during EDTA demineralization. A monoclonal antibody (9-A-2) specific for chondroitin 4-sulphate and dermatan sulphate and wheat germ agglutinin (WGA) specific for N-acetyl-D-glucosamine, N-acetylneuraminic acid, and N-acetyl-D-galactosamine were used. Bone proteins were extracted from fresh unfixed or aldehyde-fixed specimens with a three step extraction procedure, 4 M guanidine HCl (GdnCl), aqueous EDTA without GdnCl, followed by GdnCl. For comparison with the second extraction step, ethanolic trimethylammonium EDTA (ethanolic EDTA) was substituted for aqueous EDTA. Based on protein staining and Western blot analysis of SDS-polyacrylamide gel electrophoresis of each extract using 9-A-2 and WGA, retention of mineral-binding proteins extractable from fresh specimens with aqueous EDTA was greatly increased in tissue when ethanolic EDTA was used. Their retention was even greater with prior aldehyde fixation. Maximum retention with no detectable solubility of 9-A-2 and WGA reactive proteins was obtained after ethanolic EDTA extraction of aldehyde-fixed specimens, which concomitantly provided the strongest immuno- and lectin staining. These results indicate that this combined method dramatically improves retention of PGs and glycoproteins during demineralization of bone tissues and provides the best method for localizing these glycoconjugates.

Acetylgalactosamine↗

Regional trabecular bone matrix degeneration and osteocyte death in femora of glucocorticoid- treated rabbits.

Glucocorticoids at pharmacological concentrations cause osteoporosis and aseptic necrosis, particularly in the proximal femur. Several mechanisms have been proposed, but the primary events are not clear. We studied changes in the bone structure and cellular activity in femora of glucocorticoid-treated rabbits before the occurrence of fracture or collapse. In rabbits treated 28 days with 4 micromol/kg.day of methylprednisolone acetate, changes in the cortical bone were minor. However, metabolic labeling showed that bone formation was virtually absent in the subarticular trabecular bone, and scanning electron microscopy showed resorption of 50-80% of the trabecular surface. Thus, reduction in bone synthesis and increased resorption were involved in bone loss. Vascular changes, which have been hypothesized to mediate glucocorticoid damage, were not seen, but histological changes suggested that trabecular bone was damaged. Matrix integrity was examined using laser scanning confocal microscopy to detect passive tetracycline adsorption. In treated animals, but not controls, tetracycline was adsorbed, in a novel lamellar pattern, in 50--200 microm regions extending deep into trabeculae. This showed that the matrix, which is normally impervious, was exposed at these sites. TUNEL assays showed that matrix damage correlated with cell death in the subarticular trabecular bone of treated animals. The pattern of cell death involving cohorts of osteoblasts and osteocytes comprised up to half of the bone volume in affected regions and is consistent with an apoptotic mechanism. Small numbers of TUNEL-labeled osteoblasts, but no osteocytes, were detected in control bone. We conclude that exposure of bone matrix permeability and that regional cell death consistent with apoptosis is an early event in glucocorticoid-induced bone damage.

Adsorption↗

Expression of bone sialoprotein, a bone matrix protein, in human breast cancer.

Microcalcifications are often associated with human mammary lesions, particularly with breast carcinomas. To date, the molecular mechanism that leads to the deposition of hydroxyapatite in the mammary tissue has not been elucidated. Bone sialoprotein (BSP) is a glycoprotein the expression of which coincides with the appearance of the first hydroxyapatite crystals during bone development. In this study, we report the observation that BSP, a bone matrix protein, is expressed in human mammary cancer cells. Using an immunoperoxidase technique, we studied the expression of BSP in 79 breast lesions, including 28 benign and 51 malignant specimens. Two polyclonal antibodies, one directed against intact human BSP and the other against a synthetic peptide of BSP (residues 277-294), were used and gave identical results. Normal mammary glands expressed undetectable or barely detectable amounts of BSP, and the majority of the benign lesions examined were generally unstained (0) or weakly stained (1+). Most of the breast carcinoma specimens (around 87%) showed a significant increase (P = 0.0001) in BSP expression. Breast carcinomas with microcalcifications had the highest immunoreactivity (2+ or 3+) to BSP antibodies. This is the first demonstration that BSP expression is significantly increased in breast cancer. Expression of BSP by breast cancer cells could play a major role in the deposition of microcalcifications and in the preferred bone homing of breast cancer cells.

Breast↗

Effect of protein restriction on the messenger RNA contents of bone-matrix proteins, insulin-like growth factors and insulin-like growth factor binding proteins in femur of ovariectomized rats.

It has been reported that loss of ovarian oestrogen after menopause or by ovariectomy causes osteoporosis. In order to elucidate the effect of dietary protein restriction on bone metabolism after ovariectomy, we fed ovariectomized young female rats on a casein-based diet (50 g/kg diet (protein restriction) or 200 g/kg diet (control)) for 3 weeks and measured mRNA contents of bone-matrix proteins such as osteocalcin, osteopontin and alpha 1 type I collagen, insulin-like growth factors (IGF) and IGF-binding proteins (IGFBP) in femur. Ovariectomy decreased the weight of fat-free dry bone and increased urinary excretion of pyridinium cross-links significantly, although dietary protein restriction did not affect them. Neither ovariectomy nor protein restriction affected the content of mRNA of osteopontin and osteocalcin; however, ovariectomy increased and protein restriction extensively decreased the alpha 1 type I collagen mRNA content in bone tissues. Ovariectomy increased IGF-I mRNA only in the rats fed on the control diet. Conversely, protein restriction increased and ovariectomy decreased the IGF-II mRNA content in femur. Furthermore, the contents of IGFBP-2, IGFBP-4 and IGFBP-5 mRNA increased, but the content of IGFBP-3 mRNA decreased in femur of the rats fed on the protein-restricted diet. In particular, ovariectomy decreased the IGFBP-2 mRNA content in the protein-restricted rats and the IGFBP-6 mRNA content in the rats fed on the control diet. These results clearly show that the mRNA for some of the proteins which have been shown to be involved in bone formation are regulated by both quantity of dietary proteins and ovarian hormones.

Animals↗

[Use of demineralized bone matrix in children].

Eighty five clinical observations of the authors have shown autotransplants demineralized in the solution of hydrochloric acid to possess pronounced osteoinductive properties. Their ability to induce osteogenesis was mostly pronounced in simultaneous plasty with a demineralized and frozen bone. Reconstructive processes are perfectly completed within 18-24 months after operation.

Adolescent↗

Mitogenic response of cells in culture to demineralized bone matrix.

In this study the mitogenic effect of demineralized bone powder (DBP) on cells in tissue culture was evaluated. The cells studied have osteoblastic properties. At a DBP concentration of 100 micrograms/well, rat osteosarcoma cells (ROS) 17/2.8 cells, ROS 24/1 cells, and periosteal fibroblasts (POF) all showed a significant increase in proliferation in comparison to controls. The peak effect was on day two for ROS 17/2.8 (125% of control), on day three for ROS 24/1 (139% of control), and on day four for POF (145% of control). The results indicate that this in vitro system may be useful for evaluating the osteoinductive activity of DBP preparations.

Animals↗

Comparison of cell viability on anorganic bone matrix with or without P-15 cell binding peptide.

Cell-extracellular cell matrix (ECM) interactions play a vital role in development including cytoskeletal structure, growth and differentiation. A 15 amino acid, non-RGD containing, peptide that is derived from type I collagen was tested for its ability to confer cell survival. Two routinely utilized bone graft materials, anorganic bone matrix (ABM) and ABM carrying the cell binding peptide P-15 were compared for cell viability and apoptosis. Anchorage-dependent human foreskin fibroblasts (HFF) or osteogenic MC3T3-E1 cells were seeded on ABM or ABM/P-15. After serum withdrawal, viability and level of apoptosis were significantly (p<0.05) improved for cells on ABM/P-15 compared to cells on ABM. Scanning electron microscopy confirmed large, spread cells on ABM/P-15 and smaller cells with apoptotic cellular blebs on ABM. In addition, viable cell attachment was significantly greater on cells cultured on ABM/P-15 compared with demineralized freeze-dried bone allograft. This is the first report that the P-15 cell binding peptide, when complexed with ABM, promotes cell survival. The P-15 peptide may modulate cell number and tissue structure by the enhancement of viable cell attachment and via the regulation of apoptosis.

3T3 Cells↗