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PubMed1990

The role of cells versus matrix in bone induction.

Earlier observations indicated that epithelial cells of urinary bladder, and transformed epithelial cells from human amnion (FL), epidermal carcinoma (HeLa), etc. can induce ectopic endochondral bone formation when implanted into the skeletal muscle of immunosuppressed or autologous animals. Such epithelial cells are associated with little matrix. Bone-inducing activity was also demonstrated in cultured osteosarcoma cells of murine and human origin or extracts thereof, and it is notable that these bone-inducing osteosarcoma cells grow in vitro with little matrix production. Finally, electron microscopy of in vitro cartilage induction showed that decalcified rodent bone that had been extensively extracted to remove cells still contained devitalized cells and cell fragments some of which made contact with inducible, cartilage-forming mesenchymal cells that had migrated in from cocultured muscle. Suggestions: These observations suggest that: 1) the bone-inducing agent(s) of both epithelial and mesenchymal cells may reside mostly in cells rather than matrix. Thus it may be premature to assume that bone "matrix" is the major source of bone-inducing agent in decalcified bone until the osteoinductive activity of residual bone cells has been assessed; and 2) that the osteoinductive agent, whether residing in epithelial cells or bone cells, may be the same or a similar factor operating through the same mechanism.

Animals↗

PubMed2007

A proteomic analysis of adult rat bone reveals the presence of cartilage/chondrocyte markers.

The non-mineral component of bone matrix consists of 90% collagenous, 10% non-collagenous proteins. These proteins regulate mineralization, growth, cell signaling and differentiation, and provide bone with its tensile strength. Expression of bone matrix proteins have historically been studied individually or in small numbers owing to limitations in analytical technologies. Current mass-spectrometric and separations technologies allow a global view of protein expression patterns in complex samples. To our knowledge, no proteome profile of bone matrix has yet been reported. Therefore, we have used mass spectrometry as a tool to generate a profile of proteins present in the extracellular matrix of adult rat bone. Overall, 108 and 25 proteins were identified with high confidence in the metaphysis and diaphysis, respectively, using a bottom up proteomic technique. Twenty-one of these proteins were present in both the metaphysis and diaphysis including the bone specific proteins, osteocalcin, type I collagen, osteopontin, osteoregulin, and bone sialoprotein. Interestingly, type II collagen, a protein thought to be exclusively expressed in cartilage, was identified in both the metaphysis and diaphysis. This observation was validated by Western blot. Additionally, the presence of aggrecan, another protein expressed in cartilage was identified in the bone matrix extracts by Western blot. The proteome profile generated using this technology represents an initial survey of the acid soluble proteins of bone matrix which provides a reference for the analysis of deviations from the normal composition due to perturbations or disease states.

Aggrecans↗

PubMed1981

The role of fibronectin and substrate size in attachment of rat bone marrow cells to an osteoinductive matrix.

Culturing bone marrow cells in the presence of osteoinductive demineralized bone matrix particles results in increased cell proliferation. We have previously reported that this response is substrate specific and is not observed when cells are cultured with glass beads or collagenous tail tendon particles. In the present investigation, we show that a bone matrix particle size of less than 70 micrometers or greater then 420 micrometers is not supportive of cell attachment, as observed by the scanning electron microscope and that subsequent cell proliferation, as measured by DNA content, does not occur. The attachment of the bone marrow cells to bone matrix particles is dependent upon fibronectin, a serum glycoprotein. When serum was absent in the culture medium, cell attachment was still observed. Fibronectin was detected by an enzyme linked immunoabsorbent assay in cultures grown in the absence of serum. Therefore, fibronectin may be synthesized by bone marrow cells. When bone marrow cells were cultured in the presence of anti-fibronectin antibodies, the number of cells attaching to the matrix particles was greatly decreased. When cell attachment was reduced, the rate of cell proliferation as measured by DNA synthesis was also reduced. This study demonstrated that substrate size and fibronectin are important for the interaction of bone marrow cells with an osteoinductive matrix.

Animals↗

PubMed1998

Role of integrins in the attachment of metastatic follicular thyroid carcinoma cell lines to bone.

One of the principle targets for metastasis of follicular thyroid carcinoma (FTC) is the skeleton. Because no data are available on the role of the integrin adhesion molecule family in the attachment of FTC to bone, we studied the attachment characteristics of three FTC cell lines to bone and the role of integrins. Three cell lines were used from the same patient, one (FTC-133) from the primary tumor and two (FTC-236 and FTC-238) from metastases. Attachment of FTC cell lines to bone was assessed on conditioned medium of an osteoblastic cell line, coated onto plastic, as an in vitro model of bone matrix. The synthetic RGD (Arg-Gly-Asp) peptide GRGDS impaired attachment of the FTC cell lines to bone matrix, demonstrating the role of integrins in the attachment of FTC to bone. Attachment of FTC-133 to bone matrix was blocked completely by GRGDS, whereas attachment of FTC-236 and FTC-238 could not be impaired completely. Semiquantitative polymerase chain reaction (PCR) of cDNA from the cell lines indicated stronger expression of alpha5 integrin mRNA in FTC-133 than in the other cell lines. In line with this, attachment of FTC-133 to bone matrix could be inhibited almost completely by anti alpha5 and beta1 integrin antibodies, indicating the importance of the fibronectin receptor in the attachment of FTC-133 to bone. Binding of FTC-236 and FTC-238 to bone matrix could not be inhibited completely by anti-integrin antibodies, suggesting an additional role of nonintegrin adhesion molecules in the attachment of FTC-236 and FTC-238 to bone. The synthetic bone sialoprotein cyclic peptide, CNB, revealed antiadhesive effects in the binding of FTC to bone. In conclusion, integrins play an important role in the attachment of metastatic FTC to bone. Differences in the functional involvement of integrins in the attachment to bone are observed between the three cell lines studied. From the present results, antiadhesive interventions with synthetic RGD peptides in FTC may be designed.

Adenocarcinoma, Follicular↗

PubMed1998

Expression of bone microsomal casein kinase II, bone sialoprotein, and osteopontin during the repair of calvarial defects.

The temporal expression of bone microsomal casein kinase II, osteopontin, bone sialoprotein, alkaline phosphatase, and the accumulation of a solid calcium-inorganic orthophosphate mineral phase, have been charted from day 2 to day 21 during the repair of calvarial defects in rats induced by the implantation of decalcified rat bone matrix. Unlike the sequence of events that occur when the same decalcified bone matrix is implanted subcutaneously or intramuscularly, in which cases the first tissue to form in response to the implant is cartilage that subsequently calcifies and is later resorbed and replaced by bone, the repair of cranial defects is quite different. In the latter case, the first cells induced are undifferentiated mesenchymal cells and early fibroblasts followed by osteoblastic direct bone formation. Somewhat later a few small islands of cartilage are formed, widely separated and spatially distinct from the newly formed bone matrix. All of the cartilage and most of the implanted decalcified bone matrix are later resorbed and replaced by new bone by day 21. This in vivo model of the repair of a bone defect by direct bone formation has provided an excellent system to follow specific biochemical and physicochemical events. The total accumulation and rate of accumulation of the mineral and the two noncollagenous phosphoproteins (bone sialoprotein and osteopontin), as well as the activities of alkaline phosphatase, and for the first time either in vivo or in cell culture, the activity of microsomal casein kinase II, the major enzyme that phosphorylates the bone phosphoproteins, have been determined as a function of healing time in vivo. The overall general pattern of accumulation of the phosphoproteins and calcium-phosphate mineral phase and their relationships are similar to those reported in osteoblast cell cultures also monitored as a function of time.

Alkaline Phosphatase↗

PubMed2005

Effectiveness of local antibiotic delivery with an osteoinductive and osteoconductive bone-graft substitute.

BACKGROUND: The morbidity associated with open fractures and open fracture treatment is well established. An osteoinductive and osteoconductive bone-graft substitute that prevents infection would decrease the number of procedures required to treat contaminated fractures by eliminating the need for surgical removal of cement beads and perhaps autograft harvest. We hypothesized that the combination of tobramycin-impregnated calcium sulfate pellets and demineralized bone matrix would prevent the establishment of infection in a contaminated fracture model. METHODS: A unicortical 12-mm-diameter defect was created in the proximal tibial metaphysis of twenty-nine Spanish goats. After contaminating the wounds with an infective dose of Staphylococcus aureus, we divided the animals into four groups. The negative control group received no treatment, the positive control group received tobramycin-impregnated polymethylmethacrylate beads, the demineralized bone matrix group received 2.5 mL of demineralized bone matrix, and the experimental group received tobramycin-impregnated calcium sulfate pellets with 2.5 mL of demineralized bone matrix. Radiographs were made and intraosseous tissue cultures were performed on postoperative day 21. RESULTS: The cultures showed no evidence of intramedullary infection in the experimental or the positive control group, but they were positive for Staphylococcus aureus in six of the seven goats in the negative control group and seven of the eight goats in the demineralized bone matrix group. CONCLUSIONS: The combination of tobramycin-impregnated calcium sulfate pellets and demineralized bone matrix was effective in preventing intramedullary Staphylococcus aureus infection in a contaminated goat fracture model.

Animals↗

PubMed2003

Congenital lack of COX-2 affects mechanical and geometric properties of bone in mice.

We compared the mechanical properties of bones from mice lacking either a functional cycloxygenase-1 (C57BL6/DBA COX-1-/-; n = 9) or COX-2 (C57BL6/DBA COX-2-/-; n = 9) gene and wild type mice (C57BL6/DBA; n = 10). Twenty-eight right femora from 3-month-old male mice were used to determine bulk structural and material properties of bone by three-point bending. Bone matrix properties were also measured by nanoindentation to access the changes in bulk mechanical properties due to changes in bone matrix or bone geometry. The bulk material properties (elastic modulus, P < 0.05; ultimate stress, P < 0.01) of COX-2-/- bones were lower than those of wild-type mice whereas the bulk structural properties (stiffness, P > 0.2; breaking force, P > 0.1) were similar to those of the wild-type mice. COX-2-/- mice had a longer moment of inertia but their cortical bones were thinner and contained many more intra-cortical pores compared with the bones of the other two groups. Finally, the bone matrix properties of COX-1-/- mice, COX-2-/- mice and their heterozygous littermates were similar to those of C57BL6/DBA wild-type mice.

Animals↗

PubMed1984

Fetal bovine bone cells synthesize bone-specific matrix proteins.

We isolated cells from both calvaria and the outer cortices of long bones from 3- to 5-mo bovine fetuses. The cells were identified as functional osteoblasts by indirect immunofluorescence using antibodies against three bone-specific, noncollagenous matrix proteins (osteonectin, the bone proteoglycan, and the bone sialoprotein) and against type 1 collagen. In separate experiments, confluent cultures of the cells were radiolabeled and shown to synthesize and secrete osteonectin, the bone proteoglycan and the bone sialoprotein by immunoprecipitation and fluorography of SDS polyacrylamide gels. Analysis of the radiolabeled collagens synthesized by the cultures showed that they produced predominantly (approximately 94%) type I collagen, with small amounts of types III and V collagens. In agreement with previous investigators who have employed the rodent bone cell system, we confirmed in bovine bone cells that (a) there was a typical cyclic AMP response to parathyroid hormone, (b) freshly isolated cells possessed high levels of alkaline phosphatase, which diminished during culture but returned to normal levels in mineralizing cultures, and (c) cells grown in the presence of ascorbic acid and beta-glycerophosphate rapidly produced and mineralized an extracellular matrix containing largely type I collagen. These results show that antibodies directed against bone-specific, noncollagenous proteins can be used to clearly identify bone cells in vitro.

Alkaline Phosphatase↗

PubMed1986

Influence of aluminum on mineralization during matrix-induced bone development.

A model of de novo mineralization employing matrix-induced endochondral bone formation in rats was used to study the short-term effects of aluminum on the deposition of calcium and phosphate in vivo. In experiments where systemic aluminum concentrations were elevated, the cellular processes associated with bone development appeared to be normal, if somewhat delayed, however precipitation of the mineral phase was prevented. This suggests a primary direct physical chemical effect of aluminum in vivo on calcification, as suggested by in vitro studies which demonstrate that aluminum is a potent inhibitor of calcium phosphate precipitation. Aluminum salts implanted locally with the matrix appeared to be toxic to the cellular processes leading to chondrogenesis and osteogenesis.

Alkaline Phosphatase↗

PubMed2002

Smad3 promotes alkaline phosphatase activity and mineralization of osteoblastic MC3T3-E1 cells.

Transforming growth factor (TGF) beta is abundantly stored in bone matrix and appears to regulate bone metabolism. Although the Smad family proteins are critical components of the TGF-beta signaling pathways, the roles of Smad3 in the expression of osteoblastic phenotypes remain poorly understood. Therefore, this study was performed to clarify the roles of Smad3 in the regulation of proliferation, expression of bone matrix proteins, and mineralization in osteoblasts by using mouse osteoblastic cell line MC3T3-E1 cells stably transfected with Smad3. Smad3 significantly inhibited [3H]thymidine incorporation and fluorescent intensity of the MTT-dye assay, compared with empty vector. Moreover, Smad3 increased the levels of type I procollagen, osteopontin (OPN), and matrix Gla protein (MGP) mRNA in Northern blotting. These effects of Smad3 mimicked the effects of TGF-beta on the same cells. On the other hand, Smad3 greatly enhanced ALP activity and mineralization of MC3T3-E1 cells compared with empty vector, although TGF-beta inhibited ALP activity and mineralization of wild-type MC3T3-E1 cells. A type I collagen synthesis inhibitor L-azetidine-2-carboxylic acid, as well as osteocalcin (OCN), significantly antagonized Smad3-stimulated ALP activity and mineralization of MC3T3-E1 cells. In conclusion, this study showed that in mouse osteoblastic cells, Smad3 inhibited proliferation, but it also enhanced ALP activity, mineralization, and the levels of bone matrix proteins such as type I collagen (COLI), OPN, and MGP. We propose that Smad3 plays an important role in osteoblastic bone formation and might help to elucidate the transcriptional mechanism of bone formation and possibly lead to the development of bone-forming drugs.

Alkaline Phosphatase↗

PubMed1996

Ultrastructural identification of cells involved in the healing of intramembranous and endochondral bones.

This study was designed to identify the cells involved in the healing of autogenous intramembranous (IM) and of endochondral (EC) bone grafts. Thirty-six defects were created in the skull of 18 adult New Zealand White rabbits. Defects were filled with IM graft alone, EC graft alone, demineralized bone matrix (DBM) alone, or combined DBM-IM and DBM-EC bone. Cellular identification was carried out at 7 and 14 days by light and electron microscopy. In IM bone, preosteoblasts, osteoblasts, and osteocytes were observed with no cartilage intermediate stage, while in EC bone, chondroblasts and chondrocytes were observed. DBM implant and DBM-IM were characterized by the presence of a cartilage stage. In conclusion, IM bone healed through an osteogenic ossification route, while EC bone healed through an EC ossification route. In the presence of demineralized EC bone matrix, IM bone adopts an EC ossification route.

Animals↗

PubMed1993

[Purification of bone morphogenetic protein and investigation of its effects on osteoblastic cell line UMR108].

It is well known that the bone matrix contains proteins which can induce ectopic endochondral bone formation in vivo. One class of these proteins is the bone morphogenetic protein (BMP). In order to investigate the physiological function of the BMP, its purification was attempted from an extract of demineralized bone matrix and its actions on the osteoblastic cell line were investigated. To isolate the BMP, a demineralized bone matrix was extracted with 4M guanidine-HCl. A water-insoluble fraction (G-WI) was separated from the demineralized bone extract by dialysis against distilled water and centrifugation. The BMP was purified from G-WI by gel filtration on Sephacryl S-200 HR, cation exchange with Mono-S, heparin affinity column and finally by C1/8 reverse phase chromatography. Peptide sequence analysis revealed that the purified BMP fraction contained "BMP-3" reported by Wozney et al. (1988). In order to investigate its function, the BMP was applied to the rat osteogenic sarcoma cell line UMR108. The BMP inhibited the growth of the UMR108 cells and enhanced the alkaline phosphatase activity in a dose-responsive manner.

Amino Acid Sequence↗

PubMed1991

Bone growth factors.

Bone volume is determined by the relative rates of bone formation and bone resorption. Recent research in several laboratories suggests that growth factors may act locally to modulate bone formation by stimulating osteoblast proliferation and activity. A number of bone-derived growth factors have been isolated and characterized from bone matrix extracts and from media conditioned by bone cells and bone organs in culture. The growth factors found in bone matrix include insulinlike growth factors I and II, transforming growth factor-beta, acidic and basic fibroblast growth factor, platelet-derived growth factor, and bone morphogenetic proteins. Conditioned medium from bone cells contains several of these growth factors and also hematopoietic factors. These bone matrix-derived growth factors have different biologic activities, including mitogenic, differentiating, chemotactic, and osteolytic activities. Evidence suggests that bone cells produce substantial quantities of growth factors for extracellular storage in bone matrix. Apart from being produced for extracellular storage, it is possible that growth factors secreted by bone cells have acute effects on their neighboring osteoblastic cells, i.e., paracrine action, or on themselves, i.e., autocrine action. The release of matrix-stored growth factors by bone resorption may mean that growth factors act as delayed paracrine agents, e.g., osteoblasts deposit growth factors in bone and later when these growth factors are released from bone via bone resorption, the growth factors stimulate osteoblast precursors to proliferate. The findings that bone is a storehouse for growth factors and that bone cells in culture produce and respond to bone growth factors suggest bone growth factors may act as potential determinants of local bone formation. This review is focused on the structure, regulation, and biologic actions of the known bone growth factors.

Animals↗

PubMed1991

Tissue transformation into bone in vivo. A potential practical application.

The transformation of mesenchymal tissue, such as muscle, into cartilage and bone can be induced by the recently purified osteoinductive factor, osteogenin, and by its parent substratum, demineralized bone matrix. We investigated the possibility of transforming readily available muscle flaps into vascularized bone grafts of various shapes that could be used as skeletal replacement parts. In a rat experimental model, thigh adductor muscle island flaps were placed inside bivalved silicone rubber molds. Prior to closure of the mold, 18 flaps were injected with osteogenin and coated with demineralized bone matrix. Five flaps served as controls and were injected with the vehicle only, and not coated with demineralized bone matrix. The molds were implanted subcutaneously in the rats' flanks and reopened 10 days later. The control flaps consisted of intact muscle without any evidence of tissue transformation, whereas the flaps treated with osteogenin and demineralized bone matrix were entirely transformed into cancellous bone that matched the exact shape of the mold. Using tissue transformation, we were able to generate in vivo, autogenous, well-perfused bones in the shapes of femoral heads and mandibles.

Animals↗

PubMed1994

Histochemical and confocal laser scanning microscopy study of the bone-titanium interface: an experimental study in rabbits.

The aim of our study was an analysis of the presence of an unmineralized bone matrix between mineralized bone and titanium screws in rabbit tibiae. A microscopical analysis, using a histochemical technique, was performed on the titanium-bone interface of commercially pure titanium implants placed in rabbit tibiae and harvested after 2 months. Thin ground sections of the specimens were prepared by the cutting-grinding system and stained using the von Kossa method for calcium salts and basic fuchsin for osteoid. The microscopical and morphometrical evaluation showed that bone covered about 40% (+/- 7.5%) of all implants. Mineralized bone was, however, in direct contact with the titanium surface on only about 10% of the implant, while in the remaining 30% the mineralized bone was separated from the implant by an unmineralized tissue. This basophilic, probably osteoid matrix, could represent the medium that allows the biochemical exchanges between bone and cells under the influence of the implant. A small, optically translucent gap (1-5 microns), probably an artifact, was present in some areas between titanium and bone. Confocal laser scanning microscopy (CLSM) in a fluorescent mode showed the presence at the interface of a fluorescent material. Results from our study showed that light microscopy of thin ground sections allowed a good analysis of the real nature of the titanium-bone interface. Moreover, this double staining technique showed the presence of an unmineralized bone matrix at most of the bone-titanium interface.

Animals↗

PubMed1988

Osteogenesis in rats with an inductive bovine composite.

Subcutaneous (S.C.) implantation of allogeneic demineralized bone matrix in rats results in endochondral bone formation. In contrast, implants of bovine demineralized bone matrix in rat S.C. tissue show inconsistent cartilage and bone formation, presumably due to an intense inflammatory reaction at the implant site. To overcome this response, a partially purified bone inducing extract was prepared from bovine bone by a series of steps that included demineralization, guanidine/HCl extraction, gel filtration, and cation exchange chromatography. To develop a carrier, the inactive guanidine/HCl-extracted matrix was then trypsinized to remove the inflammatory and immunogenic components, thus yielding a predominantly collagenous matrix. Bovine composites were prepared by combining different amounts of the bone inducing extract with a carrier that consisted of the trypsinized bone matrix and purified soluble bovine dermal collagen. Subcutaneous implantation of the composite preparation resulted in dose-dependent endochondral bone formation in rats. The inductive activity and the low-level inflammatory response were comparable to allogeneic implants.

Animals↗

PubMed1986

Differentiation of cartilage on three substrata under the influence of an aggregate of morphogenetic protein and other bone tissue noncollagenous proteins (BMP/iNCP).

A cellulose acetate membrane was fashioned into a cone to serve as a substratum for an outgrowth of connective tissue from normal neonatal muscle, and a container for diffusion of bone morphogenetic protein (BMP) of an aggregate of BMP and cold water insoluble noncollagenous protein (BMP/iNCP). The BMP/iNCP was prepared by dissociative extraction and differential precipitation with other bone matrix proteins that slowly become soluble and diffusible in culture media at 37 degrees. The BMP/iNCP was applied either on, within, or beneath the surface of the explants or suspended in the culture medium. Under the influence of BMP in tissue cultures, without any bone matrix or bone collagen in the system, connective tissue outgrowths of muscle differentiate into cartilage on three substrata: (1) cellulose acetate membranes with pore size of 0.45-5.0 micron; (2) remnants of undissolved BMP/iNCP; and (3) degenerating myofibers. The cartilage developed in the interior of muscle, possibly by phenotypic cell transformation, when the pore size of the membrane was 0.1-0.22 micron too small to sustain anchorage of the explant. Cartilage developed on particle surfaces when the muscle tissue and BMP/iNCP particle were minced and mixed before explantation. The cartilage preferentially grew out directly onto the cellulose acetate membrane when the pore size was optimal for anchorage and the BMP/iNCP was suspended on the surface of the explant to either simultaneously percolate through the explant or diffuse through the culture medium. The biosynthetic activity of cells proliferating before and associated with cell differentiation was measured by 35S uptake in total glycosaminoglycan (GAG) per microgram of DNA. When the pore size was 8.0 micron, large enough to permit cells to migrate across the membrane, a thick plate of fibrous connective tissue developed on the undersurface of the membrane without any evidence of cartilage cell differentiation in any location. Repeated doses of BMP/iNCP with each change of culture medium produced a greater incidence and quantity of cartilage than a single dose, but the 35S incorporation into GAG always reached peak levels, in the interval between four and ten days, irrespective of the schedule of administration or dosage. These observations suggested that the exogenous or endogenous noncollagenous proteins are a carrier for BMP and can substitute for whole bone matrix or bone collagen.

Animals↗

PubMed1992

Calvarial regeneration in primates with autolyzed antigen-extracted allogeneic bone.

The limited regenerative capacity of calvarial membranous bone provides an ideal system for investigation in the comparative physiology of bone regeneration. Cranial defects, 25 mm in diameter, were created in 24 adult male baboons (Papio ursinus). In each animal, defects were implanted with chemosterilized antigen-extracted, autolyzed allogeneic (AAA) bone, grafted with iliac corticocancellous grafts, or left ungrafted to monitor the spontaneous regeneration potential of the adult baboon calvaria. Antigen-extracted, autolyzed allogeneic bone implants were prepared from donor sub-adult baboon calvariae and processed so as to retain bone morphogenetic protein (BMP) in the bone matrix. Histomorphometric analysis of undecalcified sections (7-microns thick) prepared from specimens harvested at three, six, and nine months after surgery showed superior osteogenesis in AAA bone matrix implants, compared with autografts and untreated defects. The morphogenetic response was characterized by vascular invasion and mesenchymal cell aggregation after partial resorption and dissolution of the implanted matrix, followed by bone deposition at the calvarial interfaces, the pericranial and endocranial surfaces of the implants, and within the open diploic spaces of the implanted original AAA bone matrix. The superior osteogenesis in AAA bone implants appeared consistent with extensive osteoconductive invasion from the open diploic and endosteal spaces of the recipient calvariae. In addition, the finding of a delicate, trabecularlike bone, appositional to the central areas of the implanted matrix, suggests that AAA bone implants might also have acted as inductive substratum for bone differentiation.

Analysis of Variance↗