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A role for osteocalcin in osteoclast differentiation.

Specific cellular interactions with components of the extracellular matrix can influence cellular differentiation and development of many tissues. The extracellular matrix of bone is composed of organic constituents and a solid phase of calcium and inorganic phosphate (apatite). When implanted subcutaneously in rats, particles of bone matrix (BPs) recruit progenitors that differentiate into multinucleated cells with osteoclastic features. Because BPs deficient in osteocalcin, a bone matrix protein, were less efficient at promoting osteoclast formation than were normal BPs, we directly examined the influence of osteocalcin on osteoclast differentiation. We evaluated tissue responses to particles of synthetic crystalline apatite alone (Ap), having many of the features of native apatite of mature bone, or to apatite prepared with osteocalcin (Ap/OC), bovine serum albumin (Ap/BSA) or rat bone collagen (Ap/Col). Twelve days after subcutaneous implantation in normal rats, Ap, Ap/BSA, and Ap/Col particles generated a mild foreign body reaction with multinucleated cells in direct contact with the particles; these cells were negative for tartrate-resistant acid phosphatase (TRAP) activity and lacked ruffled borders. In contrast, Ap particles containing approximately 0.1% osteocalcin were partially resorbed and they generated more multinucleated cells that were TRAP-positive, were immunoreactive with an antibody against tartrate-resistant purple acid phosphatase, and displayed ultrastructural features of active osteoclasts including ruffled borders and clear zones. These data support the hypothesis that osteocalcin may function as a matrix signal in the recruitment and differentiation of bone-resorbing cells.

Acid Phosphatase↗

Effect of sterilization on bone morphogenetic protein.

Demineralized bone matrix and bone morphogenetic protein have been used clinically to accelerate bone regeneration. However, the best method of sterilization has been the subject of controversy. Some investigators have used ethylene oxide, but others have reported that doses adequate for sterilization destroyed the osteoinductivity of demineralized bone matrix and that gamma irradiation was less harmful in this respect. We used partially purified bone morphogenetic protein and type-I collagen to investigate the effects of sterilization by ethylene oxide and gamma irradiation on the activity of bone morphogenetic protein. Osteoinductivity was reduced considerably after sterilization by gamma irradiation at 2.5 Mrad and by ethylene oxide at 37 degrees C for 4 hours and at 55 degrees C for 1 hour; however, the reduction induced by ethylene oxide at 29 degrees C for 5 hours was about half of the control values. This study showed that ethylene oxide at 29 degrees C for 5 hours can be used clinically for sterilization of bone morphogenetic protein. We also investigated the effect of gamma irradiation on bone morphogenetic protein and the collagen carrier separately and found that collagen was far more labile than bone morphogenetic protein.

Animals↗

Expression and localization of extracellular matrix metalloproteinase inducer in giant cell tumor of bone.

Matrix metalloproteinases (MMPs) are regarded as a significant regulator in tumor invasion and metastasis. Previous studies have shown that extracellular matrix metalloproteinase inducer (EMMPRIN) in tumor cells induces the synthesis of MMPs. EMMPRIN is abundantly present on the surface of tumor cells and stimulate adjacent stromal cells to synthesize MMPs to induce tumor progression. Giant cell tumor (GCT) of bone is a benign but locally aggressive primary neoplasm of bone. The spindle-shaped mononuclear stromal cells are considered to be the tumor components of GCT, which are capable of inducing osteoclast formation by recruiting the circulating monocyte and macrophage. In this study, we proposed that EMMPRIN is associated with the biological progression and aggressiveness of GCT. We have conducted semi-quantitative RT-PCR to determine the correlation of EMMPRIN expression with the clinical stage of GCT. We have also examined the cellular localization of EMMPRIN in GCT using in-situ hybridization (ISH) and Immunohistochemistry (IH). The results showed that EMMPRIN was present in GCT and its mRNA levels were associated with the clinical stage of GCT. Higher expression level of EMMPRIN was observed in GCT with advanced stage (stage III). There was a great significance (P < 0.05) of EMMPRIN expression between stage I & II and stage III GCTs. Both ISH and IH demonstrated that EMMPRIN is present at the multinuclear osteoclast-like giant cells of GCT, with strong immunostaining on the cell membrane. The stromal-like tumor cells were also positively stained but the intensity was weaker. Interestingly, the production of EMMPRIN in osteoclast-like cells of GCT seems to be regulated by stromal-like tumor cells. Receptor activator of NF-kappaB ligand (RANKL), which has been previously shown to be produced by the stromal-like tumor cells for the recruitment of osteoclast-like giant cells in GCT, enhanced the expression of EMMPRIN mRNA during the differentiation of macrophage-like RAW(264.7) cells into osteoclasts. In short, our studies suggest that EMMPRIN may be an important regulatory factor involved in the biological behaviors of GCT.

Animals↗

17 beta-Oestradiol and 1 alpha,25-dihydroxycholecalciferol modulate constitutive and bone matrix-induced interleukin-1 beta (IL-1 beta) production by peripheral blood mononuclear cells isolated from postmenopausal women.

Local production and release of interleukin-1 (IL-1) may be of importance for bone remodeling, since this cytokine is known to stimulate bone resorption. We have studied the effect of bone matrix constituents on IL-1 beta production by peripheral blood mononuclear cells (PBMCs) isolated from 20 postmenopausal non-osteoporotic women. Hydroxyapatite (0.5 mg/ml) and heat-denaturated collagen (25 micrograms/ml) stimulated IL-1 beta production 5-fold and 520-fold, respectively, compared to control (p < 0.01). In contrast, transforming growth factor-beta (TGF-beta, 10 ng/ml), a cytokine which is abundant in bone matrix, suppressed median IL-1 beta release to 13% of control value (p < 0.01). The bone matrix-induced changes in IL-1 beta production were modulated by 10 nmol/1 17 beta-oestradiol and 10 nmol/1 1 alpha,25-dihydroxy-cholecalciferol (1,25(OH)2D3). Specifically, 17 beta oestradiol stimulated constitutive IL-1 beta release with 89% (p < 0.01) and nullified the suppressive effect of TGF-beta. Moreover, 1,25(OH)2D3 had a synergistically stimulatory effect with both hydroxyapatite and collagen, although there was no effect of this hormone when added alone. The adherent cells were slightly more elongated after treatment with 1,25(OH)2D3 and collagen, while TGF-beta and 17 beta-oestradiol had no effect on cellular morphology. Addition of hydroxyapatite resulted in long and spindle-shaped cells, and phagocytosis of the particles occurred. The modulatory effects of oestrogen and vitamin D on constitutive and bone-matrix induced IL-1 beta production by PBMCs may be of importance for bone remodelling during postmenopausal bone loss and at a site of fracture.

Bone Matrix↗

Mechanism of osteoclast mediated bone resorption.

The osteoclast is the main bone resorbing cell. It has several structural features which are related to its function. In active resorption stage this multinucleated giant cell shows remarkable polarity which, with its basolateral and apical membrane surfaces, resembles a secreting epithelial cell. The third specialized membrane area, sealing zone, mediates the attachment of the osteoclast to the bone surface. The rate of bone resorption can be regulated either by changing the number or the activity of resorbing osteoclasts. These processes can be influenced by different systemic circulating factors as well as local, bone matrix or other bone cell-derived factors. After activation of stem cells, preosteoclasts are guided to the bone surface where they undergo fusion into multinucleated osteoclasts. A special type of cell attachment to mineralized bone surface precedes actual resorption process. During cell attachment cytoskeletal structures are organized into a typical belt-like structure. The actual attachment receptor as well as its counterpart in bone matrix are not yet known. Bone mineral dissolution is then initiated by active secretion of H+ through the ruffled border membrane area. Acidification of the resorption lacuna together with hydrolytic enzymes completes finally the degradation of organic matrix of bone.

Bone Matrix↗

Accelerated repair of cortical bone defects using a synthetic extracellular matrix to deliver human demineralized bone matrix.

Injectable hydrogel and porous sponge formulations of Carbylan-GSX, a crosslinked synthetic extracellular matrix (ECM), were used to deliver human demineralized bone matrix (DBM) in a rat femoral defect model. A cortical, full-thickness 5-mm defect was created in two femurs of each rat. Six rats were assigned to each of five experimental groups (thus, 12 defects per group). The defects were either untreated or filled with Carbylan-GSX hydrogel or sponges with or without 20% (w/v) DBM. Radiographs were obtained on day 1 and at weeks 2, 4, 6, and 8 postsurgery of each femur. Animals were sacrificed at week 8 postsurgery and each femur was fixed, embedded, sectioned, and processed for Masson's Trichrome staining. The bone defects were measured from radiographs and the fraction of bone healing was calculated. The average fractions of bone healing for each group were statistically different among all groups, and all treatment groups were significantly better than the control group. The Carbylan-GSX sponge with DBM was superior to the sponge without DBM and to the hydrogel with DBM. Histology showed that defects treated with the Carbylan-GSX sponge plus DBM were completely filled with newly generated bone tissue with a thickness comparable to native bone. Carbylan-GSX sponge was an optimal delivery vehicle for human DBM to accelerate bone healing.

Animals↗

Transitions in collagen types during matrix-induced cartilage, bone, and bone marrow formation.

The localization of types I, II, and III collagens during bone matrix-induced sequential differentiation of cartilage, bone, and bone marrow was studied by specific immunofluorescence. Subcutaneous transplantation of coarse powders of demineralized rat bone matrix into allogeneic recipients resulted in new bone formation. After a transient appearance of polymorphonuclear leukocytes in the implant, fibroblasts appeared in close continguity to the matrix on day 3. Type III collagen was then localized as a fine network around the invading fibroblasts. On days 4--6 smaller amounts of type I were also detected around these proliferating cells. With the onset of chondrogenesis, type II collagen was detected in the cartilage matrix on day 6 and persisted until the early stages of bone formation. Vascular invasion of the implant was accompanied by osteogenesis on day 10. Type I collagen was demonstrated in the newly deposited bone matrix coating the surfaces of cartilage spicules and particles of implanted bone powder. On day 17 and thereafter, type III collagen was localized as a fibrous array around nests of hematopoietic cells.

Animals↗

Modification of an osteoconductive anorganic bovine bone mineral matrix with growth factors.

BACKGROUND: Osteoconductive anorganic bovine bone mineral matrix material has been used clinically in bone regeneration procedures. Platelet-derived growth factor-BB (PDGF-BB) and insulin-like growth factor (IGF-I) are important anabolic growth factors for bone. It was the aim of these studies to 1) examine the interaction of this bone graft material with PDGF-BB and IGF-I and 2) determine if the combination of growth factors with the matrix could stimulate osteoblastic cell proliferation. METHODS: Adsorption of PDGF-BB and IGF-I was done using 125I radio-labeled growth factors. The PDGF-BB or IGF-I was incubated with the anorganic bovine bone matrix, and the amount of adsorbed growth factor was measured. In the desorption studies, radiolabeled growth factors were adsorbed to the matrix material. The samples were incubated in buffer for various time periods, and the amount remaining on the matrix was measured to calculate the percentage of released growth factor. The biological activity was tested in an in vitro assay with primary culture neonatal rat osteoblastic cells. Porous bone matrix with known amounts of adsorbed PDGF-BB or IGF-I was produced. The osteoblastic cells were cultured on the bone mineral matrix, with and without adsorbed growth factor, and proliferation was assessed by 3H-thymidine incorporation. RESULTS: Both PDGF-BB and IGF-I adsorbed to bone mineral matrix in a concentration-dependent fashion. The affinity of IGF-I for the material was 10-fold greater than PDGF-BB. In the experiments that measured the release of the initially adsorbed growth factors, approximately 50% of the PDGF-BB and 10% of the IGF-I were released after 10 days. PDGF-BB adsorbed to the matrix material significantly (P <0.05, ANOVA) enhanced the proliferation of cultured osteoblastic cells compared to the mineralized matrix alone. However, IGF-I adsorbed to the matrix material did not significantly enhance cell proliferation. CONCLUSIONS: These results suggest that PDGF-BB can be adsorbed to the anorganic bovine bone mineral matrix and that this growth factor subsequently enhances the osteogenic properties of this bone graft material. IGF-I also adsorbed to the graft material; however, it was not readily released and it did not produce significant effects in the biologic assay. It appears that it may be clinically feasible to adsorb PDGF to anorganic bovine bone and that this combination of bone growth factor and mineral matrix has the potential for clinical applications.

Adsorption↗

Differentiation of the organic matrix in bone repair.

This study describes the sequence of production and distribution of collagen, collagen types, proteoglycans, and calcium during bone repair in rabbits utilizing histologic and biochemical techniques. Proteoglycan content peaked 11 days following surgery and total collagen peaked 19 days after surgery, after which both levels decreased. Collagen Types I and III were present in the bone defect throughout the healing process but Type II was found only in the mid stages of repair. In the surrounding external callus Types I, II, and III were present, but Type III was not noted in the later stages of healing. These results are similar to those found in studies of fracture repair and developing bone.

Animals↗

Skeletal repair in the aged: a preliminary study in rabbits.

Bone mass loss associated with aging can lead to osteoporosis and multiple bone fractures with impaired healing requiring prolonged hospitalization and costly medical care. We have used an experimental implantation model to test the ability of old animals to form new bone. Bone repair inducers, consisting of demineralized bone matrix (DBM), bone marrow, and collagen, were implanted in the abdominal wall muscles of 1-month and 16-month old rabbits. DBM contains a bone morphogenetic protein (BMP) that induces the differentiation of primitive mesenchymal cells into bone producing cells. The stromal cells of bone marrow can differentiate into osteoblasts after implantation, while collagen could serve as a calcification nucleus or framework for new tissue formation. Animals were killed 4 to 6 weeks after implantation. Implants were X-rayed, examined histologically, and analyzed for water content, calcium, and alkaline phosphatase. Only the implants of bone marrow enclosed in filter chambers (0.45 micron pore diameter) were associated with bone formation. Intramuscular implants of DBM and bone marrow in the old animals induced the formation of new bone but contained less calcium and lower levels of alkaline phosphatase than implants in the young animals. Collagen implants were resorbed and failed to induce bone formation or calcify. The results indicate that formation of new bone, under the conditions of this study, is reduced with aging.

Aging↗

The contribution of the organic matrix to bone's material properties.

Bone is a two-phase porous composite material comprised primarily of collagen and mineral, which together provide its mechanical properties. The contribution of the mineral phase to bone's mechanical properties has dominated scientific thinking. Collagen's role has been underappreciated and not very well studied. However, there is evidence that changes in collagen content, or changes to inter- and intrafibrillar collagen cross-linking, can reduce the energy required to cause bone failure (toughness), and increase fracture risk. Although collagen may have less effect on bone's strength and stiffness than does mineral, it may have a profound effect on bone fragility. Collagen changes that occur with age and reduce bone's toughness may be an important factor in the risk of fracture in older women with low bone mass.

Animals↗

Bone graft alternatives for spinal fusion.

Bone grafting to achieve fusion is frequently performed in spinal surgery. Autograft is the gold standard bone graft material. However, due to limitations of supply and morbidity associated with the harvest of autograft, alternatives are being considered. Osteoconductive matrices, such as allograft, calcium or ceramic preparations are one such class of potential bone graft alternatives, but generally they lack osteoinductive properties. Recent attention has focused on osteoinductive materials such as demineralised bone matrix, recombinant bone morphogenetic proteins and bone marrow aspirates or blood product concentrates. These products may be combined with osteoconductive carriers and are clearly finding a place in the clinical arena.

Animals↗

Penetration and proliferation of BHK and HeLa cells into a three-dimensional collagen matrix of bone stroma type. Preliminary results.

Some preliminary data regarding the test of penetration and proliferation of BHK and HeLa cells biocompatibility to a three dimensional stroma saturated with phosphorous and calcium salts (a biomaterial replacing bone) are presented. These data suggest that the analysed biomaterial might be used in surgical practices of prosthesis appliance on interfaces of fractured bone.

Animals↗