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PubMed1991

Effects of fixation and demineralization on the retention of bone phosphoprotein and other matrix components as evaluated by biochemical analyses and quantitative immunocytochemistry.

Aqueous tissue processing and demineralization procedures may adversely affect the inorganic mineral phase of a calcified sample and, where mineral and organic constituents interact, may consequently also indirectly alter organic matrix ultrastructure and distribution. In the present work, the effects of demineralization have been investigated on the retention in chicken bone of two phosphoamino acids, O-phosphoserine and O-phosphothreonine, found in bone phosphoproteins proposed to be important in vertebrate mineralization and, more specifically, on the retention and distribution of a 66 kD bone phosphoprotein (66 kD BPP, osteopontin) also implicated in the calcification process. In tibiae fixed initially with 1% glutaraldehyde and then demineralized in 0.5 N HCl, 0.5 N acetic acid, or 0.1 M EDTA (all containing 1% glutaraldehyde), amino acid analyses and quantitative immunocytochemistry revealed that the phosphoamino acid content and the distribution of the 66 kD BPP were essentially the same as in fixed undemineralized controls. However, demineralization slightly altered the ultrastructural appearance of immunolabeled, electron-dense patches of organic material in the bone matrix. In unfixed bone demineralized with any of these acids, there was a substantial loss of phosphoamino acids and the 66 kD BPP from the bone matrix. The relative ability of these acids to extract phosphoproteins from unfixed bone was found to decrease in the order EDTA greater than HCl greater than acetic acid. These results emphasize the differential effects on structural components of various demineralization and extraction procedures for biochemical and immunocytochemical studies of biologic tissues. Furthermore, they demonstrate that initial fixation with glutaraldehyde retains phosphoproteins in bone, with or without demineralization, while being adequate for immunocytochemical localization of certain bone matrix proteins and that an understanding of the action of specimen preparation on organic constituents (as well as inorganic components) is essential for accurately describing ultrastructural matrix-mineral relationships.

Amino Acids↗
PubMed2003

Identification of the molecular mechanisms contributing to polarized trafficking in osteoblasts.

The directionality of matrix deposition in vivo is governed by the ability of a cell to direct vesicularflow to a specific target site. Osteoblastic cells direct newly synthesized bone matrix proteins toward the bone surface. In this study, we dissect the molecular mechanisms underlying the polarized trafficking of matrix protein in osteoblasts. We demonstrate using TEM, immunocytochemistry, and cDNA analysis, the ability of osteoblastic cells in culture to form tight junction-like structures and report the expression of the tight junction associated proteins occludin and claudins 1-3 in these cells. We identify intercellular contact sites and the leading edge of migratory osteoblasts as major target sites of vesicular trafficking in osteoblasts. Proteins required for this process, rsec6, NSF, VAMP1, and syntaxin 4, as well as the bone matrix protein, osteopontin, localize to these sites. We demonstrate that osteoblasts in vivo possess VAMP1 and, furthermore, report the expression of two VAMP1 splice variants in these cells. In addition, osteoblasts express the NSF attachment protein alpha-SNAP and the t-SNARE SNAP23. Thus, cell-to-cell contact sites and the leading edge of migratory osteoblasts contain a unique complement of proteins required for SNARE mediated membrane fusion.

Animals↗
PubMed1996

Expression of matrix proteins during the development of mineralized tissues.

The specific properties of mineralized tissues are defined by the composition of the fraction of the noncollagenous matrix proteins. Because these proteins play a pivotal role in the processes of cell differentiation and activation and of mineralization, their temporal and spatial expression is tightly regulated. Within this study, the expression of the enamel protein amelogenin and of the bone matrix proteins osteopontin, bone sialoprotein, osteocalcin, and osteonectin was investigated by in situ hybridization. Two models that allow observation of the formation of mineralized tissues were chosen. The development of bone and cartilage was observed on murine metatarsals from 15-day-old embryos up to 1-day-old mice. This time covers the periods of initial bone formation as well as onset of resorption of mineralized cartilage and bone. To study gene expression in the mineralized tissues of the dental organ, enamel, dentin, and cementum, developing molars ranging in age from 16-day-old embryos to 14 days after delivery were chosen. Within this time frame, the molars develop from an immature state to the differentiated organ which erupts through the mandibular bone. In the developing metatarsals, osteopontin and bone sialoprotein mRNAs were detected in osteoblasts and hypertrophic chondrocytes at the onset of mineralization. In the tooth organ, only cementoblasts expressed transcripts encoding the two proteins; odontoblasts and ameloblasts did not express these genes. Osteonectin was expressed by osteoblasts and hypertrophic chondrocytes as well, whereas in the molars it was produced exclusively by odontoblasts. Osteocalcin was expressed specifically by osteoblasts in the developing metatarsals. In tooth, osteocalcin transcripts were detected in odontoblasts. Finally, amelogenin was a specific product of ameloblasts. Thus, a sequential and cell type-restricted expression of matrix proteins takes place during the development of the mineralized tissues. The expression patterns of the transcripts encoding the bone matrix proteins suggest different biological roles depending on the time and site of expression.

Amelogenin↗
PubMed1993

Extracellular-matrix degradation at acid pH. Avian osteoclast acid collagenase isolation and characterization.

Osteoclasts degrade bone matrix, which is mainly type I collagen and hydroxyapatite, in an acidic extracellular compartment. Thus we reasoned that osteoclasts must produce an acid collagenase. We purified this enzyme, a 31 kDa protein, from avian osteoclast lysates (in 100 mM acetate/1 mM CHAPS/1 mM dithiothreitol, pH 4.4), fractionated by (NH2)2SO4 precipitation, gelatin-affinity, cation exchange, and gel filtration. Fraction activity was measured using diazotized collagen or 3H-labelled cross-linked collagen (decalcified and trypsin-treated metabolically L-[4,5-3H]proline-labelled bone) as substrates. Iodoacetate, leupeptin, antipain, pepstatin and mercurials inhibited collagenolysis by the isolated proteinase; mercurial derivatives could not be re-activated by dithiothreitol. Collagen degradation was maximal at pH 4.4; purified proteinase reproduced the collagenolytic activity of cell lysates. The N-terminal amino acid sequence from the isolated protein and its CNBr degradation fragments showed sequence similarity to mammalian cathepsin Bs, and near-identity with avian liver cathepsin B. Peptide substrate specificity of the osteoclastic enzyme resembled those of mammalian cathepsin B and its avian liver counterpart, but degradation of low-molecular-mass substrates by the osteoclastic enzyme was slower, reflecting generally lower kcat. values. Further, kcat/Km varied less between arginine-containing substrates than for previously reported cathepsin Bs, indicating different substrate specificity of the osteoclast enzyme. Polyclonal antibody raised to a 25 kDa fragment of the enzyme recognized a single 31 kDa band in SDS/PAGE of osteoclast lysates blotted to poly(vinylidene difluoride), adsorbed collagenolytic activity of osteoclast lysates, and stained avian osteoclasts in tissue sections. Degenerate sense- and antisense-oligonucleotide primers, predicted from segments of primary amino acid sequence, amplified a 486 bp DNA fragment; this was cloned and sequenced. Of 162 amino acids encoded, 77% are identical with those of human cathepsin B; hybridization identified a 2.4 kb RNA in osteoclast lysates. We conclude that the major avian osteoclast collagenolytic enzyme is a cathepsin B, whose activity varies from other enzymes of its class.

Amino Acid Sequence↗
PubMed1990

Inhibitory effects of the bone-derived growth factors osteoinductive factor and transforming growth factor-beta on isolated osteoclasts.

Demineralized bone matrix contains a number of growth factors for osteoblast-like cells. Two of these, the novel glycoprotein osteoinductive factor (OIF) and transforming growth factor-beta (TGF beta), act together to cause ectopic bone formation in vivo. Since OIF, like TGF beta, is likely released from bone when the matrix is resorbed, we examined the effects of homogeneous OIF and TGF beta on osteoclast function. Osteoclast function was tested in isolated avian osteoclasts and was measured in terms of tartrate-resistant acid phosphatase (TRAP) activity, oxygen-derived free radical production, and formation of characteristic resorption lacunae on slices of sperm whale dentine. OIF (50-100 ng/ml) inhibited the capacity of these osteoclasts to form lacunae whether assessed by the number of excavations per slice or by the total area resorbed. OIF (10-100 ng/ml) or TGF beta (10-20 ng/ml) caused a decrease in TRAP activity as well as a reduction in oxygen-derived free radical generation detected by nitroblue tetrazolium staining. TGF beta had no effect on the resorption capacity of isolated osteoclasts in concentrations that inhibited TRAP activity and nitroblue tetrazolium staining. These results suggest that growth regulatory factors, such as OIF and TGF beta, released during the resorption of bone may be endogenous inhibitors of continued osteoclastic activity. This cessation of osteoclast activity may be an essential preliminary step to the new bone formation that occurs at resorption sites during bone remodeling.

Acid Phosphatase↗
PubMed2003

Mechanisms of tumor metastasis to the bone: challenges and opportunities.

In human cancers, bone is a common site for metastasis. It is well known that metastasis is the cause of morbidity and mortality in patients with cancer. Both breast and prostate carcinomas have a propensity to metastasize to bone. In general, metastatic breast cancers result in osteolytic lesions. On the other hand, prostate cancer metastases are osteoblastic and result in osteosclerosis. Thus, bone formation and bone resorption are at the crux of the cancer metastasis problem. For example, in the prostate, there is a vicious cycle of metastasis to bone (Fig. 1). Metastases to bone causes excruciating bone pain, pathological fractures, and eventually death, and therefore is a serious challenge to both bone biologists and cancer cell biologists. The stromal-epithelial interactions in breast and prostate are critical in initiation of carcinogenesis and the progression of the metastatic cascade to bone (Fig. 2). Over a hundred years ago, Stephen Paget enunciated the seed and soil hypothesis in which seeds of metastatic cancer cells of breast preferentially settle in the soil of bone matrix. Thus, the prostate/breast cancer bone interface and continuum has continuously presented challenges and opportunities and were discussed at a recent workshop.

Animals↗
PubMed1993

Insulin-like growth factors-I and -II stimulate chemotaxis of osteoblasts isolated from fetal rat calvaria.

Repair and regeneration of damaged bone is believed to be regulated in part by growth factors stored in the bone matrix. These growth factors are synthesized and secreted by osteoblasts and are incorporated into the developing bone. This pool of stored growth factors is then released into the immediate area following resorption of the matrix. One of the initial steps in bone repair is the recruitment of osteoblasts to the repair site. Growth factors, such as TGF-beta and PDGF, which are present in bone matrix, have been shown to be chemotactic for osteoblasts. In this study, primary cultures of osteoblasts isolated from fetal rat calvaria were examined for chemotaxis in response to IGF-I and IGF-II. IGF-I stimulated a dose-dependent increase in osteoblast chemotaxis, while IGF-II stimulated chemotaxis maximally at the lowest concentration studied (0.1 ng/ml), and had no effect at the highest concentration studied (100 ng/ml). IGF-I and -II had no effect on osteoblast proliferation at any of the concentrations examined. These results indicate that IGFs may be playing an important role in the early stages of bone repair by stimulating osteoblast chemotaxis to the repair site.

Animals↗
PubMed1993

Cysteine-proteinase localization in osteoclasts: an immunocytochemical study.

Cysteine-proteinases such as cathepsin B and G were localized in rat osteoclasts, by an indirect protein A-immunogold labeling technique, on post-embedded ultrathin sections. In osteoclasts, specific immunogold labeling of both anti-cathepsin B and G was localized in Golgi vesicles, lysosomes, pale vacuoles of various sizes, and the extracellular canals of ruffled borders; no immunoreactivity was seen in the cytoplasmic matrix, mitochondria, cisterns of the rough endoplasmic reticulum, or nuclei. The presence of immunolabeling of cathepsins in osteoclasts and in the subosteoclastic compartment suggests that these enzymes are involved in the extracellular degradation of collagen and other non-collagenous bone matrix proteins.

Animals↗
PubMedDate not supplied

Chondrocyte-to-osteocyte transformation in grafts of perichondrium-free epiphyseal cartilage.

When perichondrium-free pieces of embryonic quail epiphyseal cartilage are incubated on the chorioallantoic membranes of chick embryos, 2 developmental changes are observed. First, most grafts develop a periosteum in which the osteoblasts and osteocytes are of donor, i.e., chondrocytic origin. No such periosteum is observed around explants of demineralized, inductive bone matrix. Second, the matrix surrounding some chondrocytes within the original graft became more bone-like with respect to staining pattern, birefringence and collagen morphology. We conclude that, under some conditions, the avian chondrocyte may in situ or subsequent to release from the cartilage lacuna synthesize a bone-like matrix and, in this sense, be thought to have undergone a "transformation" into an osteocytic or osteoblastic type of cell.

Allantois↗
PubMed2004

Comparative clinical evaluation of combination anorganic bovine-derived hydroxyapatite matrix (ABM)/cell binding peptide (P-15) and open flap debridement (DEBR) in human periodontal osseous defects: a 6 month pilot study.

A synthetic cell binding peptide (P-15) combined with anorganic bovine-derived hydroxyapatite bone matrix (ABM) was compared to open flap debridement (DEBR) in human periodontal osseous defects. Following initial preparation procedures, two osseous defects per patient were treated randomly with one of the two procedures after surgical preparation. Appropriate periodontal maintenance schedules were followed, and at 6 months clinical and radiographic assessments for soft tissue and hard tissue were performed for documentation and finalization of treatment. Statistical analysis using Student's paired t-test analyses of patient mean value from 10 patients revealed that the combination ABM/P-15 grafts demonstrated significantly better mean defect fill of 3.4+/-1.7 mm (73.2%) versus a mean defect fill of 0.6 mm (15.8%) for defects treated with DEBR. Soft tissue findings showed significant differences among treatments with ABM/P-15 compared to DEBR. These results suggest that the use of the P-15 synthetic cell-binding peptide combined with ABM yields better clinical results than DEBR.

Adult↗
PubMed2002

Human segmental mandibular defects treated with naturally derived bone morphogenetic proteins.

Bone induction with extracted and partially purified, naturally derived bone morphogenetic proteins (BMPs) has been demonstrated repeatedly in heterotopic and orthotopic sites of non-human primates. This spawned the investigation of bone regeneration in mandibular defects of human patients with naturally derived BMPs and was compared with osteogenesis in patients treated with autologous bone grafts (ABGs). The osteogenic device (OD) was formulated as a combination of human demineralized bone matrix as delivery system reconstituted with naturally derived BMPs. BMPs were extracted from bovine bone with chaotropic agents and purified by sequential chromatography. Thirteen patients with segmental mandibular defects were enrolled in the trial, 6 of whom received the OD and 7 the ABGs. Defects were reconstructed with a preformed titanium mesh. The OD was combined with sterile saline and applied to the defects as a paste. Autologous bone from the iliac crest was prepared as a cortico-cancellous bone graft and loaded into the titanium mesh. Patients were followed-up clinically and radiographically at 1 and 6 weeks, 3, 6, and 12-month post-implantation. A trephine biopsy of the implants was performed at 3 months post-implantation and the specimens examined on serial undecalcified sections. Histological examination showed that the OD induced bone in 2 of 6 patients treated. Histological examination of successful implanted OD exhibited mineralized bone trabeculae with copious osteoid seams lined by contiguous osteoblasts. Bone deposition directly onto non-vital matrix provided unequivocal evidence of osteoinduction. Of the 7 patients grafted with ABGs, 5 had histological evidence of osteogenesis. Morphometric analysis of the histological sections showed that, when successful, OD-treated defects had highly active osteogenesis compared with ABGs. Whilst this trial provides valuable insights for the use of BMPs in mandibular reconstruction further work is required to produce an OD that will perform reliably in clinical contexts.

Adolescent↗
PubMed1999

[EHBMP-2. Initial BMP analog with osteoinductive properties].

For the first time a non natural BMP-variant (EHBMP-2) with osteoinductive properties was produced by expression in E. coli through specific mutation of the amino acid sequence. The substitution of 12 N-terminal amino acids by a nonsense sequence results in a neglectible affinity of EHBMP-2 to the extracellular matrix. In vitro EHBMP-2 induces dose-dependent cartilage formation in neonatal muscle tissue. Single intramuscular implantation in mice results in the formation of an ossicle with functional active bone marrow. The size of the ossicle depends on the amount of implanted EHBMP-2 and can significantly be increased by the combination with a collagen carrier. The largest bone formation is observed after injection of EHBMP-2 containing collagen suspensions. In rats a stronger osteoinductive activity can be achieved by coupling of EHBMP-2 to collagen discs than by coupling natural BMP-2 to the same collagen carrier. Critical size defects in rats' mandibular angels can be restored by the combination of granular collagenous bone matrix (ICBM) with EHBMP-2. Further investigations have to show whether the altered pharmacokinetics of EHBMP-2 has advantages regarding its therapeutical use and tissue-engineering.

Amino Acid Substitution↗
PubMed1998

Bone loss (osteopenia) in old male mice results from diminished activity and availability of TGF-beta.

One of the universal characteristics of the long bones and spines of middle-age and older mammals is a loss in bone mass (osteopenia). In humans, if this bone loss is severe enough, it results in osteoporosis, a skeletal disorder characterized by a markedly increased incidence of fractures with sequelae that may include pain, loss of mobility, and in the event of hip fracture, even death within a relatively few months of injury. An important contributing factor to the development of osteoporosis appears to be a diminution in the number and activity of osteoblasts responsible for synthesizing new bone matrix. The findings in the present and other similar studies suggest that this reduction in osteoblast number and activity is due to an age-related diminution in the size and osteogenic potential of the bone marrow osteoblast progenitor cell (OPC or CFU-f) compartment. We previously postulated that these regressive changes in the OPC/CFU-f compartment occurred in old animals because of a reduction in the amount and/or activity of TGF-beta1, an autocrine growth factor important in the promotion of OPC/CFU-f proliferation and differentiation. In support of this hypothesis, we now report that (1) the osteogenic capacity of the bone marrow of 24-month-old BALB/c mice, as assessed in vivo, is markedly reduced relative to that of 3-4-month-old animals, (2) that the matrix of the long bones of old mice contains significantly less TGF-beta than that of young mice, (3) that OPC's/CFU-f's isolated from old mice produce less TGF-beta in vitro than those recovered from young mice, and (4) that OPC's/CFU-f's from old mice express significantly more TGF-beta receptor (Types I, II, and III) than those of young animals and that such cells are more responsive in vitro to exogenous recombinant TGF-beta1. We also find that colony number and proliferative activity of OPC's/CFU-f's of young mice and old mice, respectively, are significantly reduced when incubated in the presence of neutralizing TGF-beta1 antibody. Collectively, these data are consistent with the hypothesis that in old male mice the reduction in the synthesis and, perhaps, availability from the bone matrix of TGF-beta1 contributes to a diminution in the size and development potential of the bone marrow osteoprogenitor pool.

Aging↗
PubMed1977

Plutonium in bone: a high resolution autoradiographic study using plutonium-241.

Plutonium-241 citrate solution at pH 6-5 was injected intravenously into hamsters and an adult rabbit at a dose of 10 kBq g-1 (260 nCi g-1). The hamsters were killed serially at 15 min, 2 hours, 1 day, 10 days, 1 month and 6 months after injection and the rabbit at 1 week. Their knee-joints or femora were examined for plutonium-241 by autoradiography. Few differences were found between the pattern of plutonium distribution in the hamsters and the rabbit. The results showed that although plutonium is initially distributed on bone surfaces, at long periods after injection it becomes deposited throughout the bone matrix. Plutonium uptake by cells in resorbing areas of periosteum, in active osteoblasts, and in chondrocytes in regions of cartilage mineralization was rapid. Plutonium concentrated more slowly on the resting bone surfaces and at sites of low metabolic activity. In addition, some unlabelled sections of skeletal tissues were immersed in a plutonium-241 citrate solution. When autoradiographed, it was found that plutonium was bound by cell nuclei, tooth enamel matrix, dentine, predentine and bone matrix. Plutonium binding to cartilage matrix was weak. The results are discussed with reference to the literature, and a model is proposed to explain the distribution pattern and fate of plutonium deposits in bone.

Animals↗
PubMed1992

Molecular cloning and sequencing of cDNA encoding urinary stone protein, which is identical to osteopontin.

We have sequenced a cDNA of urinary stone protein. cDNA sequences show complete homology between urinary stone protein and human osteopontin (bone sialoprotein) (nucleotides 265-886 and 1183-1424). Osteopontin is a recently discovered bone matrix protein which has been implicated in mediating mineral formation within bone extracellular matrix. This result shows that osteopontin is presumably involved in stone formation as stone matrix.

Base Sequence↗
PubMed1983

Parameters influencing the sonic velocity in compact calcified tissues of various species.

Compact calcified tissues from a wide variety of species were used in a study of the dependence of sonic plesio-velocity on physical parameters. A linear dependence of velocity on wet density has been found for each of three categories of wet mineralized tissue: compact long bone measured in the axial direction, compact long bone measured in the radial direction, and hyperpycnotic mineralized tissues. A similar linear dependency was found for dry calcified tissue using the dry density. In addition to these three parameters (density, orientation, and water content) two other factors were identified. The bone fibers in long bone matrix are ordered with respect to the bone axis and the anisotropy of long bone matches that of its matrix. There is no corresponding order to the fibers in hyperpycnotic tissue matrix. The fifth parameter is believed to be the porosity. Fish bone is much more porous than other compact bone from long bone and the sonic velocity in fish bone is much lower than for other bone. These parameters are not independent.

Acoustics↗
PubMed2004

Osteopontin distribution in the canine skeleton during growth and structural maturation.

The distribution of osteopontin (OPN) was studied immunohistochemically in cells and extracellular matrix in the humerus, scapula, and lumbar vertebrae of growing (age: 6 weeks, 12 weeks, 4.5 months) and adult dogs. OPN was expressed in hypertrophic chondrocytes of epiphyseal cartilage and in chondrocytes of the deep zone of mature articular cartilage, where extracellular matrix was also stained. OPN expression was strongest in 4.5-month-old puppies in cells of the osteoblastic lineage. It also varied with microlocation and was pronounced in areas prone to resorption due to modelling and remodelling activities. Osteoclasts were always strongly labelled with OPN. OPN deposition in extracellular bone matrix was detected particularly as a delineation of cartilage cores within secondary trabeculae and as a lining of the trabecular surfaces in resorption microlocations. The OPN distribution pattern is discussed here for each cell population with regard to its functional implications.

Aging↗
PubMed2004

Tissue-engineered bone for maxillary sinus augmentation.

PURPOSE: Autologous, allogenic, and alloplastic materials for bony reconstruction in the craniomaxillofacial region have specific drawbacks stimulating the ongoing search for new materials. Cultivated skin and mucosa grafts are in clinical routine use in head and neck reconstruction but so far, to the best of our knowledge, no successful clinical application has been described of periosteum-derived tissue-engineered bone for augmentation of the edentulous posterior maxilla. PATIENTS AND METHODS: In a clinical study, augmentation of the posterior maxilla was carried out using a bone matrix derived from mandibular periosteum cells on an Ethisorb (Ethicon, Norderstedt, Germany) fleece. In this report, we show the fabrication of the matrix, clinical application, and results in 27 patients. RESULTS: In 18 patients, an excellent clinical, radiologic, and histologic result could be proved 3 months after augmentation. Histologically, the bone biopsy samples from these patients revealed mineralized trabecular bone with remnants of the biomaterial. An unsuccessful result was found in 8 cases with a more extended augmentation procedure. The clinical inspection 3 months after augmentation showed almost no formation of new bone. In contrast, a replacement resorption with connective tissue was found. This may be the result of failure of the initial supply of the cells embedded within large cell-polymer constructs with sufficient oxygen and nutrients to sustain their survival and proliferation and allow for the integration of the developing tissue within the surrounding tissue. CONCLUSION: Our achieved results suggest that periosteum-derived osteoblasts on a suitable matrix can form lamellar bone within 3 months after transplantation and provide a reliable basis for simultaneous or secondary insertion of dental implants.

Alveolar Ridge Augmentation↗