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Biochemical anatomy of human bone: comparative study of compact and spongy bone in femur, rib and iliac crest.

Using EDTA extraction procedure, compact and spongy bone from human femur, rib and iliac crest were compared in terms of their content in collagen, sialoprotein, proteoglycan and carbohydrate. The bone matrix sizes displayed significant variations, the femur having the smallest size and iliac crest the largest one. No significant difference in the matrix size has been found between the spongy and compact bone. The EDTA extractability of the spongy bone was higher than that of the compact bone, with femur showing the lowest extractability. The collagen content of the 3 bones studied was similar although the femur had slightly lower values. The sialic and uronic acids and hexose contents were higher in the femur than in the rib and iliac crest. The collagen/hexose, collagen/sialic acid and collagen/uronic acid ratios in the bone matrix were highest in the iliac crest and lowest in the femur, suggesting that alterations in the amounts of bone matrix can affect the mechanical properties of different parts of the bony skeleton and vice versa.

Bone and Bones↗

The isolation and partial characterization of a rat incisor dentin matrix polypeptide with in vitro chondrogenic activity.

In vivo implants of demineralized dentin matrix into muscle induce the formation of bone within the muscle. As with bone matrix implants, the bone induction appears to follow a chondrogenic pathway. Outgrowth cells from explants of neonatal rat muscle respond to bone matrix, in vitro, by expressing a heightened synthesis of sulfated proteoglycans and type II collagen, phenotypic of cartilage. The in vitro cell culture system has been used as an assay to monitor the isolation of the factor responsible for expression of this phenotypic transformation. Soluble proteins extracted from rat incisor dentin matrix during demineralization with EDTA, and not precipitable with 1.0 M CaCl2, were active in the in vitro system. The active extract was fractionated by Sephacryl S-100 chromatography in 6 M guanidine HCl, isoelectric focusing in Immobilines, and by reverse phase high performance liquid chromatography. All fractions were assayed for activity at every stage. The final active fraction from the reverse phase chromatography on a Zorbax Poly-F column was purified to homogeneity, and yielded a single spot on two-dimensional gel electrophoresis. The component, RP-4, had pI 5.4-5.5, and an apparent Mr 6,000-10,000, based on globular protein standards. Maximal activity with respect to both sulfate incorporation into proteoglycan and production of type II collagen was in the 1.0-10 ng/ml range. The RP-4 had a unique amino-terminal amino sequence and was rich in Gly, Pro, Glx, and Ala residues. It was different from transforming growth factor-beta and the bone morphogenetic protein family of proteins in these essential features.

Amino Acid Sequence↗

Bone formation via cartilage models: the "borderline" chondrocyte.

Increasing evidence substantiates the view that death is not necessarily the only fate of hypertrophic chondrocytes and that, when exposed to the right microenvironment, these cells can further differentiate to osteoblast-like cells and contribute to initial bone formation. In vitro, when replated as adherent cells in the presence of ascorbic acid, hypertrophic chondrocytes resume cell proliferation, switch from the synthesis of the cartilage-characteristic type II and X collagens to the synthesis of type I collagen, and organize a mineralizing bone matrix. In vivo, expression of bone specific markers by growth plate chondrocytes occurs initially in early hypertrophic cells located at the mid-diaphysis and directly facing the osteogenic perichondrium. In bones formed via cartilage models, the first mineralized bone matrix (the earliest bony collar preceding vascular invasion and the onset of endochondral bone formation) is deposited at the outer aspect of the mid-diaphysis between rows of early hypertrophic chondrocytes and osteoblasts, which are arranged in a peculiar "vis à vis" fashion. The "vis à vis" organization of perichondrial osteogenic cells and peripheral early hypertrophic chondrocytes suggests that the latter cells are exposed -- compared to their cognate, the central hypertrophic chondrocytes -- to a specific microenvironment composed of unique matrix-originating signals and cellular cross-talks. A major role in the differentiation control of, and interaction between, hypertrophic chondrocytes and osteogenic perichondrial cells is certainly played by the Indian Hedgehog/PTHrP signalling system. We propose that all early hypertrophic chondrocytes have the inherent potential to differentiate to osteoblast-like cells and to contribute to initial bone formation, but that only chondrocytes positioned at the "borderland" between cartilage and (non-cartilage) osteogenic tissues undergo further differentiation to bone producing cells. We call these hypertrophic chondrocytes "borderline chondrocytes" to emphasize both their specific location and their dual differentiation potential. Hypertrophic chondrocytes located in different cartilage areas are exposed to an inappropriate matrix and endocrine/paracrine environment, cannot differentiate to osteoblast-like cells and therefore undergo apoptosis.

Animals↗

An EDTA-KOH method to expose bone cells for scanning electron microscopy.

To observe bone cells by scanning electron microscopy (SEM), the mouse parietal bones were processed by decalcification with EDTA and digestion of collagen fibers with KOH to remove the bone matrix, in addition to the conventional preparation for SEM. The critical-point-dried specimens were split into two membranous pieces along the gaps formed by removing the bone matrix. By this method, osteoclasts showing full three-dimensional images of ruffled borders, osteoblasts showing special structures on the surfaces facing the bone matrix, and osteocytes extending many slender processes were clearly demonstrated in SEM. This new method may provide new viewpoints in bone cell biology.

Animals↗

Incorporation of nonviable bone grafts. Autoclaved autogeneic and frozen allogeneic bone grafts compared in the rabbit.

In 14 adult rabbits the middle third of the ulna was resected bilaterally followed by reimplantation of resected bone after autoclaving on one side and transplantation of allogeneic bone on the other. In 7 animals the bilateral implants were supplemented with allogeneic bone matrix. The reconstructions were studied in vivo by serial radiography, scintigraphy, and bone mineral determination. The animals were killed at 16 weeks, and the ulnar reconstructions further studied by high resolution radiography, 45Ca autoradiography, and histology. In both types of nonsupplemented reconstructions, new bone formation was poor; nonunion occurred in three out of seven autoclaved reimplants and in five out of seven allogeneic transplants. Supplemented with allogeneic bone matrix, both types of reconstructions exhibited abundant new bone formation and complete incorporation of all implants. Enhancement of new bone formation is probably more important than the type of nonviable bone graft chosen for reconstruction of large skeletal defects.

Animals↗

Effect of microcapsules containing TAK-778 on bone formation around osseointegrated implants: histomorphometric analysis in dogs.

PURPOSE: The aim of this study was to evaluate the in vivo effect of TAK-778 on osseointegration of titanium implants. MATERIALS AND METHODS: Mandibular premolars were extracted from 8 dogs. After 3 months, 2 titanium implants were bilaterally placed, and each implantation site randomly received 1 of the following treatments: sustained-release microcapsules of TAK-778, placebo microcapsules, or no treatment. At 8 and 12 weeks after implantation, the hemi-mandibles containing the implants were removed, and processed for morphologic and histomorphometric analysis. Data were submitted to 2-way analysis of variance. RESULTS: The histologic sections of the 3 experimental groups at 8 and 12 weeks did not show morphologic differences related to applied treatment. The percentage of bone-implant contact, mineralized bone matrix between implant threads, and mineralized bone matrix within mirror area were not affected either by treatments or evaluated periods. CONCLUSIONS: No effect of TAK-778 was observed on osseointegration of titanium implants, which most likely occurred because microcapsules may not be retained and, therefore, available at the implant sites. An alternative is the manufacture of a release system, which can be immobilized on implant surface, ensuring the drug permanence in the implant site at least at the initial periods of bone formation.

Animals↗

Age-related changes in insulin-like growth factor I and II in human femoral cortical bone: lack of correlation with bone mass.

The concentration of insulin-like growth factor-I (IGF-I) in human cortical bone declines with age, but the relevance of this decline for cortical bone turnover and bone mass is unknown. In the present study, we simultaneously assessed the concentration of IGF-I and -II in cortical bone matrix and histomorphometric parameters of bone mass and bone turnover in 125 samples from the proximal human femur shaft. Bone width decreased by 27% and porosity increased by 100% in female cortical bone between the fourth and the ninth decade. Similar, but weaker, changes tended to occur in male cortical bone. The concentrations of both IGF species were correlated with the percentage of osteons undergoing bone remodeling. However, despite age-related decreases in both IGF species in men and in IGF-I in women, neither of the IGFs accounted for age-related or age-independent variability in cortical porosity or bone width. In conclusion, these data suggest that the local concentrations of IGF-I and -II are related to cortical bone turnover. In contrast, our study provides no evidence for a major role of bone matrix IGF-I and -II as determinants of cortical bone mass in elderly individuals. Whether other components of the IGF system may be stronger determinants of cortical bone loss remains to be determined.

Adult↗

Noncollagenous proteins of a rat dentin matrix possessing bone morphogenetic activity.

An insoluble preparation of rat dentin matrix was shown to possess bone morphogenetic protein (BMP) activity, i.e. the capacity to induce the formation of catilage and bone when implanted intramuscularly. Since BMP activity was previously attributed to noncollagenous proteins (NCP) of bone and dentin, the nature of NCP of the rat dentin was examined. After treatment of the matrix with purified bacterial collagenase, three NCP were solubilized concomitantly with digestion of the dentin collagen to smaller peptides. The three proteins were separated by anion-exchange chromatography on DEAE-cellulose. Two of the NCP were rich in asparate, glutamate, glycine, serine, and alanine, and thus displayed compositions similar to acidic proteins of other connective tissues. The third NCP was shown by amino acid composition to be the aspartate, serine-rich phosphoprotein, which occurs mostly in a soluble form in rat dentin. This observation supports the view that a portion of dentin phosphotprotein is firmly bound.

Amino Acids↗

Demineralization for inactivation of infectious retrovirus in systemically infected cortical bone: in vitro and in vivo experimental studies.

BACKGROUND: Clinical and experimental studies have demonstrated viral transmission through the transplantation of fresh-frozen infected bone. While sterilization methods sufficient to inactivate the human immunodeficiency virus (HIV) have been shown to markedly alter osteoconductive and osteoinductive properties of bone allografts, the ability of a process for creating demineralized bone matrix to abrogate transmission of a retrovirus has not been investigated, to our knowledge. We hypothesized that a clinically accepted demineralization procedure would alter the nucleic acids of the feline leukemia virus (FeLV, a retrovirus with a structure and replication cycle similar to those of HIV), inactivating the virus in infected bone and rendering it noninfectious. METHODS: Bone infected with FeLV was demineralized with a method employed for creating demineralized bone matrix powder. The effects of demineralization on cellular and (pro)viral nucleic acids were examined with use of gel electrophoresis and quantitative polymerase chain reaction, respectively. To compare the infectivity of the demineralized bone matrix with that of mineralized bone particles in cell cultures and in animals in which they had been implanted, we measured FeLV p27 antigen and (pro)viral nucleic acids as well as antiviral antibodies. RESULTS: Demineralization of FeLV-infected bone appeared to inactivate the virus by degradation and fragmentation of the DNA, rendering it noninfectious in both in vitro and in vivo test systems. In contrast, untreated mineralized FeLV-infected bone contained intact nucleic acids and readily transmitted the virus in both test systems. CONCLUSIONS: The demineralization process inactivated infectious retrovirus in infected cortical bone, thereby preventing disease transmission.

Animals↗

Changes in subchondral bone in cartilage resurfacing--an experimental study in sheep using different types of osteochondral grafts.

OBJECTIVE: This article addresses the subchondral bone integrity in cartilage resurfacing by comparing fresh, untreated auto-, xeno-, and photooxidized osteochondral allo- and xenografts. Photooxidation was expected to improve mechanical stability of the osteochondral grafts through an improved linkage of the collagen fibers within the bone matrix. DESIGN: Untreated auto- and xenografts and with photooxidation pretreated allo- and xenografts were surgically implanted in femoral condyles of sheep (n=40). After 2, 6, 12 and 18 months results were evaluated histologically using non-decalcified bone embedded in acrylic resin. Qualitative evaluation was performed with emphasis on bone matrix, biomechanical stability of graft anchorage, formation of cystic lesions, and bone resorption and formation. Quantitative evaluation of the total subchondral bone area was conducted histomorphometrically. Statistical analysis (factorial ANOVA test) was used to compare differences between groups with respect to the percentage of bone matrix and fibrous tissue per section. RESULTS: Subchondral bone resorption was fastest in untreated, fresh autografts, followed by photooxidized allografts, untreated, fresh xenografts and last pretreated photooxidized xenografts. Cystic lesions were seen in all types of grafts, but were most pronounced at 6 months in autografts and least in photooxidized grafts. Cyst-like lesions had subsided substantially in the untreated auto- and photooxidized xenografts, if no graft dislocation occurred during the healing period. Mononuclear cell infiltration and an increase in the presence of multinuclear cells were observed at 2 months, mostly in untreated autografts, followed by photooxidized allo- and untreated xenografts. They were much higher in numbers compared to photooxidized grafts, at least in the early specimens at 2 months. Graft stability was linked to the rate of bone resorption. CONCLUSION: Substantial resorption of the subchondral bone, involving the development of cyst-like lesions, lead to dislocation and finally to cartilage matrix degradation of the grafts. The process of photooxidation decreased the speed of bone resorption in osteochondral grafts and, thus, improved graft stability and cartilage survival. These results suggest that the remodeling of the subchondral bone of the host and the graft within the first 6 months is an important factor in graft stability and overall results of cartilage resurfacing.

Animals↗

Immunoelectron microscopic studies of glycosaminoglycans in the metaphyseal bone trabeculae of growing rats.

The types and distribution of glycosaminoglycans (GAGs) were studied immunocytochemically in osteoid, mineralized bone matrix, and cartilage matrix of growing rat metaphyseal bone after aldehyde fixation and EDTA demineralization, using four monoclonal antibodies (mAbs 1-B-5, 2-B-6, 3-B-3 and 5-D-4). These mAbs specifically recognize epitopes in non-sulphated chondroitin (C0-S); chondroitin 4-sulphate (C4-S) and dermatan sulphate (DS); chondroitin 6-sulphate (C6-S) and C0-S; and keratan sulphate (KS) respectively. In osteoid, all mAbs except 1-B-5 weakly stained matrix material on and between collagen fibrils, and moderately stained organic material corresponding to bone nodules, which are known sites of mineralization. However, the staining of osteoid abruptly decreased at the mineralization front; weak staining was confined mostly to the organic material of bone nodules in mineralized bone matrix, with very weak or no staining of the rest of the bone matrix. This staining progressively decreased toward the mineralized cartilage matrix and became negative. The mineralized cartilage matrix and lamina limitans reacted strongly with all mAbs except 5-D-4. These results indicate that osteoid contains sulphated proteoglycans containing C4-S and/or DS, C6-S and KS, and subsequent bone matrix mineralization appears to require accumulation of these macromolecules within bone nodules and eventual loss of these substances for complete mineralization, whereas proteoglycans containing C0-S, C4-S and/or DS, and C6-S still exist in mineralized cartilage matrix and lamina limitans.

Animals↗

Lytic bone lesions in human neuroblastoma xenograft involve osteoclast recruitment and are inhibited by bisphosphonate.

Neuroblastoma is the second most common solid tumor in childhood and frequently metastasizes to the bone marrow and the bone matrix. The mechanism involved in bone metastasis and destruction in neuroblastoma is poorly understood. Using a model of bone invasion in immunodeficient mice, we demonstrated that neuroblastoma cells recruited osteoclasts to generate osteolytic lesions and invade the bone matrix. In further support of a contributory role for osteoclasts in neuroblastoma bone invasion, we demonstrated that treatment with the bisphosphonate compound, ibandronate, significantly delayed the progression of osteolytic lesions. The data suggest that bisphosphonates may be clinically effective in the treatment of bone metastases in neuroblastoma.

Adrenal Glands↗

Localisation and distribution of hyaluronan in normal bone marrow matrix: a novel method to evaluate impending fibrosis?

Bone marrow trephine biopsies from 30 healthy volunteers, 10 men and 20 women aged 18-60 yr were obtained for identification and localisation of hyaluronan (HYA). Fixation, decalcification and embedding were performed by two different methods, with identical results in both. For comparison bone marrow trephine biopsies from three patients with different haematological diseases and known fibrosis were studied. All bone marrow specimens were also stained for reticulin grading. HYA was found in the bone marrow specimens from healthy individuals in a pattern that was concordant with the reticulin staining, the common way of visualising bone marrow fibrosis. In bone marrow from the patients with known fibrosis the HYA and reticulin staining were both more intense and abundant. Interestingly, HYA was also found intracellularly in eosinophilic cells in normal bone marrow. HYA is a polysaccharide unique both in structural and biological properties, and in excess it may predict bone marrow fibrosis.

Adolescent↗

Immunocytochemical study of cathepsin L and rat salivary cystatin-3 in rat osteoclasts treated with E-64 in vivo.

The localization of cathepsin L and rat salivary cystatin-3 (RSC-3) in rat osteoclasts (rat femoral and alveolar bones) treated with or without E-64 (control) was examined immunocytochemically. In osteoclasts pretreated with E-64, immunoreactivity for cathepsin L was very weak extracellularly compared to that in the control osteoclasts. However, it was strong intracellularly. The localization of RSC-3 was unclear in the control osteoclasts, while in E-64 treated osteoclasts, both the clear zone and ruffled border areas showed a very strong immunoreaction. At the electron-microscopic level, in normal osteoclasts, numerous immunoreaction products for cathepsin L were found extracellularly in the bone matrix under the ruffled border, while few intracellular products were observed. In contrast, in the E-64-treated osteoclasts, only a few immunoreaction products were found extracellularly, while intracellularly cathepsin L was found in numerous endosome-lysosomal vacuoles. In the immunoreaction for RSC-3, the cytoplasm of the ruffled border was positive, and the tips of the RSC-3-positive ruffled border appeared to enter deeply into the bone matrix. Intracellularly, the granular reaction products of RSC-3 were found in the vacuoles (probably autophagolysosomes). Thus, in E-64-treated osteoclasts, inhibition of the extracellular release of cathepsin L was demonstrated. In addition, intralysosomal accumulation of RSC-3 and deep penetration of the RSC-3-positive ruffled border into the bone matrix were found. These findings suggest that RSC-3 is associated with the inhibition of cathepsin L in both the lysosomes (in the osteoclasts) and bone matrix.

Animals↗

Effect of bone matrix-derived growth factors on skull and tibia in the growing rat.

Aberration in cranial skeletal morphology during growth due to metabolic bone diseases associated with hypocalcemia is related to abnormalities in cranial suture growth. It was found in previous experimental studies that a changed cranial growth pattern induced by hypocalcemia and secondary hyperparathyroidism was dependent on a decreased bone apposition in viscerocranial growth sites. Of interest was the observation that obliterative osteogenesis occurred with this disturbance. In vitro studies have shown that when bone resorption is stimulated by parathyroid hormone, mitogenic factors for bone cells are released from the matrix. The aim of the current study was to elucidate a possible local regulatory mechanism for bone growth especially with regard to cranial growth. Two groups of young rats were injected daily with 150 micrograms and 750 micrograms respectively of bone matrix-derived growth factor preparation (BMDGF) for 2 weeks. By using cephalometric analysis, the BMDGF administration was shown to alter skull morphology during growth. Since the high doses of BMDGF caused markedly more changes than the low dose, the overall effect on craniofacial growth seemed to be dose dependent. The high dose of BMDGF induced a decrease in cortical bone apposition found in this region, as well as in the diaphysis of the tibia. Also a change in position of the viscerocranium in relation to the cranial base was found in the BMDGF-treated rats: the flexure between the viscerocranium and the cranial base being greater compared with the normal skull. This change in relationship seemed to be related to the enhanced bone apposition found in the viscerocranial sutures of BMDGF-treated rats.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

Buried alive: how osteoblasts become osteocytes.

During osteogenesis, osteoblasts lay down osteoid and transform into osteocytes embedded in mineralized bone matrix. Despite the fact that osteocytes are the most abundant cellular component of bone, little is known about the process of osteoblast-to-osteocyte transformation. What is known is that osteoblasts undergo a number of changes during this transformation, yet retain their connections to preosteoblasts and osteocytes. This review explores the osteoblast-to-osteocyte transformation during intramembranous ossification from both morphological and molecular perspectives. We investigate how these data support five schemes that describe how an osteoblast could become entrapped in the bone matrix (in mammals) and suggest one of the five scenarios that best fits as a model. Those osteoblasts on the bone surface that are destined for burial and destined to become osteocytes slow down matrix production compared to neighbouring osteoblasts, which continue to produce bone matrix. That is, cells that continue to produce matrix actively bury cells producing less or no new bone matrix (passive burial). We summarize which morphological and molecular changes could be used as characters (or markers) to follow the transformation process.

Animals↗

Osteoblasts: novel roles in orchestration of skeletal architecture.

Osteoblasts are located on bone surfaces and are the cells responsible for bone formation through secretion of the organic components of bone matrix. Osteoblasts are derived from mesenchymal osteoprogenitor cells found in bone marrow and periosteum. Following a period of secretory activity, osteoblasts undergo either apoptosis or terminal differentiation to form osteocytes surrounded by bone matrix. Osteoblasts secrete a characteristic mixture of extracellular matrix proteins including type I collagen as the major component as well as proteoglycans, glycoproteins and gamma-carboxylated proteins. Cells of the osteoblast lineage also provide factors essential for differentiation of osteoclasts (bone-resorbing cells). By regulating osteoclast differentiation and activity in response to systemic influences, osteoblasts not only play a central role in regulation of skeletal architecture, but also in calcium homeostasis. Inadequate osteoblastic bone formation in relation to osteoclastic resorption results in osteoporosis, a disease characterised by enhanced skeletal fragility. Cellfacts: Osteoblasts are the cells responsible for bone formation. Osteoblasts indirectly control levels of bone resorption. Osteoblasts play a key role in the pathophysiology of osteoporosis and the resulting fractures, which constitute a major public health burden in developed countries.

Animals↗

Influence of indomethacin on induced heterotopic bone formation in rats. Importance of length of treatment and of age.

The effect of indomethacin on heterotopic and orthotopic bone formation in rats was analyzed with respect to (1) length of treatment after implantation, (2) duration of the indomethacin induced inhibition of heterotopic bone formation, and (3) influence of age of the implant recipient. Three weeks after implantation of demineralized bone matrix, the ash weight of implants from rats receiving indomethacin 2 mg/kg body weight during the entire experiment was 31% lower than that of controls. Animals treated for only six days after implantation exhibited an almost equally pronounced inhibition. However, six weeks after implantation, the inhibition caused by six days of indomethacin treatment had almost dissipated. In older rats the implants of demineralized bone matrix induces smaller volumes of new bone than in younger rats, but indomethacin causes approximately the same degree of inhibition of osteoinduction. Orthotopic bone is not affected by indomethacin treatment. This study shows that a short period of indomethacin treatment at the time of implantation of demineralized bone matrix is sufficient to reduce experimental bone formation, but the inhibitory effect slowly diminishes if the inductive process is continuous. The results indicate that the inhibition of heterotopic new bone formation by indomethacin may be mediated through reduction of the initial inflammatory response or by reduced mesenchymal cell proliferation.

Aging↗