Surface characterization of the raw and cooked bovine cortical metatarsal bone.

PURPOSE: The purpose of this study was to quantify the elastic properties and evaluate microscopical features of raw and boiled metatarsal bovine bone. METHODS: The elastic modulus, hardness and microscopic surface of raw and cooked bovine metatarsal bone have been investigated using nanoindentation, SEM/EDX and Panasis microscope. RESULTS: Regarding raw bovine bone, the average elastic modulus was 30.515 ± 6,769 GPa, while the average hardness was 0.5683 ± 0.211 GPa. When it comes to boiled bone corresponding values were 22.298 ± 7.0303 GPa and 0.408 ± 0.199 GPa, respectively. The values for investigated parameters were significantly higher (p < 0.05) in raw bone specimens. Elastic modulus significantly correlated with hardness (p < 0.05). EDX analysis revealed significant decrease in wt% of oxygen in boiled samples (p < 0.05) No significant differences could be observed in SEM images particularly when analysing in smaller magnifications. Using higher magnification, additional branching of the existing voids as well as discrete reorganization and smoother edges of nutrient canals could be observed. The surface of boiled specimens was without the presence of crusts and layering, and no microscopical evidence of structural damage could be observed. CONCLUSIONS: This study provides detailed analysis of hardness, elastic modulus of raw and cooked bovine bone and their relation and changes during exposure to temperature. These results of elastic moduli and hardness could be comparable to similar studies of bovine and human bone tissue, but the careful analysis of experimental design, type of the bone as well as limitations of the employed techniques must be carried out before interpolation of the results to other theoretical, clinical, biomaterial and archeological studies.

The effect of hydrochloric acid on microstructure of porcine (Sus scrofa domesticus) cortical bone tissue.

We evaluated the degradation of cortical bone tissue by hydrochloric acid (HCl) since intentional bone decalcification in a forensic context has not been studied on a histomorphological level. We used 70 pig metatarsal bones split into subsamples and immersed in one of three concentrations of acidic solutions (0.5M, 1M, 2M HCl) for two and four hours. We analyzed the cortical thicknesses on transversal cross-sections, thicknesses of the three histomorphologically distinct zones present in acid-immersed bones, and number and area of crystals present in one of the zones. Furthermore, we analyzed the ratio of calcium to phosphorus (Ca:P). We observed a division of the cortical bone cross section into three distinctive zones: demineralized matrix (DM) in the periosteal part of bone, middle contact zone (CZ), and mineralized matrix (MM) in the endosteal part of bone. With increasing acid concentration and time of immersion (from 0.5M HCl for 2h to 2M HCl for 4h), the thickness of DM increased by 67%, the thickness of CZ increased by 56%, and the thickness of MM decreased by 32%. The Ca:P ratio in the contact zone of acid-treated samples did not change significantly with changing acid concentration and time of immersion. The Ca:P ratio of the CZ decreased by 10% when compared to the Ca:P ratio of MM in acid-treated samples. Moreover, we observed crystals on the outer periosteal border of the DM zone, in the CZ, and in the MM Haversian/Volkmann's canals. The size and number of the crystals in the CZ of acid-treated bones increased with acid concentration and time of acid immersion. Moreover, we also observed significant differences in all analyzed properties between anatomical regions. Due to varying reactions to acid immersion among anatomical regions, bone micro-degradation should be observed separately for each region.

Transcriptome analysis of fetal metatarsal long bones by microarray, as a model for endochondral bone formation.

Endochondral bone formation is orchestrated by mesenchymal cell condensation to form cartilage anlagen, which act as a template for bone formation and eventual mineralization. The current study performed gene expression analysis to examine pre- and post-mineralization stages (E15 and E19) of endochondral bone formation, using fetal metatarsal long bones as a model. An extensive number of genes were differentially expressed, with 543 transcripts found to have at least 2-fold up-regulation and 742 with a greater than 2-fold down-regulation. A bioinformatics approach was adopted based on gene ontology groups, and this identified genes associated with the regulation of signaling and skeletal development, cartilage replacement by bone, and matrix degradation and turnover. Transcripts linked to skeletal patterning, including Hoxd genes 10-12, Gli2 and Noggin were considerably down-regulated at E19. Whereas genes associated with bone matrix formation and turnover, ACP5, MMP-13, bone sialoprotein, osteopontin, dentin matrix protein-1 and MMP-9 all were distinctly up-regulated at this later time point. This approach to studying the formation of the primary ossification center provides a unique picture of the developmental dynamics involved in the molecular and biochemical processes during this intricately regulated process.

Osteocyte-bone lining cell system at the origin of steady ionic current in damaged amphibian bone.

A wound-generated steady electric current was measured by a two-dimensional vibrating probe system in the metatarsal bones of 22 adult frogs (Xenopus laevis) placed in amphibian Ringer. Inward currents were recorded entering a micrometric hole drilled through the cortex at middiaphyseal level. These steady state currents (mean +/- SD 8.50 +/- 2.77 microA/cm2) last approximately 2 hours, were dependent on the presence of sodium in the incubation medium, were no more detectable after fixation, and were reduced to background level when the cell membranes were solubilized. These results agree with previous recordings of metatarsal bones of weanling mice, under identical conditions. Both results suggest that the measured ionic currents have a cellular origin. Metatarsal bones of adult amphibian were purposely selected for this study because, unlike mammalian bones, their shafts are avascular and only contain an osteocyte-bone lining cell system, as documented by scanning and transmission electron observations. Thus, unlike the data from previous investigations on mammals, the results succeeded in giving the first convincing evidence that the osteocyte-bone lining cell system is the origin of damage-generated ionic currents. As damage exposes bone ionic compartment to plasma, damage-generated ionic currents are representative of ion fluxes at bone plasma interface, and cells at the origin of the current generate the driving force of such fluxes. By demonstrating that osteocytes and bone lining cells are at the origin of the current, this study suggests that the osteocyte-bone lining cell system, though operating as a cellular membrane partition, regulates ionic flow between bone and plasma. Since strain-related adaptive remodeling could also depend on ionic characteristics and flow of the bone fluid through the osteocyte lacuno-canalicular network, the results reported here support the view that osteocyte and bone lining cells may constitute a functional syncytium involved in mineral homeostasis as well as in bone adaptation to mechanical loading.

Intermittent compressive load stimulates osteogenesis and improves osteocyte viability in bones cultured "in vitro".

The effect of mechanical stresses on osteogenesis, the viability of osteocytes and their metabolic activity in organ culture of bones intermittently loaded "in vitro" are reported. Metatarsal bones, isolated from 12-day-old rats, were cultured in BGJb medium (with 10% foetal calf serum, 75 micrograms/ml of ascorbic acid, 100 U/ml of penicillin and 100 micrograms/ml of streptomycin), in humidified air enriched by 5% CO2 and 30% O2, and loaded in our original device for 1/2 an hour at 1 Hz. homotypic isolated and unloaded bones, cultured in the same medium, were taken as controls. The ALP (alkaline phophatase activity) increases in the media of loaded bones in comparison with the control bones. The percentage of viable osteocytes is significantly greater in loaded than in control bones. TEM observations demonstrate that in both loaded and control unloaded bones, osteocytes show well developed organelle machinery and several gap junctions with adjacent cellular processes. In the cells of loaded bones, however, a higher number of cytoplasmic organelles and gap junctions were found. In particular, RER increases twice, gap junctions three times. The induced osteogenesis and the TEM observations demonstrate the suitability of this experimental model and support the recent advanced hypothesis according to which the mechanical loading may exert a trophic function on osteocytes, stimulating both the proteic synthesis in the above-mentioned cells and the cell-to-cell communication. Furthermore, the loading is likely to exert a biological stimulus on osteoblasts via signalling molecules produced by osteocytes.

Pre-metatarsal skeletal development in tissue culture at unit- and microgravity.

Explant organ culture was used to demonstrate that isolated embryonic mouse pre-metatarsal mesenchyme is capable of undergoing a series of differentiative and morphogenetic developmental events. Mesenchyme differentiation into chondrocytes, and concurrent morphogenetic patterning of the cartilage tissue, and terminal chondrocyte differentiation with subsequent matrix mineralization show that cultured tissue closely parallels in vivo development. Whole mount alizarin red staining of the cultured tissue demonstrates that the extracellular matrix around the hypertrophied chondrocytes is competent to support mineralization. Intensely stained mineralized bands are similar to those formed in pre-metatarsals developing in vivo. We have adapted the culture strategy for experimentation in a reduced gravity environment on the Space Shuttle. Spaceflight culture of pre-metatarsals, which have already initiated chondrogenesis and morphogenetic patterning, results in an increase in cartilage rod size and maintenance of rod shape, compared to controls. Older pre-metatarsal tissue, already terminally differentiated to hypertrophied cartilage, maintained rod structure and cartilage phenotype during spaceflight culture.

Osteoclast development in the coculture system of periostless metatarsal bones and hemopoietic cells studied by in situ hybridization with a probe for Y chromosomes.

In the coculture system of periostless metatarsal bones of 17-day-old fetal mice and osteoclast progenitors, osteoclasts will develop. Our goal in the present report was to provide further evidence that in the coculture system of fetal metatarsal bone rudiments with hemopoietic cells, the osteoclasts developing inside the bone rudiments are exclusively derived from the cells suspended in the plasma clot and not from endogenous precursor cells of the bone explants themselves, by using the technique of in situ hybridization with a probe for the mouse Y chromosome. Osteoclast formation in unstripped male metatarsal rudiments, occurring after 3-4 days of culture, was compared with osteoclast formation in cocultures of female metatarsal rudiments and male bone marrow cells, occurring after 5-6 days of culture. Osteoclasts were recognized by their tartrate-resistant acid phosphatase activity. In paraffin sections of cultured male metatarsals, the mean percentage of microscopically identifiable osteoclast nuclei, in which the Y chromosome could be detected, was 43.1 +/- 4.2% (n = 12). For cocultures of female metatarsal bones and male bone marrow cells this mean percentage was 40.9 +/- 5.7% (n = 17). Statistical comparison by means of the two sample t-test indicated no significant difference in the percentages of osteoclast nuclei containing the Y chromosome for both groups. We concluded that the osteoclasts do derive from cocultured cells and not from precursor cells in the bone explant itself.(ABSTRACT TRUNCATED AT 250 WORDS)

Novel articular cartilage structure in the South American opossum, Monodelphis domestica.

Articular cartilage provides smooth surfaces for low-friction, unrestricted movement of opposing skeletal elements. The surface topography of articular cartilage has been the subject of numerous studies and, with few exceptions, is considered to be smooth (at least at the light microscopic level). Some studies have reported 'humps' on the articular surface which have been related to underlying chondrocytes residing very close to the surface. Here we report on a highly nodular form of articular cartilage in the distal limb joints of the South American opossum, Monodelphis domestica. Unlike previous reports, these articular 'humps' are visible under a dissecting microscope. Each 'hump' or 'nodule' represents the surrounding matrix of single or sometimes paired rounded chondrocytes. Flattened chondrocytes normally associated with mammalian articular cartilage were absent from these joints. Interestingly, the articular cartilage of the more proximal limb joints such as the knee showed more typical features of articular cartilage including flattened superficial chondrocytes.

Effect of intermittent mechanical force on bone tissue in vitro: preliminary results.

The structure of metatarsal bones from 18-day-old rats subjected to intermittent mechanical force in organ culture are reported. The application of mechanical force enhances the osteoid thickness and osteoblast number in the periosteum and increases the number of viable osteocytes. These results indicate that (1) the mature bone tissue survives in organ cultures; (2) the mechanical forces better preserve the structure of the osteocytes and stimulate the osteoblasts, and (3) stimulate the osteogenesis.

Dose-related effects of 1,25-dihydroxyvitamin D3 on growth, modeling, and morphology of fetal mouse metatarsals cultured in serum-free medium.

A serum-free, fetal bone organ culture model that permits the simultaneous determination of modeling and growth parameters was used to examine the effects of a near physiologic and a pharmacologic dose of 1,25-dihydroxycholecalciferol [1,25-(OH)2D3]. The fetuses of pregnant mice were removed on day 17 of gestation, and three medial metatarsal rudiments were cleaned and after preculturing were cultured as pair-matched groups for 4 days in MEM supplemented with 0.2% BSA. 1,25-(OH)2D3 was added to the cultures at concentrations of 10(-12) or 10(-6) M. Cultures treated with the carrier and devitalized bones served as controls. For resorption studies, pregnant mice were given 45Ca on day 17 of pregnancy and fetal metatarsals harvested 24 h later. Resorption was determined by the amount of 45Ca released into the media. DNA synthesis was estimated by determining the incorporation of [3H]thymidine, collagen synthesis by measuring the incorporation of [3H]proline, mineralization by the incorporation of 45Ca, and proteoglycan synthesis by the incorporation of 35S. The amount of radiolabel was detected in media, as well as in noncultured, dead, and cultured rudiments. The total length of the rudiments and length of the calcified diaphyses were measured daily. In addition, rudiments from all experimental groups were prepared for light and electron microscopy. The high dose (10(-6) M) of 1,25-(OH)2D3 suppressed total rudiment growth but not the growth of the calcified diaphysis, 1,25-(OH)2D3 also decreased DNA, collagen, and proteoglycan synthesis, reduced calcification, and increased bone resorption in a dose-related manner. There were morphologic and ultrastructural changes in the osseous tissues and cells, particularly with the high dose of vitamin D, that supported the biochemical findings of suppressed activity of the osteogenic and chondrogenic cells. However, the suppression of collagen production and bone cell proliferation observed with the pharmacologic dose of vitamin D may be partially attributable to the decrease in bone mass (from increased resorption), thus resulting in less osseous tissue surface for these events to occur as endochondral osteogenesis progressed. The lower dose of vitamin D, however, had effects on 35S and 45Ca incorporation that could not be attributed to a decreased osseous tissue mass. This study emphasizes the importance of measuring specialized activities of the various cell populations in bone rudiment culture models to more fully understand the changes in tissue metabolism that result in changes in rudiment growth and modeling.

Deposition of amyloid of unknown origin in articular cartilage.

Articular cartilage, obtained from the large toe during hallux valgus operations in 37 patients, was investigated for the presence of amyloid by using the Congo red staining method. Amyloid deposits were demonstrated, particularly in the superficial layer of the cartilage, in 30 cases. This amyloid did not react immunohistochemically with any of the antibodies against the known five major amyloid types (AA, A lambda, A kappa, AF, AB). From these data it is concluded that hyaline cartilage in older individuals is prone to infiltration by an amyloid of a hitherto unidentified class. From the morphological observations there seems to be no correlation between amyloid deposits and the development of osteoarthrosis.

Morphological abnormalities in vitamin B6 deficient tarsometatarsal chick cartilage.

The aim of this study was to test the hypothesis that deficiency of vitamin B6 would produce morphological characteristics of osteochondral lathyrism. To accomplish this goal, morphological characteristics of chick cartilage in which lathyrism was produced by two separate dietary regimens was compared to morphological changes encountered in vitamin B6 deficiency. Vitamin B6 deficiency should reduce activity of lysyloxidase needed for producing intermolecular cross-links. The question to be addressed was: would this latter deficiency impair collagen morphological features and secondarily other structures indirectly by reducing collagen molecular assembly? Failure of cross-linking of collagen in the positive controls was related to a lack of functional aldehyde cross-link intermediates which are blocked by homocysteine and aminoacetonitrile. Day-old-male Lohmann chicks were fed adequate (6 mg/kg) or vitamin B6-deficient diets. Cross-link defects were induced by homocysteine-rich diets (0.6% w/w) or a diet containing aminoacetonitrile (0.1% w/w). Animals were sacrificed at 6 weeks of age and Ossa tarsalia articular cartilage specimens, as well as the proximal end of tarsometatarsus were dissected from the tibial metatarsal joint, a major weight-bearing site. Light microscopic observations revealed reduction of subarticular trabecular bone formation, concurrent with overexpansion of the hypertrophic cell zone. Ultrastructural electron microscopy observation of articular fibro-cartilage indicated significant thickening of collagen fibers in vitamin B6 deficient birds, as well as the positive controls in comparison to that of cage-matched control birds. It was concluded that vitamin B6 deficient cross-linking may be responsible for the observed delay in bone development and aforementioned cartilage histological alterations.