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.

Identification of oxytetracycline as a chondrogenic compound using a cell-based screening system.

To effectively treat degenerative joint diseases including osteoarthritis (OA), small chemical compounds need to be developed that can potently induce chondrogenic differentiation without promoting terminal differentiation. For this purpose, we screened natural and synthetic compound libraries using a Col2GFP-ATDC5 system and identified oxytetracycline (Oxy) as a chondrogenic compound. Oxy induced cartilaginous matrix synthesis and mRNA expressions of chondrocyte markers in ATDC5 cells. In addition, Oxy suppressed mineralization and mRNA expressions of terminal chondrocyte differentiation markers in ATDC5 cells, primary chondrocytes, and cultured metatarsal bones. Oxy's induction of Col2 mRNA expression was decreased by the addition of Noggin and was increased by the addition of BMP2. Furthermore, Oxy increased mRNA expression of Id1, Bmp2, Bmp4, and Bmp6. These data suggest that Oxy induces chondrogenic differentiation in a BMP-dependent manner and suppresses terminal differentiation. Oxy may be useful for treatment of OA and also for regeneration of cartilage tissue.

Enlargements of the distal third metacarpus and metatarsus in Thoroughbred foals at pasture from birth to 160 days of age.

AIMS: To assess the relationship between the radiographic and microscopic appearance of the physeal regions of the distal third metacarpal (Mc3) and metatarsal (Mt3) bones of Thoroughbred foals at 160 days of age, and to clinically assess changes in contour of the distal Mc3 and Mt3 physeal regions from birth to 160 days of age. To assess relationships between maximum clinical physis scores and age, time of year, foal's sex, condition score, growth rate, copper (Cu) concentration in the liver of the foal, and supplementation of the dam with Cu in late gestation. METHODS: Dams were given Cu (n=5 and n=11, in Years 1 and 2, respectively) or saline (n=5 and n=12, in Years 1 and 2, respectively) injections in late gestation. Liver biopsies were harvested from foals in the first week of life, and the whole liver homogenised after the animals were euthanised at around 160 days of age, to determine Cu concentrations. Pasture samples were collected every 4-8 weeks for analysis of mineral composition. During Year 1, 10 foals were weighed and examined every 2 weeks from birth to 160 days of age for evidence of pain and lameness in the distal Mc3/Mt3. In Year 2, 23 foals were weighed, condition-scored and examined for evidence of pain and lameness weekly for the first 5 weeks of life, then every 2 weeks from birth to 160 days of age, and a clinical physis score for the distal Mc3/Mt3 given. Cabinet radiographs of frontal slices of the physeal region of the distal Mc3/Mt3 at around 160 days of age were given a radiographic physis score. Physes were then examined histologically for evidence of abnormal endochondral ossification. RESULTS: Gross enlargements of the distal Mc3 and Mt3 were observed in all foals in this study, but were not associated with lameness, pain or inflammation. The most severe clinical physis scores occurred over 2 months in late summer/autumn, and were not influenced by the foal's growth rate, sex, or Cu concentration in the liver, or treatment of dams with Cu in late gestation. The clinical physis score was highly correlated to radiographic evidence of shouldering in the forelimb and hindlimb (both p<0.001). Focal disturbances in endochondral ossification were evident radiographically and histologically in the some of the physes at 160 days of age. The mineral composition of pasture was similar in Years 1 and 2, and concentrations of Cu and zinc were below those currently recommended for growing horses. CONCLUSIONS: Gross enlargements of the distal Mc3 and Mt3 were not consistent with previous descriptions of physitis. Results suggest that while many Thoroughbred foals at pasture will have visible boney enlargements of the distal Mc3/Mt3 in the first 5 months of life, few have physeal cartilage abnormalities or significant compromise of endochondral ossification. The importance of these clinical swellings may be overestimated, and they may more appropriately be called physiological enlargements associated with remodelling of bone.

In vivo expression of transcripts encoding the Glvr-1 phosphate transporter/retrovirus receptor during bone development.

In vitro observations suggest that inorganic phosphate (Pi) transport plays an important functional role in osteogenic cells and in their matrix vesicles for the initiation of matrix calcification. Recent studies have shown that the type III sodium-dependent Pi transporters, Glvr-1 and Glvr-2, are expressed in human osteoblast-like cells and have suggested a potential role for type III transporters in regulated Pi handling in osteogenic cells. To address the relevance of these findings in the context of bone formation in vivo and, in particular, in relation to matrix calcification, we investigated expression of the Glvr-1 transporter by in situ hybridization in developing embryonic murine metatarsals, using human Glvr-1 cDNA as a probe. In this model of endochondral ossification, expression of transcripts encoding Glvr-1 could be detected from day 17 of embryonic development. A hybridization signal for Glvr-1 was specifically observed in a subset of hypertrophic chondrocytes and could not be detected in osteoblasts. The expression of Glvr-1 mRNA was compared with that of transcripts encoding extracellular matrix proteins. Glvr-1 mRNA expression was confined to a population of early hypertrophic chondrocytes expressing type X collagen and to slightly more mature cells that express transcripts encoding osteopontin but lack type X collagen mRNA. No Glvr-1 transcripts were detected in fully differentiated hypertrophic chondrocytes. This pattern of Glvr-1 mRNA expression was maintained throughout embryonic development until after birth. In conclusion, the Glvr-1 phosphate transporter is selectively expressed in a subset of hypertrophic chondrocytes during endochondral bone formation, in a region where matrix mineralization proceeds. This observation represents the first in vivo evidence consistent with a potential role for this phosphate transporter in matrix calcification.

Development of tissue culture techniques and hardware to study mineralization under microgravity conditions.

To study the effects of weightlessness on mouse fetal long bone rudiment growth and mineralization we have developed a tissue culture system for the Biorack facility of Spacelab. The technique uses standard liquid tissue culture medium, supplemented with NA-beta-glycerophosphate, confined in gas permeable polyethylene bags mounted inside ESA Biorack Type I experiment containers. The containers can be flushed with an air/5% CO2 gas mixture necessary for the physiological bicarbonate buffer used. Small amounts of fluid can be introduced at the beginning (e.g. radioactive labels for incorporation studies) or at the end of the experiment (fixatives). A certain form of mechanical stimulation (continuous compression) can be used to counteract the, possibly, adverse effect of microgravity. Using 16 day old metatarsals the in vitro calcification process under microgravity conditions can be studied for a 4 day period.

Different pattern of alkaline phosphatase, osteopontin, and osteocalcin expression in developing rat bone visualized by in situ hybridization.

Alkaline phosphatase (AP), osteopontin (OP), and osteocalcin (OC) are expressed during osteoblastic differentiation. However, previous studies suggested differences in the timing and possibly the site of their expression. In this study we used in situ hybridization to follow the distribution of these osteoblastic markers during bone development. Frozen sections of neonatal rat long bones and calvariae were hybridized with 35S-labeled RNA probes complementary to the AP, OP, and OC mRNAs. Controls included sections hybridized with the sense (mRNA) probes or pretreated with RNase. Positive cells were identified in all areas of bone formation of the long bones and calvariae. Based on quantitative silver grain distribution and density, high levels of OP expression were present only in osteoblasts in close proximity to bone (one to two cell layers). OC expression, apparently at lower levels than OP, was also localized to osteoblasts in contact with bone. In contrast AP, which was expressed at lower levels than OP, was present in a large number of cells, including preosteoblasts that were many layers removed from the bone-forming surface. These findings are consistent with the asynchronous expression of phenotypically related genes and suggest that AP is an earlier differentiation marker than OP and OC during the formation of endochondral and membranous bone.