Gene expression and distribution of key bone turnover markers in the callus of estrogen-deficient, vitamin D-depleted rats.

An experimental rat model was used to test the hypothesis that in osteoporosis (OP) the molecular composition of the extracellular matrix in the fracture callus is disturbed. OP was induced at 10 weeks of age by ovariectomy and a vitamin D(3)-deficient diet, and sham-operated animals fed normal diet served as controls. Three months later a closed tibial fracture was made and stabilized with an intramedullary nail. After 3 and 6 weeks of healing, the animals were killed and the fracture calluses examined with global gene expression, in situ mRNA expression, and ultrastructural protein distribution of four bone turnover markers: osteopontin, bone sialoprotein, tartrate-resistant acid phosphatase, and cathepsin K. Global gene expression showed a relatively small number of differently regulated genes, mostly upregulated and at 3 weeks. The four chosen markers were not differently regulated, and only minor differences in the in situ mRNA expression and ultrastructural protein distribution were detected. Gene expression and composition of fracture calluses are not generally disturbed in experimental OP.

Experimental osteoporosis induced by ovariectomy and vitamin D deficiency does not markedly affect fracture healing in rats.

BACKGROUND: The question of whether fracture healing and mechanical properties of the callus are influenced by osteoporosis (OP) is still not settled. We therefore studied this issue in vitamin D-depleted ovariectomized (OVX) rats, an OP model previously shown to induce weakening of the femoral neck, and thus thought to be closer to the human condition than the classic OVX rat model. METHODS: 72 female Wistar rats were randomized into two groups: ovariectomy and vitamin D-deficient diet (Ovx-D group) or sham operation and normal rat chow (Sham group). After 12 weeks, a closed tibial midshaft fracture was performed on the right side and fixed with an intramedullary nail. Bone loss and callus formation were monitored with DXA; serum levels of estradiol and vitamin D3 were measured and histomorphometric analyses were performed. Mechanical properties of callus, tibia, femoral shaft, and femoral neck were examined in 3-point cantilever bending 6 weeks after fracture. RESULTS: The Ovx-D group showed reduced BMD in the spine and femoral neck, and reduced trabecular bone volume in the femoral head. There were no differences in BMD and mechanical properties of callus between the groups. Except for reduced stiffness of the right femoral neck in the Ovx-D group (p = 0.02), no differences in the mechanical strength of long bones were detected. INTERPRETATION: Our results suggest that the systemic effects of estrogen and vitamin D deficiency are not crucial for fracture healing or mechanical properties of the callus.

Levetiracetam, phenytoin, and valproate act differently on rat bone mass, structure, and metabolism.

PURPOSE: Long-term treatment with antiepileptic drugs (AEDs) is associated with increased risk of fractures. Phenytoin (PHT) and valproate (VPA) have both been suggested to influence bone health, whereas levetiracetam (LEV) is scarcely studied. The present study compares the effect of these AEDs on bone mass, biomechanical strength, and bone turnover in rats. METHODS: Female rats received PHT (50 mg/kg), VPA (300 mg/kg), or LEV (50 and 150 mg/kg) for 90 days. Dissected femurs were analyzed using dual energy x-ray absorptiometry (DXA), three-point cantilever bending, and histomorphological evaluation. Serum levels of biochemical bone turnover markers were monitored using immunoassay quantification. RESULTS: PHT and VPA reduced bone mineral density (BMD) and content (BMC) in one or more bone compartments, whereas LEV did not. VPA induced increased bone turnover, whereas modest changes were observed for PHT. Interestingly, low-dose LEV was associated with reduced biomechanical strength of the femoral neck (mainly trabecular bone). In addition, low-dose LEV treatment resulted in significantly reduced levels of serum osteocalcin, a marker of bone formation. Histomorphological analyses indicated increased retention of cartilage remnants at the growth plate metaphysis of rats treated with low-dose LEV vs. controls. CONCLUSIONS: PHT, VPA, and LEV exert differential effects on bone mass and strength, suggesting different mechanisms of action. The weakening effect of low-dose LEV on the femoral neck, despite a constant BMD, suggests a primary effect on bone quality. These findings warrant further human studies of possible adverse effects of LEV on bone development and growth, particularly in children and adolescents.

Heat-induced retrieval of immunogold labeling for nucleobindin and osteoadherin from Lowicryl sections of bone.

The main purpose of this study was to examine whether antigens can be retrieved by heating Lowicryl sections of paraformaldehyde-fixed (PFF) tissues. Thus the intensity of the immunogold signal for two bone proteins (Nucleobindin (Nuc) and osteoadherin (OSAD)) was compared in retrieved and non-retrieved sections of PFF rat bone. As an additional experiment, the effect of antigen retrieval (for Nuc) in sections of tissue primary stabilized by high pressure freezing with subsequent freeze substitution (HPF-FS) was studied. Finally, the tissue distribution patterns of Nuc labeling were compared in non-retrieved HPF-FS sections to that of retrieved and non-retrieved PFF sections. Antigen retrieval in Lowicryl sections of PFF tissues showed significantly enhanced labeling intensity for both proteins in all compartments where they are known to occur. Retrieved PFF Lowicryl sections showed only minor ultrastructural differences compared to non-retrieved ones. Retrieval of HPF-FS sections exhibited no enhancement of labeling but rather a slight reduction, which was significant in the cytoplasm and in cartilage. Furthermore, striking ultrastructural differences were observed in retrieved HPF-FS sections compared to non-retrieved ones with loss of coherence and structure in sections subjected to heating. Comparison of the distribution patterns of Nuc in the sections of PFF and HPF-FS tissues showed discrepancy in most compartments. Antigen retrieval by heating Lowicryl sections of PFF tissues significantly enhances immunogold labeling in all cell compartments where the bone proteins are known to occur. However, the procedure may distort the tissue distribution pattern of bone proteins.