Fourier transform infrared imaging as a tool to chemically and spatially characterize matrix-mineral deposition in osteoblasts.

Mineralizing osteoblasts are regularly used to study osteogenesis and model in vivo bone formation. Thus, it is important to verify that the mineral and matrix being formed in situ are comparable to those found in vivo. However, it has been shown that histochemical techniques alone are not sufficient for identifying calcium phosphate-containing mineral. The goal of the present study was to demonstrate the use of Fourier transform infrared imaging (FTIRI) as a tool for characterizing the spatial distribution and colocalization of the collagen matrix and the mineral phase during the mineralization process of osteoblasts in situ. MC3T3-E1 mouse osteoblasts were mineralized in culture for 28 days and FTIRI was used to evaluate the collagen content, collagen cross-linking, mineralization level and speciation, and mineral crystallinity in a spatially resolved fashion as a function of time. To test whether FTIRI could detect subtle changes in the mineralization process, cells were treated with risedronate (RIS). Results showed that collagen deposition and mineralization progressed over time and that the apatite mineral was associated with a collagenous matrix rather than ectopic mineral. The process was temporarily slowed by RIS, where the inhibition of osteoblast function caused slowed collagen production and cross-linking, leading to decreased mineralization. This study demonstrates that FTIRI is a complementary tool to histochemistry for spatially correlating the collagen matrix distribution and the nature of the resultant mineral during the process of osteoblast mineralization. It can further be used to detect small perturbations in the osteoid and mineral deposition process.

Recovery of trabecular and cortical bone turnover after discontinuation of risedronate and alendronate therapy in ovariectomized rats.

Alendronate (ALN) and risedronate (RIS) are bisphosphonates effective in reducing bone loss and fractures associated with postmenopausal osteoporosis. However, it is uncertain how long it takes bone turnover to be re-established after treatment withdrawal, and whether this differs between the two drugs. The objective of this study was to determine the time required to re-establish normal bone turnover after the discontinuation of ALN and RIS treatment in an animal model of estrogen-deficiency osteoporosis. Two hundred ten, 6-mo-old female Sprague-Dawley rats were ovariectomized and 6 wk later were randomized into baseline controls (n = 10) and four treatment groups (n = 50/group): vehicle-treated controls (CON; 0.3 ml sterile water), ALN (2.4 microg/kg), low-dose RIS (RIS low; 1.2 microg/kg), and high-dose RIS (RIS high; 2.4 microg/kg). Treatments were administered 3 times/wk by subcutaneous injection. Baseline controls were killed at the initiation of treatment. Other groups were treated for 8 wk, and subgroups (n = 10/ treatment group) were killed 0, 4, 8, 12, and 16 wk after treatment was withdrawn. Static and dynamic histological analyses were performed for cortical (tibial diaphysis) and trabecular (proximal tibia and L(4) vertebrae) bone. DXA and mechanical testing was performed on the L(5) vertebra. After 8 wk of treatment, trabecular bone turnover rates were significantly suppressed in all drug-treated animals. Trabecular bone formation rate (BFR/BS) remained significantly lower than vehicle in bisphosphonate-treated animals through 12 wk. Sixteen weeks after treatment withdrawal, trabecular BFR/BS in the proximal tibia was re-established in animals treated with RIS but not in animals treated with ALN compared with controls. BMD of the fifth lumbar vertebra remained significantly higher than controls 16 wk after treatment withdrawal in ALN-treated animals but not in RIS-treated animals. Despite reductions in BMD and increases in bone turnover, ultimate force of the fifth lumbar vertebra remained significantly higher in all drug-treated animals through 16 wk after withdrawal.

Regulation of mineral-to-matrix ratio of lumbar trabecular bone in ovariectomized rats treated with risedronate in combination with or without vitamin K2.

The relationship between bone turnover and bone tissue and material properties was examined in ovariectomized (OVX) rats treated with risedronate in combination with or without vitamin K2. Seventy female rats, 18 weeks of age, were assigned to 7 groups (n=10): sham-operated + vehicle control; OVX + vehicle control; OVX + risedronate 0.1, 0.5, or 2.5 mg/kg/day po; OVX + vitamin K2 approximately 30 mg/kg/day po; OVX + vitamin K2 (approximately 30 mg/kg/day) and risedronate (0.5 mg/kg/day). Treatments were given daily for 9 months. To assess bone turnover, we measured serum osteocalcin and urinary deoxypyridinoline at 0, 3, and 9 months. To assess vertebral and femoral tissue and material properties, bone mass, bone mineral density (BMD by DXA), trabecular bone structure (vertebra: 3D-microCT), cortical bone structure (femur: histomorphometry), biomechanical properties, and mineral properties (mineral-to-matrix and carbonate-to-phosphate ratios by Fourier transform infrared microspectroscopy) were measured ex vivo at 9 months. Ovariectomy increased bone turnover and induced significant loss of bone mass/density, structure, mineral properties (mineral-to-matrix ratio), and strength. Risedronate produced dose-dependent inhibition of the ovariectomy-induced increase in turnover and loss of bone mass/density, structure, mineral-to-matrix ratio, and strength, with a lowest effective dose of 0.1-0.5 mg/kg/day. High-dose risedronate (2.5 mg/kg/day) did not induce increases in any parameter above that of sham control. Vitamin K2 had no effects. In the OVX groups, urinary deoxypyridinoline at 3 and 9 months correlated significantly with vertebral BMD, trabecular bone volume, ultimate load, stiffness, and mineral-to-matrix ratio, and with femoral BMD, cortical area, and ultimate load. These results support the concept that changes in bone tissue and material properties can result directly from changes in bone turnover. Different effects among different drugs on material properties, including mineral-to-matrix ratio, may reflect differences in the relative rate and magnitude of osteoclastic bone resorption and osteoblastic primary bone mineralization.