Effects of risedronate, alendronate, and minodronate alone or in combination with eldecalcitol on bone mineral density, quality, and strength in ovariectomized rats.

Combination therapy of active vitamin D3 with some bisphosphonates (BPs) has been reported to be clinically beneficial. However, combination therapy of eldecalcitol (ELD) with BP has to date not been validated as to whether it is beneficial in the clinical setting. Preclinical studies suggested that simultaneous treatment with ELD and some BPs is more effective than monotherapy. However, the relative potency of various BPs, when used in combination with ELD, is completely unknown. In this study, we examined and compared the effects of risedronate (RIS), alendronate (ALN), and minodronate (MIN) alone or in combination with ELD on bone mass, microarchitecture, strength, and material properties in ovariectomized Sprague-Dawley rats aged 13 weeks. RIS, ALN, MIN, and ELD were administered five times weekly for 16 weeks. Micro-computed tomography analysis, compression test, and Fourier transform infrared (FTIR) imaging analysis were performed 16 weeks after treatment initiation. Trabecular and cortical bone mineral density (BMD) in the fourth lumbar vertebra (L4) significantly increased in the RIS + ELD, ALN + ELD, and MIN + ELD groups compared with the vehicle group. Moreover, the bone microarchitecture of L4 in all the BP + ELD groups also significantly improved. On mechanical testing of L4, the maximum load was significantly increased in the RIS + ELD and ALN + ELD groups. FTIR analysis revealed that the mineral-to-collagen ratio of trabecular bone in L3 of all the BP + ELD groups was significantly increased compared with the vehicle group. By contrast, the carbonate-to-phosphate ratio, a parameter of mineral immaturity, was significantly decreased in the RIS + ELD and ALN + ELD groups. BP + ELD improved the BMD and structural properties of the bone to a similar extent. RIS + ELD and ALN + ELD also improved bone strength. Furthermore, treatment with BP + ELD improved the bone material. These results suggest that the combination therapy of BP and ELD is beneficial and warrants further clinical trials.

Degree of orientations of collagen fibers and bone apatite crystals in rat femora by infrared dichroism imaging.

OBJECTIVES: The degree of orientations of collagen fibers and bone apatite crystals affects bone strength. We demonstrated that collagen fibers were aligned along the long axis of bone and that the degree of collagen fiber orientation changed with aging using infrared (IR) dichroism imaging. In this study, we developed a technique for evaluating bone apatite crystal orientation using IR dichroism imaging to investigate the relationships between collagen fiber and bone apatite crystal orientations. METHODS: Femora were harvested from male Sprague Dawley rats of different ages (6, 12, and 33 weeks); they were then embedded in poly (methyl methacrylate) and sectioned with a microtome into 3-µm longitudinal sections. The angle-dependent Fourier transform infrared (FTIR) spectra for sections were collected using FTIR imaging, and collagen fiber and bone apatite crystal orientations in the sections were assessed using IR dichroism imaging. RESULTS: Collagen fibers and poorly crystalline apatite in the femoral cortical bone were longitudinally aligned; however, the stoichiometric hydroxyapatite crystal and all of the bone apatite were not aligned. The degree of poorly crystalline apatite orientation was higher in 33-week-old rats than in 6-week-old rats. CONCLUSIONS: Poorly crystalline apatite in the rat femoral cortical bone was aligned along the collagen fibers. The degree of poorly crystalline apatite orientation and collagen fiber orientation in the femoral cortical bone increased until at least 33 weeks; meanwhile, on aging, the stoichiometric hydroxyapatite crystal was not longitudinally aligned.

Quick and easy sample preparation without resin embedding for the bone quality assessment of fresh calcified bone using fourier transform infrared imaging.

Fourier transform infrared (FTIR) imaging is a powerful tool for the assessment of bone quality; however, it requires the preparation of thin bone sections. Conventional poly(methyl methacrylate) (PMMA) embedding for the preparation of sections takes more than two weeks and causes denaturation of the bone. Development of a quick and easy sample preparation technique without denaturation is needed for accurate clinical evaluation of fresh calcified bone using FTIR imaging. Frozen sectioning allows the quick and easy preparation of thin sections without denaturation, but it requires a substrate with good chemical resistance and improved heat shock resistance. Polypropylene (PP) film afforded both good chemical resistance and greater heat shock resistance, and the 4-µm-thick PP film coated with glue was thin enough for the IR beam to pass through it, while the optical anisotropy of infrared bands overlapping with PO43- band was negligible. The bone quality of femoral thin sections prepared by the conventional PMMA embedding and sectioning procedure (RESIN-S) or the newly developed frozen sectioning procedure (FROZEN-S) was evaluated by FTIR imaging. The mineral-to-matrix ratio and crystallinity in the RESIN-S sections were higher than those in the FROZEN-S sections, whereas the carbonate-to-phosphate ratio in the RESIN-S sections was lower than that in the FROZEN-S sections. In RESIN-S, the increased mineral-to-matrix ratio could be caused by dehydration, and the increased crystallinity and decreased carbonate-to-phosphate ratio might be consequence of dissolution of bone mineral during PMMA embedding. Therefore, the combined use of PP film coated with glue and the frozen sectioning procedure without denaturation appears well suited to the assessment of the bone quality of fresh calcified bone using FTIR imaging.

Magnesium prevents phosphate-induced vascular calcification via TRPM7 and Pit-1 in an aortic tissue culture model.

Previous clinical and experimental studies have indicated that magnesium may prevent vascular calcification (VC), but mechanistic characterization has not been reported. This study investigated the influence of increasing magnesium concentrations on VC in a rat aortic tissue culture model. Aortic segments from male Sprague-Dawley rats were incubated in serum-supplemented high-phosphate medium for 10 days. The magnesium concentration in this medium was increased to demonstrate its role in preventing VC, which was assessed by imaging and spectroscopy. The mineral composition of the calcification was analyzed using Fourier transform infrared (FTIR) spectroscopic imaging, scanning electron microscopy (SEM) and energy dispersive X-ray spectroscopy (EDX) mapping. Magnesium supplementation of high-phosphate medium dose-dependently suppressed VC (quantified as aortic calcium content), and almost ablated it at 2.4 mm magnesium. The FTIR images and SEM-EDX maps indicated that the distribution of phosphate (as hydroxyapatite), phosphorus and Mg corresponded with calcium content in the aortic ring and VC. The inhibitory effect of magnesium supplementation on VC was partially reduced by 2-aminoethoxy-diphenylborate, an inhibitor of TRPM7. Furthermore, phosphate transporter-1 (Pit-1) protein expression was increased in tissues cultured in HP medium and was gradually-and dose dependently-decreased by magnesium. We conclude that a mechanism involving TRPM7 and Pit-1 underpins the magnesium-mediated reversal of high-phosphate-associated VC.

Mineral Composition of Phosphate-Induced Calcification in a Rat Aortic Tissue Culture Model.

AIM: High phosphorus conditions promote vascular calcification (VC) in both chronic kidney disease (CKD) patients and experimental models. However, the composition of medial calcification has not been accurately determined, so the objective of this study was to evaluate the mineral composition of calcification in a tissue culture model, not a cell culture system. METHODS: Aortic rings obtained from male Sprague-Dawley rats were incubated in serum-supplemented medium for 10 days. The inorganic phosphate (Pi) concentration of the medium was increased to induce VC, which was assessed by histology, imaging, and spectroscopy. The mineral composition of the calcification was analyzed using Fourier transform infrared (FTIR) spectroscopic imaging, scanning electron microscopy (SEM), and energy dispersive X-ray spectroscopy (EDX) mapping. RESULTS: The calcium content significantly increased only in aortic rings cultured for 10 days in the high-Pi medium (HiP: 3.8 mmol/L). The concentration of the phosphate transporter Pit-1 in the aortic tissue exposed to HiP was higher than that in the control incubated sections. The FTIR images and spectra indicated that PO4(3-) was mostly distributed as hydroxyapatite in the medial calcification of aortic rings cultured in HiP. A small quantity of carbonate was identified. The SEM-EDX overlay map demonstrated that phosphorus and calcium simultaneously accumulated and localized in the area of medial calcification induced by exposure to HiP. CONCLUSION: This is the first report of accurate determination of the chemical composition of aortic medial calcification. Exposure to high Pi concentration augments aortic calcification via an increase in Pit-1, which mainly contains calcium phosphate.

Vitamin K-dependent carboxylation of osteocalcin affects the efficacy of teriparatide (PTH(1-34)) for skeletal repair.

Teriparatide (PTH1-34) promotes skeletal repair and increases bone mass. Vitamin K is involved in bone mineralization as a coenzyme of γ-carboxylase for Gla proteins, and therefore vitamin K insufficiency caused by malnutrition or therapeutic intake of the vitamin K antagonist warfarin could affect the efficacy of PTH1-34 therapy for bone repair. In the present study, we investigated whether vitamin K influences the efficacy of PTH1-34 therapy for bone repair in a rat osteotomy model. Female 12-week-old Sprague-Dawley rats were subjected to a closed midshaft osteotomy of the femur and randomized into four groups (n=10 per group): vehicle, PTH1-34 (daily 30 µg/kg/day subcutaneous injection)+solvent (orally, three times a week), PTH1-34+warfarin (0.4 mg/kg/day orally, three times a week), and PTH1-34+vitamin K2 (menatetrenone, 30 mg/kg/day orally, three times a week). Serum γ-carboxylated and uncarboxylated osteocalcin (Gla-OC and Glu-OC) levels and radiographic healing were monitored every 2 weeks. Skeletal repair was assessed by micro-computed tomography, mechanical testing, and histology at 8weeks after surgery. PTH1-34 amplified the osteotomy-induced increase in Gla-OC and improved the mechanical properties as well as the volumetric bone mineral tissue density of the fracture callus. Concurrent use of warfarin decreased the response to PTH1-34 therapy in terms of mechanical recovery, probably by impairing mineralization due to the lack of Gla-OC. Although the effects of combination therapy with PTH1-34 and vitamin K2 on bone repair did not significantly exceed those of PTH1-34 monotherapy in rats fed sufficient dietary vitamin K, postoperative Gla-OC levels were correlated with the mechanical properties of the osteotomized femur in PTH1-34-treated rats regardless of the use of warfarin or vitamin K2. These findings suggest the importance of vitamin K dependent γ-carboxylation of OC for realizing the full effects of PTH1-34 on skeletal repair.