Mineralization of three‐dimensional osteoblast cultures is enhanced by the interaction of 1α,25‐dihydroxyvitamin D3 and BMP2 via two specific vitamin D receptors

1α,25‐Dihydroxyvitamin D3 [1α,25(OH)2D3] and bone morphogenetic protein‐2 (BMP2) are both used to stimulate osteoblastic differentiation. 1α,25(OH)2D3 regulates osteoblasts through classical steroid hormone receptor mechanisms and through rapid responses that are mediated by two receptors, the traditional vitamin D receptor (VDR) and protein disulphide isomerase family A member 3 (Pdia3). The interaction between 1α,25(OH)2D3 and BMP2, especially in three‐dimensional (3D) culture, and the roles of the two vitamin D receptors in this interaction are not well understood. We treated wild‐type (WT), Pdia3‐silenced (Sh‐Pdia3) and VDR‐silenced (Sh‐VDR) pre‐osteoblastic MC3T3‐E1 cells with either 1α,25(OH)2D3, or BMP2, or with 1α,25(OH)2D3 and BMP2 together, and measured osteoblast marker expression in 2D culture and mineralization in a 3D poly(ε‐caprolactone)–collagen scaffold model. Quantitative PCR showed that silencing Pdia3 or VDR had a differential effect on baseline expression of osteoblast markers. 1α,25(OH)2D3 + BMP2 caused a synergistic increase in osteoblast marker expression in WT cells, while silencing either Pdia3 or VDR attenuated this effect. 1α,25(OH)2D3 + BMP2 also caused a synergistic increase in Dlx5 in both silenced cell lines. Micro‐computed tomography (μCT) showed that the mineralized volume of untreated Sh‐Pdia3 and Sh‐VDR 3D cultures was greater than that of WT. 1α,25(OH)2D3 reduced mineral in WT and Sh‐VDR cultures; BMP2 increased mineralization; and 1α,25(OH)2D3 + BMP2 caused a synergistic increase, but only in WT cultures. SEM showed that mineralized matrix morphology in 3D cultures differed for silenced cells compared to WT cells. These data indicate a synergistic crosstalk between 1α,25(OH)2D3 and BMP2 toward osteogenesis and mineral deposition, involving both VDR and Pdia3. Copyright © 2013 John Wiley & Sons, Ltd.     

Dioxin-induced up-regulation of the active form of vitamin D is the main cause for its inhibitory action on osteoblast activities, leading to developmental bone toxicity

Dioxin (2,3,7,8-tetrachlorodibenzo-p-dioxin, TCDD) is known to cause bone toxicity, particularly during animal development, although its action mechanism to cause this toxicity has yet to be elucidated. Mouse pups were exposed to TCDD via dam's milk that were administered orally with 15 μg TCDD/kg b.w. on postnatal day 1. Here we report that TCDD causes up-regulation of vitamin D 1α-hydroxylase in kidney, resulting in a 2-fold increase in the active form of vitamin D, 1,25-dihydroxyvitamin D₃, in serum. This action of TCDD is not caused by changes in parathyroid hormone, a decrease in vitamin D degrading enzyme, vitamin D 24-hydroxylase, or alterations in serum Ca²⁺ concentration. Vitamin D is known to affect bone mineralization. Our data clearly show that TCDD-exposed mice exhibit a marked decrease in osteocalcin and collagen type 1 as well as alkaline phosphatase gene expression in tibia by postnatal day 21, which is accompanied with a mineralization defect in the tibia, lowered activity of osteoblastic bone formation, and an increase in fibroblastic growth factor-23, a sign of increased vitamin D effect. Despite these significant effects of TCDD on osteoblast activities, none of the markers of osteoclast activities was found to be affected. Histomorphometry confirmed that osteoblastic activity, but not bone resorption activity, was altered by TCDD. A prominent lesion commonly observed in these TCDD-treated mice was impaired bone mineralization that is characterized by an increased volume and thickness of osteoids lining both the endosteum of the cortical bone and trabeculae. Together, these data suggest that the impaired mineralization resulting from reduction of the osteoblastic activity, which is caused by TCDD-induced up-regulation of vitamin D, is responsible for its bone developmental toxicity.     

Differential gene expression of bgp and mgp in trabecular and compact bone of Atlantic salmon (Salmo salar L.) vertebrae

The tissue‐specific gene expression of the vitamin K‐dependent proteins bone γ‐carboxyglutamate‐protein (BGP) and matrix γ‐carboxyglutamate‐protein (MGP) in Atlantic salmon (Salmo salar L.) was investigated. In previous studies, BGP, the most abundant non‐collagenous protein of bone, was almost exclusively associated with bone, whereas the non‐structural protein MGP has a more widespread tissue distribution. In‐situ hybridization of juvenile Atlantic salmon (～40 g, fresh water) vertebrae demonstrated expression of bgp and mgp mRNA in osteoblasts lining the trabecular bone, whereas no staining was observed in the compact bone. By separating the trabecular and compact bone of both juvenile (～40 g, fresh water) and adult (～1000 g, sea water) Atlantic salmon, we observed that the two vertebral bone compartments displayed different levels of bgp, whereas no such differences were seen for mgp. Measurements of the mineral content and Ca/P molar ratio in adult salmon revealed no significant differences between trabecular and compact bone. In conclusion, the osteoblasts covering the salmon vertebrae have unique gene expression patterns and levels of bgp and mgp. Further, the study confirms the presence of mRNA from the vitamin K‐dependent proteins BGP and MGP in the vertebrae, fin and gills of Atlantic salmon.     

Influence of the degree of dentine mineralization on pulp chamber temperature increase during resin-based composite (RBC) light-activation

Objectives

To analyse the influence of the degree of dentine mineralization on the pulp chamber temperature increase during composite light-activation.

Methods

Dentine discs (2 mm thick) obtained from recently extracted teeth or those with extensive dentine sclerosis were analysed by FT-IR spectrometry in order to choose the two discs with the greatest difference in the degree of mineralization. A model tooth was set up with the dentine discs between a molar with the pulp chamber exposed and a crown with a standardized class II cavity. A K-type thermocouple was introduced into the molar root until it came into contact with the dentine discs and the cavity was filled with P60 resin composite. The temperature rise was measured for 120 s after light-activation began: Standard (S) 600 mW/cm²/40 s; Ramp (R) 0 → 800 mW/cm²/10 s + 800 mW/cm²/10 s; Boost (B) 850 mW/cm²/10 s and LED (L) 1.300 mW/cm²/40 s (n = 10). The same protocol was repeated after grinding the dentine discs to 1.0 and 0.5 mm thickness.

Results

The temperature increase was significantly higher in dentine with high degree of mineralization (p < 0.05). With respect to the dentine thickness, the following result was found: 2 mm < 1 mm < 0.5 mm (p < 0.05). The light-activation mode also presented significant difference as follows: S > R = L > B (p < 0.05).

Conclusions

The higher the degree of dentine mineralization the greater the increase in pulp chamber temperature. The temperature increase was influenced by the light-polymerization mode and dentine thickness.     

钙磷对体外培养成骨细胞增殖、分化和矿化影响的研究

目的研究钙、磷对体外培养成骨细胞(OB)增殖、分化及矿化的作用,探讨钙、磷对骨代谢的影响。方法在体外培养OB的基础上,分别添加1、2、4mmol/L钙、1、2、4mmol/L磷及2mmol/L钙+1mmol/L磷(钙磷比2∶1)、1mmol/L钙+2mmol/L磷(钙磷比1∶2)。检测细胞增殖、碱性磷酸酶(ALP)活性、骨桥蛋白(OPN)和Ⅰ型胶原(Col-Ⅰ)含量及其mRNA表达情况。结果与对照组比,饲料中添加4mmol/L钙从D2开始促进OB增殖,差异显著(P<0.05),添加1、2mmol/L钙从D5开始差异显著(P<0.05),而钙磷比2:1与1:2到D8差异才显著(P<0.05)。除1mmol/L钙组外,各试验组从D2起均抑制细胞内ALP活性(P<0.05),在D5抑制其mRNA表达(P<0.01),在D2、D8促进其mRNA表达(P<0.05)。除1mmol/L钙组外,各试验组在D2均促进Col-Ⅰ分泌(P<0.01),但钙各组和1mmol/L磷组在D5、D8及2mmol/L磷组和钙磷比(1:2)组在D8均抑制其分泌(P<0.05)。各试验组,在D2、D8均促进Col-ⅠmRNA表达(P<0.01),在D5除钙磷比(1:2)组外均抑制其mRNA表达(P<0.01)。各试验组促进OPNmRNA表达(P<0.01),除1、2mmol/L钙组外,各实验组从D2起均促进其分泌(P<0.0)。结论添加钙及2种钙磷比(2:1,1:2)均能抑制成骨细胞早期分化,诱导其增殖及重叠生长,促进其基质成熟及矿化,而磷对其增殖作用不明显。     

Quantitative micro-computed tomography: a non-invasive method to assess equivalent bone mineral density

One of the many applications of micro computed tomography (microCT) is to accurately visualize and quantify cancellous bone microstructure. However, microCT based assessment of bone mineral density has yet to be thoroughly investigated. Specifically, the effects of varying imaging parameters, such as tube voltage (kVp), current (microA), integration time (ms), object to X-ray source distance (mm), projection number, detector array size and imaging media (surrounding the specimen), on the relationship between equivalent tissue density (rhoEQ) and its linear attenuation coefficient (micro) have received little attention. In this study, in house manufactured, hydrogen dipotassium phosphate liquid calibration phantoms (K2HPO4) were employed in addition to a resin embedded hydroxyapatite solid calibration phantoms supplied by Scanco Medical AG Company. Variations in current, integration time and projection number had no effect on the conversion relationship between micro and rhoEQ for the K2HPO4 and Scanco calibration phantoms [p>0.05 for all cases]. However, as expected, variations in scanning tube voltage, object to X-ray source distance, detector array size and imaging media (referring to the solution that surrounds the specimen in the imaging vial) significantly affected the conversion relationship between mu and rhoEQ for K2HPO4 and Scanco calibration phantoms [p<0.05 for all cases]. A multivariate linear regression approach was used to estimate rhoEQ based on attenuation coefficient, tube voltage, object to X-ray source distance, detector array size and imaging media for K2HPO4 liquid calibration phantoms, explaining 90% of the variation in rhoEQ. Furthermore, equivalent density values of bovine cortical bone (converted from attenuation coefficient to equivalent density using the K2HPO4 liquid calibration phantoms) samples highly correlated [R2=0.92] with the ash densities of the samples. In conclusion, Scanco calibration phantoms can be used to assess equivalent bone mineral density; however, they cannot be scanned with a specimen or submerged in a different imaging media. The K2HPO4 liquid calibration phantoms provide a cost effective, easy to prepare and convenient means to perform quantitative microCT analysis using any microCT system, with the ability to choose different imaging media according to study needs. However, as with any liquid calibration phantom, they are susceptible to degradation over time.