• Journal of Nutrition and Health
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• v.51 no.5
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• pp.379-385
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• 2018

Purpose: Zinc (Zn) is an essential trace element for bone mineralization and osteoblast function. We examined the effects of Zn deficiency on osteoblast differentiation and mineralization in MC3T3-E1 cells. Methods: Osteoblastic MC3T3-E1 cells were cultured at concentration of 1 to $15{\mu}M$ $ZnCl_2$ (Zn- or Zn+) for 5, 15 and 25 days up to the calcification period. Extracellular matrix mineralization was detected by staining Ca and P deposits using Alizarin Red and von Kossa stain respectively, and alkaline phosphatase (ALP) activity was detected by ALP staining and colorimetric method. Results: Extracellular matrix mineralization was decreased in Zn deficiency over 5, 15, and 25 days. Similarly, staining of ALP activity as the sign of an osteoblast differentiation, was also decreased by Zn deficiency over the same period. Interestingly, the gene expression of bone-related markers (ALP, PTHR; parathyroid hormone receptor, OPN; osteopontin, OC; osteocalcin and COLI; collagen type I), and bone-specific transcription factor Runx2 were downregulated by Zn deficiency for 5 or 15 days, however, this was restored at 25 days. Conclusion: Our data suggests that Zn deficiency inhibits osteoblast differentiation by retarding bone marker gene expression and also inhibits bone mineralization by decreasing Ca/P deposition as well as ALP activity.

• Journal of Biomedical Engineering Research
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• v.30 no.5
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• pp.444-451
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• 2009

Purpose: The aim of present study is to detect longitudinal alterations of mechanical characteristic determined by bone quality (microarchitecture and degree of mineralization) on femur trabecular bone due to metastatic bone tumor Materials and Methods: Each 6 female SD rats (12 weeks old, approximate 250g) were allocated in SHAM and TUMOR Group. W256 (Walker carcinosarcoma 256 malignant breast cancer cell) was injected into the right femur (intraosseous injection) in TUMOR Group, whereas 0.9% NaCl (saline solution) was injected in SHAM Group. The right hind limbs of all rats were scanned by in-vivo micro-CT to acquire structural parameters, bone mineral density, X-ray attenuation and bone mineralization distribution at 0 week and 4 weeks after surgery. Results: BMD, BV/TV and Tb.N of trabecular bone in TUMOR group were markedly decreased (26%, 11% and 23%) while those in SHAM group were significantly increased (34%, 48% and 11%) (p<0.05). BS/BV, Tb.Sp and SMI in TUMOR group were significantly increased (-16%, 38% and 2%) compared with those in SHAM group (-33%, 12% and -16%) (p<0.05). Additionally, bone mineralization in TUMOR group significantly decreased while those in SHAM group was significantly increased (p<0.05). Conclusion: It is identified that how much bone microarchitecture and mineralization are diminished due to the metastatic bone tumor. The results may be helpful to prediction of fracture risk by metastatic bone tumor.

• Journal of the Korean Association of Oral and Maxillofacial Surgeons
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• v.28 no.2
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• pp.114-125
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• 2002

This study was designed to evaluate the influence of fibroblasts or connective tissue from mouse oral mucosa on differentiation of neonatal mouse calvaria-derived osteoblasts and mineralization of bone nodules. Primary cell cultures from mouse calvarial osteoblasts and 2-4 passaged fibroblasts from oral mucosa were co-cultured in monolayer cultures, devided into 6 experimental group according to cell density or cell confluency. Osteoblasts were also co-cultured with fibroblasts in $Transwell^{(R)}$ culture plate with different co-cultured period according to osteoblast differentiation. The alkaline phosphatase activity were measured in monolayer cultures and cultures using $Transwell^{(R)}$. The mineralized bone nodules were presented by Von Kossa staining and density of mineralized nodules was measured by image analysis. The connective tissues with or without osteoblast seeding were cultured and examined histologically by Von Kossa and Trichrome Goldner staining. The results were as follows; 1. Prolonged maturation of matrix and delayed mineralization of bone nodules were resulted in monolayer cultures. 2. Co-culture of fibroblast with osteoblast using $Transwell^{(R)}$ during osteoblast proliferation stage stimulated proliferation of osteoblasts and increased alkaline phosphatase activity and mineralization of bone nodules. 3. Co-culture of fibroblast with osteoblast using $Transwell^{(R)}$ during matrix mineralization stage decreased and delayed mineralization of bone nodules. 4. In vitro cultured connective tissue with osteoblast seeding resulted in proliferation of osteoblasts and matrix formation with mineralization.

• Journal of the Korean Society for Precision Engineering
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• v.27 no.4
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• pp.95-101
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• 2010

The aim of this study was to detect longitudinal alterations on lumbar vertebral trabecular bone quality (microarchitecture and degree of mineralization) and bone mineral density (BMD) during pregnancy Virginal eighteen mice were used. Then, twelve mice were mated. Mice lumbar vertebrae were scanned before mate, at 7 days of pregnancy (early pregnancy, 6 mice) and immediately after delivery (late pregnancy, 6 mice) by using in-vivo micro-computed tomography Structural parameters, degree of mineralization and BMD were measured. During early pregnancy, there were no significant alterations on structural parameters, degree of mineralization and BMD. At late pregnancy, Tb.Th (11.8%) and BMD (12.7%) were significantly decreased and Tb.N (6.3%), Tb.Pf (43.0%) and BS/BV (15.1%) were significantly increased (p<0.05). Additionally, the lower degree of mineralization was increased, although, the higher degree of mineralization was decreased. These results indicated that the quality and BMD might be not affected during early pregnancy. At late pregnancy, however the bone quality and BMD were likely to be negatively affected.

• BMB Reports
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• v.45 no.10
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• pp.571-576
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• 2012

Radiotherapy is considered to cause detrimental effects on bone tissue eventually increasing bone loss and fracture risk. However, there is a great controversy on the real effects of irradiation itself on osteoblasts, and the mechanisms by which irradiation affects osteoblast differentiation and mineralization are not completely understood. We explored how X-ray radiation influences differentiation and bone-specific gene expression in mouse calvarial osteoblasts. Irradiation at 2 Gy not only increased differentiation and mineralization of the cells, but also upregulated the expression of alkaline phosphatase, type I collagen, osteopontin, and osteocalcin at early stages of differentiation. However, irradiation at higher doses (>2 Gy) did not stimulate osteoblast differentiation, rather it suppressed DNA synthesis by the cells without a toxic effect. Additional experiments suggested that transforming growth factor-beta 1 and runt-transcription factor 2 play important roles in irradiation- stimulated bone differentiation by acting as upstream regulators of bone-specific markers.

• Proceedings of the Korean Society of Precision Engineering Conference
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• 2009.10a
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• pp.31-32
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• 2009

• Journal of Acupuncture Research
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• v.22 no.2
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• pp.181-190
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• 2005

Background & Object : The differentiation of osteoblasts controlled by various growth factors and matrix proteins expression in bone. The aim of this study was to identify the Astragalus membranaceus that may induce the osteogenic activity in human osteoblast-like SaOS-2 cells. Methods : The osteogenic activity of Astragalus membranaceus were evaluated by WST-8 assay, ALP activity, RT-PCR analysis of VEGF, OCN, OPN, Col I mRNA, and ELISA or colorimetric analysis, and mineralization by Alizarin red staining in SaOS-2 cells. Results : Astragalus membranaceus had no effect on viability of osteoblastic cells, and dose dependently increased alkaline phosphatase (ALP) activity. Astragalus membranaceus markedly increased mRNA expression for vascular endothelial growth factor (VEGF), osteocalcin (OCN), osteopontin (OPN), and type I collagen (Col 1) in SaOS-2 cells. Extracellular accumulation of proteins such as VEGF, and Col I was increased in a dose-dependent manner. Also, Astragalus membranaceus significantly induced mineralization in the culture of SaOS-2 cells. Conclusion : This study showed that Astragalus membranaceus not affect on viability, but it enhanced ALP activity, VEGF, bone matrix proteins such as OCN, OPN and Col I, and mineralization in SaOS-2 cells. These results propose that Astragalus membranaceus plays an important role in osteoblastic bone formation, and possibly lead to the development of bone-forming drug.

• Journal of Periodontal and Implant Science
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• v.41 no.4
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• pp.167-175
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• 2011

Purpose: Periodontal ligament (PDL) cell differentiation into osteoblasts is important in bone formation. Bone formation is a complex biological process and involves several tightly regulated gene expression patterns of bone-related proteins. The expression patterns of bone related proteins are regulated in a temporal manner both in vivo and in vitro. The aim of this study was to observe the gene expression profile in PDL cell proliferation, differentiation, and mineralization in vitro. Methods: PDL cells were grown until confluence, which were then designated as day 0, and nodule formation was induced by the addition of 50 ${\mu}g$/mL ascorbic acid, 10 mM ${\beta}$-glycerophosphate, and 100 nM dexamethasone to the medium. The dishes were stained with Alizarin Red S on days 1, 7, 14, and 21. Real-time polymerase chain reaction was performed for the detection of various genes on days 0, 1, 7, 14, and 21. Results: On day 0 with a confluent monolayer, in the active proliferative stage, c-myc gene expression was observed at its maximal level. On day 7 with a multilayer, alkaline phosphatase, bone morphogenetic protein (BMP)-2, and BMP-4 gene expression had increased and this was followed by maximal expression of osteocalcin on day 14 with the initiation of nodule mineralization. In relationship to apoptosis, c-fos gene expression peaked on day 21 and was characterized by the post-mineralization stage. Here, various genes were regulated in a temporal manner during PDL fibroblast proliferation, extracellular matrix maturation, and mineralization. The gene expression pattern was similar. Conclusions: We can speculate that the gene expression pattern occurs during PDL cell proliferation, differentiation, and mineralization. On the basis of these results, it might be possible to understand the various factors that influence PDL cell proliferation, extracellular matrix maturation, and mineralization with regard to gene expression patterns.

• Journal of Biomedical Engineering Research
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• v.33 no.4
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• pp.213-222
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• 2012

While much has been learned about the mechanisms of metastatic spread of cancer to bone, there has been little headway in establishing guidelines for monitoring the alteration in bone quality and estimating fracture risk. The aims of this study are, therefore, 1) to evaluate bone quality induced by metastatic bone tumor by analyzing the characteristics on bone microarchitecture and degree of bone mineralization and 2) analyze fracture risk increased secondary to the bone quality changes by metastatic bone tumor through calculating mechanical rigidities based on in-vivo micro CT images. For this study, eighteen female SD rats (12 weeks old, approximate 250 g) were randomly allocated in Sham and Tumor groups. W256 (Walker carcinosarcoma 256 malignant breast cancer cell) was inoculated in the right femur (intraosseous injection) in Tumor group, while 0.9% NaCl (saline solution) was injected in Sham group. The right hind limbs of all rats were scanned by in-vivo micro-CT to acquire structural parameters and degree of bone mineralization at 0 week, 4 weeks, 8 weeks, and 12 weeks after surgery. At the same time, urine was collected by metabolic cages for a biochemical marker test in order to evaluate bone resorption. Then, bone metastasis had been directly identified by positron emission tomography. Finally, axial, bending and torsional rigidities had been calculated based on in-vivo micro CT images for predict fracture risk. The results of this study showed that metastatic bone tumor might induce significant decrease in bone quality and increase of fracture risk. This study may be helpful to monitoring a degree of bone metastasis and predicting fracture risk due to metastatic bone tumor. In addition, this noninvasive diagnostic methodology may be utilized for evaluating other bone metabolic diseases such as osteoporosis.

• Asian-Australasian Journal of Animal Sciences
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• v.27 no.4
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• pp.530-536
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• 2014

In a $2{\times}2$ factorial experiment the hypotheses tested were that the metabolic acid load caused by benzoic acid (BA) added to the feed affects bone mineralization of weanling pigs, and that a wide dietary calcium (Ca) to phosphorus (P) ratio in phytase-supplemented feeds with a marginal P concentration has a positive effect on bone mineralization. The four experimental diets, which contained 0.4% P and were supplemented with 1,000 FTU phytase/kg, contained either 5 g BA/kg or no BA and either 0.77% Ca or 0.57% Ca. The 68 four-week-old Large White pigs were fed the experimental diets ad libitum for six weeks and were then slaughtered. Benzoic acid increased feed intake (p = 0.009) and growth rate (p = 0.051), but did not influence the feed conversion ratio (p>0.10). Benzoic acid decreased the pH of the urine (p = 0.031), but did not affect breaking strength and mineralization of the tibia (p>0.10). The wide Ca:P ratio decreased feed intake (p = 0.034) and growth rate (p = 0.007) and impaired feed the conversion ratio (p = 0.027), but increased the mineral concentration in the fat-free DM of the tibia (p = 0.013) without influencing its breaking strength (p>0.10). The observed positive effect of the wide Ca:P ratio on bone mineralization may be attributed, at least in part, to the impaired feed conversion ratio, i.e. to the higher feed intake and consequently to the higher mineral intake per kg BW gain. The negative impact on animal performance of the wide dietary Ca:P ratio outweighs its potentially positive effect on bone mineralization, precluding its implementation under practical feeding conditions.