The effects of iQPR-H2O on cell proliferation, differentiation and mineral deposition as well as bone formation were analyzed in this study under in vitro condition using NIH3T3 fibroblast and MC3T3-E1 osteoblastic cell lines, and under in vivo condition using mouse model of osteoporosis as described previously . NIH3T3 cells treated with iQPR-H2O displayed an increased ability to proliferate. Both NIH3T3 and MC3T3-E1 cells in iQPR-H2O-containing medium showed higher levels of differentiation compared with control cells. In vivo studies revealed that iQPR-H2O increased BMD in OVX-SAMP8 mice, indicating that under long-term induction, osteoblasts may be capable of differentiation and improving the symptoms of osteoporosis.
MC3T3-E1 cells showed a sign of differentiation i.e. significantly higher ALP level than control, 7 days after culture in iQPR-H2O-containing medium. Further, expression of both OPN and RUNX2 mRNA was higher in the iQPR-H2O group compared with the control group after 28 days of culture. This observation is similar to that from a previous report showing that ALP levels were significantly higher 7 days after MC3T3-E1 cells were stimulated with ascorbic acid and b-glycerophosphate . OPN has been reported to modulate both mineralization and bone resorption, and RUNX2 is one of the major regulators of OPN in osteoblastic cells . Increased expression of both OPN and RUNX2 28 days after the culture suggests that iQPR-H2O may have promoted differentiation and mineralization of MC3T3-E1 cells. In contrast to MC3T3-E1 cells, increased differentiation of NIH3T3 cells was only observed in cells cultured for 14 to 28 days in the presence of iQPR-H2O. There were no statistically significant differences in ALP expression between the iQPR-H2O-treated and control cells at earlier time points (ie, within 7 days). This may be explained by the cells being in a proliferative state, which has been reported to delay differentiation , within the first 7 days of culture.
Previous studies of bone remodeling have reported that there are five stages to this process: activation, resorption, reversal, formation, and quiescence. Progression from activation to resorption was found to take place over two weeks, whereas three to four months transpired between reversal and formation, and finally quiescence [23, 24]. In the present study, OVX-SAMP8 mice exhibited increased bone density after four months treatment with iQPR-H2O. This time frame is comparable to that over which bone remodeling takes place. H&E findings corroborated the BMD analysis findings.
Bone densities in iQPR-H2O-treated OVX-SAMP8 mice declined between zero and two months and then rebounded during the third month. This presumably reflects osteoclast resorption of old bone caused by secretion of ALP, collagenase, lysosomal enzymes, and proteases, followed by stimulation and recruitment of osteoblasts into the site of osteoporosis. The formation period is a slow phase, which takes approximately 35 to 40 days, which coincides with the BMD changes observed over the four-month period in the present study.
Of note, we found that left knee BMD, as determined by bone densitometry was not significantly increased in mice treated with iQPR-H2O compared with control. This is a very puzzling finding for which we do not have an explanation. Indeed, the lack of a significant change in the left knee BMD as determined by bone densitometry is inconsistent with the findings for all other measures of BMD and bone density. We can only assume that the observed BMD findings are a statistical anomaly.
iQPR-H2O has approximately 60% less calcium than unprocessed mineral water. It is well known that inadequate intake of vitamin D and calcium can lead to reduced calcium absorption, a higher rate of bone remodeling, and increased bone loss. Although we have not assessed calcium homeostasis in mice treated with iQPR-H2O, we do not believe that a 60% reduction in the calcium content of drinking water would significantly alter calcium homeostasis given that the vast majority of calcium is obtained through the diet.
The mechanism by which iQPR-H2O influences bone remodeling remains to be investigated. iQPR-H2O may have increased the uptake of bone-associated elements from the diet in the experimental animals. As compared with unprocessed mineral water, iQPR-H2O had relatively high concentrations of magnesium, zinc, and silicon. Although the mechanism associating with the change in the level of elements is still unknown, this outcome is reproducible.
The importance of magnesium, zinc, and silicon and their deficiencies on BMD has been demonstrated in numerous studies. For example, magnesium deficiency has been associated with hypertension, cardiac arrhythmias, myocardial infarction, hypokalemia and hypocalcemia . Epidemiological studies revealed that BMD of the spine was significantly correlated with magnesium intake in premenopausal women . A cross-sectional study reported a significant correlation between magnesium intake and hip BMD men aged 69-97 years .
Several studies have assessed the effect of magnesium therapy for osteoporosis with varied results. Specifically, a prospective study in which postmenopausal osteoporotic women were given 250 mg of magnesium per day reported a significant 2.8% increase in the bone density of the distal radius at one year; however, no significant differences were shown at two years, which may be due to the small number (n = 10) of patients completing the study . A retrospective study in which postmenopausal women received 200 mg of magnesium per day resulted in a small, non significant 1.6% rise in lumbar spine bone density, and no differences were reported in the femur . In another small uncontrolled trial, a significant increase in the bone density of the proximal femur and lumbar spine was found in patients with gluten-sensitive enteropathy who received 504-576 mg of magnesium per day for two years .
Discrepant effects of zinc on bone progenitor cells have been reported. For example, in bone marrow cells, the highest ALP activity was observed when they were cultured on the ceramics with 1.26 wt% zinc; the ceramics released zinc ions at concentrations ranging from 2.2 to 7.2 μg/mL into the culture medium . Zinc ions were incorporated into mineralized areas produced by bone marrow cells. In addition, bone marrow cells cultured in medium supplemented with 100 μM ZnCl2 had significantly increased the ALP activity . However, Popp et al.  reported no differences in rat bone marrow stromal cell number, ALP activity, total protein content, collagen synthesis, or matrix mineralization in response to zinc supplementation.
Associations between silicon intake and BMD have also been seen. The most readily bioavailable source of silicon in the diet includes drinking water as well as other fluids, accounting for ≥ 20% of the total dietary intake . Beneficial effects on bone and other connective tissues have been observed upon silicon supplementation. For example, dietary silicon intake is associated with higher bone mineral density (BMD) . In osteoporotic subjects, silicon supplementation with monomethyl trisilanol resulted in increased bone volume and increases in femoral and lumbar spine BMD .
One limitation of this study is that increasing the energy state of water after processing with steady device of matter energy was not measured quantitatively, and that the mechanism associating with the change in the level of elements after the process was unknown. A further limitation is that ALP activity levels were found to increase with time in control MC3T3-E1 cells. This finding is puzzling and suggests that the cells were undergoing spontaneous differentiation under the culture conditions without extracellular stimulation.