Hyperbaric oxygen therapy exerts anti-osteoporotic effects in obese and lean D-galactose-induced aged rats.

Obesity accelerates the aging processes, resulting in an aggravation of aging-induced osteoporosis. We investigated the anti-osteoporotic effect of hyperbaric oxygen therapy (HBOT) in obese- and lean-aged rats through measurement of cellular senescence, hypoxia, inflammation, antioxidants, and bone microarchitecture. Obese and lean male Wistar rats were injected with 150 mg/kg/day of D-galactose for 8 weeks to induce aging. Then, all rats were randomly given either sham or HBOT for 14 days. Metabolic parameters were determined. Expression by bone mRNA for cellular senescence, hypoxia, inflammation, antioxidative capacity, and bone remodeling were examined. Micro-computed tomography and atomic absorption spectroscopy were performed to evaluate bone microarchitecture and bone mineral profiles, respectively. We found that HBOT restored the alterations in the mRNA expression level of p16, p21, HIF-1α, TNF-α, IL-6, RANKL, RANK, NFATc1, DC-STAMP, Osx, ALP, and Col1a1 in the bone in obese-and lean- aging rats. In obese-aging rats, HBOT increased the level of expression of Sirt1 and CuZnSOD mRNA and diminished the expression level of HIF-2α and ctsk mRNA to the same levels as the control group. However, HBOT failed to alter catalase and OCN mRNA expression in obese-aged rats. HBOT partially improved the bone microarchitecture in obese-aged rats, but completely restored it in lean-aged rats. Interestingly, HBOT protected against obesity-induced demineralization in obese-aged rats. In summary, HBOT exerts an anti-osteoporotic effect in lean-aged rats and prevents some, but not all the negative effects of obese-aged conditions on bone health. Therefore, HBOT is considered as a potential therapy for aging-induced osteoporosis, regardless of obese status.

Oral calcium and vitamin D supplements differentially alter exploratory, anxiety-like behaviors and memory in male rats.

Oral calcium and calcium plus vitamin D supplements are commonly prescribed to several groups of patients, e.g., osteoporosis, fracture, and calcium deficiency. Adequate and steady extracellular calcium levels are essential for neuronal activity, whereas certain forms of calcium supplement (e.g., CaCO3) probably interfere with memory function. However, it was unclear whether a long-term use of ionized calcium (calcium chloride in drinking water ad libitum), vitamin D supplement (oral gavage) or the combination of both affected anxiety and memory, the latter of which was probably dependent on the hippocampal neurogenesis. Here, we aimed to determine the effects of calcium and/or vitamin D supplement on the anxiety- and memory-related behaviors and the expression of doublecortin (DCX), an indirect proxy indicator of hippocampal neurogenesis. Eight-week-old male Wistar rats were divided into 4 groups, i.e., control, calcium chloride-, 400 UI/kg vitamin D3-, and calcium chloride plus vitamin D-treated groups. After 4 weeks of treatment, anxiety-, exploration- and recognition memory-related behaviors were evaluated by elevated pulse-maze (EPM), open field test (OFT), and novel object recognition (NOR), respectively. The hippocampi were investigated for the expression of DCX protein by Western blot analysis. We found that oral calcium supplement increased exploratory behavior as evaluated by OFT and the recognition index in NOR test without any effect on anxiety behavior in EPM. On the other hand, vitamin D supplement was found to reduce anxiety-like behaviors. Significant upregulation of DCX protein expression was observed in the hippocampus of both calcium- and vitamin D-treated rats, suggesting their positive effects on neurogenesis. In conclusion, oral calcium and vitamin D supplements positively affected exploratory, anxiety-like behaviors and/or memory in male rats. Thus, they potentially benefit on mood and memory in osteoporotic patients beyond bone metabolism.

Running exercise with and without calcium supplementation from tuna bone reduced bone impairment caused by low calcium intake in young adult rats.

Inadequate calcium intake during childhood and adolescence is detrimental to bone metabolism. Here, we postulated that calcium supplement prepared from tuna bone with tuna head oil should benefit for skeletal development than CaCO3. Forty female 4-week-old rats were divided into calcium-replete diet (0.55% w/w, S1, n = 8) and low-calcium groups (0.15% w/w for 2 weeks; L; n = 32). Then L were subdivided into 4 groups (8/group), i.e., remained on L, L + tuna bone (S2), S2 + tuna head oil + 25(OH)D3 and S2 + 25(OH)D3. Bone specimens were collected at week 9. We found that 2 weeks on low calcium diet led to low bone mineral density (BMD), reduced mineral content, and impaired mechanical properties in young growing rats. Intestinal fractional calcium absorption also increased, presumably resulting from higher plasma 1,25(OH)2D3 (1.712 ± 0.158 in L vs. 1.214 ± 0.105 nM in S1, P < 0.05). Four-week calcium supplementation from tuna bone further increased calcium absorption efficacy, which later returned to the basal level by week 9. Calcium supplementation successfully restored BMD, bone strength and microstructure. However, 25(OH)D3 + tuna head oil + tuna bone showed no additive effect. Voluntary running also effectively prevented bone defects. In conclusion, both tuna bone calcium supplementation and exercise are effective interventions for mitigating calcium-deficient bone loss.

Development of a mobile application to improve exercise accuracy and quality of life in knee osteoarthritis patients: a randomized controlled trial.

INTRODUCTION: Knee Osteoarthritis (OA) is a degenerative joint disease that needs consistent exercise and an accurate understanding of the condition for long-term maintenance. While the accessibility of outpatient care is essential for disease management, many patients lack the resources to receive adequate healthcare. To address this challenge, we developed a not-for-profit interactive mobile application that provides a disease-specific educational background and a structured exercise regimen to patients. MATERIAL AND METHODS: "Rak Kao" (English translation: Love-Your-Knee) mobile application was designed to analyze the questionnaire data to assess the stage of knee OA and generate a personalized recommendation of treatment and exercise type using rule-based and Artificial Intelligence (AI) techniques. A single-blinded study was conducted with patients (n = 82) who were randomly assigned to the mobile application group (M-group) and the handout group (H-group). Patient groups were controlled for age, gender, BMI, onset of pain, grade of disease, education level, and occupation. Accuracy in performance of three prescribed knee exercises (catch-bend-down, stretch-touch-feet, and sit-stretch-hold) was evaluated. Clinical outcomes were evaluated before and after the 4-weeks program to assess the range of motion, symptoms, pain, physical activity, and quality of life via the KOOS and KSS scores. RESULTS: Completion of the study led to significantly more overall exercise accuracy in the M-group (76.2%) than the H-group (52.5%). Activities of daily life, quality of life, ability to do sports and recreational activities were significantly more improved in the M-group than the H-group (p < .01). No difference in the range of motion between groups. Satisfaction of patients' experience was higher in the M-group than the H-group (p = .001) after the 4-week regimen. CONCLUSIONS: With the better accuracy and outcomes for rehabilitation in the M-group than the H-group, we strongly recommend using our mobile application as a better alternative than handouts for exercises and information for patients with knee OA. TRIAL REGISTRATION: ClinicalTrials.gov: NCT03666585.

Fe3+ opposes the 1,25(OH)2D3-induced calcium transport across intestinal epithelium-like Caco-2 monolayer in the presence or absence of ascorbic acid.

Although iron is an essential element for hemoglobin and cytochrome synthesis, excessive intestinal iron absorption-as seen in dietary iron supplementation and hereditary disease called thalassemia-could interfere with transepithelial transport of calcium across the intestinal mucosa. The underlying cellular mechanism of iron-induced decrease in intestinal calcium absorption remains elusive, but it has been hypothesized that excess iron probably negates the actions of 1,25-dihydroxyvitamin D [1,25(OH)2D3]. Herein, we exposed the 1,25(OH)2D3-treated epithelium-like Caco-2 monolayer to FeCl3 to demonstrate the inhibitory effect of ferric ion on 1,25(OH)2D3-induced transepithelial calcium transport. We found that a 24-h exposure to FeCl3 on the apical side significantly decreased calcium transport, while increasing the transepithelial resistance (TER) in 1,25(OH)2D3-treated monolayer. The inhibitory action of FeCl3 was considered rapid since 60-min exposure was sufficient to block the 1,25(OH)2D3-induced decrease in TER and increase in calcium flux. Interestingly, FeCl3 did not affect the baseline calcium transport in the absence of 1,25(OH)2D3 treatment. Furthermore, although ascorbic acid is often administered to maximize calcium solubility and to enhance intestinal calcium absorption, it apparently had no effect on calcium transport across the FeCl3- and 1,25(OH)2D3-treated Caco-2 monolayer. In conclusion, apical exposure to ferric ion appeared to negate the 1,25(OH)2D3-stimulated calcium transport across the intestinal epithelium. The present finding has, therefore, provided important information for development of calcium and iron supplement products and treatment protocol for specific groups of individuals, such as thalassemia patients and pregnant women.

Hyperbaric oxygen therapy improves age induced bone dyshomeostasis in non-obese and obese conditions.

AIMS: To investigate the effects of hyperbaric oxygen therapy (HBOT) on metabolic disturbance, aging and bone remodeling in D-galactose-induced aging rats with and without obesity by determining the metabolic parameters, aging and oxidative stress markers, bone turnover markers, bone microarchitecture, and bone biomechanical strength. MATERIALS AND METHODS: Male Wistar rats were fed either a normal diet (ND; n = 18) or a HFD (n = 12) for 22 weeks. At week 13, vehicle (0.9% NaCl) was injected into ND-fed rats (NDV; n = 6), while 150 mg/kg/day of D-galactose was injected into 12 ND-fed rats (NDD) and 12 HFD-fed rats (HFDD) for 10 weeks. At week 21, rats were treated with either sham (NDVS, NDDS, or HFDDS; n = 6/ group) or HBOT (NDDH, or HFDDH; n = 6/group) for 14 days. Rats were then euthanized. Blood samples, femora, and tibiae were collected. KEY FINDINGS: Both NDD and HFDD groups developed aging as indicated by increased AGE level, increased inflammation and oxidative stress as shown by raised serum TNF-α and MDA levels, impaired bone remodeling as indicated by an increase in levels of CTX-1, TRACP-5b, and impaired bone structure/strength, when compared with those of the NDVS group. HFD aggravated these indicators of bone dyshomeostasis in D-galactose-treated rats. HBOT restored bone remodeling and bone structure/strength in the NDD group, however HBOT ameliorated bone dyshomeostasis in the HFDD group. SIGNIFICANCE: HBOT is a potential intervention to decrease the risk of osteoporosis and bone fracture in aging with or without obesity.

Regulation of vitamin D system in skeletal muscle and resident myogenic stem cell during development, maturation, and ageing.

Skeletal muscle exhibits enormous plasticity throughout life, however, less is known regarding how the stages of growth regulate its local vitamin D system. Herein, we investigated serum 25(OH)D3 and Ca2+ levels along with the vitamin D system in skeletal muscle and resident myogenic stem cells of male C57BL/6 mice during development, maturation, and ageing. Compared with development, significant increases in vitamin D receptor (VDR) protein expression in mature and aged muscles were associated with increased serum 25(OH)D3 and centronucleated fibres, respectively. The substantial increase in VDR protein expression in aged muscle was also related to reduced downstream mTOR signalling protein expression which was more pronounced in fast-glycolytic compared to slow-oxidative muscles. Intriguingly, serum Ca2+ and vitamin D-metabolising enzyme (CYP27B1 and CYP24A1) levels in skeletal muscle were not different across age. In primary cell culture, nuclear VDR protein was expressed in undifferentiated skeletal muscle stem cells (SMSC) after 1α,25(OH)2D3 treatment. Additionally, a diminished response to 1α,25(OH)2D3 was observed with age as there was a rapid commitment of SMSC towards differentiation under growth-stimulating conditions. Collectively, understanding the local vitamin D system in skeletal muscle could help develop effective interventions for vitamin D supplementation to improve skeletal muscle mass and function during ageing.

Altered gut microbiota ameliorates bone pathology in the mandible of obese-insulin-resistant rats.

PURPOSE: The chronic consumption of a high-fat diet (HFD) induces obese-insulin resistance and impairs jawbone health via gut dysbiosis-stimulated inflammatory process. Our previous studies demonstrated that the probiotic Lactobacillus paracasei HII01, prebiotic xylooligosaccharide (XOS), and synbiotics improved several vital organ functions by reducing gut dysbiosis in HFD-induced obese rats. However, the impacts on the cellular level of jawbone microarchitecture have not been examined. Here, we hypothesized that the supplementation of L. paracasei HII01, XOS, and synbiotics ameliorated the bone microarchitectural pathology in HFD-fed rats by reducing systemic inflammation and other metabolic parameters. METHODS: The dietary regimes (normal or high-fat diet) were provided to 48 male Wistar rats throughout 24-week experiment. After week 12, rats were given either a vehicle, pro-, pre-, or synbiotic for an additional 12 weeks before being killed. Then, blood analyses and bone histomorphometry of the jawbones were performed. RESULTS: The HFD-fed rats developed obese-insulin resistance with significantly elevated systemic inflammation. Bone histomorphometry of these rats showed a decrease in trabecular thickness with increased osteoclasts and active erosion surfaces. Mineral apposition and bone-formation rates were also remarkably diminished. The treatment with pro-, pre-, and synbiotics equally improved metabolic disturbance, reduced systemic inflammation, increased trabecular thickness, decreased osteoclasts and active erosion surfaces and restored mineral apposition and bone-formation rates. CONCLUSION: The probiotic L. paracasei HII01, prebiotic XOS, and the synbiotics had similarly beneficial effects to improve jawbone microarchitecture in HFD-fed rats by possibly ameliorating osteoclast-related bone resorption and potentiating bone-formation activities.

Activation of calcium-sensing receptor by allosteric agonists cinacalcet and AC-265347 abolishes the 1,25(OH)2D3-induced Ca2+ transport: Evidence that explains how the intestine prevents excessive Ca2+ absorption.

Long-term high-calcium intake and intestinal calcium hyperabsorption are hazardous to the body. It is hypothesized that enterocytes possess mechanisms for preventing superfluous calcium absorption, including secretion of negative regulators of calcium absorption and utilization of calcium-sensing receptor (CaSR) to detect luminal calcium. Herein, Caco-2 monolayers were treated with high doses of 1,25(OH)2D3 to induce calcium hyperabsorption or directly exposed to high apical calcium. The expression of counterregulatory factor of calcium absorption, fibroblast growth factor (FGF)-23, was also investigated in the intestine of lactating rats, which physiologically exhibit calcium hyperabsorption. We found that FGF-23 expression was enhanced in all intestinal segments of lactating rats. In Caco-2 monolayers, high apical calcium and 1,25(OH)2D3 induced FGF-23 secretion into culture media. FGF-23 antagonized 1,25(OH)2D3-induced calcium transport and led to a significant, but small, change in paracellular permeability. Furthermore, high-dose 1,25(OH)2D3 upregulated FGF-23 expression, which was prevented by CaSR inhibitors. Activation of apical CaSR by cinacalcet and AC-265347 abolished 1,25(OH)2D3-induced calcium transport in a dose-dependent manner. In conclusion, the intestinal FGF-23 expression was upregulated in conditions with calcium hyperabsorption, presumably to help protect against excessive calcium absorption, while CaSR probably monitored calcium in the lumen and induced FGF-23 production for preventing superfluous calcium uptake.

Cyperenoic acid suppresses osteoclast differentiation and delays bone loss in a senile osteoporosis mouse model by inhibiting non-canonical NF-κB pathway.

Cyperenoic acid is a terpenoid isolated from the root of a medicinal plant Croton crassifolius with a wide range of biological activities. In this study, the effects of cyperenoic acid on osteoclast differentiation were investigated both in vitro and in vivo using receptor activator of nuclear factor-κB ligand (RANKL)-induced bone marrow-derived osteoclasts and senescence-accelerated mouse prone 6 (SAMP6). Cyperenoic acid significantly suppressed RANKL-induced osteoclast differentiation at the concentrations with no apparent cytotoxicity. The half maximum inhibitory concentration (IC50) for osteoclast differentiation was 36.69 µM ± 1.02. Cyperenoic acid treatment evidently reduced the expression of two key transcription factors in osteoclast differentiation, NFATc1 and c-Fos. Detailed signaling analysis revealed that cyperenoic acid did not affect MAPK pathways and canonical NF-κB pathway but impaired activation of p100/p52 in the non-canonical NF-κB pathway upon RANKL stimulation. Moreover, the expression of osteoclast-related genes, nfatc1, ctsk, irf8, acp5 and cfos were disrupted by cyperenoic acid treatment. The bone resorption activity by cyperenoic acid-treated osteoclasts were impaired. In a senile osteoporosis mouse model SAMP6, mice fed on diet supplemented with cyperenoic acid showed delay in bone loss, compared to the control. Taken together, plant-derived cyperenoic acid shows great potential as therapeutic agent for osteoporosis.

Intestinal calcium transport and its regulation in thalassemia: interaction between calcium and iron metabolism.

Osteoporosis and derangement of calcium homeostasis are common complications of thalassemia. Despite being an important process for bone and calcium metabolism, little is known about intestinal calcium transport in thalassemia. Recent reports of decreases in both intestinal calcium transport and bone mineral density in thalassemic patients and animal models suggested that defective calcium absorption might be a cause of thalassemic bone disorder. Herein, the possible mechanisms associated with intestinal calcium malabsorption in thalassemia are discussed. This includes alterations in the calcium transporters and hormonal controls of the transcellular and paracellular intestinal transport systems in thalassemia. In addition, the effects of iron overload on intestinal calcium absorption, and the reciprocal interaction between iron and calcium transport in thalassemia are elaborated. Understanding the mechanisms underlining calcium malabsorption in thalassemia would lead to development of therapeutic agents and mineral supplements that restore calcium absorption as well as prevent osteoporosis in thalassemic patients.

Prolonged exposure to 1,25(OH)2D3 and high ionized calcium induces FGF-23 production in intestinal epithelium-like Caco-2 monolayer: A local negative feedback for preventing excessive calcium transport.

Overdose of oral calcium supplement and excessive intestinal calcium absorption can contribute pathophysiological conditions, e.g., nephrolithiasis, vascular calcification, dementia, and cardiovascular accident. Since our previous investigation has indicated that fibroblast growth factor (FGF)-23 could abolish the 1,25-dihydroxyvitamin D3 [1,25(OH)2D3]-enhanced calcium absorption, we further hypothesized that FGF-23 produced locally in the enterocytes might be part of a local negative feedback loop to regulate calcium absorption. Herein, 1,25(OH)2D3 was found to enhance the transcellular calcium transport across the epithelium-like Caco-2 monolayer, and this stimulatory effect was diminished by preceding prolonged exposure to high-dose 1,25(OH)2D3 or high concentration of apical ionized calcium. Pretreatment with a neutralizing antibody for FGF-23 prevented this negative feedback regulation of calcium hyperabsorption induced by 1,25(OH)2D3. FGF-23 exposure completely abolished the 1,25(OH)2D3-enhanced calcium transport. Western blot analysis revealed that FGF-23 expression was upregulated in a dose-dependent manner by 1,25(OH)2D3 or apical calcium exposure. Finally, calcium-sensing receptor (CaSR) inhibitors were found to prevent the apical calcium-induced suppression of calcium transport. In conclusion, prolonged exposure to high apical calcium and calcium hyperabsorption were sensed by CaSR, which, in turn, increased FGF-23 expression to suppress calcium transport. This local negative feedback loop can help prevent unnecessary calcium uptake and its detrimental consequences.

Fortified tuna bone powder supplementation increases bone mineral density of lactating rats and their offspring.

BACKGROUND: Breastfeeding leads to bone calcium loss for milk production, resulting in progressive maternal osteopenia. Calcium supplement from natural sources has been postulated to be more beneficial to bone health than purified CaCO3 because natural sources also contain other nutrients such as certain amino acids that might enhance calcium metabolism. Herein, we examined the effect of calcium supplementation from tuna bone powder and CaCO3 on bones of dams and the offspring. RESULTS: Both forms of calcium supplement, i.e. tuna bone powder and CaCO3 , increased maternal bone mineral density (BMD). However, bone histomorphometry revealed that only tuna bone had beneficial effect on maternal bone microstructure, i.e. increased bone formation, decreased bone resorption and increased in bone volume. Regarding the mechanical properties, the decreased ultimate load in non-supplement lactating mothers was restored to the load seen in nulliparous animals by calcium supplementation. Moreover, both tuna bone and CaCO3 supplementation in mothers led to increased milk calcium concentration and consequently increased BMD in the growing offspring. CONCLUSION: Calcium supplement from tuna bone powder was effective in preventing maternal osteopenia. Tuna bone, which is a readily available fishing industrial waste, is a good alternative source of calcium supplement that increases BMD in both lactating mothers and the neonates. © 2017 Society of Chemical Industry.

Site-Specific Onset of Low Bone Density and Correlation of Bone Turnover Markers in Exclusive Breastfeeding Mothers.

PURPOSE: Lactation often affects calcium metabolism and induces bone loss. Calcium supplementation and a high calcium diet are recommended to prevent bone loss, especially during inadequate calcium intake. Our study aimed at determining bone loss in breastfeeding mothers, and if it occurred, whether it was site specific and there were correlations between serum bone turnover markers. MATERIALS AND METHODS: Since the 6-month exclusive breastfeeding is usually recommended in several countries, our study examined bone mineral density (BMD) in early (1-2 month), mid (3-4 month)-, and late (5-6 month) lactation compared with nonpregnant, nonlactating control women. Site-specific bone loss was monitored in lumbar vertebrae and femora. Bone turnover markers, that is, C-terminal telopeptide of type 1 collagen and N-terminal propeptide of type 1 collagen (P1NP), were determined by electrochemiluminescence immunoassays. RESULTS: The onset of bone loss in exclusive breastfeeding mothers was site specific, for example, in the lumbar bone at mid-lactation and in the femoral bone in late lactation. Serum ionized calcium levels in late lactation were lower than the normal levels. In addition, a correlation was found between bone turnover marker, P1NP, and femoral BMD. CONCLUSIONS: The onset of bone loss in exclusive breastfeeding mothers was site specific, and the lumbar bone was a vulnerable and perhaps better representative site for bone loss detection. It was suggested that the optimal starting time for calcium supplementation should be before the mid-lactation when the bone loss was observed. In addition, the biochemical marker that best predicted the onset of bone loss in lactating women was P1NP.

CFTR-mediated anion secretion across intestinal epithelium-like Caco-2 monolayer under PTH stimulation is dependent on intermediate conductance K+ channels.

Parathyroid hormone (PTH), a pleiotropic hormone that maintains mineral homeostasis, is also essential for controlling pH balance and ion transport across renal and intestinal epithelia. Optimization of luminal pH is important for absorption of trace elements, e.g., calcium and phosphorus. We have previously demonstrated that PTH rapidly stimulated electrogenic [Formula: see text] secretion in intestinal epithelial-like Caco-2 monolayers, but the underlying cellular mechanism, contributions of other ions, particularly Cl- and K+, and long-lasting responses are not completely understood. Herein, PTH and forskolin were confirmed to induce anion secretion, which peaked within 1-3 min (early phase), followed by an abrupt decay and plateau that lasted for 60 min (late phase). In both early and late phases, apical membrane capacitance was increased with a decrease in basolateral capacitance after PTH or forskolin exposure. PTH also induced a transient increase in apical conductance with a long-lasting decrease in basolateral conductance. Anion secretion in both phases was reduced under [Formula: see text]-free and/or Cl--free conditions or after exposure to carbonic anhydrase inhibitor (acetazolamide), CFTR inhibitor (CFTRinh-172), Na+/H+ exchanger (NHE)-3 inhibitor (tenapanor), or K+ channel inhibitors (BaCl2, clotrimazole, and TRAM-34; basolateral side), the latter of which suggested that PTH action was dependent on basolateral K+ recycling. Furthermore, early- and late-phase responses to PTH were diminished by inhibitors of PI3K (wortmannin and LY-294002) and PKA (PKI 14-22). In conclusion, PTH requires NHE3 and basolateral K+ channels to induce [Formula: see text] and Cl- secretion, thus explaining how PTH regulated luminal pH balance and pH-dependent absorption of trace minerals.

Pueraria mirifica alleviates cortical bone loss in naturally menopausal monkeys.

Since the in vitro and in vivo anti-osteoporotic effects of Pueraria mirifica (PM) in rodents have been verified, its activity in menopausal monkeys was evaluated as required before it can be applicable for human use. In this study, postmenopausal osteoporotic monkeys were divided into two groups (five per group), and fed daily with standard diet alone (PMP0 group) or diet mixed with 1000 mg/kg body weight (BW) of PM powder (PMP1000 group) for 16 months. Every 2 months, the bone mineral density (BMD), bone mineral content (BMC) and bone geometry parameters (cortical area and thickness and periosteal and endosteal circumference) at the distal radius and proximal tibia were determined using peripheral quantitative computed tomography together with plasma and urinary bone markers. Compared with the baseline (month 0) values, the cortical, but not trabecular, BMDs and BMCs and the cortical area and thickness at the metaphysis and diaphysis of the radius and tibia of the PMP0 group continuously decreased during the 16-month study period. In contrast, PMP1000 treatment ameliorated the bone loss mainly at the cortical diaphysis by decreasing bone turnover, as indicated by the lowered plasma bone-specific alkaline phosphatase and osteocalcin levels. Generally, changes in the cortical bone geometry were in the opposite direction to the cortical bone mass after PMP1000 treatment. This study indicated that postmenopausal monkeys continuously lose their cortical bone compartment, and they have a higher possibility for long bone fractures. Oral PMP treatment could improve both the bone quantity (BMC and BMD) and quality (bone geometry).

Anti-osteoporotic effects of Pueraria candollei var. mirifica on bone mineral density and histomorphometry in estrogen-deficient rats.

Although it has been clearly shown that Pueraria mirifica and its phytoestrogens can mimic estrogen in preventing bone loss, as osteoporosis is an asymptomatic disease, the therapeutic effects of P. mirifica should be acknowledged. In this study, 6-month-old female rats were ovariectomized, kept for 4 weeks to induce bone loss, divided into five groups, and treated with P. mirifica at doses of 0, 5, 25, and 50 mg/kg BW/day (PM0, PM5, PM25, and PM50 groups, respectively) or 7 mg/kg BW/day of puerarin (PU group) for 12 weeks. Only the trabecular bone mineral densities (BMDs) of tibia metaphysis (at the 12th, 14th, and 16th week) and total and trabecular BMDs of L4 (at the 16th week) of the PM50 group were significantly higher than those of the PM0 group. However, the BMDs of tibia metaphysis and L4 at the 16th week of the study period were kept significantly lower than those of the 0 week, and the BMD was also significantly lower than that of the 4th week for tibia metaphysis. The trabecular bone area (BV/TV), trabecular number (Tb.N), and osteoblast surface (Ob.S/BS) were significantly higher, and trabecular space (Tb.Sp) was significantly lower in the PM50 group, as compared with those of the PM0 group. This study indicates that P. mirifica could be used as an anti-osteoporotic agent for postmenopausal women. Since P. mirifica could mainly retain bone mass at the levels before bone loss is initiated, the use of other anabolic agents in combination with P. mirifica is recommended for osteoporotic patients.

Positive long-term outcomes from presuckling calcium supplementation in lactating rats and the offspring.

Adequate dietary calcium intake and the enhanced intestinal calcium absorption in lactating mothers have long been postulated to prevent maternal bone loss and benefit neonatal bone growth. We recently showed that calcium supplementation just before breastfeeding efficiently alleviated lactation-induced bone loss in dams as well as increased milk calcium concentration, which led to higher bone mineral density (BMD) in the newborns. Herein, we further elaborated in detail how presuckling calcium supplements worked in lactating rats and how they benefited bone growth in the offspring. As revealed by bone histomorphometry, presuckling supplement with calcium alone reduced the osteoclast surface and active erosion surface, leading to an increase in trabecular thickness without changes in trabecular separation or number in dams. The beneficial effects of presuckling calcium supplements, particularly the regimen containing glucose and galactose that enhanced intestinal calcium absorption, were found to last for 3 mo postweaning, although it could not restore estrogen-deficient osteopenia induced by ovariectomy. Regarding the neonatal benefits, pups nursed by calcium-supplemented dams exhibited increases in trabecular BMD, which could be observed even at the age of 27 wk. Bone elongation was also greater in pups of calcium-supplemented dams, which was due possibly to accelerated growth plate chondrocyte turnover. It could be concluded that calcium supplements markedly diminished the lactation-induced osteopenia in dams and positively affected BMD and bone elongation in growing rats. Therefore, presuckling calcium supplementation in lactating mothers is an effective strategy for promoting a long-lasting high bone density for both mother and the offspring.

Pueraria mirifica extract and puerarin enhance proliferation and expression of alkaline phosphatase and type I collagen in primary baboon osteoblasts.

Phytoestrogen-rich Pueraria mirifica (PM) tuberous extract is a promising candidate for the development of anti-osteoporosis drugs for postmenopausal women, but its action has never been validated in humans or in non-human primates, which are more closely related to humans than rodents. In vitro study of non-human primate osteoblasts is thus fundamental to prepare for in vivo studies of phytoestrogen effects on primate bone. This study aimed to establish a culture system of baboon primary osteoblasts and to investigate the effects of PM extract and its phytoestrogens on these cells. Primary osteoblasts from adult baboon fibulae exhibited osteoblast characteristics in regard to proliferation, differentiation, mineralization, and estrogen receptor expression. They responded to 17ß-estradiol by increased proliferation rate and mRNA levels of alkaline phosphatase (ALP), type I collagen, and osteocalcin. After being exposed for 48 h to 100 µg/ml PM extract, 1000 nM genistein, or 1000 nM puerarin, primary baboon osteoblasts markedly increased the rate of proliferation and mRNA levels of ALP and type I collagen without changes in Runx2, osterix, or osteocalcin expression. PM extract, genistein, and puerarin also decreased the RANKL/OPG ratio, suggesting that they could decrease osteoclast-mediated bone resorption. However, neither PM extract nor its phytoestrogens altered calcium deposition in osteoblast culture. In conclusion, we have established baboon primary osteoblast culture, which is a new tool for bone research and drug discovery. Furthermore, the present results provide substantial support for the potential of PM extract and its phytoestrogens to be developed as therapeutic agents against bone fragility.

Pre-suckling calcium supplementation effectively prevents lactation-induced osteopenia in rats.

During lactation, osteoclast-mediated bone resorption and intestinal calcium hyperabsorption help provide extra calcium for lactogenesis. Since the suckling-induced surge of pituitary prolactin (PRL) rapidly stimulates calcium absorption in lactating rats, it is hypothesized that pre-suckling oral calcium supplementation should be an efficient regimen to shift the calcium source from bone to diet, thereby slowing lactation-induced osteopenia. Our results showed that 30-min suckling markedly stimulated maternal duodenal calcium transport, which returned to the baseline at 45 min. Lactating rats given 4 mg/kg per dose calcium via a gavage tube at 90 min pre-suckling 4 doses a day for 14 days prevented a decrease in bone mineral density (BMD) of long bones and vertebrae. On the other hand, a single-dose supplementation, despite the same amount of calcium per day, appeared less effective. Because glucose and galactose further stimulated duodenal calcium transport in lactating rats, pre-suckling calcium supplement containing both sugars successfully normalized plasma ionized calcium and led to better bone gain than that with calcium alone. A histomorphometric study revealed that lactating rats given pre-suckling calcium plus monosaccharide supplement manifested greater trabecular bone volume and thickness and exhibited less eroded surface than in vehicle-treated lactating rats. Beneficial effects of the 14-day calcium supplementation persisted until 6 mo postweaning in dams and also elevated the baseline BMD of the offspring. In conclusion, our proof-of-concept study has corroborated that pre-suckling calcium supplements, especially regimens containing monosaccharides, are efficient in preventing osteopenia in lactating rats and could increase bone density in both breastfeeding mothers and neonates.