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This research focuses on the synthesis of composite CoFe2O4 (CoF) nanoparticles coated with SiO2 using hydroxyapatite (HAp) as a template (CoFSi) and the pH controlled release of indomethacin (IMC) drug from this nanocomposite. The formation of the CoFe2O4-HAp (CoFHAp) nanoparticles and of the CoFe2O4@SiO2 (CoFSi) nanoparticles were monitored using XRD, FT-IR, that TEM, SEM, and ED studies. The magnetic properties are measured with a VSM. The TEM images showed the CoFe2O4 to have a core–shell structure encapsulated by a silica coating with an average size of 50 nm. BET showed the mean pore sizes of the silica shell to be approximately 10–20 nm and the surface area to be 212.8 m2 g−1. Magnetization (Ms) and remnant (Mr) magnetic properties of CoFSi nanocomposite decreased upon silica coating (from ∼14 to 2 emu g−1 for Ms of CoF and CoFSi, respectively). In contrast, the coercivity (Hc) of the uncoated CoFe2O4 (317 Oe) is lower than that of the coated nanocomposites (1250 Oe of CoFSi). In vitro drug loading of the CoFSi nanoparticles and its pH-responsive (pH = 4.0 and 7.0) controlled release were investigated using IMC as a model drug. It was seen that the release of the IMC drug was significantly faster at a pH of 4.0 compared to that at a neutral pH 7.0. Furthermore, the lower concentration of ions release (iron and cobalt) from CoFSi was observed after aging for 50 h.

Besides being a Ca2+-regulating hormone, parathyroid hormone (PTH) has also been shown to regulate epithelial transport of certain ions, such as Cl−, HCO3−, and Na+, particularly in the kidney. Although the intestinal epithelium also expressed PTH receptors, little was known regarding its mechanism in the regulation of intestinal ion transport. We investigated the ion regulatory role of PTH in intestinal epithelium-like Caco-2 monolayer by Ussing chamber technique and alternating current impedance spectroscopy. It was found that Caco-2 cells rapidly responded to PTH within 1 min by increasing apical HCO3−secretion. CFTR served as the principal route for PTH-stimulated apical HCO3−efflux, which was abolished by various CFTR inhibitors, namely, NPPB, glycine hydrazide-101 (GlyH-101), and CFTRinh-172, as well as by small interfering RNA against CFTR. Concurrently, the plasma membrane resistance was decreased with no changes in the plasma membrane capacitance or paracellular permeability. HCO3−was probably supplied by basolateral uptake via the electrogenic Na+-HCO3−cotransporter and by methazolamide-sensitive carbonic anhydrase, while the resulting intracellular H+might be extruded by both apical and basolateral Na+/H+exchangers. Furthermore, the PTH-stimulated HCO3−secretion was markedly reduced by protein kinase A (PKA) inhibitor (PKI 14–22 amide) and phosphoinositide 3-kinase (PI3K) inhibitors (wortmannin and LY-294002), but not by intracellular Ca2+chelator (BAPTA-AM) or protein kinase C inhibitor (GF-109203X). In conclusion, the present study provided evidence that PTH directly and rapidly stimulated apical HCO3−secretion through CFTR in PKA- and PI3K-dependent manner, which was a novel noncalciotropic, ion regulatory action of PTH in the intestinal epithelium.

Parathyroid hormone (PTH) was recently demonstrated to enhance the HCO(3)(-) secretion through the apical anion channel, cystic fibrosis transmembrane conductance regulator (CFTR), but how the HCO(3)(-) entered the epithelial cells was not well understood, in part, due to the lack of specific inhibitors of the basolateral HCO(3)(-) transporters. Moreover, the function of the PTH-stimulated HCO(3)(-) secretion has never been investigated in vivo. Here, we designed three specific pairs of small interfering RNA sequences to simultaneously knockdown three variants of the electrogenic Na(+)/HCO(3)(-) co-transporter (NBCe)-1 in the intestinal epithelium-like Caco-2 monolayer. The results showed that NBCe1 mRNA levels were markedly reduced, and the PTH-induced transepithelial current and voltage changes were diminished after triple knockdown as determined by quantitative real-time PCR and Ussing chamber technique, respectively. An in vivo ligated intestinal loop study further showed that there was an increased fluid secretion, presumably driven by HCO(3)(-) transport, in the ileum, but not in jejunum or colon, of rats administered intravenously with 2 μg/kg body weight of rat PTH 1-34. Therefore, the present results suggested that PTH stimulated intestinal HCO(3)(-) secretion, particularly in the ileum, by inducing the basolateral HCO(3)(-) uptake via NBCe1.

In the present study, composite scaffolds of chitosan-graft-poly(methyl methacrylate) (Chi-g-PMMA) and mineral ions-loaded hydroxyapatite (mHA) (obtained by the hydrothermal treatment of hydroxyapatite (HA) in a simulated body fluid (SBF) solution (mHA@Chi-g-PMMA)) were prepared by the blending method. The physical properties, bioactivity, biological properties and their capabilities for sustained drug and protein release were studied. Physicochemical analysis showed a successful incorporation of the mineral ions in the HA particles and a good distribution of the mHA within the Chi-g-PMMA polymer matrix. The compressive strength and the Young's modulus were 15.760 ± 0.718 and 658.452 ± 17.020 MPa, respectively. In bioactivity studies, more apatite formation on the surface were seen after immersion in the SBF solution. In vitro growth experiments using UMR-106 osteoblast-like cells on the mHA@Chi-g-PMMA scaffold case showed that the attachment, viability and proliferation of the cells on the scaffolds had improved after 7 d of immersion. The in vitro release of two compounds (the cancer drug, doxorubicin (DOX)) and bovine serum albumin (BSA)), which had been attached to separate mHA@Chi-g-PMMA scaffolds, were studied to determine their suitability as drug delivery vehicles. It was found that the sustained release of DOX was 73.95% and of BSA was 57.27% after 25 h of incubation. These experimental results demonstrated that the mHA@Chi-g-PMMA composite can be utilized as a scaffold for bone cells ingrowth and also be used for drug delivery during the bone repairing.

Besides calcium accretion in the cortical envelope, a marked increase in the length of long bone was observed in pregnant and lactating rats, and thus the growth plate change was anticipated. Since several bone changes, such as massive trabecular bone resorption in late lactation, were found to be prolactin (PRL)-dependent, PRL may also be responsible for the maternal bone elongation. Herein, we investigated the growth plate change and possible chondroregulatory roles of PRL in the tibiae of rats at mid-pregnancy until 15 days postweaning. We found that the tibial length of lactating rats was increased and was inversely correlated with the total growth plate height, as well as the heights of proliferating zone (PZ) and hypertrophic zone (HZ), but not the resting zone (RZ). Chondrocytes in all zones expressed PRL receptors as visualized by immunohistochemistry, suggesting that the growth plate cartilage was a target of PRL action. Further investigations in lactating rats treated with an inhibitor of pituitary PRL release, bromocriptine, with or without PRL supplement, revealed the PRL-induced decreases in total growth plate height and HZ height from early to late lactation. However, decreases in RZ and PZ heights were observed only in late and mid-lactation, respectively. Thus, this was the first report on the chondroregulatory action of PRL on the growth plate of long bone in lactating rats. The results provided better understanding of the maternal bone adaptation during lactation.

The lactogenic hormone prolactin (PRL) directly regulates osteoblast functions in vitro and modulates bone remodeling in nulliparous rats, but its osteoregulatory roles in pregnant and lactating rats with physiological hyperprolactinemia remained unclear. Herein, bone changes were investigated in rats treated with bromocriptine (Bromo), an inhibitor of pituitary PRL release, or Bromo+PRL at different reproductive phases, from mid-pregnancy to late lactation. PRL receptors were strongly expressed in osteoblasts lining bone trabeculae, indicating bone as a target of PRL actions. By using dual energy X-ray absorptiometry, we found a significant increase in bone mineral density in the femora and vertebrae of pregnant rats. Such pregnancy-induced bone gain was, however, PRL independent and may have resulted from the increased cortical thickness. Bone trabeculae were modestly changed during pregnancy as evaluated by bone histomorphometry. On the other hand, lactating rats, especially in late lactation, showed massive bone loss in bone trabeculae but not in cortical shells. Further study in Bromo- and Bromo+PRL-treated rats suggested that PRL contributed to decreases in trabecular bone volume and number and increases in trabecular separation and eroded surface, as well as a paradoxical increase in bone formation rate in late lactation. Uncoupling of trabecular bone formation and resorption was evident in lactating rats, with the latter being predominant. In conclusion, pregnancy mainly induced cortical bone gain, whereas lactation led to trabecular bone loss in both long bones and vertebrae. Although PRL was not responsible for the pregnancy-induced bone gain, it was an important regulator of bone modeling during lactation.

To produce offspring, mothers require a large amount of calcium for fetal growth and milk production. Increased calcium demand leads to enhanced intestinal calcium absorption and stockpiling of bone calcium in pregnancy prior to demineralization in lactation. These coordinated events must be carefully organized by calciotropic hormone(s), but the classical hormones, namely 1,25-dihydroxyvitamin D(3), parathyroid hormone and calcitonin, do not appear to be responsible. Plasma prolactin (PRL) levels are elevated during pregnancy and, in view of the presence of PRL receptors in gut, bone and mammary glands, as well as recent evidence of the stimulatory effects of PRL on intestinal calcium transport, bone resorption and mammary calcium secretion, we postulate that PRL is the cardinal calciotropic hormone during pregnancy and lactation.

Besides enhancing osteoclast-mediated bone resorption, chronic metabolic acidosis (CMA) induces mineral efflux across the epithelial-like bone membrane formed by bone-lining cells (inactive osteoblasts), possibly via the paracellular pathway. However, there was a compensatory mechanism that restricted bone loss in the late phase of CMA, and changes in the expression of claudins, which are tight junction proteins known to regulate epithelial barrier function, were therefore anticipated in bone-lining cells. Herein, primary rat osteoblasts were found to express several transcripts of claudins, i.e., claudin-5, -11, -14, -15 and -16. Their protein expressions in bone-lining cells were demonstrated by immunohistochemistry in decalcified tibial sections. After exposure to CMA induced by oral administration of 1.5% NH(4)Cl for 21 days, expression of claudin-14, which normally seals the paracellular space and restricts ion movement, was increased, whereas that of claudin-15 and -16 which form pores for ion transport were decreased. Expressions of claudin-5 and -11 were not changed by CMA. In conclusion, the bone-lining cells of rats exposed to CMA for 21 days upregulated an ion-restrictive claudin (i.e., claudin-14), while downregulating ion-permeable claudins (i.e., claudin-15 and -16). These cellular responses might be parts of a compensatory mechanism accounting for deceleration of bone loss in late CMA.

Previous investigations suggested that prolactin (PRL) stimulated the intestinal calcium absorption through phosphoinositide 3-kinase (PI3K), protein kinase C (PKC), and RhoA-associated coiled-coil forming kinase (ROCK) signaling pathways. However, little was known regarding its detailed mechanisms for the stimulation of transcellular and voltage-dependent paracellular calcium transport. By using Ussing chamber technique, we found that the PRL-induced increase in the transcellular calcium flux and decrease in transepithelial resistance of intestinal-like Caco-2 monolayer were not abolished by inhibitors of gene transcription and protein biosynthesis. The PRL-stimulated transcellular calcium transport was completely inhibited by the L-type calcium channel blockers (nifedipine and verapamil) and plasma membrane Ca2+-ATPase (PMCA) inhibitor (trifluoperazine) as well as small interfering RNA targeting voltage-dependent L-type calcium channel Cav1.3, but not TRPV6 or calbindin-D9k. As demonstrated by45Ca uptake study, PI3K and PKC, but not ROCK, were essential for the PRL-enhanced apical calcium entry. In addition, PRL was unable to enhance the transcellular calcium transport after PKCζknockdown or exposure to inhibitors of PKCζ, but not of PKCα, PKCβ, PKCε, PKCμ, or protein kinase A. Voltage-clamping experiments further showed that PRL markedly stimulated the voltage-dependent calcium transport and removed the paracellular rectification. Such PRL effects on paracellular transport were completely abolished by inhibitors of PI3K (LY-294002) and ROCK (Y-27632). It could be concluded that the PRL-stimulated transcellular calcium transport in Caco-2 monolayer was mediated by Cav1.3 and PMCA, presumably through PI3K and PKCζpathways, while the enhanced voltage-dependent calcium transport occurred through PI3K and ROCK pathways.

Chronic metabolic acidosis (CMA) is known to have a detrimental effect on bone metabolism as a result of accelerated bone resorption and impaired bone formation. Typically, a number of compensatory adaptations must have occurred which may help palliate negative calcium balance and acidemia, e.g., increased intestinal calcium and phosphorus absorption. The final outcome with respect to bone remodeling after exposure to CMA for several months was, therefore, elusive. Herein, we investigated bone changes in male rats fed 1.5% NH(4)Cl in drinking water for up to 10 months to induce CMA with plasma pH of 7.2-7.3. Significant decreases in bone mineral density and content were detected by dual-energy X-ray absorptiometry after 6 months of CMA, whereas histomorphometric analysis revealed a significant decrease in bone volume already at week 2 after CMA induction. Exposure to CMA longer than 2 weeks also decreased trabecular number, trabecular thickness, osteoblast surface, mineral apposition rate, and bone formation rate, while increasing trabecular separation, osteoclast surface, and eroded surface. Bone resorption was rapid during weeks 2-16 (acceleration phase) and thereafter persisted at a slower rate (stationary phase) until week 40. Furthermore, CMA markedly reduced the total calcium content in bone and enhanced urinary calcium excretion as measured by atomic absorption spectrophotometry. It could be concluded that, after exposure to a long-standing acidemia, the enhanced bone resorption and suppressed bone formation led to osteopenia throughout the 10-month period, with accelerated bone loss seen only during the first 6 months. Thereafter, the compensatory adaptations appeared to help stabilize bone mass at a subnormal level.

Background Chronic metabolic acidosis (CMA) is known to induce renal phosphate wasting and hypophosphatemia by enhancing bone resorption and inhibiting renal phosphate reabsorption. However, nothing is known regarding changes in the plasma levels of phosphate-regulating hormones during CMA, especially in humans with normal kidney function. Methods Fifteen healthy Thai female volunteers were given NH (4)Cl orally for 7 days to induce CMA with or without oral phosphate supplementation. Blood and 24-h urine specimens were collected prior to and after CMA induction. Plasma concentrations and fractional excretion of calcium and inorganic phosphate as well as plasma levels of fibroblast growth factor (FGF) 23, 25(OH)D (3), 1,25(OH) (2)D (3) and intact parathyroid hormone (iPTH) were determined. Results CMA led to hypophosphatemia and hypocalcemia with increases in the fractional excretion of calcium and phosphate. Plasma concentrations of FGF23, 25(OH)D (3) and iPTH were decreased, whereas that of 1,25(OH) (2)D (3) was increased. After oral phosphate supplementation, CMA-induced changes in the concentrations of the studied ions, FGF23 and 25(OH)D (3), but not those of 1,25(OH) (2)D (3) and iPTH, were diminished. Conclusions The CMA-induced hypophosphatemia was likely to initiate a negative feedback response, thereby leading to reduction in the plasma levels of hyperphosphaturic hormones, FGF23 and PTH. An increase in the plasma 1,25(OH) (2)D (3) level, despite diminishing 25(OH)D (3) storage pool, may help enhance the intestinal phosphate absorption. Oral phosphate supplementation abolished the effects of CMA on FGF23 and 25(OH)D (3) levels, suggesting that the plasma phosphate concentration is the primary regulator of the plasma levels of these hormones during CMA.

Chronic prolactin (PRL) exposure can affect several functions of duodenal epithelia, especially those associated with fluid and electrolyte transport. However, little is known regarding its molecular mechanism. To identify PRL-regulated genes, microarray analysis was performed on RNA samples from duodenal epithelial cells of anterior pituitary (AP)-grafted hyperprolactinemic rats. Herein, we identified 321 transcripts upregulated and 241 transcripts downregulated after 4 weeks of AP transplantation. Results from real-time PCR analyses of 15 selected genes were consistent with the microarray results. Gene ontology analysis demonstrated pleiotropic effects of PRL on several cellular processes, including cellular metabolic process, cell communication and cell adhesion. Interestingly, 17 upregulated transcripts and 12 downregulated transcripts are involved in the transport of ions and nutrients, e.g., Ca(2+), Na(+), K(+), Cl(-) and glucose, thus agreeing with the established action of PRL on electrolyte homeostasis. The present results provided fundamental information for further investigations on mechanism of PRL actions in the intestine.

Hyperprolactinemia caused by physiological or pathological conditions, such as those occurring during lactation and prolactinoma, respectively, results in progressive osteopenia. The underlying mechanisms, however, are controversial. Prolactin (PRL) may directly attenuate the functions of osteoblasts, since these bone cells express PRL receptors. The present study therefore aimed to investigate the effects of PRL on the expression of genes related to the osteoblast functions by using quantitative real-time PCR technique. Herein, we used primary osteoblasts that were derived from the tibiae of adult rats and displayed characteristics of differentiated osteoblasts, including in vitro mineralization. Osteoblasts exposed for 48 h to 1000 ng/mL PRL, but not to 10 or 100 ng/mL PRL, showed decreases in the mRNA expression of Runx2, osteoprotegerin (OPG), and receptor activator of nuclear factor κB ligand (RANKL) by 60.49%, 72.74%, and 87.51%, respectively. Nevertheless, PRL did not change the RANKL/OPG ratio, since expression of OPG and RANKL were proportionally decreased. These concentrations of PRL had no effect on the mRNA expression of osteocalcin and osteopontin, nor on mineralization. High pathologic concentrations of PRL (1000 ng/mL) may downregulate expression of genes that are essential for osteoblast differentiation and functions. The present results explained the clinical findings of hyperprolactinemia-induced bone loss.

Hyperprolactinemia leads to high bone turnover as a result of enhanced bone formation and resorption. Although its osteopenic effect has long been explained as hyperprolactinemia-induced hypogonadism, identified prolactin (PRL) receptors in osteoblasts suggested a possible direct action of PRL on bone. In the present study, we found that hyperprolactinemia induced by anterior pituitary transplantation (AP), with or without ovariectomy (Ovx), had no detectable effect on bone mineral density and content measured by dual-energy X-ray absorptiometry (DXA). However, histomorphometric studies revealed increases in the osteoblast and osteoclast surfaces in the AP rats, but a decrease in the osteoblast surface in the AP+Ovx rats. The resorptive activity was predominant since bone volume and trabecular number were decreased, and the trabecular separation was increased in both groups. Estrogen supplement (E2) fully reversed the effect of estrogen depletion in the Ovx but not in the AP+Ovx rats. In contrast to the typical Ovx rats, bone formation and resorption became uncoupled in the AP+Ovx rats. Therefore, hyperprolactinemia was likely to have some estrogen-independent and/or direct actions on bone turnover. Osteoblast-expressed PRL receptor transcripts and proteins shown in the present study confirmed our hypothesis. Furthermore, we demonstrated that the osteoblast-like cells, MG-63, directly exposed to PRL exhibited lower expression of alkaline phosphatase and osteocalcin mRNA, and a decrease in alkaline phosphatase activity. The ratios of receptor activator of nuclear factor kappaB ligand (RANKL) and osteoprotegerin (OPG) proteins were increased, indicating an increase in the osteoclastic bone resorption. The present data thus demonstrated that hyperprolactinemia could act directly on bone to stimulate bone turnover, with more influence on bone resorption than formation. PRL enhanced bone resorption in part by increasing RANKL and decreasing OPG expressions by osteoblasts.

High physiological prolactin (PRL) stimulated intestinal calcium absorption and renal calcium uptake in mammals. Previous histomorphometric study revealed a significant increase in bone turnover in the trabecular part of the PRL-exposed long (cortical) bone; however, whole-bone densitometric analysis was unable to demonstrate such effect. We therefore studied differential changes in bone mineral density (BMD) and contents (BMC) of the femoral diaphysis and metaphysis in adult female rats exposed to high PRL induced by anterior pituitary (AP) transplantation. The estrogen-dependent effects of PRL on the femur were also investigated. We found that chronic exposure to PRL had no effect on BMD or BMC of the femoral diaphysis, which represented the cortical part of the long bone. It is interesting that 7 weeks after an AP transplantation, BMD and BMC of the femoral metaphysis were significantly decreased by 8% and 14%, respectively. Ovariectomy (Ovx) for 2, 5, and 7 weeks also decreased BMD and BMC in the femoral metaphysis, but not in the diaphysis. However, the AP transplantation plus Ovx (AP+Ovx) produced no additive effects. Nevertheless, 2.5 microg/kg 17beta-estradiol (E2) supplementation abolished the osteopenic effects of both Ovx and AP+Ovx on the femur. As for the L5-6 vertebrae, BMD and BMC were not affected by PRL exposure, but were significantly decreased by Ovx and AP+Ovx, and such decreases were completely prevented by E2 supplementation. It could be concluded that high physiological PRL induced a significant osteopenia in the trabecular part, i.e., the metaphysis, of the femora of adult female rats in an estrogen-dependent manner. Since PRL had no detectable effect on the vertebrae, the effects of PRL on bone appeared to be site-specific.

Previously, β-thalassemia, an inherited anemic disorder with iron overload caused by loss-of-function mutation of β-globin gene, has been reported to induce osteopenia and impaired whole body calcium metabolism, but the pathogenesis of aberrant calcium homeostasis remains elusive. Herein, we investigated how β-thalassemia impaired intestinal calcium absorption and whether it could be restored by administration of 1,25-dihydroxyvitamin D3[1,25(OH)2D3] or hepcidin, the latter of which was the liver-derived antagonist of intestinal iron absorption. The results showed that, in hemizygous β-globin knockout (BKO) mice, the duodenal calcium transport was lower than that in wild-type littermates, and severity was especially pronounced in female mice. Both active and passive duodenal calcium fluxes in BKO mice were found to be less than those in normal mice. This impaired calcium transport could be restored by 7-day 1,25(OH)2D3treatment. The 1,25(OH)2D3-induced calcium transport was diminished by inhibitors of calcium transporters, e.g., L-type calcium channel, NCX1, and PMCA1b, as well as vesicular transport inhibitors. Interestingly, the duodenal calcium transport exhibited an inverse correlation with transepithelial iron transport, which was markedly enhanced in thalassemic mice. Thus, 3-day subcutaneous hepcidin injection and acute direct hepcidin exposure in the Ussing chamber were capable of restoring the thalassemia-associated impairment of calcium transport; however, the positive effect of hepcidin on calcium transport was completely blocked by proteasome inhibitors MG132 and bortezomib. In conclusion, both 1,25(OH)2D3and hepcidin could be used to alleviate the β-thalassemia-associated impairment of calcium absorption. Therefore, our study has shed light on the development of a treatment strategy to rescue calcium dysregulation in β-thalassemia.

Prolactin has been shown to stimulate intestinal calcium absorption, increase bone turnover, and reduce renal calcium excretion. The small intestine, which is the sole organ supplying new calcium to the body, intensely expresses mRNAs and proteins of prolactin receptors, especially in the duodenum and jejunum, indicating the intestine as a target tissue of prolactin. A number of investigations show that prolactin is able to stimulate the intestinal calcium transport both in vitro and in vivo, whereas bromocriptine, which inhibits pituitary prolactin secretion, antagonizes its actions. In female rats, acute and long-term exposure to high prolactin levels significantly enhances the (i) transcellular active, (ii) solvent drag-induced, and (iii) passive calcium transport occurring in the small intestine. These effects are seen not only in pregnant and lactating animals, but are also observed in non-pregnant and non-lactating animals. Interestingly, young animals are more responsive to prolactin than adults. Prolactin-enhanced calcium absorption gradually diminishes with age, thus suggesting it has an age-dependent mode of action. Although prolactin's effects on calcium absorption are not directly vitamin D-dependent; a certain level of circulating vitamin D may be required for the basal expression of genes related to calcium transport. The aforementioned body of evidence supports the hypothesis that prolactin acts as a regulator of calcium homeostasis by controlling the intestinal calcium absorption. Cellular and molecular signal transductions of prolactin in the enterocytes are largely unknown, however, and still require investigation.

Previous investigations showed that chronic metabolic acidosis (CMA) increased the paracellular permeability of ion and neutral hydrophilic molecules in the duodenum of rats and small intestinal-like cell lines. Since proteins of the claudin family have been known to regulate the paracellular transport in several epithelia, an increase in the paracellular permeability during CMA may have resulted from changes in the pattern of claudin expression. The present study aimed to investigate the expression profile of 22 claudins in the duodenum of female Sprague-Dawley rats given 1.5% NH(4)Cl for 21 days to induce CMA. Arterial blood gas analysis revealed plasma pH values of 7.40 in normal rats and 7.31 in acidotic rats. Blood chemistry showed increases in the total plasma calcium, free-ionized calcium and magnesium, indicating a typical adaptive response of animals to CMA. RT-PCR demonstrated mRNA expressions of claudin-1 to -12, -14, -15, -17 to -20, -22 and -23 in duodenum of normal rats. Claudin-16 was not expressed in normal duodenum, but was strongly expressed in the kidney. Claudin-13 expression was seen only in the cecum, colon, liver and kidney of mice. After 21-day CMA, mRNA expressions of claudin-2, -3, -6, -8, -11, -12, -14, -19 and -22 were significantly enhanced, whereas expressions of other claudins were not changed. Confocal laser-scanning microscopy demonstrated that duodenal enterocytes of normal rats expressed claudin-3 protein on the paracellular membrane. The distribution of claudin-3 protein along the paracellular membrane was markedly increased in CMA, especially near the apical surface. Our results, therefore, provided novel evidence that 21-day CMA markedly altered claudin profile in the duodenum of rats by upregulating specific claudin expression.

In the present study, scaffolds for bone tissue engineering applications were made by immersing the inorganic phases of three different calcium phosphate (CaPs) (hydroxyapatite (HA), tricalcium phosphate (TCP), and biphasic calcium phosphate (BCP)) mixing bioactive glass (15Ca:80Si:5P) (BG) with polycaprolactone (PCL) as a binder in an organic phase of chitosan/collagen (ChiCol) matrix (CaPBG@ChiCol). Porous scaffolds were obtained by freeze drying the combinations. The mechanical properties and in vitro growth of rat osteoblast-like UMR-106 cells were investigated. The investigation indicated that the compressive strength was controlled by the types of CaP. The highest compressive modulus of the composites was 479.77 MPa (23.84 MPa for compressive strength) which is for the BCPBG@ChiCol composite. Compressive modulus of 459.01 and 435.95 MPa with compressive strength of 22.73 and 17.89 MPa were observed for the HABG@ChiCol and TCPBG@ChiCol composites, respectively. In vitro cell availability and proliferation tests confirmed the osteoblast attachment and growth on the CaPBG@ChiCol surface. Comparing the scaffolds, cells grown on the BCPBG based composite showed the higher cell density. To test its bioactivity, BCPBG@ChiCol was chosen for MTT and ALP assays on UMR-106 cells. The results indicated that the UMR-106 cells were viable and had higher ALP activity as the culturing times were increased. Therefore, ChiCol-fabricated BCPBG scaffold shows promise for bone regeneration. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 105B: 1758-1766, 2017.

The present paper studies the physico-chemical, bioactivity and biological properties of hydroxyapatite (HA) which is derived from fish scale (FS) (FSHA) and compares them with those of synthesized HA (sHA) obtained by co-precipitation from chemical solution as a standard. The analysis shows that the FSHA is composed of flat-plate nanocrystal with a narrow width size of about 15-20 nm and having a range of 100 nm in length and that the calcium phosphate ratio (Ca/P) is 2.01 (Ca-rich CaP). Whereas, synthesized HA consists of sub-micron HA particle having a Ca/P ratio of 1.65. Bioactivity test shows that the FSHA forms more new apatite than does the sHA after being incubated in simulated body fluid (SBF) for 7 days. Moreover, the biocompatibility study shows a higher osteoblast like cell adhesion on the FSHA surface than on the sHA substrate after 3 days of culturing. Our results also show the shape of the osteoblast cells on the FSHA changes from being a rounded shape to being a flattened shape reflecting its spreading behavior on this surface. MTT assay and ALP analysis show significant increases in the proliferation and activity of osteoblasts over the FSHA scaffold after 5 days of culturing as compared to those covering the sHA substrates. These results confirm that the bio-materials derived from fish scale (FSHA) are biologically better than the chemically synthesized HA and have the potential for use as a bone scaffold or as regenerative materials.

The present study aimed to test the hypothesis that testosterone deprivation impairs osteoblastic insulin signaling, decreases osteoblast survival, reduces bone density, and that obesity aggravates those deleterious effects in testosterone-deprived rats. Twenty four male Wistar rats underwent either a bilateral orchiectomy (O,n=12) or a sham operation (S,n=12). Then the rats in each group were further divided into two subgroups fed with either a normal diet (ND) or a high-fat diet (HF) for 12 weeks. At the end of the protocol, blood samples were collected to determine metabolic parameters and osteocalcin ratios. The tibiae were collected to determine bone mass using microcomputed tomography and for osteoblast isolation. The results showed that rats fed with HF (sham-operated HF-fed rats (HFS) and ORX HF-fed rats (HFO)) developed peripheral insulin resistance and had decreased trabecular bone density. In ND-fed rats, only the ORX ND-fed rats (NDO) group had decreased trabecular bone density. In addition, osteoblastic insulin resistance, as indicated by a decrease in tyrosine phosphorylation of the insulin receptor and Akt, were observed in all groups except the sham-operated ND-fed rats (NDS) rats. Those groups, again with the exception of the NDS rats, also had decreased osteoblastic survival. No differences in the levels of osteoblastic insulin resistance and osteoblastic survival were found among the NDO, HFS, and HFO groups. These findings suggest that either testosterone deprivation or obesity alone can impair osteoblastic insulin signaling and decrease osteoblastic survival leading to the development of osteoporosis. However, obesity does not aggravate those deleterious effects in the bone of testosterone-deprived rats.

As a bone-derived hormone, fibroblast growth factor-23 (FGF-23) negatively regulates phosphate and calcium metabolism, while retaining growth-promoting action for mesenchymal cell differentiation. Elevated FGF-23 levels, together with hyperparathyroidism, are often observed in chronic kidney disease, which is associated with impaired bone mineralization and enhanced bone resorption. Although overexpression of osteoblast-derived osteoclastogenic cytokines might contribute to this metabolic bone disease, whether FGF-23 alone and FGF-23 plus parathyroid hormone (PTH) directly modulated the expression of osteoblast-derived osteoclastogenic genes remained elusive. Herein, we demonstrated the direct effects of FGF-23 on proliferation and mRNA expression of osteoblast-specific differentiation and osteoclastogenic markers in rat osteoblast-like UMR-106 cells in the presence or absence of PTH. FGF-23 was found to suppress UMR-106 cell proliferation, while increasing FGF-23 expression, the latter of which suggested the presence of positive feedback regulation of FGF-23 expression in osteoblasts. FGF-23 also upregulated the mRNA expression of osteoblast differentiation markers (e.g., Runx2, osterix, AJ18, Dlx5, alkaline phosphatase, and osteopontin), osteoclastogenic factors (e.g., MCSF, MCP-1, IL-6, and TNF-α), and bone resorption regulators (RANKL and osteoprotegerin). However, combined PTH and FGF-23 exposure did not alter the levels of FGF-23-induced transcripts, suggesting that both hormones had no additive effect. In conclusion, FGF-23 directly suppressed osteoblast proliferation, while inducing osteoclastogenic gene expression in UMR-106 cells, and the FGF-23-induced transcripts were not altered by long-standing PTH exposure.

β-Thalassemia, a hereditary anemic disorder, is often associated with skeletal complications that can be found in both males and females. The present study aimed to investigate the age- and sex-dependent changes in bone mineral density (BMD) and trabecular microstructure in βIVSII-654knockin thalassemic mice. Dual-energy X-ray absorptiometry and computer-assisted bone histomorphometry were employed to investigate temporal changes in BMD and histomorphometric parameters in male and female mice of a βIVSII-654knockin mouse model of human β-thalassemia, in which impaired splicing of β-globin transcript was caused by hemizygous C→T mutation at nucleotide 654 of intron 2. Young, growing βIVSII-654mice (1 mo old) manifested shorter bone length and lower BMD than their wild-type littermates, indicating possible growth retardation and osteopenia, the latter of which persisted until 8 mo of age (adult mice). Interestingly, two-way analysis of variance suggested an interaction between sex and βIVSII-654genotype, i.e., more severe osteopenia in adult female mice. Bone histomorphometry further suggested that low trabecular bone volume in male βIVSII-654mice, particularly during a growing period (1–2 mo), was primarily due to suppression of bone formation, whereas both a low bone formation rate and a marked increase in osteoclast surface were observed in female βIVSII-654mice. In conclusion, osteopenia and trabecular microstructural defects were present in both male and female βIVSII-654knockin thalassemic mice, but the severity, disease progression, and cellular mechanism differed between the sexes.

It was previously understood that body weight gain and obesity observed in type 2 diabetes mellitus (T2DM) could be beneficial since body weight increase elevated bone mineral density and thus helped maintain the skeletal framework. However, a number of recent findings in humans and rodents have revealed that T2DM is not only associated with trabecular defects but also increases cortical porosity, and compromised bone cell function and bone mechanical properties. Hyperglycemia and insulin resistance in T2DM may further induce osteoblast apoptosis and uncoupling bone turnover. Prolonged accumulation of advanced glycation end products and diminished activity of lysyl oxidase, an essential enzyme for collagen cross-link, can lead to structural abnormalities of bone collagen fibrils, brittle matrix, and fragility fractures. Our studies in T2DM rats showed that dyslipidemia, which often occurs in T2DM, could obscure the T2DM-associated changes in bone microstructure and osteopenia. Longitudinal bone growth regulated by the growth plate chondrocytes is also impaired by T2DM since differentiation of growth plate chondrocytes is arrested and retained in the resting state while only a small number of cells undergo hypertrophic differentiation. Such a delayed chondrocyte differentiation may have also resulted from premature apoptosis of the growth plate chondrocytes. Nevertheless, the underlying cellular and molecular mechanisms of insulin resistance in osteoblasts, osteoclasts, osteocytes, and growth plate chondrocytes remain to be investigated.

For humans and rodents, duodenum is a very important site of calcium absorption since it is exposed to ionized calcium released from dietary complexes by gastric acid. Calcium traverses the duodenal epithelium via both transcellular and paracellular pathways in a vitamin D-dependent manner. After binding to the nuclear vitamin D receptor, 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] upregulates the expression of several calcium transporter genes, e.g., TRPV5/6, calbindin-D9k, plasma membrane Ca(2+)-ATPase1b, and NCX1, thereby enhancing the transcellular calcium transport. This action has been reported to be under the regulation of parathyroid-kidney-intestinal and bone-kidney-intestinal axes, in which the plasma calcium and fibroblast growth factor-23 act as negative feedback regulators, respectively. 1,25(OH)2D3 also modulates the expression of tight junction-related genes and convective water flow, presumably to increase the paracellular calcium permeability and solvent drag-induced calcium transport. However, vitamin D-independent calcium absorption does exist and plays an important role in calcium homeostasis under certain conditions, particularly in neonatal period, pregnancy, and lactation as well as in naturally vitamin D-impoverished subterranean mammals.

Chronic metabolic acidosis results in a negative calcium balance as a result of bone resorption and renal calcium loss. However, reports on the changes in intestinal calcium transport have been controversial. The present investigation therefore aimed to study the effects of chronic metabolic acidosis induced by 1.5% NH4Cl administration on the three components of duodenal calcium transport, namely, solvent drag-induced, transcellular active, and passive paracellular components, in rats using an in vitro Ussing chamber technique. The relative mRNA expression of genes related to duodenal calcium transport was also determined. We found that 21-day chronic metabolic acidosis stimulated solvent drag-induced and transcellular active duodenal calcium transport but not passive paracellular calcium transport. Our results further demonstrated that an acute direct exposure to serosal acidic pH, in contrast, decreased solvent drag-induced calcium transport in a pH-dependent fashion but had no effect on transcellular active calcium transport. Neither the transepithelial resistance nor duodenal permeability to Na+, Cl−, and Ca2+via the passive paracellular pathway were altered by chronic metabolic acidosis, suggesting that widening of the tight junction and changes in the charge-selective property of the tight junction did not occur. Thus the enhanced duodenal calcium transport observed in chronic metabolic acidosis could have resulted from a long-term adaptation, possibly at the molecular level. RT-PCR study revealed that chronic metabolic acidosis significantly increased the relative mRNA expression of duodenal genes associated with solvent drag-induced transport, i.e., the β1-subunit of Na+-K+-ATPase, zonula occludens-1, occludin, and claudin-3, and with transcellular active transport, i.e., transient receptor potential vanilloid family Ca2+channels 5 and 6 and plasma membrane Ca2+-ATPase isoform 1b. Total plasma calcium and free ionized calcium and magnesium concentrations were also increased, whereas serum parathyroid hormone and 1α,25-dihydroxyvitamin D3levels were not changed. The results indicated that 21-day chronic metabolic acidosis affected the calcium metabolism in rats partly through enhancing the mRNA expression of crucial duodenal genes involved in calcium absorption, thereby stimulating solvent drag-induced and transcellular active calcium transport in the duodenum.

Prolactin, having been shown to stimulate transcellular active and solvent drag-induced calcium transport in the duodenum of female rats, was postulated to improve duodenal calcium transport in estrogen-deficient rats. The aim of the present study was, therefore, to demonstrate the effects of long-term prolactin exposure produced by anterior pituitary (AP) transplantation on the duodenal calcium transport in young (9-week-old) and adult (22-week-old) ovariectomized rats. We found that ovariectomy did not alter the transcellular active duodenal calcium transport in young and adult rats fed normal calcium diet (1.0% w/w Ca) but decreased the solvent drag-induced duodenal calcium transport from 75.50 ± 10.12 to 55.75 ± 4.77 nmol·hr–1 cm–2 (P < 0.05) only in adult rats. Long-term prolactin exposure stimulated the transcellular active calcium transport in young and adult AP-grafted ovariectomized rats fed with normal calcium diet by more than 2-fold from 7.56 ± 0.79 to 16.54 ± 2.05 (P < 0.001) and 9.78 ± 0.72 to 15.99 ± 1.75 (P < 0.001) nmol·hr–1 cm–2, respectively. However, only the solvent drag-induced duodenal calcium transport in young rats was enhanced by prolactin from 95.51 ± 10.64 to 163.20 ± 18.03 nmol·hr–1 cm–2 (P < 0.001) whereas that in adult rats still showed a decreased flux from 75.50 ± 10.12 to 47.77 ± 5.42 nmol·hr–1 cm–2 (P < 0.05). Because oral calcium supplement has been widely used to improve calcium balance in estrogen-deficient animals, the effect of a high-calcium diet (2.0% w/w Ca) was also investigated. The results showed that stimulatory action of long-term prolactin on the transcellular active duodenal calcium transport in both young and adult rats was diminished after being fed a high-calcium diet. The same diet also abolished prolactin-enhanced solvent drag-induced duodenal calcium transport in young and further decreased that in adult AP-grafted ovariectomized rats. We concluded that the solvent drag-induced duodenal calcium transport in adult rats was decreased after ovariectomy. Long-term prolactin exposure stimulated the transcellular active duodenal calcium transport in both young and adult rats whereas enhancing the solvent drag-induced duodenal calcium transport only in young rats. Effects of prolactin were abolished by a high-calcium diet.

Prolactin has been reported to stimulate intestinal calcium absorption in young and mature, but not aging rats. The present study was performed on suckling rats to elucidate the actions of endogenous prolactin on calcium absorption in various intestinal segments. Before measuring the calcium fluxes, 9-day-old rats were administered for 7 days with 0.9% NaCl, s.c. (control), 3 mg/kg bromocriptine, i.p., twice daily to abolish secretion of endogenous pro lac tin, or bromocriptine plus exogenous 2.5 mg/kg prolactin, s.c. Thereafter, the 16-day-old rats were experimented upon by instilling the45Ca-containing solution into the intestinal segments. The results showed that, under a physiological condition, the jejunum had the highest rate of calcium absorption compared with other segments (1.4 ± 0.35 µmol·h–1·cm–1, p < 0.05). The duodenum and ileum also manifested calcium absorption, whereas the colon showed calcium secretion. Lack of endogenous prolactin decreased lumen-to-plasma and net calcium fluxes in jejunum from 2.07 ± 0.31 to 1.19 ± 0.12 and 1.40 ± 0.35 to 0.88 ± 0.18 µmol·h–1·cm–1(p < 0.05), respectively, and exogenous prolactin restored the jejunal calcium absorption to the control value. Endogenous prolactin also had an effect on the duodenum but, in this case, exogenous prolactin did not reverse the effect of bromocriptine. However, neither ileal nor colonic calcium fluxes were influenced by prolactin. Because luminal sodium concentration has been demonstrated to affect calcium absorption in mature rats, the effect of varying luminal sodium concentrations on calcium fluxes in suckling rats was evaluated. The jejunum was used due to its highest rate of calcium absorption. After filling the jejunal segments with 124 (control), 80, 40 mmol/L Na+-containing or Na+-free solution, increases in calcium absorption were found to be inversely related to luminal sodium concentrations in both control and bromocriptine-treated rats. The plasma concentration of45Ca under luminal sodium free condition was also higher than that of the control condition (2.26% ± 0.07% vs. 2.01% ± 0.09% administered dose, p < 0.05). However,3H-mannitol, a marker of the widening of tight junction that was introduced into the lumen, had a stable level in the plasma during an increase in plasma45Ca, suggesting that the widening of tight junction was not required for enhanced calcium absorption. In conclusion, calcium absorption in suckling rats was of the highest rate in the jejunum where endogenous prolactin modulated calcium absorption without increasing the paracellular transport of mannitol. Key words: calcium absorption, intestinal segments, prolactin, suckling rats.

A novel hand-held navigation combines gyroscopes, accelerometers and a secure local wireless channel to guide the distal femoral and proximal tibial cutting positions by displaying to the surgeon directly on the pods within the surgical field. No previous study has reported on its accuracy. A prospective randomized controlled trial was performed to compare radiographic outcomes in minimally invasive surgery total knee arthroplasty (MIS-TKA) with (40 patients, 40 knees) and without (40 patients, 40 knees) the novel hand-held navigation. The use of hand-held navigation resulted in fewer outliers (> ± 3° malalignment) in all frontal alignment: the hip-knee-ankle, the femoral component, and the tibial component. Tibial slope was also better achieved with the navigation. Femoral component flexion was not significantly different. Operation time and bone cutting time with the navigation were not longer than those without. Blood loss from drainage was not significantly different. The hand-held navigation improves accuracy for mechanical alignment and positioning of the prosthesis without additional surgical time.

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.

For humans and rodents, duodenum is a very important site of calcium absorption since it is exposed to ionized calcium released from dietary complexes by gastric acid. Calcium traverses the duodenal epithelium via both transcellular and paracellular pathways in a vitamin D-dependent manner. After binding to the nuclear vitamin D receptor, 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] upregulates the expression of several calcium transporter genes, e.g., TRPV5/6, calbindin-D9k, plasma membrane Ca(2+)-ATPase1b, and NCX1, thereby enhancing the transcellular calcium transport. This action has been reported to be under the regulation of parathyroid-kidney-intestinal and bone-kidney-intestinal axes, in which the plasma calcium and fibroblast growth factor-23 act as negative feedback regulators, respectively. 1,25(OH)2D3 also modulates the expression of tight junction-related genes and convective water flow, presumably to increase the paracellular calcium permeability and solvent drag-induced calcium transport. However, vitamin D-independent calcium absorption does exist and plays an important role in calcium homeostasis under certain conditions, particularly in neonatal period, pregnancy, and lactation as well as in naturally vitamin D-impoverished subterranean mammals.

A marked decrease in β-globin production led to β-thalassemia, a hereditary anemic disease associated with bone marrow expansion, bone erosion, and osteoporosis. Herein, we aimed to investigate changes in bone mineral density (BMD) and trabecular microstructure in hemizygous β-globin knockout thalassemic (BKO) mice and to determine whether endurance running (60 min/day, 5 days/wk for 12 wk in running wheels) could effectively alleviate bone loss in BKO mice. Both male and female BKO mice (1–2 mo old) showed growth retardation as indicated by smaller body weight and femoral length than their wild-type littermates. A decrease in BMD was more severe in female than in male BKO mice. Bone histomorphometry revealed that BKO mice had decreases in trabecular bone volume, trabecular number, and trabecular thickness, presumably due to suppression of osteoblast-mediated bone formation and activation of osteoclast-mediated bone resorption, the latter of which was consistent with elevated serum levels of osteoclastogenic cytokines IL-1α and -1β. As determined by peripheral quantitative computed tomography, running increased cortical density and thickness in the femoral and tibial diaphyses of BKO mice compared with those of sedentary BKO mice. Several histomorphometric parameters suggested an enhancement of bone formation (e.g., increased mineral apposition rate) and suppression of bone resorption (e.g., decreased osteoclast surface), which led to increases in trabecular bone volume and trabecular thickness in running BKO mice. In conclusion, BKO mice exhibited pervasive osteopenia and impaired bone microstructure, whereas running exercise appeared to be an effective intervention in alleviating bone microstructural defect in β-thalassemia.

Estrogen deficiency increases the risk of anxiety and mood disorders, presumably by deranging metabolism of the monoamine neurotransmitters and the expression of their reuptake transporters in the brain. Although estrogen-deficient individuals were also susceptible to stress, little was known regarding the effect of stress on the levels of transcripts related to brain monoamine metabolism. Herein, we used quantitative real-time PCR to quantify the mRNA levels of serotonin reuptake transporter (SERT), norepinephrine transporter (NET), monoamine oxidase-B (MAOB), tryptophan hydroxylase (TPH), and tyrosine hydroxylase (TH) in various brain regions of ovariectomized (OVX) rats which had been exposed for 4 weeks to chronic aversive stimuli (CAS), such as water deprivation, cage tilt, and illumination. We found that CAS induced stress responses in OVX rats as indicated by increases in the adrenal gland weight and sucrose intake. After CAS exposure, mRNA levels of SERT and NET were upregulated in the frontal cortex, hippocampus, amygdala, and periaqueductal gray. In addition, CAS also increased the mRNA levels of MAOB, an enzyme for dopamine degradation, in the same brain regions. However, CAS did not alter the mRNA levels of TPH or TH, both of which are rate-limiting enzymes for the synthesis of serotonin and norepinephrine in the dorsal raphé and locus coeruleus, respectively. Interestingly, mRNA expression of brain-derived neurotrophic factor precursor was upregulated in the hippocampus of CAS-exposed OVX rats, suggesting a compensatory mechanism which might counteract the stress-induced depression. Therefore, the present data have provided evidence to explain how stress affected brain monoamine metabolism in estrogen-deficient stressed patients.

Besides being a common haematological disorder caused by a reduction in β-globin production, β-thalassemia has been reported to impair body calcium homeostasis, leading to massive bone loss and increased fracture risk. Here, we demonstrated that heterozygous β-globin knockout thalassemic mice had a lower rate of duodenal calcium absorption compared with the wild-type littermates, whereas the epithelial electrical parameters, including transepithelial resistance, were not affected, suggesting no change in the epithelial integrity and permeability. Daily subcutaneous injection of 1 µg kg(-1) 1,25-dihydroxyvitamin D3 [1,25(OH)2 D3 ] for 3 days enhanced the duodenal calcium absorption in wild-type, but not in thalassemic mice. Although β-thalassemia increased the mRNA level of divalent metal transporter-1, an iron transporter in the duodenum, it had no effect on the transcripts of ferroportin-1 or the principal calcium transporters. In conclusion, β-thalassemia impaired the 1,25(OH)2 D3 -dependent intestinal calcium absorption at the post-transcriptional level, which, in turn, contributed to the dysregulation of body calcium metabolism and β-thalassemia-induced osteopenia.

In lactation, the intestinal absorption of nutrients and minerals, especially calcium, is markedly enhanced to supply precursors for milk production. Little is known regarding the mechanism of this lactation-induced intestinal hyperabsorption. However, it has been postulated to result from villous hypertrophy with enlarged absorptive area and the upregulation of the cation-selective tight junction protein claudin-15, which could form calcium-permeable paracellular pores, thereby enhancing the paracellular calcium absorption. Here, we demonstrated in the duodenum of 21-day lactating rats that there were increases in the villous height, villous width and crypt depth, which together led to expansion of absorptive surface area. Quantitative real-time PCR further showed that the mRNA levels of claudin-10 and -15 were increased in the duodenal mucosal cells of lactating rats as compared to age-matched unmated control rats. However, immunohistochemical analysis revealed the lactation-induced upregulation of claudin-15, but not claudin-10 protein expression in the duodenal villous cells. The present results, therefore, corroborated the previous hypothesis that lactation induced intestinal absorption of calcium and perhaps other cation minerals, in part, by increasing villous absorptive area and claudin-15 protein expression.

The aim in this research is to study the physical and biocompatible properties of hydroxyapatite (HAp) composites (HApTiR) having different amounts of titania rod (TiR) in them (10–90 wt.%). The HAp and TiR were produced using hydrothermal and co-precipitation under reflux methods, respectively. The physical properties and the in vitro biocompatibility of the composites to simulated body fluid (SBF) were investigated. They were also cultured with rat osteoblast-like UMR-106 cells. The synthesized powder showed a core-shell structure with the titania rod as the core and the apatite as the shell. The hardness of the composites of HApTiR's whisker increased from 74.8 to 92.9 MPa as the TiR content was increased from 10 to 90 wt.%. Mineralization study in SBF showed the formation of apatite crystals on the HApTiR's surface after 7 days of incubation. In vitro cell adhesion tests confirmed the osteoblast attachment and growth on the HApTiR's surface. The density of cells, spread and the production of calcium nodules on the substrate were seen to increase with increasing TiR contents except for HApTiR90 (TiR = 90 wt.%) which exhibited lesser growth. MTT tests on HApTiR70 indicated that UMR-106 cells were viable and the density of cells on the substrate was seen to increase with increasing culturing time.

Phytoestrogens have attracted attention for their potential in the prevention of postmenopausal osteoporosis. Recently, phytoestrogen-rich herb Pueraria mirifica has been demonstrated to possess an osteogenic effect on bone in ovariectomized rats, but its underlying cellular mechanism was not known. Here, we investigated the effects of P. mirifica extract and its major isoflavone compound, puerarin, on cell viability, cell proliferation and the expression of differentiation markers in rat osteoblast-like UMR106 cells. After exposure to 17β-estradiol (E2), genistein, P. mirifica extract and puerarin, proliferation but not viability of UMR106 cells was markedly decreased. Quantitative real-time PCR revealed that P. mirifica extract and puerarin significantly increased the mRNA expression of alkaline phosphatase (ALP) and osteoprotegerin, but not Runx2, osterix or osteocalcin. Puerarin also decreased the mRNA expression of receptor activator of nuclear factor-κB ligand, an osteoclastogenic factor, suggesting that it could induce bone gain by enhancing osteoblast differentiation and suppressing osteoclast function. Furthermore, after an exposure to high affinity estrogen receptor (ER) antagonist (ICI182780), the E2-, genistein-, P. mirifica extract- and puerarin-induced upregulation of ALP expressions were completely abolished. It could be concluded that P. mirifica extract and puerarin induced osteoblast differentiation rather than osteoblast proliferation in an ER-dependent manner. The present findings, therefore, corroborated the potential benefit of P. mirifica extract and puerarin in the prevention and treatment of postmenopausal osteoporosis.

A drug delivery vehicle consisting of spherical calcium phosphate-collagen particles covered by flower-like (SFCaPCol) blossoms composed of nanorod building blocks and their cellular response is studied. The spherical structure was achieved by a combination of sonication and freeze-drying. The SFCaPCol blossoms have a high surface area of approximately 280 m 2 g − 1 . The blossom-like formation having a high surface area allows a drug loading efficiency of 77.82%. The release profile for one drug, vancomycin (VCM), shows long term sustained release in simulated body fluid (SBF), in a phosphate buffer saline (PBS, pH 7.4) solution and in culture media over 2 weeks with a cumulative release ~ 53%, 75% and 50%, respectively, over the first 7 days. The biocompatibility of the VCM-loaded SFCaPCol scaffold was determined by in vitro cell adhesion and proliferation tests of rat osteoblast-like UMR-106 cells. MTT tests indicated that UMR-106 cells were viable after exposure to the VCM loaded SFCaPCol, meaning that the scaffold (the flower-like blossoms) did not impair the cell's viability. The density of cells on the substrate was seen to increase with increasing cultured time.

Despite being widely recognized as the important bone-derived phosphaturic hormone, whether fibroblast growth factor (FGF)-23 modulated intestinal calcium absorption remained elusive. Since FGF-23 could reduce the circulating level of 1,25-dihydroxyvitamin D₃ [1,25(OH)₂D₃], FGF-23 probably compromised the 1,25(OH)₂D₃-induced intestinal calcium absorption. FGF-23 may also exert an inhibitory action directly through FGF receptors (FGFR) in the intestinal cells. Herein, we demonstrated by Ussing chamber technique that male mice administered 1 μg/kg 1,25(OH)₂D₃ sc daily for 3 days exhibited increased duodenal calcium absorption, which was abolished by concurrent intravenous injection of recombinant mouse FGF-23. This FGF-23 administration had no effect on the background epithelial electrical properties, i.e., short-circuit current, transepithelial potential difference, and resistance. Immunohistochemical evidence of protein expressions of FGFR isoforms 1-4 in mouse duodenal epithelial cells suggested a possible direct effect of FGF-23 on the intestine. This was supported by the findings that FGF-23 directly added to the serosal compartment of the Ussing chamber and completely abolished the 1,25(OH)₂D₃-induced calcium absorption in the duodenal tissues taken from the 1,25(OH)₂D₃-treated mice. However, direct FGF-23 exposure did not decrease the duodenal calcium absorption without 1,25(OH)₂D₃ preinjection. The observed FGF-23 action was mediated by MAPK/ERK, p38 MAPK, and PKC. Quantitative real-time PCR further showed that FGF-23 diminished the 1,25(OH)₂D₃-induced upregulation of TRPV5, TRPV6, and calbindin-D(9k), but not PMCA(1b) expression in the duodenal epithelial cells. In conclusion, besides being a phosphatonin, FGF-23 was shown to be a novel calcium-regulating hormone that acted directly on the mouse intestine, thereby compromising the 1,25(OH)₂D₃-induced calcium absorption.

During pregnancy and lactation, prolactin (PRL) enhances intestinal absorption of calcium and other minerals for fetal development and milk production. Although an enhanced absorptive efficiency is believed to mainly result from the upregulation of mineral transporters in the absorptive villous cells, some other possibilities, such as PRL-enhanced crypt cell proliferation and differentiation to increase the absorptive area, have never been ruled out. Here, we investigated cell proliferation and mRNA expression of mineral absorption-related genes in the PRL-exposed IEC-6 crypt cells. As expected, the cell proliferation was not altered by PRL. Inasmuch as the mRNA expressions of villous cell markers, including dipeptidylpeptidase-4, lactase and glucose transporter-5, were not increased, PRL was not likely to enhance crypt cell differentiation into the absorptive villous cells. In contrast to the previous findings in villous cells, PRL was found to downregulate the expression of calbindin-D(9k), claudin-3 and occludin in IEC-6 crypt cells, while having no effect on transient receptor potential vanilloid family channels-5/6, plasma membrane Ca(2+)-ATPase (PMCA)-1b and Na(+)/Ca(2+) exchanger-1 expression. In conclusion, IEC-6 crypt cells did not respond to PRL by increasing proliferation or differentiation into villous cells. The present results thus supported the previous hypothesis that PRL enhanced mineral absorption predominantly by increasing transporter expression and activity in the absorptive villous cells.

β-thalassemia caused by the C→T mutation at nucleotide 654 of the intron 2 (β(IVSII-654)) results in aberrant splicing of β-globin RNA, leading to an almost absence of β-globin synthesis. Although trabecular and cortical bone loss was previously reported in β-thalassemic mice with deletion of β-globin gene, the microscopic changes in trabecular structure in β(IVSII-654) thalassemic mice remained elusive. Here, we investigated the macroscopic and microscopic bone changes in 12-week-old β(IVSII-654) knockin thalassemic mice by dual-energy X-ray absorptiometry (DXA) and histomorphometric analysis, respectively. DXA revealed a decrease in bone mineral density in the lumbar vertebrae and tibial metaphysis, but not in the femoral diaphysis, suggesting that β(IVSII-654) thalassemia predominantly led to osteopenia at the trabecular site, but not the cortical site. Further histomorphometric analysis of the tibial secondary spongiosa showed that trabecular bone volume was significantly decreased with the expansion of marrow cavity. Decreases in osteoblast surface, osteoid surface, mineral apposition rate, mineralizing surface, and mineralized volume were also observed. Moreover, trabecular bone resorption was markedly enhanced as indicated by increases in the osteoclast surface and eroded surface. It could be concluded that β(IVSII-654) thalassemia impaired bone formation and enhanced bone resorption, thereby leading to osteopenia especially at the trabecular sites, such as the tibial metaphysis.

Endurance swimming is known to increase duodenal calcium absorption in normal rats and bone strength in estrogen-deficient rats. Because the stress resulting from forced training often attenuates the stimulatory effect of exercise, swimming in an inescapable chamber should reveal both the positive effect of the exercise and the negative effect of stress. In the work reported herein, swimming rats showed no signs of stress during 2 weeks of training. However, stress response gradually developed thereafter and peaked at weeks 6 and 7. In rats swimming for 2 weeks, transcellular duodenal calcium transport was enhanced ~2-fold. In contrast, calcium absorption was reduced in rats swimming for 8 weeks, consistent with the absence of swimming-induced upregulation of calcium transporter genes in the 8-week group. In conclusion, prolonged stress hindered the stimulatory effect of swimming on duodenal calcium absorption, and thus endurance exercise should be performed without forced training or stress to retain its beneficial effect on calcium metabolism.

Parathyroid hormone (PTH) was recently demonstrated to enhance the HCO(3)(-) secretion through the apical anion channel, cystic fibrosis transmembrane conductance regulator (CFTR), but how the HCO(3)(-) entered the epithelial cells was not well understood, in part, due to the lack of specific inhibitors of the basolateral HCO(3)(-) transporters. Moreover, the function of the PTH-stimulated HCO(3)(-) secretion has never been investigated in vivo. Here, we designed three specific pairs of small interfering RNA sequences to simultaneously knockdown three variants of the electrogenic Na(+)/HCO(3)(-) co-transporter (NBCe)-1 in the intestinal epithelium-like Caco-2 monolayer. The results showed that NBCe1 mRNA levels were markedly reduced, and the PTH-induced transepithelial current and voltage changes were diminished after triple knockdown as determined by quantitative real-time PCR and Ussing chamber technique, respectively. An in vivo ligated intestinal loop study further showed that there was an increased fluid secretion, presumably driven by HCO(3)(-) transport, in the ileum, but not in jejunum or colon, of rats administered intravenously with 2 μg/kg body weight of rat PTH 1-34. Therefore, the present results suggested that PTH stimulated intestinal HCO(3)(-) secretion, particularly in the ileum, by inducing the basolateral HCO(3)(-) uptake via NBCe1.

Besides being a Ca²-regulating hormone, parathyroid hormone (PTH) has also been shown to regulate epithelial transport of certain ions, such as Cl, HCO₃, and Na, particularly in the kidney. Although the intestinal epithelium also expressed PTH receptors, little was known regarding its mechanism in the regulation of intestinal ion transport. We investigated the ion regulatory role of PTH in intestinal epithelium-like Caco-2 monolayer by Ussing chamber technique and alternating current impedance spectroscopy. It was found that Caco-2 cells rapidly responded to PTH within 1 min by increasing apical HCO₃- secretion. CFTR served as the principal route for PTH-stimulated apical HCOV efflux, which was abolished by various CFTR inhibitors, namely, NPPB, glycine hydrazide-101 (GlyH-101), and CFTRinh-172, as well as by small interfering RNA against CFTR. Concurrently, the plasma membrane resistance was decreased with no changes in the plasma membrane capacitance or paracellular permeability. HCOV was probably supplied by basolateral uptake via the electrogenic Na⁺-HCO₃⁻ cotransporter and by methazolamide-sensitive carbonic anhydrase, while the resulting intracellular H⁺ might be extruded by both apical and basolateral Na/H exchangers. Furthermore, the PTH-stimulated HCO₃-secretion was markedly reduced by protein kinase A (PKA) inhibitor (PKI 14-22 amide) and phosphoinositide 3-kinase (PI3K) inhibitors (wortmannin and LY-294002), but not by intracellular Ca²⁺ chelator (BAPTA-AM) or protein kinase C inhibitor (GF-109203X). In conclusion, the present study provided evidence that PTH directly and rapidly stimulated apical HCO₃- secretion through CFTR in PKA- and PI3K-dependent manner, which was a novel noncalciotropic, ion regulatory action of PTH in the intestinal epithelium.

Magnetic nanoparticle composite with a cobalt ferrite (CoFe2O4, (CF)) core and an apatite (Ap) coating was synthesized using a biomineralization process in which a modified simulated body fluid (1.5SBF) solution is the source of the calcium phosphate for the apatite formation. The core–shell structure formed after the citric acid–stabilized cobalt ferrite (CFCA) particles were incubated in the 1.5 SBF solution for 1 week. The mean particle size of CFCA-Ap is about 750 nm. A saturation magnetization of 15.56 emug-1 and a coercivity of 1808.5 Oe were observed for the CFCA-Ap obtained. Bovine serum albumin (BSA) was used as the model protein to study the adsorption and release of the proteins by the CFCA-Ap particles. The protein adsorption by the CFCA-Ap particles followed a more typical Freundlich than Langmuir adsorption isotherm. The BSA release as a function of time became less rapid as the CFCA-Ap particles were immersed in higher pH solution, thus indicating that the BSA release is dependent on the local pH.

The anxiolytic effect of fluoxetine (Flx) was often ineffective in postmenopausal and estrogen-deficient patients, but such effect had not been experimentally demonstrated, particularly in the female rat model of estrogen deficiency. Here we determined the anxiety-like behaviors in ovariectomized (Ovx) rats treated for 4weeks with 10μg/kg 17β-estradiol s.c. (Ovx+E2), 10mg/kg Flx p.o. (Ovx+Flx) or a combination of both (Ovx+E2+Flx). Since Flx is known to induce anxiolysis in males, we first evaluated the Flx regimen in male rats. The results showed that anxiety-like behaviors were reduced in Flx-treated male rats. In contrast, Ovx+Flx rats still exhibited the same anxiety-like behaviors as in Ovx rats. Both Ovx+E2 and Ovx+E2+Flx rats, however, showed comparable reductions in anxiety-like behaviors, suggesting that Flx had no anxiolytic-like effect. Furthermore, E2 and E2+Flx similarly upregulated the mRNA expression of serotonin reuptake transporter (SERT) and tryptophan hydroxylase-2 in the dorsal raphe of Ovx rats, while having no effect on SERT expression in the frontal cortex, hippocampus, septum, amygdala and periaqueductal gray. In conclusion, Flx induced anxiolytic-like action in male rats. In Ovx rats, it was E2 and not Flx that exerted the anxiolytic-like action, which was mediated, in part, by altering serotonin metabolism in the dorsal raphe.

The milk-producing hormone prolactin (PRL) increases the transcellular intestinal calcium absorption by enhancing apical calcium uptake through voltage-dependent L-type calcium channel (Ca(v)) 1.3. However, the redundancy of apical calcium channels raised the possibility that Ca(v)1.3 may operate with other channels, especially transient receptor potential vanilloid family calcium channels (TRPV) 5 or 6, in an interdependent manner. Herein, TRPV5 knockdown (KD), TRPV5/TRPV6, TRPV5/Ca(v)1.3, and TRPV6/Ca(v)1.3 double KD, and TRPV5/TRPV6/Ca(v)1.3 triple KD Caco-2 monolayers were generated by transfecting cells with small interfering RNAs (siRNA). siRNAs downregulated only the target mRNAs, and did not induce compensatory upregulation of the remaining channels. After exposure to 600 ng/mL PRL, the transcellular calcium transport was increased by ~2-fold in scrambled siRNA-treated, TRPV5 KD and TRPV5/TRPV6 KD monolayers, but not in TRPV5/Ca(v)1.3, TRPV6/Ca(v)1.3 and TRPV5/TRPV6/Ca(v)1.3 KD monolayers. The results suggested that Ca(v)1.3 was the sole apical channel responsible for the PRL-stimulated transcellular calcium transport in intestine-like Caco-2 monolayer.

Activation of adrenergic receptors (AR) was demonstrated to result in either bone gain or bone loss depending on the activated AR subtypes and concentrations of agonists used. While beta(2)-AR agonist was extensively investigated as an osteopenic agent, effects of beta(3)-AR activation on osteoblasts were still elusive. Rat osteoblast-like UMR106 cells were herein found to express several AR subtypes, including beta(3)-AR. After exposure to a low-dose beta(3)-AR agonist BRL37344 (10 nmol L(-1)), UMR106 cells downregulated the mRNA expression of transcription factors Runx2 and Dlx5, which are important for initiation of osteoblast differentiation. Low-dose BRL37344 also decreased the expression ratio of receptor activator of nuclear factor kappaB ligand (RANKL) over osteoprotegerin (OPG), suggesting the protective effect of beta(3)-AR agonist against bone resorption. Alkaline phosphatase expression was markedly decreased, whereas expressions of osteocalcin and osteopontin were increased by 100 nmol L(-1) BRL37344, indicating that beta(3)-AR activation could accelerate the transition of matrix maturation stage to mineralization stage. In conclusion, beta(3)-AR activation in rat osteoblasts induced alteration in the expression of osteoblast-related transcription factor genes as well as genes required for bone formation and resorption. The present results also suggest that, besides beta(2)-AR, beta(3)-AR is another AR subtype responsible for the sympathetic nervous system-induced bone remodeling.

As a complex Ca2+-rich fluid mixture of water, casein, lactose and several ions, milk secretion requires a number of unknown transporters, which can be identified by a genome-wide microarray study in mammary tissues of lactating animals. Ca2+ was reported to be secreted across mammary epithelial cells through the transcellular pathway, presumably involving TRPC (canonical transient receptor potential) channels. In the present study, we have used quantitative real-time PCR to demonstrate that the human mammary cell line MCF-7, as well as rat mammary tissues from pregnant and lactating rats, expressed TRPC1, TRPC5 and TRPC6. Expression of TRPC1, TRPC5 and TRPC7 were markedly up-regulated, whereas that of TRPC3 and TRPC4 was down-regulated in the early lactating period. To further identify other transporter genes affected by lactation, a highly sensitive Illumina microarray featuring Bead Array technology was performed on RNA samples from mammary tissues of lactating rats. We found that, of the 384 transcripts changed during lactation, 31 transcripts were involved in the transport of water and electrolytes, such as Ca2+, Na+, K+, Cl-, I-, Fe2+, sulfate and phosphate. The present study, therefore, provides information for further investigation of the mechanism of lactation-induced transport adaptation in mammary epithelial cells.

During pregnancy and lactation, the enhanced intestinal Ca2+absorption serves to provide Ca2+for fetal development and lactogenesis; however, the responsible hormone and its mechanisms remain elusive. We elucidated herein that prolactin (PRL) markedly stimulated the transcellular and paracellular Ca2+transport in the duodenum of pregnant and lactating rats as well as in Caco-2 monolayer in a two-step manner. Specifically, a long-term exposure to PRL in pregnancy and lactation induced an adaptation in duodenal cells at genomic levels by upregulating the expression of genes related to transcellular transport, e.g., TRPV5/6 and calbindin-D9k, and the paracellular transport, e.g., claudin-3, thereby raising Ca2+absorption rate to a new “baseline” (Step 1). During suckling, PRL surge further increased Ca2+absorption to a higher level (Step 2) in a nongenomic manner to match Ca2+loss in milk. PRL-enhanced apical Ca2+uptake was responsible for the increased transcellular transport, whereas PRL-enhanced paracellular transport required claudin-15, which regulated epithelial cation selectivity and paracellular Ca2+movement. Such nongenomic PRL actions were mediated by phosphoinositide 3-kinase, protein kinase C, and RhoA-associated coiled-coil-forming kinase pathways. In conclusion, two-step stimulation of intestinal Ca2+absorption resulted from long-term PRL exposure, which upregulated Ca2+transporter genes to elevate the transport baseline, and the suckling-induced transient PRL surge, which further increased Ca2+transport to the maximal capacity. The present findings also suggested that Ca2+supplementation at 15–30 min prior to breastfeeding may best benefit the lactating mother, since more Ca2+could be absorbed as a result of the suckling-induced PRL surge.