The clinical management of bone defects caused by trauma or nonunion fractures remains a challenge in orthopedic practice due to the poor integration and biocompatibility properties of the scaffold or implant material. In the current work, the osteogenic properties of carboxyl-modified single-walled carbon nanotubes (COOH-SWCNTs) were investigated in vivo and in vitro. When human preosteoblasts and murine embryonic stem cells were cultured on coverslips sprayed with COOH-SWCNTs, accelerated osteogenic differentiation was manifested by increased expression of classical bone marker genes and an increase in the secretion of osteocalcin, in addition to prior mineralization of the extracellular matrix. These results predicated COOH-SWCNTs' use to further promote osteogenic differentiation in vivo. In contrast, both cell lines had difficulties adhering to multi-walled carbon nanotube-based scaffolds, as shown by scanning electron microscopy. While a suspension of SWCNTs caused cytotoxicity in both cell lines at levels >20 μg/mL, these levels were never achieved by release from sprayed SWCNTs, warranting the approach taken. In vivo, human allografts formed by the combination of demineralized bone matrix or cartilage particles with SWCNTs were implanted into nude rats, and ectopic bone formation was analyzed. Histological analysis of both types of implants showed high permeability and pore connectivity of the carbon nanotube-soaked implants. Numerous vascularization channels appeared in the formed tissue, additional progenitor cells were recruited, and areas of de novo ossification were found 4 weeks post-implantation. Induction of the expression of bone-related genes and the presence of secreted osteopontin protein were also confirmed by quantitative polymerase chain reaction analysis and immunofluorescence, respectively. In summary, these results are in line with prior contributions that highlight the suitability of SWCNTs as scaffolds with high bone-inducing capabilities both in vitro and in vivo, confirming them as alternatives to current bone-repair therapies.

We report the synthesis and characterization of a novel zeolite-hydroxyapatite composite coating on titanium alloys and stainless steel. The zeolite-hydroxyapatite coating is superhydrophilic and outperforms the state-of-the-art Ti6Al4V alloys in corrosion resistance tests in aggressive pitting NaCl media, phosphate buffer solution with BSA protein, as well as highly complex DMEM cell culture media. And the composite coating also eliminates the elastic modulus mismatch between coating and bone. In addition, the composite coating has an osteoconductive and osteoinductive effect on hFOBs, indicating that it may enhance osteointegration of implants and speed up post-surgical recovery, and thus reduce the need for recurring implant replacement surgeries. Replacing titanium with zeolite-hydroxyapatite coated steel can also significantly reduce implant cost while improving implant lifespan.

Sertoli cell secretory activities are highly dependent on ion channel functions and critical to spermatogenesis. The steroid hormone 1alpha,25(OH)2-vitamin D3 (1,25(OH)2-D3) stimulates exocytosis in different cell systems by activating a nongenotropic vitamin D receptor (VDR). Here, we described 1,25(OH)2-D3 stimulation of secretion via Cl(-) channel activation in the mouse immature Sertoli cell line TM4. 1,25(OH)2-D3 potentiation of chloride currents was dependent on hormone concentration, and correlated with a significant increase in whole-cell capacitance within 20-40 min. In addition, Cl(-) currents were potentiated by the nongenomic VDR agonist 1alpha,25(OH)2 lumisterol D3 (JN), while 1,25(OH)2-D3 potentiation of channels was suppressed by nongenomic VDR antagonist 1beta,25(OH)2-vitamin D3 (HL). Treatment of TM4 cells with PKC and PKA activators PMA and forskolin respectively, increased Cl(-) currents significantly, while PKC and PKA inhibitors Go6983 and H-89, respectively, abolished 1,25(OH)2-D3 stimulation of Cl(-) currents, suggesting phosphorylation pathways in 1,25(OH))2-D3 mediated channel responses. RT-PCR demonstrated the expression of outwardly rectifying ClC-3 channels in TM4 cells. Taken together, our results demonstrate a PKA/PKC-dependent 1,25(OH)2-D3/VDR nongenotropic pathway leading to Cl(-) channel and exocytosis activation in Sertoli cells. We conclude that 1,25(OH)2-D3 appears to be a modulator of male reproductive functions at least in part by stimulating Sertoli cell secretory functions.

Biocompatible zeolite anti-corrosion coatings have potential for success as bone scaffolding materials. In this work, reported by Y. Yan and co-workers, titanium-based dental implants are covered with zeolite MFI coatings to prevent against corrosion within the dental cavity. Zeolite coatings are non-toxic, and prevent the release of toxic ions from metals into tissue. The 3D micro-topology of the zeolites also enhances cell proliferation, differentiation, and surface adhesion.

In the absence of mechanical stimulation, brief exposure of osteoblasts to 1 alpha,25(OH)(2)vitamin D-3 (1,25D) triggers plasma membrane electrical responses that couple to exocytosis. Here we describe for the first time 1,25D induction of exocytotic ATP release in static ROS 17/2.8 and SAOS-2 cells and primary calvarial osteoblasts expressing a vitamin D receptor (VDR). We found that 10 nM 1,25D optimally induced 45 +/- 1% and 40 +/- 1% of partial and complete exocytotic events, respectively, from a 1,25D-sensitive pool of ATP-containing secretory vesicles within 60 s. We measured a dose-dependent 1,25D induction of ATP secretion, with maximal response of similar to 6.2-fold (16.93 +/- 1.82 nM for SAOS-2) and 3.1-fold (18.89 +/- 1.39 nM for ROS 1.7/2.8) obtained with 10 nM 1,251) compared with basal ATP levels (2.75 +/- 0.39 nM, SAOS-2; 6.09 +/- 0.58 nM, ROS 1.7/2.8 cells). The natural metabolite 25(OH)vitamin D-3 (25D, 1.0 nM) induced a significant 3.6-fold increase of ATP release in ROS 17/2.8 cells, but there was no induction with the antagonist 1 beta,25(OH)(2)vitamin D-3 (1 beta,25D, 10 nM) or the steroid 17 beta-estradiol (10 nM). 1,25D-induced ATP secretion was abolished when cells were preincubated with inhibitors of vesicular exocytosis. siRNA VDR silencing prevented 1,25D stimulation of ATP exocytosis in ROS 17/2.8 and SAOS-2 cells. Similarly, 1,25D failed to stimulate ATP exocytosis in primary osteoblasts from a VDR knockout mouse. ATP secretion coupled to 1,251) induction of cytosolic calcium and chloride channel potentiation. Rapid 1,25D stimulation of ATP secretion involving nontranscriptional VDR functions in osteoblasts may help explain 1,25D bone anabolic properties. J Bone Miner Res 2009;24:1450-1460. Published online on March 16, 2009; doi: 10.1359/JBMR.090306

Titanium alloy, Ti6Al4V, is widely used in dental and orthopedic implants. Despite its excellent biocompatibility, Ti6Al4V releases toxic Al and V ions into the surrounding tissue after implantation. In addition, the elastic modulus of Ti6Al4V ( approximately 110GPa) is significantly higher than that of bone (10-40GPa), leading to a modulus mismatch and consequently implant loosening and deosteointegration. Zeolite coatings are proposed to prevent the release of the toxic ions into human tissue and enhance osteointegration by matching the mechanical properties of bone. Zeolite MFI coatings are successfully synthesized on commercially pure titanium and Ti6Al4V for the first time. The coating shows excellent adhesion by incorporating titanium from the substrate within the zeolite framework. Higher corrosion resistance than the bare titanium alloy is observed in 0.856M NaCl solution at pHs of 7.0 and 1.0. Zeolite coatings eliminate the release of cytotoxic Al and V ions over a 7 day period. Pluripotent mouse embryonic stem cells show higher adhesion and cell proliferation on the three-dimensional zeolite microstructure surface compared with a two-dimensional glass surface, indicating that the zeolite coatings are highly biocompatible.

The use of zeolite MFI‐coated titanium alloy for bone cell growth and new bone formation in vitro is investigated. The corrosion‐resistant MFI coating is shown to be osteoconductive and to promote proliferation of human fetal osteoblasts (hFOBs) as compared to bare titanium alloy, Ti6Al4V. The zeolite crystal microstructure appears to facilitate osteoblast adhesion and induces osteointegration, as evaluated with microscopy. In addition, the zeolite promotes the differentiation of hFOBs into mature osteoblasts, as well as the production of a mineralized matrix at earlier times in culture compared to Ti6Al4V, indicating higher osteoinductive properties of the MFI coating than titanium alone. A significant increase in the expression of the bone morphogenetic protein (BMP‐2) gene is measured in hFOBs cultured on zeolite coatings compared to bare Ti6Al4V. This is the first report on highly corrosion‐resistant zeolite MFI coatings on Ti6A14V alloys with the potential to be used as a material of improved osteointegration appropriate for bone tissue regeneration.

We demonstrated recently that CNT scaffolds support proliferation of mature osteoblasts as well as production of mineralized bone in vitro. More specifically, we showed that osteoblasts grown on single-walled (SW) CNTs retain plasma membrane electrical functions involved in secretory activities. Here we studied osteoinductive properties of SWCNT scaffolds as they support the growth of osteoblastic precursors, their differentiation into mature osteoblasts, and expression of membrane proteins involved in secretory processes. We used electrically neutral ("as prepared", AP-) SWCNTs, and SWCNTs chemically modified with carboxyl (-COOH, net negative electric charge), polyethylene glycol (PEG, electrically neutral), and poly-(m-amino-benzene sulfonic acid, PABS, net positive and negative electric charges) functional groups. We found that PEG-SWCNT and PABS-SWCNT showed the highest upregulation of ALP activity as a measure of osteoinduction. In addition, we found that AP-SWCNTs induced the expression of voltage-gated chloride channels ClC-3 and ClC-5 involved in secretory activities in hFOB osteoblasts. SWCNT preparations might be seen as osteoinductive materials with great potential for use in bone regeneration and repair.

Osteoblast apoptosis plays a crucial role in bone remodeling. Physiological doses of 1 alpha, 25(OH)(2)- vitamin D(3) (1,25D) protect osteoblasts against apoptosis by means of mechanisms only partially understood. We studied activation of an Akt survival cascade downstream of 1,25D nongenomic stimulation of phosphatidylinositide-3'-kinase (PI3K) in osteoblastic cells. We measured a dose- and time-dependent 1,25D induction of Akt phosphorylation (p-Akt) in cultured osteoblastic cells. Maximal response was achieved with 10 nM 1,25D after 5 min. We found that staurosporine (STSP)-induced apoptosis was significantly reduced in 1,25D-pretreated osteoblasts. 1,25D prosurvival effects were abolished when cells were preincubated with inhibitors of PI3K activation. By means of siRNA silencing, we proved that 1,25D induction of p-Akt requires a classic vitamin D receptor (VDR) in osteoblasts. Furthermore, non-osteoblastic CV-1 cells transfected with an enhanced green fluorescent protein (EGFP)-VDR construct responded to 1,25D treatment with a rapid p-Akt response associated with increased cell survival not detected in native, nontransfected cells. We measured increased levels of p-Akt substrates; p-Bad and p-FKHR and significantly reduced activity of caspases 8 and 3/7 after 1,25D treatment. In addition, 1,25D-induced protection against apoptosis was abolished when osteoblasts. were preincubated with pertussis toxin. We conclude that anti-apoptotic effects of 1,25D in osteoblasts occur through nongenomic activation of a VDR/PI3K/Akt survival pathway that includes phosphorylation of multiple p-Akt substrates and reduction of caspase activities.

BACKGROUND. S179D Prolactin (PRL) is a molecular mimic of naturally phosphorylated human PRL which has been shown to inhibit the growth of human Prostate cancer cells both in vitro and when grown as tumors in nude mice.METHODS. In the current study, we have investigated the potential interplay between S179D PRL and 1,25 dihydroxy vitamin D3 (1,25D) in the inhibition of prostate cancer cell growth by incubating cells under circumstances where each hormone alone has no effect.RESULTS. Incubation of DU145 or PC3 cells in 100 pM 1,25D or 10 nM S179D PRL for 3 days showed no effect of each alone on expression of the vitamin D receptor (VDR), or the cell cycle regulatory protein p21, or on cell number. Incubation in both together increased expression of the VDR and p21 two to threefold. This co-operative effect was reproduced when activation of the p21 promoter was analyzed using a p21-luciferase (p21-luc) construct. Elimination of the VDR response element from p21-luc eliminated response to the hormone combination, showing that the effect on p21 was through the VDR. Most importantly, S1 79D PRL sensitized the cells to 1,25D such that there was a concentration-related reduction in cell number versus controls between 40 and 160 pM. At least part of this effect was via the induction of cell death.CONCLUSIONS. These results suggest that combined anti-tumor therapy may be very efficacious and that the dose of 1,251) required may be below the range that results in hypercalcemia.

1alpha,25(OH)(2)-vitamin D(3) (1,25D) is considered a bone anabolic hormone. 1,25D actions leading to bone formation involve gene transactivation, on one hand, and modulation of cytoplasmic signaling, on the other. In both cases, a functional vitamin D receptor (VDR) appears to be required. Here we study 1,25D-stimulated calcium signaling that initiates at the cell membrane and leads to exocytosis of bone materials and increased osteoblast survival. We found that rapid 1,25D-induction of exocytosis couples to cytoplasmic calcium increase in osteoblastic ROS 17/2.8 cells. In addition, we found that elevation of cytoplasmic calcium concentration is involved in 1,25D anti-apoptotic effects via Akt activation in ROS 17/2.8 cells and non-osteoblastic CV-1 cells. In both cases, 1,25D-stimulated elevation of intracellular calcium is due in part to activation of L-type Ca(2+) channels. We conclude that 1,25D bone anabolic effects that involve increased intracellular Ca(2+) concentration in osteoblasts can be explained at two levels. At the single-cell level, 1,25D promotes Ca(2+)-dependent exocytotic activities. At the tissue level, 1,25D protects osteoblasts from apoptosis via a Ca(2+)-dependent Akt pathway. Our studies contribute to the understanding of the molecular basis of bone diseases characterized by decreased bone formation and mineralization.

The molecular mechanisms underlying antiproliferative actions of the steroid 1α,25-dihydroxy vitamin D3 (1,25D) in human osteosarcoma cells are known only partially. To better understand the signaling involved in 1,25D anti-tumorigenic properties in bone, we stably silenced vitamin D receptor (VDR) expression in the human osteosarcoma SaOS-2 cell line. We found that 1,25D treatment reduced cell proliferation by approximately 25% after 3 days only in SaOS-2 cells expressing native levels of VDR protein, and involved activation of MAPK/AP-1/p21waf1 pathways. Both sustained (3 days) and transient (15min) 1,25D treatment activated JNK and ERK1/2 MAPK signaling in a nongenomic VDR-dependent manner. However, only sustained exposure to hormone led to upregulation of p21 and subsequent genomic control of the cell cycle. Specific blockade of MEK1/MEK2 cascade upstream from ERK1/2 abrogated 1,25D activation of AP-1 and p21, and subsequent antiproliferative effects, even in the presence of a nuclear VDR. We conclude that 1,25D-induced inhibition of human osteosarcoma cell proliferation occurs via sustained activation of JNK and MEK1/MEK2 pathways downstream of nongenomic VDR signaling that leads to upregulation of a c-Jun/c-Fos (AP-1) complex, which in turn modulates p21waf1 gene expression. Our results demonstrate a cross-talk between 1,25D/VDR nongenomic and genomic signaling at the level of MAP kinase activation that leads to reduction of cell proliferation in human osteosarcoma cells.

Membrane-initiated cellular responses to steroids include modulation of ion channel activities via signal transduction pathways. However, the molecular mechanisms involved in nongenomic actions remain only partially understood. Our research has focused on the rapid effects of 1α,25(OH)2 Vitamin D3 [1,25D] on L-type Ca2+ [L-Ca] and DIDS-sensitive Cl− channels in osteoblasts. Physiological nanomolar concentrations of hormonally active 1,25D promote rapid (1–5min) potentiation of outward Cl− currents in osteosarcoma ROS 17/2.8 cells and mouse primary osteoblasts. In addition, 1,25D increases inward barium currents through L-Ca channels at low depolarizing potentials within seconds in a fashion similar to the 1,4-dihydropyridine [DHP] agonist Bay K8644. We found that second messenger cAMP is involved in 1,25D potentiation of Cl− and Ca2+ channels. Nongenomic 1,25D effects on ion channel activities in osteoblasts appear to involve different mechanisms that include a possible direct interaction with the L-Ca channel molecule, on one hand, and signaling through the cAMP pathway, on the other. Rapid 1,25D actions on Cl− and Ca2+ currents seem to couple to secretory activities in osteoblasts, thus contributing to bone mass formation.

A major goal in bone bioengineering is to produce artificial nanostructures with the capacity to increment or replace bone tissues. The purpose of this study is to further explore the use of carbon nanotubes (CNTs) as an adequate scaffold material for osteoblast proliferation and bone matrix formation. Ion channel activities were studied as an indirect measure of the ability of osteoblasts to carry out secretory functions. Chloride and calcium channels were measured in rat osteosarcoma 17/2.8 cells cultured on single-walled (SW) and multi-walled (MW) NTs. Osteoblasts grown on non-functionalised as prepared (AP-) and carboxyl (COOH)-modified SWNTs developed outwardly rectifying, non-inactivating voltage-gated Cl- currents that increased by 3-fold over 10 min because of mechanical stimulation. In addition, osteoblasts expressed inward Ba2+ currents through high voltage-activated L-type Ca2+ channels sensitive to the specific modulators, Bay K 8644 and nifedipine. In all cases, it was verified that COOH functionalisation of SW and MWNTs did not affect osteoblast ion channel activities. It is concluded that osteoblasts grown on AP- and COOH-modified SW and MWNTs retain electrical properties necessary for secretory activities involved in bone formation. These results confirm the suitability of NTs as a potential scaffold material in bone bioengineering.