Z Li

Strontium Promotes Osteogenic Differentiation of Mesenchymal Stem Cells Through the Ras/MAPK Signaling Pathway

Strontium ralenate is a new anti-osteoporosis agent. The cellular and molecular mechanism underlying the anabolic effect of strontium on bone remains to be elucidated. Osteoblasts, the main bone forming cells are known to be derived from bone marrow mesenchymal stem cells (MSCs). The present study therefore aimed to investigate the possible effects of strontium on MSCs and signaling pathways possibly involved. It was firstly demonstrated that strontium treatment significantly increased osteoblast-related gene expression and alkaline phosphatase (ALP) of osteogenic-differentiating MSCs. Accompanying the enhanced osteogenic differentiation, the increased phosphorylation of mitogen-activated protein kinase (MAPK) ERK1/2 and p38 was detected in strontium-treated MSCs. PD98059 and SB203580, selective inhibitors of ERK1/2 kinase and p38, attenuated the effect of strontium on osteogenesis. Furthermore, it was demonstrated that Rat Sarcoma viral oncogene homolog (RAS), an upstream regulator of ERK1/2 and p38, was activated by strontium treatment and siRNA-mediated Ras knockdown inhibited strontium-stimulated expression of osteogenic markers. Finally, the transcriptional activity and phosphorylation level of Runx2 was significantly increased in response to strontium treatment in MSCs. PD98059 and Ras siRNA inhibited the effect of strontium on Runx2 activation. Taken together, these results indicated that strontium can promote osteogenic differentiation of MSCs through activating the Ras/MAPK signaling pathway and the downstream transcription factor Runx2.

Inhibition of Sca-1-Positive Skeletal Stem Cell Recruitment by Alendronate Blunts the Anabolic Effects of Parathyroid Hormone on Bone Remodeling

The anabolic effects of parathyroid hormone (PTH) on bone formation are impaired by concurrent use of antiresorptive drugs. We found that the release of active transforming growth factor (TGF)-β1 during osteoclastic bone resorption is inhibited by alendronate. We showed that mouse Sca-1-positive (Sca-1(+)) bone marrow stromal cells are a skeletal stem cell subset, which are recruited to bone remodeling sites by active TGF-β1 in response to bone resorption. Alendronate inhibits the release of active TGF-β1 and the recruitment of Sca-1(+) skeletal stem cells for the bone formation. The observation was validated in a Tgfb1(-/-) mouse model, in which the anabolic effects of PTH on bone formation are diminished. The PTH-stimulated recruitment of injected mouse Sca-1(+) cells to the resorptive sites was inhibited by alendronate. Thus, inhibition of active TGF-β1 release by alendronate reduces the recruitment of Sca-1(+) skeletal stem cells and impairs the anabolic action of PTH in bone.

Strontium borate glass: potential biomaterial for bone regeneration

Boron plays important roles in many life processes including embryogenesis, bone growth and maintenance, immune function and psychomotor skills. Thus, the delivery of boron by the degradation of borate glass is of special interest in biomedical applications. However, the cytotoxicity of borate glass which arises with the rapid release of boron has to be carefully considered. In this study, it was found that the incorporation of strontium into borate glass can not only moderate the rapid release of boron, but also induce the adhesion of osteoblast-like cells, SaOS-2, thus significantly increasing the cyto-compatibility of borate glass. The formation of multilayers of apatite with porous structure indicates that complete degradation is optimistic, and the spread of SaOS-2 covered by apatite to form a sandwich structure may induce bone-like tissue formation at earlier stages. Therefore, such novel strontium-incorporated borosilicate may act as a new generation of biomaterial for bone regeneration, which not only renders boron as a nutritious element for bone health, but also delivers strontium to stimulate formation of new bones.

Solubility of strontium-substituted apatite by solid titration.

Solid titration was used to explore the solubility isotherms of partially (Srx-HAp, x=1, 5, 10, 40, 60 mol.%) and fully substituted strontium hydroxyapatite (Sr-HAp). Solubility increased with increasing strontium content. No phase other than strontium-substituted HAp, corresponding to the original titrant, was detected in the solid present at equilibrium; in particular, dicalcium hydrogen phosphate was not detected at low pH. The increase in solubility with strontium content is interpreted as a destabilization of the crystal structure by the larger strontium ion. Carbonated HAp was formed in simulated body fluid containing carbonate on seeding with Sr10-HAp, but the precipitate was strontium-substituted on seeding with Sr-HAp. Strontium-substituted HAp might be usable as a template for the growth of new bone, since nucleation appears to be facilitated.

The cross-talk between osteoclasts and osteoblasts in response to strontium treatment: involvement of osteoprotegerin.

BACKGROUNDnThe mechanism for the uncoupling effects of Sr on bone remains to be evaluated. Osteoblasts play important roles in osteoclastogenesis through regulating receptor activated nuclear factor kappa B (RANK) ligand (RANKL) and osteoprotegerin (OPG) expression. We hypothesize that OPG plays an important role in the cross-talk between osteoclasts and osteoblasts in response to Sr treatment.nnnMATERIALS AND METHODSnMC3T3E1 cells were treated with Sr chloride (0-3 mM) and conditioned media were collected at 24h after the treatment. The effect of conditioned media on osteoclastogenesis was evaluated by tartrate-resistant acid phosphatase (TRAP) staining and bone resorption pits analysis. OPG and RANKL mRNA expressions in osteoblastic cells and protein secretion in the conditioned media were analyzed with real-time PCR and ELISA assay, respectively. The role of OPG in Sr-mediated inhibition of osteoclastogenesis was further evaluated with anti-OPG antibody in pre-osteoclastic cells. The role of OPG in Sr-mediated uncoupling effects on osteoporotic bone was evaluated by an animal study. Ovariectomized rats were oral administrated with vehicle or Sr chloride for two months supplemented with anti-IgG antibody (control) or anti-OPG antibody. The effects of OPG neutralization after Sr treatment on bone metabolism were analyzed by microCT, bone histomorphometry and biochemical analysis.nnnRESULTSnThe conditioned media derived from Sr-treated osteoblastic cells exerted a dose-dependent inhibitory effect on osteoclastic differentiation and resorptive activity in pre-osteoclastic cells. OPG mRNA expression and protein secretion in osteoblastic cells were significantly increased after Sr treatment. Neutralization with anti-OPG antibody abolished the inhibitory effect of conditioned media on RANKL-induced osteoclastogenesis. The uncoupling effects of Sr treatment on trabecular bone were evidenced by greater bone volume and trabecular number, greater osteoid surface and bone formation rate, while less osteoclast surface. These effects were attenuated by the OPG neutralization by anti-OPG antibody injection.nnnCONCLUSIONnThe evidences from the in vitro and in vivo studies suggested that OPG played an important role in the uncoupling effect of Sr on bone metabolism, possibly by acting as a cross-talk molecule between osteoclasts and osteoblasts in response to Sr treatment.

In vivo anabolic effect of strontium on trabecular bone was associated with increased osteoblastogenesis of bone marrow stromal cells

In vitro studies have demonstrated that strontium (Sr) could increase osteogenic differentiation of bone marrow stromal cells (BMSCs). We investigated the in vivo effect of Sr on BMSCs. Thirty‐six female rats were randomly divided into the following groups: sham operated and treated with either vehicle (Shamu2009+u2009Veh) or Sr compound (Shamu2009+u2009Sr) and ovariectomized and treated with either vehicle (OVXu2009+u2009Veh) or Sr compound (OVXu2009+u2009Sr). Vehicle and Sr were orally administrated daily starting immediately after the surgery and continuing for 12 weeks. The anabolic effect of Sr on trabecular bone was determined at the structural and tissue level by microCT and histomorphometry, respectively. Colony formation assays demonstrated that BMSCs exhibited higher osteogenic colony but lower adipogenic colony in Sr‐treated versus Veh‐treated OVX rats. The mRNA level of osteogenic genes was higher, while the mRNA level of adipogenic genes was lower in BMSCs from Sr‐treated versus Veh‐treated Sham and OVX rats. The effect of Sr on rat BMSCs was reproducible in human BMSCs. Taken together, this study suggests that the anabolic effect of Sr on normal or osteoporotic bones is associated with increased osteoblastic differentiation of BMSCs.

Enhanced gene delivery by chitosan-disulfide-conjugated LMW-PEI for facilitating osteogenic differentiation

Chitosan-disulfide-conjugated LMW-PEI (CS-ss-PEI) was designed to combine the biocompatibility of chitosan and the gene delivery ability of polyethylenimine (PEI) using bio-reducible disulfide for bone morphogenetic protein (BMP2) gene delivery in mediating osteogenic differentiation. It was prepared by conjugating low molecular weight PEI (LMW-PEI) to chitosan through oxidization of thiols introduced for the formation of disulfide linkage. The structure, molecular weight and buffer capacity were characterized by Fourier transform infrared (FTIR), light scattering and acid-base titration, respectively. The reduction in molecular weight of CS-ss-PEI by the reducing agent indicated its bio-reducible property. With the increment in the LMW-PEI component, the copolymer showed increased DNA binding ability and formed denser nanocomplexes. CS-ss-PEI exhibited low cytotoxicity in COS-1, HepG2 and 293T cells over the different weight ratios. The transfection efficiency of CS-ss-PEI4 was significantly higher than that of PEI 25k and comparable with Lipofectamine in mediating luciferase expression. Its application for BMP2 gene delivery was confirmed in C2C12 cells by BMP2 expression. For inducing in vitro osteogenic differentiation, CS-ss-PEI4 mediated BMP2 gene delivery showed a stronger effect in MG-63 osteoblast cells and stem cells in terms of alkaline phosphatase activity and mineralization compared with PEI25k and Lipofectamine. This study provides a potential gene delivery system for orthopedic-related disease.

Rapid repair of rat sciatic nerve injury using a nanosilver-embedded collagen scaffold coated with laminin and fibronectin

AIMnScaffold with micro-channels has shown great promise in facilitating axonal regeneration after peripheral nerve injury. Significant research has focused on mimicking, in terms of composition and function, the ability of the basement membrane of Schwann cells to both promote and guide axonal regeneration. We aim to investigate the ability of a tissue-engineered scaffold with nanosilver and collagen to adsorb laminin and fibronectin, and the usefulness of this scaffold for repairing and regenerating a 10-mm peripheral nerve gap in rats.nnnMETHODSnIn this study, nanosilver-embedded collagen scaffolds were prepared and coated with laminin (LN) or LN plus fibronectin (FN). Scanning electron microscopy of the transverse and longitudinal sections of the scaffold revealed axially oriented microtubules ranging from 20 to 80 µm in diameter, and the internal surface of microtubules was found to be evenly coated with LN and FN. Energy dispersive spectrometry also confirmed an even distribution of nanosilver particles within the scaffold. To test its effectiveness in restoring neuronal connection, the scaffold was used in order to bridge 10 mm gaps in the severed sciatic nerve of rats. The rats were divided into an experimental group (receiving scaffold coated with LN and FN), a control group (receiving scaffold coated with LN only) and an autologous graft group. The functional recovery 40 days after surgery was examined by electrophysiology and sciatic nerve functional index (SFI) evaluation. FluoroGold™ (FG) retrograde tracing, toluidine blue staining and transmission electron microscopy were also used to examine the regenerated nerve fibers and to establish their myelination status.nnnRESULTSnThe experimental group displayed partially restored nerve function. The recovery was comparable to the effect of autologous nerve graft and was better than that observed in the control group. A better functional recovery correlated with more FG-labeled neurons, higher density of toluidine blue stained nerve fibers and thicker myelin sheath.nnnCONCLUSIONnOur results demonstrated that nanosilver-embedded collagen scaffolds with LN and FN coating is effective in aiding axonal regeneration, and recovery is comparable to the effect of an autologous nerve graft.

Osteoprotegerin deficiency attenuates strontium‐mediated inhibition of osteoclastogenesis and bone resorption

Strontium (Sr) exerts an anabolic and antiresorptive effect on bone, but the mechanism remains unknown. Osteoprotegerin (OPG) expressed by osteoblasts plays an important role in regulating bone homeostasis by inhibiting osteoclastogenesis and bone resorption. This study aims at evaluating the role of OPG in Sr‐mediated inhibition of osteoclastogenesis and bone resorption. Six‐week‐old Opg knockout (KO) male mice and their wild‐type (WT) littermates were treated orally with vehicle (Veh) or Sr compound (4u2009mmol/kg) daily for 8 weeks. Bone mass and microstructure in the lumbar spine (L4) and proximal tibia were analyzed with micro–computed tomography (µCT). Bone remodeling was evaluated with serum biochemical analysis and static and dynamic bone histomorphometry. Osteoclast differentiation potential and gene expression were analyzed in bone marrow cells. The findings demonstrate that Sr compound treatment results in greater bone volume and trabecular number than Veh treatment in WT mice. The anabolic response of trabecular bone to Sr treatment is attenuated in KO mice. Although Sr treatment significantly decreases in vitro osteoclastogenesis and bone resorption in WT mice, these effects are attenuated in KO mice. Furthermore, Sr treatment profoundly increases Opg gene expression in the tibias and OPG protein levels in the sera of WT mice. This study concludes that the inhibition of osteoclastogenesis and bone resorption is possibly associated with OPG upregulation by Sr treatment.

Strontium–calcium coadministration stimulates bone matrix osteogenic factor expression and new bone formation in a large animal model

Strontium (Sr) has become increasingly attractive for use in the prevention and treatment of osteoporosis by concomitantly inhibiting bone resorption and enhancing bone formation. Strontium shares similar chemical, physical, and biological characteristics with calcium (Ca), which has been widely used as a dietary supplement in osteoporosis. However, the effects of Sr–Ca coadministration on bone growth and remodeling are yet to be extensively reported. In this study, 18 ovariectomized goats were divided into four groups: three groups of five goats each treated with 100 mg/kg/day Ca, Ca plus 24 mg/kg/day Sr (Cau2009+u200924Sr), or Ca plus 40 mg/kg/day Sr (Cau2009+u200940Sr), and three untreated goats fed low calcium feed. Serum Sr levels increased 6‐ and 10‐fold in the Cau2009+u200924Sr and Cau2009+u200940Sr groups, respectively. Similarly, Sr in the bone increased four‐ and sixfold in these two groups. Sr–Ca coadministration considerably increased bone mineral apposition rate (MAR). The expression of insulin‐like growth factor (IGF)‐1 and runt‐related transcription factor 2 (Runx2) was significantly upregulated within the Cau2009+u200940Sr treatment group; tumor necrosis factor (TNF)‐agr; expression was significantly downregulated in the Ca and Cau2009+u200940Sr groups. The results indicate that Sr–Ca coadministration increases osteogenic gene expression and stimulates new bone formation.