Zoledronate Promotes Peri-Implant Osteogenesis in Diabetic Osteoporosis by the AMPK Pathway.

Together with diabetic osteoporosis (DOP), diabetes patients experience poor peri-implant osteogenesis following implantation for dentition defects. Zoledronate (ZOL) is widely used to treat osteoporosis clinically. To evaluate the mechanism of ZOL for the treatment of DOP, experiments with DOP rats and high glucose-grown MC3T3-E1 cells were used. The DOP rats treated with ZOL and/or ZOL implants underwent a 4-week implant-healing interval, and then microcomputed tomography, biomechanical testing, and immunohistochemical staining were performed to elucidate the mechanism. In addition, MC3T3-E1 cells were maintained in an osteogenic medium with or without ZOL to confirm the mechanism. The cell migration, cellular actin content, and osteogenic differentiation were evaluated by a cell activity assay, a cell migration assay, as well as alkaline phosphatase, alizarin red S, and immunofluorescence staining. The mRNA and protein expression of adenosine monophosphate-activated protein kinase (AMPK), phosphorylated AMPK (p-AMPK), osteoprotegerin (OPG), receptor activator of nuclear factor kappa B ligand (RANKL), bone morphogenetic protein 2 (BMP2), and collagen type I (Col-I) were detected using real-time quantitative PCRs and western blot assays, respectively. In the DOP rats, ZOL markedly improved osteogenesis, enhanced bone strength and increased the expression of AMPK, p-AMPK, and Col-I in peri-implant bones. The in vitro findings showed that ZOL reversed the high glucose-induced inhibition of osteogenesis via the AMPK signaling pathway. In conclusion, the ability of ZOL to promote osteogenesis in DOP by targeting AMPK signaling suggests that therapy with ZOL, particularly simultaneous local and systemic administration, may be a unique approach for future implant repair in diabetes patients.

Osteogenic capability of strontium and icariin-loaded TiO2 nanotube coatings in vitro and in osteoporotic rats.

Titanium (Ti) and Ti alloys are widely used biomaterials, but they lack osteogenic capability for rapid bone integration. To improve osseointegration of Ti implants, TiO2 nanotubes were prepared using the anodizing oxidation technique, and strontium (Sr) combined with icariin (ICA) was loaded on TiO2 nanotube coatings. Cell adhesion and proliferation of MC3T3-E1 cells, alkaline phosphatase (ALP) activity, mineralization of extracellular matrix, and bone formation around titanium implants in ovariectomized rats, were examined separately. The results showed that compared with pure Ti, TiO2 and Sr-loaded TiO2 coatings, the coatings loaded with both Sr and ICA showed better effect on cell adhesion and proliferation, higher ALP activity and more red-stained mineralized nodules. Furthermore, more bone was formed around implants loaded with both Sr and ICA in osteoporotic rats. Therefore, coating with Sr and ICA is valuable for clinical application to strengthen the osseointegration of titanium implants, especially in osteoporotic patients.

Evaluation of osteogenic and antibacterial properties of strontium/silver-containing porous TiO2 coatings prepared by micro-arc oxidation.

Ti and Ti alloys are bioinert materials and two frequent problems associated with them are bacterial infection and lack of osteogenic potential for rapid bone integration. To overcome the problems, the present study incorporated strontium (Sr) and silver (Ag) simultaneously into porous TiO2 coatings through a single-step technique, micro-arc oxidation (MAO). Incorporation of Sr and Ag brought no significant changes to coating micromorphology and physicochemical properties, but endowed TiO2 coatings with both strong antibacterial activity and osteogenic ability. Antibacterial activity increased with Ag contents in the coatings. When Ag content reached 0.58 wt%, the coating showed both excellent short-term (100.0%) and long-term (77.6%) antibacterial activities. Sr/Ag-containing coatings with 18.23 wt% Sr and 0.58 wt% Ag also presented good cytocompatibility for preosteoblast adhesion and proliferation, and promoted preosteoblast osteogenic differentiation both short-termly and long-termly. However, higher Ag content (1.29 wt%) showed toxic effects to preosteoblasts. In summary, MAO is a simple and effective way to incorporate Sr and Ag into porous TiO2 coatings and Sr/Ag-containing TiO2 coating with 18.5 wt% Sr and 0.58 wt% Ag has both good osteogenic activity and strong antibacterial capability short-termly and long-termly. Therefore, such coatings are valuable for clinical application to strengthen osseointegration and long-term high quality use of titanum implants.

Effectiveness of strontium-doped brushite, bovine-derived hydroxyapatite and synthetic hydroxyapatite in rabbit sinus augmentation with simultaneous implant installation.

Various bone substitutes have been applied in sinus augmentation (SA) to overcome insufficient bone height at the posterior maxilla region caused by pneumatized sinus and severe alveolar bone resorption after teeth loss. However, their effectiveness in SA needs to be further elucidated. In this study, strontium-doped brushite (Sr-DCPD), a new bone substitute, together with bovine-derived hydroxyapatite (bHA) and synthetic hydroxyapatite (sHA) was used in rabbit maxillary SA with simultaneous implant installation. The sinus space-keeping capacity, resorption rate, osteoconductivity, and mechanical properties of regenerated bone, were evaluated by micro-computed tomography (CT), histological analysis, and mechanical testing. Sr-DCPD exhibited the best osteoconductivity and new bone formation (<4 weeks), but its final bone regeneration and removal torque of implants at week 12 were the lowest, mainly due to its poor space-keeping capacity and fast resorption. bHA exhibited the best space-keeping capacity and slowest resorption rate, but relative lower final bone volume and mechanical properties, while sHA showed good space-keeping capacity, slower resorption rate, and the best final bone formation and mechanical properties. sHA was most effective for SA and bHA was also an acceptable bone substitute; however, Sr-DCPD was least effective and not suitable in SA by itself.

The Antagonist of Retinoic Acid Receptor α, ER-50891 Antagonizes the Inhibitive Effect of All-Trans Retinoic Acid and Rescues Bone Morphogenetic Protein 2-Induced Osteoblastogenic Differentiation.

BACKGROUND: Hypervitaminosis A, alcoholism or medical treatment for acute promyelocytic leukaemia may cause unphysiologically high accumulation of all-trans retinoic acid (ATRA), which could inhibit osteoblastogenesis, thereby triggering osteoporosis. We have shown that bone morphogenetic protein-2 (BMP-2) can only partially antagonize the inhibitive effects of ATRA. In this study, we hypothesized that antagonists of retinoic acid receptors (RARs) could further antagonize the inhibitive effect of ATRA and rescue BMP2-induced osteoblastogenesis. MATERIALS AND METHODS: We first screened the dose-dependent effects of the specific antagonists of RAR α, ß and γ and transforming growth factor-beta receptor (ER-50891, LE-135, MM11253, and SB-43142, respectively) on ATRA-induced inhibition of the total cell metabolic activity and proliferation of preosteoblasts. We selected ER-50891 and tested its effects on osteoblastogenesis with the presence or absence of 1 µM ATRA and/or 200 ng/mL BMP-2. We measured the following parameters: Alkaline phosphatase activity (ALP), osteocalcin (OCN) expression and extracellular matrix mineralization as well as the level of phosphorylated Smad1/5. RESULTS: ER-50891 but not LE-135, MM11253, or SB-431542 significantly antagonized the inhibition of ATRA and enhanced the total cell metabolic activity and proliferation of preosteoblasts. Dose-dependent assays show ER-50891 could also rescue ATRA inhibited OCN expression and mineralization with or without the induction of BMP. ER-50891 also suppressed the ALP activity that was synergistically enhanced by BMP and ATRA. Neither ATRA, nor ER-50891 or their combination significantly affected the level of BMP-induced phosphorylated Smad1/5. CONCLUSION: The antagonist of RARα, ER-50891 could significantly attenuate ATRA's inhibitive effects on BMP 2-induced osteoblastogenesis.

All-trans retinoic acid can antagonize osteoblastogenesis induced by different BMPs irrespective of their dimerization types and dose-efficiencies.

INTRODUCTION: Alcoholism can lead to low mineral density, compromised regenerative bone capacity and delayed osteointegration of dental implants. This may be partially attributed to the inhibitive effect of all-trans retinoic acid (ATRA), a metabolite of alcohol, on osteoblastogenesis. Our previous studies demonstrated that heterodimeric bone morphogenetic protein 2/7 (BMP2/7) was a more potent BMP than homodimeric BMP2 or BMP7, and could antagonize the inhibitive effect of ATRA to rescue osteoblastogenesis. MATERIALS AND METHODS: In this study, we compared the effectiveness of BMP2/7, BMP2 and BMP7 in restoring osteoblastogenesis of murine preosteoblasts upon inhibition with 1 µM ATRA, and we further analyzed the potential mechanisms. We measured the following parameters: cell viability, ALP, OCN, mineralization, the expression of osteogenic differentiation marker genes (Collagen I, ALP and OCN) and the expression of BMP signaling key genes (Dlx5, Runx2, Osterix and Smad1). RESULTS: BMP2/7 treatment alone induced significantly higher osteoblastogenesis compared to BMP2 and BMP7. When cells were treated by ATRA, BMP2/7 was superior only in rescuing cell viability and ALP activity, compared to BMP2 or BMP7. However, BMP2/7 was not superior to BMP2 or BMP7 in restoring OCN expression and extracellular mineralized nodules, or in rescuing expression of two key osteogenic genes, Dlx5 and Runx2. Irrespective of their dimeric types or potency, the selected BMPs could antagonize the inhibitory effect of ATRA on osteoblastogenesis. CONCLUSION: The presence of ATRA, BMP2/7 still induced significantly higher cell viability and early differentiation than the homodimers. However, ATRA significantly attenuated the advantages of BMP2/7 in inducing late and final osteoblastogenic differentiation over the homodimers.

All-trans retinoic-acid inhibits heterodimeric bone morphogenetic protein 2/7-stimulated osteoclastogenesis, and resorption activity.

BACKGROUND: Bone regenerative heterodimeric bone morphogenetic protein 2/7 (BMP2/7) enhances but all-trans retinoic acid (ATRA) inhibits osteoclastogenesis. However, the effect of ATRA on physiological and/or BMP2/7-induced osteoclastogenesis in still unclear. In this study, we aimed to test the effect of combined treatment of BMP2/7 and ATRA on osteoclastogenesis, and resorption activity. RESULTS: All-trans retinoic acid (1 µM) ± BMP2/7 (5 or 50 ng/ml) was added in murine pre-osteoclasts cell line RAW264.7 or mouse bone marrow derived macrophages (BMM) cultures. Osteoclast marker gene expression, osteoclastogenesis, and resorption activity were analyzed. BMP2/7 robustly enhanced osteoclast maker gene expression, osteoclastogenesis, and resorption activity. Interestingly, ATRA completely inhibited osteoclast formation in presence or absence of BMP2/7. Pan-antagonist of retinoic acid receptors (RARs) and antagonist of RARα, ß or γ failed to reverse the inhibitory effect of ATRA on osteoclastogenesis. ATRA strongly inhibited Rank and Nfatc1 expression. CONCLUSIONS: All-trans retinoic acid inhibits BMP2/7-induced osteoclastogenesis, and resorption activity possibly via RANKL-RANK pathway. Our findings from previous and current study suggest that combination of ATRA and BMP2/7 could be a novel approach to treat hyperactive osteoclast-induced bone loss such as in inflammation-induced severe osteoporosis and bone loss caused by cancer metastasis to bone.

Heterodimeric BMP-2/7 antagonizes the inhibition of all-trans retinoic acid and promotes the osteoblastogenesis.

OBJECTIVES: Hypervitaminosis A and alcoholism can result in a low mineral density and compromised regenerative capacity of bone, thus delaying implant osteointegration. The inhibitory effect of all-trans retinoic acid on osteoblastogenesis is considered to be one of the mechanisms. We hypothesized that heterodimeric bone morphogenetic protein-2/7 could antagonize all-trans retinoic acid and enhance osteoblastogenesis, with an aim to accelerate and enhance bone regeneration and implant osteointegration. MATERIALS AND METHODS: We applied 5 ng/ml or 50 ng/ml bone morphogenetic protein-2/7 to restore the osteoblastogenesis of pre-osteoblasts (MC3T3-E1 cell line) that was inhibited by 1 µM all-trans retinoic acid. We evaluated the efficacy by assessing cell numbers (proliferation), alkaline phosphatase activity (a marker for early differentiation), osteocalcin (a marker for late differentiation), calcium deposition (a marker for final mineralization) and the expression of osteoblastogenic genes (such as Runx2, Collagen Ia, alkaline phosphatase and osteocalcin) at different time points. RESULTS: All-trans retinoic acid significantly inhibited the expression of all the tested osteoblastogenic genes and proteins except alkaline phosphatase activity. In the presence of ATRA, 50 ng/ml bone morphogenetic protein-2/7 not only completely restored but also significantly enhanced all the osteoblastogenic genes and proteins. On the 28(th) day, mineralization was completely inhibited by all-trans retinoic acid. In contrast, 50 ng/ml BMP-2/7 could antagonize ATRA and significantly enhance the mineralization about 2.5 folds in comparison with the control treatment (no ATRA, no BMP2/7). CONCLUSIONS: Heterodimeric bone morphogenetic protein-2/7 bears a promising application potential to significantly promote bone regeneration and implant osteointegration for the patients with hypervitaminosis A and alcoholism.

Antagonistic and synergistic effects of bone morphogenetic protein 2/7 and all-trans retinoic acid on the osteogenic differentiation of rat bone marrow stromal cells.

The osteogenesis of bone marrow stromal cells (BMSCs) is of paramount importance for the repair of large-size bone defects, which may be compromised by the dietary-accumulated all-trans retinoic acid (ATRA). We have shown that heterodimeric bone morphogenetic protein 2/7 (BMP2/7) could induce bone regeneration in a significantly higher dose-efficiency in comparison with homodimeric BMPs. In this study, we evaluated the effects of ATRA and BMP2/7 on the proliferation, differentiation, mineralization and osteogenic genes. ATRA and BMP2/7 exhibited both antagonistic and synergistic effects on the osteogenesis of BMSCs. ATRA significantly inhibited proliferation and expression of osteocalcin but enhanced the activity of alkaline phosphatase of BMSCs. On day 21, 50 ng/mL BMP2/7 could antagonize the inhibitive effects of ATRA and significantly enhance osteogenesis of BMSCs. These findings suggested a promising application potential of heterodimeric BMP2/7 in clinic to promote bone regeneration for the cases with dietary accumulated ATRA.