Cbfa1 mRNA Research Articles

Cbfa1 expression in vascular smooth muscle cells may be elevated by increased nitric oxide/iNOS.

ABSTRACTIntroduction: Vascular calcification is a common complication of chronic kidney disease. Osteoblast differentiation factor (Cbfa1) is present in histologic sections of arteries from patients with end-stage renal disease. Vascular smooth muscle cells (VSMC) can dedifferentiate to osteoblast-like cells, possibly by up-regulation of Cbfa1. There is evidence that the production of nitric oxide (NO) may have an important role in the regulation of osteoblast metabolism. The aim of this study is to evaluate whether increased NO/iNOS expression causes an increase in cbfa1 expression in VSMC.Methods: VSMC were obtained from renal artery of Wistar male rats, treated for 72 hours with lipopolysaccharide (LPS), ß-glycerophosphate (BGF), a donor of phosphate and aminoguanidine (AG), an inhibitor of iNOS, in the following groups: CTL (control), LPS, BGF, LPS + BGF, and LPS + AG. NO synthesis was determined by chemiluminescence. Cbfa1 and iNOS mRNA expressions were analyzed by RT-PCR, Cbfa1 protein expression by immunohistochemistry and cellular viability by acridine orange. Results: Cbfa1 and iNOS mRNA expressions were higher in LPS and LPS+ BGF vs CTL (p < 0.05), and they were lower in LPS+AG vs LPS (p < 0.05). The Cbfa1 in the groups LPS and LPS+BGF also resulted in a higher value compared to CTL (p < 0.05), and in LPS+AG it was lower compared to LPS (p < 0.05). NO was higher in LPS and LPS+BGF compared to CTL group (p < 0.05) and lower in LPS + AG compared to LPS group (p < 0.05). Cellular viability showed no statistical difference among groups.Conclusion: This study showed that increased NO/iNOS expression causes an increase in cbfa1 expression in VSMC.

Phosphate-induced ORAI1 expression and store-operated Ca2+ entry in aortic smooth muscle cells.

Compromised renal phosphate elimination in chronic kidney disease (CKD) leads to hyperphosphatemia, which in turn triggers osteo-/chondrogenic signaling in vascular smooth muscle cells (VSMCs) and vascular calcification. Osteo-/chondrogenic transdifferentiation of VSMCs leads to upregulation of the transcription factors MSX2, CBFA1, and SOX9 as well as tissue-nonspecific alkaline phosphatase (ALPL) which fosters calcification by degrading the calcification inhibitor pyrophosphate. Osteo-/chondrogenic signaling in VSMCs involves the serum- and glucocorticoid-inducible kinase SGK1. As shown in other cell types, SGK1 is a powerful stimulator of ORAI1, a Ca2+-channel accomplishing store-operated Ca2+-entry (SOCE). ORAI1 is stimulated following intracellular store depletion by the Ca2+ sensor STIM1. The present study explored whether phosphate regulates ORAI1 and/or STIM1 expression and, thus, SOCE in VSMCs. To this end, primary human aortic smooth muscle cells (HAoSMCs) were exposed to the phosphate donor β-glycerophosphate. Transcript levels were estimated by qRT-PCR, protein abundance by western blotting, ALPL activity by colorimetry, calcification by alizarin red S staining, cytosolic Ca2+-concentration ([Ca2+]i) by Fura-2-fluorescence, and SOCE from increase of [Ca2+]i following re-addition of extracellular Ca2+ after store depletion with thapsigargin. As a result, β-glycerophosphate treatment increased ORAI1 and STIM1 transcript levels and protein abundance as well as SOCE in HAoSMCs. Additional treatment with ORAI1 inhibitor MRS1845 or SGK1 inhibitor GSK650394 virtually disrupted the effects of β-glycerophosphate on SOCE. Moreover, the β-glycerophosphate-induced MSX2, CBFA1, SOX9, and ALPL mRNA expression and activity in HAoSMCs were suppressed in the presence of the ORAI1 inhibitor and upon ORAI1 silencing. In conclusion, enhanced phosphate upregulates ORAI1 and STIM1 expression and store-operated Ca2+-entry, which participate in the orchestration of osteo-/chondrogenic signaling of VSMCs. KEY MESSAGES: • In aortic SMC, phosphate donor ß-glycerophosphate upregulates Ca2+ channel ORAI1. • In aortic SMC, ß-glycerophosphate upregulates ORAI1-activator STIM1. • In aortic SMC, ß-glycerophosphate upregulates store-operated Ca2+-entry (SOCE). • The effect of ß-glycerophosphate on SOCE is disrupted by ORAI1 inhibitor MRS1845. • Stimulation of osteogenic signaling is disrupted by MRS1845 and ORAI1 silencing.

Role of Cytosolic Serine Hydroxymethyl Transferase 1 (SHMT1) in Phosphate-Induced Vascular Smooth Muscle Cell Calcification

Background/Aims: Hyperphosphatemia promotes medial vascular calcification, at least partly, by induction of osteo-/chondrogenic transdifferentiation of vascular smooth muscle cells (VSMCs). The complex signaling pathways regulating this process are still incompletely understood. The present study investigated the role of cytosolic serine hydroxymethyl transferase 1 (SHMT1) in phosphate-induced vascular calcification. Methods: Endogenous expression of SHMT1 was suppressed by silencing in primary human aortic smooth muscle cells (HAoSMCs) followed by treatment without and with phosphate or antioxidants. Results: In HAoSMCs, SHMT1 mRNA expression was up-regulated by phosphate. Silencing of SHMT1 alone was sufficient to induce osteo-/chondrogenic transdifferentiation of HAoSMCs, as shown by increased tissue-nonspecific alkaline phosphatase (ALPL) activity and osteogenic markers MSX2, CBFA1 and ALPL mRNA expression. Furthermore, phosphate-induced ALPL mRNA expression and activity as well as calcification were augmented in SHMT1 silenced HAoSMCs as compared to negative control siRNA transfected HAoSMCs. Silencing of SHMT1 decreased total antioxidant capacity and up-regulated NADH/NADPH oxidase system components NOX4 and CYBA mRNA expression in HAoSMCs, effects paralleled by increased mRNA expression of matrix metalloproteinase MMP2 as well as BAX/BCL2 ratio. More importantly, additional treatment with antioxidants TEMPOL or TIRON blunted the increased osteogenic markers mRNA expression in SHMT1 silenced HAoSMCs. Conclusion: Silencing of SHMT1 promotes osteo-/chondrogenic signaling in VSMCs, at least in part, by inducing cellular oxidative stress. It thus aggravates phosphate-induced calcification of VSMCs. The present findings support a regulatory role of SHMT1 in vascular calcification during conditions of hyperphosphatemia such as chronic kidney disease.

Estradiol‑enhanced osteogenesis of rat bone marrow stromal cells is associated with the JNK pathway.

Bone marrow stromal cells (BMSCs) can differentiate into osteoblasts. The present study investigated the osteogenic effects of estradiol, as well as the role of the c-Jun N-terminal kinase (JNK) signaling pathway in promoting estradiol-enhanced osteogenesis of rat (r)BMSCs. rBMSCs were treated for 7 days with or without estradiol and further treated with or without the JNK-specific inhibitor SP600125. The role of estrogen during rBMSC osteogenesis was evaluated by alkaline phosphatase activity and mineralized nodule formation using the Gomori method and Alizarin red S staining, respectively. Subsequently, the mRNA expression levels of transforming growth factor-β1 (TGF-β1) and core-binding factor α1 (Cbfα1) were evaluated by reverse transcription-quantitative polymerase chain reaction, and TGF-β1, Cbfα1 and phosphorylated (p)-JNK protein expression was detected by western blotting. All groups treated with SP600125 expressed low levels of TGF-β1 and Cbfα1 mRNA and protein, and low p-JNK protein expression. Compared with the control cells, rBMSCs cultured with estradiol exhibited a significant upregulation in the expression levels of osteogenic genes and proteins. The present study demonstrated that estradiol enhanced osteogenic differentiation of rBMSCs and that the JNK signaling pathway was involved in this process, providing insights into the molecular mechanisms involved in rBMSC osteogenesis upon estradiol stimulation.

Bushen Qiangji Granule () medicated serum inhibits osteogenic differentiation of fibroblasts in ankylosing spondylitis by inhibiting the BMP/Smads signal pathway in vitro.

To explore the mechanism of Bushen Qiangji Granule (, BSQJ) in restraining the osteogenic differentiation of ankylosing spondylitis (AS) fifibroblasts. Hip joint capsules were obtained from AS patients (n=10) receiving total hip replacement and healthy hip joint capsules from patients with hip fracture (n=10) receiving surgery as a control. Finite fifibroblast lines were established from these tissue samples to observe the effect of BSQJ on suppressing osteogenic differentiation of fifibroblasts. The expression of osteogenic marker gene corebinding factor a1 (Cbfa1) and Smad family proteins were examined by Western blot and real-time quantitative polymerase chain reaction (qPCR). The mRNA expression level of Cbfa1 was significantly higher in AS fibroblasts than that in normal fibroblasts and the expression of pSmad1, pSmad5, Smad4 and Cbfa1 in AS fibroblasts was also higher, demonstrating the activation of the BMP/Smads signal pathway in AS fifibroblasts. BSQJ-medicated serum not only restrained the mRNA and protein expression levels of Cbfa1 and inhibited protein expression level of Smad4 but also decreased the expression quantities of pSmad1 and pSmad5. BSQJ can inhibit osteogenic differentiation of AS fifibroblasts in vitro by suppressing the activation of the BMP/Smads signal pathway. This may be the important molecular mechanism of BSQJ in regulating AS ossifification.

The suppressive effects of aluminum chloride on the osteoblasts function.

Aluminum (Al) exposure impairs bone formation, and bone formation is mediated by the osteoblasts. But effects of Al on the osteoblasts function remain elusive. The osteoblasts were exposed to 0, 0.0252, 0.126, 0.252mg/mL AlCl3·6H2O for 24h. The osteoblasts viability, TGF-β1, BMP-2, IGF-I and Cbfα1 mRNA expressions, and GSH-Px and SOD activities, ROS concentration were determined. The osteoblasts ultrastructural features were also observed. The results showed that AlCl3 suppressed the osteoblasts viability, TGF-β1, BMP-2, IGF-I and Cbfα1 mRNA expressions, GSH-Px and SOD activities, and elevated ROS concentration compared with the CG. The ultrastructural features of osteoblasts in the HG showed mitochondrial swelling, foam-like structure, uneven distribution of chromatin, incomplete cell membrane and cytoplasm spillover compared with the CG. It indicates that AlCl3 inhibits osteoblasts viability, growth regulation factors mRNA expressions, anti-oxidative function, and damaged the osteoblasts histology structure, impairing the osteoblasts function.

Ginsenoside Rb1 alleviates aluminum chloride-induced rat osteoblasts dysfunction.

Osteoblasts dysfunction, induced by aluminum (Al), plays a critical role in the osteoporosis etiology. Ginsenoside Rb1 (Rb1) has the therapeutic properties for osteoporosis. This study aimed to assess the efficiency of Rb1 in ameliorating Al-induced osteoblasts dysfunction. The osteoblasts were divided into four groups: Rb1-treated group (RG, 0.0145mg/mL Rb1), control group (CG, 0), AlCl3-treated group (AG, 0.126mg/mL AlCl3·6H2O), AlCl3+Rb1-treated group (ARG, 0.0145mg/mL Rb1 and 0.126mg/mL AlCl3·6H2O). After 24h of culture, the osteoblasts viability, the transforming growth factor-β1 (TGF-β1), bone morphogenetic protein-2 (BMP-2), the insulin-like growth factor I (IGF-I), core-binding factor α1 (Cbfα1) mRNA expressions, glutathione perioxidase (GSH-Px) and superoxide dismutase (SOD) activities, and reactive oxygen species (ROS) concentration were determined. The osteoblasts ultrastructural features were also observed. In the ARG, the osteoblasts viability, TGF-β1, BMP-2, IGF-I and Cbfα1 mRNA expressions and the GSH-Px and SOD activities were significantly increased, the ROS concentration was significantly decreased, and osteoblasts histology lesion was attenuated compared with the AG. These results demonstrated that Rb1 could significantly reverse osteoblasts viability and osteoblasts growth regulation factor, inhibit oxidative stress, and attenuate histology lesion in the osteoblasts with AlCl3. These results indicate that Rb1 can effectively alleviate the AlCl3-induced osteoblasts dysfunction.

Augmentation of phosphate-induced osteo-/chondrogenic transformation of vascular smooth muscle cells by homoarginine.

Reduced homoarginine plasma levels are associated with unfavourable cardiovascular outcome in chronic kidney disease (CKD). Cardiovascular events in CKD are fostered by vascular calcification, an active process promoted by hyperphosphatemia and involving osteo-/chondrogenic transformation of vascular smooth muscle cells (VSMCs). The present study explored the effect of homoarginine on phosphate-induced osteo-/chondrogenic signalling and vascular calcification. Experiments were performed in hyperphosphatemic klotho-hypomorphic mice (kl/kl), in subtotal nephrectomy and vitamin D3-overload mouse calcification models and in primary human aortic smooth muscle cells (HAoSMCs). As a result, plasma homoarginine levels were lower in kl/kl mice than in wild-type mice and in both genotypes significantly increased by lifelong treatment with homoarginine. Surprisingly, homoarginine treatment of kl/kl mice and of mice with renal failure after subtotal nephrectomy augmented vascular calcification and enhanced the transcript levels of plasminogen activator inhibitor 1 (Pai1) and of osteogenic markers Msx2, Cbfa1, and Alpl. Similarly, homoarginine treatment of HAoSMCs increased phosphate-induced calcium deposition, ALP activity, as well as PAI1, MSX2, CBFA1, and ALPL mRNA levels. Homoarginine alone up-regulated osteo-/chondrogenic signalling and indicators of oxidative stress in HAoSMCs. Furthermore, homoarginine reduced citrulline formation from arginine by nitric oxide (NO) synthase (NOS) isoforms. NO formation by NOS was reduced when using homoarginine as a substrate instead of arginine. The osteoinductive effects of homoarginine were mimicked by NOS inhibitor L-NAME and abolished by additional treatment with the NO donors DETA-NONOate and PAPA-NONOate or the antioxidants TEMPOL and TIRON. Furthermore, homoarginine augmented vascular calcification and aortic osteo-/chondrogenic signalling in mice after vitamin D3-overload, effects reversed by the NO donor molsidomine. Homoarginine augments osteo-/chondrogenic transformation of VSMCs and vascular calcification, effects involving impaired NO formation from homoarginine.

Inhibition of Phosphate-Induced Vascular Smooth Muscle Cell Osteo-/Chondrogenic Signaling and Calcification by Bafilomycin A1 and Methylamine

Background/Aims: Excessive phosphate concentrations trigger vascular calcification, an active process promoted by osteoinduction of vascular smooth muscle cells (VSMCs) with increased expression and activity of transcription factor RUNX2 (Core-binding factor α1, CBFA1), alkaline phosphatase (ALPL), TGFß1, transcription factor NFAT5, and NFAT5-sensitive transcription factor SOX9. The osteoinductive signaling and vascular calcification of hyperphosphatemic klotho-hypomorphic mice could be reversed by treatment with NH₄Cl, effects involving decrease of TGFß1 and inhibition of NFAT5-dependent osteoinductive signaling. Known effects of NH₄Cl include alkalinization of acidic cellular compartments. The present study explored whether osteo-/chondrogenic signaling could be influenced by alkalinization of acidic cellular compartments following inhibition of the vacuolar H⁺ ATPase with bafilomycin A1 or following dissipation of the pH gradient across the membranes of acidic cellular compartments with methylamine. Methods: Primary human aortic smooth muscle cells (HAoSMCs) were treated with high phosphate to trigger osteo-/chondrogenic signaling and calcification in the absence or presence of bafilomycin A1 or methylamine. Calcium content was determined using a QuantiChrom Calcium assay, ALP activity by a colorimetric assay and transcript levels by quantitative RT-PCR. Results: High phosphate increased significantly the calcium deposition, CBFA1 and ALPL mRNA expression as well as alkaline phosphatase activity in HAoSMCs, all effects ameliorated by both, bafilomycin A1 and methylamine. High phosphate further significantly up-regulated the mRNA levels of TGFB1, NFAT5 and SOX9, effects significantly blunted by additional treatment with bafilomycin A1 or methylamine. Treatment of HAoSMCs with human TGFß1 protein or high phosphate up-regulated NFAT5, SOX9, CBFA1 and ALPL mRNA expression to similarly high levels which could not be further increased by combined treatment with high phosphate and TGFß1. Bafilomycin A1 failed to reverse the osteo-/chondrogenic signaling triggered by high phosphate together with TGFß1. Conclusions: Inhibition of the vacuolar H⁺ ATPase or dissipation of the pH gradient across the membranes of acidic cellular compartments both disrupt osteo-/chondrogenic signaling and calcium deposition in VSMCs, observations supporting the hypothesis that vascular calcification requires acidic cellular compartments.

Expression of core-binding factor α1 and osteocalcin in fluoride-treated fibroblasts and osteoblasts

To study the effects and importance of fluoride on FBs in the development of extraperiosteal calcification and the ossification of skeletal fluorosis, the presence of the osteogenic phenotype, which is indicated by the expression of core-binding factor α1 (Cbfa1) and osteocalcin (OCN), in an FB cell line (L929) and in osteoblasts (OBs) exposed to fluoride was determined. Fibroblasts and osteoblasts were exposed to different concentrations of fluoride (0, 0.0001, 0.001, 0.1, 1.0, 10.0 and 20.0mg/L F−). By using RT-PCR and ELISA, the mRNA levels of Cbfa1 and OCN were measured at 48h, and the protein levels of Cbfa1 and OCN were measured at 2, 4, 24, 48 and 72h. The data demonstrated the following: (1) The Cbfa1 protein level in fluoride-treated fibroblasts clearly increased at 48h in the groups treated with 0.0001, 0.001, 0.1, 1.0 and 20.0mg/L F−. The Cbfa1 protein level of the group treated with 10mg/L F− at 72h was higher than that of the control group. The level of Cbfa1 mRNA in the fibroblasts was much higher at 48h in the group treated with 10.0mg/L F− than in the control group. (2) The OCN protein level in fluoride-treated fibroblasts was significantly higher than that of the control group in the 0.0001, 0.1, 1.0, 10.0 and 20.0mg/L F− groups at 2h, and in the 0.001 and 0.1 F− groups at 4h. A slightly higher level of OCN mRNA in fluoride-treated fibroblasts was also found in the 1.0 and 20.0mg/L F− groups compared to the control group. (3) The expressions of Cbfa1 and OCN in osteoblasts treated with the same experimental conditions as the fibroblasts were up-regulated by fluoride following the same trend as in the fibroblasts. Our results showed an increase in the expression of Cbfa1 and OCN in fibroblasts and osteoblasts exposed to fluoride and suggested that the osteogenic function of fibroblasts induced by fluoride could play an important role in the development of extraperiosteal ossification during skeletal fluorosis.

Effects of core-binding factor α1 gene silenced by siRNA on calcification of vascular smooth muscle cells induced by high phosphate

To explore the effects of core-binding factor α1 (Cbfα-1) gene silenced by siRNA on osteogenic differentiation and calcification of vascular smooth muscle cells (VSMC) induced by high phosphate in vitro. VSMC were cultured in vitro and passaged 3 to 8 times. Four pairs of Cbfα-1 siRNA were designed and synthesized. Transfection was performed with cationic lipid vectors (Lipofectamine 2000). Transfection conditions were optimized by the FAM fluorescent labeling-siRNA to screen effective siRNA sequences by reverse transcription-polymerase chain reaction (RT-PCR). After transfection with effective siRNA sequences, VSMCs were divided into 4 groups: (1) normal phosphate (Pi 1.3 mmol/L); (2) high phosphate (Pi 2.6 mmol/L); (3) siRNA transfection: high phosphate+Cbfα-1-siRNA; (4) negative transfection control: high phosphate+negative control siRNA. Cbfα-1 and osteopontin (OPN) mRNA and protein expression were detected by RT-PCR and Western blotting. Calcium deposition was visualized by Alizarin stain method. The transfection efficiency was around 55% with a concentration of Cbfα-1 siRNA 100 nmol/L and Lipo 8 µl/ well. Cbfα-1 siRNA 1952 was chosen as the effective sequence with a suppression ratio up to 81.8%. At 24 and 48 h post-transfection, the expression of Cbfα-1 mRNA was significantly lower in siRNA transfection group than that in high phosphate group (0.335 ± 0.059 vs 0.714 ± 0.106, 0.574 ± 0.036 vs 0.726 ± 0.086, all P < 0.01) . At 48 and 72 h post-transfection, the expression of Cbfα-1 protein in siRNA transfection group was significantly lower than that in high phosphate group (both P < 0.01) . While Cbfα-1 gene was silenced by siRNA in siRNA transfection group, the mRNA and protein expression of OPN significantly declined (all P < 0.05) and calcium deposition in cell layers decreased. Cbfα-1 siRNA can effectively inhibit the expression of Cbfα-1 mRNA and protein in VSMC and thus suppress the transformation of VSMC into osteoblast-like cells and calcification induced by high phosphate. Cbfα-1 may become a potential therapeutic target in vascular calcification of chronic kidney disease.

Effect of glucocorticoid-induced oxidative stress on the expression of Cbfa1

Glucocorticoids therapy is strongly limited since extended glucocorticoids can cause serious side effects, including increased susceptibility to develop the bone disease osteoporosis. Despite its side effects recognized importance to clinicians, seldom is known about how glucocorticoids directly impact bone-forming osteoblasts. Previous studies showed that dexamethasone (DEX) induces excessive production of reactive oxygen species (ROS), and causes oxidative stress in rat hippocampal slice cultures. To assess the implications and investigate the mechanisms of glucocorticoid-elicited osteoporosis, we hypothesize that DEX exposure induces oxidative stress which leads to decreased Cbfa1 mRNA expression, and predict that the antioxidant N-acetylcysteine (NAC) mitigates the damaging effects of DEX. Oxidative stress is implicated in osteoporosis. Furthermore, the osteoblast transcriptional factor Cbfa1 is reported to play a protective role against osteoporosis in postmenopausal women. Cells treated with (0.1, 1, 10μM) DEX exhibited signs of oxidative damages including depletion in total antioxidant capacity (T-AOC), increased ROS formation, and enhanced lipid peroxidation. Cbfa1 mRNA expression, by RT-PCR, was significantly reduced after exposure to (0.1, 1, 10μM) DEX. Pretreatment with the antioxidant NAC (2mM) prevented DEX-induced decrease in Cbfa1 mRNA. This study provides insight into the underlying mechanisms of high dose DEX-induced osteotoxicity. DEX (0.1, 1, 10μM) decreases the expression of Cbfa1 mRNA and inhibits differentiation and function of osteoblasts by inducing oxidative stress. The antioxidant NAC can mitigate the oxidative stress damaging effects of DEX. In addition, this study distinguishes itself by identifying Cbfa1 as a target for high dose DEX-induced osteotoxicity.

Evaluation of the proliferation and differentiation behaviors of mesenchymal stem cells with partially converted borate glass containing different amounts of strontium in vitro

The objective of this study was to examine the proliferation and differentiation behaviors of different compositions of strontium-containing (from 0-12 mol%) borate glasses with mesenchymal stem cells (MSCs). The Cell Counting Kit-8 (CCK-8) assay revealed that after three days of culturing, the 6Sr group had the highest cell growth rate. Analysis of cell morphology revealed that cells proliferated well near the particles of the samples in all the groups on day 3. On day 7, cells in the 6Sr group demonstrated a higher proliferation rate than other 4 groups under the microscope. When performing the Live-Dead staining experiment, the 6Sr group had the least number of dead cells. Total DNA qualification indicated that the 6Sr group had a statistically higher concentration compared with the remaining groups. It was found that on day 7, compared with the 0Sr group, the core binding factor α1 (Cbfa1) mRNA expression level was significantly higher in the 6Sr, 9Sr and 12Sr groups. On day 14, compared with the 0Sr group, the bone sialoprotein (BSP) mRNA level was significantly higher in the 6Sr group. Additionally, on day 21, the 6Sr and 9Sr groups demonstrated higher osteocalcin (OCN) mRNA expression levels compared with the 0Sr group. In the alkaline phosphatase (ALP) activity test, on day 21, the 6Sr group presented a higher activity than the 0Sr group. Further, the number of mineralized nodules per unit in MSCs was measured by Alizarin Red S staining. The results showed that the 6Sr and 9Sr groups had the greatest number of mineralized nodules. Therefore, it could be concluded that borate glasses containing strontium oxide of 0, 3, 6, 9 and 12 mol% demonstrate a significant level of proliferation when interacting with MSCs. The borate glass containing 6 mol% strontium oxide had the greatest level of proliferation when cultured with MSCs. The borate glass containing 6 and 9 mol% strontium oxide facilitated an improved bone formation ability compared with the remaining two compositions.

Early molecular responses of bone to obstructive nephropathy induced by unilateral ureteral obstruction in mice

This study was performed to address the bone injury and the early molecular responses of bone to obstructive nephropathy induced by unilateral ureteral obstruction in mice. The male mice were subjected to unilateral ureteral obstruction (UUO, n = 10) or sham operation (n = 10). All mice were killed on day 7 after the surgical operation. Hematoxylin and eosin and tartate-resistant acid phosphatase staining were performed on paraffin-embedded bone sections. Expression of genes and proteins was analyzed by reverse transcription-polymerase chain reaction, and Western blotting and immunohistochemistry staining, respectively. The serum calcium level was significantly reduced in UUO mice compared with that of Sham mice. The proximal tibia of UUO mice exhibited the increased expansion of chondrocytes zone, the reduction of osteoid content, and the increased separation and disconnection of woven bones. Reverse transcription-polymerase chain reaction results showed the downregulation of Cbfa1 and Col mRNA expression and the upregulation of Tgf-β, CtsK, CaII, Opg and Rankl mRNA expression in tibia of UUO mice compared to those of Sham mice. The ratio of Opg and Rankl was unchanged between Sham and the UUO group. Local protein expression of angiotensin II and its type 2 receptor was dramatically upregulated in tibia of UUO mice. Together, it is concluded that the obstructive nephropathy has defective effects on bone, and the underlying mechanisms are the reduction of bone formation and the increase of bone resorption, which is mediated, at least partially through local angiotensin II signalling.

Intermittent Traction Stretch Promotes the Osteoblastic Differentiation of Bone Mesenchymal Stem Cells by the ERK1/2-Activated Cbfa1 Pathway

Mechanical stress plays a crucial role in bone formation and absorption. We investigated the osteoblastic differentiation of bone mesenchymal stem cells (BMSCs) affected by intermittent traction stretch at different time points and explored the mechanism of osteoblastic differentiation under this special mechanical stimulation. The BMSCs and C3H10T1/2 cells were subjected to 10% elongation for 1–7 days using a Flexcell Strain Unit, and then the mRNA levels of osteoblastic genes and the expression of core-binding factor a1 (Cbfa1) were examined. Furthermore, we focused specifically on the role of the extracellular signal-regulated kinases 1/2 (ERK1/2) and Cbfa1 in the osteogenesis of BMSCs stimulated by the stretch. The results of these experiments showed that the stretch induces a time-dependent increase in the expression of osteoblastic genes. The synthesis of osteoblastic genes was downregulated after the knockdown of Cbfa1 expression by short-interfering RNA. Furthermore, the stress-induced increase in the expression of Cbfa1 mRNA and osteoblastic genes was inhibited by U0126, an ERK1/2 inhibitor. These results indicate that long periods of intermittent traction stretch promote osteoblastic differentiation of BMSCs through the ERK1/2-activated Cbfa1 signaling pathway.

The Use of Fluorescence-Labeled Mesenchymal Stem Cells in Poly(lactide-co-glycolide)/Hydroxyapatite/Collagen Hybrid Graft as a Bone Substitute for Posterolateral Spinal Fusion

Posterolateral spinal fusion is used to treat patients with degenerative spinal disorders. In this study, we investigated the effectiveness of a mesenchymal stem cell (MSC)/hydroxyapatite/type I collagen hybrid graft for posterolateral spinal fusion in a rabbit model. In vitro study, the hybrid graft was cultured in complete or osteogenic medium for 7 days and 14 days and examined by scanning electron microscopy. The alkaline phosphatase activity of the MSCs was assessed and the expression of osteogenic gene was determined by reverse transcription polymerase chain reaction. In vivo investigation, spinal fusion was examined using radiography, manual palpation, computed tomography, torsional loading tests, and histologic analysis. Furthermore, using a PKH fluorescence labeling system, we examined whether the newly formed bone was derived from the transplanted MSCs. Our data suggested that the MSCs differentiated into osteoblasts and produced extracellular matrix in the hybrid graft. Increased alkaline phosphatase activity was noted and mRNA of Cbfa-1 and osteopontin were detected. Radiographs and computed tomography images showed a continuous bone bridge and a satisfactory fusion mass incorporated into the transverse processes. The results of manual palpation and biomechanical data did not significantly differ between the two groups. Histologic examination of both groups revealed the presence of cartilage and endochondral ossification in the gaps between the grafted fragments. In situ tracing of the PKH 67-labeled MSCs indicated that the transplanted MSCs were partly responsible for the new bone formation. The hybrid graft could be effectively used to achieve posterolateral spinal fusion.

In Vitro and In Vivo Analysis of a Biodegradable Poly(lactide-co-glycolide) Copolymer Capsule and Collagen Composite System for Antibiotics and Bone Cells Delivery

The authors investigated poly(lactide-co-glycolide) (PLGA) capsule and collagen composite system for antibiotics and bone cells delivery to treat infected bone defects. Poly(lactide-co-glycolide) was mixed with vancomycin and hot compressing molded to form an antibiotic capsule. Rabbit mesenchymal stem cells (MSCs) were entrapped in collagen gel phase and dispersed throughout the void volume of capsule. In vitro study, the composite systems were cultured in complete or osteogenic medium for 21 days. The profiles of vancomycin released from the systems were evaluated using the high-performance liquid chromatography method. Relative activity of vancomycin against Staphylococcus aureus was determined by an antibiotic disk diffusion method. The expression of osteogenic gene was determined by reverse-transcription polymerase chain reaction. The alkaline phosphatase activity and calcium level of the MSCs were assessed. Analytical results demonstrated that the concentrations of vancomycin eluted from the composite system were above the minimal inhibitory concentration for 21 days. Sample inhibition zone was 10 to 24 mm, and the relative activity was 17.6% to 100%. mRNA of Cbfa1 and osteocalcin were detected, and increased alkaline phosphatase activity and calcium levels were noted. In in vivo investigation, the PKH 26-labeled MSCs and composite systems were implanted in the distal femoral cavities of four rabbits. The local concentration of vancomycin was above the minimal inhibitory concentration for 56 days. Sample inhibition zone was 9 mm to 24 mm, and the relative activity was 11.8% to 100%. Implanted PKH 26-labeled MSCs were identified in the newly formed bony trabeculae in specimens at 2 and 4 months after implantation. The results offer a potential approach to meet clinical requirements in the treatment of infected bone defects.

Effect of bone sialoprotein on proliferation and osteodifferentiation of human bone marrow-derived mesenchymal stem cells in vitro

We performed this study to investigate the effects of recombinant human bone sialoprotein (BSP) on the proliferation and osteodifferentiation of human BMSCs(hBMSCs). The hBMSC cultures were divided into 4 groups: control group, 10 −10 M BSP group (BSP group), osteogenic medium group (10 nM dexamethasone, 10 mM β-glycerophosphate, and 50 mg/L ascorbic acid, OM group) and BSP + OM group (OM plus10 −10 M BSP). Compared with the control group, cell growth of the other three groups slowed down, while fluorescence at the G 0/G 1 phase increased. After 28 days, in the OM group and the BSP + OM group, the proportion of STRO-1-positive cells decreased by 22.7% and 38.4% and ALP activity increased by 50% and 71.43%, respectively. CD271 mRNA expression decreased while Cbfa1, osteocalcin and osterix mRNA levels increased in the OM and BSP + OM groups, and the mRNA level change was greater in the BSP + OM group. After 28 days, the number of nodules in the BSP + OM group was 112.5% more than that in the OM group, but nodules did not formed in the control or BSP group. We conclude that BSP is capable of inhibiting hBMSCs proliferation and enhancing their osteogenic differentiation and mineralization in the presence of OM.

The effect of strontium incorporation in hydroxyapatite on osteoblasts in vitro

The purpose of this study was to test the effects of a series of strontium-substituted HA (Sr-HA) ceramics (0, 1, 5, and 10 mol% Sr substitution) on osteoblasts, thereby demonstrating whether strontium incorporation with HA would favor osteoblast metabolism. Rat primary osteoblasts were cultured with culture media containing ions released from the Sr-HA ceramics as they dissolved. MTT test, alkaline phosphatase activity, osteoblast transcription factor gene (cbfa1) expression and Alizarin Red staining were conducted at different time-points. There is no significant difference in cell proliferation between groups. However, compared with HA group, Sr-HA groups presented significant enhancement with regard to ALP activity, cbfa1 mRNA expression, and mineralization nodules. Among Sr-HA groups, 5 and 10% groups showed much better performances in ALP activity, cbfa1 mRNA expression, and mineralization nodules than 1% group, however, no significant difference was found between 5 and 10% groups. This study has demonstrated that Sr incorporation in HA ceramic enhanced osteoblastic cell differentiation and mineralization. However, further detailed studies are needed to understand the mechanistic effects of this Sr incorporation on osteoblastic cells and the optimal percentage of calcium should be substituted with strontium in HA.

INHIBITION OF BONE FORMATION BY HIGH INTENSITY PULSED ELECTROMAGNETIC FIELD IN MC3T3-E1 CELLS

To investigate the efiects of pulsed electromagnetic fleld (PEMF) with high electric fleld intensity on bone formation in murine osteoblast-like MC3T3-E1 cells, proliferation, alkaline phosphotase (ALP) activity, mineralized nodule formation, Collagen Type I (COL-I) and core-binding factor (Cbf)a1 mRNA expression, and bone morphogenetic protein (BMP)2/4 and mothers against decapentaplegic (Smad)1/5/8 protein expression were examined in cultured MC3T3-E1 cells after exposure to PEMF at the fleld intensities of 0kV/m, 50kV/m or 400kV/m for 400 consecutive pulses daily for 7 consecutive days. After 50kV/m of PEMF exposure, none of the above parameters of MC3T3-E1 cells changed signiflcantly when compared to the control groups. However, the proliferation, ALP activity and mineralized nodule formation of MC3T3-E1 cells in 400kV/m PEMF exposure groups decreased signiflcantly although COL-I and Cbfa1 mRNA expression and BMP2/4 and Smad1/5/8 protein expression did not change. The PEMF we used at high electric fleld intensity suppressed proliferation, difierentiation and