Activating BK channels ameliorates vascular smooth muscle calcification through Akt signaling.

Vascular calcification (VC) is characterized by pathological depositions of calcium and phosphate in the arteries and veins via an active cell-regulated process, in which vascular smooth muscle cells (VSMCs) transform into osteoblast/chondrocyte-like cells as in bone formation. VC is associated with significant morbidity and mortality in chronic kidney disease (CKD) and cardiovascular disease, but the underlying mechanisms remain unclear. In this study we investigated the role of large-conductance calcium-activated potassium (BK) channels in 3 experimental VC models. VC was induced in vascular smooth muscle cells (VSMCs) by ß-glycerophosphate (ß-GP), or in rats by subtotal nephrectomy, or in mice by high-dosage vitamin D3. We showed that the expression of BK channels in the artery of CKD rats with VC and in ß-GP-treated VSMCs was significantly decreased, which was functionally confirmed by patch-clamp recording. In ß-GP-treated VSMCs, BK channel opener NS1619 (20 µM) significantly alleviated VC by decreasing calcium content and alkaline phosphatase activity. Furthermore, NS1619 decreased mRNA expression of ostoegenic genes OCN and OPN, as well as Runx2 (a key transcription factor involved in preosteoblast to osteoblast differentiation), and increased the expression of α-SMA protein, whereas BK channel inhibitor paxilline (10 µM) caused the opposite effects. In primary cultured VSMCs from BK-/- mice, BK deficiency aggravated calcification as did BK channel inhibitor in normal VSMCs. Moreover, calcification was more severe in thoracic aorta rings of BK-/- mice than in those of wild-type littermates. Administration of BK channel activator BMS191011 (10 mg· kg-1 ·d-1) in high-dosage vitamin D3-treated mice significantly ameliorated calcification. Finally, co-treatment with Akt inhibitor MK2206 (1 µM) or FoxO1 inhibitor AS1842856 (3 µM) in calcified VSMCs abrogated the effects of BK channel opener NS1619. Taken together, activation of BK channels ameliorates VC via Akt/FoxO1 signaling pathways. Strategies to activate BK channels and/or enhance BK channel expression may offer therapeutic avenues to control VC.

PERK activator CCT020312 prevents inflammation-mediated osteoporosis in the ovariectomized rats.

OBJECTIVE: To investigate the therapeutic effects of PERK activator CCT020312 (CCT) on inflammation-mediated osteoporosis (IMO) in ovariectomized rats. METHODS: Rats were divided into Sham, IMO, IMO + 1 mg/kg CCT and IMO + 2 mg/kg CCT groups. IMO models were constructed by bilateral ovariectomy (OVX) on 1st day followed by injection with magnesium silicate (Talc) on the 59th day. Sham rats did not undergo OVX surgery and were injected with saline instead of Talc. From 60th to 79th day, rats were treated with DMSO (vehicle control) in the Sham and IMO groups, and 1 or 2 mg/kg CCT020312 in treatment groups. Osteopontin (OPN), osteocalcin (OCN), tartrate-resistant acid phosphatase (TRAP), C-terminal telopeptide of type I collagen (CTX-I), and pro-inflammatory factors were measured on the 80th day. ProdigyDEXA was used to evaluate bone mineral density and content (BMD/BMC). Bone volume/total volume (BV/TV), connectivity density (Conn.D), trabecular number (Tb.N), and trabecular separation (Tb.Sp) was assessed using 3D micro-CT scanner. RESULTS: CCT up-regulated Conn.D, BV/TV, and Tb.N, but down-regulated Tb.Sp in IMO rats. Besides, the declined femoral BMD and BMC in IMO rats were elevated after CCT treatment. Besides, IMO rats represented declined OPN and OCN, as well as increased TRAP, CTX-I, and pro-inflammatory factors, whereas those in the treatment groups were ameliorated regarding these indexes, with 2 mg/kg CCT showing better effect. CONCLUSION: PERK activator CCT020312 can be served as a new therapeutic option for the protection against bone loss in the OVX rat model associated with inflammation probably by manipulating inflammatory factors.

Imperatorin promotes osteogenesis and suppresses osteoclast by activating AKT/GSK3 ß/ß-catenin pathways.

Osteoporosis is caused by disturbance in the dynamic balance of bone remodelling, a physiological process, vital for maintenance of healthy bone tissue in adult humans. In this process, a new bone is formed by osteoblasts and the pre-existing bone matrix is resorbed by osteoclasts. Imperatorin, a widely available and inexpensive plant extract with antioxidative and apoptotic effects, is reported to treat osteoporosis. However, the underlying mechanism and specific effects on bone metabolism have not been elucidated. In this study, we used rat bone marrow-derived mesenchymal stem cells and found that imperatorin can activate RUNX2, COL1A1 and osteocalcin by promoting the Ser9 phosphorylation of GSK3ß and entry of ß-catenin into the nucleus. Imperatorin also enhanced the production of phospho-AKT (Ser473), an upstream factor that promotes the Ser9 phosphorylation of GSK3ß. We used ipatasertib, a pan-AKT inhibitor, to inhibit the osteogenic effect of imperatorin, and found that imperatorin promotes osteogenesis via AKT/GSK3ß/ß-catenin pathway. Next, we used rat bone marrow-derived monocytes, to check whether imperatorin inhibits osteoclast differentiation via AKT/GSK3ß/ß-catenin pathway. Further, we removed the bilateral ovaries of rats to establish an osteoporotic model. Intragastric administration of imperatorin promoted osteogenesis and inhibited osteoclast in vivo. Our experiments showed that imperatorin is a potential drug for osteoporosis treatment.

Raloxifene retards cartilage degradation and improves subchondral bone micro-architecture in ovariectomized rats with patella baja-induced - patellofemoral joint osteoarthritis.

OBJECTIVE: Abnormal remodeling of subchondral bone (SB) induced by estrogen deficiency has been shown to be involved in osteoarthritis (OA). Raloxifene (RAL) is commonly used to treat postmenopausal osteoporosis (OP). However, little is known about its effects on OA combined with estrogen deficiency. This study was performed to evaluate the efficacy of RAL on patella baja-induced patellofemoral joint OA (PFJOA) in an ovariectomized rat model. DESIGN: Patellar ligament shortening (PLS) and ovariectomy (OVX) were performed simultaneously in 3-month-old female Sprague-Dawley rats, which were treated with RAL (10 mg/kg/day) or vehicle at 72 h postoperatively for 10 weeks. PFJOA was assessed by immunohistochemistry (IHC), real-time polymerase chain reaction (PCR), tartrate-resistant acid phosphatase (TRAP) staining, enzyme-linked immunosorbent assay (ELISA), micro-computed tomography (µCT), histomorphology and behavioral analyses. RESULTS: X-ray examinations showed that patella baja was successfully established by PLS. Histomorphological analysis revealed that PFJOA was significantly exacerbated by OVX and markedly alleviated by RAL. Moreover, RAL improved cartilage metabolism by decreasing MMP-13, ADAMTS-4, and caspase-3 and increasing Col-II and aggrecan at both the protein and mRNA levels. Furthermore, RAL markedly improved bone mass and SB microarchitecture and reduced osteoclast numbers and the serum osteocalcin and CTX-I levels. Although RAL showed a trend toward reducing pain sensitivity based on mechanical allodynia testing, this result was not statistically significant. CONCLUSION: These findings demonstrate that RAL treatment retards PFJOA progression in an ovariectomized rat model, suggesting that it may be a potential candidate for amelioration of the progression of PFJOA accompanied by postmenopausal OP.

Effects of resveratrol on bone metabolism and bone turnover related indexes in ovariectomized osteoporosis rats.

This study aimed to investigate the effects of resveratrol (RES) on bone metabolism and bone turnover related indexes in ovariectomized osteoporosis rats. 48 clean grade adult healthy unmated female SD rats were randomly divided into 6 groups, including normal control group (NCG), osteoporosis model group (OP MG), estrogen treatment group (17ß-E2 group), RES low dose group (RES-L), RES medium-dose group (RES-M) and RES high dose group (RES-H). The rats in NCG and OP MG were given distilled water once a day and the rats in the other two groups were given 17ß-E2 and resveratrol respectively. The levels of serum calcium (S-Ca), serum phosphorus (S-P), urinary calcium (U-Ca/Cr) and urinary phosphorus (U-P/Cr) were measured with an automatic biochemistry analyzer. The levels of serum osteocalcin (BGP), alkaline phosphatase (ALP), tartrate-resistant acid phosphatase (TRAP), type I amino front-end peptide (PINP), type I collagen strong carboxyl peptide (CTX-I), urine deoxypyridinoline (DPD) and serum estrogen were detected by enzyme-linked immunosorbent assay (ELISA). In the OP group, the serum estrogen levels, S-Ca and S-P decreased significantly and the expression of U-Ca/Cr and U-P/Cr increased significantly (P< 0.05). Compared with the OP group, the expression of S-Ca and S-P increased significantly and the expression of U-Ca/Cr and U-P/Cr decreased significantly (p< 0.05) after treatment. The levels of TRAP, BGP, DPD and CTX-I in the OP group increased significantly (P< 0.05). Compared with the OP group, the levels of TRAP decreased significantly (P< 0.05). The levels of PINP and ALP in OP MG increased significantly (P< 0.05). IP and ALP increased in the middle and lower levels (P< 0.05). The bone mineral density of the OP group decreased significantly (P< 0.05). Resveratrol can affect the changes in bone turnover in ovariectomized rats, promote bone formation in low estrogen state and inhibit bone resorption. Resveratrol may have a protective effect on the bone of ovariectomized rats.

17ß-Estradiol improves osteoblastic cell function through the Sirt1/NF-κB/MMP-8 pathway.

Objective: This study aims to investigate the beneficial effects of 17ß-estradiol supplementation on the function of osteoblastic cells through the Sirtuin-1/nuclear transcription factor-κB/matrix metalloproteinase-8 (Sirt1/NF-κB/MMP-8) pathway.Methods: Mouse primary osteoblasts were obtained from neonatal mouse calvaria, and the cells were treated with or without 17ß-estradiol. We first detected the effect of 17ß-estradiol on the function of osteoblastic cells. Then, the changes in estrogen receptor-α (ERα), Sirt1, NF-κB, and MMP-8 were determined after the osteoblasts were treated with 17ß-estradiol. During supplementation with 17ß-estradiol, knockdown of Sirt1 in osteoblasts was used to further measure the changes of NF-κB and MMP-8 and observe the cell function.Results: In primary osteoblastic cells, exposure to 17ß-estradiol improved cell viability and increased the levels of bone formation biomarkers, including osteocalcin, osteoprotegerin (OPG), procollagen type 1 N-terminal propeptide (P1NP), and alkaline phosphatase (ALP). In addition, 17ß-estradiol supplement activated ERα and Sirt1 expression and inhibited NF-κB and MMP-8 expression. Moreover, these effects induced by 17ß-estradiol were reversed by knockdown of Sirt1 in mouse primary osteoblasts.Conclusion: 17ß-Estradiol replacement therapy may treat postmenopausal osteoporosis by improving osteoblastic cell function via the Sirt1/NF-κB/MMP-8 pathway.

The PERK-EIF2α-ATF4 signaling branch regulates osteoblast differentiation and proliferation by PTH.

Parathyroid hormone (PTH) and its related peptide (PTH-related peptide 1-34) are two of the Food and Drug Administration-approved bone-promoting drugs for age-related osteoporosis. Treatment with PTH stimulates bone formation. However, the molecular mechanisms of PTH-mediated osteoblast differentiation and cell proliferation are still not completely understood. In this study, we showed that PTH induced endoplasmic reticulum (ER) stress in osteoblasts through the PKR-like endoplasmic reticulum kinase (PERK)-eukaryotic initiation factor 2α (EIF2α)-activating transcription factor 4 (ATF4)-signaling pathway. After separately blocking PERK-EIF2α-ATF4 signaling with two different inhibitors [AMG'44 and integrated stress response inhibitor (ISRIB)] or specific small interfering RNA for PERK and ATF4, the following targets were all downregulated: expression of osteoblast differentiation markers [runt-related transcription factor 2 (Runx2), alkaline phosphatase (Alp), type I collagen (Col1a1), and osteocalcin (Ocn)], cell proliferation markers (CyclinE, CyclinD, and CDC2), amino acid import (Glyt1), and metabolism-related genes (Asns). Additionally, Alp-positive staining cells, Alp activity, matrix mineralization, Ocn secretion, and cell proliferation indexes were inhibited. Interestingly, we found that salubrinal enhanced PTH-induced osteoblast differentiation and proliferation by maintenance of phosphorylation of EIF2α. Furthermore, we observed that PTH increased the association between heat shock protein 90 (HSP90) and PERK and maintained PERK protein stabilization in the early stages of PTH-induced ER stress. Treatment of MC3T3-E1 cells with geldanamycin, an HSP90 inhibitor, decreased PERK protein expression and inhibited osteoblast differentiation and cell proliferation upon PTH treatment. Taken together, our data demonstrate that PTH regulates osteoblast differentiation and cell proliferation, partly by activating the HSP90-dependent PERK-EIF2α-ATF4 signaling pathway.

1α,25-Dihydroxyvitamin D3 promotes bone formation by promoting nuclear exclusion of the FoxO1 transcription factor in diabetic mice.

1α,25-Dihydroxyvitamin D3 (1,25(OH)2D3) is the active form of vitamin D, which is responsible for reducing the risk for diabetes mellitus (DM), decreasing insulin resistance, and improving insulin secretion. Previous studies have shown that 1,25(OH)2D3 inhibited the activity of FoxO1, which has been implicated in the regulation of glucose metabolism. However, its function and mechanism of action in DM-induced energy disorders and also in bone development remains unclear. Here, using in vitro and in vivo approaches including osteoblast-specific, conditional FoxO1-knock-out mice, we demonstrate that 1,25(OH)2D3 ameliorates abnormal osteoblast proliferation in DM-induced oxidative stress conditions and rescues the impaired glucose and bone metabolism through FoxO1 nuclear exclusion resulting from the activation of PI3K/Akt signaling. Using alizarin red staining, alkaline phosphatase assay, Western blot, and real-time qPCR techniques, we found that 1,25(OH)2D3 promotes osteoblast differentiation and expression of osteogenic phenotypic markers (i.e. alkaline phosphatase (1), collagen 1 (COL-1), osteocalcin (OCN), and osteopontin (OPN)) in a high-glucose environment. Moreover, 1,25(OH)2D3 increased both total OCN secretion and levels of uncarboxylated OCN (GluOC) by phosphorylating FoxO1 and promoting its nuclear exclusion, indicated by Western blot and cell immunofluorescence analyses. Taken together, our findings confirm that FoxO1 is a key mediator involved in glucose homeostasis and indicate that 1,25(OH)2D3 improves glucose metabolism and bone development via regulation of PI3K/Akt/FoxO1/OCN pathway.

MicroRNA-224 suppresses osteoblast differentiation by inhibiting SMAD4.

Osteoblast differentiation was found to be regulated by a variety of cell signaling and intracellular regulatory factors. In this study, we aimed at investigating the regulatory effect of microRNA-224 on osteoblast differentiation and its molecular mechanism. Expression of miR-224 in the osteoblasts, adipose-derived mesenchymal stem cells (MSC-A), bone marrow-derived mesenchymal stem cells (MSC-B) and mbilical cord-derived mesenchymal stem cells (MSC-U) were detected using RT-PCR. Expression of miR-224 was lower in osteoblast than in the three mesenchymal stem cells and it revealed a decreasing time-dependent trend from 0 day to 28 days during osteoblast differentiation. By using alkaline phosphatase (ALP) activity assay and alizarin red S (ARS) staining, we found that the mineralization nodules decreased in miR-224-mimics group and increased in miR-224-inhibitor group. The Western blot detection of osteoblast markers, such as osteocalcin (OCN), osteopontin (OPN), bone sialoprotein (BSP), and runt related transcription factor 2 (RUNX2), also verified that overexpression of miR-224 inhibited osteoblast differentiation, while its inhibition promoted osteoblast differentiation. Luciferase reporter assay was performed in our study, which illustrated that miR-224 regulated SMAD4 directly by targeting SMAD4 3'UTR. Then after the inhibition of SMAD4, we found that lower expression of SMAD4 suppressed the osteoblast differentiation and the related signaling pathway using RT-PCR and Western blot. Our results revealed a new mechanism of osteoblast differentiation, and provided a new therapeutic agent to promote bone anabolism by targeting miR-224.

The effects of icariin on wound healing of extraction sites with administration of zoledronic and dexamethasone: A rat model study.

OBJECTIVE: This study was intended to investigate the effects of icariin on the healing of tooth extraction sites under the systemic administration of zoledronic and dexamethasone. METHOD: Thirty female rats underwent bilateral ovariectomy and were randomly assigned to 5 groups: SS group received a weekly injection of saline, while ZD, ZD + LICA, ZD + MICA, and ZD + HICA groups received zoledronic with dexamethasone for 8 weeks. One week later, mandibular first molars were extracted in all groups. Then, 30, 60, and 120 mg/kg icariin were intragastrically given to ZD + LICA, ZD + MICA, and ZD + HICA groups daily for 10 weeks, while saline was given to SS group and ZD group. Blood samples and mandibles were harvested for examinations after 10 weeks. RESULTS: Significantly smaller wound area was noted in SS and ZD + HICA groups, but the incidence of bisphosphonate-related osteonecrosis of the jaws (BRONJ) was not significantly different. Groups injected with zoledronic and dexamethasone had higher C-terminal cross-linked telopeptide of type 1 collagen (CTX-1), tartrate-resistant acid phosphatase 5b (TRACP 5b), and the number of osteoclast cells, with less vascular endothelial growth factor (VEGF) and osteocalcin (OCN). In contrast, CTX-1, TRACP 5b, and the number of osteoclast cells declined after using icariin and promoted VEGF and OCN were noted and the effects were in a dosage-dependent manner. CONCLUSION: Concurrent use of zoledronic and dexamethasone inhibits the expression of VEGF, OCN, and wound healing and increases the number of osteoclast cells, serum CTX-1, and TRACP-5b after discontinuation for 10 weeks. Icariin weakens those effects in a dose-dependent manner but does not influence the onset of BRONJ.

Multifaceted interaction of bone, muscle, lifestyle interventions and metabolic and cardiovascular disease: role of osteocalcin.

Undercarboxylated osteocalcin (ucOC) may play a role in glucose homeostasis and cardiometabolic health. This review examines the epidemiological and interventional evidence associating osteocalcin (OC) and ucOC with metabolic risk and cardiovascular disease. The complexity in assessing such correlations, due to the observational nature of human studies, is discussed. Several studies have reported that higher levels of ucOC and OC are correlated with lower fat mass and HbA1c. In addition, improved measures of glycaemic control via pharmacological and non-pharmacological (e.g. exercise or diet) interventions are often associated with increased circulating levels of OC and/or ucOC. There is also a relationship between lower circulating OC and ucOC and increased measures of vascular calcification and cardiovascular disease. However, not all studies have reported such relationship, some with contradictory findings. Equivocal findings may arise because of the observational nature of the studies and the inability to directly assess the relationship between OC and ucOC on glycaemic control and cardiovascular health in humans. Studying OC and ucOC in humans is further complicated due to numerous confounding factors such as sex differences, menopausal status, vitamin K status, physical activity level, body mass index, insulin sensitivity (normal/insulin resistance/T2DM), tissue-specific effects and renal function among others. Current observational and indirect interventional evidence appears to support a relationship between ucOC with metabolic and cardiovascular disease. There is also emerging evidence to suggest a direct role of ucOC in human metabolism. Further mechanistic studies are required to (a) clarify causality, (b) explore mechanisms involved and

[Comparative study of proliferative and periodontal differentiation propensity of induced pluripotent stem cells at different passages].

OBJECTIVE: To compare the proliferative and periodontal specific differentiation abilities of induced pluripotent stem cells (iPSCs) at different passages, and to investigate whether long term culturing would have a negative influence on their proliferation and specific differentiation capacity, thus providing a theoretical basis for further in-depth research on periodontal regeneration and the possible clinical applications of iPSCs. METHODS: IPSCs derived from human gingival fibroblasts at passages 5, 10, 15 and 20 were recovered and cultured in vitro. Their morphology and proliferation rates were observed respectively. We further induced the iPSCs at different passages toward periodontal tissue under the treatment of growth/differentiation factor-5 (GDF-5) for 14 days through the EB routine, then compared the periodontal differentiation propensities between the different passages of iPSCs by detecting their calcified nodules formation by Alizarin red staining and assaying their relative periodontal tissue related marker expressions by qRT-PCR and immunofluorescence staining, including bone related markers: osteocalcin (OCN), bone sialoprotein (BSP); periodontal ligament related markers: periostin, vimentin; and cementum related markers: cementum attachment protein (CAP), cementum protein 1 (CEMP1). The untreated spontaneous differentiation groups were set as negative controls respectively. RESULTS: iPSCs at different passages all showed a high proliferative capacity when cultured in vitro and turned into a spindle-like shape similar to fibroblasts upon periodontal specific differentiation. All iPSCs formed typical calcified nodules upon GDF-5 induction by Alizarin red staining in comparison to their untreated controls. The relative calcium deposition at all passages had been significantly upgraded under the treatment of GDF-5 (P5: t=2.125, P=0.003; P10: t=2.246, P=0.021; P15: t=3.754, P=0.004; P20: t=3.933, P=0.002), but no significant difference in their calcium deposition were detected within passages 5, 10, 15 and 20 (periodontal differentiation: F=2.365, P=0.109; spontaneously differentiation: F=2.901, P=0.067). Periodontal tissue related marker expressions of iPSCs at all passages had also been significantly upgraded under the treatment of GDF-5 (P<0.05), but still, no significant difference in their expression levels of periodontal tissue related proteins were detected within passages (BSP: F=0.926 7, P=0.450; vimentin: F=0.917 1, P=0.455; CEMP1: F=2.129, P=0.1367). CONCLUSION: Our results preliminarily confirmed that long term culturing won't influence the proliferation capacity and periodontal specific differentiation propensity of iPSCs, as they can still proliferate and differentiate toward periodontal cells with high efficiency upon growth factor induction after continuous passaging. Therefore, iPSCs could be recognized as a promising cell source for future possible application in periodontal tissue regeneration.

[A novel tissue-engineered bone constructed by using human adipose-derived stem cells and biomimetic calcium phosphate scaffold coprecipitated with bone morphogenetic protein-2].

OBJECTIVE: To construct a novel biomimetic calcium phosphate (BioCaP) scaffold loaded with bone morphogenetic protein-2 (BMP-2), and to investigate its role in the osteogenesis of human adipose-derived stem cells (hASCs) in vitro and in vivo. METHODS: The BioCaP scaffold coprecipitated with BMP-2 (BMP-2-BioCaP) was constructed in this study. Field emission scanning electron microscopy (SEM) was used to analyze the morphology of the surfaces. The release kinetics was measured to evaluate the slow-release characteristics in vitro. BMP-2-BioCaP was immersed in proliferation medium (PM) or osteogenic medium (OM), respectively. The supernatants were collected and used to culture hASCs in vitro. Cell numbers were determined using the cell-counting kit-8 (CCK-8) to assess the cell proliferation. After 7 and 14 days, alkaline phosphatase (ALP) staining and quantification were performed to test the activity of ALP. After 14 and 21 days, the calcification deposition was determined by alizarin red S (ARS) staining and quantification. The expressions of the osteoblast-related genes were tested on day 4 and day 14. In the in vivo study, 6 nude mice were used and implanted subcutaneously into the back of the nude mice for 4 groups: (1) BioCaP scaffold only, (2) BioCaP scaffold+hASCs, (3) BMP-2-BioCaP scaffold, (4) BMP-2-BioCaP scaffold+hASCs (test group). After 4 weeks of implantation, hematoxylin-eosin (HE) staining was performed to evaluate the in vivo osteogenesis of hASCs. RESULTS: SEM observations showed that BioCaP and BMP-2-BioCaP scaffold were entirely composed of straight, plate-like and sharp-edged crystal units, and the length of the crystal units varied between 5 and 10 µm. Release kinetics analysis demonstrated that BMP-2 incorporated with BioCaP could be released at certain concentration and last for more than 21 days, and the accumulative protein release could reach 20%. CCK-8 assays showed that cell proliferation was not significantly affected by BMP-2-BioCaP. ALP activity was higher by the induction of OM+BMP-2-BioCaP than of the other groups (P<0.01). More mineralization deposition and more expressions of osteoblast-related genes such as Runt-related transcription factor 2 (RUNX2), ALP, osteopontin (OPN) and osteocalcin (OC) were determined in the OM+BMP-2-BioCaP group at different time points (P<0.01). HE staining showed that, in the test group and BMP-2-BioCaP scaffold group, the extracellular matrix (ECM) with eosinophilic staining were observed around hASCs, and newly-formed bone-like tissues could be found in ECM around the scaffold materials. Moreover, compared with the BMP-2-BioCaP scaffold group, more bone-like tissues could be observed in ECM with typical structure of bone tissue in the test groups. No obvious positive results were found in the other groups. CONCLUSION: BMP-2-BioCaP scaffold could achieve slow-release of BMP-2 and promote the osteogenic differentiation of hASCs in vitro and in vivo. The novel tissue-engineered bone composed of hASCs and BMP-2-BioCaPis promising for the repair of bone defect.

Differential effects of bisphenol A diglicydyl ether on bone quality and marrow adiposity in ovary-intact and ovariectomized rats.

Bisphenol A diglycidyl ether (BADGE), a PPARγ2 antagonist, has been shown to inhibit marrow adipogenesis and promote bone formation in intact animals. We investigated the impact of BADGE on a new and more clinically relevant physiological model, the ovariectomized (OVX) rat model. Forty female Wistar rats were divided into four treatment groups for 12 wk (n = 10/group): sham+vehicle, sham+BADGE, OVX+vehicle, and OVX+BADGE. Postmortem analyses included MRI, micro-CT, serological test, histomorphometry, biomechanical tests, RT-PCR, and Western blot. Overall, OVX induced a sequential marrow fat expansion accompanied by bone deterioration. Compared with OVX controls, BADGE reduced fat fraction of the distal femur by 36.3%, adipocyte density by 33.0%, adipocyte size by 28.6%, adipocyte volume percentage by 57.8%, and adipogenic markers PPARγ2 and C/EBPα by ∼50% in OVX rats. Similar results were observed in sham rats vs. vehicle. BADGE could promote bone quality in sham rats; however, BADGE did not significantly improve trabecular microarchitecture, biomechanical strength, and dynamic histomorphometric parameters except for trabecular separation in OVX rats. We concluded that early BADGE treatment at a dose of 30 mg/kg attenuates marrow adiposity in ovary-intact and OVX rats and stimulates bone formation in ovary-intact rats but does not significantly rescue bone quality in OVX rats.

Alendronate alters osteoblast activities.

OBJECTIVE: Due to accumulation in the bone matrix and a half-life of at least 10 years, it is important to understand the cellular impact of bisphosphonates (BPs). This study assessed the effects of alendronate (ALN) on human primary osteoblasts. MATERIAL AND METHODS: Osteoblasts were incubated with ALN (5, 20 and 100 µM), and both cells and cell culture media were harvested after d 1, 3, 7 or 14. Proliferation was evaluated by 3H-thymidine incorporation and tetrazolium dye (MTT) colorimetric assay, and viability by the lactate dehydrogenase (LDH) activity in the medium. Differentiation was evaluated using protein Luminex multiplex assays and RT-PCR. RESULTS: ALN had no significant effects on cell viability. The lower concentrations enhanced the proliferation, whereas 100 µM diminished the proliferation. mRNA expression of osteocalcin (OC), alkaline phosphatase (ALP) and α-1 type 1 collagen were reduced, whereas ALN enhanced the expression of leptin mRNA and the secretion of interleukin-8 (IL-8) and regulated on activation normal T cell expressed and secreted (RANTES). CONCLUSIONS: ALN enhanced the secretion of immune factors from human osteoblasts. Combined with a lower rate of proliferation and a decline in differentiation, this indicates that higher dosages or accumulation may cause undesirable local changes in bone.

[A preliminary study for the effect of nano hydroxyapatite on human adipose-derived mesenchymal stem cells mixture 3D bio-printing].

OBJECTIVE: To study the effect of nano hydroxyapatite on human adipose-derived mesenchymal stem cells(hASCs) mixture 3D bio-printing for cells' proliferation and osteogenesis. METHODS: P5 hASCs were used as seed cells, 10 g/L nano hydroxyapatite was added into the cell-sodium alginate-gelatin mixture (concentration: 20 g/L sodium alginate, 80 g/L gelatin; cell density: 1×106/mL), then the mixture was printed by 3D bio-printer as the experimental group. And the cell-sodium alginate-gelatin mixture without nano hydroxyapatite was printed as the control group. Respectively, both the experimental and control groups were detected by microscope, CCK-8, Western blot and PCR at certain time pointsafter being printed, whose cells' proliferation and osteogenic differentiation were analyzed. RESULTS: The microscopic observation and CCK-8 results showed that the cells of the experimental group and the control group both had a good proliferation 24 h and 7 d after being printed. The Western blot results showed that 14 d after printing, the expression of Runt-related transcription factor 2 (RUNX2) had no statistical difference between the experimental group and control group. The PCR results showed that 14 d after printing, the expression of osteogenesis-related genes (RUNX2, osterix, and osteocalcin) was significantly higher in the experimental group than in the control group. CONCLUSION: Nano hydroxyapatite can increase osteogenic differentiation of the hASCs mixture after bio-printing, in which the cells still have a good proliferation.

[Effect of miR-705 on osteogenic differentiation of mouse embryo osteoblast precursor cells MC3T3-E1].

Objective: To investigate the effect of miR-705 on osteogenic differentiation of mouse embryo osteoblast precursor (MC3T3-E1) cells. Methods: miR-705 mimics, inhibitors and negative control were transfected into MC3T3-E1 cells. Alkaline phosphates (ALP) staining were performed and quantified after 7 days of osteogenic medium induction. The mRNA and protein expression levels of runt-related transcription factor 2 (Runx2) and osteocalcin (OCN) were detected by real-time RT-PCR and Western blot after 14 days of osteogenic induction. Alizarin red staining was performed and quantified in MC3T3-E1 cells after 21 days of osteogenic induction. Results: After 7 days of osteogenic induction, ALP staining showed that overexpression of miR-705 significantly reduced ALP activity, whereas knockdown of miR-705 increased ALP activity (all P<0.05). Consistently, after 14 days of osteogenic induction, mRNA and protein expressions of Runx2 and OCN were suppressed by overexpression of miR-705, whereas they were promoted by knockdown of miR-705 (all P<0.05). After 21 days of osteogenic induction, alizarin red staining showed that overexpression of miR-705 significantly reduced the formation of mineralized node, the opposite results were found in miR-705 knockdown group (all P<0.05). Conclusion: miR-705 can inhibit the osteogenic differentiation of MC3T3-E1 cells.

[Effects of Danshensu on bone formation in ovariectomized rats].

Objective: To investigate the effects of Danshensu on bone formation in ovariectomized rats.Methods: Thirty female SD rats were randomly divided into three groups with 10 rats in each:blank control group, model control group and Danshensu group. The osteoporosis model was induced by bilateral ovariectomy and rats in Danshensu group were fed with Danshensu 12.5 mg·kg-1·d-1 by gavage after ostroporosis model induced. All animals were sacrificed after 90 days. The bone mineral density (BMD) of the whole body, femur and lumbar vertebra was measured by dual energy X-ray absorptiometry. The biomechanical properties of femur were measured by AG-IS mechanical universal testing machine. Serum osteocalcin and bone alkaline phosphates (BALP) levels were measured by ELISA. The number of osteoblasts of proximal femoral metaphysis was counted with light microscopy after HE staining. Results: Compared with blank control group, BMD, biomechanical properties of femur, serum osteocalcin and BALP levels and the number of osteoblasts were decreased in model control group (P<0.05 or P<0.01). While compared with model control group, BMDs of the whole body, femur and lumbar vertebra, the elastic modulus, maximum load, yield strength, breaking point load of femur, the serum levels of osteocalcin and BALP, and the number of osteoblasts were significantly improved in Danshensu group (P<0.05 or P<0.01). Conclusion: Danshensu can improve bone quality by increasing bone density, improving biomechanical properties, promoting the expression of osteogenesis-related factors, and increasing the number of osteoblasts.

Tridax procumbens flavonoids promote osteoblast differentiation and bone formation.

BACKGROUND: Tridax procumbens flavonoids (TPFs) are well known for their medicinal properties among local natives. Besides traditionally used for dropsy, anemia, arthritis, gout, asthma, ulcer, piles, and urinary problems, it is also used in treating gastric problems, body pain, and rheumatic pains of joints. TPFs have been reported to increase osteogenic functioning in mesenchymal stem cells. Our previous study showed that TPFs were significantly suppressed the RANKL-induced differentiation of osteoclasts and bone resorption. However, the effects of TPFs to promote osteoblasts differentiation and bone formation remain unclear. TPFs were isolated from Tridax procumbens and investigated for their effects on osteoblasts differentiation and bone formation by using primary mouse calvarial osteoblasts. RESULTS: TPFs promoted osteoblast differentiation in a dose-dependent manner demonstrated by up-regulation of alkaline phosphatase and osteocalcin. TPFs also upregulated osteoblast differentiation related genes, including osteocalcin, osterix, and Runx2 in primary osteoblasts. TPFs treated primary osteoblast cells showed significant upregulation of bone morphogenetic proteins (BMPs) including Bmp-2, Bmp-4, and Bmp-7. Addition of noggin, a BMP specific-antagonist, inhibited TPFs induced upregulation of the osteocalcin, osterix, and Runx2. CONCLUSION: Our findings point towards the induction of osteoblast differentiation by TPFs and suggested that TPFs could be a potential anabolic agent to treat patients with bone loss-associated diseases such as osteoporosis.

Comparison of the Effects of Low-Level Laser Therapy and Ozone Therapy on Bone Healing.

This study aims to compare the effect of low-level laser therapy (LLLT) and ozone therapy on the bone healing. Thirty-six adult male Wistar albino rats were used for this study. Monocortical defects were shaped in right femur of all rats. Defects were filled with nano-hydroxyapatite graft. The animals were divided into 3 groups and each group was than divided into 2 subgroups. Then, LLLT with a diode laser was applied to the first group (G1), ozone therapy was applied to the second group (G2), and no treatment was applied to the third group as a control group (G3). Animals were sacrificed after 4th and 8th weeks and the sections were examined to evaluate the density of the inflammation, the formation of connective tissue, the osteogenic potential, and osteocalcin activity. As a result, there were no significant differences among the groups of 4 weeks in terms of new bone formation. In the immunohistochemical assessment, the number of osteocalcin-positive cells was higher in the laser group compared to the other group of 4 weeks; this difference was statistically significant in the LLLT and ozone groups (P < 0.05). Histomorphometric assessment showed that the new bone areas were higher in the LLLT and ozone groups; furthermore, there was a statistically significant difference in the LLLT in comparison with the control group at 8th week (P < 0.05). At the same time immunohistochemical assessment showed that osteocalcin-positive cells were considerably higher in G2 than G1 at 8th week (P < 0.05). The findings of this study may be the result of differences in the number of treatment sessions. Further studies are therefore needed to determine the optimal treatment modality.