去卵巢对大鼠颌骨成骨细胞增殖与成骨分化能力的影响

目的探讨去卵巢对大鼠颌骨成骨细胞增殖与成骨分化能力的影响。方法建立去卵巢大鼠骨质疏松模型,分离培养假手术组和去卵巢组颌骨成骨细胞并进行形态学观察。采用CCK-8法检测假手术组和去卵巢组大鼠颌骨成骨细胞的增殖能力差异。通过碱性磷酸酶染色与活性测定、矿化结节茜素红染色与定量、成骨相关基因及蛋白表达检测,探讨去卵巢对大鼠颌骨成骨细胞增殖及成骨分化能力的影响。结果假手术组和去卵巢组大鼠均可成功分离培养颌骨成骨细胞,两组细胞均可贴壁生长,细胞形态未见显著差异。与假手术组相比,去卵巢大鼠颌骨成骨细胞的增殖能力,碱性磷酸酶染色与活性均显著增高;但去卵巢大鼠颌骨成骨细胞的Runx2和OC在mRNA与蛋白水平的表达上及体外矿化结节形成能力方面皆显著低于假手术组。结论去卵巢可影响大鼠颌骨成骨细胞的增殖与成骨分化能力,研究结果将为探讨颌面部骨组织骨质疏松的发病机制与防治提供实验依据。     

续苓健骨汤含药血清对MC3T3-E1细胞分化及增殖的影响

目的探讨续苓健骨汤含药血清对MC3T3-E1成骨细胞分化及增殖的影响。方法制备续苓健骨汤含药血清,实验分为空白对照组、含药血清低剂量组、中剂量组和高剂量组。采用CCK-8法和流式细胞术检测续苓健骨汤含药血清对MC3T3-E1细胞增殖和细胞周期的影响;碱性磷酸酶(ALP)活性测定MC3T3-E1细胞的成骨分化能力;茜素红染色检测MC3T3-E1细胞的矿化能力;实时荧光定量PCR检测成骨分化基因Runx2、OC、Bmp2、Col1a1mRNA水平。结果与空白对照组比较,中、高剂量续苓健骨汤含药血清能促进MC3T3-E1细胞增殖、S期细胞比率和细胞增殖指数,并且呈现一定的剂量依赖性;同时中高剂量续苓健骨汤含药血清组能明显提高MC3T3-E1细胞ALP活性(P0.01)和钙化能力(P0.01),促进Runx2、OC、Bmp2、Col1a1 mRNA的表达(P0.05)。结论续苓健骨汤含药血清能促进成骨细胞MC3T3-E1的增殖,并通过上调骨形成相关基因Runx2、OC、BMP2、Col1a1的表达水平,提高MC3T3-E1细胞的成骨能力。     

二甲双胍对去卵巢大鼠骨质疏松症的影响及机制研究

目的 以雌二醇为对照,观察二甲双胍(metformin, MF)对去卵巢大鼠骨密度及骨矿含量的影响,并从细胞、分子水平探究MF可能的骨保护机制。方法 将60只雌性SD大鼠随机均分4组：假手术（SHAM）组、去卵巢（OVX）组、去卵巢+二甲双胍（OVX+MF）组和去卵巢+雌二醇(OVX+E2 )组。分组灌胃给药60 d后测量大鼠右侧胫骨骨密度和骨矿含量;分离培养各组大鼠骨髓间充质干细胞（BMSCs）并诱导其向成骨细胞分化,用MTT法测定细胞活性及增殖能力;测定各组碱性磷酸酶（ALP）活性、矿化结节数目、钙含量以及I型胶原（collagen type I）、骨钙素（OC）、骨保护素 (OPG)、NFκB受体的配体 (RANKL)、白细胞介素-6（IL-6）基因表达水平。结果 与OVX组相比,OVX+MF组和OVX+E2组成骨细胞的增殖能力与ALP活性明显增强,骨密度、骨矿含量以及钙沉积量显著增加(P均<0.05),且两组collagen type I、OC、OPG mRNA的表达水平显著升高,而RANKL、IL-6mRNA表达明显受到抑制;但OVX+MF组去卵巢大鼠成骨细胞的增殖能力、ALP活性、钙沉积量、collagen type I、OC、OPG mRNA表达水平低于OVX+E2组,RANKL、IL-6mRNA表达高于OVX+E2组（P均<0.05）;与SHAM组比较,OVX+MF组的collagen type I、OC、OPG mRNA的表达水平更高（P<0.05）。结论 二甲双胍可能通过OPG/RANKL/RANK信号通路促进BMSCs向成骨细胞分化,有效逆转去卵巢大鼠骨质疏松的状态,这种潜在的骨保护作用可能会改善糖尿病引起的骨质疏松。     

Age-dependent expression of osteoblastic phenotypic markers in normal human osteoblasts cultured long term in the presence of dexamethasone

We have previously shown that osteoblasts derived from trabecular bone explants and cultured long term in 10 nM dexamethasone ((HOB+DEX) cells) exhibited properties consistent with a more differentiated phenotype compared with those grown in the absence of dexamethasone ((HOB-DEX) cells). To characterize these two cell models further, we measured the steady-state mRNA levels of the phenotypic markers alkaline phosphatase (ALP), collagen type I (COLL) and osteocalcin (OC), OC production, and the activities of ALP and parathyroid hormone (PTH)-stimulated adenylate cyclase. These findings were then correlated with the age and sex of the bone donors. Long-term culture in dexamethasone significantly increased ALP and OC mRNA levels and the activities of ALP and PTH-stimulated adenylate cyclase but not OC production, in (HOB+DEX) compared with (HOB-DEX) cells (p<0.05). When the data were examined with respect to the age of the bone donor, age-dependent differences in the expression and responses to dexamethasone were apparent. ALP and PTH-stimulated adenylate cyclase activities decreased with increasing age of the bone donor in (HOB-DEX) and (HOB+DEX) cells (p<0.05). There were no significant correlations between phenotypic marker mRNA levels and bone donor age in (HOB-DEX) and (HOB+DEX) cells. All age-dependent decreases in ALP and PTH-stimulated cyclase activities were enhanced in the (HOB+DEX) cells. However, when the data were examined according to the sex of the bone donor, there were no differences in mRNA levels, OC production, or ALP and cyclase activities between cells from male and female donors. These results indicate an age dependence in the expression of osteoblastic markers in human bone cells at different stages of differentiation: thus osteoblastic cultures derived from older donors are likely to contain fewer osteoprogenitor cells, lower levels of glucocorticoid receptors or represent more differentiated osteblasts compared with those derived from younger donors.     

Glutamate does not play a major role in controlling bone growth.

Bone cells express glutamate-gated Ca2+-permeable N-methyl-D-aspartate (NMDA) receptors and GLAST glutamate transporters. Blocking NMDA receptors has been reported to reduce the number of bone resorption pits produced by osteoclasts, and mechanical loading alters GLAST transporter expression, which should change the extracellular glutamate concentration and NMDA receptor activation. Thus, by analogy with the brain, glutamate is postulated to be an important intercellular messenger in bone, controlling bone formation and resorption. We found that activating or blocking NMDA receptors had no effect on bone formation by rat osteoblasts in culture. The number of resorption pits produced by osteoclasts was reduced by the NMDA receptor blocker MK-801 but not by another blocker AP-5, implying that this effect of MK-801 is unrelated to its glutamate-blocking action. By contrast, MK-801, AP-5, and NMDA had no consistent effect on the volume of pits. In mice with GLAST glutamate transporters knocked out, no differences were detected in mandible and long bone size, morphology, trabeculation, regions of muscle attachment, resorption lacunae, or areas of formation versus resorption of bone, compared with wild-type siblings. These data suggest that glutamate does not play a major role in controlling bone growth.     

Effect of the same mechanical loading on osteogenesis and osteoclastogenesis in vitro

Purpose: To investigate the influence of the same mechanical loading on osteogenesis and osteoclastogenesis in vitro. Methods: Primary osteoblasts, bone marrow-derived mesenchymal stem cells (BMSCs, cultured in osteoinductive medium) and RAW264.7 cells cultured in osteoclast inductive medium were all subjected to a 1000 mstrain (ms) at 1 Hz cyclic mechanical stretch for 30 min (twice a day). Results: After mechanical stimulation, the alkaline phosphatase (ALP) activity, osteocalcin protein level of the osteoblasts and BMSCs were all enhanced, and the mRNA levels of ALP and collagen type I increased. Additionally, extracellular-deposited calcium of both osteoblasts and BMSCs increased. At the same time, the activity of secreted tartrate-resistant acid phosphatase, the number of tartrate-resistant acid phosphatase-positive multinucleated cells, matrix metalloproteinase-9 protein levels of RAW264.7 cells and the extracellular calcium solvency all decreased. Conclusion: The results demonstrated that 1000 ms cyclic mechanical loading enhanced osteoblasts activity, promoted osteobla     

Supplying osteogenesis to dead bone using an osteogenic matrix cell sheet

Purpose

To evaluate whether osteogenic matrix cell sheets can supply osteogenesis to dead bone.

The P2Y13 receptor regulates extracellular ATP metabolism and the osteogenic response to mechanical loading

ATP release and subsequent activation of purinergic receptors has been suggested to be one of the key transduction pathways activated by mechanical stimulation of bone. The P2Y₁₃ receptor, recently found to be expressed by osteoblasts, has been suggested to provide a negative feedback pathway for ATP release in different cell types. Therefore, we hypothesized that the P2Y₁₃ receptor may contribute to the mediation of osteogenic responses to mechanical stimulation by regulating ATP metabolism by osteoblasts. To test this hypothesis, wild‐type (WT) and P2Y₁₃ receptor knockout (P2Y₁₃R^?/?) mice were subject to non‐invasive axial mechanical loading of the left tibiae to induce an osteogenic response. Micro‐computed tomography analysis showed mechanical loading induced an osteogenic response in both strains of mice in terms of increased total bone volume and cortical bone volume, with the P2Y₁₃R^?/? mice having a significantly greater response. The extent of the increased osteogenic response was defined by dynamic histomorphometry data showing dramatically increased bone formation and mineral apposition rates in P2Y₁₃R^?/? mice compared with controls. In vitro, primary P2Y₁₃R^?/? osteoblasts had an accumulation of mechanically induced extracellular ATP and reduced levels of hydrolysis. In addition, P2Y₁₃R^?/? osteoblasts also had a reduction in their maximal alkaline phosphatase (ALP) activity, one of the main ecto‐enzymes expressed by osteoblasts, which hydrolyzes extracellular ATP. In conclusion, deletion of the P2Y₁₃ receptor leads to an enhanced osteogenic response to mechanical loading in vivo, possibly because of the reduced extracellular ATP degradation by ALP. The augmented osteogenic response to mechanical stimulation, combined with suppressed bone remodeling activities and protection from OVX‐induced bone loss after P2Y₁₃ receptor depletion as previously described, suggests a potential role for P2Y₁₃ receptor antagonist‐based therapy, possibly in combination with mechanical loading, for the treatment of osteoporosis.     

Pulsed Electromagnetic Fields Partially Preserve Bone Mass,Microarchitecture, and Strength by Promoting Bone Formation in Hindlimb‐Suspended Rats

A large body of evidence indicates that pulsed electromagnetic fields (PEMF), as a safe and noninvasive method, could promote in vivo and in vitro osteogenesis. Thus far, the effects and underlying mechanisms of PEMF on disuse osteopenia and/or osteoporosis remain poorly understood. Herein, the efficiency of PEMF on osteoporotic bone microarchitecture, bone strength, and bone metabolism, together with its associated signaling pathway mechanism, was systematically investigated in hindlimb‐unloaded (HU) rats. Thirty young mature (3‐month‐old), male Sprague‐Dawley rats were equally assigned to control, HU, and HU + PEMF groups. The HU + PEMF group was subjected to daily 2‐hour PEMF exposure at 15 Hz, 2.4 mT. After 4 weeks, micro–computed tomography (µCT) results showed that PEMF ameliorated the deterioration of trabecular and cortical bone microarchitecture. Three‐point bending test showed that PEMF mitigated HU‐induced reduction in femoral mechanical properties, including maximum load, stiffness, and elastic modulus. Moreover, PEMF increased serum bone formation markers, including osteocalcin (OC) and N‐terminal propeptide of type 1 procollagen (P1NP); nevertheless, PEMF exerted minor inhibitory effects on bone resorption markers, including C‐terminal crosslinked telopeptides of type I collagen (CTX‐I) and tartrate‐resistant acid phosphatase 5b (TRAcP5b). Bone histomorphometric analysis demonstrated that PEMF increased mineral apposition rate, bone formation rate, and osteoblast numbers in cancellous bone, but PEMF caused no obvious changes on osteoclast numbers. Real‐time PCR showed that PEMF promoted tibial gene expressions of Wnt1, LRP5, β‐catenin, OPG, and OC, but did not alter RANKL, RANK, or Sost mRNA levels. Moreover, the inhibitory effects of PEMF on disuse‐induced osteopenia were further confirmed in 8‐month‐old mature adult HU rats. Together, these results demonstrate that PEMF alleviated disuse‐induced bone loss by promoting skeletal anabolic activities, and imply that PEMF might become a potential biophysical treatment modality for disuse osteoporosis. © 2014 American Society for Bone and Mineral Research.     

Green tea catechin enhances osteogenesis in a bone marrow mesenchymal stem cell line

Green tea has been reported to possess antioxidant, antitumorigenic, and antibacterial qualities that regulate the endocrine system. Previous epidemiological studies found that the bone mineral density (BMD) of postmenopausal women with a habit of tea drinking was higher than that of women without habitual tea consumption. However, the effects of green tea catechins on osteogenic function have rarely been investigated. In this study, we tested (-)-epigallocatechin-3-gallate (EGCG), one of the green tea catechins, on cell proliferation, the mRNA expressions of relevant osteogenic markers, alkaline phosphatase (ALP) activity and mineralization. In a murine bone marrow mesenchymal stem cell line, D1, the mRNA expressions of core binding factors a1 (Cbfa1/Runx2), osterix, osteocalcin, ALP increased after 48 h of EGCG treatment. ALP activity was also significantly augmented upon EGCG treatment for 4 days, 7 days and 14 days. Furthermore, mineralizations assayed by Alizarin Red S and von Kossa stain were enhanced after EGCG treatment for 2–4 weeks in D1 cell cultures. However, a 24-h treatment of EGCG inhibited thymidine incorporation of D1 cells. These results demonstrated that long-term treatment of EGCG increases the expressions of osteogenic genes, elevates ALP activity and eventually stimulates mineralization, in spite of its inhibitory effect on proliferation. This finding suggests that the stimulatory effects of EGCG on osteogenesis of mesenchymal stem cells may be one of the mechanisms that allow tea drinkers to possess higher BMD.     

Poncirin prevents bone loss in glucocorticoid-induced osteoporosis in vivo and in vitro

Poncirin, a flavonoid isolated from the fruit of Poncirus trifoliata, possesses anti-bacterial and anti-inflammatory activities. However, the action of poncirin in bone biology is unclear. In this study, the in vivo and in vitro effects of poncirin in a glucocorticoid-induced osteoporosis (GIO) mouse model were investigated. Seven-month-old male mice were assigned to the following five groups: (1) sham-implantation (sham), (2) prednisolone 2.1?mg/kg/day (GC), (3) GC treated with 10?mg/kg/day of genistein, (4) GC treated with 3?mg/kg/day of poncirin, (5) and GC treated with 10?mg/kg/day of strontium (GC?+?SrCl(2)). After 8?weeks, bone loss was measured by microcomputed tomography. Osteocalcin (OC) and C-terminal telopeptides of type I collagen (CTX) were evaluated in sera. Runx2 protein, OC and osteoprotegerin (OPG) mRNA expression, alkaline phosphatase (ALP) activity, and mineral nodule assay were performed in C3H10T1/2 or primary bone marrow stromal cells. Poncirin significantly increased the bone mineral density and improved the microarchitecture. Poncirin increased serum OC, Runx2 protein production, expression of OC and OPG mRNA, ALP activity, and mineral nodule formation; and decreased serum CTX. These effects were more prominent in the poncirin group compared to the other positive control groups (genistein and strontium). The poncirin-mediated restoration of biochemical bone markers, increased bone mineral density, and improved trabecular microarchitecture likely reflect increased bone formation and decreased bone resorption in GIO mice.     

Functional characterization of N-methyl-D-aspartic acid-gated channels in bone cells

Our recent identification of glutamate receptors in bone cells suggested a novel means of paracrine communication in the skeleton. To determine whether these receptors are functional, we investigated the effects of the excitatory amino acid, glutamate, and the pharmacological ligand, N-methyl-D-aspartic acid (NMDA), on glutamate-like receptors in the human osteoblastic cell lines MG63 and SaOS-2. Glutamate binds to osteoblasts, with a Kd of approximately 10(-4) mol/L and the NMDA receptor antagonist, D(L)-2-amino-5-phosphonovaleric acid (D-APV), inhibits binding. Using the patch-clamp technique, we measured whole-cell currents before and after addition of L-glutamate or NMDA and investigated the effects of the NMDA channel blockers, dizolcipine maleate (MK801), and Mg2+, and the competitive NMDA receptor antagonist, 3-((R)-2-carboxypiperazin-4-yl)-propyl-1-phosphoric acid (R-CPP), on agonist-induced currents. Both glutamate and NMDA induced significant increases in membrane currents. Application of Mg2+ (200 micromol/L) and MK801 (100 micromol/L) caused a significant decrease in inward currents elicited in response to agonist stimulation. The competitive NMDA receptor antagonist, R-CPP (100 micromol/L), also partially blocked the NMDA-induced currents in MG63 cells. This effect was reversed by addition of further NMDA (100 micromol/L). In Fura-2-loaded osteoblasts, glutamate induced elevation of intracellular free calcium, which was blocked by MK801. These results support the hypothesis that glutamate plays a role in bone cell signaling and suggest a possible role for glutamate agonists/antagonists in the treatment of bone diseases.     

Isolation and characterization of a mesenchymal cell line that differentiates into osteoblasts in response to BMP-2 from calvariae of GFP transgenic mice

We established the clonal mesenchymal cell line, GFP-C3 (C3), which differentiates into osteoblasts in response to BMP-2 from calvariae of newborn green fluorescence protein (GFP) transgenic mice. This cell line cultured with control medium expressed low levels of alkaline phosphatase (ALP) activity and osterix mRNA and undetectable ALP and osteocalcin mRNA. Incubation of these cells with rhBMP-2 increased ALP activity dose-dependently and induced substantial levels of ALP, osteocalcin and osterix mRNA expression. C3 cells infected with adenovirus vector encoding BMP-2 (AdBMP-2) or Runx2 (AdRunx2) showed greatly increased ALP mRNA expression in a time-dependent fashion. Transduction with AdRunx2-induced expression of ALP and osteocalcin mRNA, but not osterix mRNA by day 3. Transduction with AdBMP-2 induced apparent expression of ALP and osterix mRNA by day 1 after transduction, but induced only weak expression of osteocalcin mRNA day 3 after transduction. Transplantation of C3 cells transduced with AdBMP-2 into back subfascia in wild-type mice with a complex of poly-d,l-lactic-co-glycolic acid/gelatin sponge (PGS) generated ectopic bone formation involving GFP-positive osteoblasts and osteocytes 2 weeks after transplantation. C3 cells transduced with AdRunx2 or AdLacZ failed to induce ectopic bone formation. Transplantation of C3 cells transduced with AdBMP-2 into craniotomy defects in wild-type mice using PGS as a carrier induced bone formation 2 weeks after transplantation, and replaced defects 4 weeks after transplantation. C3 cells transduced with AdRunx2 failed to induce bone repair after transplantation into craniotomy defects. These results indicate that C3 cells retain differentiation potential into osteoblasts in response to BMP-2. They are useful tools for analyzing the process of osteoblast differentiation in vivo after transplantation.