Dioxins interfere with differentiation of osteoblasts and osteoclasts

We have previously shown that the environmental contaminant 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) affects bone growth, modelling and mechanical strength in vivo. In this study, we utilized differentiation of bone marrow stem cells to osteoblasts and osteoclasts as a model system to study the effects of TCDD on bones. Stem cells were isolated from bone marrow of femurs and tibias of rats and mice. Progress of osteoblastic differentiation was monitored by measuring mRNA expression levels of differentiation markers from control and TCDD-treated cells using quantitative RT-PCR. TCDD significantly and dose-dependently decreased the mRNA levels of RUNX2, alkaline phosphatase and osteocalcin. Also the activity of alkaline phosphatase was significantly inhibited in both rat and mice cells. In the case of osteoclasts, TCDD decreased the number of TRACP+ multinucleated cells, with corresponding decreases in the number of F-actin rings and the area of resorption. Studies in AHR-knockout mice indicated that TCDD has no effect on the expression of osteoblastic differentiation markers suggesting that TCDD mediates its effects by AHR. Both osteoblastic and osteoclastic effects took place at very low doses of TCDD, as in most cases 100 fM TCDD was enough to significantly affect the differentiation markers. Therefore, differentiation of osteoblasts and osteoclasts from bone marrow stem cells seems to be a very sensitive target for TCDD. Disrupting effects in osteoblastic cells, in addition to disturbed osteoclastogenesis, may thus play a role in adverse effects on bone quality in TCDD exposed animals.     

人脂肪干细胞的体外培养鉴定及分化特性

目的：在体外从脂肪块中分离出脂肪干细胞（adipose derived stem/stromal cells,ASCs）,对其进行形态观察、干细胞鉴定、增殖和分化能力检测。方法：将腹部取皮术的皮下脂肪利用胶原酶消化法,进行体外分离培养,取第3代的细胞进行细胞爬片HE染色、流式鉴定、MTT、细胞周期检测等,利用成脂和成骨培养液诱导,油红O和茜素红染色鉴定诱导结果。结果：原代培养第1次换液时细胞多呈多角形和短梭形,第3代ASCs细胞爬片HE染色显示形态多为长梭形,呈漩涡状生长;流式鉴定显示：CD29＋,CD31-,CD34-,CD44＋,CD45-,CD49＋,CD106-,CD133-;MTT显示ASCs生长增殖活性强;细胞周期检测结果显示：G1=86.8%,G2=8.77%;成脂诱导后油红O染色阳性,对照组为阴性;成骨诱导后茜素红染色阳性,对照组为阴性。结论：人ASCs具有贴壁生长、多向分化以及干细胞表型等特征,且生长增殖活性强,是一种很有应用前景的间充质干细胞。     

大鼠骨髓基质细胞体外成脂、成骨分化的研究

目的　观察体外定向诱导大鼠骨髓基质细胞分化为脂肪细胞和成骨细胞。方法　全骨髓法分离大鼠骨髓基质细胞 ,传 3代后分别在成脂、成骨诱导条件下继续培养 ,油红O染色、碱性磷酸酶染色和VonKossa染色判定其分化结果。结果　传 3代大鼠骨髓基质细胞成脂诱导 2 1d后 ,分化为脂肪细胞的阳性率为 (83. 6± 2 . 8) % ,成骨诱导 12d后碱性磷酸酶染色阳性率达 (87. 6± 2. 8) % ,连续诱导35d可见矿化结节形成。结论　随着诱导条件的不同 ,大鼠骨髓基质细胞在体外可定向分化为脂肪细胞或成骨细胞。     

成骨细胞与破骨细胞间接共培养研究进展

在骨代谢过程中,成骨细胞形成新骨,破骨细胞吸收旧骨,一旦成骨细胞介导的骨基质形成和破骨细胞介导的骨吸收失衡,则会导致骨质疏松症等危害人类健康的疾病产生。因此,不同研究者致力于开发模拟体内环境的成骨细胞与破骨细胞体外共培养模型,以进行骨代谢相关疾病的研究。间接式共培养是通过物理方式将成骨细胞与破骨细胞分隔,使二者可以进行细胞间的交流而不接触,可以针对单一的细胞进行分析,在药物筛选及研究方面,具有高通量和经济便捷等独特的优势,本文对成骨细胞和破骨细胞的间接共培养技术进行归纳和总结。     

小鼠成骨细胞和破骨细胞共培养模型的建立

目的 建立小鼠成骨细胞和破骨细胞共培养模型,为探讨成骨细胞和破骨细胞之间的相互调控作用奠定基础.方法 利用24 h内新生小鼠颅骨和4～8周龄成年小鼠四肢长骨分别分离、培养成骨细胞和破骨细胞,采用间接接触的培养模式将接种了骨髓单核细胞的玻片或骨片置入提前24 h接种了成骨细胞的培养皿中进行共培养.共培养一定时间后进行破骨细胞抗酒石酸酸性磷酸酶(tartrate-resistant acid phosphatase,TRAP)染色鉴定和骨吸收功能检测,并进一步采用RT-PCR对破骨细胞标志酶基因基质金属蛋白酶.9(MMP-9)、TRAP 和组织蛋白酶K (Cathepsin K ) 的表达进行检测.结果 共培养5天后可见TRAP( )多核细胞形成,13天TRAP( )多核细胞数目达到高峰;骨吸收陷窝在共培养7天后开始出现,随着培养时间的延长,陷窝面积呈增加趋势;破骨细胞标志基因TRAP 在共培养3天时开始表达,而MMP-9和 Cathepsin K则在共培养5天后表达.结论 共培养体系中成骨细胞对破骨细胞调控作用显著,诱导的破骨细胞具有噬骨能力,该共培养模型可用于成骨细胞和破骨细胞之间的相互调控作用研究.     

脉冲磁场对成骨细胞和破骨细胞的影响

近年来,脉冲磁场(PEMFs,pulsed electromagnetic fields)在骨质疏松治疗中已得到广泛的关注。PEMFs对参与骨重建的成骨细胞和破骨细胞都有显著影响。本文从PEMFs及其对骨质疏松的影响、PEMFs对成骨细胞和破骨细胞的影响以及可能机制方面加以综述。     

人骨髓基质细胞培养及向成骨细胞的诱导分化

目的研究人骨髓基质细胞体外培养及向成骨细胞诱导分化的实验方法。方法采用梯度离心法获得人骨髓基质细胞，细胞纯化后使用分化培养液将骨髓基质细胞向成骨细胞方向诱导分化。通过形态学观察、生化指标检测、细胞染色和矿化结节测定等方法，确定细胞的功能状态和分化程度。结果显微镜观察显示获得的人骨髓基质细胞生长状况良好，生化指标稳定；经分化培养液培养的细胞增殖速度明显减慢，生长状态平稳。细胞在分化培养过程中，上清液中碱性磷酸酶分泌量明显增加，细胞碱性磷酸酶染色明显浓染，随时间呈显著增强趋势；采用常规培养液培养的骨髓基质细胞，在汇合后不能形成明显的矿化结节。用分化培养液培养的人骨髓基质细胞，在14d时开始出现矿化结节，在21d时呈现密集的茜素红染色矿化结节。结论梯度离心法获得的人骨髓基质细胞生长情况良好，功能状态稳定；体外培养的人骨髓基质细胞在一定条件下可以向成骨细胞方向诱导分化，并具有良好的成骨细胞功能特征，可以满足进一步研究的需要。     

Lentivirus transduction of human osteoclast precursor cells and differentiation into functional osteoclasts

Gene transfer into stem cells has been an ongoing priority as a treatment for genetic disease and cancer for more than two decades. Methods described herein, form the basis for providing the cell source to determine if osteoclast precursor cells (OcP) can be used as therapeutic gene delivery systems in vivo. Osteoclasts and tumor associated macrophages or OcP, support survival, tumor progression and osteolysis in bone cancers. Two sources of precursor cells are compared: CD14+ cells, the standard OcP, found abundantly in peripheral blood and CD34+ cells, hematopoietic stem cells that are rare, but which can be expanded into OcP. Our findings characterize cell yield at each step of the transduction process and thus provide essential data for planning future in vivo experiments. In addition we demonstrate that essential functions of OcP are preserved following lentiviral transduction. Specifically, neither the transduction method nor the lentiviral transduction influence the OcP's ability to form osteoclasts, express the marker gene, EGFP, or resorb bone. Finally, we conclude that CD34+ cells yield significantly more transduced cells and form functionally superior osteoclasts in vitro. This study represents a step towards considering human gene therapy for bone cancer by demonstrating successful transduction of human OcP for use as cellular delivery vehicles to sites of bone cancer.     

(Pre-)osteoclasts induce retraction of osteoblasts before their fusion to osteoclasts.

Precursors of osteoclasts seeded on top of a confluent layer of osteoblasts/bone lining cells induced retraction of the latter cells. The (pre)osteoclasts then migrated in the formed cell-free areas and fused to form osteoclast-like cells. Retraction of the osteoblasts/bone lining cells proved to depend on activity of matrix metalloproteinases, and TGF-beta1 prevented the retraction. INTRODUCTION: It is well known that osteoblasts have a profound effect on (pre)osteoclasts in inducing the formation of bone-resorbing osteoclasts. Whether, on the other hand, (pre)osteoclasts also modulate osteoblast activity is largely unknown. Because osteoblasts/bone lining cells have to retract from the surface before resorption of bone by osteoclasts, we addressed the question of whether (pre)osteoclasts have the capacity to induce such an activity. MATERIALS AND METHODS: Rabbit calvarial osteoblasts/bone lining cells or periosteal fibroblasts were cultured until confluency, after which rabbit peripheral blood mononuclear cells (PBMCs) were seeded on top of them. The co-cultures were maintained for up to 15 days in the presence or absence of the cytokines transforming growth factor (TGF)-beta1 and TNF-alpha and selective inhibitors of matrix metalloproteinases and serine proteinases. The formation of cell-free areas and the number of TRACP+ multinucleated osteoclast-like cells were analyzed. In addition, formation of cell-free areas was analyzed in co-cultures of osteoblasts with mature osteoclasts. RESULTS: The seeding of PBMCs on a confluent layer of osteoblasts/bone lining cells resulted in the following sequence of events. (1) A low number of PBMCs strongly attached to osteoblasts. 2) At these sites of contact, the osteoblasts retracted, thus forming cell-free areas. (3) The PBMCs invaded these areas and attached to the surface of the well, after which they fused and formed multinucleated TRACP+ osteoclast-like cells. Retraction was only seen if the cells were in direct contact; conditioned media from cultured PBMCs added to osteoblasts had no effect. Mature osteoclasts seeded on osteoblasts similarly induced retraction, but this retraction occurred at a much faster rate (within 2 days) than the retraction effectuated by the osteoclast precursors (after 8 days in co-culture). Inhibition of matrix metalloproteinase activity, but not of serine proteinases, strongly reduced retraction of the osteoblasts, thus indicating that this type of cell movement depends on the activity of matrix metalloproteinases. A similar inhibitory effect was found with TGF-beta1. TNF-alpha had no effect on osteoblast retraction but enhanced the formation of multinucleated osteoclast-like cells. Addition of PBMCs to confluent layers of periosteal fibroblasts resulted in similar phenomena as observed in co-cultures with osteoblasts. However, the cell-free areas proved to be significantly smaller, and the number of multinucleated cells formed within cell-free areas was three to four times lower. CONCLUSION: Our results indicate that osteoclast precursors and mature osteoclasts have the capacity to modulate the activity of osteoblasts and that, yet unknown, membrane-bound signaling molecules are essential in inducing retraction of osteoblasts and the subsequent formation of cell-free areas.     

Multipotent menstrual blood stromal stem cells: isolation, characterization, and differentiation

The stromal stem cell fraction of many tissues and organs has demonstrated to exhibit stem cell properties such as the capability of self-renewal and multipotency, allowing for multilineage differentiation. In this study, we characterize a population of stromal stem cells derived from menstrual blood (MenSCs). We demonstrate that MenSCs are easily expandable to clinical relevance and express multipotent markers such as Oct-4, SSEA-4, and c-kit at the molecular and cellular level. Moreover, we demonstrate the multipotency of MenSCs by directionally differentiating MenSCs into chondrogenic, adipogenic, osteogenic, neurogenic, and cardiogenic cell lineages. These studies demonstrate the plasticity of MenSCs for potential research in regenerative medicine.     

共育体系中成骨细胞和破骨细胞生物学特性观察

目的建立成骨细胞和破骨细胞的体外共育体系,观察在此体系中成骨细胞和破骨细胞生物学特性的变化,探讨成骨细胞和破骨细胞间的相互作用。方法取髂骨松质骨,Ⅱ型胶原酶消化,分次获得成骨细胞和破骨细胞。建立培养上清相通但二者互不接触的成骨细胞-破骨细胞共育模型。以细胞增殖(MTT法)、碱性磷酸酶(ALP)活性代表成骨细胞的成骨活性,以抗酒石酸酸性磷酸酶(TRAP)活性、骨吸收陷窝面积代表破骨细胞的破骨能力,检测共育对成骨细胞和破骨细胞生物学特性的影响。结果成骨细胞呈饱满的梭形,ALP染色阳性;破骨细胞呈多核,TRAP染色阳性,可以吸收骨质形成骨陷窝。当成骨细胞与破骨细胞共育后,其MTT法OD值(0.60±0.08)较单独培养时(0.36±0.03)明显提高(P=0.000);其ALP活性(23.37±2.48)u/mg较单独培养时(18.33±0.34)u/mg明显提高(P=0.000)。破骨细胞与成骨细胞共育后,形成骨吸收陷窝的平均面积犤(6.55±0.34)×10-2犦μm2较单独培养时犤(5.15±0.17)×10-2犦μm2明显增大(P=0.000)。结论共育体系中成骨细胞和破骨细胞的功能相互促进,为骨组织代谢的体外研究提供了可靠的模型。     

骨髓基质干细胞在软骨组织工程中的应用

骨髓基质细胞(BMSC)是倍受关注的具有多向分化潜能的干细胞,可在体外大规模扩增而不丢失多分化潜能,异体移植时排斥反应小,是组织工程再生修复应用的重点种子细胞.近年来BMSC在软骨创伤修复中的应用较为广泛,特别是在BMSC诱导后重新分化,软骨缺损植入后刺激软骨修复再生,减缓关节损伤进一步发展,体内外特定环境下复合组织工程材料分化成骨或软骨的相容性、分化度等生物学特性等方面进行了细致研究.该文就BMSC在软骨缺损组织工程修复中应用的现状和发展方向作一综述.     

成骨与破骨细胞促红细胞生成素肝细胞受体B4/肝配蛋白B2双向信号通路

成骨细胞与破骨细胞以直接接触的方式共同调控骨重建平衡,这也决定了两者的不可分割性。最新研究表明前破骨细胞肝配蛋白(Ephrin)B2与成骨细胞膜上促红细胞生成素肝细胞受体(Eph)B4受体的直接接触来调控骨稳态平衡。具有EphrinB2配体的前破骨细胞可通过直接接触具有EphB4受体的前成骨细胞从而触发各自相应的下游信号转导分子。通过激活成骨细胞膜表面Eph受体而起正向作用,进一步去抑制下游信号转导分子RhoA活性促进前体细胞分化成熟。反之,EphrinB2配体的激活起到反向作用,抑制破骨相关转录因子的C-fos/NFATc1转录级联反应来抑制前破骨细胞的分化,磨损颗粒导致的骨溶解会使破骨细胞上EphrinB2配体表达明显升高,并且会促进NFATc1的高表达,可以通过这种双向信号的机制来减弱甚至是抑制磨损颗粒导致的破骨细胞的进一步分化。