Mechanical stretching induces osteoprotegerin in differentiating C2C12 precursor cells through noncanonical Wnt Pathways

Mechanical loading is known to be important for maintaining the formation and resorption rates of bone. To study the mechanisms by which mechanical loading regulates osteogenesis, we investigated the role of the Wnt pathway in C2C12 cells committed to osteogenic differentiation in response to cyclic mechanical stretching. Osteoprotegerin (OPG) acts as a decoy receptor for RANKL to inhibit osteoclastogenesis and resorption of bone. Our results demonstrate that stretching leads to a sustained increase in OPG expression in C2C12 cells. The expression of osteogenic marker genes, such as osteocalcin and alkaline phosphatase, was transiently decreased by stretching at 24 hours and returned to control levels at 48 hours. The addition of inhibitors of the canonical Wnt/β‐catenin pathways, such as the secreted FZD‐related peptide sRFP2, as well as siRNA‐mediated knockdown, did not inhibit the effect of stretching on OPG expression. In contrast, treatment with inhibitors of noncanonical Wnt signaling, including KN93, and siRNA for Nemo‐like kinase (NLK) blocked most of the mechanical inductive effect on OPG. Furthermore, stretching‐induced OPG production in the culture medium was able to inhibit the osteoclast formation of bone marrow macrophages. These results suggest that mechanical stretching may play an important role in bone remodeling through the upregulation of OPG and that the mechanical signaling leading to OPG induction involves the noncanonical Wnt pathway. © 2010 American Society for Bone and Mineral Research     

膜转铁蛋白1在成骨细胞上表达的初步研究

目的观察膜转铁蛋白1（FP1）基因在人成骨细胞株（hFOB1.19）内的表达情况。方法 hFOB1.19在34℃、5%CO2条件下培养,添加细胞生长营养液,细胞培养3～4d后用逆转录-聚合酶链反应（RT-PCR）检测FP1在成骨细胞内的表达情况。结果在成骨细胞株hFOB1.19内,FP1基因呈阳性表达。结论 FP1基因成功表达对＂铁调素与成骨细胞内铁离子关系＂有十分重要的解释作用。     

成骨细胞的应力调控机制

近年来,骨组织细胞的力学生物学研究逐步兴起,主要研究细胞受应力作用后的生物学反应机制,包括应力的作用效应、应力信号通路、调节机制等。由于成骨细胞易于获取,培养方便,与骨髓干细胞和骨细胞是同源细胞,在应力作用机制上具有高度的相似性,因此,研究的内容集中在成骨细胞的应力调控机制。笔者就成骨细胞的应力作用效应、调节机制、信号通路等方面的研究进展作一综述。     

股骨假体近端应力遮挡对成骨细胞增殖和凋亡的影响

目的探讨股骨假体近端不同强度的应力遮挡对成骨细胞增殖和凋亡的影响。方法根据股骨近端应变的有限元分析结果,在体外分组模拟应力遮挡的细胞力学环境,A组应变保持不变（对照组）、B～E组应力遮挡分别为25%、50%、75%和100%,观察不同强度的应力遮挡环境对成骨细胞相对活性、增殖指数和凋亡百分比的影响。结果C、D和E组成骨细胞的相对活性明显降低,分别为0.72±0.09、0.68±0.11和0.76±0.15,与对照组（0.92±0.13）相比差异有显著性（P〈0.05）;D组和E组成骨细胞增殖指数降低至47.87±6.58和51.65±7.71,与对照组（64.31±7.92）相比差异有显著性（P〈0.05）;C组和D组成骨细胞凋亡的百分比与对照组（7.36±0.59）相比差异有显著性（P〈0.05）,分别增高至18.76±2.82和16.78±1.69。B组成骨细胞的相对活性、增殖指数和凋亡百分比与对照组相比差异无显著性（P〉0.05）。结论股骨假体近端的应力遮挡能抑制成骨细胞增殖并（或）刺激其凋亡,两者皆存在“阈值”现象而缺乏强度依赖性。应力遮挡下凋亡的启动较增殖的抑制更为敏感。     

体外培养成骨细胞对自体骨髓间充质干细胞定向成骨分化的影响

目的观察体外培养成骨细胞对自体骨髓间充质干细胞（BMSC）增殖和定向成骨分化的影响．探讨此诱导方法作为骨组织工程种子细胞来源方法的可行性。方法应用密度梯度离心联合贴壁筛选法从兔股骨骨髓中分离、纯化BMSC．取第3代BMSC常规培养设为空白对照组,利用含有钙化诱导剂的DMEM培养基诱导培养第3代BMSC．设为钙化对照组。取第3代成骨细胞,1：1的比例和第3代BMSC共同培养．设为实验组。碱性磷酸酶（ALP）染色及钙结节染色鉴定诱导结果．ALP定量测定观察共同培养中成骨细胞对BMSC诱导影响。总蛋白定量测定观测诱导成骨细胞的分化活性．结果成骨细胞与BMSC共同培养24h后．在倒置光学显微镜下可见两种细胞形态混杂生长．共同培养5d后．细胞形态趋于一致,多呈长梭形。诱导培养5d后．实验组与钙化对照组ALP染色均为阳性。诱导培养21d．两组钙结节茜素红染色均呈阳性．而BMSC空白对照组并未见钙结节形成。ALP定量测定,实验组于3、5、7、9d均低于钙化对照组,且差异均具有统计学意义。实验组与空白对照组ALP定量测定,在4个时间段差异也均具有统计学意义。总蛋白定量测定显示实验组于第3天．A值高于钙化对照组,差异具有显著统计学意义（P〈0．01）,而第5天、第7天、第9天时却低于钙化对照组．差异具有显著统计学意义（P〈0．01）。实验组于各时间段均高于空白对照组．差异亦具有统计学意义。结论成骨细胞与BMSC共同培养应用于BMSC的成骨诱导．从诱导效率和诱导后成骨细胞的分化活性方面．都能证实该共同培养方法的有效性。但与钙化诱导方法相比．共同培养的诱导效果差于钙化诱导方法。     

High Cortical Bone Mass Phenotype in Betacellulin Transgenic Mice Is EGFR Dependent

Signaling through the epidermal growth factor receptor (EGFR) by ligands such as epidermal growth factor (EGF), transforming growth factor α (TGFA), and amphiregulin (AREG) has been reported to have effects on skeletal growth. The role of betacellulin (BTC), another EGFR ligand, in skeletal development and bone metabolism is unknown. In previous experiments, transgenic mice overexpressing BTC ubiquitously under the control of the chicken β‐actin promoter (BTC‐tg) exhibited stunted growth and disproportionately sized long bones. In this study, we performed a detailed phenotypic analysis of BTC‐tg mice at 3, 6, and 9 wk of age. Osteoblastic cells from transgenic mice showed strong expression of BTC as determined by Western blots and by immunohistochemistry on bone sections. In femurs of male and female BTC‐tg mice, we found reduced longitudinal bone growth and a pronounced increase in total volumetric BMD. The increased femoral BMD was mainly caused by augmented endocortical bone apposition and subsequent cortical bone thickening. In contrast, vertebral BMD was reduced in BTC‐tg mice of both sexes. An overall similar phenotype was found in 6‐mo‐old BTC‐tg mice. The increase in cortical bone mass in the appendicular skeleton of BTC‐tg mice was largely blocked when they were crossed into the Egfr^Wa5 background characterized by a dominant negative EGFR. Our study showed that overexpression of BTC results in an EGFR‐dependent upregulation of cortical bone mass in the appendicular skeleton of mice, uncovering a potential novel anabolic pathway for cortical bone.