凋亡破骨细胞的拉伸应变

背景：力学应变在骨重建中起重要作用。然而,力学应变是否影响破骨细胞的凋亡仍不清楚。 目的：观察力学应变对体外培养破骨细胞凋亡的影响。 方法：对小鼠单核细胞RAW264.7采用巨噬细胞集落刺激因子和破骨细胞分化因子诱导,抗酒石酸酸性磷酸酶染色和骨吸收实验确定成功诱导出了破骨细胞。实验共分为3组,对诱导的破骨细胞分别施加0(对照组),2 500和5 000 με的基底拉伸应变3 d,1 h/次,1次/d。 结果与结论：与对照组相比,生理强度载荷2 500 με阻止了破骨细胞的凋亡和线粒体膜电位的下降。但是,病理强度载荷5 000 με对破骨细胞的凋亡和线粒体膜电位没有影响。     

载荷作用下骨细胞分泌因子对成骨细胞和破骨细胞的调节

骨细胞是骨组织主要的力学感受及转导细胞,它们通过众多突触结构相互连接,形成庞大的骨稳态细胞调控网络,联系着成骨细胞、破骨细胞等骨基质表面细胞。骨细胞通过旁分泌途径影响成骨细胞骨形成和破骨细胞骨吸收来调节骨代谢,维持骨更新。针对骨细胞在受到力学刺激后分泌或释放的一些信号分子或蛋白因子对成骨细胞和破骨细胞生长分化的影响,本文综述近年来关于受力学刺激的骨细胞如何与成骨/破骨细胞进行通讯,为骨细胞生物力学研究提供新思路。     

周期性张应变对破骨前体细胞和破骨细胞增殖和抗酒石酸酸性磷酸酶的影响

目的研究不同强度的力学载荷对破骨细胞及其前体细胞增殖、分化和功能的影响。方法以破骨诱导液培养RAW264.7破骨前体细胞,同时施加3 d的周期性张应变,然后培养4 d;另外一组RAW264.7细胞以破骨诱导液培养4 d,将其诱导为破骨细胞,再施加3 d的周期性张应变。结果在不同张应变下,两组细胞增殖活性的变化大致相同,但细胞抗酒石酸酸性磷酸酶(tartrate-resistant acid phosphatage,TRAP)活性和破骨细胞(TRAP阳性多核细胞)数量的变化明显不同。在2 500με的中等强度张应变下,第1组的TRAP活性降幅和破骨细胞数量减幅均最高,而后者TRAP活性降幅和破骨细胞数量减幅均最低。结论不同张应变对分化初期破骨前体细胞和已分化出破骨细胞的破骨前体细胞的破骨分化和功能状态的影响有明显差异。     

破骨细胞的生物学新观

骨是一种不断进行重建的有活力组织。骨重建 (remodeling)是一个偶联过程 ,骨吸收紧随新骨形成。负责骨吸收的细胞主要是多核破骨细胞 ,关于调节破骨细胞的形成及溶骨作用的因子还有许多问题未找到答案 ,但最近在理解破骨细胞的细胞生物学和分子生物学及骨髓微环境在调节破骨细胞的形成及溶骨作用方面已获得了重大进展。1　破骨细胞形态学破骨细胞 osteoclast)是一种大的多核巨细胞 ,含 2～ 1 0 0个核 ,通常为 1 0～ 2 0个 ,直径可达 1 0 0 μm,数量极少 ,通常仅 2～ 3个 /μm3,但在骨转换活跃的部位 ,如成长中骨的干骺端 ,其数量增加。破骨…     

建立小鼠成骨与破骨细胞相互作用的共育新体系

背景：众所周知,骨重建是骨组织中重要的生物学反应过程,其中成骨细胞与破骨细胞发挥了关键作用。但目前,关于骨重建中成骨与破骨细胞间信号传递的深层机制还不清楚。 目的：利用transwell技术,在体外建立一种成骨与破骨细胞的新型共育体系,为深入研究骨重建中成骨与破骨细胞的相互作用提供成熟的实验模型。 方法：采用MC3T3-E1成骨样细胞株与RAW264.7破骨前体细胞株,进行体外成骨与破骨细胞的诱导分化,并利用Transwell共培养板(0.4 µm聚酯膜)建立成骨与破骨细胞的共育体系。共培养6 d后,通过测定细胞活性和碱性磷酸酶(ALP)活力分析成骨细胞的增殖和分化活性,利用抗酒石酸酸性磷酸酶(TRAP)染色、甲苯胺蓝(TB)染色、TRAP活性测定及扫描电镜技术观察破骨细胞的分化及骨吸收功能。 结果与结论：共培养体系中成骨样细胞的无限增殖能力减弱,而分化活性明显增强,同时破骨前体细胞被诱导分化为成熟的破骨细胞,并具有一定的骨吸收功能。因此,该共培养体系可用于骨重建中成骨与破骨细胞间信号通路的深层研究。     

骨功能适应性重建模型及数值模拟

骨骼会受到力学因素的影响和调控,发生骨功能适应性重建。建立模拟骨重建的数值模型,定量地研究骨重建过程,有着重要的临床应用价值。目前,骨功能适应性重建模型可分为两大类：力学模型和生理模型。对这两类重建模型的原理、算法和应用等做详细论述。力学模型通过假设力学环境与骨结构之间的函数关系来预测骨重建,但不考虑其真实的生物学过程。基于不同的重建激励主要有两种力学模型：骨力学稳态模型和骨损伤修复模型。生理模型则试图从微观层次阐明骨重建的力学-生物学机制,骨重建是由破骨细胞、成骨细胞等各种骨组织细胞完成,有以基本多细胞单位的形式作用或细胞独立分开作用两种观点。这些重建模型结合有限元法应用在许多有关骨重建问题的数值计算中。通过比较分析多个数值应用的模拟结果,还讨论了重建控制方程中各参数值的设置对重建结果的影响。     

骨代谢研究新进展

骨质疏松是由各种原因导致的骨代谢失衡，破骨细胞活性增强，骨吸收大于骨形成。在骨质疏松的发病过程中，破骨细胞起着重要作用。破骨细胞专司溶骨，它的活性与细胞大小、伪足运动以及溶酶体的释放密切相关。破骨细胞活性受许多因素调节，其中一些是通过升高细胞内Ｃａ２＋浓度的方式调节其活性，如降钙素、细胞因子、ＡＴＰ等。细胞内Ｃａ２＋浓度升高可使破骨细胞回缩，伪足运动减弱，溶酶体释放减少，破骨细胞活性减弱。而炎症因子一氧化氮亦与骨吸收密切相关。     

骨组织工程化培养中成骨细胞力学响应的初步研究

由于骨具有支撑、保护、运动的功能,是承力器官,力学环境对骨组织的发生、发展有着十分重要的影响。活体研究表明:载荷可促进成骨细胞的增殖、分化和细胞外基质的分泌,及骨衬细胞的生物学活动;力学因素显著影响骨细胞生物学活动,包括骨细胞的凋亡;力学环境引起骨内细胞的协同反应,对骨组织变化起整合作用。由于骨组织力学环境如此重要,力学环境就有可能是工程化骨培养中的必要因素。由此,笔者尝试建立三维立体条件下成骨细胞力学响应的模型,研究骨内细胞的力学反应。模型包括支架材料、种子细胞和有力学作用的培养环境。支架材料除具有一般生物支架材料的要求外,还应与天然松质骨有相似的结构和力学性能,这里包括生物衍生松质骨、珊瑚支架等。种子细胞可采用乳鼠分离出的成骨细胞或成骨细胞系,接种在支架上进行培养。载荷直接施加在复合体上,复合体的表观应变被精确控制,可形成与骨活体内相似的力学环境,其中载荷可采用不同形状的波形,如正弦波、方波等。加载应变可达到0～10 000,频率0～100 Hz,大大包括了活体骨组织所受的力学环境。载荷形成细胞的力学环境是以支架材料的表观应变衡量,这正对应活体骨研究的力学指标。     

成骨/基质细胞在破骨细胞分化中的作用

骨组织微环境中,成骨/基质细胞表达的骨保护素(osteoprotegerin,OPG)和骨保护素配体(osteoprotegerin ligand,OPGL)是调节骨代谢的重要因子。研究表明,成骨/基质细胞的OPG/OPGL相对表达水平与破骨细胞的分化、成熟及骨吸收直接相关。各种刺激骨吸收的生物、物理因素均通过调节成骨/基质细胞的OPG/OPGL相对表达活性,影响破骨细胞的分化和成熟。     

成骨／基质细胞在破骨细胞分化中的作用

骨组织微环境中，成骨／基质细胞表达的骨保护素(osteoprotegerin，OPG)和骨保护素配体(osteoprotegerin ligand，OPGL)是调节骨代谢的重要因子。研究表明，成骨／基质细胞的OPG／OPGL相对表达水平与破骨细胞的分化、成熟及骨吸收直接相关。各种刺激骨吸收的生物、物理因素均通过调节成骨／基质细胞的OPG／OPGL相对表达活性，影响破骨细胞的分化和成熟。     

Gap-junctional regulation of osteoclast function

Previous research has shown that bone-resorbing osteoclasts contain connexin molecules that organize to hemichannel-forming connexons, which in turn form functional gap junctions in neighboring cells. So far only little is known about the role of gap junctions in osteoclasts. However, blocking of the gap-junctional communication inhibits bone resorption in vitro. Knockout mice deficient of Connexin-43, the major connexin in bone cells, show surprisingly little skeletal manifestations. Gap-junctional communication in osteoblasts and osteocytes is well documented and seems to be essential for the integrity of bone cells, as well as for the transfer of mechanical signals of bone loading. The role of gap junction in osteoclasts is unclear, so far, but some putative roles have been suggested, including their participation in osteoclast precursor fusion to multinucleated mature osteoclasts, communication in the bone multicellular unit in bone remodeling, osteoclast survival, and apoptosis.     

Adsorption of bisphosphonate onto hydroxyapatite using a novel co-precipitation technique for bone growth enhancement

Premature bone resorption and remodeling by osteoclasts can limit the longevity of implant fixation and recovery time. Orally administered bisphosphonates (BPs) have been used to inhibit osteoclast action at the implant/bone interface. Ideally, these should be delivered at the interface with the osteoblast-active hydroxyapatite (HA) for maximum effect. This investigation introduces a novel BP loading technique to achieve improved BP release from a simulated body fluid-grown HA (SBF-HA) with the aim of improving implant fixation. A solution co-precipitation technique incorporates the BP (pamidronate) into a thin SBF-HA coating. Surface analysis, using X-ray photoelectron spectroscopy (XPS), of the resultant coating was employed to confirm the presence of the adsorbed BP on the surface of SBF-HA. XPS analysis was also used to determine the optimal adsorption process. Osteoclast cell culture experiments confirmed the biological effectiveness of BP adsorption and proved that the pamidronate was biologically active, causing both decreased osteoclast numbers and decreased resorption.     

The in vitro osteoclastic degradation of nacre

Osteoclast activity was studied on nacre, the mother of pearl (MOP) in order to assess the plasticity of bone resorbing cells and their capacity to adapt to a biomineralized material with a different organic and mineral composition from that of its natural substrate, bone. Pure MOP, a natural biomineralized CaCO(3) material, was obtained from Pinctada oyster shell. When implanted in the living system, nacre has proven to be a sustainable bone grafting material although a limited surface degradation process. Osteoclast stem cells and mature osteoclasts were cultured on MOP substrate and osteoclast precursor cells were shown to differentiate into osteoclasts capable of resorbing nacre substrate. However, analysis of the organization of the cytoskeleton showed that both a sealing zone and a podosome structure were observed on the nacre substrate. Moreover, MOP resorption efficiency was consistently found to be lower than that of bone and appeared to be a limited process.     

破骨细胞在骨组织工程中的意义及其研究策略

破骨细胞是骨吸收细胞,在骨塑造和重塑中发挥重要作用。目前在破骨细胞对成骨细胞调节方面的影响了解很少。最近的研究表明在骨发育过程中破骨细胞的缺乏会导致骨基质排列紊乱、矿化减少、成骨细胞行为异常。破骨细胞在骨组织工程的引入将会解决骨组织工程面临的许多问题。破骨细胞前体在组织工程化骨上生成破骨细胞是一条生理、实际的破骨细胞引入途径。     

Human bone marrow adipocytes support dexamethasone-induced osteoclast differentiation and function through RANKL expression

The TNF-family molecule, Receptor Activator of Nuclear factor κ B Ligand (RANKL) is known as a key regulator for bone remodeling, and is essential for the development and activation of osteoclasts. In this study, we examined the regulation of RANKL in primary human bone marrow adipocytes and the relationship between bone marrow adipocytes and bone metabolism. RANKL expression and the RANKL/osteoprotegerin (OPG) mRNA ratio in marrow adipocytes increased following dexamethasone treatment. In co-cultures of human osteoclast precursors and bone marrow adipocytes with dexamethasone, osteoclast precursors differentiated to TRAP-positive multinuclear cells. Moreover, the ability of bone resorption was confirmed in co-culture in flasks coated with calcium phosphate film. Osteoclast precursor differentiation and bone resorption were blocked by RANKL antibody pretreatment. TRAP-positive multinuclear cells did not form in coculture without cell-to-cell contact conditions. We conclude that primary human bone marrow adipocytes have the ability to promote osteoclast differentiation and activities, similar to osteoblasts and other RANKL-expressing cells.     

Evidence that protein kinase-A, calcium-calmodulin kinase and cytoskeletal proteins are involved in osteoclast retraction induced by calcitonin.

Calcitonin is a direct inhibitor of osteoclastic activity. Osteoclast retraction is readily induced by calcitonin and it is possible that calcitonin-induced inhibition of bone resorption is in part due to this effect. However, little is known of the mechanisms of this action. In these studies, we have investigated the intracellular signalling pathway of calcitonin-induced osteoclast retraction using cultures of freshly isolated rat osteoclasts. The spread area occupied by single Giemsa-stained rat osteoclasts was measured in vitro by a computer imaging analysis system and used as a quantitative parameter for calculating the degree of osteoclast retraction in response to various agents. Our results show that cAMP may be an important second messenger in the reaction of osteoclasts to calcitonin. Moreover, both protein kinase-A and calcium/calmodulin-dependent protein kinase are involved in the osteoclast retraction induced by this hormone, while cytoskeletal proteins are required for the process to occur.     

Increased apoptosis in osteoclasts and decreased RANKL immunoexpression in periodontium of cimetidine‐treated rats

It has been demonstrated that histamine interferes with the recruitment, formation and activity of osteoclasts via H₁‐ and H₂‐receptors. Cimetidine is a H₂‐receptor antagonist used for treatment of gastric ulcers that seems to prevent bone resorption. In this study, a possible cimetidine interference was investigated in the number of alveolar bone osteoclasts. The incidence of osteoclast apoptosis and immunoexpression of RANKL (receptor activator of nuclear factor κB ligand) was also evaluated. Adult male rats were treated with 100 mg kg^?1 of cimetidine for 50 days (CimG); the sham group (SG) received saline. Maxillary fragments containing the first molars and alveolar bone were fixed, decalcified and embedded in paraffin. The sections were stained by H&E or submitted to tartrate‐resistant acid phosphatase (TRAP) method. TUNEL (terminal deoxynucleotidyl transferase‐mediated dUTP nick‐end labeling) method and immunohistochemical reactions for detecting caspase‐3 and RANKL were performed. The number of TRAP‐positive osteoclasts, the frequency of apoptotic osteoclasts and the numerical density of RANKL‐positive cells were obtained. Osteoclast death by apoptosis was confirmed by transmission electron microscopy (TEM). In CimG, TRAP‐positive osteoclasts with TUNEL‐positive nuclei and caspase‐3‐immunolabeled osteoclasts were found. A significant reduction in the number of TRAP‐positive osteoclasts and a high frequency of apoptotic osteoclasts were observed in CimG. Under TEM, detached osteoclasts from the bone surface showed typical features of apoptosis. Moreover, a significant reduction in the numerical density of RANKL‐positive cells was observed in CimG. The significant reduction in the number of osteoclasts may be due to cimetidine‐induced osteoclast apoptosis. However, RANKL immunoexpression reduction also suggests a possible interference of cimetidine treatment in the osteoclastogenesis.