Mechanotransduction of bone cells<Emphasis Type="Italic">in vitro</Emphasis>: Mechanobiology of bone tissue

Mechanical force plays an important role in the regulation of bone remodelling in intact bone and bone repair. In vitro, bone cells demonstrate a high responsiveness to mechanical stimuli. Much debate exists regarding the critical components in the load profile and whether different components, such as fluid shear, tension or compression, can influence cells in differing ways. During dynamic loading of intact bone, fluid is pressed through the osteocyte canaliculi, and it has been demonstrated that fluid shear stress stimulates osteocytes to produce signalling molecules. It is less clear how mechanical loads act on mature osteoblasts present on the surface of cancellous or trabecular bone. Although tissue strain and fluid shear stress both cause cell deformation, these stimuli could excite different signalling pathways. This is confirmed by our experimental findings, in human bone cells, that strain applied through the substrate and fluid flow stimulate the release of signalling molecules to varying extents. Nitric oxide and prostaglandin E₂ values increased by between two- and nine-fold after treatment with pulsating fluid flow (0.6±0.3 Pa). Cyclic strain (1000 μstrain) stimulated the release of nitric oxide two-fold, but had no effect on prostaglandin E₂. Furthermore, substrate strains enhanced the bone matrix protein collagen I two-fold, whereas fluid shear caused a 50% reduction in collagen I. The relevance of these variations is discussed in relation to bone growth and remodelling. In applications such as tissue engineering, both stimuli offer possibilities for enhancing bone cell growth in vitro.     

Model for Intravital Microscopic Evaluation of the Effects of Arterial Occlusion-caused Ischemia in Bone

An in vivo model has been developed for chronic observation of the effects of ischemia on cortical bone remodeling and perfused vascularity. Diaphragm occluders were implanted around the right common iliac artery of four rabbits and inflated to produce 10 h of ischemia to the limb. Microcirculation was monitored with intravital microscopy of injected fluorescent microspheres and FITC-Dextran 70 through a bone window, the tibial bone chamber implant (BCI). Bone resorption and apposition in the BCI were indicated with mineralization dyes. Between 2 and 12 h following release of the occluder, secondary ischemia/no-reflow and other evidence of reperfusion injury were observed. Vessel damage was suggested by abnormally high leakage of FITC-D70 from the few vessels perfused during secondary ischemia. In the weeks following occluder release perfused vasculature increased beyond pre-occlusion levels. Net bone resorption reached a maximum when vascularity passed normal levels. In order to further validate the arterial occlusion model for osteonecrosis, techniques for (1) confirming bone death and (2) detecting increased leukocyte adherence to endothelial cells were added. The dead cell stain Ethidium homodimer-1 was used to tag dead osteocytes immediately after occlusion and produced a measure designated osteonecrosis index. To detect leukocytes adhering to vessel walls, carboxyfluorescein diacetate, succinimidyl ester was injected at occluder release. An increase in the number of adherent leukocytes was detected. © 1999 Biomedical Engineering Society. PAC99: 8764Rr, 8717-d, 8719Tt     

地塞米松促进胎儿主动脉血管CD105+间充质干细胞向脂肪细胞分化

目的:研究来自胎儿血管壁内的CD₁₀₅⁺细胞是否具有间充质干细胞的特性,地塞米松是否促进其向脂肪细胞分化。方法:免疫组化检测CD105阳性的细胞在胎儿主动脉分布,分离主动脉血管的成纤维样细胞,流式细胞仪检测细胞免疫表型,并接种在脂肪分化和成骨分化的诱导培养液中培养,油红O和VonKossa染色及透射电镜鉴定脂滴和钙化基质沉淀的形成。结果:CD105阳性的细胞分布在主动脉血管内膜的内皮细胞,部分中膜和外膜。分离到的细胞CD105、CD106、CD29、CD44阳性,CD34、CD31、CD11a、CD11b、HLA-DR为阴性。油红O和VonKossa染色分别显示脂滴和钙化基质形成。电镜下诱导的脂肪细胞胞浆中可见有包膜脂滴,诱导的成骨细胞胞浆内外电子密度高的钙盐沉积。诱导液中无地塞米松,未见含大脂滴的脂肪细胞。结论:分离到主动脉壁CD₁₀₅⁺细胞具有间充质干细胞的特性,并且地塞米松促进其向脂肪细胞分化,提示血管内的细胞可以向脂肪分化可能与动脉粥样硬化的病理发生过程可能相关。     

Transforming Growth Factor-β2 mRNA Level in Unloaded Bone Analyzed by Quantitative In Situ Hybridization

The effects of tail suspension hypokinesia on the gene expression for TGF-β₂ at different sites within bone were evaluated. TGF-β₂ mRNA signal levels were determined quantitatively by an image analysis system. The osteopenia induced by tail suspension was verified by histomorphometry. In the periosteum of nonsuspended control rats, TGF-β₂ mRNA was highly expressed in the preosteoblasts and osteoblast-rich cambial layers; very little signal was present within the middle and outer fibroblastic layers. Gene expression was significantly reduced in suspended rats, and this was evident both in terms of the number of silver grains in unit area or length of tissue and in each osteoblast and preosteoblast. Hypokinesia also reduced the expression of TGF-β₂ mRNA level in cortical and trabecular bone osteocytes, but did not adversely affect the mRNA level in chondrocytes in growth plate. The results affirm the site-specific response of TGF-β₂ gene expression in rats, and suggest that the cortical and trabecular bone osteopenia associated with hypokinesia in rats may be associated with a deficit in osteoblastic and osteocytic TGF-β₂ level. Received: 6 February 1998 / Accepted: 10 November 1998     

兔下颌骨牵张成骨组织中c—los和OPG及OPGL的表达

目的：探讨在机械张力作用下骨细胞将生物力学信号转变为生物学效应的作用机理。方法：建立兔下颌骨牵张成骨模型，用免疫组化方法检测牵张中期（牵张第4天）、牵张末期（牵张第8天）与固定期2周、4周、6周的牵张区新生骨组织中c—los、骨保护素（OPG）、破骨细胞分化因子（OPGL）的分布和表达。结果：在牵张中期和末期c—los的表达为强阳性，明显强于固定期2周、4周、6周的组织。牵张中、末期和固定期2周组织OPG的表达为强阳性，在固定期4周、6周组织中逐渐减弱。OPGL仅在固定期6周组织中有较弱表达，其余各时间点均无明显阳性表达。结论：在下颌骨牵张新骨形成改建过程中，c—los与机械力刺激关系密切，OPG能促进新骨形成，而OPGL则与骨改建有关。     

微小颗粒骨移植骨细胞活性的实验研究

目的 观察微小颗粒骨在移植修复骨缺损过程中的骨细胞存活情况和生物活性. 方法 建立大鼠桡骨骨缺损模型,近交系DA大鼠88只,其中雄性大鼠28只,作为供体;雌性大鼠60只,作为受体.将受体随机分为块状骨组(n=56)、微小颗粒骨组(n=56)和空白对照组(n=4),取雄性大鼠髂骨为供体骨,分别制成直径为2mm的骨块和直径为300～500 μm的微小颗粒骨,植入骨缺损,于术后1 d、4 d、1周、2周、4周、6周、10周取材,采用原位杂交的方法观察受体内Y染色体性别决定基因(Sry)的表达情况,应用免疫组化法观察各组骨形态发生蛋白-2(BMP～2)、转化生长因子-β1(TGF-β1)、碱性磷酸酶(ALP)和I型胶原的表达情况. 结果 块状骨组在移植早期Srv的表达逐渐减少,至1周消失,4周后再次出现,并且随时间延长表达逐渐增多;微小颗粒骨组各时间段均有Sry的表达,在同一时间点,微小颗粒骨组Sry的阳性细胞数多于块状骨组(P<0.05),两种骨移植物中参与修复骨缺损的细胞类型不同.微小颗粒骨内和周围组织中BMP-2、TGF-β1、ALP和Ⅰ型胶原的阳性细胞数在术后2周内多于块状骨组(P<0.05). 结论 微小颗粒骨与块状骨修复骨缺损时均有供体骨细胞参与,但微小颗粒骨内有更多的骨细胞存活.微小颗粒骨内存活的骨细胞具有生物学活性,合成并分泌骨生长因子和骨基质蛋白,可以加速骨缺损的修复.     

Anatomic variations of the lacunar–canalicular system influence solute transport in bone

Solute transport in the lacunar–canalicular system (LCS) is essential for bone metabolism and mechanotransduction. Using the technique of fluorescence recovery after photobleaching (FRAP) we have been quantifying solute transport in the LCS of murine long bone as a function of loading parameters and molecular size. However, the influence of LCS anatomy, which varies among animal species, bone type and location, age and health condition, is not well understood. In this study, we developed a mathematical model to simulate solute convection in the LCS during a FRAP experiment under a physiological cyclic flow. We found that the transport rate (the reciprocal time constant for refilling the photobleached lacuna) increased linearly with canalicular number and decreased with canalicular length for both diffusion and convection. As a result, the transport enhancement of convection over diffusion was much less sensitive to the variations associated with chick, mouse, rabbit, bovine, dog, horse, and human LCS anatomy, when compared with the rates of diffusion or convection alone. Canalicular density did not affect transport enhancement, while solute size and the lacunar density had more complicated, non-linear effects. This parametric study suggests that solute transport could be altered by varying LCS parameters, and that the anatomical details of the LCS need systemic examination to further understand the etiology of aged and osteoporotic bones.     

Meox2Cre-mediated disruption of CSF-1 leads to osteopetrosis and osteocyte defects

CSF-1, a key regulator of mononuclear phagocyte production, is highly expressed in the skeleton by osteoblasts/osteocytes and in a number of nonskeletal tissues such as uterus, kidney and brain. The spontaneous mutant op/op mouse has been the conventional model of CSF-1 deficiency and exhibits a pleiotropic phenotype characterized by osteopetrosis, and defects in hematopoiesis, fertility and neural function. Studies to further delineate the biologic effect of CSF-1 within various tissues have been hampered by the lack of suitable models. To address this issue, we generated CSF-1 floxed/floxed mice and demonstrate that Cre-mediated recombination using Meox2Cre, a Cre line expressed in epiblast during early embryogenesis, results in mice with ubiquitous CSF-1 deficiency (CSF-1KO). Homozygous CSF-1KO mice lacked CSF-1 in all tissues and displayed, in part, a similar phenotype to op/op mice that included: failure of tooth eruption, osteopetrosis, reduced macrophage densities in reproductive and other organs and altered hematopoiesis with decreased marrow cellularity, circulating monocytes and B cell lymphopoiesis. In contrast to op/op mice, CSF-1KO mice showed elevated circulating and splenic T cells. A striking feature in CSF-1KO mice was defective osteocyte maturation, bone mineralization and osteocyte-lacunar system that was associated with reduced dentin matrix protein 1 (DMP1) expression in osteocytes. CSF-1KO mice also showed a dramatic reduction in osteomacs along the endosteal surface that may have contributed to the hematopoietic and cortical bone defects. Thus, our findings show that ubiquitous CSF-1 gene deletion using a Cre-based system recapitulates the expected osteopetrotic phenotype. Moreover, results point to a novel link between CSF-1 and osteocyte survival/function that is essential for maintaining bone mass and strength during skeletal development.     

Osteocyte-Bone Lining Cell System at the Origin of Steady Ionic Current in Damaged Amphibian Bone

A wound-generated steady electric current was measured by a two-dimensional vibrating probe system in the metatarsal bones of 22 adult frogs (Xenopus laevis) placed in amphibian Ringer. Inward currents were recorded entering a micrometric hole drilled through the cortex at middiaphyseal level. These steady state currents (mean ± SD 8.50 ± 2.77 μA/cm²) last approximately 2 hours, were dependent on the presence of sodium in the incubation medium, were no more detectable after fixation, and were reduced to background level when the cell membranes were solubilized. These results agree with previous recordings of metatarsal bones of weanling mice, under identical conditions. Both results suggest that the measured ionic currents have a cellular origin. Metatarsal bones of adult amphibian were purposely selected for this study because, unlike mammalian bones, their shafts are avascular and only contain an osteocyte-bone lining cell system, as documented by scanning and transmission electron observations. Thus, unlike the data from previous investigations on mammals, the results succeeded in giving the first convincing evidence that the osteocyte-bone lining cell system is the origin of damage-generated ionic currents. As damage exposes bone ionic compartment to plasma, damage-generated ionic currents are representative of ion fluxes at bone plasma interface, and cells at the origin of the current generate the driving force of such fluxes. By demonstrating that osteocytes and bone lining cells are at the origin of the current, this study suggests that the osteocyte-bone lining cell system, though operating as a cellular membrane partition, regulates ionic flow between bone and plasma. Since strain-related adaptive remodeling could also depend on ionic characteristics and flow of the bone fluid through the osteocyte lacuno-canalicular network, the results reported here support the view that osteocyte and bone lining cells may constitute a functional syncytium involved in mineral homeostasis as well as in bone adaptation to mechanical loading.     

酒精性股骨头缺血性坏死发病机理的实验研究

采用灌胃法给予家兔烈性酒（含乙醇４５％）１０ｍｌ／（ｋｇ·ｄ）共１～６个月，观察其血清学、肝脏和股骨头组织学变化。结果表明血清ＬＰＯ、ＧＧＴ、ＡＬＴ、ＡＳＴ、ＴＧ和ＣＨＯ升高，ＳＯＤ降低。肝脏脂肪浸润。股骨头髓内脂肪细胞增多、增大，空骨陷窝百分比升高，骨小梁变细、稀疏，面积分数降低。透射电镜下见股骨头软骨下骨细胞内脂质沉积，甚或脂肪充满整个骨陷窝，骨细胞固缩、死亡。提示长期过量饮酒可导致股骨头缺血性坏死。