摘要
研究背景和目的
骨骼是机体中重要的承重组织，力学刺激下的骨组织主要通过动态的骨重建过程来调节其新陈代谢，从而适应新的力学环境。骨重建过程是人类进化的一种重要的生理反应，可以减少骨组织中骨量的流失，并保证骨组织结构的完整性。骨重建过程受到机体中多种因素的调控，如遗传因素、激素水平、代谢环境及力学刺激等。大量的研究表明，力学载荷在骨重建过程中发挥了重要作用：生理性动态载荷可以促进骨组织形成，而缺乏这种力学刺激会导致骨量的大量流失，如废用性骨质疏松等。成骨细胞和破骨细胞是力学环境下骨重建过程中的关键效应细胞，其中成骨细胞主要活性为促进骨形成，而破骨细胞主要活性为促进骨吸收。在不同的力学加载条件下，成骨细胞和破骨细胞均可直接感受力学信号，并转化为不同的生物学信号，导致骨重建过程向骨形成或骨吸收方向发展，从而引起骨量的重新分配和骨结构的重新排布。
目前认为，骨组织的力学生物学效应主要通过骨重建过程中成骨细胞与破骨细胞间的功能转化来进行调节，但关于力学载荷下成骨细胞与破骨细胞间的相互作用及其调控机制尚不清楚。因此，本研究通过建立成骨细胞与破骨细胞的体外共培养体系，观察不同加载强度的拉伸应变作用下，成骨细胞对破骨细胞分化与凋亡的影响，并探讨力学载荷下成骨细胞调控破骨细胞分化、凋亡的作用机制。
研究内容与方法
(1)建立成骨细胞与破骨细胞的体外共培养体系。
采用MC3T3-E1成骨样细胞株与RAW264.7破骨前体细胞株，经10-8mol/L的1α,25(OH)2维生素D3(1α,25(OH)2D3)及50ng/mL的巨噬细胞集落刺激因子(M-CSF)作用，进行体外成骨细胞与破骨细胞的诱导分化，并利用Transwell小室(口部直径2.4cm、底部面积4.67cm2、底膜为0.4μm孔径的聚酯半透膜)建立成骨细胞与破骨细胞的共培养体系。共培养6d后，通过细胞活性(MTT)实验、碱性磷酸酶(ALP)活力测定及苏木素-伊红(HE)染色鉴定共育体系中成骨细胞的增殖和分化活性；通过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(SEM)鉴定共育体系中破骨细胞的分化活性及骨吸收功能。
(2)基底拉伸应变对成骨细胞增殖与分化的影响。
采用四点弯曲力学加载装置，对共育体系中成骨细胞施加拉伸刺激，加载条件为0.5HZ、1h/天、连续3d，根据不同的加载强度将培养细胞分为0με、2500με、5000με三组，通过成骨细胞的MTT细胞活性实验、ALP活性测定及ALP染色观察不同加载强度的拉伸应变对成骨细胞的增殖与分化活性的影响。
(3)拉伸应变作用下成骨细胞对破骨细胞分化的影响及其调控机制。
采用四点弯曲力学加载装置，对共育体系中成骨细胞施加拉伸刺激，加载条件为0.5HZ、1h/天、连续3d，根据不同的加载强度将培养细胞分为0με、2500με、5000με三组，通过破骨细胞的HE染色、TRAP染色、TB染色及TRAP活性测定，观察不同加载强度的拉伸应变作用下成骨细胞对破骨细胞分化活性的调控作用；通过骨板中骨吸收陷窝的检测(TB染色)及破骨细胞中组织蛋白酶-K(Cath-k)及基质金属蛋白酶-9(MMP-9)的活性测定(免疫化学染色、ELISA法)，观察不同加载强度的拉伸应变作用下成骨细胞对破骨细胞骨吸收功能的影响。
采用四点弯曲力学加载装置，对共育体系中成骨细胞施加拉伸刺激，加载条件为0.5HZ、1h/天、连续3d，根据不同的加载强度将培养细胞分为0με、2500με、5000με三组，通过RT-PCR、Western blot及免疫化学染色方法，检测不同加载强度的拉伸应变作用下成骨细胞RANKL、OPG及EphA2的表达情况，同时观察破骨细胞中EphA2的表达及NF-κB通路中信号分子p65的磷酸化水平，探讨不同加载强度的拉伸应变作用下成骨细胞调控破骨细胞分化及骨吸收功能的分子机制。
(4)拉伸应变作用下成骨细胞对破骨细胞凋亡的影响及其调控机制。
采用四点弯曲力学加载装置，对共育体系中成骨细胞施加拉伸刺激，加载条件为0.5HZ、1h/天、连续3d，根据不同的加载强度及RANKL干预因子将培养细胞分为0με、2500με、2500με+RANKL、5000με、5000με+RANKL五组。通过流式细胞术，检测各实验组中破骨细胞的细胞凋亡率；通过Hoechst染色，观察各实验组中破骨细胞的凋亡形态学变化；通过Western blot技术，检测各实验组中破骨细胞Fas、FasL、Casepase-8及Caspase-3蛋白的表达情况，观察不同加载强度的拉伸应变作用下成骨细胞调控破骨细胞凋亡的分子机制。
研究结果
(1)成骨细胞与破骨细胞体外共育体系的建立
与单培养组比较，共培养组成骨细胞在MTT实验中的吸光度显著下降(P<0.01)；共培养组成骨细胞ALP活性显著升高(P<0.01)；HE染色后，发现共培养组成骨细胞无限增殖速度减慢，细胞数量减少。与单培养组比较，共培养组破骨细胞TRAP活性显著升高(P<0.01)，TRAP染色明显增强；共培养组可见多个成熟的破骨细胞，细胞体积增大，胞浆伸展、空泡化，并有多个细胞核；共培养组骨片上可见多个圆形的骨吸收陷窝，凹陷明显，与周边骨组织界限清楚。此结果显示，共育体系中成骨细胞与破骨细胞的分化活性均明显增强。
(2)基底拉伸应变对成骨细胞增殖与分化的影响。
对成骨细胞施加基底拉伸应变3d后，与0με组比较，2500με组可显著提高成骨细胞的增殖活性，表现为MTT实验中OD值显著升高(P<0.05)，2500με组可促进成骨细胞ALP活性的升高(P<0.05)；而5000με组则降低成骨细胞的增殖活性(P<0.05)与ALP活性(P<0.05)；各实验组中成骨细胞ALP染色结果与ALP定量分析结果一致。此结果显示，生理性拉伸应变可促进成骨细胞的增殖与分化，而超生理性拉伸应变则抑制成骨细胞的增殖与分化。
(3)拉伸应变作用下成骨细胞对破骨细胞分化的影响及其调控机制。
对成骨细胞施加拉伸应变3d后，与0με组比较，2500με组可显著抑制破骨细胞的分化和骨吸收功能，表现为多核破骨细胞的数量减少，TRAP活性降低(P<0.05)，TRAP染色强度下降(P<0.01)，骨片中骨吸收陷窝的面积减小(P<0.01)，破骨细胞中Cath-k及MMP-9的表达降低(P<0.05或P<0.01)；5000με组中破骨细胞的TRAP活性、骨吸收陷窝面积及Cath-k、MMP-9的表达也显著下降(P<0.01)。此结果显示，拉伸应变作用成骨细胞后，抑制了破骨细胞的分化与骨吸收功能。
对成骨细胞施加拉伸应变3d后，与0με组比较，2500με组可显著升高成骨细胞中OPG mRNA及蛋白表达水平(P<0.01)，而对RANKL mRNA及蛋白表达无显著影响，由于OPG的高表达，2500με组可显著上调成骨细胞中OPG/RANKL表达的比值(P<0.01)；与0με组比较，5000με组可显著升高成骨细胞中OPG mRNA及蛋白表达水平(P<0.05)，同时升高RANKL mRNA及蛋白表达水平(P<0.05)，且上调成骨细胞中OPG/RANKL表达的比值(P<0.05)；与0με组比较，2500με及5000με组对成骨细胞中EphA2的表达均无显著影响。此结果显示，拉伸应变作用成骨细胞后，对破骨细胞的抑制作用与上调成骨细胞OPG/RANKL表达的比值有关。
对成骨细胞施加拉伸应变3d后，与0με组比较，2500με组可降低破骨细胞中p65磷酸化蛋白(p-p65)的表达(P<0.05)，且OPG重组因子干预后可进一步抑制p-p65的表达(P<0.01)，并降低TRAP活性(P<0.01)；与0με组比较，2500με及5000με组对破骨细胞中EphA2的表达均无显著影响。此结果显示，拉伸应变作用成骨细胞后，对破骨细胞的抑制作用与下调破骨细胞中p65的磷酸化水平有关。
(4)拉伸应变作用下成骨细胞对破骨细胞凋亡的影响及其调控机制。
对成骨细胞施加拉伸应变3d后，与0με组比较，2500με组可升高破骨细胞的凋亡率(P<0.01)；与2500με组比较，2500με+RANKL组则降低破骨细胞的凋亡率(P<0.05)；与0με组比较，5000με组可升高破骨细胞的凋亡率(P<0.01)；与5000με组比较，5000με+RANKL组则降低破骨细胞的凋亡率(P<0.01)；各组破骨细胞的凋亡形态学变化与凋亡率的检测结果一致。此结果显示，拉伸应变作用成骨细胞后，促进了破骨细胞的凋亡发生，RANKL因子干预后可抑制破骨细胞的凋亡。
对成骨细胞施加拉伸应变3d后，与0με组比较，2500με组可显著促进破骨细胞中Fas、FasL、Casepase-8及Caspase-3蛋白的表达(P<0.01)；与2500με组比较，2500με+RANKL组则显著抑制破骨细胞中Fas、FasL、Casepase-8及Caspase-3蛋白的表达(P<0.05或P<0.01)；与0με组比较，5000με组可显著促进破骨细胞中Fas、FasL、Casepase-8及Caspase-3蛋白的表达(P<0.01)；与5000με组比较，5000με+RANKL组则显著抑制破骨细胞中Fas、FasL、Casepase-8及Caspase-3蛋白的表达(P<0.05或P<0.01)。此结果显示，拉伸应变作用成骨细胞后，对破骨细胞的促凋亡机制与激活破骨细胞中Fas/FasL介导的细胞凋亡通路有关。
研究结论
(1) Transwell共育体系中成骨样细胞的无限增殖能力减弱，而分化活性增强，同时破骨前体细胞被诱导分化为成熟的破骨细胞，并具有骨吸收功能。该共育体系可用于探讨力学环境下骨重建中成骨细胞与破骨细胞间信号调控机制的实验研究。
(2)生理性拉伸应变(2500με)可促进成骨细胞的增殖和分化，继而抑制共育体系中破骨细胞的分化及骨吸收功能，而超生理性拉伸应变(5000με)则抑制成骨细胞的增殖和分化，同时抑制共育体系中破骨细胞的分化及骨吸收功能。不同加载强度的拉伸应变作用下，成骨细胞抑制共育体系中破骨细胞分化及骨吸收功能的作用机制，可能与上调成骨细胞中OPG/RANKL表达的比值，继而抑制破骨细胞中NF-κB信号通路的激活有关。
(3)生理性拉伸应变(2500με)作用成骨细胞后，可促进共育体系中破骨细胞的凋亡发生，超生理性拉伸应变(5000με)作用成骨细胞后，亦可促进共育体系中破骨细胞的凋亡发生。不同加载强度的拉伸应变作用成骨细胞后，对共育体系中破骨细胞的促凋亡机制，可能与上调成骨细胞中OPG/RANKL表达的比值，继而激活破骨细胞中Fas/FasL介导的细胞凋亡通路有关。
Background and objective
The skeleton forms the essential load-bearing tissue that is dynamically remodeledthroughout one’s lifetime. Bone remodeling, a critical biological process formaintaining bone density and osseous integrity, is dependent on genetic, hormonal,metabolic, and age-related factors as well as mechanical forces. Growing evidencesuggests that mechanical stimuli play a pivotal role in bone remodeling becausephysiological dynamic loading can promote bone formation, whereas the absence ofmechanical forces can lead to the loss of bone mass. The mechanical response of bone ismainly affected by the coupled activities of osteoblasts and osteoclasts, two specializedcells responsible for bone formation and resorption, respectively. Additionally, animbalance between the functions of these cells can interrupt their physiological effectson bone tissue, thereby causing many bone diseases, such as osteoporosis, osteopetrosis,and arthritis.
In previous studies, research has focused of the mechanical responses ofosteoblasts and osteoclasts, but the intercellular communication between these cellsduring mechanical loading has not been fully elucidated. Therefore, the purpose of thisstudy was to examine the hypothesis that osteoblast-osteoclast communication isinvolved in mechanical responses, and we therefore investigated the effect ofstrain-loaded osteoblasts on osteoclastic differentiation and bone resorption in aco-culture system.
Methods
(1) Establishment of a co-culture system between osteoblast and osteoclast
Mouse osteoblastic cells MC3T3-E1and mouse monocyte/macrophage cellsRAW264.7, treated with1α,25(OH)2D3(10-8mol/L) and M-CSF (50ng/ml), were usedto induce the differentiation of osteoblasts and osteoclasts in a co-culture system withtranswell culture plates (6well size inserts with0.4m pores only for passage of smallsoluble factors). After co-culture for6days, cell viability, activities of alkaline phosphatase (ALP) and HE staining of osteoblasts were determined, and for osteoclasts,tartrate-resistant acid phosphatase (TRAP) staining, toluidine blue (TB) staining, TRAPactivities assay and technique of scanning electron microscope (SEM) were preformed.
(2) Effect of mechanical strain on osteoblast proliferation and differentiation
Uniaxial and homogeneous mechanical tension was generated using a speciallydesigned four-point bending device. In the present study, the MC3T3-E1cells in theco-culture system were subjected to a mechanical strain of2500με or5000με at0.5Hzonce per day with a periodicity of1h/day for3days. Simultaneously, the control cellswere incubated under the same conditions but without mechanical stimuli. Toinvestigate the effect of mechanical strain on osteoblast proliferation and differentiation,cell viability, activities of ALP and ALP staining were determined.
(3) Effect of strain-loaded osteoblast on osteoclast differentiation and its mechanism
Uniaxial and homogeneous mechanical tension was generated using a speciallydesigned four-point bending device. In the present study, the MC3T3-E1cells in theco-culture system were subjected to a mechanical strain of2500με or5000με at0.5Hzonce per day with a periodicity of1h/day for3days. Simultaneously, the control cellswere incubated under the same conditions but without mechanical stimuli. Toinvestigate the effect of strain-loaded osteoblast on osteoclast differentiation, HEsaining, TRAP staining and TRAP activities assay of osteoclasts were performed. Toinvestigate the effect of strain-loaded osteoblast on osteoclast bone resorption, TBstaining for resorption lacunae and the expressions of Cath-k and MMP-9in osteoclastswere evaluated.
Uniaxial and homogeneous mechanical tension was generated using a speciallydesigned four-point bending device. In the present study, the MC3T3-E1cells in theco-culture system were subjected to a mechanical strain of2500με or5000με at0.5Hzonce per day with a periodicity of1h/day for3days. Simultaneously, the control cellswere incubated under the same conditions but without mechanical stimuli. Toinvestigate the detailed mechanism involved in the regulatory role of strain-loadedosteoblast on osteoclast differentiation, the expressions of OPG, RANKL, and EphA2inosteoblasts and the expressions of p-p65and EphA2in osteoclasts were detected byRT-PCR, Western blot and immunochemistry staining.
(4) Effect of strain-loaded osteoblast on osteoclast apoptosis and its mechanism
Uniaxial and homogeneous mechanical tension was generated using a speciallydesigned four-point bending device. In the present study, the MC3T3-E1cells in theco-culture system were subjected to a mechanical strain of0με,2500με and5000μεrespectively, at0.5Hz once per day with a periodicity of1h/day for3days. In addtion,the recombinant RANKL was added to the culture media of2500με and5000μεgroups before application of mechanical stretch. To investigate the effect ofstrain-loaded osteoblast on osteoclast apoptosis and its mechanism, flow cytometry,Hoechst staing and the expressions of Fas, FasL, Casepase-8and Caspase-3inosteoclasts were determined.Results
(1) Establishment of a co-culture system between osteoblast and osteoclast
As for osteoblast proliferation, the results of cell viability showed that the ODvalues of pre-osteoblasts in the co-culture system were attenuated (P<0.01), comparedwith that of the pre-osteoblasts cultured alone. With regard to osteoblast differentiation,the ALP activities of pre-osteoblasts in the co-culture system were enhancedsignificantly (P<0.01), compared with that of the pre-osteoblasts cultured alone.
As for osteoclast differentiation and bone resorption function, more multinuclearcells with the characteristics of abundant cytoplasm and vacuolation, were observed inthe co-culture system compared with pre-osteoclasts cultured alone. Moreover, TRAPactivities in the culture media of co-culture system were increased remarkably (P<0.01),compared with that in the media with only pre-osteoclasts culture. In addition, moreresorption pits on bone slices with the characteristics of sealing zone and actin rings,were found in the co-culture system compared with pre-osteoclasts cultured alone.
(2) Effect of mechanical strain on osteoblast proliferation and differentiation
After application of mechanical strain on osteoblasts for three days, the OD valuesand ALP activities of osteoblasts in2500με group were increased (P<0.05), whereasthe OD values and ALP activities of osteoblasts in5000με group were decreased(P<0.05) compared with that in0με group.
(3) Effect of strain-loaded osteoblast on osteoclast differentiation and its mechanism
To explore the influence of strain-loaded osteoblasts on osteoclast differentiationand bone resorption, we first quantified TRAP activities in osteoclasts and evaluated the number of mature osteoclasts by counting TRAP-positive multinucleated cells. Theresults showed that the strain-conditioned medium in2500με group remarkablyinhibited the differentiation of pre-osteoclastic cells into mature osteoclasts anddecreased the TRAP activities (P<0.05). Second, we determined the resorptive activityof osteoclasts by counting the area of resorption lacunae in the bone slices and foundthat the strain-conditioned medium in2500με group significantly inhibited theformation of resorption pits (P<0.01). Third, we examined the expressions of Cath-Kand MMP-9and found that the levels of Cath-K and MMP-9in osteoclasts were bothdepressed by the strain-conditioned medium in2500με group (P<0.05or P<0.01).Moreover, the strain-conditioned medium in5000με group inhibited the differentiationand bone resorption function of osteoclasts compared with that in0με group.
To investigate the mechanism involved in the regulation of osteoclastdifferentiation and bone resorption by strain-stimulated osteoblasts, we focused on theexpression changes of OPG and RANKL in osteoblasts. The results showed that theexpression levels of OPG mRNA and protein were both significantly increased in2500με group (P<0.01). However, the expression levels of RANKL were not changed in2500με group. Although the expression levels of RANKL were not remarkably affectedby the mechanical strain, the ratio of OPG to RANKL expressions was significantlyup-regulated due to the enhanced OPG levels in2500με group (P<0.01). In addition,the expressions of OPG and RANKL were both significantly increased (P<0.05) and theratio of OPG to RANKL expressions was up-regulated in5000με group (P<0.05),compared with that in0με group.
To explore the potential role of OPG in the anti-osteoclastogenesis effect of loadedosteoblasts, the cells were pretreated with recombinant OPG (50ng/ml) before theapplication of mechanical stretch (2500με). The results showed that the expression ofp-p65(phosphorylation of p-p65) in osteoclasts, an activation signal for NF-κB pathway,was significantly down-regulated by the application of mechanical stretch orrecombinant OPG (P<0.05), resulting in the decreased TRAP activities (P<0.01). Thecombined application of mechanical loading and recombinant OPG can strengthen theinhibitory effect (P<0.01). Moreover, the expressions of EphA2in osteoblasts andosteoclasts were not changed in2500με and5000με groups, compared with that in0με group.
(4) Effect of strain-loaded osteoblast on osteoclast apoptosis and its mechanism
After application of mechanical strain on osteoblasts for three days, the apoptosisratio of osteoclasts in2500με and5000με groups were both increased (P<0.01),compared with that in0με group. Moreover, the apoptosis ratio of osteoclasts in2500με and5000με groups were both decreased (P<0.05or P<0.01) after the treatment ofrecombinant RANKL factor. The results of apoptotic morpholgical observation inosteoclasts were coincident with osteoclastic apoptosis ratio.
After application of mechanical strain on osteoblasts for three days, the expressionsof Fas, FasL, Casepase-8and Caspase-3in osteoclasts in2500με and5000με groupswere both increased (P<0.01), compared with that in0με group. Furthermore, theexpressions of Fas, FasL, casepase-8and caspase-3in osteoclasts in2500με and5000με groups were both decreased (P<0.05or P<0.01) after the treatment of recombinantRANKL factor.
Conclusions
(1) In the Transwell co-culture system, the proliferation activities of osteoblast-like cellsare attenuated, whereas the differentiation activities of osteoblast-like cells arepromoted, and simultaneously the pre-osteoclastic cells are induced to differentiate intothe mature osteoclasts with bone resorbing function. Thus, this co-culture system can beapplied in the study of osteoblast-osteoclast communication in bone remodeling duringmechanical loading.
(2) Physiological mechanical strain (2500με) directly activates osteoblasts and thestrain-conditioned medium inhibits the differentiation and bone resorption function ofco-cultured osteoclasts. Pathological mechanical strain (5000με) directly inhibitsosteoblast activation and the strain-conditioned medium also inhibits the differentiationand bone resorption function of co-cultured osteoclasts. The mechanism involved in theanti-osteoclastogenesis effect of strain-loaded osteoblasts is mainly dependent on theup-regulation of the OPG/RANKL expressions ratio in osteoblasts, leading to theinactivation of NF-κB signaling pathway in osteoclasts.
(3) Physiological mechanical strain (2500με) directly activates osteoblasts and thestrain-conditioned medium promotes the apoptosis of co-cultured osteoclasts.Pathological mechanical strain (5000με) directly inhibits osteoblast activation and thestrain-conditioned medium promotes the apoptosis of co-cultured osteoclasts. Thedetailed mechanism involved in the promotive effect of strain-loaded osteoblasts on osteoclast apoptosis is greatly associated with the up-regulation of the OPG/RANKLexpressions ratio in osteoblasts, resulting in the activation of Fas/FasL signalingpathway in osteoclasts.
引文
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