The Regulatory Effect And Mechanism Of Strain-loaded Osteoblasts On Osteoclasts Differentiation And Apoptosis

Background and objectiveThe 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 osteoclastMouse 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 differentiationUniaxial 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 mechanismUniaxial 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 osteoclastAs 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 differentiationAfter 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 mechanismTo 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.