Research Of Stretching Strain On Osteoclast Apoptosis And Its Mechanism

Background and objectiveAlways being in outside mechanical environment, bone is a main load-bearing andmoving organ in human body. Bone can not only bear the mechanical loads, but alsochange its own shape and function with the outside mechanical loads. Bone remodelingis an important replacement mechanism in mature bone tissue. Mechanical loads regularbone shape and function by the way of bone remodeling. In physiological conditions,bone resorption and bone formation come to a dynamic equilibrium. New bone emergeswhere it is needed, and bone disappears where it is not needed. Two parts of boneremodeling are osteoclastic bone resorption and osteoblastic bone formation. Osteoclastis a hematopoietic cell, and branches from the monocyte-macrophage lineage earlyduring the differentiation process. Being a direct participant, osteoclast plays a key rolein the progress of bone remodeling. Changes of the activity or apoptosis of osteoclastwill have a great influence on bone remodeling. But, it remains unknown whethermechanical loads have an effect on osteoclast apoptosis.To investigate the effect of substrate stretching strain on osteoclast apoptosis, cellbiology and molecular biology methods were applied. Moreover, the mechanism of thesubstrate stretching strain effect on osteoclast apoptosis was explored.Contents(1) Culture and induction of osteoclasts(2) Effect of substrate stretching strain on osteoclast apoptosis(3) Primary study of the mechanism of the substrate stretching strain effect onosteoclast apoptosisMethods(1) Murine-originated RAW264.7cells were cultured in DMEM (Dulbeccominimum essential medium) containing50ng/mL RANKL (receptor activator of NF-кBligand) and50ng/mL M-CSF (macrophage-colony stimulating factor) to induce mature osteoclasts for experiment. To identify whether the induced cells have the activity ofmature osteoclasts, observation directly and after TRAP staining under light microscopewere performed, and then toluidine blue staining and SEM (scanning electronmicroscopy) were applied to observe the resorption pits formed by the osteoclasts.(2) The induced osteoclasts were subjected to physiological2500με and pathologic5000με mechanical stretching strain for3days, once a day, once for one hour, thefrequency is0.5HZ. To detect whether mechanical strain have an influence on osteoclastapoptosis, hoechst staining, Annexin binding assay and caspase-3activity assay wereperformed to study osteoclast apoptosis.(3) The induced osteoclasts were subjected to physiological2500με and pathologic5000με mechanical stretching strain for3days, once a day, once for one hour, thefrequency is0.5HZ. To see if the mitochondrial passway involves in the regulation ofeffect of mechanical strain on osteoclast apoptosis, we measured the mitochondrialmembrane potential of the loaded osteoclast. Then, western blot was used to observe theexpression of Bcl-2and caspase-3and the release of cytochrome C.Results(1) After three days of inducing culture, multinucleated cells with an increasingvolume appeared under an optical microscope, they had an irregular shape and anunclear edge. After seven days of inducing culture, the multinucleated cells were evenmore and much bigger. After TRAP (Tartrate resistant acid phosphatase) staining, themultinucleated giant cells showed blue nuclei, and cytoplasm were stained redspecifically.Toluidine blue staining of bone resorption pits showed that bone resorption pitswith clear edge formed on bone slices. The bone resorption pits areas dyed purple blue,because toluidine blue filled the pits. Scanning electron microscopy observation showedthat obvious bone resorption pits formed on bone slices, with clear edges and an uneveninternal bottom.(2) After three days of substrate stretching strain, hoechst staining showed that allthree groups of cells underwent apoptosis. The multinucleated osteoclasts whichunderwent apoptosis appeared blue, compact, hyperchromatic nuclei. After counting,apoptosis rates of the three groups of osteoclasts were calculated. The apoptosis rate ofthe control group is21.45%±0.99%. The apoptosis rate of the2500με loading group is 15.34%±1.50%, significantly lower than the control group (p＜0.05). The apoptosisrate of the5000με loading group is21.95%±1.06%, with no significant differencescompared to the control group (p＞0.05).Annexin binding assay showed that the early apoptosis rate of the control groupwas11.80%, the early apoptosis rate of the was7.54%, significantly lower than thecontrol group (p＜0.05). The early apoptosis rate of the5000με loading group was10.30%, with no obvious differences compared to the control group (p＞0.05).Finally, caspase-3activity assay showed that compared to control, the activity ofcaspase-3of the2500με loading group was0.7046±0.0148, significantly lower thanthe control group (p＜0.05). And the activity of caspase-3of the5000με loading groupwas1.1244±0.0602, a bit higher than the control group (p＜0.05).(3) After three days of substrate stretching strain, mitochondrial membranepotential measurement assay showed that compared to control, the mitochondrialmembrane potential of the group significantly increased (p＜0.05), suggesting that thedepolarization process of the mitochondria membrane was prevented. Themitochondrial membrane potential of the5000με loading group showed no significantdifferences compared to the control group (p＞0.05).Immunoblotting experiments results showed that, compared with the control group,2500με mechanical loading upregulated the expression of Bcl-2significantly (p＜0.05).5000με mechanical loading also upregulated the expression of Bcl-2significantly (p＜0.05), but not so much as the2500με mechanical loading. At the same time, comparedwith the control group, cytosolic cytochrome C of the2500με mechanical loading groupdecreased significantly (p＜0.05). While, cytosolic cytochrome C of the5000μεmechanical loading group showed no significant differences compared to the controlgroup (p＞0.05). Finally, the expression of caspase-3of the2500με mechanical loadinggroup decreased significantly (p＜0.05), and the expression of caspase-3of the2500μεmechanical loading group showed no significant differences compared to the controlgroup (p＞0.05).Conclusions(1) In the presence of50ng/mL RANKL and50ng/mL M-CSF in DMEM, murinemonocyte/macrophage cell line RAW264.7cells can be induced to mature osteoclastswhich have the typical osteoclastic biology activity (2) Stretching strain has an effect on osteoclast apoptosis. Relative research has notbeen reported.2500με mechanical stretching strain can inhibit osteoclast apoptosissignificantly, while5000με load can not.(3) Mitochondrial passway involves in the mechanical response of osteoclastapoptosis. And similar research has not been found.