RAW264.7 cells treated with macrophage-colony stimulating factor and RANKL could be induced into mature osteoclasts

TRAP and resorption lacuna were differentiation markers for the osteoclasts.

To see if the cells were successfully induced into mature osteoclasts, TRAP staining and resorption assay were applied. We first examined TRAP staining. The result clearly confirmed our method. Osteoclasts were observed among the RAW264.7 cells. These big, multinucleated cells had irregular unclear boundaries and red-stained cytoplasm. On the contrary, the RAW264.7 cells were smaller and negatively stained (Figure 1).

We next examined the formation of resorption lacunae. They were found big, with a round or irregular shape. Contrarily, the bone lacunae in the Haversian system were much smaller (Figure 2).

Physiological strain inhibits osteoclast apoptosis, and pathological strain has nearly no impact upon osteoclast apoptosis (Figure 3)



To see if mechanical strain modulated the apoptosis rate of osteoclasts, Hoechst staining was used. As shown in Figure 3, osteoclasts displaying characteristics of apoptosis as chromatin condensation and DNA fragmentation can be easily distinguished from normal cells by Hoechst staining. We found that osteoclasts underwent apoptosis spontaneously without any mechanical strain.

Then we calculated the apoptotic rate of each group. The apoptotic rate was (21.45±0.99)% in the control group, (15.34±1.50)% in the 2 500 με group and (21.95±1.06)% in the 5 000 με group.

Compared with the control group, the apoptosis rate of osteoclasts under 2 500 με decreased significantly (P < 0.05). Whereas, the apoptosis rate of osteoclasts under 5 000 με increased back to the unstrained ones (P > 0.05).

In addition, the Annexin-V/PI binding assay was applied to further confirm this effect. As shown in Figure 4, we obtained a same tendency as the Hoechst staining test showed. These results indicate that physiological strain inhibits osteoclast apoptosis, while higher strain magnitude beyond the physiological strain has nearly no impact upon osteoclast apoptosis, suggesting that mechanical strain plays an important role in the regulation of osteoclast apoptosis.

Mitochondria play an important role in regulating cell apoptosis. To study the role of the mitochondria in the response of mechanical strain to osteoclast apoptosis, we examined the effect of mechanical strain on the mitochondrial membrane potential. After JC-1 staining, the ratio of the mitochondrial aggregates (red) to cytoplasmic monomeric form (green) of JC-1 was observed and analyzed with flow cytometry. The increase of this ratio means that fewer cells undergo depolarization of the mitochondrial membrane.

As shown in Figure 5, the red/green ratio of the cells under physiological strain increased significantly (P < 0.05), while cells under pathologic strain had almost the same level of mitochondrial membrane potential, compared to the unstrained ones (P > 0.05).

[1] Martin RB. Toward a unifying theory of bone remodeling. Bone. 2000;26(1):1-6. [2] Nakahama K. Cellular communications in bone homeostasis and repair. Cell Mol Life Sci. 2010;67(23): 4001-4009. [3] Liu H, Li B. p53 control of bone remodeling. J Cell Biochem. 2010;111(3):529-534. [4] Zelzer E, Olsen BR. The genetic basis for skeletal diseases. Nature. 2003;423(6937):343-348. [5] Rodan GA, Martin TJ. Therapeutic approaches to bone diseases. Science. 2000;289(5484):1508-1514. [6] Boyle WJ, Simonet WS, Lacey DL. Osteoclast differentiation and activation. Nature. 2003;423(6937): 337-342. [7] Quinn JM, Gillespie MT. Modulation of osteoclast formation. Biochem Biophys Res Commun. 2005;328(3):739-745. [8] Rubin J, Fan X, Biskobing DM, et al. Osteoclastogenesis is repressed by mechanical strain in an in vitro model. J Orthop Res. 1999;17(5):639-645. [9] Kurata K, Uemura T, Nemoto A, et al. Mechanical strain effect on bone-resorbing activity and messenger RNA expressions of marker enzymes in isolated osteoclast culture. J Bone Miner Res. 2001;16(4):722-730. [10] Suzuki N, Yoshimura Y, Deyama Y, et al. Mechanical stress directly suppresses osteoclast differentiation in RAW264.7 cells. Int J Mol Med. 2008;21(3):291-296. [11] Takeuchi T, Tsuboi T, Arai M, et al. Adrenergic stimulation of osteoclastogenesis mediated by expression of osteoclast differentiation factor in MC3T3-E1 osteoblast-like cells. Biochem Pharmacol. 2001;61(5):579-586. [12] Weinstein RS, Manolagas SC. Apoptosis and osteoporosis. Am J Med. 2000;108(2):153-164. [13] Ozaki K, Takeda H, Iwahashi H, et al. NF-kappa B inhibitors stimulate apoptosis of rabbit mature osteoclasts and inhibit bone resorption by these cells. FEBS Lett. 1997;410(2-3): 297-300. [14] Tang LL, Wang YL, Pan J, et al. The effect of step-wise increased stretching on rat calvarial osteoblast collagen production. J Biomech. 2004;37(1):157-161. [15] Tezuka K, Tezuka Y, Maejima A, et al. Molecular cloning of a possible cysteine proteinase predominantly expressed in osteoclasts. J Biol Chem. 1994;269(2):1106-1109. [16] Akiyama T, Dass CR, Choong PF. Novel therapeutic strategy for osteosarcoma targeting osteoclast differentiation, bone-resorbing activity, and apoptosis pathway. Mol Cancer Ther. 2008;7(11):3461-3469. [17] Hughes JM, Petit MA. Biological underpinnings of Frost's mechanostat thresholds: the important role of osteocytes. J Musculoskelet Neuronal Interact. 2010;10(2):128-135. [18] Civitelli R. Cell-cell communication in the osteoblast/ osteocyte lineage. Arch Biochem Biophys. 2008; 473(2): 188-192. [19] Jee WS. Principles in bone physiology. J Musculoskelet Neuronal Interact. 2000;1(1):11-13. [20] Chen HC, Guan JL. Association of focal adhesion kinase with its potential substrate phosphatidylinositol 3-kinase. Proc Natl Acad Sci U S A. 1994;91(21):10148-10152.

[1]	耿秋东, 葛海雅, 王和鸣, 李 楠. 基于网络药理学探讨龟鹿二仙胶治疗骨关节炎的作用及机制[J]. 中国组织工程研究, 2021, 25(8): 1229-1236.
[2]	裴丽丽, 孙贵才, 王 弟. 丹酚酸B抑制骨髓间充质干细胞氧化损伤及促进分化为心肌样细胞[J]. 中国组织工程研究, 2021, 25(7): 1032-1036.
[3]	李时斌, 赖 渝, 周 毅, 廖建钊, 章晓云, 张 璇. 激素性股骨头坏死发病机制及相关信号通路的靶点效应[J]. 中国组织工程研究, 2021, 25(6): 935-941.
[4]	徐银琴, 史红美, 王光义. 通痹方热敷联合针刺治疗对退变椎间盘细胞凋亡相关基因Caspase-3、Bcl-2 mRNA的影响[J]. 中国组织工程研究, 2021, 25(5): 713-718.
[5]	张雯雯, 金颂峰, 赵国梁, 宮丽鸿. 复方中药稳斑汤减少同型半胱氨酸诱导大鼠心肌微血管内皮细胞凋亡的作用机制[J]. 中国组织工程研究, 2021, 25(5): 723-728.
[6]	刘 青, 万碧江. 针刀治疗胶原诱导性关节炎大鼠滑膜组织Bcl-2/Bax的表达[J]. 中国组织工程研究, 2021, 25(5): 729-734.
[7]	谢崇新, 张 磊. 保留与不保留残端重建前交叉韧带术后膝关节退变的比较[J]. 中国组织工程研究, 2021, 25(5): 735-740.
[8]	白胜超, 高 扬, 王 博, 李俊平, 王瑞元. 针刺干预大负荷运动损伤模型大鼠骨骼肌线粒体功能的动态变化[J]. 中国组织工程研究, 2021, 25(23): 3648-3653.
[9]	杨彩会, 刘启成, 董 明, 王丽娜, 左美娜, 陆 颖, 牛卫东. 丝氨酸/苏氨酸蛋白激酶促进慢性根尖周炎模型小鼠的骨破坏[J]. 中国组织工程研究, 2021, 25(23): 3654-3659.
[10]	左振魁, 韩佳瑞, 计树灵, 贺璐璐. 银杏酮酯预处理减轻辐射所致模型小鼠的急性肠损伤[J]. 中国组织工程研究, 2021, 25(23): 3666-3671.
[11]	张 亮, 马晓燕, 王佳虹. 肾衰饮调控慢性肾功能衰竭大鼠肾脏细胞凋亡的机制[J]. 中国组织工程研究, 2021, 25(23): 3672-3677.
[12]	谢 阳, 吕志宇, 张淑江, 龙 婷, 李作孝. 重组腺病毒介导神经生长因子转染实验性自身免疫性脑脊髓炎模型小鼠少突胶质细胞的凋亡及髓鞘化[J]. 中国组织工程研究, 2021, 25(23): 3678-3683.
[13]	霍 花, 程余婷, 周 倩, 齐昱晗, 伍 超, 石前会, 杨童景, 廖 健, 洪 伟. 种植体表面药物涂层对骨结合的影响[J]. 中国组织工程研究, 2021, 25(22): 3558-3564.
[14]	蒋昇源, 李 丹, 姜建浩, 杨上游, 杨淑野. 假体无菌性松动过程中Co2+对成骨前体细胞的生物学反应[J]. 中国组织工程研究, 2021, 25(21): 3292-3299.
[15]	徐 彬, 杨秀书, 刘 旋, 王振兴. 尿液环境中肠上皮细胞及其凋亡因子半胱氨酸蛋白酶3、Bax和Bcl-2表达的变化[J]. 中国组织工程研究, 2021, 25(20): 3173-3177.

Mechanical forces have a great influence on bone modeling and remodeling. Physiological mechanical strain leads to bone homeostasis when bone resorption equals to bone formation; overload promotes bone gain; and disuse induces bone loss^[1]. Bone modeling and remodeling are regulated by osteoclastic bone resorption and osteoblastic bone formation^[2-3]. A balance between the resorption and the formation is of critical importance to many bone diseases such as osteoporosis and osteopetrosis^[4-6].

Osteoclast is a hematopoietic cell, and branches from the monocyte-macrophage lineage early during the differentiation process. Although it has been reported that many hormones and cytokines, such as parathormone, 1, 25-dihydroxyvitamin D3, transforming growth factor-α and interleukin-1, promote osteoclast formation, receptor activator of nudear factor-kappa B ligand (RANKL) and macrophage-colony stimulating factor are the crucial formation factors in the process of osteoclast formation^[6-7]. Mature osteoclasts are multinuclear, tartrate-resistant acid phosphatase (TRAP)-positive cells which have the ability to form bone resorption. Studies have demonstrated that mechanical forces directly affect osteoclast differentiation and resorption function^[8-10]. Osteoclasts have a finite life span, and undergo spontaneous apoptosis. Many drugs, such as bisphosphonate, which can induce osteoclast apoptosis, are used to fight against osteoporosis, because it has been demonstrated that tiny changes in osteoclast apoptosis can cause large changes in bone remodeling^[11-13]. However, it has never been illuminated whether mechanical strains are involved in the regulation of osteoclast apoptosis. We hypothesized that mechanical strains are involved in the regulation of osteoclast apoptosis. In the present study, we aimed to investigate whether there is a relationship between mechanical strain and osteoclast apoptosis.

MATERIALS AND METHODS
Design
A cellular observation.
Time and setting
The experiment was performed at the Institute of Medical Equipment of Academy of Military Medical Sciences from March to December 2011.
Materials
RAW264.7 cells were provided by Institute of Basic Medicine of Peking Union Medical College.
The main reagents and apparatus are introduced as follows:

Methods
Osteoclast culture and differentiation
RAW264.7 cells were maintained in Dulbecco minimum essential medium, supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin. The differentiation of RAW264.7 cells was induced in Dulbecco minimum essential medium containing macrophage-colony stimulating factor (50 ng/mL) and RANKL (50 ng/mL). Mature osteoclasts were observed after 3 days of stimulation.
TRAP staining
After a brief rinse in PBS, the osteoclasts were fixed with 4% paraformaldehyde in PBS, and then stained using the TRAP staining kit. The images of TRAP-positive multinucleated osteoclasts were captured under a microscope with DP Controller.
Resorption assay
Osteoclasts were seeded on bovine cortical bone slices and then cultured for 3 days. The bone slices were then removed washed and stained with 1% toluidine blue in 1% sodium borate for 10 seconds followed by extensive washing in double-distilled water. Resorption pits were observed using a light microscope.
Application of mechanical strain to cultured cells
Uniaxial and homogeneous mechanical strain was generated by a specially designed four-point bending device described previously^[14]. Osteoclasts were seeded at the density of 10⁴/cm₂ in the cell culture dishes and randomly divided into three groups. One group of cells were subjected to mechanical strain of 2 500 με at 0.5 Hz for 1 hour a day; another group of cells were subjected to mechanical strain of 5 000 με at 0.5 Hz for 1 hour a day; the third group of cells were not subjected to any mechanical strain as a control group. The mechanical loadings continued for 3 days.
Hoechst staining
Morphological changes in the nuclear chromatin of apoptotic cells were detected by staining with Hoechst 33258. After mechanical strain, osteoclasts were collected, washed twice with PBS and fixed with 4% paraformaldehyde for 10 minutes. Then the cells were washed with PBS and stained with Hoechst 33258 for 10 minutes. At last, the cells were observed by fluorescence microscopy (Olympus, Tokyo, Japan) and the images were captured.
Flow cytometric analysis of cell apoptosis
The apoptosis was measured through Annexin V-FITC apoptosis detection kit as described by the manufacture’s instruction. After mechanical strain, osteoclasts were collected, washed twice with PBS, gently resuspended in Annexin V binding buffer and incubated with Annexin V-FITC/PI in dark for 15 minutes and analyzed by flow cytometry.
Measurement of mitochondrial membrane potential
Mitochondrial activity was assessed in osteoclasts using the fluorescent probe, 5,5’,6,6’-tetrachloro- 1,1’,3, 3’-tetraethylbenzimidazolcarbocyanine iodide (JC-1). After mechanical strain, osteoclasts were incubated with JC-1 working fluid at 37 ℃ and 5% CO2 for 15 minutes, collected, washed with the staining binding buffer and analyzed by flow cytometry.
Main outcome measures
TRAP staining, bone resorption lacunae, Hoechst staining, Annexin V-FITC/PI binding assay and mitochondrial membrane potential were determined.
Statistical analysis
Data are expressed as mean ± SD. Statistical analysis was performed with analysis of variance using SPSS 13.0 software (SPSS Inc). P < 0.05 was considered statistically significant.

Although it has been reported that mature osteoclasts can be isolated from the bone successfully, the poor quantity, low purity and short survival time still cannot be satisfactory^[15-17]. To solve this problem, researchers have developed different methods to form osteoclast-like cells in vitro^[18]. In this study, the culture medium containing RANKL and macrophage-colony stimulating factor was applied to stimulate the murine monocyte-macrophage lineage RAW264.7 cells. As shown in this study, sufficient TRAP-positive multinucleated cells were obtained, and these cells were able to form resorption lacunae. These results indicated that we successfully formed osteoclasts for experiments in vitro.

It has been confirmed that osteocytes are the main cells which sense and respond to mechanical stimuli^[19]. Being the most abundant type in the bone, osteocytes disperse themselves throughout the mineralized matrix, and communicate with each other via slender cell processes connected by gap junctions[20]. Besides osteocytes, osteoblasts and osteoclasts also have the ability to respond to mechanical stimuli. The facts that mechanical forces directly affecte osteoclasts have been reported in several studies^[8-10].

Mechanical strain plays an important role in bone cells. Cellular reactions were also strongly affected by the mechanical stimulation parameters, such as strain kind, magnitude, frequency and time course. We chose the strain magnitudes of 2 500 με and 5 000 με according to Frost’s theory. The 100-2 500 microstrain span includes the adapted and mild overload windows, and these physiological strains increase bone mass and strength. The 3 000-25 000 microstrain span is called pathologic overload window or threshold range, where microdamage appears and the formation of woven bone is evoked. So, we chose 2 500 με as a representative of physiological stimulus, and 5 000 με as a representative of pathologic stimulus.

It has been recognized that mechanical forces play an important role in the regulation of cell apoptosis, and many types of cells have been reported involved. However, it has never been reported whether osteoclast apoptosis is affected by mechanical forces.

In our study, we concluded that physiological strains could inhibit osteoclast apoptosis, while the pathologic ones have nearly no impact upon osteoclast apoptosis. If the strains are physiological strains and the microdamages are under control, osteoclasts are activated and their apoptosis processes are delayed. Then, bone resorption happens and the inner microdamage is repaired by osteoblasts. This is the process of bone remodeling. If the strains are pathologic strains and repairing the microdamages takes the risk of fracture, osteoclasts are not activated and their apoptosis processes are not delayed. Osteoblasts may form the new bone rapidly to adapt the pathologic strains, leaving the inner microdamages unrepaired. However, although the rapidly formed new bone increases bone mass and strength, they do not compact the bone, but woven bones which may be replaced in future. This is the process of bone modeling. So, our results further confirmed and partially developed Frost’s theory.

In conclusion, we found that physiological strain inhibits osteoclast apoptosis, while the pathologic strain has nearly no impact upon osteoclast apoptosis. We hope our study may provide a basis for the further understanding of bone remodeling and preventing related bone diseases.

1  采用巨噬细胞集落刺激因子和破骨细胞分化因子诱导小鼠单核细胞RAW264.7，抗酒石酸酸性磷酸酶染色和骨吸收实验成功诱导出了破骨细胞。

2  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。

3  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。

1中文内容介绍：

在骨重建的过程中，破骨细胞的主要功能是参与骨的破坏和吸收。破骨细胞执行完功能后，除少部分转移到其他部位以外，大部分细胞会在一定信号提示下发生凋亡。已有大量文献报道力学应变可以直接作用于破骨细胞，并产生生物学效应。然而，力学应变对破骨细胞的凋亡是否存在调节作用尚未有过报道。

文章利用小鼠RAW264.7细胞，添加细胞因子RANKL和M-CSF诱导产生破骨细胞。实验首先对破骨细胞诱导是否成功进行了鉴定，使用的是TRAP染色和骨吸收陷窝观察的方法。TRAP是抗酒石酸酸性磷酸酶，为破骨细胞特异性表达，成熟破骨细胞有多个核，TRAP染色呈现胞质为红色。骨吸收陷窝也是鉴定破骨细胞的重要方法之一。实验结果显示诱导成功了有活性的破骨细胞。

生物力学家通常用微应变（microstrain，με）表示骨感受的力学刺激。根据Frost的力学稳态理论，骨感受到小于50με为废用状态，引起骨丢失；100-2500με为生理强度的应变，维持骨量或者轻微促进成骨；3000-25000με为病理强度的应变，骨量增加但以编制骨为主。大于25000με将导致骨折。

选择了加载强度2500με和5000με来研究生理强度和病理强度应变对破骨细胞凋亡的影响，0με作为对照组。使用的是四点弯曲基底应变加载装置，加载3d，1h/d。选择了hoechst染色、Annexin/PI结合实验和线粒体膜电位测定实验来作为检测细胞凋亡的方法。凋亡的细胞呈现hoechst染色时细胞核强烈荧光，Annexin/PI结合实验Annexin阳性、PI阴性，线粒体膜电位下降。实验结果显示，生理强度的应变抑制破骨细胞凋亡，病理强度的应变下，破骨细胞凋亡和对照组相比无显著差异。

2国内外同类研究比较：

文章的创新性体现在国内外从未有报道称做过力学刺激影响破骨细胞凋亡的研究。国内外有过研究力学刺激对破骨细胞骨吸收活性和某些破骨细胞特异性表达产物影响的报道，也有过研究化学药物影响破骨细胞凋亡的报道。但作为力学刺激影响破骨细胞凋亡的研究，尚属首次报道。

3实验设计与文章构思特点：

课题为国家自然科学基金重点资助(10832012)项目，“骨重建的生物力学机制及其在骨相关疾病诊治中的应用”。鉴于破骨细胞在骨重建过程中的重要作用，课题选择了拉伸应变对破骨细胞凋亡的影响进行了研究。

面对破骨细胞来源不足的问题，本课题采用了诱导培养法获取实验所需的破骨细胞，并对该破骨细胞进行了鉴定。在检测破骨细胞凋亡的实验中，课题选择了hoechst染色、Annexin结合实验和线粒体膜电位测定实验几种重要的凋亡检测方法。拉伸强度参数的选择参考了Frost的“力学稳态理论”。

	阅读次数
	全文
	摘要