动态牵张促进人骨髓间充质干细胞三维培养的成骨分化

Dynamic stretching promotes osteogenic differentiation of human bone marrow mesenchymal stem cells in three-dimensional culture

摘要

目的：探究动态牵张对人骨髓间充质干细胞（human bone marrow mesenchymal stem cells，hBMMSCs）三维培养中成骨分化的促进作用。方法：对载有 hBMMSCs 的三维培养体系进行动态牵张，设置动态牵张的比例（5% 、10%、20%）、频率（0.5 Hz）和时间（2 h/d），以静态培养为对照组；通过胞质荧光染色观察动态牵张引起的hBMMSCs 形变；动态牵张培养 7 d 后，通过细胞活死染色观察动态牵张对 hBMMSCs 活性的影响，通过碱性磷酸酶（alkaline phosphatase，ALP）染色及定量实验，成骨相关基因和蛋白的表达观察动态牵张对 hBMMSCs 成骨分化的影响。结果：成功构建动态牵张三维培养 hBMMSCs，可实现不同持续时间、频率和动态牵张的比例；动态牵张使得hBMMScs 发生形变，牵张比例越大，细胞形变越明显，从圆形向扁椭圆形变化；动态牵张培养7 d 后，静态对照组和动态牵张各实验组中 hBMMSCs 基本为绿染的活细胞，仅有个别红染的死细胞，活细胞比例各组之间的差异无统计学意义（P>0.05）；动态牵张各组较静态对照组 ALP 大体染色深，显微镜下 ALP 染色阳性细胞数量多，成骨相关基因和蛋白的表达增加，差异具有统计学意义（P<0.05），其中动态牵张 10% 组 ALP 大体染色最深，阳性细胞数量最多，成骨相关基因和蛋白的表达较静态对照组增加最为显著。结论：动态牵张使得 hBMMSCs 发生形变，对细胞活性无明显影响，能够有效促进 hBMMSCs 成骨向分化。

Abstract

Objective：To explore the promotion of dynamic distraction on osteogenic differentiation of human bone marrow mesenchymal stem cells（hBMMSCs）in three-dimensional culture. Methods：Dy- namic stretching in a three-dimensional culture for hBMMSCs was achieved with proportions set at 5％， 10% ，and 20% ，a frequency of 0.5 Hz ，and a dynamic stretching duration of 2 hours per day.Static culture was used as the control group.Deformation of hBMMSCs induced by dynamic stretching was ob- served through cytoplasmic fluorescence staining.After 7 days of dynamic stretching culture，the impact of dynamic stretching on the viability of hBMMSCs was observed through cell live/dead staining.The effect of dynamic stretching on the osteogenic differentiation of hBMMSCs was observed through alkaline phosphatase（ALP）staining and the expression of osteogenic related genes and proteins after 7 days of dynamic stretching culture. Results：Dynamic stretching in a three-dimensional culture for hBMMSCs was successfully constructed，which could achieve different durations，frequencies，and ratios of dynamic stretching.Dynamic stretching led to deformation of hBMMSCs，and the greater the stretching ratio，the more pronounced the cell deformation，transitioning from a circular to a flat oval shape.After 7 days of dynamic stretching culture，hBMMSCs in the static control group and dynamic stretching groups were mostly green stained live cells，with only a few red stained dead cells.The difference in the proportion of live cells between the groups was not statistically significant (P>0.05).The ALP staining in the dynamic stretching group was deeper than that in the static control group，and the number of ALP staining positive cells observed under the microscope was higher.The expression of osteogenic related genes and proteins increased after 7 days of dynamic stretching culture，and the difference was statistically signifi- cant (P<0.05).Among them，the dynamic stretching group with 10% had the deepest ALP staining， the highest number of positive cells，and the most significant increase in the expression of osteogenic re- lated genes and proteins compared with the static control group. Conclusion：Dynamic stretching caused deformation of hBMMSCs without a significant impact on cell viability，and it could effectively promote the osteogenic differentiation of hBMMSCs.

关键词

动态牵张 / 三维培养 / 人骨髓间充质干细胞 / 成骨分化

Key words

Dynamic stretching / Three-dimensional culture / Human bone marrow mesenchymal stem cells / Osteogenic differentiation

参考文献

[1] Hartmann A, Hildebrandt H, Younan Z, et al. Long-term results in three-dimensional,complex bone augmentation procedures with customized titanium meshes[J]. Clin Oral Implants Res, 2022, 33(11):1171-1181.

[2] Khachatryan L, Khachatryan G, Hakobyan G. The treatment of lower jaw defects using vascularized fibula graft and dental im- plants[J]. J Craniofacial Surg, 2018, 29(8):2214-2217.

[3] Fernandez de Grado G, Keller L, Idoux-Gillet Y, et al. Bone sub- stitutes:A review of their characteristics,clinical use,and per- spectives for large bone defects management[J]. J Tissue Eng, 2018, 9: 2041731418776819.

[4] Tang XY, Wu S, Wang D, et al. Human organoids in basic re- search and clinical applications[J]. Sig Transduct Target Ther, 2022, 7: 168.

[5] Kratochvil MJ, Seymour AJ, Li TL, et al. Engineered materials for organoid systems[J]. Nat Rev Mater, 2019, 4(9): 606 - 622.

[6] Zhang J, Griesbach J, Ganeyev M, et al. Long-term mechanical loading is required for the formation of 3D bioprinted functional osteocyte bone organoids[J]. Biofabrication, 2022, 14 (3): 035018.

[7] Boccafoschi F, Mosca C, Ramella M, et al. The effect of mechanical strain on soft(cardiovascular)and hard(bone)tis- sues:Common pathways for different biological outcomes[J]. Cell Adhes Migr, 2013, 7(2):165-173.

[8] Chen X, Yan J, He F, et al. Mechanical stretch induces antioxi- dant responses and osteogenic differentiation in human mesenchy- mal stem cells through activation of the AMPK-SIRT1 signaling pathway[J]. Free Radic Biol Med, 2018, 126:187-201.

[9] Wang L, You X, Lotinun S, et al. Mechanical sensing protein PIEZO1 regulates bone homeostasis via osteoblast-osteoclast crosstalk[J]. Nat Commun, 2020, 11: 282.

[10] Li W, Zhao J, Sun W, et al. Osteocytes promote osteoclastogene- sis via autophagy-mediated RANKL secretion under mechanical compressive force[J]. Arch Biochem Biophys, 2020, 694: 108594.

[11] Xu H, Xia M, Sun L, et al. Osteocytes enhance osteogenesis by autophagy-mediated FGF23 secretion under mechanical tension[J]. Front Cell Dev Biol, 2022, 9: 782736.

[12] Xie C, Liang R, Ye J, et al. High-efficient engineering of osteo- callus organoids for rapid bone regeneration within one month[J]. Biomaterials, 2022, 288: 121741.

[13] Sun J, Chan YT, Ho KWK, et al. ＂Slow walk＂mimetic tensile loading maintains human meniscus tissue resident progenitor cells homeostasis in photocrosslinked gelatin hydrogel[J]. Bioact Mater, 2023, 25:256-272.

[14] Wu S, Wu X, Wang X, et al. Hydrogels for bone organoid con- struction:From a materiobiological perspective[J]. J Mater Sci Technol, 2023, 136:21-31.

[15] Skerry TM. The response of bone to mechanical loading and disuse:Fundamental principles and influences on osteoblast/osteo- cyte homeostasis[J]. Arch Biochem Biophys, 2008, 473 (2): 117-123.

[16] Williams D, Kuipers A, Mukai C, et al. Acclimation during space flight:Effects on human physiology[J]. Can Med Assoc J, 2009, 180(13):1317-1323.

[17] Man J, Graham T, Squires-Donelly G, et al. The effects of micro- gravity on bone structure and function[J]. NPJ Microgravity, 2022, 8 : 9.

[18] Cooper AN, Cunningham CB, Morris JS, et al. Musculoskeletalmass and shape are correlated with competitive ability in male house mice(Mus musculus)[J]. J Exp Biol, 2020, 223(Pt 3): jeb213389.

[19] Fahy N, Alini M, Stoddart MJ. Mechanical stimulation of mesen- chymal stem cells:Implications for cartilage tissue engineering[J]. J Orthop Res, 2018, 36(1):52-63.

[20] Zhang Y, Habibovic P. Delivering mechanical stimulation to cells:State of the art in materials and devices design[J]. Adv Mater, 2022, 34(32): 2110267.

[21] Chen S, Chen X, Geng Z, et al. The horizon of bone organoid:A perspective on construction and application[J]. Bioact Mater, 2022, 18:15-25.

[22] Weivoda MM, Bradley EW. Macrophages and bone remodeling[J]. J Bone＆Mineral Res, 2023, 38(3):359-369.

[23] Yang C, Liu Y, Wang Z, et al. Controlled mechanical loading improves bone regeneration by regulating type H vessels in aS1 Pr1- dependent manner[J]. FASEB J, 2022, 36(10):e22530.

[24] Wu T, Yin F, Wang N, et al. Involvement of mechanosensitive ion channels in the effects of mechanical stretch induces osteogenic differentiation in mouse bone marrow mesenchymal stem cells[J]. J Cell Physiol, 2021, 236(1):284-293.

[25] Wang D, Cai J, Zeng Z, et al. The interactions between mTOR and NF-κB :A novel mechanism mediating mechanical stretch- stimulated osteoblast differentiation[J]. J Cell Physiol, 2021, 236 (6):4592-4603.

[26] Wang H, Yu H, Huang T, et al. Hippo-YAP/TAZ signaling in osteogenesis and macrophage polarization:Therapeutic implications in bone defect repair[J]. Genes Dis, 2023, 10 (6): 2528 - 2539.

[27] Qin L, He T, Chen S, et al. Roles of mechanosensitive channel Piezo1/2 proteins in skeleton and other tissues[J]. Bone Res, 2021, 9: 44.

[28] Fischer C, Milting H, Fein E, et al. Long-term functional and structural preservation of precision-cut human myocardium under continuous electromechanical stimulation in vitro[J]. Nat Commun, 2019, 10: 117.

[29] Mihic A, Li J, Miyagi Y, et al. The effect of cyclic stretch on maturation and 3D tissue formation of human embryonic stem cell- derived cardiomyocytes[J]. Biomaterials, 2014, 35(9): 2798 - 2808.

[30] Abdel Fattah AR, Daza B, Rustandi G, et al. Actuation enhances patterning in human neural tube organoids[J]. Nat Commun, 2021, 12: 3192.

[31] Qian X, Nguyen HN, Song MM, et al. Brain-region-specific organoids using mini-bioreactors for modeling ZIKV exposure[J]. Cell, 2016, 165(5):1238-1254.

基金资助

国家自然科学基金(82271032)

国家自然科学基金(82470943)

海南省自然科学基金优秀青年基金(825YXQN602)

北京大学临床科学家培养计划(BMU2025PYJH011)