Effects of dynamic pressure on the proliferation and mechanical properties of rabbit adipose mesenchymal stem cells transfected with insulin-like growth factor-1

doi:10.3969/j.issn.2095-4344.3514

Abstract

Abstract: BACKGROUND: Adipose mesenchymal stem cells are currently recognized as excellent seed cells for tissue engineering cartilage. Gene transfection technology can effectively induce them to differentiate into cartilage. The bioreactor is used to simulate the mechanical environment in vivo. It is a new idea for the majority of scholars to explore the construction of tissue engineering cartilage in vitro.
OBJECTIVE: To investigate the effects of cyclic dynamic compressive stress combined with insulin-like growth factor-1 gene transfection on the proliferation and elastic modulus of rabbit adipose mesenchymal stem cells implanted in chitosan/gelatin scaffold.
METHODS: Rabbit adipose mesenchymal stem cells were transfected with pcDNA3.1-IGF-1 gene mediated by liposome. The stable transfected cell lines were screened by G418. The adipose mesenchymal stem cells transfected with or without insulin-like growth factor-1 gene were inoculated in chitosan/gelatin scaffold at the density of 5×1010 L-1 for 2 days, and cultured under dynamic pressure (2% at 1 Hz, 4 hours per day) or static culture conditions for 7 days, respectively. The morphological changes of the cell/scaffold complex were observed by scanning electron microscope, Masson trichrome staining and alcian blue staining. The cell proliferation curve was drawn by MTT assay. The cell proliferation efficiency and distribution were evaluated by CM-Dil fluorescence-labeling method, and the content of total glycosaminoglycan was quantitatively determined by DMMB. The differences of type II collagen among different groups were compared with real time PCR. Compressive mechanical properties of the cell/scaffold constructs were assessed using a BioDynamicTM mechanical tester, and the corresponding elastic modulus was calculated.
RESULTS AND CONCLUSION: Dynamic pressure combined with insulin-like growth factor-1 transfection could significantly improve the cell proliferation ability of the cell/scaffold complex; the cell distribution was more uniform; glycosaminoglycan and collagen secretion in the cartilage-specific extracellular matrix were increased; the expression levels of type II collagen were up-regulated; and the mechanical properties were significantly improved. The cell proliferation and elastic modulus of insulin-like growth factor-1 group were better than those of single pressure group, but the distribution of cells in scaffolds was more uniform under dynamic pressure. The results indicate that both dynamic pressure and insulin-like growth factor-1 gene transfection can significantly improve the proliferation and mechanical properties of rabbit adipose mesenchymal stem cells; the two have synergistic effect.

Key words: stem cells, adipose mesenchymal stem cells, pressure, chondrocytes, insulin-like growth factor-1, elasticity modulus, tissue engineering

CLC Number:

Zhang Chuanhui, Li Jianjun, Yang Jun. Effects of dynamic pressure on the proliferation and mechanical properties of rabbit adipose mesenchymal stem cells transfected with insulin-like growth factor-1[J]. Chinese Journal of Tissue Engineering Research, 2021, 25(13): 1982-1987.

Figures/Tables 9

References

[1] ROSADI I, KARINA K, ROSLIANA I, et al. In vitro study of cartilage tissue engineering using human adipose-derived stem cells induced by platelet-rich plasma and cultured on silk fibroin scaffold. Stem Cell Res Ther. 2019; 10(1):369.
[2] BONASSAR LJ, GRODZINSKY AJ, FRANK EH, et al. The effect of dynamic compression on the response of articular cartilage to insulin-like growth factor-I. J Orthop Res. 2001;19(1):11-17.
[3] BRYANT SJ, NICODEMUS GD, VILLANUEVA I. Designing 3D photopolymer hydrogels to regulate biomechanical cues and tissue growth for cartilage tissue engineering. Pharm Res. 2008;25(10):2379-2386.
[4] YANG J, ZHANG Y, ZANG G, et al. Adipose-derived stem cells improve erectile function partially through the secretion of IGF-1, bFGF, and VEGF in aged rats. Andrology. 2018;6(3):498-509.
[5] OH SJ, CHOI KU, CHOI SW, et al. Comparative Analysis of Adipose-Derived Stromal Cells and Their Secretome for Auricular Cartilage Regeneration. Stem Cells Int. 2020;2020:8595940.
[6] ROBALLO KCS, DA SILVEIRA JC, BRESSAN FF, et al. Neurons-derived extracellular vesicles promote neural differentiation of ADSCs: a model to prevent peripheral nerve degeneration. Sci Rep. 2019;9(1):11213.
[7] MORSCHEID S, REY-RICO A, SCHMITT G, et al. Therapeutic Effects of rAAV-Mediated Concomittant Gene Transfer and Overexpression of TGF-β and IGF-I on the Chondrogenesis of Human Bone-Marrow-Derived Mesenchymal Stem Cells. Int J Mol Sci. 2019;20(10):2591.
[8] 杨强,丁晓明,徐宝山,等.取向性丝素蛋白支架复合脂肪干细胞体外构建组织工程软骨[J].中华骨科杂志,2015,35(12):1235-1242.
[9] AN C, CHENG Y, YUAN Q, et al. IGF-1 and BMP-2 induces differentiation of adipose-derived mesenchymal stem cells into chondrocytes-like cells. Ann Biomed Eng. 2010;38(4):1647-1654.
[10] 张传辉,杨军,李建军.调控脂肪间充质干细胞成软骨分化基因Sox-9和低氧诱导因子1α的表达[J].中国组织工程研究,2016,20(45):6766-6773.
[11] WANG X, ZHANG J, CUI W, et al. Composite Hydrogel Modified by IGF-1C Domain Improves Stem Cell Therapy for Limb Ischemia. ACS Appl Mater Interfaces. 2018;10(5):4481-4493.
[12] 赵永亮,冯军宇,王刚,等.周期性动态压力对海藻酸钠培养间充质干细胞成软骨分化的作用[J/CD].中华关节外科杂志(电子版),2016,10(4): 413-418.
[13] OKUBO R, ASAWA Y, WATANABE M, et al. Proliferation medium in three-dimensional culture of auricular chondrocytes promotes effective cartilage regeneration in vivo. Regen Ther. 2019;11:306-315.
[14] SAWATJUI N, LIMPAIBOON T, SCHROBBACK K, et al. Biomimetic scaffolds and dynamic compression enhance the properties of chondrocyte- and MSC-based tissue-engineered cartilage. J Tissue Eng Regen Med. 2018; 12(5):1220-1229.
[15] 张传辉,杨军,李建军,等.兔脂肪干细胞体外构建组织工程软骨：负载胰岛素样生长因子1基因的作用[J].中国组织工程研究与临床康复, 2010,14(28):5131-5135.
[16] PRAXENTHALER H, KRÄMER E, WEISSER M, et al. Extracellular matrix content and WNT/β-catenin levels of cartilage determine the chondrocyte response to compressive load. Biochim Biophys Acta Mol Basis Dis. 2018; 1864(3):851-859.
[17] 张云松,高建华,鲁峰,等.荧光活性染料DiI标记人脂肪干细胞[J].中国组织工程研究与临床康复,2007,11(15):2897-2899.
[18] 王一兵,张芮,冯永强,等.羰花青荧光染料示踪在大鼠胚胎皮肤成纤维细胞移植中的应用[J].中华医学杂志,2009,89(14):986-989.
[19] AZIZ AH, ECKSTEIN K, FERGUSON VL, et al. The effects of dynamic compressive loading on human mesenchymal stem cell osteogenesis in the stiff layer of a bilayer hydrogel. J Tissue Eng Regen Med. 2019;13(6):946-959.
[20] 余晓明,孟昊业,孙振,等.生物反应器中的力学刺激促进组织工程软骨再生[J].中国组织工程研究,2016,20(2):185-190.
[21] ANDERSON DE, JOHNSTONE B. Dynamic Mechanical Compression of Chondrocytes for Tissue Engineering: A Critical Review. Front Bioeng Biotechnol. 2017;5:76.
[22] KOZHEMYAKINA E, LASSAR AB, ZELZER E. A pathway to bone: signaling molecules and transcription factors involved in chondrocyte development and maturation. Development. 2015;142(5):817-831.
[23] CHEN CH, KUO CY, CHEN JP. Effect of Cyclic Dynamic Compressive Loading on Chondrocytes and Adipose-Derived Stem Cells Co-Cultured in Highly Elastic Cryogel Scaffolds. Int J Mol Sci. 2018;19(2):370.
[24] JIMI E, FEI H, NAKATOMI C. NF-κB Signaling Regulates Physiological and Pathological Chondrogenesis. Int J Mol Sci. 2019;20(24):6275.
[25] YAN C, WANG Y, SHEN XY, et al. MicroRNA regulation associated chondrogenesis of mouse MSCs grown on polyhydroxyalkanoates. Biomaterials. 2011;32(27): 6435-6444.
[26] AKIYAMA H. Transcriptional regulation in chondrogenesis by Sox9. Clin Calcium. 2011;21(6):845-851.
[27] 李昆朋,张雯,李忠,等.动态压应力促进软骨前体干细胞Sox9 和细胞外基质表达[J].中华生物医学工程杂志,2016,22(3):189-193.
[28] ZHANG Y, TANG CL, CHEN WJ, et al. Dynamic compression combined with exogenous SOX-9 promotes chondrogenesis of adipose-derived mesenchymal stem cells in PLGA scaffold. Eur Rev Med Pharmacol Sci. 2015;19(14):2671-2678.