Hypoxic precondition rescues osteogenic potential of bone marrow mesenchymal stem cells derived from ovariectomized rats

doi:10.12307/2022.329

Abstract

Abstract: BACKGROUND: Hypoxia is one of the main driving forces regulating the angiogenesis-osteogenesis differentiation process. Hypoxia treatment of cells before differentiation is considered to maintain the stemness and enhance the osteogenic potential of stem cells. However, it is not clear whether hypoxic precondition could improve the osteogenic potential of bone marrow mesenchymal stem cells derived from osteoporosis rat.
OBJECTIVE: To investigate the effects of hypoxic precondition on the osteogenic potential of bone marrow mesenchymal stem cells derived from osteoporosis rat and the role of hypoxia inducible factor-1α.
METHODS: Ovariectomized SD rat models were established. Bone marrow mesenchymal stem cells were isolated from femurs of female rats in ovariectomized group and sham operation group. The bone marrow mesenchymal stem cells were divided into three groups. In the normal group, the bone marrow mesenchymal stem cells extracted from the sham rats were cultured in the incubator with normal oxygen concentration (21% O2). In the osteoporosis group, the bone marrow mesenchymal stem cells extracted from the ovariectomized rats were cultured in the incubator with normal oxygen concentration (21% O2). In the hypoxia group, the bone marrow mesenchymal stem cells extracted from the ovariectomized rats were cultured in the hypoxic incubator with hypoxic oxygen concentration (5% O2). CCK-8 assay was used to assess the proliferation capacity of bone marrow mesenchymal stem cells by measuring absorbance values at 1, 3, 5, and 7 days. After 72 hours of incubation in hypoxic environment, cells were moved to normal incubator for osteogenic differentiation with other two groups for 14 days. Alizarin red staining and qRT-PCR were utilized to detect osteogenic related gene expression levels. Finally, the bone marrow mesenchymal stem cells of the osteoporosis group were transfected with small interfering RNA to silence the expression of hypoxia inducible factor-1α, cultured in a hypoxic environment for 72 hours, and transferred to a normal incubator for osteogenic differentiation for 14 days. The changes in osteogenic capacity were observed after silencing hypoxia inducible factor-1α. After the silence of HIF-1α, the expressions of HIF-1α and osteogenic markers were furthered detected.
RESULTS AND CONCLUSION: (1) The proliferation ability of bone marrow mesenchymal stem cells in ovariectomized rats decreased, and the osteogenic differentiation ability was impaired. (2) Hypoxia successfully accelerated the proliferation of bone marrow mesenchymal stem cells, promoted the deposition of matrix mineralization and the expression of osteogenic related genes. (3) Positive effects of hypoxic precondition on osteogenic potential after the silence of hypoxia inducible factor-1α were eliminated. (4) Results suggested that hypoxic precondition could improve the proliferation and osteogenic potential of bone marrow mesenchymal stem cells of ovariectomized rats and hypoxia inducible factor-1α activated by hypoxia may play an important effect on osteogenic differentiation.

Key words: stem cells, bone marrow mesenchymal stem cells, osteoporosis, hypoxia, cell proliferation, osteogenic differentiation, hypoxia inducible factor-1α

CLC Number:

Huang Xiaoxiong, Chen Weikai, Liu Tao, Yang Huilin, He Fan. Hypoxic precondition rescues osteogenic potential of bone marrow mesenchymal stem cells derived from ovariectomized rats[J]. Chinese Journal of Tissue Engineering Research, 2022, 26(13): 2034-2039.

Figures/Tables 6

References

[1] EASTELL R, O’NEILL TW, HOFBAUER LC, et al. Postmenopausal osteoporosis. Nat Rev Dis Primers. 2016;2:16069.
[2] BUCKLEY L, HUMPHREY MB. Glucocorticoid-Induced Osteoporosis. N Engl J Med. 2018;379(26):2547-2556.
[3] LIANG C, PENG S, LI J, et al. Inhibition of osteoblastic Smurf1 promotes bone formation in mouse models of distinctive age-related osteoporosis. Nat Commun. 2018;9(1):3428.
[4] CRANE JL, CAO X. Bone marrow mesenchymal stem cells and TGF-beta signaling in bone remodeling. J Clin Invest. 2014;124(2):466-472.
[5] RODRÍGUEZ JP, GARAT S, GAJARDO H, et al. Abnormal osteogenesis in osteoporotic patients is reflected by altered mesenchymal stem cells dynamics. J Cell Biochem. 1999;75(3):414-423.
[6] GRAYSON WL, ZHAO F, BUNNELL B, et al. Hypoxia enhances proliferation and tissue formation of human mesenchymal stem cells. Biochem Biophys Res Commun. 2007;358(3):948-953.
[7] MALDA J, KLEIN TJ, UPTON Z. The roles of hypoxia in the in vitro engineering of tissues. Tissue Eng. 2007;13(9):2153-2162.
[8] ROSOVÁ I, DAO M, CAPOCCIA B, et al. Hypoxic preconditioning results in increased motility and improved therapeutic potential of human mesenchymal stem cells. Stem Cells. 2008;26(8):2173-2182.
[9] SEMENZA GL. Hypoxia-inducible factor 1: master regulator of O2 homeostasis. Curr Opin Genet Dev. 1998;8(5):588-594.
[10] SEMENZA GL. Regulation of mammalian O2 homeostasis by hypoxia-inducible factor 1. Annu Rev Cell Dev Biol. 1999;15:551-578.
[11] BIANCO P, CAO X, FRENETTE PS, et al. The meaning, the sense and the significance: translating the science of mesenchymal stem cells into medicine. Nat Med. 2013;19(1):35-42.
[12] KULAK CA, BORBA VC, JORGETTI V, et al. Skeletal microstructural abnormalities in postmenopausal women with chronic obstructive pulmonary disease. J Bone Miner Res. 2010;25(9):1931-1940.
[13] KJENSLI A, MOWINCKEL P, RYG MS, et al. Low bone mineral density is related to severity of chronic obstructive pulmonary disease. Bone. 2007;40(2):493-497.
[14] ADAS-OKUMA MG, MAEDA SS, GAZZOTTI MR, et al. COPD as an independent risk factor for osteoporosis and fractures. Osteoporos Int. 2020;31(4):687-697.
[15] HU X, YU SP, FRASER JL, et al. Transplantation of hypoxia-preconditioned mesenchymal stem cells improves infarcted heart function via enhanced survival of implanted cells and angiogenesis. J Thorac Cardiovasc Surg. 2008;135(4):799-808.
[16] PARK IH, KIM KH, CHOI HK, et al. Constitutive stabilization of hypoxia-inducible factor alpha selectively promotes the self-renewal of mesenchymal progenitors and maintains mesenchymal stromal cells in an undifferentiated state. Exp Mol Med. 2013;45(9):e44.
[17] YAN Z, SHEN D, LIAO J, et al. Hypoxia Suppresses TGF-B1-Induced Cardiac Myocyte Myofibroblast Transformation by Inhibiting Smad2/3 and Rhoa Signaling Pathways. Cell Physiol Biochem. 2018;45(1):250-257.
[18] PRALL WC, HAASTERS F, HEGGEBÖ J, et al. Mesenchymal stem cells from osteoporotic patients feature impaired signal transduction but sustained osteoinduction in response to BMP-2 stimulation. Biochem Biophys Res Commun. 2013;440(4):617-622.
[19] EZASHI T, DAS P, ROBERTS RM. Low O2 tensions and the prevention of differentiation of hES cells. Proc Natl Acad Sci U S A. 2005;102(13): 4783-4788.
[20] POUYSSÉGUR J, DAYAN F, MAZURE NM. Hypoxia signalling in cancer and approaches to enforce tumour regression. Nature. 2006;441(7092): 437-443.
[21] KE Q, COSTA M. Hypoxia-inducible factor-1 (HIF-1). Mol Pharmacol. 2006;70(5):1469-1480.