In vitro osteogenic differentiation of bone marrow mesenchymal stem cells from ovariectomied osteoporotic rats

doi:10.3969/j.issn.2095-4344.2013.36.007

Abstract

Abstract:

BACKGROUND: Cytological studies show that bone marrow mesenchymal stem cells play an important role in postmenopausal osteoporosis mechanism.
OBJECTIVE: To study the osteogenic differentiation in vitro of bone marrow mesenchymal stem cells from ovariectomied osteoporotic rats.
METHODS: The osteoporotic animal model was established by performing ovariectomy in the 6-month-old female Sprague-Dawley rats. There were four groups: bone marrow mesenchymal stem cells control group, bone marrow mesenchymal stem cells osteoporosis group, bone marrow mesenchymal stem cells osteogenic induction group and oseogenesis induction group. Bone marrow mesenchymal stem cells were isolated from the rats of control group and oseogenesis induction group by means of the whole bone marrow adherence method and cultured to the 3rd generation. Then the bone marrow mesenchymal stem cells were used in all the experiments. Cell morphology was observed under the inverted phase contrast microscope, cell cycle and proliferation index of bone marrow mesenchymal stem cells were detected by flow cytometry. After osteogenic induction, the expression level of alkaline phosphatase was detected, and the fornation of calcium nodes of bone marrow mesenchymal stem cells were marked by alizarin red staining.
RESULTS AND CONCLUSION: The cells in the osteogenic induction group and oseogenesis induction group had the morphology of osteobalsts, and the change of morphology of the cells in the oseogenesis induction group was relatively tardiness. The proliferation index in the control group was higher than that in the osteoporosis group (P < 0.05); expression level of alkaline phosphatase in the osteogenic induction group was significantly higher than that in the oseogenesis induction group (P < 0. 05), and the control group was significantly higher than the oseogenesis group (P < 0.05). The alizarin red staining of the cells in the osteogenic induction group was positive, while negative in the control group and the oseogenesis group; the staining in the osteogenic induction group was stronger than that in the oseogenesis induction group. These findings indicate that both the proliferative potential and the osteogenic potential of bone marrow mesenchymal stem cells from the ovariectomized osteoporotic rats are decreased, which may be related with the ostoeporosis pathogensis of ovariectomied rats.

Key words: stem cells, mesenchymal stem cells, osteoporosis, ovariectomy, alkaline phosphatase

CLC Number:

R394.2

Wang Yun, Bao Xiao-ming, Hou Yong-xin, Li Jun, Zhang Min. In vitro osteogenic differentiation of bone marrow mesenchymal stem cells from ovariectomied osteoporotic rats[J]. Chinese Journal of Tissue Engineering Research, 2013, 17(36): 6423-6429.

Figures/Tables 5

References

[1] Adams JE. Advances in bone imaging for osteoporosis. Nat Rev Endocrinol. 2012; 9(1): 28-42.
[2] Coipeau P,Rosset P,Langonne A,et al.Impaired differentiation potential of human trabecular bone mesenchymal stromal cells from elderly patients.Cytotherapy.2009; 11: 584-594.
[3] Wagner W, Wein F, Seckinger A, et al. Comparative characteristics of mesenchymal stem cells from human bone marrow, adipose tissue, and umbilical cord blood. Exp Hematol. 2005;33(11): 1402-1416.
[4] Yoshimura H, Muneta T, Nimura A, et al. Comparison of rat mesenchymal stem cells derived from bone marrow, synovium, periosteum, adipose tissue, and muscle. Cell Tissue Res.2007;327 (3): 449- 462.
[5] Srouji S，Livne E. Bone marrow stem cells and biological scaffold for bone repair in aging and disease. Mech Ageing Dev.2005；126( 2): 281-287.
[6] Zhu X, Du J, Liu G. The comparison of multilineage differentiation of bone marrow and adipose-derived mesenchymal stem cells. Clin Lab. 2012;58: 897-903.
[7] 中华人民共和国科学技术部.关于善待实验动物的指导性意见. 2006-09-30.
[8] 李素萍. 骨质疏松动物模型的研究现状[J].中国组织工程研究与临床康复, 2011, 15(20): 3767-3770.
[9] Chachra D, Lee JM, Kasram, et al. Differential effects of ovariectomy on the mechanical properties of cortical and cancellous bones in rat femora and vertebrae. Biomed Sci Instrum.2000;36: 123-128.
[10] Liu Y, Wang L, Fatahi R, et al. Isolation of murine bone marrow derived mesenchymal stem cells using Twist2 Cre transgenic mice. Bone. 2010;47: 916-925.
[11] Javazon EH, Beggs K, Flake AW. Mesenchymal stem cells: paradoxes of passaging. Exp Hematol.2004;32(5): 414-425.
[12] Delmas P. Clinical usefulness of biochemical markers of bone remodeling in osteoporosis. Rev Prat. 1995;45(9):1102-1105.
[13] Bala Y, Farlay D, Chapurlat RD, et al.Modifications of bone material properties in postmenopausal osteoporotic women long-term treated with alendronate. Eur J Endocrinol. 2011; 165(4): 647-655.
[14] Pinzon R. The clinical profile and risk factors of postmenopausal lumbar osteoporosis. International Journal of Rheumatic Diseases, 2010,13:171.
[15] 中华医学会骨质疏松和骨矿盐疾病分会.原发性骨质疏松症诊治指南(2011年).中华骨质疏松和骨矿盐疾病杂志, 2011, 4: 2-17.
[16] Shil AB, Greer JR. Screening strategies for treatment of osteoporosis in long-term care residents. Am Geriatr Soc. 2012;60(12): 2383-2384.
[17] 王强,赵兵,李超,等.干细胞移植治疗骨质疏松[J].中华骨质疏松和骨矿盐疾病杂志,2012, 5(3): 225-229.
[18] Kumar S, Chanda D, Ponnazhagan S. Therapeutic potential of genetically modified mesenchymal stem cells. Gene Ther. 2008;15(10):711-715.
[19] Rosen CJ, Bouxsein ML.Mechanism of disease: Is osteoporosis the obesity of bone? Nat Clin Pract Rheumatol. 2006;2(1):35-43.
[20] Astudillo P，Rios S，Pastenes L，et al.Increased adipogenesis of osteoporotic human-mesenchymal stem cells ( MSCs) characterizes by impaired leptin action.Cell Biochem.2008; 103: 1054-1065.
[21] Kretlow JD, Jin YQ, Liu W, et al. Donor age and cell passage affects differentiation potential of murine bone marrowderived stem cells. BMC Cell Biol. 2008;9: 60.
[22] Zhou S, Greenberger JS, Epperly MW, et al. Age-related intrinsic changes in human bone-marrow-derived mesenchymal stem cells and their to osteoblasts. Aging Cell.2008 ;7: 335-343.
[23] Rodriguez JP, Montecinos L, Rios R, et al. Mesenchymal stem cells from osteoporotic patients produce a type I collagen- deficient extracellular matrix favoring adipogenic differentiation. Cell Biochem.2000;79: 557-565.
[24] Peng S, Xia R, Fang H, et al. Effect of epimedium-derived phytoestrogen on bone turnover and bone microarchitecture in OVX-induced osteoporotic rats. J Huazhong Univ Sci Technolog Med Sci. 2008;28(2): 167-170.
[25] Tahompson D，Simmons HA，Pirie CM，et al.FDA guidelines and animal models for osteopomsis.Bone.1995;17(2): 125-133.
[26] 郑良朴,李远志,胡海霞,等.不同月龄大鼠去卵巢骨质疏松模型的比较[J].福建中医药,2009,40(2):50-51.
[27] 蔡鹏,朱绍兴,苏一鸣,等.全骨髓贴壁法分离培养大鼠骨髓间充质干细胞及其诱导分化[J].中国组织工程研究与临床康复, 2009, 13(36):7073-7077.
[28] Schonau E, Rauch F. Markers of bone and collagen metabolism-problems and perspectives in paediatrics. Horm Res.1997;48: 50-59.
[29] See EY, Toh SL, Goh JC. Multilineage potential of bone-marrow-derived mesenchymal stem cell cell sheets: implications for tissue engineering. Tissue Eng Part A. 2010; 16(4): 1421-1431.
[30] 张英,袁月,孙富丽,等.成骨细胞胞内胞外碱性磷酸酶含量比较[J]. 中国医科大学学报, 2011, 40(10): 874-884.
[31] Imene B, Suganya S, William TW. Autologous bone marrow derived rat mesenchymal stem cells promote PDX-1 and insulin expression in the islets,alter T cell cytokine pattern and preserve regulatory T cells in the periphery and induce sustained normoglycemia. Autoimmun.2009;32(1)33-42.
[32] Farley JR, Stilt-Coflfing B. Apoptosis may determine the release of skeletal alkaline phosphatase activity from human osteoblast-line cell. Calcif Tissue Int. 2001;6868(1): 43-50.
[33] Ovesen O, Riegels P, Lindequist S, et al. The diagnostic value of digital substractiotn, arthrography and vadionuclide bone scan in revision hip arthroplasty. Jarthrolasty.2003;18 (6): 735- 740.
[34] Versluis RG, Papapoulos SE, Zwinderman AH, et al.Clinical risk factors as predictors of postmenopausal osteoporosis in general practice. Br J Gen Pract. 2001;51: 806-810.
[35] Tondreau T, Dejeneffe M, Meuleman N, et al. Gene expression pattern of functional neuronal cells derived from human bone marrow mesenchymal stromal cells. BMC Genomics.2008;11(9): 166-171.
[36] Yamaza T, Miura Y, Bi Y, et al. Pharmacologic stem cell based intervention as a new approach to osteoporosis treatment in rodents. Plos One.2008;3(7): 2615-2619.