Three methods to isolate osteoblasts: stem cell induction, cell line induction and primary isolation

doi:10.3969/j.issn.2095-4344.2017.17.017

Abstract

Abstract:

BACKGROUND: Osteoblasts have become a kind of important seed cells in bone tissue engineering. However, it is difficult to harvest osteoblasts, and the purity and calcification ability of osteoblasts isolated by different methods are inconsistent.
OBJECTIVE: To compare the purity and calcification ability of osteoblasts induced from mouse bone marrow mesenchymal stem cells, MC3T3 cell lines, and cultured primarily from the neonatal mouse cranium.
METHODS: Mouse bone marrow mesenchymal stem cells were isolated by differential adhesion method, and after passaing, passage 3 cells were cultured in osteogenic induction medium for 21 days. MC3T3 cell lines were cultured in osteogenic induction media 1 and 2 for 21 days. Osteoblasts were cultured primarily from neonatal mouse cranium by type I collagenase digestion method. Calcium nodules of osteoblasts obtained by three methods were observed by Alizarin red staining to detect osteogenic activity of cells.
RESULTS AND CONCLUSION: (1) There were average 16.3 calcium nodules per low-power field after osteogenic induction of bone marrow mesenchymal stem cells. (2) There were sparsely distributed calcium nodules in MC3T3 cells after induction with osteogenic induction medium 1, accounting for 1.7 calcium nodules per low-power field, while there were dense calcium nodules in MC3T3 cells after induction with osteogenic induction medium 2, accounting for 44.6 calcium nodules per low-power field. There was a significant difference in the calcium nodule formation ability between the two groups (P < 0.01). (3) After primary culture, there was only 0.6 calcium nodule per low-power field. (4) Except for the insignificant difference between osteogenic induction medium 1 and primary culture groups, there were significant differences in pair-wise comparison of any other two groups. Except the insignificant difference between group I of MC3T3 inducing conditional media and primary culture osteoblasts, there were significant differences in the osteogenic ability between groups (P < 0.01). In conclusion, it is a better method to culture MC3T3 cells in osteogenic induction medium 2 containing dexamethasone, because many uncontrollable factors are involved in the isolation and culture of bone marrow mesenchymal stem cells.

中国组织工程研究杂志出版内容重点：干细胞；骨髓干细胞；造血干细胞；脂肪干细胞；肿瘤干细胞；胚胎干细胞；脐带脐血干细胞；干细胞诱导；干细胞分化；组织工程

Key words: Bone Marrow, Mesenchymal Stem Cells, Osteoblasts, Tissue Engineering

CLC Number:

R394.2

Deng Xiang-yu, Chen Sheng, Shao Zeng-wu, Zheng Dong. Three methods to isolate osteoblasts: stem cell induction, cell line induction and primary isolation[J]. Chinese Journal of Tissue Engineering Research, 2017, 21(17): 2729-2934.

Figures/Tables 1

2.1 小鼠骨髓间充质干细胞形态及成骨诱导结果接种第2天细胞开始贴壁，细胞发亮，折光性强，此时细胞呈长椭圆形，两端有小角，培养至第3代时，细胞呈长梭形，细胞体积较大，核明显(图1A)。以C57小鼠骨髓间充质干细胞成骨诱导培养基培养至第21天时，细胞形态变大，形状不规则，可见细胞核多个，行钙结节茜素红染色，可见大颗粒状红色钙结节，分布较密集，形态不典型(图1B，C)，选取6个孔行钙结节数量统计，平均为16.3个/低倍镜视野。 2.2 MC3T3细胞形态及成骨诱导结果倒置显微镜下可见MC3T3细胞呈长梭形，细胞发亮，折光性强，核明显。细胞接种至24孔板后，以诱导培养基1和诱导培养基2分别培养，至第21天时，细胞呈层状排列生长，茜素红染色后出现红色钙结节，形态各异，大小不一，存在于细胞群中，周围细胞辐射状排列。选取6个孔行钙结节数量统计，诱导培养液1培养后钙结节数量平均为1.7个/低倍镜视野(图2)，诱导培养液2培养后钙结节数量平均为44.6个/低倍镜视野(图3)。两种诱导液相比钙结节形成能力差异有显著性意义(P < 0.01)。 2.3 成骨细胞原代培养及成骨特性小鼠成骨细胞进行吉姆萨染色后倒置显微镜观察，可见细胞折光性强，胞质染淡紫色，细胞胞核染蓝紫色，细胞形态各异，触角多，向四周延伸(图4)。钙结节染色后可见稀疏钙结节形成，选取6个孔行钙结节数量统计，平均为0.6个/低倍镜视野(图5)。 2.4 各种方法钙结节形成能力比较 3种培养或诱导方法均可得到具有成骨特性的成骨细胞，除MC3T3诱导液1诱导组与原代成骨细胞之间相比无显著差异外，其余两两相比成骨能力差异均有显著性意义(P < 0.01)，3种操作方法实践可行，见图6。"

References

[1] Corrado A, Neve A, Macchiarola A, et al. RANKL/OPG ratio and DKK-1 expression in primary osteoblastic cultures from osteoarthritic and osteoporotic subjects.J Rheumatol. 2013; 40(5):684-694.

[2] Golden D, Saria EA, Hansen MF. Regulation of Osteoblast Migration Involving Receptor Activator of Nuclear Factor-kappa B (RANK) Signaling. J Cell Physiol. 2015; 230(12):2951-2960.

[3] Drager J, Ramirez-GarciaLuna JL, Kumar A, et al. Hypoxia Biomimicry to Enhance Monetite Bone Defect Repair. Tissue Eng Part A. 2017 May 31. [Epub ahead of print]

[4] Sun C, Liu F, Cen S, et al. Tensile strength suppresses the osteogenesis of periodontal ligament cells in inflammatory microenvironments. Mol Med Rep. 2017 May 29. [Epub ahead of print]

[5] Zhang J, Yu X, Yu Y, et al. MicroRNA expression analysis during FK506-induced osteogenic differentiation in rat bone marrow stromal cells. Mol Med Rep. 2017 May 31. [Epub ahead of print]

[6] Luo G, Xu B, Huang Y. Icariside II promotes the osteogenic differentiation of canine bone marrow mesenchymal stem cells via the PI3K/AKT/mTOR/S6K1 signaling pathways. Am J Transl Res. 2017;9(5):2077-2087.

[7] Morsczeck C, Reichert TE. The dexamethasone induced osteogenic differentiation of dental follicle cells. Histol Histopathol. 2017:11907.

[8] Wei K, Xie Y, Chen T, et al. ERK1/2 signaling mediated naringin-induced osteogenic differentiation of immortalized human periodontal ligament stem cells. Biochem Biophys Res Commun. 2017 May 26. [Epub ahead of print]

[9] Genchi GG, Sinibaldi E, Ceseracciu L, et al. Ultrasound- activated piezoelectric P (VDF-TrFE)/boron nitride nanotube composite films promote differentiation of human SaOS-2 osteoblast-like cells. Nanomedicine. 2017 May 26. [Epub ahead of print]

[10] Jayakumar P, Di Silvio L. Osteoblasts in bone tissue engineering. Proc Inst Mech Eng H. 2010;224(12):1415-1440.

[11] Yin X, Chen Z, Liu Z, et al. Tissue transglutaminase (TG2) activity regulates osteoblast differentiation and mineralization in the SAOS-2 cell line. Braz J Med Biol Res. 2012;45(8): 693-700.

[12] Makowski AJ, Uppuganti S, Wadeer SA, et al. The loss of activating transcription factor 4 (ATF4) reduces bone toughness and fracture toughness. Bone. 2014;62:1-9.

[13] Guañabens N, Gifre L, Peris P. The role of Wnt signaling and sclerostin in the pathogenesis of glucocorticoid-induced osteoporosis. Curr Osteoporos Rep. 2014;12(1):90-97.

[14] Li K, Yan J, Wang C, et al. Graphene modified titanium alloy promote the adhesion, proliferation and osteogenic differentiation of bone marrow stromal cells. Biochem Biophys Res Commun. 2017 May 23. [Epub ahead of print]

[15] Cai X, Ten Hoopen S, Zhang W, et al. Influence of highly porous electrospun PLGA/PCL/nHA fibrous scaffolds on the differentiation of tooth bud cells in vitro. J Biomed Mater Res A. 2017 May 24. [Epub ahead of print]

[16] Deng Z, Han H, Yang J, et al. Fabrication and Characterization of Carbon Fiber-Reinforced Nano-Hydroxyapatite/Polyamide46 Biocomposite for Bone Substitute. Med Sci Monit. 2017;23:2479-2487.

[17] Wang X, Li G, Liu Y, et al. Biocompatibility of biological material polylactic acid with stem cells from human exfoliated deciduous teeth. Biomed Rep. 2017;6(5):519-524.

[18] Zambuzzi WF, Fernandes GV, Iano FG, et al. Exploring anorganic bovine bone granules as osteoblast carriers for bone bioengineering: a study in rat critical-size calvarial defects. Braz Dent J. 2012;23(4):315-321.

[19] Premnath P, Tan B, Venkatakrishnan K. Direct patterning of free standing three dimensional silicon nanofibrous network to facilitate multi-dimensional growth of fibroblasts and osteoblasts. J Biomed Nanotechnol. 2013;9(11):1875-1881.

[20] Gao A, Hang R, Huang X, et al. The effects of titania nanotubes with embedded silver oxide nanoparticles on bacteria and osteoblasts. Biomaterials. 2014;35(13): 4223-4235.

[21] Katsuyama M, Demura M, Katsuyama H, et al. Genistein and menaquinone-4 treatment-induced alterations in the expression of mRNAs and their products are beneficial to osteoblastic MC3T3-E1 cell functions. Mol Med Rep. 2017 May 25. [Epub ahead of print]

[22] Li JY, Liu SG, Xiao GN, et al. Fibroblast growth factor receptor 1 propagates estrogen and fluid shear stress driven proliferation and differentiation response in MC3T3-E1 cells. Mol Biol (Mosk). 2017;51(2):342-355.

[23] Yu C, Zhuang J, Dong L, et al. Effect of hierarchical pore structure on ALP expression of MC3T3-E1 cells on bioglass films. Colloids Surf B Biointerfaces. 2017;156:213-220.

[24] Gapski R, Martinez EF. Behavior of MC3T3-E1 Osteoblastic Cells Cultured on Titanium and Zirconia Surfaces: An In Vitro Study. Implant Dent. 2017;26(3):373-377.

[25] Lamoureux F, Baud'huin M, Rodriguez Calleja L, et al. Selective inhibition of BET bromodomain epigenetic signalling interferes with the bone-associated tumour vicious cycle. Nat Commun. 2014;5:3511.

[26] Wang PP, Zhu XF, Yang L, et al. Puerarin stimulates osteoblasts differentiation and bone formation through estrogen receptor, p38 MAPK, and Wnt/β-catenin pathways. J Asian Nat Prod Res. 2012;14(9):897-905.

[27] 孔祥鹤,牛银波,武祥龙,等.黄芪总黄酮对大鼠原代成骨细胞的影响及其机制研究[J].化学与生物工程, 2012,29(6): 26-30,45.

[28] Zheng D, Peng S, Yang SH, et al. The beneficial effect of Icariin on bone is diminished in osteoprotegerin-deficient mice. Bone. 2012;51(1):85-92.

[29] Wittrant Y, Theoleyre S, Couillaud S, et al. Relevance of an in vitro osteoclastogenesis system to study receptor activator of NF-kB ligand and osteoprotegerin biological activities. Exp Cell Res. 2004;293(2):292-301.

[30] 李玲慧,丁道芳,杜国庆,等.不同胶原酶消化对原代成骨细胞获得率及活性的比较[J].中国骨伤, 2013, 26(4): 328-331.

[31] Xu R, Zhang C, Shin DY, et al. c-Jun N-terminal kinases (JNKs) are critical mediators of osteoblast activity in vivo. J Bone Miner Res. 2017 May 31.[Epub ahead of print]