CONDITIONED MEDIUM OF BONE MARROW MESENCHYMAL STEM CELLS ALLEVIATE INHIBITING EFFECT OF DEXAMETHASONE ON OSTEOGENETIC CAPABILITY OF OSTEOBLAST_Chinese Journal of Reparative and Reconstructive Surgery

Authors：

 WEIBo ¹ , KANGYinhui ¹ ,  SONGLijun ² , WANGChaojun ¹ , GUOWeixiong ¹ , XIANGMin ¹ , LIGuangsheng ¹ , LINHao ¹ , ZENGRong ¹

1. Orthopedic Centre, Affiliated Hospital of Guangdong Medical College, Zhanjiang Guangdong, 524001, P.R.China;
2. Reproductive Research Centre, Affiliated Hospital of Guangdong Medical College, Zhanjiang Guangdong, 524001, P.R.China;

Corresponding author：

WEIBo, Email: webjxmc@163.com; SONGLijun, Email: 413000667@qq.com

Keywords：

Osteoblasts; Bone marrow mesenchymal stem cells; Conditioned medium; Dexamethasone; Osteogenesis; Mouse

DOI：

10.7507/1002-1892.20160155

Video：

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Objective To explore the paracrine effect of bone marrow mesenchymal stem cells (BMSCs) on dexamethasone-induced inhibition of osteoblast function in vitro. Methods The serum free conditioned medium of mouse BMSCs cultured for 24 hours was prepared for spare use. The 3rd passage of MC3T3-E1 cells were divided into 4 groups: the control group (group A), dexamethasone group (group B), dexamethasone+BMSCs conditioned medium (1:1) group (group C), and BMSCs conditioned medium group (group D). After 24 hours of culture, the alkaline phosphatase (ALP) content was determined; the protein expressions of RUNX2 and Osteocalcin were detected by Western blot; and the gene expressions of collagen type I-α 1 (COL1A1), RUNX2, ALP, and Osteocalcin were detected by real-time fluorescence quantitative PCR (RT-qPCR); alizarin red staining was used to observe calcium nodules formation at 21 days. Results After cultured for 24 hours, ALP content was significantly lower in groups B, C, and D than group A, and in group B than groups C and D (P < 0.05), but no significant difference was found between groups C and D (P > 0.05). The relative protein expression of RUNX2 of group B was significantly lower than that of groups A, C, and D (P < 0.05), but difference was not significant between groups A, C, and D (P > 0.05). The relative protein expression of Osteocalcin was significantly lower in group B than groups A, C, and D, in groups A and C than group D (P < 0.05), but difference had no significance between groups A and C (P > 0.05). The relative gene expressions of RUNX2, Osteocalcin, COL1A1, and ALP of groups B, C, and D were significantly lower than those of group A (P < 0.05); the relative gene expressions of RUNX2, Osteocalcin, and ALP were significantly higher in group D than groups B and C, in group C than group B (P < 0.05). The gene expression of COL1A1 was significantly higher in group D than group B (P < 0.05), but difference was not significant between groups B and C, and between groups C and D (P > 0.05). The cells of group A all died at 6 days after culture; at 21 days, the calcium no dule staining was positive by alizarin red in groups B, C and D, and the degree of the staining gradually increased from groups B to D. Conclusion BMSCs conditioned medium can alleviate the inhibitory effect of dexamethasone on osteoblasts function.

Citation： WEIBo, KANGYinhui, SONGLijun, WANGChaojun, GUOWeixiong, XIANGMin, LIGuangsheng, LINHao, ZENGRong. CONDITIONED MEDIUM OF BONE MARROW MESENCHYMAL STEM CELLS ALLEVIATE INHIBITING EFFECT OF DEXAMETHASONE ON OSTEOGENETIC CAPABILITY OF OSTEOBLAST. Chinese Journal of Reparative and Reconstructive Surgery, 2016, 30(6): 761-766. doi: 10.7507/1002-1892.20160155 Copy

1.	James CG, Ulici V, Tuckermann J, et al. Expression profiling of Dexamethasone-treated primary chondrocytes identifies targets of glucocorticoid signalling in endochondral bone development. BMC Genomics, 2007, 8: 205.
2.	Cui Y, Kaisaierjiang A, Cao P, et al. Association of apolipoprotein A5 genetic polymorphisms with steroid-induced osteonecrosis of femoral head in a Chinese Han population. Diagn Pathol, 2014, 9: 229.
3.	Souttou B, Raulais D, Vigny M. Pleiotrophin induces angiogenesis: involvement of the phosphoinositide-3 kinase but not the nitric oxide synthase pathways. J Cell Physiol, 2001, 187(1): 59-64.
4.	Himburg HA, Muramoto GG, Daher P, et al. Pleiotrophin regulates the expansion and regeneration of hematopoietic stem cells. Nat Med, 2010, 16(4): 475-482.
5.	Frenkel B, White W, Tuckermann J. Glucocorticoid-induced osteoporosis. Adv Exp Med Biol, 2015, 872: 179-215.
6.	Teplyuk NM, Zhang Y, Lou Y, et al. The osteogenic transcription factor runx2 controls genes involved in sterol/steroid metabolism, including CYP11A1 in osteoblasts. Mol Endocrinol, 2009, 23(6): 849-861.
7.	La Corte R, Trotta F, Adami S. Glucocorticoid receptors and bone. Gluc Curr Pharm Des, 2010, 16(32): 3586-3592.
8.	Pittenger MF, Mackay AM, Beck SC, et al. Multilineage potential of adult human mesenchymal stem cells. Science, 1999, 284(5411): 143-147.
9.	Sutton MT, Bonfield TL. Stem cells: innovations in clinical applications. Stem Cells Int, 2014, 2014: 516278.
10.	Lebouvier A, Poignard A, Cavet M, et al. Development of a simple procedure for the treatment of femoral head osteonecrosis with intra-osseous injection of bone marrow mesenchymal stromal cells: study of their biodistribution in the early time points after injection. Stem Cell Res Ther, 2015, 6: 68.
11.	Chen L, Tredget EE, Wu PY, et al. Paracrine factors of mesenchymal stem cells recruit macrophages and endothelial lineage cells and enhance wound healing. PLoS One, 2008, 3(4): e1886.
12.	Mayr-Wohlfart U, Waltenberger J, Hausser H, et al. Vascular endothelial growth factor stimulates chemotactic migration of primary human osteoblasts. Bone, 2002, 30(3): 472-477.
13.	Kinnaird T, Stabile E, Burnett MS, et al. Marrow-derived stromal cells express genes encoding a broad spectrum of arteriogenic cytokines and promote in vitro and in vivo arteriogenesis through paracrine mechanisms. Circ Res, 2004, 94(5): 678-685.
14.	Wen Q, Ma L, Chen YP, et al. Treatment of avascular necrosis of the femoral head by hepatocyte growth factor-transgenic bone marrow stromal stem cells. Gene Ther, 2008, 15(23): 1523-1535.
15.	Sodu H, Kodama HA, Amagai Y, et al. In vitro differentiation and classification in a new clonal osteogenic cell line derived from newborn mouse calvaria. J Cell Bio, 1983, 96(1): 191-198.
16.	Yeh LC, Ma X, Matheny RW, et al. Protein kinase D mediates the synergistic effects of BMP-7 and IGF-I on osteoblastic cell differentiation. Growth Factors, 2010, 28(5): 318-328.
17.	Ogoshi T, Hagino H, Fukata S, et al. Influence of glucocorticoid on bone in 3-, 6-, and 12-month-old rats as determined by bone mass and histomorphometry. Modern Rheumatol, 2008, 18(6): 552-561.
18.	Weinstein RS, Nicholas RW, Manolagas SC. Apoptosis of osteocytes in glucocorticoid-induced osteonecrosis of the hip. J Clin Endocrinol Metab, 2000, 85(8): 2907-2912.
19.	Maricic M. Update on glucocorticoid-induced osteoporosis. Rheum Dis Clin North Am, 2011, 37(3): 415-431.
20.	Hoeppner LH, Secreto FJ, Westendorf JJ. Wnt signaling as a therapeutic target for bone diseases. Expert Opin Ther Targets, 2009, 13(4): 485-496.
21.	Bonewald LF, Johnson ML. Osteocytes, mechanosensing and Wnt signaling. Bone, 2008, 42(4): 606-615.
22.	李成, 周海斌. 骨髓间充质干细胞的旁分泌能影响成骨细胞生物学功能. 中国组织工程研究, 2014, 18(10): 1477-1483.
23.	Li B, Zhang H, Zeng M, et al. Bone marrow mesenchymal stem cells protect alveolar macrophages from lipopolysaccharide-induced apoptosis partially by inhibiting the Wnt/β-catenin pathway. Cell Biol Int, 2014, 39(2): 192-200.
24.	王锋, 吴小涛, 王运涛, 等. 非接触共培养条件下人BMSCs对氧化应激环境中退变髓核细胞凋亡的保护研究. 中国修复重建外科杂志, 2010, 24(4): 391-398.
25.	Farley JR, Stilt-Coffing B. Apoptosis may determine the release of skeletal alkaline phosphatase activity from human osteoblast-line cells. Calcif Tissue Int, 2001, 68(1): 43-52.
26.	Kim MB, Song Y, Hwang JK. Kirenol stimulates osteoblast differentiation through activation of the BMP and Wnt/β-catenin signaling pathways in MC3T3-E1 cells. Fitoterapia, 2014, 98: 59-65.
27.	Huang YF, Lin JJ, Lin CH, et al. c-Jun N-terminal kinase 1 negatively regulates osteoblastic differentiation induced by BMP2 via phosphorylation of Runx2 at Ser104. J Bone Miner Res, 2012, 27(5): 1093-1105.
28.	Pradel W, Mai R, Gedrange T, et al. Cell passage and composition of culture medium effects proliferation and differentiation of human osteoblast-like cells from facial bone. J Physiol Pharmacol, 2008, 59 Suppl 5: 47-58.
29.	Zoch ML, Clemens TL, Riddle RC. New insights into the biology of osteocalcin. Bone, 2015, 82: 42-49.

1. James CG, Ulici V, Tuckermann J, et al. Expression profiling of Dexamethasone-treated primary chondrocytes identifies targets of glucocorticoid signalling in endochondral bone development. BMC Genomics, 2007, 8: 205.
2. Cui Y, Kaisaierjiang A, Cao P, et al. Association of apolipoprotein A5 genetic polymorphisms with steroid-induced osteonecrosis of femoral head in a Chinese Han population. Diagn Pathol, 2014, 9: 229.
3. Souttou B, Raulais D, Vigny M. Pleiotrophin induces angiogenesis: involvement of the phosphoinositide-3 kinase but not the nitric oxide synthase pathways. J Cell Physiol, 2001, 187(1): 59-64.
4. Himburg HA, Muramoto GG, Daher P, et al. Pleiotrophin regulates the expansion and regeneration of hematopoietic stem cells. Nat Med, 2010, 16(4): 475-482.
5. Frenkel B, White W, Tuckermann J. Glucocorticoid-induced osteoporosis. Adv Exp Med Biol, 2015, 872: 179-215.
6. Teplyuk NM, Zhang Y, Lou Y, et al. The osteogenic transcription factor runx2 controls genes involved in sterol/steroid metabolism, including CYP11A1 in osteoblasts. Mol Endocrinol, 2009, 23(6): 849-861.
7. La Corte R, Trotta F, Adami S. Glucocorticoid receptors and bone. Gluc Curr Pharm Des, 2010, 16(32): 3586-3592.
8. Pittenger MF, Mackay AM, Beck SC, et al. Multilineage potential of adult human mesenchymal stem cells. Science, 1999, 284(5411): 143-147.
9. Sutton MT, Bonfield TL. Stem cells: innovations in clinical applications. Stem Cells Int, 2014, 2014: 516278.
10. Lebouvier A, Poignard A, Cavet M, et al. Development of a simple procedure for the treatment of femoral head osteonecrosis with intra-osseous injection of bone marrow mesenchymal stromal cells: study of their biodistribution in the early time points after injection. Stem Cell Res Ther, 2015, 6: 68.
11. Chen L, Tredget EE, Wu PY, et al. Paracrine factors of mesenchymal stem cells recruit macrophages and endothelial lineage cells and enhance wound healing. PLoS One, 2008, 3(4): e1886.
12. Mayr-Wohlfart U, Waltenberger J, Hausser H, et al. Vascular endothelial growth factor stimulates chemotactic migration of primary human osteoblasts. Bone, 2002, 30(3): 472-477.
13. Kinnaird T, Stabile E, Burnett MS, et al. Marrow-derived stromal cells express genes encoding a broad spectrum of arteriogenic cytokines and promote in vitro and in vivo arteriogenesis through paracrine mechanisms. Circ Res, 2004, 94(5): 678-685.
14. Wen Q, Ma L, Chen YP, et al. Treatment of avascular necrosis of the femoral head by hepatocyte growth factor-transgenic bone marrow stromal stem cells. Gene Ther, 2008, 15(23): 1523-1535.
15. Sodu H, Kodama HA, Amagai Y, et al. In vitro differentiation and classification in a new clonal osteogenic cell line derived from newborn mouse calvaria. J Cell Bio, 1983, 96(1): 191-198.
16. Yeh LC, Ma X, Matheny RW, et al. Protein kinase D mediates the synergistic effects of BMP-7 and IGF-I on osteoblastic cell differentiation. Growth Factors, 2010, 28(5): 318-328.
17. Ogoshi T, Hagino H, Fukata S, et al. Influence of glucocorticoid on bone in 3-, 6-, and 12-month-old rats as determined by bone mass and histomorphometry. Modern Rheumatol, 2008, 18(6): 552-561.
18. Weinstein RS, Nicholas RW, Manolagas SC. Apoptosis of osteocytes in glucocorticoid-induced osteonecrosis of the hip. J Clin Endocrinol Metab, 2000, 85(8): 2907-2912.
19. Maricic M. Update on glucocorticoid-induced osteoporosis. Rheum Dis Clin North Am, 2011, 37(3): 415-431.
20. Hoeppner LH, Secreto FJ, Westendorf JJ. Wnt signaling as a therapeutic target for bone diseases. Expert Opin Ther Targets, 2009, 13(4): 485-496.
21. Bonewald LF, Johnson ML. Osteocytes, mechanosensing and Wnt signaling. Bone, 2008, 42(4): 606-615.
22. 李成, 周海斌. 骨髓间充质干细胞的旁分泌能影响成骨细胞生物学功能. 中国组织工程研究, 2014, 18(10): 1477-1483.
23. Li B, Zhang H, Zeng M, et al. Bone marrow mesenchymal stem cells protect alveolar macrophages from lipopolysaccharide-induced apoptosis partially by inhibiting the Wnt/β-catenin pathway. Cell Biol Int, 2014, 39(2): 192-200.
24. 王锋, 吴小涛, 王运涛, 等. 非接触共培养条件下人BMSCs对氧化应激环境中退变髓核细胞凋亡的保护研究. 中国修复重建外科杂志, 2010, 24(4): 391-398.
25. Farley JR, Stilt-Coffing B. Apoptosis may determine the release of skeletal alkaline phosphatase activity from human osteoblast-line cells. Calcif Tissue Int, 2001, 68(1): 43-52.
26. Kim MB, Song Y, Hwang JK. Kirenol stimulates osteoblast differentiation through activation of the BMP and Wnt/β-catenin signaling pathways in MC3T3-E1 cells. Fitoterapia, 2014, 98: 59-65.
27. Huang YF, Lin JJ, Lin CH, et al. c-Jun N-terminal kinase 1 negatively regulates osteoblastic differentiation induced by BMP2 via phosphorylation of Runx2 at Ser104. J Bone Miner Res, 2012, 27(5): 1093-1105.
28. Pradel W, Mai R, Gedrange T, et al. Cell passage and composition of culture medium effects proliferation and differentiation of human osteoblast-like cells from facial bone. J Physiol Pharmacol, 2008, 59 Suppl 5: 47-58.
29. Zoch ML, Clemens TL, Riddle RC. New insights into the biology of osteocalcin. Bone, 2015, 82: 42-49.

Chinese Journal of Reparative and Reconstructive Surgery

CONDITIONED MEDIUM OF BONE MARROW MESENCHYMAL STEM CELLS ALLEVIATE INHIBITING EFFECT OF DEXAMETHASONE ON OSTEOGENETIC CAPABILITY OF OSTEOBLAST

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