STRESS REGULATING OSTEOGENIC DIFFERENTIATION OF HUMAN INTERVERTEBRAL DISC CARTILAGE ENDPLATE-DERIVED STEM CELLS_Chinese Journal of Reparative and Reconstructive Surgery

Authors：

YUANChao ,  WANGJian , ZHUXiaolong , ZHENGYong , HUANGBo , LIChangqing , ZHOUYue

Department of Orthopedics, Xinqiao Hospital, the Third Military Medical University, Chongqing, 400037, P. R. China;

Corresponding author：

WANGJian, Email: tonywjxq@aliyun.com

Keywords：

Cartilage endplate-derived stem cells; Stretch stress; Bone morphogenetic protein 2; Osteogenic differentiation

DOI：

10.7507/1002-1892.20150074

Video：

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Objective To investigate the effect of cyclic stretch stress on the osteogenic differentiation of human cartilage endplate-derived stem cells (CESCs). Methods CESCs were isolated from the endplate cartilage tissues by the method of agarose suspension culture system. The endplate cartilage tissue was harvested for immunohistochemical staining. Flexercell-4000^TM Tension Plus system was used to apply cyclic stretch on CESCs at a frequency of 1 Hz and at a stretch rate of 10% for 1, 6, 12, or 24 hours (experimental group). No stretch stress was performed on CESCs in the same culture condition (control group). After mechanical loading, the protein expression of bone morphogenetic protein 2 (BMP-2) was measured by Western blot, and gene expressions of runt-related transcription factor 2 (Runx2), alkaline phosphatase (ALP), and SOX9 were detected by real-time fluorescent quantitative PCR. Results Immunohistochemical staining showed BMP-2 protein expression in chondrocytes. The continuous cyclic stretch stress of 10% can increase the expression of BMP-2 protein in CESCs. Significant differences were observed in the expressions of BMP-2 protein (P<0.05) between 2 groups at the other time points except at 1 hour (P>0.05), in a time-dependent manner. The real-time fluorescent quantitative PCR indicated that the gene expressions of Runx2 and ALP showed an increasing tendency with time in the experimental group when compared with the control group, but there was down-regulated expression of SOX9. Significant difference was found in mRNA expressions of Runx2 and ALP at 12 and 24 hours and in mRNA expressions of SOX9 at 6, 12, and 24 hours between 2 groups (P<0.05), in a time-dependent manner. Conclusion Cyclic stretch stress may induce osteogenic differentiation of CESCs by regulating the expressions of some genes related osteogenesis in CESCs.

Citation： YUANChao, WANGJian, ZHUXiaolong, ZHENGYong, HUANGBo, LIChangqing, ZHOUYue. STRESS REGULATING OSTEOGENIC DIFFERENTIATION OF HUMAN INTERVERTEBRAL DISC CARTILAGE ENDPLATE-DERIVED STEM CELLS. Chinese Journal of Reparative and Reconstructive Surgery, 2015, 29(3): 351-355. doi: 10.7507/1002-1892.20150074 Copy

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2.	Neidlinger-Wilke C, Galbusera F, Pratsinis H, et al. Mechanical loading of the intervertebral disc:from the macroscopic to the cellular level. Eur Spine J, 2014, 23 Suppl 3:S333-343.
3.	Maerz T, Herkowitz H, Baker K. Molecular and genetic advances in the regeneration of the intervertebral disc. Surg Neurol Int, 2013, 4(Suppl 2):S94-S105.
4.	Jain AP, Pundir S, Sharma A. Bone morphogenetic proteins:The anomalous molecules. J Indian Soc Periodontol, 2013, 17(5):583-586.
5.	Takae R, Matsunaga S, Origuchi N, et al. Immunolocalization of bone morphogenetic protein and its receptors in degeneration of intervertebral disc. Spine (Phila Pa 1976), 1999, 24(14):1397-1401.
6.	Than KD, Rahman SU, Vanaman MJ, et al. Bone morphogenetic proteins and degenerative disk disease. Neurosurgery, 2012, 70(4):996-1002.
7.	Hiyama A, Sakai D, Tanaka M, et al. The relationship between the Wnt/β-catenin and TGF-β/BMP signals in the intervertebral disc cell. J Cell Physiol, 2011, 226(5):1139-1148.
8.	Chen D, Ji X, Harris MA, et al. Differential roles for bone morphogenetic protein (BMP) receptor type IB and IA in differentiation and specification of mesenchymal precursor cells to osteoblast and adipocyte lineages. J Cell Biol, 1998, 142(1):295-305.
9.	Rui YF, Lui PP, Ni M, et al. Mechanical loading increased BMP-2 expression which promoted osteogenic differentiation of tendon-derived stem cells. J Orthop Res, 2011, 29(3):390-396.
10.	Susperregui AR, Viñals F, Ho PW, et al. BMP-2 regulation of PTHrP and osteoclastogenic factors during osteoblast differentiation of C2C12 cells. J Cell Physiol, 2008, 216(1):144-152.
11.	Huang B, Liu LT, Li CQ, et al. Study to determine the presence of progenitor cells in the degenerated human cartilage endplates. Eur Spine J, 2012, 21(4):613-622.
12.	Liu LT, Huang B, Li CQ, et al. Characteristics of stem cells derived from the degenerated human intervertebral disc cartilage endplate. PLoS One, 2011, 6(10):e26285.
13.	Burdick JA, Vunjak-Novakovic G. Engineered microenvironments for controlled stem cell differentiation. Tissue Eng Part A, 2009, 15(2):205-219.
14.	Castillo AB, Jacobs CR. Mesenchymal stem cell mechanobiology. Curr Osteoporos Rep, 2010, 8(2):98-104.
15.	Dado D, Sagi M, Levenberg S, et al. Mechanical control of stem cell differentiation. Regen Med, 2012, 7(1):101-116.
16.	Camilleri S, McDonald F. Runx2 and dental development. Eur J Oral Sci, 2006, 114(5):361-373.
17.	Kanno T, Takahashi T, Ariyoshi W, et al. Tensile mechanical strain up-regulates Runx2 and osteogenic factor expression in human periosteal cells:implications for distraction osteogenesis. J Oral Maxillofac Surg, 2005, 63(4):499-504.
18.	Stein GS, Lian JB. Molecular mechanisms mediating proliferation/differentiation interrelationships during progressive development of the osteoblast phenotype. Endocr Rev, 1993, 14(4):424-442.
19.	Sumanasinghe RD, Bernacki SH, Loboa EG. Osteogenic differentiation of human mesenchymal stem cells in collagen matrices:effect of uniaxial cyclic tensile strain on bone morphogenetic protein (BMP-2) mRNA expression. Tissue Eng, 2006, 12(12):3459-3465.
20.	Sowa G, Vadalà G, Studer R, et al. Characterization of intervertebral disc aging:longitudinal analysis of a rabbit model by magnetic resonance imaging, histology, and gene expression. Spine, 2008, 33(17):1821-1828.
21.	Yang X, Gong P, Lin Y, et al. Cyclic tensile stretch modulates osteogenic differentiation of adipose-derived stem cells via the BMP-2 pathway. Arch Med Sci, 2010, 6(2):152-159.
22.	Sato M, Ochi T, Nakase T, et al. Mechanical tension-stress induces expression of bone morphogenetic protein (BMP)-2 and BMP-4, but not BMP-6, BMP-7, and GDF-5 mRNA, during distraction osteogenesis. J Bone Miner Res, 1999, 14(7):1084-1095.
23.	Wright E, Hargrave MR, Christiansen J, et al. The Sry-related gene Sox9 is expressed during chondrogenesis in mouse embryos. Nat Genet, 1995, 9(1):15-20.
24.	Bi W, Deng JM, Zhang Z, et al. Sox9 is required for cartilage formation. Nat Genet, 1999, 22(1):85-89.
25.	Paul R, Haydon RC, Cheng H, et al. Potential use of Sox9 gene therapy for intervertebral degenerative disc disease. Spine (Phila Pa 1976), 2003, 28(8):755-763.

1. Stefanakis M, Luo J, Pollintine P, et al. IS SLS Prize winner:Mechanical influences in progressive intervertebral disc degeneration. Spine (Phila Pa 1976), 2014, 39(17):1365-1372.
2. Neidlinger-Wilke C, Galbusera F, Pratsinis H, et al. Mechanical loading of the intervertebral disc:from the macroscopic to the cellular level. Eur Spine J, 2014, 23 Suppl 3:S333-343.
3. Maerz T, Herkowitz H, Baker K. Molecular and genetic advances in the regeneration of the intervertebral disc. Surg Neurol Int, 2013, 4(Suppl 2):S94-S105.
4. Jain AP, Pundir S, Sharma A. Bone morphogenetic proteins:The anomalous molecules. J Indian Soc Periodontol, 2013, 17(5):583-586.
5. Takae R, Matsunaga S, Origuchi N, et al. Immunolocalization of bone morphogenetic protein and its receptors in degeneration of intervertebral disc. Spine (Phila Pa 1976), 1999, 24(14):1397-1401.
6. Than KD, Rahman SU, Vanaman MJ, et al. Bone morphogenetic proteins and degenerative disk disease. Neurosurgery, 2012, 70(4):996-1002.
7. Hiyama A, Sakai D, Tanaka M, et al. The relationship between the Wnt/β-catenin and TGF-β/BMP signals in the intervertebral disc cell. J Cell Physiol, 2011, 226(5):1139-1148.
8. Chen D, Ji X, Harris MA, et al. Differential roles for bone morphogenetic protein (BMP) receptor type IB and IA in differentiation and specification of mesenchymal precursor cells to osteoblast and adipocyte lineages. J Cell Biol, 1998, 142(1):295-305.
9. Rui YF, Lui PP, Ni M, et al. Mechanical loading increased BMP-2 expression which promoted osteogenic differentiation of tendon-derived stem cells. J Orthop Res, 2011, 29(3):390-396.
10. Susperregui AR, Viñals F, Ho PW, et al. BMP-2 regulation of PTHrP and osteoclastogenic factors during osteoblast differentiation of C2C12 cells. J Cell Physiol, 2008, 216(1):144-152.
11. Huang B, Liu LT, Li CQ, et al. Study to determine the presence of progenitor cells in the degenerated human cartilage endplates. Eur Spine J, 2012, 21(4):613-622.
12. Liu LT, Huang B, Li CQ, et al. Characteristics of stem cells derived from the degenerated human intervertebral disc cartilage endplate. PLoS One, 2011, 6(10):e26285.
13. Burdick JA, Vunjak-Novakovic G. Engineered microenvironments for controlled stem cell differentiation. Tissue Eng Part A, 2009, 15(2):205-219.
14. Castillo AB, Jacobs CR. Mesenchymal stem cell mechanobiology. Curr Osteoporos Rep, 2010, 8(2):98-104.
15. Dado D, Sagi M, Levenberg S, et al. Mechanical control of stem cell differentiation. Regen Med, 2012, 7(1):101-116.
16. Camilleri S, McDonald F. Runx2 and dental development. Eur J Oral Sci, 2006, 114(5):361-373.
17. Kanno T, Takahashi T, Ariyoshi W, et al. Tensile mechanical strain up-regulates Runx2 and osteogenic factor expression in human periosteal cells:implications for distraction osteogenesis. J Oral Maxillofac Surg, 2005, 63(4):499-504.
18. Stein GS, Lian JB. Molecular mechanisms mediating proliferation/differentiation interrelationships during progressive development of the osteoblast phenotype. Endocr Rev, 1993, 14(4):424-442.
19. Sumanasinghe RD, Bernacki SH, Loboa EG. Osteogenic differentiation of human mesenchymal stem cells in collagen matrices:effect of uniaxial cyclic tensile strain on bone morphogenetic protein (BMP-2) mRNA expression. Tissue Eng, 2006, 12(12):3459-3465.
20. Sowa G, Vadalà G, Studer R, et al. Characterization of intervertebral disc aging:longitudinal analysis of a rabbit model by magnetic resonance imaging, histology, and gene expression. Spine, 2008, 33(17):1821-1828.
21. Yang X, Gong P, Lin Y, et al. Cyclic tensile stretch modulates osteogenic differentiation of adipose-derived stem cells via the BMP-2 pathway. Arch Med Sci, 2010, 6(2):152-159.
22. Sato M, Ochi T, Nakase T, et al. Mechanical tension-stress induces expression of bone morphogenetic protein (BMP)-2 and BMP-4, but not BMP-6, BMP-7, and GDF-5 mRNA, during distraction osteogenesis. J Bone Miner Res, 1999, 14(7):1084-1095.
23. Wright E, Hargrave MR, Christiansen J, et al. The Sry-related gene Sox9 is expressed during chondrogenesis in mouse embryos. Nat Genet, 1995, 9(1):15-20.
24. Bi W, Deng JM, Zhang Z, et al. Sox9 is required for cartilage formation. Nat Genet, 1999, 22(1):85-89.
25. Paul R, Haydon RC, Cheng H, et al. Potential use of Sox9 gene therapy for intervertebral degenerative disc disease. Spine (Phila Pa 1976), 2003, 28(8):755-763.

Chinese Journal of Reparative and Reconstructive Surgery

STRESS REGULATING OSTEOGENIC DIFFERENTIATION OF HUMAN INTERVERTEBRAL DISC CARTILAGE ENDPLATE-DERIVED STEM CELLS

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