PROMOTION EFFECT OF NOTOCHORDAL CELLS CONDITIONED MEDIUM ON PROLIFERATION ANDDIFFERENTIATION OF BONE MARROW MESENCHYMAL STEM CELLS/_Chinese Journal of Reparative and Reconstructive Surgery

Authors：

MA Kaige , SHAO Zengwu , WANG Baichuan , ZHANG Yannan , YANG Shuhua , LIU Tao.

Department of Orthopedics, Union Hospital, Tongji Medical College, HuazhongUniversity of Science and Technology, Wuhan Hubei, 430022, P.R.China.;

Corresponding author：

Keywords：

Tissue engineering ,Bone marrow mesenchymal stem cells, Notochordal cells ,Degenerative disc disease, Rabbit

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Objective To investigate the effect of notochordal cells (NCs) conditioned medium (NCCM) on the
prol iferation and differentiation of bone marrow mesenchymal stem cells (BMSCs). Methods NCs and BMSCs were
isolated from the thoracolumbar intervertebral disc and the femurs of 4-week-old Japanese white rabbits, respectively. NCs
were cultured with DMEM/F12 medium containing 15% FBS for 5 days to prepare NCCM. The experiment consisted of
2 groups: BMSCs were cultured with NCCM in experimental group and with DMEM/F12 medium containing 15% FBS
in control group. The prol iferation of BMSCs was assessed by cell counting kit-8 at 1, 3, 5, 7, 9, and 14 days after culture;
the expression of proteoglycan and collagen type II were determined by immunofluorescence and real-time fluorescent
quantitative PCR at 7 and 14 days after culture. Results NCs and BMSCs were successfully isloated. At 5, 7, 9, and 14
days, the number of BMSCs in the experimental group was significantly more than those in the control group (P lt; 0.05).
At 7 and 14 days, there was no expression or less expression of proteoglycan and collagen type II in the control group;
however, there was a lot of expression of proteoglycan and collagen type II in the experimental group, and the expressions
were higher at 14 days than at 7 days. At 7 and 14 days after culture, the mRNA expressions of proteoglycan and collagen
type II were significantly higher in the experimental group than in the control group (P lt; 0.05), and at 14 days than at 7
days in the experimental group (P lt; 0.05). Conclusion NCCM can promote the prol iferation and the differentiation of
BMSCs into chondroyte-like cells, which provides the basis for NCs and BMSCs as seed cells in the treatment of degenerative
disc disease.

Citation： MA Kaige,SHAO Zengwu,WANG Baichuan,ZHANG Yannan,YANG Shuhua,LIU Tao.. PROMOTION EFFECT OF NOTOCHORDAL CELLS CONDITIONED MEDIUM ON PROLIFERATION ANDDIFFERENTIATION OF BONE MARROW MESENCHYMAL STEM CELLS/. Chinese Journal of Reparative and Reconstructive Surgery, 2012, 26(5): 601-606. doi: Copy

1.	Petit A, Yao G, Rowas SA, et al. Effect of synthetic link N peptide on the expression of type I and type II collagens in human intervertebral disc cells. Tissue Eng Part A, 2011, 17(7-8): 899-904.
2.	Hohaus C, Ganey TM, Minkus Y, et al. Cell transplantation in lumbar spine disc degeneration disease. Eur Spine J, 2008, 17(Suppl 4): S492-503.
3.	Bertolo A, Thiede T, Aebli N, et al. Human mesenchymal stem cell co-culture modulates the immunological properties of human intervertebral disc tissue fragments in vitro. Eur Spine J, 2011, 20(4): 592-603.
4.	Omlor GW, Nerlich AG, Wilke HJ, et al. A new porcine in vivo animal model of disc degeneration: response of anulus fibrosus cells, chondrocyte-like nucleus pulposus cells, and notochordal nucleus pulposus cells to partial nucleotomy. Spine (Phila Pa 1976), 2009, 34(25): 2730-2739.
5.	Paesold G, Nerlich AG, Boos N. Biological treatment strategies for disc degeneration: potentials and shortcomings. Eur Spine J, 2007, 16(4): 447-468.
6.	Baksh D, Song L, Tuan RS. Adult mesenchymal stem cells: characterization, differentiation, and application in cell and gene therapy. J Cell Mol Med, 2004, 8(3): 301-316.
7.	Aguiar DJ, Johnson SL, Oegema TR. Notochordal cells interact with nucleus pulposus cells: regulation of proteoglycan synthesis. Exp Cell Res, 1999, 246(1): 129-137.
8.	Johnson WE, Evans H, Menage J, et al. Immunohistochemical detection of Schwann cells in innervated and vascularized human intervertebral discs. Spine (Phila Pa 1976), 2001, 26(23): 2550-2557.
9.	Erwin WM, Las Heras F, Islam D, et al. The regenerative capacity of the notochordal cell: tissue constructs generated in vitro under hypoxic conditions. J Neurosurg Spine, 2009, 10(6): 513-521.
10.	Risbud MV, Schaer TP, Shapiro IM. Toward an understanding of the role of notochordal cells in the adult intervertebral disc: from discord to accord. Dev Dyn, 2010, 239(8): 2141-2148.
11.	Chai C, Leong KW. Biomaterials approach to expand and direct differentiation of stem cells. Mol Ther, 2007, 15(3): 467-480.
12.	严慧深, 吴小涛, 王运涛, 等. 骨髓间充质干细胞修复退变椎间盘中营养和分化效应的变化. 中华实验外科杂志, 2009, 26(6): 704-706.
13.	Blanco JF, Graciani IF, Sanchez-Guijo FM, et al. Isolation and characterization of mesenchymal stromal cells from human degenerated nucleus pulposus. Spine (Phila Pa 1976), 2010, 35(26): 2259-2265.
14.	Chen J, Yan W, Setton LA. Molecular phenotypes of notochordal cells purified from immature nucleus pulposus. Eur Spine J, 2006, 15 Suppl 3: S303-311.
15.	赵献峰, 刘浩, 丰干均, 等. 脊索细胞促进髓核软骨样细胞增殖及表型维持. 中国修复重建外科杂志, 2008, 22(8): 939-943.
16.	Yang Z, Schmitt JF, Lee EH. Immunohistochemical analysis of human mesenchymal stem cells differentiating into chondrogenic, osteogenic, and adipogenic lineages. Methods Mol Biol, 2011, 698(1): 353-366.
17.	Yang SH, Wu CC, Shih TT, et al. In vitro study on interaction between human nucleus pulposus cells and mesenchymal stem cells through paracrine stimulation. Spine (Phila Pa 1976), 2008, 33(18): 1951-1957.
18.	Strassburg S, Richardson SM, Freemont AJ, et al. Co-culture induces mesenchymal stem cell differentiation and modulation of the degenerate human nucleus pulposus cell phenotype. Regen Med, 2010, 5(5): 701-711.
19.	Erwin WM, Inman RD. Notochord cells regulate intervertebral disc chondrocyte proteoglycan production and cell proliferation. Spine (Phila Pa 1976), 2006, 31(10): 1094-1099.
20.	Steck E, Bertram H, Abel R, et al. Induction of intervertebraldisc-like cells from adult mesenchymal stem cells. Stem Cells, 2005, 23(3): 403-411.
21.	Ivkovic S, Yoon BS, Popoff SN, et al. Connective tissue growth factor coordinates chondrogenesis and angiogenesis during skeletal development. Development, 2003, 130(12): 2779-2791.
22.	Korecki CL, Taboas JM, Tuan RS, et al. Notochordal cell conditioned medium stimulates mesenchymal stem cell differentiation toward a young nucleus pulposus phenotype. Stem Cell Res Ther, 2010, 1(2): 18.
23.	Purmessur D, Schek RM, Abbott RD, et al. Notochordal conditioned media from tissue increases proteoglycan accumulation and promotes a healthy nucleus pulposus phenotype in human mesenchymal stem cells. Arthritis Res Ther, 2011, 13(3): R81.

1. Petit A, Yao G, Rowas SA, et al. Effect of synthetic link N peptide on the expression of type I and type II collagens in human intervertebral disc cells. Tissue Eng Part A, 2011, 17(7-8): 899-904.
2. Hohaus C, Ganey TM, Minkus Y, et al. Cell transplantation in lumbar spine disc degeneration disease. Eur Spine J, 2008, 17(Suppl 4): S492-503.
3. Bertolo A, Thiede T, Aebli N, et al. Human mesenchymal stem cell co-culture modulates the immunological properties of human intervertebral disc tissue fragments in vitro. Eur Spine J, 2011, 20(4): 592-603.
4. Omlor GW, Nerlich AG, Wilke HJ, et al. A new porcine in vivo animal model of disc degeneration: response of anulus fibrosus cells, chondrocyte-like nucleus pulposus cells, and notochordal nucleus pulposus cells to partial nucleotomy. Spine (Phila Pa 1976), 2009, 34(25): 2730-2739.
5. Paesold G, Nerlich AG, Boos N. Biological treatment strategies for disc degeneration: potentials and shortcomings. Eur Spine J, 2007, 16(4): 447-468.
6. Baksh D, Song L, Tuan RS. Adult mesenchymal stem cells: characterization, differentiation, and application in cell and gene therapy. J Cell Mol Med, 2004, 8(3): 301-316.
7. Aguiar DJ, Johnson SL, Oegema TR. Notochordal cells interact with nucleus pulposus cells: regulation of proteoglycan synthesis. Exp Cell Res, 1999, 246(1): 129-137.
8. Johnson WE, Evans H, Menage J, et al. Immunohistochemical detection of Schwann cells in innervated and vascularized human intervertebral discs. Spine (Phila Pa 1976), 2001, 26(23): 2550-2557.
9. Erwin WM, Las Heras F, Islam D, et al. The regenerative capacity of the notochordal cell: tissue constructs generated in vitro under hypoxic conditions. J Neurosurg Spine, 2009, 10(6): 513-521.
10. Risbud MV, Schaer TP, Shapiro IM. Toward an understanding of the role of notochordal cells in the adult intervertebral disc: from discord to accord. Dev Dyn, 2010, 239(8): 2141-2148.
11. Chai C, Leong KW. Biomaterials approach to expand and direct differentiation of stem cells. Mol Ther, 2007, 15(3): 467-480.
12. 严慧深, 吴小涛, 王运涛, 等. 骨髓间充质干细胞修复退变椎间盘中营养和分化效应的变化. 中华实验外科杂志, 2009, 26(6): 704-706.
13. Blanco JF, Graciani IF, Sanchez-Guijo FM, et al. Isolation and characterization of mesenchymal stromal cells from human degenerated nucleus pulposus. Spine (Phila Pa 1976), 2010, 35(26): 2259-2265.
14. Chen J, Yan W, Setton LA. Molecular phenotypes of notochordal cells purified from immature nucleus pulposus. Eur Spine J, 2006, 15 Suppl 3: S303-311.
15. 赵献峰, 刘浩, 丰干均, 等. 脊索细胞促进髓核软骨样细胞增殖及表型维持. 中国修复重建外科杂志, 2008, 22(8): 939-943.
16. Yang Z, Schmitt JF, Lee EH. Immunohistochemical analysis of human mesenchymal stem cells differentiating into chondrogenic, osteogenic, and adipogenic lineages. Methods Mol Biol, 2011, 698(1): 353-366.
17. Yang SH, Wu CC, Shih TT, et al. In vitro study on interaction between human nucleus pulposus cells and mesenchymal stem cells through paracrine stimulation. Spine (Phila Pa 1976), 2008, 33(18): 1951-1957.
18. Strassburg S, Richardson SM, Freemont AJ, et al. Co-culture induces mesenchymal stem cell differentiation and modulation of the degenerate human nucleus pulposus cell phenotype. Regen Med, 2010, 5(5): 701-711.
19. Erwin WM, Inman RD. Notochord cells regulate intervertebral disc chondrocyte proteoglycan production and cell proliferation. Spine (Phila Pa 1976), 2006, 31(10): 1094-1099.
20. Steck E, Bertram H, Abel R, et al. Induction of intervertebraldisc-like cells from adult mesenchymal stem cells. Stem Cells, 2005, 23(3): 403-411.
21. Ivkovic S, Yoon BS, Popoff SN, et al. Connective tissue growth factor coordinates chondrogenesis and angiogenesis during skeletal development. Development, 2003, 130(12): 2779-2791.
22. Korecki CL, Taboas JM, Tuan RS, et al. Notochordal cell conditioned medium stimulates mesenchymal stem cell differentiation toward a young nucleus pulposus phenotype. Stem Cell Res Ther, 2010, 1(2): 18.
23. Purmessur D, Schek RM, Abbott RD, et al. Notochordal conditioned media from tissue increases proteoglycan accumulation and promotes a healthy nucleus pulposus phenotype in human mesenchymal stem cells. Arthritis Res Ther, 2011, 13(3): R81.

Chinese Journal of Reparative and Reconstructive Surgery

PROMOTION EFFECT OF NOTOCHORDAL CELLS CONDITIONED MEDIUM ON PROLIFERATION ANDDIFFERENTIATION OF BONE MARROW MESENCHYMAL STEM CELLS/

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