CHONDROGENIC PHENOTYPE DIFFERENTIATION OF BONE MARROW MESENCHYMAL STEM CELLS INDUCED BY BONE MORPHOGENETIC PROTEIN 2 UNDER HYPOXIC MICROENVIRONMENT IN VITRO_Chinese Journal of Reparative and Reconstructive Surgery

Authors：

LIAO Xingen ^1,2 , WU Lihua ² , FU Mingfu ^1,2 , HE Dingwen ^1,2 , GU Yurong ¹ , CHEN Weicai ¹ , YIN Ming ¹

1First Department of Orthopedics, the Second Affiliated Hospital of Nanchang University, Nanchang Jiangxi, 330006, P.R.China;;
2Nanchang University Graduate School of Medicine.;

Corresponding author：

Keywords：

Bone morphogenetic protein 2; Hypoxia; Bone marrow mesenchymal stem cells; Chondrogenic phenotype differentiation Rat

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Objective To investigate the role of bone morphogenetic protein 2 (BMP-2) combined with hypoxic microenvironment in chondrogenic phenotype differentiation of bone marrow mesenchymal stem cells (BMSCs) of rat in vitro. Methods BMSCs were harvested from 4-week-old female Sprague Dawley rats. BMSCs at passage 2 were divided into 4 groups according different culture conditions: normoxia control group (group A), normoxia and BMP-2 group (group B), hypoxia control group (3% oxygen, group C), and hypoxia and BMP-2 group (group D). Then the cellular morphology was observed under inverted phase contrast microscope. Alcian blue immunohistochemical staining was used to detect the glycosaminoglycans (GAG), Western blot to detect collagen type II and hypoxia-inducible factor 1α (HIF-1α), and RT-PCR
to detect the expressions of chondrogenic related genes, osteogenic related genes, and hypoxia related genes. Results At 21 days after induction of BMP-2 and hypoxia (group D), BMSCs became round, cell density was significantly reduced, and lacuna-l ike cells were wrapped in cell matrix, while the changes were not observed in groups A, B, and C. Alcian blue staining in group D was significantly bluer than that in other groups, and staining became darker with induction time, and the cells were stained into pieces of deeply-stained blue at 21 days. Light staining was observed in the other groups at each time point. The expression level of collagen type II protein in group D was significantly higher than those in other groups (P lt; 0.05). HIF-1α protein expression levels of groups C and D were significantly higher than those of groups A and B (P lt; 0.05). The expressions of collagen II α1 (COL2 α1) and aggrecan mRNA (chondrogenic related genes) were highest in group D, while the expressions of COL1 α1, alkaline phosphatase, and runt-related transcri ption factor 2 mRNA (osteogenic related genes) were the highest in group B (P lt; 0.05). Compared with groups A and B, HIF-1α (hypoxic related genes) in groups C and D significantly increased (P lt; 0.05). Conclusion BMP-2 combined with hypoxia can induce differentiation of BMSCs into the chondrogenic phenotype, and inhibit osteoblast phenotype differentiation. HIF-1α is an important signaling molecule which is involved in the possible mechanism to promote chondrogenic differentiation process.

Citation： LIAO Xingen,WU Lihua,FU Mingfu,HE Dingwen,GU Yurong,CHEN Weicai,YIN Ming. CHONDROGENIC PHENOTYPE DIFFERENTIATION OF BONE MARROW MESENCHYMAL STEM CELLS INDUCED BY BONE MORPHOGENETIC PROTEIN 2 UNDER HYPOXIC MICROENVIRONMENT IN VITRO. Chinese Journal of Reparative and Reconstructive Surgery, 2012, 26(6): 743-748. doi: Copy

1.	Toh WS, Yang Z, Liu H, et al. Effects of culture conditions and bone morphogenetic protein 2 on extent of chondrogenesis from human embryonic stem cells. Stem Cells, 2007, 25(4): 950-960.
2.	Solorio LD, Vieregge EL, Dhami CD, et al. Engineered cartilage via self-assembled hmsc sheets with incorporated biodegradable gelatin microspheres releasing transforming growth factor-beta1. J Control Release, 2012, 158(2): 224-232.
3.	Danišovič L, Varga I, Polák S. Growth factors and chondrogenic differentiation of mesenchymal stem cells. Tissue Cell, 2012, 44(2): 69-73.
4.	Adams CS, Shapiro IM. The fate of the terminally differentiated chondrocyte: Evidence for microenvironmental regulation of chondrocyte apoptosis. Crit Rev Oral Biol Med, 2002, 13(6): 465-473.
5.	Herzog EL, Chai L, Krause DS. Plasticity of marrow-derived stem cells. Blood, 2003, 102(10): 3483-3493.
6.	Rocha B, Fernández-Puente P, Mateos J, et al. A quantitative proteomics approach for studying the chondrogenic differentiation process of mesenchymal stem cells. Osteoarthritis and Cartilage, 2011, 19S1: S46-S47.
7.	刘哲, 李士勇, 宋玉林, 等. 绿原酸对缺氧环境下干细胞来源软骨样细胞凋亡的影响. 中国药理学通报, 2011, 27(2): 206-210.
8.	Steinert AF, Proffen B, Kunz M, et al. Hypertrophy is induced during the in vitro chondrogenic differentiation of human mesenchymal stem cells by bone morphogenetic protein-2 and bone morphogenetic protein-4 gene transfer. Arthritis Res Ther, 2009, 11(5): R148.
9.	Rosen V. BMP2 signaling in bone development and repair. Cytokine Growth Factor Rev, 2009, 20(5-6): 475-480.
10.	Wang DW, Fermor B, Gimble JM, et al. Influence of oxygen on the proliferation and metabolism of adipose derived adult stem cells. J Cell Physiol, 2005, 204(1): 184-191.
11.	Markway BD, Tan GK, Brooke G, et al. Enhanced chondrogenic differentiation of human bone marrow-derived mesenchymal stem cells in low oxygen environment micropellet cultures. Cell Transplant, 2010, 19(1): 29-42.
12.	Kanichai M, Ferguson D, Prendergast PJ, et al. Hypoxia promotes chondrogenesis in rat mesenchymal stem cells: A role for akt and hypoxia-inducible factor (hif)-1alpha. J Cell Physiol, 2008, 216(3): 708-715.
13.	Kay A, Richardson J, Forsyth NR. Physiological normoxia and chondrogenic potential of chondrocytes. Front Biosci (Elite Ed), 2011, 3: 1365-1374.
14.	D’Ippolito G, Diabira S, Howard GA, et al. Low oxygen tension inhibits osteogenic differentiation and enhances stemness of human miami cells. Bone, 2006, 39(3): 513-522.
15.	Merceron C, Vinatier C, Portron S, et al. Differential effects of hypoxia on osteochondrogenic potential of human adipose-derived stem cells. Am J Physiol Cell Physiol, 2010, 298(2): C355-364.
16.	Singh M, Pierpoint M, Mikos AG, et al. Chondrogenic differentiation of neonatal human dermal fibroblasts encapsulated in alginate beads with hydrostatic compression under hypoxic conditions in the presence of bone morphogenetic protein-2. J Biomed Mater Res A, 2011, 98(3): 412-424.
17.	Pelaez D, Arita N, Cheung HS. Extracellular signal-regulated kinase (ERK) dictates osteogenic and/or chondrogenic lineage commitment of mesenchymal stem cells under dynamic compression. Biochem Biophys Res Commun, 2012, 417(4): 1286-1291.
18.	Sheehy EJ, Buckley CT, Kelly DJ. Oxygen tension regulates the osteogenic, chondrogenic and endochondral phenotype of bone marrow derived mesenchymal stem cells. Biochem Biophys Res Commun, 2012, 417(1): 305-310.
19.	Diaz-Prado S, Muiños-Lopez E, Hermida-Gómez T, et al. Chondrogenic differentiation of bone marrow mesenchymal stem cells (BM-MSCS) grown on collagen porous scaffolds. Osteoarthritis and Cartilage, 2011, 19S1: S221.
20.	Ronzière MC, Perrier E, Mallein-Gerin F, et al. Chondrogenic potential of bone marrow- and adipose tissue-derived adult human mesenchymal stem cells. Biomed Mater Eng, 2010, 20(3): 145-158.
21.	Bosetti M, Boccafoschi F, Leigheb M, et al. Chondrogenic induction of human mesenchymal stem cells using combined growth factors for cartilage tissue engineering. J Tissue Eng Regen Med, 2011, 6(3): 205-213.

1. Toh WS, Yang Z, Liu H, et al. Effects of culture conditions and bone morphogenetic protein 2 on extent of chondrogenesis from human embryonic stem cells. Stem Cells, 2007, 25(4): 950-960.
2. Solorio LD, Vieregge EL, Dhami CD, et al. Engineered cartilage via self-assembled hmsc sheets with incorporated biodegradable gelatin microspheres releasing transforming growth factor-beta1. J Control Release, 2012, 158(2): 224-232.
3. Danišovič L, Varga I, Polák S. Growth factors and chondrogenic differentiation of mesenchymal stem cells. Tissue Cell, 2012, 44(2): 69-73.
4. Adams CS, Shapiro IM. The fate of the terminally differentiated chondrocyte: Evidence for microenvironmental regulation of chondrocyte apoptosis. Crit Rev Oral Biol Med, 2002, 13(6): 465-473.
5. Herzog EL, Chai L, Krause DS. Plasticity of marrow-derived stem cells. Blood, 2003, 102(10): 3483-3493.
6. Rocha B, Fernández-Puente P, Mateos J, et al. A quantitative proteomics approach for studying the chondrogenic differentiation process of mesenchymal stem cells. Osteoarthritis and Cartilage, 2011, 19S1: S46-S47.
7. 刘哲, 李士勇, 宋玉林, 等. 绿原酸对缺氧环境下干细胞来源软骨样细胞凋亡的影响. 中国药理学通报, 2011, 27(2): 206-210.
8. Steinert AF, Proffen B, Kunz M, et al. Hypertrophy is induced during the in vitro chondrogenic differentiation of human mesenchymal stem cells by bone morphogenetic protein-2 and bone morphogenetic protein-4 gene transfer. Arthritis Res Ther, 2009, 11(5): R148.
9. Rosen V. BMP2 signaling in bone development and repair. Cytokine Growth Factor Rev, 2009, 20(5-6): 475-480.
10. Wang DW, Fermor B, Gimble JM, et al. Influence of oxygen on the proliferation and metabolism of adipose derived adult stem cells. J Cell Physiol, 2005, 204(1): 184-191.
11. Markway BD, Tan GK, Brooke G, et al. Enhanced chondrogenic differentiation of human bone marrow-derived mesenchymal stem cells in low oxygen environment micropellet cultures. Cell Transplant, 2010, 19(1): 29-42.
12. Kanichai M, Ferguson D, Prendergast PJ, et al. Hypoxia promotes chondrogenesis in rat mesenchymal stem cells: A role for akt and hypoxia-inducible factor (hif)-1alpha. J Cell Physiol, 2008, 216(3): 708-715.
13. Kay A, Richardson J, Forsyth NR. Physiological normoxia and chondrogenic potential of chondrocytes. Front Biosci (Elite Ed), 2011, 3: 1365-1374.
14. D’Ippolito G, Diabira S, Howard GA, et al. Low oxygen tension inhibits osteogenic differentiation and enhances stemness of human miami cells. Bone, 2006, 39(3): 513-522.
15. Merceron C, Vinatier C, Portron S, et al. Differential effects of hypoxia on osteochondrogenic potential of human adipose-derived stem cells. Am J Physiol Cell Physiol, 2010, 298(2): C355-364.
16. Singh M, Pierpoint M, Mikos AG, et al. Chondrogenic differentiation of neonatal human dermal fibroblasts encapsulated in alginate beads with hydrostatic compression under hypoxic conditions in the presence of bone morphogenetic protein-2. J Biomed Mater Res A, 2011, 98(3): 412-424.
17. Pelaez D, Arita N, Cheung HS. Extracellular signal-regulated kinase (ERK) dictates osteogenic and/or chondrogenic lineage commitment of mesenchymal stem cells under dynamic compression. Biochem Biophys Res Commun, 2012, 417(4): 1286-1291.
18. Sheehy EJ, Buckley CT, Kelly DJ. Oxygen tension regulates the osteogenic, chondrogenic and endochondral phenotype of bone marrow derived mesenchymal stem cells. Biochem Biophys Res Commun, 2012, 417(1): 305-310.
19. Diaz-Prado S, Muiños-Lopez E, Hermida-Gómez T, et al. Chondrogenic differentiation of bone marrow mesenchymal stem cells (BM-MSCS) grown on collagen porous scaffolds. Osteoarthritis and Cartilage, 2011, 19S1: S221.
20. Ronzière MC, Perrier E, Mallein-Gerin F, et al. Chondrogenic potential of bone marrow- and adipose tissue-derived adult human mesenchymal stem cells. Biomed Mater Eng, 2010, 20(3): 145-158.
21. Bosetti M, Boccafoschi F, Leigheb M, et al. Chondrogenic induction of human mesenchymal stem cells using combined growth factors for cartilage tissue engineering. J Tissue Eng Regen Med, 2011, 6(3): 205-213.

Chinese Journal of Reparative and Reconstructive Surgery

CHONDROGENIC PHENOTYPE DIFFERENTIATION OF BONE MARROW MESENCHYMAL STEM CELLS INDUCED BY BONE MORPHOGENETIC PROTEIN 2 UNDER HYPOXIC MICROENVIRONMENT IN VITRO

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