CONDITIONS OF SYNOVIAL MESENCHYMAL STEM CELLS DIFFERENTIATING INTO FIBROCARTILAGE CELLS_Chinese Journal of Reparative and Reconstructive Surgery

Authors：

FUPeiliang , CONGRuijun ^Δ , CHENSong , ZHANGLei , DINGZheru , ZHOUQi , LILintao , XUZhenyu ,  WUYuli , WUHaishan

Department of Joint Surgery, Shanghai Changzheng Hospital, Shanghai, 200003, P. R. China;

Corresponding author：

WUYuli, Email: wuyuli6019@189.cn

Keywords：

Synovial derived mesenchymal stem cells; Fibrocartilage cells; Orthogonal experiment; Differentiation condition

DOI：

10.7507/1002-1892.20150018

Video：

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Objective To explore the conditions of synovial derived mesenchymal stem cells (SMSCs) differentiating into the fibrocartilage cells by using the orthogonal experiment. Methods The synovium was harvested from 5 adult New Zealand white rabbits, and SMSCs were separated by adherence method. The flow cytometry and multidirectional differentiation method were used to identify the SMSCs. The conditions were found from the preliminary experiment and literature review. The missing test was carried out to screen the conditions and then 12 conditions were used for the orthogonal experiment, including transforming growth factor β1 (TGF-β₁), bone morphogenic protein 2 (BMP-2), dexamethasone (DEX), proline, ascorbic acid (ASA), pyruvic acid, insulin+transferrin+selenious acid pre-mixed solution (ITS), bovin serum albumin (BSA), basic fibroblast growth factor (bFGF), intermittent hydraulic pressure (IHP), bone morphogenic protein 7 (BMP-7), and insulin-like growth factor (IGF). The L60 (212) orthogonal experiment was designed using the SPSS 18.0 with 2 level conditions and the cells were induced to differentiate on the small intestinal submucosa (SIS)-3D scaffold. The CD151⁺/CD44⁺ cells were detected with the flow cytometry and then the differentiation rate was recorded. The immumohistochemical staining, cellular morphology, toluidine blue staining, and semi-quantitative RT-PCR examination for the gene expressions of sex determining region Y (SRY) -box 9 gene (Sox9), aggrecan gene (AGN), collagen type I gene (Col I), collagen type Ⅱ gene (Col Ⅱ), collagen type IX gene (Col IX) were used for result confirmation. The differentiation rate was calculated as the product of CD151/CD44⁺ cells and cells with Col I high expression. The grow curve was detected with the DNA abundance using the PicoGreen Assay. The visual observation and the variances analysis among the variable were used to evaluate the result of the orthogonal experiment, 1 level interaction was considered. The q-test and the least significant difference (LDS) were used for the variance analysis with a type Ⅲ calibration model. The test criteria (α) was 0.05. Results The cells were certified as SMSCs, the double-time of the cells was 28 hours. During the differentiation into the fibrocartilage, the volume of the SIS-3D scaffold enlarged double every 5 days. The scaffolds were positively stained by toluidine blue at 14 days. The visual observation showed that high levels of TGF-β₁ and BMP-7 were optimum for the differentiation, and BMP-7 showed the interaction with BMP-2. The conditions of DEX, ASA, ITS, transferrin, bFGF showed decreasing promotional function by degrees, and the model showed the perfect relevance. P value was 0.000 according to the variance analysis. The intercept analysis showed different independent variables brought about variant contribution; the TGF-β₁, ASA, bFGF, IGF, and BMP-7 were more remarkable, which were similar to the visual observation. Conclusion In the process of the SMSCs differentiation into the fibrocartilage, the concentrations of TGF-β₁, ASA, bFGF, and IGF reasonably can improve the conversion rate of the fibrocartilage cells. The accurate conditions of the regulatory factor should be explored further.

Citation： FUPeiliang, CONGRuijun, CHENSong, ZHANGLei, DINGZheru, ZHOUQi, LILintao, XUZhenyu, WUYuli, WUHaishan. CONDITIONS OF SYNOVIAL MESENCHYMAL STEM CELLS DIFFERENTIATING INTO FIBROCARTILAGE CELLS. Chinese Journal of Reparative and Reconstructive Surgery, 2015, 29(1): 81-91. doi: 10.7507/1002-1892.20150018 Copy

1.	Xu T, Wu M, Feng J, et al. RhoA/Rho kinase signaling regulates transforming growth factor-beta1-induced chondrogenesis and actin organization of synovium-derived mesenchymal stem cells through interaction with the Smad pathway. Int J Mol Med, 2012, 30(5):1119-1125.
2.	Fan J, Ren L, Liang R, et al. Chondrogenesis of synovium-derived mesenchymal stem cells in photopolymerizing hydrogel scaffolds. J Biomater Sci Polym Ed, 2010, 21(12):1653-1667.
3.	Vanneaux V, Farge-Bancel D, Lecourt S, et al. Expression of transforming growth factor beta receptor Ⅱ in mesenchymal stem cells from systemic sclerosis patients. BMJ Open, 2013, 3(1). pii:e001890.
4.	Schagemann JC, Paul S, Casper ME, et al. Chondrogenic differentiation of bone marrow-derived mesenchymal stromal cells via biomimetic and bioactive poly-epsilon-caprolactone scaffolds. J Biomed Mater Res A, 2013, 101(6):1620-1628.
5.	Kim M, Erickson IE, Choudhury M, et al. Transient exposure to TGF-beta3 improves the functional chondrogenesis of MSC-laden hyaluronic acid hydrogels. J Mech Behav Biomed Mater, 2012, 11:92-101.
6.	Mueller MB, Blunk T, Appel B, et al. Insulin is essential for in vitro chondrogenesis of mesenchymal progenitor cells and influences chondrogenesis in a dose-dependent manner. Int Orthop, 2013, 37(1):153-158.
7.	Cieslik KA, Trial J, Carlson S, et al. Aberrant differentiation of fibroblast progenitors contributes to fibrosis in the aged murine heart:role of elevated circulating insulin levels. FASEB J, 2013, 27(4):1761-1771.
8.	Fan J, Gong Y, Ren L, et al. In vitro engineered cartilage using synovium-derived mesenchymal stem cells with injectable gellan hydrogels. Acta Biomater, 2010, 6(3):1178-1185.
9.	Nogami M, Tsuno H, Koike C, et al. Isolation and characterization of human amniotic mesenchymal stem cells and their chondrogenic differentiation. Transplantation, 2012, 93(12):1221-1228.
10.	Shen B, Wei A, Whittaker S, et al. The role of BMP-7 in chondrogenic and osteogenic differentiation of human bone marrow multipotent mesenchymal stromal cells in vitro. J Cell Biochem, 2010, 109(2):406-416.
11.	Sekiya I, Larson BL, Vuoristo JT, et al. Comparison of effect of BMP-2, -4, and -6 on in vitro cartilage formation of human adult stem cells from bone marrow stroma. Cell Tissue Res, 2005, 320(2):269-276.
12.	Boeuf S, Richter W. Chondrogenesis of mesenchymal stem cells:role of tissue source and inducing factors. Stem Cell Res Ther, 2010, 1(4):31.
13.	Valhmu WB, Stazzone EJ, Bachrach NM, et al. Load-controlled compression of articular cartilage induces a transient stimulation of aggrecan gene expression. Arch Biochem Biophys, 1998, 353(1):29-36.
14.	Wong M, Siegrist M, Cao X. Cyclic compression of articular cartilage explants is associated with progressive consolidation and altered expression pattern of extracellular matrix proteins. Matrix Biol, 1999, 18(4):391-399.
15.	Feng JQ, Chen D, Cooney AJ, et al. The mouse bone morphogenetic protein-4 gene. Analysis of promoter utilization in fetal rat calvarial osteoblasts and regulation by COUP-TFI orphan receptor. J Biol Chem, 1995, 270(47):28364-28373.
16.	Feng JQ, Harris MA, Ghosh-Choudhury N, et al. Structure and sequence of mouse bone morphogenetic protein-2 gene (BMP-2):comparison of the structures and promoter regions of BMP-2 and BMP-4 genes. Biochim Biophys Acta, 1994, 1218(2):221-224.
17.	Rui YF, Lui PP, Ni M, et al. Mechanical loading increased BMP-2 expression which promoted osteogenic differentiation of tendonderived stem cells. J Orthop Res, 2011, 29(3):390-396.
18.	Lui PP, Fu SC, Chan LS, et al. Chondrocyte phenotype and ectopic ossification in collagenase-induced tendon degeneration. J Histochem Cytochem, 2009, 57(2):91-100.
19.	Ando K, Imai S, Isoya E, et al. Effect of dynamic compressive loading and its combination with a growth factor on the chondrocytic phenotype of 3-dimensional scaffold-embedded chondrocytes. Acta Orthop, 2009, 80(6):724-733.
20.	Chikazu D, Li X, Kawaguchi H, et al. Bone morphogenetic protein 2 induces cyclo-oxygenase 2 in osteoblasts via a Cbfa1 binding site:role in effects of bone morphogenetic protein 2 in vitro and in vivo. 2002. J Bone Miner Res, 2005, 20(10):1888-1898.
21.	Siddhivarn C, Banes A, Champagne C, et al. Mechanical loading and delta12prostaglandin J2 induce bone morphogenetic protein-2, peroxisome proliferator-activated receptor gamma-1, and bone nodule formation in an osteoblastic cell line. J Periodontal Res, 2007, 42(5):383-392.
22.	Gharibi B, Hughes FJ. Effects of medium supplements on proliferation, differentiation potential, and in vitro expansion of mesenchymal stem cells. Stem Cells Transl Med, 2012, 1(11):771-782.
23.	Liu Y, Goldberg AJ, Dennis JE, et al. One-step derivation of mesenchymal stem cell (MSC)-like cells from human pluripotent stem cells on a fibrillar collagen coating. PLoS One, 2012, 7(3):e33225.
24.	Kosmacheva SM, Volk MV, Yeustratenka TA, et al. In vitro growth of human umbilical blood mesenchymal stem cells and their differentiation into chondrocytes and osteoblasts. Bull Exp Biol Med, 2008, 145(1):141-145.
25.	Li Q, Liu T, Zhang L, et al. The role of bFGF in down-regulating alpha-SMA expression of chondrogenically induced BMSCs and preventing the shrinkage of BMSC engineered cartilage. Biomaterials, 2011, 32(21):4773-4781.
26.	Kim JH, Lee MC, Seong SC, et al. Enhanced proliferation and chondrogenic differentiation of human synovium-derived stem cells expanded with basic fibroblast growth factor. Tissue Eng Part A, 2011, 17(7-8):991-1002.
27.	Lee S, Kim JH, Jo CH, et al. Effect of serum and growth factors on chondrogenic differentiation of synovium-derived stromal cells. Tissue Eng Part A, 2009, 15(11):3401-3415.
28.	Liu Y, Wagner DR. Effect of expansion media containing fibroblast growth factor-2 and dexamethasone on the chondrogenic potential of human adipose-derived stromal cells. Cell Biol Int, 2012, 36(7):611-615.

1. Xu T, Wu M, Feng J, et al. RhoA/Rho kinase signaling regulates transforming growth factor-beta1-induced chondrogenesis and actin organization of synovium-derived mesenchymal stem cells through interaction with the Smad pathway. Int J Mol Med, 2012, 30(5):1119-1125.
2. Fan J, Ren L, Liang R, et al. Chondrogenesis of synovium-derived mesenchymal stem cells in photopolymerizing hydrogel scaffolds. J Biomater Sci Polym Ed, 2010, 21(12):1653-1667.
3. Vanneaux V, Farge-Bancel D, Lecourt S, et al. Expression of transforming growth factor beta receptor Ⅱ in mesenchymal stem cells from systemic sclerosis patients. BMJ Open, 2013, 3(1). pii:e001890.
4. Schagemann JC, Paul S, Casper ME, et al. Chondrogenic differentiation of bone marrow-derived mesenchymal stromal cells via biomimetic and bioactive poly-epsilon-caprolactone scaffolds. J Biomed Mater Res A, 2013, 101(6):1620-1628.
5. Kim M, Erickson IE, Choudhury M, et al. Transient exposure to TGF-beta3 improves the functional chondrogenesis of MSC-laden hyaluronic acid hydrogels. J Mech Behav Biomed Mater, 2012, 11:92-101.
6. Mueller MB, Blunk T, Appel B, et al. Insulin is essential for in vitro chondrogenesis of mesenchymal progenitor cells and influences chondrogenesis in a dose-dependent manner. Int Orthop, 2013, 37(1):153-158.
7. Cieslik KA, Trial J, Carlson S, et al. Aberrant differentiation of fibroblast progenitors contributes to fibrosis in the aged murine heart:role of elevated circulating insulin levels. FASEB J, 2013, 27(4):1761-1771.
8. Fan J, Gong Y, Ren L, et al. In vitro engineered cartilage using synovium-derived mesenchymal stem cells with injectable gellan hydrogels. Acta Biomater, 2010, 6(3):1178-1185.
9. Nogami M, Tsuno H, Koike C, et al. Isolation and characterization of human amniotic mesenchymal stem cells and their chondrogenic differentiation. Transplantation, 2012, 93(12):1221-1228.
10. Shen B, Wei A, Whittaker S, et al. The role of BMP-7 in chondrogenic and osteogenic differentiation of human bone marrow multipotent mesenchymal stromal cells in vitro. J Cell Biochem, 2010, 109(2):406-416.
11. Sekiya I, Larson BL, Vuoristo JT, et al. Comparison of effect of BMP-2, -4, and -6 on in vitro cartilage formation of human adult stem cells from bone marrow stroma. Cell Tissue Res, 2005, 320(2):269-276.
12. Boeuf S, Richter W. Chondrogenesis of mesenchymal stem cells:role of tissue source and inducing factors. Stem Cell Res Ther, 2010, 1(4):31.
13. Valhmu WB, Stazzone EJ, Bachrach NM, et al. Load-controlled compression of articular cartilage induces a transient stimulation of aggrecan gene expression. Arch Biochem Biophys, 1998, 353(1):29-36.
14. Wong M, Siegrist M, Cao X. Cyclic compression of articular cartilage explants is associated with progressive consolidation and altered expression pattern of extracellular matrix proteins. Matrix Biol, 1999, 18(4):391-399.
15. Feng JQ, Chen D, Cooney AJ, et al. The mouse bone morphogenetic protein-4 gene. Analysis of promoter utilization in fetal rat calvarial osteoblasts and regulation by COUP-TFI orphan receptor. J Biol Chem, 1995, 270(47):28364-28373.
16. Feng JQ, Harris MA, Ghosh-Choudhury N, et al. Structure and sequence of mouse bone morphogenetic protein-2 gene (BMP-2):comparison of the structures and promoter regions of BMP-2 and BMP-4 genes. Biochim Biophys Acta, 1994, 1218(2):221-224.
17. Rui YF, Lui PP, Ni M, et al. Mechanical loading increased BMP-2 expression which promoted osteogenic differentiation of tendonderived stem cells. J Orthop Res, 2011, 29(3):390-396.
18. Lui PP, Fu SC, Chan LS, et al. Chondrocyte phenotype and ectopic ossification in collagenase-induced tendon degeneration. J Histochem Cytochem, 2009, 57(2):91-100.
19. Ando K, Imai S, Isoya E, et al. Effect of dynamic compressive loading and its combination with a growth factor on the chondrocytic phenotype of 3-dimensional scaffold-embedded chondrocytes. Acta Orthop, 2009, 80(6):724-733.
20. Chikazu D, Li X, Kawaguchi H, et al. Bone morphogenetic protein 2 induces cyclo-oxygenase 2 in osteoblasts via a Cbfa1 binding site:role in effects of bone morphogenetic protein 2 in vitro and in vivo. 2002. J Bone Miner Res, 2005, 20(10):1888-1898.
21. Siddhivarn C, Banes A, Champagne C, et al. Mechanical loading and delta12prostaglandin J2 induce bone morphogenetic protein-2, peroxisome proliferator-activated receptor gamma-1, and bone nodule formation in an osteoblastic cell line. J Periodontal Res, 2007, 42(5):383-392.
22. Gharibi B, Hughes FJ. Effects of medium supplements on proliferation, differentiation potential, and in vitro expansion of mesenchymal stem cells. Stem Cells Transl Med, 2012, 1(11):771-782.
23. Liu Y, Goldberg AJ, Dennis JE, et al. One-step derivation of mesenchymal stem cell (MSC)-like cells from human pluripotent stem cells on a fibrillar collagen coating. PLoS One, 2012, 7(3):e33225.
24. Kosmacheva SM, Volk MV, Yeustratenka TA, et al. In vitro growth of human umbilical blood mesenchymal stem cells and their differentiation into chondrocytes and osteoblasts. Bull Exp Biol Med, 2008, 145(1):141-145.
25. Li Q, Liu T, Zhang L, et al. The role of bFGF in down-regulating alpha-SMA expression of chondrogenically induced BMSCs and preventing the shrinkage of BMSC engineered cartilage. Biomaterials, 2011, 32(21):4773-4781.
26. Kim JH, Lee MC, Seong SC, et al. Enhanced proliferation and chondrogenic differentiation of human synovium-derived stem cells expanded with basic fibroblast growth factor. Tissue Eng Part A, 2011, 17(7-8):991-1002.
27. Lee S, Kim JH, Jo CH, et al. Effect of serum and growth factors on chondrogenic differentiation of synovium-derived stromal cells. Tissue Eng Part A, 2009, 15(11):3401-3415.
28. Liu Y, Wagner DR. Effect of expansion media containing fibroblast growth factor-2 and dexamethasone on the chondrogenic potential of human adipose-derived stromal cells. Cell Biol Int, 2012, 36(7):611-615.

Chinese Journal of Reparative and Reconstructive Surgery

CONDITIONS OF SYNOVIAL MESENCHYMAL STEM CELLS DIFFERENTIATING INTO FIBROCARTILAGE CELLS

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