Tenogenic differentiation of adipose-derived stem cells in hypoxia

doi:10.3969/j.issn.2095-4344.2014.01.014

Abstract

Abstract:

BACKGROUND: Adipose-derived mesenchymal stem cells are gaining widespread interest in the Achilles tendon tissue engineering and regeneration, and an enabling environment (oxygen concentration) for cell induction and differentiation is particularly necessary.
OBJECTIVE: To co-culture adipose-derived mesenchymal stem cells with primary tenocytes in standard culture condition (20% O2 tension) and in an atmosphere of reduced oxygen (2% O2 tension) in order to determine whether the two conditions differ in their effect on tenogenic differentiation.
METHODS: Tenocytes were isolated via serial expansion in culture from several Sprague-Dawley rats’ Achilles tendons. Adipose-derived mesenchymal stem cells were purchased. After one passage, adipose-derived mesenchymal stem cells were indirectly co-cultured with tenocytes in standard culture condition (20% O2 tension) and in an atmosphere of reduced oxygen (2% O2 tension). Collagen 1, collagen 3, Tenomodulin, Thrombospondin-4, Scleraxis levels were compared for each culture condition at 7, 14 and 21 days following co-culturing. Immunofluorescence staining was performed to evaluate production of collagen 1 and Thrombospondin-4.
RESULTS AND CONCLUSION: Following indirect co-culturing, hypoxic differentiated adipose-derived mesenchymal stem cells expressed higher levels of tendon-related genes and proteins than normoxic controls, which suggest that oxygen levels can significantly affect tenogenic differentiation, and hypoxia is advantageous for efficient differentiation of adipose-derived mesenchymal stem cells in vitro for tendon tissue engineering.

中国组织工程研究杂志出版内容重点：干细胞；骨髓干细胞；造血干细胞；脂肪干细胞；肿瘤干细胞；胚胎干细胞；脐带脐血干细胞；干细胞诱导；干细胞分化；组织工程

全文链接：

Key words: stem cells, mesenchymal stem cells, Achilles tendon, anoxia

CLC Number:

R394.2

Cheng Tao, Yu Yang, Zhang Xiao-lei, Zheng Yi-jing, Lou Yu-liang, Hong Jian-jun. Tenogenic differentiation of adipose-derived stem cells in hypoxia[J]. Chinese Journal of Tissue Engineering Research, 2014, 18(1): 81-87.

Figures/Tables 3

2.2 实时荧光定量PCR检测脂肪间充质干细胞与跟腱细胞共培养第7天和第14天，常氧组(体积分数20%O2)与低氧组(体积分数2%O2)的胶原蛋白Ⅰ相对表达量分别上升为诱导前的8.6，11.9倍和11，16.8倍，2组间差异无显著性意义(P=0.37/0.16)。当第21天时，常氧组胶原蛋白Ⅰ表达量为诱导前的20倍，低氧组为33.8倍，在不同氧体积分数(2% vs. 20%)条件下的胶原蛋白Ⅰ表达量差异有显著性意义(P=0.004)(图2A)。 2.3 胶原蛋白Ⅰ与Thbs4免疫荧光染色在诱导分化的第7，14，21天，常氧组胶原蛋白Ⅲ的表达量分别为诱导前的4.6，14和19倍，而低氧组为8，21和61倍，只有在第7天时，2组差异无显著性意义(P=0.08，图2B)。常氧组的Tnmd相对表达量分别为诱导前的7.4，16.4和26.4倍，低氧组为9.8，19.7和40倍，在第21天时，2组间Tnmd的表达量差异有显著性意义(P=0.022，图2C)。常氧组的Thbs4在诱导分化第7，14，21天相对表达量分别为诱导前的5.3，11.2和18.7倍，而低氧组的表达量呈现明显上升趋势，分别达到诱导前的8.4，18.2和37.7倍；在第14和21天，2组相对表达量差异有显著性意义(P < 0.05)，(图2D)。在共培养的第14和21天，Scx相对表达量与上述Thbs4基本一致，也在第14和21天时，两组差异有显著性意义(P < 0.05)(图2E)。为进一步验证实时荧光定量PCR的结果，采用免疫荧光染色法进一步观察胶原蛋白Ⅰ和Thbs4的蛋白表达量。胶原蛋白Ⅰ与Thbs4在2种氧浓度条件下，在诱导前(第0天)脂肪间充质干细胞2个指标均阴性表达，诱导分化后的染色强度均随共培养时间的延长而逐渐增强。胶原蛋白Ⅰ在第21天时，低氧组的荧光染色强度明显要高于常氧组(图3)，这与实时荧光定量PCR结果基本相符。低氧组中的Thbs4在第14天时荧光染色就已明显呈阳性表达，在随后培养过程中逐渐增强。相比而言，常氧组Thbs4荧光染色增强幅度则比较缓和(图4)。"

References

[1] Pittenger M, Mackay A, Beck S, et al. Multilineage potential of adult human mesenchymal stem cells. Science. 1999;284 (5411):143-147.

[2] Uysal A, Mizuno H. Tendon regeneration and repair with adipose derived stem cells. Curr Stem Cell Res Ther. 2010; 5(2):161-167.

[3] Krampera M, Aprili G, Franchini M, et al. Mesenchymal stem cells for bone, cartilage, tendon and skeletal muscle repair. Bone. 2006;39(4):678-683.

[4] Barboni B, Russo V, Curini V, et al. Achilles Tendon Regeneration Can Be Improved by Amniotic Epithelial Cells Allotransplantation. Cell Transplant. 2012;21(19):2377- 2395.

[5] Obaid H, Connell D. Cell therapy in tendon disorders: what is the current evidence? Am J Sports Med. 2010; 38(10):2123- 2132.

[6] Siddiqui NA, Wong JM, Khan WS, et al. Stem cells for tendon and ligament tissue engineering and regeneration. J Stem Cells. 2010; 5(4):187-194.

[7] Yin Z, Chen X, Chen JL, et al. Stem cells for tendon tissue engineering and regeneration. Expert Opin Biol Ther. 2010; 10(5):689-700.

[8] Hogan MV, Bagayoko K, James R, et al. Tissue engineering solutions for tendon repair. J Am Acad Orthop Surg. 2011; 19(3):134-142.

[9] Ahmad Z, Wardale J, Brooks R, et al. Exploring the application of stem cells in tendon repair and regeneration. Arthroscopy. 2012; 28(7):1018-1029.

[10] Uysal AC, Mizuno H. Differentiation of adipose-derived stem cells for tendon repair. Methods Mol Biol. 2011; 702:443-51.

[11] Martinello T, Bronzini I, Volpin A, et al. Successful recellularization of human tendon scaffolds using adipose-derived mesenchymal stem cells and collagen gel. J Tissue Eng Regen Med. 2012.

[12] Raabe O, Shell K, Fietz D, et al. Tenogenic differentiation of equine adipose-tissue-derived stem cells under the influence of tensile strain, growth differentiation factors and various oxygen tensions. Cell Tissue Res. 2013; 352(3):509-521.

[13] James R, Kumbar SG, Laurencin CT, et al. Tendon tissue engineering: adipose-derived stem cell and GDF-5 mediated regeneration using electrospun matrix systems. Biomed Mater. 2011; 6(2):025011.

[14] Matsumoto A, Sowers AL, Koscielniak JW, et al. Absolute oxygen tension pO(2) in murine fatty and muscle tissue as determined by EPR. Magn Reson Med. 2005; 54(6): 1530-1535.

[15] 侯凯,李梅,杨逸昆,等.SD大鼠脂肪源间充质干细胞分离培养特性及影响因素[J].中国组织工程研究与临床康复, 2011,15(1): 29-32.

[16] 姚素艳,杨依勇,秦书俭,等.脂肪间充质干细胞的分离培养和鉴定[J].中国组织工程研究与临床康复, 2010,14(27):5067-5070.

[17] Valentina C, Russo V, Giacinto OD, et al. Tenogenic Differentiation of Ovine Amniotic Stem Cells Co-Cultured with Tenocytes. Trends Veter Sci. 2013: 45-49.

[18] Barboni B, Curini V, Russo V, et al. Indirect co-culture with tendons or tenocytes can program amniotic epithelial cells towards stepwise tenogenic differentiation. PLoS One. 2012; 7(2):e30974.

[19] Zuk P, Zhu M, Ashjian P, et al. Human adipose tissue is a source of multipotent stem cells. Mol Biol Cell. 2002; 13(12): 4279-4295.

[20] Park A, Hogan MV, Kesturu GS, et al. Adipose-derived mesenchymal stem cells treated with growth differentiation factor-5 express tendon-specific markers. Tissue Eng Part A. 2010;16(9):2941-51.

[21] Uysal CA, Tobita M, Hyakusoku H, et al. Adipose-derived stem cells enhance primary tendon repair: biomechanical and immunohistochemical evaluation. J Plast Reconstr Aesthet Surg. 2012; 65(12):1712-1719.

[22] Fehrer C, Brunauer R, Laschober G. et al. Reduced oxygen tension attenuates differentiation capacity of human mesenchymal stem cells and prolongs their lifespan. Aging Cell. 2007; 6(6):745-757.

[23] Benjamin M, Ralphs JR. Tendons and ligaments--an overview. Histol Histopathol. 1997; 12(4):1135-1144.

[24] 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.

[25] Valorani M, Montelatici E, Germani A, et al. Pre-culturing human adipose tissue mesenchymal stem cells under hypoxia increases their adipogenic and osteogenic differentiation potentials. Cell Prolif. 2012;45(3):225-238.

[26] Rosova I, Dao M, Capoccia B, et al. Hypoxic preconditioning results in increased motility and improved therapeutic potential of human mesenchymal stem cells. Stem Cells. 2008; 26(8): 2173-2182.

[27] Luo Q, Song G, Song Y, et al. Indirect co-culture with tenocytes promotes proliferation and mRNA expression of tendon/ligament related genes in rat bone marrow mesenchymal stem cells. Cytotechnology. 2009;1(1-2): 1-10.

[28] Wei Y, Gong K, Zheng Z, et al. Schwann-like cell differentiation of rat adipose-derived stem cells by indirect co-culture with Schwann cells in vitro. Cell Prolif. 2010;43(6): 606-616.

[29] Schneider PR, Buhrmann C, Mobasheri A, et al. Three-dimensional high-density co-culture with primary tenocytes induces tenogenic differentiation in mesenchymal stem cells. J Orthop Res. 2011; 29(9): 1351-1360.

[30] Gerstenfeld LC, Barnes GL, Shea CM, et al. Osteogenic differentiation is selectively promoted by morphogenetic signals from chondrocytes and synergized by a nutrient rich growth environment. Connect Tissue Res. 2003; 44 Suppl 1: 85-91.

[31] Shimode K, Iwasaki N, Majima T, et al. Bone marrow stromal cells act as feeder cells for tendon fibroblasts through soluble factors. Tissue Eng. 2007; 13(2): 333-341.

[32] Shukunami C, Takimoto A, Oro M, et al. Scleraxis positively regulates the expression of tenomodulin, a differentiation marker of tenocytes. Dev Biol. 2006; 298(1):234-247.

[33] Qi J, Dmochowski JM, Banes AN, et al. Differential expression and cellular localization of novel isoforms of the tendon biomarker tenomodulin. J Appl Physiol. 2012; 113(6): 861-871.