EXPERIMENTAL STUDY ON ADIPOSE-DERIVED STEM CELLS TRANSFECTED BY BONE MORPHOGENETIC PROTEIN 14 CO-CULTURE WITH CHONDROCYTES_Chinese Journal of Reparative and Reconstructive Surgery

Authors：

YUAN Hong ¹ , ZHANG Jieyuan ² , ZHANG Rongming ¹

1Department of Burn and Plastic Surgery, 2Department of Orthopaedic and Joint Surgery, the First Affiliated Hospital of Liaoning Medical College, Jinzhou Liaoning, 121001, P.R.China. Corresponding author: ZHANG Rongming, E-mail: mandyyh@126.com;

Corresponding author：

Keywords：

Cartilage tissue engineering; Adipose-derived stem cells; Bone morphogenetic protein 14; Chondrocytes Co-culture; Rabbit

DOI：

10.7507/1002-1892.20130080

Video：

Export PDF Favorites Scan Get Citation

Abstract Full text Figures/Tables Video References Cited by

Objective To evaluate the synergistic effect of bone morphogenetic protein 14 (BMP-14) and chondrocytes co-culture on chondrogenesis of adipose-derived stem cells (ADSCs) so as to optimize the source of seed cells for cartilage tissue engineering. Methods ADSCs and chondrocytes were isolated and cultured respectively from articular cartilage and subcutaneous fat of 2 male New Zealand white rabbits (weighing, 1.5 kg and 2.0 kg). The cells at passage 3 were harvested for experiment. ADSCs were identified by osteogenic induction (alizarin red staining), chondrogenic induction (alcian blue staining), and adipogenic induction (oil red O staining). The optimum multiplicity of infection (MOI) of transfection of adenovirus-cytomegalovirus (CMV)-BMP-14-internal ribosome entry site (IRES)-human renilla reniformis green fluorescent protein 1 (hrGFP-1) was determined and then ADSCs were transfected by the optimum MOI. The experiment was divided into 5 groups: group A, co-culture of ADSCs transfected by BMP-14 and chondrocytes (1 ∶ 1 in Transwell chambers); group B, co-culture of ADSCs and chondrocytes (1 ∶ 1 in Transwell chambers); group C, culture of ADSCs transfected by BMP-14; group D, simple chondrocytes culture; and group E, simple ADSCs culture. After 3 weeks, the glycosaminoglycan (GAG) content was detected by alcian blue staining; the expressions of collagen type II and BMP-14 protein were detected by Western blot; expression of Sox-9 gene was detected by RT-PCR. Results The cultured cells were proved to be ADSCs by identification. Inverted fluorescence microscope showed optimum transfection effect when MOI was 150. GAG content, expressions of collagen type II and BMP-14 protein, expression of Sox-9 gene were significantly higher in groups A and C than in the other 3 groups, in group A than in group C (P lt; 0.05), and groups B and D were significantly higher than group E (P lt; 0.05), but no significant difference was found between groups B and D (P gt; 0.05). Conclusion It can promote differentiation of ADSCs into chondrocytes by BMP-14 co-culture with chondrocytes, and they have a synergistic effect.

Citation： YUAN Hong,ZHANG Jieyuan,ZHANG Rongming. EXPERIMENTAL STUDY ON ADIPOSE-DERIVED STEM CELLS TRANSFECTED BY BONE MORPHOGENETIC PROTEIN 14 CO-CULTURE WITH CHONDROCYTES. Chinese Journal of Reparative and Reconstructive Surgery, 2013, 27(3): 353-357. doi: 10.7507/1002-1892.20130080 Copy

1.	15 Wang C, Ruan DK, Zhang C, et al. Effects of adeno-associated virus-2-mediated human BMP-7 gene transfection on the phenotype of nucleus pulposus cells. J Orthop Res, 2011, 29(6): 838-845. 16 Jung MR, Shim IK, Chung HJ, et al. Local BMP-7 release from a PLGA scaffolding-matrix for the repair of osteochondral defects in rabbits. J Control Release, 2012, 162(3): 485-491.
2.	17 Coleman CM, Loredo GA, Lo CW, et al. Correlation of GDF5 and connexin 43 mRNA expression during embryonic development. Anat Rec A Discov Mol Cell Evol Biol, 2003, 275(2): 1117-1121. 18 Meech R, Edelman DB, Jones FS, et al. The homeobox transcription factor Barx2 regulates chondrogenesis during limb development. Development, 2005, 132(9): 2135-2146.
3.
4.
5.	Khaled EG, Saleh M, Hindocha S, et al. Tissue engineering for bone production-stem cells, gene therapy and scaffolds. Open Orthop J, 2011, 5 Suppl 2: 289-295.
6.	Dong SW, Ying DJ, Duan XJ, et al. Bone regeneration using an acellular extracellular matrix and bone marrow mesenchymal stem cells expressing Cbfa1. Biosci Biotechnol Biochem, 2009, 73(10): 2226-2233.
7.	Raghunath J, Salacinski HJ, Sales KM, et al. Advancing cartilage tissue engineering: the application of stem cell technology. Curr Opin Biotechnol, 2005, 16(5): 503-509.
8.	任宇, 吴海青, 马玉珍, 等. 牛脂肪间充质干细胞的分离、培养与鉴定. 生物工程学报, 2010, 26(12): 1645-1651.
9.	刘振宁, 贾长青, 韩长旭, 等. 生长分化因子5诱导兔脂肪干细胞成软骨细胞分化的实验研究. 中国修复重建外科杂志, 2009, 23(4): 483-489.
10.	张勇, 赵建宁, 韩宁波. 脂肪干细胞与软骨细胞的共培养. 中国组织工程研究与临床康复, 2009, 13(24): 4632-4636.
11.	张解元, 袁虹, 李谌, 等. 腺病毒介导BMP-2联合低氧诱导因子1α突变型与单基因BMP-2分别转染BMSCs后成骨效应的比较研究. 中国修复重建外科杂志, 2012, 26(9): 1102-1106.
12.	孙磊, 綦惠, 陈磊, 等. 兔软骨细胞与骨髓基质细胞的平面共培养: 两者培养比例筛选. 中国组织工程研究与临床康复, 2010, 14(27): 4955-4659.
13.	English A, Jones EA, Corscadden D, et al. A comparative assessment of cartilage and joint fat pad as a potential source of cells for autologous therapy development in knee osteoarthritis. Rheumatology (Oxford), 2007, 46(11): 1676-1683.
14.	Bajek A, Olkowska J, Drewa T. Mesenchymal stem cells as a therapeutic tool in tissue and organ regeneration. Postepy Hig Med Dosw (Online), 2011, 65: 124-132.
15.	袁启龙, 曾晓勇, 陈亮, 等. 兔不同部位脂肪来源干细胞体外成肌诱导能力的比较. 中国修复重建外科杂志, 2010, 24(10): 1228-1232.
16.	邓春华, 孙祥宙, 高勇, 等. 大鼠脂肪组织来源干细胞的分离、培养和生物学特性的研究. 中华男科学杂志, 2008, 14(2): 99-105.
17.	Dorman LJ, Tucci M, Benghuzzi H. In vitro effects of bmp-2, bmp-7, and bmp-13 on proliferation and differentation of mouse mesenchymal stem cells. Biomed Sci Instrum, 2012, 48: 81-87.
18.	Bobinac D, Spanjol J, Marinović M, et al. Expression of bone morphogenetic proteins, cartilage-derived morphogenetic proteins and related receptors in normal and osteoarthritic human articular cartilage. Coll Antropol, 2008, 32 Suppl 2: 83-87.
19.	Vats A, Bielby RC, Tolley N, et al. Chondrogenic differentiation of human embryonic stem cells: the effect of the micro-environment. Tissue Eng, 2006, 12(6): 1687-1697.
20.	Takagi M, Umetsu Y, Fujiwara M, et al. High inoculation cell density could accelerate the differentiation of human bone marrow mesenchymal stem cells to chondrocyte cells. J Biosci Bioeng, 2007, 103(1): 98-100.
21.	Hillel AT, Taube JM, Cornish TC, et al. Characterization of human mesenchymal stem cell-engineered cartilage: analysis of its ultrastructure, cell density and chondrocyte phenotype compared to native adult and fetal cartilage. Cells Tissues Organs, 2010, 191(1): 12-20.
22.	Erickson IE, Huang AH, Chung C, et al. Differential maturation and structure-function relationships in mesenchymal stem cell- and chondrocyte-seeded hydrogels. Tissue Eng Part A, 2009, 15(5): 1041-1052.
23.	Wang L, Lin F, Wu J, et al. High efficiency adenovirus-mediated expression of truncated N-terminal huntingtin fragment (htt552) in primary rat astrocytes. Acta Biochim Biophys Sin (Shanghai), 2009, 41(4): 325-334.
24.	范先群, 肖彩雯, 周慧芳, 等. 基因修饰的组织工程骨修复眶骨缺损的实验研究. 中华眼科杂志, 2009, 45(1): 66-72.

1. 15 Wang C, Ruan DK, Zhang C, et al. Effects of adeno-associated virus-2-mediated human BMP-7 gene transfection on the phenotype of nucleus pulposus cells. J Orthop Res, 2011, 29(6): 838-845. 16 Jung MR, Shim IK, Chung HJ, et al. Local BMP-7 release from a PLGA scaffolding-matrix for the repair of osteochondral defects in rabbits. J Control Release, 2012, 162(3): 485-491.
2. 17 Coleman CM, Loredo GA, Lo CW, et al. Correlation of GDF5 and connexin 43 mRNA expression during embryonic development. Anat Rec A Discov Mol Cell Evol Biol, 2003, 275(2): 1117-1121. 18 Meech R, Edelman DB, Jones FS, et al. The homeobox transcription factor Barx2 regulates chondrogenesis during limb development. Development, 2005, 132(9): 2135-2146.
3.
4.
5. Khaled EG, Saleh M, Hindocha S, et al. Tissue engineering for bone production-stem cells, gene therapy and scaffolds. Open Orthop J, 2011, 5 Suppl 2: 289-295.
6. Dong SW, Ying DJ, Duan XJ, et al. Bone regeneration using an acellular extracellular matrix and bone marrow mesenchymal stem cells expressing Cbfa1. Biosci Biotechnol Biochem, 2009, 73(10): 2226-2233.
7. Raghunath J, Salacinski HJ, Sales KM, et al. Advancing cartilage tissue engineering: the application of stem cell technology. Curr Opin Biotechnol, 2005, 16(5): 503-509.
8. 任宇, 吴海青, 马玉珍, 等. 牛脂肪间充质干细胞的分离、培养与鉴定. 生物工程学报, 2010, 26(12): 1645-1651.
9. 刘振宁, 贾长青, 韩长旭, 等. 生长分化因子5诱导兔脂肪干细胞成软骨细胞分化的实验研究. 中国修复重建外科杂志, 2009, 23(4): 483-489.
10. 张勇, 赵建宁, 韩宁波. 脂肪干细胞与软骨细胞的共培养. 中国组织工程研究与临床康复, 2009, 13(24): 4632-4636.
11. 张解元, 袁虹, 李谌, 等. 腺病毒介导BMP-2联合低氧诱导因子1α突变型与单基因BMP-2分别转染BMSCs后成骨效应的比较研究. 中国修复重建外科杂志, 2012, 26(9): 1102-1106.
12. 孙磊, 綦惠, 陈磊, 等. 兔软骨细胞与骨髓基质细胞的平面共培养: 两者培养比例筛选. 中国组织工程研究与临床康复, 2010, 14(27): 4955-4659.
13. English A, Jones EA, Corscadden D, et al. A comparative assessment of cartilage and joint fat pad as a potential source of cells for autologous therapy development in knee osteoarthritis. Rheumatology (Oxford), 2007, 46(11): 1676-1683.
14. Bajek A, Olkowska J, Drewa T. Mesenchymal stem cells as a therapeutic tool in tissue and organ regeneration. Postepy Hig Med Dosw (Online), 2011, 65: 124-132.
15. 袁启龙, 曾晓勇, 陈亮, 等. 兔不同部位脂肪来源干细胞体外成肌诱导能力的比较. 中国修复重建外科杂志, 2010, 24(10): 1228-1232.
16. 邓春华, 孙祥宙, 高勇, 等. 大鼠脂肪组织来源干细胞的分离、培养和生物学特性的研究. 中华男科学杂志, 2008, 14(2): 99-105.
17. Dorman LJ, Tucci M, Benghuzzi H. In vitro effects of bmp-2, bmp-7, and bmp-13 on proliferation and differentation of mouse mesenchymal stem cells. Biomed Sci Instrum, 2012, 48: 81-87.
18. Bobinac D, Spanjol J, Marinović M, et al. Expression of bone morphogenetic proteins, cartilage-derived morphogenetic proteins and related receptors in normal and osteoarthritic human articular cartilage. Coll Antropol, 2008, 32 Suppl 2: 83-87.
19. Vats A, Bielby RC, Tolley N, et al. Chondrogenic differentiation of human embryonic stem cells: the effect of the micro-environment. Tissue Eng, 2006, 12(6): 1687-1697.
20. Takagi M, Umetsu Y, Fujiwara M, et al. High inoculation cell density could accelerate the differentiation of human bone marrow mesenchymal stem cells to chondrocyte cells. J Biosci Bioeng, 2007, 103(1): 98-100.
21. Hillel AT, Taube JM, Cornish TC, et al. Characterization of human mesenchymal stem cell-engineered cartilage: analysis of its ultrastructure, cell density and chondrocyte phenotype compared to native adult and fetal cartilage. Cells Tissues Organs, 2010, 191(1): 12-20.
22. Erickson IE, Huang AH, Chung C, et al. Differential maturation and structure-function relationships in mesenchymal stem cell- and chondrocyte-seeded hydrogels. Tissue Eng Part A, 2009, 15(5): 1041-1052.
23. Wang L, Lin F, Wu J, et al. High efficiency adenovirus-mediated expression of truncated N-terminal huntingtin fragment (htt552) in primary rat astrocytes. Acta Biochim Biophys Sin (Shanghai), 2009, 41(4): 325-334.
24. 范先群, 肖彩雯, 周慧芳, 等. 基因修饰的组织工程骨修复眶骨缺损的实验研究. 中华眼科杂志, 2009, 45(1): 66-72.

Chinese Journal of Reparative and Reconstructive Surgery

EXPERIMENTAL STUDY ON ADIPOSE-DERIVED STEM CELLS TRANSFECTED BY BONE MORPHOGENETIC PROTEIN 14 CO-CULTURE WITH CHONDROCYTES

Abstract Full text Figures/Tables Video References Cited by

Previous Article

Next Article

Format

Content