EFFECT OF RECOMBINANT ADENOVIRUS-BONE MORPHOGENETIC PROTEIN 12 TRANSFECTION ON DIFFERENTIATION OF PERIPHERAL BLOOD MESENCHYMAL STEM CELLS INTO TENDON/LIGAMENT CELLS_Chinese Journal of Reparative and Reconstructive Surgery

Authors：

FUWeili , CHENGang , TANGXin , LIQi ,  LIJian

Department of Orthopaedics, West China Hospital, Sichuan University, Chengdu Sichuan, 610041, P. R. China;

Corresponding?author：

LIJian, Email: hxlijian.china@163.com

Keywords：

Peripheral blood mesenchymal stem cells; Bone morphogenetic protein 12; Recombinant adenoviral vector; Tendon/ligament regeneration; Genetic engineering; Cell therapy

DOI：

10.7507/1002-1892.20150102

Video：

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Objective To research the effect of recombinant adenovirus-bone morphogenetic protein 12 (Ad-BMP-12) transfection on the differentiation of peripheral blood mesenchymal stem cells (MSCs) into tendon/ligament cells. Methods Peripheral blood MSCs were isolated from New Zealand rabbits (3-4 months old) and cultured in vitro until passage 3. The recombinant adenoviral vector system was prepared using AdEasy system, then transfected into MSCs at passage 3 (transfected group); untransfected MSCs served as control (untransfected group). The morphological characteristics and growth of transfected cells were observed under inverted phase contrast microscope. The transfection efficiency and green fluorescent protein (GFP) expression were detected by flow cytometry (FCM) and fluorescence microscopy. After cultured for 14 days in vitro, the expressions of tendon/ligament-specific markers were determined by immunohistochemistry and real-time fluorescent quantitative PCR. Results GFP expression could be observed in peripheral blood MSCs at 8 hours after transfection. At 24 hours after transfection, the cells had clear morphology and grew slowly under inverted phase contrast microscope and almost all expressed GFP at the same field under fluorescence microscopy. FCM analysis showed that the transfection efficiency of the transfected group was 99.57%, while it was 2.46% in the untransfected group. The immunohistochemistry showed that the expression of collagen type Ι gradually increased with culture time in vitro. Real-time fluorescent quantitative PCR results showed that the mRNA expressions of the tendon/ligament-specific genes (Tenomodulin, Tenascin-C, and Decorin) in the transfected group were significantly higher than those in untransfected group (0.061±0.013 vs. 0.004±0.002, t=-7.700, P=0.031; 0.029±0.008 vs. 0.003±0.001, t=-5.741, P=0.020; 0.679±0.067 vs. 0.142±0.024, t=-12.998, P=0.000). Conclusion Ad-BMP-12 can significantly promote differentiation of peripheral blood MSCs into tendon/ligament fibroblasts and enhance the expressions of tendon/ligament-specific phenotypic differentiation, which would provide the evidence for peripheral blood MSCs applied for tendon/ligament regeneration.

Citation： FUWeili, CHENGang, TANGXin, LIQi, LIJian. EFFECT OF RECOMBINANT ADENOVIRUS-BONE MORPHOGENETIC PROTEIN 12 TRANSFECTION ON DIFFERENTIATION OF PERIPHERAL BLOOD MESENCHYMAL STEM CELLS INTO TENDON/LIGAMENT CELLS. Chinese Journal of Reparative and Reconstructive Surgery, 2015, 29(4): 472-476. doi: 10.7507/1002-1892.20150102 Copy

1.	Yang G, Rothrauff BB, Tuan RS. Tendon and ligament regeneration and repair:clinical relevance and developmental paradigm. Birth Defects Res C Embryo Today, 2013, 99(3):203-222.
2.	Phinney DG. Functional heterogeneity of mesenchymal stem cells:implications for cell therapy. J Cell Biochem, 2012,113(9):2806-2812.
3.	Figueroa D, Espinosa M, Calvo R, et al. Anterior cruciate ligament regeneration using mesenchymal stem cells and collagen type Ⅰ scaffold in a rabbit model. Knee Surg Sports Traumatol Arthrosc, 2014, 22(5):1196-1202.
4.	Smith RK, Werling NJ, Dakin SG, et al. Beneficial effects of autologous bone marrow-derived mesenchymal stem cells in naturally occurring tendinopathy. PLoS One, 2013, 8(9):e75697.
5.	Bieback K, Kern S, Koca?mer A, et al. Comparing mesenchymal stromal cells from different human tissues:bone marrow, adipose tissue and umbilical cord blood. Biomed Mater Eng, 2008,18(1 Suppl):S71-76.
6.	Yang H, Gao LN, An Y, et al. Comparison of mesenchymal stem cells derived from gingival tissue and periodontal ligament in different incubation conditions. Biomaterials, 2013, 34(29):7033-7047.
7.	Martinello T, Bronzini I, Perazzi A, et al. Effects of in vivo applications of peripheral blood-derived mesenchymal stromal cells (PB-MSCs) and platlet-rich plasma (PRP) on experimentally injured deep digital flexor tendons of sheep. J Orthop Res, 2013, 31(2):306-314.
8.	Gulati BR, Kumar R, Mohanty N, et al. Bone morphogenetic protein-12 induces tenogenic differentiation of mesenchymal stem cells derived from equine amniotic fluid. Cells Tissues Organs, 2013, 198(5):377-389.
9.	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.
10.	Majewski M, Betz O, Ochsner PE, et al. Ex vivo adenoviral transfer of bone morphogenetic protein 12 (BMP-12) cDNA improves Achilles tendon healing in a rat model. Gene Ther, 2008, 15(1):1139-1146.
11.	Haddad-Weber M, Prager P, Kunz M, et al. BMP12 and BMP13 gene transfer induce ligamentogenic differentiation in mesenchymal progenitor and anterior cruciate ligament cells. Cytotherapy, 2010, 12(4):505-513.
12.	Fu WL, Zhou CY, Yu JK. A new source of mesenchymal stem cells for articular cartilage repair:MSCs derived from mobilized peripheral blood share similar biological characteristics in vitro and chondrogenesis in vivo as MSCs from bone marrow in a rabbit model. Am J Sports Med, 2014, 42(3):592-601.
13.	馬勇, 張辛, 敖英芳. 人骨形態發生蛋白 12 基因重組腺病毒載體的構建. 中國運動醫學雜志, 2008, 27(2):198-201.
14.	張辛, 楊民, 林霖, 等. 核心結合因子 (Cbfa1/Runx2) 重組腺病毒載體的構建、鑒定及在脂肪來源干細胞中的表達. 中國運動醫學雜志, 2008, 27(1):67-71.
15.	林霖, 傅欣, 張辛, 等. 骨形態發生蛋白4重組腺病毒的構建及其對NIH3T3 細胞成骨分化作用的研究. 中國運動醫學雜志, 2008, 27(3):294-297.
16.	張繼英, 劉剛, 馬棟, 等. Sprague-Dawley大鼠外周血來源間充質干細胞的動員、分離培養及鑒定. 中國運動醫學雜志, 2012, 31(9):811-816.
17.	張繼英, 付維力, 余家闊. 兔外周血 MSC 復合同種異體脫鈣皮質骨修復軟骨缺損的研究. 中國運動醫學雜志, 2013, 32(2):124-129.
18.	Ma Y, Zhang X, Wang J, et al. Effect of bone morphogenetic protein-12 gene transfer on posterior cruciate ligament healing in a rabbit model. Am J Sports Med, 2009, 37(3):599-609.
19.	Lee JY, Zhou Z, Taub PJ, et al. BMP-12 treatment of adult mesenchymal stem cells in vitro augments tendon-like tissue formation and defect repair in vivo. PLoS One, 2011, 6(3):e17531.
20.	Shen H, Gelberman RH, Silva MJ, et al. BMP12 induces tenogenic differentiation of adipose-derived stromal cells. PLoS One, 2013, 8(10):e77613.
21.	張辛, 敖英芳. 核心結合因子過表達定向誘導脂肪組織來源干細胞體內、外成骨. 中國運動醫學雜志, 2013, 32(12):1061-1067.

1. Yang G, Rothrauff BB, Tuan RS. Tendon and ligament regeneration and repair:clinical relevance and developmental paradigm. Birth Defects Res C Embryo Today, 2013, 99(3):203-222.
2. Phinney DG. Functional heterogeneity of mesenchymal stem cells:implications for cell therapy. J Cell Biochem, 2012,113(9):2806-2812.
3. Figueroa D, Espinosa M, Calvo R, et al. Anterior cruciate ligament regeneration using mesenchymal stem cells and collagen type Ⅰ scaffold in a rabbit model. Knee Surg Sports Traumatol Arthrosc, 2014, 22(5):1196-1202.
4. Smith RK, Werling NJ, Dakin SG, et al. Beneficial effects of autologous bone marrow-derived mesenchymal stem cells in naturally occurring tendinopathy. PLoS One, 2013, 8(9):e75697.
5. Bieback K, Kern S, Koca?mer A, et al. Comparing mesenchymal stromal cells from different human tissues:bone marrow, adipose tissue and umbilical cord blood. Biomed Mater Eng, 2008,18(1 Suppl):S71-76.
6. Yang H, Gao LN, An Y, et al. Comparison of mesenchymal stem cells derived from gingival tissue and periodontal ligament in different incubation conditions. Biomaterials, 2013, 34(29):7033-7047.
7. Martinello T, Bronzini I, Perazzi A, et al. Effects of in vivo applications of peripheral blood-derived mesenchymal stromal cells (PB-MSCs) and platlet-rich plasma (PRP) on experimentally injured deep digital flexor tendons of sheep. J Orthop Res, 2013, 31(2):306-314.
8. Gulati BR, Kumar R, Mohanty N, et al. Bone morphogenetic protein-12 induces tenogenic differentiation of mesenchymal stem cells derived from equine amniotic fluid. Cells Tissues Organs, 2013, 198(5):377-389.
9. 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.
10. Majewski M, Betz O, Ochsner PE, et al. Ex vivo adenoviral transfer of bone morphogenetic protein 12 (BMP-12) cDNA improves Achilles tendon healing in a rat model. Gene Ther, 2008, 15(1):1139-1146.
11. Haddad-Weber M, Prager P, Kunz M, et al. BMP12 and BMP13 gene transfer induce ligamentogenic differentiation in mesenchymal progenitor and anterior cruciate ligament cells. Cytotherapy, 2010, 12(4):505-513.
12. Fu WL, Zhou CY, Yu JK. A new source of mesenchymal stem cells for articular cartilage repair:MSCs derived from mobilized peripheral blood share similar biological characteristics in vitro and chondrogenesis in vivo as MSCs from bone marrow in a rabbit model. Am J Sports Med, 2014, 42(3):592-601.
13. 馬勇, 張辛, 敖英芳. 人骨形態發生蛋白 12 基因重組腺病毒載體的構建. 中國運動醫學雜志, 2008, 27(2):198-201.
14. 張辛, 楊民, 林霖, 等. 核心結合因子 (Cbfa1/Runx2) 重組腺病毒載體的構建、鑒定及在脂肪來源干細胞中的表達. 中國運動醫學雜志, 2008, 27(1):67-71.
15. 林霖, 傅欣, 張辛, 等. 骨形態發生蛋白4重組腺病毒的構建及其對NIH3T3 細胞成骨分化作用的研究. 中國運動醫學雜志, 2008, 27(3):294-297.
16. 張繼英, 劉剛, 馬棟, 等. Sprague-Dawley大鼠外周血來源間充質干細胞的動員、分離培養及鑒定. 中國運動醫學雜志, 2012, 31(9):811-816.
17. 張繼英, 付維力, 余家闊. 兔外周血 MSC 復合同種異體脫鈣皮質骨修復軟骨缺損的研究. 中國運動醫學雜志, 2013, 32(2):124-129.
18. Ma Y, Zhang X, Wang J, et al. Effect of bone morphogenetic protein-12 gene transfer on posterior cruciate ligament healing in a rabbit model. Am J Sports Med, 2009, 37(3):599-609.
19. Lee JY, Zhou Z, Taub PJ, et al. BMP-12 treatment of adult mesenchymal stem cells in vitro augments tendon-like tissue formation and defect repair in vivo. PLoS One, 2011, 6(3):e17531.
20. Shen H, Gelberman RH, Silva MJ, et al. BMP12 induces tenogenic differentiation of adipose-derived stromal cells. PLoS One, 2013, 8(10):e77613.
21. 張辛, 敖英芳. 核心結合因子過表達定向誘導脂肪組織來源干細胞體內、外成骨. 中國運動醫學雜志, 2013, 32(12):1061-1067.

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Chinese Journal of Reparative and Reconstructive Surgery

EFFECT OF RECOMBINANT ADENOVIRUS-BONE MORPHOGENETIC PROTEIN 12 TRANSFECTION ON DIFFERENTIATION OF PERIPHERAL BLOOD MESENCHYMAL STEM CELLS INTO TENDON/LIGAMENT CELLS

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