EXPRESSION OF HUMAN TRANSFORMING GROWTH FACTOR β<sub>1</sub> GENE MEDIATED BY ADENOVIRUS IN HAMSTRING TENDON AFTER ANTERIOR CRUCIATE LIGAMENT RECONSTRUCTION IN RABBITS_Chinese Journal of Reparative and Reconstructive Surgery

Authors：

WANGXiaoxu , HEMin , TANWenfu , TANGuanghua , YANGJuntao ,  HONGLiang

Department of Orthopedics, the Second Affiliated Hospital of Nanhua University, Hengyang Hunan, 421001, P. R. China;

Corresponding?author：

HONGLiang, Email: wxx1024@163.com

Keywords：

Human transforming growth factor β₁; Adenovirus; Hamstring tendon; Anterior cruciate ligament; Rabbit

DOI：

10.7507/1002-1892.20160252

Video：

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Objective To detect the expression of human transforming growth factor β₁ (hTGF-β₁) gene mediated by adenovirus (Ad) in hamstring tendon after anterior cruciate ligament (ACL) reconstruction in rabbits. Methods Ad-hTGF-β₁ and Ad-green fluorescent protein (GFP) were diluted to 5×10⁸ PFU/mL with DMEM. Forty-eight New Zealand white rabbits were divided into 3 groups randomly (n=16) for ACL reconstruction with hamstring tendon autograft. Hamstring tendon was cultured and transfected with Ad-hTGF-β₁ (group A) and Ad-GFP (group B) for 12 hours before ACL reconstruction, and was cultured with DMEM in group C. After 12 hours of transfection, green fluorescence was observed in groups A and B under fluorescence microscopy. At 2, 4, 6, and 8 weeks after operation, the hamstring tendon was harvested to detect the mRNA and protein expressions of hTGF-β₁ by real time fluorescence quantitative PCR and Western blot. Results Green fluorescence was observed after 12 hours of transfection in groups A and B. TGF-β₁ protein level reached (221.0±12.2) ng/mL at 12 hours in group A. The hTGF-β₁ mRNA expression could be detected in group A, but it could not be detected in group B and group C. The mRNA expression levels of hTGF-β₁ were 1.004±0.072 at 2 weeks, 0.785±0.038 at 4 weeks, 0.469±0.053 at 6 weeks, and 0.172±0.021 at 8 weeks in group A, showing significant difference (P<0.05). Western blot results showed weakly positive band in groups B and C; the protein expression of TGF-β₁ in group A was significantly higher than that in groups B and C (P<0.05), but no significant difference was found between groups B and C P>0.05). The protein expression of TGF-β₁ gradually reduced with time, showing significant difference between different time points (P<0.05). Conclusion Ad-hTGF-β₁ can transfect the hamstring tendon successfully, and it can effectively express for a long time after ACL reconstruction.

Citation： WANG Xiaoxu, HE Min, TAN Wenfu, TAN Guanghua, YANG Juntao, HONG Liang. EXPRESSION OF HUMAN TRANSFORMING GROWTH FACTOR β₁ GENE MEDIATED BY ADENOVIRUS IN HAMSTRING TENDON AFTER ANTERIOR CRUCIATE LIGAMENT RECONSTRUCTION IN RABBITS. Chinese Journal of Reparative and Reconstructive Surgery, 2016, 30(10): 1233-1237. doi: 10.7507/1002-1892.20160252 Copy

1.	Spahn G, Schiltenwolf M, Hartmann B, et al. The time-related risk for knee osteoarthritis after ACL injury. Results from a systematic review. Orthopade, 2016, 45(1):81-90.
2.	Papathanasiou I, Michalitsis S, Hantes ME, et al. Molecular changes indicative of cartilage degeneration and osteoarthritis development in patients with anterior cruciate ligament injury. BMC Musculoskelet Disord, 2016, 17:21.
3.	Murray MM, Fleming BC. Use of a bioactive scaffold to stimulateanterior cruciate ligament healing also minimizes posttraumaticosteoarthritis after surgery. Am J Sports Med, 2013, 41(8):1762-1770.
4.	翟溶凡, 王曉旭, 楊俊濤. 前交叉韌帶重建中EndoButton和RigidFix兩種股骨側固定方式比較的系統評價. 中國矯形外科雜志, 2014, 22(24):2209-2214.
5.	Negahi Shirazi A, Chrzanowski W, Khademhosseini A, et al. Anterior cruciate ligament:structure, injuries and regenerative treatments. Adv Exp Med Biol, 2015, 881:161-186.
6.	Bi F, Shi Z, Jiang S, et al. Intermittently administered parathyroid hormone 1-34 promotes tendon-bone healing in a rat model. Int J Mol Sci, 2014, 15(10):17366-17379.
7.	Bissell L, Tibrewal S, Sahni V, et al. Growth factors and platelet rich plasma in anterior cruciate ligament reconstruction. Curr Stem Cell Res Ther, 2014, 10(1):19-25.
8.	Kondo E, Yasuda K, Yamanaka M, et al. Effects of administration of exogenous growth factors on biomechanical properties of the elongation-type anterior cruciate ligament injury with partial laceration. Am J Sports Med, 2005, 33(2):188-196.
9.	王曉旭, 翟溶凡, 楊俊濤, 等. 腺病毒介導TGF-β₁基因轉染腘繩肌腱對兔前交叉韌帶重建術后腱-骨愈合的組織形態學影響. 中國修復重建外科雜志, 2015, 29(12):1488-1493.
10.	Hansen M, Boesen A, Holm L, et al. Local administration of insulin-like growth factor-I (IGF-I) stimulates tendon collagen synthesis in humans. Scand J Med Sci Sports, 2013, 23(5):614-619.
11.	Anderson K, Seneviratne AM, Izawa K, et al. Augmentation of tendon healing in an intraarticular bone tunnel with use of a bone growth factor. Am J Sports Med, 2001, 29(6):689-698.
12.	Wang Y, Tang Z, Xue R, et al. TGF-beta1 promoted MMP-2 mediated wound healing of anterior cruciate ligament fibroblasts through NF-kappaB. Connect Tissue Res, 2011, 52(3):218-225.
13.	Cheng MT, Yang HW, Chen TH, et al. Modulation of proliferation and differentiation of human anterior cruciate ligament-derived stem cells by different growth factors. Tissue Eng Part A, 2009, 15(12):3979-3989.
14.	Yamazaki S, Yasuda K, Tomita F, et al. The effect of transforming growth factor-beta1 on intraosseous healing of flexor tendon autograft replacement of anterior cruciate ligament in dogs. Arthroscopy, 2005, 21(9):1034-1041.
15.	Qin J, Hou ZQ, Wang H, et al. Effects of geneactivated matrix on autograft healing of anterior cruciate ligament. Mol Med Rep, 2013, 7(2):679-683.
16.	Moss JA. Gene therapy review. Radiol Technol, 2014, 86(2):155-184.
17.	Gao X, Du J, Fan G, et al. Adenoviral-mediated GDNF protects bonemarrow mesenchymal stem cells against apoptosis induced byhydrogen peroxide. Biomed Mater Eng, 2014, 24(6):2169-2176.
18.	Wei XL, Lin L, Hou Y, et al. Construction of recombinant adenovirus co-expression vector carrying the human transforming growth factor-beta1 and vascular endothelial growth factor genes and its effect on anterior cruciate ligament fibroblasts. Chin Med J (Engl), 2008, 121(15):1426-1432.
19.	Martinek V, Latterman C, Usas A, et al. Enhancement of tendon-bone integration of anterior cruciate ligament grafts with bone morphogenetic protein-2 gene transfer:a histological and biomechanical study. J Bone Joint Surg (Am), 2002, 84-A(7):1123-1131.
20.	Gerich TG, Kang R, Fu FH, et al. Gene transfer to the patellar tendon. Knee Surg Sports Traumatol Arthrosc, 1997, 5(2):118-123.

1. Spahn G, Schiltenwolf M, Hartmann B, et al. The time-related risk for knee osteoarthritis after ACL injury. Results from a systematic review. Orthopade, 2016, 45(1):81-90.
2. Papathanasiou I, Michalitsis S, Hantes ME, et al. Molecular changes indicative of cartilage degeneration and osteoarthritis development in patients with anterior cruciate ligament injury. BMC Musculoskelet Disord, 2016, 17:21.
3. Murray MM, Fleming BC. Use of a bioactive scaffold to stimulateanterior cruciate ligament healing also minimizes posttraumaticosteoarthritis after surgery. Am J Sports Med, 2013, 41(8):1762-1770.
4. 翟溶凡, 王曉旭, 楊俊濤. 前交叉韌帶重建中EndoButton和RigidFix兩種股骨側固定方式比較的系統評價. 中國矯形外科雜志, 2014, 22(24):2209-2214.
5. Negahi Shirazi A, Chrzanowski W, Khademhosseini A, et al. Anterior cruciate ligament:structure, injuries and regenerative treatments. Adv Exp Med Biol, 2015, 881:161-186.
6. Bi F, Shi Z, Jiang S, et al. Intermittently administered parathyroid hormone 1-34 promotes tendon-bone healing in a rat model. Int J Mol Sci, 2014, 15(10):17366-17379.
7. Bissell L, Tibrewal S, Sahni V, et al. Growth factors and platelet rich plasma in anterior cruciate ligament reconstruction. Curr Stem Cell Res Ther, 2014, 10(1):19-25.
8. Kondo E, Yasuda K, Yamanaka M, et al. Effects of administration of exogenous growth factors on biomechanical properties of the elongation-type anterior cruciate ligament injury with partial laceration. Am J Sports Med, 2005, 33(2):188-196.
9. 王曉旭, 翟溶凡, 楊俊濤, 等. 腺病毒介導TGF-β₁基因轉染腘繩肌腱對兔前交叉韌帶重建術后腱-骨愈合的組織形態學影響. 中國修復重建外科雜志, 2015, 29(12):1488-1493.
10. Hansen M, Boesen A, Holm L, et al. Local administration of insulin-like growth factor-I (IGF-I) stimulates tendon collagen synthesis in humans. Scand J Med Sci Sports, 2013, 23(5):614-619.
11. Anderson K, Seneviratne AM, Izawa K, et al. Augmentation of tendon healing in an intraarticular bone tunnel with use of a bone growth factor. Am J Sports Med, 2001, 29(6):689-698.
12. Wang Y, Tang Z, Xue R, et al. TGF-beta1 promoted MMP-2 mediated wound healing of anterior cruciate ligament fibroblasts through NF-kappaB. Connect Tissue Res, 2011, 52(3):218-225.
13. Cheng MT, Yang HW, Chen TH, et al. Modulation of proliferation and differentiation of human anterior cruciate ligament-derived stem cells by different growth factors. Tissue Eng Part A, 2009, 15(12):3979-3989.
14. Yamazaki S, Yasuda K, Tomita F, et al. The effect of transforming growth factor-beta1 on intraosseous healing of flexor tendon autograft replacement of anterior cruciate ligament in dogs. Arthroscopy, 2005, 21(9):1034-1041.
15. Qin J, Hou ZQ, Wang H, et al. Effects of geneactivated matrix on autograft healing of anterior cruciate ligament. Mol Med Rep, 2013, 7(2):679-683.
16. Moss JA. Gene therapy review. Radiol Technol, 2014, 86(2):155-184.
17. Gao X, Du J, Fan G, et al. Adenoviral-mediated GDNF protects bonemarrow mesenchymal stem cells against apoptosis induced byhydrogen peroxide. Biomed Mater Eng, 2014, 24(6):2169-2176.
18. Wei XL, Lin L, Hou Y, et al. Construction of recombinant adenovirus co-expression vector carrying the human transforming growth factor-beta1 and vascular endothelial growth factor genes and its effect on anterior cruciate ligament fibroblasts. Chin Med J (Engl), 2008, 121(15):1426-1432.
19. Martinek V, Latterman C, Usas A, et al. Enhancement of tendon-bone integration of anterior cruciate ligament grafts with bone morphogenetic protein-2 gene transfer:a histological and biomechanical study. J Bone Joint Surg (Am), 2002, 84-A(7):1123-1131.
20. Gerich TG, Kang R, Fu FH, et al. Gene transfer to the patellar tendon. Knee Surg Sports Traumatol Arthrosc, 1997, 5(2):118-123.

Chinese Journal of Reparative and Reconstructive Surgery

EXPRESSION OF HUMAN TRANSFORMING GROWTH FACTOR β₁ GENE MEDIATED BY ADENOVIRUS IN HAMSTRING TENDON AFTER ANTERIOR CRUCIATE LIGAMENT RECONSTRUCTION IN RABBITS

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EXPRESSION OF HUMAN TRANSFORMING GROWTH FACTOR β1 GENE MEDIATED BY ADENOVIRUS IN HAMSTRING TENDON AFTER ANTERIOR CRUCIATE LIGAMENT RECONSTRUCTION IN RABBITS

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EXPRESSION OF HUMAN TRANSFORMING GROWTH FACTOR β₁ GENE MEDIATED BY ADENOVIRUS IN HAMSTRING TENDON AFTER ANTERIOR CRUCIATE LIGAMENT RECONSTRUCTION IN RABBITS