PHOSPHORYLATABLE SHORT PEPTIDE CONJUGATED CHITOSAN MEDIATED GENE THERAPY FOR REPAIR OF ARTICULAR CARTILAGE DEFECT IN RABBITS_Chinese Journal of Reparative and Reconstructive Surgery

Authors：

ZHAORonglan ¹ ,  PENGXiaoxiang ¹ , CHUHairong ¹ , SONGWei ¹ , LIUQing ²

1. Department of Medical Laboratory, Shandong Provincial Key Laboratory of Clinical Laboratory Diagnostics, Weifang Medical University, Weifang Shandong, 261053, P. R. China;
2. Department of Rehabilitation, Second People's Hospital of Weifang;

Corresponding author：

PENGXiaoxiang, Email: pxx74@sina.com

Keywords：

Phosphorylatable short peptide; Chitosan; Cartilage defect; Gene therapy; Insul in-like growth factor 1; Interleukin 1 receptor antagonist; Rabbit

DOI：

10.7507/1002-1892.20140292

Video：

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Objective To investigate the effect of phosphorylatable short peptide (^pSP) conjugated chitosan (CS) (^pSP-CS) mediated insul in-l ike growth factor 1 (IGF-1) gene and human interleukin 1 receptor antagonist (IL-1Ra) gene local transfection on the repair of articular cartilage defect. Methods Co-expression plasmid pBudCE4.1-IL-1Ra+IGF-1, single gene expression plasmid pBudCE4.1-IL-1Ra and pBudCE4.1-IGF-1 were constructed and combined with ^pSP-CS to form ^pSP-CS/ pDNA complexes. Thirty 3-month-old healthy male New Zealand white rabbits, weighing 2.0-2.5 kg, double legs were randomly divided into 5 groups (n=12). Lateral femoral condyle articular surface was only exposed in sham-operated group (group A); full-thickness cartilage defects were created in the articular surface of the lateral femoral condyle of the knee in 4 intervention groups: ^pSP-CS/pBudCE4.1 (group B), ^pSP-CS/pBudCE4.1-IL-1Ra (group C), ^pSP-CS/pBudCE4.1-IGF-1(group D), and ^pSPCS/ pBudCE4.1-IL-1Ra+IGF-1 (group E). At 1 week after operation, intra-articular injection of ^pSP-CS/pDNA complexes was administrated 2 times a week for 7 weeks in each intervention group, the same volume normal sal ine in group A. The general condition of animal was observed after operation, and rabbits were sacrificed at 8 weeks. Knee joint synovial fluid was collected to measure the concentrations of the IL-1Ra and IGF-1 by ELISA; mRNA expressions of Aggrecan, matrix metalloproteinase 3 (MMP-3), and MMP inhibitor 1 (TIMP-1) were detected by real-time fluorescent quantitative PCR; the chondrogenic phenotype of nascent cells in the damage zone was identified by alcian blue-periodic acid/schiff (AB-PAS) histochemistry and Aggrecan immunohistochemistry staining. Results Thirty experimental rabbits all survived to the end of experiment, without infection and death. Large amounts of exogenous proteins of IGF-1 and IL-1Ra were detected in the synovial fluid of 4 intervention groups. There were significant differences between groups D, E and group A in IGF-1 protein expression, and between goups C, E and group A in IL-1Ra protein expression (P < 0.05). Aggrecan and TIMP-1 mRNA expressions were significantly up-regulated in group E, simultaneously MMP-3 mRNA expression was significantly down-regulated when compared with groups C and D (P < 0.05). Varying degrees of cartilage repair appeared in groups C, D, and E, showing positive staining of AB-PAS and Aggrecan, and group E had better results than groups C and D (P < 0.05); inflammatory cell infiltration and fibrous tissue prol iferation were seen in the defect region of group B, without significant cartilage repairing. Conclusion ^pSP-CS is an ideal gene del ivery system for cartilage defect gene therapy; IL-1Ra and IGF-1 double gene transfection has better biologic effect on cartilage defect repair.

Citation： ZHAORonglan, PENGXiaoxiang, CHUHairong, SONGWei, LIUQing. PHOSPHORYLATABLE SHORT PEPTIDE CONJUGATED CHITOSAN MEDIATED GENE THERAPY FOR REPAIR OF ARTICULAR CARTILAGE DEFECT IN RABBITS. Chinese Journal of Reparative and Reconstructive Surgery, 2014, 28(11): 1346-1352. doi: 10.7507/1002-1892.20140292 Copy

1.	Zhu S, Zhang B, Man C, et al. Combined effects of connective tissue growth factor-modified bone marrow-derived mesenchymal stem cells and NaOH-treated PLGA scaffolds on the repair of articular cartilage defect in rabbits. Cell Transplant, 2014, 23(6):715-727.
2.	Shi J, Zhang X, Zhu J, et al. Nanoparticle delivery of the bone morphogenetic protein 4 gene to adi pose-derived stem cells promotes articular cartilage repair in vitro and in vivo. Arthroscopy, 2013, 29(12):2001-2011. e2.
3.	Lee HH, O'Malley MJ, Friel NA, et al. Persistence, local ization, and external control of transgene expression after single injection of adenoassociated virus into injured joints. Hum Gene Ther, 2013, 24(4):457-466.
4.	Qi BW, Yu AX, Zhu SB, et al. Chitosan/poly (vinyl alcohol) hydrogel combined with Ad-hTGF-β₁ transfected mesenchymal stem cells to repair rabbit articular cartilage defects. Exp Biol Med (Maywood), 2013, 238(1):23-30.
5.	Eroglu E, Tiwari PM, Waffo AB, et al. A nonviral pHEMA+chitosan nanosphere-mediated high-efficiency gene del ivery system. Int J Nanomedicine, 2013, 8:1403-1415.
6.	Hagiwara K, Kishimoto S, Ishihara M, et al. In vivo gene transfer using pDNA/chitosan/chondroitin sulfate ternary complexes:influence of chondroitin sulfate on the stabil ity of freeze-dried complexes and transgene expression in vivo. J Gene Med, 2013, 15(2):83-92.
7.	Lu H, Dai Y, Lv L, et al. Chitosan-graft-polyethylenimine/DNA nanoparticles as novel non-viral gene delivery vectors targeting osteoarthritis. PLoS One, 2014, 9(1):e84703.
8.	Liao ZX, Peng SF, Ho YC, et al. Mechanistic study of transfection of chitosan/DNA complexes coated by anionic poly(γ-glutamic acid). Biomaterials, 2012, 33(11):3306-3315.
9.	Kong F, Liu G, Sun B, et al. Phosphorylatable short peptide conjugated low molecular weight chitosan for efficient siRNA delivery and target gene silencing. Int J Pharm, 2012, 422(1-2):445-453.
10.	Plianwong S, Opanasopit P, Ngawhirunpat T, et al. Chitosan combined with poly-L-arginine as efficient, safe, and serum-insensitive vehicle with RNase protection abil ity for siRNA delivery. BioMed Res Int, 2013, 2013:574136.
11.	Peng SF, Tseng MT, Ho YC, et al. Mechanisms of cellular uptake and intracellular trafficking with chitosan/DNA/poly(γ-glutamic acid) complexes as a gene del ivery vector. Biomaterials, 2011, 32(1):239-248.
12.	赵荣兰, 任玉平, 孙蓓, 等.壳聚糖转染胰岛素样生长因子基因促进兔关节软骨损伤修复的实验研究.中国修复重建外科杂志, 2010, 24(11):1372-1375.
13.	Sun B, Zhao R, Kong F, et al. Phosphorylatable short peptide conjugation for facil itating transfection efficacy of CS/DNA complex. Int J Pharm, 2010, 397(1-2):206-210.
14.	张平, 蔡道章, 刘斌, 等.转IL-1Ra和TGF-β₁基因治疗兔骨性关节炎的体外实验研究.中华关节外科杂志(电子版), 2011, 5(3):335-342.
15.	邬波, 马旭, 朱大木, 等.胰岛素样生长因子1对骨髓间充质干细胞软骨分化及基质金属蛋白酶表达的影响.中国组织工程研究, 2013, 17(19):3421-3429.
16.	梁东春, 郭善一, 郭刚, 等. IGF-1信号肽及成熟肽全编码序列的克隆.天津医科大学学报, 2003, 9(4):453-455.
17.	Orth P, Kaul G, Cucchiarini M, et al. Transplanted articular chondrocytes co-overexpressing IGF-I and FGF-2 stimulate cartilage repair in vivo. Knee Surg Sports Traumatol Arthrosc, 2011, 19(12):2119-2130.
18.	Zhao Q, Wang S, Tian J, et al. Combination of bone marrow concentrate and PGA scaffolds enhance bone marrow stimulation in rabbit articular cartilage repair. J Mater Sci Mater Med, 2013, 24(3):793-801.
19.	Peng XX, Zhao RL, Song W, et al. Selection of suitable reference genes for normalization of quantitative real-time PCR in cartilage tissue injury and repair in rabbits. Int J Mol Sci, 2012, 13(11):14344-14355.
20.	黄云梅, 陈文列, 黄美雅, 等.多种特殊染色法在骨关节炎组织形态学研究中的应用比较.中国比较医学杂志, 2011, 21(5):45-48.
21.	Jia X, Cheng K, Mahato RI. Coexpression of vascular endothel ial growth factor and interleukin-1 receptor antagonist for improved human islet survival and function. Mol Pharm, 2007, 4(2):199-207.
22.	Zamani S, Hashemibeni B, Esfandiari E, et al. Assessment of TGF-β3 on production of aggrecan by human articular chondrocytes in pellet culture system. Adv Biomed Res, 2014, 3:54.
23.	Williams A, Smith JR, Allaway D, et al. Carprofen inhibits the release of matrix metalloproteinases 1, 3, and 13 in the secretome of an explant model of articular cartilage stimulated with interleukin 1β. Arthritis Res Ther, 2013, 15(6):R223.
24.	贺占坤, 沈杰威. MMP-2、MMP-3、MMP-9和TIMP-1评价膝关节骨性关节炎的临床研究.重庆医学, 2013, 42(32):3872-3874.
25.	董福, 宋锦旗, 罗吉伟, 等.不同强度运动对大鼠关节软骨缺损早期修复和MMP-3、TIMP-1表达的影响.南方医科大学学报, 2014, 34(1):103-108.

1. Zhu S, Zhang B, Man C, et al. Combined effects of connective tissue growth factor-modified bone marrow-derived mesenchymal stem cells and NaOH-treated PLGA scaffolds on the repair of articular cartilage defect in rabbits. Cell Transplant, 2014, 23(6):715-727.
2. Shi J, Zhang X, Zhu J, et al. Nanoparticle delivery of the bone morphogenetic protein 4 gene to adi pose-derived stem cells promotes articular cartilage repair in vitro and in vivo. Arthroscopy, 2013, 29(12):2001-2011. e2.
3. Lee HH, O'Malley MJ, Friel NA, et al. Persistence, local ization, and external control of transgene expression after single injection of adenoassociated virus into injured joints. Hum Gene Ther, 2013, 24(4):457-466.
4. Qi BW, Yu AX, Zhu SB, et al. Chitosan/poly (vinyl alcohol) hydrogel combined with Ad-hTGF-β₁ transfected mesenchymal stem cells to repair rabbit articular cartilage defects. Exp Biol Med (Maywood), 2013, 238(1):23-30.
5. Eroglu E, Tiwari PM, Waffo AB, et al. A nonviral pHEMA+chitosan nanosphere-mediated high-efficiency gene del ivery system. Int J Nanomedicine, 2013, 8:1403-1415.
6. Hagiwara K, Kishimoto S, Ishihara M, et al. In vivo gene transfer using pDNA/chitosan/chondroitin sulfate ternary complexes:influence of chondroitin sulfate on the stabil ity of freeze-dried complexes and transgene expression in vivo. J Gene Med, 2013, 15(2):83-92.
7. Lu H, Dai Y, Lv L, et al. Chitosan-graft-polyethylenimine/DNA nanoparticles as novel non-viral gene delivery vectors targeting osteoarthritis. PLoS One, 2014, 9(1):e84703.
8. Liao ZX, Peng SF, Ho YC, et al. Mechanistic study of transfection of chitosan/DNA complexes coated by anionic poly(γ-glutamic acid). Biomaterials, 2012, 33(11):3306-3315.
9. Kong F, Liu G, Sun B, et al. Phosphorylatable short peptide conjugated low molecular weight chitosan for efficient siRNA delivery and target gene silencing. Int J Pharm, 2012, 422(1-2):445-453.
10. Plianwong S, Opanasopit P, Ngawhirunpat T, et al. Chitosan combined with poly-L-arginine as efficient, safe, and serum-insensitive vehicle with RNase protection abil ity for siRNA delivery. BioMed Res Int, 2013, 2013:574136.
11. Peng SF, Tseng MT, Ho YC, et al. Mechanisms of cellular uptake and intracellular trafficking with chitosan/DNA/poly(γ-glutamic acid) complexes as a gene del ivery vector. Biomaterials, 2011, 32(1):239-248.
12. 赵荣兰, 任玉平, 孙蓓, 等.壳聚糖转染胰岛素样生长因子基因促进兔关节软骨损伤修复的实验研究.中国修复重建外科杂志, 2010, 24(11):1372-1375.
13. Sun B, Zhao R, Kong F, et al. Phosphorylatable short peptide conjugation for facil itating transfection efficacy of CS/DNA complex. Int J Pharm, 2010, 397(1-2):206-210.
14. 张平, 蔡道章, 刘斌, 等.转IL-1Ra和TGF-β₁基因治疗兔骨性关节炎的体外实验研究.中华关节外科杂志(电子版), 2011, 5(3):335-342.
15. 邬波, 马旭, 朱大木, 等.胰岛素样生长因子1对骨髓间充质干细胞软骨分化及基质金属蛋白酶表达的影响.中国组织工程研究, 2013, 17(19):3421-3429.
16. 梁东春, 郭善一, 郭刚, 等. IGF-1信号肽及成熟肽全编码序列的克隆.天津医科大学学报, 2003, 9(4):453-455.
17. Orth P, Kaul G, Cucchiarini M, et al. Transplanted articular chondrocytes co-overexpressing IGF-I and FGF-2 stimulate cartilage repair in vivo. Knee Surg Sports Traumatol Arthrosc, 2011, 19(12):2119-2130.
18. Zhao Q, Wang S, Tian J, et al. Combination of bone marrow concentrate and PGA scaffolds enhance bone marrow stimulation in rabbit articular cartilage repair. J Mater Sci Mater Med, 2013, 24(3):793-801.
19. Peng XX, Zhao RL, Song W, et al. Selection of suitable reference genes for normalization of quantitative real-time PCR in cartilage tissue injury and repair in rabbits. Int J Mol Sci, 2012, 13(11):14344-14355.
20. 黄云梅, 陈文列, 黄美雅, 等.多种特殊染色法在骨关节炎组织形态学研究中的应用比较.中国比较医学杂志, 2011, 21(5):45-48.
21. Jia X, Cheng K, Mahato RI. Coexpression of vascular endothel ial growth factor and interleukin-1 receptor antagonist for improved human islet survival and function. Mol Pharm, 2007, 4(2):199-207.
22. Zamani S, Hashemibeni B, Esfandiari E, et al. Assessment of TGF-β3 on production of aggrecan by human articular chondrocytes in pellet culture system. Adv Biomed Res, 2014, 3:54.
23. Williams A, Smith JR, Allaway D, et al. Carprofen inhibits the release of matrix metalloproteinases 1, 3, and 13 in the secretome of an explant model of articular cartilage stimulated with interleukin 1β. Arthritis Res Ther, 2013, 15(6):R223.
24. 贺占坤, 沈杰威. MMP-2、MMP-3、MMP-9和TIMP-1评价膝关节骨性关节炎的临床研究.重庆医学, 2013, 42(32):3872-3874.
25. 董福, 宋锦旗, 罗吉伟, 等.不同强度运动对大鼠关节软骨缺损早期修复和MMP-3、TIMP-1表达的影响.南方医科大学学报, 2014, 34(1):103-108.

Chinese Journal of Reparative and Reconstructive Surgery

PHOSPHORYLATABLE SHORT PEPTIDE CONJUGATED CHITOSAN MEDIATED GENE THERAPY FOR REPAIR OF ARTICULAR CARTILAGE DEFECT IN RABBITS

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