Andrographolide-releasing collagen scaffold enhance the ability of chondrocytes to maintain their specific phenotype under inflammatory environment <i>in vitro</i> _Journal of Biomedical Engineering

Authors：

XU Linu ,  LIU Hairong , DAI Yao , LI Yongsheng , CHEN Wei

College of Materials Science and Engineering, Hunan University, Changsha 410082, P.R.China;

Corresponding author：

LIU Hairong, Email: liuhairong@hnu.edu.cn

Keywords：

andrographolide; collagen scaffold; interleukin-1β; rabbit chondrocytes; phenotypic maintenance

DOI：

10.7507/1001-5515.201804025

Video：

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The aim of this article is to study how andrographolide-releasing collagen scaffolds influence rabbit articular chondrocytes in maintaining their specific phenotype under inflammatory environment. Physical blending combined with vacuum freeze-drying method was utilized to prepare the andrographolide-releasing collagen scaffold. The characteristics of scaffold including its surface morphology and porosity were detected with environmental scanning electron microscope (ESEM) and a density instrument. Then, the release of andrographolide from prepared scaffolds was measured by UV-visible spectroscopy. Rabbit chondrocytes were isolated and cultured in vitro and seeded on andrographolide-releasing collagen scaffolds. Following culture with normal medium for 3 d, seeded chondrocytes were cultured with medium containing interleukin-1 beta (IL-1β) to stimulate inflammation in vitro for 7 d. The proliferation, morphology and gene transcription of tested chondrocytes were detected with Alamar Blue assay, fluorescein diacetate (FDA) staining and reverse-transcriptase quantitative polymerase chain reaction (RT-qPCR) test respectively. The results showed that the collagen scaffolds prepared by vacuum freeze-dry possess a high porosity close to 96%, and well-interconnected chambers around (120.7±17.8) μm. The andrographolide-releasing collagen scaffold continuously released andrographolide to the PBS solution within 15 d, and collagen scaffolds containing 2.22% andrographolide significantly inhibit the proliferation of chondrocytes. Compared with collagen scaffolds, 0.44% andrographolide-containing collagen scaffolds facilitate chondrocytes to keep specific normal morphologies following 7 d IL-1β induction. The results obtained by RT-qPCR confirmed this effect by enhancing the transcription of tissue inhibitor of metalloproteinase-1 (TIMP-1), collagen II (COL II), aggrecan (Aggrecan) and the ratio of COL II/ collagen I(COL I), meanwhile, reversing the promoted transcription of matrix metalloproteinase-1 (MMP-1) and matrix metalloproteinase-13 (MMP-13). In conclusion, our research reveals that andrographolide-releasing (0.44%) collagen scaffolds enhance the ability of chondrocytes to maintain their specific morphologies by up-regulating the transcription of genes like COL II, Aggrecan and TIMP-1, while down-regulating the transcription of genes like MMP-1 and MMP-13 which are bad for phenotypic maintenance under IL-1β simulated inflammatory environment. These results implied the potential use of andrographolide-releasing collagen scaffold in osteoarthritic cartilage repair.

Citation： XU Linu, LIU Hairong, DAI Yao, LI Yongsheng, CHEN Wei. Andrographolide-releasing collagen scaffold enhance the ability of chondrocytes to maintain their specific phenotype under inflammatory environment in vitro . Journal of Biomedical Engineering, 2018, 35(6): 905-913. doi: 10.7507/1001-5515.201804025 Copy

1.	Makris E A, Gomoll A H, Malizos K N, et al. Repair and tissue engineering techniques for articular cartilage. Nat Rev Rheumatol, 2015, 11(1): 21-34.
2.	Chen Jie, Yuan Zhaoyuan, Liu Yu, et al. Improvement of in vitro Three-dimensional cartilage regeneration by a novel hydrostatic pressure bioreactor. Stem Cells Transl Med, 2017, 6(3): 982-991.
3.	Cecil D L, Johnson K, Rediske J, et al. Inflammation-induced chondrocyte hypertrophy is driven by receptor for advanced glycation end products. Journal of Immunology, 2005, 175(12): 8296-8302.
4.	Kobayashi M, Squires G R, Mousa Aisha, et al. Role of interleukin-1 and tumor necrosis factor alpha in matrix degradation of human osteoarthritic cartilage. Arthritis Rheum, 2005, 52(1): 128-135.
5.	Chen Weiping, Tang Jingli, Bao Jiapeng, et al. Anti-arthritic effects of chlorogenic acid in interleukin-1β-induced rabbit chondrocytes and a rabbit osteoarthritis model. Int Immunopharmacol, 2011, 11(1): 23-28.
6.	Kwon H, Sun Lin, Cairns D M, et al. The influence of scaffold material on chondrocytes under inflammatory conditions. Acta Biomater, 2013, 9(5): 6563-6575.
7.	Shi Shiping, Man Zhentao, Li Wei, et al. Silencing of Wnt5a prevents interleukin-1β-induced collagen type II degradation in rat chondrocytes. Exp Ther Med, 2016, 12(5): 3161-3166.
8.	Wang Wei, Li Bo, Yang Junzhou, et al. The restoration of full-thickness cartilage defects with BMSCs and TGF-beta 1 loaded PLGA/fibrin gel constructs. Biomaterials, 2010, 31(34): 8964-8973.
9.	Zhang Ying, Pizzute T, Pei Ming. Anti-inflammatory strategies in cartilage repair. Tissue Eng Part B Rev, 2014, 20(6): 655-668.
10.	Zhao Lei, Ye Juan, Wu Guotai, et al. Gentiopicroside prevents interleukin-1 beta induced inflammation response in rat articular chondrocyte. J Ethnopharmacol, 2015, 172: 100-107.
11.	Li Feng, Yin Zhanhai, Zhou Bing, et al. Shikonin inhibits inflammatory responses in rabbit chondrocytes and shows chondroprotection in osteoarthritic rabbit knee. Int Immunopharmacol, 2015, 29(2): 656-662.
12.	Tangyuenyong S, Viriyakhasem N, Peansukmanee S, et al. Andrographolide exerts chondroprotective activity in equine cartilage explant and suppresses interleukin-1beta-Induced MMP-2 expression in equine chondrocyte culture. IS Notices, 2014, 2014: 464136.
13.	Luo Like, Wei Qingjun, Liu Lei, et al. Andrographolide enhances proliferation and prevents dedifferentiation of rabbit articular chondrocytes: an in vitro study. Evidence-Based Complementary and Alternative Medicine, 2015, (1): 984850.
14.	刘海蓉, 黄继英, 周征, 等. 透明质酸改性 PHBV 组织工程纳米纤维支架的研究. 湖南大学学报:自然科学版, 2017, 44(6): 87-95.
15.	Liu Hairong, Xia Leilei, Dai Yao, et al. Fabrication and characterization of novel hydroxyapatite/porous carbon composite scaffolds. Mater Lett, 2012, 66(1): 36-38.
16.	Dudhia J. Aggrecan, aging and assembly in articular cartilage. Cellular and Molecular Life Sciences, 2005, 62(20): 2241-2256.
17.	Stokes D G, Liu G, Dharmavaram R, et al. Regulation of type-II collagen gene expression during human chondrocyte de-differentiation and recovery of chondrocyte-specific phenotype in culture involves Sry-type high-mobility-group box (SOX) transcription factors. Biochem J, 2001, 360(2): 461-470.
18.	Huh Y H, Ryu J H, Chun J S. Regulation of type II collagen expression by histone deacetylase in articular chondrocytes. J Biol Chem, 2007, 282(23): 17123-17131.
19.	Ghayor C, Chadjichristos C, Herrouin J F, et al. SP3 represses the SP1-mediated transactivation of the human COL2A1 gene in primary and de-differentiated chondrocytes. J Biol Chem, 2001, 276(40): 36881-36895.
20.	Aida Y, Maeno M, Suzuki N, et al. The effect of IL-1β on the expression of matrix metalloproteinases and tissue inhibitors of matrix metalloproteinases in human chondrocytes. Life Sci, 2005, 77(25): 3210-3221.
21.	Vincenti M P, Brinckerhoff C E. Transcriptional regulation of collagenase (MMP-1, MMP-13) genes in arthritis: integration of complex signaling pathways for the recruitment of gene-specific transcription factors. Arthritis Res, 2002, 4(3): 157-164.
22.	Wang Xingang, You Chuangang, Hu Xinlei, et al. The roles of knitted mesh-reinforced collagen-chitosan hybrid scaffold in the one-step repair of full-thickness skin defects in rats. Acta Biomater, 2013, 9(8): 7822-7832.
23.	Wang Wei, Li Bo, Li Yanglin, et al. In vivo restoration of full-thickness cartilage defects by poly (lactide-co-glycolide) sponges filled with fibrin gel, bone marrow mesenchymal stem cells and DNA complexes. Biomaterials, 2010, 31(23): 5953-5965.
24.	Stenhamre H, Nannmark U, Lindahl A, et al. Influence of pore size on the redifferentiation potential of human articular chondrocytes in poly (urethane urea) scaffolds. J Tissue Eng Regen Med, 2011, 5(7): 578-588.
25.	Nava M M, Draghi L, Giordano C A. The effect of scaffold pore size in cartilage tissue engineering. J Appl Biomater Funct Mater, 2016, 14(3): e223-e229.

1. Makris E A, Gomoll A H, Malizos K N, et al. Repair and tissue engineering techniques for articular cartilage. Nat Rev Rheumatol, 2015, 11(1): 21-34.
2. Chen Jie, Yuan Zhaoyuan, Liu Yu, et al. Improvement of in vitro Three-dimensional cartilage regeneration by a novel hydrostatic pressure bioreactor. Stem Cells Transl Med, 2017, 6(3): 982-991.
3. Cecil D L, Johnson K, Rediske J, et al. Inflammation-induced chondrocyte hypertrophy is driven by receptor for advanced glycation end products. Journal of Immunology, 2005, 175(12): 8296-8302.
4. Kobayashi M, Squires G R, Mousa Aisha, et al. Role of interleukin-1 and tumor necrosis factor alpha in matrix degradation of human osteoarthritic cartilage. Arthritis Rheum, 2005, 52(1): 128-135.
5. Chen Weiping, Tang Jingli, Bao Jiapeng, et al. Anti-arthritic effects of chlorogenic acid in interleukin-1β-induced rabbit chondrocytes and a rabbit osteoarthritis model. Int Immunopharmacol, 2011, 11(1): 23-28.
6. Kwon H, Sun Lin, Cairns D M, et al. The influence of scaffold material on chondrocytes under inflammatory conditions. Acta Biomater, 2013, 9(5): 6563-6575.
7. Shi Shiping, Man Zhentao, Li Wei, et al. Silencing of Wnt5a prevents interleukin-1β-induced collagen type II degradation in rat chondrocytes. Exp Ther Med, 2016, 12(5): 3161-3166.
8. Wang Wei, Li Bo, Yang Junzhou, et al. The restoration of full-thickness cartilage defects with BMSCs and TGF-beta 1 loaded PLGA/fibrin gel constructs. Biomaterials, 2010, 31(34): 8964-8973.
9. Zhang Ying, Pizzute T, Pei Ming. Anti-inflammatory strategies in cartilage repair. Tissue Eng Part B Rev, 2014, 20(6): 655-668.
10. Zhao Lei, Ye Juan, Wu Guotai, et al. Gentiopicroside prevents interleukin-1 beta induced inflammation response in rat articular chondrocyte. J Ethnopharmacol, 2015, 172: 100-107.
11. Li Feng, Yin Zhanhai, Zhou Bing, et al. Shikonin inhibits inflammatory responses in rabbit chondrocytes and shows chondroprotection in osteoarthritic rabbit knee. Int Immunopharmacol, 2015, 29(2): 656-662.
12. Tangyuenyong S, Viriyakhasem N, Peansukmanee S, et al. Andrographolide exerts chondroprotective activity in equine cartilage explant and suppresses interleukin-1beta-Induced MMP-2 expression in equine chondrocyte culture. IS Notices, 2014, 2014: 464136.
13. Luo Like, Wei Qingjun, Liu Lei, et al. Andrographolide enhances proliferation and prevents dedifferentiation of rabbit articular chondrocytes: an in vitro study. Evidence-Based Complementary and Alternative Medicine, 2015, (1): 984850.
14. 刘海蓉, 黄继英, 周征, 等. 透明质酸改性 PHBV 组织工程纳米纤维支架的研究. 湖南大学学报:自然科学版, 2017, 44(6): 87-95.
15. Liu Hairong, Xia Leilei, Dai Yao, et al. Fabrication and characterization of novel hydroxyapatite/porous carbon composite scaffolds. Mater Lett, 2012, 66(1): 36-38.
16. Dudhia J. Aggrecan, aging and assembly in articular cartilage. Cellular and Molecular Life Sciences, 2005, 62(20): 2241-2256.
17. Stokes D G, Liu G, Dharmavaram R, et al. Regulation of type-II collagen gene expression during human chondrocyte de-differentiation and recovery of chondrocyte-specific phenotype in culture involves Sry-type high-mobility-group box (SOX) transcription factors. Biochem J, 2001, 360(2): 461-470.
18. Huh Y H, Ryu J H, Chun J S. Regulation of type II collagen expression by histone deacetylase in articular chondrocytes. J Biol Chem, 2007, 282(23): 17123-17131.
19. Ghayor C, Chadjichristos C, Herrouin J F, et al. SP3 represses the SP1-mediated transactivation of the human COL2A1 gene in primary and de-differentiated chondrocytes. J Biol Chem, 2001, 276(40): 36881-36895.
20. Aida Y, Maeno M, Suzuki N, et al. The effect of IL-1β on the expression of matrix metalloproteinases and tissue inhibitors of matrix metalloproteinases in human chondrocytes. Life Sci, 2005, 77(25): 3210-3221.
21. Vincenti M P, Brinckerhoff C E. Transcriptional regulation of collagenase (MMP-1, MMP-13) genes in arthritis: integration of complex signaling pathways for the recruitment of gene-specific transcription factors. Arthritis Res, 2002, 4(3): 157-164.
22. Wang Xingang, You Chuangang, Hu Xinlei, et al. The roles of knitted mesh-reinforced collagen-chitosan hybrid scaffold in the one-step repair of full-thickness skin defects in rats. Acta Biomater, 2013, 9(8): 7822-7832.
23. Wang Wei, Li Bo, Li Yanglin, et al. In vivo restoration of full-thickness cartilage defects by poly (lactide-co-glycolide) sponges filled with fibrin gel, bone marrow mesenchymal stem cells and DNA complexes. Biomaterials, 2010, 31(23): 5953-5965.
24. Stenhamre H, Nannmark U, Lindahl A, et al. Influence of pore size on the redifferentiation potential of human articular chondrocytes in poly (urethane urea) scaffolds. J Tissue Eng Regen Med, 2011, 5(7): 578-588.
25. Nava M M, Draghi L, Giordano C A. The effect of scaffold pore size in cartilage tissue engineering. J Appl Biomater Funct Mater, 2016, 14(3): e223-e229.

Journal of Biomedical Engineering

Andrographolide-releasing collagen scaffold enhance the ability of chondrocytes to maintain their specific phenotype under inflammatory environment in vitro

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