Effect of xanthohumol-loaded anti-inflammatory scaffolds on cartilage regeneration in goats_Chinese Journal of Reparative and Reconstructive Surgery

Authors：

XU Songshan ¹ , ZHAO Shaohua ² , JIAN Yanpeng ¹ , XU Yong ³ , LIU Weijie ¹ , SHAO Xinwei ¹ , FAN Jun ¹ ,  WANG Yigong ¹

1. Department of Spine and Spinal Cord Surgery, Xuchang Central Hospital Affiliated to Henan University of Science and Technology, Xuchang Henan, 461000, P. R. China;
2. Department of Plastic Surgery, Xuchang Central Hospital Affiliated to Henan University of Science and Technology, Xuchang Henan, 461000, P. R. China;
3. Department of Thoracic Surgery, Shanghai Pulmonary Hospital, Shanghai, 200433, P. R. China;

Corresponding?author：

WANG Yigong, Email: 617693188@qq.com

Keywords：

Cartilage tissue engineering; xanthohumol; scaffold; anti-inflammation; bone marrow mesenchymal stem cells; goat

DOI：

10.7507/1002-1892.202204044

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Objective To develop an anti-inflammatory poly (lactic-co-glycolic acid) (PLGA) scaffold by loading xanthohumol, and investigate its anti-inflammatory and cartilage regeneration effects in goats. Methods The PLGA porous scaffolds were prepared by pore-causing agent leaching method, and then placed in xanthohumol solution for 24 hours to prepare xanthohumol-PLGA scaffolds (hereinafter referred to as drug-loaded scaffolds). The PLGA scaffolds and drug-loaded scaffolds were taken for general observation, the pore diameter of the scaffolds was measured by scanning electron microscope, the porosity was calculated by the drainage method, and the loading of xanthohumol on the scaffolds was verified by Fourier transform infrared (FTIR) spectrometer. Then the two scaffolds were co-cultured with RAW264.7 macrophages induced by lipopolysaccharide for 24 hours, and the expressions of inflammatory factors [interleukin 1β (IL-1β) and tumor necrosis factor α (TNF-α)] were detected by RT-PCR and Western blot to evaluate the anti-inflammatory properties in vitro of two scaffolds. Bone marrow mesenchymal stem cells (BMSCs) was obtained from bone marrow of a 6-month-old female healthy goat, cultured by adherent method, and passaged in vitro. The second passage cells were seeded on two scaffolds to construct BMSCs-scaffolds, and the cytocompatibility of scaffolds was observed by live/dead cell staining and cell counting kit 8 (CCK-8) assay. The BMSCs-scaffolds were cultured in vitro for 6 weeks, aiming to verify its feasibility of generating cartilage in vitro by gross observation, histological staining, collagen type Ⅱ immunohistochemical staining, and biochemical analysis. Finally, the two kinds of BMSCs-scaffolds cultured in vitro for 6 weeks were implanted into the goat subcutaneously, respectively. After 4 weeks, gross observation, histological staining, collagen type Ⅱ immunohistochemical staining, biochemical analysis, and RT-PCR were performed to comprehensively evaluate the anti-inflammatory effect in vivo and promotion of cartilage regeneration of the drug-loaded scaffolds. Results The prepared drug-loaded scaffold had a white porous structure with abundant, continuous, and uniform pore structures. Compared with the PLGA scaffold, there was no significant difference in pore size and porosity (P>0.05). FTIR spectrometer analysis showed that xanthohumol was successfully loaded to PLGA scaffolds. The in vitro results demonstrated that the gene and protein expressions of inflammatory cytokines (IL-1β and TNF-α) in drug-loaded scaffold significantly decreased than those in PLGA scaffold (P<0.05). With the prolongation of culture, the number of live cells increased significantly, and there was no significant difference between the two scaffolds (P>0.05). The in vitro cartilage regeneration test indicated that the BMSCs-drug-loaded scaffolds displayed smooth and translucent appearance with yellow color after 6 weeks in vitro culture, and could basically maintained its original shape. The histological and immunohistochemical stainings revealed that the scaffolds displayed typical lacunar structure and cartilage-specific extracellular matrix. In addition, quantitative data revealed that the contents of glycosaminoglycan (GAG) and collagen type Ⅱ were not significantly different from BMSCs-PLGA scaffolds (P>0.05). The evaluation of cartilage regeneration in vivo showed that the BMSCs-drug-loaded scaffolds basically maintained their pre-implantation shape and size at 4 weeks after implantation in goat, while the BMSCs-PLGA scaffolds were severely deformed. The BMSCs-drug-loaded scaffolds had typical cartilage lacuna structure and cartilage specific extracellular matrix, and no obvious inflammatory cells infiltration; while the BMSCs-PLGA scaffolds had a messy fibrous structure, showing obvious inflammatory response. The contents of cartilage-specific GAG and collagen type Ⅱ in BMSCs-drug-loaded scaffolds were significantly higher than those in BMSCs-PLGA scaffolds (P<0.05); the relative gene expressions of IL-1β and TNF-α were significantly lower than those in BMSCs-PLGA scaffolds (P<0.05). Conclusion The drug-loaded scaffolds have suitable pore size, porosity, cytocompatibility, and good anti-inflammatory properties, and can promote cartilage regeneration after implantation with BMSCs in goats.

Citation： XU Songshan, ZHAO Shaohua, JIAN Yanpeng, XU Yong, LIU Weijie, SHAO Xinwei, FAN Jun, WANG Yigong. Effect of xanthohumol-loaded anti-inflammatory scaffolds on cartilage regeneration in goats. Chinese Journal of Reparative and Reconstructive Surgery, 2022, 36(10): 1296-1304. doi: 10.7507/1002-1892.202204044 Copy

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2.	陸定貴, 林佳杰, 姚順晗, 等. 關節軟骨損傷修復的臨床研究進展. 微創醫學, 2021, 16(4): 538-541.
3.	Brittberg M, Lindahl A, Nilsson A, et al. Treatment of deep cartilage defects in the knee with autologous chondrocyte transplantation. N Engl J Med, 1994, 331(14): 889-895.
4.	王新偉, 趙英杰, 常艷, 等. 間充質干細胞治療骨關節炎軟骨損傷: 作用、應用與問題. 中國組織工程研究, 2021, 25(31): 5053-5058.
5.	Liu X, Zheng L, Zhou Y, et al. BMSC transplantation aggravates inflammation, oxidative stress, and fibrosis and impairs skeletal muscle regeneration. Front Physiol, 2019, 10: 87. doi: 10.3389/fphys.2019.00087.
6.	Zhang R, Ma J, Han J, et al. Mesenchymal stem cell related therapies for cartilage lesions and osteoarthritis. Am J Transl Res, 2019, 11(10): 6275-6289.
7.	Girisa S, Saikia Q, Bordoloi D, et al. Xanthohumol from Hop: Hope for cancer prevention and treatment. IUBMB Life, 2021, 73(8): 1016-1044.
8.	林旭晨, 祝海年, 王增順, 等. 黃腐酚對骨關節炎模型小鼠炎性因子及關節軟骨的作用. 中國組織工程研究, 2022, 26(5): 676-681.
9.	臺會文, 周朝華. 用線型聚合物作致孔劑制備的大孔樹脂的形態與孔結構的研究. 河北工學院學報, 1991, (2): 95-102.
10.	Zhu Y, Zhang Y, Liu Y, et al. The influence of Chm-Ⅰ knockout on ectopic cartilage regeneration and homeostasis maintenance. Tissue Eng Part A, 2015, 21(3-4): 782-792.
11.	李強強, 謝亞東, 楊國清, 等. 骨髓間充質干細胞成骨分化的研究進展. 醫學綜述, 2022, 28(3): 434-438.
12.	Mariani E, Lisignoli G, Borzì RM, et al. Biomaterials: foreign bodies or tuners for the immune response? Int J Mol Sci, 2019, 20(3): 636. doi: 10.3390/ijms20030636.
13.	Sumayya AS, Muraleedhara Kurup G. In vitro anti-inflammatory potential of marine macromolecules cross-linked bio-composite scaffold on LPS stimulated RAW264.7 macrophage cells for cartilage tissue engineering applications. J Biomater Sci Polym Ed, 2021, 32(8): 1040-1056.
14.	Martínez-Sanmiguel JJ, G Zarate-Trivi?o D, Hernandez-Delgadillo R, et al. Anti-inflammatory and antimicrobial activity of bioactive hydroxyapatite/silver nanocomposites. J Biomater Appl, 2019, 33(10): 1314-1326.
15.	Fasolino I, Raucci MG, Soriente A, et al. Osteoinductive and anti-inflammatory properties of chitosan-based scaffolds for bone regeneration. Mater Sci Eng C Mater Biol Appl, 2019, 105: 110046. doi: 10.1016/j.msec.2019.110046.
16.	Sp?ter T, Mariyanats AO, Syachina MA, et al. In vitro and in vivo analysis of adhesive, anti-inflammatory, and proangiogenic properties of novel 3D printed hyaluronic acid glycidyl methacrylate hydrogel scaffolds for tissue engineering. ACS Biomater Sci Eng, 2020, 6(10): 5744-5757.
17.	Liang JP, Accolla RP, Jiang K, et al. Controlled release of anti-inflammatory and proangiogenic factors from macroporous scaffolds. Tissue Eng Part A, 2021, 27(19-20): 1275-1289.
18.	Go DP, Palmer JA, Gras SL, et al. Coating and release of an anti-inflammatory hormone from PLGA microspheres for tissue engineering. J Biomed Mater Res A, 2012, 100(2): 507-517.
19.	Ziadlou R, Rotman S, Teuschl A, et al. Optimization of hyaluronic acid-tyramine/silk-fibroin composite hydrogels for cartilage tissue engineering and delivery of anti-inflammatory and anabolic drugs. Mater Sci Eng C Mater Biol Appl, 2021, 120: 111701. doi: 10.1016/j.msec.2020.111701.
20.	Zheng T, Zhou Q, Huang J, et al. Xanthohumol inhibited mechanical stimulation-induced articular ECM degradation by mediating lncRNA GAS5/miR-27a axis. Front Pharmacol, 2021, 12: 737552. doi: 10.3389/fphar.2021.737552.
21.	Zhang M, Zhang R, Zheng T, et al. Xanthohumol attenuated inflammation and ECM degradation by mediating HO-1/C/EBPβ pathway in osteoarthritis chondrocytes. Front Pharmacol, 2021, 12: 680585. doi: 10.3389/fphar.2021.680585.
22.	Cho YC, Kim HJ, Kim YJ, et al. Differential anti-inflammatory pathway by xanthohumol in IFN-gamma and LPS-activated macrophages. Int Immunopharmacol, 2008, 8(4): 567-573.
23.	Rocha CV, Gon?alves V, da Silva MC, et al. PLGA-based composites for various biomedical applications. Int J Mol Sci, 2022, 23(4): 2034. doi: 10.3390/ijms23042034.
24.	Fonseca M, Macedo AS, Lima SAC, et al. Evaluation of the antitumour and antiproliferative effect of xanthohumol-loaded PLGA nanoparticles on melanoma. Materials (Basel), 2021, 14(21): 6421. doi: 10.3390/ma14216421.
25.	Wang CC, Ho YH, Hung CF, et al. Xanthohumol, an active constituent from hope, affords protection against kainic acid-induced excitotoxicity in rats. Neurochem Int, 2020, 133: 104629. doi: 10.1016/j.neuint.2019.104629.
26.	Li Z, Huang Z, Zhang H, et al. P2X7 receptor induces pyroptotic inflammation and cartilage degradation in osteoarthritis via NF-κB/NLRP3 crosstalk. Oxid Med Cell Longev, 2021, 2021: 8868361. doi: 10.1155/2021/8868361.
27.	Wu CL, Harasymowicz NS, Klimak MA, et al. The role of macrophages in osteoarthritis and cartilage repair. Osteoarthritis Cartilage, 2020, 28(5): 544-554.
28.	Li Y, Tong D, Liang P, et al. Cartilage-binding antibodies initiate joint inflammation and promote chronic erosive arthritis. Arthritis Res Ther, 2020, 22(1): 120. doi: 10.1186/s13075-020-02169-0.
29.	Kapoor M, Martel-Pelletier J, Lajeunesse D, et al. Role of proinflammatory cytokines in the pathophysiology of osteoarthritis. Nat Rev Rheumatol, 2011, 7(1): 33-42.
30.	Pozgan U, Caglic D, Rozman B, et al. Expression and activity profiling of selected cysteine cathepsins and matrix metalloproteinases in synovial fluids from patients with rheumatoid arthritis and osteoarthritis. Biol Chem, 2010, 391(5): 571-579.
31.	Shi Y, Hu X, Cheng J, et al. A small molecule promotes cartilage extracellular matrix generation and inhibits osteoarthritis development. Nat Commun, 2019, 10(1): 1914. doi: 10.1038/s41467-019-09839-x.
32.	Liu B, Yang L, Cui Z, et al. Anti-TNF-α therapy alters the gut microbiota in proteoglycan-induced ankylosing spondylitis in mice. Microbiologyopen, 2019, 8(12): e927. doi: 10.1002/mbo3.927.
33.	Lefebvre V, Peeters-Joris C, Vaes G. Modulation by interleukin 1 and tumor necrosis factor alpha of production of collagenase, tissue inhibitor of metalloproteinases and collagen types in differentiated and dedifferentiated articular chondrocytes. Biochim Biophys Acta, 1990, 1052(3): 366-378. doi: 10.1016/0167-4889(90)90145-4.

1. Boushell MK, Hung CT, Hunziker EB, et al. Current strategies for integrative cartilage repair. Connect Tissue Res, 2017, 58(5): 393-406.
2. 陸定貴, 林佳杰, 姚順晗, 等. 關節軟骨損傷修復的臨床研究進展. 微創醫學, 2021, 16(4): 538-541.
3. Brittberg M, Lindahl A, Nilsson A, et al. Treatment of deep cartilage defects in the knee with autologous chondrocyte transplantation. N Engl J Med, 1994, 331(14): 889-895.
4. 王新偉, 趙英杰, 常艷, 等. 間充質干細胞治療骨關節炎軟骨損傷: 作用、應用與問題. 中國組織工程研究, 2021, 25(31): 5053-5058.
5. Liu X, Zheng L, Zhou Y, et al. BMSC transplantation aggravates inflammation, oxidative stress, and fibrosis and impairs skeletal muscle regeneration. Front Physiol, 2019, 10: 87. doi: 10.3389/fphys.2019.00087.
6. Zhang R, Ma J, Han J, et al. Mesenchymal stem cell related therapies for cartilage lesions and osteoarthritis. Am J Transl Res, 2019, 11(10): 6275-6289.
7. Girisa S, Saikia Q, Bordoloi D, et al. Xanthohumol from Hop: Hope for cancer prevention and treatment. IUBMB Life, 2021, 73(8): 1016-1044.
8. 林旭晨, 祝海年, 王增順, 等. 黃腐酚對骨關節炎模型小鼠炎性因子及關節軟骨的作用. 中國組織工程研究, 2022, 26(5): 676-681.
9. 臺會文, 周朝華. 用線型聚合物作致孔劑制備的大孔樹脂的形態與孔結構的研究. 河北工學院學報, 1991, (2): 95-102.
10. Zhu Y, Zhang Y, Liu Y, et al. The influence of Chm-Ⅰ knockout on ectopic cartilage regeneration and homeostasis maintenance. Tissue Eng Part A, 2015, 21(3-4): 782-792.
11. 李強強, 謝亞東, 楊國清, 等. 骨髓間充質干細胞成骨分化的研究進展. 醫學綜述, 2022, 28(3): 434-438.
12. Mariani E, Lisignoli G, Borzì RM, et al. Biomaterials: foreign bodies or tuners for the immune response? Int J Mol Sci, 2019, 20(3): 636. doi: 10.3390/ijms20030636.
13. Sumayya AS, Muraleedhara Kurup G. In vitro anti-inflammatory potential of marine macromolecules cross-linked bio-composite scaffold on LPS stimulated RAW264.7 macrophage cells for cartilage tissue engineering applications. J Biomater Sci Polym Ed, 2021, 32(8): 1040-1056.
14. Martínez-Sanmiguel JJ, G Zarate-Trivi?o D, Hernandez-Delgadillo R, et al. Anti-inflammatory and antimicrobial activity of bioactive hydroxyapatite/silver nanocomposites. J Biomater Appl, 2019, 33(10): 1314-1326.
15. Fasolino I, Raucci MG, Soriente A, et al. Osteoinductive and anti-inflammatory properties of chitosan-based scaffolds for bone regeneration. Mater Sci Eng C Mater Biol Appl, 2019, 105: 110046. doi: 10.1016/j.msec.2019.110046.
16. Sp?ter T, Mariyanats AO, Syachina MA, et al. In vitro and in vivo analysis of adhesive, anti-inflammatory, and proangiogenic properties of novel 3D printed hyaluronic acid glycidyl methacrylate hydrogel scaffolds for tissue engineering. ACS Biomater Sci Eng, 2020, 6(10): 5744-5757.
17. Liang JP, Accolla RP, Jiang K, et al. Controlled release of anti-inflammatory and proangiogenic factors from macroporous scaffolds. Tissue Eng Part A, 2021, 27(19-20): 1275-1289.
18. Go DP, Palmer JA, Gras SL, et al. Coating and release of an anti-inflammatory hormone from PLGA microspheres for tissue engineering. J Biomed Mater Res A, 2012, 100(2): 507-517.
19. Ziadlou R, Rotman S, Teuschl A, et al. Optimization of hyaluronic acid-tyramine/silk-fibroin composite hydrogels for cartilage tissue engineering and delivery of anti-inflammatory and anabolic drugs. Mater Sci Eng C Mater Biol Appl, 2021, 120: 111701. doi: 10.1016/j.msec.2020.111701.
20. Zheng T, Zhou Q, Huang J, et al. Xanthohumol inhibited mechanical stimulation-induced articular ECM degradation by mediating lncRNA GAS5/miR-27a axis. Front Pharmacol, 2021, 12: 737552. doi: 10.3389/fphar.2021.737552.
21. Zhang M, Zhang R, Zheng T, et al. Xanthohumol attenuated inflammation and ECM degradation by mediating HO-1/C/EBPβ pathway in osteoarthritis chondrocytes. Front Pharmacol, 2021, 12: 680585. doi: 10.3389/fphar.2021.680585.
22. Cho YC, Kim HJ, Kim YJ, et al. Differential anti-inflammatory pathway by xanthohumol in IFN-gamma and LPS-activated macrophages. Int Immunopharmacol, 2008, 8(4): 567-573.
23. Rocha CV, Gon?alves V, da Silva MC, et al. PLGA-based composites for various biomedical applications. Int J Mol Sci, 2022, 23(4): 2034. doi: 10.3390/ijms23042034.
24. Fonseca M, Macedo AS, Lima SAC, et al. Evaluation of the antitumour and antiproliferative effect of xanthohumol-loaded PLGA nanoparticles on melanoma. Materials (Basel), 2021, 14(21): 6421. doi: 10.3390/ma14216421.
25. Wang CC, Ho YH, Hung CF, et al. Xanthohumol, an active constituent from hope, affords protection against kainic acid-induced excitotoxicity in rats. Neurochem Int, 2020, 133: 104629. doi: 10.1016/j.neuint.2019.104629.
26. Li Z, Huang Z, Zhang H, et al. P2X7 receptor induces pyroptotic inflammation and cartilage degradation in osteoarthritis via NF-κB/NLRP3 crosstalk. Oxid Med Cell Longev, 2021, 2021: 8868361. doi: 10.1155/2021/8868361.
27. Wu CL, Harasymowicz NS, Klimak MA, et al. The role of macrophages in osteoarthritis and cartilage repair. Osteoarthritis Cartilage, 2020, 28(5): 544-554.
28. Li Y, Tong D, Liang P, et al. Cartilage-binding antibodies initiate joint inflammation and promote chronic erosive arthritis. Arthritis Res Ther, 2020, 22(1): 120. doi: 10.1186/s13075-020-02169-0.
29. Kapoor M, Martel-Pelletier J, Lajeunesse D, et al. Role of proinflammatory cytokines in the pathophysiology of osteoarthritis. Nat Rev Rheumatol, 2011, 7(1): 33-42.
30. Pozgan U, Caglic D, Rozman B, et al. Expression and activity profiling of selected cysteine cathepsins and matrix metalloproteinases in synovial fluids from patients with rheumatoid arthritis and osteoarthritis. Biol Chem, 2010, 391(5): 571-579.
31. Shi Y, Hu X, Cheng J, et al. A small molecule promotes cartilage extracellular matrix generation and inhibits osteoarthritis development. Nat Commun, 2019, 10(1): 1914. doi: 10.1038/s41467-019-09839-x.
32. Liu B, Yang L, Cui Z, et al. Anti-TNF-α therapy alters the gut microbiota in proteoglycan-induced ankylosing spondylitis in mice. Microbiologyopen, 2019, 8(12): e927. doi: 10.1002/mbo3.927.
33. Lefebvre V, Peeters-Joris C, Vaes G. Modulation by interleukin 1 and tumor necrosis factor alpha of production of collagenase, tissue inhibitor of metalloproteinases and collagen types in differentiated and dedifferentiated articular chondrocytes. Biochim Biophys Acta, 1990, 1052(3): 366-378. doi: 10.1016/0167-4889(90)90145-4.

Chinese Journal of Reparative and Reconstructive Surgery

Effect of xanthohumol-loaded anti-inflammatory scaffolds on cartilage regeneration in goats

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