Chinese Journal of Tissue Engineering Research ›› 2022, Vol. 26 ›› Issue (31): 5040-5046.doi: 10.12307/2022.959

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Effect of phosphocreatine modified chitosan hydrogel on polarization and inflammatory factor expression in rat bone marrow-derived macrophages

Sheng Weibei1, 2, 3, Xiong Ao2, 3, Liu Su2, 3, Deng Jiapeng2, 3, Weng Jian2, 3, Yu Fei2, 3, Chen Yingqi2, 3, Zeng Hui2, 3   

  1. 1Weifang Medical University, Weifang 261053, Shandong Province, China; 2Department of Bone & Joint Surgery, Shenzhen Hospital of Peking University, Shenzhen 518036, Guangdong Province, China; 3National & Local Joint Engineering Research Center for Orthopedic Biomaterials, Shenzhen 518036, Guangdong Province, China
  • Received:2021-12-03 Accepted:2022-01-17 Online:2022-11-08 Published:2022-04-25
  • Contact: Zeng Hui, Chief physician, Professor, Department of Bone & Joint Surgery, Shenzhen Hospital of Peking University, Shenzhen 518036, Guangdong Province, China; National & Local Joint Engineering Research Center for Orthopedic Biomaterials, Shenzhen 518036, Guangdong Province, China
  • About author:Sheng Weibei, Weifang Medical University, Weifang 261053, Shandong Province, China; Department of Bone & Joint Surgery, Shenzhen Hospital of Peking University, Shenzhen 518036, Guangdong Province, China; National & Local Joint Engineering Research Center for Orthopedic Biomaterials, Shenzhen 518036, Guangdong Province, China
  • Supported by:
    National Natural Science Foundation of China, No. 82172432 (to ZH); National Natural Science Foundation of China, No. 82102568 (to YF); National Natural Science Foundation of China, No. 82001319 (to WJ); Guangdong Province Fundamental and Applied Basic Research Fund, No. 2019A1515011290 (to ZH); National and Local Joint Engineering Research Center for Orthopedic Biomaterials, No. XMHT20190204007 (to ZH); Shenzhen High-level Hospital Construction Fund, No. SZXK023 (to ZH); Shenzhen “Three Names” Medical Project, No. SZSM201612092 (to ZH); Shenzhen Research & Development Project, No. Z2021N054 (to ZH)

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Abstract: BACKGROUND: Macrophage polarization-mediated inflammation plays an important role in chronic inflammatory diseases such as osteoarthritis and rheumatoid arthritis. Phosphocreatine-modified chitosan hydrogels show good potential application prospects in tissue repair, but the effect of this new hydrogel on polarization and inflammatory factor expression in macrophages is still unclear. 
OBJECTIVE: To explore the effect of phosphocreatine modified methacrylic anhydride chitosan hydrogel on macrophage polarization and inflammatory factor expression. 
METHODS: One-step freeze-drying method was used to prepare phosphocreatine modified methacrylic anhydride chitosan with good water solubility. Hydrogels were further prepared under low toxicity initiators and ultraviolet irradiation. Bone marrow-derived macrophages were extracted from SD rats and cultured for 7 days, and then divided into three groups. Complete cell culture medium was added in the control group. Complete cell culture medium containing lipopolysaccharide was added in the lipopolysaccharide group. Freeze-dried hydrogel samples and cell complete medium containing lipopolysaccharide were added in the chitosan hydrogel group. Cell viability was detected using CCK-8 assay. RT-PCR, ELISA, western blot assay, and immunofluorescence staining were used to detect macrophage polarization and the expression of inflammatory factors. 
RESULTS AND CONCLUSION: (1) CCK-8 assay showed that after 1, 3, and 5 days of culture, there was no significant difference in the cell viability among the three groups (P > 0.05). (2) Western blot assay and immunofluorescence staining showed that compared with the control group, the M2 macrophage surface marker CD206 protein expression was down-regulated (P < 0.05) and the ARG1 protein expression was up-regulated (P < 0.05) in the lipopolysaccharide group after 1 and 3 days of culture. Compared with the lipopolysaccharide group, the protein expression levels of CD206 and ARG1 in the chitosan hydrogel group were up-regulated after 3 days of culture (P < 0.05). (3) ELISA and RT-PCR results showed that compared with the control group, the protein and mRNA expression levels of pro-inflammatory factors interleukin-1β and interleukin-6 in the lipopolysaccharide group increased after 1 and 3 days of culture (P < 0.05). Compared with the lipopolysaccharide group, the protein and mRNA expression levels of interleukin-1β and interleukin-6 in the chitosan hydrogel group were decreased after 1 and 3 days of culture (P < 0.05). (4) These results have shown that phosphocreatine modified methacrylic anhydride chitosan hydrogel promotes M2-type polarization of macrophages and inhibits the expression of pro-inflammatory factors, which may provide a potential treatment for immune-related diseases in the future.

Key words: chitosan hydrogel, bone marrow-derived macrophage, polarization, inflammatory factor, lipopolysaccharide

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