中国实用口腔科杂志

中国实用口腔科杂志 ›› 2022, Vol. 15 ›› Issue (6): 720-726.DOI: 10.19538/j.kq.2022.06.016

• 论著 • 上一篇    下一篇

负载地塞米松DNA水凝胶改善镁周围成骨微环境研究

廉    鑫1，2，贾    森3，薛睿喆1，2，王皓辰1，4，刘昌奎3，宋    文1   

  1. 1. 军事口腔医学国家重点实验室，口腔疾病国家临床医学研究中心，陕西省口腔医学重点实验室，空军军医大学第三附属医院修复科，陕西  西安  710032；2. 空军军医大学基础医学院学员三大队九队，陕西  西安  710032；3. 西安医学院口腔医学院，陕西  西安  710021；4. 空军军医大学基础医学院学员六大队二十四队，陕西  西安  710032
  • 出版日期:2022-11-30 发布日期:2023-01-16
  • 基金资助:
    陕西省青年创新团队建设科研计划项目（21JP111）

  • Online:2022-11-30 Published:2023-01-16

摘要： 目的    研究负载地塞米松（dexamethasone，Dex）DNA水凝胶对镁周围成骨微环境的影响。方法    研究于2019年5月至2021年5月在军事口腔医学国家重点实验室进行。将108个阳极氧化处理后的镁片按照不同表面处理方式随机分为3组，每组36个，分别记为空白对照组（未涂布任何物质）、水凝胶组（涂布DNA水凝胶）、Dex水凝胶组（涂布负载Dex的DNA水凝胶）。扫描电镜观察每组试样表面结构，同时检测培养液浸泡试样后pH值的变化及镁离子累积释放浓度；将小鼠巨噬细胞RAW264.7与试样间接共培养，应用CCK-8法测定细胞活力；应用实时荧光定量PCR检测巨噬细胞极化相关基因精氨酸酶-1（arginase-1，Arg-1）、转化生长因子-β（transforming growth factor-β，TGF-β）、诱导型一氧化氮合成酶（inducible nitric oxide synthase，iNOS）和肿瘤坏死因子-α（tumor necrosis factor-α，TNF-α）的表达，再利用流式细胞术检测极化标志物CD206和趋化因子受体7（chemokine receptor 7，CCR7）相对荧光强度；最后应用ELISA检测白介素-10（interleukin-10，IL-10）和TNF-α的分泌量。结果    扫描电镜观察显示，空白对照组表面呈现较为均匀致密的氧化层，水凝胶组和Dex水凝胶组表面被网状结构的水凝胶涂层所覆盖。水凝胶组和Dex水凝胶组pH值的升高幅度及镁离子累积释放浓度均小于空白对照组。细胞活力检测结果显示，水凝胶组及Dex水凝胶组的光密度值均高于空白对照组（均P < 0.05）。实时荧光定量PCR和流式细胞术检测结果显示，Dex水凝胶组M2极化相关标志物Arg-1、TGF-β、CD206表达均高于空白对照组和水凝胶组，而水凝胶组仅TGF-β、CD206表达高于空白对照组，差异均有统计学意义（均P < 0.05）；Dex水凝胶组M1极化相关标志物iNOS、TNF-α、CCR7表达均低于空白对照组，其中iNOS、CCR7的表达还低于水凝胶组，而水凝胶组仅TNF-α表达低于空白对照组，差异均有统计学意义（均P < 0.05）。ELISA检测发现水凝胶组和Dex水凝胶组相比空白对照组IL-10分泌量均增加，且炎症因子TNF-α的分泌量均减少；Dex水凝胶组相比水凝胶组IL-10的分泌量增加，TNF-α的分泌量减少，差异均有统计学意义（均P < 0.05）。结论    负载Dex的DNA水凝胶涂层镁片可能会诱导其周围的巨噬细胞发生M2极化，改善成骨微环境。

关键词: 引导骨再生, 屏障膜, 镁, 阳极氧化, 成骨微环境, 巨噬细胞, 地塞米松, DNA水凝胶

Abstract: Objective    To investigate the influence of dexamethasone（Dex）incorporated DNA hydrogel on the osteogenic microenvironment around magnesium. Methods    The experiments were performed from May 2019 to May 2021 in the State Key Laboratory of Military Stomatology. A total of 108 magnesium plates were anodized and randomly classified into three groups with 36 in each：blank control group （without further coating），gel group （coated with DNA hydrogel）and Dex & gel group （coated with DNA hydrogel containing Dex）. The surface structure was observed by scanning electron microscope（SEM）. The pH values and cumulative Mg2+ release profiles were monitored in cell culture medium. The transwell was used to perform indirect co-culture of RAW264.7 and magnesium samples. The cell viability was evaluated by CCK-8 test，while the polarization markers of arginase-1（Arg-1），transforming growth factor-β（TGF-β），inducible nitric oxide synthase（iNOS）and tumor necrosis factor-α（TNF-α）were measured by qPCR. The CD206 and chemokine receptor 7（CCR7）expressions were quantified by flow cytometry. The secreted interleukin 10（IL-10）and TNF-α concentrations were quantified by ELISA. Results    Under SEM observation，the surface of blank control group showed a homogeneous dense oxidative layer while surfaces of the gel and Dex & gel groups were covered by the net-like structure coating. The pH elevation and Mg2+ release were both smaller in gel and Dex & gel groups than in blank control group. The cell viability tests indicated that the OD values of gel and Dex & gel groups were significantly higher than those of blank control group（all P < 0.05）. The qPCR and flow cytometry analysis showed that the M2 polarization markers of Arg-1，TGF-β and CD206 were significantly higher in Dex & gel group compared to blank control and gel groups. Only TGF-β and CD206 were higher in gel group compared to blank control group（all P < 0.05）. The M1 polarization markers of iNOS，TNF-α and CCR7 were significantly lower in Dex & gel group compared to blank control group and the iNOS and CCR7 expression were also lower than gel group，and the TNF-α expression in gel group was also lower than blank group（all P < 0.05）. The ELISA quantification showed that the secretions of IL-10 and TNF-α were increased and decreased respectively in Dex & gel and gel groups compared to blank control group，and the same is between Dex & gel group and gel group （all P < 0.05）. Conclusion    Dex incorporated DNA hydrogel coating on magnesium surface can induce nearby macrophages M2 polarization and improve the osteogenic microenvironment.

Key words: guided bone regeneration, barrier membrane, magnesium, anodization, osteogenic microenvironment, macrophages, dexamethasone, DNA hydrogel