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Article(id=1244321216383796018, tenantId=1146029695717560320, journalId=1244284848500682798, issueId=1244321215637209904, articleNumber=null, orderNo=null, doi=10.16156/j.1004-7220.2025.05.025, pmid=null, cstr=null, oa=null, hot=null, price=null, onlineType=0, articleFormat=0, articleType=null, articleTypeStr=null, receivedDate=1742313600000, receivedDateStr=2025-03-19, revisedDate=1746201600000, revisedDateStr=2025-05-03, acceptedDate=null, acceptedDateStr=null, onlineDate=1774598896355, onlineDateStr=2026-03-27, pubDate=1759248000000, pubDateStr=2025-10-01, doiRegisterDate=null, doiRegisterDateStr=null, onlineIssueDate=1774598896355, onlineIssueDateStr=2026-03-27, onlineJustAcceptDate=null, onlineJustAcceptDateStr=null, onlineFirstDate=null, onlineFirstDateStr=null, sourceXml=null, magXml=null, createTime=1774598896355, creator=13701087609, updateTime=1774598896355, updator=13701087609, issue=Issue{id=1244321215637209904, tenantId=1146029695717560320, journalId=1244284848500682798, year='2025', volume='40', issue='5', pageStart='1079', pageEnd='1366', issueExtLink='null', onlineDate='null', pubDate='null', beforeIssueId=null, nextIssueId=null, price=null, status=1, issueComplete=1, articleOrder=1, issueType=1, specialIssue=null, createTime=1774598896178, creator=13701087609, updateTime=1774599509568, updator=13701087609, preIssue=null, nextIssue=null, ext={EN=IssueExt(id=1244323788452639476, tenantId=1146029695717560320, journalId=1244284848500682798, issueId=1244321215637209904, language=EN, specialIssueTitle=, coverIllustrator=null, specialIssueEditor=, specialIssueAbout=), CN=IssueExt(id=1244323788452639477, tenantId=1146029695717560320, journalId=1244284848500682798, issueId=1244321215637209904, language=CN, specialIssueTitle=, coverIllustrator=null, specialIssueEditor=, specialIssueAbout=)}, issueFiles=null}, startPage=1272, endPage=1280, ext={EN=ArticleExt(id=1244321217088439098, articleId=1244321216383796018, tenantId=1146029695717560320, journalId=1244284848500682798, language=EN, title=The Mechanobiological Response of Immature Dendritic Cells to Nanomorphologies with Different Crystalline Phases on Titanium Surfaces, columnId=1244321216404767539, journalTitle=Journal of Medical Biomechanics, columnName=Original Articles, runingTitle=null, highlight=null, articleAbstract=
Objective

To elucidate the regulatory effects of titanium surface modification on the immune function of immature dendritic cells (imDCs), different crystalline nanomorphologies were constructed on titanium surface to investigate the mechanobiological response of imDCs to nanomorphologies with different crystalline phases.

Methods

Nanomorphologies with different crystalline phases were constructed on the titanium surface by anodic oxidation and calcination. The changes of the cytoskeleton F-actin, cell adhesion and morphology of imDCs cultured on nanomorphologies with different crystalline phases were observed by fluorescence staining. The relative gene expression of adhesion molecules was detected by quantitative real-time PCR. The migration behaviors of imDCs were observed using real-time live-cell imaging, and the membrane fluidity was detected by fluorescence polarization.

Results

Nanomorphologies with different crystalline phases, namely amorphous phase, anatase and rutile, were obtained on the titanium surface by anodic oxidation and calcination. The cytoskeleton of imDCs on nanomorphologies with different crystalline phases was remodeled. The spreading area of cells on anatase crystalline phase was relatively small, which was (353.3±148.5) μm2. The number of adherent cells was the largest, which was 587±132. The expression of adhesion molecules such as CD11a, integrin β2, ICAM1, and VCAM1 were also increased in cells which cultured on anatase crystalline phase. The imDCs cultured on anatase crystalline phase were equipped with strong migration ability. The accumulative migration distance was (383.6±177.7) μm, and the Euclidean migration distance was (51.82±50.13) μm. The membrane fluidity was relatively weak, and the fluorescence polarization was 0.348 5±0.041 8.

Conclusions

imDCs can respond to nanomorphologies with different crystalline phases on the titanium surface and exhibit different biomechanical behaviors. The results might provide a theoretical basis for the design of titanium biomaterials with immunomodulatory functions.

, correspAuthors=Zhu ZENG, Yun WANG, authorNote=null, correspAuthorsNote=null, copyrightStatement=null, copyrightOwner=null, extLink=null, articleAbsUrl=null, sourceXml=null, magXml=null, pdfUrl=null, pdf=null, pdfFileSize=null, pdfExtLink=null, richHtmlUrl=null, mobilePdfUrl=null, reviewReport=null, pdfFirstPage=null, abstractGraph=null, abstractGraphContent=null, abstractVideo=null, citation=null, cebUrl=null, magXmlContent=null, mapNumber=null, authorCompany=null, fund=null, authors=null, authorsList=Zhaoyajing LUO, Yi WU, Hong CHEN, Jin CHEN, Zuquan HU, Zhu ZENG, Yun WANG), CN=ArticleExt(id=1244321221198857118, articleId=1244321216383796018, tenantId=1146029695717560320, journalId=1244284848500682798, language=CN, title=未成熟树突状细胞对钛表面不同晶相纳米形貌的力学生物学响应, columnId=1244321216576734006, journalTitle=医用生物力学, columnName=论著, runingTitle=null, highlight=null, articleAbstract=
目的

为阐明钛表面改性对未成熟树突状细胞（immature dendritic cells，imDCs）免疫功能的调控，在钛表面构建不同晶相纳米形貌，探究imDCs对钛表面不同晶相纳米形貌的力学生物学响应。

方法

通过阳极氧化和煅烧的方法在钛表面构建不同晶相的纳米形貌；将imDCs培养于不同晶相纳米形貌的钛基底上后，通过荧光染色观察不同晶相纳米形貌上imDCs细胞骨架F-actin、黏附及形态的变化；采用实时荧光定量PCR检测黏附分子的表达量；利用活细胞实时成像观察imDCs的迁移行为；通过荧光偏振法检测imDCs的膜流动性。

结果

经阳极氧化和煅烧在钛表面获得了不同晶相纳米形貌，分别为非晶相、锐钛矿和金红石。不同晶相纳米形貌上imDCs的细胞骨架发生重塑，锐钛矿晶相上细胞的铺展面积相对较小，为（353.3±148.5）μm2；黏附的细胞数量最多，为（587±132）个，黏附分子CD11a、integrinβ2、ICAM1、VCAM1的表达量也增加；细胞迁移能力较强，累积迁移距离为（383.6±177.7）μm，欧式迁移距离为（51.82±50.13）μm；膜流动性相对较弱，荧光偏振度为0.348 5±0.041 8。

结论

imDCs能对钛表面不同晶相纳米形貌产生响应，表现出不同的生物力学行为。研究结果为设计具有免疫调控功能的钛生物材料提供理论依据。

, correspAuthors=曾柱, 王赟, authorNote=null, correspAuthorsNote=
曾柱，教授，博士生导师，E-mail：；
王赟，副教授，青年博士生导师，E-mail：

*

为共同通信作者
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作者贡献声明：

罗赵雅靖负责实验研究、数据分析和论文撰写；吴仪、陈红和陈晋参与实验设计和数据分析；胡祖权、曾柱和王赟负责实验指导和论文修改。

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Proc Natl Acad Sci U S A, 2013, 110(29): 11875-11880., articleTitle=Plasma membrane tension orchestrates membrane trafficking, cytoskeletal remodeling, and biochemical signaling during phagocytosis, refAbstract=null)], funds=[Fund(id=1244321229998506464, tenantId=1146029695717560320, journalId=1244284848500682798, articleId=1244321216383796018, awardId=12202111; 12132006, language=CN, fundingSource=国家自然科学基金项目(12202111; 12132006), fundOrder=null, country=null), Fund(id=1244321230115946990, tenantId=1146029695717560320, journalId=1244284848500682798, articleId=1244321216383796018, awardId=黔科合基础ZK〔2022〕一般385, language=CN, fundingSource=贵州省科技计划项目(黔科合基础ZK〔2022〕一般385), fundOrder=null, country=null), Fund(id=1244321230216610295, tenantId=1146029695717560320, journalId=1244284848500682798, articleId=1244321216383796018, awardId=ZDSYS[2023]004, language=CN, fundingSource=贵州省重点实验室(ZDSYS[2023]004), fundOrder=null, country=null), Fund(id=1244321230350828033, tenantId=1146029695717560320, journalId=1244284848500682798, articleId=1244321216383796018, awardId=21NSFCP35; 校博合J字〔2021〕042号, language=CN, fundingSource=贵州医科大学基金项目(21NSFCP35; 校博合J字〔2021〕042号), fundOrder=null, country=null)], companyList=[AuthorCompany(id=1244321221404378026, tenantId=1146029695717560320, journalId=1244284848500682798, articleId=1244321216383796018, xref=1., ext=[AuthorCompanyExt(id=1244321221408572331, tenantId=1146029695717560320, journalId=1244284848500682798, articleId=1244321216383796018, companyId=1244321221404378026, language=EN, country=null, province=null, city=null, postcode=null, companyName=null, departmentName=null, remark=1.Immune Cells and Antibody Engineering Research Center of Guizhou Province, School of Biology and Engineering (School of Modern Industry for Health and Medicine), Guizhou Medical University, Guiyang 550000, China), AuthorCompanyExt(id=1244321221416960940, tenantId=1146029695717560320, journalId=1244284848500682798, articleId=1244321216383796018, companyId=1244321221404378026, language=CN, country=null, province=null, city=null, postcode=null, companyName=null, departmentName=null, remark=1.贵州医科大学　生物与工程学院（健康医药现代产业学院），贵州省普通高等学校免疫细胞与抗体工程研究中心，贵阳　550000)]), AuthorCompany(id=1244321221484069808, tenantId=1146029695717560320, journalId=1244284848500682798, articleId=1244321216383796018, xref=2., ext=[AuthorCompanyExt(id=1244321221492458417, tenantId=1146029695717560320, journalId=1244284848500682798, articleId=1244321216383796018, companyId=1244321221484069808, language=EN, country=null, province=null, city=null, postcode=null, companyName=null, departmentName=null, remark=2.Engineering Center of Cellular Immunotherapy of Guizhou Province, School of Basic Medical Sciences, Guizhou Medical University, Guiyang 550000, China), AuthorCompanyExt(id=1244321221496652722, tenantId=1146029695717560320, journalId=1244284848500682798, articleId=1244321216383796018, companyId=1244321221484069808, language=CN, country=null, province=null, city=null, postcode=null, companyName=null, departmentName=null, remark=2.贵州医科大学　基础医学院，贵州省细胞免疫治疗工程研究中心，贵阳　550000)])], figs=[ArticleFig(id=1244321226760503590, tenantId=1146029695717560320, journalId=1244284848500682798, articleId=1244321216383796018, language=EN, label=Fig. 1, caption=Characterization images of TiO2 nanomorphologies with different crystalline phases　(a) SEM images of the amorphous phase, (b) SEM images of the anatase, (c) SEM image of the rutile, (d) XRD pattern of different crystalline phases, figureFileSmall=jMKj7C/MZyptlzWN5d5Itg==, figureFileBig=/x9dKfeghXT0kPhiHIE5Fw==, tableContent=null), ArticleFig(id=1244321226869555507, tenantId=1146029695717560320, journalId=1244284848500682798, articleId=1244321216383796018, language=CN, label=图1, caption=不同晶相TiO2纳米形貌表征图片

注：比例尺=0.5 μm。

, figureFileSmall=jMKj7C/MZyptlzWN5d5Itg==, figureFileBig=/x9dKfeghXT0kPhiHIE5Fw==, tableContent=null), ArticleFig(id=1244321227154768197, tenantId=1146029695717560320, journalId=1244284848500682798, articleId=1244321216383796018, language=EN, label=Fig. 2, caption=Flow cytometry identification of DCs, figureFileSmall=4dW3f651I++42kPSK2VirA==, figureFileBig=i2ZiImQCrwEWO+EipxYRkg==, tableContent=null), ArticleFig(id=1244321227288985942, tenantId=1146029695717560320, journalId=1244284848500682798, articleId=1244321216383796018, language=CN, label=图2, caption=DCs流式鉴定结果

注：n=3；***P<0.001，****P<0.000 1。

, figureFileSmall=4dW3f651I++42kPSK2VirA==, figureFileBig=i2ZiImQCrwEWO+EipxYRkg==, tableContent=null), ArticleFig(id=1244321227406426466, tenantId=1146029695717560320, journalId=1244284848500682798, articleId=1244321216383796018, language=EN, label=Fig. 3, caption=Cytoskeletal and morphology of imDCs　(a) Immunofluorescence staining images of imDCs, (b) Morphological analysis of imDCs, (c) Statistical results for spreading area, roundness, circularity, aspect ratio of imDCs, figureFileSmall=Mm15l8t6YLyF94kb8mcPAA==, figureFileBig=IvlE/4wjzElODqMGjLHnDA==, tableContent=null), ArticleFig(id=1244321227486118249, tenantId=1146029695717560320, journalId=1244284848500682798, articleId=1244321216383796018, language=CN, label=图3, caption=imDCs细胞骨架和形态

注：图3（a）中蓝色代表细胞核，红色代表F-肌动蛋白；比例尺=100 μm；n=200；***P<0.001，****P<0.000 1。

, figureFileSmall=Mm15l8t6YLyF94kb8mcPAA==, figureFileBig=IvlE/4wjzElODqMGjLHnDA==, tableContent=null), ArticleFig(id=1244321229025427840, tenantId=1146029695717560320, journalId=1244284848500682798, articleId=1244321216383796018, language=EN, label=Fig. 4, caption=Adhesion ability of imDCs　(a) Detection of cell adhesion of imDCs by DAPI staining, (b) Statistical results of the number of adherent cells, (c) Statistical results of the relative expression of adhesion molecules, figureFileSmall=DsXcGxjK0hp8+6wX55CmqA==, figureFileBig=2ye0Ut+kk5qhoacLjRhyGA==, tableContent=null), ArticleFig(id=1244321229126091149, tenantId=1146029695717560320, journalId=1244284848500682798, articleId=1244321216383796018, language=CN, label=图4, caption=imDCs黏附能力

注：比例尺=100 μm；n黏附数量=5，nCD11a=9，nintegrin β 2=13，nICAM1=9，nVCAM1=9；*P<0.05，**P<0.01。

, figureFileSmall=DsXcGxjK0hp8+6wX55CmqA==, figureFileBig=2ye0Ut+kk5qhoacLjRhyGA==, tableContent=null), ArticleFig(id=1244321229226754457, tenantId=1146029695717560320, journalId=1244284848500682798, articleId=1244321216383796018, language=EN, label=Fig. 5, caption=Migration ability of imDCs　(a) Migration tracks, (b) Statistical results of migration velocity, cumulative migration distance and Euclidean migration distance, figureFileSmall=Zxe7ov11KMVRHYfCXOMs4A==, figureFileBig=/JsGLNovBdSz42kgMtKenQ==, tableContent=null), ArticleFig(id=1244321229407109543, tenantId=1146029695717560320, journalId=1244284848500682798, articleId=1244321216383796018, language=CN, label=图5, caption=im DCs迁移能力

注：n=50；**P<0.01，****P<0.000 1。

, figureFileSmall=Zxe7ov11KMVRHYfCXOMs4A==, figureFileBig=/JsGLNovBdSz42kgMtKenQ==, tableContent=null), ArticleFig(id=1244321229511967151, tenantId=1146029695717560320, journalId=1244284848500682798, articleId=1244321216383796018, language=EN, label=Fig. 6, caption=Fluorescence polarization degree of imDCs, figureFileSmall=5YBe3x4cLCWHpVMHjiDNwA==, figureFileBig=2X6G37lEDV55JBOjHKI5rw==, tableContent=null), ArticleFig(id=1244321229637796281, tenantId=1146029695717560320, journalId=1244284848500682798, articleId=1244321216383796018, language=CN, label=图6, caption=im DCs荧光偏振度

注：n=4；*P<0.05。

, figureFileSmall=5YBe3x4cLCWHpVMHjiDNwA==, figureFileBig=2X6G37lEDV55JBOjHKI5rw==, tableContent=null), ArticleFig(id=1244321229738459589, tenantId=1146029695717560320, journalId=1244284848500682798, articleId=1244321216383796018, language=EN, label=Tab. 1, caption=

Real time qPCR primers

, figureFileSmall=null, figureFileBig=null, tableContent=
基因序列
GAPDHF: GGTTGTCTCCTGCGACTTCA
R: TGGTCCAGGGTTTCTTACTCC
CD11aF: AGTCAGCCTACATGGTTCTAAC
R: GTAAGTGTTCTGATCGCATGTC
integrin β2F: TGGGCAATACTGCGAGTGTGA
R: TCGTAACCGGGCTTGCAACTA
ICAM1F: CTGAAAGATGAGCTCGAGAGTG
R: AAACGAATACACGGTGATGGTA
VCAM1F: GACATTTACCCAGTTTACAGGC
R: TGACGGGAGTAAAGGTTACTTC
), ArticleFig(id=1244321229851705808, tenantId=1146029695717560320, journalId=1244284848500682798, articleId=1244321216383796018, language=CN, label=表1, caption=

引物序列

, figureFileSmall=null, figureFileBig=null, tableContent=
基因序列
GAPDHF: GGTTGTCTCCTGCGACTTCA
R: TGGTCCAGGGTTTCTTACTCC
CD11aF: AGTCAGCCTACATGGTTCTAAC
R: GTAAGTGTTCTGATCGCATGTC
integrin β2F: TGGGCAATACTGCGAGTGTGA
R: TCGTAACCGGGCTTGCAACTA
ICAM1F: CTGAAAGATGAGCTCGAGAGTG
R: AAACGAATACACGGTGATGGTA
VCAM1F: GACATTTACCCAGTTTACAGGC
R: TGACGGGAGTAAAGGTTACTTC
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未成熟树突状细胞对钛表面不同晶相纳米形貌的力学生物学响应
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罗赵雅靖 1 , 吴仪 2 , 陈红 1 , 陈晋 1 , 胡祖权 1 , 曾柱 1, 2, * , 王赟 1, 2, *
医用生物力学 | 论著 2025,40(5): 1272-1280
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医用生物力学 | 论著 2025, 40(5): 1272-1280
未成熟树突状细胞对钛表面不同晶相纳米形貌的力学生物学响应
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罗赵雅靖1, 吴仪2, 陈红1, 陈晋1, 胡祖权1, 曾柱1, 2, * , 王赟1, 2, *
作者信息
  • 1.贵州医科大学　生物与工程学院（健康医药现代产业学院），贵州省普通高等学校免疫细胞与抗体工程研究中心，贵阳　550000
  • 2.贵州医科大学　基础医学院，贵州省细胞免疫治疗工程研究中心，贵阳　550000

通讯作者:

曾柱，教授，博士生导师，E-mail：；
王赟，副教授，青年博士生导师，E-mail：

*

为共同通信作者
The Mechanobiological Response of Immature Dendritic Cells to Nanomorphologies with Different Crystalline Phases on Titanium Surfaces
Zhaoyajing LUO1, Yi WU2, Hong CHEN1, Jin CHEN1, Zuquan HU1, Zhu ZENG1, 2 , Yun WANG1, 2
Affiliations
  • 1.Immune Cells and Antibody Engineering Research Center of Guizhou Province, School of Biology and Engineering (School of Modern Industry for Health and Medicine), Guizhou Medical University, Guiyang 550000, China
  • 2.Engineering Center of Cellular Immunotherapy of Guizhou Province, School of Basic Medical Sciences, Guizhou Medical University, Guiyang 550000, China
出版时间: 2025-10-01 doi: 10.16156/j.1004-7220.2025.05.025
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摘要
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目的

为阐明钛表面改性对未成熟树突状细胞（immature dendritic cells，imDCs）免疫功能的调控，在钛表面构建不同晶相纳米形貌，探究imDCs对钛表面不同晶相纳米形貌的力学生物学响应。

方法

通过阳极氧化和煅烧的方法在钛表面构建不同晶相的纳米形貌；将imDCs培养于不同晶相纳米形貌的钛基底上后，通过荧光染色观察不同晶相纳米形貌上imDCs细胞骨架F-actin、黏附及形态的变化；采用实时荧光定量PCR检测黏附分子的表达量；利用活细胞实时成像观察imDCs的迁移行为；通过荧光偏振法检测imDCs的膜流动性。

结果

经阳极氧化和煅烧在钛表面获得了不同晶相纳米形貌，分别为非晶相、锐钛矿和金红石。不同晶相纳米形貌上imDCs的细胞骨架发生重塑，锐钛矿晶相上细胞的铺展面积相对较小，为（353.3±148.5）μm2；黏附的细胞数量最多，为（587±132）个，黏附分子CD11a、integrinβ2、ICAM1、VCAM1的表达量也增加；细胞迁移能力较强，累积迁移距离为（383.6±177.7）μm，欧式迁移距离为（51.82±50.13）μm；膜流动性相对较弱，荧光偏振度为0.348 5±0.041 8。

结论

imDCs能对钛表面不同晶相纳米形貌产生响应，表现出不同的生物力学行为。研究结果为设计具有免疫调控功能的钛生物材料提供理论依据。

钛  /  晶相结构  /  树突状细胞  /  力学生物学响应
Objective

To elucidate the regulatory effects of titanium surface modification on the immune function of immature dendritic cells (imDCs), different crystalline nanomorphologies were constructed on titanium surface to investigate the mechanobiological response of imDCs to nanomorphologies with different crystalline phases.

Methods

Nanomorphologies with different crystalline phases were constructed on the titanium surface by anodic oxidation and calcination. The changes of the cytoskeleton F-actin, cell adhesion and morphology of imDCs cultured on nanomorphologies with different crystalline phases were observed by fluorescence staining. The relative gene expression of adhesion molecules was detected by quantitative real-time PCR. The migration behaviors of imDCs were observed using real-time live-cell imaging, and the membrane fluidity was detected by fluorescence polarization.

Results

Nanomorphologies with different crystalline phases, namely amorphous phase, anatase and rutile, were obtained on the titanium surface by anodic oxidation and calcination. The cytoskeleton of imDCs on nanomorphologies with different crystalline phases was remodeled. The spreading area of cells on anatase crystalline phase was relatively small, which was (353.3±148.5) μm2. The number of adherent cells was the largest, which was 587±132. The expression of adhesion molecules such as CD11a, integrin β2, ICAM1, and VCAM1 were also increased in cells which cultured on anatase crystalline phase. The imDCs cultured on anatase crystalline phase were equipped with strong migration ability. The accumulative migration distance was (383.6±177.7) μm, and the Euclidean migration distance was (51.82±50.13) μm. The membrane fluidity was relatively weak, and the fluorescence polarization was 0.348 5±0.041 8.

Conclusions

imDCs can respond to nanomorphologies with different crystalline phases on the titanium surface and exhibit different biomechanical behaviors. The results might provide a theoretical basis for the design of titanium biomaterials with immunomodulatory functions.

titanium  /  crystalline phase structure  /  dendritic cells  /  mechanobiological response
罗赵雅靖, 吴仪, 陈红, 陈晋, 胡祖权, 曾柱, 王赟. 未成熟树突状细胞对钛表面不同晶相纳米形貌的力学生物学响应. 医用生物力学, 2025 , 40 (5) : 1272 -1280 . DOI: 10.16156/j.1004-7220.2025.05.025
Zhaoyajing LUO, Yi WU, Hong CHEN, Jin CHEN, Zuquan HU, Zhu ZENG, Yun WANG. The Mechanobiological Response of Immature Dendritic Cells to Nanomorphologies with Different Crystalline Phases on Titanium Surfaces[J]. Journal of Medical Biomechanics, 2025 , 40 (5) : 1272 -1280 . DOI: 10.16156/j.1004-7220.2025.05.025
正文
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钛及其合金因良好的力学性能、优异的耐腐蚀性和生物相容性，已成为骨科和牙科缺损修复的核心植入材料，每年全球超百万患者通过钛植入体实现功能重建[1]。然而，由于植入后常引起不良炎症反应，使机体不能顺利进入增殖期和重塑期，使得钛的植入仍面临失败风险[2-3]。
树突状细胞（dendritic cells，DCs）是机体免疫系统中目前已知功能最强大的一类专职的抗原提呈细胞（antigen presenting cells，APCs），从功能上可分为未成熟DCs（immature DCs，imDCs）和成熟DCs（mature DCs，mDCs）。作为连接先天性免疫应答和适应性免疫应答的桥梁，DCs可通过表面模式识别受体识别材料植入后产生的生物材料相关模式分子，刺激初始T淋巴细胞的增殖和分化。不同的模式分子可以使DCs产生不同的响应，刺激T细胞往不同的方向分化，从而引发不同的免疫应答[4-5]。DCs的免疫学行为对植入部位免疫微环境稳态的维持至关重要[6]。研究表明，植入部位微环境中DCs的表型和功能依赖于植入生物材料表面的理化性质，如材料表面的亲疏水性、粗糙度、硬度以及拓扑结构[7-9]。
研究发现，对钛表面进行改性（如拓扑结构优化和涂层）可以影响其表面蛋白质的吸附，从而间接调控免疫细胞应答[10-11]。通过改变钛表面理化性质，可以定向调控DCs的活化状态，从而减轻钛植入后机体的排异反应[7-9]。通过阳极氧化法在钛表面制备TiO2纳米形貌是一种常用且简便的钛表面改性技术，能有效提高钛植入的生物相容性和成骨能力。但这种方法制备的表面结构以非晶相为主，此类非晶相在高温热处理过程中会发生晶相的转变，最终形成锐钛矿相或金红石相等稳定的晶相[12]。TiO2不同晶相结构可调控细胞的黏附、增殖、分化等功能，但现有研究主要集中在不同晶相对成骨细胞增殖和分化的影响[13-14]。DCs如何通过力学感知-免疫信号转导对钛表面不同晶相纳米形貌进行响应尚不清楚。
本文从材料表面改性的角度出发，通过阳极氧化与热处理相结合的方式，在钛表面构建非晶相、锐钛矿及金红石相纳米形貌层，体外研究不同晶相纳米形貌对imDCs形态、细胞骨架结构、黏附、迁移和膜流动性等力学生物学行为的调控，为明确DCs对生物材料表面改性的响应提供理论基础。
1　材料与方法
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1.1　主要试剂及仪器
RPMI-1640培养基（Gibco公司，美国）；胎牛血清（GeminiBio公司，美国）；重组小鼠粒细胞-巨噬细胞集落刺激因子（rmGM-CSF）、重组小鼠白细胞介素-4（rmIL-4）、脂多糖（Lipopolysaccharide，LPS）（Peprotech公司，美国）；CD11c+磁珠（Milteny公司，德国）；流式抗体（Thermo Fisher Scientific公司，美国）；钛片（奥秀金属材料有限公司）；乙二醇（天津市风船化学试剂科技有限公司）；PDMS（Dow Corning公司，美国）；罗丹明标记的鬼笔环肽、DAPI染料（北京索莱宝科技有限公司）；逆转录试剂盒（天根生化科技（北京）有限公司）；qPCR SYBR Green Master Mix（翌圣生物科技（上海）股份有限公司）；qPCR引物由生工生物工程（上海）股份有限公司合成；TMA-DPH（Cayman Chemical公司，美国）；直流稳定电源（北京六一仪器厂）；F-4600荧光分光光度计（Hitachi公司，日本）；流式细胞仪（Beckman Coulter公司，美国）；激光共聚焦显微镜、正置荧光显微镜（Nikon公司，日本）。
1.2　实验动物
6~8周龄SPF级C57BL/6J小鼠，购自贵州医科大学实验动物中心，常规饲养于实验室动物房。本研究获得贵州医科大学实验动物伦理委员会的批准（2200274）。
1.3　钛表面不同晶相TiO2纳米形貌的制备
配制0.25% NH4F乙二醇电解液，将钛片经超声清洗、酸蚀和抛光后，置于电解液中进行2次阳极氧化，每次氧化电压为20 V，氧化时间30 min，得到非晶相钛片。将非晶相组在450 ℃煅烧1 h得到锐钛矿相钛片，800 ℃煅烧15 min得到金红石相钛片。制备好的钛片经高温灭菌后可用于细胞培养。
1.4　小鼠骨髓来源的树突状细胞分离与培养
将C57BL/6J小鼠麻醉处死后，从胫骨和股骨中提取骨髓细胞，用红细胞裂解液去除红细胞后，将剩余细胞重悬在含10%胎牛血清、1%青霉素-链霉素、20 ng/mL rmGM-CSF和10 ng/mL rmIL-4的RPMI-1640培养基中培养，隔日半换液。第6天用小鼠CD11c+细胞分选磁珠分选获得imDCs，在imDCs中加入100 ng/mL的LPS刺激24 h获得mDCs。本文以imDCs作为研究对象。
1.5　DCs表型鉴定
离心收集细胞，用4%多聚甲醛固定20 min后PBS清洗，加入1%BSA重悬细胞，加入CD11c、CD80、CD86、CD40、MHC-Ⅱ抗体，避光孵育30 min，PBS清洗后用流式细胞仪检测上述分子的表达。
1.6　细胞的F-actin及DAPI染色
将imDCs以每孔2×105个细胞的密度接种于12孔板中培养24 h。悬浮培养的imDCs制成细胞爬片，钛片组培养的imDCs直接弃去培养基，用4%多聚甲醛固定细胞后用PBS洗3次。加入0.1% Triton X-100进行通透，PBS洗3次。加入1%BSA进行封闭，PBS洗3次。加入罗丹明标记的鬼笔环肽稀释液，室温避光孵育30 min，PBS洗3次。加入DAPI染液避光孵育5 min，PBS洗3次后，正置荧光显微镜拍照观察。用Image J软件分析各组细胞的铺展面积、圆度、圆润度、长径比（A/R）。
1.7　细胞黏附情况及黏附相关分子相对表达量
按照1.6节方法进行细胞接种、培养、DAPI染色后，正置荧光显微镜拍照观察，用Image J软件分析各组细胞黏附的情况。
将imDCs以每孔2×106个细胞的密度接种于6孔板中培养24 h（后续细胞实验中，除特殊说明外，细胞接种密度均与此相同）。将钛片上的细胞用细胞解离液解离下来后，用Trizol法提取各组细胞的总RNA，用逆转录试剂盒进行反转录为cDNA，以此作为模板，每组3个复孔，在实时荧光定量PCR系统中进行实时PCR反应。先95 ℃预变性2 min，随后进行40个扩增循环（95 ℃变性10 s，60 ℃退火/延伸30 s）。结果用2-ΔΔc（t）表示实验组目的基因相对于对照组的变化倍数。实验所用引物序列见表1。
1.8　细胞自由迁移实验
将PDMS胶的A液和B液按照3∶10比例混合后平铺在共聚焦皿中，60 ℃固化8 h；将PDMS胶浸泡在10% APTES中，50 ℃处理2 h；将PDMS胶浸泡在2.5%戊二醛中改性1 h后进行灭菌处理用于细胞培养。将钛片上的细胞解离后，接种2×105个细胞到PDMS胶上培养2 h后，用PBS洗去未贴在胶上的细胞，加入200 μL RPMI-1640培养基，用激光共聚焦显微镜活细胞成像系统记录细胞的迁移行为，每隔30 s记录1次，连续记录1 h。用Image J软件进行迁移轨迹追踪，用Chemotaxis and Migration Tool软件分析细胞的平均迁移速度、累计迁移距离及欧氏迁移距离。
1.9　细胞膜流动性检测
离心收集各组细胞，用0.5 mL RPMI-1640培养基重悬细胞，加入0.5 mL 2 μmol/L的TMA-DPH工作液混匀，37 ℃避光孵育30 min；PBS清洗后加入1 mL PBS重悬，用荧光分光光度计在时间扫描模式下检测细胞不同偏振角度时的荧光强度。其中，IVV为起偏器和检偏器光轴均在垂直方向时的荧光强度（90-90）；IVH为起偏器光轴在垂直方向、检偏器光轴在水平方向时的荧光强度（90-0）；IHH为起偏器和检偏器光轴均在水平方向时的荧光强度（0-0）；IHV为起偏器光轴在水平方向、检偏器光轴在垂直方向时的荧光强度（0-90）。荧光偏振度的计算公式为：
式中：G为校正因子（G=IHV/IHH）。
1.10　统计学分析
用GraphPad Prism对实验数据进行统计分析，结果用（均值±标准差）表示。组间比较采用t检验或单因素方差分析，以P<0.05为差异具有统计学意义。
2　结果
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2.1　不同晶相TiO2纳米形貌的制备
钛表面构建的非晶相TiO2具有不同的纳米颗粒结构[见图1（a）]。锐钛矿晶相同样具有TiO2纳米颗粒结构形貌，但颗粒致密程度比非晶相上的高[见图1（b）]。金红石晶相结构表面的颗粒变大，且更加致密光滑[见图1（c）]。XRD检测结果显示，各组样品的XRD图谱与标准卡片（441294-Ti、211272-Anatase、731765-Rutile）的特征峰吻合，说明成功制备了不同晶相的TiO2[见图1（d）]。
2.2　DCs的培养与鉴定
根据已报道的小鼠骨髓来源DCs的体外分离培养方法[15]，通过体外分离DCs的前体细胞，经诱导培养和磁珠分选后得到imDCs，经LPS诱导24 h得到mDCs。流式细胞术鉴定结果表明，imDCs的CD11c阳性率达75.7%，经过LPS诱导后，imDCs的CD80、CD86、CD40和MHC-Ⅱ表达均上调，表明成功获得了imDCs和mDCs（见图2）。
2.3　不同晶相TiO2纳米形貌上DCs的细胞骨架和细胞形态
细胞骨架F-actin染色结果显示，正常悬浮培养的imDCs形态多呈圆形，凸起较少，而不同晶相TiO2纳米形貌上培养的imDCs细胞骨架发生明显重塑，细胞出现向两级拉长的形态。此外，钛片组上imDCs铺展面积均有增大趋势，锐钛矿组的铺展面积最小。通过Image J软件统计不同晶相上培养的imDCs的圆度、圆润度和长径比。非晶相组imDCs圆度和圆润度较为分散，在0.2~1之间不等，锐钛矿组imDCs圆度和圆润度与正常悬浮培养的imDCs相似，金红石晶组的imDCs圆度和圆润度基本接近1，表明细胞更圆[16]。非晶相组imDCs细胞拉长最明显，其长径比较锐钛矿和金红石组的imDCs增加。结果表明，不同晶相TiO2纳米形貌会改变imDCs的形态（见图3）。
2.4　钛表面不同晶相TiO2纳米形貌对im DCs黏附的影响
通过计算不同晶相TiO2纳米形貌上细胞核数量，发现与金红石组相比，非晶相和锐钛矿组的imDCs黏附数量增加[见图4（a）、（b）]。实时荧光定量PCR检测结果显示，不同晶相上培养的imDCs黏附分子CD11a、integrin β2、ICAM1和VCAM1的表达较正常悬浮培养组均有不同程度上调，且锐钛矿组上imDCs的黏附分子表达量最高，与黏附数量统计的结果一致[见图4（c）]。
2.5　钛表面不同晶相TiO2纳米形貌对im DCs迁移的影响
用活细胞成像系统实时观测不同组imDCs的自由迁移情况，统计细胞的平均迁移速度、累积迁移距离及欧氏迁移距离，以此来判断不同处理组imDCs的迁移运动能力[17]。结果显示，锐钛矿组的imDCs迁移速度比悬浮培养imDCs和金红石组快，且迁移距离和欧式迁移距离也比其他两组长（见图5）。
2.6　钛表面不同晶相TiO2纳米形貌对im DCs的细胞膜流动性的影响
荧光漂白恢复（fluorescence recovery after photobleaching，FRAP）技术检测imDCs细胞膜的流动性，用荧光分光光度计检测荧光偏振度，荧光偏振度与膜流动性成反比。不同晶相纳米形貌上培养的imDCs表现出不同的细胞膜流动性，非晶相和锐钛矿上培养imDCs的荧光偏振度呈现上升趋势，而金红石组培养的imDCs的荧光偏振度下降，膜流动性增加（见图6）。
3　讨论
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由于植入后常引起不良炎症反应，使得钛的植入仍面临失败风险[18]。DCs作为连接先天性免疫应答和适应性免疫应答的桥梁，在钛植入后激发机体宿主反应中发挥着重要的作用。已有研究报道通过改变钛表面理化性质调控DCs功能从而减轻植入后不良炎症反应[7-9]。钛表面极易形成TiO2氧化层，且TiO2可形成不同的晶相[19]，具有不同的理化性质[20]。DCs是否对不同晶相的TiO2纳米形貌产生力学生物学响应进而调控其免疫学功能还鲜有报道。
从力学免疫学的角度来看，免疫细胞的生物力学特性与其免疫学功能密切相关[21]。本课题组之前的研究结果发现，肿瘤微环境来源的抑制性细胞因子或胞外力学微环境的改变，均可使DCs的细胞骨架重塑，并改变其免疫功能[6,22]。
细胞的力学性能主要由细胞骨架决定，细胞骨架连接了细胞膜上的力敏感蛋白和细胞核，是细胞产生和传导力的主要结构[23]。本文结果表明，不同晶相纳米形貌上的imDCs细胞骨架F-actin发生了明显重塑，细胞铺展面积增大，但锐钛矿组imDCs的铺展面积相对其他两组较小。研究表明，生物材料植入后，浸润的细胞通过整合素等各种表面传感器感知和响应细胞外基质，随后发生细胞骨架重塑、黏着斑形成以及细胞形态、迁移和功能的变化[24]。本文推测，imDCs细胞骨架的重塑可能是通过整合素感知并激活细胞内信号转导相关通路，进而影响imDCs的免疫功能。
本文进一步检测了不同晶相纳米形貌上imDCs的黏附和包含integrin在内的黏附分子的表达情况。结果发现锐钛矿组的imDCs黏附数量较多，且锐钛矿组上imDCs的黏附分子CD11a、integrin β2、ICAM1、VCAM1表达量较高，证明不同晶相纳米形貌上imDCs骨架重塑与细胞的黏附和黏附分子integrin等表达相关。此外，研究表明，integrin β2介导的黏附决定了材料表面DCs的成熟状态，DCs在生物材料表面的黏附越少，DCs的成熟程度越低，进而降低宿主免疫反应[4,25]。锐钛矿组的imDCs黏附数量多且黏附分子的表达量高，说明锐钛矿上的imDCs可能会被激活为mDCs，不利于钛植入的预后。
迁移能力是免疫细胞发挥功能的基础，DCs的迁移在启动免疫反应和诱导耐受性方面起着至关重要的作用[26]。已有研究表明，DCs迁移能力与其吞噬抗原能力相关，且两者相互拮抗[27]。本文检测imDCs自由迁移能力的结果显示，锐钛矿组的细胞迁移运动能力比其他组强，推测其吞噬能力弱，表明锐钛矿组imDCs可能趋于成熟[28]。
膜流动性是指生物膜中磷脂分子和膜蛋白等成分的流动能力，是生物膜的重要特性之一。研究表明，imDCs的吞噬是一个涉及细胞变形和质膜扭曲的动态过程，膜流动性增强可能使得细胞的变形性更强，这可能会使内吞能力增强[29]。本文结果显示，金红石组imDCs的荧光偏振度显著下降，膜流动性增强，而锐钛矿组的膜流动性相对较弱。由此推测，锐钛矿组上imDCs的吞噬能力可能会弱于金红石组，这也与细胞迁移能力结果相一致。
4　结论
收起
imDCs在不同晶相纳米形貌的钛基底上呈现差异化力学表型。锐钛矿相上的imDCs细胞骨架发生重塑，显著提升其黏附能力和迁移能力，但降低膜流动性，这可能导致锐钛矿上的imDCs趋于成熟，从而不利于钛的植入。本研究仅对imDCs对钛表面不同晶相纳米形貌的力学生物学响应进行了研究，后续需深入探究不同晶相纳米形貌对DCs免疫功能的调控机制，为设计具有免疫调控功能的钛基生物材料提供更全面的理论依据。
基金
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  • 国家自然科学基金项目(12202111; 12132006)
  • 贵州省科技计划项目(黔科合基础ZK〔2022〕一般385)
  • 贵州省重点实验室(ZDSYS[2023]004)
  • 贵州医科大学基金项目(21NSFCP35; 校博合J字〔2021〕042号)
文献
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文章信息
doi: 10.16156/j.1004-7220.2025.05.025
  • 接收时间：2025-03-19
  • 首发时间：2026-03-27
  • 出版时间：2025-10-01
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  • 收稿日期：2025-03-19
  • 修回日期：2025-05-03
基金
国家自然科学基金项目(12202111; 12132006)
贵州省科技计划项目(黔科合基础ZK〔2022〕一般385)
贵州省重点实验室(ZDSYS[2023]004)
贵州医科大学基金项目(21NSFCP35; 校博合J字〔2021〕042号)
作者信息
    1.贵州医科大学　生物与工程学院（健康医药现代产业学院），贵州省普通高等学校免疫细胞与抗体工程研究中心，贵阳　550000
    2.贵州医科大学　基础医学院，贵州省细胞免疫治疗工程研究中心，贵阳　550000

通讯作者:

曾柱，教授，博士生导师，E-mail：；
王赟，副教授，青年博士生导师，E-mail：

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2种不同金属材料的力学参数
科
Family		 属数
Number of
genus		 种数
Number of
species		 占总种数比例
Percentage of
total species (%)		 属
Genus		 种数
Number of
species		 占总种数比例
Percentage of total
species (%)
鹅膏菌科Amanitaceae 2 11 5.26 鹅膏菌属 Amanita 10 4.78
小菇科 Mycenaceae 2 12 5.74 丝盖伞属 Inocybe 5 2.39
多孔菌科 Polyporaceae 8 14 6.70 蜡蘑属 Laccaria 5 2.39
红菇科 Russulaceae 3 23 11.00 小皮伞属 Marasmius 6 2.87
小菇属 Mycena 11 5.26
光柄菇属 Pluteus 5 2.39
红菇属 Russula 17 8.13
栓菌属 Trametes 5 2.39
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