Fluolab【Adv.Mater.】香港中文大学李、手南方医科大学谢登辉等|仿生单原子纳米酶助力骨再生：骨体积分数提升177%的代谢重编程新策略
通讯作者： Rocky S. Tuan, Denghui Xie, Zhong Alan Li
文章概要
在这项发表于《Advanced Materials》的研究中，科研团队针对骨缺损修复中线粒体功能障碍这一核心痛点，开发了一种仿生细胞色素c氧化酶（CcO）的单原子纳米酶。该纳米酶通过负载铁、铜单原子模拟天然酶的催化中心，并结合TPP分子实现线粒体靶向，成功诱导干细胞从糖酵解向氧化磷酸化和脂肪酸氧化的代谢转变。实验结果显示，该策略显著提升了干细胞的成骨分化能力，并在大鼠大面积骨缺损模型中实现了骨体积分数177%的惊人增长，为组织再生提供了基于能量代谢调控的新思路。
Schematic diagram illustrating the ability of TPP-DMSN-Fe/Cu nanozymes to improve mitochondrial function, regulate cellular energy metabolism, and promote osteogenic differentiation of stem cells.
引言
骨组织的有效再生高度依赖于干细胞线粒体提供的高能量输出，尤其是在基质合成的初始阶段，充足的ATP供应是成骨分化的前提。然而，在临床常见的大尺寸骨缺损（CSBD） 环境中，氧化应激和氧化还原失衡往往导致线粒体呼吸链中的复合体IV（CcO） 受损，造成能量代谢枯竭并抑制骨修复。传统的生物材料多聚焦于清除活性氧（ROS），却难以从根本上修复受损的电子传递链。受生物体内高效酶催化机制的启发，研究人员致力于开发一种既能精准靶向线粒体，又能模拟CcO催化功能的人造纳米酶，以期通过重启干细胞的能量工厂来加速骨再生进程。
主要实验及结论
研究团队首先通过一步法合成了具有放射状孔道的树突状介孔二氧化硅纳米颗粒（DMSN），并精细地将铁和铜原子以单原子形式分散嵌入其中。这种结构完美模拟了天然CcO的活性中心，紫外光谱分析证实其具有优异的细胞色素c氧化酶样活性。为了实现精准打击，研究者在纳米颗粒表面修饰了亲脂性阳离子TPP。共聚焦成像和生物电镜结果清晰地展示了该纳米酶能够高效进入干细胞并精准定位于线粒体表面。生物安全性评估表明，在治疗浓度下，该材料对C3H/10T1/2干细胞表现出良好的生物相容性，为后续的功能研究奠定了基础。
Synthesis and characterization of TPP-DMSN-Fe/Cu nanozymes. (a) Schematic of the synthesis process of TPP-DMSN-Fe/Cu nanozyme. (b, c) TEM images of the synthesized nanozyme. Scale bar = 50 nm and 10 nm. (d) AC-HAADF-STEM image of TPP-DMSN-Fe/Cu nanozyme, with single iron atoms marked by red circles. Scale bar = 2 nm. (e) HAADF-STEM image showing the dendritic mesoporous structure of the synthesized nanozyme. Scale bar = 50 nm. (f) EDS elemental mapping confirming homogeneous distribution of Cu, Fe, Si, and O in TPP-DMSN-Fe/Cu nanozyme. Scale bars = 50 nm. (g) UV–vis absorbance spectra of Cyt c after reacting with different nanoparticles for 60 min. (h) TPP-DMSN-Fe/Cu nanozyme upregulated the expression of Fam36a (Cytochrome c Oxidase Assembly Factor COX20) gene. Data are presented as mean ± s.d., n = 4 biologically independent samples, by one-way ANOVA with Tukey’s post hoc test. The P value is noted. (i) Bio-TEM images showing the mitochondria-targeting ability of TPP-DMSN-Fe/Cu nanozymes after treating C3H/10T1/2 cells for 4 h (red arrows indicate mitochondria). Scale bar = 500 nm (left image) and 100 nm (magnified views on the right). (j) Representative confocal images showing that the TPP-modified nanozymes had a higher level of colocalization with C3H/10T1/2 cells compared to nanozymes without TPP modification (blue, nucleus; red, RBITC-nanoparticles; green, Mito-tracker). Scale bar = 10 µm. (k) Quantitative analysis of fluorescence intensity along the white dotted lines in j. P = Pearson's correlation coefficient.
TPP-DMSN-Fe/Cu nanozymes improved mitochondrial function and regulated energy metabolism in stem cells. (a) Schematic diagram of TPP-DMSN-Fe/Cu nanozyme-induced enhancement of OXPHOS, FAO, and TCA cycle and the changes in related target metabolites. (b–e) TPP-DMSN-Fe/Cu nanozymes treatment resulted in the highest levels of b NAD+/NADH, (c) Sdha and (d) Atp5a mRNA, and (e) ATP production in stem cells. (f) Real-time OCRs during stem cell mitochondrial stress tests and semi-quantitative analysis of basal respiration, maximal respiration, and ATP production. (g) Real-time ECARs of stem cells during the glycolytic stress test and semi-quantitative analysis of glycolysis, glycolytic capacity, and glycolytic reserve. (h) Real-time OCRs of stem cells during the substrate oxidation test and semi-quantitative analysis of maximal respiration, spare respiratory capacity, and basal respiration. (i) Representative image of JC-1 aggregates and monomers and j corresponding quantitative analysis indicating the highest mitochondrial membrane potential in cells treated by TPP-DMSN-Fe/Cu nanozymes for 7 days in osteogenic medium. Scale bar = 25 µm. Data are presented as mean ± s.d., n = 4 biologically independent samples, by one-way ANOVA with Tukey's post hoc test. The p-value is noted.
在代谢调控机制探索中，Seahorse能量代谢分析揭示了令人兴奋的发现。与对照组相比，经靶向纳米酶处理的干细胞，其基础呼吸、最大呼吸以及ATP产量分别提升了44%、89%和45%，而糖酵解水平显著下降。这表明纳米酶成功触发了能量代谢重编程，使细胞能源从低效的糖酵解转向高效的氧化磷酸化。进一步的转录组学测序（RNA-seq）深入解析了这一过程，结果显示Bmp4、Sox9以及与线粒体生物发生相关的Pgc-1α等关键基因显著上调。此外，纳米酶还激活了CAMKK-AMPK信号通路和自噬过程，这种多维度的调节共同营造了一个有利于成骨分化的能量富集型微环境。
Transcriptome analysis revealed that TPP-DMSN-Fe/Cu nanozymes reprogramed stem cell metabolism to enhance osteogenesis. (a) Schematic of TPP-DMSN-Fe/Cu-modulated mitochondrial and osteogenic pathways based on RNA sequencing results. (b) Number of differentially expressed genes in TPP-DMSN-Fe/Cu-treated stem cells versus other groups. (c) Heatmap showing upregulated osteogenesis-, chondrogenesis-, calcium influx-, and mitochondrial function-related genes for DMSN-Fe/Cu-treated stem cells. n = 3 biologically independent samples. (d–f) Gene Ontology enrichment analysis of biological process (d), cellular component (e), and molecular function (f) for up-regulated genes in TPP-DMSN-Fe/Cu nanozymes. (g) KEGG enrichment analysis showing genes upregulated by TPP-DMSN-Fe/Cu treatment. (h, i) GSEA plot showing the enrichment of the gene set of “Proton motive force driven mitochondrial ATP synthesis” and “Mitochondrial respiratory chain complex iv”. The top panels of the figures show the enrichment score (green line). The middle panels show the presence of the target gene in the gene set. The bottom panels show the drop in the ranked gene list for the ranked metric value.
TPP-DMSN-Fe/Cu nanozymes enhanced osteogenic differentiation and mitochondrial function in stem cells. (a) mRNA levels of osteogenic gene expression: Runx2, Opn, Ocn, and Alp. Data are presented as mean ± s.d., n = 4 biologically independent samples, by one-way ANOVA with Tukey's post hoc test. The p-value is noted. (b) Western blot bands for COL1A1, RUNX2, ALP, and OCN (the numbers above each band represent normalized mean band intensity ± s.d., n = 3 biologically independent samples). (c) Immunofluorescence staining of OCN and d quantification of fluorescent intensity. Data are presented as mean ± s.d., n = 4 biologically independent samples, by one-way ANOVA with Tukey's post hoc test. The p value is noted. Scale bar = 100 µm. (e) ARS staining of stem cells and f corresponding quantitative analysis after 14 days of nanoparticle treatment. Data are presented as mean ± s.d., n = 4 biologically independent samples, by one-way ANOVA with Tukey's post hoc test. The p value is noted. Scale bar = 2 mm and 100 µm. (g) mRNA levels of mitochondrial gene expression: Pgc-1α, Cpt1a, Drp1, and Opa1. Data are presented as mean ± s.d., n = 4 biologically independent samples, by one-way ANOVA with Tukey's post hoc test. The p-value is noted. (h) Western blot bands for PGC-1α, p-AMPK, AMPK, Beclin-1 and LC3-II (the numbers above each band represent normalized mean band intensity ± s.d., n = 3 biologically independent samples). (i) Quantitative analysis of p-AMPK/ AMPK, Beclin-1 and LC3-II protein expression. (j) Intracellular calcium ion levels and k corresponding quantification. Data are presented as mean ± s.d., n = 4 biologically independent samples, by one-way ANOVA with Tukey's post hoc test. The p-value is noted.
为了验证临床转化潜力，研究者将纳米酶复合在GelMA水凝胶支架中，植入大鼠股骨缺损部位。在植入4周和8周后的Micro-CT扫描中，靶向纳米酶组展现了最为卓越的修复效果。定量分析显示，其骨体积分数（BV/TV）比对照组高出177%，且骨矿物质密度明显提升。组织学染色进一步证实了缺损区形成了大量成熟且致密的骨组织。免疫组化结果清晰地标示出OCN、COL1A1和ATP5A等成骨与代谢标志物的高表达，从体内实验层面证实了该纳米酶通过优化线粒体健康和能量供应，确实有效地加速了临界尺寸骨缺损的愈合过程。
TPP-DMSN-Fe/Cu nanozymes accelerated bone regeneration in a rat model of CSBD. (a) Schematic of the experimental timeline. (b) 3D reconstructed micro-CT scan images after 4 and 8 weeks of scaffold implantation. (c, d) Quantitative micro-CT analysis of BV/TV and BMD. Data are presented as mean ± s.d., n = 3 biologically independent samples, by one-way ANOVA with Tukey's post hoc test. The p-value is noted. (e) H&E staining and f Masson's trichrome staining images of the bone defect sites at 4 and 8 weeks after the surgery. Scale bar = 100 µm. HB = host bone; NB = newly formed bone.
TPP-DMSN-Fe/Cu nanozymes upregulated the expression of markers related to osteogenesis and energy metabolism in vivo. (a) OCN IHC staining images for the femoral bone defect at 4 and 8 weeks. Immunofluorescence staining images for b COL1A1, c ATP5A, and d PGC-1α in the femoral bone defect area at 4 and 8 weeks. Scale bar = 100 µm.
总结及展望
本研究成功构建了一款具备线粒体靶向能力的仿生单原子纳米酶，通过修复电子传递链功能，不仅高效清除了有害的超氧自由基，更关键的是实现了对干细胞能量代谢的精准重塑。这种从“被动抗氧化”到“主动代谢调节”的策略转变，为解决骨再生难题提供了全新的范式。展望未来，这种单原子纳米酶的设计理念有望延伸至骨质疏松、神经损伤等其他与线粒体功能障碍相关的退行性疾病治疗中。虽然目前在小型动物模型上取得了理想效果，但未来的研究仍需在大型动物模型中验证其长效生物安全性及免疫反应，以期早日为临床骨缺失患者带来福音。