Mitochondria-targeted, Single-atom Nanozymes Accelerate Bone Regeneration by Augmenting Stem Cell Energy Metabolism

Mitochondria-targeted, Single-atom Nanozymes Accelerate Bone Regeneration by Augmenting Stem Cell Energy Metabolism | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Mitochondria-targeted, Single-atom Nanozymes Accelerate Bone Regeneration by Augmenting Stem Cell Energy Metabolism Zhong Alan Li, Yuwen Wang, Xinzhi Liang, Tiandi Xiong, Zhong Zheng, and 12 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7012382/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Critical-sized bone defects (CSBDs) do not heal spontaneously throughout a patient’s lifetime, posing a global challenge to musculoskeletal health. Resident stem cells in bone, which are indispensable in skeletal development and regeneration, undergo enhanced mitochondrial activities during osteogenic differentiation. However, accumulation of excessive reactive oxygen species (ROS) produced by injured bone tissues can lead to mitochondrial damage, which negatively affects the osteogenic differentiation of stem cells. In such an environment, it is crucial to target mitochondria in stem cells to remove ROS and restore mitochondrial homeostasis. Herein, we developed a dendritic mesoporous silica nanoparticle (DMSN)-based single-atom nanozyme, named TPP-DMSN-Fe/Cu, loaded with Fe and Cu single atoms and modified with mitochondrion-targeting triphenylphosphonium (TPP). In vitro , TPP-DMSN-Fe/Cu nanozymes were found to upregulate stem cell osteogenesis by scavenging ROS, enhancing mitochondrial function by fatty acid oxidation, and promoting autophagy of abnormal mitochondria. The nanozymes also augmented mitochondrial biogenesis via the CaMKK/AMPK/PGC-1α pathway. In vivo , TPP-DMSN-Fe/Cu nanozymes significantly enhanced mitochondrial biogenesis and bone regeneration, leading to increased bone volume and mineral density at the sites of CSBDs in rats. Taken together, these findings show that the multifunctional, mitochondria-targeting TPP-DMSN-Fe/Cu nanozymes hold promising potential in accelerating bone regeneration via regulation of cellular energy metabolism. Biological sciences/Biotechnology/Tissue engineering Biological sciences/Biotechnology/Biomaterials/Bioinspired materials Biological sciences/Biotechnology/Nanobiotechnology/Nanoparticles Health sciences/Anatomy/Musculoskeletal system/Bone Nanoparticle Stem cell Osteogenic differentiation Bone regeneration Mitochondrial biogenesis Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 1. Introduction Critical-sized bone defects (CSBDs) represent a significant clinical challenge that adversely impact musculoskeletal health, frequently resulting in impaired bone repair 1 – 3 . Stem cells are indispensable for bone repair, which rely on mitochondrial oxidative phosphorylation (OXPHOS) to meet the energy and biosynthetic demands of osteogenic differentiation 4 . In this process, the electron transport chain (ETC) chain plays a pivotal role. It is a series of protein complexes (I–IV) that drive ATP synthesis through electron transport and proton gradient formation. In healthy stem cells, ETC activity is responsible for the oxidation of nutrients (e.g., glucose, fatty acids) while maintaining redox balance to support matrix mineralization and differentiation 5 . However, under pathological conditions, such as CSBDs, stem cells exhibit impaired ETC function 6 . In CSBDs, the ETC experiences significant disruption due to (1) excessive accumulation of ROS resulting from complex I/III deficiency, (2) reduced catalytic activity of ATP synthesis/complex IV, (3) impaired mitophagy and accumulation of damaged mitochondria, and (4) inhibition of biogenesis through downregulation of PGC-1α 7–9 . Current strategies, such as gene regulation, encounter inherent biological complexity, including pathway interconnectivity, cell-type specificity, spatiotemporal delivery challenges, and off-target effects 10 . In recent years, there has been significant interest in the potential applications of biomaterials with reactive oxygen species (ROS) scavenging functions, such as polyphenols, artificial selenoenzymes, hydrogen-containing materials, catalase (CAT), polydopamine, and chiral biomaterials, for efficient bone regeneration 11 – 15 . However, many existing systems exhibit limited catalytic specificity. Inadequate mitochondrial targeting ability frequently results in antioxidant effects that extend beyond the intended field, thereby diminishing therapeutic efficacy. Moreover, while many existing material systems are capable of scavenging ROS, they are unable to address downstream mitochondrial OXPHOS dysfunction 16 , 17 . For example, among the many ROS-scavenging nanozymes developed thus far, most of them exhibit a deficiency in multimodal functionality required for the concurrent enhancement of mitochondrial biogenesis, energy metabolism, and dynamic remodeling in tissue regeneration. This underscores the pressing need for innovative therapeutic interventions capable of effectively enhancing mitochondrial function and promoting effective bone regeneration, particularly in scenarios such as CSBDs where endogenous repair mechanisms are compromised. Mitochondria are a significant source of ATP production, and recent evidence highlights their role in guiding stem cell fate during bone regeneration 18 , 19 . Stem cells rely on mitochondrial biogenesis, the process of generating new mitochondria, to satisfy the elevated energy demands during osteogenic differentiation 20 . Notably, undifferentiated stem cells, including bone marrow mesenchymal stem cells 21 , embryonic stem cells 22 , and hematopoietic stem cells 23 , exhibit a strong reliance on glycolysis to fuel rapid growth and division. However, as differentiation progresses, there is a shift in metabolic patterns: lipid metabolism becomes more prominent, while glycolytic activity declines 24 , 25 . The shift in metabolic priorities from glycolysis to lipid metabolism is a hallmark of cellular differentiation. Particularly, fatty acid β-oxidation provides energy, facilitates the synthesis of cell membranes, and contributes to signal transduction and the substrates required for OXPHOS 26 , 27 . This metabolic reprogramming from glycolysis to lipid-driven OXPHOS highlights the dynamic interplay between energy metabolism-related pathways and stem cell specialization 28 , 29 . For instance, circulating fatty acids function as signaling molecules during fracture repair, directly promoting the differentiation of bone marrow stromal cells into osteoblasts 30 . However, under pathological conditions, these processes can be disrupted, leading to mitochondrial inefficiency and ETC stress 10 . Eventually, excessive ROS accumulation can cause mitochondrial damage. The accumulation of ROS has been found to impede mitochondrial function and hinder osteoblast differentiation, thus impeding effective bone healing 7 . In instances where mitochondrial repair is not a viable option, a process known as mitophagy is initiated, to remove dysfunctional mitochondria 31 . This process is crucial for maintaining cellular homeostasis and ensuring normal cellular function. Furthermore, in the process of osteogenic differentiation, stem cells need to upregulate mitochondrial biogenesis to increase ATP production. It has been demonstrated in various studies that peroxisome proliferator-activated receptor-𝛾 coactivator-1α (PGC-1α) plays a pivotal role in mitochondrial biogenesis 32 . In addition, it has been shown that PGC-1α is induced by AMP-activated protein kinase (AMPK) 33 . Therefore, therapeutic strategies that target ROS elimination while promoting mitochondrial health and augmenting energy metabolism by, for example, enhancing mitochondrial biogenesis and OXPHOS, are highly desirable for restoring regenerative capacity of stem cells. To address these unmet needs, we developed a triphenylphosphine (TPP)-modified, dendritic mesoporous silica nanoparticle (DMSN)-based, and Fe/Cu single atom-loaded nanozyme, named TPP-DMSN-Fe/Cu, a mitochondria-targeted nanoparticle designed to mimic the activity of key enzymes in the ETC (Fig. 1 ). By replicating the ETC of OXPHOS, this nanozyme can restore the redox balance while enhancing ATP synthesis. TPP modification ensures the precise targeting of the nanozyme to the mitochondria of stem cells, where it (1) scavenges ROS, boosts glutathione levels to enhance endogenous antioxidant defenses, and enhances GTPase activity to regulate mitochondrial fission. The addition of Fe/Cu mimics the function of complex IV, cytochrome c oxidase (CcO), thereby (2) increasing the catalytic activity of ATP synthesis/complex IV. In addition to its ability to promote (3) mitochondrial autophagy, this multifunctional strategy promotes (4) mitochondrial biogenesis via the Ca 2+ /calmodulin-dependent protein kinase kinase (CaMKK)/AMPK pathway and creates a microenvironment conducive to bone formation. By focusing on energy metabolism, we targeted the root cause of stem cell dysfunction in CSBDs, i.e., mitochondrial dysfunction that precedes and impedes osteogenic function. In animal studies, TPP-DMSN-Fe/Cu nanozymes demonstrated potent bone regeneration capabilities, evidenced by enhanced bone mineral density and accelerated defect repair. Therefore, our multifunctional nanozyme system holds promising potential in bridging the gap between mitochondrial dysfunction and functional regeneration of bone defects and beyond. 2. Results 2.1. Characterization and antioxidant ability of TPP-DMSN-Fe/Cu nanozyme DMSNs were synthesized via a one-step reaction and exhibited uniform spherical morphology with well-defined mesopores, as confirmed by transmission electron microscopy (TEM) ( Supplementary Fig. 1 ). Subsequent functionalization steps involved sequential loading of Fe/Cu and covalent conjugation of mitochondria-targeting TPP through amidation, yielding the final TPP-DMSN-Fe/Cu nanozyme (Fig. 2 a-c). High-angle annular dark-field (HAADF) imaging revealed a dendritic architecture with radially oriented mesopores (Fig. 2 d), and energy-dispersive X-ray spectroscopy (EDS) elemental mapping confirmed the homogeneous distribution of Cu, Fe, Si, and O within the composite structure (Fig. 2 e). The physicochemical properties of the nanozyme were systematically examined. Zeta potential measurements demonstrated a shift from negative surface charge toward positive surface charge after TPP conjugation (Fig. 2 f), with an average surface charge value of 34.7 mV, indicating colloidal stability (absolute values > 30 mV) 34 . In addition, the use of Fourier-transform infrared (FTIR) spectroscopy further corroborated the successful synthesis of the nanozyme, with the presence of characteristic Si–O–Si stretching vibrations (410, 755, and 1019 cm − 1 ) and a C = O absorption band (1607 cm − 1 ) from L-cysteine-mediated amidation being observed (Fig. 2 g). Furthermore, nitrogen adsorption-desorption isotherms revealed a high specific surface area (592.8 m² g-1) and nanoporous structure (Fig. 2 h) for TPP-DMSN-Fe/Cu nanozyme, which are advantageous for Fe/Cu loading and catalytic activities. ROS accumulation has been demonstrated to disrupt mitochondrial function and impair tissue regeneration 35 . To evaluate the nanozyme's ROS-scavenging potential, radical elimination assays were performed. It was found that Fe/Cu loading in the nanoparticles significantly increased the rates of scavenging DPPH radical, hydroxyl radical (•OH), and hydrogen peroxide (H 2 O 2 ) in comparison with unmodified DMSN (Fig. 2 i-k and Supplementary Fig. 2, 3 ). This robust antioxidant activity positions TPP-DMSN-Fe/Cu as a potent therapeutic agent for mitigating oxidative stress and restoring redox balance in stem cells, thereby supporting mitochondrial health during osteogenesis. 2.2. In vitro biocompatibility, mitochondria-targeting ability, and ROS scavenging ability of TPP-DMSN-Fe/Cu nanozyme We used C3H/10T1/2 cells, a widely used skeletal stem cell line, in all in vitro experiments. The biocompatibility of TPP-DMSN-Fe/Cu nanozyme was assessed using the cell counting kit 8 (CCK8) assay and Live/Dead staining. Following a 24-hour incubation of the cells with different nanoparticles at varying concentrations, the half-maximum inhibitory concentration (IC50) values of DMSN, DMSN-Fe/Cu, and TPP-DMSN-Fe/Cu nanozymes were determined to be 91.71, 73.69 and 107.01 µg/mL, respectively, by fitting the dose-response curves (Fig. 3 a and Supplementary Fig. 4 ). Live/Dead staining of C3H/10T1/2 cells (a widely used skeletal stem cell line) further confirmed minimal toxicity of the nanoparticles at concentrations below 50 µg/mL (Fig. 3 b, c). After confirming the biocompatibility, the intracellular localization of the TPP-DMSN-Fe/Cu nanozymes (10 µg/mL) was examined. Bio-TEM revealed that after 4 hours of incubation, TPP modified nanozymes aggregated in proximity to the mitochondria within C3H/10T1/2 cells, with direct contact with the mitochondrial surface observed (Fig. 3 d and Supplementary Fig. 5 ). Fluorescence imaging of Rhodamine B isothiocyanate (RBITC)-labelled nanozyme and MitoTracker-stained mitochondria showed a higher level of colocalization between TPP-modified nanozymes and mitochondria than between non-targeted DMSN-Fe/Cu nanozymes and mitochondria (Fig. 3 e, f). In order to assess the intracellular ROS scavenging efficacy, quantification was performed using the fluorescent probe 2′,7′-dichlorodihydrofluorescein diacetate (DCFH-DA). In H 2 O 2 -stimulated stem cells, 10 µg/mL nanoparticles significantly reduced ROS levels, with the ROS scavenging rates of the DMSN, DMSN-Fe/Cu and TPP-DMSN-Fe/Cu groups being 1.47, 2.17 and 2.31 times those of the untreated control group, respectively (Fig. 3 g, h). This activity can protect cells from oxidative damage. Additionally, compared with the control group, the expression level of Cat mRNA increased by 47% (Fig. 3 i), indicating that TPP-DMSN-Fe/Cu nanozymes significantly up-regulated the antioxidant capacity of stem cells. In summary, TPP-DMSN-Fe/Cu nanozymes exhibit favorable biocompatibility, efficient cellular uptake, precise mitochondrial targeting, and strong ROS scavenging ability, which are crucial for their therapeutic potential in bone regeneration. 2.3. Metabolic modulation and mitochondrial enhancement by TPP-DMSN-Fe/Cu nanozyme Stem cells primarily synthesize ATP via glycolysis and oxidative phosphorylation 25 . The latter process relies on an ETC consisting of five enzyme complexes (I–V), in which complexes I–IV establish a proton gradient to drive complex V to synthesize ATP 36 . In addition to these pathways, lipid metabolism also contributes to ATP production: fatty acid oxidation (FAO) converts lipids to acetyl-CoA, which fuels the TCA 37 . Moreover, previous studies have demonstrated that osteoblasts can acquire energy from fatty acids, a process imperative for bone tissue formation 38 . In addition, it has been determined that the availability of lipids is instrumental in regulating the differentiation of skeletal progenitor cells into either chondrogenic or osteogenic lineages, a process primarily orchestrated by the transcription factor SOX9 30 . Our nanozymes exhibit CcO-like activity, as evidenced by the reduced α-band absorption (550 nm) of ferrous cytochrome c (Cyt c ) in the presence of Fe/Cu-containing nanozymes (DMSN-Fe/Cu and TPP-DMSN-Fe/Cu), indicating oxidation to the iron form (Fig. 4 b). In contrast, DMSN alone lacks this catalytic ability, confirming that the functions of Fe/Cu-integrated nanozymes as a CcO analog. Furthermore, the DMSN-Fe/Cu nanozymes exhibited dual enzymatic activities, also emulating NOX in catalyzing the conversion of NADH to NAD + . This NOX-like activity was confirmed by quantifying NAD + levels using a NAD + /NADH detection kit (Fig. 4 c). Notably, the ATP luminescence assay demonstrated that the TPP-DMSN-Fe/Cu nanozyme group exhibited a 69% increase in ATP production compared to the control group (Fig. 4 d). In addition, the expression of Atp5a mRNA was also upregulated in the treated stem cells (Fig. 4 e). During bone repair, stem cell proliferation is generally supported by glycolysis, while fatty acids can act as signaling molecules to promote osteogenic differentiation. In addition, studies have shown that fatty acid β-oxidation increases dramatically as osteoblasts mature in vitro, and anabolic Wnt signaling via LRP5 promotes FAO 39 . To evaluate potential nanomaterial-induced metabolic shift, mitochondrial respiration and glycolysis were analyzed using a Seahorse Extracellular Flux Analyzer. Initially, real-time oxygen consumption rate (OCR) was recorded after the continuous addition of oligomycin, Carbonyl cyanide-4 (trifluoromethoxy) phenylhydrazone (FCCP), and rotenone as well as antimycin A. It was found that with TPP-DMSN-Fe/Cu nanozyme treatment, the basal respiration, maximum respiration, mitochondrial ATP production, non-mitochondrial oxygen consumption, and spare respiratory capacity were increased by 44%, 89%, 45%, 137%, and 114%, respectively, for the compared with the control group (Fig. 4 f and Supplementary Fig. 6 ). These results indicate that these nanozymes could effectively enhance mitochondrial function. Subsequently, we monitored the changes in extracellular acidification rate (ECAR) after sequential addition of glucose, oligomycin, and 2-deoxy-D-glucose (2-DG). Compared with the other groups, the glycolysis, glycolytic capacity, and glycolytic reserve of the TPP-DMSN-Fe/Cu group were significantly reduced (Fig. 4 g). To elucidate metabolic reprogramming, OCR were monitored after sequential inhibition of FAO (via etomoxir), ATP synthase (via oligomycin), and mitochondrial respiration (via rotenone/antimycin A). TPP-DMSN-Fe/Cu-treated cells demonstrated a reduction in both basal and maximal respiration in comparison to the control group (Fig. 4 h). A critical observation is the decrease in OCR after etomoxir treatment (FAO inhibition) in nanozyme-treated cells, suggesting an augmented reliance on FAO for energy production. These findings suggest that TPP-DMSN-Fe/Cu nanozymes reprogram cellular metabolism by prioritizing FAO. This metabolic rewiring is concomitant with the increased levels of NADPH observed in treated cells (Fig. 4 c), a phenomenon that is imperative for the synthesis of the osteogenic matrix and the maintenance of redox homeostasis. Collectively, these results demonstrate that TPP-DMSN-Fe/Cu nanozymes promote osteogenic differentiation of stem cells by augmenting mitochondrial oxidative metabolism through FAO-driven bioenergetics. 2.4. Transcriptome sequencing analysis of TPP-DMSN-Fe/Cu nanozyme-treated stem cells In order to investigate TPP-DMSN-Fe/Cu-induced changes in metabolic and osteogenic gene expression, RNA sequencing was performed. The transcriptomic analysis revealed the key mechanisms underlying the nanozyme's ability to enhance mitochondrial function and osteogenic differentiation (Fig. 5 a). In comparison with the control group, the DMSN group, and the DMSN-Fe/Cu-treated group, the TPP-DMSN-Fe/Cu group showed 220, 216, and 157 upregulated genes, respectively (Fig. 5 b). Of note, the expression levels of Bmp4 , Sox9 , Trpc1 , and Slc25a22 were found to be significantly increased for the TPP-DMSN-Fe/Cu group (Fig. 5 c). Among these genes, the osteogenic marker Bmp4 is crucial for maintaining matrix synthesis and promote the osteogenic differentiation of stem cells 40 . Chondrogenesis, the process of cartilage formation, generates cartilage that can serve as the initial skeletal structure and as a template for endochondral ossification 41 . As a chondrogenic marker, Sox9 is the primary transcription factor necessary for the differentiation of stem cells into chondrocytes and subsequent cartilage formation 42 . Beyond its role in the initiation of chondrogenesis, Sox9 has also been shown to regulate stem cell metabolism by modulating FAO 30 . It has been established that Trpc1 , in its capacity as a functional channel for calcium ion influx, is capable of regulating calcium ion influx 43 . This, in turn, activates CAMMK/AMPK signaling pathway and promotes mitochondrial biogenesis. The mitochondrial glutamate transporter Slc25a22 is a pivotal transporter that supplies carbon substrates to the TCA cycle via glutathione synthesis 44 . Gene Ontology (GO) enrichment analysis ( Supplementary Fig. 7 ) of biological processes revealed activation of pathways linked to mitochondrial function and osteogenesis, including CAMKK-AMPK signaling cascade, positive regulation of mitochondrial fission, ATP metabolic process, glutamine metabolic process, and positive regulation of osteoblast proliferation (Fig. 5 d). These findings consistently support the nanozyme's capacity to enhance mitophagy, a selective autophagy process critical for eliminating dysfunctional mitochondria, which is closely linked to bone regeneration. Furthermore, cellular component analysis revealed an enrichment in ECM categories, including extracellular region, space, matrix, and collagen trimers, suggesting enhanced collagen biosynthesis (Fig. 5 e). Additionally, molecular function analysis identified enhanced GTP/ATP binding, NAD + activity, oxidoreductase activity, and glutathione peroxidase activity for the TPP-DMSN-Fe/Cu group, thereby supporting the nanozyme's role in mitochondrial optimization and osteogenesis (Fig. 5 f). Furthermore, Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis highlighted enriched metabolic pathways pivotal to bone formation, including alanine/aspartate/glutamate metabolism, HIF-1 signaling, glutathione metabolism, and phagosome activity, for the TPP-DMSN-Fe/Cu group (Fig. 5 g). Gene Set Enrichment Analysis (GSEA) further corroborated these findings, revealing increased mitochondrial respiratory chain complex IV activity, autophagy regulation, glutamate transport, and ATP synthesis for the TPP-DMSN-Fe/Cu-treated cells (Fig. 5 h and Supplementary Fig. 8 ). Collectively, these results demonstrate that TPP-DMSN-Fe/Cu nanozymes drive osteogenesis by augmenting mitochondrial metabolism and activating bone regeneration-related signaling pathways. 2.5. Functional validation of osteogenic differentiation and mitochondrial metabolic activation In addition to evaluating TPP-DMSN-Fe/Cu-enhanced osteogenesis by transcriptome analysis, we further validated the osteogenic properties of the nanozymes by various other methods. These findings were first confirmed by PCR-revealed mRNA expression levels. Compared with the control, the transcriptional levels of runt-related transcription factor 2 (Runx2) , osteocalcin (Ocn) , osteopontin (Opn) , Alkaline phosphatases (Alp), and collagen type I alpha 1 (Col1a1) were increased by 1.45, 4.44, 2.99, 2.79, and 2.05 folds, respectively, for the TPP-DMSN-Fe/Cu group (Fig. 6 a and Supplementary Fig. 9 ). At the protein level, TPP-DMSN-Fe/Cu treatment led to the most prominent upregulation of key osteogenic markers, with COL1A1, RUNX2, ALP, and OCN increased by 147%, 46%, 53%, and 60%, respectively (Fig. 6 b and Supplementary Fig. 10 ). Immunofluorescence staining further corroborated these findings, demonstrating heightened expression levels of osteogenic markers (OCN, COL1A1, and RUNX2) and augmented mineralization in stem cells (Fig. 6 c, d and Supplementary Fig. 11, 12 ). Alkaline phosphatase (ALP) activity is an early marker of bone formation. ALP activity was found to be significantly increased after 7 days of nanozyme treatment, with the TPP-DMSN-Fe/Cu group demonstrating the highest ALP activity ( Supplementary Fig. 13 ). Consistently, Alizarin Red S (ARS) staining revealed the highest level of mineralization for the TPP-DMSN-Fe/Cu group (Fig. 6 e, f). These results underscore the capacity of nanozymes to amplify both early- and late-stage osteogenic pathways. It is also worthy of note that genes associated with mitochondrial function was significantly upregulated after TPP-DMSN-Fe/Cu treatment. In comparison with the control group, the expression levels of Pgc-1α , Cpt1a , Dnm1l , Sdha , Fam36a , and Rpl13a increased by 41%, 52%, 63%, 59%, 30%, and 91%, respectively, for the TPP-DMSN-Fe/Cu group (Fig. 6 g and Supplementary Fig. 14 ). Among these markers, Pgc - 1α is involved in the coordination of mitochondrial biogenesis and oxidative metabolism 45 , and Cpt1a plays a regulatory role in fatty acid uptake for energy production 46 . Atp5a is a subunit of mitochondrial ATP synthase, the enzyme responsible for ATP synthesis/hydrolysis 47 . Moreover, a study has confirmed the role of Dnm1l in regulating mitochondrial fission 48 . These genes work in concert to ensure proper mitochondrial metabolism within stem cells, thereby enabling them to adapt to energy demands during osteogenic differentiation. KEGG and GO biological process enrichment (Fig. 5 d, g) suggests that CAMKK-AMPK signal cascade and autophagy are key mediators linking mitochondrial energy regulation and osteoblast differentiation. These findings are consistent with the mechanisms reported in previous studies on stem cell differentiation 49 – 51 . Furthermore, as the primary energy source for cellular activity, the status and number of mitochondria are critical for the repair of damaged tissue 52 . The regulation of mitochondrial biogenesis is a multifaceted process, with PGC-1α serving as the primary regulatory factor 53 . Additionally, AMPK has been shown to regulate PGC-1α through a process of phosphorylation, thereby enhancing mitochondrial biogenesis 54 . Therefore, we assessed the relative expression of PGC-1α, AMPK, and activated AMPK (p-AMPK) in C3H/10T1/2 cells treated with TPP-DMSN-Fe/Cu. As illustrated in Fig. 6 h, i and Supplementary Fig. 15 , TPP-DMSN-Fe/Cu significantly increased the protein expression of PGC-1α (2.14-fold) and the p-AMPK/AMPK ratio (2.09-fold) in comparison to the control group. In accordance with this observation, treatment of C3H/10T1/2 cells with TPP-DMSN-Fe/Cu resulted in a 61% and 106% increase in Beclin-1 and LC3-II protein levels, respectively, thereby confirming the activation of autophagy (Fig. 6 h, i). It is noteworthy that Beclin-1 and its binding partners regulate the activity of the Vps34 lipid kinase, which is crucial for autophagy and other membrane transport processes 55 . It has been established through previous studies that the presence of LC3-II in lipid form serves as an indicator of autophagy-related structures 56 . Moreover, it has been demonstrated that mitochondrial autophagy is implicated in the regulation of bone metabolism 51 , 57 . In addition, Ca 2+ fluorescence probe experiments demonstrated that the green fluorescence intensity in the TPP-DMSN-Fe/Cu group was considerably higher than that in the control group (Fig. 6 j, k). This finding is indicative of elevated intracellular calcium ion concentrations. Consequently, by scavenging ROS and enhancing ATP production, TPP-DMSN-Fe/Cu nanozymes create an energy-rich microenvironment that promotes osteoblast synthesis and mineralization. This multifunctional approach renders TPP-DMSN-Fe/Cu nanozymes a promising therapeutic candidate for accelerated bone regeneration. 2.6. TPP-DMSN-Fe/Cu nanozyme accelerated bone regeneration in rat CSBDs The effects of TPP-DMSN-Fe/Cu nanozyme on bone tissue regeneration in vivo were evaluated using a rat tibial defect model (Fig. 7 a). The defect sites were implanted with pure gelatin methacryloyl [GelMA, 10% (wt/v)] scaffolds and those modified with 500 µg/mL of DMSN, DMSN-Fe/Cu, and TPP-DMSN-Fe/Cu (denoted GelMA/DMSN scaffolds, GelMA/DMSN-Fe/Cu scaffolds, and GelMA/TPP-DMSN-Fe/Cu scaffolds, respectively). Bone defect specimens were harvested from the tibiae of Sprague Dawley (SD) rats after 4 and 8 weeks of treatment and evaluated using micro-CT. As demonstrated in Fig. 7 b, different amounts of newly regenerated bone were observed in the defect areas of all experimental groups following 4 weeks of treatment. After another four weeks, an increased amount of new bone was observed for all groups. Furthermore, we analyzed the bone volume fraction (BV/TV) and bone mineral density (BMD) of the newly regenerated bone tissues. In comparison with the control group, the BV/TV values for the GelMA/TPP-DMSN-Fe/Cu group were significantly higher at both four and eight weeks. At Week 4, the average BV/TV for the GelMA/TPP-DMSN-Fe/Cu group was found to be 2.77, 2.05, 1.50, and 1.49 times higher than that for the control, GelMA, GelMA/DMSN, and GelMA/TPP-DMSN-Fe/Cu groups, respectively (Fig. 7 c). At Week 8, these fold changes in BV/TV were further increased to 4.10, 1.67, 1.45, and 1.28, respectively (Fig. 7 d). Additionally, compared to the control, the scaffold groups with/without nanoparticle addition all showed significantly higher BMD values, with the largest increment observed for the GelMA/TPP-DMSN-Fe/Cu group. Furthermore, hematoxylin and eosin (H&E) staining and Masson’s trichrome staining were performed for histological analysis of the regenerated bone (Fig. 7 e, f). At both 4 and 8 weeks, the area and density of new bone for the GelMA/TPP-DMSN-Fe/Cu group were greater than those in the control group and the GelMA group. The TPP-DMSN-Fe/Cu group consistently demonstrated superior defect repair capabilities to the control group. In order to further investigate the processes of osteoblast differentiation and mitochondrial biogenesis during bone regeneration, immunohistochemical (IHC) staining for OCN and immunofluorescence staining for COL1A1, PGC-1α, and ATP5A were performed. The number of OCN-positive cells was found to be significantly higher in the GelMA/TPP-DMSN-Fe/Cu group than in the other four groups (Fig. 8 a and Supplementary Fig. 16 ). Similarly, the COL1A1 expression levels were the highest in the GelMA/TPP-DMSN-Fe/Cu group, with the control and GelMA groups showing the lowest levels of COL1A1 among all groups (Fig. 8 b and Supplementary Fig. 17 ). These results indicate that TPP-DMSN-Fe/Cu nanozymes effectively promoted osteogenic differentiation and bone regeneration. Furthermore, compared with other groups, the expression of PGC-1α, a mitochondrial biogenesis marker (Fig. 8 c and Supplementary Fig. 18 ), and ATP5A, an ATP production marker (Fig. 8 d and Supplementary Fig. 19 ), was found to be significantly increased in the GelMA/TPP-DMSN-Fe/Cu group at both 4 and 8 weeks. These findings suggest that the application of TPP-DMSN-Fe/Cu nanozymes resulted in a larger number of healthy mitochondria, thereby providing a greater energy supply for bone repair. Furthermore, the biocompatibility and biosafety of TPP-DMSN-Fe/Cu nanozymes and other nanoparticles were evaluated in vivo. No abnormalities were observed in the heart, liver, spleen, lung, and kidney tissues of rats from any groups, and no obvious difference was observed between the GelMA scaffold only and GelMA/TPP-DMSN-Fe/Cu groups, as revealed in H&E staining images ( Supplementary Fig. 20, 21 ). Moreover, a comprehensive blood analysis revealed comparable conventional blood parameters across all the groups ( Supplementary Fig. 22 ), further supporting the high biosafety of TPP-DMSN-Fe/Cu nanozymes. 3. Discussion We developed a mitochondria-targeted nanozyme platform that mimics the enzymatic activity of cytochrome c oxidase (Complex IV), pivotal components of the ETC. By enhancing FAO in stem cells, the TPP-DMSN-Fe/Cu nanozymes amplify ETC in OXPHOS and modulate the TCA cycle, restoring redox balance and boosting ATP synthesis. The nanozymes have been shown to localize to the mitochondria, where they not only function to augment the ETC, but also scavenge ROS, elevate glutathione to fortify antioxidant defenses, and enhance GTPase activity to regulate mitochondrial dynamics. This process promotes mitochondrial fission and the clearance of damaged mitochondria by autophagy. Concurrently, the nanozyme activates the CaMKK/AMPK/ PGC-1α pathway, thereby stimulating mitochondrial biogenesis and creating a microenvironment conducive to osteogenesis. In a rat model of CSBDs, the nanozyme system led to robust bone regeneration characterized with accelerated defect repair and increased bone mineral density. In injured bone microenvironments, a redox imbalance has been demonstrated to drive mitochondrial dysfunction and excessive ROS accumulation, thereby impairing osteogenesis 58 . While ROS-scavenging biomaterials have demonstrated potential, most of these materials do not regulate mitochondrial homeostasis in a targeted manner 59 , 60 . Although emerging mitochondria-targeted polyphenol/amino acid NPs (e.g., ECGG-Cys-NPs, PGA-Mn-TP04) 54 , 61 enhanced mitochondrial biogenesis and reducing ROS, they lack capacity to drive the metabolic reprogramming required for osteogenesis. The current study has significantly advanced mitochondria-targeted nanotherapeutics through a unique coupling of FAO-driven osteogenic differentiation with autophagy-driven mitochondrial dynamic mechanisms. For instance, we have identified key genes, including Pgc-1α (amplifying mitochondrial biogenesis) 45 , Slc25a22 (supplying TCA intermediates and glutathione precursors) 44 , and Cpt1a (enabling fatty acid transport for β-oxidation) 46 , that play critical roles in stem cell osteogenesis. By simultaneously supporting metabolic reprogramming, redox balance, and mitochondria homeostasis through a multifunctional nanozyme platform, our approach transcends conventional strategies that target singular pathways, establishing a versatile and effective framework for bone regeneration. Most conventional nanozymes were designed to modulate glycolysis, thereby regulating lactic acid production for tumor therapy 62 , 63 Our mitochondria-targeted TPP-DMSN-Fe/Cu nanozyme acts by a distinct mechanism. It directly enhances FAO-driven OXPHOS during stem cell osteogenesis, while concurrently inhibiting glycolysis. This distinctive metabolic reprogramming is attributable to its subcellular localization and the specific characteristics of the cellular environment. Mitochondrial localization enables TPP-DMSN-Fe/Cu to directly enhance FAO-driven OXPHOS, effectively generating acetyl-CoA and reducing reliance on glycolysis. Moreover, previous studies have predominantly focused on cancer cells or proliferating cells that rely on glycolytic anabolic metabolism for growth 64 . The process of stem cell differentiation, particularly osteogenic differentiation, has been shown to be enhanced by the inhibition of glycolysis 25 . Reduced glycolysis has been demonstrated to impede the accumulation of lactic acid and subsequent microenvironment acidification, and this microenvironmental change inhibits bone regeneration 65 . The nanozyme-enabled metabolic reprograming facilitates the redirection of energy toward collagen synthesis and mineral deposition through sustained, non-acidic OXPHOS. Furthermore, our nanozymes were capable of clearing ROS, thereby protecting the FAO enzymes and enhancing autophagy, a process that involves the removal of damaged mitochondria. This, in turn, favors an oxidative metabolic state that facilitates osteogenic differentiation. While the TPP-DMSN-Fe/Cu nanozymes hold promising potential in bone regeneration, several questions remain to be answered before the clinical translation of this technology. First, large-animal validation of long-term biocompatibility, biodistribution, and regenerative outcomes should be rigorously assessed. Second, the mechanisms underlying the body’s immune responses, to the nanozymes, such as the effects of nanozymes on macrophage polarization, remain to be clarified. Third, it is worth exploring the effects of nanozymes on stem cell subpopulations at defect sites. To quantitatively map the biodistribution of nanozymes in vivo, particularly in large animal models, isotope labeling-based tracking technology can be used in future research 66 This approach will facilitate the assessment of the long-term accumulation/excretion of nanozymes within organs. To understand the interactions between immune cells and the nanozymes, organ-on-a-chip technology can play an instrumental role in revealing the nanotechnology-biology interface by, for example, the establishment of a bone defect-on-a-chip system 67 . These systems, which may include bone cells, macrophages, and blood vessels, can be employed for real-time monitoring of the effects of nanozymes on cellular functions. Finally, stem cell lineage tracing technique can facilitate the elucidation of potentially diverse behaviors of different stem cell subpopulations 68 . In addition, emerging evidence indicates that redox dysfunction in mature osteoblasts plays a pivotal role in the development of osteoporosis. For instance, estrogen deficiency-induced downregulation of GPX4 leads to phospholipid peroxidation in osteoblasts, thereby impairing bone formation 69 . These findings indicate that our TPP-DMSN-Fe/Cu nanozymes could also be used for effective osteoporosis treatment, which necessitates both the enhancement of metabolic efficiency in stem cells and the protection of mature osteoblasts from oxidative damage. 4. Conclusion CSBDs represent a formidable clinical challenge, requiring innovative solutions to restore structural and functional integrity. In this study, a mitochondrial-targeted single-atom nanozyme platform (TPP-DMSN-Fe/Cu) was developed. This platform was designed to address the dual impediments of oxidative stress and mitochondrial dysfunction in stem cells. By scavenging ROS and enhancing mitochondrial energy metabolism, the nanozyme establishes a regenerative microenvironment that promotes osteogenesis. Mechanistic studies have revealed its capacity to augment mitochondrial biogenesis, eliminate damaged mitochondria via autophagy, and drive osteogenic differentiation. This highlights mitochondrial health as a crucial factor in bone repair. In vivo validation demonstrates accelerated regeneration, marked by significant increases in bone volume and mineral density. 5. Methods The rat experimental protocols conducted in this study were approved by the Animal Experimentation Ethics Committee of the Chinese University of Hong Kong (Approval No. (24–427) in DH/HT&A/8/2/1 Pt.63). Chemicals Triethanolamine (TEA, purity ≥ 98%), Hexadecyltrimethylammonium bromide (CTAB, purity ≥ 99%), Sodium salicylate (NaSal, purity ≥ 99%), Methanol (CH 3 OH, purity ≥ 99%), (5-carboxypentyl) (triphenyl)phosphonium bromide (TPP, purity ≥ 97% ), N-Hydroxysuccinimide (NHS, purity ≥ 97%), N-(3-Dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (EDC, purity ≥ 99%) were purchased from Sigma-Aldrich. Hydrochloric acid (HCl, 37%) was purchased from Duksan. Tetraethyl orthosilicate (TEOS, purity ≥ 98%), 1,2-bis(triethoxysilyl) ethane (BTEE, purity ≥ 95%), (3-Aminopropyl) triethoxysilane (APTES, purity ≥ 99%), Iron sulfate heptahydrate (Fe 3 O 4 -7H2O, purity ≥ 99%), Copper nitrate trihydrate ((Cu (NO 3 ) 2 -3H 2 O, purity ≥ 99%). All aqueous solutions were prepared with deionized water (18.2 MΩ; Millipore). Synthesis of TPP-Fe/Cu-DMSN Step 1: Dendritic mesoporous silicon (DMSN) Synthesis. The DMSN was synthesized in a procedure that incorporated cationic surfactants CTAB and Nasal. The silicon sources utilized were TEOS and BTEE, and the catalyst employed was TEA. Initially, 0.136 g of TEA was added to 50 ml of water and stirred gently at 80°C for 0.5 h. Subsequently, 760 mg of CTAB and 336 mg of NaSal were added to the solution and stirred at 80°C for an additional hour. The mixture of 8 ml TEOS and 3.2 ml BTEE was then subjected to gentle stirring for 6 hours at 80°C at 300 rpm. The resulting mixture was subjected to centrifugation at 10,000 rpm for 10 minutes and washed once or twice with pure ethanol. Finally, the mixture was subjected to a reflux process with HCL (3ml): methanol (60ml) solution for a duration of 2 hours at a temperature of 80°C. Subsequently, the mixture was subjected to a centrifugation step and was then washed with ethanol. Step 2: DMSN- NH 2 Synthesis In this step of the procedure, 280 mg of DMSN were dissolved in 80 ml of ethanol and sonicated. Subsequently, 7 ml of APTES were added, and the solution was refluxed for 6 hours at 70°C. Thereafter, the solution was subjected to centrifugation as previously described. Step 3: DMSN-Fe/Cu Synthesis A solution of 80 mg of DMSN-NH 2 in 20 ml of MES buffer at a pH of 6 was prepared initially. This was followed by the activation of 48 mg of EDC and 12.8 mg of NHS for a duration of 30 minutes. Subsequently, 320 mg of L-cysteine was incorporated, and the mixture was maintained at a temperature of 37°C for a period of 6 hours. The resulting nanoparticles were subjected to a centrifuge process, followed by resuspension in 9 ml of DI water. Subsequently, 1 ml of DI water containing Cu (NO 3 ) 2 -3H 2 O (41 mg) and Fe 3 O 4 -7H 2 O (100 mg) was added. Step 4: TPP-Fe/Cu-DMSN A mixture of 20 mg of DMSN/L-Cys/Fe/Cu and 24.552 mg of NHS + 49.288 mg of EDC was dispersed in 10 ml of MES buffer at pH 6.00 and stirred for 2 hours at room temperature. Subsequently, 80 mg of TPP was dispersed in 8 ml of DI water and incubated at 37°C for 6 hours. The final product was subjected to centrifugation, as previously described. Cell culture C3H/10T1/2, Clone 8 cells were procured from Oricell (Guangzhou, China). Low-glucose Dulbecco's modified Eagle's medium (DMEM; Gibco) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin was used as cell culture medium. For osteogenic differentiation, cells were transitioned to high-glucose DMEM containing 100 nM dexamethasone, 10 mM β-glycerophosphate, and 50 µg/mL L-ascorbic acid (Sigma Aldrich). The differentiation medium was refreshed every 48–72 hours to ensure consistent nutrient availability. All culture reagents, unless specified otherwise, were sourced from Gibco. Cellular uptake Nanoparticles were internalized by stem cells were observed using TEM (Hitachi H-7650; Hitachi, Tokyo, Japan). Cells were cultured in growth medium at an initial density of 5.0 × 10 5 cells/well in 6-well plates. When the cells reached 80% confluence, nanozymes were added. After 4 hours of incubation, the cells were fixed, dehydrated, embedded, cut, and then observed by TEM. For mitochondrial-targeted validation, stem cells were incubated in osteogenic differentiation DMEM medium containing nanoparticles (10 µg/mL) for 4 h. Cellular uptake of nanoparticles was observed by confocal microscopy (Leica SP8). Cells and nanoparticles were stained with Mito-tracker green and Rhodamine B isothiocyanate (RBITC), respectively. Real-time RT-PCR The stem cells were seeded into 6-well plates and allowed to proliferate to 70% confluence. Thereafter, the medium was substituted with osteogenic differentiation medium, and nanoparticles were introduced. After a 7 days incubation, total RNA was extracted using the RNA Extraction Kit (ZYMO). To obtain cDNA, total RNA was reverse transcribed using the cDNA Amplification Kit (YEASEN). The primer sequences employed in this study are listed in Supplementary Table 1 , obtained from the Primer Library ( https://www.ncbi.nlm.nih.gov/gene/ ) and synthesized by BGI Genomics. The transcript levels of the target genes were calculated using the ΔΔCt method, with GAPDH serving as the housekeeping gene. Cells not treated with nanoparticles were used as the control group. Western blotting Cells were cultured and processed in the same way as for qRT-PCR assay. On day 7, proteins were extracted using RIPA lysis buffer (Thermo Fisher) and protease and phosphatase inhibitor cocktail, MSF. After BCA (Thermo Fisher) quantification, proteins were loaded into sodium dodecyl sulphate polyacrylamide gels and transferred to Trans-Blot Turbo Midi 0.2 µm PVDF Transfer Packs (Bio-Rad). All antibodies used in this study are listed in Supplementary Table 2 . Band intensity was quantified using ImageJ software (National Institutes of Health, USA). Calcium 2+ influx Intracellular calcium ion influx imaging was performed using a Leica Thunder imager to record the intensity of the intracellular calcium dye (Fluo-4 AM, Beyotime). The calcium imaging data obtained were analyzed and visualized using ImageJ. Seahorse Seahorse XFe96 analyzed oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) to assess OXPHOS, glycolysis, and lipid metabolism. stem cells (1 × 10 4 cell/mL) were seeded onto a Seahorse XF-96 plate. For Cell Mito Stress Test, the final concentration of Oligomycin, FCCP, and Rotenone/antimycin is 2 µM, 1 µM, and 0.5 µM. For Glycolysis Stress Test, Glucose, Oligomycin, and 2-Deoxy-D-glucose is 10 mM, 1 µM, and 50 mM. For Substrate Oxidation Stress Test, the final concentration of etomoxir, Oligomycin, FCCP, and Rotenone/antimycin is 4 µM, 1.5 µM, 1.5 µM, and 0.5 µM. Therapeutic efficacy of scaffolds on critical-size bone defect rats model Adult SD rats were procured from Guangzhou Seyotin Laboratory Animal Co. The 50 rats (8–12 weeks, male, 200–220 g) were randomly divided into five groups: empty defect (control), GelMA scaffolds, DMSN + GelMA scaffolds, DMSN-Fe/Cu + SF/GelMA scaffolds, and TPP-DMSN-Fe/Cu + GelMA scaffolds. After the rats were anesthetized with intraperitoneal pentobarbital (35 mg/kg), the tibia was exposed via a surgical incision, and a 3-mm diameter and 3-mm depth bone defect was created using an electric drill. Scaffolds were then implanted into the defect. Following a 4- or 8-week treatment period, half of the rats were euthanized, and their tibiae were collected and evaluated using gross observation, micro-CT scanning, and histological staining. Histology, Immunohistochemistry, and Immunofluorescence Staining Tissue samples were decalcified in 10% EDTA-2Na (Solarbio) at 4°C for 50 days, paraffin-embedded, and sectioned (5 µm thickness). Sections underwent hematoxylin and eosin (H&E), Masson’s trichrome using commercial kits. All antibodies used in this study are listed in Supplementary Table 3 . Images intensity was semi-quantitatively analyzed using ImageJ software (NIH, USA). Micro-CT Analysis Mandibular defects were analyzed at 4- and 8-weeks post-treatment using a Quantum GX2 micro-CT system at 50 kV and 100 µA (30 µm resolution). A cylindrical volume of interest (VOI; 3 mm diameter, 0.27 mm depth) centered on the defect site was evaluated. Bone volume fraction (BV/TV) and bone mineral density (BMD) were calculated to assess regenerated bone microstructure. Statistical analysis The standard deviation (SD) of the data in each group is expressed as mean ±. Comparisons between two groups were made using Student's t-test, and comparisons between groups were made using one-way ANOVA followed by Tukey's post hoc test. All statistical analyses were performed with GraphPad Prism 8. The level of statistical significance was set at P < 0.05. At least three independent replications were performed for each experiment. Declarations Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Supplementary Information Supplementary Information 1 Acknowledgements This work was supported by (1) Shun Hing Institute of Advanced Engineering, CUHK (to ZAL, project #BME-p2-24); (2) Center for Neuromusculoskeletal Restorative Medicine (to RST and ZAL), under the Health@InnoHK program, Innovation and Technology Commission (ITC), Hong Kong SAR, China; (3) National Natural Science Foundation of China (to ZAL, 82302753); and (4) Hong Kong Research Grants Council (to ZAL, 24203523). ZAL acknowledges the support from the Vice-Chancellor Early Career Professorship Scheme of the Chinese University of Hong Kong (CUHK). RST is supported by the Lee Quo Wei and Lee Yick Hoi Lun Professorship in Tissue Engineering and Regenerative Medicine of CUHK. YW is supported by CUHK postgraduate studentship. We thank Ms. Josie Lai from the School of Biomedical Sciences at CUHK for her assistance with EM imaging. 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Kong","correspondingAuthor":false,"prefix":"","firstName":"ShangSi","middleName":"","lastName":"Chen","suffix":""},{"id":485970648,"identity":"0cdb9a83-87e5-4d32-9866-5834245ad6e0","order_by":12,"name":"Chao Zheng","email":"","orcid":"https://orcid.org/0000-0001-8530-0923","institution":"Fourth Military Medical University","correspondingAuthor":false,"prefix":"","firstName":"Chao","middleName":"","lastName":"Zheng","suffix":""},{"id":485970649,"identity":"13d981b7-3763-40ec-bd5c-75edca189c05","order_by":13,"name":"Liu Yang","email":"","orcid":"https://orcid.org/0000-0002-6498-4702","institution":"Institute of Orthopedic Surgery, Xijing Hospital, Fourth Military Medical University, Xi'an, China.","correspondingAuthor":false,"prefix":"","firstName":"Liu","middleName":"","lastName":"Yang","suffix":""},{"id":485970650,"identity":"6e6dd09d-1364-4b57-98a4-325db4aa33c0","order_by":14,"name":"Wei Huang","email":"","orcid":"","institution":"The First Affiliated Hospital of Chongqing Medical University","correspondingAuthor":false,"prefix":"","firstName":"Wei","middleName":"","lastName":"Huang","suffix":""},{"id":485970651,"identity":"27837957-3b78-4292-890c-7a6969dd182f","order_by":15,"name":"Rocky Tuan","email":"","orcid":"","institution":"The Chinese University of Hong Kong","correspondingAuthor":false,"prefix":"","firstName":"Rocky","middleName":"","lastName":"Tuan","suffix":""},{"id":485970652,"identity":"0dd4f3fc-4c17-4ae5-87b8-8e9cb3edc2e6","order_by":16,"name":"Denghui Xie","email":"","orcid":"","institution":"Department of Orthopedic Surgery, The Third Affiliated Hospital of Southern Medical University","correspondingAuthor":false,"prefix":"","firstName":"Denghui","middleName":"","lastName":"Xie","suffix":""}],"badges":[],"createdAt":"2025-06-30 15:45:55","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-7012382/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7012382/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":87606528,"identity":"368eac2b-54cf-4fad-9511-b5e6b3c5a71b","added_by":"auto","created_at":"2025-07-25 18:28:09","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":167977,"visible":true,"origin":"","legend":"\u003cp\u003eSchematic diagram illustrating the ability of TPP-DMSN-Fe/Cu nanozymes to reduce oxidative stress, improve mitochondrial function, and promote osteogenic differentiation of stem cells.\u003c/p\u003e","description":"","filename":"Onlinefloatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-7012382/v1/9d80a1eca2ee4da70d0f34fd.png"},{"id":87606526,"identity":"940408e8-a264-46a5-8837-d4a41e6cf157","added_by":"auto","created_at":"2025-07-25 18:28:09","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":234368,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSynthesis and characterization of TPP-DMSN-Fe/Cu nanozymes\u003c/strong\u003e. \u003cstrong\u003ea\u003c/strong\u003e Schematic of the synthesis process of TPP-DMSN-Fe/Cu nanozyme. \u003cstrong\u003eb, c\u003c/strong\u003e TEM images of the synthesized nanozyme. Scale bar = 50 nm and 10 nm. \u003cstrong\u003ed\u003c/strong\u003e HAADF image showing the dendritic mesoporous structure of the synthesized nanozyme. Scale bar = 50 nm. \u003cstrong\u003ee\u003c/strong\u003e EDS elemental mapping confirming homogeneous distribution of Cu, Fe, Si, and O in TPP-DMSN-Fe/Cu nanozyme. Scale bars = 50 nm. \u003cstrong\u003ef, g\u003c/strong\u003e Zeta potential values (f) and FTIR spectra (g) of different nanoparticles. \u003cstrong\u003eh\u003c/strong\u003e N\u003csub\u003e2 \u003c/sub\u003eadsorption and desorption isotherms and corresponding pore size distribution of TPP-DMSN-Fe/Cu nanozyme. \u003cstrong\u003ei-k\u003c/strong\u003e Antioxidant assays comparing the DPPH- (i), •OH- (j), and H₂O₂-scavenging (k) rates of different nanoparticles.\u003c/p\u003e","description":"","filename":"Onlinefloatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-7012382/v1/2290957d0a89052bbcf4d7a4.png"},{"id":87606527,"identity":"c495e718-02ef-4c42-a269-37c864044344","added_by":"auto","created_at":"2025-07-25 18:28:09","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":233961,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eIn vitro biocompatibility, internalization, mitochondrial targeting and intracellular ROS scavenging ability of TPP-DMSN-Fe/Cu nanozymes.\u003c/strong\u003e \u003cstrong\u003ea\u003c/strong\u003e IC50 results of stem cells co-cultured with different nanoparticles for 24 hours. \u003cstrong\u003eb, c\u003c/strong\u003eLive/dead staining images of stem cells co-cultured with nanozymes for 24 and 48 hours (b) and their corresponding fluorescence intensities (c). Scale bar = 300 \u003cem\u003eµ\u003c/em\u003em \u003cstrong\u003ed\u003c/strong\u003e Bio-TEM images of C3H/10T1/2 cells after 4 hours of treatment with TPP-DMSN-Fe/Cu nanozymes (red arrows indicate mitochondria). Scale bar = 500 nm and 200 nm. \u003cstrong\u003ee\u003c/strong\u003e Representative confocal images of C3H/10T1/2 cells co-cultured with nanozymes with or without TPP modification (blue, nucleus; red, RBITC-Nanoparticles; green, Mito-tracker). Scale bar = 10 µm \u003cstrong\u003ef\u003c/strong\u003e Quantitative analysis of fluorescence intensity along the white dotted lines in \u003cstrong\u003ee\u003c/strong\u003e. \u003cstrong\u003eg \u003c/strong\u003eROS staining, and \u003cstrong\u003eh\u003c/strong\u003e corresponding ROS scavenging ability analysis. Scale bar = 100 µm \u003cstrong\u003ei\u003c/strong\u003e mRNA expression of \u003cem\u003eCat \u003c/em\u003e(\u003cem\u003en\u003c/em\u003e = 4). One-way ANOVA with Tukey post-hoc test was used to determine statistical significance. Data presented as mean ± SD.\u003c/p\u003e","description":"","filename":"Onlinefloatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-7012382/v1/0c5c998081564cfaeafd523f.png"},{"id":87606529,"identity":"8aab21ec-e572-4b4e-88ae-7b23170d6787","added_by":"auto","created_at":"2025-07-25 18:28:09","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":298490,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTPP-DMSN-Fe/Cu nanozymes improved mitochondrial function and regulated energy metabolism in stem cells.\u003c/strong\u003e \u003cstrong\u003ea \u003c/strong\u003eSchematic diagram of TPP-DMSN-Fe/Cu-induced changes in OXPHOS, FAO, and TCA cycle-related target metabolites. \u003cstrong\u003eb\u003c/strong\u003e UV-Vis absorbance spectra of Cyt \u003cem\u003ec\u003c/em\u003e after reacting with different nanoparticles for 60 minutes. \u003cstrong\u003ec\u003c/strong\u003e NAD\u003csup\u003e+\u003c/sup\u003e/NADH levels in stem cells after treatment with different nanoparticles. \u003cstrong\u003ed\u003c/strong\u003e ATP production and \u003cstrong\u003ee\u003c/strong\u003e Relative mRNA levels of \u003cem\u003eAtp5a\u003c/em\u003e after nanoparticles treatments (\u003cem\u003en\u003c/em\u003e = 4). \u003cstrong\u003ef\u003c/strong\u003e Real-time OCRs during stem cell mitochondrial stress tests and semi-quantitative analysis of basal respiration, maximal respiration, and ATP production. \u003cstrong\u003eg\u003c/strong\u003e Real-time ECARs of stem cells during the glycolytic stress test and semi-quantitative analysis of glycolysis, glycolytic capacity, and glycolytic reserve. \u003cstrong\u003eh\u003c/strong\u003e Real-time OCRs of stem cells during the substrate oxidation test and semi-quantitative analysis of maximal respiration, spare respiratory capacity, and basal respiration (\u003cem\u003en\u003c/em\u003e = 4). One-way ANOVA with Tukey post-hoc test was used to determine statistical significance. Data presented as mean ± SD.\u003c/p\u003e","description":"","filename":"Onlinefloatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-7012382/v1/3fe905e601e9cb6ff0ba149e.png"},{"id":87606663,"identity":"71d9d250-a125-4e16-b4b2-ca2d00b8ff86","added_by":"auto","created_at":"2025-07-25 18:36:09","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":145776,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTranscriptome analysis revealed that TPP-DMSN-Fe/Cu nanozyme reprogramed stem cell metabolism to enhance osteogenesis. a\u003c/strong\u003e Schematic of TPP-DMSN-Fe/Cu-modulated mitochondrial and osteogenic pathways based on RNA sequencing results. \u003cstrong\u003eb\u003c/strong\u003e Number of differentially expressed genes in TPP-DMSN-Fe/Cu-treated stem cells versus other groups. \u003cstrong\u003ec\u003c/strong\u003eHeatmap showing upregulated osteogenesis-, chondrogenesis-, calcium influx-, and mitochondrial function-related genes for DMSN-Fe/Cu-treated stem cells (\u003cem\u003en\u003c/em\u003e= 3). \u003cstrong\u003ed-f\u003c/strong\u003e 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. \u003cstrong\u003eg\u003c/strong\u003e KEGG enrichment analysis showing genes upregulated by TPP-DMSN-Fe/Cu treatment. \u003cstrong\u003eh, i\u003c/strong\u003e 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.\u003c/p\u003e","description":"","filename":"Onlinefloatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-7012382/v1/00e097c0565316ef619a3fc0.png"},{"id":87606539,"identity":"678d4a5e-0ad4-4900-91af-8bd9fe2b90c5","added_by":"auto","created_at":"2025-07-25 18:28:09","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":269129,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTPP-DMSN-Fe/Cu nanozymes enhanced osteogenic differentiation and mitochondrial function in stem cells.\u003c/strong\u003e \u003cstrong\u003ea\u003c/strong\u003e mRNA levels of osteogenic gene expression: \u003cem\u003eRunx2\u003c/em\u003e, \u003cem\u003eOpn,\u003c/em\u003e \u003cem\u003eOcn, and Alp\u003c/em\u003e. \u003cstrong\u003eb\u003c/strong\u003eWestern blot bands for COL1A1, RUNX2, ALP, and OCN (the numbers above each band represent normalized mean band intensity ± SD; \u003cem\u003en\u003c/em\u003e = 3). \u003cstrong\u003ec\u003c/strong\u003e Immunofluorescence staining of OCN and \u003cstrong\u003ed\u003c/strong\u003e quantification of fluorescent intensity (\u003cem\u003en\u003c/em\u003e= 4). Scale bar = 100 µm. \u003cstrong\u003ee \u003c/strong\u003eARS staining of stem cells and \u003cstrong\u003ef\u003c/strong\u003e corresponding quantitative analysis after 14 days of nanoparticle treatment (\u003cem\u003en\u003c/em\u003e = 4). Scale bar = 2mm and 100 µm. \u003cstrong\u003eg\u003c/strong\u003emRNA levels of mitochondrial gene expression: \u003cem\u003ePgc-1α\u003c/em\u003e, \u003cem\u003eCpt1a\u003c/em\u003e, \u003cem\u003eDnm1l, and Fam36a \u003c/em\u003e(\u003cem\u003en\u003c/em\u003e = 4). \u003cstrong\u003eh\u003c/strong\u003e Western blot bands for PGC-1α, p-AMPK, AMPK, Beclin-1 and LC3-II (the numbers above each band represent normalized mean band intensity ± SD;\u003cem\u003e n\u003c/em\u003e = 3). \u003cstrong\u003ei \u003c/strong\u003eQuantitative analysis of p-AMPK/ AMPK, Beclin-1 and LC3-II protein expression. \u003cstrong\u003ej\u003c/strong\u003e Intracellular calcium ion levels and \u003cstrong\u003ek\u003c/strong\u003e corresponding quantification (\u003cem\u003en\u003c/em\u003e = 4). One-way ANOVA with Tukey post-hoc test was used to determine statistical significance. Data presented as mean ± SD.\u003c/p\u003e","description":"","filename":"Onlinefloatimage6.png","url":"https://assets-eu.researchsquare.com/files/rs-7012382/v1/6c6e44fc53ca931478be33c8.png"},{"id":87606664,"identity":"8594aa95-8b94-4f3e-b1cd-57bd1e4db965","added_by":"auto","created_at":"2025-07-25 18:36:09","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":457530,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTPP-DMSN-Fe/Cu nanozymes accelerated bone regeneration in a rat model of CSBD. a\u003c/strong\u003e Schematic of the experimental timeline. \u003cstrong\u003eb\u003c/strong\u003e 3D reconstructed micro-CT scan images after 4 and 8 weeks of scaffold implantation. \u003cstrong\u003ec, d\u003c/strong\u003e Quantitative micro-CT analysis of BV/TV and BMD (\u003cem\u003en\u003c/em\u003e = 3). \u003cstrong\u003ee\u003c/strong\u003e H\u0026amp;E staining and \u003cstrong\u003ef\u003c/strong\u003e Masson’s Masson 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. One-way ANOVA with Tukey post-hoc test was used to determine statistical significance. Error bars: mean ± SD.\u003c/p\u003e","description":"","filename":"Onlinefloatimage7.png","url":"https://assets-eu.researchsquare.com/files/rs-7012382/v1/31c72e657a4cfde28d832899.png"},{"id":87607011,"identity":"ef6c2118-545c-4428-9d17-c659bc19fc42","added_by":"auto","created_at":"2025-07-25 18:44:09","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":451994,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTPP-DMSN-Fe/Cu nanozymes upregulated the expression of markers related to osteogenesis and energy metabolism in vivo.\u003c/strong\u003e \u003cstrong\u003ea\u003c/strong\u003e OCN IHC staining images for the tibial bone defect at 4 and 8 weeks. Immunofluorescence staining images for \u003cstrong\u003eb\u003c/strong\u003e COL1A1, \u003cstrong\u003ec\u003c/strong\u003e ATP5A, and \u003cstrong\u003ed\u003c/strong\u003e PGC-1α in the tibial bone defect area at 4 and 8 weeks. Scale bar = 100 µm.\u003c/p\u003e","description":"","filename":"Onlinefloatimage8.png","url":"https://assets-eu.researchsquare.com/files/rs-7012382/v1/87960f9425da0c0577c8a8ff.png"},{"id":90017517,"identity":"77f242ff-b567-44fc-ac43-77004703b60c","added_by":"auto","created_at":"2025-08-27 12:13:31","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":4385760,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7012382/v1/0da6ff60-5d73-4500-b7de-1e9b4dc67459.pdf"},{"id":87606542,"identity":"a1f98890-bafb-4ea7-b5fe-e44fd27e62c0","added_by":"auto","created_at":"2025-07-25 18:28:09","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":32562308,"visible":true,"origin":"","legend":"Supplementary information1","description":"","filename":"SupplementaryinformationSubmission.docx","url":"https://assets-eu.researchsquare.com/files/rs-7012382/v1/620c3d26c31a657ed379207c.docx"}],"financialInterests":"There is \u003cb\u003eNO\u003c/b\u003e Competing Interest.","formattedTitle":"Mitochondria-targeted, Single-atom Nanozymes Accelerate Bone Regeneration by Augmenting Stem Cell Energy Metabolism","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eCritical-sized bone defects (CSBDs) represent a significant clinical challenge that adversely impact musculoskeletal health, frequently resulting in impaired bone repair\u003csup\u003e\u003cspan additionalcitationids=\"CR2\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e. Stem cells are indispensable for bone repair, which rely on mitochondrial oxidative phosphorylation (OXPHOS) to meet the energy and biosynthetic demands of osteogenic differentiation\u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e. In this process, the electron transport chain (ETC) chain plays a pivotal role. It is a series of protein complexes (I\u0026ndash;IV) that drive ATP synthesis through electron transport and proton gradient formation. In healthy stem cells, ETC activity is responsible for the oxidation of nutrients (e.g., glucose, fatty acids) while maintaining redox balance to support matrix mineralization and differentiation\u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e. However, under pathological conditions, such as CSBDs, stem cells exhibit impaired ETC function\u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e. In CSBDs, the ETC experiences significant disruption due to (1) excessive accumulation of ROS resulting from complex I/III deficiency, (2) reduced catalytic activity of ATP synthesis/complex IV, (3) impaired mitophagy and accumulation of damaged mitochondria, and (4) inhibition of biogenesis through downregulation of PGC-1α\u003csup\u003e7\u0026ndash;9\u003c/sup\u003e. Current strategies, such as gene regulation, encounter inherent biological complexity, including pathway interconnectivity, cell-type specificity, spatiotemporal delivery challenges, and off-target effects\u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eIn recent years, there has been significant interest in the potential applications of biomaterials with reactive oxygen species (ROS) scavenging functions, such as polyphenols, artificial selenoenzymes, hydrogen-containing materials, catalase (CAT), polydopamine, and chiral biomaterials, for efficient bone regeneration\u003csup\u003e\u003cspan additionalcitationids=\"CR12 CR13 CR14\" citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e. However, many existing systems exhibit limited catalytic specificity. Inadequate mitochondrial targeting ability frequently results in antioxidant effects that extend beyond the intended field, thereby diminishing therapeutic efficacy. Moreover, while many existing material systems are capable of scavenging ROS, they are unable to address downstream mitochondrial OXPHOS dysfunction\u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e,\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e. For example, among the many ROS-scavenging nanozymes developed thus far, most of them exhibit a deficiency in multimodal functionality required for the concurrent enhancement of mitochondrial biogenesis, energy metabolism, and dynamic remodeling in tissue regeneration. This underscores the pressing need for innovative therapeutic interventions capable of effectively enhancing mitochondrial function and promoting effective bone regeneration, particularly in scenarios such as CSBDs where endogenous repair mechanisms are compromised.\u003c/p\u003e \u003cp\u003eMitochondria are a significant source of ATP production, and recent evidence highlights their role in guiding stem cell fate during bone regeneration\u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e,\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e. Stem cells rely on mitochondrial biogenesis, the process of generating new mitochondria, to satisfy the elevated energy demands during osteogenic differentiation\u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e. Notably, undifferentiated stem cells, including bone marrow mesenchymal stem cells\u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e, embryonic stem cells\u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e, and hematopoietic stem cells\u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e, exhibit a strong reliance on glycolysis to fuel rapid growth and division. However, as differentiation progresses, there is a shift in metabolic patterns: lipid metabolism becomes more prominent, while glycolytic activity declines\u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e,\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e. The shift in metabolic priorities from glycolysis to lipid metabolism is a hallmark of cellular differentiation. Particularly, fatty acid β-oxidation provides energy, facilitates the synthesis of cell membranes, and contributes to signal transduction and the substrates required for OXPHOS\u003csup\u003e\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e,\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eThis metabolic reprogramming from glycolysis to lipid-driven OXPHOS highlights the dynamic interplay between energy metabolism-related pathways and stem cell specialization\u003csup\u003e\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e,\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e. For instance, circulating fatty acids function as signaling molecules during fracture repair, directly promoting the differentiation of bone marrow stromal cells into osteoblasts\u003csup\u003e\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e. However, under pathological conditions, these processes can be disrupted, leading to mitochondrial inefficiency and ETC stress\u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e. Eventually, excessive ROS accumulation can cause mitochondrial damage. The accumulation of ROS has been found to impede mitochondrial function and hinder osteoblast differentiation, thus impeding effective bone healing\u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e. In instances where mitochondrial repair is not a viable option, a process known as mitophagy is initiated, to remove dysfunctional mitochondria\u003csup\u003e\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003e. This process is crucial for maintaining cellular homeostasis and ensuring normal cellular function. Furthermore, in the process of osteogenic differentiation, stem cells need to upregulate mitochondrial biogenesis to increase ATP production. It has been demonstrated in various studies that peroxisome proliferator-activated receptor-\u0026#120574; coactivator-1α (PGC-1α) plays a pivotal role in mitochondrial biogenesis\u003csup\u003e\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e. In addition, it has been shown that PGC-1α is induced by AMP-activated protein kinase (AMPK)\u003csup\u003e\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u003c/sup\u003e. Therefore, therapeutic strategies that target ROS elimination while promoting mitochondrial health and augmenting energy metabolism by, for example, enhancing mitochondrial biogenesis and OXPHOS, are highly desirable for restoring regenerative capacity of stem cells.\u003c/p\u003e \u003cp\u003eTo address these unmet needs, we developed a triphenylphosphine (TPP)-modified, dendritic mesoporous silica nanoparticle (DMSN)-based, and Fe/Cu single atom-loaded nanozyme, named TPP-DMSN-Fe/Cu, a mitochondria-targeted nanoparticle designed to mimic the activity of key enzymes in the ETC (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). By replicating the ETC of OXPHOS, this nanozyme can restore the redox balance while enhancing ATP synthesis. TPP modification ensures the precise targeting of the nanozyme to the mitochondria of stem cells, where it (1) scavenges ROS, boosts glutathione levels to enhance endogenous antioxidant defenses, and enhances GTPase activity to regulate mitochondrial fission. The addition of Fe/Cu mimics the function of complex IV, cytochrome c oxidase (CcO), thereby (2) increasing the catalytic activity of ATP synthesis/complex IV. In addition to its ability to promote (3) mitochondrial autophagy, this multifunctional strategy promotes (4) mitochondrial biogenesis via the Ca\u003csup\u003e2+\u003c/sup\u003e/calmodulin-dependent protein kinase kinase (CaMKK)/AMPK pathway and creates a microenvironment conducive to bone formation. By focusing on energy metabolism, we targeted the root cause of stem cell dysfunction in CSBDs, i.e., mitochondrial dysfunction that precedes and impedes osteogenic function. In animal studies, TPP-DMSN-Fe/Cu nanozymes demonstrated potent bone regeneration capabilities, evidenced by enhanced bone mineral density and accelerated defect repair. Therefore, our multifunctional nanozyme system holds promising potential in bridging the gap between mitochondrial dysfunction and functional regeneration of bone defects and beyond.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"2. Results","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1. Characterization and antioxidant ability of TPP-DMSN-Fe/Cu nanozyme\u003c/h2\u003e \u003cp\u003eDMSNs were synthesized via a one-step reaction and exhibited uniform spherical morphology with well-defined mesopores, as confirmed by transmission electron microscopy (TEM) (\u003cb\u003eSupplementary Fig.\u0026nbsp;1\u003c/b\u003e). Subsequent functionalization steps involved sequential loading of Fe/Cu and covalent conjugation of mitochondria-targeting TPP through amidation, yielding the final TPP-DMSN-Fe/Cu nanozyme (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea-c). High-angle annular dark-field (HAADF) imaging revealed a dendritic architecture with radially oriented mesopores (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ed), and energy-dispersive X-ray spectroscopy (EDS) elemental mapping confirmed the homogeneous distribution of Cu, Fe, Si, and O within the composite structure (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ee). The physicochemical properties of the nanozyme were systematically examined. Zeta potential measurements demonstrated a shift from negative surface charge toward positive surface charge after TPP conjugation (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ef), with an average surface charge value of 34.7 mV, indicating colloidal stability (absolute values\u0026thinsp;\u0026gt;\u0026thinsp;30 mV)\u003csup\u003e\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e. In addition, the use of Fourier-transform infrared (FTIR) spectroscopy further corroborated the successful synthesis of the nanozyme, with the presence of characteristic Si\u0026ndash;O\u0026ndash;Si stretching vibrations (410, 755, and 1019 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) and a C\u0026thinsp;=\u0026thinsp;O absorption band (1607 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) from L-cysteine-mediated amidation being observed (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eg). Furthermore, nitrogen adsorption-desorption isotherms revealed a high specific surface area (592.8 m\u0026sup2; g-1) and nanoporous structure (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eh) for TPP-DMSN-Fe/Cu nanozyme, which are advantageous for Fe/Cu loading and catalytic activities.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eROS accumulation has been demonstrated to disrupt mitochondrial function and impair tissue regeneration\u003csup\u003e\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u003c/sup\u003e. To evaluate the nanozyme's ROS-scavenging potential, radical elimination assays were performed. It was found that Fe/Cu loading in the nanoparticles significantly increased the rates of scavenging DPPH radical, hydroxyl radical (\u0026bull;OH), and hydrogen peroxide (H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e) in comparison with unmodified DMSN (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ei-k \u003cb\u003eand Supplementary Fig.\u0026nbsp;2, 3\u003c/b\u003e). This robust antioxidant activity positions TPP-DMSN-Fe/Cu as a potent therapeutic agent for mitigating oxidative stress and restoring redox balance in stem cells, thereby supporting mitochondrial health during osteogenesis.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2. In vitro biocompatibility, mitochondria-targeting ability, and ROS scavenging ability of TPP-DMSN-Fe/Cu nanozyme\u003c/h2\u003e \u003cp\u003eWe used C3H/10T1/2 cells, a widely used skeletal stem cell line, in all in vitro experiments. The biocompatibility of TPP-DMSN-Fe/Cu nanozyme was assessed using the cell counting kit 8 (CCK8) assay and Live/Dead staining. Following a 24-hour incubation of the cells with different nanoparticles at varying concentrations, the half-maximum inhibitory concentration (IC50) values of DMSN, DMSN-Fe/Cu, and TPP-DMSN-Fe/Cu nanozymes were determined to be 91.71, 73.69 and 107.01 \u0026micro;g/mL, respectively, by fitting the dose-response curves (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea \u003cb\u003eand Supplementary Fig.\u0026nbsp;4\u003c/b\u003e). Live/Dead staining of C3H/10T1/2 cells (a widely used skeletal stem cell line) further confirmed minimal toxicity of the nanoparticles at concentrations below 50 \u0026micro;g/mL (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eb, c). After confirming the biocompatibility, the intracellular localization of the TPP-DMSN-Fe/Cu nanozymes (10 \u0026micro;g/mL) was examined. Bio-TEM revealed that after 4 hours of incubation, TPP modified nanozymes aggregated in proximity to the mitochondria within C3H/10T1/2 cells, with direct contact with the mitochondrial surface observed (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ed \u003cb\u003eand Supplementary Fig.\u0026nbsp;5\u003c/b\u003e). Fluorescence imaging of Rhodamine B isothiocyanate (RBITC)-labelled nanozyme and MitoTracker-stained mitochondria showed a higher level of colocalization between TPP-modified nanozymes and mitochondria than between non-targeted DMSN-Fe/Cu nanozymes and mitochondria (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ee, f).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eIn order to assess the intracellular ROS scavenging efficacy, quantification was performed using the fluorescent probe 2\u0026prime;,7\u0026prime;-dichlorodihydrofluorescein diacetate (DCFH-DA). In H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e-stimulated stem cells, 10 \u0026micro;g/mL nanoparticles significantly reduced ROS levels, with the ROS scavenging rates of the DMSN, DMSN-Fe/Cu and TPP-DMSN-Fe/Cu groups being 1.47, 2.17 and 2.31 times those of the untreated control group, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eg, h). This activity can protect cells from oxidative damage. Additionally, compared with the control group, the expression level of \u003cem\u003eCat\u003c/em\u003e mRNA increased by 47% (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ei), indicating that TPP-DMSN-Fe/Cu nanozymes significantly up-regulated the antioxidant capacity of stem cells. In summary, TPP-DMSN-Fe/Cu nanozymes exhibit favorable biocompatibility, efficient cellular uptake, precise mitochondrial targeting, and strong ROS scavenging ability, which are crucial for their therapeutic potential in bone regeneration.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3. Metabolic modulation and mitochondrial enhancement by TPP-DMSN-Fe/Cu nanozyme\u003c/h2\u003e \u003cp\u003eStem cells primarily synthesize ATP via glycolysis and oxidative phosphorylation\u003csup\u003e\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e. The latter process relies on an ETC consisting of five enzyme complexes (I\u0026ndash;V), in which complexes I\u0026ndash;IV establish a proton gradient to drive complex V to synthesize ATP\u003csup\u003e\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u003c/sup\u003e. In addition to these pathways, lipid metabolism also contributes to ATP production: fatty acid oxidation (FAO) converts lipids to acetyl-CoA, which fuels the TCA\u003csup\u003e\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u003c/sup\u003e. Moreover, previous studies have demonstrated that osteoblasts can acquire energy from fatty acids, a process imperative for bone tissue formation\u003csup\u003e\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e\u003c/sup\u003e. In addition, it has been determined that the availability of lipids is instrumental in regulating the differentiation of skeletal progenitor cells into either chondrogenic or osteogenic lineages, a process primarily orchestrated by the transcription factor SOX9\u003csup\u003e30\u003c/sup\u003e. Our nanozymes exhibit CcO-like activity, as evidenced by the reduced α-band absorption (550 nm) of ferrous cytochrome c (Cyt \u003cem\u003ec\u003c/em\u003e) in the presence of Fe/Cu-containing nanozymes (DMSN-Fe/Cu and TPP-DMSN-Fe/Cu), indicating oxidation to the iron form (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eb). In contrast, DMSN alone lacks this catalytic ability, confirming that the functions of Fe/Cu-integrated nanozymes as a CcO analog. Furthermore, the DMSN-Fe/Cu nanozymes exhibited dual enzymatic activities, also emulating NOX in catalyzing the conversion of NADH to NAD\u003csup\u003e+\u003c/sup\u003e. This NOX-like activity was confirmed by quantifying NAD\u003csup\u003e+\u003c/sup\u003e levels using a NAD\u003csup\u003e+\u003c/sup\u003e/NADH detection kit (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ec). Notably, the ATP luminescence assay demonstrated that the TPP-DMSN-Fe/Cu nanozyme group exhibited a 69% increase in ATP production compared to the control group (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ed). In addition, the expression of \u003cem\u003eAtp5a\u003c/em\u003e mRNA was also upregulated in the treated stem cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ee).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eDuring bone repair, stem cell proliferation is generally supported by glycolysis, while fatty acids can act as signaling molecules to promote osteogenic differentiation. In addition, studies have shown that fatty acid β-oxidation increases dramatically as osteoblasts mature in vitro, and anabolic Wnt signaling via LRP5 promotes FAO\u003csup\u003e\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e\u003c/sup\u003e. To evaluate potential nanomaterial-induced metabolic shift, mitochondrial respiration and glycolysis were analyzed using a Seahorse Extracellular Flux Analyzer. Initially, real-time oxygen consumption rate (OCR) was recorded after the continuous addition of oligomycin, Carbonyl cyanide-4 (trifluoromethoxy) phenylhydrazone (FCCP), and rotenone as well as antimycin A. It was found that with TPP-DMSN-Fe/Cu nanozyme treatment, the basal respiration, maximum respiration, mitochondrial ATP production, non-mitochondrial oxygen consumption, and spare respiratory capacity were increased by 44%, 89%, 45%, 137%, and 114%, respectively, for the compared with the control group (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ef \u003cb\u003eand Supplementary Fig.\u0026nbsp;6\u003c/b\u003e). These results indicate that these nanozymes could effectively enhance mitochondrial function. Subsequently, we monitored the changes in extracellular acidification rate (ECAR) after sequential addition of glucose, oligomycin, and 2-deoxy-D-glucose (2-DG). Compared with the other groups, the glycolysis, glycolytic capacity, and glycolytic reserve of the TPP-DMSN-Fe/Cu group were significantly reduced (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eg).\u003c/p\u003e \u003cp\u003eTo elucidate metabolic reprogramming, OCR were monitored after sequential inhibition of FAO (via etomoxir), ATP synthase (via oligomycin), and mitochondrial respiration (via rotenone/antimycin A). TPP-DMSN-Fe/Cu-treated cells demonstrated a reduction in both basal and maximal respiration in comparison to the control group (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eh). A critical observation is the decrease in OCR after etomoxir treatment (FAO inhibition) in nanozyme-treated cells, suggesting an augmented reliance on FAO for energy production. These findings suggest that TPP-DMSN-Fe/Cu nanozymes reprogram cellular metabolism by prioritizing FAO. This metabolic rewiring is concomitant with the increased levels of NADPH observed in treated cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ec), a phenomenon that is imperative for the synthesis of the osteogenic matrix and the maintenance of redox homeostasis. Collectively, these results demonstrate that TPP-DMSN-Fe/Cu nanozymes promote osteogenic differentiation of stem cells by augmenting mitochondrial oxidative metabolism through FAO-driven bioenergetics.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4. Transcriptome sequencing analysis of TPP-DMSN-Fe/Cu nanozyme-treated stem cells\u003c/h2\u003e \u003cp\u003eIn order to investigate TPP-DMSN-Fe/Cu-induced changes in metabolic and osteogenic gene expression, RNA sequencing was performed. The transcriptomic analysis revealed the key mechanisms underlying the nanozyme's ability to enhance mitochondrial function and osteogenic differentiation (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ea). In comparison with the control group, the DMSN group, and the DMSN-Fe/Cu-treated group, the TPP-DMSN-Fe/Cu group showed 220, 216, and 157 upregulated genes, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eb). Of note, the expression levels of \u003cem\u003eBmp4\u003c/em\u003e, \u003cem\u003eSox9\u003c/em\u003e, \u003cem\u003eTrpc1\u003c/em\u003e, and \u003cem\u003eSlc25a22\u003c/em\u003e were found to be significantly increased for the TPP-DMSN-Fe/Cu group (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ec). Among these genes, the osteogenic marker \u003cem\u003eBmp4\u003c/em\u003e is crucial for maintaining matrix synthesis and promote the osteogenic differentiation of stem cells\u003csup\u003e\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e\u003c/sup\u003e. Chondrogenesis, the process of cartilage formation, generates cartilage that can serve as the initial skeletal structure and as a template for endochondral ossification\u003csup\u003e\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e\u003c/sup\u003e. As a chondrogenic marker, Sox9 is the primary transcription factor necessary for the differentiation of stem cells into chondrocytes and subsequent cartilage formation\u003csup\u003e\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e\u003c/sup\u003e. Beyond its role in the initiation of chondrogenesis, \u003cem\u003eSox9\u003c/em\u003e has also been shown to regulate stem cell metabolism by modulating FAO\u003csup\u003e\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e. It has been established that \u003cem\u003eTrpc1\u003c/em\u003e, in its capacity as a functional channel for calcium ion influx, is capable of regulating calcium ion influx\u003csup\u003e\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e\u003c/sup\u003e. This, in turn, activates CAMMK/AMPK signaling pathway and promotes mitochondrial biogenesis. The mitochondrial glutamate transporter \u003cem\u003eSlc25a22\u003c/em\u003e is a pivotal transporter that supplies carbon substrates to the TCA cycle via glutathione synthesis\u003csup\u003e\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eGene Ontology (GO) enrichment analysis (\u003cb\u003eSupplementary Fig.\u0026nbsp;7\u003c/b\u003e) of biological processes revealed activation of pathways linked to mitochondrial function and osteogenesis, including CAMKK-AMPK signaling cascade, positive regulation of mitochondrial fission, ATP metabolic process, glutamine metabolic process, and positive regulation of osteoblast proliferation (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ed). These findings consistently support the nanozyme's capacity to enhance mitophagy, a selective autophagy process critical for eliminating dysfunctional mitochondria, which is closely linked to bone regeneration. Furthermore, cellular component analysis revealed an enrichment in ECM categories, including extracellular region, space, matrix, and collagen trimers, suggesting enhanced collagen biosynthesis (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ee). Additionally, molecular function analysis identified enhanced GTP/ATP binding, NAD\u003csup\u003e+\u003c/sup\u003e activity, oxidoreductase activity, and glutathione peroxidase activity for the TPP-DMSN-Fe/Cu group, thereby supporting the nanozyme's role in mitochondrial optimization and osteogenesis (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ef). Furthermore, Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis highlighted enriched metabolic pathways pivotal to bone formation, including alanine/aspartate/glutamate metabolism, HIF-1 signaling, glutathione metabolism, and phagosome activity, for the TPP-DMSN-Fe/Cu group (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eg). Gene Set Enrichment Analysis (GSEA) further corroborated these findings, revealing increased mitochondrial respiratory chain complex IV activity, autophagy regulation, glutamate transport, and ATP synthesis for the TPP-DMSN-Fe/Cu-treated cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eh \u003cb\u003eand Supplementary Fig.\u0026nbsp;8\u003c/b\u003e). Collectively, these results demonstrate that TPP-DMSN-Fe/Cu nanozymes drive osteogenesis by augmenting mitochondrial metabolism and activating bone regeneration-related signaling pathways.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.5. Functional validation of osteogenic differentiation and mitochondrial metabolic activation\u003c/h2\u003e \u003cp\u003eIn addition to evaluating TPP-DMSN-Fe/Cu-enhanced osteogenesis by transcriptome analysis, we further validated the osteogenic properties of the nanozymes by various other methods. These findings were first confirmed by PCR-revealed mRNA expression levels. Compared with the control, the transcriptional levels of runt-related transcription factor 2 \u003cem\u003e(Runx2)\u003c/em\u003e, osteocalcin \u003cem\u003e(Ocn)\u003c/em\u003e, osteopontin \u003cem\u003e(Opn)\u003c/em\u003e, \u003cem\u003eAlkaline phosphatases (Alp), and collagen type I alpha 1 (Col1a1)\u003c/em\u003e were increased by 1.45, 4.44, 2.99, 2.79, and 2.05 folds, respectively, for the TPP-DMSN-Fe/Cu group (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ea \u003cb\u003eand Supplementary Fig.\u0026nbsp;9\u003c/b\u003e). At the protein level, TPP-DMSN-Fe/Cu treatment led to the most prominent upregulation of key osteogenic markers, with COL1A1, RUNX2, ALP, and OCN increased by 147%, 46%, 53%, and 60%, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eb \u003cb\u003eand Supplementary Fig.\u0026nbsp;10\u003c/b\u003e). Immunofluorescence staining further corroborated these findings, demonstrating heightened expression levels of osteogenic markers (OCN, COL1A1, and RUNX2) and augmented mineralization in stem cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ec, d \u003cb\u003eand Supplementary Fig.\u0026nbsp;11, 12\u003c/b\u003e). Alkaline phosphatase (ALP) activity is an early marker of bone formation. ALP activity was found to be significantly increased after 7 days of nanozyme treatment, with the TPP-DMSN-Fe/Cu group demonstrating the highest ALP activity (\u003cb\u003eSupplementary Fig.\u0026nbsp;13\u003c/b\u003e). Consistently, Alizarin Red S (ARS) staining revealed the highest level of mineralization for the TPP-DMSN-Fe/Cu group (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ee, f). These results underscore the capacity of nanozymes to amplify both early- and late-stage osteogenic pathways. It is also worthy of note that genes associated with mitochondrial function was significantly upregulated after TPP-DMSN-Fe/Cu treatment. In comparison with the control group, the expression levels of \u003cem\u003ePgc-1α\u003c/em\u003e, \u003cem\u003eCpt1a\u003c/em\u003e, \u003cem\u003eDnm1l\u003c/em\u003e, \u003cem\u003eSdha\u003c/em\u003e, \u003cem\u003eFam36a\u003c/em\u003e, and \u003cem\u003eRpl13a\u003c/em\u003e increased by 41%, 52%, 63%, 59%, 30%, and 91%, respectively, for the TPP-DMSN-Fe/Cu group (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eg \u003cb\u003eand Supplementary Fig.\u0026nbsp;14\u003c/b\u003e). Among these markers, \u003cem\u003ePgc\u003c/em\u003e-\u003cem\u003e1α\u003c/em\u003e is involved in the coordination of mitochondrial biogenesis and oxidative metabolism\u003csup\u003e\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e\u003c/sup\u003e, and \u003cem\u003eCpt1a\u003c/em\u003e plays a regulatory role in fatty acid uptake for energy production\u003csup\u003e\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e\u003c/sup\u003e. \u003cem\u003eAtp5a\u003c/em\u003e is a subunit of mitochondrial ATP synthase, the enzyme responsible for ATP synthesis/hydrolysis\u003csup\u003e47\u003c/sup\u003e. Moreover, a study has confirmed the role of \u003cem\u003eDnm1l\u003c/em\u003e in regulating mitochondrial fission\u003csup\u003e\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e\u003c/sup\u003e. These genes work in concert to ensure proper mitochondrial metabolism within stem cells, thereby enabling them to adapt to energy demands during osteogenic differentiation.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eKEGG and GO biological process enrichment (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ed, g) suggests that CAMKK-AMPK signal cascade and autophagy are key mediators linking mitochondrial energy regulation and osteoblast differentiation. These findings are consistent with the mechanisms reported in previous studies on stem cell differentiation\u003csup\u003e\u003cspan additionalcitationids=\"CR50\" citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e\u003c/sup\u003e. Furthermore, as the primary energy source for cellular activity, the status and number of mitochondria are critical for the repair of damaged tissue\u003csup\u003e\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e\u003c/sup\u003e. The regulation of mitochondrial biogenesis is a multifaceted process, with PGC-1α serving as the primary regulatory factor\u003csup\u003e\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e\u003c/sup\u003e. Additionally, AMPK has been shown to regulate PGC-1α through a process of phosphorylation, thereby enhancing mitochondrial biogenesis\u003csup\u003e\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e\u003c/sup\u003e. Therefore, we assessed the relative expression of PGC-1α, AMPK, and activated AMPK (p-AMPK) in C3H/10T1/2 cells treated with TPP-DMSN-Fe/Cu. As illustrated in Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eh, i \u003cb\u003eand Supplementary Fig.\u0026nbsp;15\u003c/b\u003e, TPP-DMSN-Fe/Cu significantly increased the protein expression of PGC-1α (2.14-fold) and the p-AMPK/AMPK ratio (2.09-fold) in comparison to the control group. In accordance with this observation, treatment of C3H/10T1/2 cells with TPP-DMSN-Fe/Cu resulted in a 61% and 106% increase in Beclin-1 and LC3-II protein levels, respectively, thereby confirming the activation of autophagy (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eh, i). It is noteworthy that Beclin-1 and its binding partners regulate the activity of the Vps34 lipid kinase, which is crucial for autophagy and other membrane transport processes\u003csup\u003e\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e\u003c/sup\u003e. It has been established through previous studies that the presence of LC3-II in lipid form serves as an indicator of autophagy-related structures\u003csup\u003e\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e\u003c/sup\u003e. Moreover, it has been demonstrated that mitochondrial autophagy is implicated in the regulation of bone metabolism\u003csup\u003e\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e,\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e\u003c/sup\u003e. In addition, Ca\u003csup\u003e2+\u003c/sup\u003e fluorescence probe experiments demonstrated that the green fluorescence intensity in the TPP-DMSN-Fe/Cu group was considerably higher than that in the control group (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ej, k). This finding is indicative of elevated intracellular calcium ion concentrations. Consequently, by scavenging ROS and enhancing ATP production, TPP-DMSN-Fe/Cu nanozymes create an energy-rich microenvironment that promotes osteoblast synthesis and mineralization. This multifunctional approach renders TPP-DMSN-Fe/Cu nanozymes a promising therapeutic candidate for accelerated bone regeneration.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e2.6. TPP-DMSN-Fe/Cu nanozyme accelerated bone regeneration in rat CSBDs\u003c/h2\u003e \u003cp\u003eThe effects of TPP-DMSN-Fe/Cu nanozyme on bone tissue regeneration in vivo were evaluated using a rat tibial defect model (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003ea). The defect sites were implanted with pure gelatin methacryloyl [GelMA, 10% (wt/v)] scaffolds and those modified with 500 \u0026micro;g/mL of DMSN, DMSN-Fe/Cu, and TPP-DMSN-Fe/Cu (denoted GelMA/DMSN scaffolds, GelMA/DMSN-Fe/Cu scaffolds, and GelMA/TPP-DMSN-Fe/Cu scaffolds, respectively). Bone defect specimens were harvested from the tibiae of Sprague Dawley (SD) rats after 4 and 8 weeks of treatment and evaluated using micro-CT. As demonstrated in Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eb, different amounts of newly regenerated bone were observed in the defect areas of all experimental groups following 4 weeks of treatment. After another four weeks, an increased amount of new bone was observed for all groups. Furthermore, we analyzed the bone volume fraction (BV/TV) and bone mineral density (BMD) of the newly regenerated bone tissues. In comparison with the control group, the BV/TV values for the GelMA/TPP-DMSN-Fe/Cu group were significantly higher at both four and eight weeks. At Week 4, the average BV/TV for the GelMA/TPP-DMSN-Fe/Cu group was found to be 2.77, 2.05, 1.50, and 1.49 times higher than that for the control, GelMA, GelMA/DMSN, and GelMA/TPP-DMSN-Fe/Cu groups, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003ec). At Week 8, these fold changes in BV/TV were further increased to 4.10, 1.67, 1.45, and 1.28, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003ed). Additionally, compared to the control, the scaffold groups with/without nanoparticle addition all showed significantly higher BMD values, with the largest increment observed for the GelMA/TPP-DMSN-Fe/Cu group. Furthermore, hematoxylin and eosin (H\u0026amp;E) staining and Masson\u0026rsquo;s trichrome staining were performed for histological analysis of the regenerated bone (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003ee, f). At both 4 and 8 weeks, the area and density of new bone for the GelMA/TPP-DMSN-Fe/Cu group were greater than those in the control group and the GelMA group. The TPP-DMSN-Fe/Cu group consistently demonstrated superior defect repair capabilities to the control group.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eIn order to further investigate the processes of osteoblast differentiation and mitochondrial biogenesis during bone regeneration, immunohistochemical (IHC) staining for OCN and immunofluorescence staining for COL1A1, PGC-1α, and ATP5A were performed. The number of OCN-positive cells was found to be significantly higher in the GelMA/TPP-DMSN-Fe/Cu group than in the other four groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003ea \u003cb\u003eand Supplementary Fig.\u0026nbsp;16\u003c/b\u003e). Similarly, the COL1A1 expression levels were the highest in the GelMA/TPP-DMSN-Fe/Cu group, with the control and GelMA groups showing the lowest levels of COL1A1 among all groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eb \u003cb\u003eand Supplementary Fig.\u0026nbsp;17\u003c/b\u003e). These results indicate that TPP-DMSN-Fe/Cu nanozymes effectively promoted osteogenic differentiation and bone regeneration. Furthermore, compared with other groups, the expression of PGC-1α, a mitochondrial biogenesis marker (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003ec \u003cb\u003eand Supplementary Fig.\u0026nbsp;18\u003c/b\u003e), and ATP5A, an ATP production marker (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003ed \u003cb\u003eand Supplementary Fig.\u0026nbsp;19\u003c/b\u003e), was found to be significantly increased in the GelMA/TPP-DMSN-Fe/Cu group at both 4 and 8 weeks. These findings suggest that the application of TPP-DMSN-Fe/Cu nanozymes resulted in a larger number of healthy mitochondria, thereby providing a greater energy supply for bone repair. Furthermore, the biocompatibility and biosafety of TPP-DMSN-Fe/Cu nanozymes and other nanoparticles were evaluated in vivo. No abnormalities were observed in the heart, liver, spleen, lung, and kidney tissues of rats from any groups, and no obvious difference was observed between the GelMA scaffold only and GelMA/TPP-DMSN-Fe/Cu groups, as revealed in H\u0026amp;E staining images (\u003cb\u003eSupplementary Fig.\u0026nbsp;20, 21\u003c/b\u003e). Moreover, a comprehensive blood analysis revealed comparable conventional blood parameters across all the groups (\u003cb\u003eSupplementary Fig.\u0026nbsp;22\u003c/b\u003e), further supporting the high biosafety of TPP-DMSN-Fe/Cu nanozymes.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"3. Discussion","content":"\u003cp\u003eWe developed a mitochondria-targeted nanozyme platform that mimics the enzymatic activity of cytochrome c oxidase (Complex IV), pivotal components of the ETC. By enhancing FAO in stem cells, the TPP-DMSN-Fe/Cu nanozymes amplify ETC in OXPHOS and modulate the TCA cycle, restoring redox balance and boosting ATP synthesis. The nanozymes have been shown to localize to the mitochondria, where they not only function to augment the ETC, but also scavenge ROS, elevate glutathione to fortify antioxidant defenses, and enhance GTPase activity to regulate mitochondrial dynamics. This process promotes mitochondrial fission and the clearance of damaged mitochondria by autophagy. Concurrently, the nanozyme activates the CaMKK/AMPK/ PGC-1α pathway, thereby stimulating mitochondrial biogenesis and creating a microenvironment conducive to osteogenesis. In a rat model of CSBDs, the nanozyme system led to robust bone regeneration characterized with accelerated defect repair and increased bone mineral density.\u003c/p\u003e \u003cp\u003eIn injured bone microenvironments, a redox imbalance has been demonstrated to drive mitochondrial dysfunction and excessive ROS accumulation, thereby impairing osteogenesis\u003csup\u003e\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e\u003c/sup\u003e. While ROS-scavenging biomaterials have demonstrated potential, most of these materials do not regulate mitochondrial homeostasis in a targeted manner\u003csup\u003e\u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e,\u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e\u003c/sup\u003e. Although emerging mitochondria-targeted polyphenol/amino acid NPs (e.g., ECGG-Cys-NPs, PGA-Mn-TP04)\u003csup\u003e\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e,\u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e61\u003c/span\u003e\u003c/sup\u003e enhanced mitochondrial biogenesis and reducing ROS, they lack capacity to drive the metabolic reprogramming required for osteogenesis. The current study has significantly advanced mitochondria-targeted nanotherapeutics through a unique coupling of FAO-driven osteogenic differentiation with autophagy-driven mitochondrial dynamic mechanisms. For instance, we have identified key genes, including \u003cem\u003ePgc-1α\u003c/em\u003e (amplifying mitochondrial biogenesis)\u003csup\u003e\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e\u003c/sup\u003e, \u003cem\u003eSlc25a22\u003c/em\u003e (supplying TCA intermediates and glutathione precursors)\u003csup\u003e\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e\u003c/sup\u003e, and \u003cem\u003eCpt1a\u003c/em\u003e (enabling fatty acid transport for β-oxidation)\u003csup\u003e\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e\u003c/sup\u003e, that play critical roles in stem cell osteogenesis. By simultaneously supporting metabolic reprogramming, redox balance, and mitochondria homeostasis through a multifunctional nanozyme platform, our approach transcends conventional strategies that target singular pathways, establishing a versatile and effective framework for bone regeneration.\u003c/p\u003e \u003cp\u003eMost conventional nanozymes were designed to modulate glycolysis, thereby regulating lactic acid production for tumor therapy\u003csup\u003e\u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e62\u003c/span\u003e,\u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e63\u003c/span\u003e\u003c/sup\u003eOur mitochondria-targeted TPP-DMSN-Fe/Cu nanozyme acts by a distinct mechanism. It directly enhances FAO-driven OXPHOS during stem cell osteogenesis, while concurrently inhibiting glycolysis. This distinctive metabolic reprogramming is attributable to its subcellular localization and the specific characteristics of the cellular environment. Mitochondrial localization enables TPP-DMSN-Fe/Cu to directly enhance FAO-driven OXPHOS, effectively generating acetyl-CoA and reducing reliance on glycolysis. Moreover, previous studies have predominantly focused on cancer cells or proliferating cells that rely on glycolytic anabolic metabolism for growth\u003csup\u003e\u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e64\u003c/span\u003e\u003c/sup\u003e. The process of stem cell differentiation, particularly osteogenic differentiation, has been shown to be enhanced by the inhibition of glycolysis\u003csup\u003e\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e. Reduced glycolysis has been demonstrated to impede the accumulation of lactic acid and subsequent microenvironment acidification, and this microenvironmental change inhibits bone regeneration\u003csup\u003e\u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e65\u003c/span\u003e\u003c/sup\u003e. The nanozyme-enabled metabolic reprograming facilitates the redirection of energy toward collagen synthesis and mineral deposition through sustained, non-acidic OXPHOS. Furthermore, our nanozymes were capable of clearing ROS, thereby protecting the FAO enzymes and enhancing autophagy, a process that involves the removal of damaged mitochondria. This, in turn, favors an oxidative metabolic state that facilitates osteogenic differentiation.\u003c/p\u003e \u003cp\u003eWhile the TPP-DMSN-Fe/Cu nanozymes hold promising potential in bone regeneration, several questions remain to be answered before the clinical translation of this technology. First, large-animal validation of long-term biocompatibility, biodistribution, and regenerative outcomes should be rigorously assessed. Second, the mechanisms underlying the body\u0026rsquo;s immune responses, to the nanozymes, such as the effects of nanozymes on macrophage polarization, remain to be clarified. Third, it is worth exploring the effects of nanozymes on stem cell subpopulations at defect sites.\u003c/p\u003e \u003cp\u003eTo quantitatively map the biodistribution of nanozymes in vivo, particularly in large animal models, isotope labeling-based tracking technology can be used in future research\u003csup\u003e\u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e66\u003c/span\u003e\u003c/sup\u003eThis approach will facilitate the assessment of the long-term accumulation/excretion of nanozymes within organs. To understand the interactions between immune cells and the nanozymes, organ-on-a-chip technology can play an instrumental role in revealing the nanotechnology-biology interface by, for example, the establishment of a bone defect-on-a-chip system\u003csup\u003e\u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e67\u003c/span\u003e\u003c/sup\u003e. These systems, which may include bone cells, macrophages, and blood vessels, can be employed for real-time monitoring of the effects of nanozymes on cellular functions. Finally, stem cell lineage tracing technique can facilitate the elucidation of potentially diverse behaviors of different stem cell subpopulations\u003csup\u003e\u003cspan citationid=\"CR68\" class=\"CitationRef\"\u003e68\u003c/span\u003e\u003c/sup\u003e. In addition, emerging evidence indicates that redox dysfunction in mature osteoblasts plays a pivotal role in the development of osteoporosis. For instance, estrogen deficiency-induced downregulation of GPX4 leads to phospholipid peroxidation in osteoblasts, thereby impairing bone formation\u003csup\u003e\u003cspan citationid=\"CR69\" class=\"CitationRef\"\u003e69\u003c/span\u003e\u003c/sup\u003e. These findings indicate that our TPP-DMSN-Fe/Cu nanozymes could also be used for effective osteoporosis treatment, which necessitates both the enhancement of metabolic efficiency in stem cells and the protection of mature osteoblasts from oxidative damage.\u003c/p\u003e"},{"header":"4. Conclusion","content":"\u003cp\u003eCSBDs represent a formidable clinical challenge, requiring innovative solutions to restore structural and functional integrity. In this study, a mitochondrial-targeted single-atom nanozyme platform (TPP-DMSN-Fe/Cu) was developed. This platform was designed to address the dual impediments of oxidative stress and mitochondrial dysfunction in stem cells. By scavenging ROS and enhancing mitochondrial energy metabolism, the nanozyme establishes a regenerative microenvironment that promotes osteogenesis. Mechanistic studies have revealed its capacity to augment mitochondrial biogenesis, eliminate damaged mitochondria via autophagy, and drive osteogenic differentiation. This highlights mitochondrial health as a crucial factor in bone repair. In vivo validation demonstrates accelerated regeneration, marked by significant increases in bone volume and mineral density.\u003c/p\u003e"},{"header":"5. Methods","content":"\u003cp\u003e The rat experimental protocols conducted in this study were approved by the Animal Experimentation Ethics Committee of the Chinese University of Hong Kong (Approval No. (24\u0026ndash;427) in DH/HT\u0026amp;A/8/2/1 Pt.63).\u003c/p\u003e \u003cp\u003eChemicals\u003c/p\u003e \u003cp\u003eTriethanolamine (TEA, purity\u0026thinsp;\u0026ge;\u0026thinsp;98%), Hexadecyltrimethylammonium bromide (CTAB, purity\u0026thinsp;\u0026ge;\u0026thinsp;99%), Sodium salicylate (NaSal, purity\u0026thinsp;\u0026ge;\u0026thinsp;99%), Methanol (CH\u003csub\u003e3\u003c/sub\u003eOH, purity\u0026thinsp;\u0026ge;\u0026thinsp;99%), (5-carboxypentyl) (triphenyl)phosphonium bromide (TPP, purity\u0026thinsp;\u0026ge;\u0026thinsp;97% ), N-Hydroxysuccinimide (NHS, purity\u0026thinsp;\u0026ge;\u0026thinsp;97%), N-(3-Dimethylaminopropyl)-N\u0026prime;-ethylcarbodiimide hydrochloride (EDC, purity\u0026thinsp;\u0026ge;\u0026thinsp;99%) were purchased from Sigma-Aldrich. Hydrochloric acid (HCl, 37%) was purchased from Duksan. Tetraethyl orthosilicate (TEOS, purity\u0026thinsp;\u0026ge;\u0026thinsp;98%), 1,2-bis(triethoxysilyl) ethane (BTEE, purity\u0026thinsp;\u0026ge;\u0026thinsp;95%), (3-Aminopropyl) triethoxysilane (APTES, purity\u0026thinsp;\u0026ge;\u0026thinsp;99%), Iron sulfate heptahydrate (Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e-7H2O, purity\u0026thinsp;\u0026ge;\u0026thinsp;99%), Copper nitrate trihydrate ((Cu (NO\u003csub\u003e3\u003c/sub\u003e)\u003csub\u003e2\u003c/sub\u003e-3H\u003csub\u003e2\u003c/sub\u003eO, purity\u0026thinsp;\u0026ge;\u0026thinsp;99%). All aqueous solutions were prepared with deionized water (18.2 MΩ; Millipore).\u003c/p\u003e \u003cp\u003eSynthesis of TPP-Fe/Cu-DMSN\u003c/p\u003e \u003cp\u003eStep 1: Dendritic mesoporous silicon (DMSN) Synthesis.\u003c/p\u003e \u003cp\u003eThe DMSN was synthesized in a procedure that incorporated cationic surfactants CTAB and Nasal. The silicon sources utilized were TEOS and BTEE, and the catalyst employed was TEA. Initially, 0.136 g of TEA was added to 50 ml of water and stirred gently at 80\u0026deg;C for 0.5 h. Subsequently, 760 mg of CTAB and 336 mg of NaSal were added to the solution and stirred at 80\u0026deg;C for an additional hour. The mixture of 8 ml TEOS and 3.2 ml BTEE was then subjected to gentle stirring for 6 hours at 80\u0026deg;C at 300 rpm. The resulting mixture was subjected to centrifugation at 10,000 rpm for 10 minutes and washed once or twice with pure ethanol. Finally, the mixture was subjected to a reflux process with HCL (3ml): methanol (60ml) solution for a duration of 2 hours at a temperature of 80\u0026deg;C. Subsequently, the mixture was subjected to a centrifugation step and was then washed with ethanol.\u003c/p\u003e \u003cp\u003eStep 2: DMSN- NH\u003csub\u003e2\u003c/sub\u003e Synthesis\u003c/p\u003e \u003cp\u003eIn this step of the procedure, 280 mg of DMSN were dissolved in 80 ml of ethanol and sonicated. Subsequently, 7 ml of APTES were added, and the solution was refluxed for 6 hours at 70\u0026deg;C. Thereafter, the solution was subjected to centrifugation as previously described.\u003c/p\u003e \u003cp\u003eStep 3: DMSN-Fe/Cu Synthesis\u003c/p\u003e \u003cp\u003eA solution of 80 mg of DMSN-NH\u003csub\u003e2\u003c/sub\u003e in 20 ml of MES buffer at a pH of 6 was prepared initially. This was followed by the activation of 48 mg of EDC and 12.8 mg of NHS for a duration of 30 minutes. Subsequently, 320 mg of L-cysteine was incorporated, and the mixture was maintained at a temperature of 37\u0026deg;C for a period of 6 hours. The resulting nanoparticles were subjected to a centrifuge process, followed by resuspension in 9 ml of DI water. Subsequently, 1 ml of DI water containing Cu (NO\u003csub\u003e3\u003c/sub\u003e)\u003csub\u003e2\u003c/sub\u003e-3H\u003csub\u003e2\u003c/sub\u003eO (41 mg) and Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e-7H\u003csub\u003e2\u003c/sub\u003eO (100 mg) was added.\u003c/p\u003e \u003cp\u003eStep 4: TPP-Fe/Cu-DMSN\u003c/p\u003e \u003cp\u003eA mixture of 20 mg of DMSN/L-Cys/Fe/Cu and 24.552 mg of NHS\u0026thinsp;+\u0026thinsp;49.288 mg of EDC was dispersed in 10 ml of MES buffer at pH 6.00 and stirred for 2 hours at room temperature. Subsequently, 80 mg of TPP was dispersed in 8 ml of DI water and incubated at 37\u0026deg;C for 6 hours. The final product was subjected to centrifugation, as previously described.\u003c/p\u003e \u003cp\u003eCell culture\u003c/p\u003e \u003cp\u003eC3H/10T1/2, Clone 8 cells were procured from Oricell (Guangzhou, China). Low-glucose Dulbecco's modified Eagle's medium (DMEM; Gibco) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin was used as cell culture medium. For osteogenic differentiation, cells were transitioned to high-glucose DMEM containing 100 nM dexamethasone, 10 mM β-glycerophosphate, and 50 \u0026micro;g/mL L-ascorbic acid (Sigma Aldrich). The differentiation medium was refreshed every 48\u0026ndash;72 hours to ensure consistent nutrient availability. All culture reagents, unless specified otherwise, were sourced from Gibco.\u003c/p\u003e \u003cp\u003eCellular uptake\u003c/p\u003e \u003cp\u003eNanoparticles were internalized by stem cells were observed using TEM (Hitachi H-7650; Hitachi, Tokyo, Japan). Cells were cultured in growth medium at an initial density of 5.0 \u0026times; 10\u003csup\u003e5\u003c/sup\u003e cells/well in 6-well plates. When the cells reached 80% confluence, nanozymes were added. After 4 hours of incubation, the cells were fixed, dehydrated, embedded, cut, and then observed by TEM. For mitochondrial-targeted validation, stem cells were incubated in osteogenic differentiation DMEM medium containing nanoparticles (10 \u0026micro;g/mL) for 4 h. Cellular uptake of nanoparticles was observed by confocal microscopy (Leica SP8). Cells and nanoparticles were stained with Mito-tracker green and Rhodamine B isothiocyanate (RBITC), respectively.\u003c/p\u003e \u003cp\u003eReal-time RT-PCR\u003c/p\u003e \u003cp\u003eThe stem cells were seeded into 6-well plates and allowed to proliferate to 70% confluence. Thereafter, the medium was substituted with osteogenic differentiation medium, and nanoparticles were introduced. After a 7 days incubation, total RNA was extracted using the RNA Extraction Kit (ZYMO). To obtain cDNA, total RNA was reverse transcribed using the cDNA Amplification Kit (YEASEN). The primer sequences employed in this study are listed in \u003cb\u003eSupplementary Table\u0026nbsp;1\u003c/b\u003e, obtained from the Primer Library (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.ncbi.nlm.nih.gov/gene/\u003c/span\u003e\u003cspan address=\"https://www.ncbi.nlm.nih.gov/gene/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) and synthesized by BGI Genomics. The transcript levels of the target genes were calculated using the ΔΔCt method, with GAPDH serving as the housekeeping gene. Cells not treated with nanoparticles were used as the control group.\u003c/p\u003e \u003cp\u003eWestern blotting\u003c/p\u003e \u003cp\u003eCells were cultured and processed in the same way as for qRT-PCR assay. On day 7, proteins were extracted using RIPA lysis buffer (Thermo Fisher) and protease and phosphatase inhibitor cocktail, MSF. After BCA (Thermo Fisher) quantification, proteins were loaded into sodium dodecyl sulphate polyacrylamide gels and transferred to Trans-Blot Turbo Midi 0.2 \u0026micro;m PVDF Transfer Packs (Bio-Rad). All antibodies used in this study are listed in \u003cb\u003eSupplementary Table\u0026nbsp;2\u003c/b\u003e. Band intensity was quantified using ImageJ software (National Institutes of Health, USA).\u003c/p\u003e \u003cp\u003eCalcium\u003csup\u003e2+\u003c/sup\u003e influx\u003c/p\u003e \u003cp\u003eIntracellular calcium ion influx imaging was performed using a Leica Thunder imager to record the intensity of the intracellular calcium dye (Fluo-4 AM, Beyotime). The calcium imaging data obtained were analyzed and visualized using ImageJ.\u003c/p\u003e \u003cp\u003eSeahorse\u003c/p\u003e \u003cp\u003eSeahorse XFe96 analyzed oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) to assess OXPHOS, glycolysis, and lipid metabolism. stem cells (1 \u0026times; 10\u003csup\u003e4\u003c/sup\u003e cell/mL) were seeded onto a Seahorse XF-96 plate. For Cell Mito Stress Test, the final concentration of Oligomycin, FCCP, and Rotenone/antimycin is 2 \u0026micro;M, 1 \u0026micro;M, and 0.5 \u0026micro;M. For Glycolysis Stress Test, Glucose, Oligomycin, and 2-Deoxy-D-glucose is 10 mM, 1 \u0026micro;M, and 50 mM. For Substrate Oxidation Stress Test, the final concentration of etomoxir, Oligomycin, FCCP, and Rotenone/antimycin is 4 \u0026micro;M, 1.5 \u0026micro;M, 1.5 \u0026micro;M, and 0.5 \u0026micro;M.\u003c/p\u003e \u003cp\u003eTherapeutic efficacy of scaffolds on critical-size bone defect rats model\u003c/p\u003e \u003cp\u003eAdult SD rats were procured from Guangzhou Seyotin Laboratory Animal Co. The 50 rats (8\u0026ndash;12 weeks, male, 200\u0026ndash;220 g) were randomly divided into five groups: empty defect (control), GelMA scaffolds, DMSN\u0026thinsp;+\u0026thinsp;GelMA scaffolds, DMSN-Fe/Cu\u0026thinsp;+\u0026thinsp;SF/GelMA scaffolds, and TPP-DMSN-Fe/Cu\u0026thinsp;+\u0026thinsp;GelMA scaffolds. After the rats were anesthetized with intraperitoneal pentobarbital (35 mg/kg), the tibia was exposed via a surgical incision, and a 3-mm diameter and 3-mm depth bone defect was created using an electric drill. Scaffolds were then implanted into the defect. Following a 4- or 8-week treatment period, half of the rats were euthanized, and their tibiae were collected and evaluated using gross observation, micro-CT scanning, and histological staining.\u003c/p\u003e \u003cp\u003eHistology, Immunohistochemistry, and Immunofluorescence Staining\u003c/p\u003e \u003cp\u003eTissue samples were decalcified in 10% EDTA-2Na (Solarbio) at 4\u0026deg;C for 50 days, paraffin-embedded, and sectioned (5 \u0026micro;m thickness). Sections underwent hematoxylin and eosin (H\u0026amp;E), Masson\u0026rsquo;s trichrome using commercial kits. All antibodies used in this study are listed in \u003cb\u003eSupplementary Table\u0026nbsp;3\u003c/b\u003e. Images intensity was semi-quantitatively analyzed using ImageJ software (NIH, USA).\u003c/p\u003e \u003cp\u003eMicro-CT Analysis\u003c/p\u003e \u003cp\u003eMandibular defects were analyzed at 4- and 8-weeks post-treatment using a Quantum GX2 micro-CT system at 50 kV and 100 \u0026micro;A (30 \u0026micro;m resolution). A cylindrical volume of interest (VOI; 3 mm diameter, 0.27 mm depth) centered on the defect site was evaluated. Bone volume fraction (BV/TV) and bone mineral density (BMD) were calculated to assess regenerated bone microstructure.\u003c/p\u003e \u003cp\u003eStatistical analysis\u003c/p\u003e \u003cp\u003eThe standard deviation (SD) of the data in each group is expressed as mean \u0026plusmn;. Comparisons between two groups were made using Student's t-test, and comparisons between groups were made using one-way ANOVA followed by Tukey's post hoc test. All statistical analyses were performed with GraphPad Prism 8. The level of statistical significance was set at P\u0026thinsp;\u0026lt;\u0026thinsp;0.05. At least three independent replications were performed for each experiment.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e \u003ch2\u003eDeclaration of Competing Interest\u003c/h2\u003e \u003cp\u003eThe authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.\u003c/p\u003e \u003c/p\u003e\u003cp\u003e \u003ch2\u003eSupplementary Information\u003c/h2\u003e \u003cp\u003eSupplementary Information 1\u003c/p\u003e \u003c/p\u003e\u003ch2\u003eAcknowledgements\u003c/h2\u003e \u003cp\u003eThis work was supported by (1) Shun Hing Institute of Advanced Engineering, CUHK (to ZAL, project #BME-p2-24); (2) Center for Neuromusculoskeletal Restorative Medicine (to RST and ZAL), under the Health@InnoHK program, Innovation and Technology Commission (ITC), Hong Kong SAR, China; (3) National Natural Science Foundation of China (to ZAL, 82302753); and (4) Hong Kong Research Grants Council (to ZAL, 24203523). ZAL acknowledges the support from the Vice-Chancellor Early Career Professorship Scheme of the Chinese University of Hong Kong (CUHK). RST is supported by the Lee Quo Wei and Lee Yick Hoi Lun Professorship in Tissue Engineering and Regenerative Medicine of CUHK. YW is supported by CUHK postgraduate studentship. We thank Ms. Josie Lai from the School of Biomedical Sciences at CUHK for her assistance with EM imaging.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eLutolf MP et al (2003) Repair of bone defects using synthetic mimetics of collagenous extracellular matrices. Nat Biotechnol 21:513\u0026ndash;518\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKengelbach-Weigand A et al (2021) Personalized medicine for reconstruction of critical-size bone defects \u0026ndash; a translational approach with customizable vascularized bone tissue. 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Nat Commun 16:758\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Nanoparticle, Stem cell, Osteogenic differentiation, Bone regeneration, Mitochondrial biogenesis","lastPublishedDoi":"10.21203/rs.3.rs-7012382/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7012382/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eCritical-sized bone defects (CSBDs) do not heal spontaneously throughout a patient\u0026rsquo;s lifetime, posing a global challenge to musculoskeletal health. Resident stem cells in bone, which are indispensable in skeletal development and regeneration, undergo enhanced mitochondrial activities during osteogenic differentiation. However, accumulation of excessive reactive oxygen species (ROS) produced by injured bone tissues can lead to mitochondrial damage, which negatively affects the osteogenic differentiation of stem cells. In such an environment, it is crucial to target mitochondria in stem cells to remove ROS and restore mitochondrial homeostasis. Herein, we developed a dendritic mesoporous silica nanoparticle (DMSN)-based single-atom nanozyme, named TPP-DMSN-Fe/Cu, loaded with Fe and Cu single atoms and modified with mitochondrion-targeting triphenylphosphonium (TPP). \u003cem\u003eIn vitro\u003c/em\u003e, TPP-DMSN-Fe/Cu nanozymes were found to upregulate stem cell osteogenesis by scavenging ROS, enhancing mitochondrial function by fatty acid oxidation, and promoting autophagy of abnormal mitochondria. The nanozymes also augmented mitochondrial biogenesis via the CaMKK/AMPK/PGC-1α pathway. \u003cem\u003eIn vivo\u003c/em\u003e, TPP-DMSN-Fe/Cu nanozymes significantly enhanced mitochondrial biogenesis and bone regeneration, leading to increased bone volume and mineral density at the sites of CSBDs in rats. Taken together, these findings show that the multifunctional, mitochondria-targeting TPP-DMSN-Fe/Cu nanozymes hold promising potential in accelerating bone regeneration via regulation of cellular energy metabolism.\u003c/p\u003e","manuscriptTitle":"Mitochondria-targeted, Single-atom Nanozymes Accelerate Bone Regeneration by Augmenting Stem Cell Energy Metabolism","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-07-25 18:28:04","doi":"10.21203/rs.3.rs-7012382/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"79b41ebe-5d66-4f57-adcb-114d88f7f9ef","owner":[],"postedDate":"July 25th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":51591382,"name":"Biological sciences/Biotechnology/Tissue engineering"},{"id":51591383,"name":"Biological sciences/Biotechnology/Biomaterials/Bioinspired materials"},{"id":51591384,"name":"Biological sciences/Biotechnology/Nanobiotechnology/Nanoparticles"},{"id":51591385,"name":"Health sciences/Anatomy/Musculoskeletal system/Bone"}],"tags":[],"updatedAt":"2025-08-27T12:05:21+00:00","versionOfRecord":[],"versionCreatedAt":"2025-07-25 18:28:04","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7012382","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7012382","identity":"rs-7012382","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}