Recent Advances in Nanomaterials for Modulation of Stem Cell Differentiation and Its Therapeutic Applications

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Recent Advances in Nanomaterials for Modulation of Stem Cell Differentiation and Its Therapeutic Applications

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School of Integrative Engineering, Chung-Ang University, 84 Heukseuk-ro, Dongjak-gu, Seoul 06974, Republic of Korea

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Author to whom correspondence should be addressed.

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These authors contributed equally to this work.

Biosensors 2024, 14(8), 407; https://doi.org/10.3390/bios14080407

Submission received: 19 July 2024 / Revised: 14 August 2024 / Accepted: 20 August 2024 / Published: 22 August 2024

(This article belongs to the Special Issue Functional Materials for Biosensing Applications)

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Abstract

Challenges in directed differentiation and survival limit the clinical use of stem cells despite their promising therapeutic potential in regenerative medicine. Nanotechnology has emerged as a powerful tool to address these challenges and enable precise control over stem cell fate. In particular, nanomaterials can mimic an extracellular matrix and provide specific cues to guide stem cell differentiation and proliferation in the field of nanotechnology. For instance, recent studies have demonstrated that nanostructured surfaces and scaffolds can enhance stem cell lineage commitment modulated by intracellular regulation and external stimulation, such as reactive oxygen species (ROS) scavenging, autophagy, or electrical stimulation. Furthermore, nanoframework-based and upconversion nanoparticles can be used to deliver bioactive molecules, growth factors, and genetic materials to facilitate stem cell differentiation and tissue regeneration. The increasing use of nanostructures in stem cell research has led to the development of new therapeutic approaches. Therefore, this review provides an overview of recent advances in nanomaterials for modulating stem cell differentiation, including metal-, carbon-, and peptide-based strategies. In addition, we highlight the potential of these nano-enabled technologies for clinical applications of stem cell therapy by focusing on improving the differentiation efficiency and therapeutics. We believe that this review will inspire researchers to intensify their efforts and deepen their understanding, thereby accelerating the development of stem cell differentiation modulation, therapeutic applications in the pharmaceutical industry, and stem cell therapeutics.

Keywords:

nanomaterials; intracellular regulation; cellular adhesion; external stimulation; stem cell differentiation

1. Introduction

Stem cells are a versatile and promising class of cells that can self-renew and differentiate into various specialized cell types, which makes them a valuable tool for regenerative medicine and tissue engineering [1,2,3,4,5,6]. For example, stem cell therapy offers a new paradigm for individuals with untreatable conditions, shifting the focus of treatment from solely managing the disease to modulating immunopharmacological intervention and regeneration [7,8,9,10,11]. In recent years, research on stem cells has produced increasing evidence suggesting that stem cell transplantation is a highly effective approach for treating neurological disorders, bone injuries, and various diseases [12,13,14,15,16,17,18]. However, stem cell transplantation for clinical use has limited effectiveness in producing mature specialized cells to replace damaged cells [19,20,21]. In contrast, ex vivo differentiation of stem cells is known to have low efficiency and poor survival when transplanted into the body [22,23]. Moreover, the ability to differentiate stem cells into specific cell types of interest (e.g., bones, cartilage, and muscles) in a highly selective and efficient manner remains a significant challenge [24,25,26,27,28]. To fully realize the therapeutic potential of stem cells in the field of regenerative medicine, precise control of the fate of stem cells should be addressed [29,30,31,32,33,34,35,36,37]. The surrounding matrix can significantly influence the development and specialization of stem cells. Moreover, altering factors such as the size, hydrophilicity, roughness, and organization of the cell attachment surface can directly affect the cellular activity.

The field of nanotechnology has made significant advancements in influencing the stem cell behavior through the application of various types of nanomaterials, including metal- and carbon-based ones and nanoframeworks [38,39,40,41,42,43,44,45]. Metallic nanomaterials, such as gold nanoparticles (AuNPs), silver nanoparticles (AgNPs), and other metal-based nanoparticles, have recently gained significant attention owing to their wide range of applications, including reactive oxygen species (ROS) scavenging, autophagy, and thermoplasmonic regulation [46,47,48,49,50,51,52]. Carbon-based nanomaterials encompass fullerenes, carbon nanotubes (CNTs), graphene and its derivatives, graphene oxide (GO), nanodiamonds (NDs), and carbon-based quantum dots (CQDs) [53,54,55,56,57,58]. These materials have attracted significant interest because of their distinct structural dimensions and remarkable properties in biomedical fields, including cancer therapy and wearable device (reviewed elsewhere [59,60]). Finally, we focused the practicality of different nanomaterials in regulating biomolecule delivery and facilitating stem cell specialization, focusing on metal–organic frameworks (MOFs), zeolite imidazolate frameworks (ZIFs), and upconversion nanoparticles (UCNPs) in stem cell therapies [61,62]. In particular, nanomaterials with biodegradable and biocompatible properties can be engineered to mimic the natural extracellular matrix and provide specific chemical and physical cues to guide stem cell differentiation and proliferation [63,64].

Recent studies have investigated whether nanostructured surfaces and scaffolds can enhance the proliferation, migration, and differentiation of stem cells into specific lineages, such as osteogenic, adipogenic, or neurogenic differentiation [65,66,67]. For instance, the surface topography, stiffness, and chemical composition of nanomaterials have been shown to significantly impact stem cell differentiation [68]. Moreover, nanomaterials can serve as delivery vehicles (e.g., metal–organic framework; self-assembled, peptide-based nanodrugs) for various bioactive molecules, growth factors, and genetic materials, which further enhances stem cell differentiation and tissue regeneration [69,70,71]. This allows for the precise control and guidance of stem cell differentiation, which is crucial for the development of stem-cell-based therapies. The increased use of nanostructures in stem cell research has led to the development of several new technologies, highlighting a substantial demand for innovative therapeutic approaches.

This review provides an overview of recent advances in nanomaterials across metal-, carbon-, and peptide-based approaches, focusing on their applications in enhancing stem cell differentiation and therapeutic strategies (Figure 1). It also focuses on strategies that commonly integrate nanostructures to enhance differentiation and healing efficiency, along with descriptions of common nanomaterial fabrication approaches used in stem cell research. Finally, we conclude with a future perspective highlighting clinical applications of stem cell therapy and advancement in point-of-care treatments.

Figure 1. Schematic illustrations of various nanomaterials to modulate stem cell functions and mechanisms.

2. Metal-Based Stem Cell Differentiation Approaches and Therapeutics

Metallic nanoparticles can guide stem cell fate and influence their proliferation, migration, and differentiation. Nanomaterials such as gold nanoparticles (AuNPs), silver nanoparticles (AgNPs), and other metal-based nanoparticles have recently attracted considerable attention for potent and broad applications such as medical carriers for in regenerative medicine. These nanoparticles are used to direct stem cell differentiation toward desired lineages, thereby enhancing the therapeutic potential of these cells. The applications of metal-based nanomaterials in stem cell research extend beyond directing differentiation; they also show promise in stem cell tracking and imaging [46,72,73]. Moreover, the integration of metal-based nanomaterials with advanced biomaterials, such as hydrogels and diverse scaffolds, has further expanded the therapeutic potential of these systems [74,75]. Here, we describe the use of various metal-based nanomaterials in controlling stem cell fate and biomedical applications. (Table 1).

Table 1. Metal-based stem cell differentiation approaches and therapeutics.

2.1. Autophagy

Numerous studies have demonstrated that the autophagy process is crucial for preserving cellular homeostasis and enabling differentiation under adverse conditions [85,86,87]. Additionally, impairment of cellular autophagy can lead to metabolic disorders, such as accumulation of damaged proteins and/or organelles and inability to clear protein aggregates, leading to compromised stemness and regenerative capacity of stem cells [88,89]. Recent studies have shown that specific types of nanomaterials can be internalized in cells and can accumulate in cellular compartments, including endosomes, lysosomes, and autophagosomes, which activates autophagy through their biological effects [90]. Accordingly, for therapeutic applications, nanoparticles can be used to target the autophagy–lysosome system for stem cell rejuvenation. AuNPs have emerged as the most preferred type of nanoparticle for use in biological and pharmaceutical applications because of their unique surface plasmon resonance and optical properties, as along with their easily modifiable size, shape, functionalization, biocompatibility, and regenerative ability [91,92,93].

Yin et al. explored the potential of AuNPs to mitigate inflammation-compromised osteogenic differentiation in the periodontal ligament stem cells (PDLSCs) [94]. They evaluated the influence of AuNPs with different particle sizes on the viability and osteogenic differentiation of the PDLSCs and their inflammatory conditions (I-PDLSC). In terms of autophagy, the AuNP treatment did not change the expression of LC3 II, an indicator of the autophagic level, in I-PDLSCs during the early stage of osteogenic differentiation. This was indicated by the lack of significant difference in the LC3 II levels between the AuNPs and I-PDLSCs group. Moreover, the AuNP treatment did not change the expression of LC3 II in the I-PDLSCs during the early stage of osteogenic induction, as indicated by the lack of a significant difference in the LC3 II levels between the AuNPs and I-PDLSCs group. In contrast, the AuNP treatment increased the autophagic flux in the I-PDLSCs, as indicated by the significantly increased accumulation of LC3 II observed in the AuNP-treated I-PDLSCs compared to the I-PDLSCs group at 12 or 24 h during the early stage of osteogenic differentiation. Furthermore, quantification of cellular autophagosomes revealed an elevated proportion of RFP⁺-GFP⁺-LC3 puncta in the AuNP-treated I-PDLSCs following treatment with Bafilomycin A1 (Baf), an autophagy inhibitor, treated at 12 and 24 h during osteogenic differentiation. This is consistent with the enhanced autophagic flux observed in these cells (Figure 2A). However, the Baf treatment only enhanced the proportion of autophagosomes (RFP⁺-GFP⁺-LC3 puncta) in the I-PDLSCs during osteogenic differentiation. Moreover, the AuNP incubation increased the number of accumulated FITC-labeled LC3 puncta in the I-PDLSCs compared to that in the control I-PDLSCs group (Figure 2B). The activation of transcription factor EB (TFEB), a master regulator of the autophagy–lysosome system, and the expression of autophagy- or lysosome-related genes in I-PDLSCs were also examined after the AuNP treatment. The nuclear localization of the TFEB was enhanced in both the I-PDLSCs and AuNP-treated I-PDLSCs after osteogenic induction. However, the AuNP-treated I-PDLSCs exhibited greater nuclear TFEB levels during the osteogenic differentiation than the control I-PDLSCs. Specifically, the knockdown of the TFEB in the AuNP-treated I-PDLSCs inhibited the AuNP-induced restoration of mineralized nodule formation (Figure 2C) and osteogenesis-related protein expression (Figure 2D).

In organ transplantation, often the final therapeutic option in severe diseases, survival is often limited by immunogenic rejection and/or bacterial infection [95]. Utilizing gallium (Ga) coatings on biomedical devices can leverage their antibacterial properties, affecting various bacteria [96]. Chen et al. developed a series of Mg-Ga-layered double hydroxide (LDH) nanosheets on alkaline-treated titanium surfaces, which are composed of positively charged brucite-like layers [97]. They fabricated Mg/Ga LDH sheets on the surface of alkali-heat-treated titanium (AT) implants, which were subsequently calcinated to convert them into Mg/Ga-layered double-oxide nanosheets with enhanced alkalinity and stability. Their aim was to develop bone repair biomaterials and investigate the relationship between autophagy and the pH of the local microenvironment. Therefore, enhancing the alkalinity of implant biomaterial surfaces may prove to be an effective strategy to enhance autophagy and favor osteogenesis under osteoporotic conditions.

Through biological investigations, researchers discovered that the coating could promote autophagy by increasing the alkalinity of the surrounding environment, thereby facilitating the osteogenic differentiation of mesenchymal stem cells (MSCs) and inhibiting the bone resorption activity of osteoclasts. Scanning electron microscopy (SEM) analysis revealed the surface topographies of the samples (Figure 2E); the pure titanium and AT substrates exhibited relatively smooth surface morphologies. The Mg²⁺ and Ga³⁺ ions were incorporated into Mg-exchanged substrates through a high-pressure hydrothermal process and subsequent calcination. The resulting surface topography of the coatings displayed sheet-like structures, with the sizes of these sheets progressively decreasing from the AT-Mg to AT-Mg/Ga samples as the amount of Ga³⁺ increased. Further, to investigate the influence of pH, metal ions, and substrate topography on autophagic activity, an autophagosome formation test was performed on MSC under various conditions. The results (Figure 2F) indicate that the presence of the Mg and Ga metal ions and the topography of AT-Mg/Ga had no significant effect on the level of autophagy. However, the expression level of LC3 II in the pH 8.5 group was higher than in other groups. Interestingly, the addition of an autophagy inhibitor eliminated the differences in ALP activity and mineralization between the AT-Mg/Ga group and the control groups (Figure 2G). Subsequently, osteoclastogenesis-related genes in RAW264.7 cells were analyzed via qRT-PCR under the influence of the receptor activator of the nuclear factor kappa-B ligand (RANKL) and macrophage colony-stimulating factor (m-CSF). As shown in Figure 2H, the AT-Mg/Ga group exhibited significantly lower expression of osteoclastogenesis-related genes compared to the Ti and AT groups. To evaluate osteoclast responses, researchers quantified the tartrate-resistant acid phosphatase (TRAP) activity, a histochemical marker for osteoclasts, in the RAW264.7 cells cultured on three sample groups under the influence of RANKL and m-CSF. After 1 and 4 days of culture, the TRAP activity was significantly lower in the AT-Mg/Ga group than the control groups, particularly at day 4 (Figure 2I). Additionally, while the AT-Mg/Ga sample exhibited only a few multinuclear cells, numerous such cells were readily observed on the Ti and AT groups (Figure 2J). The results indicated that AT-Mg/Ga had significant potential to inhibit the differentiation of the RAW264.7 cells in vitro and suppress osteoclast generation and osteoclastic bone resorption in vivo under osteoporotic conditions. This suggests that AT-Mg/Ga materials could be applied in the development and research of functional orthopedic implants for patients with osteoporosis.

In conclusion, metal nanostructures, particularly AuNPs and GaNPs, have proven to be crucial for preserving the potency and enhancing the differentiation capacity of undifferentiated stem cells. Such metal-based nanomaterials are extensively utilized to facilitate and promote stem cell differentiation across diverse applications.

Figure 2. (A) AuNP treatment enhanced autophagic activity in inflammatory-conditioned periodontal ligament stem cells (I-PDLSCs) during the early phase of osteogenic differentiation represented by upregulated levels of LC3 II in the AuNP-treated I-PDLSCs and control PDLSCs. (B) Confocal images of accumulated FITC-LC3 puncta per cell in the AuNP-incubated I-PDLSCs and I-PDLSCs. (C) Knockdown of TFEB abrogated the AuNP-mediated rescue of the osteogenic potential of I-PDLSC. (D) Osteogenic protein expression in the I-PDLSCs represented by the decrease in RUNX2 expression. (E) Topographic images of different Ti substrates using SEM. (F) Quantitative analysis of the LC3 expression in MSCs treated with various samples in normal DMEM media at 1 and 7 days. (G) Related qualitative ALP activity on various sample surfaces at 7 and 14 days. (H) Relative mRNA expression of osteoclastogenesis-related genes in the RAW264.7 cells grown on different substrates. (I) quantitative TRAP activities after incubation for 1 and 4 days. (J) Confocal images of multinucleated cells on different substrates after culturing for 4 days. The asterisks and number signs indicate p-values *p and ^# p < 0.05, ** p and ^## p < 0.01, and *** p < 0.001. Reprinted with permission from [94]. Copyright 2022, Elsevier; reprinted with permission from [97]. Copyright 2022, Elsevier.

2.2. ROS Scavenger

Stem-cell-based tissue regeneration has emerged as a promising approach for the treatment of severe traumatic injuries and chronic wounds, including cardiac repair, neurological trauma, bone defects, cartilage damage, and diabetic foot complications. However, the low survival and impaired function of implanted stem cells, largely due to excessive ROS in the damaged microenvironment, have significantly limited their therapeutic efficacy [98]. To address this challenge, engineered antioxidant nanomaterials have been explored as potential strategies to enhance the resistance of stem cells to oxidative stress and promote their regenerative capacity. Accordingly, the development of ROS-scavenging nanostructures has emerged as an intriguing approach to protect and regulate stem cells, thereby facilitating tissue regeneration in high-ROS environments [99,100]. For instance, selenium, silver, and aluminum nanoparticles have been utilized to alleviate oxidative stress and enhance the stemness and stem cell differentiation. While recent progress has been made in developing catalytic materials that can scavenge ROS, designing high-performance, broad-spectrum ROS-scavenging materials with rapid enzyme-like catalytic kinetics remains a significant challenge. Therefore, it is crucial to develop suitable strategies to address the imbalanced valence states during catalytic ROS scavenging and achieve reversible catalytic cycles with high reaction activities.

Tian et al. described a novel strategy involving manganese-atom substitution to modulate the electronic structure of Co₃O₄ nanocrystals to enhance their multifaceted catalytic abilities to scavenge ROS [101]. This resulted in the Mn-Co₃O₄ material efficiently protecting human MSCs (hMSCs) from ROS-induced damage, reversing apoptotic fates, and rescuing key cellular functions, such as adhesion, spreading, proliferation, and osteogenic differentiation. The performance of hMSCs treated with Mn-Co₃O₄ was comparable to that of the hMSCs cultured in standard medium (Figure 3A). The Mn-substituted Co₃O₄ materials were synthesized with varying Mn contents and denoted as MC-0.4, MC-1.0, and MC-1.6. Because the MC-1.0 composition exhibited an optimal catalytic ROS-scavenging activity, the subsequent analysis will primarily focus on MC-1.0. All references to the Mn-Co₃O₄ materials pertain to the MC-1.0 composition and display spherical and ultrasmall nanoscale morphologies with uniform dispersal (Figure 3B). Moreover, they present clear lattice fringes, corresponding to the lattice planes of Co₃O₄. To confirm the uniform crystal structures of Mn-Co₃O₄, X-ray diffraction (XRD) was performed, as shown in Figure 3C. The increased substitution of Mn atoms in Mn-Co₃O₄ results in a slight shift in the diffraction peaks toward lower diffraction angles compared to pristine Co₃O₄. This gradual Mn atom substitution indicates a slight disorder in the Co₃O₄ crystalline structure. Subsequently, the incorporation of Mn atoms into the Co₃O₄ crystal structure results in longer interatomic distances compared to the native Co-Co bonds, as a consequence of the larger atomic radius of Mn. The electron energy-loss spectroscopy further confirmed (Figure 3D) the homogeneous distribution of both Co and Mn atoms across the surface and edges of the Mn-substituted Co₃O₄ material. These findings suggest that the substituted Mn atoms are evenly dispersed throughout the entire crystal lattice of the MC-1.0 composition. Furthermore, DPPH• is a commonly used reagent to assess the free-radical scavenging capacity of biocatalysts. As shown in Figure 3E, the MC-1.0 catalyst also effectively removes DPPH radicals. To further investigate the potential of Mn-Co₃O₄ for stem-cell-based therapeutics, researchers systematically evaluated its ability to regulate the fate of the hMSCs in high-ROS environments. Subsequently, the proliferation of the hMSCs under high-ROS conditions was studied to better understand the protective efficacy of MC-1.0 (Figure 3F). The H₂O₂-treated hMSCs exhibited the lowest cell counts, indicating that the high oxidative stress had impaired their proliferative capacity. Interestingly, the H₂O₂ + hMSCs pretreated with MC-1.0 demonstrated more efficient cell proliferation than the H₂O₂-only group. Meanwhile, the data suggest that MC-1.0 had a negligible effect on cell proliferation compared to the control, further confirming the good biocompatibility of the Mn-Co₃O₄ material. Previous studies have reported that excessive ROS can impair the osteogenic differentiation potential of the hMSCs [102]. Therefore, this study further investigated the differentiation capabilities of the hMSCs under oxidative stress conditions. It was found that the H₂O₂-induced suppression of osteogenic gene expression could be rescued by the addition of MC-1.0. Furthermore, immunofluorescence analyses revealed distinct differences in the signal intensities of the osteogenic markers osteocalcin (OCN) and osteopontin (OPN) between the H₂O₂-treated group and other groups (Figure 3G). These results suggest that H₂O₂ significantly inhibits the osteogenic differentiation of the hMSCs; however, the addition of MC-1.0 to the H₂O₂-containing media can efficiently promote the expression of osteogenic genes in the oxidative stress microenvironment. Collectively, these findings indicate that the MC-1.0 nanomaterial can effectively protect the hMSCs from ROS-induced damage and preserve their critical cellular functions, including adhesion, spreading, proliferation, and differentiation.

Figure 3. (A) Schematic illustrations of Mn-Co₃O₄ utilized as antioxidant nanostructures for regulating stem cell fates. (B) Crystal model of MC-1.0 which displays the optimized catalytic ROS-scavenging activity. (C) X-ray diffraction (XRD) analysis for the Mn-Co₃O₄ crystal structures. (D) Electron energy-loss spectroscopy (EELS) exhibiting uniformly distributed Co and Mn atoms on the surface. (E) Scavenging activities of Mn₃O₄, Co₃O₄, and MC-1.0 for DPPH radical. (F) Quantitative analysis of cell proliferation after the H₂O₂ treatment. (G) Relative fluorescence intensity of osteogenic markers. (H) Schematic diagram of the preparation of SOD-modified gold nanospheres (SOD@AuNS). (I) TEM and elemental mapping images of SOD@AuNS. (J) Histological analysis of lipid accumulation (ORO) and calcium deposition (ARS) by staining the MSCs, with and without labeling, following adipogenic and osteogenic induction, respectively. (K) Quantitative analysis of each differentiation results. (L) Cell viabilities of the MSCs labeled with SOD@Au, SOD@AuNS, and MUA@AuNS. The asterisks indicate p-values * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001 and ns represents no significant difference. Reprinted with permission from [101]. Copyright 2022, Wiley Online Library; reprinted with permission from [103]. Copyright 2023, Elsevier.

A Mn-atom-substituted Co₃O₄ nanocrystalline structure and AuNPs can affect anti-inflammation and remove ROS scavengers to protect the potential of the MSCs. Yu et al. developed superoxide dismutase (SOD)-engineered AuNPs as a comprehensive ROS scavenger. SOD is a crucial antioxidant enzyme that neutralizes intracellular ROS and computed tomography (CT) contrast agent for simultaneous protection and imaging tracking of MSCs. It was modified on the surface of the AuNPs and then encapsulated within polyphosphazene nanospheres (NS) (Figure 3H) [103]. This approach aimed to overcome the limited cell membrane penetration and chemical instability of SOD to enhance the survival of MSCs in a harsh inflammatory microenvironment through effective ROS elimination. Further, transmission electron microscopy (TEM) analysis revealed that the engineered SOD@AuNSs possessed a relatively uniform spherical morphology with an average diameter of approximately 270 nm. Elemental mapping further confirmed the presence of gold, phosphorus, nitrogen, copper, and oxygen, which originated from the AuNPs, superoxide dismutase enzyme, and polyphosphazene polymer backbone (Figure 3I). Moreover, the biocompatibility was also confirmed by CCK-8 analysis, maintained over 90% when co-incubated with SOD@AuNSs (Figure 3J). Next, the intracellular localization of SOD@AuNPs in the MSCs was investigated after nucleus staining. The results demonstrated that the SOD@AuNPs were effectively internalized by the MSCs and predominantly distributed within the cytoplasm. This suggests that the SOD@AuNSs can provide feasible cellular contrast signals through stable labeling of MSCs (Figure 3K). Assessing the multipotency of the MSCs after labeling with nanomaterials is crucial for their clinical application. For this purpose, evaluating their ability to differentiate into osteogenic and adipogenic lineages can provide insights into the maintenance of their multipotent potential. As shown in Figure 3L, the SOD@AuNS-labeled MSCs successfully differentiated into adipocytes and osteocytes. Additionally, quantitative assays confirmed that there was no significant difference in the differentiation between the labeled and unlabeled MSCs. Furthermore, protecting stem cells from oxidative stress using metal nanostructures and reversing their apoptotic fates to rescue their critical functions is a promising approach for promoting tissue regeneration. By regulating stem cell fate in microenvironments with excessive ROS, this strategy can effectively advance stem-cell- based therapeutics.

 

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