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BACKGROUND: The quality of treatment plans for breast cancer can vary greatly. This variation could be reduced by using dose prediction to automate treatment planning. Our work investigates novel methods for training deep-learning models that are capable of producing high-quality dose predictions for breast cancer treatment planning. PURPOSE: The goal of this work was to compare the performance impact of two novel techniques for deep learning dose prediction models for tangent field treatments for breast cancer. The first technique, a "glowing" mask algorithm, encodes the distance from a contour into each voxel in a mask. The second, a gradient-weighted mean squared error (MSE) loss function, emphasizes the error in high-dose gradient regions in the predicted image. METHODS: Four 3D U-Net deep learning models were trained using the planning CT and contours of the heart, lung, and tumor bed as inputs. The dataset consisted of 305 treatment plans split into 213/46/46 training/validation/test sets using a 70/15/15% split. We compared the impact of novel "glowing" anatomical mask inputs and a novel gradient-weighted MSE loss function to their standard counterparts, binary anatomical masks, and MSE loss, using an ablation study methodology. To assess performance, we examined the mean error and mean absolute error (ME/MAE) in dose across all within-body voxels, the error in mean dose to heart, ipsilateral lung, and tumor bed, dice similarity coefficient (DSC) across isodose volumes defined by 0%-100% prescribed dose thresholds, and gamma analysis (3%/3 mm). RESULTS: The combination of novel glowing masks and gradient weighted loss function yielded the best-performing model in this study. This model resulted in a mean ME of 0.40%, MAE of 2.70%, an error in mean dose to heart and lung of -0.10 and 0.01 Gy, and an error in mean dose to the tumor bed of -0.01%. The median DSC at 50/95/100% isodose levels were 0.91/0.87/0.82. The mean 3D gamma pass rate (3%/3 mm) was 93%. CONCLUSIONS: This study found the combination of novel anatomical mask inputs and loss function for dose prediction resulted in superior performance to their standard counterparts. These results have important implications for the field of radiotherapy dose prediction, as the methods used here can be easily incorporated into many other dose prediction models for other treatment sites. Additionally, this dose prediction model for breast radiotherapy has sufficient performance to be used in an automated planning pipeline for tangent field radiotherapy and has the major benefit of not requiring a PTV for accurate dose prediction.
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Acute myeloid leukemia (AML) is a deadly form of leukemia with ineffective traditional treatment and frequent chemoresistance-associated relapse. Personalized drug screening holds promise in identifying optimal regimen, nevertheless, primary AML cells undergo spontaneous apoptosis during cultures, invalidating the drug screening results. Here, we reconstitute a 3D osteogenic niche (3DON) mimicking that in bone marrow to support primary AML cell survival and phenotype maintenance in cultures. Specifically, 3DON derived from osteogenically differentiated mesenchymal stem cells (MSC) from healthy and AML donors are co-cultured with primary AML cells. The AML cells under the AML_3DON niche showed enhanced viability, reduced apoptosis and maintained CD33+ CD34-phenotype, associating with elevated secretion of anti-apoptotic cytokines in the AML_3DON niche. Moreover, AML cells under the AML_3DON niche exhibited low sensitivity to two FDA-approved chemotherapeutic drugs, further suggesting the physiological resemblance of the AML_3DON niche. Most interestingly, AML cells co-cultured with the healthy_3DON niche are highly sensitive to the same sample drugs. This study demonstrates the differential responses of AML cells towards leukemic and healthy bone marrow niches, suggesting the impact of native cancer cell niche in drug screening, and the potential of re-engineering healthy bone marrow niche in AML patients as chemotherapeutic adjuvants overcoming chemoresistance, respectively.
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Cellular, extracellular matrix (ECM), and spatial heterogeneity of tumor microenvironments (TMEs) regulate disease progression and treatment efficacy. Developing in vitro models that recapitulate the TME promises to accelerate studies of tumor biology and identify new targets for therapy. Here, we used extrusion-based, multi-nozzle 3D bioprinting to spatially pattern triple-negative MDA-MB-231 breast cancer cells, endothelial cells (ECs), and human mammary cancer-associated fibroblasts (HMCAFs) with biomimetic ECM inks. Bioprinted models captured key features of the spatial architecture of human breast tumors, including varying-sized dense regions of cancer cells and surrounding microvessel-rich stroma. Angiogenesis and ECM stiffening occurred in the stromal area but not the cancer cell-rich (CCR) regions, mimicking pathological changes in patient samples. Transcriptomic analyses revealed upregulation of angiogenesis-related and ECM remodeling-related signatures in the stroma region and identified potential ligand-receptor (LR) mediators of these processes. Breast cancer cells in distinct parts of the bioprinted TME showed differing sensitivities to chemotherapy, highlighting environmentally mediated drug resistance. In summary, our 3D-bioprinted tumor model will act as a platform to discover integrated functions of the TME in cancer biology and therapy.
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OBJECTIVES: To evaluate the dimensional accuracy of occlusal veneers printed using a novel direct ink writing (DIW) system and a clinically approved dental composite. METHODS: A novel three-dimensional printer was developed based on the extrusion-based DIW principle. The printer, constructed primarily with open-source hardware, was calibrated to print with a flowable resin composite (Beautifil Flow Plus). The feasibility of this technology was assessed through an evaluation of the dimensional accuracy of 20 printed occlusal veneers using a laboratory confocal scanner. The precision was determined by pairwise superimposition of the 20 prints, resulting in a set of 190 deviation maps used to evaluate between-sample variations. RESULTS: Without material waste or residuals, the DIW system can print a solid occlusal veneer of a maxillary molar within a 20-minute timeframe. Across all the sampled surface points, the overall unsigned dimensional deviation was 30.1 ± 20.2 µm (mean ± standard deviation), with a median of 24.4 µm (interquartile range of 22.5 µm) and a root mean square value of 36.3 µm. The pairwise superimposition procedure revealed a mean between-sample dimensional deviation of 26.7 ± 4.5 µm (mean ± standard deviation; n = 190 pairs), indicating adequate precision. Visualization of the deviation together with the nonextrusion movements highlights the correlation between high-deviation regions and material stringing. SIGNIFICANCE: This study underscores the potential of using the proposed DIW system to create indirect restorations utilizing clinically approved flowable resin composites. Future optimization holds promise for enhancing the printing accuracy and increasing the printing speed.
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BACKGROUND: It has long been of interest to characterize the components of the motor abnormality in the arm after stroke. One approach has been to decompose the hemiparesis phenotype into negative signs, such as weakness, and positive signs, such as intrusion of synergies. We sought to identify the contributions of weakness and flexor synergy to motor deficits in sub-acute stroke. METHODS: Thirty-three sub-acute post-stroke participants and 16 healthy controls performed two functional arm movements; one within flexor synergy (shoulder and elbow flexion), and the other outside flexor synergy (shoulder flexion and elbow extension). We analyzed upper limb 3D kinematics to assess both overall task performance and intrusion of pathological synergies. Weakness and spasticity were also measured. RESULTS: Both tasks produced similar impairments compared to controls. Analysis of elbow and shoulder multi-joint coordination patterns revealed intrusion of synergies in the out-of-synergy reaching task based on the time spent within a flexion-flexion pattern and the correlation between shoulder and elbow angles. Regression analysis indicated that both weakness and synergy intrusion contributed to motor impairment in the out-of-synergy reaching task. Notably, the Fugl-Meyer Assessment (FMA) was abnormal even when only weakness caused the impairment, cautioning that it is not a pure synergy scale. CONCLUSIONS: Weakness and synergy intrusion contribute to motor deficits in the sub-acute post-stroke period. An abnormal FMA score cannot be assumed to be due to synergy intrusion. Careful kinematic analysis of naturalistic movements is required to better characterize the contribution of negative and positive signs to upper limb impairment after stroke.
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OBJECTIVES: To evaluate the 3D accuracy of attachment positioning and the adaptation of aligners to attachments using in-house templates made with either polyethylene terephthalate glycol (PETG) or ethylene-vinyl acetate (EVA) and either pressure or vacuum thermoforming machines. MATERIALS AND METHODS: Overall, 140 test specimens were resin-printed. Templates for the attachment bonding were made with 1-mm EVA or 0.5-mm PETG laminates. Orthodontic aligners were manufactured with 0.75-mm PETG. The thermoplastification process was carried out using either vacuum or pressure machines. The positional differences between the virtual and bonded attachments were assessed in the X, Y and Z coordinates. The marginal adaptation between the aligners and the attachments was measured. RESULTS: Minor inaccuracies in the positioning of the attachments were observed in all combinations of thermoforming machines and plastic laminates used to fabricate the templates, mainly in the superior-inferior (Z) dimension. PETG performed better than EVA in the anterior region (p < .05). No association was found between thermoplastification machines and the accuracy of the positioning of the attachments (p > .05). While small misadaptations between the aligners and the attachments were observed, the EVA templates performed better than the PETG templates. CONCLUSIONS: The inaccuracy of the attachment positioning and the misadaptation of the aligners to the attachments were slight. The vacuum and pressure thermoplastification machines showed no difference in attachment positioning accuracy. The PETG template was better than the EVA template in the anterior region, but the EVA attachments presented a better adaptation to the aligners than the PETG attachments.
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Medical students often struggle to appreciate the anatomy of the pterygopalatine fossa. This is due to the difficulty in understanding the spatial orientation through textbook diagrams, as well as its deeper location and smaller size in cadaveric specimens. Research has proven that three-dimensional visualization enhances the spatial understanding of anatomy. However, studies have not compared the effectiveness of two different instructional designs that cater to 3D visualization. We conducted a mixed methodology (quasi-experimental pre-test/post-test control design with additional qualitative components) study to compare the effectiveness of a physical model and a 3D image in small-group teaching. The students were divided into control and intervention groups based on their roll numbers. The control group utilized a series of 3D images delivered through Microsoft PowerPoint software on computers. The students in the intervention group used a physical model made of cardboard, with colored wires representing the neurovascular structures. We used 20 spatial anatomy-based multiple-choice questions (MCQs) to assess knowledge acquisition before and after the small group discussion. Additionally, we utilized a validated 10-item feedback questionnaire to evaluate participants' perception of the teaching sessions. There was no significant difference in the knowledge gain and perception scores between the control and intervention groups. These findings suggest that a well-designed 3D image can provide an equivalent learning outcome and level of satisfaction compared to a physical model. Supplementary Information: The online version contains supplementary material available at 10.1007/s40670-024-02063-3.
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Background Knowing the characteristics of vertical patterns is crucial to provide the best orthodontic treatment. Cone beam computed tomography (CBCT) offers a valuable tool for evaluating true buccolingual inclinations. The current study investigates the buccolingual inclination of first molars in adult subjects with different vertical facial patterns. Methods CBCT scans of 66 adult patients (31 males, 35 females) with a mean age of 31.6 years (SD: 6.4 years) exhibiting skeletal class II division I maxillomandibular relationships were employed. Participants were categorized into three groups based on linear and angular measurements: normodivergent group (n=22), hypodivergent group (n=22), and hyperdivergent group (n=22). The independent samples t-test and Mann-Whitney U-test were conducted to investigate statistical differences in terms of buccolingual inclination between the three vertical patterns. Results Statistically significant differences were observed in the buccolingual inclinations of both maxillary and mandibular first molars in the hypodivergent group compared to the other groups (p<0.05). Conclusions In patients with class II division I sagittal relationships, the buccolingual inclinations of the first molars exhibit similarities between normodivergent and hyperdivergent groups. However, these inclinations differ significantly in hypodivergent adult subjects.
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Introduction: We have developed a risk-scoring model using gene expression levels related to mitotic spindle assembly (MSA) to predict the prognosis of liver cancer. Methods and results: Initially, we identified 470 genes related to MSA from public databases. Subsequently, through analysis of sequencing data from liver cancer patient samples in online databases, we identified 7 genes suitable for constructing the risk-scoring model. We validated the predictive accuracy and clinical utility of the model. Through drug sensitivity analysis, we identified SAC3D1 as a gene sensitive to the most common anti-tumor drugs among these 7 genes. We propose SAC3D1 as a significant target for future clinical treatment. Furthermore, we conducted in vivo and in vitro experiments to validate the relevance of SAC3D1 to MSA and found its significant impact on the PI3K/Akt signaling pathway and spindle function. Conclusion: Our research introduces a novel risk-scoring model that accurately predicts liver cancer prognosis. Additionally, our findings suggest SAC3D1 as a promising therapeutic target for hepatocellular carcinoma, potentially revealing new mechanisms underlying liver cancer development.
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3D visualization and segmentation are increasingly widely used in physical, biological and medical science, facilitating advanced investigative methodologies. However, the integration and reproduction of segmented volumes or results across the spectrum of mainstream 3D visualization platforms remain hindered by compatibility constraints. These barriers not only challenge the replication of findings but also obstruct the process of cross-validating the accuracy of 3D visualization outputs. To address this gap, we developed an innovative revisualization method implemented within the open-source framework of Drishti, a 3D visualization software. Leveraging four animal samples alongside three mainstream 3D visualization platforms as case studies, our method demonstrates the seamless transferability of segmented results into Drishti. This capability effectively fosters a new avenue for authentication and enhanced scrutiny of segmented data. By facilitating this interoperability, our approach underscores the potential for significant advancements in accuracy validation and collaborative research efforts across diverse scientific domains.
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We investigated the influence of the medium on the perception of depicted objects and materials. Oil paintings and their reproductions in engravings were chosen because they are vastly distinctive media while having completely identical content. A total of 15 pairs were collected, consisting of 88 fragments depicting different materials, including fabric, skin, wood and metal. Besides the original condition, we created three manipulations to understand the effect of colour (a greyscale version) and contrast (equalised histograms towards both painting and engraving). We performed rating experiments on five attributes: three-dimensionality, glossiness, convincingness, smoothness and softness. An average of 25 participants finished each of the 20 online experimental sessions (five attributes X four conditions). Besides clear correlations between the two media, the differences mainly show in their means (different levels of perceived attributes) and standard deviations (perceived range). In most sessions, paintings depict a wider range than engravings. In addition, it was the histogram equalisation (global contrast) that made the most impact on perceived attributes, rather than colour removal. This suggests that engravers compensated for the lack of colour by exploiting the possibilities of local contrast.
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The objective of this study is to collect the significant advancements of 3D printed medical devices in the biomedical area in recent years. Especially related to a range of diseases and the polymers employed in drug administration. To address the existing limitations and constraints associated with the method used for producing 3D printed medical devices, in order to optimize their suitability for degradation. The compilation and use of research papers, reports, and patents that are relevant to the key keywords are employed to improve comprehension. According to this thorough investigation, it can be inferred that the 3D Printing method, specifically Fuse Deposition Modeling (FDM), is the most suitable and convenient approach for preparing medical devices. This study provides an analysis and summary of the development trend of 3D printed implantable medical devices, focusing on the production process, materials specially the polymers, and typical items associated with 3D printing technology. This study offers a comprehensive examination of nanocarrier research and its corresponding discoveries. The FDM method, which is already facing significant challenges in terms of achieving optimal performance and cost reduction, will experience remarkable advantages from this highly valuable technology. The objective of this analysis is to showcase the efficacy and limitations of 3D-printing applications in medical devices through thorough research, highlighting the significant technological advancements it offers. This article provides a comprehensive overview of the most recent research and discoveries on 3D-printed medical devices, offering significant insights into their study.
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OBJECTIVE: To investigate the biomechanical properties of pedicle screw reinsertion along the same trajectory in a previously validated synthetic bone model. METHODS: Twenty identical acrylonitrile butadiene styrene models of a lumbar vertebrae were 3D-printed. Screws were placed in the standard fashion into each pedicle. Models were separated into two equal groups, control and experimental. Experimental group screws were completely removed from their testing block and reinserted once. All screws in both groups were then forcibly removed. Continuous torque monitoring was collected on screw insertion (IT), removal (RT), and reinsertion (RIT). Pullout strength (PO), screw stiffness (STI), and strain energy (STR) were calculated. RESULTS: There was no significant difference between control and experimental groups for PO (p = 0.26), STI (p = 0.55), STR (p = 0.50), or IT (p = 0.24). There was a significant decrease in RIT (54.5 N-cm ± 8.2 N-cm) from control IT (62.9 N-cm ± 8.4 N-cm, p = 0.045) and experimental IT (67.5 N-cm ± 7.6 N-cm, p = 0.0026). Strong correlations (Pearson's r > 0.80) were seen between control IT against STR and PO, between each of the experimental torque measurements, and between experimental PO and STI. CONCLUSION: Despite a significant decrease in insertion torque, there is no significant loss of pedicle screw performance when a screw is removed and reinserted along the same trajectory.
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Water scarcity is a pressing issue in arid and semi-arid regions, making fog harvesting a promising method for water collection. However, enhancing the rate of fog harvesting remains a challenge. Controlling the movement of droplets on functional surfaces is crucial for the development of effective water-harvesting devices. In this study, a three-dimensional (3D) fog-harvesting device with mixed wettability is fabricated using a combination of physical and chemical techniques. With inspiration drawn from natural organisms, such as the desert beetle and Nephrolepis cordifolia, which can both live in low humidity, a copper substrate with a leaf-shaped wedge superhydrophilic structure and flat superhydrophobic regions is fabricated for fog harvesting. The modified surface results in a maximum 49.89% improvement in fog-harvesting efficiency compared to the original copper substrate. The synergistic effect of the 3D structure and mixed wettability of this study offers an idea for improving fog collection efficiency, with potential implications for energy sustainability water resources.
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Lipid nanoparticles (LNPs) are gaining recognition as potentially effective carriers for delivery of therapeutic agents, including nucleic acids (DNA and RNA), for the prevention and treatment of various diseases. Much effort has been devoted to the implementation of microfluidic techniques for the production of monodisperse and stable LNPs and the improvement of encapsulation efficiency. Here, we developed three-dimensional (3D)-printed ring micromixers for the production of size-controllable and monodispersed LNPs with a high mRNA delivery efficiency. The effects of flow rate and ring shape asymmetry on the mixing performance were initially examined. Furthermore, the physicochemical properties (such as hydrodynamic diameter, polydispersity, and encapsulation efficiency) of the generated LNPs were quantified as a function of these physical parameters via biochemical analysis and cryo-electron microscopy imaging. With a high production rate of 68 mL/min, our 3D-printed ring micromixers can be used to manufacture LNPs with diameters less than 90 nm, low polydispersity (<0.2), and high mRNA encapsulation efficiency (>91%). Despite the simplicity of the ring-shaped mixer structure, we can produce mRNA-loaded LNPs with exceptional quality and high throughput, outperforming costly commercial micromixers.
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OBJECTIVE: To assess the 2D and 3D natural symmetry of the maxillary incisors. MATERIALS AND METHODS: Maxillary alginate impressions were taken of 59 young adult volunteers. Gender, orthodontic treatment history, and dominant hand were collected. For 2D analysis, a digital caliper was used for measurements on maxillary casts. For 3D analysis, the casts were scanned using a lab scanner, and the labial surface symmetry of contralateral incisors was evaluated. Mann-Whitney tests and Student's t-test were performed, α = 0.05. RESULTS: Results indicated a lack of identical lengths among contralateral central or lateral incisors, with minimal occurrences of identical width measurements. 68% of central incisors and 73% of lateral incisors showed differences in length exceeding 0.2 mm. Central incisors showed more similarities than lateral incisors in width, with 61% central incisors and 47% lateral incisors having differences under 0.2 mm. The differences between highly asymmetrical contralateral teeth are situated at the transitional lines. The influence of sexual dimorphism, orthodontic factors, and the dominant hand on incisors' symmetry was not statistically significant. CONCLUSIONS: 2D and 3D symmetry of the natural maxillary incisors are rare. 3D symmetry remains consistent across orthodontic treatment status, gender, and dominant hand, suggesting its independence from such parameters. CLINICAL SIGNIFICANCE: To achieve a natural and aesthetic smile rehabilitation, the symmetry of the maxillary incisors must be well understood. In our study, 2D and 3D symmetry of the maxillary incisors occurred rarely, but central incisors showed more similarities than lateral incisors in width, with differences under 0.2 mm. On the other hand, differences between highly asymmetrical contralateral teeth are situated at the transitional lines. These conclusions should be included in further computer-assisted three-dimensional smile designs.
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Neuroendocrine tumors (NETs) of the pancreas are rare neoplasms that present complex challenges to diagnosis and treatment due to their indolent course. The incidence of pancreatic neuroendocrine tumors has increased significantly over the past two decades. A limited number of pancreatic neuroendocrine cell lines are currently available for the research. Here, we present 3D-iNET ORION, a novel 3-dimensional (spheroid) cell line, isolated from human pancreatic neuroendocrine tumor liver metastasis. Three-dimensionally grown (3D) cancer cell lines have gained interest over the past years as 3D cancer cell lines better recapitulate the in vivo structure of tumors, and are more suitable for in vitro and in vivo experiments. 3D-iNET ORION cancer cell line showed high potential to form tumorspheres when embedded in Matrigel matrix and expresses synaptophysin and EpCAM. Electron microscopy analysis of cancer cell line proved the presence of dense neurosecretory granules. When xenografted into athymic mice, 3D-iNET ORION cells produce slow-growing tumors, positive for chromogranin and synaptophysin. Human Core Exome Panel Analysis has shown that 3DiNET ORION cell line retains the genetic aberration profile detected in the original tumor. In conclusion, our newly developed neuroendocrine cancer cell line can be considered as a new research tool for in vitro and in vivo experiments.
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One of the advancements of the transdermal drug delivery system (TDDS) is the development of microneedles (MNs). These micron-sized needles are used for delivering various types of drugs to address the disadvantage of other transdermal techniques as well as oral drug delivery systems. MNs have high patient acceptance due to self-administration with minimally invasive and pain compared to the parenteral drug delivery. Over the years, various methods have been adopted to evolve the MNs and make them more cost-effective, accurate, and suitable for multiple applications. One such method is the 3D printing of MNs. The development of MN platforms using 3D printing has been made possible by improved features like precision, printing resolution, and the feasibility of using low-cost raw materials. In this review, we have tried to explain various types of MNs, fabrication methods, materials used in the formulation of MNs, and the recent applications that utilize 3D-printed MNs.
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PURPOSE: This study evaluated the effects of artificial saliva and distilled water on the nanoindentation creep of different 3D-printed and milled CAD-CAM resin composites. MATERIAL AND METHODS: Disk-shaped specimens were subtractively fabricated from polymer-infiltrated ceramic network (EN) and reinforced resin composite (B) and additively from resin composite (C) and hybrid resin composite (VS) using digital light processing (DLP). Specimens from each material were divided into two groups according to their storage conditions (artificial saliva or distilled water for 3 months). Creep was analyzed by nanoindentation testing. Statistical analysis was done using two-way ANOVA, one-way ANOVA, Bonferroni post hoc tests, and independent t-test (α = 0.05). RESULTS: The main effects of material and storage conditions, and their interaction were statistically significant on nanoindentation (p < 0.001). Storage condition had the greatest influence (partial eta squared ηP 2 = 0.370), followed by the material (ηP 2 = 0.359), and the interaction (ηP 2 = 0.329). The nanoindentation creep depths after artificial saliva storage ranged from 0.34 to 0.51 µm and from 0.50 to 0.87 µm after distilled water storage. One of the additively manufactured groups had higher nanoindentation creep depths in both storage conditions. CONCLUSIONS: All specimens showed comparable performance after artificial saliva storage, but increased nanoindentation creep after distilled water storage for 3 months. The subtractive CAD-CAM blocks showed superior dimensional stability in terms of nanoindentation creep depths in both storage conditions. Additively manufactured composite resins had lower dimensional stability than one of the subtractively manufactured composites, which was demonstrated as having higher creep deformation and maximum recovery. However, after artificial saliva storage, one of the additively manufactured resins had dimensional stability similar to that of subtractively manufactured.
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Energy storage has become increasingly crucial, necessitating alternatives to lithium-ion batteries due to critical supply constraints. Aqueous multivalent metal-ion batteries (AMVIBs) offer significant potential for large-scale energy storage, leveraging the high abundance and environmentally benign nature of elements like zinc, magnesium, calcium, and aluminum in the Earth's crust. However, the slow ion diffusion kinetics and stability issues of cathode materials pose significant technical challenges, raising concerns about the future viability of AMVIB technologies. Recent research has focused on nanoengineering cathodes to address these issues, but practical implementation is limited by low mass-loading. Therefore, developing effective engineering strategies for cathode materials is essential. This review introduces the 3D printing-enabled structural design of cathodes as a transformative strategy for advancing AMVIBs. It begins by summarizing recent developments and common challenges in cathode materials for AMVIBs and then illustrates various 3D-printed cathode structural designs aimed at overcoming the limitations of conventional cathode materials, highlighting pioneering work in this field. Finally, the review discusses the necessary technological advancements in 3D printing processes to further develop advanced 3D-printed AMVIBs. The reader will receive new fresh perspective on multivalent metal-ion batteries and the potential of additive technologies in this field.