{"title":"Polymer-based nanocarriers to transport therapeutic biomacromolecules across the blood-brain barrier","authors":"Elena Romero-Ben , Upashi Goswami , Jackeline Soto-Cruz , Amirreza Mansoori-Kermani , Dhiraj Mishra , Sergio Martin-Saldaña , Jone Muñoz-Ugartemendia , Alejandro Sosnik , Marcelo Calderón , Ana Beloqui , Aitor Larrañaga","doi":"10.1016/j.actbio.2025.02.065","DOIUrl":"10.1016/j.actbio.2025.02.065","url":null,"abstract":"<div><div>Therapeutic biomacromolecules such as genetic material, antibodies, growth factors and enzymes represent a novel therapeutic alternative for neurological diseases and disorders. In comparison to traditional therapeutics, which are mainly based on small molecular weight drugs that address the symptoms of these disorders, therapeutic biomacromolecules can reduce undesired side effects and target specific pathological pathways, thus paving the way towards personalized medicine. However, these biomacromolecules undergo degradation/denaturation processes in the physiological environment and show poor capacity to cross the blood-brain barrier (BBB). Consequently, they rarely reach the central nervous system (CNS) in their active form. Herein, we critically overview several polymeric nanocarriers that can protect and deliver therapeutic biomacromolecules across the BBB. Polymeric nanocarriers are first categorized based on their architecture (biodegradable solid nanoparticles, nanogels, dendrimers, self-assembled nanoparticles) that ultimately determines their physico-chemical properties and function. The available polymeric formulations are then thoroughly analyzed, placing particular attention on those strategies that ensure the stability of the biomacromolecules during their encapsulation process and promote their passage across the BBB by controlling their physical (e.g., mechanical properties, size, surface charge) and chemical (e.g., surface functional groups, targeting motifs) properties. Accordingly, this review gives a unique perspective on polymeric nanocarriers for the delivery of therapeutic biomacromolecules across the BBB, representing a concise, complete and easy-to-follow guide, which will be of high interest for chemists, material scientists, pharmacologists, and biologists. Besides, it also provides a critical perspective about the limited clinical translation of these systems.</div></div><div><h3>Statement of significance</h3><div>The increasing incidence of central nervous system disorders is a major health concern. The use of therapeutic biomacromolecules has been placed in the spotlight of many investigations. However, reaching therapeutic concentration levels of biomacromolecules in the central nervous system is restricted by the blood-brain barrier and, thus, this represents the main clinical challenge when developing efficient therapies. Herein, we provide a critical discussion about the use of polymeric nanocarriers to deliver therapeutic biomacromolecules into the central nervous system, highlighting potential future directions to overcome the current challenges.</div></div>","PeriodicalId":237,"journal":{"name":"Acta Biomaterialia","volume":"196 ","pages":"Pages 17-49"},"PeriodicalIF":9.4,"publicationDate":"2025-04-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143544884","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Acta BiomaterialiaPub Date : 2025-04-01DOI: 10.1016/j.actbio.2025.03.056
Suyoung Lee , Mark Van Dyke , Minkyu Kim
{"title":"Recombinant keratin: Comprehensive review of synthesis, hierarchical assembly, properties, and applications","authors":"Suyoung Lee , Mark Van Dyke , Minkyu Kim","doi":"10.1016/j.actbio.2025.03.056","DOIUrl":"10.1016/j.actbio.2025.03.056","url":null,"abstract":"<div><div>Keratin has gained attention for its remarkable mechanical properties, thermal stability, and beneficial biological properties, such as promoting hemostasis and wound healing. Traditionally, keratin has been extracted from natural sources, including human hair, wool, and feathers, and processed into biomaterials, including films, hydrogels, and nanoparticles, primarily for biomedical applications. However, extraction methods often result in heterogeneous keratin mixtures with residual impurities and structural degradation due to harsh purification conditions, complicating efforts to understand how specific keratins and their hierarchical assemblies contribute to desired material properties. Recombinant keratin technology addresses these challenges by enabling the synthesis of individual keratin types with high purity and batch-to-batch consistency. These advancements facilitate studies on how individual and combined keratins at various assembly stages<img>from molecular components and heterodimers to intermediate filaments (IFs) and IF networks<img>impact material properties. Moreover, this technology allows for precise genetic modifications, potentially leading to engineered keratin variants with tailored characteristics for targeted applications. Despite these advantages, translating recombinant keratin into practical applications requires overcoming key manufacturing challenges, such as optimizing large-scale production and improving purification efficiency. This review presents the current state of recombinant keratin research by highlighting its advancements and exploring current biomaterial applications. While its applications remain limited compared to extracted keratin at this early stage, its potential offers future opportunities for extending its use in advanced material design and beyond biomedical fields.</div></div><div><h3>Statement of significance</h3><div>Keratin and keratinized structures provide essential protection to tissues against mechanical stress and environmental damage, serving as foundational elements across diverse biological systems. This review discusses advancements in recombinant keratin technology, enabling high-purity, reproducible synthesis with controlled composition modifications that effectively overcome the limitations of traditional extraction methods. The innovations deepen our understanding of hierarchical assembly in keratin structures across various length scales, along with their reinforcing mechanisms and mechanical and biofunctional properties. These insights lay the groundwork for biomaterials tailored to regenerative medicine, wound healing, and other biomedical applications. By focusing on the unique capabilities of recombinant keratin, this review offers a valuable resource for future advancements in high-performance biomaterials across biomedical and biotechnological fields.</div></div>","PeriodicalId":237,"journal":{"name":"Acta Biomaterialia","volume":"198 ","pages":"Pages 1-21"},"PeriodicalIF":9.4,"publicationDate":"2025-04-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143781931","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Acta BiomaterialiaPub Date : 2025-04-01DOI: 10.1016/j.actbio.2025.01.052
Linxiao Wu , Jatinder Virdee , Elizabeth Maughan , Arnold Darbyshire , Gavin Jell , Marilena Loizidou , Mark Emberton , Peter Butler , Ashley Howkins , Alan Reynolds , Ian W. Boyd , Martin Birchall , Wenhui Song
Acta BiomaterialiaPub Date : 2025-04-01DOI: 10.1016/j.actbio.2025.02.043
Yangfan Ding , Moran Huang , Pengfei Cai , Xiao Yu , Jie Cui , Binbin Sun , Xiumei Mo , Changrui Lu , Jiwu Chen , Jinglei Wu
{"title":"Inflammation-modulating elastic decellularized extracellular matrix scaffold promotes meniscus regeneration","authors":"Yangfan Ding , Moran Huang , Pengfei Cai , Xiao Yu , Jie Cui , Binbin Sun , Xiumei Mo , Changrui Lu , Jiwu Chen , Jinglei Wu","doi":"10.1016/j.actbio.2025.02.043","DOIUrl":"10.1016/j.actbio.2025.02.043","url":null,"abstract":"<div><div>Scaffold-guided meniscus repair and regeneration show promise for meniscus injuries. Desirable scaffold properties are key to promoting proper tissue remodeling and effective regeneration. Herein, we report an inflammation-modulating elastic decellularized extracellular matrix (ECM) scaffold and evaluate its biological performance on meniscus repair in a rabbit model. An elastic scaffold of decellularized meniscus ECM (dmECM) was first prepared and functionalized with chitosan (CS) and ibuprofen (IBU) to obtain dmECM/CS-IBU scaffold. Our results show that CS and IBU grafting did not affect the overall properties of the dmECM/CS-IBU scaffold, including porous structure, good mechanical strength and elasticity. It promoted chondrocyte proliferation and preserved chondrogenic properties. In addition, both <em>in vitro</em> and <em>in vivo</em> assessments indicate that the dmECM/CS-IBU scaffold showed good anti-inflammatory properties and promoted pro-healing polarization of macrophages. In a partial rabbit meniscus defect model, the dmECM/CS-IBU scaffold showed promotive effects on <em>in situ</em> meniscus repair and preserved cartilage tissue. Therefore, our study provides a feasible strategy for fabricating scaffolds with tissue-specific bioactivity and inflammation-modulating abilities that synergistically promote meniscus repair and regeneration.</div></div><div><h3>Statement of significance</h3><div>Desirable scaffold properties are key to promoting proper tissue remodeling and effective regeneration of meniscus injuries. Herein, elastic decellularized scaffolds were prepared using natural meniscus and successfully grafted with chitosan and the anti-inflammatory drug ibuprofen (dmECM/CS-IBU). The dmECM/CS-IBU scaffold showed a pro-proliferative and phenotype- preserving effect on chondrocytes. In both <em>in vitro</em> and <em>in vivo</em> models, dmECM/CS-IBU scaffolds exhibited wonderful anti-inflammatory activity. In a meniscus white zone defect model, the dmECM/CS-IBU scaffold demonstrated <em>in situ</em> repair of tissue and protection of cartilage tissue. Therefore, we provides a feasible strategy for fabricating scaffolds with tissue-specific bioactivity and inflammation-modulating abilities that synergistically promote meniscus repair and regeneration.</div></div>","PeriodicalId":237,"journal":{"name":"Acta Biomaterialia","volume":"196 ","pages":"Pages 93-108"},"PeriodicalIF":9.4,"publicationDate":"2025-04-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143485006","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
{"title":"cGAS-mediated antibacterial immunotherapy against tuberculosis by macrophage-targeted manganese dioxide nanoagonist","authors":"Kangsheng Liao , Ruihong Chen , Jinwei Zhang , Yongdui Ruan , Xueqin Huang , Yuhe Huang , Jiaojiao Xia , Daina Zhao , Lingming Chen , Yi Zhao , Fen Yang , Jun-Fa Xu , Ling Shen , Jiang Pi","doi":"10.1016/j.actbio.2025.03.002","DOIUrl":"10.1016/j.actbio.2025.03.002","url":null,"abstract":"<div><div>Tuberculosis (TB), induced by <em>Mycobacterium tuberculosis</em> (<em>Mtb</em>) infection, remains one of the top killers among infectious diseases. The pathogenesis hallmarks for TB are complex immune escape mechanisms of <em>Mtb</em> and low targeting effects of anti-TB drugs. cGAS signaling, which is responsible for triggering host antibacterial immunity against <em>Mtb</em> infection, has shown potentials to serve as targets for anti-TB immunotherapy. As cGAS agonist manganese ions (Mn<sup>2+</sup>) can activate cGAS-mediated autophagy to inhibit intracellular <em>Mtb</em> in macrophages, we constructed a functional nanoagonist targeting cGAS signaling based on manganese dioxide nanoparticles, naming Tuf-Rif@HA-MnO<sub>2</sub> NPs, for synergistic macrophage-targeted drug delivery and anti-TB immuno-therapeutics. Tuf-Rif@HA-MnO<sub>2</sub> NPs can actively target macrophages for rifampicin delivery and react with intracellular glutathione (GSH) to release Mn<sup>2+</sup> for cGAS-STING signaling activation, which further promote autophagy and antibacterial M1 polarization of <em>Mtb</em> infected macrophages to achieve synergistic intracellular <em>Mtb</em> clearance. Furthermore, Tuf-Rif@HA-MnO<sub>2</sub> NPs can potentiate dendritic cell maturation, CD4+ Th1 cell and CD8+ cytotoxic T cell activation <em>in vivo</em>, which collectively attribute to reduced <em>Mtb</em> burdens and alleviated tissue inflammations in lung of <em>Mtb</em>-infected mice without systemic toxicity. This macrophage targeted drug delivery nanoagonist system is expected to develop rational immunotherapy strategy targeting cGAS signaling against TB and drug-resistant TB.</div></div><div><h3>Statement of Significance</h3><div>cGAS-mediated autophagy plays a critical role in <em>Mtb</em> clearance in macrophages.</div><div>Tuf-Rif@HA-MnO<sub>2</sub> NPs specifically deliver rifampicin into macrophage for <em>Mtb</em> clearance.</div><div>Tuf-Rif@HA-MnO<sub>2</sub> NPs activate cGAS-mediated macrophage autophagy for <em>Mtb</em> clearance.</div><div>Tuf-Rif@HA-MnO<sub>2</sub> NPs synergize cGAS-mediated immunotherapy with targeted drug delivery for more effective anti-TB treatment.</div></div>","PeriodicalId":237,"journal":{"name":"Acta Biomaterialia","volume":"196 ","pages":"Pages 471-486"},"PeriodicalIF":9.4,"publicationDate":"2025-04-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143569260","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Acta BiomaterialiaPub Date : 2025-04-01DOI: 10.1016/j.actbio.2025.02.058
Chong-yu Zhang , Cheng Fan , Shu-hong Li , Jun Wu , Yvonne Ziyi Peng , Hsing-wen Sung , Shiming Liu , Ren-Ke Li
{"title":"A conductive polymer restores connexin43 expression through the suppression of mitogen-activated protein kinases to improve intercellular communication and alleviate atrial fibrillation","authors":"Chong-yu Zhang , Cheng Fan , Shu-hong Li , Jun Wu , Yvonne Ziyi Peng , Hsing-wen Sung , Shiming Liu , Ren-Ke Li","doi":"10.1016/j.actbio.2025.02.058","DOIUrl":"10.1016/j.actbio.2025.02.058","url":null,"abstract":"<div><div>Conductive biomaterials have shown promising results for correcting pathological cardiac electrical signaling. However, their mechanisms of operation are still largely unclear. One reason behind disrupted cardiac intercellular communication, though, is lowered expression of the gap junction protein connexin43 (Cx43), which may be alleviated by conductive biomaterials. In this study, we aimed to test this hypothesis, using the self-doping conductive biomaterial poly-3-amino-4-methoxybenzoic acid-gelatin (PAMB-G). An <em>in vitro</em> model was established, in which cardiomyocytes (CMs) were treated with anisomycin, while the <em>in vivo</em> model involved anisomycin-treated mice subjected to electrical pacing to induce atrial fibrillation (AF). Cx43 expression, Ca<sup>2+</sup> transient propagation, and CM electrical conduction <em>in vitro</em>, as well as the <em>in vivo</em> effects of PAMB-G on AF, were evaluated; additionally, the underlying molecular mechanisms were identified. We found that anisomycin, at different concentrations, down-regulated Cx43; this was counteracted by PAMB-G, which restored proper Cx43 expression, coupled with improved Ca<sup>2+</sup> signal and electrical conduction. Cx43 restoration was due to PAMB-G suppressing anisomycin-induced activation of MAPKs P38 and JNK, which are involved in phosphorylating Cx43 for degradation. Similar observations were also found <em>in vivo</em>, where a PAMB-G patch acted against anisomycin-induced Cx43 downregulation and impaired atrial cell communication, subsequently alleviating pacing-induced AF. Therefore, PAMB-G suppresses MAPKs, in turn upregulating Cx43, leading to improved electrical signal transduction. As a result, modulating the MAPK-Cx43 pathway, such as with PAMB-G, could serve as a potential therapeutic strategy for cardiac arrhythmia.</div></div><div><h3>Statement of significance</h3><div>Disruption of atrial intercellular gap junction channels, comprised of connexins, leads to atrial fibrillation (AF), the most prevalent arrhythmia, with poor clinical outcomes. Current AF treatments are associated with adverse effects, and only focus on managing symptoms, thereby necessitating innovative treatment strategies. One such strategy is conductive biomaterials, which show promising results for correcting pathological cardiac electrical signaling. We synthesized a self-doping conductive biomaterial, poly-3-amino-4-methoxybenzoic acid-gelatin (PAMB-G), and found that it counteracts against anisomycin-induced connexin43 (Cx43) downregulation, subsequently improving cardiac electrical conduction and alleviating pacing-induced AF. This is owed to PAMB-G suppressing anisomycin-associated activation of mitogen-activated kinases P38 and JNK, which are involved in phosphorylating Cx43 for degradation. Therefore, PAMB-G modulation of MAPK-Cx43 pathway could aid in cardiac arrhythmia treatment.</div></div>","PeriodicalId":237,"journal":{"name":"Acta Biomaterialia","volume":"196 ","pages":"Pages 123-135"},"PeriodicalIF":9.4,"publicationDate":"2025-04-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143538220","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Acta BiomaterialiaPub Date : 2025-04-01DOI: 10.1016/j.actbio.2025.02.053
Mohammad Tabatabaei, Lakiesha N. Williams
{"title":"Heterogeneity in cranial dura mater at the microscale: An In-situ and ex-vivo structural and mechanical investigation of sulcus and gyrus Dura","authors":"Mohammad Tabatabaei, Lakiesha N. Williams","doi":"10.1016/j.actbio.2025.02.053","DOIUrl":"10.1016/j.actbio.2025.02.053","url":null,"abstract":"<div><div>This study examines cranial dura mater's structural and mechanical heterogeneity, focusing on the distinct properties between the sulcus and gyrus regions. Microscale analyses using two-photon microscopy and atomic force microscopy (AFM) revealed significant regional differences in thickness (p < 0.05), with sulcus dura being 1.34 times thicker than gyrus dura. Differences in effective Young's modulus were observed, with values of 6.75 ± 5.12 kPa in the sulcus and 10.48 ± 7.13 kPa in the gyrus. These findings highlight the dura mater's pronounced variability in stiffness and anisotropy, with the periosteal layer being substantially stiffer than the meningeal layer. These results underscore the critical role of collagenous architecture in determining dura's mechanical behavior, particularly in the transfer of loads across the brain. This study provides valuable insights into the functional heterogeneity of the dura mater and emphasizes the importance of these variations in the design of biomimetic dural grafts. The quantitative data generated in this study has significant implications for enhancing the biofidelity of computational models used in brain biomechanics and advancing tissue engineering strategies to develop dural substitutes.</div></div><div><h3>Statement of Significance</h3><div>This study presents a comprehensive analysis of the structural and mechanical heterogeneity of cranial dura mater at the nanoscale, focusing on the differences between sulcus and gyrus regions. By employing advanced techniques such as atomic force microscopy (AFM) and two photon microscopies, the findings are crucial for understanding the dura's protective functions and its role in load transfer across the brain. The implications of this study are significant for the development of biomimetic dural grafts, as it offers detailed quantitative data necessary for designing grafts that closely mimic the native dura's structural and mechanical. Additionally, this research could help develop more accurate finite element models (FEM) to study traumatic brain injuries (TBI) and brain dynamics.</div></div>","PeriodicalId":237,"journal":{"name":"Acta Biomaterialia","volume":"196 ","pages":"Pages 222-232"},"PeriodicalIF":9.4,"publicationDate":"2025-04-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143525428","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Acta BiomaterialiaPub Date : 2025-04-01DOI: 10.1016/j.actbio.2025.02.048
Hao Zhang , Xianzhen Dong , Yuhang Liu , Ping Duan , Changjiang Liu , Kun Liu , Yifeng Yu , Xinyue Liang , Honglian Dai , Aixi Yu
{"title":"An injectable and adaptable system for the sustained release of hydrogen sulfide for targeted diabetic wound therapy by improving the microenvironment of inflammation regulation and angiogenesis","authors":"Hao Zhang , Xianzhen Dong , Yuhang Liu , Ping Duan , Changjiang Liu , Kun Liu , Yifeng Yu , Xinyue Liang , Honglian Dai , Aixi Yu","doi":"10.1016/j.actbio.2025.02.048","DOIUrl":"10.1016/j.actbio.2025.02.048","url":null,"abstract":"<div><div>The combined effects of persistent chronic inflammation, oxidative stress, microcirculation disorders, and dysregulated cellular energy metabolism often hinder the repair of diabetic skin wounds. Traditional treatment methods are typically insufficient in simultaneously addressing these complex factors, resulting in delayed wound healing and a high propensity for recurrence and chronic ulceration. This study developed an innovative strategy based on reactive oxygen species (ROS)–responsive nanoparticles loaded with an ultraviolet (UV)-light-responsive hydrogen sulfide (H<sub>2</sub>S) donor. This approach leverages the endogenous ROS present in diabetic wounds and external UV light as dual triggers to facilitate the controlled and stepwise release of H<sub>2</sub>S. The material design explicitly targets the critical challenges in diabetic wound repair, including the inhibition of chronic inflammation, oxidative stress reduction, microcirculation improvement, and support of cellular energy metabolism, thereby significantly accelerating wound healing. This adaptive release of signaling molecules effectively modulates the wound regeneration microenvironment, enhancing the repair process and offering a promising solution for diabetic skin wound management.</div></div><div><h3>Statement of significance</h3><div>This study developed an innovative strategy based on reactive oxygen species (ROS)–responsive nanoparticles loaded with an ultraviolet (UV)-light-responsive hydrogen sulfide (H<sub>2</sub>S) donor. This approach leverages the endogenous ROS present in diabetic wounds and external UV light as dual triggers to facilitate the controlled and stepwise release of H<sub>2</sub>S. The material design explicitly targets the critical challenges in diabetic wound repair, including the inhibition of chronic inflammation, oxidative stress reduction, microcirculation improvement, and support of cellular energy metabolism, thereby significantly accelerating wound healing. This adaptive release of signaling molecules effectively modulates the wound regeneration microenvironment, enhancing the repair process and offering a promising solution for diabetic skin wound management.</div></div>","PeriodicalId":237,"journal":{"name":"Acta Biomaterialia","volume":"196 ","pages":"Pages 364-379"},"PeriodicalIF":9.4,"publicationDate":"2025-04-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143494882","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}