Juan Antonio Romero-Torrecilla, José María Lamo-Espinosa, Purificación Ripalda-Cemboráin, Tania López-Martínez, Gloria Abizanda, Luis Riera-Álvarez, Sergio Ruiz de Galarreta-Moriones, Asier López-Barberena, Naiara Rodríguez-Flórez, Reyes Elizalde, Vineetha Jayawarna, José Valdés-Fernández, Miguel Echanove-González de Anleo, Peter Childs, Elena de Juan-Pardo, Manuel Salmeron-Sanchez, Felipe Prósper, Emma Muiños-López, Froilán Granero-Moltó
{"title":"An engineered periosteum for efficient delivery of rhBMP-2 and mesenchymal progenitor cells during bone regeneration.","authors":"Juan Antonio Romero-Torrecilla, José María Lamo-Espinosa, Purificación Ripalda-Cemboráin, Tania López-Martínez, Gloria Abizanda, Luis Riera-Álvarez, Sergio Ruiz de Galarreta-Moriones, Asier López-Barberena, Naiara Rodríguez-Flórez, Reyes Elizalde, Vineetha Jayawarna, José Valdés-Fernández, Miguel Echanove-González de Anleo, Peter Childs, Elena de Juan-Pardo, Manuel Salmeron-Sanchez, Felipe Prósper, Emma Muiños-López, Froilán Granero-Moltó","doi":"10.1038/s41536-023-00330-2","DOIUrl":"10.1038/s41536-023-00330-2","url":null,"abstract":"<p><p>During bone regeneration, the periosteum acts as a carrier for key regenerative cues, delivering osteochondroprogenitor cells and crucial growth factors to the injured bone. We developed a biocompatible, 3D polycaprolactone (PCL) melt electro-written membrane to act as a mimetic periosteum. Poly (ethyl acrylate) coating of the PCL membrane allowed functionalization, mediated by fibronectin and low dose recombinant human BMP-2 (rhBMP-2) (10-25 μg/ml), resulting in efficient, sustained osteoinduction in vitro. In vivo, rhBMP-2 functionalized mimetic periosteum demonstrated regenerative potential in the treatment of rat critical-size femoral defects with highly efficient healing and functional recovery (80%-93%). Mimetic periosteum has also proven to be efficient for cell delivery, as observed through the migration of transplanted periosteum-derived mesenchymal cells to the bone defect and their survival. Ultimately, mimetic periosteum demonstrated its ability to deliver key stem cells and morphogens to an injured site, exposing a therapeutic and translational potential in vivo when combined with unprecedentedly low rhBMP-2 doses.</p>","PeriodicalId":54236,"journal":{"name":"npj Regenerative Medicine","volume":"8 1","pages":"54"},"PeriodicalIF":7.2,"publicationDate":"2023-09-29","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10541910/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"41170399","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}
Devansh Agarwal, Nicholas Dash, Kevin W Mazo, Manan Chopra, Maria P Avila, Amit Patel, Ryan M Wong, Cairang Jia, Hope Do, Jie Cheng, Colette Chiang, Shawna L Jurlina, Mona Roshan, Michael W Perry, Jong M Rho, Risa Broyer, Cassidy D Lee, Robert N Weinreb, Cezar Gavrilovici, Nicholas W Oesch, Derek S Welsbie, Karl J Wahlin
{"title":"Human retinal ganglion cell neurons generated by synchronous BMP inhibition and transcription factor mediated reprogramming.","authors":"Devansh Agarwal, Nicholas Dash, Kevin W Mazo, Manan Chopra, Maria P Avila, Amit Patel, Ryan M Wong, Cairang Jia, Hope Do, Jie Cheng, Colette Chiang, Shawna L Jurlina, Mona Roshan, Michael W Perry, Jong M Rho, Risa Broyer, Cassidy D Lee, Robert N Weinreb, Cezar Gavrilovici, Nicholas W Oesch, Derek S Welsbie, Karl J Wahlin","doi":"10.1038/s41536-023-00327-x","DOIUrl":"10.1038/s41536-023-00327-x","url":null,"abstract":"<p><p>In optic neuropathies, including glaucoma, retinal ganglion cells (RGCs) die. Cell transplantation and endogenous regeneration offer strategies for retinal repair, however, developmental programs required for this to succeed are incompletely understood. To address this, we explored cellular reprogramming with transcription factor (TF) regulators of RGC development which were integrated into human pluripotent stem cells (PSCs) as inducible gene cassettes. When the pioneer factor NEUROG2 was combined with RGC-expressed TFs (ATOH7, ISL1, and POU4F2) some conversion was observed and when pre-patterned by BMP inhibition, RGC-like induced neurons (RGC-iNs) were generated with high efficiency in just under a week. These exhibited transcriptional profiles that were reminiscent of RGCs and exhibited electrophysiological properties, including AMPA-mediated synaptic transmission. Additionally, we demonstrated that small molecule inhibitors of DLK/LZK and GCK-IV can block neuronal death in two pharmacological axon injury models. Combining developmental patterning with RGC-specific TFs thus provided valuable insight into strategies for cell replacement and neuroprotection.</p>","PeriodicalId":54236,"journal":{"name":"npj Regenerative Medicine","volume":"8 1","pages":"55"},"PeriodicalIF":6.4,"publicationDate":"2023-09-29","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10541876/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"41173689","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}
Raymond M Wang, Joshua M Mesfin, Maria Karkanitsa, Jessica L Ungerleider, Emma Zelus, Yuxue Zhang, Yu Kawakami, Yuko Kawakami, Toshiaki Kawakami, Karen L Christman
{"title":"Immunomodulatory contribution of mast cells to the regenerative biomaterial microenvironment.","authors":"Raymond M Wang, Joshua M Mesfin, Maria Karkanitsa, Jessica L Ungerleider, Emma Zelus, Yuxue Zhang, Yu Kawakami, Yuko Kawakami, Toshiaki Kawakami, Karen L Christman","doi":"10.1038/s41536-023-00324-0","DOIUrl":"10.1038/s41536-023-00324-0","url":null,"abstract":"<p><p>Bioactive immunomodulatory biomaterials have shown promise for influencing the immune response to promote tissue repair and regeneration. Macrophages and T cells have been associated with this response; however, other immune cell types have been traditionally overlooked. In this study, we investigated the role of mast cells in the regulation of the immune response to decellularized biomaterial scaffolds using a subcutaneous implant model. In mast cell-deficient mice, there was dysregulation of the expected M1 to M2 macrophage transition typically induced by the biomaterial scaffold. Polarization progression deviated in a sex-specific manner with an early transition to an M2 profile in female mice, while the male response was unable to properly transition past a pro-inflammatory M1 state. Both were reversed with adoptive mast cell transfer. Further investigation of the later-stage immune response in male mice determined a greater sustained pro-inflammatory gene expression profile, including the IL-1 cytokine family, IL-6, alarmins, and chemokines. These results highlight mast cells as another important cell type that influences the immune response to pro-regenerative biomaterials.</p>","PeriodicalId":54236,"journal":{"name":"npj Regenerative Medicine","volume":"8 1","pages":"53"},"PeriodicalIF":7.2,"publicationDate":"2023-09-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10511634/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"41172747","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}
Stephanie L Tsai, Steffany Villaseñor, Rishita R Shah, Jenna L Galloway
{"title":"Endogenous tenocyte activation underlies the regenerative capacity of the adult zebrafish tendon.","authors":"Stephanie L Tsai, Steffany Villaseñor, Rishita R Shah, Jenna L Galloway","doi":"10.1038/s41536-023-00328-w","DOIUrl":"10.1038/s41536-023-00328-w","url":null,"abstract":"<p><p>Tendons are essential, frequently injured connective tissues that transmit forces from muscle to bone. Their unique highly ordered, matrix-rich structure is critical for proper function. While adult mammalian tendons heal after acute injuries, endogenous tendon cells, or tenocytes, fail to respond appropriately, resulting in the formation of disorganized fibrovascular scar tissue with impaired function and increased propensity for re-injury. Here, we show that, unlike mammals, adult zebrafish tenocytes activate upon injury and fully regenerate the tendon. Using a full tear injury model in the adult zebrafish craniofacial tendon, we defined the hallmark stages and cellular basis of tendon regeneration through multiphoton imaging, lineage tracing, and transmission electron microscopy approaches. Remarkably, we observe that zebrafish tendons regenerate and restore normal collagen matrix ultrastructure by 6 months post-injury (mpi). Tendon regeneration progresses in three main phases: inflammation within 24 h post-injury (hpi), cellular proliferation and formation of a cellular bridge between the severed tendon ends at 3-5 days post-injury (dpi), and re-differentiation and matrix remodeling beginning from 5 dpi to 6 mpi. Importantly, we demonstrate that pre-existing tenocytes are the main cellular source of regeneration, proliferating and migrating upon injury to ultimately bridge the tendon ends. Finally, we show that TGF-β signaling is required for tenocyte recruitment and bridge formation. Collectively, our work debuts and aptly positions the adult zebrafish tendon as an invaluable comparative system to elucidate regenerative mechanisms that may inspire new therapeutic strategies.</p>","PeriodicalId":54236,"journal":{"name":"npj Regenerative Medicine","volume":"8 1","pages":"52"},"PeriodicalIF":7.2,"publicationDate":"2023-09-19","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10509205/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"41106637","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}
Troy Anderson, Julia Mo, Ernesto Gagarin, Desmarie Sherwood, Maria Blumenkrantz, Eric Mao, Gianna Leon, Hailey Levitz, Hung-Jhen Chen, Kuo-Chang Tseng, Peter Fabian, J Gage Crump, Joanna Smeeton
{"title":"Ligament injury in adult zebrafish triggers ECM remodeling and cell dedifferentiation for scar-free regeneration.","authors":"Troy Anderson, Julia Mo, Ernesto Gagarin, Desmarie Sherwood, Maria Blumenkrantz, Eric Mao, Gianna Leon, Hailey Levitz, Hung-Jhen Chen, Kuo-Chang Tseng, Peter Fabian, J Gage Crump, Joanna Smeeton","doi":"10.1038/s41536-023-00329-9","DOIUrl":"10.1038/s41536-023-00329-9","url":null,"abstract":"<p><p>After traumatic injury, healing of mammalian ligaments is typically associated with fibrotic scarring as opposed to scar-free regeneration. In contrast, here we show that the ligament supporting the jaw joint of adult zebrafish is capable of rapid and complete scar-free healing. Following surgical transection of the jaw joint ligament, we observe breakdown of ligament tissue adjacent to the cut sites, expansion of mesenchymal tissue within the wound site, and then remodeling of extracellular matrix (ECM) to a normal ligament morphology. Lineage tracing of mature ligamentocytes following transection shows that they dedifferentiate, undergo cell cycle re-entry, and contribute to the regenerated ligament. Single-cell RNA sequencing of the regenerating ligament reveals dynamic expression of ECM genes in neural-crest-derived mesenchymal cells, as well as diverse immune cells expressing the endopeptidase-encoding gene legumain. Analysis of legumain mutant zebrafish shows a requirement for early ECM remodeling and efficient ligament regeneration. Our study establishes a new model of adult scar-free ligament regeneration and highlights roles of immune-mesenchyme cross-talk in ECM remodeling that initiates regeneration.</p>","PeriodicalId":54236,"journal":{"name":"npj Regenerative Medicine","volume":"8 1","pages":"51"},"PeriodicalIF":6.4,"publicationDate":"2023-09-19","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10509200/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"41157325","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}
Chi Ma, Min Sung Park, Felipe Alves do Monte, Vishal Gokani, Olumide O Aruwajoye, Yinshi Ren, Xiaohua Liu, Harry K W Kim
{"title":"Local BMP2 hydrogel therapy for robust bone regeneration in a porcine model of Legg-Calvé-Perthes disease.","authors":"Chi Ma, Min Sung Park, Felipe Alves do Monte, Vishal Gokani, Olumide O Aruwajoye, Yinshi Ren, Xiaohua Liu, Harry K W Kim","doi":"10.1038/s41536-023-00322-2","DOIUrl":"10.1038/s41536-023-00322-2","url":null,"abstract":"<p><p>Legg-Calvé-Perthes disease is juvenile idiopathic osteonecrosis of the femoral head (ONFH) that has no effective clinical treatment. Previously, local injection of bone morphogenetic protein-2 (BMP2) for ONFH treatment showed a heterogeneous bone repair and a high incidence of heterotopic ossification (HO) due to the BMP2 leakage. Here, we developed a BMP2-hydrogel treatment via a transphyseal bone wash and subsequential injection of BMP2-loaded hydrogel. In vitro studies showed that a hydrogel of gelatin-heparin-tyramine retained the BMP2 for four weeks. The injection of the hydrogel can efficiently prevent leakage. With the bone wash, the injected hydrogel had a broad distribution in the head. In vivo studies on pigs revealed that the BMP2-hydrogel treatment produced a homogeneous bone regeneration without HO. It preserved the subchondral contour and restored the subchondral endochondral ossification, although it increased growth plate fusions. In summary, the study demonstrated a promising BMP2-hydrogel treatment for ONFH treatment, especially for teenagers.</p>","PeriodicalId":54236,"journal":{"name":"npj Regenerative Medicine","volume":"8 1","pages":"50"},"PeriodicalIF":7.2,"publicationDate":"2023-09-14","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10502123/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"10320934","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}
Abigail L Lauterbach, Rachel P Wallace, Aaron T Alpar, Kirsten C Refvik, Joseph W Reda, Ako Ishihara, Taryn N Beckman, Anna J Slezak, Yukari Mizukami, Aslan Mansurov, Suzana Gomes, Jun Ishihara, Jeffrey A Hubbell
{"title":"Topically-applied collagen-binding serum albumin-fused interleukin-4 modulates wound microenvironment in non-healing wounds.","authors":"Abigail L Lauterbach, Rachel P Wallace, Aaron T Alpar, Kirsten C Refvik, Joseph W Reda, Ako Ishihara, Taryn N Beckman, Anna J Slezak, Yukari Mizukami, Aslan Mansurov, Suzana Gomes, Jun Ishihara, Jeffrey A Hubbell","doi":"10.1038/s41536-023-00326-y","DOIUrl":"10.1038/s41536-023-00326-y","url":null,"abstract":"<p><p>Non-healing wounds have a negative impact on quality of life and account for many cases of amputation and even early death among patients. Diabetic patients are the predominate population affected by these non-healing wounds. Despite the significant clinical demand, treatment with biologics has not broadly impacted clinical care. Interleukin-4 (IL-4) is a potent modulator of the immune system, capable of skewing macrophages towards a pro-regeneration phenotype (M2) and promoting angiogenesis, but can be toxic after frequent administration and is limited by its short half-life and low bioavailability. Here, we demonstrate the design and characterization of an engineered recombinant interleukin-4 construct. We utilize this collagen-binding, serum albumin-fused IL-4 variant (CBD-SA-IL-4) delivered in a hyaluronic acid (HA)-based gel for localized application of IL-4 to dermal wounds in a type 2 diabetic mouse model known for poor healing as proof-of-concept for improved tissue repair. Our studies indicate that CBD-SA-IL-4 is retained within the wound and can modulate the wound microenvironment through induction of M2 macrophages and angiogenesis. CBD-SA-IL-4 treatment significantly accelerated wound healing compared to native IL-4 and HA vehicle treatment without inducing systemic side effects. This CBD-SA-IL-4 construct can address the underlying immune dysfunction present in the non-healing wound, leading to more effective tissue healing in the clinic.</p>","PeriodicalId":54236,"journal":{"name":"npj Regenerative Medicine","volume":"8 1","pages":"49"},"PeriodicalIF":7.2,"publicationDate":"2023-09-11","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10495343/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"10605744","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}
Tristan Frum, Peggy P Hsu, Renee F C Hein, Ansley S Conchola, Charles J Zhang, Olivia R Utter, Abhinav Anand, Yi Zhang, Sydney G Clark, Ian Glass, Jonathan Z Sexton, Jason R Spence
{"title":"Opposing roles for TGFβ- and BMP-signaling during nascent alveolar differentiation in the developing human lung.","authors":"Tristan Frum, Peggy P Hsu, Renee F C Hein, Ansley S Conchola, Charles J Zhang, Olivia R Utter, Abhinav Anand, Yi Zhang, Sydney G Clark, Ian Glass, Jonathan Z Sexton, Jason R Spence","doi":"10.1038/s41536-023-00325-z","DOIUrl":"10.1038/s41536-023-00325-z","url":null,"abstract":"<p><p>Alveolar type 2 (AT2) cells function as stem cells in the adult lung and aid in repair after injury. The current study aimed to understand the signaling events that control differentiation of this therapeutically relevant cell type during human development. Using lung explant and organoid models, we identified opposing effects of TGFβ- and BMP-signaling, where inhibition of TGFβ- and activation of BMP-signaling in the context of high WNT- and FGF-signaling efficiently differentiated early lung progenitors into AT2-like cells in vitro. AT2-like cells differentiated in this manner exhibit surfactant processing and secretion capabilities, and long-term commitment to a mature AT2 phenotype when expanded in media optimized for primary AT2 culture. Comparing AT2-like cells differentiated with TGFβ-inhibition and BMP-activation to alternative differentiation approaches revealed improved specificity to the AT2 lineage and reduced off-target cell types. These findings reveal opposing roles for TGFβ- and BMP-signaling in AT2 differentiation and provide a new strategy to generate a therapeutically relevant cell type in vitro.</p>","PeriodicalId":54236,"journal":{"name":"npj Regenerative Medicine","volume":"8 1","pages":"48"},"PeriodicalIF":7.2,"publicationDate":"2023-09-09","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10492838/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"10596316","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}
Farwah Iqbal, Alexander Johnston, Brandon Wyse, Razieh Rabani, Poonam Mander, Banafshe Hoseini, Jun Wu, Ren-Ke Li, Andrée Gauthier-Fisher, Peter Szaraz, Clifford Librach
{"title":"Combination human umbilical cord perivascular and endothelial colony forming cell therapy for ischemic cardiac injury.","authors":"Farwah Iqbal, Alexander Johnston, Brandon Wyse, Razieh Rabani, Poonam Mander, Banafshe Hoseini, Jun Wu, Ren-Ke Li, Andrée Gauthier-Fisher, Peter Szaraz, Clifford Librach","doi":"10.1038/s41536-023-00321-3","DOIUrl":"10.1038/s41536-023-00321-3","url":null,"abstract":"<p><p>Cell-based therapeutics are promising interventions to repair ischemic cardiac tissue. However, no single cell type has yet been found to be both specialized and versatile enough to heal the heart. The synergistic effects of two regenerative cell types including endothelial colony forming cells (ECFC) and first-trimester human umbilical cord perivascular cells (FTM HUCPVC) with endothelial cell and pericyte properties respectively, on angiogenic and regenerative properties were tested in a rat model of myocardial infarction (MI), in vitro tube formation and Matrigel plug assay. The combination of FTM HUCPVCs and ECFCs synergistically reduced fibrosis and cardiomyocyte apoptosis, while promoting favorable cardiac remodeling and contractility. These effects were in part mediated by ANGPT2, PDGF-β, and VEGF-C. PDGF-β signaling-dependent synergistic effects on angiogenesis were also observed in vitro and in vivo. FTM HUCPVCs and ECFCs represent a cell combination therapy for promoting and sustaining vascularization following ischemic cardiac injury.</p>","PeriodicalId":54236,"journal":{"name":"npj Regenerative Medicine","volume":"8 1","pages":"45"},"PeriodicalIF":7.2,"publicationDate":"2023-08-25","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10457300/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"10096139","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}
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