Pub Date : 2024-08-11DOI: 10.1101/2024.08.10.607473
Chih-Tsung Yang, I-Chun Cho, Ching-Fang Yu, Edward Cheah, Tesi Liu, Yi-Ping Lin, Sing Yu, Jyun-Wei Jheng, Ivan Kempson, Tsi-Chian Chao, Sen-Hao Lee, Eva Bezak, Benjamin Thierry
Uncertainties on proton relative biological effectiveness (RBE) across the spread out of Bragg peak (SOBP) (typically assumed to be 1.1) may lead to suboptimal treatment plan and unwarranted toxicity to organs-at-risk. Herein, we report a reliable analytical method to determine the proton RBE along the SOBP and distal fall-off region. The 3D microtissue cassette enables the high throughput assessment of biological assays including clonogenic assay and γ-H2AX assay following a single proton irradiation. Clonogenic assay shows the RBE of 1.6 (10% cellular survival) which is consistent with the deter-mined RBE of 1.58 using the γ-H2AX assay. Besides, we also show that the high spatial resolution of the cassette can distinguish the minute but significant foci changes (number, area) in response to small proton radiation dose fraction. The results validate the reliability of our setup in addressing critical proton radiobiological questions.
{"title":"3D printed microtissue cassettes enabling high throughput proton radiobiological assays","authors":"Chih-Tsung Yang, I-Chun Cho, Ching-Fang Yu, Edward Cheah, Tesi Liu, Yi-Ping Lin, Sing Yu, Jyun-Wei Jheng, Ivan Kempson, Tsi-Chian Chao, Sen-Hao Lee, Eva Bezak, Benjamin Thierry","doi":"10.1101/2024.08.10.607473","DOIUrl":"https://doi.org/10.1101/2024.08.10.607473","url":null,"abstract":"Uncertainties on proton relative biological effectiveness (RBE) across the spread out of Bragg peak (SOBP) (typically assumed to be 1.1) may lead to suboptimal treatment plan and unwarranted toxicity to organs-at-risk. Herein, we report a reliable analytical method to determine the proton RBE along the SOBP and distal fall-off region. The 3D microtissue cassette enables the high throughput assessment of biological assays including clonogenic assay and γ-H2AX assay following a single proton irradiation. Clonogenic assay shows the RBE of 1.6 (10% cellular survival) which is consistent with the deter-mined RBE of 1.58 using the γ-H2AX assay. Besides, we also show that the high spatial resolution of the cassette can distinguish the minute but significant foci changes (number, area) in response to small proton radiation dose fraction. The results validate the reliability of our setup in addressing critical proton radiobiological questions.","PeriodicalId":501308,"journal":{"name":"bioRxiv - Bioengineering","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2024-08-11","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141941667","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-08-10DOI: 10.1101/2024.08.09.607383
Sydney Yang, Alexa Stern, Gregg Duncan
Bacterial biofilms are often highly resistant to antimicrobials causing persistent infections which when not effectively managed can significantly worsen clinical outcomes. As such, alternatives to standard antibiotic therapies have been highly sought after to address difficult-to-treat biofilm-associated infections. We hypothesized a biomaterial-based approach using the innate functions of mucins to modulate bacterial surface attachment and virulence could provide a new therapeutic strategy against biofilms. Based on our testing in Pseudomonas aeruginosa biofilms, we found synthetic mucus biomaterials can inhibit biofilm formation and significantly reduce the thickness of mature biofilms. In addition, we evaluated if synthetic mucus biomaterials could work synergistically with DNase and/or α-amylase for enhanced biofilm dispersal. Combination treatment with these antibiofilm agents and synthetic mucus biomaterials resulted in up to 3 log reductions in viability of mature P. aeruginosa biofilms. Overall, this work provides a new bio-inspired, combinatorial approach to address biofilms and antibiotic-resistant bacterial infections.
{"title":"Synthetic mucus biomaterials synergize with antibiofilm agents to combat Pseudomonas aeruginosa biofilms","authors":"Sydney Yang, Alexa Stern, Gregg Duncan","doi":"10.1101/2024.08.09.607383","DOIUrl":"https://doi.org/10.1101/2024.08.09.607383","url":null,"abstract":"Bacterial biofilms are often highly resistant to antimicrobials causing persistent infections which when not effectively managed can significantly worsen clinical outcomes. As such, alternatives to standard antibiotic therapies have been highly sought after to address difficult-to-treat biofilm-associated infections. We hypothesized a biomaterial-based approach using the innate functions of mucins to modulate bacterial surface attachment and virulence could provide a new therapeutic strategy against biofilms. Based on our testing in Pseudomonas aeruginosa biofilms, we found synthetic mucus biomaterials can inhibit biofilm formation and significantly reduce the thickness of mature biofilms. In addition, we evaluated if synthetic mucus biomaterials could work synergistically with DNase and/or α-amylase for enhanced biofilm dispersal. Combination treatment with these antibiofilm agents and synthetic mucus biomaterials resulted in up to 3 log reductions in viability of mature P. aeruginosa biofilms. Overall, this work provides a new bio-inspired, combinatorial approach to address biofilms and antibiotic-resistant bacterial infections.","PeriodicalId":501308,"journal":{"name":"bioRxiv - Bioengineering","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2024-08-10","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141941670","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-08-10DOI: 10.1101/2024.08.09.607364
Wenxi Xia, Matthew Goff, Neetu Singh, Jiemin Huang, David Gillespie, Esther Need, Randy Jensen, Mark Pagel, Amit Maity, Sixiang Shi, Shreya Goel
Tumor hypoxia leads to increased resistance to radiation therapy (RT), resulting in markedly worse clinical outcomes in the treatment and management of pediatric malignant rhabdoid tumors (MRT). To alleviate hypoxia in MRT, we repurposed an FDA approved, mitochondrial oxidative phosphorylation (OXPHOS) inhibitor, Atovaquone (AVO), to inhibit oxygen consumption and thereby enhance the sensitivity of tumor cells to low dose RT in MRT by hypoxia alleviation. Additionally, to better understand the tumor response induced by AVO and optimize the combination with RT, we employed an emerging, noninvasive imaging modality, known as multispectral optoacoustic tomography (MSOT), to monitor and evaluate real-time dynamic changes in tumor hypoxia and vascular perfusion. Oxygen-Enhanced (OE)-MSOT could measure the change of tumor oxygenation in the MRT xenograft models after AVO and RT treatments, indicating its potential as a response biomarker. OE-MSOT showed that treating MRT mouse models with AVO resulted in a transient increase in oxygen saturation (ΔsO2) in tumors when the mice were subjected to oxygen challenge, while RT or saline treated groups produced no change. In AVO+RT combination groups, the tumors showed an increase in ΔsO2 after AVO administration followed by a significant decrease after RT, that correlated with a strong anti-tumor response, demarcated by complete regression of tumors, with no relapse on long-term monitoring. These observations were histologically validated. In MRT models of acquired AVO resistance, combination therapy failed to alleviate tumoral hypoxia and elicit any therapeutic benefit. Together, our data highlights the utility of repurposing anti-malarial AVO as an anticancer adjuvant for enabling low dose RT for pediatric patients.
{"title":"Imaging-Guided Metabolic Radiosensitization of Pediatric Rhabdoid Tumors","authors":"Wenxi Xia, Matthew Goff, Neetu Singh, Jiemin Huang, David Gillespie, Esther Need, Randy Jensen, Mark Pagel, Amit Maity, Sixiang Shi, Shreya Goel","doi":"10.1101/2024.08.09.607364","DOIUrl":"https://doi.org/10.1101/2024.08.09.607364","url":null,"abstract":"Tumor hypoxia leads to increased resistance to radiation therapy (RT), resulting in markedly worse clinical outcomes in the treatment and management of pediatric malignant rhabdoid tumors (MRT). To alleviate hypoxia in MRT, we repurposed an FDA approved, mitochondrial oxidative phosphorylation (OXPHOS) inhibitor, Atovaquone (AVO), to inhibit oxygen consumption and thereby enhance the sensitivity of tumor cells to low dose RT in MRT by hypoxia alleviation. Additionally, to better understand the tumor response induced by AVO and optimize the combination with RT, we employed an emerging, noninvasive imaging modality, known as multispectral optoacoustic tomography (MSOT), to monitor and evaluate real-time dynamic changes in tumor hypoxia and vascular perfusion. Oxygen-Enhanced (OE)-MSOT could measure the change of tumor oxygenation in the MRT xenograft models after AVO and RT treatments, indicating its potential as a response biomarker. OE-MSOT showed that treating MRT mouse models with AVO resulted in a transient increase in oxygen saturation (ΔsO2) in tumors when the mice were subjected to oxygen challenge, while RT or saline treated groups produced no change. In AVO+RT combination groups, the tumors showed an increase in ΔsO2 after AVO administration followed by a significant decrease after RT, that correlated with a strong anti-tumor response, demarcated by complete regression of tumors, with no relapse on long-term monitoring. These observations were histologically validated. In MRT models of acquired AVO resistance, combination therapy failed to alleviate tumoral hypoxia and elicit any therapeutic benefit. Together, our data highlights the utility of repurposing anti-malarial AVO as an anticancer adjuvant for enabling low dose RT for pediatric patients.","PeriodicalId":501308,"journal":{"name":"bioRxiv - Bioengineering","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2024-08-10","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141969064","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-08-10DOI: 10.1101/2024.08.10.607458
Eric L Schneider, John A Hangasky, Rocio del Valle Fernandez, Gary W Ashley, Daniel V Santi
The objective of this work was to develop a long-acting form of the lipidated peptide semaglutide that can be administered to humans once-monthly. Semaglutide was attached to 50 μ diameter hydrogel microspheres by a cleavable linker with an expected in vivo release half-life of about one-month. After a single subcutaneous dose, the pharmacokinetic parameters of released semaglutide were determined in normal mice and the bodyweight loss was determined in diet induced obese mice. The results were used to simulate the pharmacokinetics of semaglutide released from the microspheres in humans.Semaglutide tethered to microspheres by a cleavable linker could be completely released with an in vitro half-life of ~55 days at pH 7.4. The in vivo half-life of released semaglutide was ~30 days, and a single dose in diet-induced obese mice resulted in a lean-sparing body weight loss of 20% over 1 month, statistically the same as semaglutide dosed twice daily. Simulations indicated the microsphere-semaglutide would permit once-monthly administration in humans. The microsphere-semaglutide conjugate described here should be suitable for once-monthly dosing in humans, and the same approach should enable conversion of other lipidated peptides from once-weekly to once-monthly administration.
{"title":"The limitation of lipidation: conversion of semaglutide from once-weekly to once-monthly dosing","authors":"Eric L Schneider, John A Hangasky, Rocio del Valle Fernandez, Gary W Ashley, Daniel V Santi","doi":"10.1101/2024.08.10.607458","DOIUrl":"https://doi.org/10.1101/2024.08.10.607458","url":null,"abstract":"The objective of this work was to develop a long-acting form of the lipidated peptide semaglutide that can be administered to humans once-monthly. Semaglutide was attached to 50 μ diameter hydrogel microspheres by a cleavable linker with an expected in vivo release half-life of about one-month. After a single subcutaneous dose, the pharmacokinetic parameters of released semaglutide were determined in normal mice and the bodyweight loss was determined in diet induced obese mice. The results were used to simulate the pharmacokinetics of semaglutide released from the microspheres in humans.Semaglutide tethered to microspheres by a cleavable linker could be completely released with an in vitro half-life of ~55 days at pH 7.4. The in vivo half-life of released semaglutide was ~30 days, and a single dose in diet-induced obese mice resulted in a lean-sparing body weight loss of 20% over 1 month, statistically the same as semaglutide dosed twice daily. Simulations indicated the microsphere-semaglutide would permit once-monthly administration in humans. The microsphere-semaglutide conjugate described here should be suitable for once-monthly dosing in humans, and the same approach should enable conversion of other lipidated peptides from once-weekly to once-monthly administration.","PeriodicalId":501308,"journal":{"name":"bioRxiv - Bioengineering","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2024-08-10","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141941668","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-08-10DOI: 10.1101/2024.08.08.607216
Yang Zhang, Maria Savvidou, Volha Liaudanskaya, Varshini Ramanathan, Thi Bui, Lindley Matthew, Ash Sze, Ugochukwu Obinna Ugwu, Fu Yuhang, Dilsizian E Matthew, Xinjie Chen, Sevara Nasritdinova, Aonkon Dey, Eric L Miller, David L Kaplan, Irene Georgakoudi
Brain metabolism is essential for the function of organisms. While established imaging methods provide valuable insights into brain metabolic function, they lack the resolution to capture important metabolic interactions and heterogeneity at the cellular level. Label-free, two-photon excited fluorescence imaging addresses this issue by enabling dynamic metabolic assessments at the single-cell level without manipulations. In this study, we demonstrate the impact of spectral imaging on the development of rigorous intensity and lifetime label-free imaging protocols to assess dynamically over time metabolic function in 3D engineered brain tissue models comprising human induced neural stem cells, astrocytes, and microglia. Specifically, we rely on multi-wavelength spectral imaging to identify the excitation/emission profiles of key cellular fluorophores within human brain cells, including NAD(P)H, LipDH, FAD, and lipofuscin. These enable development of methods to mitigate lipofuscin's overlap with NAD(P)H and flavin autofluorescence to extract reliable optical metabolic function metrics from images acquired at two excitation wavelengths over two emission bands. We present fluorescence intensity and lifetime metrics reporting on redox state, mitochondrial fragmentation, and NAD(P)H binding status in neuronal monoculture and triculture systems, to highlight the functional impact of metabolic interactions between different cell types. Our findings reveal significant metabolic differences between neurons and glial cells, shedding light on metabolic pathway utilization, including the glutathione pathway, OXPHOS, glycolysis, and fatty acid oxidation. Collectively, our studies establish a label-free, non-destructive approach to assess the metabolic function and interactions among different brain cell types relying on endogenous fluorescence and illustrate the complementary nature of information that is gained by combining intensity and lifetime-based images. Such methods can improve understanding of physiological brain function and dysfunction that occurs at the onset of cancers, traumatic injuries and neurodegenerative diseases.
{"title":"Multi-modal, Label-free, Optical Mapping of Cellular Metabolic Function and Oxidative Stress in 3D Engineered Brain Tissue Models","authors":"Yang Zhang, Maria Savvidou, Volha Liaudanskaya, Varshini Ramanathan, Thi Bui, Lindley Matthew, Ash Sze, Ugochukwu Obinna Ugwu, Fu Yuhang, Dilsizian E Matthew, Xinjie Chen, Sevara Nasritdinova, Aonkon Dey, Eric L Miller, David L Kaplan, Irene Georgakoudi","doi":"10.1101/2024.08.08.607216","DOIUrl":"https://doi.org/10.1101/2024.08.08.607216","url":null,"abstract":"Brain metabolism is essential for the function of organisms. While established imaging methods provide valuable insights into brain metabolic function, they lack the resolution to capture important metabolic interactions and heterogeneity at the cellular level. Label-free, two-photon excited fluorescence imaging addresses this issue by enabling dynamic metabolic assessments at the single-cell level without manipulations. In this study, we demonstrate the impact of spectral imaging on the development of rigorous intensity and lifetime label-free imaging protocols to assess dynamically over time metabolic function in 3D engineered brain tissue models comprising human induced neural stem cells, astrocytes, and microglia. Specifically, we rely on multi-wavelength spectral imaging to identify the excitation/emission profiles of key cellular fluorophores within human brain cells, including NAD(P)H, LipDH, FAD, and lipofuscin. These enable development of methods to mitigate lipofuscin's overlap with NAD(P)H and flavin autofluorescence to extract reliable optical metabolic function metrics from images acquired at two excitation wavelengths over two emission bands. We present fluorescence intensity and lifetime metrics reporting on redox state, mitochondrial fragmentation, and NAD(P)H binding status in neuronal monoculture and triculture systems, to highlight the functional impact of metabolic interactions between different cell types. Our findings reveal significant metabolic differences between neurons and glial cells, shedding light on metabolic pathway utilization, including the glutathione pathway, OXPHOS, glycolysis, and fatty acid oxidation. Collectively, our studies establish a label-free, non-destructive approach to assess the metabolic function and interactions among different brain cell types relying on endogenous fluorescence and illustrate the complementary nature of information that is gained by combining intensity and lifetime-based images. Such methods can improve understanding of physiological brain function and dysfunction that occurs at the onset of cancers, traumatic injuries and neurodegenerative diseases.","PeriodicalId":501308,"journal":{"name":"bioRxiv - Bioengineering","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2024-08-10","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141941673","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-08-10DOI: 10.1101/2024.08.09.606830
Ankita Pramanick, Thomas Hayes, Eoin McEvoy, Abhay Pandit, Andrew Daly
During embryogenesis, organs undergo dynamic shape transformations that sculpt their final shape, composition, and function. Despite this, current organ bioprinting approaches typically employ bioinks that restrict cell-generated morphogenetic behaviours resulting in structurally static tissues. Here, we introduce a novel platform that enables the bioprinting of tissues that undergo programmable and predictable 4D shape-morphing driven by cell-generated forces. Our method utilises embedded bioprinting to deposit collagen-hyaluronic acid bioinks within yield-stress granular support hydrogels that can accommodate and regulate 4D shape-morphing through their viscoelastic properties. Importantly, we demonstrate precise control over 4D shape-morphing by modulating factors such as the initial print geometry, cell phenotype, bioink composition, and support hydrogel viscoelasticity. Further, we observed that shape-morphing actively sculpts cell and extracellular matrix alignment along the principal tissue axis through a stress-avoidance mechanism. To enable predictive design of 4D shape-morphing patterns, we developed a finite element model that accurately captures shape evolution at both the cellular and tissue levels. Finally, we show that programmed 4D shape-morphing enhances the structural and functional properties of iPSC-derived heart tissues. This ability to design, predict, and program 4D shape-morphing holds great potential for engineering organ rudiments that recapitulate morphogenetic processes to sculpt their final shape, composition, and function.
{"title":"4D bioprinting shape-morphing tissues in granular support hydrogels: Sculpting structure and guiding maturation","authors":"Ankita Pramanick, Thomas Hayes, Eoin McEvoy, Abhay Pandit, Andrew Daly","doi":"10.1101/2024.08.09.606830","DOIUrl":"https://doi.org/10.1101/2024.08.09.606830","url":null,"abstract":"During embryogenesis, organs undergo dynamic shape transformations that sculpt their final shape, composition, and function. Despite this, current organ bioprinting approaches typically employ bioinks that restrict cell-generated morphogenetic behaviours resulting in structurally static tissues. Here, we introduce a novel platform that enables the bioprinting of tissues that undergo programmable and predictable 4D shape-morphing driven by cell-generated forces. Our method utilises embedded bioprinting to deposit collagen-hyaluronic acid bioinks within yield-stress granular support hydrogels that can accommodate and regulate 4D shape-morphing through their viscoelastic properties. Importantly, we demonstrate precise control over 4D shape-morphing by modulating factors such as the initial print geometry, cell phenotype, bioink composition, and support hydrogel viscoelasticity. Further, we observed that shape-morphing actively sculpts cell and extracellular matrix alignment along the principal tissue axis through a stress-avoidance mechanism. To enable predictive design of 4D shape-morphing patterns, we developed a finite element model that accurately captures shape evolution at both the cellular and tissue levels. Finally, we show that programmed 4D shape-morphing enhances the structural and functional properties of iPSC-derived heart tissues. This ability to design, predict, and program 4D shape-morphing holds great potential for engineering organ rudiments that recapitulate morphogenetic processes to sculpt their final shape, composition, and function.","PeriodicalId":501308,"journal":{"name":"bioRxiv - Bioengineering","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2024-08-10","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141941672","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-08-10DOI: 10.1101/2024.08.10.607417
Donia W Ahmed, Matthew L Tan, Jackson Gabbard, Yuchen Liu, Michael M Hu, Miriam Stevens, Firaol S Midekssa, Lin Han, Rachel Lynne Zemans, Brendon Baker, Claudia Loebel
Within most tissues, the extracellular microenvironment provides mechanical cues that guide cell fate and function. Changes in the extracellular matrix such as aberrant deposition, densification and increased crosslinking are hallmarks of late-stage fibrotic diseases that often lead to organ dysfunction. Biomaterials have been widely used to mimic the mechanical properties of the fibrotic matrix and study cell function. However, the initiation of fibrosis has largely been overlooked, due to the challenges in recapitulating early fibrotic lesions within the native extracellular microenvironment. Using visible light mediated photochemistry, we induced local crosslinking and stiffening of extracellular matrix proteins within ex vivo murine and human tissue. In ex vivo lung tissue of epithelial cell lineage-traced mice, local matrix crosslinking mimicked early fibrotic lesions that increased alveolar epithelial cell spreading, differentiation and extracellular matrix remodeling. However, inhibition of cytoskeletal tension or integrin engagement reduced epithelial cell spreading and differentiation, resulting in alveolar epithelial cell dedifferentiation and reduced extracellular matrix deposition. Our findings emphasize the role of local extracellular matrix crosslinking and remodeling in early-stage tissue fibrosis and have implications for ex vivo disease modeling and applications to other tissues.
{"title":"Local photo-crosslinking of native tissue matrix regulates cell function","authors":"Donia W Ahmed, Matthew L Tan, Jackson Gabbard, Yuchen Liu, Michael M Hu, Miriam Stevens, Firaol S Midekssa, Lin Han, Rachel Lynne Zemans, Brendon Baker, Claudia Loebel","doi":"10.1101/2024.08.10.607417","DOIUrl":"https://doi.org/10.1101/2024.08.10.607417","url":null,"abstract":"Within most tissues, the extracellular microenvironment provides mechanical cues that guide cell fate and function. Changes in the extracellular matrix such as aberrant deposition, densification and increased crosslinking are hallmarks of late-stage fibrotic diseases that often lead to organ dysfunction. Biomaterials have been widely used to mimic the mechanical properties of the fibrotic matrix and study cell function. However, the initiation of fibrosis has largely been overlooked, due to the challenges in recapitulating early fibrotic lesions within the native extracellular microenvironment. Using visible light mediated photochemistry, we induced local crosslinking and stiffening of extracellular matrix proteins within ex vivo murine and human tissue. In ex vivo lung tissue of epithelial cell lineage-traced mice, local matrix crosslinking mimicked early fibrotic lesions that increased alveolar epithelial cell spreading, differentiation and extracellular matrix remodeling. However, inhibition of cytoskeletal tension or integrin engagement reduced epithelial cell spreading and differentiation, resulting in alveolar epithelial cell dedifferentiation and reduced extracellular matrix deposition. Our findings emphasize the role of local extracellular matrix crosslinking and remodeling in early-stage tissue fibrosis and have implications for ex vivo disease modeling and applications to other tissues.","PeriodicalId":501308,"journal":{"name":"bioRxiv - Bioengineering","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2024-08-10","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141941669","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-08-10DOI: 10.1101/2024.08.10.607464
Tatsuya Osaki, Chloe Delepine, Yuma Osako, Devorah Kranz, April R Levin, Charles Nelson, Michela Fagiolini, Mriganka Sur
Human cerebral organoids derived from induced pluripotent stem cells can recapture early developmental processes and reveal changes involving neurodevelopmental disorders. Mutations in the X linked methyl CpG binding protein 2 (MECP2) gene are associated with Rett syndrome, and disease severity varies depending on the location and type of mutation. Here, we focused on neuronal activity in Rett syndrome patient-derived organoids, analyzing two types of MeCP2 mutations a missense mutation (R306C) and a truncating mutation (V247X) using calcium imaging with three-photon microscopy. Compared to isogenic controls, we found abnormal neuronal activity in Rett organoids and altered network function based on graph theoretic analyses, with V247X mutations impacting functional responses and connectivity more severely than R306C mutations. These changes paralleled EEG data obtained from patients with comparable mutations. Labeling DLX promoter-driven inhibitory neurons demonstrated differences in activity and functional connectivity of inhibitory and excitatory neurons in the two types of mutation. Transcriptomic analyses revealed HDAC2-associated impairment in R306C organoids and decreased GABAA receptor expression in excitatory neurons in V247X organoids. These findings demonstrate mutation-specific mechanisms of vulnerability in Rett syndrome and suggest targeted strategies for their treatment.
{"title":"Early differential impact of MeCP2 mutations on functional networks in Rett syndrome patient-derived human cerebral organoids","authors":"Tatsuya Osaki, Chloe Delepine, Yuma Osako, Devorah Kranz, April R Levin, Charles Nelson, Michela Fagiolini, Mriganka Sur","doi":"10.1101/2024.08.10.607464","DOIUrl":"https://doi.org/10.1101/2024.08.10.607464","url":null,"abstract":"Human cerebral organoids derived from induced pluripotent stem cells can recapture early developmental processes and reveal changes involving neurodevelopmental disorders. Mutations in the X linked methyl CpG binding protein 2 (MECP2) gene are associated with Rett syndrome, and disease severity varies depending on the location and type of mutation. Here, we focused on neuronal activity in Rett syndrome patient-derived organoids, analyzing two types of MeCP2 mutations a missense mutation (R306C) and a truncating mutation (V247X) using calcium imaging with three-photon microscopy. Compared to isogenic controls, we found abnormal neuronal activity in Rett organoids and altered network function based on graph theoretic analyses, with V247X mutations impacting functional responses and connectivity more severely than R306C mutations. These changes paralleled EEG data obtained from patients with comparable mutations. Labeling DLX promoter-driven inhibitory neurons demonstrated differences in activity and functional connectivity of inhibitory and excitatory neurons in the two types of mutation. Transcriptomic analyses revealed HDAC2-associated impairment in R306C organoids and decreased GABAA receptor expression in excitatory neurons in V247X organoids. These findings demonstrate mutation-specific mechanisms of vulnerability in Rett syndrome and suggest targeted strategies for their treatment.","PeriodicalId":501308,"journal":{"name":"bioRxiv - Bioengineering","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2024-08-10","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141969061","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-08-09DOI: 10.1101/2024.08.08.607163
Deepti Rana, Jeroen Rouwkema
In tissue extracellular matrix (ECM), multiple growth factors (GFs) are sequestered through affinity interactions and released as needed by proteases, establishing spatial morphogen gradients in a time-controlled manner to guide cell behavior. Inspired by these ECM characteristics, we developed an intelligent biomaterial platform that spatially controls the combined bioavailability of multiple angiogenic GFs, specifically vascular endothelial growth factor (VEGF) and platelet-derived growth factor (PDGF-BB). Utilizing aptamer affinity interactions and complementary sequences within a GelMA matrix, our platform achieves on-demand, triggered release of individual GFs which can be programmed in temporally-controlled, repeatable cycles. The platform features stable incorporation of dual aptamers specific for both GFs, functional aptamer-CS molecular recognition in a 3D microenvironment with long-term stability of at least 15 days at physiological temperature, and spatially localized sequestration of individual GFs. Additionally, the system allows differential amounts of GFs to be released from the same hydrogels at different time-points, mimicking dynamic GF presentation in a 3D matrix similar to the native ECM. This flexible control over individual GF release kinetics opens new possibilities for dynamic GF presentation, with adjustable release profiles to meet the spatiotemporal needs of growing engineered tissue.
{"title":"Spatiotemporally Programmed Release of Aptamer Tethered Dual Angiogenic Growth Factors","authors":"Deepti Rana, Jeroen Rouwkema","doi":"10.1101/2024.08.08.607163","DOIUrl":"https://doi.org/10.1101/2024.08.08.607163","url":null,"abstract":"In tissue extracellular matrix (ECM), multiple growth factors (GFs) are sequestered through affinity interactions and released as needed by proteases, establishing spatial morphogen gradients in a time-controlled manner to guide cell behavior. Inspired by these ECM characteristics, we developed an intelligent biomaterial platform that spatially controls the combined bioavailability of multiple angiogenic GFs, specifically vascular endothelial growth factor (VEGF) and platelet-derived growth factor (PDGF-BB). Utilizing aptamer affinity interactions and complementary sequences within a GelMA matrix, our platform achieves on-demand, triggered release of individual GFs which can be programmed in temporally-controlled, repeatable cycles. The platform features stable incorporation of dual aptamers specific for both GFs, functional aptamer-CS molecular recognition in a 3D microenvironment with long-term stability of at least 15 days at physiological temperature, and spatially localized sequestration of individual GFs. Additionally, the system allows differential amounts of GFs to be released from the same hydrogels at different time-points, mimicking dynamic GF presentation in a 3D matrix similar to the native ECM. This flexible control over individual GF release kinetics opens new possibilities for dynamic GF presentation, with adjustable release profiles to meet the spatiotemporal needs of growing engineered tissue.","PeriodicalId":501308,"journal":{"name":"bioRxiv - Bioengineering","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2024-08-09","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141941675","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-08-09DOI: 10.1101/2024.08.09.607294
Diane Salman, Jason Amatoury
Background and Objectives: The hyoid bone's inferior baseline position in obstructive sleep apnea (OSA) has led to surgical hyoid repositioning treatment, yet outcomes vary widely. The influence of baseline hyoid position (phenotype) and surgical hyoid repositioning on upper airway function remains unclear. We aimed to investigate their impact on the upper airway using computational modeling. Methods: A validated finite element model of the rabbit upper airway was advanced and used to simulate changes in baseline hyoid position and surgical hyoid repositioning, alone and in combination. The hyoid was displaced in cranial, caudal, anterior, anterior-cranial and anterior-caudal directions from 1-4mm. Model outcomes included upper airway collapsibility, measured using closing pressure (Pclose), cross-sectional area (CSA) and soft tissue mechanics (stress and strain). Results: Graded baseline hyoid position increments increased Pclose for all directions, and up to 29-43% at 4mm (relative to the original baseline hyoid position). Anterior-based surgical hyoid repositioning decreased Pclose (~-115% at 4mm) and increased ΔCSA (~+35% at 4mm). Cranial surgical hyoid repositioning decreased ΔPclose (-29%), minimally affecting CSA. Caudal surgical hyoid repositioning increased ΔPclose (+27%) and decreased ΔCSA (-7%). Anterior-cranial and anterior-caudal surgical hyoid repositioning produced the highest stresses and strains. Surgical hyoid repositioning effects on upper airway outcomes were dependent on baseline hyoid position, with more caudal baseline hyoid positions leading to less effective surgeries. Conclusions: Baseline hyoid position (phenotype) and surgical hyoid repositioning both alter upper airway outcomes, with effects dependent on hyoid displacement direction and magnitude. Baseline hyoid position influences the effectiveness of surgical hyoid repositioning in reducing upper airway collapsibility. These findings provide further insights into the hyoid's role in upper airway patency and suggest that considering the hyoid's baseline position and surgical repositioning direction/increment may help improve hyoid surgeries for OSA treatment.
{"title":"Hyoid bone position and upper airway patency: A computational finite element modeling study","authors":"Diane Salman, Jason Amatoury","doi":"10.1101/2024.08.09.607294","DOIUrl":"https://doi.org/10.1101/2024.08.09.607294","url":null,"abstract":"Background and Objectives: The hyoid bone's inferior baseline position in obstructive sleep apnea (OSA) has led to surgical hyoid repositioning treatment, yet outcomes vary widely. The influence of baseline hyoid position (phenotype) and surgical hyoid repositioning on upper airway function remains unclear. We aimed to investigate their impact on the upper airway using computational modeling. Methods: A validated finite element model of the rabbit upper airway was advanced and used to simulate changes in baseline hyoid position and surgical hyoid repositioning, alone and in combination. The hyoid was displaced in cranial, caudal, anterior, anterior-cranial and anterior-caudal directions from 1-4mm. Model outcomes included upper airway collapsibility, measured using closing pressure (Pclose), cross-sectional area (CSA) and soft tissue mechanics (stress and strain). Results: Graded baseline hyoid position increments increased Pclose for all directions, and up to 29-43% at 4mm (relative to the original baseline hyoid position). Anterior-based surgical hyoid repositioning decreased Pclose (~-115% at 4mm) and increased ΔCSA (~+35% at 4mm). Cranial surgical hyoid repositioning decreased ΔPclose (-29%), minimally affecting CSA. Caudal surgical hyoid repositioning increased ΔPclose (+27%) and decreased ΔCSA (-7%). Anterior-cranial and anterior-caudal surgical hyoid repositioning produced the highest stresses and strains. Surgical hyoid repositioning effects on upper airway outcomes were dependent on baseline hyoid position, with more caudal baseline hyoid positions leading to less effective surgeries. Conclusions: Baseline hyoid position (phenotype) and surgical hyoid repositioning both alter upper airway outcomes, with effects dependent on hyoid displacement direction and magnitude. Baseline hyoid position influences the effectiveness of surgical hyoid repositioning in reducing upper airway collapsibility. These findings provide further insights into the hyoid's role in upper airway patency and suggest that considering the hyoid's baseline position and surgical repositioning direction/increment may help improve hyoid surgeries for OSA treatment.","PeriodicalId":501308,"journal":{"name":"bioRxiv - Bioengineering","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2024-08-09","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141941602","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
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