Pub Date : 2024-12-01DOI: 10.1016/j.actbio.2024.10.022
Mengjun Ge , Haitao Zou , Jiahao Chen , Qinyao Zhang , Chang Li , Jiaxing Yang , Jiumei Wu , Xing Xie , Jun Liu , Lei Lei , Shaoliang Peng , Hemin Nie
Liver fibrosis is a key process in the progression of chronic liver disease to cirrhosis. Currently, early diagnosis and precise staging of liver fibrosis remain great challenges. Extracellular matrix (ECM) molecules expressed specifically during liver fibrosis are ideal targets for bioimaging and detection of liver fibrosis. Here, we report that fluorescent probes based on a nucleic acid aptamer (ZY-1) targeting cellular fibronectin (cFN), a critical ECM molecule significantly accumulating during liver fibrosis, are promising bioimaging agents for the staging of liver fibrosis. In the work, the outstanding binding affinity of ZY-1 to cFN was validated through an in vitro model of human-derived hepatic stellate cells (HSCs). Subsequently, we constructed different ZY-1-based fluorescent probes and explored the real-time imaging performance of these fluorescent probes in CCl4-induced mouse models of different liver fibrosis stages. The ZY-1-based fluorescent probes, for the first time, effectively identified and distinguished early-stage liver fibrosis (stage 3 of Ishak 6) from advanced liver fibrosis (stage 5 of Ishak 6). The proof-of-concept study provides compelling evidences that ZY-1-based probes are a promising tool for the early diagnosis and staging of liver fibrosis and paves the way for further development of clinical-related diagnosis strategies for fibrotic diseases of the liver and other organs.
Statement of significance
Currently, early diagnosis and accurate staging of liver fibrosis continue to present significant challenges. This study demonstrates that fluorescent probes based on the nucleic acid aptamer ZY-1, which targets cellular fibronectin (cFN)—a crucial extracellular matrix (ECM) molecule that significantly accumulates during liver fibrosis—are promising bioimaging agents for staging liver fibrosis. The ZY-1-based fluorescent probes effectively identified and differentiated early-stage liver fibrosis from advanced liver fibrosis. This proof-of-concept study not only provides compelling evidence that ZY-1-based probes show promise for the early diagnosis and staging of liver fibrosis but also paves the way for further investigations into the use of ZY-1 in detecting other diseases associated with cFN.
{"title":"Cellular fibronectin-targeted fluorescent aptamer probes for early detection and staging of liver fibrosis","authors":"Mengjun Ge , Haitao Zou , Jiahao Chen , Qinyao Zhang , Chang Li , Jiaxing Yang , Jiumei Wu , Xing Xie , Jun Liu , Lei Lei , Shaoliang Peng , Hemin Nie","doi":"10.1016/j.actbio.2024.10.022","DOIUrl":"10.1016/j.actbio.2024.10.022","url":null,"abstract":"<div><div>Liver fibrosis is a key process in the progression of chronic liver disease to cirrhosis. Currently, early diagnosis and precise staging of liver fibrosis remain great challenges. Extracellular matrix (ECM) molecules expressed specifically during liver fibrosis are ideal targets for bioimaging and detection of liver fibrosis. Here, we report that fluorescent probes based on a nucleic acid aptamer (ZY-1) targeting cellular fibronectin (cFN), a critical ECM molecule significantly accumulating during liver fibrosis, are promising bioimaging agents for the staging of liver fibrosis. In the work, the outstanding binding affinity of ZY-1 to cFN was validated through an <em>in vitro</em> model of human-derived hepatic stellate cells (HSCs). Subsequently, we constructed different ZY-1-based fluorescent probes and explored the real-time imaging performance of these fluorescent probes in CCl<sub>4</sub>-induced mouse models of different liver fibrosis stages. The ZY-1-based fluorescent probes, for the first time, effectively identified and distinguished early-stage liver fibrosis (stage 3 of Ishak 6) from advanced liver fibrosis (stage 5 of Ishak 6). The proof-of-concept study provides compelling evidences that ZY-1-based probes are a promising tool for the early diagnosis and staging of liver fibrosis and paves the way for further development of clinical-related diagnosis strategies for fibrotic diseases of the liver and other organs.</div></div><div><h3>Statement of significance</h3><div>Currently, early diagnosis and accurate staging of liver fibrosis continue to present significant challenges. This study demonstrates that fluorescent probes based on the nucleic acid aptamer ZY-1, which targets cellular fibronectin (cFN)—a crucial extracellular matrix (ECM) molecule that significantly accumulates during liver fibrosis—are promising bioimaging agents for staging liver fibrosis. The ZY-1-based fluorescent probes effectively identified and differentiated early-stage liver fibrosis from advanced liver fibrosis. This proof-of-concept study not only provides compelling evidence that ZY-1-based probes show promise for the early diagnosis and staging of liver fibrosis but also paves the way for further investigations into the use of ZY-1 in detecting other diseases associated with cFN.</div></div>","PeriodicalId":237,"journal":{"name":"Acta Biomaterialia","volume":"190 ","pages":"Pages 579-592"},"PeriodicalIF":9.4,"publicationDate":"2024-12-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142482757","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}
Pub Date : 2024-12-01DOI: 10.1016/j.actbio.2024.10.020
Zhendong Li , Longhui Chen , Shasha Yang , Jinzhi Han , Yunquan Zheng , Zelong Chen , Xianai Shi , Jianmin Yang
<div><div>The healing process of diabetic foot ulcers is challenging due to the presence of a complex and severe inflammatory microenvironment, characterized by hyperglycemia, low pH, susceptibility to infection, vascular dysfunction, and over-expression of reactive oxygen species (ROS), which can potentially lead to amputation or even mortality. Herein, a glucose and pH dual-responsive hydrogel was designed and prepared by crosslinking phenylboronic acid-grafted quaternary chitosan (QF, 4 wt%) with dopamine-grafted oxidized hyaluronic acid (OD, 5 wt%) through phenylboronation, schiff-base reaction, and other techniques. The multifunctional QO/@PV@AB7 hydrogel was prepared by incorporating pravastatin-loaded chitosan nanoparticles (CSNPs@PV, 2 mg/mL) and antimicrobial peptide AMP-AB7 loaded silica nanoparticles (SiO<sub>2</sub>NPs@AB7, 0.5 mg/mL). The results demonstrate that the QO/@PV@AB7 hydrogel exhibits good responsiveness to acidic conditions and high glucose levels, while effectively scavenging various types of ROS. Moreover, it exerted protective effects against oxidative stress on cells, enhanced HUVECs viability, and promoted angiogenesis. Notably, the QO/@PV@AB7 hydrogel displayed potent antibacterial activity against methicillin-resistant <em>Staphylococcus aureus</em> (MRSA) and <em>Escherichia coli</em>. Additionally, in an MRSA-infected rat model of diabetic foot wounds, administration of the QO/@PV@AB7 hydrogel led to increased secretion of pro-angiogenic factors such as vascular endothelial nitric oxide synthase (eNOS), vascular endothelial-generating factor (VEGF), and endothelial cell adhesion molecule (CD31). Furthermore, the hydrogel significantly reduced the levels of inflammatory factors such as interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α), while simultaneously increasing the levels of anti-inflammatory cytokines such as interleukin-10 (IL-10). The findings suggest that multifunctional hydrogels incorporating PV@CSNPs and SiO<sub>2</sub>NPs@AB7 demonstrate promising potential as a therapeutic approach for the treatment of diabetic foot.</div></div><div><h3>Statement of Significance</h3><div>Here, a glucose and pH dual-responsive QO/@PV@AB7 hydrogel with antimicrobial and angiogenesis-promoting properties was developed for the treatment of infected wounds in diabetic feet. Our findings demonstrate that the proposed hydrogel exhibits good responsiveness, effectively scavenges various types of reactive oxygen species (DPPH, O<sup>2-</sup>, -OH, and ABTS+), provides protection against oxidative stress, enhances HUVECs cell viability, and promotes angiogenesis. Notably, it also demonstrates potent antibacterial activity against methicillin-resistant <em>Staphylococcus aureus</em> (MRSA) and <em>E. coli</em>. Additionally, <em>in vivo</em> experiments demonstrated that the hydrogel exhibited accelerated wound healing in MRSA-infected diabetic foot ulcers, with a reduction of four days compared to the control group.</d
{"title":"Glucose and pH dual-responsive hydrogels with antibacterial, reactive oxygen species scavenging, and angiogenesis properties for promoting the healing of infected diabetic foot ulcers","authors":"Zhendong Li , Longhui Chen , Shasha Yang , Jinzhi Han , Yunquan Zheng , Zelong Chen , Xianai Shi , Jianmin Yang","doi":"10.1016/j.actbio.2024.10.020","DOIUrl":"10.1016/j.actbio.2024.10.020","url":null,"abstract":"<div><div>The healing process of diabetic foot ulcers is challenging due to the presence of a complex and severe inflammatory microenvironment, characterized by hyperglycemia, low pH, susceptibility to infection, vascular dysfunction, and over-expression of reactive oxygen species (ROS), which can potentially lead to amputation or even mortality. Herein, a glucose and pH dual-responsive hydrogel was designed and prepared by crosslinking phenylboronic acid-grafted quaternary chitosan (QF, 4 wt%) with dopamine-grafted oxidized hyaluronic acid (OD, 5 wt%) through phenylboronation, schiff-base reaction, and other techniques. The multifunctional QO/@PV@AB7 hydrogel was prepared by incorporating pravastatin-loaded chitosan nanoparticles (CSNPs@PV, 2 mg/mL) and antimicrobial peptide AMP-AB7 loaded silica nanoparticles (SiO<sub>2</sub>NPs@AB7, 0.5 mg/mL). The results demonstrate that the QO/@PV@AB7 hydrogel exhibits good responsiveness to acidic conditions and high glucose levels, while effectively scavenging various types of ROS. Moreover, it exerted protective effects against oxidative stress on cells, enhanced HUVECs viability, and promoted angiogenesis. Notably, the QO/@PV@AB7 hydrogel displayed potent antibacterial activity against methicillin-resistant <em>Staphylococcus aureus</em> (MRSA) and <em>Escherichia coli</em>. Additionally, in an MRSA-infected rat model of diabetic foot wounds, administration of the QO/@PV@AB7 hydrogel led to increased secretion of pro-angiogenic factors such as vascular endothelial nitric oxide synthase (eNOS), vascular endothelial-generating factor (VEGF), and endothelial cell adhesion molecule (CD31). Furthermore, the hydrogel significantly reduced the levels of inflammatory factors such as interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α), while simultaneously increasing the levels of anti-inflammatory cytokines such as interleukin-10 (IL-10). The findings suggest that multifunctional hydrogels incorporating PV@CSNPs and SiO<sub>2</sub>NPs@AB7 demonstrate promising potential as a therapeutic approach for the treatment of diabetic foot.</div></div><div><h3>Statement of Significance</h3><div>Here, a glucose and pH dual-responsive QO/@PV@AB7 hydrogel with antimicrobial and angiogenesis-promoting properties was developed for the treatment of infected wounds in diabetic feet. Our findings demonstrate that the proposed hydrogel exhibits good responsiveness, effectively scavenges various types of reactive oxygen species (DPPH, O<sup>2-</sup>, -OH, and ABTS+), provides protection against oxidative stress, enhances HUVECs cell viability, and promotes angiogenesis. Notably, it also demonstrates potent antibacterial activity against methicillin-resistant <em>Staphylococcus aureus</em> (MRSA) and <em>E. coli</em>. Additionally, <em>in vivo</em> experiments demonstrated that the hydrogel exhibited accelerated wound healing in MRSA-infected diabetic foot ulcers, with a reduction of four days compared to the control group.</d","PeriodicalId":237,"journal":{"name":"Acta Biomaterialia","volume":"190 ","pages":"Pages 205-218"},"PeriodicalIF":9.4,"publicationDate":"2024-12-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142482763","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}
Pub Date : 2024-12-01DOI: 10.1016/j.actbio.2024.10.015
Yuhao Zhang , Jiawen Zhang , Qiang Yang , Yao Song , Mingfei Pan , Yajing Kan , Li Xiang , Mei Li , Hongbo Zeng
Multifunctional robust protective coatings that combine biocompatibility, antifouling and antimicrobial properties play an essential role in reducing host reactions and infection on invasive medical devices. However, developing these protective coatings generally faces a paradox: coating materials capable of achieving robust adhesion to substrates via spontaneous deposition inevitably initiate continuous biofoulant adsorption, while those employing strong hydration capability to resist biofoulant attachment have limited substrate binding ability and durability under wear. Herein, we designed a multifunctional terpolymer of poly(dopamine methyacrylamide-co-2-methacryloyloxyethyl phoasphorylcholine-co-2-(dimethylamino)-ethyl methacrylate) (P(DMA-co-MPC-co-DMAEMA)), which integrates desired yet traditionally incompatible functions (i.e., robust adhesion, antifouling, lubrication, and antimicrobial properties). Direct normal and lateral force measurements, dynamic adsorption tests, surface ion conductance mapping were applied to comprehensively investigate the nanomechanics of coating-biofloulant interactions. Catechol groups of DMA act as basal anchors for robust substrate deposition, while the highly hydrated zwitterion of MPC provides apical protection to resist biofouling and wear. Moreover, the antimicrobial property is conferred through the protonation of tertiary amine groups on DMAEMA, inhibiting infection under physiological conditions. This work provides an effective strategy for harmonizing demanded yet incompatible properties in one coating material, with significant implications for the development of multifunctional surfaces towards the advancement of invasive biomedical devices.
Statement of significance
Multifunctional robust protective coatings have been widely utilized in invasive medical devices to mitigate host responses and infection. However, modified surface coatings often encounter a trade-off between robust adhesion to substrates and strong hydration capability for antifouling and antimicrobial properties. We propose a universal strategy for surface modification by dopamine-assisted co-deposition with a multifunctional terpolymer of P(DMA-co-MPC-co-DMAEMA) that simultaneously achieves robust adhesion, antifouling, and antimicrobial properties. Through elucidating the nanomechanics with fundamentally understanding the interactions between the coating and biomacromolecules, we highlight the role of DMA for substrate adhesion, MPC for biofouling resistance, and DMAEMA for antimicrobial activity. This approach presents a promising strategy for constructing multifunctional coatings on minimally invasive medical devices by tuning interfacial molecular asymmetricity to reconcile incompatible properties within one coating.
{"title":"Tuning interfacial molecular asymmetry to engineer protective coatings with superior surface anchoring, antifouling and antibacterial properties","authors":"Yuhao Zhang , Jiawen Zhang , Qiang Yang , Yao Song , Mingfei Pan , Yajing Kan , Li Xiang , Mei Li , Hongbo Zeng","doi":"10.1016/j.actbio.2024.10.015","DOIUrl":"10.1016/j.actbio.2024.10.015","url":null,"abstract":"<div><div>Multifunctional robust protective coatings that combine biocompatibility, antifouling and antimicrobial properties play an essential role in reducing host reactions and infection on invasive medical devices. However, developing these protective coatings generally faces a paradox: coating materials capable of achieving robust adhesion to substrates via spontaneous deposition inevitably initiate continuous biofoulant adsorption, while those employing strong hydration capability to resist biofoulant attachment have limited substrate binding ability and durability under wear. Herein, we designed a multifunctional terpolymer of poly(dopamine methyacrylamide-co-2-methacryloyloxyethyl phoasphorylcholine-co-2-(dimethylamino)-ethyl methacrylate) (P(DMA-co-MPC-co-DMAEMA)), which integrates desired yet traditionally incompatible functions (i.e., robust adhesion, antifouling, lubrication, and antimicrobial properties). Direct normal and lateral force measurements, dynamic adsorption tests, surface ion conductance mapping were applied to comprehensively investigate the nanomechanics of coating-biofloulant interactions. Catechol groups of DMA act as basal anchors for robust substrate deposition, while the highly hydrated zwitterion of MPC provides apical protection to resist biofouling and wear. Moreover, the antimicrobial property is conferred through the protonation of tertiary amine groups on DMAEMA, inhibiting infection under physiological conditions. This work provides an effective strategy for harmonizing demanded yet incompatible properties in one coating material, with significant implications for the development of multifunctional surfaces towards the advancement of invasive biomedical devices.</div></div><div><h3>Statement of significance</h3><div>Multifunctional robust protective coatings have been widely utilized in invasive medical devices to mitigate host responses and infection. However, modified surface coatings often encounter a trade-off between robust adhesion to substrates and strong hydration capability for antifouling and antimicrobial properties. We propose a universal strategy for surface modification by dopamine-assisted co-deposition with a multifunctional terpolymer of P(DMA-co-MPC-co-DMAEMA) that simultaneously achieves robust adhesion, antifouling, and antimicrobial properties. Through elucidating the nanomechanics with fundamentally understanding the interactions between the coating and biomacromolecules, we highlight the role of DMA for substrate adhesion, MPC for biofouling resistance, and DMAEMA for antimicrobial activity. This approach presents a promising strategy for constructing multifunctional coatings on minimally invasive medical devices by tuning interfacial molecular asymmetricity to reconcile incompatible properties within one coating.</div></div>","PeriodicalId":237,"journal":{"name":"Acta Biomaterialia","volume":"190 ","pages":"Pages 107-119"},"PeriodicalIF":9.4,"publicationDate":"2024-12-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142482780","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}
Pub Date : 2024-12-01DOI: 10.1016/j.actbio.2024.10.016
Qingwei He , Hong Lu , Yuying Chen , Huiying Zeng , Ping Hu
This study introduces a live imaging technique for real-time, non-invasive monitoring of drug release from long-acting microneedles using FRET (Fluorescence Resonance Energy Transfer). Employing Cy5.5 and Cy7 as FRET pairs and levonorgestrel as the model drug, we fabricated microneedles with varying PLGA molecular weights, demonstrating distinct release profiles. The FRET-PLGA-10-MN demonstrated a rapid drug release profile, reaching nearly complete release within a two-day period, while FRET-PLGA-30-MN showed a sustained release over four days. Sensitized Emission FRET (SE-FRET) optimized the imaging process, providing a robust correlation between FRET signals and drug absorption. This method surpasses traditional pharmacokinetic studies by offering a more efficient and comprehensive analysis of microneedle release dynamics in vivo, paving the way for enhanced long-acting microneedle design and therapeutic outcomes.
Statement of significance
1. FRET technology was applied to microneedle drug delivery system for the first time, which realized real-time, quantitative and non-invasive monitoring of drug release process.
2. The long-term microneedle technique was combined with sensitized emission method, and the FRET remaining ratio was innovatively used to investigate the FRET characteristics of microneedles, and the fluorescence ratio of FRET and donor double-channel was quantitatively calculated.
3. The correlation between visual fluorescence images of FRET effect and semi-quantitative calculation results based on fluorescence intensity and drug release in vivo with drug-loaded microneedles was analyzed.
{"title":"Visualization of the degradation of long-acting microneedles and correlation of drug release in vivo based on FRET mechanism","authors":"Qingwei He , Hong Lu , Yuying Chen , Huiying Zeng , Ping Hu","doi":"10.1016/j.actbio.2024.10.016","DOIUrl":"10.1016/j.actbio.2024.10.016","url":null,"abstract":"<div><div>This study introduces a live imaging technique for real-time, non-invasive monitoring of drug release from long-acting microneedles using FRET (Fluorescence Resonance Energy Transfer). Employing Cy5.5 and Cy7 as FRET pairs and levonorgestrel as the model drug, we fabricated microneedles with varying PLGA molecular weights, demonstrating distinct release profiles. The FRET-PLGA-10-MN demonstrated a rapid drug release profile, reaching nearly complete release within a two-day period, while FRET-PLGA-30-MN showed a sustained release over four days. Sensitized Emission FRET (SE-FRET) optimized the imaging process, providing a robust correlation between FRET signals and drug absorption. This method surpasses traditional pharmacokinetic studies by offering a more efficient and comprehensive analysis of microneedle release dynamics <em>in vivo</em>, paving the way for enhanced long-acting microneedle design and therapeutic outcomes.</div></div><div><h3>Statement of significance</h3><div>1. FRET technology was applied to microneedle drug delivery system for the first time, which realized real-time, quantitative and non-invasive monitoring of drug release process.</div><div>2. The long-term microneedle technique was combined with sensitized emission method, and the FRET remaining ratio was innovatively used to investigate the FRET characteristics of microneedles, and the fluorescence ratio of FRET and donor double-channel was quantitatively calculated.</div><div>3. The correlation between visual fluorescence images of FRET effect and semi-quantitative calculation results based on fluorescence intensity and drug release <em>in vivo</em> with drug-loaded microneedles was analyzed.</div></div>","PeriodicalId":237,"journal":{"name":"Acta Biomaterialia","volume":"190 ","pages":"Pages 152-164"},"PeriodicalIF":9.4,"publicationDate":"2024-12-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142482781","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}
Secretomes from mesenchymal stem cells (MSCs) have significant therapeutic potential and could be the basis for future MSCs treatments. Innovative design of the topology of biomaterials, which mechanically regulate cell behavior and function, can tremendously improve the efficacy of stem cell therapy. However, how spatial dimension cues derived from specific topology command cell mechanotransduction to regulate the paracrine function of MSCs remains unknown. In this study, the three-dimensional (3D) fibrous constructs with box-like pores and precise strand spacing from 150 µm down to only 40 µm were manufactured using melt electrowriting (MEW), which were used to systematically investigate the spatial dimension cues-triggered mechanotransduction of adipose-derived mesenchymal stem cells (Ad-MSCs) and their impact on the paracrine and regeneration function of Ad-MSCs. The results demonstrated that spatial instructions from the 3D fibrous constructs could influence the spatial reorganization of the cytoskeleton, resulting in cell elongation and augmented immunomodulatory and angiogenic paracrine effects of Ad-MSCs, which was most pronounced at a minimum strand spacing of 40 µm. Besides, mechanical activation of the FAK-PI3K/AKT axis significantly enhanced the paracrine function of Ad-MSCs. In vivo experiments demonstrated that the Ad-MSCs trained using well-defined 3D fibrous constructs with a strand spacing of 40 µm significantly promoted skin regeneration via paracrine signals. In conclusion, this study provides a new horizon for deciphering space dimension insights into the interactional mechanisms of mechanotransduction in regulating cell function, which has inspired innovations in biomaterials for improving tissue regeneration.
Statement of significance
This study emphasized that designing cell-scale spatial dimension cues to command mechanical activation via the FAK-PI3K/AKT axis could significantly enhance the paracrine and regenerative functions of Ad-MSCs. Paracrine signals of Ad-MSCs triggered by mechanical activation promoted skin repair and regeneration via the immunomodulation and angiogenesis. The proposed mechanobiological signal transduction triggered by spatial dimensional cues, which potentiates the paracrine and regenerative functions of Ad-MSCs, is a promising engineering strategy and is expected to provide new inspirations for the development of biomaterials based on biophysical signals for cellular behavior modulation.
{"title":"Spatial dimension cues derived from fibrous scaffolds trigger mechanical activation to potentiate the paracrine and regenerative functions of MSCs via the FAK-PI3K/AKT axis","authors":"Shixin Xu, Miaomiao Zhang, Ruoying Wang, Jinxin Zhang, Chengwei Wang, Li Xie, Wen Zhao","doi":"10.1016/j.actbio.2024.10.039","DOIUrl":"10.1016/j.actbio.2024.10.039","url":null,"abstract":"<div><div>Secretomes from mesenchymal stem cells (MSCs) have significant therapeutic potential and could be the basis for future MSCs treatments. Innovative design of the topology of biomaterials, which mechanically regulate cell behavior and function, can tremendously improve the efficacy of stem cell therapy. However, how spatial dimension cues derived from specific topology command cell mechanotransduction to regulate the paracrine function of MSCs remains unknown. In this study, the three-dimensional (3D) fibrous constructs with box-like pores and precise strand spacing from 150 µm down to only 40 µm were manufactured using melt electrowriting (MEW), which were used to systematically investigate the spatial dimension cues-triggered mechanotransduction of adipose-derived mesenchymal stem cells (Ad-MSCs) and their impact on the paracrine and regeneration function of Ad-MSCs. The results demonstrated that spatial instructions from the 3D fibrous constructs could influence the spatial reorganization of the cytoskeleton, resulting in cell elongation and augmented immunomodulatory and angiogenic paracrine effects of Ad-MSCs, which was most pronounced at a minimum strand spacing of 40 µm. Besides, mechanical activation of the FAK-PI3K/AKT axis significantly enhanced the paracrine function of Ad-MSCs. <em>In vivo</em> experiments demonstrated that the Ad-MSCs trained using well-defined 3D fibrous constructs with a strand spacing of 40 µm significantly promoted skin regeneration via paracrine signals. In conclusion, this study provides a new horizon for deciphering space dimension insights into the interactional mechanisms of mechanotransduction in regulating cell function, which has inspired innovations in biomaterials for improving tissue regeneration.</div></div><div><h3>Statement of significance</h3><div>This study emphasized that designing cell-scale spatial dimension cues to command mechanical activation via the FAK-PI3K/AKT axis could significantly enhance the paracrine and regenerative functions of Ad-MSCs. Paracrine signals of Ad-MSCs triggered by mechanical activation promoted skin repair and regeneration via the immunomodulation and angiogenesis. The proposed mechanobiological signal transduction triggered by spatial dimensional cues, which potentiates the paracrine and regenerative functions of Ad-MSCs, is a promising engineering strategy and is expected to provide new inspirations for the development of biomaterials based on biophysical signals for cellular behavior modulation.</div></div>","PeriodicalId":237,"journal":{"name":"Acta Biomaterialia","volume":"190 ","pages":"Pages 273-292"},"PeriodicalIF":9.4,"publicationDate":"2024-12-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142514435","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}
Pub Date : 2024-12-01DOI: 10.1016/j.actbio.2024.10.031
Ana Rita M.P. Santos , Bruce E. Kirkpatrick , Mirim Kim , Kristi S. Anseth , Yongdoo Park
The tumor microenvironment (TME) comprises diverse cell types within an altered extracellular matrix (ECM) and plays a pivotal role in metastasis through intricate cell-cell and cell-ECM interactions. Fibroblasts, as key constituents of the TME, contribute significantly to cancer metastasis through their involvement in matrix deposition and remodeling mechanisms, modulated by their quiescent or activated states. Despite their recognized importance, the precise role of fibroblasts in cancer cell invasion remains incompletely understood. In this study, we investigated the impact of fibroblast activity on cancer cell progression using a 2D co-culture model. Michigan Cancer Foundation-7 (MCF7) breast cancer cells were co-cultured with normal human lung fibroblasts (NHLF), both with and without transforming growth factor β (TGFβ) treatment. Traction force microscopy (TFM) was employed to quantify traction and velocity forces associated with cellular migration. We observed that TGFβ-activated fibroblasts form a distinctive ring around cancer cells in co-culture, with increased traction and tension at the cell island boundary. This force distribution is associated with the localization of force-related proteins at these boundary regions, including vinculin and E-cadherin. Metabolic profiling revealed a strong OXPHOS signal specific to the activated fibroblasts, in contrast to normal fibroblasts, which primarily display migratory behavior and a more heterogeneous pattern of forces and metabolic activity in co-culture. Our findings offer valuable insights into the mechanical forces and metabolic dynamics governing cellular migration in the tumor microenvironment, where our co-culture model could complement in vivo studies and enable researchers to explore specific microenvironmental cues for a deeper understanding of TME mechanisms.
Statement of significance
Cancer models mimicking the dynamics of tumor microenvironment (TME) are an ideal tool to study cancer mechanisms and treatment. However, the full understanding of how cancer cells interact with their surroundings and other cells is still unknown. To tackle this, we developed a simple yet effective 2D co-culture model that allows us to control the arrangement of cell cultures precisely and use various imaging techniques to study interactions between cancer cells and fibroblasts. Here we could measure cell movements, force distribution, metabolic activity, and protein localization and interplay those factors in vitro. Our model helps us observe the underlying mechanisms between cancer cells and fibroblasts, contributing to our understanding of the dynamics in the TME.
{"title":"2D co-culture model reveals a biophysical interplay between activated fibroblasts and cancer cells","authors":"Ana Rita M.P. Santos , Bruce E. Kirkpatrick , Mirim Kim , Kristi S. Anseth , Yongdoo Park","doi":"10.1016/j.actbio.2024.10.031","DOIUrl":"10.1016/j.actbio.2024.10.031","url":null,"abstract":"<div><div>The tumor microenvironment (TME) comprises diverse cell types within an altered extracellular matrix (ECM) and plays a pivotal role in metastasis through intricate cell-cell and cell-ECM interactions. Fibroblasts, as key constituents of the TME, contribute significantly to cancer metastasis through their involvement in matrix deposition and remodeling mechanisms, modulated by their quiescent or activated states. Despite their recognized importance, the precise role of fibroblasts in cancer cell invasion remains incompletely understood. In this study, we investigated the impact of fibroblast activity on cancer cell progression using a 2D co-culture model. Michigan Cancer Foundation-7 (MCF7) breast cancer cells were co-cultured with normal human lung fibroblasts (NHLF), both with and without transforming growth factor β (TGFβ) treatment. Traction force microscopy (TFM) was employed to quantify traction and velocity forces associated with cellular migration. We observed that TGFβ-activated fibroblasts form a distinctive ring around cancer cells in co-culture, with increased traction and tension at the cell island boundary. This force distribution is associated with the localization of force-related proteins at these boundary regions, including vinculin and E-cadherin. Metabolic profiling revealed a strong OXPHOS signal specific to the activated fibroblasts, in contrast to normal fibroblasts, which primarily display migratory behavior and a more heterogeneous pattern of forces and metabolic activity in co-culture. Our findings offer valuable insights into the mechanical forces and metabolic dynamics governing cellular migration in the tumor microenvironment, where our co-culture model could complement <em>in vivo</em> studies and enable researchers to explore specific microenvironmental cues for a deeper understanding of TME mechanisms.</div></div><div><h3>Statement of significance</h3><div>Cancer models mimicking the dynamics of tumor microenvironment (TME) are an ideal tool to study cancer mechanisms and treatment. However, the full understanding of how cancer cells interact with their surroundings and other cells is still unknown. To tackle this, we developed a simple yet effective 2D co-culture model that allows us to control the arrangement of cell cultures precisely and use various imaging techniques to study interactions between cancer cells and fibroblasts. Here we could measure cell movements, force distribution, metabolic activity, and protein localization and interplay those factors <em>in vitro</em>. Our model helps us observe the underlying mechanisms between cancer cells and fibroblasts, contributing to our understanding of the dynamics in the TME.</div></div>","PeriodicalId":237,"journal":{"name":"Acta Biomaterialia","volume":"190 ","pages":"Pages 264-272"},"PeriodicalIF":9.4,"publicationDate":"2024-12-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142549451","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}
Pub Date : 2024-12-01DOI: 10.1016/j.actbio.2024.10.043
Qian Qian Yang, Jing Jin, Jie Sun, Luzhong Zhang, Jin Bo Tang, You Lang Zhou
Adhesion seriously affects the recovery of tendon gliding function. Our group previously found that inhibition of TGF-β1, which is closely related to adhesion formation, effectively attenuated adhesions but did not eliminate them, suggesting that there may be other mechanisms involved in adhesion formation. In this study, we considered that uncontrolled and excessively proliferating fibroblasts undergo immune escape, which aggravates the deposition of extracellular matrix during the adhesion formation. We found that the expression of the immune checkpoint PD-L1 was significantly elevated after injury and may be involved in adhesion formation. Therefore, we intended to silence both TGF-β1 and PD-L1 to improve the immune advantage in the microenvironment after flexor tendon injury to further reduce adhesion. We constructed the nanoparticle/TGF-β1 or/and PD-L1 siRNAs complexes and verified their high biocompatibility and high transfection efficiency. We found that CD8+T cells had a greater killing effect on the excessively proliferating cells that were transfected with nanoparticle/TGF-β1 or/and PD-L1 siRNAs. The hydrogel-nanoparticle/TGF-β1 or/and PD-L1 siRNAs system could effectively improve the gliding function of the tendons without weakening the mechanical properties in injured rat FDL tendon and chicken FDP tendon models. In addition, the potential of CD8+T cells to encircle the adhesion cells on the tendon surface was observed, which resulted in increased levels of cell apoptosis. Thus, our study confirmed that combined knockdown of TGF-β1 and PD-L1 could activate immunodominance after flexor tendon repair and provided a potential treatment to limit adhesion formation and improve gliding function.
Statement of Significance
Adhesion seriously affects the recovery of tendon gliding function. TGF-β1 is related to adhesion formation as it regulates the production of extracellular matrix. We found that excessively proliferated fibroblasts might undergo immune escape, which aggravated the deposition of extracellular matrix. Therefore, we constructed a hydrogel-nanoparticle/TGF-β1 and PD-L1 siRNAs system for silencing TGF-β1 and PD-L1 to improve the immune advantage in the microenvironment after tendon injury. This system could improve the gliding function of tendons without weakening the mechanical property and increase the killing effect of CD8+T cells. Combined knockdown of TGF-β1 and PD-L1 could activate immunodominance after tendon repair and provide a potential treatment to limit adhesion formation.
{"title":"Simultaneous targeting of TGF-β1/PD-L1 via a hydrogel-nanoparticle system to remodel the ECM and immune microenvironment for limiting adhesion formation","authors":"Qian Qian Yang, Jing Jin, Jie Sun, Luzhong Zhang, Jin Bo Tang, You Lang Zhou","doi":"10.1016/j.actbio.2024.10.043","DOIUrl":"10.1016/j.actbio.2024.10.043","url":null,"abstract":"<div><div>Adhesion seriously affects the recovery of tendon gliding function. Our group previously found that inhibition of TGF-β1, which is closely related to adhesion formation, effectively attenuated adhesions but did not eliminate them, suggesting that there may be other mechanisms involved in adhesion formation. In this study, we considered that uncontrolled and excessively proliferating fibroblasts undergo immune escape, which aggravates the deposition of extracellular matrix during the adhesion formation. We found that the expression of the immune checkpoint PD-L1 was significantly elevated after injury and may be involved in adhesion formation. Therefore, we intended to silence both TGF-β1 and PD-L1 to improve the immune advantage in the microenvironment after flexor tendon injury to further reduce adhesion. We constructed the nanoparticle/TGF-β1 or/and PD-L1 siRNAs complexes and verified their high biocompatibility and high transfection efficiency. We found that CD8<sup>+</sup> <em>T</em> cells had a greater killing effect on the excessively proliferating cells that were transfected with nanoparticle/TGF-β1 or/and PD-L1 siRNAs. The hydrogel-nanoparticle/TGF-β1 or/and PD-L1 siRNAs system could effectively improve the gliding function of the tendons without weakening the mechanical properties in injured rat FDL tendon and chicken FDP tendon models. In addition, the potential of CD8<sup>+</sup> <em>T</em> cells to encircle the adhesion cells on the tendon surface was observed, which resulted in increased levels of cell apoptosis. Thus, our study confirmed that combined knockdown of TGF-β1 and PD-L1 could activate immunodominance after flexor tendon repair and provided a potential treatment to limit adhesion formation and improve gliding function.</div></div><div><h3>Statement of Significance</h3><div>Adhesion seriously affects the recovery of tendon gliding function. TGF-β1 is related to adhesion formation as it regulates the production of extracellular matrix. We found that excessively proliferated fibroblasts might undergo immune escape, which aggravated the deposition of extracellular matrix. Therefore, we constructed a hydrogel-nanoparticle/TGF-β1 and PD-L1 siRNAs system for silencing TGF-β1 and PD-L1 to improve the immune advantage in the microenvironment after tendon injury. This system could improve the gliding function of tendons without weakening the mechanical property and increase the killing effect of CD8<sup>+</sup> <em>T</em> cells. Combined knockdown of TGF-β1 and PD-L1 could activate immunodominance after tendon repair and provide a potential treatment to limit adhesion formation.</div></div>","PeriodicalId":237,"journal":{"name":"Acta Biomaterialia","volume":"190 ","pages":"Pages 447-462"},"PeriodicalIF":9.4,"publicationDate":"2024-12-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142559606","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}
Pub Date : 2024-12-01DOI: 10.1016/j.actbio.2024.10.042
Karthik R. Peddireddy, Ryan McGorty, Rae M. Robertson-Anderson
Understanding how polymers deform in response to local stresses and strains, and how strains propagate from a local disturbance, are grand challenges in wide-ranging fields from materials manufacturing to cell mechanics. These dynamics are particularly complex for blends of polymers of distinct topologies, for which several different species-dependent mechanisms may contribute. Here, we use OpTiDDM (Optical Tweezers integrating Differential Dynamic Microscopy) to elucidate deformation fields and propagation dynamics of binary blends of linear, ring and supercoiled DNA of varying sizes. We reveal robust non-monotonic dependence of strain alignment and superdiffusive transport with strain rate. However, peak alignment and superdiffusivity are surprisingly decoupled, occurring at different strain rates resonant with the distinct relaxation rates of the different topologies. Despite this universal resonance, we find that strain propagation of ring-linear blends is dictated by entanglements while supercoiled-ring blends are governed by Rouse dynamics. Our results capture critical subtleties in propagation and deformation dynamics of topological blends, shedding new light on the governing physics and offering a route towards decoupled tuning of response features. We anticipate our approach to be broadly generalizable to mapping the deformation dynamics of polymer blends, with an eye towards bottom-up bespoke materials design.
Statement of Significance
In biology and in manufacturing, biomaterials are often subject to localized and spatially nonuniform strains and stresses. Yet, understanding the extent to which strains are absorbed, distributed, or propagated across different spatiotemporal scales remains a grand challenge. Here, we combine optical tweezers with differential dynamic microscopy to elucidate deformation fields and propagation dynamics of blends of linear, ring and supercoiled DNA, revealing robust non-monotonic trends and decoupling of strain alignment and superdiffusivity, and capturing critical subtleties in propagation and deformation dynamics. Our results, shedding important new physical insight to guide decoupled tuning of response features, may be leveraged to map the deformation dynamics of wide-ranging systems of biopolymers and other macromolecules, with an eye towards bottom-up bespoke biomaterials design.
了解聚合物如何因局部应力和应变而变形，以及应变如何从局部扰动中传播，是材料制造到细胞力学等广泛领域面临的巨大挑战。对于具有不同拓扑结构的聚合物混合物而言，这些动态变化尤为复杂，其中可能存在多种不同的物种依赖机制。在这里，我们使用 OpTiDDM（光学镊子集成差分动态显微镜）来阐明不同大小的线性、环状和超卷曲 DNA 二元共混物的变形场和传播动力学。我们揭示了应变排列和超扩散传输与应变速率的非单调依赖性。然而，峰值排列和超扩散性却出人意料地解耦了，它们出现在不同的应变速率下，与不同拓扑结构的不同弛豫速率产生共振。尽管存在这种普遍共振，但我们发现环状线性混合物的应变传播是由缠结决定的，而超螺旋环状混合物则是由劳斯动力学决定的。我们的研究结果捕捉到了拓扑混合物在传播和变形动力学方面的关键微妙之处，揭示了新的物理规律，并为实现响应特征的解耦调整提供了一条途径。我们预计，我们的方法可广泛用于绘制聚合物共混物的变形动力学图，从而实现自下而上的定制材料设计。意义声明：在生物学和制造业中，生物材料经常受到局部和空间不均匀应变和应力的影响。然而，了解应变在不同时空尺度上的吸收、分布或传播程度仍然是一个巨大的挑战。在这里，我们将光学镊子与差分动态显微镜相结合，阐明了线性、环状和超螺旋 DNA 混合体的形变场和传播动力学，揭示了应变排列和超扩散性的稳健非单调趋势和解耦，并捕捉到了传播和形变动力学中的关键微妙之处。我们的研究结果为指导响应特征的解耦调整提供了新的重要物理视角，可用于绘制各种生物聚合物和其他大分子系统的变形动力学图，从而实现自下而上的定制生物材料设计。
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Pub Date : 2024-12-01DOI: 10.1016/j.actbio.2024.10.046
Ying Liu , Tianqi Liu , Zhenye Zhu , Lin Xie , De Bai , Tonglin Liu , Wenting Gu , Wei Li , Yang Shu , Jiaheng Zhang
Wound healing in diabetic patients presents a significant challenge due to delayed inflammatory responses, which obstruct subsequent healing stages. In response, we have developed a progressive, layer-by-layer responsive hydrogel, specifically designed to meet the dynamic requirements of diabetic wounds throughout different healing phases. This hydrogel initiates with a glucose-responsive layer formed by boronate ester bonds between 4-arm-poly (ethylene glycol) succinimidyl glutarate (4arm-PEG-SG) and 3-aminophenylboronic acid. This configuration ensures precise control over the physicochemical properties, facilitating accurate drug release during the healing process. Furthermore, we have incorporated an active pharmaceutical ingredient ionic liquid (API) composed of diclofenac and L-carnitine. This combination effectively tackles the solubility and stability issues commonly associated with anti-inflammatory drugs. To further refine drug release, we integrated matrix metalloproteinase-9 (MMP-9)-sensitive gelatin microcapsules, ensuring a controlled release and preventing the abrupt, uneven drug distribution often seen in other systems. Our hydrogel's rheological properties closely resemble human skin, offering a more harmonious approach to diabetic wound healing. Overall, this progressive layer-by-layer responsive wound management system, which is a safe, efficient, and intelligent approach, holds significant potential for the clinical treatment of diabetic wounds.
Statement of significance
The two main problems of diabetic wounds are the long-term infiltration of inflammation and the delayed repair process. In this experiment, a glucose-responsive hierarchical drug delivery system was designed to intelligently adjust gel properties to meet the needs of inflammation and repair stage of wound healing, accelerate the transformation of inflammation and repair stage, and accelerate the process of repair stage. In addition, in order to achieve accurate drug release in anti-inflammatory layer hydrogels and avoid sudden drug release due to poor solubility of anti-inflammatory small molecule drugs, we constructed a ionic liquid of active pharmaceutical ingredients (API-ILs) using diclofenac and L-carnitine as raw materials. It was wrapped in MMP-9 enzyme active gelatin microcapsule to construct a double-reaction anti-inflammatory layer gel to achieve accurate drug release. These findings highlight the potential of our system in treating diabetic wounds, providing a significant advance in wound treatment.
{"title":"An advanced hydrogel dressing system with progressive delivery and layer-to-layer response for diabetic wound healing","authors":"Ying Liu , Tianqi Liu , Zhenye Zhu , Lin Xie , De Bai , Tonglin Liu , Wenting Gu , Wei Li , Yang Shu , Jiaheng Zhang","doi":"10.1016/j.actbio.2024.10.046","DOIUrl":"10.1016/j.actbio.2024.10.046","url":null,"abstract":"<div><div>Wound healing in diabetic patients presents a significant challenge due to delayed inflammatory responses, which obstruct subsequent healing stages. In response, we have developed a progressive, layer-by-layer responsive hydrogel, specifically designed to meet the dynamic requirements of diabetic wounds throughout different healing phases. This hydrogel initiates with a glucose-responsive layer formed by boronate ester bonds between 4-arm-poly (ethylene glycol) succinimidyl glutarate (4arm-PEG-SG) and 3-aminophenylboronic acid. This configuration ensures precise control over the physicochemical properties, facilitating accurate drug release during the healing process. Furthermore, we have incorporated an active pharmaceutical ingredient ionic liquid (API) composed of diclofenac and L-carnitine. This combination effectively tackles the solubility and stability issues commonly associated with anti-inflammatory drugs. To further refine drug release, we integrated matrix metalloproteinase-9 (MMP-9)-sensitive gelatin microcapsules, ensuring a controlled release and preventing the abrupt, uneven drug distribution often seen in other systems. Our hydrogel's rheological properties closely resemble human skin, offering a more harmonious approach to diabetic wound healing. Overall, this progressive layer-by-layer responsive wound management system, which is a safe, efficient, and intelligent approach, holds significant potential for the clinical treatment of diabetic wounds.</div></div><div><h3>Statement of significance</h3><div>The two main problems of diabetic wounds are the long-term infiltration of inflammation and the delayed repair process. In this experiment, a glucose-responsive hierarchical drug delivery system was designed to intelligently adjust gel properties to meet the needs of inflammation and repair stage of wound healing, accelerate the transformation of inflammation and repair stage, and accelerate the process of repair stage. In addition, in order to achieve accurate drug release in anti-inflammatory layer hydrogels and avoid sudden drug release due to poor solubility of anti-inflammatory small molecule drugs, we constructed a ionic liquid of active pharmaceutical ingredients (API-ILs) using diclofenac and L-carnitine as raw materials. It was wrapped in MMP-9 enzyme active gelatin microcapsule to construct a double-reaction anti-inflammatory layer gel to achieve accurate drug release. These findings highlight the potential of our system in treating diabetic wounds, providing a significant advance in wound treatment.</div></div>","PeriodicalId":237,"journal":{"name":"Acta Biomaterialia","volume":"190 ","pages":"Pages 79-94"},"PeriodicalIF":9.4,"publicationDate":"2024-12-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142565090","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}
Pub Date : 2024-12-01DOI: 10.1016/j.actbio.2024.10.029
Yasaman Maaref , Shayan Jannati , Farah Jayousi , Philipp Lange , Mohsen Akbari , Mu Chiao , Glen F Tibbits
Human induced pluripotent stem cell-derived cardiomyocytes (hiPSCCMs) offer numerous advantages as a biological model, yet their inherent immaturity compared to adult cardiomyocytes poses significant limitations. This study addresses hiPSCCM immaturity by introducing a physiologically relevant micropatterned substrate for long-term culture and maturation. An innovative microfabrication methodology combining laser etching and casting creates a micropatterned polydimethylsiloxane (PDMS) substrate with varying stiffness, from 2 to 50 kPa, mimicking healthy and fibrotic cardiac tissue. Platinum electrodes were integrated into the cell culture chamber enable pacing of cells at various frequencies. Subsequently, cells were transferred to the incubator for time-course analysis, ensuring contamination-free conditions. Cell contractility, cytosolic Ca2+ transient, sarcomere orientation, and nucleus aspect ratio were analyzed in a 2D hiPSCCM monolayer up to 90 days post-replating in relation to substrate micropattern dimensions. Culturing hiPSCCMs for three weeks on a micropatterned PDMS substrate (2.5–5 µm deep, 20 µm center-to-center spacing of grooves, 2–5 kPa stiffness) emerges as optimal for cardiomyocyte alignment, contractility, and cytosolic Ca2+ transient. The study provides insights into substrate stiffness effects on hiPSCCM contractility and Ca2+ transient at immature and mature states. Maximum contractility and fastest Ca2+transient kinetics occur in mature hiPSCCMs cultured for two to four weeks, with the optimum at three weeks, on a soft micropatterned PDMS substrate. MS proteomic analysis further revealed that hiPSCCMs cultured on soft micropatterned substrates exhibit advanced maturation, marked by significant upregulation of key structural, electrophysiological, and metabolic proteins. This new substrate offers a promising platform for disease modeling and therapeutic interventions.
Statement of Significance
Human induced pluripotent stem cell derived cardiomyocytes (hiPSCCMs) have been transformative to disease-in-a-dish modeling, drug discovery and testing, and autologous regeneration for human hearts and their role will continue to expand dramatically. However, one of the major limitations of hiPSCCMs is that without intervention, the cells are immature and represent those in the fetal heart. We developed protocols for the fabrication of the PDMS matrices that includes variations in its stiffness and micropatterning. Growing our hiPSCCMs on matrices of comparable stiffness to a healthy heart (5 kPa) and grooves of 20 μm, generate heart cells typical of the healthy adult human heart.
{"title":"Developing a soft micropatterned substrate to enhance maturation of human induced pluripotent stem cell-derived cardiomyocytes","authors":"Yasaman Maaref , Shayan Jannati , Farah Jayousi , Philipp Lange , Mohsen Akbari , Mu Chiao , Glen F Tibbits","doi":"10.1016/j.actbio.2024.10.029","DOIUrl":"10.1016/j.actbio.2024.10.029","url":null,"abstract":"<div><div>Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC<img>CMs) offer numerous advantages as a biological model, yet their inherent immaturity compared to adult cardiomyocytes poses significant limitations. This study addresses hiPSC<img>CM immaturity by introducing a physiologically relevant micropatterned substrate for long-term culture and maturation. An innovative microfabrication methodology combining laser etching and casting creates a micropatterned polydimethylsiloxane (PDMS) substrate with varying stiffness, from 2 to 50 kPa, mimicking healthy and fibrotic cardiac tissue. Platinum electrodes were integrated into the cell culture chamber enable pacing of cells at various frequencies. Subsequently, cells were transferred to the incubator for time-course analysis, ensuring contamination-free conditions. Cell contractility, cytosolic Ca<sup>2+</sup> transient, sarcomere orientation, and nucleus aspect ratio were analyzed in a 2D hiPSC<img>CM monolayer up to 90 days post-replating in relation to substrate micropattern dimensions. Culturing hiPSC<img>CMs for three weeks on a micropatterned PDMS substrate (2.5–5 µm deep, 20 µm center-to-center spacing of grooves, 2–5 kPa stiffness) emerges as optimal for cardiomyocyte alignment, contractility, and cytosolic Ca<sup>2+</sup> transient. The study provides insights into substrate stiffness effects on hiPSC<img>CM contractility and Ca<sup>2+</sup> transient at immature and mature states. Maximum contractility and fastest Ca<sup>2+</sup>transient kinetics occur in mature hiPSC<img>CMs cultured for two to four weeks, with the optimum at three weeks, on a soft micropatterned PDMS substrate. MS proteomic analysis further revealed that hiPSC<img>CMs cultured on soft micropatterned substrates exhibit advanced maturation, marked by significant upregulation of key structural, electrophysiological, and metabolic proteins. This new substrate offers a promising platform for disease modeling and therapeutic interventions.</div></div><div><h3>Statement of Significance</h3><div>Human induced pluripotent stem cell derived cardiomyocytes (hiPSC<img>CMs) have been transformative to disease-in-a-dish modeling, drug discovery and testing, and autologous regeneration for human hearts and their role will continue to expand dramatically. However, one of the major limitations of hiPSC<img>CMs is that without intervention, the cells are immature and represent those in the fetal heart. We developed protocols for the fabrication of the PDMS matrices that includes variations in its stiffness and micropatterning. Growing our hiPSC<img>CMs on matrices of comparable stiffness to a healthy heart (5 kPa) and grooves of 20 μm, generate heart cells typical of the healthy adult human heart.</div></div>","PeriodicalId":237,"journal":{"name":"Acta Biomaterialia","volume":"190 ","pages":"Pages 133-151"},"PeriodicalIF":9.4,"publicationDate":"2024-12-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142568193","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}
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