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Reginensi D, Ortiz DA, Denis B, Castillo S, Burillo A, Khoury N, Xu J, Dam ML, Escobar AAH, Dave KR, Perez-Pinzon MA, Gittens RA. Region-specific brain decellularized extracellular matrix promotes cell recovery in an in vitro model of stroke. Sci Rep 2025; 15:11921. [PMID: 40195414 PMCID: PMC11976941 DOI: 10.1038/s41598-025-95656-w] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/21/2024] [Accepted: 03/24/2025] [Indexed: 04/09/2025] Open
Abstract
Brain decellularized extracellular matrix (ECM) can be an attractive scaffold capable of mimicking the native ecosystem of the central nervous system tissue. We studied the in vitro response of neural cultures exposed to region-specific brain decellularized ECM scaffolds from three distinct neuroanatomical sections: cortex, cerebellum and remaining areas. First, each brain region was evaluated with the isotropic fractionator method to understand the cellular composition of the different cerebral areas. Second, the cerebral regions were subjected to the decellularization process and their respective characterization using molecular, histological, and ultrastructural techniques. Third, the levels of neurotrophic factors in the decellularized brain scaffold were analyzed. Fourth, we studied the region-specific brain decellularized ECM as a mimetic platform for the maturation of PC12 cells, as a unidirectional model of differentiation. Finally, in vitro studies were carried out to evaluate the cell recovery capacity of brain decellularized ECM under stroke-mimetic conditions. Our results show that region-specific brain decellularized ECM can serve as a biomimetic scaffold capable of promoting the growth of neural lineage cells and, in addition, it possesses a combination of structural and biochemical signals (e.g., neurotrophic factors) that are capable of inducing cell phenotypic changes and promote viability and cell recovery in a stroke/ischemia model in vitro.
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Affiliation(s)
- Diego Reginensi
- Center for Biodiversity and Drug Discovery, Instituto de Investigaciones Científicas y Servicios de Alta Tecnología (INDICASAT-AIP), Panama, Panama
- Advanced Therapies, School of Medicine, Universidad de Panamá (UP), Panama, Panama
- Tissue Engineering, Faculty of Biosciences and Public Health, Universidad Especializada de las Américas (UDELAS), Panama, Panama
- Biomedical Engineering, Faculty of Health Sciences and Engineering, Universidad Latina de Panama (ULATINA), Panama, Panama
- Sistema Nacional de Investigación (SNI-SENACYT), Panama, Panama
| | - Didio Alberto Ortiz
- Center for Biodiversity and Drug Discovery, Instituto de Investigaciones Científicas y Servicios de Alta Tecnología (INDICASAT-AIP), Panama, Panama
| | - Bernardino Denis
- Center for Biodiversity and Drug Discovery, Instituto de Investigaciones Científicas y Servicios de Alta Tecnología (INDICASAT-AIP), Panama, Panama
- Sistema Nacional de Investigación (SNI-SENACYT), Panama, Panama
- MD-PhD Program in Clinical and Biomedical Research, School of Medicine, UP, Panama, Panama
| | - Solangel Castillo
- Advanced Therapies, School of Medicine, Universidad de Panamá (UP), Panama, Panama
- Tissue Engineering, Faculty of Biosciences and Public Health, Universidad Especializada de las Américas (UDELAS), Panama, Panama
| | - Andrea Burillo
- Center for Biodiversity and Drug Discovery, Instituto de Investigaciones Científicas y Servicios de Alta Tecnología (INDICASAT-AIP), Panama, Panama
| | - Nathalie Khoury
- Neuroscience Program, University of Miami Miller School of Medicine, Miami, FL, USA
- Neurology Department, The Peritz Scheinberg Cerebral Vascular Disease Research Laboratories, University of Miami Miller School of Medicine, Miami, FL, USA
| | - Jing Xu
- Neuroscience Program, University of Miami Miller School of Medicine, Miami, FL, USA
- Neurology Department, The Peritz Scheinberg Cerebral Vascular Disease Research Laboratories, University of Miami Miller School of Medicine, Miami, FL, USA
| | - Maria Lucia Dam
- Center for Biodiversity and Drug Discovery, Instituto de Investigaciones Científicas y Servicios de Alta Tecnología (INDICASAT-AIP), Panama, Panama
| | - Anthony A Hurtado Escobar
- Center for Biodiversity and Drug Discovery, Instituto de Investigaciones Científicas y Servicios de Alta Tecnología (INDICASAT-AIP), Panama, Panama
- Biomedical Engineering, Faculty of Health Sciences and Engineering, Universidad Latina de Panama (ULATINA), Panama, Panama
| | - Kunjan R Dave
- Neuroscience Program, University of Miami Miller School of Medicine, Miami, FL, USA
- Neurology Department, The Peritz Scheinberg Cerebral Vascular Disease Research Laboratories, University of Miami Miller School of Medicine, Miami, FL, USA
| | - Miguel A Perez-Pinzon
- Neuroscience Program, University of Miami Miller School of Medicine, Miami, FL, USA
- Neurology Department, The Peritz Scheinberg Cerebral Vascular Disease Research Laboratories, University of Miami Miller School of Medicine, Miami, FL, USA
| | - Rolando A Gittens
- Center for Biodiversity and Drug Discovery, Instituto de Investigaciones Científicas y Servicios de Alta Tecnología (INDICASAT-AIP), Panama, Panama.
- Sistema Nacional de Investigación (SNI-SENACYT), Panama, Panama.
- MD-PhD Program in Clinical and Biomedical Research, School of Medicine, UP, Panama, Panama.
- Instituto Técnico Superior Especializado (ITSE), Ave. Domingo Diaz, Tocumen Panama, Republic of Panama.
- Centro de Investigación e Innovación Educativa, Ciencia y Tecnología (CiiECYT-AIP), Panama, Panama.
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Stern I, Barrera V, Randles M, Rooney P. Advances in preparation of acellular human dermis for tissue banking and transplantation. Cell Tissue Bank 2024; 26:3. [PMID: 39653869 PMCID: PMC11628444 DOI: 10.1007/s10561-024-10153-0] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/23/2023] [Accepted: 11/20/2024] [Indexed: 12/12/2024]
Abstract
Non-healing wounds cost the National Health Service over £5.6 billion annually in wound management. Skin allografts are used to treat non-healing wounds, ulcers and burns, offering the best protection against infection. In order to allow host cells to repopulate and to avoid immunogenicity, cell components are removed through decellularisation. Decellularisation of human dermis has so far been performed in NHS Blood and Transplant using a combination of two enzymes (RNase T1 and the recombinant human DNase Pulmozyme)®. This study aims at validating a new method to remove DNA from donated dermis via the use of a single enzyme, Benzonase, known for its effectiveness of DNA digestion. Skin samples were decellularised by removing the epidermis, lysing of dermal cells, removal of cellular fragments by a detergent wash and removal of nucleic acids by a nuclease incubation with either Benzonase or Pulmozyme + RNase T1. DNA quantification with PicoGreen, as well as histology on wax-embedded biopsies, stained with DAPI and haemotoxylin and eosin, were performed. In vitro toxicity test on human osteosarcoma immortalised cells and skin fibroblasts, and biomechanical (tensile) testing, were also performed. The effectiveness of DNA digestion with the new methodology was comparable to previous procedure. Mean DNA removal percentage following decellularisation with Pulmozyme + RNase was 99.9% (3.83 ng/mg). Mean DNA removal percentage with Benzonase was 99.8% (9.97 ng/mg). Histology staining showed complete decellularisation following either method. Benzonase was proven to be non-toxic to both cell lines used, and a one-way Anova test showed no significant difference in neither stress nor strain between acellular dermal matrix decellularised with either Benzonase or Pulmozyme + RNase T1. Benzonase was able to effectively decellularise dermis after prior removal of epidermis. It performed just as well as the combination of Pulmozyme + RNase T1, but represents significant advantages in terms of cost effectiveness, procurement and storage; Benzonase has been successfully used in the decellularisation of other tissues, thus would be better for Tissue Banking use. Switching to this combined DNase/RNase can have far-reaching consequences in the production of acellular human dermal matrix by NHSBT and in the treatment of patients requiring it.
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Affiliation(s)
- Irit Stern
- NHS Blood and Transplant, Tissue Services, 14 Estuary Banks, Speke, Liverpool, L24 8RB, UK.
| | - Valentina Barrera
- NHS Blood and Transplant, Tissue Services R&D, 14 Estuary Banks, Speke, Liverpool, L24 8RB, UK
| | - Michael Randles
- Faculty of Medicine and Life Sciences, Chester Medical School, University of Chester, Chester, UK
| | - Paul Rooney
- NHS Blood and Transplant, Tissue Services R&D, 14 Estuary Banks, Speke, Liverpool, L24 8RB, UK
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Dortaj H, Vaez A, Hassanpour-Dehnavie A, Alizadeh AA. An update on technical method of cartilage decellularization: A physical-based protocol. BIOIMPACTS : BI 2024; 15:30047. [PMID: 40161940 PMCID: PMC11954751 DOI: 10.34172/bi.2024.30047] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 01/07/2023] [Revised: 08/12/2023] [Accepted: 09/09/2023] [Indexed: 04/02/2025]
Abstract
Introduction Despite advances in orthopedic surgery, the lack of effective conventional treatment for cartilage defects has led to research in cartilage tissue engineering. One of the interesting topics is the use of decellularized extracellular matrix (ECM) as a suitable natural scaffold that supports the growth and function of cells cultured in it. A concern with decellularization protocols, especially those with high detergent concentrations, is the disruption of native ECM, which has deleterious effects on subsequent scaffold recellularization. Therefore, this study focused on optimizing cartilage decellularization by physical methods without the use of ionic detergents. Methods The bovine tracheal cartilage fragments were decellularized by a combination of 8 cycles of freeze-thaw and ultrasound techniques. Then, the tissues were immersed and shaken in 0.25% trypsin for 24 hours. Efficient cell removal and preservation of ECM were confirmed by histological and cytocompatibility assessments. The in-vivo studies were performed to evaluate the biocompatibility and bioactivity of the scaffold. Results The histological assessments indicated the appropriate cytocompatibility and the fibroblast cell culture study demonstrated that cells were able to proliferate and migrate on the decellularized cartilage. In-vivo evaluation also showed a reduced adverse immune response, including leukocyte infiltration into the ECM. Conclusion These results suggest that a cartilage scaffold created using a physical decellularization protocol that efficiently removes cells while preserving the native ECM can be a suitable scaffold for cartilage reconstruction. The main advantage of this protocol is the absence of potentially toxic chemicals in the tissues.
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Affiliation(s)
- Hengameh Dortaj
- Department of Tissue Engineering and Applied Cell Sciences, School of Advance Medical Sciences and Technologies, Shiraz University of Medical Sciences, Shiraz, Iran
- Tissue Engineering Research Group (TERG), Department of Anatomy and Cell Biology, School of Medicine, Mashhad University of Medical Sciences, Mashhad, Iran
| | - Ahmad Vaez
- Department of Tissue Engineering and Applied Cell Sciences, School of Advance Medical Sciences and Technologies, Shiraz University of Medical Sciences, Shiraz, Iran
| | - Ashraf Hassanpour-Dehnavie
- Tissue Engineering Lab, Department of Anatomical Sciences, School of Medicine, Shiraz University of Medical Sciences, Shiraz, Iran
| | - Ali Akbar Alizadeh
- Department of Tissue Engineering and Applied Cell Sciences, School of Advance Medical Sciences and Technologies, Shiraz University of Medical Sciences, Shiraz, Iran
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Snyder Y, Jana S. Innovative Substrate Design with Basement Membrane Components for Enhanced Endothelial Cell Function and Endothelization. Adv Healthc Mater 2024; 13:e2401150. [PMID: 39021293 DOI: 10.1002/adhm.202401150] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/27/2024] [Revised: 06/28/2024] [Indexed: 07/20/2024]
Abstract
Enhancing endothelial cell growth on small-diameter vascular grafts produced from decellularized tissues or synthetic substrates is pivotal for preventing thrombosis. While optimized decellularization protocols can preserve the structure and many components of the extracellular matrix (ECM), the process can still lead to the loss of crucial basement membrane proteins, such as laminin, collagen IV, and perlecan, which are pivotal for endothelial cell adherence and functional growth. This loss can result in poor endothelialization and endothelial cell activation causing thrombosis and intimal hyperplasia. To address this, the basement membrane's ECM is emulated on fiber substrates, providing a more physiological environment for endothelial cells. Thus, fibroblasts are cultured on fiber substrates to produce an ECM membrane substrate (EMMS) with basement membrane proteins. The EMMS then underwent antigen removal (AR) treatment to eliminate antigens from the membrane while preserving essential proteins and producing an AR-treated membrane substrate (AMS). Subsequently, human endothelial cells cultured on the AMS exhibited superior proliferation, nitric oxide production, and increased expression of endothelial markers of quiescence/homeostasis, along with autophagy and antithrombotic factors, compared to those on the decellularized aortic tissue. This strategy showed the potential of pre-endowing fiber substrates with a basement membrane to enable better endothelization.
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Affiliation(s)
- Yuriy Snyder
- Department of Chemical and Biomedical Engineering, University of Missouri, 1406 Rollins Street, Columbia, MO, 65211, USA
| | - Soumen Jana
- Department of Chemical and Biomedical Engineering, University of Missouri, 1406 Rollins Street, Columbia, MO, 65211, USA
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Milton LA, Davern JW, Hipwood L, Chaves JCS, McGovern J, Broszczak D, Hutmacher DW, Meinert C, Toh YC. Liver click dECM hydrogels for engineering hepatic microenvironments. Acta Biomater 2024; 185:144-160. [PMID: 38960110 DOI: 10.1016/j.actbio.2024.06.037] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/22/2024] [Revised: 06/20/2024] [Accepted: 06/25/2024] [Indexed: 07/05/2024]
Abstract
Decellularized extracellular matrix (dECM) hydrogels provide tissue-specific microenvironments which accommodate physiological cellular phenotypes in 3D in vitro cell cultures. However, their formation hinges on collagen fibrillogenesis, a complex process which limits regulation of physicochemical properties. Hence, achieving reproducible results with dECM hydrogels poses as a challenge. Here, we demonstrate that thiolation of solubilized liver dECM enables rapid formation of covalently crosslinked hydrogels via Michael-type addition, allowing for precise control over mechanical properties and superior organotypic biological activity. Investigation of various decellularization methodologies revealed that treatment of liver tissue with Triton X-100 and ammonium hydroxide resulted in near complete DNA removal with significant retention of the native liver proteome. Chemical functionalization of pepsin-solubilized liver dECMs via 1-ethyl-3(3-dimethylamino)propyl carbodiimide (EDC)/N-hydroxysuccinimide (NHS) coupling of l-Cysteine created thiolated liver dECM (dECM-SH), which rapidly reacted with 4-arm polyethylene glycol (PEG)-maleimide to form optically clear hydrogels under controlled conditions. Importantly, Young's moduli could be precisely tuned between 1 - 7 kPa by varying polymer concentrations, enabling close replication of healthy and fibrotic liver conditions in in vitro cell cultures. Click dECM-SH hydrogels were cytocompatible, supported growth of HepG2 and HepaRG liver cells, and promoted liver-specific functional phenotypes as evidenced by increased metabolic activity, as well CYP1A2 and CYP3A4 activity and excretory function when compared to monolayer culture and collagen-based hydrogels. Our findings demonstrate that click-functionalized dECM hydrogels offer a highly controlled, reproducible alternative to conventional tissue-derived hydrogels for in vitro cell culture applications. STATEMENT OF SIGNIFICANCE: Traditional dECM hydrogels face challenges in reproducibility and mechanical property control due to variable crosslinking processes. We introduce a click hydrogel based on porcine liver decellularized extracellular matrix (dECM) that circumnavigates these challenges. After optimizing liver decellularization for ECM retention, we integrated thiol-functionalized liver dECM with polyethylene-glycol derivatives through Michael-type addition click chemistry, enabling rapid, room-temperature gelation. This offers enhanced control over the hydrogel's mechanical and biochemical properties. The resultant click dECM hydrogels mimic the liver's natural ECM and exhibit greater mechanical tunability and handling ease, facilitating their application in high-throughput and industrial settings. Moreover, these hydrogels significantly improve the function of HepaRG-derived hepatocytes in 3D culture, presenting an advancement for liver tissue cell culture models for drug testing applications.
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Affiliation(s)
- Laura A Milton
- Faculty of Engineering, School of Mechanical, Medical and Process Engineering, Queensland University of Technology, Brisbane, Australia; Centre for Biomedical Technologies, Queensland University of Technology, Brisbane, Australia; Gelomics Pty Ltd, Brisbane, Australia
| | - Jordan W Davern
- Faculty of Engineering, School of Mechanical, Medical and Process Engineering, Queensland University of Technology, Brisbane, Australia; Centre for Biomedical Technologies, Queensland University of Technology, Brisbane, Australia; Gelomics Pty Ltd, Brisbane, Australia; ARC Training Centre for Cell and Tissue Engineering Technologies, Queensland University of Technology, Brisbane, Australia
| | - Luke Hipwood
- Centre for Biomedical Technologies, Queensland University of Technology, Brisbane, Australia; Gelomics Pty Ltd, Brisbane, Australia; Faculty of Health, School of Biomedical Sciences, Queensland University of Technology, Brisbane, Australia
| | - Juliana C S Chaves
- Cell & Molecular Biology Department, Mental Health Program, QIMR Berghofer Medical Research Institute, Brisbane, Australia
| | - Jacqui McGovern
- Centre for Biomedical Technologies, Queensland University of Technology, Brisbane, Australia; ARC Training Centre for Cell and Tissue Engineering Technologies, Queensland University of Technology, Brisbane, Australia; Max Planck Queensland Centre (MPQC) for the Materials Science of Extracellular Matrices, Queensland University of Technology, Brisbane, Australia
| | - Daniel Broszczak
- Faculty of Health, School of Biomedical Sciences, Queensland University of Technology, Brisbane, Australia
| | - Dietmar W Hutmacher
- Centre for Biomedical Technologies, Queensland University of Technology, Brisbane, Australia; ARC Training Centre for Cell and Tissue Engineering Technologies, Queensland University of Technology, Brisbane, Australia; Max Planck Queensland Centre (MPQC) for the Materials Science of Extracellular Matrices, Queensland University of Technology, Brisbane, Australia; Australian Research Council (ARC) Training Centre for Multiscale 3D Imaging, Modelling and Manufacturing (M3D Innovation), Queensland University of Technology, Brisbane, Australia
| | - Christoph Meinert
- Centre for Biomedical Technologies, Queensland University of Technology, Brisbane, Australia; Gelomics Pty Ltd, Brisbane, Australia.
| | - Yi-Chin Toh
- Faculty of Engineering, School of Mechanical, Medical and Process Engineering, Queensland University of Technology, Brisbane, Australia; Centre for Biomedical Technologies, Queensland University of Technology, Brisbane, Australia; ARC Training Centre for Cell and Tissue Engineering Technologies, Queensland University of Technology, Brisbane, Australia; Max Planck Queensland Centre (MPQC) for the Materials Science of Extracellular Matrices, Queensland University of Technology, Brisbane, Australia; Centre for Microbiome Research, Queensland University of Technology, Brisbane, Australia.
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Wang X, Guo J, Yu Q, Zhao L, Gao X, Wang L, Wen M, Yan J, An M, Liu Y. Decellularized Matrices for the Treatment of Tissue Defects: from Matrix Origin to Immunological Mechanisms. Biomol Ther (Seoul) 2024; 32:509-522. [PMID: 39091238 PMCID: PMC11392660 DOI: 10.4062/biomolther.2024.050] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/23/2024] [Revised: 05/07/2024] [Accepted: 05/31/2024] [Indexed: 08/04/2024] Open
Abstract
Decellularized matrix transplantation has emerged as a promising therapeutic approach for repairing tissue defects, with numerous studies assessing its safety and efficacy in both animal models and clinical settings. The host immune response elicited by decellularized matrix grafts of natural biological origin plays a crucial role in determining the success of tissue repair, influenced by matrix heterogeneity and the inflammatory microenvironment of the wound. However, the specific immunologic mechanisms underlying the interaction between decellularized matrix grafts and the host immune system remain elusive. This article reviews the sources of decellularized matrices, available decellularization techniques, and residual immunogenic components. It focuses on the host immune response following decellularized matrix transplantation, with emphasis on the key mechanisms of Toll-like receptor, T-cell receptor, and TGF-β/SMAD signaling in the stages of post-transplantation immunorecognition, immunomodulation, and tissue repair, respectively. Furthermore, it highlights the innovative roles of TLR10 and miR-29a-3p in improving transplantation outcomes. An in-depth understanding of the molecular mechanisms underlying the host immune response after decellularized matrix transplantation provides new directions for the repair of tissue defects.
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Affiliation(s)
- Xinyue Wang
- Institute of Biomedical Engineering, College of Biomedical Engineering, Taiyuan University of Technology, Shanxi 030024, China
| | - Jiqiang Guo
- Institute of Biomedical Engineering, College of Biomedical Engineering, Taiyuan University of Technology, Shanxi 030024, China
- Shanxi Bethune Hospital, Shanxi Academy of Medical Sciences, Tongji Shanxi Hospital, Third Hospital of Shanxi Medical University, Taiyuan 030032, China
- Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030, China
| | - Qing Yu
- Institute of Biomedical Engineering, College of Biomedical Engineering, Taiyuan University of Technology, Shanxi 030024, China
| | - Luyao Zhao
- Institute of Biomedical Engineering, College of Biomedical Engineering, Taiyuan University of Technology, Shanxi 030024, China
| | - Xiang Gao
- Institute of Biomedical Engineering, College of Biomedical Engineering, Taiyuan University of Technology, Shanxi 030024, China
| | - Li Wang
- Institute of Biomedical Engineering, College of Biomedical Engineering, Taiyuan University of Technology, Shanxi 030024, China
| | - Meiling Wen
- Institute of Biomedical Engineering, College of Biomedical Engineering, Taiyuan University of Technology, Shanxi 030024, China
| | - Junrong Yan
- Shanxi Bethune Hospital, Shanxi Academy of Medical Sciences, Tongji Shanxi Hospital, Third Hospital of Shanxi Medical University, Taiyuan 030032, China
| | - Meiwen An
- Institute of Biomedical Engineering, College of Biomedical Engineering, Taiyuan University of Technology, Shanxi 030024, China
| | - Yang Liu
- Institute of Biomedical Engineering, College of Biomedical Engineering, Taiyuan University of Technology, Shanxi 030024, China
- Department of Nuclear Medicine, First Hospital of Shanxi Medical University, Taiyuan 030001, China
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Ferreira LP, Jorge C, Lagarto MR, Monteiro MV, Duarte IF, Gaspar VM, Mano JF. Photoacoustic processing of decellularized extracellular matrix for biofabricating living constructs. Acta Biomater 2024; 183:74-88. [PMID: 38838910 DOI: 10.1016/j.actbio.2024.05.054] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/22/2023] [Revised: 05/07/2024] [Accepted: 05/30/2024] [Indexed: 06/07/2024]
Abstract
The diverse biomolecular landscape of tissue-specific decellularized extracellular matrix (dECM) biomaterials provides a multiplicity of bioinstructive cues to target cells, rendering them highly valuable for various biomedical applications. However, the isolation of dECM biomaterials entails cumbersome xenogeneic enzymatic digestions and also additional inactivation procedures. Such, increases processing time, increments costs and introduces residues of non-naturally present proteins in dECM formulations that remain present even after inactivation. To overcome these limitations, herein we report an innovative conjugation of light and ultrasound-mediated dECM biomaterial processing for fabricating dECM biomaterials. Such approach gathers on ultrasound waves to facilitate dECM-in-liquid processing and visible light photocrosslinking of tyrosine residues naturally present in dECM biomaterials. This dual step methodology unlocked the in-air production of cell laden dECM hydrogels or programmable dECM hydrogel spherical-like beads by using superhydrophobic surfaces. These in-air produced units do not require any additional solvents and successfully supported both fibroblasts and breast cancer cells viability upon encapsulation or surface seeding. In addition, the optimized photoacoustic methodology also enabled a rapid formulation of dECM biomaterial inks with suitable features for biofabricating volumetrically defined living constructs through embedded 3D bioprinting. The biofabricated dECM hydrogel constructs supported cell adhesion, spreading and viability for 7 days. Overall, the implemented photoacoustic processing methodology of dECM biomaterials offers a rapid and universal strategy for upgrading their processing from virtually any tissue. STATEMENT OF SIGNIFICANCE: Leveraging decellularized extracellular matrix (dECM) as cell instructive biomaterials has potential to open new avenues for tissue engineering and in vitro disease modelling. The processing of dECM remains however, lengthy, costly and introduces non-naturally present proteins in the final biomaterials formulations. In this regard, here we report an innovative light and ultrasound two-step methodology that enables rapid dECM-in-liquid processing and downstream photocrosslinking of dECM hydrogel beads and 3D bioprinted constructs. Such photoacoustic based processing constitutes a universally applicable method for processing any type of tissue-derived dECM biomaterials.
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Affiliation(s)
- Luís P Ferreira
- Department of Chemistry, CICECO - Aveiro Institute of Materials, University of Aveiro, Campus Universitário de Santiago, 3810-193, Aveiro, Portugal
| | - Carole Jorge
- Department of Chemistry, CICECO - Aveiro Institute of Materials, University of Aveiro, Campus Universitário de Santiago, 3810-193, Aveiro, Portugal
| | - Matilde R Lagarto
- Department of Chemistry, CICECO - Aveiro Institute of Materials, University of Aveiro, Campus Universitário de Santiago, 3810-193, Aveiro, Portugal
| | - Maria V Monteiro
- Department of Chemistry, CICECO - Aveiro Institute of Materials, University of Aveiro, Campus Universitário de Santiago, 3810-193, Aveiro, Portugal
| | - Iola F Duarte
- Department of Chemistry, CICECO - Aveiro Institute of Materials, University of Aveiro, Campus Universitário de Santiago, 3810-193, Aveiro, Portugal
| | - Vítor M Gaspar
- Department of Chemistry, CICECO - Aveiro Institute of Materials, University of Aveiro, Campus Universitário de Santiago, 3810-193, Aveiro, Portugal.
| | - João F Mano
- Department of Chemistry, CICECO - Aveiro Institute of Materials, University of Aveiro, Campus Universitário de Santiago, 3810-193, Aveiro, Portugal.
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Sanguansook P, Martínez-López C, Izquierdo-Rico MJ, Martínez-Cáceres C, López-Orozco M, Chatdarong K, García-Vázquez FA. Development of decellularization protocols for female cat reproductive organs. Res Vet Sci 2024; 173:105257. [PMID: 38636324 DOI: 10.1016/j.rvsc.2024.105257] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/23/2023] [Revised: 03/27/2024] [Accepted: 04/08/2024] [Indexed: 04/20/2024]
Abstract
Decellularization is an innovative method to create natural scaffolds by removing all cellular materials while preserving the composition and three-dimensional ultrastructure of the extracellular matrix (ECM). The obtention of decellularized reproductive organs in cats might facilitate the development of assisted reproductive techniques not only in this species but also in other felids. The aim was to compare the efficiency of three decellularization protocols on reproductive organs (ovary, oviduct, and uterine horn) in domestic cats. The decellularization protocol involved 0.1% sodium dodecyl sulfate and 1%Triton X-100. Protocol 1 (P1) entailed 2-cycles of decellularization using these detergents. Protocol 2 (P2) was like P1 but included 3-cycles. Protocol 3 (P3) was similar to P2, with the addition of deoxyribonuclease incubation. Reproductive organs from nine cats were separated into two sides. One side served as the control (non-decellularized organ) while the contralateral side was the treated group (decellularized organ). The treated organs were subdivided into 3 groups (n = 3 per group) for each protocol. Both control and treated samples were analyzed for DNA content, histology (nuclear and ECM (collagen, elastin, and glycosaminoglycans (GAGs)) density), ultrastructure by electron microscopy, and cytotoxicity. The results of the study showed that P3 was the only protocol that displayed no nucleus residue and significantly reduced DNA content in decellularized samples (in all the studied organs) compared to the control (P < 0.05). The ECM content in the ovaries remained similar across all protocols compared with controls (P > 0.05). However, elastic fibers and GAGs decreased in decellularized oviducts (P < 0.05), while collagen levels remained unchanged (P > 0.05). Regarding the uterus, the ECM content decreased in decellularized uterine horns from P3 (P < 0.05). Electron microscopy revealed that the microarchitecture of the decellularized samples was maintained compared to controls. The decellularized tissues, upon being washed for 24 h, showed cytocompatibility following co-incubation with sperm. In conclusion, when comparing different decellularization methods, P3 proved to be the most efficient in removing nuclear material from reproductive organs compared to P1 and P2. P3 demonstrated its success in decellularizing ovarian samples by significantly decreasing DNA content while maintaining ECM components and tissue microarchitecture. However, P3 was less effective in maintaining ECM contents in decellularized oviducts and uterine horns.
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Affiliation(s)
- Phakjira Sanguansook
- Department of Obstetrics, Gynaecology and Reproduction, Faculty of Veterinary Science, Chulalongkorn University, Bangkok, Thailand
| | - Cristina Martínez-López
- Departamento de Biología Celular e Histología, Facultad de Medicina, Universidad de Murcia, Murcia, Spain; Instituto Murciano de Investigación Biosanitaria Pascual Parrilla (IMIB), Murcia, Spain; Departamento de Fisiología, Facultad de Veterinaria, Universidad de Murcia, Murcia, Spain
| | - Mª José Izquierdo-Rico
- Departamento de Biología Celular e Histología, Facultad de Medicina, Universidad de Murcia, Murcia, Spain; Instituto Murciano de Investigación Biosanitaria Pascual Parrilla (IMIB), Murcia, Spain
| | | | - Marina López-Orozco
- Departamento de Producción Animal, Facultad de Veterinaria, Campus de Excelencia Internacional para la Educación Superior y la Investigación "Campus Mare Nostrum", Universidad de Murcia, Murcia, Spain
| | - Kaywalee Chatdarong
- Department of Obstetrics, Gynaecology and Reproduction, Faculty of Veterinary Science, Chulalongkorn University, Bangkok, Thailand.
| | - Francisco Alberto García-Vázquez
- Instituto Murciano de Investigación Biosanitaria Pascual Parrilla (IMIB), Murcia, Spain; Departamento de Fisiología, Facultad de Veterinaria, Universidad de Murcia, Murcia, Spain.
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9
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Barajaa MA, Otsuka T, Ghosh D, Kan HM, Laurencin CT. Development of porcine skeletal muscle extracellular matrix-derived hydrogels with improved properties and low immunogenicity. Proc Natl Acad Sci U S A 2024; 121:e2322822121. [PMID: 38687784 PMCID: PMC11087813 DOI: 10.1073/pnas.2322822121] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/26/2023] [Accepted: 03/18/2024] [Indexed: 05/02/2024] Open
Abstract
Hydrogels derived from decellularized extracellular matrices (ECM) of animal origin show immense potential for regenerative applications due to their excellent cytocompatibility and biomimetic properties. Despite these benefits, the impact of decellularization protocols on the properties and immunogenicity of these hydrogels remains relatively unexplored. In this study, porcine skeletal muscle ECM (smECM) underwent decellularization using mechanical disruption (MD) and two commonly employed decellularization detergents, sodium deoxycholate (SDC) or Triton X-100. To mitigate immunogenicity associated with animal-derived ECM, all decellularized tissues were enzymatically treated with α-galactosidase to cleave the primary xenoantigen-the α-Gal antigen. Subsequently, the impact of the different decellularization protocols on the resultant hydrogels was thoroughly investigated. All methods significantly reduced total DNA content in hydrogels. Moreover, α-galactosidase treatment was crucial for cleaving α-Gal antigens, suggesting that conventional decellularization methods alone are insufficient. MD preserved total protein, collagen, sulfated glycosaminoglycan, laminin, fibronectin, and growth factors more efficiently than other protocols. The decellularization method impacted hydrogel gelation kinetics and ultrastructure, as confirmed by turbidimetric and scanning electron microscopy analyses. MD hydrogels demonstrated high cytocompatibility, supporting satellite stem cell recruitment, growth, and differentiation into multinucleated myofibers. In contrast, the SDC and Triton X-100 protocols exhibited cytotoxicity. Comprehensive in vivo immunogenicity assessments in a subcutaneous xenotransplantation model revealed MD hydrogels' biocompatibility and low immunogenicity. These findings highlight the significant influence of the decellularization protocol on hydrogel properties. Our results suggest that combining MD with α-galactosidase treatment is an efficient method for preparing low-immunogenic smECM-derived hydrogels with enhanced properties for skeletal muscle regenerative engineering and clinical applications.
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Affiliation(s)
- Mohammed A. Barajaa
- Department of Biomedical Engineering, College of Engineering, Imam Abdulrahman Bin Faisal University, Dammam34212, Saudi Arabia
| | - Takayoshi Otsuka
- The Cato T. Laurencin Institute for Regenerative Engineering, University of Connecticut, Farmington, CT06030
| | - Debolina Ghosh
- The Cato T. Laurencin Institute for Regenerative Engineering, University of Connecticut, Farmington, CT06030
| | - Ho-Man Kan
- The Cato T. Laurencin Institute for Regenerative Engineering, University of Connecticut, Farmington, CT06030
| | - Cato T. Laurencin
- The Cato T. Laurencin Institute for Regenerative Engineering, University of Connecticut, Farmington, CT06030
- Department of Biomedical Engineering, University of Connecticut, Storrs, CT06269
- Department of Orthopedic Surgery, University of Connecticut Health Center, Farmington, CT06030
- Department of Materials Science & Engineering, University of Connecticut, Storrs, CT06269
- Department of Chemical & Bimolecular Engineering, University of Connecticut, Storrs, CT06269
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10
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Gadre M, Kasturi M, Agarwal P, Vasanthan KS. Decellularization and Their Significance for Tissue Regeneration in the Era of 3D Bioprinting. ACS OMEGA 2024; 9:7375-7392. [PMID: 38405516 PMCID: PMC10883024 DOI: 10.1021/acsomega.3c08930] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Grants] [Track Full Text] [Figures] [Subscribe] [Scholar Register] [Received: 11/09/2023] [Revised: 12/19/2023] [Accepted: 01/10/2024] [Indexed: 02/27/2024]
Abstract
Three-dimensional bioprinting is an emerging technology that has high potential application in tissue engineering and regenerative medicine. Increasing advancement and improvement in the decellularization process have led to an increase in the demand for using a decellularized extracellular matrix (dECM) to fabricate tissue engineered products. Decellularization is the process of retaining the extracellular matrix (ECM) while the cellular components are completely removed to harvest the ECM for the regeneration of various tissues and across different sources. Post decellularization of tissues and organs, they act as natural biomaterials to provide the biochemical and structural support to establish cell communication. Selection of an effective method for decellularization is crucial, and various factors like tissue density, geometric organization, and ECM composition affect the regenerative potential which has an impact on the end product. The dECM is a versatile material which is added as an important ingredient to formulate the bioink component for constructing tissue and organs for various significant studies. Bioink consisting of dECM from various sources is used to generate tissue-specific bioink that is unique and to mimic different biometric microenvironments. At present, there are many different techniques applied for decellularization, and the process is not standardized and regulated due to broad application. This review aims to provide an overview of different decellularization procedures, and we also emphasize the different dECM-derived bioinks present in the current global market and the major clinical outcomes. We have also highlighted an overview of benefits and limitations of different decellularization methods and various characteristic validations of decellularization and dECM-derived bioinks.
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Affiliation(s)
- Mrunmayi Gadre
- Manipal
Centre for Biotherapeutics Research, Manipal
Academy of Higher Education, Manipal 576104, Karnataka, India
| | - Meghana Kasturi
- Department
of Mechanical Engineering, University of
Michigan, Dearborn, Michigan 48128, United States
| | - Prachi Agarwal
- Manipal
Centre for Biotherapeutics Research, Manipal
Academy of Higher Education, Manipal 576104, Karnataka, India
| | - Kirthanashri S. Vasanthan
- Manipal
Centre for Biotherapeutics Research, Manipal
Academy of Higher Education, Manipal 576104, Karnataka, India
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11
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Dhandapani V, Vermette P. Decellularized bladder as scaffold to support proliferation and functionality of insulin-secreting pancreatic cells. J Biomed Mater Res B Appl Biomater 2023; 111:1890-1902. [PMID: 37306142 DOI: 10.1002/jbm.b.35292] [Citation(s) in RCA: 2] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/02/2023] [Revised: 05/07/2023] [Accepted: 05/31/2023] [Indexed: 06/13/2023]
Abstract
Loss in the number or function of insulin-producing β-cells in pancreatic islets has been associated with diabetes mellitus. Although islet transplantation can be an alternative treatment, complications such as apoptosis, ischaemia and loss of viability have been reported. The use of decellularized organs as scaffolds in tissue engineering is of interest owing to the unique ultrastructure and composition of the extracellular matrix (ECM) believed to act on tissue regeneration. In this study, a cell culture system has been designed to study the effect of decellularized porcine bladder pieces on INS-1 cells, a cell line secreting insulin in response to glucose stimulation. Porcine bladders were decellularized using two techniques: a detergent-containing and a detergent-free methods. The resulting ECMs were characterized for the removal of both cells and dsDNA. INS-1 cells were not viable on ECM produced using detergent (i.e., sodium dodecyl sulfate). INS-1 cells were visualized following 7 days of culture on detergent-free decellularized bladders using a cell viability and metabolism assay (MTT) and cell proliferation quantified (CyQUANT™ NF Cell Proliferation Assay). Further, glucose-stimulated insulin secretion and immunostaining confirmed that cells were functional in response to glucose stimulation, as well as they expressed insulin and interacted with the detergent-free produced ECM, respectively.
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Affiliation(s)
- Vignesh Dhandapani
- Laboratoire de bio-ingénierie et de biophysique de l'Université de Sherbrooke, Department of Chemical and Biotechnological Engineering, Université de Sherbrooke, Sherbrooke, Canada
- Centre de recherche du CHUS, Faculté de médecine et des sciences de la santé, Sherbrooke, Canada
| | - Patrick Vermette
- Laboratoire de bio-ingénierie et de biophysique de l'Université de Sherbrooke, Department of Chemical and Biotechnological Engineering, Université de Sherbrooke, Sherbrooke, Canada
- Centre de recherche du CHUS, Faculté de médecine et des sciences de la santé, Sherbrooke, Canada
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12
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Afzal Z, Huguet EL. Bioengineering liver tissue by repopulation of decellularised scaffolds. World J Hepatol 2023; 15:151-179. [PMID: 36926238 PMCID: PMC10011915 DOI: 10.4254/wjh.v15.i2.151] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 10/26/2022] [Revised: 11/22/2022] [Accepted: 02/15/2023] [Indexed: 02/24/2023] Open
Abstract
Liver transplantation is the only curative therapy for end stage liver disease, but is limited by the organ shortage, and is associated with the adverse consequences of immunosuppression. Repopulation of decellularised whole organ scaffolds with appropriate cells of recipient origin offers a theoretically attractive solution, allowing reliable and timely organ sourcing without the need for immunosuppression. Decellularisation methodologies vary widely but seek to address the conflicting objectives of removing the cellular component of tissues whilst keeping the 3D structure of the extra-cellular matrix intact, as well as retaining the instructive cell fate determining biochemicals contained therein. Liver scaffold recellularisation has progressed from small rodent in vitro studies to large animal in vivo perfusion models, using a wide range of cell types including primary cells, cell lines, foetal stem cells, and induced pluripotent stem cells. Within these models, a limited but measurable degree of physiologically significant hepatocyte function has been reported with demonstrable ammonia metabolism in vivo. Biliary repopulation and function have been restricted by challenges relating to the culture and propagations of cholangiocytes, though advances in organoid culture may help address this. Hepatic vasculature repopulation has enabled sustainable blood perfusion in vivo, but with cell types that would limit clinical applications, and which have not been shown to have the specific functions of liver sinusoidal endothelial cells. Minority cell groups such as Kupffer cells and stellate cells have not been repopulated. Bioengineering by repopulation of decellularised scaffolds has significantly progressed, but there remain significant experimental challenges to be addressed before therapeutic applications may be envisaged.
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Affiliation(s)
- Zeeshan Afzal
- Department of Surgery, Addenbrookes Hospital, NIHR Comprehensive Biomedical Research and Academic Health Sciences Centre; Cambridge University Hospitals NHS Foundation Trust, Cambridge CB2 0QQ, United Kingdom
| | - Emmanuel Laurent Huguet
- Department of Surgery, Addenbrookes Hospital, NIHR Comprehensive Biomedical Research and Academic Health Sciences Centre; Cambridge University Hospitals NHS Foundation Trust, Cambridge CB2 0QQ, United Kingdom
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13
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Barbulescu GI, Bojin FM, Ordodi VL, Goje ID, Barbulescu AS, Paunescu V. Decellularized Extracellular Matrix Scaffolds for Cardiovascular Tissue Engineering: Current Techniques and Challenges. Int J Mol Sci 2022; 23:13040. [PMID: 36361824 PMCID: PMC9658138 DOI: 10.3390/ijms232113040] [Citation(s) in RCA: 30] [Impact Index Per Article: 10.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/10/2022] [Revised: 10/18/2022] [Accepted: 10/26/2022] [Indexed: 08/13/2023] Open
Abstract
Cardiovascular diseases are the leading cause of global mortality. Over the past two decades, researchers have tried to provide novel solutions for end-stage heart failure to address cardiac transplantation hurdles such as donor organ shortage, chronic rejection, and life-long immunosuppression. Cardiac decellularized extracellular matrix (dECM) has been widely explored as a promising approach in tissue-regenerative medicine because of its remarkable similarity to the original tissue. Optimized decellularization protocols combining physical, chemical, and enzymatic agents have been developed to obtain the perfect balance between cell removal, ECM composition, and function maintenance. However, proper assessment of decellularized tissue composition is still needed before clinical translation. Recellularizing the acellular scaffold with organ-specific cells and evaluating the extent of cardiomyocyte repopulation is also challenging. This review aims to discuss the existing literature on decellularized cardiac scaffolds, especially on the advantages and methods of preparation, pointing out areas for improvement. Finally, an overview of the state of research regarding the application of cardiac dECM and future challenges in bioengineering a human heart suitable for transplantation is provided.
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Affiliation(s)
- Greta Ionela Barbulescu
- Immuno-Physiology and Biotechnologies Center (CIFBIOTEH), Department of Functional Sciences, “Victor Babes” University of Medicine and Pharmacy, No 2 Eftimie Murgu Square, 300041 Timisoara, Romania
- Department of Clinical Practical Skills, “Victor Babes” University of Medicine and Pharmacy, No 2 Eftimie Murgu Square, 300041 Timisoara, Romania
| | - Florina Maria Bojin
- Immuno-Physiology and Biotechnologies Center (CIFBIOTEH), Department of Functional Sciences, “Victor Babes” University of Medicine and Pharmacy, No 2 Eftimie Murgu Square, 300041 Timisoara, Romania
- Clinical Emergency County Hospital “Pius Brinzeu” Timisoara, Center for Gene and Cellular Therapies in the Treatment of Cancer Timisoara-OncoGen, No 156 Liviu Rebreanu, 300723 Timisoara, Romania
| | - Valentin Laurentiu Ordodi
- Clinical Emergency County Hospital “Pius Brinzeu” Timisoara, Center for Gene and Cellular Therapies in the Treatment of Cancer Timisoara-OncoGen, No 156 Liviu Rebreanu, 300723 Timisoara, Romania
- Faculty of Industrial Chemistry and Environmental Engineering, “Politehnica” University Timisoara, No 2 Victoriei Square, 300006 Timisoara, Romania
| | - Iacob Daniel Goje
- Department of Medical Semiology I, “Victor Babes” University of Medicine and Pharmacy, No 2 Eftimie Murgu Square, 300041 Timisoara, Romania
- Advanced Cardiology and Hemostaseology Research Center, “Victor Babes” University of Medicine and Pharmacy, No 2 Eftimie Murgu Square, 300041 Timisoara, Romania
| | - Andreea Severina Barbulescu
- Center for Advanced Research in Gastroenterology and Hepatology, Department of Internal Medicine II, Division of Gastroenterology and Hepatology, “Victor Babes” University of Medicine and Pharmacy, 300041 Timisoara, Romania
| | - Virgil Paunescu
- Immuno-Physiology and Biotechnologies Center (CIFBIOTEH), Department of Functional Sciences, “Victor Babes” University of Medicine and Pharmacy, No 2 Eftimie Murgu Square, 300041 Timisoara, Romania
- Clinical Emergency County Hospital “Pius Brinzeu” Timisoara, Center for Gene and Cellular Therapies in the Treatment of Cancer Timisoara-OncoGen, No 156 Liviu Rebreanu, 300723 Timisoara, Romania
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14
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Whitehead KM, Hendricks HKL, Cakir SN, de Castro Brás LE. ECM roles and biomechanics in cardiac tissue decellularization. Am J Physiol Heart Circ Physiol 2022; 323:H585-H596. [PMID: 35960635 PMCID: PMC9467473 DOI: 10.1152/ajpheart.00372.2022] [Citation(s) in RCA: 8] [Impact Index Per Article: 2.7] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 07/19/2022] [Revised: 08/09/2022] [Accepted: 08/09/2022] [Indexed: 11/22/2022]
Abstract
Natural biomaterials hold enormous potential for tissue regeneration. The rapid advance of several tissue-engineered biomaterials, such as natural and synthetic polymer-based scaffolds, has led to widespread application of these materials in the clinic and in research. However, biomaterials can have limited repair capacity; obstacles result from immunogenicity, difficulties in mimicking native microenvironments, and maintaining the mechanical and biochemical (i.e., biomechanical) properties of native organs/tissues. The emergence of decellularized extracellular matrix (ECM)-derived biomaterials provides an attractive solution to overcome these hurdles since decellularized ECM provides a nonimmune environment with native three-dimensional structures and bioactive components. More importantly, decellularized ECM can be generated from the tissue of interest, such as the heart, and keep its native macro- and microstructure and tissue-specific composition. These decellularized cardiac matrices/scaffolds can then be reseeded using cardiac cells, and the resulting recellularized construct is considered an ideal choice for regenerating functional organs/tissues. Nonetheless, the decellularization process must be optimized and depends on tissue type, age, and functional goal. Although most decellularization protocols significantly reduce immunogenicity and deliver a matrix that maintains the tissue macrostructure, suboptimal decellularization can change ECM composition and microstructure, which affects the biomechanical properties of the tissue and consequently changes cell-matrix interactions and organ function. Herein, we review methods of decellularization, with particular emphasis on cardiac tissue, and how they can affect the biomechanics of the tissue, which in turn determines success of reseeding and in vivo viability. Moreover, we review recent developments in decellularized ECM-derived cardiac biomaterials and discuss future perspectives.
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Affiliation(s)
- Kaitlin M Whitehead
- Department of Physiology, The Brody School of Medicine, East Carolina University, Greenville, North Carolina
| | - Hanifah K L Hendricks
- Department of Physiology, The Brody School of Medicine, East Carolina University, Greenville, North Carolina
| | - Sirin N Cakir
- Department of Physiology, The Brody School of Medicine, East Carolina University, Greenville, North Carolina
| | - Lisandra E de Castro Brás
- Department of Physiology, The Brody School of Medicine, East Carolina University, Greenville, North Carolina
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15
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Wang X, Chan V, Corridon PR. Decellularized blood vessel development: Current state-of-the-art and future directions. Front Bioeng Biotechnol 2022; 10:951644. [PMID: 36003539 PMCID: PMC9394443 DOI: 10.3389/fbioe.2022.951644] [Citation(s) in RCA: 25] [Impact Index Per Article: 8.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/24/2022] [Accepted: 07/14/2022] [Indexed: 12/31/2022] Open
Abstract
Vascular diseases contribute to intensive and irreversible damage, and current treatments include medications, rehabilitation, and surgical interventions. Often, these diseases require some form of vascular replacement therapy (VRT) to help patients overcome life-threatening conditions and traumatic injuries annually. Current VRTs rely on harvesting blood vessels from various regions of the body like the arms, legs, chest, and abdomen. However, these procedures also produce further complications like donor site morbidity. Such common comorbidities may lead to substantial pain, infections, decreased function, and additional reconstructive or cosmetic surgeries. Vascular tissue engineering technology promises to reduce or eliminate these issues, and the existing state-of-the-art approach is based on synthetic or natural polymer tubes aiming to mimic various types of blood vessel. Burgeoning decellularization techniques are considered as the most viable tissue engineering strategy to fill these gaps. This review discusses various approaches and the mechanisms behind decellularization techniques and outlines a simplified model for a replacement vascular unit. The current state-of-the-art method used to create decellularized vessel segments is identified. Also, perspectives on future directions to engineer small- (inner diameter >1 mm and <6 mm) to large-caliber (inner diameter >6 mm) vessel substitutes are presented.
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Affiliation(s)
- Xinyu Wang
- Biomedical Engineering and Healthcare Engineering Innovation Center, Khalifa University, Abu Dhabi, United Arab Emirates
- Department of Immunology and Physiology, College of Medicine and Health Sciences, Khalifa University, Abu Dhabi, United Arab Emirates
| | - Vincent Chan
- Biomedical Engineering and Healthcare Engineering Innovation Center, Khalifa University, Abu Dhabi, United Arab Emirates
| | - Peter R Corridon
- Department of Immunology and Physiology, College of Medicine and Health Sciences, Khalifa University, Abu Dhabi, United Arab Emirates
- Center for Biotechnology, Khalifa University, Abu Dhabi, United Arab Emirates
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16
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Jain P, Rauer SB, Möller M, Singh S. Mimicking the Natural Basement Membrane for Advanced Tissue Engineering. Biomacromolecules 2022; 23:3081-3103. [PMID: 35839343 PMCID: PMC9364315 DOI: 10.1021/acs.biomac.2c00402] [Citation(s) in RCA: 32] [Impact Index Per Article: 10.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/02/2022]
Abstract
![]()
Advancements in the field of tissue engineering have
led to the
elucidation of physical and chemical characteristics of physiological
basement membranes (BM) as specialized forms of the extracellular
matrix. Efforts to recapitulate the intricate structure and biological
composition of the BM have encountered various advancements due to
its impact on cell fate, function, and regulation. More attention
has been paid to synthesizing biocompatible and biofunctional fibrillar
scaffolds that closely mimic the natural BM. Specific modifications
in biomimetic BM have paved the way for the development of in vitro models like alveolar-capillary barrier, airway
models, skin, blood-brain barrier, kidney barrier, and metastatic
models, which can be used for personalized drug screening, understanding
physiological and pathological pathways, and tissue implants. In this
Review, we focus on the structure, composition, and functions of in vivo BM and the ongoing efforts to mimic it synthetically.
Light has been shed on the advantages and limitations of various forms
of biomimetic BM scaffolds including porous polymeric membranes, hydrogels,
and electrospun membranes This Review further elaborates and justifies
the significance of BM mimics in tissue engineering, in particular
in the development of in vitro organ model systems.
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Affiliation(s)
- Puja Jain
- DWI-Leibniz-Institute for Interactive Materials e.V, Aachen 52074, Germany
| | | | - Martin Möller
- DWI-Leibniz-Institute for Interactive Materials e.V, Aachen 52074, Germany
| | - Smriti Singh
- Max-Planck-Institute for Medical Research, Heidelberg 69028, Germany
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17
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Hussain MWA, Garg P, Yazji JH, Alomari M, Alamouti-fard E, Wadiwala I, Jacob S. Is a Bioengineered Heart From Recipient Tissues the Answer to the Shortage of Donors in Heart Transplantation? Cureus 2022; 14:e25329. [PMID: 35637923 PMCID: PMC9132496 DOI: 10.7759/cureus.25329] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Accepted: 05/25/2022] [Indexed: 11/08/2022] Open
Abstract
With the increase in life expectancy worldwide, end-organ failure is becoming more prevalent. In addition, improving post-transplant outcomes has contributed to soaring demand for organs. Unfortunately, thousands have died waiting on the transplant list due to the critical shortage of organs. The success of bioengineered hearts may eventually lead to the production of limitless organs using the patient’s own cells that can be transplanted into them without the need for immunosuppressive medications. Despite being in its infancy, scientists are making tremendous strides in “growing” an artificial heart in the lab. We discuss these processes involved in bioengineering a human-compatible heart in this review. The components of a functional heart must be replicated in a bioengineered heart to make it viable. This review aims to discuss the advances that have already been made and the future challenges of bioengineering a human heart suitable for transplantation.
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18
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Naso F, Gandaglia A. Can Heart Valve Decellularization Be Standardized? A Review of the Parameters Used for the Quality Control of Decellularization Processes. Front Bioeng Biotechnol 2022; 10:830899. [PMID: 35252139 PMCID: PMC8891751 DOI: 10.3389/fbioe.2022.830899] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/07/2021] [Accepted: 01/31/2022] [Indexed: 11/13/2022] Open
Abstract
When a tissue or an organ is considered, the attention inevitably falls on the complex and delicate mechanisms regulating the correct interaction of billions of cells that populate it. However, the most critical component for the functionality of specific tissue or organ is not the cell, but the cell-secreted three-dimensional structure known as the extracellular matrix (ECM). Without the presence of an adequate ECM, there would be no optimal support and stimuli for the cellular component to replicate, communicate and interact properly, thus compromising cell dynamics and behaviour and contributing to the loss of tissue-specific cellular phenotype and functions. The limitations of the current bioprosthetic implantable medical devices have led researchers to explore tissue engineering constructs, predominantly using animal tissues as a potentially unlimited source of materials. The high homology of the protein sequences that compose the mammalian ECM, can be exploited to convert a soft animal tissue into a human autologous functional and long-lasting prosthesis ensuring the viability of the cells and maintaining the proper biomechanical function. Decellularization has been shown to be a highly promising technique to generate tissue-specific ECM-derived products for multiple applications, although it might comprise very complex processes that involve the simultaneous use of chemical, biochemical, physical and enzymatic protocols. Several different approaches have been reported in the literature for the treatment of bone, cartilage, adipose, dermal, neural and cardiovascular tissues, as well as skeletal muscle, tendons and gastrointestinal tract matrices. However, most of these reports refer to experimental data. This paper reviews the most common and latest decellularization approaches that have been adopted in cardiovascular tissue engineering. The efficacy of cells removal was specifically reviewed and discussed, together with the parameters that could be used as quality control markers for the evaluation of the effectiveness of decellularization and tissue biocompatibility. The purpose was to provide a panel of parameters that can be shared and taken into consideration by the scientific community to achieve more efficient, comparable, and reliable experimental research results and a faster technology transfer to the market.
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19
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Akbarzadeh A, Tafti SHA, Sabetkish S, Hassannejad Z, Kajbafzadeh AM. Coronary-Based Right Heart Flap Recellularization by Rat Neonatal Whole Cardiac Cells: a Viable Sheep Cardiac Patch Model for Possible Management of Heart Aneurysm. REGENERATIVE ENGINEERING AND TRANSLATIONAL MEDICINE 2022. [DOI: 10.1007/s40883-021-00238-7] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 10/19/2022]
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20
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Barbulescu GI, Buica TP, Goje ID, Bojin FM, Ordodi VL, Olteanu GE, Heredea RE, Paunescu V. Optimization of Complete Rat Heart Decellularization Using Artificial Neural Networks. MICROMACHINES 2022; 13:mi13010079. [PMID: 35056244 PMCID: PMC8778756 DOI: 10.3390/mi13010079] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 12/06/2021] [Revised: 12/30/2021] [Accepted: 12/31/2021] [Indexed: 02/01/2023]
Abstract
Whole organ decellularization techniques have facilitated the fabrication of extracellular matrices (ECMs) for engineering new organs. Unfortunately, there is no objective gold standard evaluation of the scaffold without applying a destructive method such as histological analysis or DNA removal quantification of the dry tissue. Our proposal is a software application using deep convolutional neural networks (DCNN) to distinguish between different stages of decellularization, determining the exact moment of completion. Hearts from male Sprague Dawley rats (n = 10) were decellularized using 1% sodium dodecyl sulfate (SDS) in a modified Langendorff device in the presence of an alternating rectangular electric field. Spectrophotometric measurements of deoxyribonucleic acid (DNA) and total proteins concentration from the decellularization solution were taken every 30 min. A monitoring system supervised the sessions, collecting a large number of photos saved in corresponding folders. This system aimed to prove a strong correlation between the data gathered by spectrophotometry and the state of the heart that could be visualized with an OpenCV-based spectrometer. A decellularization completion metric was built using a DCNN based classifier model trained using an image set comprising thousands of photos. Optimizing the decellularization process using a machine learning approach launches exponential progress in tissue bioengineering research.
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Affiliation(s)
- Greta Ionela Barbulescu
- Immuno-Physiology and Biotechnologies Center (CIFBIOTEH), Department of Functional Sciences, “Victor Babes” University of Medicine and Pharmacy, No. 2 Eftimie Murgu Square, 300041 Timisoara, Romania; (F.M.B.); (V.P.)
- Department of Clinical Practical Skills, “Victor Babes” University of Medicine and Pharmacy, No. 2 Eftimie Murgu Square, 300041 Timisoara, Romania;
- Center for Gene and Cellular Therapies in the Treatment of Cancer Timisoara-OncoGen, Clinical Emergency County Hospital “Pius Brinzeu” Timisoara, No. 156 Liviu Rebreanu, 300723 Timisoara, Romania; (T.P.B.); (V.L.O.)
- Correspondence: (G.I.B.); (I.D.G.); Tel.: +40-733177583 (G.-I.B.)
| | - Taddeus Paul Buica
- Center for Gene and Cellular Therapies in the Treatment of Cancer Timisoara-OncoGen, Clinical Emergency County Hospital “Pius Brinzeu” Timisoara, No. 156 Liviu Rebreanu, 300723 Timisoara, Romania; (T.P.B.); (V.L.O.)
| | - Iacob Daniel Goje
- Department of Medical Semiology I, “Victor Babes” University of Medicine and Pharmacy, No. 2 Eftimie Murgu Square, 300041 Timisoara, Romania
- Advanced Cardiology and Hemostaseology Research Center, “Victor Babes” University of Medicine and Pharmacy, No. 2 Eftimie Murgu Square, 300041 Timisoara, Romania
- Correspondence: (G.I.B.); (I.D.G.); Tel.: +40-733177583 (G.-I.B.)
| | - Florina Maria Bojin
- Immuno-Physiology and Biotechnologies Center (CIFBIOTEH), Department of Functional Sciences, “Victor Babes” University of Medicine and Pharmacy, No. 2 Eftimie Murgu Square, 300041 Timisoara, Romania; (F.M.B.); (V.P.)
- Center for Gene and Cellular Therapies in the Treatment of Cancer Timisoara-OncoGen, Clinical Emergency County Hospital “Pius Brinzeu” Timisoara, No. 156 Liviu Rebreanu, 300723 Timisoara, Romania; (T.P.B.); (V.L.O.)
| | - Valentin Laurentiu Ordodi
- Center for Gene and Cellular Therapies in the Treatment of Cancer Timisoara-OncoGen, Clinical Emergency County Hospital “Pius Brinzeu” Timisoara, No. 156 Liviu Rebreanu, 300723 Timisoara, Romania; (T.P.B.); (V.L.O.)
- Department of Applied Chemistry, Organic and Natural Compounds Engineering, Faculty of Industrial Chemistry and Environmental Engineering, “Politehnica” University Timisoara, No. 2 Victoriei Square, 300006 Timisoara, Romania
| | - Gheorghe Emilian Olteanu
- Department of Pathology, “Dr Victor Babes” Clinical Hospital of Infectious Disease and Pneumophysiology, 300041 Timisoara, Romania;
| | - Rodica Elena Heredea
- Department of Clinical Practical Skills, “Victor Babes” University of Medicine and Pharmacy, No. 2 Eftimie Murgu Square, 300041 Timisoara, Romania;
- Advanced Cardiology and Hemostaseology Research Center, “Victor Babes” University of Medicine and Pharmacy, No. 2 Eftimie Murgu Square, 300041 Timisoara, Romania
- Department of Pathology, “Louis Turcanu” Children’s Clinical Emergency Hospital, 300041 Timisoara, Romania
| | - Virgil Paunescu
- Immuno-Physiology and Biotechnologies Center (CIFBIOTEH), Department of Functional Sciences, “Victor Babes” University of Medicine and Pharmacy, No. 2 Eftimie Murgu Square, 300041 Timisoara, Romania; (F.M.B.); (V.P.)
- Center for Gene and Cellular Therapies in the Treatment of Cancer Timisoara-OncoGen, Clinical Emergency County Hospital “Pius Brinzeu” Timisoara, No. 156 Liviu Rebreanu, 300723 Timisoara, Romania; (T.P.B.); (V.L.O.)
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21
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Have we hit a wall with whole kidney decellularization and recellularization: A review. CURRENT OPINION IN BIOMEDICAL ENGINEERING 2021. [DOI: 10.1016/j.cobme.2021.100335] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/12/2022]
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22
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Mora-Navarro C, Garcia ME, Sarker P, Ozpinar EW, Enders JR, Khan S, Branski RC, Freytes DO. Monitoring decellularization via absorbance spectroscopy during the derivation of extracellular matrix scaffolds. Biomed Mater 2021; 17. [PMID: 34731852 PMCID: PMC9416610 DOI: 10.1088/1748-605x/ac361f] [Citation(s) in RCA: 9] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/15/2021] [Accepted: 11/03/2021] [Indexed: 11/12/2022]
Abstract
Extracellular matrix (ECM) is a complex structure composed of bioactive molecules representative of the local tissue microenvironment. Decellularized ECM biomaterials harness these biomolecules for regenerative medicine applications. One potential therapeutic application is the use of vocal fold (VF) specific ECM to restore the VFs after injury. ECM scaffolds are derived through a process of decellularization, which aims to remove unwanted immunogenic biomolecules (e.g. DNA) while preserving the composition of the ECM. The effectiveness of the decellularization is typically assessed at the end by quantifying ECM attributes such as final dsDNA content. However, batch-to-batch variability in ECM manufacturing remains a significant challenge for the standardization, cost-effectiveness, and scale-up process. The limited number of tools available for in-process control heavily restricts the uncovering of the correlations between decellularization process parameters and ECM attributes. In this study, we developed a technique applicable to both the classical batch method and semi-continuous decellularization systems to trace the decellularization of two laryngeal tissues in real-time. We hypothesize that monitoring the bioreactor’s effluent absorbance at 260 nm as a function of time will provide a representative DNA release profile from the tissue and thus allow for process optimization. The DNA release profiles were obtained for laryngeal tissues and were successfully used to optimize the derivation of VF lamina propria-ECM (auVF-ECM) hydrogels. This hydrogel had comparable rheological properties to commonly used biomaterials to treat VF injuries. Also, the auVF-ECM hydrogel promoted the down-regulation of CCR7 by THP-1 macrophages upon lipopolysaccharide stimulation in vitro suggesting some anti-inflammatory properties. The results show that absorbance profiles are a good representation of DNA removal during the decellularization process thus providing an important tool to optimize future protocols.
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Affiliation(s)
- Camilo Mora-Navarro
- Joint Department of Biomedical Engineering, North Carolina State and University of North Carolina-Chapel Hill, Raleigh, NC, United States of America.,Comparative Medicine Institute, North Carolina State University, Raleigh, NC, United States of America.,Department of Chemical Engineering, University of Puerto Rico, Mayaguez, PR, United States of America
| | - Mario E Garcia
- Joint Department of Biomedical Engineering, North Carolina State and University of North Carolina-Chapel Hill, Raleigh, NC, United States of America
| | - Prottasha Sarker
- Department of Chemical and Biomolecular Engineering, North Carolina State University, Raleigh, NC 27695, United States of America
| | - Emily W Ozpinar
- Joint Department of Biomedical Engineering, North Carolina State and University of North Carolina-Chapel Hill, Raleigh, NC, United States of America.,Comparative Medicine Institute, North Carolina State University, Raleigh, NC, United States of America
| | - Jeffrey R Enders
- Molecular Education, Technology and Research Innovation Center, North Carolina State University, Raleigh, NC, United States of America.,Department of Biological Sciences, North Carolina State University, Raleigh, NC, United States of America
| | - Saad Khan
- Department of Chemical and Biomolecular Engineering, North Carolina State University, Raleigh, NC 27695, United States of America
| | - Ryan C Branski
- Departments of Rehabilitation Medicine and Otolaryngology-Head and Neck Surgery, NYU Grossman School of Medicine, New York, NY, United States of America
| | - Donald O Freytes
- Joint Department of Biomedical Engineering, North Carolina State and University of North Carolina-Chapel Hill, Raleigh, NC, United States of America.,Comparative Medicine Institute, North Carolina State University, Raleigh, NC, United States of America
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23
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Ferreira LP, Gaspar VM, Mendes L, Duarte IF, Mano JF. Organotypic 3D decellularized matrix tumor spheroids for high-throughput drug screening. Biomaterials 2021; 275:120983. [PMID: 34186236 DOI: 10.1016/j.biomaterials.2021.120983] [Citation(s) in RCA: 36] [Impact Index Per Article: 9.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/18/2021] [Revised: 06/10/2021] [Accepted: 06/18/2021] [Indexed: 02/07/2023]
Abstract
Decellularized extracellular matrix (dECM) is emerging as a valuable tool for generating 3D in vitro tumor models that better recapitulate tumor-stroma interactions. However, the development of dECM-3D heterotypic microtumors exhibiting a controlled morphology is yet to be materialized. Precisely controlling microtumors morphologic features is key to avoid an inaccurate evaluation of therapeutics performance during preclinical screening. To address this, herein we employed ultra-low adhesion surfaces for bioengineering organotypic 3D metastatic breast cancer-fibroblast models enriched with dECM microfibrillar fragments, as a bottom-up strategy to include major matrix components and their associated biomolecular cues during the early stages of 3D microtissue spheroids assembly, simulating pre-existing ECM presence in the in vivo setting. This biomimetic approach enabled the self-assembly of dECM-3D tumor-stroma spheroids with tunable size and reproducible morphology. Along time, dECM enriched and stroma-rich microtumors exhibited necrotic core formation, secretion of key biomarkers and higher cancer-cell specific resistance to different chemotherapeutics in comparison to standard spheroids. Exometabolomics profiling of dECM-Spheroid in vitro models further identified important breast cancer metabolic features including glucose/pyruvate consumption and lactate excretion, which suggest an intense glycolytic activity, recapitulating major hallmarks of the native microenvironment. Such organotypic dECM-enriched microtumors overcome the morphologic variability generally associated with cell-laden dECM models, while providing a scalable testing platform that can be foreseeable leveraged for high-throughput screening of candidate therapeutics.
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Affiliation(s)
- Luís P Ferreira
- Department of Chemistry, CICECO - Aveiro Institute of Materials, University of Aveiro, Campus Universitário de Santiago, 3810-193, Aveiro, Portugal
| | - Vítor M Gaspar
- Department of Chemistry, CICECO - Aveiro Institute of Materials, University of Aveiro, Campus Universitário de Santiago, 3810-193, Aveiro, Portugal.
| | - Luís Mendes
- Department of Chemistry, CICECO - Aveiro Institute of Materials, University of Aveiro, Campus Universitário de Santiago, 3810-193, Aveiro, Portugal
| | - Iola F Duarte
- Department of Chemistry, CICECO - Aveiro Institute of Materials, University of Aveiro, Campus Universitário de Santiago, 3810-193, Aveiro, Portugal
| | - João F Mano
- Department of Chemistry, CICECO - Aveiro Institute of Materials, University of Aveiro, Campus Universitário de Santiago, 3810-193, Aveiro, Portugal.
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24
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Song YH, Maynes MA, Hlavac N, Visosevic D, Daramola KO, Porvasnik SL, Schmidt CE. Development of novel apoptosis-assisted lung tissue decellularization methods. Biomater Sci 2021; 9:3485-3498. [PMID: 33949462 DOI: 10.1039/d1bm00032b] [Citation(s) in RCA: 15] [Impact Index Per Article: 3.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/21/2022]
Abstract
Decellularized tissues hold great potential for both regenerative medicine and disease modeling applications. The acellular extracellular matrix (ECM)-enriched scaffolds can be recellularized with patient-derived cells prior to transplantation, or digested to create thermally-gelling ECM hydrogels for 3D cell culture. Current methods of decellularization clear cellular components using detergents, which can result in loss of ECM proteins and tissue architectural integrity. Recently, an alternative approach utilizing apoptosis to decellularize excised murine sciatic nerves resulted in superior ECM preservation, cell removal, and immune tolerance in vivo. However, this apoptosis-assisted decellularization approach has not been optimized for other tissues with a more complex geometry, such as lungs. To this end, we developed an apoptosis-assisted lung tissue decellularization method using a combination of camptothecin and sulfobetaine-10 (SB-10) to induce apoptosis and facilitate gentle and effective removal of cell debris, respectively. Importantly, combination of the two agents resulted in superior cell removal and ECM preservation compared to either of the treatments alone, presumably because of pulmonary surfactants. In addition, our method was superior in cell removal compared to a previously established detergent-based decellularization protocol. Furthermore, thermally-gelling lung ECM hydrogels supported high viability of rat lung epithelial cells for up to 2 weeks in culture. This work demonstrates that apoptosis-based lung tissue decellularization is a superior technique that warrants further utilization for both regenerative medicine and disease modeling purposes.
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Affiliation(s)
- Young Hye Song
- J. Crayton Pruitt Family Department of Biomedical Engineering, University of Florida, Gainesville, FL, USA. and Department of Biomedical Engineering, University of Arkansas, Fayetteville, AR, USA.
| | - Mark A Maynes
- J. Crayton Pruitt Family Department of Biomedical Engineering, University of Florida, Gainesville, FL, USA.
| | - Nora Hlavac
- J. Crayton Pruitt Family Department of Biomedical Engineering, University of Florida, Gainesville, FL, USA.
| | - Daniel Visosevic
- J. Crayton Pruitt Family Department of Biomedical Engineering, University of Florida, Gainesville, FL, USA.
| | - Kaitlyn O Daramola
- J. Crayton Pruitt Family Department of Biomedical Engineering, University of Florida, Gainesville, FL, USA.
| | - Stacy L Porvasnik
- J. Crayton Pruitt Family Department of Biomedical Engineering, University of Florida, Gainesville, FL, USA.
| | - Christine E Schmidt
- J. Crayton Pruitt Family Department of Biomedical Engineering, University of Florida, Gainesville, FL, USA.
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25
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Morrissey J, Mesquita FCP, Hochman-Mendez C, Taylor DA. Whole Heart Engineering: Advances and Challenges. Cells Tissues Organs 2021; 211:395-405. [PMID: 33640893 DOI: 10.1159/000511382] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/06/2020] [Accepted: 08/26/2020] [Indexed: 11/19/2022] Open
Abstract
Bioengineering a solid organ for organ replacement is a growing endeavor in regenerative medicine. Our approach - recellularization of a decellularized cadaveric organ scaffold with human cells - is currently the most promising approach to building a complex solid vascularized organ to be utilized in vivo, which remains the major unmet need and a key challenge. The 2008 publication of perfusion-based decellularization and partial recellularization of a rat heart revolutionized the tissue engineering field by showing that it was feasible to rebuild an organ using a decellularized extracellular matrix scaffold. Toward the goal of clinical translation of bioengineered tissues and organs, there is increasing recognition of the underlying need to better integrate basic science domains and industry. From the perspective of a research group focusing on whole heart engineering, we discuss the current approaches and advances in whole organ engineering research as they relate to this multidisciplinary field's 3 major pillars: organ scaffolds, large numbers of cells, and biomimetic bioreactor systems. The success of whole organ engineering will require optimization of protocols to produce biologically-active scaffolds for multiple organ systems, and further technological innovation both to produce the massive quantities of high-quality cells needed for recellularization and to engineer a bioreactor with physiologic stimuli to recapitulate organ function. Also discussed are the challenges to building an implantable vascularized solid organ.
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Affiliation(s)
- Jacquelynn Morrissey
- Regenerative Medicine Research Department, Texas Heart Institute, Houston, Texas, USA
| | - Fernanda C P Mesquita
- Regenerative Medicine Research Department, Texas Heart Institute, Houston, Texas, USA
| | - Camila Hochman-Mendez
- Regenerative Medicine Research Department, Texas Heart Institute, Houston, Texas, USA
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26
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Zhang J, Chan HF, Wang H, Shao D, Tao Y, Li M. Stem cell therapy and tissue engineering strategies using cell aggregates and decellularized scaffolds for the rescue of liver failure. J Tissue Eng 2021; 12:2041731420986711. [PMID: 35003615 PMCID: PMC8733710 DOI: 10.1177/2041731420986711] [Citation(s) in RCA: 30] [Impact Index Per Article: 7.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/27/2020] [Accepted: 12/18/2020] [Indexed: 12/11/2022] Open
Abstract
Liver failure is a lethal condition with hepatocellular dysfunction, and liver transplantation is presently the only effective treatment. However, due to the limited availability of donors and the potential immune rejection, novel therapeutic strategies are actively sought to restore the normal hepatic architectures and functions, especially for livers with inherited metabolic dysfunctions or chronic diseases. Although the conventional cell therapy has shown promising results, the direct infusion of hepatocytes is hampered by limited hepatocyte sources, poor cell viability, and engraftment. Hence, this review mainly highlights the role of stem cells and progenitors as the alternative cell source and summarizes the potential approaches based on tissue engineering to improve the delivery efficiency of cells. Particularly, the underlying mechanisms for cell therapy using stem cells and progenitors are discussed in two main aspects: paracrine effect and cell differentiation. Moreover, tissue-engineering approaches using cell aggregates and decellularized liver scaffolds for bioengineering of functional hepatic constructs are discussed and compared in terms of the potential to replicate liver physiological structures. In the end, a potentially effective strategy combining the premium advantages of stem cell aggregates and decellularized liver scaffolds is proposed as the future direction of liver tissue engineering and regeneration.
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Affiliation(s)
- Jiabin Zhang
- Laboratory of Biomaterials and Translational Medicine, The Third Affiliated Hospital, Sun Yat-sen University, Guangzhou, China
- Guangdong Provincial Key Laboratory of Liver Disease, Guangzhou, China
| | - Hon Fai Chan
- Institute for Tissue Engineering and Regenerative Medicine, The Chinese University of Hong Kong, Hong Kong, China
- School of Biomedical Sciences, The Chinese University of Hong Kong, Hong Kong, China
| | - Haixia Wang
- Laboratory of Biomaterials and Translational Medicine, The Third Affiliated Hospital, Sun Yat-sen University, Guangzhou, China
| | - Dan Shao
- Institutes of Life Sciences, School of Biomedical Sciences and Engineering, South China University of Technology, Guangzhou, China
| | - Yu Tao
- Laboratory of Biomaterials and Translational Medicine, The Third Affiliated Hospital, Sun Yat-sen University, Guangzhou, China
| | - Mingqiang Li
- Laboratory of Biomaterials and Translational Medicine, The Third Affiliated Hospital, Sun Yat-sen University, Guangzhou, China
- Guangdong Provincial Key Laboratory of Liver Disease, Guangzhou, China
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27
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Li T, Javed R, Ao Q. Xenogeneic Decellularized Extracellular Matrix-based Biomaterials For Peripheral Nerve Repair and Regeneration. Curr Neuropharmacol 2021; 19:2152-2163. [PMID: 33176651 PMCID: PMC9185777 DOI: 10.2174/1570159x18666201111103815] [Citation(s) in RCA: 13] [Impact Index Per Article: 3.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/02/2021] [Revised: 03/18/2021] [Accepted: 04/03/2021] [Indexed: 12/04/2022] Open
Abstract
Peripheral nerve injury could lead to either impairment or a complete loss of function for affected patients, and a variety of nerve repair materials have been developed for surgical approaches to repair it. Although autologous or autologous tissue-derived biomaterials remain preferred treatment for peripheral nerve injury, the lack of donor sources has led biomedical researchers to explore more other biomaterials. As a reliable alternative, xenogeneic decellularized extracellular matrix (dECM)-based biomaterials have been widely employed for surgical nerve repair. The dECM derived from animal donors is an attractive and unlimited source for xenotransplantation. Meanwhile, as an increasingly popular technique, decellularization could retain a variety of bioactive components in native ECM, such as polysaccharides, proteins, and growth factors. The resulting dECM-based biomaterials preserve a tissue's native microenvironment, promote Schwann cells proliferation and differentiation, and provide cues for nerve regeneration. Although the potential of dECM-based biomaterials as a therapeutic agent is rising, there are many limitations of this material restricting its use. Herein, this review discusses the decellularization techniques that have been applied to create dECM-based biomaterials, the main components of nerve ECM, and the recent progress in the utilization of xenogeneic dECM-based biomaterials through applications as a hydrogel, wrap, and guidance conduit in nerve tissue engineering. In the end, the existing bottlenecks of xenogeneic dECM-based biomaterials and developing technologies that could be eliminated to be helpful for utilization in the future have been elaborated.
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Affiliation(s)
- Ting Li
- Department of Laboratory Medicine, Shengjing Hospital of China Medical University, Shenyang, China
- Department of Developmental Cell Biology, China Medical University, Shenyang, China
| | - Rabia Javed
- Department of Developmental Cell Biology, China Medical University, Shenyang, China
| | - Qiang Ao
- Department of Developmental Cell Biology, China Medical University, Shenyang, China
- Institute of Regulatory Science for Med-ical Devices, Engineering Research Center for Biomaterials, Sichuan University, Chengdu, China
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28
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Pors SE, Ramløse M, Nikiforov D, Lundsgaard K, Cheng J, Andersen CY, Kristensen SG. Initial steps in reconstruction of the human ovary: survival of pre-antral stage follicles in a decellularized human ovarian scaffold. Hum Reprod 2020; 34:1523-1535. [PMID: 31286144 DOI: 10.1093/humrep/dez077] [Citation(s) in RCA: 86] [Impact Index Per Article: 17.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/01/2019] [Revised: 04/04/2019] [Accepted: 04/25/2019] [Indexed: 11/13/2022] Open
Abstract
STUDY QUESTION Can a reconstructed ovary using decellularized human ovarian tissue (DCT) support survival of pre-antral stage follicles? SUMMARY ANSWER We have demonstrated an effective protocol for decellularization of human ovarian tissues and successful recellularization with isolated human ovarian cells and pre-antral follicles. WHAT IS KNOWN ALREADY Survivors of leukemia or ovarian cancer run a risk of reintroducing malignancy when cryopreserved ovarian tissue is transplanted to restore fertility. A reconstructed ovary free of malignant cells could provide a safe alternative. Decellularization of ovarian tissue removes all cells from the extracellular matrix (ECM) including possible malignancies and leaves behind a physiological scaffold. The ECM offers the complex milieu that facilitates the necessary interaction between ovarian follicles and their surroundings to ensure their growth and development. Previous studies have shown that decellularized bovine ovarian scaffolds supported murine follicle growth and restoration of ovarian function in ovariectomized mice. STUDY DESIGN, SIZE, DURATION Optimizing a decellularization protocol for human ovarian tissues and testing biofunctionality of the decellularized scaffolds in vitro and in vivo by reseeding with both murine and human pre-antral follicles and ovarian cells. PARTICIPANTS/MATERIALS, SETTING, METHODS Donated human ovarian tissue and isolated pre-antral follicles were obtained from women undergoing ovarian tissue cryopreservation for fertility preservation. Ovarian cortical and medullary tissues were decellularized using 0.1% sodium dodecyl sulfate (SDS) for 3, 6, 18 and 24 hours followed by 24 hours of 1 mg/mL DNase treatment and washing. Decellularization of ovarian tissues and preservation of ECM were characterized by morphological evaluation using Periodic Acid-Schiff (PAS) staining, DNA quantification, histochemical quantification of collagen content and immunofluorescence analysis for collagen IA, laminin, fibronectin and DNA. Human ovarian stromal cells and isolated human pre-antral follicles were reseeded on the DCT and cultured in vitro. Isolated murine (N = 241) and human (N = 20) pre-antral follicles were reseeded on decellularized scaffolds and grafted subcutaneously to immunodeficient mice for 3 weeks. MAIN RESULTS AND THE ROLE OF CHANCE Incubation in 0.1% SDS for 18-24 hours adequately decellularized both human ovarian medullary and cortical tissue by eliminating all cells and leaving the ECM intact. DNA content in DCT was decreased by >90% compared to native tissue samples. Histological examination using PAS staining confirmed that the cortical and medullary tissues were completely decellularized, and no visible nuclear material was found within the decellularized sections. DCT also stained positive for collagen I and collagen quantities in DCT constituted 88-98% of the individual baselines for native samples. Human ovarian stroma cells were able to recellularize the DCT and isolated human pre-antral follicles remained viable in co-culture. Xenotransplantation of DCT reseeded with human or murine pre-antral follicles showed, that the DCT was able to support survival of human follicles and growth of murine follicles, of which 39% grew to antral stages. The follicular recovery rates after three weeks grafting were low but similar for both human (25%) and murine follicles (21%). LARGE SCALE DATA N/A. LIMITATIONS, REASONS FOR CAUTION Further studies are needed to increase recovery and survival of the reseeded follicles. Longer grafting periods should be evaluated to determine the developmental potential of human follicles. Survival of the follicles might be impaired by the lack of stroma cells. WIDER IMPLICATIONS OF THE FINDINGS This is the first time that isolated human follicles have survived in a decellularized human scaffold. Therefore, this proof-of-concept could be a potential new strategy to eliminate the risk of malignant cell re-occurrence in former cancer patients having cryopreserved ovarian tissue transplanted for fertility restoration. STUDY FUNDING/COMPETING INTEREST(S) This study is part of the ReproUnion collaborative study, co-financed by the European Union, Interreg V ÖKS. Furthermore, Project ITN REP-BIOTECH 675526 funded by the European Union, European Joint Doctorate in Biology and Technology of the Reproductive Health, the Research Pools of Rigshospitalet, the Danish Cancer Foundation and Dagmar Marshalls Foundation are thanked for having funded this study. The funders had no role in the study design, data collection and interpretation, or in the decision to submit the work for publication.
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Affiliation(s)
- S E Pors
- Laboratory of Reproductive Biology, The Juliane Marie Centre for Women, Children and Reproduction, University Hospital of Copenhagen, Faculty of Health Science, University of Copenhagen, Blegdamsvej, Copenhagen, Denmark
| | - M Ramløse
- Laboratory of Reproductive Biology, The Juliane Marie Centre for Women, Children and Reproduction, University Hospital of Copenhagen, Faculty of Health Science, University of Copenhagen, Blegdamsvej, Copenhagen, Denmark
| | - D Nikiforov
- Laboratory of Reproductive Biology, The Juliane Marie Centre for Women, Children and Reproduction, University Hospital of Copenhagen, Faculty of Health Science, University of Copenhagen, Blegdamsvej, Copenhagen, Denmark.,University of Teramo, Teramo, Via Renato Balzarini, Italy
| | - K Lundsgaard
- Laboratory of Reproductive Biology, The Juliane Marie Centre for Women, Children and Reproduction, University Hospital of Copenhagen, Faculty of Health Science, University of Copenhagen, Blegdamsvej, Copenhagen, Denmark
| | - J Cheng
- Laboratory of Reproductive Biology, The Juliane Marie Centre for Women, Children and Reproduction, University Hospital of Copenhagen, Faculty of Health Science, University of Copenhagen, Blegdamsvej, Copenhagen, Denmark.,People's Hospital of Guangxi Autonomous Region, 6 Taoyuan Rd, Qingxiu Qu, Nanning City, Guangxi province, China Via Renato Balzarini, Teramo
| | - C Yding Andersen
- Laboratory of Reproductive Biology, The Juliane Marie Centre for Women, Children and Reproduction, University Hospital of Copenhagen, Faculty of Health Science, University of Copenhagen, Blegdamsvej, Copenhagen, Denmark
| | - S G Kristensen
- Laboratory of Reproductive Biology, The Juliane Marie Centre for Women, Children and Reproduction, University Hospital of Copenhagen, Faculty of Health Science, University of Copenhagen, Blegdamsvej, Copenhagen, Denmark
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29
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Mendibil U, Ruiz-Hernandez R, Retegi-Carrion S, Garcia-Urquia N, Olalde-Graells B, Abarrategi A. Tissue-Specific Decellularization Methods: Rationale and Strategies to Achieve Regenerative Compounds. Int J Mol Sci 2020; 21:E5447. [PMID: 32751654 PMCID: PMC7432490 DOI: 10.3390/ijms21155447] [Citation(s) in RCA: 160] [Impact Index Per Article: 32.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/15/2020] [Revised: 07/25/2020] [Accepted: 07/28/2020] [Indexed: 02/07/2023] Open
Abstract
The extracellular matrix (ECM) is a complex network with multiple functions, including specific functions during tissue regeneration. Precisely, the properties of the ECM have been thoroughly used in tissue engineering and regenerative medicine research, aiming to restore the function of damaged or dysfunctional tissues. Tissue decellularization is gaining momentum as a technique to obtain potentially implantable decellularized extracellular matrix (dECM) with well-preserved key components. Interestingly, the tissue-specific dECM is becoming a feasible option to carry out regenerative medicine research, with multiple advantages compared to other approaches. This review provides an overview of the most common methods used to obtain the dECM and summarizes the strategies adopted to decellularize specific tissues, aiming to provide a helpful guide for future research development.
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Affiliation(s)
- Unai Mendibil
- Center for Cooperative Research in Biomaterials (CIC biomaGUNE), Basque Research and Technology Alliance (BRTA), 20014 Donostia-San Sebastian, Spain; (U.M.); (R.R.-H.); (S.R.-C.)
- TECNALIA, Basque Research and Technology Alliance (BRTA), 20009 Donostia-San Sebastian, Spain; (N.G.-U.); (B.O.-G.)
| | - Raquel Ruiz-Hernandez
- Center for Cooperative Research in Biomaterials (CIC biomaGUNE), Basque Research and Technology Alliance (BRTA), 20014 Donostia-San Sebastian, Spain; (U.M.); (R.R.-H.); (S.R.-C.)
| | - Sugoi Retegi-Carrion
- Center for Cooperative Research in Biomaterials (CIC biomaGUNE), Basque Research and Technology Alliance (BRTA), 20014 Donostia-San Sebastian, Spain; (U.M.); (R.R.-H.); (S.R.-C.)
| | - Nerea Garcia-Urquia
- TECNALIA, Basque Research and Technology Alliance (BRTA), 20009 Donostia-San Sebastian, Spain; (N.G.-U.); (B.O.-G.)
| | - Beatriz Olalde-Graells
- TECNALIA, Basque Research and Technology Alliance (BRTA), 20009 Donostia-San Sebastian, Spain; (N.G.-U.); (B.O.-G.)
| | - Ander Abarrategi
- Center for Cooperative Research in Biomaterials (CIC biomaGUNE), Basque Research and Technology Alliance (BRTA), 20014 Donostia-San Sebastian, Spain; (U.M.); (R.R.-H.); (S.R.-C.)
- Ikerbasque, Basque Foundation for Science, 48013 Bilbao, Spain
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30
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Moulisová V, Jiřík M, Schindler C, Červenková L, Pálek R, Rosendorf J, Arlt J, Bolek L, Šůsová S, Nietzsche S, Liška V, Dahmen U. Novel morphological multi-scale evaluation system for quality assessment of decellularized liver scaffolds. J Tissue Eng 2020; 11:2041731420921121. [PMID: 32523667 PMCID: PMC7257850 DOI: 10.1177/2041731420921121] [Citation(s) in RCA: 14] [Impact Index Per Article: 2.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/12/2019] [Accepted: 04/02/2020] [Indexed: 12/19/2022] Open
Abstract
Decellularized scaffolds can serve as an excellent three-dimensional environment for cell repopulation. They maintain tissue-specific microarchitecture of extracellular matrix proteins with important spatial cues for cell adhesion, migration, growth, and differentiation. However, criteria for quality assessment of the three-dimensional structure of decellularized scaffolds are rather fragmented, usually study-specific, and mostly semi-quantitative. Thus, we aimed to develop a robust structural assessment system for decellularized porcine liver scaffolds. Five scaffolds of different quality were used to establish the new evaluation system. We combined conventional semi-quantitative scoring criteria with a quantitative scaffold evaluation based on automated image analysis. For the quantitation, we developed a specific open source software tool (ScaffAn) applying algorithms designed for texture analysis, segmentation, and skeletonization. ScaffAn calculates selected parameters characterizing structural features of porcine liver scaffolds such as the sinusoidal network. After evaluating individual scaffolds, the total scores predicted scaffold interaction with cells in terms of cell adhesion. Higher scores corresponded to higher numbers of cells attached to the scaffolds. Moreover, our analysis revealed that the conventional system could not identify fine differences between good quality scaffolds while the additional use of ScaffAn allowed discrimination. This led us to the conclusion that only using the combined score resulted in the best discrimination between different quality scaffolds. Overall, our newly defined evaluation system has the potential to select the liver scaffolds most suitable for recellularization, and can represent a step toward better success in liver tissue engineering.
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Affiliation(s)
- Vladimíra Moulisová
- Biomedical Center, Faculty of Medicine in Pilsen, Charles University in Prague, Pilsen, Czech Republic
| | - Miroslav Jiřík
- Biomedical Center, Faculty of Medicine in Pilsen, Charles University in Prague, Pilsen, Czech Republic.,Department of Cybernetics, University of West Bohemia, Pilsen, Czech Republic
| | - Claudia Schindler
- Experimental Transplantation Surgery, Department of General, Visceral and Vascular Surgery, University Hospital Jena, Jena, Germany
| | - Lenka Červenková
- Biomedical Center, Faculty of Medicine in Pilsen, Charles University in Prague, Pilsen, Czech Republic.,Department of Pathology, Third Faculty of Medicine, Charles University in Prague, Prague, Czech Republic
| | - Richard Pálek
- Biomedical Center, Faculty of Medicine in Pilsen, Charles University in Prague, Pilsen, Czech Republic.,Department of Surgery, Faculty of Medicine in Pilsen, Charles University in Prague, Pilsen, Czech Republic
| | - Jáchym Rosendorf
- Biomedical Center, Faculty of Medicine in Pilsen, Charles University in Prague, Pilsen, Czech Republic.,Department of Surgery, Faculty of Medicine in Pilsen, Charles University in Prague, Pilsen, Czech Republic
| | - Janine Arlt
- Experimental Transplantation Surgery, Department of General, Visceral and Vascular Surgery, University Hospital Jena, Jena, Germany
| | - Lukáš Bolek
- Biomedical Center, Faculty of Medicine in Pilsen, Charles University in Prague, Pilsen, Czech Republic.,Department of Biophysics, Faculty of Medicine in Pilsen, Charles University in Prague, Pilsen, Czech Republic
| | - Simona Šůsová
- Biomedical Center, Faculty of Medicine in Pilsen, Charles University in Prague, Pilsen, Czech Republic.,Department of Toxicogenomics, National Institute of Public Health, Prague, Czech Republic
| | - Sandor Nietzsche
- Centre for Electron Microscopy, University Hospital Jena, Jena, Germany
| | - Václav Liška
- Biomedical Center, Faculty of Medicine in Pilsen, Charles University in Prague, Pilsen, Czech Republic.,Department of Surgery, Faculty of Medicine in Pilsen, Charles University in Prague, Pilsen, Czech Republic
| | - Uta Dahmen
- Experimental Transplantation Surgery, Department of General, Visceral and Vascular Surgery, University Hospital Jena, Jena, Germany
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31
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Decellularized liver matrix as substrates for rescue of acute hepatocytes toxicity. J Biomed Mater Res B Appl Biomater 2020; 108:1592-1602. [DOI: 10.1002/jbm.b.34506] [Citation(s) in RCA: 8] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/04/2019] [Revised: 08/16/2019] [Accepted: 09/22/2019] [Indexed: 12/11/2022]
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Zhao C, Li Y, Peng G, Lei X, Zhang G, Gao Y. Decellularized liver matrix-modified chitosan fibrous scaffold as a substrate for C3A hepatocyte culture. JOURNAL OF BIOMATERIALS SCIENCE-POLYMER EDITION 2020; 31:1041-1056. [PMID: 32162599 DOI: 10.1080/09205063.2020.1738690] [Citation(s) in RCA: 9] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 02/07/2023]
Abstract
A bioreactor filled with functional hepatocytes is a crucial portion of the bio-artificial liver device. However, it is a difficult task to maintain sufficient cell quantity and active hepatocellular function. In this work, we developed a promising scaffold for hepatocyte culture by coating porcine liver extracellular matrix (ECM) on chitosan (CTS) fabrics. Porcine Liver was decellularized using 1% Triton X-100. Solubilized liver ECM was immobilized on CTS fibers surface through cross linking of ECM and CTS with 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) and N-Hydroxysuccinimide (NHS). Then the scaffold was characterized by Fourier transformed infrared spectroscopy in attenuated total reflection mode (ATR-FTIR), X-photoelectron spectroscopy (XPS) and water contact angle measurement. The efficacy of modified scaffolds to maintain C3A hepatocytes adhesion, proliferation, bioactivity and functionality in vitro was detected. FTIR spectra and XPS demonstrated the presence of ECM coating on CTS fabric surface. Covalently attached coating significantly improved the binding efficiency between ECM and CTS fabrics, in comparison to the coating by physical absorption. Furthermore, C3A hepatocytes cultured on coated scaffolds showed enhanced cell bioactivity and liver-specific function, such as albumin secretion and urea synthesis, compared with those cultured on untreated scaffolds(p < 0.05). As a promising hepatocyte culture carrier, the ECM coated CTS fabrics could be applied in the biological artificial liver reactor.
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Affiliation(s)
- Chaochen Zhao
- Department of Hepatobiliary Surgery II, Guangdong Provincial Research Center for Artificial Organ and Tissue Engineering, Guangzhou Clinical Research and Transformation Center for Artificial Liver, Institute of Regenerative Medicine, Zhujiang Hospital, Southern Medical University, Guangzhou, Guangdong Province, China.,Department of Hepatobiliary Surgery, The First Affiliated Hospital of Guangzhou Medical University, Guangzhou Medical University, Guangzhou, Guangdong Province, China
| | - Yang Li
- Department of Hepatobiliary Surgery II, Guangdong Provincial Research Center for Artificial Organ and Tissue Engineering, Guangzhou Clinical Research and Transformation Center for Artificial Liver, Institute of Regenerative Medicine, Zhujiang Hospital, Southern Medical University, Guangzhou, Guangdong Province, China
| | - Gongze Peng
- Department of Hepatobiliary and Pancreatic Surgery, The Second Medical College, Shenzhen People's Hospital, Jinan University, Shenzhen, Guangdong Province, China
| | - Xiongxin Lei
- National Key Laboratory of Biochemical Engineering, Institute of Process Engineering, Beijing, China
| | - Guifeng Zhang
- National Key Laboratory of Biochemical Engineering, Institute of Process Engineering, Beijing, China
| | - Yi Gao
- Department of Hepatobiliary Surgery II, Guangdong Provincial Research Center for Artificial Organ and Tissue Engineering, Guangzhou Clinical Research and Transformation Center for Artificial Liver, Institute of Regenerative Medicine, Zhujiang Hospital, Southern Medical University, Guangzhou, Guangdong Province, China.,State Key Laboratory of Organ Failure Research, Southern Medical University, Guangzhou, Guangdong Province, China
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33
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Villamil Ballesteros AC, Segura Puello HR, Lopez-Garcia JA, Bernal-Ballen A, Nieto Mosquera DL, Muñoz Forero DM, Segura Charry JS, Neira Bejarano YA. Bovine Decellularized Amniotic Membrane: Extracellular Matrix as Scaffold for Mammalian Skin. Polymers (Basel) 2020; 12:polym12030590. [PMID: 32151022 PMCID: PMC7182835 DOI: 10.3390/polym12030590] [Citation(s) in RCA: 18] [Impact Index Per Article: 3.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/31/2019] [Revised: 11/16/2019] [Accepted: 11/23/2019] [Indexed: 12/23/2022] Open
Abstract
Decellularized membranes (DM) were obtained from bovine amniotic membranes (BAM) using four different decellularization protocols, based on physical, chemical, and mechanical treatment. The new material was used as a biological scaffold for in vitro skin cell culture. The DM were characterized using hematoxylin-eosin assay, scanning electron microscopy (SEM), Fourier transform infrared spectroscopy (FTIR-ATR), and differential scanning calorimetry (DSC). The in vitro cytotoxicity of DM was evaluated using MTT. The efficacy of decellularization process was assessed through DNA quantification and electrophoresis. All the used protocols showed a high effectiveness in terms of elimination of native cells, confirmed by DNA extraction and quantification, electrophoresis, and SEM, although protocol IV removes the cellular contents and preserve the native extracellular matrix (ECM) architecture which it can be considered as the most effective in terms of decellularization. FTIR-ATR and DSC on the other hand, revealed the effects of decellularization on the biochemical composition of the matrices. There was no cytotoxicity and the biological matrices obtained were a source of collagen for recellularization. The matrices of protocols I, II, and III were degraded at day 21 of cell culture, forming a gel. The biocompatibility in vitro was demonstrated; hence these matrices may be deemed as potential scaffold for epithelial tissue regeneration.
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Affiliation(s)
- Andrea Catalina Villamil Ballesteros
- Laboratorio de Investigaciones en Salud, Universidad Manuela Beltrán, Avenida Circunvalar No. 60-00, Bogotá 110231, Colombia; (H.R.S.P.); (D.L.N.M.); (D.M.M.F.); (J.S.S.C.); (Y.A.N.B.)
- Correspondence:
| | - Hugo Ramiro Segura Puello
- Laboratorio de Investigaciones en Salud, Universidad Manuela Beltrán, Avenida Circunvalar No. 60-00, Bogotá 110231, Colombia; (H.R.S.P.); (D.L.N.M.); (D.M.M.F.); (J.S.S.C.); (Y.A.N.B.)
| | - Jorge Andres Lopez-Garcia
- Centre of Polymer Systems, University Institute, Tomas Bata University in Zlín, Trida Tomase Bati 5678, 76001 Zlín, Czech Republic;
| | - Andres Bernal-Ballen
- Grupo de Investigación en Ingeniería Biomédica, Vicerrectoría de Investigaciones, Universidad Manuela Beltrán, Avenida Circunvalar No. 60-00, Bogotá 110231, Colombia;
| | - Diana Lorena Nieto Mosquera
- Laboratorio de Investigaciones en Salud, Universidad Manuela Beltrán, Avenida Circunvalar No. 60-00, Bogotá 110231, Colombia; (H.R.S.P.); (D.L.N.M.); (D.M.M.F.); (J.S.S.C.); (Y.A.N.B.)
| | - Diana Milena Muñoz Forero
- Laboratorio de Investigaciones en Salud, Universidad Manuela Beltrán, Avenida Circunvalar No. 60-00, Bogotá 110231, Colombia; (H.R.S.P.); (D.L.N.M.); (D.M.M.F.); (J.S.S.C.); (Y.A.N.B.)
| | - Juan Sebastián Segura Charry
- Laboratorio de Investigaciones en Salud, Universidad Manuela Beltrán, Avenida Circunvalar No. 60-00, Bogotá 110231, Colombia; (H.R.S.P.); (D.L.N.M.); (D.M.M.F.); (J.S.S.C.); (Y.A.N.B.)
| | - Yuli Alexandra Neira Bejarano
- Laboratorio de Investigaciones en Salud, Universidad Manuela Beltrán, Avenida Circunvalar No. 60-00, Bogotá 110231, Colombia; (H.R.S.P.); (D.L.N.M.); (D.M.M.F.); (J.S.S.C.); (Y.A.N.B.)
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34
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Naeem EM, Sajad D, Talaei-Khozani T, Khajeh S, Azarpira N, Alaei S, Tanideh N, Reza TM, Razban V. Decellularized liver transplant could be recellularized in rat partial hepatectomy model. J Biomed Mater Res A 2019; 107:2576-2588. [PMID: 31361939 DOI: 10.1002/jbm.a.36763] [Citation(s) in RCA: 24] [Impact Index Per Article: 4.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/24/2019] [Revised: 07/12/2019] [Accepted: 07/22/2019] [Indexed: 12/28/2022]
Abstract
In situ recellularization of the liver decellularized scaffold is a potential therapeutic alternative for liver transplantation. We aimed to develop an in situ procedure for recellularization of the rat liver using sodium lauryl ether sulfate (SLES) compared with Triton X-100/SDS. Rat liver specimens were rinsed with PBS, decellularized with either Triton X-100/SDS or SLES, and finally rinsed by distilled water. The efficiency of decellularized liver scaffolds was evaluated by histological, confocal Raman microscopy, histochemical staining, and DNA quantification assessments. Finally, in vivo studies were done to assess the biocompatibility of the liver scaffold by serum biochemical parameters and the recellularization capacity by histological and immunohistochemistry staining. Findings confirmed the preservation of extracellular matrix (ECM) components such as reticular, collagen, glycosaminoglycans, and neutral carbohydrates in both Triton X-100/SDS- and SLES-treated livers. Hoechst, feulgen, Hematoxylin and eosin, and DNA quantification assessments confirmed complete genetic content removal. The serological parameters showed no adverse impact on the liver functions. Transplantation of SLES-treated cell-free decellularized liver showed extensive neovascularization along with migration of the fibrocytes and adipocytes and some immune cells. Also, immunohistochemical staining showed that the oval cells, stellate cells, cholangiocytes and hepatocytes invaded extensively into the graft. It is concluded that SLES can be considered as a promising alternative in the liver decellularization process, and the transplanted decellularized liver can appropriately be revascularized and regenerated.
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Affiliation(s)
- Erfani M Naeem
- Department of Basic Sciences, Histology Section, Faculty of Veterinary Medicine, Shahid Chamran University of Ahvaz, Ahvaz, Iran
| | - Daneshi Sajad
- Department of Basic Sciences, Histology Section, Faculty of Veterinary Medicine, Shahid Chamran University of Ahvaz, Ahvaz, Iran
| | - Tahereh Talaei-Khozani
- Tissue Engineering Lab, Department of Anatomical Sciences, School of Medicine, Shiraz University of Medical Sciences, Shiraz, Iran.,Laboratory for Stem Cell Research, Department of Anatomical Sciences, School of Medicine, Shiraz University of Medical Sciences, Shiraz, Iran
| | - Sahar Khajeh
- Department of Biochemistry, School of Medicine, Shiraz University of Medical Sciences, Shiraz, Iran
| | - Negar Azarpira
- Transplant Research Center, Shiraz University of Medical Sciences, Shiraz, Iran
| | - Sanaz Alaei
- Department of Reproductive Biology, School of Advanced Medical Sciences and Applied Technologies, Shiraz University of Medical Sciences, Shiraz, Iran
| | - Nader Tanideh
- Stem Cells Technology Research Center, Shiraz University of Medical Sciences, Shiraz, Iran.,Department of Pharmacology, School of Medicine, Shiraz University of Medical Sciences, Shiraz, Iran
| | - Tabandeh M Reza
- Department of Biochemistry and Molecular Biology, Shahid Chamran University of Ahvaz, Ahvaz, Iran
| | - Vahid Razban
- Stem Cells Technology Research Center, Shiraz University of Medical Sciences, Shiraz, Iran.,Department of Molecular Medicine, School of Advanced Medical Sciences and Technologies, Shiraz University of Medical Sciences, Shiraz, Iran
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35
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Lindberg G, Longoni A, Lim K, Rosenberg A, Hooper G, Gawlitta D, Woodfield T. Intact vitreous humor as a potential extracellular matrix hydrogel for cartilage tissue engineering applications. Acta Biomater 2019; 85:117-130. [PMID: 30572166 DOI: 10.1016/j.actbio.2018.12.022] [Citation(s) in RCA: 18] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/09/2018] [Revised: 11/09/2018] [Accepted: 12/14/2018] [Indexed: 12/20/2022]
Abstract
Decellularisation of tissues, utilising their biochemical cues, poses exciting tissue engineering (TE) opportunities. However, removing DNA from cartilage (dCart) requires harsh treatments due to its dense structure, causing loss of bioactivity and limiting its application as a cartilaginous extra cellular matrix (ECM). In this study, we demonstrate for the first time the successful application of vitreous humor (VH), a highly hydrated tissue closely resembling the glycosaminoglycan (GAG) and collagen composition of cartilage, as an ECM hydrogel to support chondrogenic differentiation. Equine VH was extracted followed by biochemical quantifications, histological examinations, cytotoxicity (human mesenchymal stromal cells, hMSCs and human articular chondrocytes, hACs) and U937 cell proliferation studies. VH was further seeded with hACs or hMSCs and cultured for 3-weeks to study chondrogenesis compared to scaffold-free micro-tissue pellet cultures and collagen-I hydrogels. Viability, metabolic activity, GAG and DNA content, chondrogenic gene expression (aggrecan, collagen I/II mRNA) and mechanical properties were quantified and matrix deposition was visualised using immunohistochemistry (Safranin-O, collagen I/II). VH was successfully extracted, exhibiting negligible amounts of DNA (0.4 ± 0.4 µg/mg dry-weight) and notable preservation of ECM components. VH displayed neither cytotoxic responses nor proliferation of macrophage-like U937 cells, instead enhancing both hMSC and hAC proliferation. Interestingly, encapsulated cells self-assembled the VH-hydrogel into spheroids, resulting in uniform distribution of both GAGs and collagen type II with increased compressive mechanical properties, rendering VH a permissive native ECM source to fabricate cartilaginous hydrogels for potential TE applications. STATEMENT OF SIGNIFICANCE: Fabricating bioactive and cell-instructive cartilage extracellular matrix (ECM) derived biomaterials and hydrogels has over recent years proven to be a challenging task, often limited by poor retention of inherent environmental cues post decellularisation due to the dense and avascular nature of native cartilage. In this study, we present an alternative route to fabricate highly permissive and bioactive ECM hydrogels from vitreous humor (VH) tissue. This paper specifically reports the discovery of optimal VH extraction protocols and cell seeding strategy enabling fabrication of cartilaginous matrix components into a hydrogel support material for promoting chondrogenic differentiation. The work showcases a naturally intact and unmodified hydrogel design that improves cellular responses and may help guide the development of cell instructive and stimuli responsive hybrid biomaterials in a number of TERM applications.
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36
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Kim YS, Majid M, Melchiorri AJ, Mikos AG. Applications of decellularized extracellular matrix in bone and cartilage tissue engineering. Bioeng Transl Med 2019; 4:83-95. [PMID: 30680321 PMCID: PMC6336671 DOI: 10.1002/btm2.10110] [Citation(s) in RCA: 193] [Impact Index Per Article: 32.2] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/13/2018] [Revised: 07/30/2018] [Accepted: 07/31/2018] [Indexed: 12/12/2022] Open
Abstract
Regenerative therapies for bone and cartilage injuries are currently unable to replicate the complex microenvironment of native tissue. There are many tissue engineering approaches attempting to address this issue through the use of synthetic materials. Although synthetic materials can be modified to simulate the mechanical and biochemical properties of the cell microenvironment, they do not mimic in full the multitude of interactions that take place within tissue. Decellularized extracellular matrix (dECM) has been established as a biomaterial that preserves a tissue's native environment, promotes cell proliferation, and provides cues for cell differentiation. The potential of dECM as a therapeutic agent is rising, but there are many limitations of dECM restricting its use. This review discusses the recent progress in the utilization of bone and cartilage dECM through applications as scaffolds, particles, and supplementary factors in bone and cartilage tissue engineering.
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Affiliation(s)
- Yu Seon Kim
- Dept. of BioengineeringRice UniversityHoustonTX 77005
| | - Marjan Majid
- Dept. of BioengineeringRice UniversityHoustonTX 77005
| | | | - Antonios G. Mikos
- Dept. of BioengineeringRice UniversityHoustonTX 77005
- Biomaterials LabRice UniversityHoustonTX 77005
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37
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Simsa R, Padma AM, Heher P, Hellström M, Teuschl A, Jenndahl L, Bergh N, Fogelstrand P. Systematic in vitro comparison of decellularization protocols for blood vessels. PLoS One 2018; 13:e0209269. [PMID: 30557395 PMCID: PMC6296505 DOI: 10.1371/journal.pone.0209269] [Citation(s) in RCA: 65] [Impact Index Per Article: 9.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/07/2018] [Accepted: 12/03/2018] [Indexed: 01/19/2023] Open
Abstract
Decellularization of native blood vessels is a promising technology to generate 3D biological scaffolds for vascular grafting. Blood vessel decellularization has been performed in previous studies under various experimental conditions, that complicates comparison and optimization of suitable protocols. The goal of this work was to systematically compare the decellularization and recellularization efficacy of 5 different protocols utilizing the detergents sodium dodecyl sulfate (SDS), sodium deoxycholate (SDC), CHAPS and TritonX-100 together with DNA-removing enzymes on porcine vena cava in a perfusion bioreactor setup. Additionally, we tested the effect of DNase on the extracellular matrix (ECM) properties. We found that all protocols could efficiently decellularize blood vessels. Mechanical strength, collagen preservation and ECM integrity were similar among all tested detergents, yet TritonX protocols required long-term DNase application for complete decellularization. However, TritonX-based protocols showed the greatest recellularization efficacy with HUVECs in vitro. Furthermore, we developed a novel protocol for TritonX which improved recellularization and reduced total process time and ECM stiffness compared to previous protocols. SDS, SDC and CHAPS based protocols had a lower recellularization potential. In conclusion, decellularization of blood vessels can be achieved with all tested reagents, but TritonX treated ECM can be most efficiently recellularized with endothelial cells.
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Affiliation(s)
- Robin Simsa
- VERIGRAFT AB, Gothenburg, Sweden
- Department of Molecular and Clinical Medicine/Wallenberg Laboratory, University of Gothenburg and Sahlgrenska University Hospital, Gothenburg, Sweden
| | - Arvind Manikantan Padma
- Laboratory for Transplantation and Regenerative Medicine, Department of Obstetrics and Gynecology, Sahlgrenska Academy, University of Gothenburg, Gothenburg, Sweden
| | - Philipp Heher
- Ludwig Boltzmann Institute for Experimental and Clinical Traumatology/AUVA Research Center, Vienna, Austria
| | - Mats Hellström
- Laboratory for Transplantation and Regenerative Medicine, Department of Obstetrics and Gynecology, Sahlgrenska Academy, University of Gothenburg, Gothenburg, Sweden
| | - Andreas Teuschl
- Austrian Cluster for Tissue Regeneration, Vienna, Austria
- Department of Biochemical Engineering, UAS Technikum Wien, Vienna, Austria
| | | | - Niklas Bergh
- Department of Molecular and Clinical Medicine/Wallenberg Laboratory, University of Gothenburg and Sahlgrenska University Hospital, Gothenburg, Sweden
| | - Per Fogelstrand
- Department of Molecular and Clinical Medicine/Wallenberg Laboratory, University of Gothenburg and Sahlgrenska University Hospital, Gothenburg, Sweden
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38
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Abstract
In this Editor's Review, articles published in 2017 are organized by category and summarized. We provide a brief reflection of the research and progress in artificial organs intended to advance and better human life while providing insight for continued application of these technologies and methods. Artificial Organs continues in the original mission of its founders "to foster communications in the field of artificial organs on an international level." Artificial Organs continues to publish developments and clinical applications of artificial organ technologies in this broad and expanding field of organ Replacement, Recovery, and Regeneration from all over the world. Peer-reviewed Special Issues this year included contributions from the 12th International Conference on Pediatric Mechanical Circulatory Support Systems and Pediatric Cardiopulmonary Perfusion edited by Dr. Akif Undar, Artificial Oxygen Carriers edited by Drs. Akira Kawaguchi and Jan Simoni, the 24th Congress of the International Society for Mechanical Circulatory Support edited by Dr. Toru Masuzawa, Challenges in the Field of Biomedical Devices: A Multidisciplinary Perspective edited by Dr. Vincenzo Piemonte and colleagues and Functional Electrical Stimulation edited by Dr. Winfried Mayr and colleagues. We take this time also to express our gratitude to our authors for offering their work to this journal. We offer our very special thanks to our reviewers who give so generously of time and expertise to review, critique, and especially provide meaningful suggestions to the author's work whether eventually accepted or rejected. Without these excellent and dedicated reviewers the quality expected from such a journal could not be possible. We also express our special thanks to our Publisher, John Wiley & Sons for their expert attention and support in the production and marketing of Artificial Organs. We look forward to reporting further advances in the coming years.
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39
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Damania A, Kumar A, Teotia AK, Kimura H, Kamihira M, Ijima H, Sarin SK, Kumar A. Decellularized Liver Matrix-Modified Cryogel Scaffolds as Potential Hepatocyte Carriers in Bioartificial Liver Support Systems and Implantable Liver Constructs. ACS APPLIED MATERIALS & INTERFACES 2018; 10:114-126. [PMID: 29210278 DOI: 10.1021/acsami.7b13727] [Citation(s) in RCA: 45] [Impact Index Per Article: 6.4] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 06/07/2023]
Abstract
Recent progress in the use of decellularized organ scaffolds as regenerative matrices for tissue engineering holds great promise in addressing the issue of donor organ shortage. Decellularization preserves the mechanical integrity, composition, and microvasculature critical for zonation of hepatocytes in the liver. Earlier studies have reported the possibility of repopulating decellularized matrices with hepatic cell lines or stem cells to improve liver regeneration. In this work, we study the versatility of the decellularized liver matrix as a substrate coating of three-dimensional cryogel scaffolds. The coated cryogels were analyzed for their ability to maintain hepatic cell growth and functionality in vitro, which was found to be significantly better than the uncoated cryogel scaffolds. The decellularized liver matrix-coated cryogel scaffolds were evaluated for their potential application as a cell-loaded bioreactor for bioartificial liver support and as an implantable liver construct. Extracorporeal connection of the coated cryogel bioreactor to a liver failure model showed improvement in liver function parameters. Additionally, offline clinical evaluation of the bioreactor using patient-derived liver failure plasma showed its efficacy in improving liver failure conditions by approximately 30-60%. Furthermore, implantation of the decellularized matrix-coated cryogel showed complete integration with the native tissue as confirmed by hematoxylin and eosin staining of tissue sections. HepG2 cells and primary human hepatocytes seeded in the coated cryogel scaffolds implanted in the liver failure model maintained functionality in terms of albumin synthesis and cytochrome P450 activity post 2 weeks of implantation. In addition, a 20-60% improvement in liver function parameters was observed post implantation. These results, put together, suggest a possibility of using the decellularized matrix-coated cryogel scaffolds for liver tissue engineering applications.
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Affiliation(s)
- Apeksha Damania
- Department of Biological Sciences and Bioengineering, Indian Institute of Technology Kanpur , Kanpur-208016 Uttar Pradesh, India
| | - Anupam Kumar
- Institute of Liver and Biliary Sciences , Vasant Kunj, New Delhi 110070, India
| | - Arun K Teotia
- Department of Biological Sciences and Bioengineering, Indian Institute of Technology Kanpur , Kanpur-208016 Uttar Pradesh, India
| | - Haruna Kimura
- Department of Chemical Engineering, Faculty of Engineering, Kyushu University , Fukuoka 8190395, Japan
| | - Masamichi Kamihira
- Department of Chemical Engineering, Faculty of Engineering, Kyushu University , Fukuoka 8190395, Japan
| | - Hiroyuki Ijima
- Department of Chemical Engineering, Faculty of Engineering, Kyushu University , Fukuoka 8190395, Japan
| | - Shiv Kumar Sarin
- Institute of Liver and Biliary Sciences , Vasant Kunj, New Delhi 110070, India
| | - Ashok Kumar
- Department of Biological Sciences and Bioengineering, Indian Institute of Technology Kanpur , Kanpur-208016 Uttar Pradesh, India
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