Assessment of static and perfusion methods for decellularization of PCL membrane-supported periodontal ligament cell sheet constructs |
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| Institution: | 1. School of Dentistry, The University of Queensland, Oral Health Centre, Herston, Brisbane, Australia;2. Department of Basic Science, Biology Unit, Deanship of Preparatory Year and Supporting studies, Imam Abdulrahman Bin Faisal University (University of Dammam), Saudi Arabia;3. Institute of Health and Biomedical Innovation, Queensland University of Technology, 60 Musk Avenue, 4059 Kelvin Grove, Australia;4. ARC ITTC in Additive Biomanufacturing, Institute of Health and Biomedical Innovation, Queensland University of Technology, 60 Musk Avenue, 4059 Kelvin Grove, Australia;1. Center for Regenerative Medicine, Massachusetts General Hospital;2. Harvard Medical School;3. Division of Pediatric Surgery;4. Division of Thoracic Surgery, Department of Surgery, Massachusetts General Hospital;5. Harvard Stem Cell Institute, Boston, Massachusetts;1. Institute of Health and Biomedical Innovation, Queensland University of Technology, Brisbane, QLD, Australia;2. The University of Queensland, UQ Centre for Clinical Research, Brisbane, QLD, Australia;3. The University of Queensland, UQ Diamantina Institute, Translational Research Institute, Brisbane, QLD, Australia;4. Commonwealth Scientific Industrial Research Organisation, Clayton, VIC, Australia;5. Australian Regenerative Medicine Institute, Monash University, Clayton, VIC, Australia;6. Department of Functional Materials in Medicine and Dentistry, University of Würzburg, Würzburg, Germany;7. Australian Research Centre in Additive Biomanufacturing, Queensland University of Technology, Brisbane, QLD, Australia;1. School of Dentistry and Oral Health, Centre for Medicine and Oral Health, Griffith Health Institute, Griffith University (Gold Coast Campus), 16-30 High Street, Southport, QLD 4222, Australia;2. Molecular Basis of Disease Program, Griffith Health Institute, Australia;3. State Key Laboratory of Oral Diseases, West China Hospital of Stomatology, Sichuan University, No. 14, Section 3, Renminnan Road, Chengdu, Sichuan 610041, China;4. Institute of Health and Biomedical Innovation, Queensland University of Technology, 60 Musk Avenue, Kelvin Grove, QLD 4059, Australia;1. Key Laboratory of Combined Multi-organ Transplantation, Ministry of Public Health, State Key Laboratory for Diagnosis and Treatment of Infectious Diseases, Division of Hepatobiliary and Pancreatic Surgery, The First Affiliated Hospital, School of Medicine, Zhejiang University, Hangzhou 310003, China;2. Centre for Orthopaedic Research, School of Surgery, University of Western Australia, Western Australia 6009, Australia;3. Key Laboratory of Nephropathy, Zhejiang Province, Hangzhou, Zhejiang 310003, China;4. Faculty of Health Sciences and Medicine, Bond University, Queensland, Australia |
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| Abstract: | ObjectivesDecellularization aims to harness the regenerative properties of native extracellular matrix. The objective of this study was to evaluate different methods of decellularization of periodontal ligament cell sheets whilst maintaining their structural and biological integrity.DesignHuman periodontal ligament cell sheets were placed onto melt electrospun polycaprolactone (PCL) membranes that reinforced the cell sheets during the various decellularization protocols. These cell sheet constructs (CSCs) were decellularized under static/perfusion conditions using a) 20 mM ammonium hydroxide (NH4OH)/Triton X-100, 0.5% v/v; and b) sodium dodecyl sulfate (SDS, 0.2% v/v), both +/− DNase besides Freeze–thaw (F/T) cycling method. CSCs were assessed using a collagen quantification assay, immunostaining and scanning electron microscopy. Residual fibroblast growth factor (bFGF), vascular endothelial growth factor (VEGF) and hepatocyte growth factor (HGF) were assessed with Bio-plex assays.ResultsDNA removal without DNase was higher under static conditions. However, after DNase treatment, there were no differences between the different decellularization methods with virtually 100% DNA removal. DNA elimination in F/T was less efficient even after DNase treatment. Collagen content was preserved with all techniques, except with SDS treatment. Structural integrity was preserved after NH4OH/Triton X-100 and F/T treatment, while SDS altered the extracellular matrix structure. Growth factor amounts were reduced after decellularization with all methods, with the greatest reduction (to virtually undetectable amounts) following SDS treatment, while NH4OH/Triton X-100 and DNase treatment resulted in approximately 10% retention.ConclusionsThis study showed that treatment with NH4OH/Triton X-100 and DNase solution was the most efficient method for DNA removal and the preservation of extracellular matrix integrity and growth factors retention. |
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| Keywords: | Tissue engineering Periodontal ligament Cell sheet Decellularization Melt electrospinning Polycaprolactone |
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