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Protocols for Culturing and Imaging a Human Ex Vivo Osteochondral Model for Cartilage Biomanufacturing Applications
Cartilage defects and diseases remain major clinical issues in orthopaedics. Biomanufacturing is now a tangible option for the delivery of bioscaffolds capable of regenerating the deficient cartilage tissue. However, several limitations of in vitro and experimental animal models pose serious challen...
Autores principales: | , , , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
MDPI
2019
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6416585/ https://www.ncbi.nlm.nih.gov/pubmed/30791632 http://dx.doi.org/10.3390/ma12040640 |
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author | Duchi, Serena Doyle, Stephanie Eekel, Timon D. O’Connell, Cathal Augustine, Cheryl Choong, Peter Onofrillo, Carmine Di Bella, Claudia |
author_facet | Duchi, Serena Doyle, Stephanie Eekel, Timon D. O’Connell, Cathal Augustine, Cheryl Choong, Peter Onofrillo, Carmine Di Bella, Claudia |
author_sort | Duchi, Serena |
collection | PubMed |
description | Cartilage defects and diseases remain major clinical issues in orthopaedics. Biomanufacturing is now a tangible option for the delivery of bioscaffolds capable of regenerating the deficient cartilage tissue. However, several limitations of in vitro and experimental animal models pose serious challenges to the translation of preclinical findings into clinical practice. Ex vivo models are of great value for translating in vitro tissue engineered approaches into clinically relevant conditions. Our aim is to obtain a viable human osteochondral (OC) model to test hydrogel-based materials for cartilage repair. Here we describe a detailed step-by-step framework for the generation of human OC plugs, their culture in a perfusion device and the processing procedures for histological and advanced microscopy imaging. Our ex vivo OC model fulfils the following requirements: the model is metabolically stable for a relevant culture period of 4 weeks in a perfusion bioreactor, the processing procedures allowed for the analysis of 3 different tissues or materials (cartilage, bone and hydrogel) without compromising their integrity. We determined a protocol and the settings for a non-linear microscopy technique on label free sections. Furthermore, we established a clearing protocol to perform light sheet-based observations on the cartilage layer without the need for tedious and destructive histological procedures. Finally, we showed that our OC system is a clinically relevant in terms of cartilage regeneration potential. In conclusion, this OC model represents a valuable preclinical ex vivo tool for studying cartilage therapies, such as hydrogel-based bioscaffolds, and we envision it will reduce the number of animals needed for in vivo testing. |
format | Online Article Text |
id | pubmed-6416585 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2019 |
publisher | MDPI |
record_format | MEDLINE/PubMed |
spelling | pubmed-64165852019-03-29 Protocols for Culturing and Imaging a Human Ex Vivo Osteochondral Model for Cartilage Biomanufacturing Applications Duchi, Serena Doyle, Stephanie Eekel, Timon D. O’Connell, Cathal Augustine, Cheryl Choong, Peter Onofrillo, Carmine Di Bella, Claudia Materials (Basel) Article Cartilage defects and diseases remain major clinical issues in orthopaedics. Biomanufacturing is now a tangible option for the delivery of bioscaffolds capable of regenerating the deficient cartilage tissue. However, several limitations of in vitro and experimental animal models pose serious challenges to the translation of preclinical findings into clinical practice. Ex vivo models are of great value for translating in vitro tissue engineered approaches into clinically relevant conditions. Our aim is to obtain a viable human osteochondral (OC) model to test hydrogel-based materials for cartilage repair. Here we describe a detailed step-by-step framework for the generation of human OC plugs, their culture in a perfusion device and the processing procedures for histological and advanced microscopy imaging. Our ex vivo OC model fulfils the following requirements: the model is metabolically stable for a relevant culture period of 4 weeks in a perfusion bioreactor, the processing procedures allowed for the analysis of 3 different tissues or materials (cartilage, bone and hydrogel) without compromising their integrity. We determined a protocol and the settings for a non-linear microscopy technique on label free sections. Furthermore, we established a clearing protocol to perform light sheet-based observations on the cartilage layer without the need for tedious and destructive histological procedures. Finally, we showed that our OC system is a clinically relevant in terms of cartilage regeneration potential. In conclusion, this OC model represents a valuable preclinical ex vivo tool for studying cartilage therapies, such as hydrogel-based bioscaffolds, and we envision it will reduce the number of animals needed for in vivo testing. MDPI 2019-02-20 /pmc/articles/PMC6416585/ /pubmed/30791632 http://dx.doi.org/10.3390/ma12040640 Text en © 2019 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/). |
spellingShingle | Article Duchi, Serena Doyle, Stephanie Eekel, Timon D. O’Connell, Cathal Augustine, Cheryl Choong, Peter Onofrillo, Carmine Di Bella, Claudia Protocols for Culturing and Imaging a Human Ex Vivo Osteochondral Model for Cartilage Biomanufacturing Applications |
title | Protocols for Culturing and Imaging a Human Ex Vivo Osteochondral Model for Cartilage Biomanufacturing Applications |
title_full | Protocols for Culturing and Imaging a Human Ex Vivo Osteochondral Model for Cartilage Biomanufacturing Applications |
title_fullStr | Protocols for Culturing and Imaging a Human Ex Vivo Osteochondral Model for Cartilage Biomanufacturing Applications |
title_full_unstemmed | Protocols for Culturing and Imaging a Human Ex Vivo Osteochondral Model for Cartilage Biomanufacturing Applications |
title_short | Protocols for Culturing and Imaging a Human Ex Vivo Osteochondral Model for Cartilage Biomanufacturing Applications |
title_sort | protocols for culturing and imaging a human ex vivo osteochondral model for cartilage biomanufacturing applications |
topic | Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6416585/ https://www.ncbi.nlm.nih.gov/pubmed/30791632 http://dx.doi.org/10.3390/ma12040640 |
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