Cargando…

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...

Descripción completa

Detalles Bibliográficos
Autores principales: Duchi, Serena, Doyle, Stephanie, Eekel, Timon, D. O’Connell, Cathal, Augustine, Cheryl, Choong, Peter, Onofrillo, Carmine, Di Bella, Claudia
Formato: Online Artículo Texto
Lenguaje:English
Publicado: MDPI 2019
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
_version_ 1783403384762531840
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
work_keys_str_mv AT duchiserena protocolsforculturingandimagingahumanexvivoosteochondralmodelforcartilagebiomanufacturingapplications
AT doylestephanie protocolsforculturingandimagingahumanexvivoosteochondralmodelforcartilagebiomanufacturingapplications
AT eekeltimon protocolsforculturingandimagingahumanexvivoosteochondralmodelforcartilagebiomanufacturingapplications
AT doconnellcathal protocolsforculturingandimagingahumanexvivoosteochondralmodelforcartilagebiomanufacturingapplications
AT augustinecheryl protocolsforculturingandimagingahumanexvivoosteochondralmodelforcartilagebiomanufacturingapplications
AT choongpeter protocolsforculturingandimagingahumanexvivoosteochondralmodelforcartilagebiomanufacturingapplications
AT onofrillocarmine protocolsforculturingandimagingahumanexvivoosteochondralmodelforcartilagebiomanufacturingapplications
AT dibellaclaudia protocolsforculturingandimagingahumanexvivoosteochondralmodelforcartilagebiomanufacturingapplications