Protocols for Culturing and Imaging a Human <i>Ex Vivo</i> 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 <i>in vitro</i> and experimental animal models p...
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MDPI AG
2019-02-01
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| Online Access: | https://www.mdpi.com/1996-1944/12/4/640 |
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| author | Serena Duchi Stephanie Doyle Timon Eekel Cathal D. O’Connell Cheryl Augustine Peter Choong Carmine Onofrillo Claudia Di Bella |
| author_facet | Serena Duchi Stephanie Doyle Timon Eekel Cathal D. O’Connell Cheryl Augustine Peter Choong Carmine Onofrillo Claudia Di Bella |
| author_sort | Serena Duchi |
| collection | DOAJ |
| 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 <i>in vitro</i> and experimental animal models pose serious challenges to the translation of preclinical findings into clinical practice. <i>Ex vivo</i> models are of great value for translating <i>in vitro</i> 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 <i>ex vivo</i> 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 <i>ex vivo</i> tool for studying cartilage therapies, such as hydrogel-based bioscaffolds, and we envision it will reduce the number of animals needed for <i>in vivo</i> testing. |
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| format | Article |
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| institution | Directory Open Access Journal |
| issn | 1996-1944 |
| language | English |
| last_indexed | 2024-04-12T05:13:53Z |
| publishDate | 2019-02-01 |
| publisher | MDPI AG |
| record_format | Article |
| series | Materials |
| spelling | doaj.art-70743ff028194077ab079ac297b34f0b2022-12-22T03:46:42ZengMDPI AGMaterials1996-19442019-02-0112464010.3390/ma12040640ma12040640Protocols for Culturing and Imaging a Human <i>Ex Vivo</i> Osteochondral Model for Cartilage Biomanufacturing ApplicationsSerena Duchi0Stephanie Doyle1Timon Eekel2Cathal D. O’Connell3Cheryl Augustine4Peter Choong5Carmine Onofrillo6Claudia Di Bella7BioFab3D, Aikenhead Centre for Medical Discovery, St Vincent’s Hospital, Clinical Sciences Building, 29 Regent Street, 3065 Fitzroy, AustraliaBioFab3D, Aikenhead Centre for Medical Discovery, St Vincent’s Hospital, Clinical Sciences Building, 29 Regent Street, 3065 Fitzroy, AustraliaUniversity of Utrecht, Domplein 29, 3512 JE Utrecht, The NetherlandsBioFab3D, Aikenhead Centre for Medical Discovery, St Vincent’s Hospital, Clinical Sciences Building, 29 Regent Street, 3065 Fitzroy, AustraliaDepartment of Surgery, St Vincent’s Hospital, University of Melbourne, Clinical Sciences Building, 29 Regent Street, 3065 Fitzroy, AustraliaBioFab3D, Aikenhead Centre for Medical Discovery, St Vincent’s Hospital, Clinical Sciences Building, 29 Regent Street, 3065 Fitzroy, AustraliaBioFab3D, Aikenhead Centre for Medical Discovery, St Vincent’s Hospital, Clinical Sciences Building, 29 Regent Street, 3065 Fitzroy, AustraliaBioFab3D, Aikenhead Centre for Medical Discovery, St Vincent’s Hospital, Clinical Sciences Building, 29 Regent Street, 3065 Fitzroy, AustraliaCartilage 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 <i>in vitro</i> and experimental animal models pose serious challenges to the translation of preclinical findings into clinical practice. <i>Ex vivo</i> models are of great value for translating <i>in vitro</i> 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 <i>ex vivo</i> 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 <i>ex vivo</i> tool for studying cartilage therapies, such as hydrogel-based bioscaffolds, and we envision it will reduce the number of animals needed for <i>in vivo</i> testing.https://www.mdpi.com/1996-1944/12/4/640cartilage<i>ex vivo</i> modelosteochondral unithydrogel-based scaffoldhistological procedurescartilage regeneration |
| spellingShingle | Serena Duchi Stephanie Doyle Timon Eekel Cathal D. O’Connell Cheryl Augustine Peter Choong Carmine Onofrillo Claudia Di Bella Protocols for Culturing and Imaging a Human <i>Ex Vivo</i> Osteochondral Model for Cartilage Biomanufacturing Applications Materials cartilage <i>ex vivo</i> model osteochondral unit hydrogel-based scaffold histological procedures cartilage regeneration |
| title | Protocols for Culturing and Imaging a Human <i>Ex Vivo</i> Osteochondral Model for Cartilage Biomanufacturing Applications |
| title_full | Protocols for Culturing and Imaging a Human <i>Ex Vivo</i> Osteochondral Model for Cartilage Biomanufacturing Applications |
| title_fullStr | Protocols for Culturing and Imaging a Human <i>Ex Vivo</i> Osteochondral Model for Cartilage Biomanufacturing Applications |
| title_full_unstemmed | Protocols for Culturing and Imaging a Human <i>Ex Vivo</i> Osteochondral Model for Cartilage Biomanufacturing Applications |
| title_short | Protocols for Culturing and Imaging a Human <i>Ex Vivo</i> Osteochondral Model for Cartilage Biomanufacturing Applications |
| title_sort | protocols for culturing and imaging a human i ex vivo i osteochondral model for cartilage biomanufacturing applications |
| topic | cartilage <i>ex vivo</i> model osteochondral unit hydrogel-based scaffold histological procedures cartilage regeneration |
| url | https://www.mdpi.com/1996-1944/12/4/640 |
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