Micronutrient optimization for tissue engineered articular cartilage production of type II collagen

Tissue Engineering of cartilage has been hampered by the inability of engineered tissue to express native levels of type II collagen . Inadequate levels of type II collagen are, in part, due to a failure to recapitulate the physiological environment in culture. In this study, we engineered primary r...

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Bibliographic Details
Published in:	Frontiers in bioengineering and biotechnology Vol. 11; p. 1179332
Main Authors:	Cruz, Maria A, Gonzalez, Yamilet, Vélez Toro, Javier A, Karimzadeh, Makan, Rubbo, Anthony, Morris, Lauren, Medam, Ramapaada, Splawn, Taylor, Archer, Marilyn, Fernandes, Russell J, Dennis, James E, Kean, Thomas J
Format:	Journal Article
Language:	English
Published:	Switzerland Frontiers Media S.A 06-06-2023
Subjects:	Bioengineering and Biotechnology design of experiments (DoE) media optimization primary chondrocyte scaffold-free tissue engineered cartilage tissue engineered cartilage vitamins and minerals type II collagen heteropolymer design of experiments (DoE) media optimization extracellular matrix reporter vitamins and minerals scaffold-free tissue engineered cartilage tissue engineered cartilage primary chondrocyte
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Summary:	Tissue Engineering of cartilage has been hampered by the inability of engineered tissue to express native levels of type II collagen . Inadequate levels of type II collagen are, in part, due to a failure to recapitulate the physiological environment in culture. In this study, we engineered primary rabbit chondrocytes to express a secreted reporter, Luciferase, driven by the type II collagen promoter, and applied a Design of Experiments approach to assess chondrogenic differentiation in micronutrient-supplemented medium. Using a Response Surface Model, 240 combinations of micronutrients absent in standard chondrogenic differentiation medium, were screened and assessed for type II collagen promoter-driven luciferase expression. While the target of this study was to establish a combination of all micronutrients, alpha-linolenic acid, copper, cobalt, chromium, manganese, molybdenum, vitamins A, E, D and B7 were all found to have a significant effect on type II collagen promoter activity. Five conditions containing all micronutrients predicted to produce the greatest luciferase expression were selected for further study. Validation of these conditions in 3D aggregates identified an optimal condition for type II collagen promoter activity. Engineered cartilage grown in this condition, showed a 170% increase in type II collagen expression (Day 22 Luminescence) and in Young's tensile modulus compared to engineered cartilage in basal media alone.Collagen cross-linking analysis confirmed formation of type II-type II collagen and type II-type IX collagen cross-linked heteropolymeric fibrils, characteristic of mature native cartilage. Combining a Design of Experiments approach and secreted reporter cells in 3D aggregate culture enabled a high-throughput platform that can be used to identify more optimal physiological culture parameters for chondrogenesis.
Bibliography:	ObjectType-Article-1 SourceType-Scholarly Journals-1 ObjectType-Feature-2 content type line 23 Edited by: Oommen Varghese, Uppsala University, Sweden Solvig Diederichs, Heidelberg University Hospital, Germany Reviewed by: Ron June, Montana State University, United States
ISSN:	2296-4185 2296-4185
DOI:	10.3389/fbioe.2023.1179332