Micronutrient Optimization Using Design of Experiments Approach in Tissue Engineered Articular Cartilage for Production of Type II Collagen

Abstract

Tissue Engineering of cartilage has been hampered by the inability of engineered tissue to express native levels of type II collagen in vitro. 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, Gaussia 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 expression. Five conditions 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 expression. Engineered cartilage grown in this condition, showed a 170% increase in type II collagen expression (Day 22 Luminescence) and in Youngs 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.

Disease Modifying Osteoarthritis Drug Discovery Using A Temporal Phenotypic Reporter In 3D Aggregates of Primary Human Chondrocytes

“…In this work, the modified chondrocytes are used in a 3D aggregate model enabled for high throughput analysis. Published findings by our group have shown the success of this assay, by using it to identify vitamins and minerals that improve chondrogenesis in the murine chondrogenic cell line, ATDC5, and in primary rabbit chondrocytes ( 10, 14 ). This system has also been used to identify biomaterials that improve type II collagen production in human primary chondrocytes in 3D bioprinted constructs ( 13 ).…”

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

“…G, and psPAX2, were a gift from Didier Trono (Addgene plasmid # 12259; http://n2t.net/addgene:12259 ; RRID:Addgene_12259 ) and (Addgene plasmid # 12260; http://n2t.net/addgene:12260 ; RRID:Addgene_12260 ) respectively. This article was published as a pre-print on BioRxiv ( Cruz, 2022 ). Article processing charges were provided in part by the UCF College of Graduate Studies Open Access Publishing Fund.…”