Researcher(s)
- Christian Le, Biomedical Engineering, University of Delaware
Faculty Mentor(s)
- Justin Parreno, Biological Sciences, University of Delaware
Abstract
Christian Le, Justin Parreno
Articular cartilage has a limited capacity for self-repair, and damage leads to degeneration that progresses to osteoarthritis. Autologous chondrocyte implantation (ACI) is an FDA-approved cell-based cartilage repair therapy in which chondrocytes are harvested from a patient and expanded in monolayer culture to reach clinically relevant cell numbers before reimplantation. However, expansion on tissue culture polystyrene (PS) drives chondrocyte dedifferentiation. In dedifferentiation, cells flatten, downregulate chondrogenic markers such as type II collagen and aggrecan, and upregulate fibroblastic markers such as type I collagen, tenascin C, and alpha-smooth muscle actin, resulting in mechanically inferior fibrocartilage rather than hyaline cartilage.
Expansion of chondrocytes on cell-derived extracellular matrix (ECM) substrates offers an alternative to culture on polystyrene (PS) by presenting native biochemical and biomechanical cues. Prior studies show that culturing chondrocytes on chondrocyte-derived ECM reduces dedifferentiation during expansion. However, it remains unclear whether this benefit depends on the cell type used to generate the ECM. Additionally, it is unknown if ECM exposure during expansion leaves a lasting imprint that persists once cells are returned to PS. The goal of this project is to compare expansion and cartilage tissue-producing capabilities of articular chondrocytes cultured on chondrocyte-derived matrix (CM) and mesenchymal stem cell-derived matrix (MSM). We hypothesize that ECM substrates improve chondrocyte expansion and phenotype over PS, with CM outperforming MSM if the benefit is tissue-specific, and that cells primed on CM retain matrix memory that enhances redifferentiation into cartilage-like tissue.
Bovine articular chondrocytes were expanded on PS, CM, or MSM. Following expansion, we assessed phenotype by examining gene expression and the capacity to form cartilage tissue in 3D culture. To assess matrix memory, cells were expanded on PS or CM for one passage (P1). At P1, cells were placed on either PS or CM. This generated four conditions (PS→PS, CM→PS, PS→CM, CM→CM). At passage 2 (P2), we re-examined phenotype as described above.
Both ECM substrates supported expansion and preserved chondrogenic phenotype relative to PS, upregulating COL2 and PRG4 and downregulating COL1, with no significant differences between CM and MSM. After switchover, phenotype tracked the substrate present during the second passage. COL1 fell from 100% in PS→PS to 55.4% in PS→CM (p=0.0016) and 30.6% in CM→CM (p<0.0001), while CM→PS did not differ from PS→PS (p=0.21). These results indicate that cell-derived ECM improves chondrocyte expansion and phenotype, independent of matrix tissue origin, and that sustained ECM contact, rather than transient priming, drives redifferentiation.