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Tissue Engineering

Tissue Engineering - Poster Session D

Poster L4 - Recapitulating the effects of the osteoarthritic inflammatory environment on mesenchymal stem cell chondrogenesis

Friday, October 25, 2024
3:30 PM – 4:30 PM EST
Location: Exhibit Hall E, F & G

Introduction:  Osteoarthritis (OA) is a degenerative disease that involves damage to the knee joint, affecting over 240 million people worldwide. OA is characterized by a dysregulated inflammatory response in the joint, resulting in the gradual loss of cartilage and subchondral bone in a joint. In the initial stages of OA, the secretion of inflammatory cytokines shifts chondrocytes towards catabolism, which exacerbates tissue degradation and matrix breakdown. Since chondrocytes are incapable of returning to an anabolic state to repair the extracellular matrix, tissue engineering strategies are a promising option. Mesenchymal stromal cell (MSC) transplantation has been explored for cartilage regeneration due to the ability of these cells to differentiate into chondrocytes and secrete cartilaginous extracellular matrix (ECM). However, the chondrogenic capacity of these cells can be hindered by the OA inflammatory microenvironment. Therefore, the first objective of this study is to understand the effect of OA-like inflammation on MSC chondrogenesis. The second objective is to test whether dexamethasone (DEX) can protect MSC chondrogenesis from inflammation.

Materials and

Methods:  Human MSCs were seeded onto 3D-printed PCL scaffolds (filament diameter: 40 microns, shape: 3 mm diameter, and porosity: 85%). The scaffolds were cultured in chondrogenic media consisting of chemically defined media (CDM; Dulbecco's Modified Eagle Medium, penicillin-streptomycin, bovine serum albumin, and L-proline) and supplemented with dexamethasone (Dex), insulin-transferrin-selenium, transforming growth factor-β3, ascorbic acid, and linoleic acid. After 3 weeks of chondrogenic differentiation, the samples are split into chondrogenic, inflammatory, and chondro-protective media groups for an additional 2 weeks. The chondrogenic media after 3 weeks of incubation served as a baseline to indicate the completion of chondrogenesis, while the chondrogenic media at week 5 compares between the experimental groups at the end of culture. The inflammatory group consists of a 1:1 ratio of CDM and pro-inflammatory macrophage conditioned media (MCM). MCM was prepared by polarizing primary mouse macrophages towards the M1 inflammatory phenotype with colony-stimulating factor, interferon-γ, and lipopolysaccharide in Roswell Park Memorial Institute (RPMI). Conditioned media was collected by washing cells with PBS and incubating in RPMI for 24 hours. The chondro-protective group consists of MCM supplemented with 100nM Dex. To verify the impact of chondrogenesis in inflammatory media, biochemical assays were performed to characterize DNA, glycosaminoglycans (GAGs) and collagen content. Harvested samples were digested with papain enzyme for 12 hours at 60°C. Thereafter the PicoGreen, dimethyl-methylene-blue, and hydroxyproline assays were performed in order to quantify DNA, GAGs, and collagen content respectively. Finally, we collected the media to quantify the apoptotic activity using the Caspase-Glo 3/7 assay.

Results, Conclusions, and Discussions: The data suggests that inflammatory media inhibits MSC chondrogenesis, when compared with the chondrogenic group at the week 3 time point. The DNA assay showed that the chondrogenic media group at week 3 and week 5 has a higher DNA content than the inflammatory groups which suggests that the inclusion of MCM media decreases cell proliferation. The GAG and collagen data both have similar trends, the chondrogenic media group at week 5 showed increased GAG and collagen in comparison to the other groups. In comparison to week 5, only the MCM group had a significantly lower GAG and collagen content whereas all of the other groups had similar collagen and GAG levels. Finally, the caspase assay shows that Dex supplementation significantly decreases apoptotic activity since it has lower luminescence signal compared to the inflammatory groups. As we investigate ways to model inflammatory conditions, MCM shows promise to recapitulate the inflammatory environment in OA. Future work will focus on engineering strategies for the controlled delivery of anti-inflammatory molecules to reduce the effects of MCM on MSC chondrogenesis and achieve cartilage regeneration in OA models.

Acknowledgements (Optional):