(317c) Impact of Oncostatin M & Tumor Necrosis Factor-? on Chondrogenic and Immunosuppressive Capacity of Human Mesenchymal Stem Cells in Pellet Culture and 3D Hydrogel Constructs

Statement of Purpose: Mimicking the disease conditions of osteoarthritis (OA) in a laboratory setting is important to better understand biological processes and for efficacy of potential treatments. There are various ways to simulate OA in vitro. One common approach is to add inflammatory cytokines found in OA to cell culture media. This method is employed often in ex plant models and has gained traction for studying 2D cell culture and 3D hydrogels. The combination of oncostatin M (OSM) and tumor necrosis factor-α (TNF-α) at 10 ng/mL and 20 ng/mL, respectively, has been employed in ex plant models and validated in vivo. However, the impact of the combination of these cytokines on pellet culture and hydrogel systems has not been studied. Mesenchymal stem cells (MSCs) can be differentiated down cartilage lineage and are known to be immunosuppressive. Additionally, as the environment strongly impacts cell response (e.g., to mechanical stimuli), comparing MSC response in both pellet and hydrogel constructs will provide insight into contributions from more than just cytokine stimuli. In this work, we investigate MSC differentiation and response in the presence of OSM and TNF-α in both pellet culture and blended collagen I/II hydrogels, a system we previously developed and showed promoted chondrogenesis, without and with encapsulated hyaluronic acid (HA).

Results: Human MSCs were maintained in pellets and hydrogels for 2 weeks. For pellet culture, five groups were studied: 1) negative control (base media), 2) positive control (chondrogenic media with 10 ng/mL of TGF-β3), 3) OSM and TNF-α (“OT”, positive control with 10 ng/mL OSM and 20 ng/mL TNF-α), 4) TNF-α (“T”, positive control with 20 ng/mL TNF-α), and 5) OSM (“O”, positive control with 10 ng/mL OSM). Collagen I/II hydrogels (col I/II) and col I/II gels with HA (1.5 MDa) at 1 mg/mL (“HMW1”) were maintained in both positive media and OSM and TNF-α media.

After 2 weeks, pellets and constructs were harvested and analyzed for matrix production (glycosaminoglycan, GAG, content) and for cell proliferation (DNA quantification). As shown in Figure 1 below, the presence of OSM and TNF-α did not limit the amount of GAG produced (1a and 1c). The increase in GAG production for HMW1 compared to col I/II was anticipated as HA provides further cues for cartilage production. However, the similar GAG production from HMW1 with cytokines was unexpected. The most surprising result was the DNA for the hydrogel groups (Figure 1d). The DNA for gels exposed to cytokines was statistically higher and on the order of ~3x more for both col I/II and HMW1 with cytokines compared to their non-cytokine counterparts. Although not statistically significant, the DNA for positive and cytokine groups for pellet culture were also higher as compared to the negative control pellets (Figure 1b). The increase in DNA suggests that the cells are proliferating more in the cytokine-stimulated groups as compared to non-stimulated groups. Additionally, the similar GAG levels for the pellet groups in the presence of cytokines suggest that overall the MSCs were still secreting cartilage extracellular matrix components (Figure 1a).

Conclusions: In vivo, cells respond to many biological and mechanical cues simultaneously. Simplifying the system in vitro to parse out individual contributions can aid in understanding how specific factors impact cell responses. Here, we found that the addition of cytokines to pellet or hydrogel systems resulted in similar amounts of overall GAG production. However, the increase in cell proliferation suggests that the cells are more active in response to the added cytokines and were likely compensating to maintain the same amount of matrix leading to less GAG per cell – especially for the hydrogel groups. Moving forward, we plan to quantify various genes related to cartilage differentiation, inflammation, and immunosuppression to elucidate what else is happening in the system.

Figure 1. Matrix quantification and cell proliferation for pellets and hydrogels. (a) GAG content for pellets, (b) DNA content for pellets, (c) GAG content in hydrogels, and (d) DNA content for hydrogels. For hydrogels: “no” indicates no added cytokines and “cyto” indicates added OSM and TNF-α. Different letters represent statistical significance (p < 0.05).