gms | German Medical Science

Objectives: Osteoarthritis (OA) pathogenesis is strongly linked to an inflammatory milieu driven by cytokines such as interleukin-1β (IL-1β). Regarding cartilage regeneration using cell-based approaches, recently research on 3D bioprinting has attracted increased attention. However, data on the generation of cartilaginous tissue in an inflammatory milieu and the application of anti-inflammatory agents is scarce, especially in 3D printable bioinks. Therefore, in this study, we investigated the impact of anti-inflammatory therapies using hydroxysafflor yellow-A (HSYA) or leonurine (LN) in 3D bioink and pellet cultures with human mesenchymal stromal cell (MSC) stimulated with the pro-inflammatory cytokine IL-1β.

Methods: A 3D printable hyaluronic acid-based bioink or pellet culture was used for differentiation of human MSCs, performed in serum-free medium supplemented with TGF-β 1. The hydrogel was constructed using a dual-stage crosslinking system, including a pre-crosslinking of thiolated hyaluronic acid (HA-SH) with acrylated polyethylene glycol in an initial Michael addition followed by a UV-light-induced thiol-ene click reaction. Bioinks were cultured for a total period of 21 days, including 7 days of preculture and 14 days of inflammatory stimulation, using different concentrations of IL-1β (0.5 – 10 ng/ml). Pellets were also co-treated with both IL-1β and either HSYA (80 µM) or LN (50 µM). Hydrogels and pellets were analyzed using quantitative biochemical assays and histology. Statistical analysis was performed using OriginPro 2023 (OriginLab Corporation, Northampton, US).

Results and conclusion: In bioinks, treatment with different concentrations IL-1β elicited a distinct reduction in glycosaminoglycan (GAG) content in the ECM of chondrogenically differentiated MSCs, as compared to controls (Ctrl: 26.04±2.32 µg/µg; IL-1β 1ng/ml: 11.88±2.04 µg/µg; IL-1β 5ng/ml: 4.09±1.08 µg/µg; IL-1β 10 ng/ml: 4.15±0.27 µg/µg GAG/DNA; p<0.05). These results could also be validated by safranin-O staining for GAG, and further supported by immunohistochemical analyses for aggrecan, collagen II, and the ECM-degrading enzyme MMP-13. In pellets receiving IL-1β at 10 ng/ml, application of HSYA or LN showed no rescue of ECM deposition impaired by IL-1β. In contrast, pellet culture receiving lower concentrations of IL-1β exhibited a rescue effect upon HSYA or LN treatment, leading to an increase in GAG content compared to groups treated with IL-1β at 1 or 0.5 ng/ml only (e.g., IL-1β 1ng/ml: 8.97±0.59 µg/µg; IL1/HSYA: 11.86±0.52 µg/µg; IL1/LN: 13.06±0.46 µg/µg GAG/DNA; p<0.05. IL-1β 0.5 ng/ml: 12.48±1.65 µg/µg; IL1/HSYA: 15.75±1.09 µg/µg GAG/DNA; p<0.05).

In conclusion, the application of the anti-inflammatory agents HSYA and LN was demonstrated to elicit a clear rescue of ECM content in pellet culture subjected to low-grade inflammatory conditions. Future investigations will include the application of HSYA and LN in 3D printable hyaluronic acid-based hydrogels aiming at cartilage regeneration in OA.