gms | German Medical Science

Objectives: Pro-inflammatory conditions play a pivotal role in the progression of osteoarthritis (OA), particularly when aiming at cartilage regeneration in secondary post-traumatic OA. In recent studies, 3D bioprinting using a bioink platform based on thiolated hyaluronic acid (HA-SH) has shown promising results for chondrogenic differentiation of mesenchymal stromal cells (MSC) in vitro. However, effects of a pro-inflammatory environment on 3D printable bioinks in long-term culture remain largely unexplored. Therefore, in this study the effects of the pro-inflammatory cytokine interleukin-1β (IL-1β) on a 3D hyaluronic acid-based MSC-laden bioink were investigated.

Methods: Human MSC were cultured in a printable hyaluronic acid-based bioink using a dual-stage crosslinking approach. Therefore, HA-SH was pre-crosslinked with acrylated polyethylene glycol (PEG-diacryl) in an initial Michael addition. Subsequently, a UV-mediated thiol-ene reaction induced stability for long-term cell culture. For chondrogenic differentiation, 3D cell-hydrogel constructs with 8x10⁵ MSC per gel were cultured in a serum-free medium supplemented with transforming growth factor-β1 (TGF-β1) for a total period of 28 days. After a preculture for 7 days, constructs were either or not stimulated with IL-1β (10 ng/ml) for further 21 days. Constructs were analyzed using quantitative biochemical assays and histology.

Results and conclusion: As compared to controls, treatment with IL-1β (10 ng/ml) caused a distinct reduction in DNA content (Ctrl: 3.72±0.29 μg, IL-1β: 2.06±0.09 μg DNA; p<0.001). Furthermore, upon treatment with IL-1β (10 ng/ml), constructs showed drastically decreased glycosaminoglycan (GAG) content as normalized to DNA (Ctrl: 37.92±1.56 μg/μg, IL-1β: 5.70±0.65 μg/μg GAG/DNA; p<0.001) as well as per wet weight (Ctrl: 1.53±0.13 %, IL-1β: 0.39±0.09 % GAG/ww; p<0.001). Similarly, collagen content was diminished in IL-1β-stimulated constructs (Ctrl: 17.11±0.65 μg/μg, IL-1β: 5.39±1.30 μg/μg collagen/DNA; p<0.001). Histological staining for GAG deposition with safranin-O confirmed the distinct GAG depletion for constructs receiving IL-1β (10 ng/ml). Statistical analysis was performed using GraphPad Prism, version 6.0 (GraphPad Software, La Jolla, USA). Statistical significance between groups was assessed by two-way analysis of variance followed by Tukey’s post hoc test.

In conclusion, in the present study, distinct detrimental effects of IL-1β on the ECM composition in 3D hyaluronic acid-based bioink were demonstrated in long-term chondrogenic differentiation of human MSC. The established culture of 3D printable constructs in an inflammatory milieu can now be utilized to evaluate potential anti-inflammatory molecules aiming at cartilage regeneration in OA.