P042 - Covalent Protein Immobilization on Melt Electrowritten Microfiber Scaffolds for Guided Cartilage Regeneration
- J. Malda (Utrecht, NL)
- M. Ainsworth (Utrecht, NL)
- O. Lotz (Sydney, AU)
- A. Gilmour (Sydney, AU)
- A. Zhang (Sydney, AU)
- D. McKenzie (Sydney, AU)
- M. Bilek (Sydney, AU)
- J. Malda (Utrecht, NL)
- B. Akhavan (Sydney, AU)
- M. Castilho (Utrecht, NL)
Abstract
Purpose
Current tissue engineering treatments for end-stage articular cartilage fail to produce long-term functional cartilage tissue. Here, melt electrowriting (MEW) is used to fabricate 3D scaffolds with micro-resolution to mimic the properties of the native cartilage extracellular matrix (Castilho et al., 2019). These scaffolds are activated using atmospheric-pressure plasma jet (APPJ), allowing for covalent immobilization of transforming growth factor β1 (TGF), an important cytokine for the production and maintenance of cartilage (Wang, Rigueur & Lyons, 2014), onto the scaffold’s microfibers. It is hypothesized these biofunctionalized scaffolds will support differentiation of mesenchymal stromal cells (MSCs) into the chondrogenic lineage and subsequent neo-cartilage formation.
Methods and Materials
Poly-e-caprolactone MEW scaffolds were fabricated using a 3DDiscovery printer (regenHU), then activated using a computer-controlled APPJ device (Alavi et al., 2020). TGF was subsequently immobilized onto the MEW scaffolds using solution submersion (1µg/mL). Characterization of protein immobilization was performed using enzyme-linked immunosorbent assay (ELISA) and immunofluorescence detection. In vitro experiments were performed by seeding equine MSCs into the scaffolds and were cultured for 28 days. Neo-cartilage formation was quantified with dimethyl methylene blue/picogreen assays for glycosaminoglycan (GAG) production and confirmed with histological analysis.
Results
ELISA results confirmed covalent TGF concentration on the biofunctionalized scaffolds while immunofluorescently-labelled TGF was detected visually in scaffolds. The APPJ treatment caused increased hydrophilicity of the scaffolds, resulting in efficient cellular infiltration. In vitro analysis demonstrated that GAG production was significantly enhanced in both the immobilized TGF (+APPJ+TGF) and TGF (-APPJ+TGF) in medium groups, compared to the control groups without TGF supplementation (-APPJ+/-TGF). This finding was further validated by the heightened production of GAGs and collagen type II, observed in histological sections.
Conclusion
We have demonstrated that APPJ-facilitated covalent immobilization of TGF retains bioactivity and stimulates differentiation of MSCs into the chondrogenic lineage. Our results also demonstrate that the new constructs with immobilized TGF support neo-cartilage formation.