Speaker
Description
Introduction
Melt electrowriting (MEW) is an additive manufacturing technique with significant potential in cartilage tissue engineering. While osteoinductive coatings have been employed to functionalize MEW scaffolds, the efficacy of comparable chondro-inductive coatings remains unproven. Type II collagen and sGAGs are key cartilage ECM components that promote chondrogenesis when included in scaffolds [1,2]. However, conventional collagen extractions disrupt native fibril architecture, and without intact fibrils, sGAGs cannot localize in the interfibrillar spaces as in native tissue. Herein, insoluble type II collagen extraction was performed and incorporated into polydopamine coatings on melt electrowritten (MEW) scaffolds. The capacity of these coatings to enhance chondrogenesis of bone marrow derived mesenchymal stem/stromal cells (MSCs) was then assessed.
Materials & Methods
Insoluble and pepsin-soluble type II collagen were isolated from porcine cartilage, and acid-soluble type I collagen from Achilles tendon. Bulk MEW scaffolds (0.6 mm height, 40 layers) with pore sizes of 200–800 µm were printed, and 6 mm discs were punched and treated with 2 M NaOH under vacuum for 30 min to improve hydrophilicity. Scaffolds were coated in a 4 mg/mL dopamine solution (0.02 M Tris, pH 8.5), initiating polymerization, while collagen was pre-activated with EDC/NHS and added to the reaction (final: 2 mg/mL dopamine, 1.5 mg/mL collagen), which proceeded for 24 h at 4 °C. Collagen loading was assessed via hydroxyproline assay and SEM. Scaffolds were seeded with 8×10⁵ or 4×10⁵ MSCs (1.5 mm and 0.5 mm high, respectively) and cultured in chondrogenic media for 28 days.
Results
SEM revealed that collagen formed composite coatings on MEW scaffolds, predominantly as ~60 µm fibrillar networks and occasional larger D-banded fibrils (~100 µm, 65 nm periodicity). Collagen-functionalised scaffolds also exhibited abundant polydopamine nanospheres, which were sparse in polydopamine-only controls. Biochemical assays showed significantly increased sGAG deposition in collagen-functionalised scaffolds at day 3 (400 µm) and day 28 (200 µm) compared to controls. Histology confirmed widespread sGAG and collagen distribution, with intense staining around MEW fibres. Polarised-light microscopy (PLM) further revealed collagen fibril alignment along the MEW fibres in 200 µm scaffolds.
Discussion
In contrast to the population of fibrils observed in the insoluble collagen-PD coated scaffolds, pepsin-solubilised collagen-PD coatings were more amorphous nature with no clear fibrils observed, suggesting more effective biomimicry following functionalization with insoluble collagen. Polydopamine nanospheres have recently gained interest for their anti-inflammatory properties [3], and the collagen-PD film may have provided an improved pathway for their adhesion to the scaffold through their entanglement in the film compared to using polydopamine alone. The enhanced sGAG deposition in the 200 µm pore size scaffolds may be due to the increased collagen coating loading on these higher surface area scaffolds.
Conclusions
This modified PD coating method was shown to be an effective method for enhancing chondrogenesis in MEW scaffolds.
This work is supported by the European Research Council (MEMS – 101137852).
[1] D. Bosnakovski et al. Biotechnol. Bioeng. 2006, 93, 1152.
[2] J. Y. Park et al. Biofabrication 2014, 6, DOI 10.1088/1758-5082/6/3/035004.
[3] X. Bao et al. ACS Nano 2018, 12, 8882.
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