Guided cartilage formation: covalent growth factor immobilization on melt electrowritten microfiber scaffolds

Jun 28, 2022, 12:20 PM
10m
Room: S2

Room: S2

Speaker

Ainsworth, Madison J. (University Medical Center Utrecht )

Description

Introduction
Current tissue engineering treatment strategies 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 structural properties of the native cartilage extracellular matrix[1]. These scaffolds are activated using atmospheric-pressure plasma jet (APPJ), allowing for covalent immobilization of transforming growth factor beta 1 (TGF), an important cytokine for the production and maintenance of cartilage[2], 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 cartilage-like matrix deposition.

Methodology
Poly-e-caprolactone MEW scaffolds were fabricated using a 3DDiscovery printer (REGENHU), then activated using a computer-controlled APPJ device[3]. Wettability and x-ray photoelectron spectroscopy were used to assess surface chemistry changes. TGF was subsequently immobilized onto the MEW scaffolds by submersion in solution (1 µg/mL). Characterization of protein immobilization was performed using enzyme-linked immunosorbent assay (ELISA) and immunofluorescence detection. In silico modelling was performed to investigate the potential benefit of immobilizing TGF rather than supplying the TGF in the medium. In vitro experiments were performed by seeding equine-derived MSCs into the scaffolds and then culturing for 28 days. The groups investigated included APPJ-treated constructs with (+APPJ +TGF) and without (+APPJ -TGF) immobilized TGF, as well as untreated constructs with (-APPJ +TGF) and without (-APPJ -TGF) TGF supplied through the culture medium. Cartilage-like formation was quantified with dimethyl methylene blue/picogreen assays for glycosaminoglycan (GAG) production and confirmed with histological analysis, including safranin-O and collagen type I & II immunohistochemistry. Matrix deposition was additionally analyzed using compressive testing.

Results
ELISA results confirmed covalent TGF concentration on the APPJ-functionalized scaffolds while immunofluorescently-labelled TGF was detected visually in microfiber scaffolds (following 0.1% Tween20 washing). 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. In addition, in vitro and in silico analysis revealed that immobilized TGF on the scaffolds was more advantageous than TGF supplied directly through the medium. Following the 28-day culture period, the immobilized TGF (+APPJ +TGF) construct group exhibited increased compressive modulus (>3 fold) and GAG production (>5 fold) when compared to the TGF in medium (-APPJ +TGF) construct group.

Conclusions
APPJ-surface treatment facilitated covalent immobilization of TGF onto MEW scaffolds. Immobilized TGF retained bioactivity and promoted the differentiation of MSCs into the chondrogenic lineage. Our results also demonstrate that the new constructs with immobilized TGF support cartilage-like-tissue formation. These findings drive new perspectives for reagent-free, growth factor-functionalized constructs with controllable, high-resolution geometries for guided tissue regeneration.

References
[1] Castilho et al. 2019. Acta Biomaterialia.
[2] Wang, Rigueur & Lyons. 2014. Birth Defects Res C Embryo Today.
[3] Alavi et al. 2020. ACS Applied Materials & Interfaces.

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