Dr Anyu Zhang

Many biological structures such as nerves, blood and lymphatic vessels, and muscle fibres exhibit longitudinal geometries with distinct cell types extending along both the length and width of internal linear axes. Modelling these three-dimensional structures in vitro is challenging: the best-defined stem-cell differentiation systems are monolayer cultures or organoids using pluripotent stem cells. Pluripotent stem cells can differentiate into functionally mature cells depending on the signals received, holding great promise for regenerative medicine. However, the integration of in vitro differentiated cell types into diseased tissue remains a challenge. Engineered scaffolds can bridge this gap if the appropriate signalling systems are incorporated into the scaffold. Here, we have taken a biomimicry approach to generate longitudinal structures in vitro. In this approach, mouse embryonic stem cells are directed to differentiate to specific cell types on the surface of polycaprolactone (PCL) fibres treated by plasma-immersion ion implantation and to which with lineage-specifying molecules have been covalently immobilised. We demonstrate the simplicity and utility of our method for efficiently generating high yields of the following cell types from these pluripotent stem cells: neurons, vascular endothelial cells, osteoclasts, adipocytes, and cells of the erythroid, myeloid, and lymphoid lineages. Strategically arranged plasma-treated scaffolds with differentiated cell types could ultimately serve as a means for the repair or treatment of diseased or damaged tissue.

Atmospheric pressure plasma jets (APPJs) are advancing as a versatile dry technology for creating biofunctional structures. Recently, reagent-free, single-step covalent immobilization of bioactive molecules onto surfaces was demonstrated. Despite this, the mechanisms governing the covalent attachment process remain obscure. Here, we studied morphological changes, concentrations of radicals, and the formation of reactive species on APPJ-treated polymers to shed light on the underlying mechanisms of covalent attachment. The APPJ-treated polyethylene surfaces, prepared either in air or with controlled ambient gas composition, were analyzed using Fourier transform infrared, X-ray photoelectron, electron spin resonance, and fluorescence spectroscopies, as well as atomic force microscopy. It was demonstrated that only non-radical reactive oxygen species (ROS) could explain the attachment. This attachment was also demonstrated on silicone (PDMS), broadening the range of possible applications. Finally, to identify reaction pathways, fluorinated carbon brushes were used, each presenting a specific functional group. Attachment data indicated that molecules with amine or thiol groups can be covalently bound to treated surfaces. As a result, reactions involving the ROS, hydroxyl, carbonyl, carboxyl, and peroxides are potential pathways. These findings provide a means of optimizing treatment features such as binding site density in future studies and applications, thereby expanding the capabilities of APPJ treatment within 3D bioprinters.

Current biomaterial-based strategies explored to treat articular cartilage defects have failed to provide adequate physico-chemical cues in order to guide functional tissue regeneration. Here, it is hypothesized that atmospheric-pressure plasma (APPJ) treatment and melt electrowriting (MEW) will produce microfiber support structures with covalently-immobilized transforming growth factor beta-1 (TGFß1) that can stimulate the generation of functional cartilage tissue. The effect of APPJ operational speeds to activate MEW polycaprolactone meshes for immobilization of TGFß1 is first investigated and chondrogenic differentiation and neo-cartilage production are assessed in vitro. All APPJ speeds test enhanced hydrophilicity of the meshes, with the slow treatment speed having significantly less C-C/C-H and more COOH than the untreated meshes. APPJ treatment increases TGFß1 loading efficiency. Additionally, in vitro experiments highlight that APPJ-based TGFß1 attachment to the scaffolds is more advantageous than direct supplementation within the medium. After 28 days of culture, the group with immobilized TGFß1 has significantly increased compressive modulus (more than threefold) and higher glycosaminoglycan production (more than fivefold) than when TGFß1 is supplied through the medium. These results demonstrate that APPJ activation allows reagent-free, covalent immobilization of TGFß1 on microfiber meshes and, importantly, that the biofunctionalized meshes can stimulate neo-cartilage matrix formation. This opens new perspectives for guided tissue regeneration.