Development of an Integrated Melt-Electrowriting and Cell Jetting Biofabrication Platform to Generate 3d Cellular Gradients and Regional Mechanical Properties for Cartilaginous Tissue Regeneration

The meniscus plays a vital role in shock absorption and stabilisation in the knee joint. Injuries to the meniscus can lead to osteoarthritis if left untreated. Current surgical therapies provide minimal tissue regeneration and so have poor long-term success rates, thus there is a distinct clinical need for novel tissue engineered approaches. Restoring the structure-function relationship of the native meniscus is critical, but current tissue engineering approaches often fail to consider the spatial organisation of cells, or the different biomechanical requirements of the tissue. Thus, the objective of this study was to apply a sophisticated biofabrication pipeline, seamlessly integrating melt-electrowriting (MEW) and microvalve bioprinting techniques to craft meniscal tissue analogues containing cellular and mechanical gradients mirroring native tissue. MEW process parameters were optimised using a design-of-experiments approach to facilitate the deposition of layers of PCL microfibres with a range of defined geometries, creating microchambers that could reinforce a soft hydrogel matrix. The effect of process parameters on fibre geometry was assessed through SEM measurements. Microvalve jetting of alginate into MEW microchambers was the optimised to facilitate precise droplet patterning, enabling the encapsulation of human mesenchymal stem cells (MSCs) with high viability. Patterning of cellular gradients was confirmed through fluorescent cell labelling and mechanical properties of the hybrid scaffold were confirmed through mechanical testing. MSC-seeded hybrid scaffolds with varying fibre spacings were cultured in chondrogenic media with or without compressive dynamic mechanical stimulation and the resulting differentiation and matrix formation assessed using qPCR, histology, immunohistochemistry and quantitative biochemical assays. Aligned polymeric fibre walls were generated using MEW, which emulated geometries of collagen bundles in native tissues, offering tunable fibre diameters and microchamber sizes. Fine-tuning fibre diameter and spacing resulted in scaffolds approaching physiologically relevant stiffness, potentially allowing us to tailor the mechanosensory environment for encapsulated cells. Multi-nozzle microvalve jetting attained intricate patterns of bioink droplets within polymeric microchambers, showcasing high cell viability and enabling precise patterning of cellular gradients. After 28 days in culture, the reinforced scaffolds closely emulated the compressive stiffness of native cartilaginous tissue, featuring aligned collagen deposition along the polymeric fibres and glycosaminoglycan (GAG) deposition within the hydrogel matrix. These hybrid constructs exhibit significant potential for engineering mechanically robust, cartilaginous tissue analogues with defined regional properties, promising advancements in meniscus tissue regeneration and preclinical disease modelling.