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Description
Lesions in the menisci are frequently related to sports injuries and dramatically increase the risk of developing osteoarthritis. It is estimated that 1.5 million meniscal repairs are performed annually in the United States and Europe, representing one of the most common clinical procedures performed by orthopaedic surgeons. In the field of tissue engineering there is increased interest in the use of cellular aggregates, microtissues and organoids as biological building-blocks for the biofabrication of scaled-up grafts. Such microtissues can be incorporated within 3D printed polymeric scaffolds to help direct their fusion and (re)modelling and to enhance the biomechanical properties of the resulting construct, making it a promising strategy for meniscus tissue engineering. Therefore, the aim of this study was to deposit fibrocartilaginous microtissues generated using meniscus progenitor cells (MPCs) isolated from the inner and outer region of the meniscus within melt-electrowritten (MEW) scaffolds to biofabricate zonally defined meniscus grafts. We initially explored how the aspect ratio of MEW scaffolds can influence microtissue fusion and remodeling. For this, polycaprolactone (PCL) scaffolds with 0.8×0.8 mm and 0.4×1.6 mm pore sizes were fabricated using a custom-made MEW printer. Next, inner and outer MPC derived microtissues were deposited within the MEW scaffolds. After 24h, we observed that the microtissues successfully fused within the MEW scaffolds, with tissue growth modulated by the pore architecture. Scanning electron microscopic (SEM) analyses revealed the development of a dense extracellular matrix with a preferential fibre orientation when microtissues were seeded in the anisotropic (0.4:1.6 pore aspect ratio) MEW scaffolds. Histological analyses revealed intense staining for sGAG deposition when inner MPC derived microtissues were seeded into the 0.8:0.8 MEW scaffolds. Greater collagen deposition was observed when either inner or outer MPC derived microtissues were seeded in the anisotropic 0.4:1.6 MEW scaffold. All groups were negative for calcium deposition, suggesting the development of a phenotypically stable fibrocartilaginous tissue. Increased type I collagen gene expression was observed in 1.6:0.4 scaffold seeded with outer MPC derived microtissues, while increased aggrecan expression was observed in scaffolds seeded with the inner MPC derived microtissues. Next, outer MPC derived microtissues seeded in the MEW scaffolds were cultured in vitro within a caprine meniscus explant model to evaluate the integration of the constructs with the native meniscus tissue. We observed by histological analyses (hematoxylin and eosin and safranin-O staining) that the MEW-microtissue based constructs robustly integrated with the native tissue. Finally, micro extrusion-based bioprinting was used to deposit outer MPC microtissues into a 1.6:0.4 MEW scaffold, leading to robust tissue formation with substantial sGAG and collagen deposition, as confirmed by histological and biochemical analyses. Additionally, the constructs supported a uniform type I collagen deposition, resembling the outer region of the native meniscus (Fig. 1). In conclusion, this work demonstrates the successful biofabrication of zonally defined meniscus grafts using micro extrusion-based bioprinting of fibrocartilage microtissues into MEW scaffolds.
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