Speaker
Description
Introduction
Functional regeneration of musculoskeletal tissues requires engineered grafts that mimic the heterogenous and anisotropic structure and mechanics of the native tissue. Existing strategies fail to produce tissues that mimic this structural complexity, often leading to deficits in mechanical properties and repair failure in vivo. 3D bioprinting allows for the freeform patterning of cells and biomaterials at the microscale, potentially enabling the engineering of constructs that mimic the structural complexity of biological tissues. High cell density 3D embedded bioprinting has recently emerged as a versatile biofabrication tool for the generation of spatially organised tissues using high cell density hydrogel composite bioinks [1]. This technique enables the production of complex, free-form constructs using mechanically weak hydrogels. Here we hypothesise that the support bath used in such embedded bioprinting strategies can also be leveraged to direct the growth and spatial organisation of the secreted extracellular matrix (ECM). Our goal is to develop a versatile muscoskeletal bioprinting platform capable of fabricating anisotropic, mechanically functional tissues with preferential collagen alignment, leveraging the spatial cues and physical confinement provided by the embedded support bath.
Materials and methods
Goat bone marrow derived mesenchymal stem/stromal cells (MSCs) were either encapsulated within a partially crosslinked (60mM CaCl2-5min) oxidised alginate (OA) bioink at high cell densities (60x106 / 100x106 cells/mL), or bioprinted as a cell-only bioink. These bioinks were subsequently bioprinted within different concentration oxidised-methacrylated alginate (OMA) support baths using a mechanically driven syringe pump printhead, and ionically and UV crosslinked post-print and maintained in chondrogenic culture for 6 weeks. Various printing parameters such as needle size, print speed, support bath mechanical profile, and construct spatial configuration were systematically varied to in an attempt to produce cartilage and fibrocartilage tissue constructs with defined resolution and preferential alignment within the developing collagen fibres. Assessment was undertaken using brightfield microscopy, biochemical and histological evaluation, polarised light microscopy, immunofluorescence and RT-qPCR.
Results
High cell density bioinks (60/100x106 cells/ml-MSCs-OA), and cell only bioinks were bioprinted within an OMA support bath to a high degree of resolution (200-400 μm), whilst maintaining bioprint fidelity. Print parameters and the supporting bath mechanical profile could be tuned to control bioprint resolution (~200 μm), direct the phenotype of developed cartilaginous tissue, and importantly enhance the preferential alignment of developing collagen fibres (Fig. 1c) whilst maintaining cellular viability (97% viable) (Fib. 1b). MSCs were found to undergo chondrogenic differentiation, as confirmed using an assessment of sulphated-glycosaminoglycans (sGAGs) and collagen production both biochemically and histologically (Fig. 1c). Further assessment using immunofluorescence for specific collagen types highlighted differences in the resulting phenotype of engineered tissue.
Discussion
This body of work has showcased the ability to preferentially control the alignment of developing collagen fibres within cartilage constructs generated using embedded bioprinting, whilst maintaining a high print resolution and cell viability. Future work will focus on scaling up the biofabrication strategies to more native-like geometries.
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