Speaker
Description
Engineering functional articular cartilage (AC) remains a challenging goal in tissue engineering. Since the function of AC is derived from its depth-dependent organization, the field has typically focused on developing multilayered scaffolds that mimic specific zonal aspects of the native tissue. Scaffolds have succeeded in recapitulating some aspects of native AC, however, they have generally failed to regenerate its intricate architecture, including the arcade-like collagen network [1]. Computational modelling has shown that achieving this collagen organisation is the most important factor in determining the functional success of engineered AC [2]. This motivates the need for innovative strategies to direct collagen alignment. To address this challenge, this study leverages embedded 3D bioprinting to provide spatially controlled physical cues to AC progenitor cells (ACPs) facilitating their self-organization into a structurally organized tissue with an arcade-like collagen architecture.
ACPs were isolated through differential adhesion to fibronectin. Passage 4 ACPs were centrifuged and loaded into a syringe with a 25G needle without a supporting ink (cell-only bioprinting). A methacrylated xanthan gum (XGMA) support bath was prepared by dissolving xanthan gum (0.5% w/v) in deionized water, adding glycidyl methacrylate (7.41% v/v), and stirring overnight at 60°C. The solution was dialyzed (MWCO 6–8kDa), freeze-dried, and stored at -20°C. For bioprinting, 1% w/v XGMA was used, and filaments of 270, 400, and 700µm in diameter were bioprinted. Post-bioprinting, the bath was crosslinked with UV light (Fig. 1A). ACPs were bioprinted into a single-layered sheet in the XY plane with horizontal and vertical filaments to achieve a biomimetic AC collagen organization. Afterwards a two-layered graft was bioprinted with horizontal filaments in the XY plane overlaying vertical filaments in the Z-axis. Following 4 weeks of culture chondrogenesis was assessed through histology, immunohistochemistry, and biochemical assays.
All bioprinted filaments demonstrated robust chondrogenesis as evident by the positive staining for sulphated glycosaminoglycan (sGAG) and collagen. With polarized light microscopy (PLM) it was evident that the thinner bioprinted filament (270µm) supported superior collagen alignment throughout the depth of the tissue (Fig. 1B). ACPs in the bioprinted sheet also stained positive for sGAG and collagen deposition (Fig. 1C). PLM imaging revealed a horizontal collagen alignment in the superficial zone and a vertical collagen alignment in the middle/deep zones (Fig. 1C). Similarly, in the two layered graft a vertical collagen fibre alignment was observed in the vertically bioprinted filaments, while a horizontal alignment was observed in the horizontal filaments (Fig. 1D).
The findings demonstrate that external XGMA boundaries effectively guide neotissue alignment deposited by bioprinted ACPs. Thinner filaments promoted greater collagen alignment throughout the tissue. Spatial confinement of bioprinted vertical and horizontal filaments enabled the engineering of grafts recapitulating aspects of the arcade-like collagen organization of native AC. A limitation of this study is the presence of the bath in the final graft. Hence future work will investigate the use of degradable support baths. In conclusion, this approach emphasizes the potential of 3D bioprinting for replicating the collagen architecture of AC, paving the way to engineering truly functional grafts.
References
[1]doi.org/10.1089/ten.TEB.2008.0563
[2]doi.org/10.1007/s10237-012-0380-0
42705208488
