Engineering large cartilage tissues using dynamic bioreactor culture at defined oxygen conditions

Abstract

Mesenchymal stem cells maintained in appropriate culture conditions are capable of producing robust cartilage tissue. However, gradients in nutrient availability that arise during three-dimensional culture can result in the development of spatially inhomogeneous cartilage tissues with core regions devoid of matrix. Previous attempts at developing dynamic culture systems to overcome these limitations have reported suppression of mesenchymal stem cell chondrogenesis compared to static conditions. We hypothesize that by modulating oxygen availability during bioreactor culture, it is possible to engineer cartilage tissues of scale. The objective of this study was to determine whether dynamic bioreactor culture, at defined oxygen conditions, could facilitate the development of large, spatially homogeneous cartilage tissues using mesenchymal stem cell laden hydrogels. A dynamic culture regime was directly compared to static conditions for its capacity to support chondrogenesis of mesenchymal stem cells in both small and large alginate hydrogels. The influence of external oxygen tension on the response to the dynamic culture conditions was explored by performing the experiment at 20% O2 and 3% O2. At 20% O2, dynamic culture significantly suppressed chondrogenesis in engineered tissues of all sizes. In contrast, at 3% O2 dynamic culture significantly enhanced the distribution and amount of cartilage matrix components (sulphated glycosaminoglycan and collagen II) in larger constructs compared to static conditions. Taken together, these results demonstrate that dynamic culture regimes that provide adequate nutrient availability and a low oxygen environment can be employed to engineer large homogeneous cartilage tissues. Such culture systems could facilitate the scaling up of cartilage tissue engineering strategies towards clinically relevant dimensions.

Biofabrication of spatially organised tissues by directing the growth of cellular spheroids within 3D printed polymeric microchambers

“…The bioprinted constructs were then transferred to chondrogenic media and cultured in either static or dynamic conditions for 10 weeks using a custom developed bioreactor (Fig 4 a) 21 .…”

“…The smaller scale constructs developed in figures 1 were cultured in 24 well plates, with 2mls of CDM media added per construct (3% pO2). All large scale osteochondral constructs (figure 3 onwards), were cultured in a custom made bioreactor system which has been described previously 21 . The constructs were continuously translated at a speed of 0.05 mm/s and an amplitude of 6 mm for the entire culture period.…”

“…Following UV light mediated radical cross-linking of the GelMA component, the fugitive pluronic ink could be sacrificed in cold (4°C) culture media. This resulted in the formation of a network of bioink free microchannels which we have previously shown can support nutrient diffusion in larger scale tissues during bioreactor culture21 . Finally, in the last step of the biofabrication process, the articular cartilage region was developed by inkjet printing a MSC:chondrocyte co-culture into the expanded microchamber system as described above(Fig 3 d & Video 1).…”

Modeling, simulations, and optimization of smooth muscle cell tissue engineering for the production of vascular grafts

“…Under static conditions, due to the instantaneous permeation of the culture medium in the pore channels so that the nutrient concentration in the pores reaches steady state, the diffusion process still governs the transport of nutrients, as diffusion is slow and makes difficult for cells to survive if they are a few hundred micrometers away from the surface in contact with the culture medium (Rouwkema & Khademhosseini, ; Skrzypek et al, ; Stamatialis, ). Dynamic bioreactors use convection and forced permeation through porous scaffolds to facilitate and accelerate the transport of nutrients (experimental studies by Daly, Sathy, & Kelly, ; Nguyen, Ko, Moriarty, Etheridge, & Fisher, ), in which case, full transport models including the convection and permeation terms, as well as diffusion, are required and are incorporated in this study.…”

“…the transport of nutrients (experimental studies by Daly, Sathy, & Kelly, 2018;Nguyen, Ko, Moriarty, Etheridge, & Fisher, 2016), in which case, full transport models including the convection and permeation terms, as well as diffusion, are required and are incorporated in this study.…”