A Biomimetic Joint Model with Heterogenous Oxygen Environments

Crosstalk between subchondral osteoblasts and chondrocytes played significant role in OA pathogenesis. However, physiological coculture of osteoblasts and chondrocytes is limited by <i>in vitro</i> generation of differential oxygen concentrations across the cartilage-bone tissue interface. Chondrocytes in the cartilage require a low (1 %) oxygen environment while osteoblasts in bone tissues require normoxic (&gt;9%) oxygen environment to maintain their tissue-specific functions. Current approaches to control oxygen gradients in microfluidic devices rely on diffusion gradients generated across a porous matrix using active gas networks, which is limited to monocultures and cannot mimic tissue interfaces with distinct oxygen tensions. Jammed packed microgels (JPMs) are a novel class of hydrogels that are formed by swelling aqueous solution in microgels which interact with each other via weak physical forces, resulting in their shear-yielding properties. However, the use of this class of hydrogels in tissue culture remains limited.<br/>In this work, we demonstrated the use of polyacrylate JPMs to tune and mimic a heterogenous oxygen environment that mimics the tissue interface of the articular joint. We demonstrated the oxygen content tunability of the JPMs by functionalising the JPMs with two reducing agents: Oxyrase (oxygen reducing enzyme) and sodium sulphite, two common reducing agents used in anaerobic cultures. By optimizing the concentrations of reducing agents, we found that 0.1U Oxyrase (commonly used in anaerobic microbial cultures) could maintain a low oxygen level of 1.35 % for up to 48 hrs. The biocompatibilities of the JPMs for primary chondrocytes cultured in both normoxic and hypoxic JPMs demonstrated the JPMs, along with their functionalised reducing agents can support the viabilities of cell cultures. By controlling the type and concentration of the reducing agents, we demonstrated a host of JPMs with different oxygen contents, suitable for mimicking different physiological oxygen tensions. We further established patterning of JPMs with different oxygen content in a microfluidic device, mimicking a bone-cartilage oxygen tension. In situ oxygen sensing within the microfluidic device showed that we can establish and maintain physiological heterogenous oxygen level of cartilage (2% oxygen) and bone (10% oxygen) for 2 days within a cell incubator. On-chip hypoxic culture of primary human chondrocytes exhibited hypoxic response by upregulating hypoxic inducing factor alpha (HIFa), indicative of the hypoxic environment on-chip. Finally, we established co-cultures of allogenic pairs of patient-derived chondrocytes with osteoblasts under the heterogeneous oxygen environments. We showed that co-cultures under heterogenous oxygen physicochemical environment significantly enhanced chondrogenic phenotypes (i.e. increased aggrecan and decreased type I collagen expressions) as compared to coculture maintained under homogeneous oxygen levels without affecting the phenotypes of the osteoblasts within the co-cultures.<br/>This work demonstrated the first instance of osteoblast-chondrocyte microfluidic co-cultures with patterned oxygen levels for up to 4 days. Chondrocytes cocultured under low oxygen environment with osteoblast exhibited enhanced chondrogenic expression compared to monocultures and co-cultures without oxygen control. This platform can potentially be utilized for further osteoarthritis (OA) drug screening applications.