Enhancing and stabilizing oxygen supply in tissue engineered devices to promote functionality and longevity

When designing cell encapsulation devices for tissue engineering, a number of important parameters need to be considered such as immunoprotection, biodegradability, biocompatibility, mechanical strength, cell-scaffold interactions and sufficient transport of nutrients. Generally, cell encapsulation provides cells a 3D environment to mimic physiological conditions and improve cell signaling, proliferation, and tissue organization as compared to monolayer culture. However, encapsulation devices often encounter poor mass transport due to the absence of vasculature. Lack of oxygen supply to cells limits energy metabolism resulting in hypoxic conditions that lead to loss of cellular function and ultimately cell death. Because of their relatively large sizes (\u3e 1 mm in diameter), tissue engineering devices are often oxygen transport limited, reducing the maximum achievable size for clinical utility. To enhance oxygen transport we utilized perfluorocarbon (PFC) oxygen vectors, specifically perfluorooctyl bromide (PFOB) immobilized in an alginate hydrogel. PFCs have a high capacity to dissolve oxygen and we evaluated this new technology using two types of cells in terms of their oxygen requirements – immortalized human hepatocytes (HepG2 cell line) and primary bovine chondrocytes. Hepatocytes are metabolically active cells with relatively high oxygen uptake rates (OURs), whereas cartilage is avascular tissue that favors low oxygen tension. Through both experimental and theoretical approaches, we verified our hypothesis that immobilization of PFCs within hydrogel scaffolds improves effective oxygen diffusivity, resulting in a more functional homogenous engineered tissue. For hepatocytes, OURs were enhanced by 8% and 15% under both 20% and 5% O2 boundary conditions, respectively and metabolic activity was increased by up to 162%. Significant increases in both amount and homogeneous distribution of chondrocyte phenotypic markers glycosaminoglycan and type II collagen were measured by biochemical assays, qRT-PCR and histological examination. Immobilization of PFCs can be used to improve device function for both low and high oxygen demanding cells. Provision of homogeneous oxygen tension is beneficial for tissue regeneration with uniform quality as well as stem cell culture where oxygen is a critical factor for differentiation. We additionally investigated the use of a novel, synthetic, biodegradable PLLA-PEO-PLLA triblock copolymer of which the elastic modulus can be tuned by varying the chain length of the PLLA domain. Laponite is a synthetic layered silicate that serves both as a mechanical filler and pH stabilizer through cationic exchange. By creating a PLLA-PEO-PLLA/laponite composite encapsulation material, we demonstrated successful stabilization of pH induced by lactate release from hydrolysis of PLLA, resulting in extended cell viability. Because the elastic modulus is greater than 10 kPa, this material has potential as a scaffold for hard tissues such as articular chondrocytes