Oxygen consumption of chondrocytes in agarose and collagen gels: A comparative analysis
Introduction
In recent years the considerable progress made in cartilage tissue engineering in vitro has triggered hopes of overcoming the prevailing clinical limitations of traditional methods to cure osteoarthritic diseases. However, the generation of fully functional cartilage tissue in vitro is still far to come. 3D cartilage construct maintenance, survival and growth are based upon a delicate balance between chondrocyte activities and metabolism, nutrient transport and scaffold properties. The interplay between scaffold properties and metabolic demand modulates the pericellular microenvironment, which controls cellular proliferative and biosynthetic activities; the metabolic demand, in turn, modifies the local cell microenvironment. This dynamic equilibrium may vary in time and space through the cellular constructs, and failure to attain it may lead to a non-functional tissue. It has been reported that the growth of engineered cartilage tissues is impaired by an insufficient supply of nutrients throughout the constructs [1], [2]. Indeed, a non-homogeneous oxygen and nutrient distribution have often led to highly heterogeneous cell behaviour, with elevated proliferation and biosynthetic activity areas at the periphery of the constructs, and wide necrotic areas in the inner regions [3], [4]. Furthermore, the occurrence or extension of necrotic areas increases with the scaffold thickness [5], [6].
While the mechanism of oxygen and nutrient transport within 3D cellular constructs and its dependence on material structural and functional properties is well established, the mechanisms and the parameters that modulate the metabolic demand of chondrocytes seeded in 3D polymeric scaffolds are not fully understood. In particular, the influence of cell–material interaction on cellular metabolic processes is an important aspect that has not yet been completely clarified.
Current modelling approaches to describe chondrocyte oxygen consumption within engineered 3D cartilage constructs are primarily based on empirical or semi-empirical assumptions and do not provide a comprehensive description of the parameters involved in metabolic oxygen demand [7]. Most models follow a large-scale approach where spatial oxygen profiles within the constructs are derived from global measurements of oxygen uptake and overall cell density [8], [9], thus not providing information on pericellular oxygen consumption. On the other hand, the few models based on direct measurements of oxygen distribution within cellular constructs [10], [11] use a Michaelis–Menten-like equation to describe nutrient consumption that only accounts for oxygen dependence on cellular density. Experimental evidence suggests that oxygen consumption is a regulator for the biosynthetic process [2], since oxygen concentrations and consumption rates have been reported to control GAGs and type II collagen synthesis in 3D in vitro cultures [12]. Moreover, it has been suggested that cell–material interaction strongly influences cell biosynthesis [13], [14] and, therefore, it would also be expected to influence oxygen consumption. The aim of this work is to address the role of cell–material interaction in conditioning chondrocyte metabolism in 3D in vitro cultures, focusing upon the potential impact of such eventual findings on tissue engineering processes.
Section snippets
Cells
Articular bovine chondrocytes were seeded in 3D scaffolds at two different cell densities of 400,000 cells/mL and 4,000,000 cells/mL. Full depth slices of cartilage were removed from metacarpalphalangeal joints of 18-month-old steers kindly provided by a local slaughterhouse. The cartilage slices were diced finely and incubated at 37 °C for 1 h in Dulbecco's minimal essential medium, DMEM (Sigma Aldrich), supplemented with 20% (v/v) of inactivated, foetal calf serum, FCS (Sigma Aldrich), and 700
Results
In vitro oxygen metabolic consumption of primary chondrocytes was investigated by measuring oxygen partial pressure (pO2) in sandwich cultures, at two different cell densities (400,000 cells/mL and 4,000,000 cells/mL), in type VII agarose (3% w/w) and type I collagen (1.2 mg/mL) gels. Oxygen tensions were measured at 1 mm steps along the width of sandwiches at time intervals of 0, 1 h, 3 h, and 24 h. Because of the axial symmetry of the sandwich system the data reported on the x-axis of Fig. 2, Fig. 3,
Discussion
The distribution of oxygen and other metabolites within thick (>1 mm) 3D cellular constructs is determined by a complex interplay between molecular transport process and cellular uptake [4], [8], [18]. This dynamic equilibrium plays a fundamental role in the process of tissue regeneration, because it influences cell survival, proliferation, metabolism and biosynthesis [19], [20]. Scaffold material plays a central role in the establishment of this critical equilibrium because it influences both
Conclusion
In this work oxygen spatial and temporal profiles within agarose– and collagen–chondrocyte constructs were evaluated by using a non-invasive microscopic technique. Differences in oxygen profiles were interpreted as the result of different cellular OCRs. Chondrocytes seeded in agarose gels exhibited an OCR two-fold higher than those seeded in collagen gels, indicating an active role of the material in controlling cellular metabolism. Furthermore, the modulation of chondrocytes' OCR in agarose
Acknowledgements
The authors thank Francesco Travascio for his help in the PQM experimental set up and Paolo Carboni for technical support.
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