Oxygen consumption of chondrocytes in agarose and collagen gels: A comparative analysis

Abstract

The growth of engineered cartilage tissue in vitro is often impaired by the problem of insufficient oxygen and nutrient supply to cells seeded in 3D constructs. Despite its central role in controlling most cell functions, the scaffolding material has generally been thought of only as a transport barrier and its potential active role in controlling oxygen uptake has never been addressed. In this work the role of cell–material interaction on oxygen metabolism in 3D in vitro cultures was surveyed. To this aim bovine chondrocytes, at a cell density of 400,000 and 4,000,000 cells/mL, respectively, were seeded in collagen type I and in agarose, while keeping all other culture conditions constant. A unidirectional oxygen gradient was induced in the culture through the application of a “sandwich” model and the oxygen concentration at the pericellular level was measured by phosphorescence quenching microscopy. Results show that the oxygen consumption rate is two-fold higher in agarose than in collagen, which indicates that the nature of the material strongly influences cell metabolic behaviour. Moreover, since different oxygen consumption rates are linked to different cell biosynthetic activity, our findings will prove beyond any doubt the active role played by materials in tissue regeneration.

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.