Abstract
For cartilage repair in vivo or evaluation of new therapeutic approaches in vitro, the generation of functional cartilage tissue is of crucial importance and can only be achieved if the phenotype of the chondrocytes is preserved. Three-dimensional (3D) cell culture is broadly used for this purpose. However, adapting culture parameters like the oxygen tension or the osmolarity to their physiological values is often omitted. Indeed, articular cartilage is an avascular tissue subjected to reduced oxygen tension and presenting and increased osmolarity compared with most other tissues. In this study, we aimed at evaluating the effect of a physiological oxygen tension (3% instead of 21%) and physiological osmolarity (430 vs. 330 mOsm in nonadjusted DMEM) and the combination of both on the cell proliferation, matrix production, and the phenotype of porcine chondrocytes in a scaffold-free 3D culture system. We observed that a physiological osmolarity had no effect on cell proliferation and matrix production but positively influences the chondrocyte phenotype. A physiological oxygen level prevented cell proliferation but resulted in an increased matrix content/million cells and had a positive influence on the chondrocyte phenotype as well. The strongest benefit was reached with the combination of both physiological osmolarity and oxygen levels; with these conditions, type I collagen expression became undetectable. In addition, at 3% O2 the chondrocytes-matrix constructs were found to more closely resemble native cartilage regarding the matrix-to-cell ratio. In conclusion, this study clearly demonstrates the benefit of using physiological oxygen tension and osmolarity in cartilage tissue engineering with the combination of both showing the strongest benefit on the chondrocyte phenotype.
Highlights
To evaluate new therapeutic compounds in vitro and to investigate cellular mechanisms or for tissue engineering, it is of crucial importance to preserve the functional phenotype of the used cells
The goals of this study were (1) to evaluate if the benefit of a physiological oxygen tension and a physiological osmolarity are additive on both the phenotype maintenance and extracellular matrix (ECM) deposition; (2) to investigate if a potential increase of hypertrophy markers owing to the higher osmolarity could be compensated by a low oxygen tension; and (3) to compare the matrixto-cell ratio and the histological appearance of the obtained 3D constructs to those of native cartilage and determine which culture conditions lead to constructs resembling cartilage the most
We investigated the impact of physiological oxygen and osmolarity levels on primary chondrocytes in 3D culture
Summary
To evaluate new therapeutic compounds in vitro and to investigate cellular mechanisms or for tissue engineering, it is of crucial importance to preserve the functional phenotype of the used cells. It can be achieved with culture conditions that are mimicking the physiological environment. In 3D culture, chondrocytes display a similar morphology as in cartilage and produce a cartilage-like extracellular matrix (ECM), rich in type II collagen[2,3] whereas in monolayer culture they quickly dedifferentiate into fibroblast and a switch from type II to type I collagen expression occurs.[4] even in 3D culture there is some type I collagen produced and the phenotype is not totally preserved especially when serum is being used.[5]
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