[Frontiers in Bioscience E3, 1365-1374, June 1, 2011]

Physiological normoxia and chondrogenic potential of chondrocytes

Alasdair Kay1, James Richardson2, Nicholas R Forsyth1

1Guy Hilton Research Centre, Institute of Science and Technology in Medicine, Keele University, Stoke-On-Trent, ST4 7QB, UK, 2Keele University, Robert Jones and Agnes Hunt Orthopaedic Hospital, Oswestry, Shropshire, SY10 7AG, UK

TABLE OF CONTENTS

1. Abstract
2. Introduction
3. Materials and methods
3.1. Chondrocytes isolation and culture methodology
3.2. Image analysis
3.3. In vitro chondrogenic differentiation
3.4. Histochemistry.
3.5. Cartilage-linked gene expression
3.6. Immunohistochemistry
3.7. Statistical Analysis
4. Results
4.1. Influence of oxygen on chondrocyte cell size
4.2. Oxygen effects on chondrocyte growth rate
4.3. Histological determination of chondrogenic rate
4.4. Gene expression during chondrogenesis
4.5. Detection of type II collagen
5. Discussion
6. Acknowledgments
7. References

1. ABSTRACT

Cartilage is poorly vascularised with a limited capacity for repair following damage. The poor vascularisation results in cartilage tissue having a low normoxic value. This study examined and compared the effects of physiological cartilage normoxia (2% O2), hypoxia (0.2% O2), and hyperoxia (21% O2) on human articular chondrocytes (hAC) during similar time courses to those prior to transplant in cell therapy procedures. hAC were isolated and maintained at 0.2% O2, 2% O2, or 21% O2. Population doublings (PDs), cell surface area, chondrogenic differentiation potential, RT-PCR, quantitative RT-PCR and immunohistochemistry (Collagen Type II) were used to confirm chondrogenic differentiation of micromass pellets in different O2. Isolation and maintenance of hAC at less than 2% O2 resulted in significant alterations in surface area (smaller), rate of proliferation (reduced), and chondrogenic differentiation potential (enhanced). Chondrogenic gene expression appeared largely insensitive to O2 concentration. A relationship was apparent between collagen type II protein presence and O2 concentration. Oxygen concentrations of 2% O2 or less promoted retention of a dedifferentiated hAC phenotype and enhanced stability of hAC chondrogenesis.