Effects of individual control of pH and hypoxia in chondrocyte culture

Effects of oxygen tension (pO2) and pH on gene and protein expression and metabolic activity of human chondrocytes were independently assessed. Chondrocytes were cultured under a range of pH (6.4–7.4) and different pO2 (5 and 20%) during 5 days in a bioreactor. Effects on gene expression, DNA content, protein expression, and metabolic activity were determined. Linear regression analysis showed that gene expression of type I collagen (COL1), SOX9, and VEGF is significantly lower at acidic pH, while expression of aggrecan, type II collagen, and HIF1A is pH‐independent. Higher protein levels of VEGF were found under low pO2. Acidic pH severely lowered VEGF release into medium, glucose consumption, and lactate production. Extracellular pH proved to more potently influence cell function than oxygen tension, the latter showing down‐regulation of COL1 gene expression and up‐regulation of VEGF protein under hypoxia. Hypoxic culture inhibits COL1 mRNA expression pH‐dependently, while expression of SOX9 is largely hypoxia independent, but pH dependent. Expression of HIF1A and VEGF revealed divergent pH dependencies. Subtle fluctuations in extracellular pH and oxygen tension clearly influence chondrocyte metabolism and marker expression. Sophisticated pH and oxygen control not only allows study of (patho)physiological changes, but also opens new venues in cartilage tissue engineering. © 2009 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 28:537–545, 2010

[1]  Zheng Cui,et al.  Effect of Extracellular pH on Matrix Synthesis by Chondrocytes in 3D Agarose Gel , 2007, Biotechnology progress.

[2]  I. Silver.  Measurement of pH and ionic composition of pericellular sites. , 1975, Philosophical transactions of the Royal Society of London. Series B, Biological sciences.

[3]  J. Urban,et al.  Evidence for a negative Pasteur effect in articular cartilage. , 1997, The Biochemical journal.

[4]  C. L. Murphy,et al.  Effect of Oxygen Tension on Chondrocyte Extracellular Matrix Accumulation , 2001, Connective tissue research.

[5]  R. Wilkins,et al.  Mechanisms contributing to intracellular pH homeostasis in an immortalised human chondrocyte cell line. , 2004, Comparative biochemistry and physiology. Part A, Molecular & integrative physiology.

[6]  J M Bland,et al.  Statistical methods for assessing agreement between two methods of clinical measurement , 1986 .

[7]  Michael I. Wilson,et al.  Targeting of HIF-α to the von Hippel-Lindau Ubiquitylation Complex by O2-Regulated Prolyl Hydroxylation , 2001, Science.

[8]  H Weinans,et al.  In vitro expansion affects the response of chondrocytes to mechanical stimulation. , 2008, Osteoarthritis and cartilage.

[9]  R. Wilkins,et al.  Surviving in a Matrix: Membrane Transport in Articular Chondrocytes , 2000, The Journal of Membrane Biology.

[10]  J. Urban,et al.  Factors influencing the oxygen concentration gradient from the synovial surface of articular cartilage to the cartilage-bone interface: a modeling study. , 2004, Arthritis and rheumatism.

[11]  V. Harde,et al.  Vascular endothelial growth factor (VEGF) induces matrix metalloproteinase expression in immortalized chondrocytes , 2004, The Journal of pathology.

[12]  M. Schünke,et al.  [Redifferentiation of dedifferentiated joint cartilage cells in alginate culture. Effect of intermittent hydrostatic pressure and low oxygen partial pressure]. , 2000, Der Orthopade.

[13]  T Kobayashi,et al.  Hypoxia in cartilage: HIF-1alpha is essential for chondrocyte growth arrest and survival. , 2001, Genes & development.

[14]  K. Yudoh,et al.  The potential role of vascular endothelial growth factor (VEGF) in cartilage: how the angiogenic factor could be involved in the pathogenesis of osteoarthritis? , 2008, Osteoarthritis and cartilage.

[15]  R. Mason,et al.  Modulation of bovine articular chondrocyte gene expression in vitro by oxygen tension. , 2001, Osteoarthritis and cartilage.

[16]  I. Marison,et al.  The importance of ammonia in mammalian cell culture. , 1996, Journal of biotechnology.

[17]  Thomas D. Schmittgen,et al.  Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. , 2001, Methods.

[18]  D. Walsh,et al.  Inflammation and angiogenesis in osteoarthritis. , 2003, Arthritis and rheumatism.

[19]  H. Weinans,et al.  Identification of acid-sensing ion channels in bone. , 2005, Biochemical and biophysical research communications.

[20]  D. Altman,et al.  STATISTICAL METHODS FOR ASSESSING AGREEMENT BETWEEN TWO METHODS OF CLINICAL MEASUREMENT , 1986, The Lancet.

[21]  C. L. Murphy,et al.  Effect of oxygen tension and alginate encapsulation on restoration of the differentiated phenotype of passaged chondrocytes. , 2001, Tissue engineering.

[22]  J. Block,et al.  Hypoxia induces HIF‐1α and VEGF expression in chondrosarcoma cells and chondrocytes , 2004 .

[23]  R. Korenstein,et al.  The control of intracellular pH in cultured avian chondrocytes. , 1993, The Journal of physiology.

[24]  E. Goetzl,et al.  Respiratory gases of synovial fluids. An approach to synovial tissue circulatory-metabolic imbalance in rheumatoid arthritis. , 1970, The American journal of medicine.

[25]  A. Hall,et al.  Control of matrix synthesis in isolated bovine chondrocytes by extracellular and intracellular pH , 1995, Journal of cellular physiology.

[26]  B Kurz,et al.  Redifferentiation of dedifferentiated bovine articular chondrocytes in alginate culture under low oxygen tension. , 2002, Osteoarthritis and cartilage.

[27]  J. Zerwekh,et al.  Biochemical changes associated with the symptomatic human intervertebral disk. , 1993, Clinical orthopaedics and related research.

[28]  A. Beckett,et al.  AKUFO AND IBARAPA. , 1965, Lancet.

[29]  J. Abbruzzese,et al.  Regulation of interleukin-8 expression by cellular pH in human pancreatic adenocarcinoma cells. , 2000, Journal of interferon & cytokine research : the official journal of the International Society for Interferon and Cytokine Research.

[30]  Yoshiaki Toyama,et al.  Vascular endothelial growth factor isoforms and their receptors are expressed in human osteoarthritic cartilage. , 2003, The American journal of pathology.

[31]  I. Fidler,et al.  Acidic pH-induced elevation in interleukin 8 expression by human ovarian carcinoma cells. , 2000, Cancer research.

[32]  A. Hall,et al.  Measurement of intracellular pH in isolated bovine articular chondrocytes , 1992, Experimental physiology.

[33]  J. Dingle,et al.  In vitro studies on human synovial membrane; a metabolic comparison of normal and rheumatoid tissue. , 1956, British journal of experimental pathology.

[34]  V C Mow,et al.  The extracellular matrix, interstitial fluid and ions as a mechanical signal transducer in articular cartilage. , 1999, Osteoarthritis and cartilage.

[35]  D. Mccarty,et al.  Synovial fluid pH, lactate, oxygen and carbon dioxide partial pressure in various joint diseases. , 1971, Arthritis and rheumatism.

[36]  W. Solomon,et al.  Response to hypoxia involves transforming growth factor-beta2 and Smad proteins in human endothelial cells. , 2001, Blood.

[37]  R. Mason,et al.  Bovine articular chondrocyte function in vitro depends upon oxygen tension. , 2000, Osteoarthritis and cartilage.

[38]  T. Aigner,et al.  Hypoxia‐inducible factor 1α is involved in the prostaglandin metabolism of osteoarthritic cartilage through up‐regulation of microsomal prostaglandin E synthase 1 in articular chondrocytes , 2007 .

[39]  K Lund-Olesen,et al.  Oxygen tension in synovial fluids. , 1970, Arthritis and rheumatism.

[40]  B. Junker.  Foam and Its Mitigation in Fermentation Systems , 2007, Biotechnology progress.

[41]  Clemens A van Blitterswijk,et al.  Cartilage Tissue Engineering: Controversy in the Effect of Oxygen , 2003, Critical reviews in biotechnology.

[42]  H. Weinans,et al.  Glucosamine decreases expression of anabolic and catabolic genes in human osteoarthritic cartilage explants. , 2006, Osteoarthritis and cartilage.