Effects of Individual Control of pH and Hypoxia in Chondrocyte Culture

Effects of oxygen tension (pO(2)) 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 pO(2) (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), S0X9, 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 pO(2). 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. (C) 2009 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 28:537-545, 201

Comparison of Four Commercially Available cAMP Kits

Peer Reviewe

Specific leukotriene formation by purified human eosinophils and neutrophils

Human granulocytes isolated from peripheral blood have been described to synthesize both LTB4 and LTC4 from arachidonic acid. We have observed that the amount of LTC4 produced by human granulocyte preparations is strongly dependent on the relative amount of eosinophils. To investigate a possibly significant difference in leukotriene synthesis of the eosinophilic and neutrophilic granulocytes, we developed a purification method to isolate both cell types from granulocytes obtained from the blood of healthy donors. Leukotrienes were generated by incubation of the purified cells with arachidonic acid, calcium ionophore A23187, calcium-chloride and reduced glutathione. Surprisingly, eosinophils were found to produce almost exclusively the spasmogenic LTC4. In contrast, neutrophils produce almost exclusively the chemotactic LTB4, its omega-hydroxylated metabolite 20-hydroxy-LTB4 and two non-enzymically formed LTB4 isomers