Matrix-induced autologous chondrocyte implantation offers a potential cure for joint disease but is currently hindered by loss of differentiated phenotype upon culture. ECM synthesis is dependent upon phenotype whereby differentiated chondrocytes produce mainly collagen type II, which switches to collagen type I following 2D culture (1). Currently, there is limited data on how chondrocytes dedifferentiate and therefore to further our understanding of the mechanism of dedifferentiation, full depth cartilage explants were excised from 18-24month old steers (obtained from the local abattoir) and chondrocytes cultured at low density (1×104 cell/cm2) for 9 days (P1), 14 days (P2) and 21 (P3) days (2). Changes in morphology and regulatory volume increase (RVI) were determined by confocal microscopy (CLSM) and expression of key genes by RT-PCR. Data were expressed as mean ± standard error of the mean and Student’s T-Tests were performed and values deemed significant for P<0.05. A loss of a differentiated phenotype was observed in P1 with a reduction in sphericity from 0.72±0.02Au to 0.52±0.03Au as determined by CLSM (n=65 cells). Changes in cellular dimensions in response to 2D culture were additionally recorded with a ∼2-fold increase and decrease in cell length and depth, respectively, and no change in cell width, thereby yielding an overall increase in cell volume from 474.72±32.08μm3 to 725.20±35.55μm3 (P<0.05 by Students T-test; n=65 cells). Furthermore, the effect of 2D culture-induced de-differentiation on mechanotransduction was investigated in response to a 43% hypertonic challenge (380mOsm to 540mOsm; (3)) by CLSM. RVI was only observed in 2D cultured chondrocytes with linear volume recovery rates in P1 and biphasic RVI in P2 and P3 (data on rates needed here plus stats; n=132cells). The existence of a distinct phenotype upon 9 days in culture was additionally observed upon the investigation of expression of key chondrocyte genes by RT-PCR. A significant upregulation of types I and II collagen was observed in P1 chondrocytes with 2.70±0.63 and 1.58±0.15-fold increases (P<0.05 by Student’s T-Test; n=5 gels), respectively, with no further significant change in type I collagen but a return to baseline levels of type II collagen upon further culture. A transient 1.75±0.40-fold rise in SOX9, chondrocyte-specific transcription factor, was observed upon 9 days in culture (P<0.05 by Student’s T-Test; n=5 gels). Together these data indicated the presence of an intermediate phenotype, previously undefined in cultured chondrocytes and henceforth termed ‘mesodifferentiated’ phenotype, and suggested that dedifferentiation is a multi-step process in chondrocytes.