TY - JOUR

T1 - Effects of superoxide dismutase on chondrocytes in osteoarthropathy of alkaptonuria.

AU - Kammath, Vishnu

AU - Bukhari, Marwan A. S.

AU - Taylor, Adam Michael

PY - 2017/4

Y1 - 2017/4

N2 - Purpose: Alkaptonuria (AKU) is a rare autosomal recessive form of osteoarthropathy resulting from deficiency of homogentisate 1,2 dioxygenase (HGD) causing inability to metabolise the tyrosine metabolite; homogentisic acid (HGA). HGA levels in the body become elevated and show high affinity for collagenous tissues, primarily articular cartilages of load bearing joints, where it polymerises and deposits as a dark pigment; this process is ochronosis. Over time ochronosis causes rapid and early onset joint arthropathy, mimicking osteoarthritis (OA) in presentation. The ochronosis causes damage to the extracellular matrix and chondrocyte death and is reliant on the availability of oxygen radicals to proceed. One potential way to inhibit the polymerisation process is utilising antioxidants. We hypothesise that superoxide dismutase (SOD) may have a beneficial effect on chondrocytes and their extracellular matrix as well as limiting the polymerisation process in our in vitro model. This study aims to examine the effect of the superoxide dismutase on ochronosis and cell viability in an in vitro model of AKU.Methods: Cultures of immortalised human chondrocytes C20/A4 were cultured in 24 well plates in DMEM containing 0.33uM HGA/ml, 50 units of SOD/ml or a combination of both for up to 14 days. Cells were seeded at 5000/well and medium changed every 3rd day. Cell viability was assessed using trypan blue assay and expressed as number of viable cells/ml or percentage of viable cells. Statistical analysis was performed using Graphpad Prism by ANOVA and Newman-Keuls post-hoc analysis.Results: After 7 days cultures containing HGA showed a significant increase in the overall percentage of viable cells compared to control and SOD only; control vs both HGA and HGA+SOD, P<0.05 for both. The cultures containing HGA + SOD showed an increase in the overall percentage of viable cells compared to HGA only, but this was not significant. Analysis of the total number of viable cells on day 7 showed that there was no significant difference in overall total viable cell number across any of the groups; although the SOD group had the highest total number. The HGA+SOD group had a greater number of viable cells than the HGA group, although this was not significant. At day 14 cultures showed no significant difference in the percentage of viable cells across all groups, with percentage viability showing very close range across the 4 groups. The total number of viable cells was highest in the control and lowest in the SOD group.Conclusions: This model is the first to test the effect of SOD in an in vitro model of the osteoarthropathy in AKU. We demonstrate significant effects of SOD on the number of chondrocytes in this model. Prolonging the life and maintaining the number of chondrocytes in AKU, by reducing the effects of free oxygen species in tissues may be beneficial in maintaining overall cartilage health in AKU. The role of SOD in cartilage and interaction with reactive oxygen species (ROS) is well known; reduction of SOD is seen in earliest OA and results in an increase in ROS. AKU cartilage is characterised by glycosaminoglycan loss, rapid degeneration of the cartilage matrix and chondrocyte death, due to pigment formation. Whilst addition of SOD shows some chondroprotective potential in this disease, it also shows some negative effect on cell viability. These results demonstrate that antioxidant therapy, in the form of SOD, may be a beneficial treatment route in this disease. Further assessment of the role of SOD in the pigmentation process of this iconic disease is needed.

AB - Purpose: Alkaptonuria (AKU) is a rare autosomal recessive form of osteoarthropathy resulting from deficiency of homogentisate 1,2 dioxygenase (HGD) causing inability to metabolise the tyrosine metabolite; homogentisic acid (HGA). HGA levels in the body become elevated and show high affinity for collagenous tissues, primarily articular cartilages of load bearing joints, where it polymerises and deposits as a dark pigment; this process is ochronosis. Over time ochronosis causes rapid and early onset joint arthropathy, mimicking osteoarthritis (OA) in presentation. The ochronosis causes damage to the extracellular matrix and chondrocyte death and is reliant on the availability of oxygen radicals to proceed. One potential way to inhibit the polymerisation process is utilising antioxidants. We hypothesise that superoxide dismutase (SOD) may have a beneficial effect on chondrocytes and their extracellular matrix as well as limiting the polymerisation process in our in vitro model. This study aims to examine the effect of the superoxide dismutase on ochronosis and cell viability in an in vitro model of AKU.Methods: Cultures of immortalised human chondrocytes C20/A4 were cultured in 24 well plates in DMEM containing 0.33uM HGA/ml, 50 units of SOD/ml or a combination of both for up to 14 days. Cells were seeded at 5000/well and medium changed every 3rd day. Cell viability was assessed using trypan blue assay and expressed as number of viable cells/ml or percentage of viable cells. Statistical analysis was performed using Graphpad Prism by ANOVA and Newman-Keuls post-hoc analysis.Results: After 7 days cultures containing HGA showed a significant increase in the overall percentage of viable cells compared to control and SOD only; control vs both HGA and HGA+SOD, P<0.05 for both. The cultures containing HGA + SOD showed an increase in the overall percentage of viable cells compared to HGA only, but this was not significant. Analysis of the total number of viable cells on day 7 showed that there was no significant difference in overall total viable cell number across any of the groups; although the SOD group had the highest total number. The HGA+SOD group had a greater number of viable cells than the HGA group, although this was not significant. At day 14 cultures showed no significant difference in the percentage of viable cells across all groups, with percentage viability showing very close range across the 4 groups. The total number of viable cells was highest in the control and lowest in the SOD group.Conclusions: This model is the first to test the effect of SOD in an in vitro model of the osteoarthropathy in AKU. We demonstrate significant effects of SOD on the number of chondrocytes in this model. Prolonging the life and maintaining the number of chondrocytes in AKU, by reducing the effects of free oxygen species in tissues may be beneficial in maintaining overall cartilage health in AKU. The role of SOD in cartilage and interaction with reactive oxygen species (ROS) is well known; reduction of SOD is seen in earliest OA and results in an increase in ROS. AKU cartilage is characterised by glycosaminoglycan loss, rapid degeneration of the cartilage matrix and chondrocyte death, due to pigment formation. Whilst addition of SOD shows some chondroprotective potential in this disease, it also shows some negative effect on cell viability. These results demonstrate that antioxidant therapy, in the form of SOD, may be a beneficial treatment route in this disease. Further assessment of the role of SOD in the pigmentation process of this iconic disease is needed.

U2 - 10.1016/j.joca.2017.02.246

DO - 10.1016/j.joca.2017.02.246

M3 - Meeting abstract

VL - 25

SP - S148-S149

JO - Osteoarthritis and Cartilage

JF - Osteoarthritis and Cartilage

SN - 1063-4584

IS - Suppl. 1

T2 - 2017 OARSI World Congress on Osteoarthritis

Y2 - 27 April 2017 through 30 April 2017

ER -