Dual proline labeling protocol for individual “baseline” and “response” biosynthesis measurements in human articular cartilage

SummaryObjectiveThe heterogeneity of biosynthesis in human-derived cartilage explants poses a challenge to its use in experiments. The aim of this study was to determine the consistency with which two consecutive measures of biosynthesis could be made in individual human articular cartilage explants using a dual proline radiolabeling protocol.MethodsFull-thickness cartilage explants were harvested from young bovine or human (total knee replacement) tibial plateaus. Two consecutive measurements of biosynthesis were obtained by measuring 3H-proline and 14C-proline incorporation. Each sample's ratio of 14C-/3H-proline incorporation was computed. For comparison to traditional experimental designs, the 14C-proline incorporation ratio was computed for adjacent cartilage samples. The number of samples needed to observe a change in the proline incorporation ratio of 10, 20, and 50% was determined for both methods.ResultsThe dual-label ratio was consistent across samples from the same plateau [95% confidence interval (CI): ±20% (human) and ±30% (bovine) of median]. Adjacent human sample pairs had much greater variability in their 14C-proline incorporation (95% CI: ±50% of median). Adjacent bovine sample pairs had CIs that were similar in magnitude to those for the dual-label approach. In the human plateaus, ratio changes of 10, 20 and 50% could be detected using dramatically fewer samples than the adjacent pair method. For bovine samples, the two methods required a similar number of samples per group.ConclusionThe consistency of the dual-label approach may overcome the difficulties in studying the effects of interventions on biosynthesis in human cartilage in vitro.