Introduction: Longitudinal skeletal growth is achieved by enchondral ossification in epiphyseal growth plates (GP) of long bones and vertebrae. These highly organized cartilaginous tissues contain chondrocytes of all differentiational stages classified in three distinct zones named resting, proliferative and hypertrophic. Separated analysis of individual zones is essential in basic GP research thus efficiency of different zonal separation methods confers high impact on study results. Nevertheless, lack of comparative studies on commonly used separation methods complicates the interpretation and comparison of basic GP research data.
Aim: We aim to compare the efficiency of two commonly used GP separation methods, density gradient centrifugation (DGC) and laser capture microdissection (LCM), by quantitative real time PCR (qRT-PCR) of zone-specific growth plate marker genes.
Methods: Primary chondrocytes and cartilage tissues were isolated from femoral and tibial growth plates of prepubertal piglets and separated by DGC and LCM, respectively. Relative gene expressions in samples were evaluated by qRT-PCR for Secreted Frizzled Related Protein 5 (Sfrp5) and Collagen type X (ColX) as markers for resting and hypertrophic zones.
Results: Both separation methods, DGC and LCM, were capable of generating resting and hypertrophic zone samples with significantly different marker gene expression compared to the adjacent zones, with the exception of Sfrp5 expression in proliferative versus hypertrophic zone measurements by both methods and Col X expression measurements in resting versus proliferative zones by LCM. Both LCM and DGC could discriminate resting vs. proliferative zones by Sfrp5 expression values (DGC: P=0.034; LCM=0.003). Comparable results were observed for ColX gene expression levels in hypertrophic versus proliferative zones (DGC P=0.024; LCM<0.001). Two-way interaction testing between LCM and DGC revealed that, regardless of marker genes specific zonal expression difference is significantly different between two methods (P = 0.001). Similarly, marker genes expression showed a significant difference (P = 0.004) between both methods irrespective of growth plate zones investigated.
Conclusion: Taking all the above into account indicates that, while both methods are able to discriminate growth plate zones, LCM achieved substantially higher level of efficiency in zonal separation as compared to DGC. Thus, LCM is able to reduce methodological bias in and could be considered as preferred method for expressional studies on specific growth plate zones.
19 Sep 2019 - 21 Sep 2019