Searchable abstracts of presentations at key conferences on calcified tissues
Bone Abstracts (2019) 7 P219 | DOI: 10.1530/boneabs.7.P219

ICCBH2019 Poster Presentations (1) (226 abstracts)

Comparison of cell separation methods, using relative expression of specific growth plate zone markers in a pig model

Alireza Javanmardi 1 , Adalbert Raimann 1 , Monika Egerbacher 2 , Susanne Sagmeister 1 , Andreas Gleiss 3 & Gabriele Haeusler 1

1University Clinic of Pediatrics and Adolescent Medicine, Medical University of Vienna, Währinger Gürtel 18-20 1090, Vienna, Austria; 2Histology and Embryology, Department of Pathobiology, University of Veterinary Medicine Vienna, Vienna, Austria; 3Center for Medical Statistics, Informatics and Intelligent Systems, Medical University of Vienna, Vienna, Austria.

Objective: Linear growth is achieved by enchondral ossification in epiphyseal growth plates (GP) of long bones. These highly organized cartilaginous tissues contain chondrocytes of all differentiational stages classified in 3–5 specific zones. Due to their discrete characteristics, distinct analysis of each zone is essential in basic GP research. While the efficiency of zonal separation is therefore highly influencing on study results, comparative data on commonly used methods are sparse. This study aims to compare the efficiency of density gradient centrifugation (DGC) and laser capture microdissection (LCM) by quantitative real time PCR (qRT-PCR) of zone-specific growth plate samples.

Methods: Primary chondrocytes and cartilage tissues were isolated from femoral and tibial growth plates of prepubertal piglets and separated by density gradient centrifugation and Laser Microdissection (LCM) respectively. Samples were evaluated by qRT-PCR for Secreted Frizzled Related Protein 5 (Sfrp5) and Collagen type X (ColX) expression as markers for resting and hypertrophic zone, respectively.

Results: Significant differences in marker gene expression for resting and hypertrophic zones as compared with their respective adjacent zones could be found in both separation methods. Both LCM and density gradient centrifugation were able to discriminate resting vs. proliferative zones by Sfrp5 expression values, although to different levels of significance (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).

Conclusion: While both methods are able to discriminate growth plate zones, LCM achieved a higher level of significance in zonal separation as compared to DGC. Thus, LCM allows to minimize methodical bias and should be the preferred method for expressional studies on specific growth plate zones.

Disclosure: The authors declared no competing interests.

Volume 7

9th International Conference on Children's Bone Health


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