Abstract
Articular chondrocytes descend from a distinct cohort of progenitor cells located in the embryonic joint anlagen, termed interzones. Their unique lineage might explain some of the problems encountered using chondrocytes of different lineages for articular cartilage tissue engineering. While it is well established that articular chondrocytes descend from interzone cells,
... read more
the exact role the intermediate and outer layers of the interzone play in joint development and which specific articular structures they give rise to, remains equivocal. We hypothesize that new insights into the development of articular chondrocytes will further our understanding of how articular chondrocytes achieve and maintain their stable permanent chondrocytic phenotype. In this thesis, we have developed a technique to selectively harvest cells from the intermediate and outer interzone of mouse and horse as well as from the three equine articular cartilage zones in sufficient quantities for downstream microarray analysis and established the differential gene expression profile of the 2 interzone layers. The main findings of this thesis are as follows: 1) We recommend to harvest a minimum of 1 x 106μm2 of 13.5 days murine embryos and 3 x 106μm2 of 15.5 days murine embryos to obtain approximately 10 ng total RNA that can be used for linear T7-based amplification and subsequent microarray analysis. 2) Inflammatory pathways and functions were most prevalent in both the differential gene expression profile and the set of 25 genes most highly differentially up-regulated in the murine intermediate interzone. In the murine outer interzone, the 25 most highly differentially up-regulated genes play roles in cartilage matrix formation, chondrocyte differentiation (hypertrophy) and endochondral ossification. 3) At gestational day 40 the equine interzone is fully formed and consists of 3 morphologically distinct layers. 4) Laser capture microdissection of equine articular cartilage allows contact-free harvesting of zone-specific equine chondrocytes in sufficient quantities for microarray analysis from individual animals without need to pool samples. Taken together, the pathway analysis of the differentially expressed genes implies an important role for inflammatory processes in the interzone layers and hence in joint and articular cartilage development. The high representation of genes relevant to chondrocyte hypertrophy and endochondral ossification in the outer interzone suggests that the outer interzone undergoes endochondral ossification, leaving the intermediate interzone to form the articular tissues including cartilage. Further studies will be needed to look into the various pathways and genes found to be differentially expressed between the interzone layers in this study, specifically into the role of inflammatory pathways and genes in the interzone and to confirm this hypothesis
show less
