Height variation in the canine species as a model to identify new targets in bone regenerative medicine.
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In the orthopaedics regenerative medicine field, large bone defects still represent a huge challenge. The use of autografts is the current state-of-the-art treatment. However, due to disadvantages, such as the need for a large amount of donor tissue and the chance of rejection, new bone regenerative strategies concentrate on alternatives employing scaffold in combination with cells and/or modulating factors. Candidate modulating factors can be identified by studying the natural healing process of bone defects, which heal by endochondral bone formation. Even more so, studying endochondral bone formation at the growth plate level may also simplify the approach, since at the growth plate level chondrocyte proliferation and differentiation occur at an orderly manner. The overall aim of this project was to study the local pathways that enhance endochondral bone formation in order to identify new bone regenerative strategies. In this respect, this project investigated whether the naturally occurring height variation in the canine species is an appropriate model to study these pathways and set up the in vitro platform in which selected candidates can be evaluated for their efficacy to augment endochondral bone formation. GWAS and candidate-gene based approaches in human and canine populations discovered many genes that were differential regulated in individuals with different heights. However, these differences explained only 10% of human height variation. Within the canine species height variation is much more pronounced. Furthermore, the canine growth plate resembles the human growth plate in that they both close after maturity, which makes the canine species a suitable model to study the local pathways that enhance endochondral bone formation at the growth plate level. A micro-analysis was performed, comparing the growth plates of Great Danes (a large, fast-growing breed dog) with Miniature Poodles (a small, slowly growing breed dog). The micro-array detected 3010 genes that were significantly differential regulated between the two breeds of which 1193 genes were upregulated and 1817 genes were downregulated. Many of the genes that were associated by the GWAS with height variation, were also differential regulated between the GD and MP. In order to enable translation towards the clinics, the identified targets need to be evaluated in vitro followed by in vivo studies. Differentiation of MSCs towards the different lineages is a suitable in vitro model to study the role of the identified targets. Adipogenic differentiation was confirmed by Oil-red-O staining, showing red lipid droplets, and qPCR, showing an upregulation of PPARG and ADIPOQ. Osteogenic differentiation was achieved in 5 out of 9 BMSC donors and 1 out of 9 ASC donors and was confirmed by Alizarin red staining, showing red mineral deposits, and qPCR, showing an upregulation of SPP1 and Osteocalcin in the sub group of samples that stained positive with Alizarin red. Chondrogenic differentiation of the MSCs was not successful; safranin-O/fast green was negative and the qPCR did not show an upregulation of the chondrogenic markers. This may be caused by the absence of bFGF in the expansion medium and the replacement of ITS+ by ITS in the earlier experiments in combination with a less active TGF-β1 from another distributor. Altogether, the naturally occurring height variation of the canine species seems to be an appropriate model to study the local pathways that enhance the pace of endochondral bone formation in order to find new bone regenerative strategies. However, in order to fully verify this model, chondrogenic differentiation should be obtained to test the effect of the differential regulated genes of the micro-array on the chondrogenic differentiation of MSCs.