Histological analysis of calcification in explanted tibiae.

<p>(A) Midsagittal sections of tibiae were stained with Alizarin red after explantation or cultured seven days under hypoxic or normoxic conditions. (B) Image analysis was used to determine the length of the intensely calcified tissue, which was taken as the broken line indicated in ‘A’. (C) The area of calcification was used to determine relative calcification of the samples. (D) Higher magnification microphotographs were used to investigate the calcification of the hypertrophic cartilage that resides on top of the intensely stained bone. The dashed line represents the osteochondral interface. (N = 15). * = P<0.05 compared to freshly isolated tibiae. # = P<0.05 compared to normoxic condition of the same time point.</p

Explanted tibiae cultured 21 days under hypoxic or normoxic conditions.

<p>(A) Microphotographs of representative tibiae at different points in time. (B) Using image analysis the average tibiae lengths were calculated. (N = 18). * = P<0.05. ** = P<0.01.</p

Effect of hypoxic and normoxic culture conditions on Frzb and Dkk1 protein levels.

<p>(A) Frzb and Dkk1 levels were quantified in the conditioned medium of tibiae, which were cultured for 7 days without receiving new medium in either hypoxia or normoxia (N = 5). (B) The effect of oxygen levels on Frzb and Dkk1 on protein activity over time was studied by exposing culture medium containing 10% fetal bovine serum to either hypoxia or normoxia for 7 days in 37°C. (N = 4). Frzb and Dkk1 protein levels were analyzed using ELISA. * = P<0.05 compared to normoxic condition of the same time point.</p

Fetal Mesenchymal Stromal Cells Differentiating towards Chondrocytes Acquire a Gene Expression Profile Resembling Human Growth Plate Cartilage

<div><p>We used human fetal bone marrow-derived mesenchymal stromal cells (hfMSCs) differentiating towards chondrocytes as an alternative model for the human growth plate (GP). Our aims were to study gene expression patterns associated with chondrogenic differentiation to assess whether chondrocytes derived from hfMSCs are a suitable model for studying the development and maturation of the GP. hfMSCs efficiently formed hyaline cartilage in a pellet culture in the presence of TGFβ3 and BMP6. Microarray and principal component analysis were applied to study gene expression profiles during chondrogenic differentiation. A set of 232 genes was found to correlate with <em>in vitro</em> cartilage formation. Several identified genes are known to be involved in cartilage formation and validate the robustness of the differentiating hfMSC model. KEGG pathway analysis using the 232 genes revealed 9 significant signaling pathways correlated with cartilage formation. To determine the progression of growth plate cartilage formation, we compared the gene expression profile of differentiating hfMSCs with previously established expression profiles of epiphyseal GP cartilage. As differentiation towards chondrocytes proceeds, hfMSCs gradually obtain a gene expression profile resembling epiphyseal GP cartilage. We visualized the differences in gene expression profiles as protein interaction clusters and identified many protein clusters that are activated during the early chondrogenic differentiation of hfMSCs showing the potential of this system to study GP development.</p> </div

Gene expression of genes by KEGG signaling pathway.

<p>KEGG signaling pathways significantly associated with chondrogenic differentiation of hfMSCs. For each pathway, genes showing the same distinct expression profile during 5 weeks of chondrogenic differentiation are depicted as groups.</p

Analysis of changes in gene expression by microarray.

<p>Gene selection based on principal component analysis. A) variance explained by components 1–6 from principal component analysis. B) principal components 1, 2, and 3 as expression profiles. C) selection of probes based on their factor 2 and 3 scores. D) scatterplot view of gene data in respect to their correlation (factor score) to principal components 2 and 3. Subgroups 1, 2, 3, and 4 are represented by blue, green, yellow, and pink dots, respectively. Side-placed graphs depict the gene expression profiles for genes found in the four subgroups.</p

Increase in growth plate enriched genes and decrease of articular cartilage enriched genes in time.

<p>Expression of growth plate enriched genes <i>PANX3, EPYC, LEF1</i> and articular cartilage enriched genes <i>DKK1, FRZB, GREM1</i> during 4 weeks of chondrogenic differentiation of hfMSCs. The y-axis (left) indicates the qPCR results as normalized fold expression on a log-scale. The x-axis (right) indicates the time in weeks. Fold changes are calculated from qPCR data expressed as delta delta CT values corrected for the housekeeping gene <i>GAPDH</i>.</p

hfMSCs micromasses undergo chondrogenic differentiation.

<p>Expression of (A) glycosaminoglycans visualized by toluidine blue staining, nuclei are counterstained with haematoxylin, (B) collagen type II immunofluorescence, and (C) collagen type X immunohistochemistry (red-brown) during 5 weeks of chondrogenic differentiation of hfMSCs to chondrocytes. The top panel shows a magnification of the pellet cultures at week 1 and week 5 stained by toluidine blue demonstrating the change in cell morphology and the deposition of the extracellular matrix. The insets in panel B show higher magnifications of collagen type II positive chondrocytes.</p