Infectivity of dengue viruses (DENVs) for mosquito (C6/36) and human (HuH7) cell lines.

<p>DENVs isolated from clinical patients in C6/36 were infected in C6/36 and Huh7 with ten-fold dilutions to determine the relative titres in both cell types. Both cells (C6/36 and Huh7) were infected at the same time with the same dilution of DENVs to determine the relative titres.</p><p>I.C. Infectious clone derived DENV-2.</p><p>Infectivity of dengue viruses (DENVs) for mosquito (C6/36) and human (HuH7) cell lines.</p

Distribution of fitness within populations of DENV-1 from four lineages.

<p>Populations are identified as strain/year/E (extinct) or C (circulating); proportion more fit than the population average indicated horizontal hatch, same fit as the original population indicated as bold squares and less fit than the original population indicated as small squares.</p

Phylogenetic analysis of the E gene of DENV-1 showing lineage extinction and replacement of DENV-1 in Myanmar.

<p>Bootstrap values (100 replications) for key nodes are shown. A distance bar is shown below the tree. Lineage A, B and E are extinct and lineage C and D are still circulating. Strains selected for study have been highlighted.</p

Relationship between DENV lineage extinction and polarization in fitness in populations.

<p>75% of the extinct viral strains were polarized and 80% of the circulating strains were non-polarized. We have conducted a Chi-square test of independence to test the null hypothesis that there is no association between polarization and virus extinction. The test results show that there is no statistically significant association between polarization and virus extinction (Pearson Chi- square statistic = 2.7, <i>p</i> = 0.09).</p><p>Relationship between DENV lineage extinction and polarization in fitness in populations.</p

Gene expression analysis of MSC and MSC+CD macro and micropellets at day 7 and 14.

<p>Aggrecan (A), Versican (B), SOX9 (C), RUNX2 (D), Collagen II (E), Collagen I (F), Collagen X (G) and Osteocalcin (H) gene expressions were analyzed to assess the chondrogenic, hypertrophic and osteogenic characteristics of the generated macro and micropellets. Abbreviations: MSC, cell only control; MSC+CD, composite (cell and cartilage dust); macro, macropellet; micro, micropellet; d7, day 7; d14, day 14.</p

Histological assessment of MSC and MSC+CD macro and micropellets at day 14.

<p>The alcian blue staining demonstrated that MSC+CD macropellet was less intact when compared to MSC macropellet. Collagen II staining was stronger in MSC+CD macro and micropellets when compared to MSC macro and micropellets indicating that CD itself contained high quantity of collagen II. Collagen X staining was similar in all conditions. All the images were overlayed with DAPI staining. Abbreviations: MSC, cell only control; MSC+CD, composite (cell and cartilage dust); macro, macropellet; micro, micropellet. Scale bars: 100 μm.</p

Histological assessment of the assembled tissues.

<p>Alcian blue staining showed inefficient integration of the CD particles in day 0 MSC+CD assembly (equivalent to MSC+CD macropellets) and decreasing integration efficiency was observed when the assembly day increased. Collagen II staining confirmed that the quantity of collagen II in MSC+CD assembled tissues was higher than the collagen II in MSC assembled tissues. Abbreviations: MSC, cell only control; MSC+CD, composite (cell and cartilage dust); d0, day 0; d4, day 4; d7, day 7; d10, day 10; d14, day 14. Scale bars: 100 μm.</p

Cartilage dust (CD) characterization and micropellet morphology.

<p>Phase contrast image of CD demonstrated a gradient in particles size (A). The particles size distribution indicated that most of the particles had a size smaller than 10 μm and the average particle size was estimated to be 5.53 μm (B). Phase contrast images of MSC and MSC+CD micropellets on day 1 and 14 and macroscopic images of MSC and MSC+CD macropellets (C), the average diameters for MSC micropellets on day 1 was 100.8 ± 7.8 μm and on day 14 was 82.6 ± 10.8 μm, the average diameters for MSC+CD micropellets on day 1 was 176.1 ± 9.9 μm and on day 14 was 183.8 ± 16.2 μm Overall, MSC+CD macro and micropellets were greater in size. Abbreviations: MSC, cell only control; MSC+CD, composite (cell and cartilage dust). Scale bars: 500 μm.</p

Potential applications of the chondrocyte micropellets.

<p>The direct use of chondrocyte micropellets in articular cartilage defect repair (A). The use of cartilage micropellets in the manufacture of osteochondral tissues <i>in vitro</i> (B).</p

Gene expression in pellets.

<p>Aggrecan (A), collagen II (B), collagen I (C), collagen X (D), Sox9 (E), Runx2 (F), versican (G), and osteocalcin (H) expressions relative to the geometric mean of housekeeping genes cyclophilin A and GAPDH.</p