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

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

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

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

Metabolic activity, growth and sGAG production in pellets.

<p>AlamarBlue® graph for metabolic activity (A), DNA quantification (B), sGAG in construct (C) and sGAG in media (D) measurements on days 4, 7, 11, 14. The sGAG/DNA ratio (calculated by dividing the total amount of sGAG produced during the culture to the amount of DNA measured on day 14) (E) and the total sGAG graph demonstrating the total sGAG in media and in construct separately (F).</p

Hypoxic micropellets assembled into macrotissues.

<p>Alcian blue staining for hypoxic micropellets assembled at different time points (indicated days). The total culture duration was 21 days. Scale bars: 100 µm.</p

Characterisation of plasma modified surface and GFP-mMSCs expansion in our packed bed bioreactor in static and perfusion conditions.

<p>(A) Surface composition of the air plasma treated polystyrene scaffold determined by XPS. (B) GFP-mMSC attachment after one and half hours, initially seeded at 3000 cells/cm² (n = 4). (C) The growth after 3 days of culture of GFP-mMSC seeded at 1000 cells/cm² (n = 6). (D) Growth of GFP-mMSC in the bioreactor (BR) under static conditions (n = 4) and (E) under 5 mL/day perfusion (n = 4). (F) Cell harvest recovery and viablity from the bioreactor. (G, H, I and J) Fluorescent microscopy showed that the GFP-mMSC attached to the scaffold (scale bar is 500 μm). (K) IVIS imaging of the fluorescent intensity of PI stained GFP-mMSC in the bioreactor under static and (L) 5 mL/day perfusion conditions. IVIS images are a red (low) / yellow (high) heat map of fluorescent intensity.</p

Description of packed bed bioreactor and results from the steady state oxygen mass balance.

<p>(A) Process flow diagrams of the single pass small-scale 160cm² bioreactor and (C) recirculating perfusion for the large-scale 2800cm² bioreactor. (B) Picture of 160 cm² left and 2800 cm² right bioreactor scaffolds made from 3 mm polystyrene pellets (scale bar is 10 mm). (D) 2800 cm² bioreactor inside the incubator. (E) Mass transport model result for radial oxygen diffusion with cell density of 100,000 cells/cm² in small-scale (160 cm²) and (F) large-scale (2800 cm²) packed bed bioreactors.</p