Figure 1.
Fabrication of the microwell surface from PDMS replica moulding and surface modification.
A silica wafer having an array pattern of microwells was formed via deep reactive ion etching. The dimensions of the microwells on silica wafer were 360×360×180 (depth) µm (A). This surface was used to cast PDMS, generating a negative surface. PDMS mould having an inverted microwell pattern (B). This surface was then coated in 5% pluronic acid solution, which functioned as a release agent. The coated surface was used to cast a 2 mm thick PDMS sheet having a microarray pattern identical to the original silica wafer (C). Because PDMS-PDMS casting was not reproducible, the PDMS sheet with the microwells was cast with a polystyrene sheet to obtain a plastic mould (D). Using polystyrene mould PDMS sheets with microwells were produced (E). A punch was used to create 2 cm2 discs which fit snuggly into the bottom of a 24-well plate (F). Individual microwell inserts were subsequently surface modified using a CHI/HA electrostatic multilayer; see text for details (G). The chondrocytes spreading on non-modified PDMS microwell surface (cell layers marked with arrowheads) (H). Robust micropellet formation on CHI/HA multilayered PDMS surface (I). Scale bar: 200 µm.
Figure 2.
Morphology and size of the pellets.
At the end of the 14-day culture, both hypoxic macropellets (A) and micropellets (B) were bigger than the normoxic pellets. The estimated mean diameter for hypoxic micropellets was 193±20 µm (n = 20) whilst the estimated mean diameter of the normoxic micropellets was 87±10 µm (n = 20). Scale bars: 1 mm.
Figure 3.
Metabolic activity, growth and sGAG production in pellets.
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).
Figure 4.
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.
Figure 5.
Cell and matrix localization throughout pellets following 14 days of culture.
DAPI staining of nuclei in pellets (A).Immunofluorescence images for collagen I (B), collagen X (C), and collagen II (D), Alcian blue staining for sGAG (E). Scale bars: 100 µm.
Figure 6.
Hypoxic micropellets assembled into macrotissues.
Alcian blue staining for hypoxic micropellets assembled at different time points (indicated days). The total culture duration was 21 days. Scale bars: 100 µm.
Figure 7.
Potential applications of the chondrocyte micropellets.
The direct use of chondrocyte micropellets in articular cartilage defect repair (A). The use of cartilage micropellets in the manufacture of osteochondral tissues in vitro (B).