Priming Adipose-Derived Mesenchymal Stem Cells with Hyaluronan Alters Growth Kinetics and Increases Attachment to Articular Cartilage

Figure 5

MSC dispersion on cartilage with increasing concentration of HA. MSCs were cultured until the second passage before use in this experiment. Cartilage discs were used to plug the bottom of a 96-well place in the correct orientation. Media formulations were then added to each well. The media formulations used were (a) 0.5 mg/mL HA media, (b) 1 mg/mL HA media, (c) 2 mg/mL HA media, (d) 3 mg/mL HA media, (e) 4 mg/mL HA media, and (f) 5 mg/mL HA media. MSCs were then stripped using TrypLE, counted using standard enumeration technique, and then seeded onto the cartilage discs at a density of 5 × 10³ cells/disc. The 96-well plate was then incubated for 24 hours at 37°C and 5% CO₂. After 24 hours the cartilage discs were washed in PBS and then fixed in 4% paraformaldehyde. Cartilage discs were washed in PBS and then stained in using Hoechst 33342 (blue) and again washed five times in PBS. Cartilage discs were then permeabilised in 0.1% Triton X-100, washed in PBS, and blocked in 1% Bovine serum albumin in PBS before being stained with F-actin-specific Alexa Fluor 488-phalloidin (green). All images were taken using the OLYMPUS FLUOVIEW FV1000 IX81 inverted confocal microscope at 10x magnification.

(a)

(b)

(c)

(d)

(e)

(f)