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

Figure 4

Cartilage adherence time course of MSCs. MSCs were cultured until the second passage before use in this experiment. Prior to seeding cells for the time course, the monolayer was washed in PBS and then stained using CM-DiI (orange colour) membrane dye. Cartilage discs were used to plug the bottom of a 96-well plate in the correct orientation. Cells were then stripped using TrypLE, counted using the standard enumeration technique, and then seeded onto the cartilage discs at a density of 5 × 10³ cells/disc and the 96-well plate was then incubated at 37°C and 5% CO₂. At each of the time points (1, 2, 3, 4, 8, and 24 hours), the cartilage discs were washed in PBS and then fixed in 4% paraformaldehyde and then washed in PBS. Cartilage discs with attached cells 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. (a) One-hour time point at 10x magnification, (b) two-hour time point at 10x magnification, (c) three-hour time point at 10x magnification, (d) four-hour time point at 10x magnification, (e) eight-hour time point at 10x magnification, (f) 24-hour time point at 10x magnification, (g) one-hour time point at 40x magnification, (h) eight-hour time point at 40x magnification, and (i) 24-hour time point at 40x magnification.

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