The modulation of MSC integrin expression by RGD presentation

Abstract

Biomaterials designed to mimic the intricate native extracellular matrix (ECM) can use a variety of techniques to control the behavior of encapsulated cells. Common methods include controlling the mechanical properties of the material, incorporating bioactive signals, spatially patterning bioactive signals, and controlling the time-release of bioactive signals. Further design parameters like bioactive signal distribution can be used to manipulate cell behavior. Efforts on clustering adhesion peptides have focused on seeding cells on top of a biomaterial. Here we report the effect of clustering the adhesion peptide RGD on mouse mesenchymal stem cells encapsulated inside three-dimensional hyaluronic acid hydrogels. The clustered bioactive signals resulted in significant differences in both cell spreading and integrin expression. These results indicate that signal RGD peptide clustering is an additional hydrogel design parameter can be used to influence and guide the behavior of encapsulated cells.

Fig. 1

The hydrogel is composed of acrylated hyaluronic acid, MMP-degradable peptide crosslinker, and an RGD-motif containing peptide. RGD clustering is controlled by the amount of HA-AC pre-reacted with RGD. In the homogenous, or least clustered condition, the RGD is mixed with all of the HA-AC. The RGD is pre-reacted with specific percentages of the total HA-AC to create different degrees of clustering. This RGD functionalized HA-AC is mixed with un-functionalized HA-AC, if needed, peptide crosslinker and mouse mesenchymal stem cells to create the three-dimensional hydrogel.

Fig. 2

Mechanical characterization of hydrogel conditions. (A) Storage modulus of hydrogels was measured from 0.1 to 10 Hz. Three conditions from each RGD concentration were tested. (B) Mechanical properties within each RGD concentration were consistent, but increasing amounts of RGD lowered the storage modulus.

Fig. 3

Cell spreading and proliferation for 10 μm hydrogels. (A) mMSC’s within hydrogels containing 10 μm of RGD were stained with phalloidin at different time points. The most pronounced differences in spreading were observed at day 4. (B) The average cell length was quantified with the greatest spreading seen in the most clustered condition. (C) DNA quantification shows no significant difference between conditions at each time point.

Fig. 4

Cell spreading and proliferation for 100 μm hydrogels. (A) mMSC’s cells within hydrogels containing 10 μm of RGD were stained with phalloidin at different time points. The most pronounced differences in spreading were observed at day 4. (B) The average cell length was quantified with the greatest spreading seen in the middle, B3, condition. (C) DNA quantification shows no significant difference between conditions at each time point.

Fig. 5

Cell spreading and proliferation for 1000 μm hydrogels. (A) mMSC’s cells within hydrogels containing 1000 μm of RGD were stained with phalloidin at different time points. The most pronounced differences in spreading were observed at day 4. (B) The average cell length was quantified with the greatest spreading seen in the middle, C3, condition. (C) DNA quantification shows no significant difference between conditions at each time point.

Fig. 6

Integrin expression for subunits (A–B) α2, (C–D) α3, (E–F) α5, (G–H) αV was quantified for mouse mesenchymal stem cells cultured in gel conditions A1, A3, and A5 via FACS. Differences were found in the normalized mean expression for α3. Gel conditions also affected the number of cells positively expressing integrins α2 and α3.

Fig. 7

Integrin expression for subunits (A–B) β1 and (C–D) β3 for mouse mesenchymal stem cells cultured in gel conditions A1, A3, and A5 via FACS. Differences were observed in both the normalized mean fluorescence and percent of positively expressing cells for subunit β1.

Fig. 8

Time course of CD105 expression for mouse mesenchymal stem cells cultured inside hydrogels A1, A3, and A5 shows decreases across all three conditions for both the (A) normalized mean fluorescence and (B) the percent gated. (C–D) mechanical properties of hydrogel A5 was measured at days 1, 4, and 7. The storage modulus decreases over time as the cells release MMP’s which breakdown the hydrogel structure.