Effect of RGD functionalization and stiffness modulation of polyelectrolyte multilayer films on muscle cell differentiation

Abstract

Skeletal muscle tissue engineering holds promise for the replacement of muscle damaged by injury and for the treatment of muscle diseases. Although arginylglycylaspartic acid (RGD) substrates have been widely explored in tissue engineering, there have been no studies aimed at investigating the combined effects of RGD nanoscale presentation and matrix stiffness on myogenesis. In the present work we use polyelectrolyte multilayer films made of poly(L-lysine) (PLL) and poly(L-glutamic) acid (PGA) as substrates of tunable stiffness that can be functionalized by a RGD adhesive peptide to investigate important events in myogenesis, including adhesion, migration, proliferation and differentiation. C2C12 myoblasts were used as cellular models. RGD presentation on soft films and increasing film stiffness could both induce cell adhesion, but the integrins involved in adhesion were different in the case of soft and stiff films. Soft films with RGD peptide appeared to be the most appropriate substrate for myogenic differentiation, while the stiff PLL/PGA films induced significant cell migration and proliferation and inhibited myogenic differentiation. ROCK kinase was found to be involved in the myoblast response to the different films. Indeed, its inhibition was sufficient to rescue differentiation on stiff films, but no significant changes were observed on stiff films with the RGD peptide. These results suggest that different signaling pathways may be activated depending on the mechanical and biochemical properties of multilayer films. This study emphasizes the advantage of soft PLL/PGA films presenting the RGD peptide in terms of myogenic differentiation. This soft RGD-presenting film may be further used as a coating of various polymeric scaffolds for muscle tissue engineering.

FIGURE 1. Design of biomimetic thin film combining physical and biochemical cues

(A) 1- a polyelectrolyte multilayer film (PEM) is built onto a substrate by alternating deposits of PLL and of PGA. 2- The PEM film can be covalently cross-linked using a water-soluble carbodiimide to modulate its stiffness. 3- Biochemical functionality is provided by adding a final layer of PGA grafted with a RGD-containing peptide. (B) Exponential growth of the film followed by QCM-D.

FIGURE 2. Adhesion and spreading of C2C12 myoblasts at early time

Initial C2C12 cell adhesion and spreading were observed 1 h after plating the cells on NCL, CL, NCL-RGD and CL-RGD films. (A) Actin (red) and nuclei (blue) staining of C2C12 cells to visualize adhesion and spreading on the four types of films (B) Number of adherent cells. (C) Spreading area. (D) Cell circularity quantification. Error bars correspond to SD, *: p < 0.05.

FIGURE 3. Effect of film stiffness and RGD fonctionalization on cytoskeletal organization, focal adhesions and migration

(A) Staining of actin cytoskeleton (red) after 4 h of culture. (B) Staining of phosphorylated focal adhesion kinase (pFAK Y397, green) after 4 h of culture. (C) Myoblast migration measured over 5 h after seeding. (D) Effect of blocking β₁ and/or β₃ integrins using siRNA: quantification of the cell area after 4 h of adhesion (* p < 0.05 compared to scrambled siRNA). Focal adhesions/complexes are indicated by white arrowheads.

FIGURE 4. Myogenic differentiation of C2C12 myoblasts is effective on NCL-RGD films

(A) Phase contrast microscopy observations of C2C12 cell differentiation on the NCL-RGD, CL, and CL-RGD films. After 24 h of proliferation in GM (ie Day −1), the cells were put in DM (ie Day 0) and were let to differentiate until Day 9. Cell detachment was observed on CL and CL-RGD films after few days in DM. (B) Myosin heavy chain (green) and nuclei (blue) labeling (20 × and 63 × magnification). (C) Quantification of the fusion index. Error bars correspond to SD, *: p < 0.05.

FIGURE 5. Myogenin expression is decreased on stiff films

After 24 h of proliferation in GM, the medium was changed to DM and cells were let to differentiate for 2 days. (A) Myogenin labeling at day 1, 2 and 3 of differentiation. (B) Quantification of the percentage of myogenin expressing cells. Error bars correspond to SD, *: p < 0.05.

FIGURE 6. Proliferation is enhanced on CL films resulting in an increased cell number

After 24 h of proliferation in GM (Day −1), the medium was changed to DM (Day 0) and cells were let to differentiate for one day. (A) Staining of BrdU in the nuclei (in black) associated to a fluorescent labeling of total nuclei (blue). (B) Percentage of BrdU-positive cells. (C) Quantification of the total number of adherent cells. Error bars correspond to SD, *: p < 0.05.

FIGURE 7. ROCK kinase inhibition decreases myoblast proliferation and rescues differentiation on CL films

After 24 h of proliferation in GM, cells were transferred to DM and let to differentiate for 6 days. (A, B, C) Myogenin labeling at day 1, 2 and 3 in DM. (D) Percentage of BrdU-positive cells at Day 1 of differentiation. (E) Myosin heavy chain labeling at Day 6 of differentiation. Error bars correspond to SD, *: p <0.05.

FIGURE 8. Model of the interplay between mechanical and biochemical stimuli during induction of C2C12 myogenic differentiation on soft/stiff films functionalized or not with the RGD peptide

Adhesion involving β₃ integrins on NCL-RGD films (soft films with covalently attached RGD peptide) provides favorable conditions for myogenic differentiation of C2C12 cells: when the medium is changed to DM, the rate of proliferating cells decreases and that of myogenin increases. Adhesion on CL and CL-RGD films (stiff films) involving β₁ and β₃ integrins promotes ROCK activation leading to a high proliferative state even in DM and to low myogenin expression. When the cells on stiff films are treated with ROCK inhibitor during differentiation, the rate of proliferating cells decreases significantly on both CL and CL-RGD films. Additionally, on CL films, myogenin expression increases, allowing the cells to undergo myogenic differentiation. However, on CL-RGD films, ROCK inhibition was not sufficient to induce myogenin expression and to allow cell to differentiate. We suggest that mechanical signals (stiffness) on CL-RGD films may affect cell interaction with biochemical signals (RGD peptide), resulting in the inhibition of β₃ integrins by RGD peptide or by β₁ integrins.