Enhancing Schwann cell migration using concentration gradients of laminin-derived peptides

Abstract

Neuroregeneration following peripheral nerve injury is largely mediated by Schwann cells (SC), the principal glial cell that supports neurons in the peripheral nervous system. Axonal regeneration in vivo is limited by the extent of SC migration into the gap between the proximal and distal nerve, however, little is known regarding the principal driving forces for SC migration. Engineered microenvironments, such as molecular and protein gradients, play a role in the migration of many cell types, including cancer cells and fibroblasts. However, haptotactic strategies have not been applied widely to SC. Herein, a series of tethered laminin-derived peptides were analyzed for their influence on SC adhesion, proliferation, and alignment. Concentration gradient substrates were fabricated using a controlled vapor deposition method, followed by covalent peptide attachment via a thiol-ene reaction, and characterized by X-ray photoelectron spectroscopy (XPS) and MALDI-MS imaging. While tethered RGD peptides supported SC adhesion and proliferation, concentration gradients of RGD had little influence on biased SC directional migration. In contrast, YIGSR promoted less SC attachment than RGD, yet YIGSR peptide gradients directed migration with a strong bias to the concentration profile. With YIGSR peptide, overall speed increased with the steepness of the peptide concentration profile. YIGSR gradients had no haptotactic effect on rat dermal fibroblast migration, in contrast to fibroblast migration on RGD gradients. The response of SC to these tethered peptide gradients will guide the development of translationally relevant constructs designed to facilitate endogenous SC infiltration into defects for nerve regeneration.

Keywords: Cell migration; Concentration gradient; Haptotaxis; Laminin-derived peptides; Schwann cells.

Figure 1.

Functional self-assembled peptide gradients fabrication and characterization. (A) Fabrication scheme. First, one-dimensional, vinyl-terminated concentration gradient substrates are fabricated on a confined chamber by vapor deposition using 5-hexenyldimethylchlorosilane and 25 mm² glass slides as substrates. Cysteine-terminated peptide can then be attached to the surface using a thiol-ene “click” reaction. (B) The vinyl end group gradient profile was characterized by surface energy. Data was collected from five equidistant points on the surface at 5 mm intervals. (C) Average peptide concentration calculated based on XPS C_1s and N_1s high resolution peaks, based on three individually fabricated and characterized substrates for each peptide of interest. Peptides concentration increased monotonically along the x direction of the substrate. (D) The peptide gradient profile was confirmed by MALDI-MS analysis, here represented by a CQAASIKVAV concentration gradient sample. MS peaks corresponding to the tethered peptide mass and linker. (E) The choropleth intensity image corresponds to the m/z signal, which increases along the concentration gradient.

Figure 2.

SC response to peptide concentration gradients. (A) SC number on peptide uniform SAMs of RGD, YIGSR, IKVAV, CPDSGR and IIKDI, respectively. Cell number was collected at each sample position after 1 and 3 days of culture. Values are represented as means ± standard deviation, with n = 3. # represents significant difference for cell number from D1 to D3, lowercase letters compare cell number for different peptides at D1, and capital letters compare different peptides at D3 (ANOVA with Tukey’s). (B) Immunofluorescent staining of nuclei (blue), vinculin (red), and actin (green) after 3 days of culture showing cell alignment to the gradient direction. Images were taken at 20x magnification. Scale bar: 100 μm. Images indicate that SC have high proliferative potential in RGD samples, reaching confluence after 3 days of culture, while YIGSR promoted cell alignment to the gradient direction, identified by the directionality of the actin filaments. (C) SC alignment as a function of the direction of concentration gradient. A Matlab code based on an edge detection method was used to quantify actin fiber alignment. Data is given as function of percent actin aligned relative to each respective uniform control (%/%) at day 3. Values are represented as means ± standard deviation, with and n = 3. * represents significant difference for actin alignment from a certain position compared to the respective control (ANOVA with Dunnett’s). Data show that RGD concentration gradient, for example, does not promote cell alignment, as opposed to YIGSR, where cell alignment increased with peptide concentration.

Figure 3.

Migration behavior of SC in RGD and YIGSR concentration gradients. Time-lapse microscopy was used to track the position of individual cells. Images were taken every 10 min for a total of 24 h per experiment. (A, B, C, D, E) Centroid tracks from 30 representative cells in a typical experiment for YIGSR gradient, uniform YIGSR, RGD gradient, uniform RGD, and uniform laminin, respectively, with initial position of each track superimposed at 0,0 for clarity. (F) Polar plot showing the end point of each cell’s trajectory relative to its origin. Cells moving upward move toward the positive gradient of concentration of the respective peptide. (G) Mean square displacement curves for over 24 h period. (H) Overall velocity and bias velocity to the direction of the gradient μm/min (ANOVA with Tukey’s). Capital letter were used to correlate velocity in the x direction; lowercase letter were used to correlate velocity in the y direction; and Greek letters were used to correlate overall speed. Means that not share a letter were significant different. YIGSR concentration gradients had a positive influence on directing Schwann cell migration.

Figure 4.

Migration behavior of rat dermal fibroblasts in RGD and YIGSR concentration gradients. Time-lapse microscopy was used to track the position of individual cells. Images were taken every 10 min for a total of 24 h per experiment. (A, B, C, D) Centroid tracks from 50 representative cells in a typical experiment for YIGSR gradient, uniform YIGSR, RGD gradient, and uniform RGD, respectively, with initial position of each track superimposed at 0,0 for clarity. (E) Polar plot showing the end point of each cell’s trajectory relative to its origin. Cells moving upward move toward the positive gradient of concentration of the respective peptide. (F) Mean square displacement curves for over 24 h period. (G) Overall velocity and bias velocity to the direction of the gradient μ:min (ANOVA with Tukey’s). Lowercase letter were used to correlate velocity in the x direction and capital letters were used to correlate overall speed. Means that not share a letter were significant different. RGD concentration gradients had a positive influence on directing fibroblast migration, but no influence was observed for YIGSR gradient.

Figure 5.

Influence of YIGSR gradient steepness on SC migration. Time-lapse microscopy was used to capture the motility responses on individual cells. Images were taken every 10 min for a total of 24 h per experiment. (A, B, C, D, E) Centroid tracks from a minimum of 50 representative cells in a typical experiment for YIGSR shallow, medium and steep gradients, uniform YIGSR SAM, and saline gradient, respectively, with initial position of each track superimposed at 0,0 for clarity. (F) Polar plot showing the end point of each cell’s trajectory relative to its origin. Cells moving upward move toward the positive gradient of concentration of the respective peptide. (G) Mean square displacement curves for over 24 h period. (H) Overall speed and bias velocity to the direction of the gradient μm/min (ANOVA with Tukey’s). Capital letter were used to correlate velocity in the x direction; lowercase letter were used to correlate velocity in the y direction; and Greek letters were used to correlate overall speed. Means that not share a letter were significant different. These data suggest that YIGSR steep gradient promoted faster directional cell migration.

Figure 6.

Mean chemotaxis index, , over time for SC migrating in response to the concentration of RGD and YIGSR medium gradients, and YIGSR steep gradient. CI was determined by averaging the chemotaxis index obtained from at least 30 cells at each time point. Overall, cells on YIGSR gradients travelled more consistently in the direction of the gradient represented by the higher , and this distance, compared to the total travelled distance, increased as the gradient steepness increased.