Transfer of assembled collagen fibrils to flexible substrates for mechanically tunable contact guidance cues

Abstract

Contact guidance or bidirectional migration along aligned fibers modulates many physiological and pathological processes such as wound healing and cancer invasion. Aligned 2D collagen fibrils epitaxially grown on mica substrates replicate many features of contact guidance seen in aligned 3D collagen fiber networks. However, these 2D collagen self-assembled substrates are difficult to image through, do not have known or tunable mechanical properties and cells degrade and mechanically detach collagen fibrils from the surface, leading to an inability to assess contact guidance over long times. Here, we describe the transfer of aligned collagen fibrils from mica substrates to three different functionalized target substrates: glass, polydimethylsiloxane (PDMS) and polyacrylamide (PA). Aligned collagen fibrils can be efficiently transferred to all three substrates. This transfer resulted in substrates that were to varying degrees resistant to cell-mediated collagen fibril deformation that resulted in detachment of the collagen fibril field, allowing for contact guidance to be observed over longer time periods. On these transferred substrates, cell speed is lowest on softer contact guidance cues for both MDA-MB-231 and MTLn3 cells. Intermediate stiffness resulted in the fastest migration. MTLn3 cell directionality was low on soft contact guidance cues, whereas MDA-MB-231 cell directionality marginally increased. It appears that the stiffness of the contact guidance cue regulates contact guidance differently between cell types. The development of this collagen fibril transfer method allows for the attachment of aligned collagen fibrils on substrates, particularly flexible substrates, that do not normally promote aligned collagen fibril growth, increasing the utility of this collagen self-assembly system for the fundamental examination of mechanical regulation of contact guidance.

Figure 1:. Transferring assembled collagen fibrils onto alternative substrates.

(A) The schematic shows the side view of the steps involved in transferring collagen fibrils from mica to functionalized substrates with different stiffness (glass, PDMS, and PA (2000 and 200 Pa)) using the gelatin protocol described in the Experimental section. (B) Representative AFM and SHG images of aligned collagen fibrils transferred from mica substrates to functionalized glass substrates. ‘mica pre-transfer’ images indicate collagen fibrils on mica substrates before transferring. ‘mica post-transfer’ images show collagen fibrils on mica substrates after transferring. ‘glass pre-transfer’ indicate lack of collagen fibrils on glass substrates before transferring. ‘glass post-transfer’ show collagen fibrils on glass substrates after transferring. (C) Normalized collagen density and (D) collagen area fraction on mica substrates (pre- and post-transfer), glass substrates (pre- and post-transfer), PDMS, and PA (2000Pa and 200Pa). Error bars represent 95% confidence intervals. Calibration bar length for AFM images is 2 μm. Calibration bar length for SHG images is 20 μm.

Figure 2:. MDA-MB-231 cell detachment on collagen fibrils transferred from mica to alternative substrates.

(A) Normalized cell density and (B) area fraction occupied by MDA-MB-231 cells change with time on aligned collagen fibrils assembled on mica (circles). The decrease seen at 24 h is blocked when aligned collagen fibrils are transferred to glass (closed square), PDMS (closed diamond), and PA (2000Pa (open triangle) and 200 Pa (closed triangle)) as compared to aligned collagen fibrils assembled on mica (closed circle). Error bars represent 95% confidence intervals.

Figure 3:. MDA-MB-231 cell alignment on collagen fibrils transferred from mica to alternative substrates.

Images of F-actin in cells plated on aligned collagen fibrils assembled on (A) mica or transferred to (B) glass, (C) PDMS, (D) PA (2000 Pa) or (E) PA (200 Pa). (F-H) The angle distribution of cells 12 h after plating on aligned collagen fibrils assembled on mica, transferred glass and PA (2000 Pa). The sum of three von Mises distributions separated by 60 degrees is used to fit the data. (I) Aligned cell fraction and (J) cell alignment area fraction of cells adhered to aligned collagen fibrils assmbled on mica or transferred to glass, PDMS or PA (2000 Pa and 200 Pa). Error bars represent 95% confidence intervals. Calibration bar length is 30 μm.

Figure 4:. MDA-MB-231 cell alignment on collagen fibrils transferred from mica to alternative substrates at different times.

(A) Aligned cell fraction, (B) cell alignment area fraction and (C) distribution spread parameter, κ_cells as a function of time. Cells adhered to aligned collagen fibrils assmbled on mica (closed circle) or transferred to glass (closed square), PDMS (closed diamond) or PA (2000 Pa (open triangle) and 200 Pa (closed triangle)). Error bars represent 95% confidence intervals.

Figure 5:. Trajectories of fluorescent microspheres adsorbed on aligned collagen fibrils.

Collagen fibrils (A) assembled on mica substrates, (B) transferred to glass substrates or (C) transferred to 2000 Pa PA substrates and labeled with the fluorescent microspheres. Microsphere trajectories are colored differently depending on their average displacement. Blue indicates mean displacements larger than 2 μm, green indicates average displacements between 600 nm and 2 μm and red indicates average displacements smaller than 600 nm. The cell outline from one time point is shown in white. (D) Fraction of microsphere displacements when the distances between the cell edge and the microsphere is very small (0–10 μm). Arrows indicate the microsphere at which the transferred substrate crosses the mica substrate (E) Fraction of large displacements (> 6 μm). (F) Fraction of microsphere displacements when the distances between the cell edge and the microsphere is very large (180–190 μm). (G) Microsphere number per μm² on different substrates with and without fibrils. Calibration bar length is 30 μm.

Figure 6:. Angle distribution of collagen on the mica substrates, and transferred substrates (glass, PDMS, and 2000Pa and 200Pa polyacrylamide) seeded with MDA-MB-231 cells.

(A-C) The angle distribution of collagen at 0, 12, and 24 h on mica substrates, (D-F) The angle distribution of collagen at 0, 12, and 24 h after transfer to glass substrates, (G-I) The angle distribution of collagen a 0, 12, and 24 h after transfer to PDMS substrates (J-L) The angle distribution of collagen at 0, 12, and 24 h after transfer to 2000 Pa PA substrates (M-O) The angular distribution of collagen at 0, 12, and 24 h after transfer to 200 Pa PA substrates. Points represent data and the line represents the von Mises fit.

Figure 7:. Collagen fibril alignment on mica substrates and transferred substrates (glass, PDMS, and 2000Pa and 200Pa polyacrylamide) seeded with MDA-MB-231 cells.

(A) The relative intensity of the collagen fibrils on the mica-based substrates over 24 hours, normalized to the initial intensity of the control at 0 hours. Collagen fibrils on all substrates showed rapid degradation of the collagen signal over time. (B) The collagen area fraction representing the sum of both ordered and non-ordered fractions of the collagen signal on the substrates showed significant difference over 24 hours on mica, glass and PDMS substrates. (C) The non-ordered collagen fractions of the collagen signal from the different substrates showed no significant differences (D) Collagen alignment fraction, (E) Aligned collagen signal fraction, as a product of the aligned collagen fraction and the collagen area fraction and (F) distribution spread parameter, κ_collagen as a function of time on function. Cells plated on aligned collagen fibrils assembled on mica (circle), on transferred glass (closed squares), PDMS (closed diamond), and 2000Pa (open triangle) and 200Pa (closed triangle). Error bars represent 95% confidence intervals.

Figure 8:. Migration behavior of MDA-MB-231 and MTLn3 cells on mica substrates and transferred substrates (glass, PDMS, and 2000 Pa and 200 Pa polyacrylamide).

(A) speed and (B) directionality of MTLn3 cells. (C) speed and (D) directionality of MDA-MB-231 cells. Error bars are 95% confidence intervals. Solid black lines indicate that the means are statistically significant from the mica substrates (p < 0.05 using two-tailed student’s t-test).

Figure 9:. Schematic of fibrils transfer and cell behavior from mica substrates to target substrates.