Micropatterns and peptide gradient on the inner surface of a guidance conduit synergistically promotes nerve regeneration in vivo

Abstract

Both of the surface topographical features and distribution of biochemical cues can influence the cell-substrate interactions and thereby tissue regeneration in vivo. However, they have not been combined simultaneously onto a biodegradable scaffold to demonstrate the synergistic role so far. In this study, a proof-of-concept study is performed to prepare micropatterns and peptide gradient on the inner wall of a poly (D,L-lactide-co-caprolactone) (PLCL) guidance conduit and its advantages in regeneration of peripheral nerve in vivo. After linear ridges/grooves of 20/40 μm in width are created on the PLCL film, its surface is aminolyzed in a kinetically controlled manner to obtain the continuous gradient of amino groups, which are then transferred to CQAASIKVAV peptide density gradient via covalent coupling of glutaraldehyde. The Schwann cells are better aligned along with the stripes, and show a faster migration rate toward the region of higher peptide density. Implantation of the nerve guidance conduit made of the PLCL film having both the micropatterns and peptide gradient can significantly accelerate the regeneration of sciatic nerve in terms of rate, function recovery and microstructures, and reduction of fibrosis in muscle tissues. Moreover, this nerve conduit can also benefit the M2 polarization of macrophages and promote vascularization in vivo.

Keywords: Contact guidance effect; Micropatterns; Nerve guidance conduits; Nerve regeneration; Peptides gradient.

Graphical abstract

Scheme 1

Schematic drawing to delineate the preparation of micropatterned poly (D,L-lactide-co-caprolactone) (PLCL) film incorporated with a CQAASIKVAV peptide density gradient, which is manufactured into conduit for peripheral nerve regeneration. The stripe micropatterns with ridges and grooves are fabricated via polydimethylsiloxane (PDMS) template thermally pressing onto a PLCL film, which is then aminolyzed in 1,6-hexanediamine solution via a time-controlled injection method to obtain the gradient distribution of amino groups along the stripes. By covalent coupling with GA, the density gradient of amino group is transferred to the CQAASIKVAV peptides density gradient. The micropatterned PLCL film with a peptide gradient can synergistically enhance the directional migration of SCs toward to the high density region of the patterns in vitro, whereas the guidance conduit obtained thereof can significantly promote the reconstruction and functional recovery of rat sciatic nerve in vivo.

Fig. 1

SEM images of micropatterned PLCL films after being aminolyzed for (a,a1) 2, (b,b1) 4 and (c,c1) 8 min, followed by peptides grafting. (a1-c1) are higher resolution images. (d) Static water contact angles of various films. Due to the anisotropic nature of the micropatterned and/or gradient surfaces, the water contact angles were unsymmetric at different directions and they were detected paralleling to the stripes. (e) Scheme to show the relationship between the aminolysis time and the position of PLCL film. (f) The –NH₂ density on the surfaces of flat gradient PLCL and micropatterned gradient PLCL films with various aminolysis time. (g) Scheme to show the five parts of flat gradient PLCL and micropatterned gradient PLCL films for peptide detection. (h) The peptide density on the surface of various micropatterned gradient PLCL position. In this Fig. and thereafter, “F” and “MU” represent the original flat film and the micropatterned PLCL film with uniform peptide density, respectively. The “X” in the FG-X and MG-X (X = 0, 2.5, 5, 10) means the position on the flat and micropatterned PLCL films with a peptide gradient density. * indicates statistically significant difference at p < 0.05 level, n = 4.

Fig. 2

Migration of single Schwann cell. Migration traces of SCs after being seeded for 12 h at different positions on (a) flat, (b) FG-0, (c) FG-2.5, (d) FG-5, (e) FG-10, (f) MU, (g) MG-0, (h) MG-2.5, (i) MG-5, and (j) MG-10 films, respectively. (k) The statistic percentage of SCs migrating to the gradient direction, (l) migration rates parallel to the gradient direction, and (m) net displacement of SCs quantified from the migration traces. * indicates statistically significant difference at p < 0.05 level, n = 3.

Fig. 3

Optical images of regenerated nerves in vivo. The typical photos of regenerated nerves by Flat (the flat PLCL film), FG (the flat PLCL film with a peptide gradient density), MU (the micropatterned PLCL film with uniform peptide density), MG (the micropatterned PLCL film with a peptide gradient density) conduits, and autografts post-surgery for 9 d, 14 d, 8 w and 16 w.

Fig. 4

(a) Merged immunofluorescence staining images of S100β (green), NF200 (red), and DAPI (blue) for SCs, axons and cell nuclei in proximal parts of newborn nerves post surgery for 9 and 14 d, respectively. (b) Merged immunofluorescence staining images of CD163 (green), CD86 (red), and DAPI (blue) for M2, M1 and cell nuclei in the proximal segments of regenerated nerves post surgery for 9 and 14 d, respectively. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Fig. 5

In vivo nerve regenration assay. (a) CMAP amplitude and (b) NCV of regenerated nerves post surgery for 8 and 16 w. (c) Wet weight ratio, (d) percentage of collagen fibers of the gastrocnemius muscles post surgery for 9 d, 14 d, 8 w and 16 w. * indicates statistically significant difference at p < 0.05 level, n = 5.

Fig. 6

Microstructures assay in vivo. Merged immunofluorescence staining images of green S100β (SCs), red NF200 (axons), and blue DAPI (cell nuclei) of newborn nerves guided by (a,a1) Flat, (b,b1) FG, (c,c1) MU, and (d,d1) MG conduits, and (e,e1) autografts at 16 w post-surgery, respectively. The white arrows point the axon fibers wrapped by myelin sheath in the regenerated nerves. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Fig. 7

Microstructures assay in vivo. TEM images of newborn nerves guided by (a,a1; f,f1) Flat, (b,b1; g,g1) FG, (c,c1; h,h1) MU, and (d,d1; i,i1) MG conduits, and (e,e1; j,j1) autografts post surgery in vivo for (a–e) 8 and (f–j) 16 w, respectively. (k) The number of myelinated axons, (l) average myelinated axon diameter, and (m) thickness of myelin sheath in the middle part of the regenerated nerves post surgery for 8 and 16 w in vivo, respectively. The scale bars are 4 μm and 0.4 μm in (a–j) and (a1-j1), respectively. * indicates statistically significant difference at p < 0.05 level, n = 4.

Fig. 8

Vascularization assay in vivo. Immunohistochemistry staining for (a–j) CD31 (brown) and nuclei (blue) of newborn nerves guided by (a,f) Flat, (b,g) FG, (c,h) MU, and (d,i) MG conduits, and (e,j) autografts post surgery in vivo for 8 (a–e) and 16 (f–j) w, respectively. The black arrows point the vessel-like structure in the regenerated nerves. Immunofluorescence staining for (k–t) CD34 of newborn nerves guided by (k,p) Flat, (l,q) FG, (m,r) MU, and (n,s) MG conduits, and (o,t) autografts post surgery in vivo for 8 (k–o) and 16 (p–t) w, respectively. The quantification of (u) positive CD31 area fraction, (v) vessel like structure (VLS) number/ROI mm² and (w) positive CD34 area fraction in the middle parts of the regenerated nerves post surgery for 8 and 16 w in vivo, respectively. * indicates statistically significant difference at p < 0.05 level, n = 4. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)