Peripheral nerve morphogenesis induced by scaffold micropatterning

Abstract

Several bioengineering approaches have been proposed for peripheral nervous system repair, with limited results and still open questions about the underlying molecular mechanisms. We assessed the biological processes that occur after the implantation of collagen scaffold with a peculiar porous micro-structure of the wall in a rat sciatic nerve transection model compared to commercial collagen conduits and nerve crush injury using functional, histological and genome wide analyses. We demonstrated that within 60 days, our conduit had been completely substituted by a normal nerve. Gene expression analysis documented a precise sequential regulation of known genes involved in angiogenesis, Schwann cells/axons interactions and myelination, together with a selective modulation of key biological pathways for nerve morphogenesis induced by porous matrices. These data suggest that the scaffold's micro-structure profoundly influences cell behaviors and creates an instructive micro-environment to enhance nerve morphogenesis that can be exploited to improve recovery and understand the molecular differences between repair and regeneration.

Keywords: Biomaterials; Medical device; Nerve regeneration; Peripheral nervous system.

Fig. 1. Scaffolds with radially oriented porosity.

Scanning electron micrographs of MPCS#1 (A–C) and MPCS#2 (D). MPCS#1 with a gradient in radially oriented pore distribution of the wall (A): smaller porosity of the external portion (B) compared to internal portion (C); MPCS#2 with uniform and smaller pore distribution (D). Semithin sections and electron microscopy of MPCS#1 (E–G) and MPCS#2 (H–J)-implanted rats after 8 days. In MPCS#1, macrophages (E, arrowhead; F, mf) and myofibroblast (G, myo) between the pores and attached to the collagen micro-structure (E–G, arrows). In MPCS#2 few macrophages (H, arrowhead and I, μm) and myofibroblasts (J, myo) are present (H–J, arrows: collagen micro-structure). Scale bar: in A and D 100 μm; 20 μm for B, C, E and H; 2 μm for F, G, I and J.

Fig. 2. Angiogenesis, Schwann cells (SC) migration and differentiation.

Morphological analysis of MPCS (A–F) and CCNG (G and H)-implanted nerves at 8 days. Semithin sections showing the tube wall (A, asterisk) and the presence of the clot in the MPCS midgraft. Electron micrographs demonstrate well differentiated fibroblast (B, arrowhead) surrounded by organized collagen (B, arrow) and fibroblast with expanded endoplasmic reticulum in active phase of synthesis (C); many vessels are present in the midgraft (D, arrowhead), with mature endothelium connected by tight junction, confirmed by electron microscopy (E, arrowhead) and VE-cadherin immunofluorescence analysis (F, arrowhead). Semithin sections of CCNG showing the wall of collagen tube (G, asterisk) with few cells inside (H). I: GE pattern of genes involved in collagen synthesis, angiogenesis and SC migration and differentiation at the selected time points. Scale bar: 100 μm A and G; 20 μm D and H; 5 μm F; 1 μm B, C and E.

Fig. 3. Axonal growth and Schwann cells (SC)/axon interaction.

A–C: MPCS morphological analysis at 15 days after the implant. Semithin sections and immunofluorescence for neurofilaments demonstrated the presence of many axons inside MPCS (A and B, arrows). C: electron microscopy confirmed the presence inside MPCS of some axons (ax) surrounded by Schwann cell (n: SC nuclei) characterized by normal basal lamina (D, arrowhead). E: mRNA levels in each experimental group at the selected time points for keys players for SC/axon (integrin beta1) and SC/extracellular matrix (dystroglycan, laminin beta1 and laminin-alpha2) interactions. Scale bars: 10 μm A; 1 μm B and C.

Fig. 4. Myelination.

Semithin sections and electron microscopy of MPCS (A–F) and CCNG (G–K)-implanted rats after 40 days. The collagen tube is absorbed and substituted by normal endoneurium and perineurium (A, arrow); many fibers have thin myelin (B, arrows) and few normal myelin thickness (B, arrows); electron microscopy shows different stages of myelination: axons enwrapped by few myelin sheaths (D), others with compact myelin (E). F: ultrastructural analysis of perineurium displaying typical pinocytic vesicle (arrowhead). In CCNG group, the collagen tube is still present (G, asterisk), with inflammatory cells (H, arrowhead); minifascicles (H, arrows) composed by thin myelinated fibers are present; electron microscopy shows thin myelinated fibers (J) and naked axons (K, ax ¹/4 axon). L, GE pattern of molecules relevant in the early phases of myelination and structural integrity of PNS. Scale bar: 100 μm F; 20 μm A and G; 10 μm B and H; 2 μm C, D, E, I and J.

Fig. 5. Nerve regeneration.

Morphological analysis of WT sciatic nerve (A–D), MPCS (E–I) and CCNG (J-M)-implanted rats after 60 days. Semithin cross section of entire normal sciatic nerve (A): fiber density, myelinatyon and endoneurium are normal (BeD). In MPCS group, scaffold is completely substituted by normal nerve (E) with normal amount of myelinated fibers (F and G); the stromal tissues is re-established with intrafascicular septum (F, arrowhead). In the perineurium, clusters of regeneration are present (H, arrow); electron microscopy shows compacted myelin (I, asterisk) and normal non-myelinated fibers (I). In CCNG group the scaffold wall is still present (J, asterisk), the endoneurium is characterized by minifascicle formations (K, arrowhead) with thin myelinated fibers grouped in clusters of regeneration (L and M, arrows). N: fiber density and g-ratio in sciatic nerve from WT, MPCS and CCNG inside the scaffold (−I) and outside (−O) at tibial nerve (***= p < 0.0005). O: GE pattern of key protein component of myelin in PNS. Scale bar: 100 μm A, E and J; 40 μm B, F and K; 20 μm C, G, H, L, N, O and P; 2 μm D, I and M.

Fig. 6. Gene expression modulation.

A: Co-expression network of up-regulated genes in samples from MPCS-implanted rats only. The node colour corresponds to enriched biological process. Rectangular node shape corresponds to the transcription factor binding (TFBS) V$MEF2_03, which was found to be the most enriched site in the cREMaG database. Additional TFBS such as V$GATA_C is also enriched. B: cartoon model proposed for MPCS action on cells behavior and axonal regeneration. Mf: macrophage; Myf: myofibroblast; SF: soluble factor; AX/SC: axon/Schwann cell. MPCS, thanks to its micro-structure, allow macrophages and myofibroblast to leave the midgraft, avoiding scar tissue formation, while soluble factors as growth factors may enter into the scaffold and promote axonal regeneration and ultimately leading to normal myelinated fibers.