Evidence of innervation following extracellular matrix scaffold-mediated remodelling of muscular tissues

Abstract

Naturally occurring porcine-derived extracellular matrix (ECM) has successfully been used as a biological scaffold material for site-specific reconstruction of a wide variety of tissues. The site-specific remodelling process includes rapid degradation of the scaffold, with concomitant recruitment of mononuclear, endothelial and bone marrow-derived cells, and can lead to the formation of functional skeletal and smooth muscle tissue. However, the temporal and spatial patterns of innervation of the remodelling scaffold material in muscular tissues are not well understood. A retrospective study was conducted to investigate the presence of nervous tissue in a rat model of abdominal wall reconstruction and a canine model of oesophageal reconstruction in which ECM scaffolds were used as inductive scaffolds. Evidence of mature nerve, immature nerve and Schwann cells was found within the remodelled ECM at 28 days in the rat body wall model, and at 91 days post surgery in a canine model of oesophageal repair. Additionally, a microscopic and morphological study that investigated the response of primary cultured neurons seeded upon an ECM scaffold showed that neuronal survival and outgrowth were supported by the ECM substrate. Finally, matricryptic peptides resulting from rapid degradation of the ECM scaffold induced migration of terminal Schwann cells in a concentration-dependent fashion in vitro. The findings of this study suggest that the reconstruction of tissues in which innervation is an important functional component is possible with the use of biological scaffolds composed of extracellular matrix.

Figure 1

Timeline for evaluation of innervation in the rat abdominal wall model and the canine esophageal model.

Figure 2

Histologic images of remodeled UBM 28 days after repair of a 1 cm × 1 cm defect in the rat abdominal wall with specific staining for (A) neurofilament, (B) GAP43, and (C) GFAP. Specificity of staining was confirmed by primary delete negative controls for (D) neurofilament, (E) GAP43, and (F) GFAP. Positive control staining was confirmed in native rat abdominal wall muscle for (G) neurofilament, (H) GAP43, and (I) GFAP. All scale bars are 75 μm.

Figure 3

Histologic images of remodeled UBM 91 days after repair of a 5 cm long canine esophageal endomucosal resection of with removal of 70% of the muscularis externa. Specific staining is shown for (A) neurofilament, (B) GAP43, and (C) GFAP. Specificity of staining was confirmed by primary delete negative controls for (D) neurofilament, (E) GAP43, and (F) GFAP. All scale bars are 75 μm.

Figure 4

Histologic images of remodeled UBM 104 days after repair of a 5 cm long canine esophageal endomucosal resection of with removal of 70% of the muscularis externa. Specific staining is shown for (A) neurofilament, (B) GAP43, and (C) GFAP. Specificity of staining was confirmed by primary delete negative controls for (D) neurofilament, (E) GAP43, and (F) GFAP.

Figure 5

Histologic images of native esophagus displaying the intramuscular plexus of nerves, with specific staining for (A) neurofilament, (B) GAP43, and (C) GFAP.

Figure 6

β-tubulin III (green), glial fibrillary acidic protein (GFAP) (red) and DAPI (blue). (A) Immunofluorescent staining of cells seeded on poly-L-lysine control cover slips after 5 days in culture. (B) Immunofluorescent images of cells seeded on the abluminal surface of the UBM-ECM scaffolds. (C) Immunofluorescent images of seeded sections stained with secondary antibodies only. All scale bars are 50 μm.

Figure 7

Scanning electron micrographs of primary spinal cord neurons seeded on the abluminal surface of the UBM scaffold (A–C).

Figure 8

(A) Migration of Schwann cells towards matricryptic peptides resulting from rapid degradation of UBM or corresponding control. (B) Survival of Schwann cells in the presence of matricryptic peptides resulting from rapid degradation of UBM or corresponding control. (* p < 0.05 compared to control, *** p < 0.005 compared to control).