(-)-Epigallocatechin gallate-loaded polycaprolactone scaffolds fabricated using a 3D integrated moulding method alleviate immune stress and induce neurogenesis

Abstract

Objectives: In peripheral neuropathy, the underlying mechanisms of nerve and muscle degeneration include chronic inflammation and oxidative stress in fibrotic tissues. (-)-Epigallocatechin gallate (EGCG) is a major, active component in green tea and may scavenge free radical oxygen and attenuate inflammation. Conservative treatments such as steroid injection only deal with early, asymptomatic, peripheral neuropathy. In contrast, neurolysis and nerve conduit implantation work effectively for treating advanced stages.

Materials and methods: An EGCG-loaded polycaprolactone (PCL) porous scaffold was fabricated using an integrated moulding method. We evaluated proliferative, oxidative and inflammatory activity of rat Schwann cells (RSCs) and rat skeletal muscle cells (RSMCs) cultured on different scaffolds in vitro. In a rat radiation injury model, we assessed the morphological, electrophysiological and functional performance of regenerated sciatic nerves and gastrocnemius muscles, as well as oxidative stress and inflammation state.

Results: RSCs and RSMCs exhibited higher proliferative, anti-oxidant and anti-inflammatory states in an EGCG/PCL scaffold. In vivo studies showed improved nerve and muscle recovery in the EGCG/PCL group, with increased nerve myelination and muscle fibre proliferation and reduced macrophage infiltration, lipid peroxidation, inflammation and oxidative stress indicators.

Conclusions: The EGCG-modified PCL porous nerve scaffold alleviates cellular oxidative stress and repairs peripheral nerve and muscle structure in rats. It attenuates oxidative stress and inflammation in vivo and may provide further insights into peripheral nerve repair in the future.

Keywords: (-)-epigallocatechin gallate; immune milieu; integrated moulding; nerve scaffold.

Figure 1

Scheme of (‐)‐epigallocatechin gallate‐loaded polycaprolactone scaffold fabrication using integrated moulding and nerve conduit implantation in rat models (A). Characterization of an (‐)‐epigallocatechin gallate‐loaded polycaprolactone nerve conduit. The scanning electron microscopy and optical images of the EGCG/PCL nerve conduit (B‐D). CCK8 assay for RSCs and RSMCs on EGCG/PCL and PCL scaffolds at 24, 72, 120 and 168 hours. *P < .05 compared with PCL scaffolds (RSCs), ^Δ P < .05 compared with PCL scaffolds (RSMCs) (E)

Figure 2

In vitro cumulative release of EGCG from EGCG/PCL conduits in PBS by ultraviolet‐visible spectrometry

Figure 3

Ki‐67 immunofluorescent staining for RSCs on EGCG/PCL scaffolds (A, B, C) and on PCL scaffolds (D, E, F). S100 immunofluorescent staining for RSCs on EGCG/PCL scaffolds (G, H, I) and PCL scaffolds (J, K, L). Nrf2 immunofluorescent staining for RSCs on EGCG/PCL scaffolds (M, N, O) and PCL scaffolds (P, Q, R). A, D, G, J, M and P show specific marker staining (in detail, A & D: Ki‐67, G & J: S100, M & P: Nrf2). B, E, H, K, N and Q show nuclei staining. C, F, I, L, O and R show merged images of marker and nuclei staining. The scale bar is 100 μm

Figure 4

nNOS (A‐I), Nrf2 (J‐R) and MnSOD (S‐U) immunohistochemistry staining for three groups: EGCG/PCL conduit group (A‐C, J‐L, S), PCL conduit group (D‐F, M‐O, T) and sham group (G‐I, P‐R, U). The scale bar is 100 μm

Figure 5

CD68 (A‐C) and TNF‐α (D‐L) immunohistochemistry staining for three groups: EGCG/PCL conduit group (A, D‐F), PCL conduit group (B, G‐I) and sham group (C, J‐L). The scale bar is 100 μm. nNOS‐positive area (%) (M), Nrf2‐positive area (%) (N), MnSOD‐positive area (%) (O), CD68‐positive area (%) (P)and TNF‐α–positive area (%) (Q). *P < .05 compared with sham group; #P < .05 compared with PCL conduit group

Figure 6

WB for CD68, TNF‐α and IL‐6 among three groups and their relative expression (A). Gastrocnemius muscle morphology evaluation from three groups. HE staining for the EGCG/PCL conduit group (B), PCL conduit group (C) and sham group (D), and TB staining for the EGCG/PCL conduit group (E), PCL conduit group (F) and sham group (G). The scale bar is 100 μm. Muscle fibre area (%) (H), gastrocnemius muscle weight (g) (I), muscle strength (%) (J) and creatine phosphokinase level U/g (K). *P < .05 compared with sham group; ^# P < .05 compared with PCL conduit group