The Physicochemical Properties of Decellularized Extracellular Matrix-Coated 3D Printed Poly(ε-caprolactone) Nerve Conduits for Promoting Schwann Cells Proliferation and Differentiation

Abstract

Although autologous nerve grafting remains the gold standard treatment for peripheral nerve injuries, alternative methods such as development of nerve guidance conduits have since emerged and evolved to counter the many disadvantages of nerve grafting. However, the efficacy and viability of current nerve conduits remain unclear in clinical trials. Here, we focused on a novel decellularized extracellular matrix (dECM) and polydopamine (PDA)-coated 3D-printed poly(ε-caprolactone) (PCL)-based conduits, whereby the PDA surface modification acts as an attachment platform for further dECM attachment. We demonstrated that dECM/PDA-coated PCL conduits possessed higher mechanical properties when compared to human or animal nerves. Such modifications were proved to affect cell behaviors. Cellular behaviors and neuronal differentiation of Schwann cells were assessed to determine for the efficacies of the conduits. There were some cell-specific neuronal markers, such as Nestin, neuron-specific class III beta-tubulin (TUJ-1), and microtubule-associated protein 2 (MAP2) analyzed by enzyme-linked immunosorbent assay, and Nestin expressions were found to be 0.65-fold up-regulated, while TUJ1 expressions were 2.3-fold up-regulated and MAP2 expressions were 2.5-fold up-regulated when compared to Ctl. The methodology of PDA coating employed in this study can be used as a simple model to immobilize dECM onto PCL conduits, and the results showed that dECM/PDA-coated PCL conduits can as a practical and clinically viable tool for promoting regenerative outcomes in larger peripheral nerve defects.

Keywords: autologous nerve grafting; decellularized extracellular matrix; neuronal differentiation; poly(ε-caprolactone) conduits; polydopamine.

Figure 1

Schematic drawing of (A) the conduit and (B) the dumbbell-shaped sample used in the mechanical testing. The (C) top- and (D) side-view photograph of dECM/PDA-coated conduits.

Figure 2

The tensile stress–strain curve of 3D-printed PCL specimens.

Figure 3

XPS spectra obtained on PCL conduits after coating with PDA and various concentrations of dECM. The peak at 400 cm⁻¹ corresponds to the N1s from the amine group of PDA. The increase of the N–C=O component at 289.0 eV indicates the introduction of maleimide and ECM.

Figure 4

Water contact angle of various concentration dECM/PDA-coated PCL specimens.

Figure 5

Quantification of cytotoxic test of extract solutions of dECM/PDA-coated PCL conduits relative to controls on rat Schwann cells. Values not sharing a common letter are significantly different at p < 0.05.

Figure 6

(A) Col I and (B) laminin were secreted by dECM/PDA-coated PCL conduits after immersion in DMEM for 3 h. * indicates a significant difference (p < 0.05) compared to PA0.

Figure 7

(A) The adhesion of RSCs cultured with various dECM/PDA-coated PCL conduits for 3 and 6 h; (B) The pFAK expression of cells cultured on specimens for 3 h. The values shown are means ± standard errors for all the assays. Values not sharing a common letter are significantly different at p < 0.05.

Figure 8

(A) F-actin (red) and nuclei (blue) staining; and (B) the area quantification the area of RSCs seeding on dECM/PDA-coated PCL substrates for 3 h. The scale bar is 200 µm.

Figure 9

Proliferation of RSCs cultured on various dECM/PDA-coated PCL conduits for 1, 3, and 7 days. * indicates a significant difference (p < 0.05) compared to PCL. @ indicates a significant difference (p < 0.05) compared to PA0.

Figure 10

Western blot data showing various dECM/PDA-coated PCL conduits promote pERK levels in RSCs. The values shown are means ± standard errors for all the assays. Values not sharing a common letter are significantly different at p < 0.05.

Figure 11

Effects of dECM/PDA-coated PCL conduits on the neural differentiation-related proteins expressions of Nestin, TUJ1, and MAP2. * indicates a significant difference (p < 0.05) compared to PCL. # indicates a significant difference (p < 0.05) compared to PA0.