Development of ovalbumin implants with different spatial configurations for treatment of peripheral nerve injury

Abstract

Peripheral nerve injury (PNI) seriously affects the health and life of patients, and is an urgent clinical problem that needs to be resolved. Nerve implants prepared from various biomaterials have played a positive role in PNI, but the effect should be further improved and thus new biomaterials is urgently needed. Ovalbumin (OVA) contains a variety of bioactive components, low immunogenicity, tolerance, antimicrobial activity, non-toxicity and biodegradability, and has the ability to promote wound healing, cell growth and antimicrobial properties. However, there are few studies on the application of OVA in neural tissue engineering. In this study, OVA implants with different spatial structures (membrane, fiber, and lyophilized scaffolds) were constructed by casting, electrospinning, and freeze-drying methods, respectively. The results showed that the OVA implants had excellent physicochemical properties and were biocompatible without significant toxicity, and can promote vascularization, show good histocompatibility, without excessive inflammatory response and immunogenicity. The in vitro results showed that OVA implants could promote the proliferation and migration of Schwann cells, while the in vivo results confirmed that OVA implants (the E5/70% and 20 kV 20 μL/min groups) could effectively regulate the growth of blood vessels, reduce the inflammatory response and promote the repair of subcutaneous nerve injury. Further on, the high-throughput sequencing results showed that the OVA implants up-regulated differential expression of genes related to biological processes such as tumor necrosis factor-α (TNF-α), phosphatidylinositide 3-kinases/protein kinase B (PI3K-Akt) signaling pathway, axon guidance, cellular adhesion junctions, and nerve regeneration in Schwann cells. The present study is expected to provide new design concepts and theoretical accumulation for the development of a new generation of nerve regeneration implantable biomaterials.

Keywords: Casting method; Electrospinning; Freeze drying; Nerve scaffold; Ovalbumin; Peripheral nerve injury.

Graphical abstract

Fig. 1

Sketch map of the preparation of OVA implants with different spatial structures (membrane, fiber, and lyophilized scaffold).

Fig. 2

Chemical characterization of EDC cross-linked OVA scaffold. (A) FTIR Spectra of OVA Membrane Crosslinked with EDC and ethanol, A1: OVA membrane cross-linked with 5 mM EDC and different concentrations of ethanol, A2: OVA membrane crosslinked with different concentrations of EDC and 70 % ethanol, (B) FTIR spectra of OVA fiber scaffold, (C) FTIR spectra of OVA Sponge Scaffold, (D) Full spectra of XPS test, D1: High-resolution spectra of C1s and S1s elements in E10/70% group, D2: OVA group.

Fig. 3

Physical characterization of OVA implants with three forms. (A) SEM observation of OVA membrane and fiber, (B) Statistical results of OVA fiber diameter at different flow rates, n = 30 (C) Statistical results of OVA fiber diameter at different voltages, n = 30 (D) Swelling ratio test of OVA membrane, n = 3 (E) Quantitative analysis of contact angle of OVA membrane, n = 30 (F) Statistics of elastic modulus of OVA membrane before and after crosslinking, n = 5 (G) Quantitative statistical analysis of contact angle of OVA fiber scaffolds, n = 30 (H) SEM observation of 8% OVA sponge scaffolds, (I) Quantitative analysis of pore size of sponge scaffolds, (J) Elastic modulus statistics of sponge scaffolds, n = 5 (K) Quantitative statistical analysis of contact angle of OVA sponge scaffolds, n = 28 (L) Degradation rate of OVA sponge scaffolds, n = 5 (M) Swelling rate of sponge scaffolds, n = 3 (N) 50-cycle compression test of E4 sponge scaffold. Data are shown as means ± SD. Statistical analysis: ∗∗∗p<0.001, ∗∗∗∗p<0.0001, ns no significance.

Fig. 4

Biocompatibility evaluation of OVA implants with three forms. (A) Cell viability statistics of OVA scaffolds with three forms, n = 6 (B) Photo of different OVA membranes incubated with red blood cells, (C) Hemolysis rate statistics, n = 6 (D) Immunofluorescence images of RSC96 cells grown on different OVA membranes for 3 days, (E) Number of Schwann cells, n = 5 (F) Statistics of cell differentiation length, n = 30 (G) TBO staining images of RSC96 cells on OVA fiber scaffolds, (H) Number of Schwann cells, n = 5 (I) Differentiation length of Schwann cells, n = 30 (J) H&E staining images of E2 group sponge scaffolds cross section. Data are shown as means ± SD. Statistical analysis: ∗p<0.05, ∗∗∗p<0.001, ∗∗∗∗p<0.0001, ns no significance.

Fig. 5

Subcutaneous inflammation reaction of the OVA implants for 2 weeks, including E5/70%, E10/70%, 20 kV 20 μL/min, 20 kV 25 μL/min, E2, E4 and normal tissue, respectively. (A) Macrophage immunofluorescence images of subcutaneous implants for two weeks, the dashed line represents the dividing line between the scaffold and the surrounding tissue. The solid triangle and the hollow triangle represent the surrounding tissue and the implanted scaffold, respectively. (B) CD86 average immunofluorescence intensity statistics, n = 5 (C) CD163 average immunofluorescence intensity statistics, n = 5 (D) The ratio of CD86/CD163 in the tissue around the OVA scaffold. Data are shown as means ± SD. Statistical analysis: ∗p<0.05, ∗∗p<0.01, ∗∗∗p<0.001, ns no significance.

Fig. 6

Inflammatory factors and immune cells of the OVA implants for 1 week and 2 weeks, including E5/70%, E10/70%, 20 kV 20 μL/min, 20 kV 25 μL/min, E2, E4 and normal tissue, respectively. (A) The concentration of IL-10 in the tissue around the implant, n = 3 (B) The concentration of TNF-α, n = 3 (C) Immunofluorescence staining using CD3 antibody and DAPI for analyzing immune cell proliferation response, (D) Quantitative statistics of the average fluorescence intensity of CD3 in one week, n = 5 (E) Quantitative statistics of the average fluorescence intensity of CD3 in two weeks, n = 5. Data are shown as means ± SD. Statistical analysis: ∗∗∗∗p<0.0001, ns no significance.

Fig. 7

Nerve fiber of the OVA implants for 1 week and 2 weeks, including E5/70%, E10/70%, 20 kV 20 μL/min, 20 kV 25 μL/min, E2, E4 and normal tissue, respectively. (A) Immunofluorescence staining using NF200 antibody and DAPI for analyzing nerve fiber, (B) Quantitative statistics of the average fluorescence intensity of nerve fiber in one week, n = 3 (C) Quantitative statistics of the average fluorescence intensity of nerve fiber in two weeks, n = 3. Data are shown as means ± SD. Statistical analysis: ns no significance.

Fig. 8

Transcriptome profiles of Schwann cells on E5/70% (OVA membrane), E2 (OVA lyophilized scaffold), 20 kV 20 μL/min (OVA fiber scaffold), control (the pure slide) groups. (A) The concentration of NGF in cell supernatant, n = 3 (B) Quantitative statistics of up-and down-regulation of differential gene expression in OVA scaffold group samples, (C) Heatmap of DEGs of Schwann cells among different groups, (D) mRNA expressions results of the Schwann cells using RT-PCR analysis, n = 3. Data are shown as means ± SD. Statistical analysis: ∗p<0.05, ∗∗p<0.01, ∗∗∗p<0.001, ∗∗∗∗p<0.0001, ns no significance.

Fig. 9

The possible signal pathways related to cell proliferation, migration, axon growth and myelination of Schwann cells on OVA implants with different spatial structures (membrane, fiber, and lyophilized scaffold).

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