Graphdiyne-Ivy fiber neural scaffold promotes stem cell directed differentiation and neuronal maturation

Abstract

Conductive nerve scaffolds have emerged as a promising alternative to autologous grafts for promoting nerve regeneration. However, the optimization of scaffold materials and the elucidation of their regulatory mechanisms on neural stem cell (NSC) differentiation remain critical research priorities. Graphdiyne (GDY), a novel two-dimensional carbon allotrope, exhibits excellent electrical conductivity and favorable biocompatibility, yet its application in the neural field is still in its infancy. In this study, a structurally synergistic GDY/polycaprolactone (GDY/PCL) conductive composite scaffold—termed the GDY-Ivy Fiber Neural Scaffold—was fabricated using a combined electrospinning–freeze-drying strategy. This approach enabled efficient GDY loading while preserving its intrinsic properties. The resulting scaffold demonstrated superior electrical conductivity, mechanical strength, structural stability, and cytocompatibility. In vitro experiments further confirmed that the GDY-Ivy Fiber Scaffold significantly promoted NSC differentiation into neurons, inhibited glial activation, and enhanced synapse formation and the generation of functionally mature neurons. RNA-Seq analysis revealed that the scaffold orchestrated multiple key signaling pathways, including neurotrophic factor and Wnt-related pathways, thereby promoting NSC neuronal differentiation and functional maturation. In vivo experiments demonstrated that the GDY-Ivy Fiber Neural Scaffold enhances guidance for axonal oriented growth and Schwann cell activation, and promotes neovascularization, thereby improving the repair quality of peripheral nerve injury. Overall, the GDY-Ivy Fiber Neural Scaffold developed in this study establishes an optimized electrophysiological and structural microenvironment that promotes neuronal growth. These findings not only expand the application scope of carbon-based materials in neuroregenerative medicine but also offer novel design strategies for neural repair scaffolds.

Graphical abstract

1. Introduction

Peripheral nerve injury (PNI) continues to be a prevalent and clinically challenging condition, frequently characterized by suboptimal therapeutic outcomes [1]. It can lead to sensory loss, motor dysfunction, or even permanent disability, severely impairing patients’ quality of life [2]. In recent years, tissue-engineered nerve guidance scaffolds have emerged as promising alternatives, designed to mimic the structure and microenvironment of native nerve tissue and provide physical scaffolding and biochemical cues for axonal regeneration [3]. Various biodegradable and biomimetic scaffold materials have been developed for nerve repair [4]. however, their efficacy in restoring nerve function remains constrained, particularly due to insufficient conductivity and limited ability to promote cell adhesion and directed growth [5]. Therefore, the design and synthesis of advanced functional materials exhibiting excellent electrical conductivity, exceptional biocompatibility, and robust neuroinductive properties are of paramount importance for optimizing scaffold performance and accelerating progress in neural regenerative medicine [6].

Nerve regeneration is a complex, multi-step process involving neuronal polarization, axon elongation, synapse formation, and neural network reconstruction [7]. Studies have shown that the electrical microenvironment plays a pivotal role in regulating neural development and regeneration. Incorporating electrically conductive materials into scaffold holds potential to provide bioelectrical stimuli that enhance neuronal migration, axonal outgrowth, and myelination, thus accelerating the overall regenerative process [8]. NSCs, with their multipotent differentiation potential, serve as important models for evaluating the bioactivity of neural scaffolds. NSCs can differentiate into neurons, astrocytes, and oligodendrocytes, and by analyzing the expression of specific phenotypic markers, one can comprehensively assess how materials regulate neural cell fate, promote neuronal differentiation, and inhibit glial overgrowth [9,10]. Therefore, using NSC models to explore the effects of conductive scaffolds on neural differentiation and network formation offers both mechanistic insight and a theoretical foundation for combining stem cells with functional scaffolds in peripheral nerve regeneration [11]. With the advancement of stem cell therapy toward clinical translation, the development of smart biomaterials capable of simultaneously directing cellular behavior and reconstructing the neural microenvironment holds significant potential as a transformative breakthrough in regenerative medicine.

Carbon-based materials from the graphite family have garnered significant attention in neural tissue engineering due to their excellent electrical conductivity, mechanical strength, and biocompatibility [12,13]. Numerous studies have demonstrated that graphene and its derivatives (e.g., graphene oxide, reduced graphene oxide) can promote neuronal growth, induce axonal extension, and support neural differentiation [14]. Their favorable electrochemical properties mimic the natural bioelectrical microenvironment of nerve tissue, thereby facilitating the neural regeneration process [15]. Graphdiyne (GDY), a structural analog of graphene, possesses a unique sp/sp²-hybridized carbon network and intrinsic “diacetylene” linkages [16]. It features a high specific surface area, excellent conductivity, and a wealth of modifiable functional sites. Recent studies have shown that GDY, with its π-conjugated network, can facilitate electron transfer and serve as a nanocatalyst in enzyme-mimetic systems to eliminate reactive oxygen species (ROS) and alleviate oxidative stress [17]. Moreover, GDY coatings have been shown to enhance hydrophilicity and surface roughness of nanomaterials, improving cellular adhesion and spreading [18]. Its near-infrared photothermal conversion capability also offers new opportunities for neural stimulation and therapy [19]. While GDY has shown broad biomedical potential in tumor therapy, bone repair, and wound healing, its application in nerve regeneration—particularly in modulating NSC differentiation and the formation of functional neurons—remains largely unexplored [20]. Li et al. recently fabricated a GDY-loaded PCL nerve scaffold via electrospinning, demonstrating its potential to promote Schwann cell adhesion, proliferation, and myelination in vitro and in vivo [21]. However, the influence of GDY on NSC fate determination and neural functional reconstruction has not yet been systematically examined. Thus, further exploration of GDY-based conductive scaffolds in guiding NSC differentiation and promoting neural function is critical to elucidate its biological mechanisms and expand its application in neural tissue engineering.

In recent years, researchers have attempted to incorporate topographical features into biomaterials to fabricate aligned [22], biomimetic nerve scaffolds that provide physical guidance for axon growth and improve cell adhesion [23]. Oriented scaffolds not only exhibit superior mechanical properties (e.g., tensile strength and compressive resistance) but also show excellent performance in regulating cellular behavior and facilitating tissue repair [24]. Previous studies have demonstrated that, in peripheral nerve repair, aligned fibers within the lumen of nerve conduits can act as “guidance tracks,” facilitating axonal bridging across long defects and improving functional recovery. In this context, the selection of the scaffold base material is equally critical. Polycaprolactone, owing to its excellent biocompatibility and slow degradation properties, is widely regarded as an ideal scaffold substrate in neural tissue engineering [25]. However, achieving uniform and efficient integration of functional nanomaterials into aligned fiber membranes while preserving their biological activity remains a major challenge. To address this issue, we employed a freeze-drying strategy to immobilize GDY nanosheets onto polycaprolactone fibers, successfully constructing a structurally synergistic Graphdiyne-Ivy Fiber Neural Scaffold. Compared to traditional electrospinning—which may cause GDY aggregation or structural degradation—the freeze-drying process preserves the intrinsic conductivity and surface functionality of GDY, significantly improving scaffold homogeneity and interfacial stability.

Specifically, this scaffold integrates topographical and electroactive cues in a synergistic manner (Fig. 1).

• (1)This approach enabled efficient GDY loading while preserving its intrinsic properties. The resulting scaffold demonstrated superior electrical conductivity, mechanical strength, structural stability, and cytocompatibility.
• (2)The GDY-Ivy Fiber scaffolds created an ideal electrophysiological microenvironment for NSCs differentiation, significantly promoting the differentiation of NSCs into mature neurons while inhibiting their differentiation into astrocytes.
• (3)Moreover, the scaffold accelerated synapse formation and facilitated neural network construction, thereby enhancing neuronal maturation.
• (4)RNA-Seq analysis further revealed that the GDY scaffold modulates gene expression in NSCs through multi-pathway activation. This regulatory synergy not only enhances neuronal differentiation but may also improve functional integrity.
• (5)In vivo experiments further demonstrated that the GDY-Ivy scaffold can significantly promote the orderly regeneration of peripheral nerve fibers, myelin reconstruction, and angiogenesis in the regenerated region.

Fig. 1.

GDY-Ivy Fiber Neural Scaffold promotes stem cell directed differentiation and neuronal maturation.

Overall, this scaffold offers a novel strategy for directing NSC fate and enhancing neural functionality. It not only broadens the application spectrum of carbon-based materials in neuroregenerative medicine but also provides innovative design strategies for neural repair scaffolds.

2. Results

2.1. Material characterization and biocompatibility evaluation

The surface morphology of the conductive scaffolds was examined using scanning electron microscopy (SEM). As shown in Fig. 2A, the PR group displayed a randomly fiber structure, while the PO group exhibited highly aligned and oriented fibers. Previous studies have demonstrated that such aligned architectures can effectively guide axonal outgrowth and facilitate organized nerve tissue regeneration [26]. The Zeta potential analysis indicates that GDY has good dispersibility (Fig. S1). Further observation of the POGDY composite scaffold prepared via the freeze-drying method revealed a uniform distribution of GDY across the fiber surface, indicating that this technique significantly enhances GDY loading efficiency and effectively avoids nanoparticle aggregation (Fig. 2B). This provides an effective strategy for the controllable incorporation of functional nanomaterials. To verify the GDY distribution in the scaffolds, SEM-EDS elemental mapping was performed on the PR, PO, and POGDY scaffolds (Fig. 2C). The PR and PO groups exhibited similar carbon-to-oxygen (C/O) ratios, suggesting that the fiber alignment process did not alter the surface chemistry. Fourier-transform infrared (FTIR) spectroscopy was employed to characterize the chemical structure of the PO group, pure GDY powder, and the composite POGDY scaffold. The PO group exhibited a distinct peak at 1720 cm⁻¹, corresponding to the ester carbonyl (C=O) stretching vibration. GDY displayed a prominent peak at 1600 cm⁻¹ attributed to aromatic ring vibrations, and a broad absorption at 3415 cm⁻¹, indicative of hydroxyl (–OH) stretching. The POGDY spectrum retained the characteristic peaks of PCL and additionally showed GDY-related signals, especially at 1600 cm⁻¹ and 3415 cm⁻¹ (Fig. 2D), confirming that GDY is successfully loaded onto the PCL fibrous scaffold, forming the Graphdiyne-Ivy Fiber Neural Scaffold. X-ray photoelectron spectroscopy (XPS) further validated these findings. The PO group showed typical peaks for C–C/C–H (284.8 eV) and C–O/O–C=O (286.22/288.89 eV), whereas GDY exhibited sp² C–C (284.5 eV) and sp C ≡ C (285.6 eV) signals(Fig. S2). Notably, the GDY-Ivy fiber scaffold (POGDY) displayed characteristic peaks of both PCL and GDY, with a negative shift in the sp C ≡ C peak (Fig. 2E and S5). Collectively, the SEM-EDS, FTIR, and XPS results demonstrate that GDY was successfully and homogeneously loaded onto the PCL fibers and formed a stable interfacial structure (Fig. S3).

Fig. 2.

Material characterization and biocompatibility assessment of GDY-Ivy Fiber Scaffold. (A) SEM characterization: Surface morphology and local magnification of PR, PO, and POGDY scaffolds. (B) SEM image of GDY. (C) SEM-EDS elemental mapping: Distribution of C and O elements and corresponding energy spectrum analysis of the conductive scaffolds for PR, PO, and POGDY groups. (D) FTIR spectra of PO, GDY, and POGDY samples. (E) XPS spectra: High-resolution C 1s spectra of PO, GDY, and POGDY samples. (F) Data analysis of water contact angles for PR, PO, and POGDY fiber membranes, n = 5, one-way ANOVA. (G) Contact angle images of the samples, GDY0.5 specifically labeled as POGDY. (H) Live/dead cell staining of neural stem cells on the fiber conductive scaffolds from each group. (I) Quantitative analysis of cell viability for each group, n = 5. (J) CCK-8 assay: Cell viability trend at 1, 3, and 5 days of culture in different groups, two-way ANOVA. ns, not significant, ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001, n = 5.

Contact angle measurements revealed values of 135.7°, 134.6°, and 125.6° for the PR, PO, and POGDY scaffolds, respectively (Fig. 2G). Among them, the POGDY group exhibited improved hydrophilicity, which can be attributed to the inherent water dispersibility and surface-active sites of GDY. No significant difference was observed between the PR and PO groups (Fig. 2F), indicating that fiber alignment had a negligible effect on surface wettability. To evaluate the cytotoxicity and biocompatibility of the scaffolds, NSCs derived from rat hippocampi were cultured on PR, PO, and GDY-modified scaffolds (GDY0.1, GDY0.5, GDY1), and cell viability was assessed using the CCK-8 assay. As shown in Fig. 2J, NSC viability in all groups increased significantly with prolonged culture time, indicating that all materials exhibited favorable cytocompatibility. Among the tested groups, the GDY0.5 scaffold demonstrated the most pronounced promotion of NSC proliferation on days 3 and 5, suggesting optimal support for cell growth. In contrast, the PR and PO groups showed consistently lower proliferation rates, implying limited bioactivity in the absence of GDY. Although the GDY1 group also promoted proliferation, its performance was slightly inferior to GDY0.5, possibly due to mild cellular stress induced by the higher concentration of GDY. Live/Dead staining further confirmed high cell viability across all groups, with only a few dead cells observed (Fig. 2H). The GDY1 group exhibited slightly more dead cells than the others, but the overall survival rate remained high (Fig. 2I). Together, the results from the CCK-8 assay and viability staining demonstrate that all fabricated fibrous scaffolds possess good biocompatibility and cellular safety, making them suitable for subsequent applications in nerve regeneration studies.

2.2. Stability, mechanical properties, and electrical performance of GDY-Ivy Fiber Scaffolds

Thermal stability analysis revealed that the initial decomposition temperature of the POGDY scaffold was approximately 20–30 °C higher than that of the PO group, and its residual mass at 600 °C was significantly greater (Fig. 3A). Differential thermogravimetric (DTG) analysis further showed that the maximum decomposition rate of the POGDY scaffold occurred at 410 °C (−0.5%/min), in contrast to −1.5%/min at 340 °C for the PO group (Fig. 3B). The improved thermal stability of the POGDY scaffold was more likely attributable to the intrinsically high thermal stability of GDY. In terms of mechanical performance, the PR group exhibited lower tensile strength and toughness due to its disordered fiber orientation. The PO group demonstrated higher strength but reduced ductility, attributed to its aligned fibrous structure. Notably, the POGDY scaffold showed simultaneous enhancement in both strength and elongation, owing to the synergistic effects of GDY's π-conjugated structure, interfacial reinforcement, and nanoscale load transfer. This group displayed superior load-bearing capacity, particularly under conditions of large deformation (Fig. 3C ,D and E). The Young's modulus of the POGDY scaffold is approximately 8.48 MPa, showing a modest increase compared to pure PCL (Fig. S4). These findings highlight the mechanical advantages of combining fiber alignment with GDY functionalization in composite scaffold design. With respect to electrical properties, impedance measurements across the 0.1–10⁵ Hz frequency range using an electrochemical workstation revealed that the POGDY group exhibited consistently lower impedance than both the PR and PO groups (Fig. 3F), indicating enhanced electrical conductivity. Furthermore, direct conductivity testing confirmed that the POGDY scaffold possessed the highest conductivity among all groups. To evaluate the electrochemical stability of the POGDY scaffold under physiologically simulated conditions, electrochemical impedance spectroscopy measurements were performed on samples immersed for 1, 7, and 14 days. The results demonstrated highly consistent impedance spectrum profiles across all three time points (Fig. S6), thereby validating the positive role of GDY incorporation in creating an electroactive microenvironment suitable for neural applications.

Fig. 3.

Stability, mechanical properties, and electrical performance analysis of GDY-Ivy Fiber Scaffolds. (A) Thermogravimetric analysis (TGA) curve. (B) Derivative thermogravimetric (DTG) curve. (C) Static force-displacement curve of the fiber scaffold. (D) Stress-strain curve of the fiber scaffold. (E) Fracture strain statistical analysis, n = 5, one-way ANOVA. (F) Impedance test results of the fiber scaffold in the frequency range of 0.1–10⁵ Hz.

2.3. Analysis of NSC growth and differentiation induced by GDY-Ivy Fiber Scaffolds

Building on the above materials characterization, the GDY-Ivy scaffold was confirmed to possess aligned topography, good electrical conductivity, and favorable biocompatibility. We therefore proceeded to investigate its regulatory effects on NSC differentiation. Before seeding the NSCs onto the GDY-Ivy scaffold, they were subjected to Nestin staining. The results showed that the cells maintained an undifferentiated state, exhibiting typical neural stem cell characteristics (Fig. S7). Dual immunostaining of Tuj1 and NF200 was performed to label distinct stages of neuronal maturation during NSC differentiation. The GDY-Ivy group exhibited a significant increase in Tuj1-positive neurons and markedly enhanced the formation and elongation of NF200-positive axons (Fig. 4B), demonstrating a strong capacity to facilitate neuronal functional maturation. Compared to other groups, the GDY-Ivy scaffold provided a more favorable microenvironment by synergistically integrating topographical guidance and electrical conductivity, thereby offering dual support for NSC-directed differentiation and axonal network development (Fig. 4E and F). Further immunofluorescence staining of the mature neuronal marker Map2 and the astrocyte marker GFAP revealed that the GDY-Ivy scaffolds notably promoted NSC neuronal differentiation. This was evidenced by the strongest Map2 signal (Fig. 4G), a greater number of neurites, increased dendritic branching complexity, and dendrites clearly aligned along the scaffold orientation (Fig. 4C). In parallel, GFAP expression was significantly reduced, indicating suppressed glial lineage differentiation (Fig. 4D and H).

Fig. 4.

GDY-Ivy Fiber Scaffold promotes directional differentiation of NSCs. (A) Schematic illustration of NSC regulation by the GDY-Ivy Fiber Scaffold. (B) Tuj1 and NF200 immunofluorescence staining showing neuronal generation and axonal extension after seven days of culture in each group. (C) Immunofluorescence images of the astrocyte marker Map2 after seven days of culture. (D) GFAP immunofluorescence staining images. (E) Quantitative analysis of the fluorescence intensity of Tuj1. (F) Quantitative analysis of the fluorescence intensity of NF200. (G) Quantitative analysis of the fluorescence intensity of Map2. (H) Quantitative analysis of the fluorescence intensity of GFAP. Data are presented as the Mean ± SD, one-way ANOVA, ns, not significant, ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001, n = 5.

2.4. Regulatory effects of the GDY-Ivy Fiber Scaffold on synaptogenesis and neurotransmitter phenotype differentiation of NSCs

After confirming that the GDY-Ivy scaffold promotes NSC differentiation into neurons and enhances neurite outgrowth, we further evaluated neuronal functional maturation in terms of synapse formation and neurotransmitter phenotype. Dual immunofluorescence staining of SYP/Map2 and PSD95/Map2 revealed that GDY-Ivy scaffolds significantly enhanced synapse formation. Specifically, the density of PSD95-positive puncta significantly increased and displayed a typical clustered morphology (Fig. 5A), indicating synaptic maturation. Co-localized signals of SYP and Map2 were markedly enhanced, with synaptic vesicles uniformly distributed along neuronal dendrites (Fig. 5B). These findings suggest that GDY-Ivy scaffolds not only promote neuronal morphological maturation but also enhance synaptogenic capacity, thereby supporting the formation of functional neuronal networks (Fig. 5E and F). To assess neurotransmitter subtype differentiation, the expression of ChAT and GAD67 was examined. The GDY-Ivy scaffold group exhibited the highest proportion of ChAT-positive neurons, characterized by elongated and well-aligned neurites, indicating strong cholinergic differentiation potential (Fig. 5C). In addition, the proportion of GAD67-positive cells in the experimental groups showed a notable increase (Fig. 5D), suggesting that the GDY-Ivy scaffold not only promotes cholinergic neuronal differentiation but also supports the generation of GABAergic neuronal subtypes. In contrast, the PO group—despite its aligned structure—showed limited effects due to the absence of GDY's electrochemical cues and bioactive surface features, resulting in reduced synaptogenesis and neurotransmitter differentiation. The PR group, lacking both alignment and conductivity, displayed the least favorable outcomes (Fig. 5G and H).

Fig. 5.

Regulatory effects of GDY-Ivy Fiber Scaffold on neuronal maturation, synapse formation, and neurotransmitter phenotype. (A) PSD95/Map2 immunofluorescence staining images. (B) SYP/Map2 immunofluorescence staining images. (C) ChAT/Map2 immunofluorescence staining images. (D) GAD67/Map2 immunofluorescence staining images. (E) Quantitative analysis of the fluorescence intensity of PSD95. (F) Quantitative analysis of the fluorescence intensity of SYP. (G) Quantitative analysis of the fluorescence intensity of ChAT. (H) Quantitative analysis of the fluorescence intensity of GAD67. Data are presented as the Mean ± SD, one-way ANOVA, ns, not significant, ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001, n = 5.

2.5. Mechanistic insights into NSC differentiation on the GDY-Ivy scaffold

To further elucidate the molecular mechanisms by which GDY-Ivy scaffolds promote NSC differentiation toward mature neurons and account for the above phenotypic differences, we performed RNA-Seq–based transcriptomic profiling of NSCs from each group and validated representative differentially expressed genes by qPCR. Principal component analysis (PCA) revealed high intra-group correlation and clear inter-group differences among PR, PO, and POGDY samples (Fig. 6A). The Venn diagram demonstrated both overlapping and uniquely expressed genes among the three groups, with POGDY showing a substantial number of differentially regulated genes (Fig. 6B). The multi-group differential scatter plot indicated that the POGDY group had 2050 upregulated genes and 58 downregulated genes compared to the control (Fig. 6C). Gene Ontology (GO) enrichment analysis (Fig. 6D–F) revealed that GDY-Ivy scaffolds primarily activated biological processes related to nervous system development, signal regulation, and voltage-gated ion channels. These included synaptic transmission, neuronal polarity establishment, and calcium ion transmembrane transport, suggesting that the scaffold creates a microenvironment conducive to neuronal differentiation (Fig. S8). Volcano plots further visualized the magnitude and significance of gene expression changes (Fig. 6G), confirming the scaffold's ability to modulate cell fate at the transcriptomic level. KEGG pathway enrichment analysis showed marked activation of pathways closely associated with neurodevelopment, including the calcium signaling pathway, neurotrophin signaling, MAPK, PI3K-Akt, and Wnt pathways (Fig. 6H). These pathways play critical roles in neuronal maturation and network formation. Gene Set Enrichment Analysis (GSEA) further validated that the GDY-Ivy scaffold treatment significantly enriched functional pathways such as neuron-to-neuron synapse, calcium signaling, Wnt signaling, and neuroactive ligand-receptor interaction (Fig. 6I, J, S9 and S10). These findings suggest that the scaffolds enhance neuronal differentiation through the coordinated activation of multiple signaling cascades, ultimately promoting neural network reconstruction (Figs. S11–S14).

Fig. 6.

Transcriptomic sequencing results of NSCs. (A) Principal component analysis (PCA). (B) Venn diagram showing the intersection and number of specific differentially expressed genes (DEGs) between the three groups. (C) Multi-group differential scatter plot. (D–F) GO enrichment analysis (BP, CC, MF). (G) Volcano plot showing the distribution of differentially expressed genes between the groups. (H) KEGG pathway enrichment analysis. (I) GSEA analysis: GO. (J) GSEA analysis: KEGG.

To further investigate how the GDY-Ivy scaffold activates the aforementioned pathways, key genes in the Neuroactive Ligand-Receptor Interaction pathway (e.g., BDNF, NT4), the Wnt pathway (e.g., Wnt5), and the Calcium Signaling pathway (e.g., NMDAR) were validated. Gene dynamic mountain plots (Fig. 7A and B) show the expression differences of genes such as BDNF, NT4, Shc, and NMDAR across the PR, PO, and GDY-Ivy scaffold groups. The results indicate that the GDY-Ivy scaffold significantly upregulated genes involved in the Neuroactive Ligand-Receptor Interaction and Calcium Signaling pathways, suggesting enhanced neurotrophic factor signaling and synaptic activity. Genes involved in the Wnt pathway, such as Wnt5, PKC, PLA2, and PLD, were also significantly elevated in the POGDY group (Fig. 7C and D). These genes jointly regulate key processes such as neuronal polarity establishment, axon guidance, and cell migration. Mfuzz clustering analysis (Fig. 7E) identified that most target genes—including BDNF, NT4, and Wnt5—were concentrated in expression clusters (e.g., Cluster 1 and Cluster 4) showing sustained upregulation, with the highest peak in the GDY-Ivy group. A radar plot (Fig. 7F) further visualized the expression levels of eight representative genes, all showing markedly higher expression in the GDY-Ivy scaffold group. To further validate the RNA-seq results, we performed qPCR analysis on the key genes involved in the selected signaling pathways. The results showed that, compared with the PR group, the mRNA expression levels of Wnt5, PKC, PLA2, and PLD in the Wnt signaling pathway were significantly upregulated in the POGDY group. Similarly, BDNF and Shc in the neuroactive ligand–receptor interaction pathway, as well as NMDAR in the calcium signaling pathway, also exhibited a comparable upregulation trend (Fig. 7G–J and S15). Collectively, these results demonstrate that GDY-Ivy scaffolds promote efficient and directed NSC differentiation toward neurons by simultaneously activating key genes in the calcium signaling, Wnt signaling, and neuroactive ligand-receptor interaction pathways. Through enhancing neuronal polarity establishment, synaptic plasticity, and signal transduction capacity, these scaffolds accelerate the formation and reconstruction of functional neural networks.

Fig. 7.

GDY-Ivy Fiber Scaffold upregulates the expression of key genes by activating neuro-related pathways. (A) Comparison of the expression of key genes such as BDNF, NT4, Shc, and NMDAR between the PR and POGDY groups. (B) Comparison of the expression of key genes such as BDNF, NT4, Shc, and NMDAR between the PO and POGDY groups. (C) Comparison of the expression of key genes such as Wnt5, PKC, PLA2, and PLD between the PR and POGDY groups. (D) Comparison of the expression of key genes such as Wnt5, PKC, PLA2, and PLD between the PO and POGDY groups. (E) Mfuzz analysis plot: Trend analysis of differential gene expression. (F) Radar plot: Data statistics of key gene expression levels. (G) Relative expression level of BDNF. (H) Relative expression level of Shc. (I) Relative expression level of NMDAR. (J) Relative expression level of Wnt5. Data are presented as the Mean ± SD, one-way ANOVA, ns, not significant, ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001, n = 3.

2.6. In vivo evaluation of the GDY-Ivy scaffold for peripheral nerve regeneration

Given that the GDY-Ivy scaffold had already shown superior neuroregulatory effects in vitro compared with the control groups, we further evaluated its repair performance in vivo using a rat sciatic nerve defect model. By examining HE-stained sections of the regenerated sciatic nerves (Fig. 8A), the overall regeneration of the nerve bundles could be clearly evaluated in the longitudinal plane. The results showed that the repair outcome in the POGDY group was markedly superior to that in the PR and PO groups: in the PR and PO groups, nerve fibers were loosely connected and sparsely distributed, whereas in the GDY-Ivy scaffold group the regenerated nerve fibers exhibited a relatively compact structure and more orderly arrangement, indicating a more robust nerve regeneration and repair capacity. Transverse sections further supported this trend: although varying degrees of gaps and vacuolar-like defects were observed in all groups, the regenerated nerve tissue in the autograft and POGDY groups appeared more compact and better organized, with markedly fewer vacuolar defects than in the PR and PO groups. In addition, the PO group displayed slightly denser and more structurally intact nerve tissue than the PR group, suggesting that an oriented scaffold may also be beneficial for in vivo reconstruction and partial alignment of nerve fibers.

Fig. 8.

In vivo repair efficacy of the GDY-Ivy scaffold in a rat sciatic nerve defect model. (A) Longitudinal and transverse HE-stained sections of regenerated sciatic nerves in each group. (B) CD31 immunofluorescence staining images of regenerated nerves. (C) NF200/S100 immunofluorescence staining images of regenerated nerves. (D) NF200/MBP immunofluorescence staining images of regenerated nerves. (E) Quantitative analysis of CD31 fluorescence intensity. (F) Quantitative analysis of NF200 fluorescence intensity. (G) Quantitative analysis of S100 fluorescence intensity. (H) Quantitative analysis of MBP fluorescence intensity. Data are presented as the Mean ± SD, one-way ANOVA, ns, not significant, ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001, n = 5.

CD31 immunofluorescence staining revealed an increased number of newly formed blood vessels within and around the regenerated nerve in the GDY-Ivy scaffold group, with clearly defined vascular lumens, which is favorable for providing continuous nutrient and oxygen supply to the regenerating nerve; the PO group also showed a certain degree of pro-angiogenic effect compared with the PR group (Fig. 8B and E). We next evaluated the expression of NF200 and S100 in the regenerated nerve tissue (Fig. 8C). The expression levels of NF200 and S100 in the POGDY group were comparable to those in the autograft group and were significantly higher than those in the PR and PO groups (Fig. 8F and G). Moreover, nerve fibers in both the POGDY and PO groups displayed a directional growth pattern, further indicating that the combination of an oriented structure with the GDY is conducive to guiding axons to extend orderly along the scaffold. To further assess myelin regeneration, MBP immunofluorescence staining was performed on the regenerated nerve tissue (Fig. 8D). The POGDY group exhibited a more ordered and compact myelin structure, and the MBP signal intensity was significantly higher than that in the PR and PO groups (Fig. 8H), suggesting that the GDY-Ivy scaffold not only promotes axonal regeneration and Schwann cell activity, but also markedly enhances myelin reconstruction. Taken together, these results demonstrate that the GDY-Ivy scaffold promotes nerve fiber regeneration, myelin reconstruction, and angiogenesis in vivo, confirming its favorable performance in peripheral nerve repair.

3. Discussion and conclusions

Tissue-engineered nerve guidance scaffolds offer novel strategies for nerve regeneration and functional recovery by mimicking the structure and microenvironment of native nerves, representing an effective alternative to autografts [27]. However, existing scaffold materials still exhibit limitations in electrical conductivity and the ability to support neural cell maturation and differentiation [28]. NSCs, with their strong differentiation potential, can replace damaged neurons and promote nerve regeneration [29]. They also secrete neurotrophic factors in vivo to support surrounding cells, making them widely used as cellular models for nerve tissue repair [30]. Carbon-based nanomaterials, particularly those from the graphene family, have been extensively studied in neural tissue engineering due to their excellent electrical conductivity [31], high specific surface area, and favorable mechanical properties [32]. GDY, a novel carbon allotrope of graphene, exhibits outstanding conductivity and biocompatibility [33]. GDY also offers unique advantages such as regulating cell adhesion, scavenging ROS, and providing a large surface area—features that are critical for establishing a controlled microenvironment for nerve regeneration [34]. While GDY has shown promise in cancer therapy and bone repair [35], its application in NSC differentiation and functional neural network formation remains underexplored.

To address this, we successfully fabricated functional neural scaffolds by loading GDY onto aligned PCL nanofibrous membranes via a combination of electrospinning and freeze-drying. The resulting GDY-Ivy scaffolds exhibit desirable properties including electrical conductivity, fiber orientation, and excellent biocompatibility. Material characterization and biological assays demonstrate that the scaffolds effectively enhance structural performance, promote cell adhesion and proliferation, direct NSC differentiation, and support synaptic network formation. These findings underscore the promising potential of GDY-based oriented scaffolds for peripheral nerve injury repair. The freeze-drying method effectively prevented the agglomeration of GDY during electrospinning, enabling its uniform distribution on the surface of the nanofibers [36]. Compared to conventional randomly conductive scaffolds, the aligned structure provided a more ordered physical guidance cue for NSC growth. FTIR and XPS analyses confirmed the successful immobilization of GDY on the scaffold surface. The incorporation of GDY significantly enhanced the scaffold's hydrophilicity and structural stability while notably reducing its impedance, Jiang's team found that the incorporation of GDY significantly improved the thermal stability of the composite materials, demonstrated by higher initial decomposition temperatures and improved ionic conductivity [37]. Similarly, our GDY-Ivy scaffold, when compared to pure PCL scaffolds, also exhibited superior thermal stability, further confirming the role of GDY in enhancing scaffold performance. In addition, the mechanical performance tests show that the GDY Ivy fiber scaffold's mechanical properties are similar to commonly used compliant scaffolds in neural tissue engineering and exhibit good matching with the mechanical range of native neural tissue and existing neural scaffolds [38]. This suggests that the GDY Ivy fiber scaffold can provide a more suitable microenvironment for cell growth and neural regeneration [39]. Biocompatibility assays further demonstrated that the GDY-Ivy scaffold exhibited no detectable cytotoxicity and continuously promoted cell viability, establishing a solid foundation for its potential in vivo applications.

NSCs possess self-renewal and multi-lineage differentiation potential. However, in the absence of specific induction conditions, they tend to differentiate into astrocytes, which hampers the effectiveness of neural repair [40]. Therefore, the development of biomaterials with both electrical conductivity and structural guidance has become an effective strategy to direct NSCs towards neuronal differentiation [41]. Recent studies have shown that the use of multifunctional hydrogels with conductive and electroactive properties, such as BP @ Hydrogel, can significantly enhance the differentiation of NSCs into mature neurons, promoting an increase in Tuj1, Map2, and NF200 positive neurons [42]. The results of this study demonstrate that the GDY-Ivy scaffold significantly promotes the differentiation of NSCs towards neurons while inhibiting their differentiation into astrocytes. The expression of neuronal markers such as Map2, Tuj1, and NF200 was significantly enhanced in the GDY-Ivy scaffolds group [43]. Additionally, the number of neuronal processes and their branching complexity significantly increased, and the processes exhibited a clear directional growth trend, suggesting that the scaffold supports axon orientation and neural network formation. Moreover, the scaffold exhibited remarkable advantages in synapse formation. The expression levels of SYP and PSD95 were significantly elevated, indicating that the scaffold not only promotes neuronal generation but also contributes to the formation of synapses and network integration. These results are consistent with previous studies, such as those involving BTO/rGO nanostructures [44], which have been shown to enhance synapse formation and stability through their conductive properties. The balance of neurotransmitters is also crucial for the restoration of neural function. Furthermore, this study further demonstrates that the GDY-Ivy scaffold regulates neurotransmitter subtype differentiation, specifically enhancing cholinergic and GABAergic neuronal features.

The regulation of neurotrophic factor pathways plays a crucial role in determining the fate of NSCs and in the process of nerve repair. BDNF binds to its high-affinity receptor TrkB and activates multiple downstream signaling pathways, thereby regulating cell cycle progression, the expression of anti-apoptotic factors, and the determination of neuronal fate [45,46]. Studies have shown that BDNF activation not only enhances the survival of NSCs but also significantly promotes their proliferation and differentiation towards neurons [47].

In the calcium signaling pathway, N-methyl-D-aspartate receptor (NMDAR), a key protein channel with high calcium permeability, plays an important role in excitatory synaptic transmission [48]. Its activation increases intracellular Ca²⁺ influx and induces the expression of postsynaptic density protein PSD95, thereby promoting synapse maturation [49]. Ca²⁺, as a core mediator in neuronal signal transmission, plays a vital role in axon growth, synaptic plasticity, and electrophysiological activity. In the Wnt signaling pathway, the binding of Wnt5 to the Frizzled receptor triggers the activation of the non-canonical Wnt pathway, leading to the release of intracellular Ca²⁺ and the activation of downstream signaling factors such as PKC, PLA2, and PLD [50]. This signaling axis not only helps establish neuronal polarity, axon guidance, and synaptic development but also participates in regulating neurotransmitter release. Research has also shown that the expression of PKC significantly promotes axon growth in the sciatic nerve, further supporting the role of GDY scaffolds in neural repair via this pathway [51]. Our study results show that GDY-Ivy scaffolds significantly enrich and activate several key pathways, including the calcium signaling pathway, Wnt signaling pathway, and neuroactive ligand-receptor interactions. These pathways regulate the expression of multiple core genes, including BDNF, NT4, Shc, and the NMDAR subunit gene Grin2c, all of which are upregulated in the GDY scaffold-treated group. This suggests that the GDY scaffold enhances neurotrophic factor signaling and synaptic activity, supporting neuronal differentiation and maturation. Additionally, Wnt5, PKC, PLA2, and PLD form a Wnt-mediated axon guidance signaling network that promotes neuronal polarity and axon growth, enhancing the connectivity of neural networks [52]. Mfuzz analysis further revealed that these key genes showed a continuous upward expression trend under the influence of GDY, indicating that they are consistently regulated during the differentiation process. This activation and coordinated regulation not only elevate the proportion of differentiated neurons but also enhance their functional integrity.

In a rat sciatic nerve defect model, the GDY-Ivy scaffold exhibited a markedly better regenerative performance in vivo than both the PR and PO groups. In the GDY-Ivy scaffold group, the regenerated nerve bundles appeared more compact, with more orderly aligned nerve fibers, and the overall morphology was comparable to that of the autograft group. In contrast, the PR group showed relatively sparse and loosely connected nerve fibers, while the PO group displayed an intermediate morphology, suggesting that an oriented structure alone can partially improve nerve fiber reconstruction, and that the additional introduction of GDY further enhances the quality of regeneration. This conclusion was further corroborated by the immunofluorescence results. The expression levels of NF200, S100, and MBP in the POGDY group were significantly higher than those in the PR and PO groups, accompanied by a pronounced tendency toward oriented growth. These findings indicate that the GDY-Ivy scaffold is beneficial for axonal regeneration, Schwann cell activation, and myelin reconstruction, which is in agreement with previous reports that nerve scaffolds combining oriented structures with nanomaterials facilitate directional nerve fiber growth and promote nerve repair [53,54]. As angiogenesis provides essential nutritional and oxygen support for nerve regeneration, and graphene-based materials have been reported to possess pro-angiogenic potential [55], our study likewise found a clear increase in newly formed blood vessels within the regenerated area in the GDY-Ivy scaffold group [56]. Overall, the GDY-Ivy nerve scaffold demonstrates advantages in regulating cellular behavior, activating key signaling pathways, and promoting tissue reconstruction in vivo, thereby providing a strong rationale for its further development as a scaffold material for peripheral nerve repair. Meanwhile, considering the critical importance of scaffold degradation behavior in neural tissue engineering for long-term repair outcomes and clinical translation, future studies should systematically evaluate its in vivo degradation characteristics and their compatibility with the tissue regeneration process, in order to more comprehensively validate its clinical application potential.

This study developed a GDY-based composite conductive, oriented fiber scaffold by combining electrospinning and freeze-drying techniques, effectively achieving GDY loading and optimizing the scaffold's conductivity, mechanical properties, and biocompatibility. Experimental results demonstrated that the GDY-Ivy scaffolds created an ideal electrophysiological microenvironment for NSCs differentiation, significantly promoting the differentiation of NSCs into mature neurons while inhibiting their differentiation into astrocytes. Moreover, the scaffold accelerated synapse formation and facilitated neural network construction, thereby enhancing neuronal functionality and maturity. RNA-Seq analysis, together with qPCR validation, revealed that the scaffolds synergistically activate neurogenesis-related signaling pathways, promoting neuronal formation and enhancing synapse formation and axon growth, thereby supporting the stability of neural networks. Moreover, in vivo experiments further demonstrated that the GDY-Ivy scaffold can significantly promote the orderly regeneration of peripheral nerve fibers, myelin reconstruction, and angiogenesis in the regenerated region, achieving a repair efficacy comparable to that of the autograft group. In summary, the GDY-Ivy scaffold shows significant potential in regulating NSCs directional differentiation, promoting neuronal maturation, and constructing synaptic networks. These findings provide theoretical and experimental support for the design of neural repair materials in regenerative medicine, highlighting promising prospects for applications in neural tissue engineering.

4. Materials and methods

4.1. Preparation of GDY-Ivy Fiber Scaffold

A composite electroconductive fiber scaffold functionalized with GDY was fabricated using a combined electrospinning and freeze-drying process. First, a 15% (W/V) PCL solution was prepared by dissolving PCL in trifluoroethanol (Macklin, China). The solution was then sealed and stirred for 24 h in a magnetic stirrer to obtain a homogeneous polymer solution. This solution was injected into a 10 mL syringe and connected to an electrospinning device (QINGZI NANO, E05, China) to fabricate the fiber membranes. The electrospinning parameters were set as follows: a flow rate of 0.8 mL/h, an applied voltage of +12 kV (positive electrode) and −4 kV (negative electrode), and a nozzle-to-collector distance of 15 cm. To achieve an oriented fiber structure, a high-speed roller rotating at 2500 rpm was used as the collector, resulting in the PO group samples. Randomly fiber membranes were collected using a static metal collection plate, labeled as the PR group. GDY was synthesized in situ from its precursor on copper foil, followed by post-treatment to remove impurities and obtain pure GDY powder, as detailed in the supplementary materials. The PO group fiber membranes were then immersed in 0.1%, 0.5%, and 1% (W/V) GDY dispersion solutions and subjected to 48 h of treatment in a freeze-dryer (Martin Christ Alpha 1-4 LSC basic, Germany). This process ensured uniform loading of GDY onto the fiber surfaces, preventing nanoparticle aggregation and structural damage during the high-voltage electrospinning process. The resulting functionalized electroconductive scaffolds were labeled as GDY0.1, GDY0.5, and GDY1, with GDY0.5 specifically labeled as POGDY (GDY-Ivy Fiber Scaffold).

4.2. Characterization of GDY-Ivy Fiber Scaffold

The microstructure and surface morphology of the PR, PO, and POGDY fiber scaffolds were analyzed using a Focused Ion Beam Scanning Electron Microscope (FIB-SEM, Crossbeam 350, Zeiss, Germany). The particle size distribution and surface potential of the GDY dispersion were measured using a NanoSizer and Zeta potential analyzer (Zetasizer Advance Pro, UK) to evaluate dispersion stability. The wettability of the fiber scaffolds was assessed using a contact angle goniometer (OCA20), reflecting their hydrophilicity. Electrical resistance measurements were conducted using an electrochemical workstation (VersaSCAN, USA), with conductivity further analyzed through the four-point probe method. The POGDY scaffolds were immersed in PBS solution and sampled at 1, 7, and 14 days for electrochemical impedance spectroscopy (EIS) testing. The mechanical properties of the fiber scaffolds were evaluated by tensile testing using a universal material testing machine (DMA242C). Thermal stability was monitored with a Thermogravimetric Analyzer (TG209F3) to observe mass changes at various temperatures. The surface elemental composition was quantitatively analyzed using Scanning Electron Microscopy with Energy Dispersive X-ray Spectroscopy (SEM-EDS). Functional group analysis was performed by Fourier Transform Infrared Spectroscopy (FTIR, INVENIO, Bruker, USA) with a scanning range of 400–4000 cm⁻¹ and a resolution of 2 cm⁻¹. Additionally, X-ray Photoelectron Spectroscopy (XPS, AXIS SUPRA+, Japan) was used to verify the successful incorporation of GDY into the scaffold.

4.3. Isolation and culture of NSCs

NSCs were isolated from the hippocampal tissues of neonatal Sprague–Dawley (SD) rats (BesTest Bio-Tech Co., China) via mechanical dissociation to obtain primary cell suspensions. The cells were cultured in a defined medium composed of DMEM/F12 (Gibco, USA) supplemented with 20 ng/mL basic fibroblast growth factor (Stemcell Technologies, Canada), 2% B27 supplement (Gibco, USA), and 1% penicillin–streptomycin (Servicebio, China). Cultures were maintained in suspension at 37 °C in a humidified incubator with 5% CO₂, and the medium was refreshed every 48 h. Once NSCs formed neurospheres, they were dissociated into single-cell suspensions via gentle pipetting. The resulting cells were subsequently passaged for expansion or seeded for downstream experiments.

4.4. Biocompatibility assessment

The proliferative activity of NSCs cultured on different fibrous scaffolds was evaluated using the Cell Counting Kit-8 (Servicebio, China). Optical density (OD) values were measured using a microplate reader (Thermo Varioskan LUX, Singapore) to quantify cell viability. Cytotoxicity was assessed via Live/Dead double fluorescence staining (Servicebio, China), with live cells stained green and dead cells stained red. The stained samples were observed under a fluorescence microscope (Leica DMI8, Germany) to qualitatively assess cell viability and distribution on the scaffolds.

4.5. Analysis of the effects of GDY-Ivy Fiber Scaffold on NSC growth and differentiation

To evaluate the regulatory effects of the conductive fibrous scaffolds on the proliferation and differentiation of NSCs, immunofluorescence staining was performed to detect the expression of specific NSC markers. These included GFAP (astrocyte marker), Map2 (mature neuronal marker), Tuj1 (immature neuronal marker), and NF200 (axonal development marker), with all primary antibodies obtained from Abcam (USA). The experimental protocol was as follows: After co-culturing NSCs with different scaffolds for 7 days, cells were gently rinsed with PBS to remove debris, and subsequently fixed in 4% paraformaldehyde (Servicebio, China) at room temperature for 20 min. Non-specific binding sites were blocked using goat serum (Servicebio, China) for 1 h. For immunostaining, GFAP and Map2 were stained individually (1:200), while Tuj1 and NF200 were subjected to dual-staining (1:200), with primary antibody incubation carried out overnight at 4 °C. On the following day, fluorescently labeled secondary antibodies (1:500) corresponding to the primary antibody species were added and incubated at room temperature for 1 h in the dark. Finally, cell nuclei were counterstained with DAPI (1:500). All immunofluorescence images were acquired using a confocal microscope and presented through z-stack acquisition with orthogonal projection.

4.6. Regulation of synaptogenesis and neurotransmitter phenotype differentiation of NSCs by GDY-Ivy Fiber Scaffold

To systematically evaluate the regulatory effects of the GDY-Ivy Fiber Scaffold on synaptogenesis and neurotransmitter phenotype differentiation of NSCs, immunofluorescence staining was performed to assess the expression of synaptic markers (PSD95 and SYP) and neurotransmitter phenotype markers (ChAT and GAD67), all antibodies purchased from Abcam (USA). The experimental procedure was as follows: After 14 days of co-culture of NSCs with various fiber scaffolds, cells were gently rinsed with PBS to remove cellular debris. They were then fixed with 4% paraformaldehyde at room temperature (25 °C) for 20 min and blocked with goat serum for 1 h to prevent non-specific binding. Subsequently, cells were subjected to dual immunostaining with anti-PSD95 (1:200), anti-SYP (1:200), anti-ChAT (1:200), or anti-GAD67 (1:200), in combination with the neuronal marker anti-Map2 (1:200), and incubated overnight at 4 °C. On the following day, cells were incubated with the corresponding fluorescent secondary antibodies (1:500) at room temperature in the dark for 1 h. Nuclei were counterstained with DAPI (1:500). All immunofluorescence images were acquired using a confocal microscope and presented through z-stack acquisition with orthogonal projection.

4.7. RNA sequencing and bioinformatics analysis

Total RNA was extracted from NSCs cultured on PR, PO, and POGDY scaffolds, followed by RNA sequencing. Differential expression analysis was performed using DESeq2 software to identify differentially expressed genes (DEGs) between the groups. The results were visualized using volcano plots and heatmaps. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses were conducted for the DEGs, followed by Gene Set Enrichment Analysis (GSEA) for further validation. All data processing and visualizations were performed using the online platform provided by Omicsmart.

4.8. Real-time quantitative PCR (RT-qPCR)

In this study, NSCs were seeded on different scaffolds and cultured for 14 days, after which total RNA was extracted. Following quantification by NanoDrop and confirmation of RNA purity, cDNA was synthesized by reverse transcription, and gene expression levels were analyzed using RT-qPCR. Based on the RNA-seq and KEGG enrichment results, key genes were selected for validation, including Wnt5, PKC, PLA2, and PLD in the Wnt signaling pathway, BDNF and Shc in the Neuroactive ligand–receptor interaction pathway, and NMDAR in the Calcium signaling pathway. GAPDH was used as the internal reference gene, and relative mRNA expression levels were calculated using the 2^–ΔΔCt method. The primer sequences are provided in the Supplementary Materials (Table S1).

4.9. Establishment of a rat sciatic nerve defect model

All animal procedures were approved by the Animal Ethics Committee of Sun Yat-sen University and were carried out in accordance with the relevant institutional guidelines (Approval No.: SYSU-IACUC-2025-002483). Twenty adult male Sprague–Dawley rats (180–200 g) were randomly assigned to four groups: PR, PO, POGDY and autograft (n = 5 per group). Anesthesia was induced by intraperitoneal injection of 2% sodium pentobarbital. The sciatic nerve of the right hind limb was exposed via a posterior thigh incision, and a 10 mm segment was excised to establish a sciatic nerve defect model. In the PR, PO and POGDY groups, the proximal and distal nerve stumps were inserted into the corresponding scaffold conduits and fixed with interrupted 8–0 nylon sutures. In the autograft group, the excised nerve segment was rotated 180° and re-implanted, followed by end-to-end anastomosis with 8–0 nylon sutures. Muscles and skin were closed in layers with 4–0 silk sutures. Penicillin was administered intramuscularly for 7 consecutive days postoperatively to prevent infection, and the rats were housed under standard conditions for 6 weeks.

At 6 weeks post-surgery, all animals were euthanized and the regenerated sciatic nerve segments were harvested for histological analysis. Tissue samples were sectioned along and perpendicular to the longitudinal axis of the nerve to obtain longitudinal and transverse sections with a thickness of 10 μm. Both longitudinal and transverse sections were stained with hematoxylin and eosin (HE) to evaluate the overall status of nerve regeneration. Longitudinal sections were further subjected to immunofluorescence staining for the mature neuron marker NF200 and the Schwann cell marker S100. Transverse sections were double-stained for NF200 and myelin basic protein (MBP), and additionally stained for the vascular endothelial marker CD31 to assess neovascularization within the regenerated nerve tissue.

4.10. Statistical analysis

All experimental data are presented as the mean ± standard deviation (Mean ± SD), with each group comprising at least three independent replicates. Image quantification was performed using ImageJ software (National Institutes of Health, USA), and statistical analyses were conducted using GraphPad Prism 9.0. For each field of view, the mean fluorescence intensity of the target marker channel (Marker mean) and the mean fluorescence intensity of the Hoechst channel (Hoechst mean) were measured separately. The Hoechst signal was used as an internal reference to normalize the fluorescence intensity of the target marker. Depending on the data type, differences between groups were evaluated using one-way analysis of variance (ANOVA), or two-way ANOVA. The significance levels were set as follows: p < 0.05, p < 0.01, p < 0.001, where p < 0.05 indicates statistical significance.

CRediT authorship contribution statement

Haiyang Zhang: Conceptualization, Data curation, Formal analysis, Investigation, Validation, Writing – original draft. Junbo Jiang: Conceptualization, Formal analysis, Methodology, Supervision, Writing – review & editing. Cailing Zhang: Data curation, Formal analysis, Validation, Visualization. Yi Zhang: Resources, Software, Validation. Chen Gao: Validation, Visualization. Yuxing Kuang: Data curation, Investigation. Guangqing Xu: Funding acquisition, Resources, Supervision, Visualization. Yue Lan: Conceptualization, Funding acquisition, Project administration, Resources, Writing – review & editing.

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Appendix A. Supplementary data

The following is the Supplementary data to this article.

Data availability

Data will be made available on request.