
==== Front
Bioact Mater
Bioact Mater
Bioactive Materials
2452-199X
KeAi Publishing

S2452-199X(24)00289-5
10.1016/j.bioactmat.2024.07.018
Article
Biomimetic multi-channel nerve conduits with micro/nanostructures for rapid nerve repair
Wang Xinqing xinqingwang_ecust@163.com
b
Chen Shuo cshuocc@163.com
b
Chen Xiaolei cxl_jsjj@163.com
b
Wu Juan janewu1106@163.com
b
Huang Zhenhua huangzhenhua013@163.com
b
Wang Jing wangjing08@ecust.edu.cn
ab
Chen Fangping fpchen@ecust.edu.cn
ab⁎
Liu Changsheng liucs@ecust.edu.cn
ab⁎⁎
a Key Laboratory for Ultrafine Materials of Ministry of Education, School of Materials Science and Engineering, East China University of Science and Technology, Shanghai, 200237, PR China
b Engineering Research Center for Biomedical Materials of Ministry of Education, East China University of Science and Technology, Shanghai, 200237, PR China
⁎ Corresponding author. Key Laboratory for Ultrafine Materials of Ministry of Education, School of Materials Science and Engineering, East China University of Science and Technology, Shanghai, 200237, PR China. fpchen@ecust.edu.cn
⁎⁎ Corresponding author. Key Laboratory for Ultrafine Materials of Ministry of Education, School of Materials Science and Engineering, East China University of Science and Technology, Shanghai, 200237, PR China. liucs@ecust.edu.cn
24 8 2024
11 2024
24 8 2024
41 577596
14 3 2024
13 7 2024
13 7 2024
© 2024 The Authors
2024
https://creativecommons.org/licenses/by-nc-nd/4.0/ This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
Peripheral nervous system (PNS) injuries often lead to significant sensory and motor impairments. Traditional artificial nerve conduits, lacking anisotropic structures, have been associated with prolonged repair time and failures in nerve regeneration. This study aimed to address these challenges by developing a novel approach for rapid repair of peripheral nerve injuries (PNI). A 3D oriented fibers scaffold featuring distinct radial (RFs) and longitudinal (LFs) fibers orientations was engineered using coaxial electrospinning and gas directional foaming techniques. This scaffold was then integrated with a shape memory conduit to form a directional multi-channel nerve conduit with micro/nanostructures. The results revealed that the grooved surface of the fibers significantly improved cellular directional guidance, effectively facilitating the migration of SCs from the periphery towards the center and from the base to the apex of the scaffold. In a rat model with a 10 mm nerve defect, the ND-PLATMC/LF ND-PCL scaffold significantly enhanced nerve regeneration and motor function recovery within 4 weeks. These results suggest the potential of this innovative scaffold for efficient repair of the nerve injuries.

Graphical abstract

Image 1

Highlights

• Inspired by the xylem structure of Redwoods, three-dimensional double-oriented PCL/PVP fiber scaffolds with controlled micro-nano grooves were prepared by a combination of coaxial electrospinning, gas foaming and in-situ pore forming technologies.

• Radial foaming (RF) and longitudinal foaming (LF) of PCL/PVP fiber scaffolds allowed flexible adjustment of pore sizes, along with nano-scale grooves varying in width and depth within individual fibers.

• The 3D fibrous scaffold guide'd long distance (>12 mm) directional migration of cells and promoted axonal elongation with the expression of neuroblast -related genes nearly doubling that of the control group.

• The channel micro-nano composite nerve catheter demonstrates rapid and high-quality repair of a 10 mm sciatic nerve defect in rats, highlighting its significant potential for nerve regeneration and motor function recovery.
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pmc1 Introduction

Peripheral nerve injuries (PNIs) tend to cause the proximal nerve unable to contact the corresponding target organ or control muscle tissue, resulting in corresponding dysfunction. The incidence of PNIs caused by life stress, traffic accidents and natural disasters has increased significantly [1,2]. In the United States and Europe, there are more than 100,000 cases per year of PNI that are difficult or poorly repaired [3,4]. When the PNIs is longer than 5 mm, it cannot be repaired autonomously. Autologous nerve is considered as “gold standard” for nerve injuries repair [5,6]. but they are limited by secondary damage [7], nerve length and thickness disparities, and availability of nerve sources [5,[8], [9], [10]]. Therefore, artificial nerve guidance conduits (NGCs) must be implanted to promote Large-scale nerve repair.

NGCs, as a longitudinal tissue that mimics the natural structure of neural pathway, is a channel for axon extension, guiding the regenerated nerve to reconnect with the target nerve [11]. To provide adequate spatial support for axonal growth and ensure successful bridging between the injured nerves with the NGCs, NGCs should possess highly oriented structure, porosity, controllable degradation and excellent biocompatibility, appropriate mechanical properties, and suitable elasticity [[12], [13], [14], [15], [16]]. The highly directional structure is of utmost importance, as a disordered structure can result in the misalignment of regenerated nerve positioning and an inability to accurately bridge the defect's two ends. Additionally, the strong hydrophobicity caused by the disordered fiber structure may impede material exchange at the defect site, significantly impacting repair efficiency. Topographical cues (including channel fiber alignment, fiber density, micro/nano arrays, and grooves) on cell migration and growth were widely studied [17]. The nanoscale topographical cues actively stimulate cellular responses and promote tissue regeneration [[18], [19], [20]]. Particularly, nanoscale protrusions, arrays, and grooves patterns significantly enhanced cell migration, proliferation and differentiation [[21], [22], [23]]. In the process of nerve regeneration，topographic cues were reported to facilitate neural conduit interaction with the surrounding microenvironment and the formation of cones at the distal tip of nerve conduit. The adhesion of growth cones on the conduit surface is imperative for promoting axonal growth. Therefore, it is of great significance to integrate suitable topographical cues into NGC to promote peripheral nerve regeneration [[24], [25], [26], [27], [28]].

What topographic cues in NGCs are more suitable for repairing large nerve defects? Up to 50 m, the natural redwood exhibits remarkable vitality，mainly due to its intrinsic oriented microchannel structure that facilitates efficient nutrient transport from the root system to the tree crown [38]. It's intriguing that the xylem of redwood shares a similar bundle structure with nerves. Therefore, in our pursuit of biomimicry, we draw inspiration from composition of a natural redwood trunk including the outer bark and phloem, which play a protective role for the inner xylem. Within the xylem itself, there are numerous directional microchannels with rough surfaces, which are crucial factors enabling directed long-distance transport of water and nutrients [39].

In recent years, various methods, such as solvent or thermally induced phase separation, injection molding, electrospinning, and fused deposition modeling have been developed to prepare the artificial NGCs [[29], [30], [31], [32]]. However, these methods are relatively intricate and involve the mold removal, which will affect the structural integrity of the formed conduits and trigger potential collapse [33]. What's worse, most NGCs lack topographical guidance cues and thus are difficult to effectively regulate cell behaviors [12]. In addition, the prevailing artificial NGCs predominantly adopt single-channel architectures, bridging the two ends of nerve defect merely through hollow conduits. Unfortunately, this design fails to simulate the anisotropic structure inherent in natural nerves, and the large hollow lumens may produce directional impediments, and hinder the recovery of nerve function [34]. Electrospun fibrous scaffolds show great advantages in tuning the cell behaviors during tissue regeneration because these scaffolds have a high specific surface area to support cell adhesion, as well as superior biocompatibility, biodegradability, and sufficient mechanical strength to serve as matrix. Electrospinning is one of research hotspots to prepare the scaffolds with topographic clues [[35], [36], [37]]. Hence, it is urgent to develop multi-channel NGCs with rich topographical cues to maximize nerve growth while mimicking the binding structural features of natural nerves.

Hence, it is of great significance to create nanoscale topographical cues on the fiber surface. However, challenges such as how to controllably prepare nanoscale-oriented fiber membrane, how to effectively transform 2D fiber membranes into micro-nano 3D structure scaffolds, and how to optimize topographical cues to precisely regulate cell behaviors remain to be addressed.

In this investigation, we fabricate NGCs for large-sized nerve defect repair by replicate the intricate natural structure of redwood (Fig. 1). In brief, diverse nanoscale patterns are initially constructed on the fiber surfaces using coaxial electrospinning technology to simulate the rough topography of microchannels. Subsequently, a 3D oriented fiber scaffold was successfully prepared by integrating coaxial electrospinning with gas foaming technology to mimic the architecture of redwood. Moreover, by combining the shape memory properties of the polymers with electrospinning technology, we devised single-channel shape memory conduits that mimics the functions exhibited by natural bark and phloem. Finally, the adept combination of the three-dimensional oriented fiber scaffolds and shaped-memory single-channel conduits facilitated on-site assembly at 37 °C, ultimately faithfully replicating the natural redwood tree trunk structure. We investigated the roles of fiber surface micro-patterns, three-dimensional fiber scaffolds, and shape-memory single-channel nerve conduits in supporting the migration and spatial organization of nerve cells in vitro, as well as in promoting nerve cell differentiation. To demonstrate the potential of our designed materials in the field of repairing large nerve defects, we created a 10 mm defect in the rat sciatic nerve and comprehensively analyzed the role of our biomimetic multi-channel composite nerve conduit in nerve repair.Fig. 1 Schematic depicting the process of replicating the natural redwood structure using material fabrication techniques, RF: Radial foaming. LF: Longitudinal foaming.

Fig. 1

2 Results

2.1 Fabrication and characterization of nanopatterned electrospun nanofiber membranes

Homogeneous emulsion was obtained by mixing poly (ε-caprolactone) (PCL) and polyvinylpyrrolidone (PVP) with different mass ratios (1:1, 1:2 and 2:1), and then stirring overnight in a mixed solution of dichloromethane (DCM)/N, N-dimethylformamide (DMF) (4:1). After standing for 24 h, obvious phase separation occurred (Fig. 2A, Fig. S1), and PVP pockets were formed on the surface of PCL fibers. With the stretching effect of coaxial electrospinning, PVP was extended and evenly dispersed in the form of nanorods on the PCL fiber surface (Fig. 2B). PVP was then completely removed by ethanol, and the nanoscale grooves were formed on the fiber surface to create patterned PCL fibers. Interestingly, a series of patterned PCL/PVP nanofiber membranes were constructed by varying the injection speed and the ratios of PCL to PVP.Fig. 2 (A) Digital photograph of a 10 wt% PCL and PVP blended emulsion, with phase separation of PCL and PVP after 24 h of placement. (B) Schematic representation of the nanogroove pattern formed on the surface of PCL/PVP nanofibers. (C) SEM observation for the surface morphology of PCL/PVP composite fiber membranes before and after PVP removal (The sample groups are as listed in Table 1). (D) Statistical analysis of the groove width and depth dimensions of PCL/PVP fiber membranes. (E) Degradation curves (F) Degradation rates and (G) pH changes of PCL/PVP fiber membranes in serum-free α-DMEM culture medium over 1 month. (H) CCK-8 assay for the proliferation of Schwann cells (SCs) on PCL/PVP fiber membranes. (I) Live/dead fluorescence staining of SCs on PCL/PVP fiber membranes.

Fig. 2

Fig. 2C showed the SEM images of PCL/PVP oriented nanofiber membranes prepared by coaxial electrospinning. Table 1 displayed PCL/PVP composite fiber membranes prepared at different mass ratios and injection rates. For micro-patterned PCL/PVP fiber membranes with high PVP content, with the increase of external fluid flow rate, the width of grooves on the fiber surface increased while the depths decreased (Fig. 2D). Fig. 2E–G revealed that the PCL/PVP nanofiber membranes with grooves exhibited a higher degradation rate. After degradation for 30 days, the pH of all PCL/PVP nanofiber membranes remained 7–7.2, which is favor of cell adhesion and proliferation. The results of CCK-8 and cell live/dead fluorescence staining in Fig. 2H and I showed that all patterned nanofiber membranes PCL had good biocompatibility after 1, 3, 5, and 7 days of culture. The cell proliferation rates of the two groups of PCL/PVP nanofiber membranes with mass ratios of 1:2 and 1:1 was much higher than that in the control group. It was mainly attributed to the directional alignment of PCL/PVP nanofibers and the abundant pores left after the removal of PVP, which provided the cells with topographical cues and more contact sites, thereby significantly enhanced cell spreading and migration, as well as accelerated cell proliferation [[40], [41], [42]].Table 1 PCL/PVP composite fiber membranes prepared at different conditions.

Table 1Groups	PCL:PVP（mass ratio）	Flow rate of internal fluid (mL/h)	Flow rate of external fluid (mL/h)	
a	1:0	1.0	/	
b	1:2	1.0	1.0	
c	1:2	1.0	1.5	
d	1:1	1.0	1.0	
e	1:1	1.0	1.5	
f	2:1	1.0	1.0 and 1.5	

In conclusion, we successfully fabricated a series of patterned PCL nanofiber membranes characterized by distinct grooved structures through a combination of coaxial co-spinning and in-situ pore formation method with the removal of PVP. These nanofiber scaffolds demonstrated superior biocompatibility and robust capacity to facilitate cell adhesion and enhance cell proliferation.

2.2 Patterned PCL/PVP nanofiber membranes regulate cell behavior in vitro

Micro- and nano-scale structural materials were reported to facilitate neural axons extension and glial cells migration, thus accelerating the neural repair [40,[42], [43], [44], [45]]. Fig. 3 represents the migration behavior of SCs (Schwann cells) on surface-patterned PCL/PVP nanofibers based on migration times and distances. As illustrated in Fig. 3A, the migration area was divided into Region I, Region II, and Region III. Under the guidance of the contact of the underlying fibers, the cells seeded on the left side of PCL/PVP fiber membranes directionally migrate to the right side.Fig. 3 (A) Fluorescence microscopy illustrates the migration paths of stem cells cultured on PCL/PVP nanofiber membranes of Group (a) to Group (f). (B) Quantification of stem cell numbers in different migration zones. (C) Measurement of migration distances of stem cells on PCL/PVP microfiber membranes of Group (a) to Group (f). (D) Relative mRNA expression level of MBP. (E) Relative mRNA expression level of S100. (F) Relative mRNA expression level of P0.

Fig. 3

Notably, in Region II and Region III, PCL/PVP nanofiber membranes featuring nano-grooves (Group b-Group f) exhibited a substantial increase in cell migration counts compared to PCL/PVP nanofibers without grooves (Group a). Specifically, the cell migration counts in Region II and Region III for grooved nanofibers were approximately 2–3 times and over 10 times greater than those of the smooth fiber Group a, respectively (Fig. 3B). Particularly, PCL/PVP nanofiber membranes of Group d and Group e demonstrated a significant enhancement in cell migration numbers in both Region II and Region III, highlighting their excellent capability to direct cell movement. Additionally, Fig. 3C measured the migration distance of cells across patterned PCL/PVP nanofiber membranes. Among all groups, Group d displayed the most rapid cell migration, with cells covering a distance of approximately 8.68 mm, significantly higher than the 3.32 mm observed for PCL/PVP nanofibers without grooves. Similarly, on grooved and porous PCL/PVP nanofibers, Group b-f migrated to the far-right edge of the fiber membrane at distances of 10 mm, significantly greater than the control group of 7.31 mm. These results indicate that nano-patterned PCL/PVP nanofibers with grooving structure exerted a topographic guiding effect, significantly modulating cell behavior and greatly enhanced the migration capacity of SC on nanofiber membranes with groove pattern.

Directional structures and surface-patterned structures have been shown to facilitate neural cell differentiation, direct cell migration, and axonal extension [[45], [46], [47]]. To assess the neural induction ability of PCL/PVP fibers, the expression levels of neural genes in SCs cultured on nanofibers were evaluated. As shown in Fig. 3D–F, in comparison to the control group, the relative mRNA expression levels of S100, P0, and MBP were all significantly upregulated in fiber groups with surface micro-patterns (P < 0.01). Particularly, Groups d and e exhibited the most prominent upregulation, with expression levels approximately 1.5–2 times higher than those of the control group. This demonstrates that the morphological characteristics of the nanopatterned fiber membranes prepared under a PCL: PVP ratio of 1:1 with outer liquid flow rates of 1.0 mL/h and 1.5 mL/h, are optimal for guiding cell directional migration and differentiation.

In conclusion, the experimentally constructed oriented PCL/PVP microfibers with distinct groove structures has been shown to significantly enhance neural cell proliferation, migration, and neural gene expression, particularly at a PCL to PVP mass ratio of 1:1. These nanostructured patterned fiber membranes display an exceptional capacity to guide neural regeneration, as evidenced by the observed improvements in cell proliferation and directional migration. Terrain cues have attracted considerable attention in the field of neural regeneration as a potential non-pharmacological therapeutic approach. It has been confirmed that terrain cues can provide crucial bioactive cues, thereby promoting axonal extension and accelerating neural functional recovery. Furthermore, terrain cues contribute to promoting neuronal differentiation of PC12 cells, neurite outgrowth of dorsal root ganglion (DRG) neurons, and the release of neurotrophic factors from SCs. Despite the numerous advantages of terrain cues in neural regeneration, there still exist a series of formidable challenges in how to successfully construct terrain cues and how to effectively modulate cell behaviors. In response to these issues, inspired by the structure of natural redwood tree trunks, we designed a biomimetic multi-channel composite nerve conduit with micro-nanostructures.

2.3 Fabrication and characterization of 3D oriented fiber scaffolds

Traditional electrospinning techniques produce two-dimensional (2D) nanofiber membrane, which are primarily intended for the repair of neural defects [48,49]. However, the dense 2D structure of these membranes can impede cell infiltration and migration, thereby limiting their effectiveness [33,50]. To overcome these limitations, this study took inspiration from the mathematical model of the rotating bodies and introduced gas foaming technology to construct 3D fiber scaffolds. These scaffolds resembled the structure characteristics of natural redwood stems and were built upon the 2D nanofiber membrane prepared in this study.

Inspired by mathematical models of rotational bodies, Fig. 4A introduces an innovative strategy by utilizing gas foaming technology to expand selectively patterned PCL/PVP fibers in directions either parallel or perpendicular to the fiber alignment. The resulting 3D fiber scaffolds possess dimensions and porosity that are conducive to the precise guidance of cell migration and differentiation in the 3D structure, while simultaneously providing enhanced regulation of cellular behavior. The most optimal fiber membranes (a PCL: PVP mass ratio of 1:1 and outer liquid flow rates of 1.0 mL and 1.5 mL) were subjected to gas foaming experiments. Specifically, Group d was prepared under conditions of PCL/PVP mass ratio of 1:1, inner liquid flow rate of 1.0 mL/h, and outer liquid flow rate of 1.0 mL/h, yielding a fiber surface groove width of 229.54 nm and depth of 351.36 nm, hence designated as ND-PCL (Narrow and Deep) group. Conversely, Group e was prepared with a PCL/PVP mass ratio of 1:1, inner liquid flow rate of 1.0 mL/h, and outer liquid flow rate of 1.5 mL/h, resulting in a fiber surface groove width of 285.44 nm and depth of 301.56 nm, thus named WS-PCL (Wide and Shallow) group. By employing different nanopatterned designs on the 2D aligned PCL/PVP fiber membranes, radial foaming (RF) and longitudinal foaming (LF) of the 3D PCL/PVP fiber scaffolds were achieved by supercritical carbon dioxide foaming (Fig. 4B and C). RF is characterized by the fixation of the surface perpendicular to the PCL/PVP nanofiber alignment, which induces a radial expansion of the 2D fiber membrane into a 3D fiber scaffold. In contrast, LF is achieved by fixing the surface parallel to the PCL/PVP nanofiber alignment, facilitating a longitudinal expansion of the 2D fiber membrane into a 3D fiber scaffold. In summary, this novel approach to expanding patterned nanofibers utilizing gas foaming technology has successfully yielded 3D PCL/PVP fiber scaffolds. The resulting 3D fiber scaffolds exhibit pore sizes and porosities that are conducive to the precise guidance of nerve cell migration and differentiation, as well as the efficient transport of nutrients.Fig. 4 (A) Schematic representation of the preparation process for the 3D oriented composite PCL/PVP fiber scaffold. (B) Illustration of the PCL/PVP supercritical foaming process. (C) Formation of different shapes of fiber scaffolds through multiple foaming processes of 2D fiber membranes after thermal treatment. (D) Surface morphology observation of the 3D scaffold. (E) Average pore size in Region 1 and Region 2 of RF and LF areas. (F) Statistical analysis of the surface groove dimensions for foamed WS-PCL and ND-PCL fiber scaffolds.

Fig. 4

Fig. 4D presents the digital photographs and SEM images of RF and LF at various cross-sections. The transverse section of RF is composed of radially arranged nanofibers forming a radial radiating channel structure, whereas the longitudinal section exhibits a porous structure. In contrast, LF exhibits a porous structure in its transverse section and a longitudinally aligned channel structure in its longitudinal section. Fig. 4E depicts the average pore size across different regions of RF and LF scaffolds, with Region 1 (central) exhibiting smaller average pore sizes than Region 2 (peripheral). Additionally, LF displays slightly larger average pore size than those of RF. Importantly, the porosity and pore size of RF and LF can be controlled by adjusting the thickness of the PCL/PVP 2D nanofiber membranes prior to foaming. However, there is a limitation to increasing the scaffold's pore size. When the thickness of the selected 2D fiber membranes is less than 0.6 mm, the foamed 2D fiber membranes are unable to form complete cylindrical 3D scaffolds by foaming. The detailed pore control method for 3D fiber scaffolds is described in Fig. S2. SEM observes the microstructure of individual fibers in RF and LF after their transformation into 3D scaffolds. Fig. 4F provides statistical data on the groove dimensions of WS-PCL and ND-PCL fibers following foaming. The average groove width and depth of WS-PCL are 287.25 nm and 307.1 nm, respectively, while for ND-PCL, they are 240.7 nm and 353.95 nm, respectively, which is in good agreement with the previous results (Fig. 2D). These results demonstrate the PCL/PVP 2D fiber membranes can be effectively expanded into 3D RF and LF fiber scaffolds (ND-PCL and WS PCL) with controlled 3D orientation, porosity, and groove size of individual fibers.

Fig. 5A and B presents compression performance of RF and LF scaffolds. The mechanical properties of RF scaffolds were marginally inferior to those of LF scaffolds. In comparison with pure PCL, WS-PCL and ND-PCL scaffolds exhibited a modest reduction in mechanical performance without a significant difference, indicating that the nanoscale grooves on the fiber surfaces exerted a negligible impact on mechanical properties of the scaffolds. The degradation rate of scaffolds is pivotal for tissue regeneration. Fig. 5C–E showed the degradation profile of 3D LFs fiber scaffolds. The results revealed that nanoscale grooves significantly enhanced the scaffolds’ degradability. After one week of degradation, the weight loss percentages for LF PCL, LF WS-PCL, and LF ND-PCL were 4.8 wt%, 8.2 wt%, and 11.9 wt%, respectively. By the end of four weeks, LF ND-PCL exhibited a weight loss percentage of 25.6 wt%, approximately 2.5 times that of LF PCL (10.8 %). Furthermore, the pH slightly decreased over time but remained neutral (about 7.1) without statistically significant differences among the groups. These experimental results demonstrated that all LFs 3D fiber scaffolds possessed favorable biodegradability and the nanoscale grooves notably accelerated the degradation process. Fig. 5H evaluated the hydrophilicity of LF PCL, LF WS-PCL, and LF ND-PCL scaffolds. The results indicate that the time required for water droplets to achieve zero contact angle on the scaffold surface for all scaffolds is less than 1.24 s, indicative of high hydrophilicity. Specifically, the retention times for LF WS-PCL and LF ND-PCL were 0.79 s and 0.25 s, respectively, indicating a substantial enhancement in hydrophilicity. This improvement is primarily attributed to the nanoscale grooves that widened gaps between fibers, leading to increased surface roughness and enhanced capillary effect, which in turn reduces the contact time. This excellent capillary effect is beneficial for enhancing the fluid absorption capacity, cell aggregation, recruitment of microenvironmental factors, and protein adsorption_ all of which are essential for facilitating tissue regeneration. The biocompatibility of the scaffolds was evaluated by CCK-8 assay and fluorescence staining. The scaffold extracts of LF PCL, LF WS-PCL, and LF ND-PCL were co-cultured with SCs. Fig. 5F demonstrated that the scaffold extracts had no significant effect on cell growth. Fig. 5G further indicated that there was no significant difference in biocompatibility between RF and LF 3D scaffolds. Fig. 5I displayed the fluorescence staining images of SCs co-cultured with LF extracts, and indicated that LF PCL, LF WS-PCL, and LF ND-PCL scaffolds possess excellent biocompatibility.Fig. 5 (A) Stress-strain curves of RF PCL and LF PCL (n = 5). (B) Compressive stress-strain curves of LF-PCL, LF WS-PCL, and LF ND-PCL groups (n = 5). (C-E) pH values and weight changes of LF PCL, LF WS-PCL, and LF ND-PCL after 4 weeks of degradation. (F) Effects of extracts from LF PCL, LF WS-PCL, and LF ND-PCL on SCs cell proliferation measured by cck-8 assay after 1, 3, and 5 days of culture. (G) Effects of extracts from RF-PCL and LF-PCL on SCs cell proliferation measured by cck-8 assay after 1, 3, and 5 days of culture. (H) Water contact angle images of LF PCL, LF WS-PCL, and LF ND-PCL. (I) Fluorescence staining images of SCs cells cultured with extracts from LF PCL, LF WS-PCL, and LF ND-PCL for 1 and 3 days.

Fig. 5

2.4 3D oriented fiber scaffolds regulate cell behavior

The impacts of RF and LF on cell migration were investigated using two distinct infiltration models: a peripheral infiltration model and a bottom infiltration model. SCs were seeded on 8 % Gel-MA hydrogels, which were then perforated to create 10 mm diameter holes. The scaffolds were inserted into the perforated cell gels to establish a model where cells infiltrate around the scaffolds. Alternatively, the scaffolds were directly placed on the cell gels to create a bottom infiltration model. All scaffolds were cultured for 3, 5, and 7 days in both infiltration models. In the peripheral infiltration model, SCs exhibited radial distribution after 3 days of cultivation. After 5 and 7 days of culture, SCs in RF scaffolds migrated from the edges towards the center, forming a nearly complete radial front (Fig. 6A). In contrast, during the same cultivation period, SCs in LF scaffolds mainly remained at the edges (Fig. 6B). For the bottom infiltration model, SCs cells were observed to migrate from the bottom to the top of LF scaffolds after culture for 5 days (Fig. 6D). However, no significant bottom-to-top migration of SCs was observed in the RF scaffolds. Particularly, after 7 days of cultivation, some SCs had migrated to the top of LF scaffolds, while the bottom-to-top migration of SCs in RF scaffolds remained minimal (Fig. 6C). This difference was primarily attributed to the directional guidance provided by the orderly 3D oriented fiber scaffolds on cell migration. Fig. 6E shows the quantified data on SCs coverage. After 7 days of culture, the average surface coverage of RF PCL, RF WS-PCL, and RF ND-PCL scaffolds were 55.7 %, 66.5 %, and 73.2 %, respectively. The surface coverage of RF WS-PCL and RF ND-PCL scaffolds was significantly greater than that of the PCL scaffold. These results indicate that RF WS-PCL and RF ND-PCL scaffolds possess superior capabilities in guiding cell radial migration than the PCL scaffold.Fig. 6 (A) Fluorescence images showing SCs migration in the peripheral cell infiltration model after 3, 5, and 7 days of cultivation on RF PCL, RF WS-PCL, and RF ND-PCL scaffolds. (B) Fluorescence images showing SCs migration in the peripheral cell infiltration model after 5 and 7 days of cultivation on LF PCL, LF WS-PCL, and LF ND-PCL scaffolds. (C) Fluorescence images showing SCs migration in the bottom cell infiltration model after 7 days of cultivation on RF PCL, RF WS-PCL, and RF ND-PCL scaffolds. (D) Fluorescence images showing SCs migration in the bottom cell infiltration model after 5 and 7 days of cultivation on LF PCL, LF WS-PCL, and LF ND-PCL scaffolds. (E) Cell coverage on RF PCL, RF WS-PCL, RF ND-PCL, LF PCL, LF WS-PCL, and LF ND-PCL scaffolds. (F) Cell migration distance of SCs from the bottom to the top on RF PCL, RF WS-PCL, RF ND-PCL, LF PCL, LF WS-PCL, and LF ND-PCL scaffolds.

Fig. 6

Similar results were obtained in the bottom infiltration model. Fig. 6F illustrates the migration of SCs from the bottom to the top of LF PCL, LF WS-PCL, and LF ND-PCL scaffolds after 3, 5 and 7 days of cultivation. Fig. 6D displayed the migration distance of SCs from bottom to top. As the culture period extended, the average migration distance of SCs significantly increased. After 5 days, SCs exhibited obvious bottom-to-top migration on LF PCL, LF WS-PCL, and LF ND-PCL scaffolds, with average migration distances of 497.5 μm, 607.3 μm and 719.6 μm, respectively. After 7 days of cultivation, the average migration distances on LF PCL, LF WS-PCL, and LF ND-PCL scaffolds were 926.6 μm, 1084.3 μm, and 1197.3 μm, respectively. These results indicated that 3D oriented fiber scaffolds with nano-grooves exhibited superior performance in directing cell migration than smooth-surfaced PCL fiber scaffolds. The nanogrooves offered additional contact sites and topographical cues for cell growth and migration, thereby enhancing cellular growth and migration along the oriented structures. Furthermore, the width and depth of the nanogrooves also affect the cell migration behavior. ND-PCL scaffolds, with narrower and deeper grooves, exhibited a stronger guiding capability for cell migration compared to the WS-PCL scaffolds. This is mainlydue to the increased roughness of the fiber surface, which provides more sensing points and contact areas for cell growth and migration, thereby demonstrating a stronger ability to guide migration and differentiation.

3D oriented fiber scaffolds can modulate the cell morphology. As depicted in Fig. 7A, cells on the LF PCL, LF WS-PCL, and LF ND-PCL displayed distinct elongation along the direction of the scaffold, contrasting with cells that freely grow on the culture dish surface. Fig. 7B and C quantitatively analyzed the cell morphologies on different scaffolds. The results revealed that cells on 3D oriented fiber scaffold exhibited varying degrees of elongation along the directional orientation, similar to the results observed for guided migration. Remarkably, LF ND-PCL demonstrated the strongest ability to guide cell directional elongation, with a cell aspect ratio of 16:1, much higher than the SCs cell morphology grown on the culture dish surface (with an aspect ratio of 2:1). These findings demonstrate the prominent ability of the prepared 3D PCL/PVP fiber scaffolds, characterized by directional arrangement structure and micro/nano patterning, to guide neurite extension and glial cell migration to the greatest extent, which is of paramount importance for neural tissue repair. Fig. 7D–F showed that relative mRNA expression levels of S100, P0, and MBP in LF PCL, LF WS-PCL, and LF ND-PCL groups were all upregulated, with the exception of the control group. The LF ND-PCL exhibited the most pronounced effect, approximately doubling the expression intensity compared to the control group. LF ND-PCL group had the highest up-regulation in expression intensity, about twice that of control group. Considering cell proliferation, guided axonal extension, cell directional migration, and neural gene expression, the designed 3D bi-axially oriented fiber scaffolds possess superior cellular regulatory ability and play a crucial role in nerve defect regeneration.Fig. 7 (A) Immunofluorescence staining images of SCs cells cultured on LF PCL, LF WS-PCL, and LF ND-PCL scaffolds for 24 h. (B) Cell growth morphology, (C) Aspect ratio of cell morphology, (D) mRNA expression level of S100 in SCs cells, (E) mRNA expression level of MBP in SCs cells, and (F) mRNA expression level of P0 in SCs cells on different scaffolds.

Fig. 7

2.5 Shape memory single channel neural conduit and its regulation on cell behavior

The aforementioned results showed that the prepared 3D fiber scaffolds possessed exceptional capabilities in directing cell migration and differentiation. However, they encounter challenges when directly bridging nerve defects, which can result in complications such as dislocation and disintegration post-implantation. To address these challenges, we developed a shape-memory single-channel conduit with thermos responsive properties. This conduit assists the connection the nerve defect's ends to the 3D fiber scaffolds, akin to the way the he protective role of outer bark and phloem for the xylem. It firmly secures the scaffold at the defect site to prevent displacement and creates an isolated microenvironment conducive to nerve repair. Poly l-lactic acid-co-trimethylene carbonate (PLATMC) nanofibers with different copolymer ratios were prepared via electrospinning The nanofibers with the LA:TMC ratios of 80:20, 70:30, and 60:40 all exhibited uniform diameters, smooth surfaces, and lacked apparent beading structures (Fig. 8A). The average diameters of the three PLATMC nanofibers were 702.56 ± 174.36 nm (LA: TMC 80:20), 1112.97 ± 121.65 nm (LA: TMC 70:30), and 1131.12 ± 145.45 nm (LA: TMC 60:40), respectively (Fig. 8B). The data indicated that the fiber diameter significantly increased as the LA:TMC ratio decreased from 80:20 to 70:30. However，when the LA: TMC ratio further decreased to 60:40，the average diameter of PLATMC did not change significantly. Fig. 8C showed that the glass transition temperature (Tg) of PLATMC (LA: TMC 60:40), PLATMC (LA: TMC 70:30) and PLATMC (LA: TMC 80:20) were 31.2 °C, 36.7 °C and 41.2 °C. The result indicated that Tg of PLATMC gradually increased with the increased LA content in PLATMC. As a shape memory polymer, PLATMC's transition temperature is equivalent to Tg. It is evident that the transition temperature of PLATMC can be regulated by the ratio of LA to TMC. In this study, we aimed to develop PLATMC with shape-memory properties capable of forming temporary planar fiber membranes at room temperature and fully recovering their permanent tubular structure at physiological temperature. Consequently, the transformation temperature of PLATMC fiber membranes should be above room temperature but below physiological temperature. Thus, PLATMC copolymer with an LA:TMC ratio of 70:30 was selected for the preparation of neural conduits. The mechanical properties of PLATMC fiber membranes with different feed ratios were assessed. The tensile strength of membranes along the fiber orientation direction and parallel to the fiber orientation direction were displayed respectively in Fig. 8D and E. The results showed that these fiber membranes exhibited comparable mechanical properties, with a slight increase in tensile stress as the TMC content increases. These results indicated that all PLATMC fiber membranes satisfy the criteria for neural repair. Based on these results, PLATMC (LA: TMC 70:30) with a transformation temperature of 36.7 °C was selected for subsequent experiments. PLATMC fiber membranes were prefabricated into tubular configurations at elevated temperature and subsequently flattened at room temperature. These membranes could be triggered to revert to their original tubular form at physiological temperature. Upon integration with LF ND-PCL scaffolds, the PLATMC membrane could regain its permanent tubular structure upon stimulation at physiological temperature, which was enveloped around the exterior of the internal multi-channel scaffold to form a composite neural conduit (Fig. 8F). The programmed deformation and self-formation properties of the PLATMC fiber membrane are mainly attributed to its excellent shape-memory performance. The shape recovery process of PLATMC from the temporary 2D planar shape to the permanent tubular structure at 37 °C was accomplished within 12 s. After 5 deformation cycles, the shape of the PLATMC/LF ND-PCL remained indistinguishable from its initial state, indicating that the deformation process was rapid and reproducible (Video S3). Therefore, by virtue of the shape memory characteristics of PLATMC fiber membrane, the composite neural conduit was assembled by wrapping LF-ND-PCL with PLATMC.Fig. 8 (A) SEM observation the microstructure of PLATMC nanofibers. (B) Diameter distribution of PLATMC nanofibers measured with ImageJ software. (C) DSC curves of PLATMC fiber membranes. (D) Stress-strain curves of PLATMC nanofiber membranes subjected to uniaxial stretching along the fiber orientation direction (n = 6) and. (E) Stress-strain curves of PLATMC nanofiber membranes subjected to uniaxial stretching parallel to the fiber orientation direction (n = 6). (F) Assembly process of PLATMC/LF ND-PCL by wrapping multi-channel LF ND-PCL scaffolds with PLATMC.

Fig. 8

Nano-scale grooves on fiber surface have been found to exert a positive regulatory influence on cell behavior. Using coaxial electrospinning, we fabricated PLATMC-oriented fiber membranes with nano-scale grooves on the surface, designated as ND-PLATMC group, and those with a smooth surface as the PLATMC group. Cells cultured in cell culture dishes served as the control group. Fig. 9A illustrates the surface morphology of PLATMC oriented fibers observed by SEM. Prior to the removal of PVP, both groups exhibited smooth surfaces. After PVP removal, PLATMC group retained a smooth surface, while the ND-PLATMC group displayed distinct nanoscale groove structures. Quantitative analysis in Fig. 9B showed that the average width and depth of grooves in the ND-PLATMC group were 251.48 nm and 362.08 nm, respectively, consistent with our previous results (Fig. 2 D).Fig. 9 (A) SEM observation the microstructures of distinct PLATMC fibers. (B) Dimensions of surface grooves of ND-PLATMC fibers. (C-D) Weight loss and pH after PLATMC and ND-PLATMC degradation for 4 weeks. (E) CCK-8 assay the extract solutions of PLATMC and ND-PLATMC on SCs cell proliferation after incubation for 1, 3, and 5 days. (F) Fluorescence staining observation SCs cell morphology after culture with PLATMC and ND-PLATMC extract solutions for 1 and 3 days. (G) Fluorescence imaging depicting SCs cell migration and cellular morphology on the different conduits. (H) Quantitative analysis of SCs cell growth morphology on different conduits. (I) mRNA expression level of S100 in SCs cells cultured on different scaffolds. (J) mRNA expression level of MBP in SCs cells cultured on different scaffolds. (K) mRNA expression level of P0 in SCs cells cultured on different conduits.

Fig. 9

Based on the unique characteristics of ND-PLATMC structure, we hypothesized that the ND-PLATMC group may exhibit superior positive cell regulatory ability than the PLATMC group. The results confirmed that nano-patterned fiber surfaces be controllably fabricated through coaxial electrospinning, enabling the design of polymer fibers with diverse surface morphologies that are immiscible with PVP. After degradation for 4 weeks, the weight loss percentages for PLATMC group and the ND-PLATMC group were 6.4 wt% and 8.4 wt%, respectively (Fig. 9C). During the whole degradation process, the pH slightly decreased but maintained at 7.26, with no significant difference between the groups (Fig. 9D). The biocompatibility of the PLATMC nerve conduit was assessed using SCs cells co-cultured with the extracted solutions of PLATMC and ND-PLATMC fiber membranes, as evaluated by CCK-8 assay and fluorescence staining. The results demonstrated that both groups of extracted solutions did not significantly impact cell growth and had no significant difference (Fig. 9E and F), indicating their good biocompatibility. Fig. 9G showed the distribution of SCs within the conduits after 7 days of cultivation, as observed by fluorescence staining. Green-fluorescent-stained cells were evenly distributed on the inner surfaces of nerve conduits in both groups. In conduits with randomly oriented fibers, most SCs exhibited immature, round shapes with disorganized distribution. In contrast, SCs within the oriented PLATMC and ND-PLATMC conduits, and nearly all SCs showed significant oriented growth, with elongated cell bodies and axons. This is particularly true for the grooved ND-PLATMC nerve conduit, which effectively guided cell migration and axonal extension, with individual cell orientation lengths of 112.39 μm, approximately 3 times that of the control group (41.16 μm) (Fig. 9H). This is attributed to the directional arrangement of the ND-PLATMC conduit and the groove structure on the fiber, which provide additional adhesion sites for cells, thereby enhancing the ability to guide cell migration and extend neural cell synapses. In addition, compared to the control group, the relative mRNA expression levels of S100, P0, and MBP in the PLATMC and ND-PLATMC groups were upregulated, with expression levels in ND-PLATMC group being 1.4–1.8 times higher than those in the control group (Fig. 9I–K). This suggested that the oriented fiber arrangements upregulate the expression of neural genes, and the nanoscale grooves on the fiber surface further amplify these gene expression levels. In conclusion, the temperature-responsive nerve conduit ND-PLATMC developed in this study enabled uniform distribution and oriented migration of SCs on the luminal surface, effectively regulated cell extension in a predetermined direction, and upregulated the expression of relevant genes. Most importantly, the evenly distributed SCs throughout the conduit provided a favorable and independent microenvironment for cell migration, differentiation, and subsequent axonal extension in neural regeneration.

2.6 In vivo evaluation of the performance of biomimetic multi-channel neural conduits

To assess the potential of the oriented multi-channel composite conduit in nerve repair, The prepared biomimetic artificial nerve conduit was implanted into a 10 mm nerve defect of the rat sciatic nerve. Fig. 10A showed that LF ND-PCL and ND-PLATMC were in situ assembled under the trigger of physiological temperature to bridge the two ends of the nerve defect without suturing. Fig. 10B shows the digital photos of the construction process of rat nerve defect model and the surgical implantation of materials in different experimental groups. Nerve regeneration was evaluated at 1 week, 2 weeks, and 4 weeks postoperatively. All animals survived without postoperative complications.Fig. 10 (A) Schematic illustration of the sciatic nerve defect model in SD rats with implanted nerve conduits. (B) Digital photographs of surgical implantation of nerve conduits in different experimental groups. (C) Immunofluorescence images of longitudinal sections of regenerated nerves at 4 weeks post-surgery, stained for NF200 and S-100β. (D) Immunofluorescence images of cross-sections of regenerated nerves at 4 weeks post-surgery, stained for NF200 and S-100β. (E) Percentage of NF200-positive area. (F) Percentage of S-100β-positive area.

Fig. 10

At 4 weeks postoperatively, the expression levels of the nerve fiber marker NF200 and the SC-specific marker S-100β were detected by immunofluorescence staining in regenerating nerves (Fig. 10C and D). As the nerve defect length (10 mm) surpassed the autologous repair capacity in SD rats, no substantial nerve regeneration was observed occurred in the blank group's nerve defect after 4 weeks. The expression of NF200 (in green) and S-100β (in red) proteins were detected in the other groups, confirming the formation of new axons and the myelin sheaths composed of SCs at the site of nerve defect. Image-J software was utilized to quantify the percentage of NF200 and S-100β positive areas in immunostained images (Fig. 10E and F). The autograft group exhibited the highest percentage of positive area for both NF200 and S-100β. In addition, the expression levels of these markers in ND-PLATMC/LF ND-PCL composite conduit were approximately 2–3 times higher than in the ND-PLATMC single-channel conduit. The results indicate that biomimetic multi-channel conduits incorporating micro-nanostructures can effectively enhance axonal ingrowth and promote the regeneration of myelin sheath in newly formed nerve tissue, further facilitating axonal regeneration, which is of considerable importance for nerve defect repair.

Hematoxylin and Eosin (HE) staining were employed to assess the neural repair of ND-PLATMC, ND-PLATMC/LF ND-PCL and autograft groups at 2- and 4-weeks post-operation. Hematoxylin stains chromatin and nucleic acids in the cytoplasm purple-blue, while Eosin stains cellular and extracellular matrix components red. Positive HE-staining areas represent the growth of new nerve cells and tissues. After 4 weeks, the Blank group showed no connection of damaged nerves in and lacked significant histological changes. Fig. 11A depicted that HE staining positivity in ND-PLATMC/LF ND-PCL was significantly higher than that in ND-PALTMC group, suggesting more nerve cells and tissues extended into the composite conduit. The newly formed nerve tissue exhibited well-arranged fibers and channel guidance along the conduit's longitudinal axis. Compared to the ND-PLATMC conduit, the multi-channel ND-PLATMC/LF ND-PCL contained more new nerve tissue and exhibited a more regular arrangement. This study confirmed the critical role of topographical guidance cues provided by nerve conduits in guiding nerve regeneration. HE staining of the middle cross-sections of the injured nerve further investigated the neural tissue regeneration. Local inflammation, an important indicator for assessing the efficacy of nerve guidance conduits, showed no significant infiltration of inflammatory cells in all groups (Fig. 11A). The formation of new blood vessels was crucial for neural tissue regeneration. In magnified cross-sections, new blood vessels containing red blood cells (enclosed circles indicated by yellow arrows) were observed between the regenerated nerve tissues in all groups. Fig. 11C quantitatively analyzed the micro vessel density. The ND-PLATMC/LF ND-PCL group demonstrated similar neovascularization ability to the autograft group, with a significantly higher number of newly formed blood vessels per unit area compared to the ND-PALTMC single-channel conduit group. The results indicated that the ND-PLATMC/LF ND-PCL group excelled in promoting neural tissue regeneration. Cresol Purple (TB) and Luxol Fast Blue (LFB) staining were performed on the regenerated middle nerves 2- and 4-weeks post-surgery to observe the formation of myelinated nerve fibers and myelin sheath. Fig. 11B showed that SCs within the newly generated nerves were stained blue with TB, with some SCs forming closed circles and growing into myelin sheaths around nerve fibers (indicated by yellow arrows). At 4 weeks post-operation, the ND-PLATMC/LF ND-PCL conduit demonstrated a more pronounced capability for regenerating myelinated nerve fibers than the ND-PLATMC single-channel conduit. Image-J software was used to calculate the density of SCs per unit area was calculated by based on TB-stained images. The results indicated that the ND-PLATMC/LF ND-PCL composite conduit had an SCs density of approximately 5349 cells/mm2 (Fig. 11D), significantly higher than that of ND-PLATMC single-channel conduit. LFB staining revealed the bright blue myelin sheath formation within the regenerated nerves（Fig. 11B）. The percentage of the myelin sheath-positive area (bright blue area) was calculated using Image-J software based on LFB-stained images. Fig. 11E showed that the myelin sheath-positive area percentage in ND-PLATMC/LF ND-PCL composite conduit was similar to that of the autograft group, but significantly higher than that in ND-PLATMC single-channel conduit. In the single-channel conduit, positive regions were only observed near the conduit's inner wall, whereas in the ND-PLATMC/LF ND-PCL composite conduit, positive regions filled the entire lumen. These results indicate that the repair effect of the ND-PLATMC/LF ND-PCL composite nerve conduit is markedly superior to that of the single-channel nerve conduit, and the designed composite nerve conduit offers unparalleled advantages in guiding nerve regeneration.Fig. 11 (A) Histological evaluation of regenerated nerve tissue and vascular growth: Longitudinal sections of regenerated nerves stained with HE/TB at 4 weeks post-surgery, and cross-sections of mid-regenerated nerves at 2- and 4- weeks post-surgery, showing HE staining. (Yellow arrows indicate neovascularization). Histological morphological assessment of cross-sections of mid-regenerated nerves at 4 weeks post-surgery. (B) Cross-sections stained with TB/LFB. Yellow arrows indicate axons enveloped by SCs. (C) Microvascular density calculated based on HE staining. (D) SC density calculated based on TB staining. (E) Percentage of myelin sheath-positive area calculated based on LFB staining.

Fig. 11

The effect of multi-channel nerve conduits on peripheral nerve regeneration was evaluated by monitoring the restoration of motor function in rats. One month postoperatively, the 3D pressure distribution on the hind paw was shown in Fig. 12A. The hind paw pressure in the ND-PLATMC/LF ND-PCL group (the limb with the injury) was found to be comparable to that of the autograft group. Sciatic functional index (SFI) served as an effective parameter to assess the functional status of the rat's injured sciatic nerve (Fig. 12C). An SFI value approaching 0 indicates normal motor function, whereas a value close to −100 indicates complete functional impairment. 1-week post-surgery, SFI values progressively increased in all groups, with no significant inter-group differences. However, at 2- and 4-weeks post-surgery, SFI values in ND-PLATMC/LF ND-PCL group were similar to those of the autograft group and significantly higher than those in the Blank and ND-PLATMC groups, and the SFI values in the ND-PLATMC/LF ND-PCL group. At 2 weeks post-surgery, the SFI values for the ND-PLATMC/LF ND-PCL group and autologous graft were respectively approximately −58.03 and −59.13, significantly higher than those in the Blank group (−78.23) and the ND-PLATMC group (−77.53). At 4 weeks post-surgery, the SFI value in the ND-PLATMC/LF ND-PCL group increased to around −46.32, showing no significant difference from the autograft group.Fig. 12 (A) 3D plantar pressure distribution of the limb at 1-month post-surgery. (B) Digital photographs of the injured/healthy limb gastrocnemius muscle at 4 weeks post-surgery, along with cross-sectional Masson's trichrome staining of the left (injured limb) gastrocnemius muscle. (C) Sciatic Functional Index (SFI) at 1-, 2-, and 4-weeks post-surgery (n = 3). (D) The wet weight ratio of the injured limb gastrocnemius muscle compared to the healthy limb at 4 weeks post-surgery. (E) Cross-sectional area of muscle fiber (n = 3) at 4 weeks post-surgery.

Fig. 12

Sciatic nerve transection results in denervation of the gastrocnemius muscle, and inadequate nerve regeneration can prevent the gastrocnemius muscle from achieving reinnervation, leading to muscle atrophy. This study aimed to evaluate the impact of different materials on the motor function recovery in rats with nerve defects by measuring the degree of atrophy in the gastrocnemius muscle of the injured limb. The gastrocnemius muscle was subjected to cross-sectional Masson's trichrome staining and muscle wet weight ratio were analyzed on at 4 weeks post-implantation. To quantitatively assess muscle atrophy, the wet weight ratio of gastrocnemius muscle in the injured limb was compared to that of the healthy limb, as shown in Fig. 12D. The results indicated that the wet-weight ratio in the ND-PLATMC/LF ND-PCL group was about 81.3 %, which was similar to that in the autograft group and significantly higher than that observed in Blank group (32.3 %) and the single-channel conduit group (48.4 %). Cross-sectional Masson's trichrome staining images of the gastrocnemius muscle in the injured limb were presented in Fig. 12B. In the ND-PLATMC/LF ND-PCL group and the autograft group, there was a significant decrease in collagen deposition and an increase in the average cross-sectional muscle fiber area, indicating improved muscle nerve regeneration. The statistical analysis of muscle fiber cross-sectional area is shown in Fig. 12E, where the ND-PLATMC/LF ND-PCL exhibited a slightly lower muscle fiber area (approximately 1363 μm2) than the autograft group (approximately 1452 μm2), but significantly larger than the Blank group (approximately 722 μm2) and the ND-PLATMC conduit group (approximately 819 μm2). These results further underscored the positive therapeutic effect of multi-channel nerve conduits on muscle reinnervation. In conclusion, the multi-channel composite nerve conduit effectively promoted nerve defect regeneration, yielding significantly superior repair results than the single-channel conduit group, and results comparable to those of the autograft group. These findings validate our hypothesis that nerve conduits with internal oriented multi-channel structures offer advantages in the field of nerve defect repair.

3 Conclusion

Terrain cues, a significant non-pharmacological strategy in neural regeneration, have been explored for their ability to provide crucial bioactive signals that facilitate axonal elongation and neural recovery. In this study, to enhance the directional migration and differentiation of neural cells, a surface-patterned 2D oriented fiber membrane was initially fabricated using PCL and PVP as raw materials by coaxial electrospinning technology. The results showed that cellular behavior was regulated by the fiber surface patterns with various shapes and sizes.

The 2D fiber membrane was further expanded into a 3D fiber scaffold (ND-PCL) with oriented microchannels, including radial microchannels (RFs) and longitudinal microchannels (LFs), by employing gas directional foaming and in situ pore formation techniques. The 3D fibrous scaffold guided long distance (>12 mm) directional migration of cells and promoted axonal elongation, with the expression of neuroblast -related genes nearly doubling that of the control group. To address the limitation that the pure 3D fiber scaffold could not directly bridge nerve defects, a temperature-responsive shape memory nerve conduit was fabricated and assembled with the ND-PCL to form a multi-channel composite nerve conduit, incorporating micro-nanostructures. Finally, using a rat model with a 10 mm sciatic nerve defect, the study revealed the conduit's efficacy in repairing nervous defects and restoring motor function. The results indicated that the fabrication of multi-channel nerve conduit based on the oriented PCL/PVP microfibers with distinct groove structures in this study offers a novel approach for rapid PNS injury repair and held promising potential for clinical applications.

4 Methods

Materials. PCL (Mw = 80 kD a) obtained from Nantong Feiyu Biomaterial Co., Ltd. (Nantong, China). PVP (Mw ≈ 220 kDa) were purchased from Sigma-Aldrich, St. Louis, MO (Shanghai, China). Dichloromethane (DCM), N, N dimethylformamide (DMF) obtained from Aladdin Chemistry Co., Ltd. (Shanghai, China). Cell Counting Kit 8 (CCK-8), TRITC-labeled phalloidin, Triton X-100 and 4,6-diamidino-2-phenylindole (DAPI) were purchased from Beyotime Biomaterial Co., Ltd. (Shanghai, China).

Fabrication of a multi-channel composite nerve conduit. A multi-channel nerve conduit (ND-PLATMC/LF ND-PCL) with micro-nano structures was prepared as follows: (1) Preparation of patterned electrospun PCL/PVP nanofiber membranes (a-f); (2) Expansion of 2D fiber membranes into 3D fiber scaffolds (LF/RF ND-PCL); (3) Preparation of temperature-responsive shape memory fiber membranes; and (4) In-situ assembly of the multi-channel nerve conduit (ND-PLATMC/LF ND-PCL).（1）Preparation of oriented PCL/PVP nanofiber membranes: PCL (Mw ≈ 110,000) was dissolved in a 4:1 (v/v) mixture of dichloromethane (DCM) and N, N-dimethylformamide (DMF) to form the inner fluid with a final concentration of 10 wt%. PCL and PVP (Mw ≈ 130,000) were dissolved in a DCM/DMF mixture to form the outer fluid at mass ratios of 1:2, 1:1, and 2:1, each with a final concentration of 10 wt%. The inner fluid flow rate was 1.0 mL/h, and the outer fluid flow rates were 1.0 mL/h and 1.5 mL/h. A DC voltage of 12 kV was applied between the needle and the collector (rotating drum at 3000 rpm), with a collection distance of 20 cm. After electrospinning, the fiber membranes were washed 3 times with ethanol and deionized water to remove PVP, resulting in PCL/PVP nanofibers with grooves or pores. (2) Expansion into 3D fiber scaffolds: The Approximately 1 mm thick (50 mL spinning solution) uniaxially aligned nanofiber membranes were electrospinned. The nanofiber membranes were then cut into rectangles (10 × 12 mm) either aligned with the nanofiber direction or perpendicular to it, and then thermally treated at 85 °C to fix their shape. Subsequently, 1 g of dry ice and a piece of nanofiber membrane (Groups d and e） were placed in a 30 mL centrifuge tube. Upon the instant transformation of dry ice into liquid carbon dioxide, the tube cap was quickly opened to facilitate rapid depressurization, causing the liquid carbon dioxide to vaporize and foam the nanofiber (Video S1). This process was repeated until the desired shape was obtained (Video S2). The scaffold shapes were controlled by the number of pressure reductions (one reduction for wedge-shaped scaffolds, three reductions for 3/4 cylindrical scaffolds, and four reductions for complete cylindrical scaffolds). After foaming, PVP was removed, yielding PCL 3D oriented fiber scaffolds (RFs and LFs) with controlled nano-grooves on the surfaces. (3) Preparation of the shape memory single-channel nerve conduit: PLATMC copolymer was prepared with LA: TMC feed ratios of 80:20, 70:30, and 60:40 ，dissolved in hexafluoroisopropanol (HFIP) and stirred at room temperature for 12 h to obtain a 12 wt% solution. Fibers were prepared using conventional electrospinning techniques. The prepared solution was loaded in a 10 mL syringe and extruded through the peristaltic pump at a flow rate of 1.5 mL/h. A voltage of 16 kV was applied between the nozzle (22G needle) and the collector, which was either covered with aluminum foil or a high-speed rotating drum (3000 rpm). After spinning, the collected fibers were freeze-dried overnight to remove residual solvents. Based on the immiscibility of PLATMC and polyvinyl pyrrolidone (PVP), a 70:30 mass ratio of PLATMC was dissolved in HFIP to form a 12 wt% solution，serving as the inner fluid for the coaxial electrospinning. Concurrently, PLATMC and PVP (Mw = 130,000) were dissolved in HFIP at a 1:1 mass ratio to form the final 12 wt% solution as the outer fluid. The inner fluid flow rate was set at 1.0 mL/h, and the outer fluid flow rate at 1 mL/h. A voltage of 16 kV was applied between the 22G needle and the high-speed rotating drum (3000 rpm). After spinning, the fibers were treated with ethanol and deionized water to remove PVP and residual ethanol and then subjected to freeze-drying. (4) In-situ assembly of the multi-channel nerve conduit: The shape memory single-channel nerve conduit ND-PLATMC and the 3D fiber scaffold LF ND-PCL were placed at the site of the nerve defect and assembled in situ at physiological temperature (37 °C) to obtain a multi-channel composite nerve conduit with micro-nano structures.

Fiber and pore size measurements. The scaffolds were mounted on aluminum stubs, sputter-coated with gold, all scaffolds were observed by SEM (Japanese Hitachi, S-4800) at an accelerating voltage of 15 kV. For each scaffold, 5 SEM images were captured, and images (at least 30 fibers and 30 pores) were manually measured and analyzed with ImageJ software v1.5 (NIH, Maryland, US) to calculate the average fiber diameter, microchannel diameter and pore size.

Mechanical properties. The mechanical properties of 3D fibrous scaffolds (ϕ2.0 × 1.2 cm3) and PLATMC fiber membranes （1 × 1 cm2）were evaluated with a universal material testing machine at a loading rate of 1 mm/min. A 5 kN load cell was employed and stress-strain curves were recorded. 5 replicates were carried out for each group.

In vitro degradation. The degradation of samples in vitro was carried out in serum-free DMEM medium (pH = 7.4). Three parallel control samples were set for each group of samples, and the final test result was the average of the three groups. Prior to immersion, each sample was weighed and its initial mass was recorded as M0. The samples were immersed in serum-free DMEM culture solution at a ratio of 1∶100 (g/mL) and stored in a shaking incubator at 37 °C. The medium was changed every 3 days. Solution pH was measured at indicated time intervals, samples were removed and oven-dried to constant weight at 37 °C and recorded as M1. In vitro weight loss of PCL/PVP fibrous scaffolds was calculated using the following formula: Weight loss (%) = (M1-M0)/M0 × 100.

Cultivation of Schwann cells (SCs). The SCs were obtained from the Cell Bank of the Chinese Academy of Sciences. Initially, the cryopreserved cells were resuscitated by adding10 ml of culture medium to a centrifuge tube, and placing the cell vial ed in a 37 °C water bath. The cells were agitated rapidly until completely thawed, and then transferred into a laminar flow hood. The cell suspension was aspirated into a centrifuge tube containing the culture medium and centrifuged at 800 r/min for 4 min. After centrifugation, the supernatant was removed, and 5 ml of fresh culture medium was added. The cells were gently pipetted up and down for 10 times, and then the cell suspension was transferred into a culture flask. The flask was placed in a 5 % CO2, 37 °C incubator for cultivation. SCs, being adherent cells, were cultured in DMEM medium supplemented with 10 % fetal bovine serum. Routine cell culture techniques were employed for passaging and subculturing procedures.

In vitro biocompatibility: Cell viability and proliferation on samples were assessed using the cck-8 assay and fluorescence staining. SCs were seeded at a density of 3 × 103 cells per well in a 24-well plate, adhering to the sterilized 2D fiber membrane (10 × 10 mm2) surfaces. At intervals of 3, 5, and 7 days, culture medium was refreshed with fresh medium containing CCK-8 reagent. Following incubation at 37 °C for 3 h, OD values were measured at 450 nm (with a reference wavelength of 650 nm). 3D fibrous scaffolds were soaked in 0.1 g/mL serum-free α-DMEM culture medium for 24 h. The extraction culture medium was created by supplementing the soaking solution with 10 % FBS. The preparation method of material leaching solution was carried out in strict accordance with ISO_10993_12 standard. While SCs in the control group were maintained in standard medium, those in the experimental group were exposed to the extraction culture medium for 1, 3, and 5 d. Proliferation was assessed using the CCK-8 assay. Cell viability and morphology were assessed using Calcein-AM/PI live/dead staining and observed with a confocal microscope.

Cell migration directed by scaffolds. To examine the impact of nano-patterned fibers on cell directional migration, SCs were seeded on culture dishes at a density of 5 × 103 cells/cm2. After overnight incubation, cells on the right side were removed, and composite fiber membranes (Group a to f, 1 × 1 mm2) were placed on that side. The directed migration of cells from left to right was observed at designated time points using a laser confocal microscope. The cells were fixed, stained with FITC-phalloidin and DAPI, and washed before observation.

Gelatin Methacryloyl (Gel-MA) hydrogel served as a substrate for both peripheral and bottom cell infiltration models. The models were cultured for 3, 5, and 7 days. Gel-MA hydrogel was disinfected, mixed with a photo initiator, and crosslinked with UV light. SCs were seeded on the hydrogel, and after culturing, RF or LF was introduced into the models. After predefined time points, RFs and LFs were removed, fixed, stained with FITC-phalloidin and DAPI, and observed using a laser confocal microscope. To evaluate the initial adhesion ability of SCs on 3D RF and LF scaffolds, the expression of focal adhesion protein (Vinculin) was analyzed by immunofluorescence staining. After 24 h of culture, scaffolds were removed, fixed, permeabilized, and blocked. Cells were incubated with Vinculin primary antibody, followed by an Alexa Fluor®647-labeled secondary antibody. The expression of Vinculin was observed and imaged using a laser confocal microscope, and ImageJ software was used for quantitative analysis of fluorescence intensity.

Gene expression of SCs cells analyzed by PCR On the 7th day, total RNA was extracted from the scaffolds where the cells were seeded. The RNA yield was quantified using a Nano Drop spectrophotometer. PCR was performed on a CFX96 real-time PCR system (Bio-Rad, Hercules, CA). The expression of S100, P0, and MPB genes in SCs cells was detected. The relative expression levels of the corresponding mRNA were calculated using the 2−(ΔΔCT) method and normalized to the reference gene GAPDH. The primer sequences were detailed in Supplementary Table 1.

Animal selection and grouping. 8-week-old male Sprague-Dawley (SD) rats were purchased from Shanghai Jiesijie Experimental Animals Co, Ltd. The animal experimental procedures were approved by the Animal Ethics Committee of East China University of Science and Technology. A 10 mm-sciatic nerve injury model was established to study the effect of PLATMC/LF ND-PCL and ND-PLATMC nerve guidance conduits on nerve regeneration. The study included several experimental groups: A blank control group (Blank), a single-channel ND-PLATMC conduit group, a multi-channel PLATMC/LF ND-PCL conduit group, and an autograft control group. Twenty-four male SD rats weighing 200–250 mg were randomly divided into 4 groups with 6 rats in each group. Each group had two time points for sacrifice: 2 weeks and 4 weeks, with 3 rats sacrificed at each time point.

In vivo implantation. Before implantation, the rats were adaptively fed with a standard diet for 2 weeks, and the implant materials were sterilized with 75 % ethanol for 24 h. The rats were anesthetized with pentobarbital sodium (35 mg/kg) by intraperitoneal injection and fixed on the operating table. The surgical site was shaved and disinfected. A small incision was made in the left leg of each rat to expose the sciatic nerve by dissecting the surrounding muscle tissue. A 10 mm segment of the sciatic nerve was resected from each rat. Different nerve conduits/supports were assembled in situ to bridge the nerve defect. The muscle layer and skin were sutured. All experimental rats were kept under standard conditions and observed at 2- and 4-weeks post-surgery. At the predetermined time points, following the assessment the rats' motor function recovery, euthanasia was performed. The gastrocnemius muscle of the injured/healthy limb and the intermediate segment of the regenerated nerve were removed and fixed in 4 % paraformaldehyde.

Evaluation of nerve regeneration and recovery of motor ability in rats. At 2 and 4 weeks postoperatively, rat footprints were captured using the Cat Walk gait analysis system. Parameters like paw pressure, experimental toe spread (ETS), normal toe spread (NTS), experimental paw length (EPL), normal paw length (NPL), experimental intermediate toe spread (EIT), and normal intermediate toe spread (NIT) were measured. The sciatic function index (SFI) was calculated using the formula:SFI = 109.5(ETS - NTS)/NTS - 38.3(EPL - NPL)/NPL + 13.3(EIT - NIT)/NIT - 8.8.

After the assessment of motor function recovery, rats were euthanized, and the sciatic nerve defect was dissected from the nerve conduit. Histological staining analysis was performed on regenerating nerves and gastrocnemius muscles. Gastrocnemius muscles were subjected to Masson's trichrome staining for cross-sectional area analysis using Image-J software. Regenerating nerves were subjected to Hematoxylin and Eosin (HE), Toluidine Blue (TB), and Luxol Fast Blue (LFB) staining to observe tissue morphology.

For immunofluorescence analysis of regenerating nerves, nerve conduits were fixed, and cross-sections were stained with NF200 and S-100β antibodies. Sections were blocked with BSA, incubated with primary antibodies overnight, stained with secondary antibodies, and counterstained with DAPI. Stained samples were observed using a laser scanning confocal microscope. The staining procedures for HE, Masson's trichrome, Toluidine Blue, and LFB are not reiterated here.

Statistical analysis. The experimental data were expressed as mean ± standard deviation. The analysis involved the utilization of one-way analysis of variance (ANOVA) and two-way analysis of variance (two-way ANOVA), followed by statistical analysis using the student-Newman-Keuls post hoc test. In this context, significance levels were denoted as follows: *P＜0.1; **P＜0.01; ***P＜0.001. Data with p < 0.05 were considered statistically significant.

Ethics approval and consent to participate

All experimental procedures adhered to the relevant ethical guide lines, and the use of animals in this study was approved by animal Ethics Committee, East China University of Science and Technology. The ethical approval Number was ECUST-21046.

CRediT authorship contribution statement

Xinqing Wang: Writing – original draft, Validation, Methodology, Investigation, Formal analysis, Data curation. Shuo Chen: Writing – review & editing. Xiaolei Chen: Writing – review & editing. Juan Wu: Writing – review & editing. Zhenhua Huang: Writing – review & editing. Jing Wang: Writing – review & editing. Fangping Chen: Writing – review & editing, Visualization, Supervision, Resources, Project administration, Funding acquisition, Data curation, Conceptualization. Changsheng Liu: Visualization, Supervision, Project administration, Funding acquisition, Conceptualization.

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 are the Supplementary data to this article:Multimedia component 1

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Acknowledgement

This investigation was supported by the 10.13039/100014717 National Natural Science Foundation of China (No. 32171342 ; 51772100 ), 10.13039/501100012166 National Key Research and Development Program of China (2023YFC2413600 ). The authors thank ResearchCenter of Analysis and Test of East China University of Science and Technologyfor the help on the characterization.

Peer review under responsibility of KeAi Communications Co., Ltd.

Appendix A Supplementary data to this article can be found online at https://doi.org/10.1016/j.bioactmat.2024.07.018.
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