Ultrasound‐Responsive Composited Conductive Silk Conduits for Peripheral Nerve Regeneration

Peripheral nerve injuries are challenging to repair clinically due to their limited regenerative capacity. Herein, a novel poly(3,4‐ethylenedioxythiophene):poly(4‐styrene sulfonate) (PEDOT:PSS) composited conductive silk conduit with ultrasound (US)‐triggered active release for peripheral nerve regeneration is presented. The conduit is composed of silk fibroin inverse opal tubular scaffolds and the secondary hydrogel filler that contains thermosensitive material (poly(N‐isopropylacrylamide), PNIPAM), PEDOT:PSS, and nerve growth factor (NGF). Benefiting from the integration of these functional materials and active factors, the silk fibroin conduits show excellent biocompatibility, flexibility, conductivity, and bioactivity. In particular, owing to the thermal effect of US and the temperature responsiveness of PNIPAM, the secondary filling hydrogel undergoes volume shrinkage upon US triggering, allowing the responsive NGF release. Besides, the electrical conductivity of PEDOT:PSS has been affirmed to promote neuron and axon outgrowth. Through in vivo experiments, the synergistic effect of PEDOT:PSS and US‐triggered delivery of NGF on accelerating injured nerve repair are demonstrated. These results reveal the practical value of the proposed intelligent conductive silk conduits with US responsiveness for nerve regeneration in preclinical studies.

integrated into the conduits for improving nerve growth and remyelination. [6]Although with some progress, the absence of exquisite conduit microstructures usually causes the simple fusion of different polymers, resulting in unsatisfactory efficiency of their functional components.In addition, the added bioactive substances generally release simultaneously with the degradation of scaffold biomaterials, which shows the uncontrollable delivery.Therefore, new multifunctional nerve conduits with specific microstructures and controllable drug delivery capability for peripheral nerve regeneration are still anticipated.
In this article, we presented a novel poly(3,4-ethylenedioxythiophene)-polystyrene sulfonate (PEDOT:PSS) composited conductive silk conduit with ultrasound (US)-triggered nerve growth factors (NGFs) release for promoting peripheral nerve regeneration, as schemed in Figure 1.4d,6d,7] Besides, it can maintain satisfying chemical stability and electrical conductivity under physiological conditions.In contrast, US waves can be absorbed and scattered when they travel through the body and as a result, the tissue temperature increases, which is inversely proportional to US attenuation. [8]Benefitting from such a unique property, US strategy has emerged as a powerful tool for in situ drug delivery and noninvasive therapy. [9]Thus, we envisage that if PEDOT:PSS and US are integrated into the silk fibroin conduit system, it will construct an unprecedented conductive nerve conduit with controllable drug delivery ability for nerve regeneration.
Herein, we developed the desired US-responsive PEDOT:PSS composited silk conduits with inverse opal structure for controllable NGF delivery and enhanced nerve repair.As the inverse opal is a type of material with a high specific surface area and interconnected nanochannels, it allows the integration of multiple materials and sustained release of loaded drugs. [10]n this basis, silk fibroin-derived tubular inverse opal hydrogels were generated by the template replication method, while the secondary composite hydrogel of NGF, PEDOT:PSS, and poly(N-isopropylacrylamide) (PNIPAM) was introduced into the nanopores of the inverse opal scaffold.We have demonstrated that the resultant conduit exhibited prominent biological and mechanical properties.In addition, PEDOT:PSS endowed the conduits with well electrical conductivity, which contributed to nerve growth.Moreover, because of the temperature sensitivity of PNIPAM, the secondary filling hydrogel could be shrunk under the slightly increased temperature generated by US, resulting in the controllable release of the encapsulated NGF.Based on these features, through in vitro PC12 cell cultivation and in vivo animal experiments, we have revealed the positive effect of the composited conduits on peripheral nervous system regeneration.These results indicated that the presented conductive silk conduit is a promising candidate for peripheral nervous system regeneration.

Results and Discussion
In a typical experiment, a cleaned glass capillary with one blocked end was repeatedly infiltrated into the silica nanoparticles (SiO 2 NPs) solution (dispersed in ethanol).After evaporation of ethanol, the tubular SiO 2 NPs template was formed outside the capillary, as illustrated in Figure 2a.During the volatilization process of ethanol, SiO 2 NPs self-assembled into an ordered structure with a compact arrangement (Figure 2b).After that, we prepared a simple concentric glass device containing the obtained NP-assembled capillary and another capillary with larger inner diameters, as well as a slide for the fix of capillaries.Considering the slow degradation and gelation of silk fibroin, photo-crosslinkable gelatin (GelMA) hydrogel was incorporated for the optimization of hydrogel crosslinking and a suitable degradation rate to match the nerve regeneration.After the complete filling of pregel into the colloidal crystal template, the silk fibroin and GelMA hybrid hydrogel (SG hydrogel) were obtained through UV irradiation, during which UV irradiation triggered the crosslinking of the GelMA hydrogel network, as demonstrated in Figure 2c.Under the corrosion of hydrofluoric acid, the SiO 2 NPs template and glass device were removed, finally forming a tubular SG inverse opal scaffold with interconnected nanochannels (Figure 2d).Of note, to improve the mechanical performance of conduits, methanol was further utilized to induce the formation of β-sheets in silk fibroin hydrogel, resulting in the interpenetrating networks.
To endow the nerve guidance conduit with ideal electrical conductivity and controllably drug-delivery ability, conductive polymer PEDOT:PSS and thermosensitive polymer (PNIPAM) were introduced.Before integration, we explored the conductivity of composite hydrogel consisting of PNIPAM and PEDOT:PSS (0-100 vol%).It was found that the composite hydrogels showed enhanced electroconductivity with PEDOT:PSS content increasing, as demonstrated by current-voltage curves (Figure S1a,b, Supporting Information).However, the gelation time of hybrid hydrogels increased with the PEDOT:PSS content, which could be ascribed to the hindering of PEDOT:PSS on the UV light transmission (Figure S1c, Supporting Information).To avoid the negative effect of long-term UV irradiation on the active agents encapsulated in the hydrogel, 60 vol% of PEDOT:PSS was selected as the final concentration for the subsequent experiments.Additionally, the swelling performance of PNIPAM and PEDOT:PSS hybrid hydrogel was examined.It was found that introducing PEDOT:PSS slightly increased the swelling ratio of hybrid hydrogel, which was also due to its relatively poor crosslinking degree (Figure S1d, Supporting Information).
Then, the PNIPAM and PEDOT:PSS were integrated with the prepared SG inverse opal scaffold by filling the nanopores of the inverse opal scaffold (Figure 2e).As one of the most common thermosensitive polymers, PNIPAM exhibits remarkable phase transition performance when at an ambient temperature above 32 °C, which is known as the relatively low critical solution temperature (LCST).There are numerous studies on the integration of PNIPAM hydrogel to realize thermoresponsive and controllable drug delivery.However, restricted by the body temperature (37 °C) and hyperthermia temperature (42 °C), it is necessary to adjust the LCST of PNIPAM hydrogel in such a temperature range to satisfy the needs of in vivo controllable drug delivery application.Based on the previous research, acrylamide (AM) was incorporated to improve the LCST.It was observed that the LCST of AM-doped hybrid hydrogel increased to around 39 °C when the contents of AM and PNIPAM were 0.75 and 9.25 wt% (Figure S2, Supporting Information).Of note, even with the doping of PEDOT:PSS, LCST at 39 °C was kept.To explore the cytocompatibility of composite hydrogels, pheochromocytomaderived (PC12) cell line was cocultured with different substances: the glass slide (control group), the PNIPAM, and SG composite hydrogel (SN hydrogel), as well as the PNIPAM, PEDOT:PSS, and SG composite hydrogel (SNP hydrogel), and the cell viability was detected through CCK-8 assay and live/dead staining.It was found from the fluorescent images and the corresponding quantitative analysis that the majority of cells maintained good viability in all three groups (Figure S3a-d, Supporting Information), indicating the hydrogel had good biocompatibility.Furthermore, the cell proliferation experiments for 5 days also demonstrated the excellent cellular compatibility of the hydrogels that showed no inhibition on the proliferation of PC12 cells (Figure S3e, Supporting Information).
Before using SNP hydrogel as a nerve conduit, its mechanical properties should be considered.In this work, we evaluated the mechanical performance of tubular SNP hydrogel by conducting the bending, twisting, and stretching tests together with the cyclic compressing experiment.As shown in Figure 3a-c, the obtained tubular SNP hydrogel remained intact without any destruction after undergoing extreme bending, twisting, and even longitudinal stretching, showing satisfactory flexibility and durability.Besides, the resulting conduit could withstand repeated compression, which was also reflected by the compressive stress-strain and the cyclic compression curves (Figure 3d, e).After 100 compression cycles, the hydrogel conduit still showed a consistent compression force, confirming its outstanding structural integrity and mechanical durability.These results proved that our designed tubular hydrogels had excellent flexibility and durability, and could withstand various mechanical deformations during biological activities, which mainly benefited from the superior mechanical performance of silk fibroin.
Applying the US to biological tissues is able to lead to the thermal effect, which is affected by various parameters, including frequency and intensity, which is defined as the amount of the US wave-carried power divided by its applied surface (W/cm 2 ).Of note, it has been revealed that US penetration depth is inversely proportional to frequency, and US with a frequency less than 1 MHz can penetrate most tissues in the body (especially in animal models, around 10 cm).According to the rat sciatic nerve depth, we selected the frequency of 1 MHz in the following experiment.Additionally, the US with low intensity varying from 0.125 to 3 W cm À2 has been considered with reversible and acceptable changes to the tissues, whereas the US with high intensity (over 3 W cm À2 ) would result in irreversible biological damages.In our study, a piece of unpeeled pork was used as the mimic animal tissue to investigate the temperature changes caused by the US penetration.We first evaluated the thermal effects of ultrasonic waves penetrating tissues with different thicknesses.It was found that as the thickness of pork tissues decreased, a significant temperature increase occurred, and when the tissue thickness was 1 cm, the temperature increase could reach about 9 °C after 2 min (Figure 4a).In addition, it was observed that the tissue temperature increased with the US intensity, and the temperature showed the most rapid and remarkable variation when the intensity increased to 2.0 W cm À2 (Figure 4b).In consideration of actual body temperature and hyperthermia temperature, as well as the LCST of PNIPAM, we chose 1.5 W cm À2 as the final experimental intensity, at which tissue temperature increased by about 4 °C after 2 min of US treatment.Significantly, the temperature increase at the probe contact surface was found to be within an acceptable range (up to only 4.7 °C), so it would cause little harm to the rat skin (Figure 4c).Furthermore, we evaluated the effect of US treatment time on cell cultivation.The CCK-8 assay result revealed that the US had little impact on cell viability when the treatment time was limited to less than 2 min (Figure 4d).These results indicated that US could be applied to the body without significant negative effects.To verify the US-triggered temperature increment on tubular SNP hydrogel, the thermal images were obtained.It was found that the temperature of the hydrogel under US treatment could rise slightly higher than the phase transition temperature of PNIPAM within 2 min (Figure 4e).As a result, the volume shrinkage of the SNP hydrogel was successfully induced.Besides, the drug loading and release abilities of the SNP conduit were demonstrated by utilizing the fluorescein isothiocyanatelabeled bovine serum albumin (BSA-FITC) as model drug.As the fluorescence images shown in Figure 4f, it was obvious that the BSA-FITC together with thermosensitive hydrogel was confined in the nanopores of the inverse opal layer inside the SNP conduit, which contributed to the drug release in the local space.In order to evaluate the behavior of controlled drug release triggered by US, we treated the hydrogel every 1 h for about 2 min, and assayed the drug release behavior under direct thermal stimulation through the standard curve method (Figure S4, Supporting Information).As shown in Figure 4g and h, when there was no triggering, the molecules could only be passively released from the carrier under the diffusive force, and a large amount of the drug was retained.In contrast, treating the hybrid SNP hydrogel under thermal stimulation, which triggered the contraction of the polymer chains, results in the accelerated release of the encapsulated drugs.It was found that the hydrogel had similar release rates under thermal stimulation by direct heating or US irradiation.It was worth mentioning that the release rate of the US-induced group was slightly higher than that of the direct heating group, which might be caused by the probe vibration.Moreover, we examined the long-term responsibility of as-prepared SNP conduit treated with US every other day for 2 min and repeated for 2 weeks, as recorded in Figure 4i.As a result, the US-triggered conduit displayed a controllable and higher release than the control group after 2 weeks.These results illustrated the satisfactory US effects for responsive and sustained delivery of loaded drugs.
As known, PC12 cells (pheochromocytomaderived cell line) possess a unique neuronal morphogenetic response to NGF, thus being used here to validate the US-triggered controllable release ability of NGF.First, the prepared aseptic NGF-coated SNP conduits were solely placed in a 24-well plate and divided into two groups, followed by the addition of the culture medium.One group was treated with US irradiation, and the other group was not treated.After that, the medium containing releasate was collected for culturing PC12 cells, and a blank control group treated with the fresh medium was set.After 48 h, compared with the cells in the control group, the cells in the US-treated group were mostly differentiated into neurons, while the cells in the US-untreated group were slightly differentiated.Furthermore, based on statistical analysis, both the proportion of differentiated cells and the length of dendrites of the US-treated group were superior to those of the other two groups, as shown in Figure S5, Supporting Information.These results demonstrated that NGF could be released from the SNP hydrogel to stimulate PC12 cell differentiation under US irradiation, whereas the drug was rarely released without the US.
The effect of PEDOT:PSS on neurite outgrowth was also investigated from PC12 cells.PC12 cells were cultured on different substances for 7 d, namely glass slide (control group), SN hydrogel, SNP hydrogel, and NGF-loaded hydrogel (SNPN) films, respectively.As shown in Figure 5, when free NGF was added to the medium (in SN and SNP groups), both groups of PC12 cells extended distinct neurites compared to the control group, indicating that PC12 cells could be stimulated to project obvious neurites only in the presence of NGF.Besides, the lengths of neurites in the SNP group and SNPN group were longer than that in the SN group, which suggested that US could trigger the NGF release from hydrogel and PEDOT:PSS could further promote NGF-induced neurite outgrowth in PC12 cells.It was also demonstrated that the NGF encapsulated in the hydrogel was extruded to stimulate the neurite growth of PC12 cells under US stimulation, and there was no significant difference in neurite length compared with the free NGF administration group.In addition, in the SNP group and SNPN group, the expressions of postsynaptic density protein 95 (PSD95) and Synapsin I were both higher than those of the control group and SN group, as shown in Figure 5d,e.These results revealed that NGF could be released from thermosensitive hydrogels with good bioactivity under US stimulation, and neurite extension and axon-related gene expression in PC12 cells could be greatly enhanced by the synergistic promotion of released NGF and conductive polymer PEDOT:PSS.
To evaluate the feasibility of the fabricated multifunctional hydrogel conduits for the regeneration of long-segment sciatic nerve defects, we set rat models with different treatments and carried out an 8 week in vivo experiment (Figure S6, Supporting Information).At week 8 after implantation, footprint records showed the wider toe spreads in the Autograft and SNPN@US groups over that in the SNPN group, followed by the SN group (Figure 6a).Generally, the sciatic nerve index is one of the critical factors in evaluating motor function restoration, and an index of 0 indicates good motor function, while an index of 100 represents a thorough function loss.Based on the gait analysis, the sciatic nerve index was obtained, showing that the motor function of the SNPN group recovered well and was similar to the Autograft group after 8 weeks of implantation (Figure 6c).This result indicated that SNPN promoted motor recovery better than the simple SN and SNP groups, and the recovery effect was improved under US treatment, which was beneficial from the release of NGF.Gastrocnemius muscle morphological analysis and wet weight ratio were also consistent with these results (Figure 6b,d and Figure S7, Supporting Information).Afterward, the expressions of the Schwann cell-specific marker (S-100) and Neurofilament marker (NF200) in the SNPN group were close to those of the Autograft group after 8 weeks, which were higher than those of SN and SNP groups (Figure 6e-j).The above results demonstrated the successful regeneration of axons.These features proved the promotion of the effective release of NGF triggered by the US and the excellent electrical conductivity of PEDOT:PSS to sciatic nerve defect repair.

Conclusion
In conclusion, we propose a US-triggered drug delivery and conductive silk fibroin conduit for peripheral nerve regeneration.Benefiting from the porous inverse opal structures, the integration of silk scaffold with thermosensitive hydrogel, PEDOT:PSS and NGF were achieved.The fabricated conduit exhibited excellent biocompatibility and mechanical properties, such as flexibility in stretching, bending, and compression, as well as satisfactory electrical conductivity.In particular, NGF encapsulated in the thermosensitive hydrogel could be controllably released under the triggering of the US and diffused when the US was stopped.It was shown that the released NGF maintained good biological activity and synergized with PEDOT:PSS to promote neurite outgrowth.In vivo rat sciatic nerve injury model confirmed that the application of such a multifunctional conduit greatly promoted nerve regeneration, remyelination, and muscle function recovery with similar effects to autografts.These results demonstrated the potential of our multifunctional silk-based conduit to be an effective treatment for promoting peripheral nerve regeneration.
Fabrication of PEDOT:PSS Composited Conductive Silk Conduit: First, the synthetic silica nanoparticles (SiO 2 NPs) were dispersed in ethanol and the final concentration of 15 wt% was determined.After that, a cleaned hollow glass capillary (D inner = 1 mm, D outer = 1.5 mm, wherein D refers to diameter) with one blocked end was repeatedly infiltrated into the SiO 2 NPs ethanol solution and exposed to air for ethanol volatilization and self-assembly of SiO 2 NPs after each infiltration.Then, the glass capillary was put in a muffle furnace (400 °C) to further enhance the mechanical strength of SiO 2 NPs assembling system.Next, another glass capillary (D inner = 2.4 mm, D outer = 3 mm) was placed outside the above-prepared SiO 2 NPs assembling system, and the mixture of silk fibroin (4 wt%), GelMA (6 wt%) and HMPP (1 vol%) was injected into the space between two capillaries.After complete filling and UV irradiation (30 s), we utilized hydrofluoric acid to etch SiO 2 NPs and glass capillaries, successfully generating the hollow conduit with inner porous structures.Of note, methanol was utilized to induce silk fibroin gelation to further improve the mechanical performance of conduit.Finally, the mix solution consisting of NIPAM (9.25 wt%), AM (0.75 wt%), Bis (0.34 wt%), HMPP (1 vol%), PEDOT:PSS (60 vol%), and drugs was infused into the cavity of hollow conduit to obtain the drug-loaded composited conductive silk conduit after UV-triggered crosslinking.
US-Induced Temperature Change Test: In this study, we used US therapy equipment for US wave generation, while a US guiding gel was applied between the transducer and samples to avoid acoustic attenuation.Besides, the temperature variation was recorded by a thermal imager (FLIR, E5xt).Of note, for the US-triggered drug release and cell viability test, the samples (such as prepared composited conduits and cells) were placed into the 24-well plates, respectively.At each specific time point, US transducer was attached to the plates and aimed for one of the wells.During this process, the US guiding gel was also applied between transducer and plates.
Measurement of Drug Release: For a typical experiment, the FITC-BSA was selected as the model drug, which could represent proteinic drugs.First, the PBS solutions with different FITC-BSA concentrations (0, 1, 2,  3, 4, 5, 6, 7, 8, 9, 10, and 11 μg/mL) were prepared and 200 μL solutions of each concentration were collected into a 96-well plate to obtain the standard curve of FITC-BSA.Then, the FITC-BSA solution was mixed with NIPAM and PEDOT:PSS composite, and the ultimate concentration of FITC-BSA was 1 mg mL À1 .Next, the obtained mixture was injected into the cavity of the prepared GS conduit to fill the nanopores of inverse opal inner walls.After UV irradiation, the FITC-BSA loaded-composited conduit was generated and put into a 24-well plate containing PBS buffer solution (1 mL) to assess drug release behavior.Next, we collected 200 μL solution from each group into a 96-well plate at each specific time point, followed by adding 200 μL new PBS solution into a 24-well plate.Finally, the fluorescence intensity at 493 nm of collected FITC-BSA was detected through a microplate reader.The cumulative release amount of drugs was obtained based on the standard curve.
Cell Experiments: PC12 cells were cultured with RPMI 1640 medium containing penicillin-streptomycin double antibiotic (1 vol%) and fetal bovine serum (10 vol%).For the biocompatibility test, the PC12 cells cultured in a 48-well plate were divided into three groups and cocultured with diverse substances, namely, the control group (glass slide), SN group (silk fibroin and GelMA composite hydrogel film), and SNP group (SN inverse opal hydrogel film filled with PNIPAM and PEDOT:PSS).
After 5 d, the PC12 cells were incubated with live/dead staining kit for 30 min and observed through a fluorescence microscope.Besides, the CCK-8 agent was used for daily detection of cell viability.At each time point, 10 vol% CCK-8 agent was added into the culture medium, and then the well plate was placed in the cell incubator.After 3 h, 200 μL culture medium of each well was removed into another 96-well plate, and a microplate reader was utilized to detect their absorbance.To evaluate the activity of NGF released from conduits, the aseptic NGF-coated SNP conduits were solely placed in a 24-well plate containing culture medium and divided into two groups: one group was treated with US, and the other group was not treated.Then, the medium containing releasate was collected to treat PC12 cells, while a blank control group was treated with the fresh medium.To evaluate the influence of NGF-loaded composited conductive silk conduit on the PC12 cell differentiation, PC12 cells were cultured on different substances, including glass slides (control group), SN hydrogel, SNP hydrogel, and NGF-loaded hydrogel (SNPN) films, respectively, and free NGF was added to the medium of SN and SNP groups.
qRT-PCR: In order to quantitatively determine the differentiation status of PC12 cells cultured on different substrates, we used qRT-PCR to analyze the expression levels of axon-related genes, and glyceraldehyde-3phosphate dehydrogenase (GAPDH) was selected as the reference gene.On day 7, total cell RNA was extracted with the SteadyPure universal RNA Extraction kit, and then RNA was reversely transcribed into cDNA.The expression of axon-related genes including postsynaptic density protein 95 (PSD95) and Synapsin I in PC12 cells was assessed by ViiA 7 system.All primers used for quantitative analysis by qRT-PCR were provided in Table S1, Supporting Information.
Animal Experiments: Adult male Sprague-Dawley (SD) rats (200-250 g) were randomized into four groups, namely, the Autograft group (represented as Autograft), the group treated by silk composited conduit without PEDOT: PSS and NGF (represented as SN), the group treated by silk composited conduit with PEDOT:PSS and NGF (represented as SNPN), as well as SNPN-treated and US-triggered group (represented as SNPN@US).After anesthetizing rats, we exposed their left sciatic nerve and constructed the 10 mm-long segment defects.Then, the natural nerve, SN conduit, and SNPN composited conduits were sutured to the rat nerve ends in different groups, respectively.Finally, the muscle and skin of rats were sutured.All rats were observed for 8 weeks.Especially, the rats in the SNPN@US group were treated with US for 2 min every day to induce the NGF release from the conduit.To evaluate the functional recovery and nerve regeneration of rats, footprint analysis, as well as morphometric analysis of regenerated nerves and gastrocnemius muscle, was conducted.Animal experiments were conducted based on the "Guidelines for the Care and Use of Laboratory Animals in China."The Animal Experimental Ethical Inspection Committee of Southeast University has approved all animal schemes (No. 20,210,401,009).
Immunofluorescence Staining of PC12 Cells and Tissues: The samples (PC12 cells and nerve tissues) were fixed with preprepared 4 wt% PFA solution for 1 h at 4 °C.Then, the samples were washed with PBS comprising 0.1 vol% Triton X-100 solution (called PBST) 3 times and 5 min each time, aiming for the permeability of the cell membrane.Next, the samples were blocked for 1 h and incubated with primary antibody at 4 °C overnight.After discarding the primary antibody and washing samples, the secondary antibody and DAPI were simultaneously added, incubated with samples for 60 min and washed 3 times.Finally, the samples were transferred onto a clean glass slide and covered with an antifluorescence quenching agent, which were stored at 4 °C.
Characterization: The nano-and microstructures of prepared conduits were characterized by SEM.It was noted that EGDMA and PEGDA were selected to replace the first-filling hydrogel and secondary filling hydrogel, respectively, which aimed to avoid inevitable hydrogel collapse after water loss.The electrical tests were performed on a semiconductor characterization system (KEITHLEY, USA).

Figure 1 .
Figure 1.Schematic diagram of PEDOT:PSS composited conductive silk conduit with US-triggered NGF delivery ability for peripheral nerve regeneration.

Figure 2 .
Figure 2. Characterization of the composite conduit.a) Illustration diagram of the fabrication process of SG inverse opal conduit.SNP hydrogel refers to PNIPAM and PEDOT:PSS-filled SG composite hydrogel.(b-e) Scanning electron microscope (SEM) pictures of the tubular SiO 2 NPs template b), hybrid hydrogel conduit c), inverse opal conduit d), and the secondary hydrogel-filling inverse opal conduit e), respectively.Scale bars are 1, 2, and 10 μm in i-iii, respectively.

Figure 3 .
Figure 3. Evaluation of the mechanical performance and conductivity of the fabricated SNP hydrogel conduit.a-c) Bending test a), twisting test b), and stretching test of the SNP hydrogel conduit c), respectively.d, e) Cyclic fatigue test of the SNP hydrogel conduit.

Figure 4 .
Figure 4. Thermal effects of US and drug loading and release performance of fabricated conduit.a) Temperature changes of pork with different thicknesses triggered by US. b) Temperature changes of pork with 1 cm thickness triggered by US. c) Temperature changes of pork surface triggered by US. d) Cell ability affected by US treatment within 4 min.e) Thermal images of SNP hydrogel conduit treated by US (1 MHz, 1.5 W cm À2 ).f ) Fluorescent images of drug-loaded SNP hydrogel conduit.g-i) Cumulative drug release of SNP hydrogel with different treatments.

Figure 5 .
Figure 5. Neurite outgrowth of PC12 cells.a) Immunofluorescence images of PC12 cells seeded on diverse substances for 7 d.The cytoplasm and nucleus were red and blue, respectively.b,c) Statistics of differentiated cells b) and neurite length c).d, e) Quantitative real-time polymerase chain reaction (qRT-PCR) analysis of PSD95 and Synapsin I mRNA level in PC12 cells.

Figure 6 .
Figure 6.In vivo evaluation.a) Gait analysis of model rats after 8 weeks.b) Gastrocnemius muscle of model rats separated after 8 weeks.c) Sciatic nerve index of model rats after 8 weeks.d) Statistics of muscle wet weight ratios of model rats.e-h) Immunofluorescent staining images of rats with different treatments after 8 weeks.i,j) Statistics of immunofluorescent results.