The Porous SilMA Hydrogel Scaffolds Carrying Dual-Sensitive Paclitaxel Nanoparticles Promote Neuronal Differentiation for Spinal Cord Injury Repair

Abstract

BACKGROUND:

In the intricate pathological milieu post-spinal cord injury (SCI), neural stem cells (NSCs) frequently differentiate into astrocytes rather than neurons, significantly limiting nerve repair. Hence, the utilization of biocompatible hydrogel scaffolds in conjunction with exogenous factors to foster the differentiation of NSCs into neurons has the potential for SCI repair.

METHODS:

In this study, we engineered a 3D-printed porous SilMA hydrogel scaffold (SM) supplemented with pH-/temperature-responsive paclitaxel nanoparticles (PTX-NPs). We analyzed the biocompatibility of a specific concentration of PTX-NPs and its effect on NSC differentiation. We also established an SCI model to explore the ability of composite scaffolds for in vivo nerve repair.

RESULTS:

The physical adsorption of an optimal PTX-NPs dosage can simultaneously achieve pH/temperature-responsive release and commendable biocompatibility, primarily reflected in cell viability, morphology, and proliferation. An appropriate PTX-NPs concentration can steer NSC differentiation towards neurons over astrocytes, a phenomenon that is also efficacious in simulated injury settings. Immunoblotting analysis confirmed that PTX-NPs-induced NSC differentiation occurred via the MAPK/ERK signaling cascade. The repair of hemisected SCI in rats demonstrated that the composite scaffold augmented neuronal regeneration at the injury site, curtailed astrocyte and fibrotic scar production, and enhanced motor function recovery in rat hind limbs.

CONCLUSION:

The scaffold’s porous architecture serves as a cellular and drug carrier, providing a favorable microenvironment for nerve regeneration. These findings corroborate that this strategy amplifies neuronal expression within the injury milieu, significantly aiding in SCI repair.

Introduction

According to statistics, several hundred new cases of traumatic spinal occur per million individuals annually. This destructive disease causes sensory and motor impairments below the injured segment of the patient’s body [1–4]. Despite advancements in various comprehensive disciplines, clinical treatment methods for spinal cord injuries have yet to achieve satisfactory axon regeneration effects or effective motor recovery [5]. After the injury occurs, neuronal apoptosis, inflammatory cell infiltration, and complex secondary damage to the central nervous system (CNS) are significant obstacles to spinal cord injury (SCI) repair [6–10]. Injury factors increase the differentiation of endogenous neural stem cells (NSCs) and initiate neural repair. Neural stem cells can differentiate into neurons, astrocytes, and oligodendrocytes, thus providing a strategy for neural repair through multidirectional differentiation. However, the post-injury microenvironment supports the differentiation of astrocytes, hindering the regeneration of neurons, which is detrimental to neural repair [11, 12]. Therefore, transplanting exogenous NSCs and regulating their differentiation towards neurons to supplement the lost cells at the injury site and rebuild the connection between electrical signals is an effective strategy for repairing spinal cord injuries.

The transplantation of NSCs presents numerous challenges. The intricate microenvironment after injury significantly affects the physiological efficacy of these cells. Beyond the detrimental effects of the initial injury, a cascade of secondary injuries triggered by spinal cord trauma, such as the production of reactive oxygen species (ROS), inflammation, demyelination of axons, and cytotoxicity owing to ionic imbalances that lead to neuronal cell necrosis (as well as the development of glial scars and cysts) pose formidable barriers to axon regeneration and neural repair [13, 14]. Consequently, fostering a conducive microenvironment and providing the necessary support are critical for neural regeneration. Despite the inherent potential of NSCs to differentiate into various lineages, the efficiency of neuronal differentiation is compromised by the SCI microenvironment. Therefore, the use of exogenous inducers to facilitate neuronal differentiation is imperative. In this regard, Paclitaxel (PTX) emerges as a promising pro-differentiation agent capable of augmenting the yield of NSC-derived neurons [11, 15, 16]. Additionally, PTX stabilizes microtubules, safeguards the axons of compromised neurons, and aids in the restoration of spinal cord function. It can reverse the inhibitory milieu characteristics of SCI, thereby bolstering the neuronal differentiation of transplanted NSCs at the injury site [15]. However, the poor solubility of PTX diminishes its bioavailability and therapeutic efficacy. The intricate pathological microenvironment following SCI further impedes the effectiveness of pharmaceuticals. The initial pathological characteristics of ischemia and hypoxia induce an increased anaerobic metabolism at the injury site, creating an acidic microenvironment. This intense inflammatory response is primarily observed as redness, swelling, heat, and pain at the injury site. The accumulation of inflammatory cells exacerbates the formation of a local high-temperature, low-pH environment, further inhibiting cell survival and drug efficacy. In response to these complexities, we have engineered a pH- and temperature-sensitive micellar nanoparticle (NP) system to encapsulate PTX (PTX-NPs) and enhance its bioavailability. More significantly, this innovative delivery system enables responsive drug release tailored to a specific microenvironment. This is particularly important for the biological effects of chemical factors to efficiently promote the repair of SCI, as the release of drugs can be facilitated under high temperature and low pH conditions [12]. The liberated PTX further catalyzes the neuronal differentiation of implanted NSCs, compensates for the disrupted biological electrical signaling at the injury site, and ultimately facilitates motor function recovery in rats with SCI. Recovery of motor function is the ultimate goal in treating SCI. Effective functional recovery reduces risks associated with subsequent SCI, such as infection, pain, and blood clots. It enhances the patient’s quality of life and lessens societal burden. The extent of motor and sensory function recovery directly impacts the quality of life. Consequently, post-SCI behavioral assessment is a matter of concern.

The optimization of cell and molecular delivery in SCIs remains a significant challenge. Currently, the introduction of biomaterials such as hydrogels at the injury site is emerging as a promising approach. The primary advantage of hydrogel materials lies in their capacity to serve as reservoirs for sustained and localized therapeutic release of encapsulated drugs. Their biocompatibility allows them to act as an extracellular matrix that serves as a biological barrier to mitigate the effects of adverse factors. Moreover, they can function as carriers of cells and drugs, influencing cellular physiological functions, enhancing cell transplant survival rates, and controlling differentiation [17–19]. In recent years, functionalized biomaterial scaffolds have played a crucial role in bioengineering since they can integrate physical guidance signals to direct axon regeneration [20, 21] and manage immune cell polarization. Traditional methods to construct bioengineered scaffolds are limited in precision and bionics. Considering the spinal cord’s intricate anatomical structure, scaffolds must possess appropriate internal structures to mimic the anatomical structure of the spinal cord for practical axon guidance during SCI repair. As a cutting-edge solid freeform fabrication technology in tissue engineering, the latest advancements in 3D printing offer a novel and feasible method to enhance scaffold performance [22, 23]. Biomanufacturing technologies are employed to create complex three-dimensional (3D) microstructures essential for guiding cell growth and promoting tissue maturation. By printing porous hydrogel scaffolds that emulate nerve regeneration conduits, damaged host axons can easily traverse the host–scaffold interface. Under the guidance of the scaffold walls, the axons grew linearly along the rostral-caudal axis of the spinal cord. In addition to serving as a supportive platform, 3D bioscaffolds can be used as carriers for seeded cells to restore the microenvironment at the SCI site, ultimately addressing the deficiencies caused by injury [24].

Therefore, we designed a 3D bioprinted spinal cord-like neural scaffold that could be loaded with small-molecule drugs to provide a favorable microenvironment for the survival of transplanted NSCs and efficient long-term neuronal differentiation. In this study, we developed a 3D bioprinting technique to construct porous SilMA hydrogel neural scaffolds (Fig. 1) on which NSCs and PTX-NPs were loaded, investigated the neural differentiation of NSCs in bio-printed SilMA hydrogel scaffolds loaded with PTX-NPs, and elucidated the molecular mechanisms of PTX-NPs in neuronal differentiation. Neural scaffolds formed from the physiological structure of the spinal cord are expected to provide an optimal microenvironment for the growth of transplanted NSCs and neuronal differentiation, thereby promoting functional recovery in SCI rats.

Fig. 1.

Schematic diagram of functional bioprinted neural tissue constructs for in vivo SCI repair. Bioprinted 3D porous SilMA hydrogel constructs loaded with NSCs and PTX-NPs effectively induced neuronal differentiation in SCI repair

Materials and methodss

Preparing 3D-printed SilMA hydrogel scaffolds

Five grams of silk fibroin (Suzhou Yongqinquan Intelligent Equipment Co. Ltd., China) was added to 100 mL of a standard solution of phosphate-buffered saline (PBS) containing a 0.25% (w/v) photoinitiator to prepare a 5% (w/v) SilMA hydrogel. After thorough agitation, it was placed in a water bath at 60 °C until fully dissolved. The solution was then sterilized using a 0.22 μm sterile filter, resulting in a 5% SilMA hydrogel concentration. The prepared 5% SilMA hydrogel was used as bioprinting ink and maintained at 37 °C to remain as a liquid. A liquid-like bio-ink was introduced into a bio-3D printer, and a computer-generated digital pattern was employed for the 3D printing process. The scaffolds were fabricated by modulating the exposure time and light intensity utilizing a commercially available DLP 3D printer (EFL-BP8600, Suzhou Intelligent Manufacturing Research Institute, Suzhou, China) to prepare the samples [25]. Subsequently, the printed porous SilMA hydrogel scaffolds were preserved in a 4 °C refrigerator for subsequent experiments.

Preparing pH/temperature-sensitive NPs

Dual-responsive PTX-NPs were synthesized using a previously described procedure [12, 26]. Approximately 20 mg of PNIPAM75-PLA60 block polymer compound (PolymerSource, Canada) was weighed and dissolved in dimethyl sulfoxide (DMSO; Solarbio, Beijing, China). Additionally, 2 mg of PTX powder (Sunlipo Biotech Research Center for Nanomedicine, Shanghai, China) was precisely measured and uniformly mixed before dissolution in an adequate volume of DMSO. The two solutions were then thoroughly combined to form an oil phase emulsified with a ten-fold volume of ultrapure water for several hours. The resulting mixture was dialyzed overnight using a membrane with a molecular weight of 1000 Da, ultimately yielding a solution for a co-loaded sensitive drug delivery system for future use. A drug-free sensitive carrier was prepared using the same method.

Characterizing hydrogel scaffolds and dual-sensitive NPs

Hydrogel analysis by scanning electron microscopy

The fabricated 3D-printed SilMA hydrogel scaffold was subjected to a − 40 C freeze-drying process for several hours. Subsequently, the scaffold was sectioned with a scalpel to expose its cross-section. The cross-section was sputter-coated with gold for 30 s examination under a scanning electron microscope (SEM, Zeiss Gemini300, Germany).

Physical and chemical properties of dual-sensitive NPs

Ten mg of PTX, empty dual-sensitive NPs, or PTX-loaded dual-sensitive NPs (PTX-NPs) were analyzed. The samples were mixed with KBr powder. Fourier Transform infrared spectroscopy (FTIR) was used to record the infrared absorption peaks of the samples within a wavelength range of 400–4000 cm⁻¹ (Nicolet iS5, Thermo Fisher Scientific, USA).

The samples’ X-ray diffraction (XRD) patterns were determined by adding each of the three groups of samples to the grooves of the observation slides. The powder was evenly spread on the surface of the grooves with slides, and the XRD patterns were read using an X-ray diffractometer (Rigaku Ultimate IV, Japan Rigaku Corporation, Japan).

The morphology of the NPs was observed using transmission electron microscopy (TEM, JEOL2010, Japan Electronics Co., Ltd., Japan). After appropriately diluting the NPs and its PTX-NPs, 10 μL of the NP liquid was pipetted onto a carbon-film-coated copper grid, dried at 26 °C, thoroughly soaked with 2% phosphotungstic acid, and the appearance of various carriers was observed.

We dilute the NPs and PTX-NPs at room temperature 100 times with distilled water. An appropriate amount of the diluted liquid was added to a quartz dish, and a Malvern laser particle size analyzer (Zetasizer Nano ZS, Malvern Instruments, Malvern, UK) was used to detect the particle size distribution and zeta potential of the sample, respectively.

In vitro release profile

Two mL of PTX-NPs and its control solution (adjusted to 0.4 mg/mL) were placed in Millipore dialysis tubing (with a molecular weight cutoff of 8–10 kDa), which was sealed and immersed in a beaker containing 1 L of PBS (pH = 5.5/7.5). The beaker was positioned in a water bath shaker maintained at 37 °C and set to agitate at 100 rpm. The timer was initialized at 0 h. Dialysis tubes were carefully removed at predetermined times (1, 4, 8, 16, and 24 h). A pipette was used to extract 100 μL of the carrier solution into a 2 mL volumetric flask, adding acetonitrile to the volume and thorough shaking to disintegrate the lipids. The samples were then centrifuged at a speed of 1.5 × 10⁴ rpm. The supernatant obtained was filtered through a 0.45 μm microporous filter membrane and analyzed using high-performance liquid chromatography (HPLC, Agilent 1220 Infinity II, Germany).

Cytotoxicity test and culture of PC12 In vitro

The PC12 cells, purchased from Procell Life Science & Technology Co., Ltd (CL-0481)., were cultivated in RPMI-1640 complete medium supplemented with 10% fetal bovine serum (FBS) and 1% penicillin–streptomycin in a thermostat incubator set at 37 °C and 5% CO₂. The culture medium was refreshed every three days. Regular microscopic observations assessed cell density and growth status, selecting only PC12 cells exhibiting optimal growth for subsequent experiments.

PC12 cells were derived from rat adrenal pheochromocytomas, and highly differentiated PC12 cells are more phenotypically similar to neural cells, commonly used to study central nervous system diseases. The directional differentiation of NSCs is crucial for SCI repair. A specific concentration of PTX-NPs can enhance this neuronal differentiation. To determine the optimal PTX-NPs concentration, we conducted preliminary screenings to identify the most suitable drug concentration for NSCs differentiation. Subsequently, we examined the impact of this concentration on cell biocompatibility during effective differentiation. We used PTX-NPs at different concentrations (0, 5, 10, 15, and 20 ng/mL) to investigate their effects on the morphology, viability, and proliferation of PC12 cells and demonstrate the drug’s biocompatibility.

Actin filaments constitute the cell skeleton and directly affect cell morphology. Cell morphology influences extracellular matrix secretion. We cultured healthy PC12 cells at a cell density of 1 × 10³ cells/well in 96-well plates with growth media containing different PTX-NPs concentrations (0, 5, 10, 15, and 20 ng/mL) in a 37 °C, 5% CO₂ cell culture incubator for two days to detect PC12 cell morphology. After fixation with 4% paraformaldehyde (PFA; Biosharp) and permeabilization with 0.3% Triton X-100 (Biosharp), the PC12 cell skeleton was stained with TRITC phalloidin-actin filaments for 30 min. Finally, the nuclei were counterstained with 4’,6-diamidino-2-phenylindole (DAPI) (Biosharp, Beijing, China) and photographed under a fluorescence microscope (Zeiss, Oberkochen, Germany) in the dark. Inappropriate drug concentrations can impact cellular extension; hence, the biocompatibility of various drug concentrations was determined by examining alterations in PC12 cell morphology and structure when exposed to these drugs.

The live/dead state of the cells indicates the toxic effects of the drug. We cultured healthy PC12 cells at a cell density of 1 × 10³ cells/well in 96-well plates with growth media containing different PTX-NPs concentrations (0, 5, 10, 15, and 20 ng/mL) in a 37 °C, 5% CO₂ cell culture incubator. On days 1 and 3, we incubated the treated cells with a 5 mL 1 × Buffer solution containing 5 μL calcein AM and 15 μL propidium iodide at 37 °C for 20 min. We photographed them under an immunofluorescence microscope, ensuring the process was conducted in the dark according to the instructions of the live-dead staining kit. To determine the impact of unfavorable drug concentrations on cell viability, we utilized a live-dead staining reagent to assess drug-treated PC12 cells. In this assay, viable cells were stained green, while non-viable cells appeared red. The cellular activity under various drug treatments was evaluated by comparing the ratios of live to dead cells.

We performed a wound-healing assay on PC12 cells to investigate the drug’s effect on cell migration. An appropriate density of PC12 cells with good growth status was planted in 6-well plates and cultured for some time so that the cells adhered entirely to the wall. When the fusion rate between the cells reached 70%, a sterile razor blade was used to make a scratch along the center of the plate, followed by careful rinsing with PBS solution to discard the suspended cells. A small growth medium was added and photographed under a light microscope, recorded at 0 h. The incubation was continued for 24 h using a complete medium containing different PTX-NPs concentrations (0, 5, 10, 15, and 20 ng/mL), and photographs were taken under a light microscope to investigate the migration trend of PC12 cells in the presence of the drug. The wound-healing assay of PC12 cells is advantageous in determining the migratory capacity of the cells, thereby understanding their physiological impacts in vivo. The migration capability of PC12 cells, treated with various drugs, was assessed by creating uniform scratches at 0 h and measuring the distance of cell extension towards the center after a 24-h treatment period.

Extraction and culture of NSCs

The isolation of NSCs has been thoroughly documented in previous studies [27]. Briefly, the brain of a neonatal mouse was meticulously excised within 24 h postpartum under sterile conditions and homogenized using a pre-chilled basal culture medium, DMEM/F12 (Gibco, USA). After centrifugation, the resulting homogenate was resuspended in a serum-free growth medium supplemented with 20 ng/mL FGF (Peprotech, Suzhou, China), 20 ng/mL bEGF (Peprotech, Suzhou, China), 2% B27 (Gibco, USA), and 1% penicillin–streptomycin (Beyotime Biotechnology), followed by incubation at 37 °C in an atmosphere of 5% CO₂. The growth medium was replenished every three days to sustain the necessary nutrient requirements for the proliferation of NSCs.

The effect of PTX-NPs on neuronal differentiation of NSCs

Neuronal stem cells exhibiting robust growth were seeded at an appropriate density in 24-well plates coated with poly-D-lysine (Solarbio, China). After a 24-h incubation, the original growth medium was replaced with differentiation media containing varying PTX-NPs concentrations, replenishing the medium every three days. Following a seven-day culture period, immunofluorescence staining was employed to assess the extent of NSC differentiation into neurons and astrocytes using Tuj-1 and MBP as specific markers for neurons and astrocytes, respectively.

We co-cultured well-grown NSCs with 3D printed hydrogel scaffolds loaded with varying amounts of PTX-NPs to investigate the impact of drug-loaded 3D printed scaffolds on NSCs differentiation. The incorporation of PTX-NPs into hydrogel scaffolds was achieved through physical adsorption, followed by a 10-min incubation to ensure complete encapsulation, resulting in final PTX-NPs concentrations within the 3D printed hydrogel scaffolds of 0, 10, 20, 50, and 100 ng. After seven days of culture, NSC differentiation was evaluated by labeling neurons with Tuj-1 and astrocytes with GFAP.

Extracting myelin inhibitors

A previous study with modifications found that central myelin proteins were extracted by homogenizing adult rats’ brains and spinal cords [15, 28]. Briefly, the rat brain and spinal cord were thoroughly homogenized using a 0.32 M sucrose solution, then carefully added to an ultracentrifuge tube containing a 0.85 M sucrose solution, avoiding shaking and leading to the liquid’s layering. Subsequently, the prepared liquid was centrifuged at 27,000 g for 1.5 h. At this point, the sample was concentrated between the 0.32 M/0.85 M sucrose solutions. The samples were carefully collected and processed by centrifugation and sterile filtration to remove excess impurities, and the concentration was adjusted for subsequent experiments.

Western blotting analysis

The intense inflammatory milieu after SCI significantly impedes the survival and differentiation of NSCs. We examined the differentiation and signaling pathways of NSCs by introducing myelin-associated inhibitors to replicate the posttraumatic inhibitory environment. Varying concentrations of PTX-NPs solutions were absorbed into the 3D-printed hydrogel scaffolds for 1 h to ensure complete drug saturation, resulting in medicated 3D-printed hydrogel scaffolds. The concentration of myelin inhibitors was standardized to 10 μg/mL. A suitable density of NSCs mixed with myelin inhibitors was seeded into well plates containing medicated 3D-printed hydrogel scaffolds. After a 7-day incubation period, immunofluorescence staining was performed to assess the differentiation state of the NSCs. Three culture conditions were established to elucidate NSC differentiation mechanisms: control, myelin, and myelin + PTX NP. Following incubation, NSCs from each condition were harvested, and proteins were extracted and quantified using bicinchoninic acid (BCA) kits. The expression levels of Tuj-1, GFAP, ERK, and pERK proteins (Cell Signaling Technology, Danvers, MA, USA) across the different groups were detected by western blot analysis, with each group analyzed in triplicate.

Surgery for SCI and functional scaffold implantation

The establishment of a semi-sectional model of SCI has been described in detail in previous studies [29]. Adult female Sprague Dawley (SD) rats weighing 220–250 g were obtained from the Animal Breeding Center of Shushan District of Hefei. The animals were placed in a room that provided a 12/12-h cycle of light and dark conditions to allow them to adapt to the new environment. Before surgery, all experimental rats underwent 24 h of fasting and water deprivation to prevent postoperative death caused by gastrointestinal bloating. Briefly, the experimental rats were weighed, and sodium pentobarbital (50 mg/kg) was intraperitoneally administered for anesthesia. Following anesthesia, the rats were positioned prone on the operating table. After shaving and disinfecting their backs, a longitudinal incision was made at T9-T10, exposing the vertebral plate. The vertebral plate was removed using bone-biting pliers to expose the spinal cord at the T9-T10 segments. Using the median artery as a guide, ophthalmic scissors were used to make a 2 mm incision in the spinal cord to ensure complete hemostasis. The different groups received hydrogel scaffold implants in the spinal cord lesions, followed by sequential suturing of the muscle and skin. Daily doses of penicillin were subcutaneously administered to prevent infection. Manual bladder expression was performed until the rats regained their ability to urinate autonomously.

Behavioral analysis

The primary objective of addressing SCIs is to restore motor function in the hind limbs of rats. This function was assessed using the BBB (Basso, Beattie, and Bresnahan) scale, which ranges from 0 to 21 points. We initiated weekly observations of hind limb motor abilities in various groups starting the first-week post-surgery to evaluate the recovery process. These observations were made by two observers who were naïve to the experimental conditions to which the rats were subjected. The rats from each group were allowed to roam freely in an open environment, and the observers documented and scored their movements as mentioned above. This scoring process was continued weekly until the eighth week.

Tissue immunofluorescence staining analysis

The rats were processed for spinal cord tissue sampling and immunofluorescence staining of spinal cord tissue after the eighth week of SCI. Briefly, rats with SCI were perfused with normal saline, followed by 4% paraformaldehyde. Subsequently, the spinal cord was meticulously extracted from the vertebral canal, with a 0.5 cm section taken from both sides of the injury site as the center, and immersed in paraformaldehyde (PFA) for 24 h. The spinal cord samples were dehydrated using 20% and 30% sucrose solutions until they ultimately settled. The samples were then embedded in OCT (optimal cutting temperature compound, SAKURA, USA) and sectioned at − 20 °C using a frozen slicer (Leica, CM3050 S, Germany) to obtain 9 μm tissue sections. For tissue immunofluorescence staining, the tissue sections were rinsed with PBS solution (0.01 M, pH 7.4), then blocked with donkey serum at 4 °C for 2 h. Different primary antibody dilutions (1:200, including Tuj-1, GFAP, laminin, and NF) were incubated overnight at 4 °C with varying sections of the group. The antibodies were discarded, and the cells were rinsed with PBS. Sections were then incubated with a Cy3-conjugated goat anti-rabbit secondary antibody (1:400, Affinity) at room temperature for 2 h. DAPI was used to counterstain the nuclei for five minutes. Finally, the tissues were sealed with neutral resin, observed, and photographed under a fluorescence microscope. The entire process was performed in the dark.

Statistical analysis

All results were expressed as means ± standard deviation, and all data were processed using GraphPad Prism 8.0 (GraphPad Software, Inc., USA). One-way analysis of variance and two-sample independent t-tests were used to compare the data between groups. Differences were considered statistically significant at P < 0.05.

Results and discussion

Characterizing 3D hydrogel scaffolds with dual-sensitive NPs

The spinal cord has a complex physiological microstructure, and tissue-engineered bioscaffolds can be a carrier for cell growth and provide a suitable biophysical and chemical environment for the organism’s cellular components. Scanning electron microscopy images showed that the 3D printed SilMA hydrogel scaffolds had porous structures (Fig. 2A). The hydrogel scaffolds simultaneously provide a carrier for cells, and the porous structure is a nerve regeneration conduit that provides physical conditions for axonal lengthening and connectivity [30, 31]. The longitudinal section of the hydrogel scaffold exhibited a honeycomb-like structure that was patterned using a 3D printer and formed by UV cross-linking. Cellular structures provide a physical basis for exchanging substances and transporting nutrients between cells and external substances.

Fig. 2.

Characterization of hydrogel scaffolds and nanoparticles. A SEM of 3D hydrogel scaffolds. B TEM of empty-loaded NPs and PTX-NPs. C Temperature-influenced release curves of PTX vary over time. D pH-influenced release curves of PTX vary over time. E FTIR patterns of paclitaxel, empty-loaded NPs, and PTX-NPs. F XRD patterns of PTX, empty-loaded NPs, and PTX-NPs. G, H Zeta potential and particle size analyses of empty-loaded NPs and PTX-NPs

The empty-loaded NPs and PTX-NPs exhibited spherical geometries with a smooth exterior according to TEM (Fig. 2B). The diameters of NPs and PTX-NPs measured 113 ± 10 nm (with a polydispersity index (PDI) of 0.105 ± 0.02) and 134 ± 14 nm (PDI = 0.154 ± 0.01), respectively according to dynamic light scattering (Fig. 2G-H). The zeta potential values for NPs and PTX-NPs ranged between − 40 mV and − 30 mV. This potential is instrumental in fostering homogeneous dispersion among the micellar NPs, facilitated by the electrostatic repulsion related to the block copolymer PNIPAM75-PLA60 [26].

The characteristic FTIR absorption peaks of PTX included the keto carbonyl C = O peak at 1726 cm⁻¹, the amide group carbonyl peak at 1320 cm⁻¹, and the hydroxyl group O–H stretching vibration absorption peak between 3200 and 3600 cm⁻¹ (Fig. 2E) [32]. The empty-loaded NPs displayed material-specific absorption peaks: amide N–H stretching at 3000 cm⁻¹ and IR features of C–O–C, C = O, and C–N structures at 1020, 1720, and 1120 cm⁻¹, respectively. In comparison, the FTIR spectra of the PTX-NPs exhibited characteristic O–H absorption peaks of PTX (3600–3300 cm⁻¹) while the amide and C–O–C features of the empty-loaded NPs, indicating that PTX was compatible with the empty-loaded nanoparticulate preparation.

The XRD results indicated that PTX displayed subtle sharp crystalline diffraction peaks within the 5–20° range (Fig. 2F) [32, 33]. In contrast, the empty-loaded NPs exhibited broad diffraction peaks between 12–45°. Moreover, the PTX-NPs revealed a few PTX crystal diffraction peaks between 10–15°. This suggested that PTX may be encapsulated within the nano-preparations as crystalline molecules.

Composite scaffolds possess advantageous biological properties that aid in tissue repair. Hydrogels, characterized by their programmable physical attributes, controlled degradability, and capacity to stabilize unstable compounds, serve as an effective platform for drug encapsulation. This allows for a range of physicochemical interactions that regulate drug release. The efficiency of drug action is further enhanced by monitoring the release properties of nanoparticles. In the context of tissue repair, the inherent properties of composite scaffolds dictate their reparative potential. Scalability and long-term stability are particularly beneficial for transformational potential, guiding subsequent research efforts to uncover superior biological attributes.

pH / Temperature dual sensitive release characteristics

The intricate microenvironment post-SCI significantly influences drug bioavailability. Therefore, it is essential to enhance drug delivery in these challenging environments. Consequently, we conducted experiments to assess the temperature and pH sensitivity of PTX-NPs through controlled-release studies. The rate of drug release at pH 5.5 far exceeded the rate of release at pH 7.4 from 4 h onwards at 37 °C (Fig. 2B). The drug release rate at pH 5.5 was nearly 60%, which was more than 40% of that at pH 7.4 at 24 h. This indicated that an acidic environment was more likely to result in drug release at the same temperature. We also investigated the effects of different temperatures on the drug release rate under the same acidic conditions. The rate at 40 °C was much higher than the release efficiency at 37 °C at each time point of the assay (Fig. 2A). Most importantly, the release rate at 40 °C was close to complete release compared with the 30% release efficiency at 37 °C at 16 h. This suggested that drug-loaded NPs had a higher drug release rate in an acidic and relatively high-temperature environment. This indicated that the NPs encapsulating the drug have the property of double sensitivity to temperature and pH, which fits the microenvironment after SCI and is more favorable for the release of the drug, thus improving the bioavailability of PTX.

Favorable biocompatibility of specific PTX-NPs concentrations

PC12 cells derived from rat adrenal medullary pheochromocytoma cells are widely used to study Parkinson’s disease, nerve injury and regeneration, Alzheimer’s disease, and many other nervous system diseases [34–36]. Therefore, it is crucial to explore the biological properties of drugs for practical applications. In this study, the functional effect of paclitaxel NPs at an appropriate concentration was investigated on the properties of PC12 cells.

Good cytocompatibility is a prerequisite for the use of drugs to explore their toxic effects. We used different PTX-NPs concentrations to study the live and dead PC12 cells (Fig. 3A). The immunofluorescence results showed that the PC12 cells survived well on the first day of culture without dead cells in the control group, and the number of dead cells increased with higher PTX-NPs concentrations. Although some dead PC12 cells were visible in the field of view (which might have been affected by the drug concentration), the increase in drug concentration did not seriously affect cell survival, according to the number of live cells on the first day of culture. The density of live cells in each group increased with more extended cell culture, and only a few dead cells were visible in the field of view by the second day of culture, which may have been caused by the increase in cell density; the survival of PC12 cells was unaffected by this effect. PC12 cells showed sound survival effects at different PTX-NPs concentrations; the proportion of live cells showed no inhibition of cell survival on the third day, and a small number of dead cells may be the result of the normal physiological metabolism of the cell, which did not have a toxic effect for the survival of normal PC12 cells and metabolism of the material. Therefore, live-dead experiments showed that the different PTX-NPs concentrations in the experiment did not affect cell survival and material metabolism.

Fig. 3.

Good biocompatibility of PTX-NPs In vitro. Live and dead staining of PC12 cells at different concentrations of PTX-NPs during day 1, day 2, and day 3 (Green for live cells and red for dead cells). B Effect of different concentrations of PTX-NPs on the staining of the nuclear skeleton of PC12 cells during day 2. C The light microscopy of PC12 cell migration under various concentrations of PTX-NPs at 0 and 24 h

The cytoskeleton is the basis of material metabolism in cells. It is a protein fiber meshwork structure mainly composed of microfilaments, microtubules, and intermediate fibers commonly found in eukaryotic cells’ cytoplasm. It plays a vital role in cellular morphology support, morphogenesis, organization, and localization of intracellular structures, cell motility, cell division, and information transfer [37, 38]. Cytotoxic drugs can cause the cytoskeleton’s atrophy, deformation, and dissipation. Alteration of the skeleton can directly affect the secretion of the extracellular matrix; therefore, we investigated the effect of different concentrations of PTX-NPs on the adhesion of cells by nuclear skeleton staining.

PC12 cells in the control group exhibited a long strip shape, and the cells showed aggregated growth and good flexibility on the second day of cell culture (Fig. 3B). However, the morphology of PC12 cells remained as long stripes at different PTX-NPs concentrations, and there was no noticeable change in morphology. This also means that the cells can maintain a normal physiological metabolism at various drug concentrations, which is very important for cells to conduct regular material exchange. Therefore, the NPs exhibited good biocompatibility at this concentration gradient. This is favorable for cell growth; thus, a suitable drug concentration is more conducive for the cells to perform regular physiological roles.

The inflammatory microenvironment after SCI accelerates neuronal death, and the dissociation of neuronal axons is the basis for the lack of message transmission. PC12 cells that appear in the form of long strips are often used as models for probing the nervous system in basic research and wound healing tests, allowing the investigation of the effects of drug action on the migration of damaged PC12 cells. In this study, light micrographs showed that the different groups of cells appeared to have the same width of artificial scratches in the wound healing experiments at 0 h (Fig. 3C). All PC12 cells migrated to the center at 24 h at different PTX-NPs concentrations; however, there were differences in the migration efficiency of the cells under various concentrations of the drug, and the cells had the most robust migration ability at 10 ng/mL PTX-NPs. The migration efficiency of the cells was reduced at 20 ng/mL PTX-NPs compared with the control group. Normal cells did not show apparent toxic effects in this environment; however, this effect may be strengthened when the cell axons appear disjointed, thereby limiting the ability of the cell to migrate to the center. Therefore, appropriate drug concentrations are essential for the regular physiological activity of cells, which was particularly important for our in vivo investigation of SCI repair.

PTX-NPs enhance neuronal differentiation of NSCs In vitro

The direction of NSC differentiation is critical for SCI repair. Neuron formation enhances signaling at the lesion site, whereas astrocyte production leads to scarring and hinders nerve repair. Low doses of the microtubule-stabilizing drug PTX can overcome the inhibition of neuronal axonal extension and axon development, leading to functional recovery after SCI [11, 15, 39]. However, inappropriate PTX concentrations can induce multipolar mitotic spindle formation, leading to apoptosis and cell death [40]. The effect of PTX-NPs on NSC differentiation was investigated by culturing NSCs in media containing different PTX-NPs concentrations, followed by staining with Tuj-1 (a marker for neurons) and GFAP (a marker for astrocytes). Figure 4A shows the changes in Tuj-1 and GFAP-positive cells treated with different concentrations of PTX-NPs. Immunofluorescence results confirmed that the number of NSCs differentiating into neurons increased as the PTX-NPs concentration increased, and the highest number of neurons differentiated from NSCs occurred at 10 ng/mL PTX-NPs. Meanwhile, the neuron expression decreased at 20 ng/mL PTX-NPs, which may be because the high PTX-NPs concentration limited neuron differentiation of NSCs. This tendency of NSC differentiation with varying PTX-NPs concentrations matched the results of previous studies. These results suggest that appropriate PTX-NPs concentrations significantly enhanced the intrinsic neuronal differentiation of NSCs and could be used as a small-molecule inducer to modulate the differentiation of NSCs for use in SCI repair, which will be performed in subsequent experiments.

Fig. 4.

PTX-NPs promote the differentiation of NSCs In vitro. Effects of different concentrations of PTX-NPs on the differentiation of NSCs into neurons and astrocytes (green for Tuj-1, red for GFAP). B Effects of 3D-printed scaffolds encapsulated with different contents of PTX-NPs on the differentiation of NSCs into neurons and astrocytes (green for Tuj-1, red for GFAP). C, D Statistical analyses of the differentiation of NSCs into neurons and astrocytes

Another major obstacle to SCI repair is scar tissue, and glial scarring is associated with astrocytes [27, 41, 42]. Therefore, we investigated the induction of astrocytic differentiation in NSCs treated with different PTX-NPs concentrations. The number of NSCs positively stained for GFAP tended to decrease and increase after culturing in media with different PTX-NPs concentrations; this trend was most pronounced at 10 ng/mL (Fig. 4A, C). Therefore, 10 ng/mL PTX-NPs could better promote the differentiation of NSCs towards neurons, and astrocyte formation was reduced. The appropriate PTX-NPs concentration is conducive to lowering glial cell production and differentiating neurons from neural stem cells. Therefore, it can be concluded that low PTX-NPs concentrations can stimulate the differentiation of NSCs into neurons instead of astrocytes, which plays a vital role in the repair of nerves after SCI.

3D SilMA hydrogel scaffolds carrying PTX-NPs promote neuronal differentiation In vitro

The impact of porous scaffolds loaded with PTX-NPs on NSCs differentiation In vitro is significant. It offers a valuable approach to evaluate the potential of such composite scaffolds to influence NSC differentiation in the semicircular region of the rat spinal cord in vivo. Single cells were seeded on the composite hydrogel scaffolds incorporating varying PTX-NPs doses, as corroborated by the immunofluorescence data (Fig. 4B). Notably, no substantial increase in neuronal differentiation was observed at a loading of 10 ng compared to the control group. However, a significant increase in neuronal differentiation of NSCs was observed when the loading dose was increased to 20 ng and 50 ng, and the effect was more apparent at 50 ng. In contrast, the neuronal differentiation of NSCs decreased when the loading dose was 100 ng. Therefore, higher PTX-NPs doses have a specific inhibitory effect on neuronal differentiation. Choosing the appropriate drug dose to exert a physiological effect is more beneficial.

Notably, the hydrogel scaffold composite diminished the propensity of NSCs to differentiate into astrocytes relative to the control, which became more pronounced when the scaffolds were encapsulated with NPs containing 50 ng of PTX-NPs (Fig. 4B, D). Consequently, when 3D-printed hydrogel scaffolds were enveloped with 50 ng of PTX-NPs, they demonstrated an enhanced capacity to direct NSC differentiation towards a neuronal lineage while concurrently minimizing astrocyte formation.

MAPK/ERK signaling pathway involved in PTX-NPs induced NSC differentiation

The inflammatory microenvironment after SCI is a significant obstacle to nerve repair, and alleviating the harsh microenvironment to promote nerve regeneration is essential in SCI treatment [43, 44]. Composite scaffolds with different PTX-NPs concentrations were explored for the neuronal differentiation of NSCs using myelin extract to mimic the inhibitory microenvironment after SCI. A specific concentration of PTX-NPs can rescue the neuronal differentiation of myelin inhibitors, which is beneficial for nerve repair after SCI [15], thus simulating the effects of drugs in the post-injury inflammatory microenvironment by extracting myelin proteins In vitro. Immunofluorescence assays showed that composite scaffolds loaded with 20 and 50 ng of PTX-NPs led to better differentiation of NSCs into neurons using 10 μg/mL myelin inhibitor compared to that without PTX-NPs (Fig. 5A). The number of neurons was higher when the drug content reached 50 ng. This indicated that 50 ng of PTX-NPs-loaded SilMA hydrogel scaffold was more favorable for neuronal expression under inhibitory microenvironmental conditions.

Fig. 5.

Quantitative protein analysis of neuronal expression and MAPK/ERK signaling pathway in the inhibitory microenvironment. The immunofluorescence images of the effects of composite scaffolds loaded with different levels of PTX-NPs in the inhibitory microenvironment on the differentiation of NSCs into neurons (green for Tuj-1, blue for DAPI). B Protein expression levels of GFAP, Tuj-1, total ERK, and pERK in Control, Myelin, and Myelin + PTX-NPs groups. C Analysis of neuronal expression in different groups. D-F Detection of protein expression levels in the three groups. (*p < 0.05, **p < 0.01, relative to Control group)

We characterized the role of PTX-NPs by immunoblotting to investigate the potential mechanism of gene regulation that triggers neural stem cell differentiation in this environment. The MAPK/ERK signaling pathway is essential in cell proliferation and differentiation [15, 45, 46]. Total ERK and pERK levels were assayed to confirm the role of paclitaxel in NSC differentiation. Further validation was performed using the downstream expression of GFAP and Tuj-1 proteins. Western blotting analysis showed that the expression of GFAP proteins tended to decrease in the myelin + PTX-NPs group compared to the Control and Myelin groups (Fig. 5B). However, the highest GFAP expression was observed in the myelin group. In contrast, there was no significant difference between the Control and Myelin groups for the expression of Tuj-1 protein; however, the expression of Tuj-1 protein showed an increasing trend under the effect of PTX-NPs. This suggested that NSCs were more likely to differentiate into astrocytes than neurons in an inflammatory microenvironment; however, PTX-NPs could reverse this inhibitory effect to promote the neuronal differentiation of NSCs. pERK protein expression was higher in the myelin + PTX-NPs group because PTX-NPs promoted ERK phosphorylation. Thus, PTX-NPs promoted neuronal differentiation of NSCs through the MAPK/ERK signaling pathway, a link between PTX-NPs and NSC differentiation.

3D SilMA hydrogel scaffolds carrying PTX-NPs promote locomotor recovery

Hydrogel scaffolds promote NSC differentiation; therefore, we further explored the feasibility of using printed scaffolds loaded with PTX-NPs and neural stem cells to repair SCI in vivo. Patients with traumatic SCI often suffer from persistent motor dysfunction owing to an imbalance in the microenvironment, including inflammatory cell activation, scar tissue formation, and lack of neurotrophic factors after SCI, making it difficult to achieve satisfactory results in SCI repair [47, 48]. Therefore, motor function recovery is an essential criterion for evaluating treatment plans for SCI repair.

A rat spinal cord hemisection model was constructed, and a composite hydrogel scaffold was implanted into the defect. The hind limb on the injured side of the rats was paralyzed entirely (score 0) on the first day after SCI (Fig. 6C), indicating that the injury model was successfully constructed. Subsequently, the BBB scoring test was performed weekly to assess the motor recovery of the hind limbs in the SCI rats. There was minimal motor recovery in the control group. Most rats that underwent SCI exhibited complete flaccid paralysis of the hind limbs. In the initial two weeks post-injury, no significant difference was observed between the groups, albeit minor movements were discernible in the rats’ hind limbs. Interestingly, all groups of spinal cord-injured rats showed varying degrees of recovery in hind limb function over time. By the third week, a subtle enhancement in the hind limb function of rats in the SM-NSC/PTX-NPs group emerged compared to the other groups (p < 0.05). This trend became increasingly evident in the fourth week. From the onset of the fifth week through the eighth week, hind limb activity in all groups progressively improved; however, the advancement in the SM-NSC/PTX-NPs group was notably superior to the others. Importantly, the SM-NSC/PTX-NPs group significantly enhanced from the fifth week compared with the other groups. By the eighth postoperative week, the motor score of the drug-loaded composite stent group had reached 12.6 ± 0.24, significantly higher than that of the other groups. Such outcomes can be attributed to the synergistic effect of the medication and the scaffold. This indicated that the drug-loaded composite stent contributed to the recovery of motor function after SCI, revealing its potential for treating SCI.

Fig. 6.

Behavioral analysis of SCI rats. A Timeline plot of BBB scores and immunofluorescence staining. B Gross morphology of rat spinal cord sampling. C BBB scores of hind limb movements in different groups of rats. (*p < 0.05, **p < 0.01, ***p < 0.001, and ns for no significance, relative to the Control group. ^#p < 0.05, ^##p < 0.01, relative to two adjacent groups. n = 5 animals per group.)

We utilized the BBB score to evaluate hindlimb locomotor function in rats following SCI, as it more accurately reflects improvements in behavioral function. By visually assessing the rat’s hindlimb locomotor ability, the score encompasses changes in the rat’s hindlimb weight-bearing capacity, control over hindlimb placement, magnitude of hindlimb muscle force, and coordination between forelimb and hindlimb movements post-injury. This allows for the determination of locomotor function recovery and variations from the initial stages of injury to later stages, including subtle adjustments in fine locomotor movements. Elevated scores suggest enhanced weight-bearing and motor coordination recovery in the rat’s hindlimb. These data discrepancies directly result from restoring rat hindlimb motor function, which can also be observed through imprint analysis, grid movement, and neurophysiological assessments. To understand the underlying mechanism of functional recovery, histological analysis is required to examine the expression of motor-related neurons in vivo.

The BBB locomotor rating scale is a 21-point scale used to assess hind limb motor function recovery after SCI in rats. Higher scores on this scale indicate better recovery of hind limb motor ability. Scores between 0 and 7 represent the early stage of recovery, where the animal cannot support its body weight and drags its trunk, hind legs, and hips. Scores between 8 and 13 represent the middle stage of recovery, where the animal can walk and support its body weight, and coordinated movements of the front and hind limbs begin to recover. Scores between 14 and 21 represent the stage where some fine rat movements start to heal. The BBB scale includes almost all behavioral changes in the recovery process of the hind limbs of animals after SCI, and it provides a reference for the effectiveness and standard of treatment after human spinal cord injury. However, observers require rigorous training since no special equipment is needed for scoring, and the criteria are complex. Additionally, both animals and observers are susceptible to external disturbances, so minimizing subjective factors can reduce discrepancies in experimental data.

The polarization of the study of behavioral and histological analysis of rats at different time points is shown in Fig. 6A. The rat spinal cord tissues were sampled after the eighth week of SCI (Fig. 6B), which showed the site of SCI and attachment of the few repair tissues out of the injury, which were used to study the in vivo nerve repair in rats by subsequent tissue sectioning.

3D SilMA hydrogel scaffolds carrying PTX-NPs promoted neuronal differentiation

Nerve regeneration was studied two months after SCI to understand the anatomical basis of functional recovery. It is widely accepted that stem cell transplantation contributes to axonal regeneration in CNS-related diseases. Stem cell transplantation has been proposed as a scientific treatment for SCI. Briefly, transplanted stem cells are induced to differentiate into neurons, and this biologically induced effect can be integrated with the remaining neurons to reestablish neural protrusions and synapses so that neural excitatory signals can be well conducted and transmitted, leading to behavioral recovery [49]. Therefore, neurogenesis and axonal germination are crucial.

To observe the outcome of the transplanted stem cells, Tuj-1 immunostaining (Fig. 7A) was performed eight weeks postoperatively to label early neurons. Endogenous stem cells are differentiated into neurons, astrocytes, and oligodendrocytes. Among these, neurons are critical for regular electrophysiological activity in organisms. Most importantly, most neural stem cells tend to differentiate into glial cells rather than neurons in the region of injury owing to the inhibitory microenvironment, which limits functional recovery from SCI. In this current study, the expression of Tuj-1-labeled early neurons was more prevalent at the site of injury in the SM-NSC/PTX-NPs group than in the SM-NSCs, SilMA hydrogel, and injury-only groups. This is because the differentiation of NSCs into neurons is promoted under the action of the microtubule stabilizer PTX-NPs, and the newborn neurons form a connector in the injury area to transmit the electrical activity of host axons above and below the injury level. This contributes to the recovery of neural function. This result was directly reflected in the ability of the rats to recover hind limb movements.

Fig. 7.

The composite scaffold promotes neuronal expression in vivo. An Expression of neurons at the injury site in tissue sections of different groups. B Expression of motor-related neurons at the injury site in tissue sections of different groups. C-D Immunofluorescence statistical analysis of neuronal expression at the injury site (**p < 0.01, ***p < 0.001, relative to SCI group)

Axonal regeneration is essential for neural signaling in the spinal cord during injury, and NF-specific staining can be used as a biomarker for axonal repair. The nerve fibers shown in Fig. 7B allow for the assessment of nerve regeneration. The results showed that NF-positive cells were also observed in the injury-only group; however, there was a significant increase in NF-positive cells at the injury site in the SM-NSCs/PTX-NPs group compared to the other groups. This indicated that the drug-loaded composite scaffold promoted axonal repair in SCI.

3D SilMA hydrogel scaffolds carrying PTX-NPs reduce scar tissue formation

Activated macrophages and microglia contribute to scar formation, which impedes nerve regeneration and axonal outgrowth [50, 51]. Spinal cord injury scarring comprises two primary components at distinct injury sites. Scar tissue formation confines the wound and injury from further expansion but concurrently erects a barrier against axonal regeneration [52]. Hence, minimizing scar tissue development in the injured zone and fostering axonal regeneration is pivotal for functional recovery post-SCI in rats. It is widely acknowledged that the inflammatory milieu following SCI enables endogenous and transplanted NSCs to home to the injury site in vivo and differentiate into specific cell lineages, despite the majority ultimately becoming astrocytes [53–55]. GFAP differentiation was examined by immunofluorescence staining of astrocytes in injured areas. Untreated spinal cord-injured rats exhibited a robust presence of astrocytes at the injury site. In contrast, the group with PTX-NPs-containing composite scaffolds displayed significantly diminished reactive astrocyte proliferation compared to the other groups (Fig. 8A). This indicated that glial cell formation could be reversed in the presence of PTX-NPs, corroborating previous cellular experimental findings. Glial scar formation results from the secretion of extracellular matrix by astrocytes, and curtailing astrocyte formation is crucial to reducing scar tissue in the injured region.

Fig. 8.

The composite scaffold reduces the expression of scar tissue in vivo. An Expression of astrocytes at the injury site in tissue sections of different groups. B Expression of fibrous scar at the injury site in tissue sections of different groups. C-D Statistical analysis of immunofluorescence of scar tissue expression at the injury site (**p < 0.01, ***p < 0.001, relative to SCI group)

In addition to glial scarring, fibrous scarring is another integral aspect of scar tissue. Fibrous scars emerge from inflammation, rendering the post-injury inflammatory milieu suboptimal for repair and regeneration [56]. Specifically, microglia and macrophages activated by the inflammatory environment secrete copious amounts of extracellular matrix after SCI, promoting fibroblast accumulation and fibronectin deposition and culminating in fibrotic scar formation [57]. Our immunofluorescence staining assays for fibrous scarring using laminin revealed a significant increase in fibrous scarring within the injured area in the simple SCI group compared to that in the other groups (Fig. 8B). Conversely, the disparity in scarring expression between the scaffold group and the neural stem cell-laden scaffold group was insignificant and potentially linked to the use of PTX-NPs. The severe inflammatory milieu after injury expedites tissue degradation, and the absence of inducible factor stimulation exacerbates this process. Regarding the PTX-NPs-loaded composite scaffold, a notable reduction in fibrous scarring was observed on both sides of the injured tissues, suggesting that post-SCI PTX-NPs application could mitigate fibrous scarring. These findings implied that the implantation of SM-NSC/PTX-NPs scaffolds could facilitate neuronal formation, curtail scar development, and foster a conducive microenvironment for axonal regeneration.

Hence, in this study, a three-dimensional (3D) printed porous SilMA hydrogel scaffold was engineered to facilitate axonal regeneration. The capacity of the scaffold to physically adsorb dual-sensitive PTX-encapsulated micellar nanoparticles augmented the inherent neuronal differentiation potential of NSCs and alleviated the inhibitory effects of myelin. Neural stem cells were incorporated into a functionalized porous scaffold with adsorbed PTX-NPs, and the integrated scaffold was implanted into a rat spinal cord hemi-section injury model. This composite scaffold fostered a conducive microenvironment for the neuronal differentiation of NSCs and minimized scar tissue formation. Crucially, the composite scaffold aided the functional recovery of rats with SCIs. Western blotting analysis confirmed the involvement of the MAPK/ERK signaling cascade in PTX-NPs-induced neuronal differentiation. Our findings highlight the therapeutic prospects of PTX-NPs as a stem cell-based therapy for SCI repair. This study paves the way for the sophisticated design of neural scaffolds that synergistically enhance stem cell-directed differentiation and are adaptable to the microenvironment for SCI repair.

Author’s contribution

ZL (Master of Medicine), TZ (Doctor of Medicine), and ZB (Doctor of Medicine) were involved in the study design, literature research, data analysis, and writing the manuscript. MW (Doctor of Medicine) and YM (Doctor of Philosophy) were involved in the study design, data analysis, and writing the manuscript.

Declarations

Conflicts of interest

The authors declare that there is no conflict of interest regarding the publication of this paper.

Ethical Statement

This study protocol was approved by the Institutional Animal Care and Use Committee (IACUC) of Bengbu Medical University (IACUC approval No. 2020238).