Loading neural stem cells on hydrogel scaffold improves cell retention rate and promotes functional recovery in traumatic brain injury

Tiange Chen; Yuguo Xia; Liyang Zhang; Tao Xu; Yan Yi; Jianwei Chen; Ziyuan Liu; Liting Yang; Siming Chen; Xiaoxi Zhou; Xin Chen; Haiyu Wu; Jinfang Liu

doi:10.1016/j.mtbio.2023.100606

. 2023 Mar 8;19:100606. doi: 10.1016/j.mtbio.2023.100606

Loading neural stem cells on hydrogel scaffold improves cell retention rate and promotes functional recovery in traumatic brain injury

Tiange Chen ^a,^b,^c,¹, Yuguo Xia ^a,^b,^c,¹, Liyang Zhang ^a,^b,^c,¹, Tao Xu ^d, Yan Yi ^b,^e, Jianwei Chen ^d, Ziyuan Liu ^a,^b, Liting Yang ^a,^b,^c, Siming Chen ^a,^b, Xiaoxi Zhou ^a,^b, Xin Chen ^a,^b, Haiyu Wu ^a,^b, Jinfang Liu ^a,^b,^∗

PMCID: PMC10102240 PMID: 37063247

Abstract

Neural stem cell (NSC) has gained considerable attention in traumatic brain injury (TBI) treatment because of their ability to replenish dysfunctional neurons and stimulate endogenous neurorestorative processes. However, their therapeutic effects are hindered by the low cell retention rate after transplantation into the dynamic brain. In this study, we found cerebrospinal fluid (CSF) flow after TBI is an important factor associated with cell loss following NSC transplantation. Recently, several studies have shown that hydrogels could serve as a beneficial carrier for stem cell transplantation, which provides a solution to prevent CSF flow-induced cell loss after TBI. For this purpose, we evaluated three different hydrogel scaffolds and found the gelatin methacrylate (GelMA)/sodium alginate (Alg) (GelMA/Alg) hydrogel scaffold showed the best capabilities for NSC adherence, growth, and differentiation. Additionally, we detected that pre-differentiated NSCs, which were loaded on the GelMA/Alg hydrogel and cultured for 7 days in neuronal differentiation medium (NSC [7d]), had the highest cell retention rate after CSF impact. Next, the neuroprotective effects of the NSC-loaded GelMA/Alg hydrogel scaffold were evaluated in a rat model of TBI. NSC [7d]-loaded GelMA/Alg markedly decreased microglial activation and neuronal death in the acute phase, reduced tissue loss, alleviated astrogliosis, promoted neurogenesis, and improved neurological recovery in the chronic phase. In summary, we demonstrated that the integration with the GelMA/Alg and modification of NSC differentiation could inhibit the influence of CSF flow on transplanted NSCs, leading to increased number of retained NSCs and improved neuroprotective effects, providing a promising alternative for TBI treatment.

Keywords: Cerebrospinal fluid flow, Neural stem cells, Hydrogel scaffold, Traumatic brain injury, Neuroprotection

Graphical abstract

Highlights

•
CSF flow affects the retention rate of transplanted NSCs after TBI.
•
Integration with GelMA/Alg and modification of NSC differentiation mitigate CSF flow-induced loss of transplanted NSCs after TBI.
•
NSC [7d]-loaded GelMA/Alg protects against TBI by modulating neuroinflammation and promoting neurorestoration.

1. Introduction

Traumatic brain injury (TBI) typically causes devastating effects, including structural damage to the central nervous system (CNS) and permanent dysfunction. TBI has a high global morbidity rate (approximately 10 million hospitalizations/deaths per year), causing severe social and economic problems [[1], [2], [3]]. Furthermore, TBI survivors experience chronic complications, including cognitive disorders, persistent vegetative state, and disability. Surgical treatment combined with neurological rehabilitation training is used to improve the patients’ condition [4]. However, the inherently poor regenerative capability of the CNS prevents neurological recovery. Therefore, effective interventions are urgently required to treat TBI.

In the past decades, the application of stem cells, such as bone marrow mesenchymal stem cells (BMSCs) [4] and neural stem cells (NSCs), as potential regenerative therapies for neurological diseases, have been widely accepted [5]. The transplantation of BMSCs in the injured area increases the levels of neurotrophic and synaptic proteins [6,7]. Transplanted NSCs can differentiate into neurons and glia [8], as well as promote synaptic protein expression [9]. However, the effectiveness of transplanted stem cells is highly affected by the microenvironment, in which cerebrospinal fluid (CSF) is a crucial factor in the CNS. Specifically, CSF is dynamic fluid circulating in the ventricles and subarachnoid space, where it is involved in transporting hormones, fluxing away metabolic products, and therefore playing an important role in maintaining brain homeostasis [10]. It is known that fluid shear stress (FSS) generated by the in vivo circulating fluid exerts considerable influence on stem cell adhesion [11,12]. Additionally, the viability and differentiation of the transplanted stem cells are largely depend on adhesion morphology [13,14]. Thus, CSF is potentially a contributing factor affecting the therapeutic effects of stem cell transplantation in the brain. Besides, TBI can induce disorders of CSF circulation and dynamics [15], which may affect the efficacy of NSCs transplantation. Nevertheless, little is known about the influence of CSF flow on the fate of transplanted NSCs after TBI.

Bioprinting technology, which combines tissue-engineering approaches with novel biomaterials, has accelerated in recent years and demonstrated many potential prospective applications [16,17]. In our previous work, we developed a micro three-dimensional (3D) bioprinting platform that can reach a designated region for in situ 3D printing [18]. Some studies have indicated that the “bioink”-gelatin-methacrylate hydrogel (GelMA) has thixotropic and cell encapsulation properties that are naturally habituated to NSC adhesion [19]. The addition of a photoinitiator to GelMA, which is composed of collagen-derived gelatin modified with methacrylate groups, enables the formation of a photopolymerizable, patternable hydrogel that maintains the properties of gelatin. As a collagen derivative, GelMA contains cell-extracellular matrix peptides, including the Arg-Gly-Asp (RGD) tri-amino acid sequence, for recognition and binding of cell adhesion factors [20,21]. However, little advancement has been achieved on the application of GelMA-3D bioprinting techniques to injury recovery in TBI; nevertheless, it is still a valuable technique in this field.

In this study, we evaluated the influence of CSF flow on cell retention after NSC transplantation in an in vitro bionic CSF flow model. Our results demonstrate that CSF flow after TBI is a crucial factor affecting the cell retention rate of transplanted NSCs. For solving this problem, we used a hydrogel scaffold as a carrier for NSCs and modified the degree of NSC differentiation. Interestingly, loading the GelMA/Alg hydrogel scaffold with pre-differentiated NSCs cultured for 7 days in neuronal differentiation medium [7d] demonstrated improvement in cell retention after CSF flush. Additionally, NSC [7d]-loaded GelMA/Alg transplantation showed multifaceted protective effects against TBI, including inhibition of microglial activation and prevention of neuronal death during the acute phase along with a decrease in tissue loss, alleviation of astrogliosis, and promotion of neurofunctional recovery during the chronic phase. Hence, this study revealed an innovative and effective strategy to treat TBI as well as potentially other diseases in the central nervous system (CNS).

2. Materials and methods

2.1. Ethics approval and informed consent

The work involving the clinical information, MRI, and computed tomography scan data of the patients with TBI has been approved by the Medical Ethics Committee of the Xiangya Hospital of Central South University (No. 202009581) and informed consent was obtained in all cases. All animal experiments were conducted according to the standard operating procedure and complied with the rules of the Animal Care and Use Committees of the Laboratory Animal Research Center at Xiangya Medical School of Central South University, Hunan, China (No. 202110093).

2.2. Isolation and culture of NSCs

NSCs were isolated from P1 newborn rats. Briefly, the rats were euthanized and the hippocampus was dissected under a microscope. Next, the tissue was cut into small pieces with fine scissors, followed by digestion with 0.25% trypsin-EDTA (GP3108, Genview, Houston, TX, USA) at 37 °C for 15 min. The digestion step was stopped by adding a conditioned medium containing DMEM/F12 (C11330500, Gibco, Grand Island, USA) and 10% FBS (04-001-1ACS, Gibco). Thereafter, the cells were filtered through a 70 μm strainer, centrifuged, and washed with DMEM/F12 three times. Next, the cell pellets were resuspended in NSC culture medium (CM) containing the following components: DMEM/F12, 2% B27 (12,587,010, Gibco), 20 ng/ml EGF (CYT-554, ProSpec), 20 ng/ml FGF (CYT-386, ProSpec), 5 μg/ml heparin (H3149, Sigma-Aldrich, St. Louis, MO, USA), and 1% Pen-Strep (10,378,016, Thermo Fisher Scientific, Waltham, MA, USA). Finally, the cells were seeded into 25 cm² cell culture flasks (707013, Nest) at a cell density of 5 × 10⁴ cells/mL and cultured in a 37 °C incubator with 5% CO₂. The culture medium was half-changed every 2 days, and the cells were passaged every 5–6 days.

2.3. Preparation of 3D-printed hydrogel scaffolds

GelMA (EFL-GM-60) was purchased from Suzhou Intelligent Manufacturing Research Institute (Suzhou, China). Sodium alginate was procured from Shanghai Aladdin Biochemical Technology Co. Ltd. (Shanghai, China). Hydrogel scaffolds were printed using a 3D printer (Medprin Regenerative Medical Technologies Co., Ltd., Guangzhou, China), under a controlled environmental temperature (10 °C), platform temperature (10 °C), and cooling system temperature (15 °C). Three different hydrogel scaffolds were used: 10% gelatin/1% sodium alginate (Gelatin/Alg1) [22], 2.8% gelatin/2% sodium alginate (Gelatin/Alg2) [23], and 10% GelMA/1% sodium alginate (GelMA/Alg). For the preparation of GelMA hydrogel, GelMA was dissolved in phosphate-buffered saline (PBS) to obtain a 10% concentration with the addition of a photo-initiator (lithium phenyl-2,4,6-trimethylbenzoylphosphinate, LAP) at a concentration of 0.25% (w/v). For the preparation of other hydrogel, sodium alginate and gelatin were dissolved into phosphate buffered saline (PBS) according to the manufacturer's instruction. Next, the hydrogels were placed into a 1 mL syringe and loaded onto a microinjection pump. The printing parameters were as follows: nozzle diameter, 0.26 mm; scaffold size, 1 × 1 cm²; printing, 5 layers; and pump speed, 5 mm/s. Subsequently, all of the scaffolds were immersed in a calcium chloride solution (3%) to crosslink the sodium alginate. For GelMA/Alg, the scaffold was further exposed to ultraviolet light (wavelength: 405 nm; power: 3 w) for 5 min. The porous structure of the scaffold was 3D printed because good mass transportation for nutrition, cell attachment, and cell migration properties have been reported with this technique [24,25]. Finally, the scaffolds were preserved under sterile conditions until use.

2.4. In vitro CSF flush assay

The in vitro CSF flush system is composed of four major components: a solution infusion flask (material: glass), flux chamber (material: glass; height: 0.05 cm, diameter: 3.5 cm), and electrical pump, and the connecting polytetrafluoroethylene (PTFE) tubes, as illustrated in Fig. 1C–E. The basic CM (DMEM/F12) was injected into the infusion flask with a sterile 50 mL syringe, following the aseptic principle. The pump created a liquid flow at a speed of 0.3 cm/s. An inner concave chamber was used to hold the NSCs or NSCs-loaded hydrogel scaffold within the flux chamber. To test the influence of CSF flow on cell retention of the NSCs-loaded scaffold (illustrated in Fig. 1, Fig. 4C), NSCs-loaded scaffold was placed into the flux chamber, DMEM/F12 was infused, the pump was started and flushed in a 37 °C incubator with 5% CO₂ for 24 h. Finally, cryosections were cut and DAPI staining was performed as described earlier. For the in vitro CSF flush of NSCs (illustrated in Fig. 1H), the flux chamber was coated with poly-L-lysine (5 μg/mL) for 24 h at 4 °C to promote NSCs adhesion on the bottom of the chamber. Next, the flux chamber was rinsed with sterile PBS for 3 times, followed by NSCs seeding and cultured in incubator for another 24 h for cell adhesion. Next, the unattached NSCs was removed and fresh CM was added. Finally, NSCs were flushed in the devise with the same parameter as mentioned above for a certain time (3, 6, 12, and 24 h). Cells was photographed, digested using accutase (07920, STEM CELL™), and then cell number was counted using the DeNovix CellDrop BF Cell Counter. The original and final cell number was defined as A0 and A1. The cell loss rate (R) was denoted as R = (A0-A1)/A0 × 100%.

Fig. 1 — **The influence of cerebrospinal fluid (CSF) flow on neural stem cell (NSC) transplantation was detected using an *in vitro* bionic CSF flow model**. (A) Representative computed tomography (CT) images before and after surgery of patients with traumatic brain injury (TBI) showed the formation of a cavity and CSF within the cavity. The red star denotes the cavity, and the dash line indicates the border of the cavity. Scale bar: 2 cm. (B) Representative T2 magnetic resonance imaging (MRI) scanning images of the rat brain at 3 and 28 days after TBI. Scale bar: 1 mm. (C–E) Illustration of the *in vitro* bionic CSF flow model. (F) Representative T2 MRI scanning images of the patients with TBI. AM, aqueduct of the midbrain; CC, cerebellar cortex; CC-F, cerebral cortex-frontal lobe; CC-T, cerebral cortex-temporal lobe. Scale bar: 2 cm. (G) Quantification of CSF flow speed in different brain areas after TBI (n = 5/group). (H, I) Representative images (H) and quantification (I) of NSCs before and after CSF flush (n = 4/group). Scale bar: 500 μm. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Fig. 4 — **Comparison of the adhesive capacity of neural stem cells (NSCs) with different degrees of differentiation on the GelMA/Alg scaffold.** (A) Representative images of hematoxylin and eosin (HE) staining of NSCs on the GelMA/Alg scaffold before CSF flush. The dash line represents the border of the scaffold. Scale bar: 100 μm. (B) Quantification of cell number on GelMA/Alghydrogel scaffold before CSF impact in the *in vitro* CSF system (n = 5/group). (C) Illustration of the experimental design. (D) Representative images of DAPI staining after CSF flush. White dashed line represents the outline of the hydrogel. Purple arrow heads indicate retained cells after flush. Scale bar: 100 μm. (E) Quantification of DAPI⁺ cells per slide (n = 4–5/group). (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

2.5. Animals

Male Sprague-Dawley rats (age: 8–12 weeks; weight: 250–300 g) were purchased from Hunan Slack Laboratory Animal Co., Ltd. (Hunan, China). All animals were housed in the animal facility of the Laboratory Animal Research Center of Central South University under a specific pathogen-free grade environment, temperature- and humidity-controlled conditions, a 12-h light/dark cycle, and environmental enrichment. Food and water were provided ad libitum. All efforts were made to minimize animal suffering and reduce the number of animals used.

2.6. Traumatic brain injury (TBI) model

The TBI model was established using a unilateral CCI as previously described, with some modifications [26]. Briefly, rats were placed in an induction chamber with 5% isoflurane (RWD Life Science Co. Ltd, China) to induce anesthesia, which was later maintained with 1.5% isoflurane delivered during the CCI procedure via nose cones. The craniectomy and impact procedures are illustrated in Figs. S10A–B, respectively. Briefly, after the animal's head was fixed with a stereotaxic frame, a skin incision was made with a surgical knife to expose the skull, followed by craniectomy (centered 1.5 mm anterior and 3 mm lateral to the bregma, diameter of 5 mm) using an electric drill. Next, the impact was performed using a flat-tipped impactor (diameter of 5 mm) with controlled parameters (speed: 4.0 m/s, depth: 4.0 mm, and dwell time: 0.5 s). Thereafter, the skin incision was sealed, and the rat was placed back in its home cage with a warming lamp to maintain body temperature.

2.7. Animal grouping and treatment strategy

Before the CCI procedure, rats were randomly separated (using a lottery box) into five groups: control group, GelMA/Alg group, GelMA/Alg + NSCs [0d] group, GelMA/Alg + NSCs [7d] group, and GelMA/Alg + NSCs [14d]. In the control group, the GelMA/Alg hydrogel scaffold was transplanted immediately after TBI. In the GelMA/Alg + NSCs [0d] group, NSCs were mounted and cultured on the GelMA/Alg hydrogel scaffold using the NSC CM for 14 days, and the NSC-loaded GelMA/Alg was transplanted immediately after TBI. In the GelMA/Alg + NSCs [7d] group, NSCs were mounted and cultured on the GelMA/Alg hydrogel scaffold using the NSC CM for 7 days, followed by neuronal differentiation medium (neuronal basal medium [21103-049, Gibco], 2% B27 [12,587,010, Gibco], and 2 mM Glutamax supplement [35050-061, Gibco]) for another 7 days, and the NSC-loaded GelMA/Alg was transplanted immediately after TBI. In the GelMA/Alg + NSCs [14d] group, NSCs were mounted and cultured on the GelMA/Alg hydrogel scaffold using a neuronal differentiation medium as mentioned above for 14 days, and the NSC-loaded GelMA/Alg was transplanted immediately after TBI. The number of cells was counted as 4 × 10⁶ before seeding and culturing on the hydrogel scaffold. The degree of differentiation of NSCs in the different groups was evaluated by immunostaining for Nestin (ab6142, Abcam, Cambridge, MA, USA) and MAP-2 (4542, Cell Signaling Technology, Danvers, MA, USA). GelMA/Alg hydrogel scaffolds or NSC-loaded GelMA/Alg hydrogel scaffolds (size: 4.5 mm × 4.5 mm × 2 mm) were transplanted orthotopically at the injury site immediately after the impact (illustrated in Fig. S10C). The rats were observed for 28 days after TBI.

2.8. Other methods

The other methods were provided in the supporting information, including loading of NSCs on hydrogel scaffold, scanning electron microscopy (SEM) of hydrogel scaffolds and NSC-loaded hydrogel scaffolds, physical characteristics of hydrogel scaffolds, measurement of CSF velocity, HE staining, behavioral tests, immunofluorescence staining, western blotting, and evaluation of brain tissue loss.

2.9. Statistical analyses

Statistical analyses were performed using GraphPad Prism software (version 8). Two-tailed Student's t-test was used to evaluate the difference in means between two groups. One-way analysis of variance (ANOVA) was applied to compare the differences in means among three or more groups. Two-way repeated ANOVA was used to evaluate the differences in means across three or more groups over time. Bonferroni or Tukey post hoc tests were used for pairwise comparisons when significant differences were observed in the ANOVA. All data are expressed as mean ± standard error of the mean. Two-tailed Pearson correlation analyses were used for linear regression analysis. Statistical significance was set at P < 0.05. ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001. n. s. indicates no statistical significance.

3. Results

3.1. Influence of CSF flow on cell retention in NSC transplantation after TBI

During our clinical work, the formation of a cavity at the lesion site after hematoma clearance was detected in some patients diagnosed with TBI (Fig. 1A and Figs. S1A–B). Thereafter, the cavity developed into a larger CSF-containing space in the chronic phase (Fig. 1A and Fig. S1B). To confirm this finding, a rat model of TBI was constructed as previously described [26]. At 3 days after TBI, T2 magnetic resonance imaging (MRI) scan showed the formation of an obvious cavity, with its signal similar to that of CSF (Fig. 1B and Fig. S2). Consistent with the observation in the patients with TBI, the cavity became larger and filled with CSF 28 days after TBI (Fig. 1B and Fig. S2). This cavitary environment has been reported to impede stem cell-mediated tissue repair after brain injury [27] with CSF as an important component of this cavity. However, the influence of CSF dynamics on cell retention in NSC transplantation after TBI requires elucidation.

Therefore, we constructed an in vitro bionic CSF flow model to simulate CSF flow after TBI (Fig. 1C–E). The bionic model consisted of three main parts: a pump as the power system, flask for liquid infusion and air venting, and flux chamber with a concave-shaped inner chamber for cell or scaffold holding (Fig. 1C–E). First, the CSF flow speed in different brain areas was calculated by analyzing T2 MRI scanning data from five patients with TBI (Fig. 1F) whose detailed information was included in Table S1. The CSF velocities in the aqueduct of the midbrain (AM), cerebellar cortex (CC), cerebral cortex-frontal lobe (CC–F), and cerebral cortex-temporal lobe (CC-T) were 7.416 ± 2.487 mm/s, 1.517 ± 0.6902 mm/s, 0.6246 ± 0.4548 mm/s, and 0.4422 ± 0.2476 mm/s, respectively (Fig. 1G and Table S1). As most patients with TBI exhibit cerebral cortex damage and NSC transplantation is suitable in the cortex, the average CSF flow speed in the cerebral cortex was selected as the flow speed for the in vitro bionic CSF flow model. Firstly, isolated NSCs expressed typical NSC marker nestin and had the neuronal differentiation ability (Fig. S3). Next, to determine the influence of CSF flow on the cell retention rate of the transplanted cells, NSCs were seeded in the inner chamber coated with poly-L-lysine to help with cell adhesion (Fig. 1C–E and 1H) and then the cells were flushed in the model for 24 h. Results of the in vitro flush assay demonstrated that the cell loss rate relative to pre-flush gradually increased from 0 to 24 h after flush (Fig. S4), few NSCs ere visualized in the post-flush images, and the number of NSCs spheres was significantly decreased (Fig. 1H and I). Thus, these results suggest that CSF flush is an important factor affecting the cell retention rate in NSC transplantation after TBI.

3.2. Characterization of different hydrogel scaffolds

Recent evidence in material science has shown that the advancements in hydrogels allows them to be used to provide extracellular matrix and structural support [28] as well as serve as a carrier for stem cell transplantation [29], which may provide resistance to the CSF flush and thereby increase cell retention and improve functional recovery after TBI. To identify an efficient hydrogel, we compared three different types of hydrogel scaffolds reported recently, which are suitable for cell loading: 10% gelatin and 1% sodium alginate [22] (Gelatin/Alg1), 2.8% gelatin and 2% sodium alginate [23] (Gelatin/Alg2), and 10% gelatin-methacrylate (GelMA) and 1% sodium alginate [5] (GelMA/Alg). All hydrogels could be customized in shape and organized by 3D printing technology (Fig. 2A). Next, the characteristics of the 3D-printed GelMA/Alg, Gelatin/Alg1, and Gelatin/Alg2 scaffolds were compared (Fig. 2B–G). All scaffolds were printed as a multi-porous structure (Fig. 2B and C), which was conducive to cell adherence, migration, growth, and vascularization for energy supply [25]. The size of the scaffolds was moderately enlarged after hydration in a conditioned medium for 24 h (Fig. 2B). The pore size of the GelMA/Alg hydrogel scaffold was smaller than that of the other two scaffolds during dehydration and significantly increased after hydration (Fig. 2B and D). Compared to the dehydrated condition, the column width after hydration was similar in the Gelatin/Alg1 scaffold, slightly enlarged in the Gelatin/Alg2 scaffold, and significantly enhanced in the GelMA/Alg hydrogel scaffold (Fig. 2C and E). Next, the rheological properties of hydrogels were measured using a rotational rheometer. The energy store and dissipation in the hydrogel are estimated by storage modulus (G′) and loss modulus (G″), respectively. As shown in Fig. 2F, all hydrogels showed a general trend that G′ was higher in low temperature than G″, and those two parameters decreased with the rise of temperature. Besides, the plateaued storage modulus were 5907 ± 453.3, 485.0 ± 24.27, and 553.3 ± 50.05 (Pa) in Gelatin/Alg1, Gelatin/Alg2, and GelMA/Alg, respectively (Fig. 2G). As shown in Fig. 2H, the scaffold displacement was lower in Gelatin/Alg2 and GelMA/Alg than in Gelatin/Alg1 under the same mechanical force, indicating that the GelMA/Alg and Gelatin/Alg1 hydrogel scaffolds were less likely to be deformed by the compression force from the surrounding brain tissue with edema after TBI. We further measured the compressive modulus of hydrogels, and the value in gelatin/Alg1, gelatin/Alg2, and GelMA/Alg are 13.72 ± 0.3898, 35.48 ± 3.707, and 17.41 ± 2.368 (kPa), respectively (Fig. 2I), which were higher than the modulus of brain tissue. This is essential for the hydrogel scaffolds to maintain the structure of the brain. Scanning electron microscopy (SEM) of the surface showed that the Gelatin/Alg1 and Gelatin/Alg2 scaffolds were comparatively smooth, while the GelMA/Alg scaffold exhibited an interconnected porous structure (Fig. 2J), which may increase the cell adhesion capacity [24] and enhance the exchange of nutrients [30]. The controlled degradability of a biomaterial is a crucial factor in tissue repair [31]. Consequently, we detected the biodegradation of the hydrogel scaffold. Our results demonstrated that all three hydrogel scaffolds degraded gradually in both PBS and collagenase II with increasing time (Fig. S5). Compared to PBS, collagenase II results in a significantly faster degradation rate. The degradation rates of Gelatin/Alg1, Gelatin/Alg2, and GelMA/Alg in collagenase II after 72 h are 96.23 ± 1.171%, 66.80 ± 6.131%, and 95.73 ± 1.110%, respectively (Fig. S5A). In contrast, the degradation rates of Gelatin/Alg1, Gelatin/Alg2, and GelMA/Alg in PBS after 72 h are 15.49 ± 4.115%, 12.37 ± 1.118%, and 10.90 ± 1.368%, respectively (Fig. S5B).

Fig. 2 — **Characterization of hydrogel scaffold.** (A) Illustration of the generation of 3D-printed GelMA/Alg hydrogel scaffold. (B) Representative images of different hydrogel scaffolds before and after hydration. Scale bar: 200 μm. (C) Representative images showing the pores and columns of the hydrogel scaffolds. The black dash line represents the border of the pore, and the red line represents the column width. Scale bar: 500 μm. (D–E) Quantification of pore size (D) and column width (E) before and after hydration (n = 5–10/group). (F) Effect of temperature on the storage, G′, and loss, G″, modulus of the hydrogels. (G) Quantification of the storage modulus of hydrogels. (H) The displacement–strength curve of the Gelatin/Alg1, Gelatin/Alg2, and GelMA/Alg hydrogels. (I) The Compressive modulus of hydrogel scaffolds. n = 3–4/group. (J) Scanning electron microscopy (SEM) images of the hydrogel scaffolds. The red arrowhead represents small pores on the GelMA/Alg hydrogel. Scale bar: 100 μm. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

3.3. In vitro adhesion and neuronal differentiation abilities of NSCs on different hydrogel scaffolds

Cell-scaffold adherence is a crucial and fundamental characteristic that determines cell spreading, proliferation, differentiation, and most importantly, resistance to harsh cavitary environments, such as CSF flush. For understanding this characteristic, we determined the adhesive capacity of NSCs to different hydrogel scaffolds. Equal amount of NSCs (4 × 10⁶) were seeded on the scaffolds and co-cultured for 48 h. Few NSCs were attached to the Gelatin/Alg2 scaffold (black arrow in the middle panel of Fig. 3A), whereas a small number of NSCs were adhered to the Gelatin/Alg1 scaffold (yellow arrow in the left panel of Fig. 3A). Interestingly, most NSCs were attached to the GelMA/Alg scaffold (red arrow, Fig. 3A), and these cells were active in proliferation (Fig. S6). To further quantify the number of cells on the scaffolds, the NSC-loaded scaffolds after fixation and cryosection were stained with DAPI (a cell nucleus marker, Fig. 3B). Consistent with Fig. 3A, the GelMA/Alg scaffold demonstrated a significantly higher number of DAPI⁺ cells than the other two scaffolds (Fig. 3B and E). Furthermore, we determined whether incorporation of GelMA/Alg could enhance the cell retention rate after TBI. NSCs were labeled with DIR dye and transplanted after TBI (Fig. S7 A). The fluorescence signal of DIR-labeled NSCs declined significantly on day 3 compared to that of day 0 after TBI (Fig. S7), indicating NSCs loss as early as the acute phase of TBI. This in vivo result is consistent with the in vitro finding in Fig. 1H and 1I. In contrast, loading with GelMA/Alg hydrogel scaffold markedly reversed the loss of the fluorescence signal of DIR-labeled NSCs 3 days after TBI (Fig. S7), indicating that incorporation of NSCs with GelMA/Alg hydrogel scaffold could increase cell retention rate of NSCs after TBI. Next, we determined the differentiation ability of NSCs towards neurons on different hydrogel scaffolds. The results showed that NSCs on the GelMA/Alg hydrogel scaffold expressed a higher number of βIII-tubulin⁺ cells than those on the other scaffolds (Fig. 3C and Fig. S8C) and this result was further verified by Western blotting analysis (Fig. 3D). Besides, compared to NSCs on Gelatin/Alg1 and Gelatin/Alg2, NSCs on GelMA/Alg showed a significantly lower percentage of GFAP⁺ cells (Figs. S8A and S8B). Next, SEM was utilized to view the morphology of seeded NSCs on GelMA/Alg hydrogel scaffold. As shown in Fig. 3F, SEM images revealed a large number of NSCs attached to the GelMA/Alg hydrogel scaffold (Fig. 3F, a and b), which included fully attached and spreading cells (Fig. 3F, c) and synaptic connection-like structure (Fig. 3F, d). These results further confirmed the adhesion and differentiation capacity of NSCs on the GelMA/Alg hydrogel scaffold. Therefore, these results demonstrate that the GelMA/Alg hydrogel scaffold has a reliable ability for NSC adhesion and neuronal differentiation, indicating that it is a suitable carrier for NSCs. Thus, we selected the GelMA/Alg hydrogel scaffold for the subsequent experiments.

Fig. 3 — **Adhesion and neuronal differentiation capacity of neural stem cells (NSCs) on hydrogel scaffolds.** NSCs were seeded on hydrogel scaffolds for 48 h. (A) Representative images of NSCs on hydrogel scaffolds at 0 h and 48 h after culturing. The black arrowheads represent suspended NSC spheres. The yellow arrowheads represent NSC spheres that attached to the hydrogel scaffold. The red arrowheads represent NSC spheres with enlarged size and adherence to the GelMA/Alg hydrogel. Scale bar: 100 μm. (B) Representative images and quantification of DAPI staining of hydrogel cryosections with NSCs at 48 h after co-culturing. The purple frame represents the enlarged view of the staining. White dashed line represents the outline of hydrogels. The yellow star represents cell clusters. The purple arrowheads represent DAPI⁺ cells. Scale bar in upper panel: 1 mm; scale bar in lower panel: 200 μm. (C) Representative images of fluorescence staining of βIII-tubulin (red) and DAPI (blue). Scale bar: 300 μm. (D) Representative Western blotting bands of tubulin and quantification of the expression of βIII-tubulin among groups (n = 4/group). (E) Quantification of DAPI⁺ cells after culturing for 48 h (n = 3–4/group). (F) Representative scanning electron microscopy (SEM) images showing the growth and adherence of NSCs on the GelMA/Alg hydrogel scaffold. ROI, region of interest. (a) Overview image. Scale bar: 1000 μm. (b) Enlarged ROI 1 with the blue area representing NSC adherence to the GelMA/Alg hydrogel scaffold. Scale bar: 200 μm. (c) Enlarged ROI 2. The red area represents the initial condition of adherent NSCs. The yellow area represents the condition in which NSCs stretch out to adhere to the GelMA/Alg hydrogel scaffold. The green area shows the full adherence status of NSCs on the scaffold. Scale bar: 40 μm. (d) Representative image showing synaptic connection-like structure between two cells. Scale bar: 40 μm. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

3.4. Influence of cell differentiation degree on NSC attachment to GelMA/Alg hydrogel scaffold

The degree of differentiation is another important factor affecting cell adhesive capacity. To evaluate this aspect, we compared the cell retention rate of NSCs with different degree of differentiation, including NSCs cultured in NSC culture medium (CM) for 14 days (NSCs [0d], also defined as “non-differentiated group”), NSCs cultured in NSC CM for 7 days followed by another 7 days of culture in neuronal differentiation CM (NSCs [7d], also defined as “intermediately-differentiated group”), and NSCs cultured in neuronal differentiation CM for 14 days (NSCs [14d], also defined as “fully-differentiated group”) using the in vitro bionic CSF flow model (Fig. 4C). Firstly, we quantified and determined the differentiation degree of loaded NSCs on GelMA/Alg hydrogel scaffold using immunostaining of nestin (marker of NSC), MAP-2 (marker of mature neuron), and GFAP (marker for astrocyte). 89.14 ± 3.192%, 14.03 ± 2.727%, and 0.00 ± 0.000% of the cells in GelMA/Alg + NSCs [0d] group, GelMA/Alg + NSCs [7d] group, and GelMA/Alg + NSCs [14d] group expressed nestin; 0.00 ± 0.000%, 50.40 ± 4.412%, and 69.37 ± 6.838% of cells in GelMA/Alg + NSCs [0d] group, GelMA/Alg + NSCs [7d] group, and GelMA/Alg + NSCs [14d] group express MAP-2; 38.67 ± 4.712%, 20.27 ± 2.468%, and 10.99 ± 1.840% of cells in GelMA/Alg + NSCs [0d] group, GelMA/Alg + NSCs [7d] group, and GelMA/Alg + NSCs [14d] group express GFAP, respectively (Fig. S9). Next, the hematoxylin and eosin (HE) staining results pre-flush (Fig. 4A and B) showed that cells in all three groups had desired scaffold-attaching capabilities and similar cell retention rates under normal culture conditions. Next, to determine the cell retention rate under CSF flush conditions, the NSC-loaded GelMA/Alg hydrogel scaffold was placed into the CSF flush system and flushed for 24 h before DAPI staining (Fig. 4C). Interestingly, compared to the other two groups, NSCs [7d]-loaded GelMA/Alg hydrogel scaffold presented with the highest number of DAPI⁺ cells post-flush (Fig. 4D and E), suggesting that NSCs [7d] had excellent attachment capacity to the GelMA/Alg hydrogel scaffold.

3.5. NSC [7d]-loaded GelMA/Alg hydrogel scaffold reduced tissue loss and improved long-term neurological recovery after TBI

To verify the therapeutic effects of the NSC-loaded GelMA/Alg hydrogel scaffold in TBI, a controlled cortical impact (CCI) rat model was established and the NSC-loaded GelMA/Alg hydrogel scaffold was orthotopically transplanted immediately after CCI (Fig. S10). Compared to the control group (46.11 ± 5.772%), the GelMA/Alg group (41.04 ± 12.00%) showed a slight decrease while all NSC-loaded GelMA/Alg groups, including GelMA/Alg + NSCs [0d] group (30.64 ± 7.907%), GelMA/Alg + NSCs [7d] group (23.84 ± 3.876%), and GelMA/Alg + NSCs [14d] group (32.06 ± 5.383%), demonstrated a significant reduction in tissue loss 28 days after TBI, among which the tissue loss rate in the GelMA/Alg + NSCs [7d] group was the lowest (Fig. 5A–C). Generally, the CCI model induced a moderate size of tissue defect, which was significantly reduced after treatment of the NSC-loaded GelMA/Alg hydrogel scaffold (Fig. 5 A–C). Next, behavioral tests, including the beam-walking and cylinder tests, were used to evaluate the sensorimotor functions of rats after TBI (Fig. 5D and E). Treatment of GelMA/Alg without NSCs did not produce a protective effect against TBI, as beam-walking scores and asymmetric rates in the GelMA/Alg group were similar to those in the control group (Fig. 5D and E). While GelMA/Alg-loaded NSC had better functional score in the beam-walking test, in which the NSC [7d]-loaded GelMA/Alg group presented with the optimum result (Fig. 5D). Transplantation of the NSC [7d]-loaded GelMA/Alg hydrogel scaffold also lead to significantly lower asymmetric rates than the control group (Fig. 5E). Additionally, the functional score of the beam-walking test was positively correlated with the tissue loss rate on day 28 after TBI (Fig. S11A), suggesting that the beam-walking test may be a reliable indicator of tissue loss after TBI. These results confirmed that the NSC [7d]-loaded GelMA/Alg hydrogel scaffold elicited a long-term protective effect against TBI.

3.6. NSC [7d]-loaded GelMA/Alg hydrogel scaffold inhibited microglial activation and reduced neuronal death at the acute phase of TBI

NSC-loaded GelMA/Alg-induced improvement in neurological functions was detected on day 3 after TBI (Fig. 5D), indicating NSC-loaded GelMA/Alg had early protective functions against TBI. Initially, we used DiI dye to label NSCs and tracked the presence of NSCs 3 days after TBI. We demonstrated that most of the DiI-labeled NSCs situated inside GelMA/Alg hydrogel scaffold and close to the lesion border, and a few of them were found in the parenchyma (Fig. S12), indicating that the transplanted NSCs were active and tended to migrate to the lesion site during the acute phase after TBI. Neuroinflammation in the acute phase after TBI can accelerate neuronal death, resulting in worsen brain damage. As microglia is an important source of several inflammatory factors, we assessed microglial activation. Interestingly, compared to the control group, the number of Iba1⁺ microglia was significantly decreased in the NSC-loaded GelMA/Alg treatment groups (Fig. 6A and B and Fig. S13), among which NSC [7d]-loaded GelMA/Alg showed the lowest number of Iba1⁺ cells although significant difference was not reached (Fig. 6B). Next, neuronal death around the lesion border was measured by immunostaining of Tunel and NeuN. Consistently, NSC [7d]-loaded GelMA/Alg markedly decreased the number of Tunel⁺/NeuN⁺ dead neurons 3 days after TBI (Fig. 6C and D and Fig. S14). These results suggested that NSC [7d]-loaded GelMA/Alg-induced neuroprotection after TBI was partly due to the inhibition of microglial activation and the subsequent neuronal death.

Fig. 6 — **Neural stem cell (NSC)-loaded GelMA/Alg hydrogel scaffold reduces microglial activation and neuronal death 3 days after traumatic brain injury (TBI).**(A) Representative images of immunofluorescence staining of Iba1⁺ cells (red) around the lesion border. Scale bar: 100 μm. (B) Quantification of Iba1⁺ cells (n = 3/group). (C) Representative immunofluorescence images of NeuN⁺ cells (green) and Tunel⁺ cells (red) around the lesion border. Scale bar: 100 μm. (D) Quantification of NeuN ⁺/Tunel+cells (n = 3/group). (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

3.7. NSC [7d]-loaded GelMA/Alg hydrogel scaffold exerted long-term protective effects after TBI

We next determined the long-term protective effects of NSC [7d]-loaded GelMA/Alg hydrogel scaffold against TBI. Firstly, we used DiR dye to label NSCs and tracked the presence of NSCs 28 days after TBI. The result demonstrated that the radiant efficiency on day 28 relative to day 0 is 11.64 ± 1.988% (Fig. S15), suggesting the presence of NSCs at the lesion site. To further locate these NSCs, we used DiI dye to label NSCs and found that DiI-labeled NSCs were present in the brain parenchyma 28 days after TBI. Significantly, some of those DiI⁺ cells expressed neuronal marker NeuN (Fig. S16), indicating that a few of the transplanted NSCs differentiated into neurons at the chronic phase of TBI. Neurons are the fundamental units of the brain and play an essential role in the performance of sensorimotor functions. An increased number of neurons has been reported to be closely associated with improved neurological functions after brain injury [32]. Thus, we first determined neuronal loss after TBI using NeuN staining, a marker for adult neurons. The number of NeuN⁺ neurons in the cortex (CTX) was higher in the GelMA/Alg group than in the control group (Fig. 7A and B), although a significant difference was not observed. The loading of NSCs further enhanced the neuronal protection effect of GelMA/Alg, as demonstrated by the increased number of NeuN⁺ neurons in all NSC-loaded groups, among which the GelMA/Alg + NSC [7d] group showed the highest effect. Similarly, the GelMA/Alg + NSC [7d] group had a significantly higher number of NeuN⁺ neurons in the striatum (STR) than did the control group (Fig. 7B). Additionally, immunofluorescence staining of doublecortin (DCX) was performed to evaluate the immature neurons, which demonstrated that compared to the control and GelMA/Alg groups, the GelMA/Alg + NSC [0d] and GelMA/Alg + NSC [7d] groups had drastically increased number of DCX⁺ immature neurons at the lesion border 28 days after TBI (Fig. 7C–E and Fig. S17). However, the loading of NSCs [14d] with GelMA/Alg did not have a significant promotive effect on the number of DCX⁺ immature neurons. Glial scars are formed by the development of astrocytes after brain injury and create a physical barrier for the extension of neuronal connections. Next, we investigated whether these NSC transplantation treatments influence astrogliosis. The results demonstrated that among the five groups, the number of GFAP⁺ astrocytes was the lowest in the GelMA/Alg + NSC [7d] (Fig. 7F and G and Fig. S18). Additionally, angiogenesis is a key factor for effective behavioral recovery post-NSC intracerebral implantation [33,34]. Thus, we evaluated angiogenesis ability in different groups. Interestingly, we found that GelMA/Alg + NSCs [7d] showed the highest number of CD31⁺EdU⁺ cells at the lesion border 28 days after TBI among the three groups (Fig. S19), indicating the angiogenesis is improved in GelMA/Alg + NSCs [7d] group. Therefore, our results suggest that the transplantation of NSC [7d]-loaded GelMA/Alg decreased the loss of neurons, promoted the production of new neurons, increased angiogenesis, and reduced the formation of glial scars in the chronic phase of TBI.

Fig. 7 — **Neural stem cell (NSC)-loaded GelMA/Alg hydrogel scaffold reduces neuronal loss, increased immature neurons, and decreased astrogliosis 28 days after traumatic brain injury (TBI).**(A) Representative images of immunofluorescence staining of NeuN (red) around the border of the impact region. The dash line represents the injury border. CTX, cortex; STR, striatum. Scale bar: 100 μm. (B) Quantification of NeuN⁺ neurons in the CTX and STR. The upper left panel represents the image of the full coronal section scanning. IL, ipsilateral side; CL, contralateral side. The yellow dash line denotes the border of the CTX in the CL, while the white dash line represents the border of the STR in the CL. Region of interest (ROI)-C and ROI-S represent the ROIs at the CTX and STR, respectively. Scale bar: 2 mm, n = 5–8/group. (C) Representative images of doublecortin (DCX) staining around the border of the impact region. The dash line represents the injury border. Scale bar: 25 μm. (D) Representative overview images of DCX staining at the injury border 28 days after TBI. The dash line represents the injury border. The purple arrowhead indicates an accumulated DCX-positive area. Scale bar: 500 μm. (E) Quantification of DCX⁺ cells (n = 5–8/group). (F) Representative images of GFAP staining at the injury border 28 days after TBI. The dash line represents the injury border. Scale bar: 50 μm. (G) Quantification of GFAP fluorescence intensity (n = 5–8/group). (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

To determine the relationship between the behavior results and the pathological indicators (NeuN, DCX, and GFAP), correlation analyses were performed. The number of NeuN⁺ cells in CTX and STR was negatively correlated with the beam-walking score on day 28 after TBI (Figs. S20A and S20C). While no correlation was detected between the number of NeuN⁺ cells and asymmetric rate from the cylinder test (Figs. S20B and S20D). Furthermore, the number of DCX⁺ cells was negatively correlated with the beam-walking score and asymmetric rate (Figs. S20E and S20F); the number of GFAP⁺ cells was positively correlated with the beam-walking score and asymmetric rate (Figs. S20G and S20H). These results are consistent with the view that GelMA/Alg + NSC [7d]-induced neurofunctional recovery after TBI was associated with increased number of mature neurons, newborn neurons, and reduced astrogliosis.

4. Discussion

The number of people with TBI has astonishingly reached more than 50 million per year globally [35,36]. Accelerated research has led to the reduction in TBI mortality rate via advancements in neurocritical care and acute neurosurgical interventions [37]. The huge disease burden on patients with TBI has also affected their families and society. Additionally, the heterogeneity of TBI has hindered many clinical trials of treatment agents [38]. Early research has demonstrated that stem cell therapy improves recovery of cognitive and motor functions [39,40]; however, more recent evidence has suggested that transplanted cells exhibit poor survival in the physiological or pathological microenvironment of TBI, which presents with hostile conditions such as CSF disorders [41,42]. Under normal circumstances, CSF functions to maintain brain homeostasis, acts as a hormone and metabolite carrier [10], and obeys the laws of fluid mechanics. Clinical surgical intervention for TBI often involves evacuation of the subdural or intraparenchymal lesions, which inevitably disrupts CSF compartments as well as CSF dynamics (Fig. 1A). However, few studies have explored the influence of CSF flow on stem cell transplantation therapy in TBI. We suspect that transplanted cells may be fluxed away by the restoration of CSF dynamic, resulting in insufficient residual cells. Thus, in this study, we employed a bionic system that can simulate CSF circulation and dynamics in vitro (Fig. 1C–E). Using the CSF MRI data of patients with TBI, computer modeling analysis provided a CSF fluxing speed to test NSC-loaded GelMA/Alg in vitro (Fig. 1F). The CSF velocities in the AM, CC, CC-F, and CC-T were 7.416 ± 2.487 mm/s, 1.517 ± 0.6902 mm/s, 0.6246 ± 0.4548 mm/s, and 0.4422 ± 0.2476 mm/s, respectively (Fig. 1G). The NSCs were seeded, attached onto the bottom of the inner chamber of the in vitro model and flushed. Few NSCs were still visualized in the post-flush images, whereas the NSC spheres were significantly decreased (Fig. 1H and I). To further prove the influence of CSF flow on the cell retention rate of the transplanted NSCs, DIR dye was utilized to label NSCs and the cells were transplanted after TBI. Consistent with the in vitro finding (Fig. 1H and I), our in vivo result demonstrated that the fluorescence signal of DIR-labeled NSCs was significantly decreased 3 days after TBI, suggesting NSCs loss in the acute phase of TBI, which may be caused by CSF flow. In conclusion, the above data suggest that CSF flow is a crucial factor affecting the cell retention rate of NSC transplantation after TBI.

Innovative strategies, such as stem cell transplantation, have been explored to overcome TBI. Cesar V et al. reported that bone marrow mesenchymal stem cells (BMSCs) can cross the blood–brain barrier, migrate to the site of the injured area, inhibit the inflammatory response, and protect against cell death [43]. Reynolds BA et al. showed that NSCs can differentiate and mature into both neurons and glia [44]. The proliferation and differentiation of NSCs largely depend on the neuro-microenvironment [45]. In this study, we developed an efficient 3D-printed hydrogel scaffold, GelMA/Alg, which was loaded with NSCs for TBI recovery. Compared to the Gelatin/Alg1 and Gelatin/Alg2 hydrogel scaffolds, GelMA/Alg presented with better NSCs adhesion ability (Fig. 3A, B, and 3E) and NSCs on GelMA/Alg had better neuronal differentiation capability (Fig. 3C, D, and 3F). This result is consistent with the previous finding in which Fan et al. proved that GelMA/Alg hydrogel promoted cell adhesion and differentiation of induced pluripotent stem cells (iPSCs)-derived neural stem cells (iNSCs) because GelMA is enriched with the cell-adhesive Arg-Gly-Asp (RGD) peptide [46]. Thus, we selected GelMA/Alg as the carrier for NSCs loading. As expected, loading NSCs on GelMA/Alg hydrogel scaffold reversed cell loss 3 days after TBI (Fig. S7).

Numerous studies have shown that initial cell maturity is a crucial factor affecting the therapeutic effects of NSC transplantation in different diseases of the CNS [[47], [48], [49]]. For example, Marchini et al. recently compared the neuroregenerative capacities of undifferentiated human NSC (hNSC)-loaded hydrogel group and pre-differentiated hNSC-loaded hydrogel group in a rat model of spinal cord injury (SCI) [48]. Compared to the undifferentiated group, the pre-differentiated hNSC-loaded scaffold group showed better behavioral results and cell retention rate, and higher percentage of neuronal markers at 8 weeks after SCI. Next, we determined the optimal degree of differentiation of NSCs for the treatment of TBI. In this analysis, the GelMA/Alg + NSCs [7d] group showed greater benefits than the GelMA/Alg + NSCs [0d] and GelMA/Alg + NSCs [14d] groups in terms of decreased tissue loss (Fig. 5A–C), neuronal loss (Fig. 7A and B), and astrogliosis (Fig. 7F and G), as well as increased immature neurons (Fig. 7C–E) and improved long-term neurological deficits (Fig. 5D and E). These results can be explained by several reasons. First, NSCs [0d] may differentiate into unwanted cell types, such as astrocytes, which could result in glial scarring and therefore inhibit brain repair and functional recovery after brain injury [50,51]. Second, the results of the in vitro CSF flush assay revealed that the GelMA/Alg + NSCs [7d] group had higher number of remaining cells than the GelMA/Alg + NSCs [0d] and GelMA/Alg + NSCs [14d] groups (Fig. 4C–E), suggesting that CSF flow may be an important factor in cell retention after TBI. Lastly, fully differentiated donor cells have a lower survival rate after transplantation [52,53], which may be another underlying reason for the therapeutic discrepancy between the GelMA/Alg + NSCs [7d] group and GelMA/Alg + NSCs [14d] group.

Recently, basic research and clinical studies on stem cell therapy have yielded promising results in treating TBI [54,55]. Although its mechanism is not fully understood, increasing evidence has revealed that stem cell-mediated neuroprotection against brain injury mainly comprised two processes. First, neuronal replacement/differentiation by exogenous engraftment [56] and second, the promotion of endogenous neurorestorative abilities (neurogenesis and angiogenesis) and modulation of inflammatory responses by the paracrine effects of the stem cells [57]. As neurological function is the most important indicator of prognosis after TBI, two sets of behavior tasks including the beam-walking test and the cylinder test were applied up to 28 days after TBI. Beam-walking test [58] measures vestibulomotor deficits and the cylinder test [59] assesses forepaw use and rotation asymmetry after brain injuries. In the beam walking test, we found that all NSC-loaded GelMA/Alg groups had significantly better functional scores than the control group at 3 days after TBI (Fig. 5D). However, this early protective effect could not be explained by the integration of the exogenous transplant with the host circuitry, as a relatively long time frame is required [60]. Our results demonstrated that microglial activation, which is the main source of inflammatory cytokines after brain injury [61,62], was drastically reduced by the NSC-loaded GelMA/Alg transplantation 3 days after TBI (Fig. 6A and B), along with a significant decrease in neuronal death (Fig. 6C and D). These results suggest that the NSC-loaded GelMA/Alg-promoted neurofunctional recovery at the acute stage of TBI, potentially due to their anti-inflammatory effects. Our findings are consistent with previous reports showing macrophage inhibition [63] and microglial activation after NSC transplantation for CNS injury [64]. Additionally, extracellular vesicles (EVs), which are an important component of the paracrine system, were demonstrated to play a crucial role in stem cell-mediated protection against CNS diseases. Rong et al. [65] recently reported that small EVs derived from NSCs possessed a remarkable ability to attenuate neuroinflammation after traumatic SCI. However, the association between EVs and the anti-inflammatory functions of the NSC-loaded GelMA/Alg scaffold requires further investigation. In the chronic phase of TBI, the combined anti-inflammatory abilities of the GelMA/Alg hydrogel scaffold and NSCs [7d] may have transformed the unfavorable microenvironment at the lesion site into a relatively proper milieu for engraftment/differentiation of the transplanted NSCs and endogenous neurogenesis, as demonstrated by the reduced neuronal loss (Fig. 7A and B) and increased number of DCX⁺ immature neurons (Fig. 7C–E). However, several important questions remain to be answered in future studies. First, whether the transplanted NSCs could differentiate into neurons and the contribution of this effect to the improved neurological results observed in the NSC [7d]-loaded GelMA/Alg hydrogel scaffold group. Second, whether the NSC [7d]-loaded GelMA/Alg hydrogel scaffold could stimulate and promote endogenous neurorestorative capabilities after TBI. Third, elucidation of the role of EVs, a critical factor in the paracrine pathway, in the NSC [7d]-loaded GelMA/Alg hydrogel scaffold-mediated neuroprotection.

5. Conclusion

In this study, we found that CSF flow affects cell retention of transplanted NSCs after TBI. The integration of the GelMA/Alg hydrogel scaffold with pre-differentiated NSCs [7d] can alleviate the CSF flow-induced cell loss of the transplanted NSCs. Additionally, the NSC [7d]-loaded GelMA/Alg hydrogel scaffold exhibited prominent neuroprotective functions against TBI (Fig. 8). Therefore, our study suggests NSC-loaded GelMA/Alg hydrogel scaffold as a promising alternative to treat TBI and other CNS diseases, such as stroke.

Fig. 8 — Schematic illustration of mechanism for NSCs-loaded GelMA/Alg scaffold.

Author contributions

Tiange Chen: Conceptualization, Methodology, Formal analysis, Investigation, Data curation, Writing – original draft. Yuguo Xia: Conceptualization, Methodology, Formal analysis, Investigation, Data curation, Writing – original draft, Project administration, Funding acquisition. Liyang Zhang: Conceptualization, Methodology, Formal analysis, Data curation, Writing – original draft, Project administration, Funding acquisition. Tao Xu: Resources, Project administration. Yan Yi: Resources, Data curation. Jianwei Chen: Methodology, Resources, Validation, Formal analysis. Liting Yang: Validation. Siming Chen: Data curation. Xiaoxi Zhou: Validation, Data curation, Ziyuan Liu: Validation, Investigation. Xin Chen: Investigation, Data curation. Haiyu Wu: Validation, Data curation. Jinfang Liu: Validation, Data curation, Writing – review & editing, Supervision, Project administration, Funding acquisition.

Ethical statement

All of the authors declare no conflict of interest.

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.

Acknowledgement

This work was supported by the National Natural Science Foundation of China (NSFC, No. 81402249, No. 82201543), National Postdoctoral Program for Innovative Talent (No. BX20220356), China Postdoctoral Science Foundation (No. 2022M723562), Natural Science Foundation of Hunan Province (No. 2019JJ50963, No. 2021JJ31080, No. 2020SK2070, and No. 2022JJ40828), Natural Science Foundation of Changsha City (No. kq2202377), and Young Foundation of Xiangya Hospital (No. 2021Q01).

Footnotes

^{Appendix A}

Supplementary data to this article can be found online at https://doi.org/10.1016/j.mtbio.2023.100606.

Contributor Information

Tiange Chen, Email: chentgm@163.com.

Yuguo Xia, Email: xiayuguo521@csu.edu.cn.

Liyang Zhang, Email: zhangliyang@csu.edu.cn.

Tao Xu, Email: xut@tsinghua-sz.org.

Yan Yi, Email: yanyi656@csu.edu.cn.

Jianwei Chen, Email: chenjw@tsinghua-sz.org.

Ziyuan Liu, Email: ethmery@yeah.net.

Liting Yang, Email: yanglt@csu.edu.cn.

Siming Chen, Email: usccsm@126.com.

Xiaoxi Zhou, Email: zhouxiaoxi@csu.edu.cn.

Xin Chen, Email: chenxin1983@csu.edu.cn.

Haiyu Wu, Email: why653983172@163.com.

Jinfang Liu, Email: jinfang_liu@csu.edu.cn.

Appendix A. Supplementary data

The following are the Supplementary data to this article.

Multimedia component 1

mmc1.docx^{(10.6MB, docx)}

Multimedia component 2

mmc2.pdf^{(69.2KB, pdf)}

Data availability

Data will be made available on request.

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Data Availability Statement

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PERMALINK

Loading neural stem cells on hydrogel scaffold improves cell retention rate and promotes functional recovery in traumatic brain injury

Tiange Chen

Yuguo Xia

Liyang Zhang

Tao Xu

Yan Yi

Jianwei Chen

Ziyuan Liu

Liting Yang

Siming Chen

Xiaoxi Zhou

Xin Chen

Haiyu Wu

Jinfang Liu

Abstract

Graphical abstract

Highlights

1. Introduction

2. Materials and methods

2.1. Ethics approval and informed consent

2.2. Isolation and culture of NSCs

2.3. Preparation of 3D-printed hydrogel scaffolds

2.4. In vitro CSF flush assay

Fig. 1.

Fig. 4.

2.5. Animals

2.6. Traumatic brain injury (TBI) model

2.7. Animal grouping and treatment strategy

2.8. Other methods

2.9. Statistical analyses

3. Results

3.1. Influence of CSF flow on cell retention in NSC transplantation after TBI

3.2. Characterization of different hydrogel scaffolds

Fig. 2.

3.3. In vitro adhesion and neuronal differentiation abilities of NSCs on different hydrogel scaffolds

Fig. 3.

3.4. Influence of cell differentiation degree on NSC attachment to GelMA/Alg hydrogel scaffold

3.5. NSC [7d]-loaded GelMA/Alg hydrogel scaffold reduced tissue loss and improved long-term neurological recovery after TBI

Fig. 5.

3.6. NSC [7d]-loaded GelMA/Alg hydrogel scaffold inhibited microglial activation and reduced neuronal death at the acute phase of TBI

Fig. 6.

3.7. NSC [7d]-loaded GelMA/Alg hydrogel scaffold exerted long-term protective effects after TBI

Fig. 7.

4. Discussion

5. Conclusion

Fig. 8.

Author contributions

Ethical statement

Declaration of competing interest

Acknowledgement

Footnotes

Contributor Information

Appendix A. Supplementary data

Data availability

References

Associated Data

Supplementary Materials

Data Availability Statement

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