Hydrogel with cell-cell adhesion cues enhances neural regeneration

Domain: physics
Published: 2026-01-19
Editorial outcome: major_revision

Abstract
Cell-cell adhesion is crucial for maintaining cell functions and the integrity of tissues in organisms. However, cell-cell adhesion cues have not been effectively replicated in biomaterials and the associated mechanisms that enhance neural regeneration remained largely unexplored. Here, we present a diffusive N-cadherin functionalized hydrogel system, which provided cell-cell adhesion cues to modulate intercellular communications to significantly promote the formation of active neural network via thrombospondin-1 mediated neural communication and activation of TGF-β/Smad pathway. The dynamic assembly of N-cadherin at cell-hydrogel interface driven by adhered neurons effectively facilitated the reshaping of membrane protrusion to initiate intercellular adherens junctions. Further, this hydrogel system promisingly promoted neurological function recovery in rats following traumatic brain injury. Our study provides the principle of replicating diffusive cell adhesion molecules to mediate cell-cell adhesion in hydrogels, which may have broad applications in developing engineered biomaterials aimed at modulating cell fates in regeneration of various tissues.

Introduction
Besides cell-extracellular matrix (ECM) interactions, cell-cell adhesion and communication play critical roles in maintaining cellular functions and the structural integrity of tissues in organisms1–3. Cell-cell adhesion, mediated by membrane proteins, such as adhesion proteins and receptor-ligand pairs, is essential for initiating intercellular communication that sustains tissue homeostasis and drives key physiological processes, including cellular proliferation, survival, migration, and differentiation4–7. Particularly in the central nervous system, cell-cell adhesion-dependent neuron-neuron and astrocyte-neuron interactions can facilitate synaptogenesis and the functionality of neural networks, which are significant for brain functions, including motor function, memory, and learning8–12. However, the mechanisms of cell-cell adhesion cues that enhance neural regeneration remain largely unexplored.

Cell adhesion molecules are crucial in facilitating cell-cell adhesion and recognition by bringing apposed cell membranes into contact, thereby activating tissue development or regeneration processes13. For instance, favored by the dynamic flow of the membrane, diffusive cadherin monomers can assemble side-by-side to form clusters and induce intercellular adherens junctions (AJ) for establishing effective cell-cell adhesion14–16. Recently, based on the self-assembling of the phospholipids into membranes on polymer surfaces in aqueous conditions, artificial lipid bilayers mimicking cell membranes have been developed to favor protein lateral mobility, which offer possibilities for investigating interactions of cell adhesion molecules in biological process17–19. However, the inability to adapt to three-dimensional tissue structures limits the application of such artificial systems in vivo, especially in filling and repairing irregular trauma in the brain or spinal cord. On the other hand, although cell adhesion molecules, such as N-cadherin, have also been covalently conjugated to biopolymer chains without diffusivity in recent studies, the therapeutic effect on repairing CNS injury was significantly limited, due to cell-cell adhesion significantly relies on free diffusion of N-cadherin in CNS20,21. Moreover, the biological mechanism of N-cadherin based hydrogel systems mimicking cell-cell adhesion on promoting neurological function recovery was largely unexplored (Supplementary Table 1).

In this study, we present a hydrogel system characterized by dynamic diffusion of N-cadherin to modulate intercellular communications in the central nervous system. The successful cell contact-driven assembly of N-cadherin within the hydrogel provides cell-cell adhesion cues to enhance the formation of an active neural network. The neural regrowth-enhancing effects of this system are validated across various hydrogel platforms. The results of RNA-seq and Western blot indicate that this hydrogel system could effectively inhibit apoptosis by significantly down-regulating the Bax/Bcl-2 protein level; meanwhile, the TGF-β/Smad and AKT/mTOR pathways are also activated through THBS-1-mediated neural communication by significantly up-regulating phosphorylation levels of Smad, AKT, mTOR, and S6K proteins, resulting in the promotion of neural growth. Furthermore, the hydrogel system applied to traumatic brain injury (TBI) models not only effectively promotes neural regeneration and synapse formation, but also notably inhibits microglia over-activation to hinder glial scar formation, thereby facilitating neurological function recovery in rats following TBI. This finding provides a strategy for developing materials for the treatment and regeneration of the nervous system trauma. Besides, this principle of replicating diffusive cell adhesion molecules to mediate cell-cell adhesion within hydrogels can be further used to study the role of various cell surface molecules in the development and regeneration of different tissues (Fig. 1).

Results
Preparation and characterization of lipid modified hydrogel
To prepare lipid-modified hydrogel (L@SFMA), 1 mL solution of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) and 1,2-dipalmitoyl-sn-glycero-3-phosphothioethanol (DPTE) lipid mixture was mixed with 2 mL SFMA solution (15% w/w) containing photoinitiator (0.5% w/w). Upon UV irradiation (365 nm, 30 mW/cm2), methacryloyl moieties on silk fibroin underwent photo-crosslinking, facilitating the gelation of the precursor solution (Fig. 2A and B). DPTE was conjugated to the silk fibroin chain through the addition reaction between thiol groups on DPTE and methacryloyl moieties on silk fibroin. DOPC and DPTE self-assembled within the hydrogel. To demonstrate the rapid process of photo-induced formation of fluid lipid bilayers modified hydrogel, single-distilled water or lipid solution was dripped onto the surface of the SFMA hydrogel with UV irradiation (365 nm, 30 mW/cm2). The contact angles of lipid solution droplets on SFMA hydrogels significantly decreased within 30 seconds of UV irradiation compared to those in the single-distilled water group, due to the rapid photoreaction of DPTE with silk fibroin chain and the self-assembling of lipid vesicles forming lipid bilayer coating on hydrogel, which enhanced the hydrophilicity of the L@SFMA hydrogel (Fig. 2C). The gentle decrease of contact angles in lipid solution group without irradiation maybe due to physical adsorption of vesicles at the interface by silk fibroin. To further demonstrate whether the vesicles can self-assemble to form lipid bilayer in L@SFMA hydrogel upon irradiation, fluorescence correlations spectroscopy (FCS) was used to investigate the diffusion of lipid molecules. The FCS autocorrelation curve indicated that the movement of lipid molecules in the L@SFMA hydrogel conformed to a two-dimensional (2D) diffusion law22,23, demonstrating that the self-assembled lipids uniformly distributed within L@SFMA hydrogel (Fig. 2D). Three-dimensional reconstruction fluorescent images revealed that the red fluorescence dye-labeled lipids were uniformly distributed within the L@SFMA hydrogel (Fig. 2G). The scanning electron microscope (SEM) images demonstrated that L@SFMA hydrogel held interconnected microporous structures. Notably, the L@SFMA hydrogel exhibited a wrinkled surface of microporous structures (indicated by yellow pentagrams) and none lipid vesicles were found, due to the lipid bilayer uniformly distributed within L@SFMA hydrogel, while the SFMA hydrogel displayed a flat surface, indicating the successful lipid bilayer loading (Fig. 2E). The pore size of L@SFMA hydrogel was 146.3 ± 3.842 μm, providing ample space for cell growth and elongation24 (Fig. 2F). Furthermore, the introduction of lipids into the hydrogel system did not significantly affect the storage modulus (G’) and loss modulus (G”) of the hydrogel (Fig. 2H–K). The efficient photocuring process (gelation time of less than 5 min) enabled the hydrogel system to effectively fill irregular damage25. Additionally, the L@SFMA hydrogel exhibited a relatively low storage modulus of approximately 0.1 kPa, which was closely to that of brain tissue (0.1–1 kPa)26, indicating its potential application in treating traumatic brain injury.

Diffusive N-cadherin in NL@SFMA hydrogel
Cell-cell adhesion significantly relies on free diffusion of adhesion molecules based on dynamic flow of the membrane27. In our L@SFMA hydrogel system, we hypothesized that the diffusivity of lipids within the hydrogel could be modulated by altering the ratio of DPTE (non-mobile lipids, conjugated to the hydrogel network) and DOPC (mobile lipids). Therefore, lipid samples were labeled by Texas Red-DHPE and the diffusivity of lipids with various ratios (DPTE/(DPTE+DOPC): 0%, 5%, 50%, 100%) in the hydrogels was evaluated by fluorescence recovery after photobleaching (FRAP) assay. As shown in Fig. 3A and B, hydrogels containing 0% or 5% DPTE exhibited desirable fluorescence recovery after photobleaching within 20 min, indicating that lipids maintained diffusive in hydrogels containing 5% DPTE. As the content of DPTE increased to 50% or 100%, the diffusivity of lipids in hydrogels was decreased dramatically, due to the covalent binding of DPTE to the silk fibroin chain, which restricts lipid movement. Because the interaction between the lipid bilayer and the hydrogel network within the hydrogel system containing 0% DPTE was unstable, the L@SFMA hydrogel containing 5% DPTE was selected for further study.
Next, in order to equip hydrogel with cell-cell adhesion cues, N-cadherin, a transsynaptic adhesion protein, was linked to 1,2-dioleoyl-sn-glycero-3-[(N-(5-amino-1-carboxypentyl) iminodiacetic acid succinyl] (DGS-nta) via nickel-coupled His tags and introduced to L@SFMA hydrogel system through self-assembling to obtain a diffusive N-cadherin functionalized hydrogel (NL@SFMA hydrogel). Within the membrane, N-cadherin monomer can freely diffuse with the dynamic flow of the interfaces and form intercellular adhesion junctions in intercellular interactions15. Therefore, N-cadherin was labeled by Alexa Fluor™ 488 and the diffusivity of N-cadherin in NL@SFMA hydrogels was evaluated by FRAP assay. Compared to N-cadherin directly conjugated in hydrogel network (N-SFMA group), N-cadherin was freely diffusive in NL@SFMA hydrogels, which may further facilitate N-cadherin-mediated adhesive roles and intercellular interactions (Fig. 3C and D). To further evaluate the stability of this coating, NL@SFMA hydrogels were immersed in PBS for 7 days followed by fluorescence bleaching recovery assays. It was found that N-cadherin in the NL@SFMA hydrogels still maintained effective diffusivity even after 7 days, indicating the high stability of this hydrogel supports subsequent studies in biological environments, including those in the presence of cells (Supplementary Fig. 1A, B).

Cell-cell adhesion cues in hydrogel promote neural growth
N-cadherin plays a crucial role in neural development and regeneration by facilitating processes such as neuronal migration, axonal elongation, and synapse formation28. Diffusive N-cadherin on the cell surface can assemble side-by-side to form clusters via cadherin-cadherin interaction to induce cell-cell adherens junction and initiate cell-cell contact (Fig. 3E), which allows cells to migrate, differentiate, mature, and ultimately to form neural circuits29. Therefore, to demonstrate whether the diffusive N-cadherin in NL@SFMA hydrogel can form clusters at membrane protrusions to promote neuronal growth, N-cadherin in NL@SFMA hydrogel was labeled by Alexa Fluor™ 488 and PC12 cells were seeded on the hydrogel. As shown in Fig. 3F and G, N-cadherin at the cell membrane-hydrogel interface assembled into clusters. Within 30 min of incubation, dynamic formation and disassembly of N-cadherin clusters occurred, coinciding with the reshaping of membrane protrusions that facilitate neuronal migration, due to free diffusion of N-cadherin in NL@SFMA hydrogel (Fig. 3H, I and Supplementary Movie 1). Furthermore, to investigate the capacity of NL@SFMA hydrogel on promoting axonal elongation, primary cells from cerebral cortex of E16 Sprague-Dawley rat pups were seeded on SFMA, L@SFMA, N-SFMA or NL@SFMA hydrogels respectively for 3 days, and the neural cytoskeleton was labeled with NF200 antibody at 1, 2, 3 days. Attributed to cell-cell adhesions mediated by N-cadherin, neurons cultured on the N-SFMA and NL@SFMA hydrogels showed a significant increase in neurite length, which was more rapid than previously reported30,31. More importantly, compared to neurons in the N-SFMA groups, the NL@SFMA group exhibited significantly enhanced axonal elongation and improved intercellular connections between neurons during 3 days of incubation (Fig. 4A–C), indicating that the hydrogel with diffusive N-cadherins can better promote axonal elongation and neural communication. In addition, neurons in the NL@SFMA group migrated farther with a wider range of direction (Fig. 4D, E and Supplementary Movie 2–5). To further demonstrate the validity of NL@SFMA hydrogel in enhancing synaptic activity and neuronal communication, electrophysiological recordings were employed to investigate synaptic transmission between neurons32. Spontaneous excitatory postsynaptic currents (sEPSCs) were observed in autaptic cultures of cortical neurons grown on NL@SFMA hydrogels for 14 days (Fig. 4H–I). The frequency (Fig. 4J), rather than the amplitude (Fig. 4K), of sEPSCs was significantly increased compared to that of neurons cultured on N-SFMA hydrogels. This indicates that the NL@SFMA hydrogel, which incorporates diffusive N-cadherin, can effectively enhance synaptic activity and neuronal communication primarily by increasing either the presynaptic release probability or the number of synapses, rather than altering postsynaptic receptor function. It has been reported that the initial neurite outgrowth is typically determined by N-cadherin localization following cell division, and increased concentration of N-cadherin at one pole of the neuron can result in neurite formation33. Therefore, the enrichment of diffusive N-cadherin in NL@SFMA provided cell-cell adhesion cues that enhance neuronal migration, neurite sprouting, and neural communication, thereby benefiting the formation of the neural network. Moreover, it was known that the central nervous system (CNS) has a limited ability to regenerate after injury, due to the pathophysiological challenges in axonal regeneration and the reconstruction of functional connections between neurons34. Therefore, to verify the effect of this hydrogel system (NL@SFMA) on promoting the axonal regeneration of cortical neurons, SND450 neural culture plates were used to isolate neuronal cytoplasm and axons to mimic the scenario of axonal rupture after TBI. The results of axonal regeneration assay revealed that the NL@SFMA hydrogels exhibited a significantly stronger capacity for promoting axonal regeneration compared to other groups (Fig. 4F–G).
In order to further demonstrate the three-dimensional application of the hydrogel platform, PC12 cells were encapsulated in the hydrogel system and stained after 5 days of 3D cultured. The results showed that PC12 cells could survive normally in the hydrogel following 5 days of 3D culture, and almost no dead cells were observed. Moreover, the length/width ratio of PC12 cells in the NL@SFMA group was significantly higher than that in other groups, indicating that the NL@SFMA hydrogel can successfully promote the axonal growth of PC12 cells in 3D culture (Supplementary Fig. 1C-D). Next, the effect of cell-cell adhesion cues on enhancing neural growth in other commonly used light-responsive hydrogel platforms was also determined. As shown in Supplementary Fig. 2, significantly improved neurite outgrowth was observed in neurons cultured on various light-responsive hydrogels containing diffusive N-cadherin, such as gelatin, alginate, hyaluronic acid based hydrogel platforms, indicating that the principles of replicating diffusive cell adhesion molecules mediated cell-cell adhesion in hydrogels can be broadly applied to various biomaterials and tissue outcomes.

Biological mechanisms of NL@SFMA hydrogel in promoting neural growth
Next, the underlying biological mechanisms by which NL@SFMA hydrogel promotes neurite growth and neural communication were investigated using RNA-Seq analyses (Supplementary Fig. 3). A Gene Ontology (GO) Chord plot for clustering enrichment analysis revealed significantly upregulated biological processes, including synapse organization, neuron migration and cell-cell adhesion, as well as downregulated process of mitochondrial apoptosis in primary cells from the cerebral cortex of E16 Sprague-Dawley rat pups cultured on NL@SFMA hydrogel after 24 h of incubation (Fig. 5A). The proteins Bax and Bcl-2, members of the Bcl-2 family, play critical roles in regulating apoptosis by modulating mitochondrial membrane permeability35. Bax is a pro-apoptotic protein and Bcl-2 is a protein inhibiting apoptosis36. A significantly reduced ratio of Bax to Bcl-2 expression was observed in cells of the NL@SFMA group, indicating that NL@SFMA, with diffusive N-cadherin, effectively inhibited cell apoptosis by regulating mitochondrial activity (Fig. 5B and C). To further explore mitochondrial activity, mitochondrial membrane potential fluorescence staining assay and ATP content measurements were conducted. The results demonstrated that cells in the NL@SFMA group exhibited more intact mitochondria and produced greater amounts of ATP compared to other groups, suggesting that NL@SFMA hydrogel inhibits cell apoptosis by sustaining mitochondrial function (Fig. 5D–F).
Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis indicated the activation of the transforming growth factor-β (TGF-β) signaling pathway via thrombospondin-1 (THBS-1) in cells cultured on the NL@SFMA hydrogel for 24 h, compared to those cultured on the N-SFMA hydrogel with fixed N-cadherin (Fig. 5A). THBS-1, a member of the thrombospondin family, can act as an activator of latent TGF-β by the interaction of a specific sequence in THBS-1 (K412RFK415) with the latent TGF-β complex37. THBS-1 is also a synaptogenic factor that mediates cell-cell communications in the brain by engaging with membrane receptors to promote synaptogenesis38. The significantly upregulated expression of THBS-1 and TGF-β were confirmed in cells cultured on the NL@SFMA hydrogel through quantitative real-time polymerase chain reaction (qPCR) assays (Fig. 5G). To verify whether the effect of NL@SFMA hydrogel on promoting neurite growth is mediated by activating THBS-1-associated cell-cell communication, primary cortical cells were cultured on the NL@SFMA hydrogel with different concentrations of Gabapentin (an antagonist of THBS-1 receptor α2δ-1 on neurons38) or LSKL (Leu-Ser-Lys-Leu peptide, a competitive antagonist of THBS-139). As shown in Supplementary Fig. 4, the effect of NL@SFMA hydrogel on promoting neurite growth was significantly attenuated by 30 μM Gabapentin or LSKL. Therefore, 30 μM Gabapentin (Gab) was chosen for use in subsequent experiments as a THBS-1 inhibitor. To further eliminate the interference of off-target effects of inhibitors, the THBS-1 gene in neurons were knocked down using siRNA and then cultured on NL@SFMA hydrogels. As shown in Supplementary Fig. 5, the length of neurites in siTHBS-1 group was significantly reduced, which further confirms that the effect of the NL@SFMA hydrogel on promoting neurite growth was mediated by activating THBS-1-associated cell-cell communication. Given that the classical TGF-β/Smad pathway is crucial for neuronal growth and synaptogenesis40,41, the state of Smad2, Smad3, and Smad4 within this pathway was investigated. The phosphorylation of Smad2 and Smad3 was significantly activated, and the expression of Smad4 proteins was notably increased in cells cultured on the NL@SFMA hydrogel; these effects were attenuated by Gabapentin (Fig. 5H–J). Furthermore, it is well known that the AKT/mTOR signaling pathway plays a crucial role in axonal regeneration42,43 and KEGG enrichment analysis also indicated the activation of the AKT/mTOR signaling pathway in cells cultured on the NL@SFMA hydrogel for 24 h. Therefore, to further explore the underlying biological mechanisms by which NL@SFMA hydrogel promotes axonal regeneration, we investigated the status of hub molecules associated with this signaling axis. AKT is a serine/threonine-specific protein kinase highly expressed in the nervous system. Phosphorylation of Thr308, Thr309, and Thr305 sites in the kinase domain, as well as Ser473, Ser474, and Ser472 sites in the regulatory domain, can lead to complete activation of AKT. Western blot results showed that compared to the SFMA, L@SFMA, and N-SFMA groups, the expression levels of AKT and pAKT in the NL@SFMA group were significantly upregulated, with the phosphorylation level of Ser473 site (Fig. 5K) being significantly higher. mTOR (mammalian target of rapamycin protein) is one of the important downstream targets of the AKT signaling pathway. Activated AKT further phosphorylates mTOR, which participates in biological events such as neuronal development and differentiation, axonal regeneration, and synaptic plasticity by regulating overall or local protein synthesis. While studying the phosphorylation level of mTOR, we further investigated activation of its downstream signaling molecule, ribosomal protein kinase S6K. Western blot results showed that compared to the SFMA, L@SFMA, and N-SFMA groups, the pmTOR/mTOR ratio (Fig. 5L) and pS6K/S6K ratio (Fig. 5M) were significantly upregulated in the NL@SFMA group. These results indicated that NL@SFMA hydrogel can activate AKT/mTOR signaling pathway to promote axon regeneration. These results indicate that NL@SFMA hydrogel with diffusive N-cadherin can provide cell-cell adhesion cues to facilitate cell vitality and promote THBS-1 mediated neural communication and neuronal growth by activating TGF-β/Smad and AKT/mTOR pathway.

NL@SFMA hydrogel exhibits favorable biocompatibility
Prior to in vivo application, the cytocompatibility and hemocompatibility of the SFMA, L@SFMA, and NL@SFMA hydrogels were assessed. PC12 cells were seeded on hydrogels, respectively, and the cell viability was evaluated by CCK8 assay. Cells in all three hydrogel groups exhibited normal proliferation and good cell viability during 3 days of incubation. Meanwhile, all of these hydrogels displayed a hemolysis ratio of less than 1% (Supplementary Fig. 6A–C). These results indicate that these hydrogels held good blood compatibility and cytocompatibility.

NL@SFMA hydrogel enhance traumatic brain injury repair
Dysregulated interregional neural communication is a critical issue in traumatic brain injury (TBI)44. NL@SFMA hydrogel has been shown to enhance neuronal migration, neurite outgrowth, and neural communication in vitro by providing cell-cell adhesion cues. Therefore, the capacity of this hydrogel on facilitating neurological function recovery in TBI model was further examined. A TBI model was established in Sprague-Dawley (SD) rats using a free fall striking device by the modified Feeney’s free-fall method applied to the center of the right parietal bone (parameters: an impact depth of 2 mm, a weight of 40 g, and a height of 20 cm)45. All TBI rats were divided into four groups: the control group (treated with normal saline), the SFMA group (treated with SFMA hydrogel), the L@SFMA group (treated with L@SFMA hydrogel), and the NL@SFMA group (treated with NL@SFMA hydrogel) (Fig. 6A). To evaluate the neurological functional recovery, the modified neurological severity score (mNSS) and rotarod test were performed on days 1, 3, 7, 14, and 21 post treatment46. As shown in Fig. 6B and C, the mNSS scores in each group exhibited a decreasing trend over time. Notably, rats in the NL@SFMA group exhibited significantly lower mNSS scores compared to the other three groups on days 21 and 28 post treatment. Additionally, in the rotarod test, while the duration for which rats remained on the rotarod increased progressively across all groups following TBI, rats in NL@SFMA group achieved a significantly longer duration compared to the other groups on days 21 and 28 post treatment. These results indicate that NL@SFMA hydrogel effectively promoted the recovery of neurological deficits and motor dysfunction following TBI.
Traumatic brain injury (TBI) is known to cause a range of neuropsychiatric sequelae, including deficits in learning, memory, and cognitive function. Consequently, the Morris water maze (MWM) experiment was employed to assess long-term neurological recovery following TBI, and the typical movement trajectories of each group of rats were shown in Fig. 6D. Rats of the NL@SFMA group demonstrated a significantly higher frequency of visits to the target quadrant and spent more time exploring this area compared to other groups (Fig. 6E–H), demonstrating that the NL@SFMA hydrogel significantly improved learning and memory abilities during neurological recovery following TBI24.
At 28 days post treatment, the rats were sacrificed to evaluate the integrity of the brain tissue. Larger cavities were observed in the somatosensory and visual cortex of the right hemisphere in the Control, SFMA, and L@SFMA groups; however, the NL@SFMA group exhibited a significant reduction in cavity size (Fig. 7A and Supplementary Fig. 6E). Similarly, H&E staining revealed numerous defects in the Control, SFMA and L@SFMA groups; while in the NL@SFMA group, the injured areas were filled with a substantial number of regenerated cells, indicating brain tissue regeneration (Fig. 7B).
Microglia are a type of glial cell and the main immune cells in the central nervous system45. When injury occurs, microglial activation can be divided into two processes. Firstly, microglia polarize toward the pro-inflammatory (M1) phenotype, producing pro-inflammatory cytokines such as TNF-α and expressing high levels of inducible nitric oxide synthase (iNOS) for NO production. Then, microglia polarize into the M2 phenotype, expressing anti-inflammatory cytokines (IL-4, IL-10, TGF-β and etc) to promote injury recovery. However, overactivated M1 microglia can further activate reactive astrocytes by secreting factors, causing the formation of glial scars that impair recovery47,48. In order to explore the effect of these hydrogel systems on regulating microglia polarization to inhibit glial scar formation, BV2 cells were cultured on 4 types of hydrogels and observed via immunofluorescence staining. The results showed that the M2/M1 fluorescence intensity ratio in the NL@SFMA group was significantly higher than that in the other groups (Supplementary Fig. 7A, B), indicating that the NL@SFMA hydrogel can promote the polarization of BV2 cells to the M2 type. Meanwhile, qPCR results also showed that the NL@SFMA hydrogel promoted the expression of M2-related genes (IL-4 and TGF-β) but inhibited the expression of M1-related genes (IL-1β, IL-6, TNF-α) in BV2 cells (Supplementary Fig. 7C). Moreover, in vivo experiments, immunostaining assays were conducted to characterize microglial polarization in the injured area at 7 days post-treatment. Images of immunofluorescent staining for iNOS (M1 phenotype) and CD206 (M2 phenotype) revealed that the M2/M1 fluorescence intensity ratio was significantly higher in the injured area of the NL@SFMA group (Fig. 7C and D). Further immunostaining assay was conducted to characterize the regenerated cells in the injured area at 28 days post treatment. Images of immunofluorescent staining for GFAP and Tuj1 revealed a large number of Tuj1 positive neurons in the injured area of the NL@SFMA group, indicating the regeneration of dense neural networks following treatment (Fig. 7E and F). Increased astrogliosis can lead to glial scars and secondary neuronal damage following TBI49. The control group exhibited significant reactive astrocyte scarring and hippocampal tissue damage, whereas the NL@SFMA group showed significantly less reactive astrocyte scarring around the injured sites, further facilitating neurological function recovery (Fig. 7G). These results indicated that the NL@SFMA hydrogel holds a strong capacity to inhibit microglia overactivation to modulate the inflammatory microenvironment and glial scar formation, thereby facilitating neural regeneration.
Subsequently, to investigate the effect of hydrogel treatment on brain tissue apoptosis, we collected brain tissues from TBI rats at 3 days post-treatment to extract proteins and detect the expression of apoptosis-related proteins. Western blot results were consistent with those from the in vitro experiments, which showed that TBI rats treated with NL@SFMA hydrogel had significantly reduced Bax/Bcl-2 expression (Fig. 8A and 7B). Meanwhile, TUNEL staining also showed that there were significantly fewer apoptotic positive cells in the NL@SFMA group than in other groups (Fig. 8C–E). These results all proved that treatment with NL@SFMA hydrogel can effectively inhibit brain tissue apoptosis in TBI rats. Previous studies have indicated that THBS-1 may be beneficial for maintaining blood-brain barrier (BBB) integrity in the early phase and facilitating functional recovery in the later phase following traumatic brain injury (TBI)50. Given that the NL@SFMA hydrogel can promote THBS-1 medicated intracellular communication to facilitate synapse formation by providing cell-cell adhesion cues, the expression of THBS-1 was investigated in vivo at 7 and 28 days post treatment. As shown in Fig. 8F–H, the expression of THBS-1 in regenerated tissue significantly increased in NL@SFMA group, even at the early stage of 7 days post treatment, indicating that NL@SFMA hydrogel with cell-cell adhesion cues enhanced THBS-1-mediated intracellular communication to facilitate neural regeneration. The formation of synapses is crucial for establishing precise neural networks and neuronal signal transmission in the brain, and N-cadherin-mediated cell-cell adhesion can facilitate synaptogenesis51,52. To further investigate the capacity of cell-cell adhesion cues of NL@SFMA hydrogel in promoting synaptogenesis, immunofluorescent staining assays for growth-associated protein-43 (GAP43, a marker for budding axons) and synapsin I (SYN, a marker for synaptogenesis) were performed at 28 days post treatment. The density of GAP43 positive and SYN-positive cells was significantly higher in the regenerated sites of the NL@SFMA group compared to other groups, indicating that this hydrogel with cell-cell adhesion cues effectively induced synaptic formation in the regenerated neural network (Fig. 8I-K).
Here, we presented a diffusive N-cadherin-equipped hydrogel system that provides cues for cell-cell adhesion, thereby modulating intercellular communications to promote the formation of active neural networks following neural injury. The dynamic assembly of N-cadherin at the cell membrane-hydrogel interface occurred on a timescale of minutes, facilitating the reshaping of membrane protrusions that initiate intercellular adherens junctions and significantly enhance long-term cell-cell communication. Although this research primarily focuses on N-cadherin and neural regeneration, the principles of replicating diffusive cell adhesion molecules to mediate cell-cell adhesion in hydrogels may shed light on the development of engineered biomaterials aimed at modulating various cell fates in the regeneration of diverse tissues, as well as investigating the roles of other cell surface molecules across different organs during development.

Methods
Preparation of vesicle solutions
Solutions of 25 mg/mL DOPC (1,2-dioleoyl-sn-glycero-3-phosphocholine, Avanti, AL, USA), 25 mg/mL DPTE (1,2-dipalmitoyl-sn-glycero-3-phosphothioethanol sodium salt, Avanti, AL, USA), and 0.01 mg/mL DGS-nta (1,2-dioleoyl-sn-glycero-3-[(N-(5-amino-1-carboxypentyl)iminodiacetic acid) succinyl] nickel salt, Avanti, AL, USA) were prepared in chloroform. The solutions were combined in varying quantities to achieve DPTE:(DOPC+DPTE) at molar ratios of 0%, 5%, 50%, or 100%. The lipid mixtures were dried under nitrogen gas, and excess chloroform was removed by vacuum drying. The lipid mixtures were then rehydrated in 1 mL of rehydration buffer (1 mM sucrose-sodium bicarbonate solution) to a concentration of 1 mg/mL. The mixture was allowed to swell at 75 °C for 2 h and subsequently extruded through a 50 nm polycarbonate membrane (Whatman® Nucleopore Track-etched membrane, Avanti Polar Lipids, AL, USA).

Preparation of hydrogels
Raw silk fiber was obtained from Xianjida Cocoon Silk Products Co., Ltd. (Nantong, China). Following previously established procedures,53 the silk fiber was degummed in a sodium carbonate (Na2CO3) solution (0.5% w/w) (Macklin, China) for 30 min, repeated three times to effectively remove sericin proteins. A total of 10.0 g of degummed silk was dissolved in 50 mL of lithium bromide solution (9.3 M, Meryer, China) at 60 °C and stirred for 30 min. Subsequently, 2.0 mL of methacrylic anhydride was gradually added to the mixture, which was stirred for an additional 3 h and then dialyzed against distilled water for 3 days at 4 °C to yield a methacrylated silk fibroin (SFMA) solution.
SFMA hydrogel: The SFMA solution was concentrated in air at 4 °C to a concentration of 10% w/w, and then mixed with 0.5% w/w photoinitiator (2-hydroxy-1-(4-(hydroxyethoxy)phenyl)-2-methyl-1-propanone, RGACURE 2959, CIBA Chemicals, Basel, Switzerland). The mixture was irradiated with ultraviolet light (365 nm, 30 mW/cm2) for 5 min to obtain SFMA hydrogel.
SFMA hydrogel: 10 μg/mL N-cadherin was added to the above SFMA hydrogel pre-solution, then irradiated with ultraviolet light for 5 min to obtain N-SFMA hydrogel.
L@SFMA hydrogel: 1 mL vesicle solution prepared previously containing DOPC and DPTE in various ratios was mixed with 2 mL SFMA solution (15% w/w) containing photoinitiator (0.5% w/w). The mixture was irradiated with ultraviolet light (365 nm, 30 mW/cm2) for 5 min to obtain L@SFMA hydrogel.
NL@SFMA hydrogel: 30 μg of N-cadherin (His-tag) was dissolved in 1 mL lipid mixture solution containing DOPC:DPTE:DGS-nta (95:5:0.1). The lipid mixture solution was mixed with 2 mL SFMA solution (15% w/w) containing photoinitiator (0.5% w/w) and irradiated with ultraviolet light (365 nm, 30 mW/cm2) for 5 min to obtain NL@SFMA hydrogel.

Scanning electron microscope observation
A field emission scanning electron microscope (SEM, Hitachi S-3400N II, Tokyo, Japan) was employed to observe the internal architecture of the hydrogels. A representative freeze-dried sample from each group (SFMA and L@SFMA) was selected for imaging. After gold sputtering at a working voltage of 30 kV, images were captured. The pore diameter was calculated using ImageJ software (National Institutes of Health, Bethesda, MD, USA).

Rheological properties
The photocuring times of the hydrogels were assessed by rheologically determining the sol-gel transition, also referred to as the gel point, which is defined as the moment when the storage modulus (G’) exceeds the loss modulus (G”). Time sweeps were conducted at 20 °C using a Haake MARS rotational rheometer (Thermo Fisher Scientific) with P20 parallel plates and a 1.0 mm gap (CD mode), at a strain of 150%, a frequency of 1 Hz, and under UV irradiation (365 nm, 180 mW/cm²). The final moduli of the hydrogels were also determined based on the results of the rheological tests.

Photo-induced formation of lipid bilayer within the hydrogel
To demonstrate the rapid photo-reaction of DPTE with silk fibroin chain and the self-assembling of the small vesicles forming lipid bilayer on hydrogel, contact angles of single-distilled water and a lipid solution droplet on SFMA hydrogel with/without UV irradiation were recorded using a JY-PHA instrument (Chengde Jinhe Instrument Manufacturing Co. Ltd., Chengde, China). The contact angle for each sample was determined as the average value from three independent experiments.

Fluorescence Recovery after Photobleaching (FRAP) measurements
To facilitate analysis of the samples using fluorescence microscopy, lipid samples were labeled with fluorescent dye (Texas Red-DHPE or NBD-PE), or N-cadherin was labeled with Alexa Fluor™ 488. FRAP measurements were conducted using an Axioscope 5 (Carl Zeiss, Oberkochen, Germany) or a DMI3000B (Leica, Wetzlar, Germany). The initial state was recorded prior to bleaching, after which a designated region was bleached by setting the laser intensity to 100%. Images were captured immediately after bleaching and subsequently at 5-minute intervals. Fluorescence intensity was quantified using ImageJ software (National Institutes of Health, Bethesda, MD, USA).

Stability of NL@SFMA hydrogels
The NL@SFMA hydrogels with Alexa Fluor™ 488 labeled N-cadherin were immersed in PBS solution refreshed every 24 h at room temperature. Then the fluidity of N-cadherin was detected by FRAP measurement at day 0, 1, 3, 5, and 7.

Cell culture
PC12 cells (obtained from the Cell Bank of the Chinese Academy of Sciences, TCR9, China) were seeded at a density of 8000 cells/mL/well on the surfaces of NL@SFMA hydrogels with Alexa Fluor™ 488 labeled N-cadherin in DMEM (11965092, Gibco, USA) supplemented with 10% fetal bovine serum (10099-141C, Gibco, USA) and 1% penicillin-streptomycin liquid (PS, C0224, Beyotime, China). A Laser confocal microscopy LSM 900 with Airyscan 2 (Carl Zeiss, Oberkochen, Germany) was used to examine the dynamic formation and disassembly of N-cadherin clusters coinciding with the reshaping of membrane protrusions immediately after PC12 cells adhered to the hydrogels. ImageJ software was used to analyze the fluorescence intensity and cell movement.
BV2 cells (obtained from the Cell Bank of the Chinese Academy of Sciences, SCSP-5208, China) (5 × 104 cells/well) were seeded on hydrogels and cultured for 24 h in DMEM with 10% fetal bovine serum and 1% PS. Subsequently, the samples were stained with the primary antibodies iNOS (1:400, Proteintech, 18985-1-AP) and CD206 (1:400, Proteintech, 60143-1-lg), the secondary antibodies IgY Alexa Fluor 488 (1:500, Abcam, ab150113), and DAPI (1:1000, MBD0015, Sigma-Aldrich, USA), and then imaged using a fluorescence microscope.
Primary cells from the cerebral cortex of E16 SD pups (8 × 103 cells/well) were seeded on hydrogels and cultured for 3 days in Neurobasal (21103-049, Gibco, USA) with 2% B27 (17504044, Gibco, USA) and 1% PS. Subsequently, the samples were stained with the primary antibodies anti-NF200 (1:500, Proteintech, 60331-1-Ig), the secondary antibodies IgY Alexa Fluor 488 (1:500, Abcam, ab150113), and DAPI (1:1000, MBD0015, Sigma-Aldrich, USA), and then imaged using a fluorescence microscope. Subsequently, 10 neurons were randomly selected from each of the three hydrogel samples. ImageJ software was used to count the length of each neurite of these 30 neurons, and the average value was taken as the neurite length of the neuron for statistical analysis. Moreover, the ratio of neurons forming cell junctions on each hydrogel sample was calculated for statistical analysis.
After being cultured on hydrogels for 24 h, primary cells were incubated with JC-1 dye (5 μg/mL, HY-15534, MCE) at 37 °C for 30 min to observe the mitochondrial membrane potential by fluorescence microscope, and the ATP contents of them were detected using ATP assay kit (S0026B, Beyotime).
For further qPCR and Western blot analysis, 30 μM Gabapentin (HY-A0057, MCE) was used to treat primary cells to investigate the effect of THBS-1 mediated signaling pathways on neural growth, which acted as a THBS-1 inhibitor.

Microfluidic culture assay
The standard neuron device with 450 μm microgroove barrier (SND450, Xona) was used to detect the axon extension following the previous method54. The primary neurons from cerebral cortex were seeded on the somal compartment. After culturing for 7 days, the axons crossing the 450 μm-microtunel were cut off by suction. After 24 h of culture again, the regenerative axons were stained with NF200 and the total regenerative axonal length was calculated.

Three-dimensional (3D) cultured cells
For 3D cell culture, PC12 neuronal cells were mixed with the precursor solution of hydrogels with a final concentration of 1 × 106 cells mL−1. The mixture was spreaded on small round glass slides to gelate under ultraviolet light for 2 min, then placed in 24-well plates. The obtained PC12 cells encapsulated in hydrogels were cultured in DMEM supplemented with 10% FBS and 1% PS. Following the above cell culture for 5 days, live/dead staining was performed on PC12 using Calcium AM/PI (Bestbio), and imaged under confocal microscopy with ZEN 3.5 software. Images at various depths were captured for “z-stacked” compilation images.

Whole-cell Patch clamp recordings in vitro
The cultured neurons on NL@SFMA and N-SFMA hydrogels were swiftly removed and incubated in oxygenated normal ACSF (125 NaCl, 2.5 KCl, 1.25 NaHPO4, 1.3 MgSO4, 26 NaHCO3, 2.5 CaCl2, and 10 D-glucose) at a rate of 2 mL/min, maintained at 32 ± 0.5 °C. Whole-cell patch-clamp recordings were performed using borosilicate glass pipettes (Sutter Instruments), which typically exhibit a resistance of 4-6 MΩ. The internal solution employed for voltage-clamp recordings of spontaneous excitatory postsynaptic currents (sEPSCs) consisted of the following components (in mM): 125 CsMeSO3, 10 Na-phosphocreatine, 5 Tetraethylammonium chloride, 1 QX-314 Cl, 5 HEPES, 0.25 EGTA, 2 Mg ATP, and 0.5 Na GTP; the pH was adjusted to 7.25 with CsOH.
Neurons were visualized using an Olympus BX51WI microscope (Tokyo, Japan) equipped with infrared differential interference contrast optics. All images were captured with a CoolSNAP cf Photometrics camera (Roper Scientific, Rochester, NY) and displayed on a laboratory computer. Patch-clamp recordings were conducted employing an Axopatch-700B amplifier (Axon Instruments, Sunnyvale, CA), with signals transmitted to a computer via a Digidata-1440 interface (Axon Instruments) for data acquisition and analysis utilizing pClamp version 10.0 software (Axon Instruments). Whole-cell current recordings underwent low-pass filtering at 2 kHz and digitization at 10 kHz; membrane potential recordings were low-pass filtered at 5 kHz and digitized at 20 kHz. Neurons were maintained at a membrane potential of -70 mV and characterized by the injection of rectangular voltage pulses (5 mV, 50 ms) to evaluate whole-cell membrane capacitance, series resistance, and membrane resistance. Spontaneous excitatory postsynaptic currents (sEPSCs) were recorded in the presence of SR95531 (10 μM, Tocris Bioscience), which effectively blocks GABAA receptor-mediated spontaneous inhibitory postsynaptic currents (sIPSCs). Neurons exhibiting unstable series resistance or those with values exceeding 30 MΩ were excluded from the study. Additionally, neurons in which seal resistance varied by more than 30% from its initial value were also discarded from further analysis.

Inhibitor treatment
Primary cells from the cerebral cortex of E16 SD pups (8 × 103 cells/well) were seeded on NL@SFMA hydrogels, and cultured for 24 h in neuron culture medium with LSKL (L303323, Aladdin) or gabapentin (HY-A0057, MCE) of different concentrations (0, 30, 100 μM) after adhering. Subsequently, the samples were stained with NF200 and imaged by fluorescence microscope.

RNA sequencing
Primary cells cultured on different hydrogels for 24 h for the sequencing of RNA. Total RNA was extracted with Trizol (Sigma-Aldrich, USA) and assessed with NanoPhotometer® spectrophotometer (IMPLEN, CA, USA), Qubit® RNA Assay Kit in Qubit® 2.0 Flurometer (Life Technologies, CA, USA), and Agilent Fragment Analyzer 5400 system (Agilent Technologies, CA, USA). A total amount of 1.5 µg RNA per sample was used as input material for the RNA sample preparations, and the sequencing libraries were generated using NEBNext® Ultra™ RNA Library Prep Kit for Illumina® (NEB, USA). The clustering of the index-coded samples was performed on a cBot Cluster Generation System using TruSeq PE Cluster Kit v3-cBot-HS (Illumina) according to the manufacturer’s instructions. After cluster generation, the library preparations were sequenced on an Illumina NovaSeq 6000 platform, and 150 bp paired-end reads were generated.
Gene expression levels were estimated by using HTSeq v0.13.5 to count the read numbers mapped to each gene, and differential expression analysis was conducted using the DEGseq R package (1.28.0). The heatmap and volcano plot were subsequently generated to visualize the differentially expressed genes between NL@SFMA and N-SFMA groups. An adjusted P value of <0.05 and |FoldChange| ≥ 2 were used as the threshold for significance.
Subsequently, GO (Gene Ontology) analysis was employed to analyze the biological roles of core genes, while Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways analysis was employed to identify core gene signaling pathways. GO enrichment analysis was performed using clusterProfiler R package (v3.12.0), and KEGG analysis was conducted using KOBAS v3.0 software, with a significance threshold of p-value < 0.05.

Downregulation of THBS-1 by siRNA
Small-interfering RNA (siControl as the negative control and siTHBS-1) were designed by GENERAL BIOL (China). The siRNAs were transfected into primary cells from the cerebral cortex using NEPA21 efficient gene transfection instrument (NEPA GENE, Japan). 48 h after transfection, total RNA was isolated, and transfection efficiency was assessed via qPCR, as well as the cells were re-digested and inoculated onto the NL@SFMA hydrogels.
The siRNA sequences were as follows:
siControl sense, 5’-UUCUCCGAACGUGUCACGUTT-3’;
siControl antisense, 5’-ACGUGACACGUUCGGAGAATT-3’;
siTHBS-1 sense, 5’-GUAGGUUAUGAUGAGUUUAAUTT-3’;
siTHBS-1 antisense, 5’-AUUAAACUCAUCAUAACCUACTT-3’.

qPCR analysis
The TRIzol method was used to extract total RNA from primary cells or BV2 cells cultured on hydrogels for 1 day for qPCR analysis. The RNA concentration was then measured using a micro-nucleic acid protein analyzer (Thermo Scientific, USA). Subsequently, the RNA was reverse-transcribed into cDNA using the Omniscript Reverse Transcription Kit (Takara, Japan). Real-time fluorescent quantitative PCR (Applied Biosystems, USA) was then employed to evaluate gene expression of THBS1, TGF-β in primary cells, and TNF-α, IL-1β, IL-6, IL-4, IL-10 in BV2 cells. All primers were purchased from GenScript (China), with detailed sequence information provided in Supplementary Table 2.

Western blot analysis
The primary cells cultured on hydrogels for 12 or 24 h were homogenized on ice in a mixture of radioimmunoprecipitation assay (RIPA) lysis buffer (Beyotime), protease inhibitors (phenylmethanesulfonyl fluoride, PMSF, Beyotime), and phosphatase inhibitor cocktail D (Beyotime) using sonication. The cell homogenate was then centrifuged at 12,000 × g at 4 °C for 30 min, and the supernatant was collected. The protein concentration was determined using the BCA Protein Assay Kit (Beyotime). Different volumes of the mixture of RIPA, PMSF, and phosphatase inhibitor were added to the samples according to their concentration to adjust the overall sample concentration. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) sample loading buffer (Beyotime) was added to the samples, which were then boiled for 10 min. The protein samples were electrophoresed on an 8–12% SDS/PAGE gel and then electroblotted onto presoaked polyvinylidene fluoride (PVDF) membranes (Millipore). The PVDF membranes were blocked in 5% non-fat powdered milk (Beyotime) in Tris-buffered saline with Tween (TBST) for 2 h at room temperature and then separately incubated with the primary antibodies at 4 °C overnight. The PVDF membranes were washed with TBST and incubated with horseradish peroxidase-conjugated secondary antibodies for 1.5 h. After sufficient washing, the ECL chemiluminescence system (Clinx Science Instruments, China) was used to detect the immunoreactive bands. The antibody information can be found in Supplementary Table 3.

Cytotoxicity
The cytotoxicity of the hydrogels was evaluated using the Cell Counting Kit-8 (CCK-8) (Beyotime Biotechnology, China). PC12 cells were seeded in 24-well plates at a density of 50,000 cells/mL/well and incubated at 37 °C. After 4 h of incubation to allow adherence, hydrogel samples were immersed in the cell culture medium and co-cultured with the cells for 1, 2, and 3 days. And cells cultured without hydrogel samples served as the control group. At each time point, the hydrogel samples were removed, and the cell culture medium was replaced with DMEM basal medium containing CCK-8 working solution (diluted at a ratio of 1:10) for an additional 4 h of incubation. Subsequently, the absorbance of each well was measured at 450 nm using a microplate reader (ElX-800, BioTek, USA). The relative cell viability was calculated by the equation (without hydrogel samples as control):
Cell viability (%) = Asample / Acontrol × 100% (1)
Where “A” represents absorbance value, and the subscript represents its group.

Hemolysis evaluation
The hemolysis rate induced by the hydrogels was assessed according to the Chinese National Medical Products Administration (NMPA) guidelines. Specifically, a sample suspension (5 mg/mL) was prepared in saline, and 5 mL of the test sample was taken. For the negative control group, 5 mL of normal saline was added to each tube, while for the positive control group, 5 mL of distilled water was added. The samples were incubated at 37 °C for 30 min. Following this, 100 μL of preheated anticoagulant rabbit blood, diluted with physiological saline at a ratio of 1:1.25, was added to each tube and incubated at 37 °C for 1 h. Subsequently, 300 μL of supernatant was transferred to a 96-well microplate, and the absorbance was measured at a wavelength of 545 nm using a microplate reader. The hemolysis rate was calculated by the equation:
Hemolysis rate (%) = (Asample - Anegative) / (Apositive - Anegative) × 100% (2)
Where “A” represents absorbance value, and the subscript represents its group.

Animal experiments
A total of 80 Sprague-Dawley (SD) rats (females, aged 8–9 weeks, weighing 180 ± 10 g) were obtained from the Laboratory Animal Center of Nantong University. The rats were housed under controlled conditions with access to food and water ad libitum, at a humidity level of 50% and a temperature of 25 °C, during a 12-h dark-light cycle.

Traumatic brain injury (TBI) model
Traumatic brain injury (TBI) rat model was established using a free-fall striking device (ZH-ZYQ, Zhenghua Biological Instrument Equipment Co., Ltd, China) based on a modified version of the Feeney free-fall method38. Briefly, the rats were immobilized on a brain stereotaxic apparatus, and the cranial bone was exposed after After anesthetized using 0.3% pentobarbital sodium (0.3 mL/100 g). A circular bone window with a diameter of 5.00 mm was created at the center of the right parietal bone (2 mm lateral to the sagittal suture and 3 mm posterior to the coronal suture) to expose the complete dura mater. The impact parameters were set as follows: a depth of 2 mm, a weight of 40 g, and a height of 20 cm. Following the impact, the rats were randomly divided into four groups. The rats were respectively treated with saline (Control group), in situ formed SFMA, L@SFMA, or NL@SFMA hydrogel upon irradiation for 5 min to fit with the injured sites. Subsequently, the scalp was sutured and disinfected with iodophor. All procedures were conducted on a heated pad to maintain the rats’ normal body temperature. The rats were then housed in separate cages for feeding over a period of four weeks.

Modified neurological severity score (mNSS)
The motor function of SD rats was assessed on days 1, 3, 7, 14, 21, and 28 following treatment using the modified Neurological Severity Score (mNSS). Basic neurocognitive functions, including motor, sensory, and reflex capabilities, were quantified on a scale from 0 to 18 points, where a higher score indicates more severe brain damage.

Rotarod test
Three days prior to preparing the traumatic brain injury (TBI) model, rotarod training was performed to enable the rats to learn how to navigate the rotating rods. The rats underwent two trials per day, with each trial consisting of three rounds. Each training session lasted 10 min, during which the speed of the rotating rod was uniformly increased from 5 revolutions per minute (r/min) to 40 r/min over the same duration. If a rat fell off the rod during training, it was promptly placed back on the rod and allowed to continue training until the session concluded. On days 1, 3, 7, 14, 21, and 28 post-surgery, the rotarod test was administered. The settings of the rotarod apparatus during the test were consistent with those used during training. The duration of time until the rat fell from the rod was recorded, and the average score was calculated based on three trials for each rat.

Morris water maze (MWM) test
The Morris Water Maze (MWM) experiment was performed to assess the learning and memory abilities of the rats in each group following TBI. A 5+1 training protocol was employed, consisting of a positioning cruise phase lasting 5 days (from days 23 to 27 post TBI) and a space exploration phase on day 28 post TBI. During the positioning cruise phase, rats were trained to locate an underwater platform within 90 seconds from randomly assigned starting positions in four quadrants, without interruption, for 5 consecutive days. After successfully locating the platform, the rats were allowed to remain on it for 15 seconds to facilitate memorization. In contrast, rats were also guided to the platform and allowed to stay for 15 seconds. The escape latency was measured using the SLY-WMS Morris analysis system (Beijing Shuolinyuan Technology Co., Ltd, China). On day 28 post TBI, the space exploration phase was conducted by removing the platform, and rats were subjected to a probe trial lasting 90 seconds.

Histological analysis
Rats were deeply anesthetized and subsequently transcardially perfused with normal saline, followed by 4% paraformaldehyde dissolved in phosphate buffer solution. The brains were then removed and incubated in 4% paraformaldehyde for 24 h, after which gradient sucrose dehydration was performed. The brains were processed into 12 μm-thick coronal sections using a cryostat after being embedded in optimal cutting temperature (OCT) compound, followed by hematoxylin and eosin (H&E) staining.

Immunofluorescence staining
The sections were washed in phosphate-buffered saline (PBS) for 10 min and then blocked with bovine serum albumin (BSA) for 2 h at room temperature. The sections were treated with the primary antibodies (iNOS: 1:400, Proteintech, 18985-1-AP; CD206: 1:400, Proteintech, 60143-1-lg; Tuj1: 1:1000, Abcam, ab78078; GFAP: 1:1000, Abcam, ab7260; THBS1: 1:500, Proteintech, 18304-1-AP; GAP43: 1:500, Invitrogen, 33-5000; SYN: 1:1000, Proteintech, 17785-1-AP) at 4 °C overnight. After three washes with PBS, the sections were incubated with 488/CY3-conjugated secondary antibodies (1:500) at room temperature for 2 h. Following this, the sections were stained with DAPI (1:1000) for 5 min and washed three times with PBS. Images of the whole brain tissue sections were observed using a fluorescence microscope (Carl Zeiss AG, Oberkochen, Germany).

Statistical analysis
All experimental results were analyzed using ImageJ software and GraphPad Prism 10 software. Results are expressed as the mean ± standard error of the mean (S.E.M.). One-way ANOVA was used to compare means of multiple groups, n.s. indicates p > 0.05, ★ indicates p < 0.05, ★★ indicates p < 0.01, ★★★ indicates p < 0.001; whereas two-tailed, unpaired student’s t-test was used to compare means between each two groups.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "The authors developed a diffusive N-cadherin functionalized hydrogel system that effectively modulates intercellular communications by providing cell-cell adhesion cues, thereby significantly enhancing the formation of active neural networks. Both in vitro (cellular) and in vivo (rat traumatic brain injury models) studies provide compelling evidence for the therapeutic efficacy of this hydrogel system, demonstrating its advantages and underlying mechanisms. These findings hold significant implications, particularly for the development of engineered biomaterials designed to regulate cell fate in the regeneration of diverse tissues. While the study demonstrates methodological rigor, certain clarifications and minor refinements would further enhance the manuscript's quality. Therefore, I recommend acceptance after minor revisions.\nSpecific Comments:\n1. The merits of utilizing silk fibroin for scaffold development warrant further comprehensive discussion.\n2. Could you elaborate on the precise preparation methodology for the N-SFMA group that N-cadherin directly conjugated in hydrogel network?\n3. While both the methodology section and supplementary figure 3 indicate the use of two inhibitor concentrations (30 μM and 100 μM), the Western blot results exclusively present data for the NL@SFMA+Gab group treated with the inhibitor. Could you specify which concentration was employed and provide the rationale for this selection?\n4. Please maintain consistency in the expression and abbreviation of specialized terminology (eg. \"Bcl2” vs. “Bcl-2”).\n5. Please ensure the accuracy of the written content, particularly in the caption of Figure 3 (△p<0.05 compared with N-SFMA, △△p<0.01 compared with N-SFMA, △△△p<0.001 compared with N-SFMA, ☆p<0.05 compared with N-SFMA, ☆☆☆p<0.001 compared with N-SFMA).\n6. It is recommended to include a schematic diagram that delineates the design of the composite hydrogel system incorporating diffusive N-Cadherin.", "reviewer_2": "In this interesting manuscript, Tang and colleagues describe the development of a tuneable, biocompatible material that recapitulates cell-cell adhesion cues to enhance neural cell network formation. The authors highlight that most materials employed for similar applications focus on mimicking cell-extracellular matrix (ECM) cues, whereas N-cadherin-mediated cell-cell signalling provides an alternative to enhance neural network formation. This is investigated in vitro with PC12 and primary E16 cortical neurons from rat pups as well as in vivo. Overall, the article demonstrates that NL@SFMA hydrogels are a promising tool for promoting neural growth.\nHowever, several concerns should be addressed prior to accepting the study for publication:\n1. The introduction emphasizes the role of cell–cell adhesion cues in central nervous system processes and the need to recapitulate these in regenerative materials. The final paragraph introduces the hydrogel system and briefly mentions THBS-1–mediated neural communication. This section would benefit from a more comprehensive summary of the study's key findings, including a brief mention of the experimental approaches—such as RNA-seq or pharmacological inhibition—that link N-cadherin signaling to THBS-1–mediated effects.\n2. Furthermore, the novelty and broader applicability of dynamic lipid-diffusive hydrogels for tissue regeneration is only discussed in the outlook. Introducing this potential earlier in the manuscript would help the reader better contextualize the significance of this platform.\n3. Authors mention the limited application of lipid bilayers with cell-cell adhesion cues as they are limited to 2D applications, yet it remains unclear if the NL@SFMA or similar platform could be employed in 3D. It is confusing that while the scheme in Figure 1 suggests that the lipid-driven functionalisation of the underlying silk fibroin happens in 3D, then all the in vitro studies are conducted in 2D\n4. Further, the NL@SFMA hydrogel (or even the L@SFMA hydrogel) consists of lipid bilayers on polymer chains (i.e. silk fibroin). There is no mention to the stability of this coating. This is particularly relevant in biological environments and in the presence of cells. It’d be important to gather some data in this sense.\n5. Similarly, in vitro experiments employ primary rat pup and PC12cells and in vivo studies used a rat TBI model. Therefore, why were mouse fibroblasts used for cytocompatibility studies? Were similar cytocompatibility assays conducted using the rat primary neurons or PC12 cells?\n6. The authors use Gabapentin to test THBS-1 involvement, describing it as a THBS-1 inhibitor. Please provide a reference supporting this claim. Given Gabapentin’s known off-target effects (e.g., on calcium channels and neurotransmitter release), we question whether it sufficiently specific to draw conclusions about THBS-1 signaling. Authors are encouraged to use a more selective inhibitor to strengthen their mechanistic claims.\n7. The authors claim that NL@SFMA enhances intercellular communication (Fig. 2L), yet no direct recordings of synaptic activity are presented. Including functional assays—such as calcium imaging, synaptic marker immunostaining, or electrophysiological recordings— are needed to demonstrate for enhanced neuronal communication.\n8. The study highlights the importance of cell–cell adhesion cues in promoting neural network regeneration. However, it remains unclear how the proposed approach compares to existing strategies addressing the same biological challenge. The discussion should include a brief discussion on how this platform performs relative to current state-of-the-art materials would help contextualize its advantages and potential impact.\nOther minor comments include:\n9. Fig 4D is never addressed in the text and should be addressed\n10. Sample size reporting is sometimes unclear. For example, in Fig 2, ‘n=5’, is reported but it is not specified whether this refers to hydrogel replicates, cell number, neurites recorded... etc. Clarifying this across figures would improve the text.\n11. The wording and explanation of key concepts is slightly confusing, and the message would improve if these grammatical mistakes were corrected, some examples include:\na. Line 15, line 45 and so on: “cell-cell adhesion cue’ should read “cell-cell adhesion cues”\nb. Line 118 “…none vesicles were found”\nc. Line 322 “dditionaly” should be additionally, and the use of meanwhile + additionally is not necessary\nd. Line 387 medicated should be mediated\ne. Line 389 investigate should be investigated", "reviewer_3": "I co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature Communications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career Researchers who co-review manuscripts.", "reviewer_4": "This manuscript describes an N-cadherin-functionalized hydrogel (NL@SFMA) that mimics membrane-like cell-cell adhesion to promote synaptogenesis and neurological recovery after TBI. I have several major concerns:\n1.Similar hydrogel systems diffusible N-cadherin or mimicking cell-cell adhesion have already been reported to enhance neural connectivity and repair after CNS injury. The presented work does not clearly distinguish itself in design or mechanism.\n2.Mature cortical neurons in adult rats lack intrinsic regenerative ability. The described effects likely reflect synaptic remodeling or neuroprotection, not true regeneration. The authors overstate the regenerative claims.\n3.Despite proposing anti-apoptosis as a mechanism, the study lacks apoptosis validation in TBI brain tissue.\n4.The treated brains show unusually well-preserved structures without glial scar, which raises concerns about data reliability.\n5.Multiple group comparisons were analyzed using student’s t-tests instead of ANOVA, which is statistically inappropriate.\nOverall, the study lacks sufficient novelty and biological rigor to meet the publication standards of Nature Communications."}, "author_response": "Point-to-point responses on the comments\nReviewer #1 (Remarks to the Author):\nThe authors developed a diffusive N-cadherin functionalized hydrogel system that\neffectively modulates intercellular communications by providing cell-cell adhesion\ncues, thereby significantly enhancing the formation of active neural networks. Both in\nvitro (cellular) and in vivo (rat traumatic brain injury models) studies provide\ncompelling evidence for the therapeutic efficacy of this hydrogel system,\ndemonstrating its advantages and underlying mechanisms. These findings hold\nsignificant implications, particularly for the development of engineered biomaterials\ndesigned to regulate cell fate in the regeneration of diverse tissues. While the study\ndemonstrates methodological rigor, certain clarifications and minor refinements would\nfurther enhance the manuscript's quality. Therefore, I recommend acceptance after\nminor revisions.\nSpecific Comments:\n1. The merits of utilizing silk fibroin for scaffold development warrant further\ncomprehensive discussion.\nResponse: We greatly appreciate the comment. Numerous studies have shown that silk\nfibroin-based biomaterials are widely used in the field of tissue engineering—including\nbone, tendon, skin, blood vessel, and nerve repair, due to their excellent\nbiocompatibility, low immunogenicity, controllable mechanical properties,\noutstanding physicochemical properties, and biological activity1-3. In our previous\nwork, methacryloyl groups were successfully introduced into silk fibroin to obtain\nmethacrylated silk fibroin (SFMA). Double bonds on SFMA were further triggered to\ncross-link upon light to form a three-dimensional network structure and subsequently\nsolidify into gels in the presence of photoinitiator via free radical reaction. By\nincorporating bioactive molecules, the SFMA precursor solution can be delivered to the\ntarget site and cured in situ under ultraviolet light. This enables it to fill irregular\nwounds with high spatial and temporal precision, thereby promoting the regeneration\nof various tissues (e.g., spinal cord injury repair and infected skin wound healing)4, 5.\nReference\n1. Ye J, Xie B, Hu J, Xu X, Lu S, Wang J, et al. Recent advances in silk\nfibroin-based biomaterials for tissue engineering applications. International\njournal of biological macromolecules 2025, 322(pt2): 146764.\n2. Peng L, Zhou P, Liao F, Liu G, Bao S, Yang X, et al. Silk fibroin hydrogels:\nGelation mechanisms, fabrication techniques, and biomedical applications.\nInternational Journal of Biological Macromolecules 2025, 322(2): 146699.\n3. Liu Y, Zhou M, Sun J, Yao E, Xu J, Yang G, et al. Programmed BRD9\nDegradation and Hedgehog Signaling Activation via Silk-Based Core-Shell\nMicroneedles Promote Diabetic Wound Healing. Advanced Science 2024,\n11(45): 202404130.\n4. Tang X, Chen X, Zhang S, Gu X, Wu R, Huang T, et al. Silk-Inspired In Situ\nHydrogel with Anti-Tumor Immunity Enhanced Photodynamic Therapy for\nMelanoma and Infected Wound Healing. Advanced Functional Materials 2021,\n31(17): 202101320.\n5. Ling J, Huang T, Wu R, Ma C, Lin G, Zhou Z, et al. Cell Development\nEnhanced Bionic Silk Hydrogel on Remodeling Immune Pathogenesis of\nSpinal Cord Injury via M2 Polarization of Microglial. Advanced Functional\nMaterials 2023, 33(14): 202213342.\n2. Could you elaborate on the precise preparation methodology for the N-SFMA\ngroup that N-cadherin directly conjugated in hydrogel network?\nResponse: We greatly appreciate the comment. We have further provided the\npreparation methodology for the N-SFMA in methods section in the revised\nmanuscript.\n3. While both the methodology section and supplementary figure 3 indicate the use of\ntwo inhibitor concentrations (30 μM and 100 μM), the Western blot results exclusively present data for the NL@SFMA+Gab group treated with the inhibitor. Could you specify which concentration was employed and provide the rationale for this selection?\nResponse: We greatly appreciate the comment. As shown in Supplementary Figure 4,\na significant inhibitory effect on neuronal growth was observed in the group of cells\ntreated with 30 μM GAB; therefore, the concentration of 30 μM was adopted in\nsubsequent experiments.\n4. Please maintain consistency in the expression and abbreviation of specialized\nterminology (eg. \"Bcl2” vs. “Bcl-2”).\nResponse: We greatly appreciate the suggestion and we have correct it as Bcl-2 in the\nrevised manuscript.\n5. Please ensure the accuracy of the written content, particularly in the caption of\nFigure 3 (△p<0.05 compared with N-SFMA, △△p<0.01 compared with N-SFMA,\n△△△p<0.001 compared with N-SFMA, ☆p<0.05 compared with N-SFMA,\n☆☆☆p<0.001 compared with N-SFMA).\nResponse: Thank you very much for the comment and we apologize for this mistake.\nWe have further corrected the text in the caption of Figure 3 as ‘△△p<0.01 compared\nwith N-SFMA, △△△p<0.001 compared with N-SFMA, ☆p<0.05 compared with\nNL@SFMA, ☆☆☆p<0.001 compared with NL@FMA’ in the revised manuscript.\n6. It is recommended to include a schematic diagram that delineates the design of the\ncomposite hydrogel system incorporating diffusive N-Cadherin.\nResponse: We greatly appreciate the comment and we have included a schematic\ndiagram that illustrates the design and biological functions of the composite hydrogel\nsystem incorporating diffusive N-Cadherin as Scheme 1 in the revised manuscript.\nFigure 1. Schematic illustration of a hydrogel system characterized by dynamic diffusion of\nN-cadherin to modulate intercellular communications in the central nervous system.\nReviewer #2 (Remarks to the Author):\nIn this interesting manuscript, Tang and colleagues describe the development of a\ntuneable, biocompatible material that recapitulates cell-cell adhesion cues to enhance\nneural cell network formation. The authors highlight that most materials employed for\nsimilar applications focus on mimicking cell-extracellular matrix (ECM) cues,\nwhereas N-cadherin-mediated cell-cell signalling provides an alternative to enhance\nneural network formation. This is investigated in vitro with PC12 and primary E16\ncortical neurons from rat pups as well as in vivo. Overall, the article demonstrates that\nNL@SFMA hydrogels are a promising tool for promoting neural growth.\nHowever, several concerns should be addressed prior to accepting the study for\npublication:\n1. The introduction emphasizes the role of cell–cell adhesion cues in central nervous\nsystem processes and the need to recapitulate these in regenerative materials. The\nfinal paragraph introduces the hydrogel system and briefly mentions THBS-1–\nmediated neural communication. This section would benefit from a more\ncomprehensive summary of the study's key findings, including a brief mention of the\nexperimental approaches—such as RNA-seq or pharmacological inhibition—that\nlink N-cadherin signaling to THBS-1–mediated effects.\nResponse: We greatly appreciate the comment and we have modified the final\nparagraph of the introduction in the revised manuscript by providing a more\ncomprehensive summary of the study's key findings, including RNA-seq that links\nN-cadherin signaling to THBS-1-mediated effects. “In this study, we present a\nhydrogel system characterized by dynamic diffusion of N-cadherin to modulate\nintercellular communications in the central nervous system. The successful cell\ncontact driven-assembly of N-cadherin within the hydrogel provided cell-cell\nadhesion cues to enhance the formation of active neural network. The neural\nregrowth-enhancing effects of this system were validated across various hydrogel\nplatforms. The results of RNA-seq and Western blot indicated that this hydrogel\nsystem could effectively inhibit apoptosis by significantly down-regulating relative\nthe Bax/Bcl-2 protein level; meanwhile, the TGF-β/Smad and AKT/mTOR pathways\nwere also activated through THBS-1-mediated neural communication by significantly\nup-regulating phosphorylation levels of Smad, AKT, mTOR, and S6K proteins,\nresulting in the promotion of neural growth.”\n2. Furthermore, the novelty and broader applicability of dynamic lipid-diffusive\nhydrogels for tissue regeneration is only discussed in the outlook. Introducing this\npotential earlier in the manuscript would help the reader better contextualize the\nsignificance of this platform.\nResponse: We greatly appreciate the comment and we have modified the introduction\nin the revised manuscript by clarifying the novelty and broader applicability of\ndynamic lipid-diffusive hydrogels for tissue regeneration. “Furthermore, the hydrogel\nsystem was applied to traumatic brain injury (TBI) models and not only effectively\npromoted neural regeneration and synapse formation, but also notably inhibited\nmicroglia over-activation to hinder glial scar formation, thereby facilitating\nneurological function recovery in rats following TBI. This finding provides a novel\nstrategy for developing new materials for the treatment and regeneration after nervous\nsystem trauma. Besides, this principle of replicating diffusive cell adhesion molecules\nto mediate cell-cell adhesion within hydrogels can be further used to study the role of\nvarious cell surface molecules in the development and regeneration of different\ntissues (Scheme 1).”\n3. Authors mention the limited application of lipid bilayers with cell-cell adhesion\ncues as they are limited to 2D applications, yet it remains unclear if the NL@SFMA\nor similar platform could be employed in 3D. It is confusing that while the scheme in\nFigure 1 suggests that the lipid-driven functionalisation of the underlying silk fibroin\nhappens in 3D, then all the in vitro studies are conducted in 2D\nResponse: We greatly appreciate the comment. Cells were cultured on the surface of\nhydrogels to better characterize the cell features and extract RNA and proteins from\ncells for biological mechanism studies.\nIn order to further demonstrate the three-dimensional application of the hydrogel\nplatform, PC12 cells were encapsulated in the hydrogel system and stained after 5\ndays of 3D cultured. The results showed that PC12 cells could survive normally in the\nhydrogel following 5 days of 3D culture, and almost no dead cells were observed.\nMoreover, the length/width ratio of PC12 cells in the NL@SFMA group was\nsignificantly higher than that in other groups, indicating that the NL@SFMA hydrogel\ncan successfully promote the axonal growth of PC12 cells in 3D culture.\nFigure 2. A, Fluorescence images of PC12 cells after 5 days of 3D culture in hydrogels. B,\nLength/width (L/W) ratio of PC12 cells. Values represent means ± S.E.M. (n = 15 cells / 3\nhydrogels), ***p<0.001 compared with SFMA, *p<0.05 compared with SFMA, ##p<0.01\ncompared with L@SFMA, ###p<0.001 compared with L@SFMA, △p<0.05 compared with\nN-SFMA.\n4. Further, the NL@SFMA hydrogel (or even the L@SFMA hydrogel) consists of\nlipid bilayers on polymer chains (i.e. silk fibroin). There is no mention to the stability\nof this coating. This is particularly relevant in biological environments and in the\npresence of cells. It’d be important to gather some data in this sense.\nResponse: We greatly appreciate the comment. To further evaluate the stability of this\ncoating, NL@SFMA hydrogels were immersed in PBS for 7 days followed by\nfluorescence bleaching recovery assays. It was found that N-cadherin in the\nNL@SFMA hydrogels still maintained excellent diffusivity even after 7 days,\nindicating the high stability of this hydrogel supports subsequent studies in biological\nenvironments, including those in the presence of cells.\nFigure 3. Diffusivity stability of Alexa Fluor 488-labeled N-cadherin in the NL@SFMA hydrogels\nduring 7 days evaluation by FRAP assay. Values represent means ± S.E.M. (n = 3 hydrogels).\n5. Similarly, in vitro experiments employ primary rat pup and PC12cells and in vivo\nstudies used a rat TBI model. Therefore, why were mouse fibroblasts used for\ncytocompatibility studies? Were similar cytocompatibility assays conducted using the\nrat primary neurons or PC12 cells?\nResponse: We greatly appreciate the comment. To better adapt to the in vivo animal\nmodel, PC12 cells were further used to demonstrate the biocompatibility of the\nhydrogel system. PC12 cells were seeded on the hydrogels, and cell viability was\nevaluated via the CCK-8 assay. Over 3 days of incubation, cells in all three hydrogel\ngroups exhibited normal proliferation and excellent viability. These results indicated\nthat these hydrogels exhibit good cytocompatibility.\nFigure 4. A, OD at 450 nm. B, Cell viability of PC12 cells cultured with hydrogels. Values\nrepresent means ± S.E.M. (n = 5 hydrogels).\n6. The authors use Gabapentin to test THBS-1 involvement, describing it as a\nTHBS-1 inhibitor. Please provide a reference supporting this claim. Given\nGabapentin’s known off-target effects (e.g., on calcium channels and neurotransmitter\nrelease), we question whether it sufficiently specific to draw conclusions about\nTHBS-1 signaling. Authors are encouraged to use a more selective inhibitor to\nstrengthen their mechanistic claims.\nResponse: We greatly appreciate the comment. The studies reported by Naga et al.,\nCrawford et al. and Eroglu et al. showed that Gabapentin was an antagonist of\nTHBS-1 receptor α2δ-1 on neurons and was used to selectively block THBS-1\nactions1-3; Therefore, we used Gabapentin as an inhibitor in our study. According to\nyour suggestion, and we have chosen another recognized inhibitor—LSKL\n(Leu-Ser-Lys-Leu peptide), which is a competitive antagonist of THBS-14. The LSKL\npeptide can inhibit THBS-1 binding to LAP, thereby suppressing the activation of\nTGF-β pathway5. Similarly, the effect of the NL@SFMA hydrogel on promoting\nneurite growth of cortical neurons was significantly attenuated by LSKL, indicating\nthe effect of the NL@SFMA hydrogel on promoting neurite growth was mediated by\nactivating THBS-1 associated cell-cell communication. Due to time constraints, we\nhave not yet supplemented the Western blot results of the LSKL treatment group. If\nnecessary, we are more than willing to continue to continue to supplement these data.\nFigure 5. LSKL (Leu-Ser-Lys-Leu ) inhibits the growth of cortical neurons. A, The primary cells\ntreated with LSKL at different concentrations for 24 h were stained with NF-200 (green), scale bar\n= 100 μm. B, The neurite length of neurons (n = 30 neurites / 3 hydrogels). C, Ratio of neurite\nbranching (n = 6 views / 3 hydrogels). D, Ratio of cell junction (n = 6 views / 3 hydrogels). The\ndata was shown as mean ± S.E.M., *** indicates p<0.001.\nReference\n1. Nagai J, Rajbhandari AK, Gangwani MR, Hachisuka A, Coppola G,\nMasmanidis SC, et al. Hyperactivity with Disrupted Attention by Activation of\nan Astrocyte Synaptogenic Cue. Cell 2019, 177(5): 1280-1292.\n2. Crawford SE, Stellmach V, Murphy-Ullrich JE, Ribeiro SM, Lawler J, Hynes\nRO, et al. Thrombospondin-1 is a major activator of TGF-beta1 in vivo. Cell\n1998, 93(7): 1159-1170.\n3. Eroglu C, Allen NJ, Susman MW, O'Rourke NA, Park CY, Oezkan E, et al.\nGabapentin Receptor α2δ-1 Is a Neuronal Thrombospondin Receptor\nResponsible for Excitatory CNS Synaptogenesis. Cell 2009, 139(2): 380-392.\n4. Kuroki H, Hayashi H, Nakagawa S, Sakamoto K, Higashi T, Nitta H, et al.\nEffect of LSKL peptide on thrombospondin 1-mediated transforming growth\nfactor β signal activation and liver regeneration after hepatectomy in an\nexperimental model. British Journal of Surgery 2015, 102(7): 813-825.\n5. Murphy-Ullrich JE, Poczatek M. Activation of latent TGF-beta by\nthrombospondin-1: mechanisms and physiology. Cytokine & growth factor\nreviews 2000, 11(1-2): 59-69.\n7. The authors claim that NL@SFMA enhances intercellular communication (Fig. 2L),\nyet no direct recordings of synaptic activity are presented. Including functional\nassays—such as calcium imaging, synaptic marker immunostaining, or\nelectrophysiological recordings— are needed to demonstrate for enhanced neuronal\ncommunication.\nResponse: We greatly appreciate the comment and we have further demonstrated that\nNL@SFMA hydrogels enhanced synaptic activity and neuronal communication via\nelectrophysiological recordings, with a focus on investigating spontaneous excitatory\npostsynaptic currents (sEPSCs)1. When autaptic cultures of cortical neurons were\ngrown on the NL@SFMA hydrogels for 14 days, the frequency of sEPSCs was\nsignificantly increased compared to that of neurons grown on the N-SFMA hydrogels,\nindicating that NL@SFMA hydrogels with diffusive N-cadherin can effectively\nenhance synaptic activity and neuronal communication.\nFigure 6. A, Representative image of primary cultured neurons examined in this study. B, Traces\nof sEPSCs recorded from neurons cultured on NL@SFMA and N-SFMA hydrogels, respectively.\nC, The representative cumulative distributions of neuronal sEPSC intervals (upper panel) and the\ngroup data for mean frequency (lower panel) in neurons cultured on NL@SFMA and N-SFMA\nhydrogels, respectively. D, The representative cumulative distributions of neuronal sEPSC\namplitudes (upper panel) and the group data for mean amplitude (lower panel) in neurons cultured\nNL@SFMA and N-SFMA hydrogels, respectively. Values represent means ± S.E.M. (n = 10\nneurons / 3 hydrogels), two-tailed unpaired t-test, n.s. indicates p>0.05, * indicates p<0.05.\nReference\n1. Peng J-Y, Qi Z-X, Yan Q, Fan X-J, Shen K-L, Huang H-W, et al. Ameliorating\nparkinsonian motor dysfunction by targeting histamine receptors in\nentopeduncular nucleus-thalamus circuitry. Proceedings of the National\nAcademy of Sciences of the United States of America 2023, 120(17):\ne2216247120.\n8. The study highlights the importance of cell–cell adhesion cues in promoting neural\nnetwork regeneration. However, it remains unclear how the proposed approach\ncompares to existing strategies addressing the same biological challenge. The\ndiscussion should include a brief discussion on how this platform performs relative to\ncurrent state-of-the-art materials would help contextualize its advantages and potential\nimpact.\nResponse: We greatly appreciate the comment. To better clarify the novelty and\nsignificance of our design and mechanism, we have summarized studies in the\nliterature on N-cadherin-based hydrogel systems that mimic cell-cell adhesion for\nCNS injury applications. Compared to our hydrogel system with diffusible\nN-cadherin, N-cadherin were covalently conjugated to biopolymer chains without\ndiffusivity in most studies1-6, which significantly limited the therapeutic effect on\nCNS injury repair, due to cell-cell adhesion in the CNS significantly relies on free\ndiffusion of N-cadherin. Furthermore, although self-assembling phospholipid\nmembranes on 2D polymer surfaces offer possibilities for investigating interactions of\ncell adhesion molecules in biological processes7-9, their inability to adapt to\nthree-dimensional tissue structures limits the application of such systems in vivo,\nespecially in filling and repairing irregular trauma in brain or spinal cord. Moreover,\nthe biological mechanisms of N-cadherin-based hydrogel systems mimicking cell-cell\nadhesion on promoting neurological function recovery was hardly explored (Table\nS1).\nSignificance of this study:\n1. A hydrogel system with diffusible N-cadherin was successfully developed to fill\nirregular trauma upon light and modulate intercellular communication for CNS injury.\n2. Cell-cell adhesion cues provided by the hydrogel significantly enhanced axonal\nregeneration, synapse formation and neuroprotection for regenerating an active neural\nnetwork.\n3. Focusing on the mechanisms, this hydrogel system effectively inhibited apoptosis\nof neurons, and promoted axonal regeneration by activating AKT/mTOR and\nTGF-β/Smad pathways through THBS-1-mediated neural communication, resulting in\npromoting neural growth.\n4. When applied to traumatic brain injury (TBI) model, this hydrogel system not only\neffectively promoted neural regeneration and induced synapse formation, but also\npromisingly inhibited microglia over-activation to hinder glial scar formation, thereby\nfacilitating neurological function recovery in rats following TBI.\nTable S1. Summary of N-cadherin-based hydrogel systems mimicking cell-cell\nadhesion for CNS injury applications.\nImmobilization Animal\nMaterials Effects Reference\nstrategies models\nPromoting\nN-cadherin human Fc\nFibrinogen the Rat spinal\nchimeric protein\nbased aligned adhesion cord 2 mm\nimmobilized on\nfibrin and complete [1]\nhydrogel through\nnanofiber neuronal Transection\nbiochemical conjugation\nhydrogel differentia Model\nusing protein A\ntion of\nNSCs\nExtracellular domain of\nAmphiphilic N-cadherin tagged with\nCryogenic\npeptide the amphiphilic peptide Promoting\nbrain injury\n[(RADA)3-(R and selfassembled neuroblast [2]\nIschemic\nADG)] based to form an migration\nstroke\nhydrogel Ncad-mRADA\nsupramolecular hydrogel\nHyaluronic\nacid Promoting\nN-cadherin covalently Rat spinal\nderivatives, the\nconjugated to the cord 3 mm\nsodium neuronal\ncarboxyl group of Alg complete [3]\nalginate, differentia\nusing EDC and NHS as Transection\ngelatin/cellulo tion of\ncoupling agents Model\nse nanofiber NSCs\nhydrogel\nEnhancing\niPSC-deri\nN-cadherin mimic\nved\npeptides immobilized on\nneuron\nGelMA GelMA via conjugation / [4]\nmaturity\nthrough Michael\nand\naddition reaction.\nconnectivi\nty\nN-cadherin segments\ncovalently conjugated to Promoting\nthe amino sites of developme\nGelMA / [5]\nGelMA through an nt of\namide bond formation cortical\nreaction neurons\nElastin-like\nproteinvariants Maintainin\ncontaining an g neural\nNcadherin- stem cell\nderived HAVDI peptide stemness\nHyaluronic\ncovalently conjugated and / [6]\nAcid\nwith functionalized promoting\nHyaluronic Acid to form functional\nhydrogels through neuronal\ndynamic hydrazone maturation\nbonds\nReference\n1. Yang K, Yang J, Man W, Meng Z, Yang C-Y, Cao Z, et al.\nN-Cadherin-Functionalized Nanofiber Hydrogel Facilitates Spinal Cord Injury\nRepair by Building a Favorable Niche for Neural Stem Cells. Advanced Fiber\nMaterials 2023, 5(4): 1349-1366.\n2. Ohno Y, Nakajima C, Ajioka I, Muraoka T, Yaguchi A, Fujioka T, et al.\nAmphiphilic peptide-tagged N-cadherin forms radial glial-like fibers that\nenhance neuronal migration in injured brain and promote sensorimotor\nrecovery. Biomaterials 2023, 294: 122003.\n3. Li Y, Cheng S, Wen H, Xiao L, Deng Z, Huang J, et al. Coaxial 3D printing of\nhierarchical structured hydrogel scaffolds for on-demand repair of spinal cord\ninjury. Acta Biomaterialia 2023, 168: 400-415.\n4. O'Grady BJ, Balotin KM, Bosworth AM, McClatchey PM, Weinstein RM,\nGupta M, et al. Development of an N-Cadherin Biofunctionalized Hydrogel to\nSupport the Formation of Synaptically Connected Neural Networks. Acs\nBiomaterials Science & Engineering 2020, 6(10): 5811-5822.\n5. Kjar A, Haschert MR, Zepeda JC, Simmons AJ, Yates A, Chavarria D, et al.\nBiofunctionalized gelatin hydrogels support development and maturation of\niPSC-derived cortical organoids. Cell Reports 2024, 43(11): 114874.\n6. Huang MS, Lesavage BL, Ghorbani S, Gilchrist AE, Roth JG, Huerta-Lopez C,\net al. Viscoelastic N-cadherin-like interactions maintain neural progenitor cell\nstemness within 3D matrices. Nature Communications 2025, 16(1): 5213.\n7. Barcelona-Estaje E, Oliva MAG, Cunniffe F, Rodrigo-Navarro A, Genever P,\nDalby MJ, et al. N-cadherin crosstalk with integrin weakens the molecular\nclutch in response to surface viscosity. Nature Communications 2024, 15(1):\n8824.\n8. Neville GM, Dobre A-M, Smith GJ, Micciulla S, Brooks NJ, Arnold T, et al.\nInteractions of Choline and Geranate (CAGE) and Choline Octanoate (CAOT)\nDeep Eutectic Solvents with Lipid Bilayers. Advanced Functional Materials\n2024, 34(2): 2306644.\n9. Zhou X, Moran-Mirabal JM, Craighead HG, McEuen PL. Supported lipid\nbilayer/carbon nanotube hybrids. Nature Nanotechnology 2007, 2(3): 185-190.\nOther minor comments include:\n9. Fig 4D is never addressed in the text and should be addressed.\nResponse: We really appreciate the comment and we apologize for this mistake. We\nhave modified the text by addressing Fig 4D in the revised manuscript.\n10. Sample size reporting is sometimes unclear. For example, in Fig 2, ‘n=5’,\nis reported but it is not specified whether this refers to hydrogel replicates, cell number,\nneurites recorded... etc. Clarifying this across figures would improve the text.\nResponse: We greatly appreciate the comment. We have provided specific\nexplanations for the sample size of each quantitative analysis in the revised\nmanuscript.\n11. The wording and explanation of key concepts is slightly confusing, and the\nmessage would improve if these grammatical mistakes were corrected, some\nexamples include: a. Line 15, line 45 and so on: “cell-cell adhesion cue’ should read\n“cell-cell adhesion cues” b. Line 118 “…none vesicles were found” c. Line 322\n“dditionaly” should be additionally, and the use of meanwhile + additionally is not\nnecessary d. Line 387 medicated should be mediated e. Line 389 investigate should be\ninvestigated\nResponse: We greatly appreciate the commentand we apologize for this mistake. We\nhave corrected these grammatical mistakes in the revised manuscript.\nReviewer #3 (Remarks to the Author):\nI co-reviewed this manuscript with one of the reviewers who provided the listed\nreports. This is part of the Nature Communications initiative to facilitate training in\npeer review and to provide appropriate recognition for Early Career Researchers who\nco-review manuscripts.\nResponse: Thank you very much for participating in the review of our manuscript,\nand we have carefully revised and improved it.\nReviewer #4 (Remarks to the Author):\nThis manuscript describes an N-cadherin-functionalized hydrogel (NL@SFMA) that\nmimics membrane-like cell-cell adhesion to promote synaptogenesis and neurological\nrecovery after TBI. I have several major concerns:\n1. Similar hydrogel systems diffusible N-cadherin or mimicking cell-cell adhesion\nhave already been reported to enhance neural connectivity and repair after CNS injury.\nThe presented work does not clearly distinguish itself in design or mechanism.\nResponse: We greatly appreciate the comment. To better clarify the novelty and\nsignificance of our design and mechanism, we have summarized studies in the\nliterature on N-cadherin-based hydrogel systems that mimic cell-cell adhesion for\nCNS injury applications. Compared to our hydrogel system with diffusible\nN-cadherin, N-cadherin were covalently conjugated to biopolymer chains without\ndiffusivity in most studies1-6, which significantly limited the therapeutic effect on\nCNS injury repair, due to cell-cell adhesion in the CNS significantly relies on free\ndiffusion of N-cadherin. Furthermore, although self-assembling phospholipid\nmembranes on 2D polymer surfaces offer possibilities for investigating interactions of\ncell adhesion molecules in biological processes7-9, their inability to adapt to\nthree-dimensional tissue structures limits the application of such systems in vivo,\nespecially in filling and repairing irregular trauma in brain or spinal cord. Moreover,\nthe biological mechanisms of N-cadherin-based hydrogel systems mimicking cell-cell\nadhesion on promoting neurological function recovery was hardly explored (Table\nS1).\nSignificance of this study:\n1. A hydrogel system with diffusible N-cadherin was successfully developed to fill\nirregular trauma upon light and modulate intercellular communication for CNS injury.\n2. Cell-cell adhesion cues provided by the hydrogel significantly enhanced axonal\nregeneration, synapse formation and neuroprotection for regenerating an active neural\nnetwork.\n3. Focusing on the mechanisms, this hydrogel system effectively inhibited apoptosis\nof neurons, and promoted axonal regeneration by activating AKT/mTOR and\nTGF-β/Smad pathways through THBS-1-mediated neural communication, resulting in\npromoting neural growth.\n4. When applied to traumatic brain injury (TBI) model, this hydrogel system not only\neffectively promoted neural regeneration and induced synapse formation, but also\npromisingly inhibited microglia over-activation to hinder glial scar formation, thereby\nfacilitating neurological function recovery in rats following TBI.\nTable S1. Summary of N-cadherin-based hydrogel systems mimicking cell-cell\nadhesion for CNS injury applications.\nImmobilization Animal\nMaterials Effects Reference\nstrategies models\nPromoting\nN-cadherin human Fc\nFibrinogen the Rat spinal\nchimeric protein\nbased aligned adhesion cord 2 mm\nimmobilized on\nfibrin and complete [1]\nhydrogel through\nnanofiber neuronal Transection\nbiochemical conjugation\nhydrogel differentia Model\nusing protein A\ntion of\nNSCs\nExtracellular domain of\nAmphiphilic N-cadherin tagged with\nCryogenic\npeptide the amphiphilic peptide Promoting\nbrain injury\n[(RADA)3-(R and selfassembled neuroblast [2]\nIschemic\nADG)] based to form an migration\nstroke\nhydrogel Ncad-mRADA\nsupramolecular hydrogel\nHyaluronic\nacid Promoting\nN-cadherin covalently Rat spinal\nderivatives, the\nconjugated to the cord 3 mm\nsodium neuronal\ncarboxyl group of Alg complete [3]\nalginate, differentia\nusing EDC and NHS as Transection\ngelatin/cellulo tion of\ncoupling agents Model\nse nanofiber NSCs\nhydrogel\nEnhancing\nN-cadherin mimic iPSC-deri\npeptides immobilized on ved\nGelMA GelMA via conjugation neuron / [4]\nthrough Michael maturity\naddition reaction. and\nconnectivi\nty\nN-cadherin segments\ncovalently conjugated to Promoting\nthe amino sites of developme\nGelMA / [5]\nGelMA through an nt of\namide bond formation cortical\nreaction neurons\nElastin-like\nproteinvariants Maintainin\ncontaining an g neural\nNcadherin- stem cell\nderived HAVDI peptide stemness\nHyaluronic\ncovalently conjugated and / [6]\nAcid\nwith functionalized promoting\nHyaluronic Acid to form functional\nhydrogels through neuronal\ndynamic hydrazone maturation\nbonds\nReference\n1. Yang K, Yang J, Man W, Meng Z, Yang C-Y, Cao Z, et al.\nN-Cadherin-Functionalized Nanofiber Hydrogel Facilitates Spinal Cord Injury\nRepair by Building a Favorable Niche for Neural Stem Cells. Advanced Fiber\nMaterials 2023, 5(4): 1349-1366.\n2. Ohno Y, Nakajima C, Ajioka I, Muraoka T, Yaguchi A, Fujioka T, et al.\nAmphiphilic peptide-tagged N-cadherin forms radial glial-like fibers that\nenhance neuronal migration in injured brain and promote sensorimotor\nrecovery. Biomaterials 2023, 294: 122003.\n3. Li Y, Cheng S, Wen H, Xiao L, Deng Z, Huang J, et al. Coaxial 3D printing of\nhierarchical structured hydrogel scaffolds for on-demand repair of spinal cord\ninjury. Acta Biomaterialia 2023, 168: 400-415.\n4. O'Grady BJ, Balotin KM, Bosworth AM, McClatchey PM, Weinstein RM,\nGupta M, et al. Development of an N-Cadherin Biofunctionalized Hydrogel to\nSupport the Formation of Synaptically Connected Neural Networks. Acs\nBiomaterials Science & Engineering 2020, 6(10): 5811-5822.\n5. Kjar A, Haschert MR, Zepeda JC, Simmons AJ, Yates A, Chavarria D, et al.\nBiofunctionalized gelatin hydrogels support development and maturation of\niPSC-derived cortical organoids. Cell Reports 2024, 43(11): 114874.\n6. Huang MS, Lesavage BL, Ghorbani S, Gilchrist AE, Roth JG, Huerta-Lopez C,\net al. Viscoelastic N-cadherin-like interactions maintain neural progenitor cell\nstemness within 3D matrices. Nature Communications 2025, 16(1): 5213.\n7. Barcelona-Estaje E, Oliva MAG, Cunniffe F, Rodrigo-Navarro A, Genever P,\nDalby MJ, et al. N-cadherin crosstalk with integrin weakens the molecular\nclutch in response to surface viscosity. Nature Communications 2024, 15(1):\n8824.\n8. Neville GM, Dobre A-M, Smith GJ, Micciulla S, Brooks NJ, Arnold T, et al.\nInteractions of Choline and Geranate (CAGE) and Choline Octanoate (CAOT)\nDeep Eutectic Solvents with Lipid Bilayers. Advanced Functional Materials\n2024, 34(2): 2306644.\n9. Zhou X, Moran-Mirabal JM, Craighead HG, McEuen PL. Supported lipid\nbilayer/carbon nanotube hybrids. Nature Nanotechnology 2007, 2(3): 185-190.\n2. Mature cortical neurons in adult rats lack intrinsic regenerative ability. The\ndescribed effects likely reflect synaptic remodeling or neuroprotection, not true\nregeneration. The authors overstate the regenerative claims.\nResponse: We greatly appreciate the comment and we fully agree that\nthe central nervous system (CNS) has a limited ability to regenerate after injury,\ndue to the pathophysiological challenges in axonal regeneration and the reconstruction of\nfunctional connections between neurons1, 2. Therefore, to verify the effect of this\nhydrogel system (NL@SFMA) on promoting the axonal regeneration of cortical\nneurons, SND450 neural culture plates were used to isolate neuronal cytoplasm and\naxons to mimic the scenario of axonal rupture after TBI3. The results of axonal\nregeneration assay revealed that the NL@SFMA hydrogels exhibited a significantly\nstronger capacity for promoting axonal regeneration compared to other groups.\nFigure 7. A, Representative immunofluorescent images of the regenerative axons on the\nmicrofluidic culture plate (green: NF200), scale bar = 100 μm. B, Quantification of total axonal\nlength in (A). values represent means ± S.E.M. (n = 30 neurites / 3 hydrogels), ***p<0.001\ncompared with SFMA, ###p<0.001 compared with L@SFMA, △△p<0.01 compared with N-SFMA.\nFurthermore, it is well known that the AKT/mTOR signaling pathway plays a crucial\nrole in axonal regeneration4-7 and KEGG enrichment analysis also indicated the\nactivation of the AKT/mTOR signaling pathway in cells cultured on the NL@SFMA\nhydrogel for 24 hours. Therefore, to further explore the underlying biological\nmechanisms by which NL@SFMA hydrogel promotes axonal regeneration, we\ninvestigated the status of hub molecules associated with this signaling axis. AKT is a\nserine/threonine-specific protein kinase highly expressed in the nervous system.\nPhosphorylation of Thr308, Thr309, and Thr305 sites in the kinase domain, as well as\nSer473, Ser474, and Ser472 sites in the regulatory domain, can lead to complete\nactivation of AKT. Western blot results showed that compared to the SFMA,\nL@SFMA, and N-SFMA groups, the expression levels of AKT and pAKT in the\nNL@SFMA group were significantly upregulated (Figure 8A), with the\nphosphorylation level of Ser473 site (Figure 8D) being significantly higher. mTOR\n(mammalian target of rapamycin protein) is one of the important downstream targets\nof the AKT signaling pathway. Activated AKT further phosphorylates mTOR, which\nparticipates in biological events such as neuronal development and differentiation,\naxonal regeneration, and synaptic plasticity by regulating overall or local protein\nsynthesis. While studying the phosphorylation level of mTOR, we further investigated\nactivation of its downstream signaling molecule, ribosomal protein kinase S6K.\nWestern blot results showed that compared to the SFMA, L@SFMA, and N-SFMA\ngroups, the pmTOR/mTOR ratio (Figures 8B and 8E) and pS6K/S6K ratio (Figures\n8C and 8F) were significantly upregulated in the NL@SFMA group. These results\nindicated that NL@SFMA hydrogel can activate AKT/mTOR signaling pathway to\npromote axon regeneration.\nFigure 8. Representative images of western blotting assays and statistical results of pAKT/AKT\n(A, D), pmTOR/mTOR (B, E), and pS6K/S6K (C, F) protein from primary cells cultured on\nhydrogels for 12 hours, β-actin was used as a loading control. Values represent means ± S.E.M. (n\n= 5 hydrogels), *p<0.05 compared with SFMA, **p<0.01 compared with SFMA, #p<0.05\ncompared with L@SFMA, ##p<0.01 compared with L@SFMA, ###p<0.001 compared with\nL@SFMA, △p<0.05 compared with N-SFMA, △△p<0.01 compared with N-SFMA, ☆p<0.05\ncompared with NL@SFMA.\nReference\n1. He Z, Jin Y. Intrinsic Control of Axon Regeneration. Neuron 2016, 90(3):\n437-451.\n2. Zhang X, Yang C, Zhang C, Wu J, Zhang X, Gao J, et al. Functional optic\ntract rewiring via subtype- and target-specific axonal regeneration and\npresynaptic activity enhancement. Nature Communications 2025, 16(1): 2174.\n3. Qin T, Li C, Xu Y, Qin Y, Jin Y, He R, et al. Local delivery of\nEGFR+NSCs-derived exosomes promotes neural regeneration post spinal cord\ninjury via miR-34a-5p/HDAC6 pathway. Bioactive Materials 2024, 33:\n424-443.\n4. Park KK, Liu K, Hu Y, Smith PD, Wang C, Cai B, et al. Promoting Axon\nRegeneration in the Adult CNS by Modulation of the PTEN/mTOR Pathway.\nScience 2008, 322(5903): 963-966.\n5. Ma C, Wang J, Tu Q, Wu R, Lai X, Lin G, et al. Fidgetin impacts axonal\ngrowth and branching in a local mTOR signal dependent manner.\nExperimental Neurology 2023, 361: 114315.\n6. Shi Z, Mao L, Chen S, Du Z, Xiang J, Shi M, et al. Reversing Persistent\nPTEN Activation after Traumatic Brain Injury Fuels Long-Term Axonal\nRegeneration via Akt/mTORC1 Signaling Cascade. Advanced Science 2025,\n12(6): 2410136.\n7. Sarbassov DD, Guertin DA, Ali SM, Sabatini DM. Phosphorylation and\nregulation of Akt/PKB by the rictor-mTOR complex. Science 2005, 307(5712):\n1098-1101.\n4. Despite proposing anti-apoptosis as a mechanism, the study lacks apoptosis\nvalidation in TBI brain tissue.\nResponse: We greatly appreciate the comment and we have supplemented the\napoptosis validation in TBI brain tissue. To investigate the effect of hydrogel\ntreatment on apoptosis of brain tissue, we collected brain tissues from TBI rats at 3\ndays post-treatment to extract proteins and detect the expression of apoptosis-related\nproteins. Western blot results were consistent with those from the in vitro experiments,\nwhich showed that TBI rats treated with NL@SFMA hydrogels had significantly\nreduced Bax/Bcl-2 expression (Fig. 9A and 9B). Meanwhile, TUNEL staining also\nshowed that there were significantly fewer apoptotic positive cells in the NL@SFMA\ngroup than in other groups (Fig. 9C-E). These results all proved that treatment with\nNL@SFMA hydrogel can effectively inhibit apoptosis of brain tissue in TBI rats.\nFigure 9. A-B, Representative western blotting images (A) and statistical results (B) of BAX and\nBcl-2 proteins in tissues at the injury site of TBI rats treated with hydrogels for 3 days, β-actin was\nused as a loading control (n = 5 rats). C, Representative images of TUNEL (red) co-localization\nwith neurons (tuj1, green) in the brain tissue sections of rats at day 3 and 7 post-treatment. Nuclei\nwere stained with DAPI (blue). D-E, Quantitative analysis of TUNEL-positive neurons at 3 days\n(D) and 7 days (E) after TBI (n = 5 rats). Yellow squares indicate the areas enlarged, values\nrepresent means ± S.E.M., *p<0.05 compared with Control, **p<0.01 compared with Control,\n***p<0.001 compared with Control, ###p<0.001 compared with SFMA, △△△p<0.01 compared with\nL@SFMA.\n5. The treated brains show unusually well-preserved structures without glial scar,\nwhich raises concerns about data reliability.\nResponse: We greatly appreciate the comment. Microglia are a type of glial cell and\nthe main immune cells in the central nervous system1. When injury occurs, microglial\nactivation can be divided into two processes. Firstly, microglia polarize toward the\npro-inflammatory (M1) phenotype, producing pro-inflammatory cytokines such as\nTNF-α and expressing high levels of inducible nitric oxide synthase (iNOS) for NO\nproduction. Then, microglia polarize into the M2 phenotype, expressing\nanti-inflammatory cytokines (IL-4, IL-10, TGF-β and etc) to promote injury recovery.\nHowever, overactivated M1 microglia can further activate reactive astrocytes by\nsecreting factors, causing the formation of glial scars that impair recovery1-3. In order\nto explore the effect of these hydrogel systems on regulating microglia polarization to\ninhibit glial scar formation, BV2 cells were cultured on 4 types of hydrogels and\nobserved via immunofluorescence staining. The results showed that the M2/M1\nfluorescence intensity ratio in the NL@SFMA group was significantly higher than\nthat in the other groups, indicating that the NL@SFMA hydrogel can promote the\npolarization of BV2 cells to the M2 type (Fig. 10). Meanwhile, qPCR results also\nshowed that the NL@SFMA hydrogel promoted the expression of M2-related genes\n(IL-4 and TGF-β) but inhibited the expression of M1-related genes (IL-1β, IL-6,\nTNF-α) in BV2 cells (Fig. 11). Moreover, in in vivo experiments, immunostaining\nassays were conducted to characterize microglial polarization in the injured area at 7\ndays post-treatment. Images of immunofluorescent staining for iNOS (M1 phenotype)\nand CD206 (M2 phenotype) revealed that the M2/M1 fluorescence intensity ratio was\nsignificantly higher in the injured area of the NL@SFMA group (Fig. 12). These\nresults indicated that the NL@SFMA hydrogel hold a strong capacity for inhibiting\nmicroglia overactivation to modulate inflammatory microenvironment and glial scar\nformation, thereby facilitating neural regeneration.\nFigure 10. The impact of different hydrogels’ cultures on the differentiation of BV2 cells. A,\nFluorescence images of BV2 cells cultured on hydrogels for 24 hours stained with iNOS (M1, red),\nCD206 (M2, green) and DAPI (blue), scale bar = 50 μm. B) Fluorescence intensity ratio of\nCD206/iNOS. Values represent means ± S.E.M. (n = 9 views / 3 hydrogels), ***p<0.001\ncompared with SFMA, ###p<0.001 compared with L@SFMA, △△△p<0.01 compared with\nN-SFMA.\nFigure 11. TNF-α，IL-1β，IL-6, IL-4, IL-10 and TGF-β gene expression in BV2 primary cells\ncultured on hydrogels for 24 hours, 18S was used as a loading control, values represent means ±\nS.E.M. (n= 3 hydrogels), *p<0.05 compared with SFMA, **p<0.01 compared with SFMA,\n***p<0.001 compared with SFMA, #p<0.05 compared with L@SFMA, ##p<0.01 compared with\nL@SFMA, ###p<0.001 compared with L@SFMA, △△△p<0.01 compared with N-SFMA.\nFigure 12. A, Representative immunofluorescent staining images of rat brain tissue sections at day\n7 post-reatment stained with DAPI (blue), iNOS (red), and CD206 (green). B, Fluorescence\nintensity ratio of CD206/iNOS. Values represent means ± S.E.M. (n = 10 views / 5 rats),\n***p<0.001 compared with Control, ###p<0.001 compared with SFMA, △△△p<0.01 compared with\nL@SFMA.\nFigure 13. Photographs of Brain tissue of TBI rats at day 28 treatment.\nReference\n1. Liddelow SA, Guttenplan KA, Larke LEC, Bennett FC, Bohlen CJ, Schirmer\nL, et al. Neurotoxic reactive astrocytes are induced by activated microglia.\nNature 2017, 541(7638): 481-487.\n2. Bellver-Landete V, Bretheau F, Mailhot B, Vallieres N, Lessard M, Janelle\nM-E, et al. Microglia are an essential component of the neuroprotective scar\nthat forms after spinal cord injury. Nature Communications 2019, 10: 518.\n3. Li Y-F, Ren X, Zhang L, Wang Y-H, Chen T. Microglial polarization in TBI:\nSignaling pathways and influencing pharmaceuticals. Frontiers in Aging\nNeuroscience 2022, 14: 901117.\n6. Multiple group comparisons were analyzed using student’s t-tests instead of\nANOVA, which is statistically inappropriate.\nResponse: Thank you very much for the suggest. We have performed ANOVA for\nmultiple group comparisons and Student’s t-tests for pairwise comparisons for all\nquantitative data.\nPoint-to-point responses on the comments\nReviewer #1 (Remarks to the Author):\nThe authors revised the manuscript with sufficient discussion and supplemented data.\nI'm satisfied with the revision.\nResponse: Thank you very much for your comments on our manuscript, which has\nallowed us to significantly improve the quality of the work\nReviewer #2 (Remarks to the Author):\nTang and colleagues have performed revisions and clarifications of their manuscript\nthat describes a diffusive N-Cadherin functionalized hydrogel that mimics cell-cell\nadhesion cues to promote neural cell network formation. The new version of the\nmanuscript incorporates several additions, including schematic illustrations, expanded\ndiscussions of rationale and additional functional assays. However, the study still lacks\nanalytical depth and experimental rigor to substantiate its claims.\n1. Authors claim that the novelty of their work lies within the diffusivity of N-Cadherin\nin their system as well as describing a potential mechanism for neurological function\nrecovery post injury. Meanwhile, authors have cultured primary cells from the cerebral\ncortex of E16 for 24h on the compared hydrogel systems, which is a limited in-vitro\ntimepoint that can’t support claims. Authors should include more time points to follow\nthe evolution of the network formation on the hydrogels. Additionally, from the images\nprovided and the described methodology, it is also unclear on how neurites were\nidentified, analyzed and quantified. As other studies report slower neurite growth in\n24h i.e. ~ 40um at 24h in cortical neurons (Riquelme et al., 2024) or ~20um in SVZ\nneurons (Khodosevich and Monyer, 2010).\nResponse: We greatly appreciate the comment and we have performed in vitro cell\nculture experiments with more time points to follow the evolution of the network\nformation on the hydrogels.\nTo follow the evolution of the network formation, the primary cells from the cerebral\ncortex of E16 were cultured on the hydrogel systems for 3 days, aand the neural\ncytoskeleton was labeled with NF200 antibody at 1, 2, 3 days (Revised Figure 3A-C).\nSubsequently, 10 neurons were randomly selected from each of the three hydrogel\nsamples. Image J software was used to count the length of each neurite of these 30\nneurons, and the average value was taken as the neurite length of the neuron for\nstatistical analysis. Moreover, the ratio of neurons forming cell junctions on each\nhydrogel sample was calculated for statistical analysis. Attributed to cell-cell adhesions\nmediated by N-cadherin, neurons cultured on the N-SFMA and NL@SFMA hydrogels\nshowed significant increase in neurite length, which was more rapid than previously\nreported30, 31. Moreover, compared to neurons in the N-SFMA groups, the NL@SFMA\ngroup exhibited significantly enhanced axonal elongation and improved intercellular\nconnections between neurons during 3 days of incubation, indicating that the hydrogel\nwith diffusive N-cadherins can better promote axonal elongation and neural\ncommunication.\nFigure 3. Effect of NL@SFMA hydrogel on promoting neural growth. A, Fluorescence images\nof primary cells from cerebral cortex cultured on hydrogels for 3 days stained with NF200 (green),\nscale bar = 50 μm. B, The neurite length of neurons (n = 30 neurites / 3 hydrogels). C, Ratio of cell\njunction (n = 3 hydrogels). Results in B, C were analyzed by unpaired t-test, **p<0.01 compared\nwith SFMA, ***p<0.001 compared with SFMA, ##p<0.01 compared with L@SFMA, ###p<0.001\ncompared with L@SFMA, △p<0.05 compared with N-SFMA, △△p<0.01 compared with N-SFMA;\nanalyzed by One-way ANOVA, ★★★ indicates p<0.001.\nReference\n30. Sloley, S.S., Main, B.S., Winston, C.N., et al. High-frequency head impact causes\nchronic synaptic adaptation and long-term cognitive impairment in mice.\nNature Communications 2021, 12: 2613.\n31. Riquelme, D., Juanchuto-Viertel, N., Álamos, C., et al. TRPM4 inhibition slows\nneuritogenesis progression of cortical neurons. Mol Brain 2024, 17: 66.\n2. We appreciate the authors performing functional assays to sustain their claims,\nproviding new experimental data on spontaneous excitatory postsynaptic currents\n(sEPSCs). While NL@SFMA hydrogels increased the frequency of sEPSCs, results\nreported in Fig 3, H (amplitude) are never discussed, which weakens the interpretation\nof enhanced neuronal communication.\nResponse: We greatly appreciate the comment and we have further improved the\ncaption and added discussions on sEPSCs in the revised manuscript. “To further\ndemonstrate the validity of NL@SFMA hydrogel in enhancing synaptic activity and\nneuronal communication, electrophysiological recordings were employed to investigate\nsynaptic transmission between neurons32. Spontaneous excitatory postsynaptic currents\n(sEPSCs) were observed in autaptic cultures of cortical neurons grown on NL@SFMA\nhydrogels for 14 days (Fig 3H-I). The frequency, rather than the amplitude, of sEPSCs\nwas significantly increased compared to that of neurons cultured on N-SFMA\nhydrogels. This indicates that the NL@SFMA hydrogel, which incorporates diffusive\nN-cadherin, can effectively enhance synaptic activity and neuronal communication\nprimarily by increasing either the presynaptic release probability or the number of\nsynapses, rather than altering postsynaptic receptor function.”\n3. The new scheme added to the introduction and the additional paragraph have made\nit easier for the reader to contextualize the work. Still, the description of the scheme\nshould be improved so that the reader can better interpret the image.\nResponse: We greatly appreciate the comment and we have modified the description of\nthe scheme 1 in the revised manuscript. “Scheme 1. Design and proposed mechanism\nof the diffusive N-cadherin functionalized hydrogel system for neural regeneration.\nLipid bilayer containing N-cadherin was introduced to methacrylated silk fibroin\nhydrogel (SFMA) through photo-crosslinking and self-assembling to obtain a diffusive\nN-cadherin functionalized hydrogel system (above). This hydrogel system successfully\nprovided cell-cell adhesion cues to enhance the formation of active neural network\nthrough Thrombospondin-1 (THBS-1) mediated neural communication and activation\nof TGF-β/Smad pathway (middle). Furthermore, this hydrogel system promisingly\npromoted neurological function recovery in rats following traumatic brain injury\n(below).”\n4. Sample size and statistical analysis remain elusive, how many cells were used to get\nneurite tracings, what statistical test was employed to obtain the reported p values?\nEven if a statement was added to respond to Reviewer 5, comment 6, it is still unclear\nwhether authors have used t-tests to perform pairwise comparisons between group\nconditions.\nResponse: We greatly appreciate the comment. For statistical analysis, unpaired t-tests\nwere used to perform comparisons between two groups in our initial manuscript. We\nadded ANOVA for comparisons between multiple groups to address the comment—\nReviewer 4’s note that “Multiple group comparisons were analyzed using student’s t-\ntests instead of ANOVA, which is statistically inappropriate”. And we have further\nprovided specific explanations on sample size and statistical analysis in the captions of\neach statistical chart in the revised manuscript.\n5. We appreciate that the authors incorporated a new inhibitor for THBS-1 (LSKL\npeptide) and provided supporting references for the use of Gabapentin, which\nappropriately justify its use. However, the results of these additional experiments\nshould be more fully discussed to clarify how they strengthen the proposed mechanism.\nOur original concern aimed to question the specificity of the pharmacological\ninhibition—whether the reduction in neurite extension observed upon inhibitor\ntreatment truly reflects THBS-1 blockade or could instead arise from off-target effects\nof the compounds used.\nResponse: We greatly appreciate the comment. To eliminate the interference of off-\ntarget effects of inhibitors, the THBS-1 gene in neurons were knocked down using\nsiRNA (siTHBS-1) and then the neurons were cultured on NL@SFMA hydrogels. As\nshown in Supplementary Fig. 5 of the revised manuscript, the length of neurites in the\nsiTHBS-1 group was significantly reduced compared to those in siControl group, which\nfurther confirmed the effect of the NL@SFMA hydrogel on promoting neurite growth\nwas mediated by activating THBS-1 associated cell-cell communication.\nSupplementary Fig. 5 Knockdown of THBS-1 by siRNA inhibits the growth of cortical neurons. A,\nQuantification of THBS-1 gene expression by quantitative real-time PCR (n = 3 repeats). B, The\ncortical neurons were cultured on NL@SFMA hydrogels for 24 hours following siTHBS1\ntransfection, then stained with NF-200 (green), scale bar = 100 μm. C, The neurite length of neurons\n(n = 30 neurites / 3 hydrogels). The data was shown as mean ± S.E.M., *** indicates p<0.001 by\nunpaired t-test.\n6. In lines 405-408: ‘All TBI rats were divided into four groups: the control (…) Fig\n5A’ The animal groups are not shown in figure 5A, as it describes the in vivo\nexperimental timeline.\nResponse: We greatly appreciate the comment. And we have made improvements to\nFigure 5A by adding descriptions of animal grouping in the revised manuscript.\nFigure 5. Neurological functional recovery of TBI rats treated with hydrogels. A, Experimental\ntimeline of procedures of in vivo experiments.\nOverall, while the study is ambitious and has improved in response to reviewer’s\nfeedback, it remains descriptive rather than mechanistically rigorous. The range of data\nis impressive, with a significant part of the manuscript dedicated to the implantation of\nthe hydrogel in vivo which is practically useful but adds little to the conceptual novelty\nand mechanistic understanding of the new hydrogel. In summary, in our opinion the\nnarrative remains diffuse, and key experiments such as neurite quantification,\nstatistical validation, inhibitor specificity are not developed enough to support some of the\nauthor’s conclusions.\nReviewer #3 (Remarks to the Author):\nI co-reviewed this manuscript with one of the reviewers who provided the listed reports.\nThis is part of the Nature Communications initiative to facilitate training in peer review\nand to provide appropriate recognition for Early Career Researchers who co-review\nmanuscripts.\nResponse: Thank you very much for participating in the review of our manuscript, and\nwe have carefully revised and improved the quality of the work.\nReviewer #4 (Remarks to the Author):\nThe authors have addressed my questions and concerns in the revised manuscript. I\nappreciate their careful responses and the changes made. I have no further issues.\nResponse: Thank you very much for your comments on our manuscript, which has\nallowed us to significantly improve the quality of the work"}, {"round": 2, "reviewer_comments": {"reviewer_2": "Authors have addressed all the pending concerns and questions. The manuscript has significantly improved in clarity and rigor. No further questions or concerns from our side.", "reviewer_3": "I co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature Communications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career Researchers who co-review manuscripts."}, "author_response": ""}]

Decision letter