Multiplexed self-adaptable Janus hydrogels rescue epithelial malfunction to promote complete trachea repair

Chen, Yi; Tang, Hai; Zhang, Yan; Wang, Lei; Zhu, Jun; Wang, Lele; Li, Anqi; Zeng, Xiang; Yin, Binyu; Liang, Yu; Dong, Xiulin; Bai, Qingfeng; Pan, Ziyin; Wang, Long; Zhang, Lei; Yang, Minglei; She, Yunlang; Sun, Weiyan; Zhang, Kun; Chen, Chang

doi:10.1038/s41467-025-61135-z

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Published: 01 July 2025

Multiplexed self-adaptable Janus hydrogels rescue epithelial malfunction to promote complete trachea repair

Yi Chen ORCID: orcid.org/0000-0001-5391-3864¹^na1,
Hai Tang¹^na1,
Yan Zhang²^na1,
Lei Wang^1,3^na1,
Jun Zhu⁴,
Lele Wang⁵,
Anqi Li⁵,
Xiang Zeng⁶,
Binyu Yin²,
Yu Liang²,
Xiulin Dong²,
Qingfeng Bai^1,3,
Ziyin Pan^1,3,
Long Wang^1,3,
Lei Zhang^1,3,
Minglei Yang⁷,
Yunlang She^1,3,
Weiyan Sun ORCID: orcid.org/0000-0002-5397-7857^1,3,
Kun Zhang ORCID: orcid.org/0000-0002-6971-1164² &
…
Chang Chen ORCID: orcid.org/0000-0003-2841-1250^1,3

Nature Communications volume 16, Article number: 5734 (2025) Cite this article

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Abstract

Epithelial malfunction rescue is the decisive step involved in complete trachea repair; however, this step remains challenging due to the harsh tracheal environment and unclear pathogenesis, which still made current bioengineered trachea transplants receive fatal complications. Herein, bacterial infection-induced neutrophilic oxidative stress imbalance and epithelial stemness loss were identified as the pathogenic factors. Targeting pathogenesis, multiplexed hydrogels with adhesive and anti-fouling Janus sides, anti-swelling and anti-bacteria properties are constructed to adapt in mucous and causative agent-rich trachea environments. In two epithelial injury models and two tracheal transplantation-related epithelial deficiency models, the hydrogels blockade oxidative stress-innate immune cascade axis, reactivate epithelial mucociliary regenerative ability to rescue epithelial malfunction with stenosis-free mucociliary epithelium regeneration. Importantly, the versatility of hydrogel is validated via its integration with routine bioengineered vascular and cartilage transplants, wherein the regenerated pseudostratification epithelium, cartilage and vascularization resemble native-like trachea, resulting in the complete tracheal repair including structure and respiratory function reinvigoration. Our research provides insights into epithelial interface diseases and guides related biomaterials design.

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Introduction

Tracheal stenosis or injury, which represents a major public health concern, usually requires complete tracheal repair^1,2,3,4, but the tremendous difficulty in enabling the composition, structure and functions of the repaired trachea to match those of natural tracheal tissue continues to exist. Thus, complete tracheal repair remains an intractable but critical challenge. The trachea generally consists of cartilage, blood vessels and epithelial layers, which play critical roles in maintaining airway opening, supplying oxygen and nutrition and inhibiting airway bacterial adherence, respectively^5,6,7,8. To achieve complete tracheal repair, the regeneration of cartilage, blood vessels and epithelial layers needs to occur in a synchronous manner. Nevertheless, current attentions have been paid only to cartilage regeneration^9,10,11,12, and the complex pathogenesis and harsh tracheal environment of tracheal injury have discouraged researchers to carry out functional epithelial layer repair^{13,14,15,16,17}. Therefore, epithelial deficiency is ubiquitous in current bioengineered tracheal transplantation patients and in the ageing population; moreover, this deficiency often causes tracheal epithelial malfunction and severe complications and respiratory disturbances^18,19. Thus, complete trachea repair is still hard to reach, and engineered tracheal substitutes fail to meet clinical demands. In light of the vital functions^20,21,22, rescuing epithelial layer malfunctions is highly important for decreasing fatal complications and inhibiting respiratory disturbances in common clinical scenarios. Unfortunately, repeated polluted airflow stimulation and mucus layer barrier lead to the failure of current epithelial transplantation methods and drug treatment in rescuing epithelial malfunctions, as well as the failure of complete trachea repair^23,24,25,26.

In this study, we utilize clinical samples and identify innate immune and epithelial regenerative ability (or stemness) losses as two dominant pathogenesis of epithelial malfunction (Fig. 1, Supplementary Fig. 1). Building on these insights, we devise a tracheal lumen-hydrogelation strategy, in which tracheal environment-adapted and multiplexed hydrogels with Janus surfaces are constructed to target pathogenesis, rescue epithelial malfunction, and eventually achieve the complete trachea repair. In such multiplexed self-adaptable hydrogels, one Janus surface could quickly adhere to the diseased area in the fully mucous airway environment, and another Janus surface is armed with a favorable anti-fouling ability to generate a stable and degradable protection shield against causative agents (such as breathing pollutants) in the air for epithelial rehabilitation. Under this circumstance, the activation and infiltration of MPO⁺ neutrophils will be hampered, effectively restraining innate immune hyperactivation and preventing epithelial malfunction in pathogenesis. Concurrently, ROS scavenging by the L-arginine burst is expected to further resist inflammation and promote tracheal repair. Moreover, the slow release of non-covalent tannic acid is anticipated to promote the immigration and differentiation of CK14⁺ basal cells^27,28,29,30.

**Fig. 1: Neutrophilic oxidative stress hyperactivated innated immune and impaired epithelial regeneration.**

Depending on these designs, such multiplexed self-adaptable hydrogels quickly adhere to the diseased wet area, form a stable, anti-swelling, anti-bacterial, innate immune-regulating barrier, and promote the differentiation of CK14⁺ basal cells into de novo multifunctional epithelial mucociliary elevator in both rabbit acute and recurred epithelial injury model (Supplementary Fig. 1)^15,31,32,33. Especially in two rabbit bioengineered tracheal transplantation models (partial tracheal defect model and long circumferential tracheal defect model), the hydrogels also result in complete trachea repair with structural and functional reinvigoration after integration with routine vascular and cartilage repair routes. Intriguingly, the degradation of such multiplexed self-adaptable hydrogels maintains a similar pace with the trachea regeneration, thus guaranteeing no respiration disturbance and high safety during repair. The generated trachea shapes into a native-like cartilage-vessel-epithelium structure with healthy respiratory functions, and no stenosis complications are found. Overall, this pioneering work provides a solid foundation to complete structural and functional repair of trachea, which highlights epithelial malfunction rescuing, and the good compatibility with existing bioengineered tracheal construction techniques demonstrates potential for the high clinical translation potential.

Results

The screening of pathogenesis targets in clinics

To identify the pivotal pathogenesis, we compared the pathological changes between epithelial malfunction tissues in patients and normal tracheal tissues in healthy donors (Fig. 1a, Supplementary Table 1, 2). Hematoxylin eosin (HE) and immunofluorescence (IF) staining confirmed epithelial malfunction at the site of injury (Fig. 1a–f). After epithelial injury, repetitive pollutants transmitted via breathing can lead to persistent infiltration and activation of MPO⁺ neutrophils in the diseased area. The MPO⁺ neutrophils further released large amounts of reactive oxygen species (ROS), and induced CD86⁺ proinflammatory macrophage (M1 macrophage) infiltration (Fig. 1c). Under physiological conditions (Fig. 1c, d), neutrophils play an important role in immune defense, in which process neutrophils destroy phagocytosed microbes in a ROS-dependent manner and then undergo apoptosis and disappear after resolution of the inflammatory stimulus^34,35,36. While continuous infiltration and activation of neutrophils were observed in area of epithelial malfunction, thereby indicating the possibility that bacterial infection and injury-related stress induced neutrophils and downstream innate immune hyperactivation, which ultimately caused epithelial malfunction (Fig. 1b–f).

Therefore, the hypothesis was raised that due to epithelial injury or deficiency, the tracheal tissue underwent injury stress and would be constantly exposed to a polluted environment, thereby leading to recurrent infection and neutrophil infiltration. The continuous activation and infiltration of neutrophils hyperactivated the innate immune system, thus causing epithelial malfunction. To validate this hypothesis, we first analyzed the number of tracheal bacteria in tracheas in the sham group and the epithelial injury group (Supplementary Fig. 2). The results aligned with our hypothesis that the bacterial burden of both Gram⁺ and Gram^- pathogens was greater at the site of epithelial injury compared to healthy tracheas.

Furthermore, we evaluated the role of hyperactivated oxidative stress and bacterial infection via co-culture models (Supplementary Fig. 3). Pseudomonas aeruginosa (P.a.), which is a commonly observed bacterium at sites of tracheal injury, was selected for co-culture with epithelial cells either with or without oxidative stress stimulation (Supplementary Fig. 3a). An MOI (multiplicity of infection) of 5 was considered to indicate mild bacterial burden, whereas an MOI of 20 was considered to indicate severe bacterial burden. When the MOI was 5, ROS were demonstrated to exert a positive bactericidal effect and protect the function of the epithelium to some extent (Supplementary Fig. 3b, c). However, when the MOI reached 20, the bactericidal effect of ROS alone was weakened, thus indicating that it was unable to protect the epithelium (Supplementary Fig. 3b, e). Subsequently, neutrophil-bacteria-epithelial cells co-culture model was established (Supplementary Fig. 3e). Via co-culture and ROS scavenging experiments, neutrophils were demonstrated to be capable of killing bacteria. However, the ROS released during this process could also impair the function of epithelial cells (Supplementary Fig. 3f–h). At an MOI of 20, the rescue effect of ROS scavenging on epithelial function was markedly attenuated compared with that at an MOI of 5, thereby indicating that bacterial-mediated epithelial cytotoxicity also plays a substantial role in epithelial malfunction (Supplementary Fig. 3f–h).

To further determine the role of neutrophilic ROS in epithelial multifunctional differentiation (Supplementary Fig. 4a). We utilized ESR spectra and demonstrated neutrophils co-cultured with P.a. released ROS (e.g., superoxide radicals), and the addition of an ROS scavenger eliminated ROS in the culture supernatant (Supplementary Fig. 4b). The staining results demonstrated that the supernatant of the neutrophil-P.a. co-cultured medium directly impaired the differentiation of epithelial organoids, manifesting as a malfunctional phenotype (including reduced thickness, decreased basal cells, and the loss of mucociliary structures; Supplementary Fig. 4c–j). ROS scavenging protected organoid differentiation at an MOI of 5 but failed to restore functionality at an MOI of 20 (Supplementary Fig. 4d, e). The abovementioned results preliminarily confirmed our hypothesis in vitro; specifically, the increased bacterial burden observed at the injured tracheal site drove persistent ROS release by neutrophils but had limited bactericidal efficacy. Both imbalanced ROS levels and bacterial secretions were identified as two key contributors to epithelial malfunction (which included the loss of epithelial barrier/multi-differentiation function).

To further validate this hypothesis in vivo, we constructed an epithelial injury model in neutrophil function-deficient mice (C57BL/6N-Cd177^em1C/Cya, or CD177^−/− mice), where CD177 is an important surface marker for neutrophils, and its knockout impairs the functions of neutrophils and disfavors innate immune hyperactivation^37,38,39,40. It is found that CD177 knockout promoted epithelial regeneration and increased the differentiation ratio of functional cilia cells (Supplementary Fig. 5), indicating that the function-deficiency of neutrophils in a bacteria-free environment could rescue epithelial malfunction (Fig. 1g). All of the abovementioned results reveal that bacterial infection and further neutrophilic oxidative stress could hyperactivate innate immune system and impair epithelial regenerative ability to aggravate epithelial malfunction, with these effects potentially representing therapeutic targets for epithelial malfunction.

Tracheal environment-matched and multiplexed self-adaptable hydrogel synthesis

Hydrogels have shown a wide application domain after rational design and engineering procedures according to the specific demands^{41,42,43,44,45}. To target this specific pathogenesis, we designed multiplexed self-adaptable hydrogels (LA@PA-TA@C) featuring high stability, Janus adhesion, anti-swelling and anti-bacteria to reprogram the innate immune and rescue epithelial regenerative ability (Fig. 2a, Supplementary Fig. 1). Such multiplexed hydrogels adapting to tracheal environment were constructed via a hybrid crosslinking strategy combining dynamic multiple non-covalent self-assembly and photopolymerization (Fig. 2b). Specifically, the chitin (C)-assisted tannic acid (TA) self-assembly via hydrogen bonding was firstly enforced to form C@TA hydrogel. Concurrently, poly-(Aspartic acid) (PA) network is activated by NHS via replacing -COOH (Fig. 2b, c), and then loaded with L-arginine (LA) to obtain L@PA hydrogels. Immediately afterwards, unilateral photopolymerization between the C@TA hydrogel and L@PA via amide bonds was enacted to eventually obtain LA@PA-TA@C hydrogels (Fig. 2b, d). The Janus structure is observed in LA@PA-TA@C with C@TA and L@PA serving as the upper anti-adhesion side and bottom tissue-adhesion side, respectively, as indicated by the structural and composition differences between the two Janus sides (Fig. 2e, f).

**Fig. 2: Design, fabrication, and characterization of multiplexed self-adaptable LA@PA-TA@C hydrogel.**

Janus adhesion, anti-swelling and anti-bacteria assessments

In general, metabolic acid accumulation in the epithelial malfunction site presents a threat to hydrogel stability. Intriguingly, the mechanical strength of the LA@PA-TA@C hydrogels was reinforced by lactic acid (Fig. 2d, h, Supplementary Fig. 6), and the LA@PA-TA@C hydrogels were able to resist rapid degradation in vivo (Fig. 2i). Beyond that, LA@PA-TA@C hydrogels are imparted with other distinctive properties. They display good Janus adhesiveness through covalent amide cross-linking between NHS ester groups in bottom tissue-adhesive PA hydrogels and amino residues on tissues (Fig. 2j, Supplementary Fig. 7), and both LA loading and crosslinking with C@TA failed to influence the tissue adhesion strength of PA network (Fig. 2k, Supplementary Movie 1). Unilateral modification of LA@PA with TA@C imparted the two sides of LA@PA-TA@C hydrogels with different adhesive performance on tissues (Fig. 2l), in which the PA-originated Janus adhesion property was inherited, whereas C-originated anti-adhesion owning to no tissue-binding groups is introduced. All of these properties allowed the LA@PA-TA@C hydrogels to be adhered on the tracheal lumen, even if the trachea was subjected to extreme bending or stretching motions (Fig. 2m, Supplementary Movie 2).

Both the upper C and bottom PA hydrogels feature high swelling ratios^46,47, usually disfavoring repair. Appealingly, noncovalent interactions with TA inhibited C hydrogel swelling, and the cross-linking between C and PA also resisted the swelling of C and PA to some extent. Therefore, these anti-swelling actions rendered L@PA-TA@TA hydrogels to harvest a much lower swelling rate of less than 110% in different aqueous media, and such anti-swelling properties were maintained even when challenged by acidic, trypsin, or oxidative stress environments (Fig. 2n, o). Meanwhile, the LA@PA-TA@C hydrogel could be degraded in vivo within 14 days, exhibiting biodegradability (Supplementary Fig. 8).

Due to the anti-bacterial ability of C (including the effect of rupturing bacterial cell walls)⁴⁵ and TA (including the effect of interfering with bacterial cell membrane synthesis)²⁸, co-culturing and antibacterial ring experiments confirmed the antibacterial efficacy of the LA@PA-TA@C hydrogel against clinically common bacteria, such as, Gram⁺ Staphylococcus aureus, Gram^- Escherichia coli, and even multidrug-resistant Mycobacterium abscessus (Fig. 2p). The antibacterial ability of the LA@PA-TA@C hydrogel was further verified in an ex vivo trachea explant model and an in vivo epithelial injury model. The results demonstrated that after the LA@PA-TA@C hydrogel adhered to the surface of the ex vivo trachea explants, it was able to inhibit bacterial proliferation (Supplementary Fig. 9). Similarly, when the tracheal epithelial injury model treated with the LA@PA-TA@C hydrogel for 7 days was subjected to tissue homogenization and plate coating, the bacterial quantity in the LA@PA-TA@C-treated tracheas was significantly lower than that in the epithelial injury group and the sham group (Supplementary Fig. 2).

Oxidative stress-innate immune cascade axis programming

TA has been demonstrated to scavenge ROS²⁸, and LA can serve as a reducing agent^48,49. Based on these characteristics, this multiplexed Janus hydrogels exhibited a good oxidative stress scavenging ability (Supplementary Fig. 10a, b), as represented by a significant decrease in superoxide radicals and singlet oxygen signals; additionally, the results of the DPPH and ABTS scavenging tests revealed almost 100% free radical scavenging (Supplementary Fig. 10c, d). This can be attributed to the continuous release of LA and TA within 14 days (Supplementary Fig. 10e). In particular, the TA@C-LA@PA hydrogels adapted to oxidative stress environment to achieve the differential TA release manner (Supplementary Fig. 10f). To further assess the ROS scavenging ability, an in vitro oxidative stress model was established via the addition of H₂O₂. LA@PA-TA@C hydrogels were observed to downregulate intracellular ROS levels and stabilize the mitochondria membrane (Supplementary Fig. 10g–i), and the oxidative stress scavenging protected 3T3 cells and increased cell viability to above 95% (Supplementary Fig. 10j, Supplementary Fig. 11). Furthermore, we utilized macrophages to validate the regulatory capacity of LA@PA-TA@C on the innate immune system. Both IF staining (Supplementary Fig. 12a, b) and flow cytometry (Supplementary Fig. 12c, d) analyses demonstrated that LA@PA-TA@C treatment upregulated CD206 expression (Supplementary Fig. 12a, d) and induced an increased elongation ratio of morphology (Supplementary Fig. 12b), which manifested as a characteristic anti-inflammatory M2 macrophage phenotype. Additionally, when challenged under oxidative stress conditions (via the addition of 200 μM H₂O₂, Supplementary Fig. 12e–h), the LA@PA-TA@C hydrogel exhibited comparable efficacy in promoting M2 polarization.

These results indicated that LA@PA-TA@C hydrogels could remodel the oxidative stress environment, and especially modulate the phenotype of innate immune-associated cells such as macrophages, thereby successfully reprogramming oxidative stress-innate immune cascade axis.

In vitro epithelial multidirectional regenerative ability reshaping

After validating the ability of the LA@PA-TA@C hydrogel to reprogram the oxidative stress-innate immune axis, we explored whether LA@PA-TA@C hydrogel could rescue epithelial basal cells in an oxidative stress-rich environment (Fig. 3a). Consistent with the aforementioned findings, oxidative stress led to epithelial cell death and tight junction destruction, but the LA@PA-TA@C hydrogels protected the integrity and barrier function of epithelial cells (Fig. 3b, c, Supplementary Fig. 13). Scratching experiments with H₂O₂ stimulation revealed that LA@PA-TA@C hydrogels accelerated epithelial cell migration and wound healing within 48 h (Fig. 3d, e, Supplementary Fig. 14), and the IF staining of ZO-1 and Ki67 biomarkers revealed epithelial proliferation and tight junction maintenance (Fig. 3d, f, Supplementary Fig. 15). Furthermore, the results of the qPCR assay revealed the upregulation of epithelial function-associated gene upregulations (CK14, ZO-1, and E-Cadherin) and the downregulation of epithelial malfunction and apoptosis-associated genes including α-SMA and Caspase 9 in the collected epithelial basal cells (Fig. 3g–k).

**Fig. 3: LA@PA-TA@C hydrogel rescued epithelial regenerative ability and promoted mucociliary differentiation of epithelial organoids in vitro.**

To assess the epithelial regenerative ability in an oxidative stress environment, an epithelial organoid model at the air-liquid interface (ALI) was established (Fig. 3l, Supplementary Fig. 13, 16). The epithelial cells exhibited a malfunctional phenotype of squamous metaplasia, characterized by disrupted tight junction and cell apoptosis (Fig. 3m, n). By contrast, intact epithelial barriers and well-differentiated cilia were observed, wherein native-like pseudostratified columnar epithelial structures with ZO-1⁺ tight junction, AC-Tub⁺ cilia cells, PAS⁺ goblet cells, and CK14⁺ basal cells were obtained (Fig. 3o). These results demonstrated LA@PA-TA@C hydrogels activate and enhance the multidirectional regenerative ability of CK14⁺ basal cells.

Respiratory function rescue in an acute epithelial injury model

To simulate real-world clinical tracheal injury scenarios^{9,15,31,32,33}, a rabbit tracheal epithelial scraping injury model was established (Fig. 4a, b). The epithelium and most of the submucosa at the injury site were scraped away on Day 0 post operation. On Day 7, the entire trachea exhibited hyperactivated innate immunity, extensive epithelial malfunction, and stenosis (Fig. 4c), which closely resembled the pathological changes of human epithelial malfunction.

**Fig. 4: Utilizing LA@PA-TA@C hydrogel to rescue respiratory function and investigating biological mechanism in rabbit model of acute epithelial injury.**

Subsequently, corresponding treatments were initiated for the different groups. Rabbit death occured in all of the groups due to severe respiratory shortness symptoms except LA@PA-TA@C group (Fig. 4d). On day 28, bronchoscope examination shows severe stenosis and adhered sputum in the PA group (Fig. 4e, Supplementary Fig. 17a). In contrast, LA@PA-TA@C treatment resulted in good tracheal patency, native-like smooth breathing, and no sputum retention, which consequently enabling complete trachea repair including structure and function to effectively rescue respiratory function (Fig. 4f, g, Supplementary Fig. 17b, Supplementary Movie 3). The high biocompatibility and biosafety paves a solid foundation to clinical translation (Supplementary Fig. 18, 19).

LA@PA-TA@C induced trachea repair mechanism analysis

Whole transcriptome RNA sequencing (RNA-Seq) performed on Day 14 demonstrated the good agreement within the group (Fig. 4h), where a significant difference in the transcriptome profiles between LA@PA-TA@C group and blank control group (model only with no treatment) was observed (Fig. 4i). The enrichment analysis identified both upregulated and downregulated gene sets in LA@PA-TA@C group (Supplementary Fig. 20, 21), and all of these differential expression genes (DEG) were classified into four distinct clusters via machine learning-based unsupervised cluster analysis (Fig. 4j, k). Compared to the blank group, cluster 1 (purple) and cluster 4 (green) genes were downregulated genes in the LA@PA-TA@C group, while cluster 2 (pink) and cluster 3 (blue) genes were upregulated genes in the LA@PA-TA@C group. In cluster 2, the signaling pathways associated with the oxidative-reduction process and glutathione metabolism were found to be upregulated by LA@PA-TA@C. In cluster 4, there was an enrichment in biological processes that LA@PA-TA@C downregulated inflammatory responses. In cluster 3, crucial biological processes associated with the regeneration of the pseudostratified columnar ciliated epithelium were found to be upregulated by LA@PA-TA@C. These findings clearly indicated that LA@PA-TA@C hydrogels effectively fulfil their design goals by inhibiting the neutrophil-initiated oxidative stress-innate immune cascade axis, and improving epithelial regenerative ability, including that of cilium. (Fig. 4k). Furthermore, an additional cluster (cluster 1, a cluster downregulated in the LA@PA-TA@C group) of genes that was intricately involved in the biological process of collagen fibril organization closely correlates with tracheal stenosis complication (Fig. 4k, l), such as cell adhesion molecules, ECM-receptor interaction, focal adhesion, and PI3K-Akt, etc.. They indicated that LA@PA-TA@C hydrogels can inhibit ECM deposition to impede stenosis.

Further validate the mechanism involved in reprogramming the neutrophil-initiated oxidative stress-immune cascade axis using such multiplexed self-adaptable hydrogels. Many MPO⁺ROS⁺ neutrophils were observed to still be activated at the site of injury in the control group, but were extinguished in the LA@PA-TA@C group (Supplementary Fig. 22a–c). The presence of M1 macrophages (CD86⁺CD206^-) and Tunel⁺ apoptosis cells within 1 - 4 weeks indicated persistent hyperactivation of the innate immune in the control groups (Supplementary Fig. 22d–h, Supplementary Fig. 23). By contrast, the activation of neutrophils and ROS in the LA@PA-TA@C group were significantly dampened within 1 week (Supplementary Fig. 22a), and the numbers of M1 macrophages and apoptotic cells considerably decline after 4 weeks (Supplementary Fig. 23). Moreover, the up-regulation of pro-regenerative CD206⁺ M2 macrophages at 1 week and 2 weeks post-operation confirmed that the neutrophil-initiated oxidative stress-immune cascade was interrupted, thus reshaping into a pro-regenerative environment (Supplementary Fig. 22d–h).

In vivo epithelial regenerative ability reinvigoration

In addition to reprogramming the innate immune cascade axis, another pathogenesis hampering epithelial regeneration, i.e., epithelial regenerative ability or stemness impairment, was reinvigorated with such Janus hydrogels. To verify it, 42 DEGs were firstly identified after intersecting all the DEGs with the epithelial regeneration and tracheal epithelium-related gene set in public databases (Fig. 5a). Compare to the blank control, the DEGs associated with epithelial stemness (including KRT5 and TP63), epithelial barrier function (including CDH23 and SHH), and ciliated cell differentiation (ULK4) were significantly increased after LA@PA-TA@C hydrogel treatment at both the transcriptome and translational protein levels (Fig. 5b, Supplementary Fig. 24, 25). Further gene set enrichment analysis (GSEA) revealed that four pivotal signaling pathways associated with cilial cell differentiation and Notch model pathways were upregulated in LA@PA-TA@C hydrogel group (Fig. 5c), which thus undertook to regulate tracheal epithelial regeneration and differentiation.

**Fig. 5: LA@PA-TA@C hydrogel rescued epithelial regenerative ability and regulated epithelial regeneration patterns in vivo.**

The native healthy epithelium is characterized by functional cilial cells and goblet cells, and renewal-obligated basal cells (Fig. 5d). The LA@PA-TA@C hydrogel treatment resulted in the complete epithelial coverage within 1 week, featured a pseudolaminar structure. After 4 weeks, the structure, cellular composition, and thickness of regenerated epithelium resembled those of the native epithelium (Fig. 5e, f, Supplementary Fig. 26). Meanwhile, the control group failed to achieve epithelial coverage at the early stage, and eventually escalated into epithelial malfunction (Fig. 5e, f). In the LA@PA-TA@C group, residual PCNA⁺ CK14⁺ epithelial basal cells were activated, proliferated, and migrated towards the injured site (Fig. 5g–i), thereby indicating that stratified epithelial cells accumulated and communicated with ZO-1⁺ expressing tight junctions to restore the epithelial barrier function within 1 week (Fig. 5j–l). Additionally, CK14⁺ epithelial cells were observed to continuously differentiate into native-like AC-Tub⁺ cilia cells (Fig. 5m, n) and MUC5ac⁺PAS⁺ goblet cells (Fig. 5o, p), and only a small proportion of proliferatively active CK-14⁺ basal cells were observed to reside above the basement membrane for self-renewal, forming native-like epithelial regenerative patterns. Notably, the epithelial regeneration rate was consistent with LA@PA-TA@C hydrogel degradation (Fig. 2i), thus indicating that LA@PA-TA@C could perform epithelial barrier functions without respiratory disturbance before the occurrence of epithelial rescue.

Stenosis complications

Tracheal stenosis is the main fatal complication that frequently occurs after epithelial malfunction, and is characterized by α-SMA⁺ fibroblast proliferation and extracellular matrix (ECM) deposition^50,51,52. After rescuing epithelial malfunction, the ability of the multiplexed hydrogels to eliminate epithelial malfunction-caused tracheal stenosis was further assessed (Supplementary Fig. 27). The LA@PA-TA@C group demonstrated less ECM deposition (Supplementary Fig. 27a–c, Supplementary Fig. 28a–c), less α-SMA⁺ fibroblast proliferation (Supplementary Fig. 27d, e, Supplementary Fig. 28d–f), indicating with no stenosis complications via IF and Masson trichrome staining. Sirius red staining revealed that the collagen distribution direction curve exhibited a native-like bimodal pattern, indicating the ECM direction in the LA@PA-TA@C group was more regular and orderly, approaching to the collagen I/III ratio in the normal submucosa layer (Supplementary Fig. 27f–h). The other groups displayed typical remodeled fibrous scars or partially-bulging granulation with abundant isotropic and horizontally-oriented collagen bundles (Supplementary Fig. 27g), wherein α-SMA⁺ fibroblasts uncontrollably overgrew and finally led to stenosis (Supplementary Fig. 27d). RNA-seq analysis also indicated that ECM deposition related biological process was down-regulated in the LA@PA-TA@C group (Fig. 4j–l), and the key downregulated genes included COL1, VWF, THBS3, SPP1, IGF, and SOX9 (Supplementary Fig. 27i). Furthermore, the RNA-seq results were validated at the translational protein level by IF staining, and the results revealed that the expression of these genes in the submucosal region was inhibited at the protein level, which aligns with the results at the transcriptome level (Supplementary Fig. 27j, k). These results indicated that the LA@PA-TA@C hydrogels downregulated ECM deposition at the injury site and thereby significantly reduced the incidence of adverse tracheal stenosis complications.

LA@PA-TA@C rescued epithelial functions and prevented stenosis in a rabbit model of recurred circumferential epithelial injury

Tracheal injury with epithelial malfunction tends to result in nonhealing and recurrent stenosis, whereas in clinical practice, re-patency treatment via electrocautery, biopsy forceps scraping, and stent implantation can only temporarily alleviate the symptoms of stenosis. To better mimic clinical scenarios, we constructed a recurred circumferential tracheal epithelial injury model (Supplementary Fig. 29a–c). On Day 7 post-circumferential injury, the rabbits were divided into different groups: one group underwent re-patency and received LA@PA-TA@C hydrogel treatment (LA@PA-TA@C group), and the blank control group only underwent re-patency (Blank control) (Supplementary Fig. 29b). Rabbit death occurred in the blank control group, whereas the survival rate of the LA@PA-TA@C group was observed to be 100% (Supplementary Fig. 29d). Further assessment of epithelial regeneration at 2 weeks after re-patency (Supplementary Fig. 29e–h) and tissue section staining revealed that LA@PA-TA@C treatment regenerated the epithelium with a native-like pseudolaminar structure (Supplementary Fig. 29e), which featured PAS⁺ goblet cells (Supplementary Fig. 29f) and AC-Tub⁺ cilia cells (Supplementary Fig. 29g). The number of CK14⁺ epithelial basal cells was increased in the LA@PA-TA@C group, thereby indicating positive tissue regeneration (Supplementary Fig. 29e, h). These phenotypes aligned with the acute partial epithelial injury model (Fig. 5), thus indicating that LA@PA-TA@C could rescue epithelial functions in various scenarios.

We further evaluated recurrent stenosis complications in this model at 2 weeks post re-patency (Supplementary Fig. 29i, k). As previously mentioned (Supplementary Fig. 27), tracheal stenosis is a fatal complication of epithelial malfunction and is characterized by α-SMA⁺ fibroblast proliferation and ECM deposition. Tissue section staining revealed that the LA@PA-TA@C group demonstrated no stenosis complications with native-like submucosal thickness (Supplementary Fig. 29i, k). Moreover, the expression of α-SMA (Supplementary Fig. 29i) and the collagenous area (Supplementary Fig. 29j) were downregulated in the LA@PA-TA@C group, thereby indicating that the hydrogel could prevent tracheal stenosis even in recurred injured epithelial clinical scenarios.

Bioengineered tracheal transplantation for complete tracheal repair

After rescuing epithelial malfunction, we integrated the aforementioned multiplexed self-adaptable Janus hydrogels with a standardized vascular and cartilage repair protocol, and investigated whether the integrated transplants could address the long-standing challenge (i.e., epithelial malfunction) of complete tracheal repair in the thoracic field. In this standardized route, engineered cartilage (TEC) was constructed and embedded directly into the anterior cervical muscle to promote cartilage maturation and routine prevascularization for 28 days (Supplementary Fig. 30). Afterwards, the integrated trachea transplants (TEC/LA@PA-TA@C) were obtained through allowing the LA@PA-TA@C hydrogels to adhere to the vascularized TEC (Fig. 6a, Supplementary Fig. 30, Supplementary Movie 4 and Supplementary Movie 5). At 28 days post-tracheal transplantation, the integrated transplants exhibited a survival rate above 83%, while the survival rate was less than 18% in the control group (TEC group) (Fig. 6b). Simultaneously, the respiratory status of the rabbits in TEC/LA@PA-TA@C group was demonstrated to be stable, and the respiratory function of these rabbits progressively approached that of native rabbits (Fig. 6c). Notably, TEC/LA@PA-TA@C revealed tracheal patency without stenosis, and exhibits de novo structures resembling native cartilage at the transplantation site, while the control group shows stenosis with no cartilage degradation (Fig. 6d–f, Supplementary Fig. 31, Supplementary Movie 6). No neutrophil-initiated oxidative stress-immune cascade was observed in the TEC/LA@PA-TA@C group, as indicated by no neutrophil activation, lower ROS production, and less M1 macrophages (Supplementary Fig. 32). The regeneration of the tracheal pseudostratified columnar epithelium was observed (Fig. 6g–l, Supplementary Fig. 33), wherein the epithelium coverage ratio exceeded 98% (Fig. 6j) with over 85% mature cilia coverage (Fig. 6i, l). Due to the protective effect of TEC/LA@PA-TA@C and regenerated epithelial tissues, both lacunae and stroma deposits resembled to natural cartilage in tracheal grafts (Fig. 6m–o, Supplementary Fig. 34). In the TEC group, the overactivation of the innate immune system directly impaired epithelial regeneration and caused tracheal stenosis; in particular, the engineered TEC cartilage was destroyed, which further led to fatal tracheal collapse.

**Fig. 6: Multiplexed self-adaptable LA@PA-TA@C hydrogel promoted completed tracheal repair.**

The efficacy of LA@PA-TA@C was further investigated in a long circumferential tracheal defect model to confirm its clinical translational capabilities. Chondrocytes were encapsulated in a methacrylated gelatine (GelMA) hydrogel and moulded into 6 mm internal diameter rings. These rings were stacked into a tubular construct and subsequently implanted into the anterior cervical muscle to promote cartilage maturation and routine pre-vascularization for 28 days, thereby ultimately forming a tissue-engineered trachea (TET). The LA@PA-TA@C hydrogel was subsequently adhered to the TET luminal surface to create TET/LA@PA-TA@C grafts, which were then orthotopically transplanted to construct a long circumferential tracheal defect model (Fig. 7a). The survival rate of the TET/LA@PA-TA@C grafts was 100%, whereas the survival rate was 33.3% in the TET group (Fig. 7b). A comprehensive evaluation of epithelial and cartilage regeneration was performed via histological and IF staining analyses (Fig. 7, Supplementary Fig. 35). The TET/LA@PA-TA@C grafts demonstrated successful reconstruction of the tracheal wall architecture, which featured the following characteristics. 1) Complete re-epithelialization with a pseudostratified columnar epithelium lining the graft lumen was demonstrated (Fig. 7c, d). Via IF quantification, functional epithelial restoration was evidenced by increased CK14⁺ basal cells and AC-Tub⁺ ciliate cells (Fig. 7e, f). 2) Cartilage integrity preservation revealed that the chondrocyte lacunae were embedded in sulfated Col II-rich extracellular matrix, thereby phenocopying the native cartilage morphology (Fig. 7e, g). While in the TET group, no epithelial regeneration was observed, let alone cilia cells, and the cartilage was also severely degraded (Fig. 7c, e–g), indicating a lethal risk. Meanwhile, the liver function and kidney function were assessed on day 28 post orthotopic transplantation. Both liver function (AST, ALT) and kidney function (CREA and UA) were no difference with native rabbits, indicating the biosafety of LA@PA-TA@C hydrogel (Supplementary Fig. 36).

**Fig. 7: LA@PA-TA@C hydrogel realized completed tracheal repair in a long circumferential tracheal defect model.**

In brief of above two tracheal transplantation models, such multiplexed self-adaptable hydrogel represented a pioneering approach to rescue tracheal epithelial malfunction, which promoted the complete structural and functional repair of trachea through even using the simplest routine vascular and cartilage regeneration and re-epithelialization techniques.

Discussion

Complete tracheal repair with native-like structure and functions represent Goldbach’s conjecture in the field of thoracic surgery², and tissue engineered tracheal construction is considered a feasible way^9,10,11. However, there is still an unmet requirement for specialized and standardized treatment for tracheal epithelial rehabilitation after tracheal transplantation in clinical practice. Tracheal epithelial malfunction is usually accompanied by severe complications, such as sputum accumulation, tracheal stenosis, and tracheal collapse^9,10,11. Clinical treatment can only address apparent symptoms, but fails to resolve the pathogenesis of these complications^{53,54,55,56,57}. Unresolved epithelial malfunction typically induces these complications to persist and inflict patients with repeated fatal risks^13,14, thus imposing a substantial burden on medical resources. The rescue of epithelial structure and function is a precondition of complete trachea repair. Current explorations to restore the tracheal epithelium have relied on the grafts of the epidermis, buccal epithelium, bioengineered epithelium, or epithelial cells^{6,16,17,58,59,60,61,62,63,64}. However, the disputed pathogenesis of tracheal epithelial malfunction and deficiencies of targeted interventions have easily caused these epithelial grafts evolved into squamous metaplasia or other malfunctional phenotypes and shed, let alone the cost and ethical considerations. Therefore, it is necessary to find specific therapies for epithelial malfunction. In this study, we provided information on the pathogenesis of epithelial malfunction and constructed the multiplexed self-adaptable Janus hydrogels to address this issue.

Different from other epithelial interface environments^25,51, the harsh tracheal environment includes sputum, pollutants, and microbes, and was also suffered by the mechanical stress caused by inhaled air. Patients with epithelial injuries or deficiencies are unable to receive routine wound rehabilitation care and are in a persistent state of innate immune hyperactivation. In this process, the bacterial infection and neutrophil- induced oxidative stress to directly damage epithelial regenerative ability and leads to epithelial malfunction. The mechanical/physical stress caused by inhaled air could also incorporate and intensify oxidative stress and immune response. Overall, bacterial infection, oxidative stress, and innate immune hyperactivation constituted a cascade reaction, and caused a further vicious circle. Notably, the multiplexed self-adaptable hydrogels can target this pathogenesis to rescue epithelial malfunction. First, the hydrogels adapted to the full-mucus environment and formed a stable and degradable barrier for epithelial rehabilitation, which isolated foreign bodies and sterilized most of the bacteria. Second, the hydrogels adapted to the oxidative stress- innate immune cascade, blockaded breathing pollutants-caused MPO⁺ neutrophil activation, and scavenge oxidative stress. Third, the hydrogels adapted to epithelial regenerative patterns, and promoted CK14⁺ basal cells to immigrate and differentiate to a multifunctional epithelial mucociliary elevator. In this study, the multiplexed self-adaptable hydrogels effectively addressed three crucial factors in the pathogenesis, including bacterial infection, neutrophilic oxidative stress, and epithelial regenerative ability. In both two models, i.e., epithelial acute injury and recurred injury, the hydrogels successfully prevented stenosis complications and achieved functional cilia, representing a promising therapy for tracheal epithelial malfunction. In the epithelial bioengineered tracheal transplantation model, the hydrogels successfully achieved structural and functional mucociliary epithelium regeneration and promoted completed tracheal repair, thus representing a universal approach for tracheal epithelial rehabilitation after tracheal transplantation.

The activation of the innate immune system can affect the adaptive immune system; for example, neutrophils and macrophages may activate lymphocytes and plasma cells. Thus, we also examined adaptive immunity. Compared with native conditions, in both the human epithelial malfunction trachea model and the rabbit epithelial injury malfunction model, T-cell infiltration and activation were increased, as was B-cell infiltration (Supplementary Fig. 37). Furthermore, LA@PA-TA@C hydrogel treatment inhibited the activation of adaptive immunity in rabbit acute epithelial model, which was possibly due to the fact that the hydrogel blocked innate immune hyperactivation.

In summary, epithelial stemness loss and the bacterial infection-induced neutrophil-initiated innate immune hyperactivation have been identified as the pathogenesis of epithelial malfunction. With targeting them, a multiplexed self-adaptable Janus hydrogel has been constructed via the hybrid photopolymerization between assembled TA@C hydrogels and NHS-activated LA@PA hydrogels. The two Janus sides imparted LA@PA-TA@C hydrogels with high tissue adhesion and favorable anti-fouling ability, which collaborated with acid-reinforced stability, anti-swelling and oxidative stress scavenging properties to adapt harsh tracheal environment (e.g., rich mucous, pollutants, bacteria etc.) in airway. In both in vitro primary epithelial organoid model and in vivo epithelial injury model, such multiplexed self-adaptable hydrogels reprogrammed oxidative stress-innate immune cascade axis, and promoted multifunctional epithelial mucociliary regeneration. Consequently, they invigorated epithelial mucociliary regenerative ability, modulated epithelial regeneration patterns to accomplish epithelial rehabilitation. Moreover, they inhibited fibroblast activations and rearranged native-like grid ECM networks, which persistently addressed tracheal stenosis complications from the roots. Especially after integrating with a standardized routine vascular and cartilage repair protocol, a luminal hydrogel-functionalized bioengineered tracheal graft was obtained to rescue epithelial malfunctions and promote complete tracheal repair with native trachea-like structure and functions. Therefore, such multiplexed self-adaptable Janus hydrogels presented a targeted approach to various clinical scenarios of epithelial malfunction.

Methods

Ethical statement

This study protocol was approved by the Ethics Committee of Shanghai Pulmonary Hospital (K23-235), and written informed consent was obtained from all individual participants recruited in this study. The study design and implementation adhere to all relevant regulations concerning the use of human research participants and are conducted in accordance with the standards set forth in the Declaration of Helsinki.

Patient population and sample collection

Patients with tracheal epithelial malfunction and stenosis complication were diagnosed and confirmed through bronchoscopy, and biopsy samples were collected from the pathological sites for histopathological examination. The healthy control group was composed of tracheal tissue obtained from healthy lung transplant donors, with exclusion of diseases such as infection, inflammation, or sarcoidosis. Based on the observed stenosis, typing and grading were performed according to the Freitag criteria (Supplementary Table 1).

Animals

New Zealand rabbits were supplied by Shanghai Jiagan Experimental Animal Company (Shanghai, China). Both female and male New Zealand rabbits were 3 months old, with an average weight of 2 kilograms. C57BL/6N-Cd177^em1C/Cya mice were get from professor Z.J.L. of the Shanghai Tenth People’s Hospital. C57BL/6, female and male mice, 8 weeks of age, were purchased were purchased from GemPharmatech in Jiangsu, China. Mice were maintained in specific pathogen-free conditions in microisolator cages, and all the rabbits were housed under conventional (CV) level conditions. Animals were treated by following the guidelines for the care and use of animals (National Research Council and Tongji University). All animals were fed ad libitum. The procedures for the use of animals were approved by the Ethics Committee of Shanghai Pulmonary Hospital. All applicable institutional and governmental regulations concerning the ethical use of animals were followed.

Primary epithelial cell culture and epithelial organoid differentiation

The rabbit trachea was obtained from after euthanasia. Then trachea was washed three times by PBS and incubated for 30 min at 37 °C with dispase (Stem Cell, Canada). After incubation, the mucosal layer was separated from the trachea and further digested overnight. The digested liquid was collected and filtered through a 70 μm filter (Sigma-Aldrich, USA), and then cell suspension was centrifuged at 200 g for 10 min. Cells were resuspended by epithelial culture medium consisting of DMEM (Gibco, USA) and F-12 (Gibco, USA) with penicillin-streptomycin (Solarbio, Beijing, China) and 10% FBS (Gibco, USA) supplemented with 5 μm Y27632 (Macklin, China), 0.125 ng/mL EGF (PeproTech, China), 5 μg/mL insulin (Sigma, USA), 0.1 nm cholera toxin (Sigma, USA), 250 ng/mL amphotericin B (Solarbio, Beijing, China), and 10 μg/mL gentamycin (Gibco, USA), seeded in 3 cm culture dish, and cultured at 37 °C with 5% CO₂ with three changes of medium per week. Epithelial cells were passaged to the third generation (P3). Then the cells were seeded on Matrigel-coated transwell (Stem Cell, Canada) to form the air-liquid interface (ALI) culture model, and induced by PneumaCult™ culture media (Stem Cell, Canada) for 3 weeks to form epithelial organoids. In the process of organoid induction, 200 μm H₂O₂ was added to the culture medium to simulate oxidative stress.

Neutrophil isolation and culture

Neutrophils were isolated by density gradient centrifugation from the peripheral blood of rabbits with bronchiectasis (Lymphoprep 1858). Isolated rabbit neutrophils were used immediately for in vitro experiments. Neutrophils were cultured at 37 °C with 5% CO₂. The cells were seeded in 24-well flat bottom plates with a concentration of 1.5 × 10⁶/well.

3T3 cells and RAW 264.7 macrophages culture

3T3 cells (No. SCC-220911) and RAW 264.7 macrophages (No. SCC-211800) from Servicebio, China. Cells were cultured at 37 °C with 5% CO₂. The cells were seeded in 24-well flat bottom plates with a concentration of 1.5 × 10⁶/well for testing.

Bacterial strain and culture

The strain used in this study was Pseudomonas aeruginosa (P.a.) and was get from professor J.X. Department of Respiratory and Critical Care Medicine, Shanghai Pulmonary Hospital. Unless otherwise stated, all the bacteria in the represented study were cultivated in 3 ml of sterile lysogeny broth (LB) medium for 13 h (37 °C). Subsequently, they were diluted by LB at a ratio of 1:200 and agitated for an additional 2 h.

Synthesis and preparation of LA@PA-TA@C hydrogel

Chitin, tannic acid, aspartic acid, L-arginine, and other chemicals and solvents used were all purchased from MACKLIN (Shanghai, China).

First, TA@C hydrogel was prepared as previous reported. Chitin powder was immersed in 36.7 wt% NaOH solution at room temperature overnight, and subsequently, the suspension was repeated freeze-thaw to promote chitin dissolution. Then, ddH₂O was added into suspension, and freeze-thaw for serval cycles to obtain a 4.31 wt% transparent chitin solution. Besides, 10 wt% tannic acid solution were prepared. The 9:1 chitin-tannic acid solution was mixed. Tannic acid mediated chitin self-assembly and form hydrogel in 65 °C for overnight. The hydrogel was neutralized and washed thoroughly with water.

Then, N-acryloyl aspartic acid was synthesized via aspartic acid. 0.45 g N-acryloyl aspartic acid was dissolved in 0.55 g MES solutions. To replace the carboxyl group with NHS ester and form the PA precursor, 50 mg each of 1-(3-Dimethylaminopropyl)-3-ethylcarbodiimide (EDC) and N-hydroxy succinimide (NHS) were added, followed by an overnight reaction. For modification on one side of TA@C hydrogel, a mixture comprising 1 mg photoinitiator (IRGACURE 2959), 50 mg L-arginine, and 1 g PA precursor was introduced prior to inducing the reaction through UV irradiation at a wavelength of 365 nm for a duration of 30 min to yield LA@PA-TA@C hydrogel.

Characterization of LA@PA-TA@C hydrogel

Fourier transform infrared (FTIR) spectra

A FT-IR spectrometer (Nicolet iS 10, Thermo Fisher Scientific) was employed to measure the FTIR spectra of hydrogels. The hydrogels were freeze-dried, ground into powder, and pressed into tablet. The measured wavenumber ranges from 4000 to 500 cm^–1.

¹H NMR spectra

The hydrogel precursor was obtained with 5 mg, and the monomer structure was analyzed by NMR spectroscopy (AVANCENEO600M, USA).

SEM examination

The hydrogels were freeze dried and then coated with a thin gold layer by using an auto sputter fine coater (JFC 1600, JEOL, Tokyo, Japan) before imaging. The morphology of the hydrogels and simples was assessed by using field emission scanning electron microscopy (SEM, JSM-7001F, JEOL, Japan).

Element mapping and EDS examination

Element mapping was tested on both side of LA@PA-TA@C hydrogel via a field-emission Magellan microscopy (ULTIMATELYMAX 40, UK).

Compression tests of hydrogels

A nonmechanical analyzer (Instron-6800, UK) was used to evaluate the hydrogel mechanical strength via compression testing. 6 samples per group (hydrogel treated with or without 0.2% lactic acid for 1 h), which were all 1 cm in diameter were compressed to the maximum deformation strain at a rate of 2.00 mm/min. The stress-strain curve of the hydrogels plotted.

Rheology analysis

The rheological properties of different hydrogel were characterized on the stress-controlled rheometer (HAAKE RheoStress 6000, Thermo Scientific, USA). LA@PA-TA@C hydrogel diameter was 10 mm, and had been treated with or without 0.2% lactic acid for 1 h. Under the 1 Hz oscillatory frequency with a fixed 10 N shear stress, the storage (G′) and loss (G″) moduli were recorded.

Adhesive performance

The LA@PA-TA@C hydrogels (both sides were endowed adhesive ability via LA@PA) were adhered to the surfaces of different materials and recorded by iPhone camera. Adhesive strength were tests. Fresh rat skin was cut into a 3 cm × 5 mm in size, and two pieces of rat skin were adhered with hydrogel (5 mm × 5 mm in size). The adhesive stress was tested via an EZ-LX electronic universal testing machine (Shimadzu, Japan) with a speed of 10 mm/min.

Swelling ratio test

The swelling ratio were evaluated by immersing the hydrogels with PBS. The hydrogel samples (10 mm in diameter and 5 mm in height) were weighed after immersed for different time points. The swelling ratio was defined by the following formula (1). Further, the hydrogels were immersed in PBS with 5% trypsin, 0.2% lactic acid, and 200 μM H₂O₂ to test anti-swelling property.

$${{{\rm{Swelling\; ratio}}}}=\frac{{{{{\rm{Weight}}}}-{{{\rm{Weight}}}}}_{0{{{\rm{h}}}}}}{{{{{\rm{Weight}}}}}_{0{{{\rm{h}}}}}}\times 100\%$$

(1)

Anti-bacterial property test

The anti-bacterial property of hydrogel against S. aureus (CMCC26003), E. coli (CMCC44102), and multiresistant Myco. abscessus (19977) were tested. The bacteria were got from professor H.Q.C. Department of Respiratory and Critical Care Medicine, Shanghai Pulmonary Hospital. The colonies formed on the agar medium were counted, and the antibacterial rings of hydrogels were recorded.

Release curve

The hydrogel was immersed in the solution and the absorbance of the solution was measured at 200 nm and 320 nm, representing the release of LA and TA, respectively.

EPR test

Take 20 μL xanthine solution and 20 μL xanthine oxidase PBS solution, followed by the addition of 10 μL 200 mM BMPO solution. Subsequently, add 50 μL buffer or 50 μL hydrogel precursor solution. Finally, collect samples for testing after a reaction time of 10 min.

DPPH scavenging and ABTS scavenging test

The antioxidant activity of hydrogels was assessed via the scavenging ability of the 1,1-diphenyl-2-picrylhydrazyl (DPPH) and Diammonium 2,2’-azino-bis(3-ethylbenzothiazoline-6-sulfonate) (ABTS) free radical. A 100 μM DPPH or ABTS solution was prepared. The hydrogels were ground into pastes and prepared to produce a hydrogel dispersion of 100 mg/mL with ethanol. Then, a DPPH or ABTS solution and a hydrogel dispersion were mixed in the dark. After incubation for 1 h, the absorbance of the mixture was measured via a UV–vis spectrophotometer.

Cell culture and intracellular ROS scavenging

3T3 cells and RAW 264.7 macrophages were cultured via medium consisting of DMEM (Gibco, USA), penicillin-streptomycin (Solarbio, Beijing, China), and 10% FBS (Gibco, USA). The cells were co-culture with different group hydrogel via transwell (Stem Cell, Canada) for 3 days, and 200μm H₂O₂ was added to the culture medium to simulate oxidative stress.

The intracellular ROS levels were examined via ROS assay kit (Beyotime, China). fluorescence microscope (Olympus IX73; Olympus, Japan) and flow cytometry were performed to characterize the intra ROS levels by measuring the fluorescence intensity of DCF (Ex = 488 nm, and Em = 525 nm).

Scratch test

Primary epithelial cells were planted in a six-well tissue culture plate and grew for up to 70‒80% confluence in the plate. The cell monolayer in each well was then scratched with a sterile plastic tip, and the cells were washed twice with PBS and incubated in epithelial culture medium co-culture with different group hydrogel via transwell (Stem Cell, Canada). 200 μm H₂O₂ was added to the culture medium to simulate oxidative stress. The cells and scratched wound were imaged at 0, 24, and 48 h, and the area of wound closure was calculated with ImageJ 1.8 software (National Institutes of Health) for three independent replicate experiments.

Acute epithelial injury model and hydrogel treatment in rabbit

Firstly, the rabbit epithelial injury model was built and validated. After anesthesia by Zoletil (50 mg/kg, Virbac, France), the skin and fascia were sequentially separated and incised until the trachea was fully exposed in New Zealand rabbits. A nylon brush was inserted to scrape off the inner epithelium (1 cm × 1.5 cm in size) to simulate a clinical tracheal epithelium injury scenario. The incision was then closed, and the fascia and skin were sutured by layer. Trachea tissue was obtained on the 0 and 7 days after scraping and then pathological sections and HE staining were performed to validated the successful establishment of the epithelial injury model.

Hydrogel treatment: using above methods to scrape off the inner epithelium (1 cm × 1.5 cm in size) and different groups of hydrogels (1.5 cm × 2 cm in size) were subsequently adhered to cover the injury site. Then the incision was closed, and the fascia and skin were sutured by layer. Group setting: only epithelial injury model with no hydrogel treated (Blank control), PA, PA-C, LA@PA-TA@C, LA@PA-TA@C. Rabbits with an average body weight of 2.5 kg were randomly divided into different groups. n = 6 rabbits in each group for one experiment, and the experiments were repeated independently for three times.

Recurred circumferential tracheal epithelial injury model

After anesthesia by Zoletil (50 mg/kg, Virbac, France), the skin and fascia were sequentially separated and incised until the trachea was fully exposed in New Zealand rabbits. A nylon brush was inserted to scrape off the circumferential inner epithelium (1.5 cm in length) to simulate a clinical tracheal epithelium injury scenario. The incision was then closed, and the fascia and skin were sutured by layer. On day 7 post circumferential injury model, the rabbits were anesthesia, and the skin and fascia were sequentially separated and incised until the trachea was fully exposed once again. A nylon brush was inserted to scrape the inner lumen, scraping off the neoplasm at the modeling site (we called re-patency). Rabbits were then divided into different groups: one underwent re-patency and received LA@PA-TA@C hydrogel treatment, the blank control group only underwent re-patency, sham group, only received sham operation without epithelial injury. n = 6 rabbits in each group for one experiment, and the experiments were repeated independently for two times.

Bioengineered tracheal graft construction

Rabbit auricular cartilage samples, measuring 4 cm × 6 cm in size, were harvested under anesthesia induced by Zoletil (50 mg/kg, Virbac, France). The tissue was sterilized by spraying with 75% ethanol (Macklin, Shanghai, China) and subsequently washed three times with phosphate-buffered saline (PBS, Solarbio, Beijing, China) solution containing 2% penicillin-streptomycin (Solarbio, Beijing, China). On a clean bench, the attached skin and fascia were carefully peeled off from the cartilage. The cartilage was then cut into pieces and digested in 0.25% trypsin (Gibco, USA) at 37 °C for 2 h. Following neutralization and removal of trypsin, the cartilage pieces were immersed in 0.15% type II collagenase (Gibco, USA) and incubated at 37 °C overnight. The digested cartilage was filtered through a 70 μm pore filter (Sigma-Aldrich, USA) to obtain a primary chondrocyte cell suspension, which was then collected by centrifugation. Primary autologous chondrocytes (P0) were implanted at a density of 10⁶/ 10 cm diameter culture dish and passaged to the third generation (P3). The expanded chondrocytes were centrifuged at 200 g for 10 min to pellet the cells. The cells were resuspended in a 10% solution of methylacrylyl anhydride gelatin (GelMA, Engineering For Life, Suzhou, China) and then cast into specific molds. The constructs were photocrosslinked under ultraviolet light to form stable TEC. The TEC was further embedded directly into the anterior cervical muscle to promote cartilage maturation and finish prevascularization for 28 days. LA@PA-TA@C hydrogel was synthesized following a specific chemical protocol and then applied to the inner wall of the TEC to construct a bioengineered TEC/LA@PA-TA@C tracheal graft. n = 6 rabbits in each group for one experiment, and the experiments were repeated independently for two times.

The construction method of tissue-engineering trachea (TET) was basically the same as the above. The expanded P3 chondrocytes were centrifuged at 200 g for 10 min to pellet the cells. The cells were resuspended in a 10% solution of methylacrylyl anhydride gelatin (GelMA, Engineering For Life, Suzhou, China) and then cast into specific ring-shape molds with 6 mm internal diameter. These rings were stacked into a tubular construct and then implanted anterior cervical muscle to promote cartilage maturation and routine prevascularization for 28 days, ultimately forming TET. n = 3 rabbits in each group for one experiment, and the experiments were repeated independently for two times.

Rabbit trachea transplantation

After anesthesia induced by Zoletil (50 mg/kg, Virbac, France), the skin and fascia in New Zealand rabbits were sequentially separated and incised until the trachea was fully exposed.

In tracheal partial tracheal wall defect model. The TEC graft embedded before was then isolated from adjacent tissue carefully with the vascular pedicle kept at one end. Subsequently, the tracheal TEC/LA@PA-TA@C graft was prepared, and tracheal graft via only TEC was used as the control group. An anterolateral trachea was cut off (10 mm × 10 mm in size) and different groups of bioengineered graft were transplanted to defect area. The incision was then closed, and the fascia and skin were sutured by layer.

In long circumferential tracheal defect model. The TET graft embedded before was then isolated from adjacent tissue carefully with the vascular pedicle kept at one end. Subsequently, the tracheal TET/LA@PA-TA@C graft was prepared, and tracheal graft via only TET was used as the control group. A circumferential trachea was cut off (15 mm in length) and different groups of bioengineered graft were transplanted to defect area via end-to-end Anastomosis to simulate tracheal transplantation clinical scenario. The incision was then closed, and the fascia and skin were sutured by layer.

Respiratory function monitoring

Respiratory rates of the animals were monitored every 2 days. The respiratory rate was recorded at three different time points during the day: morning (8:00 AM), midday (12:00 PM), and evening (6:00 PM), to account for any diurnal variations in breathing patterns. Prior to each recording session, animals were allowed to acclimate to the testing environment for a period of 15–20 min to minimize stress and ensure they were in a calm state. Recorded the number of breaths taken by the animal over the 10 min observation period during which animals were in a quiet, undisturbed state. The total number of breaths recorded during the three time points was averaged as the respiratory rate for the day.

RNA-sequencing and data analysis

RNA-Sequencing (RNA-Seq) was finished via standard sequencing procedures (Cloud-seq Company, China) on 14 days post operation in rabbit epithelial injury model. The blank group referred specifically to the untreated blank samples (only received epithelial injury model. In Fig. 4, the upregulated genes referred to those that show significantly higher expression in the LA@PA-TA@C group relative to the blank group, while the downregulated genes were those with significantly lower expression in the LA@PA-TA@C group compared to the blank group. Genes were considered to be expressed in a sample if value was greater than or equal to that of 1 in the sample. Differentially expressed genes (DEGs) were defined as fold change ≥ 2 and P value ≤ 0.05. The raw sequencing data initially underwent quality assessment utilizing FastQC. Gene ontology analysis was performed by using DAVID and REVIGO (https://david.ncifcrf.gov; http://revigo.irb.hr/). Target gene screening was based on GeneCards dataset (https://www.genecards.org/). n = 3 biologically independent samples. The visualization of these differentially expressed genes was achieved through heatmaps generated in R and Sangerbox. Additionally, GO and pathway (KEGG) analyses were conducted with the aid of Gene Set Enrichment Analysis (GSEA) software to further elucidate the biological implications of the gene expression data.

CT scanning

Prior to the CT scanning, animals were fasted for 12 h. After being anesthetized by Zoletil (50 mg/kg, Virbac, France), the animals were positioned supine on the CT scanning table with their heads secured in a custom-made immobilization device to maintain a consistent and reproducible scanning position.

Bronchoscopy

Rabbits were fasted for 12 h prior to the bronchoscopy examination. Anesthesia was performed using Zoletil (50 mg/kg, Virbac, France) to ensure a stable plane of anesthesia throughout the procedure. The rabbits were positioned in dorsal recumbency with the neck slightly extended to facilitate access to the trachea. Then a flexible bronchoscope (diameter and length appropriate for the size of the rabbit) equipped with a light source and a camera was used for the examination. The tongue was gently pulled forward to expose the glottis. The bronchoscope was inserted through the mouth, passed over the glottis, and advanced into the trachea. The tracheal lumen and mucosa were inspected for any abnormalities and relative images and videos were captured using the bronchoscope’s camera for documentation and further analysis.

ROS production (Flow cytometry method)

Cells were digested and collected (1 × 10⁵ per group), resuspended with buffer (PBS containing 2% FBS), and collected in 1.5 ml EP tubes. 200 μL Fc-Block was firstly added and incubated at 4 °C for 10 min, and then add buffer to terminate the reaction. Subsequently, 2 μL DCFH-DA probe were added and incubated at 4 °C for 20 min. Finally, cells were washed for 2 times with buffer and resuspended. The samples were tested using a FACSVerse flow cytometer (BD Biosciences, Franklin Lakes, USA). Compare with the blank control group to distinguish the positive cells.

Quantitative real-time PCR

Total RNA was extracted from cultured cells using the TRIzol reagent (Takara Bio, Kyoto, Japan). The integrity and purity of the extracted RNA were assessed using a NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific). Complementary DNA (cDNA) was synthesized from 1 μg of total RNA using a commercial reverse transcription kit (Takara Bio). Quantitative PCR was performed to assess the expression levels of ZO-1, CK14, E-Cadherin, α-SMA, and Caspase 9. Specific primers were designed to target these genes and are listed in Supplementary Table 3.

Histological and immunohistochemical analysis

After euthanizing the animals, the samples were carefully isolated, harvested, and fixed in 4% paraformaldehyde for 24 h. The specimens were then dehydrated with a graded ethanol series and embedded in paraffin. after that, 8 μm thick sections were obtained. Hematoxylin and eosin (H&E), alcian blue, sirius red, masson trichromatic, mitochondrial membrane potential staining (JC-1), and periodic acid-schiff (PAS) staining were applied using corresponding staining kit (Solarbio, China). Staining was performed under recommendations of the kits. Using immunofluorescent staining, the expression of MPO, PCNA, TUNEL, CD86, CD206, CD80, AC-Tub, MUC5ac, ZO-1, CK14, Ki-67, α-SMA and SOX9 was detected under a standard IF protocol (Supplementary Table 4). The standard IF protocol is listed as follows: all samples were washed with PBS for 10 min, permeabilized with 1% Triton X-100 (Sigma-Aldrich, USA) for 5 min, washed with PBS, and then blocked with 5% BSA (Sangon Biotech, Shanghai, China) in PBS. The sections were incubated with primary antibodies overnight at 4 °C. They were then washed three times with PBS, followed by incubation with the fluorescent secondary antibodies for 1 h. DAPI was used to visualize the nuclei. Samples from three independent experiments were examined with a fluorescence microscope (Olympus IX73; Olympus, Japan). ImageJ 1.8 software (National Institutes of Health) was used to quantify the number of positive numbers or areas in each view.

Statistical analysis

All the results were exhibited as the mean ± standard deviation (SD) values. p values calculated by one-way ANOVA with Tukey’s post hoc test for multiple comparisons (≥3 groups) or two-tailed unpaired t-test for two-group comparisons. Statistical analysis was conducted with GraphPad Prism software (8.0), and p < 0.05 was considered to indicate statistical significance.

Reporting summary

Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.

Data availability

All data supporting the findings of this study are available within the article and its supplementary files. Any additional requests for information can be directed to, and will be fulfilled by, the corresponding authors. Source data are provided with this paper. The RNA sequence data generated in this study have been deposited in the GEO database under accession code GSE297254. Source data are provided with this paper.

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Acknowledgements

This study was supported by the Noncommunicable Chronic Diseases-National Science and Technology Major Project (2024ZD0529005, awarded to C.C.), the National Natural Science Foundation of China (82022033, awarded to K.Z.; 824B2061, awarded to Y.C.), the National Key Research and Development Program of China (2024YFC3044600, awarded to C.C.), Sichuan Science and Technology Program (2024NSFJQ0048, awarded to K.Z.), Shanghai Municipal Health Commission (2023ZZ0202, awarded to C.C.), Ningbo Top Medical and Health Research Program (No.2022030208, awarded to M.L.Y.). Thanks for the Young Elite Scientists Sponsorship Program (1st Doctoral Student Special Program) by CAST.

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These authors contributed equally: Yi Chen, Hai Tang, Yan Zhang, Lei Wang.

Authors and Affiliations

Department of Thoracic Surgery, Shanghai Pulmonary Hospital, School of Medicine, Tongji University, Shanghai, China
Yi Chen, Hai Tang, Lei Wang, Qingfeng Bai, Ziyin Pan, Long Wang, Lei Zhang, Yunlang She, Weiyan Sun & Chang Chen
Central Laboratory and Department of Medical Ultrasound, Sichuan Academy of Medical Sciences, Sichuan Provincial People’s Hospital, School of Medicine, University of Electronic Science and Technology of China, Chengdu, Sichuan, China
Yan Zhang, Binyu Yin, Yu Liang, Xiulin Dong & Kun Zhang
Shanghai Engineering Research Center of Lung Transplantation, Shanghai, China
Lei Wang, Qingfeng Bai, Ziyin Pan, Long Wang, Lei Zhang, Yunlang She, Weiyan Sun & Chang Chen
Center for Life Science, Tsinghua University, Beijing, China
Jun Zhu
Department of Respiratory and Critical Care Medicine, Shanghai Pulmonary Hospital, Institute of Respiratory Medicine, Tongji University School of Medicine, Shanghai, China
Lele Wang & Anqi Li
School of Life Sciences, Fudan University, Shanghai, China
Xiang Zeng
Department of Cardiothoracic Surgery, Ningbo No.2 Hospital, Ningbo, Zhejiang, China
Minglei Yang

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Yi Chen
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Contributions

Y.C., H.T., Y.Z. and Lei. W. contributed equally to this work. Y.C., C.C., and K.Z. provided the idea. Y.C., H.T., Y.Z. and W.Y.S. designed the experiments. Methodology: Y.C., Lei. W., L.Z., L.L.W., A.Q.L., X.L.D., L.Y., B.Y.Y, Long.W. and Y.L.S.; Visualization: Y.C., J.Z., X.Z., Lei. W., Q.F.B, and Z.Y.P.; Funding acquisition: C.C., K.Z., M.L.Y. and Y.C.; Writing: Y.C., H.T., W.Y.S. and K.Z.

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Correspondence to Weiyan Sun, Kun Zhang or Chang Chen.

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Chen, Y., Tang, H., Zhang, Y. et al. Multiplexed self-adaptable Janus hydrogels rescue epithelial malfunction to promote complete trachea repair. Nat Commun 16, 5734 (2025). https://doi.org/10.1038/s41467-025-61135-z

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Received: 30 October 2024
Accepted: 13 June 2025
Published: 01 July 2025
DOI: https://doi.org/10.1038/s41467-025-61135-z