A bio-adaptive physical hydrogel enables dynamic tissue engineering for tracheal reconstruction

Domain: biology
Published: 2025-12-14
Editorial outcome: major_revision

Abstract
Organ functionalization is inherently complex and dynamic, involving multi-layered tissue structures and continuous cellular remodeling. To address the clinical need for long-segment tracheal reconstruction, we propose a dynamic tissue engineering (DTE) strategy using a bio-adaptive physical hydrogel (BP-Gel) to emulate native tracheal development and enable dynamic regeneration of key tissue components. Here we show that chondrocytes cultured within BP-Gel form cartilage rings through an embryo-like chondrification process, during which the gel’s percolation network adapts to cell migration and aggregation. The resulting cartilage exhibits a native-like multilayered morphology that enhances mechanical stability and resists degradation. Before transplantation, BP-Gel loaded with anti-inflammatory cytokines (IL-Gel) is introduced into inter-ring spaces to suppress inflammation and promote vascularization and epithelial maturation. In a rabbit tracheal defect model, this strategy reconstructs a functional trachea mimicking native structure and physiology, offering a promising, clinically relevant route to tracheal reconstruction.

Introduction
Long-segment tracheal defects caused by tumors, infections, or congenital diseases are often associated with life-threatening complications, leading to a severe impairment in patients’ quality of life and increased mortality rates1–4. In such cases, tracheal replacement remains the sole viable therapeutic option to save lives5,6. Despite this urgent need, no clinically approved trachea substitutes are currently available. The native trachea has a complex structure featuring cartilage rings for radial support, a transmural blood supply, and a defensive airway epithelium. Although a biomimetic tracheal substitute incorporating those functional elements has been developed and has shown promising results in preclinical studies7,8, several challenges still hinder its clinical application. For example, the cartilage tissue generated via traditional scaffold-seeding methods lacks the intricate structural complexity of native cartilage, posing a risk of degradation during long-term implantation. Therefore, the trachea substitutes reconstructed from these artificial cartilages are vulnerable to loss of function due to structural collapse, which is further exacerbated by local inflammation or foreign body reactions.

In higher organisms, the formation and functional development of native organs are driven by a dynamic process in which the proliferation and migration of diverse cell types are regulated in a spatiotemporal-dependent manner, followed by the secretion of extracellular matrix (ECM) proteins and gene-phenotype transformations9–11. These dynamic processes result in continuous changes in the physical or biochemical microenvironment in the extracellular space, which in turn guide the functional development and maturation of the entire organ12,13. Therefore, we propose a dynamic tissue engineering (DTE) strategy to recapture the dynamics of native organ development by exploiting bio-adaptive biomaterials or scaffolds that can actively and continuously interact with the developing tissue throughout the regeneration window. However, to date, few biomaterials or scaffolds have been designed to coordinate with cellular or tissue dynamics in their microenvironment to enable the biomimetic reconstruction of complex organs like the trachea14,15. Hydrogels possess a three-dimensional (3D) polymer network architecture that mimics the native ECM and can serve as reservoirs for the sustained delivery of bioactive therapeutics16,17. They have been widely utilized in organ/tissue regeneration and drug delivery18,19. In contrast to stable 3D networks in chemically-crosslinked hydrogels, a gel network formed by weak physical interactions, e.g., hydrophobic interactions and host-guest interactions, easily undergoes dynamic changes in response to external stimuli20,21. The flexible, physically crosslinked network allows cells to actively remodel their niche for optimal cellular proliferation, migration, differentiation, and subsequent tissue growth and development19,22.

Inspired by these findings, a bio-adaptive physical hydrogel (BP-Gel) composed of thermosensitive and biodegradable poly(lactic acid-co-glycolic acid)-poly(ethylene glycol)-poly(lactic acid-co-glycolic acid) (PLGA-PEG-PLGA) triblock copolymers was developed to reconstruct a native-like trachea in this study. BP-Gel is a free-flowing sol at room temperature and spontaneously turns into a semi-solid physical hydrogel at body temperature mediated by enhanced hydrophobic interactions. The sol–gel transition behavior and adaptive percolated network of BP-Gel offer a thermally responsive and mechanically adaptive platform well-suited for dynamic structural rearrangement during trachea regeneration. Furthermore, BP-Gel also enables mild and efficient encapsulation of interleukin molecules while achieving sustained release in situ, thereby establishing precise immune niches conducive to trachea regeneration (Fig. 1a).

Specifically, the physical percolation network in the BP-Gel could dynamically adapt to the migration, proliferation, and aggregation of chondrocytes, thus facilitating the fusion of chondrocyte clusters into large aggregates. This procedure resembles the embryo-like chondrification process, in which undifferentiated mesenchymal cells migrate, proliferate, and condense to form cartilage primordia23–25. This dynamically adaptive culture environment induced a native-like stratified morphology in the engineered cartilage, resulting in enhanced mechanical properties and improved resistance to degradation in vivo. We refer to this type of cartilage as biomimetic cartilage (BioC) because it shares both structural and functional similarities with native tracheal cartilage. Cartilage rings derived from BioC were thus employed as the main mechanical support for trachea reconstruction. Next, to generate a dynamic regenerative niche, a second BP-Gel loaded with the anti-inflammatory cytokines interleukin-4 (IL-4)/IL-13 (denoted as IL-Gel) was applied to seal the interstices between BioC rings before heterotopic transplantation into the platysma muscle. Continuous interleukin delivery activated an immune cell-mediated cascade that substantially reduced local inflammation while stimulating coordinated regeneration of trachea components, including the formation of transmural vascular networks and the development of airway epithelium. After four weeks of dynamic reconstruction in vivo, a native-like trachea substitute was expeditiously obtained for implantation into the recipient. Finally, the long-term functionality and translational potential of the trachea substitute were extensively evaluated using a rabbit tracheal defect model.

Results
Fabrication and characterization of BP-Gel and IL-Gel
The triblock copolymer PLGA-PEG-PLGA, whose molecular structure is shown in Supplementary Fig. 1a, was synthesized by ring-opening copolymerization of D,L-lactide and glycolide using PEG as the macroinitiator. 1H nuclear magnetic resonance (1H-NMR) analysis verified that the number average molecular weight of PLGA-PEG-PLGA was 1750-1500-1750 and the ratio of lactic acid to glycolic acid in PLGA blocks was 3.2:1 (Supplementary Fig. 1b). Gel permeation chromatography (GPC) confirmed that the molar mass dispersity (Ðm) of the synthesized copolymers was 1.23 (Supplementary Fig. 1c).

The gelation mechanism of BP-Gel is attributed to the self-assembly of PLGA-PEG-PLGA polymers to micelles with PEG as the shells and PLGA as the cores in water, followed by the formation of a percolated micelle network via temperature-induced micelle aggregation17, as illustrated in Fig. 1b. This result was further confirmed by transmission electron microscopy (TEM) observations (Fig. 1c) and dynamic light scattering measurements (Supplementary Fig. 1d). When the polymer concentration reached 5 wt% or above, the PLGA-PEG-PLGA polymers were soluble in water at low or room temperatures and underwent a sol–gel transition as the temperature increased, as presented in Fig. 1d. Furthermore, dynamic rheological measurements showed that the BP-Gel system composed of 12.5 wt% polymer aqueous solution indeed exhibited a sol-to-gel transition upon heating (Supplementary Fig. 1e) and formed a stable gel at body temperature (37°C) (Supplementary Fig. 1f). Moreover, as the polymer concentration increased, the phase transition temperature decreased and the modulus of the gel formed at 37°C increased (Fig. 1d and Supplementary Fig. 1g).

Subsequently, Live/Dead staining assays demonstrated that the proliferation of cells was not affected by treatment with 1 wt% PLGA-PEG-PLGA solution, indicating the good cytocompatibility of the synthesized copolymer. Live/Dead staining with Hoechst 33342 confirmed high cell viability (Supplementary Fig. 1h) and enabled the visualization of cell nuclei. Ki-67 immunofluorescence (IF) staining on Days 1, 4, and 7 showed comparable proliferative activity among control, GelMA, and BP-Gel groups, supported by quantification of Ki-67-positive cells (Supplementary Fig. 1i,j). Cell number counting, DNA content, and Cell Counting Kit-8 (CCK-8) assays further confirmed the lack of significant differences in proliferation across groups (Supplementary Fig. 1k–m). In addition, BP-Gel with a polymer mass fraction of 25 wt% was subcutaneously injected into the dorsal region of ICR mice to assess its in vivo degradability. Anatomical observations and weighting analysis revealed that the BP-Gel system was maintained in vivo for over one month (Supplementary Fig. 1n). Non-invasive fluorescence imaging also supported these findings (Supplementary Fig. 1o).

The interleukin molecules IL-4 and IL-13, whose structures are illustrated in Supplementary Fig. 1p, were expediently encapsulated into BP-Gel composed of 25 wt% polymer solution by simply mixing them at the sol state, and the resulting system was known as IL-Gel. In vitro release tests showed that IL-4/13 could be continuously and steadily released from BP-Gel, with the cumulative release amount exceeding 80% within 4 weeks (Fig. 1e). Finally, the IL-Gel system containing Cy5.5-labeled interleukin molecules was subcutaneously injected into ICR mice, and the release of the drug in vivo was confirmed via intravital fluorescence imaging. As presented in Fig. 1f,g, the gradual decrease in fluorescence intensity indicated the slow and sustained release of interleukin molecules in vivo. In addition, routine blood and biochemical tests showed that the implantation of IL-Gel and subsequent release of interleukin molecules caused no adverse systemic effects (Supplementary Fig. 2).

Construction and characterization of BioC
BioC was constructed by seeding chondrocytes into BP-Gel (12.5 wt%) and culturing them in chondrogenic medium, with GelMA as a control. Concentration screening (6.25–25 wt%) showed that low concentrations led to structural instability and cell sedimentation, while high concentrations produced excessive stiffness and reduced porosity, resulting in scattered cells. Thus, 12.5 wt% was selected as the working concentration (Supplementary Fig. 3a). BP-Gel also maintained stable gelation at 37°C with gradual degradation, unlike Pluronic F127, which degraded completely within 72 h (Supplementary Fig. 3b). Bright-field and confocal microscopy confirmed that chondrocytes aggregated into clusters in BP-Gel, whereas they remained scattered in GelMA (Fig. 2a and Supplementary Fig. 4a). Quantitative analysis further confirmed that the number of chondrocyte clusters in BP-Gel was significantly greater than that in GelMA at all observation points (Fig. 2b). This interesting phenomenon of spontaneous chondrocyte aggregation in BP-Gel was also verified by a dynamic video (Supplementary Movie 2). Based on this, a schematic of cell growth status in the two different gel systems is proposed, as displayed in Fig. 2c.

BP-Gel was formed by the aggregation of micelles, in which hydrophilic PEG segments containing many ether bonds constituted the shells of the micelles and were exposed to the aqueous environment. We speculate that numerous water molecules bind to the PEG shells via hydrogen bonding, creating an anti-adhesion microenvironment26,27, as illustrated in Supplementary Fig. 4b. This microenvironment prevents chondrocytes from adhering to the gel network, thereby forcing them to aggregate. To verify this hypothesis, chondrocytes were cultured on the surface of BP-Gel. As expected, unlike the chondrocytes cultured on ordinary plates (2D culture), the cells grown on the BP-Gel surface did not adhere well, resulting in the formation of regular spherical shapes (Supplementary Fig. 4c).

We subsequently evaluated the expression of adhesion-related genes (Lama, Fn1, Cdh2, and Gja1) (Supplementary Fig. 4d) and cartilage-related genes (Acan, Col2a1, and Sox9) (Fig. 2d). The results showed that the expression levels of these genes in chondrocytes grown in BP-Gel were significantly up-regulated compared with those cultured on plates or in GelMA, indicating that culturing chondrocytes in BP-Gel promotes their aggregation and fusion, thus facilitating cartilage formation. Consistently, Western blotting confirmed higher levels of adhesion-related proteins in the BP-Gel group, and Hematoxylin and Eosin (H&E) and IF staining further demonstrated markedly stronger expression of Laminin subunit alpha-1 (LAMA1), Fibronectin (FN1), N-cadherin (CDH2), and Connexin-43 (GJA1), with quantitative analysis showing significant increases in all four markers (Supplementary Fig. 4e–h).

Next, we explored the process of chondrocyte development in BioC. IF staining revealed that the expression of Collagen type II alpha 1 (COL2A1) and Hypoxia-inducible factor 1-alpha (HIF-1α) was significantly increased within the chondrocyte clusters formed in BP-Gel (Fig. 2e and Supplementary Fig. 5a, showing an amplified region of Fig. 2e). Colocalization analysis based on protein expression intensity demonstrated that the expression levels of these two proteins were highly coincident (Fig. 2f and Supplementary Fig. 5b). To further substantiate this point, we examined cell spheroids, a recognized model of chondrocyte development. IF staining showed high expression of HIF-1α and COL2A1 in cell spheroids, which closely resembled the expression patterns observed in BioC cultured within BP-Gel (Supplementary Fig. 5c). These findings, combined with previous studies28–30, suggest that the formation of chondrocyte clusters creates a hypoxic environment and stimulates the expression of HIF-1α, which in turn promotes the development of chondrocytes and the secretion of the cartilage matrix. Similarly, immunohistochemical (IHC) staining manifested that Yes-associated protein 1 (YAP), a protein that regulates matrix secretion in response to mechanical signaling to promote cartilage development and tissue remodeling, was expressed at higher levels in chondrocyte clusters compared with the surrounding scattered chondrocytes (Supplementary Fig. 5d). Building on these findings, we performed additional IF staining of chondrogenic markers using GelMA as a control and cell spheroids as a reference. Chondrocytes cultured in BP-Gel showed markedly stronger SOX9 and COL2A1 expression than those in GelMA, with quantification confirming higher SOX9 and COL2A1 levels and an increased proportion of SOX9-positive cells (Supplementary Fig. 5e–h). These results further demonstrate that the BP-Gel microenvironment promotes chondrogenic differentiation and cartilage matrix deposition.

Additionally, we found that when chondrocyte clusters formed in BP-Gel, they tended to further fuse into larger chondrocyte aggregates as the incubation time extended. This fusion further promoted the secretion of the cartilage matrix, leading to the formation of native-like cartilage tissue (Supplementary Fig. 6a). To further elucidate the early chondrogenic process within BP-Gel constructs, we analyzed 2-week samples and compared them with embryonic tracheal cartilage, which represents a developmental benchmark of native chondrogenesis. Specifically, histological analyses of mice embryonic trachea at E12.5, E14.5, and E16.5 using H&E, Alcian Blue-Nuclear Fast Red (AB&NFR), Safranin O-Fast Green (SO&FG), and SOX9 IF revealed progressive mesenchymal condensation and early matrix deposition, thereby providing a reference for evaluating the developmental stage of BP-Gel-induced cartilage (Supplementary Fig. 6b). Figure 2g shows a typical fusion process where small chondrocyte clusters fused into a larger chondrocyte cluster. Conversely, the chondrocytes cultured in GelMA remained dispersed throughout a 4-week in vitro culture period (Supplementary Fig. 6a). These encouraging findings are attributed to the weak physical gel network formed in BP-Gel and the presence of micelles with numerous exposed PEG shells, which provide a dynamic adaptive environment that facilitates the aggregation and fusion of chondrocytes. In contrast, the robust chemically-crosslinked gel network in GelMA and its slow in vitro degradation result in the firm immobilization of chondrocytes within the hydrogel matrix, thereby hindering their aggregation and subsequent fusion.

Encouraged by these in vitro results, BioC samples that had been cultured in vitro for 4 weeks were implanted subcutaneously into nude mice. Following this process, BioC gradually began to resemble native cartilage. Histological analyses confirmed that after 4 weeks of in vivo culture, the collected BioC exhibited cartilage-specific extracellular matrix (ECM) deposition, as demonstrated by H&E, AB&NFR, and COL2A1 IHC staining, which qualitatively assessed tissue morphology, GAG-rich matrix deposition, and chondrogenic zonal distribution (Fig. 2h). Quantitative detection of the cartilage lacunae size in each group disclosed that the cartilage structure formed in BP-Gel was similar to that of natural cartilage (Fig. 2i). Measurements of DNA and hydroxyproline contents suggested that BioC has biochemical characteristics comparable to those of native cartilage (Fig. 2j,k).

Supplementary Fig. 7a–e presents macroscopic views, histological staining images and biochemical parameters of BioC at 2 and 4 weeks post-implantation. The continuous increase in lacunae structures and matrix content reflected the gradual maturation of cartilage in BP-Gel.

Native cartilage exhibits a distinct “cortex-medulla” structure, ensuring the structural integrity and functional adaptability required for cartilage development and load-bearing performance31,32. To further assess the structural organization of cartilage matrix, Sirius Red (SR) staining for COL2A1 and 3D reconstruction plots demonstrated that the distribution of COL2A1 within BioC based on BP-Gel closely resembled the characteristic grid-like COL2A1 arrangement in the native cartilage; however, no similar COL2A1 distribution was observed in GelMA (Fig. 2l). Moreover, the BioC samples obtained from different culture time points were also subjected to SR staining. As shown in Fig. 2m–o, SR staining of BioC at different growth stages revealed progressive fusion of chondrocyte clusters, continuous increase in COL2A1 deposition, and gradual thickening of the cortical layer, reflecting the dynamic formation of a “cortex-medulla” structure.

To elucidate the biological function of the formed cortical layer, BioC samples harvested after 4 weeks of in vivo culture were bisected and then implanted subcutaneously into nude mice (Fig. 2p). The side with an intact cortical layer exhibited well-preserved cartilaginous characteristics (Fig. 2q,r). In contrast, on the side devoid of the cortical layer, numerous surrounding cells had infiltrated into BioC, resulting in the degradation of cartilage. The results of IF staining further confirmed that the expression of COL2A1 was notably higher on the side with the retained cortex layer, while both cell density and Matrix metallopeptidase 13 (MMP13) expression markedly increased on the side where the cortex had been removed (Fig. 2s,t). These findings suggest that the formed cortical layer exerts a protective effect on inhibiting cartilage degradation.

Subsequently, tracheal BioC rings were obtained using a process similar to the construction of bulk BioC. Supplementary Fig. 7f and Supplementary Movie 1 show the macroscopic morphology of the generated tracheal BioC rings after different in vivo culture times. Mechanistic analysis further revealed that the “cortex-medulla” structure could effectively alleviate stress concentration within the ring architecture (Supplementary Fig. 7g). Notably, the stress concentration is detrimental to the stability of the trachea structure and can even lead to the collapse of the reconstructed trachea.

The ability of IL-Gel to induce macrophage polarization and modulate the local microenvironment in vivo
We designed a series of in vitro experiments to explore the local immunomodulatory effects induced by IL-Gel. M0 macrophages were utilized as the control, while the “LPS-M0 macrophage” model was used to mimic an inflammatory environment.

Cell morphology was first evaluated after various treatments (Supplementary Fig. 8a). Typically, M1 macrophages exhibit an irregular polygonal shape with many filopodia. In contrast, M2 macrophages display a spindle-like morphology characterized by prominent lamellipodia, resulting in a higher aspect ratio (Supplementary Fig. 8a–c). The results revealed that most of the macrophages exhibited an M2 phenotype after treatment with IL-Gel, with the highest aspect ratio (Supplementary Fig. 8a–c). Subsequently, we performed fluorescence staining of the cytoskeleton to visualize lamellipodia, and the statistical analysis of lamellipodium numbers was well consistent with the proportions of macrophage phenotypes in various groups (Supplementary Fig. 8d–f).

Figure 3a displays representative fluorescence micrographs of macrophages cultured in BP-Gel with or without IL-4/13, labeled with CD86 (a specific marker for M1 macrophages) and Macrophage mannose receptor (MMR) (a specific marker for M2 macrophages). Quantitative analysis revealed that the IL-Gel-stimulated macrophages exhibited reduced CD86 expression but the highest level of MMR expression (Fig. 3b). Subsequently, we analyzed these markers via flow cytometry (Fig. 3c), which demonstrated that IL-Gel induced less M1 phenotypic transformations but more M2 phenotypic transformations in macrophages (Supplementary Fig. 8g,h). Moreover, we performed PCR measurements to evaluate the expression of genes related to macrophage polarization under different induction conditions. The results showed that IL-Gel reduced the expression of M1 phenotype-related genes (Tnf and Il1b) while promoting the expression of M2 phenotype-related genes (Il10 and Mrc1) (Fig. 3d).

The in vivo functions of IL-Gel were further evaluated after subcutaneous injection into ICR mice, and the treatment timeline is presented in Supplementary Fig. 8i. Supplementary Fig. 8j shows representative micrographs of H&E-stained slices of residual hydrogels containing surrounding tissues from 14 to 28 days post-injection. BP-Gel without IL-4/13 induced an acute inflammatory response, resulting in the infiltration of inflammatory cells into the surrounding tissue. In contrast, no obvious inflammatory cell infiltration was observed in the surrounding tissue of IL-Gel.

CD86/MMR IF staining was also performed on the surrounding tissues. The results revealed a higher proportion of M1 macrophages around IL-4/13-free BP-Gel, while more M2 macrophages were observed around IL-Gel (Supplementary Fig. 8k).

Subsequently, we performed IF staining using Lymphocyte antigen 6 complex locus G6D (Ly6G) (a specific marker for neutrophils) and CD45 (a specific marker for lymphocytes). The results demonstrated that the presence of IL-Gel significantly mitigated the infiltration of both lymphocytes and neutrophils, indicating its favorable anti-inflammatory effects (Supplementary Fig. 8l).

Furthermore, IF staining for the endothelial cell-related marker (Platelet/endothelial cell adhesion molecule 1, PECAM1) was conducted on the two groups. The IL-Gel group exhibited more functional blood vessels with an orderly arrangement (Supplementary Fig. 8m–o). These findings suggest that IL-Gel promotes endothelial cell growth. The results of tissue IHC staining also coincided with those of IF staining (Supplementary Fig. 9). Taken together, IL-Gel exerts a comprehensive regulatory effect on the microenvironment at the implantation site.

Enhanced development of cartilage, endothelium and epithelium in vitro through IL-Gel-induced immune cascades
We first established a co-culture system to examine the direct effects of IL-Gel on macrophage polarization, thereby confirming the ability of IL-4/13-loaded IL-Gel. We quantified cytokine levels in the culture medium of macrophages subjected to different treatments, as illustrated in Supplementary Fig. 10a. The results demonstrated that the presence of IL-4/13 significantly increased the concentrations of TGF-β1, TGF-β3, IGF-1, and bFGF in the culture medium (Fig. 3e). Additionally, treatment with IL-4/13 also reduced the secretion of CCL2. These findings suggest that IL-4/13-treated macrophages can release beneficial factors that promote tissue regeneration while mitigating inflammatory responses.

To mimic the in vivo microenvironment, building upon this, we further established a co-culture system to co-culture macrophages with chondrocytes, endothelial cells, or epithelial cells (Fig. 3f). The results illustrate the functional consequences of IL-Gel-induced macrophage polarization, demonstrating how polarized macrophages subsequently regulate the growth and development of chondrocytes, endothelial cells, and epithelial cells. First, we stained chondrocytes in the co-culture system with toluidine blue, revealing that IL-Gel-induced macrophage polarization promoted chondroitin production (Fig. 3g–i). Further IF staining of COL2A1 demonstrated that macrophage polarization significantly enhanced the development of chondrocytes (Fig. 3j,k). Similarly, the expression of cartilage-related genes, including COL2A1 and SOX9, was significantly up-regulated following IL-Gel stimulation (Supplementary Fig. 10b). These supportive data suggest that chondrocytes can differentiate and undergo chondrogenesis in response to IL-Gel-induced M2 macrophage polarization.

Subsequently, we investigated the effects of IL-Gel-induced macrophage polarization on the growth and development of human umbilical vein endothelial cells (HUVECs). Representative fluorescence micrographs of PECAM1 demonstrated that IL-Gel-induced macrophage polarization significantly promoted endothelial cell-related protein secretion (Fig. 3l,m). Meanwhile, the expression of endothelial cell-related genes (PECAM1 and VEGFA) was also significantly up-regulated (Supplementary Fig. 10c). Moreover, scratch assays and tube formation assays discovered that the presence of IL-Gel substantially boosted the migratory ability of HUVECs (Supplementary Fig. 10d,e) and promoted angiogenesis (Supplementary Fig. 10f). These results suggest that IL-Gel facilitates well-organized angiogenesis.

Finally, we explored the effects of IL-Gel-induced macrophage polarization on the growth and development of epithelial cells in the co-culture system. Scratch assays revealed that there was no difference in the viability of epithelial cells under different co-culture conditions; however, IL-Gel-induced macrophage polarization significantly promoted the migration of epithelial cells (Supplementary Fig. 10g–j). In addition, to verify the impact of macrophage polarization on epithelial regeneration, Transwells were used to establish a air-liquid interface co-culture system (Fig. 3n). The results showed that the presence of IL-Gel significantly increased the protein expression of Keratin, type I cytoskeletal 14 (KRT14), a marker of epithelial stemness, and the expression of the epithelial ciliary marker acetylated tubulin (AC-Tub) (Fig. 3o). Scanning electron microscopy (SEM) observations also revealed enhanced ciliary coverage (Fig. 3o). Quantitative analysis further confirmed that IL-Gel significantly enhanced epithelial regeneration, characterized by the formation of a thicker membrane and more cilia (Fig. 3p,q).

Promoted the phenotype maintenance and angiogenesis of BioC through IL-Gel-induced immune cascades in vivo
After a 4-week in vitro culture of chondrocyte-laden BP-Gel (BioC), the BioC was encapsulated with IL-Gel to form the BP@IL-Gel system, which was then implanted into the platysma muscle of rabbits, as shown in Fig. 4a. Given the anti-inflammatory effects of IL-Gel, this design is expected to create an anti-inflammatory environment around BioC (Fig. 4b). We also employed BioC (the BP-Gel group), chondrocyte-laden GeMA (the GelMA group) and chondrocyte-laden GeMA combined with cell-free GelMA coverage (the GelMA@Blk group) as the control groups (Fig. 4a and Supplementary Fig. 11a).

After 2 weeks of in vivo implantation, histological staining provided clear evidence of cartilage-specific matrix deposition. AB&NFR staining and COL2A1 IHC staining consistently showed that the BP@IL-Gel composite exhibited more cartilage-specific ECM deposition among the four groups (Fig. 4c and Supplementary Fig. 11b–d). To further quantify these observations, we analyzed cartilage lacunae based on the stained tissues and found that the number of lacunae was highest in the BP@IL-Gel group (Fig. 4d and Supplementary Fig. 11d). In addition, biochemical assays of cartilage matrix demonstrated that relative glycosaminoglycan (GAG) and hydroxyproline contents were significantly elevated in the BP@IL-Gel group, indicating that this structural design effectively promoted maintenance of the cartilage phenotype and protected ectopic cartilage development (Fig. 4e and Supplementary Fig. 11e,f).

Subsequently, IF staining of CD86 and MMR was conducted to assess macrophage polarization in the vicinity of BP-Gel and BP@IL-Gel. The results demonstrated that, compared with the BP-Gel group, the BP@IL-Gel system induced a significant polarization of macrophages toward the M2 phenotype (Fig. 4f–h).

In addition, IF staining of COL2A1 and SOX9 and the corresponding quantitative analysis of COL2A1 revealed the obvious degradation of the cartilage matrix in the BP-Gel group (Fig. 4i). Conversely, the cartilage matrix was well-preserved in the BP-Gel group. SR staining further validated that the collagen arrangement in the BP@IL-Gel group exhibited a grid-like pattern, similar to that of native cartilage (Supplementary Fig. 11g, h). These results indicated that the chondrocytes in the BP@IL-Gel group were in a superior state than those in the BP-Gel group.

Staining of Actin, alpha 2, smooth muscle (α-SMA) can reflect cell infiltration and angiogenesis in the implant. As displayed in Fig. 4j, the absence of IL-4/13 cytokines in the BP-Gel group led to the growth of inflammation-induced blood vessels into the cartilage tissue, thereby promoting its degradation. Conversely, almost no blood vessels were observed within the cartilage tissue in the BP@IL-Gel group due to the sustained release of IL-4/13 from IL-Gel. Moreover, the increased vascular infiltration in the BP-Gel group made the cartilage more susceptible to ossification, as evidenced by SO&FG staining (Supplementary Fig. 11i). Collectively, these results suggest that IL-Gel facilitates well-organized angiogenesis while avoiding aberrant vascular infiltration into cartilage tissue.

Finally, we conducted an RNA-seq analysis of collected samples to determine the profile of immune-related gene expression (Fig. 4k). Pearson’s correlation analysis revealed a significant correlation between the BP-Gel and BP@IL-Gel samples (Supplementary Fig. 11j). A heatmap of the differentially expressed mRNAs is shown in Fig. 4l. The volcano maps showed the differentially expressed genes (DEGs) between the two groups (Supplementary Fig. 11k). GO enrichment analysis indicated that the biological processes involved were primarily related to the regulation of the immune microenvironment (Fig. 4m). KEGG pathway analysis yielded similar results (Supplementary Fig. 11l). The immune-related biological processes and DEGs were integrated to construct a GO Chord plot, revealing that THBS4, SLIT2, CAMK1D, and DAPK2 were the key genes in this process (Supplementary Fig. 11m). Additionally, we evaluated the relative expression of genes associated with crucial processes, including inflammation and angiogenesis. The analysis results indicate that BP@IL-Gel and BP-Gel exhibit distinct effects on modulating the inflammatory microenvironment and vascularization. The heat-map further allows a preliminary assessment of the up- or down-regulation trends of related genes (Fig. 4n).

DTE trachea-mediated segmental tracheal reconstruction
Multiple BioC rings that had been cultured in vitro for 4 weeks were superimposed and cast with IL-Gel to form a trachea substitute (called BP-Gel+IL-Gel), which was then heterotopically transplanted into the platysma muscle of rabbits for 4 weeks, achieving integration and vascularization. Afterwards, the trachea substitute was implanted to repair tracheal defects in rabbits (Fig. 5a). The trachea substitute constructed solely by superimposing multiple BioC rings served as the control group (called BP-Gel). A flowchart outlining the process for the fabrication of trachea substitute and subsequent segmental tracheal reconstruction is shown in Supplementary Fig. 12a.

Eight rabbits in each group underwent tracheal reconstruction surgery. In the BP-Gel group, none of the rabbits survived beyond 8 weeks. One rabbit died from tracheal obstruction following post-operative trachea hemorrhage, while the remaining seven succumbed to asphyxia caused by trachea collapse. In contrast, 62.5% of the rabbits in the BP@IL-Gel group survived beyond 8 weeks with stable respiration (Fig. 5b). In addition to the survival rate, the animals’ respiration rate, heart rate, and blood oxygen (SaO2) level were evaluated for up to 8 weeks post-surgery. The results indicated that the rabbits in the BP-Gel+IL-Gel group exhibited more favorable trends in these physiological parameters after tracheal reconstruction (Fig. 5c–e). Supplementary Tables 1 and 2 summarize the clinical outcomes of the DTE trachea-transplanted rabbits.

Figure 5f presents the surgical procedure, representative tracheoscopy images, and macrographs of the reconstructed trachea in the two groups. Supplementary Movies 3 and 4 demonstrate representative surgical procedures. In the BP-Gel group, the reconstructed trachea exhibited obvious collapse, whereas it remained patent in the BP-Gel+IL-Gel group. Quantitative analysis of the lumen inner diameter, tracheal length, and tracheal wall area revealed that the BP-Gel+IL-Gel group significantly outperformed the BP-Gel group and more closely resembled the native trachea (Fig. 5g, h and Supplementary Fig. 12b).

Figure 5i shows a longitudinal H&E-stained image of the native trachea. For the reconstructed trachea, longitudinal sections stained with H&E and AB&NFR are displayed in Fig. 5j. The results demonstrated that predominant cartilage degradation was found in the BP-Gel group. In contrast, the cartilage phenotype was well-preserved in the BP-Gel+IL-Gel group. Quantitative analysis of the cartilage area revealed that the cartilage retention rate in the BP-Gel+IL-Gel group was substantially higher than in the BP-Gel group (Fig. 5k). IHC staining of COL2A1 showed more deposition of the cartilage matrix in the BP-Gel+IL-Gel group (Fig. 5l, m). In addition, IF staining of epithelium-related markers (KRT14 and ZO-1) indicated significantly enhanced growth of the reconstructed tracheal epithelium in the BP-Gel+IL-Gel group compared with the BP-Gel group (Fig. 5n). Native rabbit trachea tissue stained with H&E, AB&NFR, COL2A1 (IHC), and KRT14+ZO-1 (IF) served as reference standards, demonstrating that the BP-Gel+IL-Gel group exhibited cartilage content and epithelial organization more closely resembling native tissue. Consistently, staining of Keratin, type II cytoskeletal 5 (KRT5) and Forkhead box protein J1 (FOXJ1) demonstrated the presence of basal progenitors and ciliated cells, while AC-Tub marked mature cilia in the regenerated epithelium (Supplementary Fig. 12c). Quantitative analysis further confirmed that epithelial cilia coverage was significantly restored in the BP-Gel+IL-Gel group compared with BP-Gel alone (Supplementary Fig. 12d).

Moreover, vascularization between tracheal cartilage rings was markedly improved in the BP-Gel+IL-Gel group. H&E and α-SMA IHC staining revealed abundant and organized vessels comparable to those in native trachea, whereas the BP-Gel group displayed smaller and disordered vessels (Supplementary Fig. 12e). Quantitative analysis demonstrated that both vessel diameter and vascular area in the BP-Gel+IL-Gel group closely resembled native trachea but remained significantly greater than in the BP-Gel group (Supplementary Fig. 12f).

Finally, the results of force simulation analysis demonstrated that the degradation of BioC rings in the BP-Gel group induced stress concentration within the reconstructed tracheal lumen, leading to tracheal collapse and subsequent blockage (Fig. 5o and Supplementary Fig. 12g). Figure 5p,q and Supplementary Fig. 12h, i present the maximum loads and maximum compressive values of the reconstructed trachea in both groups, indicating that the mechanical strength in the BP-Gel+IL-Gel group was superior to that in the BP-Gel group. The results of tensile experiments further supported this finding (Fig. 5r and Supplementary Fig. 12j).

In summary, our exploited trachea substitute showed significantly higher survival rates, enhanced physiological stability, superior mechanical strength, improved cartilage preservation, and robust epithelial growth. These findings underscore its potential for functional and durable tracheal reconstruction.

Methods
Study design
This study was designed to develop a DTE strategy for long-segment tracheal reconstruction using a BP-Gel. In the first stage, chondrocytes were cultured within BP-Gel to construct BioC rings, leveraging the hydrogel’s dynamic percolation network to support cell migration, proliferation, and aggregation, thereby recapitulating an embryo-like chondrification process. In the second stage, BioC rings were assembled and sealed with IL-Gel to establish a regenerative niche that reduced local inflammation and promoted vascular and epithelial regeneration. The engineered trachea constructs were first matured in a heterotopic site and subsequently implanted into a rabbit long-segment tracheal defect model to evaluate structural, functional, and survival outcomes. The main reagents used for cell culture are listed in Supplementary Table 3.

Synthesis and characterization of materials
Synthesis of materials. PLGA-PEG-PLGA triblock copolymers were synthesized by ring-opening copolymerization. Polyethylene glycol (PEG, MW=1500) and stannous octoate (Sn(Oct)2) were procured from Sigma-Aldrich, whereas glycolide (GA) and D,L-lactide (LA) were supplied by Hangzhou Medzone Biotech Ltd. Initially, 15 g of PEG1500 was placed in a three-necked flask and dried under vacuum at 120°C for 30 min. LA and GA were then introduced into the flask at a 7:2 molar ratio, followed by the addition of Sn(Oct)2 (0.4% w/w) as a catalyst. The reaction was carried out at 140°C under an inert atmosphere with constant stirring for 8 h. To remove unreacted monomers, the reaction mixture was subjected to a vacuum for an additional 30 min. The resulting copolymers were dissolved in water and precipitated at 80°C, and this process was repeated 3 times. Finally, the product was obtained by freeze-drying.

Characterization of PLGA-PEG-PLGA. The chemical structure and composition ratio of the synthesized copolymer were analyzed via proton nuclear magnetic resonance (1H NMR) spectroscopy on a 400 MHz NMR spectrometer (AVANCE IIIHD, Bruker). The 1H NMR spectrum was obtained at room temperature with CDCl3 (Sinopharm Chemical Reagent) as the solvent. The weight-average molecular weight (Mw) and molar mass dispersity (Ðm) value of the copolymer were determined via a gel permeation chromatography (GPC, Agilent 1260) system. The analysis was performed at a flow rate of 1.0 mL/min with tetrahydrofuran (THF) as the mobile phase. Polystyrene standards were used as calibration references.

Dynamic light scattering (DLS). The PLGA-PEG-PLGA copolymers were first dissolved in deionized water to prepare a 10 wt% solution, which was subsequently diluted to concentrations of 1 wt% and 0.1 wt%. The hydrodynamic diameters of micelles formed by the copolymers at various temperatures were measured via a Zetasizer Nano ZS90 (Malvern). An argon ion laser with a wavelength of 532 nm served as the light source, and the measurements were conducted at a scattering angle of 90°. Prior to analysis, the samples were subjected to 1 min of ultrasonic treatment to minimize aggregation, followed by filtration through a 0.45 μm filter and equilibration at each temperature for 10 min.

Transmission electron microscopy. The copolymers were dissolved in deionized water at a concentration of 0.1 wt%, and the resulting solution was drop-cast onto copper grids. Some of the grids were air-dried at room temperature, while others were dried in an air bath at 37°C. The dried copper grids were then observed and imaged using a transmission electron microscope (Tecnai G2 TWIN) at an accelerating voltage of 200 kV.

Phase diagram determination. The phase diagram of BP-Gel was determined via the vial inversion method50. Aqueous solutions of PLGA-PEG-PLGA at different mass ratios were prepared, and 0.5 mL of each solution was then transferred into 2-mL sample vials. The vials were immersed in a water bath with temperatures ranging from 15 to 50°C. The temperature was increased in 0.5°C increments. At each temperature, the samples were equilibrated for 10 min before being inverted. The sol–gel transition point was identified as the temperature at which the solution ceased to flow within 30 seconds upon inversion.

Rheological study. An advanced rotational rheometer (Kinexus, Malvern) was used to measure the storage modulus (G’) and loss modulus (G”) of BP-Gel across various temperatures and time intervals. The measurements were conducted with a strain amplitude of 1%, an angular frequency of 10 rad/s, and a heating rate of 1°C/min. When the storage modulus (G’) exceeded the loss modulus (G”), the BP-Gel system was classified as being in a gel state; conversely, it was considered to be in a sol state when G’ was lower than G”.

Live/Dead and nuclear staining. After an incubation with 1 wt% PLGA-PEG-PLGA solution for the indicated durations, followed by staining with a Live/Dead Cell Imaging Kit (Invitrogen) according to the manufacturer’s instructions. To improve image quality and provide clearer visualization of cell morphology and density, nuclei were counterstained with Hoechst 33342 (Yeasen). Fluorescence micrographs were acquired using a high-resolution fluorescence microscope.

In vivo BP-Gel formation and degradation. After sterilization of BP-Gel (25 wt%) via filtration, 0.2 mL of BP-Gel was subcutaneously injected into the dorsal region of ICR mice using a sterile syringe, resulting in the formation of an in situ hydrogel. At predetermined time points, the ICR mice were euthanized, and subsequent dissection was performed. The remaining BP-Gel was fully exposed and carefully peeled off, and the residual mass of BP-Gel was then measured.

Synthesis of fluorescent molecules. Rhodamine B was utilized as the fluorophore for fluorescence labeling of BP-Gel. Initially, 5 g of PLGA-PEG-PLGA was transferred into a round-bottom flask and dissolved in toluene to azeotropically remove any residual water. Subsequently, 1 g of Rhodamine B, 0.55 g of 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDCI), and 0.35 g of 4-dimethylaminopyridine (DMAP) were added. The reaction mixture was then dissolved in 20 mL of anhydrous dichloromethane and allowed to react under a nitrogen atmosphere for 36 hours. Following this, the mixture was washed with a 0.1 mol/L sodium bicarbonate (NaHCO3) aqueous solution and extracted with anhydrous dichloromethane. The organic phase was dried over anhydrous sodium sulfate. After concentration via rotary evaporation, the product was precipitated using anhydrous ether, and the resulting precipitate was collected. After freeze-drying, the final product was attained.

For fluorescence labeling of IL-13, Cy5.5 was employed as the fluorophore. A total of 5 mg of Cy5.5 N-hydroxysuccinimide (NHS) was completely dissolved in 0.5 mL of dimethyl sulfoxide (DMSO) and then added dropwise to a solution of 20 mg of IL-13 in 0.1 mol/L NaHCO3. The reaction was conducted in the dark at room temperature for 12 h. The mixture was subsequently subjected to dialysis using a dialysis bag with a MW cutoff of 3500 Da for 48 h, after which the solution was freeze-dried to yield the final product.

In vivo fluorescence imaging. After subcutaneous injection of Rhodamine B-labeled BP-Gel, ICR mice were monitored over time via an in vivo Xtreme (Bruker) multispectral optical imaging system. Anesthesia was maintained with isoflurane during the measurements. X-ray images were acquired with an exposure time of 1.2 s and an X-ray intensity of 45 kV. Fluorescence micrographs were captured in the same field of view, with an excitation wavelength of 560 nm, an emission wavelength of 600 nm, and an exposure time of 2 s. Similarly, after subcutaneous injection of IL-Gel containing Cy5.5-labeled IL-13, ICR mice were tracked over time using the same imaging system. The ICR mice were anesthetized with isoflurane, and X-ray images were captured under the same conditions as previously described. Fluorescence micrographs were also captured in the same field of view, using an excitation wavelength of 690 nm, an emission wavelength of 790 nm, and an exposure time of 2 s.

In vitro drug release. IL-Gel was prepared by mixing IL-4/13 at a concentration of 2 μg/mL with BP-Gel (PBS as the medium and the copolymer concentration of 25 wt%). Subsequently, 0.5 mL of IL-Gel was accurately transferred into the bottom of 10-mL test tubes (inner diameter of 14 mm) and incubated at 37°C to facilitate the formation of a physical hydrogel. Next, 5 mL of PBS (pH 7.4, 37°C) was added as the release medium. The in vitro release system was then placed in a shaking water bath at 37°C with a fixed shaking rate of 50 rpm. At 1-day intervals, an aliquot of the release medium was withdrawn and replaced with an equal volume of fresh PBS. The amount of drug released into the medium was quantified using ELISA kits (Yes Biotech Laboratories Ltd., Canada). Each group was composed of quadruplicate samples.

Experimental animals
Nude mice (BALB/c-nu/nu, male, 6 weeks of age) and ICR mice (male, 6 weeks of age) were purchased from Shanghai Slake Laboratory Animal Co., Ltd. New Zealand white rabbits (male, 4–8 weeks of age) were purchased from Shanghai Jiagan Biotechnology Co., LTD. Sex was controlled by using only male animals to minimize variability in hormone-related physiological responses. In addition, pregnant ICR mice at embryonic days E12.5, E14.5, and E16.5 used for embryo collection were also purchased from Shanghai Slake Laboratory Animal Co., Ltd. All animal experiments were performed in compliance with the Guidelines for the Care and Use of Laboratory Animals. The procedures were approved by the Ethics Committee of Shanghai Pulmonary Hospital, and all institutional and governmental regulations on animal ethics were strictly followed. Mice were housed in individually ventilated cages under controlled conditions (12-h light/dark cycle, 22 ± 2°C, 50 ± 10% relative humidity) with free access to standard chow and water.

In vitro construction and in vivo implantation of BioC
Chondrocyte extraction. Chondrocytes were extracted according to a previously described method7. Ear cartilage from 6-week-old New Zealand rabbits was harvested under aseptic conditions. After removing skin and fibrous membranes, cartilage was minced into 1 mm pieces and digested with 0.15% collagenase II for 24 h. The resulting suspension was filtered through a 70-µm strainer to isolate chondrocytes for culture.

Fabrication and in vitro culture of BioC. Chondrocytes were uniformly suspended with sterilized BP-Gel solution (12.5 wt%), which was filtered through 0.22 μm filters before use. Based on optimization experiments aimed at reducing chondrocyte usage while maintaining cartilage-forming capacity, the cell density was set at 4 × 106 cells/mL, which is substantially lower than that used in traditional culture conditions, to enable the formation of BioC. The chondrocyte-laden mixture was incubated at 37°C for 30 min to complete the sol–gel transition, and then the chondrocyte medium containing 1% ITS+ Liquid Medium Supplement (ITS+, Sigma) and chondrogenic growth factors, including 50 ng/mL IGF-1 (PeproTech) and 10 ng/mL TGF-β1 (PeproTech), was added for culture. The culture medium was changed every other day.

In vivo implantation of BioC. After 4 weeks of in vitro culture, the matured BioC constructs or BioC rings were harvested and thoroughly rinsed with sterile PBS. For heterotopic evaluation, BioC samples were implanted into subcutaneous pockets in the dorsal region of nude mice or ICR mice to assess cartilage maturation, vascular invasion, and immune compatibility. For orthotopic transplantation, BioC rings were assembled with IL-Gel to form a tubular structure and then implanted into tracheal defect models in New Zealand rabbits. The implantation sites were carefully sutured, and animals were monitored post-operatively for survival, respiratory function, and potential complications. Tissues were harvested at predetermined time points for histological, biochemical, and IHC analyses to evaluate the structural integrity, mechanical properties, and in vivo remodeling of BioC.

Construction of GelMA-chondrocyte composite and chondrocyte spheroids
Preparation of GelMA-chondrocyte composite. Chondrocytes were uniformly mixed with GelMA solution (10 wt%), which had been sterilized by filtration through 0.22 μm filters. The cell density was set at 4 × 106 cells/mL, consistent with that used for BioC. Other procedures for constructing the GelMA-chondrocyte composite were performed similarly to those for BioC.

Formation of chondrocyte spheroids. For the generation of chondrocyte spheroids, agarose microwell molds were prepared in six-well plates following the same approach as described in our previous study7. Approximately 1 × 105 chondrocytes were seeded per well, resulting in spheroids with an average diameter of 200 μm.

Construction and subcutaneous injection of IL-Gel
IL-4/13 was dissolved in BP-Gel (25 wt%) at a concentration of 20 μg/mL to obtain IL-gel. The resulting IL-gel was subsequently sterilized by filtration using 0.22 μm filters. Following sterilization, 0.2 mL of IL-Gel solution was subcutaneously injected into the dorsal region of ICR mice to facilitate the formation of an in situ gel. The ICR mice were euthanized via cervical dislocation at predetermined time points. The dorsal skin at the injection sites was carefully incised, and residual gels were photographed using a digital camera under standardized lighting conditions. Subsequently, the surrounding tissues containing the residual gels were harvested, fixed in 4% paraformaldehyde, and processed for histological and IHC analyses.

Construction of in vitro macrophage co-culture models
Cell sources. Endothelial cells were obtained from the human umbilical vein endothelial cell line PUMC-HUVEC-T1 (No. C01510C, Procell). Macrophages were derived from the RAW 264.7 cell line (No. SCC-211800, Servicebio). Primary chondrocytes and endothelial cells were isolated from New Zealand white rabbits. All cells were maintained at 37°C in a humidified atmosphere containing 5% CO2.

Isolation and culture of rabbit tracheal epithelial cells. The epithelial cells used for the ALI model were obtained from rabbit trachea, as previously described. Briefly, tracheal tissues were harvested following euthanasia and washed three times with PBS. The trachea was incubated at 37°C for 30 min with dispase (StemCell, Canada), after which the mucosal layer was carefully separated and further digested overnight. The digested suspension was filtered through a 70 μm filter. The resulting epithelial cells were resuspended in epithelial culture medium consisting of DMEM (Gibco) and F-12 (Gibco) supplemented with 10% FBS (Gibco), 5 μM Y-27632 (Sigma), 10 ng/mL EGF (PeproTech), 5 μg/mL insulin (Sigma), 0.1 nM cholera toxin (Sigma), 250 ng/mL amphotericin B (Solarbio), and 10 μg/mL gentamycin (Gibco). Cells were cultured in 3 cm dishes at 37°C in a humidified incubator with 5% CO2, with medium changes three times per week. Epithelial cells were expanded to the third passage before seeding.

Transwell co-culture model. As illustrated in Fig. 3f, the Transwell co-culture system was used to investigate the interactions between macrophages and chondrocytes, endothelial cells, and epithelial cells. The Transwell setup facilitates the separation of cell types while permitting the exchange of soluble factors, thereby mimicking the in vivo microenvironment. Specifically, macrophages were seeded in the upper chamber of the Transwell system and embedded in various matrices, while chondrocytes, endothelial cells, or epithelial cells were seeded in the lower chamber. This setup allowed the investigation of the effects of macrophage-secreted factors on these cell types.

Air-liquid interface (ALI) co-culture model. In addition to the Transwell system, an ALI co-culture model was established to further examine the influence of macrophage-derived soluble factors on the differentiation and barrier function of epithelial cells (Fig. 3n). In this setup, macrophages were embedded in various matrices and cultured in the lower chamber, whereas epithelial cells were seeded on the upper insert membrane and maintained under ALI conditions to promote epithelial differentiation.

Establishment of ALI epithelial culture. Third-passage epithelial cells were seeded onto Matrigel (BD Biosciences)-coated Transwell and cultured until confluence. The apical medium was then removed to expose the cell layer to air, while the basal compartment was maintained with epithelial growth medium. This ALI culture promoted epithelial stratification and mucociliary differentiation, enabling functional assessment in the co-culture model.

Construction and in vivo implantation of BioC/IL-Gel composite
To investigate the role of IL-Gel in regulating the local microenvironment and its effect on cartilage integration, various BioC composites were constructed, as illustrated in Fig. 4a and Supplementary Fig. 11a. Specifically, the experimental setup consisted of four groups: GelMA, GelMA@Blk, BP-Gel, and BP@IL-Gel. Each composite was implanted into the platysma muscle of rabbits. Periodic sampling was performed post-implantation to analyze the histological characteristics and integration efficiency of the cartilage.

Segmental tracheal reconstruction in a rabbit tracheal defect model
First, multiple BioC rings that had been cultured in vitro for 4 weeks were positioned over a silicone tube, and IL-Gel was poured into the gaps between BioC rings to obtain a DTE trachea, as shown in Supplementary Fig. 12a. Next, the platysma muscle of rabbits was used to wrap the DTE trachea. After 4 weeks of vascularization in rabbits, the DTE trachea with a pedicled muscle flap was carefully stripped from the surrounding tissues, and the vascular network was closely monitored during the operation. After that, the silicone tube was removed from the DTE trachea, and the resulting trachea was cut to approximately 2 cm. After the construction of a segmental tracheal defect model in rabbits. A 6-0 absorbable suture was used to anastomose the detached native trachea with the trachea substitute. After tracheal anastomosis, the rabbits were treated prophylactically with penicillin for 7 days. The rabbits were sacrificed at predetermined time points, and the grafts were collected for further analysis.

Morphological analyses and mechanical tests
Photographic and morphological measurements. Photographs of all harvested tracheal samples were taken under standardized conditions. The widths and thicknesses of the samples were measured using a Vernier caliper, while the mass of each sample was determined with an electronic balance.

Compression testing. The compressive strength of the reconstructed trachea was evaluated using a biomechanical analyzer (Instron-6800, Norwood, USA) equipped with a 25N load sensor. During compression testing, deformation was measured from the onset of loading until 50% of the maximal deformation was reached. The peak pressure within this range was recorded as the maximum pressure value.

Tensile testing. For tensile strength assessment, samples were subjected to uniaxial stretching until breakage. The tensile force recorded at the point of rupture was defined as the maximum sustainable tensile force of the reconstructed trachea.

Three-point bending testing. Flexural properties were assessed using a three-point bending test. Samples were positioned on two supporting anvils, with the loading point centrally located between them. A steadily increasing load was applied until either fracture occurred or the flexural angle exceeded 90°. The maximum load applied during the test was recorded as the maximum bending force.

Histological and IF & IHC analysis
Tissue fixation and section preparation. Samples were fixed in 4% paraformaldehyde for 24 h at room temperature, followed by a graded ethanol series for dehydration. Tissues were then embedded in paraffin and sectioned into 5-μm-thick slices using a microtome. The prepared sections were mounted onto glass slides for subsequent histological and IF analyses.

Histological staining. H&E staining was performed to evaluate overall tissue morphology and cellular distribution. AB&NFR and SO&FG staining were used to assess cartilage-specific ECM deposition, particularly glycosaminoglycans within the cartilage lacunae, with Fast Green serving as a counterstain for non-cartilaginous structures. SR staining was used to visualize collagen fibers. All stained sections were observed under a light microscope, and representative images were captured for comparison among groups.

IF and IHC staining. For antigen detection, tissue sections were deparaffinized, rehydrated, and subjected to antigen retrieval in citrate buffer (pH 6.0) at 95°C for 15 min. After cooling, nonspecific binding was blocked with 5% bovine serum albumin (BSA) for 1 h at room temperature. Sections were then incubated overnight at 4°C with specific primary antibodies (listed in Supplementary Table 4). For IF, sections were washed and incubated with fluorophore-conjugated secondary antibodies for 1 h at room temperature, counterstained with DAPI to visualize nuclei, and mounted with antifade reagent. Fluorescence micrographs were captured using an SP8 confocal microscope (Leica). For IHC, sections incubated with primary antibodies were subsequently treated with HRP-conjugated secondary antibodies (Abcam). Visualization was performed using diaminobenzidine (DAB) as the chromogen, and nuclei were counterstained with hematoxylin. Stained sections were observed under a bright-field microscope.

Quantitative analysis. Unless otherwise specified in the figure legends, quantification was performed by analyzing at least 5 randomly selected fields per section using ImageJ software to calculate the percentage of positive staining or fluorescence intensity.

Biochemical and molecular biology analysis
Sample preparation. Harvested tissue samples were weighed to determine wet weight, minced, and lyophilized for subsequent biochemical and molecular biology analyses. Lyophilized tissues were ground into fine powders and stored at −80°C until further analysis.

DNA quantification. DNA content was measured using a total DNA quantitative assay kit (TIANGEN) according to the manufacturer’s instructions. Briefly, tissues/cells were digested in lysis buffer (proteinase K-based) at 56°C until complete digestion, followed by brief centrifugation to remove debris. DNA concentration and purity were measured with a NanoDrop spectrophotometer (Thermo Fisher Scientific) using A260 (with A260/A280 recorded for quality control).

GAG quantification. GAG content was determined by the dimethylmethylene blue (DMMB) method (Sigma), and concentrations were calculated using a chondroitin sulfate standard curve.

Quantitative real-time PCR (qRT-PCR). Total RNA was extracted with TRIzol reagent (Invitrogen), and cDNA was synthesized using a reverse transcription kit (Takara). qRT-PCR was performed with SYBR Green PCR Master Mix (Takara) on a real-time PCR system. GAPDH served as the internal reference gene, and relative expression levels were calculated by the 2−ΔΔCt method. Primer sequences are provided in Supplementary Table 5.

Western blot (WB) analysis. Proteins were extracted using RIPA buffer containing protease inhibitors and quantified by BCA assay. Equal protein amounts were separated by SDS-PAGE, transferred to PVDF membranes, and blocked with 5% milk. Membranes were incubated overnight at 4°C with primary antibodies, followed by HRP-conjugated secondary antibodies. Signals were detected with an ECL system (Bio-Rad), and band intensities were analyzed using ImageJ software.

Flow cytometry analysis
Cells were enzymatically digested and collected at a density of 1 × 105 cells per group, then resuspended in PBS containing 2% FBS and transferred into 1.5 mL Eppendorf tubes. To block nonspecific Fc receptor binding, 200 μL of Fc-Block reagent was added and incubated for 10 min at 4°C, followed by washing with buffer to terminate the reaction. Subsequently, 2 μL of DCFH-DA fluorescent probe was added to each sample and incubated for 20 min at 4°C in the dark. After incubation, cells were washed twice with PBS containing 2% FBS and resuspended in 200 μL buffer for detection. Flow cytometric analysis was performed using a FACSVerse flow cytometer (BD Biosciences, USA). The fluorescence intensity and percentage of positive cells were determined by comparison with the blank control group using FlowJo v10.6.2 software.

RNA sequencing and data analysis
Total RNA was extracted from tissue with TRIzol reagent (TaKaRa) and subsequently depleted of rRNAs via Ribo-Zero rRNA Removal Kits (Illumina) following the manufacturer’s instructions. The RNA libraries were constructed from rRNA-depleted RNA using the TruSeq Stranded Total RNA Library Prep Kit according to the manufacturer’s instructions. Libraries were controlled for quality and quantified via the BioAnalyzer 2100 system (Agilent Technologies, Inc.). Ten picometer libraries were denatured as single-stranded DNA molecules, captured on Illumina flow cells, amplified in situ as clusters, and sequenced for 150 cycles on an Illumina HiSeq Sequencer according to the manufacturer’s instructions. Library preparation and high-throughput sequencing were performed by CloudSeq Biotech (Shanghai, China).

Statistical analysis
All statistical analyses were performed using GraphPad Prism 9.0. For comparisons between two groups, the unpaired two-tailed Student’s t-test was used. For comparisons among three or more groups, one-way ANOVA with Tukey’s post hoc test was applied. In cases involving two variables across multiple groups, two-way ANOVA followed by Bonferroni correction was employed. The log-rank test used for comparing Kaplan–Meier survival curves was performed as a two-sided test. All data are expressed as means ± standard deviations (s.d.), and p-values < 0.05 were considered statistically significant.

Ethical statement
All animal care, feeding, and research procedures were carried out following the Guidelines for the Care and Use of Laboratory Animals and were approved by the Ethics Committee of Shanghai Pulmonary Hospital (No. K23-107Y).

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

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "In this manuscript Tang et al evaluated the use of tracheal grafts generated by using bio-adaptive physical hydrogel for long-segment tracheal replacement. Applying dynamic tissue engineering techniques, the authors proposed the use of triblock copolymers (PLGA-PEG-PLGA), which promote physical and mechanical properties for chondrocyte growth and aggregation. Although the general idea would provide important aspects for tissue engineering, the study in its present form is not suitable for publication.\nMajor comments:\nIn general, there are several overarching statements in the results section.\nFor instance, the authors highlight that transitioning from a room-temperature solution to a body-temperature hydrogel (BP-gel) is a \"unique\" characteristic of their bio-adaptive hydrogel; however, Matrigel and other hydrogels demonstrate similar properties.\nThe live-dead assay does not constitute a valid evaluation of proliferation; additional experiments are necessary to substantiate claims of chondrocyte proliferation. The quality of the Live-Dead assay figures need to be improved, and nuclear staining added if possible.\nSince fibronectin binds to ECM rather than mediates cell-to-cell interactions, further experiments are required to demonstrate chondrocyte intercellular adhesion. Moreover, upregulated gene expression (the authors used RT-qPCR to measure the fn1 and lmn expression) does not necessarily translate to protein function or activity.\nIn Figure 2d, the authors suggest that culturing chondrocytes in BP-gel promotes cellular aggregation and fusion based on chondrocyte-related gene expression data; however, this conclusion lacks adequate support: while Sox-9 and its target Col2 are critical for chondrocyte differentiation, they are not directly implicated in cell adhesion mechanisms and cannot serve as a measure of aggregation and fusion.\nThe comparison of chondrocyte cartilage matrix production in BP-gel (Figure S3h) should include native tissue controls to validate the authors' conclusions.\nThe manuscript lacks clear descriptions of the statistical analyses performed across different evaluations, sometimes ANOVA is specified to be used for just 2 groups. Additionally, in multiple instances, the p-value legends in figures do not correspond to what is displayed in the associated graphs (ex. What are the significance numbers for ##, ###,#### in Fig.2o, Fig.3b etc.), please recheck the figures.\nRegarding the in vivo model (Figure 5), the authors' rationale for not using standard tracheal epithelial markers such as KRT5 and p63 remains unclear. The absence of definitive epithelial markers to delineate the epithelium from submucosa complicates interpretation. Furthermore, the authors should address whether the accumulation of K14+ layers in the BP-Gel + IL+Gel condition indicates a squamous phenotype. The substantial variation in replicate numbers across panels warrants explanation. There is no native tracheal tissue presented for panels Figure 5k-n to compare.\nSince inadequate perfusion represents a major barrier to successful long-segmental tracheal transplantation, the authors should clarify how reduced vessel formation in BOP-IL-Gel confers therapeutic benefit.\nGiven that the major findings regarding enhanced chondrocyte functional and mechanical properties resulted from aggregate formation in BP+ Gel conditions, the role of macrophage-mediated responses in this process requires further elucidation.\nSeveral parts of the methodology section lack comprehensive descriptions.\nMinor comments:\nGene/protein nomenclature usage is inconsistent across mouse, rabbit, and human studies.\nThe number of biological and technical replicates is not specified.\nInformation regarding IL-4/13 acquisition is missing\nThe authors should clarify whether they mean \"air-liquid interface\" rather than \"gas-liquid interface\".\nGEO accession number for RNA sequencing data must be provided.\nFor Figure S7, the time points for panels h and i evaluation require specification.\nAnimal protocol approval number for the studies should be included.", "reviewer_2": "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_3": "This manuscript by Tang et al. presents a well-written and conceptually innovative study that introduces the Dynamic Tissue Engineering (DTE) strategy. This approach mimics embryonic cartilage development while concurrently modulating the immune microenvironment to support coordinated epithelial and vascular regeneration. The DTE strategy utilizes a bio-adaptive physical hydrogel (BP-Gel) in combination with an interleukin-loaded gel (IL-Gel), thereby enabling precise spatiotemporal control of the regenerative process. Meanwhile, the authors developed a new in vitro cell culture model and an in vivo tissue cultivation model, which together provide robust experimental support for the DTE approach. Moreover, the successful demonstration of tracheal reconstruction with favorable survival rates and functional outcomes underscores the translational potential of this strategy. Overall, the study is supported by the well-structured experimental design and comprehensive data, representing a meaningful advancement in tracheal tissue engineering.\nHowever, the following issues need to be further clarified and supplemented by the authors to enhance the overall rigor of the manuscript and ensure accurate communication of the study’s conclusions:\n1. The authors introduced a novel paradigm for cartilage development and demonstrated its advantages over traditional cartilage culture under truly dynamic developmental conditions. However, the technical challenges associated with implementing this paradigm were not sufficiently elaborated. Compared to conventional methods, has there been any optimization in terms of culture duration or the required cell number? This should be detailed in the study.\n2. The authors mentioned that the physically crosslinked hydrogel exhibits a 3D polymer network that guides dynamic cell movement and promotes organized tissue development, which is a very novel and attractive finding. However, the background section does not clearly explain the detailed process of embryonic cartilage development. Clarifying the process of embryonic cartilage formation and highlighting both the similarities and differences between the cartilage development in BP-Gel and natural embryonic development is necessary to avoid reader confusion.\n3. The observation of dynamic cellular aggregation in the results is quite exciting. This phenomenon is likely related to the physically crosslinked structure of the material itself. However, such behavior may be highly dependent on factors such as material concentration. Yet, the manuscript only use a single material concentration. What is the basis for this choice?\n4. The BP-Gel system in this study is based on a thermosensitive PLGA-PEG-PLGA hydrogel, which undergoes a sol–gel transition near body temperature. However, the thermosensitive property does not appear to be prominently utilized or discussed as a functional feature in either in vitro or in vivo applications. The authors are encouraged to clarify whether thermosensitivity mainly serves as an operational convenience or whether it also contributes to cellular behavior modulation or tissue formation.\nRegarding specific experimental results, the following questions need to be addressed:\n1.In Fig. 1a, the conceptual illustration of the overall study is too complex. It is recommended to simplify the graphic components and add more textual explanations for better reader comprehension. For example, the representation of the inflammation microenvironment (vessels and cells in the upper right corner of BP-Gel) appears overly complicated.\n2.Based on my understanding, are Figs. 2h, 2l, and 2m derived from the same sample group? What are their differences in terms of visual presentation and quantitative analysis? What specific issues do they aim to address respectively? This should be clarified in the manuscript.\n3.In Figs. 2e–f and Figs. S3e–g, the authors attempt to interpret cartilage condensation and development using the HIF-1α and YAP signaling pathways. Is this condensation process an essential step in native cartilage development? There is a lack of control data from natural cartilage, which should be supplemented to strengthen the explanation.\n4.In Fig. 4c, the presentation of cartilage development is relatively limited. It is necessary to include additional specific staining and biochemical assays of the cartilage matrix to better validate the promoting and protective effects of the BP@IL-Gel composite structure on ectopic cartilage development in vivo.\n5.In Fig. 4m, please clearly indicate which key genes related to the biological process are upregulated or downregulated, as this is critical for the interpretation of the sequencing results.\n6.In Fig. 5, the authors suggest that IL-Gel can protect cartilage from vascular invasion. Does IL-Gel hinder capillary regeneration around the cartilage? This needs to be addressed in the manuscript, with additional data provided if necessary.\n7.In Figs. 5b–c, the authors report average values of survival and respiratory function in rabbits after orthotopic tracheal transplantation. Please provide detailed individual data for each animal model to improve data transparency.\n8.Please supplement the vascular-specific staining (e.g., CD31) of the orthotopic graft sections and include quantitative analysis, as vascularization is also crucial for tracheal regeneration.\n9.What is the difference between the results of Fig. S7 and Fig. 3f? Please further clarify their respective significance.\n10.In the Materials and Methods section, some experimental descriptions are relatively vague, which affects reproducibility. Please provide more detailed parameters and protocols for each experimental procedure."}, "author_response": "We sincerely thank the reviewer for this assessment and for recognizing the potential significance of our approach in tissue engineering. In response to the reviewer’s concerns and detailed comments, we have substantially revised the manuscript to address all scientific, methodological, and interpretational issues.\nMajor comments:\n1. In general, there are several overarching statements in the results section. For instance, the authors highlight that transitioning from a room-temperature solution to a body-temperature hydrogel (BP-gel) is a \"unique\" characteristic of their bio-adaptive hydrogel; however, Matrigor; however, Matrigel and other hydrogels demonstrate similar properties.\nReply:\nWe appreciate the reviewer’s critical observation. We agree that the sol-gel transition triggered by temperature is not unique to our BP-Gel, and recognize that similar behavior has been reported in other thermosensitive hydrogels, such as Matrigel and the hydrogel formed by Pluronic F127 aqueous solution. In response to this point, we have revised the corresponding statement in the manuscript to reflect the novelty of our material system more accurately.\nSpecifically, we have modified the phrase “unique characteristic” to “a thermally responsive and mechanically adaptive platform well-suited for dynamic structural rearrangement” (Page 3, Line 9). We have also elaborated on how the combination of thermosensitivity, physical percolation network dynamics, and PLGA-PEG-PLGA-based micelle architecture distinguishes BP-Gel from conventional hydrogel systems:\nPage 3, Line 14: “Specifically, the physical percolation network in the BP-Gel could dynamically adapt to the migration, proliferation, and aggregation of chondrocytes, thus facilitating the fusion of chondrocyte clusters into large aggregates. This procedure resembles the embryo-like chondrification process, in which undifferentiated mesenchymal cells migrate, proliferate, and condense to form cartilage primordia1-3. This dynamically adaptive culture environment induced a native-like stratified morphology in the engineered cartilage, resulting in enhanced mechanical properties and improved resistance to degradation in vivo.”\nMoreover, it is important to emphasize that, unlike Matrigel-which is derived from the ECM and contains undefined biochemical components4, PLGA-PEG-PLGA-based BP-Gel is a synthetic and well-characterized material with tunable gelation properties, and minimal batch-to-batch variability. Furthermore, its degradation rate and drug release kinetics can be precisely modulated, enhancing its suitability for clinical translation and applications. Additionally, although the hydrogel formed by Pluronic F127 exhibits thermosensitive properties, its structural integrity only lasted for 48 h due to its rapid erosion in aqueous environment (Fig. R1-1-1; Fig. S3b in the manuscript) and thus it is unable to support the full growth cycle of engineered cartilage. To clarify these points, we have added a paragraph in the Discussion section to comparatively analyze BP-Gel and other conventional system hydrogels.\nPage 27, Line 19: “Unlike conventional extracellular matrix-based hydrogels, BP-Gel based on PLGA-PEG-PLGA is a fully synthetic and well-characterized material with tunable physicochemical properties, and minimal batch-to-batch variability. Moreover, its degradation profile and drug release kinetics can be precisely controlled, making it particularly suitable for applications in tissue engineering and regenerative medicine5.”\nFigure R1-1-1 Degradation comparison between Pluronic F127 and BP-Gel. Rose red was used to trace the F127-gel, while the BP-gel was highlighted with a blue background.\nWe sincerely thank the reviewer for prompting this clarification, which has allowed us to refine the scope and highlight the distinct advantages of our hydrogel system without overstating its novelty.\n2. The live-dead assay does not constitute a valid evaluation of proliferation; additional experiments are necessary to substantiate claims of chondrocyte proliferation. The quality of the Live-Dead assay figures need to be improved, and nuclear staining added if possible.\nReply:\nWe thank the reviewer for this important and constructive suggestion. We agree that live-dead staining primarily assesses cell viability rather than cell proliferation. To substantiate our claims, we have now performed additional experiments, including:\n(1) Ki67 immunofluorescence (IF) staining\nKi67 is a well-established marker of cellular proliferation. We performed Ki67 IF staining on chondrocytes cultured in the control group (standard medium), GelMA, and BP-Gel at Days 1, 4, and 7. The proportion of Ki67-positive cells in the BP-Gel group was comparable to that in the GelMA and control groups, indicating that chondrocyte proliferation was not significantly affected in either hydrogel environment (Fig. R1-2-1; Fig. S1i-j in manuscript).\nFigure R1-2-1 Ki67 staining of chondrocytes in different hydrogels. (A) Ki-67 (green)/DAPI (blue) staining of chondrocytes cultured in control, GelMA, and BP-Gel at days 1, 4, and 7. (B) Quantification of Ki-67-positive cells showing comparable proliferation among groups.\n(2) Cell counting, analysis of the DNA content, and CCK-8 quantification\nTo further assess proliferation, we performed cell counting and DNA quantification at Day 7 and CCK-8 assays at Days 1, 4, and 7 in the control (standard medium), GelMA, and BP-Gel groups. Day-7 cell counts and DNA content were comparable across groups, while CCK-8 showed a time-dependent increase with similar proliferation rates among the three groups. These results indicate that the BP-Gel microenvironment supports chondrocyte viability and growth without compromising proliferative capacity (Fig. R1-2-2; Fig. S1k-m in manuscript).\nFigure R1-2-2 Cell counting and DNA content at Day 7, and CCK-8 assays at Days 1, 4, and 7 for chondrocytes cultured in control, GelMA, and BP-Gel groups.\n(3) Updated the Live/Dead assay figures\nTo address the reviewer’s concern regarding image quality, we have now updated the Live/Dead assay figures with higher-resolution images and added nuclear staining using Hoechst 33342 (Fig. R1-3; Fig. S1h in manuscript). While DAPI is commonly used for nuclear staining, it is impermeable to live cell membranes and therefore unsuitable for labeling nuclei in live cells. For this reason, nuclear staining was not included in our initial experiments. Following the reviewer’s suggestion, we referred to the relevant literature6 and selected Hoechst 33342, which penetrates live cell membranes and enables effective nuclear visualization. This update enables better assessment of cell morphology and density.\nFigure R1-2-3 Live/Dead staining of chondrocytes cultured in control, GelMA, and BP-Gel at days 1, 4, and 7.\nWe believe that these enhancements provide a more robust and scientifically valid demonstration of chondrocyte viability and proliferation within the BP-Gel.\n3. Since fibronectin binds to ECM rather than mediates cell-to-cell interactions, further experiments are required to demonstrate chondrocyte intercellular adhesion. Moreover, upregulated gene expression (the authors used RT-qPCR to measure the fn1 and lmn expression) does not necessarily translate to protein function or activity.\nReply:\nWe thank the reviewer for the insightful comment. We agree that while Fibronectin-1 (FN1) and Laminin (LAMA) primarily contribute to ECM-cell adhesion, it does not directly mediate intercellular junctions. We also acknowledge that mRNA expression alone cannot fully confirm functional protein activity or cell-cell adhesion. To address this concern and directly evaluate intercellular adhesion between chondrocytes, we conducted the following additional experiments:\n(1) Expression of genes related to intercellular adhesion genes\nWe fully agree with the reviewer that assessing cell aggregation solely based on FN1 and LAMA is not comprehensive. Following the reviewer’s suggestion, we conducted a literature survey and selected two representative markers of intercellular adhesion and gap junctions in chondrocytes: N-cadherin (CDH2) and Connexin-43 (GJA1)7-9. These two well-established markers mediate direct cell-cell adhesion and are critical in chondrogenesis and mesenchymal condensation. Accordingly, we performed PCR experiments targeting these genes to preliminarily validate the conclusions from our earlier analyses (Fig. R1-3-1; Fig. S4d in manuscript).\nFigure R1-3-1 Expression levels of adhesion-associated genes in chondrocytes after 3 days of culture under different conditions. 2D culture refers to chondrocytes cultured on ordinary plates.\n(2) Western blotting analysis of adhesion-related proteins\nTo assess adhesion protein expression at the protein level, we performed Western blot analysis of four markers: LAMA and FN1 (ECM-mediated adhesion) and CDH2 and GJA1 (intercellular adhesion) in chondrocytes cultured under 2D control, BP-Gel, and GelMA conditions. Protein expression was higher in both GelMA and BP-Gel groups compared with the control, indicating that 3D culture promotes enhanced cell aggregation and stronger intercellular adhesion. Notably, although s GelMA permitted limited proliferation and low-level aggregation, cell activity remained constrained. In contrast, BP-Gel fostered stronger cell–cell interactions, accompanied by markedly higher expression of adhesion-related genes (Fig. R1-3-2; Fig. S4e-f in manuscript).\nFigure R1-3-2 Western blot analysis of adhesion-related proteins in chondrocytes cultured under different conditions.\n(3) IF staining for adhesion-related proteins\nIn addition to protein quantification, we examined the spatial distribution of adhesion-related proteins by IF staining using GelMA, BP-Gel, and cell spheroids. Cell spheroids were included as a positive control, as they naturally exhibit high levels of intercellular adhesion. Chondrocytes in the BP-Gel group showed markedly stronger expression of LAMA and FN1 (ECM-mediated adhesion) as well as CDH2 and GJA1 (intercellular adhesion) compared with GelMA, reaching levels comparable to cell spheroids. This indicates that BP-Gel provides an adaptive environment that enhances intercellular connectivity. By contrast, cells in GelMA were restricted by its rigid matrix, showing only localized clusters with limited aggregation, a condition unfavorable for cartilage development (Fig. R1-3-3; Fig. S4g-h in manuscript).\nFigure R1-3-3 IF analysis of adhesion-related proteins in engineered chondrocyte tissues. (A) Representative H&E and fluorescence microscopy images illustrating the expression of LAMA1, FN1, CDH2, and GJA1 in chondrocytes cultured with GelMA, BP-Gel, or in cell spheroid form. (B) Quantification of mean gray values for LAMA1, FN1, CDH2, and GJA1 expression.\nThe corresponding paragraph in the Results section has been revised accordingly:\nPage 7, Line 28: “We subsequently evaluated the expression of adhesion-related genes (Lama, Fn1, Cdh2, and Gja1) (Fig. S4d) and cartilage-related genes (Acan, Col2a1, and Sox9) (Fig. 2d). The results showed that the expression levels of these genes in chondrocytes grown in BP-Gel were significantly up-regulated compared with those cultured on plates or in GelMA, indicating that culturing chondrocytes in BP-Gel promotes their aggregation and fusion, thus facilitating cartilage formation. Consistently, Western blotting confirmed higher levels of adhesion-related proteins in the BP-Gel group, and Hematoxylin and Eosin (H&E) and IF staining further demonstrated markedly stronger expression of Laminin subunit alpha-1 (LAMA1), Fibronectin (FN1), N-cadherin (CDH2), and Connexin-43 (GJA1), with quantitative analysis showing significant increases in all four markers (Fig. S4e-h).”\nWe thank the reviewer again for pointing out this important detail.\n4. In Figure 2d, the authors suggest that culturing chondrocytes in BP-gel promotes cellular aggregation and fusion based on chondrocyte-related gene expression data; however, this conclusion lacks adequate support: while Sox-9 and its target Col2 are critical for chondrocyte differentiation, they are not directly implicated in cell adhesion mechanisms and cannot serve as a measure of aggregation and fusion.\nReply:\nWe appreciate the reviewer’s thoughtful comment. We agree that the expression of SOX9 and COL2A1 (Col2), which are indicators of chondrogenic differentiation, are insufficient to assess chondrocyte aggregation or fusion; importantly, we did not intend to use chondrogenic gene expression alone as evidence of these processes. In our manuscript, however, we addressed this issue in two parts. First, we demonstrated that BP-Gel promotes chondrocyte clustering, which is supported by both our original data and the newly supplemented results. Second, we examined cartilage tissues formed under this growth model and compared them with chondrocytes cultured in other hydrogels (GelMA). This comparison was intended to show that the aggregation and fusion behaviors of chondrocytes are beneficial for chondrogenic differentiation, extracellular matrix secretion, and cartilage tissue formation.\n(1) Time-lapse confocal imaging of chondrocyte aggregation and fusion\nWe performed live-cell confocal microscopy to monitor chondrocyte behavior in BP-Gel over 7 days. The time-lapse sequences clearly show small chondrocyte clusters migrating toward each other and fusing into larger aggregates within BP-Gel. This process was not observed in GelMA. Representative images and videos are provided in Fig. R1-4-1 (Fig. 2a-b in manuscript) and Fig. R1-4-2 (Movie S2 in manuscript), respectively.\nFigure R1-4-1 Chondrocyte clustering and aggregation in BP-Gel versus GelMA. (A) Growth state of chondrocytes in BP-Gel and GelMA over culture time. The cells were observed under a bright-field microscope. (B) Quantitative analysis of the number of chondrocyte clusters in BP-Gel and GelMA.\nFigure R1-4-2 Spontaneous formation of chondrocyte clusters in BP-Gel. (This is an animated GIF. Please view it in a word file or refer to Movie S2)\n(2) Cluster fusion and cortical-medullary structure formation\nIn BP-Gel, small chondrocyte clusters progressively fused into larger aggregates with increasing collagen deposition and cortical thickening, ultimately forming a distinct cortex–medulla architecture over 4 weeks (Fig. R1-4-3; Fig. 2g and 2m-o in manuscript). In contrast, chondrocytes in GelMA remained dispersed without evident fusion (Fig. 2a-b and 2h in manuscript), highlighting the superior ability of BP-Gel to drive cluster fusion and organized cartilage formation.\nFigure R1-4-3 Fusion of chondrocyte clusters and cortex-medulla architecture formation in BP-Gel.. (A) Representative micrographs showing the fusion process of chondrocyte clusters in BP-Gel. (B-D) Polarizing microscope images of SR staining tissue specimens collected from different groups at designated time points, and quantitative analysis of intersphere angle of chondrocyte clusters, fluorescence intensity and thickness of cortex layer.\n(3) IF staining for chondrogenic markers (SOX9 and COL2A1)\nWe performed IF staining for SOX9 and COL2A1, using GelMA as a control and including cell spheroids as a reference (Fig. R1-4-4; Fig. S5e-h in manuscript). The results clearly showed that chondrocytes in the BP-Gel group exhibited markedly stronger SOX9 and COL2A1 staining, reflecting both the initiation of chondrogenic lineage differentiation and the enhancement of type II collagen deposition. These findings, together with previous results, demonstrate that BP-Gel promotes intercellular connectivity while supporting cartilage development and maturation.\nFigure R1-4-4 IF analysis of chondrogenic markers in cartilage tissues. (A) Representative fluorescence microscopy images of COL2A1 (red) and SOX9 (green) expression in chondrocytes cultured with GelMA, BP-Gel, or as cell spheroids. Nuclei were counterstained with DAPI (blue). (B) Quantification of SOX9 and COL2A1 mean gray values and SOX9-positive cell ratio.\nThese data provide comprehensive and multi-angle support for the claim that BP-Gel promotes chondrocyte aggregation and cluster fusion. We have revised the original paragraph and updated the relevant figure legends.\nPage 7, Line 10: “Bright-field and confocal microscopy confirmed that chondrocytes aggregated into clusters in BP-Gel, whereas they remained scattered in GelMA (Fig. 2a and Fig. S4a). Quantitative analysis further confirmed that the number of chondrocyte clusters in BP-Gel was significantly greater than that in GelMA at all observation points (Fig. 2b).”\nPage 8, Line 21: “Building on these findings, we performed additional IF staining of chondrogenic markers using GelMA as a control and cell spheroids as a reference. Chondrocytes cultured in BP-Gel showed markedly stronger SOX9 and COL2A1 expression than those in GelMA, with quantification confirming higher SOX9 and COL2A1 levels and an increased proportion of SOX9-positive cells (Fig. S5e-h).”\nPage 9, Line 4: “Fig. 2g shows a typical fusion process where small chondrocyte clusters fused into a larger chondrocyte cluster.”\nPage 10, Line 2: “As shown in Fig. 2m-o, SR staining of BioC at different growth stages revealed progressive fusion of chondrocyte clusters, continuous increase in COL2A1 deposition, and gradual thickening of the cortical layer, reflecting the dynamic formation of a “cortex-medulla” structure.”\nThe revised version now references functional imaging, protein-level adhesion markers, and blockade validation, rather than relying solely on gene expression of SOX9 and COL2A1. We thank the reviewer again for helping us improve the rigor and clarity of this key conclusion.\n5. The comparison of chondrocyte cartilage matrix production in BP-gel (Figure S3h) should include native tissue controls to validate the authors' conclusions.\nReply:\nThank you for raising this important point. We agree that including native cartilage as a benchmark is essential for validating the biomimetic quality of BioC. In the original manuscript, we already compared the morphology and biochemical composition of cartilage-like tissues formed after in vivo implantation with native cartilage (Fig. 2h), showing that the morphology and biochemical composition of BioC closely resembled those of native tissue.\nThe in vitro developmental stages were presented mainly to highlight that BP-Gel–engineered cartilage undergoes maturation processes similar to natural cartilage development. However, because immature engineered cartilage at early in vitro stages is inherently different from fully mature native cartilage, a direct comparison was not meaningful and thus not included. To address the reviewer’s concern, we have now incorporated histological sections from embryonic mice tracheal cartilage as an additional control group in our histological and biochemical analyses.\n(1) Comparison of mature engineered cartilage with native mature cartilage\nIn the current manuscript, we have already compared mature engineered cartilage with native rabbit tracheal cartilage using identical staining protocols. After 4 weeks of in vivo culture, BioC formed in BP-Gel exhibited cartilage-specific ECM deposition, confirmed by Hematoxylin and Eosin (H&E), Alcian Blue-Nuclear Fast Red (AB&NFR), and COL2A1 immunohistochemical (IHC) staining (Fig. R1-5-1A; Fig. 2h in manuscript). Quantitative analysis showed that lacunar size in the BP-Gel group was comparable to that of native cartilage. DNA and hydroxyproline measurements further indicated that BioC shared similar biochemical properties with native cartilage (Fig. R1-5-1B-D; Fig. 2i-k in manuscript). Moreover, Sirius Red (SR) staining and 3D reconstruction revealed a grid-like collagen fiber distribution in BioC, closely resembling native tissue, whereas this organization was absent in GelMA (Fig. R1-5-1E; Fig. 2l in manuscript).\nCollectively, these findings demonstrate that BioC effectively recapitulates the structural and biochemical features of native cartilage.\nFigure R1-5-1 Comparison of engineered cartilage with native tracheal cartilage. (A) Representative H&E, AB&NFR, and COL2A1 IHC staining micrographs of tissue specimens collected from different groups at 4 weeks post-implantation. (B) Quantitative analysis of the size of cartilage lacuna. (C-D) Quantitative analysis of DNA and hydroxyproline content of tissue specimens collected from different groups at 4 weeks post-implantation. (E) Representative SR-stained micrographs of tissue specimens collected from different groups at 4 weeks post-implantation.\n(2) Recapitulation of early cartilage development in BP-Gel constructs\nAt 2 weeks, BP-Gel samples displayed features reminiscent of embryonic cartilage, including tightly aggregated chondrocytes within nascent lacunae and early COL2A1 deposition, whereas GelMA constructs showed delayed organization and minimal matrix formation (Fig. R1-5-2A; Fig. S6a in manuscript). To further clarify this early chondrogenic process, we compared 2-week BP-Gel samples with embryonic tracheal cartilage as a developmental benchmark. Histological staining (H&E, AB&NFR, Safranin O–Fast Green, and SOX9 IF) of mice embryonic trachea at E12.5, E14.5, and E16.5 revealed mesenchymal condensation and early matrix deposition, providing a reference for native chondrogenesis (Fig. R1-5-2B; Fig. S6b in manuscript).\nFigure R1-5-2 Early chondrogenic features in BP-Gel constructs compared with embryonic cartilage development. (A) H&E staining of engineered cartilage formed in GelMA and BP-Gel after 2 and 4 weeks in vitro. (B) Histological analyses of mice embryonic trachea at E12.5, E14.5, and E16.5 using H&E, AB&NFR, SO&FG, and SOX9 IF staining.\nThese additional native-tissue comparisons further confirm that BP-Gel supports the formation of cartilage histologically comparable to native tracheal cartilage. We thank the reviewer again for this valuable suggestion, which has greatly strengthened the rigor of our analysis.\n6. The manuscript lacks clear descriptions of the statistical analyses performed across different evaluations, sometimes ANOVA is specified to be used for just 2 groups. Additionally, in multiple instances, the p-value legends in figures do not correspond to what is displayed in the associated graphs (ex. What are the significance numbers for ##, ###,#### in Fig.2o, Fig.3b etc.), please recheck the figures.\nReply:\nWe thank the reviewer for this important feedback on statistical analysis and figure annotations.\nIn the Statistical Analysis section, we previously stated: “For comparisons between two groups, an unpaired two-tailed Student’s t-test was used. For comparisons among three or more groups, one-way ANOVA with Tukey’s post hoc test was applied. In cases involving two variables across multiple groups, two-way ANOVA followed by Bonferroni correction was employed.”\nFollowing the reviewer’s comment, we carefully rechecked all figure legends to ensure ANOVA was not incorrectly applied to two-group comparisons and revised the descriptions accordingly. We have also specified the exact statistical method used in each figure legend. For example:\nPage 12, Line 21: “Data in (b) and (r) were analyzed by two-tailed Student’s t-test. Data in (d), (i), (j), (k), (n),and (o) were analyzed by one-way ANOVA followed by Bonferroni’s post hoc test.”\nRegarding the p-value legends, we thank the reviewer for noting inconsistencies such as the use of “##”, “###”, and “####” in earlier versions of Figures 2o and 3b. These symbols were originally used to distinguish two datasets within the same figure, but we did not clearly indicate this in the legends. All “#” markers have now been removed and replaced with the standardized “*” system across all figures. Statistical significance is consistently denoted as: *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001. We thank the reviewer for highlighting this important issue.\n7. Regarding the in vivo model (Figure 5), the authors' rationale for not using standard tracheal epithelial markers such as KRT5 and p63 remains unclear. The absence of definitive epithelial markers to delineate the epithelium from submucosa complicates interpretation. Furthermore, the authors should address whether the accumulation of K14+ layers in the BP-Gel + IL+Gel condition indicates a squamous phenotype. The substantial variation in replicate numbers across panels warrants explanation. There is no native tracheal tissue presented for panels Figure 5k-n to compare.\nReply:\nWe thank the reviewer for highlighting these important points regarding epithelial characterization and data integrity in Figure 5. Our detailed responses are as follows:\n(1) Epithelial markers: KRT5/p63 vs. KRT14/ZO-1\nWe agree that KRT5 and p63 are widely used as basal cell markers in airway epithelium and provide critical information about progenitor cell identity. In our original submission, we primarily used KRT14 (CK14) and ZO-1 to assess epithelial coverage and barrier integrity, as KRT14 labels early epithelial progenitors and ZO-1 marks tight junction integrity. In response to the reviewer’s suggestion, we have now performed additional IF staining for both KRT5 and p63 in regenerated tracheal epithelium from the BP-Gel+IL-Gel and BP-Gel groups, with native trachea as a reference. Based on a literature survey10,11, both KRT5 and p63 are standard basal markers, though KRT5 is more frequently applied for histological identification of airway structure, while p63 is often used in epithelial tumor classification. We therefore selected KRT5 as the basal marker and additionally included FOXJ1 as a marker of epithelial ciliary development.\nStrong KRT5 and nuclear p63 expression were observed in the basal layers of regenerated epithelium in the BP-Gel+IL-Gel group, confirming the presence of basal progenitors and supporting pseudostratified epithelial restoration. Expression was sparse and discontinuous in the BP-Gel group (Fig. R1-7-1A; Fig. S12c in manuscript).\nFigure R1-7-1 Regeneration of airway epithelium and cilia in trachea. (A-B) Representative fluorescence microscopy images of regenerated tracheal epithelium. KRT5 (red) and FOXJ1 (green) indicate basal progenitors and ciliated cells, while AC-Tub (green) marks mature cilia. (C) Quantification of epithelial cilia coverage confirms significant recovery in BP-Gel+IL-Gel compared with BP-Gel.\n(2) Interpretation of KRT14 expression and potential squamous metaplasia?\nWe acknowledge the reviewer’s concern that KRT14 (CK14) overexpression, particularly in multilayered formations, may suggest early squamous differentiation. To clarify, we performed additional co-staining analyses. IF staining for KRT5 and FOXJ1 confirmed the presence of basal progenitors and ciliated cells, while acetylated α-tubulin (AC-Tub) staining revealed abundant cilia formation in the BP-Gel+IL-Gel group but only minimal signals in the BP-Gel group (Fig. R1-7-1A-B; Fig. S12c in manuscript). Quantitative analysis of epithelial cilia coverage further demonstrated significant recovery in the BP-Gel+IL-Gel group (Fig. R1-7-1C; Fig. S12d in manuscript), indicating regeneration of a ciliated columnar epithelium rather than squamous metaplasia.\nAlthough KRT14 overexpression has been linked to squamous differentiation, it is also widely recognized as a marker of early epithelial progenitors12-15. Combined with the evident ciliated phenotype, these results suggest that the KRT14-positive layer reflects active basal progenitor expansion and differentiation within an immune-supportive niche, rather than pathological squamous transformation.\n(3) Explanation of differences in the replicate number across panels\nWe appreciate the reviewer’s concern regarding the variation in replicate numbers across panels. These differences reflect assay requirements and animal-use constraints. Physiological monitoring began with n = 8 rabbits per group, but numbers decreased over time due to survival differences. Histological analyses were conducted on three rabbits per group, with 6-10 random fields averaged per animal to avoid pseudoreplication. Mechanical testing was a destructive endpoint assay, and only three intact samples per group were available after allocating tissue for histology. Each reported n therefore represents independent animals, with technical replicates clearly distinguished. Despite these variations, results across survival, physiology, histology, and mechanics were consistent and statistically robust, supporting the validity of our conclusions.\nImportantly, we have now revised the figure legend to explicitly state the rationale and exact replicate numbers for each panel.\n(4) Inclusion of native trachea as control in Figure 5k-n\nAs requested, we have now included native rabbit trachea tissue sections stained for: H&E, AB&NFR, COL2A1 (IHC), KRT14+ZO-1 (IF). These are shown in Fig. R1-7-2 (Fig. 5k-n in manuscript). These serve as gold standard references for evaluating cartilage content and epithelial organization. Quantitative analyses, including cartilage area, COL2A1+ area, and epithelial thickness, have been updated and normalized to native tissue values where appropriate.\nFigure R1-7-2 Histological and IF evaluation of cartilage and epithelial regeneration in trachea. (A) Quantitative analysis of the proportion of cartilage area in reconstructed trachea in different groups. (B-C) Representative COL2A1 IHC staining micrographs of reconstructed trachea harvested from different groups, and quantitative analysis of the ratio of COL2A1+ staining area. (D-E) Representative fluorescence microscopy images of epithelial tissues labeled with KRT14 (red) and ZO-1 (green) in different groups, and quantitative analysis of epithelial thickness. The dashed lines indicate the endothelial boundary.\nWe thank the reviewer for these valuable comments, which have significantly enhanced the rigor and clarity of our epithelial analysis.\n8. Since inadequate perfusion represents a major barrier to successful long-segmental tracheal transplantation, the authors should clarify how reduced vessel formation in BOP-IL-Gel confers therapeutic benefit.\nReply:\nWe thank the reviewer for emphasizing the importance of vascularization in tissue-engineered constructs. We agree that adequate vascularization is essential for the survival of long-segment tracheal grafts. However, it is important to note that vascularization must occur in the correct tissue compartments. In our system, the reduced vascular infiltration within the cartilage core of the BP@IL-Gel group does not indicate perfusion deficiency but instead reflects a spatially selective anti-angiogenic effect that preserves cartilage integrity while supporting robust vascularization in the surrounding connective tissues.\nIn both native and engineered cartilage, excessive vascular ingrowth is detrimental, as it promotes chondrocyte apoptosis, matrix degradation, and pathological ossification or fibrotic remodeling16-18. In the BP-Gel group, histological and IF analyses showed marked vascular invasion into the cartilage rings, with upregulated MMP13 expression, reduced COL2A1 deposition, and increased α-SMA–positive vessel density in the cartilage core (Fig. R1-8-1A-E; Fig. 2q-t and 4j in manuscript). SO&FG staining further revealed a loss of proteoglycan-rich matrix, while ossification markers were elevated (Fig. R1-8-1F; Fig. S11i in manuscript). Together, these results demonstrate that uncontrolled angiogenesis within cartilage compromises structural stability and accelerates degeneration.\nFigure R1-8-1 Evaluation of cartilage matrix integrity, degradation, and vascular invasion in BioC constructs. (A) Representative H&E-stained micrograph of BioC retaining a unilateral cortical layer. (B) Quantitative analysis of the invasion depth of surrounding tissues in BioC, comparing the side with cortical layer preservation (uncut) and the side without cortical layer preservation (cut). (C) IF staining micrograph showing the distribution of MMP13 (red) and COL2A1 (green) in BioC with a unilateral cortical layer. (D) Colocalization analysis of the MMP13 and COL2A1 distribution in BioC with a unilateral cortical layer. (E) Representative α-SMA (green) labeled fluorescence microscopy images of tissue specimens collected from different groups, and quantitative analysis of the number of blood vessels. The white triangles indicate the invasive blood vessels in cartilage tissue. (F) Representative SO&FG-stained micrographs of tissue specimens collected from the GelMA and GelMA@Blk groups and quantitative analysis of SO&FG-stained area. (G) Representative fluorescence microscopy images of tissue specimens labeled with PECAM1 (red) antibody in different groups. (H-I) Quantitative analysis of vascular area in the indicated regions.\nBy contrast, in the BP@IL-Gel group, IL-4/IL-13-induced M2 macrophage polarization effectively suppressed aberrant angiogenesis inside the cartilage while preserving the chondrocyte niche. PECAM1 immunostaining and quantitative analysis of vascular area revealed that vascularization was not diminished overall (Fig. R1-8-1G-I; Fig. S8m-o in manuscript). This selective vascular pattern closely mimics the zonal avascularity of native tracheal cartilage, where cartilage is nourished by diffusion from surrounding capillaries rather than by direct vascular penetration.\nFurther evaluation in the tracheal reconstruction model supported these findings. In the BP-Gel+IL-Gel group, H&E and α-SMA staining confirmed abundant, well-organized blood vessels between cartilage rings, comparable to native trachea, whereas the BP-Gel group displayed smaller, disorganized vessels (Fig. R1-8-2A; Fig. S12e in manuscript). Quantitative analysis demonstrated that vessel diameter and vascular area in the BP-Gel+IL-Gel group closely matched native trachea and were significantly greater than in the BP-Gel group (Fig. R1-8-2B; Fig. S12f in manuscript).\nFigure R1-8-2 Vascularization in regenerated trachea. (A) Representative H&E and α-SMA IHC staining images of inter-cartilaginous tissues from the BP-Gel, BP-Gel+IL-Gel, and native groups. (B) Quantification of vascular diameter and mean vascular area.\nTogether, these results demonstrate that IL-Gel selectively modulates angiogenesis, suppressing pathological vessel ingrowth into the cartilage core while maintaining robust peripheral vascularization. This dual effect preserves cartilage integrity and matrix homeostasis while ensuring nutrient delivery, epithelial regeneration, and immune balance. In summary, BP@IL-Gel does not reduce overall perfusion but specifically prevents vascular invasion, which is critical for long-term structural stability and function. We sincerely thank the reviewer for this valuable suggestion, which has greatly improved the clarity and rigor of our analysis.\n9. Given that the major findings regarding enhanced chondrocyte functional and mechanical properties resulted from aggregate formation in BP+Gel conditions, the role of macrophage-mediated responses in this process requires further elucidation.\nReply:\nWe thank the reviewer for highlighting the need to distinguish BP-Gel–driven aggregation from macrophage-mediated modulation. Our results support a two-step mechanism: BP-Gel creates the physical basis for chondrocyte aggregation and drives cartilage formation with mechanical and structural features close to native tissue, while IL-Gel-induced macrophage polarization further strengthens this newly formed cartilage and helps maintain its maturation, thereby enhancing chondrogenesis.\n(1) Macrophage-mediated enhancement (in vitro)\nIn a transwell co-culture system, IL-Gel–polarized macrophages secreted elevated levels of trophic and angiocrine factors (TGF-β1, TGF-β3, IGF, bFGF, CCL2) after 7 days (Fig. R1-9-1A-B; Fig. 3e-f in manuscript). When chondrocytes in BP-Gel were co-cultured with these macrophages, toluidine blue staining revealed significant increases in both the number and area of cartilage matrix patches (Fig. R1-9-1C-E; Fig. 3g-i in manuscript), while IF confirmed higher COL2A1 expression with quantitative support (Fig. R1-9-1F-G; Fig. 3j-k in manuscript). In parallel, endothelial cells showed increased PECAM1 signals after 7 days of co-culture (Fig. R1-9-1H-I; Fig. 3l-m in manuscript), indicating macrophage-driven endothelial activation consistent with the establishment of a supportive perichondral niche.\nFigure R1-9-1 Effects of macrophage co-culture on chondrocyte matrix formation and endothelial cell activation. (A) Levels of TGF-β1, TGF-β3, IGF, bFGF, and CCL2 secreted in the macrophage culture medium after 7 days of various treatments. (B) Schematic diagram of the co-culture system of macrophages with other cell types. (C-E) Representative toluidine blue-stained micrographs of chondrocytes after 7 days of culture in the co-culture system and quantitative analysis of the number and area of cartilage matrix patches. The red arrows indicate accumulated patches of cartilage matrix. (F-G) Representative fluorescence microscopy images and quantitative analysis of chondrocytes labeled with COL2A1 (green) after 7 days of culture in the co-culture system. (H-I) Representative fluorescence microscopy images and quantitative analysis of endothelial cells labeled with PECAM1 (red) after 7 days of culture in the co-culture system.\n(2) Macrophage-conditioned immune niche (in vivo)\nIn the reconstruction model, BP@IL-Gel established a more pro-chondrogenic immune niche than BP-Gel alone (Fig. R1-9-2A; Fig. 4b in manuscript). Histology revealed better preserved cartilage architecture (Fig. R1-9-2B; Fig. 4c in manuscript), with higher lacunae counts and increased hydroxyproline content (Fig. R1-18C-D; Fig. 4d-e in manuscript), consistent with enhanced matrix deposition and maturation under macrophage modulation.\nFigure R1-9-2 Evaluation of cartilage lacunae and matrix deposition under different immune niches. (A) Schematic diagram of the immune niche around BioC in the BP-Gel and BP@IL-Gel groups. (B) Representative H&E-stained micrographs of tissue specimens collected from different groups. (C) Quantitative analysis of the number of cartilage lacunae in different groups. (D) Quantitative analysis of hydroxyproline content in tissue specimens collected from different groups.\nIn line with our response in Comment 8, immunophenotyping showed that IL-Gel promoted macrophage polarization toward an M2 phenotype (MMR+/CD86−) in the perichondral region. This polarization correlated with reduced expression of inflammatory mediators (IL-1β, TNF-α) and decreased infiltration of α-SMA+ inflammatory vessels into the cartilage interior, while simultaneously maintaining robust vascularization in the peripheral connective tissue. Such vascular distribution ensures adequate nutrient delivery and supports epithelial regeneration, without disrupting the intrinsic avascularity of cartilage.\nTogether, these results indicate that while BP-Gel initiates chondrocyte aggregation, IL-Gel-polarized macrophages are pivotal in (i) supplying chondrogenic factors (TGF-β, IGF, bFGF), (ii) augmenting COL2A1-rich matrix formation, and (iii) activating endothelial cells to establish a vascularized perichondral rather than intra-cartilage niche. This macrophage-driven, spatially selective support preserves cartilage integrity and promotes functional tissue maturation in vivo. We sincerely thank the reviewer for this valuable suggestion, which has helped us refine and strengthen our mechanistic interpretation.\n10. Several parts of the methodology section lack comprehensive descriptions.\nReply:\nWe thank the reviewer for highlighting this important concern. We fully agree that clear and comprehensive methodological descriptions are essential to ensure scientific reproducibility and transparency. In response, we have now thoroughly updated and expanded the Materials and Methods section to ensure that all experimental steps are fully replicable. All revisions are clearly indicated in the tracked version of the manuscript using Word’s revision mode for ease of review.\nWe thank the reviewer again for encouraging us to strengthen this crucial section.\nMinor comments:\n11. Gene/protein nomenclature usage is inconsistent across mouse, rabbit, and human studies.\nReply:\nWe thank the reviewer for highlighting the inconsistency in gene/protein nomenclature. In the revised manuscript, we have standardized the usage according to accepted conventions:\nType Human Mice/Rabbit\nGene ALL CAPS, italicized Lowercase with first letter capitalized, italicized\nProtein ALL CAPS, non-italic ALL CAPS, non-italic\nNote: In the RNA sequencing results, rabbit-specific gene nomenclature is less formally standardized; therefore, we adopted human gene naming conventions to present the data more clearly.\nAll relevant text, figures, and legends have been carefully revised to ensure consistency across species. We thank the reviewer again for identifying this important issue, which significantly improves the manuscript’s rigor and clarity.\n12. The number of biological and technical replicates is not specified.\nReply:\nWe thank the reviewer for pointing out this critical issue. To address this, we have carefully revised the figure legends to specify the replication methods and details corresponding to each result, ensuring that readers can readily understand the experimental design and reproducibility. For example:\nPage 11, Line 4: “(b) Quantitative analysis of the number of chondrocyte clusters in BP-Gel and GelMA (n = 5 samples per group, with each sample from an individual well).”\nThese revisions significantly enhance the scientific transparency and rigor of the study. We thank the reviewer again for this important reminder.\n13. Information regarding IL-4/13 acquisition is missing.\nReply:\nWe thank the reviewer for pointing out this omission. In the revised version, we have supplemented the detailed acquisition information in Table S4, where the sources are now clearly listed as follows: Interleukin-4 (IL-4), PeproTech, Cat# 214-14; Interleukin-13 (IL-13), PeproTech, Cat# 210-13.\n14. The authors should clarify whether they mean \"air-liquid interface\" rather than \"gas-liquid interface\".\nReply:\nWe thank the reviewer for this helpful clarification. The reviewer is absolutely correct, we intended to refer to the standard “air-liquid interface (ALI)” culture system, commonly used to model respiratory epithelial differentiation. The phrase “gas-liquid interface” was an error.\nWe thank the reviewer again for identifying this error. The terminology has now been corrected for accuracy and consistency with the established literature.\n15. GEO accession number for RNA sequencing data must be provided.\nReply:\nWe thank the reviewer for this important reminder regarding data accessibility. In response, we have now deposited all RNA sequencing datasets related to the perichondral immune niche analysis in the NCBI Gene Expression Omnibus (GEO) database. This information has been included in the Data and materials availability statement:\nPage 39, Line 5: “The raw RNA sequencing data have been deposited in the Gene Expression Omnibus (GEO) under accession number GSE309417.”\nWe thank the reviewer for emphasizing the importance of transparent and open data availability.\n16. For Figure S7, the time points for panels h and i evaluation require specification.\nReply:\nWe appreciate the reviewer’s attention to detail and agree that the temporal context for these panels is essential for correct data interpretation. We have now integrated panels h and i into a single figure and specified the evaluation time point, which was 6 h. The corresponding figure has been updated accordingly (Fig. R1-16-1; Fig. S10g-i in manuscript).\nFigure R1-16-1 Representative images and quantitative analysis of epithelial cell migration in different groups.\nWe thank the reviewer for helping us improve the clarity and rigor of our data presentation.\nThe specified time points have now been clearly incorporated into all relevant sections.\n17. Animal protocol approval number for the studies should be included.\nReply:\nWe thank the reviewer for this important observation. All animal experiments described in this study were conducted in full compliance with institutional and national guidelines for the care and use of laboratory animals.\nWe have now supplemented the manuscript with the relevant ethical approval information:\nPage 33, Line 2: “All animal care, feeding, and research procedures were carried out following the Guide for the Care and Use of Laboratory Animals and were approved by the Ethics Committee of Shanghai Pulmonary Hospital (No. K23-107Y).”\nWe thank the reviewer again for highlighting this critical requirement.\nReviewer #2\nI 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.\nReply:\nWe sincerely thank the co-reviewer and the primary reviewer for their time, careful reading, and valuable feedback on our manuscript. The thoughtful and constructive comments provided have significantly improved the clarity, rigor, and impact of our work. We have addressed all concerns in detail in our point-by-point responses and revised the manuscript accordingly.\nReviewer #3\nThis manuscript by Tang et al. presents a well-written and conceptually innovative study that introduces the Dynamic Tissue Engineering (DTE) strategy. This approach mimics embryonic cartilage development while concurrently modulating the immune microenvironment to support coordinated epithelial and vascular regeneration. The DTE strategy utilizes a bio-adaptive physical hydrogel (BP-Gel) in combination with an interleukin-loaded gel (IL-Gel), thereby enabling precise spatiotemporal control of the regenerative process. Meanwhile, the authors developed a new in vitro cell culture model and an in vivo tissue cultivation model, which together provide robust experimental support for the DTE approach. Moreover, the successful demonstration of tracheal reconstruction with favorable survival rates and functional outcomes underscores the translational potential of this strategy. Overall, the study is supported by the well-structured experimental design and comprehensive data, representing a meaningful advancement in tracheal tissue engineering.\nReply:\nWe sincerely thank the reviewer for their encouraging and thoughtful comments. We are particularly grateful for the recognition of the conceptual innovation behind the Dynamic Tissue Engineering (DTE) strategy, as well as the translational significance of our findings. We have carefully addressed all detailed comments and concerns raised in the review, and we believe that the revised manuscript is now significantly improved in terms of scientific rigor, clarity, and reproducibility.\nHowever, the following issues need to be further clarified and supplemented by the authors to enhance the overall rigor of the manuscript and ensure accurate communication of the study’s conclusions:\n1. The authors introduced a novel paradigm for cartilage development and demonstrated its advantages over traditional cartilage culture under truly dynamic developmental conditions. However, the technical challenges associated with implementing this paradigm were not sufficiently elaborated. Compared to conventional methods, has there been any optimization in terms of culture duration or the required cell number? This should be detailed in the study.\nReply:\nWe thank the reviewer for this important point. Indeed, the Dynamic Tissue Engineering (DTE) strategy imposes specific technical challenges compared to static or pellet-based cartilage culture methods. We have now included a detailed explanation of these aspects in the revised manuscript.\n(1) Technical challenges in the DTE paradigm\nUnlike traditional pellet culture or scaffold-free pellet models, the DTE approach relies on cell-matrix remodeling within a soft, physically adaptive hydrogel, where cell aggregation, condensation, and extracellular matrix (ECM) deposition occur in a 3D dynamic and mechanically evolving environment. The key technical challenges include: (a) Preserving chondrocyte viability while promoting cellular aggregation within a soft, shear-responsive hydrogel matrix that supports cell migration while preventing premature collapse; (b) Ensuring uniform cell seeding and homogeneous distribution in the hydrogel matrix; (c) Balancing gel mechanics by achieving stable gelation at 37℃ to maintain structural integrity during the initial stage, while allowing gradual degradation in synchrony with extracellular matrix deposition by chondrocytes at later stages. For instance, although Pluronic F127 also exhibits thermosensitive properties, its structural integrity only lasted for 48 h due to its rapid erosion in aqueous environments (Fig. R3-1-1; Fig. S3b in manuscript) and thus it is unable to support the full growth cycle of engineered cartilage.\nFigure R3-1-1 Degradation comparison between Pluronic F127 and BP-Gel. Rose red was used to trace the F127-gel, while the BP-gel was highlighted with a blue background.\n(2) Advantages over conventional cartilage culture approaches\nCurrently, the main approaches for bioengineered cartilage culture include: (a) pellet culture, in which high-density chondrocyte aggregation can generate small cartilage nodules, and some studies have further employed 3D printing of pellet units to construct larger cartilage tissues19 (Fig. R3-1-2A); (b) Another widely used and representative method is the in vitro culture of high-density chondrocytes. These cells can be stacked and shaped within molds20 (Fig. R3-1-2B), or seeded into pre-formed scaffolds, as employed in our previous study21 (Fig. R3-1-2C). Both approaches can be used to generate bioengineered cartilage.\nFigure R3-1-2 Representative strategies for bioengineered cartilage culture. (A) Pellet culture and 3D-printed pellet assemblies. (B) High-density chondrocyte stacking in molds. (C) Seeding high-density chondrocytes onto pre-formed scaffolds.\nHowever, these approaches lack the necessary cellular growth microenvironment and rely on cell stacking to promote cartilage development and ECM production. In contrast, during native cartilage development, intercellular signaling, mechanical cues, and heterogeneous oxygen distribution are critical for determining tissue-specific biological functions and microarchitectural organization. Compared with conventional cartilage tissue culture methods, the present study proposes an innovative strategy.\nMoreover, conventional culture methods require a large number of chondrocytes, leading to increased costs and prolonged culture duration. In this study, we iterated on the above research. While conventional pellet culture requires a cell density of up to 2×108 cells/mL, and the cell density for high-density scaffold implantation is at least 3×107 cells/mL. Our study reduced the cell density to 4×106 cells/mL, a 7.5-50-fold reduction. With the optimization of the culture environment and the improvement of materials, the number of cells can be further reduced, which will enhance the application and translation potential of cartilage culture technology.\nThis information has now been added to the Materials and methods section:\nPage 33, Line 12: “Chondrocytes were uniformly suspended with sterilized BP-Gel solution (12.5 wt%), which was filtered through 0.22 μm filters prior to use. Based on optimization experiments aimed at reducing chondrocyte usage while maintaining cartilage-forming capacity, the cell density was set at 4×106 cells/mL, which is substantially lower than used in traditional culture conditions, to enable the formation of BioC.”\nIn addition, we have revised the Discussion section and provided a more detailed elaboration on the relevant content:\nPage 27, Line 6: “Macrolevel structural designs are insufficient to influence matrix fiber orientation or cortical organization, and thus fail to reproduce the spatiotemporal stress patterns that guide cartilage development. Therefore, current engineered cartilage tissues remain suboptimal in terms of its retention and development in vivo 22. Increasing evidence indicates that the native cartilage formation is closely related to its growth pattern and developmental stages23,24.”\nPage 27, Line 24: “Meanwhile, within the BP-Gel, chondrocytes undergo aggregation and condensation at a significantly lower seeding density than required in conventional pellet cultures, while still supporting robust extracellular matrix deposition and cartilage-like tissue formation, thereby reducing cell consumption, minimizing culture costs, and enhancing translational potential.”\nWe thank the reviewer again for prompting this clarification. The revised text now provides a more comprehensive description of the DTE paradigm’s technical considerations and the optimizations made over traditional methods.\n2. The authors mentioned that the physically crosslinked hydrogel exhibits a 3D polymer network that guides dynamic cell movement and promotes organized tissue development, which is a very novel and attractive finding. However, the background section does not clearly explain the detailed process of embryonic cartilage development. Clarifying the process of embryonic cartilage formation and highlighting both the similarities and differences between the cartilage development in BP-Gel and natural embryonic development is necessary to avoid reader confusion.\nReply:\nWe appreciate the reviewer’s insightful suggestion. We agree that the manuscript would benefit from a clearer explanation of embryonic cartilage development, particularly as it forms the biological rationale for our Dynamic Tissue Engineering (DTE) strategy.\nFirst, we elaborate on the background of embryonic cartilage development in more detail.\nEmbryonic cartilage development is initiated through mesenchymal condensation, in which undifferentiated mesenchymal cells migrate, proliferate, and aggregate into dense clusters25. Within these condensations, cells commit to the chondrogenic lineage under the control of key transcription factors, most notably SOX926, and signaling pathways such as TGF-β, BMP, and Wnt/β-catenin27. These signals not only drive chondrocyte differentiation but also regulate extracellular matrix deposition, including type II collagen and proteoglycans, which form the structural framework of the cartilage primordium27. As development proceeds, the local microenvironment exerts profound influence25. Gradients of oxygen tension establish zonal heterogeneity, while mechanical cues derived from surrounding tissues guide matrix fiber orientation and stratified architecture28. The resulting “cortex-medulla” organization and anisotropic fiber alignment are essential for conferring mechanical resilience and load-bearing capacity29. Thus, the interplay between cellular signaling, biochemical regulation, and biophysical environment defines both the biological function and microstructure of native cartilage.\nBased on this, we have revised the Introduction section and added a comprehensive description of embryonic cartilage development in the Discussion section, as follows:\nPage 3, Line 14: “Specifically, the physical percolation network in the BP-Gel could dynamically adapt to the migration, proliferation, and aggregation of chondrocytes, thus facilitating the fusion of chondrocyte clusters into large aggregates. This procedure resembles the embryo-like chondrification process, in which undifferentiated mesenchymal cells migrate, proliferate, and condense to form cartilage primordia1-3.”\nPage 27, Line 10: “During embryonic chondrogenesis, mesenchymal cells migrate, proliferate, and condense25, followed by differentiation into chondrocytes under the regulation of key transcription factors such as SOX926 and signaling pathways including TGF-β and Wnt/β-catenin27. These coordinated events give rise to the cartilage primordium and establish the basis for matrix deposition and structural organization27. Beyond molecular regulation, the local biophysical environment plays an equally critical role. Intercellular signaling, mechanical environment, and heterogeneous oxygen distribution are critical factors in determining the biological function and microstructure of cartilage3,30.”\nSpecifically, to illustrate the process of cartilage development more directly, we further performed histological staining (H&E, AB&NFR, and SO&FG) on mice embryonic trachea at E12.5, E14.5, and E16.5, stages that are characterized by initial mesenchymal condensation and the onset of cartilage matrix deposition. We observed that cells at the sites of tracheal cartilage development exhibited pronounced condensation in E12.5 embryos, while cartilage tissue gradually began to form at E14.5 and E16.5 (Fig. R3-2-1A; Fig. S6b in manuscript). In the in vitro cartilage culture using BP-Gel, chondrocytes also exhibited aggregation and fusion, whereas in the GelMA group, the cells remained consistently scattered throughout the culture period (Fig. R3-2-1B; Fig. S6a in manuscript). This comparison helps clarify that while our system does not fully replicate all embryonic morphogen gradients, it reconstructs key biophysical and organizational dynamics, particularly mesenchymal condensation, chondrocyte-driven ECM deposition, and zonal matrix structuring.\nFigure R3-2-1 Chondrocyte condensation in embryonic trachea and in vitro cultures. (A) Mesenchymal condensation at E12.5 with cartilage formation at E14.5 and E16.5. (B) Representative histological images of chondrocytes cultured in BP-Gel and GelMA for 2 and 4 weeks in vitro.\nWe thank the reviewer again for raising this important point. By incorporating a mechanistic summary of embryonic cartilage development and drawing explicit parallels with our BP-Gel system, we believe the revised manuscript provides much clearer conceptual grounding for our DTE approach.\n3. The observation of dynamic cellular aggregation in the results is quite exciting. This phenomenon is likely related to the physically crosslinked structure of the material itself. However, such behavior may be highly dependent on factors such as material concentration. Yet, the manuscript only use a single material concentration. What is the basis for this choice?\nReply:\nWe thank the reviewer for this insightful comment. We agree that the observed dynamic cellular aggregation is closely influenced by the physical properties of the BP-Gel matrix, which are in turn governed by its polymer concentration.\nIn our previous studies, thermosensitive hydrogel materials were generally applied at concentrations of approximately 25 wt% for biological applications, with further adjustments made depending on the desired degradation time and other parameters. Building on these prior experiences and the experimental data from the present study, we elected 12.5 wt% PLGA-PEG-PLGA as the working concentration for BP-Gel after preliminary screening of concentrations ranging from 6.5 to 25 wt%. Our results demonstrated that relatively low concentrations yielded hydrogels that were too soft to maintain structural integrity under culture conditions, leading to cell sedimentation and non-uniform distribution. In contrast, relatively high concentrations produced hydrogels with excessive stiffness and reduced porosity, which hindered chondrocyte migration and aggregation, resulting in scattered cells rather than functional condensates, similar to the distribution observed in GelMA (Fig. R3-3-1 A; Fig. S3a in manuscript). Moreover, using different cell types (macrophages and chondrocytes), we verified that the 12.5 wt% BP-Gel, upon gelation, exerted a low-adhesion effect on cells, which adopted a spherical morphology. Compared to conventional 2D culture plates, this environment promoted cellular aggregation and fusion (Fig. R3-3-1 B; Fig. S4c in manuscript).\nFigure R3-3-1 Effects of BP-Gel concentration on hydrogel integrity and cell behavior. (A) Bright-field images of chondrocytes cultured in BP-Gel at different concentrations. (B) Morphology of macrophages and chondrocytes in 12.5 wt% BP-Gel compared with conventional 2D culture.\nWe thank the reviewer for prompting this clarification. By including the basis for concentration selection and new supporting data, we have strengthened the experimental rationale for our choice of BP-Gel formulation and its role in enabling dynamic cellular aggregation.\n4. The BP-Gel system in this study is based on a thermosensitive PLGA-PEG-PLGA hydrogel, which undergoes a sol-gel transition near body temperature. However, the thermosensitive property does not appear to be prominently utilized or discussed as a functional feature in either in vitro or in vivo applications. The authors are encouraged to clarify whether thermosensitivity mainly serves as an operational convenience or whether it also contributes to cellular behavior modulation or tissue formation.\nReply:\nWe greatly appreciate this insightful comment, which indeed touches on a question we frequently consider as developers of biomaterials-namely, whether the intrinsic properties of a material can fully serve the requirements of its target application. We would like to address this from three perspectives.\nFirst, the thermosensitivity of our system provides significant advantages that go beyond experimental convenience, directly contributing to the success of cartilage construction. For example, at room temperature, the system exhibits very low viscosity (G’＜1Pa), which allows chondrocytes to be homogeneously dispersed within the culture system. Upon warming to 37 ℃ (the standard cell culture temperature), the system rapidly solidifies without the need for any additional manipulation. Similarly, when constructing biomimetic connective tissue, the ability to cast IL-Gel at room temperature is not only convenient but also ensures complete filling of the inter-cartilage spaces due to its fluidity.\nSecond, we acknowledge that after the sol-gel phase transition (although this process is reversible), the subsequent cultivation and growth of engineered cartilage occur at 37 ℃, where the thermosensitive property per se does not directly influence cartilage growth. Nevertheless, we believe it is important to view material properties in an integrated rather than isolated manner, and I believe we can reach a consensus. As we have elaborated in the manuscript, the physical cross-linked network formed by PLGA-PEG-PLGA plays a vital role in supporting chondrocyte growth. The thermosensitivity, which inevitably arises from the amphiphilic micellar architecture of the polymer, was leveraged to optimize the cartilage engineering process, and we believe this represents a merit.\nFinally, it should be noted that not all thermosensitive hydrogels are suitable for cartilage tissue engineering. For instance, although Pluronic F127 is also thermosensitive, it degrades completely within 72 h (Fig. R3-4-1; Fig. S3b in manuscript) and is thus unable to support the full growth cycle of engineered cartilage.\nFigure R3-4-1 Degradation comparison between Pluronic F127 and BP-Gel. Rose red was used to trace the F127-gel, while the BP-gel was highlighted with a blue background.\nWe thank the reviewer again for the thoughtful suggestion. We have now clarified that thermosensitivity in the BP-Gel system plays both a practical and a biologically functional role in enabling dynamic tissue formation.\nRegarding specific experimental results, the following questions need to be addressed:\n5. In Fig. 1a, the conceptual illustration of the overall study is too complex. It is recommended to simplify the graphic components and add more textual explanations for better reader comprehension. For example, the representation of the inflammation microenvironment (vessels and cells in the upper right corner of BP-Gel) appears overly complicated.\nReply:\nWe thank the reviewer for this helpful suggestion regarding Figure 1a. We agree that clarity and accessibility are critical for conveying the overall concept of the Dynamic Tissue Engineering (DTE) strategy. To improve clarity and readability, we redesigned Fig. 1a by simplifying picture elements, refining labels and annotations, and harmonizing the colors and layout (Fig. R3-5-1; Fig. 1a in manuscript). We also updated the figure legend for Fig. 1a to match the simplified content and provide a concise textual summary of the conceptual diagram.\nFigure R3-5-1 DTE tracheal construction strategy.\nThe updated figure legend is as follows:\nPage 6, Line 3: “(a) DTE tracheal construction strategy: (i) The substitute consists of BioC rings (formed by culturing chondrocytes in BP-Gel) and IL-Gel (BP-Gel loaded with interleukins). (ii) The ether bonds of the BP-Gel form a surface “water film” through hydrogen bonding, which prevents adhesion and promotes cell clustering, mimicking embryonic development. (iii) IL-Gel enables slow interleukin release, inducing macrophage polarization and immune cascade activation. This sustains the cartilage phenotype, stimulates angiogenesis, and enhances epithelial migration.”\nWe appreciate the reviewer’s feedback, which has helped us improve the clarity and accessibility of our conceptual illustration. The revised Figure 1a more clearly communicates the core idea of the DTE strategy and the roles of BP-Gel and IL-Gel in orchestrating tissue regeneration.\n6. Based on my understanding, are Figs. 2h, 2l, and 2m derived from the same sample group? What are their differences in terms of visual presentation and quantitative analysis? What specific issues do they aim to address respectively? This should be clarified in the manuscript.\nReply:\nWe thank the reviewer for carefully examining these figures and raising this important point. We agree that the rationale for including Figures 2h, 2l, and 2m needs to be clarified to avoid confusion. Figures 2h, 2l, and 2m are derived from the same experimental cohort. However, each figure serves a distinct analytical and conceptual purpose, as detailed below:\n(1) Histological assessment of cartilage-like tissue and matrix deposition\nFigure 2h shows histological images stained with H&E, AB&NFR, and COL2A1 IHC to qualitatively assess cartilage-like tissue formation and GAG-rich matrix deposition. The representative sections highlight tissue morphology, matrix intensity, and zonal distribution, providing visual confirmation of successful chondrogenic matrix development in BP-Gel compared to controls (Fig. R3-6-1; Fig. 2h in manuscript).\nFigure R3-6-1 Representative H&E, AB&NFR, and COL2A1 IHC staining micrographs of tissue specimens collected from different groups at 4 weeks post-implantation.\n(2) Reticular distribution of collagen revealed by SR staining\nFigure 2l shows the distribution of collagen in cartilage using SR staining. In native cartilage, COL2A1 displays a characteristic reticular structure that provides the essential mechanical properties and architecture required for cartilage function. The BioC we cultured exhibited a morphology closely resembling that of native cartilage (Fig. R3-6-2; Fig. 2l in manuscript).\nFigure R3-6-2 Representative SR-stained micrographs of tissue specimens collected from different groups at 4 weeks post-implantation.\n(3) Development of cortex-medulla structure in BioC\nFigure 2m shows the distribution of COL2A1 in BioC at different growth stages using SR staining, reflecting the formation of the cartilage cortex-medulla structure and its morphological changes over time (Fig. R3-6-3; Fig. 3m-o in manuscript).\nFigure R3-6-3 Polarizing microscope images of SR staining tissue specimens collected from different groups at designated time points, and quantitative analysis of intersphere angle of chondrocyte clusters, fluorescence intensity and thickness of cortex layer.\nIn addition, we have highlighted the corresponding content in the Results section to indicate the characteristic key significance of each result:\nPage 9, Line 15: “Following this process, BioC gradually began to resemble native cartilage. Histological analyses confirmed that after 4 weeks of in vivo culture, the collected BioC exhibited cartilage-specific extracellular matrix (ECM) deposition, as demonstrated by H&E, AB&NFR, and COL2A1 IHC staining, which qualitatively assessed tissue morphology, GAG-rich matrix deposition, and chondrogenic zonal distribution (Fig. 2h).”\nPage 9, Line 29: “To further assess the structural organization of cartilage matrix, Sirius Red (SR) staining for COL2A1 and 3D reconstruction plots demonstrated that the distribution of COL2A1 within BioC based on BP-Gel closely resembled the characteristic grid-like COL2A1 arrangement in the native cartilage; however, no similar COL2A1 distribution was observed in GelMA (Fig. 2l).”\nPage 10, Line 3: “As shown in Fig. 2m-o, SR staining of BioC at different growth stages revealed progressive fusion of chondrocyte clusters, continuous increase in COL2A1 deposition, and gradual thickening of the cortical layer, reflecting the dynamic formation of a “cortex-medulla” structure.”\nWe appreciate the reviewer’s detailed reading of our figures. We have clarified the relationship among these panels and their distinct analytic purposes to avoid misinterpretation or perceived redundancy.\n7. In Figs. 2e-f and Figs. S3e-g, the authors attempt to interpret cartilage condensation and development using the HIF-1α and YAP signaling pathways. Is this condensation process an essential step in native cartilage development? There is a lack of control data from natural cartilage, which should be supplemented to strengthen the explanation.\nReply:\nWe thank the reviewer for this important comment. We would like to clarify that in Figs. 2e-f and S3e-g the interpretation was based on HIF-1α and COL2A1, rather than HIF-1α and YAP. Regarding the reviewer’s question, condensation is indeed an essential step in native cartilage development. During embryogenesis, undifferentiated mesenchymal cells aggregate into dense clusters, where transcription factors such as SOX9 and signaling pathways including TGF-β, BMP, and Wnt/β-catenin drive chondrocyte differentiation and matrix deposition. This process establishes the structural framework that, together with hypoxic and mechanical cues, gives rise to the “cortex-medulla” organization and anisotropic fiber alignment of functional cartilage.\nAs mentioned previously, to illustrate the process of cartilage development more directly, we further performed histological staining (H&E, AB&NFR, and SO&FG) on mice embryonic trachea at E12.5 stages that are characterized by initial mesenchymal condensation. We observed that cells at the sites of tracheal cartilage development exhibited pronounced condensation in E12.5 embryos (Fig. R3-7-1A; Fig. S6b in manuscript). In the in vitro cartilage culture using BP-Gel, chondrocytes also exhibited aggregation and fusion, whereas in the GelMA group, the cells remained consistently scattered throughout the culture period (Fig. R3-7-1B; Fig. S6a in manuscript). This comparison helps clarify that while our system does not fully replicate all embryonic morphogen gradients, it reconstructs key biophysical and organizational dynamics, particularly mesenchymal condensation, chondrocyte-driven ECM deposition, and zonal matrix structuring.\nFigure R3-7-1 Chondrocyte condensation in embryonic trachea and in vitro cultures. (A) Mesenchymal condensation at E12.5. (B) Representative histological images of chondrocytes cultured in BP-Gel and GelMA for 2 and 4 weeks in vitro.\nIn our study, HIF-1α expression and COL2A1 deposition were used as readouts to capture microenvironmental influences on condensation and cartilage characteristics. We acknowledge that including native cartilage controls could provide additional reference; however, it is important to note that during the early condensation phase, mesenchymal stem cells drive cell clustering at a stage when chondrocyte-specific markers are not yet prominent, and characteristic cartilage genes such as COL2A1 only emerge at later stages of maturation. In our culture system, since chondrocytes were used as the starting population, the expression of HIF-1α and COL2A1 appeared at earlier stages.\nMoreover, HIF-1α is the central transcription factor regulating cellular responses to hypoxia and plays a critical role in cartilage development. It is highly expressed in embryonic cartilage and growth plate cartilage, where it supports chondrocyte survival and differentiation under low-oxygen conditions. In mature cartilage, however, HIF-1α is maintained at a stable, low basal level, primarily to sustain metabolic homeostasis. Therefore, for the evaluation of HIF-1α and COL2A1 expression in this study, native tracheal cartilage does not serve as an entirely appropriate benchmark. Accordingly, we adopted a widely recognized model of chondrocyte development, namely the generation of cell spheroids, which form cartilage organoid-like aggregates. To further substantiate this point, we performed IF staining of HIF-1α and COL2A1 in cell spheroids (Fig. R3-7-2; Fig. 2e and S5c in manuscript). Consistent with developmental biology, HIF-1α was highly expressed during chondrogenic progression, accompanied by increased COL2A1 deposition. Notably, the distribution of HIF-1α differed between cell spheroids and BP-Gel constructs, which we attribute to environmental differences: BP-Gel constructs were partially surrounded by tissue, whereas cell spheroids were directly exposed to atmospheric oxygen, preventing stable retention of HIF-1α in the cytoplasm. Taken together, these results demonstrate that cartilage cultured within BP-Gel closely resembles cell spheroids, both exhibiting high expression of HIF-1α and COL2A1.\nFigure R3-7-2 Fluorescence images of HIF-1α and COL2A1 in BP-Gel-cultured versus cell spheroids.\nThese results are now included in the Results section:\nPage 8, Line 8: “Next, we explored the process of chondrocyte development in BioC. IF staining revealed that the expression of Collagen type II alpha 1 chain (COL2A1) and Hypoxia-inducible factor 1-alpha (HIF-1α) was significantly increased within the chondrocyte clusters formed in BP-Gel (Fig. 2e and S5a, showing an amplified region of Fig. 2e). Colocalization analysis based on protein expression intensity demonstrated that the expression levels of these two proteins were highly coincident (Fig. 2f and Fig. S5b). To further substantiate this point, we examined cell spheroids, a recognized model of chondrocyte development. IF staining showed high expression of HIF-1α and COL2A1 in cell spheroids, which closely resembled the expression patterns observed in BioC cultured within BP-Gel (Fig. S5c).”\nWe thank the reviewer for prompting this clarification and additional controls. By integrating native cartilage references, we believe the manuscript now provides stronger biological grounding during DTE-induced condensation.\n8. In Fig. 4c, the presentation of cartilage development is relatively limited. It is necessary to include additional specific staining and biochemical assays of the cartilage matrix to better validate the promoting and protective effects of the BP@IL-Gel composite structure on ectopic cartilage development in vivo.\nReply:\nWe sincerely thank the reviewer for this thoughtful suggestion regarding the need for additional validation of cartilage matrix deposition and phenotype maintenance. We would like to clarify that these analyses were already included in our study. Specifically, after 2 weeks of in vivo implantation, H&E, AB&NFR, and COL2A1 IHC staining of the harvested tissues confirmed that the BP@IL-Gel composite exhibited the most pronounced cartilage-specific ECM deposition among the four groups (Fig. R3-8-1A; Fig. 4c and S11b-c in manuscript). Quantitative analysis of cartilage lacunae further demonstrated that the BP@IL-Gel group showed the highest number of lacunae (Fig. R3-8-1B; Fig. S11d in manuscript).\nFigure R3-8-1 Histological staining and quantitative analysis of cartilage lacunae. (A) Representative H&E, AB&NFR and COL2A1 IHC staining micrographs of tissue specimens collected from different groups. (B) Quantitative analysis of the number of cartilage lacunae in different groups.\nIn addition, biochemical assays of cartilage matrix, including relative glycosaminoglycan (GAG) and hydroxyproline contents, revealed that the BP@IL-Gel structure design promoted both the maintenance of the cartilage phenotype and the protective effects on ectopic cartilage development (Fig. R3-8-2; Fig. S11e-f in manuscript).\nFigure R3-8-2 Biochemical evaluation of cartilage matrix components. (A) Quantitative analysis of hydroxyproline content in tissue specimens collected from different groups. (B) Quantitative analysis of GAG content in tissue specimens collected from different groups.\nWe apologize if the original presentation did not sufficiently highlight these results, and we have now emphasized them more clearly in the revised manuscript. Accordingly, the corresponding results have been re-stated in the Results section to ensure clarity for readers:\nPage 17, Line 29: “After 2 weeks of in vivo implantation, histological staining provided clear evidence of cartilage-specific matrix deposition. AB&NFR staining and COL2A1 IHC staining consistently showed that the BP@IL-Gel composite exhibited the most remarkable cartilage-specific ECM deposition among the four groups (Fig. 4c and S11b-d). To further quantify these observations, we analyzed cartilage lacunae based on the stained tissues and found that the number of lacunae was highest in the BP@IL-Gel group (Fig. 4d and S11d). In addition, biochemical assays of cartilage matrix demonstrated that relative glycosaminoglycan (GAG) and hydroxyproline contents were significantly elevated in the BP@IL-Gel group, indicating that this structural design effectively promoted maintenance of the cartilage phenotype and protected ectopic cartilage development (Fig. 4e and S11e-f).”\nWe thank the reviewer again for this constructive recommendation. The histological, immunohistochemical, and biochemical data substantiate the superior chondrogenic efficacy of the BP@IL-Gel composite.\n9. In Fig. 4m, please clearly indicate which key genes related to the biological process are upregulated or downregulated, as this is critical for the interpretation of the sequencing results.\nReply:\nWe thank the reviewer for emphasising the importance of distinguishing up‑ versus down‑regulated genes in the GO enrichment heat‑map (Fig. 4m). However, we would like to clarify that Fig. 4m in the manuscript (Fig. R3-9-1A) illustrates the correlation between biological processes and their associated molecular pathways. In this analysis, the ranking of each biological process is determined by the number of differentially expressed genes mapped to the corresponding pathway. Thus, the figure is primarily intended to reflect which pathways are most closely related to the observed alterations in local biological processes following treatment, rather than to indicate whether the associated pathways are up- or down-regulated.\nFigure R3-9-1 GO enrichment and gene expression profiling of BP-Gel and BP@IL-Gel groups. (A) GO analysis of the RNA-seq data from the BP-Gel and BP@IL-Gel groups. (B) Relative expression of genes related to crucial processes, including inflammation and angiogenesis, in the BP-Gel and BP@IL-Gel groups.\nAdditionally, we evaluated the relative expression of genes associated with crucial processes, including inflammation and angiogenesis, in the BP-Gel and BP@IL-Gel groups (Fig. R3-9-1B; Fig. 4n in manuscript). Overall, the heat map allows us to roughly determine the expression trends of specific genes based on the color patterns. For instance, inflammation-related genes such as PTK2B, ALOX5, HMGB1, BTK, and HPSE exhibit an up-regulated trend. However, the sequencing results are presented here mainly to reflect the overall regulatory tendencies of the materials. The changes in individual genes are affected by many factors, and if the role of a specific gene in a biological process is to be elucidated, further investigation would be required. We will improve on this aspect in our subsequent studies, but it is not the primary focus of the present work.\nWe apologize for not having made this point sufficiently clear in the original version. We have now emphasized it more clearly in the revised manuscript. Accordingly, the corresponding results have been re-stated in the Results section to ensure clarity for readers:\nPage 19, Line 7: “The analysis results indicate that BP@IL-Gel and BP-Gel exhibit distinct effects on modulating the inflammatory microenvironment and vascularization. The heat-map further allows a preliminary assessment of the up- or down-regulation trends of related genes (Fig. 4n).”\nWe hope this revision satisfies the reviewer’s request and improves the interpretability of our sequencing results. Thank you again for helping us strengthen the manuscript.\n10. In Fig. 5, the authors suggest that IL-Gel can protect cartilage from vascular invasion. Does IL-Gel hinder capillary regeneration around the cartilage? This needs to be addressed in the manuscript, with additional data provided if necessary.\nReply:\nWe thank the reviewer for raising this important question. We would like to emphasize that the protective effect of IL-Gel on cartilage is not achieved by indiscriminate inhibition of angiogenesis. Instead, IL-Gel reshapes the inflammatory microenvironment, selectively preventing pathological vascular invasion into cartilage while supporting the regeneration of functional capillaries in the surrounding tissue. This distinction is critical for maintaining the avascularity required for hyaline cartilage maturation, without compromising peripheral perfusion that provides nutrients and immune regulation.\n(1) IL-Gel promotes organized peripheral angiogenesis\nIF staining for PECAM1 demonstrated that blood vessels in the IL-Gel group were more abundant, evenly distributed, and structurally organized compared with BP-Gel alone (Fig. R3-10-1A; Fig. S8m in manuscript). Quantitative analysis further confirmed a significantly greater vascular area in the perichondral regions of IL-Gel constructs, comparable to native controls (Fig. R3-10-1B-C; Fig. S8n-o in manuscript). These findings indicate that IL-Gel facilitates functional angiogenesis around cartilage tissue.\nFigure R3-10-1 IL-Gel promotes functional and organized angiogenesis in vivo. (A) Representative fluorescence microscopy images of tissue specimens labeled with PECAM1 (red) antibody in different groups. (B-C) Quantitative analysis of vascular area in the indicated regions.\n(2) IL-Gel prevents pathological vascular invasion into cartilage\nIn contrast, α-SMA staining showed that in the BP-Gel group, inflammation-driven vessels penetrated the cartilage core, correlating with accelerated matrix degradation and ossification. SO&FG staining revealed a concomitant reduction in proteoglycan-rich cartilage area (Fig. R3-10-2; Fig. 4j and S11i in manuscript). By comparison, IL-Gel treatment suppressed aberrant intra-cartilage vascularization, thereby preserving cartilage lacunar structure and matrix integrity.\nFigure R3-10-2 IL-Gel prevents aberrant vascular invasion and preserves cartilage integrity. (A) Representative α-SMA (green) labeled fluorescence microscopy images of tissue specimens collected from different groups, and quantitative analysis of the number of blood vessels. The white triangles indicate the invasive blood vessels in cartilage tissue. (B) Representative SO&FG-stained micrographs of tissue specimens collected from the GelMA and GelMA@Blk groups and quantitative analysis of SO&FG-stained area.\n(3) IL-Gel exhibits intrinsic pro-angiogenic capacity in vitro\nTo further evaluate its angiogenic potential, we examined endothelial responses in vitro. IL-Gel-induced macrophage polarization significantly enhanced HUVEC migration, tube formation, and the expression of endothelial cell-related genes (PECAM1, VEGF) compared with controls (Fig. R3-10-3; Fig. 3l-m and S10c-f in manuscript). These results demonstrate that IL-Gel not only permits but actively promotes angiogenesis under controlled conditions.\nFigure R3-10-3 Pro-angiogenic effects of IL-Gel on HUVEC migration, tube formation, and gene expression. (A-B) Representative fluorescence microscopy images and quantitative analysis of endothelial cells labeled with PECAM1 (red) after 7 days of culture in the co-culture system. (C) Expression levels of endothelial cell-related genes (PECAM1 and VEGFA) in HUVECs cultured in the co-culture system for 7 days. (D-E) Representative images and quantitative analysis of endothelial cell migration in different groups. (F) Representative images and quantitative analysis of endothelial cell tube formation in different groups.\nIn summary, IL-Gel does not hinder capillary regeneration. Instead, it promotes well-organized vascularization in the perichondral niche while preventing destructive vascular ingrowth into cartilage tissue. This dual effect creates a balanced microenvironment that simultaneously preserves cartilage avascularity and structural integrity, while ensuring sufficient nutrient delivery and epithelial support. Therefore, we have highlighted this clarification in the revised manuscript, specifically in the Results section:\nPage 14, Line 7: “The IL-Gel group exhibited more functional blood vessels with an orderly arrangement (Fig. S8m-o). These findings suggest that IL-Gel promotes endothelial cell growth.”\nPage 15, Line 8: “Moreover, scratch assays and tube formation assays discovered that the presence of IL-Gel substantially boosted the migratory ability of HUVECs (Fig. S10d-e) and promoted angiogenesis (Fig. S10f). These results suggest that IL-Gel facilitates well-organized angiogenesis.”\nPage 18, Line 24: “Moreover, the increased vascular infiltration in the BP-Gel group made the cartilage more susceptible to ossification, as evidenced by SO&FG staining (Fig. S11i). Collectively, these results suggest that IL-Gel facilitates well-organized angiogenesis while avoiding aberrant vascular infiltration into cartilage tissue.”\nIn addition, we have incorporated the following statement in the Discussion section:\nPage 27, Line 30: “In addition to the intrinsic growth and developmental status of cartilage, extrinsic environmental factors also play a critical role in determining its long-term persistence in vivo. Cartilage development requires a relatively low-metabolic niche, and excessive vascular invasion can accelerate cartilage degradation. The beneficial effect of IL-Gel arises not from a direct inhibition of angiogenesis, but from reshaping the inflammatory microenvironment to prevent pathological vascular overgrowth into cartilage. Meanwhile, IL-Gel promotes functional vascularization in the surrounding tissue, ensuring adequate nutrient supply without compromising cartilage integrity.”\nWe thank the reviewer for this important comment, which has greatly assisted us in refining the presentation and clarifying the key points of our study.\n11. In Figs. 5b-c, the authors report average values of survival and respiratory function in rabbits after orthotopic tracheal transplantation. Please provide detailed individual data for each animal model to improve data transparency.\nReply:\nWe thank the reviewer for highlighting the importance of data transparency, particularly in preclinical animal studies. We agree that providing the individual values for survival and respiratory function enhances the reproducibility and credibility of the findings. We have now included detailed animal data for each group in a newly added Table S3.\nWe appreciate the reviewer’s recommendation. The individual animal data are now included as Supplementary Table S3, ensuring full transparency and reproducibility of our tracheal transplantation findings.\n12. Please supplement the vascular-specific staining (e.g., CD31) of the orthotopic graft sections and include quantitative analysis, as vascularization is also crucial for tracheal regeneration.\nReply:\nWe thank the reviewer for this constructive suggestion regarding vascularization in the orthotopic grafts. In response, we have now supplemented vascular-specific staining and quantitative analyses of the regenerated trachea.\nInstead of PECAM1 (CD31), we selected α-SMA as the primary marker to characterize vascular distribution in the grafts. The rationale is that the orthotopic grafts were harvested at relatively late stages of regeneration, by which time many vessels had already matured. While PECAM1 is well-suited for identifying newly formed endothelial cells and nascent capillaries31, α-SMA highlights mature, stabilized vessels with perivascular smooth muscle coverage32, which more accurately reflect long-term perfusion capacity and functional integration of the graft. Moreover, α-SMA staining allows clear visualization of vessel wall structure, enabling robust morphometric measurements of vascular diameter and wall thickness33. Thus, α-SMA provides a reliable representation of the functional vasculature necessary for sustained graft survival.\nOur staining results confirmed the presence of abundant vessels between the cartilage rings in the experimental group (BP-Gel+IL-Gel). Quantitative analysis demonstrated that both vessel diameter and vascular area were comparable to those observed in native trachea, indicating well-established and functional perfusion. By contrast, the BP-Gel only group also exhibited increased vascular density; however, these vessels were significantly smaller in caliber and irregularly distributed, consistent with disorganized neovascularization likely driven by inflammation rather than functional remodeling (Fig. R3-12-1; Fig. S12e-f in manuscript).\nFigure R3-12-1 Vascularization in regenerated trachea. (A) Representative H&E and α-SMA IHC staining images of inter-cartilaginous tissues from the BP-Gel, BP-Gel+IL-Gel, and native groups. (B) Quantification of vascular diameter and mean vascular area.\nTaken together, these findings demonstrate that IL-Gel not only preserves cartilage integrity but also supports the development of mature, functional vasculature in the perichondral regions of orthotopic grafts, thereby ensuring adequate nutrient supply and promoting long-term tracheal regeneration. We thank the reviewer for this important suggestion. The added vascular staining and quantification further demonstrate the functional integration and regenerative competence of our engineered tracheal grafts.\n13. What is the difference between the results of Fig. S7 and Fig. 3f? Please further clarify their respective significance.\nReply:\nWe thank the reviewer for this helpful comment. Indeed, both Fig. 3f and Fig. S7 (Now Fig. S10 in manuscript) are central to demonstrating the role of IL-Gel in activating macrophage polarization and subsequently influencing chondrocyte and endothelial development. These two figures represent two complementary but distinct models within our study.\nFig. S7 (Now Fig. S10 in manuscript) reflects the direct effects of IL-Gel on macrophages, highlighting their polarization status and cytokine secretion profile. This model is essential for confirming that IL-4/13-loaded IL-Gel can effectively drive macrophages toward an M2 phenotype.\nHowever, Fig. 3f presents the functional consequences of macrophage polarization, focusing on how IL-Gel-induced M2 macrophages influence downstream cell types, including chondrocytes, endothelial cells, and epithelial cells. Specifically, Fig. S7 (Now Fig. S10 in manuscript) shows enhanced gene expression, migration, tube formation, and chondrogenic/angiogenic potential of these target cells under co-culture with polarized macrophages.\nTaken together, Fig. 3f and Fig. S7 (Now Fig. S10 in manuscript) provide two layers of evidence: one at the level of macrophage polarization itself, and the other at the level of its impact on surrounding structural and vascular cells. This integrated approach highlights the mechanistic cascade by which IL-Gel reshapes the immune microenvironment to promote cartilage and vascular development.\nCorrespondingly, we have revised the Results section to include these findings:\nPage 14, Line 14: “We first established a co-culture system to examine the direct effects of IL-Gel on macrophage polarization, thereby confirming the ability of IL-4/13-loaded IL-Gel. We quantified cytokine levels in the culture medium of macrophages subjected to different treatments, as illustrated in Fig. S10a.”\nPage 14, Line 22: “To mimic the in vivo microenvironment, building upon this, we further established a co-culture system to co-culture macrophages with chondrocytes, endothelial cells, or epithelial cells (Fig. 3f). The results illustrate the functional consequences of IL-Gel-induced macrophage polarization, demonstrating how polarized macrophages subsequently regulate the growth and development of chondrocytes, endothelial cells, and epithelial cells.”\nWe thank the reviewer again for prompting this clarification. By clearly distinguishing the experimental context and purpose of these two figures, we ensure accurate interpretation of the immune regulatory dynamics at different phases and model systems.\n14. In the Materials and Methods section, some experimental descriptions are relatively vague, which affects reproducibility. Please provide more detailed parameters and protocols for each experimental procedure.\nReply:\nWe appreciate the reviewer’s emphasis on the importance of methodological clarity. We have now systematically revised the Materials and Methods section to ensure full reproducibility and transparency. All modifications are clearly marked in the revised manuscript using the Word track-changes mode for ease of review. We have carefully revised the Methods section to meet high standards of experimental reproducibility. We believe these additions will facilitate future replication and enhance the scientific rigor of the manuscript.\nReference：\n1. Ruscitto, A., et al. Lgr5-expressing secretory cells form a Wnt inhibitory niche in cartilage critical for chondrocyte identity. Cell Stem Cell 30, 1179-1198.e1177 (2023).\n2. Dai, M., et al. 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Dikina, A.D., et al. A Modular Strategy to Engineer Complex Tissues and Organs. Adv Sci (Weinh) 5, 1700402 (2018).\n20. Machino, R., et al. Replacement of Rat Tracheas by Layered, Trachea-Like, Scaffold-Free Structures of Human Cells Using a Bio-3D Printing System. Adv Healthc Mater 8, e1800983 (2019).\n21. Tang, H., et al. A bioengineered trachea-like structure improves survival in a rabbit tracheal defect model. Sci Transl Med 15, eabo4272 (2023).\n22. Daly, A.C. & Kelly, D.J. Biofabrication of spatially organised tissues by directing the growth of cellular spheroids within 3D printed polymeric microchambers. Biomaterials 197, 194-206 (2019).\n23. Decker, R.S., Koyama, E. & Pacifici, M. Articular Cartilage: Structural and Developmental Intricacies and Questions. Curr Osteoporos Rep 13, 407-414 (2015).\n24. Bhattacharjee, M., et al. Tissue engineering strategies to study cartilage development, degeneration and regeneration. Adv Drug Deliv Rev 84, 107-122 (2015).\n25. Yang, B., et al. Recapitulating hypoxic metabolism in cartilaginous organoids via adaptive cell-matrix interactions enhances histone lactylation and cartilage regeneration. Nat Commun 16, 2711 (2025).\n26. Yang, W. & Lefebvre, V. PTPN11 in cartilage development, adult homeostasis, and diseases. Bone Res 13, 53 (2025).\n27. Woods, J.P., Rackley, A., Kwon, H.R. & Olson, L.E. PDGFRα signaling regulates cartilage and fibrous tissue differentiation during synovial joint development. Nat Commun 16, 4041 (2025).\n28. Sunadome, K., et al. Directionality of developing skeletal muscles is set by mechanical forces. Nat Commun 14, 3060 (2023).\n29. Peters, J.R., et al. Tissue growth as a mechanism for collagen fiber alignment in articular cartilage. Sci Rep 14, 31121 (2024).\n30. Yang, S., et al. Morphogens enable interacting supracellular phases that generate organ architecture. Science 382, eadg5579 (2023).\n31. Markova, V., et al. Endothelial Cell Markers Are Inferior to Vascular Smooth Muscle Cells Markers in Staining Vasa Vasorum and Are Non-Specific for Distinct Endothelial Cell Lineages in Clinical Samples. Int Mol Sci 24(2023).\n32. Brady, E.L., et al. Engineered tissue vascularization and engraftment depends on host model. Sci Rep 13, 1973 (2023).\n33. Friend, N.E., et al. Injectable pre-cultured tissue modules catalyze the formation of extensive functional microvasculature in vivo. Sci Rep 10, 15562 (2020)."}, {"round": 2, "reviewer_comments": {"reviewer_1": "In this revised version, the authors have made substantial improvement to the manuscript in response to the feedback.\nParticularly, they conducted additional experiments to enhance evidence of the hydrogel properties and their impact on chondrocyte repair. In general, the authors have addressed my previous comments and have carried out a rigorous and more impactful study.", "reviewer_2": "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_3": "The authors have addressed all concerns in detail in their point-by-point responses and revised the manuscript accordingly.\nI have no further questions."}, "author_response": ""}]

Decision letter