Four-dimensional Hydrogel Dressing Adaptable to the Urethral Microenvironment for Scarless Urethral Reconstruction

Four-dimensional Hydrogel Dressing Adaptable to the Urethral Microenvironment for Scarless Urethral Reconstruction | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Four-dimensional Hydrogel Dressing Adaptable to the Urethral Microenvironment for Scarless Urethral Reconstruction Yujie Hua, Kai Wang, Yingying Huo, Yaping Zhuang, Yuhui Wang, and 6 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-3024823/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 22 Nov, 2023 Read the published version in Nature Communications → Version 1 posted You are reading this latest preprint version Abstract The harsh urethral microenvironment (UME) after trauma severely hinders the current hydrogel-based urethral repair. In fact, four-dimensional (4D) consideration to mimic time-dependent physiological processes is essential for scarless urethral reconstruction, which requires balancing extracellular matrix (ECM) deposition and remodeling at different healing stages. In this study, we developed a novel UME-adaptable 4D hydrogel dressing to sequentially provide an early-vascularized microenvironment and later-antifibrogenic microenvironment for scarless urethral reconstruction. With the combination of dynamic boronic ester crosslinking and covalent photopolymerization, the resultant gelatin methacryloyl phenylboronic acid/ cis -diol-crosslinked ( GMPD ) hydrogels exhibited mussel-mimetic viscoelasticity, satisfactory adhesion, and acid-reinforced stability, which could adapt to harsh UME. In addition, a temporally on-demand regulatory ( TOR ) technical platform was introduced into GMPD hydrogels to create a time-dependent 4D microenvironment. As a result, physiological urethral recovery was successfully mimicked by means of an early-vascularized microenvironment to promote wound healing by activating the vascular endothelial growth factor (VEGF) signaling pathway, as well as a later-antifibrogenic microenvironment to prevent hypertrophic scar formation by timing transforming growth factor-β (TGFβ) signaling pathway inhibition. Both in vitro molecular mechanisms of the physiological healing process and in vivo scarless urethral reconstruction in a rabbit model were effectively verified, providing a promising alternative for urethral injury treatment. Biological sciences/Biotechnology/Biomaterials/Tissues Health sciences/Urology/Urethra urethral reconstruction hydrogel four-dimensional microenvironment scarless healing Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Introduction Urethral injury is a common and complicated disease in clinical practice that inevitably destroys multiple types of urethral architecture, such as the urothelium, blood vessels and submucosal tissue 1 – 3 . Recently, three-dimensional (3D) hydrogels have attracted great attention as ideal wound dressings due to the unique advantages of wet-healing conditions, ease of carrying bioactive substances, and availiability to mimic the cell microenvironment 4 – 8 . However, severe damage to the urethral microenvironment (UME) after trauma is an inherent obstacle that hinders the current hydrogel-based urethral repair 9 – 11 . In particular, robust adhesion to dynamic wound surfaces suffering from acidic urine in a harsh UME has proven to be extremely challenging 12 – 15 . Among all types of adhesive hydrogels, boronic ester crosslinking between phenylboronic acid and cis -diol derivatives commonly exhibits mechanically dynamic self-healing and pH-dependent characteristics, as well as cis -diol-based adhesive properties analogous to those of catechol mussel adhesive protein 16 – 20 . Additionally, the combination of dynamic and covalent tandem crosslinking strategies could further enable the formation of mechanically stable hydrogels with desirable viscoelastic properties similar to those of the muscular urethra 21 , 22 . In addition to the above consideration of adaptability to the UME, ideal urethral reconstruction is a time-dependent physiological process instead of an autogenously pathological recovery, which commonly leads to serious urethral stricture due to hypertrophic scar formation 23 , 24 . As far as we know, the key point in achieving scarless urethral reconstruction is to balance extracellular matrix (ECM) deposition and remodeling at different healing stages. For example, it is necessary to build abundant vessel networks to transport blood and nutrients to support the survival of surrounding epithelial cells and fibroblasts at the early stage 25 , 26 . However, fibroblasts need to be reasonably harnessed to prevent hypertrophic scarring of obstructing the urethral lumen at the later stage 27 , 28 . To date, the current hydrogel-based treatments to efficiently promote wound healing against urethral stricture remain unreliable. Therefore, a better urethral repair strategy needs to transcend the traditional 3D hydrogel-based design and create a time-dependent four-dimensional (4D) microenvironment to fit well with different healing stages. Thus, it is necessary to effectively promote early-stage vascularized urothelium regeneration and simultaneously prevent later-stage excessive fibrogenesis. Recent advances in hydrogel-based 4D cell culture platforms with spatiotemporal tunability represent a great opportunity to mimic the dynamic heterogeneity of the UME 29 – 32 . The hydrogel design of on-demand delivery of bioactive substances, such as biological growth factors or inhibitors, has emerged as the most efficient way to precisely regulate tissue regeneration 33 – 35 . Noticeably, the vascular endothelial growth factor (VEGF) signaling pathway plays a crucial role in the process of angiogenesis and epithelialization, which has been well recognized as a key regulator for promoting wound healing at the early stage of urethral reconstruction 36 , 37 . Dysregulated transforming growth factor-β (TGFβ) signaling pathway contributes heavily to pathological wound scarring, which exhibits an adverse effect on the formation of urethral strictures 38 , 39 . As previously reported, the timing of TGFβ signaling inhibition has been proven effective for scarless wound healing 40 , which may provide a feasible approach to prevent hypertrophic scar formation at later stage of urethral reconstruction. Taken together, how to construct a time-dependent 4D modulation is essential to balance ECM deposition and remodeling for scarless urethral reconstruction. Herein, we developed a novel UME-adaptable 4D hydrogel dressing to sequentially provide an early-vascularized microenvironment and later-antifibrogenic microenvironment for scarless urethral reconstruction. (Fig. 1 ). First, gelatin methacryloyl phenylboronic acid/ cis -diol-crosslinked ( GMPD ) hydrogels were meticulously designed by a hybrid crosslinking strategy combining dynamic boronic ester crosslinking and covalent photopolymerization, which possessed mussel-mimetic viscoelasticity, satisfactory adhesion, and acid-reinforced stability adapted to the harsh UME. In addition, a temporally on-demand regulatory ( TOR ) technical platform, preferentially releasing VEGF through gelatin methacryloyl ( GM ) microgels and then releasing TGFβ inhibitor through poly-lactic-co-glycolic acid ( PLGA ) microcapsules, was introduced into GMPD hydrogels to create a time-dependent 4D microenvironment. The molecular mechanism explorations revealed that TOR -functionalized GMPD hydrogel dressing could effectively provide an early-vascularized microenvironment to promote wound healing by activating the VEGF signaling pathway, as well as a later-antifibrogenic microenvironment to prevent hypertrophic scar formation by timing TGFβ signaling pathway inhibition. In contrast to pathological wound healing repaired by traditional 3D hydrogels, our UME-adaptable 4D hydrogel dressings could successfully achieve scarless urethral reconstruction in a rabbit model. Results Fabrication of UME-adaptable hydrogels. In our design, we first synthesized two types of gelatin-derived polymers: fluorophenylboronic acid (FPBA)-modified gelatin methacryloyl ( GMP ) and cis -diol-modified gelatin methacryloyl ( GMD ). To prepare UME-adaptable viscoelastic hydrogels, GMPD + hv hydrogels (mixing GMP and GMD polymers in an equivalent ratio) were synergistically crosslinked via both boronic ester dynamic bonds (viscous segment) and photoinitiated covalent bonds (elastic segment), whereas GMPD -hv hydrogels were only crosslinked via boronic ester dynamic bonds (Fig. 2 A, B and Supplementary Fig. 1). Noticeably, GMPD hydrogels exhibited good shear-shinning and self-healing properties due to the inherent reversible crosslinked network by means of boronic ester dynamic bonds, which are conveniently injected onto the target site and completely cover the urethral defects (Supplementary Fig. 2). Furthermore, 1 H NMR spectra of GMPD hydrogels were performed to trace the crosslinking mechanism. As shown in Fig. 2 C, both FPBA-compound characteristic peaks at 7.3–7.5 ppm and cis -diol-compound characteristic peaks at 3.1 ppm simultaneously appeared in GMPD hydrogels via boronic ester bond crosslinking. Moreover, the disappearance of double bond characteristic peaks at 5.2–5.7 ppm confirmed the further photopolymerization of methacryloyl groups. The hybrid crosslinking mechanism was also monitored by X-ray photoelectron spectroscopy (XPS) and attenuated total reflection Fourier transform infrared (ATR-FTIR) spectroscopy. As shown in Fig. 2 D, the XPS results revealed that GMPD -hv hydrogels exhibited a moderate intensity of C-O (286.37 eV) species compared with GMP (lower intensity) and GMD (higher intensity) polymers, consistent with the fact that the GMD component contained more oxygen molecules in pedant alcohol groups. A new component associated with boronic ester bonds appeared clearly at 189.75 eV, close to the typical peak of boronic bonds at 191.34 eV ( GMP component), which is attributable to successful boronic ester crosslinking (Fig. 2 E). The further photopolymerization of GMPD + hv hydrogels has no significant effect on the molecular structure of the preformed dynamic boronic ester bonds according to both the C(1s) and B(1s) spectra (Supplementary Fig. 3). Additionally, ATR-FTIR spectra of GMPD + hv hydrogels showed a definite decrease in the methacryloyl-related infrared band ratio of C-H stretching vibrations at 3100 cm − 1 and C = C stretching vibrations at 1450 cm − 1 after light irradiation (Fig. 2 F), consistent with the 1 H NMR spectra trace of the photopolymerization reaction. Taken together, these results essentially confirm the hybrid crosslinking mechanism of combining dynamic boronic ester crosslinking and covalent photopolymerization. Rheological analyses were further conducted to investigate the viscoelastic properties of hybrid crosslinked hydrogels. First, frequency sweep rheological measurements were performed within the linear region to quantify the gel strength according to the crossover frequency ( ω c ) at which the storage modulus (G’) was equal to the viscous modulus (G’’). As shown in Fig. 2 G, GMPD -hv hydrogels exhibited frequency-dependent viscoelastic behavior, a typical feature of dynamic gel networks via boronic ester bonds. However, GMPD + hv hydrogels showed more elastic performance and higher mechanical strength than GMPD -hv hydrogels due to the further enhancement of gel networks via photopolymerization. Importantly, the dynamic crosslinking of boronic ester bonds exhibited acid-reinforced mechanical strength with ω c increasing as pH decreased, which is very suitable for harsh UME of low pH values (Fig. 2 H). Moreover, time sweep rheological measurements demonstrated that secondary photopolymerization could effectively improve the elasticity of GMPD hydrogels without any influence of pH values (Fig. 2 I, J). As a result, the hybrid crosslinking strategy not only provides viscous segments relying on boronic ester dynamic bonds but also offers elastic segment dependent on photoinitiated covalent bonds, and thus achieves satisfactory viscoelasticity that can closely mimic the tissue architecture of urethral mussels. Performance of tissue adhesion and acid-reinforced stability. As previously reported, phenylboronic acid has a “ cis -diol” moiety that exhibits adhesive properties similar to those of catechol mussel adhesive protein 17 , 18 . In this study, GMPD hydrogels had the potential to balance hydrogel reversibility and adhesion strength based on the hybrid crosslinking strategy (Fig. 3 A). To evaluate the tissue binding ability, GMPD hydrogels were gelled in situ on the surface of muscle upon light irradiation and then immersed in acid solution to sustain stability. Figure 3 B depicted that there was no obvious breakage or detachment of the adhesive hydrogels regardless of stretching and twisting behavior. The scanning electron microscopy (SEM) results clearly revealed a tight and seamless interface between GMP hydrogels and muscle upon photopolymerization, whereas GM hydrogels showed obvious interfacial separation against the surrounding muscle, which is consistent with the structural predictions between the “ cis -diol” moiety of GMP hydrogels and active hydrogen on the tissue surface (Fig. 3 C). To quantitatively evaluate the adhesion performance of GMPD hydrogels as wound dressings, standard lap shear and incision sealing strength were measured respectively. As shown in Fig. 3 D-I, the peak adhesive strength ( l ) and sealing strength ( i ) of GMPD -hv hydrogels ( l = 19.4 ± 2.5 kPa; i = 3.1 ± 0.6 N) were higher than those of GM hydrogels ( l = 1.1 ± 0.2 kPa; i = 0.6 ± 0.1 N) and commercially available fibrin glue ( l = 12.2 ± 1.7 kPa; i = 1.4 ± 0.3 N), which was probably attributed to the cis -diol-mediated adhesive capability. Noticeably, the tensile strength required to dislocate hydrogels from tissue significantly increased as secondary covalent stabilization via photopolymerization ( GMPD + hv hydrogels, l = 47.7 ± 4.3 kPa; i = 9.6 ± 1.2 N), indicating that the enhancement of mechanical stability could effectively improve the adhesive strength. Gel stability is another important parameter for wound dressings, especially in dynamic and acidic urethral environment. As shown in Supplementary Fig. 4, GMPD -hv hydrogels in acid solution could effectively extend the in vitro degradation time from ~ 3 days ( pH = 7.4 ) to ~ 14 days ( pH = 5.0 ), which is attributed to the acid-reinforced structural stability of boronic ester bonds (correlated with Fig. 2 H). Additionally, GMPD + hv hydrogels exhibited long-lasting stability over 14 days in any harsh UME (pH values from 6.5 to 7.4) due to the pH-independent covalent stabilization of photopolymerization. Therefore, GMPD hydrogels are suitable for dynamic and acidic urethral environment based on the unique characteristics of acid-reinforced and covalent-stabilized mechanical properties. All these results demonstrated that our GMPD hydrogels possessed facile operation, mussel-mimetic viscoelasticity, satisfactory adhesion, and acid-reinforced stability, which could serve as an ideal wound dressing applied in the harsh UME. In vitro biological evaluation of the TOR platform. To better mimic the dynamic physiological process of urethral reconstruction, it is necessary to efficiently promote wound healing at the early stage and prevent hypertrophic scarring at the later stage. In this study, a novel TOR technical platform was developed to address the above challenge by preferentially releasing VEGF from V-GM microgels and subsequently releasing a TGFβ inhibitor (SB431542) from TI-PLGA microcapsules (Fig. 4 A). The microscope and SEM examinations demonstrated that microfluidic-based GM microgels showed uniform transparent spherical morphology and the lyophilized samples displayed a typical porous structure on the microgel surface (Fig. 4 B). In addition, PLGA microcapsules prepared by the water-oil-water (W/O/W) double emulsion strategy possessed a suitable shell thickness to suppress the initial burst release and a hollow inner structure to encapsulate insoluble drugs (Fig. 4 C). The drug release kinetics were further tested to investigate the feasibility of the temporally on-demand regulatory strategy. As shown in Fig. 4 D, V -GM microgels exhibited initial burst release within ~ 3 days, while the core-shell structure of TI-PLGA microcapsules could effectively delay the release of the TGFβ inhibitor after 3 days, confirming the successful construction of the TOR platform by means of early-released V-GM microgels and later-released TI-PLGA microcapsules. Furthermore, the biological function of the TOR platform was evaluated to verify the time-dependent wound healing process. As shown in Fig. 4 E-G, cell scratch experiments demonstrated that fibroblasts in the V-GM groups reached nearly 100% coverage at 24 h and showed the highest proliferation rate due to the initial release of VEGF, whereas the TI-PLGA groups reached approximately 75% coverage at 24 h and exhibited the suppression of cell proliferation on day 7 attributed to the gradual release of the TGFβ inhibitor. In addition, HUVECs in the V-GM groups showed an accelerated healing rate, and the TI-PLGA groups showed no significant difference in HUVEC proliferation even with the release of the TGFβ inhibitor (Supplementary Fig. 5). The same results were further confirmed by both fibroblast- and HUVEC-specific fluorescence staining. As shown in Fig. 4 H-L and Supplementary Fig. 6, fibroblast-specific expression (fibronectin, α -SMA, COL1, and COL3) was significantly upregulated in the V-GM groups, whereas the corresponding expression were dramatically downregulated in the TI-PLGA groups, implying successful inhibition of fibroblast-specific function owing to the gradual release of the TGFβ inhibitor. The HUVEC-specific fluorescence staining (CD31 and VWF) showed that the initial release of VEGF effectively upregulated vascularized-related expression (Supplementary Fig. 7). In short, the meticulously designed TOR platform has a synergistic effect of promoting vascularization for wound healing at the early stage and preventing fibrogenesis at the later stage, and thus provides a 4D cell culture system combining a time-dependent TOR platform with UME-adaptable hydrogels. Scarless healing molecular mechanism of the time-dependent process. Before investigating the 4D dynamic regulation of the scarless healing process, the cytocompatibility was evaluated using CCK-8 assays. As shown in Supplementary Fig. 8, both GMPD hydrogel and TOR microsphere extracts did not show obvious cytotoxicity (> 92% cell viability) for either fibroblasts or HUVECs. To exploit the molecular mechanism involved in scarless wound healing based on TOR -functionalized hydrogel dressings, differentially expressed genes (DEGs) were identified and biological processes were investigated using RNA-seq methods (see methods). The Venn diagram showed the total changes and overlaps of fibroblast DEGs in TOR -functionalized hydrogels after 1-, 4-, and 7-days culture (Fig. 5 A). As shown in the volcano plots, there were 928 upregulated and 695 downregulated DEGs between the F1 and F4 groups at the early stage, as well as 267 upregulated and 382 downregulated DEGs between the F4 and F7 groups at the later stage (Fig. 5 B). Moreover, the volcano plots of endothelial cell DEGs showed 1989 upregulated and 2277 downregulated genes between the E1 and E4 groups at the early stage (Supplementary Fig. 9A, B). Next, DEGs of both fibroblasts and endothelial cells involved in the wound healing process at the early stage were determined to investigate the synergetic effect of the TOR platform. A total of 61 DEGs involved in the wound healing process at the early stage were dysregulated in fibroblasts (Fig. 5 C), while 82 DEGs were dysregulated in endothelial cells at day 4 (Fig. 5 D). Notably, three increased DEGs ( VEGFA , HMOX1 , and ITGA2 ) and four decreased DEGs ( HIF-1A , TPM1 , CCN1 , and COL1A1 ) involved in the wound healing process at the early stage were commonly dysregulated in both fibroblasts and endothelial cells, which are very relevant to VEGF singling pathways. The corresponding DEGs were further validated by quantitative real-time polymerase chain reaction (qRT-PCR) and western blot (WB). As shown in Fig. 5 E, gene expression ( VEGFA , HMOX1 , and ITGA2 ) was dramatically upregulated in the E4 groups compared to the E1 groups, and the same gene expression was also significantly upregulated in the F4 groups compared to the F1 groups, indicating the synergetic promotion of angiogenesis associated with fibrogenesis formation. Similarly, the gene expression levels ( HIF-1A , TPM1 , and CCN1 ) were greatly downregulated in both the E4 and F4 groups compared to the corresponding E1 and F1 groups. The same results for protein expression levels were also confirmed by WB experiments, as shown in Fig. 5 F. Additionally, the early index of the vascularized expression level (VWF) was dramatically upregulated between the E4 and E1 groups, and the later index of the vascularized expression level (CD31) were significantly upregulated between the E7 and E4 groups, demonstrating the activation of VEGF singling pathways via V-GM microgels regardless of the later release of the TGFβ inhibitor (Fig. 5 G, H). The singling pathways involved in the wound healing process were further investigated between the F1 and F4 groups at the early stage, as well as between the F4 and F7 groups at the later stage. Figure 6 A depicted the top 15 potential biological processes and signaling pathways at the early stage, according to the Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses, which were related to the terms of cell cycle, cellular component organization, and metabolic process. Notably, DEGs between the E1 and E4 groups at the early stage of the cell cycle, organelle and cellular component organization processes were commonly enriched in GO and KEGG terms of endothelial cells, revealing that these processes are critical to wound recovery (Supplementary Fig. 9C, D). At the later stage of wound healing, cell adhesion, differentiation, extracellular matrix, and structure organization, as well as tissue development and blood vessel development, were dominant, implying that ECM reconstruction is an important event during wound recovery (Fig. 6 B). Noticeably, enriched TGFβ signaling pathways were also observed by KEGG pathway analysis, which indirectly confirmed the essential regulatory roles of later-released TGFβ inhibitors in the wound healing process. As shown in Fig. 6 C, D, qRT-PCR and WB experiments demonstrated that the fibrogenic expression levels (α-SMA, elastin, COL1, COL3, and fibronectin) were significantly downregulated, which is vital for the matrix remodeling of excessive ECM at the later stage. The main explanations for the regulatory function of ECM reconstruction are attributable to the negative regulation of TGFβ2/Smad3 signaling ( TGFB2 and Smad3 downregulated) associated with the activation of MMP-related matrix degradation ( MMP1 upregulated) upon the later-released TGFβ inhibitor through TI-PLGA microcapsules. In vivo scarless urethral reconstruction in rabbits. The in vivo biological effect of the TOR -functionalized hydrogel dressings was further investigated in rabbit models. As shown in Fig. 7 A, viscoelastic hydrogel precursors were first injected to fully cover the urethral defects, followed by light irradiation (365-nm LED, 20 mW/cm 2 ) for stabilization. The recovery of injured urethras in each group was detected after 4- and 8-weeks surgery. In the ultrasound urethrograms, both the Ctrl and GMPD groups displayed a very narrow lumen caused by excessive scar formation (Fig. 7 B, C). The GMPD-V and GMPD-TI groups showed relatively wide urethral lumens to various extent, whereas the GMPD-V/TI groups exhibited a fluent lumen similar to that of a normal urethra. Gross morphology demonstrated that urethral repair in the GMPD-V/TI groups was significantly better than that in the other groups, as the repaired urethras were smooth without any hypertrophic scar formation. However, both the Ctrl and GMPD groups exhibited undesired shrinkage of urethras, showing the worst repair, while excessive scar formation could be seen in the GMPD-V groups (Fig. 7 D). Histological analyses of urethral tissue at 8 weeks after surgery were performed by hematoxylin and eosin (H&E) and Masson’s trichrome staining. The urethral condition of ECM deposition, urothelium regeneration, and tissue fibrogenesis was shown in Fig. 7 E. The repaired urethras in both the Ctrl and GMPD groups exhibited severe urethral stricture with incomplete urothelium layers, fewer blood vessels, and hypertrophic scar formation, which is mainly attributed to the lack of regenerated urothelium layers. In contrast, the repaired urethras treated by GMPD-V hydrogel dressings had almost normal urothelium layers with abundant blood vessels, but there was excessive collagen deposition under the regenerated epithelium. In the GMPD-TI groups, although there was no obvious ECM over deposition, the urothelium layers did not regenerate well. With the synergetic release of both VEGF and TGFβ inhibitor, the continued and complete urothelium layers were satisfactorily regenerated on the lumen surface without any hypertrophic scar formation in the GMPD-V/TI groups after 8-weeks surgery, which is regarded as scarless urethral reconstruction. Immunofluorescence examinations were used to further evaluate protein expression in the repaired urethras (Fig. 8 A, B). The expression level of epithelial cytokeratin AE1/AE3, an important membrane surface protein marker, was significantly higher in the GMPD-V/TI groups than in the other groups. Specifically, the regenerated urothelium layer in the GMPD-V and GMPD-TI groups was thinner than that in the GMPD-V/TI group, whereas the expression of cytokeratin in both the Ctrl and GMPD groups was hardly found. Noticeably, the GMPD-V and GMPD-V/TI groups showed large numbers of CD31-positive cells (blood vessels labelled by CD31), which were much more abundant than those in other groups, implying that the regulation of the VEGF signaling pathway has a significant role in improving angiogenesis under the epithelium. The α-SMA expression level (the marker of myofibroblasts) under the epithelium was relatively lower in the GMPD-TI and GMPD-V/TI groups than in the other groups, implying that the effective inhibition of the TGFβ signaling pathway is conductive to antifibrogenic function. However, both the Ctrl and GMPD groups showed highly expressed α-SMA protein, indicating that large numbers of myofibroblasts existed under the epithelium. Consistent with the results of α-SMA, the expression level of collagen I at the site of repaired urethras treated by GMPD-V/TI hydrogel dressing was relatively lower than that in other groups. Additionally, the positive expression of proliferating cell nuclear antigen (PCNA) was obviously increased at the site of regenerated epithelium in the GMPD-V/TI groups. Therefore, it is suggested that the synergetic release of both VEGF and TGFβ inhibitors has the potential to promote the regeneration of urothelium layers without hypertrophic scar formation. Different degrees of inflammatory responses at the injured sites appeared because of the application of the extra hydrogel dressings (Supplementary Fig. 10). The results demonstrated that the number of CD206-positive cells (M2 macrophages labelled by CD206) in the GMPD-V group was the highest, while the number of CD206-positive cells in the GMPD-TI group was significantly lower than that in the other groups, indicating the possible inhibition of inflammation by the TGFβ signaling pathway. The GMPD-V/TI hydrogel-treated urethras showed modest inflammation compared with the GMPD and GMPD-V hydrogel-treated urethras. All these results suggested that TOR -functionalized hydrogel dressings not only promoted the regeneration of vascularized urothelium layers via early-released VEGF but also effectively inhibited hypertrophic scar formation via later-released TGFβ inhibitor. Discussion Urethral injury is a common disease that is usually accompanied by severe urethral stricture due to hypertrophic scar formation. In the clinic, free autogenous lingual mucosa tissue has been widely applied in upper urinary tract repair for more than ten years, but it is extremely limited by the available sampling size, as well as adverse impacts on patients’ pronunciation, mastication and other functions 41 , 42 . Recently, the development of tissue engineering methods that combine biocompatible scaffolds with adult cells or stem cells has effectively improved urethral repair, but clinical application is difficult due to ethical, cell colonization, and local survival problems 43 , 44 . Thus, current studies have focused on delivering biological factors with biocompatible scaffolds, especially hydrogel dressings, owing to the unique advantages of wet-healing conditions, the ease of carrying bioactive substances, and availiability to mimic the cell microenvironment. Predictably, there is high clinical value in developing ideal hydrogel dressings for scarless urethral reconstruction. The primary consideration for urethral repair is to overcome the harsh UME of dynamic wound surfaces suffering from acidic urine. In this study, a hybrid crosslinking strategy combining dynamic boronic ester crosslinking and covalent photopolymerization was synergistically employed to prepare GMPD hydrogels. The hybrid design rationale is to obtain optimal physicochemical properties adaptable to harsh UME: i ) boronic ester crosslinking has good shear-shinning and self-healing features that are suitable for injecting onto the target site and completely covering the urethral defects, followed by secondary photopolymerization for post-stabilization; ii ) different from traditional elastic hydrogels, our hybrid designed hydrogels exhibit satisfactory mussel-mimetic viscoelasticity accessible to dynamic urethral environment; iii ) the inherent cis -diol-based adhesive ability derived from phenylboronic acid-modified GM together with further mechanical enhancement by covalent photopolymerization; iv ) the choice of fluorophenylboronic acid moiety is mainly related to physiological-pH crosslinking conditions and the acid-reinforced mechanical strength that could effectively resist acidic urine environment. Therefore, the hybrid crosslinking strategy provided facile operation, mussel-mimetic viscoelasticity, satisfactory adhesion, and acid-reinforced stability adapted to the harsh UME. Furthermore, the 4D design of hydrogel dressings plays a key role in achieving scarless urethral reconstruction. Fully mimicking the time-dependent physiological process requires an effective balance between ECM deposition and remodeling at different healing stages. The current results demonstrated that the TOR technical platform could simultaneously improve early-stage angiogenesis through V-GM microgels and prevent later-stage excessive fibrogenesis through TI-PLGA microcapsules. At the early stage, we found that seven genes were dominant in the wound healing process, including three upregulated DEGs ( VEGFA , HMOX1 , and ITGA2 ) and four downregulated DEGs ( HIF-1A , TPM1 , CCN1 , and COL1A1 ), which are related to angiogenesis and urothelial regeneration. At the later stage, fibrogenic gene expression ( α-SMA , elastin , COL1 , COL3 , and fibronectin ) was significantly downregulated due to the negative regulation of TGFβ2/Smad3 signaling ( TGFB2 and Smad3 downregulated) associated with the activation of MMP-related matrix degradation ( MMP1 upregulated). Taken together, the time-dependent physiological process of urethral reconstruction was successfully achieved through preferentially promoting vascularized urothelium regeneration by activating the VEGF signaling pathway and subsequently preventing excessive fibrogenesis by timing TGFβ2/Smad3 signaling pathway inhibition. The same effects of TOR -functionalized hydrogel dressing were further verified in a rabbit urethral injury model. Only the synergetic function of both VEGF and TGFβ inhibitor in the GMPD-V/TI group exhibited the optimal repair effect, showing satisfactory urothelium regeneration without hypertrophic scar formation. In summary, the current study demonstrates a novel TOR -functionalized hydrogel dressing for scarless urethral reconstruction. The UME-adaptable GMPD hydrogels possess robust adhesion to dynamic wound surfaces even when suffering from acidic urine, while the TOR platform effectively balances ECM deposition and remodeling at different healing stages. As a result, the urethral injury of rabbits was sucessfully repaired through the time-dependent physiological process of preferentially activating VEGF-related vascularized urothelium regeneration and subsequently preventing TGFβ2/Smad3-related hypertrophic scar formation. Although this work represents a proof-of-concept study, we are fully convinced of the significance of time-dependent physiological urethral reconstruction, which paves the way to improve the clinical treatment of urethral injury in the future. Methods Materials and animals. Gelatin (from porcine skin), methacrylic anhydride, sodium hydroxide, 4-carboxy-3-fluoro-phenylboronic acid, D-(+)-gluconic acid δ-lactone, 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC·HCl), N-hydroxy succinimide (NHS), triethylamine, dimethylsulfoxide (DMSO), poly(ethylene glycol) (PEG, MW: 400), polyvinyl alcohol (PVA, MW: 95, 000), and lithium phenyl-2,4,6-trimethylbenzoylphosphinate (LAP) photoinitiator were purchased from Sigma-Aldrich. Poly(lactic-co-glycolic acid) (PLGA, MW: 10 kDa, LA:GA = 75: 25) were purchased from Daigang Biotechnology Co., Ltd. The TGFβ inhibitor SB431542 was purchased from Selleck. All the other chemicals were reagent grade. New Zealand white rabbits were purchased from Shanghai Jiao Tong University School of Agriculture. All protocols for animal experiments were approved by the Animal Care and Experimental Committee of Shanghai Jiao Tong University Affiliated Sixth People’s Hospital (No: 2021 − 0154). Synthesis of GMP and GMD polymers. First, gelatin methacryloyl ( GM ) polymers were synthesized according to the previously established methods 45 . Then, 2 g of GM was dissolved in 50 mL of PBS (pH = 7.4) and stirred vigorously at 50 ℃ until complete dissolution. To synthesize GMP polymers, 92 mg (0.5 mmol) of 4-carboxy-3-fluoro-phenylboronic acid, 96 mg (0.5 mmol) of EDC·HCl, and 58 mg (0.5 mmol) of NHS were dissolved in anhydrous DMSO and sequentially added into the above GM solution. To synthesize GMD polymers, 140 mg (0.8 mmol) of D-(+)-gluconic acid δ-lactone and 0.1 mL of triethylamine were dissolved in anhydrous DMSO and sequentially added into the above GM solution. After the reaction, the solutions were collected and dialyzed against deionized water at 40 ℃ for 3 days followed by freezing and lyophilization. 1 H NMR spectra were obtained to characterize the grafted functional groups and determine the corresponding substitution degree as previously described 19 , 46 . Synthesis of V-GM microgels and TI-PLGA microcapsules. To produce V-GM microgels, 500 mg of GM and 20 mg of LAP were dissolved in 10 mL of PBS (pH = 7.4) at 40 ℃ containing 0.1 µg/mL VEGF (Dima Biotech) as the water phase. 5 mL of span-80 and 40 mL of paraffin oil were mixed with each other and preheated at 40 ℃ as the oil phase. Both water and oil fluids were injected into the microchannels by micropumps, and the water phase formed single spherical droplets under the fluid shear of the oil phase. The droplets were subsequently crosslinked by photopolymerization upon light irradiation (365-nm LED, 20 mW/cm 2 ). The formed microgels were washed with hexane and deionized water for three times, followed by freezing and lyophilization. The TI-PLGA microcapsules were produced by the W/O/W emulsion method according to the previous literature 47 . Briefly, 1 mg of TGFβ inhibitor was added into a solution of 2.5 mg PEG400/50 µL deionized water to obtain the inner water phase. The above solution was added dropwise into a solution of 100 mg PLGA/1 mL chloroform with vigorous stirring for 10 min emulsification. The resultant water-in-oil (W/O) emulsion was then added dropwise into 15 mL of 1% w/v PVA aqueous solution as an emulsion stabilizer. The double emulsion (W/O/W) was obtained using a magnetic stirrer (3000 rpm) and allowed to stand for 5 hours until solvent evaporation. The TI-PLGA microcapsules after centrifugation were washed three times, followed by freezing and lyophilization. The drug release kinetics were tested according to previously established methods 40 . Hydrogel preparation. Hydrogel precursors of GM , GMP , GMD and LAP (0.2% w/v) were mixed in certain proportions in PBS solution (pH = 7.4). The hydrogel composition in this study was as follows: GMP hydrogels: 10% w/v FPBA-modified GelMA; GM hydrogels: 10% w/v GelMA; GMPD hydrogels: 10% w/v, GMP : GMD = 1:1; GMPD -hv hydrogels: GMPD hydrogels without light irradiation; GMPD + hv hydrogels: GMPD hydrogels with light irradiation; light: 365-nm LED, 20 mW/cm 2 . XPS and ATR-FTIR experiments. First, the GMP and GMD gel precursors, as well as the GMPD -hv and GMPD + hv hydrogels, were dried at 40 ℃ for 12 hours. Then, the dry film samples were tested in an ultrahigh vacuum chamber by an ESCALAB 250Xi XPS system, and XPS spectra were analyzed by XPSPEAK software to conduct peak separation. In addition, the dry film samples were analyzed on a Nicolet 6700 FTIR spectrometer. Rheological measurements. Dynamic rheology experiments were performed on a HAAKE MARS Ⅲ photorheometer with parallel-plate (P20 TiL, 20 mm diameter) geometry and OmniCure Series 2000 (365 nm, 20 mW/cm 2 ) at 25°C. Time sweep oscillatory tests were performed at a 10% strain, a 1 Hz frequency and a 0.5 mm gap for 120 s. The gel point was determined as the time when the storage modulus (G’) surpassed the loss modulus (G’’). The elastic modulus was determined as the storage modulus (G’) reaching complete gelation. Frequency sweep oscillatory tests were performed at a 10% strain and a 0.5 mm gap from 0.5 to 100 rad/s. Strain sweeps were performed to verify the linear response. Viscosity tests were performed at a gradually increasing shear rate from 0 to 50 s − 1 . SEM examination. To evaluate the interfacial integration between muscle tissue and hydrogels, the hydrogel-muscle integrated samples were dehydrated by free drying and cut into a relatively flat interface for examination. Then, the dehydrated samples were coated with gold-palladium in a Hitachi S-3400N ion sputter for further morphological observation. Adhesion tests. For lap shear tests, fresh hot casing was attached to a glass slide with cyanoacrylate glue to prepare test samples. The hydrogel precursors were uniformly dispersed on the surface of a hot casing with or without light irradiation (365-nm LED, 20 mW/cm 2 ). For incision sealing tests, porcine muscle was cut into 2×4 cm pieces, followed by a 1-cm incision in the middle of the muscle. The hydrogel precursors were introduced into the muscle defect with or without light irradiation (365-nm LED, 20 mW/cm 2 ). Then, adhesion tests were performed on an Instron machine in tensile mode at a 5 mm/min speed. Stability tests. The hydrogel samples after complete swelling were recorded as the initial weight W 0 , followed by immersion in PBS solution (pH = 7.4). At each time point, these samples were carefully collected and recorded as dry weight for evaluating hydrogel stability. In vitro biological evaluation. For the cell scratch assay, fibroblasts or endothelial cells were manually scratched with 200 µL pipette tips, and the wound area was observed by optical microscopy after 0, 6, 12, and 24 hours in the V-GM , TI-PLGA , and Ctrl groups. For cell proliferation experiments, cell viability was examined using a CCK-8 kit (Dojindo) according to the manufacturer’s protocol, and the optical density (OD) was measured with a microplate reader (Synergy H1, BioTek). For cell expression level evaluation, the corresponding fibrogenic and angiogenic evaluations were examined via immunofluorescence staining. The fibrogenic expression levels of fibronectin, α-SMA, COL1, and COL3 in fibroblasts and the angiogenic expression levels of CD31 and VWF in endothelial cells were evaluated. The statistical data of relative fluorescent intensity were analyzed using ImageJ software. Sequencing read preprocessing. All low-quality bases and adapters of sequencing reads were trimmed using Trimmomatic (PMID: 35037208). After quality control, the reserved clean reads were mapped to the genome references of Homo sapiens (UCSC hg38) and Oryctolagus-cuniculus (Ensembl OryCun2.0) using STAR (PMID: 26334920). The gene counts and TPM (transcripts per million) values were calculated using RSEM (PMID: 21816040). The gene symbols corresponding to Ensembl gene IDs of Oryctolagus-cuniculus were retrieved from the UniProt database (PMID: 33237286). All DEGs were identified by R package DESeq2 (PMID: 25516281). An absolute value |log2FoldChange| of ≥ 1 and padj of ≤ 0.05 were considered statistically significant. Functional enrichment of DEGs. The enriched terms of Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) were summarized by using the R package clusterProfiler (PMID: 34557778). The top 15 enriched terms were plotted by using the R package ggplot2. In addition, all genes associated with wound healing (GO:0042060) were retrieved from the UniProt database (PMID: 33237286) according to their annotation. Gene Ontology (GO) and KEGG Enrichment Analysis. Gene Ontology (GO; http://www.geneontology.org ) is a systematic approach for gene and protein annotation in terms of biological process, molecular process, and cellular component. Kyoto Encyclopedia of Genes and Genomes (KEGG; http://www.genome.jp/kegg/ ) is an online database depositing biological pathways of genes and biochemicals. The enriched GO terms and KEGG pathways were annotated using the R package clusterProfiler. qRT-PCR tests. The total RNA of cell-hydrogel samples was isolated using TRIzol reagent (Life Technologies). Reverse transcription was performed with a cDNA synthesis kit (Thermo Scientific) following the manufacturer’s instructions. Gene expression was analyzed quantitatively with SYBR Green using a 7500 Real-Time PCR system (Applied Biosystems, Life Technologies). Primers and probes for VEGFA , HMOX1 , ITGA2 , HIF-1A , TPM1 , CCN1 , TGFBR2 , Smad3 , MMP1 , α-SMA , elastin , COL1 , COL3 , fibronectin , and 𝛽-actin were designed based on published gene sequences (NCBI and PubMed). The expression level for each gene was normalized to that of 𝛽-actin. Western blot tests. Cells extracted from cell-hydrogel samples were harvested, and benzosulfonylfluoride, a protease and phosphatase inhibitor, were added to dissolve tissues in RIPA lysis buffer. The cell lysates were cultured on ice for 3 h and then centrifuged to collect the supernatant. 60 µg of protein in total was loaded in each lane, and a 10% SDS-PAGE gel was used for electrophoresis. The target proteins on the SDS-PAGE gel were transferred to polyvinylidene fluoride membranes (PVDF; 0.45 µm) and then blocked with 5% blocking buffer at 37°C for 1 h. PVDF membranes were treated with primary antibodies against VEGFA, HMOX1, ITGA2, HIF-1A, TGFBR2, Smad3, MMP1, α-SMA, elastin, COL1, COL3, fibronectin, and β-actin at 4°C overnight. Subsequently, the membranes were rinsed with TBST three times, and secondary antibody was incubated for another hour. An imaging system was used to scan the membranes, and grey values were measured to present the expression of differential proteins. Urethroplasty and Postoperative Examinations in rabbit. In order to verify the feasibility of scarless urethral reconstruction, the hydrogel dressings were in situ formed in the urethral defect of rabbits for in vivo experiments. All animal experiments were performed in accordance with the guidelines for animal care. The animal protocol (SYXK 2017 − 0240) was approved by the Institutional Animal Care and Use Committee of the Shanghai Jiao Tong University Affiliated Sixth People’s Hospital. Fifteen adult male New Zealand white rabbits with an average body weight of 2.5 kg were randomly divided into 5 groups for urethral defect building and subsequent repair. The rabbits were first subjected to general anaesthesia with intravenous injection of pentobarbital, and then the rabbits’ skin and urethras were disinfected with 70% alcohol. The skin and ventral urethra were sectioned at approximately 3 cm proximal to the external urethral orifice, and the urethral lumen was exposed. A dorsal urethral defect with a mean length × width of 2.0 cm × 0.8 cm was created in the anterior urethra of rabbits. All rabbits underwent removal of the urethra near the corpus cavernosum. Rabbits in group 1 (n = 3) were not repaired as control. Rabbits in group 2 (n = 3) were repaired with GMPD hydrogels. Rabbits in group 3 (n = 3) were repaired with GMPD-V hydrogels. Rabbits in group 4 (n = 3) were repaired with GMPD-TI hydrogels. Rabbits in group 5 (n = 3) were repaired with GMPD-V/TI hydrogels. Urethrography. To observe the urethral leakage and stricture in the five groups of animals, the contrast solution was injected into the urethral lumen at 8-weeks post-surgery. Meanwhile, the rabbits underwent the urethral contrast-enhanced ultrasound test to check the condition of the scar in the urethra at 8-weeks post-surgery. The rabbits were euthanized after retrograde urethrograms, and the urethral tissue for the following histology staining was collected. Histology Assessment and Immunofluorescence. The urethral tissue was harvested 8 weeks after surgery for histology analysis. The specimens were fixed in 4% paraformaldehyde for 30 minutes at room temperature. Then, they were dehydrated with different grades of alcohol and embedded in paraffin blocks. Histological sections were prepared and observed using an optical microscope. Hematoxylin and eosin staining (H&E) and Masson’s trichrome staining tests were conducted to identify the epithelial layer and collagen distribution of the urethra. To further demonstrate the repair of urethral function, the samples were stained for immunofluorescence for epithelial cytokeratin AE1/AE3 (Santa Cruz Biotechnology, Inc.), CD31 (Proteintech Group, Inc.), α-smooth muscle actin (Proteintech Group, Inc.), COL3 (Santa Cruz Biotechnology, Inc.), En1 (Santa Cruz Biotechnology, Inc.), CD206 (Proteintech Group, Inc.), and PCNA (Proteintech Group, Inc.). Nuclei were stained with DAPI (1:500, Life Technologies). Afterwards, the specimens were imaged and observed by an optical microscope. Statistical analysis. All data are presented as the means ± SDs. Differences between the values were evaluated using one-way analysis of variance (ANOVA) with P < 0.05 considered statistically significant. Declarations Data availability The authors declare that all data supporting of results in this study are available within the paper and its Supplementary Information, or from the corresponding authors upon reasonable request. All data are available in the main text or the supplementary materials. Source data are provided with this paper. Acknowledgements This research was financially supported by Jiangsu Key Technology Research Development Program (BE2017664), Shanghai Jiao Tong University Biomedical Engineering Cross Research Foundation (YG2022ZD022 and YG2017QN15), National Natural Science Foundation of China (82072217 and 81772135), Shanghai health committee (20184Y0053), Shanghai "Rising stars of medical talent" Youth development program, Shanghai Jiao Tong University K. C. Wong Medical Fellowship Fund, Shanghai sixth people’s hospital foundational research program, National Key Research and Development Program of China (2022YFA1207500, 2018YFA0703100), and Shanghai Municipal Key Clinical Specialty (shslczdzk06601). Author contributions Y. H. and K. Z. provided the ideals. Y. H., Q. F., W. C., and K. Z. designed the experiments. Y. H., K. W., Y. H., Y. W., W. F., Y. S., and K. Z. performed the experiments. Y. H., K. W., Y. H., and K. Z. analyzed the data. Y. H. and K. Z. wrote the manuscript. Y. Z., G. Z., Q. F., and W. C. revised the manuscript. All authors commented on the manuscript and its revisions. Competing interests The authors declare no competing interests. Additional information Supplementary information The online version contains supplementary material available at. Correspondence and requests for materials should be addressed to Qiang Fu, Wenguo Cui, or Kaile Zhang. 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Supplementary Files SupplementaryInformation.docx Four-dimensional Hydrogel Dressing Adaptable to the Urethral Microenvironment for Scarless Urethral Reconstruction Cite Share Download PDF Status: Published Journal Publication published 22 Nov, 2023 Read the published version in Nature Communications → Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-3024823","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":210315270,"identity":"b3465566-38c6-4b00-af3b-175254b64a7a","order_by":0,"name":"Yujie Hua","email":"","orcid":"","institution":"Shanghai Sixth People’s Hospital Affiliated to Shanghai Jiao Tong University School of Medicine","correspondingAuthor":false,"prefix":"","firstName":"Yujie","middleName":"","lastName":"Hua","suffix":""},{"id":210315271,"identity":"f9fecc8d-e267-4263-b193-e68a585a00ea","order_by":1,"name":"Kai Wang","email":"","orcid":"","institution":"Shanghai 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A\u003c/strong\u003e Schematic of hybrid crosslinking mechanisms obtained by combining boronic ester dynamic crosslinking (viscous segment) and photopolymerization (elastic segment) to construct viscoelastic\u003cstrong\u003e GMPD\u003c/strong\u003e hydrogels. \u003cstrong\u003eB\u003c/strong\u003e Photographs show gelation steps through two-component mixing followed by light irradiation. \u003cstrong\u003eC\u003c/strong\u003e \u003csup\u003e1\u003c/sup\u003eH NMR spectra evolution of \u003cstrong\u003eGMPD\u003c/strong\u003e hydrogels with or without light irradiation. Grey arrows represent the proton peak of methacryloyl groups. \u003cstrong\u003eD, E\u003c/strong\u003e The C(1s) and B(1s) XPS regions of \u003cstrong\u003eGMP\u003c/strong\u003e, \u003cstrong\u003eGMD\u003c/strong\u003e, and \u003cstrong\u003eGMPD-\u003c/strong\u003e\u003cem\u003e\u003cstrong\u003ehv\u003c/strong\u003e\u003c/em\u003e hydrogel samples. Blue arrows represent C(1s) regions. Red arrows represent B(1s) regions. \u003cstrong\u003eF\u003c/strong\u003e ATR-FTIR spectra of \u003cstrong\u003eGMPD\u003c/strong\u003e hydrogels with or without light irradiation. \u003cstrong\u003eG\u003c/strong\u003e Representative time sweep rheological plots of \u003cstrong\u003eGMPD+\u003c/strong\u003e\u003cem\u003e\u003cstrong\u003ehv\u003c/strong\u003e\u003c/em\u003e hydrogels. \u003cstrong\u003eH\u003c/strong\u003e Representative frequency sweep rheological plots of \u003cstrong\u003eGMPD-\u003c/strong\u003e\u003cem\u003e\u003cstrong\u003ehv\u003c/strong\u003e\u003c/em\u003e and \u003cstrong\u003eGMPD+\u003c/strong\u003e\u003cem\u003e\u003cstrong\u003ehv\u003c/strong\u003e\u003c/em\u003e hydrogels. \u003cstrong\u003eI\u003c/strong\u003e The crossover frequencies (ω\u003csub\u003ec\u003c/sub\u003e) obtained from frequency sweep rheology at each pH value for \u003cstrong\u003eGMPD-\u003c/strong\u003e\u003cem\u003e\u003cstrong\u003ehv\u003c/strong\u003e\u003c/em\u003e hydrogels. \u003cstrong\u003eJ\u003c/strong\u003e Final shear moduli of \u003cstrong\u003eGMPD+\u003c/strong\u003e\u003cem\u003e\u003cstrong\u003ehv\u003c/strong\u003e\u003c/em\u003e hydrogels at each pH value. \u003cstrong\u003eGMPD\u003c/strong\u003e hydrogels: 10% w/v, \u003cstrong\u003eGMP\u003c/strong\u003e: \u003cstrong\u003eGMD\u003c/strong\u003e = 1:1; \u003cstrong\u003eGMPD\u003c/strong\u003e\u003cem\u003e\u003cstrong\u003e-hv\u003c/strong\u003e\u003c/em\u003e hydrogels: \u003cstrong\u003eGMPD\u003c/strong\u003e hydrogels without light irradiation; \u003cstrong\u003eGMPD+\u003c/strong\u003e\u003cem\u003e\u003cstrong\u003ehv\u003c/strong\u003e\u003c/em\u003e hydrogels: \u003cstrong\u003eGMPD\u003c/strong\u003e hydrogels with light irradiation; light: 365-nm LED, 20 mW/cm\u003csup\u003e2\u003c/sup\u003e.\u003c/p\u003e","description":"","filename":"floatimage2.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-3024823/v1/493bb80d3c9dd6e84994f5f6.jpeg"},{"id":38685765,"identity":"6bc1538c-45eb-43e0-b991-52e3cd9cd581","added_by":"auto","created_at":"2023-06-16 18:59:51","extension":"jpeg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":275581,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAdhesion performance of GMPD hydrogels. A\u003c/strong\u003e Schematic of \u003cem\u003ecis\u003c/em\u003e-diol-based adhesive mechanism \u003cem\u003evia\u003c/em\u003e \u003cstrong\u003eGMP \u003c/strong\u003ecomponent. \u003cstrong\u003eB\u003c/strong\u003e Photographs of \u003cstrong\u003eGMPD \u003c/strong\u003ehydrogels (stained by fast green) formed \u003cem\u003ein situ\u003c/em\u003e on muscle tissue and immersed in acid solution (pH = 6.5). \u003cstrong\u003eC\u003c/strong\u003e Representative SEM images of adhesive \u003cstrong\u003eGMP \u003c/strong\u003ehydrogel-mussel constructs compared with those of nonadhesive \u003cstrong\u003eGM \u003c/strong\u003ehydrogels. \u003cstrong\u003eD-F\u003c/strong\u003e Standard lap shear tests to determine the hydrogel-tissue binding strength of \u003cstrong\u003eGMPD+\u003c/strong\u003e\u003cem\u003e\u003cstrong\u003ehv\u003c/strong\u003e\u003c/em\u003e hydrogels compared with those of \u003cstrong\u003eGM\u003c/strong\u003e, \u003cstrong\u003eGMPD-\u003c/strong\u003e\u003cem\u003e\u003cstrong\u003ehv\u003c/strong\u003e\u003c/em\u003e, and fibrin glue. \u003cstrong\u003eG-I\u003c/strong\u003e Standard incision sealing tests to measure the hydrogel-mussel binding strength of \u003cstrong\u003eGMPD+\u003c/strong\u003e\u003cem\u003e\u003cstrong\u003ehv \u003c/strong\u003e\u003c/em\u003ehydrogels compared with those of \u003cstrong\u003eGM\u003c/strong\u003e, \u003cstrong\u003eGMPD-\u003c/strong\u003e\u003cem\u003e\u003cstrong\u003ehv\u003c/strong\u003e\u003c/em\u003e, and fibrin glue. \u003cstrong\u003eGMP\u003c/strong\u003e hydrogels: 10% w/v FPBA-modified GelMA; \u003cstrong\u003eGM\u003c/strong\u003e hydrogels: 10% w/v GelMA. The other hydrogel compositions are the same as in \u003cstrong\u003eFig. 2\u003c/strong\u003e.\u003c/p\u003e","description":"","filename":"floatimage3.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-3024823/v1/9ee7e5d3b7fafe29f1bbd99c.jpeg"},{"id":38685770,"identity":"5ddc4e80-492f-402c-b388-49f1454871da","added_by":"auto","created_at":"2023-06-16 18:59:51","extension":"jpeg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":609655,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003e\u003cstrong\u003eIn vitro\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e biological evaluation of the TOR platform. A\u003c/strong\u003e Schematic of the design of the \u003cstrong\u003eTOR\u003c/strong\u003e platform by means of early-released \u003cstrong\u003eV-GM\u003c/strong\u003e microgels and later-released\u003cstrong\u003e TI-PLGA\u003c/strong\u003e microcapsules. \u003cstrong\u003eB\u003c/strong\u003e Representative microscope (i) and SEM (ii) images of \u003cstrong\u003eV-GM\u003c/strong\u003e microgels. \u003cstrong\u003eC\u003c/strong\u003e Representative SEM images of the intact (i) and crushed (ii) \u003cstrong\u003eTI-PLGA\u003c/strong\u003e microcapsules. Blue arrows represent the shell of \u003cstrong\u003ePLGA\u003c/strong\u003e microcapsules. Red arrows represent the cargo of TGFβ inhibitor powders. \u003cstrong\u003eD\u003c/strong\u003e The daily drug release curves of \u003cstrong\u003eV-GM\u003c/strong\u003e microgels and \u003cstrong\u003eTI-PLGA\u003c/strong\u003e microcapsules. \u003cstrong\u003eE\u003c/strong\u003e Observation of the fibroblast scratch assay after 0, 6, 12, and 24 hours in the \u003cstrong\u003eV-GM\u003c/strong\u003e, \u003cstrong\u003eTI-PLGA\u003c/strong\u003e, and control (\u003cstrong\u003eCtrl\u003c/strong\u003e) groups. \u003cstrong\u003eF\u003c/strong\u003e Statistical analyses of the corresponding wound healing rates. \u003cstrong\u003eG\u003c/strong\u003e Cell proliferation rates of fibroblasts tested by CCK-8 assay after 1-, 4-, and 7-days culture in the \u003cstrong\u003eV-GM\u003c/strong\u003e, \u003cstrong\u003eTI-PLGA\u003c/strong\u003e, and \u003cstrong\u003eCtrl\u003c/strong\u003e groups. \u003cstrong\u003eH\u003c/strong\u003e Immunofluorescence staining of fibronectin (red), α-SMA (green), and cell nuclei (DAPI, blue) for fibroblast marker expression after 4- and 7-days culture in the \u003cstrong\u003eV-GM\u003c/strong\u003e and \u003cstrong\u003eTI-PLGA\u003c/strong\u003e groups. \u003cstrong\u003eI-L\u003c/strong\u003e Comparative fibrogenic expression levels (\u003cem\u003efibronectin\u003c/em\u003e, \u003cem\u003eα-SMA\u003c/em\u003e, \u003cem\u003eCOL1\u003c/em\u003e, and \u003cem\u003eCOL3\u003c/em\u003e) after 4- and 7-days culture in the \u003cstrong\u003eV-GM\u003c/strong\u003e, \u003cstrong\u003eTI-PLGA\u003c/strong\u003e, and \u003cstrong\u003eCtrl\u003c/strong\u003e groups. \u003cstrong\u003eTOR\u003c/strong\u003e: temporally on-demand regulatory;\u003cstrong\u003e TI-PLGA\u003c/strong\u003e: TGFβ inhibitor-loaded PLGA microcapsules; \u003cstrong\u003eV-GM\u003c/strong\u003e: VEGF-loaded GelMA microgels. (n = 4, \u003csup\u003e*\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05, \u003csup\u003e**\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01, \u003csup\u003e***\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001, \u003csup\u003e****\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.0001, \u003csup\u003e#\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001 compared with the corresponding group at day 4, ns = no significance)\u003c/p\u003e","description":"","filename":"floatimage4.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-3024823/v1/6a674d58598c94c34dc86168.jpeg"},{"id":38685766,"identity":"078cea64-a457-4433-b567-11a7dc56dc03","added_by":"auto","created_at":"2023-06-16 18:59:51","extension":"jpeg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":437835,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMechanistic analysis of the synergetic effect on vascularized\u003c/strong\u003e \u003cstrong\u003eurethral regeneration at the early stage. A\u003c/strong\u003e Venn diagram of fibroblast DEGs\u003cem\u003e \u003c/em\u003ein \u003cstrong\u003eTOR\u003c/strong\u003e-functionalized hydrogels after 1-, 4-, and 7-days culture. \u003cstrong\u003eB\u003c/strong\u003e The corresponding volcano plots analyzed between the F1 and F4 groups at the early stage, as well as between the F4 and F7 groups at the later stage. \u003cstrong\u003eC, D\u003c/strong\u003e Heatmaps of screened DEGs involved in the wound healing process of fibroblasts (\u003cstrong\u003eC\u003c/strong\u003e) and endothelial cells (\u003cstrong\u003eD\u003c/strong\u003e) in \u003cstrong\u003eTOR\u003c/strong\u003e-functionalized hydrogels. \u003cstrong\u003eE\u003c/strong\u003e Comparative gene expression (\u003cem\u003eVEGFA\u003c/em\u003e, \u003cem\u003eHMOX1\u003c/em\u003e, \u003cem\u003eITGA2\u003c/em\u003e, \u003cem\u003eHIF-1A\u003c/em\u003e, \u003cem\u003eTPM1\u003c/em\u003e, and \u003cem\u003eCCN1\u003c/em\u003e) of fibroblasts and endothelial cells after 1- and 4-days culture. \u003cstrong\u003eF\u003c/strong\u003e The protein expression (VEGFA, HMOX1, ITGA2, and HIF-1A) of fibroblasts and endothelial cells after 1- and 4-days culture. \u003cstrong\u003eG, H\u003c/strong\u003e The corresponding gene (\u003cstrong\u003eG\u003c/strong\u003e) and protein (\u003cstrong\u003eH\u003c/strong\u003e) expression levels (vWF and CD31) of endothelial cells\u003cem\u003e \u003c/em\u003ein \u003cstrong\u003eTOR\u003c/strong\u003e-functionalized hydrogels after 1-, 4-, and 7-days culture. DEGs: differentially expressed genes. F1, F4, and F7: fibroblasts after 1-, 4-, and 7-days culture; E1, E4, and E7: endothelial cells after 1-, 4-, and 7-days culture. (n = 4, \u003csup\u003e*\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05, \u003csup\u003e**\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01, \u003csup\u003e****\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.0001, ns = no significance)\u003c/p\u003e","description":"","filename":"floatimage5.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-3024823/v1/5e19e4dcb14d1bcd8d785aa5.jpeg"},{"id":38686024,"identity":"9596db8a-14eb-426e-aaa7-7eceedc802b1","added_by":"auto","created_at":"2023-06-16 19:07:51","extension":"jpeg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":419235,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMechanistic analysis of antifibrogenic function for scarless urethral remodeling at the later stage. A, B\u003c/strong\u003e Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses of fibroblast DEGs after mRNA sequencing in \u003cstrong\u003eTOR\u003c/strong\u003e-functionalized hydrogels between the F1 and F4 groups at the early stage (\u003cstrong\u003eA\u003c/strong\u003e), as well as between the F4 and F7 groups at the later stage (\u003cstrong\u003eB\u003c/strong\u003e), including the top 15 representative upregulated or downregulated signaling pathways. \u003cstrong\u003eC, D\u003c/strong\u003e The corresponding gene (\u003cstrong\u003eC\u003c/strong\u003e) and protein (\u003cstrong\u003eD\u003c/strong\u003e) expression levels (TGFBR2, Smad3, MMP1, α-SMA, elastin, COL1, COL3, and fibronectin) of fibroblasts\u003cem\u003e \u003c/em\u003ein \u003cstrong\u003eTOR\u003c/strong\u003e-functionalized hydrogels after 1-, 4-, and 7-days culture, revealing the molecular mechanism of scarless urethral remodeling through inhibition of the TGFβ signaling pathway. (n = 4, \u003csup\u003e***\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001, \u003csup\u003e****\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.0001, ns = no significance)\u003c/p\u003e","description":"","filename":"floatimage6.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-3024823/v1/a938acd9c27faab329d557ba.jpeg"},{"id":38685769,"identity":"42a5a1b7-54b0-4863-a937-9a928e7d82ee","added_by":"auto","created_at":"2023-06-16 18:59:51","extension":"jpeg","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":508755,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003e\u003cstrong\u003eIn vivo\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e scarless urethral reconstruction using GMPD hydrogel dressings in rabbits. A\u003c/strong\u003e Photographs of the surgical operation of \u003cem\u003ein situ\u003c/em\u003e urethral defect repair using \u003cstrong\u003eGMPD\u003c/strong\u003e hydrogel dressings. \u003cstrong\u003eB, C\u003c/strong\u003e Urethrography images (\u003cstrong\u003eB\u003c/strong\u003e) and blockage ratios (\u003cstrong\u003eC\u003c/strong\u003e) of the rabbit urethral canal in the \u003cstrong\u003eGMPD\u003c/strong\u003e, \u003cstrong\u003eGMPD-V\u003c/strong\u003e, \u003cstrong\u003eGMPD-TI\u003c/strong\u003e, \u003cstrong\u003eGMPD-V/TI\u003c/strong\u003e, and control (\u003cstrong\u003eCtrl\u003c/strong\u003e) groups after 4- and 8-weeks surgery. Red arrows represent urethral stricture. Blue arrows represent urethral patency. \u003cstrong\u003eD, E\u003c/strong\u003e Gross view (\u003cstrong\u003eD\u003c/strong\u003e) and histological examinations of H\u0026amp;E and Masson’s trichrome staining (\u003cstrong\u003eE\u003c/strong\u003e) of the rabbit urethral canal in the \u003cstrong\u003eGMPD\u003c/strong\u003e, \u003cstrong\u003eGMPD-V\u003c/strong\u003e, \u003cstrong\u003eGMPD-TI\u003c/strong\u003e, \u003cstrong\u003eGMPD-V/TI\u003c/strong\u003e, and control (\u003cstrong\u003eCtrl\u003c/strong\u003e) groups after 4- and 8-weeks surgery. \u003cstrong\u003eGMPD\u003c/strong\u003e hydrogels: 10% w/v, \u003cstrong\u003eGMP\u003c/strong\u003e: \u003cstrong\u003eGMD\u003c/strong\u003e = 1:1; \u003cstrong\u003eGMPD-V\u003c/strong\u003e hydrogels: 10% w/v \u003cstrong\u003eGMPD\u003c/strong\u003e with 1% w/v\u003cstrong\u003e V-GM\u003c/strong\u003e; \u003cstrong\u003eGMPD-TI\u003c/strong\u003e hydrogels: 10% w/v \u003cstrong\u003eGMPD\u003c/strong\u003e with 1% w/v\u003cstrong\u003e TI-PLGA\u003c/strong\u003e; \u003cstrong\u003eGMPD-V/TI\u003c/strong\u003e hydrogels: 10% w/v \u003cstrong\u003eGMPD\u003c/strong\u003e with 1% w/v\u003cstrong\u003e V-GM\u003c/strong\u003e and 1% w/v\u003cstrong\u003e TI-PLGA\u003c/strong\u003e; light: 365-nm LED, 20 mW/cm\u003csup\u003e2\u003c/sup\u003e. (n = 4, \u003csup\u003e****\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.0001)\u003c/p\u003e","description":"","filename":"floatimage7.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-3024823/v1/53c9e9a33e41348b3f01a0a7.jpeg"},{"id":38685771,"identity":"4a7e54f4-46e4-415d-a1ad-de84983c62a4","added_by":"auto","created_at":"2023-06-16 18:59:51","extension":"jpeg","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":544117,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eImmunofluorescent examinations of scarless wound healing of the rabbit urethral canal. A\u003c/strong\u003e Immunofluorescence staining of the rabbit urethral canal for evaluating epithelialization (AE1/AE3), angiogenesis (CD31), fibrogenesis (α-SMA and COL1), and cell proliferation (PCNA) after different treatments (i.e., \u003cstrong\u003eGMPD\u003c/strong\u003e, \u003cstrong\u003eGMPD-V\u003c/strong\u003e, \u003cstrong\u003eGMPD-TI\u003c/strong\u003e, \u003cstrong\u003eGMPD-V/TI\u003c/strong\u003e, and \u003cstrong\u003eCtrl\u003c/strong\u003e groups) for 8 weeks. \u003cstrong\u003eB\u003c/strong\u003e Quantitative expression levels of AE1/AE3, CD31, α-SMA, COL1, and PCNA after different treatments (i.e., \u003cstrong\u003eGMPD\u003c/strong\u003e, \u003cstrong\u003eGMPD-V\u003c/strong\u003e, \u003cstrong\u003eGMPD-TI\u003c/strong\u003e, \u003cstrong\u003eGMPD-V/TI\u003c/strong\u003e, and \u003cstrong\u003eCtrl\u003c/strong\u003e groups) for 8 weeks. The hydrogel dressing compositions are the same as in \u003cstrong\u003eFig. 7\u003c/strong\u003e. (n = 4, \u003csup\u003e*\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05, \u003csup\u003e**\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01, \u003csup\u003e***\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001, \u003csup\u003e****\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.0001, ns = no significance)\u003c/p\u003e","description":"","filename":"floatimage8.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-3024823/v1/a6f6b0e2df5ea8cca627833b.jpeg"},{"id":46961609,"identity":"009901c3-37c3-4aaf-98ab-dae746f73618","added_by":"auto","created_at":"2023-11-23 08:13:40","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2481368,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3024823/v1/988ae093-978e-49fc-9444-dc04cf377248.pdf"},{"id":38685772,"identity":"0bc739d7-62ac-478a-8aca-edcc09ab1640","added_by":"auto","created_at":"2023-06-16 18:59:51","extension":"docx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":2309103,"visible":true,"origin":"","legend":"\u003cp\u003eFour-dimensional Hydrogel Dressing Adaptable to the Urethral Microenvironment for Scarless Urethral Reconstruction\u003c/p\u003e","description":"","filename":"SupplementaryInformation.docx","url":"https://assets-eu.researchsquare.com/files/rs-3024823/v1/4ef6014415027c40f970b710.docx"}],"financialInterests":"There is \u003cb\u003eNO\u003c/b\u003e Competing Interest.","formattedTitle":"Four-dimensional Hydrogel Dressing Adaptable to the Urethral Microenvironment for Scarless Urethral Reconstruction","fulltext":[{"header":"Introduction","content":"\u003cp\u003eUrethral injury is a common and complicated disease in clinical practice that inevitably destroys multiple types of urethral architecture, such as the urothelium, blood vessels and submucosal tissue\u003csup\u003e\u003cspan additionalcitationids=\"CR2\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e. Recently, three-dimensional (3D) hydrogels have attracted great attention as ideal wound dressings due to the unique advantages of wet-healing conditions, ease of carrying bioactive substances, and availiability to mimic the cell microenvironment\u003csup\u003e\u003cspan additionalcitationids=\"CR5 CR6 CR7\" citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e. However, severe damage to the urethral microenvironment (UME) after trauma is an inherent obstacle that hinders the current hydrogel-based urethral repair\u003csup\u003e\u003cspan additionalcitationids=\"CR10\" citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e. In particular, robust adhesion to dynamic wound surfaces suffering from acidic urine in a harsh UME has proven to be extremely challenging\u003csup\u003e\u003cspan additionalcitationids=\"CR13 CR14\" citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e. Among all types of adhesive hydrogels, boronic ester crosslinking between phenylboronic acid and \u003cem\u003ecis\u003c/em\u003e-diol derivatives commonly exhibits mechanically dynamic self-healing and pH-dependent characteristics, as well as \u003cem\u003ecis\u003c/em\u003e-diol-based adhesive properties analogous to those of catechol mussel adhesive protein\u003csup\u003e\u003cspan additionalcitationids=\"CR17 CR18 CR19\" citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e. Additionally, the combination of dynamic and covalent tandem crosslinking strategies could further enable the formation of mechanically stable hydrogels with desirable viscoelastic properties similar to those of the muscular urethra\u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eIn addition to the above consideration of adaptability to the UME, ideal urethral reconstruction is a time-dependent physiological process instead of an autogenously pathological recovery, which commonly leads to serious urethral stricture due to hypertrophic scar formation\u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e. As far as we know, the key point in achieving scarless urethral reconstruction is to balance extracellular matrix (ECM) deposition and remodeling at different healing stages. For example, it is necessary to build abundant vessel networks to transport blood and nutrients to support the survival of surrounding epithelial cells and fibroblasts at the early stage\u003csup\u003e\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e. However, fibroblasts need to be reasonably harnessed to prevent hypertrophic scarring of obstructing the urethral lumen at the later stage\u003csup\u003e\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e. To date, the current hydrogel-based treatments to efficiently promote wound healing against urethral stricture remain unreliable. Therefore, a better urethral repair strategy needs to transcend the traditional 3D hydrogel-based design and create a time-dependent four-dimensional (4D) microenvironment to fit well with different healing stages. Thus, it is necessary to effectively promote early-stage vascularized urothelium regeneration and simultaneously prevent later-stage excessive fibrogenesis.\u003c/p\u003e \u003cp\u003eRecent advances in hydrogel-based 4D cell culture platforms with spatiotemporal tunability represent a great opportunity to mimic the dynamic heterogeneity of the UME\u003csup\u003e\u003cspan additionalcitationids=\"CR30 CR31\" citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e. The hydrogel design of on-demand delivery of bioactive substances, such as biological growth factors or inhibitors, has emerged as the most efficient way to precisely regulate tissue regeneration\u003csup\u003e\u003cspan additionalcitationids=\"CR34\" citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u003c/sup\u003e. Noticeably, the vascular endothelial growth factor (VEGF) signaling pathway plays a crucial role in the process of angiogenesis and epithelialization, which has been well recognized as a key regulator for promoting wound healing at the early stage of urethral reconstruction\u003csup\u003e\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e, \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u003c/sup\u003e. Dysregulated transforming growth factor-β (TGFβ) signaling pathway contributes heavily to pathological wound scarring, which exhibits an adverse effect on the formation of urethral strictures\u003csup\u003e\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e, \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e\u003c/sup\u003e. As previously reported, the timing of TGFβ signaling inhibition has been proven effective for scarless wound healing\u003csup\u003e\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e\u003c/sup\u003e, which may provide a feasible approach to prevent hypertrophic scar formation at later stage of urethral reconstruction. Taken together, how to construct a time-dependent 4D modulation is essential to balance ECM deposition and remodeling for scarless urethral reconstruction.\u003c/p\u003e \u003cp\u003eHerein, we developed a novel UME-adaptable 4D hydrogel dressing to sequentially provide an early-vascularized microenvironment and later-antifibrogenic microenvironment for scarless urethral reconstruction. (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). First, gelatin methacryloyl phenylboronic acid/\u003cem\u003ecis\u003c/em\u003e-diol-crosslinked (\u003cb\u003eGMPD\u003c/b\u003e) hydrogels were meticulously designed by a hybrid crosslinking strategy combining dynamic boronic ester crosslinking and covalent photopolymerization, which possessed mussel-mimetic viscoelasticity, satisfactory adhesion, and acid-reinforced stability adapted to the harsh UME. In addition, a temporally on-demand regulatory (\u003cb\u003eTOR\u003c/b\u003e) technical platform, preferentially releasing VEGF through gelatin methacryloyl (\u003cb\u003eGM\u003c/b\u003e) microgels and then releasing TGFβ inhibitor through poly-lactic-co-glycolic acid (\u003cb\u003ePLGA\u003c/b\u003e) microcapsules, was introduced into \u003cb\u003eGMPD\u003c/b\u003e hydrogels to create a time-dependent 4D microenvironment. The molecular mechanism explorations revealed that \u003cb\u003eTOR\u003c/b\u003e-functionalized \u003cb\u003eGMPD\u003c/b\u003e hydrogel dressing could effectively provide an early-vascularized microenvironment to promote wound healing by activating the VEGF signaling pathway, as well as a later-antifibrogenic microenvironment to prevent hypertrophic scar formation by timing TGFβ signaling pathway inhibition. In contrast to pathological wound healing repaired by traditional 3D hydrogels, our UME-adaptable 4D hydrogel dressings could successfully achieve scarless urethral reconstruction in a rabbit model.\u003c/p\u003e "},{"header":"Results","content":"\u003cp\u003e\u003cstrong\u003eFabrication of UME-adaptable hydrogels.\u003c/strong\u003e In our design, we first synthesized two types of gelatin-derived polymers: fluorophenylboronic acid (FPBA)-modified gelatin methacryloyl (\u003cstrong\u003eGMP\u003c/strong\u003e) and \u003cem\u003ecis\u003c/em\u003e-diol-modified gelatin methacryloyl (\u003cstrong\u003eGMD\u003c/strong\u003e). To prepare UME-adaptable viscoelastic hydrogels, \u003cstrong\u003eGMPD\u003c/strong\u003e\u0026thinsp;\u003cstrong\u003e+\u0026thinsp;hv\u003c/strong\u003e hydrogels (mixing \u003cstrong\u003eGMP\u003c/strong\u003e and \u003cstrong\u003eGMD\u003c/strong\u003e polymers in an equivalent ratio) were synergistically crosslinked \u003cem\u003evia\u003c/em\u003e both boronic ester dynamic bonds (viscous segment) and photoinitiated covalent bonds (elastic segment), whereas \u003cstrong\u003eGMPD\u003c/strong\u003e\u003cstrong\u003e-hv\u003c/strong\u003e hydrogels were only crosslinked \u003cem\u003evia\u003c/em\u003e boronic ester dynamic bonds (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eA, B and Supplementary Fig.\u0026nbsp;1). Noticeably, \u003cstrong\u003eGMPD\u003c/strong\u003e hydrogels exhibited good shear-shinning and self-healing properties due to the inherent reversible crosslinked network by means of boronic ester dynamic bonds, which are conveniently injected onto the target site and completely cover the urethral defects (Supplementary Fig.\u0026nbsp;2).\u003c/p\u003e\n\u003cp\u003eFurthermore, \u003csup\u003e1\u003c/sup\u003eH NMR spectra of \u003cstrong\u003eGMPD\u003c/strong\u003e hydrogels were performed to trace the crosslinking mechanism. As shown in Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eC, both FPBA-compound characteristic peaks at 7.3\u0026ndash;7.5 ppm and \u003cem\u003ecis\u003c/em\u003e-diol-compound characteristic peaks at 3.1 ppm simultaneously appeared in \u003cstrong\u003eGMPD\u003c/strong\u003e hydrogels \u003cem\u003evia\u003c/em\u003e boronic ester bond crosslinking. Moreover, the disappearance of double bond characteristic peaks at 5.2\u0026ndash;5.7 ppm confirmed the further photopolymerization of methacryloyl groups. The hybrid crosslinking mechanism was also monitored by X-ray photoelectron spectroscopy (XPS) and attenuated total reflection Fourier transform infrared (ATR-FTIR) spectroscopy. As shown in Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eD, the XPS results revealed that \u003cstrong\u003eGMPD\u003c/strong\u003e\u003cstrong\u003e-hv\u003c/strong\u003e hydrogels exhibited a moderate intensity of C-O (286.37 eV) species compared with \u003cstrong\u003eGMP\u003c/strong\u003e (lower intensity) and \u003cstrong\u003eGMD\u003c/strong\u003e (higher intensity) polymers, consistent with the fact that the \u003cstrong\u003eGMD\u003c/strong\u003e component contained more oxygen molecules in pedant alcohol groups. A new component associated with boronic ester bonds appeared clearly at 189.75 eV, close to the typical peak of boronic bonds at 191.34 eV (\u003cstrong\u003eGMP\u003c/strong\u003e component), which is attributable to successful boronic ester crosslinking (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eE). The further photopolymerization of \u003cstrong\u003eGMPD\u003c/strong\u003e\u0026thinsp;\u003cstrong\u003e+\u0026thinsp;hv\u003c/strong\u003e hydrogels has no significant effect on the molecular structure of the preformed dynamic boronic ester bonds according to both the C(1s) and B(1s) spectra (Supplementary Fig.\u0026nbsp;3). Additionally, ATR-FTIR spectra of \u003cstrong\u003eGMPD\u003c/strong\u003e\u0026thinsp;\u003cstrong\u003e+\u0026thinsp;hv\u003c/strong\u003e hydrogels showed a definite decrease in the methacryloyl-related infrared band ratio of C-H stretching vibrations at 3100 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e and C\u0026thinsp;=\u0026thinsp;C stretching vibrations at 1450 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e after light irradiation (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eF), consistent with the \u003csup\u003e1\u003c/sup\u003eH NMR spectra trace of the photopolymerization reaction. Taken together, these results essentially confirm the hybrid crosslinking mechanism of combining dynamic boronic ester crosslinking and covalent photopolymerization.\u003c/p\u003e\n\u003cp\u003eRheological analyses were further conducted to investigate the viscoelastic properties of hybrid crosslinked hydrogels. First, frequency sweep rheological measurements were performed within the linear region to quantify the gel strength according to the crossover frequency (\u003cem\u003e\u0026omega;\u003c/em\u003e\u003csub\u003ec\u003c/sub\u003e) at which the storage modulus (G\u0026rsquo;) was equal to the viscous modulus (G\u0026rsquo;\u0026rsquo;). As shown in Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eG, \u003cstrong\u003eGMPD\u003c/strong\u003e\u003cstrong\u003e-hv\u003c/strong\u003e hydrogels exhibited frequency-dependent viscoelastic behavior, a typical feature of dynamic gel networks \u003cem\u003evia\u003c/em\u003e boronic ester bonds. However, \u003cstrong\u003eGMPD\u003c/strong\u003e\u0026thinsp;\u003cstrong\u003e+\u0026thinsp;hv\u003c/strong\u003e hydrogels showed more elastic performance and higher mechanical strength than \u003cstrong\u003eGMPD\u003c/strong\u003e\u003cstrong\u003e-hv\u003c/strong\u003e hydrogels due to the further enhancement of gel networks \u003cem\u003evia\u003c/em\u003e photopolymerization. Importantly, the dynamic crosslinking of boronic ester bonds exhibited acid-reinforced mechanical strength with \u003cem\u003e\u0026omega;\u003c/em\u003e\u003csub\u003ec\u003c/sub\u003e increasing as pH decreased, which is very suitable for harsh UME of low pH values (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eH). Moreover, time sweep rheological measurements demonstrated that secondary photopolymerization could effectively improve the elasticity of \u003cstrong\u003eGMPD\u003c/strong\u003e hydrogels without any influence of pH values (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eI, J). As a result, the hybrid crosslinking strategy not only provides viscous segments relying on boronic ester dynamic bonds but also offers elastic segment dependent on photoinitiated covalent bonds, and thus achieves satisfactory viscoelasticity that can closely mimic the tissue architecture of urethral mussels.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePerformance of tissue adhesion and acid-reinforced stability.\u003c/strong\u003e As previously reported, phenylboronic acid has a \u0026ldquo;\u003cem\u003ecis\u003c/em\u003e-diol\u0026rdquo; moiety that exhibits adhesive properties similar to those of catechol mussel adhesive protein\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e. In this study, \u003cstrong\u003eGMPD\u003c/strong\u003e hydrogels had the potential to balance hydrogel reversibility and adhesion strength based on the hybrid crosslinking strategy (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eA). To evaluate the tissue binding ability, \u003cstrong\u003eGMPD\u003c/strong\u003e hydrogels were gelled \u003cem\u003ein situ\u003c/em\u003e on the surface of muscle upon light irradiation and then immersed in acid solution to sustain stability. Figure\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eB depicted that there was no obvious breakage or detachment of the adhesive hydrogels regardless of stretching and twisting behavior. The scanning electron microscopy (SEM) results clearly revealed a tight and seamless interface between \u003cstrong\u003eGMP\u003c/strong\u003e hydrogels and muscle upon photopolymerization, whereas \u003cstrong\u003eGM\u003c/strong\u003e hydrogels showed obvious interfacial separation against the surrounding muscle, which is consistent with the structural predictions between the \u0026ldquo;\u003cem\u003ecis\u003c/em\u003e-diol\u0026rdquo; moiety of \u003cstrong\u003eGMP\u003c/strong\u003e hydrogels and active hydrogen on the tissue surface (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eC). To quantitatively evaluate the adhesion performance of \u003cstrong\u003eGMPD\u003c/strong\u003e hydrogels as wound dressings, standard lap shear and incision sealing strength were measured respectively. As shown in Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eD-I, the peak adhesive strength (\u003cem\u003el\u003c/em\u003e) and sealing strength (\u003cem\u003ei\u003c/em\u003e) of \u003cstrong\u003eGMPD\u003c/strong\u003e\u003cstrong\u003e-hv\u003c/strong\u003e hydrogels (\u003cem\u003el\u003c/em\u003e\u0026thinsp;=\u0026thinsp;19.4\u0026thinsp;\u0026plusmn;\u0026thinsp;2.5 kPa; \u003cem\u003ei\u003c/em\u003e\u0026thinsp;=\u0026thinsp;3.1\u0026thinsp;\u0026plusmn;\u0026thinsp;0.6 N) were higher than those of \u003cstrong\u003eGM\u003c/strong\u003e hydrogels (\u003cem\u003el\u003c/em\u003e\u0026thinsp;=\u0026thinsp;1.1\u0026thinsp;\u0026plusmn;\u0026thinsp;0.2 kPa; \u003cem\u003ei\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.6\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1 N) and commercially available fibrin glue (\u003cem\u003el\u003c/em\u003e\u0026thinsp;=\u0026thinsp;12.2\u0026thinsp;\u0026plusmn;\u0026thinsp;1.7 kPa; \u003cem\u003ei\u003c/em\u003e\u0026thinsp;=\u0026thinsp;1.4\u0026thinsp;\u0026plusmn;\u0026thinsp;0.3 N), which was probably attributed to the \u003cem\u003ecis\u003c/em\u003e-diol-mediated adhesive capability. Noticeably, the tensile strength required to dislocate hydrogels from tissue significantly increased as secondary covalent stabilization \u003cem\u003evia\u003c/em\u003e photopolymerization (\u003cstrong\u003eGMPD\u003c/strong\u003e\u0026thinsp;\u003cstrong\u003e+\u0026thinsp;hv\u003c/strong\u003e hydrogels, \u003cem\u003el\u003c/em\u003e\u0026thinsp;=\u0026thinsp;47.7\u0026thinsp;\u0026plusmn;\u0026thinsp;4.3 kPa; \u003cem\u003ei\u003c/em\u003e\u0026thinsp;=\u0026thinsp;9.6\u0026thinsp;\u0026plusmn;\u0026thinsp;1.2 N), indicating that the enhancement of mechanical stability could effectively improve the adhesive strength. Gel stability is another important parameter for wound dressings, especially in dynamic and acidic urethral environment. As shown in Supplementary Fig.\u0026nbsp;4, \u003cstrong\u003eGMPD\u003c/strong\u003e\u003cstrong\u003e-hv\u003c/strong\u003e hydrogels in acid solution could effectively extend the \u003cem\u003ein vitro\u003c/em\u003e degradation time from ~\u0026thinsp;3 days (\u003cem\u003epH\u0026thinsp;=\u0026thinsp;7.4\u003c/em\u003e) to ~\u0026thinsp;14 days (\u003cem\u003epH\u0026thinsp;=\u0026thinsp;5.0\u003c/em\u003e), which is attributed to the acid-reinforced structural stability of boronic ester bonds (correlated with Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eH). Additionally, \u003cstrong\u003eGMPD\u003c/strong\u003e\u0026thinsp;\u003cstrong\u003e+\u0026thinsp;hv\u003c/strong\u003e hydrogels exhibited long-lasting stability over 14 days in any harsh UME (pH values from 6.5 to 7.4) due to the pH-independent covalent stabilization of photopolymerization. Therefore, \u003cstrong\u003eGMPD\u003c/strong\u003e hydrogels are suitable for dynamic and acidic urethral environment based on the unique characteristics of acid-reinforced and covalent-stabilized mechanical properties. All these results demonstrated that our \u003cstrong\u003eGMPD\u003c/strong\u003e hydrogels possessed facile operation, mussel-mimetic viscoelasticity, satisfactory adhesion, and acid-reinforced stability, which could serve as an ideal wound dressing applied in the harsh UME.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eIn vitro\u003c/strong\u003e \u003cstrong\u003ebiological evaluation of the TOR platform.\u003c/strong\u003e To better mimic the dynamic physiological process of urethral reconstruction, it is necessary to efficiently promote wound healing at the early stage and prevent hypertrophic scarring at the later stage. In this study, a novel \u003cstrong\u003eTOR\u003c/strong\u003e technical platform was developed to address the above challenge by preferentially releasing VEGF from \u003cstrong\u003eV-GM\u003c/strong\u003e microgels and subsequently releasing a TGF\u0026beta; inhibitor (SB431542) from \u003cstrong\u003eTI-PLGA\u003c/strong\u003e microcapsules (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eA). The microscope and SEM examinations demonstrated that microfluidic-based \u003cstrong\u003eGM\u003c/strong\u003e microgels showed uniform transparent spherical morphology and the lyophilized samples displayed a typical porous structure on the microgel surface (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eB). In addition, \u003cstrong\u003ePLGA\u003c/strong\u003e microcapsules prepared by the water-oil-water (W/O/W) double emulsion strategy possessed a suitable shell thickness to suppress the initial burst release and a hollow inner structure to encapsulate insoluble drugs (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eC). The drug release kinetics were further tested to investigate the feasibility of the temporally on-demand regulatory strategy. As shown in Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eD, V\u003cstrong\u003e-GM\u003c/strong\u003e microgels exhibited initial burst release within ~\u0026thinsp;3 days, while the core-shell structure of \u003cstrong\u003eTI-PLGA\u003c/strong\u003e microcapsules could effectively delay the release of the TGF\u0026beta; inhibitor after 3 days, confirming the successful construction of the \u003cstrong\u003eTOR\u003c/strong\u003e platform by means of early-released \u003cstrong\u003eV-GM\u003c/strong\u003e microgels and later-released \u003cstrong\u003eTI-PLGA\u003c/strong\u003e microcapsules.\u003c/p\u003e\n\u003cp\u003eFurthermore, the biological function of the \u003cstrong\u003eTOR\u003c/strong\u003e platform was evaluated to verify the time-dependent wound healing process. As shown in Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eE-G, cell scratch experiments demonstrated that fibroblasts in the \u003cstrong\u003eV-GM\u003c/strong\u003e groups reached nearly 100% coverage at 24 h and showed the highest proliferation rate due to the initial release of VEGF, whereas the \u003cstrong\u003eTI-PLGA\u003c/strong\u003e groups reached approximately 75% coverage at 24 h and exhibited the suppression of cell proliferation on day 7 attributed to the gradual release of the TGF\u0026beta; inhibitor. In addition, HUVECs in the \u003cstrong\u003eV-GM\u003c/strong\u003e groups showed an accelerated healing rate, and the \u003cstrong\u003eTI-PLGA\u003c/strong\u003e groups showed no significant difference in HUVEC proliferation even with the release of the TGF\u0026beta; inhibitor (Supplementary Fig.\u0026nbsp;5). The same results were further confirmed by both fibroblast- and HUVEC-specific fluorescence staining. As shown in Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eH-L and Supplementary Fig.\u0026nbsp;6, fibroblast-specific expression (fibronectin, \u003cem\u003e\u0026alpha;\u003c/em\u003e-SMA, COL1, and COL3) was significantly upregulated in the \u003cstrong\u003eV-GM\u003c/strong\u003e groups, whereas the corresponding expression were dramatically downregulated in the \u003cstrong\u003eTI-PLGA\u003c/strong\u003e groups, implying successful inhibition of fibroblast-specific function owing to the gradual release of the TGF\u0026beta; inhibitor. The HUVEC-specific fluorescence staining (CD31 and VWF) showed that the initial release of VEGF effectively upregulated vascularized-related expression (Supplementary Fig.\u0026nbsp;7). In short, the meticulously designed \u003cstrong\u003eTOR\u003c/strong\u003e platform has a synergistic effect of promoting vascularization for wound healing at the early stage and preventing fibrogenesis at the later stage, and thus provides a 4D cell culture system combining a time-dependent \u003cstrong\u003eTOR\u003c/strong\u003e platform with UME-adaptable hydrogels.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eScarless healing molecular mechanism of the time-dependent process.\u003c/strong\u003e Before investigating the 4D dynamic regulation of the scarless healing process, the cytocompatibility was evaluated using CCK-8 assays. As shown in Supplementary Fig.\u0026nbsp;8, both \u003cstrong\u003eGMPD\u003c/strong\u003e hydrogel and \u003cstrong\u003eTOR\u003c/strong\u003e microsphere extracts did not show obvious cytotoxicity (\u0026gt;\u0026thinsp;92% cell viability) for either fibroblasts or HUVECs. To exploit the molecular mechanism involved in scarless wound healing based on \u003cstrong\u003eTOR\u003c/strong\u003e-functionalized hydrogel dressings, differentially expressed genes (DEGs) were identified and biological processes were investigated using RNA-seq methods (see methods). The Venn diagram showed the total changes and overlaps of fibroblast DEGs in \u003cstrong\u003eTOR\u003c/strong\u003e-functionalized hydrogels after 1-, 4-, and 7-days culture (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eA). As shown in the volcano plots, there were 928 upregulated and 695 downregulated DEGs between the F1 and F4 groups at the early stage, as well as 267 upregulated and 382 downregulated DEGs between the F4 and F7 groups at the later stage (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eB). Moreover, the volcano plots of endothelial cell DEGs showed 1989 upregulated and 2277 downregulated genes between the E1 and E4 groups at the early stage (Supplementary Fig.\u0026nbsp;9A, B).\u003c/p\u003e\n\u003cp\u003eNext, DEGs of both fibroblasts and endothelial cells involved in the wound healing process at the early stage were determined to investigate the synergetic effect of the \u003cstrong\u003eTOR\u003c/strong\u003e platform. A total of 61 DEGs involved in the wound healing process at the early stage were dysregulated in fibroblasts (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eC), while 82 DEGs were dysregulated in endothelial cells at day 4 (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eD). Notably, three increased DEGs (\u003cem\u003eVEGFA\u003c/em\u003e, \u003cem\u003eHMOX1\u003c/em\u003e, and \u003cem\u003eITGA2\u003c/em\u003e) and four decreased DEGs (\u003cem\u003eHIF-1A\u003c/em\u003e, \u003cem\u003eTPM1\u003c/em\u003e, \u003cem\u003eCCN1\u003c/em\u003e, and \u003cem\u003eCOL1A1\u003c/em\u003e) involved in the wound healing process at the early stage were commonly dysregulated in both fibroblasts and endothelial cells, which are very relevant to VEGF singling pathways. The corresponding DEGs were further validated by quantitative real-time polymerase chain reaction (qRT-PCR) and western blot (WB). As shown in Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eE, gene expression (\u003cem\u003eVEGFA\u003c/em\u003e, \u003cem\u003eHMOX1\u003c/em\u003e, and \u003cem\u003eITGA2\u003c/em\u003e) was dramatically upregulated in the E4 groups compared to the E1 groups, and the same gene expression was also significantly upregulated in the F4 groups compared to the F1 groups, indicating the synergetic promotion of angiogenesis associated with fibrogenesis formation. Similarly, the gene expression levels (\u003cem\u003eHIF-1A\u003c/em\u003e, \u003cem\u003eTPM1\u003c/em\u003e, and \u003cem\u003eCCN1\u003c/em\u003e) were greatly downregulated in both the E4 and F4 groups compared to the corresponding E1 and F1 groups. The same results for protein expression levels were also confirmed by WB experiments, as shown in Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eF. Additionally, the early index of the vascularized expression level (VWF) was dramatically upregulated between the E4 and E1 groups, and the later index of the vascularized expression level (CD31) were significantly upregulated between the E7 and E4 groups, demonstrating the activation of VEGF singling pathways \u003cem\u003evia\u003c/em\u003e\u003cstrong\u003eV-GM\u003c/strong\u003e microgels regardless of the later release of the TGF\u0026beta; inhibitor (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eG, H).\u003c/p\u003e\n\u003cp\u003eThe singling pathways involved in the wound healing process were further investigated between the F1 and F4 groups at the early stage, as well as between the F4 and F7 groups at the later stage. Figure\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003eA depicted the top 15 potential biological processes and signaling pathways at the early stage, according to the Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses, which were related to the terms of cell cycle, cellular component organization, and metabolic process. Notably, DEGs between the E1 and E4 groups at the early stage of the cell cycle, organelle and cellular component organization processes were commonly enriched in GO and KEGG terms of endothelial cells, revealing that these processes are critical to wound recovery (Supplementary Fig.\u0026nbsp;9C, D). At the later stage of wound healing, cell adhesion, differentiation, extracellular matrix, and structure organization, as well as tissue development and blood vessel development, were dominant, implying that ECM reconstruction is an important event during wound recovery (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003eB). Noticeably, enriched TGF\u0026beta; signaling pathways were also observed by KEGG pathway analysis, which indirectly confirmed the essential regulatory roles of later-released TGF\u0026beta; inhibitors in the wound healing process. As shown in Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003eC, D, qRT-PCR and WB experiments demonstrated that the fibrogenic expression levels (\u0026alpha;-SMA, elastin, COL1, COL3, and fibronectin) were significantly downregulated, which is vital for the matrix remodeling of excessive ECM at the later stage. The main explanations for the regulatory function of ECM reconstruction are attributable to the negative regulation of TGF\u0026beta;2/Smad3 signaling (\u003cem\u003eTGFB2\u003c/em\u003e and \u003cem\u003eSmad3\u003c/em\u003e downregulated) associated with the activation of MMP-related matrix degradation (\u003cem\u003eMMP1\u003c/em\u003e upregulated) upon the later-released TGF\u0026beta; inhibitor through \u003cstrong\u003eTI-PLGA\u003c/strong\u003e microcapsules.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eIn vivo\u003c/strong\u003e \u003cstrong\u003escarless urethral reconstruction in rabbits.\u003c/strong\u003e The \u003cem\u003ein vivo\u003c/em\u003e biological effect of the \u003cstrong\u003eTOR\u003c/strong\u003e-functionalized hydrogel dressings was further investigated in rabbit models. As shown in Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e7\u003c/span\u003eA, viscoelastic hydrogel precursors were first injected to fully cover the urethral defects, followed by light irradiation (365-nm LED, 20 mW/cm\u003csup\u003e2\u003c/sup\u003e) for stabilization. The recovery of injured urethras in each group was detected after 4- and 8-weeks surgery. In the ultrasound urethrograms, both the \u003cstrong\u003eCtrl\u003c/strong\u003e and \u003cstrong\u003eGMPD\u003c/strong\u003e groups displayed a very narrow lumen caused by excessive scar formation (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e7\u003c/span\u003eB, C). The \u003cstrong\u003eGMPD-V\u003c/strong\u003e and \u003cstrong\u003eGMPD-TI\u003c/strong\u003e groups showed relatively wide urethral lumens to various extent, whereas the \u003cstrong\u003eGMPD-V/TI\u003c/strong\u003e groups exhibited a fluent lumen similar to that of a normal urethra. Gross morphology demonstrated that urethral repair in the \u003cstrong\u003eGMPD-V/TI\u003c/strong\u003e groups was significantly better than that in the other groups, as the repaired urethras were smooth without any hypertrophic scar formation. However, both the \u003cstrong\u003eCtrl\u003c/strong\u003e and \u003cstrong\u003eGMPD\u003c/strong\u003e groups exhibited undesired shrinkage of urethras, showing the worst repair, while excessive scar formation could be seen in the \u003cstrong\u003eGMPD-V\u003c/strong\u003e groups (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e7\u003c/span\u003eD).\u003c/p\u003e\n\u003cp\u003eHistological analyses of urethral tissue at 8 weeks after surgery were performed by hematoxylin and eosin (H\u0026amp;E) and Masson\u0026rsquo;s trichrome staining. The urethral condition of ECM deposition, urothelium regeneration, and tissue fibrogenesis was shown in Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e7\u003c/span\u003eE. The repaired urethras in both the \u003cstrong\u003eCtrl\u003c/strong\u003e and \u003cstrong\u003eGMPD\u003c/strong\u003e groups exhibited severe urethral stricture with incomplete urothelium layers, fewer blood vessels, and hypertrophic scar formation, which is mainly attributed to the lack of regenerated urothelium layers. In contrast, the repaired urethras treated by \u003cstrong\u003eGMPD-V\u003c/strong\u003e hydrogel dressings had almost normal urothelium layers with abundant blood vessels, but there was excessive collagen deposition under the regenerated epithelium. In the \u003cstrong\u003eGMPD-TI\u003c/strong\u003e groups, although there was no obvious ECM over deposition, the urothelium layers did not regenerate well. With the synergetic release of both VEGF and TGF\u0026beta; inhibitor, the continued and complete urothelium layers were satisfactorily regenerated on the lumen surface without any hypertrophic scar formation in the \u003cstrong\u003eGMPD-V/TI\u003c/strong\u003e groups after 8-weeks surgery, which is regarded as scarless urethral reconstruction.\u003c/p\u003e\n\u003cp\u003eImmunofluorescence examinations were used to further evaluate protein expression in the repaired urethras (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e8\u003c/span\u003eA, B). The expression level of epithelial cytokeratin AE1/AE3, an important membrane surface protein marker, was significantly higher in the \u003cstrong\u003eGMPD-V/TI\u003c/strong\u003e groups than in the other groups. Specifically, the regenerated urothelium layer in the \u003cstrong\u003eGMPD-V\u003c/strong\u003e and \u003cstrong\u003eGMPD-TI\u003c/strong\u003e groups was thinner than that in the \u003cstrong\u003eGMPD-V/TI\u003c/strong\u003e group, whereas the expression of cytokeratin in both the \u003cstrong\u003eCtrl\u003c/strong\u003e and \u003cstrong\u003eGMPD\u003c/strong\u003e groups was hardly found. Noticeably, the \u003cstrong\u003eGMPD-V\u003c/strong\u003e and \u003cstrong\u003eGMPD-V/TI\u003c/strong\u003e groups showed large numbers of CD31-positive cells (blood vessels labelled by CD31), which were much more abundant than those in other groups, implying that the regulation of the VEGF signaling pathway has a significant role in improving angiogenesis under the epithelium. The \u0026alpha;-SMA expression level (the marker of myofibroblasts) under the epithelium was relatively lower in the \u003cstrong\u003eGMPD-TI\u003c/strong\u003e and \u003cstrong\u003eGMPD-V/TI\u003c/strong\u003e groups than in the other groups, implying that the effective inhibition of the TGF\u0026beta; signaling pathway is conductive to antifibrogenic function. However, both the \u003cstrong\u003eCtrl\u003c/strong\u003e and \u003cstrong\u003eGMPD\u003c/strong\u003e groups showed highly expressed \u0026alpha;-SMA protein, indicating that large numbers of myofibroblasts existed under the epithelium. Consistent with the results of \u0026alpha;-SMA, the expression level of collagen I at the site of repaired urethras treated by \u003cstrong\u003eGMPD-V/TI\u003c/strong\u003e hydrogel dressing was relatively lower than that in other groups. Additionally, the positive expression of proliferating cell nuclear antigen (PCNA) was obviously increased at the site of regenerated epithelium in the \u003cstrong\u003eGMPD-V/TI\u003c/strong\u003e groups. Therefore, it is suggested that the synergetic release of both VEGF and TGF\u0026beta; inhibitors has the potential to promote the regeneration of urothelium layers without hypertrophic scar formation. Different degrees of inflammatory responses at the injured sites appeared because of the application of the extra hydrogel dressings (Supplementary Fig.\u0026nbsp;10). The results demonstrated that the number of CD206-positive cells (M2 macrophages labelled by CD206) in the \u003cstrong\u003eGMPD-V\u003c/strong\u003e group was the highest, while the number of CD206-positive cells in the \u003cstrong\u003eGMPD-TI\u003c/strong\u003e group was significantly lower than that in the other groups, indicating the possible inhibition of inflammation by the TGF\u0026beta; signaling pathway. The \u003cstrong\u003eGMPD-V/TI\u003c/strong\u003e hydrogel-treated urethras showed modest inflammation compared with the \u003cstrong\u003eGMPD\u003c/strong\u003e and \u003cstrong\u003eGMPD-V\u003c/strong\u003e hydrogel-treated urethras.\u003c/p\u003e\n\u003cp\u003eAll these results suggested that \u003cstrong\u003eTOR\u003c/strong\u003e-functionalized hydrogel dressings not only promoted the regeneration of vascularized urothelium layers \u003cem\u003evia\u003c/em\u003e early-released VEGF but also effectively inhibited hypertrophic scar formation \u003cem\u003evia\u003c/em\u003e later-released TGF\u0026beta; inhibitor.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eUrethral injury is a common disease that is usually accompanied by severe urethral stricture due to hypertrophic scar formation. In the clinic, free autogenous lingual mucosa tissue has been widely applied in upper urinary tract repair for more than ten years, but it is extremely limited by the available sampling size, as well as adverse impacts on patients\u0026rsquo; pronunciation, mastication and other functions\u003csup\u003e\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e, \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e\u003c/sup\u003e. Recently, the development of tissue engineering methods that combine biocompatible scaffolds with adult cells or stem cells has effectively improved urethral repair, but clinical application is difficult due to ethical, cell colonization, and local survival problems\u003csup\u003e\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e, \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e\u003c/sup\u003e. Thus, current studies have focused on delivering biological factors with biocompatible scaffolds, especially hydrogel dressings, owing to the unique advantages of wet-healing conditions, the ease of carrying bioactive substances, and availiability to mimic the cell microenvironment. Predictably, there is high clinical value in developing ideal hydrogel dressings for scarless urethral reconstruction.\u003c/p\u003e \u003cp\u003eThe primary consideration for urethral repair is to overcome the harsh UME of dynamic wound surfaces suffering from acidic urine. In this study, a hybrid crosslinking strategy combining dynamic boronic ester crosslinking and covalent photopolymerization was synergistically employed to prepare \u003cb\u003eGMPD\u003c/b\u003e hydrogels. The hybrid design rationale is to obtain optimal physicochemical properties adaptable to harsh UME: \u003cem\u003ei\u003c/em\u003e) boronic ester crosslinking has good shear-shinning and self-healing features that are suitable for injecting onto the target site and completely covering the urethral defects, followed by secondary photopolymerization for post-stabilization; \u003cem\u003eii\u003c/em\u003e) different from traditional elastic hydrogels, our hybrid designed hydrogels exhibit satisfactory mussel-mimetic viscoelasticity accessible to dynamic urethral environment; \u003cem\u003eiii\u003c/em\u003e) the inherent \u003cem\u003ecis\u003c/em\u003e-diol-based adhesive ability derived from phenylboronic acid-modified \u003cb\u003eGM\u003c/b\u003e together with further mechanical enhancement by covalent photopolymerization; \u003cem\u003eiv\u003c/em\u003e) the choice of fluorophenylboronic acid moiety is mainly related to physiological-pH crosslinking conditions and the acid-reinforced mechanical strength that could effectively resist acidic urine environment. Therefore, the hybrid crosslinking strategy provided facile operation, mussel-mimetic viscoelasticity, satisfactory adhesion, and acid-reinforced stability adapted to the harsh UME.\u003c/p\u003e \u003cp\u003eFurthermore, the 4D design of hydrogel dressings plays a key role in achieving scarless urethral reconstruction. Fully mimicking the time-dependent physiological process requires an effective balance between ECM deposition and remodeling at different healing stages. The current results demonstrated that the \u003cb\u003eTOR\u003c/b\u003e technical platform could simultaneously improve early-stage angiogenesis through \u003cb\u003eV-GM\u003c/b\u003e microgels and prevent later-stage excessive fibrogenesis through \u003cb\u003eTI-PLGA\u003c/b\u003e microcapsules. At the early stage, we found that seven genes were dominant in the wound healing process, including three upregulated DEGs (\u003cem\u003eVEGFA\u003c/em\u003e, \u003cem\u003eHMOX1\u003c/em\u003e, and \u003cem\u003eITGA2\u003c/em\u003e) and four downregulated DEGs (\u003cem\u003eHIF-1A\u003c/em\u003e, \u003cem\u003eTPM1\u003c/em\u003e, \u003cem\u003eCCN1\u003c/em\u003e, and \u003cem\u003eCOL1A1\u003c/em\u003e), which are related to angiogenesis and urothelial regeneration. At the later stage, fibrogenic gene expression (\u003cem\u003eα-SMA\u003c/em\u003e, \u003cem\u003eelastin\u003c/em\u003e, \u003cem\u003eCOL1\u003c/em\u003e, \u003cem\u003eCOL3\u003c/em\u003e, and \u003cem\u003efibronectin\u003c/em\u003e) was significantly downregulated due to the negative regulation of TGFβ2/Smad3 signaling (\u003cem\u003eTGFB2\u003c/em\u003e and \u003cem\u003eSmad3\u003c/em\u003e downregulated) associated with the activation of MMP-related matrix degradation (\u003cem\u003eMMP1\u003c/em\u003e upregulated). Taken together, the time-dependent physiological process of urethral reconstruction was successfully achieved through preferentially promoting vascularized urothelium regeneration by activating the VEGF signaling pathway and subsequently preventing excessive fibrogenesis by timing TGFβ2/Smad3 signaling pathway inhibition. The same effects of \u003cb\u003eTOR\u003c/b\u003e-functionalized hydrogel dressing were further verified in a rabbit urethral injury model. Only the synergetic function of both VEGF and TGFβ inhibitor in the \u003cb\u003eGMPD-V/TI\u003c/b\u003e group exhibited the optimal repair effect, showing satisfactory urothelium regeneration without hypertrophic scar formation.\u003c/p\u003e \u003cp\u003eIn summary, the current study demonstrates a novel \u003cb\u003eTOR\u003c/b\u003e-functionalized hydrogel dressing for scarless urethral reconstruction. The UME-adaptable \u003cb\u003eGMPD\u003c/b\u003e hydrogels possess robust adhesion to dynamic wound surfaces even when suffering from acidic urine, while the \u003cb\u003eTOR\u003c/b\u003e platform effectively balances ECM deposition and remodeling at different healing stages. As a result, the urethral injury of rabbits was sucessfully repaired through the time-dependent physiological process of preferentially activating VEGF-related vascularized urothelium regeneration and subsequently preventing TGFβ2/Smad3-related hypertrophic scar formation. Although this work represents a proof-of-concept study, we are fully convinced of the significance of time-dependent physiological urethral reconstruction, which paves the way to improve the clinical treatment of urethral injury in the future.\u003c/p\u003e"},{"header":"Methods","content":"\u003cp\u003e\u003cstrong\u003eMaterials and animals.\u003c/strong\u003e Gelatin (from porcine skin), methacrylic anhydride, sodium hydroxide, 4-carboxy-3-fluoro-phenylboronic acid, D-(+)-gluconic acid \u0026delta;-lactone, 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC\u0026middot;HCl), N-hydroxy succinimide (NHS), triethylamine, dimethylsulfoxide (DMSO), poly(ethylene glycol) (PEG, MW: 400), polyvinyl alcohol (PVA, MW: 95, 000), and lithium phenyl-2,4,6-trimethylbenzoylphosphinate (LAP) photoinitiator were purchased from Sigma-Aldrich. Poly(lactic-co-glycolic acid) (PLGA, MW: 10 kDa, LA:GA\u0026thinsp;=\u0026thinsp;75: 25) were purchased from Daigang Biotechnology Co., Ltd. The TGF\u0026beta; inhibitor SB431542 was purchased from Selleck. All the other chemicals were reagent grade. New Zealand white rabbits were purchased from Shanghai Jiao Tong University School of Agriculture. All protocols for animal experiments were approved by the Animal Care and Experimental Committee of Shanghai Jiao Tong University Affiliated Sixth People\u0026rsquo;s Hospital (No: 2021\u0026thinsp;\u0026minus;\u0026thinsp;0154).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSynthesis of GMP and GMD polymers.\u003c/strong\u003e First, gelatin methacryloyl (\u003cstrong\u003eGM\u003c/strong\u003e) polymers were synthesized according to the previously established methods\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e45\u003c/span\u003e\u003c/sup\u003e. Then, 2 g of \u003cstrong\u003eGM\u003c/strong\u003e was dissolved in 50 mL of PBS (pH\u0026thinsp;=\u0026thinsp;7.4) and stirred vigorously at 50 ℃ until complete dissolution. To synthesize \u003cstrong\u003eGMP\u003c/strong\u003e polymers, 92 mg (0.5 mmol) of 4-carboxy-3-fluoro-phenylboronic acid, 96 mg (0.5 mmol) of EDC\u0026middot;HCl, and 58 mg (0.5 mmol) of NHS were dissolved in anhydrous DMSO and sequentially added into the above \u003cstrong\u003eGM\u003c/strong\u003e solution. To synthesize \u003cstrong\u003eGMD\u003c/strong\u003e polymers, 140 mg (0.8 mmol) of D-(+)-gluconic acid \u0026delta;-lactone and 0.1 mL of triethylamine were dissolved in anhydrous DMSO and sequentially added into the above \u003cstrong\u003eGM\u003c/strong\u003e solution. After the reaction, the solutions were collected and dialyzed against deionized water at 40 ℃ for 3 days followed by freezing and lyophilization. \u003csup\u003e1\u003c/sup\u003eH NMR spectra were obtained to characterize the grafted functional groups and determine the corresponding substitution degree as previously described\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e46\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSynthesis of V-GM microgels and TI-PLGA microcapsules.\u003c/strong\u003e To produce \u003cstrong\u003eV-GM\u003c/strong\u003e microgels, 500 mg of \u003cstrong\u003eGM\u003c/strong\u003e and 20 mg of LAP were dissolved in 10 mL of PBS (pH\u0026thinsp;=\u0026thinsp;7.4) at 40 ℃ containing 0.1 \u0026micro;g/mL VEGF (Dima Biotech) as the water phase. 5 mL of span-80 and 40 mL of paraffin oil were mixed with each other and preheated at 40 ℃ as the oil phase. Both water and oil fluids were injected into the microchannels by micropumps, and the water phase formed single spherical droplets under the fluid shear of the oil phase. The droplets were subsequently crosslinked by photopolymerization upon light irradiation (365-nm LED, 20 mW/cm\u003csup\u003e2\u003c/sup\u003e). The formed microgels were washed with hexane and deionized water for three times, followed by freezing and lyophilization. The \u003cstrong\u003eTI-PLGA\u003c/strong\u003e microcapsules were produced by the W/O/W emulsion method according to the previous literature\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e47\u003c/span\u003e\u003c/sup\u003e. Briefly, 1 mg of TGF\u0026beta; inhibitor was added into a solution of 2.5 mg PEG400/50 \u0026micro;L deionized water to obtain the inner water phase. The above solution was added dropwise into a solution of 100 mg PLGA/1 mL chloroform with vigorous stirring for 10 min emulsification. The resultant water-in-oil (W/O) emulsion was then added dropwise into 15 mL of 1% w/v PVA aqueous solution as an emulsion stabilizer. The double emulsion (W/O/W) was obtained using a magnetic stirrer (3000 rpm) and allowed to stand for 5 hours until solvent evaporation. The \u003cstrong\u003eTI-PLGA\u003c/strong\u003e microcapsules after centrifugation were washed three times, followed by freezing and lyophilization. The drug release kinetics were tested according to previously established methods\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e40\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eHydrogel preparation.\u003c/strong\u003e Hydrogel precursors of \u003cstrong\u003eGM\u003c/strong\u003e, \u003cstrong\u003eGMP\u003c/strong\u003e, \u003cstrong\u003eGMD\u003c/strong\u003e and LAP (0.2% w/v) were mixed in certain proportions in PBS solution (pH\u0026thinsp;=\u0026thinsp;7.4). The hydrogel composition in this study was as follows: \u003cstrong\u003eGMP\u003c/strong\u003e hydrogels: 10% w/v FPBA-modified GelMA; \u003cstrong\u003eGM\u003c/strong\u003e hydrogels: 10% w/v GelMA; \u003cstrong\u003eGMPD\u003c/strong\u003e hydrogels: 10% w/v, \u003cstrong\u003eGMP\u003c/strong\u003e: \u003cstrong\u003eGMD\u003c/strong\u003e\u0026thinsp;=\u0026thinsp;1:1; \u003cstrong\u003eGMPD\u003c/strong\u003e\u003cstrong\u003e-hv\u003c/strong\u003e hydrogels: \u003cstrong\u003eGMPD\u003c/strong\u003e hydrogels without light irradiation; \u003cstrong\u003eGMPD\u0026thinsp;+\u003c/strong\u003e\u0026thinsp;\u003cstrong\u003ehv\u003c/strong\u003e hydrogels: \u003cstrong\u003eGMPD\u003c/strong\u003e hydrogels with light irradiation; light: 365-nm LED, 20 mW/cm\u003csup\u003e2\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eXPS and ATR-FTIR experiments.\u003c/strong\u003e First, the \u003cstrong\u003eGMP\u003c/strong\u003e and \u003cstrong\u003eGMD\u003c/strong\u003e gel precursors, as well as the \u003cstrong\u003eGMPD\u003c/strong\u003e\u003cstrong\u003e-hv\u003c/strong\u003e and \u003cstrong\u003eGMPD\u0026thinsp;+\u003c/strong\u003e\u0026thinsp;\u003cstrong\u003ehv\u003c/strong\u003e hydrogels, were dried at 40 ℃ for 12 hours. Then, the dry film samples were tested in an ultrahigh vacuum chamber by an ESCALAB 250Xi XPS system, and XPS spectra were analyzed by XPSPEAK software to conduct peak separation. In addition, the dry film samples were analyzed on a Nicolet 6700 FTIR spectrometer.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eRheological measurements.\u003c/strong\u003e Dynamic rheology experiments were performed on a HAAKE MARS Ⅲ photorheometer with parallel-plate (P20 TiL, 20 mm diameter) geometry and OmniCure Series 2000 (365 nm, 20 mW/cm\u003csup\u003e2\u003c/sup\u003e) at 25\u0026deg;C. Time sweep oscillatory tests were performed at a 10% strain, a 1 Hz frequency and a 0.5 mm gap for 120 s. The gel point was determined as the time when the storage modulus (G\u0026rsquo;) surpassed the loss modulus (G\u0026rsquo;\u0026rsquo;). The elastic modulus was determined as the storage modulus (G\u0026rsquo;) reaching complete gelation. Frequency sweep oscillatory tests were performed at a 10% strain and a 0.5 mm gap from 0.5 to 100 rad/s. Strain sweeps were performed to verify the linear response. Viscosity tests were performed at a gradually increasing shear rate from 0 to 50 s\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSEM examination.\u003c/strong\u003e To evaluate the interfacial integration between muscle tissue and hydrogels, the hydrogel-muscle integrated samples were dehydrated by free drying and cut into a relatively flat interface for examination. Then, the dehydrated samples were coated with gold-palladium in a Hitachi S-3400N ion sputter for further morphological observation.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAdhesion tests.\u003c/strong\u003e For lap shear tests, fresh hot casing was attached to a glass slide with cyanoacrylate glue to prepare test samples. The hydrogel precursors were uniformly dispersed on the surface of a hot casing with or without light irradiation (365-nm LED, 20 mW/cm\u003csup\u003e2\u003c/sup\u003e). For incision sealing tests, porcine muscle was cut into 2\u0026times;4 cm pieces, followed by a 1-cm incision in the middle of the muscle. The hydrogel precursors were introduced into the muscle defect with or without light irradiation (365-nm LED, 20 mW/cm\u003csup\u003e2\u003c/sup\u003e). Then, adhesion tests were performed on an Instron machine in tensile mode at a 5 mm/min speed.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStability tests.\u003c/strong\u003e The hydrogel samples after complete swelling were recorded as the initial weight W\u003csub\u003e0\u003c/sub\u003e, followed by immersion in PBS solution (pH\u0026thinsp;=\u0026thinsp;7.4). At each time point, these samples were carefully collected and recorded as dry weight for evaluating hydrogel stability.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eIn vitro\u003c/strong\u003e \u003cstrong\u003ebiological evaluation.\u003c/strong\u003e For the cell scratch assay, fibroblasts or endothelial cells were manually scratched with 200 \u0026micro;L pipette tips, and the wound area was observed by optical microscopy after 0, 6, 12, and 24 hours in the \u003cstrong\u003eV-GM\u003c/strong\u003e, \u003cstrong\u003eTI-PLGA\u003c/strong\u003e, and \u003cstrong\u003eCtrl\u003c/strong\u003e groups. For cell proliferation experiments, cell viability was examined using a CCK-8 kit (Dojindo) according to the manufacturer\u0026rsquo;s protocol, and the optical density (OD) was measured with a microplate reader (Synergy H1, BioTek). For cell expression level evaluation, the corresponding fibrogenic and angiogenic evaluations were examined \u003cem\u003evia\u003c/em\u003e immunofluorescence staining. The fibrogenic expression levels of fibronectin, \u0026alpha;-SMA, COL1, and COL3 in fibroblasts and the angiogenic expression levels of CD31 and VWF in endothelial cells were evaluated. The statistical data of relative fluorescent intensity were analyzed using ImageJ software.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSequencing read preprocessing.\u003c/strong\u003e All low-quality bases and adapters of sequencing reads were trimmed using Trimmomatic (PMID: 35037208). After quality control, the reserved clean reads were mapped to the genome references of \u003cem\u003eHomo sapiens\u003c/em\u003e (UCSC hg38) and \u003cem\u003eOryctolagus-cuniculus\u003c/em\u003e (Ensembl OryCun2.0) using STAR (PMID: 26334920). The gene counts and TPM (transcripts per million) values were calculated using RSEM (PMID: 21816040). The gene symbols corresponding to Ensembl gene IDs of \u003cem\u003eOryctolagus-cuniculus\u003c/em\u003e were retrieved from the UniProt database (PMID: 33237286). All DEGs were identified by R package DESeq2 (PMID: 25516281). An absolute value |log2FoldChange| of \u0026ge;\u0026thinsp;1 and padj of \u0026le;\u0026thinsp;0.05 were considered statistically significant.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunctional enrichment of DEGs.\u003c/strong\u003e The enriched terms of Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) were summarized by using the R package clusterProfiler (PMID: 34557778). The top 15 enriched terms were plotted by using the R package ggplot2. In addition, all genes associated with wound healing (GO:0042060) were retrieved from the UniProt database (PMID: 33237286) according to their annotation.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eGene Ontology (GO) and KEGG Enrichment Analysis.\u003c/strong\u003e Gene Ontology (GO; \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://www.geneontology.org\u003c/span\u003e\u003c/span\u003e) is a systematic approach for gene and protein annotation in terms of biological process, molecular process, and cellular component. Kyoto Encyclopedia of Genes and Genomes (KEGG; \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://www.genome.jp/kegg/\u003c/span\u003e\u003c/span\u003e) is an online database depositing biological pathways of genes and biochemicals. The enriched GO terms and KEGG pathways were annotated using the R package clusterProfiler.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eqRT-PCR tests.\u003c/strong\u003e The total RNA of cell-hydrogel samples was isolated using TRIzol reagent (Life Technologies). Reverse transcription was performed with a cDNA synthesis kit (Thermo Scientific) following the manufacturer\u0026rsquo;s instructions. Gene expression was analyzed quantitatively with SYBR Green using a 7500 Real-Time PCR system (Applied Biosystems, Life Technologies). Primers and probes for \u003cem\u003eVEGFA\u003c/em\u003e, \u003cem\u003eHMOX1\u003c/em\u003e, \u003cem\u003eITGA2\u003c/em\u003e, \u003cem\u003eHIF-1A\u003c/em\u003e, \u003cem\u003eTPM1\u003c/em\u003e, \u003cem\u003eCCN1\u003c/em\u003e, \u003cem\u003eTGFBR2\u003c/em\u003e, \u003cem\u003eSmad3\u003c/em\u003e, \u003cem\u003eMMP1\u003c/em\u003e, \u003cem\u003e\u0026alpha;-SMA\u003c/em\u003e, \u003cem\u003eelastin\u003c/em\u003e, \u003cem\u003eCOL1\u003c/em\u003e, \u003cem\u003eCOL3\u003c/em\u003e, \u003cem\u003efibronectin\u003c/em\u003e, and 𝛽-actin were designed based on published gene sequences (NCBI and PubMed). The expression level for each gene was normalized to that of 𝛽-actin.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eWestern blot tests.\u003c/strong\u003e Cells extracted from cell-hydrogel samples were harvested, and benzosulfonylfluoride, a protease and phosphatase inhibitor, were added to dissolve tissues in RIPA lysis buffer. The cell lysates were cultured on ice for 3 h and then centrifuged to collect the supernatant. 60 \u0026micro;g of protein in total was loaded in each lane, and a 10% SDS-PAGE gel was used for electrophoresis. The target proteins on the SDS-PAGE gel were transferred to polyvinylidene fluoride membranes (PVDF; 0.45 \u0026micro;m) and then blocked with 5% blocking buffer at 37\u0026deg;C for 1 h. PVDF membranes were treated with primary antibodies against VEGFA, HMOX1, ITGA2, HIF-1A, TGFBR2, Smad3, MMP1, \u0026alpha;-SMA, elastin, COL1, COL3, fibronectin, and \u0026beta;-actin at 4\u0026deg;C overnight. Subsequently, the membranes were rinsed with TBST three times, and secondary antibody was incubated for another hour. An imaging system was used to scan the membranes, and grey values were measured to present the expression of differential proteins.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eUrethroplasty and Postoperative Examinations in rabbit.\u003c/strong\u003e In order to verify the feasibility of scarless urethral reconstruction, the hydrogel dressings were \u003cem\u003ein situ\u003c/em\u003e formed in the urethral defect of rabbits for \u003cem\u003ein vivo\u003c/em\u003e experiments. All animal experiments were performed in accordance with the guidelines for animal care. The animal protocol (SYXK 2017\u0026thinsp;\u0026minus;\u0026thinsp;0240) was approved by the Institutional Animal Care and Use Committee of the Shanghai Jiao Tong University Affiliated Sixth People\u0026rsquo;s Hospital. Fifteen adult male New Zealand white rabbits with an average body weight of 2.5 kg were randomly divided into 5 groups for urethral defect building and subsequent repair. The rabbits were first subjected to general anaesthesia with intravenous injection of pentobarbital, and then the rabbits\u0026rsquo; skin and urethras were disinfected with 70% alcohol. The skin and ventral urethra were sectioned at approximately 3 cm proximal to the external urethral orifice, and the urethral lumen was exposed. A dorsal urethral defect with a mean length \u0026times; width of 2.0 cm \u0026times; 0.8 cm was created in the anterior urethra of rabbits. All rabbits underwent removal of the urethra near the corpus cavernosum. Rabbits in group 1 (n\u0026thinsp;=\u0026thinsp;3) were not repaired as control. Rabbits in group 2 (n\u0026thinsp;=\u0026thinsp;3) were repaired with \u003cstrong\u003eGMPD\u003c/strong\u003e hydrogels. Rabbits in group 3 (n\u0026thinsp;=\u0026thinsp;3) were repaired with \u003cstrong\u003eGMPD-V\u003c/strong\u003e hydrogels. Rabbits in group 4 (n\u0026thinsp;=\u0026thinsp;3) were repaired with \u003cstrong\u003eGMPD-TI\u003c/strong\u003e hydrogels. Rabbits in group 5 (n\u0026thinsp;=\u0026thinsp;3) were repaired with \u003cstrong\u003eGMPD-V/TI\u003c/strong\u003e hydrogels.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eUrethrography.\u003c/strong\u003e To observe the urethral leakage and stricture in the five groups of animals, the contrast solution was injected into the urethral lumen at 8-weeks post-surgery. Meanwhile, the rabbits underwent the urethral contrast-enhanced ultrasound test to check the condition of the scar in the urethra at 8-weeks post-surgery. The rabbits were euthanized after retrograde urethrograms, and the urethral tissue for the following histology staining was collected.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eHistology Assessment and Immunofluorescence.\u003c/strong\u003e The urethral tissue was harvested 8 weeks after surgery for histology analysis. The specimens were fixed in 4% paraformaldehyde for 30 minutes at room temperature. Then, they were dehydrated with different grades of alcohol and embedded in paraffin blocks. Histological sections were prepared and observed using an optical microscope. Hematoxylin and eosin staining (H\u0026amp;E) and Masson\u0026rsquo;s trichrome staining tests were conducted to identify the epithelial layer and collagen distribution of the urethra. To further demonstrate the repair of urethral function, the samples were stained for immunofluorescence for epithelial cytokeratin AE1/AE3 (Santa Cruz Biotechnology, Inc.), CD31 (Proteintech Group, Inc.), \u0026alpha;-smooth muscle actin (Proteintech Group, Inc.), COL3 (Santa Cruz Biotechnology, Inc.), En1 (Santa Cruz Biotechnology, Inc.), CD206 (Proteintech Group, Inc.), and PCNA (Proteintech Group, Inc.). Nuclei were stained with DAPI (1:500, Life Technologies). Afterwards, the specimens were imaged and observed by an optical microscope.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStatistical analysis.\u003c/strong\u003e All data are presented as the means\u0026thinsp;\u0026plusmn;\u0026thinsp;SDs. Differences between the values were evaluated using one-way analysis of variance (ANOVA) with \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05 considered statistically significant.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that all data supporting of results in this study are available within the paper and its Supplementary Information, or from the corresponding authors upon reasonable request. All data are available in the main text or the supplementary materials. Source data are provided with this paper.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis research was financially supported by Jiangsu Key Technology Research Development Program (BE2017664), Shanghai Jiao Tong University Biomedical Engineering Cross Research Foundation (YG2022ZD022 and YG2017QN15), National Natural Science Foundation of China (82072217 and 81772135), Shanghai health committee (20184Y0053), Shanghai \"Rising stars of medical talent\" Youth development program, Shanghai Jiao Tong University K. C. Wong Medical Fellowship Fund, Shanghai sixth people\u0026rsquo;s hospital foundational research program, National Key Research and Development Program of China (2022YFA1207500, 2018YFA0703100), and Shanghai Municipal Key Clinical Specialty (shslczdzk06601).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eY. H. and K. Z. provided the ideals. Y. H., Q. F., W. C., and K. Z. designed the experiments. Y. H., K. W., Y. H., Y. W., W. F., Y. S., and K. Z. performed the experiments. Y. H., K. W., Y. H., and K. Z. analyzed the data. Y. H. and K. Z. wrote the manuscript. Y. Z., G. Z., Q. F., and W. C. revised the manuscript. All authors commented on the manuscript and its revisions.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAdditional information\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSupplementary information\u003c/strong\u003e The online version contains supplementary material available at.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCorrespondence\u003c/strong\u003e and requests for materials should be addressed to Qiang Fu, Wenguo Cui, or Kaile Zhang.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePeer review information\u003c/strong\u003e\u003cem\u003eNature Communications \u003c/em\u003ethanks the anonymous reviewers for their contribution to the peer review of this work. Peer reviewer reports are available.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eReprints and permissions information\u003c/strong\u003e is available at.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePublisher\u0026rsquo;s note\u003c/strong\u003e Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eOpen Access\u003c/strong\u003e This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article\u0026rsquo;s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article\u0026rsquo;s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eManzoni, G. et al. Hypospadias Repair Failures: Lessons Learned. \u003cem\u003eEur. Urol. \u003c/em\u003e\u003cstrong\u003e49\u003c/strong\u003e, 772\u0026ndash;773 (2006).\u003c/li\u003e\n\u003cli\u003eYang, K. et al. Robotic-assisted Lingual Mucosal Graft Ureteroplasty for the Repair of Complex Urethral Strictures: Technique Description and the Medium-term Outcome. \u003cem\u003eEur. Urol.\u003c/em\u003e \u003cstrong\u003e81\u003c/strong\u003e, 533\u0026ndash;540 (2022).\u003c/li\u003e\n\u003cli\u003eMangir, N., Wilson, K. J., Osman, N. I. \u0026amp; Chapple, C. R. 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Mater.\u003c/em\u003e \u003cstrong\u003e14\u003c/strong\u003e, 97-109 (2022).\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"nature-portfolio","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"","title":"Nature Portfolio","twitterHandle":"","acdcEnabled":false,"dfaEnabled":false,"editorialSystem":"ejp","reportingPortfolio":"","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"urethral reconstruction, hydrogel, four-dimensional microenvironment, scarless healing","lastPublishedDoi":"10.21203/rs.3.rs-3024823/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-3024823/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThe harsh urethral microenvironment (UME) after trauma severely hinders the current hydrogel-based urethral repair. In fact, four-dimensional (4D) consideration to mimic time-dependent physiological processes is essential for scarless urethral reconstruction, which requires balancing extracellular matrix (ECM) deposition and remodeling at different healing stages. In this study, we developed a novel UME-adaptable 4D hydrogel dressing to sequentially provide an early-vascularized microenvironment and later-antifibrogenic microenvironment for scarless urethral reconstruction. With the combination of dynamic boronic ester crosslinking and covalent photopolymerization, the resultant gelatin methacryloyl phenylboronic acid/\u003cem\u003ecis\u003c/em\u003e-diol-crosslinked (\u003cb\u003eGMPD\u003c/b\u003e) hydrogels exhibited mussel-mimetic viscoelasticity, satisfactory adhesion, and acid-reinforced stability, which could adapt to harsh UME. In addition, a temporally on-demand regulatory (\u003cb\u003eTOR\u003c/b\u003e) technical platform was introduced into \u003cb\u003eGMPD\u003c/b\u003e hydrogels to create a time-dependent 4D microenvironment. As a result, physiological urethral recovery was successfully mimicked by means of an early-vascularized microenvironment to promote wound healing by activating the vascular endothelial growth factor (VEGF) signaling pathway, as well as a later-antifibrogenic microenvironment to prevent hypertrophic scar formation by timing transforming growth factor-β (TGFβ) signaling pathway inhibition. Both \u003cem\u003ein vitro\u003c/em\u003e molecular mechanisms of the physiological healing process and \u003cem\u003ein vivo\u003c/em\u003e scarless urethral reconstruction in a rabbit model were effectively verified, providing a promising alternative for urethral injury treatment.\u003c/p\u003e","manuscriptTitle":"Four-dimensional Hydrogel Dressing Adaptable to the Urethral Microenvironment for Scarless Urethral Reconstruction","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2023-06-16 18:59:46","doi":"10.21203/rs.3.rs-3024823/v1","editorialEvents":[],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"nature-communications","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"NCOMMS","sideBox":"Learn more about [Nature Communications](http://www.nature.com/ncomms/)","snPcode":"","submissionUrl":"https://mts-ncomms.nature.com/","title":"Nature Communications","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature Communications","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"ff4b1907-e135-4922-a6ef-3d81bfb18de7","owner":[],"postedDate":"June 16th, 2023","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":22444820,"name":"Biological sciences/Biotechnology/Biomaterials/Tissues"},{"id":22444821,"name":"Health sciences/Urology/Urethra"}],"tags":[],"updatedAt":"2023-11-23T08:13:34+00:00","versionOfRecord":{"articleIdentity":"rs-3024823","link":"https://doi.org/10.1038/s41467-023-43421-w","journal":{"identity":"nature-communications","isVorOnly":false,"title":"Nature Communications"},"publishedOn":"2023-11-22 05:00:00","publishedOnDateReadable":"November 22nd, 2023"},"versionCreatedAt":"2023-06-16 18:59:46","video":"","vorDoi":"10.1038/s41467-023-43421-w","vorDoiUrl":"https://doi.org/10.1038/s41467-023-43421-w","workflowStages":[]},"version":"v1","identity":"rs-3024823","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-3024823","identity":"rs-3024823","version":["v1"]},"buildId":"_2-kVJe1T_tPrBINL-cwx","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}