Construction of the TCDI crosslinked DHV (T-DHV) and the mechanism of H2S releaseTCDI is a versatile reagent and building block in organic synthesis. It is an efficient and widely used thiocarbonylating agent that enables the introduction of thiocarbonyl functionality into various organic molecules35. TCDI readily reacts with a variety of nucleophiles, such as amino36, alcohols37,38, and thiols39, to form thiocarbamate, thioester, and thioether derivatives, respectively40. These reactions proceed under mild conditions at room temperature with fast kinetics and high yields40.In the context of DHV crosslinking, utilizing TCDI offers several advantages. First, its ability to crosslink collagen by reacting with amino and hydroxyl groups makes it an ideal candidate for enhancing the mechanical strength of DHVs. Second, the resulting thiocarbonyl groups left after crosslinking can serve as a source for the release of the gasotransmitter H2S (Fig. 2A)41,42,43, which has potential benefits for immune modulation. Even though TCDI as a crosslinking reagent for tissue engineering has never been investigated.Fig. 2: Construction of the TCDI crosslinked DHV (T-DHV) and the mechanism of H2S release.A A schematic overview presenting the fabrication process and the mechanism of H2S release of T-DHV (DHV created with BioRender.com). B The content of free amino group on samples’ surface determined by ninhydrin staining (n = 4). C The content of free hydroxyl group on samples’ surface determined by dansyl chloride staining (n = 5). D, E Mechanical properties of DHV with different crosslinking degrees: C Strain-stress curves; D Young’s modulus (n = 4); E Ultimate tensile strength (n = 4). F H2S release equations of DETU and MECT with cysteine. G The release kinetics of DETU and MECT (n = 3). H H2S release mechanism of DETU. Int.1: MS (ESI) for C6H11N2O2S2-, [M-]: calcd.: 207.03; found: 207.07. Int.2: MS (ESI) for C4H4NO2S2-, [M-]: calcd.: 161.97; found: 161.99. Int.3: MS (ESI) for C8H10N2O6S42-, [M]2-: calcd.: 89.48; found: 89.02. I) The H2S release kinetics of DHV, GV, and T-DHV with 16 mM cysteine (n = 3). J) The H2S release kinetics of T-DHV with different concentration cysteine (n = 3). Data were expressed as mean ± SD, *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001. n means the number of independent experiments. For box plots, the lower edge denotes the first quartile (Q1, or the 25th percentile) of the data, the central line within the box signifies the median (Q2, or the 50th percentile), while the upper edge of the box corresponds to the third quartile (Q3, or the 75th percentile). The lower whisker extends to the minimum value, indicating its range of variation, and the upper whisker reaches the maximum value, demonstrating its variability. One-way ANOVA was used for analyzing the data.In this study, the DHVs were obtained by decellularizing the leaflets of procine aortic valves, which contain collagens as the main component44,45. The sufficient decellularization was confirmed by histological staining and DNA content detection as shown in Supplementary Fig. 1. When incubating DHVs with the increasing concentration of TCDI, the amino and hydroxyl groups on collagen gradually decreases, indicating successful crosslinking (Figs. 2B, C). The mechanical properties of the resulting TCDI-crosslinked DHVs (T-DHVs), such as Young’s modulus and ultimate tensile strength, exhibit an increase with higher TCDI concentration (Figs. 2D & E). For example, when the TCDI concentration was 18 mg/mL, Young’s modulus and ultimate tensile strength reached values of 20.95 ± 1.15 MPa and 11.79 ± 0.48 MPa, respectively (Figs. 2D & E). These mechanical strengths are comparable to those of commercially available glutaraldehyde-crosslinked bioprosthetic valves (GV)27. These results confirm that TCDI can serve as a fast crosslinking reagent as efficient as glutaraldehyde.Based on the reactivity of TCDI, T-DHV would contain thiourea (1) and thiocarbamate (2) linkages (Fig. 2A) after the crosslinking reaction with amino and hydroxyl groups of collagens. Thiocarbamate is well-known to slowly release H2S by hydrolysis, which would be accelerated if exposed to thiol-containing compounds such as cysteine and glutathione43. On the other hand, release of H2S using thiourea as donors is much slower and requires the presence of cysteine, and its reaction kinetics has not been studied (Fig. 2F)27,42. We investigated the release kinetics of thiourea and thiocarbamate using model compounds, N, N-diethyl thiourea (DETU) and O-(1-Methylethyl) N-ethylcarbamothioate (MECT). These studies revealed that cysteine can catalyze the degradation of thiourea and thiocarbamate, thereby accelerating release of H2S (Supplementary Figs. 3A and B, Fig. 2G). Since the cysteine-catalyzed cleavage of thiourea has not been well discussed in the literature, we have conducted a meticulous investigation into its release mechanism. Analysis of reaction mixture by mass spectroscopy suggested formation of carbamodithioate Int.2 and thiazolidine-2-thione Int.3. The latter was also evidenced in 1H nuclear magnetic resonance (NMR) spectra (Supplementary Fig. 3). Based on the experimental results, a plausible reaction pathway is proposed to account for the cysteine-catalyzed release of H2S from thioureas using the model compound DETU (Fig. 2H). The process begins with the nucleophilic addition of cysteine thiol to the thiocarbonyl group of DETU, forming thiolate Int.1, followed by elimination of ethylamine, leading to the formation of carbamodithioate Int.2. This intermediate undergoes an intramolecular ring-closing cyclization to generate thiazolidine-2-thione Int.3, accompanied by the release of a second ethylamine. The ring opening occurs through the nucleophilic addition of a basic hydroxide. The resulting thiocarboxycysteine Int.4 decomposes to produce carbonyl sulfide (COS) and regenerate cysteine46. COS is then hydrolyzed to produce H2S and CO247.The release of H2S was also observed in T-DHV. In the presence of 16 mM cysteine, significant H2S release was observed in the T-DHV samples, while the raw DHV and the clinically used glutaraldehyde-crosslinked heart valve (GV) exhibited negligible H2S release (Fig. 2I). The rate of H2S release is dependent on the concentration of cysteine. When no cysteine was added, T-DHV showed a slow release of H2S in PBS buffer. However, as the cysteine concentration increased, the rate of H2S release accelerated (Fig. 2J). Overall, these results demonstrate that TCDI efficiently crosslinks DHVs, improves their mechanical properties, and facilitates the continuous release of H2S, most likely due to the formation of thiocarbamate and thiourea linkers.Construction of double network hydrogel coated DHV (PEG/PSBMA-DHV)When the naked DHV is implanted, a cascade of coagulation reactions occurs immediately after contact with the bloodstream and absorption of circulating proteins, leading to thrombosis48. In addition, naked DHV forms a large amount of calcium and phosphorus salt deposits on the valve surface due to a large number of groups in the ECM that can bind to Ca2+ in the blood49,50. Therefore, it is necessary to construct a hydrogel coating that could provide anti-coagulation and anti-calcification functions immediately after implantation. Traditional covalent crosslinked hydrogels cannot adapt to the tissue remodeling process and are susceptible to breaking and losing of their protective effect under high shear stress in circulating blood7,28. The tiny cracks formed by hydrogel rupture may lead to more serious calcium and phosphorus salt deposition7,51.For this purpose, TCDI chemistry was further applied to construct a degradable double network hydrogel coating with anti-fatigue property on DHV. In brief, the DHV and 4-arm-PEG-NH2 (Mw = 2 kDa) were crosslinked by TCDI to immobilize the first PEG hydrogel network on DHV (PEG-DHV). Then, the second hydrogel network was constructed by soaking PEG-DHV in SBMA solution containing free radical initiator, resulting to a PEG/PSBMA dual-network hydrogel-modified DHV (PEG/PSBMA-DHV) (Fig. 3A). The PEG hydrogel alone exhibited brittleness and weak mechanical strength. The incorporation of PSBMA as the second network led to improved anti-fatigue properties (Supplementary Fig. 4A). The homogeneous PSBMA hydrogel displayed a self-recovery rate of 50% after 20 cycles of continuous stretching stimulation, demonstrating excellent self-recovery and anti-fatigue characteristics (Supplementary Figs. 4B & C). When the PSBMA hydrogel was combined with the PEG hydrogel, the performance of the PEG hydrogel was significantly enhanced, with a self-recovery rate of about 70% (Supplementary Figs. 4D & E). The fatigue resistance of the hydrogel coating is attributed to the formation of hydrogen bonds between the sulfur element and the secondary amine group in the first hydrogel network, as well as the ionic bonds formed between the SO3− and N+ groups in the second hydrogel network34,52. The presence of numerous reversible non-covalent bonds between the hydrogel networks imparts strong fatigue resistance to the hydrogel.Fig. 3: Construction and characterization of PEG/PSBMA-DHV.A A schematic overview presenting the fabrication process and structure of PEG/PSBMA-DHV. B General picture of different DHVs. C Surface and cross-section structure of the scaffolds observed by SEM and S element detected by EDS showing that TCDI achieves the cross-linking of DHV and the successful modification of SBMA. D Analysis of S element content by elemental analysis (n = 4). E, F Mechanical properties of DHV with different crosslinking degrees: (E) Ultimate tensile strength (n = 4); (F) Young’s modulus (n = 4). G Pictures of water droplet contacting scaffolds. H) Water contact angle (WCA) showing increased hydrophilicity after modification (n = 5). I The release kinetics of DHV, GV, T-DHV, PEG-DHV and PEG/PSBMA-DHV with 16 mM cysteine by WSP-1 probe (n = 4). J The thermal shrinkage temperature of scaffolds. K) Relative weight loss of DHVs after collagenase digestion (n = 5). Data were expressed as mean ± SD, *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001. n means the number of independent experiments. For box plots, the lower edge denotes the first quartile (Q1, or the 25th percentile) of the data, the central line within the box signifies the median (Q2, or the 50th percentile), while the upper edge of the box corresponds to the third quartile (Q3, or the 75th percentile). The lower whisker extends to the minimum value, indicating its range of variation, and the upper whisker reaches the maximum value, demonstrating its variability. One-way ANOVA was used for analyzing the data.From the general pictures, T-DHV and GV appear similar yellow, but gradually turn white with the modified PEG hydrogel and PEG/PSBMA hydrogel (Fig. 3B). Observations using scanning electron microscopy (SEM) revealed that the T-DHV, PEG-DHV, and PEG/PSBMA-DHV samples had smooth and flat surfaces compared to the rough surface formed by GV. Additionally, the glutaraldehyde crosslinking process led to the loss of the porous structure, whereas T-DHV, PEG-DHV, and PEG/PSBMA-DHV maintained cross-sectional structures similar to DHV (Fig. 3C). Energy-dispersive spectrum (EDS) mapping scans showed a significant increase in the sulfur element signal in T-DHV, PEG-DHV, and PEG/PSBMA-DHV compared to DHV and GV, particularly in the PEG/PSBMA-DHV. This confirmed the successful modification and crosslinking, as well as the even distribution of SBMA (Fig. 3C).Elemental analysis (EA) and Fourier-transform infrared spectrophotometer (FTIR) were used to analyze the elemental content and composition of the samples. EA results showed that the sulfur element content in T-DHV (1.50 ± 0.42%) and PEG-DHV (4.13 ± 1.24%) was higher than that in DHV (0.30 ± 0.05%) and GV (0.27 ± 0.07%) due to the TCDI crosslinking. The sulfur element content in PEG/PSBMA-DHV reached 6.61 ± 0.82% due to the further modification of PSBMA on the surface of DHV (Fig. 3D). A comparison of the FTIR spectra between PEG-DHV and PEG/PSBMA-DHV revealed an additional absorption peak at 1038 cm−1 in the latter, which can be attributed to the S = O symmetric stretching in SBMA chains. This further confirms the successful construction of the PEG/PSBMA double-network hydrogel-modified DHV (Supplementary Fig. 5A).The PEG/PSBMA-DHV exhibited a similar Young’s modulus and ultimate tensile strength to T-DHV and PEG-DHV (Supplementary Figs. 5B, Figs. 3E, F), all of which were comparable to the clinically used GV and significantly higher than unmodified DHV27. However, the hydrophilicity of the DHV was significantly improved after the application of the hydrogel coating. The water contact angle (WCA) of PEG/PSBMA-DHV decreased to 44.0 ± 9.9°, indicating a much more hydrophilic surface compared to the uncoated DHV (WCA = 80.7 ± 8.8°) and T-DHV (64.8 ± 5.2°) (Figs. 3G, H). These characterizations indicate the PEG/PSBMA-DHV could provide favorable mechanical strength and hydrophilic surface property.Furthermore, the PEG/PSBMA-DHV also exhibited a gradual release of H2S, albeit at a slower initial rate compared to T-DHV (Fig. 3I). This can be attributed to the release of H2S from the thiourea groups in the PEG hydrogel, which has slower kinetic comparing to the cleavage of thiocarbamates in T-DHV. Notably, after long-term incubation, both the PEG-DHV and PEG/PSBMA-DHV were capable of releasing more H2S than T-DHV due to the additional TCDI crosslinking sites (Fig. 3I). The presence of the PSBMA network further contributed to the slowing down of H2S release compared to PEG-DHV, resulting in less H2S release from PEG/PSBMA-DHV within the observation period. However, it is conceivable that prolonged time may lead to a further increase in H2S release from PEG/PSBMA-DHV. This observation underscores another advantage of the PEG/PSBMA hydrogel coating, which offers the potential for a continuous, slow release of H2S to regulate the immune microenvironment for an extended period.PEG/PSBMA-DHV increased thermal stability and enzymatic degradation resistanceThe introduction of the PEG/PSBMA double network hydrogel coating is expected to enhance the thermal stability of DHV and prevent enzymatic degradation. Therefore, the thermal shrinkage temperature of the samples was determined using DSC (Differential Scanning Calorimetry). The results, as shown in Fig. 3J, indicated that the thermal shrinkage temperatures of T-DHV and PEG-DHV were similar to those of clinically used GV and significantly higher than those of the raw DHV. This finding further supports the effective crosslinking achieved with TCDI. The incorporation of the second PSBMA network further improved the thermal stability of DHV. The thermal shrinkage temperature of PEG/PSBMA-DHV increased to 100.6 °C (Fig. 3J). Furthermore, the enzymatic degradation resistance of the samples was evaluated using collagenase treatment. As shown in Fig. 3K, GV barely degraded within seven days ( < 6.5 ± 2.1% weight loss), due to the non-degradable glutaraldehyde crosslinking. In contrast, DHV without modification degraded rapidly 31.1 ± 3.5% weight loss was already observed on the first day and almost complete degradation was revealed on day 7. All TCDI crosslinked samples showed improved stability comparing to DHV. T-DHV could retain half mass on the day 7, whereas PEG-DHV and PEG/PSBMA-DHV revealed even slower degradation rate (31.9 ± 6.6% and 23.1 ± 2.8% weight loss respectively on day 7). These results suggested that the TCDI crosslinking and hydrogel coating enhanced the thermal stability of DHVs and prevented the fast enzymatic degradation. The PEG/PSBMA-DHV could be relatively stable just after implantation and adaptively degrade over a longer regenerative period.The durable anti-calcification and anti-fouling property of PEG/PSBMA-DHVHeart valves require anti-calcification and anti-fouling properties to maintain their functionality and longevity. Calcification can stiffen the valve leaflets, leading to valve stenosis and obstruction50,53. Therefore, anti-calcification properties are essential to preserve the flexibility of heart valves, prevent blockages, and enhance durability49. Similarly, the absorption of blood proteins also hinders valve movement and promotes thrombus formation and inflammation49. Hence, anti-fouling properties is also requested to ensure proper valve function, reduce the risk of clot formation, and minimize inflammatory responses. Particularly, heart valves undergo continuous, cyclic mechanical stimulation due to the rhythmic contractions and relaxations of the heart muscle. Consequently, it is highly demanded to investigate the anti-calcification and anti-fouling properties even after intensive repetitive motion.For this purpose, we investigate the anti-calcification and anti-fouling property of PEG/PSBMA-DHV before and after fatigue test. The sample strips (length of 10 mm and width of 5 mm) were cut from the thickest central area of the valve belly (Fig. 4A). The thickness of the samples was measured by vernier caliper before the cyclic tensile tests to ensure the consistency among different samples. To simulate the calcification conditions, the samples, both before and after undergoing 500 fatigue tests, were immersed in a solution of CaCl2 for 24 h, followed by a dropwise addition of Na2HPO4 solution (Figs. 4A, B). The calcium deposition on the DHV’s ventricular layer was characterized using SEM and Atomic Absorption Spectrometry (AAS). PEG/PSBMA-DHV exhibited the lowest level of calcification (2.1 ± 0.6 ug/mg), confirming the desired function of the double network hydrogel coating. Remarkably, T-DHV also exhibited reduced calcification (5.2 ± 1.0 ug/mg) compared to commercially used GV (7.4 ± 1.5 ug/mg), further demonstrating the superiority of TCDI crosslinking over glutaraldehyde crosslinking. Importantly, when DHV samples underwent continuous mechanical cyclic stimulation, the degree of calcification for DHV (7.2 ± 1.1 ug/mg), GV (12.4 ± 0.7 ug/mg), T-DHV (11.2 ± 1.3 ug/mg), and PEG-DHV (7.9 ± 1.4 ug/mg) significantly increased by 1–3 times after 500 stretching cycles. However, there was no significant difference in the degree of calcification observed for PEG/PSBMA-DHV (2.3 ± 0.9 ug/mg) after the fatigue test (Fig. 4D). SEM images exhibited a similar trend (Fig. 4B). These results indicate that the hydrophilic hydrogel modification can significantly reduce calcium deposition on the valve surface, and the anti-fatigue property provided by PSBMA facilitate long-term protection even after cyclic mechanical stimulation.Fig. 4: Evaluation of durable anti-calcification and anti-fouling property.A Schematic diagram of fatigue test (created with BioRender.com). B SEM observed calcium deposition on sample surface before and after fatigue test. C Laser confocal microscopy observed sample surface protein (BSA-TXO) deposition before and after fatigue test, and the TXO group can be excited by 405 nm to emit blue fluorescence. D Quantitative analysis of calcium content in samples before and after fatigue test by atomic absorption spectrometer (n = 3). E Quantitative analysis of BSA content in samples before and after fatigue test by BCA method (n = 3). Data were expressed as mean ± SD, *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001. n means the number of independent experiments.The in vitro protein adhesion test was performed by incubating bovine serum albumin (BSA) with DHV for 12 h at 37 °C. The amount of protein absorbed on DHV was quantified by determining the BSA concentration in the solution before and after incubation with DHV samples by bicinchoninic acid (BCA) method (Fig. 4E). For visualization, the BSA was further labeled with thioxanthone (TXO) and imaged using a confocal laser scanning microscope (Fig. 4C). The results indicated that raw DHV exhibited significant protein adhesion, particularly after the fatigue test. Although the crosslinking by both glutaraldehyde and TCDI did not initially reduce protein adhesion, the amount of absorbed protein after the fatigue test was remarkably reduced due to increased mechanical stability. PEG/PSBMA-DHV showed the lowest protein adhesion both before and after the fatigue test (500 time stretching), indicating a durable anti-fouling effect after coating with double network hydrogel.In vitro hemocompatibility and biocompatibility of PEG/PSBMA-DHVExcellent blood compatibility is the most important criteria for blood-contacting devices such as heart valves. Cell damage, protein adhesion, platelet activation and other phenomena caused by blood-contacting devices can lead to serious consequences such as inflammation and thrombosis54. Thus, superior hemocompatibility is highly demanded for ideal DHV materials. As an endogenous gas signaling molecule, H2S can inhibit platelet aggregation and activation, thus inhibiting thrombus formation55. When combined with the efficient anti-fouling ability of PEG/PSBMA, the coated DHV should further improve its anti-thrombotic ability and achieve excellent blood compatibility. Therefore, the hemocompatibility of PEG/PSBMA-DHV was evaluated from three perspectives: hemolysis, platelet adhesion and thrombosis (Fig. 5A).Fig. 5: In vitro hemocompatibilities and biocompatibility of PEG/PSBMA-DHV.A Schematic diagram of hemocompatibility tests (created with BioRender.com). ACD: Acetate citrate diethylene, which is a commonly used blood anticoagulant reagent. B Platelets (false color from yellow) adhered on the scaffolds observed by SEM. C LDH assay quantified number of platelets adhered to the scaffolds (n = 6). D Quantification of the thrombus (OD540nm) by spectrophotometric method (n = 6). E Hemolysis rates of the samples (OD450nm) by spectrophotometric method (n = 3). F General picture of hemolysis experiments at different time points and thrombogenesis of the scaffolds after incubating with whole blood. *: From left to right are DHV, GV, T-DHV, PEG-DHV, and PEG/PSBMA-DHV. G Cytotoxicity of HUVECs by MTT, indicating good cytocompatibility of all scaffolds except GV group (n = 6). H) Cytotoxicity of BMSCs by MTT (n = 5). I BMSC cultured on different samples and stained by calcein to visualize the live cells. Data were expressed as mean ± SD, *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001. n means the number of independent experiments. For box plots, the lower edge denotes the first quartile (Q1, or the 25th percentile) of the data, the central line within the box signifies the median (Q2, or the 50th percentile), while the upper edge of the box corresponds to the third quartile (Q3, or the 75th percentile). The lower whisker extends to the minimum value, indicating its range of variation, and the upper whisker reaches the maximum value, demonstrating its variability. One-way ANOVA was used for analyzing the data.The platelet-rich plasma was incubated with the samples at 37 °C for 1 h, and platelet adhesion and activation were visualized using SEM and quantified through LDH (lactic dehydrogenase) analysis. Among the samples, DHV exhibited the highest number of adherent platelets (0.34 ± 0.02 OD). In contrast, T-DHV, PEG-DHV, and PEG/PSBMA-DHV showed minimal platelet adhesion, similar to GV (0.12 ± 0.01 OD) (Fig. 5C). SEM also confirmed this trend in platelet adhesion, with DHV showing a large number of pseudopodia formation indicating platelet activation. In contrast, a small number of platelets were observed on T-DHV’s surface which remained inactive (Fig. 5B). The hydrogel coating clearly reduced platelets adhesion as shown by both PEG-DHV and PEG/PSBMA-DHV.The in vitro thrombosis test was conducted by incubating the samples with recalcified whole blood at 37 °C for 30 min. The anti-thrombotic ability was analyzed using general photographs and spectrophotometry. DHV’s surface was prone to significant thrombus formation, while GV, which is clinically used with glutaraldehyde cross-linking, exhibited less thrombus formation due to its cytotoxicity (Figs. 5D, F). T-DHV had slightly higher thrombus formation than GV but significantly lower than DHV. Thrombus formation was significantly inhibited after hydrogel coating. Both PEG-DHV and PEG/PSBMA-DHV showed lower levels of thrombosis compared to clinically used GV (Figs. 5D, F).Hemolytic properties were also analyzed quantitatively using spectrophotometry and visualized through general photographs. The hemolytic rate was determined by measuring the absorbance of hemoglobin released from erythrocytes. The hemolysis rates of DHV, GV, T-DHV, PEG-DHV, and PEG/PSBMA-DHV were found to be below 2% after 1 h with no significant difference (Fig. 5E), which is consistent with the observations in general photographs (Fig. 5F).Glutaraldehyde cross-linked bioprosthetic valves have a limited lifespan due to the presence of toxic aldehyde groups that hinder cellularization. Therefore, tissue-engineered valves need to exhibit excellent biocompatibility to facilitate recellularization and accelerate endothelialization. HUVECs (Human umbilical cord blood endothelial cells) and BMSCs (Bone marrow mesenchyml stem cell) were used to evaluate the cytotoxicity of PEG/PSBMA-DHV. As depicted in Fig. 5G & H, the cell viability of HUVECs and BMSCs co-cultured with T-DHV, PEG-DHV, and PEG/PSBMA-DHV exceeded 90%, with no significant differences observed among the samples. This suggests that TCDI cross-linking and PSBMA copolymerization in DHV do not induce cytotoxicity. In contrast, GV resulted in the death of nearly half of the cells, exhibiting cell viability below 60%. Furthermore, the staining of the live cells after 48 h of co-culture indicated that all samples, except GV, maintained good cell morphology of BMSCs (Fig. 5I). These results indicate that TCDI and SBMA copolymerization cross-linked DHV demonstrated better biocompatibility compared to GV.Overall, the results presented above support the significantly improved biocompatibility provided by TCDI crosslinking compared to the clinically used glutaraldehyde crosslinking. Additionally, T-DHV demonstrated the ability to reduce platelet activation and thrombus formation compared to raw DHV, which is a favorable outcome. Moreover, the PEG/PSBMA hydrogel coating further reduced platelet adhesion and prevented thrombosis. These findings, along with the low hemolysis rate and excellent biocompatibility with HUVECs and BMSCs, highlight the superior hemocompatibility and biocompatibility of both T-DHV and PEG/PSBMA-DHV, indicating their significant potential for clinic applications.Evaluation of immunomodulatory capacity of PEG/PSBMA-DHVMacrophages, particularly the M2 phenotype, have emerged as a key focus in tissue regeneration research due to their extensive interactions with stem cells and crucial involvement in all stages of tissue repair56. In cardiovascular diseases (CVDs), the macrophage phenotype plays a pivotal role, with a predominance of pro-inflammatory M1 macrophages observed at the lesion site11,57,58. The resultant pro-inflammatory cytokines and oxidative stress generated by M1 macrophages can induce osteoblast-like differentiation of valve interstitial cells and subsequently lead to valve calcification11,57,59. However, monocytes/macrophages possess the ability to alter their phenotype in response to environmental changes, thereby modulating their function. H2S has been found to exert anti-inflammatory effects by promoting M2 macrophage polarization. It not only inhibits various inflammatory pathways, including NF-κB, JNK, and ERK signaling, through protein S-sulphydration but also functions as a potent cell redox regulator, regulating macrophage epigenetics and promoting mitochondrial biogenesis9. Furthermore, M2 macrophages secrete transforming growth factor-beta (TGF-β), vascular endothelial growth factor (VEGF), and fibroblast growth factor (FGF), which play critical roles in recruiting fibroblasts and endothelial cells, facilitating angiogenesis, collagen deposition, and wound contraction29,60,61. Therefore, it is expected that TCDI crosslinked DHVs, endowed by their H2S release ability, could promote macrophage M2 phenotypic polarization and subsequently accelerates valve endothelialization. In this section, flow cytometry and cell migration assays were used to evaluate the immunoregulatory capacity of PEG/PSBMA-DHV and its effect on cell migration (Fig. 6A).Fig. 6: Evaluation of immunomodulatory capacity of samples.A Schematic diagram of immunomodulatory capacity tests (created with BioRender.com). CM: Conditional medium. B Phenotypic analysis of RAW264.7 by flow cytometry (n = 6). C Statistics of the proportion of CD206 positive cells (n = 6). D-E) mRNA expression of anti-inflammatory factors was analyzed by RT-qPCR: (D) Arg 1 (n = 3); (E) IL-10 (n = 3). F TGF-β1 content was detected by ELLSA method (n = 3). G Scratch migration experiment of HUVECs (n = 6). H HUVECs mobility statistics (n = 6). Data were expressed as mean ± SD, *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001. n means the number of independent experiments. For box plots, the lower edge denotes the first quartile (Q1, or the 25th percentile) of the data, the central line within the box signifies the median (Q2, or the 50th percentile), while the upper edge of the box corresponds to the third quartile (Q3, or the 75th percentile). The lower whisker extends to the minimum value, indicating its range of variation, and the upper whisker reaches the maximum value, demonstrating its variability. One-way ANOVA was used for analyzing the data.To investigate the immunomodulatory function of the DHVs, we characterized the cell phenotype of RAW264.7 macrophages co-cultured with the samples for 48 h using flow cytometry (Fig. 6A). As depicted in Figs. 6B, C, the CD206 positivity of RAW264.7 cells co-cultured with T-DHV (37.05 ± 3.85%), PEG-DHV (32.19 ± 2.05%), and PEG/PSBMA-DHV (27.05 ± 1.71%) was significantly higher compared to DHV (3.86 ± 0.60%) and GV (4.85 ± 0.32%). Furthermore, we assessed the secretion of cytokines by RAW264.7 cells using enzyme-linked immunosorbent assay (ELISA) and quantitative reverse transcription PCR (RT-qPCR). TGF-β1, Arg1, and IL-10 are characteristic cytokines secreted by M2 macrophages, known for their crucial role in anti-inflammatory responses62. The relative mRNA levels of Arg1 and IL-10 were markedly upregulated in macrophages treated with these three samples, compared to DHV and GV (Fig. 6D & E). Additionally, ELISA results showed a significant promotion of TGF-β1 expression by T-DHV, PEG-DHV, and PEG/PSBMA-DHV (Fig. 6F). These findings provide evidence that T-DHV, PEG-DHV, and PEG/PSBMA-DHV effectively induce macrophage polarization toward the M2 phenotype. These observations align with the H2S release rate discussed in Fig. 2, further supporting the notion that the immunomodulatory ability is attributed to the H2S release facilitated by TCDI crosslinking. To further validate the immunomodulatory capacity independent of the modifiers, we treated RAW264.7 cells separately with 4-arm-PEG-NH2 and SBMA monomers, which resulted in no significant change in the macrophage phenotype. The CD206 positive ratios were measured as 3.73 ± 0.22% and 3.475 ± 1.00%, respectively (Supplementary Figs. 6A and C). Furthermore, no H2S production was detected from the DMEM medium with 4-arm-PEG-NH2 and SBMA monomers (Supplementary Fig. 6B). These additional experiments further confirm that the regulation of macrophage phenotype in this system is achieved through H2S release enabled by TCDI crosslinking.Furthermore, to enhance the H2S release, we added 0.8 M cysteine to the RAW264.7 cells co-cultured with 4-arm-PEG-NH2 monomer, SBMA monomer, DHV, GV, T-DHV, PEG-DHV, and PEG/PSBMA-DHV, respectively. Flow cytometry results revealed that the DHVs with TCDI crosslinking (T-DHV, PEG-DHV, and PEG/PSBMA-DHV) promoted enhanced macrophage polarization towards the M2 phenotype. In contrast, the rate of CD206-positive macrophages in the remaining samples did not change significantly (Supplementary Fig. 7A and B). These findings suggest that free cysteine in blood stream could possibly enhance the immune regulation ability due to the accelerated H2S release.In addition to evaluating the macrophage phenotype, we also investigated the recruitment and migration capabilities of differentiated macrophages towards HUVECs and BMSCs. We co-cultured the DHVs with macrophages for 48 h, and the resulting conditioned media were used to stimulate HUVECs and BMSCs. As shown in Figs. 6G, H, the conditioned media derived from T-DHV, PEG-DHV, and PEG/PSBMA-DHV significantly promoted HUVECs migration, with respective mobilities of 67.23 ± 15.03%, 61.67 ± 8.57%, and 58.43 ± 11.81% after 48 h of culture (Fig. 6H). Additionally, transwell-based cell invasion experiments also demonstrated that TCDI cross-linked DHVs promoted BMSCs recruitment (Supplementary Fig. 8). Overall, our findings indicate that the TCDI cross-linked DHVs induce macrophage polarization towards the M2 phenotype, resulting in cytokine secretion that can effectively recruit stem cells from peripheral blood and promote the migration of endothelial cells from peripheral blood vessels. These advantages are highly desirable for accelerating valve endothelialization and tissue regeneration after implantation.In vivo degradation and immunomodulatory ability of the DHVs after subcutaneous implantationA rat subdermal implantation model was established to assess the in vivo histocompatibility of the DHVs (Fig. 7A). The histological staining results are presented in Fig. 7B, and ImageJ was used to make statistics on the section area occupied by residual collagen in different sections, which was used to reflect the situation of collagen. The DHV was predominantly occupied by cells, with minimal residual collagen observed in most areas, indicating the highest degradation ratio of 69.12 ± 4.07% (Figs. 7B and D). In comparison, the collagen degradation ratios of T-DHV, PEG-DHV, and PEG/PSBMA-DHV were 29.61 ± 5.24%, 29.84 ± 7.92%, and 32.91 ± 3.72%, respectively, which demonstrated a significant deceleration in degradation compared to DHV (Fig. 7D). Notably, there was no statistical difference in degradation rate between these three samples and the GV group (24.72 ± 2.93%). Furthermore, von Kossa staining revealed the absence of calcification in all samples except the GV group, where an abundance of calcium nodules was observed throughout the entire layers.Fig. 7: In vivo degradation and immunomodulatory ability of the samples after subcutaneous implantation.A Schematic diagram of subcutaneous implantation (created with BioRender.com). B After 28 days of subcutaneous embedding, sections were stained with HE, Masson, EVG and von Kossa. HE for cell and ECM, Masson stain for collagen, EVG stain for elastin, and von Kossa stain for calcification. * indicates the position of the heart valve (n = 3). C Samples were taken at day 7, day 14 and day 28, and CD163 (green) / iNOS (red) /DAPI (blue) staining was performed on the sections to evaluate the immunomodulatory capacity of the sample (n = 3). D The degradation rate of sample was calculated by calculating the percentage of collagen in the Masson stain (n = 6). E The ratio of CD163/DAPI double-positive cells to DAPI-positive cells was calculated by Image J, representing the proportion of M2 phenotype macrophages in the sample (n = 6). Data were expressed as mean ± SD, *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001. n means the number of biologically independent samples. For box plots, the lower edge denotes the first quartile (Q1, or the 25th percentile) of the data, the central line within the box signifies the median (Q2, or the 50th percentile), while the upper edge of the box corresponds to the third quartile (Q3, or the 75th percentile). The lower whisker extends to the minimum value, indicating its range of variation, and the upper whisker reaches the maximum value, demonstrating its variability. One-way ANOVA was used for analyzing the data.We also investigated the immunomodulatory capacity of the samples in vivo, as shown in Fig. 7C. In the GV group, no significant cell infiltration was observed at any time point due to the cytotoxicity of glutaraldehyde. In the DHV group, only a small number of M2 phenotype macrophages were present over the observation period. Encouragingly, T-DHV, PEG-DHV, and PEG/PSBMA-DHV exhibited a substantial presence of M2 phenotype macrophages after 28 days. Particularly, T-DHV demonstrated 34.05 ± 3.31% M2 macrophages 7 days after implantation, and the prevalence of M2 macrophages gradually increased over time due to the rapid H2S release facilitated by the thiocarbamate groups on the surface of T-DHV. In contrast, PEG-DHV and PEG/PSBMA-DHV initially showed low amount of M2 macrophages at day 7, but the amount of M2 macrophages were increased to the comparable level with T-DHV after 28 days. This phenomenon is consistent with the in vitro DHV degradation tests (Fig. 3K). The PEG-DHV and PEG/PSBMA-DHV exhibited slower degradation rate comparing to T-DHV due to the additional hydrogel layer and the slower cleavage kinetic of the predominant thiourea linkage. This slow gradation prevented cell infiltration, but is beneficial to provide sufficient anti-calcification and anti-thrombotic function at the initial stage after implantation. As the implantation time extended, the gradual degradation facilitated continuous release of H2S and resulted in similar level of M2 macrophages infiltration comparing to DHV after 4 weeks.The results highlighted the ability of TDCI crosslinked valves to prevent rapid degradation of the collagen component and induce polarization of macrophages towards the M2 phenotype., showcasing promising biocompatibility and the potential to promote in situ regeneration of heart valves. Moreover, considering the superior anti-calcification and anti-fouling effect particularly after fatigue test as discussed in Fig. 4, PEG/PSBMA-DHV would be more attractive for in vivo application under mechanical challenges.Overall performance of PEG/PSBMA-DHV under hemodynamic environmentTraditional animal models, such as subdermal implantation or abdominal wall repair models, have limitations in simulating the in vivo environment of heart valves. These models often do not involve direct blood contact, making it difficult to assess the recellularization capability of the valves. In this study, we addressed these challenges by sewing heart valves into hollow tubes and transplanting them into the abdominal aorta of rats, partially simulating the hemodynamics of heart valves (Fig. 8A). This animal model allowed us to evaluate not only inflammation and tissue remodeling but also blood compatibility and endothelialization of the valves. While this model does not completely replicate the microenvironment of the valve in vivo, it represents a significant improvement over traditional small animal experimental models and holds great promise in evaluating the performance of tissue-engineered heart valves48.Fig. 8: Rapid endothelialization of the scaffolds under hemodynamic environment.A Schematic diagram of abdominal aorta transplantation (created with BioRender.com). B General views and doppler ultrasound images of the scaffolds implanted in the rat abdominal aorta. The model was established successfully, and DHV scaffold dilated significantly like aneurysm while the other two remained tubular structure (n = 3). C Immunofluorescence staining of endothelial cells (CD31 + , green) and all the infiltrated cells (DAPI, blue) after 7 days, 14 days and 28 days showing the endothelialization process of the scaffolds. The * represents the position of blood flow. PEG/PSBMA-DHV scaffold presented the fastest speed and highest degree of endothelialization among groups (n = 3).The performance of double network hydrogel coated PEG/PSBMA-DHV under the hemodynamic environment was evaluated in rat abdominal aorta implantation model, and compared with raw DHV and clinical used GV. Figure 8B presents photographic images of the valves after being implanted for 4 weeks. Due to the rapid degradation of DHV in vivo, the naked DHV exhibited significant dilation resembling an aneurysm, while the other samples maintained their tubular structure. Doppler ultrasound examination confirmed that all valves remained unobstructed during the study period (Fig. 8B). To assess the endothelialization capacity of the materials, we collected samples at 7 days, 14 days, and 28 days and performed CD31 fluorescence staining (green). Similar to the results in subcutaneous embedding models (Fig. 7C), no significant signs of cell infiltration were observed for PEG/PSBMA-DHV after 7 days implantation (Fig. 8C), due to the slow degradation of the thiourea based hydrogel coating. However, extensive endothelial cell coverage (approximately 50%) could be already observed after 14 days associated with the hydrogel degradation. Remarkably, at 28 days, complete endothelial cell coverage was found on the surface in contact with the blood. In contrast, the raw DHV and GV showed no signs of endothelialization at any of the observation time points, with minimal cell infiltration observed throughout the GV due to toxicity. These results indicate that the PEG/PSBMA-DHV possesses excellent blood compatibility while effectively accelerating valve endothelialization.We also investigated the degradation, calcification, immunomodulatory, and recellularization capabilities of the materials under a hemodynamic environment. As shown in Fig. 9 the histological results of sample degradation, remodeling, and calcification after 4 weeks were consistent with the outcomes of the subcutaneous implantation model mentioned above. PEG/PSBMA-DHV showed certain degradation and cell infiltration capacity, but the rate was much slower than DHV. Compared with GV, PEG/PSBMA-DHV does not have calcification and supports cell infiltration. Inflammation was further visualized using immunofluorescence imaging, focusing on the macrophage phenotype. After 28 days of implantation, DHV showed almost no macrophages, while the GV still had a significant number of M1 phenotype macrophages with a pro-inflammatory phenotype (Fig. 9). Furthermore, due to the non-degradability of the GV causing a foreign body reaction, the macrophages were present in the thick fibrous tissue surrounding the sample rather than infiltrating into the valve. In contrast to the DHV and GV, PEG/PSBMA-DHV, with its cysteine-responsive H2S release ability, exhibited a large number of M2 subtype macrophages with tissue regeneration capabilities in both the valve and surrounding tissue. Additionally, due to the controlled degradation of the covalent bonds formed by TCDI cross-linking, macrophages were found infiltrated into the valve matrix (Fig. 9).Fig. 9: Overall performance of the samples after 4-week implantation.Masson staining displayed ECM collagen fiber; von Kossa staining displayed calcification of the scaffolds; Immunofluorescence staining of iNOS (for M1 subtype, green) and CD163 (for M2 subtype, red) showed inflammatory infiltration of the scaffolds; Immunofluorescence staining of CD31 (for endothelial cells, green) and vimentin (for interstitial cells, purple) showed cellularization of the samples; Collagen type I (Col-I) was stained by immunohistochemistry to reflect ECM remodeling. All the samples exhibited different degrees of cellularization, degradation and remodeling except the GV group (n = 3). The * represents the position of blood flow.To verify the relationship between M2 phenotypic macrophages and tissue remodeling, we performed CD31 and vimentin (VIM) fluorescence staining on valve endothelial cells and valve stromal cells, respectively. The results shown in Fig. 9 revealed no cell infiltration or evident CD31+ and VIM+ cells in the GV. In the DHV, there was no complete endothelial cell layer coverage on the blood contact side, and VIM+ cells were observed within the valve tissue. However, PEG/PSBMA-DHV exhibited complete coverage of endothelial cell layers and the presence of VIM+ cells, indicating successful regeneration of valve endothelial cells and valve interstitial cells within 28 days. Collagen type I, the main component of the valve ECM, was also found regenerated, as observed through immunohistochemical staining (Fig. 9). The newly deposited collagen further demonstrated the ECM remodeling process of the materials. These results revealed that PEG/PSBMA-DHV underwent degradation and showed promising tissue remodeling, while the GV did not exhibit cellularization and DHV only permitted cell infiltration but lacked endothelialization.Overall, the degree of recellularization and tissue regeneration correlated with the content of M2 phenotype macrophages in the heart valve. PEG/PSBMA-DHV effectively induced macrophage polarization towards an anti-inflammatory and pro-tissue regeneration phenotype, while also displaying good anti-calcification and anti-thrombotic properties. Therefore, it presented favorable endothelialization, recellularization and tissue remodeling capability, which is highly challenging for heart valve development. Moreover, the bio-controllable degradation and immunoregulatory properties of the TCDI crosslinker distinguish it from traditional covalent crosslinkers, which is essential for the remarkable cell infiltration and tissue remodeling observed in vivo. It resolves the issues of unstable crosslinking and poor mechanical strength encountered with non-covalent crosslinkers, while overcoming the problems associated with non-degradable covalent crosslinkers that impede cell infiltration and hinder tissue regeneration.
