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Silk fibroin hydrogel adhesive enables sealed-tight reconstruction of meniscus tears – Nature Communications

Synthesis, preparation, and characterization of S-PIL precursors and adhesives

To integrate the excellent adhesion force, anti-swelling, and tough mechanical properties into a hydrogel adhesive, a unique design and material composition was put forward in this study. SFMA was methylated from silk fibroin referred to a previous study21, and PIL was synthesized through an alkylation reaction between 4-(bromomethyl) phenylboronic acid and 1-vinyl imidazole22 (Fig. 2A). The structure of tailor-made PIL was capable of three features: (1) the vinyl group in the imidazole cation as a monomer copolymerized with SFMA; (2) the structure of the imidazole salt generating hydrogen bonding and promoting the formation of β-sheet structures for silk fibroin; (3) The phenylboronic acid groups reacting with hydroxy groups in the chain of silk, mainly from tyrosine and serine, to form dynamic boronic ester bonds in the network23. As presented in Fig. 2B and Supplementary Fig. 1A–C, the designed chemical structure of SFMA and PIL were verified by 1H nuclear magnetic resonance (1H NMR) and Fourier transform infrared (FTIR). Their typical peaks were consistent with their chemical structures and functional groups, representing δ = 6.0 ppm and 5.6 ppm in SFMA spectrum for methacrylate vinyl group signals24. To construct the meniscus adhesive, the contents, and proportions of these synthesized S-PIL precursors were listed in Supplementary Table 1 and these adhesives were named S-Gel, S-PIL5, S-PIL10, and S-PIL15 depending on the dosage of PIL. SFMA and PIL were then mixed and irradiated under UV light, and the meniscus adhesive solidified within 5 s through photoinitiated radical polymerization (Fig. 2C). Next verified through rheological testing and under the UV irradiation (405 nm, 30 mW/cm2), the storage modulus (G′) of the adhesive increased rapidly and intersected with the loss modulus (G″), indicating instant formation of the hydrogel adhesive, which was beneficial to repair meniscus tears in-situ (Fig. 2D). Moreover, the addition of PIL did not significantly affect the gel time of the different hydrogel adhesives (Supplementary Fig. 2). Interestingly, the increasing PIL content in the hydrogel adhesives resulted in an increase in the G′ value from 35.10 kPa to 95.81 kPa, which could be attributed to the interactions between SFMA and PIL molecules and restrained movement of polymer chains in the hydrogel network25. To investigate these multiple interactions, the results of X-ray photoelectron spectroscopy (XPS) shown in Fig. 2E and Supplementary Table 2 showed a 2.38% increase in the molar percentage of B1s, suggesting the incorporation of PIL into the SFMA hydrogel network. In high-resolution XPS spectra of S-PIL10 (Fig. 2F, Supplementary Fig. 3A, B), the new signals of B-OH, B-O-C, B-C, and C=N were observed, indicating the presence of dynamic interactions between the hydroxyl groups on SFMA and the phenylboronic acid groups of PIL26. Besides, FTIR results of hydrogel adhesives were presented in Fig. 2G and Supplementary Fig. 1D, and the absorption peak at 1621 cm−1 corresponded to β-sheet structures. The secondary structure content was calculated from the amide I band, and with the addition of PIL, the molar ratio of β-sheet increased from 18% to 43% (Fig. 2H, Supplementary Fig. 4AC). Figure 2I illustrated the anatomical networks between SFMA and PIL, including these interactions between SFMA and PIL (previously mentioned covalent bonds and abundant hydrogen bonds), and the formation of many β-sheet structures, which could be attributed to the Hoffmeister effect of PIL on silk fibroin27,28. Moreover, SEM images (Fig. 2J and Supplementary Fig. 5) visualized the microporous structures in these hydrogel adhesives, which could provide a favorable space for cell growth and exchange of nutrition29. With the increase of PIL amount, the pore size becomes smaller (from 52.53 to 32.59 μm) in Supplementary Fig. 6, which could be explained that more PIL would have more interactions with SFMA and form more secondary structures and networks. Energy dispersive spectroscopy (EDS) mapping identified the presence of B element in the synthesized hydrogel adhesives, further confirming the successful construction of S-PIL hydrogel.

Fig. 2: Preparation and characterization of S-PIL precursor and hydrogel adhesives.
figure 2

A Synthesis reaction of methacrylated silk fibroin (SFMA) and PIL. B 1H NMR spectra of PIL. C S-PIL gelation after UV light. D Rheology characterizations and E XPS full spectrum of S-Gel with the different concentrations of PIL. F B1s XPS signal of S-PIL10. G Amino II of S-Gel with the different concentrations of PIL in FTIR spectra. H Quantitative analysis of secondary structures of S-PIL10 with Gaussian curve fitting in FTIR spectra. I Multiple interactions between SFMA and PIL in the network. J SEM image and corresponding EDS elemental mapping of S-PIL10. The SEM characterization experiments were conducted three times independently.

Adhesion performance and mechanical properties of S-PIL series

Based on these verified multiple interactions between SFMA and our tailor-made PIL, the amplification in adhesion strength and mechanical properties could be proposed. Hence, we moved to the experimental evaluation of the adhesion performance according to modified lap shear strength test (ASTM F2255) presented in Fig. 3A. Meniscus tissue slices were prepared and adhered through synthesized meniscus adhesive, and the resultant adhesive shear strength increased from 27.89 kPa to 113.37 kPa with the addition of PIL (Fig. 3B). Standard test methods for strength properties (ASTM F2255: Standard Test Method for Strength Properties of Tissue Adhesives in Lap Shear by Tension Loading and F2258: Standard Test Method for Strength Properties of Tissue Adhesives in Tension) were further performed. The adhesive shear strength and tensile strength of S-PIL10 were 181.81 and 170.62 kPa (Supplementary Fig. 7A, B), respectively, which were approximately 7 and 8 times larger than the commonly used Fibrin (Supplementary Fig. 8A), and the shear adhesion of the meniscus adhesive even allowed to lift a bucket (å 5 kg) (Supplementary Movie. 1). Two other ionic liquids (1-ethyl-3-methylimidazolium bromide and 1-vinyl-3-ethylimidazolium bromide named IL1 and IL2) similarly enhanced the adhesive shear strength when added to the SFMA hydrogel (Supplementary Fig. 8B–D), however, the final adhesive ability was still lower than that of S-PIL10, highlighting the superiority of tailor-made PIL, which could be attributed to their fewer interactions with SFMA because IL1 didn’t possess the vinyl group and IL2 didn’t have the phenylboronic acid groups in comparison with PIL. Moreover, anti-swelling properties are crucial for ensuring tight adherence to torn meniscus tissue for an extended period of time. As shown in Fig. 3C, S-PIL10 retained its original shape and volume after 14 days, while S-Gel presented significant swelling after only 4 h. Correspondingly, the swelling ratio decreased from 2.911 (S-Gel) to 1.02 (S-PIL10) (Fig. 3D). Compared with other reported tissue adhesives30,31,32,33,34,35,36, our meniscus adhesive S-PIL10 had superior shear adhesive strength and durable anti-swelling properties (Fig. 3E). These excellent properties can be attributed to the multiple interactions in the polymeric network, which enhance the mechanical properties to resist swelling. Additionally, the β-sheet structures and PIL in the network are hydrophobic, which contributes to the resistance against water absorption. Besides, studies have shown that the denser and smaller the pores of the polymeric network, the higher the anti-swelling properties of the hydrogel (Supplementary Fig. 6)37. Generally, other materials, such as GelMA and PEGDA, also exhibit similar anti-swelling performance due to the incorporation of PIL (Supplementary Figs. 9 and10). Notably, the anti-swelling properties of HAMA hydrogel were significantly improved, further verifying the superiority of tailor-made PIL. Then the stress-strain curves of fabricated hydrogel adhesives about compression performance supported the maximum stress and compressive modulus increased along with the introduction of PIL (Supplementary Fig. 11). To evaluate functions of the adhesive in biophysical environment of the meniscus, a 1000-cycle loading test was performed and the hydrogel didn’t show an obvious change in shape or mechanical strength (Fig. 3F), demonstrating its robust resistance to fatigue and stability of physical strength38. And with the increasing compression cycles, S-PIL10 exhibited a decline in energy dissipation (Supplementary Fig. 12), which was similar to meniscus and its derived materials. When the torn meniscus was adhered by S-PIL10, it could lift a bottle more than 1 kg (Supplementary Fig. 13) and keep robust adhesion under twisting and water-flushing conditions (Fig. 3G). We also exposed the adhered meniscus to biomechanical force to mimic the loading conditions within the knee, and showed no significant changes (Supplementary Movie. 2). The adhesive was able to withstand biomechanical force up to 80 N, meeting the experimental demands of New Zealand white rabbits (Fig. 3H). SEM images showed that S-PIL10 integrated well with the meniscus tissue, demonstrating mechanical interlocking effect between photocured hydrogel adhesives and meniscus. Furthermore, adhesion of S-PIL10 to meniscus with radial or longitudinal tears was still steady after 60 days (Fig. 3I). Based on above excellent performance, the instant robust hydrogel adhesive could be used for the subsequent repair of meniscus tears.

Fig. 3: Adhesion performance and mechanical properties of S-PIL series.
figure 3

A Schematic diagrams of lap shear testing on the meniscus slice B Load-displacement curves and lap shear strength of S-Gel with the different concentrations of PIL bonding with meniscus slice. Data are presented as mean ± SD (n = 3 independent experiments), and exact p-value was calculated with one-way ANOVA Tukey’s multiple comparison test. C Swelling conditions and D swelling ratio of S-Gel and S-PIL10 in PBS buffer. Data are presented as mean ± SD (n = 3 independent samples). E Ashby plot of S-PIL10 compared with reported adhesives. F Macrograph and stress-strain curves of S-PIL10 with cyclic compressive loading-unloading testing for 1000 cycles. G Twisting and water-flushing after meniscus adhesion by S-PIL10. H Load-displacement curves of S-PIL gel bonding meniscus tear and SEM image of interface between S-PIL10 and meniscus. I Retention condition of S-PIL bonding meniscus tear after 60 days.

Biocompatibility and ex vivo therapeutics of meniscus adhesives

The biocompatibility was evaluated to ensure the safety of the meniscus adhesives. As presented in Fig.4 A–C, rabbit meniscus cells maintained good cell viability and apparent proliferation co-cultured with S-PIL series. On Day 7, each well in the 24-well plate was overspread with rabbit meniscus cells. Similarly, L929 fibroblasts also exhibited high cell viability (>95%) on the surface of S-PIL series (Supplementary Fig. 14). However, S-PIL15 had a slight inhibition on the proliferation of rabbit meniscus cells, as PIL itself may be not very friendly to cells, and with the increase of the concentration, S-PIL series would have negative effect on the cells. PIL was added into the hydrogel adhesive and had some interactions in the polymeric network to prevent the leakage of PIL to have good biocompatibility. Integrating the above results, S-PIL10 was considered as the balanced formulation with superior mechanics and cytocompatibility, which was optimally selected in the following meniscus adhesive study. Further, in vivo biocompatibility and degradation test of meniscus adhesives was performed subcutaneously (Supplementary Fig. 15A). Silk fibroin is a well-known biomaterial with low immunogenicity and there was almost no significant difference in inflammatory response among S-Gel and S-PIL10 groups. Inflammatory cells were observed around the implants in the first week and gradually disappeared over time (Supplementary Fig. 15B), the acute inflammation disappeared and the chronic inflammation was almost invisible, suggesting that the hydrogel adhesive and its degradation product have good biocompatibility. And the morphology of S-PIL10 showed mild degradation, but the weight of S-Gel changed significantly within 8 weeks (Supplementary Fig. 15C). S-Gel first experienced significant swelling with the maximal size achieved at day 3 and then shrank in 8 weeks, and the mass of S-PIL10 in vivo increased slightly in the beginning, possibly caused by the rising of water content, consistent with previous swelling results. Subsequently, the mass of S-Gel and S-PIL10 was reduced, probably due to a slight degradation of the hydrogel adhesives. During the degradation process of S-Gel and S-PIL10, the swelling behavior of hydrogels could not be ignored. Compared with S-PIL10, S-Gel has more body fluid in the polymeric network, which contributes to the higher mass remaining of S-Gel, and the mass remaining of S-PIL10 was lighter with less water due to its excellent anti-swelling properties. These factors jointly resulted in the observed differences in degradation process between the two hydrogels. Notably, the structure of S-PIL10 at week 2 was cracked and significant cells proliferated between inter-hydrogel space, indicating the ideal biocompatibility and repair potentials of S-PIL10 (Supplementary Fig. 15C).

Fig. 4: Biocompatibility and ex vivo therapeutics of meniscus adhesives.
figure 4

A Schematic illustration of co-culture with hydrogels and live/dead cell assays. B Cell viability of rabbit meniscus cells co-cultured with hydrogels within 7 days. Data are presented as mean ± SD (n = 3 independent cell experiments). C Cell Counting Kit 8 (CCK-8) assays of rabbit meniscus cells co-cultured with hydrogels within 7 days. Data are presented as mean ± SD (n = 3 independent cell experiments). D Alcian blue (AB) staining and immunofluorescence staining of meniscus cells. E Meniscus-related gene (Sox9, Col2a1, and ACAN) expression in the S-Gel and S-PIL10 at 2 weeks. Data are presented as mean ± SD (n = 3 independent cell experiments) and exact p-value was calculated with two-tailed student’s t-tests. F TFG-β1 release-time curves of S-Gel and S-PIL10. Data are presented as mean ± SD (n = 3 independent samples). G Inflammatory condition after meniscus tears and DDPH scavenging and PTIO scavenging of S-Gel and S-PIL10. Data are presented as mean ± SD (n = 3 independent hydrogels) and exact p-value was calculated with two-tailed student’s t-tests. Scale bars are 200 μm.

The excellent mechanical properties and biocompatibilities kept adhered S-PIL10 stable on torn meniscus, in the meanwhile, growth factors or cytokines were formulated into the hydrogel to further promote tissue self-regeneration. Multiple growth factors selected from meniscus regeneration-related studies were screened according to their promoting effects on meniscus cells (Supplementary Fig. 16). Alcian blue staining and immunofluorescence staining proved transforming growth factor-beta 1 (TGF-β1) and TGF-β3 owned significant promoting effects on the cell proliferation and collage secretion of meniscus cells. TGF-β3 has widely been reported to delay the degeneration of meniscus in previous studies39,40, while the function of TGF-β1 on meniscus was still unknown that interested us profoundly41. For this purpose, TGF-β1 was included into S-PIL10 composition to fabricate the meniscus adhesive. As presented in Fig. 4D, E, TGF-β1 loaded S-PIL10 significantly enhanced the expression of meniscus-related genes in meniscus cells compared with neat S-PIL10 group, like SOX9, Col2a1, and ACAN. A long-term healing process of meniscus tears was common, so TGF-β1 continuously released for months was advantageous. The release curve of TGF-β1 loaded S-Gel and S-PIL10 demonstrated sustained release in more than 8 weeks (Fig. 4F) due to the noncovalent interactions between TGF-β1 and these polymeric networks. Also, TGF-β1 was released more slowly in S-PIL10 compared with the release performance in S-Gel due to the addition of PIL, which could be explained by anti-swelling ability of S-PIL10 and the smaller size of pores compared with S-Gel. These results were consistent with the above-mentioned physicochemical characterizations, implying that TGF-β1 loaded S-PIL10 was beneficial for in vivo repair and prognosis. Inflammation is a natural response to meniscus tears when not untreated, that will impede the repair process. The designed meniscus adhesive ideally has antioxidant capacity to achieve inflammation control42,43. The antioxidant activity was quantitated by the 1,1-diphenyl-2-picrylhydrazyl (DPPH) and 2-Phenyl-4,4,5,5-tetraMethyliMidazoline-3-oxide-1-oxyl (PITO) assay. As presented in Fig. 4G, S-PIL10 performed better scavenging ability against different free radicals compared with S-Gel, which could be attributed to the dynamic borate ester bonds of tailor-made PIL and SFMA44. To further confirm the antioxidant activity of hydrogel at the cellular level, the cell protection efficiency of prepared hydrogels by clearing ROS was evaluated through the DCFH-DA kit. As shown in Supplementary Figs. 17 and18, the S-PIL10 group exhibited the weakest green fluorescence and their lowest intracellular ROS levels, suggesting that S-PIL10 had prominent ROS scavenging activity and could protect the cells from being oxidatively damaged in vitro. Collectively, TGF-β1 loaded S-PIL10 exhibited comprehensive biological functions for meniscus repair.

The application of S-PIL10 on in vivo multiple types of meniscus tears

Among various types of meniscus tears, radial tear in white–white zone is the most challenging one due to the lack of blood flow in the region4,45. The fabricated S-PIL10 was typically applied to radial tear and the entire experimental procedures including creating meniscus tear model and in-situ adhesive operation were shown in Fig. 5A. The tear model of the medial meniscus was penetrating and the control group received no treatment. After 8 weeks, the meniscus was collected for observation and histological examination. As presented in Fig. 5B, macroscopic views of the rabbit meniscus showed no significant tear in the S-PIL10+GF group but apparent fissure could still be visualized in other experimental groups. H&E staining and immunofluorescence staining of the meniscus presented the same results (Fig. 5C). In the control group, meniscus tears were unable to be self-healed, and significant large tears remained. The S-PIL10+GF group showed successful repair of meniscus radial tears, while the group using S-PIL10 and S-Gel+GF both showed partial healing with smaller tear size, which proved the superiority of our designed meniscus adhesive. Besides, S-PIL10+GF group had the smallest area of tear based on statistical results of histological staining (Supplementary Fig. 19A). These results could be explained as followed: (1) the strong adhesion performance of S-PIL10 can adhere the meniscus tears tightly, and the insufficient adhesion strength of S-Gel itself couldn’t be maintained for a long time, which verified the importance of mechanical properties of meniscus adhesive; (2) addition of TGF-β1 can promote the regeneration of meniscus tears in white–white zone, which is also crucial for meniscus repair.

Fig. 5: Repair effect of meniscus tears and evaluation of in vivo articular cartilage wear after 8 weeks in vivo.
figure 5

A Process of meniscus tears modeling and adhesives repair: step 1: transection of the medial collateral ligament; step 2: exposure of meniscus toward the femur; step 3: incision of the meniscus through the full thickness; step 4. In-situ sealing by S-PIL adhesives; step 5: solidification by UV and wound closure. B Macrograph of meniscus after meniscus radial tears of two months C Hematoxylin & eosin (H&E) staining and immunofluorescence on collagen I and collagen II of repaired meniscus with radial tears. D Safranin O/fast green staining (SO) of femoral condyles (FCs) and the tibial plateaus (TPs) after meniscus radial tears. For C, D the animal experiment was repeated three times independently. Scale bars in B are 5 mm, in the first row of C are 1 mm, and others are 200 μm.

The purpose of meniscus tears repair is known to prevent cartilage degeneration and osteoarthritis, thus examination of articular cartilage is important in the repair process46. As shown in Fig. 5D and Supplementary Fig. 19B, meniscus tears led to local wear and tears at the site of femoral condyles (FCs) and the tibial plateaus (TPs) in the control group after 8 weeks, while there is little sign of osteochondral damage on the FCs and TPs of the S-PIL10+GF group with the lowest Osteoarthritis Research Society International (OARSI) score (Supplementary Fig. 19C), indicating the prominent osteochondral protective effects47. Lastly, the longitudinal tear is another common type of meniscus tear48. Again, S-PIL10+GF showed the repeatable excellent performance in the longitudinal tear model, indicating its suitability for various meniscus tears (Supplementary Fig. 20). Taken together, S-PIL10 meniscus adhesive can markedly repair meniscus tears and protect cartilage against wearing.