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

Despite orientationally variant tears of the meniscus, suture repair is the current clinical gold treatment. However, inaccessible tears in company with re-tears susceptibility remain unresolved. To extend meniscal repair tools from the perspective of adhesion and regeneration, we design a dual functional biologic-released bioadhesive (S-PIL10) comprised of methacrylated silk fibroin crosslinked with phenylboronic acid-ionic liquid loading with growth factor TGF-β1, which integrates chemo-mechanical restoration with inner meniscal regeneration. Supramolecular interactions of β-sheets and hydrogen bonds richened by phenylboronic acid-ionic liquid (PIL) result in enhanced wet adhesion, swelling resistance, and anti-fatigue capabilities, compared to neat silk fibroin gel. Besides, elimination of reactive oxygen species (ROS) by S-PIL10 further fortifies localized meniscus tear repair by affecting inflammatory microenvironment with dynamic borate ester bonds, and S-PIL10 continuously releases TGF-β1 for cell recruitment and bridging of defect edge. In vivo rabbit models functionally evidence the seamless and dense reconstruction of torn meniscus, verifying that the concept of meniscus adhesive is feasible and providing a promising revolutionary strategy for preclinical research to repair meniscus tears.

Glycidyl methacrylate and deuteroxide were purchased from Sigma-Aldrich.Lithium bromide was obtained from Macklin.Calcein-AM/PI double staining kit and Cell Counting Kit-8 (CCK-8) were purchased from Dojindo and Beyotime, respectively.Phosphate buffer solution and Trizol reagent were obtained from Cellmax and Invitrogen, respectively.

Cell experiments
L929 cell lines were purchased from iCell Bioscience (Shanghai, China) and derived from mice.L929 cell lines were authenticated by the supplied using Short Tandem Repeat test.
The rabbit meniscus cells were extracted from the 16-week-old New Zealand white rabbit and cultured with F12 medium.The cell line sources did not take gender into account.
Each 150 μL precursor solution formed hydrogel into each pore of a 24-well plate.The 10000 rabbit meniscus cells or L929 fibroblasts were cultured in immersed medium or on the hydrogels.The cells were observed through the live/dead staining on Day 1, Day 3 and Day 7. Live death assay and immunofluorescence images was recored by inverted microscope (Nikon-LV150N, Nikon, Japan).The 5000 rabbit meniscus cells were cultured in each pore of 96-well plates.The CCK-8 assay was performed to determine the cell viability at Day 1, Day 3, and Day 7. 50,000 rabbit meniscus cells were treated with 8 kinds of growth factors, including BMP7, PDGF-AB, IGF-1, BMP2, BMP2, TGF-β1, bFGF, CTGF and TGF-β3.After 3 days, immunofluorescence was used to verify the secretion of collagen 1 and collagen 2.About 40,000 rabbit meniscus cells were encapsulated in 200 μL S-Gel and S-PIL10+GF groups.After the culture of 14d, real-time polymerase chain reaction (PCR) was performed to detect the meniscus-related expression (Sox9, Col2a1 and ACAN).After the culture of 14d, real-time quantitative polymerase chain reaction (PCR) was performed to detect the meniscus-related expression, including type 2 Collagen (COL2A1), SRY (sex-determining region Y)-box 9 (SOX9), Agrecan(ACAN).All the primers were purchased from Beijing Tsingke Biotech Co., Ltd.used as follow: COL2A1, Forward: 5′GTC TGT GAC ACT GGG ACT GT 3′ and Reverse: 5′TCT CCG AAG GGG ATC TCA GG 3′ SOX9,Forward: 5'GGC GGA GGA AGT CGG TGA AGA A 3' and Reverse: 5′ GCT CAT GCC GGA GGA GGA GTG T 3′ ACAN, Forward: 5′ CTG CAG ACC AGG AGG TAT GTG A 3′ and Reverse: 5′ GTT GGG GCG CCA GTT CTC AAA T 3′ Controlled drug release 500 ng TGF-β1 was dissolved in 1 mL S-Gel or S-PIL10 precursor solution.Every 160 μl of solution formed hydrogel, which was then soaked in 4 mL of PBS.The 0.5 ml of liquid was taken out at each time point to determine the concentration of growth factor in the solution and meanwhile, 0.5 ml of PBS was added up to 4 mL.

Intracellular antioxidant ability of hydrogels
To investigate the intracellular oxidative stress protection of the hydrogels, we used a ROS probe DCFH-DA to assess intracellular ROS levels.Typically, L929 fibroblasts were seeded at a density of 1.5 × 10 4 cells per well in confocal dishes and incubated for 12 hours.The plates were then reinjected with a fresh medium containing the same volume of either H2O2 or H2O2 with hydrogel treatment.Next, cells were stained with DCFH-DA for 30 minutes according to the manufacturer's instructions.Finally, a confocal laser scanning microscope (A1, Nikon, Japan) was used to observe DCFH-DA fluorescence in the cells.