Fig. 1. Schematic diagram of integration and regeneration of bone-tendon interface by using a kartogenin- and polydopamine-functionalized silk fibroin nanofibrous scaffold. In the early stage of healing, the PD coating on the nanofiber surface scavenges reactive oxygen species and reduces the inflammatory response. In the middle and late stages of healing, kartogenin, released from nanofiber, promotes fibrochondrogenic differentiation. During the entire healing process, the nanofibrous scaffold provides attachment points for cell proliferation and differentiation. Multi-functionalized nanofibers create an excellent microenvironment for the regeneration of fibrocartilage at the interface, which promotes the integration and regeneration of bone-tendon interface.

Fig. 2. Fabrication and characterization of the nanofibers. (A) The schematic diagram of fabricating functionalized nanofibrous scaffold. (B) Typical 1H NMR spectra of KGN, PD-SF, and KGN-PD-SF nanofibers show successful cross-link formation of KGN to PD-SF. The mark (*) on the 1H NMR spectra of KGN-PD-SF indicates the resonance peaks (7.3-7.9 ppm) derived from KGN. (C) Scanning electron micrographs (top row) of SF, PD-SF, and KGN-PD-SF nanofibrous scaffolds. Bars represent 2 μm. Wetting behavior of a water droplet (bottom row) on different scaffolds. (D) Water contact angle measurements of SF, PD-SF, and KGN-PD-SF nanofibrous scaffolds. Modification of PD decreases water contact angle. (E) The strain-stress curves, (F) Young’s modulus and (G) elongation at breaking of different nanofiber scaffolds. Data are presented as the mean ± SD. *P < 0.05.

Fig. 3. Antioxidant activity of the nanofibrous scaffolds and the characterization of KGN released from the functionalized nanofibers. (A) Photographic images of the DPPH solution after reaction with nanofibers. (B) DPPH scavenging efficiency of nanofibrous scaffolds. DPPH scavenging efficiency of nanofiber scaffolds with (C) different concentration and (D) different incubation time. (E) Biodegradation behavior of nanofibrous scaffolds in PBS containing protease XIV (1.0 U/mL) at 37 °C. The percentages indicate the weight of scaffold after degradation divided by that before degradation. (F) In vitro cumulative release of KGN from KGN-PD-SF scaffold. Data are presented as mean ± SD. * indicates statistically significant differences (P < 0.05).

Fig. 4. Adhesion, viability, and proliferation of BMSCs on SF, PD-SF, and KGN-PD-SF scaffolds. (A) Fluorescence staining of BMSCs on scaffold following a 1, 3, or 7 d culture (bars represent 200 μm); living BMSCs were labeled with calcein-AM (green fluorescence), whereas dead BMSCs were labeled with EthD-1 (red fluorescence). (B) Quantification of cell adhesion (ratio of living cells on scaffolds). The data are shown as the mean ± SD (n = 3). (C) Proliferation of BMSCs on scaffolds following a 1, 3, or 7 d culture. Tissue culture plate (TCP) as a control group at 7 d. The data are shown as the mean ± SD (n = 3).

Fig. 5. Intracellular ROS scavenging activity of the SF, PD-SF, and KGN-PD-SF nanofiber scaffolds in the Rosup-induced damaged BMSCs. (A) Schematic illustration showing the experimental process. (B) Representative fluorescence images of BMSCs with treatment of Rosup. Green fluorescence indicates the presence of ROS in cells observed in the bright field images. Bars represent 100 μm. (C) Average optical density of cellular fluorescence. Data are presented as mean ± SD. * indicates statistically significant differences (P < 0.05).

Fig. 6. Fibrochondrogenic differentiation of BMSCs. (A) Gene expression analysis of key fibrochondrogenic markers assessed by qRT-PCR at day 7. Data are normalized to GAPDH and relative to SF. * P < 0.05, compared with SF; # P < 0.05, compared with PD-SF. Data presented are mean ± SD. (B) Immunofluorescence staining against key fibrochondrogenic markers (TNMD and Col II) in the BMSCs after 7 d culture on nanofibers. TNMD was labeled as green, Col II was labeled as red, and nucleus (blue) was stained with DAPI. Scale bar: 50 μm.

Fig. 7. Representative immunofluorescence images of inflammatory markers (COX-2 and IL-1β) within the bone tunnel from SF, PD-SF, and KGN-PD-SF groups at 1 week after surgery. COX-2 was labeled as red, IL-1β was labeled as green, and nucleus (blue) was stained with DAPI. Scale bar: 200 μm.

Fig. 8. Histological characterization of the SF group, PD-SF group, and the KGN-PD-SF group at 4 and 8 weeks after surgery. (A) Representative photomicrographs of specimens at the interface with H&E staining at 4 and 8 weeks after surgery (Scale bars: white, 50 μm). (B) Results of picrosirius red staining evaluation of the SF group, the PD-SF group, and the KGN-PD-SF group at 4 and 8 weeks after surgery. Images observed under normal condition. (C) Interface width analysis with H&E-stained slides. Smaller interface width indicates better osteointegration to the tendon. (D) Histological evaluation according to the Mankin scoring system at 8 weeks after surgery. Data are presented as mean ± SD. * indicates statistically significant differences (P < 0.05). B, bone; IF, interface; T, tendon.

Fig. 9. Integration and biomechanical function of tendon to bone in vivo. (A) Schematic diagram of the application of nanofibrous scaffold in ligament reconstruction model. (B) Gross morphology of cartilage surface of femur. (C) Biomechanical testing for tendon-to-bone integration of the SF group, the PD-SF group, and the KGN-PD-SF group at 4 and 8 weeks after surgery. The normal knee joint serves as a baseline for comparison. * indicates statistically significant differences (P < 0.05).