Multifunctional piezoelectric hydrogels under ultrasound stimulation boost chondrogenesis by recruiting autologous stem cells and activating the Ca2+/CaM/CaN signaling pathway

Abstract

Articular cartilage, owing to the lack of undifferentiated stem cells after injury, faces significant challenges in reconstruction and repair, making it a major clinical challenge. Therefore, there is an urgent need to design a multifunctional hydrogels capable of recruiting autologous stem cells to achieve in situ cartilage regeneration. Here, our study investigated the potential of a piezoelectric hydrogel (Hyd₆) for enhancing cartilage regeneration through ultrasound (US) stimulation. Hyd₆ has multiple properties including injectability, self-healing capabilities, and piezoelectric characteristics. These properties synergistically promote stem cell chondrogenesis. The fabrication and characterization of Hyd₆ revealed its excellent biocompatibility, biodegradability, and electromechanical conversion capabilities. In vitro and in vivo experiments revealed that Hyd₆, when combined with US stimulation, significantly promotes the recruitment of autologous stem cells and enhances chondrogenesis by generating electrical signals that promote the influx of Ca²⁺, activating downstream CaM/CaN signaling pathways and accelerating cartilage formation. An in vivo study in a rabbit model of chondral defects revealed that Hyd₆ combined with US treatment significantly improved cartilage regeneration, as evidenced by better integration of the regenerated tissue with the surrounding cartilage, greater collagen type II expression, and improved mechanical properties. The results highlight the potential of Hyd₆ as a novel therapeutic approach for treating cartilage injuries, offering a self-powered, noninvasive, and effective strategy for tissue engineering and regenerative medicine.

Keywords: Bioadhesiveness; Calcium ion; Chondrogenesis; Piezoelectric hydrogel; Ultrasound stimulation.

Graphical abstract

Fig. 1

Schematic illustration of PVA/PAA/CS/KNN hydrogel preparation and its synergistic effect with US-driven piezoelectric stimulation therapy in repairing cartilage defects (by Figdraw).

Fig. 2

Characterization of the prepared KNN-Hyd. (a) Photographs of liquid KNN-Hyd and solidified KNN-Hyd. (b) TEM image of the KNN nanoparticles. (c) SEM images of hydrogels with different contents of KNN. Scale bars: 20 μm. (d) Pore diameter of the hydrogels. (e) XRD patterns of KNN-Hyd. (f) FTIR spectra of the hydrogels. (g) Rheological behavior of the hydrogels. (h) Swelling ratios of hydrogels.

Fig. 3

Injectable, self-healing, adhesive and piezoelectric properties of the KNN-Hyd and electrical characterization of the nanogenerators made of Hyd₆. (a) Viscosity measurement of Hyd₆ and Hyd hydrogels. (Inset: Display of injectability of the hydrogel). (b) Time sweep testing to determine the gelation time of Hyd₆ at 37 °C. (Inset: Display of solidification of the hydrogel). (c) Strain amplitude sweep test of self-healing Hyd₆ with strains ranging from 1 % to 1000 % (1 Hz). (d) Amplitude oscillation strain test of Hyd₆ with alternating strains ranging from 1 % to 700 % for three cycles. (e) Photograph of the lap-shear test. (f) Adhesive strength of hydrogels with different contents of KNN between different substances. (g) Polarization hysteresis loops of hydrogels with different contents of KNN under the same poling electric fields. (h) Piezoelectric coefficient (d₃₃) of hydrogels with different contents of KNN as a function of the remanent polarization. (i) Schematic illustration of the electrical output of nanogenerators made of KNN-Hyd under US excitation. (j) Open-circuit voltage generated by the nanogenerator made of KNN-Hyd under US excitation (US frequency of 40 kHz, pulse width of 5 μs, pulse interval of 10 ms, and acoustic pressure of 100 kPa). (k) Short-circuit current generated by the nanogenerator made of KNN-Hyd under US excitation (US frequency of 40 kHz, pulse width of 5 μs, pulse interval of 10 ms, acoustic pressure of 100 kPa). (l) US response of nanogenerators made of Hyd₆ after implantation into a rabbit knee (US frequency of 40 kHz, pulse width of 5 μs, pulse interval of 10 ms, and acoustic pressure of 100 kPa).

Fig. 4

Biodegradable behavior and biocompatibility of KNN-Hyd and ultrasonic tolerance analysis. (a) Ion concentrations detected in the saline solution after soaking with KNN nanoparticles for different durations. (b) Time-dependent weight loss of KNN-Hyd soaked in PBS (pH 7.4, 37 °C). (c) SEM images of Hyd₆ soaked in PBS (pH 7.4, 37 °C) for different durations (0, 4, 8, and 12 weeks). Scale bars: 20 μm. (d) SEM images of the BMSCs and SMSCs seeded on Hyd₆ after 1, 3, and 5 days. Red arrows indicate filopodia. The blue arrows indicate cell adhesion. The white arrows indicate cell adhesion to the hydrogel. Scale bars: 5 μm. (e) Live/dead cell staining of the BMSCs and SMSCs cocultured with Hyd₆ after 1, 3, and 5 days. Scale bars: 100 μm (f) Schematic diagram of the BMSCs seeded on the KNN-Hyd scaffolds combined with US (by Figdraw). (g) Effect of US treatment on BMSC apoptosis on different hydrogels after 5 days. (h) Statistical analysis of the effects of US treatment on BMSC apoptosis on different hydrogels after 5 days. (i) Confocal microscopy images of the cytoskeleton of BMSCs cultured on different hydrogels after 5 days of US treatment. Red = Phalloidin and actin staining. Blue = DAPI, nucleus staining. The white arrows indicate filopodia. Scale bars: 20 μm ns p > 0.05, ∗p < 0.05, and ∗∗∗∗p < 0.0001.

Fig. 5

Piezoelectric Hyd₆ combined with US treatment induces chondrogenesis in vitro. (a) Schematic diagram of the BMSCs and SMSCs seeded on Hyd₆ scaffolds combined with US (by Figdraw). (b–d) Relative gene expression of the chondrogenic gene markers COL2A1, ACAN, and SOX9 in cultured BMSCs and SMSCs on different hydrogels after 7 days of US treatment. (e) COL2A1 visualization with immunofluorescence (red) and nuclei (blue) after 7 days of culture of BMSCs on different hydrogels subjected to US treatment. Scale bars: 50 μm. (f) SOX9 visualization with immunofluorescence (red) and nuclei (blue) after 7 days of culture of BMSCs on different hydrogels subjected to US treatment. Scale bars: 50 μm. (g) Alcian blue staining of GAGs (blue) and nuclei (pink) after 7 days of culture of BMSCs on different hydrogels subjected to US treatment. Scale bars: 50 μm. (h) Semiquantitative analysis of the mean intensity of COL2A1 and SOX9. (i) Schematic diagram of the macrophages seeded on Hyd₆ hydrogels combine with US (by Figdraw). (j) Intensity of dual iNOS/CD206 immunofluorescence staining in macrophages seeded on hydrogels and cultured for 3 days. Scale bars: 20 μm. (k) Intensity of dual iNOS/CD206 immunofluorescence staining in macrophages seeded on hydrogels and cultured for 7 days.Scale bars: 20 μm. (l) Semiquantitative analysis of the fluorescence intensity of iNOS/CD206 in macrophages seeded on hydrogels and cultured for 3 days. (m) Semiquantitative analysis of the fluorescence intensity of iNOS/CD206 in macrophages seeded on hydrogels and cultured for 7 days. ∗∗∗p < 0.001 and ∗∗∗∗p < 0.0001.

Fig. 6

Piezoelectric Hyd₆ scaffolds under US treatment recruit stem cells. (a) Schematic diagram of the hypothesis that piezoelectric Hyd6 along with US activation recruits host stem cells and promotes Ca²⁺ influx to induce chondrogenesis (by Figdraw). (b) Schematic diagram of the process by which stem cells are recruited into a hydrogel and then treated with US in a Transwell chamber (by Figdraw). (c) Counting analysis of stem cells recruited into the hydrogel after 1 and 3 days. (d) Microscopy images of the scratch assays in different groups at 0, 12, and 24 h. Scale bars: 100 μm (e) Target aggregation analysis of DIO-labeled BMSCs in vivo at different times. ∗∗p < 0.01 and ∗∗∗p < 0.001.

Fig. 7

Piezoelectric Hyd₆ scaffolds combined with US treatment induce chondrogenesis by activating the Ca²⁺/CaM/CaN signaling pathway. (a) Flow cytometry detection of the intracellular calcium content in BMSCs inside the US-activated KNN-Hyd after 3 days. (b) The ratio of Ca²⁺ in the BMSCs inside the US-activated KNN-Hyd with after 3 days. (c) Schematic diagram of the hypothesis tha piezoelectric Hyd₆ treated with US induces stem cells to activate the Ca²⁺/CaM/CaN signaling pathway, thereby enhancing cartilage regeneration (by Figdraw). (d) The Ca²⁺-CaM−CaN complex. Ca²⁺ ions are orange. CaM is yellow. The regulatory subunit of CaN is turquoise green. The catalytic subunit of CaN is blue. (e) Ca²⁺-CaM−CaN-NFATc1 complex and its core binding site. CaM is yellow.The regulatory subunit of CaN is turquoise green. The catalytic subunit of CaN is blue. NFATc1 is expressed as a magenta color. The enlarged view on the right shows the core binding site of the complex (marked in green). (f) Relative protein expression of CaM, CaN, and the chondrogenic gene markers COL2A1, ACAN, and SOX9, respectively, in stem cells inside US-activated Hyd₆ with and without the calcium channel blocker VRP after 7 days. (g) Relative protein expression of CaM, CaN, COL2A1, ACAN, SOX9 and NFATc1 from stem cells inside US-activated Hyd₆ with and without CaM inhobitor KN-93 after 7 days. (h) Relative protein expression of CaN, COL2A1, ACAN, SOX9 and NFATc1 from stem cells inside US-activated Hyd₆ with and without CaN inhibitor FK506 after 7 days. (i) Co-IP showing the binding between CaM and CaN, and between CaN and NFATc1 in BMSCs. CaM and CaN were chosen as the bait proteins. CaM, CaN, and NFATc1 were chosen as the prey proteins. β-actin is not only an internal control but also a constituent unit of the cytoskeleton. ∗∗∗p < 0.001.

Fig. 8

Piezoelectric Hyd₆ scaffolds combined with US promote cartilage regeneration in vivo. (a) Digital photographs showing the femurs of rabbits in the control, Hyd, and Hyd₆ groups with or without US activation (4–8 weeks). The red circle marks where the defect was originally created. (b) ICRS score for macroscopic cartilage evaluation with or without US activation of the knees of rabbits (4–8 weeks). (c) MR images of the femurs of rabbits in the control, Hyd, and Hyd₆ groups with or without US activation (4–8 weeks). The red circle marks where the defect was originally created. (d) H&E staining and safranin O/fast green to evaluate the newly formed articular cartilage in the control, Hyd, and Hyd₆ groups with or without US activation (4–8 weeks). Scale bars: 1 mm. Scale bar: 50 μm (magnified image). Black arrow = normal cartilage. Blue arrow = repaired tissues. Red arrow = cartilage defects. (e) ICRS histological evaluation newly formed cartilage tissues with or without US activation in rabbit knees (4–8 weeks). (f) Reduced modulus of newly formed cartilage inside the defect in the different hydrogel groups with and without US activation (4–8 weeks). Healthy normal rabbit cartilage served as a positive control. (g) Representative indentation curves for different groups after 4 and 8 weeks of treatment. ∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p < 0.001.