Janus hydrogel microrobots with bioactive ions for the regeneration of tendon-bone interface

Abstract

Regenerating natural gradients of the tendon‒bone interface (TBI) is a major challenge in the reconstruction of rotator cuff tear (RCT). In this study, magnetic Janus hydrogel microrobots to match the TBI orientation during RCT reconstruction surgery are developed via a biofriendly gas-shearing microfluidic platform. Through separate loading of Mg²⁺ and Zn²⁺, the microrobots facilitate the immediate restoration and long-term maintenance of the natural mineral gradient in the TBI after implantation and alignment through magnetic manipulation. In vitro studies confirm the spatiotemporal cell phenotype modulation effects of the microrobots. In a rat RCT model, microrobots synchronously promote the bone and tendon regeneration, and the restoration of gradient tendon‒bone transition structures in the TBI. Overall, by rebuilding the Mg²⁺/Zn²⁺ mineral gradient, cell phenotype gradient and structural gradient of the TBI, magnetic Janus microrobots loaded with dual bioactive ions represent a promising strategy for promoting TBI healing in RCT reconstruction surgery.

Fig. 1. Magnetic Janus hydrogel microrobots for the regeneration of gradients in the TBI.

A Equipment and fabrication of magnetic Janus hydrogel microrobots by gas shearing. B Motion behavior of the microrobots under an external magnetic field. C Application of the microrobots in RCT reconstruction surgery. D Therapeutic effect and underlying mechanism of the microrobots in the regeneration of gradients in the TBI. Through rebuilding of the Mg²⁺/Zn²⁺ mineral gradient, spatiotemporally modulating the cell phenotypes of BMSCs and TCs, synchronously regenerating bone and tendon, and restoring the gradient tendon‒bone transition structure of the TBI, microrobots eventually promote TBI healing. MgP: Mg₃(PO₄)₂, Fe-NPs: Fe₃O₄ nanoparticles, Zn-NPs: ZnO nanoparticles, BMSCs: bone mesenchymal stem cells, TCs: tenocytes. Created in BioRender. Ding, Z. (2025) https://BioRender.com/p43d624.

Fig. 2. Fabrication and characterization of microrobots.

A Schematic showing the microrobot fabrication process. B–D CLSM images, bright-field images, and size distributions of the microrobots prepared with various nitrogen flow rates. E Cryo-SEM image of microrobots. F SEM image (left) of microrobots and EDS images (right) of Ca (pink), Zn (red), Mg (yellow) and Fe (blue). For (B–F), experiments were repeated for 3 times independently with similar results. MgP: Mg₃(PO₄)₂, Fe-NPs: Fe₃O₄ nanoparticles, Zn-NPs: ZnO nanoparticles. (A) and left pannel of (B–D) created in BioRender. Ding, Z. (2025) https://BioRender.com/p69t917. Source data are listed in the Source Data file.

Fig. 3. Motion behavior and mineral gradient rebuilding effect of microrobots.

A Schematic illustration of the motion behavior of the microrobots under an external magnetic field. B Time-lapse images of the microrobots performing rotational motion and aligning in the same direction in a Petri dish. C In an ex vivo model, the bone side of the microrobots (nontransparent side) rotated and oriented toward the bone, whereas the tendon side of the microrobots (semitransparent side) oriented toward the tendon under an external magnetic field. D The microrobots were aligned in an in vivo RCT model after magnetic manipulation. E SEM and EDS images of the microrobots on degradation days 1, 3, 14, and 21. Experiments were repeated for 3 times independently with similar results. F Release curve of metal ions from the microrobots. Data are presented as mean ± SD (n = 3 independent experiments). G Degradation profile of the microrobots. Data are presented as mean ± SD (n = 3 independent experiments). (H) Schematic illustration of the natural mineral gradient rebuilding effect of the microrobots. A, H created in BioRender. Ding, Z. (2025) https://BioRender.com/p69t917. Source data are listed in the Source Data file.

Fig. 4. Evaluation of the ability of Zn²⁺-loaded microrobots to promote tendon regeneration in vitro.

A Live/Dead staining of TCs after coculture with the indicated microspheres for 3 and 7 days. B CCK-8 assay of TCs after coculture with the indicated microspheres for 1, 3, 5, and 7 days. C Crystal violet staining of migrated TCs in the Transwell experiment. D Scratch wound healing assays of TCs at 0 and 36 h. E Quantitative analysis of migrated TCs in the Transwell experiment. F Semiquantitative analysis of the migration area in the scratch wound healing assay at 12, 24, and 36 h. G COL-I, COL-III, and TNC immunofluorescence staining (green) of TCs. The cells were costained with DAPI (blue) and phalloidin (red). H The relative mRNA expression of COL-I, COL-III, TNC, and MKX in TCs. I Schematic illustration of Zn²⁺-loaded microrobots promoting the proliferation, migration, and tenogenesis-related functions of TCs. Data are presented as mean ± SD (n = 3 biologically independent samples). B, F two-way ANOVA with Dunnett’s multiple comparisons test and adjustment applied. E, H one-way ANOVA with Dunnett’s multiple comparisons test and adjustment applied. I created in BioRender. Ding, Z. (2025) https://BioRender.com/p69t917. Source data are listed in the Source Data file.

Fig. 5. Evaluation of the ability of Mg²⁺-loaded microrobots to promote bone formation in vitro.

A Live/Dead staining of BMSCs after coculture with the indicated microspheres for 3 and 7 days. B CCK-8 assays of BMSCs after coculture with the indicated microspheres for 1, 3, 5, and 7 days. Data are presented as mean ± SD (n = 3 biologically independent samples, two-way ANOVA with Dunnett’s multiple comparisons test and adjustment applied). C Crystal violet staining of migrated BMSCs in the Transwell experiment. D Scratch wound healing assays of BMSCs at 0 and 36 h. E ALP staining of BMSCs after coculture with the indicated microspheres for 7 and 14 days in osteogenic-conditioned medium. F ARS of BMSCs after coculture with the indicated microspheres for 14 and 21 days in osteogenic-conditioned medium. G BMP-2, RUNX-2, and OCN immunofluorescence staining (green) of BMSCs. The cells were costained with DAPI (blue) and phalloidin (red). H Relative mRNA expression of BMP-2, RUNX-2, OCN, and ALP in BMSCs. Data are presented as mean ± SD (n = 3 biologically independent samples, one-way ANOVA with Dunnett’s multiple comparisons test and adjustment applied). I Schematic illustration of Mg²⁺-loaded microrobots promoting the proliferation, migration, and osteogenesis of BMSCs. I created in BioRender. Ding, Z. (2025) https://BioRender.com/p69t917. Source data are listed in the Source Data file.

Fig. 6. Radiological evaluation, biomechanical test, and gait assessment of the rat RCT model.

A Schematic illustration of the establishment of the rat RCT model, the implantation of microrobots in RCT reconstruction surgery, and the postoperative examination methods. B Micro-CT images of the humeral head used to assess new bone formation at postoperative weeks 4 and 8. C MRI images of the supraspinatus–humerus complex used to assess tendon regeneration at postoperative weeks 4 and 8 in vivo. D, E Biomechanical evaluation of TBI tissue at postoperative weeks 4 and 8. Data are presented as mean ± SD (n = 4 biologically independent samples, two-way ANOVA with Dunnett’s multiple comparisons test and adjustment applied). F Gait analysis of rats with RCT to assess forelimb functional recovery at postoperative weeks 4 and 8, and (G, H) corresponding quantitative analyses of stride length and step length. Data are presented as mean ± SD (n = 4 biologically independent samples, two-way ANOVA with Dunnett’s multiple comparisons test and adjustment applied). RCT: rotator cuff tear, CT: computed tomography, MRI: magnetic resonance imaging. A created in BioRender. Ding, Z. (2025) https://BioRender.com/p69t917. Source data are listed in the Source Data file.

Fig. 7. Histological evaluation of the regenerated TBI.

A Histological images of the regenerated TBI and enlarged views of HE, Masson, and Sirius red staining at postoperative week 4. B Heatmap showing the specific score for each item of the modified histological scoring system among the different groups, and (C) corresponding quantitative analyses of the histological scores at postoperative week 4. Data are presented as mean ± SD (n = 4 biologically independent samples, one-way ANOVA with Dunnett’s multiple comparisons test and adjustment applied). D Histological images and enlarged views at postoperative week 8. E Heatmap scores, and (F) corresponding quantitative analyses at postoperative week 8. Data are presented as mean ± SD (n = 4 biologically independent samples, one-way ANOVA with Dunnett’s multiple comparisons test and adjustment applied). Source data are listed in the Source Data file.

Fig. 8. The underlying mechanisms of osteogenesis and tenogenesis induced by the microrobots analyzed by multiplex immunofluorescence.

A Multiplex immunofluorescence of osteogenic markers in regenerated TBI tissue at postoperative weeks 4 and 8: BMP-2 (red) and OCN (green). The cells were costained with DAPI (blue) to visualize the nuclei. Upper panel: merged images; lower panel: split images. B Quantitative analyses of BMP-2-positive cells in the TBI region at postoperative week 4. C Quantitative analyses of OCN-positive cells in the TBI region at week 4. D Proportion of BMP-2-positive cells at postoperative week 8. E Proportion of OCN-positive cells at postoperative week 8. F Multiplex immunofluorescence of the regenerated tendon for COL-I (green), COL-III (red), and TNC (yellow). The cells were costained with DAPI (blue) to visualize the nuclei. Upper panel: merged images; lower panel: split images for COL-I and COL-III. G Quantitative analyses of the COL-I mean fluorescence intensity (MFI) at postoperative week 4. H Quantitative analyses of the COL-III MFI at postoperative week 4. I MFI of COL-I at postoperative week 8. J MFI of COL-III at postoperative week 8. a. u., arbitrary units. Data are presented as mean ± SD (n = 4 biologically independent samples, one-way ANOVA with Dunnett’s multiple comparisons test and adjustment applied). Source data are listed in the Source Data file.

Fig. 9. Immunomodulatory effects of microrobots during TBI healing.

A DCFH-DA staining of intracellular ROS in LPS-stimulated macrophages. B Semiquantitative analysis of the MFI of ROS. C iNOS, CD86, and CD206 immunofluorescence staining (red) of LPS-stimulated macrophages. The cells were costained with DAPI (blue) to visualize the nuclei. D–F Semiquantitative analysis of the MFI of iNOS, CD86, and CD206. a.u., arbitrary units. G The relative mRNA expression of TNF-α, CD86, and CD206 in LPS-stimulated macrophages. H Schematic illustration of the immunomodulatory effect of microrobots. Microrobots inhibited M1 polarization and promoted M2 polarization of macrophages through the synergistic ROS-scavenging effect of Mg²⁺ and Zn²⁺. I In vivo, CD86 (red) and CD206 (green) immunofluorescence staining of the rat regenerated TBI after 4 weeks of RCT reconstruction surgery. Upper panel: merged images; lower panel: split images. Data are presented as mean ± SD (n = 3 biologically independent samples, one-way ANOVA with Dunnett’s multiple comparisons test and adjustment applied). H created in BioRender. Ding, Z. (2025) https://BioRender.com/p69t917. Source data are listed in the Source Data file.