Rehabilitation exercise-driven symbiotic electrical stimulation system accelerating bone regeneration

Abstract

Electrical stimulation can effectively accelerate bone healing. However, the substantial size and weight of electrical stimulation devices result in reduced patient benefits and compliance. It remains a challenge to establish a flexible and lightweight implantable microelectronic stimulator for bone regeneration. Here, we use self-powered technology to develop an electric pulse stimulator without circuits and batteries, which removes the problems of weight, volume, and necessary rigid packaging. The fully implantable bone defect electrical stimulation (BD-ES) system combines a hybrid tribo/piezoelectric nanogenerator to provide biphasic electric pulses in response to rehabilitation exercise with a conductive bioactive hydrogel. BD-ES can enhance multiple osteogenesis-related biological processes, including calcium ion import and osteogenic differentiation. In a rat model of critical-sized femoral defects, the bone defect was reversed by electrical stimulation therapy with BD-ES and subsequent bone mineralization, and the femur completely healed within 6 weeks. This work is expected to advance the development of symbiotic electrical stimulation therapy devices without batteries and circuits.

Fig. 1.. Schematic of the main concept for a fully implantable BD-ES system.

(A) Schematic of the reconstruction of the electrical microenvironment to accelerate bone regeneration in the bone defect region using ES. (B and C) Schematic of a fully implantable battery-free BD-ES system for patients performing active or passive functional exercise under guidance (D). Possible mechanism by which the BD-ES system promotes bone repair.

Fig. 2.. Electrical performance and biocompatibility of HTP-NG.

(A) Schematic depicting the operational principle of HTP-NG, converting applied force into real-time electron flow within the external circuit. (B) Voltage signals generated by the triboelectric module, piezoelectric module, and hybrid module of the HTP-NG. (C) The applied pressure (~6 kPa) and output voltage of the HTP-NG were monitored in real time. (D) Output voltage signals under different pressures. The inset shows the output voltage signals of HTP-NG under low pressures (≤0.5 kPa). (E) Linear fitting of peak-to-peak voltage and pressure change (n = 3). The inset shows an enlarged fitting curve for the output voltage signals of HTP-NG at low pressures (≤0.5 kPa). (F) Long-term stability test of HTP-NG. (G) Output voltage of HTP-NG in response to knee bending. (H) Fluorescence images of stained BMSCs that were cultured in a regular cell culture dish (top) and on HTP-NG (bottom). Scale bar, 100 μm. (I) Normalized viability of BMSCs after being cultured for 3 days. Data are expressed as means ± SD. One-way ANOVA with Tukey’s multiple comparisons test, not significant, n = 3. The working area of HTP-NG was set to 1 cm², and the pressure was 6 kPa unless otherwise specified.

Fig. 3.. Schematic illustration of the formation of EABP conductive hydrogels and the cytocompatibility of the BD-ES system on BMSCs.

(A) Schematic illustration of the preparation of EABP conductive hydrogels. (B) Schematic illustration of the formation of crosslinked EABP hydrogel. (C) UV-cured EABP hydrogel and SEM image. Scale bar, 100 μm. (D) Conductivity of EA and EABP hydrogels. Data are expressed as means ± SD, one-way ANOVA with Tukey’s multiple comparisons test, not significant, n = 3. (E) Schematic illustration of the stimulation on BMSCs by BD-ES system, created with BioRender.com. (F) Live/dead staining of BMSCs cultured with EA or EABP hydrogels with or without stimulation of BD-ES system. Scale bar, 300 μm. (G) Cell viability of BMSCs cultured with EA or EABP hydrogels with or without stimulation of BD-ES system. Asterisk indicates statistical significance compared to the EA group. Data are expressed as means ± SD, one-way ANOVA with Tukey’s multiple comparisons test, **P < 0.01 and *P < 0.05, n = 3. (H) Images of migrated BMSCs stained with crystal violet in Transwell assay. Scale bar, 200 μm. (I) Statistical analysis of the number of migrated cells. Asterisk indicates statistical significance between groups. Data are expressed as means ± SD, one-way ANOVA with Tukey’s multiple comparisons test, ***P < 0.001, **P < 0.01, and *P < 0.05, n = 3. (J) Immunofluorescence staining of cellular F-actin (red), nuclei (blue), and vinculin focal staining (green). Scale bars, 200 and 40 μm. (K) Quantification of the number of focal adhesions (FAs). ***P < 0.001, n = 3. All data are expressed as means ± SD.

Fig. 4.. Osteogenic differentiation of BMSCs evaluated by ALP and alizarin red staining under BD-ES system stimulation.

(A) ALP staining of BMSCs cultured with or without BD-ES system stimulation at days 7 and 14. Scale bar, 500 μm. (B) Quantitative evaluation of the ALP-positive area on days 7 and 14. Data are expressed as means ± SD. One-way ANOVA with Tukey’s multiple comparisons test, **P < 0.01 and *P < 0.05, n = 3. (C) Alizarin red staining of BMSCs on day 21. (D) Quantitative evaluation of calcium nodules in alizarin red staining on day 21. Scale bar, 200 μm. Data are expressed as means ± SD, two-way ANOVA with Tukey’s multiple comparisons test, ***P < 0.001 and *P < 0.05, n = 3.

Fig. 5.. Exploration of gene expression patterns and functional enrichment analysis.

(A) Box plot depicting FPKM values. (B) PCA illustrating the variance between the control and EABP + ES groups. (C and D) Volcano plot and heatmap showing the DEGs, including those that are up-regulated (red dots) and down-regulated (green dots). (E) GO enrichment analysis including BP (biological process), CC (cellular component), and MF (molecular function). (F to K) GSEA of osteogenesis-related signaling pathways and biological processes. (L) Western blot analysis of the expression of core proteins involved in osteogenic, mechanosensitive, and enriched signaling pathways between the control (Ctrl) group and EABP + ES group. (M) Schematic view of the possible mechanism involved in the osteogenesis process based on transcriptome sequencing results.

Fig. 6.. The implantation of the BD-ES system in vivo and its ability to promote bone regeneration were evaluated by micro-CT and histochemical staining of femoral samples.

(A) Surgical images of the implanted BD-ES system. (B) 3D reconstructive CT images of the BD-ES system 2 weeks after surgery. (C) Distribution of the electric potential in the EABP conductive hydrogel from HTP-NG calculated from finite element analysis. (D) The output voltage of HTP-NG in vivo when Sprague-Dawley rats run at a speed of 1 km/hour 2 weeks after surgery. (E) 3D reconstruction images and sagittal and transverse view images of the distal femur by micro-CT. (F to I) Micro-CT quantitative evaluation of BMD, BV/TV, Tb.N, and Tb.Th in defect areas. Data are expressed as means ± SD. One-way ANOVA with Tukey’s multiple comparisons test, ***P < 0.001 and *P < 0.05, n = 3. (J) H&E and Masson’s trichrome staining images of the rat femurs. Scale bar, 500 μm.

Fig. 7.. Immunohistochemical and immunofluorescence staining images and relative quantitative analysis of angiogenesis- and osteogenesis-related markers.

(A) Immunohistochemical staining of type I collagen. Scale bars, 400 and 100 μm. (B) Immunofluorescence staining for CD31 (red) and α-SMA (green). Scale bars, 100 and 50 μm. (C) Quantification of MVD based on the results of CD31 and α-SMA staining. Data are expressed as means ± SD. One-way ANOVA with Tukey’s multiple comparisons test, ***P < 0.001 and *P < 0.05, n = 3. (D) Immunofluorescent detection of osteogenesis-related proteins (OCN and OPN). Scale bars, 100 and 50 μm. (E and F) Quantitative expression of OCN and OPN. Data are expressed as means ± SD. One-way ANOVA with Tukey’s multiple comparisons test, ***P < 0.001, **P < 0.01, and *P < 0.05, n = 3.