Mn-containing bioceramics inhibit osteoclastogenesis and promote osteoporotic bone regeneration via scavenging ROS

Abstract

Osteoporosis is caused by an osteoclast activation mechanism. People suffering from osteoporosis are prone to bone defects. Increasing evidence indicates that scavenging reactive oxygen species (ROS) can inhibit receptor activator of nuclear factor κB ligand (RANKL)-induced osteoclastogenesis and suppress ovariectomy-induced osteoporosis. It is critical to develop biomaterials with antioxidant properties to modulate osteoclast activity for treating osteoporotic bone defects. Previous studies have shown that manganese (Mn) can improve bone regeneration, and Mn supplementation may treat osteoporosis. However, the effect of Mn on osteoclasts and the role of Mn in osteoporotic bone defects remain unclear. In present research, a model bioceramic, Mn-contained β-tricalcium phosphate (Mn-TCP) was prepared by introducing Mn into β-TCP. The introduction of Mn into β-TCP significantly improved the scavenging of oxygen radicals and nitrogen radicals, demonstrating that Mn-TCP bioceramics might have antioxidant properties. The in vitro and in vivo findings revealed that Mn²⁺ ions released from Mn-TCP bioceramics could distinctly inhibit the formation and function of osteoclasts, promote the differentiation of osteoblasts, and accelerate bone regeneration under osteoporotic conditions in vivo. Mechanistically, Mn-TCP bioceramics inhibited osteoclastogenesis and promoted the regeneration of osteoporotic bone defects by scavenging ROS via Nrf2 activation. These results suggest that Mn-containing bioceramics with osteoconductivity, ROS scavenging and bone resorption inhibition abilities may be an ideal biomaterial for the treatment of osteoporotic bone defect.

Keywords: Antioxidant biomaterials; Mn-containing bioceramics; Osteoclastogenesis; Osteoporotic bone regeneration; ROS.

Graphical abstract

Fig. 1

Mn-TCP significantly scavenged multiple freeradicals. (A) photos of Mn-TCP scavenged superoxide radical, (B) superoxide radical scavenging, (C) DPPH scavenging, (D) H₂O₂ scavenging. Mn-TCP possessed the capability to scavenge superoxide radical, DPPH free radicals and H₂O₂. CTR: The group without any treatment was used as control. Mn-TCP 6.25/50: 6.25/50 mg Mn-TCP powders were added into per mL of working solution. TCP 6.25/50: 6.25/50 mg TCP powders were added into per mL of working solution.

Fig. 2

Mn-TCP inhibits RANKL-induced osteoclast differentiation. (A-D) CCK-8 is used to detect RAW264.7 cells (A, B) and BMMs (C, D) cell viability, which are treated with different dosages of Mn-TCP and TCP extracts for 24 h and 72 h, (E) TRAP stain during osteoclastogenesis of RAW264.7 cells, which are treated with Mn-TCP and TCP extracts for 72h, (F) Quantification of TRAP(+) cells in each well, (G) TRAP stain during osteoclastogenesis of BMMs, which are treated with Mn-TCP and TCP extracts for 7 d, (H) Quantification of TRAP(+) cells in each well.

Fig. 3

Mn-TCP inhibits osteoclast function during osteoclastogenesis of BMMs which are induced by RANKL. (A) FCM shows the cell apoptosis rate during osteoclastogenesis of BMMs, which are treated with different dosages of (6.25 mg/mL, 50 mg/mL) of Mn-TCP and TCP extracts for 3d. (B) F-actin immunofluorescence staining during osteoclastogenesis of BMMs, which are stimulated with different dosage (6.25 mg/mL, 50 mg/mL) of Mn-TCP and TCP extracts for 7 d, (C) Quantitative analysis of actin ring (+) osteoclasts in each well, (D) Pit formation assay during osteoclastogenesis of BMMs at 9 d, (E) Quantitative analysis of resorption area proportion.

Fig. 4

The relative genes expression of NFATc1, c-Fos, MMP9, DC-STAMP, OC-STAMP, TRAP, and Ctsk during osteoclatogenesis of BMMs under the treatment of different dosages (6.25 mg/mL, 50 mg/mL) of Mn-TCP and TCP extracts for 3 d and 7 d.

Fig. 5

Mn²⁺ ions inhibit RANKL-induced osteoclastogenesis of BMMs. (A) TRAP stain applied to detect osteoclasts differentiation of BMMs treated with different dosage of Mn²⁺ ions (1 μM, 10 μM) for 7 d, (B) Pit formation assay is used to evaluate osteoclast function at 9 d, (C) F-actin immunofluorescence staining during osteoclastogenesis of BMMs, which are treated with different dosage of Mn²⁺ ions (1 μM, 10 μM) for 7 d, (D) Quantitative analysis of TRAP (+) cells in each well, (E) Quantitative analysis of actin ring (+) osteoclasts in each well, (F) Quantitative analysis of resorption area proportion on bone slices.

Fig. 6

Mn-TCP extracts and Mn²⁺ ions scavenge ROS generation and activate Nrf2 expression during RANKL-induced osteoclastogenesis of BMMs. (A) Fluorescence images of ROS positive during osteoclastogenesis of BMMs, which are treated with different concentrations (6.25 mg/mL, 50 mg/mL) of Mn-TCP and TCP extracts for 3 d, (B) Fluorescence images of ROS positive during osteoclastogenesis of BMMs, which are treated with different concentrations of Mn²⁺ ions (1 μM, 10 μM) for 3 d, (C) Relative mRNA expression levels of Nrf2 during osteoclastogenesis of BMMs under the treatment of different concentrations (6.25 mg/mL, 50 mg/mL) of Mn-TCP and TCP extracts for 3 d and 7 d, (D) Relative mRNA expression levels of Nrf2 during osteoclastogenesis of BMMs under the treatment of different concentrations of Mn²⁺ ions (1 μM, 10 μM) for 3 d and 7 d, (E) The protein level of Nrf2 and Keap1 during osteoclastogenesis of BMMs under the treatment of Mn-TCP and TCP extracts for 5 d.

Fig. 7

Mn-TCP bioceramics promote osteoporotic bone defect regeneration in OVX rats at 4 weeks postimplantation. (A) Schematic diagram of the surgery time points, (B) 2D images showed the normal and osteoporotic bone, (C) H&E staining and Masson staining in Sham group and OVX group, (D₁-F₄) 3D reconstruction images of Control group, Mn-TCP bioceramics group and TCP bioceramics group. D₁ and D₂: Control group without implantation, E₁-E₄: TCP bioceramics group, F₁–F₄: Mn-TCP bioceramics group. E₂ and F₂: 3D reconstruction images of bioceramics and new bone, E₃ and F₃: 3D reconstruction images of bioceramics, D₂, E₄ and F₄: 3D reconstruction images of new bone, (G–I) H&E staining for all the groups, (J–L) Masson staining for all the groups, (M–O) TRAP staining for all the groups. The red circle represents the defect area. The red arrow represents osteoclasts. The black arrow represents the host bone. The blue arrow represents the new bone (n = 3).

Fig. 8

Mn-TCP bioceramics promote osteoporotic bone defect regeneration in OVX rats at 12 weeks postimplantation. (A₁-C₄) 3D reconstruction images of Control group, Mn-TCP bioceramics group and TCP bioceramics group. A₁ and A₂: Control group without implantation, B₁–B₄: TCP bioceramics group, C₁–C₄: Mn-TCP bioceramics group. B₂ and C₂: 3D reconstruction images of bioceramics and new bone, B₃ and C₃: 3D reconstruction images of bioceramics, A₂, B₄ and C₄: 3D reconstruction images of new bone, (D-F) H&E staining for all the groups, (G-I) Masson staining for all the groups, (J-M) Micro-CT parameters of bone regeneration within femur defects. The red circle represents the defect area. The black arrow represents the host bone. The blue arrow represents the new bone (n = 3).