Strontium-incorporated hydroxyapatite nanocomposites promoting bone formation and angiogenesis by modulating M2 macrophage polarization in the bone microenvironment

Abstract

The treatment of osteoporosis is urgently needed in the clinic. Hydroxyapatite (HAP) has a bone-inducing ability on osteogenic differentiation. Especially, the presence of strontium component in HAP nanoparticles may improve the positive effect on bone regeneration and avoid undesirable bone resorption. However, the incorporating concentrations of strontium still need to be elucidated to balance the osteogenic function and side effects. Herein, a series of strontium-incorporated HAP nanocomposites (Srx-HAP) with different Sr incorporating molar ratio concentrations (0%, 1%, 2%, 5%, 10%, 20%, 50%, 80% and 100%) have been prepared by a simple hydrothermal route. The Srx-HAP samples exhibited uniform and well-dispersed rod-like morphology, mesoporous structure, eminent degradability and good biocompatibility. In particular, Sr20-HAP exhibited prominent advantages in osteogenic differentiation and mineralization of pre-osteoblasts cell line MC3T3-E1. Sr20-HAP nanoparticles were highly effective in enhancing the bone formation in the rat model of postmenopausal osteoporosis compared to the ovariectomy group. In addition, Sr20-HAP nanoparticles could regulate macrophage polarization to M2 type in vivo and in vitro, providing an anti-inflammatory bone microenvironment and promoting bone repair and angiogenesis. This study provides a new insight of strontium-incorporated hydroxyapatite nanoparticles as competent anti-osteoporotic biomaterials for bone formation.

Keywords: bone formation; bone injury microenvironment; effect of strontium concentration; strontium-incorporated hydroxyapatite nanocomposites; treatment of osteoporosis.

Graphical abstract

Figure 1.

Characterizations of Srx-HAP nanocomposites. (A) SEM, (B) TEM and SAED patterns (insets) and (C) HRTEM images of Srx-HAP. EDX elemental mapping images of (D) Sr10-HAP and (E) Sr20-HAP. (F) XRD patterns of the Srx-HAP samples. (G) N₂ adsorption/desorption isotherms and the corresponding BJH pore size distribution curves (insets) of Srx-HAP samples.

Figure 2.

Cell viability and bone formation capability of osteoporosis rat after being treated with Srx-HAP in vivo. (A) Cell viabilities of MC3T3-E1 after incubation with the samples at different concentrations. (B) Micro-CT topview images of trabecular structure of distal femur. Micrographs of (C) H&E-stained and (D) Masson-stained femur trabecular.

Figure 3.

Cell viability and biological effects of HAP and Sr20-HAP for the bone microenvironment-related cells. BMSCs: (A) ALP staining images, (B) mineralization nudes staining by alizarin red, (C) quantitative results of ALP activity and (D) quantitative results of alizarin red staining. BMMNC: (E) TRAP staining images and (F) qualitative results of TRAP-positive cells with more than two nuclei. HUVECs: (G) Cell migration images in 0, 12 and 24 h, (H) quantitative results of cell migration for 12 h, (I) quantitative results of cell migration for 24 h, (J) vascularization images, (K) quantitative results of the angiogenesis crossing points, (L) quantitative results of the total length of vascularization. Raw264.7: (M) Expression of iNOS and Arg-1 in HAP and Sr20-HAP (1 μg/mL) cultured for 6 and 72 h, (N) The fold changes of iNOS and Arg-1 in M. Statistical analysis of treated groups versus 0 μg/ml group: * 0.01 ≤ P < 0.05, ** 0.001 ≤ P < 0.01 and *** P < 0.001. Statistical analysis of Sr20-HAP group versus HAP group at the same concentration: # 0.01 ≤ P < 0.05, ## 0.001 ≤ P < 0.01 and ### P < 0.001.

Figure 4.

Osteogenic effect of BMSCs on the co-culture of Raw264.7 with HAP and Sr20-HAP for 6 and 72 h. (A) Illustration of the co-culture model on Raw264.7 and BMSCs; (B) ALP staining of BMSCs at 6H and 72H conditions; (C, D) Quantitative results of ALP activity of BMSCs; (E) Mineralization nudes staining of BMSCs at 6H and 72H conditions; (F, G) Quantitative results of alizarin red staining of BMSCs. Statistical analysis of treated groups versus 0 μg/mL group: * 0.01 ≤ P < 0.05, ** 0.001 ≤ P < 0.01 and *** P < 0.001. Statistical analysis of Sr20-HAP group versus HAP group: ### P < 0.001.

Figure 5.

Vascularization effect of HUVECs on the co-culture of Raw264.7 with HAP and Sr20-HAP for 6 and 72 h. (A) Illustration of the co-culture model on Raw264.7 and HUVEC; (B) Cell migration images at 0, 12 and 24 h at 6H and 72H conditions, (C) Quantitative results of cell migration with 6 h-Raw264.7 (6H), (D) Quantitative results of cell migration with 72 h-Raw264.7 (72H), (E) Vascularization images at 6H and 72H conditions, (F) Quantitative results of the angiogenesis crossing points and the total length of vascularization with 6 h-Raw264.7 (6H), (G) Quantitative results of the angiogenesis crossing points and the total length of vascularization with 72 h-Raw264.7 (72H). Statistical analysis of treated groups versus 0 μg/mL group: * 0.01 ≤ P < 0.05, ** 0.001 ≤ P < 0.01 and *** P < 0.001. Statistical analysis of Sr20-HAP group versus HAP group: #0.01 ≤ P < 0.05 and ## 0.001 ≤ P < 0.01.

Figure 6.

Regulation immune microenvironment in aged mice. (A) CD68 and Arg-1 immuno-fluorescence images of M2 macrophages in the bone slice. (B) CD68 and iNOS immuno-fluorescence images of M1 macrophages in the bone slice. (C) Quantitative results of Arg-1. (D) Quantitative results of iNOS. (E) Micrographs of tibias stained with IL-10 and TNF-α antibody. (F) Quantitative results of IL-10. (G) Quantitative results of TNF-α. (H) Illustration of macrophage type changes after Sr20-HAP treatment. Statistical analysis of young and treated groups versus Old-Saline group: ** 0.001 ≤ P < 0.01 and *** P < 0.001. Statistical analysis of Sr20-HAP group versus HAP group: ## 0.001 ≤ P < 0.01. Statistical analysis of HAP and Sr20-HAP groups versus young group: & 0.01 ≤ P < 0.05, && 0.001 ≤ P < 0.01 and &&& P < 0.001.

Figure 7.

Promotion on bone formation in aged mice. (A) CT images of distal tibias. (B–D) Bone parameters of the tibias. (E) Immunohistochemical staining images of CD31. (F) Quantities results of CD31. Statistical analysis of young and treated groups versus Old-Saline group: * 0.01 ≤ P < 0.05, ** 0.001 ≤ P < 0.01 and *** P < 0.001. Statistical analysis of Sr20-HAP group versus HAP group: # 0.01 ≤ P < 0.05. Statistical analysis of HAP and Sr20-HAP groups versus young group: & 0.01 ≤ P < 0.05.