MiR-21 nanocapsules promote early bone repair of osteoporotic fractures by stimulating the osteogenic differentiation of bone marrow mesenchymal stem cells

Abstract

Objective: The healing of osteoporotic fractures in the elderly patients is a difficult clinical problem. Currently, based on the internal fixation of fractures, the available drug treatments mainly focus on either inhibiting osteoclast function, such as bisphosphonate, calcitonin, oestrogen or promoting osteogenesis, such as parathyroid hormones. However, the availability of current antiosteoporotic drugs in promoting osteoporotic fracture healing is limited. The objective of the present study was to investigate the ability of the MiR-21/nanocapsule to enhance the early bone repair of osteoporotic fractures.

Methods: Based on the presence of matrix metalloproteinases that are overexpressed at the fracture site, we designed the matrix metalloproteinase-sensitive nanocapsules which were formed by in situ free radical polymerisation on the surface of MiR-21 with 2-(methacryloyloxy) ethyl phosphorylcholine and the bisacryloylated VPLGVRTK peptide. The MiR-21/nanocapsule [n (miR-21)] and O-carboxymethyl chitosan (CMCS) were mixed until they formed a gel-like material [CMCS/n (miR-21)] with good fluidity and injectability. Thirty elderly Sprague Dawley (SD) rats (female, 14-month-old, 380 ± 10 g) were subjected to bilateral removal of the ovaries (ovariectomised). All rats were subjected to bilateral bone defects (2 mm diameter) of the proximal tibia and randomly divided into three groups (groups A, B, and C): separately injected with CMCS/n (miR-21), CMCS/n (NC-miR), and saline. Micro-computed tomography (CT) imaging was performed to evaluate newly formed bone volume and connectivity. Nondecalcified histology and toluidine blue staining were performed to measure the effects of CMCS/n (miR-21) on bone repair. In vitro, the effect of n (miR-21) on osteogenic differentiation to bone marrow mesenchymal stem cells (BMSCs) which derived from the ovariectomised rat model was observed.

Results: The morphology of n (miR-21) was a regular spherical nanocapsule with a uniform small size (25-35 nm). The results confirmed that n (miR-21) could be efficiently phagocytosed by BMSCs and released in the cytoplasm to promote osteogenesis. The expression level of alkaline phosphatase and Runt-related transcription factor 2 mRNA in the n (miR-21) group was higher than that in the n (NC-miR) group. Animal experiments proved that CMCS/n (miR-21) produced better bone repair compared with the CMCS/n (NC-miR) group in the early stages of fracture healing at 4 weeks. In the late stage of fracture healing (8 weeks), micro-CT quantitative analysis showed that the new bone trabeculae in the CMCS/n (miR-21) group has decreased compared with the CMCS/n (NC-miR) group. In the CMCS/n (miR-21) group, the new cancellous bone had been absorbed, and the process of bone healing was almost completed. In contrast, the new bone in the CMCS/n (NC-miR) and the control groups was still in the healing process.

Conclusion: The cytological tests confirmed that n (miR-21) can promote osteogenic differentiation of BMSCs derived from the osteoporosis rat model. Furthermore, the results of animal tests demonstrated that local injection of CMCS/n (miR-21) promoted the early healing of osteoporotic bone defects. Consequently CMCS/n (miR-21) promoted the bone repair process to enter the moulding phase earlier.

The translational potential of this article: CMCS/n (miR-21) can be widely applied to elderly patients with osteoporotic fractures. This method can help patients with osteoporotic fractures recover earlier and avoid serious complications. It provides a potential approach for the clinical treatment of osteoporotic fractures in the elderly.

Keywords: Bone repair; MicroRNA-21; Nanocapsules; Osteoporotic fractures.

Scheme 1

A schematic illustrating the degradation and release of MMP-responsive n (miR-21) in the inflammatory environment (mainly contains MMP-2 and MMP-9) of the bone defect site. The proximal tibial bone defect model is achieved in osteoporotic rats (ovariectomised, OVX). MMP, matrix metalloproteinase.

Figure 1

(A) A schematic illustrating the synthesis mechanism of MMP-responsive miR-21 nanocapsules n (miR-21) (B) A representative TEM image of n (miR-21) (scale bar, 50 nm) (C) Size distribution of the nanocapsules measured via DSL (D and E) Agarose gel electrophoresis and zeta potential of n (miR-21) (molar ratio = 4000:1). MMP, matrix metalloproteinase; TEM, transmission electron microscopy; DSL, dynamic light scattering.

Figure 2

(A) The characterisation of the BMSCs by flow cytometry. The potential of triple lineage differentiation of BMSCs: (B) ALP staining (C) Toluidine blue staining (D) Oil red O staining. BMSCs, bone marrow mesenchymal stem cells. BMSCs, bone marrow mesenchymal stem cells.

Figure 3

(A) Intracellular uptake analysis of the nanocapsules as assessed by confocal microscopy after 24h incubation with 50 nM FAM (carboxyfluorescein)-labelled (scale bar, 200 μm) (B) Quantitative analysis of cell uptake by flow cytometry. Each experiment was performed three times (∗p < 0.05 vs. control and miR-21) (C) The cytotoxicity of the n (miR-21) in BMSCs with different concentrations of n (miR-21) (n = 5; ∗p < 0.05 vs. concentration of 100,250 and 500 nM) (D and E) The expression of early osteogenesis-related genes (ALP and RUNX-2) incubated with n (miR-21) and n (NC-miR) (∗p < 0.05). BMSCs, bone marrow mesenchymal stem cells.

Figure 4

(A) Alizarin red staining analysis was performed at days 7, 14, and 21 after treatment of the MiR-21 to visualise calcium nodule accumulation in BMSCs (40x); Quantitative analysis of alizarin red staining was performed using ImageJ (∗p < 0.05) (B) Western blot analysis of apoptosis-related caspase-3 protein expression on day 5 after miR-21 nanocapsules treated BMSCs. Quantitative analysis of the immunoblots was determined by ImageJ (∗p < 0.05). BMSCs, bone marrow mesenchymal stem cells.

Figure 5

(A) 3D images of representative lumbar cancellous bone in the SHAM and OVX group (B) Result of lumbar BMD by DEXA in the sham and OVX group (C) Micro-CT quantitative analysis of lumbar cancellous bone (BV/TV, %) (n = 10; ∗p < 0.05 vs. OVX group) (D) Fluorescence intensity of the bone defect after the 24 h injection of miR-21 labelled FITC. BMD, bone mineral density; OVX, ovariectomised; DEXA, dual energy X-ray absorptiometry; FITC, fluoresceine isothiocyanate.

Figure 6

(A) Micro CT images of representative rat tibias at 4th week: 3D architecture of trabecular within the distal tibias (B) Quantitative analysis of the new trabecular bone volume (BV/TV), trabecular thickness (Tb.Th), and trabecular number (Tb.N) of the bone defect in OVX rats by microtomography analysis (n = 5; ∗p < 0.05 vs. CMCS/n (miR-NC) group; p < 0.05 vs. control group.). OVX, ovariectomised.

Figure 7

(A) Quantitative analysis of ALP expression of BMSCs isolated from OVX rats at 4 weeks. (B) Calcium deposition of BMSCs isolated from OVX rats at 4 weeks (n = 5; ∗p < 0.05 vs. n (NC-miR) and control group). OVX, ovariectomised; BMSCs, bone marrow mesenchymal stem cells; ALP, alkaline phosphatase.

Figure 8

(A) Micro CT images of representative rat tibias at 4th week: 3D architecture of trabecular within the distal tibias (B) Quantitative analysis of the new trabecular bone volume (BV/TV), trabecular thickness (Tb.Th), and trabecular number (Tb.N) of the bone defect in OVX rats by microtomography analysis (n = 5; ∗p < 0.05 vs. CMCS/n (NC-miR) group; p < 0.05 vs. control group.). OVX, ovariectomised.

Figure 9

Nondemineralised hard tissue histology and toluidine blue stained for 4th and 8th weeks (A) At 4 weeks, the new cancellous bone (shown in the yellow frame) in the bone defect site was quantitatively analysed (BV/TV) by ImageJ (B) At 8 weeks, the thickness of the new formed cortical bone (red arrow) in the bone defect site was quantitatively analysed by ImageJ (n = 5; ∗p < 0.05 vs. CMCS/n (NC-miR) group; p < 0.05 vs. control group.