Enhanced osteogenesis and angiogenesis by mesoporous hydroxyapatite microspheres-derived simvastatin sustained release system for superior bone regeneration

Abstract

Biomaterials with both excellent osteogenic and angiogenic activities are desirable to repair massive bone defects. In this study, simvastatin with both osteogenic and angiogenic activities was incorporated into the mesoporous hydroxyapatite microspheres (MHMs) synthesized through a microwave-assisted hydrothermal method using fructose 1,6-bisphosphate trisodium salt (FBP) as an organic phosphorous source. The effects of the simvastatin-loaded MHMs (S-MHMs) on the osteogenic differentiation of rat bone marrow mesenchymal stem cells (rBMSCs) and angiogenesis in EA.hy926 cells were investigated. The results showed that the S-MHMs not only enhanced the expression of osteogenic markers in rBMSCs but also promoted the migration and tube formation of EA.hy926 cells. Furthermore, the S-MHMs were incorporated into collagen matrix to construct a novel S-MHMs/collagen composite scaffold. With the aid of MHMs, the water-insoluble simvastatin was homogenously incorporated into the hydrophilic collagen matrix and presented a sustained release profile. In vivo experiments showed that the S-MHMs/collagen scaffolds enhanced the bone regeneration and neovascularization simultaneously. These results demonstrated that the water-insoluble simvastatin could be incorporated into the MHMs and maintained its biological activities, more importantly, the S-MHMs/collagen scaffolds fabricated in this study are of immense potential in bone defect repair by enhancing osteogenesis and angiogenesis simultaneously.

Figure 1. Physical characteristics of MHMs.

(A) SEM image; (B) TEM image; (C) XRD pattern; (D) Nitrogen adsorption-desorption isotherm curve; (E) DLS size distribution; (F) BJH pore size distribution curve.

Figure 2. Simvastatin loading and release assay.

(A) FTIR spectra of FBP, MHMs S-MHMs and simvastatin; (B) UV-vis absorption spectra of the ethanol solution of simvastatin before and after loading; (C) Simvastatin release curve in NS solution of S-MHMs.

Figure 3. The effects of MHMs and S-MHMs on cell viability and ALP activity.

(A) Proliferation of rBMSCs; (B) Quantification of ALP activity; (C) ALP staining. (*Comparison between the MHMs group and the blank control, ^#comparison between MHNs and S-MHMs groups at the same concentration, p < 0.05).

Figure 4. The effects of S-MHMs on the expression of osteogenesis-related genes in rBMSCs on days 7 and 14.

(*Comparison between the MHMs group and the blank control, #comparison between MHMs and S-MHMs groups at the same concentration, p < 0.05).

Figure 5

Representative photographs showing the effects of S-MHMs on the migration of EA.hy926 cells (A,D) and rBMSCs (B,E), and the tube formation of EA.hy926 cells (C,F). (^#Comparison between the MHMs and S-MHMs at the same concentration, ^%comparison between 0.6 and 6 μg/mL of S-MHMs, 6 and 60 μg/mL of S-MHM, p < 0.05).

Figure 6

Western blot analysis for the expression of HIF-1α (A) and VEGF (B) in rBMSCs cultured with different concentrations of MHMs and S-MHMs for 48 h.

Figure 7. Scaffolds characterization and simvastatin release.

(A) SEM images of the collagen, MHMs/collagen and S-MHMs/collagen scaffolds and cell attachment (arrowheads) on the scaffolds; (B) Water absorbing capacities of the scaffolds; (C) Simvastatin release from the S-MHMs/collagen scaffold.

Figure 8. The live/dead staining and cytoskeleton staining of rBMSCs seeded on the scaffolds after culturing for 3 days.

(The viable cells were stained green whereas the dead cells were stained red; The cytoskeleton was stained red whereas the cell nuclei were stained blue).

Figure 9. New bone formation and neovascularization evaluated by micro-CT.

(A) 3-D and coronary views of reconstructed calvaria; (B,C) BMD and BV/TV in the defects; (D) New blood vessels presented by 3-D reconstruction images; (E,F) Quantitative analysis of the new blood vessel area and number. (*Comparison between collagen and other groups, ^#comparison between the MHMs/collagen and S-MHMs/collagen groups, p < 0.05).

Figure 10. Histological assessment of bone regeneration in each group.

(A) HE staining; (B,C) IHC staining for OCN and OPN.

Figure 11. Histological assessment of neovascularization in each group.

(A) IHC staining for VEGF; (B) IF staining for α-SMA (red); (C) Quantification of the brown area surface to the total surface of images; (D) Quantification of the newly formed blood vessels (red fuorescence). (*Comparison between collagen and other groups, ^#comparison between the MHMs/collagen and S-MHMs/collagen groups, p < 0.05).