Calcium Supplement by Tetracycline guided amorphous Calcium Carbonate potentiates Osteoblast promotion for Synergetic Osteoporosis Therapy

Abstract

Background: The calcium supplement is a clinically approved approach for osteoporosis therapy but usually requires a large dosage without targetability and with poor outcome. This modality is not fully explored in current osteoporosis therapy due to the lack of proper calcium supplement carrier. Methods: In this study, we constructed a tetracycline (Tc) modified and simvastatin (Sim) loaded phospholipid-amorphous calcium carbonate (ACC) hybrid nanoparticle (Tc/ACC/Sim). Results: The resulted Tc/ACC/Sim was able to enhance its accumulation at the osteoporosis site. Most importantly, the combination of calcium supplement and Sim offered synergetic osteoblast promotion therapy of osteoporosis with advanced performance than non-targeted system or mono therapy. Conclusion: This platform provides an alternative approach to stimulate bone formation by synergetic promotion of osteoblast differentiation using calcium supplement and Sim.

Keywords: amorphous calcium carbonate; calcium supplement; osteoblast promotion; osteoporosis.

Scheme 1

A biological mechanism by which calcium supplement using ACC augments Sim mediated BMP-2-induced osteoblast differentiation is shown as Runx2 expression and ALP activity through the BMP-Smad signaling pathway.

Figure 1

Characterization of Tc-SA using ¹H NMR. Inserted images were corresponding chemical structure and peak assignments.

Figure 2

Characterization of Tc/ACC from ACC using (A) dynamic light scattering (DLS) and (B) transmission electron microscopy (TEM) images. Scale bar: 100 nm.

Figure 3

In vitro cellular uptake of Tc/ACC. MC3T3-E1 cells were incubated with ODA-FITC labeled PL/ACC and Tc/ACC for different time intervals (2, 4, 6, 10 h) and then subjected to observation using confocal microscope. (A) Time-dependent cellular uptake of ODA-FITC labeled PL/ACC and Tc/ACC. Scale bar: 30 µm. (B) Semiquantitative analysis of cellular uptake by flow cytometry.

Figure 4

Bone targeting of Tc/ACC. (A) In vitro bone targeting of Tc/ACC using HAP adsorption assay. HAP was added to the ODA-FITC labeled PL/ACC and Tc/ACC and incubated for under genteel mechanical stirring for 1 hour. Afterwards, the decrease in the fluorescence of the supernatant was measured. (B) In vivo bone targeting of Tc/ACC using fluorescence imaging. ICR mice were randomly divided into two groups and intravenously injected with ICG labeled PL/ACC and Tc/ACC. At 48 h post administration, the mice were sacrificed and the quantitative accumulation of fluorescence signal in the femur, and tibia bones were calculated. Inserted image was fluorescence images of the distribution of ICG labeled PL/ACC and Tc/ACC in the femur and tibia bones. Results were expressed as mean ± S.D. (n = 3).

Figure 5

Optimization of administration dosage. MC3T3-E1 cells were exposed to a series of concentrations of PL/ACC, Tc/ACC, Sim, PL/ACC/Sim and Tc/ACC/Sim for 48 h. Afterwards, the cell viability influence of (A) Sim free carrier (PL/ACC and Tc/ACC) and (B) Sim containing formulations were evaluated using MTT assay. Results were expressed as mean ± S.D. (n = 3).

Figure 6

Effects of Sim-loaded nanoparticles on mineralized nodule formation and ALP activity in MC3T3-E1 cells. MC3T3-E1 cells were cultured in standard osteogenic differentiation and mineralization medium containing 10 mM β-glycerophosphate, 50 µg/mL ascorbic acid and different formulations at the equal Sim concentration of 10^-7 M). Afterwards, the mineralization of the extracellular matrix was evaluated by optical microscopy and macroscopic observation (A), quantitative mineralization results (B) at 14 days post incubation, and stimulation of ALP activity (C) at 7 days post incubation. Results were expressed as mean ± S.D. (n = 3). *p < 0.05, **p < 0.01, ***p < 0.001, significant differences compared with the control.

Figure 7

Female Sprague Dawley rats were randomly selected and subjected to sham operation or bilaterally oophorectomy. The treatment groups were intravenously administered with Sim, PL/ACC/Sim or Tc/ACC/Sim group at the Sim dose of 1 mg/kg/2 days for 2 months. Afterwards, the rats were sacrificed and the micro-CT analysis of bone tissue from each group were conducted. (A) Trabecular bone density (BMD); (B) Trabecular bone volume percentage (BV/TV); (C) Trabecular separation (Tb.Sp); (D) Trabecular thickness (Tb.Th) and (E) trabecular number (Tb.N) of the femur. Results were expressed as mean ± S.D. (n = 3). *p < 0.05, **p < 0.01, ***p < 0.001, significant differences compared with the control.

Figure 8

The 2D and 3D images of trabecular bone measured by micro-CT. Scale bar: 1mm.

Figure 9

(A)ALP activity (arrows) and TRAP assay results (arrowheads) of bone tissue sections. (B) Histological assessment of bone formation in each group using HE staining. (C) Immunohistochemical staining for BMP-2 in typical newly-formed bone tissue (red arrows) and immunohistochemical staining for the osteogenic markers OPN (arrows) and OCN (arrowheads). Scale bars = 100 µm.