Fabrication and evaluation of a BMP-2/dexamethasone co-loaded gelatin sponge scaffold for rapid bone regeneration

Abstract

Improving the osteogenic activity of BMP-2 in vivo has significant clinical application value. In this research, we use a clinical gelatin sponge scaffold loaded with BMP-2 and dexamethasone (Dex) to evaluate the osteogenic activity of dual drugs via ectopic osteogenesis in vivo. We also investigate the mechanism of osteogenesis induced by BMP-2 and Dex with C2C12, a multipotent muscle-derived progenitor cell. The results show that the gelatin scaffold with Dex and BMP-2 can significantly accelerate osteogenesis in vivo. It is indicated that compared with the BMP-2 or Dex alone, 100 nM of Dex can dramatically enhance the BMP-2-induced alkaline phosphatase activity (ALP), ALP mRNA expression and mineralization. Further studies show that 100 nM of Dex can maintain the secondary structure of BMP-2 and facilitate recognition of BMP-2 with its receptors on the surface of C2C12 cells. We also find that in C2C12, Dex has no obvious effect on the BMP-2-induced Smad1/5/8 protein expression and the STAT3-dependent pathway, but Runx2-dependent pathway is involved in the Dex-stimulated osteoblast differentiation of BMP-2 both in vitro and in vivo. Based on these results, a potential mechanism model about the synergistic osteoinductive effect of Dex and BMP-2 in C2C12 cells via Runx2 activation is proposed. This may provide a theoretical basis for the pre-clinical application of Dex and BMP-2 for bone regeneration.

Keywords: BMP-2; Runx2; bone regeneration; dexamethasone; pre-clinical.

Graphical abstract

Figure 1.

Effect of Dex on BMP-2-induced ectopic osteogenesis in vivo. (a) Gelatin sponge scaffold with 15 μg of BMP-2 and 0, 4, 20, 100 μg Dex was implanted into thigh muscle pouches of mice to stimulate ectopic osteogenesis. The digital pictures (b) and micro-CT images (c) of new ectopic bone after 2 weeks of surgery were displayed (n = 6). Use GE software-Microview ABA 2.1.2 to quantitatively analyze the new bone volume (d) and tissue mineral density (e) from the micro-CT image. The data presented are the average of three independent experiments. (f) Western blot analysis of Runx2 and β-actin protein abundances in ectopic bone formation. (g) Integrated in vivo Runx2 optical data analysis by ImageJ 1.42. * indicates that group with BMP-2 and 20 μg Dex contained higher bone volume than groups without Dex. # indicates that the tissue mineral density of groups with Dex and BMP-2 all higher than groups without Dex. & indicates that the Runx2 protein levels in vivo with Dex and BMP-2 higher than BMP-2 group. @ indicates that the Runx2 protein expression of the group with BMP-2 and 20 μg Dex is significantly higher than the other groups. *P, #P, &P, @P < 0.05

Figure 2.

Dex enhanced the biological activity of BMP-2. (a) ALP was measured after cells were cultured with 0.4 μg/ml of BMP-2 and 0, 5, 10, 20, 100, 200 or 500 nM of Dex for 3 days. The data were obtained from the color reaction of p-nitrophenylphosphate. (b) The staining of ALP activity was using fast blue BB and naphthol as-BI phosphate salt. (c) ALP activity of C2C12 cells for different incubation times. Cells incubated with BMP-2 (0.4 μg/ml), Dex (100 nM) or a combination of both for different incubation times. (d) The ALP activity was induced by adding inducers at different time periods. For the first 2 days of C2C12 cell culture, used BMP-2 (0.4 μg/ml), Dex (100 nM) or a mixture of two inducers as the medium, and removed the inducer after 48 h of culture. Then, we cultured the cells with the designated inducers from Day 3 to 4. ALP activity was measured at Day 4 (n = 4). (e) Effects of BMP-2 and Dex on C2C12 cells mineralization. The culture medium of C2C12 cells were BMP-2 (0.4 μg/ml), Dex (100 nM) or a mixture of two inducers. The staining of cell mineralization was detected using 1% Alizarin Red S and checked at 400× magnification. *P < 0.05. **P < 0.001

Figure 3.

(a) Effect of Dex on the binding of BMP-2 to C2C12 cell membranes. The cells were incubated with 0.4 μg/ml BMP-2 and different concentrations of Dex for 6 h. FITC-labeled phalloidin (green) was used to stain the cell cytoskeleton and DAPI (blue) was used to stain the nuclei. BMP-2 on the cell surface was detected with alexa fluor 647-labeled anti-mouse IgG (red) and anti-BMP-2 antibodies. (b) Far-UV CD spectrum of BMP-2 at different concentrations of Dex. Secondary structure of BMP-2 was detected with related software

Figure 4.

Effects of Dex on osteoblastic differentiation induced by BMP-2. (a) Effects of Dex on Smad1/5/8 phosphorylation signaling pathway induced by BMP-2. Western blotting for phosphorylated Smad1/5/8 was detected andβ-actin was set as the blank group. (b) Integrated p-Smads optical data analysis by ImageJ 1.42. (c) Western blot analysis of Runx2 and β-actin in C2C12 cells in vitro. (d) Integrated Runx2 optical data analysis by ImageJ 1.42. (e) Detection of the expression of id-1 affected by Dex and BMP-2. The mRNA expression of id-1 was measured with RT-PCR (n = 4). (f) ALP activity of C2C12 cells cultured with Dex and BMP-2 and the JAK/STAT inhibitor AG490. The concentration of BMP-2 in all experiments is 0.4 μg/ml and Dex is 100 nM. **P < 0.001, *P < 0.05

Figure 5.

The expression of osteoblast markers and osteogenic transcription factors affected by BMP-2 and Dex, (a) ALP, (b) OC, (c) collagen I and (d) Runx2, in C2C12 cells. Cells were incubated with BMP-2 (0.4 μg/ml), Dex (100 nM) or a mixture of two inducers for 1, 3 and 5 days and characterized with RT-PCR (n = 4). Control indicates the cells cultured in a medium without BMP-2 and Dex. *P < 0.05, ** P < 0.001

Figure 6.

Cartoon model showing the regulation of Dex on the binding of BMP-2 to its receptor and BMP-2-induced osteoblastic differentiation via Runx2 activation. Dex at low concentration is beneficial to the ability of BMP-2 to bind to cell membrane surface receptors and thus up-regulated the downstream signaling pathway. Dex has no obvious effect on phospho-Smad1/5/8 protein and id-1, but it can up-regulate Runx2 expression and then promote osteoblastic differentiation and inhibit myoblast differentiation in C2C12 cell model