Mesoporous silicate nanoparticles/3D nanofibrous scaffold-mediated dual-drug delivery for bone tissue engineering

Abstract

Controlled delivery systems play a critical role in the success of bone morphogenetic proteins (i.e., BMP2 and BMP7) for challenged bone repair. Instead of single-drug release that is currently and commonly prevalent, dual-drug delivery strategies are highly desired to achieve effective bone regeneration because natural bone repair process is driven by multiple factors. Particularly, angiogenesis is essential for osteogenesis and requires more than just one factor (e.g., Vascular Endothelial Growth Factor, VEGF). Therefore, we developed a novel mesoporous silicate nanoparticles (MSNs) incorporated-3D nanofibrous gelatin (GF) scaffold for dual-delivery of BMP2 and deferoxamine (DFO). DFO is a hypoxia-mimetic drug that can activate hypoxia-inducible factor-1 alpha (HIF-1α), and trigger subsequent angiogenesis. Sustained BMP2 release system was achieved through encapsulation into large-pored MSNs, while the relative short-term release of DFO was engineered through covalent conjugation with chitosan to reduce its cytotoxicity and elongate its half-life. Both MSNs and DFO were incorporated onto a porous 3D GF scaffold to serve as a biomimetic osteogenic microenvironment. Our data indicated that DFO and BMP2 were released from a scaffold at different release rates (10 vs 28 days) yet maintained their angiogenic and osteogenic ability, respectively. Importantly, our data indicated that the released DFO significantly improved BMP2-induced osteogenic differentiation where the dose/duration was important for its effects in both mouse and human stem cell models. Thus, we developed a novel and tunable MSNs/GF 3D scaffold-mediated dual-drug delivery system and studied the potential application of the both FDA-approved DFO and BMP2 for bone tissue engineering.

Keywords: Angiogenesis; Deferoxamine; Dual release system; Mesoporous silicate nanoparticles; Nanofibrous scaffold; Osteogenesis.

Fig. 1

Schematic illustration of synthesis procedure of 3D GF scaffolds.

Fig. 2

Schematic illustration of synthesis procedure of MSNs.

Fig. 3. Physicochemical characterization of MSNs

(a, b) Transmission electron micrographs, (c) Small-angle X-ray diffraction patterns, and (d) Pore size distribution of MSNs.

Fig. 4. Morphologies of prepared scaffolds

SEM images of GF, GF/MSN, and GF/MSN/CTS at low (upper panel, scale bars = 500 μm) and high (lower panel, scale bars = 20 μm) magnifications, respectively.

Fig. 5. Drug release profiles from scaffolds

(a) BMP2 and (b) DFO release behavior from GF scaffolds.

Fig. 6. Cells morphologies on scaffolds

hMSCs morphologies on (a) GF, (b) GF/MSN, and (c)and GF/MSN/CTS imaged by CLSM. Upper and lower panels are a top and z -stacked view, respectively. Scale bars = 400 μm.

Fig. 7. Cell viability and VEGF expression of hMSCs on scaffolds

(a) Cell viability of C2C12 cells on scaffolds and (b) VEGF expression in hMSCs on scaffolds. Data are expressed as mean ± SD (n = 3). *P < 0.05, **P < 0.01.

Fig. 8. Stem cells osteogenic differentiation on scaffolds

(a) RUNX2, (b) OCN (b) expression in C2C12 cells were studied by real-time PCR assay on GF/MSN, GF/MSN/CTS-DFO, GF/MSN-BMP2 and GF/MSN-BMP2/CTS-DFO scaffolds after 7 days of culture. (c)ALP activity, and (d) total calcium content produced by C2C12 and hMSCs cultured on scaffolds, respectively. Data are expressed as mean ± SD (n = 3).* P < 0.05, **P< 0.01, *** P< 0.001.

Fig. 9. Effect of free DFO on stem cells osteogenic differentiation

(a–c) stemness gene expressions (Nanog, POU5F, ITAG6) in hMSCs after 1-day culture of DFO treatment. (d) ALP staining (left panel), and Alizarin Red S staining (right panel) of hMSCs after treated with different doses of DFO in osteogenic medium. Data are expressed as mean ± SD (n = 3). *P < 0.05, **P < 0.01, ***P< 0.001.