Nanoclay Promotes Mouse Cranial Bone Regeneration Mainly through Modulating Drug Binding and Sustained Release

Abstract

Nanoclay (Nanosilicates, NS) is appearing as an intriguing 2D nanomaterial for bone tissue engineering with multiple proposed functions, e.g., intrinsic osteoinductivity, improving mechanical properties, and drug release capacity. However, the mechanism of NS for in vivo bone regeneration has been hardly defined so far. This knowledge gap will significantly affect the design/application of NS-based biomaterials. To determine the role of NS in osteoblastic differentiation and bone formation, we used the mouse calvarial-derived pre-osteoblasts (MC3T3-E1) and a clinically-relevant mouse cranial bone defect model. Instead of a hydrogel, we prepared biomimetic 3D gelatin nanofibrous scaffolds (GF) and NS-blended composite scaffolds (GF/NS) to determine the essential role of NS in critical low-dose (0.5 μg per scaffold) of BMP2-induced cranial bone regeneration. In contrast to "osteoinductivity", our data indicated that NS could enable single-dose of BMP2, promoting significant osteoblastic differentiation while multiple-dose of BMP2 (without NS) was required to achieve similar efficacy. Moreover, our release study revealed that direct binding to NS in GF scaffolds provided stronger protection to BMP2 and sustained release compared to GF/NS composite scaffolds. Consistently, our in vivo data indicated that only BMP2/NS direct binding treatment was able to repair the large mouse cranial bone defects after 6 weeks of transplantation while neither BMP2, NS alone, nor BMP2 released from GF/NS scaffolds was sufficient to induce significant cranial bone defect repair. Therefore, we concluded that direct nanoclay-drug binding enabled sustained release is the most critical contribution to the significantly improved bone regeneration compared to other possible mechanisms based on our study.

Keywords: 3D nanofibrous scaffolds; BMPs; Bone tissue engineering; Cranial bone regeneration; Nanoclays; Sustained drug release.

Fig. 1.

Cell viability and morphology of MC3T3-E1 cells after treatment by NS. Cell viability (A) was evaluated after the cells were cultured in growth medium containing with different concentrations of NS for 0, 1, and 4 days (n = 3). Confocal images were taken of MC3T3-E1 cells after treating with (50 μg/mL) and without NS for 2 hours (B1, C1) and 4 days (B2, C2), respectively. Cytoskeletons were shown in green (Fluorescein Phalloidin), while the cell nuclei were blue (DAPI). Scale bar = 50 μm. Arrows indicate aggregations of NS to the cell surface. Results are expressed as mean ± standard deviation (SD) (***p < 0.001 vs. growth medium control).

Fig. 2.

Effects of NS on MC3T3-E1 cells osteogenic differentiation. (A) ALP staining, (B) ALP quantification, (C) Alizarin Red S staining and (D) CPC quantification of MC3T3-E1 cells after treatment with different inductive factors in osteoconductive (OC) medium. Data are expressed as mean ±SD, n = 3. (*P < 0.05, **P < 0.01, ***P < 0.001 vs. OC control)

Fig.3

Osteogenic marker gene expressions (Runx2, OCN and BSP) were quantified by real-time PCR assay after 7 days’ culture. Results are presented as mean±SD (n = 3, *p < 0.05; **p < 0.01 vs. OC control)

Fig.4.

Characterization of nanofibrous scaffolds. (A-F) SEM images of GF and GF/NS scaffolds at low (scale bars=500 μm, left panel) and high (scale bars=10 μm, middle and right panel) magnifications.

Fig.5.

In vitro release curves of BMP2 from scaffolds. (A) Release profile of early-stage (3 days) (B) long term release properties (28 days). All of the results were represented as means ± SD, n = 3. (** p < 0.01, * p < 0.05 vs. GF+BMP2/NS)

Fig.6.

Digital photos and radiographic testing of the histology samples. GF, GF/NS, GF+BMP2(0.5μg), GF/NS+BMP2(0.5μg), GF+NS(1mg/mL), and GF+BMP2/NS after 6 weeks of implantation (n=4~5).

Fig.7.

H&E staining and μCT analysis of the restored calvaries after 6 weeks post-implantation in vivo. (A-F) H&E stained tissue sections acquired from the mouse cranial defects (Scale bars=200μm). (G-H) μCT top view and cross-section image of cranial defects. (I) Quantification measurement of BV/TV in vivo after implanting different scaffolds. All of the results were expressed as means ± SD (n=4~5, ***P < 0.001 vs. GF control).