Chitosan-miRNA functionalized microporous titanium oxide surfaces via a layer-by-layer approach with a sustained release profile for enhanced osteogenic activity

Abstract

Background: The biofunctionalization of titanium implants for high osteogenic ability is a promising approach for the development of advanced implants to promote osseointegration, especially in compromised bone conditions. In this study, polyelectrolyte multilayers (PEMs) were fabricated using the layer-by-layer approach with a chitosan-miRNA (CS-miRNA) complex and sodium hyaluronate (HA) as the positively and negatively charged polyelectrolytes on microarc-oxidized (MAO) Ti surfaces via silane-glutaraldehyde coupling.

Methods: Dynamic contact angle and scanning electron microscopy measurements were conducted to monitor the layer accumulation. RiboGreen was used to quantify the miRNA loading and release profile in phosphate-buffered saline. The in vitro transfection efficiency and the cytotoxicity were investigated after seeding mesenchymal stem cells (MSCs) on the CS-antimiR-138/HA PEM-functionalized microporous Ti surface. The in vitro osteogenic differentiation of the MSCs and the in vivo osseointegration were also evaluated.

Results: The surface wettability alternately changed during the formation of PEMs. The CS-miRNA nanoparticles were distributed evenly across the MAO surface. The miRNA loading increased with increasing bilayer number. More importantly, a sustained miRNA release was obtained over a timeframe of approximately 2 weeks. In vitro transfection revealed that the CS-antimiR-138 nanoparticles were taken up efficiently by the cells and caused significant knockdown of miR-138 without showing significant cytotoxicity. The CS-antimiR-138/HA PEM surface enhanced the osteogenic differentiation of MSCs in terms of enhanced alkaline phosphatase, collagen production and extracellular matrix mineralization. Substantially enhanced in vivo osseointegration was observed in the rat model.

Conclusions: The findings demonstrated that the novel CS-antimiR-138/HA PEM-functionalized microporous Ti implant exhibited sustained release of CS-antimiR-138, and notably enhanced the in vitro osteogenic differentiation of MSCs and in vivo osseointegration. This novel miRNA-functionalized Ti implant may be used in the clinical setting to allow for more effective and robust osseointegration.

Keywords: Layer-by-layer; Mesenchymal stem cells; Microarc oxidation; Sustained release; Titanium implants; microRNAs.

Scheme 1

Schematic diagram summarizing the fabrication of the CS-antimiR-138/HA PEM-functionalized microporous Ti implant through LbL with enhanced osteogenic activity

Fig. 1

Water contact angles (a) and the lateral views of water drops (b) on the MAO surface after LbL processes

Fig. 2

Morphology of the MAO Ti surfaces before and after CS-miRNA/HA PEM-functionalized inspected by FE-SEM: (a, b and c) pictures of different magnification for the naked MAO surface, (d, e and f) pictures of silane glutaraldehyde functionalized MAO surface, (g, h and i) pictures of 5 layers for the CS-miRNA/HA PEM-functionalized MAO surface

Fig. 3

Fluorescence confocal laser scanning microscope of CS-miRNA/HA PEM-functionalized MAO surface with Cy3-labeled miRNAs: a the top layer starting to display fluorescence and b–i the continuing layers from top to down with an interlayer distance of 400 nm. The fluorescence 3-D images of Cy3-labeled miRNAs on the MAO surfaces before (j) and after (k) 7 days of incubation in cell culture medium at 37 °C

Fig. 4

Quantifcation of antimiR-138 on novel CS-antimiR-138/HA PEM-functionalized MAO surface. a Accumulated antimiR-138 loading amount with increased number of biofunctional layers, b accumulated antimiR-138 release profile from the coated surface

Fig. 5

a Fluorescence images of 40× (upper) and 120× (lower) showing the uptake of Cy3-labelled miRNAs (red) by cells after 24 h of culture on the CS-miRNA/HA PEM functionalized MAO surface: Cell nucleus was visualized using DAPI (blue) and membrane using DIO (green). b Downregulation of microRNA expression by 5 layers miRNA coating. ***p < 0.001 vs the CS-antimiR-control/HA PEM-functionalized MAO surface; ^###p < 0.001 vs the CS-coated MAO surface; ^$$$p < 0.001 vs the naked MAO surface

Fig. 6

FE-SEM pictures showing the cell morphology after 24 h of incubation on different samples: a, b the CS-antimiR-138/HA PEM-functionalized MAO surface (c, d) the antimiR-control/HA PEM-functionalized MAO surface and (e, f) the naked MAO surface

Fig. 7

Relative expression of a ALP, b BMP, c Col1, d OCN, e OSX and f RUNX2 by MSCs cultured on different samples. After culturing in the growth medium for 24 h, the medium was changed to osteogenic medium for further culture of 3 and 7 days. All values are normalized to GAPDH. *, **, ***p < 0.05, 0.01 and 0.001 vs the naked MAO surface; ^#,##,###p < 0.05, 0.01 and 0.001 vs the CS-coated MAO surface; ^{@@, @@@}p < 0.01 and 0.001 vs the CS-antimiR-control/HA PEM-functionalized MAO surface

Fig. 8

The ALP product and collagen secretion after 7 days of culture as well as the ECM mineralization after 14 days of culture: (a, a’ and a’’) the CS-antimiR-138/HA PEM-functionalized MAO surface, (b, b’ and b’’) the CS-antimiR-control/HA PEM-functionalized MAO surface, (c, c’ and c’’) the CS-coated MAO surface and (d, d’ and d’’) the naked MAO surface. The bottom panel shows the semi-quantitative results. ***p < 0.001 vs the naked MAO surface; ^###p < 0.001 vs CS-coated MAO surface; ^&&&p < 0.001 vs the CS-antimiR-control/HA PEM-functionalized MAO surface

Fig. 9

a Transverse and vertical 2-D images and 3-D reconstructed views (ROI, 200 µm from the implant surface) of the Micro-CT analysis to show the new bone formation around the Ti implants at 4 weeks. Scale bar: 15 mm on the 2-D images and 1 mm on the 3-D ones. b The bone volume per total volume (BV/TV), the mean trabecular thickness (Tb.Th), the mean trabecular number (Tb.N) and the mean trabecular separation (Tb.Sp) within the ROI zone. ^{@, @@, @@@}p < 0.05, 0.01 and 0.001 vs the PT surface *, ***p < 0.05 and 0.001 vs the naked MAO surface; ^#,###p < 0.05, and 0.001 vs the CS-coated MAO surface; ^&,&&,&&&p < 0.05, 0.01 and 0.001 vs the CS-antimiR-control/HA PEM-functionalized MAO surface

Fig. 10

a Histological images of undecalcified sections of new bone formation around the implants at 4 weeks by Van Gieson staining. The bone tissue was stained in red color. Scale bar: 200 µm on the above and 100 µm on the below. b Histomorphometric measurement of the BIC in the ROI. ^@@,@@@p < 0.01 and 0.001 vs the PT surface ***p < 0.001 vs the naked MAO surface; ^###p < 0.001 vs the CS-coated MAO surface; ^&&&p < 0.001 vs CS-antimiR-control/HA PEM-functionalized MAO surface

Fig. 11

a FE-SEM pictures showing the new bone on the bone-to-implant interface of different samples. b EDX line scanning of the elements in the direction perpendicular to the bone-to-implant interface. Red frame indicates the new bone area on the implant surface