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

Background: Biofunctionalization of titanium implants for high osteogenic ability holds the hope for advanced implant of promoted osseointegration, especially in the compromised bone condition. In this study, using chitosan-miRNA (CS-miRNA) complex and sodium hyaluronate (HA) as the positively and negatively charged polyelectrolyte, the polyelectrolyte multilayers (PEMs) were fabricated using the layer-by-layer approach on the microarc oxidized (MAO) Ti surface via silane glutaraldehyde coupling. Methods: Dynamic contact angle and scanning electron microscopy were firstly analyzed to monitor the layer accumulation. RiboGreen was used to quantify the miRNA loading and release profile in phosphate-buffered saline. In vitro transfection efficiency and cytotoxicity were investigated after mesenchymal stem cells (MSCs) being seeded on the CS-antimiR-138/HA PEM functionalized microporous Ti surface. The in vitro osteogenic differentiation of MSCs and in vivo osseointegration were also inspected. Results: The surface wettability alternately changed during the formation of PEMs. The CS-miRNA nanoparticles distributed evenly along the MAO surface. The miRNA loading amount increased with the bilayer number increasing. More importantly, a sustained miRNA release of over approximately 2 week was obtained. 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 osteogenic differentiation of MSCs on it in terms of enhanced alkaline phosphatase, collagen product and extracellular matrix mineralization. In the rat model, it led to dramatically enhanced in vivo osseointegration. Conclusions: All these findings demonstrate that novel CS-antimiR-138/HA PEM functionalized microporous Ti implant exhibits sustained release of CS-antimiR-138, and obviously enhances the in vitro osteogenic differentiation of MSCs and dramatically enhanced in vivo osseointegration. This novel miRNA functionalized Ti implant may be used in clinic to allow more effective and robust osseointegration.