Surface Modification Of Titanium And Its Effect On Osteogenic Differentiation Of Mesenchymal Stem Cells

Titanium and titanium alloys have been extensively used in orthopaedic and dental fields because of their good biocompatibility and mechanical properties. However, they can only form osteointegration with bone instead of the bone-bonding due to their bioinert. In this work, TiO2 nanotubues on titanium surfaces were used as nano-reserviors for loading several bone growth fators. In vitro experiments of osteoblasts and MSCs were carried out to evaluate osteogenesis ability of surface modified titanium and study the function mechanism.This work should enrich the theory of interaction between implant material and bone tissue and also provide new experimental bases for development of osteoinductive metals.This study includes the following four parts. Firstly, titania nanotubes with different diameters on titanium surfaces were prepared and osteogenic growth peptide (OGP) was loaded onto 100 nm nanotubes. Osteobalsts were cultured on these samples in order to study effects of nano-topograph and adhered OGP on osteoblast behaviors. Loaded OGP on the surface and internal of TiO2 nanotubes layers was identified by energy-dispersive X-ray spectroscopy (EDS) and X-ray photoelectron spectroscopy (XPS). In vitro experiments showed that besides the nanotube diameter, OGP on the nanotubes improved cell adhesion,proliferation and differentiation. Therefore, this simple modified is beneficial to osteoblast growth and can stimulate bone formation.Secondly, several biofactors were loaded onto the nanotubes to investigate influences of the modified nanotubes on mesenchymal stem cells (MSCs). The biofactors used here are common added in medium of MSCs, including dexamethasone, ?-glycerophosphate,osteograwth peptide (OGP) and bone morphogenetic protein-2 (BMP-2). The four factors were respectively adsorbed onto the 100nm TiO2 nanotubues. The modified nanotubes were used for MSCs culture. The nanotubues with ?- glycerophosphate was unbeneficial to cell adhesion but increased expression of some osteogenesis genes. The nanotubes loaded dexamethasone promoted initial cell proliferation and enhanced the expression of collagen I(Col-I), osteopontin (OPN) and calcium content. The OGP modification increased cell numbers and promoted cell adhesion though showed litter obvious effect on osteogenic differentiation. The nanotubues with BMP-2 not only significantly promoted MSCs adhesion and proliferation, but also had a strong capability to induce the activity of alkaline phosphatase (ALP), up-regulate the expression of two specific proteins, osteogenesis genes and calcium content. These results confirmed that the several factors loaded on the nanotubes had different effects on MSCs by different approaches. The nanotubes with BMP-2 showed the mostest significant ability to promote proliferation and differentiation of MSCs.Thirdly, BMP-2 was adsorbed onto the nanotube with different diameters to study effect of the nanotube sizes and BMP-2 on osteogenesis differentiation of MSCs. The XPS and EDS analyses confirmed that BMP-2 was loaded onto the surface and internal of TiO2 nanotubes. The release test showed that the release amount of BMP-2 in the 100 nm nanotubes was greater than those in the 50 nm and 100 nm samples. The 50 nm nanotubes with BMP-2 promoted cell adhesion and proliferation. The 100 nm nanotubes with BMP-2 was more favorable for cell migration, the activity of ALP, the expression of specific proteins and osteogenesis genes. Therefore, BMP-2 and the topology of the 100 nm nanotubes synergistically enhanced the osteogenic differentiation of MSCs.Lastly, chitosan (CS), BMP-2 and DNA were assembled onto the 100nm nanotubes with BMP-2 via a layer-by-layer technique in order to control the release of BMP-2 and long-lasting function. The resultant multilayer film with 100 nm thickness was formed by electrostatic interaction, which can be confirmed by scanning electron microscopy (SEM),XPS, contact angle detection and the degradation of the multilayer films. The simulate degradation and release experiment showed the nano tube substrate exposed after 14 days and most of the multilayer film degraded after 21 days. The cell experiment results confirmed that the multilayer film was not bennifical to the initial cell adhesion but enhanced cell proliferation. The ALP levels, polymerase Chain Reaction (PCR) analysis and immuopluorescence staining of Col-1 and OPN revealed that the multilayer film significantly increased the osteogenic differentiation of MSCs. Moreover, the nanotubes and BMP-2 in the nanotubes could synergistically stimulated osteogenic differentiation of MSCs in the long term. This method combining nano- structurilization and layer-by-layer technique is simple and effective to prepare osteoinductive metal implants. In addition, this work may also inspire other drug controlled release system.