Surface Nanostructuring Of Titanium Mateirials And Its Effect On Mesenchymal Stem Cells

After implantation of a biomaterial into a host, the interaction betweenbiomaterials and biological system （protein adsorption, cell adhesion/proliferation, etc）occurs at the biomaterial surface. The biological behaviors of cells are mainlydominated by the local microenvironment of chemistry and topography on macroscle,mesoscale, and microscale sizes. Thus, how to construct desirable microenvironmentsonto the material surface, in turn mediating cells’ physiological functions, becomes oneof the hot topics in the related field.Titanium （Ti） and its alloys have been widely applied as bone implants in clinicalapplications because of their good mechanical properties. Nevertheless，titanium basedmaterials are surface bioinert, being lack of the potential for inducing tissue formation,which results in poor osseointegration between the implant and its surrounding naturebone tissue and short lifespan of the implant. It is the common challenge in clinicalapplication. Considering those issues, to realize surface-mediating control of thebiological behaviors of cells and induction of bone tissue formation, it is urgent todevelop novel techniques. To improve the osseointegration of titanium based implants,in this study, surface mechanical attrition treatment （SMAT） technique and anodizationwere employed to fabricate surface nanostructured titanium, and then depositedapatite/gelatin composite to construct desirable microenvironments, from theperspective of mimicking the bone-like nanoscale architecture and the components ofextracellular matrix.Main contents and conclusions of this research are listed as follows:1. Regulation of the behaviors of mesenchymal stem cells by surface nanostructuredtitaniumThe study describes the influence of surface nanostructured titanium on thebehaviors of mesenchymal stem cells （MSCs）. Surface nanostructures of titanium wereproduced with surface mechanical attrition treatment （SMAT） technique. Field emissionscanning electron microscopy （FE-SEM）, transmission electron microscopy （TEM）,atomic force microscopy （AFM）, X-ray diffraction （XRD） and contact-anglemeasurements were used to characterize the surfaces of native titanium and surfacenanostructured titanium substrates were characterized, respectively. A thinnanostructured layer was formed onto the surfaces of titanium substrates after SMAT treatment. Immunofluorescence staining of vinculin, osteocalcin （OCN）, andosteopontin （OPN）, MTT test, the levels of （Alkaline phosphatase） ALP and OCN andthe mRNA expressions of OCN, OPN, collagen type I （Col I） and Runx2（runt-relatedprotein2） were examined, in order to evaluate the effects of the surface nanostructuredtitanium substrates on the adhesion, spreading, proliferation and osteoblasticdifferentiation of MSCs at cellular and molecular levels in vitro. The results suggest thatthe surface nanostructured substrates were beneficial for the growth of MSCs, includingadhesion, filament orientation, proliferation and gene expression. This approach for thefabrication of surface nanostructured titanium may be exploited in the development ofhigh performance titanium-based implants.2. Surface functionalization of TiO₂nanotubes with bone morphogenetic protein2and its synergistic effect on the differentiation of mesenchymal stem cellsTo investigate the influence of surface-functionalized substrates withnanostructures on the behaviors of mesenchymal stem cells, we conjugated bonemorphogenetic protein2（BMP2） onto TiO₂nanotubes with different diameter sizes of30,60, and100nm for in vitro study. Polydopamine was employed as the intermediatelayer for the conjugation of BMP2. The successful conjugation of BMP2onto TiO₂nanotubes was revealed by field-emission scanning electron microscopy （FE-SEM）,X-ray photoelectron spectroscopy （XPS）, and contact angle measurements.Immunofluorescence staining of vinculin, osteocalcin （OCN）, and osteopontin （OPN）revealed that BMP2-functionalized TiO₂nanotubes were favorable for cell growth.More importantly, MSCs cultured onto BMP2-functionalized TiO₂nanotubes displayedsignificantly higher （p <0.05or p <0.01） d ifferentiation levels of ALP andmineralization after7and14day cultures, respectively. The results suggested thatsurface functionalization of TiO₂nanotubes with BMP2was beneficial for cellproliferation and differentiation. The approach presented here has potential applicationfor the development of titanium-based implants for enhanced bone osseointegration.3. Construction of microenvironment onto titanium substrates to regulate theosteoblastic differentiation of mesenchymal stem cells in vitro and osteogenesis invivoTo mimic the extracellular matrix of natural bone, apatite/gelatin composite wasdeposited onto nanostructured titanium substrates via a coprecipitation method, whichwas pretreated by potassium hydroxide and heat treatment to generate an anticorrosivenanostructured layer. The successful formation of the apatite/gelatin nanocomposite onto titanium surfaces was revealed by Fourier transform infrared spectroscopy,field-emission scanning electron microscopy, atomic force microscopy （AFM）, and thinfilm X-ray diffraction （TF-XRD） measurements, respectively. The immunofluorescencestaining of vinculin revealed that the apatite/gelatin nanocomposite deposited titaniumsubstrate was favorable for cell adhesion. More importantly, mesenchymal stem cellscultured onto the apatite/gelatin nanocomposite deposited titanium displayed higher （p<0.05or p <0.01） proliferation and osteoblastic differentiation levels of alkalinephosphatase, mRNA expressions of collagen I （Col I）, osteocalcin （OCN） andosteopontin （OPN）, and OCN content after culture for7,14, and21days, respectively,which was also revealed by the immunofluorescence analysis of OCN and OPNexpression. The deposition of apatite/gelatin nanocomposite improved bone density （p <0.05） and bone-implant contact rate （p <0.05）, which was reflected by microcomputedtomography analysis and histological evaluation in vivo using a rabbit model. This workprovides an approach to fabricate high-performance titanium-based implants withenhanced bone osseointegration.