| BackgroundTotal hip and knee arthroplasty have being successfully performed in world-wide in recent years. These procedures have been dramatically increased due to increasing aging population and higher demands regarding physical activity. Traditional orthopedic implants are mainly made by Titanium (Ti) and its alloys, because their excellent wear resistance, strong biomechanical and fine biocompatibility properties. Although the results of arthroplasty are exciting, some implants eventually failed, revision for some patients are inevitable. Over50,000revision Total hip arthroplasties are performed every year in the US with the cost of exceeding$1billion. Poor osseointegration between implant and host bone tissue is one of the main causes aseptic loosening in Ti-based materials and result in failure. There is a clinical need to explore a better implant surface which can provide better osseointegration without forming a fibrous tissue interface.Traditional prosthesis usually composed of polish surface which lack desirable bioactive properties. The influence of implant surface topography on cell behaviors has been widely explored. Nanotechnology has been applied in almost every field from drugs to machinery. Fabricating nano-topography is a promising method to improve cell behavior. Previous studies have showed sensitivity of cellular responses to nanoscale surface protrusions. Dalby showed human fibroblast rapid adhesion to27nm high nano-islands produced by polymer demixing. Another study revealed13nm islands increased adhesion, proliferation and up-regulation of gene expression while decreased on the95nm islands surface. Recently, different lengths of titania nanopillar structures were obtained via porous alumina mask. MSCs showed the greatest sensibility to15nm nano-topography. Therefore, different lengths of nanostructures may result in different reaction in cell response.Different methods may fabricate different nanostructures on metals. In previous study, the length of Ti nanorod could be tuned via tailoring the electrochemical time. Variant lengths of nanorod could be fabricated. The biological performance of materials can be quantitative researched for the precise control the length of the titanium nanorods. The purpose of this study is to determine the behavior of MSCs cultured on different lengths of nanorod and investigate the effect of such nanorod surface on bone mesenchymal stem cell (MSC) morphology, cell proliferation and differentiation.Objective1. To study the preparation methods of the Ti nanorod structure and analyze their structural characteristics, physical and chemical properties.2. To study the Cell compatibility of the Ti and Ti nanorod structure.3. To study the effects of different lengths of Ti nanorods topography on mesenchymal stem cell growth, proliferation and differentiation.4. To explore the mechanism of Ti nanorods topography on mesenchymal stem cell.5. To explore the histocompatibility and the integrity of material-bone interface after implant into rabbit.Study methods 1. Material preparation:Electrochemical anodization was conducted in a two-electrode configuration, where Ti and Cu foils worked as the working and counter electrodes, and a mixture of1.45wt%NH4F and1.45wt%H2C2O4was used as the electrolyte. Ti foils were contacted with a Cu plate and then pressed against an O-ring, leaving a dimension of3×3cm2exposed to the solution. Before electrochemical treatment, the Ti foil was placed in the fluoride-containing solution for10min. Anodization was performed by applying a constant current of200mA for different time (20min,30min and40min) at room temperature via a DC power supply. The as-prepared specimens were thoroughly washed with deionized water.2. Field emission scanning electron microscopy was employed to characterize the morphology of nanorods on titanium surface. Atomic force microscopy was carried out under phase mode with a scan rate of0.8Hz and a scan size of1*1um2. XRD was applied for analyzing the element of materials. The protein concentrations in the collected solutions were determined by using a MicroBCA protein assay kit.3. LDH test, MTT assay and Calcein-AM/EthD-lwere applied to study the Cell compatibility of the Ti and Ti nanorod structure.4. Cell morphology on sample surfaces was examined on SEM. the adhesion and spreading of the cells on the different samples were observed by SEM. Cells were seeded on the substrates and allowed to attach for30,60and120min. At each prescribed time point, the non-adherent cells were removed by rinsing with PBS solution. Cells were fixed and stained with40,60-diamidino-2-phenylindole (DAPI, Sigma). To estimate the density of viable cells, MTT assay was employed. After1,3and5days, the MTT solution was added and the specimens were incubated at37℃to form formazen. After4h of incubation in5%CO2incubator, the formazen was then dissolved using dimethyl sulfoxide (DMSO) and the optical density (OD) was measured at an ELISA plate reader at490nm. After14days culture in osteogenic medium, the ALP activities were determined by a colorimetric assay using an ALP reagent containing p-nitrophenyl phosphate (p-NPP, Amresco) as the substrate. The absorbance of p-nitrophenol formed was measured at a wavelength of405nm. The intracellular total protein content was determined using the BCA protein assay kit (Pierce) and the ALP activity OD values were finally normalized to the total protein content with OD values correspondingly. After14days culture in osteogenic medium, Mineralization of difference samples were analyzed by Alizarin Red.5. After24h of culture, TRITC-conjugated phalloidin in1×PBS was added and incubated for1h to dye the cytoskeleton. Following this, DAPI was added and incubated for30min to dye the cell nucleus, then visualized and photographed using a red (actin) and blue (DAPI) filter by a fluorescence. The long axis and wide axis across the centre of cell nucleus were measured. The length/width ration was applied to analyze the morphology of cell. After14d of incubation of cell with different samples, PCR was applied to measured the expression of the osteogenesis related gene(OCN, OPN, ALP, RUNX2COL1).6.100nm Ti and Ti samples were implanted into rabbit tibia. After4,12weeks, Micro-CT, push out forces and histology were applied to analyze the osteogenesis of biomaterial-bone interface.Results1. Surface fabrication and characterization1.1A mixture of1.45wt%NH4F and1.45wt%H2C2O4was used as the electrolyte. Anodization was performed by applying a constant current of200mA for different time at room temperature. Variant length of Ti nanorod could be tuned via tailoring the electrochemical time. The samples were observed under SEM, the result showed30nm,100nm and120nm length of nanorods were acquired after applying a constant current of200mA for20min. 1.2EDS results showed the Ti and nanorod structure were composed of Ti element.1.3The AFM results showed that the Ti nanorod structure could improve the roughness of titanium surface. The roughness of the titanium surface and Ti nanorods of different length were calculated as7.76±1.49nm,9.51±1.26nm,13.45±1.49nm and19.06±4.19nm, respectively.1.4Ti,30nm,100nm and120nm were incubated with10%FBS DMEM culture media after4h, the protein concentrations were determined by using a MicroBCA protein assay kit. The results showed no statistically among different samples.2. Cell compatibility of Ti nanorod2.1Difference samples cultured with L929after24h, LDH test showed the samples have no cell toxic.2.2Difference samples cultured with L929after1,3,5d, MTT assay showed all the four samples have no cell toxics.2.3Difference samples cultured with L929after Id, Calcein-AM/EthD-1showed the cell cultured on the all samples are alive, no cell toxic was observed.3. Effects of different lengths of Ti nanorods topography on mesenchymal stem cell growth, proliferation and differentiation.3.1In the higher magnification morphology by SEM, cells on the control Ti had a thinner and well spread shape; whereas cells attached well on nanorods surfaces in round shape, stretched more elongate (higher length/width ratio), especially on100nm nanorods:100nm Ti>30nm Ti>120nm Ti>Ti.3.2MSCs adhered on different surfaces after30min,60min and120min of incubation. The numbers of cells adhesion on surfaces of the four samples after30,60and120min of incubations were measured. At each time interval adopted in this study, the numbers of cells adhesion on30nm and100nm nanorods were statistically larger than the flat Ti. The numbers of cells on100nm surface were always the largest at all time interval among the different groups.3.3Cell proliferation at1,3and5days of incubation were measured by MTT. At day1, cells culturing on nanorods surfaces showed higher OD value than the control Ti, however, there was no statistically difference among the different groups (P>0.05). After3days incubation, the OD value of all the groups was increased when compared with day1. The OD value of30nm and100nm nanorods showed statistically significant differences compared with the control Ti (P<0.05). At day5, the cell numbers in nanorods surfaces showed statistically larger compared with the control Ti group (P<0.05). In addition, the OD value was highest for the100nm nanorods at day5.3.4The ALP activity after14days of culturing on samples were measured and the results were exhibited in Fig.8. It can be found that100nm length of nanorod elicited a significant up-regulation of ALP activity compared with the control Ti (P<0.05). There was no significant statistical differences among30nm,120nm length nanorod Ti and control Ti groups, although30nm length of nanorod enhanced the ALP activity over the control Ti. ALP activeity:100nm Ti>30nm Ti>120nm Ti>Ti.3.5Different samples incubate with MSCs after21d, the mineralization of MSCs were analyzed by Alizarin Red, the results showed100nm nanorods improved the MSCs mineralization.30nm Ti and120nm Ti were showed no statistically different when compare that with control Ti.4. To explore the mechanism of Ti nanorods topography on mesenchymal stem cell.4.1Cytoskeletal stained by TRITC-conjugated phalloidin in MSCs after24h of culture on the different surfaces. The length and width axes across the central of nuclei were measured by using of the scale bar. The length/width ratio was used as the elongation ratio to define the cell morphology changes. Control Ti samples cultured MSCs with well-defined cytoskeleton. The cells’cytoskeletons were in more elongation shape (higher length/width ratio) on30nm and100nm structures when compared with the control Ti. While MSCs cultured on the120nm high structures demonstrated less spread and decrease elongation morphology compared with that on other groups. The length/width ratio:100nm Ti>30nm Ti>120nm Ti>Ti.4.2Different samples incubate with MSCs after14d, PCR was applied to measure the osteogenesis related gene expression. The results showed100nm nanorod up-regulate the ALP, OCN and OPN gene expression. There were no statistically difference among difference samples about RUNX2and COL1.5. To explore the histocompatibility and the integrity of material-bone interface after implant into rabbit.5.1Ti and100nm Ti were implanted into rabbit tibia, all the rabbit were eat and act with normal. No wound infection and inflammation were observed. No tissue necrosis were found at biomaterial-bone interface.5.2Ti and100nm Ti were implanted into rabbit after4,12weeks, Micro-CT were taken. Ti were implanted into rabbit after4weeks, Micro-CT showed lacune exist in the bone-biomaterial interface; after12weeks, the lacune become smaller. When100nm Ti implanted after4weeks, the bone-biomaterial interface showed better osseointegration when compared that with Ti. After12weeks, the interface fused well without forming a fibrous tissue.5.3Ti and100nm Ti were implanted into rabbit after4,12weeks, Push-out force were taken. The push-out force of100nm Ti was statistically higher than Ti.100nm Ti improved the bone formation at the bone-material interface.5.4Ti and100nm Ti Masson staining after implant into rabbit4and8weeks. The surrounding of Ti was red staining, which is mature bone. Gap was observed between Ti and bone. In100nm Ti groups, red and blue staining were observed, COL1was produced after4weeks. More blue staning was observed after8weeks, the interface of bone-material was tight, new bone growed.100nm Ti improved the bone formation.Conclusion1. In present study, a mixture of1.45wt%NH4F and1.45wt%H2C2O4was used as the electrolyte. Anodization was performed by applying a constant current of200mA for different time at room temperature. Variant length of Ti nanorod could be tuned via tailoring the electrochemical time. The samples were observed under SEM, the result showed30nm,100nm and120nm length of nanorods were acquired after applying a constant current of200mA for20min. Ti,30nm,100nm and120nm were incubated with10%FBS DMEM culture media after4h, the protein concentrations were determined by using a MicroBCA protein assay kit. The results showed no statistically among different samples.2. LDH test, MTT assay and Calcein-AM/EthD-1showed Ti and Ti nanorod structure were in good cell compatibility. The results showed the samples have no cell toxic in different ways.3. In the higher magnification morphology by SEM, cells on the control Ti had a thinner and well spread shape; whereas cells attached well on nanorods surfaces in round shape, stretched more elongate (higher length/width ratio), especially on100nm nanorods. At each time interval adopted in this study, the numbers of cells adhesion on30nm and100nm nanorods were statistically larger than the flat Ti. The numbers of cells on100nm surface were always the largest at all time interval among the different groups. MTT assay results showed nanorods structures improved the MSCs proliferation. The numbers of cells on100nm surface were always the largest at all time interval among the different groups. ALP assay result showed00nm length of nanorod elicited a significant up-regulation of ALP activity compared with the control Ti. Different samples incubate with MSCs after21d, the mineralization of MSCs were analyzed by Alizarin Red, the results showed100nm nanorods improved the MSCs mineralization.4. Control Ti samples cultured MSCs with well-defined cytoskeleton. The cells’ cytoskeletons were in more elongation shape (higher length/width ratio) on30nm and100nm structures when compared with the control Ti. The length/width ratio:100nm Ti>30nm Ti>120nm Ti>Ti. Different samples incubate with MSCs after14d, PCR was applied to measure the osteogenesis related gene expression. The results showed100nm nanorod up-regulate the ALP, OCN and OPN gene expression. There were no statistically difference among difference samples about RUNX2and COL-1.5. Ti and100nm Ti were implanted into rabbit after4,12weeks, Micro-CT were taken. The bone-biomaterial interface of100nm Ti showed better osseointegration when compared that with Ti at4week and12week point. Ti and100nm Ti were implanted into rabbit after4,12weeks, Push-out force were taken. The push-out force of100nm Ti was statistically higher than Ti.100nm Ti improved the bone formation at the bone-material interface. Masson staining results showed the interface of bone-material was tight, new bone growed.100nm Ti improved the bone formation. |
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