| BackgroundThe inorganic mineral group in vivo is formed by complex biological mineralization process. From the point of view of materials science, the forming process can be seen as a structure unit of nanometer scale generated assembly gradually. The process of similar bone mineral formation, grow up and structural evolution after orthopaedic or dentistry repair material implanted in body is the core problem for material excellent fitment. To research the process of similar bone minerals the molecular recognition mechanisms of matrix protein to similar bone mineral and the accurate regulation mechanism of biological environment to similar bone calcium phosphate rock material can offer a basis for biological adaptation theory of implant material, also can provide a theoretical foundation to new orthopaedic or dentistry repair material.Now, the optimal result in tissue engineering and regenerative medicine field is that biological materials can enhance cell differentiation and promote local self-repair. Titanium (Ti) and its alloys have been widely used as implant materials for orthopedic applications owing to their excellent wear, corrosion resistance, light weight but strong mechanical and acceptable biocompatibility properties. However, many implants often clinically fail owing to some biological and mechanical factors. The most important biological factor is incomplete, insufficient prolonged osseointegration, where the implant lacks direct bonding to surrounding tissue. Severe stress shielding between an implant and bone tissue may also occur when there is an imbalance between the mechanical properties of the implant and native tissue. Moreover, there is a risk of infection in approximately1.5～2.5%of all hip and knee arthroplasties. Therefore, there is a clinical need for better implant materials that will keep Ti and its alloys intrinsic advantage and also promoting osteoblast function to sustain long-term osseointegration.Nanotechnology has been applied in different areas of the medical industry. Some researches have found that the structure of material unit (grain or hole) shrink to nanoscale, the character of material can produce great change. Not only improved original material performance, but also made materials obtain new performance and effect. These new performance may expressed on optics, catalytic thermal, photochemistry and sensitivity characteristic. Previous studies have showed sensitivity of cellular responses to nanoscale surface protrusions. Dalby showed human fibroblast rapid adhesion to nanostructure produced by polymer demixing. Sjostrom fabricate different lengths of titania nanopillar structures were obtained via porous alumina mask. MSCs showed the greatest sensibility to nano-topography. Therefore, different lengths of nanostructures may result in different reaction in cell response. The nanometer materials such as nano ceramic, nano fiber and nanometer compound had been reported implanted in human body. It can be acquired different nanostructure through different ways, however, different nanostructure may produce different effect on cells. Some scholars think the reason is that differences in surface properties may have profound effects on extracellular matrix(ECM) protein adhesion and subsequent cell attachment. Surface effects are mediated through integrins, the cell surface receptors that recognize and bind to a specific motif in ECM attachment proteins. The association of integrin receptors with the underlying cytoskeleton has been shown to influence such cell properties as cell shape, differentiation, proliferation, survival, and gene expression. In recent years, many studies have shown that variety of nanosurface materials such as:nanorods, nanotubes and others have advantage in the regulation of cell biocompatibility than titanium, different lengths of the nanorods or different diameters of nanotubes have different influence on cells, which indicated that the different nanostructure produce different influence on cells. In the previous study, most researches based on pure titanium as a control group, less literatures reported differences influence among different nanostructure on cells. Mesenchymal stem cells become a research hotspot in recent years, which can differentiate to multiple directions under different induction effect, this multi-directional differentiation ability making it become important seed cells for tissue engineering. It has been proved that the use of bone marrow mesenchymal stem cells(MSC) have better effect on tissue’s self-repair, their common applications with nano titanium material has been used in dentistry and plastic surgery treatmentsIn our study, we fabricated nanonetwork structure, nanoleaf structure, nanoneddle structure and nano wire structure through adjust the temperature by alkali heat treatment. The purpose of this study is to Co-cultured four different nanostructure with the bone marrow mesenchymal stem cells, observed cell shape and contrast the ability of cell adhesion, proliferation and osseointegration. Found the best advantage nanostructure on cell biological activity.Research objectives1. Fabricated nanonetwork structure, nanoleaf structure, nanoneddle structure and nanowire structure through adjust the temperature by alkali heat treatment. Analysis the characteristics of each nanostructure materials.2. Cultured mouse bone marrow mesenchymal stem cells in vitro, co-cultured cells with each group of materials, use of Calcein-AM/EthD-1method to stain cells on the surface of materials to detection the biological safety of materials, lay the foundation for further study of the biological activity.3. Co-cultured the mouse bone marrow mesenchymal stem cells with each group materials, observed the cell shape by scanning electron microscopy(SEM).4. Detection the ability of cell adhesion and proliferation for each group materials, observed the difference effect of biocompatibility by materials. 5. Detection the ability of cell osseointegration by co-cultured with each group materials, observed the difference effect of materials.Research methods1. Processing the titanium by alkali heat treatment, control the temperature on80℃,100℃,120℃and140℃successively. Analysis the diffraction pattern and composition of nanosurface by X-ray diffraction(XRD), observe the nanostructure on material surface by scanning electron microscope(SEM).2. Revive the bone marrow mesenchymal stem cells in vitro and Co-cultured them with each group materials, observing the material toxicity to the cells by cell staining and combined with cell proliferation, further assess the biological safety.3. Co-cultured the bone marrow mesenchymal stem cells with each group materials24h, after dehydration, sprayed gold and other treatments, observe the cell shape by SEM and contrast the difference by each group materials.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 ELIS A plate reader at490nm. Compare different effects of different types of materials biocompatibility, evaluate differences in the four materials biocompatibility aspects further.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.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 BC A 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.6. Statistical analysis:All data were "mean±standard deviation," said the application of SPSS13.0statistical analysis. Each group of data using factorial design analysis of variance, One-Way ANOVA statistical analysis, pairwise comparisons using LSD method. When heterogeneity of variance, using Dunnetts T3comparison, α=0.05, P<0.05was considered statistically significant.Results1. Processing the titanium by alkali heat treatment, acquire four nanostructure materials by control the temperature on80℃,100℃,120℃and140℃successively, observe the materials by SEM, the nanostructure were nanonetwork structure, nanoleaf structure, nanoneddle structure and nanowire structure respectively. XRD results showed the Ti and nanorod structure were composed of Ti element.2. Stained cells by Calcein-AM/EthD-1method, the result show the most cells stained green fluorescence on materials which mean cells living, indicating that all materials have no cytotoxic to cells, laid the foundation for further experiments.3. In the higher magnification morphology, cells on the nanoneedle and nanowire had a thinner and strip shape,a few uplift area, loose contact with surface; whereas cells attached well on nanonetwork and nanoleaf film surfaces in round shape, spread out more open, especially on nanonetwork film surface. We can saw lamellipodia stretched out, however, only can found filopodia on the nanoleaf film surface4. The numbers of cells adhesion on surfaces of the three samples after30,60and120min of incubations. At the30min, there were maximum numbers of cells adhesion on nanowire surface, but no statistically significant(.P>0.05). With the increase of time, the numbers of cells had significantly increased on nanonetwork surface formed compared to others. We also observed the numbers of cells were increased company with time in each group, these demonstrate show the materials had no toxic reaction.5. Cell proliferation at1,3and5days of incubation were measured by MTT. At day1, there was no statistically difference among the different groups (P>0.05). After3days and5days incubation, the OD value of all the groups was increased when compared with day1. At day3and day5, the OD value of nanonetwork surface group was greater than other groups, also had significant difference. At the same time, we found that the OD value of each group present a declining trend at day3and day5. The OD value decreased from nanoleaf surface to nanowire surface.6. cytoskeletal 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. The results were similar as SEM, cells on the nanonetwork surface spreaded larger area compared to other surfaces, particularly the nanoneedle and nanowire, cells shown long and thin or irregularity shape, in spite of filopodia formed.7. The ALP activity after14days of culturing on samples were measured. It can be found that nanonetwork surface elicited a significant up-regulation of ALP activity compared with other nanosurface(P<0.05). It also can be found that the ALP activity decreased from nanoleaf surface to nanowire surface.8. Different samples incubate with MSCs after21d, the mineralization of MSCs were analyzed by Alizarin Red, the results showed the nanonetwork structure improved the MSCs mineralization, have statistically significant with other materials(P<0.05).Conclusions1. In our study, Fabricated nanonetwork structure, nanoleaf structure, nanoneddle structure and nanowire structure through adjust the temperature by alkali heat treatment. There is a certain innovation in technology and material preparation.2. Use Calcein-AM/EthD-1method to stained cells show that all materials have no cell cytotoxic. There are important reference for subsequent batch of nano surface biocompatibility study materials, also proved nano materials by alkali heat treatment can be used as a safe implant material.3. The result of SEM shown that cells attached well on nanonetwork film surfaces in round shape, spread out more open, which shown nanonetwork structure have advantage to regulation the cell morphology.4. The result of the ability of cell adhesion and proliferation shown nanonetwork structure have advantage on regulation the bone marrow mesenchymal stem cells biocompatibility.5. Nanonetwork structure have more advantage on regulation the osteogenesis mineralization ability of bone marrow mesenchymal stem cells. This ability turn weakened from nanoleaf structure to nanowire structure. |
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