Chemical Functional Groups Effect On The Biological Behavior Of Osteosarcoma Cell

BackgroundA variety of factors including the internal and external environments of cell,may have effect on the biological behavior of cell adhesion, migration, proliferation, apoptosis and necrosis.Therefore, the form of cell on the scaffold would certainly be influenced by many factors like the physical properties of materials, surface chemistry, surface morphology and chemical composition, etc. Recently, more and more researches about the chemical modification of materials increased, and the conclusion is various because of the interference of many material features.The basic research of chemical functional groups,using self-assembled monolayers(SAMs) technology,can ensure the impact of the material surface chemistry on cells only by the single chemical character of the materials, avoiding a variety of intersect factors influence, and the conclusion probably be more reliable.Self-assembled monolayers technology developed earlier, and is a mature technology,have been widely used in many industries all over the world. The SAMs technology has been applied to much more medical basic research earlier in other countries,but which is relatively less and later in our country.Up to now,SAMs has been used to the basic research of some normal tissue cells, like the osteoclasts, human fibroblasts, messenchymal stem cells and neural stem cells. Recently, some domestic scholars began to pay much more attention to a few tumor cells.According to their report, some chemical functional groups have been found to have effect of inhibiting the adhesion and proliferation of breast cancer and hepatoma cells. These studies also found that different chemical functional groups have not the identical effects on the different biological properties of the same cell, and also on the different cell types. That is to say, some chemical functional groups can inhibit the growth of tumor cells, and some other chemical functional groups probably promote tumor cell growth on the contrary.As a kind of malignant bone tumor, osteosarcoma often occurs in young people, and has unsatisfactory treatment, and has the poor prognosis. And so far, there is no very effective way to cure this disease. Due to the fact that there is no report to explain the relationship between the chemical functional groups and the biological behaviors of the osteosarcoma cells, we are working on the research.In our study, the traditional silicon-gold was chosen as the SAMs substrate, and methyl(-CH3), amino(-NH2), carboxyl(-COOH) and hydroxyl(-OH) functional groups were selected as the influence factors.The main purpose is to find the exact effect of these chemical functional groups on these important biological behaviors of osteosarcoma cell,such as the adhesion, proliferation and apoptosis.That is to say, Learn to know what kind of chemical functional groups can give rise to what kind of behavior change of osteosarcoma cells,and learn to know whether the chemical functional groups can inhibit the tumor growth or not.ObjectiveThis topic is an exploratory study of observing the effect of chemical functional groups on the biological behaviors of osteosarcoma cells with the self-assembled monolayers technology.The main purpose will be expained explicitly from the following aspects:To clear the different physical characteristics of the self-assembled monolayers surface by measuring the contact angle of materials.To clear the impact of different chemical functional groups on osteosarcoma cell adhesion.To clear the impact of different chemical functional groups on osteosarcoma cell proliferation.To clear the impact of different chemical functional groups on osteosarcoma cell apoptosis.MethodsThis topic is an exploratory study of observing the effect of chemical functional groups on the biological behavior of osteosarcoma cells with the self-assembled monolayers technology.Therefore, the preparation of silicon-gold substrate, and its modification of chemical functional groups, and osteosarcoma cell culture, and the observation of the cells adhesion,proliferation and apoptosis on the surface of SAMs will be included in our experiment.Specific methods are shown as follows:1. Preparation of gold-silicon substrate and self-assembled with different chemical group.Using self-assembled monolayers technology, the methyl, amino, carboxyl and hydroxyl functional groups were self-assembled on the Au layers respectively. We characterized these surfaces to determine the equivalent surface densities of chemical functional groups by measuring their contact angles. Due to good self-assembled monolayers technique of co-operation unit,the further examination for the morphology of these groups surface by atomic force microscopy (AFM) and X-ray photoelectron spectroscopy (XPS) was missed in our experiment.2. Osteosarcoma cell culture.Firstly, the frozen U2-OS osteosarcoma cell lines were put into37℃water bath for resuscitation, and then shifted into25cm culture bottles. Finally the culture bottles were placed at37℃in5%CO2and95%air.24hours later, the cell culture and daily observation go on after replacing10%FBS RPMI1640medium.Culture medium was changed at any time.After cells growth to be70%to80%confluence, followed with0.25%trypsin digestion, and then passaged culture.The logarithmic growth phase cells are the best choice to do follow-up experiments.3.Adhesion of osteosarcoma cells on the surface of the SAMs.Each hole of24-well plates with a substrate assembled by these chemical functional groups, and each group with three holes. U2-OS cells were seeded on these substrates with a concentration1×104cells/ml at37℃with5%CO2.After3h,6h and9h culture, cell adhesion analyses were done under inverted microscopy. And further adhesion analysis of the cell cultured for6h was done by scanning electron microscopy (SEM).4.Proliferation of osteosarcoma cells on the surface of the SAMs.Each hole of24-well plates with a substrate assembled by these chemical functional groups, and each group with three holes. Cells were seeded on these substrates with a concentration1×104cells/ml at37℃with5%CO2. The ability of cell proliferation cultured in2d,4d and6d was checked by the MTT assay (n=3donors) in our experiments. The use of Live/Dead cell staining is to observe osteosarcoma cell viability after24hours culture on the gold substrates.The activity of LDH was measured spectrophotometrically after culturing with gold substrates in24-well plates for24hours, so as to further understand the cytotoxicity of the chemical functional groups.4. Apoptosis of osteosarcoma cells on the surface of the SAMs.Each hole of24-well plates with a substrate assembled by these chemical functional groups, and each group with three holes. Cells were seeded on these substrates with a concentration5x104cells/ml at37℃with5%CO2,and each hole with the medium500ul.The cell apoptosis was analyzed by the flow cytometry (Millipore, USA) after24hours culture on the gold substrates.Result1.Self-assembled monolayersAfter gold spraying and cutting process, the gold pieces with severe surface scratches and unqualified standard were removed.Then we can get the standard uniform gold-silicon plates without any scratches, all of these gold pieces size is1.2cm×0.8cm.Once these gold-silicon plates were immersed into different thiol solution containing methyl, amino, carboxy and hydroxy for12h, successful self-assembled process happened with the four different chemical functional groups, and an uniform surface of self-assembled monolayers was obtained. To further characterize these different surface of SAMs, we measured their contact angles. Among these test surfaces, the-CH3surface had the highest contact angle of 105°±9.1°, which was the most hydrophobic surface in these test.the-OH surface was the most hydrophilic with a contact angle of9.5°±1.2°. The surfaces of-COOH had the moderate hydrophilic with a contact angle of18.3°±3.9°.The hydrophilic of-NH2surface was less because of its contact angle of-59.7°±3.8°. Statistical analysis results showwed the difference between these chemical groups is significant(Welch=1488.41,p=0.000).2.Osteosarcoma cell culture.In the early experiments, a total of3times,6bottles of cells were contaminated by some bacterials, and the color of the medium change to muddy significantly, and original adherent osteosarcoma cell became necrosis falling into pieces. After the contamination of the cell culture, we tried to find the pollution factors timely.On summary,the first contamination happened during packing fetal bovine serum, and the second was PBS pollution, and the last happened during the period of cell passaged culture. At last,with the help of the extensive literature review related to the cell culture, and with the help of lab teachers for the relevant cell culture technology and key steps,cell pollution problems were not repeated in subsequent experiments throughout the process. After the timely exchange of medium, the passaged cells show good adhesion and proliferation,which is favourable for the follow-up tests.3. Osteosarcoma cell adhesionIn the experiment,the U2-OS cells adhesion on different chemical functional groups was checked by the phase contrast microscope and SEM. Under phase contrast microscope, after3hours culture, almost all of the U2-OS cells adhered onto the surface of the gold sustrates began to present a small round shape, and little cells became small oval. Especially,the cells on the-CH3surface seemed to be very loose.6hours later, the U2-OS cells adhered onto the surface of the gold sustrates began to present more and more morphology changes.The cells volume on-NH2,-COOH,--OH and the bare gold substrate (the control group)surface increased, and large oval, polygonal, spindle cells were seen on these surface,but the cells on-CH3surface were still small round. After9hours incubation, the cell morphology difference existed significantly. The cell on-CH3surface still remained round or oval, but more spindle and irregularly shaped cells began to appear on the other three surfaces.Under phase contrast microscope,the cell number increasing on the-CH3surface was least,and the cell number increasing on the-OH and control group surface with time changes was not so obvious as on the-NH2and-COOH surfaces,the statistical analysis result is not against the Sphericity Assumed(W=:0.813,P=0.394),and the time had cross effect on the group(F=89.315,P=0.000),and the difference between groups at each time point was significant(F=122.008,P=0.000). After3hours culture, the cell number on-NH2surface was similar as-COOH surface(P=0.786), but was higher than the other three groups (P<0.05).In addition,the cell number on-CH3,-OH and Control group surface was also similar(P>0.05).6hours later, no significant increase in cell number on-CH3surface, on the contrary, the number of cells on-NH2surface increased significantly,the difference between-NH2and the others was significant(P<0.05),but the difference between-CH3and the control group was not significant(P=0.094). After9hours incubation, for-NH2,-OH,-COOH groups, the number of cells raised accordingly.,In particular, the number on-NH2surface increased significantly, but it was no obvious on-CH3surface,and there were significant differences among these groups(P<0.05). In addition,the adhesion number of U-2OS followed the trend:-CH3<<-OH<-COOH≈-NH2.In order to further prove the different morphology changes, SEM indicated that U-2OS cells cultured on OH and COOH functional groups after6h incubation exhibitted polygonal and oval morphology,those on NH2showed a spindle shape, while cells cultured on-CH3group were smaller and in a spherical shape,.In addition, these cells on OH and COOH surfaces had bigger volume and abundant cytoplasm, and more pseudopodia extended to a larger area.But the cell on-CH3surface remained round and small volume yet.4.Osteosarcoma cell proliferation4.1MTT resultsThe U-2OS cells on different surfaces were uniformly seeded at the beginning of experiment. From day2to day6, there was a significant cell proliferation increasing on each group surface. However, the cells on-CH3surface were at a low O.D. level compared with-OH functional group, and the cell proliferation on-COOH and Control group surfaces was similar at day2(P=0.203)and at day4(P=0.700).But the cell proliferation difference between on-CH3and the other group surface at any time was also significant(P=0.000).The cell proliferation difference between on-OH surface and on the other group surfaces at day2and day4were also significant(P<0.05), especially the number increasing was more obvious at day6,but the difference was not significant compared with-NH2and -COOH surface (P=0.057and0.948respectively). The proliferation on-NH2and-COOH surface were all obvious,the difference was not significant at day2(P=0.752) and day6(P=0.051),but the difference was significant at day4(P=0.006).It appeared that-COOH、-OH and-NH2groups had a promoting effect on U-2OS proliferation in a longer culture period. On the contrary, the-CH3group had a negative effect on U-2OS proliferation. Furthermore, the proliferation capacity of U-2OS on different surfaces followed the trend:-COOH>-OH>-NH2>>-CH3.4.2cell vitality on different chemical functional groups surfaceIn this experiment, we selected24hours incubation as a time point to test the cell viability with Live/Dead cell staining kit, and the marked result was recorded under fluorescent microscope. Wherein the living cells labeled in green, dead cells marked in red. We found that there were little cells on CH3surface, which consisted most of dead cells. In contrast, on-COOH,-NH2and-OH surfaces, there were much more viable cells in a much larger contact area. The U-2OS cells cultured on OH and COOH functional groups exhibitted polygonal and oval morphology,those on NH2showed a spindle shape, which were consistent with the SEM results. The survival rates of cells on different surfaces followed the trend:-NH2>-OH>-COOH>>-CH3. These results supported the idea that the-NH2functional group diminished cell toxicity whereas-CH3exhibited cell toxicity.The result is a strong evidence that-CH3group has the effect of inhibiting osteosarcoma cell proliferation.4.3LDH analysisFrom the result of MTT and Live/Dead cell staining,we can have a hypothesis that the cells on-CH3surface died from cell membrane damaged and the outflow of intracellular substances. To test this assumption, we measured the release of the cytoplasmic enzyme LDH by adherent cells on chemical groups modified substrates. Based on the results of the cell viability test, we chose a24h incubation time as the test time point.The result proved that the largest number of methyl lactate dehydrogenase release on-CH3group, and the lowest lactate dehydrogenase activity on-NH2functional group, and the lower one on the control group. Statistical analysis result showed homogeneity of variance(F=1.716,P=0.223),and the difference between groups was significant (F=111.549,P:=0.000).Further analysis showed the difference between-CH3and the others was also significant(P=0.000),and the difference between-OH and the others was also significant(P<0.05),but the lactate dehydrogenase activity was similar among-NH2、-COOH and Control groups(P>0.05). Assays of LDH activities were observed in the followed trend:-CH3>-OH≥-COOH>-NH2, which were agreed with the MTT results. It showed the relationship between cell toxicity and the release of LDH enzymes.5.Osteosarcoma cell apoptosisU-2OS cells on different chemical groups were applied for apoptosis analysis using Annexin V-PE and7-AAD. The combination of Annexin V-PE and7-AAD is useful for apoptosis cells analysis whether in the early stage of apoptosis or not. As is shown, the-CH3group caused about7.6%apoptosis and12.5%necrosis, whereas the-NH2group caused approximately4.3%apoptosis and12.6%necrosis. The apoptosis rate on the-OH surface was only2.1%,which was the lowest,but its necrosis rate was12.3%. The-COOH group caused approximately4.8%apoptosis and11.9%necrosis. Statistical analysis result showed homogeneity of variance whether apoptosis rate(F=1.075,P=0.413) or necrosis rate(F=0.162,P=0.919),and the difference between groups was significant (F=396.20,P=0.000). Further analysis showed the difference of apoptosis rate between groups was significant(P=0.000),and the difference of necrosis rate between groups was also significant(F=7.29,P=0.01).The difference of apotosis rate between-CH3and-COOH was significant(P=0.007),and the difference of apotosis rate between-NH2and-COOH or-OH was also significant(P<0.05).The apoptosis rate was in the followed trend:-CH3>-COOH>-NH2>-OH.These results indicated that all of cells on different chemical functional groups surface had a certain degree of apoptosis, but the overall effect was not significant.From all of results aboved, we may have a conclusion that NH2surface had the best biocompatibility than-CH3surface, and-CH3can induce apoptosis and necrosis of osteosarcoma cells earlier. That is to say,-CH3group has a stronger anti-osteosarcoma cell activity.Conclusion1. Surface of self-assembled mono layers presents us an equivalent surface densities of chemical functional groups, and a three-dimensional structure. The results of contact angle measurement prove-CH3surface be a most hydrophobic group, and-OH surface be the most hydrophilic group, and-NH2,-COOH be the lower hydrophilic groups.The hydrophilic ability followed the trend:-OH>-COOH>-NH2>-CH3.2. Subculture of osteosarcoma cells belongs to adherent cell culture technology,which is a mature cell culture technology. But there may be contamination in the whole cell culture process, causing the experiment failed. It is important to find pollution factors timely and seriously, and to improve our cell culture technology continuously, which ensure the follow-up experiments effectively.3.Our work provides evidence that chemical properties of the biological material surface have an impact on the growth of osteosarcoma cells, and different chemical functional groups have not the same effects on the cells.Although-NH2and-COOH promoted cell apoptosis,but sustained visible cell adhesion and promoting cell growth. Not only-OH surfaces promoted cell proliferation,but also exhibited the weakest ability of inducing cell apoptosis. In contrast,-CH3surfaces showed anti-cancer effect from inhibiting cell growth, poor cell adhesion, high apoptosis and necrosis.