Biocompatibility of Osteoblasts in Titanium Morphology Created by Dc Voltage

Xiao-quan Mao, Department of Implantology, Central South University, China, Email: Xiaoquanmaohorse.m@163.com

Published: 31-Mar-2021

Introduction

Micro arc oxidation (MAO) was a surface treatment method that could produce well-characterized, biocompatible titanium dioxide (TiO2) morphology. Titanium and titanium alloys are widely used as dental implants because of their excellent physiochemical properties and biocompatibility. The clinical long- term success of dental implants was related to their early osseointegration, thus titanium morphology of implant plays an important role in the progression. Effects of titanium morphology treatment determine the optimum surface on the behavior of osteoblast- like cells to promote early Osseo integration.

Materials

Materials and reagents

Pure titanium matrix material: Content of titanium should be no less than 98%, which contains a small amount of impurities such as oxygen, nitrogen, hydrogen, carbon, silicon and iron. Pure titanium in China was classified into several grades, such as TA1, TA2, TA3 and TA4 according to the content of impurity elements. The matrix material of medical pure titanium was TA2 in this experiment. The chemical composition was shown in Table 1 (GB/ T13810-2007), which was provided by Hebei Xingtai Hengzhong metal material Co., Ltd. The line cutting samples would be processed into 10 × 10 × 1 mm3 [1].

Process parameters: In this study, according to previous experiments and domestic and foreign literature, titanium was treated with micro arc oxidation as single variable control method: 200 V, 250 V, 300 V, 350 V, 400 V, 450 V; treatment time: 5 S; the treatment temperature was less than 40℃, the electrolyte parameter was calcium acetate (0.075 mol/L), sodium dihydrogen phosphate (0.03 mol/L) and Ethylene diamine tetra acetic acid EDTA-2Na (10 g/L) [2].

Groups

Group Ti; Group MAO200V; Group MAO250V; Group MAO300V; Group MAO350V; Group MAO400V; Group MAO450V 3 pices respectively [3].

Micro arc oxidation process

Surface treatment of titanium: The medical pure titanium has been cut into 10 × 10 × 1 mm3 . The titanium surface was ground with 600 grit, 800 grit, 1000 grit and 1200 grit SiC papers, and then ultrasonically cleaned with acetone, absolute ethanol and distilled water for 15 min in series. Then cleaned with the acid solution (hydrofluoric acid: hydrogen nitrate: distilled water was 1:4:5). In accordance with previous work [1], the titanium was treated with MAO in an electrolyte for 5 seconds, ultrasonically rinsed with distilled water for 15 min [4].

Micro arc oxidation treatment: Electrolyte contented 0.03 mol/L calcium acetate, 0.075 mol/L sodium dihydrogen phosphate, EDTA-2Na 10 g/L. Electrolyte was mixed by electromagnetic centrifugal. Then it was poured into the electrolytic tank, and the temperature of the electrolyte was ensured less than 40℃ before the test. The pure titanium was the anode and the platinum was the cathode. The sample was soaked in the electrolytic, and the sample could not contact with tank. It was cleaned with ultrasonic wave and distilled water for 15 min after the test. Then it was dried, sealed, and stored [5].

Observation of fixed morphology of cells: MC3T3-E1 cells were transplanted into 24-well cell culture plates with different titanium materials. The number of inoculated cells was 4 × 104 per pore, and the culture was discontinued at 24th hours. After removing the culture medium, it was carefully rinsed with PBS three times for 10 min/time, and then each group of samples was transferred to a new 24-well culture plate and fixed for 24 hours in 4% glutaraldehyde solution. Remove the fluid for cell immobilization, rinse with PBS carefully three times, 10 minutes each time, dehydrate with gradient ethanol solution (30%, 50%, 70%, 80%, 90%, 100%, 100%)
twice, 10 minutes each time, and finally replace with gradient tert-butanol solution (25%, 50%, 100%) for 15 minutes each time. The samples were freeze-dried for 3 hours and sprayed with gold for 60 seconds. The samples were examined by SEM [6].

Adhesion assay: MC3T3-E1 cells were cultured according to the above methods. When the density of MC3T3-E1 cells was reached, the cells were digested by 0.25% trypsin-EDTA, and then the cell suspension was prepared. MC3T3-E1 cells were added into 24-well aseptic cell culture plates with titanium materials of each group in 4 × 104 droplets. The cells were cultured in one group with three multiple holes, respectively, adding conditioned medium, 37℃ and 5% CO2 at constant temperature, and replacing the culture medium every 2 days. Cultures were discontinued at 60 and 120 minutes respectively. CCK-8 was detected.

Cell proliferation test: MC3T3-E1 cells were cultured according to the above methods. MC3T3-E1 cells were put into 24-well aseptic culture plates containing titanium samples of each group in a number of 1 × 104.3 compound holes are a group, 37℃, 5% CO2 incubator respectively. The culture medium was replaced every 2 days. Cultures were discontinued at the 1st, 3rd, 5th and 7th day. CCK-8 was detected.

Alkaline phosphatase activity test: MC3T3-E1 cells were cultured according to the above methods. When the density of MC3T3-E1 cells was reached, the suspension was prepared by 0.25% trypsin- EDTA treatment, and then the number of cells was calculated. MC3T3-E1 cells were transferred to 24 holes of aseptic culture plate with samples of each titanium material group according to the density of 5 × 103 holes. 3 compound holes are a group and cultured in conditioned medium, 37℃ and 5% CO2 incubator, respectively. Change the medium every 2 days. At the 7th and 14th days of culture, the culture was discontinued and the ALP kit was used. The test was carried out according to the instructions.

Statistical analysis

The experimental data were expressed as mean ± standard deviation (SD). Statistical analysis was performed with SPSS 13.0 software (SPSS Inc., Chicago, USA). Paired T test was used to assess the effects of the different voltage treatments. P<0.05 was considered statistically significant.

Ethics Approval

Not applicable.

Author Contributions

The Dr xiao-quan Mao wrote the manuscript.

Acknowledgements

I would like to thank Professor Dr. Tim who modified the manuscript, Zhi-Ping Zheng who edited its grammar.

Competing Interests

The author declares that he has no competing interests.

Funding

It supported by Key projects of Haikou science and Industry Information Bureau

Availability of Data and Materials

Data sharing is applicable to this article; datasets were generated or analyzed during the current study.

References

1. Jo JI, Gao JQ, Tabata Y. Biomaterial-based delivery systems of nucleic acid for regenerative research and regenerative therapy. Regenerative therapy 2019 ;11:123-130.
2. Shen L, Wang X, Li R, Yu H, Hong H, Lin H, et al. Physicochemical correlations between membrane surface hydrophilicity and adhesive fouling in membrane bioreactors. J colloid interface sci. 2017 ;505:900-909.
3. Takamatsu S. Naturally occurring cell adhesion inhibitors. Journal of natural medicines. 2018;72(4):817-835.
4. Hu K, Olsen BR. Osteoblast-derived VEGF regulates osteoblast differentiation and bone formation during bone repair. J clin inv. 2016 ;126(2):509-526.
5. Monaco S, Mehrad M, Dacic S. Recent advances in the diagnosis of malignant mesothelioma: Focus on approach in challenging cases and in limited tissue and cytologic samples. Advances in anatomic pathology. 2018;25(1):24-30.
6. Hanna H, Mir LM, Andre FM. In vitro osteoblastic differentiation of mesenchymal stem cells generates cell layers with distinct properties. Stem cell research and therapy. 2018 ;9(1):1-1.