Proliferation and Adhesion of Endothelial Cells on the Surface of Titanium Implant

Abstract

To observe the proliferation and adhesion of umbilical cord endothelial cells (HUVEC) on the titanium (Ti) surface with large particles blasted-etched (SLA) treatments and further clarify the mechanism of action of each other between the Ti implant surface and the body. Methods: The human umbilical cord endothelial cells are cultured on the surface of SLA-Ti disks and culture plates. The proliferation of endothelial cells is detected by MTT method and adhesion of endothelial cells observed by PI fluorescence staining and western-bolt detection of protein of VE-cadherin. Results: The proliferation of endothelial cells on the titanium surface is better than the culture plate surfaces, the adhesion cells on titanium surface are more numbers, cell morphology is more extended, titanium surface expression of VE-cadherin protein higher than the culture plate surface. Conclusion: The SLA Ti surface can promote the proliferation and adhesion of endothelial cell and could promote the early healing process of osseointegration. Introduction Dental implant dentures have become the best way to repair edentulous dentition defect. Titanium implants have been used in clinical for decades and have achieved a high success rate due to its excellent mechanical properties, corrosion resistance and biocompatibility (20-month cumulative survival rate was 94.6%, 7-year survival rate 90.6%)1. Aseptic loosening of titanium implant cases occurred at intervals and the mechanism is not very clear, which are located in the interface of implants and body and caused by the wound healing process. After implantation into the body of titanium implants, new blood vessels begin to form at the wound site. The blood vessels formation, beginning from endothelial cells, is major process determine planting success2. Endothelial cell activation surrounding tissue wound healing response, the inflammatory phase of wound healing at the implantation site of vascular damage, blood flow begins to solidify and form a fibrin clot, while the activation of pro-inflammatory leukocytes such as neutrophils and monocytes cell aggregation in the wound area, leukocyte phagocytosis and by generating ROS remove cell debris and metal particles produced by corrosion3. The leukocytes from the blood to reach the wound site through the vessel wall, the production of cytokines (e.g. TNF-α), recruit more leukocytes and activation endothelial cells, stimulation of endothelial cells upregulate the expression of adhesion molecules; and binds to E-selectin surface of endothelial cells, regulating cell adhesion processes (e.g., by expression of ICAM-1); activate endothelial cells to adjust its skeletal structure, allowing leukocytes through endothelial cell between contacts (e.g. by CD31 regulation)4. In addition, activated endothelial cells release pro-inflammatory cytokines (such as E-selectin, ICAM-1 and IL-8), attracted more leukocyte activation and expand the inflammatory response. Around the implant can be observed in a transient inflammatory cytokines (such as IL-1β, IL-6 and TNF-α) expression. Endothelial cells in the wound healing process involved in the formation of early and late tissue inflammation in the proliferative phase of wound healing, fibroblasts and endothelial cells to form new blood vessels; in situ around the reconstruction of implant mineralized tissue formation, maintaining long-term implant osseointegration5. Based on the clinical use up large particles blasted processing of titanium etching (SLA-Ti) surface of cultured endothelial cells, biological response study implant surface of endothelial cells, the International Conference on Material Science and Application (ICMSA 2015) © 2015. The authors Published by Atlantis Press 9 mechanism described implant surface of endothelial cell responses. In this study, we compare the proliferation and adhesion of umbilical cord endothelial cells on the titanium (Ti) surface with large particles blasted-etched (SLA) treatments and further clarify the mechanism of action of each other between the titanium implant surface and the body. Materials and Methods Cell Culture Human umbilical vein endothelial cells were from the Shanghai Sinovac Biotech Ltd. and cultivated in 1640 medium (Hyclone) supplemented with 10% fetal bovion serum (Hyclone), 1% penicillin/streptomycin (Hyclone) in a humidified atmosphere at 5% CO2 air atmosphere at 37 °C. The medium was changed every three days, cells were subcultured after grown to 80% confluence. The endothelial cells in passage four sere seeded on Ti disks treated with sandblasting and acid etching (SLA) with or 24-well culture plates. Cell proliferation Ti disks (18 samples) were placed into 3 sterile 24-well reaction plates. Endothelial cells (500 μl, 1×105 cells/ml) were aliquoted into the three sterile 24-well plates containing the samples and placed in a 37 °C, 5% CO2 intracellular incubator with saturated humidity for 1, 2 and 3 days. At the corresponding time points, the samples were carefully transferred to unused 24-well plates and then rinsed with PBS. Subsequently, the samples were cultured with 30 μM of 5 mg/ml thiazolyl blue (MTT) per well, for 4 h under the same conditions, and the MTT then aspirated. After washing with PBS, 700 μl of dimethyl sulfoxide (DMSO) per well was added and the solution agitated for 10 min. The liquid in each well of 24-well plates was divided into four parts (150 μl) and transferred to a 96-well plate to determine the absorbance value at 490 nm using a microplate reader (Thermo Fisher, USA).The proliferation of HUVEC on the surfaces of Ti disks or culture plates incubation were protected from light, reader OD value in the detection wavelength of 450nm conditions. Cell Adhesion Ti disks were placed individually, in quadruplicate, into the sterile 24-well plates and an endothelial cells suspension at a density of 5×104 cells/ml (100 μl) was pipetted onto the surface of each sample. After 4 h, 500 μl complete medium was added to each well and cells cultured for 48 hours. The samples were rinsed three times with PBS following the removal of the medium. Samples in each well were cultured with 500 μl coloring agent (0.5 mmol/ml PI) at 37 °C for 15 min and observed under a fluorescence microscope (OLYMPUS IX71 / DP70).