| During the past decades,titanium and its alloys have become the key materials for dental implants,owing to their biocompatibility,chemical stability,and similar mechanical strength with human bones.implant denture has been widely used for the restoration of dentition defect and edentulous for its advantages of good retention,avoidance of injury,protection of adjacent teeth.But there are lacks of periodontal ligaments surround the dental implant,so it will be lack of the similar perception as natural teeth in theory.However,previor studies found that the patients with implant denture and artificial limb had good motion control,proprioception and feedback.The theory of "Osseoperception" was proposed.Because of this weak perception,it’s difficult for patients to realize the overbiting force,which may lead to the absorption of the bone around dental implant,temporomandibular joint disease or even fracture of the implant,which result in the failture of implantation.Therefore,the research of peri-implant nerve regeneration is of great significance.Due to the the approximate elastic modulus between TiO2 nanotubes to bone tissue,high surface-to volume ratio and hollow structures,TiO2 nanotubes were widely used in the construction of local drug delivery applications.Therefore,it is expected to provide new ideas and options to the research of "Osseoperception" by loading neuroprotective agents on TiO2 nanotubes.In additon,Schwann cells(SCs)are the main glial cells in peripheral nerve system and play a critical and substantial role in the peripheral nerve regeneration.How to promote the proliferation,secretion and migration of Schwann cells has profound meaning.Objective:TiO2 nanotubes with three different diameters were prepared at three different voltages.Minocycline was loaded on the wall of the nanotubes by the methods of the ultrasonic vacuum adsorption.To select the ideal diameter of the nanotubes,the drug delivery rate and release capacity of TiO2 nanotubes were investigated.To optimize the dosage of minocycline loaded on the nanotubes for osteosynthesis and neurogenesis,the osteogenic activity of MC3T3-E1 cells and the proliferation,secretion and migration performance of the schwann cells were studied.The synthesized drug carrier with minocycline was expected to construct dual functional active surfaces with both osteosynthesis and neurogenesis.Method:1.Three diameters of TiO2 nanotubes were prepared at the voltage of 30 V,40V and50 V by the methods of electrochemical anodic oxidation with glycerine system as electrolyte and.The 3 types of nanotubes were charactered as TNT30,TNT40 and TNT50.The surface morphology of TiO2 nanotubes were observed by SEM,the crystal structure was observed by X-ray diffractometer(XRD)and the contact angle was measured by contact angle tester.2.100 mg of hydrochloride minocycline and bovine serum albumin(BSA)were loaded on the surface of TiO2 nanotubes by the way of ultrasonic vacuum adsorption.The X-ray photoelectron spectroscopy analyzer(XPS)was applicated to detect whether the drug was loaded.The drug loading rate and releasing capibility of minocycline hydrochloride were detected by HPLC.Under the selected 30 V voltage,The nanotubes with the pipe diameter of 98.4 + 11.4 nm was used as the carrier of the drug delivery system.The minocycline of 5μg、20μg、50μg were respectively loaded on the nannotubes.Samples were divided into Pure titanium group(Pure titanium),TiO2 nanotubes(TNT group),TiO2 nanotube with 5μg minocycline(TNT+ 5 MC),TiO2 nanotubes with 20μg minocycline(TNT+ 20 MC)and TiO2 nanotubes with 50μg minocycline(TNT +50 MC)group.3.The MC3T3-E1 cells were inoculated to the surface of five groups of materials.The adhesion ability of cells was detected by DAPI staining.CCK-8 was used to detect the proliferation of cells.Alkaline phosphatase(ALP)activity was evaluated by thymolphthalein release from thymolphthalein monophosphate and matrix mineralization by staining with alizarin red.The collagen secretion of cells was detected by Sirius red staining,and real-time quantitative polymerase chain reaction(q RT-PCR)was used to detect the expression of osteogenic related genes.4.Schwann cells(RSC96)was inoculated to the surface of five group samples.Cell proliferation was detected by flow cytometry and cck-8.Real-time quantitative polymerase chain reaction(q rt-pcr)was used to detect the expression of the genes involved in nerve repair,and Western blot was used to detect the protein expression of the genes involved in nerve repair.5.A scratch assay was employed to analyze the influence of the five group samples on schwann cells(RSC96).Western blot was used to detect the expression of integrinβ1 and its downstream signaling molecules In this experiment,integrin β1and its downstream signal molecule Focal Adhesion Kinase(FAK),protein kinase B(Akt),p-FAK and p-Akt were detected by Western blot,in order to unveil whether the augmentation of migration was mediated by this signaling pathway.Then integrin β1 was scienced by RNA interfering technique and the changes with cell migration after integrin β1 sciencing was investigated.Results:1.TiO2 nanotubes were prepared at the voltage of 30 V,40V and 50 V by electrochemical anodizing method,with the diameter of 98.4±11.4 nm,136±21.9nm and 238±33.5 nm.As the voltage increases,the tube diameter was larger.After annealing at 400 °C for 3h,the 3 diameters of TiO2 nanotubes were anatase crystal.The contact angle of TiO2 nanotubes with all the three diameters were smaller than pure titanium,and they decreased as the diameter increased.2.Minocycline and BSA were adsorbed by ultrasonic vacuum adsorption method.XPS detected the characteristic peaks of minocycline hydrochloride and BSA.The drug delivery rate of TiO2 nanotubes were up to 75-85% and the drug sustained release capacity were better than that of pure titanium surface.3.TiO2 nanotubes loading with minocycline promoted the early adhesion and proliferation of MC3T3-E1 cells.Meanwhile,TiO2 nanotubes loading with minocycline has a positive effect on the osteogenic differentiation of MC3T3-E1 cells,but has no significant effect on the differentiation at the early age of adhesion.Moreover,the effect of TNT+5MC group and TNT+20MC group were the most obvious,and the cells on the samples loading with minocycline 5 μg and20μg had the best compatibility.4.After inoculated on the five groups of samples from 1d to 4d,RSC96 cells had no significant changes in gene expression,cell proliferation and nerve growth factor(NGF)and glia(GDNF).At 7d,TiO2 nanotube loading with minocycline was significantly promoted cell proliferation and nerve growth factor(NGF)and glia(GDNF)gene expression.In addition,TiO2 nanotubes loading with minocycline inhibits the apoptosis of RSC96 cells and promotes protein expression of NGF and GDNF.The promotion effect of TNT+5MC group and TNT+20MC group were most obvious.5.TiO2 nanotube loading with minocycline promoted the migration of schwann cells(RSC96).In this process,the expression of integrinβ1 and its downstream signaling molecule p-FAK and p-Akt increased.It is shown that the integrinβ1signaling pathway is involved in the process of promoting cell migration of TiO2 nanotube loading with minocycline.After integrinβ1 was silenced by si RNA,Transwell cell migration assay showed that TiO2 nanotube loading with minocycline could still promote schwann cells(RSC96)migration.In conclusion,TiO2 nanotubes loading with minocycline can promote osteosynthesis and acceletate the regeneration and repairing of osseoperception. |
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