Influence And Mechanism Of Titanium Nanotube Topography On The Dynamic Migration Of MBMSCs

[Background]Dental titanium implants are widely used in the restoration of missing teeth.By forming a strong osseointegration,it plays a supportive role and has chewing efficiency comparable to natural teeth.However,the problems of long healing time and poor osseointegration have always existed.Therefore,in-depth exploration of the relationship between implant surface characters and osteogenesis is of great significance for steadily improving implant osseointegration.Due to the fact that osseointegration occurs between the surface of the implant and adjacent bone tissues,the surface of the implant is a key factor affecting osseointegration.Due to the fact that bone tissue is composed of nanoscale mineralized collagen fibers,starting from the biomimetic concept,it’s an important strategy to simulating the nanoscale morphology of natural bone tissue for implant surface construction.Our research group has been committed to the construction of titanium nanotube topography and the research of its biological functions for a long time.In vitro and in vivo experiments have confirmed that titanium nanotube topography can directly promote the osteogenic differentiation ability of BMSCs,significantly enhancing the osteogenic activity of implant.However,regardless of the surface of implant,the final rate of osseointegration cannot reach 100%at the microscopic level,indicating the presence of a"no osseointegration area"on the surface of the implant,which may be closely related to the ability of cells to migrate on the surface of the implant.Actually,there is an inherent connection between various behaviors of cells,among which cell migration is closely related to cell differentiation and it’s an important ability to ensure rapid expansion of cells on the surface of materials.However,It lacks in-depth exploration of the surface of biomaterials on cell migration behavior in current research.Therefore,further exploration of the influence and mechanism of titanium topoghraphy on cell migration is of great significance for further improving the osseointegration of titanium implants.[Objective]Based on the previous research by the research group on the promotion of osteogenesis by the topography of titanium nanotubes,to clarify the role of cell migration on surfaces with different nanotube topographies in this procedure;To explore the influence of titanium nanotubes on cell migration related behaviors;To elucidate the key mechanism by which titanium nanotube regulates cell migration.[Methods]Part Ⅰ:The influence of cell migration on osteogenesis on titanium surface1.Nanotube morphology（NT）was constructed on the surface of circular titanium sheets with a diameter of 1.5cm and a thickness of 1mm,as well as pure titanium rods with a diameter of 1.5mm and a length of 5mm,using anodization method.The surfaces with different diameter nanotube topoghraphy formed under 5V and 20V voltages were named NT5 group and NT20 group,respectively.Smooth titanium（PT）surface was used as controls;Scanning electron microscopy was used to detect surface topoghraphy（SEM）;Atomic force microscopy（AFM）was used to detect roughness;Contact angle measuring instrument was used to detects surface hydrophilicity.2.The mouse bone marrow mesenchymal stem cells（mBMSCs）were separated and cultured;After osteogenic and adipogenic induction,the osteogenic and adipogenic differentiation potential of mBMSCs were detected using Alizarin Red staining and Oil Red"O"staining,respectively.In vitro experiments,migration inhibitors Blebbistatin were used to inhibit the migration of mBMSCs on titanium surfaces.Alizarin red and alkaline phosphatase（ALP）staining were applied to evaluate the effect of mBMSCs migration on osteogenesis;In SD rats,migration inhibitor Blebbistatin was used to inhibit the migration of peri implant cells,and the effect of cell migration on peri implant osseointegration was evaluated using Micro CT.Part Ⅱ:Development of titanium surface live-cell imaging attachments1.Titanium was used to make the titanium surface single field live-cell imaging attachment,"titanium cross".With the assistance of the"titanium cross",the cytoplasmic Ca²⁺was observed using an inverted laser confocal microscope.The mBMSCs on the surface of the samples in NT5 group were treated with Thapsigargin（TG）to select the optimal thickness of the"titanium cross";Using the same method to evaluate the application effect of the"titanium cross"by measuring the rate and maximum intensity of cytoplasmic Ca²⁺changes in mBMSCs.2.Stainless steel was applied to make the titanium surface multi field live-cell imaging attachment,"fixator".Firstly,the mBMSCs on the NT5 surface were labeled with live-cell membrane dye.With the assistance of the fixator,an inverted laser confocal microscope was used to observe a titanium plate at different time intervals.The application effect of the fixator was evaluated by whether there was virtual focus during the observation period.Part Ⅲ :The influence of titanium nanotube topography on the migration of mBMSCs1.After fixation with paraformaldehyde,mBMSCs were stained with phalloidin and Hoechst,and the effect of titanium nanotube topography on the cross-sectional migration behavior of mBMSCs was evaluated using an inverted laser confocal microscope;After staining the cell membrane of mBMSCs with active membrane dyes,the effect of titanium nanotube topography on the dynamic migration behavior of mBMSCs was evaluated using an inverted laser confocal microscope with the assistance of a fixator.2.Immunofluorescence staining was performed on the focal adhesion proteins FAK and Vinculin,and the effect of titanium nanotube topography on the focal adhesion proteins of mBMSCs was evaluated using an inverted laser confocal microscope;With the assistance of live cell imaging attachments,the effect of titanium nanotube topography on Calpain activity was observed and evaluated in real-time using an inverted laser confocal microscope.Part Ⅳ:Regulation and mechanisms of titanium nanotubes topography on the cytoplasmic Ca²⁺level of mBMSCs1.With the assistance of live cell imaging attachments,the effect of titanium nanotube topography on cytoplasmic Ca²⁺levels was observed and evaluated in situ real-time using an inverted laser confocal microscope;To evaluate the effect of titanium nanotube topography on the endoplasmic reticulum of mBMSCs using transmission electron microscopy（TEM）;In situ real-time observation of cytoplasmic Ca²⁺levels was conducted using an inverted laser confocal microscope,and the optimal concentration of calcium release activated calcium channel（CRACs）inhibitor CM-4620 was selected for inhibition effect;Using the same method to evaluate whether active CRACs channels are involved in the regulation of cytoplasmic Ca²⁺levels.2.RT qPCR was used to screen the most sensitive subunits of proteins Orai and Stim subunits which make up the active CRACs channels.Reuse the screened most sensitive subunits to screen for the most sensitive time points;To screen for the optimal MOI of lentivirus Orai1-GFP and Stim1-mchery infecting mBMSCs;After infecting mBMSCs with lentivirus using the optimal MOI,the co-localization between Orai1 and Stim1 was observed using fluorescence resonance energy transfer（Fret）,so as to evaluate the effect of titanium nanotube topography on the formation of active CRACs channels.Part Ⅴ:Mechanism study of titanium nanotube topography on the migration of mBMSCs through Stim11.RT-qPCR was used to screen for Stim1 knockdown lentiviruses and validate the efficiency of Stim1 knockdown and overexpression lentiviruses;With the assistance of live-cell imaging attachments,the effects of Stim1 knockdown and overexpression on the dynamic migration of single cells of mBMSCs on titanium surface were evaluated using an inverted laser confocal microscope.2.Using an inverted laser confocal microscope to evaluate the effects of Stim1knockdown and overexpression on the expression of focal adhesive proteins FAK and Vinculin;With the assistance of live-cell imaging attachments,the effect of Stim1knockdown and overexpression on Calpain activity was evaluated using an inverted laser confocal microscope.[Results]Part Ⅰ:The`influence of cell migration on osteogenesis on titanium surface1.The polished titanium surface is smooth,and nanotube arrays with diameters of approximately 30 and 100 nm are formed at voltages of 5V and 20V,respectively;The surface of the NT group can significantly increase the roughness and hydrophilicity of the titanium surface,and the degree of change is positively correlated with nanotube diameter.2.mBMSCs was successfully isolated and cultured,and mBMSCs have the potential for osteogenic and adipogenic differentiation;In vitro experiments,the NT group significantly promoted the extracellular matrix mineralization ability and ALP activity of mBMSCs on the sample surface,And compared to the NT5 group,the promotion effect of the NT20 group was much more significant;When the migration behavior of mBMSCs on the surface of samples in PT,NT5,and NT20 groups was inhibited,the extracellular matrix mineralization ability and ALP activity of mBMSCs were significantly supressed;In vivo experiments,when the migration behavior of cells on the surface of titanium implants in the NT20 group was inhibited,the osseointegration of the implants was also significantly inhibited.Part Ⅱ:Development of titanium surface live-cell imaging attachments1.Successfully developed the titanium surface single field live cell imaging attachment"Titanium Cross",with an optimal thickness of 0.45mm.During the observation period,mBMSCs can react faster to TG,and the maximum cytoplasmic Ca²⁺fluorescence intensity is also higher.2.Successfully developed a titanium surface multi field live-cell imaging attachment"fixator",which can dynamically image multiple fields of view of multiple samples simultaneously.Part Ⅲ:The influence of titanium nanotube topography on the migration of mBMSCs1.Compared with the PT group,the NT group promoted the population migration of mBMSCs;Compared with the PT group,the NT group significantly promoted the single-cell migration of mBMSCs,and the NT5 group had a stronger promoting effect than the NT20 group.2.Compared with the PT group,the NT group significantly promoted the expression of focal adhesive proteins FAK and Vinculin,and the promotion effect of the NT5 group was stronger than that of the NT20 group;Compared with the PT group,the NT group significantly promoted the increase of Calpain activity,and the promotion effect of the NT5 group was stronger than that of the NT20 group.Part Ⅳ:Regulation and mechanisms of titanium nanotubes topography on the cytoplasmic Ca²⁺level of mBMSCs1.Compared with the PT group,the NT group significantly promoted the increase of cytoplasmic Ca²⁺levels,and the promotion effect of the NT5 group was stronger than that of the NT20 group;Compared with the PT group,the NT group can put the endoplasmic reticulum in a stress state,and the stress on the surface of the NT5 group sample is more pronounced than that of the NT20 group sample;The optimal concentration for CM-4620is 10μM;CM-4620 can significantly reduce the level of cytoplasmic Ca²⁺on the surface of samples in the PT,NT5,and NT20 groups.2.Among the subunits the Orai and Stim proteins that make up the active CRACs channels,Orai1 and Stim1 are the most sensitive to time changes.At 1 day,Orai1 and Stim1 are the most sensitive to morphological factors,and the NT group significantly promotes the expression of Stim1.At the same time,the NT5 group has a stronger promoting effect than the NT20 group;The optimal MOI value for mBMSCs infecting lentivirus is 200;Compared with the PT group,the NT group can significantly promote the improvement of Fret efficiency between Orai1 and Stim1,and the promotion effect of the NT5 group is stronger than that of the NT20 group;Meanwhile,the Fret phenomenon on the surface of the PT group,NT5 group,and NT20 group can all be inhibited by CM-4620.Part Ⅴ:Mechanism study of titanium nanotube topography on the migration of mBMSCs through Stim11.The screening result of Stim1 knockdown lentivirus was shRNA2,and the knockdown efficiency of shRNA2 and the overexpression efficiency of p DNA were higher than those of the NC group;Overexpression of Stim1 can significantly promote the single-cell migration of mBMSCs on the surface of PT group and NT20 group samples,but significantly inhibit the migration of mBMSCs on the surface of NT5 group samples.Knockdown of Stim1 can significantly inhibit the migration of mBMSCs on the surface of PT group,NT5 group,and NT20 group samples.2.Overexpressed Stim1 can significantly promote the expression of Vinculin and FAK on the surface of samples in the PT and NT20 groups,but significantly inhibit their expression on the surface of samples in the NT5 group;After knocking down Stim1,the expression of Vinculin and FAK on the surface of PT,NT5,and NT20 groups of samples were inhibited;Overexpression of Stim1 can significantly increase the Calpain activity on the surface of samples in the PT,NT5,and NT20 groups,while knocking down Stim1 can inhibit the Calpain activity on the surface of samples in the PT,NT5,and NT20 groups.[Conclusions]1.The topography of titanium nanotubes can promote the osteogenic differentiation of mBMSCs,and the promotion effect of nanotubes with larger diameter is stronger;The cell migration behavior on the surface of titanium nanotubes can affect the osteogenesis on the surface.2.The topography of titanium nanotubes can enhance Calpain activity,promote the expression of focal adhesive proteins,and facilitate the migration of mBMSCs.And the promoting effect of nanotubes with small diameter is more obvious.3.The topography of titanium nanotubes promotes the formation of active CRACs channels in mBMSCs,which in turn upregulates cytoplasmic Ca²⁺levels,and the promotion effect of nanotubes with small diameter is more significant.4.The topography of titanium nanotubes promotes cell migration,focal adhesive protein expression,and Calpain activity by upregulating the expression of Stim1.