Study On The Mechanism Of Transcription Factor FOXA1 Inhibiting Proliferation And Promoting Apoptosis Of Basal Breast Cancer Cell

Objective:Bone defects caused by trauma,tumor or infection are common diseases in the clinic,and lead to a huge burden on patients and society.And new artificial bone repair materials with advanced properties have the potential to become a viable alternative to autologous and allogeneic bone transplantation.In addition,extracorporeal physical hyperthermia is a traditional and common treatment method for superficial tissues regeneration such as skin and soft tissues.Preparing photothermal bone regeneration materials will allow this noninvasive technique to be extended to deep bone tissue therapy,and will provide a new strategy to repair bone tissue.Calcium phosphate（Ca P）materials have unique superiority among the artificial bone repair materials owing to the similarity with the main inorganic components of human bones and teeth.Based on this,this study aims to the Ca P materials and focus on its microstructure and properties,via regulating and modificating the Ca P materials to explore an advanced Ca P artificial bone repair material.The research contains two mainly parts.The first part is djusting the condition of reaction system to perare the Ca P materials with special structures,then utilizing the property of the special structure of Ca P materials to construct a composite scaffold based on the Ca P and organic polymer materials,and exploring the effect of this new composite scaffold in bone regeneration.The second part is using natural small molecule drugs to modify Ca P materials for the photothermal Ca P materials preparation,and introducing the traditional physical hyperthermia therpy into the deep bone tissue repair,then evaluating the safety and effectiveness of this new type material and treatment method.Methods:（1）Preparation and modification of calcium phosphate materials.（A）.Hydroxyapatite microtubes（HAMT）with high crystallinity and uniformly dispersed property were prepared by hydrothermal solvent method.（B）.The multi-dimensional hydroxyapatite（HA）materials including one-dimensional nanoparticles（HANP）and two-dimensional nanowires（HANW）were obtained via the microwave-assisted method,and different dimensions HA materials were mpdified by gallic acid（GA）using the microwave assistance that resulting in the photothermal HA materials with photothermal property were prepared,which named photothermal HANP（GNP）,photothermal HANW（GNW）and photothermal demineralized bone matrix（GBM）.（2）Characterization of the as-prepared calcium phosphate materials.The as-prepared HAMT,HANP,HANW,GNP,GNW,demineralized bone matrix（BM）and GBM were characterilized by the scanning electron microscope,transmission electron microscope,selected area electron diffraction,energy dispersive spectroscopy,X-ray diffraction,Fourier transform infrared spectroscopy,and ultraviolet-visible spectroscopy to evaluate the physical and chemical properties of these materials.（3）Preparation and characterization of the GH/H composite scaffold.Based on the special microtube structure of HANT,the Gel MA/HAMT composite scaffolds（GH/H）were construced by combination the HAMT and Gel MA hydrogels.Then the scanning electron microscope,energy dispersive spectroscopy,X-ray diffraction and Fourier transform infrared spectroscopy were used to characterize the pore size,porosity,surface roughness and chemical composition of the GH/H composite scaffold.Besides,the mechanical properties of the GH/H composite scaffold were evaluated by the stress-strain and anti-fatigue compression expetiment.（4）Photothermal performance evaluation of photothermal HA materials.The thermal imager was used to study the photothermal performance of GNP,GNW and GBM under different conditions,including the power of near-infrared（NIR）,the concentration of photothermal HA materials,the environment（dry and liquied）and in vivo pthotothermal performance.In addition,the photothermal stability of GBM is tested also.（5）The biocompatibility of calcium phosphate materials.Bone marrow mesenchymal stem cells（BMSCs）were used to evaluate the biocompatibility of the calcium phosphate materials.After cocultured with the HAMT,GH/H composite scaffolds,GNP,GNW and GBM,the cell viability were tested by CCK8 or MTT method.The live-dead cell staining was used to observe the growth of BMSCs on GH/H composite scaffolds and GBM.And the morphology of BMSCs on the GH/H composite scaffolds was observed by the cytoskeleton staining through the confocal laser microscope.（6）The bioactivity of HAMT and GH/H composite scaffolds.The proliferation assay was performed by the BMSCs cocultured with HAMT and GH/H composites and the results were detected by CCK8 method.And the expression of the indicators associated with osteogenic differentiation of BMSCs including osteopomtin（OPN）,osteocalcin（OCN）and type I collagen（Col I）caused by the HAMT was stuied through the RT-q PCR and WB assay.（7）The effect of hypothermia stimulation for BMSCs.The CCK8 assay was used to evaluate the proliferation effect of BMSCs under 40℃hypothemia stimulation.And the level of osteogenic differentiation of BMSCs leaded by the hypothmia stimulation was assessed by the alkaline phosphatase staining and WB.（8）The performance of the GH/H composites for in vivo bone regeneration.The critical rat calvatial bone defect model was constructed and the as-prepared Gel MA hydrogels（GH）and GH/H composite scaffolds were implanted.Then,at the 4,8 and12 weeks after implantation,the calvarias were collected and fixed by the paraformaldehyde.Finally,the calvarias were scanned and analyzed by the Micro-CT and stained by HE,Masson and immunohistochemistry to ecaluate the in vivo bone regenerarion of the GH/H composites.（9）Photothermal GBM combined with near-infrared to explore the effect of the hypothermia stimulation for in vivo bone repair.The BM and GBM materials were implanted into the rat critical bone defect area,and then the 808 nm NIR was used to achieve the in vivo hypothermia stimulation environment around the bone defect area.The effect of bone repair was evaluated by Micro-CT scanning at 4-and 12-weeks post operation.Results:（1）The HAMT with a length of～30μm and a diameter of～500 nm with a special microtube structure are obtained by the one-step hydrothermal solvent method.（2）The HAMT has advanced biocompatibility and promoting osteogenic differentiation property of BMSCs.（3）The GH/H composite scaffolds are three-dimensional porous scaffolds with apore diameter of 100-200 nm,rougher internal surface,and excenllent mechanical and anti-fatigue properties.Besides,the GH/H composites has good biocompatibility and bioactivity.（4）The GH/H scaffolds can be molded and subsequently cured in situ at the defect site which enables the used scaffolds to self-fill the defect sites.And the GH/H composites have good bone regeneration and new vessel formation ability which are attributed to the excellent mechanical property and rough surface of the GH/H composites.（5）The modification of HA materials of different dimensions by gallic acid can be completed via the microwave-assisted method,then,the white HA materials is transformed into black HA materials with photothermal properties.（6）The photothermal HA materials have good photothermal response to 808 nm NIR.In addition,the photothermal performance of materials is positively correlated with the concentration of HA materials and the power of NIR.（7）The photothermal three-dimensional GBM scaffold has excelllent photothermal property and stability.And the GBM combined the low-power(1W cm^-2)NIR can achieve the in vivo hypothermia simulation（40℃±0.5）around the bone defect area.（8）The hypothermia stimulation at 40℃±0.5℃can promote the proliferation and osteogenic differentiation of BMSCs.Besieds,the degree of osteogenic differentiation of BMSCs is positively correlated with the hypothermia stimulating time.At the same time,the in vivo promoting bone regeneration ability of hypothermia stimulation is confirmed by the rat calvatial bone defect model.Conclusions:（1）In this study,the pipe framework GH/H composite scaffolds constructed based on the HAMT have good mechanical properties,biocompatibility,bioactivity and bone repair ability.（2）Multi-dimensional HA materials can be modified and modified by GA and lead to the photothermal properties for HA materials.The modified photothermal HA materials have good photothermal performance and stability.The combination of photothermal HA materials and 808 nm NIR can achieve precise temperature control in vivo which resulting in the hypothermmia stimulation of 40℃±0.5℃for bone defect area.（3）The hypothermia stimulation can promote the proliferation and osteogenic differentiation of BMSCs and bone repair in vivo.Interestingly,the degree of osteogenic differentiation is positively correlated with the stimulation time.In summary,the preparation of new bone repair biomaterials based on the special structure and modification of HA materials is expected to provide new research ideas and treatment strategies for bone defect regeneration.