| Research BackgroundBone defects are mainly caused by trauma,infection or tumor resection,and the trend is increasing year by year.If the bone defect exceeds a certain range or the bone's maximum self-healing capacity,the bone defect cannot self-healing,resulting in the formation of noncontinuous callus and nonunion.At this time,most of the patients were treated with autograft and allograft.It is reported that there are more than 2 million bone transplants every year in the world[1].Although autologous bone allograft and bone have natural mesh structure,which can well solve the problem of the porosity,pore diameter.Yet,there are varying degrees of shortcomings with the two methods of bone grafts[2-4].Therefore,to find an ideal replacement material for bone defects is a hot spot in the medical field.Bone marrow mesenchymal stem cells(BMSCs)combined with bone tissue engineering scaffolds have opened up a new approach for the treatment of bone defects.However,exogenous stem cells have problems such as limited sources,potential contamination risks,poor efficacy stability and difficult regulatory approval[5].Therefore,how to mobilize endogenous stem cells to homing to repair bone defects is a hot and difficult research topic in this field.In recent years,some non-contact micro-nano manipulation technologies have provided a new idea for the recruitment of endogenous cells in the body of bone tissue engineering scaffolds[6-9].Among them,"acoustic tweezers" can make use of the artificial structure resonance,interference and scattering to form sound field to realize the precise control of the controlled object,and because of its control required low power consumption,can produce a large force for particle control research,and the experimental equipment is relatively simple and other advantages,has been widely concerned.Study showed that the transverse wave velocity of Polylactic acid(PLA)material is less than the longitudinal wave velocity of water,which can be used to resonate under the action of acoustic radiation force to form sound field shape for acoustic control.In addition,the construction of PLA acoustic scaffolds can use three dimensional(3D)printing technology to build a complex shape matching with bone defects,and accurately regulate the morphology and size of the internal pores of the scaffolds,so as to obtain ideal bone repair materials[10-15].Therefore,the 3D-printed PLA porous scaffold based on acoustic radiation force is expected to be a new method to construct bone tissue engineering scaffold and provide a new idea for the treatment of bone defects.However,the 3D printed PLA porous scaffold can only capture cells within the range of local sound field formed near the scaffold with the help of acoustic radiation force.After the formation of bone defects,the number of stem cells that spontaneously migrated to this region was limited and insufficient to repair large bone defects.Therefore,more endogenous stem cells should be recruited to enrich in the bone defects so as to achieve more cell capture and promote bone repair[16].According to the mechanism of bone repair,in fibrovascular phase of bone repair,stromal cell derived factor-1(SDF-1)and bone morphogenetic proteins(BMPs)can chemotactic the seed cells of osteogenic repair--endogenous BMSCs that migrate and recruit from bone marrow and other parts of the body to the site of bone defect.In addition,in bone formation phase of bone repair,under the action of key bone differentiation factors such as BMPs,BMSCs differentiate into osteoblasts and chondroblasts,and eventually form bone through endochondral and endomembranous osteogenesis.In conclusion,SDF-1 and BMP-2 are the key cytokines involved in bone repair,and BMSCs are the key seed cells for bone repair.Therefore,this study intends to take bone repair mechanism as the starting point,take 3D-printed PLA porous scaffold as acoustic material,introduce cytokines--SDF-1 and BMP-2,and construct bionic scaffold complex with biological activity.To further study the effect of biomimetic scaffold complex with the help of ultrasound to mobilize endogenous BMSCs and promote bone defect repair.Chapter One Study on 3D printed polylactic acid(PLA)acoustic scaffold and its capture cell performanceObjectiveIn this chapter,PLA materials are used as acoustic materials to prepare PLA acoustic scaffolds by 3D printing technique,and it is discussed whether the PLA acoustic scaffolds can capture BMSCs under the control of "acoustic tweezers"technology and optimization of ultrasonic parameters,laying a foundation for the construction of bone tissue engineering scaffolds.Methods1.Construction and characterization of 3D-printed PLA acoustic scaffoldThe PLA acoustic scaffold was designed and prepared by 3D printing technology,and its microstructure was observed by scanning electron microscopy.The transmission spectrum of PLA acoustic support was measured.The sound field and sound radiation force are simulated by COMSOL software.2.Evaluation of thermal effect of sine Ultrasound(s-US)The 3D-printed PLA acoustic scaffold was placed on the 24-well plate,and the ultrasonic stimulation was started.Sine ultrasound(s-US)was used for ultrasound stimulation and the parameters were set at 1.5 MHz,60 mVpp,150 mVpp,and 200 mVpp for 20 minutes.Fluke thermal imager was used to measure and record the temperature every 5 minutes.3.Extraction,culture and passage of bone marrow mesenchymal stem cells(BMSCs)Bone marrow mesenchymal stem cells(BMSCs)were extracted from the femur and tibia of 4-week-old SD rats for passage,cryopreservation and resuscitation.Cell morphology was observed under light microscope.The third generation of BMSCs was selected for cell experiment.4.Verification of the ability of s-US mediated 3D PLA acoustic scaffold to capture cells in vitroThe in vitro acquisition of BMSCs by s-US mediated 3D printing PLA acoustic scaffold was dynamically observed under an optical microscope.5.s-US mediated BMSCs migration experimentSimulated cavity was constructed and BMSCs were implanted.Disc scaffolds were placed in the cavity and subjected to ultrasonic stimulation for 7 consecutive days(ultrasonic parameters setting:s-US,frequency 1.5 MHz,amplitude 150 mVpp,effect duration for 2 minutes).After treatment,BMSCs were placed in an incubator for further culture,and BMSCs migrated to the scaffold under light microscope.6.Statistical analysisStatistical Package for Social Science(SPSS)was used to process the data,and the experimental data were expressed as Mean±SD.After the homogeneity of variance was determined by comparison between measurement data,t test was used,and Mann-Whitney test was used for counting data.P<0.05 was the significance level.Results1.Characterization of 3D PLA acoustic scaffold3D PLA acoustic scaffold was a disc with a grid-like internal structure and a rough surface.The transmission spectrum of the 3D PLA acoustic scaffold was measured,and the low-order resonance frequency was 1.5 MHz.Simulation results of sound field and acoustic radiation force show that under the condition of ultrasonic resonance frequency(1.5MHz),the PLA acoustic scaffold surface forms a vibration gradient,so the acoustic radiation force is generated to promote the particles around the scaffold to move towards the scaffold.In addition,with the increase of particle size around the scaffold,the acoustic radiation force on the surface of the scaffold increases by orders of magnitude.2.Thermal effect of s-USThe temperature rise caused by thermal effect after s-US stimulation increased with the increase of amplitude(P=0.0064).When the ultrasonic amplitude of 200 mVpp was applied,the temperature induced by the thermal effect after s-US stimulation increased significantly with the extension of time(P<0.0001).3.BMSCs culture and morphological characteristicsDuring the culture of bone marrow mesenchymal cells extracted from primary SD rats,adherent growth of cells could appear about 12 hours later,and cell morphology could be clearly shown after fluid change,which was round,fusiform and triangular,with slow growth and uneven cell distribution.BMSCs proliferated obviously after the passage solution was changed,presenting various forms mainly of short spindle,and cell colonies could appear.About 80%of BMSCs could be fused to meet the passage standard after 10 days.The morphology of the second generation BMSCs was also a variety of short fusiform,and the cells were evenly distributed.The morphology of the third generation BMSCs was single and long fusiform,and some cells fused in a swirl or radial arrangement.4.s-US mediated 3D printing of PLA acoustic scaffold to capture BMSCs in vivoUnder the optical microscope,the number of captured cells of a single PLA acoustic scaffold at the resonant frequency(1.5 MHz)was significantly higher than those at the non-resonant frequency group(1.4 MHz or 1.6 MHz)after s-US stimulation for 30 seconds,60 seconds or 120 seconds(P30s=0.0014;P6Os=0.0051;P120s=0.0365).With the extension of ultrasonic time,the number of cells captured by the PLA acoustic scaffold with the resonance frequency s-US stimulation also increased gradually(N30s=2519;N60s=69±31;N120s=120±84;P=0.2341).Further,1.5 MHz S-US was used to stimulate the resonance of the 3D-printed disc PLA acoustic scaffold,and the cells moved to the four sides of the scaffold.The intensity of the ultrasonic field is 47kPa.5.s-US mediated 3D-printed PLA acoustic scaffold to promote BMSCs migrationA large number of BMSCs were observed to migrate to the surface of 3D-printed PLA acoustic scaffold after continuous ultrasound stimulation for 2 min for 7 days at a frequency of 1.5 MHz and an amplitude of 150 mVpp,and the cells were triangular,spindle or oval(Ns-US(+)=99±20;Ns-US(-)=17±9;Ps-US(+)vss-US(-)=0.0030).Conclusions1.3D PLA acoustic scaffold had acoustic characteristics,its low-order resonance frequency was 1.5 MHz.Therefore,under the action of ultrasound,the scaffold could resonate to generate a local sound field and form a gradient force.2.The 3D PLA acoustic scaffold could capture BMSCs in vitro under s-US mediated with resonant frequency conditions,and the number of captured cells increased with the extension of ultrasonic treatment time.Continuous ultrasonic resonance stimulation could promote the migration of BMSCs to the scaffold surface.3.In order to achieve the best effect of cell capture and avoid thermal damage,the ultrasonic parameters used in s-US should be 150 mVpp for 20 minutes.4.In this chapter,the 3D PLA acoustic scaffold was constructed,and it was the first time to capture near BMSCs in vitro in a non-contact way with the help of acoustic radiation force,which opened up a new way for bone defect repair with endogenous BMSCs..Chapter Two Preparation of 3D printed polylactic acid(PLA)biomimetic scaffold complex and in vitro study on homing of recruited stem cellsObjectiveIn the first chapter,we successfully constructed a 3D PLA acoustic scaffold that can capture BMSCs in a non-contact manner using "acoustic tweezers"technology.The scaffold can only capture cells at close range.However,for better bone defect repair,more distant BMSCs need to be recruited to accumulate at the bone defect site.Therefore,on the basis of the 3D-printed PLA scaffold,starting from the mechanism of bone defect repair,this chapter will introduce cytokines-SDF-1 and BMP-2 into calcium alginate hydrogel to construct a bionic scaffold complex with biological activity for bone tissue engineering,and explore its ability to optimize drug release parameters under ultrasound mediation and recruit BMSCs in vitro for homing.Methods1.Preparation of fluorescein isothiocyanate labeled serum albumin(BSA-FITC)scaffold complex and determination of its characterization and drug release propertiesBSA-FITC calcium alginate hydrogel and BSA-FITC scaffold complex were prepared successively.Scanning electron microscopy(SEM)was used to observe the microstructure changes before and after pulsed ultrasound(p-US)stimulation.The effects of p-US stimulation on drug release from BSA-FITC calcium alginate hydrogel and BSA-FITC scaffold complex were further determined.The ultrasonic parameters of drug release from BSA-FITC stent complex mediated by p-US were optimized.2.Evaluation of thermal effect of pulsed ultrasound(p-US)The calcium alginate hydrogel was placed in a 24-well plate and 1ml PBS was added into the well plate.The probe was placed at the bottom of the plate,and the ultrasonic stimulation was started.The parameters were 1.5 MHz,p-US,and the duty ratio(10%,30%,50%,80%)and amplitude(60 mVpp,150 mVpp,200 mVpp)were used to continuously ultrasonic stimulation for 20 minutes.Fluke thermal imager was used to measure and record the temperature every 5 minutes.3.Study on p-US mediated degradation of calcium alginate hydrogel in vitroThe calcium alginate hydrogels were prepared and the weight loss rate under p-US stimulation was measured in vitro.In the ultrasound group,the implanted site of hydrogel was stimulated by ultrasound every day.Ultrasonic stimulation parameters are as follows:p-US mode,frequency 1.5 MHz,amplitude 150 mVpp,duty cycle 50%,continuous action for 20 min/day.4.Study on the preparation and in vitro properties of SDF-1 and BMP-2 loaded biomimetic scaffold complexThe biomimetic scaffold complex containing SDF-1 and BMP-2 was prepared,and the final concentration of sodium alginate was 70 mg/ml,the final concentration of CaSO4 was 4 mg/ml,and the final concentration of SDF-1 and BMP-2 were 3 ?g/ml.The biomimetic scaffold complex was stimulated by p-US daily,and the release of SDF-1 and BMP-2 was measured by ELISA.Ultrasonic parameter setting:p-US mode,frequency 1.5 MHz,amplitude 150 mVpp,duty cycle 50%,continuous action for 20 minutes/day.5.Verification of SDF-1 and BMP-2 promoting differentiation of BMSCsThe experiment was divided into four groups(control group,group of SDF-1,group of BMP-2,group of SDF-1+BMP-2).Alkaline phosphatase staining and alkaline phosphatase activity test were taken 4 days after osteogenetic differentiation.Alizarin red staining was taken 2 weeks after osteogenetic differentiation.And acetic acid eluent of alizarin red staining samples was detected.Oil red O staining was taken 2 weeks after adipogenic differentiation.6.Verification of the ability of biomimetic scaffold complex with p-US stimulation to regulate the chemotaxis of BMSCs in vitroAfter taking the third-generation BMSCs for paving,they are divided into four groups:control group,bionic scaffold complex loaded with SDF-1,bionic scaffold complex loaded with BMP-2,bionic scaffold complex loaded with SDF-1 and BMP-2.Each group was divided into two subgroups:ultrasound group and non-ultrasound group.Transwell model was established,BMSCs were implanted in the upper compartment and biomimetic scaffold complex was placed in the lower compartment.After 12 hours of ultrasonic stimulation,the comparment was removed and the staining cells were fixed for counting.Ultrasonic parameters setting:p-US mode,frequency 1.5 MHz,amplitude 150 mVpp,duty cycle 50%,effect duration for 2 minutes/day.7.Statistical analysisStatistical Package for Social Science(SPSS)was used to process the data,and the experimental data were expressed as Mean±SD.After the homogeneity of variance was determined by comparison between measurement data,t test was used,and Mann-Whitney test was used for counting data.P<0.05 was the significance level.Results1.Characterization of BSA-FITC scaffold complexThe scanning electron microscopy showed that p-US promoted the opening of the hydrogel pores in the BSA-FITC scaffold complex,and with the increase of the ultrasonic amplitude and the extension of the action time,the hydrogel pores in the BSA-FITC scaffold complex increased.2.p-US mediated drug release of BSA-FITC scaffold complexWhen the concentration of calcium ions was 2 mg/ml,the release of albumin from the biomimetic scaffold with the increase of sodium alginate concentration.When the concentration of sodium alginate was 70 mg/ml,the release of albumin from the biomimetic scaffold increased with the increase of calcium ion concentration to a certain extent(4 mg/ml).When the concentration of calcium ions exceeded a certain concentration(4 mg/ml),the release of albumin from the biomimetic scaffold decreased with the increase of the concentration of calcium ions.There was no statistically significant difference in the BSA-FITC cumulative release amount between BSA-FITC calcium alginate hydrogel and BSA-FITC scaffold complex stimulated by p-US(P=0.8502).p-US promoted the release of albumin from the BSA-FITC scaffold complex.With the increase of ultrasonic amplitude,duty cycle and duration of effect,the release of albumin from the BSA-FITC scaffold complex increased.The difference in the duration of ultrasound was statistically significant between the groups(P=0.0341),and the amount of protein released after p-US duration of 15 minutes was close to the maximum,which was statistically significant compared with the group without ultrasound(P=0.0428).The ultrasonic parameter duty cycle was 50%,and the release of albumin in the BSA-FITC scaffold complex reached the maximum.3.Thermal effect of p-USThe temperature rise induced by the thermal effect after p-US stimulation increased significantly with the increase of amplitude,duty cycle and time(P<0.0001).When ultrasonic parameters duty cycle was 50%and the ultrasonic effect duration of 15 minutes,ultrasonic heat generation was significantly increased with the increase of ultrasonic amplitude(P<0.0001).4.p-US mediated the degradation of calcium alginate hydrogels in vitroThe results of in vitro degradation experiment showed that the degradation rate of calcium alginate hydrogel in the ultrasonic group was higher than that in the non-ultrasonic group,and the difference was statistically significant(P=0.0005).5.p-US mediating the release of SDF-1 and BMP-2 from the bionic scaffold complexThe daily release of BMP-2 and SDF-1 in the ultrasound-mediated SDF-1 and BMP-2 loaded biomimetic scaffold complex was higher than that in the non-ultrasound group,and the difference of BMP-2 release was statistically significant(P=0.0024).6.Effects of SDF-1 and BMP-2 on osteogenic differentiation and adipogenic differentiation of BMSCsAfter osteogenic differentiation culture,the positive rates of alkaline phosphatase staining and siderin staining of BMSCs under light microscope were as follows:SDF-1+BMP-2 group>BMP-2 group,SDF-1 group>control group.The alkaline phosphatase activity of SDF-1+BMP-2 group was significantly higher than that of control group or SDF-1 group(OD.sSDF-1+BMP-2=0.45±0.02;OD.BMP-2=0.43±0.00;OD.SDF-1=0.42±0.01;OD.Contuol=0.41±0.01;PControl vs SDF-1+BMP-2=0.0064;PSDF-1 vs SDF-1+BMP-2=0.0147).The quantitative analysis results showed that SDF-1+BMP-2 group was significantly higher than the other groups(OD.SDF-1+BMP-2=1.23 ± 0.11;OD.BMP-2=1.09±0.06;OD.SDF-1=0.79±0.14;OD.Control=0.1 6±0.06;/PControl vs SDF-1=0.0002;PControl vs BMP-2<0.0001;/PControl vs SDF-1+BMP-2<0.0001;PSDF-1 vs BMP-2=0.0245;PSDF-1 vs SDF-1+BMP-2=0.0025).After 2 weeks of adipogenic differentiation culture,the positive rate of BMSCs oil red O staining under light microscope was as follows:control group>SDF-1 group,BMP-2 group>SDF-1+BMP-2 group.7.Biomimetic scaffold complex with ultrsound regulation of the chemotaxis of BMSCs in vitroThe results of in vitro modified cell migration experiment showed that there was significant difference in the number of cell migration among all groups at 12 hours(Ncontrol=21±3;NSDF-1=150±8;NBMP-2=107±4;NSDF-1+BMP-2=301±7;P<0.0001),and ultrasound-regulated release of protein factors from the biomimetic scaffold complex could further promote cell migration(PControl=0.1662;PSDF-1=0.0102;PBMP-2=0.0022;PSDF-1+BMP-2=0.0024).Conclusions1.p-US mediated the release of protein factors in calcium alginate hydrogel,using ultrasonic parameter amplitude of 150 mVpp,duty cycle of 50%,and effect duration of 15 minutes as the most favorable factor release without causing damaging thermal effect.2.SDF-1 combined with BMP-2 could promote the migration and osteogenic differentiation of BMSCs,and inhibit the adipogenic differentiation of BMSCs.3.p-US mediated bionic scaffold composite could effectively promote the migration of BMSCs.4.This chapter,the research based on the PLA acoustic scaffold,combined with SDF-1 and BMP-2 calcium alginate hydrogel to construct the bionic scaffold complex.And this was the first time bionic scaffold complex recriuting remote BMSCs with ultrasound,which make up for the shortness of the acoustic scaffold PLA capture cell limited distance.It laid a good foundation for the use of endogenous BMSCs combining bone tissue engineering scaffolds to repair bone defect.Chapter Three Ultrasound-regulated 3D printed polylactic acid(PLA)biomimetic scaffold complex mediating endogenous stem cells to promote bone repair in vivoObjectiveIn the first two chapters,we have successfully constructed the biomimetic scaffold complex,and verified its effect of ultrasound-mediated cell recruitment and capture through in vitro experiments.In this chapter,we will further implant the biomimetic scaffold complex into animals,and discuss the efficacy and biosafety of the biomimetic scaffold complex in the ultrasound-mediated repair of bone defects.Methods1.Study on p-US mediated degradation of calcium alginate hydrogel in vivoThe hydrogels loaded with ICG calcium alginate were prepared.Furthermore,calcium alginate hydrogel loaded with ICG was implanted into subcutaneous tissues of rats.In the ultrasound group,the implanted site of hydrogel was stimulated by ultrasound every day,and the fluorescence intensity and area of ICG were observed and quantified by small animal imager.Ultrasonic stimulation parameters are as follows:p-US mode,frequency 1.5 MHz,amplitude 150 mVpp,duty cycle 50%,continuous action for 20 min/day.2.Preparation of SDF-1 and BMP-2 loaded biomimetic scafold complexThe biomimetic scaffold complex containing SDF-1 and BMP-2 was prepared,and the final concentration of sodium alginate was 70 mg/mL,the final concentration of CaSO4 was 4 mg/mL,and the final concentration of SDF-1 and BMP-2 were 3 ?g/ml.3.Verification of the ability of biomimetic scaffold complex with ultrasound to regulate the chemotaxis and capture BMSCs in vivoThe animals were divided into 4 groups:scaffold group;scaffold+s-US group;bionic scaffold complex group;bionic scaffold complex+p-US group.Treatment plans for each group:S+US(-):PLA scaffold was placed at the bone defect site after rat femoral defect modeling,without ultrasound treatment;S+s-US(+):PLA scaffold was placed at the bone defect site after rat femoral defect modeling,and S-US stimulation was used daily.SH+US(-):Biomimetic scaffold complex was placed at the bone defect site after rat femoral defect modeling,without ultrasound treatment;SH+p-US(+):The biomimetic scaffold complex was placed at the bone defect site after rat femoral defect modeling,and p-US stimulation was used daily.s-US parameter setting:1.5 MHz,effect duration for 20 minutes/day.p-US parameters setting:1.5 MHz,amplitude 150 mVpp,duty cycle 50%,effect duration for 2 minutes/day.After one week of treatment,the scaffolds were removed and placed in 24-well plates for internal fixation and staining.The migration of cells on the scaffolds was observed under microscope and the cells on the digestion-eluting scaffolds were identified by BMSCs flow cytometry.4.Treatment of femoral bone defect in rats with biomimetic scaffold complex regulated by ultrasoundThey were randomly divided into 6 groups:?SH;?SH+p-US;?SH+s-US;?SH+p-US+s-US;?S+p-US+s-US;?SHAM.Treatment plans for each group:SH:After the femoral defect modeling of rats,biomimetic scaffold complex was placed at the bone defect site,without ultrasonic irradiation treatment;SH+p-US:Biomimetic scaffold complex was placed at the bone defect site after femoral defect modeling in rats,and 1.5 MHz p-US stimulation was applied in the first 14 days;SH+s-US:The biomimetic scaffold complex was placed at the bone defect site after the femoral defect modeling in rats.After 14 days,1.5 MHz s-US stimulation was applied for 14 consecutive days;SH+p-US+s-US:The biomimetic scaffold complex was placed at the bone defect site after the femoral defect modeling in rats.During the first 14 days,the bone defects of the rats were stimulated by 1.5 MHz p-US,and 1.5 MHz s-US stimulation was applied for another 14 consecutive days;S+p-US+s-US:After the femoral defect modeling of rats,the PLA stent was placed at the bone defect site-During the first 14 days,the bone defects of the rats were stimulated by 1.5 MHz p-US,and 1.5 MHz s-US stimulation was applied for another 14 consecutive days.SHAM:No material was placed in the bone defect and no ultrasonic stimulation was used after the modeling of femoral defect in rats.p-US parameters were set as:frequency 1.5 MHz,amplitude 150 mVpp,duty cycle 50%,action duration:15 minutes;s-US parameters were set as 1.5 MHz,amplitude:150mVpp,action duration:20 minutes.After establishing the model of femoral bone defect in rats,Micro-CT(Skyscan 1176,Bruker,Madison,USA)was used to measure the new bone formation at the femoral defect site for quantitative analysis 3 months after adopting the corresponding treatment regimen.After micro-CT examination,femur specimen sections of rats were taken,and HE staining and Masson trichromatic staining were used to observe the repair of bone defect sites.The main organs of rats were observed by HE staining.5.Statistical analysisStatistical Package for Social Science(SPSS)was used to process the data,and the experimental data were expressed as Mean±SD.After the homogeneity of variance was determined by comparison between measurement data,t test was used,and Mann-Whitney test was used for counting data.P<0.05 was the significance level.Results1.p-US mediated the degradation of calcium alginate hydrogels in vivoThe area of ICG released by calcium alginate hydrogel in the ultrasonic group was significantly higher than that in the non-ultrasonic group(p=0.018).The fluorescence intensity of ICG indirectly reflected the degradation of calcium alginate hydrogel.The results showed that the degradation rate of calcium alginate hydrogel in the ultrasonic group was faster than that in the non-ultrasonic group.On the 12th day of ultrasonic stimulation,the average radiant effeciency(ARE)of the ultrasonic group and the non-ultrasonic group were ARFP-US(+)=3.25±0.54 × 106(p/s)/(u W/cm2),ARFP-US(-)=5.74±1.62×106(p/s)/(uW/cm2)respectively.2.Biomimetic scaffold complex with ultrasound regulation of the chemotaxis and captures BMSCs in vivoOne week after each group of scaffolds or biomimetic scaffold complex were implanted in the bone defect parts of rats,the scaffolds were removed,and FITC staining after cell fixation showed that the number of cells collected from the p-US-mediated biomimetic scaffold complex was most,followed by the scaffold combined with s-US group,followed by the biomimetic scaffold complex group,among which the number of cells collected from the simple scaffold group was the least.BMSCs were further identified on the recruited cells,and the results showed that the proportion of BMSCs was S+US(-)0.37%;S+s-US(+)4.96%;SH+US(-)1.95%;SH+p-US(+)17.95%.3.Efficacy of ultrasonic modulated bionic scaffold complex for repairing femoral bone defect in ratsAfter 3 months of treatment,the femoral bone defect site of rats in each group was reconstructed by CT three-dimensional reconstruction,and the repair effect of SH+p-US+s-US group was the best,and the bone defect site was almost completely repaired.Quantitative analysis of osteogenic conditions in each group showed that the bone volume fraction(BV/TV),the number of bone trabeculae(Tb.n)and bone mineral density(BMD)in SH+p-US+s-US group were significantly higher than those in the other groups(PBV/TV=0.0004;PTb.N=0.0403;PBMD=0.0014),bone trabecular separation(Tb.Sp)was significantly lower than that in other groups(PTb.Sp=0.0018).HE and Masson staining of the sections of the femur of rats showed that compared with the other groups,a large amount of new bone was formed in the defect site of the femur of rats in the SH+p-US+s-US group,forming a thicker layer of new bone.4.Evaluation of the safety of ultrasonic modulated bionic scaffold complex for repairing femoral bone defect in ratsHE staining results of histological sections showed that no significant histological morphological and pathological changes were observed in the main organs(heart,liver,spleen,lung,kidney)of each group.ConclusionsIn this chapter,we successfully make use of the biomimetic scaffold complex with ultrasound to recruit and capture endogenous BMSCs for the first time,which safely and efficiently repaired the femoral bone defect in rats,providing a new method for bone tissue engineering scaffold mediating endogenous stem cells to treat bone defect. |
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