Study On The Mechanism Of Local Rapamycin Delivery To Promote The Osseointegration Of 3D Printed Microporous Prosthetic Interface By Regulating The Osteoporotic Microenvironment

Osteointegration refers to the firm connection between the prosthesis interface and the host bone tissue after the prosthesis is implanted.Excellent osteointegration is the key factor to achieve long-term stability and prolong the service life of the prosthesis.However,most of the patients receiving joint replacement surgery in clinic are elderly patients,often accompanied by osteoporosis.After the prosthesis is implanted,the osteointegration is not ideal due to local bone recession and poor bone regeneration ability,which leads to a high incidence of complications such as prosthesis loosening,displacement and periprosthetic fractures.In order to improve the osteointegration,the personalized prosthesis prepared by the emerging 3D printing technology is highly matched with the bone defect at the implantation site in the macro,and the micro controllable microporous structure provides initial stability,reduces the elastic modulus,and induces bone ingrowth and bone integration.However,3D printed titanium alloy prosthesis,which is widely used in clinical practice,still has a certain degree of biological inertia,and the osteointegration efficiency under osteoporosis still needs to be improved.At present,some studies have shown that the insufficient ability of bone regeneration and osteointegration in osteoporosis may be partly due to the low autophagy level of osteoporotic derived bone marrow mesenchymal stem cells（OP-BMSCs）,which leads to low cell viability and proliferation,insufficient osteogenic differentiation and preferable adipogenic differentiation.Therefore,loading drugs that activate the autophagic activity of OP-BMSCs at the 3D printed microporous prosthesis interface which is conducive to bone growth is a potential strategy to restore the activity and function of OP-BMSCs and promote osseointegration.In view of the above problems,this study intends to design two delivery strategies of autophagy activator（rapamycin）to enhance the osseointegration of 3D printed prosthesis interface.The specific strategies are as follows:（1）Research system I:Percutaneous ultrasound mediated rapamycin delivery promotes osseointegration at the prosthesis interface under osteoporosis by activating autophagy and regulating the osteogenic microenvironment.（1）.Using Ti₆Al₄V powder as raw material,we have successfully prepared 3D printed titanium alloy microporous prosthesis with the pore diameter was about 800μm and porosity was about 70%.And then,the surface morphology and mechanical properties of the obtained scaffold were analyzed.（2）.The osteoporotic rabbit model was prepared by bilateral ovariectomy,and then BMSCs were extracted and cultured,and the typical cell surface antigen expression（CD45,CD90,CD105）of BMSCs were identified by flow cytometry,and the triadic differentiation ability of osteogenesis,chondrogenesis and adipogenesis was analyzed.（3）.First,the relationship between the concentration of rapamycin by ultrasound mediated delivery and the action time was detected in vitro,and FITC-rapamycin was used for delivery in vivo,and the skin,muscle and bone tissues were observed.Thus,the safety and efficient parameters of ultrasound conductance instrument to deliver rapamycin were determined,namely the frequency was 30 k Hz,the intensity was 45 W/cm²,and the time was 10 minutes.Then Western Blot was applied to evaluate the expression of the key autophagic proteins（m TOR,LC3Ⅱ/Ⅰand P62）,Ad-m Cherry-GFP-LC3B double fluorescent adenovirus infection cells,and transmission electron microscopy for observing the formation of autophagic vesicles in cells,thus comprehensively evaluating the impact of ultrasound mediated rapamycin delivery on the autophagic level of OP-BMSCs.（4）.Studies on the cell functions of OP-BMSCs regulated by ultrasound mediated rapamycin delivery,mainly including CCK-8 assay to test cell proliferation,Calcein AM/PI staining to analyze cell activity,phalloidin staining to observe the cytoskeleton,cell scratch test and Transwell test to check the cell migration ability,alizarin red staining,RT-q PCR and immunofluorescence staining to explore the osteoblastic differentiation of OP-BMSCs,and Oil Red O staining to detect the adipogenic differentiation of OP-BMSCs.（5）The 3D printed microporous prosthesis was implanted into the lateral condyle of the left distal femur of rabbits.A series of tests were used to observe the ultrasound mediated rapamycin delivery promoting osseointegration of the prosthesis interface in osteoporosis,mainly including Micro-CT,hard tissue slice,and mechanical test evaluated the osseointegration at the interface and the surrounding bone condition;detection of protein level of m TOR,LC3 II/I and P62 in the bone tissue assessed autophagic changes around the interface;the expression of osteogenic markers was observed by RT-q PCR and immunofluorescence staining in the bone around the interface.（2）Research system II:Functional hydrogel mediated local delivery of rapamycin promotes osseointegration at the prosthesis interface under osteoporosis by activating autophagy and regulating the osteogenic microenvironment.（1）.CEC-PVA-Ag NW hydrogels were synthesized by cross-linking N-carboxyethyl chitosan（CEC）,silver nanowires（Ag NW）,polyvinyl alcohol（PVA）,agarose and glycerin,and then the characteristics of rheological festures,mechanical properties,microscopic morphology,degradation,biocompatibility,and other physical and chemical properties of the hydrogels were detected.（2）.3D printed microporous prosthesis was prepared,and the 3D printed bioactive interface was constructed by compositing the rapamycin loaded hydrogel with microporous prosthesis.The drug release curve of 3D printed bioactive interface and the antibacterial performance against S.aureus and MRSA were evaluated.（3）.The expression levels of key proteins of autophagy（m TOR,LC3Ⅱ/Ⅰand P62）were evaluated by Western Blot,and Ad m Cherry GFP-LC3B double fluorescent adenovirus infection cells were used to evaluate the regulation of 3D printed bioactive interface on autophagy of OP-BMSCs.（4）.Studies on the cell functions of OP-BMSCs regulated by 3D printed bioactive interface,mainly including CCK-8 assay to test cell proliferation,Calcein AM/PI staining to analyze cell activity,phalloidin staining to observe the cytoskeleton,alizarin red staining and RT-q PCR to explore the osteoblastic differentiation of OP-BMSCs,and Oil Red O staining to detect the adipogenic differentiation of OP-BMSCs.（5）.The 3D printed bioactive prosthesis interface was implanted into the lateral condyle of the left distal femur of rabbits.A series of tests were used to observe the 3D printed bioactive prosthesis interface promoting osseointegration in osteoporosis,mainly including Micro-CT,hard tissue slice,and mechanical test evaluated the osseointegration at the interface and the surrounding bone condition;detection of protein level of m TOR,LC3 II/I and P62 in the bone tissue assessed autophagic changes around the interface;the expression of osteogenic markers was observed by RT-q PCR and immunofluorescence staining in the bone around the interface.（6）.3D printed bioactive interface was implanted into the distal femoral bone defects infected with S.aureus and MRSA,to evaluate the bone integration of the prosthesis interface under infection,including hematological examination to evaluate the status of systemic infection,and imaging,hard section histology and mechanical testing to evaluate the bone integration effect of the prosthesis interface.Through the studies of these two systems,it was found that both percutaneous ultrasound mediated rapamycin delivery and functional hydrogel mediated local delivery of rapamycin could improve the cell activity and osteogenic differentiation capacities of OP-BMSCs while inhibiting adipogenic differentiation by activating the autophagic activity,thereby promoting the bone integration of 3D printed microporous prosthesis interface in osteoporosis.In addition,the introduction of Ag NW into functional hydrogel endows 3D printed bioactive interface with unique antibacterial performance,which can effectively prevent infection around the prosthesis.