Effects Of Programmed Local Delivery From A Micro/Nano-Hierarchical Surface On Titanium Implant On Infection Clearance And Osteogenic Induction In An Infected Bone Defect

The two major causes for implant failure are postoperative infection and poor osteogenesis.Initial period of osteointegration is regulated by immunocytes and osteogenic-related cells resulting in inflammatory response and tissue healing.The healing phase can be influenced by various environmental factors and biological cascade effect.To synthetically orchestrate bone-promoting factors on biomaterial surface,built is a dual delivery system coated on a titanium surface(abbreviated as AH-Sr-Ag NPs).The results show that this programmed delivery system can release Ag+ and Sr2+ in a temporal-spatial manner to clear pathogens and activate preosteoblast differentiation partially through manipulating the polarization of macrophages.Both in vitro and in vivo assays show that AH-Sr-Ag NPs-modified surface renders a microenvironment adverse for bacterial survival and favorable for macrophage polarization(M2),which further promotes the differentiation of preosteoblasts.Infected New Zealand rabbit femoral metaphysis defect model is used to confirm the osteogenic property of AH-Sr-Ag NPs implants through micro-CT,histological,and histomorphometric analyses.These findings demonstrate that the programmed surface with dual delivery of Sr2+ and Ag+ has the potential of achieving an enhanced osteogenic outcome through favorable immunoregulation.Part ? Fabrication and characterization of AH-Sr-Ag NPsObjectives:Based on the protonation effect of alkali-heat treatment and oxidation–reduction quality of polydopamine(PDA),we aim at fabricating a controlled dual-delivery system on the implant surface with antibacterial and osteogenic properties.Methods:(1)Trabecular-like porous titanium implant was designed by computer-aided design and manufactured by selective laser melting(SLM)technology.(2)Hierarchical micro-nano structure with nanoscale needle-like structure was formed on the surface of porous titanium implants by alkali-heat treatment;The stable loading of Sr2+ on the implant surface(AH-Sr)was deposited via hydrothermal deposition because of the proton pump effect of a large number of active hydroxyl groups on this hierarchical structure.(3)Self-polymerization of PDA was performed on the surface of AH-Sr;Silver nitrate was reduced onto AH-Sr layer in form of silver nanoparticles because of bidentate chelation and oxidation–reduction property;Finally,a multifunctional implant(AH-Sr-Ag NPs)with antibacterial and osteogenic properties was formed.(4)The mechanical properties of porous titanium implants as well as the cortical and cancellous bones of New Zealand rabbits were detected by compression test using a universal mechanical tester.(5)Surface morphology and roughness of implant surface were observed by SEM and AFM,respectively.(6)The chemical composition and element distribution were detected by energy spectrometer(EDS)and X-ray photoelectron spectrometer(XPS).(7)Sessile drop method was used to detect the contact angle of implant surface.(8)Micro BCA Protein Assay was used to test the protein adsorption property of implant surface.(9)The release pattern of Sr2+ and Ag+ was detected and analyzed by inductively coupled plasma emission spectrometry(ICP-OES).Results:(1)The dual-delivery system AH-Sr-Ag NPs on implant surface was successfully fabricated.(2)Compression test results showed that AH-Sr-Ag NPs had lower elastic modulus than pure titanium,and its elastic modulus(4.88 ± 0.25GPa)was similar to that of cancellous bone(4.76 ± 0.33 GPa).(3)At high magnification,uniform and unique nanofiber texture can be observed on AH-Sr-Ag NPs coating.Moreover,silver nanoparticles(?=42.2±0.33 nm)can be clearly observed on its surface.(4)AFM results showed that the Ra of the control sample was24.028±0.01 nm,while the surface of AH-Sr-Ag NPs sample was significantly rougher(Ra = 69.594±0.03 nm).(5)The contact angle(CA)of pure titanium surface was 61.95±3.02°,while the CA of AH-Sr-Ag NPs was 11.25±2.32°.(6)Protein adsorption assay showed that the AH-Sr-Ag NPs coating could significantly absorb more fibronectin(FN).(7)The release profile showed that the release amount of Sr2+ was relatively high(> 0.05 ppm per day)in the first 4 days and the daily release amount of Sr2+ decreased during the next 3 weeks;The slow and gradual release property of Ag+ is better for long-term protection against bacterial infection;Compared with Sr2+,there was no obvious burst release for Ag+,and the daily release amount of Ag+ remained at 0.06 g/ml.Conclusions:(1)The dual-delivery system AH-Sr-Ag NPs with antibacterial and osteogenic properties was successfully fabricated.(2)The roughness of AH-Sr-Ag NPs coating increases,with higher hydrophilicity and protein adsorption capacity.(3)The AH-Sr-AgNPs coating achieved the controlled release of Sr2+and Ag+ within the theraputic window.Part ? Evaluation of antibacterial property of AH-Sr-Ag NPsObjectives:To investigate the difference of antibacterial activity between pure titanium surface and AH-Sr-Ag NPs implant surface,and further confirm the antibacterial property of AH-Sr-Ag NPs.Methods:(1)Culture and passage of the gram-negative strain Escherichia coli(E.coli,ATCC 8099)and the gram-positive strain Staphylococcus aureus(S.aureus,ATCC 25923);(2)Culture of E.coli and S.aureus on AH-Sr-Ag NPs surface;Morphological observation of bacteria on different implant surfaces and calculation of antibacterial efficacy.Results:(1)The numbers of E.coli and S.aureus on AH-Sr-Ag NPs surface decreased significantly,while there were bacteria with irregular shape and obvious pili on pure titanium surface.(2)The numbers of E.coli and S.aureus on pure titanium surface were 258.4±26.03 CFU and 460.8±13.49 CFU,respectively.There was little bacterial growth on AH-Sr-Ag NPs surface.(3)The antibacterial efficiency of AH-Sr-Ag NPs against E.coli and S.aureus was 98.66± 4.24% and 98.17± 2.31%,respectively.Conclusions:(1)AH-Sr-AgNPs coating has excellent antibacterial property and can effectively inhibit bacterial infection on implant surface.Part ? Biological cascade regulatory effect of AH-Sr-Ag NPs on bone immune cells and downstream osteogenic-related cellsObjectives:To identify the effect of locally released Sr2+ and Ag+ from AH-Sr-Ag NPs system on the phenotype of bone immune cells and downstream osteogenic-related cells.Moreover,this dual-delivery system also provides a potential surface modification strategy which can further improve implant success rate.Methods:(1)Culture and passage of macrophage cell line(Raw264.7)and pre-osteoblast cell line(MC3T3-E1);(2)Culture of Raw264.7 on AH-Sr-Ag NPs coating;Evaluate the modulation effect of AH-Sr-Ag NPs on the polarization of Raw264.7 and its effect on cell morphology;(3)Raw264.7 and MC3T3-E1 were indirectly co-cultured on AH-Sr-Ag NPs surface;Evaluate the effect of AH-Sr-Ag NPs on adhesion,extension and differentiation of downstream osteogenic-related cells through the regulation of Raw264.7.(4)Explore the molecular mechanism and key targets underlying the regulatory effect of AH-Sr-Ag NPs on cellular behavior from both m RNA and protein levels.Results:(1)The m RNA expression of CD86(M1 marker)showed no significant difference between pure titanium group and AH-Sr-Ag NPs group.m RNA expression of CD206(M2 marker)was significantly increased on the surface of AH-Sr-Ag NPs coating.(2)The protein expression of i NOS(M1 marker)significantly decreased on the surface of AH-Sr-Ag NPs coating.(3)After culture for 24 h,macrophages on pure titanium surface presented egg-like shape while macrophages on AH-Sr-Ag NPs surface were well stretched and presented spindle-like shape.(4)After indirect co-culture,the perimeter and area of MC3T3-T1 on the surface of AH-Sr-Ag NPs were significantly higher than those on the surface of pure titanium.(5)After indirect co-culture,the expression of osteogenic-related genes(eg.ALP,Runx2 and Col-1)in MC3T3-E1 incubated on AH-Sr-Ag NPs surface increased significantly compared with the control group.Conclusions:(1)The AH-Sr-Ag NPs coating can regulate the polarization of macrophages and increase the number of M2 phenotype macrophages.(2)The AH-Sr-Ag NPs coating can further regulate the adhesion,elongation and differentiation ability of downstream pre-osteoblasts via modulating the polarization of macrophages.Part ? The effect of AH-Sr-Ag NPs on the restoration of a femoral infected bone defectObjectives:Through the in vivo examination of this dual-delivery controlled release system,we aim at confirming the antibacterial and osteogenic properties of AH-Sr-Ag NPs system in an infected bone defect.Methods:(1)To establish an infected femoral metaphysis model in New Zealand rabbits;(2)Micro-CT,VG staining and histomorphometric analysis were performed to evaluate the antibacterial and osteogenic efficacy of locally released Sr2+ and Ag+ from AH-Sr-Ag NPs system.(3)By using macrophage functional inhibitor AP20187,we attempted to explore the internal mechanism of AH-Sr-Ag NPs system.Results:(1)VG staining showed that the new bone formation in AH-Sr-AgNPs group was significantly better than that in the control group.Compared with AH-Sr-Ag NPs group,the new bone formation in peri-implant region was less in AH-Sr-Ag NPs + AP20187 group.(2)Compared with the bone-implant contact rate of the control group(BIC % = 25%),the BIC % on AH-Sr-Ag NPs implant surface was more than 75% while the BIC % of the AP20187 application group was less than60%.(3)Compared with AH-Sr-Ag NPs group,the bone formation rate(BF %)in the region of interest(ROI)of the AP20187 application group was relatively low.(4)Bone formation in different groups was analyzed by continuous fluorescent injection.Compared with the control group,implants in the AH-Sr-Ag NPs group induced faster bone formation during the entire experimental period.(5)Micro-CT images showed that in the AH-Sr-Ag NPs group,it is clearly observed that almost all the peri-implant region was covered by new bone tissue and it grew into the pores of the implant,while in AP20187 application group,the bone tissue coverage around the implant and its growth into the pores of implant were reduced.Conclusion:(1)The osteogenic property of AH-Sr-Ag NPs implant system has been verified through the implantation model under infection environment.(2)By setting a group using immunosuppressant,it was further demonstrated that this AH-Sr-Ag NPs implant system could partially promote bone regeneration via modulating the polarization of macrophages.