"Dandelion" Inspired Dual-Layered Nanoarrays With Two Model Releasing Features For The Surface Modification Of 3D Printing Implants

Background and Objective: Due to the reasons of aging,sports injury and improvement of medical technology,various kinds of embedded related operations have been increased year by year.However,original implants still have certain risk of postoperative infection,which was one of the leading causes of surgical failure and medical disputes.Therefore,it is necessary to enhance their antibacterial properties for reducing embedded related infection rate through proper surface modification.In particular,the potential of antibacterial nanoparticles as clinical future agents has received much attention.Nanomaterial,especially zinc oxide(ZnO)based antibacterial materials,have the advantages of relative low dosage,large surface area and commendable biocompatibility.In the practical clinical application,the release of antibacterial components inclines to be divided into two models,quick release model(within early 48h): rapid killing the pathogenic bacteria on the implant materials' surface and surrounding tissue;and the subsequent slow release model(around approximately 2 weeks)to realize long-term inhibition.In view of the above requirements,single-structured ZnO nanoarrays were difficult to meet these two demands simultaneously.At present,3D printing technology has been widely used in the field of orthopedics,precision medical and customized equipment which are the future direction of medical development.The ZnO double-layer nanoarrays were modified on the surface of the printed orthopedic implant model which gave the customized equipment antibacterial capacity to provide new options for reducing implant-related infection rates.In this work,the specimens were detected the in vitro antibacterial activity,characteristics of release and in vivo antimicrobial activity.Methods:(1).Deposition of single ZnO nanocrystals on the substrates through coating,dropping and two kinds of ZnO nanocrystals through coating with dropping;(2).Crystalline phases of the samples were characterized by X-ray diffraction.The morphologies and structures of the as-prepared ZnO crystals were investigated by fieldemission scanning electron microscopy and transmission electron microscope(TEM);(3).The samples were soaked in 5 mL PBS for 1 day,and then retrieved to be soaked in fresh PBS of 5 mL.This procedure was repeated once a day for total 2 week.The amount of Zn filtered to the solutions were measured by inductively-coupled plasma atomic emission spectrometry;(4).The design of the PLA wafers and three different impersonative orthopaedic implantable materials were obtained by 3D software CATIA.The models were printed using a SLA 3D printer;(5).Two of the most common pathogenic bacterium of clinical orthopedic infection,Staphylococcus aureus(S.aureus,ATCC 25923)and Escherichia coli(E.coli,ATCC 25922)were used.The bacterium were co-cultured with coated-dropped pieces(the wafers were cut into eight parts evenly)for 24 and 48 hours,respectively.The specimens adhered with Zn O NR,ZnO NS and ZnO DLN were submersed in 5 mL bacterium suspension 2 h.The treated samples were then placed in a clean Petri dish.The solution of bacterial within logarithmic growth phase was added to its surface every two days for 2 weeks;(6).The test contained forty male rats(Sprague Dawley)between 224 and 263 g(8 week old,Animal science department of Jiangxi province,China)for the inflammatory reaction with S.aureus of the substrates adhered with ZnO NR,ZnO NS and ZnO DLN.The sections were stained with hematoxylin-eosin(HE)for light microscopy examination.Results:(1).The microscope observation and X-ray diffraction technique show that the surface of the material has successfully modified the ZnO nanoarrays,the Zn O nanorod arrays and the double layer(nanorods+ nanoslices)ZnO nanoarrays.After ultrasonic treatment,the double layer ZnO nanoarrays exhibit excellent adhesion to the surface of the materials;(2).The ZnO DLN modified by the surface of the material has the release characteristic of burst releasing in 48 h and slow releasing in 2 weeks,while the only modified ZnO NR has only 2 weeks slow releasing characteristics,while the only modified single ZnO NS only exists in the form of internal burst releasing.(3).The co-culture of three different morphologies of ZnO nanoarrays and Staphylococcus aureus and Escherichia coli in the 3D printed PLA model showed that the 3D printing model modified ZnO nanoarrays showed good antibacterial properties to the representatives of two Gram-positive and negative bacteria,compared with the unmodified PLA model.There were significant differences in type( < 0.001).(4).In vitro antibacterial experiments,three kinds of ZnO nanoarrays showed good antibacterial activity in 24 h and 48 h,and 48 h in them was better.The antibacterial activity of ZnO nanoarrays against Staphylococcus aureus is better than that of Escherichia coli,and the antibacterial rate of double layer ZnO nanoarrays is nearly 100%.After ultrasonic treatment,the ZnO DLN still showed good antibacterial properties,while the antibacterial properties of the single ZnO nanoarrays decreased to a certain extent.The biofilm results showed that the surface of ZnO nanoarrays only had very few bacteria and showed a good effect on destroying the formation of biofilm.(5).In vivo antibacterial experiments,the implantation incision with the surface of a ZnO DLN has healed well.The implants of the modified single ZnO nanoarrays have a certain degree of delayed healing,especially the slow healing of the unmodified implants.The count of the implant surface and the surrounding bacteria showed that the surface and surrounding bacteria of the implants modified by three different ZnO nanoarrays were greatly reduced( < 0.001),and a large number of inflammatory cells were observed in the skin and muscle tissue around the unmodified implants.On the contrary,only a few inflammatory cells were found in the surrounding tissues of three implants modified by Zn O nanoarrays.Conclusion:(1).Inspired from dandelion,ZnO DLN nanoarrays were constructed here,as a healthier alternative of antibiotics to modify the surface of 3D printing implant scaffolds.(2).In vivo and in vitro antibacterial tests showed that the proposed DLN exhibited impressive two model antibacterial releasing properties,which enabled to reduce the postoperative infective rate effectively.