Research On In-Situ Pore-Forming Mechanism And Drug Sustained-Release System Of Fluorine-Magnesium-Doped Hydroxyapatite Film

At present,the functional recovery period of the bone defect site of the implant is long.In the late stage of implantation,inflammation,infection,loosening and other symptoms may be induced,which may lead to implant failure and require a second operation.In order to better meet the requirements of orthopedic clinical implants,the most direct effective way is to perform micro-nano-functional film modifications on the surface of existing implants.In order to meet the requirements of clinical orthopedics for implants,the direct and effective way is to perform functional modifications on the surface of existing implants.In this paper,the hydroxyapatite（HA）film of the implant surface is modified by fluorine and magnesium element,and the fluorine-doped hydroxyapatite（F-HA）film,magnesium-doped hydroxyapatite（Mg-HA）film,and fluorine-magnesium co-doped hydroxyapatite（F-HA/Mg-HA）bilayered film were prepared by pulsed laser deposition technology to achieve the in-situ generated micro/nano pore structure by ion exchange in the simulated body fluid（SBF）.The sustained release system of ciprofloxacin hydrochloride（CIP）and Biotin（Biotin）was prepared by pulsed laser deposition-electron beam evaporation deposition combined technique（PLD-EBD）,which fully utilized the pore-forming properties of MG-HA film,and expanded the application potential of porous Mg-HA film in sustained release of drugs.The main conclusions are as follows:（1）Fluorine doped hydroxyapatite film was prepared by PLD.The micromorphology,structure,composition,water contact angle,biomineralization behavior,electrochemical corrosion behavior and cytocompatibility of the F-HA film was investigated.The analysis results show that the optimal deposition time of the F-HA film is 60 min,the surface roughness（Rq）of the F-HA film reaches 259±21 nm,the Ca/P ratio of the F-HA film reaches 1.73±0.05,the thickness of the F-HA film reaches 187 nm,and the water contact angle reaches 77.3±1.4°.The F-HA film can achieve rapid in-situ pore formation in SBF（1 day of immersion）,the size of the pores reaches～100 nm,and these pores can be maintained in SBF for at least 9 days.After 14 days of immersion,the micropores on the surface of the F-HA film were completely covered by a new apatite layer,and F could accelerate apatite-like deposition and induce its transition to HA.Moreover,it is found that fluorohydroxyapatite（FHA）and fluoroapatite（FA）exist in the F-HA film,accompanied by the formation of F-Ti bonds.Therefore,the in-situ pore-forming mechanism of F-HA film can be explained as follows:the films consist of low-active components and high-active components.The low-active components are regarded as the"skeleton",and the high-active components are rapidly dissolved and migrated into the SBF,thus forming a pore structure on the surface of the film.Also,the corrosion resistance,biomineralization behavior,cytocompatibility,and the effect of inducing the differentiation of pre-osteoblasts into mature osteoblasts were better than those of HA films.Compared with the heat-treated F-HA film,the loose pore structure formed by the unheat-treated F-HA film showed better cytocompatibility（p<0.05）.（2）F-HA films,Mg-HA films,and F-HA/Mg-HA bilayered films were prepared by PLD.The micromorphology,structure,composition,biomineralization behavior and biocompatibility of the three films were investigated.The analysis results show that the film in SBF（immersed for 14 days）is roughly divided into two stages of film dissolution and remineralization.The first stage is that the low-active components such as fluorapatite and hydroxyapatite in the film form the"skeleton",while the high-active components such as magnesium and tricalcium phosphate continuously migrate into the solution,thereby forming a pore structure on the surface of the film;The second stage is that the ions(Ca²⁺,Mg²⁺,etc.)released in the film seriously destroy the balance of SBF,accelerate the deposition of calcium and phosphorus substances,and form new apatite on the surface of the film,which completely covers the original film surface.The in vitro cell viability test showed that the three films all showed good cytocompatibility,and after co-cultured with SD rat bone marrow mesenchymal stem cells（r BMSCs）for 3 weeks,the cell viability of the F-HA/Mg-HA film and the blank cell group maintained a comparable level,showing higher cytocompatibility.It was also found that the biomineralization ability of the Mg-HA film was better than that of the F-HA film,which could better induce the formation of HA,and formed a micro-scale network pore structure,which can be inferred as drug transport channel.（3）Mg-HA/CIP/Mg-HA/CIP sustained release system were prepared by PLD-EBD.First,the micromorphology,composition,structure,biomineralization behavior and cytocompatibility of Mg-HA film were investigated,and then the surface morphology,structure,water contact angle and drug release behavior of Mg-HA/CIP/Mg-HA/CIP sustained release system were further analyzed.The analysis results show that elemental Mg exists in the Mg-HA film,and elemental Mg will rapidly corrode in SBF,thus forming a microporous structure on the surface of the film,which can be used as a drug diffusion channel,and the size of these channels determines the drug onset time.and release rate.Therefore,Mg-HA/CIP/Mg-HA/CIP sustained release system were designed to prolong the release period of CIP（>14 d）.Besides,the Mg-HA film expands the application of Mg in the field of bone defect repair,and provides a new strategy for continuous drug use in the repair process of bone defect patients,especially for postoperative infection treatment,which can improve the success rate of surgery.（4）In order to explore the effect of biotin,a type of vitamin related to bone health,directly used in the treatment of bone defects.First,biotin film was prepared by EBD,and the structure,morphology,osteogenic activity,and in vivo degradation behavior of biotin film was investigated;then,Mg-HA/Biotin/Mg-HA/Biotin sustained-release system were prepared by PLD-EBD.the micromorphology,structure,composition,water contact angle,drug in vivo sustained-release behavior and osteogenic activity of the sustained-release system were further investigated.The analysis results showed that:Western Blot test confirmed that the relative protein expression of bone morphogenetic protein 2（BMP2）,bone sialoprotein（BSP）and osteoprotegerin（OPG）by biotin film was significant,especially BMP2.Implanting biotin film into animals was rapid degradation,so achieving sustained release of biotin can maximize the medical value of biotin.It was also found that the Mg-HA/Biotin bilayer film hardly interfered with the release of magnesium and could improve the relative cell viability.Based on this,Sprague-Dawley（SD）rats were used as animal models for in vivo biological evaluation.Mg-HA/Biotin/Mg-HA/Biotin sustained-release system were loaded on the surface of titanium rods and implanted into the femoral medullary cavity for three weeks.Then,the osteogenic effect of the sustained-release film was observed,and the pure biotin film was used as the reference group.The results showed that a large number of trabecular bone structures were found in the bone marrow cavity of the slow-release film group,and the cancellous bone structure and complete bone marrow structure were formed in the entire observation area of the tissue section,surrounded by abundant osteoid tissue and woven bone,showing significant osteogenic effect.However,the number of animal samples studied was too small to draw conclusions about the effect of combined Mg-HA and biotin in the treatment of bone defects.