Tóm tắt Luận án Synthesis and characterization of trace elements co - Doped hydroxyapatite on 316l stainless steel application in bone implant

VIETNAM ACADEMY OF SCIENCE AND TECHNOLOGY GRADUATE UNIVERSITY SCIENCE AND TECHNOLOGY ----------------------- Vo Thi Hanh SYNTHESIS AND CHARACTERIZATION OF TRACE ELEMENTS CO-DOPED HYDROXYAPATITE ON 316L STAINLESS STEEL APPLICATION IN BONE IMPLANT Major: Theoretical and Physical Chemistry Code: 62 440119 SUMMARY OF DOCTORAL THESIS IN CHEMISTRY Hanoi – 2018 The thesis has been completed at: Department of Corrosion and Protection of Metals - Institute for Tropical Technology - Vietnam Academy of Science and Technology Scientific Supervisors: Assoc. Prof. Dr. Dinh Thi Mai Thanh, Institute for Tropical Technology - Vietnam Academy of Science and Technology Reviewer 1: Reviewer 2: Reviewer 3: The thesis was defended at Evaluation Council held at Graduate University of Science and Technology - Vietnam Academy of Science and Technology on , 2018. Thesis can be further referred at: - The Library of Graduate University of Science and Technology. - National Library of Vietnam. 1 INTRODUCTION 1. The necessary of the thesis Nowadays, 316L stainless steel (316LSS), titanium and alloys of titanium are widely used in orthopedic surgery with the purpose of splinting bone. Materials made of titanium and titanium alloy have a good mechanical properties and good biocompatibility but they have a high cost. Therefore, in Vietnam, to reduce the cost of medical services, most of the splints are made of 316L stainless steel. However, 316L stainless steel could be corroded and limited the ability of biological compatibility in the biological environment. To improve these problems, 316LSS is generally coated biomaterials such as hydroxyapatite (Ca10(PO4)6(OH)2, HAp). HAp has chemical composition and biological activity similar to the natural bone. HAp could stimulate the bonding between the host bone to implant materials and make bone healing ability faster. Moreover, HAp also protects for the metal surfaces against corrosion and prevents the release of metal ions from the substrates into the environment. However, pure HAp has been dissolved in the physiological environment which may lead to the disintegration of the coatings and affects the implant fixation. These disadvantages could deal with doping some trace elements in the HAp structure by replacing Ca 2+ ions with cations and substituting OH - group with anions. In addition, the present of trace element such as magnesium, sodium, strontium, fluorine, zinc has also the role to stimulate the new bone formation and provides minerals for bone cells to grow. Besides, the problem of postoperative infection should be concerned. Thus, antibacterial elements such as copper, silver and zinc are also being studied to incorporated into HAp. Based on the reasons mentioned above, the research topic of thesis is chosen as following: “Synthesis and characterizations of trace elements co- doped hydroxyapatite coatings on 316L stainless steel application in bone implaint” 2. The objectives of thesis: - Trace elements (sodium, magnesium, strontium, fluorine, copper, silver and zinc) doped NaHAp coatings are synthesized sucessfully on the 316LSS substrates, separately and simultaneously. - Research on the physical and chemical characteristics, cytotoxicity and antibacterial ability, biological compatibility of the NaHAp coating doping some trace elements separately and simultaneously. 3. Research contents of the thesis: - Investigating and selecting of optimal conditions for the synthesis of NaHAp coatings and NaHAp coatings doping magnesium, strontium and fluorine separately and simultaneously by cathodic scanning potential method. 2 - Investigating and selecting of optimal conditions for the synthesis of NaHAp coatings doping copper, siliver and zinc separately and simultaneously by ion exchange method. - The HAp coatings doping 7 elements simultaneously: Mg, Sr, F, Na, Cu, Ag, Zn are studied to synthesize by the combination two methods: electrodeposition and ion exchange. - Studying on the biological activity of materials: 316LSS, NaHAp/316LSS, MgSrFNaHAp/316LSS and HApđt/316LSS in simulated body fluid (SBF) solution. - Studying on the cytotoxicity ability of powder: NaHAp, MgSrFNaHAp. - Studying on antibacterial ability of powder: NaHAp, MgSrFNaHAp, AgNaHAp, CuNaHAp, ZnNaHAp và HApđt. - Evaluation of the biological compatibility of materials: 316LSS, NaHAp/316LSS, MgSrFNaHAp/316LSS on dog’s body. CHAPTER 1. OVERVIEW OF HAp AND DOPED HAp 1.1. The properties and synthesized methods of HAp and doped HAp coatings Some trace elements doped HAp coatings have more advantages than pure HAp coatings, such as: decrease of the dissolution, increase of the metabolism, antibacterial ability and compatibility. HAp coatings is deposited on the substrates by many methods: plasma, magnetron and electrodeposition These methods have advantages and disadvantages. The electrodeposition has an important technology because of the advantages: the low temperature, easily controlling the coatings thickness, the high purity, high bonding strength and low cost of the equipment. Furthermore, it is easy to substitute some trace elements ions (Mg 2+ , Na + , K + , Sr 2+ and F - ) into HAp coatings by addiction M(NO3)n or NaX into the electrolyte. Dope HAp is producted according to the chemical reaction: (10-x)Ca 2+ + 6PO4 3- + (2-y)OH - + xM 2+ + yX -  Ca10-x M x(PO4)6(OH)2-yXy 1.2. In vitro and in vivo test of HAp The compatibility of materials is studied by immersion them in SBF solution and investigate the formation of apatite on the material surface. Besides, the compatibility of materials is also studied by in vivio test on the animal. 1.4. The application of HAp, doped HAp HAp and doped HAp are used as: - The medicine of calcium supplements: the composition of HAp contains a lot of calcium and be absorbed directly without transformation. - Material for implantation: repair of the teeth and bone defects. 1.5. The situation of HAp research in the country 3 Basic on the overview of HAp and doped HAp, it can be seen that there is no published report about doped HAp coatings in our country; in the world, the trace elements doped HAp coatings have been only synthesized separately. Thus, in this doctoral thesis, some trace elements (sodium, magnesium, strontium, fluorine, copper, silver and zinc) doped HAp coatings were synthesized separately and simultaneously. The HAp obtained coatings have many good properties, such as: decrease of the dissolution and increase of the metabolism, antibacterial ability and compatibility for HAp coatings. CHAPTER 2. EXPERIMENT AND RESEARCH METHODS 2.1. Synthesis of doped HAp 2.1.1. By the electrodeposition method (cathodic scanning potential) 2.1.1.1. Electrochemical cells The electrodeposition was carried out in a three-electrode cell with 316LSS as the working electrode, platinum foil as the counter electrode and a saturated calomel electrode (SCE) as the reference electrode. 2.1.1.2. Synthesis of NaHAp coatings - NaHAp coatings were synthesized on the 316LSS by cathodic scanning potential method in 80 mL solution containing Ca(NO3)2 3×10 -2 M + NH4H2PO4 1.8×10-2 M and NaNO3 with different concentrations: 4.10 -2 M (DNa1), 6.10 -2 M (DNa2) và 8.10-2 M (DNa3). - NaHAp coatings were synthesized under following conditions as follows: the different scanning potential ranges: 0 to -1.5, 0 to -1.7, 0 to -1.9 and 0 to -2.1 V/SCE; reaction temperatures: 25, 35, 50, 60 and 70 o C; pH = 4.0, 4.5, 5.0 and 5.5; scanning time: 1, 3, 5, 7 and 10; scanning rate: 3, 4, 5, 6 and 7 mV/s. 2.1.1.3. Synthesis of Mg 2+ , Sr 2+ or F - doped NaHAp coatings (ĐNaHAp) ĐNaHAp were deposied at 50 oC in 80 mL solution containing at the Table 2.1 and under following conditions: the different scanning potential ranges: 0 to -1.5, 0 to -1.7, 0 to -1.9 and 0 to -2.1 V/SCE; scanning time: 1, 3, 5, 7 and 10; scanning rate: 3, 4, 5, 6 and 7 mV/s. Table 2.1. Chemical composition of the electrolyte ĐNaHAp Notation Chemical composition MgNaHAp DMg1 DNa2+ Mg(NO3)2 1x10 -4 M DMg2 DNa2+ Mg(NO3)2 5x10 -4 M DMg3 DNa2+ Mg(NO3)2 1x10 -3 M DMg4 DNa2+ Mg(NO3)2 5x10 -3 M SrNaHAp DSr1 DNa2 + Sr(NO3)2 1x10 -5 M DSr2 DNa2 + Sr(NO3)2 5x10 -5 M 4 2.1.3.4. Synthesis of Mg 2+ , Sr 2+ and F - co-doped NaHAp coatings (MgSrFNaHAp) MgSrFNaHAp were synthesized in 80 mL solution containing at DNa2 + NaF 2.10 -3 M + Sr(NO3)2 5.10 -5 M + Mg(NO3)2 1.10 -3 M and under following conditions as follows: the different scanning potential ranges: 0 to -1.5, 0 to - 1.7, 0 to -1.9 and 0 to -2.1 V/SCE; reaction temperatures: 25, 35, 50, 60 and 70 o C; scanning time: 3, 4, 5, 6, 7 and 10; scanning rate: 3, 4, 5, 6 and 7 mV/s. 2.1.2. By the ion exchange method Preparing material of NaHAp/316LSS: NaHAp coatings were synthesized on the 316LSS substrates by cathodic scanning potential method in the otimal condiction: the scanning potential range of 0 to -1.7 V/SCE, the reaction temperatures of 50 o C, the scanning time of 5 and the scanning rate of 5 mV/s in 80 mL DNa2 solution. 2.1.2.1. Synthesis of Cu 2+ , Ag + or Zn 2+ doped NaHAp coatings Material of NaHAp/316LSS with mass of 2.45x10 -3 g was immersed in 4 mL M(NO3)n solutions with variable concentration showed on Table 2.2 and at different time immersions: 0; 2.5; 5; 10; 20; 30; 60 and 80 minutes at room temperature. Table 2.2. The initial concentration of M n+ (mol/L) M(NO3)n Concentration (mol/L) Cu(NO3)2 0.005 0.01 0.02 0.05 0.1 AgNO3 0.0012 0.0022 0.005 0.01 - Zn(NO3)2 0.01 0.05 0.1 0.15 - 2.1.2.2. Synthesis of Cu 2+ , Ag + and Zn 2+ co-doped NaHAp coatings CuAgZnHAp coatings was synthesized by the way: immersion the material of NaHAp/316LSS about 30 minutes at room temperature in 4 mL solutions containing simultaneously: Cu(NO3)2 0.02 M + AgNO3 0.001 M + Zn(NO3)2 0.05 M. 2.1.3. Synthesis of Mg 2+ , Sr 2+ , Na + , Cu 2+ , Ag + , Zn 2+ and F - co-doped HAp coatings (HApđt) - Preparing material of MgSrFNaHAp/316LSS: MgSrFNaHAp coatings were synthesized on the 316LSS substrates by cathodic scanning potential method in the otimal condiction: the scanning potential range of 0 to -1.7 V/SCE; reaction temperatures of 50 o C; scanning time of 5; scanning rate of 5 DSr3 DNa2 + Sr(NO3)2 1x10 -4 M DSr4 DNa2 + Sr(NO3)2 5x10 -4 M FNaHAp DF1 DNa2 + NaF 5x10 -4 M DF2 DNa2 + NaF 1x10 -3 M DF3 DNa2 + NaF 2x10 -3 M 5 mV/s in 80 mL the solution containing: DNa2 + NaF 2.10 -3 M + Sr(NO3)2 5.10 -5 M + Mg(NO3)2 1.10 -3 M. - HApđt coatings was synthesized by the way: immersion the material of MgSrFNaHAp/316LSS about 30 minutes at room temperature in 4mL solutions containing simultaneously: Cu(NO3)2 0.02 M + AgNO3 0.001 M + Zn(NO3)2 0.05 M. 2.2. Research method 2.2.1. Electrochemical method Methods of scanning potential, potential applied, open circuit potential and electrochemical impedance spectra which were carried out on AUTOLAB equipment at Institute for tropical Technology. 2.2.2. Ion exchange method Ion exchange was done by immersing the meterial of NaHAp/316LSS or MgSrFNaHAp/316LSS in solution containing M n+ with different concentrations. 2.2.3. Coatings characterization The composition and structure of doped HAp obtained coatings were analyzed by the method: IR, XRD, SEM, AFM, EDX (or AAS or ICP-MS), UV-VIS. Physical properties of the coatings was determined by: mass, thickness, adhesion strength. The dissolution behavior of the coatings were studied by measuring the concentration of Ca 2+ dissolved from the coatings and iron released from 316LSS substrates when the samples immersed into the 0.9 % NaCl solution or SBF solution. 2.2.5. In vitro and in vivo Test 2.2.5.1. Invitro test in simulated body fluid (SBF) solutions The in vitro tests in SBF solution investigated by the apatite formed ability and the protection substrates ability of meterials and using the method: open circuit potential (OCP), electrochemical impedance measurements at the OCP and the polarized Tafel curves. 2.2.5.2. Cytotoxicity ability test The safety and biocompatibility of NaHAp and MgSrFNaHAp powder were tested on fibroblasts cells by two methods: the Trypan Blue and the MTT. 2.2.5.3. Antibacterial ability test The antibacterial ability of NaHAp, MNaHAp, MgSrFNaHAp and HApđt powder were tested on three strains: E.faecalis, E.coli, C.albicans và P.aerugimosa by the disk diffusion agar method. 2.2.5.4. In vivo test Healthy dogs are divided to 3 groups, each group of 6 dogs, which are implanted with 3 splint made of: 316LSS, NaHAp/316LSS and 6 MgSrFNaHAp/316LSS by two methods: implantation the materials on the thigh and on the femur. The material compatibility is evaluated by observation of the situation the incision, the general images and the microscope images at transplant location. CHAPTER 3. RESULTS AND DISCUSSION 3.1. Synthesis and characterization of doped HAp coatings 3.1.1. Electrodeposition of doped HAp coatings 3.1.1.1. NaHAp coatings a. The cathodic polarization curve The cathodic polarization curve of 316LSS substrates at the potential range 0 ÷ -2.1 V/SCE are shown in Figure 3.1. With this potential range, there are several electrochemical reactions, such as: 2H + + 2e -  H2 (3.1) O2 + 2H2O + 4e -  4OH- (3.2) 2 4H PO  + 2e -  3 4PO  + H2 (3.3) 2 2 4H PO  + 2e -  2 2 4HPO  + H2 (3.4) 2 2 4HPO  + 2e - 2 3 4PO  + H2 (3.5) 3NO  + 2H2O + 2e  2NO  + 2OH - (3.6) 2H2O + 2e -  H2 + 2OH - (3.7) 2 4H PO  + OH -  2 4HPO  + H2O (3.8) 2 4HPO  + OH -  3 4PO  + H2O (3.9) 10(Ca 2+ , Na + ) + 6 3 4PO  + 2OH − → (Ca, Na)10(PO4)6(OH)2 (3.10) -2.2 -2.0 -1.8 -1.6 -1.4 -1.2 -1.0 -0.8 -0.6 -0.4 -0.2 0.0 -6.0 -5.5 -5.0 -4.5 -4.0 -3.5 -3.0 -2.5 -2.0 -1.5 -1.0 -0.5 0.0 i (m A /c m 2 ) E (V/SCE) Figure 3.1. The cathodic polarization curve of 316LSS substrates in the DNa2 solution b. Effect of Na + concentration The ratio of (Ca+0.5Na+Mg)/P in all obtained coatings samples at DNa1, DNa2 and DNa3 solutions is the same the ratio of Ca/P in the natural bone (1.67) (Table 3.1). However, to reach the Na/Ca ratio similar to its in natural bone, the deposited NaHAp coatings in the DNa1 and DNa2 solution are suitable. Therefore, DNa2 was chosen for the next experiments. 7 Table 3.1. The component of elements of NaHAp deposited on 316L SS in DNa1, DNa2 and DNa3 solutions DD Weigh (%) Na / Ca (0.5 Na+ Ca)/ P P Ca Na DNa1 17.25 36.09 0.32 0.0155 1.63 DNa2 16.80 33.20 1.50 0.0785 1.61 DNa3 16.60 33.09 2.20 0.1156 1.58 4000 3500 3000 2500 2000 1500 1000 500 T ra n m is ta n c e ( a .u ) Wave number (cm -1 ) 4 4 7 5 6 6 6 0 3 8 7 4 1 3 8 4 1 6 4 1 3 4 4 1 P O 43 - P O 43 - C O 32 - C O 32 - O H - H 2O 1 0 3 6 10 20 30 40 50 60 70 In te n s it y degree 1 111 1 1 1 NaHAp HAp (NIST) (a) 2 3 2 11 1 1. HAp; 2. CrO.FeO.NiO; 3. Fe Figure 3.2. IR spectra and XRD patterns of NaHAp deposited in DNa2 solution Both IR spectra and XRD patterns of NaHAp deposited in DNa2 solution exhibit that NaHAp coatings have crystals structure and single phase of HAp (Figure 3.2). c. Effect of the scanning potential range The charge, mass, thickness and adhesion strength of NaHAp coatings at the different potential ranges show that the thickness and adhesion strength of NaHAp coatings reaches the maximum value at potential range of 0 ÷ -1.7 V/SCE (Table 3.2). Thus, the potential range 0 to -1.7 V/SCE is chosen for NaHAp coatings electrodeposition. Table 3.2. The variation of charge, mass, thickness and adhesion strength of obtained NaHAp coatings at the different scanning potential ranges Scanning potential ranges (V/SCE) Charge (C) Mass (mg/cm 2 ) Thickness (µm) Adhesion (MPa) 0 ÷ -1.5 0.41 1.00 3.2 - 0 ÷ -1.7 3.23 2.45 7.8 7.2 0 ÷ -1.9 4.29 1.82 5.8 7.1 0 ÷ -2.1 6.57 1.67 5.3 7.0 d. Effect of electrodeposition temperature The SEM images of NaHAp coatings deposited in DNa2 at different temperatures show that the temperature have an effected on the morphology of obtained coatings. The XRD diffraction data of NaHAp coatings at the different temperatures are shown in Figure 3.4. The typical peaks of the 316LSS substrates were 8 observed in all samples. At 25 and 35 o C, the obtained coatings is mostly dicalcium phosphate dehydrate (CaHPO4.2H2O, DCPD) with the typical peaks at 2 12o and 24o. With temperature from 50°C, the peaks of DCPD are not detected and there are only characteristic peaks of HAp phase at 2  26o (002), 32 o (211), 33 o (300), 46 o (222) and 54 o (004). Thus, 50 o C is chosen to prepare NaHAp coatings. Figure 3.3. The SEM images of deposited NaHAp coatings at different temperatures 10 20 30 40 50 60 70 degree In te ns ity 35 0 C 25 0 C2 50 0 C 60 0 C 1. HAp; 2. DCPD 3. CrO.FeO.NiO; 4. Fe 1 1 3 43 2 1 11 Figure 3.4. XRD patterns of NaHAp deposited at different temperatures e. Effect of pH Results of mass and thickness of NaHAp coatings with pH solusions from 4.0 to 5.5 show on table 3.3. The results indicate that their values reaches the highest value at pH0=4.5. Thus, pH0 is chosen for NaHAp coatings electrodeposition. Table 3.3. The variation of mass and thickness of obtained NaHAp coatings at pH solutions difference pH 4.0 4.5 5.0 5.5 Mass of NaHAp coatings (mg/cm 2 ) 2.05 2.43 1.54 1.31 Thickness of NaHAp coatings (µm) 6.55 7.80 4.92 4.19 g. Effect of the scanning times The charge, mass, thickness and adhesion strength of NaHAp coatings at the different scanning times show that the thickness and adhesion strength of NaHAp coatings are highest at 5 scanning times (Table 3.4). Table 3.4. The variation of charge, mass, thickness and adhesion strength of obtained NaHAp coatings at the different scanning times Scanning times (times) Charge (C) Mass (mg/cm 2 ) Thickness (µm) Adhesion (MPa) 9 1 0.74 0.52 1.6 - 3 2.21 1.50 4.7 7.2 5 3.23 2.45 7.8 7.2 7 4.07 1.27 4.1 6.3 10 5.20 1.05 3.4 6.0 The SEM images of obtained NaHAp coatings show that: they have slate shapes with large size at 3 scanning times; plate shapes and denser with the size of 150×25 nm at 5 scanning times; both slate and plate shapes at 7 scanning times (figure 3.5). Based on the above results, 5 scanning times is selected for NaHAp coatings electrodeposition. Figure 3.5. The SEM images of NaHAp coatings deposited at different scanning times h. Effect of the scanning rate The thickness of obtained NaHAp coatings is highest at 5 mV/s scanning rates (Table 3.5) so it is chosen to deposite the HAP coatings. Table 3.5. The variation of charge, mass, thickness and adhesion strength of obtained NaHAp coatings at the different scanning rates Scanning rates (times) Charge (C) Mass (mg/cm 2 ) Thickness (µm) Adhesion (MPa) 3 5.09 1.95 6.2 6.2 4 4.11 2.15 6.9 6.5 5 3.23 2.45 7.8 7.2 6 2.21 1.27 4.1 7.8 7 1.85 0.93 3.0 10.6 3.1.1.2. Synthesis of Mg 2+ , Sr 2+ or F - doped NaHAp coatings (ĐNaHAp) a. Effect of concentration The cathodic polarization curve of 316LSS substrates in the different solusions shows on figure 3.6. Concentration increas