Summary of Doctoral thesis in Chemistry: Synthesis and characterization of trace elements co-doped Hydroxyapatite on 316L stainless steel application in bone implant

The thesis aims to successfully fabricate NaHAp films doped separately and simultaneously with microelements: sodium, magnesium, strontium, fluorine, copper, silver and zinc on 316L stainless steel substrate to meet the requirement of screw bracing bone. Study on physicochemical characteristics, study on toxicity, antibacterial ability and bio-compatibility of NaHAp films separately and concurrently with trace elements.

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

Thesis can be further referred at:

- The Library of Graduate University of Science and Technology

- National Library of Vietnam

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 Ca2+ 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

- 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 (Mg2+, Na+, K+,

Sr2+ 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)Ca2+ + 6PO43- + (2-y)OH- + xM2+ + 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

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

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 oC; 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

ĐNaHAp were deposied at 50 o

C 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(NO ) 5x10-5 M

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 oC, the scanning time of 5 and the scanning rate of 5 mV/s

in 80 mL DNa2 solution

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

M(NO 3 ) 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 -

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

- 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 oC; 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

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)20.05 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 Mn+ 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 Ca2+ 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

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

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:

HPO + OH-  3

4

PO  + H2O (3.9) 10(Ca2+, Na+) + 6PO34 + 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

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

Table 3.1 The component of elements of NaHAp deposited on 316L SS in

DNa1, DNa2 and DNa3 solutions

NaHAp

HAp (NIST)

(a)

2 3

1 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/cm2)

Thickness

(µm)

Adhesion (MPa)

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

observed in all samples At 25 and 35 oC, the obtained coatings is mostly dicalcium phosphate dehydrate (CaHPO4.2H2O, DCPD) with the typical peaks

2

1 1

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

Mass of NaHAp coatings (mg/cm2) 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/cm2)

Thickness

(µm)

Adhesion (MPa)

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/cm2)

Thickness

(µm)

Adhesion (MPa)

In the potential range of 0 ÷ -1,7 V/SCE, the reducted reactions are listed

at section 3.1.1 Then, doped HAp coatings (MgNaHAp, SrNaHAp or FNaHAp)

is producted on the cathode substrates according to the chemical reaction:

10(Ca2+,Mg2+,Na+) + 6PO4 3−

+ 2OH− → (Ca,Mg,Na)10(PO4)6(OH)2 (3.12) 10(Ca2+,Sr2+,Na+) + 6PO43− + 2OH− → (Ca,Sr,Na)10(PO4)6(OH)2 (3.13) (Ca,Na)10(PO4)6(OH)2 + xF- + xH+  (Ca,Na)10(PO4)6(OH)2-xFx + xH2O (3.14)

DSr4 DSr3 DSr2 DSr1 DNa2

0

(c)

DF3 DF2 DF1 DNa2

Figure 3.6 The cathodic polarization curve of 316LSS substrates in the

solusions with different concentrations of Mg2+ (a), Sr2+ (b) và F

(c) ions Table 3.6 shows that the concentration of doped ions in the solution increases leading to the increase of their components and the atomic ratios of X/Ca in obtained coatings However, to reach the X/Ca similar to its in natural bone, the soluion of DMg1, DMg2, DMg3, DSr1 or DSr2 are suitable to deposite the MgNaHAp or SrNaHAp coatings The ratio of F/Ca is smaller than its in natural bone but the extension of F- concentration is more than 2.10-3 M leading to precipitation of CaF2 in the solution Therefore, DMg3 or DSr2 or DF3 is chosen to deposite the MgNaHAp or SrNaHAp or FNaHAp coatings

Table 3.6 The element components of obtained coatings at diffrent

Table 3.7 The atomic ratios of X/Ca, Y/P and the formula of

ĐNaHAp coatings

DD Na/Ca X/Ca Y/ P The formula (expectation)

DMg1 0.062 2.90x10-3 1.59 Ca9.403Mg0.027Na0.570(PO4)6(OH)2DMg2 0.056 5.70x10-3 1.58 Ca9.438Mg0.052Na0.510(PO4)6(OH)2DMg3 0.060 9.60x10-3 1.54 Ca9.378Mg0.086Na0.536(PO4)6(OH)2DMg4 0.056 1.95x10-2 1.50 Ca9.352Mg0.168Na0.480(PO4)6(OH)2DSr1 0.065 1.74x10-4 1.64 Ca9.403Sr0.002Na0.595(PO4)6(OH)2

DSr2 0.061 3.68x10-4 1.59 Ca9.447Sr0.003Na0.549(PO4)6(OH)2

DSr3 0.0605 6.30x10-4 1.57 Ca9.457Sr0.006Na0.537(PO4)6(OH)2

DSr4 0.049 1.00x10-3 1.58 Ca9.469Sr0.009Na0.521(PO4)6(OH)2

DF1 0.052 5.50x10-2 1.66 Ca9.508Na0.492(PO4)6(OH)1.477F0.523DF2 0.070 7.40x10-2 1.67 Ca9.326Na0.674 (PO4)6(OH)1.293F0.707DF3 0.099 9.90x10-2 1.67 Ca9.085Na0.915(PO4)6(OH)1.097F0.903(X/Ca = Mg/Ca or Sr/Ca or F/Ca; Y/P = (0,5Na+ Ca + Mg + Sr)/P)

b The effect of scanning potential range

Table 3.8 shows that the scanning potential range of 0 ÷ -1.7 V/SCE for MgNaHAp + SrNaHAp and at 0 ÷ -1.8 V/SCE for FNaHAp, the mass, thickness and adhension strength of obtained ĐNaHAp coatings are best Thus, the scanning potential range of 0 ÷ -1.7 V/SCE is selected to synthesize the MgNaHAp + SrNaHAp coatings and 0 ÷ -1.8 V/SCE for FNaHAp coatings

Table 3.8 The variation of charge, mass, thickness and adhesion strength

of deposited ĐNaHAp coatings at the different scanning potential ranges

ĐNaHAp

Scanning potential ranges

(V/SCE)

Charge (C)

Mass (mg/cm2)

Thickness

(µm)

Adhesion (MPa)