Properties of Binary Ti-Ag Alloy for

Biomedical Applications

Hyung-Min Shim

Department of Medical Science

The Graduate School, Yonsei University

Properties of Binary Ti-Ag Alloy for

Biomedical Applications

Directed by Professor Kyoung-Nam Kim

The Doctoral Dissertation

submitted to the Department of Medical Science,

the Graduate School of Yonsei University

in partial fulfillment of the requirements for the degree of

Doctor of Philosophy of Medical Science

Hyung-Min Shim

December 2005

This certifies that the Doctoral Dissertation

of Hyung-Min Shim is approved.

---------------------------------------------- Thesis Supervisor: Kyoung-Nam Kim

----------------------------------------------- Thesis Committee Member#1: Kyung-Ah Park

----------------------------------------------- Thesis Committee Member#2: Kwang-Mahn Kim

------------------------------------------------ Thesis Committee Member#3: Jong-Chul Park

------------------------------------------------- Thesis Committee Member#4: Keun-Taek Oh

The Graduate School

Yonsei University

December 2005

감사의감사의감사의감사의 글글글글

눈이 내린 연세대학교의 겨울 교정은 아름답습니다. 12 년간 거닐었던

백양로의 발자국들을 되새기며, 지금 제 작은 마침표 하나를 찍으려 합니다. 이

결실을 얻기까지 이끌어주시고 도움 주신 많은 분들이 생각납니다.

우선 치과대학 생체재료공학교실에서 연구하고 논문을 완성하기까지 세심한

배려와 아낌없는 관심을 주신 김경남 교수님께 진심으로 감사 드립니다.

생소했던 치과 분야에 관련해서 많은 가르침을 주시고 여러모로 챙겨주신 저희

교실의 주임교수님이신 김광만 교수님과 지금 미국에 계신 이용근 교수님,

아낌없는 조언으로 제 논문을 심사해주신 의과대학 해부학교실의 박경아 교수님,

의학공학교실의 박종철 교수님께도 감사 드립니다. 금속공학과 선배로 제가

치과대학과 연을 맺게 해주고 끊임없는 지도를 해주신 오근택 박사님께 큰

감사의 뜻을 전합니다.

여러 번의 합금 용해를 성심껏 도와주신 한국지질자원연구소의 최국선

박사님과 후배 동진, 생산기술연구소의 이진규 선배님께도 감사 드립니다. 또한

장비 사용에 있어 눈치가 보였지만 항상 친절하게 대해준 금속공학과

선후배님들께도 감사 드립니다. 금속공학과 선배로 여러모로 많은 조언을

해주셨던 덕창이형, 욱현이형, 민규형, 그 밖에 앞으로 대박터뜨릴

BMK 식구들께도 감사 드립니다.

학부시절 많은 시간을 함께 보내고 추억을 남겨준 절친한 금속공학과 친구들,

중국으로 출장 가서 인생을 즐기고 있을 희경, 멋쟁이 봉환, 일본에서

포닥하고있는 은석, 무서운 부산아그 성운에게도 고마움을 전하고 싶습니다.

대학원 생활을 함께해주고 활력이 넘치게 해준 교실 여러분들, 교실의

터줏대감 이상배 선생님, 대학원생의‘앰’이자 자상한 누님인 김남이 선생님,

애기엄마가 될 킹눈이 의국장 해경, 의국 만담꾼 세종, 막내 우현, 세포 예쁘게

잘 키워준 회영이, 김지연, 이종숙 선생님, 이지혜, 임은진양 모두에게 감사

드립니다. 박학다식한 지환형, 듬직한 대학원 동기 영일, 우수 대학원생 강서구

청년 동현, 간다무매니아 민철, 함께 제일 많이 고생한 금속전공 후배 술빵 동국,

SEM 찍기 좋아하는 과격남 재선, 호세 까를로스 애노니호 세호, 축구 좋아하는

기형, 여성스런 면이 많은 재주꾼 병현, 아줌마 대학원생 이주혜 선생님, 제도령

진아에게 고마움을 전합니다.

하늘에서 기뻐하실 할아버지와 저를 무척 사랑해주시는 할머니, 외할아버지,

외할머니, 훈훈한 격려의 말씀을 아끼지 않으셨던 큰아버지, 작은아버지, 고모들,

외삼촌께도 감사의 마음을 전합니다.

항상 아들 뒷바라지에 여념이 없으셨던, 한결 같은 마음으로 저를 지켜봐

주신 든든한 후원자이신 아버지, 어머니께 엎드려 감사 드립니다. 저를

친아들처럼 자상하게 대해주시는 장모님, 멀리 미국에서 나를 생각해주고

격려해주는 사랑하는 동생 민정과 매제 영훈, 깜찍한 조카 애쉴리, 이쁜 처제

민정에게도 감사의 마음을 전합니다.

마지막으로 이 작은 결실을 언제나 내 곁에 있어준 사랑하는 아내 김희정과

조금 있으면 세상 빛을 보게 될 나의 주니어에게 바칩니다.

2005 년 12 월

심 형 민 올림

i

TABLE OF CONTENTS

LIST OF FIGURES ································································································iv

LIST OF TABLES ·································································································vii

ABSTRACT ···············································································································1

I. INTRODUCTION ·································································································4

1. Biomaterial ············································································································4

2. Metallic biomaterials ·····························································································9

A. Stainless steels ································································································10

B. Co-Cr alloys ····································································································11

C. Titanium and its alloys ···················································································11

3. Principal requirements of metallic biomaterials as implant materials ·················13

A. Corrosion resistance ·······················································································13

B. Biocompatibility and biofunctionality ····························································14

4. Research trends of new titanium-based alloy ······················································19

II. MATERIALS AND METHODS ·······································································22

1. Materials ··············································································································22

A. Alloy design and arc melting of titanium-silver alloy ····································22

B. Heat treatment and hot rolling ········································································22

C. Specimen preparation for experiments ···························································24

2. Experimental Methods ························································································25

A. Conditions of alloys ·······················································································25

(A) Constituent analysis of alloying element and evaluation of impurity content

·····························································································································25

(B) Phase identification and microstructure observation ··································25

B. Mechanical property ······················································································25

ii

(A) Tensile test ··································································································25

(B) Bend test ·····································································································26

(C) Microhardness test ······················································································27

C. Corrosion resistance and electrochemical property ········································27

(A) Potentiodynamic test ···················································································27

(B) Potentiostatic test and open circuit potential measurement ························28

D. Surface characterizations of the alloy ····························································29

E. Effect of fluoride on the electrochemical property of the alloy ······················29

F. Electrochemical property of passive oxide film ··············································30

(A) Electrochemical Impedance Spectroscopy (EIS) ········································30

(B) Equivalent circuit model and parameter value ············································30

G. Biocompatibility evaluation ···········································································30

(A) L929 fibroblast cell culture ·········································································30

(B) Metal ion release test ··················································································31

(C) Cell adhesion: SEM observation ·································································31

(D) Agar diffusion test ······················································································32

(E) Cell viability: MTT assay ···········································································34

(F) Acute systematic toxicity test ······································································34

III. RESULTS ··········································································································36

1. Composition analysis of alloying element and evaluation of impurity content ···36

2. Phase identification and microstructure observation ··········································36

3. Mechanical property ····························································································40

A. Tensile test ······································································································40

B. Bend test ·········································································································42

C. Microhardness test ··························································································43

4. Corrosion resistance and electrochemical property ············································44

A. Potentiodynamic test ······················································································44

B. Potentiostatic test ····························································································45

C. Open circuit potential measurement ·······························································47

5. Surface characterization of the alloy ···································································48

iii

6. Effect of fluoride on the electrochemical property of the alloy ··························53

7. Electrochemical property of passive oxide film ··················································58

8. Biocompatibility evaluation ················································································63

A. Metal ion release test ······················································································63

B. Cell adhesion morphology ··············································································63

C. Agar diffusion test ··························································································69

D. Cell viability: MTT assay ···············································································72

E. Acute systematic toxicity test ·········································································72

IV. DISCUSSION ····································································································76

1. A brief overview of physical metallurgy of titanium alloy ·································76

2. Compositional analysis and phase/microstructure observation ···························77

3. Mechanical property ····························································································81

4. Corrosion resistance and electrochemical property ············································82

5. Surface characterization of the alloy ···································································87

6. Effect of fluoride on the electrochemical property of the alloy ··························91

7. Electrochemical property of passive oxide film ··················································94

8. Biocompatibility evaluation ················································································96

V. CONCLUSION ·································································································100

REFERENCES········································································································102

ABSTRACT (in KOREAN) ·················································································120

iv

LIST OF FIGURES

Figure 1. The foreign body reaction as illustrated here is the normal reaction by

higher organisms to an implanted synthetic material ··································8

Figure 2. History of metals, plastics, and ceramics for biomedical applications ········9

Figure 3. The requirements on the implant materials for orthopedic applications ···15

Figure 4. Relationship between polarization resistance and biocompatibility of

biometallic elements and alloys ································································16

Figure 5. Percentage of metal allergy caused by each metallic element ···················17

Figure 6. Simple schematic figure of stress shielding ···············································18

Figure 7. Equilibrium phase diagram for the binary titanium-silver alloy system ····23

Figure 8. Schematic diagram of specimen preparation procedure ····························24

Figure 9. Shape and size of specimen for tensile test ···············································26

Figure 10. Schematic diagram of three-point bend test ············································26

Figure 11. XRD patterns and phase identification of titanium and titanium-silver

alloys ·········································································································38

Figure 12. Microstructure of titanium and titanium-silver alloys (magnification of

×100) ········································································································39

Figure 13. Microstructure of TA5- Precipitation of Ti2Ag (white arrow) was shown

following grain boundary in TA5 under high magnification (×200) ·······40

Figure 14. Tensile strength of titanium and titanium-silver alloys ···························41

Figure 15. Elastic modulus of titanium and titanium-silver alloys from tensile test ··41

Figure 16. Bend strength of titanium and titanium-silver alloys ·······························42

Figure 17. Elastic modulus of titanium and titanium-silver alloys from bend test ·····43

Figure 18. Microhardness values of titanium and titanium-silver alloys ··················44

Figure 19. Graph of current density vs. potential of titanium and titanium-silver alloys

from potentiodynamic test ········································································45

Figure 20. Current densities of titanium and titanium-silver alloys at each potential in

artificial saliva, 37 (a) 250 mV (b) 0 mV (c) - 250 mV (SCE) ··············46

Figure 21. Open circuit potentials of titanium and titanium-silver alloys in artificial

saliva, 37 ······························································································48

v

Figure 22. XPS survey spectrum for as-polished titanium after argon ion sputtering

·····················································································································49

Figure 23. Representative high resolution spectrum of the (a) Ti 2p and (b) O 1s

regions ·······································································································51

Figure 24. Area fraction of the metallic Ti in high resolution spectrum of take off

angle 75° ···································································································53

Figure 25. Potentiodynamic curves of titanium and titanium-silver alloys when NaF

was added to artificial saliva, 37 ··························································55

Figure 26. Current densities of titanium and titanium-silver alloys at 250 mV (SCE)

when 1% NaF was added to artificial saliva, 37 ··································58

Figure 27. EIS spectra for titanium and titanium-silver alloys (a) Nyquist plot (b)

Bode phase plot ·························································································59

Figure 28. Equivalent circuit proposed for fitting EIS spectra ·································60

Figure 29. The parameter value of the equivalent circuit from Figure 25(a) after

fitting the EIS spectra. (a) Rp (passive film resistance), (b) CPE (interface

capacitance) ·······························································································62

Figure 30. Morphology evaluation of L929 fibroblast cell on the surface by SEM (a)

magnification of ×200 (b) ×500 ······························································65

Figure 31. Microvilli protrusion morphology at the leading edge of L929 fibroblast

cell (high magnification of ×5000, ×10000) ···········································68

Figure 32. Photographs of Petri-dish of each alloy after agar diffusion test (Upper left:

positive control (gutta percha), upper right: negative control (soda-lime

glass), lower: test alloys per each Petri dish) ············································70

Figure 33. Micrograph of L929 fibroblast cell morphology around titanium and

titanium-silver alloys and control ·····························································71

Figure 34. Cell (L929 fibroblasts) viability of titanium and titanium-silver alloys by

MTT assay ································································································72

Figure 35. Light microscopes of tissue from control and test group mouse (a) liver (b)

kidney ········································································································74

Figure 36. Classification scheme for binary titanium alloy phase diagrams ············78

Figure 37. Schematic view of the oxide film on pure titanium ·································90

vi

Figure 38. Illustration of XPS spectra taken from a thin oxide film on a metal at near

normal collection angle (bulk angle) and near grazing collection angle

(surface angle) ···························································································90

vii

LIST OF TABLES

Table 1. Uses for biomaterials ····················································································5

Table 2. Biocompatibility classification as proposed by Osborn and Newesely ········6

Table 3. Selective mechanical properties of metallic biomaterials ···························12

Table 4. Titanium and silver contents of each alloy ·················································23

Table 5. Constituents of artificial saliva ···································································28

Table 6. Description of decolorization index, lysis index and interpretation of

response index used in the agar diffusion test ···········································33

Table 7. Chemical compositions of titanium-silver alloys manufactured in this study

(n=3) ··········································································································36

Table 8. Impurity (C, S, O, N) contents of titanium-silver alloys manufactured in this

study (n=3) ································································································37

Table 9. Fraction of elements Ti and O in survey spectrum with change of take off

angle ··········································································································50

Table 10. Chemical compositions and their fractions of surface films on alloys ·····52

Table 11. Passive current density of alloys in 1% NaF added artificial saliva (Data

from Figure 23) ·························································································57

Table 12. Open circuit potentials of titanium and titanium-silver alloys in NaF

containing artificial saliva ·········································································57

Table 13. Titanium and silver ion release concentration (ppb) after immersion for

each period in artificial saliva, 37 ························································64

Table 14. Cytotoxicity of titanium and titanium-silver alloys evaluated by agar

diffusion test ······························································································69

Table 15. Weight changes (g) of experimental mice of each group for acute

systematic toxicity ·····················································································73

Table 16. Minimum concentration (at%) of the alloying element necessary for the

complete stabilization of the β phase in binary titanium alloys with d-metals

of 4 ~ 6 periods ·························································································79

1

ABSTRACT

Properties of Binary Ti-Ag Alloy for Biomedical Applications

Hyung-Min Shim

Department of Medical Science

The Graduate School, Yonsei University

(Directed by Professor Kyoung-Nam Kim)

A biomaterial is defined as “a nonviable material used in a medical device,

intended to interact with biological systems”. Biomaterials are inserted into or placed

onto the body with the aim of improving the function of or replacing a diseased,

damaged or lost tissue or a whole organ. Metallic biomaterials are the most suitable

for replacing failed hard tissue up to now. Among metallic biomaterials, titanium and

its alloys are used extensively in the medical and dental fields because of their good

corrosion resistance, high strength to density ratio, and specially, low elastic modulus,

and good biocompatibility compared to other metallic materials. However, the

toxicity of alloying elements has been disputed. It appears that small amounts of

metal ion or element, released in the human body, induce possible cytotoxic effect

and neurological disorders. Thus, toxicity of alloying elements like Al, V etc., and

high elastic modulus of the commercially pure (cp) titanium and Ti6Al4V alloy

compared to natural bone has required the development of new titanium based alloys.

The objective of this study is to evaluate the properties of titanium-silver alloys and

their suitability as metallic biomaterials. We selected silver as addition element to

titanium. Silver is classified as noble, to be exact precious, metal. Therefore it has a

much higher electromotive force than titanium like other noble metals and good wear

resistance and also has very soft and ductile property. We designed titanium-silver

alloys with silver contents ranging from zero to 5.0 at% in step of 1.0 at% and

designated these alloys TA1 to TA5, according to the relative silver contents of each

alloy. They were arc melted, homogenized at 950 for 72 hours, hot-rolled, and

2

solution heat-treated and quenched. First, we performed phase and microstructure

evaluation of alloys and for mechanical property evaluations, tensile test, bend test

and microhardness test were performed. Potentiodynamic test and potentiostatic test

were also performed to evaluate corrosion resistance and electrochemical property in

biological environment. From surface characterization of the alloys, relation between

silver content and surface condition of alloy was investigated. Moreover, we drew

relation between surface condition and electrochemical property, and then we also

studied effect of silver content on each property. Finally, effect of silver element on

the biocompatibility of the alloys was studied, followed by investigation of

biocompatibility and cytocompatibility of alloys in vitro and in vivo. From results of

phase identification, β phase began to appear from TA3; silver content 3.0 at%. This

3.0 at% is the minimum silver content necessary for β phase stabilization at room

temperature. In addition, TA4 and TA5 had Ti2Ag diffraction peak with small

intensity. From microstructural observation, in case of titanium, the only equiaxed α

phase was found. Besides, when silver content was over 3.0 at%, Widmanstätten α +

β phase was seen in the originally formed β matrix. The bend strength and hardness

value tended to rise with increased silver content and increased largely over 3.0 at%.

However, elastic modulus was not much different from alloys and had no relation to

silver content. From the potentiodynamic and potentiostatic test, titanium-silver alloys

showed better corrosion resistance and electrochemical property than titanium.

Titanium-silver alloys also exhibited higher open circuit potentials than pure titanium

and those of titanium-silver alloys varied directly with silver content. However, in

case of TA4 and TA5, alloys with over 3.0 at% silver content, current density of these

alloys increased. From the XPS results, it was considered that titanium-silver alloys

possessed thicker oxide films than titanium. The oxide film of TA2 and TA3

contained much TiO2, most stable oxide film. Fluoride in solution affected passive

current density and open circuit potential of alloys. The passive current densities of

titanium and titanium-silver alloys increased with increasing fluoride concentration.

TA2 and TA3 exhibited a low current density relatively and showed a stable behavior

compared to titanium. When silver content is exceeded 4.0 at%, electrochemical

stability and resistance against fluoride of titanium-silver alloy was weaken by Ti2Ag

precipitation. From the EIS data, it could be noted that titanium and titanium-silver

3

alloys showed the characteristic response of a capacitive behavior of surface film and

TA2 and TA3 had high passive film resistance. Titanium-silver alloy showed

extremely low value of metal ion release. There was no significant difference

according to silver content and immersion period. Ion release content of silver was

negligible when considering detection limit of release content. From cell adhesion

morphology on titanium and titanium-silver alloy surfaces, L929 fibroblast cells

adhered tightly, well spread and proliferated uniformly on the surface and showed

dendritic network at the leading edge of locomoting cells. Titanium-silver alloys

showed none cytotoxicity in agar diffusion test and exhibited over 95% cell viability

in MTT test. There was no difference between titanium and each titanium-silver alloy

and cytotoxicity and cell viability had no relation to silver content. From acute

systematic toxicity test, no toxic symptom or adverse reaction was discovered, and

mortality was zero. The histopathological examination of the liver and kidney of test

mice revealed no remarkable changes. From the above results, we concluded that

titanium-silver alloy had better mechanical property, corrosion resistance and

electrochemical property than pure titanium due to thick and stable passive oxide film

in biological environment but silver addition content should be limited 3.0 at% for

maintaining desirable mechanical and electrochemical property. Titanium-silver

alloys also had good biocompatibility irrelative of silver content. In conclusion, it was

regarded that titanium-silver alloys had suitable biofunctionality and biocompatibility

for biomedical applications.

Key words: acute systematic toxicity, biocompatibility, biofunctionality, corrosion

resistance, cytotoxicity, cell viability, electrochemical property, mechanical property,

metal ion release, passive oxide film, surface characterization, titanium-silver alloy

4

Properties of Binary Ti-Ag Alloy for Biomedical Applications

Hyung-Min Shim

Department of Medical Science

The Graduate School, Yonsei University

(Directed by Professor Kyoung-Nam Kim)

I. INTRODUCTION

1. Biomaterial

A commonly used definition of biomaterial is “a nonviable material used in a

medical device, intended to interact with biological systems”.1 Some scientists

defined “Biomaterial is substance, other than food or drug, introduced in therapeutic

or diagnostic systems which are in contact with the tissues or biological fluids”.2 In

the last decades a wide variety of biomaterials has been developed with different

physico-mechanical, biochemical and biological properties depending on the

biomedical application. The field of biomaterials has been consistent growth with a

steady introduction of new ideas and productive branches evolved drug delivery,

diagnostic arrays and tissue engineering.

Biomaterials are inserted into or placed onto the body with the aim of improving

the function or replacing a diseased, damaged tissue or a whole organ. Biomaterials

are categorized according to their chemical composition (polymers, ceramics, metals),

to their origin (natural or synthetic), as well as to their supramolecular structure

(porous, composites). It is this variety that provides us the almost endless

opportunities to design and produce the biomaterials we need.3 Table 1 lists some of

these applications, both medical and nonmedical.

The great complexity of conditions imposed for biomaterials makes its choice for a

certain application very difficult. Selecting biomaterials for different components

5

depends especially on several factors. First, the material must be biocompatible to the

human body.

Table 1. Uses for biomaterials3

Medical uses Non-medical uses

Artery graft Arrays for DNA and diagnostics

Breast implant Bioremediation materials

Cochlear implant Biosensors

Dental implant Bioseparations, chromatography

Ear drainage tube Biofouling-resistant materials

Feeding tube Biomimetics for new materials

Glaucoma drainage tube Cell culture

Hydrocephalous shunt Controlled release for agriculture

Intraocular lens Electrophoresis materials

Joints (hip, knee, shoulder) Fuel cells (biomass)

Keratoprosthesis MEMS

(Micro Electro-Mechanical System)

Left ventricular assist device (LVAD) Muscles (artificial) and actuators

Mechanical heart valve Nanofabrication

Nerve guidance tube NEMS

(Nano Electro-Mechanical System)

Ophthalmic drug delivery device Neural computing/biocomputer

Pacemaker Smart clothing for biowarfare

Renal dialyzer Yeast array chip

Stent

Tissue adhesive

Urinary catheter

Valve, heart

Wound dressing

X-ray guide

Zirconium knee joint

6

On top of that, it must have an excellent corrosion resistance in the body environment

and appropriate mechanical properties in service. For any material to act as a

biomaterial, it must satisfy two essential characteristics, biocompatibility and

biofunctionality. Biocompatibility refers to the ability of the device to continue to

perform that function, effectively and as long as necessary, in or on the body, while

biofunctionality is related to a set of properties which allow a device to perform a

required function. The biocompatibility of a material is affected by many factors, one

of which and the most important one, is its corrosion resistance in a highly aggressive,

high chloride containing body fluid environment. Corrosion of implant material cause

minute corrosion products to accumulate in adjacent tissues and stimulate allergy in

patients.

Biocompatibility is directly related to the chemical and biochemical characteristics

of the biomaterial and it is defined as “the quality of not having toxic or injurious

effects on biological systems” . Recently, biocompatibility has been considered as “the

ability of a material to perform with an appropriate host response in a specific

application” , taking into account the interactivity between the biomaterial and the

host.1 An important property of a biomaterial is the tissue response upon implantation.

Depending on the function of the implant, this determines the biocompatibility of a

material. The classification was proposed by Osborn and Newesely,4 and is presented

in Table 2. This classification was designed to group biomaterials as biotolerant,

bioinert and bioactive according to their tissue response.

Table 2. Biocompatibility classification as proposed by Osborn and Newesely4

Degree of compatibility Characteristics

Biotolerant Material separated from adjacent tissue by a fibrous tissue

layer along most of the interface

Bioinert

Material that retains its structure in the body after

implantation and does not induce any immunologic host

reactions

Bioactive Material that form bonds with living tissue

7

Human body is chemically, electrically, and mechanically active, and so the

interface between a biomaterial and tissue is the location of a wide range of dynamic

process and reactions. The appreciations of the host response (healing) of implanted

materials help us understand the performance of biomaterials. When a tissue is

injured, the normal healing response is initiated through a series of complex events

that include acute inflammation, the formation of granulation tissue, and eventual scar

formation.5,6 The immediate response is to flood the injured area with blood.

Fibrinogen within the blood is cleaved into fibrin to form a blood clot that promotes

platelet adhesion and aggregation. Cytokines and growth factors are released to

recruit white blood cells, mainly neutrophils. Monocytes are then called to the wound

site where they differentiate into macrophages. The macrophages are responsible for

cleaning up the wound site, which may contain foreign material, bacteria, and dead

cells, and also for recruiting cells such as fibroblasts and endothelial cells, which

convert the fibrin clot into a highly vascularized granulation tissue. The formation of

blood vessels is essential to the healing wound. The granulation tissue is subsequently

replaced by an extracellular matrix (ECM) deposited primarily by fibroblasts. The

degree of ECM remodeling depends on the extent and location of the injury. In some

cases, complete restoration of the tissue architecture is possible; however, in most

cases the granulation tissue is remodeled into scar tissue.

A biomaterial implanted into the body, however, induces a different response

termed the foreign body reaction. This response is illustrated in Figure 1. The

biological response to materials has been reviewed in detail by Anderson.7 Briefly, a

biomaterial elicits nonspecific protein adsorption immediately upon implantation.

Many different proteins adsorb to the surface in a range of conformations from native

to be denatured. Non-specific protein adsorption, however, never occurs in the normal

physiological process of wound healing. Thus, nonspecific protein adsorption may be

an instigator in the foreign body reaction.

8

Figure 1. The foreign body reaction as illustrated here is the normal reaction by

higher organisms to an implanted synthetic material.

A number of different cells, such as monocytes, leukocytes, and platelets (cells that

are key players in normal wound healing), adhere to these biomaterial surfaces and as

a result may lead to upregulation of cytokines and subsequent proinflammatory

processes. In addition, the implant is significantly larger than the adhered

macrophages, preventing them from phagocytosing the foreign body. Chronic

inflammation at the biomaterial interface ensues, and the frustrated macrophages fuse

together to form multinucleated foreign body giant cells that often persist for the

lifetime of the implant.8,9 The end stage of the foreign body reaction involves the

walling off of the device by a vascular, collagenous fibrous tissue that is typically 50

~ 200 µm thick.

9

2. Metallic biomaterials

Metallic biomaterials have the longest history among the various biomaterials.

History of representative materials for biomedical applications was shown in Figure

2.10 Stainless steel was the first successfully used implant material in surgical field.

Moreover, as the population ratio of the aged people is rapidly growing, the number

of the aged people demanding replacement of failed tissue with artificial instruments

made of biomaterials is increasing. In particular, the amount of usage of instruments

for replacing failed hard tissues such as artificial hip joints, dental implants, etc. is

increasing among the aged people. Metallic biomaterials are the most suitable for

replacing failed hard tissue up to now. These are often used to replace structural

components or reinforce the structure or function of tissues of the human body. This

is because, when compared to polymeric and ceramic materials, they possess more

superior tensile strength, fatigue strength, and fracture toughness- the very key

properties required of structural materials. As such, metallic biomaterials are used in

medical devices such as artificial joints, bone plates, screws, intramedullary nails,

spinal fixations, spinal spacers, external fixtators, pace maker cases, artificial heart

valves, wires, stents, dental implants and orthodontic wire. Main metallic biomaterials

are stainless steels, Co-Cr alloys, titanium and its alloys.

Figure 2. History of metals, plastics, and ceramics for biomedical applications.10

10

Although originally developed for industrial purposes, these materials have been

widely used for biomaterial fields due to their relatively high corrosion resistance and

excellent mechanical properties.

A. Stainless steels

The most widely used stainless steel for biomedical applications is 316L stainless

steel (16 ~ 18 wt% Cr, 12 ~ 15 wt% Ni, 2 ~ 3 wt% Mo), an austenitic stainless steel,

with the austenitic phase being stabilized by nickel (Ni). The stainless steels are the

useful materials because of their ease of fabrication and reasonable corrosion

resistance, relatively low cost compared with Co-Cr alloys, pure titanium by a factor

of one-tenth to one-fifth times.11-15

Stainless steels have been widely used for fracture fixation devices like bone plates

and screws, spinal rods, or as intravascular and ureteral stents.15 Although stainless

steels provide better biomechanical properties compared to Co-Cr alloys or titanium

and its alloys,16,17 the reduced corrosion resistance and biocompatibility restrict their

clinical application.13,18 They are prone to localized attack in long-term applications

due to the aggressive biological effects. The corrosion products include iron (Fe),

chromium (Cr), nickel (Ni) and molybdenum (Mo), etc. ions which can accumulate in

tissues surrounding the implant or be transported to distant parts of the body. Both in

vitro and in vivo studies have shown deleterious effects of stainless steel corrosion

products in several organs and tissues.19-25 Especially these may cause a negative

reaction due to their high Ni content (12~15 wt%). Ni has been reported to be the

most common metal sensitizer in humans26,27 and some concern has been expressed

regarding toxicity.28-33

Since 316L stainless steel has been shown to degrade in vivo and toxicity of Ni,

there is significant interest in developing new stainless steels with improved

properties and Ni-free. Among these new alloys, Nitrogenated 316LN stainless steels

have been researched and shown to offer improvement of strength and corrosion

resistance.34-38

11

B. Co-Cr alloys

Co-Cr based alloys are the representative Co-based alloys for biomedical

applications.39-41 They have greater tribological property such as wear resistance

compared to stainless steels and titanium alloys. Therefore, they are used extensively

for load bearing and articulating orthopedic applications such as hip and knee

prostheses. Co-Cr-Mo alloys (ASTM F-75, ASTM F-799, ASTM F-1377, and ASTM

F-1537) consist of 26 ~ 30% Cr, 5 ~ 7% Mo, and less than 0.35% carbon (C) in a base

of cobalt.

They also have some problems like metal allergy and toxicity of metal particles or

debris. There are concerns about the potential toxicity of wear particles and released

metal ions. Some studies have reported raised levels of Co and Cr ions in the body,

raising concerns over the potential toxicity of those ions at high enough levels.42,43

Further concerns have been expressed over the possible sensitivity to certain metals

and the possible links with carcinogenesis.44-46 Both in vivo and in vitro studies have

suggested that Co-Cr particles have a range of toxic effects on a variety of cells and

tissues.47-50

C. Titanium and its alloys

Titanium is the newest metallic biomaterial among the three main metallic

biomaterials (i.e., titanium, stainless steel and Co-based alloy), and these remain the

most popular metallic biomaterials. Among the main metallic materials for

biomedical applications titanium and its alloys are getting much attention in both

medical and dental fields because of good corrosion resistance, biocompatibility, light

weight and excellent balance of mechanical properties.51-55 There have been reported

that the surface oxide of titanium is inert when in contact with biological tissue and

that the formation of collagen on the surface of titanium components promotes the

growth of new bone tissues56 and also reported relatively high compatibility with bone

and tissue as compared with stainless steel.21,57 In terms of biological reactions,

stainless steels and Co-based alloys are classified as ‘biotolerant’ materials, while

titanium as ‘bioinert’ (These definitions are listed in Table 2). One of important and

12

unique property of titanium and its alloys is low elastic modulus; only about half of

elastic modulus of 316L stainless steel or Co-Cr alloy. Table 358 represents selective

mechanical properties of main metallic biomaterials. They are mainly used for

implant devices replacing failed hard tissue, for example, artificial hip joints, artificial

knee joints, bone plates, dental implants, etc. Titanium and its alloys are also used for

dental products, such as crowns, bridges and dentures.59-63

Table 3. Selective mechanical properties of metallic biomaterials58

Material

Young's

Modulus, E

(GPa)

Yield Strength,

σy (MPa)

Tensile

Strength, σUTS

(MPa)

Fatigue Limit,

σend (MPa)

Stainless steel 190 221 ~ 1213 586 ~ 1351 241 ~ 820

Co-Cr alloy 210 ~ 253 448 ~ 1606 655 ~ 1896 207 ~ 950

cp Titanium 110 485 760 300

Ti6Al4V 116 869 ~ 1034 965 ~ 1103 620

Cortical bone 15 ~ 30 30 ~ 70 70 ~ 150 -

However, they also have disadvantages, including low strength, low wear

resistance, hydrogen embrittlement, and difficulty with respect to manufacturing,

casting and machining.58,64,65 For commercially pure(cp) titanium, it showed poor

mechanical properties from its applications in the medical and dental field. It may be

cause of mechanical fracture and wear, chemical corrosion and biologically toxic

phenomena due to metal ion release. In these reasons, many titanium-based alloys

were developed and recently, most of them have been introduced for biomedical

applications, including: α + β type titanium alloys, such as Ti6Al7Nb,66 Ti5Al2.5Fe,67

Ti6Al6Nb1Ta and Ti5Al3Mo4Zr,68 near β-type alloy Ti13Nb13Zr,69 and β-type alloys

such as Ti15Mo5Zr3Al,70 Ti15Mo3Nb3Al0.2Si, Ti15Mo3Nb3Al0.3O.71

Amongst conventional titanium alloys, Ti6Al4V alloy, which was standardized

with ASTM F136 and ISO 5832-3, exhibit good properties for medical and dental

applications.11,12,39,53,63,72 By adding alloying elements to titanium, most notably

aluminum (Al) and vanadium (V), its mechanical properties can be enhanced.73,74

Ti6Al4V has been also used preferentially in orthopedic-prosthetic replacement due

13

to its added mechanical strength, while cp titanium has been employed for some

dental implants.72 Although Ti6Al4V has been widely used in biomedical fields, there

are still many unsolved questions regarding the effect of its alloying components, low

deformation strengths, such as to torsion, and the metal ions released by corrosion or

wear processes may induce aseptic loosening after long-term implantation.73-78 It

appears that small amounts of both Al and V, released in the human body, induce

possible cytotoxic effect and neurological disorders. Al is an element involved in

severe neurological, e.g. Alzheimer’s disease,79-82 and metabolic bone disease, e.g.

osteomalacia.83 Recently, locally released V ions from Ti6Al4V alloy negatively

impacted cell adhesion and inhibited expression of the osteogenic phenotype by bone

marrow stromal cells.78,84-86 Thus, toxicity of alloying elements like Al and V and

high elastic modulus compared to natural bone of the conventional Ti6Al4V alloy has

required the development of new titanium alloys with non-toxic elements.

Another popularly used titanium alloy is Ni-Ti alloy. Ni-Ti is also referred to as

Nitinol, which is an abbreviation from the words: Nickel-Titanium Naval Ordnance

Laboratory. This equiatomic Ni-Ti alloy (Ni-50 at%, Ti-50 at%) has been widely used

in the medical and dental fields; for example, intravascular stents, staples and cramps

for surgical fixation devices and orthodontic wires owing to its shape memory or

superelastic properties.87-93 The main concern about the use of this alloy derives from

the fact that it contains a large amount of Ni (more than 50 wt%), which is suspected

responsible for allergic, toxic and carcinogenic reactions.27,31,94-96

3. Principal requirements of metallic biomaterials as implant materials

A. Corrosion resistance

The physiological environment is typically modeled as a 37 aqueous solution, at

pH 7.3, with dissolved gases (such as oxygen), electrolytes, cells and proteins.

Immersion of metals in this environment can lead to corrosion, which is deterioration

and removal of the metal by chemical reactions. During the electrochemical process

of corrosion, metallic biomaterials can release ions, which may reduce the

biocompatibility of materials and jeopardize the fate of implants. For example, the

14

type and concentration of released corrosion products can alter the functions of cells

in the vicinity of implants as well as of cells at remote locations after transport of the

corrosion by products to distant sites inside the body. Even before implantation,

through chemical reaction of metals with the oxygen in ambient air or by oxidation in

an acidic solution, an oxide surface film forms on their surface. Because of these

oxides formed on surface the electrochemical reactions that lead to corrosion are

reduced or prevented. In other words, the oxidized metallic surfaces are “passivated”.

In fact, the stability of the oxides present on different metals determines their overall

corrosion resistance. For example, even though 316L stainless steel implants perform

satisfactorily in short-term applications, such as fracture fixation, they are susceptible

to crevice corrosion and pitting when implanted for longer periods. Among

commonly used metallic biomaterials, titanium and its alloys have more favorable

corrosion resistance for long-term implant applications such as joint and dental

prostheses because of stable oxide film formed on surface in various environments.

B. Biocompatibility and biofunctionality

The two primary issues in biomaterials science of new implant materials are

biocompatibility and biofunctionality; mechanical properties. The general criteria for

materials selection for implant materials are as follows:

It is highly biocompatible and does not cause an inflammatory or toxic

response beyond an acceptable tolerable level.

It has appropriate mechanical properties, closest to bone.

Manufacturing and processing methods are economically viable.97

Ideally, an implant such as hip, knee joint and dental implant should be such that it

exhibits an identical response to loading as real bone and is also biocompatible with

existing tissue. The compatibility issue involves surface compatibility, mechanical

compatibility and also osteocompatibility. More detail requirements of implant

materials were listed in Figure 3.98

15

Figure 3. Implant material requirements in orthopedic applications.98

Type 316L stainless steels, Co-Cr alloys, cp titanium, and Ti6Al4V alloys are

typical metallic biomaterials used for implants devices. However, when used as

biomaterials, these materials pose several problems as previously stated briefly.

In term of biocompatibility, these problems include toxicity of corrosion products

and fretting debris to the human body, fracture due to corrosion fatigue and fretting

corrosion fatigue, lack of biocompatibility, and inadequate affinity for cells and

tissues. In particular, the toxicity problem brought about other problems such as

allergy reaction, tumor formation, teratogenicity and inflammation. Some metal

elements are known to produce inflammatory responses in in vitro studies99-101 and

allergic reactions in vivo.102,103 The cytotoxicity of pure metals, the relationship

between biocompatibility and polarization resistance of surgical implant materials

have been reported by Steinemann. (Figure 4)77

Nowadays, metal allergy is also a significant problem. Allergy is a synonym for

hypersensitivity. It is characterized by the fact that the human organism may suffer

pathological reactions once it is repeatedly in contact with an antigenous substance.

Basically all metals may cause an allergy. Metal allergy is caused by the metallic ions,

which are released from an alloy through sweat and other body fluids.

16

Figure 4. Relationship between polarization resistance and biocompatibility of

biometallic elements and alloys.77

The percentage of metal allergy caused by each metallic element is shown in Figure

5.104 Especially in biomedical fields; Co, Cr, and Ni have been pointed out to be

highly associated with metal allergy. For example, in Europe, about 20 % of young

females and 4% of young males suffer from Ni allergy. This trend has increased

dramatically for both men and women.105 Ni ions are considered to be an especially

toxic species which causes allergy and cancer. The International Agency for Research

on Cancer (IARC) of the World Health Organization (WHO) estimates that Ni

compounds are carcinogenic, and metallic Ni and Ni contained alloys are possibly

carcinogenic to the human body.106

17

Figure 5. Percentage of metal allergy caused by each metallic element.104

As previously stated in Figure 4, V element was classified to toxic. Pure V element

impairs cell metabolism,107 affects erythrocyte and haemoglobin concentration in vivo,

inhibits enzymes including various ATPases such as those that are sodium (Na),

potassium (K) and calcium (Ca)-dependent, dynein and myosin ATPases, many

phosphatases and kinases,108-111 and influences mitotic processes.112

Al is an element involved in severe neurological, e.g. Alzheimer’s disease,79-82 and

metabolic bone disease, e.g. osteomalacia.83 In Al-related disease, the predominant

features are defective mineralization and osteomalacia that result from excessive

deposits at the site of osteoid mineralization. Al causes an oxidative stress within

brain tissue, leading to the formation of Alzheimerlike neurofibrillary tangles. Al also

has a direct effect on hematopoiesis.

For titanium, titanium is considered to be a well-tolerated and nearly inert

material113 and it induces neither toxic nor inflammatory reactions in connective or

epithelial tissues.114 In an optimal situation, titanium is capable of osteointegration

with bone.115 Doran et al.(1998)116 reported that titanium is considered one of the

best-accepted metals in vitro and in vivo. However, in vitro Ti4+ ions inhibit

18

osteoclastic activity and reduce osteoblastic protein synthesis.117 In a study using the

human osteoblastic cell line MG-63, which can be defined as proliferating osteoblasts,

titanium was shown to induce IL-6 production118 and, therefore, activate

osteoclastogenesis.119 In addition, there are reports of contact dermatitis in response to

titanium.120

In term of biofunctionality; mechanical property, the mismatch in the mechanical

properties of metallic biomaterials and the natural bone, especially in the Young’s

modulus (210 ~ 253 GPa for Co-Cr alloys, 110 GPa for cp titanium, and 15 ~ 30 GPa

for natural bone) which was listed in Table 3 leads to a so-called ‘stress shield’ during

service, which results in the implant failure.121 Figure 6122 shows simple schematic

figure of stress shielding.

Figure 6. Simple schematic figure of stress shielding.122

Long-term experience indicates that insufficient load transfer from the artificial

implant to the adjacent remodeling bone may result in bone resorption and eventual

loosening of the implant device.123,124 ‘Wolff’s law125,126 (‘the form being given, tissue

adapts to best fulfill its mechanical function’) suggests that the coupling of an implant

with a previously load bearing natural structure may result in tissue loss. Indeed, it

has been shown that when the tension-compression load or bending moment to which

living bone is exposed is reduced, decreased bone thickness, bone mass loss, and

19

increased osteoporosis ensue.127-129 This phenomenon, termed ‘stress shielding’, has

been related to the difference in flexibility or stiffness, dependent in part on elastic

moduli, between natural bone and the implant material.130

4. Research trends of new titanium-based alloy

Recently, much research effort was devoted to the study of more biocompatible,

lower modulus metallic biomaterials. As focusing on former section, current research

trends are following next two concepts

Non toxic elements should be selected for biocompatibility enhancement of

metallic biomaterials.

New metallic biomaterials should have low modulus and adequate strength.

Since Ti5Al2.5Fe and Ti6Al7Nb; V free alloys were introduced in Europe in the

mid 1980s,66,67 various kinds of new high strength α + β and low-modulus β-type

titanium alloys composed of nontoxic elements, such as niobium (Nb), tantalum (Ta),

zirconium (Zr), palladium (Pd) etc., are developed for biomedical applications. As

shown in Figure 4, Nb, Ta, Zr may be selected as the safest alloying elements to

titanium. Nb and Ta are new candidates for developing new implant materials

because they have better electrochemical properties and better biocompatibility than

titanium.131 Nb and Zr exhibit ideal passivity and are not prone to chemical

breakdown of the passive layer, exhibiting minimum passive dissolution rates. In fact,

Nb and Zr contribute to the formation of a spontaneous highly protective passive film

on titanium alloys and are not, as are Al and V, released into the environment as

dissolved metal ions, but are rather incorporated into the passive layer.132

Much research has been done to develop new β-type titanium alloys with a lower

elastic modulus.39,63 Advantages of β-type, near β-type alloys over a near-α or α + β

type alloys include their lower modulus133,134 and better formability.135-138 The most

investigated β-type titanium alloys for biomedical applications were included in the

Ti-Ta, Ti-Zr-Nb-Ta, Ti-Nb-Zr, Ti-Nb, Ti-Sn-Nb-Ta, Ti-Sn-Nb-Ta-Sb and Ti-Nb-Ta-

Mo systems. First generation alloys previously explained included Ti6Al7Nb and

20

Ti5Al2.5Fe, two alloys with properties similar to Ti6Al4V that were developed in

response to concerns relating V to potential cytotoxicity and adverse reaction with

body tissues. Further, biocompatibility enhancement and lower modulus has been

achieved through the introduction of second generation titanium based alloys

including Ti12Mo6Zr2Fe ‘TMZF’,139,140 Ti15Mo5Zr3Al,141 Ti15Mo3Nb3O

(21SRx),142 Ti15Zr4Nb2Ta0.2Pd and Ti15Sn4Nb2Ta0.2Pd alloys,143 as well as the

‘completely biocompatible’ Ti13Nb13Zr alloy.69,144,145 Finally, minimum elastic

moduli have been achieved by ‘TNZT’ alloys based on the TiNbTaZr system,

specifically by the development of the ‘biocompatible’ Ti35Nb5Ta7Zr and

Ti29Nb13Ta4.6Zr alloy.146-148 Besides other titanium based alloy, alloying with non-

toxic elements has been researched such as Ti-Ta, Ti-Hf binary alloy.149-151

We focused on new alloying elements and chose silver (Ag) element for alloying

element to titanium. Silver is listed in order of their atomic number as found in

periods 5 and 6 (Groups VIII ~ XI) of the periodic table together with ruthenium (Ru),

rhodium (Rh), palladium (Pd), osmium (Os), iridium (Ir), platinum (Pt), and gold

(Au). This is unique in nobility and for the most part offer industrial corrosion

resistance.

Some outstanding properties of silver are listed below;152

Silver is very soft and ductile. Next to Au, silver is the most easily fabricated

metal in the periodic table.

Silver has the lowest density of the noble metal group of elements.

Silver has the lowest melting point of all the noble metals.

Silver has lower elastic modulus than other noble metals

Moreover, silver also has a much higher electromotive force than titanium like

other noble metals and good wear resistance and wettability. Silver is less expensive

than other noble elements (Au, Pt, Pd, Ir, etc.), thus from the point of cost

effectiveness, silver for addition element can exhibit excellent property compared to

other noble elements. However silver is classified as moderate biocompatible element

in Figure 4.77 and this did not show excellent biocompatibility compared to Zr, Nb, Ta,

and Pt; belonging to the non-toxic group of metals.

21

The objective of this study is to evaluate the properties of titanium-silver alloys and

their suitability as metallic biomaterials. First, for mechanical property evaluations,

tensile test, bend test and microhardness test were performed. Potentiodynamic test

and potentiostatic test were also performed to evaluate corrosion resistance and

electrochemical property in biological environment. From surface characterization of

alloys, relation between silver addition and surface condition of alloy was evaluated.

Moreover, we draw relation between surface condition and electrochemical property

and studied effect of silver composition on each property. Finally, effect of silver

element on the biocompatibility of alloys was studied, followed by investigation of

biocompatibility and cytocompatibility of alloys in vitro and in vivo.

22

II. MATERIALS AND METHODS

1. Materials

A. Alloy design and arc melting of titanium-silver alloy

Titanium-silver binary alloys were designed and determined silver content: five

contents of silver were examined. According to the titanium-silver equilibrium phase

diagram (Figure 7)153, solid solubility limit of silver in titanium is about 5.1 at%. We

decided to increase the amount of silver from zero to 5.0 at%, below solid solution

limit concentration, at 1% increments. We designated these alloys TA1 to TA5,

according to the relative silver contents of each alloy. Sponge titanium (purity

99.99%) and granular silver (purity 99.99%) were used as raw materials for alloy

production. The weighing content of each element was listed in Table 4. After

weighing out the appropriate composition, each three 30 g quantity was melted using

an arc melter. Prior to melting, the chamber was evacuated to 5×10-3 torr, and high-

purity argon gas was introduced until the pressure reached 200 torr. Getter titanium

was melted before melting the material. The getter titanium was employed to

eliminate oxygen in an argon gas and a vacuum chamber. Each ingot was melted five

times to prevent the segregation.

B. Heat treatment and hot rolling

The 30 g button-shaped ingot was then heat-treated for 72 hours in a vacuum

furnace, at a constant temperature of 950 , in order to ensure compositional

homogenization. In order to maintain a constant thickness of 2 mm, the specimens

were hot-rolled at 950 . Oxide produced during the hot rolling process and other

surface contaminations were removed by the pickling (the ratio of hydrofluoric acid

to nitric acid to distilled water being 1:3:7). Finally the titanium-silver alloy

specimens were solution heat-treated at 950 for one hour in a vacuum furnace, and

then cooled in water at room temperature.

23

Figure 7. Equilibrium phase diagram for the binary titanium-silver alloy system.153

Table 4. Titanium and silver contents of each alloy

Titanium-silver alloy design (30g)

at% Ag Ti (g) Ag (g)

Ti 0 30.0000 -

TA1 1 29.3325 0.6675

TA2 2 28.6813 1.3187

TA3 3 28.0458 1.9542

TA4 4 27.4255 2.5745

TA5 5 26.8198 3.1802

24

C. Specimen preparation for experiments

The specimens were sectioned using ISOMET low speed diamond saw (Buehler,

Lake Bluff, IL, USA) for various purposes. Surfaces of alloys were mechanically

polished using ECOMET III polisher (Buehler, Lake Bluff, IL, USA) with SiC emery

paper with grits of 100, 600, 1000 and 2000, and then all were ultrasonically

degreased in 95% ethyl alcohol for 2 minutes. Schematic diagram of specimen

preparation procedures were presented in Figure 8.

Figure 8. Schematic diagram of specimen preparation procedures.

Alloy Design

& Weighing Arc Melting Hot Rolling

Test Specimen

25

2. Experimental Methods

A. Conditions of alloys

(A) Constituent analysis of alloying element and evaluation of impurity content

Energy dispersive spectroscopy (EDS) (Kevex Superdry II model, Kevex

Instruments Inc., San Carlos, CA. USA) was used to determine the amount of

titanium and silver, and CS / NO spectroscopy (CS-200, TC-300, LECO, St. Joseph,

MI, USA) was used for inspection of impurities incorporated during specimen

preparation processing, such as, carbon, sulfur, oxygen, and nitrogen.

(B) Phase identification and microstructure observation

Phase identification was conducted on X-ray diffractometer (XRD) (Dmax Rint

240, Rigaku, Tokyo, Japan). X-ray diffraction analysis was conducted over a scan

range of 25~120° and at a scan rate of 4 °/min, using the Kα1 ray (λ = 1.5406 nm) of

a Cu target. Phase was identified by matching each characteristic peak with the

JCPDS files.

To observe the alloy microstructures, the specimens were etched and placed under

an optical microscope (BX51, Olympus, Tokyo, Japan). The etchant used was Kroll’s

reagent.154,155

B. Mechanical property

(A) Tensile test

Tensile test of titanium-silver alloys was carried out using a universal testing

machine (Instron 3366, Norwood, MA, USA) with a crosshead speed of 1 mm/min at

room temperature. Test specimens were machined by wire cutting machine according

to KS standard (KSB 0801-01). Figure 9 showed shape and size of specimen for

26

tensile test. Three specimens per each alloy were used for this test. The ultimate

tensile strength and the elastic modulus of alloys were measured.

Figure 9. Shape and size of specimens for tensile test.

(B) Bend test

Three-point bend test was performed using a universal testing machine (Instron

3366, Norwood, MA, USA) with a crosshead speed of 1 mm/min at room temperature.

Test specimens and test methods were followed by ASTM E290-97a. Figure 10

showed schematic diagram of three-point bend test in this study.

Figure 10. Schematic diagram of three-point bend test.

27

The bend strength of titanium and titanium-silver alloys was determined using the

following equation,

223wt

FL=σ

σ is the bend strength (MPa); F is the load (N), L is the span length (mm), w is the

specimen width (mm), and t is the specimen thickness (mm). The dimensions of the

specimens were: L = 30 mm, w = 3 mm and t = 1 mm. The elastic modulus in bend

test is calculated from the load increment and the corresponding deflection increment

between the two points on the straight line as far apart as possible using the equation,

δ∆∆= 3

3

4wt

FLE

E is the elastic modulus (Pa), ∆F is the load increment as measured from preload

(N), and ∆δ is the deflection increment at mid-span as measured from preload.

(C) Microhardness test

The microhardness of alloys was measured 10 times on each occasion using a

MXT-α7E microhardness tester (Matsuzawa Seiki Co., Tokyo, Japan) with a load of

1000 g for 15 seconds.

C. Corrosion resistance and electrochemical property

(A) Potentiodynamic test

In order to evaluate the corrosion resistance, potentiodynamic test was performed

in artificial saliva at 37 . Composition of artificial saliva was listed at Table 5156,157

and pH of prepared artificial saliva was measured using Orion 420Aplus pH meter

(Thermo-Orion, Beverly, MA, USA). Three specimens were prepared and test was

28

performed three times using these specimens. The surface area of specimens exposed

to the electrolyte was controlled to 0.5 cm2. Before test, clean the specimen

ultrasonically for 2 minutes in ethyl alcohol, and carefully rinse with distilled water.

The specimens were installed in a corrosion cell, including the artificial saliva, which

were then connected to Electrochemical Interface (SI 1287, Solartron Instrument,

Hampshire, UK). In the case of the corrosion cell, platinum electrode was used as an

auxiliary electrode, and a saturated calomel electrode (SCE) was used as a reference

electrode.

This test was performed while increasing the potential from - 600 mV to 1600 mV

(SCE) at a scan rate of 1 mV/sec. It was conducted after removing adsorbed

impurities and scale from the specimen surfaces, under cathodic reducing conditions

using a - 1000 mV (SCE) voltage, and stabilization at open circuit potential for 10

minutes.

Table 5. Constituents of artificial saliva

Constituent Concentration(g/ℓ)

NaCl 0.40

KCl 0.40

CaCl2·2H2O 0.795

NaH2PO4·2H2O 0.780

Na2S·9H2O 0.005

CO(NH2)2(Urea) 1.0

Distilled water 1000 mℓ

(B) Potentiostatic test and open circuit potential measurement

In order to access the electrochemical behavior of alloy in biological environment,

the potentiostatic test was performed at 250, 0, - 250 mV (SCE) for 2 hours, which is

slightly higher than redox (reduction-oxidation) potential that may occur in the oral

cavity, in the artificial saliva. We assumed extreme conditions in the oral cavity and

determined test potential, 250 mV (SCE). It was reported that the oxidation potential

ranged from - 58 to 212 mV (SCE) and that the pH ranged from 6.1 to 7.9, at specific

29

sites in the oral cavity158 and Eisenbrandt reported that average initial redox potential

(Eh) of saliva was 301 mV (NHE: normal hydrogen electrode).159 The open circuit

potentials of titanium and titanium-silver alloy were also measured for 2 hours.

D. Surface characterizations of the alloy

Chemical element and chemical state of the surface of the alloys was analyzed

using X-ray photoelectron spectroscopy (XPS). Test specimens were used after

finished potentiostatic test at 250 mV (SCE) and XPS was analyzed immediately after

finishing potentiostatic test. After potentiostatic test, test specimens were removed

from the corrosion cell, and all specimens were kept in a vacuum desiccator prior to

analysis. Polished specimens were also analyzed for purposes of comparison. XPS

spectra were taken with a pass energy of 23.5 eV using Al Kα X-rays produced by the

Perkin-Elmer Φ 5700 ESCA system (Perkin-Elmer, Boston, MA, USA). The base

pressure in the chamber was maintained at 5×10-10 torr during spectra acquisition, and

binding energy shifts were referenced by setting the hydrocarbon peak in the C 1s

spectra to 285.0 eV. The accuracy of the measured binding energy was ± 0.2 eV. XPS

data was visualized using a Shirley background, and the line shape of the XPS spectra

was compiled with a mixed Gaussian-Lorentzian sum function. In order to obtain an

in-depth compositional profile difference of the oxide films, the take-off angle of the

analyzed photoelectrons with respect to the specimen surface was varied, taken at 10°

and 75°.

E. Effect of fluoride on the electrochemical property of the alloy

To investigate the effect of fluoride concentration, 0.1% or 1% NaF was added to

the artificial saliva and the above-mentioned potentiodynamic and potentiostatic tests

were performed on each NaF concentration. In addition, pH of test electrolyte:

artificial saliva containing NaF was measured using Orion 420Aplus pH meter

(Thermo-Orion, Beverly, MA, USA). All of the test condition was the same as

previous test.

30

F. Electrochemical property of passive oxide film

(A) Electrochemical Impedance Spectroscopy (EIS)

In order to evaluate electrochemical conditions of passive film formed on alloys in

artificial saliva, Electrochemical impedance spectroscopy (EIS) measurements were

performed using Electrochemical Interface (SI1287A, Solartron Instrument,

Hampshire, UK) and the Frequency Response Analyzer (SI1255B, Solartron

Instrument, Hampshire, UK) controlled by Ecorr/Zplot software. AC impedance was

measured in the frequency region of 104 ~ 10-3 Hz with an ac amplitude of 10 mV

with respect to open circuit potential. Same corrosion cell and electrode was used in

the previously mentioned potentiodynamic and potentiostatic test.

(B) Equivalent circuit model and parameter value

The EIS data were analyzed using the software Zplot2.3 software (Solartron

Instrument, Hampshire, UK). The EIS data were fit to appropriate equivalent

electrical circuit using a complex nonlinear least-square fitting routine, using both the

real and imaginary components of the data.160 Parameter values obtained from the

best fit equivalent circuit were tabulated and analyzed.

G. Biocompatibility evaluation

(A) L929 fibroblast cell culture

The mouse fibroblast cell line L929 was purchased from Korea Cell Line Bank.

The cells were cultured and maintained in RPMI Medium 1640 (Gibco, Grand Island,

NY, USA) supplemented with 10% Fetal Bovine Serum (FBS) (Gibco, Grand Island,

NY, USA) and 1% Penicillin-Streptomycin (Gibco, Grand Island, NY, USA). They

were maintained in 75 cm2 tissue culture polystyrene flasks (BD Falcon, Bedford,

MA, USA) in a 37 , 5% CO2 humidified atmosphere incubator (VS-9180MS,

31

Vision Scientific Co., Korea). All tests were performed with cultures between the 30th

and 40th passages.

(B) Metal ion release test

Metal ion release tests were performed by immersing the specimens in artificial

saliva kept in a 37 , 5% CO2 humidified atmosphere incubator. All specimens were

ultrasonically cleaned in ethanol for 1 minute and dried. After cleaning of surface, all

specimens were placed individually in the sterilized bottles with same surface area to

solution volume ratio. Artificial saliva immersed each specimen were withdrawn at

different times, ranging from 1 week to 24 weeks, A Graphite Furnace Atomic

Absorption Spectrophotometer (GFAAS, SpectrAA 220FS, Varian, Palo Alto, CA,

USA) was used to measure released metal ions concentration in the withdrawn

solution, focusing on titanium and silver content.

(C) Cell adhesion: SEM observation

L929 fibroblast cells were carefully seeded to each 10 mm × 10 mm size specimen

which was placed in the center of in 12 microwell (BD Falcon, Bedford, MA, USA)

by drop of 100 µℓ cell suspension and were kept in a 37 , 5% CO2 humidified

atmosphere incubator for 2 hours for initial cellular adhesion to the specimen surface,

and then culture medium was added to each well to completely cover the specimen.

After adding fresh medium, cell was cultured for 1 day. Each specimen was prefixed

with Karnovsky fixing solution including 0.2% GA, 2% paraformaldehyde (Sigma, St

Louis, MO, USA), and 0.5% CaCl2 (Sigma, St Louis, MO, USA) for 6 hours, washed

with pH 7.4 PBS three times to remove the first fixing solution completely, followed

by post-fixation with 1% osmium tetroxide for 1 hour, washed again with PBS three

times, and then dehydrated in graded ethanol series for 5 minutes respectively. After

treated with isoamylacetate, the fixed specimens were dried using critical point dryer

(Hitachi, Tokyo, Japan). Finally, the specimens were gold-coated with the thickness

of 300 Å using ion sputter. The cell adhesion morphology was observed with

scanning election microscopy (SEM) (S800 Hitachi, Tokyo, Japan) at an accelerating

32

voltage of 3 kV and digital images were captured at varying magnification.

(D) Agar diffusion test

To evaluate cytoxicity of titanium-silver alloys, agar diffusion test was performed.

Two specimens were prepared for each alloy. Surface of specimens was sterilized

with ethylene oxide gas, cleansed with distilled water, dried and processed so that

surface area of 0.5 cm2 came into contact with agar. Gutta-percha was used as a

positive control, and typical soda-lime glass (SiO2 71 ~ 75%, sodium oxide 12 ~ 16%,

calcium oxide 10 ~ 15%) as a negative control.

The cultured L929 fibroblast cell was used for agar diffusion test. The cells were

plated onto 90 mm2 petri dish and incubated to mitosis up to the cell concentration of

1×105 cells/mℓ in monolayer state in a 37 , 5% CO2 humidified atmosphere

incubator. The agar medium was prepared from 50% agar (Gibco BRL, Paisley,

Scotland) and 50% culture medium with 5% FBS. The culture medium was removed

from the petri dish, then 10 mℓ of agar medium at 45 ~ 50 was added to each petri

dish and left to stand at room temperature for 30 minutes. After the agar medium had

solidified, neutral red vital stain solution (10 mℓ) was added slowly to the center of

the dish and then spread over the surface and left for 30 minutes. Immediately after

removing the dyeing solution, the specimens were placed in contact with the agar and

incubated for 24 hours in a 37 , 5% CO2 humidified atmosphere incubator. First,

the Petri dish was placed on top of a white paper, then the zone index was measured

after observing the size of the discolored area and the lysis index was measured by

calculating the lysed ratio of the cells in the discolored area with an inverted phase

contrast microscope (CK2, Olympus, Tokyo, Japan). Decolorization area and cell

lysis area were measured with a ruler. Zone index and lysis index were determined by

standard (ISO 7405:1997(E)). Finally, the response index was measured by averaging

the zone and lysis indices of the specimens. Description of decolorization index, lysis

index, and criteria of cytotoxicity are listed in Table 6.

33

Table 6. Description of decolorization index, lysis index and interpretation of

response index used in the agar diffusion test

Decolorization

index Description of decolorization

0 No detectable zone around or under sample

1 Zone limited to area under sample

2 Zone not greater 5 mm in extension from sample

3 Zone not greater 10 mm in extension from sample

4 Zone greater than 10 mm in extension from sample,

but not involving entire plate

5 Zone involving entire plate

Lysis index Description of zone

0 No observable lysis

1 Up to 20% of the zone lysed

2 Over 20 % to 40 % of the zone lysed

3 Over 40 % to 60 % of the zone lysed

4 Over 60 % to 80 % of the zone lysed

5 Over 80 % lysed within the zone

Cytotoxicity Response index (zone index/lysis index)

None 0/0

Mild 1/1 ~ 1/5, 2/1

Moderate 2/1 ~ 2/3, 3/1 ~ 3/5, 4/1 ~ 4/3

Severe 4/4 ~ 4/5, 5/1 ~ 5/5

34

(E) Cell viability: MTT assay

The cell viability was determined with the MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-

diphenyltetrazolium bromide) assay.161,162 Extracts of each alloy were prepared in

accordance to international standard (ISO 10993-12, 1996). Extraction was performed

by autoclaving 121 for 1 hour; the ratio between surface of test specimen and the

volume of extraction media was 3 cm2/mℓ. Each 20 µℓ of extracts were inserted in the

cell cultured wells. As a control, 20 µℓ of medium was used. Then incubated in a

37 , 5% CO2 humidified atmosphere incubator for 24 hours. For the MTT assay, 50

µℓ of MTT solution (5 mg/mℓ) was added to each well of a 96-well plate (BD Falcon,

Bedford, MA, USA), and incubated for 4 hours. The supernatant was removed, and

the formazan crystals produced were dissolved in 200 µℓ of dimethylsulfoxide, and

quantified by measuring their optical density at 570 nm using an ELISA reader

(Precision Microplate Reader, Sunnyvale, CA, USA). Viability rates of the control

groups were set to represent 100% viability. Results of the other test materials were

expressed as a percentage of the control to yield comparable data.

(F) Acute systematic toxicity test (Intravenous injection)

For the acute systematic test, ICR mice, 7 ~ 8 weeks of age weighing 17 ~ 25 g,

were used. Ten male ICR mice were assigned for each test groups. The animals were

housed in a facility fully accredited by the Association for Assessment and

Accreditation of Laboratory Animal Care (AAALAC). The temperature and humidity

were kept constant at 22 ± 2 and 50 ± 10%, respectively. The animals were

provided food and water ad libitum. The Institutional Review Board in Yonsei

University approved all the experimental procedures and the National Institutes of

Health (NIH) guidelines were followed. All test procedure was in accordance with

ISO 10993-11. We selected two compositions among the titanium-silver alloys, TA2,

TA3, and compared the results of two different alloys to evaluate the difference of

acute toxicity relative to the silver content. Extraction media was saline. Extraction

was performed by autoclaving 121 for 1 hour; the ratio between surface of test

specimen and the volume of extraction media was 3 cm2/mℓ (ISO 10993-12). Each

35

mouse for a test group received a single intravenous injection of extract of alloys by

24 gauge needle syringe of 50 mℓ/kg body weight (in accordance with OECD

Guideline 420). Control group received a single injection of saline. Animals were

checked clinical signs such as diarrhea, piloerection, and mortality and body weight

of animals were also measured for a 3, 24, 48, 72 hours and then 1 week after

injection.

For the Histopathological examination, test animals were anesthetized after acute

systematic toxicity test and vital internal organs of each mouse were excised. Samples

of these organs were fixed in 8% formalin, embedded in paraffin wax, sectioned at 5

µm and stained with haematoxylin and eosin (H&E). Detailed microscopic

examination was carried out on organs of both control and test groups.

36

III. RESULTS

1. Composition analysis of alloying element and evaluation of impurity content

Table 7 shows the compositional analysis of the alloys produced by increasing

silver in titanium from zero to 5.0 at% in increments of 1.0 at%. Relative content of

titanium and silver in the specimen was not largely different between designed

composition and real alloy composition.

Table 7. Chemical compositions of titanium-silver alloys manufactured in this study

(n=3)

Titanium Silver Alloy

wt% at% wt% at%

Ti 99.688±0.113 99.855±0.049 - -

TA1 97.665±0.757 98.941±0.356 2.335±0.757 1.059±0.356

TA2 95.245±0.785 97.821±0.383 4.755±0.785 2.179±0.383

TA3 93.735±0.516 97.116±0.249 6.265±0.516 2.884±0.249

TA4 91.485±1.393 96.029±0.680 8.515±1.393 3.971±0.680

TA5 89.500±0.523 95.047±0.258 10.5±0.523 4.953±0.258

Table 8 shows the impurity contents of titanium-silver alloys manufactured in this

study. Oxygen incorporation, which is to be carefully treated in the production of

titanium alloys, was controlled at below 0.15 wt%. The mean oxygen content value of

each alloy was 0.1148 ~ 0.1412 wt%. Other element was controlled to below 0.03

wt%.

2. Phase identification and microstructure observation

Figure 11 shows the result of XRD phase identification. We verified α phase, β

phase and Ti2Ag of alloys with a JCPDS card. In case of titanium-silver alloy

containing silver up to 2.0 at%, the only α phase (hcp structure) was observed. At

silver levels above 3.0 at%, the β (bcc structure) and α phases co-occurred. Two

37

peaks of α-Ti (100) and β-Ti (110) overlapped each other at the range of 2θ, 38.42° to

38.49°. In view of peak intensity, main phase was α phase. In the XRD pattern of TA4

and TA5, small intensity of Ti2Ag diffraction peak, which represents an intermetallic

compound of titanium and silver, was also observed.

Table 8. Impurity (C, S, O, N) contents of titanium-silver alloys manufactured in this

study (n = 3)

Alloy Carbon (wt%) Sulfur (wt%) Oxygen (wt%) Nitrogen (wt%)

Ti 0.0273±0.0139 0.0079±0.0039 0.1251±0.0279 0.0086±0.0005

TA1 0.0283±0.0103 0.0027±0.0025 0.1148±0.0370 0.0077±0.0045

TA2 0.0209±0.0097 0.0045±0.0004 0.1333±0.0411 0.0072±0.0043

TA3 0.0226±0.0022 0.0041±0.0021 0.1297±0.0119 0.0048±0.0014

TA4 0.0369±0.0179 0.0023±0.0016 0.1314±0.0582 0.0067±0.0017

TA5 0.0308±0.0052 0.0017±0.0006 0.1412±0.0241 0.0064±0.0026

Titanium and titanium-silver alloys’ microstructures are shown in Figure 12. In

case of titanium, the only equiaxed α phase was found, and α grain boundaries were

easily seen. However, microstructure of titanium-silver alloys was not the same as

that of titanium. In microstructure of TA2, grain boundary was not easily seen and

equiaxed and acicular α phase coexisted and piled up. The acicular phase got thinner

and sharper with the increased silver content. When silver content is over 3.0 at%,

Widmanstätten α + β phase; needle like lying with long axes parallel to the constant

plane is seen into the originally formed β matrix. It can be also seen β phase grain

boundary. In addition, precipitate Ti2Ag was observed clearly in the microstructure of

TA5. Figure 13 shows the precipitation of Ti2Ag (dark region) following grain

boundary in TA5 under high magnification.

38

30 40 50 60 70 80 90 100 110 120

0

100

200

300

400

500

αααα-Ti

Inte

nsi

ty(c

ps)

Diffraction angle (2θθθθ)

Ti

30 40 50 60 70 80 90 100 110 120

0

100

200

300

400

500

TA1

αααα-Ti

Inte

nsi

ty(c

ps)

Diffraction angle (2θθθθ)

30 40 50 60 70 80 90 100 110 120

0

100

200

300

400

500

αααα-Ti

Diffraction angle (2θθθθ)

Inte

nsi

ty(c

ps)

TA2

30 40 50 60 70 80 90 100 110 120

0

100

200

300

400

500

ββββ-Tiαααα-Ti

Diffraction angle (2θθθθ)

Inte

nsi

ty(c

ps)

TA3

30 40 50 60 70 80 90 100 110 120

0

100

200

300

400

500TA4

Ti2Ag

ββββ-Ti

αααα-Ti

Diffraction angle (2θθθθ)

Inte

nsi

ty(c

ps)

30 40 50 60 70 80 90 100 110 120

0

100

200

300

400

500

Ti2Ag

ββββ-Ti

TA5

Inte

nsity

(cps

)

Diffraction angle (2θθθθ)

αααα-Ti

Figure 11. XRD patterns and phase identification of titanium and titanium-silver

alloys.

39

(a) Ti (b) TA1

(c) TA2 (d) TA3

(e) TA4 (f) TA5

Figure 12. Microstructure of titanium and titanium-silver alloys (magnification of

×100).

25 µm

40

Figure 13. Microstructure of TA5- Precipitation of Ti2Ag (white arrow) was shown

following grain boundary in TA5 under high magnification (×200).

3. Mechanical property

A. Tensile test

The tensile strength of titanium and titanium-silver alloys was shown in Figure 14.

The tensile strength of titanium-silver alloys was slightly higher than that of titanium

and TA5 had the highest value of 407.2 MPa. Although difference of the tensile

strength value was not large, the tensile strength value had a tendency to rise in

accordance with increasing silver content.

In Figure 15, elastic modulus of alloys from tensile test was shown. With regard to

elastic modulus, it revealed that there was no tendency between elastic modulus and

silver content. Elastic moduli of titanium-silver alloys were similar to that of titanium.

Among all titanium-silver alloys, TA1 had the lowest modulus of 93.17 GPa. Other

titanium-silver alloys had similar value range of 104.4 ~ 114.6 GPa.

41

Ti TA1 TA2 TA3 TA4 TA50

100

200

300

400

500T

ensi

le s

tren

gth

(M

Pa)

Alloy

Figure 14. Tensile strength of titanium and titanium-silver alloys.

Ti TA1 TA2 TA3 TA4 TA50

20

40

60

80

100

120

140

Alloy

Ela

stic

mod

ulu

s (G

Pa)

Figure 15. Elastic modulus of titanium and titanium-silver alloys from tensile test.

42

B. Bend test

Figure 16 showed the results of bend strength of titanium and titanium-silver alloys.

The bend strength of TA3, TA4, and TA5 was slightly higher than that of titanium

and TA5 had the highest value of 1413.3 MPa. The variation in alloys’ bend strength

with silver content showed a tendency similar to that in microhardness, except TA1

and TA2. These two alloys showed an unexpectedly lower strength than titanium. The

elastic modulus is calculated from the load increment and the corresponding

deflection increment between the two points on the straight line and it is plotted in

Figure 17. Among all titanium-silver alloys, TA1 had the lowest modulus of 50.58

GPa. Other titanium-silver alloys had similar value range of 61.9 ~ 69.9 GPa and their

deviation value were somewhat large. Their elastic moduli were no relation with

silver content of them.

Ti TA1 TA2 TA3 TA4 TA50

200

400

600

800

1000

1200

1400

Ben

d s

tren

gth

(M

Pa)

Alloy

Figure 16. Bend strength of titanium and titanium-silver alloys.

43

Ti TA1 TA2 TA3 TA4 TA50

10

20

30

40

50

60

70

80

Alloy

Ela

stic

mo

dulu

s (G

Pa)

Figure 17. Elastic modulus of titanium and titanium-silver alloys from bend test.

C. Microhardness test

Microhardness values in order to identify mechanical properties of titanium-silver

alloys are shown in Figure 18. The hardness value tended to rise in accordance with

increasing silver addition content. The pure titanium had microhardness of 206.7 Hv,

TA1 of 240.7 Hv, TA2 of 248.6 Hv, TA3 of 282.77 Hv, TA4 of 286.74 Hv and TA5

of 299.0 Hv. The maximum increase in the hardness value versus pure titanium was

about 44.6%.

44

MicroHardness(Hv)

0.0

50.0

100.0

150.0

200.0

250.0

300.0

350.0

Ti TA1 TA2 TA3 TA4 TA5

Hard

ness

(H

v)

Figure 18. Microhardness values of titanium and titanium-silver alloys.

4. Corrosion resistance and electrochemical property

A. Potentiodynamic test

Potentiodynamic test was performed to evaluate the corrosion resistance of

titanium-silver alloys. Graph of current density vs. potential was shown in Figure 19.

The entire alloy had a stable current density region above about 700 mV (SCE).

Pitting was not observed in all alloys. We selected current density values of each

alloy at 1 V (SCE) to compare regular passive current densities. Titanium had passive

current densities of 7.88 µА/cm2, at 1 V (SCE). Titanium-silver alloys had low

passive current density compared to titanium. When silver was added to titanium, the

passive current density decreased on the potentiodynamic curve and maintained a

stable state. Among titanium-silver alloys, TA3 had the lowest passive current density

of 2.13 µА/cm2. As silver addition was over 3.0 at%, passive current density of these

alloys decreased. TA4 and TA5 had similar passive current density to TA1.

45

1E-9 1E-8 1E-7 1E-6 1E-5 1E-4-1.0

-0.5

0.0

0.5

1.0

1.5

2.0

TA5

TA4TA2 TA1

TA3Ti

Potentiodynamic Test

E(V

vs

SC

E)

Current Density(A/cm2)

Figure 19. Graph of current density vs. potential of titanium and titanium-silver alloys

from potentiodynamic test.

B. Potentiostatic test

The each results of potentiostatic test applied 250 mV, 0 mV. - 250 mV (SCE) in

artificial saliva maintained 37 are presented in Figure 20. Three specimens of each

alloy were tested and the most representative results were plotted. As shown in Figure

20, when the specimens were immersed and potentials were then applied at each

potential, current densities decreased immediately and maintained stable. After 20

minutes, current density reached a plateau, and remained stable from that time on. All

the alloys exhibited current densities of 0.2 ~ 1.5 µA/cm2. Titanium had the highest

current density value at all potential; 0.98 µA/cm2 at 250 mV, 1.16 µA/cm2 at 0 mV,

1.05 µA/cm2 at - 250 mV (SCE).

46

0 1000 2000 3000 4000 5000 6000 7000 8000

0.0

1.0x10-6

2.0x10-6

3.0x10-6

4.0x10-6

5.0x10-6

Potentiostatic test at 250mV

TA5

TA4TA3

TA2TA1

Ti

Time(sec)

Cur

rent

den

sity

(A/c

m2 )

(a)

0 1000 2000 3000 4000 5000 6000 7000 8000

0.0

1.0x10-6

2.0x10-6

3.0x10-6

4.0x10-6

5.0x10-6

TA1

TA3 TA2

TA4TA5

Ti

Time(sec)

Cur

rent

den

sity

(A/c

m2 )

Potentiostatic test at 0mV

(b)

0 1000 2000 3000 4000 5000 6000 7000 8000

0.0

1.0x10-6

2.0x10-6

3.0x10-6

4.0x10-6

5.0x10-6

The restTA4

Ti

TA5

Potentiostatic test at - 250mV

Cu

rren

t de

nsit

y(A

/cm

2 )

Time(sec)

(c)

Figure 20. Current densities of titanium and titanium-silver alloys at each potential in

artificial saliva, 37 (a) 250 mV (b) 0 mV (c) - 250 mV (SCE).

47

Titanium-silver alloys exhibited slightly lower current density than pure titanium.

TA3 had a lowest current density. However, in case of TA4 and TA5, alloys that silver

content was over 3.0 at%, current density of these alloys increased. TA5 exhibited

somewhat unstable behavior of current density.

C. Open circuit potential measurement

Open circuit potential is potential of an electrode measured with respect to

reference electrode or another electrode when no current flows to or from it. Figure

21 shows the open circuit potentials of titanium and titanium-silver alloys in artificial

saliva at 37 . After 2 hours, all the alloys maintained stable potential. Titanium

exhibited the lowest value, at - 323.4 mV (SCE); whereas TA3 was measured to have

4 mV (SCE) of open circuit potential. In case of titanium, it took more time to get

stable potential in contrast with titanium-silver alloys. As silver content increased up

to 3.0 at%, the open circuit potential of the alloy increased. However, in case of alloys

which silver content was over 3.0 at%, TA4 and TA5, open circuit potential of these

alloys decreased. TA4 and TA5 had lower open circuit potential than TA2 and TA3.

The initial increase in open circuit potential during the early hours followed by

stabilization and remained stable during the entire measuring period.

48

0 1000 2000 3000 4000 5000 6000 7000 8000-0.8

-0.7

-0.6

-0.5

-0.4

-0.3

-0.2

-0.1

0.0

0.1TA3

TA2TA4

TA5

TA1

Ti

Open Circuit Potential

Time(sec)

E(V

vs

SC

E)

Figure 21. Open circuit potentials of titanium and titanium-silver alloys in artificial

saliva, 37 .

5. Surface characterization of the alloy

The comparative surface characteristics of titanium and the titanium-silver alloys

were evaluated by means of X-ray photoelectron spectroscopy (XPS). The XPS

results reveal differences in the chemical compositions of the surface layers of the

pure titanium and titanium-silver alloys, suggesting concomitant differences in their

protective characteristics.

After the potentiostatic test at 250 mV (SCE), the surface compositions of the

titanium-silver alloys were analyzed by XPS. Just polished specimens were also

analyzed for purposes of comparison. The survey spectrum of as-polished titanium

after 30 seconds of ion sputtering is presented in Figure 22. C 1s peak and

contamination layers were removed by argon ion sputtering. The main peak of this

survey spectrum was O 1s and Ti 2p. Table 9 shows the area fractions of elements Ti

and O at the surface, and lists the analytical data of the surface compositions of as-

polished and potentiostatic tested specimens, also with take off angles set to 10° and

75°. In comparison to as-polished specimens, the potentiostatic tested specimens

49

exhibited much higher oxygen content fractions.

In order to detect the inner surface layer, analysis was performed using a 75° take

off angle. When the take off angle was changed to 75°, area fractions of each titanium

oxide were changed, and the oxygen content decreased.

Figure 22. XPS survey spectrum for as-polished titanium after argon ion sputtering.

Figure 23(a) displays the high-resolution spectrum of the Ti 2p region, and

deconvolution. The Ti 2p spectrum (Figure 23(a)) of the specimens is dominated by a

major peak at the binding energy (458.8 eV), with a spin-orbit splitting of 5.7 eV, as

was expected for the Ti 2p3/2 and Ti 2p1/2 components of the doublet. The Ti 2p

spectrum was decomposed into four doublet spectra, originating from Ti0, Ti2+, Ti3+,

and Ti4+, according to the binding energy data.163,164 The fractions of chemical

compositions are listed in Table 10. The binding energy value of the main peak is

characteristic of titanium in its normal oxidation state, Ti4+, in agreement with data

previously reported for TiO2.163,165 A small peak at the binding energy (454.7 eV) was

also detected in the spectrum, with an energy shift of 4.1 eV from the main peak. This

peak can be assigned to the metallic titanium species,166 and the metallic contribution

in the Ti 2p peak correlates with the thickness of the oxide layer on the surface. Via

50

curve fitting analysis, the O 1s spectrum (Figure 23(b)) of all specimens can be

deconvoluted by three components, which can be assigned to oxide species (O2-),

hydroxide groups (OH-), and adsorbed water (H2O).166-168

Table 9. Fraction of elements Ti and O in survey spectrum with change of take off

angle

Take off angle

10° 75° Alloy

Ti O Ti O

Ti(a) 42.4 57.6 45.31 54.69

Ti(b) 33.29 66.71 37.31 62.69

TA1(a) 42.92 57.08 43.46 56.54

TA1 (b) 31.6 68.4 35.12 64.88

TA2 (a) 41.35 58.65 41.51 58.49

TA2 (b) 30.09 69.91 32.82 67.18

TA3 (a) 32.65 67.35 41.38 58.62

TA3 (b) 27.54 72.46 32.81 67.19

TA4 (a) 40.59 59.41 42.39 57.61

TA4 (b) 30.34 69.66 36.12 63.88

TA5 (a) 40.63 59.37 43.63 56.37

TA5 (b) 36.44 63.56 40.11 59.89

(a): As-polished, (b): After potentiostatic tested

51

(a)Ti 2p

(b)O 1s

Figure 23. Representative high resolution spectrum of the (a) Ti 2p and (b) O 1s

regions.

52

According to the results of the fractions of oxygen species, over 80% of

components consisted of oxide. According to the high resolution spectral analyses of

Ti and O, the surface film on titanium was mainly TiO2, but also contained small

amounts of hydroxyl groups and bound water.

Table 10. Chemical compositions and their fractions of surface films on alloys

Composition Ti4+ Ti3+ Ti2+ Tio(metallic Ti)

Take off angle 10° 75° 10° 75° 10° 75° 10° 75°

Ti (a) 45.06 35.21 26.21 31.47 24.76 28.37 3.97 4.95

Ti (b) 48.67 43.04 39.73 33.7 11.6 19.65 - 3.61

TA1 (a) 45.81 39.67 30.44 31.83 23.76 26.73 - 1.77

TA1 (b) 51.77 52.95 21.6 23.35 26.63 22.88 - 0.82

TA2 (a) 59.68 49.95 24.89 32.04 15.43 16.5 - 1.51

TA2 (b) 62.37 58.74 23.96 22.3 13.67 17.79 - 1.17

TA3 (a) 57.62 52.65 20.25 17.9 22.13 28.07 - 1.38

TA3 (b) 66.72 63.23 24.14 21.5 9.14 14.33 - 0.94

TA4 (a) 52.3 47.92 29.1 31.18 18.6 19.75 - 1.15

TA4 (b) 62.37 49.13 19.44 27.45 18.19 22.58 - 0.84

TA5 (a) 59.0 45.83 22.55 28.24 18.45 24.27 - 1.76

TA5 (b) 58.1 47.49 15.71 26.73 26.19 24.15 - 1.63

(a): As-polished, (b): After potentiostatic tested

In order to obtain quantitative results from XPS analysis, curve fitting was

performed, employing the Gaussian-Lorentzian method. Surface compositions of

each alloy are presented in Table 10. According to the spectral analysis from curve

fitting, almost 50% of the oxide film of alloys consisted of TiO2, and the rest of the

oxide film was composed of TiO and Ti2O3. The apparent relative intensities of the

oxides are in the order of TiO2> Ti2O3> TiO. When the take off angle was increased

from 10° to 75°, the fraction of Ti4+ decreased, demonstrating that Ti4+ was the

predominant component in the outer part of the oxide film. Concurrently, the minor

contributors, namely Ti2+ and Ti3+ (from titanium suboxides) increased. With regard

53

to the fraction of TiO2, the potentiostatic-tested specimen contains a larger amount of

TiO2 than the as-polished specimen.

Figure 24 illustrates the area fraction of the metallic Ti in the high resolution

spectrum with the take off angle set at 75°. As silver content increased, the fraction of

metallic Ti decreased. The area fraction of metallic Ti in the titanium-silver alloys

was not more than 2%. When the take off angle was 10°, no metallic Ti peak was

detected, with the exception of titanium. In comparison to the as-polished specimen,

the potentiostatic tested specimen contained a small fraction of metallic Ti.

0

1

2

3

4

5

6

Ti TA1 TA2 TA3 TA4 TA5Alloy

Are

a fr

actio

n(%

)

As-Polished

After Test

Figure 24. Area fraction of the metallic Ti in high resolution spectrum of take off

angle 75°.

6. Effect of fluoride on the electrochemical property of the alloy

Potentiodynamic test was performed to evaluate the corrosion resistance of

titanium-silver alloys in the plain artificial saliva and fluoride containing artificial

saliva. The pH of prepared artificial saliva was measured using pH meter. The each

pH values of plain artificial saliva, 0.1% NaF and 1% NaF added artificial saliva was

6.41, 6.32 and 6.09. The results of potentiodynamic polarization testing are shown in

Figure 23. This figure was representative plot of three-time test. When fluoride was

added to artificial saliva, the passive current densities on the potentiodynamic curve

54

increased. Their passive current densities increased a little in 0.1% NaF added

artificial saliva, and increased markedly in 1% NaF added. Titanium and titanium-

silver alloys exhibited the similar passive current densities in the range of 4~5 µA/cm2

(at 250 mV (SCE)) in NaF-free artificial saliva, and 8 ~ 10 µA/cm2 (at 250 mV

(SCE)) in 0.1% NaF added artificial saliva. However, in the 1% NaF added artificial

saliva; there were some differences in passive current densities in 1% added artificial

saliva between titanium and titanium-silver alloys. Titanium-silver alloys showed a

slight increase in the passive current density between NaF-free and 1% NaF added

artificial saliva compared to titanium. TA2 and TA3 showed low passive current

densities compared to titanium in 1.0% NaF added artificial saliva. Passive current

density of each alloy in 1% NaF added solution was listed in Table 11. Titanium and

titanium-silver alloys did not show any pitting corrosion to 1200 mV (SCE). As

shown in Figure 25, the addition of a high NaF concentration showed a tendency to

decrease zero current potential of these alloys.

Table 12 showed the open circuit potentials of titanium and titanium-silver alloys

in NaF containing artificial saliva. Open circuit potential measurement test was

performed for 2 hours and we chose potential value of alloys when potentials were

stable and little changed. When NaF was added to artificial saliva, the open circuit

potential decreased. In 1% NaF added artificial saliva, open circuit potential of

titanium increased to - 0.6902 V (SCE). Among titanium-silver alloys, TA3 and TA4

had low open circuit potential. Deviation of each open circuit potential was large. As

shown in Figure 25 and Table 12, fluoride exerted an influence on open circuit

potential and passive current density of alloys.

55

1E-9 1E-8 1E-7 1E-6 1E-5 1E-4 1E-3

-1.5

-1.0

-0.5

0.0

0.5

1.0

1.5

2.0

1%NaFpH=6.1

0.1%NaFpH=6.3

No additionpH= 6.4

E(V

vs

SC

E)

Current Density(A/cm2)

Ti

1E-9 1E-8 1E-7 1E-6 1E-5 1E-4 1E-3

-1.5

-1.0

-0.5

0.0

0.5

1.0

1.5

2.0

0.1%NaFpH= 6.3

No additionpH= 6.4

1%NaFpH= 6.1

TA1

Current Density(A/cm2)

E(V

vs

SC

E)

1E-9 1E-8 1E-7 1E-6 1E-5 1E-4 1E-3

-1.5

-1.0

-0.5

0.0

0.5

1.0

1.5

2.0

No additionpH= 6.4

1%NaFpH= 6.1

0.1%NaFpH= 6.3

Current Density(A/cm2)

E(V

vs

SC

E)

TA2

Figure 25. Potentiodynamic curves of titanium and titanium-silver alloys when NaF

was added to artificial saliva, 37 .

56

1E-9 1E-8 1E-7 1E-6 1E-5 1E-4 1E-3-1.5

-1.0

-0.5

0.0

0.5

1.0

1.5

2.0

1%NaFpH= 6.1

0.1%NaFpH= 6.3

No additionpH= 6.4

Current Density(A/cm2)

E(V

vs

SC

E)

TA3

1E-9 1E-8 1E-7 1E-6 1E-5 1E-4 1E-3

-1.5

-1.0

-0.5

0.0

0.5

1.0

1.5

2.0

1%NaFpH= 6.1No addition

pH= 6.4

0.1%NaFpH= 6.3

E(V

vs

SC

E)

Current Density(A/cm2)

TA4

1E-9 1E-8 1E-7 1E-6 1E-5 1E-4 1E-3

-1.5

-1.0

-0.5

0.0

0.5

1.0

1.5

2.0

No additionpH= 6.4

1%NaFpH= 6.1

0.1%NaFpH= 6.3

Current Density(A/cm2)

E(V

vs

SC

E)

TA5

Figure 25. (continued).

57

Table 11. Passive current density of alloys in 1% NaF added artificial saliva (Data

from Figure 25)

Alloy Passive current density (µA/cm2)

Ti 28.04

TA1 21.79

TA2 18.72

TA3 13.29

TA4 21.31

TA5 55.96

Table 12. Open circuit potentials of titanium and titanium-silver alloys in NaF

containing artificial saliva

Open circuit potential (V vs SCE)

No addition 0.1% NaF addition 1% NaF addition

Ti -0.3453±0.102 -0.5776±0.0507 -0.6902±0.0199

TA1 -0.1441±0.051 -0.3984±0.074 -0.5375±0.0459

TA2 -0.0261±0.092 -0.2886±0.0421 -0.5617±0.0931

TA3 -0.0137±0.0173 -0.2781±0.0703 -0.474±0.0711

TA4 -0.0567±0.0907 -0.3886±0.0681 -0.4606±0.0188

TA5 -0.1346±0.1233 -0.3764±0.068 -0.5934±0.0757

Figure 26 shows the result of potentiostatic test at 250 mV (SCE) in 1% NaF added

artificial saliva. This test was performed three times and this figure was representative

scan of repeated test. The current density of titanium fluctuated severely and was

quite unstable. On the other hand, TA2 and TA3 had a low current density and

showed a very stable behavior. TA5 showed a little fluctuation of current density and

high current density value of 12 µA/cm2. The current density of titanium shifted from

8 to 13 µA/cm2 with the change of times but TA2 and TA3 maintained a low current

58

density below 5 µA/cm2.

0 1000 2000 3000 4000 5000 6000 7000

4.0x10-6

8.0x10-6

1.2x10-5

1.6x10-5

2.0x10-5

2.4x10-5

2.8x10-5

TA1

TA4

TA3

TA2

TA5

Ti

Potentiostatic test ( 250 mV, 1% NaF) pH=6.1

Cu

rren

t Den

sity

( A/c

m2 )

Time(sec)

Figure 26. Current densities of titanium and titanium-silver alloys at 250 mV (SCE)

when 1% NaF was added to artificial saliva, 37 .

7. Electrochemical property of passive oxide film

In order to investigate the electrochemical processes at the alloy surface,

electrochemical impedance spectroscopy (EIS) was used. EIS spectra of alloys were

presented as Nyquist plot and Bode phase plot in Figure 27.

From the Nyquist plot in Figure 27(a), spectra of all the alloys showed depressed

semicircle; clearly distinguished semicircles are detectable. It is seen that diameter of

semicircle increase with increasing silver addition at the range of 1.0 ~ 3.0 at% silver.

Spectra of TA3 showed a semicircle with a larger diameter. On the other hand,

titanium had very small circle compared to titanium-silver alloys.

From the Bode phase plot in Figure 27(b), it can be noted that the phase angle

drops towards - 90° at the frequency region of 10000 ~ 100 Hz. The phase angle

remained close to - 90° over a wide range of frequency. Increase in frequency range

59

0.0 2.0x106 4.0x106 6.0x106 8.0x106 1.0x107 1.2x107

0

1x107

2x107

3x107

4x107

TA4

TA5

TA1

Ti

TA2

TA3

ZR(ohms)

-Zim

(oh

ms)

Nyquist plot

TA5 TA4 TA3 TA2 TA1 Ti

(a)

(b)

Figure 27. EIS spectra for titanium and titanium-silver alloys (a) Nyquist plot (b)

Bode phase plot.

1E-3 0.01 0.1 1 10 100 1000 10000-100

-80

-60

-40

-20

0

TA4

TA5

TA1TA3

TA2

Ti

Bode Phase plot

Frequency(Hz)

Ph

ase(

Deg

ree)

1E-3 0.01 0.1

-90

-80

-70

-60

TA4

TA5

TA1

TA3

TA2

Ti

TA5 TA4 TA3 TA2 TA1 Ti

60

over which phase angle is close to - 90° is an indicative of passive film acquiring

behavior. As shown in Figure 27(b), TA1, TA2 and TA3 had large frequency region

which phase angle is about - 90°. TA3 maintained low phase angle in the low

frequency region (confirmed from enlarged graph). EIS spectra showed a typical

behavior for a thin passive film for all alloys. Additionally, TA2 and TA3 had more

capacitive behavior than that of titanium and other titanium-silver alloys from Bode

phase plot.

In order to characterize the passive film formed on alloy surface, we proposed an

equivalent circuit (Figure 28) which took into account the structure of the passive film

and the contribution of the space charge double layer.

Re : Electrolyte resistance

Rp : Passive film resistance

Figure 28. Equivalent circuit proposed for fitting EIS spectra.

A constant phase element (CPE) was introduced in the modeling procedure

representing the electrical double layer of the surface/electrolyte interface. The use of

CPE is used to instead of pure capacitor to compensate for the non-ideal capacitive

response of the interface. CPE was given by the following expression.

[ ] 111 )()(−−− += dlf CCCPE

Cf : film capacitance

Cdl : double layer capacitance

61

To obtain the value of Rp (passive film resistance) and CPE (interface capacitance),

the EIS spectra were fitted with this equivalent circuit. Fitted data of Rp (passive film

resistance) and CPE (interface capacitance) were plotted in Figure 29.

TA1 had passive film resistance (Rp) value of 6.187×107 Ω, TA2 of 7.463×107 Ω,

TA3 of 1.048×108 Ω, TA4 of 1.789×107 Ω, and TA5 of 1.621×107 Ω. Ti had lowest

value of 7.938×106 Ω. As silver addition increase to 3.0 at%, Rp of these alloys also

increases. However, when silver addition content is over 3.0 at%, Rp showed steep

decrease. TA3 had highest value and TA4, TA5 showed similar Rp and CPE value to

titanium. CPE value is in inverse relation to Rp. TA3 had lowest CPE value of 6.015

µF/cm2.

62

Ti TA1 TA2 TA3 TA4 TA50.0

2.0x107

4.0x107

6.0x107

8.0x107

1.0x108

1.2x108

Rp

Rp(

ohm

)

Alloy

(a)

Ti TA1 TA2 TA3 TA4 TA50.0

5.0x10-6

1.0x10-5

1.5x10-5

2.0x10-5

2.5x10-5

Alloy

CPE

CP

E(F

/cm

2 )

(b)

Figure 29. The parameter value of the equivalent circuit from Figure 25(a) after fitting

the EIS spectra (a) Rp (passive film resistance), (b) CPE (interface capacitance).

63

8. Biocompatibility evaluation

A. Metal ion release test

Table 13 shows ion release concentration of titanium and silver element for all

titanium-silver alloys. Titanium ion concentration reaches a maximum of 8.9 ppb and

silver ion concentration stayed under 3 ppb even after 24 weeks and no significant

differences between titanium and titanium-silver alloys were observed.

In case of titanium ion, titanium and titanium-silver alloys showed very similar

value of 2.626 ~ 3.372 ppb for a period of 4 weeks. After 16 weeks, titanium ion

release of titanium increased largely compared to that of titanium-silver alloys.

Titanium ion release amount of titanium-silver alloy was limited below 7.7 ppb; on

the other hand, titanium had over 8.9 ppb in period of 24 weeks.

As shown in Table 14(b), released silver ions were extremely low value (< 3 ppb)

and no significant differences were observed between titanium-silver alloys. However,

there was no significant difference between alloys and immersion periods.

B. Cell adhesion morphology

To observe the typical adhesion morphology of L929 fibroblast cell, SEM studies

were carried out on cultured L929 fibroblast cell on either pure titanium or titanium-

silver alloy surfaces. Figure 30 and 31 showed morphology of L929 fibroblast cell

cultured on alloy surfaces. Cells were well spread and proliferated uniformly on each

alloy under low magnification (×200). Filopodia: a thin protrusion from a cell and

lamellipodia can be observed in cells grown on surfaces. Cells were oval or diamond-

like in shape and a lot of microvilli evidently appear and extend to all directions under

high magnification (Figure 31). Cells seemed to adhere tightly to surfaces and no

differences in detail morphology of cell were observed between titanium and titanium

alloys.

64

Table 13. Titanium and silver ion release concentration (ppb) after immersion for each

period in artificial saliva, 37

1 week 4 weeks 16 weeks 24 weeks

Ti 3.208 ± 0.091 3.020 ± 0.162 6.867 ± 0.083 8.904 ± 0.162

TA1 3.382 ± 0.118 3.372 ± 0.105 4.857 ± 0.024 7.067 ± 0.104

TA2 3.362 ± 0.116 3.169 ± 0.170 5.413 ± 0.105 7.628 ± 0.150

TA3 2.626 ± 0.093 2.835 ± 0.164 4.723 ± 0.103 6.083 ± 0.063

TA4 3.010 ± 0.090 2.978 ± 0.104 4.675 ± 0.067 6.983 ± 0.106

TA5 2.959 ± 0.107 2.987 ± 0.122 4.928 ± 0.054 6.555 ± 0.122

(a) Titanium

1 week 4 weeks 16 weeks 24 weeks

TA1 1.386 ± 0.130 1.544 ± 0.025 1.894 ± 0.110 1.885 ± 0.037

TA2 1.856 ± 0.098 1.975 ± 0.108 1.804 ± 0.138 1.638 ± 0.073

TA3 1.745 ± 0.090 1.281 ± 0.095 1.503 ± 0.153 2.066 ± 0.239

TA4 1.579 ± 0.170 1.899 ± 0.161 1.842 ± 0.060 2.078 ± 0.037

TA5 1.914 ± 0.099 1.464 ± 0.170 2.249 ± 0.100 1.910 ± 0.095

(b) Silver

65

(a)

(b)

Ti

(a)

(b)

TA1

Figure 30. Morphology evaluation of L929 fibroblast cell on the surface by SEM (a)

magnification of ×200 (b) ×500.

66

(a)

(b)

TA2

(a)

(b)

TA3

Figure 30. (continued).

67

(a)

(b)

TA4

(a)

(b)

TA5

Figure 30. (continued).

68

Figure 31. Microvilli protrusion morphology at the leading edge of L929 fibroblast

cell (high magnification of ×5000, ×10000).

69

C. Agar diffusion test

Cytotoxicity of titanium and titanium-silver alloys was evaluated using agar

diffusion test. Table 14 showed cytotoxicity of titanium and titanium-silver alloys as

well as controls determined by this test. Figure 32 showed photographs of Petri-dish

of each alloy after test and Figure 33 showed micrograph of L929 fibroblast cell

around the alloy and control. As shown in Figure 32 and 33, positive control

demonstrated severe cytotoxic results with a decolorized zone and cell lysis. On the

other hand, negative control and test alloys showed no decolorization and cell lysis.

Titanium-silver alloys showed no cytotoxicity irrespective of the silver contents.

Table 14. Cytotoxicity of titanium and titanium-silver alloys evaluated by agar

diffusion test

Alloy Zone Index Lysis Index Response Index Cytotoxicity

Ti 0 ~ 0 0 ~ 0 0 / 0 None

TA1 0 ~ 0 0 ~ 0 0 / 0 None

TA2 0 ~ 0 0 ~ 0 0 / 0 None

TA3 0 ~ 0 0 ~ 0 0 / 0 None

TA4 0 ~ 0 0 ~ 1 0 / 0 None

TA5 0 ~ 0 0 ~ 1 0 / 0 None

Positive(Gutta percha) 4 ~ 5 5 ~ 5 5 / 5 Severe

Negative(glass) 0 ~ 0 0 ~ 0 0 / 0 None

70

Ti

TA1

TA2

TA3

TA4

TA5

Figure 32. Photographs of Petri-dish of each alloy after agar diffusion test (Upper left:

positive control (gutta percha), upper right: negative control (soda-lime glass), lower:

test alloys per each Petri dish).

71

Ti

TA1

TA2

TA3

TA4

TA5

Negative control

Positive control

Figure 33. Micrograph of L929 fibroblast cell morphology around titanium and

titanium-silver alloys and control.

72

B. Cell viability: MTT assay

Figure 34 showed cell viability of titanium and titanium-silver alloys by MTT

assay. Titanium and titanium-silver alloys showed cell viability similar to control.

Titanium-silver alloys exhibited over 95% cell viability in this test. There was no

significant difference between pure titanium and titanium-silver alloys.

Ti TA1 TA2 TA3 TA4 TA550

60

70

80

90

100

110

120

Cel

l Via

bili

ty(%

)

Alloy

Figure 34. Cell (L929 fibroblasts) viability of titanium and titanium-silver alloys by

MTT assay.

C. Acute systematic toxicity test

Test group mice administered extracts did not develop any clinical signs of toxicity

either immediately or during the observation period and no mortality occurred. In the

observation period, mouse weights in test group (TA2, TA3) and control group

increase continuously; as shown in Table 15 and extract injection did not cause any

appreciable alterations in the feed intake. There was no significant difference in body

weight compared to control group and a similar pattern was observed for body weight

gains. Histopathologic evaluation was carried out on separate liver and kidney organs.

Figure 35(a) showed light micrograph of liver from a control mouse showing regular

73

Table 15. Weight changes (g) of experimental mice of each group for acute

systematic toxicity

Just after 3 hour 1 Day 2 Day 3 Day 7 Day

29.8 30.0 30.1 30.6 31.0 32.2

25.1 25.4 25.6 26.0 26.3 27.3

32.9 33.0 33.2 33.8 34.2 35.4

31.7 32.3 33.2 33.5 33.5 33.9

Control

CAGE I

25.2 25.3 25.9 26.6 27.2 30.1

28.1 28.3 29.0 29.5 30.1 30.8

29.2 29.4 29.9 30.2 31.0 32.4

26.6 26.8 27.0 27.3 27.5 28.0

28.3 28.5 28.9 29.1 29.9 30.7

Control

CAGE II

30.1 30.6 31.0 31.3 32.0 33.6

30.0 30.4 31.5 31.6 31.7 32.2

29.3 29.6 30.4 30.6 30.7 30.9

29.1 29.5 30.3 30.5 30.8 31.2

31.7 32.1 32.2 32.5 32.6 33.1

Test(TA2)

CAGE I

27.0 27.5 28.9 29.0 29.2 29.5

25.6 25.8 26.1 26.3 26.6 26.9

29.5 29.9 30.0 30.1 30.4 31.1

32.3 32.6 32.9 33.0 33.0 33.2

30.8 31.3 31.4 31.7 31.8 32.0

Test(TA2)

CAGE II

31.3 31.5 31.7 31.8 32.0 32.3

29.1 29.2 30.1 31.5 32.7 33.7

29.4 29.4 30.1 30.7 31.5 32.5

31.2 31.5 32.1 32.9 33.4 34.5

29.5 29.8 31.2 31.5 32.1 32.9

Test(TA3)

CAGE I

31.6 31.6 32.2 33.4 34.7 35.2

27.3 27.5 28.4 29.7 30.4 31.3

29.0 29.2 30.4 31.6 32.0 33.2

29.9 30.2 31.2 32.3 33.2 33.9

28.5 28.6 29.9 30.2 31.6 32.7

Test(TA3)

CAGE II

28.0 28.2 29.7 31.4 32.5 33.4

74

Control

TA2

TA3

(a) liver

Control

TA2

TA3

(b) kidney

Figure 35. Light microscopes of tissue from control and test group mouse (a) liver (b)

kidney.

75

histopathologic features and reveals no remarkable changes, such as cytoplasmic

vacuolization and karyorrhexis, of test group (TA2, TA3) versus control. In lung

tissue, some pathological changes were observed in experimental group. In case of

kidney, as shown in Figure 35(b), no atypical tubles and necrosis were observed in

test group. As illustrated in Figure 35, no microscopic changes were detected in the

kidney and liver.

76

IV. DISCUSSION

1. A brief overview of physical metallurgy of titanium alloy

Titanium is a transition metal with an incomplete shell in its electronic structure

enables it to form solid solutions with most substitutional elements having a size

factor within ± 20%. In its elemental form titanium has a high melting point

(1678 ), exhibiting an hexagonal close packed crystal structure (hcp) α up to the β

transus (882.5 ), transforming to a body centered cubic structure (bcc) β above this

temperature. The utility of titanium and its alloy is greatly enhanced by its allotropic

behavior.

Titanium alloys may be classified as either α, near-α, α + β, metastable β or stable β

depending upon their room temperature microstructure. In this regard alloying

elements for titanium fall into three categories: α-stabilizers, such as Al, O, N, C, β

stabilizers, such as Mo, V, Nb, Ta, (isomorphous), Fe, W, Cr, Si, Ni, Co, Mn, H

(eutectoid), and neutral, such as Zr. α and near-α titanium alloys exhibit superior

corrosion resistance with their utility as biomedical materials being principally

limited by their low ambient temperature strength. In contrast, α + β alloys exhibit

higher strength due to the presence of both α and β phases. Their properties depend

upon composition, the relative proportions of the α / β phases, and the alloy’s prior

thermal treatment and thermo-mechanical processing conditions. β alloys (metastable

or stable) are titanium alloys with high strength, good formability and high

hardenability. β alloys also offer the unique possibility of combined low elastic

modulus and superior corrosion resistance.

A β alloy is operationally defined that it contains sufficient total β stabilizer content

to retain 100% β upon quenching from above the β transus. Alloys lying above this

critical minimum level of β stabilizer content may still lie within a two-phase region,

with the resulting as-quenched β phase being metastable with the potential of

precipitating a second phase upon aging. Alloys with increasing alloying content

ultimately exceeding a critical value are considered stable β alloys, in which no

precipitation takes place during practical long-time thermal exposure.

Process variations are traditionally used to control the alloy microstructure and

77

therefore to optimize titanium alloys properties, i.e., ductility, strength, fatigue

resistance or fracture toughness. The effects of various microstructures are then

correlated with engineering properties, with the most common microstructural

features studied in metastable β alloys being β grain size and the size and distribution

of aged α. Apart from α phase, precipitation of transient β′ or ω phases and/or

intermetallic compounds may be observed in metastable β alloys depending upon

alloy composition, heat treatment, processing history and service conditions.

2. Compositional analysis and phase / microstructure observation

Due to the high reactivity of titanium, binary titanium-silver alloys of controlled

composition are extremely difficult to produce. Melting or other manufacturing

processes of titanium alloys should be carefully controlled for prevention of

contamination as well as precise materialization of the alloy composition.

First, a difficulty encountered in experimental work on titanium-silver alloys is the

worry of silver loss by volatilization during melting because of large melting point

difference of two elements; melting point of pure titanium is 1678 , besides, that of

pure silver is 962 . However, when the titanium-silver alloy was melted, little loss

of silver occurred.

It is thought that impurity controls and content identifications are very important.

These apparently slight concentration differences have a substantial effect on the

physical and mechanical properties for example; incorporation of impurity into the

titanium alloy increases its strength, but results in a significant decrease in corrosion

resistance. However, in this study, no severe oxidation and impurity inclusion was

encountered - the maximum amount of oxygen incorporated as impurity in the alloy

produced was 0.13% - because although various different processes were involved in

producing the alloy, most procedures were performed in high vacuum.

This can also be considered as an important finding, along with little loss of silver.

Oxygen contents of alloys were later discussed as considerable factor when discuss

mechanical property of titanium-silver alloys.

As previously mentioned, alloys of titanium can be assigned to one of two major

categories: α stabilized or β stabilized systems. Molchanova169 has offered a detailed

78

subdivision of the equilibrium phase diagrams. The schematic phase diagrams which

typify these categories and the solutes which give rise to each of them are depicted in

Figure 36. As shown in Figure 36, binary titanium-silver alloy belongs to the ‘β

eutectoid’ group in β stabilized system.

Figure 36. Classification scheme for binary titanium alloy phase diagrams.169

The transition-metal block of the periodic table may be regarded as commencing

with group III, scandium (Sc), yttrium (Y) and lanthanum (La) (or perhaps more

precisely, lutetium (Lu)). In this scheme, the alkaline-earth metals, calcium (Ca),

strontium (Sr) and barium (Ba), may be regarded as ‘pre-transition metals’, and the

noble metals, Cu, Ag, and Au, as ‘post-transition metal’. As indicated in most

periodic charts of the elements, the structures of the transition metals all change from

hcp to bcc as e / a (electron / atom ratio) increase from 4 through 6. It is possible that

stabilization of the bcc structure can be justified within the framework of a screening

79

model in terms of which a high conduction-electron concentration, which enhances

the screening of ion cores, may favor a symmetrical, hence cubic structure. Thus an

increase in electron density (as in the groups V and VI elements), which tends to

symmetrize the screening, increases the stability of the bcc structure. Symmetrization

may also be accomplished through lattice vibrations; thus, all six of the groups III and

IV elements transform to the bcc structure at high temperatures (as compared to their

Debye temperatures). With regard to alloys, the addition of transition elements to

titanium increases the electron density and consequently stabilizes the bcc or β

structure. Thus, as a general rule, the transition elements are ‘β stabilizers’. The

systematic of β stabilization by transition elements has been discussed in detail by

Ageev and Petrova;170 i) the β stabilizing action of transition metal solutes is greater

the ‘farther’ they are from titanium in the periodic table; and ii) for the retention of

the metastable β solid solution during quenching, the β stabilizers has to provide for

an e / a (electron / atom ratio) of at least 4.2.

Though silver is classified to β stabilizer, the effect of silver as β stabilizing

element was not clearly determined. Table 16 summarized results stabilization of the

β phase in quenched titanium based alloys. As shown in Table 16,171 silver

concentration was not determined.

Table 16. Minimum concentration (at%) of the alloying element necessary for the

complete stabilization of the β phase in binary titanium alloys with d-

metals of 4 ~ 6 periods171

Ti-V Ti-Cr Ti-Mn Ti-Fe Ti-Co Ti-Ni Ti-Cu

15 7 6 5 7 9 11

Ti-Nb Ti-Mo Ti-Tc Ti-Ru Ti-Rh Ti-Pd Ti-Ag

23 5 ? 33 4 11 ?

Ti-Ta Ti-W Ti-Re Ti-Os Ti-Ir Ti-Pt Ti-Au

35 10 5 3 3 7 ?

80

From XRD pattern, β phase peak began to appear from TA3; silver content 3.0 at%.

This result means 3.0 at% silver is necessary for β phase stabilization.

We could achieve microstructural observation results agreement with XRD results.

TA1 and TA2, low silver content alloys, had acicular and equiaxed α phase by

diffusional transformation, while titanium showed only fully equiaxed α phase. It was

assumed that titanium and TA1, TA2 among titanium-silver alloys preceded normal

diffusional process α phase precipitation from originally formed β phase. The

mechanism of the transformation depends on cooling rate and composition.172 Rapid

cooling rate and composition of β stabilizing element controlled diffusional phase

transformation. α phase precipitation nucleates heterogeneously from defects such as

grain boundaries, dislocations.173 Interstitial atom diffusion has an important

influence on precipitation. The element B, C, N and O are all stabilizers of the α

phase in titanium and as such are expected to encourage α phase precipitation as they

accumulate at the grain boundaries and residual dislocations. In the microstructure of

TA2, grain boundary of α or β phase was not shown. It was thought that mediate step

of β phase stabilization. In other words, α phase precipitation from defects which has

high energy such as grain boundaries, dislocations and some β phase stabilizing effect

owing to silver element occur simultaneously. However, because α phase

precipitation was dominant, β phase was not stabilized and was nonexistent.

When silver content increased to 3.0 at%, alloy structure was dominated by

acicular, to be called ‘Basketweave’ like α phase structure in β phase matrix. It was

thought that this structure was Widmanstätten structure. This structure forms as

platelets parallel to constant crystallographic orientation110 of β phase and may be

obtained due to diffusional process competes with diffusionless martensitic

transformation during quenching from β phase region. Titanium-silver alloys showed

no martensite transformation even after quenching. This result meant that silver

element effect as β phase stabilizer was not large compared to main β stabilizers such

as Mo, Nb.

81

3. Mechanical property

The interpretation of the measured strength and microhardness values could be

quite complex. Such effects as solid solution strengthening, precipitation hardening

and changes of grain size, crystal structure / phase could all affect the hardness and

strength of the alloy. The two important classes of solution strengtheners are the

interstitial elements B, C, N, and O and simple metals such as Al, Ga, and Sn.

Interstitial-element strengthening of titanium and substitutional strengthening of other

metals has been considered extensively dealing with the electronic and

thermodynamic aspects of solution strengthening.174 In case of transition element,

solid solution strengthening effect as substitutional element was little. Although some

degree of solid solution strengthening is inevitably contributed by the presence of

transition metals in β phase solid solution, the dominant strengthening mechanisms in

such alloys are precipitational effects.175 Transition elements are regarded as β

stabilizers and precipitation hardeners rather than solid solution strengtheners.

Actually, precipitation of α phase in the metastable β is a method used to strengthen

the alloys. Therefore, it was thought that main strengthening mechanism of titanium-

silver alloy was precipitation of α phase. Strength of TA1 and TA2 increased by solid

solution strengthening, but in case of TA3 ~ TA5, two effects converged:

precipitation of α phase and solid solution strengthening of silver element so

increment is large compared to TA1 and TA2. In addition, TA4 and TA5 underwent

Ti2Ag precipitation (it could be validated by XRD and microstructure), so one more

strengthening effect occurred in these alloys. It was thought that it must be calculated

to low value because width of specimens was too narrow. We must have one

important consideration for evaluation and comparison of mechanical property.

Though it was same pure titanium, strength and other mechanical property could be

altered in plentitude by manufacturing process and heat treatment. Therefore, it was

thought that relative comparison between titanium - same concept of control - and

titanium-silver alloy that we manufactured was just appropriate. The variation in alloy

bend strength with silver content had a trend similar to that in microhardness, except

for TA1 and TA2. These two alloys showed an unexpectedly lower strength than

titanium. The reason might not be certain. However, it might be considered as

82

complex atomic structure and interaction. Atomic interactions may cause complicated

changes within individual phases that could lead, on one hand, to desired

enhancement in strength, but, on the other, to unexpected lattice instabilities.

Hardness and bend strength of titanium-silver alloys increased due to solid solution

strengthening and precipitation of phase (intermetallics). Hardness value can be

related to yield strength.176,177 In this study, strength and hardness value showed

similar tendency with respect to silver content.

It has been shown that the value of elastic modulus decreased with the increase of

both the bond strength between titanium and alloying elements and the metal d-orbital

energy level (which is correlated with electronegativity and the metallic radius

elements).178 Transition metal solutes actually lower the modulus of titanium in small

content.175,179 and it is widely known that β phase titanium alloys generally have a

lower modulus level than that of α or α + β-type alloys.180 Elastic modulus of alloys

showed relatively low value compared to common data. Most common data of elastic

modulus of cp titanium is about 92 ~ 110 GPa.

Conclusively, it was thought that main strengthening mechanism of titanium-silver

alloy was precipitation of α phase. Increase mechanical properties of titanium-silver

alloy compared to titanium resulted in precipitation of α phase and solid solution

strengthening of silver element. All the alloys had similar elastic modulus. No

decrease of elastic modulus occurred with silver addition because titanium-silver

alloys showed α + β phase. It is thought that transition element with high β

stabilization ability also had great ability to decrease elastic modulus.

4. Corrosion resistance and electrochemical property

Two essential features determine how and why a metal corrodes. The first

characteristic involves thermodynamic driving forces, which cause corrosion

(oxidation and reduction) reactions, and the second involves kinetic barriers, which

limit the rate of these reactions. The thermodynamic driving forces that cause

corrosion correspond to the energy required or released during a reaction. The kinetic

barriers to corrosion are related to factors that impede or prevent corrosion reactions

from taking place. The basic underlying reaction that occurs during corrosion is the

83

increase of the valence state - that is, the loss of electrons - of the metal atom to form

an ion, as expressed by the equation: M ↔ Mn+ + ne–. This oxidation event may result

in free ions in solution, which then can migrate away from the metal surface or can

lead to the formation of metal oxides, metal chlorides, organometallic compounds, or

other chemical species. These latter forms may be soluble or may precipitate out to

form solid phases. The solid oxidation products may be subdivided into those that

form adherent compact oxide films and those that form non-adherent oxide, chloride,

phosphate, or other particles that can migrate away from the metal surface. In all of

these possible reactions, there is a thermodynamic driving force for the oxidation of

metal atoms to their ionic form. In this case, the driving force is the free energy (∆G)

resulting from these reactions, which can be calculated with use of the equation181:

nno

red eM

MRTGG

]][[][

ln −++∆=∆

where ∆Gred is the free energy for the reduction reaction, ∆G° is the free energy of

the reaction in a defined standard state, R is the gas constant, T is the temperature,

and the bracketed values are the activities (or the approximate concentrations) of the

species involved in the reaction. If ∆G is greater than zero, then the reduction process

requires energy or, alternatively, the oxidation process releases energy and will occur

spontaneously. In general, there are two sources of energy to be considered in

corrosion processes. The first is a chemical driving force that determines whether

corrosion will take place under certain conditions. If the free energy for oxidation is

less than zero, then oxidation will occur spontaneously, as in the metals in the alloys

used for orthopaedic implants. The second source of energy occurs when positive and

negative charges (metal ions and electrons, respectively) are separated from one

another during corrosion. The ions are released into solution or go on to form an

oxide or another compound, and the electrons are left behind in the metal or undergo

other electrochemical reactions, such as reduction of oxygen or hydrolysis of water.

This separation between the charges contributes to what is known as the electrical

double layer and creates an electrical potential across the metal-solution interface

(much like a capacitor), as expressed by ∆G = - nF∆E, where n is the valence of the

84

ion, F is the Faraday constant, and ∆E is the voltage or potential across the interface

between the metal and the solution. This potential is a measure of the reactivity of the

metals or the driving force for metal oxidation. The more negative the potential of a

metal in solution, the more reactive it will tend to be. At equilibrium, the chemical

energy balances with the electrical energy, yielding the Nernst equation182:

][][

lnM

M

nF

RTEE

no

+

+∆=∆

which states that there is an electrical potential across the interface between the metal

and the solution when metals are immersed in a solution. From this equation, a scale

of reactivity of the metal, known as the electrochemical series, can be established.

This scale ranks the equilibrium potential from most positive (least reactive or most

noble) to most negative (most reactive, or most base). This ranking is based only on

thermodynamic considerations - that is, if it is assumed that there is no barrier to the

oxidation of the metal, these are the potentials across the metal-solution interface.

Certain metals owe their resistance to corrosion to the fact that their equilibrium

potentials are very positive, indicating that the chemical driving force for oxidation

either is very small and negative or is positive. Therefore, there is little or no driving

force for oxidation unless the potentials of these materials are raised well above their

equilibrium potentials. Au and Pt are examples of metals that have little or no driving

force for oxidation in aqueous solutions; hence, they tend to remain in metallic form

indefinitely in the human body. However, metallic biomaterials have more negative

potentials, indicating that, from a chemical driving force perspective, they are much

more likely to corrode. For example, titanium has a very large negative potential,

indicating a large chemical driving force for corrosion (oxidation). If some other

process such as passivation does not intervene, titanium metal will react violently

with the surrounding chemical species (typically, oxygen, water, or other oxidizing

species) and will revert to its ionic form. The second factor that governs the corrosion

process of metallic biomaterials is kinetic barriers that prevent corrosion not only by

energetic mechanisms but by physical limitation of the rate at which oxidation or

reduction processes can take place. The well known process of passivation, or the

85

formation of a metal-oxide passive film on a metal surface, is an example of a kinetic

limitation to corrosion. In general, kinetic barriers to corrosion prevent the migration

of metal ions from the metal to the solution, the migration of anions from the solution

to the metal, and the migration of electrons across the metal-solution interface.

Passive oxide films are the best known forms of kinetic barriers to corrosion.

Corrosion of metallic biomaterials is a twofold problem. On the one side, it leads to

material degradation. On the other side, it produces ion release with harmful effects

on the organism. The presence or absence of a protective surface oxide film controls

behavior of the materials. The excellent corrosion resistance of titanium alloys results

from the formation of very stable, continuous, highly adherent, and protective oxide

films on metal surfaces.183-186 Because titanium is highly reactive and has an

extremely high affinity for oxygen, these beneficial surface oxide films form

spontaneously and instantly when the fresh metal surfaces are exposed to air and / or

moisture. In fact, a damaged oxide film can generally reheal itself instantaneously if

at least traces of oxygen or water are present in the environment. However, anhydrous

conditions in the absence of a source of oxygen may result in corrosion of titanium,

because the protective film may not be regenerated if damaged. Titanium alloys, like

other metals, are subject to corrosion in certain environments. The primary forms of

corrosion that have been observed on these alloys include general corrosion, crevice

corrosion, galvanic corrosion, anodic pitting, hydrogen damage, corrosion fatigue,

erosion-corrosion and stress corrosion cracking. In any contemplated application of

titanium, its susceptibility to degradation by any of these forms of corrosion should be

considered. The methods of expanding the corrosion resistance of titanium into

reducing environments include:

Increasing thickness of the surface oxide film by anodizing or thermal

oxidation

Anodically polarizing the alloy (anodic protection) by impressed anodic

current or galvanic coupling with a more noble metal in order to maintain the

surface oxide film

Applying precious metal (or certain metal oxides) surface coatings

Alloying titanium with certain elements

86

Adding oxidizing species (inhibitors) to the reducing environment to permit

oxide film stabilization

For biomedical application of metallic biomaterials, corrosion resistance and

chemical stability of materials in several environments is the most important factor.

Therefore, we evaluated corrosion resistance and electrochemical property of

titanium-silver alloys through general corrosion test: potentiodynamic and

potentiostatic test. Potentiodynamic and potentiostatic polarization techniques make it

possible to accurately predict based on electrochemical data, the corrosion resistance

of alloys. From potentiostatic test at three different potentials which can be applied in

biological environments, current densities of titanium-silver alloys were maintained

consistently without drastically increase during test periods. This result means passive

film of titanium-silver alloys is electrochemically stable for long periods. Open circuit

potential of titanium-silver alloys increased due to silver element. High open circuit

potential means that test materials are noble and stable in a certain environment.

When silver addition content was over 3.0 at%, corrosion resistance of these alloys

decreased. TA4 and TA5 have three different phases. These α, β phase and Ti2Ag has

different atomic structure, surface charge and energy. Their initial chemical or

structural inhomogeneities cause decrease of corrosion resistance. Additionally,

single phase α or β alloys have better corrosion resistance than two phase α + β

alloys.187 Moreover, from forming Ti2Ag intermetallic compound at metastable β

phase grain boundary, Ti2Ag caused compositional differences of passive film partly.

Effect of precipitate was also appeared in potentiostatic test. TA5 showed some

fluctuation of current density. Therefore, it is thought that silver content must be

limited to 3.0 at% for corrosion resistance enhancement. Titanium-silver alloy is

considered to have enhanced corrosion resistance because of its stable passive film.

We introduced selective dissolution, selective oxidation of alloying explaining for this

corrosion resistance enhancement. The difference in the electromotive force results

from selective dissolution of alloying element with a lower electromotive force

among the alloying elements.188 Theory of selective oxidation is as follows. An alloy

is selectively oxidized if one component, usually the most reactive one, is

preferentially oxidized. The simplest case would be binary alloy with a uniform scale

87

composed entirely of the only oxide that one of the components can form. This

situation will be described to illustrate the principles involved. The alloy is formed

from metal A and B where B is more reactive than A is; that is, oxide BO is

thermodynamically more stable than AO, and AO and BO are immiscible. Which

scale, AO or BO, that will form on the A-B alloy depends on the relative nobility of A

and B, their concentrations in the alloy, the oxygen pressure, and the temperature.

When alloying with a noble metal, it is an obvious example of selective oxidation

would be scale formation on alloy A-B where A is so noble that AO is not

thermodynamically stable at the environmental pressure and temperature. Then, only

BO scale can form if it is stable. From this theory, the addition of silver to titanium is

considered to improve the corrosion resistance because of easy formation of titanium

oxide. In other words, silver having a much higher electromotive force than titanium,

first facilitates the dissolution of titanium in artificial saliva which then forms a stable

titanium oxide film on the surface of titanium-silver alloy. Moreover, Stern and

Wissenberg used the mixed-potential theory to explain the electrochemical behavior

of titanium alloyed with noble or precious metal.189 When it is alloyed with noble or

precious metal, the cathodic reaction of hydrogen evolution is facilitated and the

obtained mixed-potential is in the passive region of titanium. The role of noble or

precious metal is to enhance the cathodic kinetics, shift the potential of the alloy

surface to the passive region, and promote the passivity for titanium. According to

this theory,189 it is thought that silver, classified precious metal, can promote the

passivity for titanium. There can be a reason for formation of thick titanium oxide

film on the surface of titanium-silver alloys.

5. Surface characterization of the alloy

The nature, composition, and thickness of the protective surface oxides that form

on titanium alloys depend on environmental conditions. Titanium forms several

oxides (TiO2, TiO, Ti2O3). The TiO2 oxide is the most common and most stable one

and is found in three crystalline forms: the tetragonal anatase, the rutile, and the

orthorhombic brookite. Actually, the composition of the oxide layer has not been

clearly defined, and it is unlikely that it would correspond to the stoichiometric

88

composition and, therefore, TiOx describes more accurately the oxide form. Evidence

shows that the ready formation of TiO2 in the air might be due to the low free energy

value of the reaction Ti + O2 → TiO2 which has a ∆G of 2203.8 kcal/mol (2856

kJ/mol), making the formation of TiO2 entropically favorable.190 However, a

considerable dispute exists concerning the pattern, kinetics, and direction of oxide

growth. Although some reports suggest that the oxide thickness is increased as a log

function of immersion time in electrolytes, others have noted different directions of

growth and steady-state levels of TiO2.191 When titanium is exposed to water, TiO2 is

expected to form according to the reaction Ti + 2H2O → TiO2 + 2H2 with a ∆G of

82.9 kcal/mol (348 kJ/mol). Because passivation initiates further oxidation via a

decrease in the free energy of the above reaction, the oxide’s formation is favored

thermodynamically. The Pourbaix diagram for titanium also reveals that the oxide

film is stable over a wide range of potentials.192

It is well known that the excellent corrosion resistance of titanium alloys results

from the formation of very stable, continuous, highly adherent and protective oxide

films on metal surfaces. We evaluated the corrosion resistance of titanium-silver

alloys from potentiodynamic test, etc. Their stability and electrochemical property

strongly depend on the composition, structure and thickness of the oxide film.

Therefore, the surface characterization of titanium alloys is very important to

understand the property of titanium alloys. It was investigated surface compositions

and the chemical states of passive oxide film of titanium-silver alloys after

electrochemical treatment using X-ray photoelectron spectroscopy. X-ray

photoelectron spectroscopy (XPS) and Auger electron spectroscopy (AES) provide

the chemical state information on the surface layer and the depth composition profiles.

Therefore, XPS and AES have been widely applied for the analysis of surface layer of

metals.

The XPS results show differences in the chemical composition of the titanium

alloys surface layers, suggesting differences in their protective character. The

dominant Ti and O signals of the XPS spectra of Figure 22 showed that the surface

film mainly consists of titanium oxide. In comparison to O contents between as-

polished and potentiostatic tested specimen, the latter had more O contents, these

89

results represented that immersion in artificial saliva and 250 mV load result in

passivation and formation of thick passive oxide film.

From the high resolution spectral analyses of Ti and O, the surface film on titanium

was mainly TiO2 containing small amounts of hydroxyl groups and bound water. This

result has good correlates and a thread of connection with many researchers’ reports

and explanations. The reported characteristics of oxide films grown on titanium are

schematically shown in Figure 37193 and summarized as follows:

1. The amorphous or nanocrystalline oxide film is typically 3 ~ 7 nm thick and mainly

composed of the stable oxide TiO2.

2. The TiO2 / Ti interface has an O to Ti concentration ratio that varies gradually from

2 to 1 from the TiO2 film to a much lower ratio in the bulk.

3. Hydroxide and chemisorbed water bond with Ti cations leads to weakly bound

physisorbed water on the surface. In addition, some organic species like

hydrocarbons adsorb and metal-organic species, such as alkoxides or carboxylates

of titanium also exist on the outmost surface layer whose concentrations depend on

not only the surface conditions, such as cleanliness but also the exposure time to air

as well as the quality of the atmosphere.

Angle resolved XPS is one of the ways of probing near-surface compositional

gradients. (Figure 38) When the take off angle increases from 10° to 75°- when take

off angle is low, analysis depth reduced, the fraction of Ti4+ increased, showing that

Ti4+ was the main component in the outer part of the oxide film. In the same time the

minor contributions of Ti2+, Ti3+ (from titanium suboxides) decreased. It is assumed

that TiO2 oxide layer exist in the outer surface.

90

Figure 37. Schematic view of the oxide film on pure titanium.193

Figure 38. Illustration of XPS spectra taken from a thin oxide film on a metal at near

normal collection angle (bulk angle) and near grazing collection angle (surface angle).

91

On the other hand, TiO and Ti2O3 were located in the inner layer close to the

surface. These results agreed with oxide film layer model theory: TiO(in contact with

the Ti substrate)/ Ti2O3/ TiO2(outer part).194,195 With regard to fraction of TiO2,

potentiostatic tested specimen has larger amount of TiO2 than as-polished specimen.

This result means more stable oxide was formed by potentiostatic test. Because Ti3+

or Ti2+ may have much higher solubility in solution and film will tend to dissolve

reductively. The higher TiO2 content, the more stable it forms passive film. When

viewing Figure 24, results that metallic Ti peak was small or not detected meant that

thick oxide was formed on surface. Titanium-silver alloys had thick oxide layer

compared with titanium and it was assumed that silver addition to titanium played an

important role to form a thick passive film of alloys. It was also explained by Stern

and Wissenberg’s mixed-potential theory.189 According to this theory, silver could

function as a promoter of passivity for titanium.

6. Effect of fluoride on the electrochemical property of the alloy

Fluoride has long been known to be effective in protecting the dental enamel from

caries, acting as an antibacterial agent, inhibiting dissolution and enhancing

demineralization.196 Fluoride is well documented as an anticariogenic agent. The

commercial dental gels, rinses and varnishes containing fluoride at concentrations

ranging from 0.1% to 1.0% are frequently used for caries-preventive prophylactic

applications. Common drinking water also contains a certain content of fluoride. A

number of commercially available preparations have been accepted by ADA

Acceptance Program Guidelines in American Dental Association Council on

Scientific Affairs (1998). These approved professional fluoride systems are 2.0%

sodium fluoride, 8.0% stannous fluoride and 1.23% fluoride, provided as an

acidulated phosphate fluoride preparation.197 Some dental materials like bonding

agents and restorative materials contain fluoride.198 Moreover, fluoride release is a

desirable attribute for a material used in some dental applications. In general,

materials that release larger amounts of fluoride have a greater caries preventive

potential and are desirable, so long as the physical and mechanical properties are not

adversely affected.199 However, fluoride ions in prophylactic agents have been

92

reported to cause corrosion and discoloration of titanium and its alloys used in

dentistry. It has been reported that the corrosion resistance of titanium was lost in

solution containing fluoride200,201 and in vitro studies have been reported that the

corrosion of titanium can occur in fluoride-containing prophylactic agents.202

Therefore, the corrosion resistance and stability of dental alloys against the effect of

fluoride ions are an important factor to be taken into account for dental application.

As concentration of fluoride added to the artificial saliva increased, the passive

current densities of titanium and titanium-silver alloys increased. These results mean

that titanium-silver alloys were more resistant than titanium against attack of fluoride

and had stable oxide film in fluoride containing artificial saliva.

There are two kinds of fluoride in solution, the HF molecule and the fluoride ion.

Both of two forms of fluoride in solution can affect the corrosion resistance of pure

titanium and its alloys. First, the HF molecules diffuse into the pores of the oxide

layer, attack this protective layer, and partially dissolve it. This process increases the

pore diameter and makes the unprotected titanium increasingly susceptible to attack

by the H+ ions.203 Titanium oxide easily dissolves in HF acid and makes a complex

fluoride compound such as TiF3 and TiF4. Second, the fluoride ions are aggressive

ions that degrade the protective oxide layer formed on titanium and its alloys. As a

result of complex formation of titanium-fluoride molecules such as TiOF2 on a

titanium surface, the titanium oxide is weakened and destroyed.204,205 When the pH

value of the solution approaches 7.0, few contents of the fluoride in solution are in HF

molecule form.203 Because the pH of the experimental solution was approximately 6.0,

it is thought that the largest effect of fluoride that influences corrosion resistance of

titanium and its alloys in these experiments resulted from not HF molecules but

fluoride ions. From the open circuit potential value, we can determine degree of

material’s nobleness. TA2 and TA3 alloys had little change of open circuit potential

whether fluoride exists in solution or not. It was presumed that the precious metal,

silver made an important role in rising and maintaining of the potentials. However,

when silver content exceeds 4.0 at%, there was significant difference between NaF-

free and 1% NaF concentration. As previously mentioned, it is also explained by

containing over 4.0 at% alloys precipitate intermetallic compound, Ti2Ag. The

existence of precipitates or intermetallic compounds could affect corrosion resistance

93

and induce corrosion reaction. Precipitation of Ti2Ag affected open circuit potential

and chemical stability against fluoride ions. As shown Table 11, TA5 showed increase

of passive current density in artificial saliva 1% NaF added. This result could be

explained by precipitation of Ti2Ag in TA5. Under appropriate conditions, the surface

oxide of titanium and its alloys will, if scratched or damaged, immediately reheal and

restore itself in the presence of air or even very small amounts of water.

The current density of titanium fluctuated severely and was very unstable from the

result of potentiostatic test. It is thought that titanium has a difficulty for reformation

of oxide film because fluoride ions and passive films formed on titanium were

weakened. On the other hand, titanium-silver alloys were not greatly fluctuated

compared to titanium. This difference of current behavior would be related to the

stability of passive films formed on titanium and titanium-silver alloy surface. When

silver content was low, effect of silver was so little that the behavior of current

density and open circuit potential of alloys were similar to titanium. On the other

hand, when silver content reached at 4.0 at%, silver induced Ti2Ag precipitation.

These precipitations affect open circuit potential and passive current density. It was

presumed that the oxide film of TA2 and TA3 maintained stably because silver

protects the titanium oxide layer from the attack of fluoride ions and inhibits complex

formation of titanium-fluoride molecules by binding of fluoride ions. Formation and

reaction about titanium-fluoride molecule should be clearly researched. These results

could represent that titanium-silver alloys are electrochemically stable in fluoride

containing artificial saliva and more resistant than titanium against the attack of

fluoride ions. But when silver content was exceeded 4.0 at%, electrochemical stability

and resistance against fluoride of titanium-silver alloy was weaken by Ti2Ag

precipitation. From the results mentioned above, silver element had an effect to

formation and stability of passive film in fluoride containing artificial saliva.

Additionally, it is thought that silver alloying contents must be carefully controlled in

order to increase corrosion resistance and stability of passive film.

94

7. Electrochemical property of passive oxide film

We certified titanium-silver alloys had improved corrosion resistance and formed

of thick oxide film on surface compared to titanium. We introduced electrochemical

impedance spectroscopy (EIS) to investigate resistance and capacitance of passive

film analyzing more quantitatively and evaluate interfacial reaction. EIS reveals

charge transfer processes occurring within a very narrow zone close to the electrode

surface at the electrode / electrolytic interface. This provides parameters such as

electric resistance and capacitance at the interface between metals and electrolytes. In

particular, EIS could determine the electronic properties of passive films in order to

elucidate the corrosion behavior of biomaterials in physiological media. EIS has been

used to investigate passive films on titanium and oxide film growth on a variety of

group. The analysis and interpretation of EIS data is important issue. Many

physicochemical phenomena show a resistive, capacitive or inductive behavior or a

behavior that is a combination of these. An electrical equivalent circuit, consisting of

serial and parallel combinations of resistors, capacitors and inductors and assembled

on the basis of the electrically familiar shape of spectrum, may indeed form a

reasonable model for the system investigated, translating the EIS spectrum into a

physicochemical insight. Purely capacitive behavior is experienced when metal forms

a stable and intact passive or oxide layer at the interface with the given environment.

However, an ideal capacitive spectrum is seldom found. The interface is never

completely homogenous or smooth so it was introduced EIS expression model called

a constant phase element (CPE). The use of CPE in the modeling process has been

described in detail by Esplandiu et al. 206 and Kerrec et al.207 as an effective method

for determining capacitive changes in oxide films. The Nyquist plot has the advantage

of giving an overall view of the EIS spectrum, which is convenient for acquiring

promptly an insight in the model. The Bode plot offers a complete and detailed view

of the frequency dependence of the EIS. From the EIS data, it can be noted that

titanium and titanium-silver alloys showed the characteristic response of a capacitive

behavior of surface film. The difference of passive film resistance explained with

relation to XPS results. From XPS results of Ti, TA4 and TA5 in Table 10, their

chemical composition fraction of Ti3+ and Ti2+ are higher than TA1, TA2 and TA3 -

95

they showed high passive film resistance. This could be due to the nature of reduction

process for the oxide. Reductive dissolution has been reported,208 where by Ti4+,

which is the main oxide forming species, can be reduced to Ti3+ in the active to

passive potential region and by doing so can reduce the oxide film thickness because

Ti3+ may have much higher solubility in solution and film will tend to dissolve

reductively.

From the values of the interface capacitances obtained, the thickness of passive

film can be calculated using the equation:

C

Sd oεε=

εo is permittivity of the free space, ε is dielectric constant of the oxide film, C is the

capacitance, d is the thickness of passive film and S is the surface area. (εo = 8.85×10-

12 F/m, S = 0.5 cm2) For the dielectric constant of the oxide film there are various

values reported. Di Quarto et al.209 discussed the electrical and electrochemical

behavior of anodically formed films on titanium. It stated that the electronic

conductivity of ‘TiO2 layers’ increases by up to 14 orders of magnitude with the

introduction of oxygen vacancies, and that a large variation in dielectric constant can

occur with field strength. Hollander and Castro210 found that for non-stoichiometric

single crystal rutile in a crystalline axis, a decrease in the resistivity of 8 orders of

magnitude upon the introduction of significant non-stoichiometry was accompanied

by a change in dielectric constant from 89 for stoichiometric rutile to values of

several hundreds. Blackwood and Peter211 have shown that the dielectric constant

increases from 40 to 88 with decreasing potential scan rate. Dielectric constant of the

titanium oxide film ranged between 48 and 110 according to direction of optical

axis.212 An intermediary value of 60 was used to calculate the thickness of passive

film. We could not calculate exact passive film thickness because of uncertain

dielectric constant of passive film composed of complex titanium oxides (TiO2, TiO,

and Ti2O3). As somewhat relative value, passive film of TA3 has thickness of 44.13

nm. As we could anticipate from previously presented equation, we obtained passive

film thickness and this value is in inverse relation to interface capacitance (CPE).

96

8. Biocompatibility evaluation

Metal ions are released from metallic biomaterials, such as artificial joints, bone

plates, screws and dental implants, etc., in the body. A number of approaches have

been taken to study corrosion and metal ion release from metallic biomaterials. These

studies have demonstrated that metal ions are released and transported in vivo.213,214 If

a large amount of metal ions are released, it could be generally harmful for human

health causing various phenomena: transportation, metabolism, accumulation in

organs, allergy, and carcinoma.215 These metal ions may attack at the molecular level,

affecting the ultrastructure of the cells and organelles. Released ions are able to

interfere with the osteoblast differentiation and the production of bone resorbing

agents, contributing to periprosthetic osteolysis by impairment of normal

osteogenesis.216,217 Therefore, release of metal ions from metallic biomaterials in vivo

should be understood to discuss the safety and biocompatibility of the materials.

Release of titanium ions can have a inhibiting effect on the mineralization process

during bone formation and apart from other biological and clinical effects such as

cytotoxicity, tissue lesions, metallic taste, sensitization and influences on humoral and

cellular immunity.218,219 In case of silver, alloying element of titanium-silver alloy, it

is both vital and toxic for several biological systems.220 The most evident clinical

symptom of silver intoxication is argyrism giving rise to a grey-blue color of the skin

and mucosa.221 It may be accompanied by gastro-intestinal problems, anorexia,

anemia, hepatic deficiency and respiratory insufficiency. The most toxic reactions are

found with silver chloride and sulfate. Metallic silver alloyed with other metals, in

particular with certain amounts of Au, does not show significant toxic effects.222

According to the theory of passivity, metallic biomaterials in aqueous solutions are

systems in which active and passive surfaces exist simultaneously in contact with

electrolyte.223 Therefore, it is now thought that the surface oxide film on the materials

repeats a process of partial dissolution and reprecipitation in aqueous solution. If the

dissolution rate is larger than reprecipitation rate, more metal ions gradually released.

This process is ‘anodic dissolution’ in a narrow sense. If the potential of a material

anodically changes, anodic dissolution rate increases. Metal ion release remaining

surface oxide film is relatively slow in vivo because the change in potential of

97

material in vivo is usually small. Dissolution rate of the film is accelerated by amino

acids and proteins.224 Although the mechanism of acceleration of metal ion release in

the presence of amino acid and proteins is not elucidated, equilibrium between partial

dissolution and reprecipitation in oxide film may be disturbed and ion release is

accelerated. Another mechanism of ion release is possible. When a material is

implanted, it is recognized as a foreign body by immunological processes and

macrophages adhere to the surface of the material.225 Large amounts of macrophages

and polyethylene debris are clinically observed in tissues around aseptically loosened

hip arthroplasty. Macrophage generates active oxygen species226 without response to

particles that can be phagocytosed. It produces much more active oxygen species

when they phagocytoses particles. An intracellular dismutation of O2- catalyzed by

superoxide dismutase (SOD) produces H2O2, which has a much longer lifetime and

higher permeability against cell membrane than O2-: H2O2 reaches the surface where

macrophage has adhered.226 The titanium surface is hyperoxidized by H2O2 that may

induce the release of titanium ions. H2O2 reacts the surface oxide of titanium

according to the following equation:227

Ti4+ + H2O2 → Ti5+ + OH- + OH* (where * represents radical)

With relation to above mentioned passivity theory, released titanium ions were

immediately used for regeneration of surface oxide film. Because of this reaction,

detected titanium ion was very low. Released silver ion contents are negligible when

considering detection limit of element. Passive oxide film on titanium-silver alloy

plays an important role as an inhibitor of ion release. Namely the masking effect of

the oxide film37, silver ion release was limited and negligible contents irrelevant to

silver contents of alloys. In addition, nobility of titanium-silver alloys: nobility was

determined by high open circuit potential value with silver addition, limited ‘anodic

dissolution’, which is the one cause of ion release.

The agar diffusion test detects acute cytotoxicity of leachable components through

direct contact. The presence of leachable, toxic substances results in the

decolorization in the diffusion zone. Cell lysis occurs within this zone if the

concentration of the toxic substance diffused is enough to cause cytolysis. High

98

cytotoxic as evidenced by a high degree of vacuolization, reduction in cell body size,

and disruption of the cell membrane. However, no cytotoxicity observed due to

minute amounts of silver ion leaching and diffusion through the agar. No abnormal

morphology or cellular lysis was detected. There was no diffusion of leachable

components in alloys to cause cell lysis or decolorization. Though silver content in

alloys increased, no leaching or cell lysis occurred. It is thought that this test results

are deeply related to silver ion release contents. Silver ion release was limited so

silver leaching effect did not occur.

Cells in contact with a surface will firstly attach, adhere and spread. This first

phase depends on adhesion proteins. The quality of this adhesion will influence their

morphology and their capacity for proliferation and differentiation. Fibroblasts are

slow-moving cells in which cytoskeleton plays a very important role in cell motility

and shape, among others. SEM showed the specific structures related to cell motility

of L929 fibroblast cell. Cells had a lot of microvilli, and showed dendritic network at

the leading edge of locomoting cells. Cells seemed to adhere tightly to surfaces and

grow well. No differences in detail morphology of cell were observed among the

alloys. From this result, it was thought that silver addition had no direct effect on the

fibroblast cell adhesion.

Mechanisms of the essential biological toxicity were divided into three categories:

(1) blocking of the essential biological functional groups of biomolecules, (2)

displacing the essential metal ion in biomolecules, and (3) modifying the active

conformation of biomolecules.228 The search for clinical signs and symptoms

suggestive of toxicity is helpful to ascertain the level of toxic reaction. From acute

systematic toxicity test, Mortality or clinical signs were not evident and no adverse

effects on body weight: similar pattern was observed for body weight gains. Observed

partial difference in histology of lung tissue between groups, it might need careful

consideration of experimental procedures or other effects for the evaluation of reasons.

The histopathological examination of the liver and kidney of mice revealed no

remarkable changes like degenerative alterations, cytoplasmic vacuolization and

invading of infiltrative inflammatory cells versus control animals. It means that the

extraction media of alloys do not have biological acute toxicity to the animal. More

precisely, it was summarized that the reasons for good biocompatibility stated as

99

follows; first, anodic dissolution was limited due to noble state of titanium-silver

alloys. Second, titanium-silver alloys formed thick and stable passive oxide film in

biological environment from XPS results and finally, high passive film resistance

from EIS results.

100

V. CONCLUSION

We evaluated the properties of titanium-silver alloys focusing on biofunctionality

and biocompatibility and investigated their suitability as metallic biomaterials in this

study

1. From results of phase identification, β phase began to appear from TA3; silver

content 3.0 at%. This percent might be the minimum silver content necessary

for β phase stabilization at room temperature. In addition, TA4 and TA5 had

small intensity of Ti2Ag diffraction peak.

2. From microstructural observation, in case of titanium, the only equiaxed α

phase was found. Besides, when silver content is over 3.0 at%, Widmanstätten

α + β phase is seen into the originally formed β matrix.

3. The tensile strength, bend strength and hardness value tended to rise with

increased silver content and increased largely over 3.0 at%. However, elastic

modulus was not much different from alloys and had no relation to silver

content.

4. From the potentiodynamic and potentiostatic test, titanium-silver alloys

showed better corrosion resistance and electrochemical property than titanium.

Titanium-silver alloys also exhibited higher open circuit potentials than pure

titanium and the open circuit potentials of titanium-silver alloys varied directly

with silver content. However, in case of TA4 and TA5, alloys that silver content

was over 3.0 at%, current density of these alloys increased.229

5. From the XPS results, titanium-silver alloys possessed thicker oxide films than

titanium. The oxide film of TA2 and TA3 had much TiO2, most stable oxide

film content.

6. Fluoride in solution affected passive current density and open circuit potential

of the alloys. The passive current densities of titanium and titanium-silver

alloys increased with increasing fluoride concentration. TA2 and TA3 exhibited

a low current density relatively and showed a stable behavior compared to

titanium. When silver content was exceeded 4.0 at%, electrochemical stability

and resistance against fluoride of titanium-silver alloy was weaken by Ti2Ag

101

precipitation.230

7. From the EIS data, it can be noted that titanium and titanium-silver alloys

showed the characteristic response of a capacitive behavior of surface film and

TA2 and TA3 had high passive film resistance.

8. Titanium-silver alloy showed extremely low value of metal ion release. There

was no significant difference according to silver contents and immersion

periods. Silver ions are negligible when considering detection limit of element.

9. From cell adhesion morphology on titanium and titanium-silver alloy surfaces,

L929 fibroblast cells adhered tightly, well spread and proliferated uniformly

and showed dendritic network at the leading edge of locomoting cells.

10. Titanium-silver alloys showed none cytotoxicity in agar diffusion test and

exhibited over 95% cell viability in MTT test. There was no difference between

titanium and each titanium-silver alloy and cytotoxicity and cell viability had

no relation to silver content.

11. From acute systematic toxicity test, no toxic symptom or bad reaction was

discovered, and mortality was zero. The histopathological examination of the

liver and kidney of test mice revealed no remarkable changes.

From the above results, we concluded that titanium-silver alloy had better

mechanical property than pure titanium, and corrosion resistance and electrochemical

property due to thick and stable passive oxide film in biological environment but

silver content should be limited 3.0 at% for maintaining excellent electrochemical

property. With regard to biocompatibility, the good biological safety of titanium-silver

alloys had a close relationship with their protective oxide film (TiO2) on the surface.

From this reason, titanium-silver alloys showed good biocompatibility irrelative of

silver content. Consequently, it was concluded that titanium-silver alloys had suitable

biofunctionality and biocompatibility for biomedical applications.

102

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ABSTRACTABSTRACTABSTRACTABSTRACT ((((IIIIN KOREAN)N KOREAN)N KOREAN)N KOREAN)

생체재료로의생체재료로의생체재료로의생체재료로의 적용을적용을적용을적용을 위한위한위한위한 Ti Ti Ti Ti----Ag Ag Ag Ag 이원계이원계이원계이원계 합금의합금의합금의합금의 특성특성특성특성 연구연구연구연구

<<<<지도교수지도교수지도교수지도교수 김김김김 경경경경 남남남남> > > >

연세대학교연세대학교연세대학교연세대학교 대학원대학원대학원대학원 의과의과의과의과학과학과학과학과

심심심심 형형형형 민민민민

생체재료란 생체 내에 직접 적용되는 의료용 기기에 사용되는 재료를

총칭하며 생체 내에서 삽입되어 손상된 조직이나 기관 또는 기능을 치료,

보강 또는 회복시키는데 사용되는 재료를 일컫는다. 이 중 생체금속재료는

손상된 생체 경조직을 대체하기 위한 가장 적합한 특성을 가지고 있어 이

분야에서 널리 사용되고 있다. 이런 생체금속재료 중 Ti 및 그 합금은

우수한 내식성, 비강도를 가지며 특히 다른 금속에 비해 낮은 탄성계수와

생체적합성을 가져 의료용 및 치과용 재료로 널리 사용되고 있다. 그러나

합금원소의 독성에 대한 영향이 논쟁의 대상이 되고 있다. 실제로 금속

이온이나 원소가 생체 내에 유리되었을 때 독성발현 및 신경적 이상을

유발한다는 보고가 되고 있다. 따라서 최근 연구 동향은 상용재들에

첨가되어 있는 독성원소를 배제하며 자연 골과 유사한 탄성계수를 가지는

합금을 개발하는데 초점이 맞추어 지고 있다. 이에 본 연구에서는 새로운

합금계인 Ti-Ag 합금의 특성을 연구하고 생체금속재료로서 적합한 특성을

가지는지 알아보고자 하였다. 합금원소로 택한 Ag 원소는 귀금속 원소에

속하며 기전력이 Ti에 비해 매우 높으며 우수한 마모 저항성과 연성을

가지고 있다. 합금의 조성은 Ag 함량을 1.0 at%부터 5.0 at%까지 1.0

at%씩 증가시켜 총 5가지 조성의 합금을 설계하였다(TA1~TA5). 또한 같은

공정에 의해 제조된 순수 Ti이 대조군으로 사용되었다. Ti-Ag 합금은 아크

용해로를 이용하여 용해하였고 조성의 균질화를 위해 950 의 온도를

유지하는 진공 열처리로에서 72 시간 동안 열처리하였다. 균일한 두께로

제작하기 위해 열간 압연하였고, 950 의 진공 열처리로에서 1 시간 동안

용체화 처리한 후, 상온의 수중에서 냉각하였다. 우선 합금의 상 분석 및

121

미세구조를 관찰하였고, 기계적 특성을 평가하였으며, 생체환경 내에서의

내식성과 전기화학적 특성을 고찰하고자 동전위, 정전위 실험을

시행하였다. 또한 합금의 표면 특성 및 Ag 원소가 표면특성 및 전기화학적

특성에 미치는 영향을 XPS 분석을 통해 알아 보았으며, 임피던스 분석을

통해 합금 표면과 생체용액의 전기화학적 표면 반응에 대해 고찰하였고

표면 저항 및 피막특성을 알아보았다. 마지막으로 Ti-Ag 합금의

생체적합성 및 세포독성을 in vitro, iv vivo 실험을 통해 알아 보았다.

상 분석결과 Ag가 3.0 at% 이상 첨가된 합금에서 β상이 나타났으며,

미세조직결과 Ti 경우는 구상의 α상이 나타났지만 β상이 존재하는

합금에서는 Widmanstätten조직이 관찰 되었고, TA4, TA5합금에서는 Ti2Ag

금속간 화합물이 형성되는 것을 알 수 있었다. 기계적 특성 시험결과,

강도와 경도는 Ag가 첨가됨에 따라 증가하는 경향을 나타내었으나

탄성계수는 합금 별로 큰 차이를 나타내지 않았고, Ag 첨가에 따른 영향은

없는 것으로 나타났다. 동전위, 정전위 실험결과, Ti-Ag 합금이 Ti에 비해

낮은 부동태 전류밀도, 안정된 부동태 구간을 가져 우수한 내식성 및

전기화학적 특성을 가짐을 알 수 있었다. Ag 원소가 3.0 at% 이상 첨가된

TA4, TA5 합금은 Ti2Ag가 형성되어 내식성이 저하되었으나 Ti와 유사한

내식성을 가졌다. XPS를 이용하여 합금의 표면 분석을 수행한 결과,

합금의 표면에 두꺼운 TiO2 부동태 피막이 형성되어 있음을 알 수 있었고,

임피던스 결과 합금의 피막이 축전기적 거동을 보이며 TA2, TA3의 경우

피막 저항성이 Ti에 비해 높은 결과를 얻었다. 금속 이온 용출 실험 결과,

Ag 이온용출량이 침적 기간 및 Ag 원소 함량에 따른 큰 차이점을 보이지

않았고, 원소의 검출한계를 고려해 보았을 때 무시할 수 있을 정도의

용출량이 검출되었다. 세포독성시험 결과 독성치가 none으로 나타났고 MTT

test 결과 모든 합금에서 95%이상의 세포활성도를 보였다. Ti-Ag 합금의

동물실험 결과 모든 동물에게서 실험 기간 중 어떠한 독성 징후도

나타나지 않았고 무게 감량 및 부작용이 나타나지 않았으며 적출된 간,

신장의 조직관찰 결과, 특이한 해부병리학적 소견이 발견되지 않았다.

이상의 실험결과로 Ti-Ag 합금은 Ti에 비해 강도가 높고, 표면에 두껍고

안정된 부동태 산화피막을 형성함으로 인해 생체환경에서 우수한 내식성과

전기화학적 특성을 보임을 알 수 있었다. 그러나 이러한 우수한 내식성 및

전기화학적 특성을 유지하기 위해서는 Ti2Ag가 형성되지 않는 Ag 함량, 즉

122

3.0 at%로 제한되어야 한다고 사료되었다. 생체적합성 측면에서 Ti-Ag

합금은 표면에 형성된 안정된 부동태 산화피막으로 인해 낮은 금속 이온

용출량을 보이며, 첨가원소인 Ag의 영향이 나타나지 않아 순수 Ti와

유사한 우수한 세포독성과 세포활성을 보였다. 결론적으로, Ti-Ag 합금은

생체기능적, 생체적합적인 측면을 고려해 보았을 때 생체재료로 적용될 수

있는 특성을 갖는 재료라고 사료되었다.

핵심되는 말: 급성 전신 독성, 금속 이온 용출, 기계적 특성, 내식성,

부동태 산화피막, 생체기능성, 생체적합성, 세포 독성, 세포 활성도,

전기화학적 특성, 표면 특성, Ti-Ag 합금