Cellular Response of Metallic Materials and Microstructure ...

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Cellular Response of Metallic Materials and Microstructure Cellular Response of Metallic Materials and Microstructure

Entropy Guided Understanding of Strength in Biomedical Entropy Guided Understanding of Strength in Biomedical

Austenitic Stainless Steels Austenitic Stainless Steels

Na Gong University of Texas at El Paso

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CELLULAR RESPONSE OF METALLIC MATERIALS AND MICROSTRUCTURE

ENTROPY GUIDED UNDERSTANDING OF STRENGTH IN BIOMEDICAL

AUSTENITIC STAINLESS STEELS

NA GONG

Doctoral Program in Materials Science and Engineering

APPROVED:

Devesh Misra, Ph.D., Chair

Guikuan Yue, Ph.D.

Srinivasa Rao Singamaneni, Ph.D.

Stephen L. Crites, Jr., Ph.D.

Dean of the Graduate School

Copyright ©

by

Na Gong

Summer 2020

CELLULAR RESPONSE OF METALLIC MATERIALS AND MICROSTRUCTURE

ENTROPY GUIDED UNDERSTANDING OF STRENGTH IN BIOMEDICAL

AUSTENITIC STAINLESS STEELS

by

NA GONG

DISSERTATION

Presented to the Faculty of the Graduate School of

The University of Texas at El Paso

in Partial Fulfillment

of the Requirements

for the Degree of

DOCTOR OF PHILOSOPHY

Materials Science and Engineering

THE UNIVERSITY OF TEXAS AT EL PASO

August 2020

iv

Acknowledgements

I sincerely thank my research advisor and esteemed Professor, Dr. Devesh Misra. His

continuous support and thoughtful insights helped me develop and complete this work, which has

inspired me a lot. His guidance greatly improved my skills in communication, critical thinking and

professionalism.

I would also like to express my heartfelt thanks to Dr. Srinivasa Rao Singamaneni and Dr.

Guikuan Yue, who agreed to serve as members of the dissertation committee and made suggestions

for improvements, which are critical to creating the final form of this research. I also thank Dr.

Veera Krishna Chaitanya Nune, and Dr. Yashwanth Inteti, Ivan Montes, for training me the

operations of machines. I would like to thank all the faculty members of the Department of

Metallurgical, Materials, and Biomedical Engineering for their continuous support and excellent

teaching.

I sincerely thank my beloved father, mother and brother for their spiritual support and

encouragement. Finally, I want to take this opportunity to thank all my relatives and friends who

donated directly or indirectly for successfully complete this dissertation.

v

Abstract

Austenitic stainless steels and cobalt-chrome alloys are used to fabricate biomedical devices with

good mechanical strength, excellent wear and corrosion resistance. Cellular activity of Zr-modified

Co-Cr-Mo alloys and osteoblast functions on Cu-containing and Cu-free austenitic stainless steel

were studied. Experiments on the influence of Zr addition to Co-Cr-Mo alloys and Cu-containing

austenitic stainless steel clearly demonstrated that cell adhesion, proliferation and cell-substrate

interactions were favorably modulated in the presence of Zr and Cu. Additionally, stronger

vinculin focal adhesion contact and signals associated with actin stress fibers together with

extracellular matrix protein, fibronectin, were observed. Furthermore, comparative studies on the

effect of grain size (nanograined/ultrafine-grained- NG/UFG: ~200-400 nm, coarse-grained-CG:

~55±20 µm) indicated higher cell attachment, proliferation and higher expression level of

prominent proteins, fibronection, actin and vinculin on the NG/UFG surface and was in striking

contrast with the CG counterpart. This behavior is attributed to the differences in the fraction of

grain boundaries and physicochemical properties. A concept of a phenomenological parameter,

microstructure entropy, S*, was developed using austenitic stainless steels as an example to

understand the yield strength in alloy systems with bimodal grain size distribution obtained from

large sets of experimental data. Six factors emerged from statistical data analysis in terms of grain

size and grain numbers from 60 sets of data. Microstructure entropy (S*) was obtained from the

bimodal structure data in a self-similar regime. Inverse of the square root of microstructure entropy

( ) and yield strength exhibited a linear relationship. The proposed conceptual methodology

has wide acceptance for any grain size distribution to obtain microstructure entropy (S*) of a

specific grain structure and predict yield strength. A generic equation similar to Hall-Petch

*

1

S

vi

relationship, but involving microstructure entropy is proposed to predict and understand yield

strength of metallic systems characterized by grain size distribution or microstructural evolution.

vii

Table of Contents

Acknowledgements ........................................................................................................................ iv

Abstract ............................................................................................................................................v

Table of Contents .......................................................................................................................... vii

List of Tables ...................................................................................................................................x

List of Figures ................................................................................................................................ xi

Chapter 1: Introduction ....................................................................................................................1

1.1 Biomaterials ...................................................................................................................1

1.1.1 Definition of Biomaterials ....................................................................................1

1.1.2 Properties of Biomaterials.....................................................................................1

1.1.3 Metals as biomaterials...........................................................................................2

1.1.4 Metals in orthopedics ............................................................................................6

1.2 Bone Tissue ..................................................................................................................10

1.3 Tissue-biomaterial interactions ....................................................................................11

1.3.1 Interaction between the biomaterial surface and the tissue ................................12

1.3.2 Interaction between the biomaterial surface and the cell ....................................12

1.3.3 Interaction between the cell and the cell .............................................................14

1.4 Hemocompatibility ......................................................................................................14

1.4.1 Factors affecting hemocompatibility ..................................................................15

1.4.2 Protein adsorption ...............................................................................................16

1.4.3 Surface topography .............................................................................................17

1.5 Methods of severe plastic deformation and formation of nanostructured materials ....17

1.6 Microstructure entropy.................................................................................................28

1.7 Objectives of the study.................................................................................................29

1.8 Contributions of the study ............................................................................................30

Chapter 2: Materials and methods .................................................................................................31

2.1 Materials and processing methods ...............................................................................31

2.1.1 Austenitic stainless steel (without Cu) processing and preparation methods .....31

2.1.2 Austenitic stainless steel (with Cu) processing and preparation methods ..........31

2.1.3 Co-Cr-Mo alloy (with and without Zr) processing and preparation methods ....31

viii

2.1.4 Mechanical property ...........................................................................................32

2.1.5 Metallurgical sample preparation .......................................................................32

2.1.6 Metallurgical materials characterization .............................................................34

2.2 Surface wettability .......................................................................................................36

2.3 Cell culture and cell seeding ........................................................................................36

2.3.1 Biological sample preparation ............................................................................36

2.3.2 Cleaning and sterilization ...................................................................................36

2.3.3 Cell culture ..........................................................................................................37

2.3.4 Cell seeding .........................................................................................................37

2.4 Cell attachment, viability and morphology ..................................................................37

2.5 Immunofluorescence microscopy ................................................................................38

2.6 Analysis of expression level of proteins ......................................................................39

Chapter 3: Favorable Modulation of Osteoblast Cellular Activity on Zr-modified Co-Cr-Mo

Alloy: The Significant Impact of Zirconium on Cell-Substrate Interactions .......................40

3.1 Material characterization .............................................................................................40

3.2 Cell viability and cellular response: MTT assay ..........................................................42

3.3 Immunocytochemistry .................................................................................................43

3.4 Morphology of cells .....................................................................................................47

3.5 Discussion ....................................................................................................................50

3.6 Conclusions ..................................................................................................................52

Chapter 4: Favorable modulation of osteoblast cellular activity on Cu-containing austenitic

stainless steel and comparison with the Cu-free counterpart ................................................54


4.2 Surface wettability .......................................................................................................55

4.3 Cellular response ..........................................................................................................55

4.4 Discussion ....................................................................................................................63

4.5 Conclusions ..................................................................................................................64

Chapter 5: Favorable modulation of osteoblast cellular activity on austenitic stainless steel

with nano/ultrafine grains and comparison with micrometer austenitic grains

counterpart ............................................................................................................................65


5.2 Cell viability and cellular response: MTT assay ..........................................................66

5.3 Morphology of cells .....................................................................................................68

ix

5.4 Immunocytochemistry .................................................................................................69

5.5 Discussion ....................................................................................................................70

5.6 Conclusions ..................................................................................................................72

Chapter 6: Microstructure entropy guided understanding of yield strength in alloy systems

characterized by grain size distribution ................................................................................73

6.1 Microstructure entropy - revisited ...............................................................................73

6.2 Statistical calculations based on microstructure entropy theory ..................................77

6.2.1 Bimodal distribution ...........................................................................................77

6.2.2 Self-similar regime..............................................................................................80

6.2.3 Relative grain volume .........................................................................................80

6.2.4 Microstructure entropy calculations ...................................................................81

6.3 Discussion ....................................................................................................................88

6.4 Conclusions ..................................................................................................................93

Chapter 7: Conclusions and future work .......................................................................................95

7.1 Conclusions ..................................................................................................................95

7.1.1 Favorable Modulation of Osteoblast Cellular Activity on Zr-modified Co-

Cr-Mo Alloy: The Significant Impact of Zirconium on Cell-Substrate

Interactions ..........................................................................................................95

7.1.2 Favorable modulation of osteoblast cellular activity on Cu-containing

austenitic stainless steel and comparison with the Cu-free counterpart .............96

7.1.3 Favorable modulation of osteoblast cellular activity on austenitic stainless

steel with nano/ultrafine grains and comparison with micrometer austenitic

grains counterpart................................................................................................97

7.1.4 Microstructure entropy guided understanding of yield strength in alloy

systems characterized by grain size distribution .................................................97

7.2 Future work ..................................................................................................................98

References ......................................................................................................................................99

Vita ..............................................................................................................................................118

x

List of Tables

Table 1.1: Composition of cobalt-chromium alloys (balance cobalt, wt.%) .................................. 5

Table 1.2: Surface energy and dynamic contact angle measurements for specified surfaces. ..... 16

Table 3.1: Chemical composition of Co-Cr-Mo alloy (mass %) .................................................. 42

Table 3.2: Mechanical properties of the Co-27Cr-6Mo alloy (mass %) with and without Zr ...... 42

Table 6.1: Chemical composition of stainless steels (wt.%) ........................................................ 81

Table 6.2A: Statistical analysis of microstructural entropy factors for experimental steel 1 ....... 82

Table 6.2B: Statistical analysis of microstructural entropy factors for experimental steel 2 ....... 82

Table 6.2C: Statistical analysis of microstructural entropy factors for experimental steel 3 ....... 83

Table 6.2D: Statistical analysis of microstructural entropy factors for experimental steel 4 ....... 83

Table 6.2E: Statistical analysis of microstructural entropy factors for experimental steel 5 ....... 84

Table 6.3A: Experimental yield strength of steel 1 ...................................................................... 84

Table 6.3B: Experimental yield strength of steel 2....................................................................... 84

Table 6.3C: Experimental yield strength of steel 3....................................................................... 85

Table 6.3D: Experimental yield strength of steel 4 ...................................................................... 85

Table 6.3E: Experimental yield strength of steel 5. ...................................................................... 85

Table 6.4A: Microstructure entropy S* of steel 1 ......................................................................... 86

Table 6.4B: Microstructure entropy S* of steel 2 ......................................................................... 86

Table 6.4C: Microstructure entropy S* of steel 3 ......................................................................... 86

Table 6.4D: Microstructure entropy S* of steel 4 ......................................................................... 86

Table 6.4E: Microstructure entropy S* of steel 5 .......................................................................... 87

xi

List of Figures

Figure 1.1: Use of metal alloys as biomaterials. (a) Co-Cr in knee joints, (b) Vitallium in

dentistry........................................................................................................................................... 5

Figure 1.2: (Left) the individual components of a total hip replacement. (Center) the components

merged into an implant. (Right) the implant as it fits into the hip .................................................. 8

Figure 1.3: Knee with femur, tibia and patella ............................................................................... 9

Figure 1.4: Knee replacement surgery ............................................................................................ 9

Figure 1.5: the origins and locations of bone cells ....................................................................... 10

Figure 1.6: Interaction between material surface and biological system ...................................... 11

Figure 1.7: Tissue and cell. Tissue is composed of cells and their environment. Cells are the most

basic unit of life. Cells are made up of many compartments (blue): cell membrane, cytoskeleton,

nucleus, mitochondria, endoplasmic reticulum, Golgi, and lysosome. It interacts with the

environment through molecules (yellow): extracellular matrix, integrins, soluble signals,

receptors, and cell–cell adhesion. Components involved in gene expression (black): DNA, RNA

polymerase, RNA, ribosomes, mRNA, and polypeptide chain .................................................... 13

Figure 1.8: Principles of torsion under high pressure ................................................................... 19

Figure 1.9: Principles of ECAP .................................................................................................... 20

Figure 1.10: Principle of multiple forging: (a), (b), (c) - setting and pull broaching along the first

axis; (d), (e), (f) - setting and pull broaching along the second axis; (g), (h), (i) - setting and pull

broaching along the third axis ....................................................................................................... 21

Figure 1.11: Principle of ARB ...................................................................................................... 22

Figure 1.12: Principle of twist extrusion....................................................................................... 23

Figure 1.13: A schematic representation of phase reversion concept to obtain NG/UFG structure

....................................................................................................................................................... 25

Figure 1.14: EBSD microstructures of NG/UFG specimens annealed at (a) 750°C (b) 800°C (c)

850°C (d) 950°C for 60 s .............................................................................................................. 26

Figure 1.15: TEM microstructures showing NG/UFG in specimens annealed at (a) 800°C, (b)

850°C for 60 s ............................................................................................................................... 27

Figure 1.16: Mechanical properties tested at different annealed temperature .............................. 27

Figure 2.1: Samples are mounted within a moulding compound for ease of handling ................ 33

Figure 2.2: Schematic drawing of a scanning electron beam incident on a solid sample, showing

the signals generated that can be used to help characterize the microstructure ............................ 35

Figure 3.1: Low and high magnification scanning electron micrographs representing

microstructure of Co-Cr-Mo alloy (d, e) with and (a, b) without Zr. (c, f) grain size distribution

plots with an average grain size of 104±27 µm in CCM+Zr alloy and ~53±11 µm in CCM alloy

respectively. .................................................................................................................................. 42

Figure 3.2: Histograms representing cell viability (from MTT assay) for Co-Cr-Mo alloy with

and without Zr. Zr-modified Co-Cr-Mo alloy (CCM +Zr) showed higher viability with

significant difference in comparison to conventional Co-Cr-Mo alloy (CCM) at longer time

periods. Statistical analysis (student’s t-test) was performed using IBM SPSS software, with at

least 3 replicates. (*) indicates 95% confidence level with p<0.05. ............................................. 43

Figure 3.3: The expression of actin cytoskeleton and vinculin focal adhesion proteins of

osteoblast cells cultured on Co-Cr-Mo alloy (c, d) with and (a, b) without Zr after 1 day of

incubation as imaged through fluorescence microscopy. Cell adhesion is more prominent on Zr-

xii

modified Co-Cr-Mo alloy (CCM +Zr) with extensive spreading and high density of focal

adhesion points in comparison to conventional Co-Cr-Mo alloy (CCM). .................................... 44

Figure 3.4: The expression of extracellular fibronectin protein coupled with DAPI-stained

osteoblast cell nuclei on Co-Cr-Mo alloy (c, d) with and (a, b) without Zr after 1 day of

incubation as imaged through fluorescence microscopy. Cell adhesion is more prominent on Zr-

modified Co-Cr-Mo alloy (CCM +Zr) with extensive intercellular contacts and high density of

cell nuclei in comparison to conventional Co-Cr-Mo alloy (CCM). ............................................ 45

Figure 3.5: Histograms representing expression levels of extracellular fibronectin protein (red)

and vinculin focal adhesion points (blue) using NIH Image J software for Co-Cr-Mo alloy with

and without Zr. Statistical analysis (student’s t-test) was performed using IBM SPSS software,

with at least 3 replicates. (*) indicates 95% confidence level with p<0.05. ................................. 46

Figure 3.6: Low-to-high magnification SEM micrographs representing the cell adhesion

morphology of osteoblasts cultured for 2 hours on (d-f) Zr-modified Co-Cr-Mo alloy (CCM+Zr)

and (a-c) conventional Co-Cr-Mo alloy (CCM). Cells were observed to spread with polygonal

sheet-like morphology on both the alloys with and without Zr. However, filopodia-like cellular

extensions were observed to be more on CCM+Zr alloy in comparison to CCM alloy. .............. 48


morphology of osteoblasts cultured for 24 hours on (d-f) Zr-modified Co-Cr-Mo alloy

(CCM+Zr) and (a-c) conventional Co-Cr-Mo alloy (CCM). Cells showed extensive spreading

with polygonal sheet-like morphology on CCM alloy and star-like morphology on CCM+Zr

alloy. High density of cells with multiple filopodia-like cellular extensions forming intercellular

contacts was observed on Zr-modified Co-Cr-Mo alloy (CCM+Zr) in comparison to CCM alloy,

leading to stronger adhesion with the surface. .............................................................................. 50

Figure 3.8: Histograms representing % area covered by the cells (red) and cell density (blue) as

analyzed from the Figures 6-7 area using NIH Image J software for Co-Cr-Mo alloy with and

without Zr. Statistical analysis (student’s t-test) was performed using IBM SPSS software, with

at least 3 replicates. (*) indicates 95% confidence level with p<0.05. ......................................... 50

Figure 4.1: Light micrographs representing microstructure of austenitic stainless steels with and

without Cu (Cu-containing: average grain size of ~22 µm and Cu-free: average grain size of ~55

µm, respectively). ......................................................................................................................... 55

Figure 4.2: Histograms representing cell attachment (a) after 1-4 h culture and cell viability (b)

after 1-7 days via MTT assay for austenitic stainless steels with and without Cu. ...................... 56

Figure 4.3: Low-to-high magnification scanning electron micrographs illustrating cell adhesion

morphology of osteoblasts cultured for 2 h on austenitic stainless steels with and without Cu. .. 57


morphology of osteoblasts cultured for 24 h on austenitic stainless steels with and without Cu. 58

Figure 4.5: Histograms representing % area covered by the cells as analyzed from the Figures 3-

4 area using NIH Image J software for austenitic stainless steels with and without Cu............... 59

Figure 4.6: Histograms representing cell density as analyzed from the Figures 4.3-4.4 area using

NIH Image J software for austenitic stainless steels with and without Cu. .................................. 60

Figure 4.7: Representative actin and vinculin expressed by cells incubated for 24 h on austenitic

stainless steels with and without Cu. ............................................................................................ 61

Figure 4.8: Representative immunocytochemistry of fibronectin protein combined with DAPI-

stained osteoblast cell nuclei expressed by cells incubated for 24 h on austenitic stainless steels

with and without Cu. ..................................................................................................................... 62

xiii


and vinculin focal adhesion points (blue) using NIH Image J software for austenitic stainless

steels with and without Cu. ........................................................................................................... 62

Figure 5.1: Light and TEM micrographs illustrating the microstructure of coarse-grained (CG)

and nanogrianed/ultrafine-grianed (NG/UFG) austenitic stainless steels with an average grain

size of ~55±20 µm and ~200-400 nm, respectively. .................................................................... 66


after 1-7 days via MTT assay for NG/UFG steel in comparison to the conventional CG steel. .. 68

Figure 5.3: Representative fluorescence micrographs of cells stained with nucleic acid-specific

dye after 24 h cell culture for CG and NG/UFG austenitic stainless steels, respectively. ............ 68

Figure 5.4: Scanning electron micrographs illustrating cell adhesion morphology of osteoblasts

cultured for 24 h on CG and NG/UFG austenitic stainless steels. ................................................ 69

Figure 5.5: Representative immunocytochemistry of fibronectin protein expressed by cells

incubated for 24 h on CG and NG/UFG austenitic stainless steels. ............................................. 70

Figure 5.6: Representative actin and vinculin expressed by cells incubated for 24 h on CG and

NG/UFG austenitic stainless steels. .............................................................................................. 71

Figure 6.1: Bimodal distribution for steel 1. (a) 800 °C/10s, (b) 800 °C/60s, (c) 800 °C/1000s. 78

Figure 6.2: Bimodal distribution for steel 1. (a) 800 °C/10s, (b) 800 °C/60s, (c) 800 °C/1000s,

and (d) relative grain size distribution for data presented in (a, b, c). .......................................... 79

Figure 6.3: Hall-Petch relationship for five different steels. ........................................................ 88

Figure 6.4: Relationship between microstructural entropy (S*) and average grain size (davg) for

five different steels. ....................................................................................................................... 89

Figure 6.5: Relationship between microstructural entropy (S*) and experimental yield strength

for five different steels. ................................................................................................................. 91

1

Chapter 1: Introduction

1.1 Biomaterials

1.1.1 Definition of Biomaterials

People’s expectations for biomedical materials change over time, mainly due to the

development of chemistry, materials science and engineering, and biological sciences. In 1976,

Clemson University Advisory Board for Biomaterials proposed one of the earliest formal

definitions of biomaterials as “a systemically and pharmacologically inert substance designed for

implantation within or incorporation with living systems”[1,2]. However, it is thought this

definition is incomplete because bioactive agents or biological entities carried by biomaterials or

biodegradable systems was not considered [2]. Thus, several other biomaterials definitions were

proposed later years. The European Society for Biomaterials (ESB) had the ESB Consensus

Conference I (1999) and defined biomaterial as a “nonviable material used in a medical device,

intended to interact with biological systems”[3,4]. Later in 2005, Consensus Conference II refined

it as “material intended to interface with biological systems to evaluate, treat, augment or replace

any tissue, organ or function of the body” [2]. However, it is thought the definition still lack some

aspects and can be modified as follows: “Biomaterials are substances implanted within or used in

conjunction with the body, designed to have properties closely matching that of the biological

system, be stable enough for the aimed use, have appropriate levels of bioactivity and are designed

to partially or completely fulfill the functions of the diseased, damaged or malfunctioning tissues

and organs” [2].

1.1.2 Properties of Biomaterials

Due to the intended use of biomaterials in very complex environments, it needs to meet

various requirements: (1) Be biocompatible (nontoxic, non-carcinogenic, non-allergenic, etc.); (2)

2

Have physical properties (e.g., density, form, porosity, surface roughness topography) comparable

to those of the tissue it replaces or is implanted in; (3) Have appropriate mechanical properties

(compressive, tensile, shear, impact); (4) Have appropriate service lives (stable for life or degrade

within a matter of days or weeks depending on the goal); (5) Have chemical properties similar to

that of tissues (e.g., hydrophilic or hydrophobic, have similar functional groups); (6) Be

processable and sterilizable without difficulty; (7) Have appropriate bioactivity (mostly inert, but

could have induction or conduction activities or carry bioactive agents if needed); (8) Be

economical and available [2,5].

1.1.3 Metals as biomaterials

There are many types of biological materials used to solve human health problems: (1)

natural materials, (2) synthetic polymers, (3) metals, (4) ceramics, and (5) composites[2,5].

In our subject, we use metals as research objects.

Metals are usually hard, shiny, malleable, and conductive materials. The structure of atoms

in solid metals is usually closely packed and has a crystalline structure, such as body-centered

cubic (bcc), face-centered cubic (fcc) or hexagonal close packed (hcp). Metals and alloys are

combined through metallic bonding. The outer shell electrons of the atom are easily delocalized

and move freely, forming a kind of cloud around the atom. At the same time, the atoms will stay

together with the electrostatic interaction that occurs between them. Cations are easily formed in

metals because metals can easily loosen outer shell electrons which are not firmly bonded to the

entire structure [2,5,6]. Malleability is a very important property of metals because it provides the

property to shape metal into implants. And ductility is another important factor of metals which

refers to the ability to draw out metal to wire, intramedullary rods and screws[2].

3

An alloy is formed via a combination of metals or metals combined with one or more other

elements. The formed alloys usually have enhanced properties than metal with higher hardness,

and more resistant to corrosion or have a more desirable color and luster. Since metals have higher

density and strength compared to polymers, metals and metal alloys are widely used as surgical

and dental instruments, implants, joint replacements. When metals are fabricated as biomedical

device and implant, high biocompatibility, high mechanical strength, high wear resistance, and

high corrosion resistance are required. And with high modulus of elasticity and yield strength,

metals are preferred to produce biomedical devices such as hip or knee joint replacements which

should sustain enough load. And the high strength metals are hard enough which do not deform

easily under certain load [2].

There are several important elements including Fe (iron), Cr (chromium), Co (cobalt), Ni

(nickel), Ti (titanium), Ta (tantalum), Mo (molybdenum), V (vanadium), and W (tungsten),

currently highly used in manufacturing implants in metals or alloys. With the appearance of these

metallic elements, the applications of alloys with improved properties can be extended. Since the

implant in human being is served in an aqueous environment, a low rate of corrosion and relative

inertness is essential. There are two biomedical applications for metallic implant. First, it is used

to replace a part of human body such as joints, long bones. Another application is used as fixation

devices to stabilize the broken bones [2].

Stainless Steel

Stainless steel is one of the most well-known alloys of metals which contains iron mixed

with chromium, nickel, molybdenum, and carbon elements, and also widely used in medical

applications. This strong iron alloy was first introduced to humans for fracture treatments at the

beginning of the 1900s with the application of “Sherman vanadium steel” [2,7]. Stainless steel

4

is most commonly used in implants for repairing fractures, such as orthopedic implants, joint

replacements, surgical and dental instruments, bone screws, pins, rods and coronary stents. The

good corrosion resistance of stainless steel is mainly due to the appearance of iron and other

elements such as chromium (>10.5%), cobalt, molybdenum, and carbon (<1.2%). Among these

elements, Chromium is a very reactive and has good function on preventing oxidation of the

stainless steel. The Chromium elements can promote the formation of an adherent and insoluble

film on the surface which will prevent further oxidation of the iron in the matrix [2].

In austenitic stainless steels, chromium carbides (Cr23C6) will precipitate at grain

boundaries which are more susceptible to corrosion. Thus lower carbon content is preferred which

prevents carbide formation and corrosion. In biomedical filed, low contents of carbon decrease

corrosion amounts which therefore decrease adverse tissue responses and metal allergies [2].

Microbial infections induced by medical implants and surgical instruments can also occur

leading to serious health complications, aggravating the patients to bear more pain and economic

burden [8–12]. Furthermore, the formation of bacterial biofilm on the implant surface induces

tissue infection, which is extremely difficult to be treated with conventional antibiotic therapy,

because bacteria adheres to the surface and sometimes does not respond to antibiotics [13].

To overcome the challenge of microbial infection-induced by microorganisms on

biomedical implants, antimicrobial biomaterials containing copper have been considered in recent

years [14–16]. Copper has long been considered as an antimicrobial element [17]. Cu2+ ions

released from Cu-containing alloys can damage bacteria. Copper can destroy the permeability of

the bacterial membranes, which helps the leakage of reducing sugars and proteins from the cells.

Also, they promote the formation of bacteria-killing reactive oxygen species (ROS). Thus, the

antibacterial role of Cu encourages its potential use in inhibiting bacterial infection [14].

5

Cobalt-Chromium Alloys

Cobalt-chromium (Co-Cr) alloy is a metal alloy of cobalt and chromium. Molybdenum

(Mo) is usually added to refine grain size, resulting in higher strength after casting or forging

process (Table 1.1) [2]. Cobalt-chromium (Co-Cr) alloys have high strength, high temperature

resistance and wear resistance, so they can be used in various joint replacement implants and

fracture repair implants that require a long service life. Also, it can be used in dental orthopedics,

such as knee joints and dental implants (Figure 1.1) [2]. Due to the spontaneous formation of a

protective passivation film mainly Cr2O3, Co-Cr alloy shows high corrosion resistance [18]. The

hardness range of Co-Cr alloy is 550-800 MPa, and the tensile strength range is 145-270 MPa [19].

Table 1.1: Composition of cobalt-chromium alloys (balance cobalt, wt.%).

Sample Cr Mo Ni Fe C Si

Co-Cr alloy 26–30 5–7 1 max 0.5 max 0.35 max 1 max

ASTM F75 27–30 5–7 0.5 max 0.75 max 0.35 max 1 max

Figure 1.1: Use of metal alloys as biomaterials. (a) Co-Cr in knee joints [2,20], (b) Vitallium in

dentistry [2,21].

It has been recently suggested that alloying elements, zirconium, hafnium, titanium, are

biocompatible and they increase the osseointegration ability of materials [22–25]. The beneficial

6

effect is believed to be related to the presence of passive film on the substrate surface [26–28].

Recent studies by co-authors indicated that addition of very small amounts of Zr improved the

bone bonding properties of the 3D printed alloy [29–32]. The studies shows that bone tissue grows

in direct contact with both the Co-Cr-Mo (CCM) alloys with and without Zr, and further improving

the bone anchorage of the CCM implants with low addition of Zr [29]. In another study, addition

of Zr did not alter the microstructure and extracellular matrix composition of bone formed adjacent

to the surface of electron beam melting manufactured implants [30]. Moreover, Zr forms a hard

passive surface oxide layer of ZrO2 that increases resistance to wear and corrosion of Co-Cr alloy.

The approach of cold swaging in conjunction with annealing increased the strength and ductility

of Ni-free Co-Cr-Mo (CCM) alloy [33,34].

1.1.4 Metals in orthopedics

An orthopedic implant is a device used to replace missing joints or bones or to support

damaged bones, involving the musculoskeletal system. Among the most common types of medical

implants, there are pins, rods, screws and plates for fixing fractures. Archaeological evidence

shows that orthopedic surgery was performed in many ancient civilizations. But early surgical

methods tend to amputate rather than repair [6,35]. It was until late 19th century orthopedic surgery

progressed significantly due to the development of antiseptic surgical techniques [6]. A German

surgeon named Thomas Gluck conducted hip reconstruction in ball and socket design using ivory

and other grouting agents [6,36]. In 1923, glass hip joint was introduced by Smith-Peterson, but

this implant was limited by strength and inability to withstand hip pressure [6,37]. The

development of total hip replacement promoted the advancements of materials processing, general

good understanding of physiological conditions, and orthopedic bearing combinations.

Compared with polymers, metals have higher mechanical strength, so metals are usually used to

7

treat hard tissue defects, orthopedics and dental applications. Metals as internal fixing materials

such as plates, screws, pins, rods, or hip and dental implants are common areas. In addition, with

good electrical conductivity, metals are suitable for the production of cardiovascular pacemakers

treatment [2].

Total joint replacements

Total joint arthroplasty (TJA) is a very successful surgical treatment used to treat a variety

of degenerative joint diseases, including osteoarthritis and rheumatoid arthritis. Total joint

replacement is common and very successful in restoring synovial joint function. The most

frequently replaced joints are the hips and knees, followed by the spine, shoulders, elbows and

ankles. The hip joint is the simplest of these joints. It involves a compliant ball and socket joint

with a wide range of motion (including translation and rotation) [6].

Due to the high degree of compliance and cross-shear motion in the hip joint, hip

replacement surgery is subject to relatively low contact stress of 2-5 MPa. Thus, with its relatively

simple anatomical structure (ball socket), the hip joint can rely on joint compliance to provide

most of the stability. While the anatomical structure of the knee is more complex and less

conformable, it needs to rely on the surrounding ligament structure to maintain stability. And the

knee joint involves many motions, such as rolling, sliding, and rotating due to flexion and

extension, and causes the implant to withstand high contact stress of 20-40 MPa [6,38].

American Academy of Orthopedic Surgeons has a great research on orthopedic implant.

Figure 1.2 shows the schematic of total hip replacements. First, the damaged femoral head is

remove and replaced it with a metal stem placed in the hollow center of the femur. The femoral

stem can be consolidated into the bone. Metal or ceramic ball is placed on top of the stem which

8

replaces the damaged femoral head that has been removed. The damaged cartilage surface of the

socket (acetabulum) is remove and replaced with a metal socket, secured by screws or cement [39].

Figure 1.2: (Left) the individual components of a total hip replacement. (Center) the components

merged into an implant. (Right) the implant as it fits into the hip [39].

Total knee arthroplasty

The knee is the largest synovial joint of the human body, as shown in Figure 1.3 with femur,

tibia and patella [40]. The main motion of the knee joint contains flexion and extension in the

sagittal plane with a small amount of rotation. The knee obtains great stability through the large

ligament connecting the femur, tibia and fibula. Due to ligament limitations, the movements of

knee cause a small amount of contact between femoral condyles and tibial plateaus. Compared

with the hips, knee compliance is much worse and the movement of the knee is also more

complicated, including rolling, sliding and some rotation caused by the flexion and extension of

the joint. For most people, knee replacement surgery (Figure 1.4) can relieve pain, improve

9

mobility, and improve quality of life. And most knee replacement surgery is expected to last more

than 15 years [41].

Figure 1.3: Knee with femur, tibia and patella [40].

Figure 1.4: Knee replacement surgery [41].

10

1.2 Bone Tissue

Bone is a special form of connective tissue that can be used both as tissue in higher

vertebrates and as an organ system. Its basic functions include exercise, protection and mineral

balance. The skeletal features are (spongy, trabecular) cancellous or compact cortical.

Functionally, cancellous bone is more closely related to metabolic capacity, and cortical bone

provides greater mechanical strength [42].

The four cellular elements of bone are: osteoblasts, which build bone; osteocytes, which

maintain function; bone lining cells which regulate passage of calcium into and out of the bone;

and osteoclasts which degrade bone [5,42–44]. A simple cell taxonomy consisting of bone-forming

cells and bone-resorbing cells has been proposed based on their source [45]. Osteoblasts,

osteocytes and bone lining cells originate from mesenchymal stem cells and are therefore called

osteoprogenitor cells, while osteoclasts originate from hematopoietic stem cells. The location of

these cells is also different. Bone cells found along the bone surface include osteoblasts, osteoclasts

and bone lining cells, and osteocytes are located inside the bone (Figure 1.5) [42,44,46].

Figure 1.5: the origins and locations of bone cells [42].

11

1.3 Tissue-biomaterial interactions

After a foreign body is implanted in the body, the tissue responds to this substance by

displaying an allergic, toxic, or carcinogenic reaction. At the same time, the tissue exerts various

effects on the material, such as corrosion, degradation, or deterioration. For more than 100 years,

it is clear that living cells interact and attach to material surface in different ways. The nature of

this interaction may have a huge impact on cell fate, including attachment, spreading, proliferation,

differentiation, and detachment (Figure 1.6). It is also recognized protein membranes that are

always adsorbed before the cell interacts with the surface guide and regulate the cellular response.

Thus, tissue-material interactions is critical for proper implant performance, because it determines

whether the implant is successful [2,5].

Figure 1.6: Interaction between material surface and biological system [2].

12

1.3.1 Interaction between the biomaterial surface and the tissue

Tissue is a group of cells associated with their microenvironment, which together perform

their functions. Four main types of tissue are concluded: muscle, epithelial, connective tissue and

nerve tissue. Muscle tissue generates contractile force. Epithelial tissue contains tightly packed

cells. Connective tissue contains cells wrapped by extracellular matrix (ECM). Nerve tissue

contains nerve cells that conduct nerve impulses [47].

The key parameters of the biomaterial surface are chemical properties, topography, and

physical properties. And these key parameters as well as mechanical properties of biomaterial are

the main properties of an implant that determine its biocompatibility. The first reaction between

the biomaterial surface and the tissue involves proteins and cells. If the topography, chemical

properties and mechanical properties (such as stiffness) are suitable for these events, they will

attach or adsorb on the surface. The cells in adjacent tissues begin to proliferate, differentiate, and

produce their extracellular matrix. If the interaction is unfavorable, the cells are not attached or

apoptosis may occur [2].

1.3.2 Interaction between the biomaterial surface and the cell

Cells are the most basic part of life. Millions of cells make up an organism. There are a lot

of different types of cells with different shape, distribution, and function. Cells respond to signals

from the environment. These signals may appear in the form of ECMs surrounding the cells,

soluble signals or physical interactions with neighboring cells (Figure 1.7) [47]. Cells are

functional units that detect and process signals from the environment and respond to environmental

prompts. The unit is composed of many compartments that work together to make the unit a

responsive functional unit. In the form of physical or chemical stimulation, the cell responds

locally to local stimulation outside the cell, and converts the local signal into a series of

13

intracellular signals, which are amplified and transported to a decision center, such as the nucleus.

The decision center will integrate all the signals generated by different stimuli from different parts

of the cell during a specific period to determine a series of actions [47].

Figure 1.7: Tissue and cell. Tissue is composed of cells and their environment. Cells are the most

basic unit of life. Cells are made up of many compartments (blue): cell membrane,

cytoskeleton, nucleus, mitochondria, endoplasmic reticulum, Golgi, and lysosome.

It interacts with the environment through molecules (yellow): extracellular matrix,

integrins, soluble signals, receptors, and cell–cell adhesion. Components involved

in gene expression (black): DNA, RNA polymerase, RNA, ribosomes, mRNA, and

polypeptide chain [47].

Cell attachment

Cell attachment is the first step in the interaction between cells and biological materials

and is important for cellular processes, such as cell proliferation and differentiation [48]. It is

usually that the cell proliferation of adherent cells can only occur on the surface of material. Thus,

cell-surface interactions are the basis for understanding cell behavior [5].

Spreading and migration

During the attachment process, the cells will adapt to the topography and geometry of the

surface, thereby changing to a variety of morphologies, which will affect the spreading and

migration of cells [49].

14

Metabolic activity and proliferation

Anchorage dependent cells such as osteogenic cells will not grow unless they are attached

to a surface and show changes in their metabolic activity due to inability to adhere properly or

excessively adhere to the surface. Among these activities, there are some important activities, such

as proliferation [2].

1.3.3 Interaction between the cell and the cell

Cell-cell interactions sometimes are relatively stable, such as interactions mediated by tight

junctions, adhesion junctions, desmosomes and gap junctions. But sometimes they are transient,

such as cell adhesion molecules (CAM) of the immunoglobulin and selectin family. Cell-cell

contact is very important for maintaining the structural integrity of tissues, polarization and cell

proliferation. Cell-cell interaction is very important for synchronizing responses at the tissue level

and forming communication channels between cells that do not rely on external signals. Most of

the inner cell parts of the human body that are in contact with the external environment require

strong cell-cell interaction to form a barrier. The barrier formed by the tight cell-cell connection

prevents external molecules from infiltrating the body. For many cell types, cell-cell interactions

are essential for cell survival and function. Therefore, biomaterials are designed to allow and

encourage cell-cell interactions in macroporous scaffolds or flexible nanofibers to support desired

cell and tissue functions in applications [47].

1.4 Hemocompatibility

Hemocompatibility is a specific and advanced state of biocompatibility, which is

particularly important for biological materials connected to blood. Any biomaterial proven to be

biocompatible may not necessarily be hemocompatible, but the hemocompatible material must be

biocompatible [2].

15

1.4.1 Factors affecting hemocompatibility

Hydrogen bonding

Self-assembled monolayers (SAMs) of alkanethiols with various terminating groups (such

as hydroxide, methyl and carboxyl groups (-OH, -CH3, -COOH) and their binary mixtures) was

performed in human body via in-situ testing. The results show that the adhesion of leukocytes on

the methylated (hydrophobic) surface decreases and is enhanced by hydroxylated (-OH) groups.

A very important aspect of hemocompatibility is to prevent platelets from adhesion and find that

the hydrophilic surface is better [2,50].

Surface energy and wettability

The surface energy of biological materials is characterized by general charge density and

net polarity of charge, and because cells can distinguish different surface chemistry, they can

profoundly change cell attachment and proliferation [51]. The arrangement of charges and

structures on the surface determines the ability of certain ions, proteins, minerals, biomolecules,

and cells to interact with biomaterials and thereby participate in defining biological reactions.

Surface chemistry will first alter binding orientations of cells, change in cell attachment through

cell adhesion molecules, then transform in cell shape, and ultimately cause variations in cell

behavior [49,51,52]. Table 1.2 shows the surface energy and contact angle for surface with

different chemical groups [53].

The change in molecular chemical conformation or polarity of biomaterial has great

influence on the hydrophobicity or hydrophilicity of a surface and thus modify the surface

chemistry and wettability and affect protein adsorption and cell-material interactions

[49,52,54,55]. Surface wettability plays a decisive role in cell adhesion. And it is found that 40°–

60° moderate surface wettability is suitable for cell adhesion [49,56].

16

Table 1.2: Surface energy and dynamic contact angle measurements for specified surfaces

[49,53].

Material Surface energy (mJ/m2) Contact angle

TAAB 53.39 62.16 ± 2.33

—OH 52.89 63.06 ± 2.01

—COOH 49.51 68.89 ± 4

—NH2 43.52 78.71 ± 5.69

—SH 39.6 84.96 ± 4.11

—CH3 35.53 91.36 ± 1.12

1.4.2 Protein adsorption

It is pointed out that the interaction between blood and foreign body surface starts with the

adsorption of plasma proteins, which will affect the subsequent blood-substance interaction. The

adsorption of fibrinogen in plasma plays a central role in blood coagulation, especially fibrinogen

is reported to be the main protein involved in platelet adhesion. The hydrophobic surface shows

more protein adsorption, and the affinity of fibrinogen to the methyl (hydrophobic) surface is

higher than the hydroxyl (hydrophilic) surface, which indicates that the hydrophilic surface shows

higher hemocompatibility [2,57].

The adsorption of protein indicates whether the surface is compatible with blood. Studies

have shown that the number, type, and conformation of proteins play an important role in the

interaction with platelets. For example, the conformation of fibrinogen changes after adsorption

which can bind and activate platelets. Thus the changed conformation of proteins is very important

in platelet adhesion [2].

Cell adhesion is the first phase of cell-biomaterial interactions with protein adsorption, cell-

material contact, attachment, and spreading, which have influence on subsequent cellular behavior

17

(proliferation, differentiation, and apoptosis) [58–60]. In the case of most anchor-dependent cells,

the adhesion of cells to the surface of the material is achieved by the formation of intracellular

adhesion spots [61]. Focal adhesion is a large and strong molecular complex composed of

structural proteins (including vinculin, talin, andα-actin) and signaling molecules (such as focal

adhesion kinase (FAK) and paxillin) [60,62]. Integrins are heterodimeric transmembrane

receptors, composed of α- and β-subunits. Integrins can bind to specific amino acid sequences

through their extracellular domains, such as arginine-glycine-aspartic acid (RGD) motif in

extracellular matrix (ECM) proteins (such as collagen, fibronectin, vitronectin, fibrinogen,

laminin, osteopontin, and bone sialoprotein) [60,63–65].

1.4.3 Surface topography

Surface topography plays an important role in hemocompatibility. In the design of blood

vessels, surface roughness is undesirable because any protrusions or depressions can interfere with

the flow of blood and its elements, causing them to accumulate, which may eventually cause blood

clotting. Thus, smoothness is not satisfactory for hemocompatibility. But when there are voids in

blood vessels, they may attract blood proteins to accumulate in the pores and fill them, resulting

in a very smooth and compatible surface. Therefore, the surface topography is also an important

parameter of the hemocompatibility of the material. At the same time, the surface roughness does

not cause changes in normal clotting time and thrombosis, and only exhibits minimal inflammation

[2].

1.5 Methods of severe plastic deformation and formation of nanostructured materials

It is difficult to obtain grains with nanometer (<100 nm) or sub-micrometer (<500 nm)

sizes using conventional thermomechanical treatment. Since traditional methods of severe plastic

deformation (SPD), such as rolling, drawing or extrusion cannot meet the following requirements

18

to process nanostructured materials. These are the following requirements. First, it is important to

obtain an ultrafine grain (UFG) structure with a generally high-angle grain boundary, because only

in this case can qualitative changes in material properties occur. Second, the formation of a uniform

nanostructure throughout the sample volume is necessary to provide stable processing material

properties. Third, although the samples are exposed to large plastic deformation, they should not

have any mechanical damage or cracks. At present, most of the results obtained are related to the

application of SPD methods: high-pressure torsion (HPT) process [66–75], equal channel angular

pressing [76–79], multiple forging [80–85], accumulative roll bonding (ARB) [91–93], and twist

extrusion [94]. .

High-pressure torsion (HPT) process

In SPD technology, the HPT process is one of the most powerful technologies for preparing

UFG materials, due to its uneven deformation and large strain gradient. Successful formation of

uniform nanostructures with high-angle grain boundaries through high-pressure torsion were

obtained [66,72,74]. This is a very important step, so that people can think of this procedure as a

new method for processing nanostructured materials from the aspect of severe torsion straining.

Torsional strain method under high pressure can be used to make disc-shaped samples (Figure

1.8). The ingot is fixed between the anvils and twisted and deformed under an applied pressure of

a few GPa (P in Figure 1.8). And the lower bracket rotates where a friction at the surface generates

that will deform the ingot by shearing. The disk used in traditional HPT technology is used for

small volume nanomagnets with enhanced hard and soft magnetic properties, arterial stents and

devices for microelectromechanical systems (MEMS) applications [86].

19

Figure 1.8: Principles of torsion under high pressure [66].

ECAP

Equal-Channel Angular Pressing (ECAP) is a common processing technology for

preparing ultrafine grain metallic materials. The preparation process is as follows [76–79]: the

material passes through a curved channel with a constant cross-sectional area under extrusion,

which causes the material to undergo pure shear deformation processing at the corner of the equal

channel; and through repeated operations to obtain strong strain processing, and finally ultrafine

grain structure can be obtained. The principle of the ECAP device is shown in Figure 1.9. At

present, ECAP research is mainly focused on nonferrous metals such as aluminum and copper; for

steel materials with large deformation resistance, ECAP research is still limited to industrial pure

iron and low carbon steel [87].

It is reported that ECAP method was used to conduct a study on the production of ultrafine

grain in 0.15C-1.1Mn low carbon steel [88]. The test results show that if the effective strain of

each pass is 1, and the sample is rotated 180° along the axis between each pass, the ferrite grain

size can reach 0.2~0.3 μm after 350°C and 4 passes of extrusion [88]. In addition, Park et al. [39]

20

also used the ECAP to process ultra-low carbon steel, obtaining ultrafine ferrite grains with a size

of 0.2~0.5μm [89].

Figure 1.9: Principles of ECAP [66].

Multiple forging

Another method of forming nanostructures in large blocks is multiple forging [82–85]. The

process of multiple forging is usually related to dynamic recrystallization. The principle of

multiple forging (Figure 1.10) are as follows. It is assumed that the free forging operation is

repeated many times, thus the drawing of the sitting graph is made by changing the axis of the

applied strain load. The uniformity of strain in multiple forging is relatively low compared with

HPT and ECAP technologies, so this method allows one to obtain a nanostructured state in a fairly

brittle material because the processing is started at elevated temperature and the specific load on

the tool is low [67]. The multiple forging method is used to refine the structure of various alloys,

including pure titanium [80], titanium alloy VT8 [66,83], Ti-6%Al-32%Mo [80,82], magnesium

alloy Mg-6%Zr [82], etc. The given method is usually implemented in the plastic deformation

temperature range of 0.1-0.5 Tml (Tml is the melting temperature) [66].

21

Figure 1.10: Principle of multiple forging: (a), (b), (c) - setting and pull broaching along the first

axis; (d), (e), (f) - setting and pull broaching along the second axis; (g), (h), (i) -

setting and pull broaching along the third axis [90].

Accumulative roll bonding (ARB)

ARB is first invented in 1999 and is a solid-state multi-stage process, including surface

treatment, cutting, stacking, rolling, and sometimes post-rolling heat treatment, to improve the

bonding quality of the laminate [91]. The traditional rolling technology is almost similar to the

ARB technology, in which the thickness of the material is mechanically condensed by rolling to

half the thickness of the as-received material [92]. Figure 1.11 schematically shows the process of

stacking two or more sheets together and passing through rollers to apply plastic deformation. The

deformation must be sufficient to produce a solid-state bond [93]. In order to achieve good bonding

and smooth rolling, before starting the process, the rollers are degreased and brushed, and then the

sheet of material is rolled to half the original thickness. By repeating the sequence of rolling,

22

cutting, and brushing, large strains in the material can be successfully achieved. The material can

be heated during rolling, but it should be kept below the recrystallization temperature [93].

Figure 1.11: Principle of ARB [93].

In the ARB process, there are two possible mechanisms, which may be different from other

severe straining methods. One of the most important mechanisms is the severe shear deformation

under the surface and its effects. Another method is to start a new interface, and then show the

perfect fiber structure. The bonding quality is greatly affected by the ARB parameters and the

underlying mechanism. Research has been conducted to find out the bonding mechanism during

the roll bonding process. Four main theories have been proposed to explain the binding

mechanism: movie theory: film theory; energy barrier; recrystallization; diffusion bonding [93].

And it is believed that ARB is an emerging field with huge opportunities in the industrial field and

high potential to customize microstructures for high-performance materials [93].

23

Twist extrusion

In 1999, Yan Beygelzimer proposed a severe plastic deformation process that became

known as Twist Extrusion (TE) [94]. This process can change the structure of the material, thereby

significantly improving some of its physical and mechanical properties (in some cases, new

properties can also be obtained). The principles of this process is illustrated in Figure 1.12. TE

works by extruding a prism sample through a matrix twisted at a desired angle whose outline

consists of two prismatic regions separated by twisted channels. The extruded material undergoes

severe deformation, and the characteristic of final cross-section of the sample is the same as the

initial cross-section. The twist angle is along its longitudinal axis, so it can recover enough size

and shape after each pass. Therefore, this process can be repeated multiple times to achieve

excellent grain refinement [94].

Figure 1.12: Principle of twist extrusion [94].

24

Phase Reversion Process

Through the processing of nanostructured materials, a variety of severe plastic deformation

techniques are used to successfully achieve the goal of obtaining high strength through grain

refinement. The excellent strength of the material can be obtained by reducing the ductility. The

low ductility of nanoparticle materials is believed to be the accumulation of high dislocation

density and the instability in material [95–98]. A novel phase-reversion concept (Figure 1.13) was

developed to obtain nanograined/ultrafine grained (NG/UFG) stainless steel with good

combination of high strength and high ductility [99,100]. Furthermore, the nanocrystallized

surface was observed to reduce the interaction between the material surface and the micro-

organism, thereby inhibiting the adherence of bacteria and biofilm on the surface [101–104].

Studies have shown that this kind of reversion treatment consists of the reversion

transformation of strain-induced martensite to austenite to obtain NG/UFG structures in austenitic

stainless steels [100,101,105–111]. Specifically, cold deformation plays an important role in

transforming austenite to martensite. Furthermore, the greater the degree of cold deformation,

the higher the martensite volume fraction becomes within limits. Two types of martensite, lath-

type martensite and dislocation cell-type martensite, have been proposed for a good classification

of the martensite. It is thought that martensite provides suitable sites for austenite nucleation during

annealing treatment at relatively low temperatures and short times, leading to grains with

nanometer and ultrafine sizes. Also, it is considered that the reversion process through shear

reversion or diffusional reversion largely depends on the chemical composition of the material and

its annealing temperature [99–101,109–112]. In this case, grains are generated by the recovery and

recrystallization process, forming equiaxed austenite grains or grains with defects, which is based

mostly on annealing treatment [101].

25

Figure 1.13: A schematic representation of phase reversion concept to obtain NG/UFG structure

[96].

It is proposed [101] a nano/ultrafine-grained (NG/UFG) structure was obtained by 80%

heavy cold deformation, followed by annealing in the 700-950 °C range for 60 s in austenitic

stainless steel, as shown in Figure 1.14. The results showed that the martensite was reversibly

transformed to austenite, with the accumulation of twins, dislocations, and subgrain boundaries,

as shown in TEM Figure 1.15. At 700°C, the microstructure exhibited low elongation and

consisted of 65% austenite. Over 750 °C, the amount of reversed austenite was nearly 100% [101].

26

Figure 1.14: EBSD microstructures of NG/UFG specimens annealed at (a) 750°C (b) 800°C (c)

850°C (d) 950°C for 60 s [101].

As for the mechanical properties of NG/UFG stainless steel, the tensile strength of the

sample decreased slightly whereas the elongation increased further, showing good combination of

good strength and good ductility in annealing temperature range of 750-850 °C (Figure 1.16). As

shown in Figure 16 in the annealing temperature range of 750-870°C, the tensile strength decreased

slightly, whereas the elongation to failure increased further. It is suggested that grain refinement

with the NG/UFG structure leads to a good combination of strength and toughness. Such co-

dependent strengthening and toughening can be attributed to the NG/UFG structure, which

27

decreased the nucleating defects and increased the capacity for resisting crack propagation.

Therefore, the structure exhibits a higher fracture stress [99,101].

Figure 1.15: TEM microstructures showing NG/UFG in specimens annealed at (a) 800°C, (b)

850°C for 60 s [101].

Figure 1.16: Mechanical properties tested at different annealed temperature [101].

28

1.6 Microstructure entropy

Entropy indicates system's disorder and a property that describes the thermodynamic state

of the system. In a closed thermodynamic system, it quantitatively defines the amount of energy

that is not available in the system. While discussing entropy, we generally think of configurational

entropy, which is related to discrete representative positions of its constituent particle, and follows

Boltzmann's entropy equation [113]. In 2008, a term “microstructure entropy” was proposed,

which is different from configurational entropy that is used in classical statistical mechanics for

ergodic Hamiltonian systems, while microstructure entropy corresponds to non-ergodic degrees of

freedom [114]. Microstructure entropy is viewed as an important thermodynamic parameter in the

phenomenological modeling of the behavior of random structures. The mathematical modeling of

a random structure is based on the construction of its probabilistic measure, so the entropy of

microstructure is based on the analysis of homogenization problem and selected probabilistic

measure [114]. More recently, thermodynamics of microstructure evolution for grain growth based

on microstructure entropy has been proposed [115]. It was envisaged that the macroscopic

description of microstructure evolution requires additional thermodynamic parameters, entropy of

microstructure and temperature of microstructure, leading to an additional proposition which states

that the entropy of microstructure must decay in isolated thermodynamically stable systems

[115,116]. In this regard, grain growth in polycrystalline solid was explored, which was a

modification of Hillert theory [117]. It was observed that the decay of total microstructure entropy

occurs when the system approaches the self-similar regime. The microstructure entropy per grain

was increased indicating chaos of grain size. The equations of grain boundary microstructure that

are related to the entropy of microstructure, energy of microstructure, average grain size and

29

indicative of inhomogeneity in terms of large grain size distribution [115] are discussed in Chapter

6.

1.7 Objectives of the study

There are four objectives of the study.

1. Ni-free Co-28Cr-6Mo (wt.%) alloys with Zr (CCM+0.04Zr) and without Zr were use

to explore the cellular activity, and elucidate the impact of Zr on osseointegration. It is

envisaged that the study will address the concern of Co-Cr-Mo alloys in regard to bone-

formation, and thereby contribute to further advancement and usage of their alloys.

2. Austenitic stainless steel with Cu and without Cu were used to explore the cellular

response. Cellular activity of antimicrobial activity of copper-containing steel has been

studied compared to austenitic stainless steel without Cu.

3. Biological/cellular functions on nanograined/ultrafine-grained (NG/UFG) (grain size

in the nanometer regime: ~200-400 nm) biomedical austenitic stainless steel were

studied compared with the conventional coarse-grained (grain size in the micrometer

range: ~55±20 µm) counterpart.

4. A large number of sets of grain size distribution data were obtained through the

adaptation of novel phase reversion approach involving severe cold deformation of

metastable austenite in stainless steel to martensite, followed by annealing for short

durations when martensite reverts to austenite via diffusional or shear mechanism

[118,119]. The objective of the phase reversion concept was to obtain ultrafine grain

size. The variation in grain size occurs when austenite is not completely transformed

to martensite during severe cold deformation and/or when annealing times are too short

for martensite to completely revert to austenite. In the study descried here, we apply

30

grain growth dynamics to 60 sets of bimodal structure for five different austenitic

stainless steel of chemical composition in a self-similar regime based on the

thermodynamics of microstructure evolution (grain growth) and embracing

microstructure entropy. The different grain size distribution was used to understand the

variation in microstructure entropy in the alloy system with bimodal grain size

distribution. The ultimate objective was to develop a relationship between

microstructure entropy and Hall-Petch equation, which can be successfully used to

predict yield strength.

1.8 Contributions of the study

Austenitic stainless steel with and without Cu including nano/ultrafine grain and coarse

grain and Co-Cr-Mo alloy with and without Zr were used to find the interplay between grain

structure, chemical elements and osteoblast functions on cell-substrate interactions. The presence

of Zr in Co-Cr-Mo alloys and Cu ions in austenitic stainless steel indicated favorable cellular

response of osteoblasts is a step in the right direction to promote their application for the fabrication

of biomedical devices.

Secondly, we apply grain growth dynamics to 60 sets of bimodal structure for five different

austenitic stainless steel of chemical composition in a self-similar regime based on the

thermodynamics of microstructure evolution (grain growth) and embracing microstructure

entropy. The different grain size distribution was used to understand the variation in microstructure

entropy in the alloy system with bimodal grain size distribution. The ultimate objective was to

develop a relationship between microstructure entropy and Hall-Petch equation, which can be

successfully used to predict yield strength.

31

Chapter 2: Materials and methods

2.1 Materials and processing methods

The experimental materials are commercial biomedical grade of austenitic stainless steel

18Cr-8Ni (without Cu) with nominal chemical composition of (wt.%) Fe-0.04C-1.52Mn-17.8Cr-

8.1Ni-0.005P-0.005S, austenitic stainless steel (containing Cu) with chemical composition of

(wt.%) Fe-0.023C-0.85Mn-17.40Cr-7.32Ni-3.15Cu-0.0025P-0.0011S, and Ni-free Co–28Cr–

6Mo–0.14N−0.05C (wt.%) alloy with and without Zr.

2.1.1 Austenitic stainless steel (without Cu) processing and preparation methods

Phase Reversion: To obtain the NG/UFG structure without Cu, the solution-treated

(1050 °C for 10-15 min) steel was subjected to severe cold reduction (90% reduction in thickness

to ~0.8 mm) via multiple passes. Subsequently, the strips were cut to dimensions of 0.8 mm × 70

mm × 210 mm and annealed at a temperature of 800 °C for 10 s, when the cold rolled martensite

reverts to NG/UFG austenite via diffusional reversion mechanism[100,111,112,120,121].

2.1.2 Austenitic stainless steel (with Cu) processing and preparation methods

The austenitic stainless steel with Cu was made in a laboratory using standard melting

practice. The steel was received in the form of a hot rolled sheet with about 3 mm in thickness for

cold rolling. The as-received steel sheet was then cold rolled to 1 mm thickness with 66.7%

reduction and subsequently annealed at 950 °C for 100 s to obtain the CG counterpart. The

annealing process was conducted in a Gleeble 3800 thermo-mechanical simulator. The final grain

size of CG experimental steel (950 °C for 100 s) was similar to the as-received steel.

2.1.3 Co-Cr-Mo alloy (with and without Zr) processing and preparation methods

Ni-free Co-28Cr-6Mo (wt.%) alloys with Zr (CCM+0.04Zr) and without Zr were

processed in an argon-containing environment utilizing using an induction furnace. Ingots (150

32

mm dia.) of the alloys were heat treated at 1473 K for 3 h and hot forged. The forged bars were

hot swaged to produce the rods with approximately 13 mm in diameter, followed by annealing at

1423 K for 1 h and air cooling. Samples were cut from the rod specimens with diameters of ~13

mm after annealing [34].

2.1.4 Mechanical property

Tensile test: the mechanical properties of steels were determined via CMT5605 tensile

machine with a standard corresponding to 50 mm gage length at room temperature.

Micro-hardness: Vickers micro-hardness was measured using a load of 19.6 N (2 kgf)

and hold time of 15 s. Average hardness was obtained from the measurement at ten different

locations.

2.1.5 Metallurgical sample preparation

Sampling and sectioning: It is usually necessary to check the vertical and parallel parts

of the component which are on two important axes of symmetry. In the case of a rolled sheet, a

section perpendicular to three main directions is desirable: the rolling direction, the transverse

direction, and the through-thickness direction [122]. And the specific size is required for certain

materials to be examined using various microscopes or analyzed by a variety of experimental

equipment. Based on the required size for experimental instruments, the metallographic samples

are sectioned.

Mounting and grinding: In order to facilitate surface preparation, many samples need to

be mounted for easy handling. Polymer resin or moulding compound is the most common form of

sample holder, which can be die cast or hot pressed around the sample without distorting the

sample or damaging the microstructure (Figure 2.1) [122].

33

Figure 2.1: Samples are mounted within a moulding compound for ease of handling [122].

After the sample is firmly mounted, the section can be ground flat and polished. Rough

grinding requires special care because it can easily remove too much material, overheating the

sample or causing mechanical or thermal damage to the surface. Grinding is actually a mechanical

process in which the sharp edges of the grinding media are cut parallel to the surface of the sample.

The direction of grinding may be important. For example, it is generally undesirable to grind the

area near the free surface perpendicular to the free surface, because cutting particles will almost

certainly cause extensive sub-surface damage when biting into the edge of the sample. Cutting in

the opposite direction to allow grit particles to escape from the free surface during grinding will

greatly reduce damage. The degree of sub-surface damage depends on the elastic stiffness and

hardness of the material, so soft metals are extremely difficult to grind. However, brittle materials

are prone to sub-surface cracks. It is important to recognize that sub-surface damage is always

caused during the grinding process, and to ensure that subsequent polishing can completely remove

the damaged layer [122]. The experimental specimens were mechanically ground using a series of

SiC papers (200~2000#).

Polishing and etching: the main purpose of polishing is to prepare a surface that is both

flat and free of topographic features that are not related to the bulk microstructure of the sample.

Each polishing stage is designed to remove a layer of damaged material generated in the previous

34

stage of surface preparation. There are three accepted methods of polishing samples: mechanical,

chemical, and electrochemical. Among these three, mechanical polishing is the most important. In

mechanical polishing, by using increasingly finer grit sizes, mechanical damage in the early stages

of preparation can be eliminated. The carrier of the polishing grit is usually the cloth polishing

wheel (which may be reduced to 1/4 mm diamond grit) [122].

Etching of samples refers to the selective removal of material from the surface to form

surface features related to the microstructure of the bulk material. Most etching methods involve

some form of chemical attack, which is more pronounced in surface areas with higher energy (for

example, grain boundaries). The most common etching procedure uses chemically active solutions

to chemically etch the surface and form a topology visible under the microscope. In most cases,

the sample is immersed in the solution at a carefully controlled temperature for a given period of

time, then thoroughly rinsed and dried (usually alcohol) [122]. After polishing the samples were

chemically etched with a solution containing nitric acid and hydrochloric acid (HNO3:HCl=1:3)

for 1-2 minutes.

2.1.6 Metallurgical materials characterization

Optical microscopy: optical microscopes use light rays to characterize materials. The

surface of the sample is polished to reflect light from its surface. The light rays from the mirror

finished sample will be observed at a higher magnification. The image reflected on the screen

depends on the illumination of the light, the position and characteristics of the sample. The position

of the sample should be kept perpendicular to the axis of the objective lens. In this way, the

reflected light will travel back to the lens, and then the digital image is generated as a micrograph.

Scanning electron microscopy (SEM): electron microscopes use electrons as the main

source of imaging. Electron microscopes are often used to obtain detailed information from

35

samples that cannot be observed with light/optical microscopes. The most important requirements

of electron microscopy are high resolution, large depth of field and high magnification.

Figure 2.2: Schematic drawing of a scanning electron beam incident on a solid sample, showing

the signals generated that can be used to help characterize the microstructure [122].

Generally, there are two types of scanning electron microscopes, (a) thermionic electron

microscope and (b) field emission microscope. Tungsten filament is used as the electron source of

thermionic electron microscope and field emission microscope, while the average filament life of

field emission SEM is better than that of thermionic electron SEM. The principle of SEM is that

when the incident beam hits the sample, the sample will emit secondary electrons, backscattered

electrons, X-rays and auger electrons. These electrons can be collected by the detector and

displayed on the computer screen (Figure 2.2) [122].

The structure of alloys was examined by SEM (JEOL JEM-7100F) at 15 kV after

mechanically polishing the alloys. Grain size was determined using linear intercept method.

36

Transmission electron microscopy (TEM): TEM uses a high-energy electron beam to

image the sample. The electron beam will interact with the thin foil, and the image will be projected

on the fluorescent screen. The high voltage is sent through the emission source and the filament is

heated, causing the electrons to be released. The emitted electrons have enough kinetic energy to

pass through the thin foil and project the image with high resolution. For TEM of NG/UFG steel,

the steel was metallographically ground to 50 µm thickness and 3 mm diameter disks were

punched. These disks referred as foils were electropolished in an electrolyte with 10% perchloric

acid and 90% ethanol at 25 V for 30 s.

2.2 Surface wettability

Surface wettability experiment was measured in terms of contact angle using distilled water

on steel or alloy surfaces. The experiments were repeated at least 3 times so that we can obtain

mean value and standard deviation (SD) of the hydrophilicity of samples.

2.3 Cell culture and cell seeding

2.3.1 Biological sample preparation

When preparing biological samples, it is best to do polishing in a dust-free environment,

because contaminants in the air are usually not tolerated. Cleaning between polishing stages is

critical, because residue is a big problem. Residues may come from the polishing stage or the

operator's hand contamination. Therefore, the polished surface must be thoroughly washed under

running water and wiped with a cotton cloth, and then in some cases similarly treated with alcohol

[5].

2.3.2 Cleaning and sterilization

The polished specimens were cleaned with ethanol, disinfected and sterilized. Briefly,

specimens were sonicated in an ultrasonic container with 2% RBS 35 detergent-water,

37

accompanied by rinsing with ethanol and deionized water for 1 h each. The specimens were then

subjected to steam sterilization in an autoclave at 121 ºC for 30 min. and allowed to dry under UV

light overnight.

2.3.3 Cell culture

Cell culture studies were performed using mouse pre-osteoblast MC3T3-E1 subclone 4 cell

line (American Type Cell Culture Collection, Manassas, VA, USA) in a sterilized T-flask. The

culture medium for cells was alpha minimum essential medium (α-MEM, Invitrogen Corporation,

USA) mixed with 10% fetal bovine serum (FBS) and 1% penicillin (100 U mL-1) [120]. Culture

medium in T-flask was changed every 2-3 days. Cells were cultured in an incubator at 37 ºC in a

humidified environment with 5% CO2 and 95% air. Mirror polished surface of stainless steel

samples were ultrasonic washed with acetone, ethanol and distilled water for 30 min, respectively.

Next, the samples were sterilized in an autoclave at 121 °C.

2.3.4 Cell seeding

Trypsinization was used to detach cells from T-flask. 10,000 cells/well were counted and

seeded on stainless steel surfaces. In brief, cells were first washed with phosphate buffer saline

(PBS), followed by adding 0.25% trypsin/0.53 mM EDTA into the T-flask and incubated for 5-7

min to detach the cells from the T-flask. The dispersed cells in trypsin were transferred to a

centrifuge tube and centrifuged at 2000 rpm for 5 min to obtain cell pellet, which was then re-

suspended in fresh culture medium. 10,000 cells/well were counted using hemacytometer and

pipetted onto the sterilized samples, which were then incubated in sterilized 24 well plate [120].

2.4 Cell attachment, viability and morphology

Osteoblasts (10,000 cells/well) were seeded on steel surfaces. They were then incubated

(37ºC, 5% CO2 and 95% air) for 1 and 4 h, and 1, 4, and 7 days for cell attachment and cell viability

38

studies respectively, which were measured using the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-

diphenyltetrazolium bromide) solution according to pre-determined incubation times. Given that

reductase in mitochondria of living cells can cleave the tetrazolium ring and turn the pale yellow

MTT into dark blue formazan, we can apply this principle to examine the activity of living cells.

The concentration of formazan is directly proportional to the number of metabolically active cells

[120]. In brief, the samples were incubated with 90% fresh culture medium and 10% MTT (0.5

mg/ml medium) for 3 h in dark. Next, the culture medium with 10% MTT was removed from each

the well plate and dimethyl sulfoxide (DMSO) was added on each sample to dissolve the

intracellular purple formazan product into a colored solution which was quantified by

spectrophotometer at 570 nm with a microplate reader (Bio TEK Instrument, EL307C) to measure

the absorbance of solution [120]. The experiments were repeated at least 3 times to obtain mean

value and standard deviation (SD) of cell attachment and viability.

Scanning electron microscopy (SEM, Hitachi S-4800N) was used to observe the spreading

pattern and morphology of cells on steels with and without Cu after 2 h and 24 h incubation time.

In brief, the cells on the samples were fixed with 2.5% glutaraldehyde and then rinsed with PBS,

dehydrated with a graded concentration of ethanol (10-100%) and critical point dried. The samples

were coated with a thin film of gold (~10A 。

) and observed using scanning electron microscopy.

2.5 Immunofluorescence microscopy

Immunofluorescence microscopy (Nikon H600L) was used to study the expression level

of vinculin and fibronectin proteins and actin filaments. Pre-osteoblasts (10,000 cells/well) were

seeded on steel surfaces with and without Cu and incubated (37 ºC, 5% CO2 and 95% air) for 24

h. Next, the culture medium was removed from the well plate. 4% paraformaldehyde was added

onto each sample for 20 min to fix the cells, followed by 0.1% Triton X-100 for 5 min to

39

permeabilize the cells and 0.1% bovine serum albumin (BSA) for 30 min to block the cells. To

obtain the expression level of vinculin and distribution of actin, actin and vinculin were double

stained by diluted primary antibody (anti-vinculin) in blocking solution (1:200) for 1 h, followed

by labeling with diluted secondary antibody (1:100) (goat–anti-mouse FITC conjugate) (Chemicon

International, Inc., Temecula, CA, USA), for 45 min, incubated with diluted TRITC-conjugated

phalloidin (1:400). For fibronectin protein, samples was immunostained with diluted mouse

monoclonal antibody against fibronectin (1:800) (Sigma-Aldrich, St. Louis, MO, USA ) followed

by rabbit–antimouse FITC conjugated secondary antibody at a working dilution of 1:200 (Sigma-

Aldrich, St. Louis, MO, USA). Specifically, cell nuclei were labeled with DAPI (Chemicon

International, Inc., Temecula, CA, USA, 1:1000) for 5 min at 25 ºC. Fluorescence microscope was

used to observe vinculin, fibronectin and actin filaments with excitation and emission of 346/442

nm [120].

2.6 Analysis of expression level of proteins

Image J software was used to obtain quantitative expression level of fibronectin and

vinculin focal points. The intensity of fibronectin and vinculin focal points was quantified at least

three times. Also, via Image J software, the percentage of area covered by cells was analyzed.

Cell density per square centimeter was obtained from the micrographs.

40

Chapter 3: Favorable Modulation of Osteoblast Cellular Activity on Zr-modified Co-Cr-

Mo Alloy: The Significant Impact of Zirconium on Cell-Substrate Interactions

Abstract

Cobalt-chromium-molybdenum (Co-Cr-Mo: CCM) alloys exhibit good mechanical strength and

corrosion resistance. In recent years, from the perspective of osseointegration, they are considered

to be lower in rank in comparison to the widely used titanium alloys. We elucidate here the

significant and favorable modulation of cellular activity of Zr-modified Co-Cr-Mo alloys. The

determining role of small addition of Zr to the Co-Cr-Mo alloys in favorable modulation of cellular

activity was accomplished by combining cellular biology and materials science. Experiments on

the influence of Zr addition to Co-Cr-Mo alloys clearly demonstrated that cell adhesion, spread

and cell-substrate interactions were enhanced in the presence of Zr. Additionally, stronger vinculin

focal adhesion contact and signals associated with actin stress fibers together with extracellular

matrix protein, fibronectin, were noted. It is envisaged that the excellent strength and ductility of

Co-Cr-Mo-Zr alloys combined with the observation of significantly enhanced cellular response

will encourage usage of these alloys for biomedical applications in future.

3.1 Material characterization

Ni-free Co-28Cr-6Mo (wt.%) alloys with Zr (CCM+0.04Zr) and without Zr were

processed in an argon-containing environment utilizing using an induction furnace. The chemical

compositions of the alloys are presented in Table 3.1. The processing parameters are described in

detail elsewhere [34]. In brief, ingots (150 mm dia.) of the alloys were heat treated at 1473 K for

3 h and hot forged. The forged bars were hot swaged to produce the rods with approximately 13

mm in diameter, followed by annealing at 1423 K for 1 h and air cooling. Samples were cut from

the rod specimens with diameters of ~13 mm after annealing. The structure of Co-Cr-Mo alloy

41

samples with and without Zr as imaged by SEM are presented in Figure 3.1. The microstructure

comprises of equiaxed γ–phase and annealing twins. The average grain size of Co-Cr-Mo alloy

samples with and without Zr was 104±27 and ~53±11 µm, respectively.

42

Figure 3.1: Low and high magnification scanning electron micrographs representing

microstructure of Co-Cr-Mo alloy (d, e) with and (a, b) without Zr. (c, f) grain size

distribution plots with an average grain size of 104±27 µm in CCM+Zr alloy and

~53±11 µm in CCM alloy respectively.

Table 3.1: Chemical composition of Co-Cr-Mo alloy (mass %).

Co Cr Mo Zr N Ni Mn Si Fe C

Without Zr Balance 27.8 6.03 0 0.14 <0.01 0.55 0.56 <0.10 0.05

With Zr Balance 27.6 6.04 0.04 0.12 <0.01 0.60 0.52 <0.10 0.04

The average mechanical properties of Co-Cr-Mo alloys are presented in Table 3.2. Yield

strength, ultimate tensile strength, yield ratio, elongation-to-failure, and modulus for Co-Cr-Mo

alloy samples with Zr are ~514 MPa, ~1052 MPa, ~0.49, ~42.5%, and ~225 GPa and for Co-Cr-

Mo alloy samples without Zr are ~530 MPa, ~1114 MPa, ~0.48, ~47.3%, and ~227 GPa. Data

presented here is an average of at least five set of experiments. Vickers micro-hardness

measurements also confirmed that the addition of small percentage of Zr did not impact the

hardness and was in the narrow range of 291-296 HV.

Table 3.2: Mechanical properties of the Co-27Cr-6Mo alloy (mass %) with and without Zr.

Alloy 0.2% proof

stress, MPa

Ultimate tensile

strength, MPa

Yield ratio Elongation-

to-failure, %

Young's

modulus, GPa

Without Zr 530 ± 8 1114 ± 15 0.48 47.3 ± 2 227

With Zr 514 ± 6 1052 ± 11 0.49 42.5 ± 1.5 225

3.2 Cell viability and cellular response: MTT assay

10,000 cells/well were seeded on both Co-Cr-Mo alloys with and without Zr. Figure 3.2

shows the viability and spread of cells up to 6 days, as determined by MTT assay, where the value

of optical density is a direct indication of total number of metabolically active live cells and also

43

indicative of growth and spread. The cell number density increased with increase of incubation

time and was appreciably greater after 6 days on Zr-containing Co-Cr-Mo alloy in comparison to

the conventional Co-Cr-Mo alloy.

Figure 3.2: Histograms representing cell viability (from MTT assay) for Co-Cr-Mo alloy with

and without Zr. Zr-modified Co-Cr-Mo alloy (CCM +Zr) showed higher viability

with significant difference in comparison to conventional Co-Cr-Mo alloy (CCM)

at longer time periods. Statistical analysis (student’s t-test) was performed using

IBM SPSS software, with at least 3 replicates. (*) indicates 95% confidence level

with p<0.05.

MTT data indicated that the spread/growth rate of cells was ~190 % and 120 % per day on

the Co-Cr-Mo alloy samples with and without Zr, respectively. If we compare the data of 2 days

with 4 days, there was an increase of ~3.9× and ~2.47× in cell count on Co-Cr-Mo samples with

and without Zr after 4 days and ~7.7× and ~4.73× in cell count after 6 days of culture. Statistically

significant difference with a p-value less than 0.05 is observed only after day 6 between samples

with and without Zr.

3.3 Immunocytochemistry

Given that actin and vinculin key proteins are involved in cell-alloy interactions, they

provide an idea of cell growth condition on the surface of the alloy. Thus, the expression level of

44

Figure 3.3: The expression of actin cytoskeleton and vinculin focal adhesion proteins of

osteoblast cells cultured on Co-Cr-Mo alloy (c, d) with and (a, b) without Zr after 1

day of incubation as imaged through fluorescence microscopy. Cell adhesion is

more prominent on Zr-modified Co-Cr-Mo alloy (CCM +Zr) with extensive

spreading and high density of focal adhesion points in comparison to conventional

Co-Cr-Mo alloy (CCM).

actin cytoskeleton and vinculin focal adhesion proteins of osteoblast cells cultured on Co-Cr-Mo

alloy with and without Zr after 24 hours of incubation was imaged through fluorescence

microscopy and are presented in Figure 3.3. Cellular response on Zr-modified Co-Cr-Mo alloy

(CCM+Zr) showed extensive spreading of actin stress fibers and high density of focal adhesion

points in comparison to the conventional Co-Cr-Mo alloy (CCM). Furthermore, intercellular

interaction was more pronounced with network structure of elongated cytoskeleton and focal

adhesion points on Zr-containing Co-Cr-Mo alloy (CCM+Zr). The observation of well-defined

45

actin stress fibers and high number density of small focal adhesion contacts was clearly distinct on

Zr-containing alloy and in striking contrast to Zr-free alloy.

Figure 3.4: The expression of extracellular fibronectin protein coupled with DAPI-stained

osteoblast cell nuclei on Co-Cr-Mo alloy (c, d) with and (a, b) without Zr after 1

day of incubation as imaged through fluorescence microscopy. Cell adhesion is

more prominent on Zr-modified Co-Cr-Mo alloy (CCM +Zr) with extensive

intercellular contacts and high density of cell nuclei in comparison to conventional

Co-Cr-Mo alloy (CCM).

The expression level of extracellular fibronectin protein merged with DAPI-stained

osteoblast cell nuclei on Co-Cr-Mo alloy with and without Zr after 1 day of incubation, as imaged

through fluorescence microscopy is shown in Figure 3.4. Superior cell adhesion and proliferation

within 1 h was noted on Zr-modified Co-Cr-Mo alloy (CCM+Zr) with extensive intercellular

46

network of extracellular matrix fibronectin protein and high density of cell nuclei in comparison

to conventional Co-Cr-Mo alloy (CCM).

As analyzed through Image J software, a quantitative significant difference of mean ±

standard deviation in the expression levels of extracellular fibronectin protein and vinculin focal

adhesion points was observed between Co-Cr-Mo alloy samples with and without Zr (Figure 3.5).

The pixel-based fibronectin intensity and vinculin focal adhesion points were observed to be two-

fold and three-fold times more on CCM+Zr alloy in comparison to CCM alloy.


and vinculin focal adhesion points (blue) using NIH Image J software for Co-Cr-

Mo alloy with and without Zr. Statistical analysis (student’s t-test) was performed

using IBM SPSS software, with at least 3 replicates. (*) indicates 95% confidence

level with p<0.05.

47

3.4 Morphology of cells

Representative SEM micrographs depicting attachment and morphology of cells on Co-Cr-

Mo alloy samples with and without Zr after 2 h and 24 h of culture are presented in Figures 3.6

and 3.7. After 2 h, the cells spread as a sheet with polygonal morphology on both the alloys with

and without Zr. However, filopodia-like cellular extensions were observed to be more on CCM+Zr

alloy in comparison to CCM alloy. After 24 h, cells showed extensive spreading with polygonal

sheet-like morphology on CCM alloy and elongated star-like morphology on CCM+Zr alloy. High

density of cells with multiple filopodia-like cellular extensions forming intercellular contacts were

observed on Zr-modified Co-Cr-Mo alloy (CCM+Zr) in comparison to CCM alloy. In summary,

the significant overlap of cells forming cytoplasmic bridges, and the degree of cell spread was

significantly higher on the Zr-containing alloy. This truly implied superior attachment of cells,

cell-metal surface interaction and cell-to-cell communication on the Zr-bearing alloy.

As analyzed through Image J software, the % area covered by the cells increased from

~5.1 % to ~33.6 % on CCM alloy and ~19.2 % to ~47.8 % on CCM+Zr alloy after 2 h and 24 h of

incubation. Similarly the cell density increased from ~1354 cells/cm2 to ~3424 cells/cm2 on CCM

alloy and ~3583 cells/cm2 to ~7804 cells/cm2 on CCM+Zr alloy after 2 h and 24 h of incubation

(Figure 3.8).

48


morphology of osteoblasts cultured for 2 hours on (d-f) Zr-modified Co-Cr-Mo

alloy (CCM+Zr) and (a-c) conventional Co-Cr-Mo alloy (CCM). Cells were

observed to spread with polygonal sheet-like morphology on both the alloys with

and without Zr. However, filopodia-like cellular extensions were observed to be

more on CCM+Zr alloy in comparison to CCM alloy.

49

50


morphology of osteoblasts cultured for 24 hours on (d-f) Zr-modified Co-Cr-Mo

alloy (CCM+Zr) and (a-c) conventional Co-Cr-Mo alloy (CCM). Cells showed

extensive spreading with polygonal sheet-like morphology on CCM alloy and star-

like morphology on CCM+Zr alloy. High density of cells with multiple filopodia-

like cellular extensions forming intercellular contacts was observed on Zr-modified

Co-Cr-Mo alloy (CCM+Zr) in comparison to CCM alloy, leading to stronger

adhesion with the surface.

Figure 3.8: Histograms representing % area covered by the cells (red) and cell density (blue) as

analyzed from the Figures 6-7 area using NIH Image J software for Co-Cr-Mo alloy

with and without Zr. Statistical analysis (student’s t-test) was performed using IBM

SPSS software, with at least 3 replicates. (*) indicates 95% confidence level with

p<0.05.

3.5 Discussion

In the present study, cobalt chromium-based alloys with and without Zr, processed through

hot-rolling and hot-swaging followed by annealing, were studied to evaluate the cellular response

and elucidate if the addition of Zr to the Co-Cr alloy was a step in the right direction towards

51

promoting osseointegration and bone-bonding properties. The effect of Zr on mechanical

properties of CoCrMo alloy varies with respect to its composition. Previous study by co-author on

this aspect has concluded that tensile strength decreases with increasing Zr content from 0.01%

[123]. The slight decrease in mechanical properties observed in the present study correlate with

the previous study and are also attributed to the higher grain size in 0.04Zr-added alloy.

Both the alloys with and without Zr were processed via identical processing conditions and

were mechanically polished to near identical surface finish using conventional metal polishing

procedure to maintain similar nano scale of surface topography. The data presented in Figures.

3.2-3.8 clearly suggest that the presence of Zr in the alloy was beneficial in promoting cellular

response of osteoblasts from the perspective of cell adhesion and spread, cell-to-cell interaction,

production of key proteins, namely, actin, vinculin and fibronectin. These are relevant proteins

because they impact cell-material interaction. We know that actin is the most abundant protein in

eukaryotic cells and that the network of actin filament and associated actin-linking proteins

determines the cell shape and is involved in a number of cell surface activities, including mobility,

while vinculin is involved in linkage of integrin adhesion molecules to actin cytoskeleton. While

fibronectin is a growth simulator [124–128] and is released during the early stages by osteoblast.

The properties of a surface significantly impact cellular function, including attachment,

viability, synthesis of protein etc. and primarily include surface roughness, surface chemistry, ionic

bonding, electrostatic interaction and van der Waals interactions [129,130]. Given that both the

alloys were polished to near-identical mirror finish, it is envisaged that the surface chemistry

played a major role in favorably impacting the cellular response of pre-osteoblast on Zr-containing

Co-Cr-Mo alloy. Zirconium is known to form a stable and adherent layer of ZrO2 on the surface

that prevents diffusion of ions from the environment into the alloy and release of ions of constituent

52

elements of the alloy from the bulk into the surrounding environment. Recent XPS studies

conducted by the authors indicated enrichment of Zr at the surface. Elements, Zr and Mo, are

neither toxic or have allergic nature, and are commonly used as alloying elements in the fabrication

of novel biomedical implants [131,132]. It is proposed that the oxides (ZrO2 and MoO2) of these

alloying elements enhances the corrosion resistance of the alloy and inhibit the release of metal

ions into the surrounding environment because of the compact nature of oxide film on the surface

[133,134]. In a recent study, Zr-alloyed titanium exhibited higher new bone inorganic mineral

composition and higher removal torque forces in comparison to pure titanium [135–138]. In

another study, addition of Zr led to improvement of bone anchorage properties of an electron beam

melted alloy [29,30]. The in vivo study shows that bone tissue grows in direct contact with both

the CCM alloys with and without Zr, and further improving the bone anchorage of the CCM

implants with low addition of Zr.

In summary, the zirconium-containing CCM alloy promoted enhanced cell attachment,

proliferation, and significantly greater degree and extracellular stronger signals of matrix proteins,

actin and vinculin, on the CCM+Zr alloy surface in comparison to the CCM alloy. Thus, Zr-

containing alloy promoted cellular activity and more specifically cellular responses of osteoblast.

3.6 Conclusions

1. The evidence that the presence of Zr in Co-Cr-Mo alloys indicated favorable cellular response

of osteoblasts is a step in the right direction to promote their application for the fabrication of

biomedical devices.

2. Experiments on the effect of Zr in Co-Cr-Mo alloys clearly demonstrated that adhesion, spread

and morphology of osteoblasts was favorably modulated, confirming the beneficial impact of Zr

in the alloy.

53

3. The difference in the cellular activity of Zr-free and Zr-containing alloys is attributed to the

presence of an adherent and stable ZrO2 layer on the surface that prevents release of ions from the

bulk metal to the physiological environment, consistent with our recent observation of enrichment

of Zr on the surface. This aspect is also in agreement with the thermodynamic ability of Zr to form

oxide in a manner similar to other traditional metals, Ti, Cr and Nb.

4. The cellular response additionally demonstrated stronger expression of vinculin focal adhesion

contacts and actin stress fibers, in conjunction with fibronectin.

5. The excellent strength and ductility of Co-Cr-Mo-Zr alloy combined with biological

compatibility and favorable modulation of cellular activity would encourage future application of

cobalt-based alloys.

54

Chapter 4: Favorable modulation of osteoblast cellular activity on Cu-containing austenitic

stainless steel and comparison with the Cu-free counterpart

Abstract

In the present study, we describe the favorable modulation of osteoblast functions on Cu-

containing austenitic stainless steel in comparison to the Cu-free steel. A series of biological

experiments were conducted to understand the determining role of Cu in stainless steel on cellular

activity. The study clearly demonstrated that cell attachment, proliferation and cell-material

interactions were significantly enhanced in the presence of Cu. The % area covered by cells

increased from 13.4% to 17.8% on Cu-free steel and 17.7% to 25.2% on Cu-containing steel after

2 h and 24 h of incubation, respectively. Similarly, the cell density increased from ~3845 cells/cm2

to ~5481 cells/cm2 on Cu-free steel and ~4990 cells/cm2 to ~8590 cells/cm2 on Cu-containing steel

after 2 h and 24 h of incubation, respectively. Additionally, stronger vinculin focal adhesion

contact and signals associated with actin stress fibers together with extracellular matrix protein,

fibronectin, were noted on Cu-containing steel. It is underscored that copper can be added to

biomedical alloys from the perspective of cellular functions.


The chemical composition of austenitic stainless steels with and without copper is

presented in Table 4.1. The grain structure of austenitic stainless steels with and without Cu is

presented in Figure 4.1. Figures 4.1a and 4.1b are the light micrographs of steels with and without

Cu, respectively. The average grain size of austenitic stainless steel with Cu was ~22 µm, and the

mechanical properties, namely, yield strength and elongation were ~297 MPa and ~68%,

respectively. While the average grain size of Cu-free steel was ~55 µm with yield strength of ~277

MPa and elongation of ~70%, respectively.

55

Table 4.1: Chemical composition of austenitic stainless steels (wt.%).

Steels Fe C Si Mn Cr Ni Cu P S

Cu-free Balance 0.04 0.16 1.52 17.8 8.1 0 0.005 0.005

Cu-containing Balance 0.023 0.55 0.85 17.4 7.32 3.15 0.025 0.011

Figure 4.1: Light micrographs representing microstructure of austenitic stainless steels with and

without Cu (Cu-containing: average grain size of ~22 µm and Cu-free: average

grain size of ~55 µm, respectively).

4.2 Surface wettability

The water contact angle was used to measure the surface wettability of steels with and

without Cu. The contact angle of austenitic stainless steel with and without Cu was 66.50° and

73.33°, respectively. It can be concluded that Cu-free steel shows higher contact angle than Cu-

containing steel, which suggests that Cu-containing steel was more hydrophilic than Cu-free steel.

4.3 Cellular response

The data on cells attached on steel surfaces with and without Cu and incubated for 1 and 4

h is presented in Figure 4.2a. Pre-osteoblast shows enhanced level of attachment on Cu-containing

steel surface in comparison to Cu-free surface, implying that Cu is beneficial for cell attachment.

(a) (b)

56


after 1-7 days via MTT assay for austenitic stainless steels with and without Cu.

Cell viability was measured by mitochondrial reduction of MTT assay after incubation time of 1,

4, 7 days, as shown in Figure 4.2b. It is noted that the presence of copper encouraged cell growth

57

based on the data obtained for samples incubated up to 7 days, which showed higher level of cell

viability. This again implied that Cu is a beneficial for cell proliferation and metabolism.


morphology of osteoblasts cultured for 2 h on austenitic stainless steels with and

without Cu.

Figures 3 and 4 present cell attachment and morphology after 2 h and 24 h of incubation,

respectively on steels with and without Cu. After 2 h, the pre-osteoblasts were observed to start

spreading with a star-like morphology on both the steels, but the extent of cell spreading appeared

to be greater on Cu-containing steel in comparison to Cu-free steel. Steel without Cu exhibited

58

lower cell density compared to Cu-containing steel. The cell morphology became flat after 24 h


morphology of osteoblasts cultured for 24 h on austenitic stainless steels with and

without Cu.

incubation and can be observed in Figure 4. Pre-osteoblasts appeared distinct sheet-like spreading

network and higher density of cells overlapped and higher interactions between cells were

observed on both the samples compared to 2 h incubated steels. In summary, it is clear that steel

with Cu exhibited superior cell attachment and interaction than steel without Cu.

Using Image J software, the analysis of percentage area covered by cells was estimated.

As shown in Figure 5, it is noted that the % area covered by cells increased from 17.7% to 25.2%

59

Figure 4.5: Histograms representing % area covered by the cells as analyzed from the Figures 3-

4 area using NIH Image J software for austenitic stainless steels with and without

Cu.

after 2 h and 24 h incubation for Cu-containing steel. The % area covered by cells increased from

13.4% to 17.8% after 2 h and 24 h incubation for Cu-free steel. Similarly the cell density increased

from ~4990 cells/cm2 to ~8590 cells/cm2 after 2 h and 24 h incubation on Cu-containing steel. The

cell density increased from ~3845 cells/cm2 to ~5481 cells/cm2 after 2 h and 24 h incubation on

Cu-free steel (Figure 6).

60

Figure 4.6: Histograms representing cell density as analyzed from the Figures 4.3-4.4 area using

NIH Image J software for austenitic stainless steels with and without Cu.

Individual proteins including vinculin and fibronectin that are associated with cell

attachment and proliferation were studied. Expression of vinculin with focal contacts and actin

stress fibers after 24 h cell culture are presented in Figure 7. The expression level of vinculin and

well-defined actin stress fibers on Cu-containing steel surfaces in relation to Cu-free surface was

distinct. Also, extracellular fibronectin combined with DAPI-stained cell nuclei of steels with and

without Cu are presented in Figure 8. Steel containing Cu exhibited higher expression level of

fibronectin and more cell nuclei from the observation of distinct network and fluorescence

intensity of immunostained fibronectin.

Using Image J software, the expression levels of extracellular fibronectin protein and

vinculin focal adhesion points for the two steels exhibited a significant difference, as shown in

61

Figure 9. Interestingly, the fibronectin intensity and vinclin focal adhesion points on Cu-containing

steel were about two-times greater in comparison to Cu-free steel.

Figure 4.7: Representative actin and vinculin expressed by cells incubated for 24 h on austenitic

stainless steels with and without Cu.

62

Figure 4.8: Representative immunocytochemistry of fibronectin protein combined with DAPI-

stained osteoblast cell nuclei expressed by cells incubated for 24 h on austenitic



and vinculin focal adhesion points (blue) using NIH Image J software for austenitic


63

In summary, the degree of cell attachment and viability studies, cell interaction, expression

level of fibronectin, vinculin and actin stress fibers was greater for Cu-containing steel as

compared to Cu-free steel.

4.4 Discussion

We have studied here the cellular functions of austenitic stainless steel with and without

Cu to elucidate the effect of copper on cellular response.

Under identical surface finish and incubation environment, the results of cellular response

on steels are presented in Figures 2-9. The figures clearly suggested that Cu-containing steel

exhibited better cell attachment, viability, spreading and proliferation in comparison to Cu-free

steel. Furthermore, cell-to-cell interactions and expression level of fibronectin, vinculin and actin

stress fibers were also greater on steel with Cu, indicating favorable biocompatibility.

The surface properties of steel play a significant impact on cell functions. In this study, the

two experimental steels were polished to near-identical mirror finish. Cu-containing steel exhibited

smaller contact angle as compared to Cu-free steel, which indicated that Cu-containing steel

surface is more hydrophilic than Cu-free steel surface. This may be attributed to the Cu ions in

steel. Cu loaded TiO2 exhibited smaller water contact angle, which indicated that Cu loading can

improve the hydrophilicity of metallic material by preventing electron-hole recombination [139].

This explains that steel containing Cu has smaller contact angle compared to steel without Cu

because of the function of Cu ions.

Cu-containing stainless steel has been shown to exhibit strong antibacterial activity because

of the release of Cu ions [15,140–142]. The Cu ions released from the alloy effectively inhibit the

formation of bacterial film on the surface of the alloy and do not induce toxicity. Furthermore,

the high density of grain boundaries in a nanostructured alloy provided increased diffusion path

64

for copper atoms to reach the surface to form passive film with high copper concentration, which

hindered the interaction between the material surface and microorganism. The structure of bacteria

outer cell membrane is changed after bacteria contacts with the antibacterial stainless steel surface

because it destroys cell permeability. It was observed that antibacterial stainless steel had higher

adhesion force for bacteria because of higher electrostatic forces by Cu2+ [15]. The forces between

bacteria and steel surface such as van der Waals and electrostatic forces are governed by the initial

interaction between them [143]. They are determined by the physicochemical properties of

interface, such as hydrophobicity, free energy and surface charge [144,145]. These factors are

envisaged to influence enhanced cell attachment, proliferation, and higher level of proteins in Cu-

containing steel in comparison to steel without Cu. Cu-containing steel with antibacterial

properties exhibited better biocompatibility as compared to the Cu-free steel.

4.5 Conclusions

1. The study successfully explored the biological functions of antimicrobial Cu-containing

stainless steel and compared with the Cu-free steel, in terms of cell attachment and synthesis of

proteins.

2. Cu-containing steel was beneficial for cell attachment, viability, proliferation and demonstrated

stronger expression of vinculin and fibronectin proteins and actin stress fibers in comparison to

Cu-free steel, confirming the beneficial impact of Cu in austenitic stainless steel.

3. Stainless steel with Cu is envisaged to release Cu ions and is instrumental in killing bacteria and

encouraging biological functionality.

4. The study underscores that copper can be added to biomedical alloys from the perspective of

promoting cellular functions.

65

Chapter 5: Favorable modulation of osteoblast cellular activity on austenitic stainless steel

with nano/ultrafine grains and comparison with micrometer austenitic grains counterpart

Abstract

Conventional coarse-grained (CG) biomedical austenitic stainless steel with grain size in the

micrometer range was subjected to severe cold deformation when the cold deformation martensite

reverts back to austenite with a novel phase reversion process followed by annealing for short

duration. We describe the favorable modulation of osteoblast functions on grain size in the

nanometer/ultrafine (NG/UFG) regime (~200-400 nm) in comparison to the CG (~55±20 µm)

steels. A series of biological experiments were conducted to govern the differences in the behavior

of NG/UFG and CG surfaces in terms of protein adsorption and subsequent relationship with

biological functions. Higher cell attachment, proliferation and higher expression level of

prominent proteins, fibronection, actin and vinculin were favored by a surface with grain size in

the nanometer regime and was in striking contrast with the surface with micrometer grain size.

This behavior is attributed to the differences in the fraction of grain boundaries that are high energy

region.


The microstructure of CG and NG/UFG austenitic steels is presented in Figure 5.1. The

average grain size of solution-treated CG steel was ~55±20 µm, while the NG/UFG steel (cold

rolled to 90% and annealed at 800 °C for 10 s) was in the range of ~200-400 nm. The yield strength

and tensile elongation of CG and NG/UFG steels were as follows: CG (yield strength: 277±41

MPa, elongation: 70±0.8%), NG/UFG (yield strength: 557±30 MPa, elongation: 44±1%). Thus,

through the phase reversion annealing concept there was significance increase in the strength of

steel because of refinement of grain size.

66

Figure 5.1: Light and TEM micrographs illustrating the microstructure of coarse-grained (CG)

and nanogrianed/ultrafine-grianed (NG/UFG) austenitic stainless steels with an

average grain size of ~55±20 µm and ~200-400 nm, respectively.

5.2 Cell viability and cellular response: MTT assay

Figure 5.2a shows cell attachment after culture time of 1-4 h and Figure 5.2b shows cell

viablility after culture time of 1-7 days by MTT assay on NG/UFG and CG steels. The data

presented in the form of histograms, particularly Figure 5.2b, suggests that the cell attachment on

NG/UFG austenitic stainless steel is greater than the CG counterpart. Also, cell viability

determined by mitochondrial reduction of MTT from 1 to 7 days is higher for NG/UFG steel as

compared to the CG steel. This clearly indicates that the NG/UFG steel provides conditions that

are more favorable for cell metabolism than the CG steel. We can draw an identical conclusion

from Figure 5.3 which shows flourescence images of cells stained with nucleic acid-specific dye

after 24 h of cell culture. These data (Figures 5.2 and 5.3) suggest that the finer grain size of

NG/UFG steel is at least one of the factors that contributed to higher cell attachment on the

67

NG/UFG surface. The cell attachment to the surface is important for subsequent cellular activities

that include proliferation and cell-biomaterial interactions.

68


after 1-7 days via MTT assay for NG/UFG steel in comparison to the conventional

CG steel.

Figure 5.3: Representative fluorescence micrographs of cells stained with nucleic acid-specific

dye after 24 h cell culture for CG and NG/UFG austenitic stainless steels,

respectively.

5.3 Morphology of cells

Figure 5.4 shows scanning electron micrographs of cells cultured for 24 h on NG/UFG and

CG steels. It is observed that the cells on the two steels were characterized by a polygonal

morphology, but with significant difference. The cells were widely spread on the NG/UFG surface

and formed a sheet on the surface. Furthermore, filopodia-like cellular extensions on the NG/UFG

steel surface than CG steel. The overlapping and cell-to-cell interaction was apparent on the

NG/UFG steel surface were more with the formation of cytoplasmic bridge. This implies that cell-

substrate interactions on the NG/UFG surface were remarkably greater in comparison to the CG

steel surface.

69

Figure 5.4: Scanning electron micrographs illustrating cell adhesion morphology of osteoblasts

cultured for 24 h on CG and NG/UFG austenitic stainless steels.

5.4 Immunocytochemistry

Fibronectin, vinculin and actin are important proteins that affect cell-surface interactions,

in which fibronectin protein acts as a growth stimulator [146–149] and is one of the earliest protein

released by osteoblasts during metabolism[146,150]. Fibronectin is then mineralized and

contributes to bone growth [151]. Figure 5.5 presents the immunocytochemistry of fibronectin

expressed by cells incubated for 24 h on NG/UFG and CG steels. Higher fibronectin intensity and

expanded network of fibronectin expression were observed on NG/UFG steel in relation to CG

steel. Furthermore, higher expression level of vinculin, focal adhesion contacts and actin stress

fibers were prominent on NG/UFG steel than CG steel after 24 h incubation as shown in Figure

5.6. Thus, the examination of individual proteins that are related to cell attachment and

proliferation clearly indicated that the NG/UFG surface favorably modulates cellular response and

biological functions. The aforementioned results point to the conclusion that the grain structure of

steel plays a significant role on cell attachment, proliferation and synthesis of proteins.

70

Figure 5.5: Representative immunocytochemistry of fibronectin protein expressed by cells

incubated for 24 h on CG and NG/UFG austenitic stainless steels.

5.5 Discussion

The surface properties of steel that are envisaged to have significant impact on cell

attachment, viability, expression of proteins, primarily include energetics, hydrophilicity, ionic

bonding, electrostatic and van der Waals interactions[152,153]. The hydrophilic surface enhances

the activity of alkaline phosphatases and osteocalcin and also promotes adsorption of fibronectin

and albumin[152–156]. The hydrophilicity of NG/UFG and CG steel was measured in terms of

contact angle 64.88 ± 0.85° and 73.33 ± 1.53°, respectively, and is a factor that influenced cell

attachment, proliferation and expression of proteins with grain size, keeping mind that both steels

were polished to near-identical mirror surface finish. Another factor that is responsible for

favorable modulation of cellular activity on the NG/UFG surface is surface energy. NG/UFG has

a very high faction of grain boundaries (> 50%) as compared to the CG surface (< 2%) that have

71

Figure 5.6: Representative actin and vinculin expressed by cells incubated for 24 h on CG and

NG/UFG austenitic stainless steels.

high energy. Thus, higher fraction of high energy grain boundaries must be responsible for cell

attachment and proliferation, with relatively higher expression level of proteins.

72

The study presented here advances our understanding on the biological functions by

illustrating that the cellular functionality of a biomaterial surface strongly depends on the grain

structure.

5.6 Conclusions

1. Severe cold deformation of conventional coarse-grained biomedical austenitic stainless steel

followed by annealing for short durations enabled NG/UFG stainless steel to be obtained with high

strength-high ductility combination.

2. NG/UFG austenitic stainless steel was characterized by greater attachment of cells on the surface

as compared to the CG austenitic stainless steel. Furthermore, higher fibronectin intensity and

expanded network of fibronectin, higher expression level of vinculin and actin stress fibers were

observed on NG/UFG steel in relation to the CG austenitic stainless steel. These observations

suggested that the grain size of steel plays a significant role on cell attachment, proliferation and

morphology. Smaller grain size steel favorably modulates cell development and cell attachment.

3. The underlying reason for favorable modulation of cellular response is attributed to high fraction

of high energy grain boundaries and relatively higher hydrophilic nature of NG/UFG austenitic

stainless steel.

4. The study advances our understanding on the cellular response to biomaterials.

73

Chapter 6: Microstructure entropy guided understanding of yield strength in alloy systems

characterized by grain size distribution

Abstract

We introduce and adopt the concept of a phenomenological parameter, microstructure entropy, S*,

to understand the yield strength in alloy systems with bimodal grain size distribution obtained from

large sets of experimental data. 60 groups of bimodal grain size distribution data was obtained

through the application of an ingenious concept involving severe cold deformation to obtain

dislocation cell-type of martensite, followed by annealing when martensite reverts to austenite by

shear or diffusion mechanism, with the aim to obtain ultrafine grain size. Six factors emerged from

statistical data analysis through EBSD studies in terms of grain size and grain numbers from 60

sets of data. Microstructure entropy (S*) was obtained from the bimodal structure data in a self-

similar regime. Inverse of the square root of microstructure entropy (*

1

S) and yield strength

exhibited a linear relationship. The proposed conceptual methodology has wide acceptance for any

grain size distribution to obtain microstructure entropy (S*) of a specific grain structure and predict

yield strength. A generic equation similar to Hall-Petch relationship, but involving microstructure

entropy is proposed to predict and understand yield strength of metallic systems characterized by

grain size distribution or microstructural evolution.

6.1 Microstructure entropy - revisited

It is appropriate to briefly describe and revisit the microstructure entropy theory for grain

growth dynamics that is consistent with the self-similar regime and discussed in the literature

[114–117,157] because we use the relevant equations as part of data analysis. The grain boundaries

obtained from grain growth represent a significant chaotic microgeometry of polycrystalline

metals and alloys that are a function of exponential distribution of grain volume [157]. The

74

probabilistic distribution of grain size in the self-similar regime of grain growth was observed to

be consistent and in agreement with the experimental data. Dynamic equations in phase space of

grain volumes which exhibit exponential distribution in self-similar evolution as described in the

literature are the following [115]:

Grain volume density ρ(t, ν) is defined by [115]:

ρ+ ( (t, )ρ)=0

t νV ν

(6.1)

where ρ(t, ν) is the grain volume density. ρ(t, ν)ν is the number of grains with volumes in a small

interval [ν, ν+ν]. V(t, ν) is the velocity of the phase space flow and is a linear function of ν, t is

time, and ν is grain volume. The velocity of the phase space flow V is given by [115]:

(t))((t)

1)V(t,

3/2 avg

avg

vvv

v (6.2)

Combining equations (6.1) and (6.2) [115]:

0ρ)(1

t

ρ3/2

avg

avg

vvvv (6.3)

The number of grains changing with time is given by [115]:

ν

0ρ(t, ν) νN d (6.4)

where N is set of grains with volumes ν1,…,νN, where the grain volume evolves in manner that

the total volume is conserved [115]:

1ν ... νN V (6.5)

The average change in grain volume with time is [115]:

75

(t)

(t)N

Vvavg (6.6)

The f (t, ν), which is the probability distribution of grain volume is defined by [115]:

0

1 ρ(t, ν)f (t, ν) ρ(t, ν)

(t) ρ(t, ν) νN d

(6.7)

The self-similar evolution corresponds to a stationary distribution of relative grain volume, g(u),

where u=ν/vavg (t) and is relative grain volume [115]:

)

(t)(

(t)

1)f(t,

avgavg v

vg

vv

(6.8)

In the self-similar evolution the grain volume density has the form [115]:

)

(t)(

(t))

(t)(

(t)

(t))ρ(t,

2

avgavgavgavg v

vg

v

V

v

vg

v

Nv

(6.9)

Combining equations (6.3) and (6.9) [115]:

g(u (1 q) 1) (2q 1)g

u

d

d (6.10)

where q is [115]:

)0(

13/1

avg

avg

avg

vdt

dv

vq

(6.11)

In equation (6.10), q is a constant and q cannot be negative. It determines the rate of change of

the average grain volume [115]:

t

3

2)0((t) 3/23/2 qvv avgavg

(6.12)

76

There are five distinct cases, q = 1, q > 1, 1/2 < q < 1, q = 1/2, 0 q < 1/2 [115]:

(u) n t1 co s e uq g

(6.13)

2 1

1

const(u)

(1+(q-1)u)

1:q

q

q g

(6.14)

2 1

11(u) const u1/ 2 1 -

-q:

1

q

q

q g

(6.15)

(u)1/ cons2 : tq g

(6.16)

2 1

1

const(u)

1u

1-q

0 1/ 2 :q

q

q g

(6.17)

In terms of evolution of microstructure entropy, a natural choice in self-similar regime for an

arbitrary process entropy is defined by equation (6.8) involving logarithm of phase volume [115]:

dvvvtfvtfS )),(ln(),( 0

*

(6.18)

where f (t, ν) is probability distribution of grain volume. ν0 is the smallest observable grain volume

and ν is the grain volume. Thus, the evolution equation for S* is [115]:

duuuv

vdvvvtfvtfS

avg)lng()g(ln)),(ln(),(

0

0

*

(6.19)

where g(u) is a stationary distribution of relative grain volume. u is relative grain volume, ν0 is the

smallest observable grain volume, and vavg is average grain volume. Based on the summary of the

microstructure entropy evolution for grain growth described above in references [114–117,157],

77

we collected large sets of experimental data required to calculate the microstructure entropy S*, as

described below.

6.2 Statistical calculations based on microstructure entropy theory

6.2.1 Bimodal distribution

Bimodal grain size distribution of austenitic stainless steel was obtained in the phase reversion

process. There are some aspects that governed and led to the bimodal grain size distribution in the

phase reversion concept. First, it is believed based on our processing practice that the degree of

cold reduction and nature of passes (small passes versus large passes) are important factors that

influence bimodal distribution of grain size [118,119]. It is difficult to obtain 100% stain-induced

martensite after severe cold reduction, which means that some austenite is retained. The retained

austenite will grow on annealing. Another reason is attributed to reversion mechanism. Ultrafine

austenite first nucleates at dislocation-cell type martensite, followed by nucleation of relatively

coarse or fine austenite at lath-type marteniste. In this study, 60 sets of bimodal grain size

distribution were classified into two broad categories: (a) grains less than 1 μm (ultrafine) and (b)

grains greater than 1 μm (coarse). Figure 6.1a-c shows the bimodal distribution of sample 1

annealed at 800 °C for 10s, 60s and 1000s, respectively. Figure 6.2 a-c shows the fitting bimodal

distribution of sample 1 annealed at 800 °C for 10s, 60s and 1000s, respectively. Both the

categories had good combination of high strength and high ductility (~40%), irrespective of ultra-

fine or coarse grain size. In this regard, The microstructure entropy S* was calculated for 5 different

chemical compositions of stainless steel with bimodal grain size distribution through the use of

computational tool (Matlab) in the attempt to obtain a relationship between microstructure entropy

S* and yield strength.

78

Figure 6.1: Bimodal distribution for steel 1. (a) 800 °C/10s, (b) 800 °C/60s, (c) 800 °C/1000s.

In view of the considered two categories of grain size distribution (less than 1 μm and more

than 1 μm), equation (6.6) was modified to:

fc

fc

avgNN

VV

N

Vv

(t) (6.20)

where Vc and Vf are grain volumes of coarse (micrometer region) and ultrafine (nanometer region)

grains, respectively, and Nc and Nf are grain quantities of coarse (micrometer region) and ultrafine

(near nanometer region) grains, respectively.

q is obtained from equation (6.12), and is the slope of linear function qtvtv avgavg3

2)0()( 3/23/2 .

q is a constant at a specific temperature, which was obtained by line fitting and is listed in Tables

6.2A-6.2E for different austenitic steels.

79

Figure 6.2: Bimodal distribution for steel 1. (a) 800 °C/10s, (b) 800 °C/60s, (c) 800 °C/1000s,

and (d) relative grain size distribution for data presented in (a, b, c).

80

We introduced the notation based on five distinct cases in stationary distribution g(u)

according to equations (6.13)-(6.17):

2 1

1

qn

q

(6.21)

6.2.2 Self-similar regime

It is suggested that the microstructural entropy concept pertains to self-similar regime and the

self-similar regime is described by grain size distribution f(R) (R is grain size; Ravg is average grain

size; r= R /Ravg is relative grain size). When relative grain size distributions g(r) = (1/Ravg) f(R/Ravg)

coincides for different times in a plot, the process is self-similar. According to self-similar regime

principle, the grain growth exhibited a self-similar characteristic as shown in Figure 6.2. The

relative grain size distribution g(r) (Figure 6.2d) shows the same bimodal distribution as steel 1 at

different time instances while at same temperature (Figure 6.2a-c).

6.2.3 Relative grain volume

Given that u= ν/vavg(t) is relative grain volume, we modified u based on bimodal grain

structure. We also need to consider the fraction of ultrafine grains and coarse grains into

consideration, which is described by the factor m (fraction of ultrafine grains or coarse grains in

the entire set of bimodal grains). Hence, for ultrafine grains, the relative grain volume is defined

by:

(t)

ff

f

avg

avg

v

vmu

(6.22)

where mf is the fraction of ultrafine grains in the entire set of bimodal grains. vavgf is the average

ultrafine grain volume. vavg is average grain volume change with time.

Similarly, for coarse grains, the relative grain volume is defined by:

81

(t)avg

cavgc

cv

vmu

(6.23)

where mc is the fraction of coarse grains in the entire set of bimodal grains. vavgc is the average

coarse grain volume. vavg is average grain volume change with time.

6.2.4 Microstructure entropy calculations

We considered 6 factors (ν0, vavg, q, n, uf, uc) defined above for 5 steels to calculate

microstructure entropy S* for different experimental conditions.

The chemical composition of steels with bimodal structures is shown in Table 6.1.

Table 6.1: Chemical composition of stainless steels (wt.%).

Steels C Si Mn Cu Cr Ni Nb

1 0.095 0.35 10.1 0.90 13.8 1.25 -

2 0.089 0.35 9.92 0.87 12.8 1.27 0.018

3 0.088 0.35 9.98 0.90 12.7 1.24 0.034

4 0.088 0.35 9.82 0.90 12.5 1.20 0.045

5 0.13 0.31 8.68 0.88 11.5 1.23 0.11

All the five austenitic stainless steels were subjected to 70% cold reduction to obtain

marteniste followed by annealing at 800-1000 °C for 10-1000 s, when marteniste reverts to

austenite with bimodal grain size distribution with different fraction of ultrafine and coarse grains

via phase reversion [118]. Tensile tests were conducted at room temperature using a CMT5105

tensile machine at a strain rate of 5 × 10-4 s-1. The experiments were repeated at least 3 times to

obtain average yield strength, as shown in Tables 6.3A-6.3E, with an experimental error of 0-4%.

EBSD studies were conducted on 5 steels. The specimens after mechanical polishing were

electrolytically polished in a solution containing 20 vol.% of perchloric acid and 80 vol.% of

ethanol at a voltage of 15 V for 30 s. HKL-Channel 5 system was used to collect grain size

distribution and grain number. 6 factors (ν0, vavg, q, n, uf, uc) were statistically obtained based on

bimodal structure, as shown in Tables 6.2A-6.2E. Average grain volume vavg comes from average

82

Table 6.2A: Statistical analysis of microstructural entropy factors for experimental steel 1.

Microstructural Entropy Factors

ν0 νavg q n uf uc Annealing Temperature

(°C)/Time (s)

800 °C /10 s 0.004 1.144 0.0033 0.997 0.156 0.719

800 °C /60 s 0.006 2.212 0.0033 0.997 0.174 0.522

800 °C /300 s 0.0013 2.770 0.0033 0.997 0.222 0.809

800 °C /1000 s 0.0013 5.947 0.0033 0.997 0.056 0.847

900 °C /10 s 0.006 14.250 0.0054 0.995 0.102 0.559

900 °C /60 s 0.003 18.420 0.0054 0.995 0.046 0.658

900 °C /300 s 0.0013 19.890 0.0054 0.995 0.110 0.650

900 °C /1000 s 0.001 27.198 0.0054 0.995 0.121 0.325

1000 °C /10 s 0.0008 150.845 1.4019 4.488 0.0079 0.0227

1000 °C /60 s 0.0005 3800.247 1.4019 4.488 0.0007 0.0014

1000 °C /300 s 0.0002 8000.648 1.4019 4.488 0.0004 0.0011

1000 °C /1000 s 0.0004 31425.701 1.4019 4.488 0.0002 0.0007

Table 6.2B: Statistical analysis of microstructural entropy factors for experimental steel 2.

Microstructural Entropy

Factors ν0 νavg q n uf uc

Annealing Temperature

(°C)/Time (s)

800 °C /10 s 0.010 0.862 0.003 0.997 0.097 0.573

800 °C /60 s 0.008 0.983 0.003 0.997 0.057 0.422

800 °C /300 s 0.008 2.339 0.003 0.997 0.065 0.616

800 °C /1000 s 0.010 4.567 0.003 0.997 0.035 0.562

900 °C /10 s 0.013 9.120 0.013 0.987 0.107 0.358

900 °C /60 s 0.010 15.365 0.013 0.987 0.037 0.218

900 °C /300 s 0.008 25.248 0.013 0.987 0.110 0.525

900 °C /1000 s 0.008 45.128 0.013 0.987 0.041 0.227

1000 °C /10 s 0.005 350.265 1.836 3.196 0.004 0.014

1000 °C /60 s 0.005 1626.387 1.836 3.196 0.0014 0.0061

1000 °C /300 s 0.005 7500.254 1.836 3.196 0.0004 0.0020

1000 °C /1000 s 0.005 45000.847 1.836 3.196 0.0002 0.0006

83

Table 6.2C: Statistical analysis of microstructural entropy factors for experimental steel 3.




(°C)/Time (s)

800 °C /10 s 0.015 0.525 0.0006 0.9994 0.112 0.418

800 °C /60 s 0.019 0.933 0.0006 0.9994 0.093 0.399

800 °C /300 s 0.019 0.946 0.0006 0.9994 0.117 0.359

800 °C /1000 s 0.019 1.139 0.0006 0.9994 0.146 0.398

900 °C /10 s 0.030 3.021 0.0060 0.9940 0.175 0.755

900 °C /60 s 0.022 7.069 0.0060 0.9940 0.156 0.849

900 °C /300 s 0.015 10.256 0.0060 0.9940 0.162 0.819

900 °C /1000 s 0.022 14.845 0.0060 0.9940 0.154 0.336

1000 °C /10 s 0.0118 94.610 0.1520 0.8208 0.0155 0.0161

1000 °C /60 s 0.0060 400.365 0.1520 0.8208 0.0070 0.0093

1000 °C /300 s 0.0013 537.899 0.1520 0.8208 0.0060 0.0098

1000 °C /1000 s 0.0013 1394.615 0.1520 0.8208 0.0022 0.0077

Table 6.2D: Statistical analysis of microstructural entropy factors for experimental steel 4.




(°C)/Time (s)

800 °C /10 s 0.040 0.421 0.0012 0.9988 0.193 0.263

800 °C /60 s 0.040 0.601 0.0012 0.9988 0.150 0.359

800 °C /300 s 0.040 0.708 0.0012 0.9988 0.132 0.373

800 °C /1000 s 0.070 1.548 0.0012 0.9988 0.128 0.388

900 °C /10 s 0.150 3.202 0.0038 0.9962 0.119 0.353

900 °C /60 s 0.200 6.408 0.0038 0.9962 0.132 0.367

900 °C /300 s 0.400 12.776 0.0038 0.9962 1.475 4.609

900 °C /1000 s 1.800 139.611 0.0038 0.9962 0.016 0.027

1000 °C /10 s 0.800 33.808 0.0641 0.9316 0.043 0.066

1000 °C /60 s 0.800 67.586 0.0641 0.9316 0.027 0.036

1000 °C /300 s 0.900 145.031 0.0641 0.9316 0.012 0.033

1000 °C /1000 s 1.200 382.843 0.0641 0.9316 0.007 0.015

84

Table 6.2E: Statistical analysis of microstructural entropy factors for experimental steel 5.




(°C)/Time (s)

800 °C /10 s 0.060 0.826 0.0015 0.9985 0.007 0.569

800 °C /60 s 0.060 0.886 0.0015 0.9985 0.084 0.255

800 °C /300 s 0.060 1.147 0.0015 0.9985 0.128 0.254

800 °C /1000 s 0.0857 2.257 0.0015 0.9985 0.061 0.445

900 °C /10 s 0.070 1.565 0.0050 0.9950 0.114 0.403

900 °C /60 s 0.070 1.850 0.0050 0.9950 0.166 0.354

900 °C /300 s 0.080 3.902 0.0050 0.9950 0.147 0.328

900 °C /1000 s 0.090 9.670 0.0050 0.9950 0.156 0.287

1000 °C /10 s 0.150 6.818 0.0215 0.9781 0.123 0.152

1000 °C /60 s 0.200 12.180 0.0215 0.9781 0.081 0.105

1000 °C /300 s 0.300 41.298 0.0215 0.9781 0.035 0.060

1000 °C /1000 s 0.400 74.344 0.0215 0.9781 0.023 0.046

Table 6.3A: Experimental yield strength of steel 1.


(T, °C) 800 °C 900 °C 1000 °C

Annealing time, (t, s)

10 s 738 MPa 587 MPa 454 MPa

60 s 717 MPa 558 MPa 377 MPa

300 s 684 MPa 532 MPa 358 MPa

1000 s 658 MPa 499 MPa 354 MPa

Table 6.3B: Experimental yield strength of steel 2.


(T, °C) 800 °C 900 °C 1000 °C


10 s 751 MPa 563 MPa 388 MPa

60 s 683 MPa 528 MPa 362 MPa

300 s 648 MPa 498 MPa 324 MPa

1000 s 618 MPa 461 MPa 299 MPa

85

Table 6.3C: Experimental yield strength of steel 3.


(T, °C) 800 °C 900 °C 1000 °C


10 s 722 MPa 639 MPa 444 MPa

60 s 706 MPa 584 MPa 347 MPa

300 s 693 MPa 556 MPa 332 MPa

1000 s 650 MPa 495 MPa 316 MPa

Table 6.3D: Experimental yield strength of steel 4.


(T, °C) 800 °C 900 °C 1000 °C


10 s 792 MPa 639 MPa 522 MPa

60 s 755 MPa 558 MPa 462 MPa

300 s 698 MPa 531 MPa 423 MPa

1000 s 663 MPa 508 MPa 394 MPa

Table 6.3E: Experimental yield strength of steel 5.


(T, °C) 800 °C 900 °C 1000 °C


10 s 782 MPa 633 MPa 534 MPa

60 s 725 MPa 606 MPa 499 MPa

300 s 650 MPa 547 MPa 410 MPa

1000 s 639 MPa 453 MPa 393 MPa

grain diameter (davg). Using computation tools (Matlab programming), the microstructure entropy

(S*) of steels 1-5 were calculated using the factors in Tables 6.2A-6.2E and presented in Tables

6.4A-6.4E.

Additionally, based on Hall-Petch relationship, the plots for average grain diameter (davg) and

yield strength for five steels are presented in Figure 6.3. Equations (6.24)-(6.28) are the

86

Table 6.4A: Microstructure entropy S* of steel 1.


(T, °C) 800 °C 900 °C 1000 °C


10 s 4.88 7.46 12.15

60 s 5.66 8.17 15.84

300 s 6.36 9.11 17.50

1000 s 6.72 10.14 18.18

Table 6.4B: Microstructure entropy S* of steel 2.


(T, °C) 800 °C 900 °C 1000 °C


10 s 4.11 6.47 11.16

60 s 4.67 7.31 12.69

300 s 5.23 7.81 14.22

1000 s 5.79 8.61 16.01

Table 6.4C: Microstructure entropy S* of steel 3.


(T, °C) 800 °C 900 °C 1000 °C


10 s 3.42 3.69 8.99

60 s 3.77 4.09 11.11

300 s 3.82 5.15 12.93

1000 s 3.98 6.45 13.89

Table 6.4D: Microstructure entropy S* of steel 4.


(T, °C) 800 °C 900 °C 1000 °C


10 s 2.33 2.98 3.74

60 s 2.62 3.38 4.44

300 s 2.78 4.00 5.08

1000 s 2.99 4.35 5.77

87

Table 6.4E: Microstructure entropy S* of steel 5.


(T, °C) 800 °C 900 °C 1000 °C


10 s 2.27 2.99 3.81

60 s 2.65 3.20 4.11

300 s 2.92 3.82 4.92

1000 s 3.10 4.64 5.22

corresponding linear relationship between avgd

1 (davg: average grain diameter) and yield

strength for the five experimental steels.

)(

)(409)(241)(:1Steel

2/1

md

mMPaMPaMPa

avg

S

(6.24)

)(

)(398)(208)(:2Steel

2/1

md

mMPaMPaMPa

avg

S

(6.25)

)(

)(407)(175)(:3Steel

2/1

md

mMPaMPaMPa

avg

S

(6.26)

)(

)(357)(248)(:4Steel

2/1

md

mMPaMPaMPa

avg

S

(6.27)

)(

)(505)(58)(:5Steel

2/1

md

mMPaMPaMPa

avg

S

(6.28)

88

6.3 Discussion

The grain growth state in self-similar regime is defined by microstructure entropy in terms of

grain growth dynamics. We used the microstructure entropy theory in terms of bimodal grain

Figure 6.3: Hall-Petch relationship for five different steels.

89

Figure 6.4: Relationship between microstructural entropy (S*) and average grain size (davg) for

five different steels.

growth structure to calculate the microstructure entropy S* of steels with bimodal structure. The

aim was to explore the relationship between microstructure entropy S* and yield strength. The

90

application of thermodynamics of microstructural evolution applied to bimodal grain size

distribution is envisaged to predict yield strength, which can be successfully used to design steels

and heat treatment, enabling prediction of yield strength. Now we summarize the microstructure

entropy in terms of grain growth dynamics that occurs at higher temperature and longer times and

modify some factors in our bimodal grain structure distribution. The computation (Matlab

programming) involved modified factors (relative grain volume) and equations. For 5 different

chemical compositions of steels with bimodal grain structure, 6 factors (ν0, vavg, q, n, uf, uc) were

statistically obtained from EBSD and HKL-Channel 5 system in terms of grain size and grain

numbers. In this case, 60 sets of data presented in Tables 6.2A-6.2E were analyzed. Additionally,

the relationship between average grain diameter (davg) and yield strength for five steels is presented

in Figure 6.3 and equations (6.24)-(6.28), which suggested that the five steels followed Hall-Petch

relationship. 60 microstructure entropy S* were obtained for the bimodal structure (Tables 6.4A-

6.4E). The data confirmed that grain growth that occurs during annealing for different chemical

compositions and temperature-time combination is a self-similar evolution.

The relation between microstructure entropy S* versus average grain diameter (davg) of

steels 1-5 is presented in Figure 6.4. Based on line fitting results, it is suggested that *

1

S and

avgd

1have a linear relationship. Thus, we can elucidate the relation between microstructure

entropy S* and yield strength. It is pertinent to mention that the plot of S* and davg indicated a non-

linear relationship (not presented). Thus, S* is not linearly related to davg.

91

Figure 6.5: Relationship between microstructural entropy (S*) and experimental yield strength

for five different steels.

92

Experimental yield strength versus *

1

S is shown in Figure 6.5 for the 5 steels.

Interestingly, we also observe a linear relationship between *

1

S and yield strength based on

line fitting results. Equations (6.30)-(6.34) are the corresponding linear relationship between

*

1

S and yield strength. We know that yield strength is inversely proportional to the square-root

of grain diameter, as described by the Hall-Petch relationship [158,159], and is given by:

)(

)()()(

2/1

0md

mMPakMPaMPaS

(6.29)

where d is the average grain diameter of a polycrystalline material, 0 and k are constants.

*

190175)(:1Steel

SMPaS (6.30)

*

1853164)(:2Steel

SMPaS (6.31)

*

141862)(:3Steel

SMPaS (6.32)

*

1729344)(:4Steel

SMPaS (6.33)

*

1741361)(:5Steel

SMPaS (6.34)

From equations (6.29)-(6.34), we modify the well-known Hall-Petch equation from the

perspective of bimodal grain structure. Thus, we propose equation (6.35), when the microstructure

entropy S* of a grain structure is known, the yield strength can be predicted if the constant can be

93

estimated. Specifically, we take the fraction of ultrafine and coarse grains into consideration

because their fraction governs the yield strength of steel. High fraction of grain boundary provides

higher strength. We modify the Hall-Petch relationship and incorporate microstructure entropy S*

as follows:

*)(

S

kCMPaS

(6.35)

where S* is microstructure entropy, C and k′ are constant.

We are currently focusing on developing an understanding of constant C and k′ in equation

(6.35).

6.4 Conclusions

1. We adopted the concept of microstructure entropy, S*, a phenomenological thermodynamic

parameter to understand yield strength in metallic alloy systems with bimodal grain size

distribution that can be extended to alloy systems other than bimodal distribution (i.e. tri-modal

grain size distribution).

2. Six factors namely, ν0, vavg, q, n, uf and uc, were derived from the theory of microstructure

entropy and calculated using the experimental data to derive an alternate form of Hall-Petch

relationship for understanding yield strength in metallic systems with distribution of grain size.

3. Thermodynamics of microstructure evolution for grain growth based on microstructure entropy,

S*, was used to develop an understanding of microstructure entropy in systems with varying grain

size.

4. Computational tool (Matlab programming) was used to calculate the microstructure entropy

from the statistical analysis of 60 sets of bimodal structure in a self-similar regime.

94

5. A linear relationship between yield strength and inverse of square-root of microstructure entropy

was obtained, similar to Hall-Petch relationship.

6. A generic equation *

)(S

kCMPaS

is proposed that can be widely used for any grain

distribution to predict the yield strength in future.

95

Chapter 7: Conclusions and future work

7.1 Conclusions

Austenitic stainless steel with and without Cu including nano/ultrafine grain and coarse

grain and Co-Cr-Mo alloy with and without Zr were used to find the interplay between grain

structure, chemical elements and osteoblast functions on cell-substrate interactions.

Secondly, we apply grain growth dynamics to 60 sets of bimodal structure for five different

austenitic stainless steel of chemical composition in a self-similar regime based on the

thermodynamics of microstructure evolution (grain growth) and embracing microstructure

entropy. The different grain size distribution was used to understand the variation in microstructure

entropy in the alloy system with bimodal grain size distribution. The ultimate objective was to

develop a relationship between microstructure entropy and Hall-Petch equation, which can be

successfully used to predict yield strength.

7.1.1 Favorable Modulation of Osteoblast Cellular Activity on Zr-modified Co-Cr-Mo

Alloy: The Significant Impact of Zirconium on Cell-Substrate Interactions

The evidence that the presence of Zr in Co-Cr-Mo alloys indicated favorable cellular

response of osteoblasts is a step in the right direction to promote their application for the

fabrication of biomedical devices.

Experiments on the effect of Zr in Co-Cr-Mo alloys clearly demonstrated that adhesion,

spread and morphology of osteoblasts was favorably modulated, confirming the beneficial

impact of Zr in the alloy.

The difference in the cellular activity of Zr-free and Zr-containing alloys is attributed to

the presence of an adherent and stable ZrO2 layer on the surface that prevents release of

ions from the bulk metal to the physiological environment, consistent with our recent

96

observation of enrichment of Zr on the surface. This aspect is also in agreement with the

thermodynamic ability of Zr to form oxide in a manner similar to other traditional metals,

Ti, Cr and Nb.

The cellular response additionally demonstrated stronger expression of vinculin focal

adhesion contacts and actin stress fibers, in conjunction with fibronectin.

The excellent strength and ductility of Co-Cr-Mo-Zr alloy combined with biological

compatibility and favorable modulation of cellular activity would encourage future

application of cobalt-based alloys.

7.1.2 Favorable modulation of osteoblast cellular activity on Cu-containing austenitic

stainless steel and comparison with the Cu-free counterpart

The study successfully explored the biological functions of antimicrobial Cu-containing

stainless steel and compared with the Cu-free steel, in terms of cell attachment and

synthesis of proteins.

Cu-containing steel was beneficial for cell attachment, viability, proliferation and

demonstrated stronger expression of vinculin and fibronectin proteins and actin stress

fibers in comparison to Cu-free steel, confirming the beneficial impact of Cu in austenitic

stainless steel.

Stainless steel with Cu is envisaged to release Cu ions and is instrumental in killing bacteria

and encouraging biological functionality.

The study underscores that copper can be added to biomedical alloys from the perspective

of promoting cellular functions.

97

7.1.3 Favorable modulation of osteoblast cellular activity on austenitic stainless steel with

nano/ultrafine grains and comparison with micrometer austenitic grains counterpart

Severe cold deformation of conventional coarse-grained biomedical austenitic stainless

steel followed by annealing for short durations enabled NG/UFG stainless steel to be

obtained with high strength-high ductility combination.

NG/UFG austenitic stainless steel was characterized by greater attachment of cells on the

surface as compared to the CG austenitic stainless steel. Furthermore, higher fibronectin

intensity and expanded network of fibronectin, higher expression level of vinculin and actin

stress fibers were observed on NG/UFG steel in relation to the CG austenitic stainless steel.

These observations suggested that the grain size of steel plays a significant role on cell

attachment, proliferation and morphology. Smaller grain size steel favorably modulates

cell development and cell attachment.

The underlying reason for favorable modulation of cellular response is attributed to high

fraction of high energy grain boundaries and relatively higher hydrophilic nature of

NG/UFG austenitic stainless steel.

The study advances our understanding on the cellular response to biomaterials.

7.1.4 Microstructure entropy guided understanding of yield strength in alloy systems

characterized by grain size distribution

We adopted the concept of microstructure entropy, S*, a phenomenological thermodynamic

parameter to understand yield strength in metallic alloy systems with bimodal grain size

distribution that can be extended to alloy systems other than bimodal distribution (i.e. tri-

modal grain size distribution).

98

Six factors namely, ν0, vavg, q, n, uf and uc, were derived from the theory of microstructure

entropy and calculated using the experimental data to derive an alternate form of Hall-

Petch relationship for understanding yield strength in metallic systems with distribution of

grain size.

Thermodynamics of microstructure evolution for grain growth based on microstructure

entropy, S*, was used to develop an understanding of microstructure entropy in systems

with varying grain size.

Computational tool (Matlab programming) was used to calculate the microstructure

entropy from the statistical analysis of 60 sets of bimodal structure in a self-similar regime.

A linear relationship between yield strength and inverse of square-root of microstructure

entropy was obtained, similar to Hall-Petch relationship.

A generic equation *

)(S

kCMPaS

is proposed that can be widely used for any

grain distribution to predict the yield strength in future.

7.2 Future work

Microstructure entropy guided understanding of yield strength can be applied in alloy

systems.

99

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Vita

Na Gong is presently pursuing her Doctor of Philosophy in Department of Metallurgical,

Materials and Biomedical Engineering, with an emphasis in Material Science and Engineering at

the University of Texas at El Paso. She earned her Bachelor of Material Forming and Control

Engineering from Wuhan University of Science and Technology, China in 2015. In January of

2018, she received her Master of Material Science and Engineering from University of Science

and Technology Beijing, China.

Until now, she has authored and co-authored over 20 peer-reviewed publications in

international journals.

The publications related to her dissertation topic are as follows:

1 Gong, N., Misra, R. D. K., Niu, G., & Wu, H. (2020). Acta Materialia.

2 Gong, N., & Misra, R. D. K. (2019). Materials Technology: Advanced Performance

Materials, 1-10.

3 Gong, N., Montes, I., Nune, K. C., Misra, R. D. K., Yamanaka, K., Mori, M., & Chiba, A.

(2020). Journal of Biomedical Materials Research Part B: Applied Biomaterials, 108(4), 1518-

1526.

4 Gong, N., Hu, C., Hu, B., An, B., & Misra, R. D. K. (2020). Journal of the mechanical

behavior of biomedical materials, 101, 103433.

Contact Information: [email protected]

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