ABSTRACT
The kinetics of adsorption of mucin onto titanium surface was investigated. The amount of
titanium, concentration of mucin solution, reaction temperature and pH were optimised by
preliminary experiments as 0.02 mg, 0.1 mg/ml, 35 °C and 6.4, respectively. The amount of
mucin adsorbed increased with time until equilibrium was attained after about 115 minutes
with the maximum concentration of mucin adsorbed being 0.0603 mg/ml for Ca-Ti followed
by that of unto K-Ti implying that the adsorption is not influenced by the presence of Ca²
ions. Analysis of kinetic data obtained with Lagergren, Bhattacharya/Venkobacharya and
Flick’s models revealed that the adsorption is first order with respect to mucin molecules and
that diffusion is the predominant mechanism for the adsorption.
TABLE OF CONTENTS
itle page i
Declaration ii
Certification iii
Acknowledgements iv
Dedication v
Abstract vi
Table of Contents vii
List of Figures x
List of Appendices xii
Abbreviations xiv
CHAPTER ONE
1.0 INTRODUCTION 1
1.1 Adsorption of Protein on Solid Substrates 1
1.2 Biomaterials Application 2
1.3 Biocompatibility of Implants 3
1.4 Titanium: Occurrence, Chemistry and Biomaterial Application 4
1.5 Mucin 7
1.6 Research Objective 9
CHAPTER TWO
2.0 LITERATURE REVIEW 11
2.1 Protein Adsorption and Interfaces 11
2.2 Surface Charge 12
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2.3 Hydrophobic Effects 13
2.4 Protein Structure and Stability 13
2.5 Adsorption Measurement 14
2.6 Proteins-Dye Binding 15
2.7 Adsorption of Proteins on Surfaces 16
2.8 Rate Expression and Order of Reaction 19
2.9 Kinetic Rate Constant 21
CHAPTER THREE
3.0 MATERIALS AND METHODS 24
3.1 Reagents 24
3.1.1 Phosphate Buffer Saline Solutions (0.02 M, pH 3 – pH 8) 24
3.1.2 Mucin Solutions 24
3.1.3 Bovine Serum Albumin Solution 24
3.1.4 Calcium Chloride Solution 25
3.1.5 Potassium Chloride Solution 25
3.2 Standard Calibration Curve 25
3.3 Optimisation of Variables Used in the Adsorption Studies 25
3.3.1 Optimisation of Mass of Ti 25
3.3.2 Optimisation of the Concentration of Mucin Solution 26
3.3.3 Optimisation of Temperature for the Adsorption of Mucin onto Ti 26
3.3.4 Optimisation of pH for the Adsorption of Mucin 27
3.4 Pre-treatment of Ti 28
3.5 Kinetic Studies: Effect of Contact Time on Adsorption of Mucin onto
Treated and Untreated Ti (Rates of Adsorption) 28
3.6 Adsorption Rate Constants 28
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CHAPTER FOUR
4.0 RESULTS AND DICUSSION 31
4.1 Standard Calibration Curve of BSA 31
4.2 Optimisation of Variables Used in the Kinetic Studies 31
4.2.1 Optimisation of Mass of Ti 31
4.2.2 Optimisation of the Concentration of Mucin in the
Adsorption of Mucin onto Ti 33
4.2.3 Optimisation of Temperature for the Adsorption of Mucin
onto Ti 35
4.2.4 Optimisation of pH for the Adsorption of Mucin onto Ti 37
4.3 Kinetics Study of Adsorption of Mucin onto Ti 39
4.3.1 Effect of Incubation Time and pH on the Adsorption
of Mucin onto Un-Ti, Ca–Ti and K–Ti (Rates of Adsorption) 39
4.3.2 Adsorption Rate Constant 42
CHAPTER FIVE
5.0 SUMMARY, CONCLUSION AND RECOMMENDATION 55
5.1 Summary 55
5.2 Conclusion 55
5.3 Recommendation 56
REFERENCES 57
APPENDICES 67
CHAPTER ONE
INTRODUCTION
1.1 Adsorption of Protein on Solid Substrates
The adsorption of dissolved proteins on solid substrates is a widespread phenomenon
(Kasemo, 1998). For many applications, such as protein chromatography, the use of medical
implants or preparation of solid- phase immunoassays for medical diagnostic tests, it is
important to understand the driving forces for the protein adsorption process and the effect of
adsorption on the protein conformation and activity. This may help to develop new
biocompatible materials or coatings which prevent or favour protein adsorption (Horbett and
Brash 1995).
Surface-protein adsorption is being studied with regard to the total amount and composition
of adsorbed protein, the thickness and uniformity of the surface coverage, the kinetics of the
protein- adsorption process, the preferential adsorption of certain protein species from
complex mixtures such as desorption, organisation and arrangement of adsorbed protein and
the denaturation of adsorbed proteins by interfacial forces (Brash, 1981). However, the
relationship of a surface toward protein adsorption and the significance of the adsorbed
protein layer in a material’s thrombogenic nature still remain ill-defined and need to be
studied (Andrade and Hlady, 2004).
It is now widely recognised that biocompatibility in a number of situations is controlled to a
large extent by the initial interfacial reactions that occur when the biomaterial comes into
contact with tissues, especially on the protein adsorption process that takes place at an
interface. This is more significantly demonstrated with the thrombogenesis that occurs when
a foreign material comes in contact with blood (Lee and Kim, 1974). A considerable amount
of data is available concerning the role of protein adsorption in the initiation of clotting on
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polymer surfaces (Brash, 1981). Adsorption is an important factor in the context of response
of soft tissues to implanted polymers (Bagnash, 1977).
The adsorption of proteins onto titanium is important in a variety of oral biological events.
Salivary proteins (e.g.mucin) are selectively adsorbed onto tooth surfaces forming the
acquired enamel pellicle (Mayhall, 1970). The pellicle influences the initial attachment of
microorganisms to the tooth surface and remains interposed between the enamel and dental
(Wang et al., 1997). In research, proteins are receiving increased interest as potential
inhibitors of enamel or dentine demineralisation (Diana et al., 1995).
1.2 Biomaterials’ Application
Biomaterials are used to repair, restore or replace a damaged or diseased tissue. Biomaterials
may be of natural origin, such as purified collagen, protein fibres, polysaccharides and treated
tissues. They could also be synthetic biomaterials, such as metals, ceramics or polymers
which are often called biomedical biomaterials to differentiate them from natural biomaterials
(Yannas and Burke, 1980).
The earliest recorded use of biomaterial was nearly 4000 years ago, as sutures’ for wound
repair. Ancient Indians of about 2,500 years ago used hair, leather, cotton, animal sinew and
tree bark as suture materials. The use of gold wire suture in 1550 was the first recorded
application of a biomedical material and was followed by increasingly wide use of metal
plates and pin to repair broken bones (Lyman et al., 1974).
Recent advances in materials science and surgery now make it possible to rebuild many parts
of the human body. The synthetic polymer industry has expanded and polymeric materials
with vast properties are available. Some of these polymers have mechanical properties that
resemble those of natural tissues, making them suitable as biomedical materials with many
applications such as intracorporeal (implanted) materials and extracorporeal materials.
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Biomaterial science encompasses aspects of physical sciences, engineering, biology and
medicine (Black, 1998).
1.3 Biocompatibility of Implants
Traditionally, the primary focus of the search for biomedical materials has been on finding
biocompatible materials. A biocompatible biomaterial is one that is inert toward the
physiological environment (Black, 2006). This has been modified to include materials having
minimal interaction with the environment (Hench, 1991). A biomaterial may be compatible
in one application for a specific purpose but not in a similar application at a different site
(Kamimski et al., 1981). Therefore, successful application of a biomaterial as a medical
device requires both an acceptable biological response to the implanted materials and absence
of physiologically induced damage. This is the measure of the overall interaction between
biomaterials and living systems (Black, 1984).
Adverse biological responses to an implanted biomedical material including excessiveforeign
body response, thrombogenicity, and immunogenic, antileukotactic (predisposition
towards infection) and mutagenic complications have seen reported (kambie, 1993.)
The defence mechanisms of the body must be considered in the application of implant
materials. This is because physiological responses do not exhibit uniquely differentiable
stages. The responses are continuous and often integrated among various defence
mechanisms against injury or invasion of foreign materials (Williams and Bagnall, 1981).
The correlation between biomaterial surface and blood compatibility has been much studied.
A review by Andrade (1985 a) indicates that no foreign material can ever be truly blood
compatible, but that differences in the extent of the response and its relationship to proper
device function could be exploited.
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Performances in an implant or extracorporeal devices are the ultimate tests of a biomaterial.
In addition to performance, it is the evaluation that should determine any adverse reactions
are short- and long-term effects of the physiological environment on the implant. The
reactions of implants to their host depend on the chemical and physico- chemical properties
of the material as well as on the site ions and type of implantation. The biocompatibilities of
biomaterials also include the entire field of bio-safety and bio-functionality (Williams and
Bagnall, 1981).
The international standard organisation (ISO), in directive 10993 (ISO, 1992), described the
methods for biological evaluation of medical devices as the bases for assessment of
biocompatibility. Also, the guidelines for physicochemical characterisation of biomaterials, in
directive 80-216 (NIH, 1980), explained the level –II surface characterisation test. This is the
technique that provides information on surface chemistry morphology and interfacial
properties of implants to be used to screen biomedical applications.
1.4 Titanium: Occurrence, Chemistry and Biomaterial Application
Titanium (Ti) with atomic number 22 and atomic weight 47.90 is a member of the first series
of transition elements of group 4A of the periodic table. It is the ninth most plentiful in the
lithosphere and the fourth most common structural metal following aluminium, iron and
magnesium. It constitutes 0.63% of the earth’s crust and has been detected in sea water, most
plants and in all forms of animal life.
Titanium was discovered by Williams Gregory, an English mineralogist in 1790. Five years
later Martins Heinrich Klaproth found that a Hungarian mineral, rutile, was the oxide of a
new element which proved to be identical to that of Gregory. The name, titanium, from the
Titans of mythology, was assigned to the new element of Klaproth (Noe, 1972). More than
one hundred years passed before any use was made of the new element. It was first employed
5
in the United States as an additive to iron and steel; titanium (IV) oxide pigments became
available in 1918.
Titanium has five naturally occurring isotopes and four artificial radioactive ones. It is widely
distributed in rocks and minerals; the common commercial sources of minerals are rutile and
ilment. Titanium occurs as titanium (IV) oxide (TiO2) in nature as the mineral rutile, brookite
and anatase which have the same formula TiO2, but vary in crystal habit. Titanium also
occurs in the form of titanates, the most extensive deposits being ferrous titanate, (Fe Ti03),
ilment or calcium titanate (perovskite) (CaTiO3) and calcium titanium silicate (CaO.
TiO2.SiO) (Bamberger, 1980; Deeson, 1973).
Titanium, despite its common occurrence, proved difficult to prepare in a pure state. This is
because, though it is electrochemically active (Barksdale, 1980), it cannot be electrodeposited
from aqueous solution. The high strength to weight ratio and good corrosion resistance make
it attractive for aeroplanes and chemical application and technology have advanced rapidly in
the last decade (Scheffer, 1981). It has good resistance to air at about 650 °C; at higher
temperatures, it is contaminated by oxygen and becomes a brittle metal. It does not react with
nitrogen.
Powerful oxidants such as hydrogen per-oxide (70-90%) attack titanium. It is resistant to
aqueous solutions of oxidizing agents like nitric acid, chromic and hypochlorous acids.
Titanium resists most aqueous salt solutions like chlorides, hypochlorite, sulphides, sulphates,
nitrates, phosphates and others. Resistance is limited to fluorides, hot concentrated solutions
of aluminium, calcium, magnesium and zinc chlorides (Jaffee, 1973). These chlorides
hydrolyse and become acidic and since they are none oxidising, do not favour the formation
of a protective film. Although, it resists most neutral and oxidising environments, it has little
resistance to non-oxidising acids. Reducing conditions can impair the protective films and
break them down with a resultant corrosion if hydrogen ions are reduced on the surface.
6
Many materials act as inhibitors against corrosive attack and several titanium alloys have
better resistance to specific corrodents than the pure metals (Noe, 1972).
Metallic titanium is readily soluble in hydrofluoric acid but violent if the acid added is much.
With fluoroboric acid the reaction is complete and less violent if the acid is cold. Sulphuric
acid reacts with it slowly at room temperature and faster at elevated temperatures.
Hydrochloric acid dissolves titanium slowly at room temperature and more rapidly at
elevated temperature. Titanium and its inorganic compounds are non –toxic, unless the
associated radicals are toxic or the compounds are strongly acidic or basic (Scheffer, 1981).
According to Byan (2006), the high strength, low weight outstanding corrosion resistance
possessed by titanium and titanium alloys have led to a wide range of successful applications
which demand high levels of reliable performance in surgery and medicines as well as in
aerospace, automotive, chemical plant, power generation, oil and gas extraction, sports and
other industries.
More than 1,000 tonnes (2.2 million pounds) of titanium devices of every description and
function are implanted in patients worldwide every year. Requirements for joints replacement
continue to grow as people live longer or damage themselves more through hard sports or are
seriously injured in road traffic and other accidents. By being light, strong and totally
biocompatible, titanium is one of few materials that naturally match the requirement for
implantation in the human body.
‘Fit and forget’ is an essential requirement where equipment in critical applications, once
installed, cannot readily be maintained or replaced. There is no more challenging use in this
respect than implants in human body. The effectiveness and reliability of the implants, and
medical and surgical instruments and devices are essential factors in saving lives and in the
long term, relief suffering and pain. Titanium is judged to be completely inert and immune to
corrosion by all body fluids and tissue, and is, thus, wholly – bio-compatible.
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The natural selection of titanium for implantation is determined by a combination of
favourable characteristics which include immunity to corrosion, bio-compatibility, strength,
low modulus and density and the capacity for joining with bone and other tissue
(osseointegration). The chemical and physical properties of titanium alloys combine to
provide implants which are highly damage-tolerant.
Forms and materials specifications are detailed in a number of international and domestic
specifications, including ASTM and BS7252/ISO05832. Other applications of titanium
implants are in bone and joint replacement, dental implants, Maxillofacial and craniofacial
treatments, cardio-vascular devices, external prosthesis and surgical instruments (Byan,
2006).
1.5 Mucin
Mucin is a glycoprotein with two major characteristics:
(1) A high content of O-linked oligosaccharides (the carbohydrate content of mucin is
generally more than 50%), and (2) the presence of repeated amino acid sequences (tandem
repeats) in the centre of their polypeptide backbones, to which the o-glycan chains are
attached in clusters. These sequences are rich in serine, threonine and proline (Brockhausen
and Kuhns, 1997).
Both secretory and membrane –bound mucin occur. The former is found in the mucus present
in the secretion of the gastrointestinal (pancreatic, small intestine and salivary), respiratory
and reproductive tracts (Rosalind and Stuart, 1980). Mucus consists of about 94% water and
5% mucin, the remainder being a mixture of various cell molecules, electrolytes and
remnants of cells. Secretory mucin generally has an oligometric structure and often has a very
high molecular mass. The oligomers are composed of monomers linked by disulphide bonds.
Mucus exhibits a high viscosity and, often is a gel. These qualities are a function of its mucin
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content. The conformations of mucin often become those of rigid rods. The high content of
sulphate residues found in many mucins confers a negative charge on them. With regards to
function, mucins help to lubricate and form a protective physical barrier on epithelial
surfaces.
Membrane- bound mucin participates in various cell–cell interactions. The density of
oligosaccharide chains makes it difficult for proteases to approach their polypeptide
backbones, making mucin to be resistant to their action. Mucin tends to “mask” certain
surface antigens. Many cancer cells form excessive amounts of mucin; perhaps the mucin
masks certain surface antigens on such cells and thus protects the cells from immune
surveillance. Mucin also carries cancer-specific peptides and carbohydrate epitopes (an
epitope is a site on an antigen recognised by an antibody, also called antigenic determinant).
Some of these epitopes have been used to stimulate an immune response against cancer cells
(Robert and Murray, 2000).
1.6 Research Objective
It is well known that serum proteins adsorb to inorganic surfaces almost immediately on
contact with blood following surgical implantation and that the original surfaces are therefore
no longer in contact with the host tissue (Albrektsson and Sennerby, 1990). However, little is
known about the specificity of this process and still less is known about the proteins that
adsorb on the titanium surfaces. In terms of the host tissue-biomaterial interaction, the role of
adsorbed proteins is an important one since subsequent events depend on the composition and
conformation of the protein layer and its tendency to change over time (Kotharis, 1995).
Lori and Hanawa (2004) monitored the adsorption of the simplest amino acid, glycine, on
gold and titanium surfaces in Hank’s solution using electrochemical quartz crystal
microbalance (EQCM) and observed adsorption of more glycine on titanium than on gold.
9
Adsorption of protein has been thoroughly studied by the use of different techniques
(Ivarsson and Lundstrom, 1986). Bentaleb et al. (1998) studied the kinetics of the
homogeneous, exchange of alpha-lactalbumine protein adsorbed on titanium oxide surface
with radio-labelling technique and summarised that the protein release rates were higher than
the pure desorption rates.
It has already been demonstrated that proteins are able to influence the rate of metal ion
release (Clark and Williams, 1982). There are many ways in which the chemistry of the metal
could control the biological responses, for example, by the influence of released metal ions
on the immune system (McNamara and Williams, 1982), on chemotaxis (McNamara et al.,
1983), on the integrity of cell membranes (Rae, 1975) and on the activity of intracellular
enzymes (Rae, 1978). One further possibility is that the proteins present in the exudates that
accumulate around the implant could be adsorbed onto the metal surface and thereby
influence the subsequent development of the biological response (Williams et al., 1985). It is
a reasonable hypothesis that protein- metal interactions are important in the determination of
the tissue response. Although several studies have been made on the Ti-protein interactions
for the development of body implants, for example, studies of the mechanism of adsorption
of mucin on Ti surface reported by Lori and Nok (2004); Kinetic study of the adsorption of
lactalbumine on TiO2 was reported by Bentaleb et al. (1998). Kotharis (1995) investigated
the adsorption of several serum proteins on TiO2. There has not been any report however, on
the kinetics of adsorption of mucin on Ti metal surface. Since mucin is a glycoprotein present
in the saliva and damaged parts where titanium metal is mostly used as implant, it is very
necessary to study the kinetics of mucin adsorption onto Ti so as to know the correlation
between it and the Ti in terms of the kinetic parameters. Due of the dearth of published work
on this to confirm or deny the above hypothesis; this research work is carried out with the
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aim of kinetically studying the adsorption of mucin onto the Ti surface. The following
objectives will achieve the above aim through investigation of
1) rate constants,
2) rates of adsorption,
3) the effect of pH on rates and rate constants,
4) the effect of Ca2+ ion on rates and rate constants, and
5) the effect of K ion on rates and rate constants.
Scientific data generated will help in understanding the adsorptive rates of mucin on titanium
metal as a biomedical material.
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