POLITEHNICA University of Bucharest

Faculty of Medical Engineering

MASTERAL THESIS

Development of Ceramic Engineered Surfaces and Analysis of Their

Suitability for Biomedical Applications

Master Student:
Michael SKOKIN

Scientific Coordinator:
Lecturer Dr. Eng. Cristina BUSUIOC

July 2019
Bucharest
 POLITEHNICA University of Bucharest
Faculty of Medical Engineering

ABSTRACT

To increase their suitability for implant materials, titanium-based alloys have received a
superficial vitroceramic coating. This study describes the methods of fabrication and tests the
biocompatibility and bioactivity of surfaces as to improve a material suitability as an implantable
device. Titanium targets were coated with a ceramic material mix composed of SiO2, P2O5, CaO,
MgO, ZnO and CaF2, deposited upon the targets as a ceramic coating via Pulsed Laser
Deposition. It was found that a higher substrate temperature and a lower work pressure resulted
in the highest quality film. The final product was characterized via SEM, EDX and FTIR. The
SEM pictures were used in quantifying the quality of the surfaces though the use of JIMAGE, a
machine vision application. Finally, the products’ biocompatibility was assessed and they were
found to be biocompatible by several tests.

ACKNOWLEDGEMENTS

I would like to thank the entire teaching staff of the Faculty of Medical Engineering for
providing genuinely useful ideas. Special thanks go to Project Coordinator, Lecturer Dr. Eng.
Cristina BUSUIOC, for her encouragement, patience, valuable advice and technical expertise in
ceramic coated thin films and directing this project and the staff of National Institute for Laser
Plasma and Radiation Physics for contributing with the deposition experiments, as well as the
researchers from “Horia Hulubei” National Institute for Physics and Nuclear Engineering for
assisting in evaluating the biological side of the study.

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TABLE OF CONTENTS

ABSTRACT ................................................................................................................................................................... 2
ACKNOWLEDGEMENTS .............................................................................................................................................. 2
1) INTRODUCTION .............................................................................................................................................. 4
2) PULSED LASER DEPOSITION ....................................................................................................................... 6
Principles .................................................................................................................................................................... 7
Equipment .................................................................................................................................................................. 9
Variables................................................................................................................................................................... 13
Method of characterization of surface growth resulting from PLD ......................................................................... 14
Medical applications................................................................................................................................................. 16
3) BIOCOMPATIBILITY ANALYSIS METHODOLOGY ............................................................................... 17
Biocompatibility and cytotoxicity assay .................................................................................................................. 18
In vitro cell culture analysis ..................................................................................................................................... 20
Alamarblue™ proliferation assay............................................................................................................................. 20
Immunofluorescence of vinculin and actin .............................................................................................................. 20
4)THIN FILM CERAMIC COATING METHODOLOGY .................................................................................... 22
5) RESULTS AND DISCUSSION ...................................................................................................................... 27
Results ...................................................................................................................................................................... 27
SEM imaging of obtained ceramic thin films ........................................................................................................... 27
EDX analysis of obtained ceramic thin films ........................................................................................................... 30
FTIR analysis of obtained ceramic thin films .......................................................................................................... 33
XRD analysis of obtained ceramic thin films........................................................................................................... 36
SEM Image Processing ............................................................................................................................................ 37
Biocompatibility evaluation of obtained ceramic thin films .................................................................................... 38
Discussion ................................................................................................................................................................ 40
6) FUTURE RESEARCH..................................................................................................................................... 42
7) CONCLUSION ................................................................................................................................................ 42
BIBLIOGRAPHY .................................................................................................................................................... 43
ACRONYM LIST .................................................................................................................................................... 46

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1) INTRODUCTION

The field of biomaterials is continuously increasing due to the high demand of an aging
population, as well as the increasing average weight of people. Biomaterials are artificial or
natural materials that are used to restore or replace the loss or failure of a biological structure to
recover its form and function in order to improve the quality and longevity of human life [1].

Biomaterials are used for various applications in different parts of the human body, as
artificial valves in the heart, stents in blood vessels, and mainly as hard tissues as replacement
implants in shoulders, knees, hips, elbowsand dental structures. Thin coatings are studied to
improve implant fixation of metallic or ceramic materials in bone, connective tissue, oral mucosa
or skin[2].

Among all implants, the highest number of applications is for spinal, hip and knee
replacements. It is estimated that by the end of 2030, the number of total hip replacements will
rise by 174% (572,000 procedures) and total knee arthroplasties are projected to grow by 673%
from the present rate (3.48 million procedures). This is due to the fact that human joints suffer
from degenerative diseases, such as osteoarthritis (inflammation in the bone joints), osteoporosis
(weakening of the bones) and trauma leading to pain or loss in function. The degenerative
diseases lead to degradation of the mechanical properties of the bone due to excessive loading or
absence of normal biological self-healing process. Artificial biomaterials are the solution to these
problems and the surgical implantation of these artificial biomaterials of suitable shapes help
restore the function of the otherwise functionally compromised structures.

Until recently, titanium (Ti) was considered to be one of the best available materials to be
used in repairing injured and diseased bone tissue. Prostheses and similar artifacts were used for
implant devices to replace patients’ hard tissues. This preference was afforded based upon a
number of in vivo and in vitro experiments with various grades of Ti that concluded that
commercially pure Ti is a biocompatible material because its surface properties result in the
spontaneous build-up of a stable and inert oxide layer. The main physical properties of Ti
responsible for the biocompatibility are:
• low level of electronic conductivity;
• high corrosion resistance;
• thermodynamic state at physiological pH values (7.4);

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• low ion-formation tendency in aqueous environments;

• an isoelectric point of the oxide of 5-6.

Additionally, the passive-film-covered surface is only slightly negatively charged at

physiological pH, and Ti has a dielectric constant comparable to that of water with the
consequence that the Coulomb interaction of charged species is similar to that in water[3].Initial
studies found that Ti has the rare property of osseointegration, allowing bone to firmly grow over
and attach itself to the implant and securing it to the body, which is especially important in load-
bearing implants. However, further studies found that the initial studies were optimistic. Several
drawbacks, like fibrous tissue growth, slow osteointegration and reduced close contact with the
bone were noticed upon wider use of Ti implants.

Ti-based alloys

Originally, implant devices were manufactured from pure Ti. Although for some
applications pure Ti was a suitable material, for many others it was found that it did not have the
required mechanical properties. Thus occurred the need for alloys with superior mechanical and
biological properties. For some time, Ti-6Al-4V [4]was favored for biomedical applications.
However, this material was observed to have biological drawbacks when used for permanent
implant applications because of possible toxic effect resulted from released vanadium and
aluminum into the body.

Some vanadium- and aluminum-free alloys have been considered for implant
applications, these new alloys being Ti-13Nb-13Zr [4] and Ti-12Mo-6Zr[4].

Although mechanical properties can be improved by alloying Ti, biocompatibility must

always be kept in mind. There have been found cases where alloying element leached form
implant to human body, causing harm. The current wide use of Au-based implant devices use its
already proven biocompatibility and corrosion resistance. The ability of this material to adhere to
ceramic parts can reduce both the weight and the cost of medical components [5].

The need for finding a solution to preventing the leaking of metallic ions from implants
into a biological medium is still ongoing. The direction researchers worldwide are looking at
currently as being the most promising is the coating of implantable artifacts made of pure Tiwith
a thin layer of glass, ceramic or vitroceramic materials.

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A team of researchers, among which Dr. Sorin Jinga and Dr. Cristina Busuioc [6], have
pioneered a method of coating pure Ti substrates with various vitroceramic materials by using
the pulsed laser deposition method.

2) PULSED LASER DEPOSITION

In the field of thin films deposition, there are several methods that produced quality
results. Somewhat related methods are molecular beam epitaxy and sputter deposition [7]. The
Pulsed Laser Deposition (PLD) technique was invented to use elements from botht hese methods
and combines laser physics with plasma physics to irradiate a target with a laser, transform it into
plasma, and then, deposit it on the substrate in a controlled manner [7]. By controlling the
parameters that influence the process, the desired outcome can also be controlled.

In spite of these significant advantages, industrial uptake of PLD has been slow and to
date most applications have been confined to the research environment. There are basically three
main reasons for this:

1. The plasma plume created during the laser ablation process is highly forward-directed,
therefore the thickness of material collected on a substrate is highly non-uniform and
the composition can vary across the film. The area of deposited material is also quite
small, typically ~1cm2, in comparison to that required for many industrial applications
which require area coverage of ~(7.5×7.5) cm.
2. The ablated material contains macroscopic globules of molten material, up to ~10μm
diameter. The arrival of these particulates at the substrate is obviously detrimental to
the properties of the film being deposited.
3. The fundamental processes, occurring within the laser-produced plasmas, are not fully
understood. Thus, deposition of novel materials usually involves a period of empirical
optimization of deposition parameters. [8]

PLD has several characteristics that make it the choice technique in oxide thin film
research. The main features that make PLD capable for complex material film growth are
stoichiometric transfer of material from the target, generation of energetic species, hyperthermal
reaction between the ablated cations and the background gas in the ablation plasma and

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compatibility with background pressures ranging from ultrahigh vacuum to 1 Tor. The
equipment is relatively simple and the initial setup is easy to perform [7].

However, PLD applications are limited in size by the fact that they must be enclosed
inside the vacuum chamber.

When compared with other thin layer, PLD-deposited films have shown superior results.
In an experiment [9][2], PLD of hydroxyapatite (HA) was compared with Plasma Spraying (PS).
The Ti rods used were grit-blasted to increase surface roughness, and then, HA coated using
PLD or PS. The rods were implanted in the tibias of rabbits and harvested 24 weeks after
implantation with the surrounding bone. HA-PLD showed the highest percentage of bone contact
and bone linear density and the lowest lacunae contact and lacunae linear density (Fig. 1).

Fig. 1. SEM image of HA-PLD implant surface in contact with surrounding bone [9].

Principles

PLD is a physical vapor deposition process, carried out in a vacuum system. Fig. 2 shows
the principles behind PLD.
A pulsed laser is focused onto a target of the material to be deposited. High laser energy
density is necessary, so each laser pulse ablates a small amount of the material, creating a plasma
plume. The plasma is a cloud of free electrons and negatively charged ions.

The ablated material is ejected from the source in a highly forward-directed plume. The
ablation plume provides the material flux for film growth. Ablation conditions are chosen such
that the ablation plume consists primarily of atomic, diatomic, and other low-mass species. This

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is typically achieved by using an ultraviolet (UV) laser wavelength and nanosecond pulse width
that is strongly absorbed by a small volume of the target material [7].

Fig. 2. Principle and steps of PLD process [7].

The thickness distribution of material on target from a stationary plume is lacking

uniformity due to the highly forward-directed nature of the ablation plume. In its simplest form,
the distribution of material deposited from the ablation plume is symmetric with respect to the
target surface normal and can be described in terms of a cosine distribution.

Raster scanning of the ablation beam over the target and/or rotating the substrate produce
much more uniform film coverage over larger areas.

The detailed mechanisms of PLD are very complex, including the ablation process of the
target material by the laser irradiation, the development of a plasma plume with high energetic
ions, electrons, as well as neutrals and the crystalline growth of the film itself on the heated
substrate. The process of PLD can generally be divided into four stages:
• laser absorption on the target surface and laser ablation of the target material and

creation of a plasma;
• dynamic of the plasma;

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• deposition of the ablation material on the substrate;

• nucleation and growth of the film on the substrate surface.

Each of these steps is crucial for the crystallinity, uniformity and stoichiometry of the
resulting film. The most used methods for modelling the PLD process are Monte Carlo
techniques.

Equipment

To a large extent, the first two problems have been solved. Films of uniform thickness
and composition can be produced by rastering the laser spot across the target surface and/or
moving the substrate during deposition. Line-focus laser spots have also been used to obtain
large area coverage. The particulate material was initially removed from the plume using a
mechanical velocity filter, although recently more elaborate techniques, involving collisions
between two plasma plumes or off-axis deposition, have been used to successfully grow
particulate-free films. The third problem is resolved by the development of computer simulations
to describe PLD and will further our understanding of the fundamental physics and chemistry
involved in the deposition process. However, a large amount of experimental data, obtained
under accurately controlled and defined conditions, is required to assist the verification of such
models. Fig. 3 displays a representative image of a PLD equipment, device found at Nebraska
Center for Materials and Nanoscience, USA.

Fig. 3. Image of a PLD equipment [10].

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A number of different deposition techniques are used for the production of thin films.
Each technique has some limitations, such as the pressure region, ability to deposit metallic or
dielectric materials, restricted use of reactive gases, problems with the homogeneity of the films
created, limited deposition process control and differing kinetic energies in the active species.
Some techniques can also struggle with crystallinity, the stoichiometric transfer of complicated
materials, and the creation of highly oriented structures, nanocomposites and nanocrystals. To
overcome some of the limitations and to find new possible deposition methods, several
combined deposition techniques are used.

Hybrid technologies are used, for example, to improve surface structure, stoichiometry,
morphology and adhesion, and to enable the fabrication of nanocomposites. Some laser-based
hybrid techniques used in biomaterials research are discussed below.

Matrix Assisted Pulsed Laser Evaporation (MAPLE)

Matrix Assisted Pulsed Laser Evaporation(MAPLE) is a relatively new method of

physical vapor deposition, similar to PLD. MAPLE is used for the fabrication of organic layers.
One drawback of using standard PLD to produce films is that the direct ablation of the target can
be stressful to fragile materials, which may consequently break during the process. MAPLE was
developed to decrease the photochemical damage caused by the direct interaction of the UV laser
light with the organic or biomaterial target and to overcome the difficulties in solvent-based
coating technologies, such as inhomogeneous films, inaccurate placement of material and
difficult or incorrect cryogenically frozen composite target consisting of a dilute mixture of the
material to be deposited and a high vapor- pressure solvent. The low-fluence laser pulse interacts
mainly with the volatile solvent, causing it to evaporate. During the process, the solute desorbs
without any significant decomposition, and is then uniformly deposited on the substrate[11].

Several different polymeric and biomaterial films have been prepared in various studies
using the MAPLE technique and their applications tested in electronic devices, passivation
coatings, chemical and biological sensors, tissue engineering, pharmaceutical and bioengineering
applications, drug delivery systems or wound healing [12].

PLD + Radiofrequency (RF) + Hollow Cathode Discharge

Hybrid PLD + Radiofrequency (RF) discharge were first used for the synthesis of
superhard carbon nitride films. PLD was combined with capacitively or inductively coupled RF

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discharge (13.56 MHz), which was sustained between the ‘life electrode’ (substrate holder)and
the chamber (‘ground electrode’), as illustrated in Fig. 4. A graphite target was used and the
chamber was filled with nitrogen. In addition, two flat rectangular electrodes were tested, which
were situated between the target and substrate, parallel to the plasma plume. The same scheme
was used for the low temperature production of crystalline TiO2 layers for use in a urethral
catheter. In order to combine PLD with RF and hollow cathode discharge, one electrode was
fabricated in the shape of a cylinder and RF power, when applied to the hollow cylindrical
electrode, produced a stream of excited nitrogen gas.

Fig. 4. Scheme of PLD + Radiofrequency + Hollow Cathode Discharge [13].

PLD + Magnetron Sputtering (MS)

Another example of hybrid PLD is the combination of PLD and Magnetron Sputtering
(MS), known as PLD-MS deposition and shown in Fig. 5. The streams of materials from the
PLD target and the MS target intersect on the substrate. The combination of high energetic
material flow from PLD (kinetic energy of species up to 1 keV) and low energetic material flow
from MS (around 5 eV) allows the synthesis of materials with new properties under
technologically reasonable conditions. By changing the laser repetition rate and the magnetron
power, the flows of materials on the target can be modified and gradient layers created; these are
layers with a special material distribution along a thickness profile and nanostructured
multilayers. PLD-MS was used to synthesize TiC, TiCN and SiC films [13].

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Fig. 5. Scheme and image of PLD + Magnetron Sputtering [13].

PLD + PLD

Double PLD hybrid systems are based on the simultaneous application of two lasers and
involve the deposition of material from two different targets, as depicted in Fig. 6.

Fig. 6. Scheme and image of PLD + PLD [13].

By changing the repetition rate and energy density of the lasers, multilayer systems can
be easily fabricated, along with doped and graded layers. The quality of the films can also be
influenced by the time synchronization of both streams of materials from the targets. If careful
synchronization is carried out, both streams of material can reach the implant (substrate) at the
same time, and the surface chemistry and properties of the layers are different from those
produced when the material streams are not synchronized. The double PLD technique was tested
inthe fabrication of Cr-doped double-layer coatings [13].

PLD + Ion Gun

All PLD systems can be combined with an ion beam gun. Ion energy of up to 300 eV is
usually applied and the process can contribute to grow modifications and subsequent increases in

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film density and crystallinity, as well as improvements in morphology. The parameters involved,
such as ion gas, ion energy and ion current, can be changed to achieve a film with optimum or
specifically required properties. Fig. 7 shows the use of the ion gun in the modification of
double-layer coating films [13].

Fig. 7. Scheme and image of PLD + Ion Gun [13].

Variables

There are numerous factors that may affect the quality of the thin film deposited through
PLD. These variables may be classified into material variables and equipment variables.

Material variables:
- target material chemistry; type of material (metal, polymer, ceramic, vitroceramic);
policrystal vs. single crystal; melting point; vaporization point; thermal conductivity;
surface energy; surface reflectivity; homogeneity (possible mass defects - closed
pores), thickness;
- substrate on which the target material is to be deposited; chemistry; surface roughness;
thickness; thermal conductivity; heat capacity;
- working gas (chemistry, temperature, gas pressure); in [14], Si-substituted
hydroxyapatite (Si-HA) films were deposited on Si and Ti substrates by PLD in the
presence of water vapor atmosphere.

Equipment Variables can be subdivided into laser, and enclosure and auxiliary
equipment.

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Laser. It is desirable to have a high energy laser operating as close to the target as
possible, with a small spot to ensure maximum energy density on target. It is natural that this
application would use pulsed lasers rather than continuous wave lasers. Thus, the following
parameters are to be considered: maximum energy per pulse; total number of pulses; pulse
frequency; pulse shape; pulse time, spot area; laser wavelength (UV is preferred in many cases);
distance.

Equipment variables: operating pressure (high vacuum) or working gas pressure;

energy losses within the system; angle of incidence for laser; substrate temperature; heat post-
treatment; ability to rotate the target; rotation speed of target; ability to raster the substrate.

For ceramic targets, PLD is most easily achieved by lasers operating in the UV spectrum.
High-energy ultraviolet laser pulses can be readily provided via excimer lasers or frequency-
tripled or quadrupled Nd:YAG solid-state lasers. The amount of film growth per laser pulse will
depend on multiple factors, including target-substrate separation, background gas pressure, laser
spot size and laser energy density. Under typical conditions, the deposition rate per laser pulse
can range from 0.001 to 1 Å per pulse.

Method of characterization of surface growth resulting from PLD

The rate and quality of growth can be seen by applying Reflection High-Energy
Electron Diffraction (RHEED). RHEED provides a means of determining the crystallinity and
smoothness of a surface, and oscillations in the intensity of diffraction spots during film growth
correlate to the atomic layer-by-layer growth of the material.

Fig. 8. RHEED measurements performed on a PLD deposited film [7].

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The two graphs above (Fig. 8) represent the deposition of single unit cells of the oxide
film. Specular RHEED intensity is dependent on the spatial coherence of the surface atoms. As
layer-by-layer growth cycles through submonolayer coverage of the surface, RHEED intensity
decreases, while for completed layers, the intensity is high. The oscillations indicate that unit cell
by unit cell growth on an atomically flat surface is occurring. The superimposed time-dependent
substructure in the RHEED intensity corresponds to surface redistribution of ablation plume
species that have condensed on the surface from an individual ablation pulse. The time
dependence of this structure yields insight into the nucleation and growth of the film at the
submonolayer level for the arrival of each ablation plume [7].

Infrared (IR) Spectroscopy is a fast, simple chemical analysis technique based on the
interaction of IR light with matter. This technique is sensitive to the presence of chemical
functional groups in a sample, thereby allowing identification of structural fragments of the
molecules.

Infrared spectroscopy is based on the interaction of electromagnetic radiation with

molecules. Two conditions need to be met for molecules to absorb IR light and cause absorption
in the IR spectrum:
1. Molecules must possess functional groups with a permanent dipole moment, resulting
from electric charge asymmetry of the molecule. The alternating electric field vector
of the IR light induces vibration of the bond by the transfer of energy to the molecule.
The absorption of the radiation intensity at certain frequency can be observed in the IR
spectrum. Molecules without a permanent dipole moment can also interact with IR
radiation, as the interaction of the electric field vector makes the charge of the
molecule asymmetrical, leading to temporary formation of the so-called induced
dipole moment.
2. The quantum theory implies that molecules can have only specifically allowed or
quantized vibrational energies. As a result, a necessary condition for IR radiation
absorption to occur is that the energy of the IR light impinging on a molecule must be
equal to a vibrational energy level difference.

Most molecules have functional groups that absorb radiation in the mid-IR range, which
is found between 4000 and 400 cm-1. It should be mentioned that for spectroscopy, the

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wavelength of the light is usually expressed in wave numbers (cm-1), defined as the reciprocal of
the wavelength.

Most of the intense features found in IR spectra can be ascribed to fundamental

transitions of the molecule, and the correlation between these bands and the chemical structure is
the basis of IR spectroscopy being a powerful tool for chemical analysis. For the identification of
unknown molecules from their IR spectra, several characteristics of the absorption bands should
be carefully considered (the position, intensity and full width at half maximum) [2].

Medical applications

Thin calcium phosphate (CaP) coatings improve the drawbacks of the first-generation
biomedical materials that are, by definition, inert materials that are used to replace diseased or
missing hard tissue.

Second-generation biomedical materials elicit a controlled action in the physiological

environment. Second-generation materials are systematically designed as bioactive glasses. The
first such mineral - composed of Na2O-CaO-P2O5-SiO2 - was synthesized by Hench et al.[15] in
1971. These materials rely on a concept opposite to that for inert materials. They are bioactive
and display mechanisms of controlled dissolution and stepwise ion exchange at the surface,
leading to a cascade of steps. They lead to the deposition of carbonated hydroxyapatite (HA) at
the implant surface and the development of a bond to collagen fibers and mineralized tissue in
the extracellular matrix (ECM). The principal phenomenon exhibited by these bioactive glasses
and glass-ceramics is the strong bond between the implanted material and the surrounding tissue
(the mineralized or unmineralized ECM)[2].

Some studies [16] obtained Si-substituted HA thin films on Ti surfaces in situ by

magnetron sputtering deposition, a technique that predates PLD + Magnetron Sputtering, and
they were tested in vitro with excellent results.

In [17], octacalcium phosphate and Mn2+-doped carbonate HA thin films were deposited
by PLD on pure, highly polished and chemically etched Ti substrates. Obtained data
demonstrated that both CaP coatings favor osteoblasts proliferation, activation of their
metabolism and differentiation.

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In [18], a study on amorphous CaP coatings revealed that the crystallinity of amorphous
CaP coatings that were produced by PLD from targets of nonstoichiometric HA (Ca/P=1.70)
improved as different post-deposition heat treatment temperatures were used, starting from 300
to 450, 525 and 650 °C.

Bioactive materials include dopants. One that was found to produce particularly good
results is magnesium (Mg) ions present in several bioactive glasses. Mg already exists in the
human body. Dentin and bone contain 0.44, 1.23 and 0.72 wt.% of Mg, respectively. Moreover,
Mg not only is essential for cellular and enzymatic reactions, but also directly affects the
bonemineralization process and mechanical properties of human bone. Glass samples with
composition of 28CaO-10MgO-4P2O5-58SiO2 (mol%) were synthesized by sol-gel method and
deposited upon a substrate of Ti6Al4V by PLD. The results of in vitro assays showed that with
increased immersion time, the lathlike and spherical apatite particles were gradually precipitated,
indicating that the prepared Mg containing glass film provided a promising candidate as implant
materials [19].

3) BIOCOMPATIBILITY ANALYSIS METHODOLOGY

It is of utmost importance that the materials created are biocompatible. The two main
factors that influence the biocompatibility of a material are the host response induced by the
material and the material degradation in the biological system environment. According to the
tissue reaction phenomena, the biocompatibility of orthopedic implant materials was classified
into three categories by Heimke [20], such as biotolerant, showing distant osteogenesis (bone
formation with indirect contact to the material), bioinert, showing contact osteogenesis (bone
formation with direct contact to the material), and bioactive, showing bonding osteogenesis
(bone formation with chemical or biological bonding to the material) [1].

When implants are exposed to human tissues and fluids, several reactions take place
between the host and the implant material and these reactions dictate the acceptability of these
materials by the organism (Fig. 9). The issues with regard to biocompatibility are: thrombosis,
which involves blood coagulation and adhesion of blood platelets to biomaterial surface, and the
fibrous tissue encapsulation of biomaterials that are implanted in soft tissues.

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Fig. 9. Biological effects of a biomaterial [1].

Biocompatibility and cytotoxicity assay

Rafieerad et al.[21] describe a technique applied in studying the biocompatibility and in

vitro cytotoxicity of coated composite films. This in vitro cell adhesion test was performed on
the HA-coated and HA-ZrO2-coated samples, using well-characterized hFOB cell lines
[hFOB1.19SV40 transfected osteoblast] provided by the American Type Culture Collection
(ATCC, Rockville, MD). The cells were seeded at an initial density of 1×104 cells/mL on
samples, in minimum essential medium supplemented with 10% human fetal bovine serum
(Gibco, NY, U.S.A), under standard cell culture conditions (100 U/mL penicillin and 100 μg/mL
streptomycin in 5% CO2 and 95% air atmosphere at 37°C), in an incubator. The samples were
autoclaved for sterilization purposes at 121°C for 20 min. After 48 h, the specimens were rinsed
with 0.1 M phosphate buffer saline solution to remove the non-adherent cells, while the adherent
cells were fixed with 4% glutaraldehyde for 2 h at room temperature. The fixed samples were
then dehydrated with graded concentration of ethanol 40, 50, 60, 70, 80, 90 and 100% for three
times, followed by the addition of 0.5 mL of hexamethyldisilane (HMDS) to maintain the
original morphology of the cell. The dried samples were sputter-coated with platinum and were
subjected to SEM examination(Fig. 10) to evaluate the adhesion of the hFOB cells [21].

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Fig. 10. SEM image of cell attachment on HA substrate [24].

The apatite formation of the HA-coated and HA-ZrO2-coated samples can be appraised
by soaking the samples in SBF. The type of ions and their concentrations in SBF mimic those in
the human blood plasma and is prepared by referring to the method developed by Kokubo
[22](Table 1) in his study on the chemistry of bioactive glass ceramics.

Table 1. Composition of SBF developed by Kokubo [22].

This medium contains reagent grade CaCl2, K2HPO4·3H2O, NaCl, KCl,MgCl2·6H2O,

NaHCO3 and Na2SO4 dissolved in distilled water and the pH adjusted to 7.4. The samples are
immersed in solution and kept at 37°C for 5 days at a surface area to volume ratio of 1 cm2/mL.
The SBF solution is refreshed every 24 h and is kept colorless and stable without any deposit
during the immersion period to maintain the ionic concentration. After immersion for 5 days, the
samples are removed from the SBF solution, washed with distilled water dried in air, stored in
vacuum at 80°C and analyzed by SEM [21].

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In vitro cell culture analysis

Thin CaP coatings of implant material can be investigated in in vitro test systems using
primary cells or cell lines in culture and even more reliable results would be produced from in
vivo investigations on animal models.

Human osteoblast-like (HOB) cells, obtained from human hipbone by surgical operations
(Promocell, UK) were used to study the biocompatibility of the uncoated Ti substrates, as-
deposited and heat-treated Si-HA thin films. These cells were cultured in McCoy’s 5A medium
containing 10% foetal calf serum, 1% glutamine and Vitamin C (30 mg/mL). Before seeding, all
the specimens were soaked in ethanol for 48 h before treatment with UV radiation for 30 min as
a sterilization procedure. A specimen was placed in each well of the24-wells tissue culture plate
(TCP) before seeding with 1×104 cells/test. HOB cells seeded on TCP were used as a test
control. The cells were then incubated at 37 °C in a humidified atmosphere of 95% air and 5%
carbon dioxide.

Alamarblue™ proliferation assay

The ability of uncoated Ti substrates, as-deposited and heat-treated Si-HA thin films to
support the growthof HOB cells was evaluated by the alamarBlueTM proliferation assay at days
3, 7 and 14. After each time point, cells were cultured in 10% alamarBlueTM medium (Serotec,
Oxford, UK) for 4 h. The absorbance of the specimens was measured on a plate reader at a
wavelength of 570nm with a reference wavelength of 600 nm. A total of 6 replicates was
performed for each specimen.

Immunofluorescence of vinculin and actin

After 4 days of culturing, the cells on the uncoated Ti substrates, as-deposited and heat-
treated Si-HA thin films were fixed with 4% paraformaldehyde/phosphate buffer solution (PBS)
with 1% sucrose for 15 min before washing with PBS and finally, permeated at 4 °C for 5 min.
The specimens were then incubated with 1% bovine serum albumin (BSA)/PBS at 37 °C for 5
min, followed by the addition of anti-vinculin primary antibody (1:100 in 1% BSA/PBS,
monoclonal anti-human raised in mouse (IgG1), Sigma, Poole, UK) at 37 °C for 1 h. FITC

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conjugated phalloidin (1:100 in 1% BSA/PBS, Sigma, Poole, UK) was concurrently added at this
incubation duration. After washing with 0.5% Tween 20/PBS for 5 min, a secondary biotin
conjugated antibody (1:50 in 1% BSA/PBS, monoclonal anti-mouse raised in rabbit (IgG),
Vector Laboratories, Peterborough, UK) was added at 37 °C for 1 h. The specimens were again
washed three times with 0.5% Tween 20/PBS for 5 min before a Texas red conjugated
stretavidin (1:50 in 1% BSA/PBS, Vector Laboratories, Peterborough, UK) was added at 4 °C
for 30 min. The specimens were given a final wash (5 min) before mounting on the fluorescent
mountant (Vector Laboratories, Peterborough, UK) and viewed under a Leica SP2 Confocal
Laser Scanning Microscopy (CLSM).

CLSM images illustrating actin cystoskeleton and vinculin focal contacts on uncoated Ti
substrates, as-deposited and heat-treated Si-HA thin films at culture day 4 are presented in Fig.
11. FITCphalloidin stains the actin cytoskeleton (green) and Texas red stretavidin stains the
vinculin focal contacts (red) [16]. FITC is a small organic molecule used as a biomolecular
reagent, and is typically conjugated to proteins via primary amines (i.e., lysines).

Fig. 11. CLSM images illustrating actin cystoskeleton and vinculin

focal contacts on uncoated Ti substrates, as-deposited and heat-treated
Si-HA thin films at culture day 4 [16].

The morphology of HOB cells and the specimen surfaces on the uncoated Ti substrates,
as-deposited and heat-treated Si-HA thin films after 4, 20 and 56 days of culture were subjected
to SEM examination. After each incubation time point, the specimens were rinsed with PBS after
decanting the media and finally freeze-dried. The specimens were then coated with a thin layer
of carbon before examination using SEM.

In their assay of biocompatibility and toxicological response, [23] used the MTT assay
and GSH assay luminescence test. The MTT assay is a colorimetric assay for assessing cell

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metabolic activity. NAD(P)H-dependent cellular oxidoreductase enzymes may, under defined

conditions, reflect the number of viable cells present. These enzymes are capable of reducing the
tetrazolium dye MTT 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide to insoluble
formazan, which has a purple color. The GSH assay is an indicator of the oxidative stress that
can lead to apoptosis or cell death. Reduced glutathione (GSH) is a tripeptide (γ-
glutamylcysteinylglycine) that contains a free thiol group. GSH is a major tissue antioxidant that
provides reducing equivalents for the glutathione peroxidase (GPx) catalyzed reduction of lipid
hydroperoxides to their corresponding alcohols and hydrogen peroxide to [Link] the GPx
catalyzed reaction, the formation of a disulfide bond between two GSH molecules gives rise to
oxidized glutathione (GSSG). The enzyme glutathione reductase (GR) recycles GSSG to GSH
with the simultaneous oxidation of β-nicotinamide adenine dinucleotide phosphate (β-
NADPH2).When cells are exposed to increased levels of oxidative stress, GSSG will accumulate
and the ratio of GSH to GSSG will decrease. Therefore, the determination of the GSH/GSSG
ratio and the quantification of GSSG are useful indicators of oxidative stress in cells and tissues
[24].

4)THIN FILM CERAMIC COATING METHODOLOGY

A thin film ceramic coating applied to a hard tissue takes advantage of the fact that
ceramics are, generally speaking, considered biologically inert. This can be an advantage, but, to
a certain, it can also be a hindrance, because the substrate-biological system does not facilitate
the biointegration of such a composite system. The essential requirement for an artificial material
to bond to living bone is the formation of bone-like apatite on its surface when implanted in the
living body and that this in vivo apatite formation can be reproduced in a simulated body fluid
(SBF) with ion concentrations nearly equal to those of human blood plasma. This means that in
vivo bone bioactivity of a material can be predicted from the apatite formation on its surface in
SBF [25].

This drawback is resolved by a novel technique called Self-Assembled Monolayers

(SAM) that allows the chemical functionalization of the ceramic thin film to create a biologically
active tissue-facing surface layer. The direct functionalization of the ceramic that may promote
tissue-interaction is highly beneficial [26].

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Material target was used from a previous experiment, documented by Izabela

CONSTANTINOIU [27] in her Bachelor Degree Dissertation, under the supervision of Dr.
Cristina BUSUIOC.

For the manufacture of thin films by PLD, the thin film to be deposited must have a
precise composition, treated to a well-controlled densification process.

This material was obtained through the sol-gel method, which gives the material several
advantages compared with the traditional method of melting followed by controlled
crystallization. Thus, the material will have in-depth porosity, aiding the Si-OH functional
groups that decorate the surfaces to fix HA deposition.

The chemical composition of the material, together with all the precursors, are listed in
Table 2 below.

Table 2. Target composition and used precursors [22].

• SiO2 helps by forming a vitreous structure with crystalline phases, useful for
material biocompatibility;
• P2O5 helps by forming a vitreous structure; it is a basic component of natural bone
and it has a strong influence on biointegration;
• CaO helps by acting as a network modifier and by generating crystalline phases,
which have positive effects upon biocompatibility and bioactivity;
• MgO helps by acting as a network modifier, thus improving the material
mechanical properties in a biological setting;

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• ZnO, in trace quantities, positively affects crystallization processes and increases

the material opacity;
• CaF2 is a filling additive and it helps with biocompatibility and bioactivity.

According to the relationships of thermal equilibrium and phase that exist in the SiO2-
CaO-MgO system (Fig. 12) and accounting for the proposed mix, it is expected that the mix of
crystalline phases obtained will be akermanite (Ca2MgSi2O7) and/or diopsid (CaMgSi2O6).

Fig. 12. Phase diagram of the SiO2-CaO-MgO oxide system [22].

The TEOS precursor was hydrolyzed in acidic medium (the pH was adjusted with HNO3
to between 1 and 2) while magnetic stirring was applied. CaF2 was added to the mix and stirred
until the materials dissolved. The TEP precursor was hydrolyzed separately, stirred and then
mixed with a solution used for dissolving nitrates in a small quantity of water. The two solutions
were mixed and stirred for 1 h. The product was allowed to jellify for 12 h. The gel was aged by
drying it at 80°C for 24 h. Next, the gel was calcined at 600 °C for 2 h, for the purpose of
eliminating all organic residues and obtaining only the amorphous or crystalline phases. The
temperature and time were selected based upon previous thermal analysis of the material.

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For the purpose of attaining densities as high as possible for the final substrate, the
calcined powder was molded in 13 mm diameter cylinders by uniaxial pressing at 50 MPa. Next,
the cylinders were pre-sintered at 1000 °C for 2 h. Upon cooling, the material was crushed to a
fine powder. The powder was granulated with a 2% polyvinyl alcohol solution. The powder was
molded and pressed into pill-like disks, some with 13 mm diameter and others with 25 mm
diameter by uniaxial pressing at 150 MPa. The 13 mm disk probes were used for optimizing the
parameters of the sintering thermal treatment process. Thus, three combinations of sintering
parameters were tested:1100 °C / 2 h,1200 °C / 2 h and 1200 °C / 10 h. All with a heating
increase rate of 2 °C/min and natural cooling.

Following the analysis of the result, it was decided to use the sintering treatment with the
highest temperature and for the longest period: 1200 °C for 10 h.

Thus, the XRD pattern of the sintered target (Fig. 13a) indicates the presence of
akermanite as major crystalline phase and diopside as minor crystalline phase; the specimen
contains crystallites of akermanite with an average size of 40 nm. The sintering temperature
promotes the emergence of compact blocks with irregular shape; the high magnification image
reveals both the intergranular and intragranular porosity (Figs. 3b and c). [26]

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Fig. 13. (a) XRD pattern and (b and c) SEM images at different magnifications of the sintered target [26].

The material was deposited upon thin, pure Ti substrates with a laser using the constant
and variable parameters listed in Tables 3 and 4.

Table 3. Values of constant parameters.

Constant parameter Value

Laser wavelength 532 nm

Pulsed laser frequency 10 Hz

Total number of pulses per sample 26,000 pulses

Energy/pulse 81 mJ

Table 4. Values of variable parameters.

Sample Substrate temperature (°C) Atmosphere pressure O2(mTorr)

Film A 250 100

Film B 350 100

Film C 250 200

Film D 350 200

Fig. 14 shows the four physical samples, or films, as they are prepared to be observed
under Scanning Electron Microscopy (SEM).The films have been sputtered with gold to make
them conductive and thus visible under SEM.

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Fig. 14. Image of the four thin film to be observed in SEM.

5) RESULTS AND DISCUSSION

Results

SEM imaging of obtained ceramic thin films

Upon SEM analysis, it was relevant to note that the samples show evidence of the
deposition process, with spherical agglomerations of particles, consistent with the laser
sputtering process (Figs. 15, 16, 17, 18). Based upon visual interpretation of the SEM images,
compared to the image of the bare Ti strip, Film B at 350 °C and 100 mTor shows the best
growth. Film D shows the most agglomeration. At lower magnifications, the substrate may be
visible and the distribution of the ceramic material seems uneven in places.

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Fig. [Link] images of Film A at two different magnification levels, before and after SBF immersion.

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Fig. [Link] images of Film B at two different magnification levels, before and after SBF immersion.

Fig. [Link] images of Film C at two different magnification levels, before and after SBF immersion.

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Fig. [Link] images of Film D at two different magnification levels, before and after SBF immersion.

EDX analysis of obtained ceramic thin films

EDX analysis shows that all the films show identical composition, as can be seen from
the spectra below (Figs. 19, 20, 21, 22).

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Fig. 19. Chemical composition of Film A.

Fig. 20. Chemical composition of Film B.

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Fig. 21. Chemical composition of Film C.

Fig. 22. Chemical composition of Film D.

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FTIR analysis of obtained ceramic thin films

Fourier Transform Infrared Spectroscopy (FTIR) is a technique that can identify the
composition of a material or the presence of contaminants in a given material. It can also be used
for identifying specific functional groups that may exist at the surface of the sample, for example
carbonyl, carboxyl, hydroxyl or thiols.

Below, in Fig. 23, there is an image with all four samples FTIR spectra, obtained on a
Thermo Scientific Nicolet 6700 spectrophotometer. The information used in processing these
graphs was provided. The information was modified through the signal processing filter, a filter
used in Origin 8 to smooth out this type of information.

Fig. 23. Overlapped FTIR spectra of the four samples with labeled bands.

FTIR analysis of the diopside powders sintered at 900 °C for 3 h (Fig. 24) show the O-
Mg-O (470, 510 cm-1) and O-Ca-O (418 cm-1) non-bridging bending vibrational modes in the
range of 400 - 600 cm-1. The bands at 620 and 674 cm-1 are related to the O-Si-O bending mode.
The stretching modes of the Si-O bond were also seen at 857, 908 and 963 cm-1. The intensely
sharp peak at 1075 cm-1 corresponds to the symmetric stretching mode of Si-O-Si [28].

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Fig. 24. FTIR spectrum of diopside nanopowder [28].

In addition, according to [14], which tested the composition of films of HA, HASi50 and
HASi100 deposited on Ti, a signal at wavenumber 1,000 cm-1may represent PO43-ions (Fig. 25).

According to [29], the characteristic bands for PO43- appear at 470, 568, 602, 964, 1041
and 1093 cm-1, a result of asymmetric P-O stretching vibration. Together with the sharp peaks at
633, 602 and 568 cm-1, it corresponds to the triply degenerate bending vibrations of PO43- in HA
(Fig. 25).

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Fig. 25. FTIR spectrum of human bone [29].

In [30], it is shown the chemical composition of human bone examined by FTIR (Fig.
26). It is shown that the peaks corresponding to wavenumber 1,000 cm-1 represent phosphate
groups - acid phosphate, crystalline phosphate and non-crystalline phosphate.

Fig. 26. FTIR spectrum of coatings reinforced with various carbon nano structured materials
(exfoliated grapheme, reduced grapheme oxide and carbon nanotubes) [31].

In [31], which studied coatings reinforced with various carbon nanostructured materials,
a similar pattern emerges at wavenumber 1,000 cm-1 and it is being identified as PO43- bending,
resulting from HA deposited on the coatings.

Below (Fig. 27) is a diagram from a FTIR spectrum recorded in the Bachelor Degree
Dissertation of Izabela CONSTANTINOIU [22] that shows that indeed, upon being subjected to

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a process of calcination, the material surface loses most of its functional groups and the
dominant features are Si-O bonds.

Fig. 27. FTIR spectrum of project precursor materials [22].

XRD analysis of obtained ceramic thin films

Fig. 28 displays the result of the XRD on the samples, showing that a non-stoichiometric
transformation occurred, more powerful in Film D. This is possibly from crystals of Ca3(PO4)2
(ICDD 00-003-0713) that did not fully undergo vitrification. The peak placed around 17 ° could
not be entirely identified through the methods used (Match! software). Otherwise, two crystalline
phases were identified, mainly the substrate, titanium (Ti) with hexagonal crystal system and
P63/mmc space group (ICDD 00-044-1294), since the thickness of the deposition is so reduced
that the most intense diffraction peaks are attributed to the substrate. Probably, akermanite

(Ca2MgSi2O7, 2CaO・MgO・2SiO2, C2MS2) with tetragonal crystal system and P-421m space

group (ICDD 00-083-1815) is also present in the samples, but in a small quantity. However, the
films are most probably composed of a vitreous matrix and at least two families of crystalline
domains spread throughout the volume. The glassy character is confirmed by the presence of a
diffraction bump at low angles.

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Fig. 28. XRD patterns of the four samples.

SEM Image Processing

Last, a method of quantifying the deposition level on top of the substrates observed in
SEM was conducted, with the idea that a machine vision application will produce an unbiased
analysis of the amount of deposition and the quality of the surface of a sample, before and after
immersion in SBF. It was made use of the observation that upon the SEM images, there were
found a lot of spherical artifacts that can be identified and quantified by a JIMAGE, a public
domain software produced in the United States of America with the purpose of counting red
blood cells in a blood sample. The software is capable of applying various filters, identifying
artifacts within given sizes, and determining an artifact level of circularity. With JIMAGE help,
the relative differences in surface roughness were compared. Table 5 below presents the results
that were found.
For the JIMAGE quantification, the metrics considered the most relevant were Difference
in Number of Artifacts and their respective Average and Standard Deviation variation. These
metrics were chosen because they are independent of the quality of the image.
According to Table 6, Film B, Film C and Film D recorded a decrease in the number of
artifacts after exposure to SBF. Film A recorded a small increase. All three films recorded a
somewhat similar decrease, the strongest decrease being with Film C.

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Table 5. JIMAGE-based results of surface artifacts before and after immersion in SBF.

The average artifact size presented a decrease across all four films, the strongest decrease
being with Film B. In addition, the decrease in the standard deviation of the recorded artifacts is
recorded in Film B, also. This may show that Film B experienced the strongest deposition of
material upon its surface, while the other films experienced some intermediate level of materials
deposition.

Table 6. JIMAGE-based results interpretation.

Biocompatibility evaluation of obtained ceramic thin films

The metallic samples coated with a thin layer of vitroceramic material were tested in
terms of biocompatibility in the presence of BJ epithelial cells. Healthy BJ epithelial cells were
grown on the samples in the presence of DMEM cellular growth medium, on 24 wells plates.
After 24/48 h of incubation, a solution of MTT (10 % MTT, 90 % DMEM) was applied. The
samples were incubated for 3 h, after which DMSO is added and the absorbance of the
specimens is being measured on the Mithras plate reader at 570 nm wavelength.

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The viability was 10-20 % smaller than the Control sample, but this does not indicate that
the samples are toxic, just the movement of the samples led to the peeling off of the cells (Fig.
29). The viability was calculated as (OD sample - OD medium)/(OD Control - OD medium) ×
100 %, where OD represents the optical density and OD medium is OD of MTT solution (10 %
MTT, 90 % DMEM).

Fig. 29. Viability assay at 24 h of the four samples in contact with cells.

From the acquired microscopy images (Fig. 30), it can be clearly seen that the cells
adhered well at the surface of the samples and those around the edges of the samples (before the
test) have appropriate characteristics and maintain their initial oblong shape morphology similar
to those on the Control sample.

CONTROL

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Film A Film B

Film C Film D
Fig. 30. Optical microscopy images of the four samples in contact with cells.

Discussion

According to the FTIR and SEM images, a clear pattern emerges that the films produced
with a higher substrate temperature and the films produced at the lower pressure show better
results for the deposition of HA.

Before immersion in SBF fluid, Film A (250 °C, 100 mTor) shows a uniform pattern of
even-sized grains that are well coalesced and without any visible surface defects like pore holes,
uneven grain boundaries or gas pockets at lower magnification. At higher magnification, the
spherical sputtered ceramic grains are visible, together with grains of higher density that appear
as white spots due to being of higher density. They seem to be unevenly distributed across the
field of observation. Spherical ceramic particles do not have very strong adhesion to the substrate
due to their geometry and the fact that the substrate did not fully connect with the metallic
substrate. It is expected that this surface has a higher roughness, and it can accommodate
deposits of HA better than surfaces with low roughness.

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After immersion in SBF for a period of 14 days, there are signs of unevenness in the
shape of the surface particles, indicating that there was a mineral deposition that altered their
shape. It is seen that the surface is more coalesced and, thus, of lower roughness.

Film B (350 °C, 100mTor) at lower magnification shows some layering and directionality
(diagonal across the field of view) and grains that seem to have coalesced between those layers
and within the substrate. Some minor surface defects are also present, but do not constitute an
outstanding feature. At a higher magnification, the surface appears to be smooth, with
directionality and very few spherical ceramic particles that did not entirely coalesce into the
substrate.

The post-immersion SEM shows clearly visible and substantial formations of HA that is
deposited along geometric patterns running diagonal across the field of view. The deposited
formations do not form a continuous film across the sample, rather, they seem to be in the
process of developing and growth even across uneven areas.

Film C (250 °C, 200mTor) shows a surface with some subdued directional features,
surface defects and unmelted, sputtered spherical particles scattered unevenly across the surface.
This feature becomes more obvious at higher magnification, which shows even smaller spherical
particles that are not fully coalesced to form a smooth surface. Surface defects are visible in the
form of porosity as particles agglomerated during the process of deposition.

Post-immersion, the surface is seen being loaded with minerals in a disorganized fashion,
probably due to the original unevenness of the surface. Particles appear to be larger in size, and
there are numerous porosities and areas of uneven growth.

Film D (350 °C, 200 mTor) presents a surface with several surface defects and a subdued
level of directionality. The surface is decorated with granular and spherical particles in an
uneven manner. At higher magnification, the unevenness of the particles is even more
pronounced, even though particles seem to have coalesced into a solid surface. A few surface
defects that increase porosity are present.

Post-immersion SEM shows a surface that is, in general, uneven and rough, but in certain
areas, the HA deposited itself into flat, even layers. Surface defects are still present among the
islands of flat deposits.

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According to the images obtained, the substrate temperature is the underlying factor that
dictates the quality of the initial surface and subsequently, that of the deposited HA. Chamber
pressure appears to be of secondary importance.

The biocompatibility of all four samples was proven to be acceptable and the best
viability values were obtained for the films deposited at a higher substrate temperature (350 °C).

6) FUTURE RESEARCH

The results presented herein look promising for future research. One direction of research
might be in the direction of applying the materials presented here on ceramic surfaces via laser
sputtering to create engineered surfaces with minimal roughness and thus prevent surface
degradation by friction, biological activity and long-term use. Obviously, cellular adhesion
would be undesirable on such surface, but cellular adhesion would be necessary on other areas of
the implant.

Another direction might be in the biofunctionalization of a deposited film

biofunctionalized with highly active cyclo RGD-thiol polypeptides, as it is able to fixate integrin,
among other proteins, which has proven beneficial for the osteoblast attachment process and
further aids in osteointegration of an implant. Thiols have been previously shown to readily bond
to Au-based substrates.

7) CONCLUSION

This project shows that the materials that were tested are, indeed, biocompatible, and
have potential in the field of biomedicine. Many issues need to be resolved before this project
may turn into a commercial application, such as the scale and the geometric complexity of the
samples to be processed, while keeping in mind that some parts of a bone implant need
frictionless surfaces while others need solid osteointegration with the surrounding tissue.
However, these challenges are not insurmountable and can be resolved in further projects.

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ACRONYM LIST

CLSM – Confocal Laser Scanning Microscopy

CNT – carbon nanotubes
ECM – extracellular matrix
ExG – exfoliated graphene
FESEM – Field Emission Scanning Electron Microscopy
GSH – Gluthatione (an antioxidant peptide found in plants)
HA – hydroxyapatite
MAPLE – Matrix-Assisted Pulsed Laser Evaporation
MS – Magnetron Sputtering
MTT (assay) – tetrazolium dye
OD – optical density
PLD – Pulsed Laser Deposition
RF – Radiofrequency
RHEED – Reflection High-Energy Electron Diffraction
RGD – (protein amino sequence: Arg-Gly-Asp)
RGO – Reduced Graphene Oxide
SBF – simulated body fluid
TCP – tissue culture plate

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