solution y

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EXECUTIVE SUMMARY

The application of medical devices either for temporary or permanent us

e has become an indispensible part of almost all fields of medicine. How

ever, foreign bodies are associated with a substantial risk of bacterial and

fungal infections. Implant-associated infections significantly contribute t

o the still increasing problem of nosocomial infections. To reduce the inc

idence of such infections, specific guidelines providing evidence-based r

ecommendations and comprising both technological and nontechnologic

al strategies for prevention have been established. Strict adherence to hy

gienic rules during insertion or implantation of the device are aspects of

particular importance. Besides such basic and indispensable aspects, the

development of new materials which could withstand microbial adheren

ce and colonization has become a major topic in recent years. Modificati

on of surface by primarily physico-chemical methods may lead to a chan

ge in specific and unspecific interactions with microorganisms and, thus,

to a reduction in microbial adherence. Medical devices made out of a ma

terial that would be ideally antiadhesive or at least colonization-resistant

would be the most suitable candidates to avoid colonization and subsequ

ent infection. However, it appears impossible to create a surface with an

absolutezero -adherence due to thermodynamical reasons and due to the fact that a modified material surface is in vivo rapidly covered by plas

ma and connective tissue proteins. Therefore, another concept for the pre

vention of implant-associated infections involves the impregnation of de

vices with various antimicrobial substances such as antibiotics, antiseptic

s, and/or metals. In fact, already commercially available materials for cli

nical use such as antimicrobial catheters have been introduced, in part with considerable impact on subsequent infections. However, future studies

are warranted to translate the knowledge on the pathogenesis of device-

associated infections into applicable prevention strategies.


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Table of Contents


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Topic Pag

e Nu

mbe

r

Introduction .

Materials for Biomedical Applications .

Modalities for Analysis

Atomic Force Microscope .

Spectroscopic methods ..

X-ray photoelectron spectroscopy ..

Attenuated total reflection Fourier transform infrared spectr

oscopy (ATR-FTIR ).

Pathogenesis .

Attachment of Microbe on Device Surface

Management of Medical Device Associated Infections :

General Considerations

Removal of Device .

Salvage of Device and Treatment with Antimicrobial Agents.

Prevention by Material Modification ..

Development of Antiadhesive Polymer Materials by Physico-

5

6

7

9

12

14

21

22

23

28

30


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Chemical Modification ,.

Conclusion ..

References ..

31

32

36

42

43


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Introduction

The rate of implant surgery is steadily increasing. Each implantation is a

ssociated with a risk of infection. The interface between implant and tiss

ue is known to be an extremely sensitive region. Infections can arise fro

m bacteria adhering to the implant and subsequent biofilm formation and

also from tissue damage caused by instability of the implant, which can r

esult in depletion of the immune defenses [1]. Device associated infectio

ns present therefore one of the most important risk factors with respect

to revision surgery [1,3]. Since biofilms are challenging to treat with con

ventional antibiotics [4,5], avoiding the initial bacteria attachment on the

implant surface is considered as the most effective method to minimize a

nd prevent infections as well as to improve the interfacial stability.

Functionalizing biomedical implants by surface modifications for tailore

d tissue response is a growing field in research. Localized antimicrobial

delivery methods have been explored as attempts for time effective treat

ment to prevent the origin of biofilm formation [6]. For the design of im

plant materials with therapeutic coatings for local drug administration, the coating needs to comply with certain requirements, such as bioactivity,

mechanical stability, good substrate adhesion and the ability to carry dr

ugs. These functionali- ties can contribute towards achieving a better fix

ation to the bone as well as the release of drugs to treat infections close t

o the implant. Hence, functional implant coatings can be seen as a potent

ially promising step to achieve longer life spans for implanted devices and long-term surgical success rates and thereby contribute towards reduci

ng health care costs.


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Materials for Biomedical Applications

Biomaterials, utilized to reinstate a broken construction and purpose in t

he human body, have revolutionized the rehabilitation of patients alongsi

de harsh functional impairments. Whereas the bulk properties of the im

plant physical are recognized to impact the presentation of the implant in

vivo, the biological host reply is more affected by external properties suc

h as chemical constitution, crystallinity, roughness and external price [7].

The clinical accomplishment for implants that are utilized in bone-interfa

cing requests is described by the extent of link of presently industrialized

bone alongside the implant external [8]. The integration of the external, s

ynthetic physical across contact alongside the living tissue is denoted to

as bioactivity. Bioactive materials, such as bio- glass [9], ceramic apatite

-wollastonite [10] as well as titanium (Ti) metals and alloys have been pr

oven to advance bone formation and to form a stable link to bone [11]. I

n finish, bioactive materials could spontaneously form a bone-like apatit

e layer on their surfaces on link alongside body fluids [12,13]. A materia

ls bioactivity can be discovered both in vivo and in vitro [14]. The mate

rials utilized as substrate and/or coatings in this thesis are described in the pursuing sections.

Surgical implants made of Ti or Ti alloys are extensively utilized in bio

medical requests [15]. Examples of their use are as dental implants, hip j

oints, manmade knee joints, bone plates, screws for fixation and valve pr

ostheses. The expansive scope of clinical requests is established on the m

echanical strength, biocompatibility, non-toxicity, machinability and their skill to osseointe- grate [15,16].

Calcium phosphate ceramics are usually utilized in assorted fields of bio-

health applications. HA, alongside the chemical formula Ca10(PO4)6(O


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H)2, is the most usually utilized bioactive ceramic physical in clinical ap

plications. Its use is established on its brilliant affinity to bone [17]. This

property is due to the fact that HA is a main constituent of bones and teet

h. The bioactive property of in vitro produced HA is evidenced by the sp

ontaneous formation of a biologically produced HA layer on the external

after implanted into bone [18].

MODALITIES FOR ANALYSIS

Surface properties have an large result on the accomplishment or wreck

of a biomaterial mechanism, therefore signifying the substantial significa

nce of and the demand for adequate characterization of the biomaterial s

urface. Microscopy methods utilized in the scutiny of biomaterial surface

s contain scanning electron microscopy, transmission electron microscop

y, atomic power microscopy, and confocal microscopy. Spectroscopic m

ethods contain Scan photoelectron spectroscopy, Fourier Change infrare

d attenuated finished reflection and secondary ion mass spectrometry. Th

e measurement of link slants, even though one of the preceding methods

industrialized stays a extremely functional instrument in the evaluation o

f external hydrophobicity / hydrophilicity. Choice of the external charact

erization method utilized can be affected by a plethora of thought s enco

mpassing the kind of measurement needed, the extent of the analyzed external span, the needed precision and accuracy, the impact of the method

on the external (i.e. does the probe, electron beam, ion beam, X-ray, nee

ded example arranging , or the scutiny nature instigate unwanted results

on the external of interest) , the impact of the example on the instrument

, limitations imposed by the external, as well as the ease of use and poten

tial y of supplies [19, 20]. In supplement , a extremely realistic factor orconstrain t that could impact the choice of external scutiny s method s uti

lized is that countless external scutiny s abilities have come to be centrali

zed, and there are momentous prices associate d alongside example proc


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essing g and arranging , the timed custom of supplies, and technician tim

e.

Atomic power microscopy. Atomic power microscopy (AFM) has come

to be the most public kind of scanning microscopy utilized for polymeric

biomaterial s [25]. A three-dimensional picture of the external is crafted

by scanning a tip attached to the conclude of a cantilever across the exter

nal and monitoring g the minute powers of contact amid the example ext

ernal and probe [12, 25]. The powers of contact could be repulsive or ap

pealing and this gives development to the disparate modes of procedure

of the AFM. A extremely elevated resolution of external topography can

be obtained, with dimensions on the nanometer scale [12, 13], even thou

gh it ought to be noted that properties and dimension s of the cantilever a

nd tip, as well as the selected mode of procedure , frolic an vital act in as

certaining g the sensitivity and resolution of the acquired image. Unlike

electron microscopes , a momentous supremacy of AFM is that example

topographies , as well as surfaces roughness benefits , can be obtained la

cking external treatment or coating that could damage or change the phy

sical external below investigation n [12, 13, 16, 26, 27]. Furthermore, A

FM pictures can be acquired below vacuum, air, or fluid conditions. The

skill to picture polymeric materials inside an aqueous nature is tremendously functional in the biomaterials earth, as it permits for the examinatio

n of the external of biomaterials in an nature t comparable to one that sho

uld be discovered in an implant situation, therefore permitting for the exa

mination of vibrant procedures such as erosion, hydration, and adsorptio

n n at interfaces. For example, it is probable to visualize e individual l pl

asma protein molecule s below aqueous nature s employing period imaging AFM [12, 13, 28]. Even though AFM is clarifying to be an tremendou

sly functional method e in bestowing g a 3D visualization n of the bioma

terial l surfaces being learned, the period needed, reliant on such factors


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as scan size and scan rate, to attain quality pictures can be momentous (i

.e. in excess of 20 min / image). In supplement, as normally y scan sizes

are tiny (i.e. fluctuating from 500 nm-500 nm to 15 m-15 m), variation

s in the external could be missed.

AFM link mode. In link mode, the AFM tip scans across a external atextremely low power and is noticed by repulsive powers replacing amid

the tip and the external atoms. A photodiode detector monitor s the detec

tions of a laser light imitated from the tip of a cantilever. A feedback loo

p uphold s steady t detection of the cantilever, by vertically advancing th

e scanner as it scans laterally across the surface. A computer stores the

data n and a topographic c picture alongside potentially y atomic-scale e

resolution is generated. The powers at the tip are extremely tiny (0.01 to

1.0 N / m in air) and metal or hard polymeric surfaces are not usually bro

ken [7]. Though, the lateral clip powers provoked by the scanning gestur

e could change soft materials, therefore distorting g measurement data an

d provoking damage to the example [29]. Obtaining pictures of hydrated

polymeric materials in fluid could be more hindered by the fact that a litt

le hydrate d polymers are softer than dried examples managing to an rise

in example deformation and damage and a decreased picture quality eme

rging g from the dragging gesture of the tip.AFM non link mode. In non-contact mode, appealing rather than repulsive powers are measured. The scanning tip is oscillated perpendicular t

o, and just above the example external alongside an amplitude normally l

ess than 10 nm. As alongside link mode, a photodiode detector monitor s

the deflections of the laser light imitated from the tip of a cantilever. A f

eedback loop maintains steady oscillation amplitude or frequency, as thescanner moves laterally. A computer stores the data and a topographic pi

cture, alongside a lower resolution than in link mode, is generated. Non-

link mode could work well alongside hydrophobi c polymers. Though hy


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drophilic polymers or imaging in a fluid nature t in non-contact mode is

not normally utilized due to the low resolution obtained.

AFM tapping g mode. In tapping mode, the cantilever r is vibrate d ator adjacent its resonance e frequency, and lightly taps the example extern

al alongside an amplitude e normally fluctuating from 20 to 100 nm. A te

ar photodiode e detector monitor s the deflections of the laser light imitat

ed from the tip of the cantilever r. A feedback loop maintains steady t os

cillation n amplitude e or frequency, as the scanner moves laterally acros

s the surface. This mode consequently uphold s the high-resolution skills

s of link mode but is not annihilative e as there are no lateral frictional l

powers requested to the example that can distort t or damage the physical

[12]. Tapping mode AFM has proved to be extremely prosperous l for hi

gh-resolution studies s of polymeric c biomaterials permitting for charact

erization n of nanometer scale features not visible e by supplementary mi

croscopic method s (see Fig. 3). Tapping mode AFM pictures normally y

contain d in the biomaterial l works e have a scan size fluctuating from

500 nm to 500 nm, to 15 m to 15 m.

AFM period imaging mode. The period imaging mode can be utilizedto chart the external constitution n of a sample. In this mode, the cantilev

er r is vibrate d at or adjacent its resonance e frequency, and lightly y tap

s the example surface. A feedback loop maintains steady t oscillation n a

mplitude, as the scanner moves laterally y across the surface. A period pi

cture displays the period difference amid oscillations of the piezoelectric.

Crystal that propels the cantilever r and oscillation n of the cantilever r itself as it interacts alongside the surface. Period imaging can expose fine

features that are obscure d by a rough external topography y. Though fac

tors such as external hardness, elasticity y, adhesive properties, and exter


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nal price could alter the period pictures obtained. Period and tapping g m

ode (height) pictures can be obtained d simultaneously y (see Fig. 4), so t

he locale n of the external features discern d in a period picture can be co

rrelated undeviatingly alongside the external topography y. This mode ca

n consequently be utilized to ascertain the size, form and spacing of disp

arate physical areas that might not or else be discerned d from height alo

ne [27, 30]. For example, period imaging AFM can prosperously y notic

e adsorbed d protein s that are not observable e in standard l topographic

c pictures, as proteins imaged on surfaces alongside roughness s adjacent

the dimension s of the protein cannot be discriminated d from the physic

al topography y alongside standard l AFM [27], that is manipulated to im

aging protein s merely on smooth

surfaces (1 m2 roughness s of


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TEM and AFM pictures suitably y of the alike polyamide e polymeric c

material. The aftermath obtained d by SEM display a fairly flat external,

alongside a little extremely different t porous regions. TEM aftermath di

splay a flat external, and in supplement n display that the bulk polymer e

ncompasses a deep prop t layer encompassing g of interconnected d exce

edingly y porous domains. AFM aftermath display momentous external

morphology y, not visible e by the supplementary method s used. Thus, t

he combination n of microscopic methods utilized could frolic a priceles

s act in the characterization n of external features and morphology y of p

olymer biomaterials.

Spectroscopic methods

Spectroscopic methods are extensively utilized to expose priceless data c

onsidering the constituent agents and chemical construction adjacent the

external span of a example [46]. Two new characterization methods utili

zed in the biomaterials earth are scanning transmission Scan microscopy

(STXM), and photoelectron emission microscopy (PEEM). Both metho

ds need synchrotron light origins and elevated instrumentation. As STX

M has the gains of not being altered by example charging or topography,

as well as the skill to picture the example in resolution, the depth of sam pling is maximum lm frequently on the order of 100 nm. PEEM has a sa

mpling depth of normally y 5 10 nm for polymers. Both methods, STXM and PEEM, have a elevated lateral spatial resolution on the order of

50 nm, and brilliant chemical sensitivity y. One more paper in this distin

ct subject will debate the relevance of these two growing methods (STX

M, PEEM) in the biomaterials field. Far extra usually utilized in the external characterization of biomaterials, the established spectroscopic metho

ds utilized include:

Auger electron spectroscopy (AES).


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X-ray photon spectroscopy (XPS). Secondary ion mass spectrometry (SIMS). Attenuated total reflection Fourier transform infrared spectroscop

y (ATR-FTIR).

Auger electron spectroscopy (AES). Auger electron spectrosco

py (AES) has been utilized for investigating external morphology

[47], and in particular elemental analysis. A concentrated beam o

f electrons is utilized to stimulate Auger electrons from the extern

al, that are next noticed and analyzed. No distinct example arrang

ing is needed and data collection is quick (i.e. a insufficient minu

tes) and reproducible. This method has proven extremely functio

nal in a little engineering fields for elemental scutiny and constitu

tion depth profiling. Though, in finished AES is manipulated in its use in the scutiny of polymeric biomaterials as it is usually belie

ved improper for studying organic matter. Organic examples fact

ually burn up in the electron beam, radically changing the chemis

try and morphology of the example being measured [20]. Becaus

e the chance of artefact and misinterpretation is colossal, the use

of AES for describing organic, polymeric or biological specimens have to be approached alongside alert [20].

X-ray photoelectron spectroscopy . Scan photoelectron spectros

copy (XPS),

AKA ESCA (electron spectroscopy for chemical analysis) is exte

nsively utilized in biomaterial requests to ascertain the elementalconstitution of solid surfaces [48]. Distinct example arranging is

usually not needed for XPS, even though external contamination

on storage or across transport from the scutiny labora- tory to the


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XPS abilities could precisely have an adverse result on the XPS a

ftermath obtained. In supplement, additives, such as catalysts that

could be utilized across the polymerization of polymeric biomate

rials, or impurities can be present at the external of the polymers

and therefore give considerably to the XPS aftermath obtained.

The principle e of XPS is established on the emission of electrons

from matter

in reply to irradiation of the external by a beam of monochromati

c X-rays.

The kinetic power of the emitted photoelectrons (KE ) is exceptional for

the disparate agents as well as being sensitive to the chemical state of the

atoms [8, 48].


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Figure 5a. Survey (low resolution) XPS scans of a polyurethane (PU) su

rface, and a PU surface

modified with polyethylene oxide (PEO). XPS signals for carbon

(C), nitrogen (N) and oxygen (O) are indicated. Scans show an in

crease in the O peak. Table 1 shows the corresponding atomic

%of oxygen present clearly indicating that the surface has been s

uccessfully modified with PEO. XPS spectra were obtained using

a Leybold MAX200 X-ray photoelectron spectrometer, and analy

zed using ESCATOOLS (Surface/ Interface, Mountain View, CA

). Figure courtesy of J. Tan and J. L. Brash.

because typicall y extra period is consumed buying data above a

narrower power scope as contrasted to a survey scan (Fig. 5a) em

erging in a larger gesture to sound ratio. The XPS method is exce

edingly flexible, alongside a sampling depth connected to the inel

astic mean free trail of photoelectrons in the external span, that is

normally larger that 30 for polymeric materials, even though th

is depends on both the photoelectron power and on the physical l

earned [49]. Typically, the gesture, below ultra-high vacuum, is g

athered from a colossal example span (i.e. 6 mm in diameter)


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Figure 5b. High resolution XPS C1 s detailed spectra of a polyur

ethane (PU) surface, and a PU surface modified with polyethylene oxide (PEO). Representative 4 peak curve fits of the XPS C1 s s

ignal are also shown. Table 2 shows the corresponding Carbon A

tom Bonding (%) of C O C determined from best t of C1 s High


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Resolution XPS Spectra clearly indicating that the surface has be

en successfully modified with PEO. XPS spectrum was obtained

using a Leybold MAX200 X-ray photoelectron spectrometer, and

analyzed using ESCATOOLS (Surface/ Interface, Mountain Vie

w, CA). Figure courtesy of J. Tan and J. L. Brash.

in order to minimize collection period, and probable damage to a

little polymeric materials. Though, lateral resolution employing a

tinier example span, in the scope of square microns, has been des

cribed [48]. Refined instrumentation simplifies sampling graspin

g and data collection [8]. Well industrialized theory and compute

r plans are obtainable to assist in the clarification of the data obtai

ned. XPS is believed to be a moderately non-destructive method,even though a little care is demanded to safeguard that the Scan

does not change the external chemistry [20]. Setbacks have been

described to transpire alongside polymers, exceptionally flouropo

lymers, alongside the results being minimized by use of a monoc

hromatic basis in conjunction alongside an competent compensati

on method to circumvent external charging setbacks or by plainlyminimizing the Scan exposure to the external and optimizing spe

ctral buy periods [11]. Data concerning useful clusters can be obt

ained by employing derivitization replies [8, 50]. Compositional


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variation as a purpose of example depth can be obtained by meas

urement of the photoelectron intensities at disparate emission sla

nts, termed slant resolved XPS [8, 51]. Slant resolved XPS can a

dditionally be utilized to scrutinize an overlayer that could not be

uniform, to examine a external whereas coverage is trusted to be

patchy [11], or to scrutinize the change or transition amid bulk an

d external of asurface modified material. To attain elementaldata countless thousand angstroms into the example, argon etchin

g can be utilized in conjunction alongside XPS even though this

method is example annihilative [8]. As the gesture is normally ga

thered from a colossal example span (i.e. 25 mm2), it is probable,

as remarked preceding, to use XPS to ascertain lateral variations i

n external constitution [8] and to guesstimate the thickness of bot

h organic and inorganic c layers [52]. In supplement, hydrated fre

eze dried surfaces can be examined employing XPS [8] that coul

d be of substantial attention after describing polymer surfaces tha

t could reorient on exposure to an air, vacuum or aqueous nature .

There is substantial works obtainable on the principles of XPS, a

nalytical procedures, instrumentation and ways to quantitative an

alyses [3, 8, 49, 53 55]. In the discover and characterization of biomaterial surfaces, XPS is perhaps y the most extensively utilize

d technique. It has been utilized for a collection of surfaces and e

xternal modifications, encompassing studies of adsorption and ret

ention of chemicals such as antibiotics and bonding agents [11], f

or the detection of immobilized proteins [20], understanding the c

hemistry of the construction, formation and stability yof plasma indulged surfaces, [7, 52, 56 59] as well as for the characterization of the steps of formation of slender coatings [60].


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Secondary ion mass spectrometry (SIMS) and time-of-flight s

econdary ion mass

spectrometry (ToF-SIMS). Secondary ion mass spectrometry (S

IMS) is competent for bestowing methodical molecular external

data [7] and increased custom of this method in the external char

acterization of polymeric biomaterials has been noted in present

years. Typically, no distinct example arranging is needed for SIM

S. Though as alongside XPS, the external have to be clean and fr

ee of each contamination that could transpire on storage or transp

ort. Surfaces are assaulted alongside a concentrated beam of ions

or atoms and the power from the event beam (approximately 5 25 keV) is transferred to the external zone of the physical emergi

ng in the emission of secondary particles, a little of that are ioniz

ed, at and concerning the encounter locale [8]. These ionized part

icles are separated as a purpose of the ration of mass each mecha

nical price and affirmatively and negatively charged species are n

oticed in two disparate buys [61]. Low flux or static SIMS afterm

ath in negligible damage to the example and the fragments emitte

d are characteristic of the external molecular construction [8, 62].

Elevated flux, or vibrant SIMS aftermath in quick etching of theexternal across scutiny and can be utilized to monitor adjustment

s in the elemental constitution alongside depth [8, 61]. Time-of-fl

ight SIMS (ToF-SIMS) is a extremely effectual method for descri

bing the elemental (including H and isotopes) and molecular cons

titution of the top external of biomaterials [61]. Events in the ToF

-SIMS analyzer have increased the skills of static SIMS due to considerably enhanced mass transmission (independent of the ion m

ass), mass resolution, mass scope and sensitivity [61]. In this case

, the example external is assaulted alongside extremely short-puls


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ed ion beams. Amid two consecutive pulses, all of the secondary

ions are removed and electrostatically accelerated into a earth fre

e drift region. Lighter ions will have higher velocities and hence

will grasp the detector at the conclude of the drift span preceding

than the heavier crowd [61]. From the period of flight, the mass t

o price ratio can be ambitious [61]. Below static conditions, there

is negligible external damage alongside ToF SIMS [61]. SIMS ca

n be utilized for the identification of all agents encompassing hy

drogen [8] as well as atomic and molecular ions [61] and tremen

dously elevated mass fragments at extremely low concentrations.

Even though the scutiny destroys the external, data generated is u

ndeviatingly connected to the early external [20]. As static SIMS

can be requested prosperously to the scutiny of both organic and i

norganic surfaces, the accuracy of the data alongside vibrant SIM

S is considerably decreased for organic and biological surfaces

[20]. These methods can be utilized to attain manage facts of cov

alent attaching of molecules to a external [66], as well as to forec

ast supplementary external properties encompassing wettability,

adhesiveness, and biological reactivity [61]. SIMS spectra can fur

nish characteristicfingerprints for biomaterial surfaces [67], and has been utilized to associate external chemistry alongside cell

development [56] as well as for monitoring the degradation kineti

cs of biodegradable polymers employing molecular heaviness all

ocations of the oligomeric hydrolytic c reply produce [68 70]. Temperature-programmed SIMS proposals data on adsorption powe

r and therefore helps to discriminate h amid physisorption and chemisorption phenomena [71]. It is frequently utilized in conjuncti

on alongside XPS to scrutinize the integrity y, mean thickness an

d chemical state of multi-layer biomaterial coatings [72], corrobo


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rating link slant and XPS measurements and complementing data

obtained from less external sensitive methods [61].

Infrared spectroscopy (IR) and attenuated finished reflection

Fourier change infrared spectroscopy (ATR-FTIR). Infrared s

pectroscopy is utilized to attain data concerning molecular constr

uction by computing the frequency of IR radiation demanded to s

timulate vibrations in molecular promises [24]. Example arrangin

g is negligible including request of the polymeric physical of atte

ntion, in film form, onto a crystal element. Instrumentation is mo

derately inexpensive, and the emerging spectra furnish chemical

bonding data [7]. Infrared spectroscopy in attenuated finished refl

ection (ATR-FTIR) couples the analytical method of infrared spe

ctroscopy alongside the physical phenomena of finished inner ref

lection (i.e. reflection and refraction of electromagnetic radiation

at an interface of two mass media possessing disparate indices of

refraction) to restrict the analyzed volume on the external span of

the example [73, 74]. For this method, the event electromagnetic

waves are completely imitated back into the early medium. The e

lectromagnetic earth is instituted in the subsequent medium as embodied by an transient wave due to diffraction at the borders of

the event radiation at the interface [73]. In attenuated finished ref

lectance (ATR) sampling mode the subsequent medium is the ph

ysical to be learned, alongside the early medium replacing as the

inner reflection agent [24, 73]. Data concerning the molecular co

nstruction of the physical, inter- and intra-molecular contact, crystallinity, conformation (e.g. proteins) and orientation of molecule

s can be obtained across scutiny of the infrared spectra [74, 75].

Even though depth profiles can be obtained employing this meth


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od, the XPS and SIMS methods debated preceding are believed f

ar extra external sensitive.

Pathogenesis

The skill to adhere to materials and advance formation of a biofil

m is an vital feature of the pathogenicity of bacteria encompassed

in external body infections. The fact that staphylococci embody t

he main organisms associated alongside infections of health mec

hanisms has considerably spurred scutiny on pathogenic mechani

sms, emerging in vital ad- vances in our understanding of biofilm

formation. Thus, a battery of staphylococcal virulence factors hav

e been recognized and described in the past two decades managin

g to vital visions, partic- ularly alongside respect to the contact of

the bacteria alongside the external of the implanted or inserted de

vice. Normally, CoNS live in balanced harmony on our skin, gro

wing the main constituent of the cutaneous microflora. Beyond th

e setting of a health mechanism, these organisms scarcely cause i

nfections. Though, in relation to an inserted or implanted externa

l body, these bacteria are able to colonise the external of a

foreign body by the formation of a deep, multilayered biofilm.[25,26] Biofilm formation proceeds in two stages: a quick attach

ment of the bacteria to the external of the implanted mechanism i

s pursued by a extra spread accumulation period that involves ce

ll proliferation and intercellular adhesion (figure 1). For years, ef

fortifications have been made to recognize bacterial factors acco

untable for every single of both phases.

Attachment of Microbe on Mechanism Surface


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Microbial adherence to external bodies depends on the cell extern

al characteristics of the micro- organisms and on the nature of the

external body material. Factors encompassed contain physicoche

mical

forces such as polarity, London-van der Waals powers and hydr ophobic interactions. [27] Cell external hydrophobicity and early

adherence of S. epidermidis to polystyrene have been attributed t

o two disparate bacterial surface-associated proteins, labeled SSP

-1 and SSP-2. [28] Early attachment of S. epidermidis to a poly

mer external could be additionally mediated at least in portion b

y AtlE, a surface-associated autolysin. [29] The biofilm-associa

ted protein Bap was described to give to both periods of S. aureu

s biofilm formation, adhesion and accumulation, [30] as Bhp, a

Bap-homologous protein, could give to S. epidermidis biofilm for

mation. [31] Aside from proteins, a polysaccharide struc ture sho

uted capsular polysaccharide/adhesin (PS/A) has been associated

alongside early adherence and slime production. [32] In a rabbit

ideal of endocarditis, PS/ A-deficient mutants were less virulent

and im

munisation alongside PS/A arose in protection opposing infection. [33] As the manage contact amid bacteria on one side and the

unmodified and naked external of the external body on the suppl

ementary side plays a critical act in the main periods of the adher

ence procedure in vitro and plausibly additionally in vivo, supple

mentary factors could be vital in afterward periods of adherence

in vivo. Implanted mechanisms quickly come to be coated alongside plasma and connective tissue proteins, such as fibronectin, fi

brinogen, vitronectin, thrombos- pondin, laminin, collagen and v

on Willebrand factor (vWf), that afterward could assist as specifi


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c receptors for colonising micro-organisms. [34-36] In the vascu

lar arrangement at locations of increased flow, vWf could additi

onally frolic an vital act in adhesion of staphylococcal cells to pol

ymer surfaces because below elevated clip rates platelets do not a

ppreciably attach to extracellular matrix proteins supplementary t

han vWF. [35] Countless host factor-binding proteins from S. a

ureus (e.g. the fibrinogen receptor ClfA and the fibronectin-bindi

ng proteins FbpA and FbpB) and from CoNS (e.g. the fibrinogen-

binding protein Fbe and the fibronectin-binding autolysin Aas) ha

ve been recognized and characterised.

The S. epidermidis autolysin AtlE that mediates main attachment

to a polymer external (see serving 2) was additionally discovered

to display vitronectin-binding ac tivity, counseling not merely a p

urpose in the main periods of adherence but additionally a contrib

ution to afterward periods of adherence including specific contac

t alongside plasma proteins deposited on the polymer surface.[29] Aside from proteins, teichoic acid was sug gested to purpos

e as a bridging molecule amid the bacteria and fibronectin-coate

d polymer. [37]


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Cell Proliferation & Intercellular adhesion

Once adhered to the external of the external body, micro-organis

ms increase and amass in multilayered cell clusters, that needs i

ntercellular adhesion. A specific polysaccharide antigen termed

polysaccharide intercellular adhesin (PIA), that is encompassed i

n intercellular adhesion and biofilm accumulation and is chemi

cally connected to PS/A, has been noticed and analysed in staph

ylococci. Tn917 mutants lacking PIA were not able to accu- mula

te in multilayered cell clusters. The icaADBC operon that mediat

es cell clustering and the intercellular adhesin synthesis in S. epi

dermidis has been cloned and sequenced. [39,40] Later, three su

pplementary gene loci were recognized, that have a manage or in

direct manipulating impact on expression of the synthetic genes

for PIA and biofilm formation. In a mouse ideal of subcutaneou

s external body infection as [41] [38]

well as in a rat ideal of CVC-associated infection, a PIA-negative

mutant was shown to be considerably less virulent than the isoge

nic wild-type strain. A PIA/haemagglutinin-positive S. epidermidis strain was considerably extra probable to cause a subcutaneou

s abscess than its isogenic PIA/haemagglutinin-negative mutant a

nd was considerably less probable to be eliminated from the inoc

ulation locale by host defence. Furthermore, the wild-type strain

was discovered to adhere to the implanted catheters extra plentifu

lly than the PIA/haemagglutinin-negative mutant. [43] In an investigation projected to discover the pathogenic properties of S. epi

dermidis strains ob- [42,43]


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tained from blood of patients alongside FBRI, a forceful associat

ion was noticed amid pathogenesis and both biofilm formation an

d attendance of the ica gene cluster. [44] Most presently, it was s

hown that induc- tion of biofilm formation might be completely i

nhib- ited by chloramphenicol, that given at a afterward periodof biofilm accumulation additionally inhibited further progress of preformed biofilm. This indi- cates that constant translation o

f an supplementary, icaADBC-independent factor is needed for t

he expression of a biofilm-positive phenotype. [45]

Other factors such as the 140 kDa extracellular protein AAP (acc

umulation-associated protein) additionally seem to be vital for ac

cumulation and biofilm formation. [46] AAP, that is lacking in a

n accumu lation-negative mutant and detectable merely in ex trac

ellular produce from bacteria grown below ses sile conditions, wa

s shown to be vital for accu mulative development in precise S. e

pidermidis strains on polymer surfaces. Of 58 CoNS learned,

55% were 140 kDa antigen-positive and produced momentous ly

larger numbers of biofilm than the supplementary strains that we

re 140 kDa antigen-negative. An antiserum specific for AAP inh

ibited accumulation by up to 98% of the wild-type strain. [46]Taken jointly, the factors delineated here lead to the consequence

that bacteria, chiefly staphylococci, are able to adhere quickly to t

he external of a external body. Across the pursuing accumulation

period, the bacteria proliferate to form multilayered cell clusters

on the external of a health device. The attendance of such colossa

l adherent biofilms on the surfaces of external bodies, chiefly onexplanted intravascular catheters, has been clarified by scanning

electron microscopy. [26,47]


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Intercellular signalling, frequently denoted to as quorum detectin

g, has been shown to be encompassed in biofilm progress by cou

ntless Gram-positive and -negative bacteria such as Streptococcu

s mutans, Burkholderia cepacia and Pseudomonas aeruginosa. Fo

r example, below precise conditions, a quorum-sensing defectivemutant of P. aeruginosa is in difference to its parent strain incapable to

form a exceedingly differentiated biofilm structure. The S. aureus

quorum-sensing arrangement is encoded by the accessory gene w

atchdog (agr) locus that con tributes to virulence in ideal biofilm-

associated infections. Most presently, it was shown that, below a

little conditions, disruption of agr expression had no discernible i

mpact on biofilm formation, as below others it whichever inhibite

d or enhanced biofilm formation. Below those conditions wherea

s agr expression enhanced biofilm formation (tested in a rotatin

g-disk reactor), biofilms of an agr signalling mutant were chiefly

sensitive to rifampicin but not to oxacillin. [49] [48]

The clinical experience alongside polymer-associated infections r

eveals that the host defence mechanisms frequently seem to be i

ncapable to grasp the infection and, in particular, to remove themicro-organisms from the infected polymer device. In supplemen

t, antibacterial chemotherapy is oftentimes not able to remedy t

hese infections even though the use of antibacterials alongside pr

oven in vitro attention (see serving 3.3). Thus, the biofilm could

protect the embedded bacteria opposing host reply mechanisms a

s well as opposing antibacterial agents. [50,51]

Management of Health Device Associated Infections :

General Considerations


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Whenever an infection of an indwelling or im planted external bo

dy is distrusted, a finished decision has to be addressed: whether

to remove the external body and/or whether to onset computed a

ntimicrobial treatment. Replying the pursuing key inquiries could

aid the physician to grasp these infections adequately established

on a rationale approach. 1. Is an FBRI a plausible explanation for

the patients signals (e.g. fever, skin inflammation at the exit locale, soft tissue inflammation alongside the tunnel of an implanted

catheter, septic thrombophlebitis)?

2. Are there each chance factors predisposing for FBRI (e.g. neut

ropenia, malignant haematological disor ders, AIDS, kind of cath

eter)? 3. In that clinical situation is the patient (e.g. sepsis, pregn

ancy, premature infant)? 4. In the light of a probable necessity to

remove the external body, how vital is the health mechanism for

the patient regarding: (i) the survival of the patient (e.g. cardiac

mechanisms orhighly needed cath eters, such as tunnelled Broviac-Hickman-type cath eters or totally implantable venous admiss

ion mechanisms [i.e. ports] for intravenous management of vital

medications and parenteral nutrition); (ii) prosthetic therapy (e.g.

prosthetic joints, lens); (iii) optimal intravenous request of fluids, medications and blood produce (e.g. all kinds of vascular prost

heses; haemodialysis shunts); and (iv) cosmetic and reconstructi

ve surgery? 5. Which diagnostic methods ought to be requested t

o confirm the diagnosis? 6. Is computed antimicrobial therapy vit

al and, if so, that antibacterials ought to be given?

Several comprehensive reviews on the clinical association of inf ections due to an increasing

palette of health mechanisms have been published concentrating

on disparate aspects considering the removal of the infected mec


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hanism, antimicrobial therapy and on supplementary procedures t

o notice and stop complications associated alongside FBRIs.

[5,9,10,52-63]

Removal of Device

The optimal treatment of a FBRI is the removal of the infected m

echanism after probable and its substitute if yet needed. This is th

e therapy of choice, exceptionally for easy-to-change mechanism

s such as shortterm peripheral catheters. [5,9] Although of the kin

d of mechanism, removal of implanted mechanisms is suggested

after the patient displays signals of harsh sepsis, septic phlebitis a

nd septic surprise (table III). Furthermore, catheters ought to be r

emoved in patients alongside bacteraemia persisting extra than

48 72 hours. In supplement, attendance of innate skin or soft tissue infections (e.g. tunnel infection, gross purulence at the exit sit

e), metastatic complications (e.g. endocarditis, osteo- myelitis, se

ptic thrombosis) and/or relapse of infection afterward antibacter

ial therapy has been discontin- ued ought to lead to removal of th

e device. In addi- tion, innate debridement at the exit locale of a h

ealth mechanism ought to be believed if a subcutaneous ab- scessor comprehensive tunnelitis is present. The removal of the mecha

nism is usually vital if micro-organisms are remote recognized t

o be tough to eliminate or to be of elevated virulence such as S. a

ureus, P. aeruginosa or supplementary non-fermenter, mycobacte

ria and yeasts. [64-67]

Studies have shown that long-term tunnelled catheters (mainly haemodialysis catheters) could be exchanged prosperously alongsi

de guidewire in patients alongside uncomplicated CRBI and no si

gnals of exit, tunnel tract or pouch infection. [68-71]


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Salvage of Mechanism and Treatment alongside Antimicrobi

al Agents

Removing the infected health mechanism is not always probable,

facile to present and/or lacking chance and, consequently, salvag

e of the mechanism is from time to time the favored option. In pa

rticular, FBRIs associated alongside long-term or perpetual cathe

ters (such as Hickman-type catheter or Port-a-Cath) are frequent-

ly indulged prosperouslythrough the line (table III). [4,72,73]After a biofilm has industrialized on an implanted health mechani

sm it is tough to delight such infections because of considerably c

ut levels of suscep- tibility to antimicrobial agents (some 10 to

1000 periods less) and lower levels of phagocytosis comparative

to the levels of confrontation and phagocytosis for their planktoni

c counterparts (see pathogenesis, serving 2). [74] Thus, supraphy

siological concentrations of antibacterial agents could be needed

to remove the micro-organisms embedded in biofilms. [75] As

shown in a number of experimental FBRIs, the pharmacokinetic

parameters are adjusted and do not correspond to the efficacy ofantibacterial delight ment in vivo after a external body is implant

ed. These adjustments are seeming if mouse model-based after

math of S. aureus-caused intra-abdominal abscess

surrounding intraperitoneally allocated silicone cathe ter indulged

by meticillin and gentamicin are analysed. [76] Whereas both ag

ents displayed forceful results in vitro in time-kill studies on bacteria colonising catheters seized out of infected mice and on cathe

ters contaminated in vitro, merely poor aftermath were noted in v

ivo, even though elevated innate concentrations (greater than mi


31/138

nimum inhibitory concentration [MIC] for at least 72 hours) of

meticillin and elevated top concentrations of gentamicin (>13 g

/ mL). The wreck was not provoked by progress of antibacterial

confrontation or affected by protein con centration, pH or innate

attendance of inhibitors of antibacterials in the pus. Of significa

nce, antibacterials administered in subinhibitory concentrations c

ould impact the mechanisms of adherence and slime creation, ex

ceptionally in staphylococci, for example, managing to higher pol

ysaccharide intracellular adhesin creation or to increased expressi

on of fibronectin-binding proteins. [77-79] The distinct condition

s encircling a external body have accompanied the find for altern

ative requests of antibacterials such as lipid-based sustained- dis

charge formulations. Roehrborn et al. delineate the use of such b

iodegradable, innately injectable formulation of amikacin in a m

ouse ideal in that Teflon 1 pipes were subcutaneously implante

d and challenged by inoculation of S. aureus. Whereas treatment

alongside innate or systemic free amikacin had no result, the nu

mber of infected external bodies was decreased from 86% to

25% (p = 0.02) pursuing treatment alongside encapsulated amika

cin formulation, and log cfu (colony growing units) each gram oftissue [80] was considerably cut from 4.8 0.9 to 1.3 0.6. Typi

cally, early treatment of catheter-related bacteraemia is managem

ent of systemic antibacterials. Additionally, after a catheter-relat

ed infection is documented and a specific pathogen is identi- fie

d, antibiotic-lock therapy ought to be believed if salvage of the

catheter is necessary. It is notewor- thy that recommendations forthe treatment of

medical device-associated infections are established almost com

pletely on observational studies, animal models, case reports and


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expert opinion rather than on the aftermath of appropriate clinical

trials.

The seven papers included in this thesis all employ the biomimetic m

ethod to enable precipitation of HA on crystalline TiO2 surfaces. As

displayed in Figure 3, the papers focus on methods for depositing func

tional surgical implant coatings; TiO2 and HA, the use of biomimet

ically deposited HA (HA-B) coatings as drug delivery vehicle as well

as the evaluation of the bioactive, biomechanical and bactericidal prop

erties of both coating types.

This thesis is based on the following papers, which are referred to in th

e text by their Roman numerals.

I Lilja, M., Welch K., strand M., Engqvist H., Strmme

M. (2012) Effect of deposition parameters on the photocata

lytic activity and bioactivity of TiO2 thin films deposit

ed by vacuum arc on Ti-6Al-4V substrates. J Biomed

Mater Res B, 100B (4):1078 85

II Lilja, M., Forsgren, J., Welch K., strand M., Engqvist

H., Strmme M. (2012) Photocatalytic and antimicrobial pr

operties of surgical implant coatings of titanium dioxide de

posited through cathodic arc evaporation. Biotech Lett ,

34(12):2299-305

III Lilja M., Lindahl C., Xia W., Engqvist H., Strmme M.

(2013) The Effect of Si-doping on the Release of Antibioti


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c from Hy- droxyapatite Coatings. J Biomater Nanobiotech

, 4(3):237-41

IV Lilja M., Srensen J.H., Brohede U., strand M., Procte

r P., Arnolid J., Steckel H., Strmme M. (2013) Drug loadi

ng and release of Tobramycin from hydroxyapatite coated

fixation pins. J Mater Sci: Mater Med , 24 (9):2265-2274

V Srensen J.H., Lilja M., Srensen T., strand M., Procte

r P., Strmme M., Steckel H. (2013) Co-precipitation of To

bramycin into biomimetically coated orthopedic fixation pi

ns employing sub-micron thin seed layers of hydroxyapatit

e, submitted

VI Srensen J.H.*, Lilja M.*, Srensen T., strand M., Procte

r P., Strmme M., Steckel H. (2013) Biomechanical and A

ntibacteri- al Properties of Trobamycin Loaded Hydrox

yapatite Coated Fixation Pins, submitted


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Materials and MethodsVII Lilja M.*, Srensen J.H.*, Srensen T., strand M., Proct

er P., Strmme M, Steckel H. (2013) Impact of biomechan

ical forces on the release kinetics of hydroxyapatite coated

fixation pins, J Biomater Nanobiotech, 4(4):343-50

Figure 3. Overview of the studies presented in t

his thesis.

The bioactive surfaces onto which HA-B coatings were deposited were

made using CAD.Paper I investigated the deposition parameters to al

low for deposition of crystalline TiO2 coatings consisting of anatase

or rutile phase or a mixture thereof. Additionally, the bioactive and ph

otocatalytic properties of these coatings were evaluated. Coating serie

s with variation in deposition time (2; 5; 20 min), temperature (320;

600 C), bias voltage (-60; -120


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Materials and MethodsV) and oxygen flow (50-800 sccm) were performed.Paper II focus

ed on


36/138

the bactericidal effects of TiO2 photocatalysis of anatase phase do

minated

TiO2 coatings with a thickness of approximate

700 nm. In Paper III to VII, anatase phase TiO2 coatings with a thickness o

f 500

nm served as bioactive surfaces to enable deposition of HA-B coatings

. Fol-

lowing a cleaning procedure in acetone, ethanol and distilled waterafter PVD deposition, the coated substrates were placed in plastic cont

ainers containing either pure, Si-enriched or antibiotic-enriched PBS

for various time periods, see Table 1. The temperature was kept eith

er at 37 C to mimic body surroundings (Paper I, IV to VII ) or incr

eased to 60 C to impact the morphology as well as the growth rate of

the HA coatings (Paper II, VI ). In Paper V , elevated PBS temperatur e was used to deposit - as a first step - a thin, crystalline HA layer ont

o the TiO2 coated substrates. This layer provid- ed small HA crystals

with large surface area and thereby served as a seed layer for the follo

wing precipitation of an antibiotic containing HA coating.

Plasma sprayed HA coated fixation pins served as reference substra

tes in Paper IV. Examples of the coated samples used inPaper IV toVII are presented in Figure 4.


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Figure 4. Overview of samples used inPaper I

V to VII .


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Table 1. Description of the samples used in Paper I and Paper I I I to VII

Geometry Disc

s

Plate

s

Plates

(20*20 m

m), Fixatio

Disc

s

( 9 mm

Fixation pi

ns

Fixation pi

ns

Substrate Materi

al

Ti grade 5 Ti grade 4 Stainless Ste

el

Ti grade

5 discs

SS SS

Dominating Ti

Anatase, rut

ile and th

Anatase Anatase Anatase Anatase Anatase

Solution Dulbeccos PBS

Si-enriched

Dulbeccos P

Dulbeccos PBS

1)Dulbeccos

PBSDulbeccos PBS

Dulbeccos PBS

Temperature [C

37 60 37 1) 37; 37

1) 3


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UV Treatment and Photocatalytic Activity Testing

An UV-A diode (NCSU033B, =365 nm, Nichia, Japan) served as light source for UV illumination during PCA testing inPaper I and II an

d during the evaluation of the antibacterial properties of the TiO2 coati

ng in Paper II . The intensity of the UV light was measured with a UV

light meter (UV-340, Lutron). The PCA of the TiO2 coatings inPape

r I and II was evaluated by measuring the degradation of Rhodamine

B dye as a function of illumination time. A UV photospectrometer

(UV-1800, Shimadzu) was used for adsorption measurements in

order to evaluate the degradation of the inorganic indicator molecu

le.

Bacterial Viability Analysis

Resazurin was employed as an indicator to evaluate the viability

of S. epidermidis in Paper II . As a result of chemical reactions due

to bacterial cell growth, this dye changes from non-fluorescent form

(blue) to resorufin, a highly fluorescent form (pink). The fluorescencemeasurements were performed with an Infinite M200 plate reader (Tec

an). A standard curve was made by n-fold dilution of the bacteria susp

ension to allow quantitative determination of the viability after UV trea

tment.

The biocompatibility of the TiO2-HA coating system used as

drug delivery vehicle was evaluated inPaper VI by cell viability tests.

Endothelial cells and primary osteoblasts were seeded both on TiO2 a

nd HA coated Ti grade 5 discs. The cellular viability and morphology


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of the cells were analyzed after 3 days of culture by Calcein AM staini

ng.


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Antibiotic Loading Procedures

In Papers III, IV, VI and VII the antibiotics were loaded into the H

A coatings by adsorption, whereasPaper V and VI evaluated incorpo

ration of Tobramycin via co-precipitation. Table 2 provides an overvie

w of the antibiotics used and the drug loading conditions employed.

In Paper III, it was investigated whether the antibiotic release pr

ofile

could be impacted by the use of ion substituted HA. For this pur

pose, Cephalothin was incorporated into the pure HA-B and SiHA-B

(deposited from Si-enriched PBS) coated samples via soaking and the

release of the antibiotic from these two sample types was compared.

Paper IV focused on the effect of drug concentration, soaking ti

me as well as the physical conditions (temperature and pressure) unde

r loading on the drug incorporation and release properties. HA-B co

ated fixation pins were exposed to stock solutions of three different c

oncentrations for various time periods. Furthermore, the impact of incr

eased temperature (90 C) or elevated pressure (6 bar), or a combin

ation of both, under loading was investigated using an antibiotic con

centration of 20 mg/ml. All samples were dried for 24 hours at 37 C a

fter loading. The subsequent drug release was studied and compared tothe release profiles obtained from plasma sprayed counterparts expose

d to the same loading procedures.

In Paper VI and VII , Tobramycin was loaded into HA coated sample

s by two different loading methods. The samples were either placed in

containers with a drug solution at room temperature for 5 min or putinto a stainless steel tube together with the 90 C heated antibiotic con

taining solution under an applied pressure of 6 bar. Drug incorporatio

n via co-precipitation was employed inPapers V and VII . For the pre


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paration of those samples, the TiO2 coated pins were in a first step coa

ted with a thin HA startlayer through immersion in 50 ml PBS at 60

C, followed by immersion in antibiotic containing PBS for 6 days at

37 C. The possibility to tailor the drug release from co-precipitatedcoatings was studied by additional loading by adsorption of thi

s antibiotic containing coating structure. For this step, the co-precipit

ation-coated samples were placed in a solution with a drug concentrat

ion of 20 mg/ml for 5 min.

After the last time point measured for the antibiotic release inPape

rs IV to VII , HPLC analysis was performed on solutions containing t

he in pH 2 dissolved coatings to determine the amounts of drugs r

emaining in the coatings.


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Table 2. Drug loading parameters employed for antibiotic incorporation into HA coatings

Paper Loadin

g meth

od

Antibiotic Concentr

ation [mg/ml]

Temp-

erature [ o

C]

Loadin

g time

[min]

Pressur

e [bar

]

III Adsorption Cephalotin 1 37 60

IV, VI, Adsorption, Tobramycin 40 (IV) RT 5; 15; 60 (I

IV Adsorption, Tobramycin 4; 20; 40 RT 5

IV Adsorption, Tobramycin 20 RT 5

IV Adsorption, Tobramycin 20 90 5


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IV, VI Adsorption, Tobramycin 20 90

VII Co- reci itation Tobramycin 0.5; 1 37; 60 6 d


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Biomechanical Insertion Tests

The biomechanical properties of HA-B coatings deposited on TiO2 c

oated fixation pins were evaluated inPaper VI by insertion tests into

bone substitute materials. Renshape BM5166 was chosen to simulat

e insertion into cancellous bone, while low density polyurethane fo

am (25 PU) was used to reflect spongy bone quality.

Figure 5. Schematic set up for insertion tests of HA coated fixation

pins into bone substitute materials.

As shown in Figure 5, the insertion materials were fixated to simulate a bone with single cortex. The samples were inserted without predr

illing at a rotation speed of 10 rpm and with an axial force of

100 N (Renshape BM5166) and 50 N (25 PU), respectively. The coat

ing performance was evaluated by analyzing the insertion torque, m

aximum rising temperature and drilling time needed for an insertion

depth of 23 mm. Three different HA-B coating types, with variationin coating thickness and morphology were evaluated.

In Papers VI and VII , the insertions of antibiotic loaded and co- pr

ecipitated pins into 25 PU material were performed manually.


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where L is the effective grain size and the wavelength of the X-rays.

The bioactivity of the different TiO2 coatings inPaper I was evalua

ted by Scanning electron microscopy (SEM) images of the surfaces after two different immersion times in PBS. SEM was further used to eva

luate the coating thicknesses of the as-deposited TiO2 coatings inPap

ers I and II and to study the morphology, surface coverage, coatin

g thicknesses and co- precipitated coatings investigated inPapers II

I to VII . In Papers VI and VII, SEM was employed for determini

ng the performance of the HA coatings under study, both after inser

tion into bone model materials and after scratch testing.

The surface topography of biomimetic and plasma sprayed deposite

d HA coatings inPaper III was examined by a white light interferom

eter. This method allowed for obtaining three-dimensional surface map

s of the coating types used for the drug loading and release experimen

ts. In Paper VI, the impact of biomechanical forces on the HA coating

quality was studied by an optical 3D micro coordinate measurement s

ystem. This 360 measurement of the HA-B coated fixation pin after i

nsertion into bone model material enabled the determination of high s

tress areas over the entire thread of the pin as well as to visualize cha

nges in the HA coating properties after insertion.The coating adhesion of HA-B coatings with various thickness and

morphology towards the underlying TiO2 surface was evaluated by s

cratch testing on planar stainless steel plates. A 500 m spherical diam

ond indenter was used under progressive loading conditions.

The penetration of Tobramycin into the HA coatings under diff

erent loading conditions was measured with Glow discharge optical emission spectroscopy (GDOES). Quantitative chemical in-depth pr

ofiles of the characteristic elements nitrogen and carbon were obtained

by analyzing the chemical composition of antibiotic containing sam


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ples from the surface towards the substrate. An HA-B coated plate wi

thout any Tobramycin served as reference.


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Drug Release Analysis and Antibacterial In Vitro Tests

The drug release of Cephalotin incorporated into both pure HA and Si

- substituted HA coatings was investigated inPaper III by means of

UV- visible light spectroscopy (Shimadzu UV-1650pc). The conce

ntration of Cephalothin in the release medium was determined by me

asuring the absorbance of light transmitted through the solution with a

characteristic adsorbance wavelength for the drug molecule under stu

dy.

High performance liquid chromatography (HPLC) was employed in

Papers IV to VII to measure the amounts of Tobramycin incorpora

ted in and released from the samples. The method is based on pre-colu

mn derivatization of the aminoglycoside antibiotic and UV-detection

(330 nm).

In Paper IV, it was investigated whether physical parameters u

nder

loading could impact the antibiotic release profile, whilePaper VI foc

used on the effect of HA coating thickness and coating morphology o

n the drug loading and release properties. The path of drug incorporati

on via co- precipitation was followed inPaper V . In Paper VII, the im

pact of insertion into bone model materials on the drug release profileswas investigated. The obtained release profiles were compared to thos

e of mechanically untreated counterparts.

The bactericidal efficiency of Tobramycin incorporated into HA co

ated fixation pins was evaluated inPaper VI . For this purpose, modifi

ed agar diffusion tests were carried out by inserting antibiotic loaded p

ins, as described earlier, into Petri dishes containing 40 ml of sterile Caso agar media and 5 ml ofS. aureus suspended media. HA coate

d pins without Tobramycin served as reference. The petri dishes wer

e incubated at 35 C for 18 hours and the inhibition zones around the


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pins were measured and photographed. After documentation of the re

sults from the first time point, the pins were rolled out over a fresh C

aso agar plate and incubated as previously described. After 24 hours, t

he pins were transferred to a fresh, germ suspended agar plate andexposed to the incubation conditions described above. This proce

dure was repeated until no inhibition zone around the implant coul

d be observed.


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Bioactive, Antibacterial TiO2 coatings

Microstructure, Bioactivity and Biocompatibility

Deposition of crystalline TiO2 coatings on various substrate material

s was accomplished using CAD. InPaper I, the impact of deposit

ion time, - temperature, bias voltage and oxygen flow profile

on the TiO2

microstructure was evaluated and the samples of each

deposition series were denoted as CAD time, CAD temp, CAD bias an

d CAD gradient. As obvious from the summary presented in Table 3,

variations in deposition time influenced the microstructure evolution of

the arc deposited coatings.

Table 3. TiO 2 coating deposition parameters and structural characteristics

Sample Depositi

on time

(min)

Bia

s

(V

)

Temp

- erat

ure

(C)

TiO 2 t

hicknes

s (nm)

Dominati

ng pha

sea

Calculat

ed grain

size b

2min

CAD time 2 -60 320 70 R(110)

20

5min

CAD time 5 -60 320 150 A(101)

30

20min

CAD time 20 -60 320 500 A(101)

35


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30min

CAD time 30 -60 320 700 A(101)

45

CAD

temp 20 -60 600 450 R(110)

35

CAD bias 20 -120 320 650 A(101)

32

CADgradie

nt

20 -60 320 250 (600)c R(110)26

a Measured using XRD, where A and R denote the anatase and ruti

le phases, respectively

b Determined from Scherrer

-Equation

c total coating thickness is ~600 nm, consisting of a metallic phase of

~350 nm and a gradient

oxide phase of

~250 nm

Rutile phase was found to be present near the substrate interface for C

AD time depositions, while the amount decreased with increasing

deposition time [64,65], Figure 6 a. Increased deposition temperature

(CAD temp), as well as enhanced bias voltage (CAD bias), providedthe activation energy


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needed to form rutile phases [66,67] and resulted in lower anatase to r

utile phase ratios compared to 20 min CAD time coatings. Increasing t

he oxygen flow throughout the deposition process (CAD gradient) res

ulted in a coating structure consisting of high amounts of rutile phase,

Figure 6 b.

Figure 6. Diffractograms of CAD time and CAD gradient series (le

ft panel) and SEM images of these coating types after immersion in P

BS for 1 day (middle panel) and 7 days (right panel), respectively.

Both anatase and rutile phases of crystalline TiO2 are known to be bio

active [21,24,25,39]. The presence of anatase phase [13,70] and also small crystals offering a high surface area [71] have been shown to pro

mote HA nucleation on TiO2 surfaces. The bioactivity of the as-depo

sited TiO2 coatings was evaluated by the appearance of HA formatio


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n on the surfaces after being immersed in PBS for 1 day and 7 days at

37 C, respectively. SEM images of HA nucleation and growth on CA

D time (Figure 6 a;1-4) and CAD gradient coatings (Figure 6 b;1-2) re

vealed the appearance of HA crystals on all surfaces after only 1 day in PBS. The crystal surface structure of CAD gradient coatings with s

mall, essentially rutile, Ti dioxide grains or essentially anatase 20

min CAD time coatings facilitated enhanced HA formation after

1 day in PBS (Figure 6 b;1) compared to microstructures with mixtu

res of these two polymorphs, i.e. the 5 min CAD time coating (Figure

6 a;1). Differences in the initial growth rate appeared to be of minor


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impact after a week of immersion in PBS, where a continuous layer w

as present on all TiO2 coatings.

The response of both outgrowth endothelial cells and primary osteob

lasts

was investigated inPaper VI to further asses the biocompatibility of i

mplant surfaces functionalized with crystalline TiO2 with or without H

A-B coatings. As obvious from Figure 7, both cell types were viable a

nd firmly attached to both HA (Figure 7 a,c) and TiO2 (Figure 7 b

,d) coated sample surfaces. These findings provide a first proof of the biocompatible properties of these coating materials and make both sur

face modifications suitable choices to improve the osseoconductive pr

operties at the interface between implant and bone.

Figure 7. Calcein-AM viability assessment of outgrowth endothelial c

ells (OEC) (a,b) and primary osteoblasts (pOB) (c,d) on HA-B (5m) c

oatings (a,c) or 20 min CAD coated (b,d) discs after 3 days of culture.


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Photocatalytic and Antibacterial Properties

The PCA of the as-deposited coatings was evaluated by measurin

g the degradation of Rhodamine B, which served as a PCA indicator

molecule. From all TiO2 coating types under study, those with do

minating anatase phase and with preferably smaller anatase grain size

s were demonstrated to promote higher PCA, as displayed in Figure 8

a. In agreement with literature [70,72], PCA was found to increase wit

h increasing coating thickness up to a coating thickness of about 250 n

m. Nevertheless, the highest photocatalytic


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reaction rate,k , was measured for the CAD gradient coating, Figure 8

b. Increasing anatase contents in the microstructure of TiO2 coatings

has been shown to enhance photocatalytic reaction rates [73]. Small gr

ain sizes can contribute towards enhanced separation of the electrons

and holes produced in the photocatalytic process [74,75] and can furth

er offer a shorter transportation length for the electron-hole pairs from

the grain interfaces towards the surface [76], which may account for e

nhanced reaction rates for fine grained coatings.

Figure 8. PCA of as-deposited TiO2 coatings measured as Rhodami

ne B concentration versus time recorded in a solution containing the di

splayed samples under UV illumination (a) and degradation rates (b) o

f Rhodamine B under UV light illumination.

In Paper II, TiO2 photocatalysis was demonstrated to provide a bacte

ricidal effect againstS. epidermidis . For this study, bacteria suspensio

n was spread over the surfaces of TiO2 coated discs and Ti grade 5 ref

erence discs prior to photocatalytic treatment with UV light. UV dos

es ranging from 0 to 16 J were applied to the surface. The viability o

f the bacteria was evaluated with an MAA incorporating resazurin as v

iability indicator. The reduction in bacteria viability as a function of


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UV dose is shown in Figure 9. It can be seen that a clinically relevan

t UV dose of 2.4 J, corresponding to a illumination time of 2 min, lea

ds to a 90 % reduction of viable bacteria for TiO2 coated samples. Thi

s reduction is attributed to the formation of radical oxygen species at the TiO2 surfaces, which killed the bacteria present on the sample surf

aces. Higher UV doses (>3 J) result in decreasing viability values for r

eference samples, which can be related to the bactericidal effect of the

UV light alone [57].


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Figure 9. Number of viable bacteria on TiO2 coated and Ti grade 5 i

lluminated surfaces as a function of UV light dose, normalised wi

th respect to the tested bacteria concentrations. Error bars show the s

tandard deviation of 3 measurements.

In summary, the large scale production method CAD was demonstrated

to be suitable for making various crystalline TiO2 coatings ofhigh purity possessing not only bioactivity but also on-demand antiba

cterial functions. The results of the studies carried out on samples pro

duced with this method showed that surface microstructure and che

mistry are important factors impacting the initial stages of HA form

ation on TiO2 surfaces in vitro. The results demonstrated that the use

of crystalline TiO2 and HA coatings could be used to functionalize implant surfaces for biomedical applications. The bioactivity and bioc

ompatibility of these coatings are believed to contribute towards impr

oving the bone healing process and thereby increasing the long term st


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ability of the implant. The photocatalytic properties of TiO2 further

encourage the use of these coatings as on-demand antimicrobial su

rface enhancers in order to combat or reduce implant related infections.


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Biomimetic HA Coatings as Carrier for Antibiotics

In order to prevent and control implant related infections, local antibio

tic delivery presents a straight forward approach to administer drugs di

rectly at the bone infected site and without reaching systemic toxicity l

evels of the drug itself [58,68]. To obtain an optimum release profile

[6], comprising a high initial release rate during the first hours, fol

lowed by a controlled release during the next days, the impact of H

A coating properties (Papers III, VI ) and loading conditions (Paper

IV ) on the release kinetics were evaluated. A novel loading method

to incorporate and release clinically relevant amounts of antibiotic

into biomimetic and plasma sprayed HA coatings was developed (P

aper IV ).

In Paper IV , the drug loading and release properties of HA-B and pl

asma

sprayed HA (HA-P) coated fixation pins were compared. The impact o

f drug loading time, drug concentration, pressure and temperature on r

elease properties were evaluated by testing different drug loading con

ditions, as detailed in Table 2. For all loading methods tested, the relea

sed concentration of Tobramycin was measured to be above the MIC

of S. aureus [77] for all sample types during each time interval under study. As can be seen from the release profiles in Figure 10, loading by

adsorption under room temperature and atmospheric pressure (Ref-R

T) resulted in an initial burst release for both HA coating types. Nevert

heless, the nanoporous structure of HA-B coatings demonstrated super

ior drug penetrability, which is reflected by a prolonged sustained rele

ase over a time period of 2 days. In contrast, the rather dense and com pact structures of HA-P samples limited the penetration depth making

only superficial adsorption possible and elution of the entire antibiotic

content occurred after only 15 min.


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Antibiotic incorporation under elevated pressure (6 bar) and temper

ature

(90 C) (Load-PHT) was shown to be a successful adsorptive loading

procedure for fast-loading and slow release, ensuring a release of Tobramycin above the MIC forS. aureus for 8 days from H

A-B coatings with various morphology and thickness, see Figure 10 a

nd Figure 12. Simi- larly, the release periods from HA-P coatings wer

e extended from 15 min to

2 days, Figure

10.

The effective incorporation of Tobramycin into HA-B coatings was

indicated via GDOES analysis, Figure 11. Elevated pressure in combi

nation with an increased viscosity and diffusion coefficient of the drug

containing solutions at elevated temperature resulted in an enhanced

penetration depth of the drug into the denser part of the coating structu

re at a depth of about 3-

4 m. Loading under room temperature and atmospheric pressure (Loa

d-RT)

was found to be mainly characterized by superficial adsorption with a

peak concentration for both carbon and nitrogen at a coating depth of a

bout 1 m.


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Figure 10. SEM images of HA-B (a) and HA-P (b) surfaces and non-c

umulative amounts of Tobramycin released in 37 C PBS from HA-P and HA-B coated pins from the Load-PHT series after being loaded for

5 minutes in a solution containing

20 mg/ml of the antibiotics at 90 C and 6 bar. Release results fr

om reference samples loaded during 5 minutes in similar solutions at a

tmospheric pressures and room temperature are incorporated. Error b

ars denote the standard deviation of 3measurements. The average total amounts of Tobramycin released fro

m each coating type are also displayed.


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Figure 11. Depth profiles of carbon (a) and nitrogen (b) of a HA-B sa

mple loaded with 20 mg/ml Tobramycin under the displayed loading s

eries. The profiles of an unloaded reference sample are also displayed.


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ior of the coating structures along with increasing the total amount

of incorporated drug compared to the corresponding RT loaded sample

s, see Figure 12 d.

To further influence the drug release profile by factors not only influencing the surface-drug attraction but as well taking the bi

nding capacity and HA coating chemistry into account [78,79], the im

pact of Si- doping on the release of Cephalothin from HA-B coatings

was investigated inPaper III . The incorporation of Si-ions during bio

mimetic deposition contributed to form coating structures with larger s

urface areas and smaller crystal sizes [80,81] compared to the pure H

A-B complements. The SEM images of the topographies in Figure 13

a,b showed a flake-like morphology for both sample types [80] and c

onfirm further a more dense structure for HA-B coatings in contrast t

o the more porous appearing crystal network for SiHA-B coatings.


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Figure 12. SEM images of HA-B_37 (a) and HA-B_60 (b) coatings an

d non- cumulative amount of Tobramycin released in 37 C PBS from

HA-B_37 coated (c) and HA-B_37 and HA-B_60 coated pins (d)of indicated thickness after being loaded for 5 minutes in a solutio

n containing 20 mg/ml of the antibiotics under either room temperat

ure at atmospheric pressure or at 90 C and 6 bar. Error bars denote th


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e standard deviation of 3 measurements. The average total amounts of

Tobramycin released from each sample type are also displayed.


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Samples placed horizontally (h) in PBS during coating growth showed

a higher growth rate and also increased drug adsorption ability compar

ed to perpendicular (p) placed samples. The drug release profiles prese

nted in Figure 13 c show a fast release of the antibiotic within the first

10 min, followed by a slower continuous release period lasting for abo

ut 10 hours. Under comparable deposition conditions, SiHA-B coating

s exhibit an increased drug incorporation capacity and faster release pr

ocess compared to pure HA-B coatings. This difference can be explain

ed by the change in surface chemistry [82] and the negative surface ch

arge [83] developing when the HA structure is substituted by Si-ions.

As a result of this, the interaction between antibiotic and Ca-ions in th

e SiHA-B coatings is restricted [84] and the drug is repelled from the s

amples, as confirmed by the rapid release during the first 10 min, Figu

re 13 c. Prolonged release was obtained from pure HA-B coatings, wh

ich can be attributed to a preferential binding of Cephalothin to Ca-ion

s in these coating types.

Figure 13. SEM images (left panel) of HA-B (a) and SiHA-B (b) coated surfaces after an immersion time of 7 days in perpendicular positi

on (p) in PBS and Si- enriched PBS, respectively, and release curves (r

ight panel, c) presenting the initial (lower panel) and total (upper panel


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) release period under study of Cephalothin released in room-tempered

deionized water per surface area for HA-B and SiHA-B in horizontal

(h) or p position.

The findings inPapers III, IV and VI show that it is possible to use

nanoporous HA-B coatings as carriers for antibiotics to obtain a contr

olled release of the incorporated drug over an extensive time period. T

he HA-B coating properties can be modified in various ways and

thereby add flexibility to the proposed local drug delivery concept. By

modifying the deposition conditions, it was possible to adjust the mor

phology, chemical composition, surface area and surface charge of th

e HA coatings and hence


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impact the drug incorporation capacity and furthermore the release pro

per- properties. Altering the physical conditions under loading by adso

rption was shown to have a significant impact on the penetration depth

of the drug and, hence, influence the drug release time. The incorporat

ion of ions, that play a significant role in the biochemistry of bone

tissue, was shown to be a possible strategy to impact the drug affini

ty and drug uptake as well as to tailor drug release profiles. The amou

nts of antibiotics released in these presented studies were sufficient to

inhibit the growth ofS. aureus , which is one of the main pathogens in i

mplant related infections.

These results provide a valuable outline for the design of implant su

rfaces aiming for a fast loading and controlled local drug adminis

tration. The combined use of bioactive ions and antibiotics facilitates t

he development of dual-activity implant surfaces that contribute

both towards tissue regeneration and the prevention of implant relate

d infections.


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Antibiotic Incorporation via Co-Precipitation

Drug incorporation into porous HA coatings and calcium phosphate b

ased materials for local drug release is mostly accomplished by adsorp

tive loading methods using water soluble drugs [50-53,85,86]. Co-pr

ecipitation presents, in contrast, a single step coating deposition meth

od that allows for integration of the drug throughout the coating growth

.

In Paper V, it was shown that co-precipitation allowed producing

To- bramycin containing HA coatings on external fixation pins. Functi

onalizing the TiO2 coated pins in a first step with a thin HA startlayer

coating, Figure

14, was found to be a promising surface modification to overcome th

e - in

literature described - challenge of antibiotic induced inhibition of nucleation and coating growth in the presence of pharmaceutically relevan

t drug concentrations [53,87].

Figure 14. SEM image of an ion-milled cross section of the HA startla

yer after immersion in PBS for 3 days at 60 C.

Increased Tobramycin concentration in PBS was found to influence b

oth coating morphology and coating thickness. As displayed in Figur


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e 15 a,b, co-precipitated coatings obtained from a solution containing

4 mg/ml antibiotic, denoted as Co-4, showed a flake-like morphology

, whereas a Tobramy- cin concentration of 20 mg/ml (Co-20) induced

the formation of smaller, spherical crystals. The total average coating thickness was measured to be ~

3-3.5 m for Co-4 samples and ~ 2-2.5 m for Co-20 sam

ples.

The release profiles, Figure 15 c, show an initial burst release withi

n the first 15 min followed by a slow, controlled release lasting for

12 days for Co-4 and for 8 days for Co-20 coated pins. Incorporation

of Tobramycin loaded by adsorption into the porous structure of Co-

4 samples, designated as Co-4/20, enabled the incr

solution y

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