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Title: PowerPointPrsentation


1
biofilm on dental material
2
overview
  • biofilms in general
  • protein adsorbtion
  • botrytis cinerea and dental materials
  • atomic force microscopy (AFM)
  • AFM as sensor to analyze biofilms

3
definition biofilm
  • can be composed of bacterials, fungis, proteins
  • always on interfaces
  • are present in all our daily life
  • efficient way of life with division of work

4
phases of biofilm development
  • accumulation bacterials are aggregating on the
    basical film and develop a bulk-layer and a
    surface film

1. induction basical film with slimy texture
within some hours
1
3. enlarging the volume because of cell division
and protein production
5. complete/fractional decomposition
4. existence-/plateauphase balance between
growing and fading away
5
structure of a biofilm
washing some cells away to build new biofilm
surface-biofilm compact/loose but also regular
and frayed form possible
compact basical film
pores, caverns and lodes mass transfer between
the cells is possible supply with water and
nutrients optimized
6
what keeps them together?
  • cell communication (quorum sensing
    communication depending on concentration of
    cells)
  • changes in gene expression (signaling cascade
    starts because of surface contact)
  • adhesives
  • protein adsorption/adhesion

7
parameters of protein adsorption 1
charged surfaces effect of electrostatics
ambient conditions (pH, ionic strength, buffer,
)
surface characteristics (hydrophobicity of the
interface)
thermodynamics
protein adsorption
texturing of the proteinfilm
protein characteristics (charge, conformation,
interior stability, )
diffusion velocity (small molecules are faster
than large molecules)
concentration of different proteins in solution
(competition on binding sites)
8
parameters of proteinadsorption 2
  • In general applies the following
  • hydrophilic surface no or low proteinadsorption
    and celladhesion
  • hydrophobic surface good proteinadsorption and
    celladhesion
  • hydrophobie/hydrophilie and interior stability of
    the protein
  • - hydrophobic proteins bind irreversibel on
    hydrophobic
  • surfaces and reversibel on hydrophilic
    surfaces
  • - proteins with low interior stability adsorb
    on all surfaces
  • - proteins with high interior stability
    adsorb in general on
  • hydrophobic surfaces

9
steps of protein adsorption
  • transport/diffusion in direction of the
    interface/surface (2 versions)
  • very fast diffusion-controlled process protein
    adsorps directly when reaching the surface
  • 2. slow process because of occupied binding
    sites
  • bonding on the interface/surface
  • structural rearrangement of the protein
  • desorption from the interface/surface
  • evacuation from the interface/surface

10
kinetics of protein adsorption
adsorbed amount shows a dependen of protein
concentration kinetics are diffusion-controlled
(diffusion according to Fick Dt1/2)
11
competition of proteins
  • Sequential adsorption
    VROMAN-EFFECT 1
  • at the beginning small proteins adsorb to the
    surface (high diffusion velocity)
  • In the course of time displacement of the small
    proteins by large proteins (low diffusion
    velocity)

12
pH and concentration dependency
lysozyme on SiO2 a 0,03 g/l b 1g/l
  • pH 7 shows a significant intenser adsorption as
    pH4
  • the larger the concentration the intenser the
    adsorbed amount of proteins
  • orientation of adsorbed molecules is dependent
    on pH when the concentration of protein is high

13
forces in proteinadsorption
  • Van-der-Waals-interactions
  • Coulomb-interactions between proteins and
    electrical charged surfaces
  • hydrophobic effect
  • changing of conformation

14
thermodynamics
  • protein adsorption if
  • Van-der-Waals-interactions
    DHlt0
  • Coulomb interactions
    DHlt0
  • release of watermolecules (hydrophobic effect)
    DSgt0
  • changing of conformation
    DSgt0

15
Van-der-Waals interactions
  • If DHlt0 then DGlt0 protein
    adsorption

protein
attractive interaction
H2O
VdW-interaction between -0,6 kT and -4,6 kT
2 (k Boltzmann constant T temperature)
H2O
r
H2O
H2O
d
Si-oxid
r 2 nm d 0,1 bis 0,5 nm
16
coulomb interactions
If DHlt0 then DGlt0 protein
adsorption
coulomb interaction between -0,8 kT and -1,9 kT
2 (dependent on orientation of the protein and
a supposed distance of 0,8 nm between protein and
interface/surface)
Si-oxid
Charge density -0,14 e nm-2 (e elementary
charge)
maximum adsorption if net charge is 0
(isoelectric point) because the repulisve
electrosatic forces are minimized within the
protein
17
hydrophobic effect
  • contact of two hydrophobic surfaces
    release of surface water DSlt0 (water
    molecules get back their degrees of freedom)
  • dehydration of protein with 10 hydrophobic
    side chains
  • DG decreases in the range -3,4kT and -7,3kT
    2 (2 because of dehydration of the surface)
  • no hydrogen bonds between water molecules and
    hydrophobic surface DSlt0 no
    protein adsorption

H2O
H2O
H2O
H2O
H2O
H2O
18
changing of conformation
  • changings in protein structure produce an
    optimization of interactions
  • losing a secondary structure involves DSgt0
  • BSA reduces his a-helices on Si from 74 to 38
  • 2 conformations accepted and 210 rests of amino
    acids involved
  • interaction energy about -146kT
    3

19
comparison
20
biofilms on dental materials
  • pellicle
  • natural biofilm on enamel
  • thin (about 1 mm) invisible permanent film
  • is composed of saliva constituents (water, mucin,
    proteins, salts, enzymes)
  • function pH-regulation, lubricant for
    tooth-tooth-friction, depot of remineralization,
    caries protection
  • build within 30 minutes and washable

21
biofilms on dental materials
  • plaque
  • bacterials attach to the pellicle
  • consists of several complex layers
  • in addition to components of pellicle it
    contains microorganisms (bacterial biofilm hold
    together by factors described)
  • to be removed with toothbrush
  • consequences are caries and parodontitis or
    white coationg on dental implants

22
formation of pellicle/plaque
proteins
23
biofilms on fruits
  • Botrytis cinerea is a pathogen of grey mould on
  • strawberries/grapes/tomatos (over 200 fruits
  • can be attacked)
  • germination depends on hydrophobicity and
  • hardness of the surface in addition to nutrients
  • after germination a homogeneous biofilm is
  • build (mould)

24
analyzing biofilms
  • atomic force microscope (AFM) is a versatile tool
    with a
  • large application area
  • for example analyzing biofilms
  • forces in the biofilm (force-distance-curves)
  • structure of the biofilm (height, surface
    structure,)
  • characterization of biofilm formation on a
    molecular scale

25
principle of AFM
different applications
force-measurements surface strucuture
piezo
26
force-measurements
  • proteins are covalently bound on the tip of the
  • Cantilever via
  • crosslinkers
  • Pegylation
  • Botrytis is bound via Poly-D-Lysin on the tip of
  • the cantilever

27
force-measurements
force-distance-curve
28
substrate dependency
  • Botrytis on different substrates 4

the larger the contact angle the more hydrophobic
is the surface
29
pH and substrate dependency
lysozyme on enamel and dyract pH 5 and pH 7 5
30
time and substrate dependency
BSA on 2 different dental materials 4
31
pellicle on enamel
after 60 min
32
pellicle on dyract
after 60 min
33
take-home message
Coca cola lightTM etching 5
pH-value 2,7 Residence time 60min
untreated enamel
34
from idea to market
  • Botrytis cinerea
  • surface coating for fruits???
  • .
  • dental materials
  • new surfaces where no plaque can be generated
  • upgrading of existing materials or enamel to
    invoid
  • plaque (surface coating?)

35
literature
1 Vroman et al. 1977, Lu et al 1994 2 Chem.
Unserer Zeit, 2006, 40, 238-245 3
http//eldorado.uni-dortmund.de/bitstream/2003/215
20/2/Jackler.doc 4 Schmitt Felix 2006,
Cantilevermodifizierungen zur Untersuchung der
Biofilmbildung auf Implantatmaterialien und der
Bestimmung der Adhäsion von Botrytis cinerea
Konidien auf Modelloberflächen mit Hilfe der
Rasterkraftmikroskopie 5 Schwender et al. 2005,
initial bioadhesion on surfaces in the oral
cavitiy investigated by scanning force
microscopy 6 Lu et al. 2004 proteinadsorption
at interfaces
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