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