BE 527 / 727 / MS 727
Exam 1 Review
Preparation for Exam 1
(discussion in lectures before Exam; preparation for
Exam 1)
• State clearly and succinctly motivation of each
article (shared drive: Folder - Exam 1
homework)
• Be able to describe succinctly the fundamental
basis of all of the experimental methods and
biological processes
• Comment on the interpretation of the results
• What experiment(s) would you do next?
*Note:
1) May require researching related/cited reference list
2) Serves also to prepare you for the ‘mini grant’
Biomaterials Science and Engineering
is highly interdisciplinary
We will be covering topics in diverse fields such as:
• biochemistry
• biophysics
• anatomy
• structural biology
• immunology
• histology
• pathobiology
• engineering
• materials science (polymers)
• physical chemistry (colloid & surface science)
Blood-Material Interactions
• The Players
• Proteins
• Cells
• Surfaces
• Biological Processes
• Physiology
• Molecular level
Motivation
• Implants can often lead to blood clotting
• Design of biomaterials requires a
fundamental understanding of hemostatic
mechanisms and how these impact response
to biomaterials
Tom Horbett (Univ of Washington)
Physiology
Sequence/Continuum of Host Reactions
Following Implantation of Medical Devices
• Injury
• Blood-material interactions
• Provisional matrix formation
• Acute inflammation
• Chronic inflammation
• Granulation tissue
• Foreign-body reaction
• Fibrosis/fibrous capsule development
Physiology
Hemostasis: Control of Blood Flow
after Vascular Injury
• vascular constriction (limit blood flow)
• platelet activation, aggregation, formation of platelet
plug
• fibrin mesh, clot formation
• dissolution of clot triggered by plasmin
Same process can occur when artificial surfaces
(biomaterials) are placed in contact with blood!
Blood clotting
• Components:
– Platelets; fibrinogen; vWF
• Phases in blood clotting pathway: platelet
activation, adhesion, aggregation, clot
retraction, clot dissolution
• Crosslinking between platelets (via cell
surface receptors) and serum proteins
• Serum proteins can adsorb onto
biomaterial surfaces
How do we study protein adsorption?
Properties Properties
of of
Surface Protein(s)
Organization of
Adsorbed Protein Layer
Cellular Response
to Surface
Properties of the “Big 12” plasma proteins
Properties of Material Surfaces
• Many types of biomaterials exist with specific properties
– Metals
– Ceramics
– Polymers
– Composites
• Material surface properties
– Topography and roughness
– Charge
– Hydrophobicity
– Specific chemistry
Key
Defini)ons
• Adsorp)on:
molecule
adheres
to
the
surface
• Absorp)on:
molecules
go
into
the
interior
of
the
surface
• Physical
(physisorp)on):
molecule
is
bound
by
physical
forces
• Chemical
(chemisorp)on):
chemical
bond
is
formed
*How
can
you
tell
the
difference
between
physical
and
chemical
adsorp6on?
Why is fibrinogen adsorption assessment
required for new biomaterials?
Protein Adsorption Experimental Techniques
Imamura 2001
Additional techniques include SPR, ESCA-XPS, SIMS, SERS, SFA
Langmuir Adsorption Model
Assump)ons
of
Langmuir
model
• All
surface
sites
have
same
ac)vity
for
adsorp)on
• Adsorbed
species
do
not
interact
with
each
other
à
only
a
monolayer
can
form
• Monolayer
is
an
ideal
2-‐d
solu)on
of
equal
sized
solute
and
solvent
molecules
• Compe))on
between
solute
and
solvent
to
occupy
a
site
on
the
surface
Desirable outcomes from model:
• Information about adsorption isotherms
• Adsorption kinetics (in situ)
• Conformation of adsorbed proteins
• Number and character of surface bound protein
segment
• Physical parameters describing the adsorbed
protein layer
*How would you validate these predictions
experimentally?
Derivation: Key Relations
b s s b
x +x ↔x +x
2 1 2 1
s b
aa2 1
K= s b
aa1 2
x: mole fraction
a: activity
1: solvent
2: solute
s: surface
b: bulk
Derivation
Assume
that
surface
layer
is
ideal.
Ac6vity
for
the
surface
can
be
replaced
with
mole
frac6on.
s b
xa 2 1
K= s b
xa 1 2
Surface
contains
only
two
components:
solvent
and
solute.
b
a
s b K 2
b
xa s 2 1 a 1
K= b
⇒ x2 = s b
(1 − x )a2 a
K 2 b +1
2
a1
Derivation
Define
a
new
constant
b
=
K/a1b
Define θ = fraction of surface occupied by solute
b
s 2 ba
x =
2 b
=θ
ba + 1
2
For
ideal
solu6ons,
a=c
bc kc
θ= =
bc + 1 kc + 1
k: equilibrium constant for
adsorption
Langmuir Adsorption Isotherm
• Case
1:
kc
is
large
(kc>>1)
kc – Θ
approaches
1,
surface
is
saturated
θ= • Case
2:
kc
is
small
(kc<<1)
kc + 1 – Θ
~
kc
for
low
c
Surface concentration initially increases linearly with solute
concentration and switches to an asymptotic approach to
monolayer saturation
CharacterizaEon
of
biomaterials
• Why
characterize
materials?
• What
properEes
are
we
interested
in?
• What
techniques
are
available
to
measure
these
properEes?
• What
are
the
advantages
and
shortcomings
of
available
techniques?
• CharacterizaEon
in
literature:
Hirsh
et
al.
Mo)va)on
for
Biomaterials
Characteriza)on
• Develop
quality
control
criteria
• Analysis
of
surface
modifica)on
• Assess
the
impact
of
steriliza)on
on
biomaterials
surfaces
• Evaluate
oxida)on,
degrada)on,
corrosion,
etc.
• Compare
process
methods
• Monitor
surface
ac)ve
leachables
exuded
from
the
bulk
• Correlate
surface
chemistry
and
biological
response
Why
Characterize
a
Surface
• Surface
of
a
biomaterial
is
oLen
different
from
the
bulk.
• Surface
atoms
have
different
reac)vity
than
bulk
material.
• Molecules
at
the
surface
of
a
material
are
what
determine
the
biological
response
• Methods
for
bulk
characteriza)on
are
oLen
not
sensi)ve
enough
to
analyze
only
the
small
amount
of
material
at
a
surface
• Surfaces,
due
to
posi)on
and
chemistry,
readily
contaminate
with
vapor
phase
components
• Surface
structure
is
oLen
mobile
• Many
previous
studies
have
shown
importance
of
many
factors
–
roughness,
weOability,
chemistry,
mobility,
charge,
etc.
on
biological
response.
Technique Physical Principle Measured Quantity Depth of Analytical
Analysis Sensitivity
Photoelectron Effect: Kinetic energy ( or binding
XPS (ESCA) incident X-rays cause energy) of emitted core- 10-250 Å 0.1 At%
emission of electrons level electrons
Incident electrons (e-) cause Kinetic energy of Auger
AES emission of Auger e-’s electrons 50-100 Å 0.1 At%
Incident ions emit ions (2˚) Mass/charge ratio of
SIMS of different mass emitted ions 10 Å - 1µm 0.001 amu
IR radiation is absorbed by Absorbance as function of
ATR-FTIR molecules leading to wave number 1-5 µm 1 mol %
unique interatomic
vibrations
Photons created by visible Intensity as a function of
SERS light yield wavelength- frequency Thin films Low
shifted visible light
Surface topography Quantitative three-
AFM induced deflection of a dimensional images and depth/lateral Atoms and
cantilever is detected by surface physical properties resolution: molecules
reflected laser light and a 1Å
photodiode
Ellipsometry Detection of polarization Overlayer thickness,
(SE) change of light reflected on surface coverage, and Å to µm 2Å
a surface adsorption kinetics
• And others: Contact angle, QCM, etc.
Choosing
Techniques
• Different
techniques
require
different
sample
prepara)on
• Some
techniques
are
more
destruc)ve
to
the
surface
than
others
• Some
techniques
offer
greater
sensi)vity
than
others
• Cost,
)me,
availability…
The
Vroman
Effect:
CompeEEve
Protein
Exchange
with
Dynamic
MulElayer
Protein
Aggregates
Hirsh
et
al.
2013
• Apply surface characterization techniques at varying time
points following material contact with a protein mixture in an
attempt to better understand the Vroman Effect.
• Utilize AFM, QCM-D, TOF-SIMS, in-solution TOF-MS to
demonstrate that protein exchange can occur by the turning of
multilayer protein aggregates.
• Results consistent with earlier postulated “transient complex”
models.
embeds itself in the initially adsorbed layer o
ustrating three different processes proposed for the change in composition of a layer adsorbed from a mixture solution by exch
roteins. (A)• Initially
Howadsorbed
was surface
protein characterization useda to
1 (blue) desorbs, leaving support
vacancy the Transient
for protein 2 (red) to Complex Modeladsorbed
adsorb. (B) Initially ?
stronger binding affinity to the surface. (C) Protein 2 embeds itself in previously adsorbed protein 1 to form a transient com
protein 1 to solution (middle); protein 1 desorbs into the solution and protein 2 remains on the surface (bottom). (For interpr
• Motivation for studying
intermolecular and surface forces
• Surface Forces Apparatus
• van der Waals forces
– Molecular description
– Going from molecular to macroscopic
• Hamaker constant
Motivation for studying DLVO theory
How can we describe electrostatic, van der Waals,
hydrophobic interactions between surfaces, particles, and
particles and surfaces?
Why should we be interested in studying
intermolecular forces?
We would like to answer questions such as...
• Will a molecule (protein) be adsorbed or repelled from a surface?
• Under what conditions will it desorb from the surface? (pH, [salt], T)
• Under what conditions can I release it from the surface?
• How weakly/strongly is something adsorbed to the surface?
• Under what conditions will my particles aggregate or remain
dispersed?
Under what conditions would I want aggregated nano/microparticles?
Under what conditions would I want dispersed nano/microparticles?
Example: Stability against aggregation
Theoretical prediction: Interaction between two MBs
Free energy of two interacting microbubbles (MBs) with a polymeric shell
• WPEGW5KPEGMB W
much WElectextended than PEG 2K MB.
VdW more
• PEG 5K MBs: 1
AH R marginally stable against
s æ k BT ö
aggregation æ e 0 ö
WVdW = . WElect = RZe , Z = 64 0ç ÷ tanh ç ÷ ,
æ ps ö 2 è e ø è 4kBT ø
• 12s çè
PEG 2K MBs: 1+ ÷ø
unstable at physiological conditions.
WPEG from a molecular theory
Polymer volume fraction Free energy
Park et al. (2012 epub) Langmuir
Conventions for force-distance curves:
DLVO example
1. Interactions b/w Macroscopic Bodies
•Interaction free energy
Electrostatic repulsion
W(D) WE (D) WA (D)
W(D)
D(nm)
2o
van der Waals
1o attraction
* Need to understand van der Waals and electrostatic interactions
to develop curves based on DLVO theory
Surface Forces Apparatus
D. Leckband (UIUC)
Classes of Intermolecular Forces
• electrostatic: Coulomb force between charges, permanent
dipoles (ion-ion, ion-dipole, dipole-dipole)
• polarization forces: arise from dipole moments induced by
E of nearby charges and permanent dipoles (all interactions
in a solvent medium involve polarization effects)
• quantum mechanical: covalent or chemical bonding or
charge transfer; and repulsive steric or overlap interactions
(occurs when the electron clouds of two closed-shell
molecules overlap – Pauli exclusion principle) that balance
attractive forces at very short distances
• dispersion or fluctuation interactions: induced dipole
induces a dipole in another molecule to form another
induced dipole
Difference between physical and
chemical bonds
• covalent bonds: electron charge distribution
changes completely and merge during binding
• physical ‘binding’: electron charge distribution is
only perturbed and atoms remain distinct
entities
• physical ‘binding’ forces can be as strong as
covalent bonds; weakest force is strong to hold
most atoms and molecules together in solids
and liquids at room temperature (eg:
streptavidin-biotin (35kT) )
van der Waals forces
• exhibit common power law dependence ~ 1/r6
• Lennard-Jones potential
W(r) = A/r12 – B/r6
• three components: Keesom, Debye and London
– Keesom: dipole-dipole
– Debye: dipole-induced dipole
– London: dispersion
Hamaker Constant (A)
Α = π2Cρ1ρ2 (See table of geometries)
• Hamaker constant is closely related to range and
the strength of the vdW contribution.
• Hamaker constant depends on the dielectric
properties of the interacting materials and the
intervening medium.
• Hamaker constant of proteins in water ~ 3 – 10
k BT
DLVO is sum of vdW and e-statics
1. Interactions b/w Macroscopic Bodies
•Interaction free energy
Electrostatic repulsion
W(D) = WE (D) + WA (D)
W(D)
D(nm)
2o
van der Waals
1o attraction
* Need to understand van der Waals and electrostatic interactions
to develop curves based on DLVO theory
Review from Last Lecture
Diffuse Electrical Double Layer
(How do surfaces
acquire charge?)
We have a framework to describe vdW interaction. How do we describe the electrical
potential as a function of distance from a surface??
Poisson equation
• gives relationship between electrical potential
and charge density in a vacuum
• Assumptions:
• ion-ion interactions dominate
• Average over solvent degree of freedom
• Eliminate ion-dipole and dipole-dipole interactions
Boltzmann Equation
• Need to know potential as a function of x
• Boltzmann equation gives charge density per unit volume r
at any location in the solution
Need fx= f(x)
Use Poisson-Boltzman equation
Solved gives r(x), f(x), and
d f
2 E= df/dx at any point
2 zer / between surfaces
dx o
Poisson-Boltzmann (P-B) Equation
Electrostatic interactions require solving the
P-B equation
2 1
n z e exp( z i e / kT )
o
i i
o i
• 2nd order nonlinear differential equation
• gives
• r(x),
• f(x), and
• E = df/dx at any point between surfaces
K-1= Debye length (L)
Macroscopic vdW and electrostatics
