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