1

Characterization of Cell-Based Adhesive Biointerfaces

September 11 (Mon) and 14 (Thurs)Dr. David Shreiber

Dept. of Biomedical Engineering

Prof. Moghe

125:583 Biointerfacial Characterization

2

SIGNAL TRANSDUCTIONGENERAL CONTROL MECHANISM

Stimulus

Sensor

Signal Transducer

Regulator

Effector(Response)

ECMAnother CellForce(Form junctions/adhesion complexes)

IntegrinsCadherinsIg Superfamily of cell adhesion molecules (CAMs)Selectins

ProliferationTissue FormationTractionMigrationApoptosisDifferentiation

Cytoskeletal ElementsKinasesG-Proteins

3

CELL ADHESION

• Most cells demonstrate anchorage-dependent growth.

• Adhesion can also affect migration, differentiation, and apoptosis.– Includes adhesion to materials for tissue engineering

4

FORCES IN BIOLOGICAL INTERACTIONS

Electrostatic double layer: even though cell and bacterial membranes are negatively charged, and you think they’d repel each other, ions can associate with the cell membranes to form a kind of cloud around it. If two cells share the same cloud, there is measurable adhesion between them.

5

FORCES IN BIOLOGICAL INTERACTIONS

Electrostatic double layer: even though cell and bacterial membranes are negatively charged, and you think they’d repel each other, ions can associate with the cell membranes to form a kind of cloud around it. If two cells share the same cloud, there is measurable adhesion between them.

6

FORCES IN BIOLOGICAL INTERACTIONS

Of course, cells are not in water, and this force is sensitive to the ionic strength of the extracellular environment in vivo and culture medium in vivo.

7

FORCES IN BIOLOGICAL INTERACTIONS

Then are the Van der Waals attractive forces, which are due to interactions between oscillating dipoles of the surface molecules.

These forces are very powerful, but only for a small distance.

8

FORCES IN BIOLOGICAL INTERACTIONS

If we combine these forces, we can describe the net forces acting on, essentially, colloidal particles. At least this is what the first people were studying who described this theory.

It’s called DLVO theory, named after people from two research groups: Deryagin and Landau (group one) and Verway and Overbeek (group two).

SUMMARY

1.Most cells and biological surfaces are negatively charged

2.In a fluid environment the surface negative charge attracts a layer of mobile counterions (cations) which is known as the "Electrical Double Layer"

3.As bacteria approach a surface or another cell the electric double layers begin to overlap causing an electrostatic repulsive force

4.As bacteria approach a surface they also experience an attractive force known as van der Waals force

5.The combination of these two forces dictates how a bacterial cell adheres and is described by the DLVO theory

 6.The DLVO theory predicts that in a solution of physiological ionic strength bacterial cells will be held about 10nm away from a surface and will be unable to approach closer.

7.The electrical double layer shrinks if the ionic strength of the surrounding fluid is increased which allows cells to get nearer to surfaces. This is the reason why adding salt to a bacteria suspension causes the cells to flocculate

8.The stability of bacteria in suspension is very sensitive to the valence of the counterion, therefore, calcium has a greater effect on bacterial adhesion than sodium.

http://www.ncl.ac.uk/dental/oralbiol/oralenv/tutorials/electrostatic.htm

9

THERMODYNAMIC ASPECT OF CELL ADHESION

10

TISSUE SORTING

What is the work of adhesion for a single liquid?

Wo/o = Eo/A + Eo/A - Eo/o= Eo/A + Eo/A – 0 = 2Eo/A

Suppose

Wo/w >= Wo/o and Wo/w >= Ww/w

Add these to get

Wo/w >= (Wo/o + Ww/w)/2 or >=Eo/A + EW/A

Recall from previous slide that:

Wo/w = EW/A+ Eo/A - Eo/w

So in this case, in order for

EW/A+ Eo/A - Eo/w >= Eo/A + EW/A,

Eo/w must be <= 0….the liquids are totally miscible

If we change the conditions in blue above, we can get separation or sorting.

O

W

WW

W

W

O

O

O OO

W

W

W

OOO

OO

O

W

W

W

W

O

O

OO

O

W

W

W

O

OO

O OOO O

WW

W

WW

W

W

SORTING:Wo/o>Wo/w>=Ww/w

RANDOM:Wo/w>Wo/o>=Ww/w

SEPARATION:Ww/w>=Wo/o>>Wo/w

11

CELL/TISSUE SORTING

12

FORCES IN CELL ADHESION

13

CELL ADHESION: STRENGTH

From Sundar

14

ADHESIVE DYNAMICS

From Sundar

15

ADHESIVE DYNAMICS

16

ADHESIVE DYNAMICS

17

MEASURING ADHESION

18

ATOMIC FORCE MICROSCOPY

19

SURFACE FORCE APPARATUS

20

OPTICAL TWEEZERS(LASER TRAP)

A particle encountering the laser beam will be pushed towards the center of the beam, if the particle's index of refraction is higher than that of the surrounding medium.

In a ray optics picture we realize how light is deflected in the particle, resulting in a gradient force that pushes the particle vertically to the propagation of the laserbeam, towards the largest intensity of light (the middle of the laserbeam).

By focusing the light, the gradient force pushes the particle backwards as well. If this force overcomes the propagation force of the laserbeam, the particle is trapped.

21

OPTICAL TRAP

22

MICROPIPETTE ASPIRATION

23

controlled conditions

MEASURING CELL ADHESION

24

HOW CAN YOU MEASURE BULK ADHESION STRENGTH IN VITRO?

• Parallel plate flow chamber

• Cone and plate viscometer

• Rotating cylinder

25

SCHEMATICS OF ASSEMBLIES

26

CELLS CAN EXERT FORCES ON A SUBSTRATE THROUGH ADHESIONS

27

TENSEGRITY AND CELL ADHESION

28

FORCES DURING MIGRATION

29

TISSUE EQUIVALENTS

• Self-assembled biopolymer networks with entrapped tissue cells of interest.

• Cells exert traction on network resulting in compaction of the network and/or locomotion of the cells.

Fibroblast

Cell traction-induced alignment of network

30

ICTA - Methods

Lexan Mold

Medium with defined soluble factors

Coverslip

Stainless Steel Well

31

FREELY COMPACTING VS. CONSTRAINEDGELS

Freely CompactingUnstressed

Constrained at EndsStressed

Presence of stress -> network alignment -> cell alignment

32

DYNAMIC REGULATION OF ADHESION

33

IN WHAT TISSUES DO CELLS REGULARLY EXPERIENCE

MECHANOTRANSDUCTION?

34

MECHANOTRANSDUCTION

It’s well known that shear flow affects endothelial cell behavior.

How can you study this mechanotransduction system in a

controlled manner?

This would be the intima later of a vessel, which is lined by a confluent layer of endothelial cells.

35

LAMINAR FLOW IN A PIPE

( )

L

pRQ

x

pRQ

rdrRrx

prdrudQ

L

p

L

pp

x

p

RR

A

μ

πμ

π

πμ

π

8

8

24

12

)(

4

40

22

0

12

Δ=

⎟⎠⎞

⎜⎝⎛∂

∂−=

−⎟⎠⎞

⎜⎝⎛∂

∂==⋅=

Δ−=

−=

∂

∂

∫∫∫ Au

:is c)(volumetri rateflow The

constant. is gradient pressure the flow, developed fully For

Anyone know what this flow rate law is commonly called?

36

SHEAR FLOW ON ENDOTHELIAL CELLS

3

4

2

R

Qô

x

pr

dr

du

R

rx

πμ

μτ

=

=

⎟⎠⎞

⎜⎝⎛∂∂

==

reside. cells lendothelia where R,r at stress shear the need wecourse, Of

:by given is ondistributi stress shear The

Anyone know what this flow rate law is commonly called?

This defines the shear stress experienced by endothelium as a function of vessel radius and flow rate.

37

LAMINAR VS. TURBULENT

•

Re low = laminar

Re high = turbulent

μρVD

=Re

Laminar

Turbulent

• D is on the order of microns• Small reagent volume, Shorter

reaction times• “Low Reynolds number flow”

All mixing is through diffusion

In ‘Microfluidics’ …

38

IN VIVO FLOW PREDICTIONS

39

MECHANOTRANSDUCTION

40

HOW CAN YOU MEASURE BULK ADHESION STRENGTH IN VITRO?

• Parallel plate flow chamber

• Cone and plate viscometer

• Rotating cylinder

41

FLOW AFFECTS CELL SHAPE

42

RESPONSE OF ENDOTHELIAL CELLS TO SHEAR

Just about every aspect of endothelial cell behavior can be regulated in part by mechanical signals.

43

RESPONSE OF ENDOTHELIAL CELLS TO SHEAR

Just about every aspect of endothelial cell behavior can be regulated in part by mechanical signals.

44

MECHANOTRANSDUCTION IN VITRO

eME

bubble

medium

stir bar

45

ECMCELLS

• Cells adhere to matrix by specific receptors that, just by binding, can initiate specific signal cascades

• Forces exerted on ECM are transduced to the cells, and forces by cells can remodel the matrix

• The distribution and strength of adhesion sites (either on cells or matrix/biomaterials) can control cell growth, differentiation, migration, traction etc…

46

CELL ADHESION AND BIOMATERIAL DESIGN

• Obviously the type and number of cell adhesion sites can be critical in optimizing a tissue engineered product

• Biomaterials can, therefore, be more than just “biocompatible/bioinert” – they can be “bioactive”.

• Fortunately, or unfortunately, after most cells adhere to a substrate, they begin to secrete their own matrix (especially fibronectin), which pretty much wipes out any specificity the biomaterial may provide.