Lecture3: PhysicsofCellAdhesion · Lecture3: PhysicsofCellAdhesion Prof. Dr. Thomas Groth...

Martin Luther University Halle-Wittenberg

Lecture 3:Physics of Cell Adhesion

Prof. Dr. Thomas Groth

Biomedical Materials



Content

• Role of cell adhesion in growth, differentiation, function andsurvival of cells.

• Understanding physicochemical base of cell adhesion on biomedical materials and other surfaces.

• Brief introduction DLVO Theory

• Brief introduction thermodynamic approach

• Examples how surface properties of biomedical materialsinfluence adhesion of cells but also phagocytosis of bacteriaor particles.


___________________ ___________________

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adhesion-dependent adhesion-independent

Two Different Kinds of Body Cells

-Epithelia-connective tissue-muscles-nerves

4 different types of tissues:

Blood cells


Adhesion-Dependent Cells

• Adhesion-dependent cells tissue formation

• Basis tissue structure cell-matrix and cell-cell-adhesions

• Adhesion control of cell growth (contact inhibition) Loss of control cancer.

• Loss of adhesion normal cells controlled cell death (apoptosis)

• Loss of adhesion cancer cells survival, growth,metastasis

• Cell adhesion prerequisite wound healing, integration of implants & tissue engineering scaffolds

Types of connective tissue

edinformatics.com


Adhesion

Adhesion-Dependent Cells on Surfaces

Loss ofadhesion

Migration Proliferation

Differentiation


Scratch Wound Healing Assay ShowingMigration of Cells

Provided by courtesy of Jose Ballester-Beltran


Adhesion-Independent Cells

• Adhesion-independent cells originbone marrow blood cells.

• Lack of adhesion no effect on survival.

• However, cell-matrix and cell-cell adhesion control of programming, function and life-cycle

• Adhesion prerequisite fortransmigration from blood into tissues

• Adhesion on biomedical materials surfaces induction of inflammatory responses and thrombus formation.

Red blood cells Leukocytes

Process of transmigration of leukocytes from blood into tissue

Thrombus (bloodclot) formation


Why is Cell Adhesion Important in Application of Biopharmaceuticals?

Phagocytosis of micro and nanoparticles in transfection or deliveryof other biopharmaceuticals

In-situ transfection of cells on tissue engineering scaffoldsrequires adhesion (e.g. HeLAcells transfected with GFP) © Uni Hamburg AG Stefan Förster

200 µm

Colonisation of scaffolds

b


Theory of Cell Adhesion

I. Cell adhesion is dependent on the physicochemical properties of the substratum (long& short range interaction forces, hydrophobic effectdominates).(Curtis, 1960)

II. Cell adhesion is based on receptor-ligand interactions (specific) short-range interaction forces dominate).(Grinnell 1980, Yamada, 1986, Hynes, 1990)

//////////////////////////////- - - - - - - - - - - - -

Cell

//////////////////////////////

Cell

Substratum

ReceptorsAdsorbed proteins(ligands)

Long-range forces Short-range forces


Sequence of Cell Attachment

Biomaterial Biomaterial

Biomaterial

Cell

Approaching surface by Brownianmotion, convection orsedimentation.

Biomaterial

CellCell-surface interaction bylong and short rangeforces.

Cell

Minimum of free energy withstable adhesion.

Cells increase contact areaby „spreading as activeprocess.

1

2

3

4


Process of Cell Adhesion Time-LapseMicroscopy

Adhesion of fibroblasts in PBS + CaCl2 at pH 7.4

Adhesion of fibroblasts in PBS + CaCl2 at pH 6.0 (inflammatory model)

D. Guduru, Biomedical Materials Group


Physics of Cell Adhesion

• Cellular polysaccharides and membrane proteins negative surface charge by carboxylic and sulfate groups

• Cellular glycocalyx (lipopolysaccharides and glycoproteins) mobile hydrophilic surface layer

• Chemical composition of biomedical materials negative or positive surface charge, hydrophilic or hydrophobic

• Interaction between cells and surfaces in the absence of proteins Description by DLVO theory (XDLVO) or thermodynamic approach

hyperphysics.phy-astr.gsu.ed

eng.thesaurus.rusnano.com


Cell Surface and Interaction with Biomaterials


Properties of an Average (Hypothetical) Cell

• Shape: spherical

• Density: 1.025 kg/m³

• Radius: 5 x 10-6 m

• Volume 5.24 x 10-16 m³

• Surface charge:-1.6 x 10-2 C/m²

• Thickness of glycokalyx: 10 nm or more

Bongrand et al. Progress in Surface Science 12 (1982) 217-286.


Overview on Interaction Forces BetweenCells and the Surface

• Long-range electrostatic (Coloumb) interactions (repulsive or attractive)

• van der Waals forces (attractive).

• Short range: Hydrophobic interaction (attractive).

Steric interaction of macromolecules (repulsive).

Hydration forces (repulsive).


Coulomb Interaction


Coulomb Force (Comparison of Two Spheres)

• Coulomb force F Coulomb (d) = (2k-1s1s2/ ee0) e-kd

e0 as dielectrical constant of vacuum, e as dielectrical constant of the medium, s charge density of cell and surface, and k-1 as Debey-Hueckellength.

s1 > 0 s 2 > 0 repulsive forces s1 < 0 s 2 > 0 attractive forces

• Range of electrostatic force highly dependent on environmental condition particularly ionic strength because

• k-1 = (ere0kbT/2Nae²I)1/2 with I as ionic strength of the electrolyte, and here the unit should be mol/m3, ε0 is the permittivity of free space, εr is the dielectric constant, kB is the Boltzmann constant, T is the absolute temperature in Kelvins, NA is the Avogadro number. e is the elementary charge k-1 controls decay of electrostatic force


van der Waals Forces• The attractive force between uncharged atoms and molecules (covalent

compounds) in solid, liquid or gaseous phase van der Waal’s force

• (Short-range) forces and thus only interactions between nearest neighbors should be considered.

• Origin and nature of van der Waals forces The origin and nature of three possible sources of van der Waals interactions

• Dipole-dipole interactions (longer range)

• Dipole-induced dipole interactions (short range)

• Induced dipole-induced dipole interactions (short range)

Example Adhesion of Gecko feet due to van der Waals Forces


Dipol-Dipol Interaction (Keesom Force)

• Interaction between molecules with permanent dipole moments e.g. H2O, HF, HCl, NF3 and OF2.

• Centres of positive and negative charge are different in these molecules arrange themselves that opposite end of two molecules come nearer to each other.

• Dipole-dipole interaction or Keesom forces are weak. They are predominant in solids and weak in liquids.


Dipol-Induced Dipole (Debye) Interaction

• A dipole can induce a dipole in a non-polar molecule.

• The electric fields associated with a dipole causes slight displacements of the electrons and nuclei of surrounding molecules, which lead to induced dipoles.

• These interactions are weak in nature because the polarizability of non-polar compounds is not large. The energy of the induction force is always small.

http://ashish.org.in/ACEL/Chemical_Bonding/introduction.html


Induced Dipole-Induced Dipole Attraction or London Forces

• Existence of intermolecular forces in nonpolar materials

• Presence of a third type attractive van der Waals force called London dispersion force.


DLVO Theory and Cell Adhesion

• The interaction force F as function of distance d between cells and surface is:

• Ftotal (d) = F vdW (d) + F Coulomb (d) with

• van der Waals force F vdW (d) = - A/12pd² with A as Hamaker constant (positive in aqueous fluids) always attractive force components

• And Coulomb force F Coulomb (d) = (2k-1s1s2/ ee0) e-kd with e0 as dielectricalconstant of vacuum, e as dielectrical constant of the medium, s charge density of cell and surface, and k-1 as Debey-Hueckel length as repulsive or attractive force.

• (Negative force balance – attractive; positive – repulsive force)


Cell Adhesion and DLVO Theory

Ftotal > 0 repulsion

Ftotal < 0 attraction

Interaction of negatively charged cells with negatively charged surface at high (solid line), inter-mediate (dotted) und low (dashed) salt

concentration.

Distance d

Inte

ract

ion

fo

rce

F

Ftotal (d) = F vdW (d) + F Coulomb (d)

Biomaterial

Cell

- - - - - - - - - - - - - - - - - - - -

-- -

F Coulomb

0Distance DForce F


Example: Dependence of Cell Adhesionon Ionic Strength

Percentage of adherent red blood cells in an inverted sedimentation chamber (F = 1x g) as function of ionic

strength at pH 7.4. Trommler et al. Biophysical Journal 48 (1985).

nature.com

Simple assay to measure adhesion/detachment by gravitational force on cells byinversion of support after seeding andincubation.Adhesion of negatively charges cells on anegatively charges susbtratum like glass.


Distance and Contact Area of Cells

Trommler et al. Biophysical Journal 48 (1985).

Interference reflection microscopy of adherent red blood cells black areas show closest contact

Increase in ionic strength decrease in electrostatic repulsion dominance of van der Waals attraction decrease of separation distance between cell membrane and surface and increase of contact area


Quantitative Estimates of Contact Area and Separation Distance

Trommler et al. Biophysical Journal 48 (1985).

Contact area and separation distance of red blood cells on glass as function of ionic strength of the medium at pH 7.4.


Impact of Comonomer Charge on Bacterial Adhesion

1

2 1) and 2) Increase in content of positivelycharged comomomer(dimethyl aminoethylmethacrylate) enhancessurface coverage withbacteria.

3) + 4) increase in contentof negatively chargedcomonomer (acrylic acid) reduces surface coverage.

3 + 4

0% 10% 20% 30%


The Electrical Surface Potential ofMaterials and Adhesion of Fibroblasts

R2 = 0,6378

0

0,1

0,2

0,3

0,4

0,5

0,6

0,7

0,8

0 10 20 30 40 50 60 70

Zeta potential at pH 7.0 [mV]

Cell a

dh

esio

n a

t 24h

[O

D]

Predicted behaviour up to – 40mV, then increased adhesion through electrostatic repulsionshould increases. Reasons presence of proteins that adsorb also at negative surface charge –Specific interaction between cells & proteins (see next lectures).

Follows theoretical

predictions !

Contradictory !


Cell Adhesion in Terms of Surface Energy (I)

Neglecting specific forces as well as receptor-ligand interactions net change in free energy function per unit surface area :

DGadh = gCS - gCW – g SW

with gCS is cell-substrate interfacial energy, gCWcell-water interfacial energy, and g SW substrate water interfacial energy and DGadh as free energy or work of adhesion. Adhesion if DGadh < 0 requires gCS < gCW + g SW

Experimental data for interfacial energy of substrate-water g SW and cell-water gCW by Youngs equation g SV - g SW = g WV cos q

Propensity for adhesion higher if interfacial energy solid-water is high: Example g SW ~ 40 mN/m for polymers, gCW > 0, if gCS smaller than 40 mN/m, then adhesion but dependent on cell type

Vitte et al. 2004

However, for solid-liquid interfaces approximately g12 = g1 – g2 like for non-miscible water-CCL4 system


Experimental Proof Thermodynamic Approach for Red Blood Cells (RBC)

10 20 30 40 50 60

16

00

12

00

80

0 4

00

Substratum surface energy (mJ/m²)

Erythro

cytes/m

m²

gLV = 72.8 mJ/m² (Water)

gLV = 64.6 mJ/m² surface energy RBC

gLV = 69.5

Absolom et al. 1985

gCS < gCL + g SL

Washed red blood cells adhesion on substrata with different surface energy (C – cell, S – solid, L –liquid)

Suspension of RBC in liquids with different surface tension gLV

Increase gLV increase of gSL for substrata with low surface energy surfaces (higher water contact angle, higher solid-liquid interfacial energy cell adhesion increases

(“hydrophobic effect”)


Lowered Surface Energy as Driving Force for Bacterial Adhesion

20 40 80 120

Biomaterial water contact angle

60

40

20

Nu

mb

ero

fad

her

ent

Bac

teri

a/1

00

µm

²

Experimental estimate ofbacterial adhesionin dependence on water contactangle ofbiomaterials

Fletcher und Loeb 1979.

Hydrophobicity


Role of Bacteria Hydrophobicity High orLow Hydrophobic Polymers and Shear Stress

PE – polyethylene (hydrophobic polymer), PBS – phosphate buffered saline,

PPP – blood plasma

Low


Tissue Cells (HeLa) do not Follow Predictions on Adhesion by Thermodynamic Approach

Tamada und Ikada 1984

Lower cell adhesion with high water contact angle (hydrophilicity of tissue cells).

Decrease in cell adhesion at lower water contact angles (9, 10, 15) water uptake and chain mobility (hydration forces, steric repulsion).

Soft materials(hydrogels)


Adhesion and Spreading of Cells Reduced on Low Energy Substrata

Glass

20°

APS

60 °

ODS

80°

Hydrophobicity (WCA)

Cell adhesion, cell spreading


Spreading of Tissue Cells is Reduced on Less Polar Surfaces of Low Surface Energy


gP1W

Why do Hydrophobic Particles Tend toAggregate in Aqueous Systems?

• DGadh = gP1P2 – gP1W – g P2W (1)

• gP1P2 becomes zero because surface of particle 1 and 2 are the same interfacial energy gP1P2 = 0

• gP1W and g P2W are identical, hence (1) becomes

• DGadh = - 2 gPW

• Ideally hydrophilic particles interfacial energy gPW becomes zero DGadh = 0 no driving force for aggregation

• The more hydrophobic particles become, the larger the interfacial energy between water and particles surfaces gPW >> 0 (DGadh < 0) adhesion/aggregation of particles is promoted

ParticleP1

ParticleP2

Water

gP2W


b c

Bottom: Effect of hydrophilic coatings on hydrophobic PLGA nanoparticle endocytosisby cells. (b) low coating stability particlemore hydrophobic (c) high coating stabilitymaking more hydrophilic morehydrophobic particles tend to beendocytosed easier by non-specificmechanism as indicated by red colour in (b)Ref. Beesher et al.

Little phagocytosiswhen interfacialenergy is zero (WCA of phagocytes & bacteria are thesame)

Increasedphagocytosis wheninterfacial energybetween phagocytesand bacteria increasesWCA phagocytes < WCA bacteria

Phagocytosis/Endocytosis of Particles isalso Related to Free Energy Decrease


Steric Repulsion/Stabilisation

Mixing or compression of surface-boundchains Free energy increase by decreaseof available polymer configurations supresses adhesion/cohesion.

DGmix = 2 kT(VS²/Vl) (0.5-X1)∫p1p2 dv

Vs -the volume of the polymer segment, Vl

the volume of solvent molecule,

Xl solvent-polymer interaction,

p1 and p2 densities of chain segments ofinteracting surfaces.

Free energy increase with rise in chainvolume (Vs) and chain densities (p1, p2)

repulsive effect

Cell Surface

Intermixing of chains

Glycokalyx of cells; Surface modficationwith mobile, hydrophilicmacromoles.


Schematic Representation of Steric Repulsion


Example of Steric Repulsion

Biomaterial

Cell

Repulsive force on cells upon approach of surface due to energetically non-favoured compression of hydrophilic mobile macromolecules on the material and cell surface.Increasing

concentration of hydrophilic macromolecules (PEG) Decrease of cell adhesion (blood platelets stained with antibody).

Poly-ethylenglycol


Lowered Tissue Cell Adhesion by IncreasedDensity of Pluronics PEO-PPO-PEO Block Copolymer cell adhesion on ODS with F68

0

50

100

150

200

250

0g 0.001g 0.01g 0.1g 1g 10g

concentration of pluronics (g\L)

cells p

er

are

a

control

serum

fibronectin

Increasing density of polyethylenglycol – steric repulsion

De

creasin

gad

he

sion

of

cells

Nadia Lias

Masterthesis BMM

PEO – Polyethylene oxidePPO – Polypropylene oxide


Hydration Forces

• Repulsive forces between hydrophilic surfacesat very small distances.

• Caused by the necessary dehydration of polar groups upon approach of both surfaces (celland biomaterials)

• Empirical formula

FHyd = F0Hyd e –D/l with D as distance between

cell and surface and l as decline distance offorce about 0.2-0.3 nm. Lipid double-layer with adsorbed

water molecules (green)

Polar phosphatidylcholine headgroups bind water molecules tightly


Effect of Lipid-Coating on Cell Adhesion

Increasing density of phophorylcholinemoieties

Increasing concentration ofphosphorylcholine group (head group ofouter red blood cell membrane)

reduction of cell adhesion by increasedhydration force


Magnitude of Interaction Forces

Nature ofInteraction

Force (in Newton/m²) at distance Sign of Inter-action

100 A 75 A 50 A 20 A 10 A

Electrostatic 2.7 66.2 1.4x10³ 7.1x104 4.1x105 Repulsive/

Attractive

Electro-dynamic

2.7 x10² 6.3x10² 2.0x10² 3.3x104 2.7x105 Attractive

Stericstabilisation

2.8 x105 5.0x105 1.1x106 7.0x106 2.8x107 Repulsive

Hydration force

0 0 2.3 2.8x105 1.4x107 Repulsive

Bongrand et al.Optional forces –depending on composition of surface

Depending on salt concentration (Debye-Hueckel-Length)


Cell Adhesion and XDLVO Theory


Summary

• Adhesion of cells on materials surfaces is ruled by a number of interaction forces.

• Theoretical models can describe cell adhesion under selected conditions but approximate only reality.

• Interaction of cells with surfaces is a multi step process at which distance dependent specific interaction forces can dominate (e.g. long range versus short-range).

• Forces between cells and surfaces are dependent on environmental conditions like ionic strength (Debey-Hueckel length).

• Thermodynamic approach has only predicitive value, difficult to interpret for tissue cells, easier for bacterial adhesion.

• In reality situation more complex due to protein adsorption from surrounding media or secreted by cells.


Literature

• Bongrand et al. Physics of cell adhesion, Progress in surface science Vol. 12 (1982) 217-286.

• Trommler et al. Red blood cells experience electrostatic repulsion but make molecular adhesions with glass Vol 48 (1985) 835-841.

• Absolom et al. Erythrocyte adhesion to polymer surfaces. Journal ofColloid and Interface Science Vol. 104 (1985) 51-59.

• Curtis and Lackie, Measuring Cell Adhesion. Wiley 1991.

• Dubiel et al. Bridging the gap between physicochemistry andinterpretation prevalent in cell-surface interactions. Chemical Reviews 2011, 11, 2900-2936

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