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Lecture 3:Physics of Cell Adhesion
Prof. Dr. Thomas Groth
Biomedical Materials
Martin Luther University Halle-Wittenberg
Martin Luther University Halle-Wittenberg
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
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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
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Adhesion
Adhesion-Dependent Cells on Surfaces
Loss ofadhesion
Migration Proliferation
Differentiation
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Scratch Wound Healing Assay ShowingMigration of Cells
Provided by courtesy of Jose Ballester-Beltran
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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
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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
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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)
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Cell
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Cell
Substratum
ReceptorsAdsorbed proteins(ligands)
Long-range forces Short-range forces
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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
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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
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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
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Cell Surface and Interaction with Biomaterials
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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.
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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).
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Coulomb Interaction
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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
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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
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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.
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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
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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.
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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)
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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
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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.
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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
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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.
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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%
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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 !
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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
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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”)
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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
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Role of Bacteria Hydrophobicity High orLow Hydrophobic Polymers and Shear Stress
PE – polyethylene (hydrophobic polymer), PBS – phosphate buffered saline,
PPP – blood plasma
Low
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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)
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Adhesion and Spreading of Cells Reduced on Low Energy Substrata
Glass
20°
APS
60 °
ODS
80°
Hydrophobicity (WCA)
Cell adhesion, cell spreading
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Spreading of Tissue Cells is Reduced on Less Polar Surfaces of Low Surface Energy
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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
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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
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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.
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Schematic Representation of Steric Repulsion
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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
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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
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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
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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
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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)
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Cell Adhesion and XDLVO Theory
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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.
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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