Cell Adhesion to Polymer Surfaces

1
Cell Adhesion to Polymer Surfaces

• George Tulevski
• Colloids and Surface phenomena

2
Introduction

• Cell adhesion to polymer surfaces has obvious
  implications in the field of tissue engineering
• Facilitating cellular adhesion, growth and
  differentiation onto a surface can aid in wound
  healing and tissue growth
• a polymer can provide mechanical stability for
  the newly forming tissue
• understanding adhesion mechanisms can prevent
  bacterial cell adhesion and aid in infection
  control

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Composition of biomaterial surfaces1

• Substrate - polymer surface
• Conditioning layer - adsorbed macromolecues (i.e.
  polysaccharides, proteins
• Cells - adhesion of cells to conditioned
  biosurface
• Needs to be a clear understanding of composition
  of the three components and how they interact

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Surface Characterization3

• Use contact angle measurements to obtain surface
  energy of substrate
• surface energy of substrate, along with that of
  the protein, governs the protein adsorption,
  including which proteins adsorb and to what
  degree

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Surface spectroscopy General Method4

• The solid sample is irradiated with a primary
  beam of electrons, photons, ions or molecules
• The impact of this beam, then generates a
  secondary beam which is sent to the analyzer

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Surface spectroscopy Examples4
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XPS4

• X-ray photoelectron spectroscopy
• a photon of a monochromatic x-ray beam displaces
  an electron from an orbital of the atom
• Eb hv EK w
• The ejected electrons kinetic energy, Ek,, is
  measured and can be used to determine Eb,, which
  is characteristic of the atom.
• 
  
  
  
• Elemental composition, functionality, oxidation
  states and structure can be elucidated

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SIMS and SEM4

• SIMS - secondary ion mass spectrometry
• the surface is bombarded with beams of 5 to 20
  keV ions, such as Ar, Cs, N2 or O2 by an ion
  gun.
• The surface is stripped of its molecules and the
  resultant charged molecules are sent to the mass
  analyzer (i.e. quadropole, flight tube)
• SEM - scanning electron microscopy
• surface is scanned with a beam of energetic
  electrons
• the secondary beam is created and consists of
  backscattered electrons and secondary electrons
• the secondary beam is then sent to the analyzer
  and morphological and topographical information
  is obtained

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Formation of the conditioning film2

• The interaction between proteins on the surface
  and the cells has an enormous impact on cell
  adhesion
• Adhesion of animal cells is mediated by adhesion
  receptor proteins in the cells membrane (the
  integrin family is primarily responsible for
  cell/foreign substance adhesion)
• Adhesion proteins bind to ligand proteins
  adsorbed to the surface (i.e. fibrinogen, von
  Willebrand, vitronectin and fibronectin)
• Adsorbing these proteins to a surface and
  maintaining their bio-activity can facilitate
  cell growth

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Protein adsorption3

• Proteins are surfactants- they have hydrophobic
  and hydrophilic portions and adsorb to virtually
  any surface
• the degree of adsorption is governed by the
  interfacial free energy between the protein and
  substrate
• ?GIF1w2 ?GLW1w2 ?GAB1w2
• A favorable interaction corresponds to a negative
  value of ?GIF1w2
• this value can be obtained from tensiometric
  measurements such as contact angle or wicking

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Vroman Effect

• Protein adsorption is essentially irreversible if
  no other proteins are present
• Proteins can replace themselves at the surface
• Different proteins have different affinities for
  a surface, proteins with a stronger affinity can
  displace proteins with a weaker affinity at the
  surface
• When proteins are allowed to adsorb to steady
  state, and isotherm is formed in which (1) an
  adsorption plateau occurs at high bulk
  concentrations (2) at lower concentrations,
  adsorption is linear until it reaches a plateau

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Protein adsorption (van Oss 1991)14

• Maximum cell (absence of protein) and protein
  adhesion occurs at surface tension of 33 mJ/M2
• Despite this, minimal cell adhesion occurs at
  this surface tension
• since proteins adhere first, the hydrated surface
  of the adsorbed protein is hydrophilic in nature
  and results in minimal cell adhesion
• correlates well with previous data showing that
  cells adhere least to polymer surfaces of this
  energy in vivo (Baier 1984)10

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Protein adsorption and cell adhesion (Busscher
1989)6

• This work displays that cell adhesion increases
  with increasing surface energy
• cell adhesion moves from poor to good at around
  ?s 55 mJ/m2
• higher surface energy facilitates cell growth
  (fibroblasts, smooth muscle cells, epithelial
  cells
• bulk measurements can elucidate macroscopic
  interactions, but there needs to be a correlation
  between surface chemistry and cell adhesion to
  develop control over the system. Conformational
  changes occur regularly upon adsorption of a
  protein and can alter bio-activity. This also
  effects cell adhesion and cannot be explained by
  energetics alone.

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Microscopic correlation (van Oss 1995)14

• The adsorption of H.S.A. onto glass is
  energetically disfavored
• despite this, the elbow can overcome the
  macroscopic repulsion and obtain a local point of
  attachment via a divalent metal cation (i.e.
  Ca2) electron acceptor interacting with an
  electron donor in the protein with little
  conformational changes
• Determined by adding EDTA and then observing
  detachment of the protein

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Minimal Peptide Sequences

• One can circumvent problems with conformational
  changes in the protein by using minimal peptide
  sequences.
• The most influential sequence in many ligand
  proteins is the RGDS (arg-gly-asp-ser) sequence
• this sequence can be covalently linked to the
  surface and be used to bind to the adhesion
  receptor protein in the cells membrane and
  encourage adhesion
• Example Brandley and Schnaar 1988
• used (tyr-ala-val-gly-arg-gly-asp-ser) sequence
  and covalently immobilized it to a polyacrylamide
  gel surface
• the surface was coated with a density of 2 nmol
  peptide/cm2 and supported long-term fibroblast
  growth
• tertiary structure must have some importance and
  a role in mediating cell adhesion.

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Specific case (Busscher 1992)12

• The author used e-PTFE vascular grafts that were
  luminally modified and implanted into rabbit
  carotid artery
• the interior was modified by ion etching and was
  followed by oxygen glow-discharge to render the
  surface highly hydrophobic
• the water contact angle went from 109 to 140-150
  degrees for the modified surface
• the graft was implanted and then removed after
  one week and inspected with SEM
• the lumen had some platelet adhesion, but did not
  experience a cascade of platelet adhesion which
  would have led to clotting
• the outside, anastomic side, had some endothelial
  outgrowth, as desired
• the biomaterial succeeded on both fronts in
  providing a pathway for blood flow and tissue
  growth around the outside.

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SEM images12
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Conclusions

• Four aspects to address
• Biomaterial substrate must be well characterized
  including its chemical composition, morphology
  and structure.
• .A firm understanding of macromolecular film
  conditioning structure, conformation and
  interaction with the substrate.
• Cell processes such as retention, adhesion,
  differentiation and growth should be
  quantitatively measured.
• Short and long term effects should be evaluated
• Future trend ability to control formation of
  conditioning layer with selectivity and
  conformational control of adsorbed protein.
  Correlations between surface chemistry and
  protein conformation and how to modify the
  surface chemistry to obtain desired results