Protein-based Biointerfaces to Control Stem Cell Differentiation - Biointerfaces: Where Material Meets Biology - page 16

oxygen in the environment 98 and create an elevated negative potential on the
surface. This effect generates a strong double layer in solution and attracts
charged protein molecules to the surface. The radicals in the interface also
allow the covalent binding of biomolecules without the need for any add-
itional linkers. 38 It is believed that the reactive radicals act as anchors to
immobilize biomolecules through the substitution of mobile hydrogen
atoms bonded to carbon, nitrogen or oxygen atoms in amino acids on the
outer surface of the protein molecule. At the same time, the radicals and
surface oxygen groups interact with water molecules due to strong hydrogen
bonds providing a hydrophilic surface. It is known that an adsorbed water
layer prevents direct physical contact of the protein molecule with the sur-
face creating a water shell around the protein and maintaining the natural
conformation of the protein molecule despite the covalent bonding with the
surface. 99 This approach has been proven effective for both polymers and
metals and immobilization of different types of proteins, peptides and yeast
has been demonstrated.
The pros and cons of the linker-free approach are:

d n 3 r 4 n g | 1

Pros
J Single step method
J Applicable to the broad range of biomolecules
J Applicable for all types of polymers
J Significantly increases the amount of immobilized protein
Cons
J Not applicable for 3D structures
J Activity of the surface decreases with time
J Requires a specialized facility

.

1.2.2 Determination of Successful Functionalization of
Proteins, Growth Factors or Peptides
1.2.2.1 Determination of Successful Functionalization of
Proteins, Growth Factors or Peptides in Three
Dimensions
The determination of successful covalent functionalization of growth factors
or peptides in a 3D microenvironment (synthetic or naturally derived scaf-
folds) is not easy. As explained above, growth factors and small peptides can
elicit specific cellular responses in encapsulated cells to recapitulate a par-
ticular tissue, and promote their proliferation or differentiation. Addition-
ally, growth factors and peptides show short half-lives when injected in vivo,
and their functionalization within a 3D matrix extends their functionality
and protects them from degradation enzymes in the host environment. 100,101
In practice, we have found that the assessment of covalent functionalization
of growth factors or peptides in naturally derived scaffolds made of type I
collagen, gelatin, alginate, hyaluronic acid, or silk fibroin cannot be done