Question

Receptor binding motifs such as RGD have highly selective recognition for cell adhesion. Outline a strategy for incorporating a cell adhesion. Outline a strategy for incorporating a cell adhesion recognition domain to a biomaterial. Address the problems surrounding domain quantity, type and affinity in your discussion.

Answer 1

RGD is a tri amino acid peptide made up of: Arginine-Glycine-Aspartate. It is most commonly used adhesive peptide which is much effective in attaching various cell tyoes to plethora of biomaterials. Extracellular matrix proteins such as integrins, osteopontin, fibrinogen, fibronectin, bone sialoproteina nd vibronectin alongwith collagens and laminins contains a prime binding domain that is RGD. Thus the artificial peptide, RGD can bind to many integrin species. Due to this recognition benefit, the artificial RGD maintains its functionality during the sterilization and processing steos of biomaterial synthesis. Again this property nominalises the immne reactivity and transfer of pathogens during xenograft etc.

Conformation of artificial RGD can modulate affinity.

In vitro loading of cells on the surface of material too determines the efficiency of RGD.

In the in vivo models, the moleculars events transpires its efficacy during its usage as a scaffold.

Now a critical factor that determines the activity of artificial RGD is that it cannot function properly in isolation. It is seen that cells secrete variou sintegrin binding proteins which has a better efficiency as stimulating the integrin signaling than the isolated RGD. And thus most of the biomaterials will adsorb these proteins than RGD. Thus in most of the in vitro studies serum free media is used.

also since it is a recognition sequence of integrin and an antagonist too, the soluble RGD can inhibit cell adhesion, thus it is imperative to immobilize RGD on the surface.

Thus to minimize the variation caused by the native proteins, the biomaterials are loaded with RGD alongwith nonfouling polymers for example polyethylene glycol.

Addition of extra amino acids to RGD based on the natural sequences can add to the biological activity of RGD and it can then help in the new tissue formation and induction of cellular responses. This peptide RGD links to the surface of biomaterial through the covalent bond formation between the amino terminus of the peptide and the carboxyl group of the surface material.

Several cell adhesion material for example, poly-L-lysine, mussels adhesive protein (MAP) and extracellular matrix proteins are used to make cell adhesion biomaterials. Here RGD is associated with either of these to increase its capacity for cell adhesion as MAPs are rich in Dihydroxyphenylalanine and lysine. These can thus help attaching to wet surfaces too. It can bind strongly to glass, metals and plastics too.

Also introduction of thiol groups to the peptide can help in orientation and increased stability of the peptide.

The spacing and the density of this peptide sequence on the biomaterial surface can be controlled through the patterning strategy at the micro and nano scale range, which also brings precision of ligand binding. It also controls the cellular responses and cell behaviour. Size of integrin receptors have been shown to be between 9-12 nm hence surface patterning at nano sacle is important ascpect.

Hence the above methods acts as a strategy for incorporation of cell adhesion recognition domain to a biomaterial considering all the factors affecting its quantity, type and affinity to the surface for attachment and proliferation of cells after adhesion not as a monolayer but as a functioning tissue.