SYNTHESIS AND IN VITRO/IN VIVO RESPONSE TO PEPTIDEPOLYMER CONJUGATES - Biologically-Responsive Hybrid Biomaterials - page 113

Direct contact is the most widely applied test in cell-material in vitro
analysis, and it involves placing the material directly on top of a confluent layer
of cells or directly placing cells on top of the material. After incubation, cells are
microscopically evaluated for toxicity (through viability staining), adherent
density and morphology. Direct cell-material contact studies are the most
prevalent in testing material biocompatibility and can reveal the intermediate
zone of damaged cells. Especially when testing for cell response to peptide
conjugated materials, the assessment of the cells in direct interaction with the
peptide presented on the material surface is imperative. Cell adhesion and
spreading as modulated by various peptides are central to the study of
biologically responsive peptide conjugated materials. Since its discovery by
Pierschbacher and Ruoslahti in 1984, the arginine-glycine-aspartic acid (RGD)
sequence has been the most effective peptide sequence for enhanced integrin-
mediated cell adhesion and has been applied to a myriad of materials and
assessed for enhanced cell adhesion and wound repair [4]. The tripeptide
sequence has been shown to enhance osteoblast adhesion in vivo and a myriad of
cells in vitro , including osteoblasts, chondrocytes, human umbilical vein
endothelial cells, fibroblasts and monocytes [5-13]. Table 1 shows increased
monocyte adherent cell density on RGD-PEG grafted on a semi-interpenetrating
network (sIPN) of gelatin and PEGdiacrylate (PEGdA) compared to that on
sIPNs grafted with control ligands triglycine (GGG) or methoxy [13].

Table 1. Adherent monocyte density on RGD-PEG grafted sIPN and control ligand-PEG grafted
sIPN (adapted from [13]).

Adherent Monocyte Density (cells/mm 2 )
Culture Time:

Ligand-PEG grafted
onto gelatin of sIPN 2 h 24 h 96 h 168 h

GGG 328 ± 69 444 ± 16 207 ± 52 74 ± 40

Methoxy 145 ± 18 233 ± 14 106 ± 2 95 ± 12

1321 ± 74 * 1071 ± 190 * 350 ± 42 * 157 ± 8 *

RGD

* Significantly higher than all other ligand-PEG grafted sIPN (p<0.001)

Table 2. Synthetic peptides derived from ECM components and biological activities of peptide-
chitosan membranes (adapted from [14]).

Fibroblast Neurite

Peptide Sequence Protein Source (Residues) Attachment Outgrowth Ref

531 GEFYFDLRLKGDKY Human collagen alpha1, type IV ++ 25,26

−

AG73 RKRLQVQLSIRT Murine laminin alpha1 chain ++ + 23