SYNTHESIS AND IN VITRO/IN VIVO RESPONSE TO PEPTIDEPOLYMER CONJUGATES - Biologically-Responsive Hybrid Biomaterials - page 122

demonstrated by Glass and others [32]. Hyaluronic acid, a non-immunogenic
polysaccharide found in many human tissues, is upregulated during repair
and naturally cleared by the host. Though hyaluronic acid modifications with
synthetic polymers have been commonly reported, studies on peptide conjugation
on the polysaccharide are to date elusive. Hyaluronic acid crosslinked with a
diepoxide is reacted with periodate to create reactive aldehydes by peroxidate
oxidation of the polyhydroxyls of glucuronic acid (Figure 8A). A RGD
containing 18 amino acid peptide sequence, Gly-Arg-Arg-Arg-Arg-Arg-Gly-
Gly-Gly-Arg-Gly-Asp-Ser-Pro-Ala-Ser-Ser-Lys (GR 5 G 3 RGDSPASSK, or WH18),
was then conjugated to hyaluronic acid through sodium cyanoborohydride
reduction. Primary amines of the peptide sequence serve as nucleophiles to form
secondary amine bonds, which are reduced by sodium cyanoborohyride, creating
a more stable primary amine, and covalently linking the peptide to the carbonyl
chains on hyaluronic acid (Figure 8B). The WH18 peptide sequence, while
designed to present the RGD sequence recognized by the integrin receptors, was
also designed with the D-isomer or arginine to protect it against a number of
serine proteases known to be present in wounds.

(A)

(B) (C)

Fig. 9. Photomicrograph of 8-day-old burn wound (A) no treatment, air-exposed control, (B)
treated with hyaluronic acid, and (C) treated with RGD-hyaluronic acid matrix. Note the very thin
layer of loose edematous connective tissue subepidermally in the air-exposed controls, the wider
subepidermal zone with some cellular infiltrate in the hyaluronic acid treated wound, and the
majority of the dermis appearing as granulation tissue with marked cellular infiltrate in the RGD-
hyaluronic matrix treated wound. (Hematoxylin and eosin, original magnification x100) [32].

In vitro , the conjugation of the RGD containing WH18 peptide sequence
onto hyaluronic acid resulted in a 90% or greater attachment and subsequently
supported spreading of human osteosarcoma cells (MG63). On surfaces that were
treated with periodate in the absence of peptide, or that were not treated with
periodate resulted in very little attachment of the osteosarcoma cells and no cell
spreading on the material. They also described enhanced attachment of human
colon fibroblasts, primary bovine epithelial cells and human gingival fibroblasts.