| Glycosaminoglycans (GAGS), including hyaluronic acid (HA), heparin, heparan sulfate (HS), chondroitin sulfate (CS), dermatan sulfate (DS), and keratan sulfate (KS) are natural polysaccharides widely distributed in the extracellular matrix (ECM), cell surface, and the basement membrane. GAGs bind numerous proteins, which integrate them into many essential biological pathways. Therefore, studying GAGs and their binding proteins is important for the application of GAGs in biotechnology, pharmaceutical, and medical fields. This dissertation mainly discusses HA and heparin, with some biochemical studies with their binding proteins and potential applications to develop wound-healing biomaterials.; An HA binding protein, RHAMM (receptor of HA-mediated motility), was engineered to generate a recombinant protein (HB3) with excellent heparin affinity and specificity. Therapeutically relevant heparin (both unfractionated and low molecular weight) can be measured using HB3 protein in a competition assay modified from an enzyme-linked immunosorbent assay (ELISA). This heparin assay has advantages in high consistency and low cost over current methods of activated partial thromboplastin time (APTT) and anti-Xa assays.; Another important HA binding protein, SPACRCAN (sialoproteoglycan associated with cones and rods) in retinal areas, was found to bind with heparin and CS. The binding domain was identified to be RHAMM-like BX7B motifs (where B is a basic amino acid residue and X is any nonacidic amino acid residue). Cross-reactivity of SPACRCAN with other GAGs suggests the involvement of SPACRACN in ECM networks to help maintain the structure of interphotoreceptor matrix (IPM).; Many growth factors (such as basic fibroblast growth factor, bFGF) bind heparin to execute their bioactivities. Thus, thiol-modified heparin was covalently crosslinked into GAG hydrogels (composed of thiol-modified HA or CS crosslinked by poly (ethylene glycol) diacrylate (PEGDA)) to control bFGF release. Release of bFGF could be extended to 4 weeks with only 1% heparin (w/w to all GAGs) included, and bioactivity of bFGF was well maintained, as demonstrated by promoted neovascularization in Balb/c mice and improved wound healing in diabetic mice. Therefore, GAG-based biomaterials can be potentially developed into synthetic ECM (sECM) with additional necessary factors (such as laminin, which is also discussed in this dissertation) included to better mimic natural ECM for applications in tissue engineering. |
