**6. Heparan Sulfate and Heparin**

Heparan sulfate's dominant repeating disaccharide unit is composed of glucuronic acid linked to N-acetylglucosamine [1]. Heparan sulfate is considered the most complex GAG and medical uses of this GAG are currently few and far between. Heparin, on the other hand has seen a myriad of medical applications. It is composed primarily of iduronic acid-N-sulfoglucosamine disaccharide units and is heavily sulfated. Many naturally occurring GAGs display a hybrid structure that blurs the line between HS and heparin. It has been proposed that the name heparin be only applied to GAGs containing more N-sulfate groups than N-acetyl groups. This falls in line with the generally accepted distinction that heparin is more highly sulfated than HS [106].

Pan, et al. have combined CS-chitosan scaffolds with heparin-gelatin microspheres to utilize the growth factor sequestering properties of heparin. These gels were formed from oxidized CS and carboxymethyl chitosan using the schiff's base reaction similar to the gels mentioned previously. Doping with these microspheres accelerated gelation, slowed weight loss, increased water uptake, and increased the compressive modulus over controls. Adipose-derived stem cells showed good viability as they did with the CS-chitosan gels, but had the added benefit of controlled release of incorporated growth factors such as insulin-like growth factor 1, while gels without heparin-gelatin microbeads exhibited burst release. These gels also showed the same injectability of CS-chitosan only gels for non-invasive tissue engineering therapies [107].

Tissue engineering research using heparan sulfate and, in some cases heparan sulfate mimetics [108], has recently increased as it became clear that HS can be administered to injury sites to support bone healing [109] and angiogenesis [110] and might, therefore, confer benefits to tissue engineering constructs. Lee, et al. recently investigated the binding affinity of a myriad of growth factors including TGF-β1, BMP-2, FGF-2, PDGF-BB, and VEGF165 and found it binds them all but with varying affinities, that may depend on the sulfation pattern and composition of the HS used. Further, in a mouse model of osteochondral defect, HA gels loaded with HS and no growth factors or stem cells showed recovery to normal or near normal as measured using the International Cartilage Regeneration and Joint Preservation Society cartilage injury evaluation scoring system. Gels containing HS were also the only to support regeneration of bone and cartilage, while HA only gels did not support bone regeneration [111]. Sefkow-Werner, et al. also noted that HS as part of a gel construct including bone morphogenic protein 2 and cyclic RGD worked synergistically with the growth factor and cell adhesion molecule in eliciting osteogenic differentiation and promoting enhanced and sustained signaling [112]. Possibly more importantly, they developed a streptavidin-based system that allows for tunable amounts of each ligand to be immobilized in a gel to investigate how their relative densities affect cell behavior with the potential to further the use of GAGs to improve tissue engineering constructs. While work with HS and heparin in tissue engineering is nascent with the exception of controlled release of growth factors, as more is learned about these important extracellular matrix components we anticipate that, as with other GAGs, their uses will increase.
