**6. Hyaluronan**

trans with an average molecular weight of 1000 to 2 million g/mol are commercially available for research purposes [59]. Two preparations of dextran with lower fractions (40000 and 70000 g/mol) are suitable for nontoxic clinical use [60]. However, high fractions of dextrans can produce erythrocyte aggregation, impaired microcirculation, and a clinical

**Figure 3.** Eleven-day Masson's trichrome (MT) staining sections of a fibrin scaffold. (A) Untreated defects and (B) de‐ fects containing empty scaffolds were filled with new bone tissue. However, no reparative bone was observed in the center of defects containing (C) scaffolds filled with fibrin (low T) and (D) scaffolds filled with fibrin (high T). (Inset) Patches of multinucleated giant cells (striped arrow) were observed at the scaffold interface in all scaffold-containing groups. Black arrows point to areas occupied by the scaffold, whereas white arrows point to the advancing bone

During 1990-1994, extensive toxicologic evaluations indicate that small-volume infusions of 7.5% NaCl/6% dextran 70 (HSD) at the proposed therapeutic dose of 4 mL/kg, present little risk as implantable biomaterials [61,62]. Dextran hydrogels have offered good opportunities as protein delivery systems or tissue engineering scaffolds because of an inherent biocom‐ patibility [63]. The hydrophilic, soft and rubbery properties of the dextran hydrogels ensure minimal tissue irritation and a low tendency of cells and proteins to adhere to the hydrogel surface [59]. Althogh dextran itself is not toxic, some of the methods used for crosslinking the polymer may result in toxic byproducts. For example, the toxicity of dialdehyde cross‐ linked dextran/gelatin hydrogel can be detected in fibroblast and endothelial cell cultures. Subcutaneous implantation studies in mice showed that the foreign body reaction seen around the implanted hydrogel samples was moderate and became minimal upon increas‐

picture akin to shock and certain other diseases.

118 Advances in Biomaterials Science and Biomedical Applications

front. Field width 5.2 mm, inset field width 0.2 mm [46].

Hyaluronan (also called hyaluronic acid or hyaluronate, HA) is a natural anionic, viscoelas‐ tic and hygroscopic glycosaminoglycan, discovered in 1934, by Karl Meyer and his assistant, John Palmer in the vitreous of bovine eyes [71]. As one of the chief components of the ECM, hyaluronan distributes widely throughout connective, epithelial, and neural tissues. It is unique among glycosaminoglycans in that it is nonsulfated, forms in the plasma membrane instead of the Golgi, and can be very large in molecular weight (often reaching the millions) [72]. HA plays several important organizational roles in the ECM by binding with cells and other protein components through specific and nonspecific interactions [73] and is responsi‐ ble for various functions within the ECM such as cell growth, proliferation, differentiation, migration [74], and even some malignant tumors [76].

Basically hyaluronan is a highly non-toxic, non-antigenic and non-immunogenic polysac‐ charide, owing to its high structural homology across species, and poor interaction with blood components [77,78]. The FDA in American has approved the use of hyaluronic acid for certain eye surgeries, such as cataract removal, corneal transplantation, and de‐ tached retina [79]. People take hyaluronic acid for various joint disorders (lubricant agents), lip fillers, "youth fountains", and even wound healing catalysts [80]. Nowadays various hyaluronan hydrogels have been used to delivery drugs and cell growth factors [81,82]. There are some evidence show that fragmented hyaluronan stimulates the ex‐ pression of inflammatory genes by a variety of immune cells at the injury site. With the protein-bonding abilities, hyaluronan fragments signal through both Toll-like receptor (TLR) 4 and TLR2 as well as CD44 to stimulate inflammatory genes in inflammatory cells. Hyaluronan presents on the epithelial cell surface can provide protection against tissue damage from the environment by interacting with TLR2 and TLR4 [83-85]. It is well known that accumulation and turnover of ECM components are the hallmarks of tissue injury. Current model of hyaluronic acid appear in the early stages of wound healing is to physically make room for white blood cells, which mediate the immune re‐ sponse and at least in part, reduce collagen deposition and therefore lead to reduced scarring [86]. This hypothesis is in agreement with the research of West and coworkers, who have showed that in adult and late gestation fetal wound healing process, removal of HA results in fibrotic scarring [87].

HA can be modified through several different ways, such as chemically esterify its car‐ boxylic groups with some types of alcohol. The physico-chemical properties of the new biopolymers allow the preparation of many biomaterials with different biocompatibilities for various medical applications [88]. Shen and coworkers implanted hyaluronan hydro‐ gel and periodate oxidated hyaluronan hydrogel in ischemic myocardium and found rapid degradation rates, low quantity of inflammation-mediating cells, thin fibrous cap‐ sules with dense blood vessels around the hydrogels at week 2 [89]. Praveen and cow‐ orkers used HA/polyvinyl alcohol (PVA) coating membrane to minimize the problems related to protein deposition and fibrous tissue formation on an implanted glucose sen‐ sor [90]. HA hydrogels modified with laminin could support cell infiltration, angiogene‐ sis, and simultaneously inhibit the formation of glial scar after being implanted into the lesion of the cortex [91]. Compared with pure gelatin hydrogen, HA/gelatin composite has a better compatibility and contiguity with the surrounding brain tissue with no in‐ flammatory reaction and fibrous encapsulation [92]. Intravitreal implants of hyaluronic acid esters represent useful biocompatible and biodegradable properties for a potential drug delivery system in the treatment of posterior segment ocular diseases [93]. A crosslinked HA hydrogel that contained a covalently bound derivative of the anti-prolifera‐ tive drug MMC was synthesized and evaluated *in vitro* and *in vivo*. This hydrogel has strong potential as anti-fibrotic barriers for the prevention of post-surgical adhesions [94]. Two injectable thiolated HA derivatives were coupled to four alpha, beta-unsaturat‐ ed ester and amide derivatives of poly(ethylene glycol) (PEG) 3400 and were found that the encapsulated cells can retained their original fibroblast phenotype and secreted ECM in vivo [95]. A fibrin/HA composite gel with autologous chondrocytes has been synthe‐ sized for tracheal reconstruction. Histologically, the grafts showed no signs of inflamma‐ tory reaction and were covered with ciliated epithelium [96].

#### **7. Heparin**

Heparin (from Ancient Greek ηπαρ (hepar), liver), a highly sulfated glycosaminoglycan, is widely used as an injectable anticoagulant, and has the highest negative charge densi‐ ty of any known biological molecule [97]. Heparins are involved in different pathways of the coagulation cascade with anticoagulant, antithrombotic, profibrinolytic, anti-aggre‐ gative, as well as anti-inflammatory effects [98]. As stated in the fibrin section, the pri‐ mary anticoagulant effect of heparin is through the suppression of thrombin-dependent amplification of the coagulation cascade, and inhibition of thrombin-mediated conversion of fibrinogen to fibrin [99].

Heparin holds the ability to relieve pain, inhibit clotting and inflammation, restore blood flow, enhance healing, and can be a useful addition to a range of available treatments for burn wounds [100]. Unfractioned heparin exhibits a broad spectrum of immunomodulating and anti-inflammatory properties, by inhibiting the recruitment of neutrophils and reducing pro-inflammatory cytokines in the treatment of inflammatory bowel disease [101]. Low-mo‐ lecular-weight heparin can reduce or prevent development of signs/symptoms associated with post-thrombotic syndrome [102]. Heparin has been widely used to form an inner anti‐ coagulant surface on various experimental and medical devices such as membranes [103,104], tubes and renal dialysis machines [105,106].

Although heparin is used principally in medicine for anticoagulation, its true physiological role in the body remains unclear. Blood anti-coagulation is usually achieved by heparan sul‐ fate proteoglycans which derive from endothelial cells stored within the secretory granules of mast cells and only released into the vasculature at sites of tissue injury [107]. Rather than anticoagulation, the main role of heparin may be defense at such sites against invading bac‐ teria and other foreign materials [108]. A thiol-modified heparin in the Extracel-HP® mimics heparan sulfate proteoglycans also normally presents in the ECM and regulates the *in vivo* growth factor release for a functional microvessel network development [109]. A wellknown adverse effect of heparin therapy is thrombocytopenia, a serious, immune system– mediated complication with significant mortality (Figure 4) [110-112].
