## 3 Chemical Modification of Hyaluronan
### Oxidation of Hyaluronan  
The introduction of aldehydes can be used for functionalization via reductive amantion or chemical crosslinking. The regioselective oxidation of HA at the primary alcohol located at position C6 of N-acetyl glucosamine is obtained by using 4-Ac-TEMPO or Dess-Martin periodinane (DMP) as depicted in Figure 12. The oxidation is performed in water mediated by 4-acetamido-TEMPO, NaBr, and the oxidant NaOCl and is carried out at 4 \({}^{\circ}\)C to slow down the degradation of the skeletal. This reaction is performed in DMSO mediated by DMP for a higher oxidation degree. DMP yielded a high substitution degree (up to 48%) [122]. However, the use of organic solvent increases the price of this type of modification. [PERSON] et al. reported that the oxidation of HA proceeds with a quantitative yield to the preparation of carboxylated HA (HA-COOH). The reaction was mediated via oxoammonium salt TEMPO\({}^{+}\)Cl\({}^{-}\) obtained by oxidation of (2,2,6,6-tetramethylpiperidin-1-yl) oxyl, commonly referred to as TEMPO. TEMPO\({}^{+}\)Cl\({}^{-}\) is prepared by reacting TEMPO radical with chlorine in carbon tetrachloride. The oxidation of 1.5 \(\times\) 10\({}^{6}\) g/mol and 4-6 \(\times\) 10\({}^{4}\) was compared. The degree of oxidation values (60 and 72%) depended on the molar equivalents of the oxidant [123]. The starting molecular weight of HA (7.3 \(\times\) 10\({}^{4}\) g/mol) decreased to 3.4 \(\times\) 10\({}^{4}\) at 4 \({}^{\circ}\)C, but the degradation was more pronounced at 25 \({}^{\circ}\)C (2.7 \(\times\) 10\({}^{4}\) g/mol). The use of a tamponed buffer (such as phosphate buffer) also diminished the degradation of HA during oxidation. The authors reported that the isolation of the HA-COH in the degree of substitution was up to 15%. However, the reaction is extremely degradative of the HA-skeletal, thus, the Mw decreased from 5.4 \(\times\) 10\({}^{5}\) to 1.3 \(\times\) 10\({}^{4}\) g/mol. Depending on the conditions for oxidation, the reaction could give a mixture of HA-COH and HA-COOH. The oxidation of HA is highly degradative, but the Mw of HA is usually not characterized. By controlling the temperature, time, and pH of the reaction, the degradation of HA skeletal can be diminished, allowing control of the Mw and degree of substitution. Furthermore, the isolation of intermediate HA-aldehyde can be easily reached. Unfortunately, the oxidation of HA not only reduces the Mw, but it also affects the interaction of the polysaccharide with CD44 receptors, so that it might interrupt the transduction signal [124]. The oxidation of HA can be mediated by sodium meteoriodate (NaIO\({}_{4}\)). In this case, vicinal hydroxyl groups presented at the second and third carbon atoms (C-2 and C-3) of glucuronic acid are oxidized. The reaction breaks C-C bonds and leads to the formation of two aldehydes at the monomeric sugar units (HA-di-CHO) [125]. This type of modification of HA can be used for self-crosslinking by reaction with diamino linkersor functionalization by reaction with primary amines. [PERSON] et al. tested the pro-healing effects of tetramethylpyrazine loaded in (HA-di-CHO) of different molecular weights (Mw = 1.0 \(\times\) 10\({}^{4}\)-1 \(\times\) 10\({}^{5}\) g/mol, 8.0 \(\times\) 10\({}^{5}\)-1.0 \(\times\) 10\({}^{6}\), and 1.8 \(\times\) 10\({}^{6}\)-2.2 \(\times\) 10\({}^{6}\) g/mol) and self-crosslinked by ADH [126]. Natural or synthetic polymers containing amino functional groups can be used to form Schiff base bonds with HA. These types of reactions are useful for chemical crosslinking of the polysaccharide. The prepared hydrogels can be used as reservoirs of active pharmaceutical ingredients. Chitosan [71] or gelatin [128] have been used due to their adequate biocompatibility. In this idea, [PERSON] et al. prepared a hydrogel formed by Schiff base linkage between oxidized HA (HA-di-CHO) and carboxymethyl chitosan. A dressing was prepared by adding active polypeptides extracted from _Periplaneta American_ for diabetic wound healing.