## 3 Chemical Modification of Hyaluronan
### Amidation of Hyaluronic Acid
Hydroxyethyl cellulose (HEC) was oxidized by NaIO4; thus, the oxidation degree increased from 30.3 to 68.5% by increasing the oxidation agent. The authors crosslinked the HA-ADH derivative (DS = 30%) with oxidized hydroxyethyl cellulose without using any catalyst. The swelling degree, porosity, and hemolysis rate were controlled by the degree of oxidation of HEC. The swelling degree decreased from 2888% to 2040% as the degree of oxidation increased from 30.3% to 68.5%. The hemolysis was higher (4.0%) for the hydrogel with a lower oxidation degree [64]. Unfortunately, the gels were slightly cytotoxic. [PERSON] et al. reported the synthesis of injectable HA hydrogels with redox and pH dual-responsive properties for protein and cell delivery. Three different HAs (Mw = 1.0 \(\times\) 10\({}^{5}\), 3.0 \(\times\) 10\({}^{5}\), and 1.0 \(\times\) 10\({}^{6}\) g/mol, respectively) were modified by am  
Figure 4: The amidation reaction of HA is mediated by (**a**) 1-ethyl-3[-(3-dimethylaminopropyl)] carbon-iodimide (EDC); (**b**) 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (DMTMM); (**c**) diisopropylcarbodiimide (DIC)/Ethyl cyanohydroyiminoacetate (Oxyma) in water, buffers (pH 4–6) or water/DMSO. The amidation reaction of HA is carried out in DMSO by using (**d**) 2-bromo-1-ethyl pyridinium tetrafluoroborate (BEP); (**e**) Ethyl chloroform (ECF); (**f**) 1,1’-carbonyldiimidazole (CDI). with oxidized HA. The substitution degree with 3,3'-dithiobis (propanoic dihydrazide)-modified HA (HA-TPH) was determined as 36.1% by nuclear magnetic resonance (NMR). In addition, this derivative was crosslinked by using HA-di-CHO (Figure 5). The oxidation degree was quantified using the trinitrobenzene sulfonate assay as 23.5, 34.9, and 48.3%, respectively. The Mw decreased due to oxidation from 5.5 to 7.8 \(\times\) 105 by increasing the molar ratio of NaIO4. By increasing the oxidation degree, the porosity and pore size decreased (from 87.6 to 54.5 \(\upmu\)m). Furthermore, the higher the oxidation degree was, the greater the mechanical strength due to an increased crosslinking density [67]. This kind of chemical modification has different applications in regenerative medicine. The authors have not reported potential degradation of the polysaccharide after amidation. The mechanical strength of this hydrogel was up to 4 kPa. Thus, it is not easy to handle it without the use of injection. [PERSON] and collaborators modified HA (3.7 \(\times\) 105 g/mol, 1.0 \(\times\) 106 g/mol, and 2.0 \(\times\) 106 g/mol) by EDC with cysteine hydrochloride (CYS) in (2-(N-morpholino) ethanesulfonic acid) MES buffer for 2 h at pH 4.75. The DS was determined around 40%. Self-crosslinked HA was incorporated with collagen I to fabricate scaffolds towards chondrogenic differentiation of mesenchymal stem cells. Bone marrow mesenchymal stem cell suspension isolated from neonatal New Zealand white rabbits was dropped on the surface of the materials (2D scaffold). These results were compared with 3D scaffolds prepared by dispersion of the cells [68]. Even though the hydrogels presented poor mechanical properties, the scaffolds promoted cell proliferation in vitro. [PERSON]'s group described the use of amidation of HA with dithiobis-propanoic dihydrazide) followed by cleavage of the disulfide bonds by dithiothreitol. Thiolated HA was crosslinked with polyethylene glycol diacrylate (PEGDA) via Michael addition. These materials were used for the encapsulation of ECM-derived peptides, including RGD, IKVAV, or VFDNFVLK, to control myoblast behavior. Particularly, HA hydrogels loaded with the laminin peptide (IKVAVV) promoted GFP + cell attachment and spreading, promoted migration of GFP cells, enhanced the proliferation, and up-regulated transcription factors (MPCs, Myod1, and Pax7), which are responsible for muscle repair [69].