## 4 Biofabrication Considerations  
The physicochemical and biological properties of chitosan rely on the degree of acetylation (DA) and degree of polymerization (DP), which also determines the molecular weight of the polymer. Chitosan is the deacetylated form of chitin, but the deacetylation process is incomplete or partial. This causes chitosan to exist as a copolymer of N-acetylglucosamine units and D-glucosamine units. Chitosan differs in molecular weight, sequencing of the acetylated residues in the structure, degree of acetylation, and \(\mathrm{pk_{a}}\) properties. As chitin deacetylation progresses, the important change that occurs is that chitosan becomes polycationic in acidic media. This is due to the exposure of the amino groups on the molecule that are ionizable. The molar fraction of N-acetylglucosamine units present in the chain are defined in the terms of degree of deacetylation (DDA). In the literature, degree of acetylation (DA) has been used, \(\mathrm{DA}=100-\mathrm{DDA}\). The physicochemical properties of chitosan such as solubility [21], viscosity [22], and biocompatibility [23] are suggested to increase as the DA decreases. On the other hand, properties like crystallinity and biodegradability have a direct relation to the DA. The faster rate of degradation causes the breakdown and accumulation of amino sugars which are responsible for eliciting inflammatory responses. This faster degradation rate also affects the biocompatibility of chitosan [24]. Biological properties such as mucoadhesive, antimicrobial, hemostatic, antioxidant, and analgesic properties increase once the degree of acetylation is lower [25]. The degree of acetylation is a fundamental feature that needs to be accurately estimated for the characterization of chitosan. The most widely used techniques to determine DA include H-NMR, UV-Vis spectroscopy, infrared spectroscopy and elemental analysis [26]. A facile and efficient deacetylation method of chitosan using 1-butyl-3-methylimidazolium acetate (BMIMOAc) as the reaction medium and catalyst has been studied in the literature. The process involves hydrothermally treated chitosan in BMIMOAc which is then recovered by dialysis followed by repetitive washings with methanol and acetone. Degree of deacetylation (DDA), estimated by colloid titration technique, was enhanced from 77% to 86% for commercial chitosan. The extent of DDA enhancement was significantly higher than the conventional deacetylation method using aqueous sodium acetate. The DDA of chitosan treated with acetate (CH\({}_{3}\)COO-) and formate (HCOO-) anions suggested that the acetate anion catalyzes the hydrolysis of the residual acetamido group in chitosan. In contrast, the formate anion did not act as the deacetylation catalyst [27]. Figure 2: Chitosan production. The molecular weight is a fundamental characteristic of chitosan polymer and has a great effect on its biomedical properties. It has a partial influence on hydrated colloidal forms and the viscoelastic properties of solutions. Techniques like gel permeation chromatography (GPC), size exclusion chromatography, static light scattering (SLS), intrinsic viscosity measurements can aid to determine the molecular weight distribution [28]. Lower molecular weight chitosan is generally preferred, as it increases the transfection efficiency of chitosan pDNA complexes and permeation characteristics. This is usually achieved by physical, chemical or enzymatic degradation [29]. The viscosity development of the aqueous solution is impacted by the molecular weight, thus playing a significant role in the biopharmaceutical aspect of chitosan [30]. The chemical structure elucidation of chitosan suggests the presence of reactive hydroxyl and primary amino groups which are less crystalline in nature compared to chitin. The pKa value of primary amino groups is 6.3, so it is considered a strong base. The amino groups of chitosan become deprotonated at pH above 6; the polymer tends to lose its charge and solubility.