## 4 Current Challenges for the Wound Healing Applications of HA
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**Biological Testing In Vitro** & **Physicochemical Characterization** & **Form** & **Composition** & **Major Outcomes** & **Ref.** \\ \hline Cytotoxicity tested on NIH-3T3, biodegradation study. & Adhesion, haemostasis and wound reduction. & Rheology & Hydrogels & Chitosan-Tannic acid/oxidized HA & Enhanced wound closure rate after 13 days. Not cytotoxic material. Observed a low haemolysis (\(\leq\)1.6\%). Low blood loss was 20\% & [71] \\ \hline Antioxidant activity, Anti-inflammatory. & 75 & Lap-shear test, rheology & Hydrogels & Oxidized HA, modified with DOPA, carboxymethyl chitosan, and PVA + NIR & \(\uparrow\) IL-4 and IL-10, \(\downarrow\) IL-18, IL-6, and TNF\(\alpha\) on 7 th day. F4/80 and CD206 & [72] \\ \hline L929 fibroblast cells for cytotoxicity, ROS assessment & Wound healing & Lap-shear test, rheology & Hydrogels & Gallic acid-collagen/HA-dopamine/\(\gamma\)-PGA grafted with \(\text{-}\)3-APB. & [75] \\ \hline \hline \end{tabular}
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**In vitro** & **Haemostatic effect** tested in rabbit model & Lap-shear test & Hydrogels & Dopamine/maleic acid-HA & Rapid gelation and strong tissue adhesion (257.4 \(\pm\) 86 J/m\({}^{2}\)) \\ \hline Biocompatibility, cell activity (CCK-8), cell migration. Angiogenesis (HUVECs), Antibacterial effect. & Infected wound & & Electrospun modified & Enhanced wound contraction compared brous scaffold & [88] \\ \hline Cytotoxicity tested on NIH-3T3, biodegradation study. & In vivo treated on reduction. & In vivo haematogenesis & & Enhanced therapeutic efficacy in diabetic wound, hepatic haemorrhage, and post-surgical adhesion. Clot formation within 4–6 min, non-cytotoxic effects of degradation products and extracted gel. Haemolysis ratio \(\leq\) 1\% & [117] \\ \hline \hline \end{tabular}
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Table 2: _Cont._The Michael addition reaction is useful for the functionalization of HA as depicted in Figure 13. Using this concept, [PERSON] et al. prepared a protein vector containing an AKPSYPP-TYK motif and a C-terminal cysteine residue, which could impart adhesive properties to a dressing. The bioactivity of maleimide-HA, which was crosslinked by cysteine-modified MAPC (DOPA), was evaluated via blood clotting index (BCI) experiments. The material showed antibacterial properties and enhanced adhesion. At a concentration of 10 mg/mL, the hydrogel significantly induced platelet aggregation, showing hemostasis. After contact with the material, the release of platelet factor 4 (PF4) promotes blood clotting [152]. In adult wound healing, elastin is lacking, and only a disorganized elastic fiber network is present after scar formation. In this idea, [PERSON] et al. prepared electrospun scaffolds as wound dressing materials containing elastin. The HA-elastin conjugate was prepared via a thiol-ene reaction mediated by tris (2-carboxy) phosphine hydrochloride. The synthesis started with the amidation of HA with 2-aminoethylmethacrylamidehydrochloride  
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**Biological Testing** **In Vitro** & **Physicochemical** **Characterization** & **Form** & **Composition** & **Major Outcomes** & **Ref. ** \\ \hline Antioxidant test. & In vivo wound rheogenicity test (blood clotting test). & Water uptake, wound rheoculation & Membranes & Chitosan/HA/phosphatidylcholine/ dihydroercetin, glycerol. & Reduction in the wound (51.9 \(\pm\) 2.6\%) compared to 22.15 \(\pm\) 1.1\% on control and 15.01 \(\pm\) 0.8\% in cotton g  
containing a terminal double bond, which was reacted in a second step with an elastin-derived peptide via Michael addition. The HA derivative was electrospun with poly-D, L-lactide in a ratio of 1:50 using HFP [153]. The last challenge is to develop hydrogels with mechanical integrity without compromising their biocompatibility.