## 3 Chemical Modification of Hyaluronan
### _Esertification of HA with Thiols_  
The preparation of sulfated HA (HA-O-SO\({}_{3}\)), as it is shown in Figure 11, has been reported by several groups [116; 117]. [PERSON] used HMW-HA (1.1 \(\times\) 10\({}^{6}\) g/mol) for the preparation of HA-TBA salt. After that, the fragmented HA underwent acrylation/sulfation. The degree of sulfation varied from 3.0 to 3.4, but the degree of substitution (DS) did not significantly affect macrophages. The authors studied the immunomodulatory effect of acrylated HA and collagen-based hydrogels containing highly substituted sulfated HA. Sulfated HA modulates the M1 and M2 repolarization via suppression of the NFkB pathway [116]. [PERSON] et al. reported that sulfated HA reduced the inflammatory activities of macrophages, impeding TLR signaling. Sulfated HA-mediated blockage of NFkB and subsequent impaired release of pro-inflammatory cytokines such as IL-6, IL-8, CXCL, CCL2, and PGE2 in human mesenchymal stromal cells [118].  
[PERSON] et al. used a double modification of HA of two different Mw (1.5 \(\times\) 10\({}^{6}\) g/mol and 4 \(\times\) 10\({}^{4}\) g/mol). The authors carried out the sulfation of HA mediated by H\({}_{2}\)SO\({}_{4}\)/DCC using HA-TBA salt. The hydrogel was formed via crosslinking of HA-DOPA after amidation of HMW-HA and LMW-HA mediated by EDC and sulfo-NHS. The degree of chemical modification varied 31.7% for HMW-HA and 12.0% for LMW-HA, using 13.1% for sulfated HA. The crosslinking of catechol via oxidation was mediated by sodium hydroxide. Due to the highly acidic conditions, the Mw of HA drastically decreased. Catechol makes the derivative adhere to the surface of bleeding wounds. To compare the hemostatic abilities of the materials, in vitro blood clotting tests were performed. Thrombin, sulfated HA/HA-DOPA patch, and thrombin-loaded patch were treated with recalcified citrated whole blood for various time intervals (30 s-10 min). The adhesive patches produced using the derivative stopped bleeding by absorbing blood and thrombin-supporting hemostasis. The dry patch prevented post-surgical adhesion by forming an anti-inflammatory physical barrier. This anti-inflammatory effect was confirmed by using RAW264.7 macrophages for 24 h stimulated by LPS (100 ng/mL). The effect of this patch was tested as an adhesion barrier compared to the commercial product Sepraflim(r) [117].  
[PERSON] et al. reported the synthesis of sulfated HA via chemical modification of HA (Mw = 1 \(\times\) 10\({}^{5}\) g/mol). The synthesis is performed in DMF using HA-TBA salt, with  
Figure 11: Preparation of sulfated HA is performed via HA-TBA salt.  
degradation of the skeletal. After that, the reaction with SO\({}_{3}\) in pyridine yielded sulfated HA. The amount of sulfur in the polymer can be varied from 2 to almost 10% by using an excess of this reagent from 8 to 32 molar eq. Interestingly, human umbilical vein endothelial cells (HUVECs) grow as a function of increasing the sulfur content [119].  
[PERSON] et al. used similar conditions to prepare HA-SO\({}_{3}\) derivatives (DS = 11.6%) and HA of Mw = 1 \(\times\) 10\({}^{5}\) g/mol. The HA-SO\({}_{3}\) derivatives promoted the growth and migration of endothelial cells, regulating the transformation of vascular smooth muscle cells from synthetic phenotype to contractile phenotype and inhibiting the adhesion/aggregation of macrophages. Furthermore, the derivative improved the transformation of macrophages from M1 to M2 and inhibited hemolytic reaction and platelet aggregation [120].  
[PERSON] et al. combined sulfated HA (sHA) with methacrylated gelatin and modified graphene. The hydrogel sHA/gelatin/G was applied to the rabbit ear wound model to evaluate the role of the material during scar formation. The material promoted scarless wound healing via fibrosis suppression. The hydrogels increased the expression of CD206 and IL-10 and decreased TNF-\(\alpha\), IL-1, and TGF-\(\beta\)[121].