DEVELOPMENT OF CARBOXYMETHYLCELLULOSE WITH PHENOL MOIETIES FOR TISSUE ENGINEERING Yuko Ogushi, Shinji Sakai, Koei Kawakami Department of Chemical Engineering, Faculty of Engineering, Kyushu University, 744 Motooka, Nishiku, Fukuoka, Japan firstname.lastname@example.org 【Introduction】 Hydrogels are insoluble three-dimensional polymeric networks that absorb and retain a significant amount of water. The wide interest in the application of hydrogels in the biomedical field continues to grow. Thus, creating new materials that form hydrogels is a challenging and new area of biomedical engineering. We now report novel hydrogels prepared by crosslinking of cellulose derivatives by enzymatic reaction under mild conditions for mammalian cells. In this study, we developed an enzymatically-crosslinkable cellulose derivative prepared from carboxymethylcellulose (CMC) and tyramine using aqueous-phase carbodiimide activation chemistry. CMC is a water-soluble derivative of cellulose produced by the introduction of carboxymethyl groups along the polymer backbone. Especially, due to the biodegradability and non-toxicity associated with this polymer, CMC can be found in an increasing number of applications for food, cosmetic and pharmaceutical products as a viscosifier, emulsion stabilizer, and as a means to improve texture. Tyramine is an amine found in ripe cheese, wine and fermented or aged foods. Recently, it was reported that hyaluronic acid and alginate conjugated with tyramine formed hydrogels by a horseradish peroxidase (HRP)-catalyzed oxidation reaction. HRP functions as an oxidoreductase that catalyzes the oxidation of donors using hydrogen peroxide (H2O2) as the oxidizing agent. This enzyme has been used for the oxidative polymerization of phenol derivatives under mild reaction conditions. The attractive point of CMC compared with hyaluronic acid is its low cost. In addition, the easily available of the CMC with high purity in low cost is the attractive point compared with alginate. Impurity content in most commercial alginates has been a problem in the field of biomedical applications. 【Materials and Methods】 We synthesized a carboxymethylcellulose with phenol moieties (CMC-Ph) by covalently incorporating tyramine into carboxymethylcellulose using aqueous-phase carbodiimide activation chemistry. The resulting hydrogel was prepared from an aqueous solution of the conjugate via the HRP-catalyzed oxidation reaction of phenols by consuming H2O2. The dependence of gelation time on catalysts (HRP and H 2O2) was studied for the CMC with defferent content of phenols. The cytotoxicity of the gelation process via HRP-catalyzed oxidation reaction and the gelated conjugate was evaluated for cells enclosed in a thin gel sheet of conjugate. After subsequent 24 h of incubation, live and dead cells were stained using a fluorescence double staining kit. 【Results and Discussion】 CMC-Ph conjugates were synthesized by a general carbodiimide/active ester-mediated coupling reaction and hydrogels were successfully obtained by the enzymatic oxidative reaction of tyramine moieties using HRP and H2O2. The gelation time was controlled by changing the concentration of H2O2 and HRP. Within 10 seconds of gelation was achieved under 5 units/ml HRP and 1 mM H2O2. The viability of the mammalian cells enclosed within the hydrogel was 80% after 24 h. 【Conclusion】 In this work, we developed enzymatically gellable CMC-Ph. The CMC-Ph could be gelated with enclosing living mammalian cells. We concluded that the hydrogels prepared from CMC-Ph crosslinkable by enzymatic reaction has the potential for biomedical applications including tissue-engineering.