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International Symposium Hyaluronic Acid


International Symposium Hyaluronic Acid

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                                FOR TISSUE ENGINEERING

                                      Yuko Ogushi, Shinji Sakai, Koei Kawakami
                 Department of Chemical Engineering, Faculty of Engineering, Kyushu University,
                                       744 Motooka, Nishiku, Fukuoka, Japan
 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

【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.

 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.

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