Two types of CHP nanogels were synthesized as previously described [27 (link)]. Both CHP nanogel materials were dissolved in super dehydrated dimethylsulfoxide (DMSO; FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan), followed by the addition of 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methyl-morpholinium chloride (DMT-MM; Tokyo Chemical Industry Co., Ltd., Tokyo, Japan). Then, N,N-Diisopropylethylamine and acrylic acid (DIPEA; Tokyo Chemical Industry Corp.) were added, and the mixture was stirred for 22 h. After stirring, the liquid was collected and dialyzed with MilliQ water, and DMSO for 4 days. The degree of substitution of the acryloyl groups in both CHP nanogel materials is as follows: The CHP-A and the CHP-OA nanogels (21 acryloyl groups per 100 monosaccharides) and PEG-SH were dissolved in 10× phosphate-buffered saline (PBS; FUJIFILM Wako Pure Chemical Corp.), then mixed and gelatinized to form the gels (Figure 1 B). A cylindrical mold with a 6 mm diameter and 3 mm thickness was used to fabricate both CHP scaffolds. A 4 mm diameter mold was used, and solidified hydrogels were used to make the mold porous by the freeze–thaw method so that cells could easily penetrate inside, as previously described [27 (link)].
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