EGCG was purchased from Bio Verde Inc. (Kyoto, Japan). 4-(4,6-Dimethoxy-1,3,5-triazin-2-yl)-4-methyl-morpholinium chloride (DMT-MM) and N-methylmorpholine (NMM) were purchased from Tokyo Chemical Industry Co., Ltd. (Tokyo, Japan) and Nacalai Tesque Inc. (Kyoto, Japan), respectively. Gelatin extracted from porcine skin in acidic conditions (Type A gelatin) was purchased from Sigma-Aldrich (St. Louis, MO, USA). EGCG-GS was prepared by an aqueous synthesis method reported previously [23 (link)]. In brief, gelatin (100 mg) was dissolved in warm Milli-Q water (5 mL) at 50 °C. A solution with NMM (27.5 µL), EGCG (0.07, 0.7 or 6.7 mg), and DMT-MM (69.2 mg) was stirred for 24 h at room temperature in the dark. The products were purified by dialysis (Spectra/Por7 MWCO 1000; Spectrum Labs, Rancho Dominguez, CA, USA) in Milli-Q water in the dark. The same conditions but without EGCG, DMT-MM, and NMM were used to prepare the gelation solution. After dialysis, the resulting solution was diluted to 10 mL with Milli-Q water and was poured in φ5-mm silicon tubes, followed by lyophilization with DC800 (Yamato Co., Ltd., Tokyo, Japan) to produce the EGCG-GSs or GS. To fabricate vhEGCG-GSs and vhGS, EGCG-GSs and GS were treated by vacuum heating using ETTAS AVO-250NS (AS ONE, Osaka, Japan) at 150 °C for 24 h with a gauge pressure of −0.1 MPa. All sponges were stored at 4 °C in the dark until use.
Dmt mm
Manufactured by Tokyo Chemical Industry
Sourced in Japan
DMT-MM is a chemical reagent used in organic synthesis. It is primarily employed as a coupling agent to facilitate the formation of amide bonds between carboxylic acids and amines. The core function of DMT-MM is to activate carboxylic acids, enabling their efficient reaction with amine compounds to produce amide products.
Automatically generated - may contain errors
Lab products found in correlation
Ettas avo 250ns, by As One (2 mentions)
Gelatin type a, by Merck Group (2 mentions)
N methylmorpholine, by Tokyo Chemical Industry (1 mentions)
Fitc labelled bsa, by Merck Group (1 mentions)
β galactosidase from aspergillus oryzae, by Merck Group (1 mentions)
Va 044, by Fujifilm (1 mentions)
N 2 hydroxyethyl acrylamide, by Tokyo Chemical Industry (1 mentions)
Dipea, by Tokyo Chemical Industry (1 mentions)
Phosphate buffered saline (pbs), by Welgene (1 mentions)
Cell counting kit 8 cck 8, by Dojindo Laboratories (1 mentions)
Fetal bovine serum (fbs), by Welgene (1 mentions)
Dulbecco s modified eagle s medium dmem, by Welgene (1 mentions)
Dimethyl sulfoxide (dmso), by Fujifilm (1 mentions)
Acetonitrile, by Fujifilm (1 mentions)
Formic acid, by Fujifilm (1 mentions)
7 protocols using dmt mm
1
Fabrication of EGCG-Gelatin Sponges
2
Synthesis and Characterization of Acrylate Monomers
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Gal and 2,6-lutidine were purchased from Nacalai Tesque, INC. (Kyoto, Japan). DMT-MM, p NP-Gal, and N -(2-hydroxyethyl)acrylamide ( 1a ) were purchased from Tokyo Chemical Industry Co., Ltd. (Tokyo, Japan). N -(2-Hydroxyethyl)methacrylamide ( 1b ), 2-hydroxyethyl acrylate ( 1c ), and 2-hydroxyethyl methacrylate ( 1d ) were purchased from Combi-Blocks Inc. (San Diego, USA), Nacalai Tesque, INC. and FUJIFILM Wako Pure Chemical Corporation (Osaka, Japan), respectively. 1a and 1b were used after purification by activated alumina column. 1c and 1d were used after purification by washing using hexane and then through activated alumina column according to a literature. 33) The radical initiator VA-044 was purchased from FUJIFILM Wako Pure Chemical Corporation. β-galactosidase from Aspergillus oryzae , FITC-labelled PNA from Arachis hypogaea , and FITC-labelled BSA were purchased from Sigma-Aldrich Co. LLC. (St. Louis, USA). All other reagents were commercially available and used without further purification.
3
Synthesis and Characterization of Acryloylated Chitosan Polymer
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CHPA was synthesized as previously described [18 (link)]. Briefly, CHP was dissolved in super dehydrated dimethylsulfoxide (DMSO), followed by the addition of 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methyl-morpholinium chloride (DMT-MM). Then, N,N-diisopropylethylamine, and acrylic acid (DIPEA) 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 the CHPA was 9.3 acryloyl groups per 100 glucoside units (Supplementary Figure S2 ). DMT-MM and DIPEA were purchased from Tokyo Chemical Industry Co., LTD (Tokyo, Japan). Dehydrated DMSO were purchased from Wako Pure Chemical Industries Ltd. (Osaka, Japan).
4
Carboxyl NDs and DMTMM-mediated Alendronate Conjugation
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Carboxyl NDs and 4-(4,6-dimethoxy-1,3,5-triazine-2-yl)-4-methylmorpholinium chloride (DMTMM) were purchased from Tokyo Chemical Industry Co., Ltd. (TCI, Tokyo, Japan). Alendronate (Alen) was provided by Samjin Pharm. Co. Ltd. (Seoul, Korea). Dulbecco’s Modified Eagle’s Medium (DMEM), phosphate-buffered saline (PBS), and fetal bovine serum (FBS) were purchased from Welgene (Gyeongsan, Korea). Formaldehyde solution (4%) was purchased from T&I (Chuncheon, Korea). Cell Counting Kit-8 (CCK-8) was purchased from Dojindo Laboratories Co. Ltd. (Kumamoto, Japan).
5
Fabrication and Characterization of EGCG-Loaded Gelatin Sponges
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The EGCG-GS was prepared by the aqueous synthesis method reported previously [29 (link)]. In brief, type A gelatin (100 mg; Sigma–Aldrich, St. Louis, MO, USA) was dissolved in warm Milli-Q water (5 mL) at 50 °C. The solution with 27.5 µL N-methylmorpholine (NMM), 0.07 mg EGCG, and 69.2 mg 4-(4,6-Dimethoxy-1,3,5-triazin-2-yl)-4-methyl-morpholinium chloride (DMT-MM) was stirred for 24 h at room temperature in the dark. The EGCG was purchased from Bio Verde Inc. (Kyoto, Japan), DMT-MM from Tokyo Chemical Industry Co., Ltd. (Tokyo, Japan), and NMM from Nacalai Tesque Inc. (Kyoto, Japan). The products were dialyzed with Spectra/Por7 MWCO 1000 (Spectrum Labs, Rancho Dominguez, CA, USA) in Milli-Q water in the dark for purification. To prepare the gelatin solution, the same conditions, but without EGCG, DMT-MM, and NMM were used. After dialysis, the resulting solution was diluted to 10 mL with Milli-Q water and was poured in the mold (5 mm diameter, 3 mm height) in a polytetrafluoroethylene plate, followed by pre-freezing for 24 h at −30 °C and lyophilization with DC800 (Yamato Co., Ltd., Tokyo, Japan) to obtain the EGCG-GS or gelatin sponge (GS). The EGCG-GS and GS were treated by vacuum heating using ETTAS AVO-250NS (AS ONE, Osaka, Japan) at 150 °C for 24 h with a gauge pressure of −0.1 MPa to fabricate vhEGCG-GS and vhGS. All sponges were stored at 4 °C in the dark until use.
6
Synthesis and Fabrication of CHP Nanogel Scaffolds
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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)].
7
Sialic Acid Oxidation Protocol
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Reagents and chemicals NANA, acetonitrile, formic acid, and H2O2 were purchased from Wako Pure Chemical Industries, Ltd. (Osaka, Japan). DBD-PZ and DMT-MM were purchased from Tokyo Chemical Industry Co., Ltd. (Tokyo, Japan). NANA was dissolved and diluted with distilled water to a concentration of 1 mM and used as the standard. The standard of ADOA was prepared by mixing NANA (pH 7.3) with H2O2 to a final concentration of 100 mM, and shaking the mixture at 37°C for 24 h. The standard solutions were stored at -30°C, and were suitably diluted during usage. These solutions were stable for 2 weeks at 4°C, and 3 months at -30°C.
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