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7 protocols using hypermer b246

1

Synthesis and Characterization of High Internal Phase Emulsion Polymers

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A HIPE is formed by mixing two immiscible liquids to form a stable emulsion, and the EHA/IBOA copolymer HIPE was prepared according to a published procedure.29 (link) Briefly, a HIPE was prepared by mixing the monomers isobornyl acrylate (IBOA, 3.66 g) and 2-ethylhexyl acrylate (EHA, 1.56 g) with the crosslinker trimethylolpropane triacrylate (TMPTA, 1.41 g) using an overhead stirrer. The surfactant Hypermer B246 (0.21 g, Croda) was added, and the solution was mixed until it was dissolved. The photoinitiator diphenyl(2,4,6-trimethylbenzoyl)phosphine oxide/2-hydroxy-2-methylpropiophenone blend (0.35 ml) was added before the addition of water.
To prepare a HIPE with an aqueous content of 80%, 28 ml of deionised water was added over 5 min in a drop-wise fashion into a 100 ml capacity beaker containing the oil phase, stirred at a specific stirring speed using an overhead stirrer (Lab Egg, IKA). The emulsion was stored in an amber glass vial (Supelco) and used within 6 h of preparation. The stirring speeds used to investigate processing conditions were 320, 540, 765, 870, and 1260 rpm. To investigate the effect of temperature on porosity, the aqueous temperature was stabilised at 4, 15, and 30 °C before addition to the monomer.
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2

Synthesis of Hydrophobic Polymer Microspheres

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The monomer, 2,2,2-trifluoroethyl methacrylate (TFEMA, hydrophobic monomer), was purchased from Harbin Xeogia Fluorine-Silicon Chemical CO., Ltd. (Harbin, China), the cross-linker, divinylbenzene (DVB, technical grade, 80%), azobisisobutryonitrile (AIBN), and calcium chloride dihydrate (CaCl2·2H2O) were purchased from Sigma-Aldrich (USA). Span 80 (sorbitanmonooleate, HLB (hydrophilic–lipophilic balance) = 4.3), Span 85 (sorbitantrioleate, HLB = 2.1), Span 60 (sorbitan stearate, HLB = 4.7) were bought from Sigma-Aldrich (USA). Tween-80 (polyoxyethylene sorbitanmonooleate, HLB = 15.0), cationic surfactant CTAB (HLB = 15.8), anionic surfactant sodium dodecyl sulfate (SDS, HLB = 40), and Sudan III dyes were provided by Sinopharm Chemical Reagent Co., Ltd (Shanghai, China). Non-ionic surfactant Hypermer B246 (HLB = 4.9), a block copolymer of polyhydroxystearic acid and polyethylene glycol was supplied by Croda (USA). Toluene (>97%) was supplied by Tianjin Fuyu Fine Chemical Company (China).
The 2,2,2-trifluoroethyl methacrylate (TFEMA) and divinylbenzene (DVB) were filtered over basic alumina columns to remove the inhibitor and any other acidic impurities prior to use. azobisisobutryonitrile (AIBN, ≥98%) was recrystallized from methanol and dried under reduced pressure for 12 h. Deionized water was used in all experiments. All other materials were used as received.
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3

Synthesis of Polymeric Microspheres

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Styrene
(St), divinylbenzene (DVB), azobis(isobutyronitrile)
(AIBN), 2-ethylhexyl acrylate (EHA), and CaCl2·2H2O were purchased from Sigma-Aldrich. Polyurethane diacrylate
(PUDA) Ebecryl 8402 was kindly supplied by Allnex (Netherlands) and
the surfactant Hypermer B246 by Croda (Spain). All chemicals were
used as received.
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4

Glycan Analysis with Enzyme-Linked Assay

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Reagents Pentaerythritol-tetrakisIJ3-mercaptopropionate) ("tetrathiol"), triallyl-1,3,5-triazine-2,4,6IJ1H,3H,5H)-trione ("triallyl"), 2-(boc amino)ethanethiol, L-ascorbic acid (ASA), galactose-oxidase from Dactylium dendroides [50 U ml -1 ], horseradish peroxidase (HRP, lyophilized powder, 150 U mg -1 ), PNGase F, ninhydrin, D-galactose, D-IJ+)-glucose, ribonuclease B (RNase B from bovine pancreas (50 Kunitz units per mg protein)), 10acetyl-3,7-dihydroxyphenoxazine (ADHP), Tween 20 and 5,5′-dithiobisIJ2-nitrobenzoic acid) (DTNB) were obtained from Sigma Aldrich (Brøndby, DK). Lucirin TPO-L (ethyl-2, 4, 6-trimethylbenzoylphenyl phosphinate) was obtained from BASF (Hardmatt, CH). Sylgard 184 -polyIJdimethylsiloxane) (PDMS) elastomer kit was obtained from Dow Corning (Midland, MI, USA). Hypermer B246 and Span 80 surfactants were obtained from Croda International Plc (Snaith, UK)
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5

Synthesis of Polymerized High Internal Phase Emulsions

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All reagents and solvents used in polyHIPE synthesis were obtained at the highest purity available from Sigma-Aldrich and used without further purification, apart from the surfactant Hypermer B246 (a block copolymer of polyhydroxystearic acid and polyethylene glycol), which was obtained from Croda International.
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6

Biomimetic Bone Tissue Engineering

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Trimethylolpropane tris(3-mercaptopropionate) (TMPTMP), dipentaerythritol penta-/hexa-acrylate (DPEHA), 1,2-dichloroethane, dichloromethane, diphenyl (2,4,6-trimethylbenzoyl) phosphine oxide/2-hydroxy-2-methylpropiophenone blend, Pluronic F108, strontium hexachloride, nitric acid and hydroxyapatite were purchased from Sigma-Aldrich and used without modification. Hypermer B246 was obtained from Croda while Alvetex® 3D scaffolds were purchased from Reinnervate Ltd. Complete media for culture experiments was formulated using Dulbecco's Modified Eagle Medium 11995-065 with 10% foetal bovine serum and 1% penicillin/streptomycin purchased from Life Technologies through Thermo Fisher Scientific. Alkaline phosphatase (ALP) activity was determined using an ALP assay kit (Abcam).
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7

Synthesis of PLLA-PTFEMA Particles

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d,l-Lactide (dimer of lactic acid) was ordered from Jinan Daigang Biological Engineering Co., Ltd. (Jinan, China); 2,2,2-trifuoroethyl methacrylate (TFEMA, hydrophobic monomer) was from Harbin Xeogia Fluorine–Silicon Chemical CO., Ltd. (Harbin, China); DVB (technical grade, 80%), azobisisobutryonitrile (AIBN), calcium chloride dihydrate (CaCl2–2H2O), Span 80 (sorbitanmonooleate), Span 85 (sorbitantrioleate), Span 60 (sorbitan stearate) were all from Sigma-Aldrich (USA); Tween-20 (polyoxyethylene (20) sorbaitan monolaurate), Tween-80 (polyoxyethylene sorbitanmonooleate), Sudan III dyes were provided by Sinopharm Chemical Reagent Co., Ltd (Shanghai, China); non-ionic surfactants Hypermer-B246 and Hypermer A70 were from Croda (USA); toluene and xylene (>97%) was supplied by Tianjin Fuyu Fine Chemical Company (China). The nonionic surfactant Hypermer-B246 is a polyhydroxystearic acid/polyethylene oxide/polyhydroxystearic acid ABA block copolymer.32–35 (link) TFEMA and DVB were purified by passing through a column of basic alumina to remove the inhibitor. Technical grade AIBN was recrystallized from methanol and dried under reduced pressure for 12 h to yield AIBN (98%). Deionized water was used in all experiments. All other materials were used as received.
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