The bone graft material used for the BCP group in this study was BCP (OSTEON, Genoss, Seoul, Korea). The ratio of β-TCP to HA was 3:7. The bone graft material used for the mBCP group was prepared with a coating procedure on BCP, for which ErhBMP-2 (0.05 mg/mL, Cowellmedi, Busan, Korea) and EGCG (5 mg/mL, purity >90%, Sigma-Aldrich, Ishikari, Hokkaido, Japan) was used [13 (link)17 (link)]. The process was conducted as follows [19 20 (link)]: 1) the hydroxide ion of HA was combined with the silane coupling agent (3-aminopropyltriethoxysilane, Sigma-Aldrich), 2) amino radicals were combined with a bifunctional cross-linker (N-succinimidyl-3-maleimidopropionate [SMP], Sigma-Aldrich), 3) SMP was combined with ErhBMP-2 and EGCG, 4) lyophilization was performed, 5) the material was dried (2 hours in a pressure chamber at 7–10 mTorr and more than 2 hours at −20 to 20°C), and 6) sterilization was performed using ethylene oxide.
N succinimidyl 3 maleimidopropionate smp
Manufactured by Merck Group
Sourced in United States, Spain
N-succinimidyl-3-maleimidopropionate [SMP] is a chemical compound used in various research and laboratory applications. It functions as a heterobifunctional crosslinking reagent, containing both an N-hydroxysuccinimide ester and a maleimide group. This compound can be utilized for the covalent attachment of proteins, peptides, or other biomolecules.
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Lab products found in correlation
4 protocols using n succinimidyl 3 maleimidopropionate smp
1
Preparation of Modified Biphasic Calcium Phosphate Bone Graft
2
Immobilization of ErhBMP-2 and EGCG on BCP Surfaces
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ErhBMP-2 (Cowellmedi, Busan, Korea) and EGCG (Sigma-Aldrich) immobilized grafting materials were stored at 70°C in a deep-freezer. When required, the frozen solution was placed in a lyophilizer (Ilshin Laboratory, Seoul, Korea) for 1 day to sublimate the liquid phase in a vial without denaturation of ErhBMP-2 and EGCG. The BCP surface (OSTEON, Dentium, Seoul, Korea) was coated with a combination of ErhBMP-2 and EGCG using a 3-step technique [23 24 (link)]. The first step was 3-aminopropyltriethoxysilane (Sigma-Aldrich), in which the OH− of HA was reacted with a silane coupling agent. The second step was to combine N-succinimidyl-3-maleimidopropionate (SMP; Sigma-Aldrich) with amino radicals. The last step was to combine EGCG and ErhBMP-2 with SMP. The success of the surface treatment was confirmed through field-emission scanning electron microscopy (FE-SEM; S4800, Hitachi/Horiba, Japan) (Figure 1 ).
3
FGF-2 Coated Biphasic Calcium Phosphate Granules
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The biphasic calcium phosphate (BCP) granule (Osteon GBC0305,) was obtained from Dentium Corporation, Suwon, Korea, and FGF-2 was obtained from the Genoss Corporation, Suwon, Korea, respectively. BCP is a 70% hydroxyapatite (HAp) scaffold coated with 30% β-tricalcium phosphate (β-TCP) with a particle size of 0.3–0.5 μm. As previously reported [22 (link)], the procedure for preparing FGF-2 coated BCP (see Figure 1 ) includes the grafting of 3-aminopropyl triethoxysilane (APTES, Sigma, MO, USA) onto the BCP surface and the replacement of the terminal amine by a maleimide functional group that reacts with FGF-2 via a heterobifunctional cross-linker (N-Succinimidyl-3-maleimidopropionate: SMP, Sigma, MO, USA). Briefly, 1.0 g of BCP powder was silanized by 10 mM APTES dissolved in hexane (Sigma, MO, USA) for 2 h. The silanized BCP powder was substituted for maleimide groups by 2 mM SMP dissolved in anhydrous dimethylformamide (DMF, Sigma, MO, USA) for 2 h. Then, FGF-2 dissolved in anhydrous DMF was immobilized on BCP by stirring for 2 h. All experimental procedures were performed at 25°C to avoid the risk of FGF-2 denaturation.
4
Titanium Surface Functionalization for Cell Studies
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Commercially pure Ti grade II disks of 10mm diameter and 2mm height (Technalloy S.A., Sant Cugat del Vallès, Spain) were polished, extensively washed and activated by oxygen plasma [25] .
After reaction with (3-aminopropyl)triethoxysilane (Sigma-Aldrich) and the bifunctional crosslinker N-succinimidyl-3-maleimidopropionate (SMP, Sigma-Aldrich), disks were functionalized with Cys B27(1-18), or B27(1-18) Cys . The coupling procedure [25] and the designations of titanium samples are reported in Fig. S1A (Supplementary material). The wettability and surface chemical composition of titanium samples were evaluated by Static Contact Angle Measurements (CA) and X-ray photoelectron spectroscopy (XPS) [25] .
After reaction with (3-aminopropyl)triethoxysilane (Sigma-Aldrich) and the bifunctional crosslinker N-succinimidyl-3-maleimidopropionate (SMP, Sigma-Aldrich), disks were functionalized with Cys B27(1-18), or B27(1-18) Cys . The coupling procedure [25] and the designations of titanium samples are reported in Fig. S1A (Supplementary material). The wettability and surface chemical composition of titanium samples were evaluated by Static Contact Angle Measurements (CA) and X-ray photoelectron spectroscopy (XPS) [25] .
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