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Biomaterials of Natural Origin in Regenerative Medicine
Published in Severian Dumitriu, Valentin Popa, Polymeric Biomaterials, 2020
Vijay Kumar Nandagiri, Valeria Chiono, Piergiorgio Gentile, Franco Maria Montevecchi, Gianluca Ciardelli
In the field of tissue engineering, fibrin has been attempted for a variety of tissue engineering applications such as to fabricate sealants in surgery and porous scaffold for bone regeneration (Eyrich et al. 2007). Biomaterials produced from fibrin display varying mechanical and structural properties depending on the initial fibrinogen concentrations, and hence, they are applied for various tissue regeneration applications. For instance, biomaterials produced by using higher concentrations of fibrinogen have been used as tissue sealants (Traver and Assimos 2006), whereas fibrin hydro-gels, which have been produced at lower fibrinogen concentrations, have been studied as cardiovascular grafts (Tiwari et al. 2002, del Mel et al. 2008) and bioengineered cartilages. For use in tissue engineering applications, fibrin gels have been prepared by combining fibrinogen and thrombin solutions containing calcium ions. These preparations are not suitable in conditions where the scaffolds are needed to be intact for longer durations. One of the major challenges in using fibrin as a scaffold relays on its rapid degradation nature. However, the rate and extent of its degradation varies depending on cell type and culture conditions; in some cases, it is too rapid as compared to the rate at which ECM is produced by the cells. For instance, this happens in case of smooth muscle cells which rapidly degrade the fibrin, leading to the complete degradation of scaffold before significant ECM is produced to replace it (Ahmed et al. 2008). Rapid degradation of the fibrin matrix can be controlled by using serine protease inhibitor aprotinin. Tuan et al. have reported the successful inhibition of fibrin degradation by using aprotinin in concentrations of 500 KIU/mL (Tuan et al. 1996).
Production, purification and characterization of a novel antithrombotic and anticoagulant serine protease from food grade microorganism Neurospora crassa
Published in Preparative Biochemistry & Biotechnology, 2022
Yajie Duan, Priti Katrolia, Ailing Zhong, Narasimha Kumar Kopparapu
Effect of metal ions on fibrinolytic activity was also studied. Metal ions such as Cu2+, Na+, and Zn2+ enhanced the fibrinolytic activity, while catalysis was strongly compromised by Fe2+. Similar results were reported from Pleurotus ostreatus and Xylaria curta where Fe2+ reduced fibrinolytic activities.[31,39] Based on their inhibitor specificity, fibrinolytic enzymes can be grouped as serine proteases, metalloproteases, and others. In this study, serine protease inhibitors such as PMSF, SBTI and aprotinin completely inhibited fibrinolytic activity, which indicates the purified enzyme could be a serine protease. Metalloprotease inhibitor EDTA did not inhibit the activity, which indicates metal ions are not involved in catalysis. Effect of other reagents on fibrinolytic activity was also studied. 0.25% β-mercaptoethanol (130%) enhanced the activity of the enzyme, which is similar to another study.[34] Denaturing agents such as 8 M urea (54%) and 0.25% SDS (100%) reduced the fibrinolytic activity, which showed they might have denatured the enzyme. Denaturing agents may denature the protein and change its structure which results in decreased or complete loss of activity. Similar results were reported for fibrinolytic enzyme from Agrocybe aegerita.[36]
Purification of chitosanases produced by Bacillus toyonensis CCT 7899 and functional oligosaccharides production
Published in Preparative Biochemistry & Biotechnology, 2022
Julia Maria de Medeiros Dantas, Nathália Kelly de Araújo, Nayara Sousa da Silva, Manoela Torres-Rêgo, Allanny Alves Furtado, Cristiane Fernandes de Assis, Renata Mendonça Araújo, José António Teixeira, Leandro de Santis Ferreira, Matheus de Freitas Fernandes-Pedrosa, Everaldo Silvino dos Santos
The elution samples to the best purification factor obtained were selected to undergo 12% polyacrylamide electrophoresis gel in the presence of sodium dodecyl sulfate (SDS) to qualitatively estimate their purity.[19] The samples, previously solubilized in sample buffer, were heated at 100 °C for five min, then 20 µL of each sample and 10 µL of molecular weights standard were applied to the gel. The marker used was the SigmaMarker Electrophoresis (Sigma-Aldrich) containing the following standard proteins (kDa): aprotinin, bovine lung (6.5); α-lactalbumin, bovine milk (14.2); trypsin inhibitor, soybean (20.0); trypsinogen, bovine pancreas (24.0); carbonic anhydrase, bovine erythrocytes (29.0); glyceraldehyde-3-phosphate dehydrogenase, rabbit muscle (36.0); ovalbumin, chicken egg (45.0); albumin, bovine serum (66.0). After the analysis, the gel was stained using Coomassie Blue allowing the protein bands visualization.
Natural latex serum: characterization and biocompatibility assessment using Galleria mellonella as an alternative in vivo model
Published in Journal of Biomaterials Science, Polymer Edition, 2022
Giovana Sant’Ana Pegorin Brasil, Patrícia Pimentel de Barros, Matheus Carlos Romeiro Miranda, Natan Roberto de Barros, Juliana Campos Junqueira, Alejandro Gomez, Rondinelli Donizetti Herculano, Ricardo José de Mendonça
NLS was analyzed by SDS-PAGE, according to Laemmli [57]. This procedure was performed using discontinuous gels (10 × 10 cm) with a 15% separating gel and a 5% stacking gel. The protein samples were boiled for 5 min in 10 mM TrisHCl buffer (pH 7.0) containing 1% Sodium Dodecyl Sulphate (SDS), 80 mM 2-mercaptoethanol and 15% glycerol. The protein bands were visualized by staining with 0.025 coomassie brilliant blue R250 in the mixture of 50% methanol and 10% acetate. Bovine serum albumin (66.000); Ovalbumin (45.000); Glyceraldehyde-3-phosphate dehydrogenase of rabbit muscle (36.000 Da); Carbonic anhydrase (29.000); Trypsinogen (24.000 Da); Trypsin inhibitor (20.100); α-Lactalbumin (14.400) and Aprotinin (6.500 Da) were used as standards of molecular markers.