by Keyword: Recombinant protein
Arista-Romero M, Delcanale P, Pujals S, Albertazzi L, (2022). Nanoscale Mapping of Recombinant Viral Proteins: From Cells to Virus-Like Particles Acs Photonics 9, 101-109
Influenza recombinant proteins and virus-like particles (VLPs) play an important role in vaccine development (e.g., CadiFluS). However, their production from mammalian cells suffers from low yields and lack of control of the final VLPs. To improve these issues, characterization techniques able to visualize and quantify the different steps of the process are needed. Fluorescence microscopy represents a powerful tool able to image multiple protein targets; however, its limited resolution hinders the study of viral constructs. Here, we propose the use of super-resolution microscopy and in particular of DNA-point accumulation for imaging in nanoscale topography (DNA-PAINT) microscopy as a characterization method for recombinant viral proteins on both cells and VLPs. We were able to quantify the amount of the three main influenza proteins (hemagglutinin (HA), neuraminidase (NA), and ion channel matrix protein 2 (M2)) per cell and per VLP with nanometer resolution and single-molecule sensitivity, proving that DNA-PAINT is a powerful technique to characterize recombinant viral constructs.
JTD Keywords: dna-paint, hemagglutinin, influenza, neuraminidase, paint, recombinant proteins, single-molecule localization microscopy, single-particle analysis, virus-like particles, Dna-paint, Hemagglutinin, Influenza, Neuraminidase, Paint, Recombinant proteins, Single particle analysis, Single-molecule localization microscopy, Single-particle analysis, Super-resolution microscopy, Superresolution microscopy, Virus-like particles
Vidal, E., Torres, D., Guillem-Marti, J., Scionti, G., Manero, J. M., Ginebra, M. P., Rodríguez, D., Rupérez, E., (2020). Titanium scaffolds by direct ink writing: Fabrication and functionalization to guide osteoblast behavior Metals 10, (9), 1156
Titanium (Ti) and Ti alloys have been used for decades for bone prostheses due to its mechanical reliability and good biocompatibility. However, the high stiffness of Ti implants and the lack of bioactivity are pending issues that should be improved to minimize implant failure. The stress shielding effect, a result of the stiffness mismatch between titanium and bone, can be reduced by introducing a tailored structural porosity in the implant. In this work, porous titanium structures were produced by direct ink writing (DIW), using a new Ti ink formulation containing a thermosensitive hydrogel. A thermal treatment was optimized to ensure the complete elimination of the binder before the sintering process, in order to avoid contamination of the titanium structures. The samples were sintered in argon atmosphere at 1200 °C, 1300 °C or 1400 °C, resulting in total porosities ranging between 72.3% and 77.7%. A correlation was found between the total porosity and the elastic modulus of the scaffolds. The stiffness and yield strength were similar to those of cancellous bone. The functionalization of the scaffold surface with a cell adhesion fibronectin recombinant fragment resulted in enhanced adhesion and spreading of osteoblastic-like cells, together with increased alkaline phosphatase expression and mineralization.
JTD Keywords: Direct ink writing, Osseointegration, Recombinant protein, Thermoresponsive binder, Titanium, Titanium scaffold
Guillem-Marti, J., Gelabert, M., Heras-Parets, A., Pegueroles, M., Ginebra, M. P., Manero, J. M., (2019). RGD mutation of the heparin binding II fragment of fibronectin for guiding mesenchymal stem cell behavior on titanium surfaces ACS Applied Materials and Interfaces 11, (4), 3666-3678
Installing bioactivity on metallic biomaterials by mimicking the extracellular matrix (ECM) is crucial for stimulating specific cellular responses to ultimately promote tissue regeneration. Fibronectin is an ECM protein commonly used for biomaterial functionalization. The use of fibronectin recombinant fragments is an attractive alternate to the use of full-length fibronectin because of the relatively low cost and facility of purification. However, it is necessary to combine more than one fragment, for example, the cell attachment site and the heparin binding II (HBII), either mixed or in one molecule, to obtain complete activity. In the present study, we proposed to install adhesion capacity to the HBII fragment by an RGD gain-of-function DNA mutation, retaining its cell differentiation capacity and thereby producing a small and very active protein fragment. The novel molecule, covalently immobilized onto titanium surfaces, maintained the growth factor-binding capacity and stimulated cell spreading, osteoblastic cell differentiation, and mineralization of human mesenchymal stem cells compared to the HBII native protein. These results highlight the potential capacity of gain-of-function DNA mutations in the design of novel molecules for the improvement of osseointegration properties of metallic implant surfaces.
JTD Keywords: Fibronectin, Growth factor, Mutation, Osseointegration, Recombinant protein, Titanium
Guillem-Marti, J., Boix-Lemonche, G., Gugutkov, D., Ginebra, M.-P., Altankov, G., Manero, J.M., (2018). Recombinant fibronectin fragment III8-10/polylactic acid hybrid nanofibers enhance the bioactivity of titanium surface Nanomedicine 13, (8), 899-912
Aim: To develop a nanofiber (NF)-based biomimetic coating on titanium (Ti) that mimics the complex spatiotemporal organization of the extracellular matrix (ECM). Materials & methods: Recombinant cell attachment site (CAS) of fibronectin type III8-10 domain was co-electrospun with polylactic acid (PLA) and covalently bound on polished Ti discs. Osteoblast-like SaOS-2 cells were used to evaluate their complex bioactivity. Results: A significant increase of cell spreading was found on CAS/PLA hybrid NFs, followed by control pure PLA NFs and bare Ti discs. Cell proliferation showed similar trend being about twice higher on CAS/PLA NFs. The significantly increased ALP activity at day 21 indicated an enhanced differentiation of SaOS-2 cells. Conclusion: Coating of Ti implants with hybrid CAS/PLA NFs may improve significantly their osseointegration potential.
JTD Keywords: Electrospinning, Fibronectin, Hybrid nanofibers, Osseointegration, PLA, Recombinant protein