Development of a Versatile Procedure for the Biofunctionalization of Ti-6Al-4V Implants

Significance Statement

The use of titanium alloy despite their advantages in biomedical applications such as replacement of hard tissues faces certain challenges due to dissimilarities in the surface features of the alloys and bone tissues.

A commonly used silanization technique which involves biofunctionalization of prosthetic surface layers, immersion silanization, also needs to be worked upon due to a need for surface preparation coupled with its uneven distribution of lowly concentrated amine functional groups.

In a recent research led by Professor Gustavo Guinea and published in the journal, Applied Surface Science, investigations were made on a newly developed activated vapor silanization technique for the effective biofunctionalization with amine layers of Ti-6Al-4V alloy.

The authors used atomic force microscopy to characterize the topography of the bare and biofunctionalized amine layer on the Ti-6Al-4V alloy. A fluorescent microscopy, which involves the use of fluorescein isothiocynate,  was used to determine the density of the amines at the surface. Osteoblast-like murine MC3T3-31 cells were modeled to investigate their compatibility response to the bare and biofunctionalized amine layer of the Ti-6Al-4V alloy. The stability of the modeled cells was also checked after cell culturing for a seven-day period.

Results observed from cell structures in the non-functionalized samples of Ti-6Al-4V at three different degrees of roughness showed attached cells which were scantily spread on the bare Ti-6Al-4V substrates at 4 h. However, at a longer period of 48 h, a completely spread polygon-shaped cells on the surface were observed. The cells implanted on the polystyrene wells which serve as control had similar features but higher density compared to the three samples of the Ti-6Al-4V substrates. The control samples also had a higher number of viable cells and similar number of dead cells compared to the three samples of the Ti-6Al-4V substrates.

At different degrees of roughness, the same proliferation rate of cells observed on each sample of the Ti-6Al-4V substrates for seven days and the authors considered the smoothest Ti-6Al-4V samples for functionalization.

Results from biofunctionalization with amine layers on the surface of the 4000 Ti-6Al-4V samples by means of the activated vapor silanization technique confirmed the deposition of amine layers on the Ti-6Al-4V samples, which were uniformly spread on the surface, thereby reducing the roughness values. Further results from the atomic force microscopy measurements confirmed the presence of high density amines with higher contact angle values and a perfect reproducibility of the deposited biofunctional layers.

Cell structures on the functionalized Ti-6Al-4V samples showed an adhered and completely spread cells on the surface. They also had a higher number of viable cells compared to the non-functionalized samples at the same period of 4 h. The number of dead cells was also considerably lower compared to the non-functionalized samples.

Similar number of viable cells and responses was also found in both functionalized and non-functionalized samples at 48 h, which indicates that the deposited functional amine layer does not inhibit cell proliferation. After cell culturing, for a period of seven days, the biofunctionalized amine layer of Ti-6Al-4V substrates maintained its integrity with the presence of a high density of amines.

This study was able to generate a homogenous and stable biofunctional amine layer on Ti-6Al-4V alloy surface with the use of the activated vapor silanization technique. The biofunctional layer may be of relevance to subsequent covalent immobilization of biomolecules, in order to improve the biocompatibility of Ti6Al4V prostheses.

Versatile Procedure for Biofunctionalization of Ti-6Al-4V Implants - Advances in Engineering

Development of a Versatile Procedure for the Biofunctionalization of Ti-6Al-4V Implants - Advances_in_Engineering

About the author

Gustavo V. Guinea is professor of materials science, Head of the Biomaterials & Regenerative Engineering Lab and Director of the Center for Biomedical Engineering (CTB) at the Universidad Politécnica de Madrid. Among other prizes, he was awarded the RILEM’s Robert L’Hermite Medal in 1993, and in 2006 he was appointed to National Correspondent Member of the Royal Spanish Academy of Sciences. Expert on mechanical behavior of biological materials, Prof. Guinea has served as President of the Spanish Structural Integrity Society in the period 2001-2009.

His research is focused in the development of new bioinspired fibers for scaffolding and tissue regeneration and on the characterization and modeling of mechanical properties and microstructure of biological fibers and soft biological tissues. He has authored more than a hundred indexed scientific papers, half of them in Q1. H index is 30. He has also been PI of 14 national and EU projects with more than 2M€ budget.

About the author

José Pérez-Rigueiro obtained his Degree in Physics (Solid State Physics) in 1991 at Universidad Autónoma de Madrid (UAM). He made his doctorate work in Departamento de Física Aplicada of the UAM, where he obtained his PhD on metallizations on silicon for microelectronics in 1995. In the same year he obtained a Degree in Biochemistry and Molecular Biology at UAM. He started as Deputy Associate Professor in the Technical University of Madrid (Departamento de Ciencia de Materiales, ETSI Caminos, Canales y Puertos), where he has been Lecturer since 1996.

His specialty is Biomaterials and Regenerative Engineering and he has worked mainly in the fields of biofunctionalization of biomaterials and in the characterization and biomedical applications of silks. At present he is also a Researcher in the Center for Biomedical Engineering (CTB-UPM).

About the author

Manuel Elices PhD. in Civil Engineering, Graduated in Physics, Full professor of Materials Science and Technology in the Polytechnic University of Madrid (UPM). Professor Elices’ professional and research work has always centred upon Materials Science and Engineering. Main areas of research are mechanical performance of materials, in particular cracking and fracture of materials, and mechanical behaviour of biological materials. He is foreign member of the National Academy of Engineering (NAE) (USA).

About the author

María Arroyo-Hernández defend her Thesis in the Universidad Autónoma de Madrid (UAM) in 2007. Her Thesis Project was about surface modification of silicon and porous silicon based materials, receiving outstanding cum laude. Her work was funded by the Education Spanish Ministry. Her first postdoctoral work was performed in the Microelectronics Institute of Madrid (IMM-CSIC) on nanomechanical biosensors. A second postdoctoral position was performed in the same institute, working on the development of zinc oxide structures for biosensing applications. These two postdoctoral positions were funded under Juan de la Cierva and JAE-DOC contracts, respectively. Subsequently, she joint the “Biomaterial, Biomimetics and Tissue Engineering” at Center for Biomedical Technology (CTB-UPM) as Research Associate.

At present, she is working at Francisco de Vitoria University as Associate Professor. She has published 19 papers in international journals, 11 of them as first author and 14 in Q1 journals, she has presented 29 contributions in national and international congresses.

About the author

Milagros Ramos received the B.S. degree in Biology and the Ph.D. degree in Molecular Biology, both from Universidad Autónoma de Madrid, Spain, in 1991 and 1996, respectively. Since 2008, she has been working as an Associate Professor of Biomedical Engineering and as researcher at the Centre for Biomedical Technology in the Universidad Politécnica de Madrid, Spain. She has published more than 35 papers in indexed journals.

Her research focuses on the development of new functionalized nanoparticles and materials obtained from bioresidues for several applications in biomedicine, especially focused on regenerative therapies for bone repair and neurodegenerative diseases.

About the author

Parsa Rezvanian graduated with a Bachelor of Science in Materials Science and Engineering from Isfahan University of Technology, Iran in 2009. He then took the nationwide Materials Science and Engineering MSc entrance exam and was ranked 20th among more than four thousand participants. Afterwards, he continued his graduate studies in the same university working on bioceramic composite foams for bone reconstruction applications and obtained a Master of Science degree in Materials Science and Engineering in 2012. His MSc thesis was accepted and funded by Iran Nanotechnology Initiative Council (INIC). Subsequently, in 2014 he joined the Biomaterials and Regenerative Engineering group at Center for Biomedical Technology, Universidad Politecnica de Madrid as a PhD candidate and undertook his studies under the supervision of Prof. Jose Perez-Rigueiro on protein immobilization techniques on biofunctionalized titanium-based materials which still continues to this date.

His research interests include protein conjugation techniques, surface biofunctionalization of biomaterials and calcium-phosphate bioceramics for life science applications.

About the author

Rafael Daza finished his degree in Physics at the Complutense University of Madrid in 2008. One year later, he joined the Biomaterials and Regenerative Engineering group where he began his PhD, mainly focused in the topographical and mechanical characterization of silks by means of atomic force microscopy. One year later, he did an internship at the CIC Biomagune (San Sebastian, Spain) in the José Luis Toca-Herrera group where he came into contact with cells mechanical characterization. In 2014 he obtained his PhD in cell mechanical characterization with AFM. Since 2015, he is Assistant Professor at the Technical University of Madrid (UPM).

He is author of eight articles based on the topographical and/or mechanical characterization of different biological entities by means of atomic force microscopy. Also he has attended to more than five international congresses regarding to biomechanics and has taken part in different research projects.


Rezvanian, P1,2, Arroyo-Hernández, M1,2, Ramos, M1, Daza, R1,2, Elices, M1,2, Guinea, G.V1,2, Pérez-Rigueiro, J1,2. Development of a Versatile Procedure for the Biofunctionalization of Ti-6Al-4V Implants, Applied Surface Science 387 (2016) 652–660.

Show Affiliations
  1. Centro de Tecnología Biomédica, Universidad Politécnica de Madrid, 28223 Pozuelo de Alarcón, Madrid, Spain
  2. Departamento de Ciencia de Materiales, ETSI Caminos, Canales y Puertos, Universidad Politécnica de Madrid, 28040 Madrid, Spain


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