Garrido Dominguez, Beatriz

Gil Mur, Francisco Javier

Mas Moruno, Carlos

Rodríguez Rius, Daniel

Rupérez de Gracia, Elisa

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Immobilization Strategies to Functionalize Tantalum Surfaces with Cell-Adhesive Peptides: Physicochemical and Biological Characterization   Carlos Mas-Moruno1,2, Beatriz Garrido1, Daniel Rodriguez,1,2 Elisa Ruperez,1,2 F. Javier Gil1,2    1Biomaterials, Biomechanics and Tissue Engineering Group, Department of Materials Science and Metallurgical Engineering, Universitat Politècnica de Catalunya, Spain 2Research Networking Center in Bioengineering, Biomaterials and Nanomedicine (CIBER-BBN), Spain carles.mas.moruno@upc.edu   INTRODUCTION Tantalum (Ta)-based implants are commonly used in orthopedics to replace trabecular bone tissues. This is due to the combination of optimal mechanical properties and excellent biocompatibility displayed by this material1. Moreover, biomimetic approaches aiming at conferring a superior bioactivity on Ta surfaces could increase osteointegrative rates and improve the efficacy of such materials2. However, to date only a few studies have attempted the biofunctionalization of this material with cell-adhesive molecules3,4. The objective of this work was thus to investigate immobilization strategies to functionalize Ta surfaces with an RGD peptide. The process of biofunctionalization was characterized by means of physicochemical methods and cell adhesion assays.   EXPERIMENTAL METHODS General: Smooth (Ra ≈ 40 nm) Ta disks were used as model material. A linear RGD peptide containing a bioactive sequence, a spacer unit and an anchoring group (Figure 1) was synthesized in solid-phase and used as cell adhesive molecule. Biofunctionalization: Ta samples were activated (passivation with HNO3 or treatment by UV/ozone) and the RGD peptide immobilized either by physical adsorption or silanization (APTES). Surface characterization: the physicochemical properties of the samples were analyzed by means of contact angle measurements and surface energy calculations, white light interferometry, scanning electron microscopy (SEM) and X-ray photoelectron spectroscopy (XPS). Biological characterization: Cell adhesion studies were conducted using human osteogenic sarcoma (Saos-2) cells. The number of cells attached on the functionalized Ta samples was measured by LDH assays, and their spreading by immunofluorescent methods. Proliferation of adherent cells was also evaluated by LDH activity. Statistics: Significant differences were analyzed either by ANOVA or by non-parametric Mann-Whitney test. Confidence levels were set at 95 %.    Fig 1. Chemical structure of the cell adhesive peptide containing the GRGDS active motif, three units of aminohexanoic acid as spacer and a mercaptopropionic acid as anchoring unit.  RESULTS AND DISCUSSION Physicochemical characterization: Passivation with HNO3 only reduced slightly the water contact angle of Ta samples. In contrast, activation with ozone drastically increased the wettability of the samples, resulting in highly hydrophilic surfaces. 1. Physical adsorption of the RGD peptide on Ta/Ta HNO3  samples was analyzed by the increase in the N1s signal by XPS. The peptide was physisorbed at a similar rate in both surfaces. However, peptide attachment was less efficient in ozone-activated samples. 2. Silanization with APTES proved to be more efficient on ozone-treated Ta samples than on Ta/Ta HNO3 samples, probably due to the difference in the presence of hydroxyl groups on the surfaces. The final amount of peptide bound to silanized-surfaces was nonetheless similar to all samples, regardless of the activation method used. Biological characterization: 1. Physisorption of the RGD peptide on Ta and Ta-HNO3 samples resulted in a significant increase in the surface’s cell adhesive capacities, displaying higher number of cells attached and improved cellular spreading compared to non-functionalized samples. However, this effect was not observed on ozone-activated samples, due to the lower efficiency of peptide attachment observed for these samples. 2. The covalent immobilization of the peptide (silanization) rendered also increased cell adhesion activities. Interestingly, the extent of cell adhesion was similar for all functionalized surfaces, consequent with an optimal silanization for all the activation condition tested.   CONCLUSION We have characterized two methods of peptide immobilization to functionalize Ta. Both strategies have increased the number and spreading of Saos-2 cells onto the surfaces, thus representing viable approaches to increase the osteointegrative capacities of this material.  REFERENCES [1] Levine B. R. et al., Biomaterials 27:4671-81, 2006; [2] Shekaran A. et al. J. Biomed. Mater. Res. Part A 96:129-35, 2011; [3] Li Y. et al. Colloid Surf. B-Biointerfaces 95:10-5, 2012; [4] McNichols C. et al. J. Biomed. Mater. Res. Part A 102:532-9, 2014. ACKNOWLEDGMENTS CMM thanks the Generalitat de Catalunya and the European Commission (FP7-PEOPLE-2012-CIG) for funding.     26th European Conference on Biomaterials, Liverpool, 31st August – 3rd September 2014 

Immobilization strategies to functionalize tantalum surfaces with cell-adhesive peptides: physicochemical and biological characterization

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