Biofunctionalization strategies on tantalum-based materials for osseointegrative applications
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The use of tantalum as biomaterial for orthopedic applications is gaining considerable attention in the clinical practice because it presents an excellent chemical stability, body fluid resistance, biocompatibility, and it is more osteoconductive than titanium or cobalt-chromium alloys. Nonetheless, metallic biomaterials are commonly bioinert and may not provide fast and long-lasting interactions with surrounding tissues. The use of short cell adhesive peptides derived from the extracellular matrix has shown to improve cell adhesion and accelerate the implant’s biointegration in vivo. However, this strategy has been rarely applied to tantalum materials. In this work, we have studied two immobilization strategies (physical adsorption and covalent binding via silanization) to functionalize tantalum surfaces with a cell adhesive RGD peptide. Surfaces were used untreated or activated with either HNO3 or UV/ozone treatments. The process of biofunctionalization was characterized by means of physicochemical and biological methods. Physisorption of the RGD peptide on control and HNO3-treated tantalum surfaces significantly enhanced the attachment and spreading of osteoblast-like cells; however, no effect on cell adhesion was observed in ozone-treated samples. This effect was attributed to the inefficient binding of the peptide on these highly hydrophilic surfaces, as evidenced by contact angle measurements and X-ray photoelectron spectroscopy. In contrast, activation of tantalum with UV/ozone proved to be the most efficient method to support silanization and subsequent peptide attachment, displaying the highest values of cell adhesion. This study demonstrates that both physical adsorption and silanization are feasible methods to immobilize peptides onto tantalum-based materials, providing them with superior bioactivity.
KeywordsContact Angle Tantalum White Light Interferometry Static Contact Angle Measurement Cell Adhesion Study
Metallic biomaterials are nowadays commonly used for bone replacing applications due to their unique combination of optimal mechanical properties, resistance to corrosion in biological environments and excellent biocompatibility [1, 2]. This alliance of properties has been described for stainless steel, cobalt–chromium (Co–Cr) alloys and titanium (Ti). In particular, Ti and its alloys (e.g. Ti–6Al–4V) are currently the major choice for dental and orthopedic applications . Another biomaterial that is attracting a great deal of attention from both researchers and clinicians is tantalum (Ta). Ta unites mechanical strength, ductility and high chemical stability with an outstanding in vitro and in vivo biocompatibility, and very good osteoconductivity [4, 5, 6, 7], thus offering interesting potential for orthopedic reconstructive applications. Moreover, in vivo studies have demonstrated no dissolution of Ta metal after several weeks of implantation and no evidence of inflammatory reaction was detected in tissues surrounding Ta implants .
Nevertheless, the use of Ta as implant material has been limited because of its elevated cost of production and difficult processing: it has a high melting point and it easily reacts with oxygen. Its high density is also a major drawback, preventing the elaboration of massive implants. For this reason, many studies have focused on the deposition of thin films of Ta onto other surfaces to confer its excellent properties to these materials without increasing their density. In this regard, the deposition of Ta coatings onto metallic substrates has been shown to improve the corrosion resistance and biocompatibility of stainless steel , Co–Cr alloys  and Ti-based materials . Interestingly, Ta coatings on Ti/TiO2 surfaces were shown to improve the adhesion and proliferation of human osteoblasts , as well as their production of alkaline phosphatase and mineralization , compared to untreated Ti. Likewise, in a series of recent studies the osteogenic differentiation of human mesenchymal stem cells was significantly enhanced on Ta surfaces in comparison with Ti surfaces [13, 14, 15].
Furthermore, the introduction of porous Ta implants (80–85 % porosity), which show an elastic modulus of ~3 GPa (i.e. very close to that of trabecular bone) , represents a powerful alternative to classical metallic implants because it facilitates implant stability and allows a closer contact between the implant and living tissues [17, 18, 19]. The favorable pore size and the desirable biomechanical compatibility of porous Ta has resulted in numerous applications in joint replacements such as knee [20, 21, 22], hip [23, 24, 25] and shoulder .
Besides the excellent mechanical and biological properties exhibited by Ti and Ta, the success of these materials as orthopedic and/or dental implants relies on their capacity to establish an optimal osseointegration with peri-implant bone right after the implant surgery . However, both Ti and Ta are biologically inert materials and in vivo may not elicit the specific cellular responses required for a fast and reliable bone regeneration. Such minimal biological interaction with the surrounding tissues might jeopardize the long-term stability of the implant, especially in patients with compromised clinical scenarios .
Thus, surface modifications aiming at increasing the bioactivity of implant materials are regarded as promising approaches to accelerate their osseointegrative capacity [1, 28, 29, 30]. In regard to this, the immobilization of cell adhesive molecules from the extracellular matrix (ECM) onto Ti-based materials has been thoroughly investigated for repairing and regenerating bone tissues, with encouraging outcomes both in vitro and in vivo [1, 30, 31, 32]. Such biomimetic strategies to functionalize Ti include the use of native ECM proteins and their recombinant fragments [33, 34, 35, 36], peptides [37, 38, 39, 40] and peptidomimetics [41, 42, 43].
However, this strategy has been rarely applied to Ta materials. Whereas NaOH/thermal treatments (i.e. bone-like apatite formation) [44, 45] and coatings/growth of calcium phosphate layers [46, 47, 48] onto Ta substrates have been vastly explored, the literature has only documented a few examples on the biofunctionalization of Ta with cell adhesive ECM molecules. The physical adsorption of fibronectin on nanostructured Ta surfaces efficiently enhanced the proliferation of mesenchymal stem cells compared to uncoated samples . A positive effect on the proliferation of this cell type was also observed onto tantalized steel surfaces upon covalent immobilization of type-I collagen . Nonetheless, the only report studying the functionalization of Ta surfaces with a synthetic cell adhesive peptide corresponds to a recent work from McNichols et al., in which Ta substrates were coated with a cyclic RGD peptide to improve vascular endothelialization . To the best of our knowledge, the use of RGD peptides for osseointegrative applications on Ta has not yet been investigated.
In this work, we proposed the biofunctionalization of Ta surfaces with a synthetic cell adhesive peptide (i.e. an RGD peptide) as a feasible and inexpensive approach to increase the bioactivity of this material and thus improve its efficacy for application in bone regeneration. In addition, the use of short synthetic peptides offers several advantages over the use of proteins, including higher stability to temperature and pH changes, lack of immunogenicity, ease of preparation, well-defined chemical compositions, and capacity to be immobilized on surfaces at high densities with an optimal orientation [1, 51, 52].
Thus, we have investigated the activation and biofunctionalization of Ta surfaces with a short synthetic RGD peptide by either physical adsorption or covalent binding via silane chemistry. These two methods of immobilization have been physicochemically characterized at the surface level by means of contact angle measurements, surface energy calculations, white light interferometry, scanning electron microscopy (SEM) and X-ray photoelectron spectroscopy (XPS). The biological performance of the resulting surfaces has also been evaluated by cell adhesion studies using human osteogenic sarcoma (Saos-2) cells.
2 Materials and methods
2.1 Biofunctionalization of Ta samples
2.1.1 Preparation of Ta disks
Ta disks (3 mm thick, 6.4 mm diameter) were obtained from Ta bars of 99.95 % purity (Alfa Aesar, Karlsruhe, Germany). Samples were polished to achieve mirror-like, smooth surfaces, (average surface roughness, Ra, ≈40 nm) by grinding with abrasive SiC papers (Presi, Oxford, UK) of decreasing grit size (from P800 to P4000—European P-grade standard), followed by polishing with a diamond suspension (Presi) (1.0 µm particle size) on cotton clothes. After polishing, samples were ultrasonically rinsed with cyclohexane, isopropanol, distilled water, ethanol and acetone, and stored dry under vacuum.
2.1.2 Synthesis of the cell-adhesive peptide
2.1.3 Activation of samples
Prior to functionalization, samples were either passivated with HNO3 or activated by UV/ozone treatment. Passivation with HNO 3 : Ta samples were immersed in a 32.5 % (v/v) solution of HNO3 and treated for 10 min under sonication. After this treatment, samples were thoroughly washed with distilled water, ethanol and acetone, and dried with nitrogen gas. UV/ozone treatment: samples were placed in a UVO-Cleaner® (model 42-220, Jelight Company, Inc., Irvine, CA, USA) and treated with UV/ozone for 2 h. Samples were then kept under vacuum.
2.1.4 Silanization of the samples
Activated samples were silanized with (3-aminopropyl)triethoxysilane (APTES) (2 %, v/v) (Sigma-Aldrich, St Louis, MO, USA) in anhydrous toluene for 1 h at 70 °C under nitrogen atmosphere. After this time, Ta disks were subjected to sonication for 10 min to remove non-covalently bound silanes, and washed with toluene, isopropanol, distilled water, ethanol and acetone, and dried with nitrogen. Aminosilanized samples were then further modified by reaction with 2 mg/mL of the bifunctional crosslinker 3-maleimidopropionic acid N-hydroxysuccinimide ester (Alfa Aesar) in N,N-dimethylformamide (DMF) for 1 h at room temperature. Samples were finally washed with DMF, distilled water, ethanol and acetone, and dried with nitrogen. This method was adapted, with some modifications, from previously published protocols [53, 54].
2.1.5 Immobilization of RGD peptide onto Ta samples
Ta: Non-treated tantalum
Ta HNO3: Tantalum passivated with nitric acid
Ta ozone: Tantalum treated with UV/ozone
Ta + RGD: Tantalum coated with 100 µM of RGD peptide
Ta HNO3 + RGD: Tantalum passivated with nitric acid and coated with 100 µM of RGD peptide
Ta ozone + RGD: Tantalum treated with UV/ozone and coated with 100 µM of RGD peptide
Ta + APTES: Tantalum silanized with APTES
Ta HNO3 + APTES: Tantalum passivated with nitric acid and silanized with APTES
Ta + ozone + APTES: Tantalum treated with UV/ozone and silanized with APTES
Ta + APTES + RGD: Tantalum silanized with APTES and coated with 100 µM of RGD peptide
Ta HNO3 + APTES + RGD: Tantalum passivated with nitric acid, silanized with APTES and coated with 100 µM of RGD peptide
Ta + ozone + APTES + RGD: Tantalum treated with UV/ozone, silanized with APTES and coated with 100 µM of RGD peptide
2.2 Physicochemical characterization of biofunctionalized samples
2.2.1 Static contact angle measurements and surface energy calculations
2.2.2 Topographical analysis
The topographical features (morphology and roughness) of the samples were studied by means of scanning electron microscopy (SEM) and white light interferometry. SEM analysis was conducted on a Zeiss Neon40 microscope (Carl Zeiss, Jena, Germany). For each sample, five images were taken at a working distance of 7 mm and a potential of 5 kV. The surface roughness of the samples was determined by interferometry using a Wyko NT9300 Optical Profiler microscope (Veeco Instruments, New York, NY, USA) in vertical scanning interferometry mode. Data analysis was performed with Wyko Vision 4.10 software (Veeco Instruments). The average roughness (Ra) was measured by triplicate for each sample. For each surface treatment three disks were analyzed.
2.2.3 X-ray photoelectron spectroscopy (XPS)
The chemical composition of the functionalized Ta samples was analyzed using an XPS equipment (SPECS Surface Nano Analysis GmbH, Berlin, Germany) with a Mg anode XR50 source operating at 150 W and a Phoibos 150 MCD-9 detector. High resolution spectra were recorded with a pass energy of 25 eV at 0.1 eV steps at a pressure below 7.5 × 10−9 mbar. Binding energies were referred to the C 1 s signal at 284.8 eV. Each sample series was studied by duplicate. Data was analyzed using CasaXPS software (Version 2.3.16, Casa Software Ltd., Teignmouth, UK).
2.3 Biological characterization of biofunctionalized samples
2.3.1 Cell culture
Cellular experiments were conducted using the human osteogenic sarcoma (Saos-2) cell line as osteoblast-like cellular model. Saos-2 cells were cultured in McCoy’s 5A medium (Sigma-Aldrich) supplemented with 10 % (v/v) fetal bovine serum (FBS), 1 M 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), 1 % (w/v) sodium pyruvate, 50 U/mL penicillin, 50 μg/mL streptomycin and 1 % (w/v) l-glutamine. Cells were maintained at 37 °C, in a humidified atmosphere containing 5 % (v/v) CO2, changing culture medium every 2–3 days. Upon reaching confluence, cells were detached by trypsin–EDTA and subcultured into a new flask. All experiments were performed using cells at passages between 25 and 35.
2.3.2 Cell adhesion studies: number of cells attached
Functionalized Ta samples were transferred into 48-well plates and blocked with 1 % (w/v) bovine serum albumin (BSA) in PBS for 40 min at room temperature. This step was done to reduce non-specific interactions between the cells and the surface. Next, Saos-2 cells were seeded at a density of 50,000 cells/mL (500 µL/disk) and allowed to attach in serum free medium. After 4 h of incubation at 37 °C, samples were rinsed twice with PBS to remove non-adherent cells. To determine the number of adherent cells, cells were lysed with 300 µL/disk of mammalian protein extraction reagent (M-PER) and the activity of lactate dehydrogenase (LDH) enzyme was measured by means of a conventional colorimetric assay (Cytotoxicity Detection Kit (LDH), Roche Diagnostics, Mannheim, Germany) using a multimode microplate reader (Infinite M200 PRO, Tecan Group Ltd., Männedorf, Switzerland). To convert the absorbance read-out of the test into cell numbers, a standard curve of defined cell concentrations was applied.
2.3.3 Cell adhesion studies: immuno-staining of nuclei and actin fibers
Saos-2 cells were incubated for 4 h onto Ta samples as explained above. After this time, cells attached to the surfaces were fixed for 30 min with 4 % (w/v) paraformaldehyde (PFA). Next, cells were permeabilized with 500 µL/disk of 0.05 % (w/v) triton X-100 in PBS for 20 min, and blocked with 1 % BSA (w/v) in PBS for 30 min. Washings between steps were all performed with PBS-Gly (PBS containing 20 mM of glycine) for 3 × 5 min. Then, 100 µL/disk of phalloidin-rodhamine (1:300) were incubated in triton 0.05 % (w/v) in PBS for 1 h in the dark. In a final step, nuclei of cells were also stained with 500 µL/disk of 4′,6-diamidino-2-phenylindole (DAPI, 1:1,000) in PBS-Gly for 2 min in the dark. Metallic disks were then mounted on microscope slides and analyzed by fluorescence microscopy (Nikon E600, Tokyo, Japan). The spreading of cells attached on each surfaces was assessed using ImageJ 1.46R software (NIH, Bethesda, MD, USA). Spreading of adherent cells was measured for at least 10 cells for each sample and averaged for three samples for each condition. All cellular studies were done using triplicates and repeated at least in two independent assays to ensure reproducibility.
2.4 Statistical analysis
All data presented in this study are given as mean values ± standard deviations. Significant differences between group means were analyzed either by ANOVA test followed by post hoc pairwise comparisons using Tukey’s test, or by Kruskal–Wallis non-parametric test followed by Mann–Whitney test. Confidence levels were set at 95 % unless otherwise stated.
3 Results and discussion
3.1 Functionalization strategy
The aim of the present work was to investigate and characterize the biofunctionalization of Ta surfaces with a well-known cell adhesive peptide in order to enhance the adhesion of osteoblast-like cells onto these materials. Improving the bioactivity of Ta holds great potential to improve the biological performance of Ta-based implant materials. As previously introduced, such approach, widely studied on Ti and other materials, has rarely been explored on Ta substrates.
To establish a reliable functionalization protocol to immobilize bioactive peptides, two classical approaches were considered: physical adsorption and covalent binding via silanization (Fig. 2). Physical adsorption is a simple and inexpensive method, and usually does not require to chemically modify the surface of the material. Its main disadvantage, though, is that the binding of synthetic oligopeptides by physisorption is commonly less stable than that achieved by covalent methods such as silanization, in which the peptides are irreversibly bound to the surfaces [32, 39]. Silanization, however, often requires the activation of the surface to generate accessible hydroxyl groups that will allow a successful binding and polymerization of siloxane layers. To this end, we subjected Ta samples to either passivation with HNO3  or UV/ozone treatment . Both methods do not modify the topography (i.e. roughness) of the surfaces, an advantage over other activation methods that use stronger acids or alkaline etchings . Silanization was performed with APTES following well-established protocols [53, 54]. The conditions used in this study were optimized to yield a sub- to monolayer of silane on the surface [55, 56]. The reactive amino groups of the silane layer were further modified with 3-maleimidopropionic acid N-hydroxysuccinimide ester. The presence of maleimide groups on the surfaces is useful to attach thiol-bearing peptides through a Michael addition (Fig. 2). As a model of cell-adhesive peptide, the well-known RGD sequence was selected . This ECM-motif has affinity for integrin receptors expressed in a large number of cells and has been described to efficiently promote cell adhesion and proliferation [31, 52, 58]. In our study, the RGD peptide was designed with three main characteristics: The bioactive sequence, an Ahx-based spacer, and an anchoring unit (Fig. 1). The spacer system is important to ensure an adequate accessibility of the RGD motif and efficiently interact with cell receptors (i.e. integrins) [38, 43]. The MPA group has a thiol functionality that can chemoselectively bind to maleimide groups, thereby providing a selective and stable anchorage to the surfaces.
3.2 Physicochemical characterization of the surfaces
Average roughness values (Ra) of Ta surfaces
912 ± 172 (*)
40.6 ± 8.0
47.9 ± 6.3
45.5 ± 8.2
Upon peptide binding, Ta/Ta HNO3 surfaces render surfaces with very similar hydrophilicity. This observation could be related to a similar extent of peptide attachment on these surfaces
On the contrary, the binding of the RGD peptide on Ta ozone surfaces yields surfaces that are much more hydrophilic. This effect could be attributed to lower peptide coverage, due to electrostatic repulsions between these highly negatively charged Ta surfaces and the peptides, which bear an overall negative charge (−1) under the coating conditions (pH 7.4) [28, 39].
The covalent binding of the RGD peptide through silanization was also studied with contact angle measurements (Fig. 4b). The introduction of APTES resulted in a clear increase in the contact angle of all samples (P < 0.05), in agreement with the hydrophobic nature of silane molecules. This effect was more remarkable for ozone-treated samples. These results suggest that UV/ozone treatments might be more effective in generating hydroxyl groups on the surface than the other methods, therefore yielding a most efficient silanization. In addition, the positive charge present in APTES molecules may promote in this case a higher number of electrostatic interactions with surface silanol groups . The attachment of the RGD peptide on silanized samples decreased their contact angle values to a similar level for all conditions (though slightly higher values were observed on ozone-treated samples, P < 0.05). Thus, the amount of peptide bound on the surfaces via silanization might be similar for Ta + APTES + RGD and Ta HNO3 + APTES + RGD, but slightly higher for Ta ozone + APTES + RGD.
Analysis of the chemical composition (atomic %) of Ta surfaces by XPS
Composition (atomic %)
32.8 ± 1.0
47.1 ± 2.2
1.6 ± 0.1
2.2 ± 0.2
16.3 ± 1.1
36.1 ± 8.3
45.5 ± 6.2
2.1 ± 0.1
2.4 ± 0.5
14.0 ± 2.4
19.0 ± 1.1
60.3 ± 1.1
0.9 ± 0.2
1.9 ± 0.6
17.9 ± 0.5
Ta + RGD
36.2 ± 4.5
46.3 ± 3.7
3.1 ± 0.1
1.4 ± 0.5
13.1 ± 0.4
Ta HNO3 + RGD
36.3 ± 3.3
45.9 ± 3.8
3.0 ± 0.4
2.2 ± 0.0
12.7 ± 0.1
Ta ozone + RGD
25.5 ± 5.3
55.8 ± 3.9
0.9 ± 0.2
1.9 ± 0.3
15.8 ± 1.9
Ta + APTES + RGD
41.4 ± 0.3
39.8 ± 0.1
4.5 ± 0.2
4.1 ± 0.2
10.2 ± 0.1
Ta HNO3 + APTES + RGD
42.4 ± 3.0
39.1 ± 1.6
4.5 ± 0.4
3.6 ± 0.1
10.4 ± 0.9
Ta ozone + APTES + RGD
44.7 ± 1.2
35.5 ± 0.4
5.8 ± 0.2
6.2 ± 0.1
7.8 ± 0.4
The RGD peptide can be physisorbed on Ta and HNO3-treated Ta surfaces, but this method of immobilization is not effective on very hydrophilic, ozone-activated samples.
The covalent attachment of the peptide through silanization is achieved on all surfaces, although this process seems to be more efficient on ozone-treated surfaces.
3.3 Biological characterization of the surfaces: adhesion of osteoblast-like cells
The effect of the biofunctionalization strategies on the biological performance of Ta surfaces was analyzed by studying the adhesion of sarcoma osteogenic (Saos-2) cells to these substrates. The RGD sequence, originally identified in fibronectin , is a common cell adhesive sequence found in many proteins of bone ECM. This cell-binding domain interacts with integrin receptors expressed by osteoblasts such as αvβ3, αvβ5 and α5β1 that trigger the adhesion, spreading and proliferation of these cells [31, 52, 58].
In this work we have explored the functionalization of Ta samples with a cell adhesive peptide to improve Ta’s bioactivity. Two methods were studied: physical adsorption and covalent binding via silanization with APTES. Both methods were shown to be efficient in increasing the number and area of adherent cells. However, interesting differences were also observed. Whereas the physical adsorption of an RGD peptide was easily carried out on control and HNO3-treated Ta surfaces, binding of this peptide on highly hydrophilic, UV/ozone-treated Ta surfaces proved to be inefficient and had no effect in cell activity. On the contrary, UV/ozone-activated surfaces promoted the most efficient silanization and yielded the highest values of cell adhesion compared to the other strategies of silanization. Thus, both physical adsorption and silanization are feasible methods to anchor bioactive peptides on Ta surfaces, provided that the appropriate activation methods are used. Moreover, silanization methods displayed higher values of cell attachment than physisorption. Immobilization of peptides with other biofunctionalities (e.g. antibacterial, osteogenic properties, etc.) may be pursued to confer additional biological functions to Ta. These strategies could be easily applied to porous Ta implants and other Ta-based scaffolds to increase the osseointegrative properties of such materials.
The authors acknowledge the Spanish Government for financial support through Project MAT2012-30706, co-funded by the European Union through European Regional Development Funds, and the Government of Catalonia (SGR2009 1039). Carlos Mas-Moruno thanks the support of the Secretary for Universities and Research of the Ministry of Economy and Knowledge of the Government of Catalonia (2011-BP-B-00042) and the People Programme (Marie Curie Actions) of the European Union’s Seventh Framework Programme (FP7-PEOPLE-2012-CIG, REA Grant Agreement 321985). The authors thank Mrs. Montse Dominguez and Mr. Trifon Trifonov (CRNE) for their excellent technical support with the XPS and SEM measurements, respectively.
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