Materials Science and Engineering C 27 (2007) 83 89 www.elsevier.

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Investigation of the effects of alkane phosphonic acid/RGD coatings on cell spreading and the interfacial strength between human osteosarcoma cells and Ti6Al4V
R.A. Bly, Y. Cao, W.A. Moore, W.O. Soboyejo
Princeton Institute for the Science and Technology of Materials (PRISM), and The Department of Mechanical and Aerospace Engineering, Princeton University, Princeton, NJ 08544, USA Received 28 October 2005; received in revised form 23 January 2006; accepted 5 February 2006 Available online 19 April 2006

Abstract This paper presents the results of an experimental study of the effects of alkyl phosphonic acid/RGD complexes on cell spreading and the interfacial strength between human osteosarcoma (HOS) cells and Ti6Al4V surfaces. The initial stage of cell spreading is shown to be accelerated by the coatings in 9-day cell culture experiments. The adhesion between human osteosarcoma (HOS) cells and alkyl phosphonic acid/ RGD-coated surfaces is also quantified by performing shear assay experiments on coated and uncoated Ti6Al4V specimens. These show that the interfacial shear strengths required to detach the HOS cells from the coated/uncoated specimens, increase from approx. 407 Pa (in the case of the uncoated samples) to 80 Pa in the case of the of alkyl phosphonic acid/RGD coated samples. The increase is attributed to the tethering effect of the of alkyl phosphonic acid/RGD complexes on the HOS cells. The implications of the results are then discussed for improving the wound healing and the osseointegration of biomedical implants fabricated from Ti6Al4V. 2006 Elsevier B.V. All rights reserved.
Keywords: RGD peptide; Shear assay; Interfacial strength; HOS cells; Cell spreading; Ti6Al4V

1. Introduction Piershbacher and Rouslahti discovered that the Arg-Gly-Asp (RGD) tripeptide sequence is a necessary cell adhesion peptide sequence for fibronectin's primary receptor, the 51 integrin [1]. The discovery of the ability to mimic fibronectinintegrin interaction by using the simple RGD peptide suggests a method to achieve increased cellular adhesion on biomaterials. A variety of cell types has demonstrated increased focal adhesions on a variety of surfaces functionalized with a biomimetic coating of the RGD sequence [24]. Several sources suggest functionalizing biocompatible surfaces with the RGD sequence in order to improve adhesion [25]. Mwemfumbo et al. reported that osteoblast cells mechanically pulled from Ti6Al4V surfaces leave behind focal adhesion proteins and suggested that increasing focal adhesion concentration could increase cellular adhesion strength [5]. Increasing osteoblast focal point adhesion on Ti6Al4V ELI
Corresponding author. Tel.: +1 609 258 5609; fax: +1 609 258 5877. E-mail address: soboyejo@princeton.edu (W.O. Soboyejo). 0928-4931/$ - see front matter 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.msec.2006.02.005

(surgical grade titanium alloy) utilizing an RGD-peptide coating could enhance implant osseointegration, thereby increasing implant working life and ultimately patient quality of life. The surface chemistry of interest in terms of covalently attaching organic molecules to titanium alloys is the native oxide layer [6]. X-ray photoelectron spectroscopy (XPS) reveals that the majority of the oxide layer consists primarily of -oxygens (85%) as well as some hydroxyl groups (15%) [7]. -oxygens are ether functional groups linked to the surface of titanium. Most methods for attaching organic molecules to the titanium surface, such as salinization, utilize the hydroxyl groups as anchoring points. However, the surface density of these coatings can be poor due to the low percentage of native titanium hydroxyl coverage with which to react [8]. In addition, coatings achieved through such methods have low physiological stability. Gawalt et al. [9] have developed a coating method utilizing the self-assembly of an alkyl phosphonate. The resulting self-assembled monolayer (SAM) exhibits high coverage density and physiological stability. This method is well suited for attaching the RGD protein sequence to titanium alloys.

84 Table 1 Ti6Al4V polishing procedure Roughness (grit) 300 600 800 1200 Colloidal Silica (0.05 m) a On TEXMET cloth
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Time (min) 1025 7 7 6

RPM 170 170 170 170

Approximate force (Newton/sample) 27 27 27 22

140

18

Done in two segments with rinsing and sonication.

In vitro cellular adhesion analysis can be divided into static and dynamic analysis systems. Static analysis typically involves studies of cell spreading. Gawalt et al. [6] has grown human fetal osteoblasts on RGD coated Ti6Al4V and determined that RGD coating promotes extensive cellular proliferation and spreading in the short term and remains viable under physiological conditions. The Schwartz study combined cell spreading studies with immunofluorescent (IF) staining for the cytoskeletal proteins, actin and/or vinculin, allowing visualization of internal cellular organization and cell attachment. After 90 min cells grown on control Ti6Al4V exhibited little spreading and no organized actin cytoskeleton, while more than 90% of cells seeded on the RGD coated surfaces became well spread and organized their actin filaments into robust stress fibers. The development of stress fibers indicates that the cells are forming focal adhesions and will be better able to distribute applied stresses and adhere more tightly to the substrate. These results suggest improved adhesion characteristics on Ti6Al 4V as a result of the RGD surface coating. In addition, staining for hydroxyapatite, signifying bone formation, revealed that the RGD coating also accelerates bone mineralization and therefore could improve osseointegration in the long term. Dynamic cellular analysis yields quantitative evaluation of the effect of RGD coating on cellular adhesion strength. These methods include: micropipette manipulation [10], atomic force microscopy [11], laser tweezers [12], and shear assay

techniques [13]. Shear assay devices such as spinning disks and cone plates simultaneously apply a range of shear stress, but do not allow continuous visualization of single cells [14]. Micropipette aspiration, atomic force microscopy, laser tweezers, and other similar methods have the advantage of being able to study a large number of cells, but studying the detachment of cells in different stages of cell spreading has not been described, detachment times are short, and no quantitative shear stress data can be calculated [15]. The shear assay technique overcomes the difficulties encountered in these other methods. Using a laminar flow of a viscous fluid through a parallel plate flow chamber in order to shear cells from a particular surface allows visualization of multiple cells at single cell resolution while applying a uniform shear stress to attached, spread cells. The wall shear stress can be expressed as the following [15]: sw 6lQ=wh2 1

where is the dynamic viscosity, Q is the volumetric flow rate, and w and h are the width and height of the chamber, allowing the following assumptions: the fluid is incompressible and viscous, the flow plane is steady and horizontal between the two parallel plates, and the only body force is gravity. While the static adhesion analysis performed by Gawalt et al. [6] suggests that the RGD coating should increase cellular adhesion, no quantitative data exists examining the effects of an RGD or alkyl phosphonate (AP) surface treatment on the interfacial shear strengths between human bone cells and Ti 6Al4V surfaces. This paper presents shear assay analysis on control, AP-coated, and RGD-coated Ti6Al4V surfaces seeded with human osteosarcoma cells after 1 h of growth. No previous quantitative data exists quantifying the strength of attachment between bone cells and titanium alloys or protein coated titanium alloys. The work presented here produced such data in the short growth phase. Additional conventional cell analysis techniques complimented these results, rendering a more fully developed picture of cellular adhesion on titanium.

Fig. 1. Control (left) and RGD-coated (right) 20 20 m area. RMS surface roughness values of 5 and 7 nm, respectively.

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2. Materials and methods 2.1. Titanium alloy preparation A 3.49 cm diameter rod of Ti6Al4V ELI (Grade 23) was obtained (Titanium Industries, Parsippany, NJ) and cut into 75 disks (0.635 cm thickness) via EDM cutting (New Jersey Precision Technologies, Mountainside, NJ). All samples were polished under a developed protocol: 300, 600, 800, 1200 grit, and finally on a 0.05 m colloidal silica suspension using TEXMET cloth for 10, 7, 7, 6, and 6 min, respectively (Buehler, Lake Bluff, IL) (Table 1). Polishing was performed using 8-in. Ecomet wheels and Automet power heads (Buehler, Lake Bluff, IL). Surface topologies were characterized via atomic force microscopy; The root mean square (RMS) surface roughness values were determined to be on the order of 5 nm (Fig. 1). An RGD coating was applied to the polished Ti6Al4V disks. The RGD coating applied reacts and bonds with the oxide layer on Ti6Al4V via a cysteine amino acid and alkyl phosphonate (AP) linker. 15 samples were functionalized with the RGD amino acid sequence via an alkyl phosphonate (AP) tether as per the procedure of Gawalt et al. [9]. In order to properly isolate the effects of RGD, and not the potential effects of simply applying organic materials to the surface, a control group consisting of only the AP molecule functionalized on the surface was also produced. Chemical structures of RGD-coated and AP coated TiO2 surfaces are shown in Fig. 2. Once polished and cleaned, Ti6Al4V samples were suspended vertically in a solution of 0.1 mM 11-Hydroxyundecylphosphonate (AP) in Tetrahydrofuran (THF) as per the T-bag method [21] to set up a self assembled monolayer (SAM). It is hypothesized that the high

(a)

10 m

(b)

Detachment

10 m

(c)

C-

DG

10 m

Fig. 3. Sequence of initial cell detachment. (a) t = 0 s, (b) t = 2 s, (c) t = 4 s.

O N O

OH
O O C

energy associated with the meniscus attracts a higher concentration of solute locally, leaving behind a monolayer (after rinsing) of the AP as the solution evaporates down the sample. Following evaporation, samples were heated for 24h at 120 C and then rinsed with sonication in Methanol (2 15 min) and 18 M deionized water (1 20 min), removing all but the monolayer. IR spectroscopy analysis was performed to verify the presence an alkyl-chain ordered film indicative of the AP molecule. This marked the completion of the blank AP control samples. 2.2. Cell culture Surfaces were sterilized through sonication in distilled water, acetone, 30% nitric acid, and finally 100% ethanol at 20 min each. Human osteosarcoma (HOS) cells were seeded onto polished control, AP-coated, and RGD-coated surfaces with serum-free Dulbecco's Modified Eagle's Medium (DMEM) (Quality Biological, Gaithersburg, MD). Cells were incubated

O
O P O O

P O O

TiO2 Surface

TiO2 Surface

RGD-Coated

AP-Coated

Fig. 2. Chemical structures of RGD-coated and AP coated TiO2 surfaces.

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Fig. 4. HOS cells stained for actin (red) and vinculin (green). (a) control1h, (b) RGD1 h, (c) AP1h. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

for 1 h at 37 C. In the cases of the samples prepared for experiments requiring an incubation time of greater than 2 h, 10% Fetal Bovine Serum and 1% penicillin/streptomycin/ amphotericin B (Quality Biological, Gaithersburg, MD) were added to the DMEM at 2 h. Initial seeding concentration was carefully controlled to avoid confluent cells, permitting the analysis of isolated cells required for an effective shear assay test. 2.3. Immunofluorescence (IF) staining and microscopy Cells from each of the experimental groups were stained for visualization of actin and vinculin under immunofluorescent light. After 1 h, samples from each experimental group were rinsed with a solution of 50 mL phosphate buffer solution and 25 L 1 M MgCl (Quality Biological, Gaithersburg, MD). The cells were then fixed for 15 min with 3.7% formaldehyde in the MgCl solution (Quality Biological, Gaithersburg, MD) prior to rinsing with MgCl solution. Cell membranes were permeated by soaking with 0.5% Triton-X100 solubilizing agent for 15 min (Quality Biological, Gaithersburg, MD) and then rinsed with the MgCl solution. Cells were stained with vinculin antibodies: anti-vinculin antibody (Sigma, Mouse anti-vinculin, stock # V9132), secondary vinculin antibody (Molecular Probes, Goat anti-mouse stock # F2761) and rhodamine phalloidin (Molecular Probes, stock # R-415), incubating at 37 C during each step and rinsing with MgCl solution between each step.

2.4. Shear assay measurement of interfacial strength Shear assay tests were performed on 5 control, AP-coated, and RGD-coated surfaces using a parallel plate flow chamber (Glycotech Corporation, Rockville, Maryland) and a viscous flow medium of 60% Methylcellulose and 40% DMEM ( = 3.34 10 2 kg/m s). The viscous flow rates were increased in steps of 2550 mL/h (flow rates varied from 25 to 500 mL/h) until cell detachment was observed to occur using an in situ optical microscope (200 magnification) attached to a CCD video camera. A total of 3060 isolated, single cells were detached and recorded in each trial, which lasted 38min. It

Cell Area Compared vs Time

1000 900 800 Cell Area (m2) 700 600 500 400 300 200 100 0 0.5 1 2 6 Time (hours) 12 48 Control RGD AP

Fig. 5. The average cell area compared across experimental groups and incubation times.

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magnification for each growth time, and images were input into a MATLAB program, where cell areas were calculated in terms of square pixels, and were then converted into square microns through calibration. Each image had 3060 cells in the field of view, cell areas were averaged. 2.6. Environmental scanning electron microscopy (ESEM) Images were obtained on a Philips XL30 field emission ESEM (Lehigh University, Bethlehem, PA) on control and RGD-coated surfaces after 48 h of incubation. Samples were prepared as per a procedure modified from that described in McKinlay et al. [16] and Cyster et al. [17]. Samples were washed with phosphate buffered saline (PBS) and then fixed for 2h with 2.5% gluteraldehyde while buffered at pH = 7.2 with 0.1 M Sodium Cacodylate (Electron Microscopy Sciences, Hatfield, PA). For image acquisition, the chamber was at a pressure of 2.5 Torr and the Peltier cooling stage was maintained at 0.2 C. 3. Results Significant differences were observed between the experimental groups at the 1 h incubation in the IF staining images (Fig. 4). Cells on the RGD-coated surface displayed increased cytoskeleton organization at the early stage of attachment. Cells on the control surface were circular in shape and the vinculin (green) was highly concentrated in the center. The RGD-coated surface showed cells at a seemingly later stage of attachment in which the actin (red) has significantly spread, and the vinculin has formed points of concentration on the perimeter of the cell. Cells on the AP-coated sample indicated decreased organization, in which the actin was spread, almost to the extent observed on the RGD-coated sample, but the vinculin was highly concentrated in the center, as seen in the control sample cells. The increase in cellular spreading observed was quantified; Fig. 5 shows the average cell area compared across experimental

Fig. 6. Images acquired on an ESEM at the 48h incubation time. (a) RGD2 days, (b) control2 days.

was necessary to perform shear assay trials only on surfaces in which cells were isolated, that is, not confluent or in contact with a neighboring cell. This was accomplished in the initial seeding concentration, ensuring that after the incubation time, the cells would not grow into one another. The accurate calculation of shear strength would be compromised, should a particular cell detachment be influenced by a neighboring cell. If a cell is detached while adhered to a neighboring cell, it will pull the other cell off with it, rendering erroneously low shear strength calculations. The trials were analyzed to determine the flow rate at which 50% of cells were sheared off, from which the average wall shear stress was calculated using Eq. (1). Cells typically detach at focal adhesion points; and from the time of the first point of detachment, within a few seconds, the entire cell is often detached. A further magnified sequence of an individual cell shows a typical process by which cells detach (Fig. 3). For data collecting, the lesser magnified view of 200 was chosen to accommodate larger data samples, that is, more cells in the field of view. 2.5. Measurement of cell spreading Average cell area was quantified for incubation times of 0.5, 1, 2, 6, 12, 24, and 48h. Digital images were captured at 200

Average Shearing Stress

120.00 Control 100.00 Average Shearing Stress (Pa) AP coated RGD coated 80.00

60.00

40.00

20.00

0.00 12 4 Incubation Time (hr) 48

Fig. 7. The average shear stress at which HOS cells detached from control Ti 6Al4V and RGD-coated surfaces at different incubation time.

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groups and incubation times. The RGD-coated surfaces showed increased cell area for all incubation times except at 48h, at which time the control surface converged with the RGD-coated in terms of cell area. Images acquired on an ESEM at the 48 h incubation time (Fig. 6) showed surface characteristics at a high resolution, but revealed little differences between experimental groups. Both surfaces show no evidence of filopodia, indicating the completion of the spreading process. Fig. 7 shows the average shear stress at which HOS cells detached from control Ti6Al4V and RGD-coated surfaces at the incubation time of 1, 24, and 48h. The RGD-coated surfaces indicated a nearly twofold increase in the interfacial strength over the control and AP samples at the incubation time of 1h. This is clearly much greater than the scatter in the experimental data, which are characterized by the error bars in Fig. 7. However, the RGD coated samples showed a smaller increase in the interfacial strength over the control and AP samples at 24 and 48h. Those results indicated that the effect on increased cellular adhesion that the RGD coated samples had was significantly reduced after about 2 days. 4. Discussion An effort was made to fully understand the processes of cellular attachment with an array of experiments ranging from adhesion strength quantification to IF staining and spectroscopy. The results obtained from the IF staining and cellular spreading results agreed with the study of Gawalt et al. [6]. In the cases of the novel aspects of the study, such as the shear assay experiment to measure cellular interfacial shear strength, the results were as expected and in accordance with biological understanding. The results from the shear assay experiment revealed shear stresses comparable with previous studies done on polystyrene and pure titanium-coated polystyrene [18]. A protocol was established to obtain images of a living cell on an ESEM, which indicated a resolution comparable with previous HOS cell imaging on titanium. In order to isolate the effects of the RGD peptide being tested, serum-free DMEM was used. Since cells cannot survive longer than 2h without serum, a 1h incubation time was selected for most of the experimentation, permitting the exclusive use of serum-free DMEM. In the cell spreading results and the ESEM images, where the incubation times exceeded 2 h, some variability in comparison exists in that additional proteins were available in the DMEM solution after that time, when the media change-out was performed. This is a possible explanation to the convergence of the control and RGD-coated samples at 48h, observed in the cell area versus time graph (Fig. 5). Adhesion factors such as the RGD sequence have been known to be instrumental in cellular adhesion and attachment [25]. Hence, with the ability to chemically bond RGD to a titanium surface with physiological stability [1] and the increased initial interfacial strength in the presence of RGD coatings observed in the current study, it is tempting to suggest that RGD coatings could reduce micro-motion, and hence reduce the fibrous

encapsulation that can occur during the early stages of implantation of biomedical devices. Also, the combination of increased initial bonding strength, as well as accelerated stages of cellular attachment make RGDcoated titanium attractive for a wide range of implantable devices that are currently fabricated from Ti and its alloys [19]. RGD coated Ti coatings can also be envisaged for the design of a wide range of bio-micro-electro-mechanical systems (bioMEMS) and microelectronic systems that are being explored for potential applications in the body [19]. In some cases, such coatings may be applied to micro-grooved geometries that promote contact guidance [20], and thereby reduce scar tissue formation. These are clearly the opportunities and challenges for future work. 5. Conclusions A nearly twofold increase in cell interfacial shear strength was observed for cells seeded on the RGD-coated versus control surfaces at the 1h incubation time. The entire process of cellular attachment was accelerated with the RGD coating in terms of cell spreading and internal organization of the cytoskeleton, as indicated in the results from the IF staining as well as the cell area quantification throughout the spreading process. The increased interfacial shear strength, the faster cell spreading and the improved cytoskeletal organization suggest that RGD coated Ti surfaces can be used to enhance the initial stages of cell/surface integration and wound healing. Acknowledgements The authors thank the National Science Foundation (Grant no. DMR-0231418), Dr. Carmen Huber, as well as the following individuals for making this work possible: Steve Mwenifumbo, Chris Milburn, Prof. Craig Arnold, Michael Carolus, and Prof. Jeffrey Schwartz. References
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