investigation of the effects of alkane phosphonic acid rgd coatings on cell

8/12/2019 Investigation of the Effects of Alkane Phosphonic Acid RGD Coatings on Cell

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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 80Pa 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 methodto

achieve increased cellular adhesion on biomaterials. A variety of

cell types has demonstrated increased focal adhesions on a varietyof surfaces functionalized with a biomimetic coating of the RGD

sequence[24]. Several sources suggest functionalizing biocom-

patible 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

(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 areether

functional groups linked to the surface of titanium. Most methodsfor 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 methodutilizing 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.

Materials Science and Engineering C 27 (2007) 8389

www.elsevier.com/locate/msec

Corresponding author. Tel.: +1 609 258 5609; fax: +1 609 258 5877.

E-mail address: [email protected](W.O. Soboyejo).

0928-4931/$ - see front matter 2006 Elsevier B.V. All rights reserved.doi:10.1016/j.msec.2006.02.005
mailto:[email protected]://dx.doi.org/10.1016/j.msec.2006.02.005http://dx.doi.org/10.1016/j.msec.2006.02.005http://dx.doi.org/10.1016/j.msec.2006.02.005mailto:[email protected]


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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 cellspreading studies with immunofluorescent (IF) staining for the

cytoskeletal proteins, actin and/or vinculin, allowing visualiza-

tion 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 theRGD 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 twee-

zers, 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 hasnot 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,

andw 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 1h of growth. Noprevious 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.

Table 1

Ti6Al4V polishing procedure

Roughness (grit) Time (min) RPM Approximate force

(Newton/sample)

300 1025 170 27

600 7 170 27

800 7 170 271200 6 170 22

Colloidal Silica

(0.05 m) a

On TEXMET cloth 6 140 18

a Done in two segments with rinsing and sonication.

Fig. 1. Control (left) and RGD-coated (right) 2020 m area. RMS surface roughness values of 5 and 7 nm, respectively.

84 R.A. Bly et al. / Materials Science and Engineering C 27 (2007) 8389


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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 JerseyPrecision 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 oxidelayer 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 inFig. 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

energy associated with the meniscus attracts a higher concentra-

tion 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 (215 min) and 18 Mdeionized water (120 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

(a)

10 m

(b)

Detachment

(c)

10 m

10 m

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

C-DGR

N

O

OO

TiO2Surface

OO

C

P

O

O

O

H

OO

C

OH

P

O

TiO2Surface

RGD-Coated AP-Coated

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

85R.A. Bly et al. / Materials Science and Engineering C 27 (2007) 8389


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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 and25L 1 M MgCl (Quality Biological, Gaithersburg, MD). The

cells were then fixed for 15min 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

15min (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

(=3.3410 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

Fig. 4. HOS cells stained for actin (red) and vinculin (green). (a) control1h, (b) RGD1h, (c) AP1h. (For interpretation of the references to colour in this figure

legend, the reader is referred to the web version of this article.)

Cell Area Compared vs Time

0

100

200

300

400

500

600

700

800

900

1000

0.5 1 2 6 12 48

Time (hours)

CellArea(m

2)

Control

RGD

AP

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



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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 wallshear 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

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 48h 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 experi-

mental groups at the 1h 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 organiza-tion, 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. 5shows 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.

Average Shearing Stress

0.00

20.00

40.00

60.00

80.00

100.00

120.00

12 4 48

Incubation Time (hr)

AverageShearingStress(Pa)

Control

AP coated

RGD coated

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 48h incubation time

(Fig. 6) showed surface characteristics at a high resolution, but

revealed little differences between experimental groups. Bothsurfaces 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 increasedcellular 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 spectrosco-

py. 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 biologicalunderstanding. 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 theincubation times exceeded 2h, 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 RGD-

coated 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 ofa wide range of bio-micro-electro-mechanical systems (bio-

MEMS) 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 controlsurfaces at the 1 h 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 (Grantno. 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.

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