Quantifying Inter-Cellular Forces in Bioadhesion:

Examination of Sialyl Lewis X and Selectin Interactions with Atomic Force Microscopy

Introduction

Engineering biointerfaces requires an understanding of the interactions between cells and materials and the changes that occur to both. For materials placed in a biological environment, the adhesion between the material surface and cells is a critical performance factor. Whether the application is an appliqué on a marine hull to prevent hard and soft fouling or a non-thrombogenic coating on an artificial vascular graft to prevent clotting, many of the cellular adhesion mechanisms are the same. When a cell approaches a surface the immediate environment is on the order of 100 microns and characterization of the surface energetics and cellular adhesion requires probing at these same dimensions. The atomic force microscope (AFM) provides the means to do this through a combination of tip functionalities and imaging modalities[1-8]. Elasticity, adhesion and topography may be examined by AFM in order to determine mechanical, chemical and physical properties respectively. Relevant to biointerfaces is the development of an AFM based biochemical probe to determine adhesion bond strength and interaction distance[9-11]. In order to establish the AFM biochemical probe as a means to measure cellular adhesion to surfaces, a model cell system was selected. Leukocyte (white blood cell) rolling on endothelial cells in the human vasculature, at sites of inflammation, was a physiologically relevant example to demonstrate the application of AFM (illustrated in Figure 1). In this example the low strength and rapid interaction between sLeX and selectins on the cell surface was investigated. While this interaction has been investigated for selectins grafted to a glass substrate, the experiments performed here were unique by measuring the interaction on living endothelial cells[10]. Porcine vascular endothelial cells (PVECs) were cultured on biopolymer surfaces. Sialyl Lewis X (sLeX) functionalized AFM tips were used to probe the strength, density and distance of specific ligand bondings to P-Selectin on the membrane of the PVECs as a function of the underlying polymer substrate. By quantifying the adhesion of cells on a surface at high spatial and force resolution, one can fine-tune biomaterials for anti-fouling and foul release application. Thus, it becomes possible to monitor and control cellular adhesion on the nanometer scale.

Materials and Methods

The biomaterials studied were polydimethylsiloxane (PDMS) elastomers with varying elastic moduli of 0.5, 1 and 3 MPa. The specific siloxane elastomer used was Silastic T-2 (Dow Corning) with varied crosslink densities; increased by adding a linear vinyl terminated PDMS oil and decreased by adding a bulky oil vinyl tris(trimethylsiloxy)-silane (Gelest Inc.). Samples of the unmodified Silastic T-2 formulation were also subjected to an Argon plasma treatment after cure to increase the hydrophilicity of the surface.

The model cell system studied was porcine vascular endothelial cells (PVECs) harvested from the pulmonary artery. PVECs were obtained from Dr. Edward R. Block's group at the Malcolm Randall VAMC. The cells were cultured on the PDMS elastomer substrates in 10% FBS supplemented RPMI media at 37C and 5% CO₂ for a period of 2 to 5 days. Samples were immediately transferred to the AFM for observation in HBSS or fixed in formalin and stained with crystal violet for light microscopy.

Measurements were performed on a Dimension 3100 atomic force microscope with a Nanoscope IIIa controller (Digital Instruments, Veeco Metrology Group). The AFM was equipped with a fluid tip holder for use in liquid. AFM cantilevers with a spring constant of 0.06 N/m were used (Model DNP, Digital Instruments, Veeco Metrology Group). For specific force measurements, the AFM probes were modified by attaching 3-aminopropyl triethoxysilane (APTES) coated silica microspheres, ∼5.9 μm in diameter, to the end of the cantilever using epoxy. Glutaraldehyde was reacted to the tip in order to bind bovine serum albumin linked sLeX. The experiments were conducted in HBSS at ambient temperatures of ∼22°C in the same 35 mm petri dish cells were cultured in. The AFM was operated in contact mode in HBSS at ambient temperatures to determine the topography of the PDMS elastomer before and after culture with PVECs, see figure 1 for a schematic. Elastic modulus, of both the PDMS elastomer and PVECs, was recorded in the force volume mode and analyzed using a modified Hertz model developed by Radmacher et al[7,12]. Topographic and force data were analyzed using Nanoscope v5r12 software.

Results & Discussion

Topographical images of 2500 mm² areas for the PDMS cast against the silicon wafers showed very smooth surfaces with an RMS roughness of ∼5 nm. Figure 2 shows the topography, force curves and PVEC morphology and Table 1 lists the elastic modulus for the biopolymers used in this study as measured by AFM to a depth of ∼20 nm. AFM elasticity measurements found elastic modulus for the PDMS formulations to be very similar at the surface varying from 1 to 2 MPa, and a force curve indicative of a soft surface. In contrast, the plasma treate PDMS elastomer had a higher elastic modulus of 2.96 ± 0.84 MPa with a force curve indicative of a hard sample. An attractive force was seen during tip approach to PDMS samples believed to be caused by PDMS oligomer oils not tied into the elastomer network coating the polymer surface and the tip. This attraction was not seen in the plasma treated surfaces.

Material	Bulk Elastic Modulus [MPa]	Elastic Modulus [MPa]	Adhesion Force [nN]
Polystyrene Culture Dish	∼3000	4.42 ± 1.39	-47.3 ± 23.4
PDMS Low Modulus	1.02 ± 0.05	2.30 ± 0.86	-82.1 ± 19.9
PDMS Medium Modulus	1.26 ± 0.35	1.52 ± 0.79	-74.3 ± 17.2
PDMS High Modulus	2.34 ± 0.49	1.26 ± 0.57	-98.2 ± 72.2
PDMS Plasma Treated	1.26 ± 0.35	2.96 ± 0.84	-34.3 ± 16.8

Material	Maximum Force [nN]	Jump-On Deflection [nm]
Polystyrene Culture Dish	9.6 ± 2.1	2.5±2.0
PDMS Low Modulus	9.9 ± 2.1	96.6 ± 26.0
PDMS Medium Modulus	15.9 ± 4.3	42.2 ± 11.1
PDMS High Modulus	15.2 ±3.8	96.0 ± 49.1
PDMS Plasma Treated	13.7 ± 4.1	N/A

Table 1 - Elastic modulus and adhesion of the PS and PDMS elastomer formulation evaluated with AFM.

Analysis of AFM force curves demonstrated specific interactions between the sLeX functionalized tip and the endothelial cells. As seen in figure 4, there is primary interaction at ∼15 nm separation followed by a secondary interaction at ∼100 nm separation. It is believed that the primary interaction is a nonspecific interaction due to uncoiling of the macromolecules and that the secondary interaction is due to breaking of th specific chemical bond. Table 2 lists the measured forces for the different biochemical tip/PVEC interactions. AFM measurements of the bonding between the sLeX functionalized tip and the PVECs indicated a specific interaction of 460 ± 80 pN/mm at a distance of 106.7 ± 26 nm for PVECs on PDMS at ambient temperatures. This was independent of the nonspecific interaction determined to be 210 ± 40 pn/mm at a distance of 14.2 ± 3.1 nm from the cell surface. A closer examination of the non-contact region of the force curve in figure 4 shows a separation of the extend and retract curves at >200 nm separation. This difference is currently being investigated; however, it may be due to cellular material adhering to the tip or possibly a hydrodynamic drag effect due to the liquid environment.

Microsphere Type	Glutaraldehyde	SLeX in 7M Urea
Substrate	PVECs	PVECs
Distance (nm)	16.2 ± 2.9	21.3 ± 6.1
Force (nN/μm)	-0.27 ± 0.06	-0.28± 0.07

Microsphere Type	SLeX-BSA, 1st interaction	SLeX-BSA, 2nd interaction
Substrate	PVECs	PVECs
Distance (nm)	14.2 ± 3.1	106.7 ± 26.0
Force (nN/μm)	-0.21 ± 0.04	-0.46± 0.08

Table 2 - Force and distance interactions between sLeX modified microspheres and PVECs.

Contact AFM of PVECs on the biopolymers was done in order to determine the morphology of the cells. By examining the cellular topography, it is hoped that the specific chemical bonding can be spatially correlated with cellular membrane structures. The defection scans in figure 5 are an example of an AFM images of living cells. Many structures including the nucleus and stress fibers can be seen. Scans of smaller areas are also capable of resolving what are believed to be cytoplasmic organelles. Figure 5 clearly shows two of these objects within a PVEC. Research is currently ongoing to see if these structures are correlated with specific membrane bound proteins.

Conclusions

These experiments have shown the applicability of AFM to investigate inter-cellular forces and the celluluar environment. The AFM was used in contact mode in a HBSS to simulate physiologic fluids and was able to accurately measure the topography and mechanical properties of the biomaterial and cells. To measure inter-cellular force the AFM tip was biochemically modified with sLeX to measure the specific interaction with selectins on the surface of endothelial cells. During separation, these measurements showed both an initial interaction believed to be due to non-specific bonding and chain unraveling and a secondary interaction believed to be due to the specific bonding. Future studies are planned to investigate the strength, distance and density of the specific interactions as a function of the biomaterial on which the cells are grown. In this way it will be possible to use the AFM to determine the strength of specific inter-cellular interactions, the spatial location of these interactions and the environmental factors that affect them.

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Acknowledgements

The authors acknowledge the financial support of the Office of Naval Research to fund this research. We thank the research group of Dr. Edward R. Block for supplying endothelial cells, and Dr. C. Keith Ozaki and Mr. Zaher Abouhamze for technical assistance and advice.