Excimer laser microstructuring finds use in modifying cell adhesion.
Ralph Delmdahl, Coherent Inc.
Teflon’s nonstick properties make it ideal for more than pots and pans. The material, also known as poly-tetrafluoroethylene, is a good choice for use in direct contact with blood because its high bond energies render it biologically inert, which means it won’t cause a clotting reaction. Consequently, it is widely used for temporary and permanent medical implants and disposable devices, including vascular prostheses, tubes for nerve regeneration, subcutaneous augmentation materials, and for appliances related to oral and maxillofacial surgery.
But sometimes the body will require a little stickiness from Teflon devices. Regenerated tissue often needs to adhere to a target area on the implant, requiring that the surface property of the implant be modified. It can be coated locally with peptides containing a “sticky” amino acid sequence, with proteins such as fibronectin or other extracellular-matrix proteins, or with albumin. Another choice is depositing multiple-layer combinations of polysaccharides and proteins on the surface.
An alternative to these techniques is microstructuring.
It is well known that adhesion of cells to other implant materials, such as titanium, can be influenced directly by microstructuring the surface – creating periodic or random features on the same-size scale (microns) as features of key scar formation cell types such as fibroblasts.
However, the same high bond energies that make Teflon inert also make it a notoriously difficult substance to microstructure. It is available with a host of various-colored and colorless additives, and many of these variants can be laser-micromachined. But in addition to the basic issue of the additive, laser machining also transforms the surface chemically – through oxidation of the additive, for example.
So, until recently, a fundamental question has gone unanswered: “Can modifying surface topography alone affect how cells adhere to pure Teflon?”
Excimer laser microstructuring
Researchers in Jena, Germany, recently set out to find an answer. The key team members were Dr. J. Reichert and Prof. K.D. Jandt at Friedrich Schiller University, Institute of Materials Science and Technology, together with S. Brückner and Prof. H. Bartelt of the Institute of Photonic Technology Jena.
The team used the pulsed (~10 ns) output of a 157-nm fluorine excimer laser (Coherent LPFPro 220) to directly micromachine Teflon sample disks under several sets of conditions. This particular laser can deliver average output power at 200 Hz in excess of 8 W at 157 nm (See Figure 1) and 40 W at 193 nm, making it a popular choice for large-area micromachining of optically transparent polymers and glasses. The short wavelength of 157 nm was critical in this study because high photon energy of ~8 eV is required to directly break the strong bonds in the Teflon material by cold photoablation.
Figure 1. Shown is the typical output power range of the excimer laser (Coherent LPFPro 220) when operated at a 157-nm wavelength and a 200-Hz repetition rate.
The team then characterized the surfaces optically, topographically and chemically. Specifically, the investigators passed a laser beam through a mask containing a circular aperture and focused it onto the Teflon surface with a demagnification as high as 20×. The sample was translated onto an X-Y stage to create patterns both of linear and square crossed grooves. In this way, several 1 × 1-mm areas on each polished sample disk were laser-machined with high lateral and depth control.
By varying the laser fluence and X-Y motion, the researchers created pattern features of assorted sizes, characterizing the final patterns using an atomic force microscope.
Because 157 nm is strongly absorbed by oxygen in the air, the ablation was performed in a vacuum chamber under two conditions: at a pressure of 0.1 Pa, and under a nitrogen atmosphere at a pressure of 110 kPa. This was to determine whether the surrounding atmosphere causes a change in the chemical composition of the Teflon during the laser structuring process. The investigators carried out the assessment using x-ray photoelectron spectroscopy. Surprisingly, the vacuum-processed material showed a slight chemical transformation in the form of oxidation, presumed to be the result of outgassing of materials trapped in the polished substrates.
Testing cell adhesion
To test for cell adhesion, 3T3 Swiss albino mouse fibroblasts were cultured on the disks for 72 hours. The disks were gently washed with a phosphate-buffered saline to remove any nonadhering cells; then the disks were chemically treated to fix the remaining attached cells, which were stained with fluorophores targeted at various cell components, including actin fibers, critical to the function of fibroblasts.
Figure 2. A scanning electron microscope image is shown of a patterned Teflon sample with a grid size of 4 × 4 μm, an interval of 1 μm and a feature depth of 0.5 μm. Most of the adhered cells are confined to this patterned area, with very few on the polished area.
The treated samples were imaged using a scanning electron microscope and a confocal microscope equipped with laser excitation at 488-, 543- and 638-nm wavelengths.
Figure 2 is a typical scanning electron image showing preferential adhesion. Specifically, the middle of this image contains a square pattern with a grid size of 4 × 4 µm, an interval of 1 µm and a feature depth of 0.5 µm. Most of the adhered cells are confined to this patterned area; very few adhered to the polished area. The results are summarized in Figure 3.
Figure 3. “+” indicates preferential cell adhesion to the patterned area; “–” indicates no preferential adhesion.
These experiments show that (a) cells adhere preferentially to the textured surface, and (b) the adhesion is definitely a function of the size and spacing of the surface features. As the feature width increases from 1 μm, preferential cell adhesion is lost.
“We believe,” Reichert said, “these results provide potentially very important information for developers of medical implants and possibly for scientists investigating replacement tissue/micro-organ growth by culturing cells in the lab on supporting substrates and synthetic matrices.”
Meet the author
Ralph Delmdahl is the product marketing manager at Coherent GmbH in Göttingen, Germany; e-mail: firstname.lastname@example.org.