Nuclear Radiation Unit Department of Physics and Materials Science City University of Hong Kong Tat Chee Ave, Kowloon Tong, Hong Kong Email: apnru@cityu.edu.hk
		Page last modified on 7-Aug-2008

iointerfaces

Epithelial cell behavior on PADC films with etched alpha-particle tracks

Nuclear track materials as cell-culture substrates

Surface energy gradient surfaces

Epithelial cell behavior on PADC films with etched alpha-particle tracks

It is well established that pores introduce topographies onto the substrates, while substrate topographies will control the nature and degree of cell-cell and cell-matrix interactions and determine the morphology and functional induction of cultured cells in vivo. However, it would be sometimes difficult to separate the relative contribution of topography and porosity from pores. Track-etch pits commonly encountered in ion-irradiated and chemically etched solid-state nuclear track detectors (SSNTDs) provide unique opportunities to determine the topographical effects alone.

The objective of the present project was to explore the feasibility of using pits created on the surface of a polymer (polyallyldiglycol carbonate or PADC) by alpha-particle irradiation and subsequent chemical etching to study the substrate topographical effects on behaviors of cells (HeLa cervix cancer cells). The pits were purposely not etched-through, so that topographical effects alone (excluding porosity effects) can be studied. The average opening diameter and depth of the track-etch pits after etching in NaOH/H₂O with the additional etching by NaOH/ethanol for 5 min determined from their lateral images after polishing the edge of the film were 4.8±0.1 and 6.6±0.3 mm, respectively.

Fig. 1. Representative images showing the spatial expression of vinculin on HeLa cells on (a) raw and unetched PADC film; (b) etched blank PADC film; (c) PADC film irradiated by 3 MeV alpha particles for 3 h and etched; (d) PADC film irradiated by 3 MeV alpha particles for 6 h and etched. For (b), (c) and (d), etching was performed for 3 h in 6.25 N aqueous NaOH at 70 oC and then for 5 min in 1 N NaOH/ethanol at 40 oC. Bar = 20 mm; n = 3; (a) to (d) are at identical magnification. For (a) to (d), upper left: images from confocal microscope; upper right: transmission mode optical images; lower left: superposition of the upper left and upper right images.

            Fig. 1 above shows the expression of vinculin on HeLa cells cultured on the PADC films subjected to different treatments. Here, the images from confocal microscope, the transmission mode optical images as well as their superpositions are shown. We observe overall increases in vinculin expression for HeLa cells cultured on PADC films with track-etch pits. On the contrary, vinculin-rich regions were not revealed in cells cultured on the raw (unetched) as well as the blank (etched) PADC films. Fig. 2 below illustrates the expression of vinculin. Here, representative images from western blotting are shown, where the size of vinculin protein is about 117 kDa. The respective bands which represent the expression of vinculin from proteins sampled from HeLa cells cultured on PADC films with track-etch pits were stronger than those for cells cultured on the raw (unetched) as well as on the blank (etched) PADC films. The results for cells cultured on track-etch pits and those cultured on the raw detectors were found to be statistically significant (p<0.05).

Fig. 2. Vinculin detected by immunoblot presented as fold changes. Upper image lanes: 1, blank and unetched film; 2, 3h etched blank film; 3, etched film with 3 h irradiation; 4, etched film with 6 h irradiation. Right margin arrow, molecular weight standard. Below, identically loaded Coomassie-stained gel (loading controls). 3i3 and 3i6 showed statistically significant increases compared with raw (* p < 0.05, mean+SD, n=3).

Moreover, a closer look at the cells cultured on PADC films with track-etch pits revealed that the cells were largely contained by the track-etch pits (see Fig. 3). In other words, the cell membrane edges tended to be in contact with the pits. By comparing the correlation between the positions of HeLa epithelial cells and the track-etch pits on PADC films, and that between the positions of cells and computer-simulated pits, the tendency for membrane edges of HeLa epithelial cells to be in contact with the track-etch pits could indeed be recognized. A higher etch-pit density also resulted in a higher number of etch pits in contact with the cell membrane edges, but the percentage of these etch pits to the total number of etch pits remained more or less the same. Inhibition of membrane protrusion at the pores could explain this phenomenon. 

Fig. 3. An image (200´ in the transmission mode) showing the superposition of HeLa cells cultured for 3 d and track-etch pits generated from irradiation of 3 MeV alpha particles for 3 h, etched for 3 h in 6.25 N aqueous NaOH at 70 oC and then for 5 min in 1 N NaOH/ethanol at 40 oC.

Recent Publication:

Ng, C.K.M., Poon, W.L., Li, W.Y., Cheung, T., Cheng, S.H., Yu, K.N., "Study of substrate topographical effects on epithelial cell behavior using etched alpha-particle tracks on PADC films", 2008, Nuclear Instruments and Methods in Physics Research Journal (Section B), 266, 3247-3256.

Ng, C.K.M., Chan, K.F., Li, W.Y., Tse, A.K.W., Fong, W.F., Cheung, T., Yu, K.N., "Biocompatibility enhancement of chemically etched CR-39 SSNTDs through superficial pore formation by alpha-particle irradiation", 2008, Radiation Measurements, 43 (Suppl. 1), S537-S540.

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Nuclear track materials as cell-culture substrates

Alpha-particle radiobiological experiments involve irradiating cells or zebrafish embryos with alpha particles and require accurate positions where the alpha particles hit the cells. We have successfully developed procedures to prepare thin (< 20 μm) polyallyldiglycol carbonate films (CR-39 films). These thin CR-39 films have relatively small roughness as revealed by atomic force microscopy, and thus provide transparent detectors for radiobiological experiments. The colorless cellulose nitrate films (commercially available as LR 115 films) have also been proposed as cell-culture substrates for alpha-particle radiobiological experiments. Cytocompatibility of the substrate is a key factor to the success of such experiments. We have also investigated the cytocompatibility of surface-treated cellulose nitrate films by using plasma immersion ion implantation-deposition. Our tests showed that some plasma-treated films are more cytologically compatible.

Fig. 4. The image of a HeLa cell monolayer on a thin CR-39 SSNTD with revealed alpha-particle tracks under optical microscope with a magnification of 200x (taken from the side of the cell monolayer). The 5 MeV alpha-particle tracks appear as black dots, which are developed on the underside of the CR-39 SSNTD after chemical etching in (while floating on) a 14 N KOH solution at 37 °C for 4 h.

Recent Publications:

Chan, K.F., Tse, A.K.W., Fong, W.F., Yu, K.N., “Feasibility studies of colorless LR 115 SSNTD for alpha-particle radiobiological experiments”, 2006, Nuclear Instruments and Methods in Physics Research Journal (Section B), 247, 307-312.

Li, W.Y., Chan, K.F., Tse, A.K.W., Fong, W.F., Yu, K.N., “Studies of biocompatibility of chemically etched CR-39 SSNTDs in view of their applications in alpha-particle radiobiological experiments", 2006, Nuclear Instruments and Methods in Physics Research Journal (Section B), 248, 319-323.

Chan, K.F., Ho, J.P.Y., Li, W.Y., Lau, B.M.F., Tse, A.K.W., Fong, W.F., Bilek, M.M.M., McKenzie, D.R., Chu, P.K., Yu, K.N., "Investigation of cytocompatibility of surface-treated cellulose nitrate films by using plasma immersion ion implantation", 2007, Surface & Coatings Technology, 201, 6897-6900. 

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Surface energy gradient surfaces

Surface energy gradient surfaces with changes in the ratio between the polar component (g_s^p) to the dispersive component (g_s^d) of the surface energy g_s have been successfully fabricated by irradiating CR-39 solid-state nuclear track detectors (SSNTDs) with 3 MeV alpha-particles with different fluence followed by irradiation with ultraviolet photons with 257 nm. The alpha-particle source has an activity of 0.1 mCi and the irradiation time ranges from 1 to 7 d. The contact angles for doubly distilled water, glycerin and ethylene glycol, as well as g_s do not vary significantly with the alpha-particle irradiation. In contrast, g_s^p decreases steadily while g_s^d increases steadily, and the ratio g_s^p/g_s^d decreases significantly with the alpha-particle fluence. Such surface energy gradient surfaces are of particular interest for establishing the relationship between the g_s^p/g_s^d ratio and the biocompatibility.

Table 1. The surface energy (g_s), its polar component (g_s^p), its dispersive component (g_s^d) and the ratio (g_s^p/g_s^d) (all with the unit mJ/m²) determined for different regions on the CR-39 detector, namely, UVC only, and 1, 3, 5, 7 d of alpha irradiation + UVC. The values determined for the raw CR-39 detector (without alpha and without UVC irradiation) are also shown for comparison.

Fig. 5. A CR-39 detector with a size of 2 cm x 5 cm separated into five regions from top to bottom, each having a dimension of 2 cm x 1 cm. The top region was not irradiated with alpha particles, while the second to the fifth regions (from top to bottom) were irradiated with the same alpha-particle source and the same source to detector distance for 1, 3, 5 and 7 d, respectively. The images show the track densities in different regions after chemically etching in aqueous 6.25 N NaOH for 10 mins at 70oC. Different track densities are clearly noticeable in different regions.

Recent Publications:

Li, W.Y., Yu, K.N., "Surface energy gradient surfaces created by latent alpha-particle tracks in SSNTDs", 2008, Radiation Measurements, 43 (Suppl. 1), S558-S590.

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