Excimer laser texturing
CR-39 polymer sheets of laboratory grade are supplied from Sigma company. At first, CR39 samples (200 µm thick, 1cm × 1cm area) have been treated by high dose of UV laser. In the course of laser exposure, plenty of parallel regular micro channels are created on the surface. ArF excimer laser at 193nm benefits the following properties, i.e. 15 nsec duration and spatial distribution of ∼1 cm × 1 cm with transverse and vertical divergence of 1 and 3 mrad in orthogonal directions respectively 38–41. Fig.8 depicts the sequential steps of PC12 cell transformation and subsequent nerve outgrowth, according to the systematic experiments.
The optimal energy density is tuned by varying the output energy through a Ca lens (f=20 cm). The target is placed on a computer assisted translational mount such that the focusing area, power, and energy densities are well controlled according to the fine displacement of the target in 1 µm spatial resolution to adjust the proper clearance from the lens. A UV pulsed energy of 300 mJ is tuned during the series of experiments to irradiate the targets. The laser energy is kept constant during UV laser treatments. The samples are treated with 300 mJ/shot and 1000 laser shots delivering max energy dose up to 300 J/. Figure 9 illustrates the treatment arrangement using UV laser irradiation in order to create the regular self-pattern in the form of parallel microchannels on CR-39 foils. Fig 9. a depicts experimental set up for UV laser irradiation of CR39 target using ArF laser at 193nm in order to create plenty of parallel microchannels. Fig 9.b displays a typical induced grooves on CR-39 foil under UV laser dose of 300 J/ cm2 ,1000 shot, with 1 µm spacing and 0.3 µm groove width. After laser treatment, the CR39 morphology is inspected by means of SEM model XL30-Philips, with max 400,000 magnifications. The gold coating is essential using a polaran SC7620 sputter coater (Baltek, 80s). Furthermore, the hydrophilicity properties are examined according to the contact angle measurements using a camera from CAG-20, Jikan Co.38.
Note that the UV laser texturing of CR39 takes place at high UV doses i.e) large number of laser shots (up to 1000 shots) are applied leading to reduce the surface contact angle by (from initial angle down to final value of ), resulting in an enhanced hydrophilic surface38.
Hydrophilicity
The hydrophilicity tests of treated/untreated CR39 scaffolds are performed by measuring the corresponding contact angles. Accordingly, the contact angle of the fluid droplet on the solid surface has been measured. This determines the degree of hydrophilicity based on the sessile drop method. A drop of specified size (4 microliters) is placed on the sample and the contact angle is measured by means of a high-precision camera at the point where the droplet touches the surface and the subsequent images are taken using an image processing software Fig 9.b displays a typical UT CR-39 scaffold.
The excimer laser induces self-lining microstructures on some polymers such as PMMA and CR-39. As a consequence, the surface cross-linking takes place due to high dose UV exposure. The hydrophilicity refers to a physical skin property of a material that can transiently attach to water through hydrogen bonding. The latter is mainly due to the skin softening/hardening mechanisms during the laser-polymer interactions 38. In fact, UV laser creates a cross-linked surface improving the wettability alongside the formation of many microchannels. In this regard, the reduction of contact angle increases the hydrophilicity. Surface wettability elevates the protein uptake and cell adhesion subsequently. According to the previous articles, the cells are able to adhere much better to the hydrophilic surface 5,27,28. Simultaneously, the induced microchannels provide a suitable bed for oxygen and water diffusion providing better condition to feed the cells during the outgrowth. Furthermore, the parallel microchannels conduct the nerve axonal elongation to provide a suitable scaffold for further cell proliferation. Fig 9.c illustrates the contact angle of treated/ untreated samples.
PC12 cell culture/ assay
PC12 cells have been supplied from Pasteur Institute of Iran. In our series of experiments carried out in this university, we have not examined any human/ animal and only the cells of interest were supplied by the cell bank. Then they are cultured in T25 flask in RPMI medium containing 10% fetal bovine serum and 1% penicillin-streptomycin at 37 ° C in 5% . The culture medium is refreshed every 2 to 3 days and the cells are passaged when those reach ~ 80% confluent.
Several scaffolds with different size of parallel microchannels are initially examined. The PC12 cell proliferation on the treated CR39 scaffold is optimal with micro channels in submicron dimensions of 300nm width and 1 µm spacing. A similar result was previously reported, where PC12 outgrowth is investigated on a carbon surface with pores in few micrometer 28,29.
The treated/untreated scaffolds are immersed in 70% ethanol for 30 minutes. Then, those are sterilized for 2 hours under UV irradiation, and finally are washed 3 times with PBS containing 10% pen-strep in 30 minutes. Afterwards, the treated/untreated CR39 samples (1cm ×1cm) are placed at the bottom of a 24 well plate. Fig 9.d shows the scheme of 24 well plate 1.9 and ~1cm diameter. In the following, the nutrition liquid used to support cell viability medium (RPMI), are poured in all wells. Afterwards, the plate is placed in incubator for 24 hours. A certain initial member of 25000 cells is seeded in each well. The cell growth is pronounced for 1, 4 and 7 days of incubation following three MTT tests respectively. Some wells are labeled as control group occupied by cells but without the attendance of UT CR-39 scaffold. After cell culturing, the cell orientation on the scaffold is examined. Most of the cells are somehow aligned along the axis of parallel microchannels. Fig 9.e) represents the position of treated/untreated CR39 scaffolds inside each well.
The MTT assay is widely used to determine the cell viability. It is based on a colorimetric reaction of a yellow tetrazolium salt 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenylterazolium bromide to purple formazan crystals by metabolically active cells. The proliferation and cytotoxicity of PC12 cells are examined according to the MMT assay in three-time intervals. First, the UT-CR39 scaffolds+ cells are washed with PBS solution, then 450 μl of RPMI 1640 and 50 μl of MTT reagent are added on each scaffold and incubated for further 4 hours. In the next step, the previous solution is removed to dissolve the formazan crystals and 500 μl of DMSO solution is added to each sample. The resulting solution is transferred to a 96-well plate in order to read the optical density at 570 nm by making use of Eliza Reader. In fact, each test is repeated 3 times taking average and standard deviation for the statistical assessment and the subsequent p-values would be obtained.
The cells are provoked by laser at three wavelengths i.e,450nm, 660nm and 808nm under variant laser exposure times of 3 sec, 9 sec, 3 min, 6 min and 12 min to obtain the optimal wavelength and exposure time. After examining a number of options, we came to conclusion that the red diode laser gives out an optimal light after 12 min exposure to the cell filled wells. Thus, we have ruled out other options and irradiated the cell colonies by coherent photons at 660 nm. Note that this wavelength appears within the therapeutic window, suitable for the bio stimulation.
The cell adhesion is also inspected counting the number of cells attached to the scaffold. The numbers of cells are measured according the trypan blue staining test. After placing the cells on the scaffold, those usually attach to the scaffold in 12 hours. The medium is then removed and the cells on the scaffold are washed three times with PBS to collect the non-adherent cells. The number of cells linked to the scaffold is stained and counted with trypan blue in order to measure the live cells attached to the UT CR-39 plate.
According to MAP2 marker expression, the cell differentiation takes place in favor of neurons. The UT-CR39 scaffold and UT-CR39 scaffold+ laser demonstrate lucid cell proliferation against the control group. The regular 3D microchannels attest a pivotal role in the cell adhesion, proliferation and differentiation. The cell adhesion to the grooved scaffold is essential for initiating the cell proliferation/ differentiation 27,42. It is worth nothing that laser power and its wavelength notably effect on the cell outgrow, adhesion and differentiation too.