Endothelial cells form the inner lining of the blood vessel wall. They act as an interface between blood and the vessel wall. It is very crucial to maintain a healthy endothelium layer to avoid any kind of vascular dysfunction. Extensive studies have been carried out to understand the function and the behaviour of the endothelial cells. Recent studies have shown that the endothelial cells alter their morphology and phenotype in response to the blood flow. Endothelial cells demonstrated healthy elongated morphology under unidirectional smooth flow whereas under disturbed flow, endothelial cells exhibited thrombogenic cobblestone morphology (Uttayarat et al., 2008; Anderson and Hinds, 2011). In the last few years, micropatterning techniques have been utilised to alter the morphology of the endothelial cells under in vitro conditions. This technique involves the fabrication of the scaffold or the cell-substrate by creating definite microtopographical features on the surface that allows the cells to adhere and migrate in an aligned pattern (Thery, 2010). From literature, it is known that the surface topography determines the biocompatibility of a biomaterial. Surface topography and chemistry govern various important processes such as protein adsorption, cell adhesion and migration (Duncan et al., 2007). The micropatterning technique creates a tissue like environment within the in vitro culture. It has made it easier for the researchers to study the interaction between cells and the surface topography which acts as the microenvironmental cue. Apart from causing a change in the morphology of the endothelial cells, micropatterning technique can also modify other cell processes such as their adhesion, proliferation, and their function. This technique has been very useful in studying the interaction between the cells and their method of proliferation. The use of this technique in the development of tissue engineered constructs is still in its initial phase (Thery, 2010; Anderson and Hinds, 2011). At present, there are several micropatterning techniques that are being utilised to understand the morphology of the cells such as microcontact printing, photopatterning and laser micropatterning.
• Microcontact printing: This is one of the most commonly used micropatterning technique. It utilises cytophobic and cytophilic adsorbed coatings to direct the attachment of the cells on the substrate. Microcontact printing involves the use of polydimethylsiloxane (PDMS) with the desired surface features as a stamp. This patterned PDMS stamp is then pressed against scaffold or the cell substrate to transfer the pattern. The endothelial cells are then cultured on these patterned substrates. Even though this technique has been widely used, it has several drawbacks such as its inconsistency in the quality of the protein transfer and the requirement of an additional step of fabricating the stamp prior to the final printing step (Thery, 2010; Anderson and Hinds, 2011).
• Photopatterning: This method uses Ultraviolet (UV) light to fabricate the surface of the scaffold used for cell culture. UV irradiation causes the polymerization or the detachment of different molecules present on the surface of the substrate. In order to control the UV exposure spatially, a photo mask is placed on top of the substrate. This photo mask is also used to filter the UV light to prevent over exposure of the sample to UV light. This technique is often very flexible, reproducible and a large area of the substrate can be fabricated at a time. However, it also has some drawbacks such as the requirement of specialized equipment which are not available readily and the requirement of photosensitisers which are highly incompatible (Thery, 2010; Anderson and Hinds, 2011).
• Laser micropatterning: This technique has gained popularity in the recent years. It involves the use of UV light with a wavelength around 200nm from the laser source. One of the greatest advantages of this technique is that it does not require any photosensitisers and hence allows direct patterning of the biomaterials. The only disadvantage of using this technique is the requirement of expensive specialized equipment (Thery, 2010; Anderson and Hinds, 2011).
1.9.1 Use of laser micropatterning for medical applications
Laser micropatterning is one of the well established techniques used for surface fabrication. It has been used to fabricate different vascular grafts, stents and tissue engineering scaffolds. The use of this technique in medical research has gained momentum in the last few years due to its ease of handling, time efficiency and its ability to produce various micropatterns. Several investigations have been made to determine the role of laser micropatterning technique in understanding the cell- substrate relationship.
Li et al., used laser micropatterning to fabricate the thermanox film for their potential application in the development of small diameter vessels. Grooves of width 1.2 µm – 9.7 µm, groove depth and the ridge depth of 0.4 µm – 1.3 µm were created. Mouse fibroblast cell line L929 was grown on these laser modified surface.
They observed that the cells exhibited elongated morphology in narrow grooves compared to the cells in wider grooves that exhibited triangular morphology. They concluded that the microstructures on the surface of the film had an effect on the morphology and the orientation of the cells (Li et al., 2003). In another similar experiment, Rebollar et al., used KrF laser of 248 nm wavelength to fabricate the surface of polystyrene. The laser modified surface was analysed using Atomic force microscopy. They cultured Human embryonic kidney cells (HEK-293) on the modified as well as the unmodified polystyrene surface. They observed that cell adhesion on the micropatterned surface was significantly higher compared to the unmodified surface. Along with this, they also carried out an experiment on cell alignment by culturing Chinese Hamster Ovary cells (CHO-K1) as well as skeletal myoblast cells. They observed that the cells aligned themselves following the micropattern on the polystyrene surface. They concluded that the micropatterns on the cell substrate affect the adhesion, proliferation and alignment (Rebollar et al., 2008).
Mikulikova et al., carried out an experiment where they modified the surface of polytetrafluroethylene (PTFE) using a laser beam of the wavelength 172 nm. They cultured Human embryonic kidney cells (HEK) on these laser modified surfaces to study the behaviour of the cells on the micropatterned substrate. The cell proliferation was measured using the Neutral Red Assay. They observed that the cells adhered on the micropatterned surfaces and proliferated. They stated that the
increase in hydrophilicity of the laser modified surface resulted in an increase in the protein adsorption, hence lead to increased cell proliferation (Mikulikova et al., 2005). Yu et al., studied the interaction of the cells with a micropatterned surface.
They altered the surface of the polycarbonate substrate using the Laser ablation technique. They generated two different types of the micropatterns, line micropatterns and the point micropatterns. On these micropatterned surfaces they cultured Human pulmonary fibroblast cells. They made an interesting observation that cells cultured on the line micropatterns exhibited alignment following the micropatterns whereas the cells cultured on the point micropatterns had no definite orientation (Yu et al., 2005). Similar observation was made by Duncan et al., when they carried out an experiment where they modified the surface of PTFE disks using computer controlled cold laser KrF beam coupled with a microlithographic projection technique. They designed parallel microgrooves of 3- -30 µm deep, 1-10 µm wide and 3-30 µm apart. They cultured Human umbilical vein endothelial cells (HUVEC) on these laser modified PTFE surfaces to determine their potential for applications such as biosensors, tissue engineering scaffolds and vascular grafts. They observed that the cells aligned themselves based on the microgrooves on the modified surface (Duncan et al., 2007).
Isenberg et al., used micropatterned polystyrene substrates to generate cell sheets with definite extracellular matrix and cellular organization. They cultured vascular smooth muscle cells on the micropatterned substrate to generate intact cell sheets.
They observed that the cells demonstrated strong alignment based on the micropattern and successfully generated cell sheets (Isenberg et al., 2008).
Laser micropatterning has thus proven to be a valuable technique for various issues associated with the conventional dosage system is a requirement of higher dosage of drug to elicit a pharmacological response causing a number of side effects (Pouton and Akhtar, 1996).