Artificial Extracellular Matrix

Cells normally only attach poorly onto glass, plastic or metal surfaces,¹⁹ which are common materials that are easy to engineer. To culture cells in vitro the substrates on which the cells should grow have to be coated.^20-22 Even if cells are grown on unmodified substrates, cells secrete proteins which are absorbed onto the substrate and binding of the cells is mediated by this protein layer, but these processes are generally very slow.²³

The simplest way to improve cell attachment to an artificial substrate is to subject polystyrene to plasma treatment,^24-25 so the polystyrene is oxidized.

Hydroxyl- and carboxyl-groups are introduced, thereby rendering the hydrophobic plastic more hydrophilic. The negative charges of the carboxyl groups have a similar effect as the acidic glycosaminoglycans of the ECM.

The negative charges bind sodium ions and thereby hydrate the surface by osmosis. Additionally, the adsorption of secreted proteins is improved on this more hydrophilic surface.²⁴

Still, this surface’s properties can be improved.19, 21-22, 24, 26 Fragmented proteins of the ECM, for example collagen, can be adsorbed onto the surface

and thereby facilitate cell adhesion and cell growth. Another approach is to use poly-lysine peptides.²⁷ These positively charged polymers interact electrostatically with the negatively charged cell surfaces and thereby mediate cell adhesion. These generic modifications are commonly used to culture many cell lines, for example HeLa or HEK cells. However, many cell lines require more sophisticated growth substrates. These simple substrates additionally lack any kind of selectivity. For tissue engineering purposes, more specific approaches have to be developed. Especially, in medical applications, for example for the transplantation of artificial heart valves or stents, selective cell adhesion to the artificial material is necessary to prevent side effects.²⁸ For these kind of application, the above mentioned solutions do not suffice.²⁸

1.2.1 Applications of ECMs

The nonphysiological character of “easy-to-manipulate” materials, like metals, plastic, glass and others, leads to problems,²⁶ when these materials are transplanted into the body. Inflammation, encapsulation of the artificial material and thrombogenesis are common side effects of artificial transplants.²⁹ However, during treatment of some severe malignancies, for example arteriosclerosis, or the transplantation of artificial heart valves, such artificial materials are necessarily introduced into the human body.^30-31 Especially, if these materials have direct contact to the blood, they can cause many severe side effects.²⁹

Atherosclerosis and related coronary heart diseases are the most predominant diseases in developed countries.³² Atherosclerosis is a disease characterized by the deposition of excess lipids in the arterial vessels.³³ This depositions lead to the recruitment of macrophages to clean the excess of extracellular lipids in the arterial intima, the innermost cell layer. The macrophages fill with lipid droplets, giving a foamy appearance under the microscope, these altered cells are called “foam cells”, which release cytokines, stimulating the proliferation of smooth muscle cells.^34-36 Altogether, these processes lead to the formation of atherosclerotic plaques which reduce the diameter of the affected vessel, thereby impeding the blood transport. Furthermore, these plaques can disrupt spontaneously and thereby completely block smaller vessels, for example the coronary heart vessels, inducing a heart stroke.

The affected vessels can be treated by opening the obstruction with a balloon, which is introduced into the vessel via a catheter and the balloon is inflated at the side of obstruction.³⁷ To prevent restenosis,³⁸ the reclosing of

the vessel via the same processes, stents are used. Bare metal stents suffer from low biocompatibility.³⁹ Bare metal surfaces are thrombogenic, leading to blood clotting and thereby reclose the stent. Even worse, these thrombi can be transported via the blood system to smaller vessels, which are then blocked completely. Furthermore, the presence of the artificial surface leads to an inflammation response.³⁸ Proinflammatory cytokines are released at the site and smooth muscle cells grow into the stent to encapsulate the artificial material, thereby again reducing the vessel diameter.

Different strategies are followed to minimize the side effects, for example drug eluting stents⁴⁰ are used, which release compounds, which for example abolish blood clotting at the stent, but have also adverse effect on endothelialisation. These stents are often made of bare metal with a polymer-coating. This polymer is loaded with the drug, which slowly is passively released to the surrounding.⁴¹

Besides the negative reactions of the blood on the artificial material, it is slowly covered by proteins of the ECM.²³ To this coating, epithelial cells can slowly attach. When the surface is completely covered with ECM-proteins, the side effects are diminished. To improve biocompatibility, this process has to be as fast as possible.

Fig 1.01: Concept of in vivo grafting. A biocompatible material is coated onto the metal surface of the transplant. This biocompatible material bears bait molecules that interact with surface markers of target cells (epithelial progenitor cells, EPCs). These cells adhere to the surface and attach to it. Finally, the EPCs differentiate into epithelial cells (ECs). Adapted from ref.²⁸

One interesting strategy is so called in vivo grafting of the stent (Fig.1.01).^28,

42 The metal surface is covered with a biocompatible material, which also bears molecules, that interact with a special, desired cell type. These cells should be recruited to the stent and grow on the artificial material. Target cells are for example epithelial progenitor cells (EPCs).^43-48 EPCs are adult stem cells, which can only differentiate into cells of the epithelial-lineage and

occur naturally in the blood. Furthermore, EPCs partake in repair of blood-vessel damage.^49-50

Stents coated with polyethylene glycol presenting an antibody against CD34, a protein present on EPCs²⁸ but also on many other cells, are transplanted.

Indeed, cells are attracted to the stent, but in addition to the wanted recruitment of EPCs, cells of the immune system are recruited which deteriorate the above mentioned side effects.²⁸ Currently, the unspecificity of the bait molecule (antibody against CD34) limits this approach. Another improvement of the coating material would be the tunability of the physical properties. As cells grow differently depending on the stiffness and other parameters, like charge and hydration state of the surrounding,²⁰ the coating would optimally be tuneable to achieve best growing conditions for different target cells.

A molecule, which possesses the property to be manipulated on the sub-nanometer scale is DNA, rendering it a promising material for the development of a new generation of artificial transplant-coating material.