**5. Interaction between plasma induced amino groups and cells**

In this section, an overview of literature on plasma treatments used for the incorporation of amine functionalities and their influence on cell-material interactions will be given. It is important to note that each precursor compound and each plasma treatment is unique and will yield different surface chemical and physical properties. Hence, for the same plasma media, a different plasma treatment will yield specific surface characteristics. And for the same plasma treatment, a different plasma media will also yield specific surface characteristics. Moreover, a specific type of cell reacts differently with specific surface properties.

Cell adhesion and proliferation on amine plasma polymer films deposited onto different substrates have been widely investigated. Different plasma methods have been used and different cells and biomolecules interactions with the treated surfaces have been investigated for various biomedical applications. In the following, only some of the main studied bio applications will be explored. An overview of the various cited works on cell-material interactions for different amine plasma treatments is given in table 1.

One important studied bio application is hemocompatibility of biomaterials used in blood contacting devices [74]. Hemocompatibility is considered to be one of the most critical aspects of biocompatibility. In order to achieve better hemocompatibility, a common approach is to immobilize heparin on the surface of the implant. Heparin is a strongly acidic, negatively charged polysaccharide (see figure 8) used in preventing thromboembolic complications due to its high affinity binding to antithrombin III (ATIII) resulting in its conformational change thus accelerating its ability to inactivate the coagulation enzymes [75, 76]. Metallic biomaterials used for vascular devices have excellent mechanical properties and corrosion resistance but have insufficient long-term hemocompatibility. Yang et al. [77] modified stainless steel using radio frequency plasma and a mixed gas of allylamine vapor and NH3. After plasma treatment, an FTIR and XPS study of the deposited film showed a good retention of the allylamine structure and thus the presence of primary amine functionalities on the surface. Nevertheless, the appearance of some new peaks in the spectra of the deposited film suggests that the primary amine groups are partially transformed into nitrile, amide or imine functional groups. As a consequence, the hydrophilicity was greatly improved. The water contact angle decreased from 70.8 to 62.7°. Hemocompatibility improvement was successfully achieved by heparin immobilization on the plasma polymerized allylamine. Cell culture tests were also conducted using endothelial cells which were found to adhere and proliferate in a better way on the plasma polymerized allylamine coating. Both improved hemocompatibility, adhesion and proliferation of endothelial cells were attributed to a combined effect of increased wettability and amine based surface chemistry. In another study [78], the improvement of hemocompat‐ ibility of polymeric vascular prosthesis such as polytetrafluoroethylene (PTFE), polystyrene and silicon was investigated. After depositing diamond-like carbon (DLC) films on the polymer substrates via acetylene plasma, functionalization was done using ammonia plasma. The NH3 plasma exposure time was varied from 0 to 300 s resulting in a different heparin coverage. For the different treatment times, heparin was successfully immobilized on the functionalized DLC leading to an extended blood coagulation time. The maximum of bound heparin was obtained at a 30 s treatment. Moreover, different heparin coverages were obtained for the different substrates. Based on these observations, the authors suggested that the initial surface roughness and the plasma treatment time i.e. the surface chemical structure influence the final heparin coverage.

**Figure 8.** Chemical structure of heparin molecule [95]

which influence to a great extent the selectivity of the fragmentation processes in the plasma. Therefore, depending on the kind of monomer used, various chemical compositions are obtained after each plasma treatment [18, 25, 55]. For example, Hamerli et al. [47] used ammonia and allylamine as plasma precursors. A higher amine concentration was found on the allylamine modified samples. Mangindaan et al. [60] used allylamine, propylamine and propargylamine (another unsaturated monomer) as precursors. XPS coupled to derivatization showed that allylamine incorporates the highest amount of amine functionalities into the corresponding thin films compared to those synthesized from the two other precursors.

34 Advances in Bioengineering

The precursor type also influences the growth mode and thickness of the deposited layers. Michelmore et al. [66] noticed that films grown from n-heptylamine initially show "islandlike" growth before a continuous smooth film is formed. In contrast, films from allylamine grow smoothly from the very earliest stages. Moreover, it has been found that monomers containing double bonds polymerize faster in plasma than their saturated counterparts. Gancarz et al. [25] have investigated the plasma polymerization of n-butylamine and allyla‐ mine and observed that the deposited layers are much thicker for allylamine plasmas. This

observation has also been confirmed in a study performed by Mangindaan et al. [60].

In this section, an overview of literature on plasma treatments used for the incorporation of amine functionalities and their influence on cell-material interactions will be given. It is important to note that each precursor compound and each plasma treatment is unique and will yield different surface chemical and physical properties. Hence, for the same plasma media, a different plasma treatment will yield specific surface characteristics. And for the same plasma treatment, a different plasma media will also yield specific surface characteristics.

Cell adhesion and proliferation on amine plasma polymer films deposited onto different substrates have been widely investigated. Different plasma methods have been used and different cells and biomolecules interactions with the treated surfaces have been investigated for various biomedical applications. In the following, only some of the main studied bio applications will be explored. An overview of the various cited works on cell-material

One important studied bio application is hemocompatibility of biomaterials used in blood contacting devices [74]. Hemocompatibility is considered to be one of the most critical aspects of biocompatibility. In order to achieve better hemocompatibility, a common approach is to immobilize heparin on the surface of the implant. Heparin is a strongly acidic, negatively charged polysaccharide (see figure 8) used in preventing thromboembolic complications due to its high affinity binding to antithrombin III (ATIII) resulting in its conformational change thus accelerating its ability to inactivate the coagulation enzymes [75, 76]. Metallic biomaterials used for vascular devices have excellent mechanical properties and corrosion resistance but have insufficient long-term hemocompatibility. Yang et al. [77] modified stainless steel using

**5. Interaction between plasma induced amino groups and cells**

Moreover, a specific type of cell reacts differently with specific surface properties.

interactions for different amine plasma treatments is given in table 1.

In another important studied bio application related to biomedical implant devices, cell adhesion is very important because it is considered to be the determinant of the success or failure of implantation. Anchorage-dependent cells such as fibroblasts and osteoblasts need the adhesion for cell growth, division and spreading [79]. It was found that attachment, proliferation and function of these anchorage-dependent cells are highly dependent on the surface properties of biomaterials [80]. The variation of biocompatibility after amine plasma treatment of many biomaterials has been studied by the means of fibroblast and osteoblast cell cultures. Wen-Juan et al. [71] and Ren et al. [81] modified silicon surfaces with allylamine dielectric barrier discharge and microwave plasma respectively. After plasma exposure, the contact angle decreased considerably due to the formation of various nitrogen functionalities as determined by FTIR. Cell culture tests with fibroblasts showed that both cell adhesion as well as cell proliferation could be improved by allylamine plasma treatment. Comparable results were obtained by Zelzer et al. [82] using glass substrates.

In a study by Hamerli et al. [43], the surface of polyethylene terephtalate (PET) was modified via allylamine microwave plasma polymerization. Plasma process parameters such as power (MW power), monomer flow rate (ØAllylamine) and duty cycle were varied which allowed the formation of different film chemical compositions. FTIR and XPS indicated that nitrogen as well as oxygen functionalities were incorporated which resulted in an increased hydrophilic‐ ity. Pictures from scanning electron microscopy showed that homogeneous pinhole-free allylamine plasma polymer (PPAa) films were obtained. Cell tests revealed improved cell attachment and spreading on PPAa coated PET compared to plain PET (see figure 9) with a greater improvement of biocompatibility on plasma polymerized allylamine coated surfaces containing amine functionalities in relatively high concentration. This is in agreement with other researches indicating that amino groups rather than others are favorable for protein adhesion [83, 84].

**Figure 9.** Photomicrographes of fibroblasts adhering on PET and PPAa membrane surfaces; (a,c) 4h of cultivation, (b,d) 24h of cultivation, (a,b) PET membrane, and (c,d) PPAa coated PET membrane "MW power of 1200W and ØAllylamine of 30 sccm" [96]

In another study by Hamerli et al. [47], PET membranes were modified by ammonia and allylamine microwave plasma treatments. Both plasma treatments yielded an approximately similar decrease in contact angle. Cell tests showed that fibroblast adhesion and spreading was improved for both plasma treatments compared to plain PET with higher proliferation on allylamine-modified samples in comparison to ammonia-plasma modified samples. This is mainly attributed to the higher amine concentration on allylamine modified PET.

contact angle decreased considerably due to the formation of various nitrogen functionalities as determined by FTIR. Cell culture tests with fibroblasts showed that both cell adhesion as well as cell proliferation could be improved by allylamine plasma treatment. Comparable

In a study by Hamerli et al. [43], the surface of polyethylene terephtalate (PET) was modified via allylamine microwave plasma polymerization. Plasma process parameters such as power (MW power), monomer flow rate (ØAllylamine) and duty cycle were varied which allowed the formation of different film chemical compositions. FTIR and XPS indicated that nitrogen as well as oxygen functionalities were incorporated which resulted in an increased hydrophilic‐ ity. Pictures from scanning electron microscopy showed that homogeneous pinhole-free allylamine plasma polymer (PPAa) films were obtained. Cell tests revealed improved cell attachment and spreading on PPAa coated PET compared to plain PET (see figure 9) with a greater improvement of biocompatibility on plasma polymerized allylamine coated surfaces containing amine functionalities in relatively high concentration. This is in agreement with other researches indicating that amino groups rather than others are favorable for protein

**Figure 9.** Photomicrographes of fibroblasts adhering on PET and PPAa membrane surfaces; (a,c) 4h of cultivation, (b,d) 24h of cultivation, (a,b) PET membrane, and (c,d) PPAa coated PET membrane "MW power of 1200W and ØAllylamine of

In another study by Hamerli et al. [47], PET membranes were modified by ammonia and allylamine microwave plasma treatments. Both plasma treatments yielded an approximately

results were obtained by Zelzer et al. [82] using glass substrates.

adhesion [83, 84].

36 Advances in Bioengineering

30 sccm" [96]

Other studies involving osteoblast cell tests were also conducted on amine plasma polymer‐ ized films resulting in an improved cell adhesion and proliferation [39, 59, 65].



**Table 1.** Representative overview of plasma processes for the incorporation of amine groups and their influence on cell-material interactions
