**2. Plasma generation**

#### **2.1. Plasma sources**

The success of an implant is determined by the response of its surrounding biological envi‐ ronment. This is governed to a large extent by the surface properties of the biomaterial where the interaction happens. Correspondingly, considerable efforts have been focused on surface engineering of biomaterials in order to give them the ability to promote cell adhesion, proliferation and to maintain cell functions. Therefore, a clear characterization of the physical and chemical properties of the biomaterial surface has a major scientific importance on cellbiomaterial interactions allowing the evaluation of the bioactivity of the surface engineered

Up to present, a large number of surface engineering techniques for improving biocompati‐ bility have been well established. The work generally contains three main steps: after the surface modification of the biomaterial is done, chemical and physical characterizations are conducted followed by a biocompatibility assessment through in vitro cell culture [5, 6].

In the past decades, surface treatment of biomaterials with plasma has been extensively studied [7-9]. Plasma modification of biomaterials gives the opportunity to change the surface characteristics to achieve better biocompatibility without altering the bulk properties. At the same time, plasma surface modification is a very versatile technology: the results of plasma modification can be easily controlled by choosing suitable gases or monomers and the

In order to introduce plasma it is often stated that plasma is the fourth state of matter in the sequence: solid, liquid, gas, and plasma. The state of matter changes from solid to liquid to gas to plasma by increasing the temperature of the material under consideration (see figure 1).

When gas atoms are subjected to energy (thermal, electrical or light) they become ions by releasing some of their electrons. Collisions between electrons and molecules and bond breaks in molecules create radicals. Energy will also create excited species that will generate photons. This is how plasma is created with a unique mixture of electrons, ions, radicals, photons and

biomaterial.

22 Advances in Bioengineering

**1.2. Plasma**

appropriate conditions in the plasma [10].

neutral atoms and molecules [11, 12].

**Figure 1.** States of matter [90]

As already mentioned, for surface modification treatments, various plasma sources are available. Each of them has its own characteristics such as density, temperature, chemical composition, etc., and leads to different results. The choice of the proper source for the specific task requires the study of the characteristics of the various plasmas. In this section, a brief summary of the most common plasma sources used for the incorporation of amine functional groups in tissue engineering will be given.

#### *2.1.1. Microwave (MW) plasma*

MW discharges are electrical discharges generated by electromagnetic waves with frequencies between 300 MHz and 10 GHz. MW discharges represent a simple way of plasma generation both with high (> 100 W/cm3 ) and low (< 1 W/cm3 ) power levels and can be used over a wide region of operating pressures (from 10-3 Pa up to atmospheric pressure). Nowadays, these discharges are widely used for generation of quasi-equilibrium and non-equilibrium plasmas for different applications because of the simplicity of control of the plasma internal structure by means of changes of the plasma characteristics and the possibility of plasma generation both in small and large chambers. The plasma absorbed power can be high enough and runs up to 90% of the incident power [14, 15].

#### *2.1.2. Radiofrequency (RF) plasma*

RF discharges usually operate in the frequency range f=1–100 MHz. The power coupling in RF discharges can be accomplished in different ways: capacitively coupled discharges and inductively coupled discharges.

**•** Capacitively coupled plasma (CCP)

CCP is generated with high-frequency RF electric fields, typically 13.56 MHz. In its simplest form, the RF voltage is applied across two parallel metal plates, generating an oscillating electric field between them. This field accelerates electrons leading to an ionization avalanche. The parallel electrodes which are separated by a distance of a few centimeters may be in contact with the discharge or insulated from it by a dielectric. Gas pressures are typically in the range 1–103 Pa. In a capacitively coupled RF discharge, the electron density is in the range ne=109 -1010 cm-3 and densities up to 1011 cm-3 are possible at higher frequencies.

**•** Inductively coupled plasma (ICP)

ICP is similar to CCP but the electrode consists of a coil wrapped around the discharge volume that inductively excites the plasma. ICP is excited by an electric field generated by a trans‐ former from an RF current in a conductor. The changing magnetic field of this conductor induces an electric field in which the plasma electrons are accelerated. ICPs can achieve high electron densities (ne=1012 cm-3) at low ion energies [15, 16].

#### *2.1.3. Dielectric Barrier Discharge (DBD)*

Dielectric barrier discharges (silent discharges) are non-equilibrium discharges that can be conveniently operated over a wide temperature and pressure range. DBDs are characterized by the presence of one or more insulating layers in the current path between metal electrodes in addition to the discharge space. At a sufficient AC voltage, electrical breakdown occurs in many independent thin current filaments. These short-lived microdischarges have properties of transient high pressure glow discharges with electron energies ideally suited for exciting or dissociating background gas atoms and molecules.

Due to charge build up on the dielectric, the field at the location of a microdischarge is reduced within a few nanoseconds after breakdown thus terminating the current flow at this location. The current density in a microdischarge channel can reach 100 to 1000 Acm-2. Due to the short duration, this normally results in very little transient gas heating in the remaining channel. The dielectric barrier limits the amount of charge and energy deposited in a microdischarge and distributes the microdischarges over the entire electrode surface. As long as the external voltage is rising, additional microdischarges will occur at new positions because the presence of residual charges on the dielectric has reduced the electric fields at positions where micro‐ discharges have already occurred. When the voltage is reversed, however, the next microdi‐ scharges will form in the old microdischarge locations.

**Figure 2.** Primary amine, hydroxyl and carboxyl functionalities

Its flexibility with respect to geometrical configuration, operating medium and operating parameters is unprecedented. Conditions optimized in laboratory experiments can easily be scaled up to large industrial installations.

Although DBD configurations can be operated between line frequency and microwave frequencies the typical operating range for most technical DBD applications lies between 500 Hz and 500 kHz [17].

Plasma can be used in the continuous wave (CW) or pulsed mode. In the continuous wave mode, plasma with a specific power is turned on for a specific amount of time. In the pulsed mode, plasma is intermittently generated with a fixed duty cycle (Δ).

$$
\Delta = \mathbf{t}\_{\rm on} / \left(\mathbf{t}\_{\rm on} + \mathbf{t}\_{\rm off}\right) \tag{1}
$$

where ton is the time during which the plasma is turned on and toff is the time during which the plasma is turned off. The mean power (Pmean) is then defined by equation (2) and represents the average energy dissipated in the plasma period, with Ppeak the power injected during ton[18].

$$\mathbf{P}\_{\text{mean}} = \Delta^\ast \mathbf{P}\_{\text{peak}} \tag{2}$$

#### **2.2. Plasma media**

both in small and large chambers. The plasma absorbed power can be high enough and runs

RF discharges usually operate in the frequency range f=1–100 MHz. The power coupling in RF discharges can be accomplished in different ways: capacitively coupled discharges and

CCP is generated with high-frequency RF electric fields, typically 13.56 MHz. In its simplest form, the RF voltage is applied across two parallel metal plates, generating an oscillating electric field between them. This field accelerates electrons leading to an ionization avalanche. The parallel electrodes which are separated by a distance of a few centimeters may be in contact with the discharge or insulated from it by a dielectric. Gas pressures are typically in the range

Pa. In a capacitively coupled RF discharge, the electron density is in the range

ICP is similar to CCP but the electrode consists of a coil wrapped around the discharge volume that inductively excites the plasma. ICP is excited by an electric field generated by a trans‐ former from an RF current in a conductor. The changing magnetic field of this conductor induces an electric field in which the plasma electrons are accelerated. ICPs can achieve high

Dielectric barrier discharges (silent discharges) are non-equilibrium discharges that can be conveniently operated over a wide temperature and pressure range. DBDs are characterized by the presence of one or more insulating layers in the current path between metal electrodes in addition to the discharge space. At a sufficient AC voltage, electrical breakdown occurs in many independent thin current filaments. These short-lived microdischarges have properties of transient high pressure glow discharges with electron energies ideally suited for exciting or

Due to charge build up on the dielectric, the field at the location of a microdischarge is reduced within a few nanoseconds after breakdown thus terminating the current flow at this location. The current density in a microdischarge channel can reach 100 to 1000 Acm-2. Due to the short duration, this normally results in very little transient gas heating in the remaining channel. The dielectric barrier limits the amount of charge and energy deposited in a microdischarge and distributes the microdischarges over the entire electrode surface. As long as the external voltage is rising, additional microdischarges will occur at new positions because the presence of residual charges on the dielectric has reduced the electric fields at positions where micro‐ discharges have already occurred. When the voltage is reversed, however, the next microdi‐


up to 90% of the incident power [14, 15].

*2.1.2. Radiofrequency (RF) plasma*

24 Advances in Bioengineering

inductively coupled discharges.

1–103

ne=109

**•** Capacitively coupled plasma (CCP)

**•** Inductively coupled plasma (ICP)

*2.1.3. Dielectric Barrier Discharge (DBD)*

electron densities (ne=1012 cm-3) at low ion energies [15, 16].

dissociating background gas atoms and molecules.

scharges will form in the old microdischarge locations.

Among other physical and chemical techniques to improve the surface biocompatibility [19-22], plasma surface modification is used to adapt the surface properties by functionaliza‐ tion of the material surface to control the biological response. Different functionalities have been investigated, such as carboxyl [23], hydroxyl [24] and primary amine [25] (see figure 2). In this chapter, we will focus on plasma surface modification of biomaterials by means of primary amine group incorporation.

Amino groups are usually incorporated on the surface using either non-polymer-forming gases (amine plasma activation) such as ammonia (NH3) or nitrogen (N2), or amine monomers (amine plasma polymerization) in the plasma medium. The former etches the biomaterial surface and introduces nitrogen functionalities; the latter deposits a plasma polymer layer containing nitrogen groups on the surface [26].

Plasma polymerization of amine-based monomers is an efficient way to prepare bioactive amino functionalized polymer surfaces. Amine-functionalized surfaces have previously been obtained through plasma polymerization using different monomers such as allylamine [27, 28], ethylenediamine [29-31], n-heptylamine [32, 33], propylamine [34, 35], cyclopropylamine [18], diaminocyclohexane [36, 37], and butylamine [26].

In the biomedical field, the most common used plasma media are ammonia as non-polymer forming precursor and allylamine as polymer forming precursor.

Primary amine (-NH2) functional groups can promote covalent immobilization with biomo‐ lecules such as protein like antibodies, collagen and DNA [28, 38]. Moreover, protonated amines can introduce a localized positive charge in aqueous solution at physiological pH value, which can potentially be used for electrostatic interactions with negatively charged cells and proteins and is propitious to promote cell adhesion and proliferation [39, 40].

In this chapter we will focus more on amine plasma polymerization since this is the most commonly used plasma technique for the incorporation of primary amines. Plasma polymer‐ ization has unique practical advantages which include (i) ultra-thin film deposition, (ii) good adhesion to the substrate material, and (iii) chemically stable and physically durable nature of the polymers [38].
