**4. Examples of plasma-assisted surface modification of biodegradable polyesters**

In this last section, we will give some examples of the above-mentioned approaches of plasma-assisted surface modification of biodegradable aliphatic polyesters. Due to the introductory nature of this chapter, not all literature will be discussed in detail. For a more complete overview of literature, the reader could consult the review paper on the same subject of our research group (Morent et al., 2011). Plasma treatment as described in section 3.2.1 is by far the most occurring approach used to modify the surface of biodegradable polyesters and numerous examples can be found in literature. In this chapter, we will try to give some examples for all the biodegradable polyesters discussed in section 2. Therefore, section 4.1 will be much more extensive than the other sections (4.2 up to 4.4) since the availability of literature on these latter approaches is less pronounced.

### **4.1 Plasma treatment of biodegradable polymers**

Hirotsu et al. published in 1997 one of the first studies on plasma modification of biodegradable polymers and treated PLA fabrics with a low pressure radio frequent (RF) discharge generated in pure oxygen and nitrogen (Hirotsu et al., 1997). The same group reported in 2002 about an enhancement of the wettability of PLLA sheets and showed a strong decrease in water contact angle from 80° to approximately 55° after 30 seconds of oxygen and helium plasma treatment (Hirotsu et al., 2002). They suggested that this increased wettability was only due to chemical changes of the surface, since pronounced etching is not likely to happen after such short treatment times. However, they were not able to determine the groups incorporated at the surface.


Table 2. Atomic composition of untreated and air plasma-treated PLA films (De Geyter et al., 2010).

To identify the functionalities incorporated at the surface, De Geyter et al. did detailed XPS studies on PLA sheets plasma-treated with a medium pressure dielectric barrier discharge (DBD) sustained in air. Table 2 shows the atomic composition of the PLA films plasmatreated in air. This table suggests that air plasma mainly adds oxygen atoms to the PLA surfaces. From high-resolution XPS scans, the authors have concluded that after plasma treatment in air, the concentration of C-O and O-C=O groups increases, while the C-C and C-H functional groups decrease. Hirtosu et al. observed a gradual increase in water contact angle when PLA samples were kept in dry air (Hirotsu et al., 2002). A similar hydrophobic recovery has recently been examined in detail by Morent et al., who employed a medium pressure DBD in different atmospheres for the surface modification of PLA. They concluded that during storage in air, the induced polar chemical groups reorientate or migrate to the bulk of the material (Morent et al., 2010). The introduction of specific functional groups on the surface of PLA samples and the accompanying increase in wettability often have the aim to improve the cell-material interactions. These interactions between B65 nervous tissue cells and oxygen plasma-treated PLLA films were studied by Khorasani et al. (Khorasani et al., 2008). Figure 2 shows optical photomicrographs of B65 cell attachment and growth on untreated and plasma-modified PLLA surfaces and it can clearly be observed that this oxygen plasma treatment substantially improves cell attachment and growth. The authors concluded that plasma-modified PLLA surfaces are very suitable for nervous tissue engineering purposes.

The majority of the above-discussed research is on flat 2D PLLA surfaces. However, from biomedical point of view, 3D porous polymer scaffolds are needed in the field of tissue engineering in order to offer sufficient support for tissue growth (Djordjevic et al., 2008). Only few authors have worked with 3D structures because of two reasons: (1) the insufficient knowledge on the penetration of plasma into porous structures and (2) the difficulty of characterisation of the interior surface with classical surface analytical tools. Wan et al. have modified 4 mm thick PLLA scaffolds with an ammonia plasma (Wan et al., 2006). To examine the plasma effect, they have immersed the scaffolds in blue ink after treatment to demonstrate the influence of treatment time on the modifying depth. Figure 3 shows that due to the poor hydrophilicity of the internal surface of the untreated PLLA sample only the most outside layer is dyed. However, with increasing treatment time, the ink increasingly penetrates the PLLA scaffold and after a treatment of half an hour the interior part of the scaffold is fully dyed.

Fig. 2. B65 attachment on (a) untreated PLLA and (b) oxygen plasma-treated PLLA (magnification 400x) (Reprinted from (Khorasani et al., 2008) with permission of Elsevier).

treated in air. This table suggests that air plasma mainly adds oxygen atoms to the PLA surfaces. From high-resolution XPS scans, the authors have concluded that after plasma treatment in air, the concentration of C-O and O-C=O groups increases, while the C-C and C-H functional groups decrease. Hirtosu et al. observed a gradual increase in water contact angle when PLA samples were kept in dry air (Hirotsu et al., 2002). A similar hydrophobic recovery has recently been examined in detail by Morent et al., who employed a medium pressure DBD in different atmospheres for the surface modification of PLA. They concluded that during storage in air, the induced polar chemical groups reorientate or migrate to the bulk of the material (Morent et al., 2010). The introduction of specific functional groups on the surface of PLA samples and the accompanying increase in wettability often have the aim to improve the cell-material interactions. These interactions between B65 nervous tissue cells and oxygen plasma-treated PLLA films were studied by Khorasani et al. (Khorasani et al., 2008). Figure 2 shows optical photomicrographs of B65 cell attachment and growth on untreated and plasma-modified PLLA surfaces and it can clearly be observed that this oxygen plasma treatment substantially improves cell attachment and growth. The authors concluded that plasma-modified PLLA surfaces are very suitable for nervous tissue

The majority of the above-discussed research is on flat 2D PLLA surfaces. However, from biomedical point of view, 3D porous polymer scaffolds are needed in the field of tissue engineering in order to offer sufficient support for tissue growth (Djordjevic et al., 2008). Only few authors have worked with 3D structures because of two reasons: (1) the insufficient knowledge on the penetration of plasma into porous structures and (2) the difficulty of characterisation of the interior surface with classical surface analytical tools. Wan et al. have modified 4 mm thick PLLA scaffolds with an ammonia plasma (Wan et al., 2006). To examine the plasma effect, they have immersed the scaffolds in blue ink after treatment to demonstrate the influence of treatment time on the modifying depth. Figure 3 shows that due to the poor hydrophilicity of the internal surface of the untreated PLLA sample only the most outside layer is dyed. However, with increasing treatment time, the ink increasingly penetrates the PLLA scaffold and after a treatment of half an hour the

(a) (b)

Fig. 2. B65 attachment on (a) untreated PLLA and (b) oxygen plasma-treated PLLA (magnification 400x) (Reprinted from (Khorasani et al., 2008) with permission of Elsevier).

engineering purposes.

interior part of the scaffold is fully dyed.

Fig. 3. Effect of plasma treatment time on the modifying depth of PLLA scaffolds (Reprinted from (Wan et al., 2006) with permission of Elsevier).

To our knowledge, no literature on plasma treatment of polyglycolic acid has been published so far. However, quite a few research articles deal with plasma modification of the copolymer PLGA (Khorasani et al., 2008, Hasirci et al., 2010, Khang et al., 2002, Park et al., 2007, Park et al., 2010, Safinia et al., 2007, Safinia et al., 2008, Shen et al., 2008, Wang et al., 2004). 50/50 PLGA films were modified in an oxygen plasma at low pressure and a decrease of the contact angle from 67° to below 40° after plasma treatment was observed. XPS revealed that oxygen containing functionalities are introduced and cell culture tests (3T3 fibroblasts) showed a higher cell attachment and proliferation on oxygen plasma-treated PLGA surfaces. As discussed in the previous paragraph on PLLA treated surfaces, Khorasani et al. also investigated in the same paper the interaction between nervous tissue cells and plasma modified PLGA samples (Khorasani et al., 2008). Figure 4 shows that oxygen plasma treatment clearly improves attachment and growth of B65 cells, however, the effect of oxygen plasma treatment seems less pronounced as was the case for PLLA surfaces (see Figure 2). Khang et al. studied and compared several modification methods including chemical methods (sulphuric acid, chloric acid, sodium hydroxide) as well as physical methods (atmospheric pressure air discharge) for the surface treatment of PLGA (Khang et al., 2002). Their results clearly evidenced that both chemical methods and plasma treatment could enhance cell attachment and growth. The high potential of non-thermal plasma for the surface modification of biodegradable polymers was clearly demonstrated since plasma treatment showed to be almost as efficient in increasing cell-material interactions as a chloric acid treatment and more efficient than sulphuric acid and sodium hydroxide treatments.

Fig. 4. B65 cell attachment on (a) untreated PLGA and (b) oxygen plasma-treated PLGA (magnification 400x) (Reprinted from (Khorasani et al., 2008) with permission of Elsevier).

Surface modification of PCL with oxygen, helium and air plasmas has resulted into similar effects as on PLA and PLGA: an increased hydrophilicity, a higher oxygen amount and consequently an enhanced cell attachment and proliferation (Yildirim et al., 2008, Hirotsu et al., 2000a, Lee et al., 2009, Prabhakaran et al., 2008, Little et al., 2009). Lee and co-workers treated PCL with atmospheric pressure plasmas with different discharge gases (Lee et al., 2008). Figure 5 shows the enhancement in hydrophilicity (Figure 5 (a)), the increased cell attachment (Figure 5 (b)) and the increased cell proliferation (Figure 5 (c)).

Fig. 5. Plasma treatment of PCL: (a) contact angle (b) cell attachment and (c) cell proliferation of human epithelial cells (Reprinted from (Lee et al., 2008) with permission of Elsevier).

The most common polyhydroxyalkanoate subjected to plasma treatments is the co-polymer poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) and oxygen plasmas have been widely employed to modify this co-polymer (Wang et al., 2006, Hasirci et al., 2003, Tezcaner et al., 2003, Kose et al., 2003b, Kose et al., 2003a, Ferreira et al., 2009). A low pressure oxygen plasma was employed to PHBV films containing 8% hydroxyvalerate in its structure by Hasirci et al. (Hasirci et al., 2003). A decrease in water contact angle upon oxygen plasma treatment was observed which was attributed to the incorporation of oxygen-containing functional groups on the PHBV surface. A subsequent study showed that O2 plasma treatment significantly enhanced the interaction between retinal pigment epithelium (RPE) cells and PHBV (Tezcaner et al., 2003).

Hirotsu et al. published an interesting article on the plasma modification of self-made PBS sheets in different discharge atmospheres (O2, N2 and helium) (Hirotsu et al., 2000b). Contact angle measurements on the plasma-modified samples clearly showed that plasmas are able to increase the hydrophilicity, however, it was not stated which chemical groups contributed to this increased wettability.

### **4.2 Plasma post-irradiation grafting of biodegradable polyesters**

Section 4.1 clearly focussed on the observation that plasma treatment can easily induce desired functionalities onto the surface of biodegradable polymers resulting in an improved cell affinity. However, hydrophobic recovery acts as a brake on practical applications of plasma-treated polyesters. Nevertheless, this drawback can be solved by covalently immobilizing bioactive molecules on plasma-treated surfaces (Gupta et al., 2002). Typically extracellular matrix (ECM) proteins such as gelatine, collagen or fibrin have been grafted on the surface of biodegradable polyesters since these proteins are known to enhance cell adhesion and proliferation (Ma et al., 2007). Different authors have studied the immobilization of collagen on PCL films (Ma et al., 2007, Chong et al., 2007,

Surface modification of PCL with oxygen, helium and air plasmas has resulted into similar effects as on PLA and PLGA: an increased hydrophilicity, a higher oxygen amount and consequently an enhanced cell attachment and proliferation (Yildirim et al., 2008, Hirotsu et al., 2000a, Lee et al., 2009, Prabhakaran et al., 2008, Little et al., 2009). Lee and co-workers treated PCL with atmospheric pressure plasmas with different discharge gases (Lee et al., 2008). Figure 5 shows the enhancement in hydrophilicity (Figure 5 (a)), the increased cell

(a) (b) (c)

proliferation of human epithelial cells (Reprinted from (Lee et al., 2008) with permission of

The most common polyhydroxyalkanoate subjected to plasma treatments is the co-polymer poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) and oxygen plasmas have been widely employed to modify this co-polymer (Wang et al., 2006, Hasirci et al., 2003, Tezcaner et al., 2003, Kose et al., 2003b, Kose et al., 2003a, Ferreira et al., 2009). A low pressure oxygen plasma was employed to PHBV films containing 8% hydroxyvalerate in its structure by Hasirci et al. (Hasirci et al., 2003). A decrease in water contact angle upon oxygen plasma treatment was observed which was attributed to the incorporation of oxygen-containing functional groups on the PHBV surface. A subsequent study showed that O2 plasma treatment significantly enhanced the interaction between retinal pigment epithelium (RPE)

Hirotsu et al. published an interesting article on the plasma modification of self-made PBS sheets in different discharge atmospheres (O2, N2 and helium) (Hirotsu et al., 2000b). Contact angle measurements on the plasma-modified samples clearly showed that plasmas are able to increase the hydrophilicity, however, it was not stated which chemical groups

Section 4.1 clearly focussed on the observation that plasma treatment can easily induce desired functionalities onto the surface of biodegradable polymers resulting in an improved cell affinity. However, hydrophobic recovery acts as a brake on practical applications of plasma-treated polyesters. Nevertheless, this drawback can be solved by covalently immobilizing bioactive molecules on plasma-treated surfaces (Gupta et al., 2002). Typically extracellular matrix (ECM) proteins such as gelatine, collagen or fibrin have been grafted on the surface of biodegradable polyesters since these proteins are known to enhance cell adhesion and proliferation (Ma et al., 2007). Different authors have studied the immobilization of collagen on PCL films (Ma et al., 2007, Chong et al., 2007,

Fig. 5. Plasma treatment of PCL: (a) contact angle (b) cell attachment and (c) cell

Elsevier).

cells and PHBV (Tezcaner et al., 2003).

contributed to this increased wettability.

**4.2 Plasma post-irradiation grafting of biodegradable polyesters** 

attachment (Figure 5 (b)) and the increased cell proliferation (Figure 5 (c)).

Cheng & Teoh, 2004). Firstly, an argon plasma is applied to a PCL film to generate radicals on the polyester surface. Exposure to the atmosphere for several minutes leads to the formation of functionalities such as surface peroxides and hydroperoxides that will be employed as initiator sites for UV-induced graft polymerization of acrylic acid. To preactivate the carboxyl groups, the grafted substrates are immersed into a carbodiimide solution. In a final step, the material is immersed into a collagen solution leading to the production of a collagen-immobilized biodegradable polyester. These collagen-modified PCL surfaces have been tested with a diversity of cells including human dermal fibroblasts, human myoblasts, human endothelial cells and human smooth muscle cells and all demonstrated favourable response from these cells (Ma et al., 2007, Chong et al., 2007, Cheng & Teoh, 2004). Next to collagen, Kang et al. also immobilized insulin on the surface of a PHBV co-polymer and observed that the proliferation of human fibroblasts was significantly accelerated on these films compared to the untreated samples (Kang et al., 2001).
