**3.1. Wound dressing**

*2.3.1. Plasma cleaning and etching*

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*2.3.2. Plasma activation*

implant material [11, 36, 37].

preservation of even the most delicate structures.

*2.3.3. Plasma grafting and polymerization*

During the production process and storage of (bio)materials, they can be exposed to a number of solvents, greases, volatiles components etc. These contaminants will adsorb and accumulate on the material surface over time, resulting in an altered, non-reproducible surface with a likely reduced product performance. A typical example in the biomedical field, is the adsorption of low molecular weight carbon species onto a pristine titanium sample, when exposed to ambient air. When used as an implant material, this surface pollution results in a reduced cell adhesion, proliferation and growth and in some cases even results in cell death [26, 27].

Any volatile surface contamination that is exposed to a non-thermal plasma, will be removed in a few seconds [28]. Prolonged exposure to the plasma will not only result in the removal of the adsorbed contamination but will cause etching of the top layers of the material surface [29-32]. Depending on the density and hardness of the exposed material, more intense discharges and/or extended exposure are required to obtain a notable effect. As (biomedical) textiles are in most cases build out of relatively soft materials, the etching effect cannot be overseen and will introduce a certain nano-roughness on the fiber surface. For in-vitro and invivo applications this change in surface topography can have a benign effect, as it can amplify

Plasma activation or plasma treatment is the exposure of a surface to the reactive particles present in the plasma. This mixture of reactive particles will result in the incorporation of radical sites on the surface, up to the depth of a few 10 nm. Depending on the gas used to maintain the plasma, these sites will react (in)directly with other radicals present, recombining into a broad variety of functional groups. These new functional groups have a high impact on surface properties such as wettability and surface free energy, which in turn might have a

In most cases an increase in hydrophilicity is pursued to enhance the materials histological performance. For some applications such as the surface of heart valves, the insides of needles and tubes or artificial stents, any adhesion of cells and proteins is highly unwanted, as it can lead to blockages resulting in premature failure of the biomedical device. Instead of using typical gas feeds for plasma treatment (noble gasses, oxygen, dry air, nitrogen...), fluorinated gasses such as CF4 are used which result in the formation of super hydrophobic surfaces with water contact angles of 150° and higher. These fluorinated surfaces prevent cells and proteins from effectively adhering on the surface and thus guaranteeing an optimal performance of the

Plasma activation is definitely not the only technique available for the introduction of new functional groups onto a surface, but as it is non-invasive and chemical-free, it guarantees the

Non-thermal plasmas are not only applied for plasma treatments, but can also be used as an initiation medium for radical polymerization, resulting in the deposition of a wide variety of

the other effects plasma has on cell adhesion and proliferation [33-35].

positive effect on material-material and material-cell interactions.

Optimal modern wound dressings should assure a moisture wound bed, help drainage, remove debris of the wound surface, provide optimal thermal stability, might be removed without trauma of the wound bed and wound edge, be antiallergenic and without immuno‐ genicity [38]. Over the years wound dressings have experienced a continuous development stimulated by a better understanding of wound healing and bacterial growth mechanisms. In more recent years research has shifted to targeted therapy by including different pharmaceut‐ ical compounds (e.g., antiseptics, analgetics, or growth factors) in to wound dressings. The continuous presence, or controlled release of active substances, can tremendously stimulate the healing process.

Non-thermal plasma technology has been part of this development process in many different aspects of wound healing treatments. The review of the literature dealing with the use of plasma technology for the enhancement of wound dressings will be divided according to the purpose of the treatment: wound monitoring, enhancement antimicrobial properties, inter‐ mediate bonding, and adhesion.

#### *3.1.1. Antimicrobial properties*

Successful wound treatment cannot be achieved without keeping its two major aspects in mind: maintaining a decent hydrophilicity and a high antimicrobial efficiency.

In two different papers Persin et al. compared a few different treatment methods on viscose fibers to address both of these aspects [39, 40]. In a first reaction pathway, a two-step process consisting of an oxygen plasma treatment, followed by the immobilization of AgCl particles, is followed. The alternative pathway consists of a single-step treatment of the cellulose fibers with an ammonia plasma. The single-step treatment resulted in a 30-fold increase in water uptake while the immobilization of the silver particles only had a marginal effect. For the antimicrobial properties of the wound dressings, the reverse trend was found. The single-step treatment only had an effect on Gram-positive bacteria, while the steady release of silver ions resulted in a quasi-complete destruction of both Gram-positive and Gram-negative bacteria. The authors therefore suggest to use a different treatment for different wound-healing applications, depending on the risk of infection. In a third paper from the same authors, a closer look is taken on the ageing of the plasma treated surfaces and the effect on the water up-take [35]. The study showed that 4 days after the treatment, the water contact angle increased with 15° and the oxygen concentration decreased with less than 1%. These numbers suggest that there is a limited shelf-lifetime of the modified wound dressings. Hacker et al. followed an alternative strategy for the immobilization of Ag particles by plasma polymerizing PEG onto electrospun PU mats, followed by soaking in AgNO3 and UV treatment, resulting in the incorporation of metallic silver [41]. Water-uptake tests revealed a significant increase of hydrosorption after 24 hours. The steady release of silver particles had a detrimental effect on the viability of both Gram-positive and –negative bacteria while at the same time no cytotoxic effects were noted on the adhesion and proliferation of fibroblasts. Further on in the chapter, other strategies will be discussed to obtain antibacterial properties for other applications, but it is already clear that the incorporation of silver particles with the help of plasma technology is a successful pathway for the improvement of wound dressings.

**Figure 1.** SEM micrographs (3000×) of the (a) plasma modified nonwoven, (b) AAc coated nonwoven, (c) PP-g-collagen nonwoven, and (d) PP-g-collagen-g-PNIPAAm nonwoven [126].

### *3.1.2. Adhesion and intermediate bonding*

In two different papers Persin et al. compared a few different treatment methods on viscose fibers to address both of these aspects [39, 40]. In a first reaction pathway, a two-step process consisting of an oxygen plasma treatment, followed by the immobilization of AgCl particles, is followed. The alternative pathway consists of a single-step treatment of the cellulose fibers with an ammonia plasma. The single-step treatment resulted in a 30-fold increase in water uptake while the immobilization of the silver particles only had a marginal effect. For the antimicrobial properties of the wound dressings, the reverse trend was found. The single-step treatment only had an effect on Gram-positive bacteria, while the steady release of silver ions resulted in a quasi-complete destruction of both Gram-positive and Gram-negative bacteria. The authors therefore suggest to use a different treatment for different wound-healing applications, depending on the risk of infection. In a third paper from the same authors, a closer look is taken on the ageing of the plasma treated surfaces and the effect on the water up-take [35]. The study showed that 4 days after the treatment, the water contact angle increased with 15° and the oxygen concentration decreased with less than 1%. These numbers suggest that there is a limited shelf-lifetime of the modified wound dressings. Hacker et al. followed an alternative strategy for the immobilization of Ag particles by plasma polymerizing PEG onto electrospun PU mats, followed by soaking in AgNO3 and UV treatment, resulting in the incorporation of metallic silver [41]. Water-uptake tests revealed a significant increase of hydrosorption after 24 hours. The steady release of silver particles had a detrimental effect on the viability of both Gram-positive and –negative bacteria while at the same time no cytotoxic effects were noted on the adhesion and proliferation of fibroblasts. Further on in the chapter, other strategies will be discussed to obtain antibacterial properties for other applications, but it is already clear that the incorporation of silver particles with the help of plasma technology

124 Advances in Bioengineering

is a successful pathway for the improvement of wound dressings.

nonwoven, and (d) PP-g-collagen-g-PNIPAAm nonwoven [126].

**Figure 1.** SEM micrographs (3000×) of the (a) plasma modified nonwoven, (b) AAc coated nonwoven, (c) PP-g-collagen

Next to increasing the hydrophilicity of surfaces, plasma are also known to promote the adhesion between layers. Gajanan et al. published two papers where plasma technology was used to improve the adhesion between an electrospun material and a woven support structure [42, 43]. In the first article chitosan was used as a raw material for the electrospinning process. In the second article, the chitosan was mixed with silk fibroin. For both cases a 100% cotton gauze was used as support material. Out of the different DBD plasma treatments, using He +1% O, the combination of a pre-and post-treatment gave the best results. Flex durability tests showed that there was a 4-fold increase in adhesion between the electrospun material and the cotton gauze. On top of that SEM images showed that the plasma treatment resulted in a reduction of the fibers' delamination after repetitive flexing.

As explained earlier in section 2.3, plasma can be used to graft new side chains on polymer substrates. The goal of grafting can either be to introduce new functional groups and use their properties as such, or to introduce them as intermediates that can be used in consecutive reactions. In biomedical surface engineering the latter is quite popular as the list of products that can be used is limited, meaning that in most cases there is a sub-optimal affinity between coating and substrate. Chen et al. used a PP-non-woven as a substrate on which acrylic acid was grafted. The newly introduced carboxylic acid functionalities were used to either cova‐ lently bond collagen or chitosan, on which the thermo sensitive polymer PNiPAAm was immobilized of which each step is depicted in figure 1. Above 32° C, the wound dressing becomes hydrophobic and releases the stored moisture, which in turn resulted in an enhanced wound healing [44, 45]. Lin et al. used an oxygen low pressure plasma to activate a PE nonwoven substrate, followed by the grafting of N-isopropyl acrylamide [46]. This intermediate was then used for the covalent bonding of bovine gelatin. In-vivo tests revealed that the covered wound healed completely (reached the maturation phase) and much faster compared to the PE control.

The results discussed above show that plasma technology can be a valid, solvent-free alterna‐ tive for the permanent fixation of technically advanced layers onto standard substrates. Most of the studies on adhesion and intermediate bonding are relatively new and more research is needed to see if the obtained results can be extrapolated to other materials and applications.

#### *3.1.3. Wound monitoring*

In modern society the development of 'smart' materials is a hot topic (crf smartphones, smartwatches, smart-TV's etc.). In the wound care industry similar developments are taking place and several groups are doing research on so-called 'smart bandages'. The idea behind smart bandages is that they autonomously could monitor the wound and signal in case of infection or other irregularities. A few groups working on biosensors have successfully incorporated plasma technology in their biosensor production process.

Phair et al. used a corona discharge in ambient air to activate carbon fiber threads in order to improve their electro-analytical performance [47]. Via a redox probe set, the ion exchange rate was determined as a function of plasma treatment time. The results showed that the incorpo‐ ration of carboxyl groups resulted in better electron transfer kinetics. Exposing the prototype bandage to whole blood proved that they were capable of detecting urates.

Zhou et al. developed a biosensor based on self-quenching fluorocarbons [48]. These fluoro‐ carbons were stored in lipid vessels which were immobilized on a pp-non-woven that was plasma coated with maleic anhydride using a commercial RF discharge system. Upon contact with both Gram positive and negative bacteria, the lipid vessels released the fluorocarbons, allowing for a visual infection detection mechanism. In combination with the simultaneous release of antibacterial products using the same release system, they believe it is possible to make a quasi-autonomous or 'smart' bandage.

The collection of reviewed papers shows that non-thermal plasma technology can play an important role in the development of smart bandages, allowing for low-cost personalized wound care treatment.

#### **3.2. Antimicrobial functionality**

Within the section on wound dressings already a part on antibacterial properties of plasma treated fibers has been included, but the applicability of these modified textiles goes much further than wound treatment alone and is highly wanted for other (bio)medical applications such as surgical gowns, sutures etc. as well as in the food industry and catering business [49]. The study on antimicrobial finishes of fibers and meshes has been quite extensive and between 25 and 30 papers will be reviewed here. A wide variety of substrates has been modified, ranging from natural-based products such as cellulose and wool to PP, polyesters, polysulfons, polyamides, carbon composites etc.

#### *3.2.1. Ag immobilization*

As for the modification itself, a distinction can be made between alterations involving the immobilization of metal nanoparticles (Ag, Cu) and all other treatments. To this day, the controlled release of silver particles is by far the most effective technique to inhibit the attachment and growth of both Gram positive and negative bacteria [50-65]. Most of the research groups use a non-thermal plasma to increase the wettability and nano-roughness of the textile substrate in order to enhance the uptake of silver nanoparticles (either via AgNO3 or Agn) or a plasma polymer coating with controlled release properties. Antibacterial tests involving micro-organisms such as E. Coli, S. Aureus and C. Albicans show in most cases a bactericidal efficiency of more than 99.9%.

#### *3.2.2. Plasma activation, grafting and intermediate bonding*

Other research groups followed less known pathways with mixed success. Yao et al. did an Ar low pressure plasma pretreatment of PVDF-HFP/PU fibrous membranes, followed by the UV grafting of 4-vinylpyridine and a quaternization of the introduced pyridine group using hexyl bromide [66]. Anti-bacterial essays showed a killing efficiency of 99.9999 % for both E-Coli and S. Aureus strains (See figure 2). Despite the excellent performance of the fibers, the commercial applicability remains limited due to relatively expensive production process in a competitive business environment.

ration of carboxyl groups resulted in better electron transfer kinetics. Exposing the prototype

Zhou et al. developed a biosensor based on self-quenching fluorocarbons [48]. These fluoro‐ carbons were stored in lipid vessels which were immobilized on a pp-non-woven that was plasma coated with maleic anhydride using a commercial RF discharge system. Upon contact with both Gram positive and negative bacteria, the lipid vessels released the fluorocarbons, allowing for a visual infection detection mechanism. In combination with the simultaneous release of antibacterial products using the same release system, they believe it is possible to

The collection of reviewed papers shows that non-thermal plasma technology can play an important role in the development of smart bandages, allowing for low-cost personalized

Within the section on wound dressings already a part on antibacterial properties of plasma treated fibers has been included, but the applicability of these modified textiles goes much further than wound treatment alone and is highly wanted for other (bio)medical applications such as surgical gowns, sutures etc. as well as in the food industry and catering business [49]. The study on antimicrobial finishes of fibers and meshes has been quite extensive and between 25 and 30 papers will be reviewed here. A wide variety of substrates has been modified, ranging from natural-based products such as cellulose and wool to PP, polyesters, polysulfons,

As for the modification itself, a distinction can be made between alterations involving the immobilization of metal nanoparticles (Ag, Cu) and all other treatments. To this day, the controlled release of silver particles is by far the most effective technique to inhibit the attachment and growth of both Gram positive and negative bacteria [50-65]. Most of the research groups use a non-thermal plasma to increase the wettability and nano-roughness of the textile substrate in order to enhance the uptake of silver nanoparticles (either via AgNO3 or Agn) or a plasma polymer coating with controlled release properties. Antibacterial tests involving micro-organisms such as E. Coli, S. Aureus and C. Albicans show in most cases a

Other research groups followed less known pathways with mixed success. Yao et al. did an Ar low pressure plasma pretreatment of PVDF-HFP/PU fibrous membranes, followed by the UV grafting of 4-vinylpyridine and a quaternization of the introduced pyridine group using hexyl bromide [66]. Anti-bacterial essays showed a killing efficiency of 99.9999 % for both E-Coli and S. Aureus strains (See figure 2). Despite the excellent performance of the fibers, the

bandage to whole blood proved that they were capable of detecting urates.

make a quasi-autonomous or 'smart' bandage.

wound care treatment.

126 Advances in Bioengineering

**3.2. Antimicrobial functionality**

polyamides, carbon composites etc.

bactericidal efficiency of more than 99.9%.

*3.2.2. Plasma activation, grafting and intermediate bonding*

*3.2.1. Ag immobilization*

Different research groups did a plasma pretreatment on cotton and polyproplyne (PP) nonwovens respectively, followed by the immobilization of natural products [67-69]. Vaideki et al compared the uptake of neem leaf extract before and after RF air plasma treatment and found that both the increase in wettability as well as the surface etching resulted in a superior adsorption of the extract [67]. Nitkyakalyani et al made a mix of herbal plant parts that were dried and grinded after which they were dissolved so the PP could be soaked in it [68]. A wide variety of both Gram positive and negative bacteria were tested and excellent antimicrobial activity was noted. Although the use of natural products is a pro in some cases, it is difficult to obtain a certain consistency in the production process and there is always a certain risk of product pollution. Strnad et al. treated cellulose fibers with an RF O2 plasma to increase the adsorption of chitosan [69]. The antimicrobial tests revealed a modest effect against S. Aureus and no effect against E-Coli. Antifungal tests using several fungi strings resulted in modest antifungal behavior.

Some groups simply applied a plasma treatment as such, using reactive gasses such as CF4 and hydrazine to obtain the desired antimicrobial effect. Uygun et al. did a pretreatment of chitosan powder using a RF hydrazine plasma [70]. Chemical analysis shows a significant increase in the number of primary amines present. Using the modified chitosan for the electrospinning of nano-fibers results in a nonwoven that has a better moisture uptake and an enhanced antibacterial effect for Gram-positive bacteria. Canal et al. and Virk et al. used an Ar-CF4 post discharge plasma with mixed results. The treatment of wool, polyamide and Sontara® resulted in a heightened bacterial resistance, while the treatment of cotton had no effect at all [71, 72].

Just as described in the intermediate bonding paragraph of the wound treatment section, plasma is used for the grafting of intermediate layers that are used for the immobilization of antibacterial components. Degoutin et al. used a low pressure RF Ar plasma to graft acrylic acid onto a PP nonwoven [73]. The carboxylic acid functionalities were used for the immobi‐ lization of gentamicin, which is known to be 99% bactericidal as was confirmed by the testing with E. Coli. Gawish et al. used a He plasma for the grafting of glycylmethacrylate, which was used for the covalent bonding of cyclodextrines [74]. These macromolecules are known for the controlled release of active components, in this case biocides with antimicrobial and insect repellent properties. These experiments confirm that plasma grafting is a versatile technique that forms the basis for subsequent reactions.

In general it can be concluded that the incorporation of silver micro-and nano-particles is still the golden standard for the production of antibacterial textiles. Non-thermal plasma technol‐ ogy can be a useful tool for the immobilization of the metal particles and plasma deposited coatings are ideal for the controlled release. Plasma treatments as such can generate highly antimicrobial surfaces, as was proven, amongst others, by Yao et al. [66], but only after the right selection of precursors and discharge gasses.

**Figure 2.** SEM images of (a) and (d) filter paper (control), (b) and (e) pristine, and (c) and (f) modified PU fibrous mem‐ branes after immersed in PBS suspension of (a)–(c) S. Aureus, or (d)–(f) E. Coli at 107 cells/mL for 4 h. PU fibrous mem‐ branes were electrospun from 10% (w/v) solutions in THF and DMF (1:1, v/v) [127].

#### **3.3. Cell culture and soft tissue engineering**

Technical textiles for tissue engineering applications are one of the youngest branches in the textile industry, but in a just a few decades they have become a major player on the biomedical market and the number of publications dealing on the theme of tissue engineering applications has exponentially grown. The idea behind tissue engineering is that (stem) cells are extracted from a patient with a malfunctioning organ. The retrieved healthy cells are seeded on a culture plate and grown to a full culture [3, 4, 75]. Once enough cells can be harvested, they are seeded onto a 3D nanofibrous scaffold. After a certain incubation period, allowing the cells to grow and differentiate into the scaffold, the nonwoven is implanted into the patient in order to restore the organ functionality or replace the organ as such. By treating patients with their own cells, immuno-response can be reduced to an absolute minimum and it is considered to be a constructive solution for the transplant waiting list issue.

In this specific chapter part, both the enhancement of cell culture applications as well as soft tissue engineering will be discussed together, as they are closely related and in some cases even overlap. This chapter part will therefore be subdivided into a first section dealing with plasma treatment as such and its effect on cell growth, adhesion, proliferation and differen‐ tiation and a second section handling plasma grafting and polymerization, talking in more detail about adhesion and homogeneity of the deposited coatings as well as the histological properties.

#### *3.3.1. Plasma treatment*

**Figure 2.** SEM images of (a) and (d) filter paper (control), (b) and (e) pristine, and (c) and (f) modified PU fibrous mem‐ branes after immersed in PBS suspension of (a)–(c) S. Aureus, or (d)–(f) E. Coli at 107 cells/mL for 4 h. PU fibrous mem‐

Technical textiles for tissue engineering applications are one of the youngest branches in the textile industry, but in a just a few decades they have become a major player on the biomedical market and the number of publications dealing on the theme of tissue engineering applications has exponentially grown. The idea behind tissue engineering is that (stem) cells are extracted

branes were electrospun from 10% (w/v) solutions in THF and DMF (1:1, v/v) [127].

**3.3. Cell culture and soft tissue engineering**

128 Advances in Bioengineering

The most widely used polymer collection for the production of nonwoven scaffolds for tissue engineering is the biodegradable polymer family. Polylactic acid(PLLA), polylactic-glycolic acid (PLGA), Polycaprolacton (PCL)... are well established biomaterials due to the fact that after implantation they get broken down by the body in harmless end products (ideally CO2 and H2O) that can be secreted by the body, making a second surgery no longer necessary [13, 76]. The mechanical and structural properties of these materials are sufficient for their field of applications. The biocompatibility and bioactivity on the other hand are mediocre at best and often inhibit the migration and differentiation of cells into the textile scaffold structure [77, 78]. The surface treatment of these scaffolds is rather complicated, as the pore size limits the infiltration efficiency of (wet)-chemical treatments and often degrade the structural stability of the delicate nano-fibers. Gas-based treatments such as non-thermal plasmas are promising to penetrate more easily into the electrospun scaffold structure and are at the same time known for the fact that they only alter the surface without affecting the bulk, guaranteeing the mechanical and structural integrity of the modified biomaterial [14, 79].

The contact angle of PCL electrospun scaffolds lies between 120° and 140°, indicating a hydrophobic surface, which is not well liked by most cells. After treatment with either air, Ar, NH3 or O2 plasmas, all research groups were able to reduce the contact angle to less than 5° [80-85]. The XPS results reveal an increase in the oxygen content with the incorporation of a mixture of C-O, C=O and O-C=O functional groups. Prabhakaran et al. seeded neurolemmo‐ cytes (or Schwann) cells onto the nanofibrous scaffolds and found an increase in proliferation rate at all times, with a maximum of 17% compared to the untreated material after 10 days, compared to the untreated scaffolds [80]. They claim that the treatment is as effective as a collagen coating, making it a cost-effective alternative for nerve cell regeneration applications. Yan et al. found that after plasma treatment with NH3+O2 had no influence on the mechanical properties of the non-woven. After seeding with MC3T3 osteoblasts a 2-3 times increase in cell adhesion was found in the first 24 hours and after 7 days the proliferation was increased by a factor of 6, making the plasma treatment an excellent tool for the introduction of osteoinductive properties [81, 84]. Martins et al. came to the same conclusions after treating their PCL nano-textile scaffolds both with Ar and O2 plasma [82]. Seeding 3 different cell lines (L929, ATDC5 and Saos-2) covering a wide variety of cell-types, resulted in a significant increase in both adhesion and proliferation for all cells. Min et al. tested the O2 plasma treated PCL nanotextile with primary astrocytes and noted an increase in adherence and viability in the first 24 hours [83]. Jeon et al. used a nano-sized template to enhance the nano-roughness introduced by the plasma [85]. After seeding MG63 osteoblasts a dramatic increase in cell adhesion and proliferation were noted as well as an elongated morphology compared to both untreated PCL and PCL treated without the nano-sized template, showing that both the surface chemistry and topography have a significant influence on the histological performance of the PCL electrospun scaffolds. Finally Blackstone et al. used a CF4 plasma to further increase the contact angle of the non-woven in order to obtain a superhydrophobic scaffold [86]. After seeding a mixture of fibroblasts, keranocytes and MCF-7 cancer cells, they were able to sort out the cancer cells by applying a fixed amount of stress to the textile. The recovered cancer cells did not change in morphology, allowing for post-sorting analysis, making the development of a low cost cancer detection device possible.

**Figure 3.** Photographs of water droplets taken immediately after contacting (a) non-treated, (b,c) oxygen plasma-treat‐ ed and (d,e) ammonia plasma-treated PLGA nanofibers. Treatment time was varied from (b,d) 30 to (c,e) 180 s [128].

PLLA and PLGA both exhibit the same hydrophobic properties as PCL non-woven, giving water contact angles situated between 130° and 150°. After a plasma treatment with the typical discharge gasses (O2, Ar, NH3...) a decrease in contact angle was found to a minimum, ranging between 20° and 45° as depicted in figure 3 [87-89]. XPS reveals that PLLA is less robust compared to PCL as the initial increase in oxygen content is reversed when the substrate is over-treated, resulting in etching/degradation of the polymer structure [88]. Both Park et al. and Dolci et al. seeded fibroblasts (NIH 3T3 and MEF) after NH3 and air treatments respectively and similar results for the increase in viability and elongated morphology were found [88, 90]. Liu et al. did a study on the adhesion behavior before and after O2 plasma treatment of pMSC cells in the first hour after seeding [89]. Results revealed that both the adhesion and cell morphology were greatly improved as can be seen from figure 4. These results show that plasma treatment is indeed a valid option for the culturing of stem cells.

factor of 6, making the plasma treatment an excellent tool for the introduction of osteoinductive properties [81, 84]. Martins et al. came to the same conclusions after treating their PCL nano-textile scaffolds both with Ar and O2 plasma [82]. Seeding 3 different cell lines (L929, ATDC5 and Saos-2) covering a wide variety of cell-types, resulted in a significant increase in both adhesion and proliferation for all cells. Min et al. tested the O2 plasma treated PCL nanotextile with primary astrocytes and noted an increase in adherence and viability in the first 24 hours [83]. Jeon et al. used a nano-sized template to enhance the nano-roughness introduced by the plasma [85]. After seeding MG63 osteoblasts a dramatic increase in cell adhesion and proliferation were noted as well as an elongated morphology compared to both untreated PCL and PCL treated without the nano-sized template, showing that both the surface chemistry and topography have a significant influence on the histological performance of the PCL electrospun scaffolds. Finally Blackstone et al. used a CF4 plasma to further increase the contact angle of the non-woven in order to obtain a superhydrophobic scaffold [86]. After seeding a mixture of fibroblasts, keranocytes and MCF-7 cancer cells, they were able to sort out the cancer cells by applying a fixed amount of stress to the textile. The recovered cancer cells did not change in morphology, allowing for post-sorting analysis, making the development of a low

**Figure 3.** Photographs of water droplets taken immediately after contacting (a) non-treated, (b,c) oxygen plasma-treat‐ ed and (d,e) ammonia plasma-treated PLGA nanofibers. Treatment time was varied from (b,d) 30 to (c,e) 180 s [128].

PLLA and PLGA both exhibit the same hydrophobic properties as PCL non-woven, giving water contact angles situated between 130° and 150°. After a plasma treatment with the typical discharge gasses (O2, Ar, NH3...) a decrease in contact angle was found to a minimum, ranging between 20° and 45° as depicted in figure 3 [87-89]. XPS reveals that PLLA is less robust compared to PCL as the initial increase in oxygen content is reversed when the substrate is over-treated, resulting in etching/degradation of the polymer structure [88]. Both Park et al. and Dolci et al. seeded fibroblasts (NIH 3T3 and MEF) after NH3 and air treatments respectively and similar results for the increase in viability and elongated morphology were found [88,

cost cancer detection device possible.

130 Advances in Bioengineering

As PLLA, PLGA and PCL all show similar histological effects after exposure to a wide variety of plasmas, it is no surprise that blends of PLLA and PCL exhibit similar behavior. Chanda‐ sekaran et al. exposed such a co-polymerized fiber mesh to an air plasma treatment and studied the effects on fibroblast growth and proliferation [91]. In both cases a significant increase was noted and stimulation of extra cellular matrix formation was found, opening up the possibility for skin tissue regeneration applications.

**Figure 4.** SEM images of pMSCs on Plasma treated PLLA nanofibers (NFS) and pristine PLLA NFS. (A–D) pMSCs on PLLA NFS after cultured for 10 min, 20 min, 30 min,60 min, respectively; (E–H) pMSCs on P-PLLA NFS after cultured for 10 min, 20 min, 30 min,60 min, respectively; (a–h) higher magnification for (A–H) [129].

The biodegradability of the textile scaffold material is not required in every case and sometimes has to be avoided all together (tissue culture 'plates', vascular grafts...) as the loss of the mechanical framework would result in the permanent failure of the implant. A polystyrene electrospun scaffold was treated by Baker et al. with a low pressure Ar plasma, followed by the seeding of smooth muscle cells [92]. The in-vitro tests revealed a significant increase in cells and an alignment with the electrospun material. The excellent results show that the polystyr‐ ene electrospun scaffold could be a valid alternative for 2D tissue culture plates. Zandén et al. treated a PU fiber mesh with an oxygen plasma in an attempt to improve the interaction with red blood cells [93]. SEM images showed that prolonged exposure to the plasma resulted in a reduction of the fiber diameter and finally the degradation of the structure. As was the case with PLLA and PCL, a significant increase in hydrophilicity was noted due to the incorporation of polar functional groups. The in-vitro tests revealed that there was no significant difference between the adhesion of red blood cells before and after treatment. This shows that not all plasma treatments have a positive effect on biocompatibility and that the treatment gas and operation parameters should be carefully selected.

The final paragraph of this chapter part on plasma treatment for tissue engineering will go over the possibilities to use non-thermal plasmas to stimulate the formation of apatite on flexible scaffold structures. Yang et al. immersed an Ar plasma treated PCL fiber structure in an SBF 10 solution for a period of 7 days [94]. In the first 24 hours, already a CaP coating had grown on the fibers, consisting of nano-apatite and dicalcium phosphate dehydrate. After 7 days a structure closely resembling bioapatite was found. Luo et al. performed the same analysis on air plasma treated PEEK (reinforced with carbon fiber) and whereas the untreated PEEK resulted in no apatite formation, a fully grown apatite layer could be found on the treated nonwoven scaffold [95]. Other research groups made a solution of PCL mixed with hydrox‐ yapatite/CaCO3 which was electrospun, resulting in a composite nano-textile which was followed by an oxygen/air plasma treatment [96, 97]. After seeding hFOB osteoblasts, a significant proliferation rate was noted, as well at the first signs of mineralization similar to human bone as depicted in figure 5. These papers show that plasma activation of polymeric nonwoven scaffolds is an excellent tool for the promotion of apatite growth.

**Figure 5.** Mineralization of hFOB on PCL/HA-P nanofibrous scaffolds at different magnifications: (a) mineral deposi‐ tion 5000× (6 days), (b) mineral deposition 10000× (6 days), (c) mineral deposition 15000× (6 days), (d) apatite-like mor‐ phology of natural bone 15000× (10 days) [130].

In general it can be concluded that non-thermal plasma treatments, both at lower and elevated pressures, are excellent tools for the stimulation of the histological properties of a wide variety of cells seeded onto flexible scaffolds for tissue culture and tissue engineering applications.

#### **3.4. Plasma grafting, polymerization and immobilization**

with PLLA and PCL, a significant increase in hydrophilicity was noted due to the incorporation of polar functional groups. The in-vitro tests revealed that there was no significant difference between the adhesion of red blood cells before and after treatment. This shows that not all plasma treatments have a positive effect on biocompatibility and that the treatment gas and

The final paragraph of this chapter part on plasma treatment for tissue engineering will go over the possibilities to use non-thermal plasmas to stimulate the formation of apatite on flexible scaffold structures. Yang et al. immersed an Ar plasma treated PCL fiber structure in an SBF 10 solution for a period of 7 days [94]. In the first 24 hours, already a CaP coating had grown on the fibers, consisting of nano-apatite and dicalcium phosphate dehydrate. After 7 days a structure closely resembling bioapatite was found. Luo et al. performed the same analysis on air plasma treated PEEK (reinforced with carbon fiber) and whereas the untreated PEEK resulted in no apatite formation, a fully grown apatite layer could be found on the treated nonwoven scaffold [95]. Other research groups made a solution of PCL mixed with hydrox‐ yapatite/CaCO3 which was electrospun, resulting in a composite nano-textile which was followed by an oxygen/air plasma treatment [96, 97]. After seeding hFOB osteoblasts, a significant proliferation rate was noted, as well at the first signs of mineralization similar to human bone as depicted in figure 5. These papers show that plasma activation of polymeric

**Figure 5.** Mineralization of hFOB on PCL/HA-P nanofibrous scaffolds at different magnifications: (a) mineral deposi‐ tion 5000× (6 days), (b) mineral deposition 10000× (6 days), (c) mineral deposition 15000× (6 days), (d) apatite-like mor‐

phology of natural bone 15000× (10 days) [130].

nonwoven scaffolds is an excellent tool for the promotion of apatite growth.

operation parameters should be carefully selected.

132 Advances in Bioengineering

The tissue engineering nanofibrous scaffold materials subjected to plasma grafting and plasma polymerization are, in the majority of the papers reviewed, more or less the same ones used for plasma activation, PCL being the most popular one. A minority of papers investigated less obvious material choices that are inherently not (sufficiently) biocompatible, but once coated exhibit sufficient bioactive properties, as will be discussed in the last paragraph of this chapter part.

Guex et al. coated a PCL mesh using a combination of ethylene and CO2 in an Ar discharge in order to restore the functionality of damaged myocardium [98]. After seeding extracted mesenchymal stem cells onto the modified electrospun scaffold, it was implanted in a rodent model. Post-mortem analysis revealed a stabilized cardiac functionality as well as an attenu‐ ated dilation. Zander et al. covalently bonded lamilin proteins onto an air plasma treated PCL scaffold [99]. PC12 neuron-like cells were seeded onto the modified substrate and analysis showed a positive correlation between the neuron outgrowth and the concentration of the immobilized proteins. Xie et al followed a similar strategy, immobilizing dopamine [100]. The dopamine coating itself was then used to immobilize fibronectin, which significantly stimu‐ lated the attachment, spreading and cytoskeletal development of NIH 3T3 cells. Furthermore it was proven that the coatings could be used for the controlled release of active substances. Ma et al. used a combination of air plasma and wet carboiimide chemistry to graft gelatin onto both random and aligned PCL fiber meshes [101]. The spreading and proliferation of endo‐ thelial cells was greatly enhanced and the cells aligned themselves along the fibers, which was not the case for the untreated material. Finally Hegeman et al. performed a degradation study of PCL nanofibrous scaffolds coated with amine containing polymers and showed that the incorporation of oligomers in the deposited films can leach out, causing cell death [102]. Storage of the coated nano-textiles in liquid media removed the low molecular weight residue, solving the problem. Overall, the literature shows that PCL nonwoven scaffolds, either activated or coated, can be used for a wide variety of tissue engineering applications, exhibiting excellent bioactive properties. It is essential though to use the right set of parameters and avoid the inclusion of unreacted products as this can have a detrimental effect on the histological performance of the 3D electrospun scaffold.

Several research teams used non-thermal plasmas to immobilize bioactive macromolecules (collagen, cRGD peptides and heparin respectively) onto a PLLA nano-textile scaffold, after which they were seeded with either BOECs, hMSC or endothelial cells [78, 103-106]. In all cases a positive influence was found on the scaffold's histological properties. Park et al. also obtained an increase in adhesion and proliferation after seeding NIH 3T3 fibroblasts onto PLLA nanotextile scaffolds that were grafted with an acrylic acid coating using a low pressure O2 plasma [107]. He et al and Chan et al. performed a similar procedure compared to Feng et al. to immobilize collagen onto PLLA-PCL electrospun scaffolds [108, 109]. The first group success‐ fully seeded hCAEC's, showing the possibilities for vascular grafts (see figure 6), while the Chan et al. studied the enhancement of the adsorption properties of MSC cells in the first hour after seeding, proving that coated nonwoven scaffolds are more efficient than plasma treated samples, which in turn are more effective then untreated samples.

A rather large number of publications can be found on a variety of other biomaterials that are not always biodegradable such as PDMS, PU, PET, silk fibroin, cellulose, PHBV... Most of these textile scaffolds get coated with well-known bioactive macromolecules such as collagen, galactose, lamilin, peptides, or polymer films containing functional groups that are well-liked by cells, such as primary amines or carboxylic acids [92, 110-118]. A whole spectrum of cells is seeded on the coated textile scaffolds, ranging from osteoblasts and fibroblasts to endothelial cells, nerve cells and even stem cells. Discussing all of them again would lead to far, especially as the effects are similar to the histological performance of the coated PCL and PLLA textiles. Therefore the results have been summarized in table 1.

**Figure 6.** Fluorescent micrographs of HUVECs cultured on PU (A and B), plasma treated PU (P-PU) (C and D), P-PU/ PLGA (E and F), and plasma treated (P-PU/PLGA) films for 3 s (G and H). Cells were stained with Texas-Red Malei‐ mide C2 for cell membrane and nuclei were stained with Hoechst33258. Images are 40× (A, C, E, and G) and 400× (B, D, F, and H) magnified [131].



**Table 1.** Overview of papers on the histological performance of plasma coated 3D electrospun scaffolds (no PCL and PLLA)

#### **3.5. Sutures**

fully seeded hCAEC's, showing the possibilities for vascular grafts (see figure 6), while the Chan et al. studied the enhancement of the adsorption properties of MSC cells in the first hour after seeding, proving that coated nonwoven scaffolds are more efficient than plasma treated

A rather large number of publications can be found on a variety of other biomaterials that are not always biodegradable such as PDMS, PU, PET, silk fibroin, cellulose, PHBV... Most of these textile scaffolds get coated with well-known bioactive macromolecules such as collagen, galactose, lamilin, peptides, or polymer films containing functional groups that are well-liked by cells, such as primary amines or carboxylic acids [92, 110-118]. A whole spectrum of cells is seeded on the coated textile scaffolds, ranging from osteoblasts and fibroblasts to endothelial cells, nerve cells and even stem cells. Discussing all of them again would lead to far, especially as the effects are similar to the histological performance of the coated PCL and PLLA textiles.

**Figure 6.** Fluorescent micrographs of HUVECs cultured on PU (A and B), plasma treated PU (P-PU) (C and D), P-PU/ PLGA (E and F), and plasma treated (P-PU/PLGA) films for 3 s (G and H). Cells were stained with Texas-Red Malei‐ mide C2 for cell membrane and nuclei were stained with Hoechst33258. Images are 40× (A, C, E, and G) and 400× (B, D,

> L929 EVC

Galactose HepG2/C3A Improved

**Cell line Histological**

**Effects**

Better

Smooth muscle cell formation

albumin secretion

**Authors Reference**

al. [111]

Chien et al. [112]

proliferation Wang et al. [110]

Rayatpishesh et

samples, which in turn are more effective then untreated samples.

Therefore the results have been summarized in table 1.

F, and H) magnified [131].

134 Advances in Bioengineering

**Plasma treatment**

RF Ar low pressure

RF Ar low pressure

RF O2 low pressure

**Deposited coating**

Heparin

pNIPAm aoSMC

**Scaffold material**

**Silk fibroin**

**PDMS**

**PU**

Of all the textiles for biomedical applications, sutures are probably the most low-tech. The amount of research conducted to improve the performance of surgical sutures is therefore not as extensive. Traditionally sutures were either non-biodegradable, requiring removal after‐ wards or biodegradable, but lacking the necessary mechanical strength and flexibility [49]. Eventually glycolide and lactide polymers such as PLLA and PLGA found their way into the suture market, introducing the required mechanical properties combined with biodegrada‐ bility. Yet, as has been discussed earlier in the chapter, these biodegradable polymers do not always exhibit the wanted bioactive surfaces. A small number of research groups have investigated if non-thermal plasma technology can help to further improve the performance of medical sutures, of which a brief overview will be given.

Loh et al. performed a study, using both activation and deposition, analyzing the hydrolytic degradation rate of commercially available synthetic absorbable sutures [119, 120]. Dexon (PGA), Vicryl (PGLA), PDS11 (PpDO) and Maxon were either coated with parylene or treated by a number of different plasma gasses. Using the right set of plasma treatment parameters significantly increased the degradation rate of Vicryl and PDS11, while for Dexon and Maxon only marginal differences were found. The plasma coating process, using parylene, resulted in an increase in tensile strength, most likely due to the hydrophobic character of the coating. Saxena et al. published 3 papers on plasma grafting of PP sutures [121-123]. In all the articles an RF O2 plasma was used to activate the monofilament, followed by the immersion in an acrylic acid solution. The introduced carboxylic acid groups were then used to successfully immobilize chitosan. While the first 2 articles elaborates on the preservation of the mechanical properties and the surface chemical characterization, the second article focusses more on the antimicrobial, in-vitro and in-vivo properties. The viability of both E. Coli and S. Aureus were reduced with more than 90%. The in-vitro studies revealed excellent adhesion and prolifera‐ tion of MC3T3 cells and the in-vivo use in a rodent resulted in a better quality of tissue integration and a minimal inflammatory response. The grafting of acrylic acid onto a mono‐ filament for improved antimicrobial functionality was inspired by Gupta at al. who grafted acrylic acid onto PET monofilaments, obtaining similar antimicrobial results [124, 125].

**Figure 7.** Left: Zone of inhibition against E. Coli (a) control PP suture and (b) drug-loaded PP suture (degree of graft‐ ing, 5%). Middle: Zone of inhibition against K. Pneumonia (a) control PP suture and (b) drug-loaded PP suture (degree of grafting, 5%). Right: Zone of inhibition against S. Aureus (a) control PP suture and (b) drug-loaded PP suture (de‐ gree of grafting, 5%) [132].

Albeit being a rather low-tech application, plasma technology is still able to improve the performance of medical sutures. If the results found for tissue engineering applications would be applied for monofilament applications, it is beyond doubt that the biomedical properties could be further enhanced.
