**6. Plasma polymer gradients**

After accomplishing significant advancements in the biofunctionalization of surfaces by different chemical and physical homogeneous modifications, a growing research interest is being shifted toward the development of gradient surfaces presenting graded wettability, chemistry, biomolecule density and nanoparticle distribution [84]. This interest stems from the fact that many essential and poorly understood biological activities are driven by such gradients. For instance, chemotaxis mediate a number of physiological processes such as leukocyte recruitment to the infection site, guiding of neuronal and glial cells during nervous system development or regeneration and cancer metastasis. Moreover well-ordered gradient distribution of specific functional groups, extracellular matrix components, signaling biomolecules and even topographical cues induce particular cell type proliferation, migration and differentiation [85–87]. Besides their biological importance, gradients are also powerful for high throughput screening in several applications such as biomaterial development, tissue engineering and sensors, in the sense that a single sample designed with a gradient surface is used to procure multiple data points. This reduces dramatically the number of samples and cells, eliminates inaccuracies triggered by sample reproducibility and speeds up the analysis [84, 88, 89].

Different approaches are commonly adopted to create surface gradients such as self-assembled monolayers (SAMs), grafting on hydrogels and Boyden chambers and filters. Several limitations are associated with these traditional methods including the substrate dependency (e.g., gold-coated surface is required for SAMs), the short term gradient "shelf-life," the restricted chemistries that can be obtained and the long experimental timing [46, 90, 91]. As an alternative, applying high energy source plasmas that are associated with many advantages such as the absence of solvent, the specificity and the substrate independency has shown great successes in the generation of gradient surfaces. In 1989, Witt el al. were one of the first groups if not the first, to generate wettability gradients on polyethylene, polystyrene, polydimethylsiloxane, and polytetrafluoroethylene by a radio frequency (RF) plasma activation operating in oxygen, ammonia and sulfur dioxide atmospheres. A special gradient apparatus consisting of an aluminum box with a translating cover and two aluminum plates serving as electrodes, was designed for this purpose. During the treatment, the cover is retracted with a constant velocity automatically controlled by a microprocessor driving the stepping motor. This movement linearly increases the plasma exposure time over the sample length. As a result, water contact angles (WCA) increased along the length of the sample thus ensuring the presence of a wettability gradient. Moreover, a wide range of wettability gradients is obtained by varying the gas, the radio frequency power, the cover retraction velocity and the plasma exposure time. This study highlighted the high flexibility of the plasma treatment to generate gradients with defined length and magnitude and pointed out, by using several substrates, that the process is substrate independent [92]. Therefore, a steep rise in literature focusing on the generation of gradients by plasma activation followed. However, the different plasma activation methods that were described are mainly limited to the production of wettability gradients with a relatively restricted control over the chemical group specific incorporation. Other concerns include the aging effect of the treated surfaces due to the reorientation of the incorporated groups away from the surface when the environment is thermodynamically unfavorable and the roughening of the surface due to the plasma etching effect [92, 93]. Consequently, the interest was shifted toward the generation of polymer gradients via plasma polymerization to be able to control more precisely the functional group nature and densities, the gradient stability and the gradient shape. Nevertheless, it was until 2003 that the first method enabling the deposition of controllable horizontal plasma chemical gradients was described by Whittle et al. [93] and was subsequently adopted as it is or with some amendments by several other groups [91, 94]. In their study, Whittle et al. created hydrocarbon/carboxyl and amine/carboxyl functionality gradients on glass substrates over a distance of 11 mm. Instead of using the traditional cylindrical plasma reactor, a RF glow discharge T-shaped reactor presenting a drawer as sample holder was used [93, 95]. As a first step, an amine coating was deposited on the whole glass substrate by performing a plasma polymerization using allylamine (Aam) monomers as precursors and a continuous power of 10 W while the drawer was fully extended. An underlayer presenting a good adhesion was thus formed for the subsequent gradient deposition. In the second step, the power was decreased to 5 W and a plasma polymerization was performed while the drawer was slowly closed at a constant velocity of 1 mm/min along with a controlled change in the plasma composition over time. This was performed by introducing acrylic acid (Aac) as the second monomer while decreasing instantaneously the flow rate of Aam by 4 cm3 stp/min. For the deposition of hydrocarbon/carboxyl gradients, the same procedure was followed but with the use of octa-1,7-diene instead of Aam precursors. The obtained plasma polymerized surfaces were characterized by X-ray photoelectron spectroscopy (XPS) and chemical derivatization of acid functionalities using trifluoroethanol. A gradual increase in the concentration of acid functionalities was observed in the case of hydrocarbon/carboxyl gradients and an increase of acid and amine functionalities was attained in opposite directions in the case of the amine/carboxyl gradients. These findings demonstrated the power of this first-hand methodology to successfully generate plasma polymer gradients that can subsequently allow the grafting of a broad range of biochemical entities in a spatially structured manner [93]. Surface engineers waited around 3 years after the study of Whittle to begin their investigations regarding the grafting of biomolecules and the cell-biomaterial interactions when a plasma polymer gradient is implicated. Moreover, several other methods generating plasma polymer gradients were

Major advancements in the understanding of oxidative reaction mechanisms that occur dur-

This was done using XPS, FTIR spectroscopy and contact angle goniometry characterization techniques which allowed significant perception of the eventual surface compositional changes. Their studies included detailed oxidation investigations of hydrocarbon based plasma polymers [80], fluorocarbon coatings [81], nitrogen coatings [82] and other plasma deposited films [83]. Results showed that the aging process was due to the reaction of ambient oxygen with the residual radicals present in plasma polymers; ESR spectroscopy showed that the free radicals detected in freshly deposited plasma films slowly disappear upon storage in air. Results also showed that, the kinetics, mechanisms and formed oxidative products during aging depend on many factors, such as the structure of the film, the type of functional groups and the mobility of the surface.

After accomplishing significant advancements in the biofunctionalization of surfaces by different chemical and physical homogeneous modifications, a growing research interest is being shifted toward the development of gradient surfaces presenting graded wettability, chemistry, biomolecule density and nanoparticle distribution [84]. This interest stems from the fact that many essential and poorly understood biological activities are driven by such gradients. For instance, chemotaxis mediate a number of physiological processes such as leukocyte recruitment to the infection site, guiding of neuronal and glial cells during nervous system development or regeneration and cancer metastasis. Moreover well-ordered gradient distribution of specific functional groups, extracellular matrix components, signaling biomolecules and even topographical cues induce particular cell type proliferation, migration and differentiation [85–87]. Besides their biological importance, gradients are also powerful for high throughput screening in several applications such as biomaterial development, tissue engineering and sensors, in the sense that a single sample designed with a gradient surface is used to procure multiple data points. This reduces dramatically the number of samples and cells, eliminates inaccuracies triggered by sample reproducibility and speeds up the analysis [84, 88, 89].

Different approaches are commonly adopted to create surface gradients such as self-assembled monolayers (SAMs), grafting on hydrogels and Boyden chambers and filters. Several limitations are associated with these traditional methods including the substrate dependency (e.g., gold-coated surface is required for SAMs), the short term gradient "shelf-life," the restricted chemistries that can be obtained and the long experimental timing [46, 90, 91]. As an alternative, applying high energy source plasmas that are associated with many advantages such as the absence of solvent, the specificity and the substrate independency has shown great successes in the generation of gradient surfaces. In 1989, Witt el al. were one of the first groups if not the first, to generate wettability gradients on polyethylene, polystyrene, polydimethylsiloxane, and polytetrafluoroethylene by a radio frequency (RF) plasma activation operating in oxygen, ammonia and sulfur dioxide atmospheres. A special gradient apparatus consisting of an aluminum box

ing plasma polymer aging have been made by Gengenbach et al. [80–83].

**6. Plasma polymer gradients**

78 Recent Research in Polymerization

described with a distinctive focus on amine and carboxylic acid being the most 2 extensively considered functionalities in the subsequent literature of gradient plasma polymerization. In what follows, an overview on the achievements of these carboxylic acid and amine plasma gradients in several tissue engineering and biomedical applications will be given.

#### **6.1. Surface plasma polymer gradient of carboxylic acid functionalities**

In 2006, Parry et al. [91] performed a plasma copolymerization of Aac and octadiene (OD) based on the mechanism described by Whittle et al. [93] but with a modification of the setup in a way allowing the production of 20 similar gradients at a time. Up to 20 substrates could thus be placed in the redesigned RF plasma reactor and moved under a slot by an automated stepper motor in 250 μm paces at a rate of 750 μm/min. Simultaneously, a controlled composition of the monomer mixture is sent to the chamber via two computer-regulated valves. A thorough characterization of the surface gradient was executed by angle resolved x-ray photoelectron spectroscopy (ARXPS) that showed in great details how acid functionalities changed on different positions of the gradient and highlighted the presence of vertical changes especially when it comes to the plasma polymer thickness. An assay investigating the passive adsorption of immunoglobulin G (IgG) as a function of the acid surface density was supplemented to the study to be, to the best of our knowledge, the first reported biological assay done on plasma polymer gradients. ARXPS measurements showed that IgG was by far more absorbed on the OD gradient end and that IgG amount decreased gradually as the concentration of Aac increased thus creating an IgG gradient [91]. In 2009, Walker et al. [96] also deposited a gradient of OD/Aac on coverslips using the plasma deposition/masking method of Whittle but this time with a renovated protocol permitting the generation of submillimeterscale gradients instead of millimeter scale length. In the updated method, OD was constantly fed to the reactor as the slot moves across the substrate surface, then it was brusquely turned off and a pulse of Aac was launched. The scale length and density of the carboxylic groups were thus tailored by varying the pulse width of Aac. The obtained gradient surface was used to immobilize the intercellular signaling molecule delta-like-1 Dll 1, a factor enhancing stem cells self-renewal and preventing cell differentiation which is an issue to be considered when developing cell therapy technologies. Since tiny changes in surface properties can considerably affect the stem cell behavior either by enhancing the commitment path toward their differentiation to particular cell types or by maintaining and stabilizing the stem cell pluripotent phenotype, concentration-based factor and chemical group gradients are highly expedient to study stem cells. Instead of directly grafting Dll 1 factor on the generated gradient, a mouse monoclonal antimyc-tag (9E10) antibody is covalently coupled, then Dll 1 is immobilized on the antibodies thus avoiding the alteration of its biological activity by separating it from the solid surface. A visualization of the Dll 1 gradients was performed by binding a rabbit anti-Dll-1 antibody and then introducing a colloidal gold-conjugated secondary antibodies. Several Dll 1 gradients with different slopes and end points were obtained depending on the plasma Aac pulse width adopted during the plasma polymerization (**Figure 4**). During the same year, the first cell tests on plasma gradients were performed by Wells et al. using mouse embryonic stem (ESC) cell lines E14 and R1 in order to examine their pluripotency [97]. OD/ Aac gradients were deposited on coverslips using the same setup described by Parry et al. [91]. The degree of cell spreading was studied in function of COOH concentration. Alkaline

described with a distinctive focus on amine and carboxylic acid being the most 2 extensively considered functionalities in the subsequent literature of gradient plasma polymerization. In what follows, an overview on the achievements of these carboxylic acid and amine plasma gradients

In 2006, Parry et al. [91] performed a plasma copolymerization of Aac and octadiene (OD) based on the mechanism described by Whittle et al. [93] but with a modification of the setup in a way allowing the production of 20 similar gradients at a time. Up to 20 substrates could thus be placed in the redesigned RF plasma reactor and moved under a slot by an automated stepper motor in 250 μm paces at a rate of 750 μm/min. Simultaneously, a controlled composition of the monomer mixture is sent to the chamber via two computer-regulated valves. A thorough characterization of the surface gradient was executed by angle resolved x-ray photoelectron spectroscopy (ARXPS) that showed in great details how acid functionalities changed on different positions of the gradient and highlighted the presence of vertical changes especially when it comes to the plasma polymer thickness. An assay investigating the passive adsorption of immunoglobulin G (IgG) as a function of the acid surface density was supplemented to the study to be, to the best of our knowledge, the first reported biological assay done on plasma polymer gradients. ARXPS measurements showed that IgG was by far more absorbed on the OD gradient end and that IgG amount decreased gradually as the concentration of Aac increased thus creating an IgG gradient [91]. In 2009, Walker et al. [96] also deposited a gradient of OD/Aac on coverslips using the plasma deposition/masking method of Whittle but this time with a renovated protocol permitting the generation of submillimeterscale gradients instead of millimeter scale length. In the updated method, OD was constantly fed to the reactor as the slot moves across the substrate surface, then it was brusquely turned off and a pulse of Aac was launched. The scale length and density of the carboxylic groups were thus tailored by varying the pulse width of Aac. The obtained gradient surface was used to immobilize the intercellular signaling molecule delta-like-1 Dll 1, a factor enhancing stem cells self-renewal and preventing cell differentiation which is an issue to be considered when developing cell therapy technologies. Since tiny changes in surface properties can considerably affect the stem cell behavior either by enhancing the commitment path toward their differentiation to particular cell types or by maintaining and stabilizing the stem cell pluripotent phenotype, concentration-based factor and chemical group gradients are highly expedient to study stem cells. Instead of directly grafting Dll 1 factor on the generated gradient, a mouse monoclonal antimyc-tag (9E10) antibody is covalently coupled, then Dll 1 is immobilized on the antibodies thus avoiding the alteration of its biological activity by separating it from the solid surface. A visualization of the Dll 1 gradients was performed by binding a rabbit anti-Dll-1 antibody and then introducing a colloidal gold-conjugated secondary antibodies. Several Dll 1 gradients with different slopes and end points were obtained depending on the plasma Aac pulse width adopted during the plasma polymerization (**Figure 4**). During the same year, the first cell tests on plasma gradients were performed by Wells et al. using mouse embryonic stem (ESC) cell lines E14 and R1 in order to examine their pluripotency [97]. OD/ Aac gradients were deposited on coverslips using the same setup described by Parry et al. [91]. The degree of cell spreading was studied in function of COOH concentration. Alkaline

in several tissue engineering and biomedical applications will be given.

80 Recent Research in Polymerization

**6.1. Surface plasma polymer gradient of carboxylic acid functionalities**

**Figure 4.** (a) Densitometry results of 9E10 antibodies immobilized on the gradient surface (3 different Aac pulse durations) and visualized by FITC-conjugated secondary antibodies. Horizontal lines show the results of homogeneous surface treatments (b) false color heat maps of the 9E10 antibody gradients. Homogeneous surfaces are presented for comparison. Scale bars: 100 μm.

phosphatase staining showed that cell capacity of self-renewal is preserved when the cell spreading is still below 120 μm2 [97]. In 2012, in an attempt aiming to make a sweeping statement about this result, Harding et al. [98] used polyethylene oxide (PEO) that is well-known in the biomaterials field to limit protein adsorption and thus cell adhesion and spreading, together with Aac to produce two counter gradients. A RF apparatus consisting of a cylindrical glass chamber was used for the plasma copolymerization. As a first step, an OD layer then an Aac layer were deposited on the substrate since a unique Aac deposition resulted in the coating dissolution in water. Then a mask 12 ̊titled in respect to the surface was employed to deposit a PEO-like gradient by using the monomer diethylene glycol dimethyl ether (DG) as a precursor. A successful fabrication of AA-DG plasma polymer gradient was revealed by XPS, profilometry and infrared microscopy mapping. The gradient could be easily altered by changing the plasma process parameters. Mouse ESC were cultured on the gradient surfaces, then immunocytochemical stainings of the stem cell markers Oct4 and alkaline phosphatase were performed. Results showed a low cell adhesion and colony formation on the DG rich end and an increased colony size and decreased stem cell marker expression on the COOH rich end, thus supporting the hypothesis stating that cellular spreading influences the fate toward cell differentiation or self-renewal. The same method using a tilted mask was then applied by Wang et al. [99] in 2014 to create the same Aac-DG gradients but also Aac-OD gradients by firstly depositing OD uniformly then using the tilted mask to deposit Aac. Attachment and differentiation of rat bone marrow mesenchymal stem cells (rBMSCs) into adipogenic and osteogenic lineages were investigated on both gradients. After 24 h of cell culture, a gradient in cell density was observed on the substrate with a decreased cell adhesion on DG and OD rich ends. The obtained cell density gradient vanished on Aac-OD gradient after 6 days but not on Aac-Dg gradient, thus suggesting the long-term efficacy of the later gradient. Cell colonies containing bone nodules were detected on this gradient especially on the Aac rich ends but not on the DG rich end. Moreover, proteins and calcium were not secreted on the DG end implying that osteogenic differentiation is influenced by local cell densities. However, the induction of the cells toward an adipogenic lineage showed that this differentiation is cell density insensitive.

#### **6.2. Surface plasma polymer gradient of amine functionalities**

In addition to COOH functionalities, NH<sup>2</sup> groups were also shown to be very powerful in influencing a wide range of particular cell type performances such as adhesion, proliferation, migration and differentiation. Therefore, when the research community started investigating surface gradients, a distinctive focus was directed toward the production of amine gradients and their use in several biomaterial and tissue engineering applications [100]. To the best of our knowledge, all COOH plasma polymer gradients described so far were only deposited on flat substrates, however some amine plasma polymer gradients were deposited on 3D scaffolds. For instance, in 2006 Barry et al. [101] thought of generating an amine gradient on poly(D,L-lactic acid) 3D porous scaffolds in order to solve the common problem of the highly disproportionate cell colonization on the scaffold periphery in comparison to the hardly accessible scaffold center that remains poorly colonized and supplied by nutrients. This issue was solved by plasma polymerizing hexane, known to be resistant to cellular adhesion, on the periphery of the scaffold while generating an amine plasma polymer coating on the central surface. To do so, a first plasma polymerization step using Aam monomers as precursors was performed, then a second polymerization using the cell-repellent hexane was achieved at lower deposition rate. XPS measurements throughout the whole scaffold showed that when the second hexane polymerization step is absent, a decrease in amine functionalities is observed toward the center. However, when hexane polymerization is introduced, the nitrogen concentration is reduced by 1 to 2% in the periphery thus creating a reversed gradient. After seeding 3 T3 fibroblasts on the treated scaffolds, X-ray micro-computed tomography and scanning electron microscopy revealed a uniform cell distribution throughout the whole scaffold with well spread cells in the center associated with a high production of extracellular matrix (ECM) components. The use of hexane and Aam to create amine gradients was also considered by Zelzer et al. [102] in the subsequent year, but this time on flat glass coverslips. The idea behind the study was to compare between mammalian cell interactions on gradient and on uniformly treated surfaces. A T-shaped borosilicate RF reactor was used to plasma polymerize uniformly an amine coating on glass coverslip using Aam as precursors. Afterward, a poly-hexane was deposited on the poly-Aam coated surface after placing a mask either directly or making use of a spacer clamping the mask at a distance of 0.04 mm from the surface. The direct positioning of the mask resulted in steep gradients while the use of a spacer gave more shallow gradients. Wettability gradients were detected by WCA measurements showing a gradual decrease from 93̊ to 66 ̊, thus correlating with the gradual increase of N/C ratio. NIH 3 T3 fibroblasts cultured on the gradients surfaces were preferentially adhered and proliferated on the N-rich end with a gradual cell density decrease toward the poly-hexane rich end. Surprisingly, experiments performed on uniform surfaces revealed significant differences in cellular behavior compared to the gradient surfaces, leaving question marks on the use of gradients for high throughput screening. The cell signaling and the protein synthesis might be different between gradient and uniform surfaces since the cell neighboring environment differs. Several subsequent studies involving amine plasma polymer gradients and their general results are summarized in **Table 2**.

the fate toward cell differentiation or self-renewal. The same method using a tilted mask was then applied by Wang et al. [99] in 2014 to create the same Aac-DG gradients but also Aac-OD gradients by firstly depositing OD uniformly then using the tilted mask to deposit Aac. Attachment and differentiation of rat bone marrow mesenchymal stem cells (rBMSCs) into adipogenic and osteogenic lineages were investigated on both gradients. After 24 h of cell culture, a gradient in cell density was observed on the substrate with a decreased cell adhesion on DG and OD rich ends. The obtained cell density gradient vanished on Aac-OD gradient after 6 days but not on Aac-Dg gradient, thus suggesting the long-term efficacy of the later gradient. Cell colonies containing bone nodules were detected on this gradient especially on the Aac rich ends but not on the DG rich end. Moreover, proteins and calcium were not secreted on the DG end implying that osteogenic differentiation is influenced by local cell densities. However, the induction of the cells toward an adipogenic lineage showed that this

in influencing a wide range of particular cell type performances such as adhesion, proliferation, migration and differentiation. Therefore, when the research community started investigating surface gradients, a distinctive focus was directed toward the production of amine gradients and their use in several biomaterial and tissue engineering applications [100]. To the best of our knowledge, all COOH plasma polymer gradients described so far were only deposited on flat substrates, however some amine plasma polymer gradients were deposited on 3D scaffolds. For instance, in 2006 Barry et al. [101] thought of generating an amine gradient on poly(D,L-lactic acid) 3D porous scaffolds in order to solve the common problem of the highly disproportionate cell colonization on the scaffold periphery in comparison to the hardly accessible scaffold center that remains poorly colonized and supplied by nutrients. This issue was solved by plasma polymerizing hexane, known to be resistant to cellular adhesion, on the periphery of the scaffold while generating an amine plasma polymer coating on the central surface. To do so, a first plasma polymerization step using Aam monomers as precursors was performed, then a second polymerization using the cell-repellent hexane was achieved at lower deposition rate. XPS measurements throughout the whole scaffold showed that when the second hexane polymerization step is absent, a decrease in amine functionalities is observed toward the center. However, when hexane polymerization is introduced, the nitrogen concentration is reduced by 1 to 2% in the periphery thus creating a reversed gradient. After seeding 3 T3 fibroblasts on the treated scaffolds, X-ray micro-computed tomography and scanning electron microscopy revealed a uniform cell distribution throughout the whole scaffold with well spread cells in the center associated with a high production of extracellular matrix (ECM) components. The use of hexane and Aam to create amine gradients was also considered by Zelzer et al. [102] in the subsequent year, but this time on flat glass coverslips. The idea behind the study was to compare between mammalian cell interactions on gradient and on uniformly treated surfaces. A T-shaped borosilicate RF reactor was used to plasma polymerize uniformly an amine coating on glass coverslip using Aam as precursors. Afterward, a poly-hexane was deposited on the poly-Aam coated surface after placing a mask either directly or making

groups were also shown to be very powerful

differentiation is cell density insensitive.

82 Recent Research in Polymerization

In addition to COOH functionalities, NH<sup>2</sup>

**6.2. Surface plasma polymer gradient of amine functionalities**



**Table 2.** Overview of literature on amine gradient obtained by plasma polymerization and not discussed in the text.

**Figure 5.** Schematic representation of the creation of two-protein gradient. Step 1. PEG grafting on the amine plasma polymer gradient to generate a PEG density gradient. Step 2. Large proteins adsorption. Step 3. Small protein adsorption.

Since the biological systems in vivo are much more complex than in vitro assays, some authors considered a closer mimicking of the real systems by designing, instead of one dimensional or single protein gradients, 2 protein and 2 dimensional gradients. For instance, in 2009 Vasilev et al. [94] created an Aam-OD gradients on SPRchips or on silicon wafers based on the method described by Whittle et al. [93]. Afterwards, polyethylene glycol (PG), known to be resistant to protein adsorption, was grafted on the amine gradient thus generating a PEG density gradient. The obtained density gradient was then benefited to control the deposition of 2 proteins, namely the large protein fibrinogen and the small protein lysozyme, by differential passive adsorption. A first incubation with the larger protein led to its adsorption on low PEG density regions, then a second incubation with the small lysozyme led to its adsorption only where there is still a "room" for it to adsorb since the previous fibrinogen adsorption passivated gradually the surface. As a result, 2 reversed gradients of 2 proteins could be designed and the method could be generalized to other pairs of small and large proteins (**Figure 5**). In 2013, Mangindaan et al. [90] designed a 2 dimensional amine gradient by performing firstly a plasma polymerization of Aam on a propylene membrane while a mask is placed on top with a gap distance of 1 mm. Subsequently, the same procedure is repeated but after rotating the sample by 90 ̊. WCA measurements showed that both gradients were well controlled by varying the plasma treatment exposure time in each step. L-929 fibroblasts seeded on the treated surfaces adhered and grew proportionally with the amine content on the 2 dimensional gradient with a predominant effect of the gradient created during the initial plasma deposition.
