Surface Modification of Polyamide 6 Immobilized with ...

International Journal of Polymeric Materials, 60:907–921, 2011 Copyright # Taylor & Francis Group, LLC ISSN: 0091-4037 print=1563-535X online DOI: 10.1080/00914037.2010.551370

Surface Modification of Polyamide 6 Immobilized with Collagen: Characterization and Cytocompatibility Juan Shen,1,2 Yubao Li,1 Yi Zuo,1 Qin Zou,1 Li Zhang,1 and Haohuai Liu1 1

The Research Center for Nano-biomaterials, Analytical and Testing Center, Sichuan University, Chengdu, China 2 Department of Chemistry, Southwest University of Science and Technology, Mianyang, China Polyamide 6 (PA6) membranes were exposed to Ar plasma to produce peroxides on their surfaces, followed by grafting polymerization of methacrylic acid (MAA) introducing -COOH on surfaces. PA6 membranes immobilized with collagen I were obtained by coupling collagen to the MAA graft chains. The physicochemical properties were characterized by contact angle measurement, ATR-FTIR, XPS, and AFM. The results showed that the hydrophilicity of the surface improved after surface modification. The surface topography of the original and the modified PA 6 membranes showed an increase in roughness. Moreover, collagen immobilized onto PA 6 membranes showed enhanced growth in ROB culture tests. Keywords covalent linkage, cytocompatibility, plasma, polyamide 6, surface modification

Received 30 August 2010; accepted 22 December 2010. This work was financially supported by China 973 fund (No. 2007CB936102) and China 863 fund (No. 2007AA03Z328766). Address correspondence to Yubao Li, The Research Center for Nano-biomaterials, Analytical and Testing Center, Sichuan University, Chengdu 610064, China. E-mail: [email protected]

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INTRODUCTION Polyamide is an important engineering material with its wide range of applications in automobile and engineering components due to excellent mechanical properties [1]. Recently, polyamide 6 has attracted attention as a hard tissue engineering material due to its excellent mechanical property and biocompatibility. For example, polyamide 6 matrix-filled hydroxyapatite has been used to build a three-dimensional scaffold for load-bearing repair of vertebral column or for repair of large area skulls [2–4]. Nevertheless, PA6 is often assigned to the lack of cell recognition sites, which restricts their future applications in tissue engineering. There is an ongoing interest in modifying implant surfaces to improve their cell adhesive properties and tissue integration capacity [5,6]. Recently, the pivotal strategy of functionalized polymer surfaces for biomaterial applications has been the attachment of proteins or peptides to mimic the extracellular matrix that promotes receptor-mediated cell adhesion, obtaining an optimal cell-surface interaction [7,8]. Previous studies have shown that covalent immobilization can provide specific sites for focal adhesion molecules, thus allowing the cells to adhere for continued bioactivity of in-dwelling devices [9]. Consequently, in this study, a PA6 surface covalent immobilized with collagen, so as to endow the PA6 material with the capacity to regulate such cell behaviors as adhesion, spreading, and growth, has been used to enhance cell-materials interactions for both in vivo and in vitro applications. In addition, many materials used in surface engineering lack reactive groups for the covalent attachment of biologically active molecules. Therefore, much effort has gone into exploring techniques such as wet chemical [10], ionized gas treatments [11], and UV irradiation [12,13] to introduce reactive groups onto the biopolymer. In particular, plasma-surface modification (PSM) as an economical and effective materials processing technique has been found to be applied in the biomedical field. Plasma can provide modification of the top nanometer of a polymer surface, which is possible to change in continuum the chemical composition and properties such as wettability, chemical inertness, lubricity, and biocompatibility of materials surfaces [14]. Moreover, plasma is obviously attractive to industrial users due to low engineering costs [15]. At present, plasma techniques are widely used for surface modification in the treatment of polymers for plasma coating, thin-film deposition, cleaning and activation of substrates, and sterilization of surfaces and materials [16–18]. Many kinds of plasma treatments used to modify surface properties of biopolymers have been studied [19,20]. For example, cell-affinity stimulating proteins immobilized on poly(lactide-coglycolide) have been prepared by the treatment of oxygen plasma followed by a coupling reaction of gelatin to enhance the growth of 3T3 fibroblasts [21]. Recently, a desired methacrylic acid (MAA) monomer was polymerized onto the surface of plasma-activated polyamide 6 membranes, forming a grafted brush layer on top of the surface, and then covalent immobilization of bioactive

Polyamide 6 Immobilized with Collagen

surface was obtained by coupling collagen to the MAA graft chains with the aid of a water-soluble carbodiimide. The surface hydrophilicity, chemical structures, and surface roughness were studied, and the in vitro cytocompatibility of PA6 and modified PA6 membranes was evaluated. Moreover, the correlation between the surface properties and the cytocompatibility was also discussed.

EXPERIMENTS Materials Polyamide 6 (PA6) grains with a viscosity-average molecular weight of 19 kDa were supplied by DuPont Company, USA. The PA6 membrane was prepared by hot compression molding, and 5 cm 5 cm and 1 mm of the PA6 membrane was obtained. The membrane was then washed with 75% ethanol to remove surface impurities, and dried by vacuum. Methacrylic acid was purified by distillation under vacuum. Collagen I was purchased from Sigma.

Plasma Treatment PA6 membrane was activated by Ar-plasma treatment. A glow discharge reactor (Model PD-2-plasma deposition system) with a bell-jar-type reactor cell was used. PA6 membranes were placed on the electrode in the plasma chamber. After pressure of the chamber was stabilized to 20 Pa, glow discharge plasma was created by controlling electrical power at 60 W and radio frequency of 13.56 MHz for a predetermined time. After the treatment, PA6 membrane was taken out and exposed to the air for about 30 min for peroxide formation [22].

Grafted Polymerization The Ar-plasma treated PA6 membrane was immersed in the reaction bottles filled with the 10 wt% monomer solution of MAA. The bottles were degassed, completely sealed, and kept at 70 C for 20 h. Samples were taken out and washed thoroughly in 70 C distilled water to remove the monomer and possible homopolymer on the surface, dried under vacuum at 60 C for 24 h, and stored in desiccators.

Collagen I Immobilization The PA6-g-PMAA samples were carried out in 2-morpholinoethanesulfonic acid buffered solution (MES, pH ¼ 5.5, Aldrich) and were treated with 5 mg=ml solution of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (WSC, Aldrich) at 4 C for 24 h, then subsequently rinsed with deionized water. The membranes

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were then reacted with collagen solution (2.5 mg=ml, in 0.3 v% acetic acid) at 4 C for 24 h. After being fully rinsed with deionized water to remove free collagen, the modified PA6 membrane was dried in a vacuum oven at 30 C to constant weight.

Characterization of Modified PA6 Surface Contact angle measurement: The wettability of original and treated PA6 membranes was measured by means of the sessile drop method. A drop shape analysis system (DSA100, Kruss Co., Germany) was used for the measurements, i.e., with a software-controlled multi-dosing system DS3228 and drop analysis software DSA3. An averaged contact angle was determined by averaging the results obtained from at least five independent specimens. Attenuated total reflection Fourier transform infrared (ATR-FTIR): The chemical structure of original and treated PA6 membranes was tested by a Fourier transform infrared (FT-IR) spectrophotometer (Nicolet-5700, Thermo Nicolet Co., USA) in a wave-number range from 4000 cm1 to 600 cm1 at 4 cm1 resolution. X-ray photoelectron spectroscopy (XPS): The chemical compositions of the original and the treated PA6 membrane surfaces were determined by X-ray photoelectron spectroscopy (XPS). The XPS measurements were made by an X-ray photoelectron spectrometer (XSAM800, KRATOS, England) with a monochromatized Al Ka X-ray source (1486.6 eV photons). All specimens were analyzed at a photoelectron take-off angle of 20 . The XPS spectra were referenced with respect to the 286.4 eV C1 s (CC) core level to eliminate the charging effects. Atomic force microscope (AFM): The surface morphology and roughness of the original and the grafted PA6 membrane were analyzed by atomic force microscopy (SPA400, Seiko, Japan) in tapping mode at room temperature, and images were taken in a dynamic force mode (DFM mode) at an optimal force. The surface roughness was expressed as the root mean square (RMS) roughness.

Cytocompatibility in Vitro Cell culture: The cell culture system used in this study was rat embryo osteoblastsic cells (ROBs). Bone cells were isolated from neonatal rat calvaria according to a standard procedure [23]. Osteoblastic stromal cells were isolated from the calvarias of 3-day-old Sprague-Dawley rats using sequential enzymatic digestion. Cells were collected from the third and fourth digestions. Osteoblastic stromal cells were cultured in Ham’s F-12 media supplemented with 10% FBS and 1% penicillin=streptomycin and maintained in a humidified incubator at 37 C (5% CO2=balanced air). The medium was changed every third day. The passage 2 was used for matrix seeding.


Cell seeding: The original and treated PA6 membranes (u 12 mm 1 mm) were sterilized by ethylene oxide gas and incubated in culture media for 24 h before cell seeding. The original and treated PA6 membranes were seeded with osteoblasts (4 104 cells=cm2) by direct pipetting of the cell suspension and incubated at 37 C=5% CO2 in 1 ml of cell culture medium in 24-well dishes. The cell culture medium was changed every 3 days. Cell attachment and proliferation: The cell attachment of osteoblasts cultured with the original and treated PA6 membranes was determined using the MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenylte-2H-tetrazolium bromide) assay after incubation for 4 h. Cell proliferation was evaluated after seeding for 1, 4, 7, and 11 day(s), with the medium replaced every second day. Adhesion and viable cells on substrates were assessed quantitatively using the MTT assay. Samples=cell constructs were placed in a culture medium containing 45 ml MTT solution at 37 C for 4 h. After removal of supernatants, 450 ml=well of DMSO (Dimethyl sulfoxide) was added and mixed. After complete solubilization of the MTT formazan, the absorbance of the contents of each well was measured at 570 nm with a microplate spectrophotometer (Perkin Elmer Co). Cell morphological assessment: Osteoblasts were seeded on the original and treated PA6 membranes at a density of 6 104 cells=cm2 for 72 h in a humidified atmosphere at 37 C and 5% CO2. After incubation, the medium was removed and the specimens were rinsed with PBS, fixed with 2.5% volume fraction of glutaraldehyde, subjected to graded alcohol (30, 50, 75, 83, 95 and 100 v=v%) dehydration, rinsed with isoamyl acetate, and observed with SEM (JEOL, JEM-100CX, Japan). The morphology, cytoskeleton, and spreading of osteoblasts cultured with the pure PA 6 and PA6-g-PMAA-collagen membranes were observed by fluorescent staining. Cells were fluorescently dyed with DiI (1,10 -dioctadecyl-3,3,30 ,30 tetramethylindocarbocyanine) after being cultured for 24 h, and the images were recorded using a fluorescence inverted microscope (Nikon TE300, Japan).

Statistical Analysis The quantitative data were expressed as mean standard deviation (S.D.). The significance of differences in means was determined using student’s t-test between experimental groups; a value of p < 0.05 was considered to be statistically significant.

RESULTS Ar Plasma-Treated and Grafting of PMAA onto PA6 Surfaces Polymers may be treated with an inert-gas plasma and then exposed to oxygen to generate hydroperoxide active species that may initiate the grafting

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of a desired monomer [20], then provide active sites for the binding of protein molecules. This method is highly surface-selective, where the surface modification is confined to a depth of a few nanometers without modification of the bulk properties. Moreover, plasma processing is an extremely useful technique to design PA6 membrane for the immobilization of collagen and cell growth on the modified surfaces [21]. Therefore, we used plasma technology to activate the surface of PA6. Upon Ar plasma treatment radicals are formed on the polymer surface, which react with oxygen in contact with air and form hydroperoxides. The subsequent thermal decomposition of the hydroperoxides produces secondary radicals that are able to initiate the polymerization of MAA. Figure 1 shows the changes of the water contact angle (h) and O=C atomic ratio of PA sample versus Ar plasma treatment time. The modified PA6 membrane shows an increase in the O=C ratio from 0.18 for the untreated polymer to the limiting value of 0.59. The simultaneous increase and saturation of the O=C ratios obtained by XPS also reflects this improvement in the hydrophilicity. It is apparent that the PA6 surfaces were easily modified with the Ar plasma, and a treatment time of 6 s was sufficient to decrease the contact angle from 70.0 2.0 to 22.5 3.0 and then decrease slightly to 90 s, which suggests that the changes on the surface of the treated PA6 samples mainly happened within the first 10 treatment times. The increased surface wettability may be correlated with both the introduction of hydrophilic groups due to Ar plasma oxidation of the surface and the change in surface morphology. Ar-plasma treated PA6 membrane for 10 s, then the contact angle of PA6-g-PMAA changed to 49.5 1.5 due to the introduction of some functional carboxyl groups onto the PA6 surface. The water contact angle of PA6-g-PMAA-collagen changed to 55.0 2.5 after collagen immobilization for 24 hours.

Figure 1: The variation curves of water contact angle and O=C versus treating time at 60 W.


Surface Characterization ATR-FTIR Analysis The ATR-FTIR spectra in Figure 2 show the change of chemical structure on the original and treated PA6 membranes. In Fig. 2a, the characteristic adsorption peak at 3302 cm1 is for NH vibration, and bands around 2935 and 2861 cm1 represent CH2 vibration. The amide I absorption (n C=O) at 1635 cm1 and the amide II absorption (d NH) at 1538 cm1 can be also observed. Compared with original PA6 in Fig. 2a, a new peak at 1704 cm1 in Fig. 2b is ascribed to the grafted PMAA, resulting from the absorption of carboxylic groups. After immobilization of collagen, the peak of PMAA carboxylic groups at 1704 cm1 disappears (Fig. 2c) due to the reaction between carboxyl groups of PMAA and amino groups of collagen through the coupling agent (EDAC). The spectrum of PA6-g-PMAA-collagen was almost the same as that of the PA6 control; in contrast, the amide I absorption (n C=O) shifts at 1649 cm1 and the amide II absorption (d NH) shifts at 1553 cm1 due to the collagen immobilized onto the PA6 membrane surface.

XPS Analysis Figure 3 shows the XPS wide scan spectra and the corresponding O1 s peak of the original and treated PA6 membrane. The peaks are obtained by the ‘‘XPS peak’’ software affixed to the ESCA instrument. The XPS wide scan spectrum of the PA6 surface is shown in Fig. 3a. It reveals that carbon,

Figure 2: ATR-FTIR spectra of (a) PA6, (b) PA6-g-PMAA, and (c) PA6-g-PMAA-collagen membranes.

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Figure 3: XPS wide scan spectra of the pure and modified PA6 membranes: (a) pure PA6; (b) PA6-g-PMAA; and (c) PA6-g-PMAA-collagen, and inserted spectra show the corresponding oxygen signal at high resolution.

oxygen, and nitrogen signals are present. The spectra of PA6-g-PMAA and PA6-g-PMAA-collagen membranes (Fig. 3b and c) also show the same peaks as pure PA6 surface corresponding to C1 s, O1 s, and N1 s. In contrast, the relative intensity of carbon, oxygen, and nitrogen peaks varied after grafting with methacrylic acid and immobilizing with collagen. In Fig. 3a, the inserted O1 s spectrum of PA6 can be considered as the contribution of oxygen in the amide units (CONH). There is one expected peak located at 531.8 eV in Fig. 3a. In contrast, a new peak appearing at 532.9 eV is attributed to an oxygen atom with a double bond to the carbon atom in C(=O)OR coming from the PMAA in Fig. 3b. The appearance of the peak at 533.0 eV is ascribed to an oxygen atom with a double bond to carbon atom in CONH2 coming from the collagen in Fig. 3c [24,25]. The C1 s XPS spectra are shown in Fig. 4. The binding energies are 284.8 eV for CCC, 286.0 eV for CCN, and 287.8 eV for CC¼O, respectively. After the grated modification procedure, there is a surface chemistry change. As shown in Fig. 4b, the appearance of the peak at 288.8 eV indicates the existence of carbon atoms with a double bond to oxygen in carbonyl groups C(=O)OR on the surface of the PA6 substrate, which verifies the chemical bond between PMAA and PA6 [24,25]. Compared with the original PA6, the C1 s of carbon atoms with a double bond to oxygen in the amide group shifts to 288.2 eV due to the collagen immobilized on the PA6 surface in Fig. 4c.

Figure 4: XPS spectra of the C1 s peak of the pure and modified PA6 membranes: (a) pure PA6; (b) PA6-g-PMAA; and (c) PA6-g-PMAA-collagen.


These results demonstrate the grafting occurrence of PMAA on the PA6 surface, and there is immobilized collagen present on the PA6 membrane. AFM Measurement Topographic images from the surfaces of the original PA6, PA6-g-PMAA, and PA6-g-PMAA-collagen membranes are shown in Fig. 5. It can be seen that the pure PA6 surface was almost smooth at a scale of 5.0 mm (Fig. 5a). The surfaces of the treated PA6 membranes in Fig. 5b and 5c become rougher than that of the original PA6 membrane. The root mean square (RMS) roughness is 22.05 nm for original PA6, 35.51 nm for PA6-g-PMAA, and 53.70 nm for PA6-g-PMAA-collagen based on a 5.0 5.0 mm2 scan area. It further indicates the successful grafting of PMAA and collagen on the PA6 surface. That is to say, after grafting PMAA and collagen on PA6 membrane, the surface topography of PA6 has undergone significant changes.

Cell Culture Cell Proliferation The results of the proliferation of cells cultured on the pure PA6 and PA6-gPMAA-collagen membranes are shown in Fig. 6. It can be seen that the cell proliferation on all three kinds of materials increased with culture time. This shows that all samples do not display significant cytotoxicity against cells. After culturing for 11 days, statistical analysis indicated that the proliferation of cells on PA6-g-PMAA-collagen is both significantly higher than on the pure PA6 sample (p < 0.05). The results indicate that cell proliferation on modified PA6 membranes is superior to that on the untreated PA6 sample. Cell Morphology It has been reported that surface characteristics of material govern cell growth, gene expression, extracellular matrix metabolism, and differentiation

Figure 5: Atomic force micrographs of the original and the modified PA membranes: (a) PA6; (b) PA6-g-PMAA; and (c) PA6-g-PMAA-collagen membranes.

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Figure 6: Proliferation rate of ROBs on PA6 and PA6-g-PMAA-collagen.

[26,27]. In Fig. 7, osteoblasts show morphological details and the spindleshaped cells attached to the pure PA6 membranes and PA6-g-PMAA-collagen membranes cultured for 24 h. Osteoblasts attached on pure PA6 membranes displayed a spindle-like morphology (Fig. 7a). Compared with that of pure PA6 membrane, osteoblasts attached on PA6-g-PMAA-collagen membranes preserve a morphology, while developing numerous cellular processes (lamellipodia and filopodia) to facilitate cell–substrate and cell–cell interactions (Fig. 7b) [28]. These results suggest that there were different cellular behaviors between the pure and modified polyamide 6 membranes. Figure 8 shows SEM micrographs of morphologies of ROBs cultured on pure PA6 and PA6-g-PMAA-collagen samples at 72 h after seeding the cells. As shown in Fig. 8a, the ROBs started to spread and exhibited a spindle-like shape on the

Figure 7: Fluorescence images of osteoblasts adhered on: (a) the PA6; and (b) PA6-gPMAA-collagen membranes after 24 h incubation ( 100). (Figure is provided in color online.)


Figure 8: SEM micrographs of the osteoblasts cultured on: (a) the PA6; (b) and PA6-gPMAA-collagen membranes after 72 h cell culture.

pure PA6 sample surface through the 72 h, while on the PA6-g-PMAA-collagen membrane in Fig. 8b the cells were spindle-like in shape and more spread out. Cells firmly attached, spread well and formed a confluent layer in intimate contact with the modified PA6 surface; however, the cells also spread unevenly on the pure PA6 sample in Figure 8a. A large amount of lamellipodia structure was noticed in this study, which indicates the preferable, strong interactions between cells and the PA6-g-PMAA-collagen membrane surface.

DISCUSSION Usually, the surface modification of biomaterials with bioactive molecules is a simple way to make biomimetic materials. Cell adhesion to materials is mediated by cell-surface receptors, interacting with cell adhesion proteins bound to the material surface. In aiming to promote receptor-mediated cell adhesion, the polymer surface should mimic the extracellular matrix (ECM), because the ECM plays a major role in regulating growth factor signaling, acting as a local reservoir for latent forms, and rapidly releasing and activating them on demand [29,30]. Herein, biomolecules such as fibronectin, collagen, insulin, and the epidermal growth factor have often been introduced on polymer surfaces to enhance cell attachment or cell proliferation [31]. For example, collagen type I, the main extracellular matrix protein of bone tissue, has been widely investigated in biomolecular surface modification of implant devices. Polyamide 6 exhibits excellent mechanical properties, mainly used in engineering components [32]. In the biomedical field, polyamide 6 has been widely used for artificial ligaments, tendons, inguinal meshes, and joints. As indicated by a previous study [33], polyamide can easily be modified by an atmospheric pressure air dielectric barrier discharge. However, it has rarely been found in practical application. Plasma are shown to be very convenient

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for the activation or modification of polymer surfaces, and such discharges provide chemically mild and mechanically nondestructive means for surface modification and produce desirable surface characteristics. The experimental results showed that a short time Ar plasma treatment led to morphological, wettability, and chemical changes in the PA6 membranes. In order to examine the in vitro biological property of the difference between PA6 and PA6-g-PMAA-collagen membranes, a cytocompatibility test has been performed, focusing on attachment, proliferation, and cell morphology by direct contact with the target rat calvarial osteoblasts (ROBs). The proliferation of cells on PA6-g-PMAA-collagen membranes increases significantly during 11 days (Fig. 6). Cell proliferation data showed that osteoblasts could grow on PA6 matrices for 11 days and collagen immobilized onto PA6 is a more suitable material for osteoblast growth than the untreated one. Moreover, the cells incubated with PA6-g-PMAA-collagen membranes show a well-preserved morphology and cells dramatically reproduce and aggregate to form stratified cell layers at one day (Fig. 7). In contrast, the rate of cell growth was high enough and the cell confluence was observed within three days where the PA6-g-PMAA-collagen membrane surface was completely covered with cells (Fig. 8). In the present study, ROBs on the surface of the PA6-g-PMAA-collagen membranes can largely spread out, suggesting good cell adhesion capability. Both the PA6 and PA6-g-PMAA-collagen membranes have no negative effect on the cell morphology, cell viability, and proliferation. In contrast, the PA6-g-PMAA-collagen membranes, on the other hand, show excellent adherence and rapid cell growth. There are some basic factors governing cellular proliferation, migration, and physiological response of biomaterial surfaces, such as the hydrophilic property, structure, and chemical composition of the membrane surface, etc. [34–36]. Appropriate modification of the surface characteristics of the membrane increases cell adhesion, proliferation, migration, and differentiation [37]. On the one hand, the hydrophilicity and topography can influence the attachment of cells on materials. It is known that surface wettability plays an important role in cell-material interaction and modulates both cell adhesion and protein adsorption, where cell adhesion and motility are improved on more hydrophilic surfaces [38,39]. The average contact angle of a water droplet on an osteoblast monolayer is reported to be 26.9 0.3 . In this paper, the water contact angles of PA6 and PA6-g-PMAA-collagen are 70.0 2.0 and 55.0 2.5 , respectively. It was found that the higher hydrophilic surface of PA6-gPMAA-collagen was better for cell growth and proliferation than the pure PA6 surface. Surface topography and roughness are also important factors in determining the response of cells to a material [40]. Surfaces with grooves can induce ‘‘contact guidance’’ and difference in roughness can affect adsorption of fibronectin and albumin in vitro to influence cell attachment and adhesion. The topography and roughness (53.70 nm) of PA6-g-PMAA-collagen are


different from the topography and roughness (22.05 nm) of the pure PA6 membrane, corresponding to different cell proliferation behavior between them. An obvious enhancement of cytocompatibility on the collagen-immobilized surfaces should result from the improvement of surface chemical composition, hydrophilicity, and topography. On the other hand, in the attachment assay, osteoblasts attached most easily to the collagen-treated membranes, but significantly less easily to the pure polyamide 6. This is not surprising, since collagen as the main extracellular matrix protein of bone tissue contains the arginine–glycine– asparagine (RGD) peptide sequence of amino acids, which helps osteoblasts attach to the membranes [41]. Moreover, the covalent attachment of collagen to the biomaterial surface brings a reasonable stabilization by crosslinking, which achieves the desired enhancement in biocompatibility [31]. Compared with the original polyamide 6 membrane, the PA6-g-PMAA-collagen membrane has increased hydrophilicity, surface roughness, and surface chemical composition. An obvious improvement of cytocompatibility is observed on the collagen-existing PA6 surfaces. These results indicate that this convenient and effective surface modification method does improve the surface properties of polyamide 6 membranes, thereby making them more suitable for tissue engineering. As a next step, we are still in the process of optimization of the possible influence of the graft densities and surface morphology on the osteoblasts and the functionalization of PA6 may further improve their effectiveness in new approaches towards the controlled engineering of selected tissues.

CONCLUSION Surface-linked cell-signaling biological molecules play an important role in biological interactions at the tissue=implant material interface. Therefore, a surface modification method by plasma and grafting polymerization was performed to introduce carboxyl groups (COOH) on the polyamide 6 membrane surface. A spacer arm (EDAC) was used to connect the immobilizing collagen and the PA6 substrate. The hydrophilicity and roughness of both grafted polyamide 6 membranes increased compared with untreated PA6 membrane. ROBs were cultured on original and modified polyamide 6 samples. The polyamide 6 surface immobilized with collagen promoted a higher attachment and proliferation of ROBs in comparison to the untreated PA6 sample. Therefore, hydrophilic collagen introduced onto the polyamide 6 surface supplied a better cell growth environment. It can be inferred that the surface chemical structure, hydrophilicity, and surface topography play an important role in improving the cytocompatibility. In addition, suitable surface modification may have broad application for biomaterials, which can further covalently bind adhesion mediating factors for specific cells. Thus, functionalized polyamide 6 may be more efficient to use in tissue engineering.

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REFERENCES [1] Singhal, J.P.; Ray, A.R. Biomaterials 2002, 23(4), 1139–1145. [2] Wang, H.; Li, Y.; Zuo, Y.; Li, J.; Ma, S.; Cheng, L. Biomaterials 2007, 28(22), 3338–3348. [3] Wang, H.; Zuo, Y.; Zou, Q.; Cheng, L.; Huang, D.; Wang, L.; Li, Y. J. Polym. Sci., Part A: Polym. Chem. 2009, 47(3), 658–669. [4] Menon, D.; Anoop Anand, K.; Anitha, V.C.; Nair, S. Int. J. Polym. Mater. 2010, 59(7), 498–509. [5] Mu¨ller, R.; Abke, J.; Schnell, E.; Scharnweber, D.; Kujat, R.; Englert, C.; Taheri, D.; Nerlich, M., et al. Biomaterials 2006, 27(22), 4059–4068. [6] Puleo, D.A.; Nanci, A. Biomaterials 1999, 20(23–24), 2311–2321. [7] Kim, Y.J.; Kang, I.K.; Huh, M.W.; Yoon, S.C. Biomaterials 2000, 21(2), 121–130. [8] Shin, H.; Jo, S.; Mikos, A.G. Biomaterials 2003, 24(24), 4353–4364. [9] Goddard, J.M.; Hotchkiss, J.H. Prog. Polym. Sci. 2007, 32(7), 698–725. [10] Tao, G.; Gong, A.; Lu, J.; Sue, H.-J.; Bergbreiter, D.E. Macromolecules 2001, 34(22), 7672–7679. [11] Vasilets, V.N.; Hermel, G.; Ko¨ig, U.; Werner, C.; Mu¨ler, M.; Simon, F.; Grundke, K.; Ikada, Y., et al. Biomaterials 1997, 18(17), 1139–1145. [12] Shen, J.; Li, Y.; Zuo, Y.; Zou, Q.; Li, J.; Huang, D.; Wang, X. J. Biomed. Mater. Res. Part B: Appl. Biomater. 2009, 91B(2), 897–904. [13] Michael, M.N.; El-Zaher, N.A.; Ibrahim, S.F. Polym. Plast. Technol. Eng. 2004, 43(4), 1041–1052. [14] Chu, P.K.; Chen, J.Y.; Wang, L.P.; Huang, N. Materials Science and Engineering: R: Reports 2002, 36(5–6), 143–206. [15] Kogelschatz, U. Plasma Sources Sci. Technol. 2002, 11(3A), A1–A6. [16] Behnisch, J.; Holla¨nder, A.; Zimmermann, H. Int. J. Polym. Mater. 1994, 23(3), 215–224. [17] Gawish, S.M.; Ramadan, A.M.; Matthews, S.R.; Bourham, M.A. Polym. Plast. Technol. Eng. 2008, 47(5), 473–478. [18] Li, J.T. Polym. Plast. Technol. Eng. 2010, 49(2), 204–207. [19] Ratnam, C.T.; Hill, D.J.T.; Rasoul, F.; Whittaker, A.K.; Keen, I. Polym. Plast. Technol. Eng. 2010, 49(1), 1–7. [20] Gupta, B.; Plummer, C.; Bisson, I.; Frey, P.; Hilborn, J. Biomaterials 2002, 23(3), 863–871. [21] Shen, H.; Hu, X.; Yang, F.; Bei, J.; Wang, S. Biomaterials 2007, 28(29), 4219–4230. [22] Gupta, B.; Hilborn, J.G.; Bisson, I.; Frey, P. J. Appl. Polym. Sci. 2001, 81(12), 2993–3001. [23] Schwartz, E. Culture of Animal Cells; Wiley: New York, 1987. [24] Beamson, G.; Briggs, D. High Resolution XPS of Organic Polymers the Scientia ESCA300 Database; John Wiley & Sons: New York, 1992, 210, 212. [25] Briggs, D.; Seach, M.P. Practical Surface Analysis, Vol. 1. Auger and X-ray Photoelectron Spectroscopy; JohnWiley & Sons: Chichester, England, 1996.

Polyamide 6 Immobilized with Collagen [26] Ma, Z.; Gao, C.; Gong, Y.; Shen, J. Biomaterials 2003, 24(21), 3725–3730. [27] Chen, C.S.; Mrksich, M.; Huang, S.; Whitesides, G.M.; Ingber, D.E. Science 1997, 276(5317), 1425–1428. [28] Cai, K.; Rechtenbach, A.; Hao, J.; Bossert, J.; Jandt, K.D. Biomaterials 2005, 26(30), 5960–5971. [29] Klee, D.; Ademovic, Z.; Bosserhoff, A.; Hoecker, H.; Maziolis, G.; Erli, H.-J. Biomaterials 2003, 24(21), 3663–3670. [30] Ma, Z.; Gao, C.; Ji, J.; Shen, J. Eur. Polym. J. 2002, 38(11), 2279–2284. [31] Kang, I.K.; Choi, S.H.; Shin, D.S.; Yoon, S.C. Int. J. Biol. Macromol. 2001, 28(3), 205–212. [32] Vyas, H.; Shrinet, V.; Murthy, C.N.; Jain, R.C.; Singh, A.K. Polym. Plast. Technol. Eng. 2008, 47(12), 1231–1241. [33] Cui, N.Y.; Upadhyay, D.J.; Anderson, C.A.; Brown, N.M.D. Surf Coat Technol. 2005, 192(1), 94–100. [34] Tidwell, C.D.; Ertel, S.I.; Ratner, B.D.; Tarasevich, B.J.; Atre, S.; Allara, D.L. Langmuir 1997, 13(13), 3404–3413. [35] Stevens, M.M.; George, J.H. Science 2005, 310(5751), 1135–1138. [36] Wang, Y.-X.; Robertson, J.L.; Spillman, W.B.; Claus, R.O. Pharm. Res. 2004, 21(8), 1362–1373. [37] Garcia, A.J.; Ducheyne, P.; Boettiger, D. J. Biomed. Mater. Res. 1998, 40(1), 48–56. [38] van Wachem, P.B.; Hogt, A.H.; Beugeling, T.; Feijen, J.; Bantjes, A.; Detmers, J.P.; van Aken, W.G. Biomaterials 1987, 8(5), 323–328. [39] He, Z.; Xiong, L. Polym. Plast. Technol. Eng. 2009, 48(10), 1020–1024. [40] Brunnette, D.M. Int. J. Oral. Maxillofac. Implants. 1988, 3, 231–246. [41] Grzesik, W.J.; Robey, P.G. J. Bone. Miner. Res. 1994, 9, 487–496.

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