Acta Biomaterialia 3 (2007) 183–190 www.actamat-journals.com

Cytocompatibility of poly(acrylonitrile-co-N-vinyl-2-pyrrolidone) membranes with human endothelial cells and macrophages Ling-Shu Wan a, Zhi-Kang Xu a

a,*

, Xiao-Jun Huang a, Xiao-Dan Huang b, Ke Yao

b

Institute of Polymer Science, and Key Laboratory of Macromolecular Synthesis and Functionalization (Ministry of Education), Zhejiang University, Hangzhou 310027, China b College of Medicine, Zhejiang University, Hangzhou 310009, China Received 25 May 2006; received in revised form 13 September 2006; accepted 15 September 2006

Abstract Polyacrylonitrile modified with N-vinyl-2-pyrrolidone (NVP) shows good hemocompatibility. This work, which aims to evaluate the cytocompatibility of membranes fabricated from poly(acrylonitrile-co-N-vinyl-2-pyrrolidone) (PANCNVP), studied the adhesion of macrophages and endothelial cell (EC) cultures. It was found that PANCNVP membranes with higher NVP content decreased the adhesion of both macrophages and ECs. Compared with polyacrylonitrile and tissue culture polystyrene control, however, these PANCNVP membranes promoted the proliferation of ECs. Furthermore, the viability of ECs cultured on the PANCNVP membrane surfaces was also relatively competitive. Both static and dynamic water contact angle measurements were conducted to explain the nature of cell adhesion to the PANCNVP membranes. On the basis of these results and the phenomena of water swelling and water states reported previously, it was presumed that the coexistence of large amounts of bound water and free water induced by NVP moieties are responsible for the lower adhesion and better function of cells adhering to the PANCNVP membranes.  2006 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved. Keywords: Membranes surface; Cytocompatibility; Endothelial cell; Macrophage; Copolymer

1. Introduction Poly(N-vinyl-2-pyrrolidone) (PVP), a known watersoluble and biocompatible polymer, has been widely used as a blood plasma substitute, additive and surface modifier [1–12]. In particular, PVP ensures excellent hemocompatibility for many kinds of dialysis membranes, e.g. polyacrylonitrile and polysulfone membranes. PVP is usually blended with other polymers for membrane preparation, but its subsequent elution from the membrane is disadvantageous during hydraulic permeation [8]. Therefore, we synthesized copolymers of acrylonitrile and N-vinyl-2pyrrolidone (NVP) by a simple process, and our previous work has confirmed the hemocompatibility of the corresponding copolymer membranes [13,14]. The introduction *

Corresponding author. Fax: +86 571 8795 1773. E-mail address: [email protected] (Z.-K. Xu).

of NVP into polyacrylonitrile (PAN) can suppress blood platelet adhesion and increase plasma recalcification time as well as reduce bovine serum albumin adsorption on the PAN-based membranes. In addition, these copolymers have superior membrane-forming properties. Therefore, this type of membrane appears suitable for hemodialysis. Generally speaking, microporous polymer membranes, except those being considered for tissue engineering, should inhibit protein adsorption as well as cell adhesion [15]. It is believed that the behavior of cells on a specific biomaterial surface may vary greatly with the type of cell investigated [15,16]. Macrophages are a kind of immune cell and perform various functions in living bodies, such as migration, phagocytosis, secretion, antigen presentation and survival through precisely modulated adhesion [17,18]. Preventing the activation of immune cells is a requirement for biomedical membranes. In contrast, endothelial cells (ECs) constitute the natural antithrombotic surface contacting the

1742-7061/$ - see front matter  2006 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.actbio.2006.09.007

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blood in native vessels. Endothelialization is an approach to produce an antithrombotic surface for blood-contacting devices, such as vascular grafts. Many efforts have been made to generate EC-seeded surfaces, including surface plasma treatment [19], patterned or phase-separated surface construction [20–24], protein/peptide coating or immobilization [25–35], chemical modification [36,37] and other methods [38–40]. However, results show that blood platelet adhesion to the endothelialized surface is affected not only by the seeding of ECs but also by the underlying substrate. The nature of the substrate affects the coagulant functions of ECs, such as the production of plateletactivating factor and tissue plasminogen activator [41]. Therefore, studying the interactions of materials with ECs that have special significance for hemocompatibility may help in understanding the hemocompatibility of such materials. In this work, macrophage adhesion and EC culture were performed to evaluate the cytocompatibility of poly(acrylonitrile-co-N-vinyl-2-pyrrolidone) (PANCNVP) membranes. 2. Materials and methods 2.1. Membrane preparation PANCNVP and PAN were synthesized in our laboratory. Details of the synthesis and characterization of the polymers were described previously [13,14]. The NVP content of the copolymers, calculated from 1H NMR spectra, was 7, 15, 22 or 31 wt.% and those copolymers were denoted as PANCNVP7, PANCNVP15, PANCNVP22 and PANCNVP31, respectively. Membranes were prepared by casting the polymer solutions in DMF (8 wt.%) onto clean glass plates which were then dried for 24 h at 100 C under vacuum to remove the residual solvent. After immersion in pure water for another 24 h, the resultant membranes were finally dried at 60 C under vacuum. All membranes for contact angle measurement, macrophage adhesion and EC culture were treated according to the same procedure. The thickness of these membranes was approximately 18 ± 2 lm. 2.2. Water contact angle measurement The water contact angles of the membranes were measured by the sessile drop method with a contact angle goniometer (OCA20, Dataphysics, Germany) at room temperature. In a typical sessile drop method, a water drop (5 ll) was placed on the dry membrane surface and the static water contact angle was determined after 10 s. The dynamic contact angle – in our case, the dependence of contact angle on time – was recorded every 30 s for 30 min. 2.3. Macrophage adhesion The murine macrophage suspension was prepared using the method reported previously [17]. This suspen-

sion was isolated from freshly killed mice. After killing the animals with chloroform, the skin was sprayed with alcohol and the abdomen was opened. A 10 ml sample of Roswell Park Memorial Institute (RPMI) 1640 containing 10% fetal bovine serum (FBS), 100 U/ml penicillin and 100 lg/ml streptomycin was injected into the peritoneal cavity, and then the abdomen was gently massaged manually for 5 min. The peritoneum was carefully punctured, and then the washings were removed with a sterile pipet and placed in a sterile container which was centrifuged at 1000 rpm. for 10 min to collect the macrophages. The macrophages were grown in RPMI 1640 to obtain a macrophage suspension in which the cell concentration was 1 · 106 cells/ml. The membrane (10 · 10 mm2) was cleaned sequentially in an ultrasonic bath of ethanol solution for 10 min and rinsed in PBS. The sample was then immersed in physiological saline (pH 7.4) to recondition for 2 h. The cell suspension was inoculated on the surface of the membrane to assess the cell adhesion. The incubation period was 48 h for the cell adhesion test in a humidified atmosphere of 5% CO2 in air at 37 C. The supernatant was then removed, and the membrane was washed cautiously five times using PBS (pH 7.2), and the adherent cells were fixed by the addition of methanol for 5 min. The adherent cell density on the membrane was quantified on the basis of measurements obtained visually from at least five randomly selected fields (0.24 · 0.36 mm2) using an Olympus TE300 phase-contrast optical microscope. The mean values and standard deviation of triplicate samples for each membrane were reported. 2.4. Endothelial cell culture Human umbilical vein ECs were isolated from the human umbilical cord veins with 1.0 mg/ml collagenase (type I, Sigma)/phosphate buffer solution (PBS, pH 7.4) for 20 min at room temperature. The isolated ECs were routinely seeded at a density of 100,000 cells/cm2 on the membranes laid in the cell culture well. As a control, the ECs were also directly seeded on tissue culture polystyrene (TCPS, Nunc, Denmark). The ECs were incubated in a culture medium consisting of 20% (v/v) FBS and 80% RPMI 1640 supplemented with 100 U/ml of penicillin and 100 lg/ ml of streptomycin in humidified air containing 5% CO2 at 37 C. The morphology of living cells after 96 h of incubation was observed under an optical microscope (BX51T3200, Olympus). After 12 h and 4 days of incubation, the medium was discarded. The samples were washed three times with PBS, which removed the poorly adhering and suspended cells. Then 100 ll 0.25% trypsin solution was added to each well. After a 15 min digestion, 100 ll of media was added and the cells were resuspended. The cells were counted with a hemocytometer and a flow cytometer. The adhesion density of cells on the TCPS surface was used as a standard. We define the cell adhesion ratio at t = 12 h or 4 days as

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r(t) = Nsample(t)/NTCPS(t), where N is the adhesion density of cells. The cell viability was measured by the methylthiazoletetrazolium (MTT) assay. After 4 days of incubation, 20 ll of MTT (5 mg/ml in PBS) was added to each well, followed by another 4 h incubation at 37 C. The supernatants were then removed and 100 ll of DMSO added to dissolve the tiny crystals. The absorbance at 490 nm was recorded and four parallel measurements were averaged for each sample. 3. Results Fig. 1 shows the water contact angles on the membrane surfaces. It was found that all the original static contact angles of PAN and PANCNVPs were around 65. This result was in agreement with that reported by Groth et al. [42]. In view of the hydrophilic characteristics of NVP, dynamic contact angle measurements were conducted, as this angle reflects the water-absorbing ability of the membranes. Advancing and receding contact angles are named ‘‘dynamic’’ contact angles, and can be measured by adding/withdrawing pure water to/from the water drop, as reported in a previous paper [13]. Unfortunately, the measured advancing and receding contact angles did not reflect the changes induced by NVP in this study, a finding that might be attributed to the moderate hydrophilicity of PAN. Therefore, the dynamic water angle was measured by monitoring the age of the water drop on the sample surface. It can be seen from Fig. 1 that the introduction of NVP caused a large decrease in water contact angle with time on the membrane surface, which was mainly due to water absorption into the membrane. In fact, PVP is a kind of versatile hydrogel material with a proven capability for water absorption [9,10,12].

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Macrophage adhesion on the membrane surface was performed. Although the molecular mechanism of macrophage adhesion is complex, dynamic and not yet fully understood, it is generally accepted that the fewer macrophages adhere, the better the biocompatibility of the material. Fig. 2 shows micrographs of macrophages adhering to the membrane surface. The average numbers of adhered macrophages are shown in Fig. 3. Compared with PAN membrane, PANCNVP membranes – especially those with high NVP content, i.e. PANCNVP 22 and PANCNVP 31 – could greatly suppress the adhesion of macrophages. ECs were also cultured on the membrane surfaces, and TCPS was used as a control. Fig. 4 presents the growth behaviors of ECs on different membrane surfaces. It was found that the number of attached ECs decreased with the increase of NVP content in the PANCNVP membranes. On the control TCPS, the number of attached ECs was the largest after culture for 12 h or even 96 h. It seems that the introduction of NVP could reduce the adhesion of ECs, even if ECs are a kind of natural bloodcontacting cell. Nevertheless, ratio for the number of attached ECs at 96 h to that at 12 h, otherwise known as the cell proliferation, increased with the NVP content of the membranes. As we know, attached cells may partly maintain their functions and secrete proteins that can modify the underlying substratum to promote cell adhesion. It might be speculated that the PANCNVP membranes could favor the functions of adhered cells in comparison with PAN membranes. Similar results have been reported by Groth and co-workers [15]. The viability of attached ECs over a period of 96 h was investigated using MTT assay. Four parallel samples for each membrane were tested and the results are shown in Fig. 5. It was found that the values of the MTT assay of ECs adhering to TCPS and PAN membranes were higher than those adhering to PANCNVP membranes. But, in view of the relatively lower EC adhesion on the PANCNVP membranes, the MTT viability of ECs attached to PANCNVP membranes (especially PANCNVP22 and PANCNVP31) was competitive. This was confirmed by the MTT assay value divided by the value of EC adhesion ratio as shown in Fig. 5. The morphologies of ECs cultured for 96 h were observed by optical microscopy. As can be seen from Fig. 6, many of the ECs homogeneously attached to the TCPS surface are well spread. Similar adhesion occurred for ECs on the PAN or PANCNVP7 membrane surfaces on which ECs spread in a polygon fashion. However, ECs adhered to the PANCNVP22 and PANCNVP31 membrane surfaces poorly and most of them remained in a rounded-up shape.

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4. Discussion

Time (min)

Fig. 1. Typical curves of the time dependence of water contact angle on the membrane surfaces measured by the sessile drop method. The inset shows the contact angle change (D(CA)) after 30 min.

Krasteva et al. [15] discussed in detail the reasons for the poor adhesion of human hepatoblastoma C3A cells to the polyetherimide (PEI) and PANCNVP membrane surfaces.

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Fig. 2. Optical micrographs of macrophages adhering to the membrane surfaces: (a) PAN; (b) PANCNVP7; (c) PANCNVP15; (d) PANCNVP22; and (e) PANCNVP31.

However, as they pointed out, different cells might show diverse behaviors. Therefore, we explored the cytocompatibility of PANCNVP membranes with ECs and macrophages. Although many works have been published upon the promotion of EC adhesion, proliferation and viability, most of these concentrated on biologically active materials. The results reported in this work indicate the low cell adhesion (for both macrophages and ECs) and relatively high proliferation ratio and MTT viability of ECs on the PANCNVP membranes. It seems that the NVP-modified materials studied were to some extent inert. Why, therefore, does this type of material achieve good hemocompatibility? There is considerable interest in PVP- or NVP-containing materials and some results have also been reported

concerning cell culture on them. Kottke-Marchant et al. [41,43,44] obtained plasma polymer films of NVP and c-butyrolactone by the radiofrequency glow discharge method. They found these films to be highly hydrophilic and suitable for seeding ECs. Groth et al. [15,42,45] reported the culture of human skin fibroblasts, hepatocyte and hybridoma cells on PANCNVP membrane surfaces. It was found that the cells attached and spread better on the PAN membrane surface than on the PANCNVP ones, while the functions of the attached cells were just the reverse, e.g. the secretion of human serum albumin for the attached human skin fibroblasts and the production of monoclonal antibodies for the hybridoma cells were better on the PANCNVP membrane surfaces. PVP-based hydrogel was also studied by Risbud et al. [12]. They

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Fig. 3. Adhesion density of macrophages on the membrane surfaces.

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Fig. 4. Endothelial cell adhesion ratio of different samples after incubating for 12 h (shadow column) and 96 h (gray column). All data presented are simple mean values from the results of four parallel samples, and the bars represent standard deviations.

proposed that the hydrogel did not activate macrophages and alter the functions of the ECs. Nevertheless, the hydrogel did not allow the majority of cells to adhere well. In fact, the effects of the wettability of substrates on the behaviors of cultured cells are very complex. Krasteva et al. [15] mentioned that, as a general rule, cells adhered less efficiently on hydrophobic surfaces. Furthermore, cells could not adhere well to either hydrophilic and hydrophobic material surfaces at the initial stage. However, Meinhold et al. [9] pointed out that modifications with water-soluble polymers such as PVP and poly(ethylene glycol) (PEG) could strongly attenuate the adsorption of proteins and the adhesion of cells. Furthermore, as shown in this paper, PAN, a moderately hydrophilic polymer (the water contact angle for PAN is about 65, Fig. 1), was a good substrate for ECs and macrophages to adhere to, while PANCNVP was not. Thus, there are no simple relations between the

Fig. 5. MTT assay after the endothelial cells were cultured on the membrane and TCPS surface for 96 h (shadow column), and divided by the value of endothelial cell adhesion ratio shown in Fig. 4 (96 h) (gray column). All data presented are simple mean values from the results of four parallel samples, and the bars represent standard deviations.

wettability of the substrates and their ability to support cell adhesion [15]. Based on the results of dynamic water contact angles (Fig. 1) and the swelling behaviors reported previously [14], one could envisage that the capability of water absorption might play an important role for materials such as PANCNVP. That is to say, materials absorbing more water might decrease the adhesion of cells. It should be noted that the hydrophilic NVP film prepared by KottkeMarchant et al. [41,43,44] was deposited on glass coverslips that had a hydrophobic coating and the film thickness was less than 100 nm. It could be speculated that the water absorption of their films was tiny and so the ECs adhered well. Furthermore, we found that, though fewer ECs adhered, the MTT viability of ECs adhering to PANCNVP22 and PANCNVP31 was relatively high, which was in accordance with previous reports [12,15,42,45]. It is generally accepted that the physicochemical properties of interfacial water profoundly influence the biological response to materials [46]. Wang et al. reported that the stability of the interfacial water layer between the PEG-modified surface and bulk water could prevent the direct contact of the surface with proteins [47]. On the basis of the results of thermal analysis on poly(2-methoxyethyl acrylate), it was also proposed by Tanaka et al. [48] that the freezing bound water, which is located between nonfreezing water and free water, can shield proteins or cells from direct contact with nonfreezing water (or material surface). Recently, Kitano et al. [49] pointed out that the carboxybetaine monomer residues with a zwitterionic structure did not significantly disturb the hydrogen bonding between water molecules in both aqueous solution and thin film systems, and thus this type of polymer showed excellent hemocompatibility. We have previously examined the water states in PAN

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Fig. 6. Optical micrographs (10·) of endothelial cells cultured on the membrane and TCPS surfaces for 96 h: (a) PAN; (b) PANCNVP7; (c) PANCNVP15; (d) PANCNVP22; (e) PANCNVP31; and (f) TCPS.

and PANCNVP membranes and found that the introduction of NVP could greatly increase the content of bound water and free water at the same time [14]. Given that free water is compatible with biological systems, it was presumed that the bound water mainly induced by NVP builds a stable layer and separates the PANCNVP membranes from the free water. The stable layer hindered the direct attachment of cells to membranes; therefore, the cells remained in free water and functioned well. Moreover, the weaker cell–substrate interaction on the water-swollen membrane could strengthen cell–cell interactions, which promote some functional features for the cells. Therefore, PANCNVP membrane could reduce the adhesion of cells (proteins) while favoring the functions of adhered cells.

5. Conclusion PAN-based membranes with various NVP contents were fabricated. The cytocompatibility of these membranes with macrophages and ECs were studied. The numbers of both macrophages and cells adhering to the membranes decreased with increasing NVP content. However, proliferation rates of ECs adhering to the PANCNVP membrane surfaces were higher than those adhering to the PAN or even the TCPS surfaces. MTT assay also indicated the relatively higher viabilities of ECs adhering to the PANCNVP membranes. Based on the dynamic water contact angle measurements and the investigation of water states for the studied membranes, as well as some findings reported elsewhere, we speculate that the coexistence of large

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