Co-culture of vascular endothelial cells and smooth muscle cells by hyaluronic acid micro-pattern on titanium surface

Applied Surface Science 273 (2013) 24–31

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Co-culture of vascular endothelial cells and smooth muscle cells by hyaluronic acid micro-pattern on titanium surface Jingan Li a , Guicai Li a,b , Kun Zhang a , Yuzhen Liao a , Ping Yang a,∗ , Manfred F. Maitz a,c , Nan Huang a a b c

Key Lab. for Advanced Technologies of Materials, Ministry of Education, School of Material Science and Engineering, Southwest Jiaotong University, Chengdu 610031, PR China Jiangsu Provincial Key Laboratory for Interventional Medical Devices, Huaiyin Institute of Technology, Huaian, Jiangsu Province 223003, PR China Leibniz Institute of Polymer Research Dresden, Max Bergmann Center of Biomaterials Dresden, Dresden 01069, Germany

a r t i c l e

i n f o

Article history: Received 5 November 2012 Received in revised form 6 January 2013 Accepted 9 January 2013 Available online 17 January 2013 Keywords: Co-culture Micro-pattern Hyaluronic acid Endothelial cells Biomaterials

a b s t r a c t Micro-patterning as an effective bio-modification technique is increasingly used in the development of biomaterials with superior mechanical and biological properties. However, as of now, little is known about the simultaneous regulation of endothelial cells (EC) and smooth muscle cells (SMC) by cardiovascular implants. In this study, a co-culture system of EC and SMC was built on titanium surface by the high molecular weight hyaluronic acid (HMW-HA) micro-pattern. Firstly, the micro-pattern sample with a geometry of 25 ␮m wide HMW-HA ridges, and 25 ␮m alkali-activated Ti grooves was prepared by microtransfer molding (␮TM) for regulating SMC morphology. Secondly, hyaluronidase was used to decompose high molecular weight hyaluronic acid into low molecular weight hyaluronic acid which could promote EC adhesion. Finally, the morphology of the adherent EC was elongated by the SMC micro-pattern. The surface morphology of the patterned Ti was imaged by SEM. The existence of high molecular weight hyaluronic acid on the modified Ti surface was demonstrated by FTIR. The SMC micro-pattern and EC/SMC co-culture system were characterized by immunofluorescence microscopy. The nitric oxide release test and cell retention calculation were used to evaluate EC function on inhibiting hyperplasia and cell shedding, respectively. The results indicate that EC in EC/SMC co-culture system displayed a higher NO release and cell retention compared with EC cultured alone. It can be suggested that the EC/SMC co-culture system possessed superiority to EC cultured alone in inhibiting hyperplasia and cell shedding at least in a short time of 24 h. © 2013 Published by Elsevier B.V.

1. Introduction Neointima hyperplasia and thrombosis are the main problems for cardiovascular implants [1,2]. It has been reported that biomaterials with in vitro endothelialization can effectively inhibit thrombosis and intimal hyperplasia [3,4] after implantation into the body. However, endothelial cells (EC) cultured in vitro do not exhibit the physiological function due to the lack of pericytes as would be under normal physiological conditions. EC and smooth muscle cells (SMC) are the major cellular components of the vessel wall [5]. EC regulate the vascular tonus, permeability, inflammation, thrombosis, and fibrinolysis through the expression and secretion of a series of molecules [6]. While SMC play an important role for EC in the maintenance of normal cell morphology and function [7], such as the anticoagulant properties of EC and the release of biofunctional factors for inhibiting hyperplasia [8]. Therefore,

∗ Corresponding author. E-mail address: [email protected] (P. Yang). 0169-4332/$ – see front matter © 2013 Published by Elsevier B.V. http://dx.doi.org/10.1016/j.apsusc.2013.01.058

it may be a promising method to build a co-culture system of EC and SMC on biomaterials surface to regulate the cell behavior, and further to investigate in vitro interactions of EC and SMC. Several co-culture systems have been developed to investigate the interactions between SMC and EC, including culture of SMC and EC on opposite sides of semipermeable membranes [9], culture of EC on collagen gels containing SMC [10], culture of EC directly on SMC [11] and culture of EC on media layer which is covered by SMC [12]. Co-culture of EC and SMC on opposite sides of a thin membrane stimulated SMC proliferation [13], but the effects on EC has not been reported. Culturing EC directly on SMC also changes EC from the normal polygonal morphology in vitro to an elongated shape [14], whereas EC presents disordered behavior because the SMC below is also disordered. The above mentioned studies are helpful for understanding the interaction of biomaterials-EC-SMC to some extent, but few of the studies have been performed on an ordered surface. As known to all, the distribution of EC and SMC in human vascular wall is ordered and this distribution guarantees normal physiological functions of the two cell types. Thus, a coculture system should be built on an ordered surface to study the

J. Li et al. / Applied Surface Science 273 (2013) 24–31

7.2, respectively. Fibronectin (Fn, Sigma–Aldrich) was diluted to a concentration of 100 ␮g/ml with PBS at pH 7.2. Cell TrackerTM Orange CMTMR and Green CMFDA (Invitrogen, USA) were diluted to a concentration of 1 mmol/ml with sterile dimethyl sulfoxide, respectively. Medium F12, medium 199 (M199), fetal calf serum and type II collagenase were purchased from Gibco BRL. EC growth factor, trypsinization, Griess reagent, rabbit antihuman actin antibody, goat anti-rabbit IgG antibody and dimethyl sulfoxide were purchased from Sigma–Aldrich. All the other reagents were analytical of grade and used as received.

O

HO

OH O

O HO

OH

OH O HO

HN

OH

O H3C

25

n

2.2. Methods

Fig. 1. Structure of hyaluronic acid.

interaction of biomaterials-EC-SMC under biomimetic conditions. The micro-pattern technology of biomolecule offers a promising approach to study the cell behavior on biomaterial surface [15]. Several kinds of biomolecule micro-pattern have been manufactured on biomaterial surface including groove/ridge stripes [16], square or round micro-domians [17], micro-networks [18], microtips [19], etc. Biomolecules such as heparin [20], fibronectin [21], collagen [22], silk fibroin [18] and arginine-glycine-aspartic acid (RGD) [23], etc. were used as single material or compounds to fabricate micro-pattern for promoting or inhibiting cell adhesion. Here, hyaluronic acid (HA) has been chosen to fabricate a stripe micro-pattern, because HA from human tissue induces only weak immune reactions, additionally, the high molecular weight hyaluronic acid (HMW-HA) inhibits cell adhesion [24], while the low molecular weight hyaluronic acid (LMW-HA) promotes cell adhesion [25]. The key substance that makes the former into the latter is hyaluronidase (HAa). The chemical structure of HA [26] is shown in Fig. 1. It is reported that a parallel groove/ridge stripes micro-pattern can guide cell growth along the grooves [27], which are considered closest to physiological EC status exposed to blood flow shear stress [28]. Thus, HMW-HA micro-pattern can be fabricated to guide SMC growth along the grooves. Moreover, after HAa cleaves HMW-HA to LMW-HA, EC can adhere on the surface and grow on the ridge of the structure parallel to the SMC in the grooves. Thus the EC/SMC co-culture system can be applied to enhance the adhesion of EC and further promote the secretion of bioactive mediators by EC. In the present work, we describe the construction, morphology, size and hydrophilicity of the HMW-HA micro-pattern on titanium (Ti) plates, and construct an EC/SMC co-culture system by the micro-pattern template for cardiovascular implants. As a preliminary study of co-culturing these two cells, the focus of functional assessment here is on EC nitric oxide (NO) release and anti-shedding that describes the stability of EC adhesion to the surfaces, which guarantee vascular endothelium anti-hyperplasia function and structure integrity [29]. Ti plates are chosen as the substrates because Ti and its alloys are the most widely used biomaterials for cardiovascular implants [30]. The modification of Ti substrate is characterized by scanning electron microscopy (SEM), Fourier transform infrared spectroscopy (FTIR) and water contact angle. To access the adhesion and anticoagulant function of EC in co-culture systems, the shear stress offered by a flow chamber is used and NO release amount is measured.

2.2.1. The preparation of HMW-HA micro-pattern on TiOH surface The fabrication strategy of the model micro-patterned surface consists of three stages. The first stage was to fabricate the polydimethylsiloxane (PDMS) stamp. PDMS prepolymer was poured on a silicon surface with stripe micro-pattern (width of ridge 25 ␮m, width of groove 25 ␮m, 25 ␮m/25 ␮m), and then the PDMS stamp was peeled off from the silicon master after vacuum drying at 80 ◦ C for 2 h. The PDMS stamp got a complementary relief structure against the silicon master. Secondly, 99.5% pure Ti plates (Baoji, China) were cut into round foils whose diameters were 10 mm and polished, then cleaned ultrasonically three times with acetone, ethanol, deionized water, and dried at room temperature. The cleaned Ti plates were soaked in a 1 M NaOH solution at 80 ◦ C for 24 h, then rinsed thoroughly with deionized water (sample labeled as TiOH). Subsequently, 2 ␮l HMW-HA solution was added onto each TiOH surface (sample labeled as TiOH/HA), and subsequently the PDMS stamps were pressed down on the TiOH/HA surfaces by the force of 8 Newton for 12 h. Finally, micro-pattern on silicon master was moved to the TiOH/HA surface (sample labeled as TiOH/HAP) by PDMS stamp. All the preparation process is shown in Fig. 2. 2.2.2. Surface characterization The morphologies of bare Ti, TiOH and TiOH/HAP samples were observed by scanning electron microscopy (SEM, JSM-7001F, Japan), and the surface chemical composition was examined using Fourier transform infrared spectrometry (FTIR, NICOLET 5700, USA) with reflectance mode. The wettability of the surface was assessed

2. Materials and methods 2.1. Materials HMW-HA (Sangon Biological Engineering Co. Ltd., China) and HAa (Sigma–Aldrich) were diluted to a concentration of 5 mg/ml and 10 ␮g/ml with phosphate buffered saline (PBS) solution at pH

Fig. 2. Sketch map of the preparation of hyaluronic acid micro-pattern on TiOH surfaces.

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by water contact angle measurement (DSA 100, Krüss, GmbH, Germany) [31]. 2.2.3. Determination of HAa hydrolysis time The HAa hydrolysis time was the time during which HAa could completely transform the HMW-HA into the LMW-HA so that the fibronectin adhesion amount on the surfaces could reach the maximum. The fibronectin adhesion amount on the surfaces was determined by ELISA. 0.5 ml HAa (2000 U/ml) solution was dropped on TiOH/HA surfaces and incubated at 37 ◦ C for 1.5 h, 2 h and 3 h. Then 0.05 ml fibronectin solution (100 ␮g/ml, pH 7.2) was dropped on the surfaces and incubated at 37 ◦ C for 20 min and rinsed with PBS twice. After that the surfaces was blocked with 1% BSA at 37 ◦ C for 30 min and rinsed with PBS three times. Subsequently, 20 ml mouse monoclonal antihuman fibronectin antibody (Sigma–Aldrich, diluted 1:250 in PBS) was added and incubated at 37 ◦ C for 1 h, then the samples were thoroughly rinsed three times with PBS. Thereafter, 20 ␮l HRP conjugated goat anti-mouse IgG antibody (diluted 1:100 in PBS) was added and incubated at 37 ◦ C for another 1 h. The samples were washed three times again with PBS. Further, 100 ␮l TMB solution was added onto the sample surfaces and reacted in the dark for 10 min (blue color), and then 50 ␮l 1 M H2 SO4 was used to stop the peroxidase catalyzed reaction (yellow color). Finally, 130 ␮l supernatant was transferred to a 96-well plate and the absorbance at 450 nm was measured on a microplate reader (BIO-TEK Instruments, USA) [32]. 2.2.4. Cell culture SMC derived from human umbilical artery were isolated and cultured using the following method: the human umbilical cord was washed thoroughly with PBS to remove the blood outside, and then the artery was excised from the umbilical cord and opened at its length. Connective tissue outside and fibroblast layer outside were peeled off. The EC inside were gently scraped by a sharp tweezer. The muscle tissue was washed thoroughly with PBS and cut into small fragments. The fragments were then seeded in a single-used culture flask filled with 4 ml medium F12 and 1 ml fetal calf serum (FCS, Gibco BRL) mixture, and incubated at 37 ◦ C in a humidified atmosphere containing 95% air and 5% CO2 . The fragments were removed after SMC migrated to the culture flask. Replicated cultures were performed by trypsinization when cells were approaching confluence. Cells were fed with freshly prepared growth medium every 24 h. The second passage of SMC was used to build SMC micro-stripes on samples and co-culture with EC [33]. EC was derived from human umbilical vein and cultured using the method reported by Li et al. [34]: briefly, the umbilical cord was cannulated and washed thoroughly with PBS to remove the blood inside the lumen. Then 0.1% type II collagenase (Gibco BRL, USA) in M199 was introduced and incubated at 37 ◦ C for 10 min. The digested cells were washed in serum-free medium and separated from the supernatant by a centrifugation at 1200 rpm, and then collected in complete M199 containing 15% fetal calf serum, 50 mg/ml EC growth factor (ECGS, Sigma), 100 mg/ml heparin, 20 mmol/l HEPES, 2 mmol/l L-Gln. The suspended cells were then seeded in a cell culture flask and incubated at 37 ◦ C in an atmosphere containing 5% CO2 . Replicated cultures were performed by trypsinization when cells were approaching confluence. Cells were fed with fresh complete M199 every 48 h. The second passage was used to coculture with SMC on samples and evaluate the cell function. Both SMC and EC were obtained from newborn umbilical cord (Maternal and Child Health Hospital in Chengdu, China) per protocols approved by the Maternal and Child Health Hospital Institutional Review Board. Our research complied with Helsinki Declaration. To investigate the regulation effect of HA micro-pattern on SMC, SMC was seeded on the TiOH/HAP, TiOH and Ti samples at the

concentration of 5 × 104 cells/ml, respectively. After incubation at 37 ◦ C for 4 h and rinse with 37 ◦ C warm PBS, all the samples were fixed with 2.5% glutaraldehyde for 2 h and detected by immunofluorescence staining and optical microscope. For immunofluorescence staining, the prepared samples were firstly rinsed with PBS, blocked with 1% BSA at 37 ◦ C for 30 min and rinsed with PBS again three times. Subsequently, rabbit antihuman actin antibody (1:100 diluted in PBS) was added and incubated at 37 ◦ C for 30 min, then rinsed with PBS for three times again, then goat antirabbit IgG antibody (1:100 diluted in PBS) was added as second antibody and incubated at 37 ◦ C for 30 min, subsequently rinsed with PBS for three times again. Finally, FITC labeled antibody was introduced, incubated at 37 ◦ C for 30 min and rinsed with PBS for four times. The stained samples were observed under an inverted fluorescence microscope (OLIMPUS-IX51, Japan). 2.2.5. Observation of EC/SMC adhesion on micro-patterned surfaces In order to distinguish SMC and EC on the co-culture samples clearly, Cell Trackers with two different colors (Orange and Green) were used in the whole co-culture process. The Tracker with Orange color was used for SMC while the Green one was used for EC. The cells were firstly marked by the trackers before being seeded on the samples, and the co-culture method was presented as follow: firstly, SMC was seeded on the patterned samples at the concentration of 5 × 104 cells/ml and incubated at 37 ◦ C for 4 h, then rinsed with 37 ◦ C warm PBS, then 30 ␮l HAa was dropped on the samples and incubated at 37 ◦ C for 2 h, rinsed with 37 ◦ C warm PBS. Thereafter, EC were seeded on the same samples at the concentration of 5 × 104 cells/ml and incubated at 37 ◦ C for 1 day and 3 days. The samples were rinsed thoroughly with PBS for three times to remove non-adherent cells and fixed with 2.5% glutaraldehyde for 2 h. Finally, the EC/SMC co-cultured samples and control samples were examined by fluorescence microscope. EC/TiOH samples were used as control. 2.2.6. NO release measurements NO released from EC/SMC, EC/TiOH and patterned SMC samples were measured using Griess Reagent method. The supernatant from the co-culture samples and reference samples was collected after EC culture on the samples for 16 h, 20 h and 24 h, respectively. 100 ␮l medium supernatant and 100 ␮l Griess solution were put into a 96-well culture plate and the absorbance was measured by a microplate reader at 540 nm. The amount of NO released from the samples was calculated according to a calibration curve which was prepared by series of decreasing concentration of Griess reagent as 50%, 25%, 12.5%, 6.25%, 3.125%, 1.5625%, 0.78125%. 2.2.7. Calculation of retention of endothelial cells under shear stress The shear stress for steady flow conditions which was offered by a flow chamber and peristaltic pump (Masterflex7518-10, Cole Parmer, USA) was used to evaluate retention of EC. Samples were put into the flow chamber after EC culture for 1 day, 3 days separately and immersed in EC medium as described earlier (Section 2.2.4). Afterwards the samples were washed under the shear stress 15 dyn/cm2 for 0.5 h, and then stained by actin immunofluorescence staining as described in Section 2.2.5. The retention of endothelial cells was calculated as following: Retention (%) = Ct/Co × 100% where Co and Ct were the EC amount on the surface before and after scouring with medium, respectively.

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The data were analyzed with the software SPSS 11.5 (Chicago, IL). The data are expressed as mean ± SD. The statistical analysis between different groups was tested by one factor or two-factor analysis of variance. p < 0.05 was considered as a significant difference. All experiments were repeated three times at least. 3. Results and discussion

Reflectance(%)

2.3. Statistical analysis

27

100

Ti TiOH TiOH/HAP

80

3400cm

-OH

-COOH-1 1750cm

C-O

-1

1100cm

60

40

3.1. Surface characterization of the micro-patterned TiOH

-NH

1600cm

Fig. 3A depicts the SEM images of the bare Ti, TiOH and TiOH/HAP surfaces. Compared with the Ti surface (Fig. 3Aa), the TiOH surface (Fig. 3Ab) showed more roughness beneficial for biomolecules adsorption, and HA as a biomolecule with high viscosity preferentially adsorbed to a rough surface compared to a smooth one [25,35]. Thus, HMW-HA micro-pattern was prepared on TiOH surface by a method of physical imprint. After patterning, the TiOH/HAP surface (Fig. 3Ac) had a homogeneous morphology of stripes with 25 ␮m wide ridges and 25 ␮m wide grooves. The size of EC and SMC in vivo was about 25 ␮m wide [20]. Therefore, EC and SMC could be co-cultured and realized their bionic

-1

20 4000

3500

3000

2500

2000

-CH3

-1

1450cm

1500

-1

1000

500

Wavenumber(cm-1) Fig. 4. Characterization of FTIR spectra of: Ti, TiOH and TiOH/HAP.

shape by 25 ␮m/25 ␮m micro-grooves of the stripes of hyaluronic acid. Fig. 3B shows the results of the water contact angles. Compared with the bare Ti, the water contact angles dramatically decreased to about 13.6 ± 3.5◦ after activation by NaOH due to the introduction of hydroxyl groups. After fabricating the parallel groove/ridge stripes micro-pattern of HA on the TiOH surface the water contact angles increased to 33.3 ± 4.5◦ . The reason might be ascribed to the exposure of hydrophobic groups such as CO CH3 . However, the TiOH/HAP samples still exhibited more hydrophilic character than the bare Ti samples, which might be beneficial for cell adhesion. Fig. 4 shows the FTIR spectra of all the samples. Compared with the bare Ti sample, the TiOH sample showed a broad OH peak near 3400 cm−1 . Compared with the Ti and TiOH samples, a significant increase of COOH, NH, CH3 and saturated C O bond vibration peaks were observed at 1750 cm−1 , 1600 cm−1 , 1450 cm−1 and 1100 cm−1 on the TiOH/HAP sample, and abroad OH peaks were also found near 3400 cm−1 . Because the COOH and NH peaks were too close, the two peaks appeared as one broad peak containing 1750 cm−1 and 1600 cm−1 . The appearance of COOH, NH, CH3 and saturated

*

2.5

OD(450nm)

2.0

*

1.5

1.0

0.5

0.0 Fig. 3. Characterization of (A) SEM images of: (a) Ti, (b) TiOH and (c) TiOH/HAP; (B) the water contact angle measurement of Ti, TiOH and TiOH/HAP (mean ± SD, n = 3, *p < 0.05).

TiOH

TiOH/HA

1.5h

2h

3h

Fig. 5. Amount of adherent Fn after enzymatic degradation of HA by HAa for 1.5 h, 2 h, 3 h (mean ± SD, n = 3,*p < 0.05).

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Fig. 6. Immunofluorescence staining micrographs of SMC actin after culture on TiOH/HAP, TiOH and Ti for 4 h.

C O bond vibration peaks indicated the existence of HA on the TiOH. 3.2. HAa hydrolysis time The adsorption of fibronectin on different samples is shown in Fig. 5. Compared with TiOH surfaces, the adsorption of fibronectin on TiOH/HA surfaces was significantly reduced. The result above displayed that high molecular weight HA impeded the adhesion of fibronectin on the surfaces, which was consistent with the result by Kaph et al. [36]. The results also showed that adsorption of fibronectin after HAa digested HA for 1.5 h significantly increased compared to the non-digested HA. This indicated that enzymatic degradation of HA by HAa could turn off its anti-adhesive effect. As the hydrolysis time increased, the adsorption of fibronectin increased again. However, the amount between 2 h and 3 h showed little difference, therefore 2 h was chosen as the suitable time for HAa to turn off the anti-adhesive effect of HA.

3.3. SMC adhesion The morphology and cytoskeleton arrangement of SMC on different samples was observed using immunofluorescence microscopy after staining of filamentary actin. Fig. 6 depicts the cytoskeleton of SMC after incubation for 4 h. SMC seeded on all samples exhibited long spindle-shaped morphology. On TiOH/HAP surfaces they grew along with the TiOH grooves, while SMC seeded on TiOH and Ti surfaces exhibited random orientation. These results indicated that SMC were inhibited by HMW-HA and the growth behavior of SMC on the TiOH/HAP surfaces was controlled by the HMW-HA micro-pattern. 3.4. Co-culture of EC and SMC The equal examination was performed with EC seeded on TiOH/HAP surfaces secondary to seeding SMC. As shown in Fig. 7, the SMC (Orange) on all samples in the co-culture exhibited long

Fig. 7. Fluorescence micrographs of EC (Green) and SMC (Orange) after co-cultured for 24 h and 72 h, respectively. EC cultured alone on the TiOH samples were used as control. (A and C) EC cultured alone on the TiOH samples; (B and D) EC and SMC co-culture system. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

J. Li et al. / Applied Surface Science 273 (2013) 24–31

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spindle-shaped morphology and formed several long stripes, and EC (Green) grew between two adjacent SMC stripes and exhibited polarized form along the stripes direction. The physiological regulation of EC morphology might be attributed to the ability of LMW-HA in promoting cell adhesion, proliferation and migration [37]. However, the EC/SMC system could not be controlled when samples were incubated for 72 h. The SMC did not grow in orientation without the limit of the HMW-HA micro-pattern. The EC in co-culture system was compressed in limited areas. The EC on control samples exhibited polygonal morphology and disordered distribution. The results demonstrated that co-culture of EC and SMC for 24 h was realized. Cell compatibility is very important for cardiovascular implanted biomaterials. EC morphology and peripheral environment on the surfaces of implants determine their anti-shedding ability and normal physiological function. EC cultured alone cannot fully copy the properties of normal blood vessel intima in vivo, due to a lack of direct and paracrine interaction with SMC. The morphology of EC cultured in vitro without blood flow shear stress environment differs substantially from human blood vessel intima, leading to significant functional differences. The presence of SMC can provide pericytes environment close to normal physiological conditions, and micro-stripe material surface can control EC to mimic the physiological morphology. So far, many studies have reported both the EC and SMC co-culture system [38], or the effects of surface texture on EC morphology [39], respectively. However, the presented approach for achieving EC with near-physiological morphology and SMC in a co-culture system via the control of micro-patterned surface exceeds all other studies in this field. It is considered that primary conditions for the realization of a bionic EC/SMC membrane are provided; however, there is a lot of work to be done to improve this system. 3.5. NO release Release of NO has a significant impact for the inhibition of platelets [40], SMC proliferation [41] and adhesion of leukocytes to the endothelium [42], in addition to causing smooth muscle relaxation. Decreased NO production or availability has been linked to endothelial dysfunction [43]. Release of NO from the endothelium has been thought to be responsible for SMC relaxation which lead to changes in shear stress and vasodilatation [44], while the former changes the morphology of endothelial cells, the latter is related to smooth muscle cells. The method of Griess reagent [45] has been used to measure NO release in this work, and the results are shown in Fig. 8A. NO released by EC in EC/SMC co-culture system was 11.87 ± 0.82 nmol/103 cells compared with the amount of 1.56 ± 0.05 nmol/103 cells released by EC alone at 16 h, NO released by EC in EC/SMC was 17.50 ± 2.72 nmol/103 cells compared with the amount of 2.17 ± 0.10 nmol/103 cells released by EC alone at 20 h, and NO released by EC in EC/SMC was 43.02 ± 4.41 nmol/103 cells compared with the amount of 8.03 ± 4.55 nmol/103 cells released by EC alone at 24 h. Obviously, EC/SMC released larger amount of NO than EC alone. Little NO released by SMC was detected during the culture periods. It was supposed that the SMC micro-stripes regulate the morphology of EC similar to the shear stress of flowing fluids, therefore the EC in co-culture system were stimulated to release more NO. 3.6. Retention of endothelial cells under shear stress It has been reported that flaking and damage of EC on the vascular implants play an important role in restenosis [46]. The fundamental reason is that EC adhesion on the surface is not

Fig. 8. Detection of (A) NO release; and (B) retention of EC in the EC/SMC coculture system. EC cultured alone on TiOH samples were used as control (mean ± SD, *p < 0.05).

sufficient; the adhesion is closely related to interaction between EC and the surfaces cell morphology, and it is well-known that cell morphology depends on the distribution of the cytoskeleton which can be controlled by micro-patterns. Determination of the retention of EC on the surface after application of a defined shear stress was used as a method to evaluate indirectly the interaction between EC and the surfaces. The retention of EC on the surfaces washed by flow shear stress is shown in Fig. 8B. Retention of EC in EC/SMC system was 33.3 ± 9.3% compared with 16.0 ± 6.3% of EC alone, significantly higher than the latter at 1 day. The reason might be the regulation of SMC micro-stripes and the effect of LMW-HA in promoting cell adhesion, and the former had effect on EC cytoskeleton, where the actin became sturdier [47] and cells could withstand the shear stress better. Biomolecules secreted by the SMC such as fibronectin, collagen, and laminin also enhance the ability of EC adhesion [48]. However, the retention of EC in EC/SMC became 46.1 ± 6.5% compared with 56.9 ± 8.6% of EC alone, a little lower than the latter at the 3rd day. The reason of that might be the formation of EC monolayer completed in the latter, but not in the former. It was thought that EC cultured alone were connected into a single layer membrane at this time, and the cell gap narrowed, cytoskeleton of different EC connected tightly, while EC from the co-culture system did not contact with each other closely because of barrier by SMC. In short, the result showed not only interaction between the cells and the substrate, but also interaction between cells and cells. However, more work should be performed to verify the results in future.

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4. Conclusions A parallel micro-stripes of high molecular weight hyaluronic acid (HMW-HA) was successfully prepared on NaOH-activated Ti surface. The HMW-HA stripes could effectively regulate the morphology and behavior of SMC. EC could be cultured along the low molecular weight hyaluronic acid (LMW-HA) strips and regulated by SMC stripes after HMW-HA degradaton by HAa for 3 h. The 24 h micro-patterned EC/SMC co-culture system could be realized, and the system showed higher NO release and lower EC shedding than EC alone. In our opinion, the present surface micropattern fabrication and EC/SMC co-culture technology may offer a potential application for investigating the interactions of EC and SMC on the surface of cardiovascular implants used in tissue engineering. Acknowledgments We acknowledge the financial support of the National Physiological Science Foundation of China (No. 30870629), Key Basic Research Project (No. 2011CB606204), Foundation of Jiangsu Provincial Key Laboratory for Interventional Medical Devices (JR1205) and Fundamental Research Funds for the Central universities (SWJTU11ZT11). References [1] A. Andukuri, M. Kushwaha, A. Tambralli, J.M. Anderson, D.R. Dean, J.L. Berry, Y.D. Sohn, Y.S. Yoon, B.C. Brott, H.W. Jun, A hybrid biomimetic nanomatrix composed of electrospun polycaprolactone and bioactive peptide amphiphiles for cardiovascular implants, Acta Biomaterialia 7 (2011) 225–233. [2] U. Baber, R. Mehran, S.K. Sharma, S. Brar, J. Yu, J.W. Suh, H.S. Kim, S.J. Park, A. Kastrati, A. Waha, P. Krishnan, P. Moreno, J. Sweeny, M.C. Kim, J. Suleman, R. Pyo, J. Wiley, J. Kovacic, A.S. Kini, G.D. Dangas, Impact of the everolimus-eluting stent on stent thrombosis, Journal of the American College of Cardiology 58 (2011) 1569–1577. [3] R.E. Unger, K. Peters, M. Wolf, A. Mottab, C. Migliaresi, C.J. Kirkpatrick, Endothelialization of a non-woven silk fibroin net for use in tissue engineering: growth and gene regulation of human endothelial cells, Biomaterials 25 (2004) 5137–5146. [4] A. Lichtenberg, I. Tudorache, S. Cebotari, S.R. Lichtenberg, G. Sturz, K. Hoeffler, C. Hurscheler, G. Brandes, A. Hilfiker, A. Haverich, In vitro re-endothelialization of detergent decellularized heart valves under simulated physiological dynamic conditions, Biomaterials 27 (2006) 4221–4229. [5] G. Krenning, J.A. Moonen, M.J. van Luyn, M.C. Harmsen, Vascular smooth muscle cells for use in vascular tissue engineering obtained by endothelial-tomesenchymal transdifferentiation (EnMT) on collagen matrices, Biomaterials 29 (2008) 3703–3711. [6] K.B. Vartanian, S.J. Kirkpatrick, S.R. Hanson, M.T. Hinds, Endothelial cell cytoskeletal alignment independent of fluid shear stress on micropatterned surfaces, Biochemical and Biophysical Research Communications 371 (2008) 787–792. [7] C. Cheung, S. Sinha, Human embryonic stem cell-derived vascular smooth muscle cells in therapeutic neovascularisation, Journal of Molecular and Cellular Cardiology 51 (2011) 651–664. [8] S.H. Hagvall, G. Helenius, B.R. Johansson, J.Y. Li, E. Mattsson, B. Risberg, Co-culture of endothelial cells and smooth muscle cells affects gene expression of angiogenic factors, Journal of Cellular Biochemistry 89 (2003) 1250–1259. [9] J.J. Chiu, L.J. Chen, C.N. Chen, P.L. Lee, C.I. Lee, A model for studying the effect of shear stress on interactions between vascular endothelial cells and smooth muscle cells, Journal of Biomechanics 37 (2004) 531–539. [10] T. Ziegler, R.W. Alexander, R.M. Nerem, An endothelial cell-smooth muscle cell co-culture model for use in the investigation of flow effects on vascular biology, Annals of Biomedical Engineering 23 (1995) 216–225. [11] K. Niwa, T. Kado, J. Sakai, T. Karino, The effects of a shear flow on the uptake of LDL and acetylated LDL by an EC monoculture and an EC-SMC coculture, Annals of Biomedical Engineering 32 (2004) 537–543. [12] M.D. Lavender, Z.Y. Pang, C.S. Wallace, L.E. Niklason, G.A. Truskey, A system for the direct co-culture of endothelium on smooth muscle cells, Biomaterials 26 (2005) 4642–4653. [13] M.F. Fillinger, L.N. Sampson, J.L. Cronenwett, R.J. Powell, R.J. Wagner, Coculture of endothelial cells and smooth muscle cells in bilayer and conditioned media models, Journal of Surgical Research 67 (1997) 169–178.

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