Guiding the behaviors of human umbilical vein endothelial cells with patterned silk fibroin films

Colloids and Surfaces B: Biointerfaces 122 (2014) 79–84

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Guiding the behaviors of human umbilical vein endothelial cells with patterned silk fibroin films Xuejiao Du a,b , Yanyun Wang b , Lin Yuan b,∗ , Yuyan Weng a,b , Gaojian Chen a,b,∗ , Zhijun Hu a,b,∗ a

Center for Soft Condensed Matter Physics and Interdisciplinary Research, Soochow University, Suzhou 215006, China The Key Lab of Health Chemistry and Molecular Diagnosis of Suzhou, Department of Polymer Science and Engineering, College of Chemistry, Chemical Engineering and Materials Science, Soochow University, Suzhou 215123, China b

a r t i c l e

i n f o

Article history: Received 22 April 2014 Received in revised form 31 May 2014 Accepted 23 June 2014 Available online 28 June 2014 Keywords: Silk fibroin film Surface pattern Endothelial cell Cell morphology Cell proliferation

a b s t r a c t Silk fibroin is an ideal blood vessel substitute due to its advantageous qualities including variable size, good suture retention, low thrombogenicity, non-toxicity, non-immunogenicity, biocompatibility, and controllable biodegradation. In this study, silk fibroin films with a variety of surface patterns (e.g. square wells, round wells plus square pillars, square pillars, and gratings) were prepared for in vitro characterization of human umbilical vein endothelial cell’s (HUVEC) response. The affects of biomimetic length-scale topographic cues on the cell orientation/elongation, proliferation, and cell-substrate interactions have been investigated. The density of cells is significantly decreased in response to the grating patterns (70 ± 3 nm depth, 600 ± 8 nm pitch) and the square pillars (333 ± 42 nm gap). Most notably, we observed the contact guidance response of filopodia of cells cultured on the surface of round wells plus square pillars. Overall, our data demonstrates that the patterned silk fibroin films have an impact on the behaviors of human umbilical vein endothelial cells. © 2014 Elsevier B.V. All rights reserved.

1. Introduction Extracellular matrix (ECM) consisting of cell-secreted proteins, polysaccharides, and complex three-dimensional micro/nanoscale topographies plays an important role in affecting the behaviors of cells [1–4]. It is well known that cells respond to micro/nano-scale topographies and exhibit different behaviors in many aspects including adhesion, proliferation, migration and apoptosis [5–9]. Studies of cellular responses to topographies of varying geometry and length scale are thus of great importance to understand cell biology and to promote applications of patterned substrates in tissue engineering [10,11]. Vascular endothelial cells are critical for forming the inner lining of major blood vessel which plays an important role in regulating blood pressure and preventing coagulation. A feature of vascular endothelial cells is that they fasten themselves to the underlying stroma through a particular specialized ECM, the basement

∗ Corresponding authors at: Soochow University, Center for Soft Condensed Matter Physics and Interdisciplinary Research, Suzhou 215006, China. Tel.: +8651265882467. E-mail addresses: [email protected] (L. Yuan), [email protected] (G. Chen), [email protected] (Z. Hu). http://dx.doi.org/10.1016/j.colsurfb.2014.06.049 0927-7765/© 2014 Elsevier B.V. All rights reserved.

membrane [12,13]. The abundant features of basement membranes, which include cell-secreted proteins, displayed functional groups, a battery of trophic agents and other cytoactive factors, can regulate the fundamental behaviors of endothelial cells [8,14–16]. In order to replicate native basement membrane and eventually improve vascular prosthetics, many synthetic or biologically derived substrates that simulate the native basement membrane have been developed to regulate endothelial cell behavior [8,17,18]. Surface topography is one of the key parameters that have been focused on to investigate the endothelial cell responses. Different substrates such as polydimethylsiloxane (PDMS) [8,19–21] and poly-(methyl methacrylate) (PMMA) [22–24], which were patterned by lithographic techniques, have been explored for in vitro applications. Biodegradable polymers such as poly(l-lactic acid) (PLLA) are being explored for potential in vivo applications [25]. Most of them, however, have significant drawbacks such as bulk degradation upon implantation and rigid mechanical properties. Rigid mechanical materials can cause localized inflammation in the dynamic in vivo environment [26–28]. Silk fibroin from Bombyx mori silkworm cocoons is biocompatible and possesses excellent features such as tunable mechanical properties and ambient aqueous processing. In addition, silk fibroin is implantable due to its non-immunogenic response and controllable degradation rates [29–31]. Thus, silk fibroin has been

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increasingly used as a biomaterial in a wide range of forms such as films, sponges, hydrogels and solid blocks for applications in tissue engineering and regenerative medicine [32,33]. Furthermore, silk fibroin film is able to be easily patterned with soft or nanoimprint lithography techniques [34,35]. The successful design of blood vessels should critically consider the blood pressure, the compatibility with adjacent host vessels, and the ability of sustaining cyclic loading and anti-thrombotic lining [36]. In this aspect, silk fibroin is an ideal blood vessel substitute and demonstrates an advantage over other anti-thrombotic materials with its excellent resistance to high shear stress and blood flow pressure [37,38]. In fact, it has been formed into microtubules with different inner diameters, porosities, mechanical strengths, and diffusivities for blood vessel engineering [31,36]. Additionally, the silk fibroin’s elastic modulus is within a range that does not cause deleterious effects for cells. Other materials used for similar purposes with more rigid mechanical properties have been shown to cause localized inflammation in vivo [26–28]. Therefore, it would be advantageous to design silk fibroin films with micro/nano-scale topographies that are appropriate for future in situ tissue integration and regeneration. Such films have the potential to become the preferred vascular stents for in vivo implantation. It has been reported that patterned silk fibroin films can be developed for corneal tissue engineering applications, and the micro/nano-scale topographies play important roles in the wound-healing responses, such as corneal epithelial and fibroblast attachment, proliferation and alignment [29,30,39]. However, it is not clear regarding the vascular endothelial growth on silk substrates, especially taking into consideration the varying geometries and sizes of substrate topography. The purpose of this study is to investigate the relationship between the various micro/nano-scale topographical structures of silk fibroin films and the behavior of human umbilical vein endothelial cells (HUVECs). Silk fibroin films containing different surface topographic features have been prepared in this study. Such patterned silk films have never before been used as a culture substrate for human umbilical vein endothelial cells. The design of silk fibroin films with micro/nano-scale topography allows for the study of fundamental endothelial cell behaviors including orientation and alignment, proliferation, and attachment. This will aid in the development of novel strategies in tissue engineering and will ultimately advance the development of cardiovascular prosthetics. 2. Materials and methods 2.1. Preparation of aqueous silk solutions Aqueous silk solutions were obtained using previously published protocols with slight modifications [40]. Cocoons from B. mori silkworm were boiled twice for 30 min in an aqueous solution of 0.02 M Na2 CO3 (Sigma-Aldrich). The silk fibers were rinsed thoroughly with distilled water three times with room temperature water to remove the glue-like sericin proteins and then dried at room temperature overnight. The purified silk fibers were dissolved in a mixed solution of CaCl2 (Sigma-Aldrich): water: ethanol (in a 1:8:2 molar ratio) at 72 ◦ C. The solution was put into a dialysis cassette (3.5k MWCO, 5–15 ml capacity) and dialyzed against distilled water for three days. The distilled water was replaced for at least five times during the dialysis. The solution was centrifuged at 10,000 rpm for 20 min at 4 ◦ C. The final solution had a relative concentration of 4–5% (w/v), and was stored at 4 ◦ C. 2.2. Fabrication of silk fibroin films with micro/nano-scale surface topographies Homemade substrates with micro/nano-scale patterns were prepared by electron beam lithography using

Table 1 Average dimensions of the structured surfaces for the replicated pattern types, n = 5. Types

Width (nm ± SD)

Gap (nm ± SD)

Depth (nm ± SD)

Square wells Round wellsa plus square pillarsb Square pillars

1033 ± 57 291 ± 31a 486 ± 24b 1167 ± 57

967 ± 57 513 ± 47.9a 333 ± 42b 293 ± 11

357 ± 40 97 ± 15a 380 ± 20b 373 ± 25

a b

The size of round wells. The size of square pillars in Round wells plus square pillars.

polymethylmethacrylate (PMMA) as photoresist. The thickness of PMMA resist on silicon wafer was 400 nm. Each patterned field consists of an array of features over a total area of 16 mm2 . To generate clean silk fibroin films, the aqueous silk solution was filtered through a 0.45 ␮m pore size syringe filter before using. Patterned silk films with controllable film thickness were prepared by pouring aqueous silk solution onto the patterned silicon substrates bearing PMMA micro/nano-structures and drying overnight at room temperature. The replicated pattern types include square wells, square pillars, round wells plus square pillars, and gratings. The geometries of the patterns are summarized in Tables 1 and 2. In order to make the films water insoluble, the silk fibroin films were treated with 90% methanol for about 5 h to induce ␤-sheet transition. The silk fibroin films were subsequently degassed for 1 h under vacuum and dried for at least 24 h. The patterned silk fibroin films were placed into 48-well plates. The plates were sterilized with 75% ethanol for 30 min. Each film was washed with three separate aliquots of aseptic dH2 O. The samples were left in the final aseptic dH2 O wash until ready for cell seeding. 2.3. Cell culture HUVECs were cultured in RPMI medium 1640 (Hyclone, UT, USA) containing 10% FBS, 100 U/ml penicillin, and 0.1 mg/ml streptomycin at 37 ◦ C with 98% humidity and 5% CO2 in air. Cells were harvested by trypsinization at approximately 80–90% confluence. 2.4. Cell alignment and orientation analysis HUVECs were seeded on the silk films at a density of 8000 cells/cm2 as previously described [29]. Briefly, to characterize the alignment and orientation, microscopic images were acquired on the second day with an inverted fluorescence microscope (IX71, Olympus). The orientations of the HUVECs were analyzed with ImagePro 6 software. The orientation angle was determined by measuring the angle differences between the longest axis within the cell borders and the orientation of gratings. Over one day in culture, the angle difference between the longest axis of the cell boundary and the groove direction was measured from all existing cells shown in 10× microscopic images. Cells were considered aligned with gratings when this angle was between 0 and 10◦ . Cell elongation is defined as the ratio between the length and breadth of each cell. Cells were considered elongated if this factor was higher than 1.3. 2.5. Cell proliferation HUVECs were plated at a density of 10,000 cells/cm2 . One day after plating, cells on each pattern were imaged and counted at 10× magnification using an inverted microscope (IX-71, Olympus). Cells were cultured for 3 days at which time the cells were imaged using an inverted microscope (IX-71, Olympus). Cell counts were obtained from images using ImagePro 6 software. Each experiment was repeated for at least three times.

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Table 2 Average dimensions of the nano-structured surfaces for the various gratings, n = 5. Gratings

Groove width (nm ± SD)

Ridge width (nm ± SD)

Pitch (nm ± SD)

Depth (nm ± SD)

A B C D E

356 ± 5 315 ± 26 610 ± 17 678 ± 20 880 ± 20

251 ± 2 522 ± 26 589 ± 30 722 ± 30 783 ± 23

600 ± 8 812 ± 10 1151 ± 50 1420 ± 30 1690 ± 15

70 ± 3 400 ± 44 399 ± 31 410 ± 20 417 ± 18

2.6. Imaging cells’ morphology with scanning electron microscopy (SEM) The samples were washed with PBS and fixed with paraformaldehyde (Sigma-Aldrich) for 5 min at room temperature after 3 days. The samples were processed through serial EtOH dehydration baths (30%, 50%, 70%, 90% and 100% EtOH) for 5 min per bath concentration. Samples were further dried using hexamethyldisilazane (Sigma-Aldrich) solvent to remove residual water saturation for 2 min, and then dried for 2 h in a desiccator. A thin layer of gold was coated on the samples by sputtering before SEM (Hitachi S-4700) observation.

To quantitate the morphological observations, the percentage of HUVECs that demonstrated orientation and alignment response occurring on gratings was analyzed. Quantitative results of our analysis are shown in Fig. 3. HUVECs exhibited a different response depending on the size of the topographic features. About 42% of the HUVECs population demonstrated orientation and alignment response on the smallest feature size of 600 ± 8 nm pitch and 70 ± 3 nm depth, significantly higher than the unpatterned control (p < 0.001). The cells exhibited a higher percentage of alignment on the pitch size from 800 to 1700 nm (about 400 nm depth) compared to the grating A. 3.3. Filopodial observation

2.7. Statistical analysis Statistical analysis was performed using Student’s t-test. All data are presented as mean ± SD unless specified. Within the figures significance is denoted by the following: * p < 0.05, ** p < 0.01, *** p < 0.001. 3. Results 3.1. Characterization of patterned silk fibroin films Some representative SEM images of patterned silk fibroin films are shown in Fig. 1. All the rest were shown in Fig. S1. The pitch (the sum of ridge and groove width) of gratings ranges from 600 to 1700 nm. The silk fibroin films largely replicate the features defined on the substrates.

SEM images revealed the types of interaction between cells and patterned substrates, as shown in Fig. 4. HUVECs are aligned parallel to gratings (Fig. 4A). Lamellipodia descended into the grooves or aligned parallel to the ridges. Interestingly, filopodia demonstrated different behavior on gratings. Some filopodia were found to be perpendicular to the grooves, indicating that the filopodia alter their primary growth orientation on the ridges in order to choose the shortest pathway for crossing the grooves (Fig. 4B and C). It can be seen that the filopodia of the cells cultured on round wells plus square pillars grew along the ridge of the round wells when they reached the borders of the round wells, and altered their original orientation (Fig. 4D and E). However, the filopodia of the cells could not alter their original orientation when they reached the borders of the square wells (Fig. 4F). 3.4. Cell proliferation on patterned silk fibroin films

3.2. The orientation of HUVECs on patterned silk fibroin films We first investigated the orientation and alignment of HUVECs on silk fibroin substrates with different patterns. The cells remained flat and circular on the unpatterned control, and exhibited no observable alignment or orientation on the patterns such as square wells, round wells plus square pillars or square pillars (Fig. 2A–D). However, the cells exhibited morphological response to the patterns with the grating topographies. Representative images of HUVECs on the gratings are shown in Fig. 2E. To further investigate whether the orientation response is specific to the dimensions of the gratings, we analyzed the orientation and alignment on a series of gratings ranging from 600 to 1700 nm pitch. Cell alignment was evident in all the substrates with grating topographic patterns.

We also performed experiments to characterize the effects of micro/nano-scale topography on the proliferation of HUVECs (Fig. 5). The overall cellular densities upon the patterns were similar to the unpatterned control after 1 day (Fig. 5F). Microscopic imaging after 3 days in culture revealed that cells have grown readily on most of the patterns (Fig. 5A–C), except for the square pillars and the gratings A (Fig. 5D and E), on which the cell densities were found to be significantly lower than those on the unpatterned control (Fig. 5F). Interestingly, square wells induced higher proliferation rates as compared to the unpatterned control (N = 3, n = 9, p < 0.001). To further investigate whether the size of gratings is responsible for the significant decrease in HUVEC proliferation, additional experiments were also conducted with the other gratings. No

Fig. 1. Scanning electron microscopy characterization of the patterned silk films. (A) Square wells. (B) Round wells plus square pillars. (C) Square pillars. (D) Gratings C.

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Fig. 2. Inverted microscope of HUVECs grown on the patterned silk films. (A) Unpatterned control. (B) Square wells (C) Round wells plus square pillars. (D) Square pillars. (E) Gratings C. For 1 day in culture. Scale bars are 60 ␮m.

significant difference was observed between unpatterned control and the other gratings (800 to 1700 nm pitch, 400 nm depth) (Fig. 5G). 4. Discussion Self-standing silk fibroin films of various geometries and feature dimensions were patterned and utilized to affect various aspects of cell function. Silk fibroin was chosen for patterning due to its advantageous material properties as well as its simple and inexpensive production methods [31,36,41,42]. Endothelial cell behaviors including orientation and alignment response and proliferation are important for the remodeling of extracellular matrix during the formation of new vessels. These are applied to both wound healing and for the study of

Fig. 3. HUVECs demonstrate a heterogeneous orientation and alignment response to gratings of silk films. Nine Images (10× magnification) were taken on each topographic surface for calculation of the percentage of cell orientation (<10◦ ). Significant differences between unpatterned control and topography are marked as follows: * p < 0.05, ** p < 0.01, *** p < 0.001. The significant differences were also observed when comparing gratings A (600 ± 8 nm pitch, 70 ± 3 depth) to gratings B (812 ± 10 nm pitch, 400 ± 44 nm depth) through gratings E (1690 ± 15 nm pitch, 417 ± 18 nm depth) and are marked # p < 0.05, ## p < 0.01. (Data is shown as mean ± SEM.).

interactions between native endothelial cells and implanted prosthetic scaffolds. These essential processes may be driven by changes in endothelial cell shape [43]. For example, changes in cell shape from orientation and alignment of cells have been associated with alterations in cell cycle that would directly influence the proliferative state of the cell [8]. Fluid shear forces have been found to influence endothelial cell shape and alignment both in vitro and in vivo [44]. In our studies, we investigated the single biophysical cue from surface-patterned silk films to explore their impact on cell behaviors. We have found during the course of our studies that the gratings with varying sizes and the same depth have a similar effect on orientation and alignment of HUVECs. Recent reports demonstrate that human umbilical vein endothelial cells exhibit similar orientation with the greatest alignment response occurring on patterned polyurethane surface features between 800 and 1200 nm pitch [8]. Other studies have reported that deeper and narrower gratings have resulted in more human corneal fibroblast orientation. These results have indicated the depth of the gratings was more important for cell alignment than width [29,45–47]. In addition, this may also help explain the phenomenon that a lower percentage of human umbilical vein endothelial cells were observed to be aligned with the smallest size of gratings in our studies. It is well known that there is no one-size-fits-all approach and each cell-type will have a unique response to topographic cues. Feature shape, size scale, surface order, depth, and other natural properties of a selected substrate should all be taken into account as being important elements in the future design of vascular prosthetics. A cell probes its environment and moves using nano-scale processes such as lamellipodia and filopodia upon adhesion to a substrate. It has been reported that filopodia play a sensory or exploratory role with diameters of 250–400 nm [41,42]. Teixeira et al. found that human corneal epithelial cells cultured in Epilife medium elongate and align perpendicular to patterns of gratings of the nano-scale dimension (70 nm wide ridges on a 400 nm pitch) [43,44]. Our studies demonstrated two distinct results: The first result was that some filopodia of the cells align perpendicular to the grooves but not to the ridges. The second result was that the filopodia of cells grew along the borders of the round wells or stride over the wells when cultured on round wells plus square pillars.

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Fig. 4. SEM images of HUVECs on the patterned silk films. (A) Cell aligned along nanostructured substrate on gratings. (B and C) Detail of previous cell, lamellipodia descended into the grooves or aligned parallel to the ridges. Some filopodia were perpendicular to the grooves. (D and E) On round wells plus square pillars, filopodia grew along the ridge of the round wells. (F) On square wells, the cells could stride over the wells and not alter their original orientation.

However, the cells could not grow inside 300 nm wells. This phenomenon was quite similar to the “contact guidance” caused by “groove-ridges” observed in the literature [48]. Cells preferred to grow along the borders of small round wells (i.e. those with diameter less than 2.2 ␮m) due to the diameter of the small pits being too small to provide enough room for the deformation of the filopodia of cells [49]. The filopodia of cells on square wells could stride over the wells but not grow along the borders. Our data reinforces the notion that filopodia play an important role in the capacity of cells to perceive different topographic features [8].

The regulation on the proliferation of cells by incorporating biologically active molecules such as peptides, antibodies and growth factors with biomaterials has been widely studied. The influence of topographic features in natural materials without attaching additional molecules is another important parameter and essential in the design of improved prosthetics. In our studies, HUVECs demonstrated a decrease in proliferation only on the patterned surfaces with smaller size. Similar phenomenon was also observed on the smallest features by Murphy and coworkers [8].

Fig. 5. Screening for geometric patterns that can influence HUVECs cell densities. Inverted microscope images of HUVECs grown on (A) unpatterned control, (B) square wells (SW), (C) round wells plus square pillars (RWSP), (D) square pillars (SP) and (E) gratings A silk substrates after 3 days in culture. Scale bars are 60 ␮m. (F) Cells on patterned features including square wells, round wells plus square pillars, square pillars, gratings A were counted after 1 day and 3 days in culture. (G) No significant differences were observed between unpatterned control and gratings (from about 800 to 1700 nm pitch, about 400 nm depth). Significant differences between unpatterned controls and topography are marked as follows: * p < 0.05, ** p < 0.01, *** p < 0.001.

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5. Conclusion This study demonstrated that different substrate topographies of thin silk films influenced HUVECs behaviors, including cell orientation and alignment, filopodial interactions with surface topography, and proliferation. A similar response in orientation and alignment of HUVECs was observed on topographic gratings greater than 800 nm pitch (about 400 nm depth). The cells demonstrated a decrease in orientation and alignment with response to the smallest topographic features containing grating patterns. Interestingly, we concluded that filopodia altered their primary growth orientation on the ridges to choose the shortest pathway for crossing the grooves. “Contact guidance” effects have been observed for HUVECs cultured on round wells plus square pillars. In addition, cell densities were also observed to be lower on the smallest topographic features containing square pillars (about 300 nm gap) as well as the gratings A (70 ± 3 nm depth, 600 ± 8 nm pitch) patterns when compared to the unpatterned control. These studies would be useful to understand the importance of incorporating topographic cues within substrates used for in vitro experimentation and for the development of artificial tissue-engineered blood vessels. Moreover, this study suggested that patterned silk films could be used for the engineering of highly orientated tissue structures including not only in the realm of corneal tissue engineering but also vascular tissue engineering or even neural and muscular tissues, due to the predominate importance of structures in the function of these tissues. Acknowledgments

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This work was financially supported by the National Natural Science Foundation of China (Nos. 91027040, 21074084, 21374070, 21374069 and 21204058), the National Basic Research Program of China (No. 2012CB821500), the Scientific Research Foundation for the Returned Overseas Chinese Scholars, State Education Ministry, and the Project of Scientific and Technologic Infrastructure of Suzhou (SZS201207).

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