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A Bio-inspired 3D Multilayered Shape Memory Scaffold with a Hierarchically Changeable Micropatterned Surface for Efficient Vascularization Dian Liu, Tao Xiang, Tao Gong, Tian Tian, Xian Liu, and Shaobing Zhou ACS Appl. Mater. Interfaces, Just Accepted Manuscript • Publication Date (Web): 25 May 2017 Downloaded from http://pubs.acs.org on May 26, 2017

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ACS Applied Materials & Interfaces

A Bio-inspired 3D Multilayered Shape Memory Scaffold with a Hierarchically Changeable Micropatterned Surface for Efficient Vascularization

Dian Liu, Tao Xiang, Tao Gong, Tian Tian, Xian Liu, Shaobing Zhou* Key Laboratory of Advanced Technologies of Materials, Ministry of Education, School of Materials Science and Engineering, Southwest Jiaotong University, Chengdu 610031, China

Keywords: Shape memory; micropattern; biodegradable polymer; co-culture; three-dimension

ABSTRACT: How to achieve three-dimensional (3D) cell alignment and subsequent prompt tissue regeneration remains a great challenge. Here, inspired by the interior 3D architecture of native arteries, we develop a new 3D multilayered shape memory vascular scaffold with a hierarchically changeable micropatterned surface for vascularization. The shape memory function renders the implantation of the scaffold safe and convenient via minimally invasive surgery. By co-culturing endothelial cells (ECs) and vascular smooth muscle cells (VSMCs) on the 3D multilayered structure, the inner monolayer, which has a square micropatterned surface, can promote EC adhesion and migration, resulting in rapid endothelialization; the outer multilayers, which have rectangular micropatterned surfaces, can induce a circumferential alignment of VSMCs. After implantation in the cervical artery of a New Zealand rabbit for 120 days, the graft developed a good capacity for modulating cellular 3D alignment to generate a neonatal functional blood vessel with an endothelium layer in the

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inner layer and multilevel VSMC circumferential alignments in the outer layers.

INTRODUCTION Cardiovascular diseases, principally those of small-diameter arteries (< 6 mm), are major diseases and threaten public health and even cause death and disability worldwide.1 Smalldiameter vascular grafts (SDVGs) (Diameter < 6 mm) are increasingly needed for clinical surgical intervention. Autologous grafts, such as arteries and saphenous veins, can be beneficial, but these substitutes may be unavailable due to insufficient sources, morbidity and multiple surgeries.2 Commercial artificial poly(ethylene terephthalate) and poly(tetrafluoroethylene) materials have been successfully applied in large-diameter vascular grafts, but often lead to failure because of thrombus, intimal hyperplasia and aneurysm formation.2 Vascular tissue engineering has been developed and is considered to be a promising approach to address these defects.3 To guarantee the sustainable and functional compliance of vascular grafts, regeneration of a patient's neonatal well-organized vascular tissue through vascular remodeling is considered paramount.3 Cell three-dimensional (3D) alignment exists extensively in various native organs and tissues and exerts crucial effects on tissue regeneration and physiological function.4 Natural arteries mainly comprise endothelial cells (ECs) and vascular smooth muscle cells (VSMCs); in arteries, ECs are located in the innermost layer to form monolayer endothelium.4 ECs secrete both endothelium-derived relaxing factors and endothelium-derived contracting factors, which play important roles in vascular relaxation, contraction and platelet disaggregation.5 Several


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layers of VSMCs circumferentially wrap the endothelium perpendicular to the vessel long axis. VSMCs produce extracellular matrix (ECM) and provide an appropriate physiological and functional vascular tunica media.4 Researchers have expended a great deal of effort on directing the use of various approaches to engineer vascular grafts in an attempt to mimic native vascular 3D architecture. Several groups have developed circumferentially aligned fiber vascular grafts and circular microchannels to guide VSMC behavior.6,

7

Both vascular

substitutes have achieved a good arrangement of VSMCs in a circumferential orientation with high viability in vitro or in vivo, and some substitutes can function in contraction and relaxation. These studies extensively document the availability and promise of using 3D vascular grafts, which mimic the native 3D structure of arteries to regulate VSMC alignment and regenerate functional tunica media. However, both ECs and VSMCs form typical 3D cell alignments in natural arteries. The studies mentioned above merely mimic the VSMC layer, and the endothelium is not involved. Recently, Jiang et al.8 described a general stress-induced rolling membrane technique to imitate the 3D structures of blood vessels. In this method, the 3D alignments of ECs and VSMCs can be accurately controlled; the ECs are located in the innermost layer, and the VSMCs are circumferentially aligned and located in the interlayer region. However, this study was limited by using an in vitro model, and regeneration of the neonatal artery in vivo using 3D biomimetic grafts remains unknown. In addition, the role of topographical patterning cues in cell fate has been widely recognized, and many attempts have been made to project an appropriate topography to regulate cell alignment.9 Topography determines cell behavior and function through a mechanism known as mechanotransduction.10 Kim et al. 11 engineered lattice arrays to investigate cell migration and



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organization and found that square and rectangular lattice arrays promote cell migration. Moreover, cells were oriented along the long axis of the rectangle. The size of the topographical patterning is another factor that significantly influences cell fate. Other studies have reported that square wells with lengths ranging from 4 μm to 25 μm allow cells to achieve better attachment and deposition.12, 13 Grooves with widths ranging from 1 μm to 10 μm are available and have an optimal size for inducing the directional alignment of cells through contact guidance.14 Moreover, dense ridges have a positive effect on cell alignment and migration.11 In our previous work,15 we found that the use of a dynamic surface topography with a combination of concentric circular microgrooves and radial, straight microgrooves on a monolayer tunable graft effectively captured vascular cells and precisely guided the distribution and alignment of ECs and VSMCs. These encouraging results reveal the great effects of surface topography and its promise for using in future applications. However, 2D topographical patterning cues may be weakened when used to regulate multilayered cells because the upper cells cannot contact the bottom topography.16 In addition, the structure of two-dimensional grafts does not correspond with actual 3D vascular construction. Thus, 3D multilayered structures are urgently needed to address these limitations. Shape memory polymers (SMPs) have been widely used in biomedical applications, particularly in minimally invasive surgery, due to their exciting property of temporarily “memorizing” a macroscopic shape and then recovering to their permanent shape via an external stimulus.17 In our previous work, a porous polymer scaffold was fabricated that could be utilized for minimally invasive implantation and matched the contour of damaged tissue.18 Additionally, a biodegradable polylactide-co-poly(ε-caprolactone) polymer stent with a


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temperature-switched shape memory function was manufactured for treating esophageal stenosis as it returned to a ring shape.19 Inspired by the interior 3D architecture of native arteries, in this work, we develop a new 3D multilayered shape memory vascular scaffold with a hierarchically changeable micropatterned surface. The surface of the scaffold is decorated with two typical topological structures: square patterns (15 × 15 μm, spacing 5 μm, depth 10 μm) in the inner monolayer and rectangular patterns (5 × 20 μm, spacing 5 μm, depth 10 μm) in the outer multilayer. The inner monolayer, which has a square pattern, can promote EC adhesion and migration, resulting in rapid endothelialization. The outer multilayers, which have rectangular surfaces, can induce VSMC circumferential alignment. We selected multilayer rectangular structures because 1) VSMCs form multilayers in native arteries and 2) 2D topography has a weak impact on multilayered cells. Thus, a biomimetic 3D vascular graft would not only reconstruct the 3D vascular architecture but would also regulate EC and VSMC alignment through the use of different topological patterns. The vascular graft substrate is composed of a crosslinked 6-arm poly(ethylene glycol)-poly(ε-caprolactone) (c-6a PEG-PCL) polymer, which has an excellent shape memory function, biocompatibility and biodegradability. 20 Therefore, minimally invasive surgery can be accomplished by exploiting the shape memory effect; simultaneously, the graft can support the blood vessel by preventing its blockage. The in vitro behaviors of ECs and VSMCs on the square and rectangular topological structures were investigated first; later, to confirm the in vivo vascularization capacity of the 3D multilayered shape memory scaffold with a hierarchically changeable micropatterned surface, implantation and long-term biological evaluation were performed in a rabbit cervical artery model.



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EXPERIMENTAL SECTION Preparation of the 3D Multilayered Shape Memory Vascular Graft. As shown in Figure 1A, a mixture of 6a PEG-PCL-AC and 10 wt% phosphine oxide (TPO) was melted at 65 oC; then, the molten mixture was cast on a micropatterned polydimethylsiloxane (PDMS) mold. The sample was then photocrosslinked by irradiation with ultraviolet light (360 nm, 110 W) for 40 min. The patterned c-6a PEG-PCL membrane was then carefully detached from the PDMS replica. Finally, the c-6a PEG-PCL membrane (7 ×15 mm, with a thickness of 85 ±25 μm), which had a square pattern on the inner layer and rectangular pattern on the outer layer, was rolled up to form a solid tube at 60 oC to decrease its volume for minimally invasive implantation and then cooled to 0 oC to fix the temporary shape. The topography of the surface patterns and cross-section of the multilayered vascular graft were observed under scanning electron microscopy (SEM). In vitro Shape Memory Effect of the Vascular Graft. To verify the shape memory effect of the vascular graft, which is required for minimally invasive implantation, an in vitro simulation was conducted. A hollow red plastic tube was used to simulate a native artery. The vascular graft was tightly rolled up at 60 oC and plugged into the red plastic tube after fixing the deformed temporary shape at 0 oC. The specimen was then heated to 37 oC to test whether the vascular scaffold could recover its permanent shape. All processes were recorded under a stereoscopic microscope. Quantitative analyses of the shape memory fixity ratio and recovery ratio were measured using a dynamic mechanical analysis (DMA) (TA, DMA-Q 800) instrument. Shape memory


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effects were implemented for the samples in both dry and wet states using a typical investigation procedure. The stress was increased at a frequency of 1 Hz and was fixed at 0.8 MPa while the temperature was cooled to 0 oC. The temperature was then equilibrated at 0 oC for 10 min, and the stress was released. Finally, the specimens were heated to the temperature corresponding to shape recovery. For the wet state, the specimens were immersed in phosphatebuffered saline (PBS) for 24 h before testing. The shape memory fixity ratio (Rf) and recovery ratio (Rr) were calculated according to the following equations: Rf 

 

1

Rr 

2

   1

2

1

where εx is the strain of each shape obtained from the DMA strain curve. The mechanical properties of the c-6a PEG-PCL membranes were measured using a universal mechanical testing machine (5567Q4052, Instron, USA). The samples were pulled longitudinally at a rate of 1 mm/min until rupture. Young’s modulus was calculated by measuring the slope of the stress-strain curve. Vascular Graft Implantation and Doppler Ultrasound. New Zealand White rabbits (2.2– 3.0 kg) were selected as the experimental animals; approximately 15 rabbits were used for the in vivo experiments. All animals were cared for in conformity with the protocol of the Animal Care Committee of Sichuan University. Before implantation, the vascular grafts was tightly rolled at 60 oC and the temporarily deformed shape was fixed at 0 oC. Vascular grafts (temporary shape) were engineered (7 ×1 mm; length ×radius). The graft was carefully inserted into the cervical artery and the details of the experimental process are shown in Figure S10. After 7, 30 and 120 days, the animals were euthanized, and the vascular tissues containing the



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grafts were harvested for further histological analysis. Doppler ultrasonography (Z6Vet, Mindray, China) was used to monitor the shape recovery of the grafts after 14 days of implantation, and artery patency was determined using color Doppler analysis. Additional methods are in the Supporting Information RESULTS Characterization of the 3D Multilayered Shape Memory Scaffold. The chemical structure and composition of the 6a PEG-PCL-AC copolymer was confirmed based on FT-IR and 1H NMR spectra (Figure S1B, C). The gel content of the chemically crosslinked polymer was approximately 86.5%. The surface micropatterns, square (15 × 15 μm) and rectangular (5 × 20 μm) with a spacing of 5 μm and a height of 10 μm, were transferred from PDMS replicas to the polymer membrane (Figure 1A). The transferred micropatterns showed uniform alignment (Figure 1B, C). A 3D multilayered tubular architecture was constructed in which the micropatterned membrane was rolled up such that the square micropatterns were located in the luminal layer and the rectangular micropatterns were located in the outer layers (Figure 1D, E). The 3D multilayered structure of the graft cross-section was apparent in SEM images (Figure 1D); moreover, at greater magnification (Figure 1E), the surface micropatterns were clearly seen on each layer. Shape Memory Function and Mechanical Performance of the Vascular Graft. To confirm the shape memory function of the vascular grafts, quantitative dynamic mechanical analyses (DMA) of c-6a PEG-PCL were conducted under dry and wet conditions. According to the stress-strain-temperature curves, c-6a PEG-PCL possesses excellent shape memory


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effects under both dry and wet conditions (Figure 2 and Figure S2). The shape fixed ratio (Rf) surpasses 98% under both conditions, and the recovery ratio (Rr) was 87% under the dry conditions and 81% under the wet conditions. The recovery temperature of dry sample was 41 o

C (Figure S2), and this was reduced to 37 oC under the wet conditions. This decrease in

temperature was mainly because water acts as plasticizer, increasing the flexibility of the macromolecular chains.20 The shape memory effect of the vascular scaffold at body temperature is used to facilitate minimally invasive therapy and to support the blood vessels and prevent restenosis when the scaffold is implanted into a blood vessel and recovers to its original shape. An in vitro simulation experiment was first carried out; as shown in Figure 2C, a hollow red plastic tube was used to mimic an artery. The shape memory vascular graft was deformed by tightly rolling, and its volume was decreased to approximately 25% of the original value, indicating that minimally invasive surgery is feasible. After fixing the temporary shape at 0 oC, the vascular graft was implanted into the simulated blood vessel. With reheating to 37 o

C (close to body temperature), the vascular graft spontaneously recovered its tubular structure

and supported the blood vessel mimic. The mechanical properties of c-6a PEG-PCL were characterized by axial tensile testing, and the resulting stress-strain curves are shown in Figure 2B. According to the stress-strain curves, the Young’s modulus of this material is 111.9 ± 2.3 MPa, a value that almost matches the mechanical property of a native vascular graft under arterial pressure.21 Degradation of the Vascular Graft. To ensure the sustainable and functional compliance of vascular grafts, the regeneration of a patient's neonatal well-organized vascular tissue through vascular remodeling is required. Biodegradability is required since timely scaffold



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degradation is essential for host cell remodeling and the synthesis of significant amounts of ECM.2 The degradation properties of the scaffold in vitro and in vivo were investigated (Figure S3). Both the mass and gel content of the scaffolds were gradually reduced as degradation time increased; the residual mass percentage was 88.9 ±4.1 % and the gel content was 55.1 ±5.8 % at 150 days. The surface micropatterns were also pictured by SEM at different degradation times (Figure S3); the surface topology did not obviously change after 150 days of degradation. However, the in vivo degradation of the scaffold was remarkably more rapid than the in vitro degradation (Figure S4). The patterned surface has only little effect on the degradability of the scaffold. The mass residual percentage and the gel content percentage were 92.3 ± 4.6 % and 68.8 ±5.8 %, respectively, at 30 days after implantation in a rabbit carotid artery. The vascular grafts were completely degraded at 120 days after implantation due to the presence of enzymes and monocytes in vivo, which accelerate the degradation of materials.22 There has been reported that rapid graft degradation within 90 days can result in rapid graft remodeling and subsequent neo-artery regeneration.21 EC and VSMC Behavior on the Surface Micropatterns. Hydrophilicity is very important for inducing specific cellular responses, and weak hydrophilicity limits cell attachment. As shown in Figure S5, the contact angles of the scaffolds having flat, square and rectangular patterns were 54 ± 3.4 °, 60.1 ±4.5 °and 50.4 ±3.8 °, respectively, indicating that the surface micropatterns exhibit the appropriate hydrophilicity. EC and VSMC proliferation and viability on different surface topologies (square, rectangular and flat) were studied using the Alamar Blue (AB) assay. As shown in Figure S6A and B, the viabilities of both VSMCs and ECs increased with time on all micropatterns. EC viability was


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97.2 ±2.7 % on the square pattern and 80.7 ±2.3 % on the rectangular pattern on day 7. VSMC viability was 97.5 ±2.1 % on the rectangular pattern and 82.7 ±3.5 % on the square pattern on day 7. Moreover, live-dead staining was further adopted to assess the cytocompatibility of the materials (Figure S6C); most of cells were stained green, indicating their survival. Interestingly, the square patterns facilitated EC proliferation while the rectangular patterns promoted VSMC proliferation after 6 days. These results demonstrated that these materials possess good cytocompatibility and that the square and rectangular patterns have a selectively positive effect on the proliferation of different cell types. The numbers of cells on the square and rectangular patterns were further compared under confocal microscopy (Figure 3C); significantly more ECs were found on square patterns (square 48 ±10; rectangle: 26 ±5 / cell number) and more VSMCs (square 17 ± 4; rectangle: 30 ± 7 / cell number) were found on rectangular patterns at day 6, consistent with the results of the AB assay. Importantly, cell shape decides cell fate and function.23, 24 The corresponding cellular cytoskeleton and morphologies of ECs and VSMCs on different micropatterns were further revealed by immunofluorescence at 3 and 6 days (Figure 3A and B), respectively. ECs were tightly deposited in the square wells at 3 days, suggesting that the square wells effectively captured ECs; the cells gradually sprawled out and covered the substrate surface at 6 days. Notably, most ECs preferentially extended lamellipodia from the edge of the square, indicating that the cells could rapidly migrate along tension field lines of the extended lamellipodia.25 Abundant lamellipodia and filopodia, which extended outward from the leading edges of the ECs and along the ridge of square, were also observed under SEM (Figure S7). This behavior can cause the fast dissemination of ECs in vivo, leading to



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rapid endothelial coverage. However, ECs on the rectangular and flat patterns showed inanimate pseudopodia or no lamellipodia, indicating low mobility, at both 3 and 6 days. More importantly, the cells were forced to adopt a depauperate shape (small with no pseudopodia) on the rectangles compared with cells on the flat surface and on the squares, implying that the ECs underwent apoptosis.26 All VSMCs cultured on rectangles presented a perfectly directional arrangement and were coerced to adopt a highly stretched and thin morphology at all of time points because of the well-known effect of contact guidance

10

(Figure 3B and Figure S8). Remarkably, the cell

nuclei were highly elliptical, and the cytoskeletons showed parallel and dense F-actin bundles along the long axis of the rectangles. Highly aligned F-actin bundles are important for cell contraction. Moreover, elongated morphology not only promotes the proliferation of VSMCs but can also affect myogenic protein expression in the cells.27 However, VSMCs on both the squares and the flat surface were randomly oriented and expressed randomly aligned F-actin bundles. The angular distribution of the VSMCs was analyzed after 6 days of culture on the squares and rectangles (Figure 3D, E). Alignment angle was defined as the cell’s long axis between rectangle’s long axis (Figure 3D); more than 90% of the VSMCs had angles of less than 20°on the rectangular patterns (Figure 3E), while the cells on the square patterns were randomly oriented (Figure 3F and Figure S8). That is, the rectangular micropatterns effectively induced VSMC proliferation and arrangement to form a circular smooth muscle layer, whereas the square patterns encouraged the ECs to proliferate and spread out. EC & VSMC Co-culture and Platelet Adhesion. To further study vascular cell behavior and function, cross-sections of ECs and VSMCs that had been co-cultured for 7 days on curly


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micropatterned vascular grafts were stained with laminin (green) and α-SMA (red), which specifically label ECs and VSMCs, respectively. As shown in Figure 4A, the cells rapidly proliferated and completely covered the entire cross-sections, possibly leading to rapid endothelialization in vivo. As expected, the cells on the scaffolds with rectangular patterns showed elongated nuclei and a significantly circumferential alignment compared with the square patterns in the lumen, a pattern that is similar to that seen in smooth muscle cell layers in natural blood vessels. Additionally, based on the semiquantitative fluorescence intensity of laminin and α-SMA (Figure 4A), it is apparent that more laminin is expressed in cells on the square, while more α-SMA is expressed in cells on the rectangular patterns. The result in cocultured cells is consistent with the results obtained for the single-cell culture. The hierarchically changeable micropatterned surface exhibits a great capacity for separating the co-cultured ECs and VSMCs. Moreover, ECs on the square pattern exhibited rapid proliferation, contributing to in vivo endothelialization; VSMCs, which exhibited good proliferation, displayed a circumferential arrangement on the rectangular pattern, which is conducive to forming a circular smooth muscle layer in vivo. A previous report showed that an elongated morphology can up-regulate relevant myogenesis genes, such as GATA-4 and Myoblast differentiation protein 1 (MyoD1), possibly explaining our result.27 To determine the effect of surface topography on blood platelet adhesion, in vitro platelet adhesion was performed and characterized by SEM (Figure 4B and Figure S9). The results showed that the platelets were round and had fewer pseudopodia, suggesting that the adhered platelets are in an inactivated state (Figure 4B). The SEM images presented in Figure S8 show that these platelets do not abundantly aggregate on the surface and that they are inactivated,



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indicating that our material and surface topographies are inert with respect to platelets, a property that is crucial for vascular grafts. Operative and Patency of the Implanted Vascular Grafts. An in vivo experiment was carried out to confirm whether the 3D multilayer vascular grafts with the hierarchically changeable micropatterned surface could promote the regeneration and reconstruction of neonatal blood vessel tissue after implantation into the cervical artery of New Zealand rabbits (Figure S10A, B). The patency of the implanted micropatterned grafts was examined by color Doppler ultrasound at 15 days (Figure S10C), and the result indicated that the grafts retained good patency and showed uniform blood flow profiles. In addition, the inner diameter of the grafts was 1.5 mm according to Doppler ultrasound; this result proved that after minimally invasive implantation of the temporarily smaller shape, the exposure of the shape memory vascular grafts to body temperature (37.5 oC, similar to the transition temperature of the vascular grafts; 37 °C) caused the grafts to spontaneously recover their original shape. In situ Vascular Tissue Remodeling and Regeneration. After implantation of these vascular grafts for 7, 30, and 120 days, the rabbits were euthanized, and the carotid arteries containing the implants were examined by Masson trichrome staining to visualize the neovascularization, and vessel permeability was further confirmed. As shown in Figure 5A, in all groups, no occlusion-related phenomena occurred, and the lumens of the grafts remained clean and free of platelet aggregates and thrombi due to the hydrophilicity and biocompatibility of the PEG component of the scaffolds.28 In the micropatterned groups (Figure 5 A), a distinct attached cell layer was observed on the surface of each layer. The thickness of the cell layer was considerably increased after implantation for 1 month, forming a continuous and dense


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new tissue layer. The thickness of the new vessel-like tissue was 12 ± 5.6 μm at 7 days; this was dramatically increased to 79.6 ± 21.4 μm at 30 days, finally reaching 324.3 ± 37 μm at 120 days with obvious dense collagen deposits, a value that is very close to that of native arteries (Figure 5A). After the scaffolds had been implanted for 30 days, the micropatterns remained, according to the Masson staining, demonstrating that the patterns can persist and continue to aid the vascular tissue regeneration process over long periods. On the flat surface, there was almost no cell deposition on the surface of the scaffolds (Figure S11 B) because this surface does not facilitate cell adhesion, and cells may be washed away by the blood flow in vivo in the absence of topological structure.15 To further ascertain whether the neonatal cell layers resembled the construction of native artery layers, immunofluorescence staining was performed using CD31 (green) to label ECs and α-SMA (red) to label VSMCs (Figure 5B). Natural autologous vascular tissues are layered. Both markers are visible on the inner wall of the scaffolds, but few of the cells were positively stained at 7 days. After 30 days, circumferential regeneration was detected, and the grafts were almost fully covered by newly regenerated tissues in each layer. ECs formed a continuous and homogeneous endothelium at the innermost square layer. The intimal endothelial coverage ratio was 4.1 ±2.3 % at 7 days but sharply increased to 100% at 30 days (Figure 5D), indicating that endothelialization was complete by 30 days. The outer multilayer was also covered by ECs, effectively reducing the contact of the grafts with blood, leading to a decrease of thrombus and possibly indicating a beneficial interaction between the ECs and VSMCs. On the other hand, few α-SMA+ cells were detected at 7 days and were dispersed in each layer; however, the early cells developed into neo-tissue with an ordered circumferential arrangement in outer



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rectangular layers at 30 days; the cells exhibited positive α-SMA staining, which is characteristic of a typical VSMC phenotype. At 30 days, the scaffold material continued to exist, and the topology of the structure persistently regulated cell alignment. Finally, at 120 days, when the scaffold had completely degraded, the neonatal VSMC layers developed into mature, organized filamentous fiber tissues, which were relatively homogenous the with native arteries. More importantly, the neo-vessel was layered, showing a remarkable 3D cell alignment similarity with native arteries. However, free α-SMA+ or CD31+ cells were present on the flat surface (Figure S11A) because it did not facilitate cells adhesion under blood flow.15 It is well known that inflammation plays an important role in tissue repair.6 To reveal inflammatory cell infiltration and the graft response, the grafts were staining against CD68, and the macrophage phenotype was further confirmed by CD206 staining, which indicates M2 macrophages (Figure 5C). A large number of cells including ECs, VSMCs and macrophages were recruited in the grafts at 14 days (Figure 5E). A strong inflammatory reaction was detected, due to the occurrence of foreign bodies (Figure 5C, F). More macrophages were subsequently recruited at the graft surface at 30 days, and these macrophages developed into M2 macrophages (Figure 5F). The CD206＋macrophages were uniformly located in each layer of the grafts and markedly increased at 30 days (Figure 5C, F). Plentiful pro-healing M2 macrophages recruited in the vascular graft resulted in marked tissue repair, which is beneficial for revascularization.6, 22 Extracellular Matrix Deposition in the Regenerated Vessel. A proper ECM is crucial for the long-term stability of arteries. After implantation for 4 months, the explanted vascular scaffolds were harvested. The expression levels of the selected marker genes were evaluated,


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and a natural carotid artery was chosen as a control. Figure 6D shows high expression of αSMA, laminin, collagen-I and collagen-IV in both the vascular grafts and natural artery. Western blotting analysis further quantitated the changes in responsive protein expression (Figure 6E and F). The results showed that the abundant ECM deposition was similar to the variations seen in the native artery. Despite slight differences in matrix quantity, no significant difference in the mechanical properties was found. The burst pressure of the neonatal functional blood vessel was 260 ± 57 kPa, a value that is similar to that of natural carotid arteries (330 ± 42.42 kPa) (Figure 6B).

DISCUSSION Tissue engineering offers an attractive option to create durable and available vascular prostheses. Two strategies have been developed for producing vascular grafts, including cellcontaining and cell-free vascular grafts. Cell-containing vascular grafts are produced by seeding ECs and VSMCs (and even stem cells) on natural or synthetic materials and regenerating neo-vascular tissue in a special bioreactor system in vitro. Some functional vascular substitutes have been developed using this strategy.29 However, it can take several weeks to gather the necessary cells and culture tissues. Long fabrication times and high production costs limit the application of these grafts. By contrast, cell-free vascular grafts recruit autologous cells after implantation, and these cells regenerate neonatal vascular tissues. Therefore, composite scaffolds can provide a favorable environment for the capture of vascular cells and create effective elements to induce 3D cell alignment. The substrate material is another important element of vascular grafts. Non-degradable or



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slowly degradable grafts prolong the presence of foreign materials, not only limiting the diffusion of nutrients but also leading to chondroid metaplasia and even calcification due to the hypoxic conditions.30 Therefore, the materials used to create vascular grafts should be biodegradable. Such materials are timely degraded after implantation and can be substituted by autologous cells, which secrete an ECM and promote neo-angiogenesis.21 In this study, crosslinked 6a-PEG-PCL, a typical biodegradable shape memory biomaterial, was used as a vascular graft substrate. The degradation of this vascular graft during cellular ingrowth provides a space for ECM deposition, nutrient transport and oxygen diffusion, and the grafts could completely degraded in vivo within 120 days (Figure S4). Several previous works have reported that the neo-vascular tissue could regenerate in 90 days, which contains well threedimensional structure and physiological function.2,6 Even there has been reported that rapid graft degradation within 90 days can result in rapid graft remodeling and subsequent neo-artery regeneration.21 In addition, the material demonstrated excellent biocompatibility with ECs, VSMCs (Figure S5) and blood platelets (Figure S9). Because of its excellent shape memory effects and distinct recovery temperature close to body temperature in the wet state (Figure 2, Figure S2), this material can be used for minimally invasive surgery in vivo, and the grafts can recover their tubular shape to support blood vessels and prevent restenosis (Figure S10). The artery exhibits a 3D structural organization typically including endothelium, tunica media and adventitia, consequently providing both physiological functions and tensile strength.5 Monolayer endothelium contributes significantly to both critical vasoactive response and artery patency. Therefore, it is important to achieve a uniform endothelium for EC adhesion and proliferation on the lumen surface of grafts. In the tunica media, multiple layers of VSMCs


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circumferentially wrap the endothelium. Both EC and VSMC layers play vital roles in maintaining vessel architecture and mechanical properties and the response to various biochemical and mechanical stimuli.5 Inspired by cell alignment in vivo, numerous groups have designed various vascular grafts to mimic 3D blood vessel construction.4, 6 Although some encouraging results have been achieved, these grafts merely adjusted the alignment of either ECs or VSMCs and failed to concurrently regulate and control ECs and VSMCs. The effect of topographical cues on cell behavior and fate has long been recognized. 25-27 Through a mechanism known as mechanotransduction, a variety of physical stimuli in the environment surrounding cells are integrated and converted to biochemical signals, leading to changes in cell behavior.10 Inspired by the 3D cell alignment of native arteries and based on the influence of topological structure on cell behavior as mentioned above, a new 3D vascular graft was developed in this work. This graft contains two different topological structures that specifically modulate corresponding vascular cell behaviors based on surface topology. As expected, our in vitro experiment confirmed that square patterns stimulate EC migration and adhesion (Figure 3). These EC behaviors are favorable for the rapid endothelialization of these vascular grafts in vivo. Furthermore, ECs express suitable amounts of laminin (Figure 4A), an important glycoprotein that can produce simultaneous interactions between cells and other ECM components via specific integrin receptors.31 Conversely, rectangular patterns facilitate VSMC proliferation.32 In addition, VSMCs adopt an elongated morphology, an oriented alignment and the up-regulation of myogenic protein when located on the rectangular surface (Figure 3B and Figure 4A). Appropriate cell morphology is vital for DNA synthesis, which directly determines cells fate.23 It is well known that interactions between ECs and VSMCs are



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fundamental for arteriogenesis, and these cells communicate through the synthesis and secretion of mediators into the surrounding medium and through the direct transmission of information via gap junctions.5 We found an evident physical contact between the ECs and VSMCs in each layer after 30 days of implantation (Figure 5B). During vasculogenesis, angiogenic factors, such as platelet-derived growth factor BB, which is released from ECs, recruit undifferentiated mesenchymal cells; once these cells contact the ECs, they are induced to differentiate into VSMCs.33 ECs direct the migration and proliferation of VSMCs and directly induce SMCs to adopt a differentiated phenotype by activating the phosphoinositide 3-kinase pathways of VSMCs.34 As the ECs and VSMCs interact, a statistically significant increase in the gene expression of angiogenic factors occurs, including increases in the expression of vascular endothelial growth factor, platelet-derived growth factor BB and transforming growth factor-β.35 With time, ECs restrict the over-proliferation of VSMCs,36 and VSMCs can guide the alignment of ECs.37 It is evident that inflammation is a significant physiological response and plays an important role during tissue regeneration.6,

22

Macrophages are quite abundant and show diverse

phenotypes in inflammatory and anti-inflammatory environments. Macrophages arrive at a wound within 48 to 96 hours after injury and participate in removing debris, releasing proinflammatory cytokines, producing growth factors, and remodeling the ECM.38 It has been revealed that M2 macrophages, which can be activated by cytokines such as interleukin-4 and interleukin-10, promote tissue repair by producing anti-inflammatory cytokines that modulate cell replacement, angiogenesis, and matrix remodeling.39 We found that early macrophages were recruited to the grafts and might be intricately involved in postnatal blood vessel


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formation.40 Moreover, M2 macrophages were observed at 14 days, and both macrophages and M2 macrophages progressively increased in number over time (Figure 3C, F). These macrophages may motivate cell migration, proliferation, secretion in the grafts, and mediate tissue repair and ECM deposition. The immunofluorescence results showed that endothelial cell coverage was achieved in the intimal layer within 30 days after implantation (Figure 3B, D) due to the assistance of the square patterns. Furthermore, our data showed that a circumferential smooth muscle layer was present in each rectangle layer resulting from the facilitation of VSMC proliferation and the alignment induced by the rectangle patterns. These cells stained positive for α-SMA, indicating that the VSMCs had a contractile phenotype (Figure 5). Due to the timely degradation of the grafts, the VSMCs produced intermixed collagen (mainly types I and III), elastin, and proteoglycans. These components not only provide appropriate stiffness, elasticity and compressibility but also influence cell adhesion, migration, and phenotype. Abundant ECM components were detected in the neo-artery, particularly α-SMA, laminin, collagen-I and collagen- IV (Figure 6D, E). Collagen-I not only constitutes 60% of collagens in natural arteries but also inhibit excessive VSMC proliferation through the regulation of cdk2 inhibitors; this inhibition may be helpful for preventing intimal hyperplasia.41 CONCLUSIONS In summary, inspired by the interior 3D architecture of native arteries, we constructed a 3D multilayered shape memory vascular scaffold with a hierarchically adjustable micropatterned surface. The surface of the scaffold was decorated with square patterns in the inner layer and rectangle patterns in the outer layers. The shape memory function was used to



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support the blood vessels by preventing their blockage via a minimally invasive implantation. The different topographical structures used selectively affected vascular cell behavior. The square patterns encouraged proliferation and spread of ECs, while the square patterns facilitated proliferation of VSMCs and guided the ordered alignment of VSMCs. More importantly, the 3D micropatterned grafts exhibited a good ability to modulate the 3D alignment of the cells in vivo to form a neonatal functional blood vessel with an innermost endothelium layer and multilevel circumferentially aligned VSMCs in the outer layers after the grafts were implanted for 120 days. Therefore, when an artery is damaged by disease or operation, the 3D multilayered shape memory vascular scaffold is very suitable for the timely blocking of leaks because of its shape memory effect and for regenerating neonatal wellorganized vascular tissue that can replace and repair the damaged vessels.

ASSOCIATED CONTENT

Supporting Information. Synthesis, characterization of the polymer including Figure S1, DMA measurement in Figure S2, in vitro degradation of the grafts in Figure S3, in vivo degradation of the grafts in Figure S4, water contact angles in Figure S5, the ECs and VSMCs proliferation in Figure S6, ECs and VSMCs morphology and distribution observed with SEM in Figure S7 and S8, platelet adhesion in Figure S9, implantation and patency of the grafts in Figure S10, in vivo histological staining of the vascular grafts in Figure S11 as described in the text. The Supporting Information is available free of charge on the ACS Publications website.


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CONFLICT OF INTEREST The authors declare no conflict of interest. ACKNOWLEDGMENT This work was partially supported by the National Natural Science Foundation of China (Grant Nos. 51373138, 21574105), the National Basic Research Program of China (973 Program, 2012CB933600) and the Sichuan Province Youth Science and Technology Innovation Team (Grant No.2016TD0026). AUTHOR INFORMATION

Corresponding Author *(S.Z.) E-mail: [email protected]; [email protected]

Author Contributions D.L. and T.X. contributed equally to this work.

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Figure 1. (A) Scheme of the preparation of micropatterned shape memory c-6a PEG-PCL vascular grafts. (B, C) SEM images show the square patterns and rectangular patterns on c-6a PEG-PCL membranes. (D, E) SEM images of cross-sections of the surface micropatterns and corresponding magnified images of the vascular graft.


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Figure 2. (A) Quantitative thermal mechanical demonstration of wet c-6a PEG-PCL membranes as assessed by dynamic mechanical analysis (DMA). (B) Tensile strain-stress curve of a micropatterned c-6a PEG-PCL membrane. (C) In vitro simulation of the shape memory process.



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Figure 3. Confocal images of F-actin organization in ECs (A) and (B) VSMCs on different surface topologies (squares, rectangles, and a flat surface) at 3 and 6 days, respectively. F-actin is labeled in red, and nuclei are labeled in blue. (C) The average number of cells on the square and rectangular patterns was calculated based on confocal images (200×) after 6 days of cultivation (at least 4 pictures was used). (D) Scheme showing the definition of cell orientation. (E-F) Polar plots showing the quantification of the angle distribution of VSMCs after 6 days of culture on the square and rectangular patterns.


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Figure 4 (A) Immunofluorescence staining and semiquantitative fluorescence intensity of protein expression for laminin (green), α-SMA (red) and counterstained for nuclei (blue) taken at section of vascular grafts, when SMCs/ECs co-cultured on curly vascular grafts for 7 days. (B) SEM images of platelet adhesion on the square, rectangle pattern and flat. The platelets maintained less activated. (*P

Bioinspired 3D Multilayered Shape Memory ... - ACS Publications

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