Heterotypic Scaffold Design Orchestrates Primary Cell Organization and Phenotypes in Cocultured Small Diameter Vascular Grafts

Advanced Functional MaterialsVolume 29, Issue 43 1905987 Full PaperOpen Access Heterotypic Scaffold Design Orchestrates Primary Cell Organization and Phenotypes in Cocultured Small Diameter Vascular Grafts Tomasz Jungst, Tomasz Jungst Department for Functional Materials in Medicine and Dentistry and Bavarian Polymer Institute, University of Würzburg, Pleicherwall 2, 97070 Würzburg, GermanySearch for more papers by this authorIris Pennings, Iris Pennings orcid.org/0000-0003-0372-7398 Department of Oral and Maxillofacial Surgery and Special Dental Care, University Medical Center Utrecht, Regenerative Medicine Utrecht, Utrecht University, Heidelberglaan 100, 3508 GA Utrecht, The NetherlandsSearch for more papers by this authorMichael Schmitz, Michael Schmitz Department for Functional Materials in Medicine and Dentistry and Bavarian Polymer Institute, University of Würzburg, Pleicherwall 2, 97070 Würzburg, GermanySearch for more papers by this authorAntoine J. W. P. Rosenberg, Antoine J. W. P. Rosenberg Department of Oral and Maxillofacial Surgery and Special Dental Care, University Medical Center Utrecht, Regenerative Medicine Utrecht, Utrecht University, Heidelberglaan 100, 3508 GA Utrecht, The NetherlandsSearch for more papers by this authorJürgen Groll, Corresponding Author Jürgen Groll juergen.groll@fmz.uni-wuerzburg.de Department for Functional Materials in Medicine and Dentistry and Bavarian Polymer Institute, University of Würzburg, Pleicherwall 2, 97070 Würzburg, GermanyE-mail: d.gawlitta@umcutrecht.nl, juergen.groll@fmz.uni-wuerzburg.deSearch for more papers by this authorDebby Gawlitta, Corresponding Author Debby Gawlitta d.gawlitta@umcutrecht.nl Department of Oral and Maxillofacial Surgery and Special Dental Care, University Medical Center Utrecht, Regenerative Medicine Utrecht, Utrecht University, Heidelberglaan 100, 3508 GA Utrecht, The NetherlandsE-mail: d.gawlitta@umcutrecht.nl, juergen.groll@fmz.uni-wuerzburg.deSearch for more papers by this author Tomasz Jungst, Tomasz Jungst Department for Functional Materials in Medicine and Dentistry and Bavarian Polymer Institute, University of Würzburg, Pleicherwall 2, 97070 Würzburg, GermanySearch for more papers by this authorIris Pennings, Iris Pennings orcid.org/0000-0003-0372-7398 Department of Oral and Maxillofacial Surgery and Special Dental Care, University Medical Center Utrecht, Regenerative Medicine Utrecht, Utrecht University, Heidelberglaan 100, 3508 GA Utrecht, The NetherlandsSearch for more papers by this authorMichael Schmitz, Michael Schmitz Department for Functional Materials in Medicine and Dentistry and Bavarian Polymer Institute, University of Würzburg, Pleicherwall 2, 97070 Würzburg, GermanySearch for more papers by this authorAntoine J. W. P. Rosenberg, Antoine J. W. P. Rosenberg Department of Oral and Maxillofacial Surgery and Special Dental Care, University Medical Center Utrecht, Regenerative Medicine Utrecht, Utrecht University, Heidelberglaan 100, 3508 GA Utrecht, The NetherlandsSearch for more papers by this authorJürgen Groll, Corresponding Author Jürgen Groll juergen.groll@fmz.uni-wuerzburg.de Department for Functional Materials in Medicine and Dentistry and Bavarian Polymer Institute, University of Würzburg, Pleicherwall 2, 97070 Würzburg, GermanyE-mail: d.gawlitta@umcutrecht.nl, juergen.groll@fmz.uni-wuerzburg.deSearch for more papers by this authorDebby Gawlitta, Corresponding Author Debby Gawlitta d.gawlitta@umcutrecht.nl Department of Oral and Maxillofacial Surgery and Special Dental Care, University Medical Center Utrecht, Regenerative Medicine Utrecht, Utrecht University, Heidelberglaan 100, 3508 GA Utrecht, The NetherlandsE-mail: d.gawlitta@umcutrecht.nl, juergen.groll@fmz.uni-wuerzburg.deSearch for more papers by this author First published: 16 August 2019 https://doi.org/10.1002/adfm.201905987Citations: 56 The copyright line for this article was changed on 3 January 2020 after original online publication. AboutSectionsPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinkedInRedditWechat Abstract To facilitate true regeneration, a vascular graft should direct the evolution of a neovessel to obtain the function of a native vessel. For this, scaffolds have to permit the formation of an intraluminal endothelial cell monolayer, mimicking the tunica intima. In addition, when attempting to mimic a tunica media-like outer layer, the stacking and orientation of vascular smooth muscle cells (vSMCs) should be recapitulated. An integral scaffold design that facilitates this has so far remained a challenge. A hybrid fabrication approach is introduced by combining solution electrospinning and melt electrowriting. This allows a tissue-structure mimetic, hierarchically bilayered tubular scaffold, comprising an inner layer of randomly oriented dense fiber mesh and an outer layer of microfibers with controlled orientation. The scaffold supports the organization of a continuous luminal endothelial monolayer and oriented layers of vSM-like cells in the media, thus facilitating control over specific and tissue-mimetic cellular differentiation and support of the phenotypic morphology in the respective layers. Neither soluble factors nor a surface bioactivation of the scaffold is needed with this approach, demonstrating that heterotypic scaffold design can direct physiological tissue-like cell organization and differentiation. 1 Introduction Vascular grafts with improved long-term efficacy are a great clinical demand. Especially the replacement of small diameter blood vessels (<6 mm) remains a major challenge, due to problems associated with occlusion of the grafts, intimal hyperplasia, or thrombosis.1 The current clinical gold standard is to use autologous vessels, which is restricted by limited availability. Due to this and other limitations, biofabrication is a promising alternative approach for creating vascular grafts. Natural blood vessels are, inter alia, comprised of a luminal layer (tunica intima) containing a single antithrombogenic monolayer of endothelial cells (ECs) attached to their basement membrane,1 and a secondary contractile medial layer containing near-circumferentially oriented vascular smooth muscle cells (vSMCs) which are embedded in an independent basement membrane matrix (tunica media).2 Efforts for tissue engineering of vascular grafts are mostly focused on recreating these two layers and result in bilayered scaffolds. Approaches for creating vascular grafts can rely on scaffolds, on soft hydrogels, or composites thereof3 and numerous studies have demonstrated promising results in the field of vascular tissue regeneration. Examples for studies that have shown promising in vivo performance include completely biological hydrogels in the form of human cell-based sheets4 and on biopolymer-based attempts in the form of hydrogels.5 Despite the promising performance, those approaches rely on time-consuming and often manual fabrication. Here, scaffold-based approaches are of advantage, which also offer the possibility to improve cell–material interactions and true hierarchical biomimicry of the native morphology in the constructs. Among the applied fabrication methods, electrospinning of polymer solutions (solution electrospinning, SES), a process that predominantly yields nonwoven mats of fibers with diameters in a nanometer to micrometer range, has received considerable attention.1, 6 The morphology of SES scaffolds can be adjusted as the fiber diameter influences the pore size of the nonwoven mats. Besides biochemical cues, the pore size is the main factor that promotes endothelialization of the luminal layer.7 It was suggested that to achieve a mono layer of endothelial cells, pore sizes should not be greater than the size of a cell.7, 8 Especially for small diameter vascular grafts, this monolayer is critical to avoid intimal hyperplasia and thrombosis. Recently, a new fabrication technique called melt electrowriting (MEW) that employs controllable polymer melts instead of solutions has emerged.9 Due to the viscoelastic properties of the polymer melt, the chaotic instabilities, which occur during SES, are suppressed. The stretched polymer jet can, in combination with an automated collector plate, be used for direct writing of structures9, 10 composed of polymer fibers with diameters in the range of several hundreds of nanometers11 to micrometers.12 Compared to traditional additive manufacturing approaches like fused deposition modelling, the reduction in fiber diameter in combination with the control of deposition of the fibers enables generating constructs with a higher fiber density, a higher surface to volume ratio and a better control over pore architecture at a micrometer level. Using cylindrical targets and dedicated software, we have shown that it is possible with MEW to create tubular constructs with precise control over the fiber angle relative to the longitudinal axis of the tube.13 Altering the fiber orientation is beneficial for the creation of biofabricated vascular grafts, as the orientation of the vSMCs and the extracellular matrix (ECM) in the tunica media is important for the contractile function of blood vessels.1, 3, 14 In the tunica media, collagen fibers predominately run in the circumferential direction but can also be oriented helically which is decisive for the circumferential mechanical properties of the vessel.15 Recapitulating the regulation of cellular orientation in the tunica media of biofabricated vascular grafts has already been the point of focus in several studies but was performed with limited control over fiber density in the selected fabrication techniques.14, 16 This often resulted in medial layers with high fiber density, lacking in space for adequate cellular interactions and resulting in slow vSMC colonization.16, 17 In the native situation, cells residing in the tunica media and tunica intima have extensive crosscommunication. Therefore, a major advantage of tissue mimetic bilayered constructs is the introduction of cocultures with associated cellular crosstalk. The adequate function of a blood vessel is based on an EC monolayer that can stimulate the vSMCs by specific signaling pathways, such as the Alk1/Alk5/transforming growth factor (TGF) β pathway for steering the plastic phenotype of the vSMCs, or the secretion of nitric oxide (NO) via endothelial nitric oxide synthase for vasoconstriction and dilatation of the blood vessel.18 Despite its relevance, the interplay between the cell types in an engineered blood vessel are, rarely addressed. So far, most studies only have reported bilayered electrospun scaffolds supporting the culture of endothelial cells and vSM-like cells separately.19 Only few have shown simultaneous culturing of these cell types on bilayered tubular scaffolds in vitro7, 16, 20 but fall short in extensive phenotypical characterization of the cell layers and none have taken into account clinically relevant cell sources for the perspective of an eventual clinical application. To integrate that in the experiments from the beginning, the use of autologous cells, in case of vascular grafts, vSMCs, would be the best option. Still, in vitro this has been reported as a challenge due to their limited availability and proliferative capacity, and their switch to the synthetic phenotype, which challenges clinical translation.21 Therefore, multipotent mesenchymal stromal cells (MSCs), which have the ability to differentiate into vSMCs are an appropriate alternative for vSMCs.22 Further, as a source for endothelial cells, endothelial progenitor cells (EPCs) can be used, which can also be harvested from an easily obtainable cell source (e.g., human umbilical cord blood and peripheral blood). Among the EPCs, a subgroup named “endothelial colony forming cells” (ECFCs) show high expansion potential and inherent vasculogenic and angiogenic capacity.23 Both, MSCs and ECFCs, can originate from autologous sources and thus are suitable for clinical translation of biofabricated vascular grafts. Following an analysis of the architecture of a human artery, the underlying hypothesis of this study was that, if an advanced scaffold can be fabricated with tissue-mimetic layered hierarchy combined with a heterotypic topology, this scaffold can direct the formation of vessel-like cell organization, orientation, and differentiation upon seeding of ECFCs and MSCs onto the respective layers. Heterotypic topology means a basal-membrane like morphology at the inner lumen for endothelial cells and an adhesion and migration guidance for vSMCs or their progenitors in the outer layer in a tissue-analogous orientation toward the circumferential axis of the constructs. We further hypothesized that such scaffolds may result in tissue-analogous cell organization and phenotype evolution without the need for additional soluble factors or a surface bioactivation of the scaffolds. 2 Results and Discussion Tubular scaffolds as a basis for the biofabricated vascular grafts were thus fabricated by combining SES and MEW of poly(ε-caprolactone) (PCL) through consecutive fiber deposition (first SES, then MEW) onto a cylindrical target with an outer diameter of 3 mm. These scaffolds were then seeded with ECFCs and MSCs. The SES nonwoven inner layer enabled the ECFCs to organize into a continuous endothelium. Furthermore, the ECFCs expressed signals associated with crosscommunication toward vSMCs. The MEW layer controlled the orientation of the MSCs, was fully populated with cells, facilitated close cell–cell contacts, and accelerated the differentiation of MSCs into vSM-like cells. Along, it was shown that the scaffold could support both cell types when cocultured, providing a platform in which cellular crosscommunication can be studied. In order to know how to design biomimetic bilayered scaffolds with heterotypic topology, first the architecture and phenotypic aspects of a human muscular artery were established by analyzing the presence of a selection of contractile vSMC markers and associated ECM components (Figure S1, Supporting Information). Calponin, α-smooth muscle actin (α-SMA), smooth muscle myosin heavy chain (SMMHC), laminin α5, and collagen type IV were identified and located in the natural vessel tissue (Figure S1B–F, Supporting Information). Also, overall tissue hierarchy was established (H&E staining, Figure S1A, Supporting Information). The tissue layers could be distinguished clearly, with the relatively thin tunica intima (10–20 µm) on the luminal side of the vessel with its collagen type IV-positive basement membrane and a fine network of connective tissue with elastic fibers. The native tissue stained positive throughout the whole thickness of the tunica media (≈400 µm) for the contractile vSMC and ECM markers of interest. Additionally, the vSMCs were organized in an elongated, concentric, and multilayered manner. Based on the analysis of the human blood vessel and on the literature search presented in the introduction, design criteria for the biofabricated vascular grafts-scaffolds were formulated. The newly designed bilayered small diameter vascular grafts (3 mm Ø) should provide both the ECFCs and the MSCs with the appropriate environment mimicking and instructing the cells to organize and deposit matrix in the native architecture. To achieve this, we defined the following key consideration points: The scaffold should provide a substrate on which the ECFCs form a continuous monolayer. The endothelial monolayer should express mature EC markers, components of the basement membrane and ECM, and signals associated with crosscommunication toward vSMCs. The scaffold should provide a porous outer layer that vSMCs can migrate in and fill to achieve a multilayered elongated cell organization. The vSMCs should align in a near-circumferential orientation, specifically controlled by the MEW fiber orientation. The scaffold should provide an environment for the MSCs to differentiate into the vSMCs contractile phenotype. As these design criteria could not be met by any single fabrication technique, we developed a new fabrication procedure by combining two techniques, SES and MEW (Figure 1A,B). This enabled the fabrication of scaffolds with so far unachievable biomimetic and heterotypic structural features as shown in Figure 1C. The scaffolds were composed of an inner cylindrical nonwoven (80% ± 5% porosity, inner diameter 3 mm) of solution electrospun PCL fibers with a diameter of 1.4 ± 0.2 µm and a random orientation. The same material was used to create a layer of MEW fibers (15.2 ± 4.8 µm) with a winding angle (Figures S2 and S3, Supporting Information) between 30° and 70° and controllable large open pores. As the material deposited onto the SES nonwoven by MEW was still at a temperature above its melt point, it could fuse with the fibers of the inner nonwoven as shown in Figure 1C. This is crucial to avoid delamination of the layers during cell culture and when removing the samples from the cylindrical collector they are deposited onto. As revealed via balloon inflation burst experiments, the burst pressures of the (cell-free) constructs was 2400 ± 75 mmHg. This value exceeded the required pressure for transition to clinical translation based on measurements of the saphenous vein of 1700 mmHg24 (Figure S4, Supporting Information). Figure 1Open in figure viewerPowerPoint Preparation of bilayered tubular scaffolds. A) Solution electrospinning is used to generate a tubular nonwoven inner layer. B) The rod with the nonwoven is transferred to a melt electrowriting device and oriented fibers are deposited on top of the nonwoven luminal layer. C) The final construct is removed from the cylindrical collector. Bilayered scaffolds are made from one material and the fibers with different dimensions fuse, which prevents delamination. The heterotypic electrospinning approach supported endothelialization on the inside of the dense luminal nonwoven layer as illustrated by the presence of a confluent endothelial monolayer in scaffolds with ECFC monocultures (Figure S6, Supporting Information). Importantly, this advanced scaffold design also supported so far unreached endothelialization in coculture conditions of ECFCs with MSCs (Figure 2A) and showed no infiltration of the ECFCs into the SES layer (Figure 2A and Figure S8C,D, Supporting Information). The formation of a continuous monolayer meets the achievement of key point 1. Scanning electron microscopy (SEM) demonstrated the establishment of connections between neighboring ECs (black arrows) with their extrusions (white arrows), indicative of a restrictive endothelial barrier and low permeability (Figure 2B,C). The low permeability and functional integrity of the monolayer was mostly supported by the specific redistribution of the mature endothelial cell markers CD31 and vascular endothelial cadherin (VE-cadherin) toward the cell periphery (Figure 2D,E), as validated previously.25 The tight connections between the ECs via these interactions, are a prerequisite for an endothelium to form a semi-permeable barrier and are essential for their interaction in signaling pathways regarding endothelial plasticity, vascular integrity as well as sensing mechanical tensions, such as shear stress and to inhibit the activation of platelets and leukocytes.25, 26 Reproducing the functional integrity of the endothelium with barrier function is essential to resist thrombosis following introduction in vivo and is thus required to be present in biofabricated vascular grafts to pass functionality and for long term patency.24 The capability of the generated monocultured endothelium to prevent platelet adhesion was shown, while platelets did aggregate on the exposed scaffold surface (Figure S6D–F, Supporting Information). With this, a lining with anticoagulative properties was produced, resembling the properties of a native endothelium. Likewise, the monolayer showed positive endothelial marker staining for the platelet adhesion glycoprotein von Willebrand factor (vwF) (Figure 2D) and was supported by a collagen type IV-positive matrix (Figure 2E), as also found in the native basement membrane2, 27 supporting the biomimetic properties of the scaffold. Expression of these EC-related markers and ECM/basement membrane components were also confirmed on a gene expression level, both in mono and cocultures (Figures S6 and S7, Supporting Information). Figure 2Open in figure viewerPowerPoint Cell culture on bilayered heterotypic vascular grafts. A) Layered organization and distinctive phenotypes of simultaneously cultured ECFCs (CD31+) and vSM-like cells (αSMA+) after 17 d (cross-sectional view). B) Endothelialization of the SES layer on the luminal side with C) tight cell–cell connections (black arrows) and cell extrusions (white arrows), D) positive staining for the endothelial cell markers von Willebrand factor and VE-cadherin, the latter of which was located at the cell periphery, and E) CD31; also, a collagen type IV-positive basement membrane-like matrix was detected. F) The endothelial cells produced nitric oxide (NO) for signaling to the vSM-like cells (MSC donors n = 3, M1–M3). G) vSM-like cells covered the medial layer in an aligned fashion, H) with elongated αSMA+ cells following the MEW fibers with I) a collagen type IV deposition in the direction of the cell alignment. J) vSM-like cells filled the whole thickness of the MEW layer and showed a circumferential orientation in monoculture after 7 d. Scale bars represent 100 µm unless stated otherwise. To meet the second key consideration point, signaling pathways for vSMC-EC communication were examined via assessment of NO secretion into the culture medium and via gene expression of the Alk1/Alk5/TGFβ pathway for vSMC differentiation in cocultured biofabricated vascular grafts. For MSCs of all three donors, combined with ECFCs, the product NO was detected in the medium (Figure 2F). Also, the presence of mRNA of the Alk1/Alk5/TGFβ genes was confirmed after coculturing (Figure S7, Supporting Information). This indicated the presence of the cellular crosscommunication for vasoactivity, which is required for the development of a functional endothelium in biofabricated vascular grafts.24 Especially the production of NO, which is considered one of the predominant vasodilators and is involved in inhibition of platelet aggregation is necessary for a functional endothelium.28 The measured synthesis of NO in our culture system gives indications that the formed endothelium holds functionality and possesses the ability to signal to vSM-like cells when cultured on our biomimetic bilayered scaffolds. Altogether, the expression of the markers for mature ECs, for components of the basement membrane and ECM markers, together with the indicated presence of the cellular crosscommunication shows that also the second key consideration point was met. Further, we demonstrated that the large pores and MEW fiber alignment could induce a fast infiltration by the vSM-like cells to meet the third key consideration point in both MSC/ECFC cocultures (Figure 2J) and MSC monocultures (Figure S8, Supporting Information). The seeded MSCs covered the whole outer surface of the scaffold (Figure 2G) and appeared in an aligned fashion (Figure 2H). Moreover, the initially seeded MSCs showed the desired arrangement, indicated by the elongated cytoskeletons and αSMA-positive structures. Also collagen type IV was synthesized (Figure 2I), which was enclosing the individual vSMCs as a basement membrane-like matrix (Figure S9, Supporting Information), mimicking the native situation.2, 27, 29 To prove that the high control over MEW fiber placement could also guide the orientation of the MSCs, they were monoseeded (n = 4 MSC donors) on scaffolds i) without MEW layer, ii) an MEW layer with fiber winding angle of 30° (programmed angle 30°, measured angle 35.3° ± 0.8°), and iii) samples with MEW layer at an angle of 70° (programmed angle 70°, measured angle 71.6° ± 0.6°). The cellular orientation in the whole MEW layer was analyzed based on immunofluorescence of F-actin stained cells by determining the pixel orientation of the F-actin fibers throughout the thickness of the MEW layer (Figure 3 and Figure S10 and detailed description in Supporting information). Cells seeded on the nonwoven mesh (i) were mainly oriented in the longitudinal direction of the tubular scaffold (2.3° ± 1.4°). With increasing winding angles of the scaffold fibers, the cellular orientation changed into a near-circumferential direction (Figure 3A) as present in the native tunica media. The cells seeded on the constructs with a 70° winding angle of the MEW fibers were oriented in an angle that was larger (77.7° ± 3.4°) than the angle of the fibers. This winding angle was used in constructs for further cell culture experiments, as this met the fourth key consideration point of (near) circumferentially arranged cells and mimicked the helically orientated collagen fibrils as in the native arteries14, 15 and also the orientation of the cell nuclei of vSMC in a human aortic tunica media.30 Taken together, we thereby proof and verify our hypothesis that the orientation of vSMCs on tubular scaffolds can be guided in a tissue-mimetic manner by controlled orientation of fibers in the micrometer range. The open porous structure of the outer layer facilitated a fast cellular ingrowth and resulted in several layers of orientated aligned cells with close cellular interactions. We could achieve different cell orientations and found one that is potentially exploitable for facilitating vasodilation and constriction for next generation bioengineered vascular grafts. Figure 3Open in figure viewerPowerPoint Influence of the orientation of melt electrowritten fibers on the orientation of MSCs. A) Representative scanning electron microscopy images of the scaffolds before seeding (top row) and a projection of F-actin stained cells from a 3D stack of scaffolds after seeding and culture for 7 d (bottom row). B) The 3D projection was used to analyze the mean cell orientation throughout the thickness of the MEW layer and showed the average orientation of the melt electrowritten fibers as well. The last key consideration point for aiming at engineering of the tunica media is to realize the SMC phenotype switch to the contractile phenotype at the proper stage during their maturation process on the scaffold. Generally, differentiation of MSCs into contractile vSMCs is accomplished by the addition of biochemical factors associated with differentiation, such as TGF-β1 or platelet-derived growth factor subunit β.22, 29, 31 Interestingly, we observed that by expanding bone marrow-derived MSCs in a culture plate, supplemented with the proliferation-associated basic fibroblast growth factor (bFGF), also an induction of differentiation was observed after reaching confluency. This differentiation was accompanied by protein upregulation of the contractile vSMC marker proteins αSMA and calponin. In addition, the cells showed capacity for contraction of a collagen lattice (Figures S11A–C and S14, Supporting Information) as well as elevated gene expression levels of additional contractile vSMC markers (Figure S11D, Supporting Information). The induction of differentiation in postconfluent vSMC cultures has been reported previously32 and several groups showed very promising results and could, for example, use the fiber orientation to control the 2D orientation of a monolayer of vSMCs.33 It could be shown that the orientation, alignment and confluency can be utilized to control the phenotype of vSMCs.34 The effect of confluency was also reported to be existent in MSC cultures by Alimperti et al., where the induction of differentiation of MSCs into contractile vSM-like cells was described, without the use of above-mentioned differentiation growth factors.35 They hypothesized that the observed differentiation is a cell–cell junction-mediated process through the adherens junction cadherin-11, normally found on MSCs, with an associated autocrine action of TGF-β1,35 or possibly by the secretion of a basement membrane-like matrix associated with the contractile vSMC phenotype.14 In addition, the MSCs used here appeared to be positive for CD146, a marker associated with perivascular cells and were therefore more prone to vSMC differentiation (Figure S12, Supporting Information).36 To more closely investigate the effect of increased cell–cell interactions on the differentiation of MSCs in our scaffold with open pores, we compared the differentiation on constructs without or with MEW layer, as the latter enhances stacking of MSCs and thereby increases cell–cell contacts in a 3D setting. To do so, we first showed that the SES layer was a sufficient substrate for the MSCs to proliferate and differentiate on, to reach the same confluency as in a culture plate, with similar expression of contractile vSMC markers on both gene and protein levels (ACTA2 – αSMA; CNN1 – Calponin; Transgelin – SM22α and the extracellular matrix markers LAMA4 – Laminin subunit α4 and ELN – Elastin) (Figure 4A, no significant differences) as found in culture plates, after both 7 and 14 d. In this setup, the MSCs were monocultured on the single-layered SES scaffold without the MEW fibers and compared to the differentiation status of MSCs cultured in plates (control group) with correspo