Gelfoam scaffolds were purchased from Pfizer and were cut into pieces 1 cm long, 0.5 cm wide, and 1.5 mm thick pieces. PLLA/PLGA scaffolds were prepared as followed: 0.4 g NaCl particles were covered with 0.24 ml PLLA/PLGA solution, which was dissolved in chloroform, and evaporated overnight. Salt was then leached out by four washes, leaving behind pores within the scaffold. Scaffolds were then cut into 6 mm diameter circles with the thickness of 0.8 mm. Fibrin gel was obtained by mixing thrombin (20 U/ml, Sigma-Aldrich) with fibrinogen (15 mg/ml, Sigma-Aldrich).

Human adipose microvascular endothelial cells (HAMECs; ScienceCell), lentivirally transduced with ZsGreen fluorescent protein, were grown in endothelial cell medium (ScienceCell), supplemented with 5% FBS (ScienceCell), and endothelial cell growth supplement (ScienceCell), and used for five to nine passages. Neonatal normal human dermal fibroblasts (HNDFs) (Lonza Walkersville Inc.) were grown in Dulbecco’s modified Eagle medium (DMEM) (Gibco), supplemented with 10% FBS (HyClone), 1% non-essential amino acids (NEAAs), 0.2% β-mercaptoethanol (Sigma-Aldrich), and 1% penicillin–streptomycin solution (PEN STREP) (Biological Industries). Dental pulp stem cells (DPSCs) (Lonza) were cultured in low glucose DMEM (Gibco), supplemented with 10% FBS (HyClone), 1% NEAAs, 1% GlutaMAX (Gibco), and 1% penicillin–streptomycin-nystatin solution (Biological Industries). Three dimensional, vascularized constructs were obtained by coseeding endothelial cells (3 × 10⁵ cells) and DPSCs (9 × 10⁵) or fibroblasts (0.6 × 10⁵ cells) in 20 µl medium on the gelfoam or in 7 µl fibrin on the PLLA/PLGA scaffolds followed by incubation of 30 min, before addition of medium. For the mitomycin experiments, 5 ml mitomycin (Sigma-Aldrich) was applied to the fibroblast cells 2 h before seeding, and washed twice with PBS.

The Lenti-X HTX Packaging System (Clontech, a fourth generation) was used to generate recombinant, replication-incompetent VSV-G pseudotyped lentiviruses, according to the manufacturer’s instructions. Transfection of the expression vector into the Lenti-X 293 T packaging cells (Clontech) was performed in a Lenti-X HTX Packaging mix. The supernatants of transfected packaging cells were collected 72 h later and filtered through a 45-μm filter before being added to the HAMECs, with 6 mg/ml polybrene (Sigma-Aldrich). The transduction medium was replaced by culture medium 24 h thereafter, and cells were then cultured for 72 h to allow gene product accumulation in the cells. Cells were then selected using 1 mg/ml puromycin (Takara Bio Company).

Whole constructs were fixated in paraformaldehyde (4%), for 20 min, and then permeabilized with 0.3% Triton X-100 (Bio Lab Ltd.), for 10 min. Constructs were then washed with PBS and immersed overnight in BSA solution (5%; Millipore). Samples were then incubated with goat antihuman VE-cadherin (1:100; Santa Cruz), mouse antihuman Yes-associated protein (YAP) (1:100; Santa Cruz), mouse antihuman NG2 (1:100; Santa Cruz), rabbit antihuman vWF (1:150; Abcam), rabbit antihuman β-catenin (1:100; Sigma-Aldrich), or mouse antihuman Ki67 (1:20, DAKO) antibodies, overnight, at 4°C. Constructs were then treated with Cy3-labeled (1:100; Jackson Immunoresearch Laboratory), Cy5-labeled (1:100; Jackson Immunoresearch Laboratory), or Alexa-488 (1:400; ThermoFisher Scientific) antibodies, mixed with DAPI (1:1000; Sigma-Aldrich), for 2 h, at room temperature. For the phalloidin staining, constructs were treated with FITC phalloidin (1:100; Sigma-Aldrich) and DAPI for 20 min. For the mitomycin experiment, mitomycin-treated cells and control cells without mitomycin were fixated in paraformaldehyde (4%), for 20 min on day 10 of culture, and then incubated in a 30% (wt/vol) sucrose solution overnight, embedded in optimal cutting temperature compound (Tissue-Tek) and frozen for subsequent cryosectioning (5–20 µm). Standard protocols were used for H&E and Masson’s trichrome staining of the sections.

Whole vascularized constructs were imaged using a confocal microscope (LSM700, Zeiss), equipped with 20× and 63× oil immersion lenses. Three-dimensional images were projected into 2D images, using maximum intensity projection, and the stacks were then separated into three main regions: the surface (0–10 µm), middle (10–20 µm), and deeper (20 μm-end) areas of the scaffold. Confocal images were then analyzed using a self-written algorithm in MATLAB, for ki67 quantification: images were transformed into binary images and pixel density was calculated. Vessel quality was determined using a self-written algorithm in MATLAB: images were transformed into binary images and the eccentricity parameter, an indicator of the deviation of an element from circularity, was calculated for each separate element in the image. Elongated vessels received a higher eccentricity score, whereas, cells clusters and disrupted vessels, which are more circular, receive lower eccentricity scores. Imaris software (BITPLANE) was used to detect YAP and β-catenin localization through the 3D image.

Presented data include the mean ± SD. Two-way analysis of variance was performed to examine the influence of two independent categorical variables, followed by Bonferroni’s multiple comparison tests. Results were considered significant for p < 0.05. Statistical analysis was performed using a computerized statistical program (GraphPad Software). Experiments were repeated three times.

To follow vessel formation dynamics within 3D structures, a coculture of HAMECs and HNDFs was seeded into a gelfoam scaffold. After 7 days of culturing, cells were stained for VE-cadherin and were imaged using a confocal microscope. A sheet-like structure of an endothelial cell monolayer was observed on the surface of the scaffold, whereas in the scaffolds depth, cells began to form microvessels (Figure 1A). On day 14 postseeding, the endothelial cell monolayer structure was no longer observed and only microvessels were observed in the scaffolds depth; 3D confocal imaging showed that these vessels were lumenalized. In addition, at the same time point, a dense fibroblast layer was apparent on the scaffold surface and around the vessels (Figure 1B; Figures S1 and S2 in Supplementary Material). To confirm that this phenomenon was not limited to a certain scaffold or cell type, dTomato HAMECs were seeded with DPSCs into a PLLA/PLGA scaffold, which was then stained with DAPI and phalloidin. DPSCs were mainly observed on the scaffold surface and endothelial micro-vessels were seen within the scaffold depth (Figure 2).

We hypothesize that EC migration toward the scaffolds interior might be due to hypoxia. It is recognized that secretion of VEGF increases in hypoxic conditions (Shweiki et al., 1992) which consequently triggers EC migration. Transversed cryosections were stained with PHD2 (prolyl hydroxylase domain 2), which flags hypoxia inducing factor alpha subunits for ubiquitin-proteasome degradation under normoxic conditions (Carmeliet and Jain, 2011). PHD2 expression was higher at the scaffold surface as compared to the scaffolds depth (Figures 3A,B). Hence, the increasing hypoxic conditions at the scaffold depths may have triggered the ECs to form vessels there.

To examine fibroblast characteristics in the 3D constructs, a HAMEC and HNDF coculture was seeded into the gelfoam scaffold and grown for 14 days, fixated and stained for CD31, to mark the ECs, and for PDGFRβ and αSMA, to mark the pericytes; expression of the two proteins has been shown to increase during the recruitment of pericytes to stabilize the forming vessels (Welti et al., 2013). Confocal imaging revealed that PDGFRβ-expressing cells were located both on the scaffold surface and in the scaffold depths (Figure 4), whereas αSMA-expressing cells were mainly located around the vessel network in the scaffold depth. To assess the degree of cell proliferation at the various scaffold locations, samples were stained for Ki67. Ki67-expressing fibroblasts were significantly more frequent on the scaffold surface compared to the scaffold depth (Figure 5), suggesting that cell proliferation decreased within the deeper layers of the scaffold, closer to the forming vessels.

Since canonical Wnt signaling has been implicated in cell proliferation (Clevers, 2006), and is involved in supporting cell recruitment to forming vessel (Reis and Liebner, 2013), we next examined the expression patterns of proteins which play a central role in the Wnt canonical pathway within the cells composing the forming vessels. Cells grown on the scaffolds for 14 days were stained for YAP, β-catenin, and VE-cadherin. Localization of YAP in the fibroblast nucleus was only observed in the layers closer to the scaffold surface and its levels decreased with closer proximity to the scaffold core, where the endothelial cells were localized (Figure 6A). Fibroblasts β-catenin showed cytoplasmic localization, which also decreased in the scaffold depths (Figure 6B). For the cells grown for 4 days, cytoplasmic β-catenin and nuclear YAP were observed within the ECs sheets located on the surface of the scaffold, which formed at the earlier stages of culturing (Figure 6C).

The heightened expression of nuclear YAP and cytoplasmic β-catenin and more extensive cell proliferation on the scaffold surface, led us to investigate the role of Wnt signaling and specifically, of fibroblast proliferation in vasculogenesis. To this end, fibroblasts were treated with mitomycin prior to seeding and cell-embedded constructs were then grown for 7 or 10 days before being transversely cryosectioned. Confocal images of the various constructs revealed that the ECs seeded with mitomycin-treated fibroblasts, failed to form vessel networks (Figures 7A,B). In addition, the ECs in these constructs did not penetrate into the scaffold depths (Figure 7C), and remained localized in the same plane as the fibroblasts. Hematoxylin and eosin and trichrome stainings revealed a dense, thick layer of cells and collagen on the surface of scaffolds containing untreated fibroblasts, whereas the constructs with mitomycin-treated cells showed a thin layer, with less collagen deposition (Figures 8A,B, dashed box).

Following the observed migration of ECs from the scaffold surface to its depth, when coseeded with fibroblasts, we set out to determine whether this migration occurs in the presence of a preexisting fibroblast layer. To this end, RFP-expressing HNDFs were seeded onto a gelfoam scaffold, and cultured for 12 days before Zs-green-expressing HAMECs were added to the culture. Despite the dense fibroblast layer, the endothelial cells migrated toward the inner part of the scaffold, where they formed a vessel network within 5 days (Figure 9). This migration might be due to the previously described hypoxic conditions in the scaffold depth.