*4.5. Immunocytochemistry*

After in vitro culture, constructs were fixed in a 10% (*v/v*) buffered formalin solution. A 3% (*w/v*) BSA was used to block non-specific binding, and cells were incubated overnight at 4 ◦C with primary antibody mouse anti-human CD31 (M0823) (1:30) (Dako, Cambridge, UK) and rabbit anti-human CD146 (ABCAAB75769) (1:50) (VWR, Lutterworth, UK). After repeated washes in PBS, secondary antibody Alexa Fluor 594 donkey anti-mouse (1:500) and AlexaFluor 488 donkey anti-rabbit (Molecular probes, Eugene, OR, USA) were incubated with cells for 4 h at room temperature. After washing with PBS, cell nuclei were counterstained with DAPI. The presence of capillary-like structures was verified in a confocal laser scanning microscope (SP8 Leica Microsystems CMS GmbH, Wetzlar, Germany).

### *4.6. Secretome Analysis*

In order to analyze the expression profile of angiogenesis-related proteins during the prevascularization process, conditioned media were collected and centrifuged to remove cell debris after 5 and 7 days of culture. A control with basal media was also set up. Secreted proteins were analyzed using a Proteome Profiler human angiogenesis array (R&D Systems; Minneapolis, MN, USA) in accordance with manufacturer guidelines. Briefly, conditioned media were incubated with an assay-specific detection antibody cocktail for 1 h at room temperature. After a membrane blocking step, samples containing antibody cocktail were added to the respective membrane and incubated overnight at 4 ◦C under shaking. Membranes were then washed with 1× wash buffer, incubated with streptavidin-HRP for 30 min, and washed again. Membranes were incubated with Chemi Reagent Mix for 1 min, and spots detected by using chemiluminescence in an Odyssey Fc Imaging System

(LI-COR, Lincoln, NE, USA) and densitometry were quantified using Image studio 5.2 software (LI-COR, Lincoln, NE, USA)

### *4.7. In ovo Implantation*

A CAM assay was performed as previously described [57,63]. White fertilized chicken eggs were incubated at 37 ◦C in a temperature incubator (Termaks KB8000, Bergen, Norway) for 3 days. After this, a window was opened into the shell to evaluate embryo viability. Prevascularized sponges (*n* = 12) were implanted on the CAM at day 10 of embryonic development, and the eggs returned to the incubator at 37 ◦C. Control groups with empty materials (*n* = 12) and without the material (*n* = 12) were also set up. After 4 days of implantation, embryos were sacrificed with4%(*v/v*) paraformaldehyde and subsequent incubation at −80 ◦C for 10 min. Then, the implanted materials with adjacent portions of CAM were cut and fixed with 4% (*v/v*) paraformaldehyde. Ex ovo images were captured using a Stemi 2000-C stereo microscope (Zeiss, Oberkochen, Germany).

### *4.8. New Vessel Quantification*

The obtained ex ovo images were processed using ImageJ 1.52a (National Institutes of Health, Bethesda, MD, USA). Images were cropped to a defined area of 500 × 500 pixels, considering the implanted construct in the center of the image. The number of new/recruited vessels growing radially towards the constructed area was quantified by manual counting, in a blind fashion, by three independent operators.

### *4.9. Histological Analysis*

After formalin fixation, CAM explants were processed in a MICRON STP120-2 spin tissue processor (MICRON, Walldorf, Germany), embedded in paraffin (Thermo Scientific, Waltham, MA, USA), and serially sectioned into 4 μm-thick sections.

### *4.10. Hematoxylin and Eosin Staining*

Hematoxylin and eosin (H&E) staining was performed in CAM sections. Briefly, sections were deparaffinized with xylene, rehydrated in graded ethanol series and stained with hematoxylin and eosin in an MICROM HMS740 automatic stainer (MICROM, Walldorf, Germany). Afterwards, sections were dehydrated and mounted with resinous mounting medium Entellan® (Merck, Darmstadt, Germany). Histological sections were analyzed under a Leica DM750 microscope (Leica, Wetzlar, Germany).
