**2. Results**

### *2.1. Generation of Prevascularized Collagen Sponges in an Extrinsic Angiogenic Growth Factor Free Manner*

We investigated the potential of cryopreserved SVF to create a prevascular network, in the absence of extrinsic angiogenic growth factors, after seeding in a blue shark collagen sponge (Figure 1A). Highly interconnected microporous sponges were produced by resorting to a cryogelation method, as previously described [27] (Figure 1B). SVF was isolated from human adipose subcutaneous tissue and cryopreserved in 10% DMSO in FBS for at least 7 days. SVF was then thawed and seeded on collagen sponges and cultured for 7 days without angiogenic growth factor supplementation, as previously described for fresh SVF [13] (Figure 1A). After that period, the expression pattern of endothelial marker CD31 and pericytes CD146 revealed the presence of endothelial cells together with pericytes organized in a complex and interconnected capillary-like network, confirming that SVF maintained its capacity to create a prevascular network without angiogenic growth factor supplementation even after cryopreservation (Figure 1C).

**Figure 1.** Generation of pre-vascularized collagen sponges. (**A**) In vitro experimental design. (**B**) Representative macroscopic images of shark skin collagen sponge's macroporosity after freeze drying. Scale bar: 1000 μm. (**C**) Representative immunocytochemistry images of the network-like organization of SVF-derived CD31-expressing cells (white) interconnected with pericytes CD146-expressing cells (green), within collagen sponges after 7 days of culture in the absence of extrinsic angiogenic growth factors. Cell nuclei were counterstained with DAPI (blue). Scale bar: 75 μm (**top**) and 25 μm (**bottom**). (**D**) Angiogenic secretome profile of SVF cells seeded in collagen sponges at different culture periods. Conditioned media were collected at days 5 and 7 for dot blot analysis of angiogenesis-related factors. Protein expression profiles were measured using mean intensity and normalized to the reference spots. Data are presented as mean ± std dev and were analyzed using a paired *t*-test (\* *p* < 0.0332, \*\* *p* < 0.0021, \*\*\* *p* < 0.0002, and \*\*\*\* *p* <0.0001).

### *2.2. Profiling of Angiogenesis-Related Proteins in Prevascularized Collagen Sponges Secretome*

Given the confirmation of prevascular network formation, we sought to understand if and how the secretome profile changed throughout the culture time. To achieve this, a multiplex analysis of secretome targeting angiogenesis-related proteins was performed on secretome samples collected after 5 and 7 days of culture (Figure 1D). The selection of these timepoints was based on previous studies from our lab [13]. The secretion of important angiogenic modulators such as VEGF and MMP-9 remained unchanged from one time point to the other. However, an increase in the secretion of several factors specifically involved in ECM remodeling (uPA, PAI-1, and TIMP-1) was verified from day 5 to 7, while macrophage-related factors decreased over culture time (IL-8, MCP-1). Interestingly, no expression of angiogenic proteins other than the ones detected for the first timepoint was found for the later timepoint.

### *2.3. In Ovo Evaluation of Angiogenic Potential*

Upon the in vitro confirmation of prevascular network formations, the in ovo angiogenic potential of the prevascularized collagen sponge was assessed by using a chick CAM assay (Figure 2A). After prevascularization, collagen sponges were implanted into the CAM of chicken eggs. A control group consisting of sponges without seeded cells was also implanted. For the evaluation and quantification of angiogenesis, the area around the implantation site was fixed, photographed, and finally excised and paraffin embedded. Results demonstrate host vessel recruitment in both the prevascularized and control sponges (Figure 2B). However, vessel quantification demonstrated a significantly higher number of recruited vessels for prevascularized sponges when comparing with sponges without SVF cells (Figure 2C), strongly suggesting a beneficial role of prevascularization with SVF in post-implantation vascularization. Concurrently, histological analysis after H&E staining clearly presented a higher CAM tissue ingrowth towards the bulk of prevascularized sponges in contrast with the control group with empty sponges where host tissue was very much limited to the outside of the sponge's structure (Figure 2D). Importantly, no significant immune reaction was visible for both groups. Together with the higher number of recruited vessels, these results strongly sugges<sup>t</sup> a positive effect of prevascularization with SVF upon the integration of implanted collagen sponges with the CAM tissue. In situ hybridization results show that human origin cells from the SVF persist in the CAM tissue after 4 days of implantation (Figure 3A) and, importantly, incorporate new vessels, suggesting a net contribution to the higher vessel density determined above. The contribution of implanted SVF cells to neo-vessel formation was further confirmed after immunohistochemistry for human CD31, which clearly demonstrates CD31-positive cells lining blood vessel walls in the interface between CAM tissues and collagen sponges (Figure 3B).

**Figure 2.** In ovo angiogenic potential upon implantation in Chick Chorioallantoic Membrane (CAM). (**A**) In vivo experimental design. (**B**) Representative micrographs of recruited vessels after 4 days of implantation of collagen sponges with and without SVF. Scale bar: 2000 μm. (**C**) Quantification of recruited vessels after 4 days of implantation of collagen sponge with and without SVF. Data are presented as violin plot illustrating the kernel density distribution frequency of recruited vessels and analyzed using an unpaired *t*-test (\*\* *p* < 0.0021). (**D**) Representative micrographs of hematoxylin and eosin staining in collagen sponges with and without SVF. Scale bar: 500 μm (**left**) and 50 μm (**right**).

**Figure 3.** In ovo angiogenic potential upon implantation in Chick Chorioallantoic Membrane (CAM). (**A**) Representative images of the in situ hybridization performed with a DNA probe that stains human cellular nuclei (blue, arrows) in contrast with chicken nuclei (pink). The implanted cells infiltrated the host tissue and vasculature as highlighted by black arrows. Chicken erythrocytes identified by orange arrows. Scale bars: 200 μm (**left**) and 50 μm (**right**). (**B**) Representative immunohistochemistry images of the collagen sponge after 4 days of implantation showing human CD31-positive cells (brown). Human CD31 expression patterns demonstrated the integration of the pre-vascular network in the CAM, as highlighted by black arrows. Chicken erythrocytes identified by orange arrows. Scale bars: 500 μm (**left**) and 20 μm (**right**) and 50 μm (**inset right**).
