**1. Introduction**

Vascularization is a critical aspect of every tissue engineering (TE) approach for thick perfused tissues. A comprehensive network of capillaries is necessary to ensure, upon anastomosis with the host's circulation, the proper delivery of nutrients and oxygen to all cells of the engineered tissue, avoiding necrotic events and promoting integration with the surrounding tissue. TE approaches for thick tissues not specifically addressing vascularization rely, in an initial phase after implantation, on passive diffusion, which is limited to ~150 μm [1]. Although the hypoxic environment created by the lack of oxygen potentiates the host vessel's invasion towards the implanted construct, the rate of spontaneous vascular

**Citation:** Freitas-Ribeiro, S.; Diogo, G.S.; Oliveira, C.; Martins, A.; Silva, T.H.; Jarnalo, M.; Horta, R.; Reis, R.L.; Pirraco, R.P. Growth Factor-Free Vascularization of Marine-Origin Collagen Sponges Using Cryopreserved Stromal Vascular Fractions from Human Adipose Tissue. *Mar. Drugs* **2022**, *20*, 623. https://doi.org/10.3390/ md20100623

Academic Editors: Sik Yoon and Azizur Rahman

Received: 28 September 2022 Accepted: 30 September 2022 Published: 30 September 2022

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**Copyright:** © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

ingrowth is slow [2] and does not satisfy the metabolic needs of cells, leading to necrosis at the bulk of the graft. As a result, the successful use of TE constructs in the clinic is mainly limited to thin engineered constructs in which this rate of neovascularization from the host combined with diffusion is sufficient [3,4]. Therefore, most current strategies for engineering thick 3D constructs encompass a prevascularization step. Prevascularization uses endothelial cells (ECs) to form capillary-like networks before implantation, which will ideally anastomose with the circulation of the host tissue after implantation [5]. ECs are seeded alone or in combination with other supportive cell types and most frequently require supplementation with angiogenic growth factors [6,7]. However, the sourcing of ECs is an issue since macrovascular cells such as HUVECs are often used but are arguably not the most suited for capillary network formation [8]. Moreover, supplementation with extrinsic angiogenic growth factors fails to reproduce the growth factor kinetics involved in native vasculogenesis, which often leads to the formation of a non-mature network [9]. In this context, adipose tissue's stromal vascular fraction (SVF) may be extremely important due to its intrinsic angiogenic potential [10]. This fraction can be isolated after the enzymatic digestion of adipose tissue obtained from liposuction or abdominoplasties, which otherwise would be discarded. Several cell populations encompass the SVF, namely endothelial and hematopoietic cells, mesenchymal and endothelial progenitors, pericytes, fibroblasts, and preadipocytes [10–12]. As previously reported, the pericytes and endothelial progenitors present in this fraction have the potential to spontaneously assemble in capillary-like networks in vitro without the need for angiogenic growth factor supplementation, both in 2D [13] and 3D settings [14]. However, freshly isolated SVF is usually used for this purpose, which may be disadvantageous for widespread clinical applications. The use of preserved SVF is therefore underexplored and warrants further investigation. Another important factor for the vascularization of 3D TE constructs is the biomaterial used. The use of natural origin polymers with intrinsic biocompatible properties has revealed a promising approach [15]. [15] Of these polymers, collagen is the most broadly used, as it is the major structural component of the native extracellular matrix (ECM) in living tissues and provides several cues for directing cellular behavior. Among the latter, adhesive motifs are included, which are powerful regulators of cell responses, such as cell spreading or stem cell differentiation [16]. Although collagen from mammalian sources is mostly used to produce TE constructs [17–21], regulatory and religious issues [22] have boosted the search for other collagen sources. Marine collagen is one such source. This type of collagen can be isolated from a number of marine species. In particular, the skin and bones of fish, sea urchin waste, jellyfish, and starfish have high collagen contents [15] with similar properties to mammalian collagen type I [23]. Due to by-catch, the blue shark is one of the most caught shark species [24], and its by-products are highly available and, thus, desirable for collagen extraction [25,26]. We have previously developed blue shark skin collagen sponges with interconnected micro-porous structures that promote not only human adipose stem cells adhesion but also ECM production and cell proliferation and infiltration within scaffolds, indicating a grea<sup>t</sup> potential for vascularization purposes [27].

Considering all of the above, blue shark collagen sponges were used as 3D matrices to explore the capacity of cryopreserved SVF to spontaneously yield a prevascular network in vitro in the absence of extrinsic angiogenic growth factors. This was achieved by assessing the spontaneous formation of capillary-like structures in vitro and by evaluating vessel recruitment, constructing the integration of the prevascular network with the host tissue after in ovo implantation using a chick chorioalantoic membrane (CAM) model.
