"Introduction: Vascularization is a critical aspect of every tissue engineering (TE) approach, especially in 3D constructs. The formation of a network of capillaries is necessary to ensure adequate delivery of nutrients and oxygen to cells within the constructs, as well as fast anastomosis with the host’s vasculature after implantation. Pre-vascularization of these constructs before implantation can be a solution. However, cell sourcing is a limiting issue. Adipose tissue is regarded as a privileged source of mesenchymal progenitor cells due to its easy accessibility and abundancy. This tissue hosts adipocytes, as well as a stromal vascular fraction (SVF) comprising several other cell types including fibroblasts, endothelial progenitors, endothelial cells and hematopoietic cells. Due to this composition, the SVF of adipose tissue is highly angiogenic and has been proposed for the growth factor-free vascularization of TE constructs. To produce such constructs, collagen from mammalian sources is widely used. However, regulatory issues associated with the risk of disease transmission have boosted the search for new collagen sources such as from marine organisms. Collagen from otherwise wasted blue shark skin was herein used to produce sponges that were then seeded with cryopreserved SVF for growth factor-free vascularization.
Methodology: A blue shark skin collagen hydrogel was created by acid solubilization of blue shark skin collagen, followed by cryogelation with crosslinking reaction carried out at low temperatures using 1-[3-(dimethylamino) propyl]-3-ethylcarbodiimide hydrochloride. Finally, cryogels were freeze dried to form the collagen sponge. SVF was isolated from human adipose tissue subcutaneous tissue and cryopreserved in 10% DMSO in FBS with a controlled freeze rate of 1ºC/min, for at least 7 days. SVF was then seeded on the collagen sponges and cultured for 7 days to create a pre-vascularized construct. Sponges’ pre-vascularization was assessed in vitro by immunohistochemistry and secretome profiling and their functionality was tested in ovo using a chick chorioallantoic membrane (CAM) assay.
Results: After 7 days of in vitro culture, CD31 expression pattern demonstrated the formation of a vessel like network. The secretome profile of angiogenic-related factors changed with culture time. From 5 to 7 days of culture, there was an increase in the secretion of both pro angiogenic proteins (VEGF, MMP-9, IL-8) and angiogenesis inhibitors (TIMP-1, SERPIN E1, Thrombospondin-1). Upon in ovo implantation, vessel number quantification demonstrated an increase in vessel recruitment in pre-vascularized sponges when comparing with sponges without SVF cells. CD31 expression pattern demonstrated the integration of the pre-vascular network within the CAM, while in situ hybridization confirmed the presence of the seeded human cells.
Conclusions: These results demonstrate the potential of cryopreserved SVF to assist in the vascularization of TE constructs in an extrinsic growth factor-free manner, allowing a simplified and cost-efficient methodology to ensure construct integration after implantation.
- Costa, M et al, Acta Biomater. 2017 Jun 1;55:131–43.
Acknowledgements: EU Horizon2020 ERC grant CapBed (805411); FCT fellowships PD/BD/135252/2017, IF/00347/2015; INTERREG España-Portugal 2014-2020 project 0474_BLUEBIOLAB_1_E; Atlantic Area Programme project BLUEHUMAN (EAPA_151/2016) and NORTE2020/PT2020 project ATLANTIDA (Norte-01-0145-FEDER-000040). Dr. Cármen G. Sotelo (IIM-CSI, Vigo, Spain), for the kind offer of blue shark skin collagen."