Engineering vessel-like networks within multicellular fibrin-based constructs
Introduction
Promotion of vascularization, the process in which new blood vessels assemble, is critical for a number of pathological conditions and surgical interventions as well as for the construction of viable, engineered tissue constructs. Wound healing, myocardial ischemia, peripheral vascular disease, and plastic reconstruction surgery are a few of the many clinical situations that benefit from enhanced vascularization [1]. In the context of tissue engineering, vascularization maintains cell viability during in-vitro construct growth, induces tissue organization and differentiation, and promotes graft survival upon implantation [2], [3].
Self-assembly of vessel networks can be stimulated in-vitro within 3D scaffolds by means of multicellular culturing of endothelial cells (ECs), vascular mural cells and cells specific to the tissue of interest [4], [5], [6], [7]. The vascular mural cells are smooth muscle cells or pericytes, which provide physical support to ECs, generate extracellular proteins (e.g. collagen, laminin, fibronectin) and release proangiogenic growth factors (GFs) (e.g. VEGF, FGF, TGF, angiopoietin) which induce vascularization [8], [9]. Embryonic fibroblasts and mesenchymal precursor cells are extensively used in multicellular tissue modeling protocols, due to their capacity to differentiate into mural cells [4], [5], [6], [10]. The multicellular culturing approach both supports formation of endothelial vessels and promotes EC and tissue specific cell interactions, suggested to play a key role in further tissue construct development and differentiation [4], [5], [11], [12]. Such endothelial vessels consisting of tube-like openings are presumed to form the basis for improved media penetration to the inner regions of thick 3D constructs, enhancing construct survival and enabling effective engineering of large complex tissues in the lab. Upon implantation, the pre-existing blood vessels can anastomose with host vasculature, enhancing graft perfusion and accelerating host neovascularization via graft-driven paracrine signaling pathways [4], [6], [10], [13], [14].
The choice of scaffold biomaterial onto which cells are seeded, grow and proliferate, is central to the tissue engineering efficacy. Biomaterial properties dictate those of the new tissue substitute and have been shown to directly affect cell organization and differentiation [15], [16], [17], [18]. Thus, manipulation of scaffold composition can provide a powerful approach to explore and enhance vascularization both in-vitro and in-vivo. Choice of scaffold biomaterial must consider both requisites for structural support of cells (typically provided by synthetic scaffolds) and for biological interactions (usually provided by naturally harvested materials) allowing for cell adhesion, migration, and differentiation into functional tissue [19]. The fibrous and insoluble fibrin matrix, naturally formed upon thrombin-driven proteolysis of fibrinogen during blood clotting, with proven angiogenesis-promoting activity [20] presents an attractive scaffold candidate for promotion of vascularization when prepared in combination with the synthetic PLLA/PLGA scaffold. The PLLA/PLGA material has been approved for clinical applications and has been shown to support cell growth and vascularization [4], [5], [13], [17], [21]. In addition, this scaffold provides mechanical strength, tunable by changing the PLLA versus PLGA ratios within the scaffolds, which we have previously reported as influential in regulating cell organization and differentiation [17].
In this study, we aimed to generate vascular networks by applying the multicellular culturing technique within 3D fibrin-based constructs, and to explore the environmental factors influencing vascularization. To this end, various fibrin concentrations and quantities were used to create fibrin matrices alone or in combination with PLLA/PLGA synthetic sponges (will be referred as fibrin + PLLA/PLGA constructs). Constructs were then embedded with multicellular preparations of ECs and fibroblasts in the presence or absence of tissue specific skeletal myoblast cells. Skeletal myoblast cells were chosen due to their ability to differentiate into skeletal muscle tissue [4] in-vitro with the objective of constructing engineered muscle tissue embedded with vessel-like network.
The process of vascular formation was then monitored in-vitro using advanced confocal imaging. Thereafter, implantation studies were performed utilizing an in-vivo model allowing for quantitative evaluation of graft neovascularization and perfusion. In addition, the fate of the implanted EC cells was assessed as was their spatial 3D organization toward formation of vascular network in-vivo.
Section snippets
Human umbilical vein endothelial cells (HUVECs)
HUVECs (passage 4–6, Clonetics) were grown on tissue culture plates in EGM-2 medium supplemented with 2%FBS and endothelial cell growth medium BulletKit®-2 (EGM-2® BulletKit®). To closely monitor the temporal development of vascularization in-vitro and in-vivo, HUVEC-GFP and HUVEC-RFP (passage 4–6, Angio-Proteomie) were also used.
Human foreskin fibroblast cells (HFF)
Primary cultures of HFF cells were extracted, in our laboratory, from newborns’ foreskins and used until passage 10. HFF cells were cultured in Dulbecco’s Modified
The multicellular culturing strategy
Using the multicellular strategy, formation of vascular networks within engineered skeletal [4] and cardiac [5] muscle tissues were successfully generated in our laboratory. Fig. 1 illustrates procedure in which vascular cells (ECs and fibroblasts cells) are simultaneously cultured with tissue specific myoblast cells within 3D scaffolds to spontaneously organize into vessel-like networks. In this study, we evaluate the ability of fibrin and the hybrid fibrin + PLLA/PLGA scaffolds (Fig. 1, Fig. 2
Discussion
The present study explored the efficacy of fibrin matrices when applied toward multicellular cultures for the purpose of engineering complex vascularized constructs.
To date, fibrin matrices have been widely studied in engineering of soft tissues, such as cartilage [26], [27], or used as an injectable carrier material for cell therapy procedures [28], [29]. In addition, investigation of angiogenesis have been described for fibrin matrices [14], [30] or synthetic scaffolds [31], but has never
Conclusions
This study presented a detailed analysis of the impact of fibrin and synthetic PLLA/PLGA scaffolds on regulation of 3D vessel network formation in-vitro and on neovascularization upon implantation. The results described herein demonstrate that fibrin concentrations and quantities influence both the degree of vascular maturity as well as network morphology in-vitro. The impact of fibrin on vascular maturity was further enhanced by addition of synthetic PLLA/PLGA sponges through provision of
Acknowledgments
We would like to thank Prof. David Yaffe (Weizmann Institute, Faculty of Biology, Israel) for providing the C2 cell line. We would like to acknowledge Shirley Rachel Rochman for the graphic assistance, and Yehudit Posen for editing the manuscript. The research was funded by The Israeli Ministry of Industry & Trade, Nofar program and Omrix Biopharmaceuticals Ltd.
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