Tissue Engineering of Axially Vascularized Soft Tissue Flaps with a Poly-e-Caprolactone Nanofiber Hydrogel Composite
Dominic Henn1, Annika Rauh, MD1, Katharina Fischer, BS1, Ulrich Kneser, MD1, Yoo-Jin Kim, MD2, Hai-Quan Mao, PhD3, Volker J. Schmidt, MD1, Justin M. Sacks, MD, MBA4.
1Heidelberg University, BG Trauma Center Ludwigshafen, Ludwigshafen, Germany, 2Institute of Pathology, Kaiserslautern, Germany, 3Department of Materials Science & Engineering, Whiting School of Engineering, Translational Tissue Engineering Center, School of Medicine, Institute for NanoBioTechnology, Johns Hopkins University, Balitmore, MD, USA, 4Department of Plastic and Reconstructive Surgery, Johns Hopkins University, Balitmore, MD, USA.
Donor sites for autologous free flaps are limited, especially in severe burn and trauma patients; moreover, donor site morbidity of free flaps can cause significant wound complications and functional impairments. Here we propose a novel approach for tissue engineering of axially vascularized soft tissue flaps suitable for free microsurgical transfer using a flowable nanofiber hydrogel composite vascularized by an arteriovenous (AV) loop and compare it to a standard collagen-elastin based scaffold.
AV loops were microsurgically created in 15 rats and placed into subcutaneous isolation chambers, which were filled with either a composite hydrogel of hyaluronic acid and poly-e-caprolactone nanofibers (NF-HA), a hyaluronic acid-only hydrogel (HA), or two sheets of a collagen-elastin (CE) scaffold. Chambers filled with NF-HA or CE without an AV loop served as controls. Neoangiogenesis and cellular infiltration were compared in histologic sections of the constructs after explantation on postoperative day 14 using bright field light microscopy and scanning electron microscopy (SEM). The expression of angiogenesis regulating proteins, namely synaptojanin-2 binding protein (SYNJ2BP), ephrin receptor kinase 2 (EPHA2), and forkhead box C1 (FOXC1), which have been shown to be involved in AV loop associated angiogenesis in previous studies by our group, were analyzed in endothelial cells (ECs) and within the scaffolds by immunohistochemistry and quantified using standardized photometric analysis.
A functional neovascular network was evident in NF-HA flaps after 15 days with the amount of neoangiogenesis being comparable to CE constructs. SEM revealed a strong mononuclear cell infiltration along the nanofibers in NF-HA constructs. HA alone was not suitable for AV loop based tissue engineering, leading to microvascular thrombosis and shrinking. Theexpression of the anti-angiogenic protein SYNJ2BP as well as EPHA2 and FOXC1 expression was significantly reduced in vascularized AV loop constructs compared to native scaffolds. Within ECs of AV loop constructs, SYNJ2BP and EPHA2 expression was significantly decreased in NF-HA constructs, whereas FOXC1 expression was significantly increased compared to CE constructs.
Hyaluronic acid hydrogels with poly-e-caprolactone nanofibers are suitable for tissue engineering of axially vascularized soft tissue flaps with a solid neovascularization and strong cellular infiltration. Our data indicate that SYNJ2BP, EPHA2, and FOXC1 are involved in AV loop associated angiogenesis and that the presence of nanofibers has an impact on the expression of these proteins in ECs, thus potentially increasing their angiogenic capacity.
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