Implantation of a Pre-vascularized Tissue-Engineered Human Vaginal Mucosa in an animal model
Weronika Jakubowska, MD-MSc student, Stéphane Chabaud, PhD., Ingrid Saba, PhD., Stéphane Bolduc, MD.
LOEX/ Université Laval, Quebec, QC, Canada.
Tissue engineering of autologous vaginal mucosa opens the door to new surgical applications for vaginal reconstruction for paediatric patients with congenital urogenital abnormalities such as the Mayer-Rokitansky-Küster-Hauser syndrome (MRKH). Vascularization of tissue-engineered constructs represents a major challenge as graft survival and success rate highly depend on it. This study aims at reconstructing a capillary network within a tissue-engineered vaginal mucosa that is functional in vivo.
Vaginal stromal and epithelial cells were isolated from healthy donors' biopsies. Vaginal mucosa constructs free of exogenous materials were produced using the self-assembly technique which relies on the intrinsic properties of fibroblasts to secrete their own extracellular matrix in the presence of ascorbic acid. Previously, our team has determined that a hybrid reseeding and sheet-stacking approach is best adapted for the endothelialization of vaginal mucosa constructs while retaining interesting mechanical properties for surgical handling. Vaginal stromal cells were co-seeded with endothelial cells derived from a human umbilical cord vein (HUVEC) that were transduced with a vector that induces GFP and luciferase gene expression. The formation of a capillary-like network within the constructs was monitored using a fluorescent inverted microscope. Two reconstructed vaginal mucosa grafts, one with endothelial cells and one control, were implanted subcutaneously on the back of 12 female nude mice. Each graft was made of two constructs that were sutured together along the periphery with an opening at one end. The vaginal epithelium of the constructs was facing the lumen. An agarose hydrogel stent was inserted into this newly created pocket in order to maintain the opening of the graft and prevent contraction. A Premarin cream that is frequently used in clinic and that contains conjugated estrogens was applied twice daily to prevent dryness. The stability of endothelial cells in vivo was assessed with the IVIS microscope as HUVEC GFP/luciferase generate bioluminescence in the presence of luciferine. The mice were sacrificed by groups of four after 3, 7, 14 and 21 days. Samples were analyzed with Masson's trichrome staining and immunofluorescence: blood perfusion was assessed by the presence of mouse red blood cells within human GFP+ capillaries, while angiogenesis by the invasion of mouse endothelial cells inside the graft.
The presence of a pseudo-capillary network within the vaginal mucosa constructs was confirmed in vitro with a fluorescence microscope before implantation. All vaginal constructs were easily handled by a surgeon and resistant to sutures. Human endothelial cells were present within grafts in vivo until 21 days after surgery. Overall, endothelialized grafts demonstrated signs of earlier vascularization. The finding of mouse red blood cells within GFP positive capillaries 7 days after implantation confirms the capacity of the reconstructed capillary-like network to sustain blood perfusion, thus its functionality. Furthermore, there has been inosculation between GFP capillaries and mouse endothelial cells.
Our results have demonstrated that the addition of endothelial cells to our tissue engineered vaginal mucosa model generates a capillary-like network that is mature enough to become functional in vivo. Such pre-vascularized model can improve surgical outcomes of vaginal reconstruction.
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