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Tissue engineering offers novel therapies for vaginal reconstruction in patients with congenital vaginal agenesis such as Mayer-Rokitansky-Kuster-Hauser (MRKH) syndrome. This study aims to reconstruct a prevascularized tissue-engineered model of human vaginal mucosa (HVM) using the self-assembly approach, free of exogenous materials. In this study, a new cell culture method was used to enhance microcapillary network formation while maintaining sufficient biomechanical properties for surgical manipulation. Human vaginal fibroblasts were coseeded with human umbilical vein endothelial cells (HUVECs). Transduction of HUVEC with a vector that allows the expression of both green fluorescent protein (GFP) and luciferase allowed the monitoring of the formation of a microvascular network in vitro and the assessment of the viability and stability of HUVEC in vivo. Two reconstructed vaginal mucosa grafts, a prevascularized, and a nonvascularized control were implanted subcutaneously on the back of 12 female nude mice and monitored for up to 21 days. Prevascularized grafts demonstrated signs of earlier vascularization compared with controls. However, there were no differences in graft survival outcomes in both groups. The finding of mouse red blood cells within GFP-positive capillaries 1 week after implantation demonstrates the capacity of the reconstructed capillary-like network to connect to the host circulation and sustain blood perfusion in vivo. Furthermore, sites of inosculation between GFP-positive HUVEC and mouse endothelial cells were observed within prevascularized grafts. Our results demonstrate that the addition of endothelial cells using a hybrid approach of self-assembly and reseeding generates a mature capillary-like network that has the potential to become functional in vivo, offering an optimized prevascularized HVM model for further translational research. Impact statement This study introduces a prevascularized tissue-engineered model of human vaginal mucosa (HVM), which is adapted for surgical applications. The prevascularization of tissue-engineered grafts aims to enhance graft survival and is an interesting feature for sexual function. Various scaffold-free cell culture methods were tested to reconstruct a mature microcapillary network within HVM grafts while meeting biomechanical needs for surgery. Moreover, this animal study assesses the vascular functionality of prevascularized grafts in vivo, serving as a proof of concept for further translational applications. This research underlines the continuous efforts to optimize current models to closely mimic native tissues and further improve surgical outcomes.

The study of neurodegenerative diseases (such as Alzheimer’s disease, Parkinson’s disease, Huntington’s disease, or amyotrophic lateral sclerosis) is very complex due to the difficulty in investigating the cellular dynamics within nervous tissue. Despite numerous advances in the in vivo study of these diseases, the use of in vitro analyses is proving to be a valuable tool to better understand the mechanisms implicated in these diseases. Although neural cells remain difficult to obtain from patient tissues, access to induced multipotent stem cell production now makes it possible to generate virtually all neural cells involved in these diseases (from neurons to glial cells). Many original 3D culture model approaches are currently being developed (using these different cell types together) to closely mimic degenerative nervous tissue environments. The aim of these approaches is to allow an interaction between glial cells and neurons, which reproduces pathophysiological reality by co-culturing them in structures that recapitulate embryonic development or facilitate axonal migration, local molecule exchange, and myelination (to name a few). This review details the advantages and disadvantages of techniques using scaffolds, spheroids, organoids, 3D bioprinting, microfluidic systems, and organ-on-a-chip strategies to model neurodegenerative diseases.

One of the major challenges in the development of a larger and longer nerve conduit for peripheral nerve repair is the limitation in oxygen and nutrient diffusion within the tissue after transplantation preventing Schwann cell and axonal migration. This restriction is due to the slow neovascularization process of the graft starting from both nerve endings. To overcome this limitation, we propose the design of a living tissue-engineered nerve conduit made of an internal tube with a three-dimensional structure supporting axonal migration, which is inserted inside a hollow external tube that plays the role of an epineurium and is strong enough to be stitched to the severed nerve stumps. The internal tube is made of a rolled living fibroblast sheet and can be seeded with endothelial cells to promote the formation of a network containing capillary-like structures which allow rapid inosculation with the host nerve microvasculature after grafting. Human nerve conduits were grafted in immunodeficient rats to bridge a 15 mm sciatic nerve gap. Human capillaries within the pre-vascularized nerve conduit successfully connected to the host circulation 2 weeks after grafting. Twenty-two weeks after surgery, rats transplanted with the nerve conduits had a similar motor function recovery compared to the autograft group. By promoting rapid vascularization of the internal nerve tube from both ends of the nerve stumps, this endothelialized nerve conduit model displays a favorable environment to enhance axonal migration in both larger caliber and longer nerve grafts.

Regeneration of lymphatic vessels is important for treatment of various disorders of lymphatic system and for restoration of lymphatic function after surgery. We have developed a method for generating a human 3D lymphatic vascular construct. In this system, human lymphatic endothelial cells, co-cultured with fibroblasts, spontaneously organized into a stable 3D lymphatic capillary network without the use of any exogenous factors. In vitro-generated lymphatic capillaries exhibited the major molecular and ultra-structural features of native, human lymphatic microvasculature: branches in the three dimensions, wide lumen, blind ends, overlapping borders, adherens and tight junctions, anchoring filaments, lack of mural cells, and poorly developed basement membrane. Furthermore, we show that fibroblast-derived VEGF-C and HGF cooperate in the formation of lymphatic vasculature by activating ERK1/2 signaling, and demonstrate distinct functions of HGF/c-Met and VEGF-C/VEGFR-3 in lymphangiogenesis. This lymphatic vascular construct is expected to facilitate studies of lymphangiogenesis in vitro and it holds promise as a strategy for regeneration of lymphatic vessels and treatment of lymphatic disorders in various conditions.

There is a clinical need for better blood vessel substitutes, as current surgical procedures are limited by the availability of suitable autologous vessels and suboptimal behavior of synthetic grafts in small caliber arterial graft (5 mm) applications. The aim of the present study was to compare the mechanical properties of arterial and venous tissue-engineered vascular constructs produced by the self-assembly approach using cells extracted from either the artery or vein harvested from the same human umbilical cord. The production of a vascular construct comprised of a media and an adventitia (TEVMA) was achieved by rolling a continuous tissue sheet containing both smooth muscle cells and adventitial fibroblasts grown contiguously in the same tissue culture plate. Histology and immunofluorescence staining were used to evaluate the structure and composition of the extracellular matrix of the vascular constructs. The mechanical strength was assessed by uniaxial tensile testing, whereas viscoelastic behavior was evaluated by stepwise stress-relaxation and by cyclic loading hysteresis analysis. Tensile testing showed that the use of arterial cells resulted in stronger and stiffer constructs when compared with those produced using venous cells. Moreover, cyclic loading demonstrated that constructs produced using arterial cells were able to bear higher loads for the same amount of strain when compared with venous constructs. These results indicate that cells isolated from umbilical cord can be used to produce vascular constructs. Arterial constructs possessed superior mechanical properties when compared with venous constructs produced using cells isolated from the same human donor. This study highlights the fact that smooth muscle cells and fibroblasts originating from different cell sources can potentially lead to distinct tissue properties when used in tissue engineering applications.

Delayed or absence of vascularization is one of the major reasons for skin engraftment failure in patients with extensive burns. For such trauma victims, the best alternative to a split-thickness graft would be wound coverage with an autologous in vitro reconstructed skin (RS) combining dermis and epidermis with an appropriate microvascularization. We have developed an endothelialized RS based on our self-assembly approach, which is generated from autologous cultured cells without any exogenous angiogenic growth factor or scaffold. After transplantation in athymic mice, an early inosculation between the graft and host vasculatures occurred within 4 days. We also concurrently detected an active invasion of the dermis by host capillaries sprouting from the wound bed. Thus, the microvascular network constructed in vitro within our three-dimensional skin substitute did not only develop functional anastomoses with the host's blood vessels but also promoted a rapid, complete, and optimal vascularization of the implanted tissues by exerting an angiogenic effect compared with control RS. Our model may bring about interesting possibilities for regenerative medicine by leading to faster vascularization in clinical applications. In addition, the endothelialized RS can be a useful in vitro angiogenesis model.