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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.

In vitro prevascularization has the potential to address the challenge of maintaining cell viability at the core of engineered constructs, such as bone substitutes, and to improve the survival of tissue grafts by allowing quicker anastomosis to the host microvasculature. The self-assembly approach of tissue engineering allows the production of biomimetic bone-like tissue constructs including extracellular matrix and living human adipose-derived stromal/stem cells (hASCs) induced towards osteogenic differentiation. We hypothesized that the addition of endothelial cells could improve osteogenesis and biomineralization during the production of self-assembled human bone-like tissues using hASCs. Additionally, we postulated that these prevascularized constructs would consequently improve graft survival and bone repair of rat calvarial bone defects. This study shows that a dense capillary network spontaneously formed in vitro during tissue biofabrication after two weeks of maturation. Despite reductions in osteocalcin levels and hydroxyapatite formation in vitro in prevascularized bone-like tissues (35 days of culture), in vivo imaging of prevascularized constructs showed an improvement in cell survival without impeding bone healing after 12 weeks of implantation in a calvarial bone defect model (immunocompromised male rats), compared to their stromal counterparts. Globally, these findings establish our ability to engineer prevascularized bone-like tissues with improved functional properties.

Tissue engineering offers novel therapies for vaginal reconstruction in patients with congenital vaginal agenesis such as Mayer-Rokitansky-Küster-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

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.

We examined the lifetime prevalence of anxiety disorders (ADs) among adolescents with lifetime intermittent explosive disorder (IED), as well as the impact of co-occurring ADs on anger attack frequency and persistence, additional comorbidity, impairment, and treatment utilization among adolescents with IED. IED was defined by the occurrence of at least three anger attacks that were disproportionate to the provocation within a single year. Data were drawn from the National Comorbidity Survey-Adolescent Supplement (N = 6,140), and diagnoses were based on structured lay-administered interviews. Over half (51.89%) of adolescents with IED had an AD, compared to only 22.88% of adolescents without IED. Compared to adolescents with IED alone, adolescents with IED and comorbid ADs: (a) were more likely to be female; (b) reported greater impairment in work/school, social, and overall functioning; (c) were more likely to receive an additional psychiatric diagnosis, a depressive or drug abuse diagnosis, or diagnoses of three or more additional disorders; and (d) had higher odds of receiving any mental/behavioral health treatment as well as treatment specifically focused on aggression. Adolescents with IED alone and those with comorbid ADs did not differ in the number of years experiencing anger attacks or the highest number of anger attacks in a given year. ADs frequently co-occur with IED and are associated with elevated comorbidity and greater impairment compared to IED alone. Gaining a better understanding of this comorbidity is essential for developing specialized and effective methods to screen and treat comorbid anxiety in adolescents with aggressive behavior problems.

Recently, the tubular shape has been suggested as an effective geometry for tissue-engineered heart valves, allowing easy fabrication, fast implantation, and a minimal crimped footprint from a transcatheter delivery perspective. This simple design is well suited for the self-assembly method, with which the only support for the cells is the extracellular matrix they produce, allowing the tissue to be completely free from exogenous materials during its entire fabrication process. Tubular constructs were produced by rolling self-assembled human fibroblast sheets on plastic mandrels. After maturation, the tubes were transferred onto smaller diameter mandrels and allowed to contract freely. This precontraction phase thickened the tissue and prevented further contraction, while improving fusion between the self-assembled layers and aligning the cells circumferentially. When mounted in a pulsed-flow bioreactor, the valves showed good functionality with large leaflets coaptation and opening area. Although physiological aortic flow conditions were not reached, the leaflets could withstand a 1 Hz pulsed flow with a 300 mL/s peak flow rate and a 70 mmHg peak transvalvular pressure. This study shows that the self-assembly method, which has already proven its potential for the production of small diameter vascular grafts, could also be used to achieve functional tubular heart valves.

Socially anxious college students are at increased risk for engaging in problematic drinking (i.e. heavy or risky drinking) behaviors that are associated with the development of an alcohol use disorder. The present study examined whether post-event processing (PEP), repeatedly thinking about and evaluating one's performance in a past social situation, strengthens the association between social anxiety and vulnerability to problematic drinking among college students. Eighty-three college drinkers with high or low social anxiety participated in a social interaction task and were exposed to a manipulation that either promoted or inhibited PEP about the social interaction. Among participants randomized to the PEP promotion condition, those with high social anxiety exhibited a greater urge to use alcohol after the social interaction and greater motivation to drink to cope with depressive symptoms over the week following the manipulation than did those with low social anxiety. These findings suggest that targeting PEP in college drinking intervention programs may improve the efficacy of such programs for socially anxious students.

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 strong clinical need to develop small-caliber tissue-engineered blood vessels for arterial bypass surgeries. Such substitutes can be engineered using the self-assembly approach in which cells produce their own extracellular matrix (ECM), creating a robust vessel without exogenous material. However, this approach is currently limited to the production of flat sheets that need to be further rolled into the final desired tubular shape. In this study, human fibroblasts and smooth muscle cells were seeded directly on UV-C-treated cylindrical polyethylene terephthalate glycol-modified (PETG) mandrels of 4.8 mm diameter. UV-C treatment induced surface modification, confirmed by Fourier-transform infrared spectroscopy (FTIR) analysis, was necessary to ensure proper cellular attachment and optimized ECM secretion/assembly. This novel approach generated solid tubular conduits with high level of cohesion between concentric cellular layers and enhanced cell-driven circumferential alignment that can be manipulated after 21 days of culture. This simple and cost-effective mandrel-seeded approach also allowed for endothelialization of the construct and the production of perfusable trilayered tissue-engineered blood vessels with a closed lumen. This study lays the foundation for a broad field of possible applications enabling custom-made reconstructed tissues of specialized shapes using a surface treated 3D structure as a template for tissue engineering.

Background Evidence suggests that impulsive aggression and explosive anger are common among individuals with anxiety disorders; yet, the influence of intermittent explosive disorder (IED) on the onset, course, consequences, and patterns of comorbidity among those with anxiety disorders is unknown. Methods Data were drawn from the National Comorbidity Survey Replication (N = 9,282) and Adolescent Supplement (N = 9,632), nationally representative surveys conducted between 2001 and 2004. Diagnoses were based on structured lay-administered interviews. Lifetime diagnoses were assessed with structured instruments. Outcomes included comorbidity, functional and role impairment, and treatment utilization. Results Adolescents with a lifetime anxiety disorder had a higher prevalence of a lifetime anger attacks (68.5%) and IED (22.9%) than adolescents without a lifetime anxiety disorder (48.6 and 7.8%, respectively), especially social phobia and panic disorders. Similar elevation was found for adults. Age of onset and course of anxiety disorders did not differ by IED. Severe functional impairment associated with anxiety was higher among adolescents (39.3%) and adults (45.7%) with IED than those without IED (29.2 and 28.2%, respectively). Comorbidity for all other disorders was elevated. However, individuals with anxiety disorders and IED were no more likely to use treatment services than those with anxiety disorders without IED. Conclusions Individuals with IED concomitant to anxiety disorder, especially social phobia and panic, are at marked risk for worse functional impairment and a higher burden of comorbidity, but onset and course of anxiety disorder do not differ, and those with anxiety and IED are no more likely to utilize treatment services. Assessment, identification, and specialized treatment of anger in the context of anxiety disorders are critical to reducing burden.

This protocol describes a unique in vitro method for the generation of a 3D human lymphatic network within native connective tissue devoid of any exogenous material such as scaffolds or growth factors. In this five-stage protocol, human lymphatic endothelial cells (LECs) cocultured with dermal fibroblasts spontaneously organize into a stable 3D lymphatic capillary network. Stage 1 involves the isolation of primary fibroblasts and LECs from human skin. Fibroblasts are then cultured to produce connective tissue rich in extracellular matrix (stage 2), onto which LECs are seeded to form a network (stage 3). After stacking of tissue layers and tissue maturation at the air-liquid interface (stage 4), the 3D construct containing the lymphatic microvascular network can be analyzed by microscopy (stage 5). Lymphatic vasculature generated by this approach exhibits the major cellular and ultrastructural features of native in vivo human dermal lymphatic microvasculature and is stable over many weeks. The protocol for generating a 3D construct takes 6 weeks to complete, and it requires experience in cell culture techniques. The system described here offers a unique opportunity to study the mechanisms underlying lymphatic vessel formation, remodeling and function in a human cell context.

Achieving optimal bone defect repair is a clinical challenge driving intensive research in the field of bone tissue engineering. Many strategies focus on seeding graft materials with progenitor cells prior to in vivo implantation. Given the benefits of closely mimicking tissue structure and function with natural materials, the authors hypothesize that under specific culture conditions, human adipose-derived stem/stromal cells (hASCs) can solely be used to engineer human reconstructed osseous tissues (hROTs) by undergoing osteoblastic differentiation with concomitant extracellular matrix production and mineralization. Therefore, the authors are developing a self-assembly methodology allowing the production of such osseous tissues. Three-dimensional (3D) tissues reconstructed from osteogenically-induced cell sheets contain abundant collagen type I and are 2.7-fold less contractile compared to non-osteogenically induced tissues. In particular, hROT differentiation and mineralization is reflected by a greater amount of homogenously distributed alkaline phosphatase, as well as higher calcium-containing hydroxyapatite (P < 0.0001) and osteocalcin (P < 0.0001) levels compared to non-induced tissues. Taken together, these findings show that hASC-driven tissue engineering leads to hROTs that demonstrate structural and functional characteristics similar to native osseous tissue. These highly biomimetic human osseous tissues will advantageously serve as a platform for molecular studies as well as for future therapeutic in vivo translation.

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.

Substance use has been identified as a major problem on college campuses across the country, with excessive use often leading to unintended and unwanted negative health outcomes. Sensation seeking has been shown to be a consistent predictor of engagement in various health risk behaviors, including substance use. Religiosity has been shown to negatively predict substance use. However, there is mixed evidence on the relations among these risk and protective factors. This may be due to the operational definitions of religiosity in previous research. The current study investigated religiosity as a moderator of the relation between sensation seeking and substance use using robust measures of religiosity. The primary hypotheses were (a) sensation seeking would be positively associated with higher levels of heavy episodic drinking and marijuana use; (b) religiosity would be negatively associated with higher levels of substance use; and (c) religiosity would moderate the relation between sensation seeking and substance use such that, when religiosity was high, there would be no association between sensation seeking and substance use, but at low and moderate levels of religiosity, there would be a positive association between them. Religiosity was a significant moderator of the relation between risk seeking and marijuana use (p < .01), but it was less effective as a moderator between sensation seeking and heavy episodic drinking. Religiosity appears to have a stronger buffering effect for illegal drug use compared with alcohol use, perhaps in part because of the relative acceptance of alcohol consumption across major U.S. religious orientations.

Purpose. It is a common preconception that supercoiledplasmid DNA is more desirable for the transfection of cells that the relaxedform of the plasmid. This notion has led to the recommendation that aspecification for the minimum amount of plasmid in the supercoiled formshould exist in a gene therapy product. We have tested this notion byexamining the effects of the degree of supercoiling on cationiclipid-mediated gene transfer in vitro and in vivo.

Social anxiety disorder (SAD) and antisocial personality disorder (ASPD) are not often thought of as being comorbid. However, recent research suggests the existence of a SAD subtype with characteristics atypical of SAD but common to ASPD. Thus, we explored two competing hypotheses: (1) SAD and ASPD represent opposite ends of a single dimension, or (2) SAD and ASPD exist on two separate dimensions that may be positively correlated. Data were obtained from the National Epidemiological Survey on Alcohol and Related Conditions. SAD-ASPD was related to greater impairment and psychiatric comorbidity than either disorder alone. The SAD-ASPD group was also more likely to seek treatment for their SAD symptoms and to drink before/during antisocial acts than the SAD only group. The presence of SAD for individuals with ASPD (and vice versa) does not appear to provide any "protective benefits." SAD and ASPD appear to be two separate but correlated disorders.

The human umbilical cord (UC) has attracted interest as a source of cells for many research applications. UC solid tissues contain four cell types: epithelial, stromal, smooth muscle and endothelial cells. We have developed a unique protocol for the sequential extraction of all four cell types from a single UC, allowing tissue reconstruction using multiple cell types from the same source. By combining perfusion, immersion and explant techniques, all four cell types have been successfully expanded in monolayer cultures. We have also characterised epithelial and Wharton’s jelly cells (WJC) by immunolabelling of specific proteins. Epithelial cell yields averaged at 2.3 × 105 cells per centimetre UC, and the cells expressed an unusual combination of keratins typical of simple, mucous and stratified epithelia. Stromal cells in the Wharton’s jelly expressed desmin, α-smooth muscle actin, elastin, keratins (K12, K16, K18 and K19), vimentin and collagens. Expression patterns in cultured cells resembled those found in situ except for basement membrane components and type III collagen. These stromal cells featured a sustained proliferation rate up to passage 12 after thawing. The mesenchymal stem cell (MSC) character of the WJC was confirmed by their expression of typical MSC surface markers and by adipogenic and osteogenic differentiation assays. To emphasise and demonstrate their potential for regenerative medicine, UC cell types were successfully used to produce human tissue-engineered constructs. Both bilayered stromal/epithelial and vascular substitutes were produced, establishing the versatility and importance of these cells for research and therapeutic applications.

Cutaneous malignant melanomas represent an important clinical problem because they are highly invasive, they can metastasize to distant sites and are typically resistant to available therapy. The precise molecular determinants responsible for melanoma progression and chemo-resistance are not yet known, in part due to lack of pertinent experimental models that mimic human melanoma progression. Accordingly, we developed a complex human microvascularized reconstructed skin substitute in which the organized three-dimensional (3D) architecture of the native skin is reproduced. Human melanoma cell lines derived from primary and metastatic sites were added to this 3D model. Our results demonstrate that histological features and behavior of melanoma cells applied in our skin substitute model are specific to their site of origin. In particular, the ability of melanoma cells to cross the dermal–epidermal junction correlates with their metastatic potential. In addition, a potent angiogenic effect was detected for an aggressive metastatic cell line that produces VEGF. The presence of a microvascular network within this model will allow studying a crucial step of the metastatic process. We conclude that such an in vitro human tumor microvascularized reconstructed skin substitute promises to be a versatile and efficient model to investigate skin cancer progression and to screen new anticancer drugs to improve currents clinical treatments.

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.

The incorporation of pegylated lipid into Lipid-Protamine-DNA (LPD-PEG) lipopolyplexes causes a decrease of their in vitro transfection activity. This can be partially attributed to a reduction in particle binding to cells. To restore particle binding and specifically target LPD formulations to tumor cells, the lipid-peptide conjugate DSPE-PEG5K-succinyl-ACDCRGDCFCG-COOH (DSPE-PEG5K-RGD-4C) was generated and incorporated into LPD formulations (LPD-PEG-RGD). LPD-PEG-RGD was characterized with respect to its biophysical and biological properties. The Incorporation of DSPE-PEG5K-RGD-4C ligands into LPD formulations results in a 5 and a 15 fold increase in the LPD-PEG-RGD binding and uptake, respectively, over an LPD-PEG formulation. Enhancement of binding and uptake resulted in a 100 fold enhancement of transfection activity. Moreover, this transfection enhancement was specific to cells expressing appropriate integrin receptors (MDA-MB-231). Huh7 cells, known for their low level of alphavbeta3 and alphavbeta5 integrin expression, failed to show RGD mediated transfection enhancement. This transfection enhancement can be abolished in a competitive manner using free RGD peptide, but not an RGE control peptide. Results demonstrated RGD mediated enhanced LPD-PEG cell binding and transfection in cells expressing the integrin receptor. These formulations provide the basis for effective, targeted, systemic gene delivery.