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A major roadblock in implementing engineered tissues clinically lies in their limited vascularization. After implantation, such tissues do not integrate with the host's circulation as quickly as needed, commonly resulting in loss of viability and functionality. This study presents a solution to the vascularization problem that could enable the survival and function of large, transplantable, and vascularized engineered tissues. The technique allows vascularization of a cell laden hydrogel through angiogenesis from a suturable tissue-engineered vascular graft (TEVG) constructed from electrospun polycaprolactone with macropores. The graft is surrounded by a layer of cell-laden gelatin-methacryloyl hydrogel. The constructs are suturable and possess mechanical properties like native vessels. Angiogenesis occurs through the pores in the graft, resulting in a hydrogel containing an extensive vascular network that is connected to an implantable TEVG. The size of the engineered tissue and the degree of vascularization can be increased by adding multiple TEVGs into a single construct. The engineered tissue has the potential to be immediately perfused by the patient's blood upon surgical anastomosis to host vessels, enabling survival of implanted cells. These findings provide a meaningful step to address the longstanding problem of fabricating suturable pre-vascularized tissues which could survive upon implantation in vivo. STATEMENT OF SIGNIFICANCE: Creating vascularized engineered tissues that can be transplanted and rapidly perfused by the host blood supply is a major challenge which has limited the clinical impact of tissue engineering. In this study we demonstrate a technique to fabricate vascularized tissue constructs via angiogenesis from a suturable tissue-engineered vascular graft. The macroporous graft is surrounded with hydrogel, allowing endothelial cells to migrate from the lumen and vascularize the hydrogel layer with capillary-like structures connected to the macrovessel. The graft has comparable mechanical properties to native blood vessels and larger constructs can be fabricated by incorporating multiple grafts. These constructs could potentially be connected surgically to the circulation at an implantation site to support their immediate perfusion and survival., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Background & Aims: Liver sinusoidal endothelial cells (LSECs) are important in liver development, regeneration, and pathophysiology, but the differentiation process underlying their tissue-specific phenotype is poorly understood and difficult to study because primary human cells are scarce. The aim of this study was to use human induced pluripotent stem cell (hiPSC)-derived LSEC-like cells to investigate the differentiation process of LSECs., Methods: hiPSC-derived endothelial cells (iECs) were transplanted into the livers of Fah -/- / Rag2 -/- / Il2rg -/- mice and assessed over a 12-week period. Lineage tracing, immunofluorescence, flow cytometry, plasma human factor VIII measurement, and bulk and single cell transcriptomic analysis were used to assess the molecular and functional changes that occurred following transplantation., Results: Progressive and long-term repopulation of the liver vasculature occurred as iECs expanded along the sinusoids between hepatocytes and increasingly produced human factor VIII, indicating differentiation into LSEC-like cells. To chart the developmental profile associated with LSEC specification, the bulk transcriptomes of transplanted cells between 1 and 12 weeks after transplantation were compared against primary human adult LSECs. This demonstrated a chronological increase in LSEC markers, LSEC differentiation pathways, and zonation. Bulk transcriptome analysis suggested that the transcription factors NOTCH1 , GATA4 , and FOS have a central role in LSEC specification, interacting with a network of 27 transcription factors. Novel markers associated with this process included EMCN and CLEC14A . Additionally, single cell transcriptomic analysis demonstrated that transplanted iECs at 4 weeks contained zonal subpopulations with a region-specific phenotype., Conclusions: Collectively, this study confirms that hiPSCs can adopt LSEC-like features and provides insight into LSEC specification. This humanised xenograft system can be applied to further interrogate LSEC developmental biology and pathophysiology, bypassing current logistical obstacles associated with primary human LSECs., Impact and Implications: Liver sinusoidal endothelial cells (LSECs) are important cells for liver biology, but better model systems are required to study them. We present a pluripotent stem cell xenografting model that produces human LSEC-like cells. A detailed and longitudinal transcriptomic analysis of the development of LSEC-like cells is included, which will guide future studies to interrogate LSEC biology and produce LSEC-like cells that could be used for regenerative medicine., Competing Interests: None to declare. Please refer to the accompanying ICMJE disclosure forms for further details., (© 2024 The Author(s).)

Background: Sexual assault (SA) is experienced by a substantial proportion of emerging adult college students and is associated with elevated rates of posttraumatic stress disorder (PTSD) and alcohol use. This study examines the mediating role of posttraumatic stress symptoms (PTSS) in the associations among SA severity, drinking to cope with anxiety, and average weekly drinks while considering the moderating roles of gender identity and sexual orientation., Methods: A total of 2160 college students who were diverse in gender (cisgender women, 64.4%; cisgender men, 30.6%, and transgender and gender diverse [TGD] individuals = 4.9%) and sexual orientation (heterosexual = 68.0%, LGBQ+ = 32.0%) completed measures of SA severity, PTSS, drinking to cope with anxiety motives, and average weekly drinks., Results: The mediation model for the full sample indicated significant indirect effects of SA severity on drinking to cope with anxiety through PTSS, but not on average weekly drinks. Moderation analyses revealed differential relationships between the variables based on both gender identity and sexual orientation. For instance, the association between SA severity and PTSS was stronger for cisgender women and TGD individuals than cisgender men, and for LGBQ+ individuals than heterosexual individuals. While the association between PTSS and average weekly drinks was only significant for cisgender men, the association between PTSS and drinking to cope with anxiety was significant for both cisgender men and women but not TGD individuals. Furthermore, the association between SA severity and drinking to cope with anxiety was stronger for cisgender women than cisgender men., Conclusions: Findings from this study demonstrate sexual orientation and gender identity differences and similarities in the associations of SA severity, PTSS, drinking to cope with anxiety, and alcohol use. Results are discussed in relation to the self-medication hypothesis and tailoring interventions for diverse groups., (© 2023 Research Society on Alcohol.)

Dynamic positioning of endothelial tip and stalk cells, via the interplay between VEGFR2 and NOTCH signaling, is essential for angiogenesis. VEGFR2 activates PI3K, which phosphorylates PI(4,5)P 2 to PI(3,4,5)P 3 , activating AKT; however, PI3K/AKT does not direct tip cell specification. We report that PI(4,5)P 2 hydrolysis by the phosphoinositide-5-phosphatase, INPP5K, contributes to angiogenesis. INPP5K ablation disrupted tip cell specification and impaired embryonic angiogenesis associated with enhanced DLL4/NOTCH signaling. INPP5K degraded a pool of PI(4,5)P 2 generated by PIP5K1C phosphorylation of PI(4)P in endothelial cells. INPP5K ablation increased PI(4,5)P 2 , thereby releasing β-catenin from the plasma membrane, and concurrently increased PI(3,4,5)P 3 -dependent AKT activation, conditions that licensed DLL4 / NOTCH transcription. Suppression of PI(4,5)P 2 in INPP5K -siRNA cells by PIP5K1C -siRNA, restored β-catenin membrane localization and normalized AKT signaling. Pharmacological NOTCH or AKT inhibition in vivo or genetic β-catenin attenuation rescued angiogenesis defects in INPP5K-null mice. Therefore, PI(4,5)P 2 is critical for β-catenin/DLL4/NOTCH signaling, which governs tip cell specification during angiogenesis.

Due to a relative paucity of studies on human lymphatic assembly in vitro and subsequent in vivo transplantation, capillary formation and survival of primary human lymphatic (hLEC) and blood endothelial cells (hBEC) ± primary human vascular smooth muscle cells (hvSMC) were evaluated and compared in vitro and in vivo. hLEC ± hvSMC or hBEC ± hvSMC were seeded in a 3D porous scaffold in vitro, and capillary percent vascular volume (PVV) and vascular density (VD)/mm 2 assessed. Scaffolds were also transplanted into a sub-cutaneous rat wound with morphology/morphometry assessment. Initially hBEC formed a larger vessel network in vitro than hLEC, with interconnected capillaries evident at 2 days. Interconnected lymphatic capillaries were slower (3 days) to assemble. hLEC capillaries demonstrated a significant overall increase in PVV ( p = 0.0083) and VD ( p = 0.0039) in vitro when co-cultured with hvSMC. A similar increase did not occur for hBEC + hvSMC in vitro, but hBEC + hvSMC in vivo significantly increased PVV ( p = 0.0035) and VD ( p = 0.0087). Morphology/morphometry established that hLEC vessels maintained distinct cell markers, and demonstrated significantly increased individual vessel and network size, and longer survival than hBEC capillaries in vivo, and established inosculation with rat lymphatics, with evidence of lymphatic function. The porous polyurethane scaffold provided advantages to capillary network formation due to its large (300-600 μm diameter) interconnected pores, and sufficient stability to ensure successful surgical transplantation in vivo. Given their successful survival and function in vivo within the porous scaffold, in vitro assembled hLEC networks using this method are potentially applicable to clinical scenarios requiring replacement of dysfunctional or absent lymphatic networks., Competing Interests: The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article., (© The Author(s) 2022.)

Objective: Synovial fibrosis contributes to osteoarthritis (OA) pathology, but the underlying mechanisms remain unknown. We have observed increased microRNA-27b-3p (miR-27b-3p) levels in synovial fluid of patients with late-stage radiographic knee OA. Here, we investigated the contribution of miR-27b-3p to synovial fibrosis in patients with severe knee OA and in a mouse model of knee OA., Methods: We stained synovium sections obtained from patients with radiographic knee OA scored according to the Kellgren/Lawrence scale and mice that underwent destabilization of the medial meniscus (DMM) for miR-27b-3p using in situ hybridization. We examined the effects of intraarticular injection of miR-27b-3p mimic into naive mouse knee joints and intraarticular injection of a miR-27b-3p inhibitor into mouse knee joints after DMM. We performed transfection with miR-27b-3p mimic and miR-27b-3p inhibitor in human OA fibroblast-like synoviocytes (FLS) using reverse transcriptase-quantitative polymerase chain reaction (RT-qPCR) array, RNA sequencing, RT-qPCR, Western blotting, immunofluorescence, and migration assays., Results: We observed increased miR-27b-3p expression in the synovium from patients with knee OA and in mice with DMM-induced arthritis. Injection of the miR-27b-3p mimic in mouse knee joints induced a synovial fibrosis-like phenotype, increased synovitis scores, and increased COL1A1 and α-smooth muscle actin (α-SMA) expression. In the mouse model of DMM-induced arthritis, injection of the miR-27b-3p inhibitor decreased α-SMA but did not change COL1A1 expression levels or synovitis scores. Transfection with the miR-27b-3p mimic in human OA FLS induced profibrotic responses, including increased migration and expression of key extracellular matrix (ECM) genes, but transfection with the miR-27b-3p inhibitor had the opposite effects. RNA sequencing identified a PPARG/ADAMTS8 signaling axis regulated by miR-27b-3p in OA FLS. Human OA FLS transfected with miR-27b-3p mimic and then treated with the PPARG agonist rosiglitazone or with ADAMTS8 small interfering RNA exhibited altered expression of select ECM genes., Conclusion: Our findings demonstrate that miR-27b-3p has a key role in ECM regulation associated with synovial fibrosis during OA., (© 2022 The Authors. Arthritis & Rheumatology published by Wiley Periodicals LLC on behalf of American College of Rheumatology.)

Rationale: ICU discharge delay occurs when a patient is considered ready to be discharged but remains in the ICU. The effect of discharge delay on patient outcomes is uncertain. Objectives: To investigate the association between discharge delay and patient outcomes including hospital mortality, readmission to ICU, and length of hospital stay after ICU discharge. Methods: Data were accessed from the Australian and New Zealand Intensive Care Society Adult Patient Database between 2011 and 2019. Descriptive analyses and hierarchical logistic and Cox proportional hazards regression were used to examine association between discharge delay and adjusted outcomes. Patients were stratified and analyzed by categories of mortality risk at ICU admission. Measurements and Main Results: The study included 1,014,540 patients from 190 ICUs: 756,131 (75%) were discharged within 6 hours of being deemed ready, with 137,042 (13%) discharged in the next 6 hours; 17,656 (2%) were delayed 48-72 hours; 31,389 (3.1%) died in hospital; and 45,899 (4.5%) patients were readmitted to ICU. Risk-adjusted mortality declined with increasing discharge delay and was lowest at 48-72 hours (adjusted odds ratio, 0.87; 95% confidence interval, 0.79-0.94). The effect was seen in patients with predicted risk of death on admission to ICU of greater than 5% (adjusted odds ratio, 0.77; 95% confidence interval, 0.70-0.84). There was a progressive reduction in adjusted odds of readmission with increasing discharge delay. Conclusions: Increasing discharge delay in ICUs is associated with reduced likelihood of mortality and ICU readmission in high-risk patients. Consideration should be given to delay the discharge of patients with high risk of death on ICU admission.

The structural and physiological complexity of currently available liver organoids is limited, thereby reducing their relevance for drug studies, disease modelling, and regenerative therapy. In this study we combined mouse liver progenitor cells (LPCs) with mouse liver sinusoidal endothelial cells (LSECs) to generate hepatobiliary organoids with liver-specific vasculature. Organoids consisting of 5x10 3  cells were created from either LPCs, or a 1:1 combination of LPC/LSECs. LPC organoids demonstrated mild hepatobiliary differentiation in vitro with minimal morphological change; in contrast LPC/LSEC organoids developed clusters of polygonal hepatocyte-like cells and biliary ducts over a 7 day period. Hepatic (albumin, CPS1, CYP3A11) and biliary (GGT1) genes were significantly upregulated in LPC/LSEC organoids compared to LPC organoids over 7 days, as was albumin secretion. LPC/LSEC organoids also had significantly higher in vitro viability compared to LPC organoids. LPC and LPC/LSEC organoids were transplanted into vascularised chambers created in Fah -/- /Rag2 -/- /Il2rg -/- mice (50 LPC organoids, containing 2.5x10 5 LPCs, and 100 LPC/LSEC organoids, containing 2.5x10 5 LPCs). At 2 weeks, minimal LPCs survived in chambers with LPC organoids, but robust hepatobiliary ductular tissue was present in LPC/LSEC organoids. Morphometric analysis demonstrated a 115-fold increase in HNF4α+ cells in LPC/LSEC organoid chambers (17.26 ± 4.34 cells/mm 2 vs 0.15 ± 0.15 cells/mm 2 , p = 0.018), and 42-fold increase in Sox9+ cells in LPC/LSEC organoid chambers (28.29 ± 6.05 cells/mm 2 vs 0.67 ± 0.67 cells/mm 2 , p = 0.011). This study presents a novel method to develop vascularised hepatobiliary organoids, with both in vitro and in vivo results confirming that incorporating LSECs with LPCs into organoids significantly increases the differentiation of hepatobiliary tissue within organoids and their survival post-transplantation., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Alloying is well-known to improve the dehydrogenation selectivity of pure metals, but there remains considerable debate about the structural and electronic features of alloy surfaces that give rise to this behavior. To provide molecular-level insights into these effects, a series of Pd intermetallic alloy catalysts with Zn, Ga, In, Fe and Mn promoter elements was synthesized, and the structures were determined using in situ X-ray absorption spectroscopy (XAS) and synchrotron X-ray diffraction (XRD). The alloys all showed propane dehydrogenation turnover rates 5-8 times higher than monometallic Pd and selectivity to propylene of over 90%. Moreover, among the synthesized alloys, Pd 3 M alloy structures were less olefin selective than PdM alloys which were, in turn, almost 100% selective to propylene. This selectivity improvement was interpreted by changes in the DFT-calculated binding energies and activation energies for C-C and C-H bond activation, which are ultimately influenced by perturbation of the most stable adsorption site and changes to the d-band density of states. Furthermore, transition state analysis showed that the C-C bond breaking reactions require 4-fold ensemble sites, which are suggested to be required for non-selective, alkane hydrogenolysis reactions. These sites, which are not present on alloys with PdM structures, could be formed in the Pd 3 M alloy through substitution of one M atom with Pd, and this effect is suggested to be partially responsible for their slightly lower selectivity., Competing Interests: There are no conflicts to declare., (This journal is © The Royal Society of Chemistry.)

Poor vascularization remains a key limiting factor in translating advances in tissue engineering to clinical applications. Vascular pedicles (large arteries and veins) isolated in plastic chambers are known to sprout an extensive capillary network. This study examined the effect vascular pedicles and scaffold architecture have on vascularization and tissue integration of implanted silk scaffolds. Porous silk scaffolds with or without microchannels are manufactured to support implantation of a central vascular pedicle, without a chamber, implanted in the groin of Sprague Dawley rats, and assessed morphologically and morphometrically at 2 and 6 weeks. At both time points, blood vessels, connective tissue, and an inflammatory response infiltrate all scaffold pores externally, and centrally when a vascular pedicle is implanted. At week 2, vascular pedicles significantly increase the degree of scaffold tissue infiltration, and both the pedicle and the scaffold microchannels significantly increase vascular volume and vascular density. Interestingly, microchannels contribute to increased scaffold vascularity without affecting overall tissue infiltration, suggesting a direct effect of biomaterial architecture on vascularization. The inclusion of pedicles and microchannels are simple and effective proangiogenic techniques for engineering thick tissue constructs as both increase the speed of construct vascularization in the early weeks post in vivo implantation., (© 2019 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Tissue flaps are used to cover large/poorly healing wounds, but involve complex surgery and donor site morbidity. In this study a tissue flap is assembled using the mammalian body as a bioreactor to functionally connect an artery and vein to a human capillary network assembled from induced pluripotent stem cell-derived endothelial cells (hiPSC ECs). In vitro: Porous NovoSorb™ scaffolds (3 mm × 1.35 mm) were seeded with 200,000 hiPSC ECs ± 100,000 human vascular smooth muscle cells (hvSMC), and cultured for 1-3 days, with capillaries formed by 24 h which were CD31 + , VE-Cadherin + , EphB4 + , VEGFR2 + and Ki67 + , whilst hvSMCs (calponin + ) attached abluminally. In vivo: In SCID mice, bi-lateral epigastric vascular pedicles were isolated in a silicone chamber for a 3 week 'delay period' for pedicle capillary sprouting, then reopened, and two hiPSC EC ± hvSMCs seeded scaffolds transplanted over the pedicle. The chamber was either resealed (Group 1), or removed and surrounding tissue secured around the pedicle + scaffolds (Group 2), for 1 or 2 weeks. Human capillaries survived in vivo and were CD31 + , VE-Cadherin + and VEGFR2 + . Human vSMCs remained attached, and host mesenchymal cells also attached abluminally. Systemically injected FITC-dextran present in human capillary lumens indicated inosculation to host capillaries. Human iPSC EC capillary morphometric parameters at one week in vivo were equal to or higher than the same parameters measured in human abdominal skin. This 'proof of concept' study has demonstrated that bio-engineering an autologous human tissue flap based on hiPSC EC could minimize the use of donor flaps and has potential applications for complex wound coverage. STATEMENT OF SIGNIFICANCE: Tissue flaps, used for surgical reconstruction of wounds, require complex surgery, often associated with morbidity. Bio-engineering a simpler alternative, we assembled a human induced pluripotent stem cell derived endothelial cell (hiPSC ECs) capillary network in a porous scaffold in vitro, which when transplanted over a mouse vascular pedicle in vivo formed a functional tissue flap with mouse blood flow in the human capillaries. Therefore it is feasible to form an autologous tissue flap derived from a hiPSC EC capillary network assembled in vitro, and functionally connect to a vascular pedicle in vivo that could be utilized in complex wound repair for chronic or acute wounds., (Copyright © 2019 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.)

Type 1 diabetes (T1D) is characterized by the loss of insulin-producing β-cells in the pancreas. T1D can be treated using cadaveric islet transplantation, but this therapy is severely limited by a lack of pancreas donors. To develop an alternative cell source for transplantation therapy, we carried out the epigenetic characterization in nine different adult mouse tissues and identified visceral adipose-derived progenitors as a candidate cell population. Chromatin conformation, assessed using chromatin immunoprecipitation (ChIP) sequencing and validated by ChIP-polymerase chain reaction (PCR) at key endocrine pancreatic gene promoters, revealed similarities between visceral fat and endocrine pancreas. Multiple techniques involving quantitative PCR, in-situ PCR, confocal microscopy, and flow cytometry confirmed the presence of measurable (2-1000-fold over detectable limits) pancreatic gene transcripts and mesenchymal progenitor cell markers (CD73, CD90 and CD105; >98%) in visceral adipose tissue-derived mesenchymal cells (AMCs). The differentiation potential of AMCs was explored in transgenic reporter mice expressing green fluorescent protein (GFP) under the regulation of the Pdx1 (pancreatic and duodenal homeobox-1) gene promoter. GFP expression was measured as an index of Pdx1 promoter activity to optimize culture conditions for endocrine pancreatic differentiation. Differentiated AMCs demonstrated their capacity to induce pancreatic endocrine genes as evidenced by increased GFP expression and validated using TaqMan real-time PCR (at least 2-200-fold relative to undifferentiated AMCs). Human AMCs differentiated using optimized protocols continued to produce insulin following transplantation in NOD/SCID mice. Our studies provide a systematic analysis of potential islet progenitor populations using genome-wide profiling studies and characterize visceral adipose-derived cells for replacement therapy in diabetes.

The success of tissue engineering hinges on the rapid and sufficient vascularization of the neotissue. For efficient vascular network formation within three-dimensional (3D) constructs, biomaterial scaffolds that can support survival of endothelial cells as well as formation and maturation of a capillary network in vivo are highly sought after. Here, we outline a method to biofabricate 3D porous collagen scaffolds that can support extrinsic and intrinsic vascularization using two different in vivo animal models-the mouse subcutaneous implant model (extrinsic vascularization, capillary growth within the scaffold originating from host tissues outside the scaffold) and the rat tissue engineering chamber model (intrinsic vascularization, capillary growth within the scaffold derived from a centrally positioned vascular pedicle). These in vivo vascular tissue engineering approaches hold a great promise for the generation of clinically viable vascularized constructs. Moreover, the 3D collagen scaffolds can also be employed for 3D cell culture and for in vivo delivery of growth factors and cells.

Vascularisation is key to developing large transplantable tissue constructs capable of providing therapeutic benefits. The vascularised tissue engineering chamber originates from surgical concepts in tissue prefabrication and microsurgery. It serves as an in vivo bioreactor in the form of a closed, protected space surgically created and embedded within the body by fitting a noncollapsible chamber around major blood vessels. This creates a highly angiogenic environment which facilitates the engraftment and survival of transplanted cells and tissue constructs. This article outlines the chamber concept and explores its application in the context of recent advances in biomedical engineering, and how this can play a role in the future of cell therapies and regenerative medicine., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Background: The liver sinusoidal capillaries play a pivotal role in liver regeneration, suggesting they may be beneficial in liver bioengineering. This study isolated mouse liver sinusoidal endothelial cells (LSECs) and determined their ability to form capillary networks in vitro and in vivo for liver tissue engineering purposes., Methods and Results: In vitro LSECs were isolated from adult C57BL/6 mouse livers. Immunofluorescence labelling indicated they were LYVE-1 + /CD32b + /FactorVIII + /CD31 - . Scanning electron microscopy of LSECs revealed the presence of characteristic sieve plates at 2 days. LSECs formed tubes and sprouts in the tubulogenesis assay, similar to human microvascular endothelial cells (HMEC); and formed capillaries with lumens when implanted in a porous collagen scaffold in vitro. LSECs were able to form spheroids, and in the spheroid gel sandwich assay produced significantly increased numbers (p = 0.0011) of capillary-like sprouts at 24 h compared to HMEC spheroids. Supernatant from LSEC spheroids demonstrated significantly greater levels of vascular endothelial growth factor-A and C (VEGF-A, VEGF-C) and hepatocyte growth factor (HGF) compared to LSEC monolayers (p = 0.0167; p = 0.0017; and p < 0.0001, respectively), at 2 days, which was maintained to 4 days for HGF (p = 0.0017) and VEGF-A (p = 0.0051). In vivo isolated mouse LSECs were prepared as single cell suspensions of 500,000 cells, or as spheroids of 5000 cells (100 spheroids) and implanted in SCID mouse bilateral vascularized tissue engineering chambers for 2 weeks. Immunohistochemistry identified implanted LSECs forming LYVE-1 + /CD31 - vessels. In LSEC implanted constructs, overall lymphatic vessel growth was increased (not significantly), whilst host-derived CD31 + blood vessel growth increased significantly (p = 0.0127) compared to non-implanted controls. LSEC labelled with the fluorescent tag DiI prior to implantation formed capillaries in vivo and maintained LYVE-1 and CD32b markers to 2 weeks., Conclusion: Isolated mouse LSECs express a panel of vascular-related cell markers and demonstrate substantial vascular capillary-forming ability in vitro and in vivo. Their production of liver growth factors VEGF-A, VEGF-C and HGF enable these cells to exert a growth stimulus post-transplantation on the in vivo host-derived capillary bed, reinforcing their pro-regenerative capabilities for liver tissue engineering studies.

Nonvascularized fat grafting is a valuable technique for soft tissue reconstruction but poor survival of fat in the host environment remains a problem. A process known as cell-assisted transfer is used to enhance fat graft retention by adding stromal vascular fraction, an adipose-derived stem cell (ASC) rich content to lipoaspirate. We have recently shown that the use of melatonin, a reactive oxygen species scavenger, protects human ASCs from hydrogen peroxide-induced oxidative stress and cell death in vitro but its role as a pharmacological adjunct in clinical fat grafting has not been studied. Herein, the effect of melatonin was examined on human ASCs in vitro using survival and functional assays including the MTT assay, CellTox Green assay, monolayer scratch assay as well as a human cytokine chemoluminescence, and tumour necrosis factor-α assay. Further, the effect of melatonin-treated fat grafts was tested in vivo with a murine model. Haematoxylin and eosin staining, perilipin and CD31 immunostaining were performed with morphometric analysis of adipose tissue. The results demonstrate that, in vitro, the addition of melatonin to ASCs significantly improved their cell-viability, promoted cell migration and preserved membrane integrity as compared to controls. In addition, it induced a potent anti-inflammatory response by downregulating acute inflammatory cytokines particularly tumour necrosis factor-α. For the first time, it is demonstrated in vivo that melatonin enhances fat graft volume retention by reducing inflammation and increasing the percentage of adipose volume within fat grafts with comparable volumes to that of cell-assisted lipotransfer. Based on these novel findings, melatonin may be a useful pharmacological adjunct in clinical fat grafting., (Copyright © 2017 John Wiley & Sons, Ltd.)

Vascularization is a major hurdle for growing three-dimensional tissue engineered constructs. This study investigated the mechanisms involved in hypoxic preconditioning of primary rat myoblasts in vitro and their influence on local angiogenesis postimplantation. Primary rat myoblast cultures were exposed to 90 min hypoxia at <1% oxygen followed by normoxia for 24 h. Real time (RT) polymerase chain reaction evaluation indicated that 90 min hypoxia resulted in significant downregulation of miR-1 and miR-206 (p < 0.05) and angiopoietin-1 (p < 0.05) with upregulation of vascular endothelial growth factor-A (VEGF-A; p < 0.05). The miR-1 and angiopoietin-1 responses remained significantly downregulated after a 24 h rest phase. In addition, direct inhibition of miR-206 in L6 myoblasts caused a significant increase in VEGF-A expression (p < 0.05), further establishing that changes in VEGF-A expression are influenced by miR-206. Of the myogenic genes examined, MyoD was significantly upregulated, only after 24 h rest (p < 0.05). Preconditioned or control myoblasts were implanted with Matrigel™ into isolated bilateral tissue engineering chambers incorporating a flow-through epigastric vascular pedicle in severe combined immunodeficiency mice and the chamber tissue harvested 14 days later. Chambers implanted with preconditioned myoblasts had a significantly increased percentage volume of blood vessels (p = 0.0325) compared with chambers implanted with control myoblasts. Hypoxic preconditioned myoblasts promote vascularization of constructs via VEGF upregulation and downregulation of angiopoietin-1, miR-1 and miR-206. The relatively simple strategy of hypoxic preconditioning of implanted cells - including non-stem cell types - has broad, future applications in tissue engineering of skeletal muscle and other tissues, as a technique to significantly increase implant site angiogenesis., (Copyright © 2017 John Wiley & Sons, Ltd.)

Background: Cell-assisted lipotransfer has been promisingly applied to restore soft-tissue defects in plastic surgery; however, the harvesting of stromal vascular fraction increases morbidity and poses potential safety hazards. The authors investigated whether adding indomethacin, an antiinflammatory proadipogenic drug, to the fat graft at the time of transplantation would enhance the final graft volume compared with cell-assisted lipotransfer., Methods: In vitro, human adipose-derived stem cells were cultured in conditioned growth media supplemented with various doses of indomethacin to investigate adipogenesis and the expression of the adipogenic genes. In vivo, lipoaspirate mixed with stromal vascular fractions or indomethacin was injected into the dorsum of mice. Tissues were harvested at weeks 2, 4, and 12 to evaluate histologic changes., Results: In vitro, polymerase chain reaction analysis revealed that increased up-regulation of adipogenic genes and activation of the peroxisome proliferator-activated receptor-γ pathway. In vivo, the percentage volume of adipocytes in the indomethacin-assisted groups was higher than that in the lipoaspirate-alone (control) group at 12 weeks (p = 0.016), and was equivalent to the volume in the cell-assisted groups (p = 1.000). Indomethacin improved adipose volumes but had no effect on vascularity. A larger number of small adipocytes appeared in the treatment samples than in the controls at 2 weeks (p = 0.044) and 4 weeks (p = 0.021)., Conclusions: Pretreating lipoaspirate with indomethacin enhances the final volume retention of engrafted fat. This result is explained in part by increased adipogenesis and possibly by the inhibition of inflammatory responses.

'Off-the-shelf' tissue-engineered skin alternatives for epidermal and dermal skin layers are available; however, no such alternative for the subdermal fat layer exists. Without this well-vascularized layer, skin graft take is variable and grafts may have reduced mobility, contracture and contour defects. In this study a novel adipose-derived acellular matrix (Adipogel) was investigated for its properties to generate subdermal fat in a rat model. In a dorsal thoracic site, a 1 × 1 cm Adipogel implant was inserted within a subdermal fat layer defect. In a dorsal lumbar site, an Adipogel implant was inserted in a subfascial pocket. Contralateral control defects remained empty. At 8 weeks wound/implant sites were evaluated histologically, immunohistochemically and morphometrically. Identifiable thoracic Adipogel implants lost volume in vivo over 8 weeks. Neovascularization and adipogenesis were evident within implants and adipocyte percentage volume was 33.07 ± 6.55% (mean ± SEM). A comparison of entire cross-sections of thoracic wounds demonstrated a significant increase in total wound fat in Adipogel-implanted wounds (37.19 ± 4.48%, mean ± SEM) compared to control (16.53 ± 4.60%; p = 0.0092), indicating that some Adipogel had been completely converted to normal fat. At the lumbar site, Adipogel also lost volume, appearing flattened, although fat generation and angiogenesis occurred. At both sites macrophage infiltration was mild, whilst many infiltrating cells were PDGFRβ-positive mesenchymal cells. Adipogel is adipogenic and angiogenic and is a promising candidate for subcutaneous fat regeneration; it has the potential to be a valuable adjunct to wound-healing therapy and reconstructive surgery practice. Copyright © 2015 John Wiley & Sons, Ltd., (Copyright © 2015 John Wiley & Sons, Ltd.)

In reconstructive surgery, there is a clinical need for an alternative to the current methods of autologous reconstruction which are complex, costly and trade one defect for another. Tissue engineering holds the promise to address this increasing demand. However, most tissue engineering strategies fail to generate stable and functional tissue substitutes because of poor vascularization. This paper focuses on an in vivo tissue engineering chamber model of intrinsic vascularization where a perfused artery and a vein either as an arteriovenous loop or a flow-through pedicle configuration is directed inside a protected hollow chamber. In this chamber-based system angiogenic sprouting occurs from the arteriovenous vessels and this system attracts ischemic and inflammatory driven endogenous cell migration which gradually fills the chamber space with fibro-vascular tissue. Exogenous cell/matrix implantation at the time of chamber construction enhances cell survival and determines specificity of the engineered tissues which develop. Our studies have shown that this chamber model can successfully generate different tissues such as fat, cardiac muscle, liver and others. However, modifications and refinements are required to ensure target tissue formation is consistent and reproducible. This article describes a standardized protocol for the fabrication of two different vascularized tissue engineering chamber models in vivo.

A transient ischemic attack (TIA) significantly increases a person's risk of ischemic stroke. However, little is known about the experiences of these people or what influences their decision to access care. This article explores the stories of three people who experienced a TIA. All participants were interviewed after receiving a diagnosis of TIA by a specialist neurologist. By utilizing a framework of narrative inquiry, several storylines were revealed across temporal, social, and geographical landscapes. The main storylines include: bodily disruption, time stasis, altered temporal perceptions, the roles of others, and help-seeking behavior. The primary author also reflects upon his own origin and explores how this inquiry has influenced his perceptions, career, and nursing practice. This inquiry may assist healthcare workers to gain insight into the experiences of people with TIA and suggests how involvement of significant others is beneficial when educating people at risk of TIA., (© 2015 Wiley Publishing Asia Pty Ltd.)

Here, we describe a porous 3-dimensional collagen scaffold material that supports capillary formation in vitro, and promotes vascularization when implanted in vivo. Collagen scaffolds were synthesized from type I bovine collagen and have a uniform pore size of 80 μm. In vitro, scaffolds seeded with primary human microvascular endothelial cells suspended in human fibrin gel formed CD31 positive capillary-like structures with clear lumens. In vivo, after subcutaneous implantation in mice, cell-free collagen scaffolds were vascularized by host neovessels, whilst a gradual degradation of the scaffold material occurred over 8 weeks. Collagen scaffolds, impregnated with human fibrinogen gel, were implanted subcutaneously inside a chamber enclosing the femoral vessels in rats. Angiogenic sprouts from the femoral vessels invaded throughout the scaffolds and these degraded completely after 4 weeks. Vascular volume of the resulting constructs was greater than the vascular volume of constructs from chambers implanted with fibrinogen gel alone (42.7±5.0 μL in collagen scaffold vs 22.5±2.3 μL in fibrinogen gel alone; p<0.05, n = 7). In the same model, collagen scaffolds seeded with human adipose-derived stem cells (ASCs) produced greater increases in vascular volume than did cell-free collagen scaffolds (42.9±4.0 μL in collagen scaffold with human ASCs vs 25.7±1.9 μL in collagen scaffold alone; p<0.05, n = 4). In summary, these collagen scaffolds are biocompatible and could be used to grow more robust vascularized tissue engineering grafts with improved the survival of implanted cells. Such scaffolds could also be used as an assay model for studies on angiogenesis, 3-dimensional cell culture, and delivery of growth factors and cells in vivo.

Tissue engineering is a complex and dynamic process that requires varied biomolecular cues to promote optimal tissue growth. Consequently, the development of delivery systems capable of sequestering more than one biomolecule with controllable release profiles is a key step in the advancement of this field. This study develops multilayered polyelectrolyte films incorporating alpha-melanocyte stimulating hormone (α-MSH), an anti-inflammatory molecule, and basic fibroblast growth factor (bFGF). The layers were successfully formed on macroporous poly lactic-co-glycolic acid microspheres produced using a combined inkjet and thermally induced phase separation technique. Release profiles could be varied by altering layer properties including the number of layers and concentrations of layering molecules. α-MSH and bFGF were released in a sustained manner and the bioactivity of α-MSH was shown to be preserved using an activated macrophage cell assay in vitro. The system performance was also tested in vivo subcutaneously in rats. The multilayered microspheres reduced the inflammatory response induced by a carrageenan stimulus 6 weeks after implantation compared to the non-layered microspheres without the anti-inflammatory and growth factors, demonstrating the potential of such multilayered constructs for the controlled delivery of bioactive molecules., (© 2014 Wiley Periodicals, Inc.)

The extracellular matrix (ECM) Matrigel™ has frequently and successfully been used to generate new adipose tissue experimentally, but is unsuitable for human application. This study sought to compare the adipogenic potential of a number of alternative, biologically derived or synthetic ECMs with potential for human application, with and without growth factors and a small fat autograft. Eight groups, with six severe combined immunodeficient (SCID) mice per group, were created with bilateral chambers (silicone tubes) implanted around the epigastric vascular pedicle, with one chamber/animal containing a 5mg fat autograft. Two animal groups were created for each of four ECMs (Matrigel™, Myogel, Cymetra® and PuraMatrix™) which filled the bilateral chambers. One group/ECM had no growth factors added to chambers whilst the other group had growth factors (GFs) (vascular endothelial growth factor-A (VEGF-A) plus fibroblast growth factor-2 (FGF-2) plus platelet-derived growth factor-BB (PDGF-BB)) added to both chambers. At 6weeks, chamber tissue was morphometrically assessed for percent and absolute adipose tissue volume. Overall, the triple GF regime significantly increased percent(∗) and absolute(#) adipose tissue volume (p<0.0005(∗#)) compared to chambers without triple GF treatment. The fat autograft also significantly increased percent (p<0.0005) and absolute (p<0.011) adipose tissue volume. Cymetra® (human collagen) constructs yielded the largest total tissue and absolute adipose tissue volume. We found that the pro-angiogenic FGF-2, VEGF-A and PDGF-BB combination in ECMs of synthetic and biological origin produced an overall significantly increased adipose tissue volume at 6weeks and may have clinical application, particularly with Cymetra., (Copyright © 2013 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.)

Macrophages predominate among the cells that directly interact with biomaterials and are key orchestrators of host-biomaterial interactions. However, the macrophage response to synthetic scaffolds in particular has not been well studied. The aim of this study was therefore to characterise the macrophage response to several synthetic scaffolds in the rat using immunohistological techniques for a panel of markers of macrophage subclass or activation, including ED1 (CD68), ED2 (CD163), CD80, mannose receptor and inducible nitric oxide synthase (iNOS). Materials were implanted subcutaneously and collected after 6-8 weeks during the chronic phase of the host response. Unmodified polycaprolactone scaffolds uniquely demonstrated a total lack of both macrophage adherence to surfaces and a wider foreign body response compared to scaffolds composed of poly(lactic-co-glycolic acid) (PLGA) and polyurethanes (PURs), with those macrophages present having a clear M2 (MR+, CD80-, iNOS-) phenotype. PLGA scaffolds displayed an M1-dominant (CD80+, iNOS+, MR-) response with substantial foreign body giant cell (FBGC) formation, whilst PUR scaffold FBGCs had a more mixed M1 (CD80+, iNOS+) and M2 (MR+) phenotype. The study also identified that the use of the ED1 antibody in the rat as a pan-macrophage marker is problematic as there is a separate and substantial ED2-positive macrophage population that it does not label, both in response to biomaterials and in normal tissues. The biomaterial-dependent nature of activation for both macrophages and FBGCs was confirmed, and nuanced M1/M2 phenotypes were described., (© 2014 S. Karger AG, Basel.)

Human lymphatic vascular malformations (LMs), also known as cystic hygromas or lymphangioma, consist of multiple lymphatic endothelial cell-lined lymph-containing cysts. No animal model of this disease exists. To develop a mouse xenograft model of human LM, CD34(Neg)CD31(Pos) LM lymphatic endothelial cells (LM-LEC) were isolated from surgical specimens and compared to foreskin CD34(Neg)CD31(Pos) lymphatic endothelial cells (LECs). Cells were implanted into a mouse tissue engineering model for 1, 2 and 4 weeks. In vitro LM-LECs showed increased proliferation and survival under starvation conditions (P < 0.0005 at 48 h, two-way ANOVA), increased migration (P < 0.001, two-way ANOVA) and formed fewer (P = 0.029, independent samples t test), shorter tubes (P = 0.029, independent samples t test) than foreskin LECs. In vivo LM-LECs implanted into a Matrigel™-containing mouse chamber model assembled to develop vessels with dilated cystic lumens lined with flat endothelium, morphology similar to that of clinical LMs. Human foreskin LECs failed to survive implantation. In LM-LEC implanted chambers the percent volume of podoplanin(Pos) vessels was 1.18 ± 2.24 % at 1 week, 6.34 ± 2.68 % at 2 weeks and increasing to 7.67 ± 3.60 % at 4 weeks. In conclusion, the significantly increased proliferation, migration, resistance to apoptosis and decreased tubulogenesis of LM-LECs observed in vitro is likely to account for their survival and assembly into stable LM-like structures when implanted into a mouse vascularised chamber model. This in vivo xenograft model will provide the basis of future studies of LM biology and testing of potential pharmacological interventions for patients with lymphatic malformations.

Liver tissue engineering is hampered by poor implanted cell survival due to inadequate vascularization and cell-cell/cell-matrix interactions. Here, we use liver progenitor cell (LPC) spheroids to enhance cell-cell/cell-matrix interactions, with implantation into an angiogenic in vivo mouse chamber. Spheroids were generated in vitro in methylcellulose medium. Day 2 spheroids were optimal for implantation (22,407 +/-645 cells/spheroid), demonstrating maximal proliferation (Ki67 immunolabeling) and minimal apoptosis (caspase-3 immunolabelling). In vivo chambers established bilaterally on epigastric vessels of immunodeficient mice were implanted with equivalent numbers of LPCs as a cell suspension (200,000 cells), or spheroids (9 spheroids). At day 14, a trend of increased LPC survival was observed in spheroid-implanted chambers [pan-cytokeratin (panCK+) cells, p = 0.38, 2.4 fold increase)], with significantly increased differentiation [cytokeratin 18 (CK18+) cells, p < 0.002, 5.1 fold increase)] compared to cell suspension-implanted chambers. At day 45, both measures were significantly increased in spheroid-implanted chambers (panCK, p < 0.006, 16 fold increase) (CK18, p < 0.019, 6 fold increase). Hepatic acini/plates of CK18 + cells expressed hepatocyte nuclear factor 4-α and β-catenin, indicating ongoing hepatic differentiation. Spheroid cell-delivery significantly increased LPC survival and differentiation compared to conventional cell suspensions. This LPC spheroid/vascularized chamber model has clinical potential to generate three-dimensional vascularized liver tissue for liver replacement., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

The effects of in vitro preconditioning protocols on the ultimate survival of myoblasts implanted in an in vivo tissue engineering chamber were examined. In vitro testing: L6 myoblasts were preconditioned by heat (42 °C; 1.5 h); hypoxia (<8% O(2); 1.5 h); or nitric oxide donors: S-nitroso-N-acetylpenicillamine (SNAP, 200 μM, 1.5 h) or 1-[N-(2-aminoethyl)-N-(2-aminoethyl)amino]-diazen-1-ium-1,2-diolate (DETA-NONOate, 500 μM, 7 h). Following a rest phase preconditioned cells were exposed to 24 h hypoxia, and demonstrated minimal overall cell loss, whilst controls (not preconditioned, but exposed to 24 h hypoxia) demonstrated a 44% cell loss. Phosphoimmunoblot analysis of pro-survival signaling pathways revealed significant activation of serine threonine kinase Akt with DETA-NONOate (p < 0.01) and heat preconditioning (p < 0.05). DETA-NONOate also activated ERK 1/2 signaling (p < 0.05). In vivo implantation: 100,000 preconditioned (heat, hypoxia, or DETA-NONOate) myoblasts were implanted in SCID mouse tissue engineering chambers. 100,000 (not preconditioned) myoblasts were implanted in control chambers. At 3 weeks, morphometric assessment of surviving myoblasts indicated myoblast percent volume (p = 0.012) and myoblasts/mm(2) (p = 0.0005) overall significantly increased in preconditioned myoblast chambers compared to control, with DETA-NONOate-preconditioned myoblasts demonstrating the greatest increase in survival (p = 0.007 and p = 0.001 respectively). DETA-NONOate therefore has potential therapeutic benefits to significantly improve survival of transplanted cells., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Huntington's disease (HD) is a fatal, autosomal dominant neurodegenerative disorder caused by an expanded trinucleotide (CAG) repeat in exon 1 of the huntingtin gene (Htt). This expansion creates a toxic polyglutamine tract in the huntingtin protein (HTT). Currently, there is no treatment for either the progression or prevention of the disease. RNA interference (RNAi) technology has shown promise in transgenic mouse models of HD by reducing expression of mutant HTT and slowing disease progression. The advancement of RNAi therapies to human clinical trials is hampered by problems delivering RNAi to affected neurons in a robust and sustainable manner. Mesenchymal stem cells (MSC) have demonstrated a strong safety profile in both completed and numerous ongoing clinical trials. MSC exhibit a number of innate therapeutic effects, such as immune system modulation, homing to injury, and cytokine release into damaged microenvironments. The ability of MSC to transfer larger molecules and even organelles suggested their potential usefulness as delivery vehicles for therapeutic RNA inhibition. In a series of model systems we have found evidence that MSC can transfer RNAi targeting both reporter genes and mutant huntingtin in neural cell lines. MSC expressing shRNA antisense to GFP were found to decrease expression of GFP in SH-SY5Y cells after co-culture when assayed by flow cytometry. Additionally MSC expressing shRNA antisense to HTT were able to decrease levels of mutant HTT expressed in both U87 and SH-SY5Y target cells when assayed by Western blot and densitometry. These results are encouraging for expanding the therapeutic abilities of both RNAi and MSC for future treatments of Huntington's disease., (Copyright © 2011 Elsevier Inc. All rights reserved.)

There is much interest in the use of mesenchymal stem cells/marrow stromal cells (MSC) to treat neurodegenerative disorders, in particular those that are fatal and difficult to treat, such as Huntington's disease. MSC present a promising tool for cell therapy and are currently being tested in FDA-approved phase I-III clinical trials for many disorders. In preclinical studies of neurodegenerative disorders, MSC have demonstrated efficacy, when used as delivery vehicles for neural growth factors. A number of investigators have examined the potential benefits of innate MSC-secreted trophic support and augmented growth factors to support injured neurons. These include overexpression of brain-derived neurotrophic factor and glial-derived neurotrophic factor, using genetically engineered MSC as a vehicle to deliver the cytokines directly into the microenvironment. Proposed regenerative approaches to neurological diseases using MSC include cell therapies in which cells are delivered via intracerebral or intrathecal injection. Upon transplantation, MSC in the brain promote endogenous neuronal growth, encourage synaptic connection from damaged neurons, decrease apoptosis, reduce levels of free radicals, and regulate inflammation. These abilities are primarily modulated through paracrine actions. Clinical trials for MSC injection into the central nervous system to treat amyotrophic lateral sclerosis, traumatic brain injury, and stroke are currently ongoing. The current data in support of applying MSC-based cellular therapies to the treatment of Huntington's disease is discussed.

MicroRNAs (miRNAs) are a class of short, single-stranded non-protein coding gene products which can regulate the gene expression through post-transcriptional inhibition of messenger RNA (mRNA) translation. They are known to be involved in many essential biological processes including development, insulin secretion, and adipocyte differentiation. miRNAs are involved in complex metabolic processes, such as energy and lipid metabolism, which have been studied in the context of diabetes and obesity. Obesity, hyperlipidemia (elevated levels of blood lipids), and insulin resistance are strongly associated with the onset of type 2 diabetes. These conditions are also associated with aberrant expression of multiple essential miRNAs in pancreatic islets of Langerhans and peripheral tissues, including adipose tissue. A thorough understanding of the physiological role these miRNAs play in these tissues, and changes to their expression under pathological conditions, will allow researchers to develop new therapeutics with the potential to correct the aberrant expression of miRNAs in type 2 diabetes and obesity.

Background: Inducible nitric oxide synthase (nitric oxide synthase 2, NOS 2) inhibition significantly suppresses chronically ischaemic skin flap survival, possibly because of reduced angiogenesis., Objectives: To investigate the effect of genetic NOS 2 inhibition on cutaneous wound angiogenesis in two in vivo murine models. The impact of NOS 2 manipulation on vascular endothelial growth factor (VEGF)-A stimulated and fibroblast growth factor (FGF)-2 stimulated angiogenesis was also investigated in the Matrigel(®) plug assay., Methods: (i) Matrigel plugs/incisional wounds: two groups of NOS 2-/- mice and two groups of wild-type (WT) mice had bilateral Matrigel plugs containing 500 ng mL(-1) VEGF-A or 1000 ng mL(-1) FGF-2 injected subcutaneously in the abdomen. A 2·5 cm long dorsal incisional skin wound was created and sutured closed in the same animals. Wounds and plugs were explored at 7 or 12 days. (ii) Excisional wounds: dorsal 0·5 × 1·0 cm excisional skin wounds were created in four groups (two NOS 2-/- and two WT) and explored at 7 or 14 days. Wounds and Matrigel plugs were examined histologically and morphometrically for determination of percentage vascular volume (PVV)., Results: The PVV in NOS 2-/- incisional wounds and excisional wounds was significantly less than in WT wounds (P = 0·05 and P < 0·001, respectively). The PVV was significantly less in VEGF-A stimulated Matrigel plugs compared with FGF-2 stimulated plugs in NOS 2-/- mice (P < 0·01), but not in WT mice., Conclusions: NOS 2 is significantly involved in angiogenic signalling in healing skin wounds, particularly within the first 7 days. However, Matrigel plug vascularization suggests that the role of NOS 2 in angiogenesis is related to VEGF-A but not FGF-2 stimulated angiogenesis., (© 2011 The Authors. BJD © 2011 British Association of Dermatologists.)

The physiological processes involved in tissue development and regeneration also include the parallel formation of blood and lymphatic vessel circulations which involves their growth, maturation and remodelling. Both vascular systems are also frequently involved in the development and progression of pathological conditions in tissues and organs. The blood vascular system circulates oxygenated blood and nutrients at appropriate physiological levels for tissue survival, and efficiently removes all waste products including carbon dioxide. This continuous network consists of the heart, aorta, arteries, arterioles, capillaries, post-capillary venules, venules, veins and vena cava. This system exists in an interstitial environment together with the lymphatic vascular system, including lymph nodes, which aids maintenance of body fluid balance and immune surveillance. To understand the process of vascular development, vascular network stability, remodelling and/or regression in any research model under any experimental conditions, it is necessary to clearly and unequivocally identify and quantify all elements of the vascular network. By utilising stereological methods in combination with cellular markers for different vascular cell components, it is possible to estimate parameters such as surface density and surface area of blood vessels, length density and length of blood vessels as well as absolute vascular volume. This review examines the current strategies used to visualise blood vessels and lymphatic vessels in two- and three-dimensions and the basic principles of vascular stereology used to quantify vascular network parameters.

Objective: The U.S. military has traditionally had high rates of alcohol misuse and alcohol-related problems, necessitating effective treatment programs that minimize participant burden. Web-based interventions have shown promise as efficient treatment options for college students and adults but have not been widely evaluated in the military. This study evaluated the efficacy of two web-based alcohol interventions originally created for civilians and then adapted for U.S. military personnel., Method: Two web-based alcohol interventions, Alcohol Savvy and Drinker's Check-Up, were adapted for use among military populations. The interventions were evaluated using a convenience sample of 3,070 active-duty military personnel at eight installations. Following a baseline survey, participants were assigned to one of three treatment conditions: (a) Alcohol Savvy, (b) Drinker's Check-Up, or (c) control (no program participation). Follow-up surveys were completed by 1,072 participants 1 month following baseline and by 532 participants 6 months following baseline., Results: At 1-month follow-up, participants who completed the Drinker's Check-Up intervention had significant reductions in multiple measures of alcohol use relative to controls. Positive outcomes were found for average number of drinks consumed per occasion, frequent heavy episodic drinker status, and estimated peak blood alcohol concentration. These reductions in alcohol use at the 1-month follow-up were maintained at the 6-month follow-up. There were no statistically significant changes in alcohol use for participants who completed Alcohol Savvy., Conclusions: This study expands the literature on the effectiveness of web-based treatment for alcohol misuse. Findings indicate that web-based programs (Drinker's Check-Up in particular) can significantly decrease several indicators of alcohol use in U.S. military personnel.

Current cell-based treatment alternatives to organ transplantation for liver failure remain unsatisfactory. Hepatocytes have a strong tendency to dedifferentiate and apoptose when isolated and maintained in culture. In contrast, liver progenitor cells (LPCs) are robust, easy to culture and have been shown to replace damaged hepatocytes in liver disease. In this study we investigate whether isolated LPCs can survive and differentiate toward mature hepatocytes in vivo when implanted into a heterotopic mouse tissue engineering chamber model. Healthy Balb/c mice and those put on a choline-deficient ethionin-supplemented diet to induce chronic liver disease were implanted with a tissue engineering chamber based on the epigastric flow through pedicle model, containing either 1 × 10(6) LPCs suspended in Matrigel, or LPC-spheroids produced by preculture for 1 week in Matrigel. Four weeks after implantation the chamber contents were harvested. In all four groups, progenitor cells persisted in large numbers to 4 weeks and demonstrated evidence of considerable proliferation judged by Ki67-positive cells. Periodic acid Schiff staining demonstrated differentiation of some cells into mature hepatocytes. Constructs grown from LPC-spheroids demonstrated considerably greater LPC survival than those from LPCs that were grown as monolayers and implanted as dissociated cells. The combined use of LPC spheroids and the vascularized chamber model could be the basis for a viable alternative to current treatments for chronic liver failure.

Objectives: Dense angiogenic sprouting occurs from arteriovenous loops (AVLs) incorporating autologous vein grafts inserted into empty plastic chambers in vivo. The purpose of this study was to determine if angiogenesis from the AVL was limited by substituting an "off the shelf" cold-stored allograft vein instead of an autologous vein., Methods: Four Sprague Dawley rat groups (two AVL configurations × two chamber types) were established for both 2-week and 6-week harvest. Control AVLs were autologous femoral vein grafts harvested from the left femoral vein that were surgically inserted between the cut femoral artery and vein on the right side. Experimental "allograft" AVLs were rat femoral veins cold-stored (4°C, sterile) for 4 to 7 weeks and then microsurgically interposed between the right femoral artery and vein of an unrelated rat. The two AVL types were inserted in one of two plastic chamber types--smooth or perforated. At harvest, the AVL constructs were checked for patency, weighed, their volume determined, and histology undertaken. Morphometric assessment of percent and absolute volume of major tissue components (including blood vessels) at 6 weeks was completed., Results: There were no significant differences between autograft and allograft groups in construct weight, volume, or morphology at 2 or 6 weeks. No statistical differences occurred in the percent or absolute vascular volume of AVLs incorporating a cold-stored allograft vs autologous vein grafts at 6 weeks regardless of the chamber type. However, perforated chambers caused significant increases in construct weight (P = .015), volume (P = .006), and percent and absolute connective tissue volume at 6 weeks (P = .001) compared to smooth chamber constructs, regardless of the graft type., Conclusion: Cold-stored small-caliber allografts interposed in AVLs do not inhibit microcirculatory development and can be used in composite tissue engineering., (Copyright © 2011 Society for Vascular Surgery. Published by Mosby, Inc. All rights reserved.)

Recreating an environment that supports and promotes fundamental homeostatic mechanisms is a significant challenge in tissue engineering. Optimizing cell survival, proliferation, differentiation, apoptosis and angiogenesis, and providing suitable stromal support and signalling cues are keys to successfully generating clinically useful tissues. Interestingly, those components are often subverted in the cancer setting, where aberrant angiogenesis, cellular proliferation, cell signalling and resistance to apoptosis drive malignant growth. In contrast to tissue engineering, identifying and inhibiting those pathways is a major challenge in cancer research. The recent discovery of adult tissue-specific stem cells has had a major impact on both tissue engineering and cancer research. The unique properties of these cells and their role in tissue and organ repair and regeneration hold great potential for engineering tissue-specific constructs. The emerging body of evidence implicating stem cells and progenitor cells as the source of oncogenic transformation prompts caution when using these cells for tissue-engineering purposes. While tissue engineering and cancer research may be considered as opposed fields of research with regard to their proclaimed goals, the compelling overlap in fundamental pathways underlying these processes suggests that cross-disciplinary research will benefit both fields. In this review article, tissue engineering and cancer research are brought together and explored with regard to discoveries that may be of mutual benefit., (Copyright 2010 S. Karger AG, Basel.)

In in vivo tissue engineering, many implanted cells die because of hypoxic conditions immediately postimplantation. The aim of this study was to determine whether delayed myoblast implantation, at day 4 or 7, improves myoblast survival compared with implantation at day 0 in an in vivo arterio-venous loop (AB loop) chamber model. In adult inbred Sprague-Dawley rats, an AB loop was inserted into a plastic chamber (day 0). In Group I, day 0, two million DiI-labeled (neonatal inbred) myoblasts were implanted around the AB loop. In Groups II and III, day 0, the AB loop was created and inserted into a novel delayed cell seeding chamber, and 4 (Group II) or 7 days (Group III) later the delay chamber was seeded with 2 million DiI-labeled myoblasts. Constructs were harvested 7-day postmyoblast implantation, for morphometric determination of DiI/DAPI-positive myoblasts/mm(2), and percent vascular volume on Griffonia simplicifolia lectin (endothelial cell marker)-labeled tissue sections. Control (nonmyoblast seeded) and experimental (myoblast seeded) constructs demonstrated similar capillary and tissue growth patterns. DiI/DAPI-labeled myoblasts/mm(2) appeared in similar numbers in constructs implanted at days 0 and 4, but increased markedly in day-7 implanted constructs. The percent vascular volume increased significantly (p = 0.03) over time. A positive correlation existed between myoblast survival and construct vascularity (p = 0.017). In conclusion, delaying myoblast implantation to 7-day postconstruct assembly, when new capillary growth is well established, significantly correlates with increased myoblast survival and indicates that cell seeding in regenerative procedures should always occur into an established vascular bed.

Tissue engineering of vascularized constructs has great utility in reconstructive surgery. While we have been successful in generating vascularized granulation-like tissue and adipose tissue in an in vivo tissue engineering chamber, production of other differentiated tissues in a stable construct remains a challenge. One approach is to utilize potent differentiation factors, which can influence the base tissue. Endothelial precursor cells (EPCs) have the ability to both carry differentiation factors and home to developing vasculature. In this study, proof-of-principle experiments demonstrate that such cells can be recruited from the circulation into an in vivo tissue engineering chamber. CXC chemokine ligand 12 (CXCL12)/stromal cell-derived factor 1 was infused into the chamber through Alzet osmotic pumps and chamber cannulation between days 0 and 7, and facilitated recruitment of systemically inoculated exogenous human EPCs injected on day 6. CXCL12 infusion resulted in an eightfold increase in EPC recruitment, 2 (p = 0.03) and 7 days postinfusion (p = 0.008). Delivery of chemotactic/proliferation and/or differentiation factors and appropriately timed introduction of effective cells may allow us to better exploit the regenerative potential of the established chamber construct.

Summary Background: Autogenous fat injection is widely used for the correction of acquired and congenital soft tissue defects. However, the high absorption rate results in the need for over-correction of the defect and repeat procedures. We hypothesised that platelet-derived growth factor (PDGF), a potent mitogen and known stimulant for murine preadipocytes, would improve fat graft survival when concentrations were sustained with a gelatine microsphere delivery system., Methods: Abdominal fat was harvested from an otherwise healthy 43-year-old woman during a breast reconstruction. Prior to subdermal injection into severe combined immunodeficient (SCID) mice, the fat grafts were divided into 1-ml aliquots, mixed with microspheres bound to PDGF, free PDGF, or nothing depending on its experimental group, and weighed. The following experimental groups were thus created (minimum n=8 per group): (1) fat graft control, (2) fat graft with free PDGF, (3) fat graft with blank microspheres, and (4) fat graft with microspheres bound to PDGF. After 12 weeks, the fat xenografts were harvested for analysis of weight maintenance and histological and morphometric evaluation., Results: The addition of PDGF bound to gelatine microspheres was effective in improving xenograft weight maintenance (P=0.018) and preservation of adipose tissue architecture (P<0.0005) compared to controls at 3 months. The microspheres were completely absorbed at 12 weeks., Conclusions: Sustained, local delivery of PDGF via a gelatine microsphere delivery system resulted in improved weight maintenance of the xenografts with greater preservation of adipose tissue architecture at 3 months compared to controls.

Background: An arteriovenous loop (AVL) enclosed in a polycarbonate chamber in vivo, produces a fibrin exudate which acts as a provisional matrix for the development of a tissue engineered microcirculatory network., Objectives: By administering enoxaparin sodium - an inhibitor of fibrin polymerization, the significance of fibrin scaffold formation on AVL construct size (including the AVL, fibrin scaffold, and new tissue growth into the fibrin), growth, and vascularization were assessed and compared to controls., Methods: In Sprague Dawley rats, an AVL was created on femoral vessels and inserted into a polycarbonate chamber in the groin in 3 control groups (Series I) and 3 experimental groups (Series II). Two hours before surgery and 6 hours post-surgery, saline (Series I) or enoxaparin sodium (0.6 mg/kg, Series II) was administered intra-peritoneally. Thereafter, the rats were injected daily with saline (Series I) or enoxaparin sodium (1.5 mg/kg, Series II) until construct retrieval at 3, 10, or 21 days. The retrieved constructs underwent weight and volume measurements, and morphologic/morphometric analysis of new tissue components., Results: Enoxaparin sodium treatment resulted in the development of smaller AVL constructs at 3, 10, and 21 days. Construct weight and volume were significantly reduced at 10 days (control weight 0.337 +/- 0.016 g [Mean +/- SEM] vs treated 0.228 +/- 0.048, [P < .001]: control volume 0.317 +/- 0.015 mL vs treated 0.184 +/- 0.039 mL [P < .01]) and 21 days (control weight 0.306 +/- 0.053 g vs treated 0.198 +/- 0.043 g [P < .01]: control volume 0.285 +/- 0.047 mL vs treated 0.148 +/- 0.041 mL, [P < .01]). Angiogenesis was delayed in the enoxaparin sodium-treated constructs with the absolute vascular volume significantly decreased at 10 days (control vascular volume 0.029 +/- 0.03 mL vs treated 0.012 +/- 0.002 mL [P < .05])., Conclusion: In this in vivo tissue engineering model, endogenous, extra-vascularly deposited fibrin volume determines construct size and vascular growth in the first 3 weeks and is, therefore, critical to full construct development.

The ultimate survival of tissue-engineered constructs in vivo depends on the provision of an adequate blood supply to the engineered tissue and the capacity of the engineered microcirculation to connect with the existing recipient circulation. Techniques for the vascularization of tissue-engineered constructs can be broadly grouped into in vitro and in vivo approaches that rely on the presence of a pro-angiogenic microenvironment. Significant advances have been made in resolving the problem of microcirculatory network formation for large 3-dimensional constructs; however, issues concerning construct-host vessel connection, expansion of vascular volume accompanying growing tissue, and prevention of premature or excessive vascular regression remain to be resolved. This review provides an overview of current approaches to creating microcirculatory networks with respect to the cells involved, growth factors, growth factor delivery systems, and scaffold properties required to engineer a permanent microcirculatory network for tissue-engineered constructs. In addition, the review examines concerns related to vascular remodeling and regression reported in some tissue-engineering models.