Host Vessels Research Articles

Towards reconstruction of epithelialized cartilages from autologous adipose tissue-derived stem cells.

Deformities of the upper airways, including those of the nose and throat, are typically corrected by reconstructive surgery. The use of autologous somatic stem cells for repair of defects could improve quality and outcomes of such operations. The present study explored the ability of paediatric adipose-derived stem cells (pADSCs), a readily available source of autologous stem cells, to generate a cartilage construct with a functional epithelium. Paediatric ADSCs seeded on the biodegradable nanocomposite polymer, polyhedral oligomeric silsesquioxane poly(ϵ-caprolactone-urea) urethane (POSS-PCL), proliferated and differentiated towards mesenchymal lineages. The ADSCs infiltrated three-dimensional POSS-PCL nanoscaffold and chondroid matrix was observed throughout chondrogenically induced samples. In ovo chorioallantoic membrane-grafted ADSC-nanoscaffold composites were enwrapped by host vessels indicating good compatibility in an in vivo system. Furthermore, pADSCs could be induced to transdifferentiate towards barrier-forming epithelial-like cells. By combining differentiation protocols, it was possible to generate epithelial cell lined chondrogenic micromasses from the same pADSC line. This proof-of-concept study appears to be the first to demonstrate that individual pADSC lines can differentiate towards two different germ lines and be successfully co-cultured. This has important implications for bioengineering of paediatric airways and further confirms the plastic nature of ADSCs. Copyright © 2016 John Wiley & Sons, Ltd.

Vascularization of plastic calcium phosphate cement in vivo induced by in-situ-generated hollow channels

Despite calcium phosphate cement (CPC) is promising for bone repair therapy, slow biodegradation and insufficient vascularization in constructs negatively impacts its clinical application. A self-setting CPC composited with gelatin fiber is investigated to test the utility of this tissue engineering strategy to support rapid and extensive vascularization process. The interconnected hollow channels in CPC are formed after dissolution of gelatin fibers in vivo. The CPC-gelatin samples exhibit relatively decent/enhanced mechanical property, compared to the control. When implanted in vivo, the pre-established vascular networks in material anastomose with host vessels and accelerate vascular infiltration throughout the whole tissue construct. Different channel sizes induce different vascularization behaviors in vivo. Results indicate that the channel with the size of 250μm increases the expression of the representative angiogenic factors HIF1α, PLGF and migration factor CXCR4, which benefit the formation of small vessels. On the other hand, the channel with the size of 500μm enhances VEGF-A expression, which benefit the development of large vessels. Notably, the intersection area of channels has high invasive, sprouting and vasculogenesis potential under hypoxic condition, because more HIF1α-positive cells are observed there. Observation of the CD31-positive lumen in the border of scaffold indicates the ingrowth of blood vessels from its host into material through channel, benefited from gradually increased HIF1α expression. This kind of material was suggested to promote the effective application of bone regeneration through the combination of in situ self-setting, plasticity, angiogenesis, and osteoconductivity.

Effect of dual growth factor delivery using poly(lactic-co-glycolic acid) mesh on neovascularization in a mouse skin flap model

Skin flaps have the crucial issue that skin necrosis can occur due to tissue ischemia in the distal parts of flaps. To prevent tissue necrosis, skin flaps need to be supplied with sufficient blood perfusion through quick vascularization in the ischemic area. In this study, we fabricated poly(lactic-co-glycolic acid) (PLGA) meshes that had shaped random or alignments using electrospinning and modified the surface of the PLGA mesh, using 3,4-dihydroxy- L-phenylalanine (L-DOPA), for drug delivery. We carried out X-ray photoelectron spectroscopy and contact angle analysis to confirm the L-DOPA coating on the surface of PLGA meshes. Then, we loaded vascular endothelial growth factor (VEGF) and fibroblast growth factor 2 (FGF2) on the PLGA meshes for quicker angiogenesis through synergistic effect of the two growth factors. We investigated whether dual growth factor-loaded PLGA meshes on skin flaps promoted angiogenesis during 7-day treatment. When implanted into ICR mice models for skin flaps, the PLGA random mesh led to a result value of significantly smaller than the PLGA aligned mesh in skin flap necrosis and the epidermal thickness with the PLGA random mesh was much thicker than other groups in the necrotic area. Furthermore, coadministration of the two growth factors with the PLGA random meshes resulted in significantly higher blood vessel density than PLGA aligned mesh by ingrowth of host vessels at the edge of the skin flap. These results indicated that dual growth factor-loaded PLGA meshes could contribute to reducing the necrosis by dual growth factor delivery and geometric cues.

Mathematical Modelling of a Brain Tumour Initiation and Early Development: A Coupled Model of Glioblastoma Growth, Pre-Existing Vessel Co-Option, Angiogenesis and Blood Perfusion.

We propose a coupled mathematical modelling system to investigate glioblastoma growth in response to dynamic changes in chemical and haemodynamic microenvironments caused by pre-existing vessel co-option, remodelling, collapse and angiogenesis. A typical tree-like architecture network with different orders for vessel diameter is designed to model pre-existing vasculature in host tissue. The chemical substances including oxygen, vascular endothelial growth factor, extra-cellular matrix and matrix degradation enzymes are calculated based on the haemodynamic environment which is obtained by coupled modelling of intravascular blood flow with interstitial fluid flow. The haemodynamic changes, including vessel diameter and permeability, are introduced to reflect a series of pathological characteristics of abnormal tumour vessels including vessel dilation, leakage, angiogenesis, regression and collapse. Migrating cells are included as a new phenotype to describe the migration behaviour of malignant tumour cells. The simulation focuses on the avascular phase of tumour development and stops at an early phase of angiogenesis. The model is able to demonstrate the main features of glioblastoma growth in this phase such as the formation of pseudopalisades, cell migration along the host vessels, the pre-existing vasculature co-option, angiogenesis and remodelling. The model also enables us to examine the influence of initial conditions and local environment on the early phase of glioblastoma growth.

Methacrylic acid- containing beads modulate macrophage polarization in a vascularizing subcutaneous mouse model

Event Abstract Back to Event Methacrylic acid- containing beads modulate macrophage polarization in a vascularizing subcutaneous mouse model Kongyu Zhang1 and Michael Sefton1, 2* 1 University of Toronto, Institute of Biomaterials and Biomedical Engineering, Canada 2 University of Toronto, Chemical Engineering and Applied Chemistry, Canada Previously, polymeric beads containing methacrylic acid (MAA) have been used to accelerate vascularization in mice. However, the mechanism(s) of this highly beneficial phenomenon are unclear. Studies in a diabetic wound healing model in vivo showed augmented vessel formation and accelerated wound closure, despite no apparent increase in macrophage infiltration. Vascularization has been linked to macrophage polarization and function[1],[2], suggesting that perhaps an advantageous shift in macrophage polarization may be the impetus for this favorable response. Given this, we hypothesized that MAA beads shift the dynamic sequence in macrophage polarization towards the vascular reparative phenotype to promote vessel formation. To test our hypothesis, we subcutaneously injected MAA beads and control methyl methacrylate (MM) beads into CD1 mice for 1-7 days (using PEG avg. wt. 1450 as a vehicle). The beads and surrounding tissue were explanted and analyzed for the infiltration of inflammatory cells and the formation of vessels by flow cytometry and histology, respectively. MAA beads induced a polarization bias towards MHCII-CD206+ (“M2”) macrophages (Fig. 1); which was accompanied by significantly more CD31+ vessels (Fig. 2), compared to controls. To fully demonstrate that the newly formed MAA-mediated vessels were perfusable and anastomosed with the host vasculature, we injected fluorophore-conjugated mouse GSL-1 lectin into mice 7 days after bead injection and visualized the mouse vasculature using confocal microscopy following CLARITY, a tissue preparation technique that enabled high-resolution imaging of intact tissues[3]. Fluorophore staining was observed surrounding MAA beads but not MM beads in CLARITY-processed tissues, suggesting that vessels were anastomosed to host vessels and perfusable (Fig. 2). These data support the hypothesis that macrophage polarization is a key element of MAA-mediated vessel formation. This information is expected to facilitate the development of advanced therapeutic biomaterials with vascular regenerative properties. Ontario Research Foundation; Natural Sciences and Engineering Research Council (NSERC); NSERC Collaborative Research and Training Experience (CREATE) in Manufacturing Materials and Mimetics (M3) training program; Province of Ontario; University of Toronto

A Coupled Mathematical Model of Cell Migration, Vessel Cooption and Tumour Microenvironment during the Initiation of Micrometastases.

We propose a coupled mathematical model for the detailed quantitative analyses of initial microtumour and micrometastases formation by including cancer cell migration, host vessel cooption and changes in microenvironment. Migrating cells are included as a new phenotype to describe the migration behaviour of malignant tumour cells. Migration probability of a migrating cell is assumed to be influenced by local chemical microenvironment. Pre-existing vessel cooption and remodelling are introduced according to the local haemodynamical microenvironment, such as interstitial pressure and vessel wall permeability. After the tumour cells and tumour vessels distribution are updated, the chemical substances are coupled calculated with the haemodynamical environment. The simulation results clearly reproduce the tumour cells migrate and proliferate along the pre-existing vessels at the very early stage of growth, which are consistent with many published experimental observations. In addition, the model demonstrates the interactions of tumour cells with the pre-existing vessels, which are believed to be essential for initial adhesion, proliferation, invasion, and micrometastases establishment. Quantitative analysis of tumour expansion in longitudinal and transverse directions shows that the cooption and migration along host vessels will be inhibited once angiogenesis phase occurs. The influences of the ability of cell migration and the inclusion of vessel cooption on the formation of micrometastases are discussed.

Characterization of vasculogenic potential of human adipose-derived endothelial cells in a three-dimensional vascularized skin substitute.

The need for clinically applicable skin substitutes continues to be a matter of fact. Hypothetically, a laboratory grown autologous skin analog with near normal architecture might be a suitable approach to yield both satisfactory functional and cosmetic long-term results. In this study, we explored the use of human endothelial cells derived from freshly isolated adipose stromal vascular fraction (SVF) in a three-dimensional (3D) co-culture model of vascularized bio-engineered skin substitute. The SVF was isolated from human white adipose tissue samples and keratinocytes from human skin biopsies. The SVF, in particular endothelial cells, were characterized using flow cytometry and immuofluorescence analysis. Endothelial and mesenchymal progenitors from the SVF formed blood capillaries after seeding into a 3D collagen type I hydrogel in vitro. Subsequently, human keratinocytes were seeded on the top of those hydrogels to develop a vascularized dermo-epidermal skin substitute. Flow cytometric analysis of surface markers of the freshly isolated SVF showed the expression of endothelial markers (CD31, CD34, CD146), mesenchymal/stromal cell-associated markers (CD44, CD73, CD90, CD105), stem cell markers (CD49f, CD117, CD133), and additionally hematopoietic markers (CD14, CD15, CD45). Further analysis of white adipose-derived endothelial cells (watECs) revealed the co-expression of CD31, CD34, CD90, CD105, and partially CD146 on these cells. WatECs were separated from adipose-stromal cells (watASCs) using FACS sorting. WatASCs and watECs cultured separately in a 3D hydrogel for 3weeks did not form any vascular structures. Only if co-cultured, both cell types aligned to develop a ramified vascular network in vitro with continuous endothelial lumen formation. Transplantation of those 3D-hydrogels onto immuno-incompetent rats resulted in a rapid connection of human capillaries with the host vessels and formation of functional, blood-perfused mosaic human-rat vessels within only 3-4days. Adipose tissue represents an attractive cell source due to the ease of isolation and abundance of endothelial as well as mesenchymal cell lineages. Adipose-derived SVF cells exhibit the ability to form microvascular structures in vitro and support the accelerated blood perfusion in skin substitutes in vivo when transplanted.

Bone Tissue Engineering with Multilayered Scaffolds-Part I: An Approach for Vascularizing Engineered Constructs In Vivo.

Obtaining functional capillaries through the bulk has been identified as a major challenge in tissue engineering, particularly for critical-sized defects. In the present study, a multilayered scaffold system was developed for bone tissue regeneration, designed for through-the-thickness vascularization of the construct. The basic principle of this approach was to alternately layer mesenchymal stem cell-seeded nanofibers (osteogenic layer) with microfibers or porous ceramics (osteoconductive layer), with an intercalating angiogenic zone between the two and with each individual layer in the microscale dimension (100-400 μm). Such a design can create a scaffold system potentially capable of spatially distributed vascularization in the overall bulk tissue. In the cellular approach, the angiogenic zone consisted of collagen/fibronectin gel with endothelial cells and pericytes, while in the acellular approach, cells were omitted from the zone without altering the gel composition. The cells incorporated into the construct were analyzed for viability, distribution, and organization of cells on the layers and vessel development in vitro. Furthermore, the layered constructs were implanted in the subcutaneous space of nude mice and the processes of vascularization and bone tissue regeneration were followed by histological and energy-dispersive X-ray spectroscopy (EDS) analysis. The results indicated that the microenvironment in the angiogenic zone, microscale size of the layers, and the continuously channeled architecture at the interface were adequate for infiltrating host vessels through the bulk and vascularizing the construct. Through-the-thickness vascularization and mineralization were accomplished in the construct, suggesting that a suitably bioengineered layered construct may be a useful design for regeneration of large bone defects.

Abstract 5216: Potential use of saliva and hair samples to identify genetic markers for anti-VEGF therapy

Abstract Anti-angiogenic therapy to block and disrupt tumor angiogenesis has been used in clinical treatment of various malignancies including: glioblastoma, colorectal, lung and kidney cancer. Currently, the most clinically effective anti-angiogenic agents are the humanized monoclonal antibody (bevacizumab) against vascular endothelia growth factor (VEGF) and the small molecule receptor-tyrosine-kinase inhibitors (RTKIs) of VEGF receptors. However, the therapeutic benefit varies substantially for many of these agents. Predictive biomarkers that are required for personalized treatment with these agents are currently not available. Recently the role of germline single-nucleotide polymorphisms (SNPs) has been investigated as potential biomarkers for anti-angiogenic treatments. The rationale is that these agents target the vasculature in host (vessels) rather than tumor tissue, which could mean that much of the variability is dictated by the host. Several studies reported the association of SNPs in VEGFA with differential outcomes from clinical trials with these agents. SNP genotyping assays in these studies used germline DNA extracted from blood. However, collection of peripheral blood is invasive. Saliva and hair that are known to contain genomic DNA can be used as noninvasive and convenient DNA sampling resources, but the limited quantity of DNA in the hair and the low quality of DNA in the saliva make genotyping assays non-reproducible. We have sought to improve SNP genotype assays with DNA from saliva and hair by applying SNP target pre-amplification for genotyping five SNPs (rs699947, rs833061, rs1570360, rs201093, rs3025039) in VEGFA. The concordance of genotyping calls of individual SNP using DNA from blood, saliva and hair from the same volunteer was compared. Our preliminary results demonstrated that pre-amplification substantially improved genotyping calls with saliva DNA and hair DNA samples. The average genotyping call rate with saliva and hair samples was about 45% and 10%, respectively, without pre-amplification. The pre-amplification improved the call rate with saliva and hair samples to 100% and 94%, respectively. The current report may facilitate easier clinical sample collection for genetic tests for these SNPs. Citation Format: Gloria R. Xue, Crystal R. Xue, Fei Shen, Bryan P. Schneider. Potential use of saliva and hair samples to identify genetic markers for anti-VEGF therapy. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5216. doi:10.1158/1538-7445.AM2015-5216

Analyzing Structure and Function of Vascularization in Engineered Bone Tissue by Video-Rate Intravital Microscopy and 3D Image Processing.

Vascularization is a key challenge in tissue engineering. Three-dimensional structure and microcirculation are two fundamental parameters for evaluating vascularization. Microscopic techniques with cellular level resolution, fast continuous observation, and robust 3D postimage processing are essential for evaluation, but have not been applied previously because of technical difficulties. In this study, we report novel video-rate confocal microscopy and 3D postimage processing techniques to accomplish this goal. In an immune-deficient mouse model, vascularized bone tissue was successfully engineered using human bone marrow mesenchymal stem cells (hMSCs) and human umbilical vein endothelial cells (HUVECs) in a poly (D,L-lactide-co-glycolide) (PLGA) scaffold. Video-rate (30 FPS) intravital confocal microscopy was applied in vitro and in vivo to visualize the vascular structure in the engineered bone and the microcirculation of the blood cells. Postimage processing was applied to perform 3D image reconstruction, by analyzing microvascular networks and calculating blood cell viscosity. The 3D volume reconstructed images show that the hMSCs served as pericytes stabilizing the microvascular network formed by HUVECs. Using orthogonal imaging reconstruction and transparency adjustment, both the vessel structure and blood cells within the vessel lumen were visualized. Network length, network intersections, and intersection densities were successfully computed using our custom-developed software. Viscosity analysis of the blood cells provided functional evaluation of the microcirculation. These results show that by 8 weeks, the blood vessels in peripheral areas function quite similarly to the host vessels. However, the viscosity drops about fourfold where it is only 0.8 mm away from the host. In summary, we developed novel techniques combining intravital microscopy and 3D image processing to analyze the vascularization in engineered bone. These techniques have broad applicability for evaluating vascularization in other engineered tissues as well.

A hydrogel-endothelial cell implant mimics infantile hemangioma: modulation by survivin and the Hippo pathway

Microvascular endothelial cells cultured in three-dimensional hydrogel scaffolds form a network of microvessel structures when implanted subcutaneously in mice, inosculate with host vessels, and over time remodel into large ectatic vascular structures resembling hemangiomas. When compared with infantile hemangiomas, similarities were noted, including a temporal progression from a morphological appearance of a proliferative phase to the appearance of an involuted phase, mimicking the proliferative and involutional phases of infantile hemangioma. Consistent with the progression of a proliferative phase to an involuted phase, both the murine implants and human biopsy tissue exhibit reduced expression of Ajuba, YAP, and Survivin labeling as they progressed over time. Significant numbers of CD45+, CD11b+, Mac3+ mononuclear cells were found at the 2-week time point in our implant model that correlated with the presence of CD45+, CD68+ mononuclear cells observed in biopsies of human proliferative-phase hemangiomas. At the 4-week time point in our implant model, only small numbers of CD45+ cells were detected, which again correlated with our findings of significantly diminished CD45+, CD68+ mononuclear cells in human involutional-phase hemangiomas. The demonstration of mononuclear cell infiltration transiently in the proliferative phase of these lesions suggests that the vascular proliferation and/or regression may be driven in part by an immune response. Gross and microscopic morphological appearances of human proliferative and involutional hemangiomas and our implant model correlate well with each other as do the expression levels of Hippo pathway components (Ajuba and YAP) and Survivin and correlate with proliferation in these entities. Inhibitors of Survivin and Ajuba (which we have demonstrated to inhibit proliferation and increase apoptosis in murine hemangioendothelioma cell tissue culture) may have potential as other beneficial treatments for proliferating infantile hemangiomas. This implant model may have potential as a modest through-put screen for testing and development of therapeutics targeted at the proliferative phase of infantile hemangiomas, reducing the subsequent postinvolutional scarring or deformities sometimes associated with these lesions.

Inosculation of blood vessels allows early perfusion and vitality of bladder grafts--implications for bioengineered bladder wall.

Bioengineered bladder tissue is needed for patients with neurogenic bladder disease as well as for cancer. Current technologies in bladder tissue engineering have been hampered by an inability to efficiently initiate blood supply to the graft, ultimately leading to complications that include graft contraction, ischemia, and perforation. To date, the biological mechanisms of vascularization on transplant have not been suitably investigated for urologic tissues. To better understand the mechanisms of neovascularization on bladder wall transplant, a chimeric mouse model was generated such that angiogenesis and vasculogenesis could be independently assessed in vivo. Green fluorescence protein (GFP) transgenic mice received bone marrow transplants from β-galactosidase (LacZ) transgenic animals and then subsequent bladder wall transplants from wild-type donor mice. Before euthanization, the aorta was infused with fluorescent microbeads (fluorospheres) to identify perfused vessels. The contributions of GFP (angiogenesis) and LacZ (vasculogenesis) to the formation of CD31-expressing blood vessels within the wild-type graft were evaluated by immunohistochemistry at different time points and locations within the graft (proximal, middle, and distal) to provide a spatiotemporal analysis of neovascularization. The GFP index, a measure of angiogenic host ingrowth, was significantly higher at proximal versus mid or distal regions in animals 2-16 weeks post-transplant. However, GFP index did not increase over time in any area. Within 7 days post-transplant, perfusion of primarily wild-type, donor blood vessels in the most distal areas of the graft was observed by intraluminal fluorospheres. In addition, chimeric host-donor (GFP-wild type) blood vessels were evident in proximal areas. The contribution of vasculogenesis to vascularization of the graft was limited, as LacZ cells were not specifically associated with the endothelial cells of blood vessels, but rather found primarily in areas of inflammation. The data suggest that angiogenesis of host blood vessels into the proximal region leads to inosculation between host and donor vessels and subsequent perfusion of the graft via pre-existing graft vessels within the first week after transplant. As such, the engineering of graft blood vessels and the promotion of inosculation might prevent graft contraction, thereby potentiating the use of bioengineered bladder tissue for transplantation.

Vascularization of hollow channel-modified porous silk scaffolds with endothelial cells for tissue regeneration

Despite the promise for stem cell-based tissue engineering for regenerative therapy, slow and insufficient vascularization of large tissue constructs negatively impacts the survival and function of these transplanted cells. A combination of channeled porous silk scaffolds and prevascularization with endothelial cells was investigated to test the ability of this tissue engineering strategy to support rapid and extensive vascularization process. We report that hollow channels promote in vitro prevascularization by facilitating endothelial cell growth, VEGF secretion, and capillary-like tube formation. When implanted in vivo, the pre-established vascular networks in the hollow channel scaffolds anastomose with host vessels and exhibit accelerated vascular infiltration throughout the whole tissue construct, which provides timely and sufficient nutrients to ensure the survival of the transplanted stem cells. This tissue engineering strategy can promote the effective application of stem cell-based regeneration to improve future clinical applications.

In vivo generation of a mature and functional artificial skeletal muscle.

Extensive loss of skeletal muscle tissue results in mutilations and severe loss of function. In vitro-generated artificial muscles undergo necrosis when transplanted in vivo before host angiogenesis may provide oxygen for fibre survival. Here, we report a novel strategy based upon the use of mouse or human mesoangioblasts encapsulated inside PEG-fibrinogen hydrogel. Once engineered to express placental-derived growth factor, mesoangioblasts attract host vessels and nerves, contributing to in vivo survival and maturation of newly formed myofibres. When the graft was implanted underneath the skin on the surface of the tibialis anterior, mature and aligned myofibres formed within several weeks as a complete and functional extra muscle. Moreover, replacing the ablated tibialis anterior with PEG-fibrinogen-embedded mesoangioblasts also resulted in an artificial muscle very similar to a normal tibialis anterior. This strategy opens the possibility for patient-specific muscle creation for a large number of pathological conditions involving muscle tissue wasting.

Oxygen transport in a three-dimensional microvascular network incorporated with early tumour growth and preexisting vessel cooption: numerical simulation study.

We propose a dynamic mathematical model of tissue oxygen transport by a preexisting three-dimensional microvascular network which provides nutrients for an in situ cancer at the very early stage of primary microtumour growth. The expanding tumour consumes oxygen during its invasion to the surrounding tissues and cooption of host vessels. The preexisting vessel cooption, remodelling and collapse are modelled by the changes of haemodynamic conditions due to the growing tumour. A detailed computational model of oxygen transport in tumour tissue is developed by considering (a) the time-varying oxygen advection diffusion equation within the microvessel segments, (b) the oxygen flux across the vessel walls, and (c) the oxygen diffusion and consumption within the tumour and surrounding healthy tissue. The results show the oxygen concentration distribution at different time points of early tumour growth. In addition, the influence of preexisting vessel density on the oxygen transport has been discussed. The proposed model not only provides a quantitative approach for investigating the interactions between tumour growth and oxygen delivery, but also is extendable to model other molecules or chemotherapeutic drug transport in the future study.

Tuning graft- and host-derived vascularization in modular tissue constructs: a potential role of HIF1 activation.

A better understanding of the factors governing the vascularization of engineered tissues is crucial for their advancement as therapeutic platforms. Here, we studied the effect of implant volume and cell densities on the in vivo vascularization of modular engineered tissue constructs. Sub-millimeter collagen modules containing adipose-derived mesenchymal stromal cells (adMSC) and enveloped by human umbilical vein endothelial cells (HUVEC) were subcutaneously implanted in severe-combined immunodeficient mice with a beige-mutation (SCID-bg) mice. Implant volume and cell density was varied relative to a base case, defined as a 0.01 mL implant containing 1.5×10(7) adMSC/mL and 3.9×10(6) HUVEC/mL. At 7 and 14 days post-transplantation, the constructs were harvested for immunohistochemical analysis of total (CD31(+)) and graft-derived (UEA1(+)) vessel formation, hypoxia-inducible factor 1-alpha (HIF1α) expression, infiltration of host-derived leukocytes (CD45), and macrophages (F4/80). Implant volume and cell density affected the relative contributions of host- versus graft-derived vascularization, highlighting that different mechanisms underlie the two processes. Graft-derived vessel formation was most rapid and robust in implants with high HIF1α expression, namely large volume implants and implants with high adMSC and HUVEC density (p<0.01 compared to base case at day 7). Many HIF1α(+) cells were vessel-lining HUVEC, suggesting that HIF1 activation may be key to vessel assembly in the graft. Host vessel ingrowth, however, dominated the vascularization of small volume implants (of high and low adMSC density alike), which showed low HIF1α expression at day 7. Host vessels were sustained to day 14 when adMSC density alone was increased, presumably due to increased paracrine secretions. This study points to a potential role of HIF1 activation in the vascularization of tissue constructs, which may be harnessed to engineer robust vessels for therapeutic applications.

Abstract 96: A novel in vivo preclinical model for the study of human tumor angiogenesis with human vessels

Abstract The preclinical assessment of anti-angiogenic drug efficacy is hampered by the lack of availability of suitable model systems to study anti-human therapies. Good in vitro assays exist for testing anti-human-specific therapies but these are let down by the lack of robust translatable in vivo models. Currently available preclinical models such as the matrigel plug assay and the window chamber assay focus on the angiogenesis of mouse vessels which may not be truly reflective of the influence of test items on human vasculature, and in vivo models to study the angiogenesis of human tumors by human vessels are unfortunately lacking. We have developed a sophisticated model to address this using adult human skin engrafted onto immunocompromised CB17-SCID mice. Angiogenesis of the skin follows, with human and host vessels anastomosing, resulting in a viable skin graft containing functioning human CD31-labelled vessels. This skin graft can be intradermally injected with a cell line of interest, such as the human melanoma cell line A375, and the ensuing tumors contain human vessels as evidenced by immunohistochemistry with a human-specific anti-CD31 antibody. Using the Aperio® Scanscope and FDA-approved microvessel analysis algorithm, 5-10 human vessels per mm2 of human tumor are observed. Furthermore, the model has the potential to be used in combination with non-invasive in vivo imaging techniques to assess tumor growth inhibition in response to therapies in a longitudinal fashion. This novel technique could potentially enable the rapid preclinical testing of anti-angiogenic anti-human therapies to help provide a go/no-go decision prior to costly clinical trials. Citation Format: Kirsty A. Holden, Michael A. Batey, Martyn Bottomley, Catherine Booth. A novel in vivo preclinical model for the study of human tumor angiogenesis with human vessels. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 96. doi:10.1158/1538-7445.AM2014-96

Triple-Layered Cell Sheet for Tissue-Engineering the Synovial Membrane of the Temporomandibular Joint

Objective: Temporomandibular disorder causes the dysfunction of fibroblast-like synoviocytes (FLSs) which are predominant in the lining layer (LL) of synovial membrane (SM) and responsible for the secretion function of the SM of the temporomandibular joint (TMJ). This study aimed to construct a triple-layered cell sheet (CS) for tissue-engineering the SM. Methods: FLSs were harvested and identified immunocytochemically. A triple-layered CS was fabricated by an original method of combining type I collagen and FLSs. Staining and a transmission electron microscope were used to compare the morphological similarities between the CS and the natural LL. Hyaluronic acid (HA) production and HA synthase 2 (HAS2) gene expression were assessed by ELISA and PCR, respectively. Transplantation of triple-layered CSs into nude mice was performed and examined by staining and immunohistochemical methods. Results: FLSs expressed vimentin, CD44 and heat shock protein 27. The triple-layered CS possessed a structure similar to natural LL. No tight conjunction was observed between adjacent FLSs. The triple-layered CS secreted HA at a quantity about 3 times that of the single-layered CS. The triple-layered structure induced higher expression of HAS2 in FLSs. No difference in HAS2 expression between the triple-layered CS and natural SM was observed. Multiple-layered FLSs and invasion of host fibroblasts and vessels were observed 2 weeks after transplantation. HAS2 and HA were expressed in surface cells and extracellular matrix, respectively. Conclusion: FLSs of the TMJ were type B synoviocytes. The triple-layered CS mimicked natural SM morphologically and functionally. The CS survived for 2 weeks in vivo. Therefore, triple-layered CS might be highly competent for tissue-engineered SM.

Whole body magnetic resonance angiography and computed tomography angiography in the vascular mapping of head and neck: an intraindividual comparison.

IntroductionThe aim of the study was to compare the detectability of neck vessels with contrast enhanced magnetic resonance angiography (MRA) in the setting of a whole-body MRA and multislice computed tomography angiography (CTA) for preoperative vascular mapping of head and neck.MethodsIn 20 patients MRA was performed prior to microvascular reconstruction of the mandible with osteomyocutaneous flaps. CTA of the neck served as the method of reference.1.5 T contrast enhanced magnetic resonance angiograms were acquired to visualize the vascular structures of the neck in the setting of a whole-body MRA examination. 64-slice spiral computed tomography was performed with a dual-phase protocol, using the arterial phase images for 3D CTA reconstruction. Maximum intensity projection was employed to visualize MRA and CTA data. To retrieve differences in the detectability of vessel branches between MRA and CTA, a McNemar test was performed.ResultsAll angiograms were of diagnostic quality. There were no statistically significant differences between MRA and CTA for the detection of branches of the external carotid artery that are relevant host vessels for microsurgery (p = 0.118). CTA was superior to MRA if all the external carotid artery branches were included (p < 0.001).ConclusionsMRA is a reliable alternative to CTA in vascular mapping of the cervical vasculature for planning of microvascular reconstruction of the mandible. In the setting of whole-body MRA it could serve as a radiation free one-stop-shop tool for preoperative assessment of the arterial system, potentially covering both, the donor and host site in one single examination.

Manipulation of a VEGF-Notch signaling circuit drives formation of functional vascular endothelial progenitors from human pluripotent stem cells.

Human pluripotent stem cell (hPSC)-derived endothelial lineage cells constitutes a promising source for therapeutic revascularization, but progress in this arena has been hampered by a lack of clinically-scalable differentiation protocols and inefficient formation of a functional vessel network integrating with the host circulation upon transplantation. Using a human embryonic stem cell reporter cell line, where green fluorescent protein expression is driven by an endothelial cell-specific VE-cadherin (VEC) promoter, we screened for > 60 bioactive small molecules that would promote endothelial differentiation, and found that administration of BMP4 and a GSK-3β inhibitor in an early phase and treatment with VEGF-A and inhibition of the Notch signaling pathway in a later phase led to efficient differentiation of hPSCs to the endothelial lineage within six days. This sequential approach generated > 50% conversion of hPSCs to endothelial cells (ECs), specifically VEC+CD31+CD34+CD14−KDRhigh endothelial progenitors (EPs) that exhibited higher angiogenic and clonogenic proliferation potential among endothelial lineage cells. Pharmaceutical inhibition or genetical knockdown of Notch signaling, in combination with VEGF-A treatment, resulted in efficient formation of EPs via KDR+ mesodermal precursors and blockade of the conversion of EPs to mature ECs. The generated EPs successfully formed functional capillary vessels in vivo with anastomosis to the host vessels when transplanted into immunocompromised mice. Manipulation of this VEGF-A-Notch signaling circuit in our protocol leads to rapid large-scale production of the hPSC-derived EPs by 12- to 20-fold vs current methods, which may serve as an attractive cell population for regenerative vascularization with superior vessel forming capability compared to mature ECs.