Fabrication of biodegradable synthetic vascular networks and their use as a model of angiogenesis

One of the greatest challenges currently faced in tissue engineering is the incorporation of vascular networks within tissue-engineered constructs. The aim of this study was to develop a technique for producing a perfusable, three-dimensional cell friendly model of vascular structures that could be used to study the factors affecting angiogenesis and vascular biology in engineered systems in more detail. Initially, biodegradable synthetic pseudo-vascular networks were produced via the combination of robocasting and electrospinning techniques. The internal surfaces of the vascular channels were then recellularized with human dermal microvascular endothelial cells (HDMECs) with and without the presence of human dermal fibroblasts (HDFs) on the outer surface of the scaffold. After 7 days in culture, channels that had been reseeded with HDMECs alone, demonstrated irregular cell coverage. However when using a co-culture of HDMECs inside and HDFs outside the vascular channels, coverage was found to be continuous throughout the internal channel. Using this cell combination, collagen gels loaded with vascular endothelial growth factor were deposited onto the outer surface of the scaffold and cultured for a further 7 days after which endothelial cell (EC) outgrowth from within the channels into the collagen gel was observed showing the engineered vasculature maintains its capacity for angiogenesis. Furthermore the HDMECs appeared to have formed perfusable tubules within the gel. These results show promising steps towards the development of an in vitro platform upon which to study angiogenesis and vascular biology in a tissue-engineering context

Bioengineering vascular networks to study angiogenesis and vascularisation of physiologically relevant tissue models in vitro

Angiogenesis assays are essential for studying aspects of neovascularisation and angiogenesis and investigating drugs that stimulate or inhibit angiogenesis. To date, there are several in vitro and in vivo angiogenesis assays that are used for studying different aspects of angiogenesis. Although in vivo assays are the most representative of native angiogenesis, they raise ethical questions, require considerable technical skills, and are expensive. In vitro assays are inexpensive and easier to perform, but the majority of them are only two-dimensional cell monolayers which lack the physiological relevance of three-dimensional structures. Thus, it is important to look for alternative platforms to study angiogenesis under more physiologically relevant conditions in vitro. Accordingly, in this study, we developed polymeric vascular networks to be used to study angiogenesis and vascularisation of a 3D human skin model in vitro. Our results showed that this platform allowed the study of more than one aspect of angiogenesis, endothelial migration and tube formation, in vitro when combined with Matrigel®. We successfully reconstructed a human skin model, as a representative of a physiologically relevant and complex structure, and assessed the suitability of the developed in vitro platform for studying endothelialisation of the tissue-engineered skin model

Decellularised extracellular matrix decorated PCL PolyHIPE scaffolds for enhanced cellular activity, integration and angiogenesis

Wound healing involves a complex series of events where cell–cell and cell-extracellular matrix (ECM) interactions play a key role. Wounding can be simple, such as the loss of the epithelial integrity, or deeper and more complex, reaching to subcutaneous tissues, including blood vessels, muscles and nerves. Rapid neovascularisation of the wounded area is crucial for wound healing as it has a key role in supplying oxygen and nutrients during the highly demanding proliferative phase and transmigration of inflammatory cells to the wound area. One approach to circumvent delayed neovascularisation is the exogenous use of pro-angiogenic factors, which is expensive, highly dose-dependent, and the delivery of them requires a very well-controlled system to avoid leaky, highly permeable and haemorrhagic blood vessel formation. In this study, we decorated polycaprolactone (PCL)-based polymerised high internal phase emulsion (PolyHIPE) scaffolds with fibroblast-derived ECM to assess fibroblast, endothelial cell and keratinocyte activity in vitro and angiogenesis in ex ovo chick chorioallantoic membrane (CAM) assays. Our results showed that the inclusion of ECM in the scaffolds increased the metabolic activity of three types of cells that play a key role in wound healing and stimulated angiogenesis in ex ovo CAM assays over 7 days. Herein, we demonstrated that fibroblast-ECM functionalised PCL PolyHIPE scaffolds appear to have great potential to be used as an active wound dressing to promote angiogenesis and wound healing

Porous microspheres support mesenchymal progenitor cell ingrowth and stimulate angiogenesis

Porous microspheres have the potential for use as injectable bone fillers to obviate the need for open surgery. Successful bone fillers must be able to support vascularisation since tissue engineering scaffolds often cease functioning soon after implantation due to a failure to vascularise rapidly. Here, we test the angiogenic potential of a tissue engineered bone filler based on a photocurable acrylate-based high internal phase emulsion (HIPE). Highly porous microspheres were fabricated via two processes, which were compared. One was taken forward and investigated for its ability to support human mesenchymal progenitor cells and angiogenesis in a chorioallantoic membrane (CAM) assay. Porous microspheres with either a narrow or broad size distribution were prepared via a T-junction microfluidic device or by a controlled stirred-tank reactor of the HIPE water in oil in water (w/o/w), respectively. Culture of human embryonic stem cell-derived mesenchymal progenitor (hES-MP) cells showed proliferation over 11 days and formation of cell-microsphere aggregates. In-vitro, hES-MP cells were found to migrate into microspheres through their surface pores over time. The presence of osteoblasts, differentiated from the hES-MP cells, was evidenced through the presence of collagen and calcium after 30 days. Microspheres pre-cultured with cells were implanted into CAM for 7 days and compared with control microspheres without pre-cultured cells. The hES-MP seeded microspheres supported greater angiogenesis, as measured by the number of blood vessels and bifurcations, while the empty scaffolds attracted host chick cell ingrowth. This investigation shows that controlled fabrication of porous microspheres has the potential to create an angiogenic, bone filling material for use as a cell delivery vehicle

Assessment of the angiogenic potential of 2-deoxy-D-ribose using a novel in vitro 3D dynamic model in comparison with established in vitro assays

Angiogenesis is a highly ordered physiological process regulated by the interaction of endothelial cells with an extensive variety of growth factors, extracellular matrix components and mechanical stimuli. One of the most important challenges in tissue engineering is the rapid neovascularization of constructs to ensure their survival after transplantation. To achieve this, the use of pro-angiogenic agents is a widely accepted approach. The study of angiogenesis has gained momentum over the last two decades. Although there are various in vitro, ex vivo, and in vivo angiogenesis models that enable testing of newly discovered pro-angiogenic agents, the problem with researching angiogenesis is the choice of the most appropriate assay. In vivo assays are the most representative and reliable models, but they are expensive, time-consuming and can cause ethical concerns whereas in vitro assays are relatively inexpensive, practical, and reproducible, but they are usually lack of enabling the study of more than one aspect of angiogenesis, and they do not fully represent the complexity of physiological angiogenesis. Therefore, there is a need for the development of an angiogenesis model that allows the study of angiogenesis under physiologically more relevant, dynamic conditions without causing ethical concerns. Accordingly, in this study, we developed 3D in vitro dynamic angiogenesis model, and we tested the angiogenic potential of 2-deoxy-D-ribose (2dDR) in comparison with vascular endothelial growth factor (VEGF) using newly developed in vitro 3D dynamic model and well-established in vitro models. Our results obtained using conventional in vitro assays demonstrated that 2dDR promoted proliferation, migration and tube formation of human aortic endothelial cells (HAECs) in a dose-dependent manner. Then, the angiogenic activity of 2dDR was further assessed using the newly developed 3D in vitro model, which enabled the monitoring of cell proliferation and infiltration simultaneously under dynamic conditions. Our results showed that the administration of 2dDR and VEGF significantly enhanced the outgrowth of HAECs and the cellular density under either static or dynamic conditions

The use of microfabrication techniques for the design and manufacture of artificial stem cell microenvironments for tissue regeneration.

The recapitulation of the stem cell microenvironment is an emerging area of research that has grown significantly in the last 10 to 15 years. Being able to understand the underlying mechanisms that relate stem cell behavior to the physical environment in which stem cells reside is currently a challenge that many groups are trying to unravel. Several approaches have attempted to mimic the biological components that constitute the native stem cell niche, however, this is a very intricate environment and, although promising advances have been made recently, it becomes clear that new strategies need to be explored to ensure a better understanding of the stem cell niche behavior. The second strand in stem cell niche research focuses on the use of manufacturing techniques to build simple but functional models; these models aim to mimic the physical features of the niche environment which have also been demonstrated to play a big role in directing cell responses. This second strand has involved a more engineering approach in which a wide set of microfabrication techniques have been explored in detail. This review aims to summarize the use of these microfabrication techniques and how they have approached the challenge of mimicking the native stem cell niche

Economic value for the trait tick count in Brangus cattle.

Profitability of a beef cattle system may be significantly reduced by the effects of tick (Rhipicephalus (Boophilus) microplus).IMAS. Pôster 45209

Delivery of bioactive compounds to improve skin cell responses on microfabricated electrospun microenvironments

The introduction of microtopographies within biomaterial devices is a promising approach that allows one to replicate to a degree the complex native environment in which human cells reside. Previously, our group showed that by combining electrospun fibers and additive manufacturing it is possible to replicate to an extent the stem cell microenvironment (rete ridges) located between the epidermal and dermal layers. Our group has also explored the use of novel proangiogenic compounds to improve the vascularization of skin constructs. Here, we combine our previous approaches to fabricate innovative polycaprolactone fibrous microtopographical scaffolds loaded with bioactive compounds (2-deoxy-D-ribose, 17β-estradiol, and aloe vera). Metabolic activity assay showed that microstructured scaffolds can be used to deliver bioactive agents and that the chemical relation between the working compound and the electrospinning solution is critical to replicate as much as possible the targeted morphologies. We also reported that human skin cell lines have a dose-dependent response to the bioactive compounds and that their inclusion has the potential to improve cell activity, induce blood vessel formation and alter the expression of relevant epithelial markers (collagen IV and integrin β1). In summary, we have developed fibrous matrixes containing synthetic rete-ridge-like structures that can deliver key bioactive compounds that can enhance skin regeneration and ultimately aid in the development of a complex wound healing device

An “off-the shelf” Synthetic Membrane to Simplify Regeneration of Damaged Corneas

yesOur overall aim is to develop a synthetic off-the-shelf alternative to human amniotic membrane which is currently used for delivering cultured limbal stem cells to the cornea in patients who suffer scarring of the cornea because of the loss of limbal stem cells. We have recently reported that both cultured cells and limbal explants grow well on electrospun Poly(D,L-lactide-co-glycolide) (PLGA) (44 kg/mol) with a 50:50 ratio of lactide and glycolide and sterilized with γ-irradiation. Prior to undertaking a clinical study our immediate aim now is to achieve long term storage of the membranes in convenient to use packaging. Membranes were electrospun from Poly(D,L-lactide-co-glycolide) (44 kg/mol) with a 50:50 ratio of lactide and glycolide and sterilized with γ-irradiation and then stored dry (with desiccant) for several months at -80°C and -20°C , Room temperature (UK and India), 37°C and 50°C. We explored the contribution of vacuum sealing and the use of a medical grade bag (PET/Foil/LDPE) to achieve a longer shelf life. Confirmation of membranes being suitable for clinical use was obtained by culturing tissue explants on membranes post storage. When scaffolds were stored dry the rate of breakdown was both temperature and time dependent. At -20°C and -80°C there was no change in fiber diameter over 18 months of storage, and membranes were stable for 12 months at 4°C while at 50°C (above the transition temperature for PLGA) scaffolds lost integrity after several weeks. The use of vacuum packaging and a medical grade bag both improved the storage shelf-life of the scaffolds. The impact of temperature on storage is summarized beneath. We report that this synthetic membrane can be used as an off-the-shelf or-out-of-the freezer alternative to the amniotic membrane for corneal regeneration

Combination of Microstereolithography and Electrospinning to Produce Membranes Equipped with Niches for Corneal Regeneration

We report a technique for the fabrication of micropockets within electrospun membranes in which to study cell behavior. Specifically, we describe a combination of microstereolithography and electrospinning for the production of PLGA (Poly(lactide-co-glycolide)) corneal biomaterial devices equipped with microfeatures