Osteosarcoma growth on trabecular bone mimicking structures manufactured via laser direct write

This paper describes the direct laser write of a photocurable acrylate-based PolyHIPE (High Internal Phase Emulsion) to produce scaffolds with both macro- and microporosity, and the use of these scaffolds in osteosarco-ma-based 3D cell culture. The macroporosity was introduced via the application of stereolithography to produce a clas-sical woodpile structure with struts having an approximate diameter of 200 ?m and pores were typically around 500 ?m in diameter. The PolyHIPE retained its microporosity after stereolithographic manufacture, with a range of pore sizes typically between 10 and 60 ?m (with most pores between 20 and 30 ?m). The resulting scaffolds were suitable substrates for further modification using acrylic acid plasma polymerisation. This scaffold was used as a structural mimic of the trabecular bone and in vitro determination of biocompatibility using cultured bone cells (MG63) demon-strated that cells were able to colonise all materials tested, with evidence that acrylic acid plasma polymerisation im-proved biocompatibility in the long term. The osteosarcoma cell culture on the 3D printed scaffold exhibits different growth behaviour than observed on tissue culture plastic or a flat disk of the porous material; tumour spheroids are ob-served on parts of the scaffolds. The growth of these spheroids indicates that the osteosarcoma behave more akin to in vivo in this 3D mimic of trabecular bone. It was concluded that PolyHIPEs represent versatile biomaterial systems with considerable potential for the manufacture of complex devices or scaffolds for regenerative medicine. In particular, the possibility to readily mimic the hierarchical structure of native tissue enables opportunities to build in vitro models closely resembling tumour tissue

Osteosarcoma growth on trabecular bone mimicking structures manufactured via laser direct write

This paper describes the direct laser write of a photocurable acrylate-based PolyHIPE (High Internal Phase Emulsion) to produce scaffolds with both macro- and microporosity, and the use of these scaffolds in osteosarco-ma-based 3D cell culture. The macroporosity was introduced via the application of stereolithography to produce a clas-sical woodpile structure with struts having an approximate diameter of 200 ?m and pores were typically around 500 ?m in diameter. The PolyHIPE retained its microporosity after stereolithographic manufacture, with a range of pore sizes typically between 10 and 60 ?m (with most pores between 20 and 30 ?m). The resulting scaffolds were suitable substrates for further modification using acrylic acid plasma polymerisation. This scaffold was used as a structural mimic of the trabecular bone and in vitro determination of biocompatibility using cultured bone cells (MG63) demon-strated that cells were able to colonise all materials tested, with evidence that acrylic acid plasma polymerisation im-proved biocompatibility in the long term. The osteosarcoma cell culture on the 3D printed scaffold exhibits different growth behaviour than observed on tissue culture plastic or a flat disk of the porous material; tumour spheroids are ob-served on parts of the scaffolds. The growth of these spheroids indicates that the osteosarcoma behave more akin to in vivo in this 3D mimic of trabecular bone. It was concluded that PolyHIPEs represent versatile biomaterial systems with considerable potential for the manufacture of complex devices or scaffolds for regenerative medicine. In particular, the possibility to readily mimic the hierarchical structure of native tissue enables opportunities to build in vitro models closely resembling tumour tissue

Imaging of 3D tissue-engineered models of oral cancer using 890 and 1300 nm optical coherence tomography

© 2015, Nizhny Novgorod State Medical Academy. All rights reserved. Optical coherence tomography (OCT) generates its primary form of contrast from elastic backscatter. It is now the gold standard technique for retinal screening and is emerging rapidly in cardiovascular research however it remains a research goal to establish it to the same degree in epithelial cancer detection and diagnosis. In this report we compare two different OCT systems: an 890 nm spectrometer-based OCT system with 2.5 µm axial resolution and a 1300 nm swept-source OCT system with 7.5 µm axial resolution to determine the effect of these different OCT parameters on the endogenous backscatter contrast of dysplastic/malignant oral mucosa models relative to normal mucosa models. Tissueengineered oral mucosa models constructed with a dysplastic cell line (DOK), a malignant cell line (Cal27) and normal cell were imaged with both of these OCT platforms and comparisons made with regard to apparent epithelial thickness and the visibility of the epithelium relative to the underlying stroma. For the Cal27’s, hematoxylin and eosin staining confirmed the formation of a keratinized layer superficial to a thickened layer of viable cells on top of the stroma. The keratinized layer presented as a hyperreflective thickened layer superficial to a darker region on both OCT platforms. The keratinized layer caused a steep fall in signal at 890 nm, making it difficult to visualise underlying structures, whereas 1300 nm OCT clearly visualized both the epithelial cells and the stroma lying beneath. For the DOK cells, hematoxylin and eosin staining confirmed the formation of an epithelial layer frequently presenting an abnormal morphology especially at the epidermal/stromal junction, with features such as infiltrating, bulbous rete pegs. These were more clearly visualized under 890 nm OCT. These observations show that 890 nm OCT retains some of its known advantages of higher contrast between anatomical tissue layers when used to observe dysplastic and malignant 3D oral mucosa constructs. However 1300 nm OCT is confirmed to possess a greater ability to image the full thickness of the model epithelia and in particular it is more suited to imaging through the keratinized layer

A methodology for the production of microfabricated electrospun membranes for the creation of new skin regeneration models

The continual renewal of the epidermis is thought to be related to the presence of populations of epidermal stem cells residing in physically protected microenvironments (rete ridges) directly influenced by the presence of mesenchymal fibroblasts. Current skin in vitro models do acknowledge the influence of stromal fibroblasts in skin reorganisation but the study of the effect of the rete ridge-microenvironment on epidermal renewal still remains a rich topic for exploration. We suggest there is a need for the development of new in vitro models in which to study epithelial stem cell behaviour prior to translating these models into the design of new cell-free biomaterial devices for skin reconstruction. In this study we aimed to develop new prototype epidermal-like layers containing pseudo-rete ridge structures for studying the effect of topographical cues on epithelial cell behaviour. The models were designed using a range of 3D electrospun microfabricated scaffolds. This was achieved via the utilisation of polyethylene glycol diacrylate (PEDGA) to produce a reusable template over which Poly (3-hydrroxybutyrate-co-3-hydroxyvalerate) (PHBV) was electrospun. Initial investigations studied the behaviour of keratinocytes cultured on models using plain scaffolds (without the presence of intricate topography) versus keratinocytes cultured on scaffolds containing micro-features

Raman spectroscopy detects melanoma and the tissue surrounding melanoma using tissue-engineered melanoma models

Invasion of melanoma cells from the primary tumour involves interaction with adjacent tissues and extracellular matrix. The extent of this interaction is not fully understood. In this study Raman spectroscopy was applied to cryo-sections of established 3D models of melanoma in human skin. Principal component analysis was used to investigate differences between the tumour and normal tissue and between the peri-tumour area and the normal skin. Two human melanoma cells lines A375SM and C8161 were investigated and compared in 3D melanoma models. Changes were found in protein conformations and tryptophan configurations across the entire melanoma samples, in tyrosine orientation and in more fluid lipid packing only in tumour dense areas, and in increased glycogen content in the peri-tumour areas of melanoma. Raman spectroscopy revealed changes around the perimeter of a melanoma tumour as well as detecting differences between the tumour and the normal tissue

Ubiquitination of basal VEGFR2 regulates signal transduction and endothelial function

Cell surface receptors can undergo recycling or proteolysis but the cellular decision-making events that sort between these pathways remain poorly defined. Vascular endothelial growth factor A (VEGF-A) and vascular endothelial growth factor receptor 2 (VEGFR2) regulate signal transduction and angiogenesis, but how signaling and proteolysis is regulated is not well understood. Here, we provide evidence that a pathway requiring the E1 ubiquitin-activating enzyme UBA1 controls basal VEGFR2 levels, hence metering plasma membrane receptor availability for the VEGF-A-regulated endothelial cell response. VEGFR2 undergoes VEGF-A-independent constitutive degradation via a UBA1-dependent ubiquitin-linked pathway. Depletion of UBA1 increased VEGFR2 recycling from endosome-to-plasma membrane and decreased proteolysis. Increased membrane receptor availability after UBA1 depletion elevated VEGF-A-stimulated activation of key signaling enzymes such as PLCγ1 and ERK1/2. Although UBA1 depletion caused an overall decrease in endothelial cell proliferation, surviving cells showed greater VEGF-A-stimulated responses such as cell migration and tubulogenesis. Our study now suggests that a ubiquitin-linked pathway regulates the balance between receptor recycling and degradation which in turn impacts on the intensity and duration of VEGF-A-stimulated signal transduction and the endothelial respons

Regulation of VEGFR1 localisation and trafficking in human endothelial cells

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