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Bone diseases such as osteoporosis, delayed or impaired bone healing, and osteoarthritis still represent a social, financial, and personal burden for affected patients and society. Fully humanized in vitro 3D models of cancellous bone tissue are needed to develop new treatment strategies and meet patient-specific needs. Here, we demonstrate a successful cell-sheet-based process for optimized mesenchymal stromal cell (MSC) seeding on a β-tricalcium phosphate (TCP) scaffold to generate 3D models of cancellous bone tissue. Therefore, we seeded MSCs onto the β-TCP scaffold, induced osteogenic differentiation, and wrapped a single osteogenically induced MSC sheet around the pre-seeded scaffold. Comparing the wrapped with an unwrapped scaffold, we did not detect any differences in cell viability and structural integrity but a higher cell seeding rate with osteoid-like granular structures, an indicator of enhanced calcification. Finally, gene expression analysis showed a reduction in chondrogenic and adipogenic markers, but an increase in osteogenic markers in MSCs seeded on wrapped scaffolds. We conclude from these data that additional wrapping of pre-seeded scaffolds will provide a local niche that enhances osteogenic differentiation while repressing chondrogenic and adipogenic differentiation. This approach will eventually lead to optimized preclinical in vitro 3D models of cancellous bone tissue to develop new treatment strategies. [ABSTRACT FROM AUTHOR]

3D culture systems have the ability to mimic the natural microenvironment by allowing better cell-cell interactions. We have prepared an in vitro 3D osteogenic cell culture model using human adipose derived stem cells (hASCs) cultured atop recombinant elastin-like polypeptide (ELP) conjugated to a charged polyelectrolyte, polyethyleneimine (PEI). We demonstrate that hASCs cultured atop the ELP-PEI coated tissue culture polystyrene (TCPS) formed 3D spheroids and exhibited superior differentiation toward osteogenic lineage compared to the traditional two dimensional (2D) monolayer formed atop uncoated TCPS. Live/dead viability assay confirmed >90% live cells at the end of the 3-week culture period. Over the same culture period, higher protein content was observed in 2D monolayer than 3D spheroids, as the 2D environment allowed continued proliferation, while 3D spheroids underwent contact-inhibited growth arrest. The normalized alkaline phosphatase (ALP) activity, which is an indicator for early osteogenic differentiation was higher for 3D spheroids. The normalized osteocalcin (OCN) production, which is an indicator for osteogenic maturation was also higher for 3D spheroids while 2D monolayer had no noticeable OCN production. On day 22, increased Alizarin red uptake by 3D spheroids showed greater mineralization activity than 2D monolayer. Taken together, these results indicate a superior osteogenic differentiation of hASCs in 3D spheroid culture atop ELP-PEI coated TCPS surfaces than the 2D monolayer formed on uncoated TCPS surfaces. Such enhanced osteogenesis in 3D spheroid stem cell culture may serve as an alternative to 2D culture by providing a better microenvironment for the enhanced cellular functions and interactions in bone tissue engineering. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 105A: 1230-1236, 2017. [ABSTRACT FROM AUTHOR]

In the last few years microfluidics and microfabrication technique principles have been extensively exploited for biomedical applications. In this framework, organs-on-a-chip represent promising tools to reproduce key features of functional tissue units within microscale culture chambers. These systems offer the possibility to investigate the effects of biochemical, mechanical, and electrical stimulations, which are usually applied to enhance the functionality of the engineered tissues. Since the functionality of muscle tissues relies on the 3D organization and on the perfect coupling between electrochemical stimulation and mechanical contraction, great efforts have been devoted to generate biomimetic skeletal and cardiac systems to allow high-throughput pathophysiological studies and drug screening. This review critically analyzes microfluidic platforms that were designed for skeletal and cardiac muscle tissue engineering. Our aim is to highlight which specific features of the engineered systems promoted a typical reorganization of the engineered construct and to discuss how promising design solutions exploited for skeletal muscle models could be applied to improve cardiac tissue models and vice versa. [ABSTRACT FROM AUTHOR]

An in vitro three-dimensional (3D) cell culture system that can mimic organ and tissue structure and function in vivo will be of great benefit for drug discovery and toxicity testing. In this study, the neuroprotective properties of the three most prevalent flavonoid monomers extracted from EGb 761 (isorharmnetin, kaempferol, and quercetin) were investigated using the developed 3D stem cell-derived neural co-culture model. Rat neural stem cells were differentiated into co-culture of both neurons and astrocytes at an equal ratio in the developed 3D model and standard two-dimensional (2D) model using a two-step differentiation protocol for 14 days. The level of neuroprotective effect offered by each flavonoid was found to be aligned with its effect as an antioxidant and its ability to inhibit Caspase-3 activity in a dose-dependent manner. Cell exposure to quercetin (100 µM) following oxidative insult provided the highest levels of neuroprotection in both 2D and 3D models, comparable with exposure to 100 µM of Vitamin E, whilst exposure to isorhamnetin and kaempferol provided a reduced level of neuroprotection in both 2D and 3D models. At lower dosages (10 µM flavonoid concentration), the 3D model was more representative of results previously reported in vivo. The co-cultures of stem cell derived neurons and astrocytes in 3D hydrogel scaffolds as an in vitro neural model closely replicates in vivo results for routine neural drug toxicity and efficacy testing. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:735-744, 2016 [ABSTRACT FROM AUTHOR]

Angiopoietin-like 4 (ANGPTL4) is a target of hypoxia that accumulates in the endothelial extracellular matrix. While ANGPTL4 is known to regulate angiogenesis and vascular permeability, its context-dependent role related to vascular endothelial growth factor (VEGF) has been suggested in capillary morphogenesis. We here thus develop in vitro 3D models coupled to imaging and morphometric analysis of capillaries to decipher ANGPTL4 functions either alone or in the presence of VEGF. ANGPTL4 induces the formation of barely branched and thin endothelial capillaries that display linear adherens junctions. However, ANGPTL4 counteracts VEGF-induced formation of abundant ramified capillaries presenting cell–cell junctions characterized by VE-cadherin containing reticular plaques and serrated structures. We further deciphered the early angiogenesis steps regulated by ANGPTL4. During the initial activation of endothelial cells, ANGPTL4 alone induces cell shape changes but limits the VEGF-induced cell elongation and unjamming. In the growing sprout, ANGPTL4 maintains cohesive VE-cadherin pattern and sustains moderate 3D cell migration but restricts VEGF-induced endothelium remodeling and cell migration. This effect is mediated by differential short- and long-term regulation of P-Y1175-VEGFR2 and ERK1-2 signaling by ANGPTL4. Our in vitro 3D models thus provide the first evidence that ANGPTL4 induces a specific capillary morphogenesis but also overcomes VEGF effect. [ABSTRACT FROM AUTHOR]

The role of astrocytes in the glioblastoma (GBM) microenvironment is poorly understood; particularly with regard to cell invasion and drug resistance. To assess this role of astrocytes in GBMs we established an all human 2D co-culture model and a 3D hyaluronic acid-gelatin based hydrogel model (HyStem™-HP) with different ratios of GBM cells to astrocytes. A contact co-culture of fluorescently labelled GBM cells and astrocytes showed that the latter promotes tumour growth and migration of GBM cells. Notably, the presence of non-neoplastic astrocytes in direct contact, even in low amounts in co-culture, elicited drug resistance in GBM. Recent studies showed that non-neoplastic cells can transfer mitochondria along tunneling nanotubes (TNT) and rescue damaged target cancer cells. In these studies, we explored TNT formation and mitochondrial transfer using 2D and 3D in vitro co-culture models of GBM and astrocytes. TNT formation occurs in glial fibrillary acidic protein (GFAP) positive "reactive" astrocytes after 48 h co-culture and the increase of TNT formations was greater in 3D hyaluronic acid-gelatin based hydrogel models. This study shows that human astrocytes in the tumour microenvironment, both in 2D and 3D in vitro co-culture models, could form TNT connections with GBM cells. We postulate that the association on TNT delivery non-neoplastic mitochondria via a TNT connection may be related to GBM drug response as well as proliferation and migration. [ABSTRACT FROM AUTHOR]