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Tissue engineered cardiac grafts (TECG) have been proposed as a platform for systematic in vitro studies of complex stimuli that occur in vivo, and as a means to repair damaged or congenitally defective myocardium. However, clinical application of TECG is currently limited by suboptimal cardiomyocyte (CM) survival, differentiation, and contractility. We tested the hypothesis that insulin-like growth factor-I (IGF) and perfused culture can enhance formation of TECG. Method: Three-dimensional porous scaffolds were placed individually in either Petri dishes or the chamber of a novel modular, perfused bioreactor, seeded with primary neonatal rat heart cells, and cultured in 10 mL of media supplemented or not with 100 ng/mL of IGF for 8 days. Two-factor ANOVA was used to assess significant effects of IGF and perfusion acting individually and in concert. Results: IGF significantly enhanced contractile properties of TECG compared to unsupplemented controls, as assessed by contractile amplitude (p Conclusion: Insulin-like growth factor-I and perfusion culture can improve the viability, differentiation, and contractility of cardiomyocytes in tissue engineered cardiac grafts.

We tested the hypothesis that supplemental regulatory factors can improve the contractile properties and viability of cardiac tissue constructs cultured in vitro. Neonatal rat heart cells were cultured on porous collagen sponges for up to 8 days in basal medium or medium supplemented with insulin-like growth factor-I (IGF), insulin-transferrin-selenium (ITS), platelet-derived growth factor-BB (PDGF), or angiopoietin-1 (ANG). IGF and ITS enhanced contractile properties of the 8-day constructs significantly more than with unsupplemented controls according to contractile amplitude and excitation threshold, and IGF also significantly increased the amount of cardiac troponin-I and enhanced cell viability according to different assays (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), lactate dehydrogenase (LDH), and terminal deoxynucleotidyl transferase biotin-2'-deoxyuridine 5'-triphosphate nick end labeling (TUNEL)). PDGF significantly increased the contractile amplitude of 4-day constructs and enhanced cell viability according to MTT, LDH, and TUNEL; ANG enhanced cell viability according to the LDH assay. Our results demonstrate that supplemental regulatory molecules can differentially enhance properties of cardiac tissue constructs and imply that these constructs can provide a platform for systematic in vitro studies of the effects of complex stimuli that occur in vivo to improve our basic understanding of cardiogenesis and identify underlying mechanisms that can potentially be exploited to enhance myocardial regeneration.

Cancer cell metastases arise due to a series of biological processes starting from malignant tumor cells invading from primary to distal organs. Bone is one of the preferred sites of metastasis for various tumors including breast cancer. Understanding metastatic invasion to the secondary site requires studying the individual steps in metastasis, particularly those leading up to and immediately following extravasation of a circulating tumor cell from a blood vessel into the remote tissue. Microfluidic systems can provide cells with a controllable and reproducible 3D bone micro-environment to study cancer extravasation to a degree not possible by standard tissue culture methods while also simultaneously providing in situ imaging capabilities for visualization. We investigate the critical steps of cancer extravasation: tumor cells adhering to the endothelium and subsequently transmigrate across it into a hydrogel model for the bone extracellular space. Our model system consists of a functional microvascular network generated through vasculogenesis in a bone-mimicking microenvironment all within a microfluidic system. The microfluidic system contains a central hydrogel region flanked by two lateral media channels. The hydrogel region is filled with cells dispersed inside a fibrin gel. The two channel system enables easy access to the microvascular network for the addition of cancer cells for extravasation. Cells of passage 6 or lower MSCs, harvested from patients undergoing hip arthroplasty, were differentiated to osteo lineage. GFP-human umbilical vein endothelial cells (HUVECs) were suspended at 2·107 cells/ml in EMG2 + thrombin and combined with osteo-differentiated and non-differentiated MSCs mixed at a 9:1 ratio, and inserted into the gel channel to complete the seeding. RFP-expressing BOKL, which is an MDA-MB-231 cell line specific to bone metastasis enabled live-cell imaging via fluorescent microscopy. We created a functional microvascular network in a bone cell-conditioned microenvironment with the addition of osteo-differentiated and non-differentiated MSCs. Immunofluorescent imaging of α-smooth muscle actin in the system confirmed the presence of mural cells differentiated from MSCs wrapped around the generated vascular network. Visual confirmation of 3μm microspheres flowing through the vessels confirmed that the vascular network generated in this system was indeed perfusable. Finally, the osteo-cell conditioned microenvironment was confirmed by staining for osteocalcin. BOKL were then perfused into the microvasculature to model the extravasation process. The percentage of cancer cell extravasation in a device containing a microvascular network seeded with osteo-differentiated MSCs was 3.8 fold higher (56.5±4.8%) than the case with HUVECs alone (14.7±3.7%). Permeability values of the vasculature were analyzed by monitoring the leakage of fluorescent 70kDa dextran from the microvessels over time. Vasculature in the bone cell-conditioned microenvironment exhibited a higher permeability (4.12±0.75)·10-6 cm/s compared to the HUVEC only condition (0.89±0.31)·10-6 cm/s. While the increase in extravasation rates may due to a combination of factors, the higher permeability of the vasculature in osteo-cell conditioned environment may also be one of the contributing factors. Microfluidics offers the capability to create organ-specific microenvironments that can capture extravasation events, leading to tumor metastasis. This system therefore captures certain aspects of cell-cell and cell-matrix interactions for preferential metastasis to bone, offering potential for drug screening. Citation Format: Jessie S. Jeon, Simone Bersini, Gabriele Dubini, Joseph Charest, Matteo Moretti, Roger D. Kamm. Extravasation of breast cancer cells to a bone-cell conditioned microenvironment in functional 3D microvascular networks generated by vasculogenesis in a microfluidic system. [abstract]. In: Abstracts: AACR Special Conference on Cellular Heterogeneity in the Tumor Microenvironment; 2014 Feb 26-Mar 1; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2015;75(1 Suppl):Abstract nr B23. doi:10.1158/1538-7445.CHTME14-B23

We investigated whether, and under which conditions (i.e., cell-seeding density, medium supplements), in vitro preculture enhances in vivo development of human engineered cartilage in an ectopic nude mouse model. Monolayer-expanded adult human articular chondrocytes (AHACs) were seeded into Hyalograft C disks at 1.3 x 10(7) cells/cm3 (low density) or 7.6 x 10(7) cells/cm3 (high density). Constructs were directly implanted subcutaneously in nude mice for up to 8 weeks or precultured for 2 weeks before implantation. Preculture medium contained either transforming growth factor-beta1 (TGF-beta1, 1 ng/mL), fibroblast growth factor-2, and platelet-derived growth factor (proliferating medium) or TGF-beta1 (10 ng/mL) and insulin (differentiating medium). Both in vitro and after in vivo implantation, constructs derived by cell seeding at high versus low density and precultured in differentiating versus proliferating medium generated more cartilaginous tissues containing higher amounts of glycosaminoglycan and collagen type II and lower amounts of collagen type I, and with higher equilibrium moduli. As compared with direct implantation of freshly seeded scaffolds, preculture of AHAC-Hyalograft C constructs in differentiating medium, but not in proliferating medium, supported enhanced in vivo development of engineered cartilage. The effect of preculture was more pronounced when constructs were seeded at low density as compared with high density. This study indicates that preculture of human engineered cartilage in differentiating medium has the potential to provide grafts with higher equilibrium moduli and enhanced in vivo developmental capacity than freshly seeded scaffolds. These findings need to be validated in an orthotopic model system.

Structural and functional characterization of integrative cartilage repair in controlled model systems can play a key role in the development of innovative strategies to improve the long-term outcome of many cartilage repair procedures. In this work, we first developed a method to reproducibly generate geometrically defined disk/ring cartilage composites and to remove outgrown fibrous layers which can encapsulate cartilaginous tissues during culture. We then used the model system to test the hypothesis that such fibrous layers lead to an overestimation of biomechanical parameters of integration at the disk/ring interface. Transmission electron microscopy images of the composites after 6 weeks of culture indicated that collagen fibrils in the fibrous tissue layer were well integrated into the collagen network of the cartilage disk and ring, whereas molecular bridging between opposing disk/ring cartilage surfaces was less pronounced and restricted to regions with narrow interfacial regions (< 2 microm). Stress-strain profiles generated from mechanical push-out tests for composites with the layers removed displayed a single and distinct peak, whereas profiles for composites with the layers left intact consisted of multiple superimposed peaks. As compared to composites with removed layers, composites with intact layers had significantly higher adhesive strengths (161+/-9 vs. 71+/-11 kPa) and adhesion energies (15.0+/-0.7 vs. 2.7+/-0.4 mJ/mm2). By combining structural and functional analyses, we demonstrated that the outgrowing tissue formed during in vitro culture of cartilaginous specimens should be eliminated in order to reliably quantify biomechanical parameters related to integrative cartilage repair.

Cartilage integration in vivo does not occur, such that even cartilage fissures do not heal. This could be due not only to the limited access of chondrocytes to the wound, but also to exogenous factors. In this paper, we tested the hypothesis that lubricin, a lubricating protein physiologically present in the synovial fluid, reduces the integrative cartilage repair capacity. Disk/ring composites of bovine articular cartilage were prepared using concentric circular blades and cultured for 6 weeks with or without treatment with 250 microg/ml lubricin applied three times per week. Following culture, the percentage of contact area between the disks and the rings, as assessed by light microscopy, were equal in both groups. The adhesive strength of the integration interface, as assessed by push-out mechanical tests, was markedly and significantly lower in lubricin-treated specimens (2.5 kPa) than in the controls (28.7 kPa). Histological observation of Safranin-O stained cross-sections confirmed the reduced integration in the lubricin treated composites. Our findings suggest that the synovial milieu, by providing lubrication of cartilage surfaces, impairs cartilage--cartilage integration.

We used anti-α7 and anti-α8 subunit specific antibodies to study a-bungarotoxin (α-BTX) receptors at different developmental stages in chick optic lobe and retina. We found that only the α7 and α7-α8 subtypes are present at all developmental stages in chick optic lobe, whereas in addition to these two subtypes, chick retina contains an α8 subtype representing 50% of all α-BTX receptors at 1 day post hatching. By using an immunoaffinity column, we purified these subtypes and studied their biochemical and functional properties by reconstituting them in planar lipid bilayer. The a7 and α7–α8 receptor subtypes are made up of two subunits: a 57000 dalton subunit which binds α-BTX, and a 52000 dalton subunit whose function and structure is unknown. The three subtypes differ in pharmacological and biophysical properties.

Publisher Summary Neurotoxins play a fundamental role in isolating nicotinic receptors and in understanding their key features. This chapter summarizes some of those properties that are relevant to the isolation of α -bungarotoxin receptors (BgTXRs). It also describes a method for the purification of relatively large amounts of chick optic lobe (COL) BgTXR in a very pure form. Several factors are critical for obtaining high yields of pure BgTXRs—the blocking of proteolysis by using protease inhibitors of different specificities, and the elimination of tubulin and probably other contaminations by repeated washings with large volumes of high ionic strength buffer and pre-incubation of the extract with cytochrome c -sepharose. Tubulin contamination is a serious problem in BgTXR purification from nervous tissues and can lead to tubulin peptides being mistaken for BgTXR peptides. When neuronal tissues other than COL are used, the contaminants of the BgTXR can be slightly different and thus the isolation procedure should be tailored to each type of tissue.

Cell-based therapies have recently been proposed for the treatment of degenerative articular pathologies, such as early osteoarthritis, with an emphasis on autologous mesenchymal stem cells (MSCs), as an alternative to terminally differentiated cells. In this study, we performed a donor-matched comparison between infrapatellar fat pad MSCs (IFP-MSCs) and knee subcutaneous adipose tissue stem cells (ASCs), as appealing candidates for cell-based therapies that are easily accessible during surgery. IFP-MSCs and ASCs were obtained from 25 osteoarthritic patients undergoing total knee replacement and compared for their immunophenotype and differentiative potential. Undifferentiated IFP-MSCs and ASCs displayed the same immunophenotype, typical of MSCs (CD13+/CD29+/CD44+/CD73+/CD90+/CD105+/CD166+/CD31-/CD45-). IFP-MSCs and ASCs showed similar adipogenic potential, though undifferentiated ASCs had higher LEP expression compared to IFP-MSCs (p < 0.01). Higher levels of calcified matrix (p < 0.05) and alkaline phosphatase (p < 0.05) in ASCs highlighted their superior osteogenic commitment compared to IFP-MSCs. Conversely, IFP-MSCs pellets showed greater amounts of glycosaminoglycans (p < 0.01) and superior expression of ACAN (p < 0.001), SOX9, COMP (p < 0.001) and COL2A1 (p < 0.05) compared to ASCs pellets, revealing a superior chondrogenic potential. This was also supported by lower COL10A1 (p < 0.05) and COL1A1 (p < 0.01) expression and lower alkaline phosphatase release (p < 0.05) by IFP-MSCs compared to ASCs. The observed dissimilarities between IFP-MSCs and ASCs show that, despite expressing similar surface markers, MSCs deriving from different fat depots in the same surgical site possess specific features. Furthermore, the in vitro peculiar commitment of IFP-MSCs and ASCs from osteoarthritic donors towards the chondrogenic or osteogenic lineage may suggest a preferential use for cartilage and bone cell-based treatments, respectively.

Cardiac toxicity still represents a common adverse outcome causing drug attrition and post-marketing withdrawal. The development of relevant in vitro models resembling the human heart recently opened the path towards a more accurate detection of drug-induced human cardiac toxicity early in the drug development process. Organs-on-chip have been proposed as promising tools to recapitulate in vitro the key aspects of the in vivo cardiac physiology and to provide a means to directly analyze functional readouts. In this scenario, a new device capable of continuous monitoring of electrophysiological signals from functional in vitro human hearts-on-chip is here presented. The development of cardiac microtissues was achieved through a recently published method to control the mechanical environment, while the introduction of a technology consisting in micro-electrode coaxial guides allowed to conduct direct and non-destructive electrophysiology studies. The generated human cardiac microtissues exhibited synchronous spontaneous beating, as demonstrated by multi-point and continuous acquisition of cardiac field potential, and expression of relevant genes encoding for cardiac ion-channels. A proof-of-concept pharmacological validation on three drugs proved the proposed model to potentially be a powerful tool to evaluate functional cardiac toxicity.

Background Bone metastases arise in nearly 70% of patients with advanced breast cancer, but the complex metastatic process has not been completely clarified yet. RANKL/RANK/OPG pathway modifications and the crosstalk between metastatic cells and bone have been indicated as potential drivers of the process. Interactions between tumor and bone cells have been studied in vivo and in vitro, but specific effects of the direct contact between human metastatic cells and human bone cells on RANKL/RANK/OPG pathway have not been investigated. Findings We directly co-cultured bone metastatic human breast cancer cells (BOKL) with osteo-differentiated human mesenchymal cells (BMSCs) from 3 different donors. BMSCs and BOKL were then enzymatically separated and FACS sorted. We found a significant increase in the RANKL/OPG ratio as compared to control, which was not observed in BOKL cultured in medium conditioned by BMSCs, neither in BOKL directly cultured with fibroblasts or medium conditioned by fibroblasts. Direct co-culture with osteo-differentiated BMSCs caused BOKL aggregation while proliferation was not affected by co-culture. To more specifically associate RANKL expression to osteogenic differentiation degree of BMSCs, we determined their osteogenic markers expression and matrix calcification relative to osteoblasts and fibroblasts. Conclusions In conclusion, our co-culture model allowed to demonstrate for the first time that direct contact but not paracrine interactions between human metastatic breast cancer cells and bone cells has a significant effect on RANKL/OPG expression in bone metastatic cells. Furthermore, only direct contact with the bone microenvironment induced BOKL clustering without however significantly influencing their proliferation and migration. Electronic supplementary material The online version of this article (doi:10.1186/1476-4598-13-238) contains supplementary material, which is available to authorized users.