Influence of multi-axial dynamic constraint on cell alignment and contractility in engineered tissues

AbstractIn this study an experimental rig is developed to investigate the influence of tissue constraint and cyclic loading on cell alignment and active cell force generation in uniaxial and biaxial engineered tissues constructs. Addition of contractile cells to collagen hydrogels dramatically increases the measured forces in uniaxial and biaxial constructs under dynamic loading. This increase in measured force is due to active cell contractility, as is evident from the decreased force after treatment with cytochalasin-D. Prior to dynamic loading, cells are highly aligned in uniaxially constrained tissues but are uniformly distributed in biaxially constrained tissues, demonstrating the importance of tissue constraints on cell alignment. Dynamic uniaxial stretching resulted in a slight increase in cell alignment in the centre of the tissue, whereas dynamic biaxial stretching had no significant effect on cell alignment. Our active modelling framework accurately predicts our experimental trends and suggests that a slightly higher (3%) total SF formation occurs at the centre of a biaxial tissue compared to the uniaxial tissue. However, high alignment of SFs and lateral compaction in the case of the uniaxially constrained tissue results in a significantly higher (75%) actively generated cell contractile stress, compared to the biaxially constrained tissue. These findings have significant implications for engineering of contractile tissue constructs.

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Biological cell alignment for electrofusion

IEE Proceedings A Science Measurement and Technology ◽

10.1049/ip-a-3.1992.0020 ◽

1992 ◽

Vol 139 (3) ◽

pp. 112

Author(s):

S.J. MacDonald ◽

P.S. Bodger ◽

P.A. Elder

Keyword(s):

Biological Cell ◽

Cell Alignment

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Long-Term Severe In Vitro Hypoxia Exposure Enhances the Vascularization Potential of Human Adipose Tissue-Derived Stromal Vascular Fraction Cell Engineered Tissues

International Journal of Molecular Sciences ◽

10.3390/ijms22157920 ◽

2021 ◽

Vol 22 (15) ◽

pp. 7920

Author(s):

Myroslava Mytsyk ◽

Giulia Cerino ◽

Gregory Reid ◽

Laia Gili Sole ◽

Friedrich S. Eckstein ◽

...

Keyword(s):

Adipose Tissue ◽

Therapeutic Potential ◽

Stromal Vascular Fraction ◽

Human Adipose Tissue ◽

Bioreactor Culture ◽

Proliferating Cells ◽

Angiogenic Potential ◽

Engineered Tissues

The therapeutic potential of mesenchymal stromal/stem cells (MSC) for treating cardiac ischemia strongly depends on their paracrine-mediated effects and their engraftment capacity in a hostile environment such as the infarcted myocardium. Adipose tissue-derived stromal vascular fraction (SVF) cells are a mixed population composed mainly of MSC and vascular cells, well known for their high angiogenic potential. A previous study showed that the angiogenic potential of SVF cells was further increased following their in vitro organization in an engineered tissue (patch) after perfusion-based bioreactor culture. This study aimed to investigate the possible changes in the cellular SVF composition, in vivo angiogenic potential, as well as engraftment capability upon in vitro culture in harsh hypoxia conditions. This mimics the possible delayed vascularization of the patch upon implantation in a low perfused myocardium. To this purpose, human SVF cells were seeded on a collagen sponge, cultured for 5 days in a perfusion-based bioreactor under normoxia or hypoxia (21% and <1% of oxygen tension, respectively) and subcutaneously implanted in nude rats for 3 and 28 days. Compared to ambient condition culture, hypoxic tension did not alter the SVF composition in vitro, showing similar numbers of MSC as well as endothelial and mural cells. Nevertheless, in vitro hypoxic culture significantly increased the release of vascular endothelial growth factor (p < 0.001) and the number of proliferating cells (p < 0.00001). Moreover, compared to ambient oxygen culture, exposure to hypoxia significantly enhanced the vessel length density in the engineered tissues following 28 days of implantation. The number of human cells and human proliferating cells in hypoxia-cultured constructs was also significantly increased after 3 and 28 days in vivo, compared to normoxia. These findings show that a possible in vivo delay in oxygen supply might not impair the vascularization potential of SVF- patches, which qualifies them for evaluation in a myocardial ischemia model.

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Neovascularization of engineered tissues for clinical translation: Where we are, where we should be?

APL Bioengineering ◽

10.1063/5.0044027 ◽

2021 ◽

Vol 5 (2) ◽

pp. 021503

Author(s):

Muhammad Anwaar Nazeer ◽

Ismail Can Karaoglu ◽

Onur Ozer ◽

Cem Albayrak ◽

Seda Kizilel

Keyword(s):

Clinical Translation ◽

Engineered Tissues

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Perfusion and endothelialization of engineered tissues with patterned vascular networks

Nature Protocols ◽

10.1038/s41596-021-00533-1 ◽

2021 ◽

Author(s):

Ian S. Kinstlinger ◽

Gisele A. Calderon ◽

Madison K. Royse ◽

A. Kristen Means ◽

Bagrat Grigoryan ◽

...

Keyword(s):

Vascular Networks ◽

Engineered Tissues

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Thermoplastic Elastomer (TPE)–Poly(Methyl Methacrylate) (PMMA) Hybrid Devices for Active Pumping PDMS-Free Organ-on-a-Chip Systems

Biosensors ◽

10.3390/bios11050162 ◽

2021 ◽

Vol 11 (5) ◽

pp. 162

Author(s):

Mathias Busek ◽

Steffen Nøvik ◽

Aleksandra Aizenshtadt ◽

Mikel Amirola-Martinez ◽

Thomas Combriat ◽

...

Keyword(s):

Methyl Methacrylate ◽

Drug Testing ◽

Low Cost ◽

Thermoplastic Elastomer ◽

Bonding Process ◽

Cell Alignment ◽

Poly Methyl Methacrylate ◽

Bonding Parameters ◽

Organ On A Chip ◽

Hybrid Devices

Polydimethylsiloxane (PDMS) has been used in microfluidic systems for years, as it can be easily structured and its flexibility makes it easy to integrate actuators including pneumatic pumps. In addition, the good optical properties of the material are well suited for analytical systems. In addition to its positive aspects, PDMS is well known to adsorb small molecules, which limits its usability when it comes to drug testing, e.g., in organ-on-a-chip (OoC) systems. Therefore, alternatives to PDMS are in high demand. In this study, we use thermoplastic elastomer (TPE) films thermally bonded to laser-cut poly(methyl methacrylate) (PMMA) sheets to build up multilayered microfluidic devices with integrated pneumatic micro-pumps. We present a low-cost manufacturing technology based on a conventional CO2 laser cutter for structuring, a spin-coating process for TPE film fabrication, and a thermal bonding process using a pneumatic hot-press. UV treatment with an Excimer lamp prior to bonding drastically improves the bonding process. Optimized bonding parameters were characterized by measuring the burst load upon applying pressure and via profilometer-based measurement of channel deformation. Next, flow and long-term stability of the chip layout were measured using microparticle Image Velocimetry (uPIV). Finally, human endothelial cells were seeded in the microchannels to check biocompatibility and flow-directed cell alignment. The presented device is compatible with a real-time live-cell analysis system.

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Akt signaling is activated by TGFβ2 and impacts tenogenic induction of mesenchymal stem cells

Stem Cell Research & Therapy ◽

10.1186/s13287-021-02167-2 ◽

2021 ◽

Vol 12 (1) ◽

Cited By ~ 1

Author(s):

Sophia K. Theodossiou ◽

Jett B. Murray ◽

LeeAnn A. Hold ◽

Jeff M. Courtright ◽

Anne M. Carper ◽

...

Keyword(s):

Stem Cells ◽

Mesenchymal Stem Cells ◽

Signaling Pathways ◽

Transforming Growth Factor ◽

Akt Signaling ◽

Cell Alignment ◽

Limited Information ◽

Akt Inhibitor ◽

Tenogenic Differentiation

Abstract Background Tissue engineered and regenerative approaches for treating tendon injuries are challenged by the limited information on the cellular signaling pathways driving tenogenic differentiation of stem cells. Members of the transforming growth factor (TGF) β family, particularly TGFβ2, play a role in tenogenesis, which may proceed via Smad-mediated signaling. However, recent evidence suggests some aspects of tenogenesis may be independent of Smad signaling, and other pathways potentially involved in tenogenesis are understudied. Here, we examined the role of Akt/mTORC1/P70S6K signaling in early TGFβ2-induced tenogenesis of mesenchymal stem cells (MSCs) and evaluated TGFβ2-induced tenogenic differentiation when Smad3 is inhibited. Methods Mouse MSCs were treated with TGFβ2 to induce tenogenesis, and Akt or Smad3 signaling was chemically inhibited using the Akt inhibitor, MK-2206, or the Smad3 inhibitor, SIS3. Effects of TGFβ2 alone and in combination with these inhibitors on the activation of Akt signaling and its downstream targets mTOR and P70S6K were quantified using western blot analysis, and cell morphology was assessed using confocal microscopy. Levels of the tendon marker protein, tenomodulin, were also assessed. Results TGFβ2 alone activated Akt signaling during early tenogenic induction. Chemically inhibiting Akt prevented increases in tenomodulin and attenuated tenogenic morphology of the MSCs in response to TGFβ2. Chemically inhibiting Smad3 did not prevent tenogenesis, but appeared to accelerate it. MSCs treated with both TGFβ2 and SIS3 produced significantly higher levels of tenomodulin at 7 days and morphology appeared tenogenic, with localized cell alignment and elongation. Finally, inhibiting Smad3 did not appear to impact Akt signaling, suggesting that Akt may allow TGFβ2-induced tenogenesis to proceed during disruption of Smad3 signaling. Conclusions These findings show that Akt signaling plays a role in TGFβ2-induced tenogenesis and that tenogenesis of MSCs can be initiated by TGFβ2 during disruption of Smad3 signaling. These findings provide new insights into the signaling pathways that regulate tenogenic induction in stem cells.

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