38 research outputs found

    Modulating stem cell differentiation via cell-material interaction

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    Human mesenchymal stem cells (hMSCs) have become the popular candidate in the field of regenerative medicines due to unique properties like multipotency, ease of availability and nonimmunogenicity. In the past, various biochemical methods have been employed to regulate the stem cell differentiation but shortcomings such as tumorigensis and cell death are associated with these methods. Therefore, in recent years, biophysical induction methods are emerging rapidly to control the stem cell lineage commitment. In biophysical induction methods, stem cell behavior can be modulated by regulating the cell-material interactions but detailed study of these cell-material interactions is still limited to date. Here, in the first part, we studied the cell-material interactions by investigating the spatial distribution of integrin-β1 receptors (ITG-β1) at micro- and nanoscale level and systematic study of the relationship between spatial distribution of ITG-β1 and stem cell differentiation (cardiomyogenesis) was performed. We observed the distinct recruitment of ITG-β1 in hMSCs when hMSCs were committed to myocardial lineage induced by cell patterning. We investigated the spatial distribution of ITG-β1 using super resolution imaging in those committed hMSCs. Aligned and elongated ITG-β1 focal adhesions (ITG-β1 FAs) were found in those committed patterned hMSCs in contrast to short and nonaligned ITG-β1 FAs in unpatterned hMSCs. Nanoscale distribution study of integrins revealed that ITG-β1 clusters were uniformly spread within FAs of patterned hMSCs, whereas ITG-β1 clusters were expressed at the periphery of FAs of unpatterned hMSCs. Further, we deciphered the decisive role of cell patterning in generating the optimal cytoskeletal tension in hMSCs to induce cardiomyogenic differentiation via mechanotransduction pathways. The cell’s mechanical properties (cell stiffness and traction forces) which are indicator of cell cytoskeletal tension were drastically reduced in the committed hMSCs as compared to the non-committed unpatterned hMSCs. This fact suggested the positive correlation between the cell patterning-triggered myocardial differentiation and actomyosin-generated optimal cytoskeletal tension within patterned cells. In the next part, we utilized the same dimensions and spatial distribution data of ITG-β1 FAs to design the unique biofunctionalized gold micropatterned platform and reverse engineer the hMSCs differentiation process. The platform was fabricated by following standard photolithography, bioinert polyethylene glycol (PEG) passivation and precise immobilization of ITG-β1 antibodies to gold pattern lanes. Aligned and elongated morphology was shown by hMSCs cultured on this platform and later these patterned hMSCs displayed end to end fusion to form multinucleated myotubes with continuous actin cytoskeleton after two weeks of culture. Aforementioned results illustrated that cell patterning and ITG-β1 mediated signaling synergistically promoted the myotubes formation from patterned hMSCs. This ITG-β1 antibody immobilized micropatterned platform together with hMSCs is a tissue engineered construct and in future, may find use for a wide range of applications, right from muscle tissue engineering to the investigation of stem cell-material interactions to gain insights into signaling pathways involved in stem cell myogenesis.DOCTOR OF PHILOSOPHY (MSE

    Contact guidance for cardiac tissue engineering using 3D bioprinted gelatin patterned hydrogel

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    Here, we have developed a 3D bioprinted microchanneled gelatin hydrogel that promotes human mesenchymal stem cell (hMSC) myocardial commitment and supports native cardiomyocytes(CMs) contractile functionality. Firstly, we studied the effect of bioprinted microchanneled hydrogel on the alignment, elongation, and differentiation of hMSC. Notably, the cells displayed well defined F-actin anisotropy and elongated morphology on the microchanneled hydrogel, hence showing the effects of topographical control over cell behavior. Furthermore, the aligned stem cells showed myocardial lineage commitment, as detected using mature cardiac markers. The fluorescence-activated cell sorting analysis also confirmed a significant increase in the commitment towards myocardial tissue lineage. Moreover, seeded CMs were found to be more aligned and demonstrated synchronized beating on microchanneled hydrogel as compared to the unpatterned hydrogel. Overall, our study proved that microchanneled hydrogel scaffold produced by 3D bioprinting induces myocardial differentiation of stem cells as well as supports CMs growth and contractility. Applications of this approach may be beneficial for generating in vitro cardiac model systems to physiological and cardiotoxicity studies as well asin vivo generating custom designed cell impregnated constructs for tissue engineering and regenerative medicine applications.NRF (Natl Research Foundation, S’pore)Published versio

    A Solvent-Free Surface Suspension Melt Technique for Making Biodegradable PCL Membrane Scaffolds for Tissue Engineering Applications

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    In tissue engineering, there is limited availability of a simple, fast and solvent-free process for fabricating micro-porous thin membrane scaffolds. This paper presents the first report of a novel surface suspension melt technique to fabricate a micro-porous thin membrane scaffolds without using any organic solvent. Briefly, a layer of polycaprolactone (PCL) particles is directly spread on top of water in the form of a suspension. After that, with the use of heat, the powder layer is transformed into a melted layer, and following cooling, a thin membrane is obtained. Two different sizes of PCL powder particles (100 µm and 500 µm) are used. Results show that membranes made from 100 µm powders have lower thickness, smaller pore size, smoother surface, higher value of stiffness but lower ultimate tensile load compared to membranes made from 500 µm powder. C2C12 cell culture results indicate that the membrane supports cell growth and differentiation. Thus, this novel membrane generation method holds great promise for tissue engineering

    A solvent-free surface suspension melt technique for making biodegradable PCL membrane scaffolds for tissue engineering applications

    No full text
    In tissue engineering, there is limited availability of a simple, fast and solvent-free process for fabricating micro-porous thin membrane scaffolds. This paper presents the first report of a novel surface suspension melt technique to fabricate a micro-porous thin membrane scaffolds without using any organic solvent. Briefly, a layer of polycaprolactone (PCL) particles is directly spread on top of water in the form of a suspension. After that, with the use of heat, the powder layer is transformed into a melted layer, and following cooling, a thin membrane is obtained. Two different sizes of PCL powder particles (100 µm and 500 µm) are used. Results show that membranes made from 100 µm powders have lower thickness, smaller pore size, smoother surface, higher value of stiffness but lower ultimate tensile load compared to membranes made from 500 µm powder. C2C12 cell culture results indicate that the membrane supports cell growth and differentiation. Thus, this novel membrane generation method holds great promise for tissue engineering.ASTAR (Agency for Sci., Tech. and Research, S’pore)Published versio

    Modulating Human Mesenchymal Stem Cell Plasticity Using Micropatterning Technique

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    <div><p>In our previous work, we have reported that enforced elongation of human mesenchymal stem cells (hMSCs) through micropatterning promoted their myocardial lineage commitment. However, whether this approach is robust enough to retain the commitment when subsequently subjected to different conditions remains unsolved. This de-differentiation, if any, would have significant implication on the application of these myocardial-like hMSCs either as tissue engineered product or in stem cell therapy. Herein, we investigated the robustness of micropatterning induced differentiation by evaluating the retention of myocardial differentiation in patterned hMSCs when challenged with non-myocardial differentiation cues. Altogether, we designed four groups of experiments; 1) Patterned hMSCs cultured in normal growth medium serving as a positive control; 2) Patterned hMSCs cultured in normal growth medium for 14 days followed by osteogenic and adipogenic media for next 7 days (to study the robustness of the effect of micropatterning); 3) Patterned hMSCs (initially grown in normal growth medium for 14 days) trypsinized and recultured in different induction media for next 7 days (to study the robustness of the effect of micropatterning without any shape constrain) and 4) Patterned hMSCs cultured in osteogenic and adipogenic media for 14 days (to study the effects of biochemical cues versus biophysical cues). It was found that hMSCs that were primed to commit to myocardial lineage (Groups 2 and 3) were able to maintain myocardial lineage commitment despite subsequent culturing in osteogenic and adipogenic media. However, for hMSCs that were not primed (Group 4), the biochemical cues seem to dominate over the biophysical cue in modulating hMSCs differentiation. It demonstrates that cell shape modulation is not only capable of inducing stem cell differentiation but also ensuring the permanent lineage commitment.</p></div

    Immunostaining of β-MHC and osteocalcin after 21 days of hMSCs culture (14 days in normal growth medium +7 days in osteogenic medium).

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    <p>Patterned cells showed positive expression of β-MHC (<b>a</b>), whereas unpatterned cells stained negatively for β-MHC expression (<b>c</b>). Patterned cells showed negligible signs of osteocalcin expression (<b>b</b>) but significant increase in the level of osteocalcin expression was visualized in unpatterned cells (<b>d</b>). The scale bar is 100 µm.</p

    Validation of tissue-lineage commitment of hMSCs.

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    <p>Tissue-lineage specific markers; osteocalcin and β-MHC along with oil red O dye were used to check hMSCs commitment grown in osteogenic (<b>a, b, e, f</b>) and adipogenic medium (<b>c, d, g, h</b>) for 14 days. Immunostaining results revealed that patterned cells displayed osteocalcin expression (<b>a</b>) and oil globules formation (<b>c</b>) cultured in osteo- and adipogenic medium respectively, while patterned cells grown in osteo- and adipogenic medium failed to express cardiac marker (<b>b, d</b>). Unpatterned cells followed similar trend by showing osteocalcin expression (<b>e</b>), distinct oil globules formation (<b>g</b>) and no indication of β-MHC expression (<b>f, h</b>). The scale bar is 100 µm.</p

    Immunostaining of β-MHC and osteocalcin along with oil droplets detection after 2 weeks of hMSCs culture in normal growth medium.

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    <p>Distinct up-regulation of β-MHC, a cardiac marker (green) was observed in the patterned cells (<b>a</b>) but not for the unpatterned cells (<b>d</b>). No signs of osteocalcin expression (<b>b</b>) and oil droplet formation (<b>c</b>) were observed in cells from patterned group. Unpatterned cells expressed osteocalcin abundantly (red) (<b>e</b>), while no oil droplets formation was observed in unpatterned cells (<b>f</b>). The scale bar is 100 µm.</p
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