Introducing height to mechanobiology:A tissue engineering perspective

Cell behavior is relatively well studied in 2D petridishes, but not in a 3D environment. Increasing evidence shows a large difference in cell behavior in 3D vs 2D. This thesis is focussed on exploring the extent and consequences of these differences in tissue stiff engineering scaffolds. A large influence of the 3D environment on mechanobiological proteins was found and it was shown that these proteins have a different role in 3D than in 2D. On top of this, several novel scaffolds were developed that can help to advance 3D biological research

Fiber diameter, porosity and functional group gradients in electrospun scaffolds

Developing, homeostatic, and regenerating tissues are full of various gradients, including mechanical, chemical, porosity and growth-factor gradients. However, it remains challenging to replicate these gradients using common tissue engineering approaches. Here, we use electrospinning to create scaffolds with in-depth gradients. We created a fiber diameter gradient and pore size gradient throughout the depth of electrospun (ESP) scaffolds by a continuous gradient of polymer concentration. As an alternative to this established method, we developed a novel method to create fiber diameter gradients by changing the voltage on both needle and collector, keeping the total voltage constant. In this way, fiber diameter could be changed in a gradient matter by focusing the electrospinning spot. Using this method, we created a fiber diameter and pore size gradient, while keeping all other parameters constant. Lastly, we developed a novel method to create functional group gradients, which can potentially be used in a wide variety of polymer solutions to couple peptides and proteins to ESP scaffolds. A scaffold with an in-depth gradient of functional groups was created by adding functionalized poly(ethylene glycol) additives to the polymer solution, a novel method with potentially wide applications. The techniques demonstrated here could be applied to a wide variety of polymers and applications and can aid in developing physiologically relevant gradient scaffolds.</p

Dimensionality changes actin network through lamin A/C and zyxin

Mechanosensing proteins have mainly been investigated in 2D culture platforms, while understanding their regulation in 3D enviroments is critical for tissue engineering. Among mechanosensing proteins, the actin cytoskeleton plays a key role in human mesenchymal stromal cells (hMSCs) activity, but its regulation in 3D tissue engineered scaffolds remains poorly studied. Here, we show that human mesenchymal stromal cells (hMSCs) cultured on 3D electrospun scaffolds made of a stiff material do not form actin stress fibers, contrary to hMSCs on 2D films of the same material. On 3D electrospun and additive manufactured scaffolds, hMSCs also displayed fewer focal adhesions, lower lamin A and C expression and less YAP1 nuclear localization and myosin light chain phosphorylation. Together, this strongly suggests that dimensionality prevents the build-up of cellular tension, even on stiff materials. Knock down of either lamin A and C or zyxin resulted in fewer stress fibers in the cell center. Zyxin knock down reduced lamin A and C expression, but not vice versa, showing that this signal chain starts from the outside of the cell. Lineage commitment was not affected by the lack of these important osteogenic proteins in 3D, as all cells committed to osteogenesis in bi-potential medium. Our study demonstrates that dimensionality changes the actin cytoskeleton through lamin A and C and zyxin, and highlights the difference in the regulation of lineage commitment in 3D enviroments. Together, these results can have important implications for future scaffold design for both stiff- and soft tissue engineering constructs

Mechanosensitive regulation of stanniocalcin-1 by zyxin and actin-myosin in human mesenchymal stromal cells

Stanniocalcin-1 (STC1) secreted by mesenchymal stromal cells (MSCs) has anti-inflammatory functions, reduces apoptosis, and aids in angiogenesis, both in vitro and in vivo. However, little is known about the molecular mechanisms of its regulation. Here, we show that STC1 secretion is increased only under specific cell-stress conditions. We find that this is due to a change in actin stress fibers and actin-myosin tension. Abolishment of stress fibers by blebbistatin and knockdown of the focal adhesion protein zyxin leads to an increase in STC1 secretion. To also study this connection in 3D, where few focal adhesions and actin stress fibers are present, STC1 expression was analyzed in 3D alginate hydrogels and 3D electrospun scaffolds. Indeed, STC1 secretion was increased in these low cellular tension 3D environments. Together, our data show that STC1 does not directly respond to cell stress, but that it is regulated through mechanotransduction. This research takes a step forward in the fundamental understanding of STC1 regulation and can have implications for cell-based regenerative medicine, where cell survival, anti-inflammatory factors and angiogenesis are critical

Immunodeficiency in Bloom’s Syndrome

Bloom’s syndrome (BS) is an autosomal recessive disease, caused by mutations in the BLM gene. This gene codes for BLM protein, which is a helicase involved in DNA repair. DNA repair is especially important for the development and maturation of the T and B cells. Since BLM is involved in DNA repair, we aimed to study if BLM deficiency affects T and B cell development and especially somatic hypermutation (SHM) and class switch recombination (CSR) processes. Clinical data of six BS patients was collected, and immunoglobulin serum levels were measured at different time points. In addition, we performed immune phenotyping of the B and T cells and analyzed the SHM and CSR in detail by analyzing IGHA and IGHG transcripts using next-generation sequencing. The serum immunoglobulin levels were relatively low, and patients had an increased number of infections. The absolute number of T, B, and NK cells were low but still in the normal range. Remarkably, all BS patients studied had a high percentage (20–80%) of CD4+ and CD8+ effector memory T cells. The process of SHM seems normal; however, the Ig subclass distribution was not normal, since the BS patients had more IGHG1 and IGHG3 transcripts. In conclusion, BS patients have low number of lymphocytes, but the immunodeficiency seems relatively mild since they have no severe or opportunistic infections. Most changes in the B cell development were seen in the CSR process; however, further studies are necessary to elucidate the exact role of BLM in CSR

Actomyosin and the MRTF-SRF pathway downregulate FGFR1 in mesenchymal stromal cells

Both biological and mechanical signals are known to influence cell proliferation. However, biological signals are mostly studied in two-dimensions (2D) and the interplay between these different pathways is largely unstudied. Here, we investigated the influence of the cell culture environment on the response to bFGF, a widely studied and important proliferation growth factor. We observed that human mesenchymal stromal cells (hMSCs), but not fibroblasts, lose the ability to respond to soluble or covalently bound bFGF when cultured on microfibrillar substrates. This behavior correlated with a downregulation of FGF receptor 1 (FGFR1) expression of hMSCs on microfibrillar substrates. Inhibition of actomyosin or the MRTF/SRF pathway decreased FGFR1 expression in hMSCs, fibroblasts and MG63 cells. To our knowledge, this is the first time FGFR1 expression is shown to be regulated through a mechanosensitive pathway in hMSCs. These results add to the sparse literature on FGFR1 regulation and potentially aid designing tissue engineering constructs that better control cell proliferation. Zonderland et al discover that fibroblasts and mesenchymal stromal cells (MSCs) differ in their response to bFGF when grown on microfibrillar substrates. They find that MSCs cultured on this substrate leads to reduced FGFR levels, as does inhibition of actomyosin contractility or MRT/SRF, suggesting mechanosensitive control of FGFR

Mechanosensitive regulation of stanniocalcin-1 by zyxin and actin-myosin in human mesenchymal stromal cells

Stanniocalcin-1 (STC1) secreted by mesenchymal stromal cells (MSCs) has anti-inflammatory functions, reduces apoptosis, and aids in angiogenesis, both in vitro and in vivo. However, little is known about the molecular mechanisms of its regulation. Here, we show that STC1 secretion is increased only under specific cell-stress conditions. We find that this is due to a change in actin stress fibers and actin-myosin tension. Abolishment of stress fibers by blebbistatin and knockdown of the focal adhesion protein zyxin leads to an increase in STC1 secretion. To also study this connection in 3D, where few focal adhesions and actin stress fibers are present, STC1 expression was analyzed in 3D alginate hydrogels and 3D electrospun scaffolds. Indeed, STC1 secretion was increased in these low cellular tension 3D environments. Together, our data show that STC1 does not directly respond to cell stress, but that it is regulated through mechanotransduction. This research takes a step forward in the fundamental understanding of STC1 regulation and can have implications for cell-based regenerative medicine, where cell survival, anti-inflammatory factors and angiogenesis are critical