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Stability of bradykinin-and compression-induced F-actin protrusions. (AeH) F-actin (red) and DAPI (blue) labeling in hMSCs encapsulated in collagen constructs at different time points (AeD) after bradykinin treatment and (EeH) after 9 h of cyclic compression. The cells were fixed and the F-actin labeled (A, E) immediately; or (B, F) 15 min; (C, G) 30 min; or (D, H) 1 h after the cessation of either bradykinin treatment or compression loading; (IeJ) Number of actin protrusions per cell induced by bradykinin (I) and dynamic compression (J). Scale bars: 7 mm for a; 5 mm (BeH); and. White arrows indicate F-actin protrusions while white asterisks indicate F-actin patches. Images A1, A2, B1, E1, F1, F2, G1, G2 and G3 show magnified views of the F-actin protrusions in panels A, B, E, F and G, respectively. Black asterisks indicate statistical significant difference (p < 0.05). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
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Cells are known to respond to multiple niche signals including extracellular matrix and mechanical loading. In others and our own studies, mechanical loading has been shown to induce the formation of cell alignment in 3D collagen matrix with random meshwork, challenging our traditional understanding on the necessity of having aligned substrates as...
Contexts in source publication
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... distinguishing feature of different types of actin-based protrusions is their stability. Both filopodia [14] and podosomes [18,24] have a short life span that they can last for minutes, while invadopodia have much longer life span, i.e., up to a few hours [13,31,35]. In order to investigate the identity of the compression- induced actin protrusions, the duration that these structures last post-induction was studied and compared with that of bradykinin- induced filopodia (Fig. 7). Fig. 7AeH shows F-actin staining in both bradykinin-and compression-induced protrusions. Immediately after bradykinin treatment, filopodia were clearly identified (see white arrows in Fig. 7AeA2) but these structures were significantly reduced within 15 min after the withdrawal of the drug (Fig. 7BeB1), and in some cells, they had completely disappeared after 30 min (Fig. 7CeD). On the other hand, compression-induced F-actin protrusions were shown to be more stable than the bradykinin-induced filopodia. These protrusions were identified immediately after compression (see white arrows in Fig. 7EeE1) and the majority of them were still apparent at 15 min (Fig. 7FeF2) and 30 min (Fig. 7GeG3), but significantly reduced at 1 h (see white arrows in Fig. 7H) after the withdrawal of compression although their disappearance coincided with the appearance of numerous F- actin patches, which started to be identified at 1 h (see white as- terisks in Fig. 7H). Quantitative analyses of the number of actin protrusions induced by dynamic compression and bradykinin were shown in Fig. 7IeJ. Around 50 actin protrusions per cell have been induced by the dynamic compression. Although the number of these protrusions was decreasing over 15 and 30 min, statistical significant decrease (p ¼ 0.001) was only detected at 60 min after removal of the compression. While for bradykinin-induced filopo- dia, around 50 actin protrusions per cell have been induced. Upon removal of bradykinin, there was significant reduction in the number of filopodia at 15, 30 and 60 min (p 0.021), suggesting a shorter stability of the bradykinin-induced filopodia than that of the compression-induced actin ...
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... distinguishing feature of different types of actin-based protrusions is their stability. Both filopodia [14] and podosomes [18,24] have a short life span that they can last for minutes, while invadopodia have much longer life span, i.e., up to a few hours [13,31,35]. In order to investigate the identity of the compression- induced actin protrusions, the duration that these structures last post-induction was studied and compared with that of bradykinin- induced filopodia (Fig. 7). Fig. 7AeH shows F-actin staining in both bradykinin-and compression-induced protrusions. Immediately after bradykinin treatment, filopodia were clearly identified (see white arrows in Fig. 7AeA2) but these structures were significantly reduced within 15 min after the withdrawal of the drug (Fig. 7BeB1), and in some cells, they had completely disappeared after 30 min (Fig. 7CeD). On the other hand, compression-induced F-actin protrusions were shown to be more stable than the bradykinin-induced filopodia. These protrusions were identified immediately after compression (see white arrows in Fig. 7EeE1) and the majority of them were still apparent at 15 min (Fig. 7FeF2) and 30 min (Fig. 7GeG3), but significantly reduced at 1 h (see white arrows in Fig. 7H) after the withdrawal of compression although their disappearance coincided with the appearance of numerous F- actin patches, which started to be identified at 1 h (see white as- terisks in Fig. 7H). Quantitative analyses of the number of actin protrusions induced by dynamic compression and bradykinin were shown in Fig. 7IeJ. Around 50 actin protrusions per cell have been induced by the dynamic compression. Although the number of these protrusions was decreasing over 15 and 30 min, statistical significant decrease (p ¼ 0.001) was only detected at 60 min after removal of the compression. While for bradykinin-induced filopo- dia, around 50 actin protrusions per cell have been induced. Upon removal of bradykinin, there was significant reduction in the number of filopodia at 15, 30 and 60 min (p 0.021), suggesting a shorter stability of the bradykinin-induced filopodia than that of the compression-induced actin ...
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... distinguishing feature of different types of actin-based protrusions is their stability. Both filopodia [14] and podosomes [18,24] have a short life span that they can last for minutes, while invadopodia have much longer life span, i.e., up to a few hours [13,31,35]. In order to investigate the identity of the compression- induced actin protrusions, the duration that these structures last post-induction was studied and compared with that of bradykinin- induced filopodia (Fig. 7). Fig. 7AeH shows F-actin staining in both bradykinin-and compression-induced protrusions. Immediately after bradykinin treatment, filopodia were clearly identified (see white arrows in Fig. 7AeA2) but these structures were significantly reduced within 15 min after the withdrawal of the drug (Fig. 7BeB1), and in some cells, they had completely disappeared after 30 min (Fig. 7CeD). On the other hand, compression-induced F-actin protrusions were shown to be more stable than the bradykinin-induced filopodia. These protrusions were identified immediately after compression (see white arrows in Fig. 7EeE1) and the majority of them were still apparent at 15 min (Fig. 7FeF2) and 30 min (Fig. 7GeG3), but significantly reduced at 1 h (see white arrows in Fig. 7H) after the withdrawal of compression although their disappearance coincided with the appearance of numerous F- actin patches, which started to be identified at 1 h (see white as- terisks in Fig. 7H). Quantitative analyses of the number of actin protrusions induced by dynamic compression and bradykinin were shown in Fig. 7IeJ. Around 50 actin protrusions per cell have been induced by the dynamic compression. Although the number of these protrusions was decreasing over 15 and 30 min, statistical significant decrease (p ¼ 0.001) was only detected at 60 min after removal of the compression. While for bradykinin-induced filopo- dia, around 50 actin protrusions per cell have been induced. Upon removal of bradykinin, there was significant reduction in the number of filopodia at 15, 30 and 60 min (p 0.021), suggesting a shorter stability of the bradykinin-induced filopodia than that of the compression-induced actin ...
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... distinguishing feature of different types of actin-based protrusions is their stability. Both filopodia [14] and podosomes [18,24] have a short life span that they can last for minutes, while invadopodia have much longer life span, i.e., up to a few hours [13,31,35]. In order to investigate the identity of the compression- induced actin protrusions, the duration that these structures last post-induction was studied and compared with that of bradykinin- induced filopodia (Fig. 7). Fig. 7AeH shows F-actin staining in both bradykinin-and compression-induced protrusions. Immediately after bradykinin treatment, filopodia were clearly identified (see white arrows in Fig. 7AeA2) but these structures were significantly reduced within 15 min after the withdrawal of the drug (Fig. 7BeB1), and in some cells, they had completely disappeared after 30 min (Fig. 7CeD). On the other hand, compression-induced F-actin protrusions were shown to be more stable than the bradykinin-induced filopodia. These protrusions were identified immediately after compression (see white arrows in Fig. 7EeE1) and the majority of them were still apparent at 15 min (Fig. 7FeF2) and 30 min (Fig. 7GeG3), but significantly reduced at 1 h (see white arrows in Fig. 7H) after the withdrawal of compression although their disappearance coincided with the appearance of numerous F- actin patches, which started to be identified at 1 h (see white as- terisks in Fig. 7H). Quantitative analyses of the number of actin protrusions induced by dynamic compression and bradykinin were shown in Fig. 7IeJ. Around 50 actin protrusions per cell have been induced by the dynamic compression. Although the number of these protrusions was decreasing over 15 and 30 min, statistical significant decrease (p ¼ 0.001) was only detected at 60 min after removal of the compression. While for bradykinin-induced filopo- dia, around 50 actin protrusions per cell have been induced. Upon removal of bradykinin, there was significant reduction in the number of filopodia at 15, 30 and 60 min (p 0.021), suggesting a shorter stability of the bradykinin-induced filopodia than that of the compression-induced actin ...
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... distinguishing feature of different types of actin-based protrusions is their stability. Both filopodia [14] and podosomes [18,24] have a short life span that they can last for minutes, while invadopodia have much longer life span, i.e., up to a few hours [13,31,35]. In order to investigate the identity of the compression- induced actin protrusions, the duration that these structures last post-induction was studied and compared with that of bradykinin- induced filopodia (Fig. 7). Fig. 7AeH shows F-actin staining in both bradykinin-and compression-induced protrusions. Immediately after bradykinin treatment, filopodia were clearly identified (see white arrows in Fig. 7AeA2) but these structures were significantly reduced within 15 min after the withdrawal of the drug (Fig. 7BeB1), and in some cells, they had completely disappeared after 30 min (Fig. 7CeD). On the other hand, compression-induced F-actin protrusions were shown to be more stable than the bradykinin-induced filopodia. These protrusions were identified immediately after compression (see white arrows in Fig. 7EeE1) and the majority of them were still apparent at 15 min (Fig. 7FeF2) and 30 min (Fig. 7GeG3), but significantly reduced at 1 h (see white arrows in Fig. 7H) after the withdrawal of compression although their disappearance coincided with the appearance of numerous F- actin patches, which started to be identified at 1 h (see white as- terisks in Fig. 7H). Quantitative analyses of the number of actin protrusions induced by dynamic compression and bradykinin were shown in Fig. 7IeJ. Around 50 actin protrusions per cell have been induced by the dynamic compression. Although the number of these protrusions was decreasing over 15 and 30 min, statistical significant decrease (p ¼ 0.001) was only detected at 60 min after removal of the compression. While for bradykinin-induced filopo- dia, around 50 actin protrusions per cell have been induced. Upon removal of bradykinin, there was significant reduction in the number of filopodia at 15, 30 and 60 min (p 0.021), suggesting a shorter stability of the bradykinin-induced filopodia than that of the compression-induced actin ...
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... distinguishing feature of different types of actin-based protrusions is their stability. Both filopodia [14] and podosomes [18,24] have a short life span that they can last for minutes, while invadopodia have much longer life span, i.e., up to a few hours [13,31,35]. In order to investigate the identity of the compression- induced actin protrusions, the duration that these structures last post-induction was studied and compared with that of bradykinin- induced filopodia (Fig. 7). Fig. 7AeH shows F-actin staining in both bradykinin-and compression-induced protrusions. Immediately after bradykinin treatment, filopodia were clearly identified (see white arrows in Fig. 7AeA2) but these structures were significantly reduced within 15 min after the withdrawal of the drug (Fig. 7BeB1), and in some cells, they had completely disappeared after 30 min (Fig. 7CeD). On the other hand, compression-induced F-actin protrusions were shown to be more stable than the bradykinin-induced filopodia. These protrusions were identified immediately after compression (see white arrows in Fig. 7EeE1) and the majority of them were still apparent at 15 min (Fig. 7FeF2) and 30 min (Fig. 7GeG3), but significantly reduced at 1 h (see white arrows in Fig. 7H) after the withdrawal of compression although their disappearance coincided with the appearance of numerous F- actin patches, which started to be identified at 1 h (see white as- terisks in Fig. 7H). Quantitative analyses of the number of actin protrusions induced by dynamic compression and bradykinin were shown in Fig. 7IeJ. Around 50 actin protrusions per cell have been induced by the dynamic compression. Although the number of these protrusions was decreasing over 15 and 30 min, statistical significant decrease (p ¼ 0.001) was only detected at 60 min after removal of the compression. While for bradykinin-induced filopo- dia, around 50 actin protrusions per cell have been induced. Upon removal of bradykinin, there was significant reduction in the number of filopodia at 15, 30 and 60 min (p 0.021), suggesting a shorter stability of the bradykinin-induced filopodia than that of the compression-induced actin ...
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... [18,24] have a short life span that they can last for minutes, while invadopodia have much longer life span, i.e., up to a few hours [13,31,35]. In order to investigate the identity of the compressioninduced actin protrusions, the duration that these structures last post-induction was studied and compared with that of bradykinininduced filopodia (Fig. 7). Fig. 7AeH shows F-actin staining in both bradykinin-and compression-induced protrusions. Immediately after bradykinin treatment, filopodia were clearly identified (see white arrows in Fig. 7AeA2) but these structures were significantly reduced within 15 min after the withdrawal of the drug (Fig. 7BeB1), and in some cells, they had ...
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... a short life span that they can last for minutes, while invadopodia have much longer life span, i.e., up to a few hours [13,31,35]. In order to investigate the identity of the compressioninduced actin protrusions, the duration that these structures last post-induction was studied and compared with that of bradykinininduced filopodia (Fig. 7). Fig. 7AeH shows F-actin staining in both bradykinin-and compression-induced protrusions. Immediately after bradykinin treatment, filopodia were clearly identified (see white arrows in Fig. 7AeA2) but these structures were significantly reduced within 15 min after the withdrawal of the drug (Fig. 7BeB1), and in some cells, they had completely ...
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... staining in both bradykinin-and compression-induced protrusions. Immediately after bradykinin treatment, filopodia were clearly identified (see white arrows in Fig. 7AeA2) but these structures were significantly reduced within 15 min after the withdrawal of the drug (Fig. 7BeB1), and in some cells, they had completely disappeared after 30 min (Fig. 7CeD). On the other hand, compression-induced F-actin protrusions were shown to be more stable than the bradykinin-induced filopodia. These protrusions were identified immediately after compression (see white arrows in Fig. 7EeE1) and the majority of them were still apparent at 15 min (Fig. 7FeF2) and 30 min (Fig. 7GeG3), but significantly ...
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... F-actin protrusions were shown to be more stable than the bradykinin-induced filopodia. These protrusions were identified immediately after compression (see white arrows in Fig. 7EeE1) and the majority of them were still apparent at 15 min (Fig. 7FeF2) and 30 min (Fig. 7GeG3), but significantly reduced at 1 h (see white arrows in Fig. 7H) after the withdrawal of compression although their disappearance coincided with the appearance of numerous Factin patches, which started to be identified at 1 h (see white asterisks in Fig. 7H). Quantitative analyses of the number of actin protrusions induced by dynamic compression and bradykinin were shown in Fig. 7IeJ. Around 50 ...
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... 7EeE1) and the majority of them were still apparent at 15 min (Fig. 7FeF2) and 30 min (Fig. 7GeG3), but significantly reduced at 1 h (see white arrows in Fig. 7H) after the withdrawal of compression although their disappearance coincided with the appearance of numerous Factin patches, which started to be identified at 1 h (see white asterisks in Fig. 7H). Quantitative analyses of the number of actin protrusions induced by dynamic compression and bradykinin were shown in Fig. 7IeJ. Around 50 actin protrusions per cell have been induced by the dynamic compression. Although the number of these protrusions was decreasing over 15 and 30 min, statistical significant decrease (p ¼ 0.001) was ...
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... at 1 h (see white arrows in Fig. 7H) after the withdrawal of compression although their disappearance coincided with the appearance of numerous Factin patches, which started to be identified at 1 h (see white asterisks in Fig. 7H). Quantitative analyses of the number of actin protrusions induced by dynamic compression and bradykinin were shown in Fig. 7IeJ. Around 50 actin protrusions per cell have been induced by the dynamic compression. Although the number of these protrusions was decreasing over 15 and 30 min, statistical significant decrease (p ¼ 0.001) was only detected at 60 min after removal of the compression. While for bradykinin-induced filopodia, around 50 actin protrusions ...
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Citations
... Moreover, while short term loading may promote actin production, it decreases with loading time, as it has been shown on myocyte 3D cultures [52]. Interestingly, it has been shown that actin is not produced, and even degraded, during loading, but that if resting periods are allowed, actin production is promoted sometime after the loading [53]. ...
While periodontal ligament cells are sensitive to their 3D biomechanical environment, only a few 3D in vitro models have been used to investigate the periodontal cells mechanobiological behavior. The objective of the current study was to assess the capability of a 3D fibrous scaffold to transmit a mechanical loading to the periodontal ligament cells. Three-dimensional fibrous polycaprolactone (PCL) scaffolds were synthetized through electrospinning. Scaffolds seeded with human periodontal cells (103 mL−1) were subjected to static (n = 9) or to a sinusoidal axial compressive loading in an in-house bioreactor (n = 9). At the end of the culture, the dynamic loading seemed to have an influence on the cells’ morphology, with a lower number of visible cells on the scaffolds surface and a lower expression of actin filament. Furthermore, the dynamic loading presented a tendency to decrease the Alkaline Phosphatase activity and the production of Interleukin-6 while these two biomolecular markers were increased after 21 days of static culture. Together, these results showed that load transmission is occurring in the 3D electrospun PCL fibrous scaffolds, suggesting that it can be used to better understand the periodontal ligament cells mechanobiology. The current study shows a relevant way to investigate periodontal mechanobiology using 3D fibrous scaffolds.
... En los últimos años, la investigación ambiental se ha centrado cada vez más en cuantificar y pronosticar los impactos del cambio climático en los ecosistemas forestales, por lo que la demanda de información a gran escala y a largo plazo sobre el crecimiento de árboles y el clima, más allá de la disponibilidad de los datos instrumentales está aumentando rápidamente (Babst et al., 2018). Los árboles, como organismos de larga vida, registran información ecológicamente relevante en sus anillos anuales por tal razón, son reservorios indirectos para el estudio de los cambios globales durante los últimos siglos o incluso milenios (Trouet et al., 2009;Fonti et al., 2010;Zhang, 2015). La dendrocronología como disciplina ha sido utilizada para registrar la variabilidad ambiental en la estructura de la madera de los árboles que crecen en climas estacionales, así como en las latitudes medias y altas, y en algunos árboles tropicales que crecen en ambientes con una pronunciada estación húmeda o seca (Speer, 2009). ...
... El colágeno está constituido por moléculas de tropocolágeno, es un agente encapsulante empleado en medicina para reparar daños o traumas químico-mecánicos en piel o mucosas, debido a su biocompatibilidad y capacidad para promover la cicatrización de heridas (Quek et al., 2004, Lee et al., 2012, Ding et al., 2014, Ho et al., 2015. ...
... Mechanical loading can induce polarization and alignment in both 2D [20] and 3D cell-models [21][22][23]. Our group has demonstrated mechanical loading-induced reorientation of mesenchymal stem cells (MSCs) in 3D micro-tissues from a random distribution to a direction along the loading axis [24]. ...
... In order to delineate the mechanisms of the mechanical loadinginduced cell reorientation and alignment, we recently investigated the actin-cytoskeleton organization and dynamics of MSCs encapsulated 3D collagen micro-tissues. Interestingly, we discovered for the first time that cyclic compression has induced numerous omni-directional actin protrusions, namely compression-induced actin protrusions (CAPs), at the interface between MSCs and collagen matrix [21]. These CAPs co-localized with CTTN and MMP14 and showed pericellular proteolytic digestion zone at the cell-matrix interface [21]. ...
... Interestingly, we discovered for the first time that cyclic compression has induced numerous omni-directional actin protrusions, namely compression-induced actin protrusions (CAPs), at the interface between MSCs and collagen matrix [21]. These CAPs co-localized with CTTN and MMP14 and showed pericellular proteolytic digestion zone at the cell-matrix interface [21]. Nevertheless, the mechanistic pathways regulating the formation of CAPs and hence their identity are not known. ...
Cells can sense mechanical signals through cytoskeleton reorganization. We previously discovered the formation of omni-directional actin protrusions upon compression loading, namely compression-induced actin protrusions (CAPs), in human mesenchymal stem cells (MSCs) in 3D micro-tissues. Here, the regulatory roles of three RhoGTPases (CDC42, Rac1 and RhoA) in the formation of CAPs were investigated. Upon compression loading, extensive formation of CAPs was found, significantly associated with an upregulated mRNA expression of Rac1 only, but not CDC42, nor RhoA. Upon chemical inhibition of these RhoGTPase activity during compression, only Rac1 activity was significantly suppressed, associating with the reduced CAP formation. Silencing the upstream regulators of these RhoGTPase pathways including Rac1 by specific siRNA dramatically disrupted actin cyto-skeleton, distorted cell morphology and aborted CAP formation. Silencing cortactin (CTTN), a downstream effector of the Rac1 pathway, induced a compensatory upregulation of Rac1, enabling the MSCs to respond to the compression loading stimulus in terms of CAP formation, although at a reduced number. The importance of Rac1 signalling in CAP formation and the corresponding upregulation of lamellipodial markers also suggest that these CAPs are lamellipodia in nature. This study delineates the mechanism of compression-induced cytoskeleton reorganization, contributing to rationalizing mechanical loading regimes for functional tissue engineering.
... Nevertheless, how active mechanical stimulation can be used to modulate these cell-matrix interactions in 3D for regulating cell fate has not been studied. Our group has been investigating the role of different types of mechanical stimulation, including tension [25], torsion [26], and compression [27,28], in regulating cell alignment [28] and actin protrusion formation [27] in Dynamic compression (cylic loading with a frequency of 1 Hz), was applied the hMSCs embed in collagen microtissues. C.W. Li et al. collagen microtissues. ...
... Nevertheless, how active mechanical stimulation can be used to modulate these cell-matrix interactions in 3D for regulating cell fate has not been studied. Our group has been investigating the role of different types of mechanical stimulation, including tension [25], torsion [26], and compression [27,28], in regulating cell alignment [28] and actin protrusion formation [27] in Dynamic compression (cylic loading with a frequency of 1 Hz), was applied the hMSCs embed in collagen microtissues. C.W. Li et al. collagen microtissues. ...
... Collagen microencapsulation was used to encapsulate human MSCs to form the microtissues as previously described [27]. In brief, a custom-made cylindrical container with the non-adhesive surface was used to cast the microtissues. ...
... Given that CDC42 has been identified as a contributor to cellular morphology [39], we investigated the simultaneous effect of CDC42 inhibition and drug treatment on cell shape ( Figure S5). The most significant changes were observed with simultaneous inhibitor and drug treatment, with cellular area significantly increasing for both control and compressed cells. ...
This report investigates the role of compressive stress on ovarian cancer in a 3D custom built bioreactor. Cells within the ovarian tumor microenvironment experience a range of compressive stimuli that contribute to mechanotransduction. As the ovarian tumor expands, cells are exposed to chronic load from hydrostatic pressure, displacement of surrounding cells, and growth induced stress. External dynamic stimuli have been correlated with an increase in metastasis, cancer stem cell marker expression, chemoresistance, and proliferation in a variety of cancers. However, how these compressive stimuli contribute to ovarian cancer progression is not fully understood. In this report, high grade serous ovarian cancer cell lines were encapsulated within an ECM mimicking hydrogel comprising of agarose and collagen type I, and stimulated with confined cyclic or static compressive stresses for 24 and 72 h. Compression stimulation resulted in a significant increase in proliferation, invasive morphology, and chemoresistance. Additionally, CDC42 was upregulated in compression stimulated conditions, and was necessary to drive increased proliferation and chemoresistance. Inhibition of CDC42 lead to significant decrease in proliferation, survival, and increased chemosensitivity. In summary, the dynamic in vitro 3D platform developed in this report, is ideal for understanding the influence of compressive stimuli, and can be widely applicable to any epithelial cancers. This work reinforces the critical need to consider compressive stimulation in basic cancer biology and therapeutic developments.
... Cells at passage 2 were thawed and expanded in a monolayer using a 100 mm culture dish for three days until at 70-80% confluence. Cells were then detached using 0.25% trysin/EDTA (Gibco) and encapsulated in a collagen construct, as described previously 21,38 . In brief, rat tail collagen type I (BD Biosciences, San Jose, CA, USA) was neutralized with 1N NaOH and diluted with culture medium before being mixed with the cells, after which 50 μ L aliquots of the mixture were transferred into cylindrical molds and incubated in a humidified incubator at 37 °C and 5% CO 2 for 1 h to form disc-shaped gels. ...
... To study the effect of loading, a micromanipulator-based loading device was used, as described previously [38][39][40] . In brief, the disc-shaped constructs were transferred into a two-compartment Petri dish and covered with culture media. ...
Cells protect themselves from stresses through a cellular stress response. In the interverebral disc, such response was also demonstrated to be induced by various environmental stresses. However, whether compression loading will cause cellular stress response in the nucleus pulposus cells (NPCs) is not well studied. By using an in vitro collagen microencapsulation model, we investigated the effect of compression loading on the stress response of NPCs. Cell viability tests, and gene and protein expression experiments were conducted, with primers for the heat shock response (HSR: HSP70, HSF1, HSP27 and HSP90), and unfolded protein response (UPR: GRP78, GRP94, ATF4 and CHOP) genes and an antibody to HSP72. Different gene expression patterns occurred due to loading type throughout experiments. Increasing the loading strain for a short duration did not increase the stress response genes significantly, but over longer durations, HSP70 and HSP27 were upregulated. Longer loading durations also resulted in a continuous upregulation of HSR genes and downregulation of UPR genes, even after load removal. The rate of apoptosis did not increase significantly after loading, suggesting that stress response genes might play a role in cell survival following mechanical stress. These results demonstrate how mechanical stress might induce and control the expression of HSR and UPR genes in NPCs.
... Cells at passage 2 were thawed and expanded in a monolayer using a 100 mm culture dish for three days until at 70-80% confluence. Cells were then detached using 0.25% trysin/EDTA (Gibco) and encapsulated in a collagen construct, as described previously 21,38 . In brief, rat tail collagen type I (BD Biosciences, San Jose, CA, USA) was neutralized with 1N NaOH and diluted with culture medium before being mixed with the cells, after which 50 μ L aliquots of the mixture were transferred into cylindrical molds and incubated in a humidified incubator at 37 °C and 5% CO 2 for 1 h to form disc-shaped gels. ...
... To study the effect of loading, a micromanipulator-based loading device was used, as described previously [38][39][40] . In brief, the disc-shaped constructs were transferred into a two-compartment Petri dish and covered with culture media. ...
The cause of degenerative disc disease is multi-factorial and is often associated with mechanical loading. However, mechanism of the cells' response to compressive loading is not fully known. It was demonstrated that in mechanical force can induce chondrocytes to upregulate expression of heat shock proteins (HSPs) as a form of cellular stress response.¹ Similarly, stretching annulus fibrosus cells was found to upregulate endoplasmic reticulum stress markers.² However, it is unclear that whether cells in the intervertebral disc (IVD) would experience compressive force as cellular stress in 3D environment. The objective of this study is to investigate the stress response of nucleus pulposus cells (NPCs) during compression in 3D culture environment.
... Understanding the disease mechanisms underlying these clinical complications is of utmost importance, especially considering that these symptoms can manifest prior to the onset of skeletal manifestations and can lead to an initial misdiagnosis. Moreover, mesenchymal tissues are highly mechanically responsive and able to regulate gene expression depending upon the mechanical stimulus [116,117]. It is therefore interesting to speculate that the understanding of the musculoskeletal complications associated with GSDs may lead to a better physiotherapy regime and management of the patients in the future. ...
Introduction: Genetic skeletal diseases (GSDs) are a diverse and complex group of rare genetic conditions that affect the development and homeostasis of the skeleton. Although individually rare, as a group of related diseases, GSDs have an overall prevalence of at least 1 per 4,000 children. There are currently very few specific therapeutic interventions to prevent, halt or modify skeletal disease progression and therefore the generation of new and effective treatments requires novel and innovative research that can identify tractable therapeutic targets and biomarkers of these diseases.
Areas covered: Remarkable progress has been made in identifying the genetic basis of the majority of GSDs and in developing relevant model systems that have delivered new knowledge on disease mechanisms and are now starting to identify novel therapeutic targets. This review will provide an overview of disease mechanisms that are shared amongst groups of different GSDs and describe potential therapeutic approaches that are under investigation.
Expert opinion: The extensive clinical variability and genetic heterogeneity of GSDs renders this broad group of rare diseases a bench to bedside challenge. However, the evolving hypothesis that clinically different diseases might share common disease mechanisms is a powerful concept that will generate critical mass for the identification and validation of novel therapeutic targets and biomarkers.
... For stem cells, stiffer matrices result in a higher actin concentration near the cell cortex [100]. In 3D collagen gels under dynamic compression, actin protrusions are correlated to matrix remodeling [176]. ...
... Vimentin increases cell stiffness and can protect the cell against compressive loads [202]. In 3D collagen gels, the vimentin and MT network persists after dynamic compression, while the actin forms local patches to remodel the ECM [176]. Intriguingly, on 2D substrates, the solubility of vimentin depends on the underlying substrate stiffness [171], which may contribute to stiffness adaption of cells to their substrate. ...
Cells actively sense and process mechanical information that is provided by the extracellular environment to make decisions about growth, motility and differentiation. It is important to understand the underlying mechanisms given that deregulation of the mechanical properties of the extracellular matrix (ECM) is implicated in various diseases, such as cancer and fibrosis. Moreover, matrix mechanics can be exploited to program stem cell differentiation for organ-on-chip and regenerative medicine applications. Mechanobiology is an emerging multidisciplinary field that encompasses cell and developmental biology, bioengineering and biophysics. Here we provide an introductory overview of the key players important to cellular mechanobiology, taking a biophysical perspective and focusing on a comparison between flat versus three dimensional substrates.
Copyright © 2015. Published by Elsevier B.V.
... Cells at passage 2 were thawed and expanded in a monolayer using a 100 mm culture dish for three days until at 70-80% confluence. Cells were then detached using 0.25% trysin/EDTA (Gibco) and encapsulated in a collagen construct, as described previously 21,38 . In brief, rat tail collagen type I (BD Biosciences, San Jose, CA, USA) was neutralized with 1N NaOH and diluted with culture medium before being mixed with the cells, after which 50 μ L aliquots of the mixture were transferred into cylindrical molds and incubated in a humidified incubator at 37 °C and 5% CO 2 for 1 h to form disc-shaped gels. ...
... To study the effect of loading, a micromanipulator-based loading device was used, as described previously [38][39][40] . In brief, the disc-shaped constructs were transferred into a two-compartment Petri dish and covered with culture media. ...
Introduction
Previous research has been done to study the effect of mechanical loading on intervertebral disc (IVD) cells. However, few studies have investigated in whether the IVD cells perceive mechanical loading as stress and respond by expression of stress response proteins such as heat shock proteins (HSP). Studies have shown that stress response can be seen in cell line chondrocytes under hydrostatic pressure. On the other hand, studies have also shown that expression of heat shock protein-72 (HSP72) and HSP27 was associated with disc degeneration and IVD cells can secrete HSP70 in response to oxidative stress. This study aims to study the stress response in the IVD in response to compressive loading and whether the disc cells are able to adapt to the loading. The outcome of the study will help to understand how the disc cells adapt or cope with mechanical stress.
Materials and Methods
Fresh adult bovine caudal discs were harvested and cultured with dynamic compressive loading applied at physiological range magnitude, 0.1 to 0.6 MPa. The culture condition was such that the discs underwent 2 hours of dynamic loading, followed by 22 hours of resting for 2 days. Samples were retrieved at different time points: right after loading (Dyna) and right after resting (DyNa ⁺ rest). Positive control discs were put under static loading (0.35 MPa, static) and heat shock (43°C, HS) exposed for 2 h/d during 2 days and gene expression was quantified right after the treatments. Both nucleus pulposus (NP) and annulus fibrosus (AF) were retrieved for gene expression study of the cellular stress response genes. HSP72 and heat shock factor-1 (HSF1). HSP72 is the general stress response protein which is upregulated in the cell in response to stress while HSF1 is the transcriptional factor of HSP72. The expression was normalized to free swelling control.
Results
In the NP of the bovine disc, both positive controls (HS and static) expressed high level of HSP72, confirming their expression in the NP tissues and their response to stress. For the experimental groups, the expression of HSP72 was upregulated after loading, decreased after resting but was again increased after second round of loading at day 2. On the other hand, HSF1 expression increased after resting in the day 1 loading and peaked at day 2 after loading. For the AF tissues, the expression of HSF1 was low in most of the groups including the positive control, even the HSP72 expression was high in these two groups. The expression of HSP72 in AF tissues was decreasing with both resting and an additional round of loading. The pattern of HSF1 expression of AF tissues was similar to the NP tissues where the expression was the highest 2 days after loading.
Conclusion
This study showed that the IVD cells do upregulate the stress response proteins expression in response to loading induced stress. The cells express HSP72 in response to the stress while HSF1 may have a slower and transient expression. The increase in HSP72 and HSF1 expression after two rounds of loading may indicate more cycles are needed to see whether there is adaptation in stress response induced by mechanical loading. Disclosure of Interest None declared
