a-novel-stretching-platform-for-applications-cell-tissue

A Novel Stretching Platform for Applications in Cell and Tissue Mechanobiology

Tools that allow the application of mechanical forces to cells and tissues or that can quantify the mechanical properties of biological tissues have ...

decellularized-apple-derived-scaffolds-for-bone-tissue-engineering

Decellularized Apple-Derived Scaffolds for Bone Tissue Engineering <em>In Vitro</em> and <em>In Vivo</em>

Plant-derived&#160;cellulose biomaterials have been employed in various tissue engineering applications. In vivo studies have shown the remarkable ...

University of Ottawa

Eukaryotic cells were individually transfected using commercially available atomic force microscope tips decorated with plasmidic DNA encoding for the fluorescent protein EGFP. In a typical transfection attempt, the tip is forcibly incorporated into the cell thus allowing for the transfer of the genetic material through the cell membrane. A sharp discontinuity, corresponding to the passage of the tip through the cell membrane can be easily detected when monitoring the cellular deformation as a function of the applied force. In order for the transfection to be successful, the tip must reversibly penetrates the membrane without causing disturbance or damage to the cell. Transfection success rate (30%), cell survival, and growth are confirmed by epifluorescence microscopy. This technique provides an alternative tool to the transfection toolbox, allowing the transfection of specific individual cells with minimal disturbance.

Single cell transfection using plasmid decorated AFM probes.

The exploration of molecular processes governing physiological functions has significantly benefited from the emergence of novel nanoscaled techniques. Atomic force microscopy in force measurement mode can be used to investigate a multitude of cellular events in individual living cells with great sensitivity. Precise regions of the plasma membrane can be examined in relation to particular signalling pathways activated by a mechanical stimulus. Similarly, subtle cellular movements induced by biochemical activation of specific membrane receptors can be detected in real time with excellent temporal and spatial resolution. The possibility to challenge locally and mechanically cell surface receptors also provides information on the control exerted by a cell over individual adhesion sites. Overall, this information is vital for an in-depth understanding of mechanisms related to cellular movement and morphological regulation. Lastly, atomic force microscope-based nanomanipulations on living cells have recently been proposed as a tool to influence and monitor cellular homeostasis by introducing specific molecular entities into or extracting them from the cytoplasm of individual cells. This review provides detailed examples on how such atomic force microscopy experiments can be conducted to investigate processes relevant to cell physiology.

AFM as a tool to probe and manipulate cellular processes.

We propose the combination of surface plasmon resonance (SPR) with living cells as a biosensing method. Our detection scheme is based on the premise that cellular activity induced by external agents is often associated with changes in cellular morphology, which in turn should lead to a variation of the effective refractive index at the interface between the cell membrane and the metal layer. We monitored surface plasmon resonance signals originating from a gold surface coated with cells on a custom apparatus after injection of various agents known to influence cellular activity and morphology. Specifically, we evaluated three types of stimulation: response to an endotoxin (lipopolysaccharides), a chemical toxin (sodium azide) and a physiological agonist (thrombin). A comparison with phase contrast microscopy reveals that SPR signal variations are associated with the induction of cell death for lipopolysaccharides treatment and a contraction of the cell body for sodium azide. Thrombin-induced cellular response shows a rapid decrease of the measured laser reflectance over 5min followed by a return to the original value. For this treatment, phase contrast micrographs relate the first phase of the SPR variation to cell contraction and increase of the intercellular gaps, whereas the recovery phase can be associated with a spreading of the cell on the sensing surface. Hence, the SPR signal is very consistent with the cellular response normally observed for these treatments. This confirms the validity of the biosensing method, which could be applied to a large variety of cellular responses involving shape remodeling induced by external agents.

Biosensing based on surface plasmon resonance and living cells.

Physiological processes, occurring as a result of specific receptor stimulation, are generally assessed via molecular biology techniques and microscopic approaches with the involvement of specific molecular markers. The recent progress in experimental approaches, allowing the mechanical characterization of individual biological entities, now makes it possible to address cellular processes occurring in individual cells as a result of their stimulation by hormones. Here, we demonstrate that the atomic force microscope (AFM) can be used to mechanically probe individual cells following the activation of the angiotensin-1 receptor, a receptor well known for its role in cell homeostasis regulation. Our goal is to demonstrate that the measurement of cantilever deflection can be used to quantify in real time the mechanical and morphological cell activity associated with the activation of the receptor. By combining the AFM with time-lapse sequences of phase-contrast and confocal micrographs, we show that the angiotensin-1 receptor stimulation with 100 nM angiotensin II produces an actin-dependent contractile response with an amplitude of 262 +/- 52 nm. We validated the mechanical origin of the responses by measuring the elastic modulus of the cell from indentation experiments performed at 30-s intervals. Additionally, nanoscaled height fluctuations of the cell membrane occurring after the initial contraction response could be attributed to an increased actin cytoskeleton activity and remodeling detected by confocal microscopy. Finally, by using inhibitors for specific elements of the angiotensin-1 receptor signaling pathways, we demonstrate that AFM real-time height monitoring allows a read out of the molecular processes responsible for the cell mechanical response.

Real-time monitoring of angiotensin II-induced contractile response and cytoskeleton remodeling in individual cells by atomic force microscopy.

The endothelium is closely implicated in the control and maintenance of the vascular homeostasis. The functions of endothelial cells are highly regulated by several agonists of G protein-coupled receptors (GPCR), which can mediate signals involved in morphological remodeling. Here, we evaluated the mechanical properties of human umbilical vein endothelial cells (HUVEC) in responses to two physiological agonists namely thrombin and bradykinin. We used the atomic force microscopy (AFM) technique to study changes in cell membrane stiffness and interaction between the actin cytoskeleton and the cell membrane. HUVEC stimulated with thrombin (10 nM) and bradykinin (1 microM) showed a temporal increase in their membrane stiffness from 5.0 +/- 0.1 kPa (control) to 8.2 +/- 0.4 kPa (thrombin) and 7.3 +/- 0.5 kPa (bradykinin) and in membrane tethers elongation forces from 43.9 +/- 0.9 pN (control) to 49.5 +/- 0.8 pN (thrombin) and 53.1 +/- 0.8 pN (bradykinin). These results were consistent with the reorganization of the actin cytoskeleton observed in fluorescence microscopy. This study demonstrates that these agonists induce important modifications of the cell membrane properties that can be directly linked to the reorganization and the interaction of the actin cytoskeleton near the apical side of the membrane. These changes in the mechanical properties of endothelial cells provide relevant informations in the biological and pathophysiological behaviors of endothelial cells.

Effect of thrombin and bradykinin on endothelial cell mechanical properties monitored through membrane deformation.

The morphological and functional integrity of the microcirculation is compromised in many cardiovascular diseases such as hypertension, diabetes, stroke, and sepsis. Angiotensin converting enzyme inhibitors (ACEi), which are known to favor bradykinin (BK) bioactivity by reducing its metabolism, may have a positive impact on preventing the microvascular structural rarefaction that occurs in these diseases. Our study was designed to test the hypothesis that BK, via B2 receptors (B2R), protects the viability of the microvascular endothelium exposed to the necrotic and apoptotic cell death inducers H(2)O(2) and LPS independently of hemodynamics. Expression (RT-PCR and radioligand binding) and functional (calcium mobilization with fura-2AM, and p42/p44MAPK and Akt phosphorylation assays) experiments revealed the presence of functional B2R in pig cerebral microvascular endothelial cells (pCMVEC). In vitro results showed that the cytocidal effects of H(2)O(2) and LPS on pCMVEC were significantly decreased by a BK pretreatment (MTT and crystal violet tests, annexin-V staining/FACS analysis), which was countered by the B2R antagonist HOE 140. BK treatment coincided with enhanced expression of the cytoprotective proteins COX-2, Bcl-2, and (Cu/Zn)SOD. Ex vivo assays on rat brain explants showed that BK impeded (by approximately 40%) H(2)O(2)-induced microvascular degeneration (lectin-FITC staining). The present study proposes a novel role for BK in microvascular endothelial protection, which may be pertinent to the complex mechanism of action of ACEi explaining their long-term beneficial effects in maintaining vascular integrity.

Bradykinin protects against brain microvascular endothelial cell death induced by pathophysiological stimuli.

Morphological changes occurring in individual cells largely influence the physiological functions of various cell layers. The control of barrier function of epithelia and endothelia is a prime example of processes highly dependent on cellular morphology and cell layer integrity. Here, we applied the surface plasmon resonance (SPR) technique to the quantification of cellular activity of an epithelial cell monolayer stimulated by angiotensin II. The analysis of the SPR signal shows reproducible concentration-dependent biphasic responses after cell activation with angiotensin II. Phase-contrast and confocal microscopy imaging was performed to link the SPR signal to molecular and global morphological remodeling. The SPR signal was observed to be in relation with the rapid cell contraction and the subsequent cell spreading observed by phase-contrast microscopy. Additionally, the temporal redistribution of actin, observed by confocal microscopy after angiotensin II stimulation, was also found to be consistent with the SPR signal variation. The modulation of signaling pathways involved in actin-myosin driven cell contraction confirms the direct implication of actin structures in the SPR response. Additionally, we show that the intracellular calcium mobilization associated with angiotensin II stimulation did not produce any significant SPR signal variation. Altogether, our results demonstrate that SPR is a rapid label-free method to study cellular activity and molecular mechanisms implicated in the modulation of the integrity of a cell monolayer in relation to cytoskeleton remodeling with associated cell morphological changes.

Surface Plasmon Resonance Monitoring of Cell Monolayer Integrity: Implication of Signaling Pathways Involved in Actin-Driven Morphological Remodeling.

Heat shock protein 27 (HSP27) shows attenuated expression in human coronary arteries as the extent of atherosclerosis progresses. In mice, overexpression of HSP27 reduces atherogenesis, yet the precise mechanism(s) are incompletely understood. Inflammation plays a central role in atherogenesis, and of particular interest is the balance of pro- and anti-inflammatory factors produced by macrophages. As nuclear factor-kappa B (NF-κB) is a key immune signaling modulator in atherogenesis, and macrophages are known to secrete HSP27, we sought to determine if recombinant HSP27 (rHSP27) alters NF-κB signaling in macrophages. Treatment of THP-1 macrophages with rHSP27 resulted in the degradation of an inhibitor of NF-κB, IκBα, nuclear translocation of the NF-κB p65 subunit, and increased NF-κB transcriptional activity. Treatment of THP-1 macrophages with rHSP27 yielded increased expression of a variety of genes, including the pro-inflammatory factors, IL-1β, and TNF-α. However, rHSP27 also increased the expression of the anti-inflammatory factors IL-10 and GM-CSF both at the mRNA and protein levels. Our study suggests that in macrophages, activation of NF-κB signaling by rHSP27 is associated with upregulated expression and secretion of key pro- and anti-inflammatory cytokines. Moreover, we surmise that it is the balance in expression of these mediators and antagonists of inflammation, and hence atherogenesis, that yields a favorable net effect of HSP27 on the vessel wall.

Extracellular HSP27 acts as a signaling molecule to activate NF-κB in macrophages.

Expression of Heat Shock Protein-27 (HSP27) is reduced in human coronary atherosclerosis. Over-expression of HSP27 is protective against the early formation of lesions in atherosclerosis-prone apoE(-/-) mice (apoE(-/-)HSP27(o/e)) - however, only in females. We now seek to determine if chronic HSP27 over-expression is protective in a model of advanced atherosclerosis in both male and female apoE(-/-) mice.

Chronic over-expression of heat shock protein 27 attenuates atherogenesis and enhances plaque remodeling: a combined histological and mechanical assessment of aortic lesions.

The aim of this study was to evaluate the potential of serum heat shock protein 27 (HSP27) as a therapeutic target in coronary artery disease.

Serum heat shock protein 27 levels represent a potential therapeutic target for atherosclerosis: observations from a human cohort and treatment of female mice.

Previously, we showed an inverse correlation between HSP27 serum levels and experimental atherogenesis in ApoE(-/-) mice that over-express HSP27 and speculated that the apparent binding of HSP27 to scavenger receptor-A (SR-A) was of mechanistic importance in attenuating foam cell formation. However, the nature and importance of the interplay between HSP27 and SR-A in atheroprotection remained unclear. Treatment of THP-1 macrophages with recombinant HSP27 (rHSP27) inhibited acLDL binding (-34%; p<0.005) and uptake (-38%, p<0.05). rHSP27 reduced SR-A mRNA (-39%, p=0.02), total protein (-56%, p=0.01) and cell surface (-53%, p<0.001) expression. The reduction in SR-A expression by rHSP27 was associated with a 4-fold increase in nuclear factor-kappa B (NF-κB) signaling (p<0.001 versus control), while an inhibitor of NF-κB signaling, BAY11-7082, attenuated the negative effects of rHSP27 on both SR-A expression and lipid uptake. To determine if SR-A is required for HSP27 mediated atheroprotection in vivo, ApoE(-/-) and ApoE(-/-) SR-A(-/-) mice fed with a high fat diet were treated for 3weeks with rHSP25. Compared to controls, rHSP25 therapy reduced aortic en face and aortic sinus atherosclerotic lesion size in ApoE(-/-) mice by 39% and 36% (p<0.05), respectively, but not in ApoE(-/-)SR-A(-/-) mice. In conclusion, rHSP27 diminishes SR-A expression, resulting in attenuated foam cell formation in vitro. Regulation of SR-A by HSP27 may involve the participation of NF-κB signaling. Lastly, SR-A is required for HSP27-mediated atheroprotection in vivo.

Heat shock protein-27 attenuates foam cell formation and atherogenesis by down-regulating scavenger receptor-A expression via NF-κB signaling.

Physical forces arising in the extra-cellular environment have a profound impact on cell fate and gene regulation; however the underlying biophysical mechanisms that control this sensitivity remain elusive. It is hypothesized that gene expression may be influenced by the physical deformation of the nucleus in response to force. Here, using 3T3s as a model, we demonstrate that extra-cellular forces cause cell nuclei to rapidly deform (<1 s) preferentially along their shorter nuclear axis, in an anisotropic manner. Nuclear anisotropy is shown to be regulated by the cytoskeleton within intact cells, with actin and microtubules resistant to orthonormal strains. Importantly, nuclear anisotropy is intrinsic, and observed in isolated nuclei. The sensitivity of this behaviour is influenced by chromatin organization and lamin-A expression. An anisotropic response to force was also highly conserved amongst an array of examined nuclei from differentiated and undifferentiated cell types. Although the functional purpose of this conserved material property remains elusive, it may provide a mechanism through which mechanical cues in the microenvironment are rapidly transmitted to the genome.

Extracellular Forces Cause the Nucleus to Deform in a Highly Controlled Anisotropic Manner.

There is intense interest in developing novel biomaterials which support the invasion and proliferation of living cells for potential applications in tissue engineering and regenerative medicine. Decellularization of existing tissues have formed the basis of one major approach to producing 3D scaffolds for such purposes. In this study, we utilize the native hypanthium tissue of apples and a simple preparation methodology to create implantable cellulose scaffolds. To examine biocompatibility, scaffolds were subcutaneously implanted in wild-type, immunocompetent mice (males and females; 6-9 weeks old). Following the implantation, the scaffolds were resected at 1, 4 and 8 weeks and processed for histological analysis (H&E, Masson's Trichrome, anti-CD31 and anti-CD45 antibodies). Histological analysis revealed a characteristic foreign body response to the scaffold 1 week post-implantation. However, the immune response was observed to gradually disappear by 8 weeks post-implantation. By 8 weeks, there was no immune response in the surrounding dermis tissue and active fibroblast migration within the cellulose scaffold was observed. This was concomitant with the deposition of a new collagen extracellular matrix. Furthermore, active blood vessel formation within the scaffold was observed throughout the period of study indicating the pro-angiogenic properties of the native scaffolds. Finally, while the scaffolds retain much of their original shape they do undergo a slow deformation over the 8-week length of the study. Taken together, our results demonstrate that native cellulose scaffolds are biocompatible and exhibit promising potential as a surgical biomaterial.

Biocompatibility of Subcutaneously Implanted Plant-Derived Cellulose Biomaterials.

Plant-derived cellulose scaffolds constitute a highly viable and interesting biomaterial. They retain a high flexibility in shape and structure, present the ability to tune surface biochemistry, display a high degree of biocompatibility, exhibit vascularization, and are widely available and easily produced. What is also immediately clear is that pre-existing cellulose structures in plants can also provide candidates for specific tissue engineering applications. Here, we report a new preparation and fabrication approach for producing large scale scaffolds with customizable macroscopic structures that support cell attachment and invasion both in vitro and in vivo. This new fabrication method significantly improves cell attachment compared to that in our previous work. Moreover, the materials remain highly biocompatible and retain vascularization properties in vivo. We present proof-of-concept studies that demonstrate how hydrogels can be temporarily or permanently cast onto the macroscopic scaffolds to create composite plant-derived cellulose biomaterials. This inverse molding approach allows us to provide temporary or permanent biochemical cues to invading cells in vitro. The development of a new-generation of rapidly and efficiently produced composite plant-derived biomaterials provides an important proof that such biomaterials have the potential for numerous applications in tissue engineering.

ArticleTotal : 2	Year
Publication title Cited by 6	2014
Publication title Cited by 5	2024

Article	Year
Single cell transfection using plasmid decorated AFM probes. Biochemical and biophysical research communications\| PubMed ID: 17316557	2007
AFM as a tool to probe and manipulate cellular processes. Pflügers Archiv : European journal of physiology\| PubMed ID: 18080817	2008
Biosensing based on surface plasmon resonance and living cells. Biosensors & bioelectronics\| PubMed ID: 18845432	2009
Real-time monitoring of angiotensin II-induced contractile response and cytoskeleton remodeling in individual cells by atomic force microscopy. Pflügers Archiv : European journal of physiology\| PubMed ID: 18953565	2009
Effect of thrombin and bradykinin on endothelial cell mechanical properties monitored through membrane deformation. Journal of molecular recognition : JMR\| PubMed ID: 19415761	2009
Bradykinin protects against brain microvascular endothelial cell death induced by pathophysiological stimuli. Journal of cellular physiology\| PubMed ID: 19780024	2010
Surface Plasmon Resonance Monitoring of Cell Monolayer Integrity: Implication of Signaling Pathways Involved in Actin-Driven Morphological Remodeling. Cellular and molecular bioengineering\| PubMed ID: 21052479	2008
Extracellular HSP27 acts as a signaling molecule to activate NF-κB in macrophages. Cell stress & chaperones\| PubMed ID: 22851137	2013
Chronic over-expression of heat shock protein 27 attenuates atherogenesis and enhances plaque remodeling: a combined histological and mechanical assessment of aortic lesions. PloS one\| PubMed ID: 23409070	2013
Serum heat shock protein 27 levels represent a potential therapeutic target for atherosclerosis: observations from a human cohort and treatment of female mice. Journal of the American College of Cardiology\| PubMed ID: 23764828	2013
Heat shock protein-27 attenuates foam cell formation and atherogenesis by down-regulating scavenger receptor-A expression via NF-κB signaling. Biochimica et biophysica acta\| PubMed ID: 23939398	2013
Extracellular Forces Cause the Nucleus to Deform in a Highly Controlled Anisotropic Manner. Scientific reports\| PubMed ID: 26892269	2016
Biocompatibility of Subcutaneously Implanted Plant-Derived Cellulose Biomaterials. PloS one\| PubMed ID: 27328066	2016
Customizing the Shape and Microenvironment Biochemistry of Biocompatible Macroscopic Plant-Derived Cellulose Scaffolds. ACS biomaterials science & engineering\| PubMed ID: 33429594	2018

Charles M. Cuerrier

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