scholarly journals Nanoanalytical analysis of bisphosphonate-driven alterations of microcalcifications using a 3D hydrogel system and in vivo mouse model

2021 ◽  
Vol 118 (14) ◽  
pp. e1811725118
Author(s):  
Jessica L. Ruiz ◽  
Joshua D. Hutcheson ◽  
Luis Cardoso ◽  
Amirala Bakhshian Nik ◽  
Alexandra Condado de Abreu ◽  
...  
Keyword(s):  
Vascular Calcification ◽  
Cardiovascular Events ◽  
Plaque Rupture ◽  
Three Dimensional ◽  
Element Analysis ◽  
Fibrous Cap ◽  
3D Collagen ◽  
Atherosclerotic Plaque Rupture

Vascular calcification predicts atherosclerotic plaque rupture and cardiovascular events. Retrospective studies of women taking bisphosphonates (BiPs), a proposed therapy for vascular calcification, showed that BiPs paradoxically increased morbidity in patients with prior acute cardiovascular events but decreased mortality in event-free patients. Calcifying extracellular vesicles (EVs), released by cells within atherosclerotic plaques, aggregate and nucleate calcification. We hypothesized that BiPs block EV aggregation and modify existing mineral growth, potentially altering microcalcification morphology and the risk of plaque rupture. Three-dimensional (3D) collagen hydrogels incubated with calcifying EVs were used to mimic fibrous cap calcification in vitro, while an ApoE−/− mouse was used as a model of atherosclerosis in vivo. EV aggregation and formation of stress-inducing microcalcifications was imaged via scanning electron microscopy (SEM) and atomic force microscopy (AFM). In both models, BiP (ibandronate) treatment resulted in time-dependent changes in microcalcification size and mineral morphology, dependent on whether BiP treatment was initiated before or after the expected onset of microcalcification formation. Following BiP treatment at any time, microcalcifications formed in vitro were predicted to have an associated threefold decrease in fibrous cap tensile stress compared to untreated controls, estimated using finite element analysis (FEA). These findings support our hypothesis that BiPs alter EV-driven calcification. The study also confirmed that our 3D hydrogel is a viable platform to study EV-mediated mineral nucleation and evaluate potential therapies for cardiovascular calcification.

Abstract 658: Unraveling the Controversy of Bisphosphonates as Vascular Calcification Therapy Using a Nanoanalytical Approach

2016 ◽  
Vol 36 (suppl_1) ◽  
Author(s):  
Jessica L Ruiz ◽  
Joshua D Hutcheson ◽  
Elena Aikawa
Keyword(s):  
Vascular Calcification ◽  
Plaque Rupture ◽  
Three Dimensional ◽  
Building Blocks ◽  
Underlying Mechanism ◽  
Pharmacological Intervention ◽  
Fibrous Cap ◽  
Collagen Hydrogel ◽  
Increased Risk

Vascular calcification significantly predicts atherosclerotic plaque rupture and cardiovascular events. Retrospective studies of women taking bisphosphonates, a proposed therapy for vascular calcification, paradoxically indicated increased risk in patients with prior acute events. We recently demonstrated that calcifying extracellular vesicles (EVs) released by cells within the plaque aggregate and nucleate calcific mineral, but the underlying mechanism and the potential for pharmacological intervention remain poorly understood. We hypothesize that bisphosphonates block EV aggregation and arrest existing mineral growth, freezing calcifications in a high-risk morphology that hastens plaque rupture. This study visualized for the first time EV aggregation and calcification at single-EV resolution, via scanning electron microscopy. Three-dimensional (3-D) collagen hydrogels incubated with calcifying EVs modeled fibrous cap calcification, serving as an in vitro platform to image mineral nucleation and test candidate drugs for the potential to inhibit or reverse vascular calcification. EVs aggregated along and between collagen fibrils. Energy-dispersive x-ray spectroscopy (EDS) confirmed that EV aggregates contained calcium and phosphorous, the building blocks of calcific mineral (vs. internal collagen control, p<0.001). The addition of the bisphosphonate ibandronate decreased the EDS-detected amount of calcium (4.32% by weight (wt%) vs. 2.36 wt%, p<0.001) and phosphorous (4.26 wt% vs. 1.94 wt%, p<0.001) comprising EV aggregates. Further, ibandronate reduced the size (21.5 μm 2 vs. 14.2 μm 2 , p=0.012) and changed the morphology of calcific EV aggregates (Figure). These findings agree with our hypothesis that bisphosphonates alter EV-driven calcification, and confirm that our 3-D collagen hydrogel system is a viable platform to study EV-mediated mineral nucleation and evaluate potential therapies for cardiovascular calcification.

Local, Three-Dimensional Strain Measurements Within Largely Deformed Extracellular Matrix Constructs

2004 ◽  
Vol 126 (6) ◽  
pp. 699-708 ◽  
Author(s):  
Blayne A. Roeder ◽  
Klod Kokini ◽  
J. Paul Robinson ◽  
Sherry L. Voytik-Harbin
Keyword(s):  
Extracellular Matrix ◽  
Local Level ◽  
Three Dimensional ◽  
Collagen Matrices ◽  
Mechanical Loads ◽  
Strain Measurements ◽  
3D Collagen ◽  
Vitro Model

The ability to create extracellular matrix (ECM) constructs that are mechanically and biochemically similar to those found in vivo and to understand how their properties affect cellular responses will drive the next generation of tissue engineering strategies. To date, many mechanisms by which cells biochemically communicate with the ECM are known. However, the mechanisms by which mechanical information is transmitted between cells and their ECM remain to be elucidated. “Self-assembled” collagen matrices provide an in vitro-model system to study the mechanical behavior of ECM. To begin to understand how the ECM and the cells interact mechanically, the three-dimensional (3D) mechanical properties of the ECM must be quantified at the micro-(local) level in addition to information measured at the macro-(global) level. Here we describe an incremental digital volume correlation (IDVC) algorithm to quantify large (>0.05) 3D mechanical strains in the microstructure of 3D collagen matrices in response to applied mechanical loads. Strain measurements from the IDVC algorithm rely on 3D confocal images acquired from collagen matrices under applied mechanical loads. The accuracy and the precision of the IDVC algorithm was verified by comparing both image volumes collected in succession when no deformation was applied to the ECM (zero strain) and image volumes to which simulated deformations were applied in both 1D and 3D (simulated strains). Results indicate that the IDVC algorithm can accurately and precisely determine the 3D strain state inside largely deformed collagen ECMs. Finally, the usefulness of the algorithm was demonstrated by measuring the microlevel 3D strain response of a collagen ECM loaded in tension.

Can We Use In Vivo MRI and FEA to Determine Peak Cap Stress in Carotid Plaques? MRI Simulations Provide Answers

2013 ◽  
Author(s):  
Harm A. Nieuwstadt ◽  
Jolanda W. Wentzel ◽  
Aad van der Lugt ◽  
Anton F. W. van der Steen ◽  
Marcel Breeuwer ◽  
...  
Keyword(s):  
Plaque Rupture ◽  
Important Determinant ◽  
Peak Stress ◽  
Necrotic Core ◽  
Rupture Risk ◽  
Resonance Imaging ◽  
Element Analysis ◽  
Fibrous Cap ◽  
Magnetic Resonance Imaging Mri

Vulnerable plaques are characterized by a large lipid-rich necrotic core (LRNC) separated by a thin fibrous cap (FC) from the lumen. Plaque rupture occurs when the peak stress in the FC exceeds its strength. Carotid in vivo magnetic resonance imaging (MRI) data can be segmented to obtain the plaque geometry noninvasively. An increasing number of studies use MR imaging for biomechanical finite element analysis (FEA) to compute peak cap stresses [1, 2]. Previous studies have shown that the thickness of the FC is an important determinant of peak cap stress: the thinner the FC, the higher the stress, the higher the plaque rupture risk [3].

Identification of a 2-stage platelet aggregation process mediating shear-dependent thrombus formation

Blood ◽  
2006 ◽  
Vol 109 (2) ◽  
pp. 566-576 ◽  
Author(s):  
Mhairi J. Maxwell ◽  
Erik Westein ◽  
Warwick S. Nesbitt ◽  
Simon Giuliano ◽  
Sacha M. Dopheide ◽  
...  
Keyword(s):  
Platelet Aggregation ◽  
Plaque Rupture ◽  
Thrombus Formation ◽  
Imaging Techniques ◽  
Platelet Aggregates ◽  
Adhesive Interactions ◽  
Membrane Tethers ◽  
Atherosclerotic Plaque Rupture

Abstract Disturbances of blood flow at sites of atherosclerotic plaque rupture are one of the key pathogenic events promoting platelet activation and arterial thrombus formation. Shear effects of platelets have been extensively investigated in vitro; however, the mechanisms by which shear promotes platelet aggregation in vivo remain incompletely understood. By employing high-resolution imaging techniques to in vitro and in vivo thrombosis models, we demonstrate a unique mechanism initiating shear-dependent platelet aggregation involving aggregate formation between discoid platelets. These discoid platelet aggregates are initially unstable and result from the development of membrane tethers between coadhering platelets. Tether formation involves the adhesive function of GPIb/V/IX and integrin αIIbβ3, and conversion of discoid platelet aggregates into stable aggregates requires released ADP. The efficiency of this process is regulated by 3 independent variables, including the reactivity of the adhesive substrate, the level of shear flow, and the platelet density at the adhesive surface. These studies identify a new mechanism initiating platelet aggregation that is critically influenced by shear, physical proximity between translocating platelets, and membrane tether formation. Moreover, they provide a model to explain how the discoid morphology of platelets facilitates the maintenance of adhesive interactions with thrombogenic surfaces under high shear stress conditions.

scholarly journals A mechanistic analysis of the role of microcalcifications in atherosclerotic plaque stability: potential implications for plaque rupture

2012 ◽  
Vol 303 (5) ◽  
pp. H619-H628 ◽  
Author(s):  
Natalia Maldonado ◽  
Adreanne Kelly-Arnold ◽  
Yuliya Vengrenyuk ◽  
Damien Laudier ◽  
John T. Fallon ◽  
...  
Keyword(s):  
Plaque Rupture ◽  
Tissue Sample ◽  
Circumferential Stress ◽  
Three Dimensional ◽  
Surrounding Tissue ◽  
Element Analysis ◽  
Clinical Criteria ◽  
Fibrous Cap ◽  
Three Dimensional Finite Element

The role of microcalcifications (μCalcs) in the biomechanics of vulnerable plaque rupture is examined. Our laboratory previously proposed (Ref. 44 ), using a very limited tissue sample, that μCalcs embedded in the fibrous cap proper could significantly increase cap instability. This study has been greatly expanded. Ninety-two human coronary arteries containing 62 fibroatheroma were examined using high-resolution microcomputed tomography at 6.7-μm resolution and undecalcified histology with special emphasis on calcified particles <50 μm in diameter. Our results reveal the presence of thousands of μCalcs, the vast majority in lipid pools where they are not dangerous. However, 81 μCalcs were also observed in the fibrous caps of nine of the fibroatheroma. All 81 of these μCalcs were analyzed using three-dimensional finite-element analysis, and the results were used to develop important new clinical criteria for cap stability. These criteria include variation of the Young's modulus of the μCalc and surrounding tissue, μCalc size, and clustering. We found that local tissue stress could be increased fivefold when μCalcs were closely spaced, and the peak circumferential stress in the thinnest nonruptured cap (66 μm) if no μCalcs were present was only 107 kPa, far less than the proposed minimum rupture threshold of 300 kPa. These results and histology suggest that there are numerous μCalcs < 15 μm in the caps, not visible at 6.7-μm resolution, and that our failure to find any nonruptured caps between 30 and 66 μm is a strong indication that many of these caps contained μCalcs.

scholarly journals Comparative Evaluation of Stress developed on Rotary Retreatment Instruments during Retrieval of Gutta-percha

2017 ◽  
Vol 18 (6) ◽  
pp. 484-489 ◽  
Author(s):  
Sushmita Shivanna ◽  
Dhanasekaran Sihivahanan ◽  
T Vinay Kumar Reddy ◽  
Anchu Rachel Thomas ◽  
Natarajan Senthilnathan ◽  
...  
Keyword(s):  
Stress Distribution ◽  
Comparative Evaluation ◽  
Structural Characteristics ◽  
Three Dimensional ◽  
Element Analysis ◽  
Basic Model ◽  
Gutta Percha ◽  
Group I

ABSTRACT Aim The aim of the study is to compare the maximum stress distribution on the rotary retreatment instruments within the root canal at cervical, middle, and the apical one-third during retreatment of gutta-percha. Materials and methods A human mandibular premolar was scanned, and three-dimensional geometry of the root was reconstructed using finite element analysis (FEA) software package (ANSYS). The basic model was kept unchanged; tooth models were created using the same dimensions and divided into two groups as follows: Group I: ProTaper Universal retreatment system and group II: Mtwo rotary retreatment system. The stress distribution on the surface and within the retreatment files was analyzed numerically in the FEA package (ANSYS). Results The FEA analysis revealed that the retreatment instruments received the greatest stress in the cervical third, followed by the apical third and the middle third. The stress generated on the ProTaper Universal retreatment system was less when compared with the Mtwo retreatment files. Conclusion The study concludes that the retreatment instruments undergo higher stress in the cervical third region, and further in vivo and in vitro studies are necessary to evaluate the relationship between instrument designs, stress distribution, residual stresses after use, and the torsional fracture of the retreatment instrument. Clinical significance The stress developed on the rotary retreatment instruments during retrieval of gutta-percha makes the instrument to get separated. There is no instrument system, i.e., suitable for all clinical situations and it is important to understand how the structural characteristics could influence the magnitude of stresses on the instrument to prevent its fracture in use. How to cite this article Sihivahanan D, Reddy TVK, Thomas AR, Senthilnathan N, Sivakumar M, Shivanna S. Comparative Evaluation of Stress developed on Rotary Retreatment Instruments during Retrieval of Gutta-percha. J Contemp Dent Pract 2017;18(6):484-489.

Remote Determination of Time-Dependent Stiffness of Surface-Degrading-Polymer Scaffolds Via Synchrotron-Based Imaging

2017 ◽  
Vol 139 (4) ◽  
Author(s):  
N. K. Bawolin ◽  
X. B. Chen
Keyword(s):  
Mechanical Properties ◽  
Three Dimensional ◽  
Tissue Scaffolds ◽  
Time Dependent ◽  
Element Analysis ◽  
Surface Degradation ◽  
Direct Measurements ◽  
Nonlinear Stress

Surface-degrading polymers have been widely used to fabricate scaffolds with the mechanical properties appropriate for tissue regeneration/repair. During their surface degradation, the material properties of polymers remain approximately unchanged, but the scaffold geometry and thus mechanical properties vary with time. This paper presents a novel method to determine the time-dependent mechanical properties, particularly stiffness, of scaffolds from the geometric changes captured by synchrotron-based imaging, with the help of finite element analysis (FEA). Three-dimensional (3D) tissue scaffolds were fabricated from surface-degrading polymers, and during their degradation, the tissue scaffolds were imaged via the synchrotron-based imaging to characterize their changing geometry. On this basis, the stiffness behavior of scaffolds was estimated from the FEA, and the results obtained were compared to the direct measurements of scaffold stiffness from the load–displacement material testing. The comparison illustrates that the Young's moduli estimated from the FEA and characterized geometry are in agreement with the ones of direct measurements. The developed method of estimating the mechanical behavior was also demonstrated effective with a nondegrading scaffold that displays the nonlinear stress–strain behavior. The in vivo monitoring of Young's modulus by morphology characterization also suggests the feasibility of characterizing experimentally the difference between in vivo and in vitro surface degradation of tissue engineering constructs.

In vitro and in vivo polysaccharides assembly: ultrastructural and cytochemical study of growing plant cell wall components.

1978 ◽  
Vol 36 (2) ◽  
pp. 434-435
Author(s):  
D. Reis ◽  
B. Vian ◽  
J. C. Roland
Keyword(s):  
Cell Wall ◽  
Self Assembly ◽  
Plant Cell Wall ◽  
Higher Plants ◽  
Three Dimensional ◽  
Cytochemical Study ◽  
Wall Components

Wall morphogenesis in higher plants is a problem still open to controversy. Until now the possibility of a transmembrane control and the involvement of microtubules were mostly envisaged. Self-assembly processes have been observed in the case of walls of Chlamydomonas and bacteria. Spontaneous gelling interactions between xanthan and galactomannan from Ceratonia have been analyzed very recently. The present work provides indications that some processes of spontaneous aggregation could occur in higher plants during the formation and expansion of cell wall.Observations were performed on hypocotyl of mung bean (Phaseolus aureus) for which growth characteristics and wall composition have been previously defined.In situ, the walls of actively growing cells (primary walls) show an ordered three-dimensional organization (fig. 1). The wall is typically polylamellate with multifibrillar layers alternately transverse and longitudinal. Between these layers intermediate strata exist in which the orientation of microfibrils progressively rotates. Thus a progressive change in the morphogenetic activity occurs.

Epithelial Organotypic Cultures: A Viable Model to Address Mechanisms of Carcinogenesis by Epitheliotropic Viruses

2018 ◽  
Vol 18 (4) ◽  
pp. 246-255 ◽  
Author(s):  
Lara Termini ◽  
Enrique Boccardo
Keyword(s):  
Three Dimensional ◽  
Cell Types ◽  
Experimental Models ◽  
Biological Research ◽  
Specific Gene ◽  
Specific Cell ◽  
Organotypic Cultures ◽  
Skin Physiology

In vitro culture of primary or established cell lines is one of the leading techniques in many areas of basic biological research. The use of pure or highly enriched cultures of specific cell types obtained from different tissues and genetics backgrounds has greatly contributed to our current understanding of normal and pathological cellular processes. Cells in culture are easily propagated generating an almost endless source of material for experimentation. Besides, they can be manipulated to achieve gene silencing, gene overexpression and genome editing turning possible the dissection of specific gene functions and signaling pathways. However, monolayer and suspension cultures of cells do not reproduce the cell type diversity, cell-cell contacts, cell-matrix interactions and differentiation pathways typical of the three-dimensional environment of tissues and organs from where they were originated. Therefore, different experimental animal models have been developed and applied to address these and other complex issues in vivo. However, these systems are costly and time consuming. Most importantly the use of animals in scientific research poses moral and ethical concerns facing a steadily increasing opposition from different sectors of the society. Therefore, there is an urgent need for the development of alternative in vitro experimental models that accurately reproduce the events observed in vivo to reduce the use of animals. Organotypic cultures combine the flexibility of traditional culture systems with the possibility of culturing different cell types in a 3D environment that reproduces both the structure and the physiology of the parental organ. Here we present a summarized description of the use of epithelial organotypic for the study of skin physiology, human papillomavirus biology and associated tumorigenesis.

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