scholarly journals Cyclically stretched ACL fibroblasts emigrating from spheroids adapt their cytoskeleton and ligament-related expression profile

2021 ◽  
Author(s):  
Clemens Gögele ◽  
Christina Hoffmann ◽  
Jens Konrad ◽  
Rudolf Merkel ◽  
Silke Schwarz ◽  
...  
Keyword(s):  
Expression Profile ◽  
Cruciate Ligament ◽  
Focal Adhesions ◽  
Cyclic Stretch ◽  
Type I ◽  
Mechanical Stimuli ◽  
Cell Nuclei ◽  
Polydimethyl Siloxane ◽  
3D Microenvironment ◽  
3D Spheroids

AbstractMechanical stress of ligaments varies; hence, ligament fibroblasts must adapt their expression profile to novel mechanomilieus to ensure tissue resilience. Activation of the mechanoreceptors leads to a specific signal transduction, the so-called mechanotransduction. However, with regard to their natural three-dimensional (3D) microenvironment cell reaction to mechanical stimuli during emigrating from a 3D spheroid culture is still unclear. This study aims to provide a deeper understanding of the reaction profile of anterior cruciate ligament (ACL)-derived fibroblasts exposed to cyclic uniaxial strain in two-dimensional (2D) monolayer culture and during emigration from 3D spheroids with respect to cell survival, cell and cytoskeletal orientation, distribution, and expression profile. Monolayers and spheroids were cultured in crosslinked polydimethyl siloxane (PDMS) elastomeric chambers and uniaxially stretched (14% at 0.3 Hz) for 48 h. Cell vitality, their distribution, nuclear shape, stress fiber orientation, focal adhesions, proliferation, expression of ECM components such as sulfated glycosaminoglycans, collagen type I, decorin, tenascin C and cell–cell communication-related gap junctional connexin (CXN) 43, tendon-related markers Mohawk and tenomodulin (myodulin) were analyzed. In contrast to unstretched cells, stretched fibroblasts showed elongation of stress fibers, cell and cytoskeletal alignment perpendicular to strain direction, less rounded cell nuclei, increased numbers of focal adhesions, proliferation, amplified CXN43, and main ECM component expression in both cultures. The applied cyclic stretch protocol evoked an anabolic response and enhanced tendon-related marker expression in ACL-derived fibroblasts emigrating from 3D spheroids and seems also promising to support in future tissue formation in ACL scaffolds seeded in vitro with spheroids.

scholarly journals Matrix mechanotransduction mediated by thrombospondin-1/integrin/YAP in the vascular remodeling

2020 ◽  
Vol 117 (18) ◽  
pp. 9896-9905 ◽  
Author(s):  
Yoshito Yamashiro ◽  
Bui Quoc Thang ◽  
Karina Ramirez ◽  
Seung Jae Shin ◽  
Tomohiro Kohata ◽  
...  
Keyword(s):  
Blood Vessels ◽  
Intracellular Signaling ◽  
Vascular Remodeling ◽  
Vessel Wall ◽  
Focal Adhesions ◽  
Small Gtpase ◽  
Cyclic Stretch ◽  
Matricellular Protein ◽  
Mechanical Stimuli ◽  
Thrombospondin 1

The extracellular matrix (ECM) initiates mechanical cues that activate intracellular signaling through matrix–cell interactions. In blood vessels, additional mechanical cues derived from the pulsatile blood flow and pressure play a pivotal role in homeostasis and disease development. Currently, the nature of the cues from the ECM and their interaction with the mechanical microenvironment in large blood vessels to maintain the integrity of the vessel wall are not fully understood. Here, we identified the matricellular protein thrombospondin-1 (Thbs1) as an extracellular mediator of matrix mechanotransduction that acts via integrin αvβ1 to establish focal adhesions and promotes nuclear shuttling of Yes-associated protein (YAP) in response to high strain of cyclic stretch. Thbs1-mediated YAP activation depends on the small GTPase Rap2 and Hippo pathway and is not influenced by alteration of actin fibers. Deletion of Thbs1 in mice inhibited Thbs1/integrin β1/YAP signaling, leading to maladaptive remodeling of the aorta in response to pressure overload and inhibition of neointima formation upon carotid artery ligation, exerting context-dependent effects on the vessel wall. We thus propose a mechanism of matrix mechanotransduction centered on Thbs1, connecting mechanical stimuli to YAP signaling during vascular remodeling in vivo.

scholarly journals Maintenance of Ligament Homeostasis of Spheroid-Colonized Embroidered and Functionalized Scaffolds after 3D Stretch

2021 ◽  
Vol 22 (15) ◽  
pp. 8204
Author(s):  
Clemens Gögele ◽  
Jens Konrad ◽  
Judith Hahn ◽  
Annette Breier ◽  
Michaela Schröpfer ◽  
...  
Keyword(s):  
Gene Expression ◽  
Cell Suspension ◽  
Connexin 43 ◽  
Type I Collagen ◽  
Cruciate Ligament ◽  
Cyclic Stretch ◽  
Type I ◽  
Tenascin C ◽  
Gene Expression Control ◽  
Anterior Cruciate

Anterior cruciate ligament (ACL) ruptures are usually treated with autograft implantation to prevent knee instability. Tissue engineered ACL reconstruction is becoming promising to circumvent autograft limitations. The aim was to evaluate the influence of cyclic stretch on lapine (L) ACL fibroblasts on embroidered scaffolds with respect to adhesion, DNA and sulphated glycosaminoglycan (sGAG) contents, gene expression of ligament-associated extracellular matrix genes, such as type I collagen, decorin, tenascin C, tenomodulin, gap junctional connexin 43 and the transcription factor Mohawk. Control scaffolds and those functionalized by gas phase fluorination and cross-linked collagen foam were either pre-cultured with a suspension or with spheroids of LACL cells before being subjected to cyclic stretch (4%, 0.11 Hz, 3 days). Stretch increased significantly the scaffold area colonized with cells but impaired sGAGs and decorin gene expression (functionalized scaffolds seeded with cell suspension). Stretching increased tenascin C, connexin 43 and Mohawk but decreased decorin gene expression (control scaffolds seeded with cell suspension). Pre-cultivation of functionalized scaffolds with spheroids might be the more suitable method for maintaining ligamentogenesis in 3D scaffolds compared to using a cell suspension due to a significantly higher sGAG content in response to stretching and type I collagen gene expression in functionalized scaffolds.

The molecular mechanism of mechanotransduction in vascular homeostasis and disease

2020 ◽  
Vol 134 (17) ◽  
pp. 2399-2418
Author(s):  
Yoshito Yamashiro ◽  
Hiromi Yanagisawa
Keyword(s):  
Shear Stress ◽  
Blood Vessels ◽  
Vessel Wall ◽  
Vascular Diseases ◽  
Cell Interactions ◽  
Aortic Aneurysms ◽  
Cyclic Stretch ◽  
Mechanical Stimuli ◽  
Vascular Homeostasis ◽  
Matrix Cell

Abstract Blood vessels are constantly exposed to mechanical stimuli such as shear stress due to flow and pulsatile stretch. The extracellular matrix maintains the structural integrity of the vessel wall and coordinates with a dynamic mechanical environment to provide cues to initiate intracellular signaling pathway(s), thereby changing cellular behaviors and functions. However, the precise role of matrix–cell interactions involved in mechanotransduction during vascular homeostasis and disease development remains to be fully determined. In this review, we introduce hemodynamics forces in blood vessels and the initial sensors of mechanical stimuli, including cell–cell junctional molecules, G-protein-coupled receptors (GPCRs), multiple ion channels, and a variety of small GTPases. We then highlight the molecular mechanotransduction events in the vessel wall triggered by laminar shear stress (LSS) and disturbed shear stress (DSS) on vascular endothelial cells (ECs), and cyclic stretch in ECs and vascular smooth muscle cells (SMCs)—both of which activate several key transcription factors. Finally, we provide a recent overview of matrix–cell interactions and mechanotransduction centered on fibronectin in ECs and thrombospondin-1 in SMCs. The results of this review suggest that abnormal mechanical cues or altered responses to mechanical stimuli in EC and SMCs serve as the molecular basis of vascular diseases such as atherosclerosis, hypertension and aortic aneurysms. Collecting evidence and advancing knowledge on the mechanotransduction in the vessel wall can lead to a new direction of therapeutic interventions for vascular diseases.

scholarly journals Role of Curing Temperature of Poly(Glycerol Sebacate) Substrates on Protein-Cell Interaction and Early Cell Adhesion

Polymers ◽  
2021 ◽  
Vol 13 (3) ◽  
pp. 382
Author(s):  
Rubén Martín-Cabezuelo ◽  
José Carlos Rodríguez-Hernández ◽  
Guillermo Vilariño-Feltrer ◽  
Ana Vallés-Lluch
Keyword(s):  
Protein Interactions ◽  
Chemical Properties ◽  
Focal Adhesions ◽  
Collagen Type I ◽  
Inhibitory Effect ◽  
Synthesis Temperature ◽  
Curing Temperature ◽  
Cell Protein ◽  
Preliminary Treatment ◽  
Type I

A novel procedure to obtain smooth, continuous polymeric surfaces from poly(glycerol sebacate) (PGS) has been developed with the spin-coating technique. This method proves useful for separating the effect of the chemistry and morphology of the networks (that can be obtained by varying the synthesis parameters) on cell-protein-substrate interactions from that of structural variables. Solutions of the PGS pre-polymer can be spin-coated, to then be cured. Curing under variable temperatures has been shown to lead to PGS networks with different chemical properties and topographies, conditioning their use as a biomaterial. Particularly, higher synthesis temperatures yield denser networks with fewer polar terminal groups available on the surface. Material-protein interactions were characterised by using extracellular matrix proteins such as fibronectin (Fn) and collagen type I (Col I), to unveil the biological interface profile of PGS substrates. To that end, atomic force microscopy (AFM) images and quantification of protein adsorbed in single, sequential and competitive protein incubations were used. Results reveal that Fn is adsorbed in the form of clusters, while Col I forms a characteristic fibrillar network. Fn has an inhibitory effect when incubated prior to Col I. Human umbilical endothelial cells (HUVECs) were also cultured on PGS surfaces to reveal the effect of synthesis temperature on cell behaviour. To this effect, early focal adhesions (FAs) were analysed using immunofluorescence techniques. In light of the results, 130 °C seems to be the optimal curing temperature since a preliminary treatment with Col I or a Fn:Col I solution facilitates the formation of early focal adhesions and growth of HUVECs.

scholarly journals Cruciate Ligament Cell Sheets Can Be Rapidly Produced on Thermoresponsive poly(glycidyl ether) Coating and Successfully Used for Colonization of Embroidered Scaffolds

Cells ◽  
2021 ◽  
Vol 10 (4) ◽  
pp. 877
Author(s):  
Ingrid Zahn ◽  
Daniel David Stöbener ◽  
Marie Weinhart ◽  
Clemens Gögele ◽  
Annette Breier ◽  
...  
Keyword(s):  
Gene Expression ◽  
Type I Collagen ◽  
Cruciate Ligament ◽  
Glycidyl Ether ◽  
Stable Gene ◽  
Type I ◽  
Cell Sheets ◽  
Thermoresponsive Polymers ◽  
Related Phenotype ◽  
Anterior Cruciate

Anterior cruciate ligament (ACL) cell sheets combined with biomechanically competent scaffolds might facilitate ACL tissue engineering. Since thermoresponsive polymers allow a rapid enzyme-free detachment of cell sheets, we evaluated the applicability of a thermoresponsive poly(glycidyl ether) (PGE) coating for cruciate ligamentocyte sheet formation and its influence on ligamentocyte phenotype during sheet-mediated colonization of embroidered scaffolds. Ligamentocytes were seeded on surfaces either coated with PGE or without coating. Detached ligamentocyte sheets were cultured separately or wrapped around an embroidered scaffold made of polylactide acid (PLA) and poly(lactic-co-ε-caprolactone) (P(LA-CL)) threads functionalized by gas-phase fluorination and with collagen foam. Ligamentocyte viability, protein and gene expression were determined in sheets detached from surfaces with or without PGE coating, scaffolds seeded with sheets from PGE-coated plates and the respective monolayers. Stable and vital ligamentocyte sheets could be produced within 24 h with both surfaces, but more rapidly with PGE coating. PGE did not affect ligamentocyte phenotype. Scaffolds could be colonized with sheets associated with high cell survival, stable gene expression of ligament-related type I collagen, decorin, tenascin C and Mohawk after 14 d and extracellular matrix (ECM) deposition. PGE coating facilitates ligamentocyte sheet formation, and sheets colonizing the scaffolds displayed a ligament-related phenotype.

STRETCHABLE Lab-on-Chip Device With Impedance Spectroscopy Capability for Mammalian Cell Studies

2016 ◽  
Author(s):  
Xudong Zhang ◽  
Anis Nurashikin Nordin ◽  
Fang Li ◽  
Ioana Voiculescu
Keyword(s):  
Impedance Spectroscopy ◽  
Mammalian Cells ◽  
Dynamic Environment ◽  
Cyclic Strain ◽  
Cyclic Stretch ◽  
Mechanical Stimuli ◽  
Aortic Endothelial Cells ◽  
Lab On Chip

This paper presents the fabrication and testing of electric cell-substrate impedance spectroscopy (ECIS) electrodes on a stretchable membrane. This is the first time when ECIS electrodes were fabricated on a stretchable substrate and ECIS measurements on mammalian cells exposed to cyclic strain of 10% were successfully demonstrated. A chemical was used to form strong chemical bond between gold electrodes of ECIS sensor and polymer membrane, which enable the electrodes keep good conductive ability during cyclic stretch. The stretchable membrane integrated with the ECIS sensor can simulate and replicate the dynamic environment of organism and enable the analysis of the cells activity involved in cells attachment and proliferation in vitro. Bovine aortic endothelial cells (BAEC) were used to evaluate the endothelial function influenced by mechanical stimuli in this research because they undergo in vivo cyclic physiologic elongation produced by the blood circulation in the arteries.

Intrinsic mechanical properties of the extracellular matrix affect the behavior of pre-osteoblastic MC3T3-E1 cells

2006 ◽  
Vol 290 (6) ◽  
pp. C1640-C1650 ◽  
Author(s):  
Chirag B. Khatiwala ◽  
Shelly R. Peyton ◽  
Andrew J. Putnam
Keyword(s):  
Mechanical Properties ◽  
Type I Collagen ◽  
Focal Adhesions ◽  
Microscopic Analysis ◽  
Maximal Activity ◽  
Type I ◽  
Cell Functions ◽  
Collagen Density ◽  
Cells Cultured ◽  
In Cells

Mechanical cues present in the ECM have been hypothesized to provide instructive signals that dictate cell behavior. We probed this hypothesis in osteoblastic cells by culturing MC3T3-E1 cells on the surface of type I collagen-modified hydrogels with tunable mechanical properties and assessed their proliferation, migration, and differentiation. On gels functionalized with a low type I collagen density, MC3T3-E1 cells cultured on polystyrene proliferated twice as fast as those cultured on the softest substrate. Quantitative time-lapse video microscopic analysis revealed random motility speeds were significantly retarded on the softest substrate (0.25 ± 0.01 μm/min), in contrast to maximum speeds on polystyrene substrates (0.42 ± 0.04 μm/min). On gels functionalized with a high type I collagen density, migration speed exhibited a biphasic dependence on ECM compliance, with maximum speeds (0.34 ± 0.02 μm/min) observed on gels of intermediate stiffness, whereas minimum speeds (0.24 ± 0.03 μm/min) occurred on both the softest and most rigid (i.e., polystyrene) substrates. Immature focal contacts and a poorly organized actin cytoskeleton were observed in cells cultured on the softest substrates, whereas those on more rigid substrates assembled mature focal adhesions and robust actin stress fibers. In parallel, focal adhesion kinase (FAK) activity (assessed by detecting pY397-FAK) was influenced by compliance, with maximal activity occurring in cells cultured on polystyrene. Finally, mineral deposition by the MC3T3-E1 cells was also affected by ECM compliance, leading to the conclusion that altering ECM mechanical properties may influence a variety of MC3T3-E1 cell functions, and perhaps ultimately, their differentiated phenotype.

An Uncommon Trauma: Hoffa's Fracture Associated with Tibial Avulsion Fracture of the Posterior Cruciate Ligament

2021 ◽  
Author(s):  
Bertan Cengiz ◽  
Sinan Karaoglu
Keyword(s):  
Posterior Cruciate Ligament ◽  
Avulsion Fracture ◽  
Cruciate Ligament ◽  
Easy Access ◽  
Lateral Condyle ◽  
Type I ◽  
Hoffa Fracture ◽  
Tibial Avulsion Fracture ◽  
Additional Stability ◽  
Buttress Plate

Abstract BackgroundSince Hoffa fractures are usually the result of high-energy injuries, many additional accompanying injuries have been described. This is the first paper representing the lateral condyle Hoffa fracture accompanying tibial avulsion fracture of the posterior cruciate ligament (PCL).Case PresentationA 42-years-old male presented with swelling and instability in his left knee after falling during parachute landing. He was diagnosed with simultaneous Letenneur Type I lateral condyle Hoffa fracture and tibial avulsion fracture of the PCL. He was operated on with a single posterior incision for both fractures. Multiple cannulated lag screws were used for the fixation of the Hoffa fracture, and a buttress plate was used for additional stability. PCL avulsion fracture was fixed with a cannulated screw with a washer. The patient was allowed for full-weight-bearing and range of motion at the sixth week after the operation. No complications occurred during follow-up.ConclusionCare should be taken in terms of additional injuries that may accompany Hoffa fractures. The posterior approach allows easy access to both fractures with a single incision. Using a buttress plate after the fixation of the Hoffa fracture with multiple lag screws provides additional stability.

scholarly journals Arthroscopic Primary Repair of Proximal Anterior Cruciate Ligament Tears: With or Without Additional Suture Augmentation?

2018 ◽  
Vol 6 (7_suppl4) ◽  
pp. 2325967118S0006 ◽  
Author(s):  
Anne Jonkergouw ◽  
Jelle P. van der List ◽  
Gregory S. DiFelice
Keyword(s):  
Range Of Motion ◽  
Patient Reported Outcomes ◽  
Suture Anchor ◽  
Primary Repair ◽  
Cruciate Ligament ◽  
Type I ◽  
Acl Repair ◽  
Patient Reported ◽  
Anterior Cruciate

Objectives: Over the last years, arthroscopic primary repair of anterior cruciate ligament (ACL) tears has shown excellent results owing to appropriate patient selection (only repairing proximal ACL tears and good tissue quality), minimal invasive surgery (arthroscopy) and focus on early range of motion. Some surgeons have repaired proximal ACL tears without suture augmentation while others have used internal suture augmentation to reinforce and thus protect the repaired ligament during range of motion. No studies have yet compared the two surgical techniques. The objective of this study was to compare failure rates, reoperation rates and patient-reported outcomes of arthroscopic primary repair with versus without suture augmentation. Methods: A retrospective search for all patients treated with suture anchor arthroscopic primary ACL repair between April 2008 and June 2016 was performed. All patients with isolated proximal ACL tears (type I) were included. Since the development of internal suture augmentation, this reinforcement was added to the repaired ACLs. Minimum follow-up length was 1.0 years. Results: A total of 56 patients were included (mean age 33 years (range: 14 - 57), 59% male) of which 28 (50%) patients received additional suture augmentation. Mean follow-up was 2.3 years (range: 1.0-9.2). Six of all patients had reruptured their repaired ACL (10.7%), of which four underwent uncomplicated ACL reconstruction and two were treated conservatively. Four reruptures were initially treated with primary repair only (4/28, 14.3%) and two patients with additional suture augmentation (2/28, 7.1%; p = 0.431). During follow-up, three patients underwent reoperation (5.4%; two for medial meniscus tear (one in each group) and one for tibial suture anchor removal of the suture augmentation). Patient-reported outcomes have so far been collected in 20 patients without reruptures (currently collecting), with mean Lysholm score of 96, modified Cincinnati 94, SANE 93, pre-injury Tegner 6.7, postoperative Tegner 6.3 and subjective IKDC 91. Objective IKDC was A in 90%, B in 5%, C in 5%. Conclusion: In this study, the total failure rate of arthroscopic primary ACL repair was 10.7% and was lower with additional suture augmentation (7.1%) than primary repair alone (14.3%). Patients with failed ACL repair underwent uncomplicated primary ACL reconstruction. We recommend adding suture augmentation in high-risk patients (i.e. adolescents, ones with hyperlaxity, high contact sports), to protect the repaired ligament, especially during early range of motion. These data support treating type I proximal ACL tears with arthroscopic primary repair.

scholarly journals A Dynamic Stochastic Model of Frequency-Dependent Stress Fiber Alignment Induced by Cyclic Stretch

2009 ◽  
Author(s):  
Hui-Ju Hsu ◽  
Chin-Fu Lee ◽  
Roland Kaunas
Keyword(s):  
Cell Shape ◽  
Principal Direction ◽  
Focal Adhesions ◽  
Cyclic Stretch ◽  
Cell Alignment ◽  
Cellular Functions ◽  
Fiber Alignment ◽  
Dynamic Stochastic ◽  
Dynamic Structures ◽  
Point Level

Actin stress fibers (SFs) are bundles of actin filaments anchored at each end via focal adhesions. Myosin-generated contraction leads to the development of tension, which extends SFs beyond their unloaded lengths. In human aortic ECs, the level of SF extension is maintained at a set-point level of ∼1.10 (1). SFs are also dynamic structures and their continuous assembly and disassembly is critical to cellular functions involving changes in cell shape. Further, deformation of the extracellular matrix perturbs SF extension, leading to compensatory responses such as the gradual alignment of SFs perpendicular to the principal direction of cyclic stretch. The extent of cell alignment has been shown to depend on the pattern of matrix stretch; however, it is unclear how cells distinguish between different patterns of stretch to determine their unique responses.

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