Mechanical Properties of the Extracellular Matrix Influence Fibronectin Fibril Assembly in Vitro

1995 ◽  
Vol 217 (1) ◽  
pp. 109-117 ◽  
Author(s):  
Nancy L. Halliday ◽  
James J. Tomasek
Keyword(s):  
Mechanical Properties ◽  
Extracellular Matrix ◽  
Matrix Influence

Development Of 3D Models For Studying Megakaryopoiesis In Vitro

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 3696-3696
Author(s):  
Lindsay Wray ◽  
Christian A Di Buduo ◽  
David L. Kaplan ◽  
Alessandra Balduini
Keyword(s):  
Mechanical Properties ◽  
Bone Marrow ◽  
Extracellular Matrix ◽  
Endothelial Cells ◽  
Peripheral Blood ◽  
Platelet Production ◽  
Vascular Niche ◽  
Proplatelet Formation ◽  
Bone Marrow Vascular Niche

Abstract Introduction Silk fibroin, derived from Bombyx mori silkworm cocoons, is used extensively in biomaterials and regenerative medicine. The useful characteristics of this protein include self-assembly, robust mechanical properties, biocompatibility and biodegradability. Moreover, silk can be enhanced through a variety of chemical modifications that affect cell attachment, growth and differentiation. Thrombocytopenia occurs when a patient suffers from an abnormally low platelet count in the peripheral blood; usually a result of disease, trauma, or cancer treatment. To treat these patients, it is estimated that two million platelet transfusions are performed in the U.S. each year. This high demand for platelets has created a clinical demand for studying the causes of thrombocytopenia and alternative routes for treatment. Platelets are anuclear cells that are released into the bloodstream in the bone marrow by megakaryocytes via the extension of long filaments called proplatelets. It is hypothesized that platelet production from megakaryocytes is regulated by environmental factors at the site of bone marrow vascular niche. Studies of megakaryopoiesis are typically performed on extracellular matrix protein-coated culture plates and transwell membranes. While these initial studies have provided invaluable insight into the process of megakaryopoiesis, the goal of the present project was to create a bone marrow model that mimics the vascular niche for functional in vitro platelet production. We hypothesized that a silk-based in vitro tissue model would allow the effects of substrate surface properties and endothelial co-culture on megakaryopoiesis to be studied in a holistic manner, thereby enabling further elucidation of the mechanisms involved in the process of platelet production. Results In order to more closely mimic the bone marrow vascular niche structure, a porous silk sponge was assembled around the silk vessel-like tubes. Megakarycytes seeded in the porous silk sponge migrated toward the silk tube and released platelets into the tube lumen. The perfusion bioreactor moved the platelets into the platelet collecting bags. After perfusion the platelets were collected and analyzed by flow cytometry. The bioreactor platelets exhibited similar morphology, CD41 positive staining, and activation compared to peripheral blood platelet controls. Megakaryocyte attachment and proplatelet formation through the silk vascular wall were improved by altering the silk properties. Silk functionalized by entrapping extracellular matrix proteins within the tube membrane resulted in increased megakaryocyte attachment and proplatelet compared to unfunctionalized silk tube controls. Silk surface roughness improved megakaryocyte attachment compared to the control but did not affect proplatelets. Decreasing the silk stiffness improved proplatelets, but did not significantly affect megakaryocyte attachment. Co-culture with endothelial cells improved megakaryocyte attachment while maintaining a high level of proplatelet formation. Additionally, megakaryocyte and endothelial cell co-culture on the silk vessel model resulted in an icreased platelet production compared to megakaryocytes cultured alone. Conclusions The goal of this project was to develop an in vitro model of megakaryopoiesis using a tissue engineering approach. Using human megakaryocytes and endothelial cells, we demonstrate the following advanced features of the silk-based model: (1) immobilization of extracellular matrix components within the membrane, (2) tunable surface topography, (3) tunable mechanical properties, (4) physiologically relevant thickness for appropriate proplatelet extension, and (5) controlled localization of a vascular endothelium. Thus, by functionalizing silk, we can control megakaryocyte function on silk. The broader impact of this work offers a versatile new tool for studying megakaryocyte development and platelet production in vitro. Disclosures: No relevant conflicts of interest to declare.

Natural Scaffold, from Bovine Bone Marrow, Reproduces Native Microenvironment and Supports CD34+ and Stromal Cells

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 2400-2400
Author(s):  
Renata Giardini Rosa ◽  
Juares E. Romero Bianco ◽  
Gabriela Pereira dos Santos ◽  
Stephen D. Waldman ◽  
Joanna Weber ◽  
...  
Keyword(s):  
Mechanical Properties ◽  
Bone Marrow ◽  
Extracellular Matrix ◽  
Tissue Regeneration ◽  
Stromal Cells ◽  
Bovine Bone ◽  
Ecm Proteins ◽  
Histological Staining

Abstract Background: The idea of studying bone marrow outside its native environment is attractive and ideal. Due to the many functions of extracellular matrix (ECM), currently there is an interest in creating an environment that mimics the ECM present in the tissue, similar to the microenvironment in vivo. Molds replacing the ECM (scaffolds) have a porous structure and may assist the tissue regeneration by forming a suitable environment for adhesion, migration, proliferation and cellular differentiation. The appropriate ECM is a key factor as ECM proteins are site-specific and provide protein 'footprints' of previous resident cells. Because ECM proteins are among the most conserved proteins, the removal of xenogenic/allogenic cellular contents via decellularization could theoretically produce an essentially minimally immunogenic scaffold with a native intact structure for new tissue regeneration. Thus, the search for a scaffold that could be used to assess the behavior of cells and their interactions with the ECM in vitro/in vivo, and has different niches in its composition is highly desirable. Aims: In recent years, a large number of molecular and cytogenetic abnormalities have been identified in AML, MDS and multiple myeloma, many of these defects can serve as markers for diagnosis/prognosis or as therapeutic targets. However, there are still many unknown molecular factors involved in genetic abnormalities or signaling pathways that contribute to the pathogenesis of the disease. Another very important aspect of these diseases is that they all are related to the mutual interaction of neoplastic cells and the microenvironment of bone marrow. In the absence of an ideal model or even the difficulty in reproduce a native environment, we proposed the characterization of a natural scaffold, from bovine bone marrow, which can be used as a study model, previously patented by our laboratory. Materials and Methods: Bone marrow was decellularized by one or more incubations in an enzymatic digestion solution and polar solvent extractions, comprising an extracellular matrix with well-preserved 3D structure. Scaffolds were analyzed after the decelularization process for potential changes in structure (TEM, SEM, Histological staining, and immunohistochemistry for collagen III, IV, fibronectin) and mechanical properties. To verify if the scaffold would hold and support cell survival and extracellular matrix production, an in vitro study was performed using CD34+ (non-stromal) and HS-5 (stromal) cells. Cell-seeded decellularized scaffolds were cultured for 7-14 days and analyzed for Histological staining. Results: Histology sections (H&E staining), TEM and SEM demonstrated the structure and ultrastructure of the processed matrix and confirmed both cellular extraction and preservation of the macroscopic 3-D architecture of the collagen fibers, blood vessels, and preservation of an organized matrix. Also, the decellularized scaffold was quite comparable to the native tissue in terms of its mechanical properties. Immunohistochemistry of the scaffold showed that the main components of the ECM were preserved. The in vitro experiments of both stromal cells (HS-5) and non-stromal cells (CD34+) demonstrated that they were able to adhere and in the HS-5 case also produce ECM during 7-14 days of culture. In both cases, an increase in cell number was observed and CD34+ overtime formed cluster and with 14 days of culture the cluster formation increased in size. Conclusions: The results demonstrated that the decellularization process was efficient in keeping a 3-D structure and mechanical properties with a well-organized-preserved ECM. In vitro experiments showed that both CD34+ and HS-5 were able to proliferate and adhere in specific sites of the scaffold, suggesting that they were able to recognize their native environment. HS-5 produced ECM indicating that the scaffold worked as an optimal microenvironment. In conclusion, the scaffold could be used as a model, which has the potential to mimic the native microenvironment to enable research/studies of factors that are involved in self-renewal and maintenance of neoplastic cells in bone marrow. Also, this model could be very useful for pharmacological testing of bone marrow in vitro. Disclosures No relevant conflicts of interest to declare.

scholarly journals An Electromagnetic System for Inducing a Localized Force Gradient in an ECM and Its Influence on HMVEC Sprouting

2017 ◽  
Vol 23 (1) ◽  
pp. 70-82 ◽  
Author(s):  
Hian Hian See ◽  
Sahan C. B. Herath ◽  
Rerngchai Arayanarakool ◽  
Yue Du ◽  
Evan Tan ◽  
...  
Keyword(s):  
Mechanical Properties ◽  
Extracellular Matrix ◽  
Endothelial Cells ◽  
Experimental Results ◽  
Microvascular Endothelial Cells ◽  
Electromagnetic System ◽  
Magnetic Forces ◽  
Subsequent Response ◽  
Microvascular Endothelial

Mechanical properties of the extracellular matrix (ECM) have been observed to influence the behavior of cells. Investigations on such an influence commonly rely on using soluble cues to alter the global intrinsic ECM properties in order to study the subsequent response of cells. This article presents an electromagnetic system for inducing a localized force gradient in an ECM, and reports the experimentally observed effect of such a force gradient on in vitro angiogenic sprouting of human microvascular endothelial cells (HMVECs). This force gradient is realized through the induction of magnetic forces on the superparamagnetic microparticle–embedded ECM ( sECM). Both analytical and statistically meaningful experimental results demonstrate the effectiveness of this approach in influencing the behavior of a targeted HMVEC sprout without affecting that of other sprouts nearby. These results suggest the possibility of selectively controlling the in vitro behavior of cells by the induction of a localized force gradient in the ECM.

Smart Molecules: Organization and Morphology of the Self-Assembled Collagen Fibrils Formed From a Solution of Densly Packed Collagen Monomers

2008 ◽  
Author(s):  
Nima Saeidi ◽  
Jeffrey W. Ruberti
Keyword(s):  
Mechanical Properties ◽  
Extracellular Matrix ◽  
Liquid Crystalline ◽  
Alternative Hypothesis ◽  
Collagen Fibrils ◽  
The Self ◽  
Collagen Molecules ◽  
Cholesteric Liquid Crystalline

Load-bearing tissues owe their mechanical properties to the presence of highly-organized arrays of collagen fibrils. Aligned lamellae in cornea and aligned fascicles in tendon are the best examples of collagen fibrillar organization at the macroscopic level. The process by which collagen is organized in the extracellular matrix (ECM) is still unclear. But it is generally thought to be facilitated locally via “fibripositors” or cell surface “crypts”. According to this theory, fibroblasts create bounded “compartments” in the ECM through which they deposit organized groups of fibrils (in the form of lamellae in the cornea and in the form of fascicles in the tendon) [1, 2]. An alternative hypothesis proposed by Marie Giraud-Guille suggests that fibroblasts concentrate collagen monomers to form cholesteric liquid crystalline patterns that resemble those found in collagenous matrices in vivo [3–8]. Such organization has been demonstrated in vitro using extracted collagen monomers. However, the data presented in these studies focuses principally on the alignment of the collagen molecules and not on the organization and resulting morphology of condensed collagen fibrils. Considering that matrix mechanical properties in vivo are the result of the fibrillar alignment and not the alignment of individual molecules, further investigation of cholesterically organized condensed fibrils and their morphology is necessary.

Abstract 373: Impacts of Harvesting Sites on Cardiac Extracellular Matrix Hydrogels Properties

2020 ◽  
Vol 127 (Suppl_1) ◽  
Author(s):  
Emily Mulvany ◽  
Sara McMahan ◽  
Jiazhu Xu ◽  
Narges Yazdani ◽  
Rebecca Willits ◽  
...  
Keyword(s):  
Mechanical Properties ◽  
Extracellular Matrix ◽  
Chemical Composition ◽  
Cell Viability ◽  
Complex Modulus ◽  
Gelation Time ◽  
Human Mscs ◽  
Vascular Differentiation ◽  
Human Cardiomyocytes

Introduction: Cardiac extracellular matrix (cECM) hydrogel has been explored to treat myocardial infarction (MI). It possesses organ specific microenvironmental cues for resident cells and can be minimally invasive delivered into the heart using a transendocardial catheter. Recent clinical studies demonstrated the safety and feasibility of using cECM for treating MI patients. However, little is known about whether the region of cardiac tissue harvesting is critical for downstream applications. We performed in vitro studies to compare cECM hydrogels derived from porcine whole heart (WH), left ventricle (LV), and right ventricle (RV). Materials and Methods: Adult porcine hearts were obtained from a local USDA approved abattoir. The tissues were separated into 3 major categories: LV, RV, and WH tissues. Following our published protocol, tissues were decellularized and solubilized. The complete decellularization was confirmed by H&E staining and DNA content measurement. Chemical composition was examined using SDS-PAGE electrophoresis as well as Collagen and GAGs measurements. The gelation time, SEM (hydrogel microstructures), and mechanical properties (storage and complex modulus) were studied. Human cardiomyocytes (CMs) and mesenchymal stem cells (MSCs) were separately seeded on the hydrogels to examine their responses. Cell viability and vascular differentiation of human MSCs were examined. Results and Discussion: The cECM from WH has more complex chemical composition compared with cECM from LV and RV. All three types of cECM hydrogels share many similarities in terms of their microstructure, gelation time and mechanical properties. WH-derived cECM hydrogels have larger variations in storage modulus (G’) and complex modulus (G*) compared with the other two types of cECM hydrogels. Both human CMs and MSCs could maintain high cell viability on all hydrogels. Regardless of harvesting sites, all cECM hydrogels have equivalent potency in directing vascular differentiation of seeded human MSCs. Conclusions: The cECM hydrogels from WH, LV and RV exhibited similarity in material properties and cell response in vitro . Future fabrication of cECM hydrogels from WH would increase the yield and decrease production cost.

Cellular Deformations in Microintegrated Electrospun Poly (Ester Urethane) Urea Scaffolds Under Biaxial Stretch

2007 ◽  
Author(s):  
Jun Liao ◽  
W. David Merryman ◽  
Yi Hong ◽  
John Stella ◽  
Simon Watkins ◽  
...  
Keyword(s):  
Mechanical Properties ◽  
Extracellular Matrix ◽  
Smooth Muscle ◽  
Soft Tissue ◽  
Aspect Ratio ◽  
Cellular Interactions ◽  
Cardiovascular Tissues ◽  
Scaffold Properties ◽  
Cellular Deformation

Recently, vascular smooth muscle cells (SMCs) have been electrosprayed concurrently with electrospun biodegradable elastomeric poly (ester urethane) urea (PEUU) to form cell-microintegrated scaffolds [1]. These scaffolds exhibit soft tissue-like elastomeric mechanical properties [2], and are thus promising candidates for repair or replacement of diseased cardiovascular tissues. The level of cellular deformation during in vitro mechanical training will likely influence the extracellular matrix formation. However, these deformations are likely complex and dependent upon both scaffold properties and the cellular interactions with the local fibers. Our objective is to quantify microintegrated cellular deformations in response to biaxial scaffold stretches, using the nuclear aspect ratio (NAR) as an index of overall cellular deformation.

scholarly journals Cardiac Extracellular Matrix Hydrogel Enriched with Polyethylene Glycol Presents Improved Gelation Time and Increased On-Target Site Retention of Extracellular Vesicles

2021 ◽  
Vol 22 (17) ◽  
pp. 9226
Author(s):  
Lidia Gómez-Cid ◽  
María Luisa López-Donaire ◽  
Diego Velasco ◽  
Víctor Marín ◽  
María Isabel González ◽  
...  
Keyword(s):  
Mechanical Properties ◽  
Extracellular Matrix ◽  
Polyethylene Glycol ◽  
Extracellular Vesicles ◽  
Therapeutic Efficacy ◽  
Gelation Time ◽  
Target Site ◽  
Fourfold Increase

Stem-cell-derived extracellular vesicles (EVs) have demonstrated multiple beneficial effects in preclinical models of cardiac diseases. However, poor retention at the target site may limit their therapeutic efficacy. Cardiac extracellular matrix hydrogels (cECMH) seem promising as drug-delivery materials and could improve the retention of EVs, but may be limited by their long gelation time and soft mechanical properties. Our objective was to develop and characterize an optimized product combining cECMH, polyethylene glycol (PEG), and EVs (EVs–PEG–cECMH) in an attempt to overcome their individual limitations: long gelation time of the cECMH and poor retention of the EVs. The new combined product presented improved physicochemical properties (60% reduction in half gelation time, p < 0.001, and threefold increase in storage modulus, p < 0.01, vs. cECMH alone), while preserving injectability and biodegradability. It also maintained in vitro bioactivity of its individual components (55% reduction in cellular senescence vs. serum-free medium, p < 0.001, similar to EVs and cECMH alone) and increased on-site retention in vivo (fourfold increase vs. EVs alone, p < 0.05). In conclusion, the combination of EVs–PEG–cECMH is a potential multipronged product with improved gelation time and mechanical properties, increased on-site retention, and maintained bioactivity that, all together, may translate into boosted therapeutic efficacy.

scholarly journals Mechanical Cues: Bidirectional Reciprocity in the Extracellular Matrix Drives Mechano-Signalling in Articular Cartilage

2021 ◽  
Vol 22 (24) ◽  
pp. 13595
Author(s):  
Sophie Jane Gilbert ◽  
Cleo Selina Bonnet ◽  
Emma Jane Blain
Keyword(s):  
Mechanical Properties ◽  
Extracellular Matrix ◽  
Articular Cartilage ◽  
Proteolytic Enzymes ◽  
Type Ii Collagen ◽  
Cartilage Degradation ◽  
Effect Change ◽  
Physiological Loading

The composition and organisation of the extracellular matrix (ECM), particularly the pericellular matrix (PCM), in articular cartilage is critical to its biomechanical functionality; the presence of proteoglycans such as aggrecan, entrapped within a type II collagen fibrillar network, confers mechanical resilience underweight-bearing. Furthermore, components of the PCM including type VI collagen, perlecan, small leucine-rich proteoglycans—decorin and biglycan—and fibronectin facilitate the transduction of both biomechanical and biochemical signals to the residing chondrocytes, thereby regulating the process of mechanotransduction in cartilage. In this review, we summarise the literature reporting on the bidirectional reciprocity of the ECM in chondrocyte mechano-signalling and articular cartilage homeostasis. Specifically, we discuss studies that have characterised the response of articular cartilage to mechanical perturbations in the local tissue environment and how the magnitude or type of loading applied elicits cellular behaviours to effect change. In vivo, including transgenic approaches, and in vitro studies have illustrated how physiological loading maintains a homeostatic balance of anabolic and catabolic activities, involving the direct engagement of many PCM molecules in orchestrating this slow but consistent turnover of the cartilage matrix. Furthermore, we document studies characterising how abnormal, non-physiological loading including excessive loading or joint trauma negatively impacts matrix molecule biosynthesis and/or organisation, affecting PCM mechanical properties and reducing the tissue’s ability to withstand load. We present compelling evidence showing that reciprocal engagement of the cells with this altered ECM environment can thus impact tissue homeostasis and, if sustained, can result in cartilage degradation and onset of osteoarthritis pathology. Enhanced dysregulation of PCM/ECM turnover is partially driven by mechanically mediated proteolytic degradation of cartilage ECM components. This generates bioactive breakdown fragments such as fibronectin, biglycan and lumican fragments, which can subsequently activate or inhibit additional signalling pathways including those involved in inflammation. Finally, we discuss how bidirectionality within the ECM is critically important in enabling the chondrocytes to synthesise and release PCM/ECM molecules, growth factors, pro-inflammatory cytokines and proteolytic enzymes, under a specified load, to influence PCM/ECM composition and mechanical properties in cartilage health and disease.

Neutrophil migration through in vitro interstitial matrix

1992 ◽  
Vol 50 (1) ◽  
pp. 894-895
Author(s):  
J. Roemer ◽  
S.R. Simon
Keyword(s):  
Extracellular Matrix ◽  
Smooth Muscle ◽  
Cell Migration ◽  
Smooth Muscle Cells ◽  
Inflammatory Cell ◽  
Neutrophil Migration ◽  
Culture Conditions ◽  
Aortic Smooth Muscle ◽  
Specific Culture

We are developing an in vitro interstitial extracellular matrix (ECM) system for study of inflammatory cell migration. Falcon brand Cyclopore membrane inserts of various pore sizes are used as a support substrate for production of ECM by R22 rat aortic smooth muscle cells. Under specific culture conditions these cells produce a highly insoluble matrix consisting of typical interstitial ECM components, i.e.: types I and III collagen, elastin, proteoglycans and fibronectin.

Development of an in vitro Model for the Study of the Response of Equine Tendon Fibroblasts to Injury and Medication

1997 ◽  
Vol 10 (01) ◽  
pp. 6-11 ◽  
Author(s):  
R. F. Rosenbusch ◽  
L. C. Booth ◽  
L. A. Dahlgren
Keyword(s):  
Electron Microscopy ◽  
Extracellular Matrix ◽  
Light Microscopy ◽  
Light And Electron Microscopy ◽  
Tendon Healing ◽  
Matrix Proteins ◽  
Isolated Cells ◽  
Superficial Digital Flexor Tendon ◽  
Vitro Model

SummaryEquine tendon fibroblasts were isolated from explants of superficial digital flexor tendon, subcultured and maintained in monolayers. The cells were characterized by light microscopy, electron microscopy and radiolabel studies for proteoglycan production. Two predominant cell morphologies were identified. The cells dedifferentiated toward a more spindle shape with repeated subcultures. Equine tendon fibroblasts were successfully cryopreserved and subsequently subcultured. The ability to produce proteoglycan was preserved.The isolated cells were identified as fibroblasts, based on their characteristic shape by light microscopy and ultrastructure and the active production of extracellular matrix proteins. Abundant rough endoplasmic reticulum and the production of extracellular matrix products demonstrated active protein production and export. Proteoglycans were measurable via liquid scintillation counting in both the cell-associated fraction and free in the supernatant. This model is currently being utilized to study the effects of polysulfated glycosaminoglycan on tendon healing. Future uses include studying the effects of other pharmaceuticals, such as hyaluronic acid, on tendon healing.A model was developed for in vitro investigations into tendon healing. Fibroblasts were isolated from equine superficial digital flexor tendons and maintained in monolayer culture. The tenocytes were characterized via light and electron microscopy. Proteoglycan production was measured, using radio-label techniques. The fibroblasts were cryopreserved and subsequently subcultured. The cells maintained their capacity for proteoglycan production, following repeated subculturing and cryopreservation.

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