Capacitively coupled electric fields accelerate proliferation of osteoblast-like primary cells and increase bone extracellular matrix formation in vitro

2000 ◽  
Vol 29 (7) ◽  
pp. 499-506 ◽  
Author(s):  
Mareke Hartig ◽  
Ulrich Joos ◽  
Hans-Peter Wiesmann
Keyword(s):  
Extracellular Matrix ◽  
Electric Fields ◽  
Primary Cells ◽  
Capacitively Coupled

scholarly journals A Computational Model for Cardiomyocytes Mechano-Electric Stimulation to Enhance Cardiac Tissue Regeneration

Mathematics ◽  
2020 ◽  
Vol 8 (11) ◽  
pp. 1875
Author(s):  
Pau Urdeitx ◽  
Mohamed H. Doweidar
Keyword(s):  
Extracellular Matrix ◽  
Computational Model ◽  
Electric Fields ◽  
Electric Stimulation ◽  
Computational Models ◽  
Cell Maturation ◽  
Cardiac Cell ◽  
Cell Orientation ◽  
Aspect Ratios

Electrical and mechanical stimulations play a key role in cell biological processes, being essential in processes such as cardiac cell maturation, proliferation, migration, alignment, attachment, and organization of the contractile machinery. However, the mechanisms that trigger these processes are still elusive. The coupling of mechanical and electrical stimuli makes it difficult to abstract conclusions. In this sense, computational models can establish parametric assays with a low economic and time cost to determine the optimal conditions of in-vitro experiments. Here, a computational model has been developed, using the finite element method, to study cardiac cell maturation, proliferation, migration, alignment, and organization in 3D matrices, under mechano-electric stimulation. Different types of electric fields (continuous, pulsating, and alternating) in an intensity range of 50–350 Vm−1, and extracellular matrix with stiffnesses in the range of 10–40 kPa, are studied. In these experiments, the group’s morphology and cell orientation are compared to define the best conditions for cell culture. The obtained results are qualitatively consistent with the bibliography. The electric field orientates the cells and stimulates the formation of elongated groups. Group lengthening is observed when applying higher electric fields in lower stiffness extracellular matrix. Groups with higher aspect ratios can be obtained by electrical stimulation, with better results for alternating electric fields.

scholarly journals Daratumumab, a Novel Anti-CD38 Monoclonal Antibody Shows Anti-Tumor Activity in CLL and hampers Leukemia-Microenvironment Interactions

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 4680-4680 ◽  
Author(s):  
Alba Matas-Céspedes ◽  
Anna Vidal-Crespo ◽  
Vanina Rodriguez ◽  
Julio Delgado ◽  
Neus Villamor ◽  
...  
Keyword(s):  
Extracellular Matrix ◽  
Cell Migration ◽  
Cell Adhesion ◽  
Tumor Microenvironment ◽  
Primary Cells ◽  
Isotype Control ◽  
Mmp9 Expression ◽  
Anti Tumor Activity

Abstract Daratumumab (DARA) is a anti-human CD38 antibody with Fc-mediated cell killing activity. DARA induces killing of tumor cells, mainly via complement-dependent cytotoxicity (CDC), antibody-dependent cell-mediated cytotoxicity (ADCC) (de Weers M. J Immunol 2011), and antibody-dependent cellular phagocytosis (ADCP) by macrophages (mΦ), both murine and human in multiple myeloma (MM) and Burkitt lymphoma cells. DARA is currently being evaluated in phase III clinical trials in patients with MM. We have previously reported that DARA induces cytotoxic activity in vitro via ADCC in primary cells and cell lines from Chronic Lymphoctic Leukemia (CLL), and significantly prolongs overall survival of animals in a systemic CLL mouse model. Here, we present additional data on in vivo mechanism of DARA and its effect on tumor-microenvironment interactions in CLL. We first evaluated whether ADCP contributes to DARA activity both in vitro and in vivo. For in vitro ADCP, mΦ were generated from monocytes of normal PBMCs and stimulated with GM-CSF (10ng/mL, 7 days). CLL cell lines and primary cells were labeled with calcein and incubated for 4h with mΦ at an effector:target ratio of 2:1 in the presence of a fixed mAb concentration of 1 μg/mL, followed by flow cytometric analysis. The amount of remaining CLL target cells (CD19+, CD11b-) was reduced by 3-16%. ADCP defined as percentage of mφ which had phagocytosed, referred to as double positive mΦ (CD11b+, calcein+, CD19-), ranged from 3-10%. To analyze ADCP in vivo, SCID beige mice, devoid of NK cells but with active macrophages, were inoculated intraperitoneally with CLL cells (20×106) and simultaneously treated with a single dose of DARA or isotype control (20mg/kg, n=3-5 per group). Forty-eight hours later, CLL cells were recovered from the intraperitoneal cavity and counted in a flow cytometer (identified as human CD45+/CD19+/CD5+cells). In DARA-treated mice the number of CLL cells recovered was reduced by 42% (n=2, p<0.05) compared to the isotype control group. Remarkably, the decrease in cell number was already detectable 2h after DARA administration. CLL pathogenesis relies on supportive tumor-microenvironment interactions both in the bone marrow (BM) and in the lymph node (LN), and CD38 constitutes a molecular hub integrating proliferative and migratory signals for CLL (Malavasi, F. Blood 2011). We evaluated the effect of DARA on migration and adhesion. In in vitro migrations assays, we have demonstrated that DARA (10-30 μg/mL) inhibited CXCL12/SDF1α-mediated migration up to 70% (n=5). In addition, DARA reduced up to 55% (n=2) of downstream pERK activation, that peaked after 5min of CXCL12/SDF1α stimulation. We analyzed the effect of DARA on primary CLL cell migration from Peripheral Blood (PB) to BM and spleen in vivo, using NOD/SCID/gamma (NSG) null mice (lacking NK cells and effective macrophages). In this system, NSG mice were pretreated (day 0) with DARA, control IgG or anti-CXCR4 as positive control for inhibition of cell homing, prior to injection of fresh primary CLL cells (50×106 cells/per mice) on day 1. PB, BM and spleen cells were isolated on day 2 and CLL cells were identified by staining for human CD45/CD19/CD5 and counted using a flow cytometer. Cell counting showed that CLL cells mainly migrate to the spleen, and that DARA significantly reduced this migration (55% inhibition on average, p<0.05). In addition to migration, CD38 also plays a key role in cell adhesion through interaction with integrins (CD49d/CD29) and with extracellular matrix proteins. We analyzed the effect of DARA on the adhesion of CLL cells to the extracellular matrix vascular-cell adhesion molecule-1 (VCAM-1) mediated by CD49d/CD29. DARA reduced adhesion of CLL cells (n=4), to VCAM-1 by 46±13% (range 27-57) compared to isotype control. By RT-PCR we observed an up-regulation of MMP9 transcripts (average 2 fold, n=2), and DARA abrogated both constitutive MMP9 expression (90% reduction) and VCAM-derived (94% reduction) MMP9 expression. In summary, DARA shows a positive effect on ADCP-mediated anti-tumor activity on CLL cells both in vitro and in vivo. In addition DARA exhibits a strong effect on CLL cell migration and adhesion. Based on these data, we hypothesize that DARA may exert unique and substantial effects on CLL tumor cell growth and contributes to potent therapeutic efficacy in a clinical setting. Disclosures Doshi: Janssen R&D: Employment. Parren:Genmab: Employment, Equity Ownership. Lammerts van Bueren:Genmab : Employment. Pérez-Galán:Genmab: Research Funding.

scholarly journals Re-Differentiation Capacity of Human Chondrocytes in Vitro Following Electrical Stimulation with Capacitively Coupled Fields

2019 ◽  
Vol 8 (11) ◽  
pp. 1771 ◽  
Author(s):  
Simone Krueger ◽  
Sophie Achilles ◽  
Julius Zimmermann ◽  
Thomas Tischer ◽  
Rainer Bader ◽  
...  
Keyword(s):  
Electrical Stimulation ◽  
Electric Fields ◽  
Ex Vivo ◽  
Human Chondrocytes ◽  
Capacitively Coupled ◽  
Differentiation Capacity ◽  
Cartilage Lesions ◽  
Gene And Protein Expression ◽  
Chondrocytic Differentiation

Treatment of cartilage lesions remains a clinical challenge. Therefore, biophysical stimuli like electric fields seem to be a promising tool for chondrocytic differentiation and treatment of cartilage lesions. In this in vitro study, we evaluated the effects of low intensity capacitively coupled electric fields with an alternating voltage of 100 mVRMS (corresponds to 5.2 × 10−5 mV/cm) or 1 VRMS (corresponds to 5.2 × 10−4 mV/cm) with 1 kHz, on human chondrocytes derived from osteoarthritic (OA) and non-degenerative hyaline cartilage. A reduction of metabolic activity after electrical stimulation was more pronounced in non-degenerative cells. In contrast, DNA contents in OA cells were significantly decreased after electrical stimulation. A difference between 100 mVRMS and 1 VRMS was not detected. However, a voltage-dependent influence on gene and protein expression was observed. Both cell types showed increased synthesis rates of collagen (Col) II, glycosaminoglycans (GAG), and Col I protein following stimulation with 100 mVRMS, whereas this increase was clearly higher in OA cells. Our results demonstrated the sensitization of chondrocytes by alternating electric fields, especially at 100 mVRMS, which has an impact on chondrocytic differentiation capacity. However, analysis of further electrical stimulation parameters should be done to induce optimal hyaline characteristics of ex vivo expanded human chondrocytes.

scholarly journals Establishment and Evaluation of an In Vitro System for Biophysical Stimulation of Human Osteoblasts

Cells ◽  
2020 ◽  
Vol 9 (9) ◽  
pp. 1995
Author(s):  
Martin Stephan ◽  
Julius Zimmermann ◽  
Annett Klinder ◽  
Franziska Sahm ◽  
Ursula van Rienen ◽  
...  
Keyword(s):  
Electrical Stimulation ◽  
Bone Remodeling ◽  
Electric Fields ◽  
Metabolic Activity ◽  
In Vitro System ◽  
Human Osteoblasts ◽  
Capacitively Coupled ◽  
Procollagen Type ◽  
Bone Remodeling Markers

While several studies investigated the effects of mechanical or electrical stimulation on osseointegration and bone fracture healing, little is known about the molecular and cellular impact of combined biophysical stimulation on peri-implant osseointegration. Therefore, we established an in vitro system, capable of applying shear stress and electric fields simultaneously. Capacitively coupled electric fields were used for electrical stimulation, while roughened Ti6Al4V bodies conducted harmonically oscillating micromotions on collagen scaffolds seeded with human osteoblasts. Different variations of single and combined stimulation were applied for three days, while samples loaded with Ti6Al4V bodies and untreated samples served as control. Metabolic activity, expression of osteogenic markers and bone remodeling markers were investigated. While combined stimulation showed no substantial benefit compared to sole mechanical stimulation, we observed that 25 µm micromotions applied by roughened Ti6Al4V bodies led to a significant increase in gene expression of osteocalcin and tissue inhibitor of metalloprotease 1. Additionally, we found an increase in metabolic activity and expression of bone remodeling markers with reduced procollagen type 1 synthesis after 100 mVRMS electrical stimulation. We were able to trigger specific cellular behaviors using different biophysical stimuli. In future studies, different variations of electrical stimulation will be combined with interfacial micromotions.

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.

Perturbation of Platelet Adhesion to Endothelial Cells by Plasminogen Activation In Vitro

1997 ◽  
Vol 78 (02) ◽  
pp. 934-938 ◽  
Author(s):  
Hsiun-ing Chen ◽  
Yueh-I Wu ◽  
Yu-Lun Hsieh ◽  
Guey-Yueh Shi ◽  
Meei-Jyh Jiang ◽  
...  
Keyword(s):  
Extracellular Matrix ◽  
Endothelial Cells ◽  
Platelet Adhesion ◽  
Treatment Time ◽  
Shape Change ◽  
Umbilical Vein ◽  
Tissue Type ◽  
Apical Surface ◽  
Plasminogen Activation

SummaryTo investigate whether the endothelium-platelet interactions may be altered by plasminogen activation, cultured human umbilical vein endothelial cells (ECs) were treated with tissue-type plasminogen activator (t-PA) in the presence of plasminogen, and platelet adhesion to ECs was subsequently measured by using a tapered flow chamber. Our results demonstrated that platelets adhered more readily to t-PA treated EC monolayer than to the control monolayer at all shear stress levels tested. This phenomenon was treatment time-dependent and dose-dependent, and it could be blocked by adding plasmin inhibitors, such as e-amino caproic acid and aprotinin. Adherent platelets on t-PA treated EC monolayer underwent more severe shape change than those on the control monolayer. While the extracellular matrix directly treated with t-PA attracted less platelets than the control matrix did, platelet adhesion to the matrix that was produced by t-PA-treated ECs was unaltered. These data suggest that t-PA treatment on ECs compromised antiplatelet-adhesion capability on their apical surface without altering the reactivity of their extracellular matrix towards platelets.

Desmopressin (DDAVP) Enhances Platelet Adhesion to the Extracellular Matrix of Cultured Human Endothelial Cells through Increased Expression of Tissue Factor

1997 ◽  
Vol 77 (05) ◽  
pp. 0975-0980 ◽  
Author(s):  
Angel Gálvez ◽  
Goretti Gómez-Ortiz ◽  
Maribel Díaz-Ricart ◽  
Ginés Escolar ◽  
Rogelio González-Sarmiento ◽  
...  
Keyword(s):  
Extracellular Matrix ◽  
Endothelial Cells ◽  
Tissue Factor ◽  
Platelet Adhesion ◽  
Umbilical Vein ◽  
Procoagulant Activity ◽  
Experimental Conditions ◽  
Chromogenic Assay

SummaryThe effect of desmopressin (DDAVP) on thrombogenicity, expression of tissue factor and procoagulant activity (PCA) of extracellular matrix (ECM) generated by human umbilical vein endothelial cells cultures (HUVEC), was studied under different experimental conditions. HUVEC were incubated with DDAVP (1, 5 and 30 ng/ml) and then detached from their ECM. The reactivity towards platelets of this ECM was tested in a perfusion system. Coverslips covered with DD A VP-treated ECMs were inserted in a parallel-plate chamber and exposed to normal blood anticoagulated with low molecular weight heparin (Fragmin®, 20 U/ml). Perfusions were run for 5 min at a shear rate of 800 s1. Deposition of platelets on ECMs was significantly increased with respect to control ECMs when DDAVP was used at 5 and 30 ng/ml (p <0.05 and p <0.01 respectively). The increase in platelet deposition was prevented by incubation of ECMs with an antibody against human tissue factor prior to perfusion. Immunofluorescence studies positively detected tissue factor antigen on DDAVP derived ECMs. A chromogenic assay performed under standardized conditions revealed a statistically significant increase in the procoagulant activity of the ECMs produced by ECs incubated with 30 ng/ml DDAVP (p <0.01 vs. control samples). Northern blot analysis revealed increased levels of tissue factor mRNA in extracts from ECs exposed to DDAVP. Our data indicate that DDAVP in vitro enhances platelet adhesion to the ECMs through increased expression of tissue factor. A similar increase in the expression of tissue factor might contribute to the in vivo hemostatic effect of DDAVP.

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