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.

scholarly journals Establishment of a New Device for Electrical Stimulation of Non-Degenerative Cartilage Cells In Vitro

2021 ◽  
Vol 22 (1) ◽  
pp. 394
Author(s):  
Simone Krueger ◽  
Alexander Riess ◽  
Anika Jonitz-Heincke ◽  
Alina Weizel ◽  
Anika Seyfarth ◽  
...  
Keyword(s):  
Electrical Stimulation ◽  
Electric Fields ◽  
Cartilage Tissue ◽  
Type I ◽  
Dependent Manner ◽  
New Device ◽  
Alternating Electric Fields ◽  
Cartilage Cells ◽  
Stimulation Of

In cell-based therapies for cartilage lesions, the main problem is still the formation of fibrous cartilage, caused by underlying de-differentiation processes ex vivo. Biophysical stimulation is a promising approach to optimize cell-based procedures and to adapt them more closely to physiological conditions. The occurrence of mechano-electrical transduction phenomena within cartilage tissue is physiological and based on streaming and diffusion potentials. The application of exogenous electric fields can be used to mimic endogenous fields and, thus, support the differentiation of chondrocytes in vitro. For this purpose, we have developed a new device for electrical stimulation of chondrocytes, which operates on the basis of capacitive coupling of alternating electric fields. The reusable and sterilizable stimulation device allows the simultaneous use of 12 cavities with independently applicable fields using only one main supply. The first parameter settings for the stimulation of human non-degenerative chondrocytes, seeded on collagen type I elastin-based scaffolds, were derived from numerical electric field simulations. Our first results suggest that applied alternating electric fields induce chondrogenic re-differentiation at the gene and especially at the protein level of human de-differentiated chondrocytes in a frequency-dependent manner. In future studies, further parameter optimizations will be performed to improve the differentiation capacity of human cartilage cells.

scholarly journals Antioxidant Role of PRGF on RPE Cells after Blue Light Insult as a Therapy for Neurodegenerative Diseases

2020 ◽  
Vol 21 (3) ◽  
pp. 1021 ◽  
Author(s):  
Carlota Suárez-Barrio ◽  
Susana del Olmo-Aguado ◽  
Eva García-Pérez ◽  
María de la Fuente ◽  
Francisco Muruzabal ◽  
...  
Keyword(s):  
Ex Vivo ◽  
Human Cell Line ◽  
Antioxidant Effect ◽  
Age Related Macular Degeneration ◽  
Strong Impact ◽  
Retinal Diseases ◽  
Age Related ◽  
Gene And Protein Expression

Oxidative stress has a strong impact on the development of retinal diseases such as age-related macular degeneration (AMD). Plasma rich in growth factors (PRGF) is a novel therapeutic approach in ophthalmological pathologies. The aim of this study was to analyze the antioxidant effect of PRGF in retinal epithelial cells (EPR) in in vitro and ex vivo retinal phototoxicity models. In vitro analyses were performed on ARPE19 human cell line. Viability and mitochondrial status were assessed in order to test the primary effects of PRGF. GSH level, and protein and gene expression of the main antioxidant pathway (Keap1, Nrf2, GCL, HO-1, and NQO1) were also studied. Ex vivo analyses were performed on rat RPE, and HO-1 and Nrf2 gene and protein expression were evaluated. The results show that PRGF reduces light insult by stimulating the cell response against oxidative damage and modulates the antioxidant pathway. We conclude that PRGF’s protective effect could prove useful as a new therapy for treating neurodegenerative disorders such as AMD.

scholarly journals Influence of Conditioned Media on the Re-Differentiation Capacity of Human Chondrocytes in 3D Spheroid Cultures

2020 ◽  
Vol 9 (9) ◽  
pp. 2798
Author(s):  
Annett Klinder ◽  
Sophie Kussauer ◽  
Bettina Hiemer ◽  
Andreas Wree ◽  
Rainer Bader ◽  
...  
Keyword(s):  
Conditioned Medium ◽  
Culture Media ◽  
Ex Vivo ◽  
Cartilage Matrix ◽  
Human Chondrocytes ◽  
Growth Media ◽  
Cell Cultivation ◽  
Cell Based Therapy ◽  
Tgf Β1

A major challenge of cell-based therapy for cartilage lesions is the preservation of the chondrogenic phenotype during ex vivo cell cultivation. In this in vitro study, the chondro-inductive capacity of two different hyaline cartilage-conditioned cell culture media on human chondrocytes in 3D spheroids was determined. Media were conditioned by incubation of 200 mg/mL vital or devitalized cartilage matrix in growth media over 35 days. The media were analyzed for the content of soluble procollagen type (Col) II and glycosaminoglycans (GAGs) as well as released TGF-β1, IGF-1 and IGFBP3. Unconditioned medium served as a negative control while the positive medium control was supplemented with TGF-β1 and IGF-1. Spheroid cultures prepared from human chondrocytes were cultivated at 37 °C, 5% CO2 and 21% O2 in the respective media and controls. After 14 and 35 days, the deposition of ECM components was evaluated by histological analysis. Vital cartilage-conditioned medium contained significantly higher levels of Col II and active TGF-β1 compared to medium conditioned with the devitalized cartilage matrix. Despite these differences, the incubation with vital as well as devitalized cartilage conditioned medium led to similar results in terms of deposition of proteoglycans and collagen type II, which was used as an indicator of re-differentiation of human chondrocytes in spheroid cultures. However, high density 3D cell cultivation showed a positive influence on re-differentiation.

scholarly journals In Vitro Analysis of the Differentiation Capacity of Postmortally Isolated Human Chondrocytes Influenced by Different Growth Factors and Oxygen Levels

Cartilage ◽  
2017 ◽  
Vol 10 (1) ◽  
pp. 111-119 ◽  
Author(s):  
Anika Jonitz-Heincke ◽  
Annett Klinder ◽  
Diana Boy ◽  
Achim Salamon ◽  
Doris Hansmann ◽  
...  
Keyword(s):  
Growth Factor ◽  
Transforming Growth Factor ◽  
Hyaline Cartilage ◽  
Human Chondrocytes ◽  
Knee Cartilage ◽  
Differentiation Markers ◽  
Differentiation Capacity ◽  
Oxygen Levels ◽  
Tgf Β1

Objective In the present in vitro study, we analyzed the chondrogenic differentiation capacity of human chondrocytes postmortally isolated from unaffected knee cartilage by the addition of transforming growth factor–β1 (TGF-β1) and/or insulin-like growth factor–1 (IGF-1) and different oxygen levels. Design After 14 and 35 days, DNA concentrations and protein contents of Col1, Col2, aggrecan as well as glycosaminoglycans (GAGs) of chondrocytes cultivated as pellet cultures were analyzed. Additionally, expression rates of mesenchymal stem cell (MSC)–associated differentiation markers were assessed in monolayer cultures. Results All cultivated chondrocytes were found to be CD29+/CD44+/CD105+/CD166+. Chondrocytic pellets stimulated with TGF-β1 showed enhanced synthesis rates of hyaline cartilage markers and reduced expression of the non-hyaline cartilage marker Col1 under hypoxic culture conditions. Conclusions Our results underline the substantial chondrogenic potential of human chondrocytes postmortally isolated from unaffected articular knee cartilage especially in case of TGF-β1 administration.

Effect of electrical stimulation on biological cells by capacitive coupling – an efficient numerical study considering model uncertainties

2020 ◽  
Author(s):  
Julius Zimmermann ◽  
RIchard Altenkirch ◽  
Ursula van Rienen
Keyword(s):  
Electrical Stimulation ◽  
Cell Membrane ◽  
Electric Fields ◽  
Biological Samples ◽  
Numerical Study ◽  
Cell Activity ◽  
Cellular Level ◽  
Capacitive Coupling ◽  
Fe Method

Electrical stimulation of biological samples such as tissues and cell cultures attracts growing attention due to its capability of enhancing cell activity, proliferation and differentiation. <br>Eventually, profound knowledge of the underlying mechanisms paves the way for innovative therapeutic devices. <br>Capacitive coupling is one option of delivering electric fields to biological samples and has advantages with regard to biocompatibility.<br>However, the mechanism of interaction is not well understood.<br>Experimental findings could be related to voltage-gated channels, which are triggered by changes of the transmembrane potential (TMP).<br>Numerical simulations by the Finite Element method (FEM) provide a possibility to estimate the TMP.<br>For realistic simulations of <i>in vitro</i> electric stimulation experiments, a bridge from the mesoscopic level down to the cellular level has to be found.<br>A special challenge poses the ratio between the cell membrane (a few <i>nm</i>) and the general setup (some <i>cm</i>).<br>Hence, a full discretization of the cell membrane becomes prohibitively expensive for 3D simulations.<br>We suggest using an approximate FE method that makes 3D multi-scale simulations possible.<br>Starting from an established 2D model, the chosen method is characterized and applied to realistic <i>in vitro</i> situations.<br>A to date not investigated parameter dependency is included and tackled by means of Uncertainty Quantification (UQ) techniques.<br>It reveals a strong, frequency-dependent influence of uncertain parameters on the modeling result.<br><br>

scholarly journals Tegavivint and the β-Catenin/ALDH Axis in Chemotherapy-Resistant and Metastatic Osteosarcoma

2019 ◽  
Vol 111 (11) ◽  
pp. 1216-1227 ◽  
Author(s):  
Motonari Nomura ◽  
Nino Rainusso ◽  
Yi-Chien Lee ◽  
Brian Dawson ◽  
Cristian Coarfa ◽  
...  
Keyword(s):  
Mrna Expression ◽  
Cell Lines ◽  
Ex Vivo ◽  
Statistical Tests ◽  
Control Group ◽  
Gene And Protein Expression ◽  
Relative Mrna Expression ◽  
Pdx Models

Abstract Background The Wnt/β-catenin pathway is closely associated with osteosarcoma (OS) development and metastatic progression. We investigated the antitumor activity of Tegavivint, a novel β-catenin/transducin β-like protein 1 (TBL1) inhibitor, against OS employing in vitro, ex vivo, and in vivo cell line and patient-derived xenograft (PDX) models that recapitulate high risk disease. Methods The antitumor efficacy of Tegavivint was evaluated in vitro using established OS and PDX-derived cell lines. Use of an ex vivo three-dimensional pulmonary metastasis assay assessed targeting of β-catenin activity during micro- and macrometastatic development. The in vivo activity of Tegavivint was evaluated using chemoresistant and metastatic OS PDX models. Gene and protein expression were quantified by quantitative Reverse transcription polymerase chain reaction or immunoblot analysis. Bone integrity was determined via microCT. All statistical tests were two-sided. Results Tegavivint exhibited antiproliferative activity against OS cells in vitro and actively reduced micro- and macrometastatic development ex vivo. Multiple OS PDX tumors (n = 3), including paired patient primary and lung metastatic tumors with inherent chemoresistance, were suppressed by Tegavivint in vivo. We identified that metastatic lung OS cell lines (n = 2) exhibited increased stem cell signatures, including enhanced concomitant aldehyde dehydrogenase (ALDH1) and β-catenin expression and downstream activity, which were suppressed by Tegavivint (ALDH1: control group, mean relative mRNA expression = 1.00, 95% confidence interval [CI] = 0.68 to 1.22 vs Tegavivint group, mean = 0.011, 95% CI = 0.0012 to 0.056, P < .001; β-catenin: control group, mean relative mRNA expression = 1.00, 95% CI = 0.71 to 1.36 vs Tegavivint group, mean = 0.45, 95% CI = 0.36 to 0.52, P < .001). ALDH1high PDX-derived lung OS cells, which demonstrated enhanced metastatic potential compared with ALDHlow cells in vivo, were sensitive to Tegavivint. Toxicity studies revealed decreased bone density in male Tegavivint-treated mice (n = 4 mice per group). Conclusions Tegavivint is a promising therapeutic agent for advanced stages of OS via its targeting of the β-catenin/ALDH1 axis.

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