Discrimination between the effects of pulsed electrical stimulation and electrochemically conditioned medium on human osteoblasts.

BACKGROUND: Electrical stimulation is used for enhanced bone fracture healing. Electrochemical processes occur during the electrical stimulation at the electrodes and influence cellular reactions. Our approach aimed to distinguish between electrochemical and electric field effects on osteoblast-like MG-63 cells. We applied 20 Hz biphasic pulses via platinum electrodes for 2 h. The electrical stimulation of the cell culture medium and subsequent application to cells was compared to directly stimulated cells. The electric field distribution was predicted using a digital twin. RESULTS: Cyclic voltammetry and electrochemical impedance spectroscopy revealed partial electrolysis at the electrodes, which was confirmed by increased concentrations of hydrogen peroxide in the medium. While both direct stimulation and AC-conditioned medium decreased cell adhesion and spreading, only the direct stimulation enhanced the intracellular calcium ions and reactive oxygen species. CONCLUSION: The electrochemical by-product hydrogen peroxide is not the main contributor to the cellular effects of electrical stimulation. However, undesired effects like decreased adhesion are mediated through electrochemical products in stimulated medium. Detailed characterisation and monitoring of the stimulation set up and electrochemical reactions are necessary to find safe electrical stimulation protocols.

Contributions of deep learning to automated numerical modelling of the interaction of electric fields and cartilage tissue based on 3D images.

Electric fields find use in tissue engineering but also in sensor applications besides the broad classical application range. Accurate numerical models of electrical stimulation devices can pave the way for effective therapies in cartilage regeneration. To this end, the dielectric properties of the electrically stimulated tissue have to be known. However, knowledge of the dielectric properties is scarce. Electric field-based methods such as impedance spectroscopy enable determining the dielectric properties of tissue samples. To develop a detailed understanding of the interaction of the employed electric fields and the tissue, fine-grained numerical models based on tissue-specific 3D geometries are considered. A crucial ingredient in this approach is the automated generation of numerical models from biomedical images. In this work, we explore classical and artificial intelligence methods for volumetric image segmentation to generate model geometries. We find that deep learning, in particular the StarDist algorithm, permits fast and automatic model geometry and discretisation generation once a sufficient amount of training data is available. Our results suggest that already a small number of 3D images (23 images) is sufficient to achieve 80% accuracy on the test data. The proposed method enables the creation of high-quality meshes without the need for computer-aided design geometry post-processing. Particularly, the computational time for the geometrical model creation was reduced by half. Uncertainty quantification as well as a direct comparison between the deep learning and the classical approach reveal that the numerical results mainly depend on the cell volume. This result motivates further research into impedance sensors for tissue characterisation. The presented approach can significantly improve the accuracy and computational speed of image-based models of electrical stimulation for tissue engineering applications.

Experimental and numerical methods to ensure comprehensible and replicable alternating current electrical stimulation experiments.

Electrical stimulation has received increasing attention for decades for its application in regenerative medicine. Applications range from bone growth stimulation over cartilage regeneration to deep brain stimulation. Despite all research efforts, translation into clinical use has not yet been achieved in all fields. Recent critical assessments have identified limited documentation and monitoring of preclinical in vitro and in vivo experiments as possible reasons hampering clinical translation. In this work, we present experimental and numerical methods to determine the crucial quantities of electrical stimulation such as the electric field or current density. Knowing the stimulation quantities contributes to comprehending the biological response to electrical stimulation and to finally developing a reliable dose-response curve. To demonstrate the methods, we consider a direct contact electrical stimulation experiment that stands representative for a broad class of stimulation experiments. Electrochemical effects are addressed and methods to integrate them into numerical simulations are evaluated. A focus is laid on affordable lab equipment and reproducible open-source software solutions. Finally, clear guidelines to ensure replicability of electrical stimulation experiments are formulated.

Long-term stimulation with alternating electric fields modulates the differentiation and mineralization of human pre-osteoblasts.

Biophysical stimulation by electric fields can promote bone formation in bone defects of critical size. Even though, long-term effects of alternating electric fields on the differentiation of osteoblasts are not fully understood. Human pre-osteoblasts were stimulated over 31 days to gain more information about these cellular processes. An alternating electric field with 0.7 Vrms and 20 Hz at two distances was applied and viability, mineralization, gene expression, and protein release of differentiation factors were analyzed. The viability was enhanced during the first days of stimulation. A higher electric field resulted in upregulation of typical osteogenic markers like osteoprotegerin, osteopontin, and interleukin-6, but no significant changes in mineralization. Upregulation of the osteogenic markers could be detected with a lower electric field after the first days of stimulation. As a significant increase in the mineralized matrix was identified, an enhanced osteogenesis due to low alternating electric fields can be assumed.

Pulsed Electrical Stimulation Affects Osteoblast Adhesion and Calcium Ion Signaling.

An extensive research field in regenerative medicine is electrical stimulation (ES) and its impact on tissue and cells. The mechanism of action of ES, particularly the role of electrical parameters like intensity, frequency, and duration of the electric field, is not yet fully understood. Human MG-63 osteoblasts were electrically stimulated for 10 min with a commercially available multi-channel system (IonOptix). We generated alternating current (AC) electrical fields with a voltage of 1 or 5 V and frequencies of 7.9 or 20 Hz, respectively. To exclude liquid-mediated effects, we characterized the AC-stimulated culture medium. AC stimulation did not change the medium's pH, temperature, and oxygen content. The H2O2 level was comparable with the unstimulated samples except at 5 V_7.9 Hz, where a significant increase in H2O2 was found within the first 30 min. Pulsed electrical stimulation was beneficial for the process of attachment and initial adhesion of suspended osteoblasts. At the same time, the intracellular Ca2+ level was enhanced and highest for 20 Hz stimulated cells with 1 and 5 V, respectively. In addition, increased Ca2+ mobilization after an additional trigger (ATP) was detected at these parameters. New knowledge was provided on why electrical stimulation contributes to cell activation in bone tissue regeneration.

Numerical study on the effect of capacitively coupled electrical stimulation on biological cells considering model uncertainties.

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. Eventually, a profound knowledge of the underlying mechanisms paves the way for innovative therapeutic devices. Capacitive coupling is one option of delivering electric fields to biological samples that has advantages regarding biocompatibility. However, its biological mechanism of interaction is not well understood. Experimental findings could be related to voltage-gated channels, which are triggered by changes of the transmembrane potential. Numerical simulations by the finite element method provide a possibility to estimate the transmembrane potential. Since a full resolution of the cell membrane within a macroscopic model would lead to prohibitively expensive models, we suggest the adaptation of an approximate finite element method. Starting from a basic 2.5D model, the chosen method is validated and applied to realistic experimental situations. To understand the influence of the dielectric properties on the modelling outcome, uncertainty quantification techniques are employed. A frequency-dependent influence of the uncertain dielectric properties of the cell membrane on the modelling outcome is revealed. This may have practical implications for future experimental studies. Our methodology can be easily adapted for computational studies relying on experimental data.

Using a Digital Twin of an Electrical Stimulation Device to Monitor and Control the Electrical Stimulation of Cells in vitro.

Electrical stimulation for application in tissue engineering and regenerative medicine has received increasing attention in recent years. A variety of stimulation methods, waveforms and amplitudes have been studied. However, a clear choice of optimal stimulation parameters is still not available and is complicated by ambiguous reporting standards. In order to understand underlying cellular mechanisms affected by the electrical stimulation, the knowledge of the actual prevailing field strength or current density is required. Here, we present a comprehensive digital representation, a digital twin, of a basic electrical stimulation device for the electrical stimulation of cells in vitro. The effect of electrochemical processes at the electrode surface was experimentally characterised and integrated into a numerical model of the electrical stimulation. Uncertainty quantification techniques were used to identify the influence of model uncertainties on relevant observables. Different stimulation protocols were compared and it was assessed if the information contained in the monitored stimulation pulses could be related to the stimulation model. We found that our approach permits to model and simulate the recorded rectangular waveforms such that local electric field strengths become accessible. Moreover, we could predict stimulation voltages and currents reliably. This enabled us to define a controlled stimulation setting and to identify significant temperature changes of the cell culture in the monitored voltage data. Eventually, we give an outlook on how the presented methods can be applied in more complex situations such as the stimulation of hydrogels or tissue in vivo.

Ambiguity in the interpretation of the low-frequency dielectric properties of biological tissues.

The frequency-dependent behaviour of the dielectric properties of biological tissues in the frequency range below 1 kHz has been under debate since the past century. Here, we reanalyse the raw data of the main resource of the dielectric properties of biological tissues in impedance representation. Employing a Kramers-Kronig validity test and parameter estimation techniques, we can describe the data by two physical parametric models that correspond to opposing biophysical interpretations: on the one hand the data can be explained only by intrinsic tissue properties, but on the other hand evidence for electrode-specific effects can be found for all tissues under investigation. The first interpretation would justify the continued use of a parametric model comprising four Cole-Cole dispersions, which describe the dielectric properties from extremely low to very high frequencies. As an alternative that is in accordance with the second interpretation, we suggest to omit the slowest of the four dispersions in the model and increase the static conductivity to account for a frequency-independent conductivity below 1 kHz.

Numerical Simulations as Means for Tailoring Electrically Conductive Hydrogels Towards Cartilage Tissue Engineering by Electrical Stimulation.

Cartilage regeneration is a clinical challenge. In recent years, hydrogels have emerged as implantable scaffolds in cartilage tissue engineering. Similarly, electrical stimulation has been employed to improve matrix synthesis of cartilage cells, and thus to foster engineering and regeneration of cartilage tissue. The combination of hydrogels and electrical stimulation may pave the way for new clinical treatment of cartilage lesions. To find the optimal electric properties of hydrogels, theoretical considerations and corresponding numerical simulations are needed to identify well-suited initial parameters for experimental studies. We present the theoretical analysis of a hydrogel in a frequently used electrical stimulation device for cartilage regeneration and tissue engineering. By means of equivalent circuits, finite element analysis, and uncertainty quantification, we elucidate the influence of the geometric and dielectric properties of cell-seeded hydrogels on the capacitive-coupling electrical field stimulation. Moreover, we discuss the possibility of cellular organisation inside the hydrogel due to forces generated by the external electric field. The introduced methodology is easily reusable by other researchers and allows to directly develop novel electrical stimulation study designs. Thus, this study paves the way for the design of future experimental studies using electrically conductive hydrogels and electrical stimulation for tissue engineering.

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

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.

Computational study on electromechanics of electroactive hydrogels for cartilage-tissue repair.

BACKGROUND AND OBJECTIVE: The self-repair capability of articular cartilage is limited because of non-vascularization and low turnover of its extracellular matrix. Regenerating hyaline cartilage remains a significant clinical challenge as most non-surgical and surgical treatments provide only mid-term relief. Eventually, further pain and mobility loss occur for many patients in the long run due to further joint deterioration. Repair of articular cartilage tissue using electroactive scaffolds and biophysical stimuli like electrical and osmotic stimulation may have the potential to heal cartilage defects occurring due to trauma, osteoarthritis, or sport-related injuries. Therefore, the focus of the current study is to present a computational model of electroactive hydrogels for the cartilage-tissue repair as a first step towards an optimized experimental design. METHODS: The multiphysics transport model that mainly includes the Poisson-Nernst-Planck equations and the mechanical equation is used to find the electrical stimulation response of the polyelectrolyte hydrogels. Based upon this, a numerical model on electromechanics of electroactive hydrogels seeded with chondrocytes is presented employing the open-source software FEniCS, which is a Python library for finite-element analysis. RESULTS: We analyzed the ionic concentrations and electric potential in a hydrogel sample and the cell culture medium, the osmotic pressure created due to ionic concentration variations and the resulting hydrogel displacement. The proposed mathematical model was validated with examples from literature. CONCLUSIONS: The presented model for the electrical and osmotic stimulation of a hydrogel sample can serve as a useful tool for the development and analysis of a cartilaginous scaffold employing electrical stimulation. By analyzing various parameters, we pave the way for future research on a finer scale using open-source software.

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

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.

Numerical Simulation of Electroactive Hydrogels for Cartilage-Tissue Engineering.

The intrinsic regeneration potential of hyaline cartilage is highly limited due to the absence of blood vessels, lymphatics, and nerves, as well as a low cell turnover within the tissue. Despite various advancements in the field of regenerative medicine, it remains a challenge to remedy articular cartilage defects resulting from trauma, aging, or osteoarthritis. Among various approaches, tissue engineering using tailored electroactive scaffolds has evolved as a promising strategy to repair damaged cartilage tissue. In this approach, hydrogel scaffolds are used as artificial extracellular matrices, and electric stimulation is applied to facilitate proliferation, differentiation, and cell growth at the defect site. In this regard, we present a simulation model of electroactive hydrogels to be used for cartilage-tissue engineering employing open-source finite-element software FEniCS together with a Python interface. The proposed mathematical formulation was first validated with an example from the literature. Then, we computed the effect of electric stimulation on a circular hydrogel sample that served as a model for a cartilage-repair implant.

Requirements for Documenting Electrical Cell Stimulation Experiments for Replicability and Numerical Modeling∗.

Thorough documentation of biological experiments is necessary for their replicability. This becomes even more evident when individual steps of in vitro wet-lab experiments are to be incorporated into computer simulation models. In the highly interdisciplinary field of electrical stimulation of biological cells, not only biological but also physical aspects play a crucial role. Simulations may help to identify parameters that influence cells and thereby reveal new insights into mechanisms of the cell biological system. However, missing or misleading documentation of the electrical stimulation step within wet-lab experiments may lead to discrepancies between reported and simulated electrical quantities. In addition, this threatens the replicability of electrical stimulation experiments. Thus, we argue that a minimal set of information is needed to enable a translation of electrical stimulation experiments of biological cells into computer simulation experiments and to support replicability. This set includes detailed information about the electronic devices and components, their set-up as well as the applied stimulus and shall be integrated into an existing guideline for cell biological experiments. Ideally, the documentation should also contain measured properties of the cellular and experimental environment. Furthermore, a realization of our proposed documentation requirements within electronic lab notebooks may provide a crucial step toward a more seamless integration of wet-lab data into simulations. Based on two exemplary studies, we demonstrate the relevance of our claim.