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.

Biomimetic Calcium Phosphate Coatings for Bioactivation of Titanium Implant Surfaces: Methodological Approach and In Vitro Evaluation of Biocompatibility.

Ti6Al4V as a common implant material features good mechanical properties and corrosion resistance. However, untreated, it lacks bioactivity. In contrast, coatings with calcium phosphates (CaP) were shown to improve cell-material interactions in bone tissue engineering. Therefore, this work aimed to investigate how to tailor biomimetic CaP coatings on Ti6Al4V substrates using modified biomimetic calcium phosphate (BCP) coating solutions. Furthermore, the impact of substrate immersion in a 1 M alkaline CaCl2 solution (pH = 10) on subsequent CaP coating formation was examined. CaP coatings were characterized via scanning electron microscopy, x-ray diffraction, energy-dispersive x-ray spectroscopy, and laser-scanning microscope. Biocompatibility of coatings was carried out with primary human osteoblasts analyzing cell morphology, proliferation, collagen type 1, and interleukin 6 and 8 release. Results indicate a successful formation of low crystalline hydroxyapatite (HA) on top of every sample after immersion in each BCP coating solution after 14 days. Furthermore, HA coating promoted cell proliferation and reduced the concentration of interleukins compared to the uncoated surface, assuming increased biocompatibility.

miR-130b and miR-128a are essential lineage-specific codrivers of t(4;11) MLL-AF4 acute leukemia.

t(4;11) MLL-AF4 acute leukemia is one of the most aggressive malignancies in the infant and pediatric population, yet we have little information on the molecular mechanisms responsible for disease progression. This impairs the development of therapeutic regimens that can address the aggressive phenotype and lineage plasticity of MLL-AF4-driven leukemogenesis. This study highlights novel mechanisms of disease development by focusing on 2 microRNAs (miRNAs) upregulated in leukemic blasts from primary patient samples: miR-130b and miR-128a. We show that miR-130b and miR-128a are downstream targets of MLL-AF4 and can individually drive the transition from a pre-leukemic stage to an acute leukemia in an entirely murine Mll-AF4 in vivo model. They are also required to maintain the disease phenotype. Interestingly, miR-130b overexpression led to a mixed/B-cell precursor (BCP)/myeloid leukemia, propagated by the lymphoid-primed multipotent progenitor (LMPP) population, whereas miR-128a overexpression resulted in a pro-B acute lymphoblastic leukemia (ALL), maintained by a highly expanded Il7r+c-Kit+ blast population. Molecular and phenotypic changes induced by these two miRNAs fully recapitulate the human disease, including central nervous system infiltration and activation of an MLL-AF4 expression signature. Furthermore, we identified 2 downstream targets of these miRNAs, NR2F6 and SGMS1, which in extensive validation studies are confirmed as novel tumor suppressors of MLL-AF4+ leukemia. Our integrative approach thus provides a platform for the identification of essential co-drivers of MLL-rearranged leukemias, in which the preleukemia to leukemia transition and lineage plasticity can be dissected and new therapeutic approaches can be tested.

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.

Alternating Electric Fields Modify the Function of Human Osteoblasts Growing on and in the Surroundings of Titanium Electrodes.

Endogenous electric fields created in bone tissue as a response to mechanical loading are known to influence the activity and differentiation of bone and precursor cells. Thus, electrical stimulation offers an adjunct therapy option for the promotion of bone regeneration. Understanding the influence of electric fields on bone cell function and the identification of suitable electrical stimulation parameters are crucial for the clinical success of stimulation therapy. Therefore, we investigated the impact of alternating electric fields on human osteoblasts that were seeded on titanium electrodes, which delivered the electrical stimulation. Moreover, osteoblasts were seeded on collagen-coated coverslips near the electrodes, representing the bone stock surrounding the implant. Next, 0.2 V, 1.4 V, or 2.8 V were applied to the in vitro system with 20 Hz frequency. After one, three, and seven days, the osteoblast morphology and expression of osteogenic genes were analysed. The actin organisation, as well as the proliferation, were not affected by the electrical stimulation. Changes in the gene expression and protein accumulation after electrical stimulation were voltage-dependent. After three days, the osteogenic gene expression and alkaline phosphatase activity were up to 2.35-fold higher following the electrical stimulation with 0.2 V and 1.4 V on electrodes and coverslips compared to controls. Furthermore, collagen type I mRNA, as well as the amount of the C-terminal propeptide of collagen type I were increased after the stimulation with 0.2 V and 1.4 V, while the higher electrical stimulation with 2.8 V led to decreased levels, especially on the electrodes.