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      Optimal parameters for the enhancement of human osteoblast-like cell proliferation in vitro via shear stress induced by high-frequency mechanical vibration

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          Abstract

          Abstract Introduction: Biomechanical stimulation of cultured human osteoblast-like cells, which is based on controlled mechanical vibration, has been previously indicated, but the exact mechanical parameters that are effective for cells' proliferation enhancement are still elusive due to the lack of direct data recordings from the stimulated cells in culture. Therefore, we developed a low friction tunable system that enables recording of a narrow range of mechanical parameters, above the infrasonic spectrum, that applied uniformly to human osteoblast-like cells in monolayer culture, aiming to identify a range of mechanical parameters that are effective to enhance osteoblast proliferation in vitro. Methods: Human osteoblast-like cells in explant monolayer culture samples were exposed to mechanical vibration in the 10-70Hz range of frequencies for two minutes, in four 24 hours intervals. Cell numbers in culture, cellular alkaline phosphatase activity (a marker of cell maturation), and lactate dehydrogenase activity in culture media (representing cell death) were measured after the mechanical stimulation protocol application and compared statistically to the control cell cultures kept in static conditions. The cell proliferation was deduced from cell number in culture and cell death measurements. Results: We found that 50-70 Hz of vibration frequency protocol (10-30 µm of maximal displacement amplitude, 0.03g of peak-to-peak acceleration) is optimal for enhancing cells' proliferation (p<0.05), with a parallel decrease of their maturation (p<0.01). Discussion: We detected the optimal mechanical parameters of excitation protocol for induction of osteoblast proliferation in vitro by a mechanical platform, which can be used as a standardized method in the research of mechanotransduction in human osteoblast.

          Translated abstract

          Resumen Introducción: La estimulación biomecánica de células similares a osteoblastos humanos cultivadas, que se basa en vibraciones mecánicas controladas se ha demostrado anteriormente, pero los parámetros mecánicos exactos que son efectivos para la mejora de la proliferación de células aún son difíciles de alcanzar debido a la falta de registros de datos directos de las células estimuladas en cultivo. Por lo tanto, desarrollamos un sistema sintonizable de baja fricción que permite el registro de un rango estrecho de parámetros mecánicos, por encima del espectro infrasónico, que se aplica de manera uniforme a células similares a osteoblastos humanos en cultivo monocapa, con el objetivo de identificar un rango de parámetros mecánicos que son efectivos para mejorar la proliferación de osteoblastos in vitro. Métodos: Se expusieron células similares a osteoblastos humanos en muestras de cultivo de monocapa de explante a vibración mecánica en el rango de frecuencias de 10-70 Hz durante dos minutos, en cuatro intervalos de 24 horas. El número de células en cultivo, la actividad de la fosfatasa alcalina celular (un marcador de maduración celular) y la actividad de la lactato deshidrogenasa en los medios de cultivo (que representa la muerte celular) se midieron después de la aplicación del protocolo de estimulación mecánica y se compararon estadísticamente con los cultivos de células de control mantenidos en condiciones estáticas. La proliferación celular se dedujo del número de células en cultivo y mediciones de muerte celular. Resultados: Encontramos que 50-70 Hz de protocolo de frecuencia de vibración (10-30 µm de amplitud de desplazamiento máxima, 0,03 g de aceleración de pico a pico) es óptimo para mejorar la proliferación de células (p <0,05), con una disminución paralela de su maduración (p <0.01). Discusión: Detectamos los parámetros mecánicos óptimos del protocolo de excitación para la inducción de la proliferación de osteoblastos in vitro mediante una plataforma mecánica, que puede utilizarse como método estandarizado en la investigación de la transducción mecánica en osteoblastos humanos.

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          Most cited references23

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          Osteoblast Differentiation at a Glance

          Ossification is a tightly regulated process, performed by specialized cells called osteoblasts. Dysregulation of this process may cause inadequate or excessive mineralization of bones or ectopic calcification, all of which have grave consequences for human health. Understanding osteoblast biology may help to treat diseases such as osteogenesis imperfecta, calcific heart valve disease, osteoporosis, and many others. Osteoblasts are bone-building cells of mesenchymal origin; they differentiate from mesenchymal progenitors, either directly or via an osteochondroprogenitor. The direct pathway is typical for intramembranous ossification of the skull and clavicles, while the latter is a hallmark of endochondral ossification of the axial skeleton and limbs. The pathways merge at the level of preosteoblasts, which progress through 3 stages: proliferation, matrix maturation, and mineralization. Osteoblasts can also differentiate into osteocytes, which are stellate cells populating narrow interconnecting passages within the bone matrix. The key molecular switch in the commitment of mesenchymal progenitors to osteoblast lineage is the transcription factor cbfa/runx2, which has multiple upstream regulators and a wide variety of targets. Upstream is the Wnt/Notch system, Sox9, Msx2, and hedgehog signaling. Cofactors of Runx2 include Osx, Atf4, and others. A few paracrine and endocrine factors serve as coactivators, in particular, bone morphogenetic proteins and parathyroid hormone. The process is further fine-tuned by vitamin D and histone deacetylases. Osteoblast differentiation is subject to regulation by physical stimuli to ensure the formation of bone adequate for structural and dynamic support of the body. Here, we provide a brief description of the various stimuli that influence osteogenesis: shear stress, compression, stretch, micro- and macrogravity, and ultrasound. A complex understanding of factors necessary for osteoblast differentiation paves a way to introduction of artificial bone matrices.
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            A method for the rapid determination of alkaline phosphates with five cubic millimeters of serum.

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              Optimum reaction conditions for human lactate dehydrogenase isoenzymes as they affect total lactate dehydrogenase activity.

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                Author and article information

                Journal
                ijm
                Iberoamerican Journal of Medicine
                Iberoam J Med
                Hospital San Pedro (Logroño, La Rioja, Spain )
                2695-5075
                2695-5075
                2021
                : 3
                : 3
                : 204-211
                Affiliations
                [3] Haifa orgnameTechnion-Israel Institute of Technology orgdiv1Department Faculty of Aerospace Engineering Israel
                [1] Haifa orgnameTechnion-Israel Institute of Technology orgdiv1Ruth and Bruce Rappaport Faculty of Medicine Israel
                [2] Haifa orgnameSheltagen Medical Ltd Israel
                Article
                S2695-50752021000300005 S2695-5075(21)00300300005
                10.5281/zenodo.4746404
                70a4265f-b275-4266-8d92-f970f245e9c2

                This work is licensed under a Creative Commons Attribution 4.0 International License.

                History
                : 05 May 2021
                : 23 March 2021
                Page count
                Figures: 0, Tables: 0, Equations: 0, References: 23, Pages: 8
                Product

                SciELO Spain


                Mechanotransduction,Vibration,Mechanical stimulation,Osteoblast,Transducción mecánica,Vibración,Estimulación mecánica,Osteoblasto

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