Combined Effects of Cyclic Hypoxic and Mechanical Stimuli on Human Bone Marrow Mesenchymal Stem Cell Differentiation: A New Approach to the Treatment of Bone Loss

Abstract

Background: The prevention and treatment of bone loss and osteoporotic fractures is a public health challenge. Combined with normobaric hypoxia, whole-body vibration has a high clinic potential in bone health and body composition. The effect of this therapy may be mediated by its action on bone marrow mesenchymal stem cells (MSCs). Objectives: Evaluate the effects of cyclic low-vibration stimuli and/or hypoxia on bone marrow-derived human MSC differentiation. Methods: MSCs were exposed four days per week, two hours/day, to hypoxia (3% O₂) and/or vibration before they were induced to differentiate or during differentiation into osteoblasts or adipocytes. Gene and protein expression of osteoblastic, adipogenic, and cytoskeletal markers were studied, as well as extracellular matrix mineralization and lipid accumulation. Results: early osteoblastic markers increased in undifferentiated MSCs, pretreated in hypoxia and vibration. This pretreatment also increased mRNA levels of osteoblastic genes and beta-catenin protein in the early stages of differentiation into osteoblasts without increasing mineralization. When MSCs were exposed to vibration under hypoxia or normoxia during osteoblastic differentiation, mineralization increased with respect to cultures without vibrational stimuli. In MSCs differentiated into adipocytes, both in those pretreated as well as exposed to different conditions during differentiation, lipid formation decreased. Changes in adipogenic gene expression and increased beta-catenin protein were observed in cultures treated during differentiation. Conclusions: Exposure to cyclic hypoxia in combination with low-intensity vibratory stimuli had positive effects on osteoblastic differentiation and negative ones on adipogenesis of bone marrow-derived MSCs. These results suggest that in elderly or frail people with difficulty performing physical activity, exposure to normobaric cyclic hypoxia and low-density vibratory stimuli could improve bone metabolism and health.

Keywords: adipocyte; bone; hypoxia; mechanical stimuli; mesenchymal stem cells; osteoblast.

Figure 1

Temporal distribution of MSCs subjected to low-intensity vibration and cyclic hypoxia treatments. (a) Human MSCs were exposed to four different conditions during their expansion before confluence (seven days). Then, cultures were maintained in a conventional incubator during 14 (adipocytes and undifferentiated MSCs) or 21 (osteoblasts) days. (b) When MSCs were induced to differentiate into osteoblasts or adipocytes, tissue cultures were exposed to four different conditions during 14 (adipocytes) or 21 (osteoblast) days. Credit: illustration created with BioRender <https://www.biorender.com>.

Figure 2

Quantification of gene expressions in MSCs pretreated with low-intensity vibration stimuli and/or hypoxia. The ones of osteoblastic gene markers (runt-related transcription factor 2 and SP7), periostin, and glucose transporter 3 were measured in MSCs after their expansion under different treatments: normoxia (N), cyclic hypoxia (H), low-intensity vibration (NV), and cyclic-hypoxia combined with low-intensity vibration (HV), the next day (0 d), 7 and 14 days after application of treatments. * p < 0.05.

Figure 3

Effects of pretreatment of cyclic hypoxia and low-intensity vibration on MSCs induced to differentiate into osteoblasts. MSCs were expanded and induced to differentiate into osteoblasts under different conditions. See legend of Figure 2. The ones of osteoblastic markers (runt-related transcription factor 2, SP7, collagen type I alpha 1, osteocalcin, and integrin-binding sialoprotein) were measured at days 7 and 14 after being induced to differentiate into osteoblasts. At day 21, the extracellular matrix mineralization of the cultures was stained with alizarin red. (a) Representative images of alizarin-red staining of cultures differentiating into osteoblasts under the different conditions; (b) Alizarin-red quantification; (c) Quantification of osteoblastic-marker gene expressions in induced osteoblasts of preconditioned MSCs. * p < 0.05.

Figure 4

Quantification of gene expressions and protein synthesis in MSCs pretreated and then induced to differentiate into osteoblasts. (a) Periostin and glucose transporter 3 were measured at days 7 and 14, and (b) β-catenin, COL1A1, β-actin, and α-tubulin were determined by Western blot at day 14 in induced osteoblasts from mesenchymal-stem cells after their expansion under different treatments. See legend of Figure 2. * p < 0.05.

Figure 5

Effects of pretreatment of cyclic hypoxia and low-intensity vibration on MSCs and induced differentiation into adipocytes. MSCs were pretreated under different conditions and induced to differentiate into adipocytes. Genes encoding adipogenic markers (peroxisome proliferator-activated receptor gamma 2, lipoprotein lipid, fatty acid synthase, fatty acid-binding protein 4, and glycerol-3-phosphate dehydrogenase 1) were measured at days 7 and 14 after being induced to differentiate into adipocytes. Cultures were stained with oil-red O to reveal lipid droplets at day 14. (a) Representative images of oil-red O staining of cultures differentiating into adipocytes under the different conditions; (b) Oil-red quantification; (c) Quantification of adipogenic-marker gene expressions in induced adipocytes of preconditioned MSCs. * p < 0.05.

Figure 6

Effects of the combination of cyclic hypoxia and low-intensity vibration pretreatment on protein synthesis in MSCs differentiated into adipocytes. β-catenin and β-actin were quantified by Western blot at day 14, after being induced to differentiate into adipocytes. * p < 0.05.

Figure 7

Effects of a cotreatment of cyclic hypoxia and low-intensity vibration on differentiation of induced osteoblasts from MSCs. Mesenchymal stem cells were induced to differentiate into osteoblasts under different conditions, 4 days per week. Osteoblastic markers were measured at days 7 and 14. At day 21, the extracellular matrix mineralization of the cultures was stained with alizarin red. (a) Representative images of alizarin-red staining of cultures differentiating into osteoblasts under the different conditions; (b) Alizarin-red quantification; (c) Quantification of osteoblastic-marker gene expressions in induced osteoblasts of MSCs under different conditions. * p < 0.05.

Figure 8

Quantification of gene expression and protein synthesis in MSCs differentiated into osteoblasts under different treatments of cyclic hypoxia and low-intensity vibration. (a) Periostin and glucose transporter 3 gene expression were measured at days 7 and 14; (b) β-catenin, COL1A1, β-actin, and α-tubulin were quantified by Western blot at day 14. * p < 0.05.

Figure 9

Effects of cyclic hypoxia and low-intensity vibration on MSCs differentiated into adipocytes under different conditions. Genes encoding adipogenic markers were measured at days 7 and 14. Cultures were stained with oil-red O to reveal lipid droplets at day 14. (a) Representative images of oil-red O staining of cultures differentiating into adipocytes under the different conditions; (b) Oil-red quantification; (c) Quantification of adipogenic-markers gene expressions in induced adipocytes from MSCs. * p < 0.05.

Figure 10

Effects of combination of cyclic hypoxia and low-intensity vibrations on protein synthesis in MSCs differentiated into adipocytes. β-catenin and β-actin were determined by Western blot at day 14 under different conditions. * p < 0.05.