Co-Culture with Human Osteoblasts and Exposure to Extremely Low Frequency Pulsed Electromagnetic Fields Improve Osteogenic Differentiation of Human Adipose-Derived Mesenchymal Stem Cells

Abstract

Human adipose-derived mesenchymal stem cells (Ad-MSCs) have been proposed as suitable option for cell-based therapies to support bone regeneration. In the bone environment, Ad-MSCs will receive stimuli from resident cells that may favor their osteogenic differentiation. There is recent evidence that this process can be further improved by extremely low frequency pulsed electromagnetic fields (ELF-PEMFs). Thus, the project aimed at (i) investigating whether co-culture conditions of human osteoblasts (OBs) and Ad-MSCs have an impact on their proliferation and osteogenic differentiation; (ii) whether this effect can be further improved by repetitive exposure to two specific ELF-PEMFs (16 and 26 Hz); (iii) and the effect of these ELF-PEMFs on human osteoclasts (OCs). Osteogenic differentiation was improved by co-culturing OBs and Ad-MSCs when compared to the individual mono-cultures. An OB to Ad-MSC ratio of 3:1 had best effects on total protein content, alkaline phosphatase (AP) activity, and matrix mineralization. Osteogenic differentiation was further improved by both ELF-PEMFs investigated. Interestingly, only repetitive exposure to 26 Hz ELF-PEMF increased Trap5B activity in OCs. Considering this result, a treatment with gradually increasing frequency might be of interest, as the lower frequency (16 Hz) could enhance bone formation, while the higher frequency (26 Hz) could enhance bone remodeling.

Keywords: extremely low frequency pulsed electromagnetic fields (ELF-PEMF); primary human adipose-derived mesenchymal stem cells (Ad-MSCs); primary human osteoblasts (OBs); primary human osteoclasts (OCs).

Figure 1

Increased proliferation in co-cultures of human osteoblasts (OBs) and adipose-derived mesenchymal stem cells (Ad-MSCs). OBs and Ad-MSCs (N = 6, n ≥ 3) were plated as mono- as well as co-cultures with a 3:1, 1:1 or 1:3 ratio and osteogenically differentiated as indicated in the materials and methods section. On days 0, 7 and 14 of differentiation (a) total protein content was determined by sulforhodamine B (SRB) staining and (b) mitochondrial activity was determined by Resazurin conversion. Results are given as fold of control (average of day 0). * p < 0.05, ** p < 0.01 and *** p < 0.001 as compared to day 0. ° p < 0.05 and °° p < 0.01 as indicated.

Figure 2

Increased AP activity and matrix mineralization in co-cultures of OBs and Ad-MSCs. OBs and Ad-MSCs (N = 6, n ≥ 3) were plated as mono- as well as co-cultures with a 3:1, 1:1, or 1:3 OB/Ad-MSC ratio, and osteogenically differentiated as indicated in the Materials and Methods section. On days 0, 7, and 14 of differentiation (a) AP activity was measured and (b) von Kossa and alizarin red staining was performed to visualize matrix mineralization (100× magnification; representative figure for day 14). (c) Matrix mineralization was quantified by resolving the alizarin red staining. * p < 0.05 and ** p < 0.01 as compared to day 0. ° p < 0.05, °° p < 0.01 and °°° p < 0.001 as indicated. Scale bar = 400 μm.

Figure 3

ELF-PEMF application with the Somagen^® device improved function of OBs and Ad-MSCs co-culture. OBs and Ad-MSCs (N = 6, n ≥ 3) were plated as co-culture with a 3:1 ratio. During the osteogenic differentiation process, cells were exposed to two specific ELF-PEMFs (16 and 26 Hz) 5 times per week, for 7 min each day. ELF-PEMFs were generated using the Somagen^® device. On days 0, 7, and 14 of differentiation (a) mitochondrial activity (resazurin conversion) and (b) AP activity were measured. (c) von Kossa and alizarin red staining were performed to visualize matrix mineralization (100× magnification). (d) Matrix mineralization was quantified by resolving the alizarin red staining. * p < 0.05, ** p < 0.01 and *** p < 0.001 as compared to day 0. ° p < 0.05 and °° p < 0.01 as indicated. Scale bar = 400 μm.

Figure 4

Osteoclast (OC) function is induced by repetitive exposure to 26 Hz ELF-PEMF but not 16 Hz ELF-PEMF. To investigate the effect of the two specific ELF-PEMFs (16 and 26 Hz) on OC function, we differentiated human peripheral blood mononuclear cells (N = 6, n = 3) to OCs. Following differentiation, OCs were exposed to the two ELF-PEMFs for 7 min each day. TRAP staining and activity was performed, not only to confirm the phenotype of generated OCs, but also to assess the effect of ELF-PEMFs exposure on their function. Thus, after the 5th exposure (day 5) (a) mitochondrial activity (resazurin conversion) and (b) Trap5B activity were measured. (c) Trap5B staining was performed to visualize OCs (100× magnification). ° p < 0.05 as indicated. Scale bar = 400 μm.

Figure 5

Experimental setup. (a) OBs and Ad-MSCs: Cells were plated either as mono- or co-culture with different ratios. After complete adherence (3 days), growth medium was replaced by osteogenic differentiation medium. Medium was replaced every 3–4 days. During the entire differentiation period, cells were exposed daily to ELF-PEMF (16 or 26 Hz) for 7 min (for 5 consecutive days). On day 0, 7, and 14, total protein content (SRB staining), mitochondrial activity (resazurin conversion), AP activity, and matrix mineralization (alizarin red and von Kossa staining) were assessed. The red numbers 75% (OB) and 25% (Ad-MSCs) indicate the co-culture ratio with the best results. (b) Generated OCs: Mononuclear cells were isolated from peripheral blood via density gradient centrifugation. After 24 h, non-adherent cells were washed off the plate, and cells were exposed to osteoclastic (de-)differentiation medium, as indicated in Materials and Methods. Medium was replaced every 3–4 days over the course of 21 days. After 21 days (indicated as d21/0), the generated OCs were exposed daily to ELF-PEMF (16 or 26 Hz) for 7 min each day for the following 5 days. Before and after ELF-PEMF exposure, mitochondrial activity and Trap5B activity were measured. “X” indicates the daily 7 min exposure of the cells or cell cultures to ELF-PEMF.