Pulsed electromagnetic fields (PEMFs) play a regulatory role on osteoblast activity and are clinically beneficial during fracture healing. Human mesenchymal stem cells (MSCs) derived from different sources have been extensively used in bone tissue engineering. Compared with MSCs isolated from bone marrow (BMSCs), those derived from adipose tissue (ASCs) are easier to obtain and available in larger amounts, although they show a less osteogenic differentiation potential than BMSCs. The hypothesis tested in this study was to evaluate whether PEMFs favor osteogenic differentiation both in BMSCs and in ASCs and to compare the role of PEMFs alone and in combination with the biochemical osteogenic stimulus bone morphogenetic protein (BMP)-2. Early and later osteogenic markers, such as alkaline phosphatase (ALP) activity, osteocalcin levels, and matrix mineralization, were analyzed at different times during osteogenic differentiation. Results showed that PEMFs induced osteogenic differentiation by increasing ALP activity, osteocalcin, and matrix mineralization in both BMSCs and ASCs, suggesting that PEMF activity is maintained during the whole differentiation period. The addition of BMP-2 in PEMF exposed cultures further increased all the osteogenic markers in BMSCs, while in ASCs, the stimulatory role of PEMFs was independent of BMP-2. Our results indicate that PEMFs may stimulate an early osteogenic induction in both BMSCs and ASCs and they suggest PEMFs as a bioactive factor to enhance the osteogenesis of ASCs, which are an attractive cell source for clinical applications. In conclusion, PEMFs may be considered a possible tool to improve autologous cell-based regeneration of bone defects in orthopedics.

Pulsed electromagnetic fields (PEMFs) have been used to treat bone diseases, particularly nonunion healing. Although it is known that PEMFs promote the osteogenic differentiation of human mesenchymal stem cells (hMSCs), to date PEMF molecular mechanisms remain not clearly elucidated. The Notch signalling is a highly conserved pathway that regulates cell fate decisions and skeletal development. The aim of this study was to investigate if the known PEMF-induced osteogenic effects may involve the modulation of the Notch pathway. To this purpose, during in vitro osteogenic differentiation of bone marrow hMSCs in the absence and in the presence of PEMFs, osteogenic markers (alkaline phosphatase activity, osteocalcin and matrix mineralization), the messenger ribonucleic acid expression of osteogenic transcription factors (Runx2, Dlx5, Osterix) as well as of Notch receptors (Notch1-4), their ligands (Jagged1, Dll1 and Dll4) and nuclear target genes (Hes1, Hes5, Hey1, Hey2) were investigated. PEMFs stimulated all osteogenic markers and increased the expression of Notch4, Dll4, Hey1, Hes1 and Hes5 in osteogenic medium compared to control. In the presence of DAPT and SAHM1, used as Notch pathway inhibitors, the expression of the osteogenic markers, including Runx2, Dlx5, Osterix, as well as Hes1 and Hes5 were significantly inhibited, both in unexposed and PEMF-exposed hMSCs. These results suggest that activation of Notch pathway is required for PEMFs-stimulated osteogenic differentiation. These new findings may be useful to improve autologous cell-based regeneration of bone defects in orthopaedics.

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Osteogenic differentiation is a multi-step process controlled by a complex molecular framework. Notch is an evolutionarily conserved intercellular signaling pathway playing a prominent role in cell fate and differentiation, although the mechanisms by which this pathway regulates osteogenesis remain controversial. This study aimed to investigate, in vitro, the involvement of Notch pathway during all the developmental stages of osteogenic differentiation in human osteosarcoma cell line MG63. Cells were cultured in basal condition (control) and in osteoinductive medium (OM). Notch inhibitors were also added in OM to block Notch pathway. During osteogenic differentiation, early (alkaline phosphatase activity and collagen type I) and late osteogenic markers (osteocalcin levels and matrix mineralization), as well as the gene expression of the main osteogenic transcription factors (Runx2, Osterix, and Dlx5) increased. Time dependent changes in the expression of specific Notch receptors were identified in OM versus control with a significant reduction in the expression of Notch1 and Notch3 receptors in the early phase of differentiation, and an increase of Notch2 and Notch4 receptors in the late phase. Among Notch nuclear target genes, Hey1 expression was significantly higher in OM than control, while Hes5 expression decreased. Osteogenic markers were reduced and Hey1 was significantly inhibited by Notch inhibitors, suggesting a role for Notch through the canonical pathway. In conclusion, Notch pathway might be involved with a dual role in osteogenesis of MG63, through the activation of Notch2, Notch4, and Hey1, inducing osteoblast differentiation and the depression of Notch1, Notch3, and Hes5, maintaining an undifferentiated status. J. Cell. Physiol. 231: 2652-2663, 2016. © 2016 Wiley Periodicals, Inc.

Objective. Osteochondral allograft (OCA) transplantation has demonstrated good long-term outcomes in treatment of cartilage defects. Viability, a key factor in clinical success, decreases with peri-implantation storage at 4°C during pathogen testing, matching logistics, and transportation. Modern, physiologic storage conditions may improve viability and enhance outcomes. Design. Osteochondral specimens from total knee arthroplasty patients (6 males, 5 females, age 56.4 ± 2.2 years) were stored in media and incubated at normoxia (21% O2) at 22°C or 37°C, and hypoxia (2% O2) at 37°C. Histology, live-dead staining, and quantitative polymerase chain reaction (qPCR) was performed 24 hours after harvest and following 7 days of incubation. Tissue architecture, cell viability, and gene expression were analyzed. Results. No significant viability or gene expression deterioration of cartilage was observed 1-week postincubation at 37°C, with or without hypoxia. Baseline viable cell density (VCD) was 94.0% ± 2.7% at day 1. At day 7, VCD was 95.1% (37°C) with normoxic storage and 92.2% (37°C) with hypoxic storage ( P ≥ 0.27). Day 7 VCD (22°C) incubation was significantly lower than both the baseline and 37°C storage values (65.6%; P < 0.01). COL1A1, COL1A2, and ACAN qPCR expression was unchanged from baseline ( P < 0.05) for all storage conditions at day 7, while CD163 expression, indicative of inflammatory macrophages and monocytes, was significantly lower in the 37°C groups ( P < 0.01). Conclusion. Physiologic storage at 37°C demonstrates improved chondrocyte viability and metabolism, and maintained collagen expression compared with storage at 22°C. These novel findings guide development of a method to optimize short-term fresh OCA storage, which may lead to improved clinical results.