Electromagnetic field-stimulated microreactor system with biosensing potential of stem cell-secreted proteins = Electromagnetic field-stimulated microreactor system with biosensing potential of stem cell-secreted proteins

다국어 초록 (Multilingual Abstract)

Most bone tissue-engineering models fail to demonstrate the complex cellular functions of living bone; therefore, most translational studies on bone tissue are performed in live models. To reduce the need for live models, we developed a stimulated micro-chip model for monitoring protein secretion during osteogenesis using human mesenchymal stem cells (hBMSCs). We established a bone micro-chip system for monitoring the in vitro differentiation and sensing the secreted proteins of hMSCs under a sinusoidal electromagnetic field (SEMF), which ameliorates bone healing in a biomimetic natural bone matrix. A 3V-1Hz SEMF biophysically stimulated osteogenesis by activating ERK-1/2 and promote phosphorylation of p38 MAPK kinases. Exposure to a 3V-1Hz SEMF upregulated the expression of osteogenesis-related genes, and enhanced the expression of key osteoregulatory proteins. hBMSCs undergoing osteoblastic differentiation are responsive to a well-defined SEMF stimulation at 3 V and 1 Hz, with an amplitude of 0.1 mT, for 20 minutes once a day. SEMF-treated plates contained the highest number of viable cells (>11%) compared to the other groups. Constant treatment with higher voltage, amplitude, or for a longer duration is generally counterproductive, suggesting that these parameters are optimized for this cell type. Our findings suggest that low voltage-frequency SEMF-guided osteoblast differentiation is not only triggered by the exposure time or dosage, but is also affected by various physicochemical factors. The activation of ERK1/2 and phosphorylation of p38 factors regulates the osteogenic differentiation during SEMF stimulation. Moreover, the bioinformatics analysis revealed the activation of various genes that are differentially expression only during SEMF stimulation but not in unstimulated condition. Notably, the gene expression of ALP (1.3 fold) and OCN (5.2 fold) was significantly higher (*p<0.05, **p<0.01) in cells receiving 3 V-1 Hz (0.1 mT, 20 min/day) stimulation compared to in the control. Significant increases in the expression of osteogenic gene markers were also observed for Runx2, OSX, BSP, OPN, and COL1, compared to in the unstimulated cells (*p<0.05). Six secretome proteins were upregulated in stimulated hBMSCs compared to in unstimulated hBMSCs. Overall, 23 proteins were either upregulated or downregulated by SEMF stimulation. These proteins are predicted to be secreted or included in secretory vesicles according to annotations in the Uniport database. Out of the upregulated proteins in SEMF-stimulated hBMSCs, 36% or 50% are involved in the 'immune response' or 'extracellular matrix function,' respectively. The unprecedented efficacy of our low-voltage-frequency SEMF exposure protocol for achieving hBMSC osteogenesis has broad clinical and practical implications, and could form the basis of SEMF-based therapeutic strategies for stem cell-based bone tissue regeneration. Our on-chip stimulation technology is easy to use, versatile, and non-disruptive, and should have diverse applications in regenerative medicine and cell-based therapies.