Abstract
Pulsed-electromagnetic-field (PEMF) treatment was found to enhance cellular differentiation of the mouse preosteoblast, MC3T3-E1, to a more osteoblastic phenotype. Differentiation genes such as Alp, BSPI, cFos, Ibsp, Osteocalcin, Pthr1 and Runx2 showed increased expression in response to PEMF stimulation. Detailed molecular mechanisms linking PEMF to the activation of these genes are limited. Two adenosine receptors known to be modulated in response to PEMF, Adora2A and Adora3, were functionally impaired by CRISPR-Cas9-mediated gene disruption, and the consequences of which were studied in the context of PEMF-mediated osteoblastic differentiation. Disruption of Adora2A resulted in a delay of Alp mRNA expression, but not alkaline phosphatase protein expression, which was similar to that found in wild type cells. However, Adora3 disruption resulted in significantly reduced responses at both the alkaline phosphatase mRNA and protein levels throughout the PEMF stimulation period. Defects observed in response to PEMF were mirrored using a chemically defined growth and differentiation-inducing media (DM). Moreover, in cells with Adora2A disruption, gene expression profiles showed a blunted response in cFos and Pthr1 to PEMF treatment; whereas cells with Adora3 disruption had mostly blunted responses in AlpI, BSPI, Ibsp, Osteocalcin and Sp7 gene activation. To demonstrate specificity for Adora3 function, the Adora3 open reading frame was inserted into the ROSA26 locus in Adora3 disrupted cells culminating in rescued PEMF responsiveness and thereby eliminating the possibility of off-target effects. These results lead us to propose that there are complementary and parallel positive roles for adenosine receptor A2A and A3 in PEMF-mediated osteoblast differentiation.
Highlights
Bone fracture is the most common musculoskeletal injury, and healing of such injury involves complex cellular and molecular processes [1,2]
To make the donor plasmid, the A3 open reading frame (ORF) was subcloned in the SparQ-T2A plasmid downstream of the cumate promoter, Adenosine receptors are required from PEMF-mediated osteoblast differentiation and left and right homology arms for mouse ROSA26 locus were synthesized from GenScript and were subcloned upstream of cumate promoter and downstream of SV40 polyA sequence, respectively. pX330-Cas9-ROSA26gRNA was made by subcloning ROSA26gRNA sequence in pX330-Cas9 (Addgene #42230)
We examined the effect that PEMF elicited on the expression of select genes known to be associated with the early stages of pre-osteoblast to osteoblast differentiation
Summary
Bone fracture is the most common musculoskeletal injury, and healing of such injury involves complex cellular and molecular processes [1,2]. Fracture healing is a multistep biological process, involving temporally defined stages including hematoma, inflammation, fibro-vasculature, bone formation and remodeling. Intermittent pulsed-electromagnetic-field (PEMF) stimulation has long been reported to heal bone fractures, especially delayed and non-unions [3,4,5,6,7,8,9]. Proliferation of different cell types, including neural stem cells [10], bone marrow mesenchymal stem cells [11], intervertebral disc cells [12], tendon cells [13], myoblasts [14], and osteoblasts [15] have been reported to be enhanced by PEMF stimulation. Reinforcement of intracellular calcium transients [15] and activation of the mammalian target of rapamycin (mTOR) signaling pathway leading to proliferation [16] of osteoblast cells have been described as possible effector pathways activated through intermittent treatment with electromagnetic pulses. PEMF has been reported to modulate cAMP levels [17] and to induce an anti-inflammatory response in a variety of cell types [18,19,20,21]
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