Abstract
Oxidative stress from high levels of intracellular reactive oxygen species (ROS) has been linked to various bone diseases. Previous studies indicate that mesenchymal stem cells (MSC) secrete bioactive factors (conditioned medium (MSC-CM)) that have antioxidant effects. However, the antioxidant role of MSC-CM on osteogenesis has not been fully studied. We aimed to identify antioxidant proteins in MSC-CM using mass spectrometry-based proteomics and to explore their effects on osteogenic differentiation of human bone marrow mesenchymal stem cells (hBMSC) exposed to oxidative stress induced by hydrogen peroxide (H2O2). Our analysis revealed that MSC-CM is comprised of antioxidant proteins that are involved in several biological processes, including negative regulation of apoptosis and positive regulation of cell proliferation. Then, hBMSC exposed to H2O2 were treated with MSC-CM, and the effects on their osteogenic differentiation were evaluated. MSC-CM restored H2O2-induced damage to hBMSC by increasing the antioxidant enzyme-SOD production and the mRNA expression level of the anti-apoptotic BCL-2. A decrease in ROS production and cellular apoptosis was also shown. MSC-CM also modulated mRNA expression levels of osteogenesis-related genes, runt-related transcription factor 2, collagen type I, bone morphogenic protein 2, and osteopontin. Furthermore, collagen type I protein secretion, alkaline phosphatase activity, and in vitro mineralization were increased. These results indicate that MSC-CM contains several proteins with antioxidant and anti-apoptotic properties that restored the impaired hBMSC osteogenic differentiation associated with oxidative stress.
Highlights
Bone is a complex, mineralized connective tissue made up of different types of cells that constantly interact together
A total of 2218 proteins were identified by mass spectrometry (MS), and the data were subsequently investigated for differentially expressed proteins (DEPs) between Mesenchymal stem cells (MSC)-CM and control-DMEM
We found a decrease in Bcl-2 Associated X-protein (BAX)/B-cell lymphoma 2 (BCL-2) ratio to more than 1/10 in human bone marrow mesenchymal stem cells (hBMSC) exposed to H2O2 after treatment with MSC-conditioned media (MSC-CM)
Summary
Bone is a complex, mineralized connective tissue made up of different types of cells that constantly interact together. As critical mediators of pathophysiological responses, reactive oxygen species (ROS), including hydrogen peroxide (H2O2), superoxide, and hydroxyl radicals, have been considered as negative regulators of bone homeostasis via reducing osteoblast number and differentiation [3,4]. Based on these considerations, various antioxidants have been recommended to prevent the detrimental effects of ROS on osteogenic progenitors, promoting proper bone healing [5]. Current evidence indicates that ROS in the inflamed bone tissue may lead to impaired cell metabolism and decreased cell viability of engrafted MSC, which limits their therapeutic effects and delays osteogenesis [9,10]. Protecting cells in vivo from apoptosis, together with enhancing their ability to survive under oxidative stress and promoting the endogenous healing process, is crucial
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