Platelet-released factors stimulate mobility and PI3K/AKT/mTOR pathway in bone marrow mesenchymal stromal cells

Introduction Mobilization of bone-residing progenitors is an important event in bone injury and during bone regeneration. The bone healing is initiated by inflammatory hematoma formation where the functional state of bone marrow mesenchymal stromal cells (BM-MSC) governs the final bone tissue restoration. Since our previous findings demonstrated augmenting role of platelet-released factors on immuno-regulatory functions of BM-MSCs, here we investigated how the factors derived from hematoma-mimicking hydrogels containing platelet rich plasma (PRP) or fibrin (FBR) impact BM-MSC motility and signaling pathways.

Methods BM-MSCs were isolated from BM of OA patients undergoing hip arthroplasty (Ethical approval 187/18). PRP was collected from thrombocyte concentrates and hydrogel formation was induced by addition of thrombin. Conditioned media (CM) was collected after incubating activated PRP hydrogels for 24 h and BM-MSCs were exposed to 10% CM, FBR hydrogels served as control. BM-MSC were investigated for metabolic activity and ATP content. To test cellular motility and migration, we performed scratch assays, and applied a transwell culture system where BM-MSCs were co-cultured with endothelial cells (HUVEC-GFP). Cell cycle, apoptosis and mitochondrial membrane potential were assessed by flow cytometry. Gel zymography, western blot and qPCR were used for enzyme, protein and gene expression analysis values are given as mean±standard deviation.

Results PRP-CM showed moderate effects on viability and ATP content, without influence on cell cycle and apoptosis, suggesting that BM-MSC survival was not challenged. BM-MSC motility and migration were stimulated in presence of PRP-CM (cell free area relative to control (CFR): 63.1327.98%) along with decreased mitochondrial membrane potential and cytoskeletal rearrangement. Interestingly, the presence of Rapamycin inhibited PRP-CM-stimulated motility of BM-MSCs (CFR:128.643.55%). Results indicate that stimuli derived from PRP-hydrogels regulate components of PI3K/AKT/mTOR pathway, leading to the slightly reduced mTOR phosphorylation at Ser-2448 in BM-MSCs, followed by a modest increase in LC3B I and II protein expression in both conditions, which suggests initiation of autophagic process. In line with this, PRP-CM upregulated gene expression of anti – apoptotic (Bcl-2) and autophagy markers (Atg7, Beclin-1) in a time-dependent manner. Although both PRP- and FBR-derived CM could stimulate urokinase-type plasminogen activator (uPA), matrix metalloproteinase MMP-2 and MMP-9 gene expression, only FBR-CM augmented MMP2 enzyme activity.

Discussion Our findings indicate that changes in BM-MSC mobility mediated by PRP-CM might be controlled by PI3K/AKT/mTOR cascade. Obtained results can contribute to the understanding of BM-MSC mobility in the hematoma microenvironment and its control, with implications for further bone tissue regeneration approaches.

Keywords MSCs, PRP, Bone

Korrespondenzadresse Magdalena Stöckl, University Hospital Wuerzburg, IZKF Group Tissue Regeneration in Musculoskeletal Diseases, Bernhard-Heine-Center for Locomotion Research, Röngtenring 11, 97070 Würzburg, Deutschland, E-Mail: [e-mail]

Publication History

Article published online:
08 September 2022