Today, the tool that is most commonly used to evaluate the osteogenic differentiation of bone marrow stromal cells (BMSCs) in vitro is the demonstration of the expression of multiple relevant markers, such as ALP, RUNX2 and OCN. However, as yet, there is no single surface marker or panel of markers which clearly defines human BMSCs (hBMSCs) differentiating towards the osteogenic lineage. The aim of this study was therefore to examine this issue. Stable isotope labeling by amino acids in cell culture (SILAC)-based quantitative proteomics was utilized to investigate differently expressed surface markers in osteogenically differentiated and undifferentiated hBMSCs. Labeled membrane proteins were analyzed by mass spectrometry (MS) and 52 proteins with an expression ratio above 2, between osteogenically differentiated and undifferentiated cells, were identified. Subsequent validation, by flow cytometry and ELISA, of the SILAC expression ratios for a number of these proteins and investigations of the lineage specificity of three candidate markers were performed. The surface markers, CD10 and CD92, demonstrated significantly increased expression in hBMSCs differentiated towards the osteogenic and adipogenic lineages. In addition, there was a slight increase in CD10 expression during chondrogenic differentiation. Furthermore, the expression of the intracellular protein, crystalline-αB (CRYaB), was only significantly increased in osteogenically differentiated hBMSCs and not affected during differentiation towards the chondrogenic or adipogenic lineages. It has been concluded from the present results that CD10 and CD92 are potential markers of osteogenic and adipogenic differentiation and that CRYaB is a potential novel osteogenic marker specifically expressed during the osteogenic differentiation of hBMSCs in vitro.

Inflammation and regeneration at the implant-bone interface are intimately coupled via cell-cell communication. In contrast to the prevailing view that monocytes/macrophages orchestrate mesenchymal stem cells (MSCs) and progenitor cells via the secretion of soluble factors, we examined whether communication between these different cell types also occurs via exosomes. LPS-stimulated human monocytes released exosomes, positive for CD9, CD63, CD81, Tsg101 and Hsp70, as determined by flow cytometry and Western blot. These exosomes also contained wide size distribution of RNA, including RNA in the size of microRNAs. The exosomes were shown to interact with human mesenchymal stem cells. After 24 h of culture, a considerable portion of the MSCs had internalised PKH67-labelled exosomes. Furthermore, after 72 h, the gene expression of the osteogenic markers runt-related transcription factor 2 (RUNX2) and bone morphogenetic protein-2 (BMP-2) had increased in comparison with control medium, whereas no significant difference in osteocalcin (OC) expression was demonstrated. The present results show that, under given experimental conditions, monocytes communicate with MSCs via exosomes, resulting in the uptake of exosomes in MSCs and the stimulation of osteogenic differentiation. The present observations suggest that exosomes constitute an additional mode of cell-cell signalling with an effect on MSC differentiation during the transition from injury and inflammation to bone regeneration.

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