Vascularization is important for bone development, fracture healing and engineering of artificial bone tissue. In the context of bone tissue engineering, it was shown that coimplantation of human primary umbilical vein endothelial cells (HUVECs) and human osteoblasts (hOBs) results in the formation of functional blood vessels and enhanced bone regeneration. Implanted endothelial cells do not only contribute to blood vessel formation, but also support proliferation, cell survival and osteogenic differentiation of coimplanted hOBs. These effects are partially mediated by direct heterotypic cell contacts. In a previous report we could show that cocultivated hOBs strongly increase the expression of genes involved in extracellular matrix (ECM) formation in HUVECs, suggesting that ECM may be involved in the intercellular communication between hOBs and HUVECs. The present study aimed at investigating whether comparable changes occur in hOBs. We therefore performed a microarray analysis of hOBs cultivated in direct contact with HUVECs, revealing 1,004 differentially expressed genes. The differentially expressed genes could be assigned to the functional clusters ECM, proliferation, apoptosis and osteogenic differentiation. The microarray data could be confirmed by performing quantitative real time RT-PCR on selected genes. Furthermore, we could show that the ECM produced by HUVECs increased the expression of the osteogenic differentiation marker alkaline phosphatase (ALP) in hOBs. In summary, our data demonstrate that HUVECs provoke complex changes in gene expression patterns in cocultivated hOBs and that ECM plays and important role in this interaction. J. Cell. Biochem. 117: 1869-1879, 2016. © 2016 Wiley Periodicals, Inc.

Monocytes are the third most frequent type of leukocytes in humans, linking innate and adaptive immunity and are critical drivers in many inflammatory diseases. Based on the differential expression of surface antigens, three monocytic subpopulations have been suggested in humans and two in rats with varying inflammatory and phenotype characteristics. Potential intervention strategies that aim to manipulate these cells require an in-depth understanding of monocyte behavior under different conditions. However, monocytes are highly sensitive to their specific activation state and expression of surface markers, which can change during cell isolation and purification. Thus, there is an urgent need for an unbiased functional analysis of activation in monocyte subtypes, which is not affected by the isolation procedure. Here, we present a flow cytometry-based protocol for evaluating subset-specific activation and cytokine expression of circulating blood monocytes both in humans and rats using small whole blood samples (50 - 100 μL). In contrast to previously described monocyte isolation and flow cytometry visualization methods, the presented approach virtually leaves monocyte subsets in a resting state or fixes them in their current state and allows for an unbiased functional endpoint analysis without prior cell isolation. This protocol is a comprehensive tool for studying differential monocyte regulation in the inflammatory and allogeneic immune response in vitro and vivo.

Coculturing of bone-forming and blood vessel-forming cells is a strategy aimed at increasing vascularity of implanted bone constructs in tissue-engineering applications. We previously described that the coculture of primary human osteoblasts (hOBs) and human umbilical vein endothelial cells (HUVECs) improves the differentiation of both cell types, leading to the formation of functional blood vessels and enhanced bone regeneration. The objective of this study was to further delineate the multifaceted interactions between both cell types. To investigate the proteome of hOBs after cocultivation with HUVECs we used stable isotope labeling by amino acids in cell culture, revealing 49 significantly upregulated, and 54 significantly downregulated proteins. Amongst the highest regulated proteins, we found the proteins important for osteoblast differentiation, cellular adhesion, and extracellular matrix function, notably: connective tissue growth factor, desmoplakin, galectin-3, and cyclin-dependent kinase 6. The findings were confirmed by enzyme-linked immunosorbent assays. We also investigated whether the mRNA transcripts correlate with the changes in protein levels by quantitative real-time reverse transcription polymerase chain reaction. In addition, the data was compared to our previous microarray analysis of hOB transcriptome. Taken together, this in-depth analysis delivers reliable data suggesting the importance of coculturing of hOBs and HUVECs in tissue engineering.