Increased differentiation and production of extracellular matrix components of primary human osteoblasts after cocultivation with endothelial cells: A quantitative proteomics approach

• Published in: Journal of cellular biochemistry Vol. 120; no. 1; pp. 396 - 404
• Main Authors: Simunovic, F., Winninger, O., Strassburg, S., Koch, H. G., Finkenzeller, G., Stark, G. B., Lampert, F. M.
• Format: Journal Article
• Language: English
• Published: United States Wiley Subscription Services, Inc 01.01.2019
• Subjects: Addition polymerization; Amino acids; angiogenesis; Biocompatibility; Blood vessels; Bone growth; Bone Regeneration; bone tissue engineering; Cell culture; Cell Differentiation - physiology; Cells, Cultured; Coculture Techniques - methods; Connective tissue growth factor; Connective Tissue Growth Factor - genetics; Connective Tissue Growth Factor - metabolism; Connective tissues; Cyclin-Dependent Kinase 6 - genetics; Cyclin-Dependent Kinase 6 - metabolism; Data processing; Desmoplakins - genetics; Desmoplakins - metabolism; Differentiation; DNA microarrays; Down-Regulation - genetics; Endothelial cells; Extracellular matrix; Extracellular Matrix - metabolism; Galectin 3 - genetics; Galectin 3 - metabolism; Gene expression; Growth factors; human umbilical vein endothelial cells (HUVECs); Human Umbilical Vein Endothelial Cells - metabolism; Humans; Immunoassays; Kinases; Osteoblastogenesis; Osteoblasts; Osteoblasts - metabolism; Osteogenesis; Polymerase chain reaction; primary human osteoblasts (hOBs); Proteins; Proteomics; Proteomics - methods; quantitative proteomics; Regeneration; Regeneration (physiology); Reverse transcription; RNA, Messenger - genetics; stable isotope labeling by amino acids in cell culture (SILAC); Stable isotopes; Tissue engineering; Tissue Engineering - methods; Transcription, Genetic; Umbilical vein; Up-Regulation - genetics; stable isotope labeling by amino acids in cell culture (SILAC); bone tissue engineering; primary human osteoblasts (hOBs); angiogenesis; human umbilical vein endothelial cells (HUVECs); quantitative proteomics
• ISSN: 0730-2312; 1097-4644
• DOI: 10.1002/jcb.27394

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. We performed stable isotope labeling by amino acids in cell culture (SILAC), revealing 49 significantly upregulated and 54 significantly downregulated proteins in osteoblasts after coculture with endothelial cells. Among the highest regulated proteins, we found proteins important for osteoblast differentiation, cellular adhesion, and extracellular matrix function, notably: connective tissue growth factor (CTGF), desmoplakin (DSP), galectin‐3 and cyclin‐dependent kinase 6 (CDK6). These findings, which were replicated by enzyme‐linked immunosorbent assays (ELISA), real‐time quantitative reverse transcription polymerase chain reaction (RT‐qPCR) and former microarray studies, have implications for bone tissue engineering.