The development of novel bioactive biomaterials is urgently needed to meet the 
needs of an aging population. Both sulfated hyaluronic acid and dexamethasone 
are candidates for the functionalization of bone grafts, as they have been shown 
to enhance the differentiation of osteoblasts from bone marrow stromal cells in 
vitro and in vivo. However, the underlying mechanisms are not fully understood. 
Furthermore, studies combining different approaches to assess synergistic 
potentials are rare. In this study, we aim to gain insights into the mode of 
action of both sulfated hyaluronic acid and dexamethasone by a comprehensive 
analysis of the cellular fraction, released matrix vesicles, and the 
extracellular matrix, combining classical biochemical assays with mass 
spectrometry-based proteomics, supported by novel bioinformatical computations. 
We found elevated differentiation levels for both treatments, which were further 
enhanced by a combination of sulfated hyaluronic acid and dexamethasone. Single 
treatments revealed specific effects on osteogenic differentiation. 
Dexamethasone activates signalling pathways involved in the differentiation of 
osteoblasts, for example, CXC-motif chemokine receptor type 4 and 
mitogen-activated protein kinases. The effects of sulfated hyaluronic acid were 
predominantly linked to an alteration in the composition of the extracellular 
matrix, affecting the synthesis, secretion, and/or activity of fibrillary 
(fibronectin and thrombospondin-2) and nonfibrillary (transglutaminase-2, 
periostin, and lysyloxidase) extracellular matrix components, including 
proteases and their inhibitors (matrix metalloproteinase-2, tissue inhibitor of 
metalloproteinase-3). The effects were treatment specific, and less additive or 
contrary effects were found. Thus, we anticipate that the synergistic action of 
the treatment-specific effects is the key driver in elevated osteogenesis.