J. van de Peppel

IFNβ impairs extracellular matrix formation leading to inhibition of mineralization by effects in the early stage of human osteoblast differentiation.

Osteoimmunology is an emerging field of research focused on the interaction of the immune system and bone. In this study we demonstrate that human osteoblasts are sensitive to the immune cytokine interferon (IFN)β. Osteoblasts respond to IFNβ as shown by the induction of several known IFN target genes such as interferon‐induced (IFI) proteins (IFIT1, IFI44L), interferon‐stimulated gene factor 3 (ISGF3) complex and the induction of IFNβ itself. We demonstrated that IFNβ has severe inhibitory effects on mineralization of osteoblast‐derived extracellular matrix (ECM). Analysis of the timing of the IFNβ effects revealed that committed osteoblasts in early stage of differentiation are most sensitive to IFNβ inhibition of mineralization. A single IFNβ treatment was as effective as multiple treatments. During the progress of differentiation osteoblasts become desensitized for IFNβ. This pinpoints to a complex pattern of IFNβ sensitivity in osteoblasts. Focusing on early osteoblasts, we showed that IFNβ decreased gene expression of ECM‐related genes, such as type I Collagen (COL1A1), fibronectin (FN1), fibullin (FBLN1), fibrillin (FBN2), and laminin (LAMA1). Additionally, ECM produced by IFNβ‐treated osteoblasts contained less collagen protein. IFNβ stimulated gene expression of osteopontin (OPN), annexin2 (ANXA2), and hyaluronan synthase 1 (HAS1), which are important factors in the adhesion of hematopoietic stem cells (HSC) in the HSC niche. In conclusion, IFNβ directly modifies human osteoblast function by inhibiting ECM synthesis eventually resulting in delayed bone formation and mineralization and induces a HSC niche supporting phenotype. These effects are highly dependent on timing of treatment in the early phase of osteoblast differentiation. J. Cell. Physiol. 227: 2668–2676, 2012.

Evidence of vitamin D and interferon-β cross-talk in human osteoblasts with 1α,25-dihydroxyvitamin D3 being dominant over interferon-β in stimulating mineralization.

It is well established that 1α‐25‐dihydroxyvitamin D3 (1,25D3) regulates osteoblast function and stimulates mineralization by human osteoblasts. The aim of this study was to identify processes underlying the 1,25D3 effects on mineralization. We started with gene expression profiling analyses of differentiating human pre‐osteoblast treated with 1,25D3. Bioinformatic analyses showed interferon‐related and ‐regulated genes (ISG) to be overrepresented in the set of 1,25D3‐regulated genes. 1,25D3 down‐regulated ISGs predominantly during the pre‐mineralization period. This pointed to an interaction between the vitamin D and IFN signaling cascades in the regulation of osteoblast function. Separately, 1,25D3 enhances while IFNβ inhibits mineralization. Treatment of human osteoblasts with 1,25D3 and IFNβ showed that 1,25D3 completely overrules the IFNβ inhibition of mineralization. This was supported by analyses of extracellular matrix gene expression, showing a dominant effect of 1,25D3 over the inhibitory effect of IFNβ. We identified processes shared by IFNβ‐ and 1,25D3‐mediated signaling by performing gene expression profiling during early osteoblast differentiation. Bioinformatic analyses revealed that genes being correlated or anti‐correlated with interferon‐induced protein with tetratricopeptide repeats 1 (IFIT1) were associated with osteoblast proliferation. In conclusion, the current study demonstrates a cross talk between 1,25D3 and IFNβ in osteoblast differentiation and bone formation/mineralization. The interaction is complex and depends on the process but importantly, 1,25D3 stimulation of mineralization is dominant over the inhibitory effect of IFNβ. These observations are of potential clinical relevance considering the impact of the immune system on bone metabolism in conditions such as rheumatoid arthritis. J. Cell. Physiol. 227: 3258–3266, 2012.

High content imaging in the screening of biomaterial-induced MSC behavior

Upon contact with a biomaterial, cells and surrounding tissues respond in a manner dictated by the physicochemical and mechanical properties of the material. Traditionally, cellular responses are monitored using invasive analytical methods that report the expression of genes or proteins. These analytical methods involve assessing commonly used markers for a predefined readout, masking the actual situation induced in the cells. Hence, a broader expression profile of the cellular response should be envisioned, which technically limits up scaling to higher throughput systems. However, it is increasingly recognized that morphometric readouts, obtained non-invasively, are related to gene expression patterns. Here, we introduced distinct surface roughness to three PLA surfaces, by exposure to oxygen plasma of different duration times. The response of mesenchymal stromal cells was compared to smooth untreated PLA surfaces without the addition of differentiation agents. Morphological and genome wide expression profiles revealed underlying cellular changes which was hidden for the commonly used gene markers for osteo-, chondro- and adipogenesis. Using 3 morphometric parameters, obtained by high content imaging, we were able to build a classifier and discriminate between oxygen plasma-induced modified sheets and non-modified PLA sheets where evaluating classical candidates missed this effect. This approach shows the feasibility to use noninvasive morphometric data in high-throughput systems to screen biomaterial surfaces indicating the underlying genetic biomaterial-induced changes.