Rational synthesis of a heparan sulfate saccharide that promotes the activity of BMP2.

Heparan sulfate (HS) is a glycosaminoglycan (GAG) found throughout nature and is involved in a wide range of functions including modulation of cell signalling via sequestration of growth factors. Current consensus is that the specificity of HS motifs for protein binding are individual for each protein. Given the structural complexity of HS the synthesis of libraries of these compounds to probe this is not trivial. Herein we present the synthesis of an HS decamer, the design of which was undertaken rationally from previously published data for HS binding to the growth factor BMP-2. The biological activity of this HS decamer was assessed in vitro, showing that it had the ability to both bind BMP-2 and increase its thermal stability as well as enhancing the bioactivity of BMP-2 in vitro in C2C12 cells. At the same time no undesired anticoagulant effect was observed. This decamer was then analysed in vivo in a rabbit model where higher bone formation, bone mineral density (BMD) and trabecular thickness were observed over an empty defect or collagen implant alone. This indicated that the HS decamer was effective in promoting bone regeneration in vivo.

Micro-Topographies Promote Late Chondrogenic Differentiation Markers in the ATDC5 Cell Line.

Chemical and mechanical cues are well-established influencers of in vitro chondrogenic differentiation of ATDC5 cells. Here, we investigate the role of topographical cues in this differentiation process, a study not been explored before. Previously, using a library of surface micro-topographies we found some distinct patterns that induced alkaline phosphatase (ALP) production in human mesenchymal stromal cells. ALP is also a marker for hypertrophy, the end stage of chondrogenic differentiation preceding bone formation. Thus, we hypothesized that these patterns could influence end-stage chondrogenic differentiation of ATDC5 cells. In this study, we randomly selected seven topographies among the ALP influencing hits. Cells grown on these surfaces displayed varying nuclear shape and actin filament structure. When stimulated with insulin-transferrin-selenium (ITS) medium, nodule formation occurred and in some cases showed alignment to the topographical patterns. Gene expression analysis of cells growing on topographical surfaces in the presence of ITS medium revealed a downregulation of early markers and upregulation of late markers of chondrogenic differentiation compared to cells grown on a flat surface. In conclusion, we demonstrated that surface topography in addition to other cues can promote hypertrophic differentiation suitable for bone tissue engineering.

High-Throughput Screening Assay for the Identification of Compounds Enhancing Collagenous Extracellular Matrix Production by ATDC5 Cells.

Extracellular matrix (ECM) provides not only structural support to the cells but also signals that regulate their fate. In many tissue engineering approaches, it remains challenging to achieve the right amount and type of ECM secreted by the cells to faithfully mimic the native tissue. In this article, we describe how to design and perform a high-throughput assay to screen for molecules capable of enhancing collagenous ECM (cECM) production. We chose ATDC5 cells to validate the assay since we want to use this chondrogenic cell line later for tissue engineering of hypertrophic cartilage. We used a fluorescently labeled collagen-binding probe to quantify total collagen content in ATDC5 cultures. The LOPAC(1280) library of pharmaceutically active compounds was screened using insulin (a known inducer of cECM in ATDC5 cells) as positive control. After screening and validation, the small-molecule tetradecylthioacetic acid (TTA) was shown to enhance cECM production by ATDC5 cells at both gene expression and protein level. Moreover, when combined with insulin, TTA showed a synergistic effect on cECM production. In contrast, exposure of human primary chondrocytes and human mesenchymal stromal cells to TTA did not induce cECM secretion. In conclusion, we have developed an HTS assay to screen for compounds capable of enhancing cECM production and discovered TTA as a potent enhancer of cECM secretion by ATDC5 cells.

A small molecule approach to engineering vascularized tissue.

The repertoire of growth factors determines the biological engagement of human mesenchymal stromal cells (hMSCs) in processes such as immunomodulation and tissue repair. Hypoxia is a strong modulator of the secretome and well known stimuli to increase the secretion of pro-angiogenic molecules. In this manuscript, we employed a high throughput screening assay on an hMSCs cell line in order to identify small molecules that mimic hypoxia. Importantly, we show that the effect of these small molecules was cell type/species dependent, but we identified phenanthroline as a robust hit in several cell types. We show that phenanthroline induces high expression of hypoxia-target genes in hMSCs when compared with desferoxamine (DFO) (a known hypoxia mimic) and hypoxia incubator (2% O(2)). Interestingly, our microarray and proteomics analysis show that only phenanthroline induced high expression and secretion of another angiogenic cytokine, interleukin-8, suggesting that the mechanism of phenanthroline-induced hypoxia is distinct from DFO and hypoxia and involves the activation of other signaling pathways. We showed that phenanthroline alone was sufficient to induce blood vessel formation in a Matrigel plug assay in vivo paving the way to its application in ischeamic-related diseases.