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Galectins,β-galactoside binding proteins, function in several physiological and pathological processes. The further evaluation of these processes as well as possible applications of galectins in diagnosis and therapy has raised high scientific interest. Therefore, easy and reliable test systems are necessary. Here we present the simple and cost-efficient production of recombinant human galectins as fusion proteins with SNAP-tag and fluorescent proteins. These constructs show binding specificities and oligomerisation properties generally comparable to recombinant galectins. Their direct fluorescence signal was utilised by ELISA-type assay and flow cytometry analysis with human and ovine mesenchymal stem cells (MSC). Flow cytometry demonstrated glycan mediated binding of His6-SNAP-YFP-Gal- 3 to both MSC types, which was specifically inhibited by lactose. Moreover, directed immobilisation by SNAP-tag technology onto benzylguanine- activated sepharose was utilised to prepare galectin affinity columns for glycoprotein analysis and purification. The SNAPtag directed coupling yielded up to three-fold higher binding capacities for the glycoprotein standard asialofetuin compared to nondirected coupled galectin suggesting improved functionality following directed coupling.

Human adult mesenchymal stem cells (MSC) can be readily harvested from bone marrow through aspiration. MSC are involved in tissue regeneration and repair, particularly in wound healing. Due to their high self-renewal capacity and excellent differentiation potential in vitro, MSC are ideally suited for regenerative medicine. The complex interactions of MSC with their environment and their influence on the molecular and functional levels are widely studied but not completely understood. MSC secrete, for example, hepatocyte growth factor (HGF), whose concentration is enhanced in wounded areas and which is shown to act as a chemoattractant for MSC. We produced HGF-loaded biomaterials based on collagen and fibrin gels to develop a recruitment system for endogenous MSC to improve wound healing. Here, we report that HGF incorporated into collagen or fibrin gels leads to enhanced and directed MSC migration in vitro. HGF-loaded biomaterials might be potentially used as in vivo wound dressings to recruit endogenous MSC from tissue-specific niches towards the wounded area. This novel approach may help to reduce costly multistep procedures of cell isolation, in vitro culture, and transplantation usually used in tissue engineering.

Mesenchymal stem cells (MSC) are precursor cells of mesodermal tissue and, because of their trophic phenotype, they are known to play beneficial roles in wound healing. In addition, various tissue engineering strategies are based on MSC/biomaterial constructs. As the isolation and expansion of MSCs is a long-term process, a major goal is to develop an endogenous stem cell recruitment system that circumvents all ex vivo steps generally used for tissue engineering. Therefore collagen and silk fibroin were loaded with hepatocyte growth factor (HGF), a chemoattractant for MSCs. Collagen was mixed with HGF during polymerization, while silk fibroin and HGF were produced as fusion proteins by transgenic silkworms. To demonstrate release of active HGF, enzyme-linked immunosorbent assay, in vitro migration assays and animal studies were performed to demonstrate MSC migration in vivo, followed by detailed examinations of the immunological effects of the biomaterials. Hepatocyte growth factor was released burst-like, both from silk fibroin and collagen during the first 8 h and gradually for up to 168 h in vitro. Directed migration in vitro was demonstrated when MSCs were exposed to HGF. In vivo, HGF-loaded collagen and silk fibroin were tolerated as subcutaneous implants. In addition, it was proved that endogenous MSCs were recruited from the local environment. These results show for the first time recruitment of endogenous MSCs to HGF-loaded collagen (fast degradable) and silk fibroin scaffolds (long-term degradable) in vitro and in vivo. This knowledge could be applied to make off-the-shelf, readily available constructs for use in patients with chronic wound or burns. Copyright © 2016 John Wiley & Sons, Ltd.

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