Fucoidans are polysaccharides from brown macroalgae, showing multiple bioactivities important for bone regeneration and bone health. However, the use of fucoidans in medical applications remains sparse due to the heterogeneity in their chemical properties and unclear structure-function relationships. Innovations in extraction techniques and post processing steps are needed to produce homogeneous fucoidan molecules with tailorable bioactivities. Here, we applied enzyme-assisted extraction coupled with enzymatic hydrolysis by Fhf1 fucoidanase to generate low (LMW) and medium molecular weight (MMW) fucoidans from Fucus evanescens. In contrast to the anti-angiogenic properties of the high molecular weight fucoidan, LMW and MMW no longer suppressed the production of pro-angiogenic molecules by bone stem cells, nor impaired the formation of prevascular structures in vitro. In contrast to LMW, a pro-inflammatory response of OEC was observed after treatment with high concentrations of MMW. Thus, fucoidanase hydrolysis could be a useful tool to tailor the bioactivity of fucoidans.

Fucoidans, sulfated polysaccharides from brown algae, possess multiple bioactivities in regard to osteogenesis, angiogenesis, and inflammation, all representing key molecular processes for successful bone regeneration. To utilize fucoidans in regenerative medicine, a delivery system is needed which temporarily immobilizes the polysaccharide at the injured site. Hydrogels have become increasingly interesting biomaterials for the support of bone regeneration. Their structural resemblance with the extracellular matrix, their flexible shape, and capacity to deliver bioactive compounds or stem cells into the affected tissue make them promising materials for the support of healing processes. Especially injectable hydrogels stand out due to their minimal invasive application. In the current study, we developed an injectable thermosensitive hydrogel for the delivery of fucoidan based on chitosan, collagen, and β-glycerophosphate (β-GP). Physicochemical parameters such as gelation time, gelation temperature, swelling capacity, pH, and internal microstructure were studied. Further, human bone-derived mesenchymal stem cells (MSC) and human outgrowth endothelial cells (OEC) were cultured on top (2D) or inside the hydrogels (3D) to assess the biocompatibility. We found that the sol-gel transition occurred after approximately 1 min at 37 °C. Fucoidan integration into the hydrogel had no or only a minor impact on the mentioned physicochemical parameters compared to hydrogels which did not contain fucoidan. Release assays showed that 60% and 80% of the fucoidan was released from the hydrogel after two and six days, respectively. The hydrogel was biocompatible with MSC and OEC with a limitation for OEC encapsulation. This study demonstrates the potential of thermosensitive chitosan-collagen hydrogels as a delivery system for fucoidan and MSC for the use in regenerative medicine.

Angiogenesis, the formation of new blood vessels from existing ones, is an essential process for successful bone regeneration. Further, angiogenesis is a key factor for the development of bone-related disorders like osteosarcoma or arthritis. Fucoidans, sulfated polysaccharides from brown algae, have been shown to affect angiogenesis as well as a series of other physiological processes including inflammation or infection. However, the chemical properties of fucoidan which define the biological activity vary tremendously, making a prediction of the bioactivity or the corresponding therapeutic effect difficult. In this study, we compare the effect of four chemically characterized high molecular weight fucoidan extracts from Fucus distichus subsp. evanescens (FE_crude and fractions F1, F2, F3) on angiogenic and osteogenic processes in bone-related primary mono- and co-culture cell systems. By determining the gene expression and protein levels of the regulatory molecules vascular endothelial growth factor (VEGF), angiopoietin-1 (ANG-1), ANG-2 and stromal-derived factor 1 (SDF-1), we show that the extracted fucoidans negatively influence angiogenic and osteogenic processes in both the mono- and co-culture systems. We demonstrate that purer fucoidan extracts with a high fucose and sulfate content show stronger effects on these processes. Immunocytochemistry of the co-culture system revealed that treatment with FE_F3, containing the highest fucose and sulfate content, impaired the formation of angiogenic tube-like structures, indicating the anti-angiogenic properties of the tested fucoidans. This study highlights how chemical properties of fucoidan influence its bioactivity in a bone-related context and discusses how the observed phenotypes can be explained on a molecular level—knowledge that is indispensable for future therapies based on fucoidans.

Fucoidans, sulfated polysaccharides extracted from brown algae, are marine products with the potential to modulate bone formation and vascularization processes. The bioactivity and safety of fucoidans are highly associated with their chemical structure, which may vary with algae species and extraction method. Thus, in depth evaluation of fucoidan extracts in terms of endotoxin content, cytotoxicity, and their detailed molecular biological impact on the individual cell types in bone is needed. In this study, we characterized fucoidan extracts from three different Fucus species including Fucus vesiculosus (Fv), Fucus serratus (Fs), and Fucus distichus subsp. evanescens (Fe) for their chemical features, endotoxin content, cytotoxicity, and bioactive effects on human outgrowth endothelial cells (OEC) and human mesenchymal stem cells (MSC) as in vitro models for bone function and vascularization. Extracts contained mainly high molecular weight (HMW) fucoidans and were free of endotoxins that may cause inflammation or influence vascularization. OEC tolerated fucoidan concentrations up to 200 µg/mL, and no indication of cytotoxicity was observed. The inflammatory response, however, investigated by real-time PCR (RT-PCR) and enzyme-linked immunosorbent assay (ELISA) and endothelial barrier assessed by impedance measurement differed for the individual extracts. MSC in comparison with endothelial cells were more sensitive to fucoidans and showed partly reduced metabolic activity and proliferation at higher doses of fucoidans. Further results for MSC indicated impaired osteogenic functions in alkaline phosphatase and calcification assays. All tested extracts consistently lowered important molecular mediators involved in angiogenesis, such a VEGF (vascular endothelial growth factor), ANG-1 (angiopoietin 1), and ANG-2 (angiopoietin 2), as indicated by RT-PCR and ELISA. This was associated with antiangiogenic effects at the functional level using selected extracts in co-culture models to mimic bone vascularization processes during bone regeneration or osteosarcoma.