Influence of Fucoidan Extracts from Different Fucus Species on Adult Stem Cells and Molecular Mediators in In Vitro Models for Bone Formation and Vascularization

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

Forces during cellular uptake of viruses and nanoparticles at the ventral side

Many intracellular pathogens, such as mammalian reovirus, mimic extracellular matrix motifs to specifically interact with the host membrane. Whether and how cell-matrix interactions influence virus particle uptake is unknown, as it is usually studied from the dorsal side. Here we show that the forces exerted at the ventral side of adherent cells during reovirus uptake exceed the binding strength of biotin-neutravidin anchoring viruses to a biofunctionalized substrate. Analysis of virus dissociation kinetics using the Bell model revealed mean forces higher than 30 pN per virus, preferentially applied in the cell periphery where close matrix contacts form. Utilizing 100 nm-sized nanoparticles decorated with integrin adhesion motifs, we demonstrate that the uptake forces scale with the adhesion energy, while actin/myosin inhibitions strongly reduce the uptake frequency, but not uptake kinetics. We hypothesize that particle adhesion and the push by the substrate provide the main driving forces for uptake

Fucoidan in der Knochenregeneration: Von Zellspezifischen Bioaktivitäten zur Entwicklung eines Hydrogelbasierten Trägersystems

Fucoidans are sulfated polysaccharides found in the cell wall of brown algae. Due to their ample effects on angiogenesis, osteogenesis and inflammation, these polysaccharides are promising bioactive compounds for the application in regenerative medicine. However, the chemical structure of fucoidan which determines the bioactivities is dependent on many parameters such as algae species, location, season and extraction technique and can therefore vary tremendously between extracts. Due to the heterogeneity of fucoidan and the largely unknown relation between chemical structure and function, it remains challenging to use fucoidan in a medical context. The presented dissertation investigates the biological activity of different fucoidan extracts on molecular processes relevant for bone tissue regeneration and relates chemical properties of the extracts to the observed bioactivities. Mono- and co-cultures of human outgrowth endothelial cells (OEC) isolated from peripheral blood and human mesenchymal stem cells isolated from cancellous bone and differentiated towards an osteoblast lineage (MSC) were used as cell model systems to mimic the bone tissue environment. To come one step closer to the utilization of fucoidan as a therapeutic agent, a thermosensitive chitosan-collagen hydrogel-based delivery system with incorporated fucoidan was developed

Injectable Thermosensitive Chitosan-Collagen Hydrogel as A Delivery System for Marine Polysaccharide Fucoidan

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

Effect of Enzymatically Extracted Fucoidans on Angiogenesis and Osteogenesis in Primary Cell Culture Systems Mimicking Bone Tissue Environment

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

Impact of Enzymatically Extracted High Molecular Weight Fucoidan on Lipopolysaccharide-Induced Endothelial Activation and Leukocyte Adhesion

The endothelial cell lining creates an interface between circulating blood and adjoining tissue and forms one of the most critical barriers and targets for therapeutical intervention. Recent studies suggest that fucoidans, sulfated and fucose-rich polysaccharides from brown seaweed, show multiple promising biological effects, including anti-inflammatory properties. However, their biological activity is determined by chemical characteristics such as molecular weight, sulfation degree, and molecular structure, which vary depending on the source, species, and harvesting and isolation method. In this study, we investigated the impact of high molecular weight (HMW) fucoidan extract on endothelial cell activation and interaction with primary monocytes (MNCs) in lipopolysaccharide (LPS)-induced inflammation. Gentle enzyme-assisted extraction combined with fractionation by ion exchange chromatography resulted in well-defined and pure fucoidan fractions. FE_F3, with a molecular weight ranging from 110 to 800 kDa and a sulfate content of 39%, was chosen for further investigation of its anti-inflammatory potential. We observed that along with higher purity of fucoidan fractions, the inflammatory response in endothelial mono- and co-cultures with MNCs was reduced in a dose-dependent manner when testing two different concentrations. This was demonstrated by a decrease in IL-6 and ICAM-1 on gene and protein levels and a reduced gene expression of TLR-4, GSK3β and NF-kB. Expression of selectins and, consequently, the adhesion of monocytes to the endothelial monolayer was reduced after fucoidan treatment. These data indicate that the anti-inflammatory effect of fucoidans increases with their purity and suggest that fucoidans might be useful in limiting the inflammatory response of endothelial cells in cases of LPS-induced bacterial infection