Cellular adhesion and chondrogenic differentiation inside an alginate-based bioink in response to tailorable artificial matrices and tannic acid treatment.

Many established bioinks fulfill important requirements regarding fabrication standards and cytocompatibility. Current research focuses on development of functionalized bioinks with an improved support of tissue-specific cell differentiation. Many approaches primarily depend on decellularized extracellular matrices or blood components. In this study, we investigated the combination of a highly viscous alginate-methylcellulose (algMC) bioink with collagen-based artificial extracellular matrix (aECM) as a finely controllable and tailorable system composed of collagen type I (col) with and without chondroitin sulfate (CS) or sulfated hyaluronan (sHA). As an additional stabilizer, the polyphenol tannic acid (TA) was integrated into the inks. The assessment of rheological properties and printability as well as hydrogel microstructure revealed no adverse effect of the integrated components on the inks. Viability, adhesion, and proliferation of bioprinted immortalized human mesenchymal stem cells (hTERT-MSC) was improved indicating enhanced interaction with the designed microenvironment. Furthermore, chondrogenic matrix production (collagen type II and sulfated glycosaminoglycans) by primary human chondrocytes (hChon) was enhanced by aECM. Supplementing the inks with TA was required for these positive effects but caused cytotoxicity as soon as TA concentrations exceeded a certain amount. Thus, combining tailorable aECM with algMC and balanced TA addition proved to be a promising approach for promoting adhesion of immortalized stem cells and differentiation of chondrocytes in bioprinted scaffolds.

Bone marrow mesenchymal stromal cell-derived extracellular matrix displays altered glycosaminoglycan structure and impaired functionality in Myelodysplastic Syndromes.

Myelodysplastic syndromes (MDS) comprise a heterogeneous group of hematologic malignancies characterized by clonal hematopoiesis, one or more cytopenias such as anemia, neutropenia, or thrombocytopenia, abnormal cellular maturation, and a high risk of progression to acute myeloid leukemia. The bone marrow microenvironment (BMME) in general and mesenchymal stromal cells (MSCs) in particular contribute to both the initiation and progression of MDS. However, little is known about the role of MSC-derived extracellular matrix (ECM) in this context. Therefore, we performed a comparative analysis of in vitro deposited MSC-derived ECM of different MDS subtypes and healthy controls. Atomic force microscopy analyses demonstrated that MDS ECM was significantly thicker and more compliant than those from healthy MSCs. Scanning electron microscopy showed a dense meshwork of fibrillar bundles connected by numerous smaller structures that span the distance between fibers in MDS ECM. Glycosaminoglycan (GAG) structures were detectable at high abundance in MDS ECM as white, sponge-like arrays on top of the fibrillar network. Quantification by Blyscan assay confirmed these observations, with higher concentrations of sulfated GAGs in MDS ECM. Fluorescent lectin staining with wheat germ agglutinin and peanut agglutinin demonstrated increased deposition of N-acetyl-glucosamine GAGs (hyaluronan (HA) and heparan sulfate) in low risk (LR) MDS ECM. Differential expression of N-acetyl-galactosamine GAGs (chondroitin sulfate, dermatan sulfate) was observed between LR- and high risk (HR)-MDS. Moreover, increased amounts of HA in the matrix of MSCs from LR-MDS patients were found to correlate with enhanced HA synthase 1 mRNA expression in these cells. Stimulation of mononuclear cells from healthy donors with low molecular weight HA resulted in an increased expression of various pro-inflammatory cytokines suggesting a contribution of the ECM to the inflammatory BMME typical of LR-MDS. CD34+ hematopoietic stem and progenitor cells (HSPCs) displayed an impaired differentiation potential after cultivation on MDS ECM and modified morphology accompanied by decreased integrin expression which mediate cell-matrix interaction. In summary, we provide evidence for structural alterations of the MSC-derived ECM in both LR- and HR-MDS. GAGs may play an important role in this remodeling processes during the malignant transformation which leads to the observed disturbance in the support of normal hematopoiesis.

Artificial Extracellular Matrices Containing Bioactive Glass Nanoparticles Promote Osteogenic Differentiation in Human Mesenchymal Stem Cells.

The present study analyzes the capacity of collagen (coll)/sulfated glycosaminoglycan (sGAG)-based surface coatings containing bioactive glass nanoparticles (BGN) in promoting the osteogenic differentiation of human mesenchymal stroma cells (hMSC). Physicochemical characteristics of these coatings and their effects on proliferation and osteogenic differentiation of hMSC were investigated. BGN were stably incorporated into the artificial extracellular matrices (aECM). Oscillatory rheology showed predominantly elastic, gel-like properties of the coatings. The complex viscosity increased depending on the GAG component and was further elevated by adding BGN. BGN-containing aECM showed a release of silicon ions as well as an uptake of calcium ions. hMSC were able to proliferate on coll and coll/sGAG coatings, while cellular growth was delayed on aECM containing BGN. However, a stimulating effect of BGN on ALP activity and calcium deposition was shown. Furthermore, a synergistic effect of sGAG and BGN was found for some donors. Our findings demonstrated the promising potential of aECM and BGN combinations in promoting bone regeneration. Still, future work is required to further optimize the BGN/aECM combination for increasing its combined osteogenic effect.

Genome-wide screens uncover KDM2B as a modifier of protein binding to heparan sulfate.

Heparan sulfate (HS) proteoglycans bind extracellular proteins that participate in cell signaling, attachment and endocytosis. These interactions depend on the arrangement of sulfated sugars in the HS chains generated by well-characterized biosynthetic enzymes; however, the regulation of these enzymes is largely unknown. We conducted genome-wide CRISPR-Cas9 screens with a small-molecule ligand that binds to HS. Screening of A375 melanoma cells uncovered additional genes and pathways impacting HS formation. The top hit was the epigenetic factor KDM2B, a histone demethylase. KDM2B inactivation suppressed multiple HS sulfotransferases and upregulated the sulfatase SULF1. These changes differentially affected the interaction of HS-binding proteins. KDM2B-deficient cells displayed decreased growth rates, which was rescued by SULF1 inactivation. In addition, KDM2B deficiency altered the expression of many extracellular matrix genes. Thus, KDM2B controls proliferation of A375 cells through the regulation of HS structure and serves as a master regulator of the extracellular matrix.

Hyaluronan/Collagen Hydrogels with Sulfated Glycosaminoglycans Maintain VEGF165 Activity and Fine-Tune Endothelial Cell Response.

In order to restore the regeneration capacity of large-size vascularized tissue defects, innovative biomaterial concepts are required. Vascular endothelial growth factor (VEGF165) is a key factor of angiogenesis interacting with sulfated glycosaminoglycans (sGAG) within the extracellular matrix. As this interplay mainly controls and directs the biological activity of VEGF165, we used chemically modified sGAG derivatives to evaluate the structural requirements of sGAG for controlling and tuning VEGF165 function and to translate these findings into the design of biomaterials. The in-depth analysis of this interaction by surface plasmon resonance and ELISA studies in combination with molecular modeling stressed the relevance of the substitution position, degree of sulfation, and carbohydrate backbone of GAG. Acrylated hyaluronan (HA-AC)/collagen (coll)-based hydrogels containing cross-linked acrylated, sulfated hyaluronan (sHA-AC) derivatives with different substitution patterns or an acrylated chondroitin sulfate (CS-AC) derivative function as multivalent carbohydrate-based scaffolds for VEGF165 delivery with multiple tuning capacities. Depending on the substitution pattern of sGAG, the release of biologically active VEGF165 was retarded in a defined manner compared to pure HA/coll gels, which further controlled the VEGF165-induced stimulation of endothelial cell proliferation and extended morphology of cells. This indicates that sGAG can act as modulators of protein interaction profiles of HA/coll hydrogels. In addition, sHA-AC-containing gels with and even without VEGF165 strongly stimulate endothelial cell proliferation compared to gels containing only CS-AC or HA-AC. Thus, HA/coll-based hydrogels containing cross-linked sHA-AC are biomimetic materials able to directly influence endothelial cells in vitro, which might translate into an improved healing of injured vascularized tissues.

Dual Action of Sulfated Hyaluronan on Angiogenic Processes in Relation to Vascular Endothelial Growth Factor-A.

Pathological healing characterized by abnormal angiogenesis presents a serious burden to patients' quality of life requiring innovative treatment strategies. Glycosaminoglycans (GAG) are important regulators of angiogenic processes. This experimental and computational study revealed how sulfated GAG derivatives (sGAG) influence the interplay of vascular endothelial growth factor (VEGF)165 and its heparin-binding domain (HBD) with the signaling receptor VEGFR-2 up to atomic detail. There was profound evidence for a HBD-GAG-HBD stacking configuration. Here, the sGAG act as a "molecular glue" leading to recognition modes in which sGAG interact with two VEGF165-HBDs. A 3D angiogenesis model demonstrated the dual regulatory role of high-sulfated derivatives on the biological activity of endothelial cells. While GAG alone promote sprouting, they downregulate VEGF165-mediated signaling and, thereby, elicit VEGF165-independent and -dependent effects. These findings provide novel insights into the modulatory potential of sGAG derivatives on angiogenic processes and point towards their prospective application in treating abnormal angiogenesis.

Hyaluronan/collagen hydrogels containing sulfated hyaluronan improve wound healing by sustained release of heparin-binding EGF-like growth factor.

Functional biomaterials that are able to bind, stabilize and release bioactive proteins in a defined manner are required for the controlled delivery of such to the desired place of action, stimulating wound healing in health-compromised patients. Glycosaminoglycans (GAG) represent a very promising group of components since they may be functionally engineered and are well tolerated by the recipient tissues due to their relative immunological inertness. Ligands of the Epidermal Growth Factor (EGF) receptor (EGFR) activate keratinocytes and dermal fibroblasts and, thus, contribute to skin wound healing. Heparin-binding EGF-like growth factor (HB-EGF) bound to GAG in biomaterials (e.g. hydrogels) might serve as a reservoir that induces prolonged activation of the EGF receptor and to recover disturbed wound healing. Based on previous findings, the capacity of hyaluronan (HA) and its sulfated derivatives (sHA) to bind and release HB-EGF from HA/collagen-based hydrogels was investigated. Docking and molecular dynamics analysis of a molecular model of HB-EGF led to the identification of residues in the heparin-binding domain of the protein being essential for the recognition of GAG derivatives. Furthermore, molecular modeling and surface plasmon resonance (SPR) analyses demonstrated that sulfation of HA increases binding strength to HB-EGF thus providing a rationale for the development of sHA-containing hydrogels. In line with computational observations and in agreement with SPR results, gels containing sHA displayed a retarded HB-EGF release in vitro compared to pure HA/collagen gels. Hydrogels containing HA and collagen or a mixture with sHA were shown to bind and release bioactive HB-EGF over at least 72 h, which induced keratinocyte migration, EGFR-signaling and HGF expression in dermal fibroblasts. Importantly, hydrogels containing sHA strongly increased the effectivity of HB-EGF in inducing epithelial tip growth in epithelial wounds shown in a porcine skin organ culture model. These findings suggest that hydrogels containing HA and sHA can be engineered for smart and effective wound dressings. STATEMENT OF SIGNIFICANCE: Immobilization and sustained release of recombinant proteins from functional biomaterials might overcome the limited success of direct application of non-protected solute growth factors during the treatment of impaired wound healing. We developed HA/collagen-based hydrogels supplemented with acrylated sulfated HA for binding and release of HB-EGF. We analyzed the molecular basis of HB-EGF interaction with HA and its chemical derivatives by in silico modeling and surface plasmon resonance. These hydrogels bind HB-EGF reversibly. Using different in vitro assays and organ culture we demonstrate that the introduction of sulfated HA into the hydrogels significantly increases the effectivity of HB-EGF action on target cells. Therefore, sulfated HA-containing hydrogels are promising functional biomaterials for the development of mediator releasing wound dressings.

Increased pore size of scaffolds improves coating efficiency with sulfated hyaluronan and mineralization capacity of osteoblasts.

BACKGROUND: Delayed bone regeneration of fractures in osteoporosis patients or of critical-size bone defects after tumor resection are a major medical and socio-economic challenge. Therefore, the development of more effective and osteoinductive biomaterials is crucial. METHODS: We examined the osteogenic potential of macroporous scaffolds with varying pore sizes after biofunctionalization with a collagen/high-sulfated hyaluronan (sHA3) coating in vitro. The three-dimensional scaffolds were made up from a biodegradable three-armed lactic acid-based macromer (TriLA) by cross-polymerization. Templating with solid lipid particles that melt during fabrication generates a continuous pore network. Human mesenchymal stem cells (hMSC) cultivated on the functionalized scaffolds in vitro were investigated for cell viability, production of alkaline phosphatase (ALP) and bone matrix formation. Statistical analysis was performed using student's t-test or two-way ANOVA. RESULTS: We succeeded in generating scaffolds that feature a significantly higher average pore size and a broader distribution of individual pore sizes (HiPo) by modifying composition and relative amount of lipid particles, macromer concentration and temperature for cross-polymerization during scaffold fabrication. Overall porosity was retained, while the scaffolds showed a 25% decrease in compressive modulus compared to the initial TriLA scaffolds with a lower pore size (LoPo). These HiPo scaffolds were more readily coated as shown by higher amounts of immobilized collagen (+ 44%) and sHA3 (+ 25%) compared to LoPo scaffolds. In vitro, culture of hMSCs on collagen and/or sHA3-coated HiPo scaffolds demonstrated unaltered cell viability. Furthermore, the production of ALP, an early marker of osteogenesis (+ 3-fold), and formation of new bone matrix (+ 2.5-fold) was enhanced by the functionalization with sHA3 of both scaffold types. Nevertheless, effects were more pronounced on HiPo scaffolds about 112%. CONCLUSION: In summary, we showed that the improvement of scaffold pore sizes enhanced the coating efficiency with collagen and sHA3, which had a significant positive effect on bone formation markers, underlining the promise of using this material approach for in vivo studies.

Role of WNT5A receptors FZD5 and RYK in prostate cancer cells.

Prostate cancer is the most common malignancy in men and has a high propensity to metastasize to bone. WNT5A has recently been implicated in the progression of prostate cancer, however, the receptors that mediate its effects remain unknown. Here, we identified Wnt receptors that are highly expressed in prostate cancer and investigated which of these receptors mediate the anti-tumor effects of WNT5A in prostate cancer in vitro. Extensive in vitro analyses revealed that the WNT5A receptors FZD5 and RYK mediate the anti-tumor effects of WNT5A on prostate cancer cells. Knock-down of FZD5 completely abrogated the anti-proliferative effect of WNT5A in PC3 cells. In contrast, knock-down of RYK and FZD8 did not rescue the inhibition of proliferation after WNT5A overexpression. In contrast, RYK knock-down inhibited the pro-apoptotic effect of WNT5A in PC3 cells by 60%, whereas the knock-down of either FZD5 or FZD8 further stimulated apoptosis after WNT5A overexpression (by 33% and 234%, respectively). Surface plasmon resonance analysis indicated that WNT5A has a 30% stronger binding response to FZD5 than to RYK. Further investigations using a tissue microarray revealed that expression of RYK is increased in advanced prostate cancer tumor stages, but is not associated with survival of prostate cancer patients. In contrast, patients with low local FZD5 expression, in particular in combination with low WNT5A expression, showed a longer disease-specific survival. In conclusion, WNT5A/FZD5 and WNT5A/RYK signaling are both involved in mediating the pro-apoptotic and anti-proliferative effects of WNT5A in prostate cancer.

Reciprocal influence of hMSCs/HaCaT cultivated on electrospun scaffolds.

Here, we investigated the synergistic effect of electrospun nanofibrous scaffolds made of gelatin /sulfated hyaluronan (sHA) or native hyaluronan (HA)/chondroitin sulfate (CS) and, keratinocytes (HaCaT)-human mesenchymal stem cells (hMSCs) contact co-culture on epithelial differentiation of hMSCs. The hMSCs were co-cultured in contact with HaCaT cells for 5 days on electrospun scaffold. Results show that electrospun scaffolds containing sulfated glycosaminoglycans (GAGs) stimulate epithelial differentiation in terms of various protein expression markers (keratin 14, ΔNp63α and Pan-cytokeratin) and gene expression of several dermal proteins (keratin 14, ΔNp63α). Electrospun scaffold independent of GAGs alone did not affect the epithelial differentiation of hMSCs but combination of keratinocyte-hMSC contact co-culture and electrospun scaffold promotes the epithelial differentiation of hMSCs.

Sulfated Hyaluronan Alters Endothelial Cell Activation in Vitro by Controlling the Biological Activity of the Angiogenic Factors Vascular Endothelial Growth Factor-A and Tissue Inhibitor of Metalloproteinase-3.

Several pathologic conditions such as rheumatoid arthritis, ocular neovascularization, cancer, or atherosclerosis are often associated with abnormal angiogenesis, which requires innovative biomaterial-based treatment options to control the activity of angiogenic factors. Here, we studied how sulfated hyaluronan (sHA) and oversulfated chondroitin sulfate derivatives as potential components of functional biomaterials modulate vascular endothelial growth factor-A (VEGF-A) signaling and endothelial cell activity in vitro. Tissue inhibitor of metalloproteinase-3 (TIMP-3), an effective angiogenesis inhibitor, exerts its activity by competing with VEGF-A for binding to VEGF receptor-2 (VEGFR-2). However, even though TIMP-3 and VEGF-A are known to interact with glycosaminoglycans (GAGs), the potential role and mechanism by which GAGs alter the VEGF-A/TIMP-3 regulated VEGFR-2 signaling remains unclear. Combining surface plasmon resonance, immunobiochemical analysis, and molecular modeling, we demonstrate the simultaneous binding of VEGF-A and TIMP-3 to sHA-coated surfaces and identified a novel mechanism by which sulfated GAG derivatives control angiogenesis: GAG derivatives block the binding of VEGF-A and TIMP-3 to VEGFR-2 thereby reducing their biological activity in a defined, sulfation-dependent manner. This effect was stronger for sulfated GAG derivatives than for native GAGs. The simultaneous formation of TIMP-3/sHA complexes partially rescues the sHA inhibited VEGF-A/VEGFR-2 signaling and endothelial cell activation. These results provide novel insights into the regulation of angiogenic factors by GAG derivatives and highlight the potential of sHA derivatives for the treatment of diseases associated with increased VEGF-A and VEGFR-2 levels.

Sulfated Hyaluronan Alters the Interaction Profile of TIMP-3 with the Endocytic Receptor LRP-1 Clusters II and IV and Increases the Extracellular TIMP-3 Level of Human Bone Marrow Stromal Cells.

Sulfated glycosaminoglycans (sGAGs) modulate cellular processes via their interaction with extracellular matrix (ECM) proteins. We revealed a direct binding of tissue inhibitor of metalloproteinase-3 (TIMP-3) to the endocytic receptor low-density lipoprotein receptor-related protein (LRP-1) clusters II and IV using surface plasmon resonance. Sulfated hyaluronan (sHA) and chondroitin sulfate (sCS) derivatives interfered with TIMP-3/LRP-1 complex formation in a sulfation-dependent manner stronger than heparin. Electrostatic potential calculations suggested a competition between negatively charged GAGs and highly negatively charged complement-like domains of LRP-1 for the binding to a positively charged area of TIMP-3 as an underlying mechanism. In vitro studies revealed increased amounts of pericellular TIMP-3 in the presence of sHA as a consequence of the blocked protein uptake. GAG derivatives as part of biomaterials might post-translationally modulate TIMP-3 levels stronger than native GAGs, thus exhibiting catabolic effects on the ECM, which could prevent extensive pathological matrix degradation and promote wound healing.

Bioinspired Collagen/Glycosaminoglycan-Based Cellular Microenvironments for Tuning Osteoclastogenesis.

Replicating the biocomplexity of native extracellular matrices (ECM) is critical for a deeper understanding of biochemical signals influencing bone homeostasis. This will foster the development of bioinspired biomaterials with adjustable bone-inducing properties. Collagen-based coatings containing single HA derivatives have previously been reported to promote osteogenic differentiation and modulate osteoclastogenesis and resorption depending on their sulfation degree. However, the potential impact of different GAG concentrations as well as the interplay of multiple GAGs in these coatings is not characterized in detail to date. These aspects were addressed in the current study by integrating HA and different sulfate-modified HA derivatives (sHA) during collagen in vitro fibrillogenesis. Besides cellular microenvironments with systematically altered single-GAG concentrations, matrices containing both low and high sHA (sHA1, sHA4) were characterized by biochemical analysis such as agarose gel electrophoresis, performed for the first time with sHA derivatives. The morphology and composition of the collagen coatings were altered in a GAG sulfation- and concentration-dependent manner. In multi-GAG microenvironments, atomic force microscopy revealed intermediate collagen fibril structures with thin fibrils and microfibrils. GAG sulfation altered the surface charge of the coatings as demonstrated by ζ-potential measurements revealed for the first time as well. This highlights the prospect of GAG-containing matrices to adjust defined surface charge properties. The sHA4- and the multi-GAG coatings alike significantly enhanced the viability of murine osteoclast-precursor-like RAW264.7 cells. Although in single-GAG matrices there was no dose-dependent effect on cell viability, osteoclastogenesis was significantly suppressed only on sHA4-coatings in a dose-dependent fashion. The multi-GAG coatings led to an antiosteoclastogenic effect in-between those with single-GAGs which cannot simply be attributed to the overall content of sulfate groups. These data suggest that the interplay of sGAGs influences bone cell behavior. Whether these findings translate into favorable biomaterial properties needs to be validated in vivo.

Sulfated hyaluronan influences the formation of artificial extracellular matrices and the adhesion of osteogenic cells.

The aim of this study is to compare differentially sulfated hyaluronan (sHA) derivatives and chondroitin sulfate (CS) with respect to their ability to influence the formation of artificial extracellular matrices (aECMs) during in vitro-fibrillogenesis of collagen type I at high- and low-ionic strength. Analysis is performed using turbidity, biochemical assays, atomic force (AFM), and transmission electron microscopy (TEM). In general, high-sulfated glycosaminoglycans (GAGs) associate to a higher amount with collagen than the low-sulfated ones. The addition of GAGs prior to fibrillogenesis at low-ionic strength results in a dose-dependent decrease in fibril diameter. At high-ionic strength these effects are only obtained for the sHA derivatives but not for CS. Likewise, increasing concentrations and degree of GAG sulfation strongly affected the kinetics of fibrillogenesis. The impact of sulfation degree on F-actin location and fiber formation in SaOS-2 cells implies that adhesion-related intracellular signaling is influenced to a variable extent.