Self-Assembled Sulfated Hyaluronan Coating Modulates Transforming Growth Factor-Beta1 Penetration for Corneal Scarring Alleviation.

Corneal scarring caused by epithelial-stromal injury impairs corneal transparency and visual acuity. Excess secretion of transforming growth factor-beta 1 (TGF-ß1), which promotes wound closure, penetrates the corneal stroma via defects in the epithelial basement membrane and induces the differentiation of corneal fibroblasts to myofibroblasts, leading to scar formation. Modulating TGF-ß1 penetration might alleviate corneal scar formation and restore transparency. In this study, sulfated hyaluronan (sHA) coatings were self-assembled above wounded corneal stroma to modulate TGF-ß1 penetration, and their ability to alleviate corneal scarring was investigated. The formation of sHA coatings was rapid (within 30 s), and the high-sulfated hyaluronan coating efficiently blocked penetration by TGF-ß1 and reduced the concentration of TGF-ß1 in the corneal stroma. Further investigation showed that the ability of TGF-ß1 to induce differentiation of corneal fibroblasts into myofibroblasts was inhibited by sHA binding. Evaluation of corneal scarring with sHA coating in a rabbit model of lamellar resection indicated that a sHA (high sulfation) coating effectively reduced scar formation. Immunohistochemical staining of α-smooth muscle actin and optical coherence tomography of the anterior segment showed minimal scar tissue formation in the sHA group. This work presents a promising alternative to alleviate scarring in corneal epithelial-stromal injury.

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.

Impact of Sulfated Hyaluronan on Bone Metabolism in Diabetic Charcot Neuroarthropathy and Degenerative Arthritis.

Bone in diabetes mellitus is characterized by an altered microarchitecture caused by abnormal metabolism of bone cells. Together with diabetic neuropathy, this is associated with serious complications including impaired bone healing culminating in complicated fractures and dislocations, especially in the lower extremities, so-called Charcot neuroarthropathy (CN). The underlying mechanisms are not yet fully understood, and treatment of CN is challenging. Several in vitro and in vivo investigations have suggested positive effects on bone regeneration by modifying biomaterials with sulfated glycosaminoglycans (sGAG). Recent findings described a beneficial effect of sGAG for bone healing in diabetic animal models compared to healthy animals. We therefore aimed at studying the effects of low- and high-sulfated hyaluronan derivatives on osteoclast markers as well as gene expression patterns of osteoclasts and osteoblasts from patients with diabetic CN compared to non-diabetic patients with arthritis at the foot and ankle. Exposure to sulfated hyaluronan (sHA) derivatives reduced the exaggerated calcium phosphate resorption as well as the expression of genes associated with bone resorption in both groups, but more pronounced in patients with CN. Moreover, sHA derivatives reduced the release of pro-inflammatory cytokines in osteoclasts of patients with CN. The effects of sHA on osteoblasts differed only marginally between patients with CN and non-diabetic patients with arthritis. These results suggest balancing effects of sHA on osteoclastic bone resorption parameters in diabetes.

Sulfated Hyaluronan Binds to Heparanase and Blocks Its Enzymatic and Cellular Actions in Carcinoma Cells.

We examined whether sulfated hyaluronan exerts inhibitory effects on enzymatic and biological actions of heparanase, a sole endo-beta-glucuronidase implicated in cancer malignancy and inflammation. Degradation of heparan sulfate by human and mouse heparanase was inhibited by sulfated hyaluronan. In particular, high-sulfated hyaluronan modified with approximately 2.5 sulfate groups per disaccharide unit effectively inhibited the enzymatic activity at a lower concentration than heparin. Human and mouse heparanase bound to immobilized sulfated hyaluronan. Invasion of heparanase-positive colon-26 cells and 4T1 cells under 3D culture conditions was significantly suppressed in the presence of high-sulfated hyaluronan. Heparanase-induced release of CCL2 from colon-26 cells was suppressed in the presence of sulfated hyaluronan via blocking of cell surface binding and subsequent intracellular NF-κB-dependent signaling. The inhibitory effect of sulfated hyaluronan is likely due to competitive binding to the heparanase molecule, which antagonizes the heparanase-substrate interaction. Fragment molecular orbital calculation revealed a strong binding of sulfated hyaluronan tetrasaccharide to the heparanase molecule based on electrostatic interactions, particularly characterized by interactions of (-1)- and (-2)-positioned sulfated sugar residues with basic amino acid residues composing the heparin-binding domain-1 of heparanase. These results propose a relevance for sulfated hyaluronan in the blocking of heparanase-mediated enzymatic and cellular actions.

The effects of sulfated hyaluronan in breast, lung and colorectal carcinoma and monocytes/macrophages cells: Its role in angiogenesis and tumor progression.

Hyaluronan (HA) is a component of the extracellular matrix (ECM) it is the main non-sulfated glycosaminoglycan able to modulate cell behavior in the healthy and tumor context. Sulfated hyaluronan (sHA) is a biomaterial derived from chemical modifications of HA, since this molecule is not naturally sulfated. The HA sulfation modifies several properties of the native molecule, acquiring antitumor properties in different cancers. In this study, we evaluated the action of sHA of ~30-60 kDa with different degrees of sulfation (0.7 sHA1 and 2.5 sHA3) on tumor cells of a breast, lung, and colorectal cancer model and its action on other cells of the tumor microenvironment, such as endothelial and monocytes/macrophage cells. Our data showed that in breast and lung tumor cells, sHA3 is able to modulate cell viability, cytotoxicity, and proliferation, but no effects were observed on colorectal cancer cells. In 3D cultures of breast and lung cancer cells, sHA3 diminished the size of the tumorsphere and modulated total HA levels. In these tumor models, treatment of monocytes/macrophages with sHA3 showed a downregulation of the expression of angiogenic factors. We also observed a decrease in endothelial cell migration and modulation of the hyaluronan-binding protein TSG-6. In the breast in vivo xenograft model, monocytes/macrophages preincubated with sHA1 or sHA3 decreased tumor vasculature, TSG-6 and HA levels. Besides, in silico analysis showed an association of TSG-6, HAS2, and IL-8 with biological processes implicated in the progression of the tumor. Taken together, our data indicate that sHA in a breast and lung tumor context is able to induce an antiangiogenic action on tumor cells as well as in monocytes/macrophages (Mo/MØ) by modulation of endothelial migration, angiogenic factors, and vessel formation.

Covalent linkage of sulfated hyaluronan to the collagen scaffold Mucograft® enhances scaffold stability and reduces proinflammatory macrophage activation in vivo.

Sulfated glycosaminoglycans (sGAG) show interaction with biological mediator proteins. Although collagen-based biomaterials are widely used in clinics, their combination with high-sulfated hyaluronan (sHA3) is unexplored. This study aims to functionalize a collagen-based scaffold (Mucograft®) with sHA3 via electrostatic (sHA3/PBS) or covalent binding to collagen fibrils (sHA3+EDC/NHS). Crosslinking without sHA3 was used as a control (EDC/NHS Ctrl). The properties of the sHA3-functionalized materials were characterized. In vitro growth factor and cytokine release after culturing with liquid platelet-rich fibrin was performed by means of ELISA. The cellular reaction to the biomaterials was analyzed in a subcutaneous rat model. The study revealed that covalent linking of sHA3 to collagen allowed only a marginal release of sHA3 over 28 days in contrast to electrostatically bound sHA3. sHA3+EDC/NHS scaffolds showed reduced vascular endothelial growth factor (VEGF), transforming growth factor beta 1 (TGF-ß1) and enhanced interleukin-8 (IL-8) and epithelial growth factor (EGF) release in vitro compared to the other scaffolds. Both sHA3/PBS and EDC/NHS Ctrl scaffolds showed a high proinflammatory reaction (M1: CD-68+/CCR7+) and induced multinucleated giant cell (MNGC) formation in vivo. Only sHA3+EDC/NHS scaffolds reduced the proinflammatory macrophage M1 response and did not induce MNGC formation during the 30 days. SHA3+EDC/NHS scaffolds had a stable structure in vivo and showed sufficient integration into the implantation region after 30 days, whereas EDC/NHS Ctrl scaffolds underwent marked disintegration and lost their initial structure. In summary, functionalized collagen (sHA3+EDC/NHS) modulates the inflammatory response and is a promising biomaterial as a stable scaffold for full-thickness skin regeneration in the future.

Sulfated Hyaluronan Modulates the Functional Properties and Matrix Effectors Expression of Breast Cancer Cells with Different Estrogen Receptor Status.

Hyaluronan (HA) is an extracellular matrix glycosaminoglycan (GAG) that plays a pivotal role in breast cancer. While HA is the only GAG not normally substituted with sulfate groups, sulfated hyaluronan (sHA) has previously been used in studies with promising antitumor results. The aim of the present study was to evaluate the effects sHA fragments have on breast cancer cells with different estrogen receptor (ER) status. To this end, ERα-positive MCF-7, and ERß-positive MDA-MB-231 cells were treated with non-sulfated HA or sHA fragments of 50 kDa. The functional properties of the breast cancer cells and the expression of key matrix effectors were investigated. According to the results, sHA attenuates cell proliferation, migration, and invasion, while increasing adhesion on collagen type I. Furthermore, sHA modulates the expression of epithelial-to-mesenchymal transition (EMT) markers, such as e-cadherin and snail2/slug. Additionally, sHA downregulates matrix remodeling enzymes such as the matrix metalloproteinases MT1-MMP, MMP2, and MMP9. Notably, sHA exhibits a stronger effect on the breast cancer cell properties compared to the non-sulfated counterpart, dependent also on the type of cancer cell type. Consequently, a deeper understanding of the mechanism by which sHA facilitate these processes could contribute to the development of novel therapeutic strategies.

Sulfated Hydrogels in Intervertebral Disc and Cartilage Research.

Hydrogels are commonly used for the 3D culture of musculoskeletal cells. Sulfated hydrogels, which have seen a growing interest over the past years, provide a microenvironment that help maintain the phenotype of chondrocytes and chondrocyte-like cells and can be used for sustained delivery of growth factors and other drugs. Sulfated hydrogels are hence valuable tools to improve cartilage and intervertebral disc tissue engineering. To further advance the utilization of these hydrogels, we identify and summarize the current knowledge about different sulfated hydrogels, highlight their beneficial effects in cartilage and disc research, and review the biofabrication processes most suitable to secure best quality assurance through deposition fidelity, repeatability, and attainment of biocompatible morphologies.

Impact of binding mode of low-sulfated hyaluronan to 3D collagen matrices on its osteoinductive effect for human bone marrow stromal cells.

Synthetically sulfated hyaluronan derivatives were shown to facilitate osteogenic differentiation of human bone marrow stromal cells (hBMSC) by application in solution or incorporated in thin collagen-based coatings. In the presented study, using a biomimetic three-dimensional (3D) cell culture model based on fibrillary collagen I (3D Col matrix), we asked on the impact of binding mode of low sulfated hyaluronan (sHA) in terms of adsorptive and covalent binding on osteogenic differentiation of hBMSC. Both binding modes of sHA induced osteogenic differentiation. Although for adsorptive binding of sHA a strong intracellular uptake of sHA was observed, implicating an intracellular mode of action, covalent binding of sHA to the 3D matrix induced also intense osteoinductive effects pointing towards an extracellular mode of action of sHA in osteogenic differentiation. In summary, the results emphasize the relevance of fibrillary 3D Col matrices as a model to study hBMSC differentiation in vitro in a physiological-like environment and that sHA can display dose-dependent osteoinductive effects in dependence on presentation mode in cell culture scaffolds.

Collagen/hyaluronan based hydrogels releasing sulfated hyaluronan improve dermal wound healing in diabetic mice via reducing inflammatory macrophage activity.

Sustained inflammation associated with dysregulated macrophage activation prevents tissue formation and healing of chronic wounds. Control of inflammation and immune cell functions thus represents a promising approach in the development of advanced therapeutic strategies. Here we describe immunomodulatory hyaluronan/collagen (HA-AC/coll)-based hydrogels containing high-sulfated hyaluronan (sHA) as immunoregulatory component for the modulation of inflammatory macrophage activities in disturbed wound healing. Solute sHA downregulates inflammatory activities of bone marrow-derived and tissue-resident macrophages in vitro. This further affects macrophage-mediated pro-inflammatory activation of skin cells as shown in skin ex-vivo cultures. In a mouse model of acute skin inflammation, intradermal injection of sHA downregulates the inflammatory processes in the skin. This is associated with the promotion of an anti-inflammatory gene signature in skin macrophages indicating a shift of their activation profile. For in vivo translation, we designed HA-AC/coll hydrogels allowing delivery of sHA into wounds over a period of at least one week. Their immunoregulatory capacity was analyzed in a translational experimental approach in skin wounds of diabetic db/db mice, an established model for disturbed wound healing. The sHA-releasing hydrogels improved defective tissue repair with reduced inflammation, augmented pro-regenerative macrophage activation, increased vascularization, and accelerated new tissue formation and wound closure.

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.

The influence of different artificial extracellular matrix implant coatings on the regeneration of a critical size femur defect in rats.

Resorbable biomaterials based on artificial extracellular matrices (aECM) represent promising scaffolds for the treatment of large bone defects. Here, we investigated various glycosaminoglycan (GAG) derivatives of varying sulfation degree with respect to their influence on in vivo bone healing. The materials used in this study consisted of GAG-coated degradable polycaprolactone-co-lactide (PCL). Critical size femur defects in rats were filled with autologous bone serving as positive control or the respective coated or uncoated PCL scaffolds. After 2 and 12 weeks, progress in the healing process was investigated by analyzing the new bone matrix formation, the collagen content and hydroxyapatite formation by using micro-computed tomography (µCT), biomechanical testing, nuclear magnetic resonance spectroscopy (NMR) and histology. The sulfated GAG coating contributed substantially to bone regeneration, increased collagen synthesis and initiated mineralization of the organic matrix. Most substantial collagen production was detected in scaffolds coated with chondroitin sulfate. Scaffolds coated with hypersulfated hyaluronan induced formation of new bone volume comparable to what was observed in the positive control. GAG differing in the sugar backbone and degree of sulfation modulate the healing process at different times, eventually leading to improved bone healing.

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.

Molecular size-dependent specificity of hyaluronan on functional properties, morphology and matrix composition of mammary cancer cells.

High levels of hyaluronan (ΗΑ), a major extracellular matrix (ECM) glycosaminoglycan, have been correlated with poor clinical outcome in several malignancies, including breast cancer. The high and low molecular weight HΑ forms exert diverse biological functions. Depending on their molecular size, ΗΑ forms either promote or attenuate signaling cascades that regulate cancer progression. In order to evaluate the effects of different ΗΑ forms on breast cancer cells' behavior, ΗΑ fragments of defined molecular size were synthesized. Breast cancer cells of different estrogen receptor (ER) status - the low metastatic, ERα-positive MCF-7 epithelial cells and the highly aggressive, ERß-positive MDA-MB-231 mesenchymal cells - were evaluated following treatment with HA fragments. Scanning electron microscopy revealed that HA fragments critically affect the morphology of breast cancer cells in a molecular-size dependent mode. Moreover, the ΗΑ fragments affect cell functional properties, the expression of major ECM mediators and epithelial-to-mesenchymal transition (ΕΜΤ) markers. Notably, treatment with 200 kDa ΗΑ increased the expression levels of the epithelial marker Ε-cadherin and reduced the expression levels of HA synthase 2 and mesenchymal markers, like fibronectin and snail2/slug. These novel data suggest that the effects of HA in breast cancer cells depend on the molecular size and the ER status. An in-depth understanding on the mechanistic basis of these effects may contribute on the development of novel therapeutic strategies for the pharmacological targeting of aggressive breast cancer.

Hyaluronan/Collagen Hydrogels with Sulfated Hyaluronan for Improved Repair of Vascularized Tissue Tune the Binding of Proteins and Promote Endothelial Cell Growth.

Innovative biomaterial-based concepts are required to improve wound healing of damaged vascularized tissues especially in elderly multimorbid patients. To develop functional hydrogels as 3D cellular microenvironments and as carrier or scavenging systems, e.g., for mediator proteins or proinflammatory factors, collagen fibrils are embedded into a network of photo-crosslinked acrylated hyaluronan (HA), chondroitin sulfate (CS), or sulfated HA (sHA). After lyophilization, the gels show a porous structure and an improved stability against degradation via hyaluronidase. Gels with CS and sHA bind significantly more lysozyme than HA/collagen gels and retard its release. The proliferation and metabolic activity of endothelial cells are significantly increased on sHA gels compared to CS- or only HA-containing hydrogels. These findings highlight the potential of HA/collagen hydrogels with sulfated glycosaminoglycans to tune the protein binding and release behavior and to directly modulate cellular response. This can be easily translated into biomimetic biomaterials with defined properties to stimulate wound healing.

Sulfated hyaluronan attenuates inflammatory signaling pathways in macrophages involving induction of antioxidants.

It is well recognized that high molecular weight hyaluronan (H-HA) exerts potent anti-inflammatory effects while its fragmentation into low molecular weight HA (L-HA) is discussed to promote inflammation. Chemical modification of HA with sulfate groups has been shown to foster its anti-inflammatory activity which seems to be maintained in sulfated low molecular weight HA derivatives (sL-HA). However, the molecular mechanisms by which sL-HA produces its anti-inflammatory activity are not understood. In this study, we used global quantitative proteomics combined with targeted analysis of key proteins to characterize the effect of sL-HA on fully differentiated human inflammatory macrophages (iMФ). Culture of iMФ with sL-HA did not affect cell viability but resulted in a reduced pro-inflammatory cytokine response of iMФ after activation indicating a profound counter-regulation of their initial inflammatory phenotype. Rapid internalization of sL-HA involving CD44 and scavenger receptors was observed. Furthermore, an upregulation of the antioxidants SOD2 and SOD3 was found while no oxidative stress was induced. Consequently, activity of transcription factors for inflammatory gene expression was downregulated in iMФ with sL-HA after activation whereas anti-inflammatory proteins were induced. This study proves anti-inflammatory properties of sL-HA and provides information on its regulatory mode of action on iMФ.

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.

Evaluation of cell-surface interaction using a 3D spheroid cell culture model on artificial extracellular matrices.

Since decades, cell-surface interactions are studied in 2D cell culture approaches, but cells organized in 3D (spheroids) reflect the normal situation of cells in tissues much better due to intense cell-cell-contacts. Accordingly, this study aimed to prove, if spheroids could be used to study cell-surface interaction. Spheroids consisting of fibroblasts and/or osteoblasts were seeded on artificial extracellular matrices. Here, non-sulfated hyaluronan as a biological relevant compound of the extracellular matrix was chemically sulfated to different extents and co-fibrillised with collagen. The changes of the spheroid diameters and the migration distance of outgrown cells after seeding on the matrices were used as parameters to evaluate cell-surface interaction quantitatively. Fibroblast-based spheroids reacted in the initial phase of adhesion with different spheroid sizes on the contact with the matrices. In contrast, the reaction of osteoblasts was more pronounced at later time points exhibiting a decrease of the size of the spheroids with increasing sulfation degree of the matrix. The migration of the cells was impaired by increasing sulfation degree, which might be caused by an increased expression of focal adhesion relevant proteins. In summary, spheroids can be used in cell-surface interaction studies and additional analytical tools could be implemented.

Structural and functional insights into the interaction of sulfated glycosaminoglycans with tissue inhibitor of metalloproteinase-3 - A possible regulatory role on extracellular matrix homeostasis.

An imbalance between tissue-degrading matrix metalloproteinases (MMPs) and their counterparts' tissue inhibitors of metalloproteinases (TIMPs) causes pathologic extracellular matrix (ECM) degradation in chronic wounds and requires new adaptive biomaterials that interact with these regulators to re-establish their balance. Sulfated glycosaminoglycans (GAGs) and TIMP-3 are key modulators of tissue formation and remodeling. However, little is known about their molecular interplay. GAG/TIMP-3 interactions were characterized combining surface plasmon resonance, ELISA, molecular modeling and hydrogen/deuterium exchange mass spectrometry. We demonstrate the potential of solute and surface-bound sulfated hyaluronan (sHA) and chondroitin sulfate (sCS) derivatives to manipulate GAG/TIMP-3 interactions by varying GAG concentration, sulfation degree and chain length. Three GAG binding sites in the N- and C-terminal domains of TIMP-3 were identified. We reveal no overlap with the matrix metalloproteinases (MMP)-binding site, elucidating why GAGs did not change MMP-1/-2 inhibition by TIMP-3 in enzyme kinetics. Since we prove that GAGs alone have a low impact on MMP activity, sHA and sCS offer a promising strategy to possibly control ECM remodeling via stabilizing and accumulating TIMP-3 by maintaining its MMP inhibitory activity under GAG-bound conditions. Whether GAG-based functional biomaterials can be applied to foster chronic wound healing by shifting the MMP/TIMP balance to a healing promoting state needs to be evaluated in vivo. STATEMENT OF SIGNIFICANCE: Increased levels of tissue-degrading matrix metalloproteinases (MMPs) lead to pathologic matrix degradation in chronic wounds. Therefor functional biomaterials that restore the balance between MMPs and tissue inhibitors of metalloproteinases (TIMPs) are required to promote wound healing. Since sulfated glycosaminoglycan (GAG) derivatives demonstrated already to be e.g. anti-inflammatory and immunomodulatory, and native GAGs interact with TIMP-3 the former are promising candidates for functionalizing biomaterials. We identified the GAG binding sites of TIMP-3 by combining experimental and molecular modeling approaches and revealed that GAG derivatives have a higher capacity to sequester TIMP-3 than native GAGs without altering its inhibitory potential towards MMPs. Thus GAG derivative-containing biomaterials could protect tissue from excessive proteolytic degradation e.g. in chronic wounds by re-establishing the MMP/TIMP balance.

Interplay of Substrate Conductivity, Cellular Microenvironment, and Pulsatile Electrical Stimulation toward Osteogenesis of Human Mesenchymal Stem Cells in Vitro.

The influences of physical stimuli such as surface elasticity, topography, and chemistry over mesenchymal stem cell proliferation and differentiation are well investigated. In this context, a fundamentally different approach was adopted, and we have demonstrated the interplay of inherent substrate conductivity, defined chemical composition of cellular microenvironment, and intermittent delivery of electric pulses to drive mesenchymal stem cell differentiation toward osteogenesis. For this, conducting polyaniline (PANI) substrates were coated with collagen type 1 (Coll) alone or in association with sulfated hyaluronan (sHya) to form artificial extracellular matrix (aECM), which mimics the native microenvironment of bone tissue. Further, bone marrow derived human mesenchymal stem cells (hMSCs) were cultured on these moderately conductive (10(-4)-10(-3) S/cm) aECM coated PANI substrates and exposed intermittently to pulsed electric field (PEF) generated through transformer-like coupling (TLC) approach over 28 days. On the basis of critical analysis over an array of end points, it was inferred that Coll/sHya coated PANI (PANI/Coll/sHya) substrates had enhanced proliferative capacity of hMSCs up to 28 days in culture, even in the absence of PEF stimulation. On the contrary, the adopted PEF stimulation protocol (7 ms rectangular pulses, 3.6 mV/cm, 10 Hz) is shown to enhance osteogenic differentiation potential of hMSCs. Additionally, PEF stimulated hMSCs had also displayed different morphological characteristics as their nonstimulated counterparts. Concomitantly, earlier onset of ALP activity was also observed on PANI/Coll/sHya substrates and resulted in more calcium deposition. Moreover, real-time polymerase chain reaction results indicated higher mRNA levels of alkaline phosphatase and osteocalcin, whereas the expression of other osteogenic markers such as Runt-related transcription factor 2, Col1A, and osteopontin exhibited a dynamic pattern similar to control cells that are cultured in osteogenic medium. Taken together, our experimental results illustrate the interplay of multiple parameters such as substrate conductivity, electric field stimulation, and aECM coating on the modulation of hMSC proliferation and differentiation in vitro.