Microdialysis Reveals Anti-Inflammatory Effects of Sulfated Glycosaminoglycanes in the Early Phase of Bone Healing.

Although chronic inflammation inhibits bone healing, the healing process is initiated by an inflammatory phase. In a well-tuned sequence of molecular events, pro-inflammatory cytokines are secreted to orchestrate the inflammation response to injury and the recruitment of progenitor cells. These events in turn activate the secretion of anti-inflammatory signaling molecules and attract cells and mediators that antagonize the inflammation and initiate the repair phase. Sulfated glycosaminoglycanes (sGAG) are known to interact with cytokines, chemokines and growth factors and, thus, alter the availability, duration and impact of those mediators on the local molecular level. sGAG-coated polycaprolactone-co-lactide (PCL) scaffolds were inserted into critical-size femur defects in adult male Wistar rats. The femur was stabilized with a plate, and the defect was filled with either sGAG-containing PCL scaffolds or autologous bone (positive control). Wound fluid samples obtained by microdialysis were characterized regarding alterations of cytokine concentrations over the first 24 h after surgery. The analyses revealed the inhibition of the pro-inflammatory cytokines IL-1ß and MIP-2 in the sGAG-treated groups compared to the positive control. A simultaneous increase of IL-6 and TNF-α indicated advanced regenerative capacity of sGAG, suggesting their potential to improve bone healing.

Sulfated glycosaminoglycans inhibit transglutaminase 2 by stabilizing its closed conformation.

Transglutaminases (TGs) catalyze the covalent crosslinking of proteins via isopeptide bonds. The most prominent isoform, TG2, is associated with physiological processes such as extracellular matrix (ECM) stabilization and plays a crucial role in the pathogenesis of e.g. fibrotic diseases, cancer and celiac disease. Therefore, TG2 represents a pharmacological target of increasing relevance. The glycosaminoglycans (GAG) heparin (HE) and heparan sulfate (HS) constitute high-affinity interaction partners of TG2 in the ECM. Chemically modified GAG are promising molecules for pharmacological applications as their composition and chemical functionalization may be used to tackle the function of ECM molecular systems, which has been recently described for hyaluronan (HA) and chondroitin sulfate (CS). Herein, we investigate the recognition of GAG derivatives by TG2 using an enzyme-crosslinking activity assay in combination with in silico molecular modeling and docking techniques. The study reveals that GAG represent potent inhibitors of TG2 crosslinking activity and offers atom-detailed mechanistic insights.

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.

Identification of intracellular glycosaminoglycan-interacting proteins by affinity purification mass spectrometry.

Glycosaminoglycans (GAGs) are essential functional components of the extracellular matrix (ECM). Artificial GAGs like sulfated hyaluronan (sHA) exhibit pro-osteogenic properties and boost healing processes. Hence, they are of high interest for supporting bone regeneration and wound healing. Although sulfated GAGs (sGAGs) appear intracellularly, the knowledge about intracellular effects and putative interaction partners is scarce. Here we used an affinity-purification mass spectrometry-based (AP-MS) approach to identify novel and particularly intracellular sGAG-interacting proteins in human bone marrow stromal cells (hBMSC). Overall, 477 proteins were found interacting with at least one of four distinct sGAGs. Enrichment analysis for protein localization showed that mainly intracellular and cell-associated interacting proteins were identified. The interaction of sGAG with α2-macroglobulin receptor-associated protein (LRPAP1), exportin-1 (XPO1), and serine protease HTRA1 (HTRA1) was confirmed in reverse assays. Consecutive pathway and cluster analysis led to the identification of biological processes, namely processes involving binding and processing of nucleic acids, LRP1-dependent endocytosis, and exosome formation. Respecting the preferentially intracellular localization of sGAG in vesicle-like structures, also the interaction data indicate sGAG-specific modulation of vesicle-based transport processes. By identifying many sGAG-specific interacting proteins, our data provide a resource for upcoming studies aimed at molecular mechanisms and understanding of sGAG cellular effects.

Peptide-mediated surface coatings for the release of wound-healing cytokines.

Supporting the wound healing process by sending the appropriate cytokine signals can shorten healing time and overcome chronic inflammation syndromes. Even though adhesion peptides consisting of Arg-Gly-Asp (RGD) are commonly used to enhance cell-surface interactions, peptide-mediated cytokine delivery has not been widely exploited so far. Cytokines interact with high affinity with their cognitive receptors but also with sulfated glycosaminoglycans (GAGs), both of which form a base for incorporation of cytokines into functional biomaterials. Here, we report on a mussel-derived surface coating as a prospective cytokine delivery system using covalently bound heparin mimetics, receptor-derived chemokine-binding peptides, and heparin-binding peptides (HBP). The latter enabled non-covalent immobilization of heparin on the surface followed by chemokine binding and release, whereas the former allowed direct non-covalent chemokine immobilization. The peptide displayed excellent binding to custom-made polystyrene 96-well plates, enabling convenient testing of several compounds. Released chemokine successfully induced migration in Jurkat cells, especially for the non-covalent heparin immobilization approach using HBPs as evaluated in a transwell assay. In comparison, heparin-mimetic coatings, comprised of sulfated peptides and GAG derivatives, proved less efficient with respect to amount of immobilized chemokine and migratory response. Thus, our study provides a roadmap for further rational optimization and translation into clinics.

Biomimetic tissue models reveal the role of hyaluronan in melanoma proliferation and invasion.

Interactions of hyaluronan (HA) and tumor and stromal cells are highly discussed as one of the major contributors in tumor progression and metastasis. The balance of HA in the tissue is highly regulated by two key enzyme classes; hyaluronan synthases (HAS) and hyaluronidases (HYAL). Current reports hint that the HA amount in the tissue is correlated with poor prognosis in melanoma, the most life-threatening skin tumor. In this work, we generated in vivo mouse models with low and high expression of Has2 and used the models for studying melanoma proliferation of the B78D14 melanoma cell line. We found that a strong reduction of HA amount in the skin was correlated to decreased tissue stiffness and a reduction in tumor weight. Since tumor cells have a direct contact to the HA in the tumor and at the stroma interface, we reconstituted different biomimetic in vitro models using fibroblasts derived from a mouse model to recapitulate melanoma cell behavior at the tumor boundary, namely, (i) decellularized fibroblast matrix (FbECM), (ii) fibroblast embedded into 3D collagen matrices (FbColl), and (iii) well-defined HA-functionalized 3D collagen matrices (HAColl). We found no considerable effect of high and low amounts of fibroblast-derived HA in the matrices on melanoma proliferation and invasion. However, HYAL1-treated FbECM and FbColl, as well as HAColl functionalized with low molecular weight HA (34 kDa) promoted proliferation and invasion of melanoma cells in a concentration dependent manner. Our results emphasize the molecular weight specific effects of HA in regulation of melanoma behavior and provide an alternative explanation for the in vivo observation of HA dependent tumor growth.

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.

Glycosaminoglycans influence enzyme activity of MMP2 and MMP2/TIMP3 complex formation - Insights at cellular and molecular level.

The extracellular matrix (ECM) is a highly dynamic network constantly remodeled by a fine-tuned protein formation and degradation balance. Matrix metalloproteinases (MMPs) constitute key orchestrators of ECM degradation. Their activity is controlled by tissue inhibitors of metalloproteinases (TIMPs) and glycosaminoglycans (GAG). Here, we investigated the molecular interplay of MMP2 with different GAG (chondroitin sulfate, hyaluronan (HA), sulfated hyaluronan (SH) and heparin (HE)) and the impact of GAG on MMP2/TIMP3 complex formation using in vitro-experiments with human bone marrow stromal cells, in silico docking and molecular dynamics simulations. SH and HE influenced MMP2 and TIMP3 protein levels and MMP2 activity. Only SH supported the alignment of both proteins in fibrillar-like structures, which, based on our molecular models, would be due to a stabilization of the interactions between MMP2-hemopexin domain and TIMP3-C-terminal tail. Dependent on the temporal sequential order in which the final ternary complex was formed, our models indicated that SH and HA can affect TIMP3-induced MMP2 inhibition through precluding or supporting their interactions, respectively. Our combined experimental and theoretical approach provides valuable new insights on how GAG interfere with MMP2 activity and MMP2/TIMP3 complex formation. The results obtained evidence GAG as promising molecules for fine-balanced intervention of ECM remodeling.

Synergistic effect of bimodal pore distribution and artificial extracellular matrices in polymeric scaffolds on osteogenic differentiation of human mesenchymal stem cells.

The main objective of this study was to enhance the biological performance of resorbable polymeric scaffolds for bone tissue engineering. Specifically, we focused on both microstructure and surface modification of the scaffolds to augment adhesion, proliferation and osteogenic differentiation of human mesenchymal stem cells (hMSC). Moreover, a new cell seeding method assuring 90% seeding efficiency on the scaffolds was developed. Poly(l­lactide­co­glycolide) (PLGA) scaffolds with monomodal and bimodal pore distribution were produced by solvent casting/phase separation followed by porogen leaching and modified with artificial extracellular matrices (aECM) consisting of collagen type I and high sulphated hyaluronan (sHya). The application of two porogens resulted in bimodal pore distribution within the PLGA scaffolds as shown by scanning electron microscopy and microcomputer tomography. Two types of pores with diameters 400-600 µm and 2-20 µm were obtained. The scaffolds were successfully coated with a homogenous layer of aECM as shown by Sirius red and toluidine blue staining. In vitro study showed that presence of bimodal pore distribution in combination with collagen/sHya did not significantly influence hMSC proliferation and early osteogenic differentiation compared to scaffolds with monomodal pore distribution. However, it enhanced mineralization as well as the expression of Runt-related transcription factor 2, osteopontin and bone sialoprotein II. As a result PLGA scaffolds with bimodal pore distribution modified with collagen/sHya can be considered as prospective material promoting bone regeneration.

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.

Molecular weight specific impact of soluble and immobilized hyaluronan on CD44 expressing melanoma cells in 3D collagen matrices.

Hyaluronan (HA) and its principal receptor CD44 are known to be involved in regulating tumor cell dissemination and metastasis. The direct correlation of CD44-HA interaction on proliferation and invasion of tumor cells in dependence on the molecular weight and the presentation form of HA is not fully understood because of lack of appropriate matrix models. To address this issue, we reconstituted 3D collagen (Coll I) matrices and functionalized them with HA of molecular weight of 30-50kDa (low molecular weight; LMW-HA) and 500-750kDa (high molecular weight; HMW-HA). A post-modification strategy was applied to covalently immobilize HA to reconstituted fibrillar Coll I matrices, resulting in a non-altered Coll I network microstructure and stable immobilization over days. Functionalized Coll I matrices were characterized regarding topological and mechanical characteristics as well as HA amount using confocal laser scanning microscopy, colloidal probe force spectroscopy and quantitative Alcian blue assay, respectively. To elucidate HA dependent tumor cell behavior, BRO melanoma cell lines with and without CD44 receptor expression were used for in vitro cell experiments. We demonstrated that only soluble LMW-HA promoted cell proliferation in a CD44 dependent manner, while HMW-HA and immobilized LMW-HA did not. Furthermore, an enhanced cell invasion was found only for immobilized LMW-HA. Both findings correlated with a very strong and specific adhesive interaction of LMW-HA and CD44+ cells quantified in single cell adhesion measurements using soft colloidal force spectroscopy. Overall, our results introduce an in vitro biomaterials model allowing to test presentation mode and molecular weight specificity of HA in a 3D fibrillar matrix thus mimicking important in vivo features of tumor microenvironments. STATEMENT OF SIGNIFICANCE: Molecular weight and presentation form (bound vs. soluble) of hyaluronan (HA) are intensively discussed as key regulators in tumor progression and inflammation. We introduce 3D fibrillar collagen matrices with defined microstructure and stiffness allowing the presentation of specific molecular weight forms of HA in soluble and bound manner. Mimicking in that way important in vivo features of tumor microenvironments, we found that only low molecular weight HA (LMW-HA) in soluble form promoted proliferation of a melanoma cell line (BRO), while it enhanced cell invasion in bound form. The molecular weight specificity of LMW-HA was verified to be CD44 receptor dependent and was correlated to adhesive ligand-receptor interactions in quantitative colloidal force spectroscopy at single cell level.

Collagen/glycosaminoglycan coatings enhance new bone formation in a critical size bone defect - A pilot study in rats.

Bone regeneration in critical size bone defects still represents an important but unsolved clinical problem. Glycosaminoglycans (GAGs) like chondroitin sulfate (CS) or hyaluronan (HA) are important multifunctional components of the extracellular matrix (ECM) in bone and may stimulate bone healing by recruitment of mesenchymal stromal cells and by supporting their differentiation. Sulfation of GAGs affects their biological activity and thus their interactions with growth factors and/or cells involved in the bone healing process. The aim of this pilot study was to evaluate the osteogenic capacity of chemically high-sulfated chondroitin sulfate (sCS3) and hyaluronan (sHA3) with an average degree of sulfation DS≈3 on bone healing. Titanium-coated polyetheretherketone (Ti-PEEK) plates were coated with collagen type I (col), collagen-based artificial ECMs containing CS or HA and compared to col/sCS3 and col/sHA3 coatings bridging a critical size bone defect in rat femur. After 4weeks the gap size of 5.1mm±0.1mm following surgery was significantly reduced to 1.4mm±0.9mm for col/sHA3 and to 0.9mm±0.7mm for col/CS. The highest amount of newly formed bone was detected for col/CS (79%±30%) and col/sHA3 (36%±20%) compared to uncoated plates (13%±3%) or col-coated plates (18%±16%). Enchondral ossification could be confirmed for col/CS, col/HA, and col/sHA3 by positive staining for Alcian blue and collagen type II. These results suggest that an artificial ECM has osteogenic effects and is able to enhance bone healing in critical situations.

Sulfated hyaluronan alters fibronectin matrix assembly and promotes osteogenic differentiation of human bone marrow stromal cells.

Extracellular matrix (ECM) composition and structural integrity is one of many factors that influence cellular differentiation. Fibronectin (FN) which is in many tissues the most abundant ECM protein forms a unique fibrillary network. FN homes several binding sites for sulfated glycosaminoglycans (sGAG), such as heparin (Hep), which was previously shown to influence FN conformation and protein binding. Synthetically sulfated hyaluronan derivatives (sHA) can serve as model molecules with a well characterized sulfation pattern to study sGAG-FN interaction. Here is shown that the low-sulfated sHA (sHA1) interacts with FN and influences fibril assembly. The interaction of FN fibrils with sHA1 and Hep, but not with non-sulfated HA was visualized by immunofluorescent co-staining. FRET analysis of FN confirmed the presence of more extended fibrils in human bone marrow stromal cells (hBMSC)-derived ECM in response to sHA1 and Hep. Although both sHA1 and Hep affected FN conformation, exclusively sHA1 increased FN protein level and led to thinner fibrils. Further, only sHA1 had a pro-osteogenic effect and enhanced the activity of tissue non-specific alkaline phosphatase. We hypothesize that the sHA1-triggered change in FN assembly influences the entire ECM network and could be the underlying mechanism for the pro-osteogenic effect of sHA1 on hBMSC.

Short-range cytokine gradients to mimic paracrine cell interactions in vitro.

Cell fate decisions in many physiological processes, including embryogenesis, stem cell niche homeostasis and wound healing, are regulated by secretion of small signaling proteins, called cytokines, from source cells to their neighbors or into the environment. Concentration level and steepness of the resulting paracrine gradients elicit different cell responses, including proliferation, differentiation or chemotaxis. For an in-depth analysis of underlying mechanisms, in vitro models are required to mimic in vivo cytokine gradients. We set up a microparticle-based system to establish short-range cytokine gradients in a three-dimensional extracellular matrix context. To provide native binding sites for cytokines, agarose microparticles were functionalized with different glycosaminoglycans (GAG). After protein was loaded onto microparticles, its slow release was quantified by confocal microscopy and fluorescence correlation spectroscopy. Besides the model protein lysozyme, SDF-1 was used as a relevant chemokine for hematopoietic stem and progenitor cell (HSPC) chemotaxis. For both proteins we found gradients ranging up to 50µm from the microparticle surface and concentrations in the order of nM to pM in dependence on loading concentration and affinity modulation by the GAG functionalization. Directed chemotactic migration of cells from a hematopoietic cell line (FDCPmix) and primary murine HSPC (Sca-1(+) CD150(+) CD48(-)) toward the SDF-1-laden microparticles proved functional short-range gradients in a two-dimensional and three-dimensional setting over time periods of many hours. The approach has the potential to be applied to other cytokines mimicking paracrine cell-cell interactions in vitro.

Artificial extracellular matrices with oversulfated glycosaminoglycan derivatives promote the differentiation of osteoblast-precursor cells and premature osteoblasts.

Sulfated glycosaminoglycans (GAG) are components of the bone marrow stem cell niche and to a minor extent of mature bone tissue with important functions in regulating stem cell lineage commitment and differentiation. We anticipated that artificial extracellular matrices (aECM) composed of collagen I and synthetically oversulfated GAG derivatives affect preferentially the differentiation of osteoblast-precursor cells and early osteoblasts. A set of gradually sulfated chondroitin sulfate and hyaluronan derivatives was used for the preparation of aECM. All these matrices were analysed with human bone marrow stromal cells to identify the most potent aECM and to determine the influence of the degree and position of sulfate groups and the kind of disaccharide units on the osteogenic differentiation. Oversulfated GAG derivatives with a sulfate group at the C-6 position of the N-acetylglycosamine revealed the most pronounced proosteogenic effect as determined by tissue nonspecific alkaline phosphatase activity and calcium deposition. A subset of the aECM was further analysed with different primary osteoblasts and cell lines reflecting different maturation stages to test whether the effect of sulfated GAG derivatives depends on the maturation status of the cells. It was shown that the proosteogenic effect of aECM was most prominent in early osteoblasts.

Artificial matrices with high-sulfated glycosaminoglycans and collagen are anti-inflammatory and pro-osteogenic for human mesenchymal stromal cells.

Bone healing has been described to be most efficient if the early inflammatory phase is resolved timely. When the inflammation elevates or is permanently established, bone healing becomes impaired and, moreover, bone destruction often takes place. Systemic disorders such as diabetes and bone diseases like arthritis and osteoporosis are associated with sustained inflammation and delayed bone healing. One goal of biomaterial research is the development of materials/surface modifications which support the healing process by inhibiting the inflammatory bone erosion and suppressing pro-inflammatory mediators and by that promoting the bone repair process. In the present study, the influence of artificial extracellular matrices (aECM) on the interleukin (IL)-1ß-induced pro-inflammatory response of human mesenchymal stromal cells (hMSC) was studied. hMSC cultured on aECM composed of collagen I and high-sulfated glycosaminoglycan (GAG) derivatives did not secrete IL-6, IL-8, monocyte chemoattractant protein-1, and prostaglandin E2 in response to IL-1ß. The activation and nuclear translocation of nuclear factor κBp65 induced by IL-1ß, tumor necrosis factor-α or lipopolysaccharide was abrogated. Furthermore, these aECM promoted the osteogenic differentiation of hMSC as determined by an increased activity of tissue non-specific alkaline phosphatase (TNAP); however, the aECM had no effect on the IL-1ß-induced TNAP activity. These data suggest that aECM with high-sulfated GAG derivatives suppress the formation of pro-inflammatory mediators and simultaneously promote the osteogenic differentiation of hMSC. Therefore, these aECM might offer an interesting approach as material/surface modification supporting the bone healing process.

Sulfated hyaluronan containing collagen matrices enhance cell-matrix-interaction, endocytosis, and osteogenic differentiation of human mesenchymal stromal cells.

Inorganic-organic composite implant materials mimicking the environment of bone are promising applications to meet the increasing demands on biomaterials for bone regeneration caused by extended life spans and the concomitant increase of bone treatments. Besides collagen type I (Col-I) glycosaminoglycans (GAG), such as hyaluronan, are important components of the bone extracellular matrix (ECM). Sulfated GAGs are potential stimulators of bone anabolic activity, as they are involved in the recruitment of mesenchymal stromal cells (MSCs) to the site of bone formation and support differentiation to osteoblasts. Nevertheless, no consecutive data is currently available about the interaction of hyaluronan or sulfated hyaluronan derivatives with hMSCs and the molecular processes being consequently regulated. We applied quantitative proteomics to investigate the influence of artificial ECM composed of Col-I and hyaluronan (Hya) or sulfated hyaluronan (HyaS3) on the molecular adaptation of osteogenic-differentiated human MSCs (hMSCs). Of the 1,370 quantified proteins, the expression of 4-11% was altered due to both aECM-combinations. Our results indicate that HyaS3 enhanced multiple cell functions, including cell-matrix-interaction, cell-signaling, endocytosis, and differentiation. In conclusion, this study provides fundamental insights into regulative cellular responses associated with HyaS3 and Hya as components of aECM and underlines the potential of HyaS3 as a promising implant-coating-material.

Over-sulfated chondroitin sulfate derivatives induce osteogenic differentiation of hMSC independent of BMP-2 and TGF-ß1 signalling.

Natural glycosaminoglycans (GAGs) and chemically modified GAG derivatives are known to support osteogenic differentiation of mesenchymal stromal cells (MSC). This effect has mainly been described to be mediated by increasing the effectiveness of bone anabolic growth factors such as bone morphogenetic proteins (BMPs) due to the binding and presentation of the growth factor or by modulating its signal transduction pathway. In the present study, the influence of chondroitin sulfate (CS) and two chemically over-sulfated CS derivatives on osteogenic differentiation of human mesenchymal stromal cells (hMSC) and on BMP-2 and transforming growth factor ß1 (TGF-ß1) signalling was investigated. Over-sulfated CS derivatives induced an increase of tissue non-specific alkaline phosphatase (TNAP) activity and calcium deposition, whereas collagen synthesis was slightly decreased. The BMP-2-induced Smad1/5 activation was inhibited in the presence of over-sulfated CS derivatives leading to a loss of BMP-2-induced TNAP activity and calcium deposition. In contrast, the TGF-ß1-induced activation of Smad2/3 and collagen synthesis were not affected by the over-sulfated CS derivatives. BMP-2 and TGF-ß1 did not activate the extracellular signal-regulated kinase 1/2 or mitogen-activated protein kinase p38 in hMSC. These data suggest that over-sulfated CS derivatives themselves are able to induce osteogenic differentiation, probably independent of BMP-2 and TGF-ß1 signalling, and offer therefore an interesting approach for the improvement of bone healing.

Artificial extracellular matrices composed of collagen I and high-sulfated hyaluronan promote phenotypic and functional modulation of human pro-inflammatory M1 macrophages.

The sequential phases of biomaterial integration and wound healing require different macrophage functions mediated by distinct macrophage subsets. During the initial phase of healing, pro-inflammatory M1 macrophages (MΦ1) are required to clear the wound from microbes and debris; however, their unopposed, persistent activation often leads to disturbed integration of biomaterials and perturbed wound healing. Here we investigated whether pro-inflammatory macrophage functions are affected by immunomodulatory biomaterials based on artificial extracellular matrices (aECM). To address this issue, we tested the capacity of two-dimensional aECM consisting of collagen I and hyaluronan or sulfated derivatives of hyaluronan to affect functions of in vitro polarized human pro-inflammatory MΦ1. The aECM containing high-sulfated hyaluronan substantially decreased inflammatory macrophage functions, including pathogen uptake and release of the pro-inflammatory cytokines tumor necrosis factor alpha and interleukin-12 due to impaired activation of nuclear factor "kappa-light-chain-enhancer" of activated B-cells. Moreover, these macrophages secreted immunregulatory IL-10 and showed reduced activity of the transcription factors signal transducer and activator of transcription 1 and interferon-regulating factor 5, both controlling macrophage polarization to MΦ1 subsets. Our data reveal that the collagen I matrix containing high-sulfated hyaluronan possesses immunomodulating properties and dampens inflammatory macrophage activities by impeding signaling pathways crucial for polarization of pro-inflammatory MΦ1. We therefore suggest this aECM as a promising coating for biomaterials to modulate inflammatory macrophage functions during the healing response and recommend its further testing as a three-dimensional construct and in in vivo models.

Artificial extracellular matrices composed of collagen I and sulfated hyaluronan with adsorbed transforming growth factor ß1 promote collagen synthesis of human mesenchymal stromal cells.

Sulfated glycosaminoglycans (GAG) are multifunctional components of the extracellular matrix and are involved in the regulation of adhesion, proliferation and differentiation of cells. The effects of GAG are mediated in general by their interactions with cations and water, and in particular by their binding to growth factors. The aim of this study was to generate artificial extracellular matrices (aECM) containing collagen I and hyaluronan sulfate (HyaS), which are capable of adsorbing and releasing transforming growth factor ß1 (TGF-ß1), and to promote collagen synthesis of cultured human mesenchymal stromal cells (hMSC). For the preparation of aECM, monosulfated Hya (HyaS1) or trisulfated Hya (HyaS3) were used; the natural chondroitin-4-sulfate was used as a control. As applied for the in vitro experiments, the resulting matrices were composed of 93-98% collagen I and 2-7% GAG derivative. Adsorption of TGF-ß1 to the aECM and release from the aECM was dependent on the degree of sulfation of hyaluronan. Collagen synthesis of hMSC was promoted only by aECM with adsorbed TGF-ß1; the bare aECM had a slightly inhibitory effect on collagen synthesis. The promoting effect did not correlate either to the amount of adsorbed TGF-ß1 nor to the release of TGF-ß1, indicating that the correct presentation of TGF-ß1 to the cells might be critical. The results indicate that sulfated hyaluronan-containing aECM have the potential to control both the adsorption and release of TGF-ß1, and thereby promote collagen synthesis of hMSC. Thus, these aECM might be a useful tool for different tissue-engineering applications to enhance bone formation when used for biomaterial coating.