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.

The interplay of collagen/bioactive glass nanoparticle coatings and electrical stimulation regimes distinctly enhanced osteogenic differentiation of human mesenchymal stem cells.

Increasing research has incorporated bioactive glass nanoparticles (BGN) and electric field (EF) stimulation for bone tissue engineering and regeneration applications. However, their interplay and the effects of different EF stimulation regimes on osteogenic differentiation of human mesenchymal stem cells (hMSC) are less investigated. In this study, we introduced EF with negligible magnetic field strength through a well-characterized transformer-like coupling (TLC) system, and applied EF disrupted (4/4) or consecutive (12/12) regime on type I collagen (Col) coatings with/without BGN over 28 days. Additionally, dexamethasone was excluded to enable an accurate interpretation of BGN and EF in supporting osteogenic differentiation. Here, we demonstrated the influences of BGN and EF on collagen topography and maintaining coating stability. Coupled with the release profile of Si ions from the BGN, cell proliferation and calcium deposition were enhanced in the Col-BGN samples after 28 days. Further, osteogenic differentiation was initiated as early as d 7, and each EF regime was shown to activate distinct pathways. The disrupted (4/4) regime was associated with the BMP/Smad4 pathways that up-regulate Runx2/OCN gene expression on d 7, with a lesser effect on ALP activity. In contrast, the canonical Wnt/ß-Catenin signaling pathway activated through mechanotransduction cues is associated with the consecutive (12/12) regime, with significantly elevated ALP activity and Sp7 gene expression reported on d 7. In summary, our results illustrated the synergistic effects of BGN and EF in different stimulation regimes on osteogenic differentiation that can be further exploited to enhance current bone tissue engineering and regeneration approaches. STATEMENT OF SIGNIFICANCE: The unique release mechanisms of silica from bioactive glass nanoparticles (BGN) were coupled with pulsatile electric field (EF) stimulation to support hMSC osteogenic differentiation, in the absence of dexamethasone. Furthermore, the interplay with consecutive (12/12) and disrupted (4/4) stimulation regimes was investigated. The reported physical, mechanical and topographical effects of BGN and EF on the collagen coating, hMSC and the distinct progression of osteogenic differentiation (canonical Wnt/ß-Catenin and BMP/Smad) triggered by respective stimulation regime were not explicitly reported previously. These results provide the fundamentals for further exploitations on BGN composites with metal ions and rotation of EF regimes to enhance osteogenic differentiation. The goal is sustaining continual osteogenic differentiation and achieving a more physiologically-relevant state and bone constructs in vitro.

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.

Structural insights into the modulation of PDGF/PDGFR-ß complexation by hyaluronan derivatives.

Angiogenesis is an important physiological process playing a crucial role in wound healing and cancer progression. Vascular endothelial growth factor (VEGF) and platelet derived growth factor (PDGF) are key players in angiogenesis. Based on previous findings regarding the modulation of VEGF activity by glycosaminoglycans (GAG), here we explore the interaction of hyaluronan (HA)-based GAG with PDGF and its receptor PDGFR-ß by applying molecular modeling and dynamics simulations in combination with surface plasmon resonance (SPR). Computational analysis on the interaction of oligo-hyaluronan derivatives with different sulfation pattern and functionalization shows that these GAG interact with PDGF in relevant regions for receptor recognition, and that high sulfation as well as modification with the TAMRA group convey stronger binding. On the other hand, the studied oligo-hyaluronan derivatives are predicted to scarcely recognize PDGFR-ß. SPR results are in line with the computational predictions regarding the binding pattern of HA tetrasaccharide (HA4) derivatives to PDGF and PDGFR-ß. Furthermore, our experimental results also show that the complexation of PDGF to PDGFR-ß can be modulated by HA4 derivatives. The results found open the path for considering HA4 derivatives as potential candidates to be exploited for modulation of the PDGF/PDGFR-ß signaling system in angiogenesis and related disease conditions.

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.

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.

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.

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.

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 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.

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 glycosaminoglycans support osteoblast functions and concurrently suppress osteoclasts.

In order to improve bone regeneration, development and evaluation of new adaptive biomaterials is warranted. Glycosaminoglycans (GAGs) such as hyaluronan (HA) and chondroitin sulfate (CS) are major extracellular matrix (ECM) components of bone, and display osteogenic properties that are potentially useful for biomaterial applications. Using native and synthetic sulfate-modified GAGs, we manufactured artificial collagen/GAG ECM (aECMs) coatings, and evaluated how the presence of GAGs and their degree of sulfation affects the differentiation of murine mesenchymal stem cells to osteoblasts (OB) cultivated on these aECMs. GAG sulfation regulated osteogenesis at all key steps of OB development. Adhesion, but not migration, was diminished by 50% (P < 0.001). Proliferation and metabolic activity were slightly (P < 0.05) and cell death events strongly (P < 0.001) down-regulated due to a switch from proliferative to matrix synthesis state. When exposed to sulfated GAGs, OB marker genes, such as alkaline phosphatase, osteoprotegerin (OPG), and osteocalcin increased by up to 28-fold (P < 0.05) and calcium deposition up to 4-fold (P < 0.05). Furthermore, GAG treatment of OBs suppressed their ability to support osteoclast (OC) differentiation and resorption. In conclusion, GAG sulfation controls bone cell homeostasis by concurrently promoting osteogenesis and suppressing their paracrine support of OC functions, thus displaying a favorable profile on bone remodeling. Whether these cellular properties translate into improved bone regeneration needs to be validated in vivo.

The promotion of osteoclastogenesis by sulfated hyaluronan through interference with osteoprotegerin and receptor activator of NF-κB ligand/osteoprotegerin complex formation.

In order to improve bone regeneration, in particular in aged and multimorbid patients, the development of new adaptive biomaterials and their characterization in terms of their impact on bone biology is warranted. Glycosaminoglycans (GAGs) such as hyaluronan (HA) are major extracellular matrix (ECM) components in bone and may display osteogenic properties that are potentially useful for biomaterial coatings. Using native and synthetically derived sulfate-modified HA, we evaluated how GAG sulfation modulates the activity of two main regulators of osteoclast function: receptor activator of NF-κB ligand (RANKL) and osteoprotegerin (OPG). GAGs were tested for their capability to bind to OPG and RANKL using surface plasmon resonance (SPR), ELISA and molecular modeling techniques. Results were validated in an in vitro model of osteoclastogenesis. Sulfated GAGs bound OPG but not RANKL in a sulfate-dependent manner. Furthermore, OPG pre-incubated with different GAGs displayed a sulfate- and dose-dependent loss in bioactivity, possibly due to competition of GAGs for the RANKL/OPG binding site revealing a potential GAG interaction site at the RANKL/OPG interface. In conclusion, high-sulfated GAGs might significantly control osteoclastogenesis via interference with the physiological RANKL/OPG complex formation. Whether these properties can be utilized to improve bone regeneration and fracture healing needs to be validated in vivo.

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.

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.