Response of Osteoblasts on Amine-Based Nanocoatings Correlates with the Amino Group Density.

Increased life expectancy in industrialized countries is causing an increased incidence of osteoporosis and the need for bioactive bone implants. The integration of implants can be improved physically, but mainly by chemical modifications of the material surface. It was recognized that amino-group-containing coatings improved cell attachment and intracellular signaling. The aim of this study was to determine the role of the amino group density in this positive cell behavior by developing controlled amino-rich nanolayers. This work used covalent grafting of polymer-based nanocoatings with different amino group densities. Titanium coated with the positively-charged trimethoxysilylpropyl modified poly(ethyleneimine) (Ti-TMS-PEI), which mostly improved cell area after 30 min, possessed the highest amino group density with an N/C of 32%. Interestingly, changes in adhesion-related genes on Ti-TMS-PEI could be seen after 4 h. The mRNA microarray data showed a premature transition of the MG-63 cells into the beginning differentiation phase after 24 h indicating Ti-TMS-PEI as a supportive factor for osseointegration. This amino-rich nanolayer also induced higher bovine serum albumin protein adsorption and caused the cells to migrate slower on the surface after a more extended period of cell settlement as an indication of a better surface anchorage. In conclusion, the cell spreading on amine-based nanocoatings correlated well with the amino group density (N/C).

Collagen/glycosaminoglycan-based matrices for controlling skin cell responses.

Wound healing and tissue regeneration are orchestrated by the cellular microenvironment, e.g. the extracellular matrix (ECM). Including ECM components in biomaterials is a promising approach for improving regenerative processes, e.g. wound healing in skin. This review addresses recent findings for enhanced epidermal-dermal regenerative processes on collagen (coll)/glycosaminoglycan (GAG)-based matrices containing sulfated GAG (sGAG) in simple and complex in vitro models. These matrices comprise 2D-coatings, electrospun nanofibrous scaffolds, and photo-crosslinked acrylated hyaluronan (HA-AC)/coll-based hydrogels. They demonstrated to regulate keratinocyte and fibroblast migration and growth, to stimulate melanogenesis in melanocytes from the outer root sheath (ORS) of hair follicles and to enhance the epithelial differentiation of human mesenchymal stem cells (hMSC). The matrices' suitability for delivery of relevant growth factors, like heparin-binding epidermal growth factor like growth factor (HB-EGF), further highlights their potential as bioinspired, functional microenvironments for enhancing skin regeneration.

Chemically modified glycosaminoglycan derivatives as building blocks for biomaterial coatings and hydrogels.

Tissue regeneration is regulated by the cellular microenvironment, e.g. the extracellular matrix. Here, sulfated glycosaminoglycans (GAG), are of vital importance interacting with mediator proteins and influencing their biological activity. Hence, they are promising candidates for controlling tissue regeneration. This review addresses recent achievements regarding chemically modified GAG as well as collagen/GAG-based coatings and hydrogels including (i) chemical functionalization strategies for native GAG, (ii) GAG-based biomaterial strategies for controlling cellular responses, (iii) (bio)chemical methods for characterization and iv) protein interaction profiles and attained tissue regeneration in vitro and in vivo. The potential of GAG for bioinspired, functional biomaterials is highlighted.

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.

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.

Glycosaminoglycan-Based Cryogels as Scaffolds for Cell Cultivation and Tissue Regeneration.

Cryogels are a class of macroporous, interconnective hydrogels polymerized at sub-zero temperatures forming mechanically robust, elastic networks. In this review, latest advances of cryogels containing mainly glycosaminoglycans (GAGs) or composites of GAGs and other natural or synthetic polymers are presented. Cryogels produced in this way correspond to the native extracellular matrix (ECM) in terms of both composition and molecular structure. Due to their specific structural feature and in addition to an excellent biocompatibility, GAG-based cryogels have several advantages over traditional GAG-hydrogels. This includes macroporous, interconnective pore structure, robust, elastic, and shape-memory-like mechanical behavior, as well as injectability for many GAG-based cryogels. After addressing the cryogelation process, the fabrication of GAG-based cryogels and known principles of GAG monomer crosslinking are discussed. Finally, an overview of specific GAG-based cryogels in biomedicine, mainly as polymeric scaffold material in tissue regeneration and tissue engineering-related controlled release of bioactive molecules and cells, is provided.

A Novel Resorbable Composite Material Containing Poly(ester-co-urethane) and Precipitated Calcium Carbonate Spherulites for Bone Augmentation-Development and Preclinical Pilot Trials.

Polyurethanes have the potential to impart cell-relevant properties like excellent biocompatibility, high and interconnecting porosity and controlled degradability into biomaterials in a relatively simple way. In this context, a biodegradable composite material made of an isocyanate-terminated co-oligoester prepolymer and precipitated calcium carbonated spherulites (up to 60% w/w) was synthesized and investigated with regard to an application as bone substitute in dental and orthodontic application. After foaming the composite material, a predominantly interconnecting porous structure is obtained, which can be easily machined. The compressive strength of the foamed composites increases with raising calcium carbonate content and decreasing calcium carbonate particle size. When stored in an aqueous medium, there is a decrease in pressure stability of the composite, but this decrease is smaller the higher the proportion of the calcium carbonate component is. In vitro cytocompatibility studies of the foamed composites on MC3T3-E1 pre-osteoblasts revealed an excellent cytocompatibility. The in vitro degradation behaviour of foamed composite is characterised by a continuous loss of mass, which is slower with higher calcium carbonate contents. In a first pre-clinical pilot trial the foamed composite bone substitute material (fcm) was successfully evaluated in a model of vertical augmentation in an established animal model on the calvaria and on the lateral mandible of pigs.

Fabrication and Plasma Surface Activation of Aligned Electrospun PLGA Fiber Fleeces with Improved Adhesion and Infiltration of Amniotic Epithelial Stem Cells Maintaining their Teno-inductive Potential.

Electrospun PLGA microfibers with adequate intrinsic physical features (fiber alignment and diameter) have been shown to boost teno-differentiation and may represent a promising solution for tendon tissue engineering. However, the hydrophobic properties of PLGA may be adjusted through specific treatments to improve cell biodisponibility. In this study, electrospun PLGA with highly aligned microfibers were cold atmospheric plasma (CAP)-treated by varying the treatment exposure time (30, 60, and 90 s) and the working distance (1.3 and 1.7 cm) and characterized by their physicochemical, mechanical and bioactive properties on ovine amniotic epithelial cells (oAECs). CAP improved the hydrophilic properties of the treated materials due to the incorporation of new oxygen polar functionalities on the microfibers' surface especially when increasing treatment exposure time and lowering working distance. The mechanical properties, though, were affected by the treatment exposure time where the optimum performance was obtained after 60 s. Furthermore, CAP treatment did not alter oAECs' biocompatibility and improved cell adhesion and infiltration onto the microfibers especially those treated from a distance of 1.3 cm. Moreover, teno-inductive potential of highly aligned PLGA electrospun microfibers was maintained. Indeed, cells cultured onto the untreated and CAP treated microfibers differentiated towards the tenogenic lineage expressing tenomodulin, a mature tendon marker, in their cytoplasm. In conclusion, CAP treatment on PLGA microfibers conducted at 1.3 cm working distance represent the optimum conditions to activate PLGA surface by improving their hydrophilicity and cell bio-responsiveness. Since for tendon tissue engineering purposes, both high cell adhesion and mechanical parameters are crucial, PLGA treated for 60 s at 1.3 cm was identified as the optimal construct.

Hyaluronan/collagen hydrogel matrices containing high-sulfated hyaluronan microgels for regulating transforming growth factor-ß1.

Hyaluronan (HA)-based microgels generated in a microfluidic approach, containing an artificial extracellular matrix composed of collagen and high-sulfated hyaluronan (sHA3), were incorporated into a HA/collagen-based hydrogel matrix. This significantly enhanced the retention of noncrosslinked sHA3 within the gels enabling controlled sHA3 presentation. Gels containing sHA3 bound higher amounts of transforming growth factor-ß1 (TGF-ß1) compared to pure HA/collagen hydrogels. Moreover, the presence of sHA3-containing microgels improved the TGF-ß1 retention within the hydrogels. These findings are promising for developing innovative biomaterials with adjustable sHA3 release and growth factor interaction profiles to foster skin repair, e.g., by rebalancing dysregulated TGF-ß1 levels.

The effect of interleukin-8 truncations on its interactions with glycosaminoglycans.

The chemokine interleukin-8 (IL-8, CXCL8) plays an important role in inflammatory processes and consecutive wound healing. It recruits primarily neutrophils to infection sites and stimulates their degranulation and phagocytosis in effector cells. IL-8 binds glycosaminoglycans (GAGs), a class of complex linear anionic polysaccharides often organized into diversely sulfated micro-domains, that enriches the protein concentration locally and so facilitate the formation of stable concentration gradients. In this study, we applied experimental and computational techniques to investigate the binding of wild type and truncated IL-8 variants to natural and chemically modified GAGs to gain further insight into the IL-8/GAG interaction. Circular dichroism spectroscopy of IL-8 variants did not reveal major structural changes upon GAG binding. Heparin affinity chromatography clearly demonstrates that gradual truncation of the C-terminal helix leads to decreasing affinities. Similarly, surface plasmon resonance indicates participation of both IL-8 termini in GAG binding, which strength is dependent on GAG sulfation degree. Molecular modeling suggests that C-terminal truncation of IL-8 weakens the interaction with GAGs by an alteration of IL-8 GAG binding site. Our study provides more detailed understanding of the IL-8/GAG interaction and contributes to the data of potential use for the development of biomedical implications in tissue regeneration.

Binding Kinetics and Lateral Mobility of HSV-1 on End-Grafted Sulfated Glycosaminoglycans.

Many viruses, including herpes simplex (HSV), are recruited to their host cells via interaction between their envelope glycoproteins and cell-surface glycosaminoglycans (GAGs). This initial attachment is of a multivalent nature, i.e., it requires the establishment of multiple bonds between amino acids of viral glycoproteins and sulfated saccharides on the GAG chain. To gain understanding of how this binding process is modulated, we performed binding kinetics and mobility studies using end-grafted GAG chains that mimic the end attachment of these chains to proteoglycans. Total internal reflection fluorescence microscopy was used to probe binding and release, as well as the diffusion of single HSV-1 particles. To verify the hypothesis that the degree of sulfation, but also the arrangement of sulfate groups along the GAG chain, plays a key role in HSV binding, we tested two native GAGs (chondroitin sulfate and heparan sulfate) and compared our results to chemically sulfated hyaluronan. HSV-1 recognized all sulfated GAGs, but not the nonsulfated hyaluronan, indicating that binding is specific to the presence of sulfate groups. Furthermore we observed that a notable fraction of GAG-bound virions exhibit lateral mobility, although the multivalent binding to the immobilized GAG brushes ensures firm virus attachment to the interface. Diffusion was faster on the two native GAGs, one of which, chondroitin sulfate, was also characterized by the highest association rate per GAG chain. This highlights the complexity of multivalent virus-GAG interactions and suggests that the spatial arrangement of sulfates along native GAG chains may play a role in modulating the characteristics of the HSV-GAG interaction. Altogether, these results, obtained with a minimal and well-controlled model of the cell membrane, provide, to our knowledge, new insights into the dynamics of the HSV-GAG interaction.

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.

Embryonic Stem Cells for Tissue Biocompatibility, Angiogenesis, and Inflammation Testing.

AIM: To introduce embryoid bodies derived from mouse embryonic stem (ES) cells, which differentiate blood vessel-like structures and leukocytes, as a novel in vitro model system for biocompatibility, inflammation, and angiogenesis studies. METHODOLOGY/RESULTS: Punched spherical discs of bioabsorbable polymers (ε-caprolactone and L-lactide in different compositions) with a diameter of 2 mm and a thickness of 0.2 mm were inoculated with embryoid bodies for cocultivation. As reference material for biocompatible, nonbioabsorbable, and bioincompatible materials, polymer punched discs of petriPERM (PP) membrane (polytetrafluoroethylene) as well as polyvinylchloride (PVC) were used. Tissue outgrowth on the polymer discs decreased and cell toxicity increased upon confrontation on bioabsorbable biomaterials and PVC. Bioabsorbable polymers as well as PVC decreased the branching points and total tube length of CD31-positive vascular structures in embryoid bodies. With the exception of PP, all applied materials increased the differentiation of CD68-positive macrophages and the generation of reactive oxygen species, which is indicative of proinflammatory processes upon contact of tissue with biomaterials. Consequently, cocultivation with polymers increased secretion of the cytokines interleukin-6, monocyte chemotactic protein-1, and tumor necrosis factor-α. CONCLUSION: Three-dimensional tissues cultivated from ES cells are well-suited for testing the biocompatibility, the vascular response, and the inflammatory reaction towards bioabsorbable and nonbioabsorbable polymers.

Dual-Functional Hydrazide-Reactive and Anhydride-Containing Oligomeric Hydrogel Building Blocks.

Biomimetic hydrogels are advanced biomaterials that have been developed following different synthetic routes. Covalent postfabrication functionalization is a promising strategy to achieve efficient matrix modification decoupled of general material properties. To this end, dual-functional macromers were synthesized by free radical polymerization of maleic anhydride with diacetone acrylamide (N-(1,1-dimethyl-3-oxobutyl)acrylamide) and pentaerythritol diacrylate monostearate. Amphiphilic oligomers (Mn < 7.5 kDa) with anhydride contents of 7-20% offered cross-linking reactivity to yield rigid hydrogels with gelatinous peptides (E = 4-13 kPa) and good cell adhesion properties. Mildly reactive methyl ketones as second functionality remained intact during hydrogel formation and potential of covalent matrix modification was shown using hydrazide and hydrazine model compounds. Successful secondary dihydrazide cross-linking was demonstrated by an increase of hydrogel stiffness (>40%). Efficient hydrazide/hydrazine immobilization depending on solution pH, hydrogel ketone content as well as ligand concentration for bioconjugation was shown and reversibility of hydrazone formation was indicated by physiologically relevant hydrazide release over 7 days. Proof-of-concept experiments with hydrazido-functionalized hyaluronan demonstrated potential for covalent aECM immobilization. The presented dual-functional macromers have perspective as reactive hydrogel building blocks for various biomedical applications.

Reciprocal influence of hMSCs/HaCaT cultivated on electrospun scaffolds.

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

Mucin-like Region of Herpes Simplex Virus Type 1 Attachment Protein Glycoprotein C (gC) Modulates the Virus-Glycosaminoglycan Interaction.

Glycoprotein C (gC) mediates the attachment of HSV-1 to susceptible host cells by interacting with glycosaminoglycans (GAGs) on the cell surface. gC contains a mucin-like region located near the GAG-binding site, which may affect the binding activity. Here, we address this issue by studying a HSV-1 mutant lacking the mucin-like domain in gC and the corresponding purified mutant protein (gCΔmuc) in cell culture and GAG-binding assays, respectively. The mutant virus exhibited two functional alterations as compared with native HSV-1 (i.e. decreased sensitivity to GAG-based inhibitors of virus attachment to cells and reduced release of viral particles from the surface of infected cells). Kinetic and equilibrium binding characteristics of purified gC were assessed using surface plasmon resonance-based sensing together with a surface platform consisting of end-on immobilized GAGs. Both native gC and gCΔmuc bound via the expected binding region to chondroitin sulfate and sulfated hyaluronan but not to the non-sulfated hyaluronan, confirming binding specificity. In contrast to native gC, gCΔmuc exhibited a decreased affinity for GAGs and a slower dissociation, indicating that once formed, the gCΔmuc-GAG complex is more stable. It was also found that a larger number of gCΔmuc bound to a single GAG chain, compared with native gC. Taken together, our data suggest that the mucin-like region of HSV-1 gC is involved in the modulation of the GAG-binding activity, a feature of importance both for unrestricted virus entry into the cells and release of newly produced viral particles from infected cells.

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.

Glycosaminoglycan derivatives: promising candidates for the design of functional biomaterials.

Numerous biological processes (tissue formation, remodelling and healing) are strongly influenced by the cellular microenvironment. Glycosaminoglycans (GAGs) are important components of the native extracellular matrix (ECM) able to interact with biological mediator proteins. They can be chemically functionalized and thereby modified in their interaction profiles. Thus, they are promising candidates for functional biomaterials to control healing processes in particular in health-compromised patients. Biophysical studies show that the interaction profiles between mediator proteins and GAGs are strongly influenced by (i) sulphation degree, (ii) sulphation pattern, and (iii) composition and structure of the carbohydrate backbone. Hyaluronan derivatives demonstrate a higher binding strength in their interaction with biological mediators than chondroitin sulphate for a comparable sulphation degree. Furthermore sulphated GAG derivatives alter the interaction profile of mediator proteins with their cell receptors or solute native interaction partners. These results are in line with biological effects on cells relevant for wound healing processes. This is valid for solute GAGs as well as those incorporated in collagen-based artificial ECM (aECMs). Prominent effects are (i) anti-inflammatory, immunomodulatory properties towards macrophages/dendritic cells, (ii) enhanced osteogenic differentiation of human mesenchymal stromal cells, (iii) altered differentiation of fibroblasts to myofibroblasts, (iv) reduced osteoclast activity and (v) improved osseointegration of dental implants in minipigs. The findings of our consortium Transregio 67 contribute to an improved understanding of structure-function relationships of GAG derivatives in their interaction with mediator proteins and cells. This will enable the design of bioinspired, functional biomaterials to selectively control and promote bone and skin regeneration.

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.

Sulfated glycosaminoglycans exploit the conformational plasticity of bone morphogenetic protein-2 (BMP-2) and alter the interaction profile with its receptor.

Sulfated glycosaminoglycans (GAGs) can direct cellular processes by interacting with proteins of the extracellular matrix (ECM). In this study we characterize the interaction profiles of chemically sulfated hyaluronan (HA) and chondroitin sulfate (CS) derivatives with bone morphogenetic protein-2 (BMP-2) and investigate their relevance for complex formation with the receptor BMPR-IA. These goals were addressed by surface plasmon resonance (SPR) and ELISA in combination with molecular modeling and dynamics simulation. We found not only the interaction of BMP-2 with GAGs to be dependent on the type and sulfation of GAGs but also BMP-2/GAG/BMPR-IA complex formation. The conformational plasticity of the BMP-2 N-termini plays a key role in the structural and thermodynamic characteristics of the BMP-2/GAG/BMPR-IA system. Hence we propose a model that provides direct insights into the importance of the structural and dynamical properties of the BMP-2/BMPR-IA system for its regulation by sulfated GAGs, in which structural asymmetry plays a key role.