Sandra Rother

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.

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 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.

Biomimetic electrospun scaffolds from main extracellular matrix components for skin tissue engineering application – The role of chondroitin sulfate and sulfated hyaluronan

Incorporation of bioactive components like glycosaminoglycans (GAGs) into tissue engineering scaffolds, is a promising approach towards developing new generation functional biomaterial. Here, we have designed electrospun nanofibrous scaffolds made of gelatin and different concentrations of chemically sulfated or non-sulfated hyaluronan (sHA or HA) and chondroitin sulfate (CS). Evenly distributed fiber morphology was observed with no differences between varying concentrations and types of GAGs. In vitro release kinetics revealed that GAGs release is driven by diffusion. The effects of these scaffolds were analyzed on human keratinocyte (HaCaT), fibroblast (Hs27) and mesenchymal stem cells (hMSCs) adhesion and proliferation. A significant increase in cell number (~5 fold) was observed when cultivating all three cell types alone on scaffolds containing sHA and CS. These findings suggest that sulfated GAG-containing electrospun nanofibrous scaffolds might be beneficial for the development of effective skin tissue engineered constructs by stimulating cellular performance and therefore accelerate epidermal-dermal regeneration processes.

Sulfated Hyaluronan Derivatives Modulate TGF-β1:Receptor Complex Formation: Possible Consequences for TGF-β1 Signaling

Glycosaminoglycans are known to bind biological mediators thereby modulating their biological activity. Sulfated hyaluronans (sHA) were reported to strongly interact with transforming growth factor (TGF)-β1 leading to impaired bioactivity in fibroblasts. The underlying mechanism is not fully elucidated yet. Examining the interaction of all components of the TGF-β1:receptor complex with sHA by surface plasmon resonance, we could show that highly sulfated HA (sHA3) blocks binding of TGF-β1 to its TGF-β receptor-I (TβR-I) and -II (TβR-II). However, sequential addition of sHA3 to the TβR-II/TGF-β1 complex led to a significantly stronger recruitment of TβR-I compared to a complex lacking sHA3, indicating that the order of binding events is very important. Molecular modeling suggested a possible molecular mechanism in which sHA3 could potentially favor the association of TβR-I when added sequentially. For the first time bioactivity of TGF-β1 in conjunction with sHA was investigated at the receptor level. TβR-I and, furthermore, Smad2 phosphorylation were decreased in the presence of sHA3 indicating the formation of an inactive signaling complex. The results contribute to an improved understanding of the interference of sHA3 with TGF-β1:receptor complex formation and will help to further improve the design of functional biomaterials that interfere with TGF-β1-driven skin fibrosis.

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

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

Sulfation degree not origin of chondroitin sulfate derivatives modulates keratinocyte response

Chondroitin sulfate (CS) sulfation-dependently binds transforming growth factor-β1 (TGF-β1) and chronic wounds often accompany with epidermal hyperproliferation due to downregulated TGF-β signaling. However, the impact of CS on keratinocytes is unknown. Especially biotechnological-chemical strategies are promising to replace animal-derived CS. Thus, this study aims to evaluate the effects of CS derivatives on the interaction with vascular endothelial growth factor-A (VEGF-A) and on keratinocyte response. Over-sulfated CS (sCS3) interacts stronger with VEGF-A than CS. Furthermore, collagen coatings with CS variants are prepared by in vitro fibrillogenesis. Stability analyses demonstrate that collagen is firmly integrated, while the fibril diameters decrease with increasing sulfation degree. CS variants sulfation-dependently decelerate keratinocyte (HaCaT) migration and proliferation in a scratch assay. HaCaT cultured on sCS3-containing coatings produced increased amounts of solute active TGF-β1 which could be translated into biomaterials able to decrease epidermal hyperproliferation in chronic wounds. Overall, semi-synthetic and natural CS yield to comparable responses.

Nanofibrous artificial skin substitute composed of mPEG–PCL grafted gelatin/hyaluronan/chondroitin sulfate/sericin for 2nd degree burn care: in vitro and in vivo study

The aim of this study was to investigate the efficacy of a skin substitute composed of mPEG–PCL–grafted-gelatin (Bio-Syn)/hyaluronan/chondroitin sulfate/sericin and to study its in vitro biocompatibility with human fibroblasts, human keratinocytes and hMSCs in terms of cellular adhesion and proliferation (∼5–6 fold). mPEG–PCL was grafted into a gelatin backbone via a Michael addition reaction to prepare Bio-Syn and it was characterized using ATR-FTIR, 1H NMR and TNBS assay. Additionally, keratinocyte–hMSC contact co-culture studies showed that Bio-Syn composite scaffolds loaded with sericin promote hMSCs’ epithelial differentiation with regard to qRT-PCR gene expression (ΔNp63α and keratin 14) and expression of various epithelial markers (Pan-cytokeratin, ΔNp63α and keratin 14). In vivo efficacy studies on a 2nd degree burn wound model in Wistar rats showed an improved rate of wound contraction, histology (H&E and Van Gieson’s staining) and pro-healing marker (hexosamine, hydroxyproline, etc.) expression in granular tissue compared to using the commercial dressing Neuskin™ and a cotton gauze control.

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.Graphical abstract

MEHP and MEOHP but not DEHP bind productively to the peroxisome proliferator-activated receptor γ

RATIONALE The most frequently occurring phthalate, di(2-ethylhexyl) phthalate (DEHP), causes adverse effects on glucose homeostasis and insulin sensitivity in several cell models and epidemiological studies. However, thus far, there is no information available on the molecular interaction of phthalates and one of the key regulators of the metabolism, the peroxisome proliferator-activated receptor gamma (PPARγ). Since the endogenous ligand of PPARγ, 15-deoxy-delta-12,14-prostaglandin J2 (15Δ-PGJ2 ), features structural similarity to DEHP and its main metabolites produced in human hepatic metabolism, mono(2-ethylhexyl) phthalate (MEHP) and mono(2-ethyl-5-oxohexyl) phthalate (MEOHP), we tested the hypothesis of direct interactions between PPARγ and DEHP or its transformation products. METHODS Hydrogen/deuterium exchange mass spectrometry (HDX-MS) and docking were conducted to obtain structural insights into the interactions and surface plasmon resonance (SPR) analysis to reveal information about binding levels. To confirm the activation of PPARγ upon ligand binding on the cellular level, the GeneBLAzer® bioassay was performed. RESULTS HDX-MS and SPR analyses demonstrated that the metabolites MEHP and MEOHP, but not DEHP itself, bind to the ligand binding pocket of PPARγ. This binding leads to typical activation-associated conformational changes, as observed with its endogenous ligand 15Δ-PGJ2 . Furthermore, the reporter gene assay confirmed productive interaction. DEHP was inactive up to a concentration of 14 μM, while the metabolites MEHP and MEOHP were active at low micromolar concentrations. CONCLUSIONS In summary, this study gives structural insights into the direct interaction of PPARγ with MEHP and MEOHP and shows that the DEHP transformation products may modulate the lipid metabolism through PPARγ pathways.