Improvement of the Digestibility of Sulfated Hyaluronans by Bovine Testicular Hyaluronidase: a UV Spectroscopic and Mass Spectrometric Study

Glycosaminoglycans (GAGs) such as hyaluronan (HA) and chondroitin sulfate (CS) are important, natural polysaccharides which occur in biological (connective) tissues and have various biotechnological and medical applications. Additionally, there is increasing evidence that chemically (over)sulfated GAGs possess promising properties and are useful as implant coatings. Unfortunately, a detailed characterization of these GAGs is challenging: although mass spectrometry (MS) is one of the most powerful tools to elucidate the structures of (poly)saccharides, MS is not applicable to high mass polysaccharides, but characteristic oligosaccharides are needed. These oligosaccharides are normally generated by enzymatic digestion. However, chemically modified (particularly sulfated) GAGs are extremely refractive to enzymatic digestion. This study focuses on the investigation of the digestibility of GAGs with different degrees of sulfation by bovine testicular hyaluronidase (BTH). It will be shown by using an adapted spectrophotometric assay that all investigated GAGs can be basically digested if the reaction conditions are carefully adjusted. However, the oligosaccharide yield correlates reciprocally with the number of sulfate residues per polymer repeating unit. Finally, matrix-laser desorption and ionization (MALDI) MS will be used to study the released oligosaccharides and their sulfation patterns

Podoplanin immunopositive lymphatic vessels at the implant interface in a rat model of osteoporotic fractures

Insertion of bone substitution materials accelerates healing of osteoporotic fractures. Biodegradable materials are preferred for application in osteoporotic patients to avoid a second surgery for implant replacement. Degraded implant fragments are often absorbed by macrophages that are removed from the fracture side via passage through veins or lymphatic vessels. We investigated if lymphatic vessels occur in osteoporotic bone defects and whether they are regulated by the use of different materials. To address this issue osteoporosis was induced in rats using the classical method of bilateral ovariectomy and additional calcium and vitamin deficient diet. In addition, wedge-shaped defects of 3, 4, or 5 mm were generated in the distal metaphyseal area of femur via osteotomy. The 4 mm defects were subsequently used for implantation studies where bone substitution materials of calcium phosphate cement, composites of collagen and silica, and iron foams with interconnecting pores were inserted. Different materials were partly additionally functionalized by strontium or bisphosphonate whose positive effects in osteoporosis treatment are well known. The lymphatic vessels were identified by immunohistochemistry using an antibody against podoplanin. Podoplanin immunopositive lymphatic vessels were detected in the granulation tissue filling the fracture gap, surrounding the implant and growing into the iron foam through its interconnected pores. Significant more lymphatic capillaries were counted at the implant interface of composite, strontium and bisphosphonate functionalized iron foam. A significant increase was also observed in the number of lymphatics situated in the pores of strontium coated iron foam. In conclusion, our results indicate the occurrence of lymphatic vessels in osteoporotic bone. Our results show that lymphatic vessels are localized at the implant interface and in the fracture gap where they might be involved in the removal of lymphocytes, macrophages, debris and the implants degradation products. Therefore the lymphatic vessels are involved in implant integration and fracture healing

Instructing human macrophage polarization by stiffness and glycosaminoglycan functionalization in 3D collagen networks

Dynamic alterations of composition and mechanics of the extracellular matrix (ECM) are suggested to modulate cellular behavior including plasticity of macrophages (MPhs) during wound healing. In this study, engineered 3D fibrillar matrices based on naturally occurring biopolymers (collagen I, glycosaminoglycans (GAGs)) were used to mimic matrix stiffening as well as modification by sulfated and non-sulfated GAGs at different stages of wound healing. Human MPhs were found to sensitively respond to these microenvironmental cues in terms of polarization towards pro-inflammatory or wound healing phenotypes over 6 days in vitro. MPhs exhibited a wound healing phenotype in stiffer matrices as determined by protein and gene expression of relevant cytokines (IL10, IL12, TNF). Presence of sulfated and non-sulfated GAGs inhibited this polarization effect. Furthermore, control experiments on 2D matrices stressed the relevance of using stiffness-controlled 3D matrices, as MPhs showed a reciprocal polarization behavior depending on GAG presence. Hence, the results indicate a strong influence of dimensionality, stiffness, and GAG presence of the biomaterial scaffold on MPh polarization and emphasize the need for matrices closely mimicking the 3D in vivo context with a variable stiffness and GAG composition in in vitro studies

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

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

Biomimetic PLGA 3D Scaffold Potentiate Amniotic Epithelial Stem Cells Biological Capability for Tendon Tissue Engineering Applications

INTRODUCTION: Tendon tissue engineering represents a promising solution to deal with tendinopathies and aims to develop effective implantable 3D biomimetic scaffolds with ideally native tissue’s physical, mechanical, biological, and functional qualities. These constructs can be engineered with stem cells to potentiate their teno-inductive and immunomodulatory properties (El Khatib, Mauro, Di Mattia, et al., 2020; El Khatib, Mauro, Wyrwa, et al., 2020; Russo et al., 2020). In this context, amniotic epithelial stem cells (AECs) have recently received much attention in the field of regenerative medicine due to their capacity to differentiate into the tenogenic lineage and to their immunomodulatory profile (Barboni et al., 2012, 2018; Mauro et al., 2016). The focus of this research was to create bundle tendon-like PLGA 3D scaffolds, which mimic tendon macro and micro-architecture and biomechanics, and to assess their impacts on AECs’ biological potential. METHODS: PLGA fleeces, with highly aligned fibers, were fabricated via electrospinning technique through a rotatory collector. The obtained fleeces were then wrapped manually to form 3D tendon-like scaffolds, which were evaluated in terms of structure, mechanical characteristics, and biological influence on AECs by conducting in vitro experiments. Indeed, ovine AECs, seeded on the PLGA 3D scaffolds and fleeces, were compared for their morphological changes and for the cytoplasmic expression of TNMD, a mature tendon protein, respect to cells cultured on Petri dishes (CTR), after 48h and 7d of culture through a confocal microscope. Moreover, the teno-differentiative potential and immunomodulatory properties of the produced constructs were assessed by analyzing the gene expression of tendon related markers (early: SCX, late: COL1 and TNMD) and of anti- (IL10) and pro- (IL12) inflammatory cytokines respectively. Moreover, the present research evaluated YAP protein activation in the engineered AECs through immunofluorescence assay by assessing its cellular localization. RESULTS: The produced PLGA 3D scaffolds, analyzed though a scanning electron microscope, showed high fiber alignment, which closely resemble the architecture, both macroscopically and microscopically, and the biomechanical properties of native tendon tissue. AECs seeded on the produced constructs exhibited an elongated tenocyte-like morphology already after 24 hours, while AECs cultivated on petri dishes (CTR) retained their characteristic polygonal morphology. The engineered AECs' phenotypic change was also confirmed by visualizing the cytoplasmic expression of TNMD protein and supported by tendon-related genes (SCX, COL1, and TNMD) upregulation at 7-day culture respect to CTR cells (p<0.05), which showed no TNMD protein expression or significant increase in tendon-related genes. Moreover, AECs seeded on 3D PLGA scaffolds showed an anti-inflammatory profile, with a significant higher IL10/IL12 ratio respect to the CTR (p<0.05). Finally, 3D scaffolds with highly aligned fibers stimulated AECs in terms of cell cytoskeleton stress, activating their mechanosensitive YAP pathway by significantly increasing YAP nuclear localization compared to the CTR (p<0.05), in which YAP was instead localized in the cytoplasm. DISCUSSION & CONCLUSIONS: Overall, these results support the biomimicry of the fabricated scaffolds in terms of structure and biomechanics and reveal their great teno/immuno-inductive potential and mechanosensing stimulus on AECs, thus standing biomimetic PLGA 3D scaffolds as a potential candidate for tendon regeneration

A Versatile Macromer-Based Glycosaminoglycan (sHA3) Decorated Biomaterial for Pro-Osteogenic Scavenging of Wnt Antagonists

High serum levels of Wnt antagonists are known to be involved in delayed bone defect healing. Pharmaceutically active implant materials that can modulate the micromilieu of bone defects with regard to Wnt antagonists are therefore considered promising to support defect regeneration. In this study, we show the versatility of a macromer based biomaterial platform to systematically optimize covalent surface decoration with high-sulfated glycosaminoglycans (sHA3) for efficient scavenging of Wnt antagonist sclerostin. Film surfaces representing scaffold implants were cross-copolymerized from three-armed biodegradable macromers and glycidylmethacrylate and covalently decorated with various polyetheramine linkers. The impact of linker properties (size, branching) and density on sHA3 functionalization efficiency and scavenging capacities for sclerostin was tested. The copolymerized 2D system allowed for finding an optimal, cytocompatible formulation for sHA3 functionalization. On these optimized sHA3 decorated films, we showed efficient scavenging of Wnt antagonists DKK1 and sclerostin, whereas Wnt agonist Wnt3a remained in the medium of differentiating SaOS-2 and hMSC. Consequently, qualitative and quantitative analysis of hydroxyapatite staining as a measure for osteogenic differentiation revealed superior mineralization on sHA3 materials. In conclusion, we showed how our versatile material platform enables us to efficiently scavenge and inactivate Wnt antagonists from the osteogenic micromilieu. We consider this a promising approach to reduce the negative effects of Wnt antagonists in regeneration of bone defects via sHA3 decorated macromer based macroporous implants. © 2020 by the authors. Licensee MDPI, Basel, Switzerland

CUSTOMIZATION OF ELECTROSPINNING FOR TISSUE ENGINEERING

This paper deals with two electrospinning technologies: the melt electrospinning with a customized jet head, adapted from the fused deposition modeling (FDM) 3D printer, in comparison with the standard solution electrospinning, aiming at fabrication of tissue engineering scaffolds. The resulting fibers are compared. The influence of the collector properties on those of the fabricated scaffold is investigated. The resulting electrospun fibers exhibit different characteristics such as morphology and thickness, depending on the technology. The micro-fibers are produced by the melt electrospinning with an inbuilt 3D printer jet head, whereas the solution electrospinning has produced nano- and micro-fibers. The scaffolds fabricated on the rotating drum collector exhibit a more ordered structure as well as thinner fibers than those produced on the stationary plate collector. Further investigations should aim at fabrication of porous hollow fibers and tissue engineering scaffolds with controlled porosity and properties