Elastin is heterogeneously cross-linked

Elastin is an essential vertebrate protein responsible for the elasticity of force-bearing tissues such as those of the lungs, blood vessels, and skin. One of the key features required for the exceptional properties of this durable biopolymer is the extensive covalent cross-linking between domains of its monomer molecule tropoelastin. To date, elastin's exact molecular assembly and mechanical properties are poorly understood. Here, using bovine elastin, we investigated the different types of cross-links in mature elastin to gain insight into its structure. We purified and proteolytically cleaved elastin from a single tissue sample into soluble cross-linked and noncross-linked peptides that we studied by high-resolution MS. This analysis enabled the elucidation of cross-links and other elastin modifications. We found that the lysine residues within the tropoelastin sequence were simultaneously unmodified and involved in various types of cross-links with different other domains. The Lys-Pro domains were almost exclusively linked via lysinonorleucine, whereas Lys-Ala domains were found to be cross-linked via lysinonorleucine, allysine aldol, and desmosine. Unexpectedly, we identified a high number of intramolecular cross-links between lysine residues in close proximity. In summary, we show on the molecular level that elastin formation involves random cross-linking of tropoelastin monomers resulting in an unordered network, an unexpected finding compared with previous assumptions of an overall beaded structure

Effect of metal ions on the physical properties of multilayers from hyaluronan and chitosan, and the adhesion, growth and adipogenic differentiation of multipotent mouse fibroblasts

[EN] Polyelectrolyte multilayers (PEMs) consisting of the polysaccharides hyaluronic acid (HA) as the polyanion and chitosan (Chi) as the polycation were prepared with layer-by-layer technique (LbL). The [Chi/HA](5) multilayers were exposed to solutions of metal ions (Ca2+, Co2+, Cu2+ and Fe3+). Binding of metal ions to [Chi/HA](5) multilayers by the formation of complexes with functional groups of polysaccharides modulates their physical properties and the bioactivity of PEMs with regard to the adhesion and function of multipotent murine C3H10T1/2 embryonic fibroblasts. Characterization of multilayer formation and surface properties using different analytical methods demonstrates changes in the wetting, surface potential and mechanical properties of multilayers depending on the concentration and type of metal ion. Most interestingly, it is observed that Fe3+ metal ions greatly promote adhesion and spreading of C3H10T1/2 cells on the low adhesive [Chi/HA](5) PEM system. The application of intermediate concentrations of Cu2+, Ca2+ and Co2+ as well as low concentrations of Fe3+ to PEMs also results in increased cell spreading. Moreover, it can be shown that complex formation of PEMs with Cu2+ and Fe3+ ions leads to increased metabolic activity in cells after 24 h and induces cell differentiation towards adipocytes in the absence of any additional adipogenic media supplements. Overall, complex formation of [Chi/HA](5) PEM with metal ions like Cu2+ and Fe3+ represents an interesting and cheap alternative to the use of growth factors for making cell-adhesive coatings and guiding stem cell differentiation on implants and scaffolds to regenerate connective-type of tissues.This work was part of the High-Performance Center Chemical and Biosystems Technology Halle/Leipzig, supported by the European Regional Development Fund (ERDF), and a grant to HK from the Martin Luther University Halle-Wittenberg for female PhD students. The work was further supported by the Fraunhofer Internal Programs under Grant No. Attract 069-608203 (CEHS). TG acknowledges the kind support by the Ministry of Science and Higher Education of the Russian Federation within the framework of state support for the creation and development of World-Class Research Centers ``Digital biodesign and personalized healthcare'' 075-15-2020926. GGF acknowledges funding by the State Research Agency. Ministry of Science and Innovation of Spain, grant PID2019106000RB-C21/AEI/10.13039/501100011033 project. We are grateful for the kind support by Christian Willems for the help in formatting and proof reading the manuscript.Kindi, H.; Menzel, M.; Heilmann, A.; Schmelzer, CEH.; Herzberg, M.; Fuhrmann, B.; Gallego-Ferrer, G.... (2021). Effect of metal ions on the physical properties of multilayers from hyaluronan and chitosan, and the adhesion, growth and adipogenic differentiation of multipotent mouse fibroblasts. Soft Matter. 17(36):8394-8410. https://doi.org/10.1039/d1sm00405k83948410173

Lipoplex-functionalized thin-film surface coating based on extracellular matrix components as local gene delivery system to control osteogenic stem cell differentiation

A gene-activated surface coating is presented as a strategy to design smart biomaterials for bone tissue engineering. The thin-film coating is based on polyelectrolyte multilayers composed of collagen I and chondroitin sulfate, two main biopolymers of the bone extracellular matrix, which are fabricated by layer-by-layer assembly. For further functionalization, DNA/lipid-nanoparticles (lipoplexes) are incorporated into the multilayers. The polyelectrolyte multilayer fabrication and lipoplex deposition are analyzed by surface sensitive analytical methods that demonstrate successful thin-film formation, fibrillar structuring of collagen, and homogenous embedding of lipoplexes. Culture of mesenchymal stem cells on the lipoplex functionalized multilayer results in excellent attachment and growth of them, and also, their ability to take up cargo like fluorescence-labelled DNA from lipoplexes. The functionalization of the multilayer with lipoplexes encapsulating DNA encoding for transient expression of bone morphogenetic protein 2 induces osteogenic differentiation of mesenchymal stem cells, which is shown by mRNA quantification for osteogenic genes and histochemical staining. In summary, the novel gene-functionalized and extracellular matrix mimicking multilayer composed of collagen I, chondroitin sulfate, and lipoplexes, represents a smart surface functionalization that holds great promise for tissue engineering constructs and implant coatings to promote regeneration of bone and other tissues.publishe

Extracellular Matrix Stiffness and Composition Regulate the Myofibroblast Differentiation of Vaginal Fibroblasts

Fibroblast to myofibroblast differentiation is a key feature of wound-healing in soft tissues, including the vagina. Vaginal fibroblasts maintain the integrity of the vaginal wall tissues, essential to keep pelvic organs in place and avoid pelvic organ prolapse (POP). The micro-environment of vaginal tissues in POP patients is stiffer and has different extracellular matrix (ECM) composition than healthy vaginal tissues. In this study, we employed a series of matrices with known stiffnesses, as well as vaginal ECMs, in combination with vaginal fibroblasts from POP and healthy tissues to investigate how matrix stiffness and composition regulate myofibroblast differentiation in vaginal fibroblasts. Stiffness was positively correlated to production of α-smooth muscle actin (α-SMA). Vaginal ECMs induced myofibroblast differentiation as both α-SMA and collagen gene expressions were increased. This differentiation was more pronounced in cells seeded on POP-ECMs that were stiffer than those derived from healthy tissues and had higher collagen and elastin protein content. We showed that stiffness and ECM content regulate vaginal myofibroblast differentiation. We provide preliminary evidence that vaginal fibroblasts might recognize POP-ECMs as scar tissues that need to be remodeled. This is fundamentally important for tissue repair, and provides a rational basis for POP disease modelling and therapeutic innovations in vaginal reconstruction

Intrinsically Cross‐Linked ECM‐Like Multilayers for BMP‐2 Delivery Promote Osteogenic Differentiation of Cells

Abstract Surface coatings prepared by layer‐by‐layer technique permit loading of growth factors (GFs) and their spatially controlled release. Here, native chondroitin sulfate (nCS), oxidized CS (oCS100), or mixture of both (oCS50) are combined with collagen I (Col I) to fabricate polyelectrolyte multilayers (PEMs) that exhibit structural, mechanical, and biochemical cues like the natural extracellular‐matrix. The use of oCS enables intrinsic cross‐linking of PEM that offers higher stability, stiffness, and better control of bone morphogenetic protein‐2 (BMP‐2) release compared to nCS. oCS100 PEMs have enhanced stiffness, promote Col I fibrillization, and present BMP‐2 in a matrix‐bound manner. oCS50 PEMs show intermediate effects on osteogenesis, soft surface, high water content but also moderately slow BMP‐2 release profile. C2C12 myoblasts used for osteogenesis studies show that oCS PEMs are more stable and superior to nCS PEMs in supporting cell adhesion and spreading as well as in presenting BMP‐2 to the cells. oCS PEMs are triggering more osteogenesis as proved by the quantitative real‐time polymerase chain reaction, immune and histochemical staining. These findings show that intrinsic cross‐linking in oCS/Col I multilayers provides a successful tool to control GFs delivery and subsequent cell differentiation which opens new opportunities in regenerative therapies of bone and other tissues