Stable Scaffold Research Articles

Proliferation and differential regulation of osteoblasts cultured on surface-phosphorylated cellulose nanofiber scaffolds

Phosphorus-containing polymers have received much attention for their excellent ability to regulate bone cell differentiation and calcification. Given the increasing concern about environmental issues, it is promising to utilize “green” biomaterials to construct novel cell culture scaffolds for bone tissue engineering. Herein, surface-phosphorylated cellulose nanofibers (P-CNFs) were fabricated as a novel green candidate for osteoblast culture. Compared with native CNF, P-CNFs possessed shorter fiber morphology with tunable phosphate group content (0–1.42 mmol/g). The zeta-potential values of CNFs were enhanced after phosphorylation, resulting in the formation of uniform and stable scaffolds. The cell culture behavior of mouse osteoblast (MC3T3-E1) cells showed a clear phosphate content-dependent cell proliferation. The osteoblast cells adhered well and proliferated efficiently on P-CNF0.78 and P-CNF1.05, with phosphate contents of 0.78 and 1.05 mmol/g, respectively, whereas the cells grown on native CNF substrate formed aggregates due to poor cell attachment and exhibited limited cell proliferation. In addition, the P-CNF substrates with optimal phosphate content provided a favorable cellular microenvironment and significantly promoted osteogenic differentiation and calcification, even in the absence of a differentiation inducer. The bio-based P-CNFs are expected to mimic the bone components and provide a means to regulate osteoblast proliferation and differentiation in bone tissue engineering.

Physicochemical properties of a novel chiral self-assembling peptide R-LIFE-1 and its controlled release to exosomes

This research aims to investigate the encapsulation and controlled release effect of the newly developed self-assembling peptide R-LIFE-1 on exosomes. The gelling ability and morphological structure of the chiral self-assembling peptide (CSAP) hydrogel were examined using advanced imaging techniques, including atomic force microscopy, transmission electron microscopy, and cryo-scanning electron microscopy. The biocompatibility of the CSAP hydrogel was assessed through optical microscopy and fluorescent staining. Exosomes were isolated via ultrafiltration, and their quality was evaluated using Western blot analysis, nanoparticle tracking analysis, and transmission electron microscopy. The controlled release effect of the CSAP hydrogel on exosomes was quantitatively analyzed using laser confocal microscopy and a BCA assay kit. The results revealed that the self-assembling peptide R-LIFE-1 exhibited spontaneous assembly in the presence of various ions, leading to the formation of nanofibers. These nanofibers were cross-linked, giving rise to a robust nanofiber network structure, which further underwent cross-linking to generate a laminated membrane structure. The nanofibers possessed a large surface area, allowing them to encapsulate a substantial number of water molecules, thereby forming a hydrogel material with high water content. This hydrogel served as a stable spatial scaffold and loading matrix for the three-dimensional culture of cells, as well as the encapsulation and controlled release of exosomes. Importantly, R-LIFE-1 demonstrated excellent biocompatibility, preserving the growth of cells and the biological activity of exosomes. It rapidly formed a three-dimensional network scaffold, enabling the stable loading of cells and exosomes, while exhibiting favorable biocompatibility and reduced cytotoxicity. In conclusion, the findings of this study support the notion that R-LIFE-1 holds significant promise as an ideal tissue engineering material for tissue repair applications.

Adipose Mesenchymal Stem Cell Derived Exosomes Promote Keratinocytes and Fibroblasts Embedded in Collagen/Platelet-Rich Plasma Scaffold and Accelerate Wound Healing.

Engineered skin substitutes derived from human skin significantly reduce inflammatory reactions mediated by foreign/artificial materials and are consequently easier to use for clinical application. Type I collagen is a main component of the extracellular matrix during wound healing and has excellent biocompatibility, and platelet-rich plasma can be used as the initiator of the healing cascade. Adipose mesenchymal stem cell derived exosomes are crucial for tissue repair and play key roles in enhancing cell regeneration, promoting angiogenesis, regulating inflammation, and remodeling extracellular matrix. Herein, Type I collagen and platelet-rich plasma, which provide natural supports for keratinocyte and fibroblast adhesion, migration, and proliferation, are mixed to form a stable 3D scaffold. Adipose mesenchymal stem cell derived exosomes are added to the scaffold to improve the performance of the engineered skin. The physicochemical properties of this cellular scaffold are analyzed, and the repair effect is evaluated in a full-thickness skin defect mouse model. The cellular scaffold reduces the level of inflammation and promotes cell proliferation and angiogenesis to accelerate wound healing. Proteomic analysis shows that exosomes exhibit excellent anti-inflammatory and proangiogenic effects in collagen/platelet-rich plasma scaffolds. The proposed method provides a new therapeutic strategy and theoretical basis for tissue regeneration and wound repair.

Biodegradable piezoelectric skin-wound scaffold

Electrical stimulation (ES) induces wound healing and skin regeneration. Combining ES with the tissue-engineering approach, which relies on biomaterials to construct a replacement tissue graft, could offer a self-stimulated scaffold to heal skin-wounds without using potentially toxic growth factors and exogenous cells. Unfortunately, current ES technologies are either ineffective (external stimulations) or unsafe (implanted electrical devices using toxic batteries). Hence, we propose a novel wound-healing strategy that integrates ES with tissue engineering techniques by utilizing a biodegradable self-charged piezoelectric PLLA (Poly (l-lactic acid)) nanofiber matrix. This unique, safe, and stable piezoelectric scaffold can be activated by an external ultrasound (US) to produce well-controlled surface-charges with different polarities, thus serving multiple functions to suppress bacterial growth (negative surface charge) and promote skin regeneration (positive surface charge) at the same time. We demonstrate that the scaffold activated by low intensity/low frequency US can facilitate the proliferation of fibroblast/epithelial cells, enhance expression of genes (collagen I, III, and fibronectin) typical for the wound healing process, and suppress the growth of S. aureus and P. aeruginosa bacteria in vitro simultaneously. This approach induces rapid skin regeneration in a critical-sized skin wound mouse model in vivo. The piezoelectric PLLA skin scaffold thus assumes the role of a multi-tasking, biodegradable, battery-free electrical stimulator which is important for skin-wound healing and bacterial infection prevention simultaneuosly.

A Chicken Tapasin ortholog can chaperone empty HLA-B∗37:01 molecules independent of other peptide-loading components

Human Tapasin (hTapasin) is the main chaperone of MHC-I molecules, enabling peptide loading and antigen repertoire optimization across HLA allotypes. However, it is restricted to the endoplasmic reticulum (ER) lumen as part of the protein loading complex (PLC), and therefore is highly unstable when expressed in recombinant form. Additional stabilizing co-factors such as ERp57 are required to catalyze peptide exchange invitro, limiting uses for the generation of pMHC-I molecules of desired antigen specificities. Here, we show that the chicken Tapasin (chTapasin) ortholog can be expressed recombinantly at high yields in a stable form, independent of co-chaperones. chTapasin can bind the human HLA-B∗37:01 with low micromolar-range affinity to form a stable tertiary complex. Biophysical characterization by methyl-based NMR methods reveals that chTapasin recognizes a conserved β2m epitope on HLA-B∗37:01, consistent with previously solved X-ray structures of hTapasin. Finally, we provide evidence that the B∗37:01/chTapasin complex is peptide-receptive and can be dissociated upon binding of high-affinity peptides. Our results highlight the use of chTapasin as a stable scaffold for protein engineering applications aiming to expand the ligand exchange function on human MHC-I and MHC-like molecules.

Epitopes Displayed in a Cyclic Peptide Scaffold Bind SARS-COV-2 Antibodies.

The SARS-CoV-2 virus that causes COVID-19 is a global health issue. The spread of the virus has resulted in seven million deaths to date. The emergence of new viral strains highlights the importance of continuous surveillance of the SARS-CoV-2 virus by using timely and accurate diagnostic tools. Here, we used a stable cyclic peptide scaffolds to present antigenic sequences derived from the spike protein that are reactive to SARS-CoV-2 antibodies. Using peptide sequences from different domains of SARS-CoV-2 spike proteins, we grafted epitopes on the peptide scaffold sunflower trypsin inhibitor 1 (SFTI-1). These scaffold peptides were then used to develop an ELISA to detect SARS-CoV-2 antibodies in serum. We show that displaying epitopes on the scaffold improves reactivity overall. One of the scaffold peptides (S2_1146-1161_c) has reactivity equal to that of commercial assays, and shows diagnostic potential.

Silk sericin-hydroxyapatite nanoribbons toward structurally stable osteogenic scaffolds

Silk sericin-hydroxyapatite nanoribbons toward structurally stable osteogenic scaffolds

A Stable Hydrogel Scaffold with Anti-Inflammatory Effects Treats Intervertebral Disc Degeneration

A Stable Hydrogel Scaffold with Anti-Inflammatory Effects Treats Intervertebral Disc Degeneration

Schiff base reaction induced densification of chitosan-derived microporous carbon for compact capacitive energy storage

Preparing carbon materials with both high surface area and density is crucial for improving the volumetric performance of supercapacitors. Herein, we show a novel phenomenon that chitosan can go through spontaneous densification during chemical activation, enabling the microporous carbon with large surface area and extremely high compaction density (2,007 m2 g−1, 1.21 g cm−3). This is quite different from direct pyrolysis and/or activation of many other carbon precursors, which usually form loose structure due to the serious foaming. The mechanism is revealed as the formation of highly crosslinked and stable scaffold by the thermally induced Schiff base reaction between amino and aldehyde groups, which not only inhibits the foaming phenomenon, but also facilitates further aromatization of carbon scaffold. The optimized sample shows high gravimetric and volumetric capacitances (280 F g−1 and 339 F cm−3 at 1 A g−1) and good rate performance (58% retention at 100 A g−1) for aqueous supercapacitors, outperforming most of the reported carbon materials. Considering the low-cost and simple process, this work may provide a new way for structural design and practical application of dense carbon materials.

Dual-Crosslinking of Gelatin-Based Hydrogels: Promising Compositions for a 3D Printed Organotypic Bone Model.

Gelatin-based hydrogels have emerged as a popular scaffold material for tissue engineering applications. The introduction of variable crosslinking methods has shown promise for fabricating stable cell-laden scaffolds. In this work, we examine promising composite biopolymer-based inks for extrusion-based 3D bioprinting, using a dual crosslinking approach. A combination of carefully selected printable hydrogel ink compositions and the use of photoinduced covalent and ionic crosslinking mechanisms allows for the fabrication of scaffolds of high accuracy and low cytotoxicity, resulting in unimpeded cell proliferation, extracellular matrix deposition, and mineralization. Three selected bioink compositions were characterized and the respective cell-laden scaffolds were bioprinted. Temporal stability, morphology, swelling, and mechanical properties of the scaffolds were thoroughly studied and the biocompatibility of the constructs was assessed using rat mesenchymal stem cells while focusing on osteogenesis. Experimental results showed that the composition of 1% alginate, 4% gelatin, and 5% (w/v) gelatine methacrylate, was found to be optimal among the examined, with shape fidelity of 88%, large cell spreading area and cell viability at around 100% after 14 days. The large pore diameters that exceed 100 µm, and highly interconnected scaffold morphology, make these hydrogels extremely potent in bone tissue engineering and bone organoid fabrication.

A bilayer biocompatible polycaprolactone/zinc oxide/Capparis spinosa L. ethyl acetate extract/polylactic acid nanofibrous composite scaffold for novel wound dressing applications

Capparis spinosa L. (CSL) is used in traditional medicinal purposes for wound dressing because it contains natural phenolic and flavonoid active compounds. In the current study, a bilayer of biocompatible and mechanically stable nanofiber scaffolds with polycaprolactone (PCL)/zinc oxide and Capparis spinosa L. ethyl acetate extract (CSLE)/polylactic acid (PLA) layers was successfully prepared by an electrostatic spinning technique. Microstructural observations carried out by scanning electron microscopy (SEM) have shown that the nanofibers with a smooth surface are continuous and bead-free, and that the size distribution is uniform, with an average diameter of 314.15 nm. The results of careful observation further suggested that polymers in the nanofibers have excellent compatibility with drugs. The results of Fourier transform infrared (FTIR) spectroscopy suggested that CSLE and zinc oxide nanoparticles (ZnO) were successfully loaded in the nanofiber membranes. Water contact angle measurements revealed that the bilayer nanofiber membranes exhibited satisfactory wettability (outside layer, 130°; inner layer, 72.4°). Tensile testing showed that the bilayer PCL/ZnO-CSLE/PLA nanofibers remained unbroken until reaching 10.69 MPa, which is much higher than the tensile strengths of the individual layers or the individual components. Moreover, agar disk diffusion assessment confirmed that the bilayer nanofiber membranes obviously hindered bacterial growth. Cytotoxicity studies showed that the bilayer nanofiber membranes effectively accelerated cell proliferation. The investigated PCL/ZnO-CSLE/PLA bilayer nanofibers have potential for use as membranes for wound dressing applications.

Generation of Haploid Spermatids on Silk Fibroin-Alginate-Laminin-Based Porous 3D Scaffolds.

In vitro production of sperm is a desirable idea for fertility preservation in azoospermic men and prepubertal boys suffering from cancer. In this study, a biocompatible porous scaffold based on a triad mixture of silk fibroin (SF), alginate (Alg), and laminin (LM) is developed to facilitate the differentiation of mouse spermatogonia stem cells (SSCs). Following SF extraction, the content is analyzed by SDS-PAGE and stable porous 3D scaffolds are successfully prepared by merely Alg, SF, and a combination of Alg-SF, or Alg-SF-LM through freeze-drying. Then, the biomimetic scaffolds are characterized regarding the structural and biological properties, water absorption capacity, biocompatibility, biodegradability, and mechanical behavior. Neonatal mice testicular cells are seeded on three-dimensional scaffolds and their differentiation efficiency is evaluated using real-time PCR, flow cytometry, immunohistochemistry. Blend matrices showed uniform porous microstructures with interconnected networks, which maintained long-term stability and mechanical properties better than homogenous structures. Molecular analysis of the cells after 21 days of culture showed that the expression of differentiation-related proteins in cells that are developed in composite scaffolds is significantly higher than in other groups. The application of a composite system can lead to the differentiation of SSCs, paving the way for a novel infertility treatment landscape in the future.

A cellulose reinforced polymer composite electrolyte for the wide-temperature-range solid lithium batteries

The lightweight solid electrolyte design in replacement of the flammable liquid electrolyte and polyolefin separator is the key of the energy-dense all-solid-state batteries (ASSBs) construction. However, the technological barriers of scalable manufacturing, retarded ionic conductivity at room temperature as well as the mechanical fragility upon the high-temperature operation restrict the ASSB deployment of practical relevance. In this study, an ultra-lightweight (1.67 mg cm−2), thin (25 μm), high strength and thermally robust (stability up to 180 °C) composite solid electrolyte (CSE) has been designed to address the dilemma by the rational integration of polyethylene glycol monomethyl ethers coated Li6.4La3Zr1.4Ta0.6O12 nanofillers (MPEG@LLZTO) with lightweight nanocellulose scaffold. The MPEG coating layer enhances the interfacial compatibility and homogeneous dispersibility of LLZTO nanofiller within the poly (ethylene oxide) (PEO) matrix, while the mechanically flexible and thermally stable nanocellulose scaffold guarantees the formation of the high-temperature endurance and structural robustness for the as-formed CSE layer. Upon the solvent-free, layer stacked-up assembly of the PEO/MPEG@LLZTO-Nanocellulose (PLCN) CSE film with the high-mass-loading LiFePO4 cathode and thin-layer lithium anode, the ASSB prototype could simultaneously realize the high gravimetric energy density (323 Wh kg−1), cycling stability as well as operation reliability within a wider temperature range (25 °C ∼ 130 °C).

Molecular Modeling of Self-Assembling Peptides.

Peptide epitopes mediate as many as 40% of protein-protein interactions and fulfill signaling, inhibition, and activation roles within the cell. Beyond protein recognition, some peptides can self- or coassemble into stable hydrogels, making them a readily available source of biomaterials. While these 3D assemblies are routinely characterized at the fiber level, there are missing atomistic details about the assembly scaffold. Such atomistic detail can be useful in the rational design of more stable scaffold structures and with improved accessibility to functional motifs. Computational approaches can in principle reduce the experimental cost of such an endeavor by predicting the assembly scaffold and identifying novel sequences that adopt said structure. Yet, inaccuracies in physical models and inefficient sampling have limited atomistic studies to short (two or three amino acid) peptides. Given recent developments in machine learning and advances in sampling strategies, we revisit the suitability of physical models for this task. We use the MELD (Modeling Employing Limited Data) approach to drive self-assembly in combination with generic data in cases where conventional MD is unsuccessful. Finally, despite recent developments in machine learning algorithms for protein structure and sequence predictions, we find the algorithms are not yet suited for studying the assembly of short peptides.

Conformational Dynamics of an α-Synuclein Fibril upon Receptor Binding Revealed by Insensitive Nuclei Enhanced by Polarization Transfer-Based Solid-State Nuclear Magnetic Resonance and Cryo-Electron Microscopy.

Many amyloid fibrils associated with neurodegenerative diseases consist of an ordered fibril core (FC) and disordered terminal regions (TRs). The former represents a stable scaffold, while the latter is rather active in binding with various partners. Current structural studies mainly focus on the ordered FC since the high flexibility of TRs hinders structural characterization. Here, by combining insensitive nuclei enhanced by polarization transfer-based 1H-detected solid-state NMR and cryo-EM, we explored the intact structure of an α-syn fibril including both FC and TRs and further studied the conformational dynamics of the fibril upon binding to lymphocyte activation gene 3 (LAG3)─a cell surface receptor that is involved in α-syn fibril transmission in brains. We found that both the N- and C-TRs of α-syn are disordered in free fibrils featuring similar conformation ensembles as those in soluble monomers. While in the presence of the D1 domain of LAG3 (L3D1), the C-TR directly binds to L3D1, meanwhile the N-TR folds into a β-strand and further integrates with the FC, which leads to alteration of the overall fibril structure and surface property. Our work reveals synergistic conformational transition of the intrinsically disordered TRs of α-syn, which sheds light on mechanistic understanding of the essential role of TRs in regulating the structure and pathology of amyloid fibrils.

Application of 3D-printed tissue-engineered skin substitute using innovative biomaterial loaded with human adipose-derived stem cells in wound healing.

Large-scale skin injuries are usually accompanied by impaired wound healing, resulting in scar formation, or significant morbidity and mortality. The aim of this study is to explore the in vivo application of 3D-printed tissue-engineered skin substitute using innovative biomaterial loaded with human adipose-derived stem cells (hADSCs) in wound healing. Adipose tissue was decellularized, and extracellular matrix components were lyophilized and solubilized to obtain adipose tissue decellularized extracellular matrix (dECM) pre-gel. The newly designed biomaterial is composed of adipose tissue dECM pre-gel, methacrylated gelatin (GelMA), and methacrylated hyaluronic acid (HAMA). Rheological measurement was performed to evaluate the phase-transition temperature and the storage and loss modulus at this temperature. Tissue-engineered skin substitute loaded with hADSCs was fabricated by 3D printing. We used nude mice to establish full-thickness skin wound healing model and divided them into four groups randomly: (A) Full-thickness skin graft treatment group, (B) 3D-bioprinted skin substitute treatment group as the experimental group, (C) microskin graft treatment group, and (D) control group. The amount of DNA in each milligram of dECM was 24.5 ± 7.1 ng, fulfilling the currently accepted decellularization criteria. The solubilized adipose tissue dECM was thermo-sensitive biomaterial and underwent a sol-gel phase transition when temperature rises. The dECM-GelMA-HAMA precursor undergoes a gel-sol phase transition at 17.5°C, where the storage and loss modulus of the precursor is about 8 Pa. The scanning electron microscope showed that the interior of crosslinked dECM-GelMA-HAMA hydrogel is 3D porous network structure with suitable porosity and pore size. The shape of the skin substitute is stable with regular grid-like scaffold structure. Wound healing in the experimented animals was accelerated after being treated with 3D-printed skin substitute, which attenuate inflammatory response, increase blood perfusion around the wound, as well as promote re-epithelialization, collagen deposition and alignment, and angiogenesis. In summary, 3D-printed dECM-GelMA-HAMA tissue-engineered skin substitute loaded with hADSCs, which can be fabricated by 3D printing, can accelerate wound healing and improve healing quality by promoting angiogenesis. The hADSCs and the stable 3D-printed stereoscopic grid-like scaffold structure play a critical role in promoting wound healing.

Biocompatibility of silk methacrylate/gelatin-methacryloyl composite hydrogel and its feasibility as a vascular tissue engineering scaffold

Silk methacrylate (SilMA) has been studied extensively due to its ability to modify Silk fibroin (SF) by increasing the water solubility and enhancing the mechanical properties of SF hydrogels. However, SilMA hydrogels are generally soft with weak mechanical properties. In order to enhance the mechanical properties of hydrogel scaffolds, we used liquid nitrogen to modify SilMA to obtain a novel N2-SilMA/gelatin-methacryloyl (GelMA) composite hydrogel. N2-SilMA was successfully detected by Fourier transform infrared (FTIR) spectroscopy and 1H nuclear magnetic resonance. Scanning electron microscope showed that the composite hydrogel still had certain arrangement characteristics of SF and dense pores which met the necessary conditions for the cell scaffold. The mechanical tests showed that the mechanical properties of SilMA were greatly enhanced after modification at ultra-low temperature. We evaluated its cytocompatibility and biocompatibility, and the results showed that the composite scaffold promoted the growth of cells. Different types of composite hydrogels were injected into ICR mice and the results showed a stable scaffold structure in vivo, suggesting their ability to promote angiogenesis. In conclusion, the N2-SilMA/GelMA composite hydrogel had better mechanical properties, excellent cytocompatibility, and biological properties compared to the other groups.

Distance-Triggered Distinct Aryl Migrations on Azidodiboranes.

Derived from structurally similar precursors, two different azidodiboranes went through distinct aryl migration reactions triggered by different boron-boron separation distances. Biphenylene based diborane with a shorter boron-boron distance underwent heterolateral aryl migration to form a seven-membered azadiborepin, while xanthrene based diborane with a longer boron-boron distance afforded a stable bis-azidoborane scaffold. The pyrolysis of such a bis-azidoborane led to eight-membered oxazadiborocine through homolateral aryl migration and subsequent [3+2] cycloaddition. Density functional theory (DFT) calculations unveiled that the boron-boron separation distances were the intrinsic factors for the distinct migrations.

Catanionic Vesicles as a Facile Scaffold to Display Natural N-Glycan Ligands for Probing Multivalent Carbohydrate-Lectin Interactions.

Multivalent interactions are a key characteristic of protein-carbohydrate recognition. Phospholipid-based liposomes have been explored as a popular platform for multivalent presentation of glycans, but this platform has been plagued by the instability of typical liposomal formulations in biological media. We report here the exploitation of catanionic vesicles as a stable lipid-based nanoparticle scaffold for displaying large natural N-glycans as multivalent ligands. Hydrophobic insertion of lipidated N-glycans into the catanionic vesicle bilayer was optimized to allow for high-density display of structurally diverse N-glycans on the outer membrane leaflet. In an enzyme-linked competitive lectin-binding assay, the N-glycan-coated vesicles demonstrated a clear clustering glycoside effect, with significantly enhanced affinity for the corresponding lectins including Sambucus nigra agglutinin (SNA), concanavalin A (ConA), and human galectin-3, in comparison with their respective natural N-glycan ligands. Our results showed that relatively low density of high-mannose and sialylated complex type N-glycans gave the maximal clustering effect for binding to ConA and SNA, respectively, while relatively high-density display of the asialylated complex type N-glycan provided maximal clustering effects for binding to human galectin 3. Moreover, we also observed a macromolecular crowding effect on the binding of ConA to high-mannose N-glycans when catanionic vesicles bearing mixed high-mannose and complex-type N-glycans were used. The N-glycan-coated catanionic vesicles are stable and easy to formulate with varied density of ligands, which could serve as a feasible vehicle for drug delivery and as potent inhibitors for intervening protein-carbohydrate interactions implicated in disease.

Incorporation of a molybdenum atom in a Rubredoxin-type Centre of a de novo-designed α3DIV-L21C three-helical bundle peptide

The rational design and functionalization of small, simple, and stable peptides scaffolds is an attractive avenue to mimic catalytic metal-centres of complex proteins, relevant for the design of metalloenzymes with environmental, biotechnological and health impacts. The de novo designed α3DIV-L21C framework has a rubredoxin-like metal binding site and was used in this work to incorporate a Mo-atom. Thermostability studies using differential scanning calorimetry showed an increase of 4 °C in the melting temperature of the Mo-α3DIV-L21C when compared to the apo-α3DIV-L21C. Circular dichroism in the visible and far-UV regions corroborated these results showing that Mo incorporation provides stability to the peptide even though there were almost no differences observed in the secondary structure. A formal reduction potential of ∼ −408 mV vs. NHE, pH 7.6 was determined. Combining electrochemical results, EPR and UV–visible data we discuss the oxidation state of the molybdenum centre in Mo-α3DIV-L21C and propose that is mainly in a Mo (VI) oxidation state.