Crosslinking Conditions Research Articles

Non-invasive MRI Assessments of Tissue Microstructures and Macromolecules in the Eye upon Biomechanical or Biochemical Modulation.

The microstructural organization and composition of the corneoscleral shell (CSS) determine the biomechanical behavior of the eye, and are important in diseases such as glaucoma and myopia. However, limited techniques can assess these properties globally, non-invasively and quantitatively. In this study, we hypothesized that multi-modal magnetic resonance imaging (MRI) can reveal the effects of biomechanical or biochemical modulation on CSS. Upon intraocular pressure (IOP) elevation, CSS appeared hyperintense in both freshly prepared ovine eyes and living rat eyes using T2-weighted MRI. Quantitatively, transverse relaxation time (T2) of CSS increased non-linearly with IOP at 0–40 mmHg and remained longer than unloaded tissues after being unpressurized. IOP loading also increased fractional anisotropy of CSS in diffusion tensor MRI without apparent change in magnetization transfer MRI, suggestive of straightening of microstructural fibers without modification of macromolecular contents. Lastly, treatments with increasing glyceraldehyde (mimicking crosslinking conditions) and chondroitinase-ABC concentrations (mimicking glycosaminoglycan depletion) decreased diffusivities and increased magnetization transfer in cornea, whereas glyceraldehyde also increased magnetization transfer in sclera. In summary, we demonstrated the changing profiles of MRI contrast mechanisms resulting from biomechanical or biochemical modulation of the eye non-invasively. Multi-modal MRI may help evaluate the pathophysiological mechanisms in CSS and the efficacy of corneoscleral treatments.

Preparation of Biologically Active Film-Forming Materials Based on Polyphosphate-Modified Chitosan

The processes of preparing film-forming materials based on chitosan with surface modification and ionic cross-linking using sodium tripolyphosphate and potassium pyrophosphate respectively were studied. Optimum cross-linking conditions were identified by studying the interactions of chitosan with different levels of amino group protonation with polyphosphates with different numbers of ionized groups. These studies showed that changes in the duration of treatment of films allowed the level of swelling to be varied over the range 165% to 148% for potassium pyrophosphate and from 196% to 80% for sodium tripolyphosphate. Studies of the kinetics of study drug release showed that changes in the conditions used for modification with polyphosphates could increase the duration of release of the biologically active components to 8 h.

Preparation and thermal stability of fluoroalkyl end-capped vinyltrimethoxysilane oligomeric silica/poly(acrylonitrile-co-butadiene) nanocomposites—application to the separation of oil and water

Fluoroalkyl end-capped vinyltrimethoxysilane oligomeric silica nanocomposites-encapsulated poly(acrylonitrile-co-butadiene) (NBR) [RF-(VM-SiO2) n -RF/NBR] were prepared by the sol-gel reactions of the corresponding oligomer [RF-(VM) n -RF] in the presence of NBR under alkaline conditions. Interestingly, it was demonstrated that these obtained fluorinated nanocomposites can afford no weight loss corresponding to the contents of NBR in the composites even after calcination at 800 °C. On the other hand, the nanocomposites, which were prepared under the cross-linking conditions at 150 °C for 30 min by using NBR containing zinc oxide and sulfur (S8) as the cure activator and the cross-linker, respectively, were found to exhibit a clear weight loss corresponding to the contents of the NBR after calcination at 800 °C. The fluoroalkyl end-capped oligomeric silica nanocomposites-encapsulated the cross-linked NBR were applied to the surface modification of glass to supply the superoleophilic/superhydrophobic characteristic on the surface. The nanocomposites possessing such superoleophilic/superhydrophobic characteristic were applied to the packing material for the column chromatography to separate not only the mixture of oil and water but also the W/O emulsion. In contrast, the nanocomposites, which were prepared under no cross-linking conditions, were not applicable to the packing materials for the separation of these mixtures due to the oleophilic characteristic of the encapsulated NBR in the nanocomposite cores.

In planta chemical cross-linking and mass spectrometry analysis of protein structure and interaction in Arabidopsis.

Site-specific chemical cross-linking in combination with mass spectrometry analysis has emerged as a powerful proteomic approach for studying the three-dimensional structure of protein complexes and in mapping protein-protein interactions (PPIs). Building on the success of MS analysis of in vitro cross-linked proteins, which has been widely used to investigate specific interactions of bait proteins and their targets in various organisms, we report a workflow for in vivo chemical cross-linking and MS analysis in a multicellular eukaryote. This approach optimizes the in vivo protein cross-linking conditions in Arabidopsis thaliana, establishes a MudPIT procedure for the enrichment of cross-linked peptides, and develops an integrated software program, exhaustive cross-linked peptides identification tool (ECL), to identify the MS spectra of in planta chemical cross-linked peptides. In total, two pairs of in vivo cross-linked peptides of high confidence have been identified from two independent biological replicates. This work demarks the beginning of an alternative proteomic approach in the study of in vivo protein tertiary structure and PPIs in multicellular eukaryotes.

Biopolymer-based hydrogels for cartilage tissue engineering

Hydrogels hold a macromolecular structure comparable to that of native tissues and thus are very attractive materials for tissue engineering. The authors prepared three-dimensional bioartificial matrices based on methacrylated gelatin and chondroitin sulfate that can be tuned to closely mimic the natural environment of specific cell types – for example, chondrocytes. The authors investigated the hydrogel’s gel yield, swellability, mechanical strength, cytocompatibility, degradation and effect on chondrocyte redifferentiation. Furthermore, porcine chondrocytes were photoencapsulated into hydrogels and cultivated for 21 d. It was found that the methacrylation of chondroitin sulfate is crucial for the generation of stable hydrogels with methacrylated gelatin. Compared to pure gelatin, hybrid hydrogels possessed significantly higher swellability, while the mechanical strength remained constant. The hydrogel properties could be controlled by the mass fraction and the cross-linking density. The hydrogels as well as the cross-linking conditions were proven to render cytocompatible. Furthermore, it was found that the addition of chondroitin sulfate promoted a spherical morphology and thus chondrocyte phenotype retention. Thus, it is suggested that chondroitin sulfate is a potential redifferentiating agent of articular chondrocytes. In summary, hydrogels based on both chondroitin sulfate and gelatin hold tunable physical and biological properties and are preferable matrices for cartilage tissue engineering.

Synthesis and characterization of crosslinked gellan/PVA nanofibers for tissue engineering application

Electrospun nanofibers based on gellan are considered as promising biomaterial for tissue engineering and wound healing applications. However, major hurdles in usage of these nanofibers are their poor stability and deprived structural consistency in aqueous medium which is a prerequisite for their application in the biomedical sector. In this investigation, three dimensional nanofibers, consisting of gellan and PVA have been fabricated and then stabilized under various crosslinking conditions in order to improve their physiochemical stability. The impacts of different crosslinking procedures on the gellan/PVA nanofibers were examined in terms of changes in morphological, mechanical, swelling and biological properties. Superior tensile strength and strain was recorded in case of crosslinked nanofibers as compared to non-crosslinked nanofibers. Contact angles and swelling properties of fabricated gellan/PVA nanofibers were found to vary with the crosslinking method. All crosslinking conditions were evaluated with regard to their response towards human dermal fibroblast (3T3L1) cells. Biocompatibility studies suggested that the fabricated crosslinked gellan/PVA nanofibers hold a great prospective in the biomedical engineering arena.

Controlled Drug Release Formulation by Sequential Crosslinking of Multilayered Electrospun Gelatin Nanofiber Mat

ABSTRACTThe major aim of the present study is to develop and explore the potential of large surface area electrospun polymer nanofabric as a carrier for controlled and sustained release, in particular for hydrophobic drugs. Gelatin (type A), FDA approved natural polymer was electrospun in a mixture of solvent (20% acetic acid in water) to yield long, continuous and uniform fibers with average diameter ∼ 200 nm. Piperine was chosen as a model hydrophobic drug in this study. As gelatin is highly soluble in aqueous medium, we crosslinked electrospun gelatin nanofibers using saturated GTA vapor to increase the water resistive properties. For controlled release over a period of 12 h, we devised several strategies to vary the crosslinking conditions and accordingly understand their effect on drug release mechanism. One of such successful efforts was based on deposition of multiple layers of electrospun fabric by sandwiching between drug loaded gelatin nanofibers and without drug gelatin nanofibers from both sides. Not only the layer by layer deposition, we also crosslinked the different layer in the same sequential way. Sequential crosslinking using GTA vapor in different layers of the fabric, helped in uniform crosslinking throughout the thickness compared to crosslinking after final deposition in the form of a single layer. Effect of different crosslinking strategies was investigated in terms of surface morphology and drug stability. Finally,in-vitrorelease study was performed maintaining the physiological conditions mimicking GI tract to analyze the effect of crosslinking on the drug release profile. Thein-vitrostudies concluded that the controlled drug release can be achieved by tuning the thickness of individual fabric layer followed by their sequential crosslinking, which finally affects the diffusional barrier for drug release. Interestingly, we also found that only 6 min exposure to saturated GTA vapor is sufficient to provide the required drug release in contrast to up to 24 h as reported in literature. This finding also addresses the toxicity problem associated with the use of GTA as a cross-linker.

ChemInform Abstract: Regioselectivity of the Insertion of RNSO(S) and RCN into the M—L Bonds of the Tungsten Chlorocomplexes

AbstractReview: 39 refs.

Tendon Collagen Crosslinking Offers Potential to Improve Suture Pullout in Rotator Cuff Repair: An Ex Vivo Sheep Study.

The suture-tendon interface is often the weakest link in tendon to bone repair of massive rotator cuff tears. Genipin is a low-toxicity collagen crosslinker derived from the gardenia fruit that has been shown to augment collagen tissue strength and mechanically arrest tendon-tear progression. The purpose of the current study was to evaluate whether genipin crosslinking can sufficiently augment the suture-tendon interface to improve suture pullout strength using simple single-loop sutures and the modified Mason-Allen technique. The study also aimed to assess whether time of genipin treatment is a relevant factor in efficacy. In an ex vivo (cadaveric) sheep rotator cuff tendon model, a total of 142 suture pullout tests were performed on 32 infraspinatus tendons. Each tendon was prepared with three single-loop stitches. Two groups were pretreated by incubation in genipin solution for either 4 hours or 24 hours. Two corresponding control groups were incubated in phosphate buffered saline for the same periods. The same test protocol was applied to tendons using modified Mason-Allen technique stitch patterns. Each suture was loaded to failure on a universal materials testing machine. Suture pullout force, stiffness, and work to failure were calculated from force-displacement data, and then compared among the groups. Median single-loop pullout force on tendons incubated for 24 hours in genipin yielded an approximately 30% increase in maximum pullout force for single-loop stitches with a median of 73 N (range, 56-114 N) compared with 56 N (range, 40-69 N; difference of medians = 17 N; p = 0.028), with corresponding increases in the required work to failure but not stiffness. Genipin treatment for 4 hours showed no added benefit for suture-pullout behavior (46 N, [range, 35-95 N] versus 45 N, [range, 28-63 N]; difference of medians, 1 N; p = 1). No tested genipin crosslinking conditions indicated benefit for tendons grasped using the modified Mason-Allen technique after 4 hours (162 N, [range, 143-193 N] versus 140 N, [range, 129-151 N]; difference of medians, 22 N; p = 0.114) or after 24 hours of crosslinking (172 N, [range, 42-183 N] versus 164 N [range, 151-180 N]; difference of medians, 8 N; p = 0.886). Exogenous collagen crosslinking in genipin can markedly improve resistance to pullout at the tendon-suture interface for simple stitch patterns while the modified Mason-Allen stitch showed no benefit in an ex vivo animal model. Tendon strength augmentation by genipin pretreatment offers the potential to improve suture retention properties. Future studies are warranted for the development of clinically viable intraoperative delivery strategies and in vivo testing for safety and efficacy.

Tuning the Range of Polyacrylamide Gel Stiffness for Mechanobiology Applications.

Adjusting the acrylamide monomer and cross-linker content in polyacrylamide gels controls the hydrogel stiffness, yet the reported elastic modulus for the same formulations varies widely and these discrepancies are frequently attributed to different measurement methods. Few studies exist that examine stiffness trends across monomer and cross-linker concentrations using the same characterization platform. In this work, we use Atomic Force Microscopy and analyze force-distance curves to derive the elastic modulus of polyacrylamide hydrogels. We find that gel elastic modulus increases with increasing cross-link concentration until an inflection point, after which gel stiffness decreases with increasing cross-linking. This behavior arises because of the formation of highly cross-linked clusters, which add inhomogeneity and heterogeneity to the network structure, causing the global network to soften even under high cross-linking conditions. We identify these inflection points for three different total polymer formulations. When we alter gelation kinetics by using a low polymerization temperature, we find that gels are stiffer when polymerized at 4 °C compared to room temperature, indicating a complex relationship between gel structure, elasticity, and network formation. We also investigate how gel stiffness changes during storage over 10 days and find that specific gel formulations undergo significant stiffening (1.55 ± 0.13), which may be explained by differences in gel swelling resulting from initial polymerization parameters. Taken together, our study emphasizes the importance of polyacrylamide formulation, polymerization temperature, gelation time, and storage duration in defining the structural and mechanical properties of the polyacrylamide hydrogels.

Crosslinking of chitosan fiber by a water-soluble diepoxy crosslinker for enhanced acid resistance and its impact on fiber structures and properties

Chitosan fiber for functional textile applications has drawn great attention due to its naturally occurring antibacterial activity, moisture-retaining and no allergenic reaction. However chitosan fiber has the weight loss defect under acidic processing conditions and during peroxide bleaching, which restricts its application and decreases its functionalities. In the present work, chitosan fiber was crosslinked by a water-soluble diepoxy compound with the aim of enhancing its acid resistance. The effects of crosslinking conditions (sodium carbonate dosage, crosslinker dosage and temperature) on the weight loss of crosslinked chitosan fiber in acid solution were studied. Additionally, the effects of crosslinking on the structures, resistance to peroxide bleaching, tensile strength, thermal stability and dye adsorption properties of chitosan fiber were also discussed. The results showed that the crosslinking by the diepoxy crosslinker was able to greatly reduce the weight loss of chitosan fiber in acid solution, and slightly reduce the weight loss during hydrogen peroxide bleaching. Crosslinking treatment slightly improved the thermal stability of chitosan fiber, and had no impact on the crystalline structure and dye adsorptions of chitosan fiber. However, the dry tensile strength of chitosan fiber decreased linearly with increasing diepoxy crosslinker dosage.

Dicarboxylic acids crosslinked graphene oxide membranes for salt solution permeation

Abstract Graphene oxide (GO) membranes were crosslinked by dicarboxylic acids in order to improve the mechanical strength, stability and permeation performance. The effects of crosslinking conditions (temperature, catalyst concentration, time, crosslinker concentration) on the mechanical performance, along with the influences of crosslinker molecular structure on the thermal stability and swelling degree of GO membranes, were studied. The thermal pyrolysis temperature of crosslinked GO membranes increased about 2–8 °C while the swelling degree averagely decreased about 66% in DI water and 40% in 0.05 mol L−1 of KCl solution in comparison with those of pristine one. In the permeation of single salt solutions, the fluxes are positively correlated to the swelling degree of GO membranes. The effects of swelling degree on the permeation fluxes were firstly noticed. The crosslinked GO membranes show high permeation fluxes (more than 2 times) along with low swelling degree, demonstrating its potential application in ion dialysis.

Guidelines for standardization of bioprinting: a systematic study of process parameters and their effect on bioprinted structures

AbstractBiofabrication techniques including three-dimensional bioprinting could be used one day to fabricate living, patient-specific tissues and organs for use in regenerative medicine. Compared to traditional casting and molding methods, bioprinted structures can be much more complex, containing for example multiple materials and cell types in controlled spatial arrangement, engineered porosity, reinforcement structures and gradients in mechanical properties. With this complexity and increased function, however, comes the necessity to develop guidelines to standardize the bioprinting process, so printed grafts can safely enter the clinics. The bioink material must firstly fulfil requirements for biocompatibility and flow. Secondly, it is important to understand how process parameters affect the final mechanical properties of the printed graft. Using a gellan-alginate physically crosslinked bioink as an example, we show shear thinning and shear recovery properties which allow good printing resolution. Printed tensile specimens were used to systematically assess effect of line spacing, printing direction and crosslinking conditions. This standardized testing allowed direct comparison between this bioink and three commercially-available products. Bioprinting is a promising, yet complex fabrication method whose outcome is sensitive to a range of process parameters. This study provides the foundation for highly needed best practice guidelines for reproducible and safe bioprinted grafts.

Nanofibrous clinical-grade collagen scaffolds seeded with human cardiomyocytes induces cardiac remodeling in dilated cardiomyopathy

Limited data are available on the effects of stem cells in non-ischemic dilated cardiomyopathy (DCM). Since the diffuse nature of the disease calls for a broad distribution of cells, this study investigated the scaffold-based delivery of human induced pluripotent stem cell-derived cardiomyocytes (hiPS-CM) in a mouse model of DCM. Nanofibrous scaffolds were produced using a clinical grade atelocollagen which was electrospun and cross-linked under different conditions. As assessed by scanning electron microscopy and shearwave elastography, the optimum crosslinking conditions for hiPS-CM colonization proved to be a 10% concentration of citric acid crosslinking agent and 150 min of post-electrospinning baking. Acellular collagen scaffolds were first implanted in both healthy mice and those with induced DCM by a cardiac-specific invalidation of serum response factor (SRF). Seven and fourteen days after implantation, the safety of the scaffold was demonstrated by echocardiography and histological assessments. The subsequent step of implantation of the scaffolds seeded with hiPS-CM in DCM induced mice, using cell-free scaffolds as controls, revealed that after fourteen days heart function decreased in controls while it remained stable in the treated mice. This pattern was associated with an increased number of endothelial cells, in line with the greater vascularity of the scaffold. Moreover, a lesser degree of fibrosis consistent with the upregulation of several genes involved in extracellular matrix remodeling was observed. These results support the interest of the proposed hiPS-CM seeded electrospun scaffold for the stabilization of the DCM outcome with potential for its clinical use in the future.

The effects of different cross-linking conditions on collagen-based nanocomposite scaffolds—an in vitro evaluation using mesenchymal stem cells

Nanocomposite scaffolds which aimed to imitate a bone extracellular matrix were prepared for bone surgery applications. The scaffolds consisted of polylactide electrospun nano/sub-micron fibres, a natural collagen matrix supplemented with sodium hyaluronate and natural calcium phosphate nano-particles (bioapatite). The mechanical properties of the scaffolds were improved by means of three different cross-linking agents: N-(3-dimethylamino propyl)-N’-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide in an ethanol solution (EDC/NHS/EtOH), EDC/NHS in a phosphate buffer saline solution (EDC/NHS/PBS) and genipin. The effect of the various cross-linking conditions on the pore size, structure and mechanical properties of the scaffolds were subsequently studied. In addition, the mass loss, the swelling ratio and the pH of the scaffolds were determined following their immersion in a cell culture medium. Furthermore, the metabolic activity of human mesenchymal stem cells (hMSCs) cultivated in scaffold infusions for 2 and 7 days was assessed. Finally, studies were conducted of cell adhesion, proliferation and penetration into the scaffolds. With regard to the structural stability of the tested scaffolds, it was determined that EDC/NHS/PBS and genipin formed the most effectively cross-linked materials. Moreover, it was discovered that the genipin cross-linked scaffold also provided the best conditions for hMSC cultivation. In addition, the infusions from all the scaffolds were found to be non-cytotoxic. Thus, the genipin and EDC/NHS/PBS cross-linked scaffolds can be considered to be promising biomaterials for further in vivo testing and bone surgery applications.

Capillary condensation-induced anomalous water sorption in urethane-based coatings exposed to high humidity conditions

In this work, the water uptake of crosslinked urethane-based coatings in high humidity conditions as a function of crosslinking temperature was investigated. The water uptake values were measured using an electrochemical impedance spectroscopy technique by applying the Brasher–Kingsbury procedure. Water uptake values as high as 200% were observed in the current research. These anomalous results were explained based on capillary condensation of diffused water molecules in the nano-metrically (or nano-scale) rough hydrophilic surfaces of the pores formed by thermal degradation of the polyurethane network. The water uptake value is at its minimum when an effectively pore-free tight network formed at moderate crosslinking conditions.

Facile method for forming ionically cross-linked chitosan microcapsules from Pickering emulsion templates

Microcapsules with an oil-core hold great promise as an effective delivery system for lipophilic active ingredients used in myriad applications. Herein, we describe a facile technique for preparing chitosan microcapsules (CMCs) containing tocotrienol using oil-in-water (O/W) Pickering emulsions droplets as templates. The CMCs were fabricated under mild and safe processing conditions i.e., at ambient temperature and in the absence of organic solvents. Chitosan (CS) was precipitated above its pKa value to form a particulate emulsifier that could assemble at the O/W interface. The adsorbed particles were subsequently ionically cross-linked with sodium tripolyphosphate (TPP) at different pH values (i.e., 2–8.5) to form oil-core microcapsules. The Pickering emulsions (CPEs) and CMCs were characterized using laser light scattering, optical microscopy, confocal laser scanning microscopy (CLSM), and Fourier transform infrared spectroscopy (FTIR). The results showed that the size of the CMCs and oil leakage was affected by the pH used in the cross-linking process. The increase in the CMCs' sizes was attributed to the extended conformation of CS polymer chains at low cross-linking pH. The oil leakage was ascribed to the CMCs' porous microstructures, which resulted from the swelling and/or dissolution of some parts of the shell at low cross-linking pH. The varied porous microstructure exhibited by the CMCs was found to be dependent on the cross-linking pH. Therefore, by tuning the cross-linking conditions, one may obtain tailor-made microcapsules that are well suited for the rapid or controlled release of lipophilic compounds.

Design, synthesis and characterization of a pH-sensitive DS/GM hydrogel

Dextran sulfate/glycidyl methacrylate (DS/GM) hydrogels were synthesized by intermolecular side-chain reaction of DS hydroxyl groups with monomeric crosslinking agent, GM in aqueous solutions at 75 °C in the basic medium in this study. The optimum conditions of effective crosslinking for DS/GM system were determined with changing amounts of crosslinker and initiator (benzoyl peroxide, BPO). The swelling abilities of the hydrogels were explained with determining of certain structural parameters for the hydrogel such as equilibrium water content, percentages of gellation, and swelling ratio. pH dependent swelling behaviors of hydrogels were investigated and swelling ability at basic medium was higher than that at acidic medium. Surface morphologies of DS and its hydrogels were examined by scanning electron microscope (SEM) and they compared. Structures and thermal stabilities of DS/GM hydrogels were characterized by infrared (FTIR), thermal gravimetric analysis (TGA), differential thermal analysis (DTA), and Differential Scanning Calorimeter (DSC); effect of GM on crosslinking was investigated. The viscosities of DS/GM reaction products were recorded continuously as a function of time with rotary viscometer and gellation points of binary system reaction between DS and GM were determined via observed viscosity numbers, changes during reaction.

Biological and mechanical characterization of chitosan-alginate scaffolds for growth factor delivery and chondrogenesis.

Chitosan-alginate (Ch-Al) natural polysaccharide blends have been used for wound healing, tissue engineering, and drug delivery due to their ability to form pH-dependent ionic chain-chain interactions. Yet, the biomechanical properties and growth factor (GF) release kinetics of Ch-Al, which are important in controlling the microenvironment during tissue regeneration, have not been fully explored. This study examines the compressive elastic modulus of many Ch-Al scaffold formulations and crosslinking conditions, and also the strain recovery after compressive deformation of Ch-Al scaffolds, both of which make Ch-Al an attractive composite for reproducing articular cartilage's resistance to and resiliency under compression. Cell viability, proliferation, and in vitro cartilaginous matrix production (collagen type II, glycosaminoglycans, aggrecan) without supplemental GFs are also investigated, demonstrating the polymer blend's inherent chondrogenic properties. Additionally, this study explores the ability of Ch-Al chain functional groups to control and extend GF delivery and minimize GF burst release, using model proteins BSA and histone at high loading dose and chondrogenic protein TGF-β1 at low loading dose in complete media. Expedited cartilaginous matrix synthesis on Ch-Al with low dose TGF-β1 release is evaluated, with Ch-Al supporting homogeneous matrix deposition and lacunae formation as early as 3 weeks due to Ch-Al's maintenance of GF bioactivity and sustained GF delivery. These results illustrate the potential to focus the formulational range of Ch-Al to provide enhanced mechanical performance and controlled, bioactive GF release to cooperatively promote cartilage regeneration. © 2015 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 105B: 272-282, 2017.

Regioselectivity of the insertion of RNSO(S) and RCN into the M–L bonds of the tungsten chlorocomplexes

The results of studying the reaction behavior of tungsten hexachloride toward organic iso(thio)cyanates and their combinations with nitriles are generalized. The factors determining the multiplet insertion of iso(thio)cyanates at the W–Cl bond and the conditions of symmetrical or asymmetrical crosslinking of the inserted fragments into heterocycles upon their trimerization (on the basis of the X-ray structural data on the products of the partial or complete hydrolysis of the complexes) are discussed. The reasons determining the direction of the combined insertion of heterocumulenes and nitriles at the W–Cl or W–N bond are considered, which is important for the targeted design of complexes of various architecture and new poorly accessible related heterocyclic compounds.