A conserved strategy to attack collagen: The activator domain in bacterial collagenases unwinds triple-helical collagen.

Bacterial collagenases are important virulence factors, secreted by several pathogenic Clostridium, Bacillus, Spirochaetes, and Vibrio species. Yet, the mechanism by which these enzymes cleave collagen is not well understood. Based on biochemical and mutational studies we reveal that collagenase G (ColG) from Hathewaya histolytica recognizes and processes collagen substrates differently depending on their nature (fibrillar vs. soluble collagen); distinct dynamic interactions between the activator and peptidase domain are required based on the substrate type. Using biochemical and circular dichroism studies, we identify the presumed noncatalytic activator domain as the single-domain triple helicase that unwinds collagen locally, transiently, and reversibly.

Swim bladder-derived biomaterials: structures, compositions, properties, modifications, and biomedical applications.

Animal-derived biomaterials have been extensively employed in clinical practice owing to their compositional and structural similarities with those of human tissues and organs, exhibiting good mechanical properties and biocompatibility, and extensive sources. However, there is an associated risk of infection with pathogenic microorganisms after the implantation of tissues from pigs, cattle, and other mammals in humans. Therefore, researchers have begun to explore the development of non-mammalian regenerative biomaterials. Among these is the swim bladder, a fish-derived biomaterial that is rapidly used in various fields of biomedicine because of its high collagen, elastin, and polysaccharide content. However, relevant reviews on the biomedical applications of swim bladders as effective biomaterials are lacking. Therefore, based on our previous research and in-depth understanding of this field, this review describes the structures and compositions, properties, and modifications of the swim bladder, with their direct (including soft tissue repair, dural repair, cardiovascular repair, and edible and pharmaceutical fish maw) and indirect applications (including extracted collagen peptides with smaller molecular weights, and collagen or gelatin with higher molecular weights used for hydrogels, and biological adhesives or glues) in the field of biomedicine in recent years. This review provides insights into the use of swim bladders as source of biomaterial; hence, it can aid biomedicine scholars by providing directions for advancements in this field.

Investigation of the Presence of Fibrillin in Sea Cucumber (Apostichopus japonicus) Body Wall by Utilizing Targeted Proteomics and Visualization Strategies.

Fibrillin is an important structural protein in connective tissues. The presence of fibrillin in sea cucumber Apostichopus japonicus is still poorly understood, which limits our understanding of the role of fibrillin in the A. japonicus microstructure. The aim of this study was to clarify the presence of fibrillin in the sea cucumber A. japonicus body wall. Herein, the presence of fibrillin in sea cucumber A. japonicus was investigated by utilizing targeted proteomics and visualization strategies. The contents of three different isoforms of fibrillin with high abundance in A. japonicus were determined to be 0.96, 2.54, and 0.15 µg/g (wet base), respectively. The amino acid sequence of fibrillin (GeneBank number: PIK56741.1) that started at position 631 and ended at position 921 was selected for cloning and expressing antigen. An anti-A. japonicus fibrillin antibody with a titer greater than 1:64 000 was successfully obtained. It was observed that the distribution of fibrillin in the A. japonicus body wall was scattered and dispersed in the form of fibril bundles at the microscale. It further observed that fibrillin was present near collagen fibrils and some entangled outside the collagen fibrils at the nanoscale. Moreover, the stoichiometry of the most dominant collagen and fibrillin molecules in A. japonicus was determined to be approximately 250:1. These results contribute to an understanding of the role of fibrillin in the sea cucumber microstructure.

Sulfated hyaluronic acid/collagen-based biomimetic hybrid nanofiber skin for diabetic wound healing: Development and preliminary evaluation.

Diabetic foot ulcers (DFUs) are one of the most serious and devastating complication of diabetes, manifesting as foot ulcers and impaired wound healing in patients with diabetes mellitus. To solve this problem, sulfated hyaluronic acid (SHA)/collagen-based nanofibrous biomimetic skins was developed and used to promote the diabetic wound healing and skin remodeling. First, SHA was successfully synthetized using chemical sulfation and incorporated into collagen (COL) matrix for preparing the SHA/COL hybrid nanofiber skins. The polyurethane (PU) was added into those hybrid scaffolds to make up the insufficient mechanical properties of SHA/COL nanofibers, the morphology, surface properties and degradation rate of hybrid nanofibers, as well as cell responses upon the nanofibrous scaffolds were studied to evaluate their potential for skin reconstruction. The results demonstrated that the SHA/COL, SHA/HA/COL hybrid nanofiber skins were stimulatory of cell behaviors, including a high proliferation rate and maintaining normal phenotypes of specific cells. Notably, SHA/COL and SHA/HA/COL hybrid nanofibers exhibited a significantly accelerated wound healing and a high skin remodeling effect in diabetic mice compared with the control group. Overall, SHA/COL-based hybrid scaffolds are promising candidates as biomimetic hybrid nanofiber skin for accelerating diabetic wound healing.

Chromatographic separation by RPLC-ESI-MS of all hydroxyproline isomers for the characterization of collagens from different sources.

During collagen biosynthesis, proline is post-translationally converted to hydroxyproline by specific enzymes. This amino acid, unique to collagen, plays a crucial role in stabilizing the collagen triple helix structure and could serve as an important biomarker for collagen content and quality analysis. Hydroxyproline has four isomers, depending on whether proline is hydroxylated at position 4 or 3 and on whether the cis- or trans- conformation is formed. Moreover, as extensive hydrolysis of collagen is required for its amino acid analysis, epimerization may also occur, although to a lesser extent, giving a total of eight possible isomers. The aim of the present study was to develop a reversed-phase high-performance liquid chromatography-UV-mass spectrometry (RPLC-UV-MS) method for the separation and quantification of all eight hydroxyproline isomers. After the chiral derivatization of the hydroxyproline isomers with Nα-(2,4-dinitro-5-fluorophenyl)-L-valinamide (L-FDVA), to enable their UV detection, the derivatized diastereoisomers were separated by testing different C18 column technologies and morphologies and optimizing operative conditions such as the mobile phase composition (solvent, additives), elution mode, flow rate and temperature. Baseline resolution of all eight isomers was achieved on a HALO® ES-C18 reversed-phase column (150×1.5 mm, 2.7 µm, 160 Å) using isocratic elution and MS-compatible mobile phase. The optimized method was validated for the quantification of hydroxyproline isomers and then applied to different collagen hydrolysates to gain insight and a deeper understanding of hydroxyproline abundances in different species (human, chicken) and sources (native, recombinant).

Plasma-Activated Polydimethylsiloxane Microstructured Pattern with Collagen for Improved Myoblast Cell Guidance.

We focused on polydimethylsiloxane (PDMS) as a substrate for replication, micropatterning, and construction of biologically active surfaces. The novelty of this study is based on the combination of the argon plasma exposure of a micropatterned PDMS scaffold, where the plasma served as a strong tool for subsequent grafting of collagen coatings and their application as cell growth scaffolds, where the standard was significantly exceeded. As part of the scaffold design, templates with a patterned microstructure of different dimensions (50 × 50, 50 × 20, and 30 × 30 µm2) were created by photolithography followed by pattern replication on a PDMS polymer substrate. Subsequently, the prepared microstructured PDMS replicas were coated with a type I collagen layer. The sample preparation was followed by the characterization of material surface properties using various analytical techniques, including scanning electron microscopy (SEM), energy-dispersive X-ray spectroscopy (EDS), and X-ray photoelectron spectroscopy (XPS). To evaluate the biocompatibility of the produced samples, we conducted studies on the interactions between selected polymer replicas and micro- and nanostructures and mammalian cells. Specifically, we utilized mouse myoblasts (C2C12), and our results demonstrate that we achieved excellent cell alignment in conjunction with the development of a cytocompatible surface. Consequently, the outcomes of this research contribute to an enhanced comprehension of surface properties and interactions between structured polymers and mammalian cells. The use of periodic microstructures has the potential to advance the creation of novel materials and scaffolds in tissue engineering. These materials exhibit exceptional biocompatibility and possess the capacity to promote cell adhesion and growth.

Synthesis and Characterization of Amine and Aldehyde-Containing Copolymers for Enzymatic Crosslinking of Gelatine.

In tissue engineering (TE), the support structure (scaffold) plays a key role necessary for cell adhesion and proliferation. The protein constituents of the extracellular matrix (ECM), such as collagen, its derivative gelatine, and elastin, are the most attractive materials as possible scaffolds. To improve the modest mechanical properties of gelatine, a strategy consists of crosslinking it, as naturally occurs for collagen, which is stiffened by the oxidative action of lysyl oxidase (LO). Here, a novel protocol to crosslink gelatine has been developed, not using the commonly employed crosslinkers, but based on the formation of imine bonds or on aldolic condensation reactions occurring between gelatine and properly synthesized copolymers containing amine residues via LO-mediated oxidation. Particularly, we first synthesized and characterized an amino butyl styrene monomer (5), its copolymers with dimethylacrylamide (DMAA), and its terpolymer with DMAA and acrylic acid (AA). Three acryloyl amidoamine monomers (11a-c) and their copolymers with DMAA were then prepared. A methacrolein (MA)/DMAA copolymer already possessing the needed aldehyde groups was finally developed to investigate the relevance of LO in the crosslinking process. Oxidation tests of amine copolymers with LO were performed to identify the best substrates to be used in experiments of gelatine reticulation. Copolymers obtained with 5, 11b, and 11c were excellent substrates for LO and were employed with MA/DMAA copolymers in gelatine crosslinking tests in different conditions. Among the amine-containing copolymers, that obtained with 5 (CP5/DMMA-43.1) afforded a material (M21) with the highest crosslinking percentage (71%). Cytotoxicity experiments carried out on two cell lines (IMR-32 and SH SY5Y) with the analogous (P5) of the synthetic constituent of M21 (CP5/DMAA) had evidenced no significant reduction in cell viability, but proliferation promotion, thus establishing the biocompatibility of M21 and the possibility to develop it as a new scaffold for TE, upon further investigations.

Enhanced dietary reconstruction of Korean prehistoric populations by combining δ13C and δ15N amino acids of bone collagen.

Compound specific stable isotope analysis of amino acids (CSIA-AA) is a powerful tool for determining dietary behaviors in complex environments and improving dietary reconstructions. Here, we conducted CSIA-AA on human (n = 32) and animal (n = 13) remains from two prehistoric archaeological sites (Mumun, Imdang) to assess in more detail the dietary sources consumed by prehistoric Korean populations. Results of estimated trophic position (TP) using Δ15NGlx-Phe show that the Imdang individuals consumed aquatic resources, as well as terrestrial resources. Principal component analysis (PCA) using δ13C and δ15N essential amino acid (EAA) values show that the Imdang humans closely cluster with game birds and terrestrial herbivores, whilst the Mumun humans closely cluster with C4 plants. Quantitative estimation by a Bayesian mixing model (MixSIAR) indicates that the Imdang humans derived a large proportion of their proteins from terrestrial animals and marine fish, whereas the main protein sources for the Mumun humans were C4 plants and terrestrial animals. Additionally, the comparison between the EAA and bulk isotope models shows that there is a tendency to overestimate the consumption of plant proteins when using bulk isotopic data. Our CSIA-AA approach reveals that in prehistoric Korea there were clear differences in human diets through time. This study adds to a growing body of literature that demonstrates the potential of CSIA-AA to provide more accurate estimations of protein consumption in mixed diets than previous bulk isotopic studies.

Micromechanical modelling of transverse fracture behaviour of lamellar bone using a phase-field damage model: The role of non-collagenous proteins and mineralised collagen fibrils.

At the tissue-scale and above, there are now well-established structure-property relationships that provide good approximations of the biomechanical performance of bone through, for example, power-law relationships that relate tissue mineral density to elastic properties. However, below the tissue-level, the individual role of the constituents becomes prominent and these simple relationships tend to break down, with more detailed theoretical and computational models are required to describe the mechanical response. In this study, a two-dimensional micromechanics damage-based representative volume element (RVE) of lamellar bone was developed, which included a novel implementation of a phase-field damage model to describe the behaviour of non-collagenous proteins at mineral-mineral and mineral-fibril interface regions. It was found that, while the stiffness of the tissue was governed by the relative proportion of extra-fibrillar mineral and mineralised collagen fibrils, the strength and toughness of the tissue in transverse direction relied on the interactions occurring at mineral-mineral and mineral-fibril interfaces, highlighting the prominence of non-collagenous proteins in determine fracture-based processes at this scale. While fractures tended to initiate in mineral rich areas of the extra-fibrillar mineral matrix, it was found that the presence of mineralised collagen fibrils at low density did not provide a substantial contribution to crack propagation behaviour under transverse loading. However, at physiological volume fraction (VfMCF=50%), different scenarios could arise depending on the relative strength value of the interphase around the MCFs ( [Formula: see text] ) to the interphase between individual minerals ( [Formula: see text] ): (i) When [Formula: see text] , MCFs appear to facilitate crack propagation with MCF-mineral debonding being the dominant failure mode; (ii) once γ>1, the MCFs hinder the microcracks, leading to inhibition of crack propagation, which can be regarded as an energy dissipation mechanism. The effective fracture properties of the tissue also experience a sudden increase in fracture work density (J-integral) once the crack is arrested by MCFs or severely deflected. Collectively, the predicted behaviour of the model compared well to those reported through experimental and computational methods, highlighting its potential to provide further understanding into the mechanistic response of bone ultrastructure alterations related to the structural and compositional changes resulting from disease and aging.

Exploring the hierarchical structure of lamellar bone and its impact on fracture behaviour: A computational study using a phase field damage model.

Bone is a naturally occurring composite material composed of a stiff mineral phase and a compliant organic matrix of collagen and non-collagenous proteins (NCP). While diverse mineral morphologies such as platelets and grains have been documented, the precise role of individual constituents, and their morphology, remains poorly understood. To understand the role of constituent morphology on the fracture behaviour of lamellar bone, a damage based representative volume element (RVE) was developed, which considered various mineral morphologies and mineralised collagen fibril (MCF) configurations. This model framework incorporated a novel phase-field damage model to predict the onset and evolution of damage at mineral-mineral and mineral-MCF interfaces. It was found that platelet-based mineral morphologies had superior mechanical performance over their granular counterparts, owing to their higher load-bearing capacity, resulting from a higher aspect ratio. It was also found that MCFs had a remarkable capacity for energy dissipation under axial loading, with these fibrillar structures acting as barriers to crack propagation, thereby enhancing overall elongation and toughness. Interestingly, the presence of extrafibrillar platelet-based minerals also provided an additional toughening through a similar mechanism, whereby these structures also inhibited crack propagation. These findings demonstrate that the two primary constituent materials of lamellar bone play a key role in its toughening behaviour, with combined effect by both mineral and MCFs to inhibit crack propagation at this scale. These results have provided novel insight into the fracture behaviour of lamellar bone, enhancing our understanding of microstructure-property relationships at the sub-tissue level.

Fabrication and characterization of a bioactive composite scaffold based on polymeric collagen/gelatin/nano ß-TCP for alveolar bone regeneration.

One strategy to correct alveolar bone defects is use of bioactive bone substitutes to maintain the structure of defect site and facilitate cells and vessels' ingrowth. This study aimed to fabricate and characterize the freeze-dried bone regeneration scaffolds composed of polymeric Type I collagen, nano Beta-tricalcium phosphate (ß-TCP), and gelatin. The stable structures of scaffolds were obtained by thermal crosslinking and EDC/NHS ((1-ethyl-3-(3-dimethylaminopropyl) carbodiimide)/(N-hydroxysuccinimide)) chemical crosslinking processes. Subsequently, the physicochemical and biological properties of the scaffolds were characterized and assessed. The results indicated the bioactive composite scaffolds containing 10% and 20% (w/v) nano ß-TCP exhibited suitable porosity (84.45 ± 25.43 nm, and 94.51 ± 14.69 nm respectively), a rapid swelling property (reaching the maximum swelling rate at 1 h), excellent degradation resistance (residual mass percentage of scaffolds higher than 80% on day 90 in PBS and Type I collagenase solution respectively), and sustained calcium release capabilities. Moreover, they displayed outstanding biological properties, including superior cell viability, cell adhesion, and cell proliferation. Additionally, the scaffolds containing 10% and 20% (w/v) nano ß-TCP could promote the osteogenic differentiation of MC3T3-E1. Therefore, the bioactive composite scaffolds containing 10% and 20% (w/v) nano ß-TCP could be further studied for being used to treat alveolar bone defects in vivo.

Structural parameters defining distribution of collagen fiber directions in human carotid arteries.

Collagen fiber arrangement is decisive for constitutive description of anisotropic mechanical response of arterial wall. In this study, their orientation in human common carotid artery was investigated using polarized light microscopy and an automated algorithm giving more than 4·106 fiber angles per slice. In total 113 slices acquired from 18 arteries taken from 14 cadavers were used for fiber orientation in the circumferential-axial plane. All histograms were approximated with unimodal von Mises distribution to evaluate dominant direction of fibers and their concentration parameter. 10 specimens were analyzed also in circumferential-radial and axial-radial planes (2-4 slices per specimen in each plane); the portion of radially oriented fibers was found insignificant. In the circumferential-axial plane, most specimens showed a pronounced unimodal distribution with angle to circumferential direction µ = 0.7° ± 9.4° and concentration parameter b = 3.4 ± 1.9. Suitability of the unimodal fit was confirmed by high values of coefficient of determination (mean R2 = 0.97, median R2 = 0.99). Differences between media and adventitia layers were not found statistically significant. The results are directly applicable as structural parameters in the GOH constitutive model of arterial wall if the postulated two fiber families are unified into one with circumferential orientation.

Effect of mechanical fatigue on commercial bioprosthetic TAVR valve mechanical and microstructural properties.

Valvular structural deterioration is of particular concern for transcatheter aortic valve replacements due to their suspected shorter longevity and increasing use in younger patient populations. In this work we investigated the mechanical and microstructural changes in commercial TAVR valves composed of both glutaraldehyde fixed bovine and porcine pericardium (GLBP and GLPP) following accelerated wear testing (AWT) as outlined in ISO 5840 standards. This provided greater physiological relevance to the loading compared to previous studies and by utilizing digital image correlation we were able to obtain strain contours for each leaflet pre and post fatigue and identify sites of fatigue damage. The areas of greatest change in mechanical strain for each leaflet were then further probed using biaxial tensile testing, confocal microscopy, and electron microscopy. It was observed that overall strain decreased in the GLPP valves following AWT of 200 million cycles while the GLBP valve showed an increase in overall strain. Biaxial tensile testing showed a statistically significant reduction in stress for GLPP while no significant changes were seen for GLBP. Both confocal and electron microscopy showed a disruption to the gross collagen organization and fibrillar structure, including fragmentation, for GLPP but only the former for GLBP. However, further test data is required to confirm these findings and to provide a better understanding of this fatigue pathway is required such that it can be incorporated into both valve design and selection processes to improve overall longevity for both GLPP and GLBP devices.

Versatile Triblock Peptide Self-Assembly System to Mimic Collagen Structure and Function.

The construction of collagen mimetic peptides has been a hot topic in tissue engineering due to their attractive advantages, such as virus-free nature and low immunogenicity. However, all of the reported self-assembled peptides rely on the inclusion of risky elements of potential safety concerns or lack the capability of incorporating critical functional motifs. A versatile self-assembly design of pure synthetic peptides that can mimic the collagen structure and function remains an insurmountably challenging target. We have herein created a type of triblock peptide consisting of a central triple helical block and N-terminal/C-terminal blocks with oppositely charged amino acids. Favorable electrostatic interactions between the two terminal blocks have been demonstrated to trigger the triblock peptides to form collagen-like nanofibers with a distinct D-banding pattern. A length of 3 or above charged amino acid pairs as well as the maintenance of the triple helical conformation are required for the self-assembly of triblock peptides. Notably, integrin and discoidin domain receptor (DDR) binding sequences GFOGER and GVMGFO have been well demonstrated as vivid examples of convenient incorporation of functional motifs into the triblock peptides without interfering with their self-assembly. These triblock peptides provide a robust and versatile strategy to create next-generation peptide-based biomaterials that can recapitulate the structure and function of collagen, which have promising applications in the fields of tissue engineering and regenerative medicine.

Controlled Release of Drugs from Extracellular Matrix-Derived Peptide-Based Nanovesicles through Tailored Noncovalent Interactions.

Elastin-collagen nanovesicles (ECnV) have emerged as a promising platform for drug delivery due to their tunable physicochemical properties and biocompatibility. The potential of nine distinct ECnVs to serve as drug-delivery vehicles was investigated in this study, and it was demonstrated that various small-molecule cargo (e.g., dexamethasone, methotrexate, doxorubicin) can be encapsulated in and released from a set of ECnVs, with extents of loading and rates of release dictated by the composition of the elastin domain of the ECnV and the type of cargo. Elastin-like peptides (ELPs) and collagen-like peptides (CLPs) of various compositions were produced; the secondary structure of the corresponding peptides was determined using CD, and the morphology and average hydrodynamic diameter (∼100 nm) of the ECnVs were determined using TEM and DLS. It was observed that hydrophobic drugs exhibited slower release kinetics than hydrophilic drugs, but higher drug loading was achieved for the more hydrophilic Dox. The collagen-binding ability of the ECnVs was demonstrated through a 2D collagen-binding assay, suggesting the potential for longer retention times in collagen-enriched tissues or matrices. Sustained release of drugs for up to 7 days was observed and, taken together with the collagen-binding data, demonstrates the potential of this set of ECnVs as a versatile drug delivery vehicle for longer-term drug release of a variety of cargo. This study provides important insights into the drug delivery potential of ECnVs and offers useful information for future development of ECnV-based drug delivery systems for the treatment of various diseases.

Inhibition of platelet adhesion to exposed subendothelial collagen by steric hindrance with blocking peptide nanoparticles.

The inhibition of platelet adhesion to collagen in exposed vessels represents an innovative approach to the treatment of atherosclerosis and thrombosis. This study aimed to engineer peptide-based nanoparticles that prevent platelet binding to subendothelial collagen by engaging with collagen with high affinity. We examined the interactions between integrin α2/ glycoprotein VI/ von Willebrand factor A3 domain and collagen, as well as between the synthesized peptide nanoparticles and collagen, utilizing molecular dynamics simulations and empirical assays. Our findings indicated that the bond between von Willebrand factor and collagen was more robust. Specifically, the sequences SITTIDV, VDVMQRE, and YLTSEMH in von Willebrand factor were identified as essential for its attachment to collagen. Based on these sequences, three peptide nanoparticles were synthesized (BPa: Capric-GNNQQNYK-SITTIDV, BPb: Capric-GNNQQNYK-VDVMQRE, BPc: Capric-GNNQQNYK-YLTSEMH), each displaying significant affinity towards collagen. Of these, the BPa nanoparticles exhibited the most potent interaction with collagen, leading to a 75% reduction in platelet adhesion.

Collagen-decorated electrospun scaffolds of unsaturated copolyesters for bone tissue regeneration.

Many efforts have been devoted to bone tissue to regenerate damaged tissues, and the development of new biocompatible materials that match the biological, mechanical, and chemical features required for this application is crucial. Herein, a collagen-decorated scaffold was prepared via electrospinning using a synthesized unsaturated copolyester (poly(globalide-co-pentadecalactone)), followed by two coupling reactions: thiol-ene functionalization with cysteine and further conjugation via EDC/NHS chemistry with collagen, aiming to design a bone tissue regeneration device with improved hydrophilicity and cell viability. Comonomer ratios were varied, affecting the copolymer's thermal and chemical properties and highlighting the tunable features of this copolyester. Functionalization with cysteine created new carboxyl and amine groups needed for bioconjugation with collagen, which is responsible for providing biological and structural integrity to the extra-cellular matrix. Bioconjugation with collagen turned the scaffold highly hydrophilic, decreasing its contact angle from 107 ± 2° to 0°, decreasing the copolymer crystallinity by 71%, and improving cell viability by 85% compared with the raw scaffold, thus promoting cell growth and proliferation. The highly efficient and biosafe strategy to conjugate polymers and proteins created a promising device for bone repair in tissue engineering.

Stiffness assisted cell-matrix remodeling trigger 3D mechanotransduction regulatory programs.

Engineered matrices provide a valuable platform to understand the impact of biophysical factors on cellular behavior such as migration, proliferation, differentiation, and tissue remodeling, through mechanotransduction. While recent studies have identified some mechanisms of 3D mechanotransduction, there is still a critical knowledge gap in comprehending the interplay between 3D confinement, ECM properties, and cellular behavior. Specifically, the role of matrix stiffness in directing cellular fate in 3D microenvironment, independent of viscoelasticity, microstructure, and ligand density remains poorly understood. To address this gap, we designed a nanoparticle crosslinker to reinforce collagen-based hydrogels without altering their chemical composition, microstructure, viscoelasticity, and density of cell-adhesion ligand and utilized it to understand cellular dynamics. This crosslinking mechanism utilizes nanoparticles as crosslink epicenter, resulting in 10-fold increase in mechanical stiffness, without other changes. Human mesenchymal stem cells (hMSCs) encapsulated in 3D responded to mechanical stiffness by displaying circular morphology on soft hydrogels (5 kPa) and elongated morphology on stiff hydrogels (30 kPa). Stiff hydrogels facilitated the production and remodeling of nascent extracellular matrix (ECM) and activated mechanotransduction cascade. These changes were driven through intracellular PI3AKT signaling, regulation of epigenetic modifiers and activation of YAP/TAZ signaling. Overall, our study introduces a unique biomaterials platform to understand cell-ECM mechanotransduction in 3D for regenerative medicine as well as disease modelling.

[The Three-dimensional Environment of Type â Collagen Gels With Varying Stiffness Modulates the Immunological Functions of NK Cells]. / 不同刚度â 型胶原凝胶三维环境调控NKç»†èƒžå ç–«åŠŸèƒ½.

Objective: To construct type Ⅰ collagen gels with different stiffness and to investigate the effects of three-dimensional (3D) culture environments of the gels on the morphology, free migration ability, and cell killing function of natural killer (NK) cells. Methods: Type Ⅰ collagen was isolated from the tails of Sprague Dawley (SD) rats and collagen gels with different levels of stiffnesses were prepared accordingly. The microstructure of the collagen gels was observed by laser confocal microscopy. The stiffness of the collagen gels was assessed by measuring the plateau modulus with a rheometer. NK-92MI cells were cultured in collagen gels with different levels of stiffness. The morphology of NK-92MI cells was observed by inverted microscope. High content imaging system was used to record the free migration process of NK-92MI cells and analyze the migration speed and distance. NK-92MI cells were cultured with type Ⅰ collagen gels with different levels of stiffness for 24 h and 48 h and, then, co-cultured with human colorectal DLD-1, a human adenocarcinoma epithelial cell line. CCK8 assay was performed to determine the proliferation rate of DLD-1 cells and analyze the cell killing ability of NK-92MI cells. Results: Low-stiffness type Ⅰ collagen gel and high-stiffness type Ⅰ collagen gel with the respective stiffness of (10.970±2.10) Pa and (114.50±3.40) Pa were successfully prepared. Compared with those cultured with the low-stiffness type Ⅰ collagen gel, the NK-92MI cells in the high-stiffness type Ⅰ collagen gel showed a more elongated shape (P<0.05), the mean area of the cells was reduced ([69.88±26.97] µm2 vs. [46.59±21.62] µm2, P<0.05), the roundness of the cells decreased (0.82±0.12 vs. 0.78±0.18, P<0.05), cell migration speed decreased ([2.50±0.91] µm/min vs. [1.70±0.72] µm/min, P<0.001) and the migration distance was shortened ([147.10±53.74] µm vs. [98.03± 40.95] µm, P<0.0001), with all the differences being statistically significant. Compared with those cultured with the low-stiffness type Ⅰ collagen gel, NK-92MI cells cultured with high-stiffness type Ⅰ collagen gel for 24 h could promote DLD-1 cell proliferation, with the proliferation rate being (46.39±12.79)% vs. (65.87±4.45)% (P<0.05) and reduce the cell killing ability. Comparison of the cells cultured for 48 h led to similar results, with the proliferation rates being (31.36±2.88)% vs. (74.57±2.16)% (P<0.05), and the differences were all statistically significant. Conclusion: The 3D culture environment of type Ⅰ collagen gels with different levels of stiffness alters the morphology, migration ability, and killing function of NK-92MI cells. This study provides the research basis for exploring and understanding the mechanisms by which the biomechanical microenvironment affects the immune response of NK cells, as well as laying the theoretical foundation for optimizing immunotherapy protocols.

Structure and function of engineered stromal cell-derived factor-1α.

To design biologically active, collagen-based scaffolds for bone tissue engineering, we have synthesized chimeric proteins consisting of stromal cell-derived factor-1α (SDF) and the von Willebrand factor A3 collagen-binding domain (CBD). The chimeric proteins were used to evaluate the effect of domain linkage and its order on the structure and function of the SDF and CBD. The structure of the chimeric proteins was analyzed by circular dichroism spectroscopy, while functional analysis was performed by a cell migration assay for the SDF domain and a collagen-binding assay for the CBD domain. Furthermore, computational structural prediction was conducted for the chimeric proteins to examine the consistency with the results of structural and functional analyses. Our structural and functional analyses as well as structural prediction revealed that linking two domains can affect their functions. However, their order had minor effects on the three-dimensional structure of CBD and SDF in the chimeric proteins.