Iron-Confined CRISPR/Cas9-Ribonucleoprotein Delivery System for Redox-Responsive Gene Editing.

Clustered regularly interspaced short palindromic repeat (CRISPR)-associated protein 9 (Cas9) is a promising gene editing tool to treat diseases at the genetic level. Nonetheless, the challenge of the safe and efficient delivery of CRISPR/Cas9 to host cells constrains its clinical applicability. In the current study, a facile, redox-responsive CRISPR/Cas9-Ribonucleoprotein (RNP) delivery system by combining iron-coordinated aggregation with liposomes (Fe-RNP@L) is reported. The Fe-RNP is formed by the coordination of Fe3+ with amino and carboxyl groups of Cas9, which modifies the lipophilicity and surface charge of RNP and alters cellular uptake from primary endocytosis to endocytosis and cholesterol-dependent membrane fusion. RNP can be rapidly and reversibly released from Fe-RNP in response to glutathione without loss of structural integrity and enzymatic activity. In addition, iron coordination also improves the stability of RNP and substantially mitigates cytotoxicity. This construct enabled highly efficient cytoplasmic/nuclear delivery (≈90%) and gene-editing efficiency (≈70%) even at low concentrations. The high payload content, high editing efficiency, good stability, low immunogenicity, and ease of production and storage, highlight its potential for diverse genome editing and clinical applications.

Black phosphorous-based biomaterials for bone defect regeneration: a systematic review and meta-analysis.

Critical-sized bone defects are always difficult to treat, and they are associated with a significant burden of disease in clinical practice. In recent decades, due to the fast development of biomaterials and tissue engineering, many bioinspired materials have been developed to treat large bone defects. Due to the excellent osteoblastic ability of black phosphorous (BP), many BP-based biomaterials have been developed to treat bone defects. Therefore, there are abundant studies as well as a tremendous amount of research data. It is urgent to conduct evidence-based research to translate these research data and results into validated scientific evidence. Therefore, in our present study, a qualitative systematic review and a quantitative meta-analysis were performed. Eighteen studies were included in a systematic review, while twelve studies were included in the meta-analysis. Our results showed that the overall quality of experimental methods and reports of biomaterials studies was still low, which needs to be improved in future studies. Besides, we also proved the excellent osteoblastic ability of BP-based biomaterials. But we did not find a significant effect of near-infrared (NIR) laser in BP-based biomaterials for treating bone defects. However, the quality of the evidence presented by included studies was very low. Therefore, to accelerate the clinical translation of BP-based biomaterials, it is urgent to improve the quality of the study method and reporting in future animal studies. More evidence-based studies should be conducted to enhance the quality and clinical translation of BP-based biomaterials.

Single SNP- and pathway-based genome-wide association studies for beak deformity in chickens using high-density 600K SNP arrays.

BACKGROUND: Beak deformity, typically expressed as the crossing of upper and lower mandibles, is found in several indigenous chicken breeds, including the Beijing-You chickens studied here. Beak deformity severely impairs the birds' growth and welfare. Although previous studies shed some light on the genetic regulation of this complex trait, the genetic basis of this malformation remains incompletely understood. RESULTS: In this study, single SNP- and pathway-based genome-wide association studies (GWASs) were performed using ROADTRIPS and SNP ratio test (SRT), respectively. A total of 48 birds with deformed beaks (case) and 48 normal birds (control) were genotyped using Affymetrix 600 K HD genotyping arrays. As a result, 95 individuals and 429,539 SNPs were obtained after quality control. The P-value was corrected by a Bonferroni adjustment based on linkage disequilibrium pruning. The single SNP-based association study identified one associated SNP with 5% genome-wide significance and seven suggestively associated SNPs. Four high-confidence genes, LOC421892, TDRD3, RET, and STMN1, were identified as the most promising candidate genes underlying this complex trait in view of their positions, functions, and overlaps with previous studies. The pathway-based association study highlighted the association of six pathways with beak deformity, including the calcium signaling pathway. CONCLUSIONS: Potentially useful candidate genes and pathways for beak deformity were identified, which should be the subject of further functional characterization.

Omnidirectional Shape Memory Effect via Lyophilization of PEG Hydrogels.

Device applications of shape memory polymers demand diverse shape changing geometries, which are currently limited to non-omnidirectional movement. This restriction originates from traditional thermomechanical programming methods such as uniaxial, biaxial stretching, bending, or compression. A solvent-modulated programming method is reported to achieve an omnidirectional shape memory behavior. The method utilizes freeze drying of hydrogels of polyethylene glycol networks with a melting transition temperature around 50 °C in their dry state. Such a process creates temporarily fixed macroporosity, which collapses upon heating, leading to significant omnidirectional shrinkage. These shrunken materials can swell in water to form hydrogels again and the omnidirectional programming and recovery can be repeated. The fixity ratio (R f ) and recovery ratio (R r ) can be maintained at 90% and 98% respectively upon shape memory multicycling. The maximum linear recoverable strain, as limited by the maximum swelling, is ≈90%. Amongst various application potentials, one can envision the fabrication of multiphase composites by taking advantages of the omnidirectional shrinkage from a porous polymer to a denser structure.

Extracellular matrix-coating pedicle screws conduct and induce osteogenesis.

BACKGROUND: The purpose of the present study was to study the effects of the extracellular matrix-coating pedicle screws on the conduction and induction of bone formation in young sheep. METHODS: Pedicle screws [coated with collagen/chondroitin sulfate (coll/CS), hydroxyapatite (HA), and coll/CS/HA or uncoated] were randomly implanted into the L2-L5 pedicles of sheep. In the first stage, a static experiment was performed. In the second stage, a loading test was performed by implanting connecting rods. After 3 months, the lumbar vertebrae with the screws were removed and examined by micro-CT, histological, and biomechanical analyses. RESULTS: Under non-loading conditions, there is bone formation around the surfaces of coated screws. Bone formation on the surface of the coll/CS/HA coating of pedicle screws was the highest. In terms of the trabecular bone morphology parameters of the region of interest around the surface of the pedicle screws, those associated with coll/CS/HA coatings were highest under non-loading conditions, the pullout strength of the coll-/CS-/HA-coated screws was the highest and that of the uncoated screws was minimal. Under loading conditions, the maximum pullout strength of each group of pedicle screws was less than that of the pedicle screws in the non-loading state. CONCLUSIONS: Under non-loading conditions, the organic and inorganic components of the titanium pedicle screw coatings can conduct or induce bone formation around the surface of the screws. The ability of the coll/CS/HA coating to induce bone formation was the strongest. Under loading conditions, a large amount of connective tissue formed around the surface of the screws in each group.

Promising applications of human-derived saliva biomarker testing in clinical diagnostics.

Saliva testing is a vital method for clinical applications, for its noninvasive features, richness in substances, and the huge amount. Due to its direct anatomical connection with oral, digestive, and endocrine systems, clinical usage of saliva testing for these diseases is promising. Furthermore, for other diseases that seeming to have no correlations with saliva, such as neurodegenerative diseases and psychological diseases, researchers also reckon saliva informative. Tremendous papers are being produced in this field. Updated summaries of recent literature give newcomers a shortcut to have a grasp of this topic. Here, we focused on recent research about saliva biomarkers that are derived from humans, not from other organisms. The review mostly addresses the proceedings from 2016 to 2022, to shed light on the promising usage of saliva testing in clinical diagnostics. We recap the recent advances following the category of different types of biomarkers, such as intracellular DNA, RNA, proteins and intercellular exosomes, cell-free DNA, to give a comprehensive impression of saliva biomarker testing.

Effects of the Number of Crested Cushions in Runzhou White-Crested Ducks on Serum Biochemical Parameters.

We investigated the effects of crest cushions in Runzhou white-crested (RWC) ducks. A total of 322 duck eggs were collected for incubation; 286 eggs were fertilized, and 235 RCW ducks were hatched. All the RWC ducks were weighed after 100 days and counted, and the volume of the crest cushion was measured. The number of crest cushions was positively correlated with the body weight, volume of the crest cushion, and distance from the mouth (p < 0.05). The serum Ca, Mg, Fe, Cu, Zn, and Se contents in the multiple-crest-cushion group were significantly higher (p < 0.05), as were the levels of triglycerides, immunoglobulin A, immunoglobulin G, immunoglobulin M, and immunoglobulin D (p < 0.01). The opposite results were seen for glycosylated low-density lipoprotein (p < 0.01). Propionic acid and acetic acid contents differed significantly between the two groups (p < 0.05), as did butyric acid content (p < 0.01), being higher in the multiple-crest-cushion group. Thus, an increase in the number of crest cushions coincided with a change in various serum biochemical indicators. The number of crest cushions might be involved in regulating various mechanisms of RWC ducks and might have an immunoregulatory effect.

Preparation of polyvinyl alcohol/chitosan nanofibrous films incorporating graphene oxide and lanthanum chloride by electrospinning method for potential photothermal and chemical synergistic antibacterial applications in wound dressings.

Electrospun fibres have been widely used as skin dressings due to their unique structur. However, due to the lack of intrinsic antimicrobial activity, it is easy for the wound to become infected. Bacterial infection, which leads to chronic inflammation, severely hinders the normal process of skin regeneration. In this study, a polyvinyl alcohol/chitosan (PVA/CS) composite films with chemical sterilization and near-infrared (NIR) photothermal antibacterial activity was fabricated by electrospinning. Graphene oxide (GO), a photosensitiser, was incorporated into the films, and lanthanum chloride (Lacl3) as a chemical antibacterial agent was also doped in the electrospun films. The structure, morphology, mechanical properties, wettability, and antimicrobial and photothermal antibacterial activity of the PVA/CS-based fibre films were investigated. The results showed that the addition of Lacl3 to the PVA/CS/GO nanofibres (PVA/CS/GO-La) improved the hydrophilicity, tensile strength and resistance to elastic deformation of the nanofibres. The PVA/CS/GO-La12.5 mM sample exhibited the best antibacterial performance, showing high inhibition against Staphylococcus aureus (82% antibacterial efficacy) and Escherichia coli (99.7% antibacterial efficacy). Furthermore, the antibacterial efficacy of the films surface was further enhanced after exposure to NIR light (808 nm, 0.01 W) for 20 min. In addition, the nanofibre films showed no cytotoxicity against human skin fibroblasts (HSFs), indicating its potential application in the field of broad-spectrum antibacterial materials.

Wireless, battery-free, multifunctional integrated bioelectronics for respiratory pathogens monitoring and severity evaluation.

The rapid diagnosis of respiratory virus infection through breath and blow remains challenging. Here we develop a wireless, battery-free, multifunctional pathogenic infection diagnosis system (PIDS) for diagnosing SARS-CoV-2 infection and symptom severity by blow and breath within 110 s and 350 s, respectively. The accuracies reach to 100% and 92% for evaluating the infection and symptom severity of 42 participants, respectively. PIDS realizes simultaneous gaseous sample collection, biomarker identification, abnormal physical signs recording and machine learning analysis. We transform PIDS into other miniaturized wearable or portable electronic platforms that may widen the diagnostic modes at home, outdoors and public places. Collectively, we demonstrate a general-purpose technology for rapidly diagnosing respiratory pathogenic infection by breath and blow, alleviating the technical bottleneck of saliva and nasopharyngeal secretions. PIDS may serve as a complementary diagnostic tool for other point-of-care techniques and guide the symptomatic treatment of viral infections.

Large-scale fabrication of bioinspired fibers for directional water collection.

Spider-silk inspired functional fibers with periodic spindle-knots and the ability to collect water in a directional manner are fabricated on a large scale using a fluid coating method. The fabrication process is investigated in detail, considering factors like the fiber-drawing velocity, solution viscosity, and surface tension. These bioinspired fibers are inexpensive and durable, which makes it possible to collect water from fog in a similar manner to a spider's web.

Mechanically Efficient Cellular Materials Inspired by Cuttlebone.

Cellular materials with excellent mechanical efficiency are essential for aerospace structures, lightweight vehicles, and energy absorption. However, current synthetic cellular materials, such as lattice materials with a unit cell arranged in an ordered hierarchy, are still far behind many biological cellular materials in terms of both structural complexity and mechanical performance. Here, the complex porous structure and the mechanics of the cuttlebone are studied, which acts as a rigid buoyancy tank for cuttlefish to resist large hydrostatic pressure in the deep-sea environment. The cuttlebone structure, constructed like lamellar septa, separated by asymmetric, distorted S-shaped walls, exhibits superior strength and energy-absorption capability to the octet-truss lattice and conventional polymer and metal foams. Inspired by these findings, mechanically efficient cellular materials are designed and fabricated by 3D printing, which are greatly demanded for many applications including aerospace structures and tissue-engineering-scaffold. This study represents an effective approach for the design and engineering of high-performance cellular materials through bioinspired 3D printing.

Analysis of DNA Methylation Profiles in Mandibular Condyle of Chicks With Crossed Beaks Using Whole-Genome Bisulfite Sequencing.

Crossed beaks have been observed in at least 12 chicken strains around the world, which severely impairs their growth and welfare. To explore the intrinsic factor causing crossed beaks, this study measured the length of bilateral mandibular ramus of affected birds, and investigated the genome-wide DNA methylation profiles of normal and affected sides of mandibular condyle. Results showed that the trait was caused by impaired development of unilateral mandibular ramus, which is extended through calcification of mandibular condyle. The methylation levels in the CG contexts were higher than that of CHG and CHH, with the highest methylation level of gene body region, followed by transcription termination sites and downstream. Subsequently, we identified 1,568 differentially methylated regions and 1,317 differentially methylated genes in CG contexts. Functional annotation analysis of Gene Ontology and Kyoto Encyclopedia of Genes and Genomes showed that these genes were involved in bone mineralization and bone morphogenesis. Furthermore, by combining the WGBS and previous RNA-Seq data, 11 overlapped genes were regulated by both long non-coding RNA and DNA methylation. Among them, FIGNL1 is an important gene in calcification of mandibular condyle. Generally, because the affected genes play key roles in maintaining mandibular calcification, these changes may be pivotal factors of crossed beaks.

Absorptive and expansive behaviors of poly(methyl methacrylate-co-acrylic acid) bone cement.

Some additives had provided the expansion capacity to the polymethylmethacrylate (PMMA) bone cement and also reduced its maximum reaction temperature. However, the corresponding modified bone cement displayed inferior simulated body fluid (SBF) absorption capacity and expansion behavior, the mechanism of SBF absorption and the trend of expansion stress were ignored additionally. In this study, a homogeneous distribution of poly (methyl methacrylate-co-acrylic acid) [P(MMA-AA)] microspheres led to the formation of microchannels that favored the delivery of SBF to the interior, causing an increased absorption capacity and enhanced expansion behavior before solidification of the bone cement, with the maximum equilibrium absorption ratio and the expansion ratio reaching 27.3 % and 26.3 %, respectively, at an AA content of 50 %. In addition, the expansion stress induced by the expansion behavior experienced a gradual increase from the 0 s to 2590s, followed by a sharp climbed in a short period ranging from 2590s to 2900s, finally reaching maximum stress of 82.1 MPa. Furthermore, the expansion stress within the maximum value could be obtained by controlling the AA content in the P(MMA-AA) bone cement. With the above characteristics, the prepared P(MMA-AA) bone cement has potential applications as a filling and adhesive material in arthroplasties, vertebroplasties, joint replacements, bone screws, and dentistry.

Structural analysis and allergenicity assessment of an enzymatically cross-linked bovine α-lactalbumin polymer.

Enzymatic cross-linking is frequently used in bio-processing of dairy products since it could change the physiochemical and functional characterization. In our study, bovine α-lactalbumin was cross-linked by polyphenol oxidase from Agaricus bisporus and the changes in the structure, digestibility and allergenicity of α-lactalbumin were explored after cross-linking, and the structural alterations of the polymers were analyzed by circular dichroism spectroscopy, ultraviolet absorption spectroscopy and fluorescence spectroscopy. The digestibility of cross-linked α-lactalbumin was evaluated by simulated digestion in vitro. After that, the allergenicity of α-lactalbumin polymers was evaluated by detection of the specific IgE binding ability using an animal model. The results showed that the secondary and tertiary structures of various α-lactalbumin polymers exhibited a significant variation compared with those of untreated α-lactalbumin, and the cross-linked α-lactalbumin was relatively less susceptible to digestion. Moreover, the allergenicity of cross-linked polymers decreased significantly. These results suggested that there was a direct correlation between a loss of an α-helix and IgE binding to α-lactalbumin, which indicated that enzymatic cross-linking might be an efficient approach to reduce the allergenicity of bovine α-lactalbumin.

Phenotype characterization of crossed beaks in Beijing-You chickens based on morphological observation.

The prevalence of crossed beaks ranging from 0.2 to 7.4% was documented in at least 12 chicken strains. Previous studies focused largely on candidate molecules, whereas the morphological observation was missing. This study reported a detailed phenotype and prevalence of crossed beaks based on morphological observation in nine thousand nine hundred 1-day-old female Beijing-You chicks. Affected chicks were classified into 2 categories based on the direction of the mandibular deformation: left and right. Each category was selected to sacrifice for the measurement of length, width, and thickness of the bilateral mandibular ramus (MR). The normal chicks were used as controls. Paraffin section was made for the bilateral MR of a crossed beak and a normal control for histology analysis. A total of 97 out of 9,900 chickens showed beak deformity including 71 crossed beaks (0.72%) and 26 side beaks (0.26%) for which the upper and lower beak were both bent in the same direction. There was no difference in the direction of the bend of the lower beak in crossed beaks (P > 0.05). The incidence of crossed beaks increased quickly from 0 to 56 d and no new incidence after 56 d. The angle of the crossed beaks was below 5° in the first week and had grown more severe with age until 56 d. The mandible structure showed that condyle served as a growth center for the MR extension. The short-side MR of crossed beaks was thicker than normal ones (P < 0.05) and caused the mandible deviated to the same direction. Meanwhile, the short-side MR prevented the occlusion, leading the jugal arch deformity, which in turn resulted in a bent maxillary horizontally. Similarly, chicks with side beaks also had asymmetry in MR length and the deformities of the jugal arch after dissection. In summary, asymmetric growth of bilateral MR induced crossed beaks and side beaks; the mandibular condyle could be an ideal sample for the related molecular mechanism studies underlying this trait.

Antiplatelet and thermally responsive poly(N-isopropylacrylamide) surface with nanoscale topography.

Nanoscale topography was constructed on a thermally responsive poly(N-isopropylacrylamide) (PNIPAAm) surface by grafting the polymer from silicon nanowire arrays via surface-initiated atom transfer radical polymerization. The as-prepared surface showed largely reduced platelet adhesion in vitro both below and above the lower critical solution temperature (LCST) of PNIPAAm ( approximately 32 degrees C), while a smooth PNIPAAm surface exhibited antiadhesion to platelets only below the LCST. Contact angle and adhesive force measurements on oil droplets (1,2-dichloroethane) in water demonstrated that the nanoscale topography kept a relatively high ratio of water content on the as-prepared surface and played a key role in largely reducing the adhesion of platelets; however, this effect did not exist on the smooth PNIPAAm surface. The results can be used to extend the applications of PNIPAAm in the fields of biomaterials and biomedicine under human physiological temperature and provide a new strategy for fabricating other blood-compatible materials.

Fabrication, microstructure, and properties of a biodegradable Mg-Zn-Ca clip.

An Mg-Zn-Ca alloy biodegradable clip was fabricated by combining hot extrusion and blanking processing. Microstructure evolution was investigated by optical microscopy and electron backscattering diffraction and the occlusion properties of Mg-Zn-Ca alloy clip were evaluated in vitro and in vivo. It was found that the as-extruded Mg-Zn-Ca alloy exhibited a typical fiber microstructure. After blanking, the basal texture intensity increased because of the work hardening effect. Subsequent annealing treatment of the blanking clip can significantly weaken the texture while improving the ductility of the Mg-Zn-Ca alloy. It was found that Mg-Zn-Ca clips can maintain closure performance for 2 weeks in in vitro immersion tests while in vivo tests indicated that the Mg-3Zn-0.2Ca alloy clips fabricated by this preparation processing successfully occluded the blood vessels. These results suggest that the developed Mg-3Zn-0.2Ca alloy clip is a suitable candidate for biodegradable soft tissue fixation devices such as surgical clips. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater 107B: 1741-1749, 2019.

Fabrication of the antibiotic-releasing gelatin/PMMA bone cement.

High mechanical property especially the exorbitant elastic modulus is the common complication of the clinical polymethylmethacrylate (PMMA) bone cement which will generate the fracture of the adjacent bone and even aseptic loosening, other side effects including excess thermal temperature and low efficiency of the drug release bother the development of the PMMA bone cement. The present study aims to investigate the optimum dosage of gelatin as the porogen, which reduced the elastic modulus of the bone cement to the relatively close level of the cancellous bone. Meanwhile, better antibiotic release profile was introduced by enhancing the specific surface area and interconnectivity than the neat PMMA bone cement. Compared to the PMMA bone cement, the mechanical and thermal property was successfully reduced by the porous structure, the component with 200-400 µm gelatin has the better porosity which resulted in the increasing drug release amount and rate than that of the PMMA bone cement. Furthermore, data analysis and fitting curve could guide experiments, in turn, to obtain the PMMA bone cement with specific requirements of the mechanical properties by the addition of gelatin as the pore-forming agent, and in some cases for predictive purposes, to estimate how a change of gelatin may affect the porosity, mechanical properties, and drug release profiles.

Promoting Cell Migration in Tissue Engineering Scaffolds with Graded Channels.

Ideal bone scaffolds having good biocompatibility, good biodegradability, and beneficial mechanical properties are the basis for bone tissue engineering. Specifically, cell migration within 3D scaffolds is crucial for bone regeneration of critical size defects. In this research, hydroxyapatite scaffolds with three different types of architectures (tortuous, parallel, and graded channels) are fabricated using the freeze-casting (ice-templating) method. While most studies promote cell migration by chemical factors, it can be greatly enhanced by introducing only graded channels as compared with tortuous or parallel channels. The results provide insights and guidance in designing novel scaffolds to enhance cell migration behavior for bone tissue regeneration.