High Cell Viability Research Articles

Optimizing extrusion-based 3D bioprinting of plant cells with enhanced resolution and cell viability

3D bioprinting of plant cells has emerged as a promising technology for plant cell immobilization and related applications. Despite the numerous progress in mammalian cell printing, the bioprinting of plant cells is still in its infancy and needs further investigation. Here, we present a systematic study on optimizing the 3D bioprinting of plant cells, using carrots as an example, towards enhanced resolution and cell viability. We mainly investigated the effects of cell cluster forms and nozzle size on the rheological, extrusion, and printability properties of plant cell bioinks, as well as on the resultant cell viability and growth. We found that when the printing nozzle is larger than 85% of the cell clusters embedded in the bioink, smooth extrusion and good printability can be achieved together with considerable cell viability and long-term growth. Specifically, we optimized a bioink composited with suspension-cultured carrot cells, which exhibited better uniformity, smoother extrusion, and higher cell viability over 1 month culture compared to those with the regular callus or fragmented callus. This work provides a practical guideline for optimizing plant cell bioprinting from the bioink development to the printing outcome assessment. It highlights the importance of selecting a matched nozzle and cell cluster and might provide insights for a better understanding and exploitation of plant cell bioprinting.

Hydroelectric simulation of the phreatic water response of mining cracked soil based on microbial solidification

Abstract Coal mining in ecologically fragile areas results in the failure of aquiclude layers and the loss of surface water bodies. Herein, research was conducted on the microbial solidification of cracked soils and the corresponding response of the ecological water table. A simulation of mining-induced cracked soils was performed via microbial solidification. The mechanical and hydrological properties of cracked soil samples repaired with different filling materials were compared via unconfined compressive strength and falling head permeability tests. Hydraulic-electric similarity modeling techniques were employed to evaluate the effectiveness of microbial solidification in the aquiclude layers. After low-temperature acclimation, Bacillus megaterium adapted to the geological environment of the study area, exhibiting a high viable cell density. When the cracked soil was filled with a 1:1 ratio of aeolian sand to clay particles, the microbially remediated soil demonstrated optimal mechanical and hydraulic properties. Hydraulic-electric similarity numerical simulations revealed that the ecological water table at the coalface remained within a reasonable range following microbial solidification, suggesting that microbial solidification achieved water-preserving coal mining. These findings provide a reference for restoring aquiclude layers damaged by coal mining.

Silk-fibroin chitosan film for palatal wounds: Material development, in vitro study, and pilot clinical trial.

In the present study, we aim to assess a novel silk-fibroin (SF) chitosan (CH) film to treat oral mucosa wounds. The SF/CH films, sterilized with 130Gy in a gamma cell, were subjected to tests for thickness, water vapor permeability, tensile strength, elongation, and swelling as well as scanning electron microscopy. Additionally, in vitro cytotoxicity and genotoxicity were evaluated using human epidermal keratinocytes, human foreskin fibroblasts, and human gingival fibroblasts. A pilot clinical trial was conducted with 10 patients who underwent a free gingival graft procedure for socket preservation. The palatal wound healing was evaluated through clinical, patient-centered, immunological, and histological assessments. The SF/CH film exhibited a thickness of 187.5±8.0µm and water vapor permeability of 15.87±7.52gmmm2day-1kPa-1, and microscopy revealed a uniformly rough surface. A flexible scaffold was observed (Young's modulus=1.74±0.90MPa) with 1% of water absorption within 2h. The in vitro analysis showed increased cell viability and low genotoxicity for epithelial and fibroblast cells. For the clinical assessments, wound closure was 28.06±4.3mm2 and 0.95±1.9mm2 after 7 and 14 days, respectively. The immunological assay exhibited a decrease in tissue inhibitor of metalloproteinase-1 (TIMP-1) (p=0.02) and an increase in macrophage inflammatory protein-1 alpha (MIP-1α) (p=0.008) from Day 3 to Day 7. Histology showed the absence of residual biomaterial after 12 months. Complete epithelialization was reached on Day 21, and no tissue thickness loss was observed after 90 days. Patients reported low discomfort and minimal analgesic intake. Within the present study's limits, SF/CH film may be useful to help in the healing of wounds on the palatal mucosa. Further clinical investigations are required. This study aimed to evaluate a novel silk fibroin and chitosan film for treating palatal mucosa wounds. The films were sterilized, tested for physical properties, such as thickness, tensile strength, elongation, water vapor permeability, and swelling, and subjected to scanning electron microscopy. In vitro tests using human oral and skin cells assessed the film's toxicity. Ten patients undergoing graft-harvesting procedures received the film, and their healing was monitored clinically, immunologically, and histologically. The film demonstrated appropriate physical properties, and laboratory results indicated high cell viability and low toxicity. Clinically, the wounds demonstrated considerable closure by Day 7 and almost full closure by Day 14. Immunological assessments indicated elevation in some healing markers, and histology revealed no residual biomaterial at 12months post treatment. Complete epithelialization occurred by Day 21, with no tissue thickness loss at 90days, and patients reported low discomfort and minimal analgesic use. These findings suggest that the silk-fibroin/chitosan film may be beneficial for oral wound healing, warranting further clinical studies to confirm its efficacy.

3D Printed Bioelectronic Scaffolds with Soft Tissue‐Like Stiffness

Abstract3D printing is a leading technique for fabricating tissue engineering scaffolds that facilitate native cellular behavior. Engineering scaffolds to possess functional properties like electronic conductivity is the first step toward integrating new technological capabilities like stimulating or monitoring cellular activity beyond the traditionally presented biophysical and biochemical cues. However, these bioelectronic scaffolds have been largely underdeveloped since the majority of electrically conducting materials possess high stiffness values outside the physiological range and that may negatively impact desired cell behavior. Here, methods of 3D printing poly(3,4‐ethylenedioxythiophene):poly(styrene sulfonate) (PEDOT:PSS) hydrogel scaffolds and techniques to achieve stiffness relevant to many soft tissues (<100 kPa) are reported. Structures are confirmed as ideal tissue scaffolds by maintaining biostability, promoting high cell viability, as well as appropriate cell morphology and proliferation. These findings present a customizable 3D platform that provides favorable soft cellular microenvironments and this is envisioned to be adaptable to several bioelectronic applications.

All-Cellulose Hydrogel-Based Bioinks for the Versatile 3D Bioprinting of Different Cell Lines.

The development of bioink formulations with suitable properties is fundamental for the progress of 3D bioprinting. The potential of cellulose, the most abundant biopolymer, in this realm has often been underestimated, relegating it essentially to a reinforcement additive of bioinks. In this work, cell-laden bioink formulations, composed exclusively of cellulose, viz., "all-cellulose bioinks", were developed by combining carboxymethyl cellulose (CMC) and nanofibrillated cellulose (NFC) in different mass proportions (90/10, 80/20, and 70/30%). The incorporation of NFC increases the printability of the inks (from Pr = 0.7 to 0.9) while maintaining their shear-thinning behavior, and increasing contents of NFC also decrease the degradation rate of the hydrogels after 7 days. The bioprinting of the cell-laden formulations, with HaCaT (keratinocyte) and ATDC5 (chondrogenic) cells, resulted in high (>80%) cell viabilities for up to 7 days, corroborating the versatility of the bioinks and their potential to originate distinct 3D living structures for biomedical applications.

Gold Nanorod Nanolayer-Mediated Photothermal Transfection Promotes DNA Nanomachine Crossing of Cell Membranes for Sensitive Cell Imaging.

The transmembrane efficiency of DNA nanomachines greatly affects the performance of cell imaging. The current transmembrane methods suffer from long time incubation, low efficiency, and potential cyto-toxicity. Here, we report on a novel gold nanorod nanolayer-mediated photothermal transfection (AuNRs-NMPT) method to deliver a DNA nanomachine across the cell membrane. Due to good photothermal effects, AuNRs nanolayer-functionalized slide (AuNRs-NFS) converted light energy into local heat. Local heat disrupted the fluidity of the cell membrane for perforation. The DNA nanomachine penetrates the cell membrane rapidly and efficiently via those holes under the optimal laser power and irradiation time. The AuNRs-NMPT exhibited a 12-fold lower incubation time and 13-fold higher fluorescent intensity compared to that of common direct incubation. The cells also exhibited a high cell viability after being treated with the AuNRs-NMPT, due to cell membrane self-repair. Due to efficient, rapid, and low cytotoxic transmembrane transfection, the AuNRs-NMPT ensures sensitive cell imaging of miRNA-21. The proposed AuNRs-NMPT will promote the application of DNA nanomachines for clinical cancer diagnosis in the future.

Highly efficient one-pot synthesis of polyesteramides from ɛ-caprolactone and l-phenylalanine with high cell-viability and chemical stability

Highly efficient one-pot synthesis of polyesteramides from ɛ-caprolactone and l-phenylalanine with high cell-viability and chemical stability

Pneumatic conveying inkjet bioprinting for the processing of living cells

Inkjet printing techniques are often used for bioprinting purposes because of their excellent printing characteristics, such as high cell viability and low apoptotic rate, contactlessmodus operandi, commercial availability, and low cost. However, they face some disadvantages, such as the use of bioinks of low viscosity, cell damage due to shear stress caused by drop ejection and jetting velocity, as well as a narrow range of available bioinks that still challenge the inkjet printing technology. New technological solutions are required to overcome these obstacles. Pneumatic conveying printing, a new type of inkjet-based printing technique, was applied for the bioprinting of both acellular and cellular fibrin-hydrogel droplets. Drops of a bioink containing 6 × 106HEK293H cells ml-1were supplied from a sterile nozzle connected to a syringe pump and deposited on a gas stream on a fibrinogen-coated glass slide, here referred to as biopaper. Fibrinogen film is the substrate of the polymerization reaction with thrombin and Ca2+present in the bioink. The pneumatic conveying printing technique operates on a mechanism by which drop ejection and deposition in a stream of gas occurs. The percentage of unprinted and printed dead HEK293H cells was 5 ± 2% and 7 ± 4%, respectively. Thus, compared to normal handling, pneumatic conveying printing causes only little damage to the cells. The velocity of the drop approaching the biopaper surface is below 0.2 m s-1and does not cause any damage to the cells. The cell viability of printed cells was 93%, being an excellent value for inkjet printing technology. The HEK293H cells exhibited approximately a 24 h lag time of proliferation that was preceded by intense migration and aggregation. Control experiments proved that the cell migration and lag time were associated with the chemical nature of the fibrin hydrogel and not with cell stress.

Chitosan-DNA nanoparticles: synthesis and optimization for long-term storage and effective delivery.

Chitosan nanoparticles (CsNPs) are an effective and inexpensive approach for DNA delivery into live cells. However, most CsNP synthesis protocols are not optimized to allow long-term storage of CsNPs without loss of function. Here, we describe a protocol for CsNP synthesis, lyophilization, and sonication, to store CsNPs and maintain transfection efficiency. The size and zeta potential of CsNPs were analyzed by dynamic light scattering (DLS) and the morphology of CsNPs was assessed by transmission electron microscopy (TEM). HEK293 cells were transfected with CsNPs, and expression of H2B-CMV-mScarlet plasmid was assessed by flow cytometry. Confocal microscopy was used to visualize post-transfection gene expression. Time, volume, and effect of sonication were tested to optimize the lyophilization process. DLS and TEM analysis indicated amine groups on chitosan to phosphate groups on DNA (N:P) ratios yielded smaller CsNPs sizes. Transfection efficiency, measured by FACS and confocal microscopy, peaked at N:P ratios of 2:1 and 3:1 for both fresh and lyophilized CsNPs. Chitosan/DNA complexes remained stable in solution for at least 72 h at a ratio ≥2:1 as assessed by agarose gel electrophoresis. A lower surface charge with lower N:P ratios was indicated by zeta potential measurements. Lyophilized CsNPs lost 50% transfection efficiency compared to those freshly made. In contrast, sonication of lyophilized CsNPs restored their transfection efficiency to the level of fresh CsNPs. Sonicated CsNPs maintained spherical morphology, while unsonicated CsNPs showed aggregates. Cytotoxicity assays revealed high cell viability (>90%) after CsNPs transfection for a ratio of 2:1 or 3:1. This optimized CsNPs synthesis protocol opens the possibility of long-term storage for CsNPs, which would provide broader applications of this technology.

Bioprinted optoelectronically active cardiac tissues.

Electrical stimulation of existing three-dimensional bioprinted tissues to alter tissue activities is typically associated with wired delivery, invasive electrode placement, and potential cell damage, minimizing its efficacy in cardiac modulation. Here, we report an optoelectronically active scaffold based on printed gelatin methacryloyl embedded with micro-solar cells, seeded with cardiomyocytes to form light-stimulable tissues. This enables untethered, noninvasive, and damage-free optoelectronic stimulation-induced modulation of cardiac beating behaviors without needing wires or genetic modifications to the tissue solely with light. Pulsed light stimulation of human cardiomyocytes showed that the optoelectronically active scaffold could increase their beating rates (>40%), maintain high cell viability under light stimulation (>96%), and negligibly affect the electrocardiogram morphology. The seeded scaffolds, termed optoelectronically active tissues, were able to successfully accelerate heart beating in vivo in rats. Our work demonstrates a viable wireless, printable, and optically controllable tissue, suggesting a transformative step in future therapy of electrically active tissues/organs.

Open Microfluidic Cell Culture in Hydrogels Enabled by 3D-Printed Molds

Cell culture models with tissue-mimicking architecture enable thein vitro investigation of cellular behavior and cell–cell interactions. These models can recapitulate the structure and function of physiological systems and can be leveraged to elucidate mechanisms of disease. In this work, we developed a method to create open microfluidic cell cultures in vitro using 3D-printed molds. The method improves sample accessibility, is simpler to manufacture than traditional closed microfluidic cell culture systems and requires minimal specialized equipment, making it an attractive method for cell culture applications. Further, these molds can generate multiple tissue-mimicking structures in various hydrogels, including blood vessel mimics using endothelial cells (HUVECs). Various geometries were patterned into agarose, gelatin, and collagen type I hydrogels, including star-shaped wells, square wells, round wells, and open channels, to demonstrate the versatility of the approach. Open channels were created in collagen with diameters ranging from 400 µm to 4 mm and in multiple collagen densities ranging from 2 mg/mL to 4 mg/mL. To demonstrate the applicability of our approach for tissue modeling, blood vessel mimics were generated in open channels with diameters of 800 µm and 2 mm, with high cell viability (>89%) for both dimensions. The vessel mimics were used to study the effects of hypoxia on cell viability and CD31 expression by subjecting them to a reduced-O2 environment (∼16% O2). As compared to normoxia conditions, vessel mimics under hypoxia had a reduction in cell viability by 8.3% and CD31 surface expression by 7.4%. Overall, our method enables the generation of different geometries in hydrogels and the development of in vitro tissue mimics for biological applications.

Automated Laser-Assisted Single-Cell Sorting for Cell Functional and RNA Sequencing.

Accurate and efficient sorting of single target cells is crucial for downstream single-cell analysis, such as RNA sequencing, to uncover cellular heterogeneity and functional characteristics. However, conventional single-cell sorting techniques, such as manual micromanipulation or fluorescence-activated cell sorting, do not match current demands and are limited by low throughput, low sorting efficiency and precision, or limited cell viability. Here, we report an automated, highly efficient single-cell sorter, integrating laser-induced forward transfer (LIFT) with a high-throughput picoliter micropore array. The micropore array was surface-functionalized to manipulate liquid surface tension, facilitating the formation of single-cell picoliter droplets in the micropores to realize automated and highly efficient (>80%) single-cell isolation. Using an in-house built microscopic system, rare target cells were identified and automatically retrieved by LIFT with precise sorting efficiency (about 100%) for downstream single-cell analysis while maintaining high cell viability (about 80%). As a case demonstration, we demonstrated the accurate sorting of rare transfected PC-9 cells and post-transfection cell culture, minimizing cell loss and the risk of contamination. Furthermore, we performed single-cell RNA sequencing and showed that high-quality single-cell transcriptome information was efficiently and reliably obtained during cell sorting, preventing additional costs due to low sorting accuracy. The single-cell sorter will become invaluable for single-cell analysis, laying the foundation for multiomics analysis and precision medicine research.

Formation and stability of green and low-cost magnetoliposomes of the soy lecithin, stigmasterol, and β-sitosterol for hyperthermia treatments

Magnetoliposomes containing magnetite, soy lecithin, stigmasterol, and beta-sitosterol of the mean size minor than 160 nm were obtained by a scalable and green process using autoclave and sonication without organic solvents. The formation, size of the liposome, linkage, and encapsulation of the magnetite were evaluated by Cryo-TEM. The stability of magnetoliposomes after storage for 6 months at 4 °C was improved by liposome size, the ability of soy lecithin to preserve the magnetite phase against oxidation, pH, polydispersity index, and zeta potential. The iron oxide phase stability was assessed using no conventional X-ray diffraction (high-resolution transmission electron microscopy), energy loss electron spectroscopy, and selected area electron diffraction) in time zero (fresh sample) and 6 months. The high zeta potential measured for magnetoliposomes, │53│ mV, indicated a low tendency to agglomerate. Lip-Fe3O4@lecithin with concentrations of 0.58 mg mL−1 of liposome showed high cell viability and are potential candidates for drug delivery and hyperthermia treatments in 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays.

Biofabricated tissue model for determining biocompatibility of metallic coatings.

Metallic biomaterials are extensively used in orthopedics and dentistry, either as implants or coatings. In both cases, metal ions come into contact with surrounding tissues causing a particular cell response. Here, we present a biofabricated in vitro tissue model, consisting of a hydrogel reinforced with a melt electrowritten mesh, to study the effects of bound and released metal ions on surrounding cells embedded in a hydrogel matrix. We evaluate the biocompatibility, bioactivity, and antibacterial properties of these metal coatings. Our approach involves integrating physical vapour deposition coating technology with 3D bioprinting methods. To produce tissue models, melt electrowritten (MEW) meshes composed of polycaprolactone (PCL) were printed and integrated into cell-laden methacrylated galatin (GelMa). The mouse embryonic fibroblast cell line (NIH3T3) was used. GelMa concentration and printing parameters for MEW were adjusted and mechanical analysis of the models was performed to find the optimal material composition. Optimized models were placed on the glass slide surfaces coated with typically non-toxic metals, i.e. titanium (Ti), tantalum (Ta), zirconium (Zr), silver (Ag), tungsten (W), and niobium (Nb). Except for W, all other coatings were stable in a physiological wet environment, as studied by SEM. The viability of the cells at different distances from the coated surface was analyzed. Antibacterial tests against pathogens Staphylococcus aureus and Escherichia coli were used to assess the models' resistance, important for infection control. While Ag coatings showed toxicity, Nb, Ta, Ti, and Zr coatings promoted fibroblast growth, with the highest cell viability after 14 days of culture revealed for Ta and Nb. The strongest antimicrobial effect against E. coli and S. aureus was observed for Ag and W, while Ta exhibited antibacterial activity only against S. aureus. From a broader perspective, our work offers an effective 3D in vitro model for an in-depth characterization of the biocompatibility of metals and metal coatings.

Enhancing elemental release and antibacterial properties of resin-based dental sealants with calcium phosphate, bioactive glass, and polylysine

BackgroundThis study aimed to develop ion-releasing and antibacterial resin-based dental sealants comprising 3 to 6 wt% monocalcium phosphate monohydrate (MCPM, M), 3 to 6 wt% bioactive glass (BAG, B), and 3 to 6 wt% polylysine (PLS, P). The physical properties, mechanical performance, cytotoxicity, and inhibition of S. mutans biofilm by these materials were subsequently evaluated.MethodsFive experimental dental sealants were formulated as follows: F1 (M6B6P6), F2 (M6B6P3), F3 (M3B3P6), F4 (M3B3P3), and F5 (M0B0P0, serving as the control). ClinproXT (CP, 3 M, Saint Paul, MN, USA) was used for commercial comparison. The degree of monomer conversion (DC) was determined using attenuated total reflectance-Fourier transform infrared spectroscopy (n = 5). The biaxial flexural strength (n = 6) and Vickers surface microhardness (n = 5) of the materials were evaluated after a 24-hour immersion in water. The element release over 4 weeks was measured using inductively coupled plasma-optical emission spectrometry (ICP-OES) (n = 3). The cell viability of mouse fibrosarcoma cells exposed to the extract was assessed via an MTT assay (n = 3). Additionally, the inhibition of S. mutans biofilm was tested (n = 3). Statistical analysis was conducted using one-way ANOVA and the Tukey HSD test.ResultsThe lowest DC among experimental sealants was obtained from F1 (66 ± 4%), which was significantly higher than CP (54 ± 2%, p < 0.001). The lowest biaxial flexural strength was obtained from F3 (131 ± 47 MPa). This was comparable to that of CP (140 ± 58 MPa, p = 0.992). The lowest surface microhardness among experimental materials was detected with F2 (19 ± 2 Vickers hardness number), which was higher than that of CP (12 ± 1 Vickers hardness number, p = 0.003). Furthermore, high cell viability of > 90% after exposure to extracts from the experimental materials was detected, which was similar to that observed with CP. Additionally, the experimental materials exhibited higher Ca and P release compared to CP and showed a potential trend for reducing S. mutans biofilm formation. Increasing additive concentrations exhibited minimal effects on material properties, except for enhanced elemental release and a slight reduction in BFM with higher PLS content.ConclusionThe experimental sealants provided sufficient physical and mechanical strength and maintained cell viability and bacterial inhibition with higher elemental release than the commercial product.

Digital light processing 3D bioprinting of biomimetic corneal stroma equivalent using gelatin methacryloyl and oxidized carboxymethylcellulose interpenetrating network hydrogel.

Corneal blindness, a leading cause of visual impairment globally, has created a pressing need for alternatives to corneal transplantation due to the severe shortage of donor tissues. In this study, we present a novel interpenetrating network hydrogel composed of gelatin methacryloyl (GelMA) and oxidized carboxymethyl cellulose (OxiCMC) for bioprinting a biomimetic corneal stroma equivalent. We tested different combinations of GelMA and OxiCMC to optimize printability and subsequently evaluated these combinations using rheological studies for gelation and other physical, chemical, and biological properties. Using digital light processing (DLP) bioprinting, with tartrazine as a photoabsorber, we successfully biofabricated three-dimensional constructs with improved shape fidelity, high resolution, and excellent reproducibility. The bioprinted constructs mimic the native corneal stroma's curvature, with central and peripheral thicknesses of 478.9 ± 56.5 µm and 864.0 ± 79.3 µm, respectively. The dual crosslinking strategy, which combines Schiff base reaction and photocrosslinking, showed an improved compressive modulus (106.3 ± 7.7 kPa) that closely matched that of native tissues (115.3 ± 13.6 kPa), without relying on synthetic polymers, toxic crosslinkers, or nanoparticles. Importantly, the optical transparency of tartrazine-containing corneal constructs was comparable to the native cornea following phosphate-buffered saline washing. Morphological analyses using scanning electron microscopy confirmed the improved porosity, interconnected network, and structural integrity of the GelMA-OxiCMC hydrogel, facilitating better nutrient diffusion and cell viability. In vitro biological assays demonstrated high cell viability (>93%) and desirable proliferation of human corneal keratocytes within the biofabricated constructs. Our findings indicate that the GelMA-OxiCMC hydrogel system for DLP bioprinting presents a promising alternative for corneal tissue engineering, offering a potential solution to the donor cornea shortage and advancing regenerative medicine for corneal repair.&#xD.

One-step bioprinting of endothelialized, self-supporting arterial and venous networks.

Advances in biofabrication have enabled the generation of freeform perfusable networks mimicking vasculature. However, key challenges remain in the effective endothelialization of these complex, vascular-like networks, including cell uniformity, seeding efficiency, and the ability to pattern multiple cell types. To overcome these challenges, we present an integrated fabrication and endothelialization strategy to directly generate branched, endothelial cell-lined networks using a diffusion-based, embedded 3D bioprinting process. In this strategy, a gelatin microparticle sacrificial ink delivering both cells and crosslinkers is extruded into a crosslinkable gel precursor support bath. A self-supporting, perfusable structure is formed by diffusion-induced crosslinking, after which the sacrificial ink is melted to allow cell release and adhesion to the printed lumen. This approach produces a uniform cell lining throughout networks with complex branching geometries, which are challenging to uniformly and efficiently endothelialize using conventional perfusion-based approaches. Furthermore, the biofabrication process enables high cell viability (>90%) and the formation of a confluent endothelial layer providing vascular-mimetic barrier function and shear stress response. Leveraging this strategy, we demonstrate for the first time the patterning of multiple endothelial cell types, including arterial and venous cells, within a single arterial-venous-like network. Altogether, this strategy enables the fabrication of multi-cellular engineered vasculature with enhanced geometric complexity and phenotypic heterogeneity.

Fabrication of nanoparticles with precisely controllable plasmonic properties as tools for biomedical applications.

Metal nanoparticles are established tools for biomedical applications due to their unique optical properties, primarily attributed to localized surface plasmon resonances. They show distinct optical characteristics, such as high extinction cross-sections and resonances at specific wavelengths, which are tunable across the wavelength spectrum by modifying the nanoparticle geometry. These attributes make metal nanoparticles highly valuable for sensing and imaging in biology and medicine. However, their widespread adoption is hindered due to challenges in consistent and accurate nanoparticle fabrication and functionality as well as due to nanotoxicological concerns, including cell damage, DNA damage, and unregulated cell signaling. In this study, we present a fabrication approach using nanoimprint lithography in combination with thin film deposition which yields highly homogenous nanoparticles in size, shape and optical properties with standard deviations of the main geometry parameters of less than 5% batch-to-batch variation. The measured optical properties closely match performed simulations, indicating that pre-experimental modelling can effectively guide the design of nanoparticles with tailored optical properties. Our approach also enables nanoparticle transfer to solution. Particularly, we show that the surface coating with a PEG polymer shell ensures stable dispersions in buffer solutions and complex cell media for at least 7 days. Furthermore, our in vitro experiments demonstrate that these nanoparticles are internalized by cells via endocytosis, exhibit good biocompatibility, and show minor cytotoxicity, as evidenced by high cell viability. In the future, our high-precision nanoparticle fabrication method together with tunable surface plasmon resonance and reduced nanotoxicity will offer the possibility to replace conventional nanomaterials for biomedical applications that make use of an optical response at precise wavelengths. This includes the use of the nanoparticles as contrast agents for imaging, as probes for targeted photothermal cancer therapy, as carriers for controlled drug delivery, or as probes for sensing applications based on optical detection principles.

Improved Mesenchymal Stem Cell Viability in High-Stiffness, Translational Cartilage Matrix Hydrogels.

Scaffolds made from cartilage extracellular matrix are promising materials for articular cartilage repair, attributed to their intrinsic bioactivity that may promote chondrogenesis. While several cartilage matrix-based scaffolds have supported chondrogenesis in vitro and/or invivo, it remains a challenge to balance the biological response (e.g., chondroinductivity) with structural (e.g., robust mechanical performance, >1 MPa in compressive stiffness) and translational (e.g., ease of surgical implantation) considerations. Few studies have evaluated encapsulated cell viability within high-stiffness (>1 MPa) hydrogels. We previously fabricated one formulation of a high-stiffness (>3 MPa) pentenoate-functionalized, solubilized, devitalized cartilage (PSDVC) hydrogel that possessed an injectable, paste-like precursor for easy surgical application. In the current study, the characterization of the PSDVC material was expanded by varying the degree of functionalization (i.e., 0.45-1.09 mmol/g) and amount of crosslinker, dithiothreitol (DTT), to improve the reproducibility of the high compressive moduli and evaluate the viability of encapsulated human bone marrow-derived mesenchymal stem cells (hBMSCs) in high-stiffness cartilage matrix hydrogels. Prior to crosslinking, specific formulations functionalized with 0.80 mmol/g or less of pentenoate groups retained a paste-like precursor rheology. After crosslinking, these formulations produced hydrogels with greater than 1 MPa compressive stiffness. However, hBMSCs encapsulated in PSDVC hydrogels with lower functionalization (i.e., 0.57 mmol/g, no crosslinker) had a higher stiffness (i.e., 1.4 MPa) but the lowest viability of encapsulated hBMSCs (i.e., 5%). The middle PSDVC functionalization (i.e., 0.70 mmol/g) with DTT (i.e., 0.50 mmol thiols/g) demonstrated high cell viability (77%), high mechanical performance (1.65 MPa, 31% failure strain), and translational features (i.e., paste-like precursor, 1.5 min crosslinking time). For future evaluations of PSDVC hydrogels in cartilage repair, a middle functionalization (i.e., 0.70-0.80 mmol/g) with the addition of a crosslinker (i.e., 0.50 mmol thiols/g) had a desirable balance of high mechanical performance (i.e., >1 MPa compressive stiffness), high viability, and paste-like precursor for surgical translation.

Exploring Liraglutide’s mechanism in reducing renal fibrosis: the Fsp1-CoQ10-NAD(P)H pathway

Studies have confirmed that elevated glucose levels could lead to renal fibrosis through the process of ferroptosis. Liraglutide, a human glucagon-like peptide-1 (GLP-1) analogue, is a potential treatment option for diabetes. This study aimed to examine the potential of liraglutide (LIRA) in inhibiting ferroptosis and reducing high glucose-induced renal fibrotic injury in mice, and whether the Fsp1-CoQ10-NAD(P)H signal pathway is a mechanism for this effect. In our study, we used db/db mice to simulate Type 2 diabetes mellitus (T2DM). The mice were intraperitoneally injected with LIRA (200 µg/kg/d) daily for 6 weeks. Renal function, pathologic changes, lipid peroxidation levels, iron levels, and ferroptosis were assessed. First, LIRA ameliorated renal dysfunction and fibrosis in db/db mice. Second, LIRA inhibited lipid peroxidation by up-regulating T-SOD, GSH-Px, and GSH activities as well as down-regulating the levels of 8-OHDG, MDA, LPO, 4-HNE, 12-Lox, and NOX4 in db/db mice. In addition, LIRA attenuated iron deposition by decreasing the expression of TfR1 and increasing the expression of FPN1. Meanwhile, LIRA reduced high levels of high glucose-induced cell viability decline and intracellular lipid peroxidation. Furthermore, LIRA inhibited ferroptosis by adjusting the Fsp1-CoQ10-NAD(P)H pathway in vivo and in vitro. These findings suggested that LIRA attenuated kidney fibrosis injury in db/db mice by inhibiting ferroptosis through the Fsp1-CoQ10-NAD(P)H pathway.