In Situ Polymerization for Manufacture of Multifunctional Delivery Systems for Transcellular Delivery of Nucleic Acids.

Electrostatic self-assembly between negatively charged nucleic acids and cationic materials is the basis for the formulation of the delivery systems. Nevertheless, structural disintegration occurs because their colloidal stabilities are frequently insufficient in a hostile biological environment. To overcome the sequential biological barriers encountered during transcellular gene delivery, we attempted to use in situ polymerization onto plasmid DNA (pDNA) with a variety of functional monomers, including N-(3-aminopropyl)methacrylate, (aminopropyl)methacrylamide hydrochloride, 1-vinylimidazole, and 2-methacryloyloxyethylphosphorylcholine and N,N'-bis(acryloyl) cystamine. The covalently linked monomers could polymerize into a network structure on top of pDNA, providing excellent structural stability. Additionally, the significant proton buffering capacity of 1-vinylimidazole is expected to aid in the release of pDNA payloads from acidic and digestive endolysosomes. In addition, the redox-mediated cleavage of the disulfide bond in N,N'-bis(acryloyl)cystamine allows for the selective cleavage of the covalently linked network in the cytosolic microenvironment. This is due to the high intracellular level of glutathione, which promotes the liberation of pDNA payloads in the cell interiors. The proposed polymerization strategies resulted in well-defined nanoscale pDNA delivery systems. Excellent colloidal stabilities were observed, even when incubated in the presence of high concentrations of heparin (10 mg/mL). In contrast, the release of pDNA was confirmed upon incubation in the presence of glutathione, mimicking the intracellular microenvironment. Cell transfection experiments verified their efficient cellular uptake and gene expression activities in the hard-transfected MCF-7 cells. Hence, the polymerization strategy used in the fabrication of covalently linked nonviral gene delivery systems shows promise in creating high-performance gene delivery systems with diverse functions. This could open new avenues in cellular microenvironment engineering.

Preparation and application of a pseudo-templated multi-monomer aflatoxins imprinted polymer.

A functional material was developed with specific recognition properties for aflatoxins for pre-processing enrichment and separation in the detection of aflatoxins in Chinese herbal medicines. In the experiment, ethyl coumarin-3-carboxylate, which has a highly similar structure to the oxonaphthalene o-ketone of aflatoxin, was selected as a pseudo-template, zinc acrylate, neutral red derivative, and methacrylic acid, which have complementary functions, were selected as co-monomers to prepare a pseudo-template multifunctional monomer molecularly imprinted polymer (MIP). The MIP obtained under the optimal preparation conditions has a maximum adsorption capacity of 0.036 mg/mg and an imprinting factor of 3.67. The physical property evaluation of the polymers by Fourier infrared spectrometer, scanning electron microscopy, pore size analyzer, thermogravimetric analyzer, and diffuse reflectance spectroscopy showed that the MIP were successfully prepared and porous spherical-like particles were obtained. The synthesized polymer was used as a solid-phase extraction agent for the separation of aflatoxins from the extract of spina date seed. The linear range of the developed method was 10-1000 ng/mL, the limit of detection was 0.36 ng/mL, the limit of quantification was 1.19 ng/mL, and the recoveries of the extracts at the concentration level of 0.2 µg/mL were in the range 88.0-93.4%, with relative standard deviations (RSDs) of 1.97% (n). The results showed that the preparation of MIPs using ethyl coumarin-3-carboxylate as a template was simple, economical, and convenient. It is expected to become a promising functional material for the enrichment and separation aflatoxins from complex matrices.

Highly precise strategy of polygalacturonic acid microcarriers functionalized with zwitterions and specific peptides for MSC screening.

Microcarriers for large-scale cell culture have a broader prospect in cell screening compared with the traditional high cost, low efficiency, and cell damaging methods. However, the equal biological affinity to cells has hindered its application. Therefore, based on the antifouling strategy of zwitterionic polymer, we developed a cell-specific microcarrier (CSMC) for shielding non-target cells and capturing mesenchymal stem cells (MSCs), which has characteristics of high biocompatibility, low background noise and high precision. Briefly, [2-(methacryloyloxy) ethyl] dimethyl-(3-sulfopropyl) ammonium hydroxide and glycidyl methacrylate were grafted onto polygalacturonic acid, respectively. The former built a hydration layer through solvation to provide an excellent antifouling surface, while the latter provided active sites for the click reaction with sulfhydryl-modified cell-specific peptides, resulting in rapid immobilization of peptides. This method is applicable to the vast majority of polysaccharide materials. The accurate capture ratio of MSCs by CSMC in a mixed multicellular environment is >95 % and the proliferation rate of MSCs on microcarriers is satisfactory. In summary, this grafting strategy of bioactive components lays a foundation for the application of polysaccharide materials in the biomedical field, and the specific adhesive microcarriers also open up new ideas for the development of stem cell screening as well.

Synthesis and biological applications of nanocomposite hydrogels based on the methacrylation of hydroxypropyl methylcellulose and lignin loaded with alpha-pinene.

Oral conditions, such as recurrent aphthous stomatitis, are chronic inflammatory disorders that significantly affect the life quality. This study aims to develop a novel buccal mucoadhesive based on methacrylate hydroxypropyl methylcellulose (M-HPMC) and methacrylate lignin (M-SLS) encapsulated with nanostructured lipid carriers (NLCs) for controlled release of alpha-pinene (α-pinene). NLCs with particle sizes of 152 ± 3 nm were prepared by using stearic acid and oleic acid as solid and liquid lipids, respectively. Following the successful synthesis of M-HPMC and M-SLS, various concentrations of α-pinene loaded NLCs (0, 18, 38, and 50 wt%) were encapsulated in M-HPMC/M-SLS hydrogel. It was demonstrated that the physiological and mechanical performances of hydrogels were changed, depending on the NLC content. Remarkably, the incorporation of 18 wt% NLC improved the compressive strength (143 ± 14 kPa) and toughness (17 ± 1 kJ/m3) of M-HPMC/M-SLS hydrogel. This nanocomposite hydrogel considerably decreased dissipated energy from 1.64 kJ/m3 to 0.99 kJ/m3, after a five-cycle compression test. The nanocomposite hydrogel exhibited controlled α-pinene release for up to 96 h which could significantly improve the antioxidant activity of M-HPMC/M-SLS matrix. Moreover, the reinforcing M-HPMC/M-SLS hydrogel with α-pinene-loaded NLCs resulted in increased adhesive strength (113.5 ± 7.5 kPa) to bovine buccal mucosa and cytocompatibility in contact with fibroblasts.

Multiresponsive 4D Printable Hydrogels with Anti-Inflammatory Properties.

Multiresponsive hydrogels are valuable as biomaterials due to their ability to respond to multiple biologically relevant stimuli, i.e., temperature, pH, or reactive oxygen species (ROS), which can be present simultaneously in the body. In this work, we synthesize triple-responsive hydrogels through UV light photopolymerization of selected monomer compositions that encompass thermoresponsive N-isopropylacrylamide (NIPAM), pH-responsive methacrylic acid (MAA), and a tailor-made ROS-responsive diacrylate thioether monomer (EG3SA). As a result, smart P[NIPAMx-co-MAAy-co-(EG3SA)z] hydrogels capable of being manufactured by digital light processing (DLP) 4D printing are obtained. The thermo-, pH-, and ROS-response of the hydrogels are studied by swelling tests and rheological measurements at different temperatures (25 and 37 °C), pHs (3, 5, 7.4, and 11), and in the absence or presence of ROS (H2O2). The hydrogels are employed as matrixes for the encapsulation of ketoprofen (KET), an anti-inflammatory drug that shows a tunable release, depending on the hydrogel composition and stimuli applied. The cytotoxicity properties of the hydrogels are tested in vitro with mouse embryonic fibroblasts (NIH 3T3) and RAW 264.7 murine macrophage (RAW) cells. Finally, the anti-inflammatory properties are assessed, and the results exhibit a ≈70% nitric oxide reduction up to base values of pro-inflammatory RAW cells, which highlights the anti-inflammatory capacity of P[NIPAM80-co-MAA15-co-(EG3SA)5] hydrogels, per se, without being necessary to encapsulate an anti-inflammatory drug within their network. It opens the route for the fabrication of customizable 4D printable scaffolds for the effective treatment of inflammatory pathologies.

High-Quality Three-Dimensionally Cultured Cells Using Interfaces of Diblock Copolymers Containing Different Ratios of Zwitterionic N-Oxides.

To control three-dimensional (3D) cell spheroid formation, it is well-known the surface physicochemical and mechanical properties of cell culture materials are important; however, the formation and function of 3D cells are still unclear. This study demonstrated the precise control of the formation of 3D cells and 3D cell functions using diblock copolymers containing different ratios of a zwitterionic trimethylamine N-oxide group. The diblock copolymers were composed of poly(n-butyl methacrylate) (PBMA) as the hydrophobic unit for surface coating on a cell culture dish and stabilization in water, and poly(2-(dimethylamino)ethyl methacrylate) (PDMAEMA) as the precursor of N-oxide. The zwitterionic N-oxide converted from 0 to 100% using PDMAEMA. The wettability and surface zeta potential varied with different ratios of N-oxide diblock copolymer-coated surfaces, and the amount of protein adsorbed in the cell culture medium decreased monotonically with increasing N-oxide ratio. 3D cell spheroid formations were observed by seeding human umbilical cord mesenchymal stem cells (hUC-MSCs) in diblock copolymer-coated flat-bottom well plates, and the N-oxide ratio was over 40%. The cells proliferated in two-dimensions (2D) and did not form spheroids when the N-oxide ratio was less than 20%. Interestingly, the expression of undifferentiated markers of hUC-MSCs was higher on surfaces that adsorbed proteins to some extent and formed 50-150 µm spheroids in the range of 40-70% of N-oxide ratio. We revealed that a moderately protein-adsorbed surface allows precise control of spheroid formation and undifferentiated 3D cells and has potential applications for high-quality spheroids in regenerative medicine and drug screening.

Antibacterial properties of photo-crosslinked chitosan/methacrylated hyaluronic acid nanoparticles loaded with bacitracin.

The current treatments for wounds often fail to induce adequate healing, leaving wounds vulnerable to persistent infections and development of drug-resistant microbial biofilms. New natural-derived nanoparticles were studied to impair bacteria colonization and hinder the formation of biofilms in wounds. The nanoparticles were fabricated through polyelectrolyte complexation of chitosan (CS, polycation) and hyaluronic acid (HA, polyanion). UV-induced photo-crosslinking was used to enhance the stability of the nanoparticles. To achieve this, HA was methacrylated (HAMA, degree of modification of 20 %). Photo-crosslinked nanoparticles obtained from HAMA and CS had a diameter of 478 nm and a more homogeneous size distribution than nanoparticles assembled solely through complexation (742 nm). The nanoparticles were loaded with the antimicrobial agent bacitracin (BC), resulting in nanoparticles with a diameter of 332 nm. The encapsulation of BC was highly efficient (97 %). The BC-loaded nanoparticles showed significant antibacterial activity against gram-positive bacteria Staphylococcus aureus, Methicillin-resistant S. aureus and S. epidermidis. Photo-crosslinked HAMA/CS nanoparticles loaded with BC demonstrated inhibition of biofilm formation and a positive effect on the proliferation of mammalian cells (L929). These crosslinked nanoparticles have potential for the long-term treatment of wounds and controlled antibiotic delivery at the location of a lesion.

Stability of the Dentin-Bonded Interface Using Self-Etching Adhesive Containing Diacrylamide after Bacterial Challenge.

Purpose/Aim: Acrylamides are hydrolytically stable at pH lower than 2, and were shown to preserve bonded interface integrity with two-step, total etch adhesives. The objective of this study was to leverage those two characteristics in self-etching primers containing the acidic monomer 10-MDP and test the microtensile bond strength before and after incubation with S. mutans incubation. Materials and Methods: Acidic primers (10 wt % 10-methacryloyloxydecyl dihydrogen phosphate─10-MDP; 45 wt % N,N-diethyl-1,3-bis(acrylamido)propane─DEBAAP, or 2-hydroxyethyl methacrylate─HEMA; 45 wt %, glycerol-dimethacrylate─GDMA) and adhesives (DEBAAP or HEMA/10-MDP/UDMA 45/10/45 wt %) were made polymerizable by the addition of 0.2 wt % camphorquinone, 0.8 wt % ethyl-4-dimethylaminobenzoate, 0.4 wt % diphenyliodonium hexafluorophosphate, and 0.1 wt % butylhydroxytoluene. Nonsolvated materials were characterized for flexural strength (FS), modulus (E), toughness, water sorption/solubility (WS/SL), contact angle, and vinyl conversion (DC). Viscosity was evaluated after adding 20 and 40 vol % ethanol to the primer and adhesive, respectively. The experimental materials or Clearfil SE Bond (CC─commercial control) were used to bond a commercial composite (Filtek Supreme) to the flat surface of human dentin. Microtensile bond strength (MTBS) was tested in 1 mm2 sticks for the 5 primer/bond combinations: CC (Clearfil Bond Primer and Bond), HH (HEMA/HEMA), DD (DEBAAP/DEBAAP), HD (HEMA/DEBAAP), and DH (DEBAAP/HEMA). Prior to testing, sticks were stored in water or biofilm-inducing culture medium with S. mutans for 1 week. Confocal images and FTIR-ATR evaluation evaluated the hybrid layer of the adhesives. Results were analyzed using Student's t-test (WS, SL, DC, contact angle, FS, E, toughness), one-way ANOVA/Tukey's test for viscosity, and two-way ANOVA/Tukey's test for MTBS (95%). Results: HEMA-based materials had lower contact angle (p = 0.004), higher WS (p < 0.001), and similar SL values compared to DEBAAP (p = 0.126). FS (p = 0.171) and E (p = 0.065) dry values were similar, but after one week of water storage, FS/E dropped more significantly for HEMA materials. Dry and wet toughness was greater for DEBAAP (p < 0.001), but it also had the greatest drop (46%). Clearfil bonds had the highest viscosity, followed by DEBAAP and HEMA, respectively (p = 0.002). For the primers, HEMA had the lowest viscosity (p = 0.003). As far as MTBS, all groups tested in water were statistically different when compared with HH (p < 0.001). After storage in biofilm, DH had the highest MTBS value, being statistically different from HH (p = 0.002), CC (p = 0.015), and DD (p = 0.027). Conclusions: The addition of a diacrylamide and its association with HEMA in self-etching adhesive systems provided greater bonding stability after bacterial challenge.

Functional poly(e-caprolactone)/SerMA hybrid dressings with dimethyloxalylglycine-releasing property improve cutaneous wound healing.

Medical dressings with multifunctional properties, including potent regeneration capability and good biocompatibility, are increasingly needed in clinical practice. In this study, we reported a novel hybrid wound dressing (PCL/SerMA/DMOG) that combines electrospun PCL membranes with DMOG-loaded methacrylated sericin (SerMA) hydrogel. In such a design, DMOG molecules are released from the hybrid dressing in a sustained mannerin vitro. A series ofin vitroassays demonstrated that DMOG-loaded hybrid dressing has multiple biological functions, including promotion of human umbilical vein endothelial cells proliferation and migration,in vitrovascularization, and the generation of intracellular NO. When applied to the cutaneous wound, the PCL/SerMA/DMOG dressing significantly accelerated wound closure and tissue regeneration by promoting angiogenesis in the wound area, collagen deposition, and cell proliferation within the wound bed. These results highlight the potential clinical application of PCL/SerMA/DMOG hybrid dressings as promising alternatives for accelerating wound healing via improved biocompatibility and angiogenesis amelioration.

Photo-crosslinking methacrylated-amylopectin/polyacrylamide hydrogels loading curcumin for applications as degradable, injectable, and antibacterial wound dressings.

The preparation of biodegradable and antibacterial hydrogels has important clinical value. In this work, a novel strategy has been developed to prepare degradable hydrogel dressings without chemical crosslinking agent using methacrylate anhydride (MA)-modified amylopectin (APMA) and polyacrylamide (PAM). After introducing CC bonds, APMA/PAM hydrogels can be formed under light irradiation. This strategy improves the gelling ability of AP and degradation properties of the hydrogel by avoiding the addition of crosslinking agent. The degradation rate of APMA/PAM hydrogel is 74.04 ± 0.69 % within 12 weeks, while that of APMA/PAM hydrogel containing crosslinking agent is only 38.5 ± 0.1 %. The APMA/PAM hydrogel loading curcumin (Cur) (APMA/PAM-Cur) exhibits high antibacterial efficiency of 98.29 ± 0.41 % and 97.18 ± 0.81 % against S. aureus and E. coli, respectively, with light irradiation. Animal experiments show that the APMA/PAM-Cur hydrogel reduces the infiltration of inflammatory factors, increases the density of collagen, and makes the newly formed granulation tissue thicker and tighter. This study not only proves the promising potential of the APMA/PAM-Cur hydrogel as degradable and antibacterial wound dressing for clinical treatment, but also provides a new strategy for developing low-cost, degradable, and antibacterial wound dressings and reducing antibiotic abuse and environmental pollution caused by medical waste.

Arginine-loaded globular BSAMA/fibrous GelMA biohybrid cryogels with multifunctional features and enhanced healing for soft gingival tissue regeneration.

Mucogingival surgery has been widely used in soft gingival tissue augmentation in which autografts are predominantly employed. However, the autografts face grand challenges, such as scarcity of palatal donor tissue and postoperative discomfort. Therefore, development of alternative soft tissue substitutes has been an imperative need. Here, we engineered an interconnected porous bovine serum albumin methacryloyl (BSAMA: B, as a drug carrier and antioxidant)/gelatin methacryloyl (GelMA: G, as a biocompatible collagen-like component)-based cryogel with L-Arginine (Arg) loaded as an angiogenic molecule, which could serve as a promising gingival tissue biohybrid scaffold. BG@Arg cryogels featured macroporous architecture, biodegradation, sponge-like properties, suturability, and sustained Arg release. Moreover, BG@Arg cryogels promoted vessel formation and collagen deposition which play an important role in tissue regeneration. Most interestingly, BG@Arg cryogels were found to enhance antioxidant effects. Finally, the therapeutic effect of BG@Arg on promoting tissue regeneration was confirmed in rat full-thickness skin and oral gingival defect models. In vivo results revealed that BG@Arg2 could promote better angiogenesis, more collagen production, and better modulation of inflammation, as compared to a commercial collagen membrane. These advantages might render BG@Arg cryogels a promising alternative to commercial collagen membrane products and possibly autografts for soft gingival tissue regeneration.

Anti-inflammatory potential of casein enzymatic hydrolysate/gelatin methacryloyl scaffolds for vital pulp therapy.

OBJECTIVES: To synthesize casein enzymatic hydrolysate (CEH)-laden gelatin methacryloyl (GelMA) fibrous scaffolds and evaluate the cytocompatibility and anti-inflammatory effects on dental pulp stem cells (DPSCs). MATERIALS AND METHODS: GelMA fibrous scaffolds with 10%, 20%, and 30% CEH (w/w) and without CEH (control) were obtained via electrospinning. Chemo-morphological, degradation, and mechanical analyses were conducted to evaluate the morphology and composition of the fibers, mass loss, and mechanical properties, respectively. Adhesion/spreading and viability of DPSCs seeded on the scaffolds were also assessed. The anti-inflammatory potential on DPSCs was tested after the chronic challenge of cells with lipopolysaccharides (LPS), followed by treatment with extracts obtained after immersing the scaffolds in α-MEM. The synthesis of the pro-inflammatory cytokines IL-6, IL-1α, and TNF-α was measured by ELISA. Data were analyzed by ANOVA/post-hoc tests (α = 5%). RESULTS: CEH-laden electrospun fibers had a larger diameter than pure GelMA (p ≤ 0.036). GelMA scaffolds laden with 20% and 30% CEH had a greater mass loss. Tensile strength was reduced for the 10% CEH fibers (p = 0.0052), whereas no difference was observed for the 20% and 30% fibers (p ≥ 0.6736) compared to the control. Young's modulus decreased with CEH (p < 0.0001). Elongation at break increased for the 20% and 30% CEH scaffolds (p ≤ 0.0038). Over time, DPSCs viability increased across all groups, indicating cytocompatibility, with CEH-laden scaffolds exhibiting greater cell viability after seven days (p ≤ 0.0166). Also, 10% CEH-GelMA scaffolds decreased the IL-6, IL-1α, and TNF-α synthesis (p ≤ 0.035). CONCLUSION: CEH-laden GelMA scaffolds facilitated both adhesion and proliferation of DPSCs, and 10% CEH provided anti-inflammatory potential after chronic LPS challenge. CLINICAL RELEVANCE: CEH incorporated in GelMA fibrous scaffolds demonstrated the potential to be used as a cytocompatible and anti-inflammatory biomaterial for vital pulp therapy.

Extracellular matrix regulation of cell spheroid invasion in a 3D bioprinted solid tumor-on-a-chip.

Tumor organoids and tumors-on-chips can be built by placing patient-derived cells within an engineered extracellular matrix (ECM) for personalized medicine. The engineered ECM influences the tumor response, and understanding the ECM-tumor relationship accelerates translating tumors-on-chips into drug discovery and development. In this work, we tuned the physical and structural characteristics of ECM in a 3D bioprinted soft-tissue sarcoma microtissue. We formed cell spheroids at a controlled size and encapsulated them into our gelatin methacryloyl (GelMA)-based bioink to make perfusable hydrogel-based microfluidic chips. We then demonstrated the scalability and customization flexibility of our hydrogel-based chip via engineering tools. A multiscale physical and structural data analysis suggested a relationship between cell invasion response and bioink characteristics. Tumor cell invasive behavior and focal adhesion properties were observed in response to varying polymer network densities of the GelMA-based bioink. Immunostaining assays and reverse transcription-quantitative polymerase chain reaction (RT-qPCR) helped assess the bioactivity of the microtissue and measure the cell invasion. The RT-qPCR data showed higher expressions of HIF-1α, CD44, and MMP2 genes in a lower polymer density, highlighting the correlation between bioink structural porosity, ECM stiffness, and tumor spheroid response. This work is the first step in modeling STS tumor invasiveness in hydrogel-based microfluidic chips. STATEMENT OF SIGNIFICANCE: We optimized an engineering protocol for making tumor spheroids at a controlled size, embedding spheroids into a gelatin-based matrix, and constructing a perfusable microfluidic device. A higher tumor invasion was observed in a low-stiffness matrix than a high-stiffness matrix. The physical characterizations revealed how the stiffness is controlled by the density of polymer chain networks and porosity. The biological assays revealed how the structural properties of the gelatin matrix and hypoxia in tumor progression impact cell invasion. This work can contribute to personalized medicine by making more effective, tailored cancer models.

Flexural Properties, Wear Resistance, and Microstructural Analysis of Highly Filled Flowable Resin Composites.

OBJECTIVE: This study aimed to evaluate the flexural properties and two-body wear resistance of nine highly filled flowable resin composites relative to those of viscous and conventional low-filled flowable composites. In addition, scanning electron microscopy (SEM) analysis of the microstructures was performed. METHODS AND MATERIALS: For each resin composite group (n=12), 12 specimen bars (25 mm × 2 mm × 2 mm) were fabricated using a silicon mold for performing flexural strength (FS), flexural modulus (E), flexural toughness (FT), Weibull modulus (m) tests, and SEM microstructural analysis. For each group, ten bars were tested using a three-point flexural test on a universal testing machine, while the other two were embedded in acrylic resin before being observed by SEM for structural analysis. During the two-body wear test with a chewing simulator, 8 specimens (12 groups, n=8) of each resin composite group were manufactured in a specific mold and subjected to 120,000 cycles of wear against a steatite ball, and the depth loss was measured. Three one-way ANOVA tests followed by Tukey's post hoc tests were conducted to compare the flexural and wear properties among the different groups. RESULTS: The majority of highly filled composites tested in this study exhibited similar flexural strengths (between 105.68 MPa and 135.49 MPa) and superior wear resistance to those of viscous composites. The flexural moduli (between 5.12 GPa and 9.62 GPa) of these composites were in between those of the viscous and low-filled composites tested in this study. CONCLUSIONS: The highly filled flowable composites tested in this study exhibited different in vitro properties but were often superior to those of viscous resin composite suggesting their possible use for posterior restorations.

In Vitro Model Integrating Substrate Stiffness and Flow to Study Endothelial Cell Responses.

We present an innovative in vitro model aimed at investigating the combined effects of tissue rigidity and shear stress on endothelial cell (EC) function, which are crucial for understanding vascular health and the onset of diseases such as atherosclerosis. Traditionally, studies have explored the impacts of shear stress and substrate stiffness on ECs, independently. However, this integrated system combines these factors to provide a more precise simulation of the mechanical environment of the vasculature. The objective is to examine EC mechanotransduction across various tissue stiffness levels and flow conditions using human ECs. We detail the protocol for synthesizing gelatin methacrylate (GelMA) hydrogels with tunable stiffness and seeding them with ECs to achieve confluency. Additionally, we describe the design and assembly of a cost-effective flow chamber, supplemented by computational fluid dynamics simulations, to generate physiological flow conditions characterized by laminar flow and appropriate shear stress levels. The protocol also incorporates fluorescence labeling for confocal microscopy, enabling the assessment of EC responses to both tissue compliance and flow conditions. By subjecting cultured ECs to multiple integrated mechanical stimuli, this model enables comprehensive investigations into how factors such as hypertension and aging may affect EC function and EC-mediated vascular diseases. The insights gained from these investigations will be instrumental in elucidating the mechanisms underlying vascular diseases and in developing effective treatment strategies.

Porous Gelatin Methacrylate Gel Engineered by Freeze-Ultraviolet Promotes Osteogenesis and Angiogenesis.

Alveolar bone defect reconstruction is a common challenge in stomatology. To address this, a thermosensitive/photosensitive gelatin methacrylate (GelMA) gel was developed based on various air solubilities and light-curing technologies. The gel was synthesized by using a freeze-ultraviolet (FUV) method to form a porous and quickly (within 15 min) solidifying modified network structure. Unlike other gel scaffolds limited by complex preparation procedures and residual products, this FUV-GelMA gel shows favorable manufacturing ability, promising biocompatibility, and adjustable macroporous structures. The results from a rat model suggested that this gel scaffold creates a conducive microenvironment for mandible reconstruction and vascularization. In vitro experiments further confirmed that the FUV-GelMA gel promotes osteogenic differentiation of human bone marrow mesenchymal stem cells and angiogenesis of human umbilical vein endothelial cells. Investigation of the underlying mechanism focused on the p38 mitogen-activated protein kinase (MAPK) pathway. We found that SB203580, a specific inhibitor of p38 MAPK, abolished the therapeutic effects of the FUV-GelMA gel on osteogenesis and angiogenesis, both in vitro and in vivo. These findings introduced a novel approach for scaffold-based tissue regeneration in future clinical applications.

In vitro analysis of a novel dimethylaminododecyl methacrylate modification of dental acrylic soft liner material.

Soft denture liners have limitations like short lifespan and increased microbial buildup. Despite promise as a non-leaching antimicrobial polymer in dentistry, the impact of dimethylaminododecyl methacrylate (DMADDM) on soft liner performance remains unexplored. This study aimed to evaluate the effect of integrating different concentrations of DMADDM to cold cure acrylic resin soft liner, on its antimicrobial activity, cytotoxicity, and physical properties. The same properties were compared to a conventional commercially available denture soft liner. The study employed a control group (conventional soft liner) and three test groups containing 3.3%, 6.6%, and 10% (total mass fraction) DMADDM, respectively. Antimicrobial activity against Candida albicans and Streptococcus mutans was assessed through colony counts and biofilm biomass. Cytotoxicity was evaluated using an oral epithelial cell line. Additionally, wettability and hardness were measured to assess physical properties. Incorporation of DMADDM significantly reduced Candida albicans and Streptococcus mutans counts, and biofilm biomass, compared to the control. Additionally, DMADDM improved the soft liner's wettability and mitigated long-term hardness increase. In conclusion, DMADDM holds promise in enhancing soft liner performance. However, careful selection of its optimum concentration is crucial to ensure both safety and efficacy for future clinical use.

Massive fabrication of functional hepatic cancer spheroids by micropatterned GelMA hydrogel chip for drug screening.

Since hepatic cancer incidence and mortality continue to grow worldwide, it is necessary to develop the biomimetic tumor models for drug development and tumor therapeutics. Cellular spheroids as an excellent simple 3D model can bridge the gap between 2D cell culture and live tissue. In this study, we proposed a biological methacrylated gelatin (GelMA) hydrogel-based microplatform for the massive generation of hepatocellular spheroids and downstream investigation of drug resistance. Micropatterned GelMA hydrogel microwell chip (GHM-chip) with tunable array was easily achieved in standard 24-culture well plates through the micro-molding fabrication strategy. The fabricated GHM-chip induced multicellular self-assembly behavior within the defined topography and further formed spheroidal structure. By regulating cell seeding density and designing microwell size, uniform hepatic cancer spheroids with tunable diameters were obtained in a simplicity, stability and controllable manner. In addition, the screening chemotherapy study of anti-cancer drug was completed through non-destructive recovery of spheroids from the GHM-chip. Beyond that, the recovered functional spheroids have potential application value in various biomedical fields such as tumor biology, pharmacology, and tissue microengineering. Finally, the proposed GHM-chip incorporated into standard cell culture plates with easy to manufacture and operate properties, may be an efficient culture microplatform for cancer research applications.

GelMA loaded with platelet lysate promotes skin regeneration and angiogenesis in pressure ulcers by activating STAT3.

Pressure ulcers (PU) are caused by persistent long-term pressure, which compromises the integrity of the epidermis, dermis, and subcutaneous adipose tissue layer by layer, making it difficult to heal. Platelet products such as platelet lysate (PL) can promote tissue regeneration by secreting numerous growth factors based on clinical studies on skin wound healing. However, the components of PL are difficult to retain in wounds. Gelatin methacrylate (GelMA) is a photopolymerizable hydrogel that has lately emerged as a promising material for tissue engineering and regenerative medicine. The PL liquid was extracted, flow cytometrically detected for CD41a markers, and evenly dispersed in the GelMA hydrogel to produce a surplus growth factor hydrogel system (PL@GM). The microstructure of the hydrogel system was observed under a scanning electron microscope, and its sustained release efficiency and biological safety were tested in vitro. Cell viability and migration of human dermal fibroblasts, and tube formation assays of human umbilical vein endothelial cells were applied to evaluate the ability of PL to promote wound healing and regeneration in vitro. Real-time polymerase chain reaction (PCR) and western blot analyses were performed to elucidate the skin regeneration mechanism of PL. We verified PL's therapeutic effectiveness and histological analysis on the PU model. PL promoted cell viability, migration, wound healing and angiogenesis in vitro. Real-time PCR and western blot indicated PL suppressed inflammation and promoted collagen I synthesis by activating STAT3. PL@GM hydrogel system demonstrated optimal biocompatibility and favorable effects on essential cells for wound healing. PL@GM also significantly stimulated PU healing, skin regeneration, and the formation of subcutaneous collagen and blood vessels. PL@GM could accelerate PU healing by promoting fibroblasts to migrate and secrete collagen and endothelial cells to vascularize. PL@GM promises to be an effective and convenient treatment modality for PU, like chronic wound treatment.

Surface roughness of different types of resin composites after artificial aging procedures: an in vitro study.

BACKGROUND: The temperature changes, chemical agents, and brushing activity that resin composite restorations are exposed to in the oral environment can cause changes in surface roughness. In this study, the aim was to investigate in vitro the clinical one-year surface roughness changes of different types of composites (flowable or conventional) from the same companies by subjecting them to immersion in solutions, brushing, and thermal cycling procedures to simulate intraoral conditions. METHODS: Four different resin composite brands were included in the study using both their conventional (Charisma Smart, 3M Filtek Ultimate Universal, Omnichroma, Beautifil II) and flowable resin composites (Charisma Flow, 3M Filtek Ultimate Flowable, Omnichroma Flow, Beautifil Flow Plus F00), giving 4 groups with 2 types of resin composite in each. 40 samples were prepared for each group/resin type, for a total of 320 samples. After initial surface roughness measurements by a mechanical profilometer, the samples were divided into 4 subgroups (n = 10) and immersed in solutions (distilled water, tea, coffee, or wine) for 12 days. The samples were then subjected to 10,000 cycles of brushing simulation and 10,000 cycles of thermal aging. Surface roughness measurements were repeated after the procedures. For statistical analysis, the 3-way analysis of variance and the Tukey test were used (p < 0.05). RESULTS: It was concluded that composite groups and types had an effect on surface roughness at time t0 (p < 0.001). At time t1, the highest surface roughness value was obtained in the Beautifil-conventional interaction. When the surface roughness values between time t0 and t1 were compared, an increase was observed in the Beautifil II and Beautifil Flow Plus F00, while a decrease was observed in the other composite groups. CONCLUSION: Composite groups, types, and solutions had an effect on the surface roughness of resin composites. After aging procedures, it was concluded that the Beautifil group could not maintain the surface structure as it exceeded the threshold value of 0.2 µm for bacterial adhesion.