Culture Plates Research Articles

A Low‐Cost, User‐Friendly Rheo‐Optical Compression Assay to Measure Mechanical Properties of Cell Spheroids in Standard Cell Culture Plates

AbstractThe mechanical characterization of cell spheroids, one of the most widely used 3D biology models in vitro, is a hotspot of current research on the role played by the mechanical response of cells and tissues. The techniques proposed so far in the literature, while providing important scientific insights, require specialized equipment and technical skills that are not usually available in cell biology facilities. Here, an innovative rheo‐optical compression assay is presented: it is based on microscopy glass coverslips as the load is applied to cell spheroids in standard cell culture plates and on image acquisition with an optical microscope or even a smartphone equipped with adequate magnification lenses. Mechanical properties can be simply obtained by correlating the applied load to the deformation of cell spheroids measured by image analysis. The low‐cost, user‐friendly features of the proposed technique can boost mechanobiology research making it easily affordable to any biomedical lab equipped with cell culture facilities.

Saliva biofilm-derived outer membrane vesicles regulate biofilm formation and immune response of oral epithelial cells on titanium surfaces.

While the significant roles of outer membrane vesicles (OMVs) from individual oral bacterial species in bacterial-host interactions are known, the involvement of saliva biofilm-derived OMVs in peri-implant disease pathogenesis remains unclear. This study aimed to investigate the effect of saliva biofilm-derived OMVs on regulating saliva biofilm formation and modulating the immune response of the epithelial cells on titanium surfaces. Saliva derivedbiofilms were cultured on tissue culture plates (TCP) for 4 days using pooled saliva from four healthy donors. OMVs secreted from the TCP bound biofilm (referred to as OMVs or healthy saliva biofilm OMVs) were enriched using the size-exclusion chromatography method. We then evaluated the effects of these OMVs on theviability, metabolic activity, and the presence oforal pathogens in saliva biofilm grownon titanium discs for 24 h and 72 h. Furthermore, the impact of OMVs on the mRNA expression and inflammatory cytokines [interleukin (IL)-6, IL-1α, and monocyte chemoattractant protein-1 (MCP-1)] in human oral epithelial cells (OKF6/TERT-2) was investigated using RT-qPCR and enzyme-linked immunosorbent assay(ELISA), respectively. Healthy saliva biofilm OMVs improved the biomass and activity of saliva biofilm cultured on the titanium surfaces, with inhibited Porphyromonas gingivalis and Fusobacterium nucleatum, and enhanced Streptococcus mutansexpression. Additionally, OMVs increased pro-inflammatory cytokine IL-6 mRNA and IL-6cytokine expression in human oral epithelial cells. However, IL-1α and MCP-1 cytokines were inhibited 24-hour post-incubation with OMVs. Healthy saliva biofilm derivedOMVs regulate the activity and pathogen composition of biofilms formed on titanium, while modulating the secretion of pro-inflammation factors of oral epithelial cells grownon titanium surfaces. Healthy saliva biofilm OMVs may regulate the early biofilm formation on abutment surfaces and modulate epithelial cell immune response, which may alter the peri-implant niche and participate in the pathogenesis of peri-implant disease.

Enhanced burn wound healing by controlled-release 3D ADMSC-derived exosome-loaded hyaluronan hydrogel.

Adipose mesenchymal stem cell (ADMSC)-derived exosomes (ADMSC-Exos) have shown great potential in regenerative medicine and been evidenced benefiting wound repair such as burns. However, the low yield, easy loss after direct coating, and no suitable loading system to improve their availability and efficacy hinder their clinical application for wound healing. And few studies focused on the comparison of biological functions between exosomes derived from different culture techniques, especially in exosome-releasing hydrogel system. Therefore, we designed a high-performance exosome controllable releasing hydrogel system for burn wound healing, namely loading 3D-printed microfiber culture-derived exosomes in a highly biocompatible hyaluronic acid (HA). In this project, we compared the biological functions in vitro and in a burn model among exosomes derived from the conventional two-dimensional (2D) plate culture (2D-Exos), microcarrier culture (2.5D-Exos), and 3D-printed microfiber culture (3D-Exos). Results showed that compared with 2D-Exos and 2.5D-Exos, 3D-Exos promoted HACATs and HUVECs cell proliferation and migration more significantly. Additionally, 3D-Exos had stronger angiogenesis-promoting effects in tube formation of (HUVECs) cells. Moreover, we found HA-loaded 3D-Exos showed better burn wound healing promotion compared to 2D-Exos and 2.5D-Exos, including accelerated burn wound healing rate and better collagen remodeling. The study findings reveal that the HA-loaded, controllable-release 3D-Exos repair system distinctly augments therapeutic efficacy in terms of wound healing, while concurrently introducing a facile application approach. This system markedly bolsters the exosomal loading efficiency, provides a robust protective milieu, and potentiates the inherent biological functionalities of the exosomes. Our findings provide a rationale for more efficient utilization of high-quality and high-yield 3D exosomes in the future, and a novel strategy for healing severe burns.

Indirubin induces apoptosis in ovarian cancer cells via the mitochondrial pathway.

To investigate the pro-apoptotic effects of Indirubin, a traditional Chinese medicine, on ovarian cancer SKOV3 cell line and to explore its underlying mechanisms. Ovarian cancer SKOV3 cells were divided into a control group (cells cultured normally) and an experimental group (cells cultured in medium containing Indirubin). SKOV3 cells at the logarithmic phase were treated with Indirubin at various concentrations. Cell proliferation was assessed using the Cell Counting Kit-8 (CCK-8) assay and 3-(4,5)-dimethylthiahiazo (-z-y1)-3,5-di-phenytetrazoliumromide (MTT) assay, while apoptosis was detected by flow cytometry, TdT-mediated dUTP Nick-End Labeling (TUNEL) staining, and immunofluorescence. Transcriptome sequencing was conducted to screen for apoptosis-related factors. qPCR and western blot were used to detect the mRNA and protein expression on added of molecules related to mitochondrial permeability transition pores. MTT assay showed that Indirubin inhibited the growth of SKOV3 cells in both plate and sphere cultures, with IC50 values of 3.003 μM (plate culture) and 4.253 μM (sphere culture), respectively. Indirubin showed a lower inhibitory concentration than cisplatin (IC50 3.687 μM in plate culture and 7.023 μM in sphere culture), and its effect was comparable to adriamycin. Flow cytometry revealed an increase in apoptosis rates in SKOV3 cells treated with Indirubin. Transcriptome sequencing indicated significant changes in the transcription of various apoptosis-related genes, particularly those involved in the mitochondrial apoptosis pathway, such as Bcl-2-associated X protein (Bax) and Bcl2 associated agonist of cell death (Bad). After Indirubin treatment, the mRNA and protein expression levels of mitochondrial channel-related genes Cyclophilin D (CyPD), adenine nucleotide translocator 1 (ANT1), and voltage-dependent anion channel (VADC) were significantly elevated (all P < 0.05). By regulating the mitochondrial membrane permeability through the Bcl-2 family members, Indirubin promoted apoptosis in SKOV3 cells. Indirubin inhibits the proliferation and promotes the apoptosis of ovarian cancer cells, exerting an anti-tumor effect. Its pro-apoptotic action is closely related to the mitochondrial apoptosis pathway.

Cell growth monitoring in a tetrapolar electrode configuration.

There are various methods for cell growth monitoring. However, most of these methods have drawbacks, such as being invasive, not providing real-time results, or being costly. In this study, we present an alternate method of cell growth monitoring, which is low-cost, non-invasive, real-time, and uses Electrical Impedance Spectro-scopy (EIS). In this work, commercially available culture plates were fitted with custom tetrapolar electrodes, and mouse cells were cultured on them. The variation of culture media impedance, resulting from cell growth, proliferation and other metabolic activities, was recorded over a period of seven days. The results demonstrated an initial increase in impedance corresponding with the cell growth phase, followed by a decrease during the cell death (apoptosis) phase, as confirmed by microscope images. Overall, the results show that our method to monitor cell growth using tetrapolar electrodes is promising and can be further refined for related applications.

Ceratocystis fimbriata Causing Postharvest Fruit Rot of Pear in Punjab, Pakistan.

Pear (Pyrus communis) is an important deciduous fruit cultivated on a worldwide scale including Pakistan. During August 2021, a postharvest fruit rot disease was observed on several pears at various farmers market in Okara- a district of Punjab Province, Pakistan. The incidence of the disease varied from 7 to 20% with 35% disease severity. Necrotic spots (10 to 20 mm diameter) were first observed on the infected pear fruit. The spots enlarged gradually and developed into a brown, water-soaked and rotted lesion. Eventually, the whole fruit became soft, rotted and covered with a gray-brown mycelium. The isolates were obtained from the symptomatic tissues (n = 18) incubated on carrot discs that had been surface sterilized in 100-ppm streptomycin solution. After consistent sporulation of a fungus on the carrot discs, the ascospore masses formed at the tip of perithecia were transferred to malt extract agar (MEA). Primary conidia were cylindrical and hyaline (7 to 11 × 4 to 7 μm) and secondary conidia were hyaline and barrel-shaped (7 to 12 × 5 to 8 μm). Endoconidiophores with primary conidia were (12 to 27 × 2.6 to 5.5 μm). Perithecia produced on carrot discs were dark brown to black, and the base was 157 to 278 μm in diameter. Ascomatal necks were 512 to 656 µm long, dark brown to black, lighter in color at apices, tapering from base (23 to 45 μm diameter) to apex (13 to 24 μm diameter). Ostiolar hyphae were 41 to 79 μm long. Ascospores were hyaline, hat shaped, 3 to 4 μm long, and accumulated in a sticky matrix at the tips of perithecial necks. Mycelium was initially hyaline but became dark greenish brown after 7 days. Dark brown, thick-walled aleuroconidia (13 to 19.5 × 9 to 14 μm) appeared on culture plates after 2 months. Based on morphological characteristics, the fungus was identified as Ceratocystis fimbriata (Engelbrecht, 2005; Suwandi et al. 2021). To further confirm species identification, genomic DNA of two representative isolates (UO-05 and UO-06) was obtained using an extraction kit. The internal transcribed spacer (ITS) region was amplified using ITS1/4 (White et al. 1990). A BLAST search with GenBank accession nos. OR185451 and OR185456 indicated 99 to 100% identity with several C. fimbriata including type species (MH856050.1; KC493160.1; MT560374.1). Pathogenicity tests were conducted by inoculating nine disease-free pear (cv. Concord) fruit after disinfesting in 75% ethanol. A prepared spore suspension (1.0 × 106 spores/ml) was dropped on the wounds (a depth of 1 mm diameter) on the pear surface, which were made by a sterilized needle. 10 μl of a prepared spore suspension was dropped onto nine pears. Sterile water (10 μl) was dropped on the wounded sites of nine pear fruits as negative controls and all fruits were incubated in a growth chamber at 30/26°C (day/night, 90% relative humidity). Symptoms similar to those on the naturally infected fruits began after 4 to 5 days of inoculation, while controls remained healthy. The fungal isolates recovered from inoculated pears were morphologically identical to the C. fimbriata isolates originally recovered from symptomatic fruits fulfilling Koch's postulates. The pathogen has been reported to cause postharvest fruit rot of passion fruit and cucumber (Firmino et al. 2016; Li et al. 2019). To our knowledge, this is the first report of C. fimbriata causing fruit rot of pear in Punjab Province. The detection of this disease will help pear growers to take actions to monitor and prevent disease outbreak as well as develop an effective management practice when it occurs.

Reversible bonding in thermoplastic elastomer microfluidic platforms for harvestable 3D microvessel networks.

Transplantable ready-made microvessels have therapeutic potential for tissue regeneration and cell replacement therapy. Inspired by the natural rapid angiogenic sprouting of microvessels in vivo, engineered injectable 3D microvessel networks are created using thermoplastic elastomer (TPE) microfluidic devices. The TPE material used here is flexible, optically transparent, and can be robustly yet reversibly bonded to a variety of plastic substrates, making it a versatile choice for microfluidic device fabrication because it overcomes the weak self-adhesion properties and limited manufacturing options of poly(dimethylsiloxane) (PDMS). By leveraging the reversible bonding characteristics of TPE material templates, we present their utility as an organ-on-a-chip platform for forming and handling microvessel networks, and demonstrate their potential for animal-free tissue generation and transplantation in clinical applications. We first show that TPE-based devices have nearly 6-fold higher bonding strength during the cell culture step compared to PDMS-based devices while simultaneously maintaining a full reversible bond to (PS) culture plates, which are widely used for biological cell studies. We also demonstrate the successful generation of perfusable and interconnected 3D microvessel networks using TPE-PS microfluidic devices on both single and multi-vessel loading platforms. Importantly, after removing the TPE slab, microvessel networks remain intact on the PS substrate without any structural damage and can be effectively harvested following gel digestion. The TPE-based organ-on-a-chip platform offers substantial advantages by facilitating the harvesting procedure and maintaining the integrity of microfluidic-engineered microvessels for transplant. To the best of our knowledge, our TPE-based reversible bonding approach marks the first confirmation of successful retrieval of organ-specific vessel segments from the reversibly-bonded TPE microfluidic platform. We anticipate that the method will find applications in organ-on-a-chip and microphysiological system research, particularly in tissue analysis and vessel engraftment, where flexible and reversible bonding can be utilized.

Immunohistochemical characterization of pluripotency of an iPSC line derived from a patient with cone‐rod dystrophy related to a mutation in the PROM1 gene

Purpose: PROM1 gene encodes the protein Prominin‐1 which plays a critical role in the morphogenesis of photoreceptors outer segments. The c.1354dupT mutation in PROM1 causes a premature stop codon and it has been related with different inherited retinal diseases (IRD) phenotypes. The aim of this study was to demonstrate the expression of key pluripotency genes by studying protein transcription factors and surface markers on the iPSC line [DCB]‐FiPSC1‐Ep5F‐2.Methods: Our group generated the iPSC line [DCB]‐FiPSC1‐Ep5F‐2 from a patient with Cone‐rod dystrophy (CRD) related to the mutation c.1354dupT in the PROM1 gene. iPSC was grown on matrigel coated 15 μ‐slide 8‐well culture plates and fixed with 10% formalin solution. Plates were washed with Tris Buffered Saline and blocked (0.3%Triton X‐100 and 3%Donkey serum) for 60 min. Then, different combinations of primary antibodies related to pluripotency (OCT4, SSEA3, SOX2, SSEA4, TRA1‐60, NANOG and TRA1‐81) were incubated at 4°C overnight. Afterwards, species‐specific secondary antibodies were conjugated and immunostaining with DAPI was used. Finally, the samples were evaluated by confocal microscopy.Results: Immunofluorescence analysis of the iPSC line [DCB]‐FiPSC1‐Ep5F‐2 revealed the expression of the common embryonic stem cell markers such as the transcription factors OCT4, NANOG and SOX2, and the surface markers SSEA3, TRA‐1‐81, SSEA4 and TRA‐1‐60. Transcriptional markers stain cell nuclei, while surface markers stain cell membranes showing that iPSCs have the potential for pluripotency and multilineage differentiation.Conclusions: Although molecular characterization is necessary to completely elucidate iPSC pluripotency and bank this line in an international depository, the [DCB]‐FiPSC1‐Ep5F‐2 patient derived iPSC line could be useful as a disease model for the development of personalized therapies.

Engineered Heart Tissues for Standard 96-Well Tissue Culture Plates.

Engineered heart tissues (EHTs) have been shown to be a valuable platform for disease investigation and therapeutic testing by increasing human induced pluripotent stem cell-derived cardiomyocyte (hiPSC-CM) maturity and better recreating the native cardiac environment. The protocol detailed in this chapter describes the generation of miniaturized EHTs (mEHTs) incorporating hiPSC-CMs and human stromal cells in a fibrin hydrogel. This platform utilizes an array of silicone posts designed to fit in a standard 96-well tissue culture plate. Stromal cells and hiPSC-CMs are cast in a fibrin matrix suspended between two silicone posts, forming an mEHT that produces synchronous muscle contractions. The platform presented here has the potential to be used for high throughput characterization and screening of disease phenotypes and novel therapeutics through measurements of the myocardial function, including contractile force and calcium handling, and its compatibility withimmunostaining.

Quantitative mechanical stimulation of GPR68 using a novel 96 well flow plugin.

Mechanosensitive proteins play a crucial role in a range of physiological processes, including hearing, tactile sensation and regulating blood flow. While previous work has demonstrated the mechanosensitivity of several proteins, the ability to apply precisely defined mechanical forces to cells in a consistent, replicable manner remains a significant challenge. In this work we present a novel 96-well plate-compatible plugin device for generating highly-controlled flow-based mechanical simulation of cells, which enables quantitative assessment of mechanosensitive protein function. The device is used to mechanically stimulate HEK 293T cells expressing the mechanosensitive protein GPR68, a G protein-coupled receptor. By assaying intracellular calcium levels during flow-based cell stimulation, we determine that GPR68 signalling is a function of the applied shear-force. As this approach is compatible with conventional cell culture plates and allows for simultaneous readout in a conventional fluorescence plate reader, this represents a valuable new tool to investigate mechanotransduction.

Substrate softness increases magnetic microdiscs-induced cytotoxicity

Cytotoxicity of nanoparticles is primarily assessed on cells grown in plastic culture plates, a mechanical environment that is a million times stiffer than most of the human tissues. Here we...

Collagen/polyester-polyurethane porous scaffolds for use in meniscal repair.

Focusing on the regeneration of damaged knee meniscus, we propose a hybrid scaffold made of poly(ester-urethane) (PEU) and collagen that combines suitable mechanical properties with enhanced biological integration. To ensure biocompatibility and degradability, the degradable PEU was prepared from a poly(ε-caprolactone), L-lysine diisocyanate prepolymer (PCL di-NCO) and poly(lactic-co-glycolic acid) diol (PLGA). The resulting PEU (Mn = 52 000 g mol-1) was used to prepare porous scaffolds using the solvent casting (SC)/particle leaching (PL) method at an optimized salt/PEU weight ratio of 5 : 1. The morphology, pore size and porosity of the scaffolds were evaluated by SEM showing interconnected pores with a uniform size of around 170 μm. Mechanical properties were found to be close to those of the human meniscus (Ey ∼ 0.6 MPa at 37 °C). To enhance the biological properties, incorporation of collagen type 1 (Col) was then performed via soaking, injection or forced infiltration. The latter yielded the best results as shown by SEM-EDX and X-ray tomography analyses that confirmed the morphology and highlighted the efficient pore Col-coating with an average of 0.3 wt% Col in the scaffolds. Finally, in vitro L929 cell assays confirmed higher cell proliferation and an improved cellular affinity towards the proposed scaffolds compared to culture plates and a gold standard commercial meniscal implant.

Myrtenol's Effectiveness against Hospital-Acquired Methicillin-Resistant Staphylococcus aureus: Targeting Antibiofilm and Antivirulence Properties.

The emergence of methicillin-resistant Staphylococcus aureus (MRSA) several years ago highlighted the challenge of multidrug-resistant infections, emphasizing the critical need for innovative treatment approaches. Myrtenol, known for its antibacterial and antibiofilm properties, holds promise as a potential treatment option. This study aimed to evaluate the effectiveness of myrtenol against MRSA. The collected MRSA isolates were assessed for antimicrobial susceptibility following the Clinical and Laboratory Standards Institute (CLSI) guidelines 2023. Biofilm formation by MRSA was evaluated using the tissue culture plate (TCP) technique. The minimal inhibitory concentration (MIC), minimal bactericidal concentration (MBC), and minimal biofilm inhibitory concentration (MBIC) of myrtenol against MRSA were determined both individually and in combination with antibiotics. Real-time PCR was employed to investigate the impact of myrtenol on the expression of virulence genes (sarA, agrA, and icaD) across the isolates. In this study, MRSA was identified in 90 out of 400 cases (22.5%) of hospital-acquired pathogens. Among the collected MRSA isolates, 53 out of 90 (59%) were found to produce biofilms. The MIC of myrtenol was comparable to the MBC across all tested isolates, they were almost the same. Combinations of myrtenol with most tested antibiotics exhibited synergistic effects exceeding 60%. Among the 53 biofilm-producing isolates, 45 isolates (85%) expressed the sarA gene, 49% expressed the agrA gene, and all biofilm-producing MRSA isolates (100%) expressed the icaD gene. A notable reduction in the relative quantity (RQ) values of virulence gene expression was observed after treatment with the MBIC of myrtenol across all tested isolates. Myrtenol demonstrated strong antimicrobial activity against MRSA, notably reducing the expression of key virulence genes linked to biofilm formation. This suggests its potential as a therapeutic agent for treating biofilm-associated MRSA infections.

Comparison of two invitro phenotypic methods (Tissue Culture plate and Congo Red Agar) for Detection of Biofilm Formation by Enterococci

Comparison of two invitro phenotypic methods (Tissue Culture plate and Congo Red Agar) for Detection of Biofilm Formation by Enterococci

Optimal amount of Bacillus amyloliquefaciens FH1-RS1 and Bacillus cereus R19 to inhibit the growth of Verticillium dahliae

Biological control of Verticillium wilt caused by Verticillium dahliae is a green and sustainable strategy. However, the antifungal activities of different amounts of biocontrol bacterial are still not understand. This study investigates the antifungal activity of different amount of Bacillus amyloliquefaciens FH1-RS1 and Bacillus cereus R19 against Verticillium dahliae V991 by dual culture plate. The results showed that both the fungal inhibition rate (FIR) and far fungal inhibition rate (FFIR) of B. amyloliquefaciens FH1-RS1 elevated with the increasing volume. The highest FIR (77.78%) and FFIR (58.33%) of B. amyloliquefaciens FH1-RS1 were both in 22 μL volume (7.2 × 108 CFU/mL). The highest FIR of B. cereus R19 was 48.15% in 12, 18 and 20 μL volume (1.2 × 108 CFU/mL). The highest FFIR of B. cereus R19 was 38.89% in 6 μL volume. This suggests that not all biocontrol bacteria have higher antifungal activity in higher amounts.

Sensitivity analysis and quality indicators for an in vitro oral irritation assay.

Biocompatibility testing using in vivo tests is often one of the final evaluations of new dental materials. To reduce the likelihood of failure at this late stage, predictive biocompatibility testing using in vitro methods is needed. In this study, we describe a sensitivity analysis of an oral irritation test by evalu­ating changes in the viability, using the MTT assay, of 3-D models with EpiOral constructs as a case study. Experiments that tested sources of variability in the assay led to recommendations regarding the storage of the constructs after arrival, pipetting procedure, use of MTT reagents from different vendors, use of transepithelial electrical resistance measurements, and statistical analyses. A statis­tical model was proposed to evaluate whether test substances yield a positive or negative result and the associated statistical confidence. Testing several test compounds such as the Y-4 polymer, which contains a known irritant, and dentally relevant substances such as sodium dodecyl sulfate (SDS) at varying concentrations revealed statistically significant results as expected. Lastly, a software app was designed to support a multiwell culture plate layout design. Overall, the findings and sugges­tions documented here will support the further development and potential standardization of this assay system and may be useful for the development of other assays using 3-D constructs.

Caffeine exposure causes downregulation of the expression of genes related to osteogenesis and chondrogenesis in Zebrafish (Danio rerio)

With the aim of evaluating the effects of caffeine on the expression of gene transcripts related to osteogenesis and chondrogenesis, during zebrafish embryonic development the embryos were divided into five groups: control (without caffeine), 0.25 mM caffeine, 0.5 mM caffeine, 1.0mM caffeine, and 2.0mM caffeine. Embryos were placed in cell culture plates with aquarium water at 280 C for 72 hours post-fertilization (hpf). Embryonic development was followed at times of six, 12 hpf, 24 hpf, 30 hpf, 48 hpf, 54 hpf, and 72 hpf. At 72 hpf, the expression of Sox9a, runx2b, osteocalcin, osteopontin, collagen 2a1, and bmp2a was analyzed using real-time RT-PCR. The 0.25mM caffeine group showed embryonic development that was visually similar to the control group despite having a significantly lower relative expression of bmp2a, runx2b, and collagen 2a1. In the 0.5mM and 1mM caffeine groups, morphological alterations such as tail folding, pericardial edema, and yolk sac deformation were observed, and the relative expression levels of bmp2a, runx2b, and collagen 2a1 were significantly lower. In contrast, osteocalcin expression was significantly higher than in the control group. In the 2mM caffeine group, the embryos did not hatch until 72 hpf and showed tail docking and pericardial and yolk sac edema. The relative expression of bmp2a and runx2b was significantly lower, whereas that of osteocalcin, osteopontin, and collagen 2a1 did not differ from that of the control group. We conclude that caffeine altered the expression of gene transcripts related to osteogenesis and chondrogenesis in zebrafish embryos, even in embryos without visible morphological changes.

Increased Resolvin E1 Production by Peripheral Blood Mononuclear Cells in Periodontitis Patients: Pilot Study.

This study quantified the production of the pro-resolving agent Resolvin E1 by peripheral blood mononuclear cells (PBMC) from 20 systemically healthy volunteers with and without periodontitis after stimulation with lipopolysaccharide (LPS) from Porphyromonas gingivalis (Pg). Ten periodontitis patients and 10 healthy volunteers (30-50 years old), matched by age and sex, were recruited. Peripheral blood mononuclear cells were isolated and stimulated in culture plates for 24 hours with Pg LPS. Resolvin E1 levels were measured in the supernatants by enzyme-linked immunosorbent assay. Significantly higher production of Resolvin E1 was observed in both groups when stimulated with LPS compared to baseline levels (p<0.001). A significant increase in Resolvin E1 was observed in the presence of Lipopolysaccharide in the patients with periodontitis compared to the healthy group (p=0.0019). Resolvin E1 levels may reflect a measure of resolution of inflammation that warrants further clinical investigation.

Static magnetic field enhances the bone remodelling capacity of human demineralized bone matrix in a rat animal model of cranial bone defects.

The regeneration of bone defects that exceed 2 cm is a challenge for the human body, necessitating interventional therapies. Demineralized bone matrices (DBM) derived from biological tissues have been employed for bone regeneration and possess notable osteoinductive and osteoconductive characteristics. Nevertheless, their efficiency in regenerating critically sized injuries is limited, and therefore additional signaling cues are required. Thanks to the piezoelectric properties of the bone, external physical stimulation is shown to accelerate tissue healing. We have implanted human DBM in critically sized cranial bone defects in rat animal models and exposed them to an external magnetic field (1 T) to enhance endogenous bone formation. Our in vitro experiments showed the superior cytocompatibility of DBM compared to cell culture plates. Furthermore, alkaline phosphatase activity after 14 days and Alizarin red staining at 28 days demonstrated differentiation of rat bone marrow mesenchymal stem cells into bone lineage on DBM. Computer tomography images together with histological analyses showed that implanting DBM in the injured rats significantly enhanced bone regeneration. Notably, combining DBM transplantation with a 2 h daily exposure to a 1 T magnetic field for 2 weeks (day 7 to 21 post-surgery) significantly improved bone regeneration compared to DBM transplantation alone. This research indicates that utilizing external magnetic stimulation significantly enhances the potential of bone allografts to regenerate critically sized bone defects.

Antimicrobial and Antibiofilm Activity of Monolaurin against Methicillin-Resistant Staphylococcus aureus Isolated from Wound Infections.

Methicillin-resistant Staphylococcus aureus (MRSA) is one of the major pathogens associated with life-threatening infections, showing resistance to various antibiotics. This study aimed to assess the influence of monolaurin on biofilm-forming MRSA. The agar dilution method determined the minimum inhibitory concentration (MIC) of monolaurin against MRSA isolates and explored its impact on the resistance profile of selected antibiotics. The assessment of combined therapy involving monolaurin and antibiotics was conducted using fractional inhibitory concentration (FIC). The tissue culture plate strategy appraised monolaurin's antibiofilm activity and its inhibitory concentration (IC50), with assessment via scanning electron microscopy. Reverse transcription polymerase chain reaction (RT-PCR) discerned a monolaurin effect on the expression of the icaD gene. Monolaurin exhibited MIC values ranging from 500 to 2000 μg/mL. FIC index showed a synergistic effect of monolaurin with β-lactam antibiotics ranging from 0.0039 to 0.25 (p < 0.001). Among the 103 investigated MRSA isolates, 44 (44.7%) displayed moderate biofilm formation, while 59 (55.3%) were strong biofilm producers. Antibiofilm activity demonstrated concentration dependence, confirming monolaurin's capacity to inhibit biofilm formation and exhibited strong eradicating effects against preformed MRSA biofilms with IC50 values of 203.6 μg/mL and 379.3 μg/mL, respectively. Scanning electron microscope analysis revealed reduced cell attachments and diminished biofilm formation compared to the control. The expression levels of the icaD gene were remarkably reduced at monolaurin concentrations of 250 and 500 μg/mL. Monolaurin had significant inhibitory effects on MRSA pre-existing biofilms as well as biofilm development. So, it can be employed in the treatment of severe infections, particularly those associated with biofilm formation including catheter-associated infections.