Lactobacillus rhamnosus GG microcapsules inhibit Escherichia coli biofilm formation in coculture.

OBJECTIVES: Microbial biofilms have become one of the most significant causes of nosocomial infections. The aim of this study was to examine the potential quorum sensing inhibitor activities of Lactobacillus rhamnosus GG microcapsules. RESULTS: Lactobacillus rhamnosus GG microcapsules effectively inhibited initial biofilm formation at a concentration of 2.5 × 108 CFU/mL. Furthermore, the inhibition rate was increased to 79% in the Lactobacillus rhamnosus GG microcapsules group, resulting in a reduction in the biofilm maturation stage. In addition, real-time PCR analysis revealed that the LGG microcapsules can act as effective inhibitors of transcriptional activators of the quorum sensing circuit in E.coli, luxS, lsrK, and lsrR. CONCLUSIONS: Lactobacillus rhamnosus GG microcapsules can effectively inhibit biofilm formation and disturb mature biofilms.

Silybin Modulates Collagen Turnover in an In Vitro Model of NASH.

Silybin has been proposed as a treatment for nonalcoholic steatohepatitis (NASH). In this study, we assessed the effect of Silybin in a well-established in vitro coculture model of early-stage NASH. LX2 and Huh7 cells were exposed to free fatty acid (FFA) and Silybin as mono- or coculture (SCC). Cell viability, LX2 activation, collagen deposition, metalloproteinase 2 and 9 (MMP2-9) activity, and ROS generation were determined at 24, 96, and 144 h. Exposure to FFA induced the activation of LX2 as shown by the increase in cell viability and upregulation of collagen biosynthesis. Interestingly, while cotreatment with Silybin did not affect collagen production in LX2, a significant reduction was observed in SCC. MMP2-9 activity was reduced in FFA-treated Huh7 and SCC and cotreatment with Silybin induced a dose-dependent increase, while no effect was observed in LX2. Silybin also showed antioxidant properties by reducing the FFA-induced production of ROS in all the cell systems. Based on these data, Silybin exerts its beneficial effects by reducing LX2 proliferation and ROS generation. Moreover, MMP2-9 modulation in hepatocytes represents the driving mechanism for the net reduction of collagen in this NASH in vitro model, highlighting the importance of hepatic cells interplay in NASH development and resolution.

Brentuximab vedotin exerts profound antiproliferative and pro-apoptotic efficacy in CD30-positive as well as cocultured CD30-negative germ cell tumour cell lines.

Prognosis in patients suffering from high-risk, refractory and relapsed germ cell tumours (GCT) often comprising of CD30-positive embryonal carcinoma (EC) components remains poor. Thus, novel treatment strategies are warranted. The antibody-drug conjugate (ADC) brentuximab vedotin delivers the potent antimitotic drug monomethyl auristatin E (MMAE) to CD30-expressing tumour cells. After CD30 binding, internalization and intracellular linker cleavage cytotoxic MMAE can efflux and eradicate neighbouring CD30-negative cells. To analyse cytotoxicity and a potential bystander effect of brentuximab vedotin in GCT, we established an in vitro coculture model mimicking GCT of heterogeneous CD30 positivity and measured cell viability, proliferation and apoptosis after exposure to brentuximab vedotin and unbound MMAE by MTS- and flow cytometry-based CFSE/Hoechst assay. CD30 expression being assessed by quantitative RT-PCR and immunohistochemistry was apparent in all EC cell lines with different intensity. Brentuximab vedotin abrogates cell viability of CD30-positive GCT27 EC line exerting marked time-dependent antiproliferative and pro-apoptotic activity. CD30-negative JAR cultured alone barely responds to brentuximab vedotin, while in coculture with GCT27 brentuximab vedotin induces clear dose-dependent cytotoxicity. Cellular proliferation and cell death are significantly enhanced in CD30-negative JAR cocultured with CD30-positive GCT27 compared to JAR cultured alone in proof of substantial bystander activity of brentuximab vedotin in CD30-negative GCT. We present first evidence that in an in vitro model mimicking GCT of heterogeneous histology, brentuximab vedotin exerts potent antiproliferative and pro-apoptotic activity against both CD30-positive as well as CD30-negative GCT subsets. Our results strongly support translational efforts to evaluate clinical efficacy of brentuximab vedotin in high-risk GCT of heterogeneous CD30 positivity.

In vitro short-term exposure to air pollution PM2.5-0.3 induced cell cycle alterations and genetic instability in a human lung cell coculture model.

Although its adverse health effects of air pollution particulate matter (PM2.5) are well-documented and often related to oxidative stress and pro-inflammatory response, recent evidence support the role of the remodeling of the airway epithelium involving the regulation of cell death processes. Hence, the overarching goals of the present study were to use an in vitro coculture model, based on human AM and L132 cells to study the possible alteration of TP53-RB gene signaling pathways (i.e. cell cycle phases, gene expression of TP53, BCL2, BAX, P21, CCND1, and RB, and protein concentrations of their active forms), and genetic instability (i.e. LOH and/or MSI) in the PM2.5-0.3-exposed coculture model. PM2.5-0.3 exposure of human AM from the coculture model induced marked cell cycle alterations after 24h, as shown by increased numbers of L132 cells in subG1 and S+G2 cell cycle phases, indicating apoptosis and proliferation. Accordingly, activation of the TP53-RB gene signaling pathways after the coculture model exposure to PM2.5-0.3 was reported in the L132 cells. Exposure of human AM from the coculture model to PM2.5-0.3 resulted in MS alterations in 3p chromosome multiple critical regions in L132 cell population. Hence, in vitro short-term exposure of the coculture model to PM2.5-0.3 induced cell cycle alterations relying on the sequential occurrence of molecular abnormalities from TP53-RB gene signaling pathway activation and genetic instability.

Biodegradable Film for the Targeted Delivery of siRNA-Loaded Nanoparticles to Vaginal Immune Cells.

The goal of this study was to develop and characterize a novel intravaginal film platform for targeted delivery of small interfering RNA (siRNA)-loaded nanoparticles (NP) to dendritic cells as a potential gene therapy for the prevention of sexually transmitted human immunodeficiency virus (HIV) infection. Poly(ethylene glycol) (PEG)-functionalized poly(D, L-lactic-co-glycolic acid) (PLGA)/polyethylenimine (PEI)/siRNA NP (siRNA-NP) were fabricated using a modified emulsion-solvent evaporation method and characterized for particle size, zeta potential, encapsulation efficiency (EE), and siRNA release. siRNA-NP were decorated with anti-HLA-DR antibody (siRNA-NP-Ab) for targeting delivery to HLA-DR+ dendritic cells (DCs) and homogeneously dispersed in a biodegradable film consisting of poly vinyl alcohol (PVA) and λ-carrageenan. The siRNA-NP-Ab-loaded film (siRNA-NP-Ab-film) was transparent, displayed suitable physicomechanical properties, and was noncytotoxic. Targeting activity was evaluated in a mucosal coculture model consisting of a vaginal epithelial monolayer (VK2/E6E7 cells) and differentiated KG-1 cells (HLA-DR+ DCs). siRNA-NP-Ab were rapidly released from the film and were able to penetrate the epithelial layer to be taken up by differentiated KG-1 cells. siRNA-NP-Ab demonstrated higher targeting activity and significantly higher knockdown of synaptosome-associated 23-kDa protein (SNAP-23) mRNA and protein when compared to siRNA-NP without antibody conjugation. Overall, these data suggest that our novel siRNA-NP-Ab-film may be a promising platform for preventing HIV infection within the female genital tract.

Analysis of the response to cigarette smoke exposure in cell coculture and monoculture based on bionic-lung microfluidic chips.

BACKGROUND: In vitro toxicity assessment studies with various experimental models and exposure modalities frequently generate diverse outcomes. In the prevalent experimental, aerosol pollutants are dissolved in culture medium through capture for exposure to two-dimensional planar cellular models in multiwell plates via immersion. However, this approach can generate restricted and inconclusive experimental data, significantly constraining the applicability of risk assessment outcomes. Herein, the in vitro cocultivation of lung epithelial and/or vascular endothelial cells was performed using self-designed bionic-lung microfluidic chip housing a gas-concentration gradient generator (GCGG) unit. Exposure experiments involving a concentration gradient of cigarette smoke (CS) aerosol were then conducted through an original assembled real-time aerosol exposure system. RESULTS: Transcriptomic analysis revealed a potential involvement of the cGMP-signaling pathway following online CS aerosol exposure on different cell culture models. Furthermore, distinct responses to different concentrations of CS aerosol exposure on different culture models were highlighted by detecting inflammation- and oxidative stress-related biomarkers (i.e., cell viability, reactive oxygen species, nitric oxide, IL-6, IL-8, TNF-α, GM-CSF, malondialdehyde, and superoxide dismutase). SIGNIFICANT: The results underscore the importance of improving chip biomimicry while addressing multi-throughput demands, given the substantial influence of the coculture model on cellular responses triggered by CS. Furthermore, the coculture model exhibited a mutually beneficial protective effect on cells at low CS concentrations within the GCGG unit, yet revealed a mutually amplified damaging effect at higher CS concentrations in contrast to the monoculture model.

Fabrication of a Coculture Organoid Model in the Biomimetic Matrix of Alginate to Investigate Breast Cancer Progression in a TAMs-Leading Immune Microenvironment.

The current research models of breast cancer are usually limited in their capacity to recapitulate the tumor microenvironment in vitro. The lack of an extracellular matrix (ECM) oversimplifies cell-cell or cell-ECM cross-talks. Moreover, the lack of tumor-associated macrophages (TAMs), that can comprise up to 50% of some solid neoplasms, poses a major problem for recognizing various hallmarks of cancer. To address these concerns, a type of direct breast cancer cells (BCCs)-TAMs coculture organoid model was well developed by a sequential culture method in this study. Alginate cryogels were fabricated with appropriate physical and mechanical properties to serve as an alternative ECM. Then, our previous experience was leveraged to polarize TAMs inside of the cryogels for creating an in vitro immune microenvironment. The direct coculture significantly enhanced BCCs organoid growth and cancer aggressive phenotypes, including the stemness, migration, ECM remodeling, and cytokine secretion. Furthermore, transcriptomic analysis and protein-protein interaction networks implied certain pathways (PI3K-Akt pathway, MAPK signaling pathway, etc.) and targets (TNF, PPARG, TLR2, etc.) during breast cancer progression in a TAM-leading immune microenvironment. Future studies to advance treatment strategies for BCC patients may benefit from using this facile model to reveal and target the interactions between cancer signaling and the immune microenvironment.

Generation of fluorescently labeled cell lines, C3A hepatoma cells, and human adult skin fibroblasts to study coculture models.

Hepatic/nonhepatic cell cocultures are widely used in studies on the role of homo- and heterotypic interactions in liver physiology and pathophysiology. In this article, for the first time, establishment of the coculture model employing hepatoma C3A cells and human skin fibroblasts, stably expressing fluorescent markers, is described. Suitability of the model in studying coculture conditions using fluorescence microscopy and flow cytometry was examined. C3A cells spontaneously formed island-like growth patterns surrounded by fibroblasts. The "islands" size and resulting intensity of the homo- and heterotypic interactions can easily be tuned by applying various plated cells ratios. We examined the capability of the hepatoma cells to produce albumin in hepatic/nonhepatic cell cocultures. The enzyme-linked immunosorbent assay (ELISA) tests showed that greater number of fibroblasts in coculture, resulting in smaller sizes of hepatoma "islands," and thus, a larger heterotypic interface, promoted higher albumin synthesis. The use of fluorescently labeled cells in flow cytometry measurements enabled us to separately gate two cell populations and to evaluate protein expression only in/on cells of interest. Flow cytometry confirmed ELISA results indicating the highest albumin production in hepatoma cells cocultured with the greatest number of fibroblasts and the inhibited protein synthesis in coculture with osteosarcoma cells.

Anti-Inflammatory and Transepithelial Transport Activities of Rapeseed (Brassica napus) Napin-Derived Dipeptide Thr-Leu in Caco-2 and RAW264.7 Cocultures.

This study aimed to investigate the anti-inflammatory molecular activity of rapeseed napin-derived dipeptide Thr-Leu (TL) using Caco-2/RAW264.7 cell cocultures. This in vitro coculture intestinal inflammation model was used to assess the absorption, evolution, and anti-inflammatory effects of peptides. TL was absorbed by the intestinal epithelial cells with an apparent permeability of (2.48 ± 0.18) × 10-6 cm/s, primarily through the PepT1 pathway. TL treatment exerted anti-inflammatory and restorative effects on the impaired intestinal barrier function by enhancing the expression levels of occludin and ZO-1 in lipopolysaccharide (LPS)-induced Caco-2 cells. No significant change (P < 0.05) was detected in claudin-1 expression levels; however, the occludin expression levels were upregulated through the protein kinase C (PKC) signaling pathway. Compared with the LPS-induced group, TL (2.0 mM) reduced the levels of intracellular inflammation-related enzymes (iNOS: by 50.84%; COX-2: by 49.64%) on the coculture cell model. In addition, the interleukin (IL)-1ß, IL-6, and TNF-α levels in RAW264.7 cells were significantly (P < 0.05) downregulated following TL treatment (2.0 mM) due to the suppression of the phosphorylation of the JNK-independent pathway on the basolateral side of the coculture cell model. These findings highlight the potential use of TL in functional foods or nutraceuticals to prevent intestinal inflammation.

Generation of Multispecies Oral Bacteria Biofilm Models.

It is well-recognized that oral biofilms that occur in health and disease have a polymicrobial composition, though these are poorly reflected in the literature, with many studies focussing on simple mono-species biofilm model systems. The utility of polymicrobial biofilm model systems is that they more accurately reflect the oral cavity and allow researchers to ask relevant questions in basic science studies, pharmaceutical screening, and investigating inflammatory interactions. Here we describe the detailed methodology of how to sequentially construct and maintain polymicrobial biofilm models pertinent to caries, periodontal disease, and denture stomatitis.

IL18 Receptor Signaling Inhibits Intratumoral CD8+ T-Cell Migration in a Murine Pancreatic Cancer Model.

In pancreatic ductal adenocarcinoma (PDAC), the infiltration of CD8+ cytotoxic T cells (CTLs) is an important factor in determining prognosis. The migration pattern and interaction behavior of intratumoral CTLs are pivotal to tumor rejection. NLRP3-dependent proinflammatory cytokines IL-1ß and IL-18 play a prominent role for CTL induction and differentiation. Here, we investigate the effects of T-cellular IL-1R and IL-18R signaling for intratumoral T-cell motility. Murine adenocarcinoma cell line Panc02 was stably transfected with ovalbumin (OVA) and fluorophore H2B-Cerulean to generate PancOVA H2B-Cerulean tumor cells. Dorsal skinfold chambers (DSFC) were installed on wild-type mice, and PancOVA H2B-Cerulean tumor cells were implanted into the chambers. PancOVA spheroids were formed using the Corning® Matrigel®-based 3D cell culture technique. CTLs were generated from OT-1 mice, Il1r-/- OT-1 mice, or Il18r-/- OT-1 mice and were marked with fluorophores. This was followed by the adoptive transfer of CTLs into tumor-bearing mice or the application into tumor spheroids. After visualization with multiphoton microscopy (MPM), Imaris software was used to perform T-cell tracking. Imaris analysis indicates a significantly higher accumulation of Il18r-/- CTLs in PancOVA tumors and a significant reduction in tumor volume compared to wild-type CTLs. Il18r-/- CTLs covered a longer distance (track displacement length) in comparison to wild-type (WT) CTLs, and had a higher average speed (mean track speed). The analysis of instantaneous velocity suggests a higher percentage of arrested tracks (arrests: <4 µm/min) for Il18r-/- CTLs. Our data indicate the contribution of IL-18R signaling to T-cell effector strength, warranting further investigation on phenomena such as intratumoral T-cell exhaustion.

Human Endometrial Organoids: Recent Research Progress and Potential Applications.

Since traditional two-dimensional (2D) cell culture cannot meet the demand of simulating physiological conditions in vivo, three-dimensional (3D) culture systems have been developed. To date, most of these systems have been applied for the culture of gastrointestinal and neural tissue. As for the female reproductive system, the culture of endometrial and oviductal tissues in Matrigel has also been performed, but there are still some problems that remain unsolved. This review highlights recent progress regarding endometrial organoids, focusing on the signal for organoid derivation and maintenance, the coculture of the epithelium and stroma, the drug screening using organoids from cancer patients, and provides a potential guideline for genome editing in endometrial organoids.

Polyphenol Content of Green Pea (Pisum sativum L.) Hull under In Vitro Digestion and Effects of Digestive Products on Anti-Inflammatory Activity and Intestinal Barrier in the Caco-2/Raw264.7 Coculture Model.

Green pea hulls are a byproduct of the processing of green pea and are rich in phenolic substances. In the present study, in vitro digestion, human colonic adenocarcinoma cell line (Caco-2) monolayer, and the Caco-2/macrophage cell lines of the murine origin (Raw264.7) coculture model were established to investigate the release of polyphenols, absorption, and transport of digestive products and their effects on inflammation and intestinal barrier. During the digestive process, polyphenols were constantly released from the pea hulls, reaching the maximum amount in the small intestine (total phenolic content (TPC): 5.41 ± 0.04 mg gallic acid (GAE)/g dry weight (DW)), and the digestive products (800 µg/mL) could reduce the secretion of NO (50.9%), IL-6 (50.6%), and TNF-α (24.6%) and inhibit the mRNA expression of cyclooxygenase-2 (COX-2) (37.2%) and inducible nitric oxide synthase (iNOS) (91.1%) compared with the lipopolysaccharide (LPS) group. A total of 12 phenolic components were quantified by ultraperformance liquid chromatography-linear ion trap orbitrap tandem mass spectrometry (UHPLC-LTQ-OrbiTrap-MS) technology. Kaempferol trihexoside in digestive products could be absorbed and transported (1.25 ± 0.13 ng quercetin/mL). The digestive products could promote the expression of claudin-1 (210.8%), occludin (64.9%), and zonulin occludin-1 (ZO-1) (52.0%) compared with the LPS group and exert anti-inflammatory effects after being absorbed. The results indicated that pea hull polyphenols could be continuously released and absorbed to play a positive role in protecting the intestinal barrier and anti-inflammatory activity.

Endothelial-Smooth Muscle Cell Interactions in a Shear-Exposed Intimal Hyperplasia on-a-Dish Model to Evaluate Therapeutic Strategies.

Intimal hyperplasia (IH) represents a major challenge following cardiovascular interventions. While mechanisms are poorly understood, the inefficient preventive methods incentivize the search for novel therapies. A vessel-on-a-dish platform is presented, consisting of direct-contact cocultures with human primary endothelial cells (ECs) and smooth muscle cells (SMCs) exposed to both laminar pulsatile and disturbed flow on an orbital shaker. With contractile SMCs sitting below a confluent EC layer, a model that successfully replicates the architecture of a quiescent vessel wall is created. In the novel IH model, ECs are seeded on synthetic SMCs at low density, mimicking reendothelization after vascular injury. Over 3 days of coculture, ECs transition from a network conformation to confluent 2D islands, as promoted by pulsatile flow, resulting in a "defected" EC monolayer. In defected regions, SMCs incorporated plasma fibronectin into fibers, increased proliferation, and formed multilayers, similarly to IH in vivo. These phenomena are inhibited under confluent EC layers, supporting therapeutic approaches that focus on endothelial regeneration rather than inhibiting proliferation, as illustrated in a proof-of-concept experiment with Paclitaxel. Thus, this in vitro system offers a new tool to study EC-SMC communication in IH pathophysiology, while providing an easy-to-use translational disease model platform for low-cost and high-content therapeutic development.

Investigation of metabolic crosstalk between host and pathogenic Clostridioides difficile via multiomics approaches.

Clostridioides difficile is a gram-positive anaerobic bacterium that causes antibiotic-associated infections in the gut. C. difficile infection develops in the intestine of a host with an imbalance of the intestinal microbiota and, in severe cases, can lead to toxic megacolon, intestinal perforation, and even death. Despite its severity and importance, however, the lack of a model to understand host-pathogen interactions and the lack of research results on host cell effects and response mechanisms under C. difficile infection remain limited. Here, we developed an in vitro anaerobic-aerobic C. difficile infection model that enables direct interaction between human gut epithelial cells and C. difficile through the Mimetic Intestinal Host-Microbe Interaction Coculture System. Additionally, an integrative multiomics approach was applied to investigate the biological changes and response mechanisms of host cells caused by C. difficile in the early stage of infection. The C. difficile infection model was validated through the induction of disaggregation of the actin filaments and disruption of the intestinal epithelial barrier as the toxin-mediated phenotypes following infection progression. In addition, an upregulation of stress-induced chaperones and an increase in the ubiquitin proteasomal pathway were identified in response to protein stress that occurred in the early stage of infection, and downregulation of proteins contained in the electron transfer chain and ATP synthase was observed. It has been demonstrated that host cell energy metabolism is inhibited through the glycolysis of Caco-2 cells and the reduction of metabolites belonging to the TCA cycle. Taken together, our C. difficile infection model suggests a new biological response pathway in the host cell induced by C. difficile during the early stage of infection at the molecular level under anaerobic-aerobic conditions. Therefore, this study has the potential to be applied to the development of future therapeutics through basic metabolic studies of C. difficile infection.

On the origin of germ cell neoplasia in situ: Dedifferentiation of human adult Sertoli cells in cross talk with seminoma cells in vitro.

Germ cell neoplasia in situ (GCNIS) is the noninvasive precursor of testicular germ cell tumors type II, the most common cancer in young men, which originates from embryonic germ cells blocked in their maturation. GCNIS is associated with impaired Sertoli cells (SCs) that express fetal keratin 18 (KRT18) and the pluripotency factor SRY-Box transcription factor 2 (SOX2). According to the current theory concerning the origin of GCNIS, these SCs are prepubertal cells arrested in their maturation due to (epi)genetic anomalies and/or environmental antiandrogens. Thus, they are unable to support the development of germ cells, which leads to their maturational block and further progresses into GCNIS. Alternatively, these SCs are hypothesized to be adult cells dedifferentiating secondarily under the influence of GCNIS. To examine whether tumor cells can dedifferentiate SCs, we established a coculture model of adult human SCs (FS1) and a seminoma cell line similar to GCNIS (TCam-2). After 2 wk of coculture, FS1 cells showed progressive expression of KRT18 and SOX2, mimicking the in vivo changes. TCam-2 cells showed SOX2 expression and upregulation of further pluripotency- and reprogramming-associated genes, suggesting a seminoma to embryonal carcinoma transition. Thus, our FS1/TCam-2 coculture model is a valuable tool for investigating interactions between SCs and seminoma cells. Our immunohistochemical and ultrastructural studies of human testicular biopsies with varying degrees of GCNIS compared to biopsies from fetuses, patients with androgen insensitivity syndrome, and patients showing normal spermatogenesis further suggest that GCNIS-associated SCs represent adult cells undergoing progressive dedifferentiation.

Inflammatory stress induced by a combination of cytokines (IL-6, IL-17, TNF-α) leads to a loss of integrity on bEnd.3 endothelial cells in vitro BBB model.

The brain is a complex organ protected by the blood-brain barrier (BBB), which also has a complex organization. To play its protective role, the BBB acts by limiting the paracellular passage of potentially cytotoxic compounds through tight junctions as well as limiting transcellular passage by efflux pumps (ABC transporters). In many conditions such as sleep apnea or Alzheimer's disease, there is chronic inflammation, resulting in the presence of pro-inflammatory cytokines in the bloodstream. The effect of this chronic inflammation on the integrity of the BBB has been studied mainly through a single inflammatory molecule; but in physiological and pathological conditions, it is a combination of inflammatory cytokines. We investigated the effect of three major pro-inflammatory cytokines (IL-17, IL-6, TNF-α) used alone or in combination on the integrity of an in vitro model of BBB. Our study showed 24 h of inflammatory stress led to a BBB's opening, reflected by a significant increase of permeability, which was correlated to a significant decrease of tight junction protein expressions (ZO-1, claudin-5), involving a possible entry of cytotoxic compounds into the brain. To compensate the loss of integrity, one of defense mechanism of endothelial cells was efflux transport, which showed a significant increase in expression and functionality of ABC transport proteins (MRP-1, Pgp). This opening of the BBB was more important when pro-inflammatory cytokines were combined, which could be explained by the interaction between cytokines and the potentiation of their effect.

Eradicating Infecting Bacteria while Maintaining Tissue Integration on Photothermal Nanoparticle-Coated Titanium Surfaces.

Photothermal nanoparticles locally release heat when irradiated by near-infrared (NIR). Clinical applications initially involved tumor treatment, but currently extend toward bacterial infection control. Applications toward much smaller, micrometer-sized bacterial infections, however, bear the risk of collateral damage by dissipating heat into tissues surrounding an infection site. This can become a complication when photothermal nanoparticle coatings are clinically applied on biomaterial surfaces requiring tissue integration, such as titanium-made, bone-anchored dental implants. Dental implants can fail due to infection in the pocket formed between the implant screw and the surrounding soft tissue ("peri-implantitis"). We address the hitherto neglected potential complication of collateral tissue damage by evaluating photothermal, polydopamine nanoparticle (PDA-NP) coatings on titanium surfaces in different coculture models. NIR irradiation of PDA-NP-coated (200 µg/cm2) titanium surfaces with adhering Staphylococcus aureus killed staphylococci within an irradiation time window of around 3 min. Alternatively, when covered with human gingival fibroblasts, this irradiation time window maintained surface coverage by fibroblasts. Contaminating staphylococci on PDA-NP-coated titanium surfaces, as can be per-operatively introduced, reduced surface coverage by fibroblasts, and this could be prevented by NIR irradiation for 5 min or longer prior to allowing fibroblasts to adhere and grow. Negative impacts of early postoperative staphylococcal challenges to an existing fibroblast layer covering a coated surface were maximally prevented by 3 min NIR irradiation. Longer irradiation times caused collateral fibroblast damage. Late postoperative staphylococcal challenges to a protective keratinocyte layer covering a fibroblast layer required 10 min NIR irradiation for adverting a staphylococcal challenge. This is longer than foreseen from monoculture studies because of additional heat uptake by the keratinocyte layer. Summarizing, photothermal treatment of biomaterial-associated infection requires precise timing of NIR irradiation to prevent collateral damage to tissues surrounding the infection site.

Fabrication of a Perfusable 3D In Vitro Artery-Mimicking Multichannel System for Artery Disease Models.

Fabrication of a 3D in vitro model that mimics the artery takes an important role in understanding pathological cell behaviors and mechanisms of vascular diseases by proposing an advanced model that can recapitulate a native vessel condition in a controlled manner. Because a model geometry and the structure of cells are significant for the recapitulation of the hemodynamics of arterial and cell functions, it is necessary to mimic geometries and to induce the proper morphology and orientation of the cells when fabricating a model. In this study, smooth muscle cells (SMCs) and endothelial cells (ECs), which were the main elements in the arterial wall, were cocultured in a multichannel device connected with polydimethylsiloxane (PDMS) fluidic chamber modules to parallelly fabricate a pefusable 3D in vitro human artery-mimicking multichannel system. In the coculture model, a circular PDMS channel with a wrinkled-surface guided directionality and contractile morphology to SMCs, and media perfusion induced directionality to a confluent EC layer as in vivo. Protein markers of cells and synthesized extracellular matrices were demonstrated. Because multichannels were connected to a microfluidic module in a device, it was possible to easily control the microenvironmental conditions and to fabricate coculture models in parallel with a single flow system. Coculture models that can be tuned in designs such as diameter, wall shear stress, and geometry of artery disease were constructed by 3D-printed molds to recapitulate various cellular microenvironments and to model vessels effectively. Finally, the effect of wall shear stress on cells was compared using a device with four different degrees of stenosis channels and investigated in parallel.

The Short Lipopeptides (C10)2-KKKK-NH2 and (C12)2-KKKK-NH2 Protect HaCaT Keratinocytes from Bacterial Damage Caused by Staphylococcus aureus Infection in a Co-Culture Model.

The search for new antimicrobial strategies is of major importance since there is a growing resistance of both bacteria and fungi to existing antimicrobials. Lipopeptides are promising and potent antimicrobial compounds. For translation into clinically useful molecules, effectiveness of peptide treatment against human infections must be proved in complex in vitro wound models. The aim of this study was to examine if the synthesized short lipopeptides (C10)2-KKKK-NH2 and (C12)2-KKKK-NH2 can protect HaCaT keratinocytes from bacterial damage caused by Staphylococcus aureus infection in a coculture model. After 1 h, 24 h, and 48 h incubation, cellular ATP level and release of the cytotoxicity marker LDH as well as the proinflammatory cytokines interleukin-6 and interleukin-1α were measured. Infection of the keratinocytes resulted in strong bacterial damage of HaCaT cells along with low cellular ATP levels and high release of LDH, IL-6, and IL-1α after 24 h and 48 h. Incubation of the infected human keratinocytes with (C10)2-KKKK-NH2 and (C12)2-KKKK-NH2 resulted in protection of the keratinocytes from bacterial damage caused by Staphylococcus aureus infection with ATP, LDH, IL-6, and IL-1α levels comparable to the untreated control. Hence, both synthesized lipopeptides are promising candidates with high therapeutic potential in dermatology for the treatment of topical infections.