Greater Cell Viability Research Articles

Hydrothermal extraction and thorough characterization of carrageenans and proteins from Gigartina pistillata

Carrageenan, an interesting biopolymer from red seaweed, possesses a myriad of applications in food, nutraceutical, or pharmaceutical industries. Although its conventional extraction used to be performed with alkaline solvents, water extraction under subcritical conditions is an alternative eco-friendly technique that has gained popularity in recent years. This work evaluates the recovery and characterization of carrageenan and proteins from Gigartina pistillata. The highest number of proteins was identified at the lowest processing tempeartures. Extracted carrageenan exhibits molecular weights greater than 500 kDa and good rheological behaviour, with interest for food applications and for the formulation of hydrogels when mixed with KCl. The carrageenans and carrageenan hydrogels presented interesting properties such as good thermal stability until 170 °C (TGA-DTG) and show characteristic bands of kappa/iota carrageenans in FTIR studies. Greater cell viability than 70% were achieved on NIH/3T3 fibroblast at carrageenan concentrations of 0.05 and 0.025%, whereas carrageenan extracted at 160 °C (concentration of 0.025%) displays a lower inflammatory action than other samples. In this sense, in this work the eco-friendlier extracted carrageenan was thoroughly characterized and its potentiality to be used in the biomedical field was evaluated.

Premixed calcium silicate-based root canal sealers have better biological properties than AH Plus: A systematic review and meta-analysis of in vivo animal studies and in vitro laboratory studies.

The aim was to determine whether premixed calcium silicate-based root canal sealers have better biological properties than AH Plus. Searches of studies published up to January 2023 were performed in the PubMed/MEDLINE and EMBASE and via other methods (databases of the International Endodontic Journal, Journal of Endodontics, and gray literature). The inclusion criteria were in vivo animal and in vitro studies that analyzed the response in the dorsal subcutaneous tissue of rats, cell viability, and genotoxicity. Systematic Review Centre for Laboratory Animal Experimentation Risk of Bias (RoB) tool for in vivo studies and modified CONSORT checklist for in vitro were appraised. Meta-analysis was performed using the Stata. Fifty-two studies were included. In the RoB, in vivo studies fulfilled 20%-50% of the items and in vitro 60%-100%. The studies included in the meta-analysis demonstrated better histocompatibility with the premixed calcium silicate-based sealers at 30 days and greater cell viability with these sealers when used in undiluted extracts in experimental period of 72 h and in extracts with 1:2 and 1:4 dilution in 24 and 72 h. In contrast, no difference between materials was found concerning genotoxicity. Premixed calcium silicate-based root canal sealers have better histocompatibility and are less cytotoxic than the epoxy resin-based sealer AH Plus, demonstrating favorable biological behavior.

Biofilm formation on metal alloys and coatings, zirconia, and hydroxyapatite as implant materials in vivo.

Composition of implant material and its surface structure is decisive for oral biofilm accumulation. This study investigated biofilm formation on eight different materials. Eighteen healthy subjects wore intraoral splints fitted with two sets of eight materials for 24 h: zirconia [ZrO2 ]; silver-gold-palladium [AgAuPd]; titanium zirconium [TiZr]; Pagalinor [PA]; hydroxyapatite [HA]; silver-platinum [AgPt]; titanium aluminum niobium [TAN]; titanium grade4 [TiGr4]. Total biomass was stained by safranin to assess plaque accumulation while conventional culturing (CFU) was conducted to investigate viable parts of the biofilm. Cell viability of human gingival fibroblasts (HGF-1) was assessed in vitro. Statistical evaluation was performed with linear mixed-effects models to compare materials (geometric mean ratios, 95% CI), with the level of significance set at ɑ = .05. Less biofilm mass and CFU were found on noble metal alloys (AgPt, AgAuPd, and PA). Compared to AgPt, PA had 2.7-times higher biofilm mass value, AgAuPd was 3.9-times, TiGr4 was 4.1-times, TiZr was 5.9-times, TAN was 7.7-times, HA was 7.8-times, and ZrO2 was 9.1-times higher (each p < .001). Similarly, CFU data were significantly lower on AgPt, AgAuPd had 4.1-times higher CFU values, PA was 8.9-times, TiGr4 was 11.2-times, HA was 12.5-times, TiZr was 13.3-times, TAN was 16.9-times, and ZrO2 was 18.5-times higher (each p < .001). HGF-1 viability varied between 47 ± 24.5% (HA) and 94.4 ± 24.6% (PA). Noble alloys are considered as beneficial materials for the transmucosal part of oral implants, as less biofilm mass, lower bacterial counts, and greater cell viability were detected than on titanium- or zirconia-based materials.

The Combined Effects of Hydraulic Calcium Silicate Cement and Enamel Matrix Derivative Regarding Osteogenic and Dentinogenic Differentiation on Human Dental Pulp Stem Cells.

The ideal treatment option for immature necrotic permanent teeth is regeneration of the pulp-dentin complex. Mineral trioxide aggregate (MTA), the conventional cement used for regenerative endodontic procedures, induces hard tissue repair. Various hydraulic calcium silicate cements (HCSCs) and enamel matrix derivative (EMD) also promote osteoblast proliferation. The purpose of the present study was to determine the osteogenic and dentinogenic potential of commercially distributed MTA and HCSCs when applied in combination with Emdogain gel on human dental pulp stem cells (hDPSCs). The presence of Emdogain resulted in greater cell viability, and higher alkaline phosphatase activity was detected in the Emdogain-supplemented groups in the early days of cell culture. On qRT-PCR, the groups treated, respectively, with Biodentine and Endocem MTA Premixed in the presence of Emdogain showed an increased expression of the dentin formation marker DSPP, and the group treated with Endocem MTA Premixed in the presence of Emdogain showed an upregulated expression of the bone formation markers OSX and RUNX2. In an Alizarin Red-S staining assay, all of the experimental groups exhibited a greater formation of calcium nodules when treated in combination with Emdogain. Overall, the cytotoxicity and osteogenic/odontogenic potential of HCSCs were similar to that of ProRoot MTA. The addition of the EMD increased the osteogenic and dentinogenic differentiation markers.

Injectable Microporous Annealed Particle Hydrogel Based on Guest-Host-Interlinked Polyethylene Glycol Maleimide Microgels.

Microporous annealed particle (MAP) hydrogels have emerged as a versatile biomaterial platform for regenerative medicine. MAP hydrogels have been used for the delivery of cells and organoids but often require annealing post injection by an external source. We engineered an injectable, self-annealing MAP hydrogel with reversible interparticle linkages based on guest-host functionalized polyethylene glycol maleimide (PEG-MAL) microgels. We evaluated the effect of guest-host linkages on different types of microgels fabricated by either batch emulsion or mechanical fragmentation methods. Batch emulsion generated small spherical microgels with controllable 10-100 μm diameters and mechanical fragmentation generated irregular microgels with larger diameters (100-200 μm). Spherical microgels (15 μm) showed self-healing behavior and completely recovered from high strain while fragmented microgels (133 μm) did not recover. Guest-host interactions significantly contributed to the mechanical properties of spherical microgels but had no effect on fragmented microgels. Spherical microgels were superior to the fragmented microgels for co-injection of immune cells and pancreatic islets due to their lower force of injection, demonstrating more homogeneously distributed cells and greater cell viability after injection. Based on these studies, the spherical guest-host MAP hydrogels provide a controllable, injectable scaffold for engineered microenvironments and cell delivery applications.

P-170 Comparison of the effect of human polarized and non-polarized Granulosa cells co-culture on mouse embryo development

Abstract Study question Do human polarized Granulosa cells (GCs), improve the development of mouse embryos after co-culture, compared to human non-polarized GCs and standard culture? Summary answer Yes, human polarized GCs improve blastulation and hatching rate and also embryos quality after co-culture, compared to human non-polarized GCs and standard embryo culture. What is known already Co-culture embryo with the cells of the female reproductive tract is one of the methods that, in addition to meeting the metabolic needs of the embryo, compensate for the lack of interactions between the embryo and the female reproductive tract during in vitro culture. Various studies have shown that co-culturing of embryos with GCs improves embryo quality and clinical outcomes. However, GCs morphology is essential for proper function changes during in vitro culture to fibroblast-like appearance. Matrigel can effectively maintain epithelial cells' physiology, morphology, and polarity like GCs in vitro and provide more efficient feeder cells. Study design, size, duration GCs were cultured on Matrigel to maintain epithelial nature. The effect of Matrigel on GCs morphology, ultrastructure, and the transcription level of specific and apoptotic genes was studied following one and six days of culture and compared to culturing without Matrigel. Then Polarized and non-polarized GCs were applied for co-culturing with embryos. Blastulation and hatching rate, cell count in the blastocyst stage, and expansion were compared between two co-culture groups and standard embryo culture. Participants/materials, setting, methods GCs were enzymatically isolated from oocyte surroundings of fertile women who had undergone IVF treatment due to male factors and cultured with or without Matrigel overnight. Pronucleus embryos were extracted from adult female NMRI mice and cultured randomly for up to five days in three groups: standard culture, co-culture with non-polarized GCs, and co-culture with polarized GCs. Main results and the role of chance Histological results showed that Matrigel maintains the round shape of GCs, but in the group without Matrigel, GCs were slender and spindle-shaped. Ultrastructure sections showed in culture on Matrigel, Multiple cell junctions connected GCs, lipid droplets were homogeneously spread on the cytoplasm, and Cylinder- shape mitochondria were also detected. GCs culture without Matrigel showed thick cell extensions and gaps between cells and also clustered lipid droplets on one side of the cytoplasm. Gene expression studies indicated culturing GCs on Matrigel is associated with maintaining function and greater cell viability, which had a positive effect on co-culture with embryos; In the co-culture group with polarized GCs, the blastulation rate, as well as hatching or hatched blastocyst, was significantly higher compared to the control group (P-value&amp;lt;0.05). Also, the number of blastomeres increased dramatically in both co-culture groups compared to the standard culture (P-value&amp;lt;0.05). The approximate ratio of the area of inner cell mass to the total area of blastocytes was also significantly lower in the co-culture group with polarized GCs compared to the control group and culture with non-polarized GCs (P-value&amp;lt;0.05), indicating more blastocyst expansion in the co-culture group with polarized GCs. Limitations, reasons for caution Because of ethical issues, mice embryos were used in this study instead of human embryos. Wider implications of the findings In the presence of polarized granulosa cells, the number of embryonic cells, especially trophectoderm cells, blastocyst expansion, blastulation, and hatching rate increase. It seems that granulosa cell culture on Matrigel can effectively simulate the in vivo environment for the embryo and lead to more successful assisted reproductive techniques. Trial registration number 98000112

Crystalline phase of TiO2 nanotube arrays on Ti–35Nb–4Zr alloy: Surface roughness, electrochemical behavior and cellular response

An investigation was made into the electrochemical, structural and biological properties of self-organized amorphous and anatase/rutile titanium dioxide (TiO 2 ) nanotubes deposited on Ti–35Nb–4Zr alloy through anodization-induced surface modification. The surface of as-anodized and heat-treated TiO 2 nanotubes was analyzed by field emission scanning electron microscopy (FE-SEM), revealing morphological parameters such as tube diameter, wall thickness and cross-sectional length. Glancing angle X-ray diffraction (GAXRD) was employed to identify the structural phases of titanium dioxide, while atomic force microscopy (AFM) was used to measure surface roughness associated with cell interaction properties. The electrochemical stability of TiO 2 was examined by electrochemical impedance spectroscopy (EIS) and the results obtained were correlated with the microstructural characterization. The in vitro bioactivity of as-anodized and crystallized TiO 2 nanotubes was also analyzed as a function of the presence of different TiO 2 polymorphic phases. The results indicated that anatase TiO 2 showed higher surface corrosion resistance and greater cell viability than amorphous TiO 2 , confirming that TiO 2 nanotube crystallization plays an important role in the material's electrochemical behavior and biocompatibility.

P1 - Calcium Silicate Loaded-Porous Chitosan Scaffold for Pulp-Dentin Complex Regeneration

Innovative pulp therapy strategies based on the use of biomaterials capable of stimulating dental pulp cells to participate effectively of the complete pulp-dentin complex regeneration is a challenge in Dentistry. Therefore, the aim of this study was to develop and characterize several formulations of calcium silicate (SiCa)-loaded porous chitosan (CH) scaffolds, and evaluate their bioactive and chemotactic potential on human dental pulp cells (hDPCs). Different concentrations of SiCa suspensions (0.5, 1.0 and 2.0%, w/v) were either incorporated (1:5; v/v) or not to 2% CH solution, establishing the following groups: CH (control); CH+0.5SiCa; CH+1.0SiCa; CH+2.0SiCa. The resulting solutions were submitted to thermally induced phase separation followed by freeze drying to obtain porous scaffolds. The topography and chemical characterization (SEM/EDS) of the scaffolds, as well as their pH and calcium release kinetics were assessed. Next, hDPCs were seeded onto the scaffolds to assess their bioactivity potential according with the parameters of cell viability (Live/Dead assay; 1, 7 and 14-days), proliferation (AlamarBlue assay; 1, 7 and 14-days), adhesion and spreading (F-actin assay; 1, 7 and 14-days). The pulp cells were also evaluated for total protein synthesis (Lowry Protein assay; 14-days), alkaline phosphatase activity (ALP Assay Kit; 14-days), mineralized matrix deposition (Alizarin Red assay; 21-days), and gene expression of odontogenic differentiation markers (ALP, DSPP, and DMP-1; RT-qPCR assay, 21-days). The chemotactic stimulus of the scaffolds on pulp cells (Transwell assay; 24 h) was also assessed. Data were analyzed by ANOVA and Tukey post-hoc (?=5%). The incorporation of SiCa in the chitosan scaffold formulations increased the pore diameter in comparison to control (p<0.05). High amounts of SiCa changed the surface nano-topography of the chitosan scaffolds (Image 1) and increased the pH and Ca release (p<0.05). Greater cell viability, proliferation, adhesion and spreading, as well as expression of odontogenic markers and mineralized matrix deposition was observed in the CH+1.0SiCa and CH+2.0SiCa groups (p<0.05). However, increased synthesis of total protein and mineralized matrix, as well as expression of DMP-1 and DSPP occurred only in group CH+2.0SiCa (p<0.05). Based on the fact that porous chitosan scaffolds enriched with 2% of calcium silicate showed improved bioactive and chemotactic potential on hDPCs, it should be considered as a potential biomaterial for cell-free tissue engineering strategy to drive pulp-dentin complex regeneration.

Co-supplementation of freezing media with trehalose and vitamin C on cell viability and apoptotic gene expression in ovine spermatogonial stem cells

The purpose of this research was to investigate the trehalose and vitamin C (Vit C) co-supplementation of freezing media to create a successful cryopreservation protocol for conservation of ovine spermatogonial stem cells (SSCs). SSCs were cryopreserved and cultured with an essential freezing medium containing 200 mM trehalose, 40 µg/mL Vit C, and a combination of both for 3 weeks. Cell viability, colony number and diameter and mRNA levels of Bax, and Bcl-2 genes were evaluated before and after cryopreservation with quantitative real-time PCR. The results showed that cells cryopreserved in freezing medium containing 200 mM of trehalose plus 40 µg/mL Vit C had considerably greater cell viability than the control group (P&lt;0.0001). Up to the 3rd week of cell culture, supplementation of freezing medium with 200 mM trehalose resulted in significantly lower colonies diameters than in the control group. No significant differences were observed during the 1st to 2nd weeks in colony diameter and number of colonies between cells cryopreserved in the freezing medium containing either Vit C or trehalose compared with the control groups. Following thawing, the mRNA level of Bax in the Vit C + trehalose group was drastically lower than in those treated with trehalose or Vit C only (P&lt;0.0001). High expression of Bcl-2 in the 40 µg/mL Vit C group was recorded in the thawed cells compared to the control group (P&lt;0.0001). These findings indicate that the concomitant use of antioxidants and sugar in the freezing medium can improve the quality and viabi­lity of SSCs after freezing via different mechanisms. Further studies are needed to clarify apoptosis and cell biomarkers in SSCs during freezing and thawing.

Styrene-Maleimide/Maleic Anhydride Alternating Copolymers: Recent Advances and Future Perspectives.

Alternating sequencing of styrene-maleimide/maleic anhydride (S-MI/MA) in the copolymer chain is known for a long time. But since early 2000, this class of copolymers has been extensively studied using various living/controlled polymerization techniques to design S-MI/MA alternating copolymers with tunable molecular weight, narrow dispersity (Ð), and precise chain-end functionality. The widespread diverse applications of this polymeric backbone are due to its ease of synthesis, cheap starting materials, high precision in alternating sequencing, and facile post-polymerization functionalization with simple organic reactions. Recently, S-MI/MA alternating copolymers have been rediscovered as novel polymers with unprecedented emissive behavior. It outperforms the traditional fluorophores with no aggregation caused quenching (ACQ), aqueous solubility, and greater cell viability. Herein, the origin of alternating sequence, synthesis, and recent (2010-Present) developments in applications of these polymers in different fields are elaborately discussed, including the advantages of the unconventional luminogenic property. This review article also highlights the future research directions of the versatile S-MI/MA copolymers.

Histological evaluation of thermal damage to Osteocytes: A comparative study of conventional and ultrasonic-assisted bone grinding

This paper addresses an important issue faced by neurosurgeons during surgical skull bone grinding, a common process used to remove bone in skull base tumour removal surgery to enable the neurosurgeon to reach the target region. The heat generated during bone grinding could harm the soft tissues and can lead to osteonecrosis and cell death. In the present study, a novel process of rotary ultrasonic bone grinding (RUBG) was proposed for osteotomy to limit the temperature to a safe level. A systematic investigation was conducted to determine the effect of varying process parameters on osteonecrosis at the cut surface. Three input parameters - rotational speed, feed rate and frequency – were investigated (at three levels) in terms of change in temperature and thermal biological damage. A sterile solution was used as a coolant to irrigate the grinding zone. Viable lacunae (filled osteocytes), non-viable lacunae (empty lacunae), necrosed tissues, and Haversian canal were found during the histological examination. Statistical analysis revealed that feed rate (45.43%) had the highest contribution towards temperature rise during grinding, followed by ultrasonic frequency (23.87%), and rotational speed (12.85%). The optimal machining parameters to avoid osteonecrosis and thermal trauma were rotational speed 35,000 rpm, feed rate 20 mm/min and ultrasonic frequency 20 kHz. Furthermore, histograms revealed that ultrasonic skull bone grinding was associated with greater cell viability and reduced temperature compared with conventional bone grinding.

Influences of acid catalysts on the microstructure, bioactivity and cytotoxicity of bioactive glass nanoparticles prepared by spray pyrolysis

Bioactive glasses (BGs) have received considerable attention in the fields of medical and material science and have been applied in various applications due to their superior properties. Although studies have demonstrated the fabrication of BG by various approaches, the effects of acid catalysts in the synthesis of spray pyrolyzed BG remain unclear. Therefore, in this study, we aim to synthesize BG nanoparticles using spray pyrolysis technique and treated them with different acid catalysts of hydrochloric acid, lactic acid and acetic acid. By characterizing the phase information and morphologies of the BG specimens, three morphologies of smooth, rough, and porous spheres were found and their corresponding formation mechanisms were discussed. Finally, evaluations of both bioactivity and cytotoxicity were carried out, and the results suggested that all BG specimens are bioactive while BG treated with hydrochloric acid was associated with greater cell viability than the lactic acid- and acetic acid-treated specimens.

Cytotoxic, Migration, and Angiogenic Effects of Photodynamic Therapy and Photobiomodulation Associated with a Revascularization Protocol

IntroductionThis study evaluated photodynamic therapy (PDT) and photobiomodulation therapy (PBM) as adjuncts to pulp revascularization using cultures of apical papilla cells (APCs) and endothelial cells (HUVECs). MethodsThe root canal and apical foramen of 2 mandibular first premolars were enlarged to simulate immature teeth. The canal of 1 tooth was filled with 1 mL 0.005% methylene blue (MB). After that, the canals of both teeth were irrigated with 20 mL 1.5% sodium hypochlorite (NaOCl) and 20 mL 17% EDTA. The resulting solutions were diluted in cell culture media at a concentration of 0.5% (0.5% MB + NaOCl + EDTA and 0.5% NaOCl + EDTA). After PDT (0.5% MB + NaOCl + EDTA + PDT) and PBM (0.5% NaOCl + EDTA + PBM) applications, the effects were evaluated to determine cytotoxicity, polarity index, APC migration, and HUVEC sprouting, and results were compared with those of their controls (solutions without laser application). Cell culture media (CT) was also used as a control. Data were analyzed using 1-way analysis of variance and the Tukey post hoc test (P ≤ .05). ResultsPDT and PBM promoted greater APC viability than their controls, and PDT had greater cell viability than CT (P < .05). All protocols reduced APC migration when compared with CT (P < .05). HUVEC sprouts grown out of spheroids in PBM had a greater ratio area than their control (P ≤ .01), and the PDT ratio of the spheroid area was similar to that of its control (P > .05). ConclusionsPBM and PDT seem to be potentially effective adjuncts to revascularization in nonvital immature teeth.

Effect of Iodine and Selenium on Proliferation, Viability, and Oxidative Stress in HTR-8/SVneo Placental Cells.

Adequate maternal micronutrition is vital for placental formation, fetal growth, and development. Oxidative stress adversely affects placental development and function and an association between deficient placental development, oxidative stress, and micronutrient deficiency has been observed. Selenium and iodine are two essential micronutrients with antioxidant properties. Epidemiological studies have shown that poor micronutrient status in pregnant women is associated with a higher incidence of pregnancy complications. The aim of this study was to determine how selenium, iodine, and their combination impact oxidative stress in placental trophoblast cells. HTR8/SVneo extravillous trophoblasts were supplemented with a concentration range of organic and inorganic selenium, potassium iodide, or their combination for 24h. Oxidative stress was then induced by treating cells with menadione or H2O2 for 24h. Cell viability and lipid peroxidation as the biomarker of oxidative stress were assessed at 48h. Both menadione and H2O2 reduced cell viability and increased lipid peroxidation (P < 0.05). Greater cell viability was found in selenium-supplemented cells when compared with vehicle treated cells (P < 0.05). Selenium and iodine supplementation separately or together were associated with lower lipid peroxidation compared with vehicle control (P < 0.05). Supplementation with the combination of selenium and iodine resulted in a greater reduction in lipid peroxidation compared with selenium or iodine alone (P < 0.05). Oxidative stress negatively impacts trophoblast cell survival and cellular integrity. Selenium and iodine protect placental trophoblasts against oxidative stress. Further research is warranted to investigate the molecular mechanisms by which selenium and iodine act in the human placenta.

Characterization and engineering control of the effects of reactive oxygen species on the conversion of sterols to steroid synthons in Mycobacterium neoaurum

The conversion of sterols to steroid synthons by engineered mycobacteria comprises one of the basic ways for the production of steroid medications in the pharmaceutical industry. Here, we revealed that high amounts of reactive oxygen species (ROS) generate during the conversion process of sterols, which impairs the cell viability of mycobacterial cells and thus hinders the conversion of sterols to steroid synthons. Accordingly, the endogenous antioxidants for detoxifying ROS in mycobacteria, ROS scavenging enzymes and low molecular weight thiols, were examined. The results revealed that three antioxidants, catalase (CAT), mycothiol (MSH), and ergothioneine (EGT), demonstrated efficacy toward neutralizing the excessive ROS produced during sterol metabolism. CAT overexpression or MSH or EGT augmentation enhanced the conversion of phytosterols to 22-hydroxy-23,24-bisnorchol-4-ene-3-one (4-HBC) by 18.9%, 23.8%, and 32.1%, respectively, and also enhanced the cell viability, indicating the benefits of these antioxidants in reducing ROS-induced stress. Further combinatorial augmentation of CAT, MSH, and EGT demonstrated enhanced effects toward intracellular ROS scavenging, resulting in 54.2% greater cell viability and 47.5% enhancement in 4-HBC production. These findings indicated that the excessive ROS induces cell stress, in turn limiting the conversion of sterols, whereas neutralization of the excessive ROS by combined control of CAT, MSH, and EGT serves as an effective strategy to boost the conversion productivity of sterols to steroid synthons.

A magnetic three-dimensional levitated primary cell culture system for the development of secretory salivary gland-like organoids.

Salivary gland (SG) hypofunction and oral dryness can be induced by radiotherapy for head and neck cancers or autoimmune disorders. These are common clinical conditions that involve loss of saliva-secreting epithelial cells. Several oral complications arise with SG hypofunction that interfere with routine daily activities such as chewing, swallowing, and speaking. Hence, there is a need for replacing these saliva-secreting cells. Recently, researchers have proposed to repair SG hypofunction via various cell-based approaches in three-dimensional (3D) scaffold-based systems. However, majority of the scaffolds used cannot be translated clinically due to the presence of non-human-based substrates. Herein, saliva-secreting organoids/mini-glands were developed using a new scaffold/substrate-free culture system named magnetic 3D levitation (M3DL), which assembles and levitates magnetized primary SG-derived cells (SGDCs), allowing them to produce their own extracellular matrices. Primary SGDCs were assembled in M3DL to generate SG-like organoids in well-established SG epithelial differentiation conditions for 7days. After such culture time, these organoids consistently presented uniform spheres with greater cell viability and pro-mitotic cells, when compared with conventional salisphere cultures. Additionally, organoids formed by M3DL expressed SG-specific markers from different cellular compartments: acinar epithelial including adherens junctions (NKCC1, cholinergic muscarinic receptor type 3, E-cadherin, and EpCAM); ductal epithelial and myoepithelial (cytokeratin 14 and α-smooth muscle actin); and neuronal (β3-tubulin and vesicular acetylcholine transferase). Lastly, intracellular calcium and α-amylase activity assays showed functional organoids with SG-specific secretory activity upon cholinergic stimulation. Thus, the functional organoid produced herein indicate that this M3DL system can be a promising tool to generate SG-like mini-glands for SG secretory repair.

Evaluation of apoptosis/necrosis and cytokine release provoked by three root canal sealers in human polymorphonuclears and monocytes.

To evaluate the invitro cytotoxicity and cytokine release of three fresh root canal sealers and to determine the type of cell death they induce. The sealers tested were Sealer 26 (S26), AH Plus (AHP), and Endosequence BC Sealer (END). Fresh sealers were cultivated in contact with monocytes and polymorphonuclears (PMNs) obtained from the peripheral blood of humans. Cell viability, apoptosis and necrosis were analysed at 4h (PMNs) or 24h (monocytes) using Annexin-V and propidium iodide in a cytometer. The supernatants were used to quantify Interleukin (IL)-4, IL-6, IL-10, IL-12 and tumour necrosis factor-α (TNF-α) in monocytes and IL-8 in PMNs by ELISA. One-way ANOVA and the Tukey post-test were used to compare data for cytotoxicity, and the multiple T-test was used to determine the differences between sealers in the release of cytokines that were statistically significant. After 4h of treatment, S26 was associated with greater cell viability than the other sealers (P<0.05) in the PMN culture and had similar values of necrosis as END (P>0.05). After 24h of treatment, AHP and END had greater monocyte cell viability than S26 (P<0.05), which had more necrosis (P<0.05). END had the lowest levels of IL-12 compared to the other sealers (P<0.05) and higher levels of IL-6 compared to S26 (P<0.05). The tested sealers did not differ in the release of IL-8, IL-10, TNF-α and IL-4 (P>0.05). The effect of toxic agents released varied depending on the cell type studied. The composition of the sealers appeared to alter the form of self-regulation in the production of these cytokines by cells.

Sequential broncho-alveolar lavages reflect distinct pulmonary compartments: clinical and research implications in lung transplantation

BackgroundBronchoalveolar lavage (BAL) has proven to be very useful to monitor the lung allograft after transplantation. In addition to allowing detection of infections, multiple BAL analytes have been proposed as potential biomarkers of lung allograft rejection or dysfunction. However, BAL collection is not well standardized and differences in BAL collection represent an important source of variation. We hypothesized that there are systematic differences between sequential BALs that are relevant to BAL analysis.MethodsAs part of 126 consecutive bronchoscopies in lung transplant recipients, two sequential BALs (BAL1 and BAL2) were performed in one location during each bronchoscopy by instilling and suctioning 50 ml of normal saline twice into separate containers. Cell concentration, viability and differentials, Surfactant Protein-D (SP-D), Club Cell Secretory Protein (CCSP), and levels of CXCL10, IL-10, CCL2, CCL5, VEGF-C, RAGE, CXCL9, CXCL1, IL-17A, IL-21, PDGF, and GCSF were compared between BAL1 and BAL2.ResultsTotal cell concentration did not differ between BAL1 and BAL2; however, compared to BAL2, BAL1 had more dead cells, epithelial cells, neutrophils, and higher concentrations of airway epithelium-derived CCSP and inflammatory markers. BAL2 had a higher concentration of SP-D compared to BAL1.ConclusionIn this study performed in lung transplant recipients, we show that sequential BALs represent different lung compartments and have distinct compositions. BAL1 represents the airway compartment with more epithelial cells, neutrophils, and epithelium-derived CCSP. Conversely, BAL2 samples preferentially the distal bronchoalveolar space with greater cell viability and higher SP-D. Our findings illustrate how the method of BAL collection can influence analyte concentrations and further emphasize the need for a standardized approach in translational research involving BAL samples.

Abstract 500: Endothelial Specific Meis1 Knockout Protects Cells from Doxorubicin-Induced Apoptosis

Introduction: Meis1 belongs to the TALE (three amino-acid loop extension) subclass of the homeobox gene families. It is a highly conserved transcription factor in all eukaryotes and plays a crucial role in anatomical development during embryogenesis and maintaining homeostasis after birth. However, little is known about the role of Meis1 gene on regulating functions of endothelial cells (ECs). Our preliminary study found that EC-specific knockout (KO) of Meis1 showed protective benefits in response to hindlimb ischemia, such as increased blood flow and decreased toe apoptosis. We hypothesize that deletion of Meis1 may enhance EC viability or resistance to harsh environment. The present study aims to explore the potential underlying mechanisms using an in vitro apoptotic condition induced by doxorubicin (Dox). Methods: EC-specific deletion of Meis1 gene was generated by crossbreeding Meis1 flox/flox mice with Tie2-Cre mice. Following confirmation of genotyping, ECs were isolated from the lungs of 3-5 WT and KO newborn pups using CD31 microbeads. Cells were then plated and treated with 0, 0.5, and 1 μM Dox for 24 hours prior to various assays. Results: Our qPCR data indicated that KDR expression was ~100-times more in CD31 + fraction than in CD31 - fraction both in WT and KO groups (n=4); while in KO group, Meis1 expression was significantly decreased to 20% of WT control, suggesting the successful isolation of Meis1-KO ECs. Further analysis with immunocytochemistry indicated that 83.1±2.3% CD31 + cells were also KDR + , compared to 8.3±1.2% KDR + cells in CD31 - fraction (n=7-8). Using EC-specific dye uptake assay, we found that 84.6±2.4% CD31 + cells were positive for Dil-Ac-LDL, compared to 7.9±3.2% in CD31 - cells (n=6), which appears to correspond to more tube formation observed in KO than WT groups. As we expected, Meis1-KO ECs demonstrated greater cell viability (81.2±6.1%) than WT cells (58.0±1.6%) under 0.5 μM Dox treatment (n=5-6). The increased viability in KO ECs may be due to the decreased expression of Bax , a pro-apoptotic factor. Conclusions: We conclude that activation of Meis1 gene may facilitate EC death under ischemic condition while deletion of Mei1 gene promote protective effects on ECs.

Fatigue resistance, electrochemical corrosion and biological response of Ti-15Mo with surface modified by amorphous TiO2 nanotubes layer.

The objective of this work was a systemic evaluation of the anodizing treatment in a β-type Ti-15Mo alloy to grow a TiO2 nanostructured layer for osseointegration improvement. The technical viability of the surface modification was assessed based on the resistance to mechanical fatigue, electrochemical corrosion, and biological response. By using an organic solution of NH4 F in ethylene glycol, a well-organized array of 90 nm diameter nanotubes was obtained with a potential of 40 V for 6 h, while undefined nanotubes of 25 nm diameter were formed with a potential of 20 V for 1 h. Nevertheless, the production of the 90 nm diameter nanotubes was followed by micrometer pits that significantly reduced the fatigue performance. The undefined nanotubes of 25 nm diameter, besides the greater cell viability and improved osteoblastic cell differentiation in comparison to the as-polished surface, were not deleterious to the fatigue and corrosion properties. This result strengthens the necessity of an overall evaluation of the anodizing treatment, particularly the fatigue resistance, before suggesting it for the design of implants. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 107B: 86-96, 2019.