Longer Incubation Times Research Articles

Extracellular vesicles production and proteomic cargo varies with incubation time and temperature

Extracellular vesicles (EVs) are heterogenous populations of proteolipid bi-layered vesicles secreted by cells as an important biological process. EVs cargo can reflect the cellular environmental conditions in which cells grow. The use of serum-free conditioned media to harvest EVs leads to stress-mediated cellular changes with longer incubation time and impacts EV production and functionality. This study aims to explore the role of incubation time and temperature on EV production and proteomic cargo. For this purpose, an optimized ultrafiltration-size exclusion chromatography-based technique is developed, which isolates small EVs ranging from 130 to 220 nm. The result shows higher EVs production in cancerous cells (K7M2) compared to noncancerous cells (NIH/3T3), which increases with longer incubation time and elevated temperature. Mass spectrometry-based proteomic characterization of EVs showed incubation time and temperature-dependent proteomic profile. A set of enriched EV proteins were identified in EVs isolated at nutrient-stress (72 h incubation time) and heat-stress (40 °C incubation temperature) environment. Enrichment of Serpinb1a in EVs isolated in heat stress was further validated via immunoblot. Gene enrichment analysis revealed that enriched EV proteins following nutrient stress were involved in negative regulation of transcription, response to oxidative stress, and protein folding. Likewise, enriched EV proteins following heat stress were involved in oxaloacetate and aspartate metabolism, and glutamate catabolic process. EVs isolated under nutrient stress showed pro-proliferative activity whereas EVs isolated under heat stress showed anti-proliferative activity. Our results show that incubation time and temperature can alter EV production, its proteomic cargo, and functionality, which can be used to design need-based standard isolation parameters for reproducible EV research.

The Effect of a Rotating Cone on Horseradish Peroxidase Aggregation on Mica Revealed by Atomic Force Microscopy.

Our study reported herein aims to determine whether an electromagnetic field, induced triboelectrically by a metallic cone, rotating at a frequency of 167 Hz, has an effect on the properties of the horseradish peroxidase (HRP) enzyme. Atomic force microscopy (AFM) was employed to detect even the most subtle effects on single enzyme molecules. In parallel, a macroscopic method (spectrophotometry) was used to reveal whether the enzymatic activity of HRP in solution was affected. An aqueous solution of the enzyme was incubated at a distance of 2 cm from the rotating cone. The experiments were performed at various incubation times. The control experiments were performed with a non-rotating cone. The incubation of the HRP solution was found to cause the disaggregation of the enzyme. At longer incubation times, this disaggregation was found to be accompanied by the formation of higher-order aggregates; however, no change in the HRP enzymatic activity was observed. The results of our experiments could be of interest in the development of enzyme-based biosensors with rotating elements such as stirrers. Additionally, the results obtained herein are important for the correct interpretation of data obtained with such biosensors.

The propofol-induced mitochondrial damage in fetal rat hippocampal neurons via the AMPK/P53 signaling pathway.

Propofol is a commonly used general anesthetic that may cause neuronal damage, especially in infants and young children. Mitochondria play an essential role in cellular metabolism and signal transduction. Propofol may cause neurotoxicity by inhibiting mitochondrial function, but the mechanism by this which occurs remains unclear. First, the primary rat hippocampal neurons were cultured for 7 days in vitro. The neurons were incubated with propofol at different times or different concentrations, and then the adenosine triphosphate (ATP), reactive oxygen species (ROS), mitochondrial membrane potential, and apoptosis-related proteins were analyzed. Based on the results of the 1st phase, the neurons were then incubated with propofol (100 µM) or corresponding reagents, including 5-aminoimidazole-4-carboxamide ribonucleotide, tenovin-1, and pifithrin-α. Subsequently, the ATP, ROS, mitochondrial membrane potential, phospho-adenosine 5'-monophosphate-activated protein kinase (p-AMPK), protein 53 (p53), and related apoptosis proteins were analyzed. Higher propofol concentrations or longer incubation times were associated with more pronounced decreases in ATP, B-cell lymphoma 2 (Bcl-2), and mitochondrial membrane potential, and more pronounced increases in ROS, BCL2-associated X (Bax), Cytochrome C (CytC), and cleaved caspase-9. Additionally, after incubation with propofol (100 µM), neuronal Bcl-2, p-AMPK, ATP, and mitochondrial membrane potential were downregulated, and ROS, p53, CytC, Bax, cleaved caspase-3, and cleaved caspase-9 were upregulated. AMPK activators or p53 inhibitors reversed the above-mentioned changes. Propofol (100 µM)-induced mitochondrial damage in fetal rat hippocampal neurons may be mediated by the AMPK/p53 signaling pathway. Propofol (100 µM) was shown to inhibit the activity of AMPK in neurons, upregulate the expression of p53, and then activate the mitochondrial-dependent apoptosis pathway, which may lead to neuronal apoptosis.

Reversible and Irreversible HAuCl4 Binding to DNA for Seeded Gold Nanoparticle Growth and Opposite DNA and Aptamers Colorimetric Sensing Outcomes

AbstractDNA‐directed seeded growth of gold nanoparticles has been used for the development of aptamer‐based biosensors with the assumption that target analytes can modulate the adsorption of aptamers to the gold seeds and thus affect the growth reaction. To understand the reaction, the effect of single‐ and double‐stranded DNA is first examined, and it is found that they have a similar promotion effect of anisotropic growth, suggesting that DNA cannot be detected using this method. By studying the interaction between HAuCl4 and DNA, both weak reversible binding and strong irreversible binding are identified, with the latter becoming dominating with a longer incubation time. Single‐ and double‐stranded DNA have similar weak binding to HAuCl4, and this weak binding is more important for the growth reaction. Then three aptamers are tested, where only cortisol appeared to modulate its aptamer adsorption and the growth reaction reflected aptamer binding. Hg2+ shows no advantage for its aptamer, and quinine induced aggregation of AuNPs cannot be detected by this reaction either. Therefore, each aptamer target needs to be individually studied to test if this method is applicable. It is also noted that DNA and aptamers have opposite outcomes for the target‐dependent growth reactions.

Biocorrosion Behaviour of Carbon Steels by Tropical Microbes in the Presence of Corrosion-Inhibiting Bacterium

A set of microbiologically induced corrosion was carried out on different types of carbon steels: AISI 1006 and API 5L X52, iron-oxidizing bacterium and sulfur-oxidizing bacterium, a mixed culture of Alicyclobacillus ferrooxydans SKC/SAA-2 and Aspergillus niger and Comamonas thiooxydans SKC/SAA-1, in the presence of the corrosion-inhibiting bacterium, Pseudomonas plecoglossicida. According to the immersion test experiments, weight loss of API 5L X52 was lower (0.06-0.27%) than AISI 1006 (0.14-0.32%). AISI 1006 showed more detrimental localized pitting corrosion than API 5L X52. During the longer incubation time, the corrosion-inhibiting bacterium was more homogenous and compact, which affected the specimen surface more protective characteristics. The 2-week-old biofilm effectively protected the API 5L X52, as indicated by the low amount and more negligible pitting corrosion. This study will be the first report on the biocorrosion behaviour of carbon steels using different corrosion-causing microbes in the presence of the corrosion-inhibiting bacterium.

Characterization of Protease Enzyme Produced from Locally Isolated Bacillus sp.

This study aimed to characterization of protease enzyme purified from locally isolated Bacillus sp.. A total of 50 soil samples were taken for this purpose. Only 35 (or 70%) of the sample were found to have given rise to bacterial growth. As the first phase in the cultivation of bacteria, the samples were grown on mannitol egg yolk polymyxin agar and nutrition agar. More than 20 bacterial isolates were discovered, according to the findings. The results of the cultural, microscopic, and biochemical examinations used to identify these isolates revealed that 15 of them were Bacillus spp. Additionally, the use of the Vitek 2 technique ensured that each of the 15 isolates belonged to this species. Examining these isolates' capacity to create proteases revealed that all 15 of the Bacillus sp. isolates were capable of doing so, albeit to varying degrees. The isolate with the number S9 was the best at producing proteases, with a specific activity value of 40.19 U/mg proteins. On a casein hydrolyzed medium plate with a diameter of 3.0 mm and 2.2 mm, respectively, they demonstrated a clean zone. After 42 hours and 72 hours of incubation at 37°C, respectively, the strain 9 produces 1.01 units of proteolytic enzyme per milliliter. Proteolytic activity decreases with longer incubation times. It has potential for industrial use because the isolated Bacillus sp. was generating protease.

PLGA Nanoparticles Uptake in Stem Cells from Human Exfoliated Deciduous Teeth and Oral Keratinocyte Stem Cells.

Polymeric nanoparticles have been introduced as a delivery vehicle for active compounds in a broad range of medical applications due to their biocompatibility, stability, controlled release of active compounds, and reduced toxicity. The oral route is the most used approach for delivery of biologics to the body. The homeostasis and function of oral cavity tissues are dependent on the activity of stem cells. The present work focuses, for the first time, on the interaction between two types of polymeric nanoparticles, poly (lactic-co-glycolic acid) or PLGA and PLGA/chitosan, and two stem cell populations, oral keratinocyte stem cells (OKSCs) and stem cells from human exfoliated deciduous teeth (SHEDs). The main results show that statistical significance was observed in OKSCs uptake when compared with normal keratinocytes and transit amplifying cells after 24 h of incubation with 5 and 10 µg/mL PLGA/chitosan. The CD117+ SHED subpopulation incorporated more PLGA/chitosan nanoparticles than nonseparated SHED. The uptake for PLGA/chitosan particles was better than for PLGA particles with longer incubation times, yielding better results in both cell types. The present results demonstrate that nanoparticle uptake depends on stem cell type, incubation time, particle concentration, and surface properties.

Polymer Texture Influences Cell Responses in Osteogenic Microparticles.

Polymer materials used in medical devices and treatments invariably encounter cellular networks. For the device to succeed in tissue engineering applications, the polymer must promote cellular interactions through adhesion and proliferation. To predict how a polymer will behave in vitro, these material-cell interactions need to be well understood. To study polymer structure-property relationships, microparticles of four chemically distinct tyrosol-derived poly(ester-arylate) polymers and a commercially available poly(lactic acid-co-glycolic acid) (PLGA) copolymer were prepared and their interactions with cells investigated. Cell loading concentration was optimized and cell adhesion and proliferation evaluated. Particles were also tested for their ability to adsorb bone morphogenetic protein-2 (BMP-2) and differentiate a myoblast cell line towards an osteoblast lineage through BMP-2 loading and release. While cell adhesion was observed on all particles after 24 h of incubation, the highest degree of cell adhesion occurred on polymers with smaller crystallites. At longer incubation times, cells proliferated on all particle formulations, regardless of the differences in polymer properties. High BMP-2 loading was achieved for all particle formulations and all formulations showed a burst release. Even with the burst release, cells cultured on all formulations showed an upregulation in alkaline phosphatase (ALP) activity, a measure of osteoblast differentiation. As with cell adhesion, the polymer with the smaller crystallite showed the most ALP activity. We suggest that smaller crystallites serve as a proxy for topographical roughness to elicit the observed responses from cells. Furthermore, we have drawn a correlation between the polymer crystallite with the hydration potential using surface analysis techniques. The online version contains supplementary material available at 10.1007/s12195-022-00729-9.

The Effects of Transforming Growth Factor-β1 on the Differentiation of Cell Organoids Composed of Gingiva-Derived Stem Cells.

This study was aimed at evaluating the effects of transforming growth factor-β on the differentiation and mRNA expression of organoids made out of human mesenchymal stem cells. Cell organoids composed of gingiva-derived stem cells were cultured in the presence of transforming growth factor-β1 at concentrations ranging from 0, 1, 10, to 20 ng/ml. Evaluations of the cell morphology of the organoids were performed on days 7, 9, 11, and 14. Quantitative cellular viability was completed on day 14. Alkaline phosphatase activity assays were performed to evaluate the differentiation of stem cells on day 14. Real-time polymerase chain reactions were used to determine the expression levels of TGF-β1, RUNX2, OCN, SOX9, and COL1A1 mRNA on day 14. The stem cells produced well-formed organoids on day 7, and the addition of transforming growth factor-β1 did not result in relevant changes in their shape. The organoids grew in size and became more intact with longer incubation times. On day 14, the diameters were 222.2 ± 9.6, 186.1 ± 4.8, 197.2 ± 9.6, and 211.1 ± 19.2 m for transforming growth factor-β1 at final concentrations of 0, 1, 10, and 20 ng/ml, respectively. Quantitative cell viability results from day 14 exhibited no significant difference between the groups (P > 0.05). There was significantly higher alkaline phosphatase activity with the addition of transforming growth factor-β1 with the highest value for the 1 ng/ml group (P < 0.05). Real-time polymerase chain reaction results demonstrated that the mRNA expression levels of RUNX2, OCN, and SOX were higher in 1 ng/ml but did not reach statistical significance. Treatment with 1 ng/ml of transforming growth factor-β1 significantly increased COL1A1 mRNA expression at day 14. The application of transforming growth factor-β1 increased differentiation, which was confirmed by alkaline phosphatase activity and mRNA expression while maintaining cell viability.

Nonspecific Proteolysis-Based Glycomics Strategy for Bacterial Glycoproteins.

Protein glycosylation is one of the most common and complex post-translational modifications. Many techniques have been developed to characterize the specific roles of glycans, the relationship between their structures and their impact on the functions of proteins. A common method for glycan analysis is to employ exoglycosidase cleavage to release N-linked glycans from glycoproteins or glycopeptides using Peptide-N-Glycosidase F (PNGase F). However, the glycan-protein linkages in bacteria are different and there is no enzyme available to release glycans from bacterial glycoproteins. In addition, free glycans have also been described in mammalian cells, bacteria, yeast, plants, and fish. In this article, we present a method that can characterize the N-linked glycosylation system in Campylobacter jejuni by detecting asparagine (Asn)-linked and free glycans that are not attached to their target proteins. In this method, total proteins from C. jejuni were digested by Pronase E with a higher enzyme to protein ratio (2:1-3:1) and a longer incubation time (48-72 h). The resulted Asn-glycans and free glycans were then purified using porous graphitic carbon cartridges, permethylated, and analyzed by mass spectrometry.

Biological Resistance of Elm (Ulmus carpinifolia var. Umbelifera) Trees against Fungal Endophytes and White Rot Decay Fungi

Urban trees are one of the valuable storage in metropolitan areas. Nowadays, a particular attention is paid to the trees and spends million dollars per year to their maintenance. Trees are often subjected to abiotic factors, such as fungi, bacteria, and insects, which lead to decline mechanical strength and wood properties. The objective of this study was to determine the potential degradation of Elm tree wood by Phellinus pomaceus fungi, and Biscogniauxia mediteranae endophyte. Biological decay tests were done according to EN 113 standard and impact bending test in accordance with ASTM-D256-04 standard. The results indicated that with longer incubation time, weight loss increased for both sapwood and heartwood. Fungal deterioration leads to changes in the impact bending. In order to manage street trees, knowing tree characteristics is very important and should be regularly monitored and evaluated in order to identify defects in the trees.

The Effect of Hydrolysis Time with Trichoderma Reesei on Soluble Sugar Concentration in Sweet Sorghum Stalk Pulp and Alcohol Concentration of Its Fermentation Results

It has been done a study on the use of Trichoderma reesei in the hydrolysis of sweet sorghum stalk pulp continued to fermentation by Saccharomyces cerevisiae to produce bioethanol. The purpose of this study was to examine the effect of hydrolysis time by T. reesei on the sugar content of sweet sorghum stalk pulp and the ethanol concentration obtained after its fermentation using Saccharomyces cerevisiae. The sweet sorghum stalks were cut into small pieces, blended until crushed, incubated with T. reesei at various times. and then fermented using S. cerevisiae for 24 hours after the incubation period was complete. The results showed that the soluble sugar concentration in the sweet sorghum stalk pulp increased after the incubation, which occurred from the 8th to the 24th hour. Furthermore, it continued to decrease until the longest incubation time. The alcohol concentration produced after the fermentation period changed according to the pattern of changes in sugar concentration in the substrate. These results suggest that to increase bioethanol production from sweet sorghum stalk, it will be better to incubate the pulp of stalk first with T. reesei for approximately 24 hours before fermented with S. cerevisiae.

On the role of strain hardening and mechanical properties in water droplet erosion of metals

The role of strain hardening on the resistance of metals to water droplet erosion is investigated for the first time. A stainless steel (17–4PH) was heat treated to various conditions to achieve different sets of mechanical properties. Erosion tests were then performed at 250 and 300 m/s impact speeds and incubation time and erosion rate were measured. It was found that higher strain hardening is conducive to higher erosion resistance as demonstrated by longer incubation time. Strain hardening was also found to dictate the influence of other mechanical properties, such as yield strength and resilience, on the initial stages of erosion. It is concluded that erosion resistance is a synergistic combination of mechanical properties that includes a stain hardening parameter.

Evaluation of lactic acid fermentation in a dairy and non-dairy beverage using two commercial starter cultures

Background: Lactic fermentations are a catabolic process in which biochemical transformations of different organic products occur. Sugars are mainly converted into organic acids, increasing viscosity, acid taste, aroma, and flavor. Lactic acid bacteria provide probiotic characteristics if they reach counts of 106 CFU*g-1 (Colony Forming Units) in the final product, which can generate wellness for consumers. Objective: This research aimed to compare the lactic fermentation process in three substrates using two commercial cultures. Methods: Whole milk (control), aqueous extract of oats flakes, and an aqueous extract of a mixture of oats flakes with mashua pulp were used. The whole milk was heated, and the aqueous extracts were prepared. All samples were divided into two parts, keeping the temperature at 42°C, and then inoculated with Yomix y Choozit. Each fermentation lasted 6 hours at 42°C. Fermentation samples were taken each hour, and pH, titratable acidity, and Brix degrees were determined. Results: Total lactic acid bacteria were counted at the end of each fermentation. The final product was evaluated with sensory analysis. As expected, there was an increase in titratable acidity and a decreased pH and Brix degrees. It was observed that the dairy product showed the most significant changes. Fermentations performed with Yomix presented a higher count of lactic bacteria. Conclusion: It is possible to carry out lactic fermentation using substrates that do not contain milk, requiring higher initial soluble solids and a longer incubation time.

The Influence of Selected Antipsychotic Drugs on Biochemical Aspects of Alzheimer's Disease.

The aim of this study was to assess the potency of selected antipsychotic drugs (haloperidol (HAL), bromperidol (BRMP), benperidol (BNP), penfluridol (PNF), pimozide (PIM), quetiapine (QUET) and promazine (PROM)) on the main pathological hallmarks of Alzheimer’s disease (AD). Binary mixtures of donepezil and antipsychotics produce an anti-BuChE effect, which was greater than either compound alone. The combination of rivastigmine and antipsychotic drugs (apart from PNF) enhanced AChE inhibition. The tested antipsychotics (excluding HAL and PNF) significantly reduce the early stage of Aβ aggregation. BRMP, PIM, QUET and PROM were found to substantially inhibit Aβ aggregation after a longer incubation time. A test of human erythrocytes hemolysis showed that short-term incubation of red blood cells (RBCs) with QUET resulted in decreased hemolysis. The antioxidative properties of antipsychotics were also proved in human umbilical vein endothelial cells (HUVEC); all tested drugs were found to significantly increase cell viability. In the case of astrocytes, BNP, PNF, PIM and PROM showed antioxidant potential.

Interleukins Profiling in Umbilical Cord Mesenchymal Stem Cell-Derived Secretome.

BackgroundUmbilical cord mesenchymal stem cells (UC-MSCs)-derived secretome is currently used in regenerative therapy. MSCs are believed to secrete a wide spectrum of bioactive molecules which give paracrine effects in immunomodulation and regenerative capacities. One group that was found in secretome is interleukins (ILs), a cytokine that plays an essential role in the process of proliferation, differentiation, maturation, migration, and adhesion of immune cells. However, as there are many types of ILs, the profile of ILs in the UC-MSCs-derived secretome has been limitedly reported. Therefore, in this study, we would like to profile and detect the interleukin concentration secreted by UC-MSCs.MethodsUC-MSCs-derived secretome was collected from UC-MSCs passage 5 after 24- and 48-hour incubation (n=9). Secretome was filtered using 0.2 µm and stored at −80°C for further detection. All samples were normalized before the interleukin (IL-2, IL-4, IL-6, IL-9, IL-10, IL-12, IL-17A) detection using a MACSPlex Cytokine Kit.ResultsThe IL-6 has the highest concentration among other interleukins in both groups and increases significantly (p<0.003) after incubation for 48 hours. The pro-inflammatory factors are decreasing while anti-inflammatory factors are increasing after 48-hour incubation.DiscussionOur studies show that the UC-MSCs secrete pro- and anti-inflammatory interleukins. The concentration of anti-inflammatory interleukins shows to be increasing, while the pro-inflammatory interleukins are decreasing within the longer incubation time, but this not be applicable for IL-10 and IL-6. IL-6 has the highest concentration among other ILs. These results may provide important clues regarding when is the right time for secretome to be used in therapy patients, because all the molecules in the secretome can lead to many clinical manifestations.

Understanding Consequences and Tradeoffs of Melt Processing as a Pretreatment for Enzymatic Depolymerization of Poly(ethylene terephthalate).

Melt extrusion pretreatment of poly(ethylene terephthalate) (PET) prior to enzymatic depolymerization with an unpurified leaf branch compost cutinase enzyme cocktail is explored to ascertain the efficiency gained by different processing methods on the enzymatic depolymerization of PET. Specific surface area (SSA) is investigated as a key factor in reducing depolymerization time. Higher SSA substrates (>5.6mm2 mg-1 ) show higher depolymerization rates (≈0.88gL-1 terephthalic acid [TPA] per day) and no induction phase, while lower SSA substrates (≈4.3, 4.4, and 5.6mm2 mg-1 ) show, after an initial induction phase, similar depolymerization rates (≈0.46, 0.45, and 0.44gL-1 TPA per day) despite increases in SSA of up to 30%. The mechanism of enzymatic depolymerization manifests in the appearance of anisotropic pitting. Longer incubation time used to overcome the induction phase in low SSA substrates allows for nearly full recovery of monomeric products, but manual pregrinding of extruded PET sharply increases SSA, depolymerization rate, and substrate crystallinity which may decrease the maximum recycled yield of the product materials. An estimate of the energy cost of increasing SSA is made and its effects on material properties are discussed. This work highlights key material structure and pretreatment aspects influencing the enzymatic recycling of PET.

Secretomics-A Key to a Comprehensive Picture of Unconventional Protein Secretion.

For a long time, leaderless secreted proteins (LLSP) were neglected as artifacts derived from dying cells. It is now generally accepted that secretion of LLSP–as a part of the collective term unconventional protein secretion (UPS) - is an evolutionarily conserved process and that these LLSP are actively and selectively secreted from living cells bypassing the classical endoplasmic reticulum-Golgi pathway. However, the mechanism of UPS pathways, as well as the number of LLSP and which part of a protein is involved in the selection of LLSPs for secretion, are still enigmatic and await clarification. Secretomics-a proteomics-based approach to identify and quantify all proteins secreted by a cell-is inherently unbiased toward a particular secretion pathway and offers the opportunity to shed light on the UPS. Here, we will evaluate and present recent results of proteomic workflows allowing to obtain high-confident secretome data. Additionally, we address that cell culture conditions largely affect the composition of the secretome. This has to be kept in mind to control cell culture induced artifacts and adaptation stress in serum free conditions. Evaluation of click chemistry for secretome analysis of cells under serum-containing conditions showed a significant change in the cellular proteome with longer incubation time upon treatment with non-canonical amino acid azidohomoalanine. Finally, we showed that the number of LLSP far exceeds the number of secreted proteins annotated in Uniprot and ProteinAtlas. Thus, secretomics in combination with sophisticated microbioanalytical and sample preparation methods is well suited to provide a comprehensive picture of UPS.

Differences in susceptibility of deciduous and permanent teeth to erosion exist, albeit depending on protocol design and method of assessment

Controversial results showing that deciduous teeth are more susceptible to erosion than permanent teeth might be related to study designs. We investigated how different conditions (pH: 3.0, 4.0, 5.0; acid agitation: gentle or vigorous; acid exposure times: 1–5 min) affect the susceptibility of both teeth to erosion. Enamel specimens (90 deciduous, 90 permanent) were distributed into groups (n = 15 permanent, n = 15 deciduous) according to acid pH (pH 5, 4 or 3) and agitation (gentle or vigorous) during erosive challenge. Both milder (less incubation time, gentle agitation, and higher pH) and more severe (longer incubation times, vigorous shaking, and lower pH) conditions were used. Demineralization was measured by relative surface microhardness (rSMH) and calcium released to the acid. Demineralization increased gradually for both teeth with increasing incubation time, agitation (gentle or vigorous), and with decreasing acid pH. The differences between deciduous and permanent teeth depended on the protocol design and assessment method. Under milder conditions, demineralization was better detectable with rSMH. Under more severe conditions, differences were more perceptible with calcium analyses. Differences exist in the susceptibility to erosion between deciduous and permanent teeth, but they are only distinguishable when the appropriate assessment method is used for the specific erosive condition.

In Vitro Direct and Indirect Cytotoxicity Comparative Analysis of One Pre-Hydrated versus One Dried Acellular Porcine Dermal Matrix.

Aim: The aim of the present study was to compare the direct and indirect cytotoxicity of a porcine dried acellular dermal matrix (PDADM) versus a porcine hydrated acellular dermal matrix (PHADM) in vitro. Both are used for periodontal and peri-implant soft tissue regeneration. Materials and methods: Two standard direct cytotoxicity tests—namely, the Trypan exclusion method (TEM) and the reagent WST-1 test (4-3-[4-iodophenyl]-2-[4-nitrophenyl]-2H-[5-tetrazolio]-1,3-benzol-desulphonated)—were performed using human primary mesenchymal stem cells (HPMSCs) seeded directly onto a PDADM and PHADM after seven days. Two standard indirect cytotoxicity tests—namely, lactate dehydrogenase (LTT) and MTT (3-[4,5-dimethyl-2-thiazolyl]-2,5-diphenyl-2H-tetrazoliumbromide)—were performed using HPMSCs cultivated in eluates from the matrices incubated for 0.16 h (10 min), 1 h, and 24 h in a serum-free cell culture medium. Results: The WST and the TEM tests revealed significantly lower direct cytotoxicity values of HPMSCs on the PHADM compared with the PDADM. The indirect cytotoxicity levels were low for both the PHADM and PDADM, peaking in short-term eluates and decreasing with longer incubation times. However, they were lower for the PHADM with a statistically significant difference (p < 0.005). Conclusions: The results of the current study demonstrated a different biologic behavior between the PHADM and the PDADM, with the hydrated form showing a lower direct and indirect cytotoxicity.