The mitochondrial membrane potential (∆Ψ) is the driving force providing the electrical component of the total transmembrane potential of hydrogen ions generated by proton pumps, which is utilized by the ATP synthase. The role of ∆Ψ is not limited to its role in bioenergetics since it takes part in other important intracellular processes, which leads to the mandatory requirement of the homeostasis of ∆Ψ. Conventionally, ∆Ψ in living cells is estimated by the fluorescence of probes such as rhodamine 123, tetramethylrodamine, etc. However, when assessing the fluorescence, the possibility of the intracellular/intramitochondrial modification of the rhodamine molecule is not taken into account. Such changes were revealed in this work, in which a comparison of normal (astrocytic) and tumor (glioma) cells was conducted. Fluorescent microscopy, flow cytometry, and mass spectrometry revealed significant modifications of rhodamine molecules developing over time, which were prevented by amiodarone apparently due to blocking the release of xenobiotics from the cell and their transformation with the participation of cytochrome P450. Obviously, an important role in these processes is played by the increased retention of rhodamines in tumor cells. Our data require careful evaluation of mitochondrial ∆Ψ potential based on the assessment of the fluorescence of the mitochondrial probe. The mitochondrial membrane potential (∆ψ) is a highly important part of the mitochondrial existence and functioning . For a lEarlier, we have overviewed known options of utilizing ∆ψ for non-energetic needs of mitochondria and cells . Among tIn 1982, the study made in Lan Bo Chen’s group established a new background for the development of the mitochondrial approach in anti-cancer strategy ,11. HoweOn the other hand, the detailed analysis of this data has called into question not only the interpretation of the data but also the adequacy of determination of mitochondrial membrane potential in cells of various types by the ability to distribute the potential-sensitive probe truly reflecting the value of the membrane potential ,12. ThisIt should be noted that fluorescent probes for mitochondrial ∆ψ have become the main tool for evaluating the functional activity of mitochondria in cells . Theoretin/Fout = 10) of the fluorescent cationic probe in the mitochondrial matrix compared to the extra-mitochondrial volume. However, the discrepancy between theory and practice and the ambiguous interpretation of changes in the probe fluorescence in response to a challenge when working with cells is caused by a set of possibilities that we had considered earlier. They are determined by the following factors:Binding of the fluorescent probe to cellular elements and structures of the mitochondrial matrix. Theoretically, positively charged hydrophobic/amphiphilic drugs can be bound to intracellular anionic constituents such as phospholipids and nucleic acids. In addition, the electrostatic interaction of cationic probes with the anionic moiety of proteins must also be considered and among those, mitochondrial proteins, so-called chargerins were proposed to play a role as binding partners of mitochondrial probes ,15,16.Transformation of the permeable form of the probe into an impermeable form with the preservation of fluorescent properties. For example, as a result of deesterification by intracellular and intramitochondrial esterases, the highly membrane-permeable Rh123  was proposed to be converted into the corresponding zwitterion , in contrast to the parental cationic form, having a very low membrane permeability  thus posIntracellular/intramitochondrial degradation of the probe. We admit that not much data exists on this issue, but all probes introduced into the cell can be considered as xenobiotics, which obey the common cellular rule to be expelled from the cell either in intact form or after its modification, often with the involvement of cytochrome P450 ,19. TherThe change in the contribution of the membrane potential on the cell membrane to the total fluorescence of the probe in the mitochondrial matrix .+) [Changes in the values of ∆Ψ on the inner mitochondrial membrane due to the possible transformation of membrane potential to the concentration difference of pH values (∆pH) while maintaining the values of the transmembrane potential of hydrogen ions (∆µH+) ,8.Activity of non-specific pumps in the cell and mitochondrial membrane efflux of xenobiotics in cells and mitochondria. The protein-meditated transport of xenobiotics  across membranes is predominantly executed by the solute carrier (SLC) and ATP-binding cassette (ABC) superfamilies of proteins ,23,24. WHence, the potential difference on the inner mitochondrial membrane of 59 mV (minus inside) determines a ten-fold accumulation  can lead to a false interpretation of the values of fluorescence as an index of the membrane potential, the purpose of this study is to explore the possibility of such modifications for Rh123.At the first step of the study, we compared the degree of release of Rh123 from glioma cells and astrocytes after the elimination of the proton-motive force by adding an uncoupler CCCP in concentrations that deliberately eliminate the electrochemical potential of hydrogen ions in mitochondria (10 µM). The fundamental difference from the conditions for the release of Rh123 from mitochondria and cells observed in  consisteThe fluorescence microscopy data was confirmed by flow cytometry. A brief analysis of the populations of glioma cells and astrocytes stained with Rh123 and subjected to 15 min treatment of the CCCP shows that initially the population of astrocytes is heterogeneous in terms of the membrane potential of mitochondria with the presence of a small shoulder on the histogram of distribution, while the histogram of fluorescence of glioma cells shows homogeneous fluorescence with a peak in the region of high values . A 15-miSince our main goal was to determine the possibility and location of the formation of modified forms of Rh123 when comparing cultured normal astrocytes and the C6 glioma tumor cells, butanol extracts from the cells and isolated mitochondria were analyzed by the express thin layer chromatography (TLC). The cells were preincubated for two hours with Rh123 (2 µM). This concentration is slightly higher than the concentrations of Rh123 used for imaging mitochondria in the cell. However, by using these concentrations it was easier to detect low levels of modified products by TLC and mass spectrometry, and additionally, the different toxicities of Rh123 were assessed in different cell types, and the contribution of dye release from the cell by non-specific pumps was insignificant. The separation into fractions was carried out in order to determine the localization of modifications, if any occurred. In an attempt to directly assess the role of mitochondria in the modification of rhodamine molecules, mitochondria were isolated from cells, which were incubated with a probe in a buffer solution in the presence of oxidative substrates. In another set of experiments, mitochondria were isolated from intact culture and separately incubated for two hours with Rh123. From the comparison of lanes 1 and 6 and simultaneously lanes 2 and 7 in During the mitochondria isolation procedure, we also analyzed intermediate fractions, namely, butanol extract, which was obtained from the pellet obtained after cell homogenization and centrifugation at 1200g . This fraction contains intact cells, nuclei, and plasma membrane fragments. The resulting supernatant  contains mitochondria and other miniature components of the cytoplasm. A comparison of cytoplasmic fractions from astrocytes and glioma cells (lanes 3 and 4) shows a high level of Rh110 in both extracts, apparently due to its presence in the incubation medium as an impurity in the Rh123 samples. However, a significant difference was found in the presence in cytoplasmic glioma samples of products other than Rh123 and Rh110 (shown by the arrow), which was completely absent in the astrocyte samples. Another comparison of lanes 1 and 6 and separately 2 and 7 in Given that glioma cells acquire an enhanced retention of Rh123, in order to explore the dependence of modifications on time, we incubated cells with Rh123 for 2 h and placed them for 22 h in a Rh123-free medium. We found that in glioma cells, after 24 h of cultivation, essential levels of fluorescent products different from Rh123 and Rh110 were observed  value of 345.28 corresponding to a mixture of Rh123 and 110. In addition, two more minor peaks were resolved with molecular masses of 327.44 and 151.94. The presence of amiodarone in the medium led to abrupt changes in the mass spectrum  coincide with the components found in extracts from glioma cells, which, after a standard 2 h incubation with the Rh123 probe, were additionally incubated in pure medium for 22 h. This experimental observation supports the formation of Rh123 modifications in glioma through the step of deesterification and the formation of Rh110.An analysis of a sample from glioma cells, which, after a standard 2-h incubation with Rh123 incubated in a cultivation medium for an additional 22 h, showed the presence of five components, presumably derivatives of Rh123 . It is nThe direct and main, though not the only, aim of this work was to evaluate methods of measuring the membrane potential using fluorescent dyes, which are positively charged fluorescent molecules in which the positive charge is delocalized by a system of conjugated double bonds, as a result of which, it becomes permeable to the inner membrane of mitochondria, in which the fluorescent cation accumulates in accordance with the values of the membrane potential. The process of cation accumulation in mitochondria stops when the steady state level of cation distribution on both sides of the inner membrane  theoretically corresponds to the membrane potential generated by mitochondrial proton pumps. The ratio of the cation in the intermembrane space with respect to the matrix is determined by the Nernst equation given in the Introduction. According to the theory, for every 59 mV, one order of magnitude of the concentrations of the penetrating cation accumulates in the matrix, and if the membrane potential is 177 mV, then the concentration of the cation in the mitochondrial matrix will be 1000 times higher in relation to cytoplasmic levels. However, all this is true if we are dealing with a single molecule, as the law of distribution applies only to this situation. If a molecule is modified in the mitochondrial matrix, and the modified species retains the original fluorescent characteristics, then the membrane potential will continue to drive the initial cation in, as a result of which an additional number of fluorescent molecules will appear in the matrix, exceeding that which should be there in accordance with the theory. By itself, the process of an exceedingly long accumulation of fluorescent cation in cells, which almost does not reach a steady state, should already alert the researcher.Theoretically, it is possible to search for potential candidates for the presented modifications based on the obtained molecular weights. However, a huge number of variants, in particular with there being possible products of multiple cytochrome P450 activities, including oxidation, reduction, desaturation, ester cleavage, ring expansion, ring formation, aldehyde scission, dehydration, etc.  make theTwo cell cultures were used in the study. The culture of rat astroglial cells was obtained with the modified method of McCarthy and de Vellis  from theg (2 °C), respectively.Mitochondria were isolated from cell cultures with the standard method of differential centrifugation. For this purpose, cells dissociated from cultural flasks by 2 mL of trypsin were centrifuged in an Eppendorf 5702 centrifuge , and the precipitate was diluted in 2 mL of cooled non-ionic medium for mitochondrial isolation . Next, the cells were homogenized and sequentially centrifuged at 1200 and 8000× Butanol extracts were obtained by adding 96% butanol to the vial containing a pellet obtained after centrifugation in an equal volume or a supernatant in the ratio of 1 volume of butanol to 5 volumes of the supernatant. After gently shaking for 2 min, the system was left for the time necessary for the formation of the butanol fraction at the top of the vial. Butanol extracts were removed from the vial, evaporated in a rotary evaporator RV 3 Flex at a temperature of 40°C and rotation mode 5.5 until the liquid was completely evaporated. The dry residue was dissolved in 50% ethanol. The resulting solution was analyzed by thin-layer chromatography on silica gel plates ). As a mobile phase, a mixture of butanol–water–ethanol was selected in a ratio of 9: 2: 1. Initially, samples with a volume of 1 µL were deposited at a level of 1 cm from the lower edge of the plate, which was immersed in a solvent system. Furthermore, in some cases, the application was normalized; for each sample, such a volume of application was selected in order to unify the detected area of the component corresponding to Rh123. This allowed us to estimate the relative contribution of other detectable components. Next, the membranes were dried and photographed on a ChemiDoc MP device with proper software. To detect Rh123, fluorescence was excited at a wavelength of 488 nm and collected emission at >530 nm.2 incubator at 37 °C and 5% CO2. Before confocal microscopy, the cells were placed in a bicarbonate-free medium buffered with Hepes.The study assessed the fluorescence levels of various fluorescent mitochondrial probes, as well as the morphological structure of the mitochondrial network of astrocyte cells and glioma C6 under the influence of various agents. When using the Axiovert conventional fluorescence microscope , visualization was performed using ventilated cultural flasks , and the cells were previously plated onto glass-bottom Petri dishes. Cells were grown in a COEach sample was imaged 3 times on a fluorescence microscope, whereas with confocal scanning, each sample was imaged 4 times. The final images obtained from the confocal microscope are the result of averaging four scans. The scanning speed, signal amplification level, and resolution of the resulting image were the same for all experiments in each series. The thickness of the confocal plane was set at 5 microns (pinhole 150). To excite the fluorescence, an argon laser with excitation at 488 nm was used, and the fluorescence was collected in the range above 530 nm. Image processing was carried out in the Image Lab™ Software and Fotor Photo Editor Software.Cells incubated with various agents for different times depending on the task were separated from the cultural flasks as described above. Next, the cells were transferred to test tubes, where complete medium was added in the ratio of cell suspension: complete medium = 1:3, centrifuged in an Eppendorf 5702 centrifuge for 5 min, 600 rpm, and the precipitate was dissolved in 1 mL of saline solution. Samples were analyzed on a CyFlow flow cytometer, Sysmex-Partec. For each sample, 50,000 events were recorded. To detect the fluorescence of the analyzed samples, a laser excitation at 488 nm was used collecting emission above 530 nm.For the analysis of samples obtained by the method described above for TLC, reverse-phase high-performance liquid chromatography was used for product separation and mass spectrometry for more accurate identification of possible modifications of fluorescent dyes that occurred in the cell.The Agilent 1260 instrument  equipped with a PDA detector and a mass spectrometric detector, and a Zorbax SB-C8 column of 5 µm  with an acetonitrile gradient were used. In the experiments, electrospray ionization was employed. Only substances that fluoresce at wavelengths greater than 500 nm when excited with a wavelength of 488 nm were analyzed. The results were processed in MassLynx and ChemDoodle software.Statistical analysis was carried out in the Flowing Software 2.5.1 and Origin Pro 2016 software. The Wilcoxon test was used for quantitative comparison of two dependent samples . The Mann-Whitney U-test was used to compare two independent samples, both astrocyte cultures and gliomas treated with the same agent. The significance level was set at 0.05.


The effect of reinforcements and thermal exposure on the tensile properties of aluminium AA 5083–silicon carbide (SiC)–fly ash composites were studied in the present work. The specimens were fabricated with varying wt.% of fly ash and silicon carbide and subjected to T6 thermal cycle conditions to enhance the properties through “precipitation hardening”. The analyses of the microstructure and the elemental distribution were carried out using scanning electron microscopic (SEM) images and energy dispersive spectroscopy (EDS). The composite specimens thus subjected to thermal treatment exhibit uniform distribution of the reinforcements, and the energy dispersive spectrum exhibit the presence of Al, Si, Mg, O elements, along with the traces of few other elements. The effects of reinforcements and heat treatment on the tensile properties were investigated through a set of scientifically designed experimental trials. From the investigations, it is observed that the tensile and yield strength increases up to 160 °C, beyond which there is a slight reduction in the tensile and yield strength with an increase in temperature . Additionally, the % elongation of the composites decreases substantially with the inclusion of the reinforcements and thermal exposure, leading to an increase in stiffness and elastic modulus of the specimens. The improvement in the strength and elastic modulus of the composites is attributed to a number of factors, i.e., the diffusion mechanism, composition of the reinforcements, heat treatment temperatures, and grain refinement. Further, the optimisation studies and ANN modelling validated the experimental outcomes and provided the training models for the test data with the correlation coefficients for interpolating the results for different sets of parameters, thereby facilitating the fabrication of hybrid composite components for various automotive and aerospace applications. The investigation on the necessity for materials for explicit applications to support the interaction capacities has assisted the examination of the most recent advancements for the evolution of composites. Aluminium composites are used profusely in aircraft owing to the higher strength-to-weight ratio, especially with regard to tensile and yield strength, along with the corrosion resistance characteristics ,2. These2O3 in the range of 2 to 8 and reported that the tensile characteristics improve due to particle diffusion and particle–matrix bonding. Bienia et al. [The composites essentially have two constituents, i.e., the hypo-eutectic blends of Al alloys and the reinforcements. The morphology of the carbide particles is pivotal for deciding the tensile characteristics of the composites ,16. The a et al.  carried a et al.  fabricatRohatgi et al.  conducteFrom the review of the research findings related to the scope of the present work, it is evident that the motivation for carrying out the research on the influence of thermal cycle on the mechanical properties of aluminium AA 5053–SiC–fly ash composites arises from the fact that the inclusion of the reinforcements and the treatment of the composites for the range of thermal cycle is found to enhance the tensile strength required for potential applications in automotive and aerospace components. Further, it is evident from the literature that there is sufficient scope for evaluating the effect of heat treatment on the properties of the aluminium composites, especially the effect of inoculation brought about by fly ash on the hybrid aluminium composites.Aluminium AA 5083 alloys in billet form were procured from Perfect Metal Corporation, Bengaluru, Karnataka State, India, alongside SiC of an average size of 35 to 70 microns procured from Snam abrasives, Bengaluru, Karnataka State, India, and C-type, and fly ash procured from Karnataka Power Corporation, Raichur, Karnataka State, India, which were used as reinforcements. The selection of the matrix and reinforcement phases was carried out based on the property requirements and ground survey on the availability and requirements matrix; the composition and property of the matrix and reinforcements ascertained from the supplier specifications are given in The micrographic images of fly ash flakes and SiC are observed using a Hitachi make SU-8000 SEM and their microstructural images are obtained at 500× magnification and 15 kV acceleration voltage. The size of fly ash flakes varies in the range of 45–125 µm , whereasIn SEM images, the particle size and shape of the fly ash and SiC are distinctly visible, and it is observed that the fly ash flakes and SiC are almost homogenous in size and have quasi-cubic and polyhedral sizes which are found to enhance the properties of the composite materials synthesised en route stir casting. The uniform dispersion of reinforcement particles can be attained by this shape of the particles and thus helps in strong bonding.2Cl6) tablets were plunged into the molten metal to remove the entrapped air. Subsequently, the temperature of molten metal was sustained at 750 °C, followed by a second round of stirring ceaselessly for a duration of 10 min before pouring the melt to the preheated die. The stir cast composites with varying wt.% of SiC and fly ash were subjected to varying temperatures, i.e., 40 °C, 80 °C, 120 °C, 160 °C, and 200 °C. The photographic images of the stir casting process, pouring of molten metal into the die-set, and cast specimens are given in The stir casting was accomplished to fabricate AA 5083-SiC-fly ash composites because of their capability to deliver high-performance composites. The weighed amount of cut pieces of aluminium AA 5083 were fed into the furnace, and the temperature was raised to 770 °C, following the works of Santhosh et al. ,33. The The ‘T6-grade’ heat treatment cycle comprised of two stages, i.e., the solution treatment carried out at 530 °C for a duration of 2 h, followed by water quenching and ageing at a temperature of 150 °C for a duration of 6 h and the thermal treatment at temperatures of 40 °C, 80 °C, 120 °C, 160 °C, and 200 °C in Heattek make Oven, with time and temperature controls available at University, Visvesvaraya College of Engineering, Bangalore University, Bengaluru, Karnataka State, India, at 0.5 MPa operating pressure and 10 h soak time in still air, with time and temperature controls as per the AMS-2771 specifications. This process of solutionising, ageing, and thermal exposure facilitate the dissolution of intermetallic phases.The composites are characterised for tensile characteristics, which are prepared in accordance with the ASTM E8-95 standards , with a The tensile test specimens were characterised using a FINE INSTRUMENTS  make TFUN-600 Universal Testing Machine with a strain rate of 0.00025/s (1.5%/min) available at MVJ College of Engineering, Bengaluru, Karnataka State, India, and the values of the tensile test were tabulated.2 to substantiate the findings by evaluating the presence of reinforcements. The SEM image depicts particle distribution in the matrix phase, while the EDS gives the elemental point analysis to significantly understand the homogenous dispersion of the SiC and fly ash flakes in the matrix facilitated by stir casting and thermal treatment.The microstructural evaluation of the specimens was accomplished using a Hitachi make SU-8000 series SEM  available at the University Visvesvaraya College of Engineering, Bangalore University, Bengaluru, Karnataka State, India, with a scanning electron voltage of 15 keV at magnifications of 500× to study the particle distribution of the reinforcements in the matrix; in addition, the elemental analysis from the energy dispersive spectrum was performed using the silicon drift detector with the detection area of 100 mmY = responses for the given factor level combination and n = number of responses in the factor level combination.Taguchi’s method is a useful statistical tool for studying the optimisation of the process. The study encompassed a design of experiments based on the orthogonal arrays (OA) and subsequent simplification of the experimental plan and the feasibility study involving the interaction between different parameters selected for the experimental trials. In the present work, optimisation studies were carried out in Minitab software based on the “larger is better” condition for ultimate tensile strength (UTS) and yield strength (YS), and “smaller is better” condition for the % elongation; the characteristic formulae for both conditions for each factor level combination are given in Equations (1) and (2), respectively.A neural network is a layered computer program with layers linked to nodes, and it resembles the network architecture of neurons of the human brain. Artificial neural network (ANN) can be taught to identify patterns, categorise information, and forecast future outcomes based on data . The datThe characterisation of the composites was accomplished to evaluate the tensile properties and determine the variation of these properties with temperature and wt.% of reinforcements.The SEM images were analysed to examine the microstructure and the elemental distribution using the energy dispersive spectrum of the chosen region of interest. The SEM images in Further, the EDS of the composites clearly affirm the presence of Si, O, and Al-Mg elements in the composite, which ascertain the presence of matrix of aluminium AA 5083, and the SiC and fly ash reinforcements. 2O3) and silicates (SiO2) in the fly ash particulates.The EDS spectrums  clearly ➢Ultimate Tensile Strength (UTS)2O3 particle dispersions in the matrix; the Al2O3 particles dispersed in the matrix phase and exposed to a temperature of 150 °C had better strength characteristics, as compared to those aluminium composites with Al2O3 particle dispersions in the matrix subjected to a temperature above 150 °C which is in line with the findings of the present work.The comparative evaluation of the ultimate tensile strength of the composite specimens for different compositions of the reinforcements and heat treatment temperatures is given in 2O3 by 8.5%, while an increase in yield strength by 36.8% mainly due to the adhesive bonding between the matrix and reinforcements attributed to uniform mixing and distribution of the reinforcements.Further, the contour and 3D surface plots  depict tSanthosh et al.  reportedThe influence of heat treatment on the strength of these composites is of significant importance to enhance the characteristics of the composites for real-time engineering applications. In this regard, the contour plots and surface plots help us to understand the influence of heat treatment on the ultimate tensile strength of the composite materials. The contour and 3D surface plots in Further, 2 reinforced Al 2024 composites provided a fundamental basis for the thermal exposure of the composites, and the results reveal that the addition of TiB2 results in significant grain refinement with the random orientation of θ-Al2Cu phases.The findings are in line with the research outcomes of Tiwari et al. , who stu➢Yield StrengthThe comparative evaluation of the yield strength of the composite specimens for different heat treatment temperatures and composition of reinforcements is presented in Further, the contour and 3D surface plots  are cons2Si precipitates attributed to precipitation hardening.From the contour and 3D surface plots for variation of YS with varying wt.% of reinforcements at different heat treatment temperatures, it is clearly evident that the reinforcements and heat treatment increases the yield strength of the composites, attributed to Orowan strengthening facilitated by precipitation hardening. Shin et al.  identifi➢% Elongation% Elongation is an important attribute for determining the ductility of the composites; specifically, it gives an overview of the length up to which the material elongates in the plastic zone before fracturing. The bar chart in The comparative evaluation of the % elongation of the composite specimens for different heat treatment temperatures is interpreted from the graph . From thFurther, the contour and 3D surface plots  depict tFrom the contour and 3D surface plots for variation of % elongation with varying wt.% of reinforcements at different heat treatment temperatures, it is clearly evident that the reinforcements and heat treatment decreases the % elongation of the composites, thereby reducing the ductility of the composites attributed to strain hardening and embrittlement. The findings are in line with the results reported by Gan et al. . The res➢Optimisation Studies for Ultimate Tensile Strength (UTS)Optimisation studies by Taguchi methods were carried out to find the optimised wt.% of fly ash and SiC and the temperature of the exposure for maximising the UTS of the composite specimens fabricated en route stir casting. Additionally, the optimisation results give us an overview of the influence of the reinforcements and heat treatment on the ultimate tensile strength of the composites developed. From the response tables and main-effect plots for S/N Ratios and means, it is evident that the wt.% of SiC has a major influence on the UTS, followed by the wt.% of fly ash and heat treatment temperature. Among these factors, level 4 (6 wt.%) for fly ash and level 4 (160 °C) for HT temperature are found to be the optimised set of parameters for maximising the UTS, while the inclusion of silicon carbide in the increments of 2 wt.% is found to outrightly improve the UTS, beginning from 3 wt.% up to the maximum limit of 9 wt.% chosen for the present work.2 composites; the parametric studies helped optimise the cutting forces and surface roughness during the drilling of composites, and the methodology adopted by the authors are in line with the statistical studies carried out in the present work to optimise the process parameters for stir casting the composites, subsequently followed by post-treatment to obtain maximum strength.Parasuraman et al.  studied Park et al.  reportedThe ANN method is considered to be the optimal option for any problem involving nonlinear and complex relationships between the variables. The neurons are organised via layers in the multi-layer perceptron (MLP) structure, with one input neurons layer, another output neuron layer, and one or more hidden layers. The hidden layers are made up of numerous interconnected neurons, as shown in The ANN used 520 experimental results, split into 3 segments: the training set (65% data), the test set (25% data), and the validation set (10% data). The importance of determining the best ANN architecture is critical because it has a significant impact on the results ,51,52. TThe ANN model thus provided a framework for prediction of the tensile strength characteristics of the composites developed for different compositions of the reinforcements and the heat treatment conditions, thus evolving a scientific database for further experimental trials on the composites for need-based fabricate on of components from the given combination of constituents for automotive and aerospace applications .The review of the outcomes of the experiments and the statistical validations yielded optimum parameters for maximising the strength of the composites developed. The distribution of the reinforcements was ascertained from the SEM images. The composites subjected to T6 tempering with solution treatment, subsequently followed by the precipitation hardening, enhanced the characteristics, especially the tensile and yield strength of the composites, facilitated by the thermal exposure. However, the percentage elongation of the composites was reduced significantly with the increase in reinforcements owing to inherent bonding, which was further accelerated by the T6 treatment.The findings for tensile strength were further optimised using Taguchi methods, and the optimised set of parameters for obtaining maximum ultimate tensile strength, yield strength, and stiffness for the composites were determined. The ANN modelling yielded modelling coefficients achieved by minimising the MSE after examining a large number of distinctly configured neural networks; the appropriate ANN framework that was chosen in this analysis has 30 neurons in the hidden layer for the prediction of UTS. The test data were compared to the expected UTS data obtained from ANN, and it is evident from the findings that the ANN’s estimation of the results is accurate, with the error % between the test results and the predictions less than +/−2%.Thus, the findings of the experiment and statistical validations provide optimised set of parameters for fabrication and post0treatment of AA 5083/SiC/fly ash composites for high-performance applications, especially as structural components in automotive and aerospace domains, wherein the composites have to possess high tensile, yield strength, and stiffness. The present work can be extended to the fabrication of real-time applications based on the post-treatment conditions optimised for the set of parameters considered in this research.


Background: The anastomosis leak in colon resections is a crucial post-operative complication with significant morbidity and mortality.
Methods: Forty (40) Wistar rats were allocated in two groups. In SHAM group only anastomosis was performed. In ILEUS group anastomosis was performed following one day of ileus. Animals in both groups were subdivided in two groups according to the day they were sacrificed, 4th or 8th post-operative day. A number of variables between the groups were estimated.
Results: Body weight loss was higher following obstructive ileus on both days. Adhesion score in 4th and 8th post-operative day was higher in ILEUS1, ILEUS2 groups compared to SHAM1, SHAM2 groups respectively (p<0.001 for both). Neovascularization decreased following obstructive ileus compared to control on the 4th day . Bursting pressure was lower in ILEUS2 group than SHAM2 group (p<0.001). The number of fibroblasts decreased following obstructive ileus compared to control on the 4th and 8th day . Hydroxyproline concentration was decreased in ILEUS2 group compared to SHAM2 group (p<0.001).
Conclusions: The balance of collagenolysis and collagenogenesis plays a decisive role in the healing of anastomoses following bowel obstruction. Under those circumstances, anastomosis’ bursting pressure is reduced owning to decreased neovascularization, reduced fibroblast presence and lower hydroxyproline concertation. In our study, local inflammation, neocollagen concentration and collagenase activity were not associated with this adverse effect. However, further research should delineate the mechanisms of healing of colonic anastomoses and identify those factors that can improve our outcomes. A prerequisite for the success of surgery and the reduction of post-operative complications is the creation of safe anastomosis following surgical resection3,4. The healing of anastomoses represents a complex process of repairing tissue damage, the end result of which depends on several local and systemic factors. The healing process is the subject of many clinical and experimental studies, as it is an important prognostic factor of postoperative morbidity and mortality in colorectal cancer interventions5-7.Colonic surgery is attracting increasing interest due to its particular anatomical and pathophysiological characteristics and the increased incidence of colonic malignancy. Colorectal cancer is the third most common cancer in the world, and surgery is the mainstay of treatment. It is estimated that 1 out of 20 people over 75 will develop colon cancer4,8-10. In particular, it ranges from more than 17% in selective to 30% in urgent interventions. It is also known that more than 30% of patients with colorectal cancer first present with obstructive ileus symptomatology and 10% of them need urgent surgical treatment, which is accompanied by an increased incidence of anastomosis leakage11-13.Anastomosis leakage in colon resections, especially for cancer, remains a very serious post-operative complication with significant morbidity and mortality. Despite advances in surgical techniques, suture materials, adequate preoperative preparation, and perioperative chemoprophylaxis, the rate of leakage from anastomosis has remained high (3-30%) over the last few decades14.The healing of anastomosis after colon resection following obstructive ileus is difficult. On the one hand, the large proximal dilation of the intestine causes wall ischemia, on the other hand, the large difference in the diameters of the two parts that are anastomosed leads to an increased likelihood of anastomosis dehiscence. Additionally, ischemia of the intestinal wall, hypovolemia and electrolyte disturbances can inhibit anastomotic healing. The creation of protective ileostomy or colostomy proximal of the anastomosis, although it does not appear to affect healing, seems to reduce postoperative morbidity and mortality by protecting against possible anastomotic dehiscenceThe aim of the current experimental study was to evaluate the healing of colonic anastomosis following large bowel obstruction. Anastomosis healing was evaluated by measuring anastomosis ruptures and the gravity of adhesions by measuring the bursting pressure of intact anastomoses, quantifying hydroxyproline and collagenase and evaluating histological parameters of the healing process in the area of anastomosis.An experimental animal protocol was designed to minimize discomfort and pain to the animals. For this research, forty male rats of the Wistar species were used, each weighing 200-300 g. The Ethical Committee of the Department of Veterinary Services of the Prefecture of Thessaloniki approved this research (S.N.: 13/11872/11-09-08) and all necessary approved protocols for laboratory animal care were applied. The rats were under a 12-hour cycle of light and dark conditions for seven to ten days prior to the operation. Each animal was individually housed and had unlimited access to both food and water before and after the operation. No antibiotics were administrated. The sacrifice was performed using intracardial administration of KCL solution of 10% concentration.The animals were divided randomly into 2 groups of 20 rats each.ILEUS GROUP : Anastomosis was performed following one day of obstructive ileus:th post-op dayILEUS1 : the sacrifice took place on the 4th post-op dayILEUS2 : the sacrifice took place on the 8SHAM1 : the sacrifice took place on the 4th post-op day SHAM2 : the sacrifice took place on the 8th post-op daySHAM GROUP : Only anastomosis was performed. th post-op daySHAM1 : the sacrifice took place on the 4th post-op daySHAM2 : the sacrifice took place on the 8The surgery was initiated using a 3cm incision at the midline. The anaesthesia was administered intraperitoneally using thiopental at a 40mg per kilogram concentration. A 1cm part of the colon was resected at a distance of 5cm from the rectum followed by an end-to-end anastomosis. Eight polypropylene sutures were used for the anastomosis, with 6-0 at a single layer. The obstructive ileus was initially “created” using 3-0 silk suture at a 5cm distance from the rectum. 24 hours later, a 1cm part of the large intestine around the ligation area was excised and an end-to-end anastomosis was performed. The abdominal wall was closed using 3-0 silk sutures.All animals were weighed at the start of the experiment and before sacrifice. Measurement was done with precision scales according to a code assigned to each animal after the anaesthesia was administered.15. Score 0 represented no adhesion formation, score 1 a minimum number of adhesions, score 2 moderate adhesion between the anastomosis and, for example, an intestine loop and score 3 represented severe adhesion formation plus the presence of abscess.On the sacrifice day, the animals were subjected to laparotomy after proper administration of anaesthesia. During the operation, the anastomotic parts of the intestine were dissected and examined macroscopically. The presence of abscess near the anastomosis or peritonitis, the presence of adhesions and anastomotic integrity were evaluated using the van der Ham scaleex vivo. A 2.5cm part of the colon containing the anastomoses was removed alongside the attached adhesions. A catheter was inserted into the distal part of the removed colon after the proximal part was closed with a silk 3-0 suture. The catheter was secured at the bursting pressure measuring device. The catheter was fixed to the bursting pressure apparatus, as described in previous research16-18. Through this, the lumen of the bowel was filled with normal saline solution at a 1ml/min rate. The pressure, measured in mmHg, at which a leakage of normal saline or rupture occurred was defined as the bursting pressure. The exact point of a possible leak or rupture during the measurement of bursting pressure was noted, due to the fact that in some rats that point was the site of the anastomosis and in others was at another point.Bursting pressure was evaluated 19. The parameters included: the infiltration of inflammatory cells, the formation of new blood vessels, fibroblast activity and the deposition of collagen20. These parameters were evaluated individually using a numbered scale: 0 (-) = no evidence; 1 (+) = occasional evidence; 2 (++) = light scattering; 3 (+++) = abundant evidence; and 4 (++++) = confluent fibres or cells.After the bursting pressure measurement, the resected part of the intestine was separated from the mesentery and the fat then cleaned with normal saline. The segment of the colon containing the anastomosis was resected, with half-cm parts on each side. This was divided into two parts in the vertical level. One part of the segmented colon was placed in a formaldehyde solution of 4% concentration for histology examination and then stained with eosin-haematoxylin stain. Under a light microscope, the anastomosis was evaluated histologically using the 0-4 Erlich-Hunt grading scale with Phillips et al.’s modification21,22. The samples were first lyophilized, then the tissue specimens were homogenized using a polytron homogenizer in distilled water. The collagen that was soluble to acid was taken from the tissue of the specimens using incubation overnight with an acetic acid solution of 0.5mol/l at 4°C. 70 μL of standard/test sample was hydrolysed in 30 μL NaOH 10.125 mol/L for 25 min at 120℃ by autoclaving. After hydrolysation, the sample was blended with a chloramines-T reagent buffer (0.056mol/L) at room temperature. The oxidation processed lasted for 25 minutes. The chromophore growth was followed by adding Ehrlich’s reagent. The absorption was assessed at 550nm using a Biotek μQuant™ spectrophotometer. The presence of hydroxyproline in unknown tissue extracts was determined from the standard curve after absorbance values were plotted against the concentration of standard hydroxyproline. The outcomes were evaluated in μg per tissue gram21.The evaluation of collagen deposition in the anastomosis was performed by evaluating the hydroxyproline percentage. For that purpose, the second part of the resected colon with the anastomosis was weighed and kept at -20°C after being cut into two segments vertically. The hydroxyproline quantity in the tissues was measured with modified methods from other experiments23.The density of type I collagenase was also measured in another part of the anastomotic area using an ELISA kit (USCNLIFEm E0212r). A plate was precoated with specific antibodies for type I collagenase . The control sample was then added. Avidin, which was also conjugated to hydroxyproline, was also added to the plate and hatched. The development of chromophore occurred after adding TMB substrate solution. The reaction was terminated using a sulphuric acid solution. Using spectrophotometry, the change in colour was assessed at 450nm (by Stat Fax-210™ spectrophotometer (Awareness Technology Inc.)) The density of type I collagenase in random tissue specimens was extracted from the standard curve. The results were measured in μg per gram of wet tissueStatistical analysisThe data extracted from the experiment were summarized using statistical descriptive indices of central tendency and dispersion. Data appear as mean value +/- standard deviation as they were normally distributed after the Shapiro-Wilk test for normality. ANOVA was performed to compare all the study groups. For the post hoc pairwise comparisons the independent t-test were used with the Bonferoni correction Paired before-and-after observations on the same subjects were analyzed using the paired t-test. Percentages were compared using the Fisher’s Exact Test. The level of statistical significance was set at p value <0.05 for the comparisons between the groups. All the statistical analyses were performed using the IBM SPSS Statistics (Version 22) enhanced with the module Exact Tests.th and 8th postoperative days). The difference between the mean values was evaluated. According to the statistical analysis, the mean weight between the subgroups did not present a statistically significant difference at the beginning of the experiment (p=0.707). However, the mean body weight differed between groups at the end of the experiment (p<0.001), as the weight in group ILEUS2 was significantly lower than in group ILEUS1 (p=0.003). Bodyweights at the beginning and end of the experiment within the same group did not differ significantly between group SHAM1 (p=0.168) and SHAM2 (p=0.09) but did differ in groups ILEUS1 (p<0.001) and ILEUS2 (p<0.001). The mean body weight was calculated both preoperatively and on the day of sacrifice . In particular, significant differences in bodyweight change were found between subgroups SHAM1 and ILEUS1 (p < 0.001), SHAM2 and ILEUS2 (p < 0.001), and ILEUS1 and ILEUS2 (p=0.001). th and 8th postoperative days, according to the protocol of the experiment. The colon segment that was removed was examined macroscopically to determine whether the anastomosis was intact. The frequency of rupture of the anastomosis was evaluated on both the 4th and 8th postoperative days. On the 4th postoperative day in the SHAM1 group, no rupture occurred, while in the ILEUS1 group there were 2 animals with anastomotic dehiscence (20%). However, there was no statistically significant difference in the rupture percentages (p=0.474). At the 8th postoperative day, no anastomotic leak occurred in the SHAM2 group, while 3 animals presented with anastomotic leak in the ILEUS2 group (30%). However, there was no statistically significant difference in the dehiscence percentages (p=0.211). The burst of the anastomosis resulted in perianastomotic abscess and peritonitis. In addition, no difference in dehiscence percentages was noted between the SHAM1 and SHAM2 groups and the ILEUS1 and ILEUS2 groups (p=1).All animals survived the experiment. Sacrifice occurred at the 4. According to this scale, on the 4th postoperative day, 90% of the rats showed no adhesions in subgroup SHAM1, while 10% had grade 1 adhesions. In contrast, in subgroup ILEUS1, all rats exhibited adhesions, with 80% being grade 1 and 20% grade 3. At the 8th postoperative day, 90% of the SHAM2 subgroup rats showed no adhesions, while 10% had grade 1 adhesions. In the subgroup ILEUS2, all rats showed adhesions, with 80% of them grade 2 and 20% grade 3. The adhesion formation scores differed significantly between groups . Also, while the adhesion formation score didn’t differ in the SHAM group between the 4th and 8th days, in the ILEUS group, adhesions increased significantly from the 4th to the 8th day (p=0.015).On both the 4th postoperative day, the ILEUS1 group presented lower mean bursting pressure values compared to SHAM1, but the difference was not significant (p=1). However, on the 8th day, the bursting pressure in the ILEUS2 group was significantly lower than in SHAM2 , and not for the ILEUS group (p=0.202).The bursting pressure at the ruptured anastomosis was set at 0mm Hg. There was a significant difference in bursting pressure among groups (p < 0.001). On the 4p<0.001; . Comparith day, the rupture rate at the anastomotic site was greater in the ILEUS1 group (90%) compared to the SHAM1 group (50%); however, the difference was not statistically significant (p=0.141). On the 8th day, the results were analogous to the previous ones, with the ILEUS2 group presenting with a greater incidence of rupturing at the anastomotic site (80%) compared with the SHAM2 group (0%), but the difference was statistically significant (p=0.005). Comparing the leakage site on the 4th and 8th days within each group, it was observed that while leakage at the anastomotic site decreased from the 4th to the 8th day in both groups, this difference was significant for the SHAM group (p=0.033), but not for the ILEUS group (p=1).During the evaluation of bursting pressure, the examined parts of the colon ruptured at the anastomotic site or elsewhere. On the 419. The analysis was done in a blind fashion by an experienced pathologist. In each group, the evaluated parameters were leukocytosis, neovascularization, fibroblasts and neo-collagen generation.The segments from the anastomotic region were assessed microscopically after haematoxylin and eosin staining. The classification was semiquantitative/ categorical and was based on the Erlich-Hunt scale, as amended by Philipsth and 8th postoperative days, but none of the differences were significant. Comparing leukocytosis on the 4th and 8th days within each group revealed that a significant decrease from the 4th to the 8th day occurred in both groups . Leukocytosis increased following obstructive ileus, compared to the control group, both on the 4th day (ILEUS1 vs. SHAM1 with p=0.038), but not on the 8th day (ILEUS2 vs. SHAM2 with p=0.559). Comparing neovascularization on the 4th and 8th days within each group revealed that a significant increase from the 4th to the 8th day occurred in both groups . Neovascularization significantly decreased following obstructive ileus, compared to the control, on the 4th and on the 8th days (ILEUS1 vs. SHAM1 with p=0.001 and ILEUS2 vs. SHAM2 with p=0.016). Comparing fibroblasts on the 4th and 8th days within each group revealed that a significant increase from the 4th to the 8th day occurred in both groups . Fibroblasts significantly decreased following obstructive ileus, compared to the control, both on the 4th day and slightly decreased, but not significantly, on the 8th day compared to the control (p=0.287). Comparing neocollagen on the 4th and 8th days within each group showed that a significant increase from the 4th to the 8th day occurred in both groups . Neocollagen was not affected by obstructive ileus on the 4th day in the ILEUS1 and SHAM1 groups (p=1), but significantly decreased on the 8th day following obstructive ileus, compared to the control .Hydroxyproline concentration differed significantly between groups (p<0.001). Specifically, it was similar on the 4Collagenase concentration did not differ significantly between groups (p=0.221). Specifically, while it decreased on the 4th day in the ILEUS1 group, compared to the SHAM1 group, the difference was not significant (p=0.928);furthermore, it increased on the 8th day following obstructive ileus, compared to the control, but the difference was not significant (p=0.1). Comparing collagenase on the 4th and 8th days within each group revealed that while an increase from the 4th to the 8th day occurred in both the control group and after obstructive ileus, no significant difference was observed .8,9. This risk is particularly high in the case of emergency surgical treatment of obstructive colorectal ileus due to neoplasm, during which the section of the intestine carrying the neoplasm is excised, followed by primary anastomosis24,25.The healing of anastomosis is a complex biological procedure of restoring tissue damage. The final result depends on both regional and systemic factors. Insufficiency of the mechanisms of the anastomosis healing process results in anastomotic dehiscence, which increases postoperative morbidity and mortality26,27. The ileus causes disturbances in the intestinal wall perfusion above the obstruction, resulting in poor blood supply to the anastomosis and increased risk of leaking28,29.The prognosis of patients undergoing urgent surgery is worse than those undergoing elective surgery30-32. It is generally believed that excision and anastomosis at one time in the occluded colon is feasible for most patients, except those who are hemodynamically unstable or present with peritonitis24,30. Recent animal studies on minimizing the risk of anastomotic leak provided favourable results. Omiganan, an anti-microbial peptide, enhanced the safety of gastrointestinal anastomoses in rats with intraperitoneal sepsis108, while selective inhibition of hydroxylases proved beneficial105. However, this increases the likelihood of anastomotic dehiscence and, consequently, the development of intra-abdominal abscesses and wound infection33,34.It is well known that a high proportion of colorectal cancer (8-30%) initially manifests with obstructive ileus symptomatology33. The frequency of leakage varies from 0.2 to 4% for colon anastomoses and from 3 to 21% for rectal anastomoses33. This postoperative complication leads to prolonged hospital stays, increased hospitalization costs and increased mortality (from 2.7% to 10-16%)35.Despite the improvement in surgical techniques, colorectal surgery is accompanied by significant post-operative morbidity and mortality. The presence of anastomotic leakage or rupture is the most common complication and the most important cause of post-operative morbidity and mortality after colectomy. A rupture is defined as the complete or partial separation of the anastomosis labia, while a leak refers to the presence of intraluminal intestinal contents in the extraluminal area36. In the first 3 to 5 days, collagen degradation is prominent 2.5cm on both sides of the anastomosis, compared to its composition. After 5-7 days, there is an increase in inflammatory response, neovascularization, fibroblasts and collagen fibres. More specifically, in the first week, the synthesis of type III collagen and fibronectin predominates, followed by the synthesis of type I collagen37,38. Experimental studies have shown a significant reduction in collagen concentrations in the anastomosis area during the early postoperative period39,40. Thus, anastomotic leakage usually occurs between 5 and 7 postoperative days (remodeling phase).Escape from anastomosis is a multifactorial process that involves local and / or general factors, as mentioned above. All these factors have a common feature: they affect the composition of the new or the degradation of the old collagen, which is a key point in the healing process. In the early phase of healing, anastomosis has increased destruction of old collagen fibres and a reduced composition of new ones. This period is very important because the integrity and strength of the anastomosis depends on the complementary strength of the sutures, as the fibrin layer is very thin and cannot keep the flaps united24,41. Excessive growth of bacteria in the occluded part of the intestine is also due to the loss of self-purification capacity. The effect of all this is the concentration of a significant amount of intestinal, fluid and air content in the part of the bowel before the obstruction42. That also results in increased pressure before occlusion. While normal intraluminal pressure ranges between 2 and 4 mm Hg, during occlusion it may reach up to 30 mm Hg. This increased intraluminal pressure causes both the coincidence of veins and an increase in hydrostatic pressure in the blood capillaries, with consequent increased fluid output to the intestinal lumen and to the peritoneal cavity12,43. Rapid growth of pathogenic microbial flora (mainly bacteroides) in the supernatant of the obstruction results in increased production of NH4, H2 and CO2. The concentration of these intestinal gasses causes further bowel dilatation44. Compression of the intestinal wall veins, which leads to increased diffusion of fluids from the capillaries to the intercellular space, is followed by an increase in edema of the intestinal wall and consequently the obstruction of arterioles. This results in ischemia, which is more pronounced initially in the intestinal mucosa. The prolongation of ischemia leads to necrosis, perforation of the intestine, excretion of intestinal contents in the peritoneal cavity and peritonitis44,45. The ileus also disrupts the intestinal barrier, resulting in the entry of microbes and endotoxins into systemic circulation via the portal vein. Within 6-12 hours of the occlusion, microorganisms are found within the peritoneal cavity , and systemic endotoxemia begins on the 4th day. Endotoxemia may lead to the development of Systemic Inflammatory Reaction Syndrome (SIRS) or Multidrug Disorder Syndrome (MODS)46,47.In cases of obstructive ileus, the observed pathophysiological disorders are: 1) changes in intestinal mobility, 2) intestinal content concentration prior to obstruction, 3) distension of the central part of the intestine by the addition of gases and liquids, 4) multiplication of microorganisms and bacterial translocation, 5) changes in blood flow, 6) homeostasis disturbances and 7) systematic disorders29,42. The ileus causes the reported local and systemic disorders, which inhibit the anastomosis healing process and lead to an increased frequency of dehiscence of the anastomosis. More specifically, the ileus causes the following disorders in the healing of anastomoses: disruption of anastomosis circulation and disruption of bowel wall. Mucosal haemorrhage, especially of the submucosal layer, determines perfusion of the anastomosis. It is known that the capillary network of the colon is less developed than that of the small intestine, which also explains its greater susceptibility to decreased blood flow13,22. In the obstructive ileus, the increased intraluminal pressure causes ischemia of the intestinal wall. In particular, the mucosa is edematous and hypo-perfused, and extensive swelling develops in the submucosal layer. Also, as the ischaemia is prolonged, ulcerations in the intestinal mucosa develop due to destruction of the epithelial cells. Ischemic lesions at this stage are usually reversible. Over the time, however, the lesions extend to the musculoskeletal and the entire thickness of the wall and are irreversible, which can lead to perforation of the intestine41,43. After the cause of the obstructive ileus has been removed, the ischemic bowel is reperfused, and there is a possibility of ischemia-reperfusion injuries43,44. This results in severe tissue alterations in the intestinal epithelium and capillary endothelium due to the release of oxygen free radicals. Ischemic lesions, destruction of the parietal capillaries and secretion of vasoconstrictor substances result in disorder of the anastomosis infusion and inhibit its healing45,48. Experimental studies have shown that ileus increases the activity of metalloproteinases, which reduce the deposition of new collagen in the anastomosis and increase the degradation of the old. Their greater concentration and consequent greater collagen degradation is observed near the staple line. The end result is a delay in the healing of anastomosis and an increased chance of leakage42. Newer studies demonstrated that selective matrix metalloproteinases (MMP)’ inhibition increased anastomotic breaking strength106 and the the application of protective local agents, such as albumin/glutaraldehyde, in colonic anastomoses leads to better outcomes109.It is known that 8-30% of patients with colorectal cancer first appear with obstructive ileus symptomatology and 10% of these require urgent surgical treatment. Obstruction is usually found in the left colon, as its diameter is shorter and its content denser49,50. It has also been found that subnutrition and hypoproteinaemia inhibit healing of anastomoses, as they reduce collagen synthesis51. In an experimental study of obstructive ileus and tacrolimus, Raptis et al.23 observed greater reduction in the body weights of the experimental animals in the ileus group than in the other group. In our study, both on the 4th and 8th postoperative days, the presence of obstructive ileus led to significant body weight loss compared to the control.Patients with colorectal cancer, particularly those with an obstructive ileus condition, show a decrease in body weight52-55. In occlusive ileus, the risk of anastomotic leakage is greater28,56. Raptis et al.23 found that leaks were more common in the ileus group than in the control group. Similarly, in our study, the frequency of rupture from the anastomosis increased in obstructive ileus conditions. Specifically, on the 4th postoperative day, anastomosis leakage was observed in 20% with obstructive ileus, but the difference with the control was not statistically significant. Similarly, on the 8th postoperative day, anastomotic leakage presented in 30% of rats with ileus, but this difference was also not statistically significant.The rate of anastomotic leakage is the most important indicator for the inadequacy of the mechanisms for healing bowel anastomoses, ranging from 3 to 30% 56,57. Adhesion formation is determined by the severity of intestinal trauma, the activation of fibroblasts, the action of metalloproteinases and the adequacy of the fibrinolytic mechanism58-60. The quantitative evaluation of adhesions in our study was done according to the van der Hamm scale in all subgroups 61. Hoffman et al.57 also reported that adhesions occur more frequently in obstructive ileus. In the current experimental study, both in the 4th and 8th postoperative days, the obstructive ileus subgroup showed significantly more adhesions compared to the control group.Adhesions occur when the organs of the peritoneal cavity and/or the peritoneal wall are traumatized. These are fibrous bands that connect the organs of the peritoneal cavity, either with each other or with the peritoneal peritoneum, and can contribute to microleaks from the anastomosis site, while improving perfusion62. During the early post-operative period (between day 3 and day 5), there is a dramatic reduction in the collagen concentration of up to 40%, due to increased collagenolysis, closer to the anastomosis site. This explains why intestinal disruption in these cases occurs in the area of reperfusion59,63. The occurrence of rupture outside of anastomosis occurs in labile areas or in focal necrosis sites. With the progressive increase in collagen synthesis from day 5, the strength of anastomosis increases, resulting in the seventh postoperative day to gain 50% of its measured potency. After the 14th postoperative day, the sutures no longer contribute to the strength of the anastomosis and rupture usually occurs away from the anastomosis39. Measurement of anastomosis bursting pressure is the most reliable method of assessing mechanical strength in the first postoperative week and is affected by the amount of collagen deposited37,53,64,65. Obstructive ileus also affects the mechanical strength of anastomoses27,65. Raptis et al.23 found a decrease in mean bursting pressure in the ileus group relative to the control group. In our study, on the 4th postoperative day, while the obstructive ileus subgroup showed lower mean bursting pressure compared to the control, the difference was not significant. However, on the 8th postoperative day, mean bursting pressure in the ileus subgroup was statistically significantly lower compared to control subgroup.The adequacy of colonic anastomosis healing is assessed by measuring the anastomosis bursting pressure, which is the most reliable method of mechanical power assessment. The bursting pressure is measured by the resistance of the intestinal wall to increased intraluminal pressure10,66. Ruptures in the area of the anastomosis occur in less-powerful mechanical anastomoses or anastomoses found in early healing (3rd to 5th day). In contrast, ruptures away from anastomosis occur in very strong mechanical anastomoses or anastomoses, where the healing process is at an advanced stage, particularly in the remodeling phase67,68. Raptis et al.23 found that the rupture rate on anastomosis was higher in the ileus group relative to the control group. In our study, on the fourth postoperative day, the highest incidence of anastomosis rupture was observed after obstructive ileus conditions compared to the control group, but this difference was not statistically significant. However, on the 8th postoperative day, a statistically significantly highest incidence of anastomosis rupture was observed after obstructive ileus conditions compared to the control group.An essential element for evaluating the effects of bursting pressure and assessing the adequacy of healing mechanisms is the point of the bowel part where a rupture occurs in the performance of the corresponding test69-71. The most important stage of the healing process is the inflammation phase, in which a fibrin clot is formed. That clot bridges the trauma gap. Inflammatory cells  also accumulate in the area. Neutrophils remove foreign bodies and necrotized tissues by releasing proteolytic factors like collagenase. Also, macrophages produce growth factors, which are responsible for the migration of fibroblasts and epithelial and endothelial cells to the wound site, resulting in the formation of granulomatous tissue69,70,72. The extension or inhibition of this phase may cause disruption of the anastomosis healing mechanisms. Although the process of healing the anastomoses of the colon is similar to that of skin, there are several differences particular to the gastrointestinal tract73-75. First, the intestinal mucosal epithelium has greater regenerative capacity, leading to faster wound repair. Anastomoses of the digestive tract acquire their maximum strength in 14-21 days, while skin sutures can take as long as the 120th day70,74,76. During the first 3-5 days, collagenolysis is predominant in the area of anastomosis. This period is the most critical, as the sheared ends of the bowel are held together only by sutures or clips. The strength of the anastomosis gradually increases until the 12th-14th days77. Also, smooth muscle cells and fibroblasts are responsible for the production of collagen in the intestine, as opposed to the skin, where these cells do not play such a role. Between the 3rd and 5th days, an abundance of undifferentiated mesenchymal cells accompanies the invasion of the capillaries in the muscle layer. These cells are transformed into smooth muscle cells and phagocytes77,78. The formation of smooth muscle cells at the wound edges is responsible for the reshaping of the lost smooth muscle tissue. Additional factors that differentiate the intestine from the skin and other tissues are its multi-layered architecture, its high content of micro-organisms, the effect of serum on the formation of a barrier on the staple line and its particular vascularization74.Healing consists of chained biological reactions that lead to collagen deposition at the anastomosis70,76,79. It evolves immediately after trauma for the structural and functional repair of the colon tissues. All processes involve the epithelial and fibrous connective tissue with its three components—cells, matrix and fibres—and aim to synthesize new connective tissue and heal the anastomosis73,79-82. The mechanisms of healing are distinguished in four overlapping phases:80-88 A) haemostasis phase, B) inflammatory phase, C) production phase and D) remodeling phase. Haemostasis is distinguished in the vascular phase where there is a contraction of the bleeding vessels, the platelet phase, where platelets accumulate and the platelet or temporal thrombus is formed, and the coagulation phase89. The inflammation phase follows the haemostasis phase, beginning 24 hours after the wound and lasting for 3-4 days. The accumulation of platelets is followed by the infiltration of the area by white blood cells, which arrive after their dissection from the vascular wall90. The transudation is neutrophilic polymorphonuclear, mononuclear macrophages and lymphocytes. Immune system cells significantly contribute to the regulation of the anastomosis healing process, through the secretion of molecular signals, such as cytokines, lymphokines and growth factors91. The activity of polymorphonuclear cells involves the phagocytosis of bacteria (by opsonization), release of collagenase and elastase and the degradation of elastin, the basal membrane of the vessels. In addition, they produce IL-1 and TNF-α, which activate fibroblasts. In the current study, while leukocytosis increased following obstructive ileus, none of the differences observed were significant compared to the control.The healing of the anastomoses of the colon involves succession and complex processesrd day and ends on the 12th day of healing. It involves the synthesis of new collagen, angiogenesis and the replacement of temporary fibrous tissue from granular tissue. Granular tissue is a loose connective tissue of fibrin, fibronectin, collagen fibres, glycosaminoglycans and hyaluronic acid38,92. In addition, it contains macrophages, fibroblasts and neoplastic vessels. Its presence lasts from day 3 to day 21 and is determined by various substances produced in the inflammatory phase37,93. In the productive phase of healing, which begins 6-7 days after anastomosis and is completed in 12-16 days, neovascularization also plays an important role. In particular, endothelial cells from the adjacent capillaries invade the anastomosis and form tubular formations surrounded by collagen fibres and the primary capillaries. This process is regulated by the action of promoters and inhibiters. Promoter agents include FGF, TGF-α and VEGF. Turlu et al. showed that TGF-β1 represents a potential therapeutic agent for the prevention of anastomotic leakage by increasing collagen synthesis and collagen deposition104. Positive effects also have local factors, such as hypoxia, low pH and high levels of lactic acid89. It has also been found that the ileus causes disturbances in the intestinal wall perfusion. Raptis et al.23 found a significant decrease in neovascularization in the ileus group compared to the control group. In our study, on the 4th postoperative day, the obstructive ileus group showed lower neovascularization compared to the control and this difference was statistically significant. However, on the 8th postoperative day, while neovascularization was reduced in ileus subgroup, the decrease was not significant compared to the control.The proliferation phase begins after the 375,94. An important factor directly affecting collagenase activity is the presence of bacteria in the anastomosis. Bacteria are sources of proteolytic enzymes that produce collagenase and can cause increased collagen breakdown at the site of anastomosis95,96. Studies on mice suggested that drinking a phosphate-based polymer can achieve the goal of preventing anastomotic leak by suppressing collagenase production in E. faecalis107. After the early phase, the production phase of healing is followed by the production of collagen by the fibroblasts, which increases the strength of the anastomosis39,40,92. For this reason, the production and deposition of collagen in the area of anastomosis is an important indicator of the adequacy of the healing process52,63. Raptis et al.23 found a significant reduction in the number of fibroblasts in the ileus group compared to the control group. In our study, on the 4th postoperative day, a reduced number of fibroblasts, compared with the control, was observed in the ileus subgroup and this difference was statistically significant. Similarly, on the 8th postoperative day in the ileus subgroup, a decreased number of fibroblasts was observed, compared to the control, and this difference was also statistically significant.During the production phase, the area of anastomosis is predominantly occupied by endothelial cells and fibroblasts. The most important processes, both in this phase and in the remodeling phase, are collagenolysis and collagenogenesis, the balance of which plays a decisive role in the healing of anastomoses. A significant increase in collagenase was found in the gastrointestinal tract following anastomosis in the colon38. Additionally, as the inflammatory elements recede, angiogenesis is disrupted, fibroblasts are produced, the old type III collagen degrades and the novel type I collagen is synthesized. The strength of the anastomosis is determined by the quality and amount of the newly formed collagen. This increases from the 2nd to the 6th week of healing, then stays constant and reaches its maximum in 2-3 years. At the same time, tissue remodeling (after three weeks), which lasts up to three years, can also cause anastomosis constriction after a significant period of time (up to six months). The end result is the formation of scarring connective tissue59,83,97. In experimental studies, occlusive ileus has been shown to increase the activity of metalloproteinases (between day 1 and day 5), which reduces the deposition of new collagen and increases the degradation of the old, both in the anastomosis region and away from it96,98,99. Raptis et al.23 found significant reduction in neocollagen formation in the ileus group compared to the control group. Also, Moran et al.100, in an experimental study on rats, after erythropoietin administration under ileus conditions, found that the formation of neo-collagen decreased in the ileus group compared to all other groups. However, in our study, we didn’t observe any significant differences in neocollagen synthesis among the experimental groups.At the remodeling phase, collagenolysis progressively decreases, whereas collagen deposition, which begins on days 7-8 from the formation of anastomosis, increases and may take up to 90 days. The collagen fibres intertwine with each other and, in combination with the connective tissue, provide elasticity, durability and integrity. In the remodeling phase, the most important event is the collagen deposition and the gradual replacement of granular tissue with connective tissue, which consists of a network of collagen and elastic fibres101. It was also found that obstructive ileus causes disorders in the structure of the intestinal wall, resulting in a decrease in the concentration of hydroxyproline63,101. Raptis et al.23 found that the ileus group had a decreased concentration of hydroxyproline relative to all other groups. In our study, we found that on the 4th postoperative day, after obstructive ileus, there was a decreased concentration of hydroxyproline compared to the control, but this difference was not statistically significant, while on the 8th postoperative day, there was a significant reduction in hydroxyproline concertation in the ileus group compared to the control.The mechanical strength of anastomosis is dependent on connective tissue cell proliferation, collagen molecule synthesis and collagen fibril formation. The synthesis and deposition of collagen are complex biological processes that are affected by a variety of factors. Experimental models are necessary to investigate the deposition of collagen during the anastomosis healing process. The most common method is to determine hydroxyproline, as a unit of total collagen count, in colorectal cells from the anastomosis site. Hydroxyproline is one of the key amino acids components of collagen of all types and is a product of proline hydroxylation metabolism. The main feature of this amino acid is that it is almost exclusively found in connective tissue collagen. The measurement of hydroxyproline is quantitative and does not give information on the quality of collagen and its identification in fractions . However, its measurement is now the only reliable quantitative method for determining collagen in the anastomosis region46,60,66. Various experimental studies have shown that the intense activity of MMPs is one of the major pathogenetic factors of postoperative attenuation of the anastomosis. Other studies have found that the levels of MMPs on the suturing line increased during the first three days after anastomosis. In addition, other studies have shown that MMP-1 and MMP-13 are found in all patients with leakage, suggesting a potential predisposition for collagen metabolism disorder. These findings suggest that treatment with synthetic inhibitors of MMPs contributes to maintaining mature collagen fibres in the submucosal layer and, consequently, the integrity of anastomosis98,102.Collagen is an abundant extracellular substance and determines the non-mechanical stability of connective tissue during healing. The most important types are type I collagen, which is responsible for the formation of mature collagen, and type III collagen, which occurs mainly during the early phase of healing and has less mechanical strength. Their content and proportion determine the mechanical strength and stability of the anastomosis. The total degradation of collagen is the result of the synergistic action of various MMPs42,96,98. Collagen is resistant to the action of proteolytic enzymes and is degraded by collagenase. A significant increase in collagenase was found in the gastrointestinal tract following anastomosis in the colon96. Collagenase plays an important role in determining the integrity of anastomosis and the ability to hold sutures in the early postoperative days of healing. In addition, immunohistochemical studies have shown that collagenase and other metalloproteinases are proximal to the anastomosis stapling line. Collagenase belonging to the metalloproteinase family is considered to be responsible for the loss of mature collagen of the submucosal layer and the consequent breakdown of anastomosis103. This is supported by experimental data indicating maximum collagenolytic activity in anastomosis in rats on the 3rd postoperative day. Other experimental studies in rats using aprotinin, which is a protein inhibitor, showed a significant increase in the breakdown pressure of the anastomoses of the colon52. Collagenase is the major breakdown enzyme of mature collagen, belongs to the family of metalloproteinases and plays an important role in the integrity of anastomosis. The factors that affect the activity of collagenase are bacteria and the inflammatory reaction around the anastomosis. It has also been found that occlusive ileum increases the enzymatic activity of metalloproteinases and causes rapid degradation of collagen in the intestinal wall. Therefore, the collagenase concentration assay provides a better measure of the balance between collagenogenesis and collagenolysis28,46,60. In our study, on the 4th postoperative day in the ileus subgroup, the collagenase concentration decreased in comparison with the control group, but this difference was not statistically significant. On the 8th postoperative day, the ileus subgroup showed a higher collagenase concentration compared to control subgroup, but this difference was also not statistically significant.Experimental studies have shown that ileus increases the activity of metalloproteinases, which reduces the deposition of new collagen in the anastomosis and increases the degradation of the old one. Their greater concentration and greater collagen degradation are observed near the staple line. The end result is a delay in the healing of anastomosis and an increased chance of leakageAs a high proportion of colorectal cancer cases initially manifest with obstructive ileus symptomatology, there is a need for improved outcomes and safety of colonic anastomosis. The balance of collagenolysis and collagenogenesis plays a decisive role in the healing of anastomoses. Anastomotic healing in the early period seems to be impaired following obstructing ileus conditions, as anastomotic dehiscence percentages did not significantly increase, the bursting pressure significantly decreased, and the majority of leakages occurred at the anastomotic site. This reduction was not affected by local inflammatory reaction, neocollagen concentration and collagenase activity, but rather by decreased neovascularization, reduced fibroblast presence and lower hydroxyproline concertation. Thus, extreme caution should be exercised in the construction of an anastomosis following obstructive ileus and there is a need for measures, either local or systemic, to enhance the anastomotic healing process. Future studies should focus on the delineation of the mechanisms of healing of colonic anastomoses and identify those factors that can be altered to minimize the incidence of the anastomosis rupture.◊ The balance of collagenolysis and collagenogenesis plays a decisive role in the healing of anastomoses◊ Obstructive ileus subgroup presented with decreased number of fibroblasts (production and deposition of collagen) and decreased concentration of hydroxyproline (reliable quantitative method for determining collagen)◊ In obstructive ileus subgroup, higher collagenase concentration and lower neovascularization were observed◊ The highest incidence of anastomosis rupture was seen after obstructive ileus conditions compared to the control group◊ Future studies should focus on the delineation of the mechanisms of healing of colonic anastomoses and identifying those factors that can be altered to minimize the incidence of anastomosis rupture


Buffalo rats for testing. Selected morphological, chemical and physical parameters of the teeth of the offspring of female rats from the experimental groups were analyzed. The analysis showed the effect of vitamin E and ASA for the content of Mg, Ca and P. In combination with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), vitamin E and ASA, they positively inhibit the inflammatory process, preventing the leaching of Ca and Mg from the bones. ASA counteracted this phenomenon much more effectively than vitamin E. An increase in the number of dentiform cells was observed, but a slightly lower Ca content, which means that the tooth walls in this group have a lower density. Despite the use of compounds protecting the teeth against the harmful effects of TCDD, disturbances in the structure of the tooth crown cusps were still observed.The present study aimed to determine the possibility of limiting potential dioxin disorders of the structure of hard tissues in the offspring of intoxicated rat mothers by simultaneous administration of α-tocopherol (vitamin E) or acetylsalicylic acid (ASA). The levels of magnesium (Mg), calcium (Ca) and phosphorus (P) contained in bone tissue as indicators of the process of mineralization of hard tissues were also determined. The chaps were harvested from the offspring of eight female p-dioxin (TCDD) and acetylsalicylic acid or α-tocopherol. The experiment was carried out on eight female rats of the Buffalo strain divided into four groups. From the offspring of eight females, the mandibles were removed from the mandibular joints, and then, after the removal of soft tissues, they were prepared for individual tests. Selected morphological, chemical and physical parameters of the teeth of the offspring of female rats from the experimental groups were analyzed. The analysis showed the effect of vitamin E and ASA on the content of Mg, Ca and P. In combination with TCDD, vitamin E and ASA, they positively inhibit the inflammatory process, preventing the leaching of Ca and Mg from the bones. ASA counteracted this phenomenon much more effectively than vitamin E. Detailed analysis of the tooth morphology showed that the molars’ crowns exhibit shape disturbances under the influence of TCDD. Individual nodules in teeth T1, T2, T3 did not fuse, and the roots showed signs of hypertrophy. The study confirmed the negative effect of TCDD on tooth development. Teeth arising early in development are the most sensitive to the disorders, while the later ones are less exposed to the toxic effects of TCDD transmitted by the mother.The studies conducted so far indicate a negative effect of dioxins on the structure of the alveolar bone and teeth, especially in the developmental period in rats. The research aimed to analyze the indirect effect of dioxins contained in the body of female rats on the structure of the dental organ in their offspring in the neonatal period and to determine the possibility of reducing potential dioxin disorders of the structure of hard tissues in the offspring of intoxicated mothers by simultaneous administration of vitamin E or acetylsalicylic acid (ASA). Another goal of the research was to determine the level of magnesium, calcium and phosphorus contained in bone tissue as indicators of the mineralization process of hard tissues in rats, in the case of using 2,3,7,8-tetrachlorodibenzo- Based on the results of experimental studies carried out in the Wrocław Center, it has been shown that 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) causes, by inducing inflammation, the simultaneous destruction of connective tissue [Dioxins are formed in any thermal process and are characterized by the presence of organic matter, chlorine, high temperature and low oxygen concentration. Dioxins are emitted not only during the incineration and disposal of waste but also in various technological processes. Important routes of penetration of dioxins into the body are the digestive system, respiratory tract and skin. Dioxins have a high affinity for fats and can dissolve in them, thanks to which they can be easily supplied and stored in the body’s adipose tissue. Dioxins circulate in various food chains, including their presence in organisms living in water . The mose tissue ,5. The se tissue ,5,6.Therefore, it was assumed that dioxin may affect the bone structure of the alveolar process and teeth, especially in the developmental period. This seems to be confirmed by the observed changes in the external appearance of the offspring of rats administered TCDD ,8. The cThe rat is a commonly used mammal for odontogenesis studies. The molars of these mammals are very similar in terms of odontogenesis, physiology and histological structure to human milk teeth ,14. The Assessment of the impact of specific metabolic changes in the body on the odontogenesis process should be performed in very young animals. In rats, the odontogenesis process ends around the 35th day of individual life, after which catabolic processes begin to dominate over anabolic processes and may disturb the proper interpretation of even subtle consequences of developmental conditions, making it unreliable or even impossible ,18,19.Taking all of this under consideration, the aim of this study was to compare tooth malformation in rat offspring whose mothers were intoxicated by TCDD and possible prevention by known antioxidants such as α-tocopherol and acetylsalicylic acid using the micro CT technique with histological analysis.Buffalo strain  were used in the experiment. The animals were housed in polystyrene cages with "Labofeedh" food and water.Eight female rats of the I.A control group of females (C), not treated with any chemicals;II.p-dioxin (TCDD) in a single dose of 5 μg/kg BW them;A group of females (TCDD) who received a solution of 2,3,7,8-tetrachlorodibenzo-III.p-dioxin (TCDD) in a single dose of 5 μg/kg BW them. and a solution of α-tocopherol acetate at the dose of 30 mg/kg BW/day s.c. was administered for a period of 3 weeks;A group of females (TCDD + E) administered a solution of 2,3,7,8-tetrachlorodibenzo-IV.p-dioxin (TCDD) in a single dose of 5 μg/kg BW them and administration of acetylsalicylic acid suspension in starch solution at a dose of 50 mg/kg BW/day per os (p.o.) for a period of 3 weeks.A group of females (TCDD + ASA) administered a solution of 2,3,7,8-tetrachlorodibenzo-The females were randomized into 4 groups, each consisting of 2 animals:After a period of 3 weeks after administration of TCDD in individual groups , the above-mentioned groups of females and group C were matched with randomly selected males from the same strain, not treated with any chemical substances. After the mating period, pregnant females were placed in separate cages.Newborns (NC)—10 individuals ;Newborns (NTCDD)—4 individuals ;Newborns (NTCDD + E)—7 individuals ;Newborns (NTCDD + ASA)—6 individuals .TCDD females’ deliveries were extended over 3 weeks compared to the control group in which deliveries occurred within 4 days. The number of offspring born to females in individual groups was different:All newborns were kept in cages for another 30 days, after which they were sacrificed, and the material was collected for testing  was injected intraperitoneally at a dose of 120 mg/kg of BW).The mandibles were dissected from the temporomandibular joints, and then, after the removal of soft tissues, they were prepared for individual examinations.α-tocopherol acetate  (vitamin E);Acetylsalicylic acid—Aspirin (Bayer) ;Thiopental ;p-dioxin (TCDD) ;Standard solution of 2,3,7,8-tetrachlorodibenzo-Pharmacological agents and chemicals used in the experiment:The research material consisted of 16 prepared fragments of the mandibles with incisors and molars.The collected mandibles were divided into two from each individual, and one-half underwent the microCT and chemical spectroscopic analysis , and the second half underwent the histological analysis. The purpose of microCT analysis was to measure the density and geometric parameters of the tissues of rats’ teeth.The mandibles of rats immediately after extraction were fixed in a 4% formalin solution, pH 7.2–7.4 for 48 hours, then rinsed in running water for 24 hours. After rinsing, the material was decalcified in a mixture of concentrated acids: hydrochloric and formic (for 24 hours), dehydrated in an alcohol series and embedded in paraffin. The blocks were cut in the midplane of the tooth, longitudinally to the mandible edge into sections 7–9 μm thick and then dewaxed in xylene and stained with hematoxylin and eosin according to Delafield. The slides were observed using a Nikon Eclipse 80i  optical microscope in transmitting light.3 of concentrated nitric acid (V) p.a. was added to an aliquot of a homogeneous sample (from 0.1 to 0.5 g) and 1 cm3 of concentrated hydrogen peroxide p.a., then the samples were mineralized in the microwave MARS 5  sample preparation system. The minerals were quantitatively transferred to 10 cm3 measuring vessels with redistilled water. The mineralization was carried out by the Polish Standard PN-EN 13805: 2003 "Foodstuffs. Determination of trace elements. Pressure mineralization" [The samples were "wet" mineralized in a closed microwave system. A total of 5 cm‑Determination of calcium content by atomic emission spectrometry:Determination of the calcium content in an acetylene/air flame was carried out by atomic emission spectrometry using a SpectraAA atomic absorption spectrometer Cal-L Enterprises  with a Varian AA240FS flame attachment.The Ca content was determined by the Research Procedure PB-01/AAS developed and used in the Research Laboratory for Atomic Absorption Spectrometry at the University of Life Sciences in Wroclaw.‑Determination of magnesium content by atomic absorption spectrometry:Determination of the magnesium content in an acetylene/air flame was carried out by atomic absorption spectrometry using a SpectraAA atomic absorption spectrometer  with a Varian AA240FS flame attachment.The determination of Mg content was performed by the Research Procedure PB-02/AAS developed and used in the Research Laboratory for Atomic Absorption Spectrometry at the University of Life Sciences in Wroclaw.‑Determination of phosphorus content by atomic emission spectrometry:The phosphorus content of the acetylene/air flame was determined by atomic emission spectrometry using a SpectraAA atomic absorption spectrometer  with a Varian AA240FS flame attachment.The P content was determined by the Research Procedure PB-03/AAS developed and used in the Research Laboratory for Atomic Absorption Spectrometry at the University of Life Sciences in Wroclaw.Measurements of the density and geometrical parameters of individual teeth were carried out with the use of CTAn and DataViewer programs on reconstructions obtained with the use of the 1172 SkyScan, Bruker®  computer microtomography. Each of the preparations was recorded with a resolution of 9 µm, with the lamp parameters: 80 kV/124 mA, Al filter. (0.5 mm), with a unit rotation angle of 0.4 degrees. The analysis occurred in the Department of Mechanics, Materials and Biomedical Engineering, Faculty of Mechanical Engineering, Wroclaw University of Science and Technology.‑Estimating descriptive statistics ;‑Checking the normality of the distribution of features using the Shapiro–Wilk test;‑Verification of hypotheses about the equality of the level of measurable features with a normal distribution in more than two groups using the analysis of variance (ANOVA).The research results were based on the statistical analysis of the measurement results, which consisted of:p < 0.05.All hypotheses were verified at the significance level of The statistical analysis was carried out using the statistical software package Prism 9.3.1, GraphPad Software . Histological examination revealed a decrease in the number of odontoblasts and ameloblasts in both the root formation zone and the dental body in the TCDD group compared to the control groups A,B. ThisThe analysis showed the effect of tocopherol and acetylsalicylic acid on the content of calcium and magnesium A,B. ThisAs a result of the action of TCDD in the pre- and postnatal period, the odontogenesis of the tooth crown and roots is disturbed. This effect persists despite the protective effect of vitamin E and ASA. Visible thinning and change of incisor geometry in TCDD and its clear improvement in group E. Moreover, a significant enlargement of the tooth cavity is observed in the TCDD group .In incisors, a change in the form of flattening of the arch geometry is observed in the teeth of the experimental groups . Teeth fFor each of the incisors, cross-sections were prepared for the transaxial (XY) and frontal coronary (XZ) planes, for which the values of the orientation angle of the examined cross-section (T.Or (°)) were determined . Teeth t3, which together with the individual preparations were scanned in an 1172 SkyScan, Bruker®  computer microtomography. The measurement of the density of individual teeth, i.e., incisors and cheek teeth, is shown in The mineral density measurement was carried out using density phantoms with known values of 0.25 and 0.75 g/cmIn this evaluation, the highest disturbances were observed within M3, which in the TCDD group is composed of the crown only with a visible decrease in bone density beneath the roots. In all TCDD-treated groups, enlargement of incisors cavity in the mandible is observed. In addition, teeth nodules on M1, M2 and M3 do not show abrasion, which means disturbance of their growth.Understanding the course of the odontogenesis process, the biological mechanisms controlling it, and the assessment of the influence of certain innate and acquired factors on this process are possible thanks to experimental studies on animals .The most important features of incisor odontogenesis are ,17,18,19There are four phases in the process of rat molar odontogenesis ,17,18,19Several factors can disturb tooth formation ,30,31,32The observed changes are the more important because TCDD can accumulate in the environment and be passed down from generation to generation. This will result in an increase in tooth development disorders in future generations unless the increase in the number of dioxins in the environment is significantly slowed down or even stopped completely. The possibilities of counteracting this effect may be largely limited by the use of antioxidants, but they do not reduce the amount of dioxins in the environment. This requires, however, further research.The study confirmed the negative effect of TCDD on tooth development. Developmental disorders are primarily molars and incisors, in which there is a decrease in the number of odontoblasts and ameloblasts, and thus the thickness of dentin and enamel. Teeth arising early in development are the most sensitive to the disorders, while the later ones are less exposed to the toxic effects of TCDD transmitted by the mother.