Cracks are one of the main distresses that occur on concrete surfaces. Traditional methods for detecting cracks based on two-dimensional (2D) images can be hampered by stains, shadows, and other artifacts, while various three-dimensional (3D) crack-detection techniques, using point clouds, are less affected in this regard but are limited by the measurement accuracy of the 3D laser scanner. In this study, we propose an automatic crack-detection method that fuses 3D point clouds and 2D images based on an improved Otsu algorithm, which consists of the following four major procedures. First, a high-precision registration of a depth image projected from 3D point clouds and 2D images is performed. Second, pixel-level image fusion is performed, which fuses the depth and gray information. Third, a rough crack image is obtained from the fusion image using the improved Otsu method. Finally, the connected domain labeling and morphological methods are used to finely extract the cracks. Experimentally, the proposed method was tested at multiple scales and with various types of concrete crack. The results demonstrate that the proposed method can achieve an average precision of 89.0%, recall of 84.8%, and F1 score of 86.7%, performing significantly better than the single image (average F1 score of 67.6%) and single point cloud (average F1 score of 76.0%) methods. Accordingly, the proposed method has high detection accuracy and universality, indicating its wide potential application as an automatic method for concrete-crack detection. Cracks are one of the main types of distress in concrete surfaces, with a significant impact on the bearing capacity and durability of concrete structures. Therefore, an automatic and accurate method for recognizing them is crucial for the monitoring and maintenance of concrete structures. In the past few years, various technologies have been developed for automatic concrete distress detection. According to the principles of detection, we can divide these into three main categories: 2D image-based detections, 3D laser-scanning methods, and 2D–3D combination techniques. Many researchers have proposed various crack-edge detection algorithms based on 2D images, such as Laplacian, Sobel, Canny, and Prewitt ,2,3,4. TWith the recent developments and innovations in hardware, 3D-laser disease-detection technology has become a research hotspot ,16,17,18Considering the complex conditions of concrete surfaces, such as the presence of stains and shadows, it is difficult to achieve high accuracy using a 2D-image method or a 3D point clouds separately. Therefore, some scholars have proposed methods combining 2D and 3D crack extraction . ValençaTo overcome the discussed limitations for high precision concrete-surface crack detection based on single 2D images and 3D point clouds methods, an automatic method fusing 3D point clouds and 2D images based on an improved Otsu’s method is proposed in this paper. In this method, depth information from point clouds and gray information is fused at the pixel level; then based on an improved Otsu segmentation method and fine detection of cracks, high-precision crack detection, is achieved. The main innovations of the proposed method in this paper are as follows: (1) a new fusion method of 3D point clouds and 2D image data is proposed; (2) proposing an improved Otsu crack detection method; (3) discussing the application of two different types of 3D laser scanners in the field of crack detection. The rest of the article is organized as follows: the proposed fusion detection method is explained in detail in In this study, the overall framework of the proposed method is mainly composed of the following steps: (1) data preprocessing; (2) the fusion of 3D point clouds and 2D images; (3) crack detection based on an improved Otsu’s method; (4) denoising and repair of cracks. Among them, the fusion of 3D point clouds and 2D images and the crack detection based on the improved Otsu’s method are the two most important parts. The former determines the quality of the fusion image and is the basis for high-precision crack detection, while the latter determines the accuracy with which cracks are extracted. A flow chart summarizing the process is presented in 8)3 = 16,777,216 colors) is converted into 8-bit gray-level (28 = 256 levels) data. Image noise is then removed using the median filter to avoid its interference in crack detection, (the median filter was selected after comparing various filtering methods). For the 3D point clouds, the preprocessing mainly includes two steps: (1) the filtering and denoising of the point clouds; and (2) depth-image acquisition. Before fusing the 2D and 3D data, the point clouds need to be reduced into 2D depth images. However, any noise in the original point clouds greatly impacts the quality of the generated depth images; therefore target-area point clouds are extracted from the original point clouds and then denoised with the median filter.Due to interference from the sensor itself, the environment, and human factors, there is a certain amount of noise in the collected point cloud and image data, so they need to be preprocessed separately. For the images, the three-color component weighted average method is first applied for image graying: the true color image  is converted into grayscale by removing a lot of color information that is not good for crack detection, which reduces the image data. Specifically, the 24-bit RGB data  is used to fit the point clouds and calculate the parameters of the fitting plane. Next, the rotation and translation matrix of the fitting plane transformed into the XOY plane is calculated. Then, the coordinate of all the point clouds is transformed using the calculated rotation-translation matrix. The points from plane fitting contain crack points, which cause errors in the plane fitting, so the Z coordinate threshold was used to remove the crack point clouds in this study; then, a second plane fitting for the remaining non-crack point clouds was carried out by the PCA method, and the corresponding rotation translation matrix was calculated again. Finally, the rotation and translation matrix was used to transform the coordinates of all the point clouds including the cracked and non-cracked point clouds.(1)An image grid of size (2)The number of points falling into the grid is calculated, and the average of their coordinate values is taken as the depth.(3)The depth is normalized to a 0–255 scale and then the image grid matrix is output as the depth image.The depth of a crack point can be directly expressed by the Z coordinate value of point clouds in the new coordinate system, so point clouds can be projected into depth images as follows: The fusion of 3D point clouds and 2D images is one of the key aspects of our method. The purpose is to generate a fused image with low noise and prominent cracks by fusing the depth of the point clouds and the image gray information. In this study, the point clouds were projected into depth images, and then, the latter and the 2D gray images were fused by an image processing method mainly consisting of: (1) the registration of the depth and gray image; (2) the fusion of the depth and gray image.Image registration means overlaying two or more images of the same scene taken at different times, from different viewpoints, and/or by different sensors. It geometrically aligns two images—moving and fixed images. The key is to find the best transformation matrix for maximizing the degree of alignment between the two images, and it mainly involves the calculation of the control points coordinates and geometric transformation. The advantage of the method in this study is that it can not only achieve accurate image registration but also use the target to determine the ground sampling distance (GSD) in the images . Firstly, we selected circular targets uniformly distributed on the surface of the concrete structure as the control points and calculated their image coordinates The Hough transform circle detection algorithm  was usedAfter obtaining the precise control point coordinates of the two images, the precise registration of the depth image and the 2D image can be realized through geometric transformation between the two images, which includes spatial geometric transformation and grayscale interpolation. The former defines the pixel distribution of the image in the new coordinate system; the latter assigns the pixel gray level of the image after spatial transformation . This study used a bilinear interpolation method whose principle is shown in To following measures were taken to ensure the accuracy of the registration: (1) a certain number of targets were evenly distributed on the surface of concrete; (2) the center coordinates of the target were obtained by using the circular detection algorithm and fine scan; (3) the two images after registration were overlapped and displayed to ensure that the crack edges completely overlapped. The registration results for the depth and gray image are shown in The fusion of depth and gray images is based on their complementarity for crack detection. For example, in a background area with stain interference, the grayscale of the gray image is smaller than the normal grayscale value, but the grayscale of the depth image is not affected; in an area with muddy landfill or shallow cracks, the grayscale of the depth image is larger than the normal grayscale value, but the grayscale of the gray image is not affected. Harnessing the advantage of the two types of data, and according to the concrete surface background interference and the depth and width of the cracks, we used the weighted-average method of pixel-level fusion to assign different weights to the depth and gray image for data fusion. The fusion image generated by the above steps is a composite of gray information and depth information. Compared with a single image or depth image, it can have reduced background noise and enhanced crack information, providing a solid foundation for subsequent crack detection research.The comparison of the different images is shown in Otsu’s method is an adaptive threshold crack-detection method that is widely employed. The basic principle is dividing the image into background and target according to a threshold. The best segmentation threshold is the pixel value that maximizes the variance between the two kinds of pixel. Although a fusion image has more advantages for crack detection than a single image or depth image, with the traditional Otsu method, it still presents certain limitations in complex backgrounds and images with unclear background contrasts.To overcome the shortcomings of the traditional Otsu method in this regard, this study proposes an improved Otsu crack-detection method that incorporates several constraints for the image-segmentation algorithm, which makes the block-based crack segmentation more accurate. This method first divides the fused image into blocks and calculates the segmentation thresholds for different image sub-blocks. Next, several constraints are introduced to divide the image sub-blocks into the crack and background sub-blocks, and then, the crack sub-blocks are roughly extracted through the steps described above; lastly, all the binary sub-block images are merged, and the fine detection of cracks is realized by denoising and repairing cracks. A flow chart summarizing the improved Otsu crack detection method is shown in Firstly, the fused image is divided into blocks, and then, the Otsu’s threshold for all the image sub-blocks are calculated. Compared with a single threshold for the whole image, different thresholds for different image sub-blocks can allow the extraction of the cracks from the background with more accuracy.However, the forced binarization to the background sub-blocks would result in errors. Therefore, we introduced local constraints related to the image sub-blocks such as the pixel ratio After the rough crack detection, some crack burrs, noises, and fractures may still exist, which could seriously affect the accuracy. Hence, denoising and connection of cracks are needed to refine the final detection. The cracks in the fusion image mainly present the following two characteristics: (1) generally linear shape; (2) areas are large relative to the noises. Therefore, the connected component labeling method can be used to remove noises based on the shape and area feature constraint of the connected component. Then the morphology method is used to remove the crack burrs and connect the broken cracks. The specific process is shown in Firstly, connected region analysis is performed on the binary image after the implementation of the improved Otsu’s method. First, employing the algorithm used in the connected-region labeling function “bwlabel” in MATLAB, the pixels that conform to the four-connection relation are classified into the same connected domain. For extracting the crack, the connected area and minimum length /width ratio of the circumscribed rectangle of the crack should meet the conditions of Equations (9) and (10).After the noise is removed based on the above constraints, the open and close operations for morphology can be used to remove the burr and connect the fractured cracks. The crack burrs and isolated noise are removed using the open operation, and the holes in the cracks are filled and the cracks in adjacent areas are connected using the closed operation. A comparison of before and after the denoising and connection of cracks is shown in The concrete crack data in this experiment were collected on different pavements and walls at a university covering an area of 3 square kilometers. The cracks include 3D point clouds and 2D-image data. The 3D point clouds were collected by a handscan700 handheld 3D laser scanner and RIEGL VZ-2000 terrestrial 3D laser scanner. The two scanners have different spatial resolutions: the minimum for the handheld scanner is 0.2 mm (the minimum distance between two points), while the point cloud of the terrestrial laser scanner is related to the scanning distance and angular resolution. The shorter the distance, the smaller the angular resolution and the higher the accuracy. To obtain as much 3D information of the crack as possible, we used the highest accuracy that the scanner can achieve, and its corresponding minimum point cloud resolution was 1 mm. The specific technical parameters of the scanner are shown in The 2D images were captured by a camera with a resolution of 7952 × 5304, orthogonally to the concrete surface. The 3D laser scanner and camera were completely separated, each acquiring data separately, so that high-resolution concrete-crack images could be obtained at a close distance. Three typical cracks were included: transverse, longitudinal, and inclined. The length and width of the crack concrete area measured by the hand-held 3D laser scanner are about 0.5 m, and the grid spacing of the depth image of the point clouds collected with the handheld 3D laser scanner is 0.5 mm. Therefore, the length and width of the corresponding depth image are about 500 × 500. The length and width of the crack concrete area measured by the terrestrial 3D laser scanner are about 0.6–1.5 m, and the grid spacing of the depth image of the point clouds collected by the terrestrial 3D laser scanner is 1 mm in the experiment. Thus, the length and width of the corresponding depth image were about 600–1500. The relevant parameter settings used for this method are shown in To verify the feasibility and universality of the proposed method, it was compared with a single 3D point clouds method and a single 2D-images method, respectively. All the real cracks were obtained manually for evaluation. Some of the crack detection results are as follows: To illustrate the superiority of the proposed method, a set of cracks was randomly selected from large experimental samples, and the deep learning crack detection algorithm called DeepCrack  and the In this study, the precision, recall, and F1-measure  were used to quantitatively analyze and evaluate the performance of the proposed method. The precision measures the exactness or fidelity of detection, while the recall describes the completeness of detection. We used a common evaluation standard for the proposed method, the F1-measure , which combines precision and recall. The closer the F1 value is to 1, the better the method performs. P, R, and F1 were calculated using Equations (11)–(13), respectively. The quantitative results detected by the deep learning and the proposed method are shown in The crack-detection results for the handheld and the terrestrial 3D laser scanners are shown in Pixel calibration was realized by using a circular target. Then, the corresponding true distances (GSDs) of pixels for six groups of crack images were obtained, as shown in The weighted average pixel-level image fusion method is used to fuse the depth and gray image, so the selection of the factor weighting greatly impacts the final detection accuracy. If the depth image is overweighted, it becomes difficult to effectively take advantage of the high resolution of the image, which can allow the identification of tiny cracks. By contrast, if the weight of the image is too large, then stains and uneven illumination in the image can greatly interfere with crack detection. Therefore, appropriately setting these factors according to the situation of the concrete crack and the actual measured performance of the scanner will improve the accuracy of crack detection. The weighting factors in The scanner distance, incident angle, and other factors have a certain impact on the accuracy of crack detection. The existing research of Kim  and the The algorithm used for crack extraction has a great influence on the accuracy of detection. The proposed method, fusing point clouds and images based on an improved Otsu algorithm, not only obtains width and depth information for cracks but is also strongly resistant to interference from stains. It should be mentioned that the development of computer vision technology has made deep learning in crack-detection research a hotspot in recent years. Therefore, in the future, we will conduct in-depth research on crack detection with the fusion of point clouds and images based on deep learning.To deal with the disadvantages of low accuracy in extracting concrete surface cracks based on single 3D point-cloud and 2D-image data, this paper proposes an automatic concrete crack detection method fusing 3D point clouds and 2D images based on an improved Otsu’s algorithm. Firstly, depth images from point clouds and 2D images are fused at the pixel level to generate a fused image, and then accurate crack detection is realized based on an improved Otsu method. The main contributions of this paper to the research on crack detection, regarding the proposed method, are as follows: (1) the point clouds are first processed to reduce dimensionality not only does this significantly reduces the data and necessary calculations, but mature 2D image-processing technology can be used to detect cracks from the fusion image; (2) the fusion of 3D point clouds and 2D images is realized at the pixel level. The fusion image combines the grayscale information from the high-resolution images and the depth information from the point clouds, fully harnessing the advantages and compensating for the shortcomings of both; (3) an improved Otsu crack-detection method is proposed. By dividing the image into blocks and introducing constraints related to the image sub-blocks, the error that would be introduced by the forced binarization of the image sub-blocks is avoided, thereby improving the crack detection accuracy; (4) the effects on the detection accuracy of different methods for acquiring the point clouds were studied, and methods are proposed according to the scenarios and precision requirements for crack detection. Further research will seek to improve the image segmentation algorithm.


Travel restrictions in and out of Wuhan were too late to prevent the spread of the virus to 262 cities within 28 days. However, the epidemic peaked in Hubei province on 4 February 2020, indicating that measures such as closing citywide public transport and entertainment venues and banning public gatherings combined to avert hundreds of thousands of cases of infection. It is unlikely that this decline happened because the supply of susceptible people was exhausted, so relaxing control measures could lead to a resurgence.By 23 February 2020, China had imposed a national emergency response to restrict travel and impose social distancing measures on its populace in an attempt to inhibit the transmission of severe acute respiratory syndrome–coronavirus 2 (SARS-CoV-2). However, which measures were most effective is uncertain. Tian Science, this issue p. 638 Travel restrictions and the national emergency response delayed the growth and limited the size of the COVID-19 epidemic in China. Responding to an outbreak of a novel coronavirus [agent of coronavirus disease 2019 (COVID-19)] in December 2019, China banned travel to and from Wuhan city on 23 January 2020 and implemented a national emergency response. We investigated the spread and control of COVID-19 using a data set that included case reports, human movement, and public health interventions. The Wuhan shutdown was associated with the delayed arrival of COVID-19 in other cities by 2.91 days. Cities that implemented control measures preemptively reported fewer cases on average (13.0) in the first week of their outbreaks compared with cities that started control later (20.6). Suspending intracity public transport, closing entertainment venues, and banning public gatherings were associated with reductions in case incidence. The national emergency response appears to have delayed the growth and limited the size of the COVID-19 epidemic in China, averting hundreds of thousands of cases by 19 February (day 50). In an attempt to prevent further dispersal of COVID-19 from its source, all transport was prohibited in and out of Wuhan city from 10:00 a.m. on 23 January 2020, followed by the whole of Hubei Province a day later. In terms of the population covered, this appears to be the largest attempted cordon sanitaire in human history.On 23 January, China also raised its national public health response to the highest state of emergency: Level 1 of 4 levels of severity in the Chinese Emergency System, defined as an “extremely serious incident” . In The dispersal of COVID-19 from Wuhan was rapid . A totalr) = 0.98, P < 0.01; excluding Hubei, r = 0.69, P < 0.01]  compared with cities that started control later , with a statistically significant difference between the two groups . A separate analysis using regression models shows that among specific control measures, cities that suspended intracity public transport and/or closed entertainment venues and banned public gatherings, and did so sooner, had fewer cases during the first week of their outbreaks (Cities that implemented a Level 1 response (any combination of control measures) (figs. S2 and S4) preemptively, before discovering any COVID-19 cases, reported 33.3%  fewer laboratory-confirmed cases during the first week of their outbreaks  was 3.15 before the implementation of the emergency response on 23 January (C1R0) in three groups of provinces, depending on the rate of implementation in each group (C2R0), far below the replacement rate (≪1) and consistent with the rapid decline in incidence (The reported daily incidence of confirmed cases peaked in Hubei province (including Wuhan) on 4 February  and in all other provinces on 31 January  (fig. S1). The low level of peak incidence per capita, the early timing of the peak, and the subsequent decline in daily case reports suggest that transmission control measures were associated not only with a delay in the growth of the epidemic but also with a marked reduction in the number of cases. By fitting an epidemic model to the time series of cases reported in each province (fig. S3), we estimate that the (basic) case reproduction number  measures initiated during the Chinese Spring Festival holiday, including the unprecedented Wuhan city travel ban and the Level 1 national emergency response, were strongly associated with, although not necessarily the cause of, a delay in epidemic growth and a reduction in case numbers during the first 50 days of the COVID-19 epidemic in China.The number of people who have developed COVID-19 during this epidemic, and therefore the number of people who were protected by control measures, is not known precisely, given that cases were almost certainly underreported. However, in view of the small fraction of people known to have been infected by 19 February , it is unlikely that the spread of infection was halted and epidemic growth reversed because the supply of susceptible people had been exhausted. This implies that a large fraction of the Chinese population remains at risk of COVID-19; control measures may need to be reinstated, in some form, if there is a resurgence of transmission. Further investigations are needed to verify that proposition, and population surveys of infection are needed to reveal the true number of people who have been exposed to this novel coronavirus.We could not investigate the impact of all elements of the national emergency response because many were introduced simultaneously across China. However, this analysis shows that suspending intracity public transport, closing entertainment venues, and banning public gatherings, which were introduced at different times in different places, were associated with the overall containment of the epidemic. However, other factors are likely to have contributed to control, especially the isolation of suspected and confirmed patients and their contact, and it is not yet clear which parts of the national emergency response were most effective. We did not find evidence that prohibiting travel between cities or provinces reduced the numbers of COVID-19 cases outside Wuhan and Hubei, perhaps because such travel bans were implemented as a response to, rather than in anticipation of, the arrival of COVID-19.This study has drawn inferences not from controlled experiments but from statistical and mathematical analyses of the temporal and spatial variation in case reports, human mobility, and transmission control measures. With that caveat, control measures were strongly associated with the containment of COVID-19, potentially averting hundreds of thousands of cases by 19 February, day 50 of the epidemic. Whether the means and the outcomes of control can be replicated outside China and which of the interventions are most effective are now under intense investigation as the virus continues to spread worldwide.


In vitro viability results, scanning electron and fluorescence microscope imaging analysis show that the HS-5 preferentially grows on regular microstructured surfaces, while the THP-1 on irregular microstructured ones.Polycaprolactone (PCL) is a biocompatible and biodegradable polymer widely used for the realization of 3D scaffold for tissue engineering applications. The hot embossing technique (HE) allows the obtainment of PCL scaffolds with a regular array of micro pillars on their surface. The main drawback affecting this kind of micro fabrication process is that such structural superficial details can be damaged when detaching the replica from the mold. Therefore, the present study has focused on the optimization of the HE processes through the development of an analytical model for the prediction of the demolding force as a function of temperature. This model allowed calculating the minimum demolding force to obtain regular micropillars without defects. We demonstrated that the results obtained by the analytical model agree with the experimental data. To address the importance of controlling accurately the fabricated microstructures, we seeded on the PCL scaffolds human stromal cell line (HS-5) and monocytic leukemia cell line (THP-1) to evaluate how the presence of regular or deformed pillars affect cells viability.  In tissue engineering field, the scaffolds represent a three-dimensional (3D) structure able to assist and tune cell/tissue morphogenesis . These cell type represents a minor subpopulation in solid tumors able to drive tumor progression and metastatization. CSCs are difficult to culture in 2D systems and in vitro and in vivo anti-tumor activity applications   is an efin vitro experiments were performed by growing monocytes (THP-1) and mesenchymal stem cells (HS-5) on both deformed and regular PCL microstructures to demonstrate how the micro texture of scaffolds could be crucial for regenerative medicine and tissue-engineering applications.The purpose of this work is to understand which parameters affect the micro texture of HE structures, and how imperfections on microtextured scaffolds could affect cells’ viability. The HE process was examined through the implementation of an analytical model able to predict the demolding force. By using this kind of fabrication technique, the replica microstructures stuck on the silicon mold and a demolding force should be applied to detach the replica from the mold. If this force produces stretching pressures on the just realized polymer microstructures, these will be deformed. In this work, we investigated the parameters related to the demolding force and the possibility to minimize them. In particular, the developed model was able to predict the demolding force as a function of the temperature of the mold and the replica. It was validated by using an experimental set-up to allow the measure of the demolding force of the embossed PCL microstructures. Finally, preliminary Dem) was composed of three components: adhesion (FAd), deformation (FDef), and friction forces (FAttr)  .It is known that the pressure generated during the embossing creates adhesion between the horizontal surfaces at the base of the mold microcavities and the tips of the micropillars of the replica.sad):The resulting adhesion force was calculated as a function of the adhesion surface (A) and adhesion strength  known; rc is the hydraulic radius of the cavity, calculated as half of the diameter (d) of the circular micropillars. The second part of the equation concerns the deformation of the polymer and the third is the contact area between polymer and the side walls of the cavity (Aw). The frictional force is the product of the coefficient of friction (μ), the contact stress on the side walls of the cavities (σd) and the area of the walls themselves (Aw):The first part of the equation (6) considers the uneven profile of the cavities. The etching process yields a sinusoidal profile with a peak-to-valley distance (δ) and a peak-to-peak distance  is released, p is considered constant in each point and is equal to the force applied by the thermal press divided by the surface A. In this condition, the friction, the adhesion and pressure counteract the shrinkage. Then, σbd can be calculated asσT) is a result of the polymer shrinkage during cooling:The thermal stress  replicas was performed by using a Lab press (Presstronic P/O/Weber). The molds used for the replica were composed of two parts . The firAfter the HE process, the mold was detached from the replica by using an experimental set-up by which we measured the demolding force and the temperature of the replica during the process . In addiThe detached PCL replica from the mold was characterized by using a Dual Beam (SEM- FIB) FEI Nova 600 NanoLab system to measure the pillars deformation. Longitudinal and transverse deformations of scaffolds detached from the mold at different temperatures  were measured. For the SEM acquisition, a beam energy of 30 kV and an electron current of 98 pA were used.2 atmosphere. When the cells reached confluence, were washed in phosphate buffered saline solution (PBS), treated with trypsin-EDTA 0.25% , centrifuged , re-suspended in fresh medium and seeded on the scaffold by using the “drop seeding” method that allow to cells to get better contact with the surface of the devices in the early stages of growth (The fabricated scaffolds were used to carry out studies of cell growth using the THP-1 and the HS-5-GFP (transduced to express the green fluorescent protein). Control cells were seeded in standard Sigma polystyrene cell culture plate. All the scaffolds, used for the study, were first sterilized by keeping them for 2 h in 1 ml of a solution composed of 70% of ethanol  and at 30% of sterile DI water. Then, they were dried under a hood, washed twice in PBS and dried again. THP-1 is a human monocytic cell line derived from an acute monocytic leukemia patient and HS-5 is a human bone marrow stromal cell line. The used cells were provided from the laboratory of Medical Oncology at the University Magna Graecia of Catanzaro. The THP-1 cell line was cultured in RPMI-1640 medium with 1X L-glutamine  and supplemented with PMA  which stimulates the differentiation of monocytes into macrophages, 10% fetal bovine serum . All cell cultures were maintained in incubator at 37°C in a 5% COf growth .Cell viability was performed by seeding the cells on fragments of four different types of scaffolds: 3D micro-structured devices with regular micropillars, 3D micro-structured scaffolds with irregular deformed pillars, 2-dimensional (2D) rough devices and 2D flat devices. Previous studies  showed t2). The proliferation rate was performed with the cell counting kit CCK 8 (Dojindo) in combination with 450 nm absorbance measurements on a plate reader. Data are expressed as mean ± standard error, the number of samples used in each experiment is provided in figure legend and statistical comparison between two measurements were made using t-tests.The different types of supports were all made performing the HE process and using different demolding temperatures. While, to fabricate the flat devices, a flat glass support replaced the silicon support and, after the demolding step, some of these scaffolds were sanded to get specific roughness values. The roughness of these scaffolds was measured with a profilometer (Dektak) and it is 945 nm. Each scaffold fragment was placed in a multiwell plate and for each cell line, three experimental tests were carried out. On each scaffold they were seeded about 2000 cells for THP-1 and 5000 cells for HS-5. The cultures were kept for 48 h in an incubator  and the images were then processed using the ImageJ software.SEM images of both cell lines, cultured for 48 h, were acquired. To verify cell adhesion on the four devices, each scaffold was loaded with 50000 cells and, after 48 h of incubation, placed on a glass slide and washed twice in PBS. The samples were washed with PBS and then fixed using a solution of 1.2% glutaraldehyde (G5882 Sigma Aldrich) in 0.1 M sodium cacodylate (C0250 Sigma Aldrich), for 1 h at room temperature. After the fixing, the cell supports were washed with sodium cacodylate buffer solution , subsequently these were treated for 1 h with a solution of 1% osmium tetroxide  in 0.1 M sodium cacodylate. Subsequently, three washes in distilled water were performed, followed by washes of 5 min each in increasing concentrations of ethanol . After that, the cell supports were washed two times for 15 min each in a 100% ethanol solution, followed by gradual replacement of ethanol with the hexamethyl-disilazane . To perform this step, the samples were first immersed in a solution of ethanol/HMDS at a ratio of 3:1, for 10 min; then at a ratio of 1:1 for 10 min; and at a ratio of 1:3 for 30 min. Finally, the samples were left under a hood to let the solution evaporate completely and sputtered with gold for the SEM visualization. The scaffolds were observed in a Dual Beam (SEM- FIB) FEI Nova 600 NanoLab system. For the SEM acquisition, a beam energy of 30 kV and an electron current of 98 pA were used.p < 0.05), with a slight margin of error, probably due to the approximations made in the model. If the demolding temperature decreases, the demolding force decreases as well in the range of temperature considered, for the specific used material, and microstructures replicated. The model allowed calculating the proper value of demolding temperature by which we obtained a value of demolding force correspondent to tensions lower than the material strength. For such value of temperature, it was possible to obtain regular and not deformed PCL microstructures.The comparison between the experimentally measured demolding force and that resulting from the calculation with the analytical model are shown in the left panel of The scaffolds obtained by the HE process were observed by means of an optical  and a scThe viability data of cells seeded on PCL scaffolds  after 48 h of incubation are plotted in 2 scaffold. Here, considering GFP fluorescence (green) and nuclear DAPI signal (blue) it is possible to note that cells prefer grow on the uniform and regular 3D structures  while they are less viable or dead (white asterisks) in areas with irregular pillars . Unlike 5–2 × 106 cells/ml, if they get denser they may slow down or stop dividing and may clump or have an irregular or blubbing appearance  showed to grow on regular microstrucured surfaces, while the lymphocyte cell lines (THP-1) showed a preference toward irregular microstructured surfaces.The possibility of building complex tissues, All datasets generated for this study are included in the article/TL and GP: conceptualization. EL and CB: methodology. BS, MG, MCu, and CC: validation. PC and MCo: formal analysis. ED and BS: investigation. MCo: data curation. GP: writing and original draft preparation. ED, CC, SP, GP, and TL: writing, review, and editing. GP, AF, ED, EL, PTs, and PTg: supervision. All authors provided approval for the publication of the content.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Recently, concerns have been rising about the impact of increasing the depletion of natural resources and the relevant generation of construction and demolition waste, on the environment and economy. Therefore, several efforts have been made to promote sustainable efficiency in the construction industry and the use of recycled aggregates derived from concrete debris for new concrete mixtures  is one of the most promising solutions. Unfortunately, there are still gaps in knowledge regarding the durability performances of RAC. In this study, we investigate durability of structural RAC subjected to wet-dry cycles. We analyze the results of an experimental campaign aimed at evaluating the degradation process induced by wetting and drying cycles on the key physical and mechanical properties of normal- and high-strength concrete, produced with coarse recycled concrete aggregates (RCAs) of different sizes and origins. On the basis of the results we propose a degradation law for wetting and drying cycles, which explicitly makes a possible correlation between the initial concrete porosity, directly related to the specific properties of the RCAs and the resulting level of damage obtained in RAC samples. The construction sector is one of the most environmentally impactful industrial activities in the world, as it requires a significant amount of natural resources and energy and generates an enormous volume of waste ,2. ThereIn a historical context, interest in RCAs began in the 1970s ,7. SinceThe main difference between RCA and ordinary aggregate is the presence of an aged mortar, the so-called attached mortar (AM), on the recycled aggregates . This diConsequently, recycled aggregate concrete (RAC) could also present higher absorption and higher porosity in relation to conventional concrete produced with natural aggregates. This makes durability a problem, since aggressive agents find it easier to enter concrete ,14,15,16The investigation of durability is certainly a very important aspect for the development and application of a new material. Durability can be defined as the ability of concrete to resist the actions of external agents without compromising the performance for which it was designed during its useful life. Therefore, the durability of isolated concrete and concrete structures depends on the proper selection of materials, mix-design, production and curing, and also depends on the interactions of the final material with its exposed environment . TemperaWu et al.  explaineHowever, there is no single parameter to determine the durability of concrete structures, especially when subjected to the actions of potentially aggressive environments. It is possible to take into account durability issues through mechanical properties, such as compressive strength and elastic modulus. Most of the structures in service experience wetting and drying cycles and temperature variation and, as a result, concrete structures are rarely completely dry, fully saturated, or in constant thermal conditions . To studThe influence of wetting and drying cycles on concrete was analyzed by numerical resolutions in a study by Li et al. , and it In the case of RCAs, according to Diagne et al. , the higObserving the absorption results of RAC samples, Oliveira et al.  reportedQi et al.  evaluateAlthough studies on the durability of concrete produced with RCAs have already been published, the main topics involving RAC subjected to wetting and drying cycles are mainly cases of exposure to sulfates and chlorides, and therefore little information on the exposure of recycled concrete to isolated wet and dry cycles can be found in the literature. In this context, there is still a knowledge gap about the influence of rain effects of typical tropical zones on the durability of RAC, in particular, in relation to processes of alternating cycles of wetting and drying together with temperature variations.The present study reports on an experimental investigation aimed at analyzing the influence of the use of RCAs of different origins and sizes on wet-dry durability of normal (35 MPa) and high strength (60 MPa) concrete. Through a scientific mix-design technique for RACs previously developed by the authors , which c3, 28-day compressive strength of 40.6 MPa, and actual packing density of 0.67.The cement used, in this study, was “high initial strength Portland cement” , labeled CPV-ARI, according to the National Brazilian Standard (NBR) 16697 , with th3, solid concentration content of 35%, and saturation dosage of 1.5% (solids of SP based on the cement weight).The superplasticizer used in all mixes for workability control was a polycarboxilate superplasticizer, labeled “MC Powerflow 1180” , with the following main properties: specific gravity of 1070 kg/m3 [Natural quartz sand was used in this study as natural fine aggregate (NFA), with the following characterization: maximum grain size of 4.75 mm, specific gravity of 2447 kg/m3 , water a3 , and act3 .Granite type stones were used as the natural coarse aggregate (NCA) in the following two types of sizes: the first type was “NCA_C0” with a nominal diameter ranging from 4.75 to 9.5 mm (size named by “coarse aggregate 0”); the second type was “NCA_C1” with a nominal diameter ranging from 9.5 to 19 mm (size named by “coarse aggregate 1”).The recycled concrete aggregates (RCAs), employed herein, were obtained from the following two different sources : concretIn order to characterize both natural and recycled coarse aggregates, several tests were carried out and the main results are shown in The reported data showed that the properties of the aggregates depended mainly on the characteristics of the original concretes (L-waste and D-waste). Although the RCA particles obtained from the two sources presented similar properties for the fraction C0, for the class C1 this difference was more evident. In fact, since the L-waste had a maximum dimension of the original aggregate of 9.5 mm, generating aggregates of 19 mm led to greater amounts of attached mortar to the grains . As expeIn this study, a scientific mix design for the produced concrete mixtures was performed using the so-called compressive packing model (CPM) . This meThe optimization method was performed by maximum granular compactness of concrete, using the above-presented data of the actual packing density of the raw materials. The recycled materials were added to the mixture in a dry condition and the singular high absorption of the RCAs was considered during the definition of the mix proportioning. The absorption value of 50% of the total absorption obtained experimentally was used, since this value was previously shown  water did not take into account the absorption water of the aggregates. The aggregate proportion was fixed at 50 for sand, 25 for class C0, and 25% for class C1 in relation to the overall volume of aggregates (fine and coarse). For each class, five concrete mixtures were designed, varying the coarse aggregate, as follows:(1)Reference with only natural aggregates (NCA_C0 and NCA_C1);(2)100% RCA_L_C0 in fraction C0 (RCA_L_C0 and NCA_C1);(3)100% of RCA_L_C1 in fraction C1 (NCA_C0 and RCA_L_C1);(4)100% RCA_D_C0 in fraction C0 (RCA_D_C0 and NCA_C1);(5)100% of RCA_D_C1 in fraction C1 (NCA_C0 and RCA_D_C1).The concrete mixtures were developed for the following two classes of compressive strength: normal strength with 35 MPa and high performance with 60 MPa. All mixtures were produced with 30% effective paste, and “effective paste” was the volume of free paste in the mixture  in relation to the total volume of concrete, in which the mixing  of each mixture (AM) and the new mortar volume  given by the sum of sand, cement, water, and superplasticizer:i represents the weight used for each fraction  developed and improved for cases of RAC by Tam et al.  and Tam The mixing procedure was performed in the following order: Initially, homogenization of all coarse and fine aggregates by mixing for 1 min; followed by the addition of half the total amount of water and mixing for 1 min for better absorption by RCAs; then, the cement was added and mixed for 1 min with the aggregates; finally, the rest of the water  and the superplasticizer were added and mixed for 8 min (superplasticizer action time), concluding the concrete mixing process. The casting of the concrete was carried out in two layers, followed by mechanical compaction for 30 s. The samples were demolded after 24 h and the cure was carried out in a wet chamber with relative humidity of 100% and temperature of 21 °C.The degradation process by wetting and drying cycles started at the age of 28 days. Cylindrical samples, 75 mm in diameter and 150 mm in height, were used. Initially, the samples were saturated in water for 48 h, and then the cycles started. The durability test of concrete samples subjected to accelerated wetting and drying cycles was based on NBR 13554  and ASTMAfter the degradation process, the concrete samples were characterized by determining their relevant mechanical properties, such as compressive strength, elastic modulus, tensile strength, and mass loss. The weighing of the samples was carried out in order to follow the mass variation. The weight loss test was performed on three samples of each mixture for each level of wet-dry degradation. The samples were weighed in an oven-dried condition (after mass consistency) before the degradation process. After 25 cycles, the same samples were weighed again in an oven-dried condition. The same procedure was performed for the samples of 50 wet-dry cycles. The calculated mass loss is the average of the percentage differences between the initial mass and the mass after degradation of each sample. It is worth mentioning that, for all mixtures, non-degraded samples were kept protected in a wet chamber (21 °C temperature and 100% humidity) and tested as reference at the same age as the samples degraded by 25 and 50 cycles.The characterization of all mixtures was carried out through the tests detailed in This section presents the results of the experimental characterization of the ten concrete mixtures produced in this study. Many authors have stated that the presence of RCA caused a reduction in the workability of concrete, caused mainly by the higher absorption capacity of these materials ,47. Howeopen), higher void index, and lower specific gravity as compared with natural concrete. These results are consistent with the conclusions that have been obtained in several studies available in the literature [The results of physical properties show that recycled concrete has higher total water absorption , the higher the open porosity of concrete (wopen) . This is explained by the fact that the open porosity of concrete also depends on the raw materials employed within the mixture, and, in the specific case of RACs, it is influenced by the presence of the porous attached mortar inside or around the RCAs particles. Thus, the strong connection between these two parameters, that is, the trend of the increasing curves in 1, k2, and VM,0 represent numerical parameters calibrated for both normal and high strength classes:1 was calibrated with 7.3 for C35 and 4.2 for C60, k2 with 30.0 for both classes, and VM,0 with 45.0 for C35 and 55.0 for C60.Meanwhile, from the percentage of total mortar volume of each mixture , the following relationship is shown in c,28) prove that all recycled concretes obtained similar values to natural concretes for the studied classes, according to the initial proposal of this study, that is, the premise to understand the influence of the presence of RCAs in concretes of the same strength subjected to degradation by repetitive wet-dry cycles. In fact, the maximum difference between the expected compressive strength and the experimentally obtained compressive strength was 4% for both normal strength and high-performance mixtures as compared with the predicted values of 35 and 60 MPa. These results reinforce the excellent capacity of the specific mix design methodology described (based on [The results obtained from compressive strength at 28 days (fbased on ) to predc,28), as compared with the values obtained for concretes with 100% NCA, there was a maximum variation of 1.8% decrease to 3.7% increase in RACs of C35 , and a maximum variation of 1.3% to 6.5% increase in RACs of C60 . Finally, the results of splitting tensile strength at 28 days  of C35 showed a variation of 3% decrease to a 7% increase in RACs in relation to the natural mixture . The results of C60 expressed a maximum increase of 12% (from both C60-L-C1 and C60-D-C0) as compared with the result of the natural concrete. Therefore, in the high strength class, all RACs obtained a better result than the natural mixture for these two main mechanical properties.For elastic modulus at 28 days  the more porous structure ” and “REF(WD50)”, according to the samples to which they serve as reference.The physical and mechanical degradation of concrete subjected to wetting and drying cycles can be associated with the different hygrometric profile which is generated within the concrete element. As a matter of principle, during the wetting phase, the open pores are filled by water during drying, depending on the concrete size and the duration of each cycle, only the cortical area of the element is able to be fully dried. This phenomenon promotes the shrinkage of the external layers while the internal core  locks the volume change . This reThe mass loss results for natural aggregate concrete (NAC) and RAC after 25 and 50 wet-dry cycles are shown in In both strength classes, the mass loss rates do not show significant differences with an increased number of cycles. In general, the change in the mass of concrete  occurs due to mortar fragmentation during wet-dry cycles. In the drying stage, a pressure arises due to the shrinkage of the concrete by the evaporation of the internal water, thus, the concrete becomes weaker and fragmented with the creation and evolution of micro cracks. This aspect is also confirmed by the fact that during the wet-dry tests there were small components of the concrete in the water tank. Consequently, the mass of the samples decreases. With the repetition of the cycles, this process repeats, causing more and more reductions in the mass. However, as shown in the results, this occurs at a (approximately) constant rate of change.Finally, in both C35 and C60, mixtures produced with the RCA of class size C1 from laboratory waste (C35-L-C1 and C60-L-C1) stand out for higher mass loss as compared with other mixtures, while natural mixtures (C35-NAT and C60-NAT) have the lowest mass loss results. Therefore, with the joint action of wetting and drying processes, the results show that these interactions modify the properties and the microstructure of RACs more clearly than natural concretes. This fact can be explained as follows: Natural concrete presents only the new mortar in concrete undergoing this aggressive process, while RACs undergo these processes in the new mortar, and also in the old mortar attached to the grains of RCAs, consequently RACs suffer further damage.The typical stress-strain responses obtained in compressive strength tests of normal and high strength classes in the wet-dry degradation study of RACs are presented in Samples of the typical rupture that occurred in each class in the compressive tests after wetting-drying degradation are shown in This variation in the cracking pattern of the samples after wet-dry cycles can be explained by the fragmentation of the mortar and the increase in the volume of pores, previously described. This modification in the internal structure of the concrete, caused by the degradation process, can result in a weaker “line” in the concrete , which becomes the “path” of the cracking process during the application of stress in the compressive test.The results confirmed that all samples from both classes suffered a loss in compressive strength when subjected to repetitive wetting and drying cycles. The normal strength class reached degradation percentage decreases from 6.9% to 7.8% at the end of 25 cycles and from 14.5% to 20% after 50 cycles a. For loThe percentage decrease in compressive strength was higher for the normal strength class than for the high-performance class for both two levels of cycles. This fact was expected due to the previously explained mass loss, i.e., it was related to the higher porosity (associated with greater water penetration in the wetting phase) and the lower resistance (associated with the lower capacity to withstand the stresses generated by the shrinkage in the drying phase) of C35. For C60, the compressive strength percentage decreases ranged from 3.2% to 4.6% after 25 wet-dry cycles and from 9.7% to 13.5% after 50 wet-dry cycles a. The saFor C35, the values of elastic modulus decrease of samples degraded by 25 wet-dry cycles ranged from 3.8% to 6.7%, and, after 50 cycles, the values increased for 13.8% to 18.1% as compared with control samples b. For thFor the normal strength class, tensile strength values showed a decrease of 6.6% to 9.0% for samples degraded by 25 cycles, and a decrease of 17.4% to 20.3% for RACs, and 13.5% for natural concrete after 50 cycles c. Therefopen), and the variations obtained in the main mechanical properties of concrete after wet-dry degradation, that is, compressive strength (fc), elasticity modulus (Ec), and tensile strength (ft). The three proposed relationships are described in the following equations:With the experimental results obtained for the properties discussed above, a generalized degradation law is proposed for RAC subjected to wet-dry cycles. The law represents the relationships between an initial property of the intact concrete, in this case, the concrete open porosity , there would be no degradation of concrete, since the water would not flow within the concrete structure and it would not cause damage to the material.In all graphs, the following can be observed: On the one hand, the points above are the results for 25 wet-dry cycles (WD25) of the ten concrete mixtures and they indicate that the influence of this degradation level is less relevant; on the other hand, the observation of the lower points, which represent 50 wet-dry cycles (WD50), highlights two very evident trend lines, one for each strength class, where the right trend line represents C35 (higher porosity) and the left trend line represents C60 (lower porosity). Thus, two numbers (one for each resistance class) were calibrated for parameter a for compressive strength a, followIn the three graphics, in addition, the points more to the left of each of the four groups of points refer to the mixtures of natural concrete (C35-NAT and C60-NAT), since these concretes have lower porosity in each class, because they have a lower total mortar volume than RACs  there is a greater variation in durability capacity (y-axis) than for the normal strength class. This confirms that the presence of a more porous aggregate causes a greater impact on the wet-dry resistance of C60. In other words, as the porosity of the aggregate is directly related to the attached mortar content, the durability of C60 is more sensitive to the presence of AM than C35.The shape of the curves shows that the graph of the high-performance class is more vertical for all three properties, which indicates that for a smaller variation in concrete porosity  showed decreases in compressive strength, elastic modulus, tensile strength, and mass value, when evaluating the results obtained for samples degraded by 25 and 50 wet-dry cycles.The percentage decreases in the properties studied were greater for the normal strength class than for the high-performance class. This evidence is associated with the higher porosity of C35 mixtures.For higher number of cycles, it is evident that the capacity of the natural mixture to resist degradation by wetting-drying cycles is greater than RACs, for both C35 and C60. It is believed that this occurs because the natural concrete presents only the fragmentation of the new mortar when subjected to degradation, while RACs undergo this process in both the new mortar and the mortar attached to the RCA particles, suffering, therefore, greater damages.A wet-dry degradation law for RAC is proposed in this study for the key mechanical properties after wetting and drying degradation  as a function of the initial open porosity of the concrete, which is also directly related to the total mortar volume of concrete.Finally, as the RAC resistance to wet-dry degradation is governed by the initial open porosity of the concrete, it would be possible to use RAC in structures subjected to environmental conditions similar to the ones analyzed herein through the control of this physical property in the mix design stage.This study reports the results of an experimental study on the physical and mechanical performance of structural concrete made with either natural or recycled aggregates and subjected to degradation processes similar to environmental conditions of temperature and humidity variation. Specifically, it summarizes the results of an experimental investigation aimed at analyzing one of the aspects related to the durability of normal and high strength concretes produced with different types of RCAs, after being subjected to different levels of wetting-drying cycles. Particularly, the following considerations can be highlighted:


On this basis, wear marks and elements on the wear surfaces after friction were analyzed, and then the anti-wear and mechanism effects of WS2 of different forms in the lubrication process were discussed. Meanwhile, the Stribeck curve was used to study the lubrication state of the lubricating oil in the friction process. It was revealed that the COF of lubricating oil containing lamellar WS2 decreased by 29.35% at optimum condition and the minimum COF was concentrated at around 100 N. The COF of lubricating oil containing spherical WS2 decreased by 30.24% and the minimum coefficient was concentrated at 120 N. The extreme pressure property of spherical WS2 was better than that of lamellar WS2, and the wear resistance of spherical WS2 was more stable when the load was over 80 N. The different morphology of WS2 additives can play anti-wear and anti-friction roles within a wide range of sliding speeds.In the present work, the relationship curve of the coefficient of friction (COF) with varying loads of different morphology WS It is one of the most popular new functional materials in China and abroad . WS2 nanttention ,4.2 as a lubricant additive. It was found that WS2 could maintain excellent lubrication performance in the range of −273–425 °C, which was significantly better than traditional lubricating oil [2 as a lubricant additive can significantly improve the anti-wear and anti-friction properties of base oils [2 nanomaterials as additives in paraffin oil by Universal Micro-Tribotester (UMT). The experimental results show that the addition of WS2 nanomaterials can greatly improve the tribological properties of paraffin oil. When the concentration of WS2 nanomaterial additive is 0.5 wt%, the tribological performance of lubricating oil is the best [2 nanoparticles by adding WS2 nanoparticles into poly alpha olefin (PAO) base oil containing a zinc dialkyl dithiophosphate (ZDDP) additive, and found that the presence of WS2 nanoparticles can improve friction and wear under boundary lubrication conditions, and friction film containing WS2 is formed on the friction surface [In recent years, researchers have tested the lubricating properties of WSting oil ,7,8,9. Nase oils ,11,12,13the best . Aldana  surface .2 is related to the contact force. With the same nano-WS2 content, WS2 can show good lubrication performance within a certain force range, and the higher the force, the better the lubrication performance. However, once the contact force is exceeded, the effect of WS2 as a lubricant addition is greatly weakened [At the same time, the lubrication performance of WSweakened .2 particles in the medium have different effects on anti-wear and anti-friction performance [2 was added to the lubricating oil at a mass fraction of 0.01%, it showed excellent anti-wear and anti-friction performance, and the tribological performance of layered nanometer tungsten disulfide was better than that of lamellar tungsten disulfide [It has been found that the size and crystal structure of WSformance ,21,22,23isulfide .2 are usually limited to the lubricating effect as a lubricant additive, and few studies have been conducted on the tribological properties of micro-nano lubricant additives in different forms under different loads and speeds. According to the literature, the tribological properties of different forms of micro-nano additives are different under different loads and speeds. For example, the performance of MoS2 in different forms as a lubricant additive is quite different. Under the conditions of low loads and low speeds, nano-MoS2 can improve the anti-friction and anti-wear performance of base oil, while micro-MoS2 can increase the wear. Under high loads and high speeds, micro-MoS2 exhibits certain anti-friction and anti-wear ability, while nano-MoS2 easily leads to lubrication failure [However, studies on WS failure .2 with different morphology under different loads and speeds were studied, and the surface morphology and element composition of wear scars were also analyzed. At the same time, the anti-wear and anti-friction properties of two kinds of WS2 as lubricating additives were discussed, and the mechanism was also investigated.In this paper, the tribological properties of WS2, lamellar WS2 and spherical WS2, were selected as lubricating additives in this experiment. These WS2 powders were characterized through X-ray diffraction (XRD) , Scanning Electron Microscopy (SEM) , and Transmission Electron Microscopy (TEM)  to obtain their physical and chemical properties, such as surface morphology, particle size, and chemical composition. The XRD patterns of two kinds WS2 are shown in 2 is shown in 2 is a layered structure, its thickness is about 2 μm and the lateral dimension is between 5–10 μm. The TEM diagram of spherical WS2 is shown in 2 is 90 nm. In this experiment, low-viscosity PAO6 was selected as the base oil. Lamellar WS2 and spherical WS2 were added to base oil PAO6 at a concentration of 1.5 wt% to prepare sample oil #1 and #2. The sample oil was dispersed by mechanical stirring and strong ultrasonic compounding for 1 h, and finally the sample oil with WS2 evenly dispersed was obtained.Two forms of WS2 in different forms as lubricant additives under different sliding speeds and loads were tested using a UMT-2 tribometer. In the experiment, the kinematic form was reciprocating linear motion, and the single-stroke of the scratch was set to 15 mm. The contact form of friction pair was ball-on-disc. The upper sample was a steel ball processed from high-carbon chromium bearing steel GCr15 with a diameter of 10 mm, a hardness of HRC58–65, and a surface roughness of Ra ≤ 0.02 μm. The lower sample was also a steel disc processed by high-carbon chromium bearing steel GCr15. The diameter of the sample was 25 mm, the thickness was 10 mm, the hardness was HRC60–63, and the surface roughness was Ra ≤ 0.05 μm. Before the experiment, the samples were cleaned with ultrasonic cleaner for 15 min and dried. Six sliding speeds (10–35 mm/s) and six loads (20–120 N) were set for the tests. The specific set values are shown in The tribological properties of WSAfter the friction experiment, the steel disks were cleaned with acetone for 10 min and dried. The depth and width of the wear scars of the disks were measured using a SV-3000 Ultra-Depth Three-Dimensional Microscope . The wear scars on the steel disks were then analyzed with SEM and an electron-probe micro-analyzer (EPMA) to determine the anti-wear properties of lubricating oil. The anti-wear properties of additives can be determined according to the size of the wear scar, wear depth, and material loss.2 with increased loads at given sliding speeds is shown in The average COF curve of the sample oil with different forms of WSIt can be seen from As can be seen from the Stribeck curve in 2 particles is better than that containing lamellar WS2 in this load range.In addition to the change in the lubrication state caused by the thinning of the oil film, it may also be because the frictional heat inside the lubricant causes the bearing capacity of the lubrication film not to increase infinitely. When it increases to the maximum, the oil film will break. However, the lubrication state of sample oil #2 did not change significantly within the same load range. This indicates that the bearing capacity of sample oil containing spherical WS2 transitioned from a fluid lubrication state to a mixed lubrication state, while the lubrication state of spherical WS2 remained basically in a fluid lubrication state and did not changed. According to the experimental result, it can be found that both forms of WS2 as additives can affect the anti-friction performance and bearing capacity of lubricating oil, and the spherical WS2 shows more outstanding anti-friction performance and bearing capacity.In conclusion, within the range of experimental load, the lubrication state of lamellar WSThe three-dimensional morphology and wear trace diagram of wear scars on steel disks under four different loads  are shown in −4 mm3⋅N−1 m−1, and the minimum wear rate of sample oil #2 was 10.9 × 10−4 mm3⋅N−1 m−1, indicating that the abrasion resistance of the two kinds WS2 was similar at this time. However, when it exceeded 80 N, the wear rate of sample oil #2 increased slowly compared with that of sample oil #1, which indicates that the abrasion resistance of spherical WS2 was more stable under large loads.From 2 can improve the anti-wear performance of lubricating oil within a certain range of the load. When the load exceeded a certain value, the anti-wear performance was limited and the anti-wear ability was weakened.In order to understand the wear form of the wear scars on the surface of the steel ball, the wear scars were characterized. The composition and distribution of elements on the surface of wear marks were analyzed using EPMA to grasp the chemical changes on the worn surface. A wear scar of sample oil #1 with a 100 N load and 15 mm/s sliding speed, and a wear scar of sample oil #2 with a 120 N load and 20 mm/s sliding speed were used as examples. The results of their characterization are shown in 2O3 or FeO, and the worn surface appeared on FeO and Fe2O3 film. The XPS data indicate that a frictional chemical reaction occurs on the surface of the wear steel during the friction process, thus forming the boundary lubrication film composed of WO3, Fe2O3, and FeO. Therefore, it can be speculated that the friction surface is most likely to form a dense chemical reaction film containing Fe2O3 and FeO through chemical reactions [2 on the friction surface.It can be found from the distribution diagram of each element in eactions ,33,34,352O3, FeO, and WO3 is formed on the friction surface. In addition, the distribution diagram of elements W and S indicates that some nano-WS2 remained in the grooves on both sides and cracks of the wear scars.From the distribution diagram of each element in 2O3, FeO, and WO3 on the surface. WS2 will be deposited on the friction surface to form a physical adsorption film filled in time if there is damage on the surface. Under the combined action of physical deposition film and chemical reaction film, the anti-wear and anti-friction properties of the sample oil were improved. In addition, the residue amount of spherical WS2 was larger than that of lamellar WS2, which may be because the small size of WS2 makes it more prone to deposit in furrows and grooves, and timely fill and repair when surface damage occurs.It can be inferred from the elemental analysis of the wear surface that during the lubrication process, the sample oil is likely to form a dense layer of chemical reaction film containing Fe2 as an additive is shown in 2 dispersed in the lubricating oil is partially deposited on the surface of the friction pair to form a physical deposition film under the action of frictional shear and normal load. Due to the crystal structure of lamellar WS2, the layer to layer is connected by weak van der Waals force, and the surface is easy to slip, which leads to the reduction of COF. Moreover, the deposition film prevents the direct contact of the friction surface; therefore, the wear is reduced, and the anti-wear performance of the lubricating oil is improved.The lubrication mechanism of lamellar WS2 particles, the lubricating mechanism is roughly shown in 2 dispersed in the lubricating oil will slowly be deposited on the surface of the friction pair to form a certain thickness of physical deposited film during the process of friction, preventing direct contact of the friction pair surface. The spherical structure of WS2 can be regarded as a ‘small ball’, which can play a similar role as a ‘micro-bearing’ on the friction surface, changing the sliding friction between the surfaces into rolling friction, thereby reducing the COF. With the action of frictional shear force and normal load, the spherical WS2 will be deformed by squeezing and even peeling off. The exfoliated WS2 molecular layer will remain on the pits and peak tips of the friction surface, covering the contact point, thereby reducing the direct contact of friction pair, and then reducing friction and wear. In addition, the peaks of some micro-convex bodies on the surface of the friction pair will penetrate the lubricating oil film and directly contact, collide, and break, accompanied by abrasive particles and new wear scars. With the generation of new wear scars, spherical WS2 with small sizes will fill in the scratches generated by grinding, playing a role in repairing the friction surface and making the surface smooth: thus, reducing the COF and reducing wear.For spherical WS2 with different forms as lubricant additives under different loads were studied, and the anti-friction performance and lubrication states of sample oil were analyzed through the COF curve and the Stribeck curve. The anti-wear performance of the sample oil was analyzed by the surface morphology and wear form of the wear scars, and the anti-wear and anti-friction mechanism of WS2 with different forms in the lubrication process was discussed. The conclusions are as follows:(1)2 decreased first and then increased with the increase of load, and the COF was reduced by a maximum of 29.35%, and the minimum COF was concentrated around 100 N. The average COF of lubricating oil containing spherical WS2 gradually decreased with the increase of load, and the COF was reduced by a maximum of 30.24%. In the range of the experimental load, the minimum COF was concentrated around 100 N. In addition, the extreme pressure performance of the spherical WS2 was superior to that of the lamellar WS2.The average COF of lubricating oil containing lamellar WS(2)2 transitioned from a fluid lubrication state to a mixed lubrication state, while the lubrication state of the spherical WS2 basically remained in a fluid lubrication state without any change.According to the Stribeck curve, within the range of the experimental load, the lubrication state of the lamellar WS(3)2 was more stable.With the increase of the load, the average depth and width of the wear track were continuously expanding, and the wear rate of the two sample oils tended to decrease first and then increase with the increase of the load. The minimum wear rate occurred when the load was around 80 N, and the two values were close, indicating that the anti-wear performance of the sample oil was similar at this time. However, when it exceeded 80 N, the wear resistance of spherical nano-WS(4)2 with different forms was that, for lamellar WS2, its lamellar structure made it easy to slip between layers and the surfaces of layers, leading to the reduction of COF. For spherical WS2, its spherical structure can be regarded as a ‘small ball’, which can play a role similar to ‘micro-bearing’ on the friction surface, changing the sliding friction between surfaces into rolling friction: thus, reducing the COF.The main difference in the anti-wear and anti-friction mechanism of WSIn this paper, the tribological properties of WS


The aim of the study was to compare the prognostic significance of lymph node status of patients with lung cancer analyzed by three different methods: hematoxylin and eosin (H&E), immunohistochemistry of cytokeratin 19 (IHC CK19), and One-Step Nucleic Acid Amplification (OSNA). The clinical relevance of the results was evaluated based on relation to prognosis; the disease-free interval (DFI) and overall survival (OS) were analyzed. During radical surgical treatment, a total of 1426 lymph nodes were obtained from 100 patients, creating 472 groups of nodes (4–5 groups per patient) and examined by H&E, IHC CK19 and OSNA. The median follow-up was 44 months. Concordant results on the lymph node status of the H&E, IHC CK19 and OSNA examinations were reported in 78% of patients. We recorded shorter OS in patients with positive results provided by both OSNA and H&E. The study demonstrated a higher percentage of detected micrometastases in lymph nodes by the OSNA method. However, the higher sensitivity of the OSNA, with the cut-off value 250 copies of mRNA of CK19/µL, resulted in a lower association of OSNA positivity with progress of the disease compared to H&E. Increasing the cut-off to 615 copies resulted in an increase in concordance between the OSNA and H&E, which means that the higher cut-off is more relevant in the case of lung tumors. Today, the standard detection of tumor cells in hilar and mediastinal lymph nodes (LNs) removed during radical surgical treatment of primary and secondary lung tumors is performed using histopathological methods, i.e., by the microscopic examination of specimens stained using hematoxylin–eosin (H&E) by pathologists. However, this approach is limited, in particular in the case of micrometastases, clusters (clusters of tumor cells), or isolated tumor cells, where, according to certain studies, false negative results of the examination are produced in up to 20% of cases . One of A number of studies comparing the OSNA method with the standard histopathological examination in patients with breast cancer and/or colorectal carcinoma (CRC) have been published. The conclusions of these studies presented comparable results  , gastricIn the case of lung tumors, the OSNA method has been the subject of several studies, the purpose of which was both to ascertain the presence of CK19 in these tumors and offer a comparison of OSNA to H&E ,15,16,17The aim of the present study was to analyze, by carefully monitoring patients, the relationship between micrometastases detected in LNs using the OSNA, H&E, and IHC CK19 methods and the progress of the disease (the median follow-up was 44 months) in a group of 100 patients.The inclusion of patients with operable non-small cell lung carcinoma (NSCLC) and pulmonary metastases of colorectal carcinoma in the prospective study was performed in the period of 2015–2017. The study was approved by the Ethics Committee of the Faculty Hospital in Pilsen (No. 20150604). All patients provided their informed consent for inclusion in the study. The detailed characteristics of the group of 100 patients are provided in During the surgery of 100 patients, a total of 1426 lymph nodes were removed, with the average value being 14.3 LNs/person (5–32 LNs). Each LN was dissected into four parts, whereby parts one and three were examined using the H&E and IHC CK19 method, and parts two and four were examined using the OSNA assay. For methodological reasons, parts of LNs for OSNA method  were pooled into 3–5 groups of LNs within the framework of one nodal zone based on the IASLC LNs classification developed from the Mountain–Dresler classification [The removal of lymph nodes, the scheme for dissecting and pooling, as well as the histopathological and immunohistochemical examinations, CK19 detection and the OSNA method, are described in detail in our previous article .Briefly, LNs of one group were homogenized and lysed according to the manufacturer’s instructions. Determination was performed using the diagnostic kit LYNOAMP BC OSNA . Determination of the presence of mRNA CK19, as the marker of epithelial cells, was executed using the RD100i instrument . The net time of amplification reaction is 16 minutes, up to four groups of pooled lymph nodes can be analyzed in parallel. The cut-off values for the OSNA method were determined as specified in the LYNOAMP BC OSNA manufacturer’s manual and were used in the previous studies of tumor cells in LNs in patients with lung cancer  ,15,20,21The statistical analysis was calculated using the SAS software . Essential descriptive statistics for all variables of interest were prepared based on the clinical and pathological data of the patients. Categorical variables were reported as absolute and percentage values. Venn diagrams were used for comparing the results of H&E, IHC CK19 and OSNA method. Kaplan–Meier survival curves for overall survival (OS) and disease-free interval (DFI) were generated to compare prognostic significance of results obtained by H&E and OSNA method. The statistical significance of differences in CK19 mRNA copies assessed by OSNA between H&E-positive and H&E-negative samples was calculated using t test for independent samples. The statistical significance level was determined at the alpha limit = 5%.The concordance of the H&E, IHC CK19 and OSNA examinations was evaluated in 472 groups of lymph nodes. Full concordance of the three methods was recorded in 432 groups of lymph nodes (91.5%), 400 groups of lymph nodes were concordantly negative, and 32 groups of lymph nodes were concordantly positive, see After evaluating the groups of lymph nodes for a given patient , concordance was recorded in 78 patients (78%), of whom 59 patients had all examined LNs concordantly negative and 19 patients concordantly positive. Micrometastases in the lymph nodes were detected in 16 patients (16%) by the OSNA method, with negative results for the other methods. In three cases (3%), the H&E and IHC CK19 examinations were positive while the OSNA method produced negative results, see The clinical significance of the presence of tumor cells in lymph nodes determined by the H&E and OSNA methods was evaluated by analyzing the relation to DFI and OS. The Kaplan–Meier curves comparing NSCLC patients with positive and negative LNs detected concordantly by the H&E and OSNA methods are provided in In patients with discordant results (17 out of 80 NSCLC patients), the relevance to prognosis spoke in favor of the H&E method see c. PatienIn the clinical use, the status of lymph nodes itself is just a part of the TNM score, based on which the stage of the disease is classified. The following charts show the relevance between the stage of the disease based on the TNM score to the prognosis for patients with NSCLC, where the status of the lymph nodes was evaluated either by the H&E method or by the OSNA method .p < 0.0001) than in the groups of lymph nodes that were positive only according to the OSNA method . Determination of tumor cells in the lymph nodes was carried out using the standard method employed by pathologists (preparation staining by H&E), IHC CK19, and by the OSNA molecular biology method. Considering the fact that the results of the IHC CK19 method were concordant with those ascertained by the H&E staining method, with only a single exception, the survival charts include only the comparison of the H&E and OSNA methods. The methodological approach is completely different for H&E and OSNA, in terms of both the quantity of analyzed tissue, and the rate of processing. Therefore, each method has its advantages and disadvantages. It is obvious that evaluation of clinical benefits also depends on priorities. The OSNA method allows the entire lymph node to be analyzed, and compared to the H&E method, it requires less experience from the health care professional.Compared to certain other tumors, examination of LNs in the case of lung tumors is also specific due to the fact that the concept of the first regional (sentinel) node cannot be applied. One of the reasons is a high percentage of so-called skip metastases ,23. ThisConcordance of the H&E and IHC CK19 examinations was ascertained in 99.4% groups of nodes . Out of 100 patients, OSNA and H&E were concordant in 80 (80%) of the patients in N-staging; OSNA detected tumor cells in an additional 17 (17%) patients who were H&E-negative. On the other hand, H&E revealed micrometastases in three 3%) patients where the result of the OSNA method was negative. These results are similar to those previously published by other authors. Masai et al. monitored the CK19 expression in primary pulmonary tumors and breast cancer pulmonary metastases. CK19 was detected in 88% of patients with a pulmonary tumor and in nearly 91% of patients with breast cancer metastases . Hayama % patientFrom our point of view, the most relevant criterion for correctly detecting tumor cells (the CK 19 expression) for the purposes of N-staging as a part of TNM classification is correspondence with the progress of the disease, vesting the result with clinical significance. The clinical significance of the higher sensitivity of OSNA compared to H&E was assessed based on the analysis of the examination result’s relevance to disease-free interval (DFI) and overall survival (OS). In patients with concordant results detected by both methods (80% of patients), it was observed that positivity of lymph nodes was associated with worse prognosis for both subgroups of patients . From the point of view of our research aims, patients for whom discordance between H&E and OSNA was observed, were key for further analysis. There were 20 such patients out of 100, 17 with NSCLC and three with pulmonary metastases of CRC. For each patient, 3–5 groups of lymph nodes were examined with differences detected in only 39 out of 472 groups of lymph nodes. It was observed that the further progress of the disease better correlated with the examination based on the H&E method c,d.To explain this finding, it is necessary to focus on patients with LN positivity detected by H&E but not by OSNA. An explanation could be offered that tumor cells present in LNs of these patients do not express CK19. CK19 is an intermediate filament of the cytoskeleton, which is present in the cells of epithelial origin, but not in LN tissue. The presence of CK19 in an LN is therefore an indicator of the metastatic involvement by tumor cells of epithelial origin . HoweverFurther progress of the disease is affected not only by the positivity of the lymph nodes themselves, but also other parameters assessed by the TNM score, and/or the stage of the disease. More relevant to the progress of the disease is the stage classification based on the N-staging according to results obtained by the H&E method compared to the OSNA method . In thisIn the OSNA molecular genetic method, the key parameter is the cut-off value of the number of copies of mRNA of CK19. This value determines what number of copies will be analyzed as positive, i.e., interpreted as the presence of tumor cells in the LN. When the study was implemented, the kit for the assay of lung cancer LNs was not yet available and that was why published studies, including ours, employed kits for assays of LNs obtained from patients with breast cancer, including the set cut-off value, which was 250 copies of mRNA of CK19/µL.The instrument RD100i (and currently available RD210) used for the OSNA method to quantify mRNA of CK19 makes it possible to export the numbers of copies for individual assays. We compared the numbers of copies of mRNA of CK19 for the results concordantly positive with those obtained by the H&E method and for the positive results, which were negative if the H&E method was used. The results concordantly positive with the H&E method had statistically significantly more copies of mRNA of CK19 compared to the OSNA-positive results which were H&E-negative. None of the results concordantly positive with the H&E method had a number of copies lower than 620 copies of mRNA of CK19/µL. In the case of results which were OSNA-positive and H&E-negative, 53% of the specimens had a number of copies lower than 620 copies of mRNA of CK19/µL. The increase in the cut-off value to 615 copies of mRNA of CK19/µL proposed by us would increase the theoretical concordance between the OSNA and H&E methods for the analysis of groups of lymph nodes from 91.7% to 95.1%. With these results reflected in the complete evaluation of the patients’ lymph nodes (N0 vs. N1 or N2), concordance would increase from 80% to 87%.Studies aiming to define a new cut-off value for the OSNA method better reflecting clinical outcomes have been already published for sentinel lymph node examination in patients with breast cancer ,27. TerrThe high value of sensitivity and the cut-off value of the OSNA method are closely related to the clinical relevance of clusters and/or solitary tumor cells, which may or may not have potential for further progression, and/or may be eliminated by the immune system. The study carried out by Ren et al. has proven that the quantity of residual tumor cells in the lymph nodes of patients with bronchogenic adenocarcinoma is relevant to prognosis. The employed method was IHC anti-CK detection, while micrometastases referred to sets of cells—the largest dimension of which was smaller than 2 mm. The authors described that patients with micrometastases had significantly shorter recurrence-free survival and overall survival compared to N0, but significantly longer than for those with N1 macrometastases .We can conclude that for patients who were OSNA-positive and at the same time H&E-negative, the higher sensitivity of OSNA was not reflected in a less favorable progress of the disease. On the other hand, it must be noted that the OSNA method allows the entire lymph node to be analyzed, and, compared to the H&E method, requires less experience from the health care professional. However, the aforementioned advantage is paid by the lower association of OSNA positivity with the progress of the disease . The increase in the cut-off value to 615 copies of mRNA of CK19/µL increased concordance between the OSNA and H&E methods.The results of the study demonstrated a higher percentage of detected micrometastases in hilar and mediastinal lymph nodes when the OSNA method was used for examination. However, the higher sensitivity of the OSNA method in our set of patients corresponded less with the progress of the disease  when compared to the H&E method. The less time-consuming OSNA method was associated with an 8.3% discordance of results between OSNA and H&E during the analysis of LNs. The study suggests that an increase in the cut-off value of the OSNA method results in significantly higher concordance between the two methods.


Prionace glauca) led to the generation of a large number of central skeletons of low interest to fishmeal plants handling such wastes. In this context, the present study describes the optimization of the hydrolysis process  of alcalase and t = 4 h) to produce chondroitin sulfate (CS) together with the recovery of bioapatites. Then, that hydrolysate was chemically treated with an optimal alkaline-hydroalcoholic-saline solution  and finally purified by ultrafiltration-diafiltration (30 kDa) to obtain glycosaminoglycan with a purity of 97% and a productive yield of 2.8% (w/w of skeleton). The size of the biopolymer (CS) was of 58 kDa with prevalence of 6S-GalNAc sulfation (4S/6S ratio of 0.25), 12% of GlcA 2S-GalNAc 6S and 6% of non-sulfated disaccharides. Crude bioapatites were purified by pyrolysis and FT-Raman and XRD techniques confirm the presence of hydroxyapatite [Ca5(PO4)3(OH)], with a molar mass of 502.3 g/mol, embedded in the organic matrix of the skeleton. The mineralized tissues of blue shark are promising marine sources for the extraction of high value biomaterials with clinical application in bone and tissue regeneration and are still completely unexplored.The industrial filleting of blue shark ( Thousands of tons of fish by-products are generated around the world in the filleting of commercial fish species for the production of marine foods. In many cases those wastes are managed as solid urban fraction and in other occasions they are substrates for the production of fish meal ,2, althoPrionace glauca), after muscle separation, central skeletons are disposed in the plants as residues. This by-product supposes around 10–20% of the total weight of the shark and they are formed of vertebral discs and intervertebral fibrocartilage , corresponding, respectively, to , , , , , , and  diffraction planes  embedded in an organic matrix. The central skeleton of blue shark is a promising new marine source for the extraction of high value ceramic biomaterials. Further experiments must be performed to validate the capacity of these CS and bioapatites to be included in scaffolds, composites or nanodevices for the regeneration of cartilaginous and bone tissues.The optimal recovery of CS and bioapatites from skeleton wastes of blue shark was studied here. The three main processes involved  were optimized using response surface methodology and non-linear function modelling. Thus, the isolation of CS and bioapatite was maximized by alcalase hydrolysis of skeleton at 1% (