Erosion and the stripping effect of moisture on asphalt mixtures is one of the main reasons for the shortened service life of asphalt pavements. The common mean of preventing asphalt pavements from being damaged by moisture is adding anti-stripping agents (ASAs) to asphalt mixtures. However, the effect regularity and mechanism of anti-stripping agents on the physicochemical properties of asphalt is not exactly defined. This study compared the physical properties of ASA-modified asphalt (AMAs) to determine the optimal dosage and investigated the rheological and adhesion properties. Based on the roller bottle method and water immersion method, the moisture susceptibility of AMAs with three particle sizes was investigated. The results showed that the modification of asphalt using anti-stripping agents was a physical modification. At the optimum dosage of anti-stripping agents (0.3%), the basic physical properties of AMA1 were the most desirable. ASA2 increased the resistance of asphalt for deformation at high temperature by 46%, and AMA3 had the best low-temperature performance. ASAs enhanced the dispersed and polar components in the asphalt binder, improving the adhesion energy of asphalt. AMA3 had the strongest adhesion to the aggregate, with an increase in adhesion work by 2.8 times and a 45% of increase in ER value. This was attributed to ASA3 containing with a large number of metal cations and polar functional groups. It was shown that ASAs provided the most improvement in the anti-stripping performance of asphalt mixtures with 9.5–13.2 mm particles. The amide ASA, phosphate ASA and aliphatic amine ASA improved the water damage resistance of asphalt by 65%, 45% and 78%, respectively. This study can help engineers realize the effects of different types of ASAs on the physicochemical properties of asphalt and select the most suitable type of ASAs according to the service requirements. Asphalt mixtures are exposed to moisture and atmospheric rainfall for long periods, and their interaction with water leads to bond failure, which in turn causes the asphalt mixture to be stripped . Further2O3 > SiO2 [2 content  have worse adhesion properties than those with low SiO2 content . This is attributed to the weak bonding ability of the anionic component present in asphalt with siliceous aggregates, which can be easily replaced by water in combination with the aggregates [Aggregate type is associated with asphalt mixture wettability and contributes critically to the asphalt adhesion characteristics. Asphaltene is an asphalt component that plays a key role in the asphalt–aggregate interface performance, as it interacts with the mineral components in the aggregate to varying degrees: MgO > CaO > Al3 > SiO2 . Aggregagregates . Under wgregates  and polygregates , may alsNew liquid ASAs have been widely used in pavement engineering in recent years because of their small dosage, low cost, ease of construction, and high-temperature stability. These materials are organic polymer compounds, which are mainly divided into amine surfactants and non-amine surfactants , and theTherefore, amide, phosphate ester, and fatty amine ASAs were selected in this study, and the chemical composition of these three ASAs was investigated using X-ray fluorescence spectroscopy (XRF) and Fourier transform infrared spectrometer (FTIR). The physical properties of asphalt, including the softening point, penetration, and ductility, were used to determine the optimum dosage of ASAs. By using dynamic shear rheometer (DSR) and bending beam rheometer (BBR), the rheological properties of AMAs at high and low temperatures were evaluated. Based on the surface free energy principle, the enhancement mechanism on the anti-stripping performance of asphalt was analyzed. The moisture susceptibility tests (rolling bottle method and water immersion method) of the asphalt mixture were combined with digital image processing to assess the performance of AMA under dynamic water and high-temperature conditions, respectively.The material used was an asphalt binder from Ezhou Kepai Asphaltic Products Co., Ltd.  with Pen 60/80 (abbreviated as AB). Three commercial anti-stripping agents (ASAs) with different major components were used to modify the asphalt binder properties. ASA1, ASA2, and ASA3 refer to amide, phosphate ester, and aliphatic amine ASAs, respectively. The characteristics of ASAs were listed in The melt blending method was used to prepare the AMA. According to the manufacturer’s recommendation, the four dosages of ASAs in the asphalt were set at 0.2%, 0.3%, 0.4%, and 0.5%, respectively. The asphalt binder was heated to a flowing state at 140 °C. Thereafter, ASA was slowly dripped into the asphalt binder through a dropper with a rubber tip and stirred at 1000 r/min. At a constant temperature of 140 °C, the ASA and asphalt binder were mixed by a high-speed shearing machine at 4000 r/min for 30 min. To control the experimental variables, a blank sample was created using the same procedure for the base asphalt binder.Powdered basaltic aggregates and three ASAs samples were subjected to qualitative and semi-quantitative chemical analysis using Zetium XRF spectrometer . The test was performed by the pressed slice method, which is based on the principle of X-ray fluorescence spectroscopy. The elements were determined from F to U by also using OMNIC 9.2 standardless analysis software, and the content was determined in the identification range from ppm to 100%. The test results are presented in oxide mode.−1, the infrared spectra of the samples were obtained using Nicolet iS5  with the parameters of scan number and resolution setting at 32 and 4 cm−1, respectively. After that, the position, shape, and width of the peaks of the infrared spectra were observed in conjunction with Omnic 9.2 software for functional group analysis.FTIR is a molecular functional group identification method that is commonly used to identify organic compounds, such as asphalt . The mecThe sessile drop method is widely used by researchers as an SFE test method because of its simplicity and the reliability of the results . On the Based on Fowkes’s theory , the SFEThe interface free energy of the solid and liquid is expressed in Equation (3) ,27.(3)γFollowing that, Equation (4) is established from Equations (1) and (3) to calculate the SFE components of the solid-phase . Three eIn addition, the effect of ASA was quantified by calculating the adhesion energy and the stripping energy. When THE asphalt binder is mixed with the aggregate, the energy released by the formation of the bonding interface is adhesion energy. Its calculation formula is shown in Equation (5) ,30. The The adhesion-stripping energy ratio (ER) was used to visually characterize the surface energy differences of different asphalt samples. The The rheological properties of asphalt materials vary significantly under different loads and temperatures when they are in the linear viscoelastic range, and the rheological properties of asphalt binders at high temperatures can provide a rough performance indication in the production and usage of asphalt binders . A DSR tThe cracking resistance of asphalt at low temperatures was characterized by the BBR test, which tests the flexural creep modulus of stiffness (S) and the slope of the double logarithmic curve of S versus the load action time  of the matrix asphalt and the three AMAs, as shown in Equation (1) ,36. The A modified rolling bottle test (RBT) based on BS EN 12697-11  was deveUnder the combined effect of high temperature and water conditions, the stripping resistance of the AMA was evaluated using an improved high-temperature water immersion test . First, In the studies of moisture damage on asphalt mixtures, researchers usually use a visual method to evaluate the asphalt stripping rate, which is subject to human factors and excessive errors . By intrtion (9) :(9)RMS=The area stripping rate is a measure of the change in the asphalt coating area on the loose mixture and characterizes the asphalt–aggregate interface adhesion. Images of the tested loose mixtures were taken in a well-lit environment and imported into Image J software to analyze the stripping area ,44. Figu2 content ratio) was used as a parameter for the aggregate affinity to asphalt. The basalt had a low alkalinity of 0.27, which indicated that the basalt aggregates are acidic and may have weak bonding to the asphalt in a moisture-heavy environment [The performance of asphalt mixtures is largely influenced by the chemical composition of the aggregates. The chemical composition of the ASA plays a crucial role in asphalt modification. ironment . It is aironment . The ionironment . ASA2 ma−1, 1350–1450 cm−1, and 723 cm−1 wavenumber ranges. The characteristic peaks at 2920 cm−1, 2852 cm−1, and 1461 cm−1 were attributed to the occurrence of the asymmetric and symmetric stretching and vibration as well as the in-plane bending vibration of C−H in methylene, respectively. The asymmetric stretching vibration and symmetric bending vibration of C−H in methyl can be identified at 2959 cm−1 and 1373 cm−1, respectively. The methylene chain segment (−(CH2)n−) (n ≥ 4) at 723 cm−1 undergo synergistic motion. The result indicates that there may exist alkanes, aromatic hydrocarbons, and their derivatives among the three types of ASAs.−1 band. ASA1 belongs to the amide ASAs, and the vibration of C=O in tertiary amines corresponded to the peak centered at 1660 cm−1. The absorption peaks of both the amine groups and C=O in tertiary amide were weaker than in those with methylene, indicating the presence of a long alkyl chain of amide in ASA, which can have better compatibility with asphalt. The adsorption of polar groups in amide played a role in forming a high-strength bond in the asphalt–aggregate interface. Moreover, the remaining absorption peaks in ASA1 imply carboxylic acids, benzene rings, aromatic oxides, and aliphatic ethers might be present. ASA2 is a phosphate ester ASA, the absorption peaks of which are at 1200 cm−1, 1013 cm−1, and 808 cm−1, corresponding to the infrared characteristic absorption peaks of phosphate esters. Further evidence that aromatic esters might be exist in ASA2 come from the absorption peaks at 3028, 1741, 1604, and 872 cm−1. ASA3 is an aliphatic secondary amine ASA, the characteristic functional groups of ASA3 corresponded to the peaks centered at 3310 cm−1, 1554 cm−1, and 1095 cm−1. The presence of carboxylic acid and amide in ASA3 was indicated by the typical peaks for C=O at 1643 and 1719 cm−1. Additionally, the high-frequency peak bands at 1182–1315 cm−1 were attributed to the stretching vibration of C−N in the benzene ring. The stretching mode of the 1,4 para-substituted benzene ring was detected at 871 cm−1. Furthermore, ASA3 was shown to potentially contain carboxylic acids and amides. These polar groups contribute the most to the electrostatic interaction at the asphalt–aggregate interface and can prevent the fracture of the interface [In addition, nterface .as (P−O−C of phosphate ester) in the modified asphalt with the addition of ASA2 can be observed compared to AB. Moreover, the fatty secondary amine ASA is more similar to the asphalt composition, and only the stretching vibration of the para-substituted benzene ring can be observed, which can be better integrated with the asphalt. Overall, no additional absorption bands were found in the modified asphalt other than those already present in the asphalt binder and ASAs. This indicated that there is basically no chemical reaction within them.The infrared spectrogram of asphalt, which was modified with a large dosage of ASA was investigated to study the mutual reaction between the two substances. Compared to the FTIR spectra of AB, the FTIR spectra of the asphalt containing ASAs were more complex. In ASA was added to asphalt as a modifier, which has a noteworthy impact on the rheological properties of asphalt . DSR tes2, respectively, and the polarity component had a higher incremental than the dispersion component [The modified asphalt was prepared based on the optimal dosage, and surface free energy was applied to analyze the mechanism of the improved adhesion properties at the asphalt–aggregate interface by the ASAs. omponent . It is w2. The results indicated that ASA3 could promote both the internal cohesive properties of asphalt and its interfacial adhesion with the aggregate. 2, which meant that AMA3 was less prone to stripping compared to AMA1 and AMA2. When comparing the ER values of the AB and AMAs, the ER values of AMA1 and AMA2 increased by 7% and 3%, respectively, and that of AMA3 increased by 45%. At the molecular level, this might be explained by the adhesion between the amine group of ASA3 and the silanol group on the surface of the aggregate. The silanol groups on the surface of the aggregate produced a stronger connection between the asphalt and the aggregate [ggregate . The SFESince the ASAs had the most significant effect on 9.5–13.2 mm particle size asphalt mixtures, 9.5–13.2 mm particle size aggregate was used as the research object to further determine the effect of ASAs on the stripping resistance of asphalt mixtures at high temperatures. The main elements of ASA1 are Fe, S and Cl. ASA2 is mainly composed of non-metallic elements such as P and S. ASA3 contains up to 96% Ca. Based on the results of infrared spectra, it is known that there is almost no chemical reaction between the AB and ASA, and the mechanism of the ASA can be classified as a physical modification.The optimum dosage of ASAs is 0.3%, as determined by the physical properties of asphalt. ASA1 exhibits the best basic physical properties, and ASA2 shows the best high-temperature deformation resistance, with a 46% increase compared to the base asphalt. ASA3 significantly improves the low-temperature performance of the asphalt, with a 5% improvement.ASAs increases the dispersion and polarity components in asphalt. Asphalt adhesion energy is improved and the stripping energy is reduced. ASA3 contains a large amount of metal cations and polar amine groups, which increased the polar component by 2.8 times and the ER value by 45%. The increase in these indexes is significantly higher than that of ASA1 and ASA2.Compared to other particle sizes of coarse aggregates, ASAs have the best modification effect on the moisture resistance of basalt with a particle size of 9.5–13.2 mm. Under different testing conditions, the adhesion properties of ASA1, ASA2, and ASA3 modifying the asphalt–aggregate interface are enhanced by 65%, 45%, and 70%, respectively.In this study, the main aims were to investigate the chemical composition of amide, phosphate ester, and fatty amine ASAs, and to analyze their anti-stripping mechanisms. The effect of ASA dosage on the physical and rheological properties of asphalt was discussed, and the anti-stripping performance of AMAs under dynamic water and high-temperature conditions was characterized. The following conclusions can be drawn:The findings of this study provide a basis for investigating the mechanism of action of each component of the anti-stripping agent and its modified asphalt performance under multiple conditions, which can help engineers understand the effects of different types of anti-stripping agents on the physicochemical properties of asphalt and select the right type of anti-stripping agent according to the service requirements. However, the lack of anti-stripping agents and their associated adhesion models makes this paper incomplete in exploring the adhesion mechanism of anti-stripping agents in the asphalt–aggregate interface. In future research, it is recommended to conduct further studies on the effect of each component of the anti-stripping agent on the properties of asphalt and its coupling mechanism in combination with molecular dynamics simulations and other computer tools.


In recent years, diatomite has been successfully adopted in asphalt modification to overcome the problems of polymer modified asphalt, because of the advantages in wide sources, low price, and worthy technical characteristics. Although the improvement of the high-temperature performance of the modified diatomite asphalt has been verified in previous studies, the diatomite will bring negative impact on the low-temperature resistance. Hence, the objective of this study is to seek a new channel to improve the comprehensive performance of the diatomite modified asphalt binder. Considering the advantage of the SBR in improving the low-temperature performance of asphalt binder, the diatomite/SBR composite modified asphalt binder (DSA) and the corresponding preparation technology are developed to obtain an improved comprehensive performance via the orthogonal experiment method in this study. Moreover, the modification mechanisms of the DSA are revealed using fluorescence microscopy (FM) tests, Gel permeation chromatography (GPC) tests, and Fourier transform infrared spectroscopy (FTIR) tests. By the end of 2020 in China, the length of highways had reached 161,000 km, of which more than 90% are asphalt pavement . ObviousDiatomite is a kind of biogenic siliceous sedimentary rock and mainly composed of silica. Owing to the advantages in wide sources, low price, and worthy technical characteristics  , 12, theTherefore, there have been some studies focusing on diatomite composite modified to improve the comprehensive performance of the diatomite modified asphalt binder. For example, Liu et al. , 19 founThe previous studies are mainly focused on the diatomite/RP composite modified asphalt binder. However, they are not able to tackle the lack of the low-temperature resistance of diatomite modified. Hence, the objective of this study is to seek a new channel to improve the comprehensive performance of the diatomite modified asphalt binder. From the above, it can be understood that the improvement of high temperature performance of diatomite is due to the addition of diatomite itself to absorb the easy flowing and small molecular weight aromatic and saturated fractions in asphalt, but also causes the imbalance of the four components of asphalt . SBR modThe list of abbreviations which used in the following text, as shown in In this study, Donghai 90# base asphalt binder, 90# Polymerized Styrene Butadiene Rubber (SBR) asphalt modifier, and diatomite were mixed to produce the DSA. Their technical indexes are presented in Tables The DSA is prepared according to the following steps.Heat the base asphalt to an oven at 140°C until it has good fluidity.Mix the SBR and diatomite with the base asphalt and stir to swell for 20 minutes.According to the set shear temperature, shear number rate and shear time to complete the shear mixing.-1 to complete the preparation of the DSA.After the shearing was completed, the development was stirred at 160°C for 3 hours at 600 r·minTest Regulations for Asphalt and Asphalt Mixture in Highway Engineering (JTGE20-2011) .Softening point reflects the temperature sensitivity of asphalt, ductility at 5°C reflects the plasticity ability of asphalt at low temperatures, Brinell viscosity at 135°C reflects the flow resistance of asphalt at high temperatures. These three performance indicators can well characterize the high and low temperature performance of asphalt at the macro level. The softening point, ductility at 5°C and Brinell viscosity at 135°C tests of asphalt shown in the -1, and the resolution is 0.4 cm-1. The FTIR sample of the asphalt binder is approximatively circular disc with the diameter of 1–1.5 cm, as shown in The Nicolet 5700 Fourier transform infrared spectrometer as shown in The Waters 515–410 gel permeation chromatograph as shown in The LW100 FT/B fluorescence microscope as shown in Heat the asphalt binders and the glass slide. The asphalt binders must achieve a flow state. The surface temperature of the glass slide should be 150–160°C.Drip 1–2 g asphalt onto the heated glass slide, and put the cover glass down from one side of the glass slide quickly and gently.After the asphalt on the glass slide is completely covered by the cover glass, gently rock the glass slide to distribute the asphalt evenly. The FM samples of the asphalt can be obtained after the glass slide is cooled for 30 min, as shown in The softening point, ductility at 5°C and Brinton viscosity at 135°C of the DSA are mainly affected by five factors: diatomite content, SBR content, shear temperature, shear rate and shear time. In order to cut the test work scientifically and reveal the complex relevance of these factors, the orthogonal test method is adopted in this study. The factors and levels of the orthogonal test are shown in Technical Specifications for Construction of Highway Asphalt Pavement (JTG F40-2004) . Therefore, only the softening point and ductility at 5°C are analyzed in the follow-up. The range method is used to analyze the range value of each factor on the performance of the DSA. A higher range of one factor represents a higher influence degree of the factor. The meanings of computational symbols Ki, ki and R are as follows.As shown in Ki represents the sum of the corresponding test results when the level number on any column is i.ki = Ki / S, S is the frequency of occurrence of each level in any column.R = max {k1, k2, k3}- min {k1, k2, k3} on any of the columns.Range Analysis of the softening point range results based on orthogonal test is shown in -1, and the shear time is 50 ~ 60 min, the softening point of the DSA is better.As shown in Analysis of the ductility at 5°C range results based on orthogonal test is shown in -1, and the shear time is 40 ~ 50 min, the ductility at 5°C of the DSA is better.As shown in -1, and 50 min to carry out the conventional performance test of the DSA , the principle of the highest softening point is taken as the premise that the ductility at 5°C is greater than 40cm, and the final diatomite content is 9%. Therefore, the optimal blending scheme is proposed as follows: the diatomite content is 9%, the SBR content is 2.5%, the shear temperature is 170°C, the shear rate is 4500 r•min-1, the shear time is 50 min. The DSA has superior and stable performance.As shown in The subsequent the DSA refers to No.27 in As shown in The FM images of the base asphalt, diatomite modified asphalt, SBR modified asphalt, SBS modified asphalt and DSA magnified 100 time are shown in As shown in d, as expressed in Eqs , an, and, asd in Eqs , respectNi is the number of molecules with molecular weight Mi, Wi is the weight of the component with molecular weight Mi.Where Mp of the diatomite modified asphalt, SBS modified asphalt, and DSA are smaller than that of base asphalt. This indicates that the incorporation of the diatomite modifier, SBS modifier and Diatomite/SBR composite modifier decreases the content of small molecular weight components in asphalt, while the phase pair of large molecular weight components increases, so the interaction between molecules increases, which makes the high-temperature stability of the modified asphalt better than that of the base asphalt.As shown in MW in MW of the diatomite modified asphalt, SBS modified asphalt and DSA increases by 5% and 6% respectively, which indicating that the shear failure resistance of the diatomite modified asphalt, SBS modified asphalt and DSA are all enhanced. Among them, the DSA and SBS modified asphalt have the best high temperature stability.As shown the data of d in d of diatomite modified asphalt, the SBS modified asphalt and DSA are all greater than those of base asphalt. The molecular weight distribution becomes more dispersed with the increase of polydispersity coefficient d. In other words, the components in a certain range of molecular weight are fewer. Considering the phase transition interval of molecular weight is consistent with the temperature interval of the transformation of asphalt aggregation components, the heat absorption of asphalt binder  will be reduced.As shown the data of In order to further explore the influence of the composite modifier on asphalt, the functional groups of the base asphalt, diatomite modified asphalt and the DSA via the FTIR tests, as shown in -1, 1460 cm-1, 1375 cm-1 and 785 cm-1 in all the spectrograms. The absorption peaks at 1600 cm-1, 1460 cm-1 and 1375 cm-1 are caused by the stretching of the C = C in benzene ring, the stretching of the CH3, deformation of the CH3, and the stretching of the S = O (sulfoxide), respectively. The absorption peaks at 785 cm-1 are caused by the rocking of the -CH2 in benzene ring of polystyrene.As shown in -1 is the only difference between the IR spectrums of the base asphalt and diatomite modified asphalt, which is proving the existence of the diatomite. Therefore, the addition of diatomite does not produce new functional groups, but only a physical blending process. Compared with base asphalt, the IR spectrum of the DSA generates two new characteristic peaks near 1030 cm-1 and 966cm-1. The absorption peak near 1030cm-1 should be the stretching of the S = O (sulfoxide) vibration absorption peak of diatomite, and the absorption peaks at 966 cm-1 is caused by the wagging of the trans-C-H-disubstituted -CH = CH2 in styrene-butadiene, which should be the characteristic absorption peak of trans-butadiene of the SBR. The generation of the S = O will increase the intermolecular forces. The corresponding macroscopic performance presents the increase of hardness and the shear failure resistance of the diatomite modified asphalt and the DSA.Moreover, the appearance of S = O stretching vibration absorption peak near 1076cmIn general, the absorption peak of the DSA is the same as that of the base asphalt. It can be determined that the chemical composition and relative content of the DSA and the base asphalt are basically similar, and when the diatomite and SBR are added to the base asphalt at the same time, it is mainly physical blending, and no complex chemical reaction occurs.This study investigated the performance of the DSA via the orthogonal experiment method, and revealed the modification mechanisms of the DSA by FM tests, GCP tests, and FTIR tests. It greatly expands the applicability of SBR modified asphalt, and this experiment provides a reference for future research on dosage and blending method through complicated orthogonal test. On the other hand, through the micro-level verification of the modification effect of diatomite, it also provides technical support for promoting the wide use of this cheap material. Based on the findings of this study, the following conclusions can be drawn.-1, and shear time is 50 min.The optimal preparation parameters of the DSA are put forward as follows: shear temperature is 170°C, shear rate is 4500 r·minThe content of diatomite and SBR have a significant effect on the ductility at 5°C and the softening point of the DSA. When the content of diatomite and SBR is equal to 9% and 2.5%, respectively, the comprehensive performance of the DSA is appropriate.The modified particles are small and evenly distributed in the DSA, and form a certain network structure with the base asphalt. Compared with the base asphalt, the average molecular weight and polydispersity coefficient of the DSA increased, which means that the DSA has good shear deformation resistance and low temperature sensitivity. And only two vibration absorption peaks in the infrared spectrum are different from base asphalt, and the overall trend is consistent. The comprehensive performance of the DSA is improved by physical blending of diatomite and SBR.Finally, the research on the pavement performance of diatomite /SBR modified asphalt combined aggregate remains to be carried out. The composite modified asphalt prepared in this test is based on the conventional performance index of asphalt as the standard, so the dosage and mixing conditions obtained are only applicable to the road conditions under general and ordinary environmental conditions. In the case of complex load or extreme conditions, the reference index should be appropriately adjusted according to the situation. Secondly, due to the porous structure of diatomite, it should have great research potential in the future to improve the aging properties of asphalt materials.S1 File(DOCX)Click here for additional data file.


Bone defects lead to the structural loss of normal architecture, and those in the field of bone tissue engineering are searching for new alternatives to aid bone regeneration. Dental pulp-mesenchymal stem cells (DP-MSC) could provide a promising alternative to repair bone defects, principally due to their multipotency and capacity to fabricate three-dimensional (3D) spheroids. The present study aimed to characterize the 3D DP-MSC microsphere and the osteogenic differentiation capacity potential cultured by a magnetic levitation system. To achieve this, the 3D DP-MSC microsphere was grown for 7, 14, and 21 days in an osteoinductive medium and compared to 3D human fetal osteoblast (hFOB) microspheres by examining the morphology, proliferation, osteogenesis, and colonization onto PLA fiber spun membrane. Our results showed good cell viability for both 3D microspheres with an average diameter of 350 μm. The osteogenesis examination of the 3D DP-MSC microsphere revealed the lineage commitment, such as the hFOB microsphere, as evidenced by ALP activity, the calcium content, and the expression of osteoblastic markers. Finally, the evaluation of the surface colonization exhibited similar patterns of cell-spreading over the fibrillar membrane. Our study demonstrated the feasibility of forming a 3D DP-MSC microsphere structure and the cell-behavior response as a strategy for the applications of bone tissue guiding. Bone/periodontal defects caused by trauma or diseases increase each year, causing major health problems when the defects exceed the self-regenerative capacities of the tissues. The current therapies are not sufficient for solving this problem, and the need to look for new strategies to aid in regenerating bone tissue is challenging ,2.One strategy that has been used in regenerative medicine is the use of mesenchymal stem cells (MSC) because they represent a heterogeneous population of undifferentiated cells capable of self-renewal and multipotentiality . MSC havTo avoid the aforementioned drawbacks, the search for novel options for this type of cell has turned to the oral cavity, where there are sources of MSC, such as those isolated from the periodontal ligament, gum, pulp, and dental follicles, and all of the latter are accessible without costly or invasive surgeries ,9,10,11.Another important aspect to consider in MSC cell-based studies is the method employed for the cell culture, in that the differentiation potential must be preserved . RecentlWithin 3D cultures, what is sought is simplicity, reproducibility, and scalability, all of which allow for the facilitation of interaction and increase in cell aggregation, such as those based on low-adherence plates and rotational and suspension systems ,19. HoweMagnetic levitation is considered a guided self-assembly technique. The technique is based on the interaction of a non-toxic bioinorganic hydrogel of iron oxide and gold nanoparticles with the cells, permitting their manipulation through the use of magnets. Due to its magnetic force, this system has facilitated the aggregation to form a 3D cellular structure and co-cultures of different cell lines. The 3D cellular spheroids showed good proliferation and protein expression similar to that in vivo and had clear differences when compared to 2D traditional culture, rendering it suitable for high-throughput screening studies ,22,23,24Therefore, this study aimed to report the characterization of DP-MSC microsphere formation, the osteogenic differentiation capacity, and the colonization onto fiber spun mats cultured by the magnetic levitation system and compared to 3D human fetal osteoblast microsphere in order to provide an assessment of their suitability for fabricating 3D cellular structures for bone tissue purposes.The cell line used in this study was human fetal osteoblast cells , as well as the human dental pulp mesenchymal stem cell line previously reported [TM-PL; Greiner Bio-One, Houston, TX, USA) in accordance with the manufacturer’s instruction and as previously reported [−7 M of dexamethasone), which was maintained for 7, 14, and 21 days under the magnetic levitation system by the Bio-assembler Kit . A resazurin assay was performed to quantify the cell viability on both 3D microspheres. Briefly, 10 μL of resazurin solution was added, and after 6 h of incubation, 100 μL of the medium was read at 570 nm .The 3D DP-MSC and 3D hFOB microsphere were fabricated using a magnetic nanoparticle solution  according to the manufacturer’s protocol. After 3D microspheres were observed utilizing inverted epifluorescence microscopy . All microspheres were counterstained with DAPI for the nucleus. The diameter of the microsphere was obtained from the average of joining two points of the microsphere imaging and passing through its center. The experiments were conducted in triplicate and the medium was changed every 2 days.The osteogenic differentiation of the 3D DP-MSC and 3D hFOB microspheres was evaluated by ALP activity, calcium deposition by Alizarin Red staining , and the expression of osteoblast-related markers by qPCR.The intracellular ALP activity of the 3D microspheres was determined by an ALP assay kit  according to the manufacturer’s protocol. Briefly, on the day of the test , the microsphere was lysed and incubated with 50 μL of 5 mM of p-nitrophenol phosphate solution (pNP), and the absorbance was read at 405 nm after 1 h of incubation.Extracellular mineralization related to calcium deposits was detected by employing an osteogenesis quantification kit based on alizarin red staining . Representative images of the 3D microsphere were captured after 21 days with a light microscope. For quantitative analysis, the alizarin red staining of both 3D microspheres was extracted by 10% of acetic acid and the eluted concentration was correlated to a standard curve of known ARS dye concentrations.The transcriptional level of bone-related genes  were quantified by using qPCR. The 3D DP-MSC and 3D hFOB microsphere were collected after 14 days for total RNA isolation by TRIzol (Invitrogen). The cDNA was synthesized using the Improm-IIReverse Transcription system and amplified by employing the appropriated primers  and GAPDw/v) were synthesized by air-jet spinning, as reported previously [Fiber-spun membranes of PLA 10%  indicates significant differences between conditions.The results were expressed as the mean ± standard deviation (SD). For comparison between groups, Student’s In order to assess the suitability as a facile methodology to fabricate 3D microsphere structures by a magnetic levitation system, the cell line derived from dental pulp mesenchymal stem cells (hDP-MSC) was used as a model to study the osteogenic differentiation capacity. Moreover, to assess whether the interaction of incubating hDP-MSC cells with the nanoshuttle magnetic nanoparticle solution to induce 3D microsphere aggregation under the magnetic system affects cell viability, we carried out a comparison with the human fetal osteoblasts (hFOB) cell line utilizing the resazurin assay.As shown in The morphology of the 3D DP-MSC and 3D hFOB microspheres were analyzed by fluorescently labeled cells after 21 days of culture . After tThe quantification of the intracellular presence of the ALP enzyme as an early marker for osteogenic differentiation is presented in Extracellular matrix mineralization of the 3D DP-MSC microsphere and the 3D hFOB microsphere was analyzed using Alizarin Red staining after 7, 14, and 21 days . Color sThe qPCR analysis of osteoblast-related genes in the 3D DP-MSC and 3D hFOB microspheres such as RUNX-2, OCN, ALP, and Col1 was evaluated after 14 days of culture . RegardiThe evaluation of the interaction of the 3D DP-MSC and 3D hFOB microspheres on the surface of the PLA fiber-spun membrane was carried out using live and dead staining assays after 3 and 7 days .The morphological analysis of the 3D microsphere after 3 days of being in contact with the surface of the PLA fiber membrane showed that 3D hFOB B and 3D On the other hand, the 3D hFOB microsphere, after 7 days of cultivation, exhibits an increase in the colonization of the surface of the PLA fiber membrane D–F. In tA similar behavior could be observed in the 3D DP-MSC microsphere, whereby the cells that move away from the periphery remain viable and colonize the surface topography of the membrane at approximately 193.84 ± 7.22 μm D–F. MoreIn our results, it is possible to observe the core center related to the senescent and dead cells associated with the limited diffusion of oxygen and nutrients, decreasing cell viability in the inner layers; however, this reduction in the viability does not affect the growth, interaction, and colonization behavior of the cells on the topographical surface of the fibrillar PLA membrane. Moreover, in the micrographs, some zones of the internal structure related to the core center are not completely stained when using DAPI for nuclear localization. A possible explanation could be related to the poor penetration of DAPI staining in the microsphere. This penetration depth can be affected depending on various factors such as the size of the microsphere, the density and compaction of the cell layers, the incubation period used for the labeling, the concentration of the dye, and due to the light-scattering in thick specimens as the microsphere. In addition, we used epifluorescent microscopy as a strategy for end-point fluorescence images of fixed microspheres that limited the analysis. A possible strategy to solve this limitation could be the microsphere clearing technique that enables 3D volumetric imaging by reducing light-scattering and improving the penetration of label dye staining ,28.Bones perform important functions in life, such as providing mechanical support for locomotion, protecting organs, and controlling mineral homeostasis. These functional properties are affected by diseases or traumas that cause bone defects so that the bone itself cannot be regenerated. To solve this issue of diminished capacity induced by bone defects, bone tissue engineering is seeking new alternatives to achieve or help the regeneration of bone tissue, and one of those strategies is through the use of mesenchymal stem cells (MSC).One of the advantages of using MSC cells is that their multipotency can help to regenerate bone defects through differentiation into bone-forming cells called osteoblasts, which are responsible for secreting, forming, and mineralizing the bone matrix. Thus, the successful use of MSC depends on the delivery method for the reconstruction of bone tissue, and the most common of these is through the use of biomaterials in order to provide an ideal microenvironment for harnessing the ability to form immature osteoblasts .Recently, an alternative method related to the formation of 3D spheroid culture models has been introduced as a strategy for local delivery during the process of regenerating bone tissue. This strategy has advantages over the monolayer cultures used to seed onto the surface of biomaterials because its three-dimensionality can provide a microenvironment that mimics the interactions that take place between cells and the extracellular matrix as closely as possible to what occurs naturally .In our study, we present a set of experiments to support the concept that three-dimensionality enhanced the osteogenic potential of MSC. For this, we formed a 3D microsphere using dental pulp mesenchymal stem cells that were under the stimulation of biomineralization-inducing media and cultured using a magnetic levitation system. Our data are in agreement with previous studies that have reported that the use of magnetic nanoparticles to manipulate cell aggregation by magnetic force does not cause any toxicity effects ,31,32,33Likewise, our data revealed that the 3D microspheres of both cell lines present good cell viability throughout the culture time and that this may be related to the microenvironment that is favored by the 3D microspheres, due to the cells interacting more and increased signaling, as has been reported via cell–cell and cell-ECM ,34. MoreIn tissue engineering, 3D spheroids have become increasingly important as biofabrication models; however, their application, efficiency, or improvement in creating a 3D structure with highly uniform size, shape (geometry), and morphology presents a challenge in controlling the manipulation during the experiment . In our In bone/periodontal regeneration, it is recognized that DP-MSC possesses several immunomodulatory, paracrine, and multipotency properties that make it a candidate for exploring its differentiation toward osteogenic lineage .In our study, we compared the commitment toward bone differentiation of the 3D DP-MSC microsphere against the 3D hFOB microsphere under osteogenic induction medium by calcium deposition through red alizarin staining, ALP activity, and determined gene expression profiles via qPCR analysis.Our results showed that Col1 and ALP increase in hFOB compared to DP-MSC, while RUNX2 was comparable among groups. RUNX 2 is a key transcription factor involved in the early phase of osteogenesis and remodeling, whereas ALP and COL1 are reported to increase prior to the onset of mineralization ,42. The As for the OCN gene, our results revealed that DP-MSC has more upregulated expression, and this confirmed the commitment toward osteoblastic lineage because OCN is considered a marker involved in bone formation and the mineralization of the extracellular matrix. The differences in expression could be related to the proposed function as a late-stage marker of mineralization, which is consistent with our result, where the 3D hFOB microsphere enters more rapidly into the differentiation and mineralization stimulus than the 3D DP-MSC microsphere, supporting a more mineral deposition via alizarin red staining in the osteoblast microsphere. Moreover, the OCN upregulation in DP-MSC is considered a marker of osteoblast commitment because its expression is a marker of early osteoblast differentiation and is involved in the signaling pathways that regulate differentiation and maturation ,43.Our data support that the 3D microspheres in both lines possess a great capacity to respond to osteogenic induction under magnetic levitation system conditions. This typical pattern related to bone differentiation has been reported as being regulated by multiple factors and signaling pathways . This siOur findings demonstrate that there is a synergistic effect between the osteogenic media and the magnetic nanoparticles. This synergism could regulate cell viability, proliferation, and differentiation in both 3D microspheres and could be influenced by the 3D culture system.Overall, the osteoblast-markers in both 3D microspheres demonstrated that the microenvironment regulates the expression of the ALP molecule, and its activity could provide a high concentration of phosphates in the earlier stages of extracellular matrix mineral deposition onto the 3D microspheres. In the continued process of biomineralization, the presence of the non-collagenous protein OCN allows for the mineralization of the ECM collagen fiber, regulating the strength and mechanical properties of the microsphere during the culture time. Moreover, the biomolecular mechanism involved in the biomineralization of the 3D microsphere requires further study.Guide tissue engineering (GTE) is mainly based on using resorbable and non-resorbable membranes to guide the response of progenitor cells at the site of the defect in order to regenerate the tissue. One resorbable membrane that is being used in GTE is made from polylactic acid (PLA). PLA is an aliphatic polymer approved by the U.S. Federal Drug Administration (FDA) due to its excellent physical, thermal, biocompatibility, and biodegradability properties, which have rendered it ideal for use as a scaffold ,47. For Recently, a concept has been introduced into the field of tissue engineering denominated mini-scaffolding, which seeks to combine the principles of scaffold-base and scaffold-free approaches to obtain a combinational synergy that could open a strong field toward translational medicine because it could be used as a source of cellular release at a damage site. Likewise, it is important to note that an important challenge comes in the form the cellularization of the mini-scaffold and that this could be solved by employing cell aggregates or spheroids, where it is proposed that the mini-scaffold could be seeded directly or have an entrance for the insertion of the spheroids .With this idea in mind, we analyzed interaction and colonization by directly seeding the 3D DP-MSC and 3D hFOB microspheres onto the surface of the PLA fiber-spun mat in an attempt to contribute to the concept of the mini-scaffold.From our preliminary analysis, both lines under osteogenic stimulation demonstrated a similar spreading and colonization response. This response could be regulated by the topography of the PLA membrane. The surface could be sensed through the 3D microsphere, and the cell on the periphery could be receiving the information cues to begin to spread and colonize the membrane following the random orientation of the PLA fiber. These cell behaviors are in agreement with previous studies reporting that the microenvironment, the physical signals mediated by the fibers mimicking the ECM, and the surface energy regulating the adhesive properties of the spreading and migration of cells could be utilized as a quantitative indicator of tissue biocompatibility ,54,55.However, at this stage of the study, we were unable to know whether the thickness of the mini-scaffold could affect the cellular response of the 3D microsphere. This is because a study that explored the responses to microfibrous scaffolds indicated that cell adhesion and proliferation could be regulated by the microstructure of the scaffold associated with the size and density of the pores .It is essential to mention that 3 D microspheres are recognized for their ability to more accurately reproduce the gradients of micronutrients, including metabolites and oxygenation found in the tumor microenvironment. In this regard, flow cytometry is the standard method used to quantify the protein expression of 3D microspheres. However, the technique requires disruption of the spheroid, which results in the loss of spatial information from the microenvironment. Techniques in which 3D microspheres are embedded in hydrogel–protein hybrids, followed by clearing and denaturation, have permitted high-resolution imaging via an epifluorescent microscope . In thisMoreover, additional studies are necessary to understand the cellular response influenced by the PLA fiber-spun mat to generate the signal related to topotaxis and mechanotaxis, which contributes to instructing the 3D microsphere to colonize by analyzing the focal adhesion point, the cytoskeletal organization, and the evaluation of the physicochemical composition of the tissue deposited by the 3D microsphere in the in vitro model of biomineralization, as well as the application of the 3D microsphere-PLA construct in the in vivo bone calvaria defect model.This study shows that DP-MSC has the potential to form a 3D microsphere through the magnetic levitation system, maintaining good morphology, viability, and differentiation ability similar to the 3D microsphere of hFOB, which is more committed to an osteogenic lineage. Moreover, the colonization and spreading properties of both 3D microspheres revealed similar behavior when cultured above the PLA fiber-spun membranes. In addition, our data suggested that 3D microsphere fabrication could be a tool and alternative strategy for the treatment of bone/periodontal defects or diseases that affect the musculoskeletal system.


However, these studies are limited and have not been discussed in detail. In this regard, this research work was designed to evaluate the contribution of the scattering effect in different systems of carbon quantum dots used in a possible luminescent solar concentrator. To carry out this study, C-QDs and SiO2 Nps were synthesized by hydrothermal methods and the Stober method, respectively. We used different concentrations of both materials to fabricate film LSCs (10 × 10 cm2). The results show that the light scattered by the SiO2 Nps has a double contribution, in terms of light redirected towards the edges of the window and as a secondary source of excitation for the C-QDs; thus, an improvement in the performance of the LSC is achieved. The best improvement in photoluminescence is achieved when the films are composed of 20% wt carbon quantum dots and 10% wt SiO2 Nps, reaching a gain of 16% of the intensity of the light incident on the edges of the window with respect to the LSCs where only C-QDs were used.Luminescent solar concentrators (LSCs) have become an attractive way to produce green energy via their integration into buildings as photovoltaic windows. Recently, carbon quantum dots (C-QDs) have become the most studied luminescent material for the manufacture of luminescent solar concentrators due to their advantages, such as low toxicity, sustainability, and low cost. Despite the advantages of carbon quantum dots, they remain a low-efficiency material, and it is difficult to fabricate LSCs with a good performance. To address this problem, some of the research has used SiO Luminescent solar concentrators (LSCs) have become an attractive technological area for solar energy conversion devices that can integrate with modern urban architecture ,2,3. GenDespite their many advantages, the quantum emission yield and brightness of organic-based quantum dots suffer due to their high sensitivity to contaminants in their environment; this is due to the presence of carboxyl groups that form non-radiative recombination centers. To deal with this issue, surface passivations have had very favorable results; for this strategy, a wide variety of both organic and inorganic materials has been used; in addition, different polymers have been used that have the function of forming a thin insulating capping layer that shields C-QDs from the adhesion of impurities and further improves their fluorescence ,16. It hG factor is defined as the ratio of the area of the top surface and the surface of the edge that is favored; thus, it is possible to improve the optical efficiency [LSCs based on C-QDs have been reported in different configurations, including the single-layer configuration, which is the most basic; it involves incorporating the C-QDs and embedding them into a polymer matrix . Anotherficiency . In addificiency ,23,24.On the other hand, there are different loss mechanisms in LSCs; among the most common are front surface reflection, transparency to long-wavelength incident light, reabsorption, escape cone losses, and scattering loss ; the lat2 nanoparticles in a polymethyl methacrylate (PMMA) matrix into the system. With this configuration, a part of the solar radiation is transmitted, while another part is redirected towards the edge of the window due to the light scattering caused by the TiO2 Nps. Using the same idea, Feng Zhang et al. [2 aerogel.In 2010, Chau et al.  defined g et al.  report a2 or TiO2 nanoparticles as scattering material to increase the probability of the light captures and absorbance by QDs. Our system involves the use of a nanocomposite polymeric film with C-QDs as a fluorescent emitter and SiO2 nanoparticles as light-scattering material so that the carbon quantum dots receive double excitation. The first is by the direct incidence of solar radiation, and the second is due to the scattering effect produced by the silicon particles. In addition, the scattered light also contributes as an incident light at the edges of the LSCs. In this way, using SiO2 particles can enhance the LSC devices´ efficiency. In the previous research, they were interested in quantifying the efficiency of luminescent solar concentrators using light-scattering particles, such as TiO2 or SiO2 Nps [2 Nps. These nanomaterials were used in different concentrations to fabricate polyvinylpyrrolidone (PVP) nanocomposite films; deposited on a glass substrate, the different LSCs were placed vertically by means of a support, and a halogen lamp was an external light source placed in front of the LSCs. In order to evaluate the light-scattering effect of the SiO2 Nps, the different wavelengths incident at the edge of the window were collected using a collimating lens coupled to an optical fiber connected to a spectrofluorometer. Under this experimental arrangement, the contribution of the different optical phenomena of the system is discussed.In the case of solar concentrators, this strategy has been underexplored; Debije et al.  report aSiO2 Nps ,33. Howe2H4), citric acid (C6H8O7), dimethylformamide (DMF), hexane (C6H14), methanol (CH3OH), sodium hydroxide (NaOH), 2-propanol (C3H8O), deionized water (H2O), ammonium hydroxide (NH4OH), tetraethoxysilian (TEOS) (purity ≥ 99.0%), and polyvinylpyrrolidone (PVP). All the reagents were purchased from Sigma-Aldrich.To carry out this research, the following materials were used: urea  was used. First, we prepared a solution of PVP (PVP K30 concentration: 200 mg mL) in 7 mL of methanol with moderate agitation. Then, different concentrations of C-QDs were dispersed in the PVP solution and sonicated for 10 min to homogenize. Finally, to obtain different LSCs based on C-QDs, the mixture was drop-casted onto the surface of a glass substrate. Before their characterization, the different samples obtained were dried at room temperature for 4 h in order to evaporate the solvent. For the LSCs based on SiO2 Nps and based on a mix of C-QDs and SiO2 Nps, different volumes of C-QDs and SiO2 Nps were dispersed in 7 mL of PVP solution. Then, the same steps explained above were followed. Thus, three different types of LSCs  with different volume concentrations were obtained.Three different types of luminescent solar concentrators were fabricated: LSCs based on C-QDs, LSCs based on SiO2 Nps were analyzed using a TESCAN model Mira 3 LMU scanning electron microscope. A Cary-700 UV–Vis-NIR spectrophotometer was used to obtain the characterization of the absorption and transmission spectra. Photoluminescence (PL) of the C-QDs was performed using a spectrofluorometer . The analysis of the optical phenomena of the different systems was carried out by placing the LSCs vertically using a homemade support placed on the lower edge of the substrate. In order to collect incident light at the edges of the LSCs, on the dais support, we adapted a collimating lens connected to the fiber optic cable of the spectrofluorometer (Silver Nova-Stellar), and as an external light source, a lamp was used , placed in the front at a distance of 20 cm. All the experimentation was conducted in a dark room at room temperature. 2 Nps, a large part of the light that falls on them is scattered in all directions; in the same way, this scattered light reaches the edge directly or by total internal reflection. However, the scattered light can be found in Q-CDs in the path; therefore, the Q-CDs receive double excitation, one from the external light and the other from the scattering effect produced by the SiO2 Nps. Finally, all these wavelengths that reach the edges of the device are collected by the optical fiber and sent to the computer for further analysis.The morphologies of the C-QDs and SiO2 Nps synthesized by the Stöber method are shown in the SEM micrographs in the inset of 2 spheres good candidates as light-scattering agents in different systems [The spherical SiO systems ,38.2. For the reference sample (PVP film without Nps), the spectrum of light in the region from 380 nm to 700 nm exhibits almost the same structure as the radiation spectrum of the external light source (dotted curve); therefore, it is assumed that a part of the light from the incident source travels towards the edge of the window due to the phenomenon of total internal reflection. Subsequently, due to the light-scattering phenomenon, the intensity of the curve increases as the volume of the SiO2 Nps in the LSC film increases, until reaching a maximum of 10% SiO2 Nps. Subsequently, the intensity of the curve begins to decrease due to the saturation of the Nps, which propitiate an obstruction of the free passage of the scattered light towards the edge of the LSC. It can be seen that the main peak of radiation from the lamp, centered at 800 nm, decreased considerably, which indicates that this region is mostly transmitted through the LSC, which is consistent with the reflectance spectrum discussed in As described in the experimental section, the optical fiber mounted in the LSC system is responsible for collecting the different light beams that are directed towards one of the edges of the LSC, for which the emission spectra obtained are the enveloping of the overlap of the different optical phenomena participating in the system. In order to break down the contribution of each of the elements of the composite films, n → π* transition of the C=O bond in the carbon cores and the transition from the surface state with lone electron pairs, respectively [The absorption and emission spectra of the C-QDs are shown in ectively ,40. The ectively ,42,43.2 systems, the C-QD concentration with the best performance (LSC-CQD 20%) was chosen in combination with different concentrations of SiO2 Nps. The emission spectra of these systems are shown in 2(5%), the intensity has a slight increase with respect to the sample without SiO2 Nps; later, for LSC-CQD(20%)-SiO2(10%), the maximum intensity is achieved. However, as the SiO2 amount increases, the system is affected, and the intensities decrease. This behavior is attributed to the saturation of the system, where the same particles represent a blockage that decreases the amount of light that reaches the edges of the glass; however, it can be observed that the quenching of the curves occurs at a different rate between the bands’ centers at 550 nm and 750 nm, which is due to the fact that the 550 nm band corresponds to the emission of the Q-CDs that receive double excitation, from the incident light from the external source and from the light scattered by the SiO2 particles.To analyze the contribution of the light-scattering phenomenon in the LSC-CQD-SiO2(10%), reaching an increase of 16% of the total area under the curve with respect to the LSC-CQD(20%) sample. In the same way, it can be observed that the curve centered at 500 nm on the LSC-SiO2 spectra is not present in the PL curves of the LSC-CQD and LSC-CQD-SiO2 samples: this corroborates the fact that this band has been absorbed by the C-QDs as a second source of excitation, thus causing an increase in the intensity of the sample LSC-CQD(20%)-SiO2(10%).In order to obtain a better comparison, 2 particles.According to what has just been discussed, it is important to note that the spectrum of light scattered by the silicon particles must coincide with the C-QDs’ excitation region; in this way, the versatility of what is presented in this work is remarkable since, according to the Mie theory in corroboration with different works ,45, the 2 systems are shown in 2 particles into the system  the transmittance decreases only 3% with respect to the LSC-CQD. The inset shows the qualitative transparency of the sample, which makes it viable for use in buildings. However, it is known that one of the greatest difficulties of the LSCs is the scaling of the system since the photons that contribute to the efficiency of the CSSL at greater travel distances increase the probability of falling into the escape cone; recently, however, in addition to the application in buildings, other types of applications have been proposed for LSCs, where large-scale configurations are not required, such as photochemical or electrochemical reactors and microalgal production [On the other hand, the transmittance spectra of the LSC-CQD and LSC-CQD-SiOoduction ,47. With2 Nps. The experimental arrangement allowed the breaking down of the incident radiation at the edge of the LSC; this was attributed to different optical phenomena, including the light scattering due to the SiO2 Nps, the total internal reflection, and the broadening of the C-QD emission, due to a double excitation by the external source and by the light scattered from the SiO2 Nps.In summary, in this study we analyzed the enhancement of the photoluminescence in LSCs sensitized by C-QDs through the scattering effect produced by SiO2(10%) systems reached a 16% increase in the intensity of incident light at the edge of the LSC with respect to the sample where only C-QDs were used. Therefore, the use of the phenomenon of scattering is a strategy with great potential in LSC applications. In order to achieve a better understanding of this study, different characterizations, such as the scattering loss, the quantum efficiency, the scaling of the LSCs´ sizes, and their long-term stability, among others, should be analyzed in future work.Understanding the contribution of these phenomena will allow a better exploration of these systems with other types of light-scattering particles. The results of this work showed that the LSC-CQD(20%)-SiO


The results showed that the aromatic carbon content of Tiebei lignite was 51.98%, the aromatic carbon structure was mainly composed of benzene and naphthalene, and the ratio of aromatic bridgehead carbon to surrounding carbon Xbp was 0.14. Oxygen existed in phenol, ether, carbonyl, and carboxyl; nitrogen-containing structures mainly existed in the form of pyrrole and pyridine; sulfur mainly existed in thiophene sulfur; and aromatic substitution was mainly in the form of trisubstitution. The molecular formula of the macromolecular structure model of Tiebei lignite was C190H161O57N2, and the 13C NMR spectrum of the model was in good agreement with the experimental results, which fully verified the accuracy of the macromolecular structure model of Tiebei lignite. The construction of a macromolecular structure model of Tiebei lignite is essential to intuitively understand the molecular structure characteristics of Tiebei lignite and to provide theoretical support and guidance for the micromechanism research and prevention of lignite spontaneous combustion and other disasters.Mastering the molecular structure of coal is important for the effective utilization of coal. For a detailed study of the microstructural characteristics of Tiebei lignite, its molecular structure was characterized by elemental analysis, solid  Coal is a kind of abundant fossil energy distributed around the world that plays an important role in the fields of global industry, fuel, and energy . In rece13C nuclear magnetic resonance (13C NMR) experiments to analyze and construct the coal molecular model and clarified the mechanism of Dense medium component scaffold (DMC-S) aggregation. Zhang et al.  fro fro27] f13C NMR analysis, aromatic carbons accounted for 51.98%, and so, the total number of carbons in the molecular structure of lignite was 190. According to the elemental analysis of coal samples, we determined that the carbon content of lignite was 58.93%, the oxygen content was 23.55%, the nitrogen content was 0.79%, and the sulfur content was 0.11%. We calculated that the structure had 57 oxygen and 2 nitrogen in the model. Because of the low sulfur content, the number was less than one, and so, the molecular structure of Tiebei lignite constructed in this study did not contain sulfur. According to XPS analysis, the main forms of N in lignite were pyridine nitrogen and pyrrole nitrogen, and the quantity ratio was about 1:1, and so, there was one pyridine nitrogen and one pyrrole nitrogen.The ratio of bridge carbon to peripheral carbon of lignite was 0.14, which showed that the aromatic carbon structure in the model was mainly composed of benzene and naphthalene, supplemented by anthracene. Through a Matlab calculation , we obta13C NMR spectrum of the model with the experimental 13C NMR spectrum, as shown in 13C NMR spectrum of the macromolecular model of Tiebei lignite established in this study was in good agreement with the experimental 13C NMR spectrum. Finally, we determined the molecular formula of lignite in the Tiebei Coal Mine to be C190H161O57N2. We drew a molecular structure model of Tiebei lignite using KingDraw software, as shown in According to the analysis results, we established the macromolecular model of Tiebei lignite, and then, we compared the We introduced the two-dimensional plane molecular model of Tiebei lignite into the Materials Studio molecular simulation software . We geomBased on the results of this study, we drew the following conclusions:13C NMR. The results showed that the ratio of aromatic bridgehead carbon to peripheral carbon Xbp was 0.14; the aromatic carbon rate was 51.98%; the aromatic carbon structure was mainly composed of benzene and naphthalene; the oxygen element existed in phenol, ether, carbonyl group, and carboxyl group; the nitrogen structure mainly existed in the form of pyrrole and pyridine; the sulfur element mainly existed in thiophene sulfur; and the aromatic hydrocarbon substitution was mainly in the form of trisubstitution.The molecular structure of Tiebei lignite was characterized by elemental analysis, FT-IR, XPS, XRD, and 190H161O57N2. We optimized the macromolecular model of Tiebei lignite using the Materials Studio software, and the total energy of molecular structure after annealing optimization was 3577.233 kJ/mol. van der Waals energy was the main component of nonbonding energy, and the energy value changed the most before and after optimization, which was the main factor that kept the molecular structure stable.According to the experimental analysis results, we drew the molecular structure model of Tiebei lignite using KingDraw software, and we determined the molecular formula of the molecular structure model of Tiebei lignite as CThe optimized macromolecular structure model of Tiebei lignite was more three-dimensional. This will help us to intuitively understand the molecular structure characteristics of Tiebei lignite and holds important guiding significance for the mechanism research of coal and gas outburst, coal spontaneous combustion, and other disasters.


Artificial insemination (AI) is the first and the most used biotechnologies in reproduction all over the world. Many studies reported the beneficial role of gonadotropin-releasing hormone (GnRH) administrated some hours before the AI or at the artificial insemination time. This study aimed to assess the effect of GnRH analogs given at the time of insemination on the first, second, and third AIs and to assess the economical implications of GnRH administration. We hypothesized that administration of GnRH at the time of insemination would increase ovulation and pregnancy rate. The study was conducted on small farms in northwestern Romania and included animals of the Romanian Brown and Romanian Spotted breeds. Animals in estrous at the first, second, and third inseminations were randomly divided into groups that received GnRH at insemination and groups that did not. A comparison between the groups was performed and the cost of GnRH administration to obtain one gestation was calculated. The GnRh administration increased the pregnancy rate at the first and the second inseminations by 12 and 18%, respectively. For one pregnancy, the cost of GnRH administration was approximately 49 euros for the first insemination group and around 33 euros for the second insemination group. No improvement of the pregnancy rate was observed after the GnRH administration for the cows at the third insemination, so, for this group, no economic statistics were performed. Artificial insemination is the first and the most used biotechnologies in reproduction all over the world. One of the main objectives for both dairy and beef farm cows is to have high reproductive performances . TherefoNumerous studies have documented the beneficial effects of administering gonadotropin-releasing hormone (GnRH) at or just before the time of AI –16. It wThe majority of cattle in Romania are raised in extensive breeding systems with tie-stalls; in small farms with a number of cows <30, and in medium-sized farms with a number of cows <150 . During This study aimed to assess the effect of GnRH analogs given at the time of AI on the first, second, and third AIs and the economical implication of the GnRH administration. The study involved both Romanian brown and Romanian spotted breeds of cattle, and it was conducted on small farms in northwest Romania.n = 58), farms with several animals that range between 3 and 124. The data were collected from three AI operators that performed 1,322 inseminations in the interval between January 2022 and October 2022. The cows that were included in the study were females with condition body scores between 2.5 and 3.5 out of 5 (n = 420), second insemination group , and third insemination group . All three groups were equally and randomly divided into another two groups. FIG was divided into FIG 1 (n = 210) and FIG 2 (n = 210); for the animals included in FIG 1, no treatment was performed on the AI day, and for the animals included in FIG 2, 100 μg D-Phe 6-gonadorelin  were administrated at the AI time. SIG was divided into Sig 1 (n = 305) and Sig 2 (n = 305). No treatment was performed for Sig 1 group at the AI time, and 100 μg D-Phe 6-gonadorelin  was administrated for Sig 2 group at the AI time. TIG was divided into TIG 1 (n = 146) and TIG 2 (n = 146); for TIG 1, no treatment at the AI time, and 100 μg D-Phe 6-gonadorelin  for TIG 2 group at the AI time  was estabilished the percent of gestation, and the pregnancy was diagnosed by ultrasonography 35–45 days after the insemination. Females diagnosed empty from FIG 1 and FIG 2 groups were included in the following groups, SIG groups and TIG groups, respectively. Empty animals in FIG 2 and SIG 2 were not included in other groups.For all groups  = cost for 1 gestation6 (Euros) = price for GnRH administrationIG2 (number) = number of inseminated animals for the second group Pd (number) = pregnancy differences between Group 2 and 1 t-test was used.All data were entered into Microsoft Excel and statistically analyzed to demonstrate the favorable impact of GnRH at the AI time on small farms in the northwest of Romania. A comparison between the groups was done. To compare the groups and find the significant variation, the The overall pregnancy rate in this research was 53% (704/1320), 47% (313/661) for the groups in which ovarian stimulation with GnRH was not conducted, and 59% (391/661) for the groups where it was performed.t-test value of 0.0045 at p = 0.5) was found.In the case of the cows at the first insemination, the GnRH hormonal stimulation increased the pregnancy rate. From 210 cows who were artificially inseminated without the use of GnRH (group FIG1), 113 animals (54%) had gestation confirmed. Of the 210 animals who were inseminated for the first time using GnRH injection (group FIG2), 139 (66%) had gestation confirmed . After cThe economic statistics showed that for improving the pregnancy with one gestation, it is necessary to spend approximately 49 Euros.t-test determination (0.00011 at p < 0.5). The cost to improve the pregnancy rate with one gestation for the SIG2 group was somewhere around 33 Euros.The administration of GnRH at the AI time in the case of second insemination was likewise linked to a rise in the pregnancy rate. From 305 inseminations performed for the SIG1 group, a total number of 146 pregnancies (48%) was obtained. In the case of the SIG2 group, in which 201 pregnancies were obtained from 305 inseminations, the rate of pregnancy was 66% . A signit-test value of 0.35 at p < 0.5) after the comparison of these two groups. This group did not have economic statistics run because there was no improvement in the pregnancy rate.In our investigation, the third insemination did not have the same effects as the first two groups when GnRH was administered. The pregnancy rate for the TIG1 group was 37%, with 54 of the 146 inseminated cows having gestations that were confirmed. Pregnancy was confirmed in 51 cases in the TIG2 group, which had a slightly lower pregnancy rate (35%) than the TIG 1 group . No signTo achieve the goal of annual calving in cattle, the open days may only be split by a maximum of 90 days . NumerouAccording to Kim and Jeong , cows thSimilar to the results of our investigation, Kaim et al.  observedt-test value 0.000011). The price with the GnRH administration was less than for the first group, at roughly 33 Euros  on the farm. In our study, the pregnancy rate for the second insemination group was similar to that of the first insemination group (66%), but it was lower for the unstimulated group (SIG1) (48 vs. 54%). When GnRH was administered during AI, the pregnancy rate for the SIG2 group vs. the SIG 1 group increased considerably , defined as cows' failure to conceive from three or more regularly spaced services in the absence of detectable abnormalities, is a costly problem for cows' farmers . Repeat The pregnancy rate in the group of cows at the first and second services (12 and 18%) increased as a result of the administration of GnRH at the time of AI. The cost with the GnRH administration to obtain one pregnancy was 49 Euros at the first insemination at 33 Euros at the second insemination. In the case of cows at the third service, no advantage was seen following the GnRH administration. In addition, a thorough examination of the reproductive system is required during the third insemination to determine the cause of infertility.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The animal study was reviewed and approved by the Bioethics Commission of the USAMV CLUJ NAPOCA. Written informed consent was obtained from the owners for the participation of their animals in this study.DB and LB: conceptualization, formal analysis, and writing—review and editing. DB and PO: methodology and investigation. RC: statistical analysis, writing, and validation. DB: writing—review and editing. LB: conceptualization, formal analysis, supervision, methodology, and writing—original draft. All authors contributed to the article and approved the submitted version.


Anatomical diversity of the hard palate in the population involves the need to perform bone and mucosa thickness measurements before palatal micro-implant placement.Since class II malocclusion and lack of space within the dental arch due to early loss of deciduous molars is a common orthodontic problem in the Polish population, bone-anchored distalizers are becoming more and more popular. The aim of the present study was to evaluate palatal soft and hard tissue thickness using cone beam computed tomography (CBCT) at the area of micro-implant placement of two appliances for maxillary first molar distalization: Beneslider and TopJet distalizer. The study data were 100 consecutively selected CBCT images (53 of men and 47 of women). Measurements of bone and mucosa thickness were performed at six locations in the palate and tested according to their correlation with sex and age. The biggest bone and mucosa thickness were recorded in the insertion site of the TopJet miniscrew. Bone thickness in all points of paramedian insertion was significantly greater in males and the mean difference was approximately 1–1.8 mm. Age correlates significantly ( Skeletal anchorage irreversibly changed the process of orthodontic treatment planning enabling tooth movements, previously considered impossible or at least difficult. It also minimized the need for patient cooperation in terms of wearing extraoral appliances or intermaxillary elastics commonly recommended as a method of anchorage reinforcement . Molar dSince class II malocclusion and lack of space within the dental arch due to early loss of deciduous molars is a common orthodontic problem in the Polish population, bone-anchored distalizers are becoming more and more popular ,13. MaxiThe aim of the present study was to evaluate palatal soft and hard tissue thickness using cone beam computed tomography (CBCT) at the area of micro-implant placement of two appliances for maxillary first molar distalization, that are successfully used in the Department of Prosthodontic and Orthodontics of Jagiellonian University in Cracow: Beneslider  and TopJet distalizer  .Beneslider is a distalization appliance supported with two orthodontic mini-implants  positioned medially or paramedially in the anterior palate ,3. The TThe source of data for this cross-sectional retrospective research was cone-beam computed tomography scans of the maxilla of patients of University Dental Clinic in Krakow performed for any reason between 2018 and 2021. The study data were 100 consecutively selected CBCT images (53 of men and 47 of women) that met the inclusion criteria: age of the patient > 11 years, presence of upper first and second premolars, absence of pathologies in the anterior part of the maxilla , and absence of clefts and maxillofacial syndromes. Patients with a history of facial surgery or facial trauma and images with artifacts were excluded from the study. The CBCT scans were obtained using the OP 3D Pro . Average parameters: field of view 130 × 150 mm, average exposure time 8.5 s, average scanning time 39 s, and average voxel size 380 μm–5 mA. All the images were analyzed by a single trained and calibrated senior postgraduate trainee in orthodontics. Intra-examiner error was calculated by repetitive measurements within a 3-week interval on 10 randomly selected scans to determine the reliability. For the analysis of the CBCT images, a medical diagnostic monitor  and InViVo Dental Viewer  were used.the horizontal plane passing through the right and left Orbitale and right and left Porion points;the frontal plane passing through the right and left Porion point, perpendicular to the horizontal plane; andthe sagittal plane passing through the Nasion point, perpendicular to the horizontal and frontal planes.The measurements were performed in three planes of space: Measurements of bone and mucosa thickness were performed at six locations in the palate. In the first part of the study, tissue thickness for TopJet miniscrew was measured as presented in In the second part of the study, tissue thickness for paramedian insertion of Benefit micro-implants was investigated as presented in In the last part of the study, tissue thickness for the median pattern of insertion of Benefit miniscrews was determined. According to the authors of the method, micro-implants should be placed along the x-axis 7–14 mm apart . For theThe abovementioned measurements were tested according to their correlation with sex and age.The study was approved by the bioethics committee of Jagiellonian University (number 1072.6120.132.2020).Data analysis was performed using R software version 4.2.1 .The normality of the distribution of the various groups was tested by means of a Shapiro–Wilk test. Analysis of quantitative variables was performed by calculating mean, standard deviation, median, and quartiles. Qualitative variables were analyzed by calculating the number and percentage of occurrences of each value. A Mann–Whitney test was used to compare quantitative variables between two groups. The relationship between quantitative variables was assessed with Spearman’s coefficient of correlation. A comparison of the values of quantitative variables in three repeated measurements (measurement points) was made using the Friedman test. After detecting statistically significant differences, a post-hoc analysis (Wilcoxon matched-pairs test with Bonferroni correction) was performed in order to identify statistically significantly different measurements. The significance level for all statistical tests was set to 0.05.A total of 100 consecutively selected CBCT images of the maxilla that met the inclusion criteria were assessed in this study. The scans came from 47 women and 53 men with a mean age of 31.9 years for women and 31.7 years for men . The ageTen randomly selected scans were subjected to repeated measurements within a 3-week interval. The concordance of the measurements of quantitative variable assessment with intraclass correlation coefficient type 2 (according to the Shrout and Fleiss classification) indicated excellent accordance between the first and second measurements.Values of the thickness of hard and soft tissues as well as general tissue depth are presented in In the case of median insertion sites, the biggest general tissue thickness was recorded at the level of point B (6.68 ± 1.67 mm) but the greatest bone thickness was observed at B14 point (5.01 ± 2.06 mm). Mucosa thickness was gradually decreasing from point B (1.91 ± 0.7 mm) to point B14 (1.39 ± 0.96 mm).Considering the correlation with the sex of the patients, bone thickness in all points of paramedian insertion was significantly greater in males and the mean difference was approximately 1–1.8 mm .In the case of mucosa thickness, the correlation was not so clear, and a statistically significant difference was found only in the case of B7 and B14 points .p < 0.05) and negatively (r < 0) with the bone thickness only at the B14 point, so the older the age, the thinner the bone at this point (p < 0.05) and positively (r > 0) with the thickness of the mucosa at all points: the older the patient, the thicker the mucosa at each measurement point . Significantly thinner bone levels were located at points B2.5 and B5 . Amri et al., who measures bone thickness 3 mm and 6 mm laterally to the midsagittal line at the level of contact points of first and second premolars, obtained similar results (4.51 mm and 3.65 mm) and concluded that palatal bone thickness tends to decrease from medial to lateral region . NeverthTo minimize the potential risk of incisor root damage, median location of palatal micro-implants along the midpalatal suture is also recommended . HoweverThere are theories that micro-implant resistance to lateral load depends on the ratio of insertion depth to total miniscrew length . As a reConsidering the abovementioned results, it can be summarized that the initial hypothesis of our research was confirmed only to some extent. The thickness of the palatal mucosa tends to increase with the growing distance from the midpalatal suture and decrease from anterior to posterior areas, but in the case of bone thickness, correlation is not so obvious. Indeed, the greatest values were recorded laterally, in the middle of the palate, but bone at the suture and 2.5 mm laterally was thicker than 5 mm from the midline. Conversely, in the sagittal dimension, bone depth in the midpalatal suture seemed to increase with the growing distance from p2 line.We were able to find a correlation between the gender of patients and palatal depth in points M4, B2.5, and B5 , which is in line with other studies ,11. ThisMost of the analyzed studies found no correlation between age and palatal bone thickness, which partially confirms the results of our study indicating poor negative age correlation only in the case of point B14 ,20,21,23In light of the general guidelines regarding recommended minimum bone thickness equal 5 mm , accordiThe results of our study provide information regarding average bone thickness in the area of application of popular bone-borne distalizers; nevertheless, it should be interpreted in the light of certain limitations. One of them is a correlation between palatal bone thickness and skeletal relation in the sagittal and vertical plane: palatal bone thickness seems to be thinner in class III and high-angle patients compared to class I and normal and low-angle cases ,29,30. AThe anatomy of the hard palate presents a considerable variety of shapes. In the studied group, the most preferable conditions for micro-implants were found at the point of insertion of the TopJet miniscrew (8.01 mm) and the thinnest bone was localized 5 mm laterally to the midpalatal suture (4.28 mm). In the case of 15 patients, bone depth did not reach a minimum of 5 mm at any of the analyzed points. Greater values of bone thickness in the paramedian areas were observed in the case of males. While no clear correlation was found between age and bone thickness, the thickness of the mucosa tends to increase with age. Although CBCT use in dentistry is sometimes controversial due to the relatively large radiation dose, anatomical diversity of the hard palate in the population involves the need to perform bone and mucosa thickness measurements before palatal micro-implant placement to select the appropriate size of the miniscrew and suitable area of the hard palate.