Polydimethylsiloxane (PDMS) has many advantages, but the friction coefficient generated by contact with the counter material is high. The purpose of this study is to reduce the friction coefficient by forming hierarchical micro/nanopatterns on the PDMS surface using the imprinting method. In addition, the optimum conditions for reducing the friction coefficient by controlling the sliding speed and normal load were determined. After contacting flat bare PDMS and hierarchical micro/nanostructured PDMS with a counter tip made of polyurethane (PU), the change in friction with sliding speed and vertical load was evaluated. Under normal load conditions, the average friction coefficient of the bare PDMS decreased as the sliding speed increased, and that of the patterned PDMS slightly increased. Regardless of the sliding speed, the friction coefficient decreased as the normal load increased for both specimens. At a sliding speed of 4 mm/s under a load of 10 mN, the friction reduction effect of the pattern structure was the largest at 79%. Overall, the greatest friction reduction effect (84%) was confirmed in patterned PDMS with the lowest friction coefficient under the conditions of 4 mm/s, 50 mN, compared to bare PDMS with the highest friction coefficient under the conditions of 4 mm/s, 10 mN. PDMS has high elasticity and rigidity, and is known to be a transparent, flexible, and biocompatible material . Owing tVarious experimental methods have been used to improve the friction properties of PDMS ,8,9. As For this reason, to eliminate the disadvantages caused by other materials mixed with PDMS, we attempted to form a specific pattern on the surface of PDMS that could improve the friction properties using only PDMS itself without adding other materials . First, In this study, a relatively simple etching method that can form random nanoscale morphologies on the surface of a metal plate by immersing it in a chemical solution was utilized. Since the surface of the metal plate is etched by a chemical reaction simply by immersing the metal plate, it is easy to form a pattern on the surface of an object with a complicated shape. In addition, because of the chemical reaction between the atoms of the metal plate and the chemical solution, the size of the pattern formed on the surface to be etched is of a micro/nanoscale, and a very small contact area with the counter surface can be expected. After the PDMS solution poured on the master surface is cured, it is peeled off, and a pattern with random micro/nanoscale morphologies, which is the opposite shape of the master surface, can be formed on the cured PDMS surface. For this hierarchical micro/nanopatterned PDMS, the friction properties were analyzed by changing the sliding speed and normal load.n-Hexane (C6H14) were mixed at 1:1000 was used to form a superhydrophobic self-assembled monolayer (SAM) coating on the patterned surface to make it easy to peel off. It was completely dried in an oven at 40 °C for 40 min. The PDMS solution, in which the PDMS base and curing agent were mixed in a weight ratio of 10:1, was poured onto the hierarchical micro/nanopatterned master on which the oxide film and SAM coating were formed, and then cured in an oven at 60 °C for 3 h. The hierarchical micro/nanopatterned PDMS, which has a shape opposite to the surface pattern of the master, was peeled off. To evaluate the effect of hierarchical micro/nanopatterns on the friction properties of PDMS, bare PDMS cured on a flat Al plate was also prepared as a comparative specimen.To form hierarchical micro/nanopatterns on the PDMS surface, an aluminum (Al) plate  was used to fabricate a master for use in the imprinting process. The surface morphologies of the flat bare PDMS and hierarchical micro/nanopatterned PDMS specimens were measured using a high-resolution field emission scanning electron microscopy (FE-SEM) . The surface roughness values of the two specimens were obtained using a 3D laser scanning confocal microscope .The friction properties of the flat bare PDMS and hierarchical micro/nanopatterned PDMS specimens were measured using a tribotester  driven by a reciprocating sliding motion ,13,14,15As a result of the friction test conducted for 1000 cycles at sliding speeds of 4, 12, and 20 mm/s under a normal load of 10 mN, the change in the friction coefficient can be confirmed, as shown in Bare PDMS exhibits a high friction coefficient of 3.84 at a sliding speed of 4 mm/s under a normal load of 10 mN because strong adhesion occurs when it is in contact with the PU film on the counter tip, owing to the strong adhesive properties of the polymer. At a sliding speed of 12 mm/s, the friction coefficient decreased to 3.07, and at 20 mm/s, the friction coefficient was significantly reduced to 2.67. Compared with 4 mm/s, at sliding speeds of 12 and 20 mm/s, the friction coefficient decreased by approximately 20% and 30%, respectively. In the case of normal loads of 30 and 50 mN, the reduction amount of the friction coefficient according to the increase in the sliding speed was smaller than in the case where a normal load of 10 mN was applied. In bare PDMS, under the same normal load, when the sliding speed increased, the adhesion between the PDMS and PU counter tip surfaces was momentarily detached, and the counter tip passed as it was before the compressed surface of the PDMS recovered . AccordiOverall, for bare PDMS, the highest friction coefficient (3.84) was generated when the reciprocating sliding motion was performed at a sliding speed of 4 mm/s under a normal load of 10 mN, and the lowest friction coefficient (1.82) was observed for sliding at 20 mm/s under a normal load of 50 mN. That is, it was confirmed that the friction reduction effect of bare PDMS increased as the sliding speed and normal load increased. The friction coefficients of the patterned PDMS showed the highest value (1.00) at a sliding speed of 20 mm/s under a normal load of 10 mN and the lowest value (0.60) at a sliding speed of 4 mm/s under a normal load of 50 mN. In other words, the friction reduction effect of the patterned PDMS increased as the sliding speed decreased and the normal load increased. Therefore, the lowest friction coefficient (0.60) of the patterned PDMS was for a sliding speed of 4 mm/s and normal load of 50 mN, a reduction by 84% compared to the highest friction coefficient (3.84) of the bare PDMS for a sliding speed of 4 mm/s and normal load of 10 mN.In this study, the friction properties of flat bare PDMS and hierarchical micro/nanopatterned PDMS specimens were analyzed. Both specimens were prepared using an imprinting method. Bare PDMS was imprinted by pouring the PDMS solution onto a flat Al plate, and patterned PDMS was imprinted by pouring the PDMS solution onto hierarchical micro/nanopatterns formed on the Al plate surface through surface treatment processes comprising etching, oxidation, and SAM coating. It was confirmed that the surface morphology of the patterned PDMS was rougher than that of bare PDMS. For the two specimens with different surface properties, the changes in friction properties according to the sliding speed  and normal loads  were compared. As the sliding speed increased, regardless of the normal load, the friction coefficient of the bare PDMS decreased, while that of the patterned PDMS increased. Under the same experimental conditions, the friction reduction effect of the patterned PDMS was the highest compared to that of the bare PDMS, with a sliding speed of 4 mm/s and a normal load of 10 mN, and the friction coefficient was reduced by 79%. Overall, the friction reduction effect of 84% under the condition of 50 mN, 4 mm/s, showing the lowest friction coefficient in the patterned PDMS compared to the condition of 10 mN, 4 mm/s, showing the highest friction coefficient in the bare PDMS, has appeared. Through this study, it was confirmed that a large reduction in friction can be obtained by controlling the surface patterning and contact conditions . The results of this study are expected to be helpful in solving the problems in various fields caused by the intrinsic friction properties of PDMS.


The recycling and reuse of construction waste have not only effectively protected natural resources but also promoted the sustainable development of the environment. Therefore, in this article, waterborne polyurethane (WPU) as a promising new polymer reinforcement material was proposed to reinforce the road demolition waste (RDW), and the mechanical performance of WPU-reinforced RDW (named PURD) was investigated using triaxial unconsolidated and undrained shear (UU) and Scanning Electron Microscope (SEM) tests. The results showed that under the same curing time and confining pressure, the shear strength of PURD increased with the increase in WPU content. When the WPU content was 6%, the WPU presented the best reinforcement effect on RA. The failure strain of PURD increased with the increase in confining pressure, but increased first and then reduced with the increase in WPU content. The specimens with 5% WPU content showed the best ductility. At the curing time of 7 and 28 days, the internal friction angle and cohesion of PURD increased with the increase in WPU content, and they reached a maximum when the WPU content was 6%. The internal friction angle barely budged, but the cohesion increased obviously. The enhancement effect of WPU was attributed to the spatial reticular membrane structure produced by wrapping and bonding particles with the WPU film. Microscopic analysis showed that with the increase in WPU content, the internal pore and crack size of PURD gradually decreased. As the WPU content increased, the WPU film became increasingly thicker, which increased the adhesion between WPU and RA particles and made the structure of PURD become increasingly denser. Over the past few decades, infrastructure construction has flourished due to widespread urbanization, the world population soaring, and the changing economic landscape of the developing countries ,2. This In the process of pursuing sustainable development in the construction industry, cement production consumes a lot of energy and emits a large amount of greenhouse gases, and the total emission accounts for about 8% of the total global carbon dioxide emissions ,17,18. TCurrently, the high-molecular polymers have been applied in the reinforcement of building materials, among which polyurethane is particularly prominent in improving strength and stability ,28,29,30In summary, it is found that due to the pollution problems caused by the use of cement and the accumulation of construction waste, as well as the shortcomings of high energy consumption in the mining of construction materials, the current society urgently needs to find new environmental protection reinforcement materials and resource utilization methods of construction waste to alleviate the pressure on the environment and resources. At present, a large number of research works mainly focuses on natural building materials such as sand, marine clay, gravel, and concrete, while the research on recycled building materials is relatively rare. Therefore, using waterborne polyurethane as a new environmental protection reinforcing material to study its effect on the mechanical properties of recycled aggregate can realize the engineering recycling of building materials, to achieve the social benefits of protecting the environment and saving resources. Meanwhile, the abundant research work has only explored the compressive strength, flexural strength, shear modulus, and damping ratio of various building materials, and has not yet conducted in-depth research on shear strength.The objective of this paper was to explore the effect of WPU on the triaxial shear strength of RA by unconsolidated undrained shear (UU) tests on the WPU-reinforced RA (PURD) specimens under different ages, confining pressures, and contents of waterborne polyurethane. Meanwhile, the microstructure of PURD was characterized by scanning electron microscope (SEM) tests to explore the WPU reinforcement mechanism on RA.2 and CaCO3. The acquisition process of RA required for the test is shown in The RA was collected from the abandoned road section of Erhuan North Road, Shaoxing City, Zhejiang Province, which was the mixed gravel after the crushing of the roadbed base material. The main chemical components of RA are SiOThe RA was reinforced by WPU with four contents. The WPU solutions were diluted with water to a solution with a low content, and then the RA was mixed with WPU. The specific test scheme is shown in The WPU content in the test scheme is the mass ratio of WPU and RA, and the solid content of the solution needs to be considered. The moisture content of the specimen was set to 16%, the main purpose was to keep consistent with the natural moisture content of the RA, and the moisture contained in the WPU solution should be considered. The calculation process of the test mix proportion is shown in Equations (1) and (2).The preparation process of the UU test specimens in this study is divided into the following steps see  as per t(1)The RA was placed in a constant-temperature oven at 105 °C for 24 h to ensure that the moisture in the RA was completely dried, and then cooled to room temperature.(2)The corresponding qualities of the RA, WPU solution, and water were weighed according to the mix proportion set in (3)An amount of 154 g of the mixture stirred evenly was shaped in a cylindrical mold with a height of 80 mm and a diameter of 39.1 mm. It must be fully compacted with a jack and statically pressed for 10 min.(4)The specimens were demolded after being fully compacted. Then, the specimens were cured in a standard curing box for 7 and 28 days, respectively. The curing temperature was controlled at 20 ± 2 °C, and the relative humidity was >95%.The samples for the SEM test were from the dried sample blocks of broken sample in the UU test. A layer of conductive adhesive was pasted tightly on the sample with a thin rod, and then the specimen particle powder on the surface was blown away with a suction balloon to avoid polluting the instrument lens.The instrument used in the UU test was a fully automatic triaxial shearing instrument (TKA.TTS.3S) produced by Nanjing, China, TKA Technology Co., Ltd. The four confining pressures  were applied, and the loading rate was set to 1 mm/min. According to the relevant provisions of the Standard for geotechnical testing method (GB/T 50123-2019) , after tThe equipment used for the SEM test was a high- and low-vacuum scanning electron microscope (JSM-6360LV), which is produced by Tokyo, Japan, Electronics Co., Ltd. The specimens damaged from the UU test were placed in an oven for 24 h, and the test was performed after the specimen was dry. Finally, the SEM images were taken under different magnifications, and the magnifications of this study were selected as 500× and 2000×.According to the deviatoric stress q and the axial strain ε obtained from the test, the deviatoric stress–strain relationship curves of PURD specimens are drawn in qmax, kPa, which can reflect the shear strength of PURD. According to the deviatoric stress–strain curves of PURD from By further analyzing The curing time of cement-based materials has an important influence on the strength of the specimen. Cement usually completes almost all hydration reactions when curing for 28 days, and the strength of the specimen will increase significantly compared with that under the curing time of 7 days. Therefore, it is necessary to explore the effect of curing time on WPU-strengthening RA ,45. CompThe strain corresponding to the peak strength of PURD in the deviatoric stress–strain curve in According to Comparing the failure strain of PURD with different WPU contents, it can be found that the failure strain of PURD increases first and then decreases with the increase in WPU content under the curing time of 7 and 28 days. Under two curing times and four confining pressures, the failure strain of PURD-5 reaches the maximum value. Compared with PURD-3, the failure strain increases by 41%, 50%, 124%, and 99% at the curing time of 7 days, and increases by 61%, 37%, 31%, and 41% at the curing time of 28 days, respectively. The growth rate is relatively large. The above analysis shows that PURD-5 exhibits the best ductility, mainly because the increase in WPU content will make the cementation between the RA particles more obvious, and there will be a certain “pulling” effect between particles to prevent brittle failure . Howeverσ as the abscissa, the shear stress τ as the ordinate, (σ1f + σ3f)/2 as the center, and (σ1f − σ3f)/2 as the radius, the limit Mohr stress circle is drawn on the τ-σ stress plane diagram. Then, the common tangents of the Mohr circle under four different confining pressures are drawn, which are the shear strength envelopes of the limit Mohr stress circle, where σ1 represents the large principal stress, σ3 represents the small principal stress, and the subscript f represents the limit equilibrium state, that is, the molar stress circle is tangent to the shear strength envelope, which means that the shear stress on the τ-σ stress plane is equal to the shear strength. Based on this, the limit Mohr stress circles and shear strength envelopes of PURD with different WPU contents under 7 and 28 days of curing time were drawn, respectively WPU can obviously improve the shear strength of RA. The shear strength of PURD increases with the increase in WPU content. When the WPU content is 6%, the shear strength reaches the maximum value, which is nearly 30% higher than that of PURD-5. However, the curing time has little effect on the reinforcement effect of WPU, and the increase in shear strength can be ignored. Therefore, WPU can directly exert all strength effects in a short curing time, and is relatively flexible in engineering application, which is a very effective and promising new type of high-molecular polymer reinforcement material.(2)WPU has great influence on the ductility of RA. The failure strain of PURD increases with the increase in confining pressure, and first increases and then decreases with the increase in WPU content. The failure strain of PURD-5 reaches the maximum value. Compared with PURD-3, the failure strain of PURD-5 increases by 41%, 50%, 124%, and 99% at 7 days and 61%, 37%, 31%, and 41% at 28 days, respectively. The growth range is large, so PURD-5 shows the best ductility.(3)WPU can obviously increase the cohesion of RA. The internal friction angle and cohesion of PURD increase with the increase in WPU content, which is highly consistent with the change law of shear strength. However, the increase in internal friction angle is small, only about 5%, and the increase in cohesion is obvious. At the curing time of 7 days, the cohesion of PURD-6 increases by 144%, 142%, and 91%, respectively, compared with those of PURD-3, PURD-4, and PURD-5. At the curing time of 28 days, the cohesion of PURD-6 increases by 141%, 134%, and 77%, respectively, compared with those of PURD-3, PURD-4, and PURD-5.(4)The enhancement effect of the polymer can be attributed to the spatial reticular membrane structure produced by wrapping and bonding particles with the polymer film. With the increase in WPU content, the internal pore and crack size of PURD gradually decreases, and the number of them also decreases. Meanwhile, the polyurethane-wrapped RA particles gradually form agglomerates and finally produce a large number of polyurethane films. With the gradual increase in WPU content, the polyurethane film becomes increasingly thicker, which makes the bonding effect of the polyurethane on the RA particles become increasingly stronger, and the overall structure of the PURD becomes increasingly denser.(5)In this test, the mechanical strength of RA is obtained on the basis that the mud content is 20.5%. If different practical projects need to obtain higher mechanical parameters, it is necessary to reduce the mud content of RA. Similarly, if the engineering has low requirements for mechanical parameters, the requirements for mud content can be appropriately relaxed. Thus, the processing steps for RA can be appropriately reduced.To explore new environmentally friendly reinforcement materials and realize the recycling and utilization of construction waste, a new reinforcement method using PU to enhance RDW was proposed in this study, thus reducing the environmental pressure and resource shortage. The UU test and SEM test were conducted to investigate the reinforcement performance and mechanism of PU. The main conclusions are presented as follows:


The effects of PHAs film surface modification, depending on the composition and ratio of monomers according to the results of the study of SEM and AFM, contact angles of wetting with water, adhesion and growth of fibroblasts have been revealed for the laser radiation regime used. Under continuous irradiation with vector lines, melted regions in the form of grooves are formed on the surface of the films, in which most of the samples have increased values of the contact angle and a decrease in roughness. The quasi-pulse mode by the raster method causes the formation of holes without pronounced melted zones, the total area of which is lower by 20% compared to the area of melted grooves. The number of viable fibroblasts NIH 3T3 on the films after the quasi-pulse mode is 1.5–2.0 times higher compared to the continuous mode, and depends to a greater extent on the laser treatment mode than on the PHAs’ composition. The use of various modes of laser modification on the surface of PHAs with different compositions makes it possible to influence the morphology and properties of polymer films in a targeted manner. The results that have been obtained contribute to solving the critical issue of functional biodegradable polymeric materials.The results of surface modification of solvent casting films made from polyhydroxyalkanoates (PHAs) of various compositions are presented: homopolymer poly-3-hydroxybutyrate P(3HB) and copolymers comprising various combinations of 3-hydroxybutyrate (3HB), 3-hydroxyvalerate (3HV), 4-hydroxybutyrate(4HB), and 3-hydroxyhexanoate (3HHx) monomers treated with a CO The intensive use of non-destructible synthetic plastics and their accumulation in the biosphere is a global environmental problem ,2, the sThe first discovered and massively studied representative of PHAs is )—a homopolymer of 3-hydroxybutyric acid. Despite the biodegradability, the application potential of this representative is sharply limited due to the high crystallinity (above 70%) and hydrophobicity, the tendency to “physical aging”, and disordered crystallization ,28. In tMethods of laser action for the modification of polymer products have been used relatively recently. The main advantage of this method lies in the selective nature of the change in the surface properties of the product without violating the integrity of the material itself and without the accumulation of toxic substances. The papers on this topic describe the results of using various types of lasers  to modify the surface of various biopolymers—polylactide, polycaprolactone, polyglycolide, chitosan, etc., by various methods  ,36,37,38The effect of laser processing is determined by the type and composition of the material, laser parameters, and the processing mode . DependiThere are few studies characterizing the potential of laser surface treatment of PHAs polymer products. Using homogeneous poly-3-hydroxybutyrate P(3HB) as an example, the possibility of changing the surface and bulk properties of products made from this polymer by laser treatment has been shown ,45,46,472 laser) or ultraviolet spectral regions are suitable for their processing  2 laser) PHAs is a family of polymers of different chemical compositions, in which, depending on the conditions of biosynthesis, the set and ratio of monomers in the C-chain can vary over a wide range, which affects the properties of the polymers themselves, as well as the properties of the resulting polymer products ,10,11,12In the present work, for the first time, the effect of laser treatment is researched towards the microstructure, surface properties, and biological properties of films obtained from various types of PHAs copolymers: P(3HB-co-3HV), P(3HB-co-4HB), and P(3HB-co-3HHx), in which the ratio of monomers differ significantly. As noted above, despite the fact that research on laser processing for the modification of various materials is very active, in relation to PHAs, these studies are very limited. In this case, P(3HB) homopolymer and P(3HB-co-3HV) copolymers with a low monomer content, about 8–11 mol.%, have been studied ,48,49. I3)-CH2-CO-] and copolymers containing the 3HB monomer and another monomer in various proportions. The second monomers differ in chemical structure and C-chain length: 4-hydroxybutyrate (4HB) [-O-CH2-CH2-CH2-CO-], 3-hydroxyvalerate (3HV) [-CH(C2H5)-CH2-CO-], and 3-hydroxyhexanoate (3HHx) [-O-CH(C3H7)-CH2-CO-]. The biosynthesis of polyhydroxyalkanoates has been carried out using the bacterial strain Cupriavidus necator B-10646 according to our own developed technology 2-laser rvely. In , Michaljrate. In , Çatıker3 mm2 in area, and up to 200 µm apart from each other is observed on samples of this copolymer with a 3HV monomer content of 11 mol.% after Nd:YAG laser irradiation [+ plasma. This causes a decrease in the contact angles from 60–68° to 40–54° and significantly increases the surface roughness [Regarding the laser modification of the surface of films made of P(3HB-co-3HV) copolymers, several works are known in which samples with a low (about 10 mol.%) content of 3HV monomers are used. The formation of micropores 150 × 100 µm in size, 11.7 × 10adiation . On filmadiation . Films ooughness .2 laser treatment has changes in roughness and wettability; Femtosecond laser modification of thin chitosan films increases the surface roughness from 0.5 to almost 3 μm [The results of studying the consequences of laser processing of polymer products made from copolymers P(3HB-co-4HB) and P(3HB-co-3HHx) in the available literature have not been found. At the same time, the effect of laser processing on the change in the morphology and surface roughness is shown on other polymeric materials. Thus, on the surface of films obtained from carbon-coated polyethylene, the wettability and roughness indices increase after laser treatment . The modost 3 μm ,61.The revealed opposite changes in the angle in response to laser irradiation in the studied film samples obtained from PHAs of various chemical compositions, apparently, can be legitimately associated with differences in their properties, particularly the degree of crystallinity . As is kThe obtained results of changes in the roughness of PHAs polymer films of various chemical compositions and the value of the contact angle of wetting with water after continuous laser irradiation do not correspond to the established ideas and models of Cassie Baxter and Wenzel, which characterize changes in the wettability of surfaces in relation to roughness. Thus, using the Cassie-Baxter model, amorphization and an increase in the hydrophobicity of the surface are explained as the polar components of the free surface energy increase and the roughness indices change. In this case, the increase in hydrophobicity is due to the formation of a Cassie−Baxter state in which the liquid does not penetrate into the hollows of the corrugated surface and, consequently, faces a composite interface consisting of both solid and vapor. In this case, the increase in hydrophobicity is due to the formation of a Cassie−Baxter state in which the liquid does not penetrate into the hollows of the corrugated surface and, consequently, faces a composite interface consisting of both solid and vapor ,64,65. TIt should be noted that the development of the theoretical concepts of the models of Cassie–Baxter and Wenzel reveals the presence, in a number of cases, of limitations for their application. So, A. Giacomello and colleagues have used atomistic simulations aimed at computing the free energy of the stable and metastable states of the system as well as the intermediate states separating them, and have found that the usual description in terms of the Cassie−Baxter and Wenzel states is insufficient, as the system presents two states of the Cassie−Baxter type. These states are characterized by different curvatures of the meniscus. The measured free energy barrier separating the Cassie−Baxter from the Wenzel state (and vice versa) largely exceeds the thermal energy, attesting the existence of Cassie−Baxter/Wenzel metastabilities. Finally, it has been found that the Cassie−Baxter/Wenzel transition follows an asymmetric path, with the formation of a liquid finger on one side of the groove and a vapor bubble on the opposite side .It should be emphasized that the wettability of the surface, which changes as a result of the formation of laser-induced periodic surface structures (LIPSS), depends not only on the microrelief and roughness indices, but also on other factors.As shown by A. B. D. Cassie and S. Baxter , in the Busscher et al.  have detThe change in the wettability of surfaces is also influenced by the regime itself and the conditions of the medium during laser irradiation. Changes in the input parameters  lead to a change in the surface and the magnitude of the angles. Scaffolds that have been treated in air or oxygen show an increased concentration of atomic oxygen and also ablation of the material. Oxygen concentrations tend to be higher and contact angles are smaller for surfaces where ablation is more pronounced. This effect is independent of the fact that the ablation is caused by higher laser power, reduced scan space, or shorter defocus distance . It is s2 laser processing films are obtained from various polymers (PE), polypropylene (PP), polytetrafluoroethylene (PTFE), polystyrene (PS), and polyethylene terephthalate (PET), it is found that the chemical composition affects the ablation and degradation of the surface layer of the polymer and, consequently, the influence on the contact angle of the liquid with the surface and the value of the contact angle [2 laser against the background of a change in surface roughness, no change in wettability is detected, which indicates the absence of the formation of polar functional groups [There are works that show the influence of the chemical composition of the material on the consequences of laser processing. For example, when Fct angle . On the l groups .In general, these published data, as well as the complexity and ambiguity of the results obtained during laser processing of PHA films with different chemical compositions and properties, indicate the need for special theoretical and experimental studies to understand the patterns of changes in the surface structure of polymer films obtained from these semi-crystalline and degradable polymers.The second studied mode of processing polymer films from PHAs of various compositions  is quasiAs a result of the processing of films in the quasi-pulse mode, SEM images  show theThe hole sizes (192.0–177.0 µm) and their total area (11.3–9.9%) in two samples obtained from P(3HB-co-3HV) copolymers with an average and the highest content of 3HV monomers are higher than for films from P(3HB), but comparable. That is, they do not depend on the ratio of monomers in this type of copolymer. A similar effect of the absence of influence of the content of second monomers on the sizes of formed holes after laser irradiation in the quasi-pulse mode is typical for films obtained from another type of PHAs—copolymers P(3HV-co-3HHx) with the lowest (9.0 mol.%) and the highest (38.0 mol.%) content of 3HH monomers. The diameter of the wells in these two samples and their total area are the same, about 160 µm and 8.3–8.3%. It should also be noted that the dimensions of the holes and their total area in copolymer samples from PHAs with an average content of second monomers (close to 30 mol.%) are similar to the previously obtained results in the quasi-pulse mode of processing copolymer films of a similar composition, but at a lower radiation power (13.5 W) .The mode of quasi-pulse laser radiation, similarly to the continuous processing mode, has an impact on the surface roughness of polymer films . The surThe effect of quasi-pulse radiation on the surface roughness indices of copolymer films is ambiguous and depends not only on the chemical composition of the copolymers and on the type of the second monomer, but also on its content in the copolymers. All roughness indices  for films obtained from the P(3HB-co-3HV) copolymer with a low (15.0 mol.%) and medium (27.2 mol.%) content of 3HV monomers decrease by 3 or more times relative to untreated films, amounting to arithmetic mean and root mean square roughness of the order of 100 microns or less; the Sz values are also low, on the order of 600–690 µm. However, with an increase in the content of 4HV monomers to 65 mol.%, the roughness indices Sa, Sq, and Sz increase, respectively, to 419; 486, and 2,284 µm. In the continuous processing mode, the roughness indices of this sample, on the contrary, decrease significantly.A change in the content of 4HB monomers in the P(3HB-co-4HB) copolymers has the same effect on the film surface roughness parameters. Thus, upon irradiation of the films, regardless of the monomers containing 4HB  after the quasi-pulse treatment, the values of Sa increase, respectively, to 275, 442, and 5,153 µm; Sq—up to 442, 561, and 388 µm; Sz—up to 305, 388, and 2,612 microns, which is higher compared to untreated films (from 10–15 to 100% or more) and several times higher than all these indicators for films after processing in continuous mode, which has the opposite effect—causing a decrease in roughness.For films obtained from the P(3HB-co-3HHx) copolymer, the effect of the quasi-pulse processing mode is generally comparable with the effect of the continuous processing mode; the quantitative indicators of roughness are basically similar. All measured roughness indices for film samples, regardless of the content of 3HHx monomers in the copolymer, are low and close to those for untreated films, except for the value of Sz, which decreases to 869 and 972 μm at a 3HHx content of 16.4 and 38.0 mol.%, which is inferior to the values of untreated samples and after continuous irradiation (1200–1600 µm).Thus, the quasi-pulse mode of laser processing increases the roughness indices for all types of P(3HB-co-4HV) copolymer films and the P(3HB-co-3HV) sample with the highest content of 3HV monomers. In relation to the rest of the samples, the effect is generally the opposite, and the roughness indices decrease relative to the original films. The complexity and ambiguity of the influence of the composition of polymers on the surface roughness indices of PHAs films has been noted in a number of works. For example, it has been shown in  that theThe results of the effect of quasi-pulse laser treatment on the value of the contact angle are shown in Similar to the effect of the continuous treatment mode, as a result of quasi-pulse treatment of the surface of films made from the P(3HB) homopolymer, the value of the contact angle significantly decreases, amounting to 74.9°, relative to 92° for the initial films. The angle of the initial copolymer films is lower than that of the homopolymer (it is noted above). A significant change in the angle value after irradiation of films from all types of P(3HB-co-3HV) copolymers is not observed; values remain close to those of the untreated films. Surface hydrophilization takes place during irradiation of films obtained from the P(3HB-co-4HB) copolymer with a low (14 mol.%) and medium (35.5 mol.%) content of 4HB monomers, for which a significant decrease in the angle values is recorded . At the Differences in the degree of crystallinity in the studied samples of films obtained from PHAs of various compositions, most likely, affect the formation of the melt and the evaporation processes in the zone of exposure to the laser beam, as well as the subsequent crystallization of the material as it cools. These processes could cause differences in surface formation in the formation of LIPSS ,79. In tIn principle, taking into account the larger size of the obtained structures both in terms of period and depth, these results may be consistent to some extent with the Cassie–Baxter model for explaining the increase in surface hydrophobicity after laser irradiation of individual film samples obtained from the P(3HB) homopolymer and P(3HB-co-3HHx) copolymers of these samples. According to this model, the contact angle can increase even if the contact angle of the liquid on the original (unmodified) surface is less than 90° .As noted above, in the present work for the processing of polymer films, a quasi-pulse mode has been used using an optical system of mirrors, which interrupts continuous laser irradiation, which makes it difficult to discuss the results of colleagues who have used the true pulsed mode of laser irradiation. Thus, in a series of deep and fundamental works by colleagues from Spain, the regularities of the formation of LIPSS on the surface of various materials under various modes of pulsed irradiation with a pulse duration in the nanosecond, picosecond, and femtosecond ranges, as well as in the range of lengths waves from ultraviolet to infrared. At the same time, it has been found that, depending on the type of initial material and the degree of wettability of the surface of products made from it, as well as the mode of laser radiation, the consequences of laser processing can be different and lead both to the hydrophilization of the surface of the original samples, and, conversely, to an increase in their hydrophobicity ,81,82,83Thus, this research team, through using the example of thin films of PET and PET reinforced with foamed graphite (EG) under laser irradiation with ultraviolet (265 nm) and near-infrared (795 nm) femtosecond laser pulses, shows that LIPSS forms in both materials, but by different mechanisms . In someIn general, the changes in the structure and properties of the surface of the studied polymer films obtained from PHAs of various compositions, with different ratios of monomers and irradiated with two different laser treatment modes, obtained in different directions and very difficult to interpret, should be considered today as preliminary results requiring the carrying out of special and theoretical, as well as additional experimental, studies.The studies have been performed in a culture of mouse fibroblasts of the NIH 3T3 line. The biocompatibility of PHA polymer films of various compositions, depending on the laser irradiation mode, has been assessed by the results of fibroblast staining with fluorescent dyes DAPI (marker of nuclear DNA) and phalloidin conjugated with fluorescein FITC (marker of cell cytoplasm) , as wellThe difficulty in identifying the effects of laser treatment itself on cell adherence and proliferation lies in the fact that various PHAs compositions also affect cellular processes on the sample’s surface. In our previous study  in a culIt should be noted that a change in the cellular response can be associated with even minor changes in the surface profile at the nano and micrometer levels, which mediates the processes of adhesion and further proliferation of cells on the surface. However, the produced effect is not universal, since morphologically different types of cells differ in their sensitivity to changes in surface topography, which requires additional experiments on mammalian cell cultures of various origins in vitro.The results show that the laser treatment mode has a different effect on the number of fibroblasts cultivated on polymer films of different composition. As an example, Due to the fact that the total modified area, depending on the processing mode, differs significantly and is significantly higher for films after continuous processing  compared to the quasi-impulse mode (8–9%), this significantly affects the number of viable cells, determined in the MTT test .It should be noted that, regardless of the laser treatment mode and the PHAs composition used to obtain the films, no negative effect on the development of fibroblasts during their direct contact with the surfaces of all the studied samples, including all types of initial, untreated films, as well as both groups of irradiated films, has been detected. The number of viable metabolically active cells in the control (polystyrene) is lower to the indicators in all variants of the studied PHAs films .3/cm2). When analyzing the result of the MTT test when growing fibroblasts on films from P(3HB-co-4HB), a higher number (18.28·103/cm2 and 17.5·103/cm2) is obtained on films obtained from the polymer of this type with the highest content of 4HB monomers, respectively 35.5 and 75 mol.% This is 29 and 38% higher than in the control and even more significantly higher by 134 and 93%, compared with the number of cells on similar samples of films, but processed in a continuous mode. Similarly, the highest number of fibroblasts is recorded on all films of P(3HB-co-3HHx) copolymers, regardless of the content of medium-chain 3HHx monomers. The number of cells in this variant is 49–50% higher than the control and 100% higher than the result on films of these types processed in the continuous mode of laser radiation.PHAs films processed in the quasi-pulse mode are the most favorable as cell carriers. The number of fibroblasts on films of this type of all studied PHAs compositions is higher than the control (polystyrene) and depends very significantly on the composition of PHAs. Thus, on P(3HB) films, the number of fibroblasts is 85% higher than in the control, and even more significant (by 122%) is the number of cells on films irradiated in a continuous mode. The number of cells on films of P(3HB-co-3HV) copolymers is comparable with P(3HB), and is inferior to the values obtained on all films of other types of PHAs copolymers. At the same time, the lowest number of cells in the MTT test is recorded on films with the lowest content (15 mol.%) of 3HV monomers  . The lowThus, the number of metabolically active fibroblasts and those grown on the studied polymer films is most pronouncedly affected by the laser treatment mode and, to a lesser extent, by the composition of PHAs.2 laser in a quasi-pulse mode. This confirms the results obtained earlier on the example of three types of copolymer PHAs films (P(3HB-co-3HV), P(3HB-co-4HB), and P(3HB-co-3HHx) with one content of second monomers at the level of 30 mol.%) [In general, we can state a more favorable effect on the development of fibroblasts of the studied line of films of all the studied types of PHAs after treatment of films with a CO0 mol.%) .As is known, laser processing is considered one of the most suitable methods for modifying the surface of cell matrices. This is due to the fact that laser processing makes it possible to form surface reliefs of complex shape, while providing high structuring accuracy, allowing for the creation of complex biomedical devices with high biocompatibility without the use of toxic chemicals ,85.The positive effect of laser treatment on the development of eukaryotic cells has been described in a series of papers. Thus, the modification of films from the P(3HB-co-3HV) copolymer using Nd:YAG laser treatment provides stronger attachment and development of keratinocytes on the matrix compared to the original films . A similSimilar positive results of laser processing have been described for other polymers. For example, femtosecond laser treatment of thin films of chitosan and a composition of chitosan with ceramics favorably influences the attachment and development of osteoblasts isolated from the calvaria of mice and mesenchymal stem cells of adipose tissue . The favThus, multicenter studies include (1) a set of films obtained from four types of PHAs (10 variants); (2) including copolymer films (nine variants) are obtained from three different types of PHAs with different ratios of monomers; (3) all versions of the films are treated with two different laser radiation regimes.Due to the multifactorial nature of the objects of study and indicators for assessing the consequences of laser radiation, an integrated approach has been used to statistically process the results obtained in the form of plotted radar diagrams to characterize the complex effect of two types of laser processing on the total modified surface area of the films, the average roughness, the value of the contact angle, and the number of viable cells, according to the MTT test . This tyIn these works, the use of such a mathematical model makes it possible to reveal the regularities of the complex multifunctional impact of the studied processing mechanisms on the properties of the resulting products.It can be seen that for all film samples processed in the quasi-pulse regime of laser radiation, which is accompanied by a reduction in the total modified surface and an increase in the arithmetic mean roughness for a group of samples from P(3HB-co-4HB) and a sample of P(3HB-co-3HV) with a maximum inclusion of 3HV and a decrease in this parameter in samples from P(3HB-co-3HV) with low and medium inclusions of 3HV monomers, it increases the biological compatibility of polymer films and provides an increase in the number of physiologically active fibroblasts. In contrast to these results, the use of continuous laser radiation is accompanied by an increase in melted modified areas. At the same time, it is not observed that there are changes in the average roughness and the value of the contact angle between the samples. This is accompanied by a decrease in the biological compatibility of the surface of irradiated polymer films and the number of viable fibroblasts. From this point of view, the use of this analysis of the results in the form of radar diagrams makes it possible to more correctly identify indicators that positively affect the biological compatibility of cellular matrices.It can be seen that for the films treated in the quasi-pulse regime of laser radiation, which is accompanied for all samples by reduced values of the total modified surface and an increase in the arithmetic mean roughness for a group of samples from P(3HB-co-4HB) and a sample of P(3HB-co-3HV) with a maximum incorporation of 3HV, and a decrease in roughness in the P(3HB-co-3HHx) group and samples from P(3HB-co-3HV) with low and medium incorporation of 3HV monomers, biocompatibility increases and an increase in the number of physiologically active fibroblasts is provided. In contrast to these results, the use of continuous laser radiation is accompanied by an increase in melted modified areas and, in general, the area of modifications. In this case, no significant changes in the contact angle between the samples are observed, with the exception of the P(3HB-co-3HV) copolymer with low 3HV inclusion and the P(3HB-co-4HB) copolymers with medium and high 4HB inclusions, and while processing these samples quasi-pulse mode reduces the values of the contact angle. This is accompanied by a decrease in the biological compatibility of the surface of the irradiated polymer films and the number of viable fibroblasts. The use of this analysis of the results in the form of radar diagrams makes it possible to more correctly identify indicators that positively affect the biological compatibility of cell matrices.2 laser irradiation in continuous or quasi-pulsed modes, have been studied. The PHAs samples used to prepare the films are P(3HB) homopolymer and three types of copolymers formed by 3-hydroxybutyrate monomers with 4-hydroxybutyrate, or 3-hydroxyvalerate, or 3-hydroxyhexanoate. The ratio of monomers «3HB:other types» varies from 75:15 to 25:75 mol.%For the first time, the properties of solvent casting films obtained from PHAs, not only with a different set of monomers, but with a different ratio of them, the surface of which is modified by COThe consequences of laser treatment of the surface of films obtained from various types of PHAs includes the study of the structure of the surface microrelief (SEM and AFM), roughness indices, water contact angles, adhesion, and fibroblast growth. The consequences of laser treatment are determined depending on the radiation regime and the composition of monomers in PHAs. The surface modification after continuous treatment consists in the formation of melted areas in the form of grooves, the total area of which is 11% of the total area of films for P(3HB) and from 12 to 15% for copolymer films. The quasi-pulse mode by the raster method causes the formation of holes without pronounced melted zones, the total area of which is lower by 20% compared to the area of melted grooves. The processing mode affects the surface properties, cell adhesion, and fibroblast growth. The number of viable fibroblasts on the films after the quasi-pulse mode is 1.5–2.0 times higher compared to the continuous mode, and depends to a greater extent on the laser treatment mode than on the PHAs’ composition.The presence of PHAs of various compositions and the use of continuous and quasi-pulsed laser radiation regimes make it possible to influence the morphology and surface properties of polymer films in a targeted manner. Changes in the structure and properties of the surface of the studied polymer films obtained from PHAs of various compositions, with different ratios of monomers and irradiated with two different laser treatment modes, in our opinion, should be considered today as preliminary results requiring special theoretical studies, as well as additional experimental studies. The results make it possible for direct modification of the surface of polymer products made from PHAs, a promising family of biodegradable polymers.


To determine the economic impact of the incremental consumption of resources for the diagnosis and treatment of anastomotic leak (AL) in patients after resection with anastomosis for colorectal cancer compared to patients without AL on the Spanish health system.This study included a literature review with parameters validated by experts and the development of a cost analysis model to estimate the incremental resource consumption of patients with AL versus those without. The patients were divided into three groups: 1) colon cancer (CC) with resection, anastomosis and AL; 2) rectal cancer (RC) with resection, anastomosis without protective stoma and AL; and 3) RC with resection, anastomosis with protective stoma and AL.The average total incremental cost per patient was €38,819 and €32,599 for CC and RC, respectively. The cost of AL diagnosis per patient was €1018 (CC) and €1030 (RC). The cost of AL treatment per patient in Group 1 ranged from €13,753 (type B) to €44,985 (type C + stoma), that in Group 2 ranged from €7348 (type A) to €44,398 (type C + stoma), and that in Group 3 ranged from €6197 (type A) to €34,414 (type C). Hospital stays represented the highest cost for all groups. In RC, protective stoma was found to minimize the economic consequences of AL.The appearance of AL generates a considerable increase in the consumption of health resources, mainly due to an increase in hospital stays. The more complex the AL, the higher the cost associated with its treatment.it is the first cost-analysis study of AL after CR surgery based on prospective, observational and multicenter studies, with a clear, accepted and uniform definition of AL and estimated over a period of 30 days.The online version contains supplementary material available at 10.1186/s13561-023-00425-y. Cancer is one of the diseases with the greatest impact on public health and is the second leading cause of death in Europe . In SpaiOne of the complications with the greatest clinical repercussions after colorectal cancer surgery is anastomotic leak (AL), defined as a defect of the intestinal wall at the site of the anastomosis, which involves communication between the intra- and extraluminal space . AlthougDue to the high incidence of CRC and the proportion of patients who undergo resection surgery, two multicentre and prospective studies have been conducted at the national level to determine the risk factors related to AL and the real incidence rates in patients with colon cancer (CC)  and rectPatients with AL require greater postoperative follow-up, since they may require one or more surgical interventions, be admitted to an intensive care unit (ICU), or even require a stoma . This geThe average cost generated by patients with CRC, depending upon the cancer stage, has been estimated at between €8813 (in situ stage) and €49,518 (advanced stage) including all the costs generated from the treatment of the disease for 1 year. The cost of the interventions and the hospital stay are the greatest expenses, accounting for 55.2 and 72.0% of the total, respectively .The present analysis examines the economic impact of CRC complications on the health system in Spain. Specifically, we estimated the incremental economic burden for the diagnosis and treatment of AL in adult patients following a resection with anastomosis due to CRC with regards to patients who do not develop ALs.For the development of the analysis, a panel of experts devised parameters for a literature review, which was then conducted. Next, a cost analysis model was developed to estimate the economic impact of the increased consumption of health resources for the diagnosis and treatment of patients with AL compared to CRC patients without this complication.The literature review was performed with a structured search of the electronic database Medline (PubMed), scientific societies and associations, such as the Spanish Society of Medical Oncology , Spanish Association of Coloproctology  and the Spanish Association of Surgeons . In addition, a grey literature search was carried out to obtain all reports outside of the databases consulted. The search strategy in PubMed was performed using the following terms: “cost”, “anastomotic leak”, “resection”, “cancer”, “colon”, “rectal” and “colorectal”. This search was limited to articles published in English or Spanish with no limit on the year of publication. To obtain data on the epidemiology of CRCs in Spain, the incidence of AL and its classification according to the treatment of the patient, articles with data referring to Spain were prioritized , 11.For the validation of the parameters and the conceptualization of the analysis, several online and face-to-face sessions were held with a panel of five experts in colorectal surgery , who are members of scientific committees or boards of the AECP and the AEC.The panel validated epidemiological data and the cost estimates for the diagnosis and treatment of AL.Defect of the integrity of the intestinal wall at the colorectal or coloanal anastomotic site  that leads to communication between the intra- and extraluminal compartments. A pelvic abscess near the anastomosis is also considered an anastomotic leak, according to the International Study Group of Rectal Cancer (ISREC) . In addiDoes not require an active therapeutic intervention. Normally, type A is detected by routine radiological examinations since the appearance of clinical symptoms or alteration of analytical parameters is not common . TherefoThis type can present clinical symptoms such as distress or abdominal or pelvic pain, and it can even produce air, purulent or faecal material through the drainage, wound or rectum. In addition, these patients usually present with leukocytosis and increased C-reactive protein (CRP). Therefore, they require active therapeutic intervention (percutaneous drainage or antibiotic treatment) without the need for a second surgery .Requires urgent reoperation since it generally presents purulent or faecal drainage and/or clinical symptoms of peritonitis and/or laboratory signs of infection .GROUP 1: patients with resection and anastomosis due to CC and suffering from AL.GROUP 2: patients with resection and anastomosis due to RC, without protective stoma and experiencing AL.GROUP 3: patients with resection and anastomosis due to RC, with protective stoma and experiencing AL.A model was developed using the Microsoft Excel programme to estimate the costs generated for the diagnosis and treatment of AL in adult patients with CRC Fig. . The patIn the cost estimations for the diagnosis and treatment of AL, only those resources that are modified due to AL have been considered. Therefore, GROUPS 1, 2 and 3 reflect the increased costs incurred when CRC patients experience AL, as determined by the Spanish National Health System.For the estimation of costs, we considered adult patients with CRC who underwent resection of the colon or rectum with anastomosis and developed AL. Based on the total Spanish adult population, epidemiological data and clinical management were applied to obtain statistics on CRC patients Fig. .The population of Spain, as determined by the National Institute of Statistics , reported that the adult population ≥18 years) residing in Spain on January 1, 2021, was 39,154,892, of whom 20,176,435 were women and 18,978,459 were men  years re, which iThe time horizon established for the cost analysis was 1 year after the first resection with anastomosis due to CRC.The perspective established for the analysis was that of the Spanish National Health System.In accordance with the perspective chosen for the analysis, only direct health costs were considered . Further, only those resources that were increased as a result of the occurrence of AL were identified and quantified  and €20,769,368 for RC (GROUPS 2 and 3). The average per patient with CC was €38,819 and €32,599 for patients with RC. Among patients with RC, the average cost for patients without protective stoma was €34,704 compared to €29,792 for patients with stoma, (22.8% reduction).The cost of AL diagnosis for CC GROUP 1) was €1,932,982 and €633,050 for RC (GROUPS 2 and 3)  and type of AL , the hospital stay  represents the single greatest expense in the diagnosis and treatment of AL, a conclusion similar to that of other studies . In patiIn a 2021 retrospective, single-centre observational study, Capolupo et al.  examinedA study by Hammond et al.  in the UAs demonstrated in multiple studies, performing a protective stoma minimizes the consequences of AL and even decreases the rate of reoperations but not its occurrence . In our The hospital costs obtained are similar to those reported by the observational study conducted by Ashraf et al.  in the UIn other retrospective publication, such as the one carried out in the USA by Lee et al. , it is eThe comparison of the economic impact of AL referred to in the publications discussed above is very complex, due to differences in the definition of AL and the methodology of estimation and evaluation of costs. Standardization in the definition of AL is fundamental in this process , hence tOur analysis is not without limitations. To obtain data related to patient flow, literature from various sources was used. In addition, it was necessary to make some assumptions to determine health resources and hospital stay in patients with AL due to the absence of data in the literature. In any case, these were validated by the expert panel based on their experience. Another limitation is the possible overestimation of costs due to the adjustment of patients with CC (GROUP 1) according to the Rahbari et al.  classifi. Protective ileostomy in patients with RC minimizes the clinical consequences of AL and, consequently, the economic costs.After resection surgery with anastomosis in patients with CRC, the appearance of AL implies an increase in the consumption of health resources compared to patients without AL. Likewise, the greater the complexity of AL, the greater the cost associated with its treatment. In addition, AL can lead to other complications, generating even higher costs. The resources that are increased in greater amounts are hospital stays, and in type C AL, the cost of interventions and stoma closure is addedThese conclusions are of great importance for surgeons and health managers to include AL in internal audits that validate good work in the services or units of colorectal surgery and dedicate more resources to the training of surgeons and the technological development that reduces the incidence of anastomotic leaks.Additional file 1: Table S1. Consumption of resources for the diagnosis of anastomotic leak (AL). Table S2. Consumption of resources for the treatment of AL in GROUP 1 (patients with CC and AL) vs. patients without AL. Table S3. Consumption of resources for the treatment of AL in GROUP 2 (patients with RC and AL) vs. patients without AL. Table S4. Consumption of resources for the treatment of AL in GROUP 3 (patients with RC and with a protective stoma suffering from AL) vs. patients without AL.Additional file 2: Table S5. Unit cost of resources for the diagnosis and treatment of AL.


The formation of pharmacobezoars from suspensions of spray-dried amorphous solid dispersions (SD-ASDs) of new chemical entities (NCEs) and hydroxypropyl methylcellulose acetate succinate (HPMC-AS) represents a non-compound related adverse effect in preclinical oral toxicity studies in rodents. Whereas the contribution of the insolubility of the carrier polymer to this process taking place in the acidic environment of the rodent stomach is conclusive, unawareness of the extent of in vivo pharmacobezoar formation is adverse. In order to evaluate the risk of pharmacobezoar formation before in vivo administration, we subsequently introduce an in vitro model to assess the agglomeration potential of solid dispersions. To verify that the pharmacobezoar formation potential can be assessed based on the observed agglomeration potential, we conducted a sequence of experiments with two HPMC-AS-based SD-ASD formulations. In vitro, we found their different in vivo pharmacobezoar formation potential reflected by a significantly increased agglomerated mass of formulation 1 per day compared to formulation 2. In order to find an approach to reduce the agglomeration potential of solid dispersion from suspensions, we further applied the model to investigate the impact of the viscosity of the vehicle used to prepare suspensions on agglomerate formation. Appropriate bioavailability is a prerequisite for adequate preclinical testing of new chemical entity (NCE) drug candidate’s safety and efficacy . If suffThe lumen of the rodent stomach is separated into two parts, divided by a visually prominent line extending from the esophagus to the greater curvature known as the limiting ridge. It separates the nonglandular part of the stomach from the glandular. The nonglandular section, representing the fundus region of the rat stomach, is grossly characterized by a translucent, elastic wall with a thin layer of smooth muscle ,9,10. FoGiven the anatomical and physiological heterogeneity within the rat stomach, biorelevant in vitro simulation of the variable, complex dynamic environment is challenging. A series of sophisticated dynamic rat stomach devices has been developed previously to investigate digestion processes in the rat stomach in vitro ,17,18. TIn this work, we present an in vitro model capable of individual testing of the in vitro agglomeration potential of ASD formulations. First, mechanical function and experimental procedures are introduced in detail, rationales of model development are described and discussed subsequently. To investigate the extent of correlation between in vitro agglomeration potential and the pharmacobezoar formation potential in vivo, we applied the in vitro model to comparatively assess the agglomeration potential of two HPMC-AS-based SD-ASDs that revealed a different potential to form pharmacobezoars in preclinical testing. Commonly used to improve stability of suspensions, this sequence of experiments also included testing of the influence of enhanced viscosity of the vehicle used to prepare suspensions in order to find an approach to reduce the agglomeration potential of both formulations.®, B.Braun Melsungen AG, Melsungen, Germany) marked yellow in The central compartments of the in vitro model are the cavities of the 18 syringe cylinders .The 3D printed parts were designed using FreeCAD 0.18. Stereolithography files were imported into the slicing software Cura (Version 3.6.0) and printed via an UltimakerVolume changes in the cavities of the syringes where suspensions were once daily administered to, having been generated by movement of the plunger in the syringe cylinder, so that medium was drawn up and emptied together with non-agglomerated SD-ASD to the reservoirs directly attached to the syringes. To repeatably execute volume changes, we programmed a 23.5 h volume pattern using SyringePumpProV1 (Version 1.6.4.7). The frequency and dimension of motions and rests in this pattern shown in The starting volume of the 23.5 h pattern was set at 3.5 mL and the end volume at 0.5 mL. Contrary to the constant medium uptake rate of 1.5 mL/min, we decreased the rate of medium emptying stepwise. Whereas 3% of the initial volume at the beginning of the emptying cycle were emptied per minute for the first 15 min, the rate decreased to 1.5%/min from 15–30 min and to 0.5%/min until the aspired volume of this emptying step was reached. In our experiments, suspensions were drawn up once daily to the syringes, where 30 min per day were planned, before the next cycle of 23.5 h could be started. Media in the reservoirs, deionized water adjusted to pH 3 using 1 N HCl, was replaced following daily administration of suspensions.® AS-LG)-based SD-ASDs were provided for in vitro testing by Boehringer Ingelheim Pharma GmbH & Co. KG, Biberach, Germany. Formulation 1 contained a drug load of 70% (w/w) BI 1026706 and formulation 2 of 80% (w/w) BI 1026891. Physicochemical properties of both crystalline NCEs and HPMC-AS-based SD-ASD formulations are given in Two HPMC-AS  1 N hydrochloric acid in deionized water. In case of viscosity-enhanced vehicles, hydroxyethyl cellulose  was added to the vehicle under rapid magnetic stirring prior to manufacturing the ASD suspension. Both the intermediate viscous vehicle containing HEC 0.5% (w/w) and the high viscous vehicle containing HEC 1% (w/w) were allowed to swell for 24 h in the fridge at 7 °C until both viscosity-enhanced vehicles were clear and homogenous. Suspensions with a concentration of 100 mg/mL SD-ASD were prepared similar to the suspensions for preclinical testing in rodents. The appropriate amount of SD-ASD was weighed into a glass beaker, wetted by addition of a small amount of vehicle , and manually stirred using a glass stick until a homogenous paste was formed. The remaining amount of vehicle was added and suspensions were further stirred at 200 rpm for 5 min using a magnetic stirrer (Heidolph MR 3001) and under continuous stirring drawn up into appropriate syringes. Vehicle was prepared every 4th day with 1% , it had to be brought back into the starting position (volume of 3.5 mL per syringe). Therefore, the pump executed a 3 mL draw up program. The plungers of syringes that were already dosed with 2 mL suspension slipped through the retainer bar until the endcaps were adjacent to the backside of the retainer bar. From there on, the plungers had to follow the movement of the retainer bar, thereby drawing up 1.5 mL medium from the reservoirs to the syringes (2 mL → 3.5 mL). Once back in starting position, springs had to be attached on the plunger’ endcaps and the volume pattern program was uploaded to the syringe pumps’ mainboard before the next 24 h cycle could be started. The experiment was terminated for those syringes in which an agglomerate in the syringe cylinder led to spring compression of at least 2 mm at position of minimal volume after running through the volume pattern. At the end of the pump cycle in which spring compression of at least 2 mm was observed, generated agglomerates were removed from the syringe. Therefore, syringe cylinders were cut open circumferentially with a scalpel as indicated in t-test using GraphPad Prism 5.01 and considering a significance level of p < 0.05.The mass of the agglomerates per day as main comparative criterion was calculated by dividing the total mass of the dried in vitro agglomerate by the number of dosing days. As experiments were conducted in triplicate, results are given as the mean of agglomerated masses of formulation per day from three units The first step of the model development was to identify a cavity variable in volume, that would allow the simulation of a dynamic environment with adjustable fluid uptake and emptying pattern. Thus, three major reasons favored the application of a less complex approach regarding anatomical and morphological features compared to established in vitro models ,17,18. First, the comparative assessment of agglomeration potential prior to in vivo studies requires a high degree of repeatability, which should be achieved by reduction of complexity of the cavities’ spatial shape in which the agglomeration potential of solid dispersions is studied. Second, preliminary testing revealed that removal of agglomerates from cavities for further characterization would be either destructive to the compartment itself or likely cause unfavorable damage to the agglomerate formed. Third, valid statements about differences of SD-ASD agglomeration potential ideally require performing multiple experiments in parallel at an overall reasonable timescale to compare the agglomeration potential of different suspensions as a part of preclinical formulation development. Complex manufacturing of model components or complex preparation steps, as described for other rat stomach models, are therefore unsuitable and may furthermore transfer variability of model components, such as their volume ,17,18, tCompact cylindrical shape, variability in volume, and commercial availability in standardized quality plus an outlet diameter of 2 mm, that is comparable to the maximum size of particles which are able to pass the rat’s pyloric sphincter physiologically , led to The rationale for the design of the volume pattern was to transpose in vivo intragastric volume changes to media draw up and emptying processes in the model. In total, 37.5 mL medium was drawn up to each syringe per day. The first half of the volume pattern simulated the photophase, where activity of nocturnal rodents is low and thus frequency and amplitude of fluid uptake reduced. Fluid uptake of rodents in this period accounts for 20% of the daily intake, whereas in the active scotophase, simulated in the second half of the profile, the residual 80% is consumed . As mentioned, the amplitude and sequence of medium uptake and emptying steps were free chosen, however, with the total volume drawn up and the distribution to the simulated day (photophase) and night phase (scotophase) corresponding to physiological parameters . BesidesContrary to the burst and rest process of in vivo fluid uptake, gastric emptying of fluids from the stomach is a continuous process with a decreasing rate. To simulate the in vivo fluid emptying kinetics in vitro, we derived the percentage of fluid remaining in the stomach over time from in vivo profiles of fluid emptying from the rat stomach ,20,21,22The minimal volume of 0.5 mL, reached at several points of the volume pattern, was introduced to prevent complete emptying of syringes and to allow agglomeration of SD-ASD particles. If an agglomerate in the syringe cavity prevented a plunger from reaching the position of 0.5 mL, the respective spring, which transferred the forward movement of the movement transmission bar to the plunger endcaps, compensated for the resistance in the syringe cavity by compression . Spring Deionized water adjusted to pH 3 with 1 N hydrochloric acid was used as medium in the reservoirs. This pH was chosen to simulate the acidic intragastric fluids as environment where in vivo pharmacobezoar formation takes place in, even though at the lower intragastric pH range. With respect to composition, a more sophisticated medium simulating the physiologically relevant fed state of rodents would be desirable ,27.In preclinical rodent studies, dose and administered volume are related to body weight of the animals. In vitro, we adopted this approach and standardized the dose of SD-ASD and the corresponding volume of suspension to simulate a rat with a body weight of 200 g. As standard dose, 1000 mg/kg/day SD-ASD suspended in 10 mL/kg vehicle, respectively 2 mL of a suspension containing 100 mg/mL SD-ASD in 0.01 N HCl was used as dose volume per syringe. Whereas the dosing volume meets recommendations for good practice in animal science ,29, the In order to prove the capability of the in vitro model to detect differences of the agglomeration potential of solid dispersions, we tested suspensions of formulation 1 and formulation 2 in a sequence of three repetitive experiments with three units per formulation per experiment. p < 0.0006) (Formulation 1 completed the formation of agglomerates within three days and obtained an agglomerated dry mass of 149.6 ± 19.2 mg/day (n = 9). Intraexperimental standard deviation of the three simultaneously investigated syringes was found between 1.2% to 5.3%. Spring compression indicated completion of agglomerate formation for formulation 2 after 4 days, with a mean agglomerated dry mass of 115.9 ± 11.3 mg/day (n = 9) and intraexperimental standard deviations of 5.6% to 7.8% . InterexIn addition, we investigated the impact of the viscosity of the vehicle on the agglomeration potential of both formulations (each n = 3). Using the intermediate viscous vehicle (0.5% HEC in 0.01 N HCl) to suspend SD-ASD formulations, the in vitro agglomeration potential of formulation 1 was found to be reduced by 36%, and 24% for formulation 2. The effect was even more pronounced using the high viscous vehicle (1% HEC in 0.01 N HCl), resulting in reduction of agglomerated mass per day of 88% for formulation 1 and 87% for formulation 2. In case of the latter, formation of agglomerates inducing spring compression took up to 14 days.Differences of agglomerated masses per day between both formulations were especially pronounced without viscosity enhancer added to the vehicle and are in line with the in vivo observed higher pharmacobezoar formation potential of formulation 1 compared to formulation 2 . This diIn terms of transferability of in vitro results to in vivo, the fact that agglomerates were obtained with formulation 2 as well shows that the processes of pharmacobezoar formation and in vitro agglomeration of SD-ASDs might not be exactly transferable to each other. In vivo, pharmacobezoar formation taking place in a continuously changing environment in terms of shape, motility, pH, and presence of gastric contents until end of dosing has been highly variable from one nonclinical study to the other . To obtaReferring to the physical background of agglomeration processes, it is known that enhanced particle motion and accompanying collisions enhance aggregation of particles . ThroughUnawareness of the pharmacobezoar formation potential of SD-ASD-containing suspensions in the stomach of rodents in nonclinical safety studies includes the risk of adverse events affecting animal welfare and thus, the potential to misinterpret experimental results. Addressing this problem, we developed an in vitro model enabling parallel comparative assessment of the agglomeration potential of up to 6 different suspensions, each in triplicate.Based on the study design of preclinical oral toxicity studies, in vivo pharmacobezoar formation in the rat stomach following once daily oral administration of suspensions has been transposed to once daily administration of suspensions to syringes, whose volume in the cylinder is changed by uptake and emptying of medium. Draw up and emptying processes were adapted to physiological volumes and rates. To allow formation of agglomerates in the syringe cylinder, a minimum volume of 0.5 mL is mandatory. Concentration and volume of SD-ASD suspensions administered daily to the syringe cavities were adapted from standards of preclinical practice and related to a hypothetical body weight of 200 g per rat. When agglomerates in the syringes exceeded the minimal volume and thus induced spring compression, syringe cylinders were cut open to extract the agglomerates. Their dry mass divided by the number of daily dosings of SD-ASD suspensions equaled the agglomerated mass per day as outcome criterium.To prove the in vitro model’s ability to detect differences in agglomeration potential, a sequence of three experiments on two HPMC-AS-based SD-ASD formulations has been conducted to investigate their agglomeration potential and its intra- and interexperimental variability. We found that the in vitro agglomeration potential of formulation 1 and formulation 2 correlated with their in vivo pharmacobezoar formation potential observed in previous preclinical testing. Subsequently, we tested the influence of the vehicles’ viscosity, used to suspend the SD-ASDs, on in vitro agglomeration of both formulations. We observed a significant decrease of agglomerated mass/day with increasing concentration of viscosity enhancer in the vehicle used to prepare suspensions for both formulations. Thus, increasing the vehicles’ viscosity might be an approach to reduce the pharmacobezoar formation potential of SD-ASDs in vivo.The developed in vitro model should be considered as a tool to assess the pharmacobezoar formation potential of suspensions of solid dispersions based on their in vitro agglomeration potential so that action can be taken to prevent pharmacobezoar-induced adverse effects on animal welfare.


Due to the special geographical location, climatic characteristics, special soil properties and the flood zone of the Yellow River in Kaifeng, the groundwater level in the lower reaches of the river basin is high and contains much salt. The matrix suction and surface free energy of the Kaifeng city wall earthen site changed under capillary action, resulting in cracking, peeling and efflorescence to varying degrees. In order to reduce the deterioration of the Kaifeng city wall caused by environmental erosion, a select lime with excellent mechanical properties and waterproof methanesiliconic acid sodium salt with excellent water resistance were chosen to reinforce the earthen sites. In this paper, 0%, 3%, 5% and 7% lime, and 0%, 1%, 2% and 3% waterproof materials were selected to determine four types of imitation site soil with 16 different mix proportion samples. Further, samples with different mixing ratios were subjected to direct shear, a disintegration test, and a microscopic scanning electron microscope test. The results show that under different normal stresses, with the increase in waterproof material content, the growth rate of shear strength of imitation site soil ranges from 1.82% to 10.81%. With the increase in lime content, the shear strength of imitation site soil increases rapidly, up to 38.16%. Both materials can improve the shear strength of the soil site. Under reinforcement with the two materials, the cohesive force of the imitation site soil can be improved at a maximum rate of 59.23%, and the internal friction angle changes in the range of 36.72°–41.61°. Compared with the sample without waterproof material, the mass water absorption rate of the sample with waterproof material decreases in the range of 2.76–27.77, and with the increase in waterproof material, the mass water absorption rate of the sample gradually decreases. The chemical reaction products of the waterproof materials and lime can play a filling role in silty clay; filling micro-pores and micro-cracks between soil particles. Most of the earthen sites in Henan Province are open-air, above-ground and alternating wet and dry nature sites; they are precious immovable cultural relics with high historical value, such as the Kaifeng city wall earthen sites , city st2 to generate Ca(OH)2 and CaCO3; thus, enhance the cohesion between soil particles.In recent years, domestic and foreign scholars have conducted extensive research on materials that can be used to reinforce earthen sites, including lime, glutinous rice slurry, and microbial mineralization, which are effective in resisting weathering and improving mechanical properties ,8,9,10. In this paper, we used a combination of organic material lime and a methyl silicic acid type waterproof agent to restore the site of the Henan section of the Yellow River Basin, and investigated the mechanism by which improvement of the mechanical and hydraulic properties of the Kaifeng city wall was achieved using indoor direct shear tests, a disintegration test, and microscopic analysis. The research methods and results will provide new ideas for moisture proofing and waterproofing of earthen sites and ancient city wall foundations in the Henan section of the Yellow River Basin.The working principle of the X-ray fluorescence analysis method is that the incident X-ray interacts with the substance to produce the characteristic X-ray fluorescence intensity. According to the relationship between the fluorescence intensity and the content of the elements to be analyzed, the content of each element to be measured can be obtained by using the corresponding working curve. X-ray fluorescence sample processing does not require wet digestion. The spectral lines are very simple. More than 90 kinds of elements can be analyzed, and no pollution will occur after the sample measurement. Many elements can be determined. It has many advantages, such as rapid analysis, no damage to the sample attributes, a wide analysis range, stable and reliable results, rapid simultaneous analysis of multiple elements, simple operation, etc. The equipment used in this section is the X-ray fluorescence spectrum analyzer (ARLAdvantX Intellipower TM3600) produced by the Thermo Flying Company, as shown in 2, Al2O3, CaO, Fe2O3 and MgO, and their mass proportions are 57.79%, 16.05%, 10.52%, 6.16% and 3.11%, respectively. The main elements are Si, Al, Ca, Fe and Mg, and their mass proportions are 27.02%, 8.49%, 7.52%, 4.31% and 1.89%, respectively. As other substances and elements only account for a small proportion, they are not included in the calculation and research.It can be seen from 3, and the optimum moisture content was 14.32%. The particle sieving test was carried out by using an LP-100D digital display soil liquid plastic limit tester, and the gradation of the four parallel groups of specimens was calculated to be basically the same; the test results are shown in A powder clay with similar basic physical properties to those of the Kaifeng city wall was selected for the Limit water content test to obtain its basic physical properties. The plastic limit was 21.23%, the liquid limit was 37.33%, the plasticity index was 16.1, the maximum dry density was 1.68 g/cmLime was purchased from Tianjin Comio Chemical Reagent Co., Ltd. and the main physicochemical indexes are as follows. The relative molecular mass is 56.08, CaO content is greater than or equal to 98%, cauterization loss is 2%, and the state is powder.The waterproof material selected was a methyl silicic acid type waterproof agent; a colorless transparent liquid, soluble in water, having good waterproof and weathering resistance itself, and widely used in waterproofing, moisture and material protection, etc.; the pH value was between 12–14.2O and CO2, and consume a part of pure water, which has a great influence on the water content. Therefore, compaction tests were conducted on soil materials mixed with 0%, 3%, 5% and 7% lime, and three parallel specimens were set for each mixture. The results of the compaction test are shown in Lime can chemically react with HThe retrieved powdered clay was dried, crushed, sieved to remove impurities and prepared for use. After the pre-test trial, we selected 0%, 3%, 5% and 7% of lime, and 0%, 1%, 2% and 3% of waterproofing materials, to determine the four types of sixteen different ratios of specimens of imitation site soil; the specific formula is shown in For the direct shear test, an automatic straight shear instrument produced by Beijing Huakan Technology Co. was used in accordance with The Standard for Geotechnical Test Methods . We set The disintegration test complied with The Standard for Geotechnical Test Methods (GB/T50123-2019)  by usingThis test was observed via scanning electron microscope test equipment used for the FEI Quanta 250 environmental scanning electron microscope, before filming the sample after maintenance sampling. We roughly cut out of the sample a side length of 5 mm cubic pieces, which were placed at a constant temperature of 105 °C and oven baked for 8 h until thoroughly dried. Then, conductive adhesive was used to paste the specimen on the copper table, before plating with conductive film, and applying a gold spraying treatment. 2 crystals and CaCO3 precipitates generated by the chemical reaction of lime can fill the pores between soil particles, reduce the porosity between soil particles, and increase the compactness of the Kaifeng city wall repair soil. Sodium methylsilicate can fully react with water and CO2 to form enough polysiloxane films.The shear strength of each ratio specimen under different normal stresses is shown in When the amount of lime is constant, the shear strength of the imitation site soil gradually increases with the increase in the amount of waterproofing material; the growth rate is in the range of 1.82–10.81% and it can be judged, again, that the waterproofing material is effective in improving the shear strength of the imitation site soil. The waterproofing material used has excellent mechanical properties and can improve the overall stability of the soil sample, which is consistent with the results obtained by relevant researchers ; when th2 crystal and CaCO3 precipitation generated by the chemical reaction of lime in the sample can greatly improve the shear strength, cohesion and internal friction angle of the sample.This is because the Ca(OH)Disintegration test process: The specimen is completely placed in the hydrostatic water for wet disintegration, and the rate of disintegration of the specimen in the hydrostatic water is measured; the greater the rate of disintegration, the weaker the water corrosion resistance of the specimen’s dispersion performance—a specimen impacted by water erosion and latent corrosion is more serious ,24.2 crystals and CaCO3 precipitation generated by the chemical reaction of lime can fill the pores between soil particles, reduce the porosity between soil particles, and increase the compactness of the imitation site soil, thus improving the capillary action and disintegration resistance of the imitation site soil.During the disintegration test, only specimen A00 disintegrated and became a pile of loose soil, so it was impossible to weigh its mass to calculate its disintegration rate. None of the other compound specimens disintegrated and they only slightly absorbed water. The two materials had obvious effects on improving the disintegration resistance of the imitation site soil. In order to express the disintegration resistance and water stability of different ratio combinations more accurately, the recorded data were plotted against the disintegration test time as the horizontal coordinate, and the mass water absorption rate as the vertical coordinate, as shown in 3 [3 precipitation, and the residual Ca(OH)2 will increase the capillary effect of the imitation site soil. With the increase in waterproofing material, the water absorption of the specimen’s mass gradually decreased; it can be seen that waterproofing materials are more effective in improving the water resistance of the imitation site soil. The overall water absorption rate of the specimen’s mass mixed with only water repellent material is lower, compared to the specimen’s mass absorption rate without water repellent material, which is reduced in the range of 2.76–27.77%. When lime and water repellent materials were incorporated at the same time, the water resistance of the soil was slightly affected because the CO2 concentration in the maintenance environment was insufficient, which affected the chemical reaction between the water repellent materials and the lime in the soil; the effect of the maintenance environment on the water resistance of the soil could be investigated in the next stage of the experiment. From 3 , and at The properties of soil sites in the Henan section of the Yellow River Basin are mostly powder clay, which has the characteristics of easy water absorption and weathering, vigorous capillary action, and fast salt transport. Capillary water can have a large deterioration effect on the soil site, and the physical–chemical interaction between the water molecules and soil particles leads to changes in the physical properties of the soil’s skeleton and the pore water, resulting in dry shrinkage and cracking of the soil site, a reduction of free energy, and the destruction of the internal structure of the soil site, which leads to the reduction in the strength of the soil site.The morphology of water droplets on the surface of the site soil is shown in In order to observe the changes in particle morphology and pore characteristics inside the modified specimens, SEM was used to analyze the specimens A00, A03, A70 and A73 after 1000 times magnification to further investigate the mechanism of the improved shear strength, shear strength index and disintegration resistance of the modified materials used to improve the imitation site soil.2 crystals and CaCO3 precipitates inside the imitation site soil filled the micro-pores and micro-cracks between the soil particles. In addition, the pore structure of the soil particles in specimen A73 was most obviously improved, and the soil particles were more closely connected to each other, the Ca(OH)2 crystals, and the CaCO3 precipitation, which can improve the shear strength, cohesion and internal friction angle of the specimens.Through the study of this paper, the following conclusions can be obtained:(1) The shear strengths of the specimens with different ratio combinations under normal stresses of 50 kPa, 100 kPa, 150 kPa and 200 kPa were improved to different degrees compared with A00, ranging from 10.53–75% under 50 kPa, 4.17–46.53% under 100 kPa, 6.71–39.02% under 150 kPa and 10.29–56% under 200 kPa. Both lime and waterproofing materials are beneficial to the improvement of the shear strength of soil sites, and the effect of lime is greater than that of waterproofing materials.(2) With the increase in lime and waterproofing materials, the overall trend of the cohesion and internal friction angle of the imitation site soil increased. The cohesive force and internal friction angle of the imitation site soil increased when the lime mixture was 3%, 5% and 7%, and the cohesive force of the imitation site soil increased at the maximum rate of 59.23% when the water repellent material was 2% and the lime mixture was 7%; the internal friction angle of the imitation site soil varied in the range of 36.72°–41.61° when the lime and water repellent materials were used to improve the imitation site soil at the same time. The variation is small.(3) The soil group placed in static water disintegrated within a short period of time; the water stability and disintegration resistance was poor. In the other ratio specimens, no disintegration phenomenon occurred, and the water stability and disintegration resistance performance was good. During the disintegration test process, there was slight water absorption, which led to the quality of the specimen being increased, not reduced. Only when the specimens were mixed with waterproof material was the surface smooth and complete, with no disintegration phenomena, and the water absorption phenomena was not obvious, indicating that the waterproof material improves the crumbling resistance of imitation site soil performance and has more effective water stability. When the specimens were only mixed with lime, during the disintegration test process there were tiny bubbles, indicating that the sample had a small amount of water absorption phenomena; however, an overall collapse did not occur, and the disintegration resistance was good.2 crystals and CaCO3 precipitation, which can improve the shear strength, cohesion and internal friction angle of the specimen.(4) The most obvious improvement in the internal pore structure of the modified imitation site soil is the tighter connection between the soil particles and the surface of the specimen, which had more obvious flocculation. This is due to the chemical reaction of lime inside the specimen generating Ca(OH)