Combinations of different biomaterials with certain formulations may lead to improved properties and have significant potential for use in tissue regeneration applications. However, previously reported studies comparing biomaterials often suffered from inconsistent processing methods or inadequate comprehensive application research, hindering a comprehension of their efficacy in tissue engineering. This report explores the significance of screening the combination of gelatine with polysaccharide materials, specifically hyaluronic acid (HA) and carboxymethyl cellulose (CMC), using the same crosslinking method used for tissue regeneration. Hydrogel scaffolds (Gel/HA and Gel/CMC) at various concentrations were developed and characterized to assess their physiochemical properties. The results demonstrated that the hydrogels exhibited desirable mechanical properties, appropriate swelling behaviour, suitable porosity, and excellent cytocompatibility. In particular, the Gel1HA1 and Gel1CMC1 hydrogels showed remarkable cellular proliferation and aggregation. Further, we performed animal studies and explored the tissue regeneration effects of the Gel1HA1 and Gel1CMC1 hydrogels. Both hydrogels exhibited an accelerated wound closure rate and promoted vessel formation in a rodent full-thickness skin excisional model. Additionally, the subcutaneous implantation model demonstrated the induction of angiogenesis and collagen deposition within the implanted hydrogel samples. Overall, the hydrogels developed in this study demonstrated promising potential for use in the regeneration of soft tissue defects and this study emphasizes the significance of screening biomaterial combinations and formulations for tissue regeneration applications. Tissue engineering is a promising field that aims to develop functional tissues for the regeneration and repair of damaged or diseased tissue . BiomateNumerous biomaterials have been manufactured for hydrogel fabrication in attempts to regenerate different tissues and organs, including naturally derived materials , synthetic materials (poly(ethylene glycol), poly-l-lactic acid, etc.), and hybrid hydrogels that combine both synthetic and natural materials ,4,5. HydTo create effective hydrogel scaffolds, it is crucial to carefully select biomaterial component based on their fundamental bioactivity and characteristics. Among the various biomaterials, natural biomaterials, such as collagen-derived materials and polysaccharides, have garnered significant attention due to their biocompatibility, biodegradability, and ability to mimic the natural extracellular matrix (ECM) ,10. GelaResearchers have studied the physiochemical properties of materials comprising gelatine and polysaccharides using different crosslinking approaches and explored their potential in tissue engineering ,21,22,23In this report, we investigated the significance of screening the combination of gelatine and polysaccharide materials, specifically HA and CMC, for their use in tissue regeneration. To achieve this objective, we developed Gel/HA and Gel/CMC hydrogel scaffolds using varying concentrations. We employed 1-ethyl-3-[3-dimethylaminopropyl] carbodiimide hydrochloride (EDC) to effectively crosslink gelatine with either HA, CMC, or gelatine alone. The resulting hydrogels displayed proper mechanical properties, swelling behaviour, and porosity, demonstrating excellent cytocompatibility. Furthermore, the Gel1HA1 and Gel1CMC1 hydrogels exhibited remarkable cellular proliferation and aggregation. To explore the tissue regeneration effect of the hydrogels, we first used a full-thickness skin excisional wound model in vivo as a representative tissue defect model to test tissue repair efficacy. Furthermore, we performed a subcutaneous implantation model in vivo to explore the tissue integration and regeneration effect of the hydrogels. Various gelatine-based hydrogels have been extensively developed and studied for use in tissue engineering due to their feasible fabrication methods and advantageous formulation. In general, it is considered appropriate to utilize a relatively low concentration of gelatine in order to fabricate soft tissue engineering scaffolds. By incorporating an additional material that can crosslink with the amine or carboxyl groups present in gelatine, the mechanical properties, degradability, and porosity of the resultant hydrogels will be adjusted, effectively overcoming the limitations associated with hydrogel scaffolds composed solely of gelatine. We selected hyaluronic acid (HA) and (CMC) as representative polysaccharides, each possessing carboxyl groups, for effective crosslinking with the amine groups present in gelatine using the EDC method . As a gew/v) was used for all the hydrogels. CMC or HA was chemically incorporated at 0.1% and 0.2%, respectively, and crosslinked with 1.58% gelatine. As the concentration of CMC or HA increased, the storage modulus of the resulting hydrogels also increased, as shown in The mechanical properties of the hydrogels were evaluated and presented in The swelling behaviour of the hydrogels over 48 h is shown in Morphological analysis was performed using SEM detection, as depicted in The cytotoxicity and proliferation effect of the hydrogels were assessed using a CCK-8 assay with 3T3 fibroblasts. The cells were seeded ono the surface of the gels to mimic the in vivo microenvironment of an implanted gel and replicate the natural progression of cell interactions that occur during the tissue regeneration process. Cells cultured with a full cell culture media were referred to as the “control” group. As shown in These physiochemical and in vitro findings emphasise the distinctive attributes of Gel1HA1 and Gel1CMC1 hydrogels, suggesting their versatility for use in a range of biomedical applications beyond tissue regeneration. To confirm the potential of the hydrogels prepared in this work, we first verified it using an excisional full-thickness skin wound model. Based on the physiochemical and in vitro results, Gel1CMC1 and Gel1HA1 were selected as the experimental group. As shown in Angiogenesis plays critical roles in the wound healing process by facilitating the delivery of nutrients and oxygen to the injured tissue and draining out the wastes. We conducted an immunofluorescent assay to observe the CD31- and α-SMA-positive stained cells A. FurtheTo explore the potential applicability of the hydrogels in other medical scenarios that require angiogenesis, we used a subcutaneous implantation model in rats to evaluate tissue integration with hydrogel and neovascularisation . This moMixtures of hydrogels composed of gelatine with various polysaccharides have been explored in previous studies, demonstrating potential utility in various tissue engineering strategies. However, it should be noted that for translational medicine, the formulation and screening processes should be consistent and of significant relevance. For example, researchers investigated widely used biomaterials like alginate, hyaluronic acid and gelatine in a variety of concentrations and hydrogel formulations to obtain a comprehensive understanding of their behaviours . NeverthIn conclusion, hydrogels formed by chemically crosslinking between gelatine and polysaccharides were successfully developed to function as tissue engineering scaffolds. These hydrogels exhibited favourable physiochemical properties, including suitable mechanical properties, appropriate swelling behaviour, and adequate pore structure, ensuring their compatibility with soft tissue engineering requirements. The in vitro cell experiments confirmed the biocompatibility and the ability of the promotion of cell proliferation. In the in vivo experiment, optimised hydrogels accelerated skin wound healing process by promoting re-epithelialisation, angiogenesis, and granulation formation, as evidenced by histological and immunochemical analysis. The subcutaneous implantation of the hydrogels in vivo demonstrated excellent tissue integration and neovascularisation, along with minimal immune response and favourable hydrogel biocompatibility. These findings present the hydrogel formulated in this study, demonstrating the advantageous features of each active component and highlighting the potential synergistic effects that can be harnessed for more effective clinical outcomes.Gelatine  and carboxymethyl cellulose (50–200 mPa.s) were purchased from Sigma-Aldrich . Hyaluronic acid (Mw 10 kDa) was purchased from Focuschem, Qufu, China. N-(3-dimethylaminopropyl)-N-ethylcarbodiimide hydrochloride (EDC) was purchased from Sigma-Aldrich. All other chemicals were of analytical grade and used without further purification. Dulbecco’s Modified Eagle’s Medium (DMEM), foetal bovine serum (FBS), and penicillin/streptomycin were purchased from Invitrogen . All scaffold samples were prepared according to the concentrations described in To prepare the self-crosslinked Gel1 sample, gelatine was added to deionised water at 50 °C and stirred until fully dissolved. Then EDC/NHS solution was added to crosslink gelatine at room temperature for 24 h. The concentration of the EDC/NHS used in our study was 0.2% for EDC and 0.1% for NHS. To prepare Gel1CMC1 and Gel1CMC2 samples, gelatine was firstly dissolved in deionised water at 50 °C. The CMC solution was then added to the gelatine solution and stirred for full mixing. Subsequently, an EDC/NHS solution in MES was added to the mixture dropwise to crosslink the materials at room temperature for 24 h. The hydrogel precursor solutions were sterilized via filtration through a sterile filter with 0.22 μm, which could eliminate the microbial contaminants. The hydrogels were formed within a cylindrical plastic model with a diameter of 2.5 cm. n = 3). In total, 100 μL of the mixed hydrogel precursor was add on the plate and the test was performed at 25 °C. The hydrogel samples were tested using the following regimes: (1) time sweeps with a frequency of 1 Hz and a strain of 1%; and (2) strain sweeps ranging from 0.1 to 100% at 1 Hz. The storage modulus (G′) and loss modulus (G″) were monitored with time. G′ and G″ values on various strain sweeps were also measured. Rheological properties were analysed with a TA rheometer equipped with an 8 mm parallel plate (n = 3), which were immersed in PBS at 37 °C for 48 h. The wet weight of the samples was measured, and the swelling degree was calculated as follows:A swelling test was performed using freeze-dried hydrogel samples . ImageJ 1.53t software was used to determine the pore size of the hydrogel scaffold (n = 3).Scanning electron microscopy (SEM) was used to characterise the morphology and porosity of the freeze-dried hydrogels. The samples were cut in half using a razor blade and mounted onto an aluminium stub. Prior to imaging, the samples were coated with a layer of gold and examined using an AMETEK3T3 cells were used to analyse the cytocompatibility and cell proliferation of the hydrogel scaffolds using CCK8 (Invitrogen). The hydrogel samples were set as experimental groups and were immersed in DI water to exclude the uncrosslinked agents.n = 3). Samples weighing 20 μg and a 10 μL cell suspension with a concentration of 1 × 106 cells/mL were added to the top of the hydrogels and allowed to settle for 2 h before the addition of 1 mL full cell culture media to the well. Cells with a concentration of 10,000 cells per well were used as the control group and allowed to attach overnight. The cells were incubated at 37 °C and the test following standard protocol was performed at 24, 48, and 72 h after coculture. All groups were added to each well and co-cultured with the cells in triplicate. Each hydrogel group was added to a 24-well plate  and ethidium homodimer-1 for dead cells (red colour). Images were taken using an inverted fluorescence microscope .Male C57BL/6 mice weighing 18–25 g were utilized to create an excisional skin wound model and to test the wound treatment efficacy. Sprague Dawley (SD) male rats 6–8 weeks in age with body weights ranging from 200 to 240 g were used for subcutaneous hydrogel implantation. The animals were fed ad libitum water and a rodent diet. All procedures were approved by the Experimental Animal Welfare Ethics Committee of Hangzhou Medical College (China). n = 5). Full-thickness excisional wounds with a diameter of 1 cm were created on the dorsal region of mice. Wounds were treated using Gel1CMC1, Gel1HA1, and saline (Blank), respectively. At regular time points, gross images of wounds were taken using a digital camera. The wound area was measured using ImageJ and the remaining percentage (%) was calculated as follows:Wound tissues were sampled at day 14 and fixed with 4% paraformaldehyde. Then, the samples were embedded in paraffin and cut into sections with 5 μm thickness. Hematoxylin and eosin  were used to assess the tissue integrity and re-epithelialisation. Masson’s trichrome (Sigma-Aldrich) staining was conducted to detect the neo-dermis formation. In total, 10 sections were assessed per wound, and 5 wounds were examined per time point per group. Immunohistochemical staining of the sections was conducted. Sections were deparaffinized and washed three times in PBS for 5 min. Then, the sections were blocked with 5% serum for 30 min. Next, the slides were incubated in primary antibodies, VEGF (1:50), TNF-a (1:200), and IL-1β. Immunofluorescence was processed after the sections’ fixation in aceton at −20 °C. Sections were stained with antibodies of CD31 and α-SMA. After PBS washing, sections were incubated using secondary antibodies. The positive signals were further treated using DAB + kit . For double immunofluorescent staining of CD31 and α-SMA, paraffin sections were first deparaffinized and washed with PBS. Then, the sections were blocked by serum and incubated with anti-CD31 primary antibody  and anti-α-SMA antibody . Lastly, sections were stained with DAPI (Abcam). Immunofluorescence images were taken via an AxioCam HRm camera mounted on a Zeiss Imager M2 microscope.+-stained vessels and the mature vessels stained by both CD31 and α-SMA were counted using ImageJ. All tissues were imaged and five high-power microscopic fields for each separate wound were sampled by three blinded evaluators. The number of CD31The in vivo biocompatibility, tissue integration and effect of vascularisation of the hydrogels were evaluated in the subcutaneous implantation model in SD rats. The animals were allowed ad libitum feeding and were anesthetized via an intraperitoneal injection of 10% chloral hydrate. To create the model, four subcutaneous pockets were made on the dorsum of each animal by blunt dissection, with two on each side. One hydrogel weighing 200 mg was implanted in each pocket, and the incision was sutured with silk stitches. Implants were sampled every week to visualise adverse effects after implantation and degradability was observed until there was no obvious implant (five samples for each time point). At regular time points, animals were euthanised and implants were exposed by cutting the skin. Gross images of the implants were recorded to observe the remaining hydrogels. The samples of the hydrogel implants were fixed in a 4% formaldehyde/PBS solution for analysis. H&E and Masson’s trichrome were used to visualise the in vivo degradation, tissue integration, and neo-vessel formation of the samples following the manufacturer’s instruction for use. After staining, the prepared slides were examined under an optical microscope to gain insight into the structure and composition of the tissue. t test was employed. A p value of less than 0.05 was considered statistically significant.The values are reported as mean ± standard deviation (SD). To determine statistical differences between the two groups, the unpaired Student’s


The demand for materials with improved properties and less negative impact on the environment is growing. Artificial stones are examples of these materials produced with up to 90% of particulate material joined by a binder. This article evaluates the physical and mechanical properties of two artificial stones produced with processing steel residue (blast furnace dust waste) and quartz powder. Two binders were used: pure epoxy resin, denoted as ASPB100, or a mixture of 70 wt% epoxy resin with 30 wt% cashew nut shell oil, denoted as ASPB7030. The process took place under vibration, compression (3 MPa/20 min and 90 °C) and vacuum (80 Pa). ASPB100 showed water absorption of 0.07%, while for ASPB7030, it was 0.54%. They were classified as having high mechanical strength associated with bending stress values equal to 32 and 25 MPa, respectively. Stain resistance indicated that both artificial stones had their stains removed with the tested cleaning agents. In this way, the novel artificial stones produced are sustainable alternatives for the application of blast furnace waste and cashew nut shell oil, reducing their negative impacts on the environment. Artificial stone is defined as a composite material made from a mixture of stone aggregates and other minerals bonded together with a polymeric resin, which allows a material with mechanical properties close to or superior to natural stones. Aggregates normally represent between 90 and 94 wt% of artificial stones .Faced with the growing demand for diversified materials with improved physical and mechanical properties, artificial stones have caught the interest of consumers due to the advantages they have over natural stones, such as the minimum amount of pores, which contributes to a more compact and durable material. Artificial stones might have better mechanical resistance, lower density (due to the polymeric matrix) and the possibility of being manufactured from industrial waste, such as those from the ornamental stone industry as well as glass and steel industries, among others, with different textures, colors and shapes ,3,4.Due to the advantages presented by artificial stones, they have been widely used in decoration and construction. Although they originated in the 1960s in Italy, artificial stones became popular in the rest of Europe and North America only in the 1990s .Owing to the control of the process and the inputs used in production, this material has a slightly higher market price than natural stones. The average price of artificial stones is currently USD 540/t, while for natural stones, this is USD 527/t [Today, the artificial stone industry’s challenge is to make this material increasingly sustainable, economically viable and with improved mechanical properties, thus leading producers to seek alternative fillers to replace conventional aggregates. There are studies reporting the use of different solid wastes as mineral fillers in the production of artificial stones. Lee et al.  and PeixThe addition of mineral filler in the production of polymeric composites contributes to an improvement in mechanical resistance. Anastasios et al.  added flOther additives such as natural fibers also bring improvements in mechanical properties. Rajawat et al.  producedPolymer resin has the advantage of being lighter, thus reducing transportation costs; however, degradation in external environments is a disadvantage. This disadvantage can be improved through additives that act as inhibitors of its accelerated degradation, but more studies in this area are required .As industrialization increases worldwide, wastes are becoming a modern environmental problem. Poor handling intensifies their improper disposal, thus jeopardizing landscapes, fauna, flora and populational health and leading to air and soil contamination. Hence, there is an urgent need to propose new sustainable strategies aiming to lessen these impacts through more effective waste management .An alternative path to reduce the amount of improperly discarded waste while enhancing the mechanical properties of a commercial material is to produce artificial stones based on waste materials, which can be achieved by replacing part of the raw material aggregates (such as quartz) with these wastes. In this scenario, siderurgic waste has potential to be employed in artificial stone’s development since it has a chemical composition close to that of the conventional aggregates. In particular, the presence of metallic oxides, such as iron, contributes to increasing the mechanical strength of the final product .Despite its economic importance, the steelmaking industry is a major waste generator by virtue of each stage of the siderurgic production process. According to the Steel Institute, in 2018, for each 1 ton of crude steel produced, 628.5 kg of waste and co-products was generated. Notwithstanding its 85% reuse potential, the remaining 15% still does not have an adequate destination, thus lingering as stock or discarded in industrial landfills .Another of the artificial stone’s development challenges is the polymeric resin. Currently, the industry uses mostly epoxy or polyester, both derived from petroleum. Seeking bio-based resins as a substitute to make the material more sustainable, this study used a mixture of 70 wt% epoxy resin with 30 wt% cashew nut shell oil (CNSL). CNSL is an inedible agricultural waste widely available, with a production in the order of 4,450,000 tons per year. Cardanol is a CNSL major constituent and is characterized by a phenol ring connected to C15 alkyd chains at the meta position, which have different degrees of unsaturation. The unique structure of cardanol makes this material a promising candidate for a variety of applications and for many routes of chemical modifications, exhibiting excellent thermal and mechanical properties owing to its unique structural features ,23.Unlike previously published studies, this work produced an environmentally friendly artificial rock by modifying the binder through the addition of CNSL to the epoxy resin. This work’s objective was to produce and characterize a novel artificial stone based on blast furnace dust waste and conventional aggregate as fillers agglomerated by a mixture of epoxy resin with CNSL. Characterization was performed by evaluating the physical, chemical and mechanical properties of the artificial stones produced.The blast furnace dust was supplied by ArcelorMittal, located in Serra, Espírito Santo, Brazil. The other congregate mineral material, quartz sand, was supplied by EcologicStone, located in Cachoeiro de Itapemirim, Espírito Santo, Brazil. Both wastes were applied to manufacture the artificial stones proposed. The quartz sand was supplied as particles of both medium and coarse granulometry, ranging from 2 to 0.42 mm and 0.42 to 0.075 mm. The blast furnace waste was supplied as fine granulometric particles of less than 0.075 mm.3.As the binder, we used the epoxy resin type diglycidyl ether of bisphenol A (DGEBA) mixed with the triethylenetetramine (TETA) hardener, both supplied by Epoxyfiber, Brazil. The supplier indicated the density of the epoxy was 1.15 g/cmThe CNLS was supplied by Resibras, Fortaleza, Ceará, Brazil.The blast furnace dust waste was subjected to an X-ray fluorescence (XRF) analysis for chemical characterization using a wavelength distribution XRF Spectrometer , max. 1 kW, voltage 20–50 kV, current: 5–50 mA). The mineralogical composition was studied in a SHIMADZU diffractometer operating with copper radiation (Cu-Kα) and a 2θ scanning ranging from 5° to 90°.−1 rate. The composition with the highest amount of CNLS presented the best bend strength and was thus chosen for the manufacturing of the artificial stones.Three compositions were prepared for the epoxy resin: CNLS: EPOJU8020 (80–20 wt%), EPOJU7030 (70–30 wt%) and EPOJU6040 (60–40 wt%). In order to evaluate their mechanical properties, 3-point bend tests were performed as per the UNEEN ISO 178 standard (for plastics) , using tTen different particle size compositions were proposed using three particle sizes: large (L), medium (M) and fine (F). The mixture with the highest packing was determined as per the ABNT/MB-3388—“Determination of the minimum index void ratio of non-cohesive” . Each cop ≤ 0.05) to corroborate the statistical significance. Then, the Tukey test (p ≤ 0.05) was performed to compare the averages and verify the one with the best results. All calculations were performed using Microsoft Excel from Office, 2010.The data obtained above the underwent analysis of variance (ANOVA) of the completely randomized design (CRD) (VV) of the mixture, using [With the highest package composition vibrated density, it is possible to calculate the void volume VV%=1VV% = Void volume present in the mixture of particles;Qρ = Mineral charge density, calculated via pycnometry;After that, to determine the minimum content of resin to fill the void volume and, therefore, produce the artificial stones, Equation (2) was used:MAR% = Minimum amount of resin to fill the void volume;Qρ = Mineral charge density, calculated using pycnometry.MAR of epoxy resin was calculated to fill the void volume between the waste mixture particles.In this work, an 18% Artificial stones were produced with epoxy resin and CNSL, in the composition determined via ANOVA and the resin content determined via MAR in Equation (2). Stones sized 100 × 100 × 15 mm were processed using vacuum, vibration and compression methods, with pure epoxy resin (ASPB100) and with 70 wt% epoxy resin and 30 wt% CNLS (ASPB7030). The stones were developed based on blast furnace dust waste and quartz sand.Initially, the waste was dried in an oven at 100 °C to remove moisture, and then it was weighed and placed inside the mixer. The resin and the CNLS were added to the mixer and, after homogenizing, the resulting mass was deposited in the mold on a vibrating table. The vibration and vacuum were important to ensure that all mass was well spread in the mold cavity and avoid leaving air bubbles trapped in the mass. The mold was then placed in a hydraulic press (brand Marcone model MA 098-A) for 20 min while subjected to a compression under a pressure of 3 MPa, and heated at 90 °C.The mold was disconnected from the vacuum system and cooled to room temperature (RT). The stones were removed and put in an oven at 90 °C for post-curing. At the end, they were sanded and cut according to the sizes recommended for each test.The values of density, water absorption and apparent porosity were obtained from the standard tests according to the ABNT/NBR standard ,30. For Ten prismatic specimens, cut from the ASPB100 and ASPB7030 stones, sized with dimensions of 10 × 100 × 25 mm, were three-point bend tested in a model 5582 Instron machine under a 0.25 mm/min loading rate, 100 KN load cell and 80 mm two-point distance, following the NBR 15845-6 standard, to evaluate the maximum bending stress ,31.Wear resistance was measured in terms of thickness loss with the Amsler wear test (in Amsler equipment branded MAQTEST). Two samples measuring 70 × 70 × 40 mm were subjected to wear on a 500 and 1000 m circular running track, respectively, according to the ABNT/NBR standard .The microstructure of the fracture region after bending tests was evaluated by using scanning electron microscopy (SEM) for the analysis of the adhesion of particles to the DEGBA-TETA system. The equipment used was the SSX-550 SHIMADZU model operated by using secondary electrons.2O3 and red Fe2O3), an oxidizing agent (iodine) and a film-forming agent (olive oil). Domestic products such as wine, ketchup, mustard, lemon and coffee were also used. For each coloring agent, a specimen was tested and the staining agent remained on its surface for 24 h. The material was then classified according to the ease of stain removal.Stain resistance was tested according to an adaptation of the NBR 10545-14 standard . This teTo remove stains, the specimens were subjected to the following cleaning procedures: (i) washing with hot water; (ii) manual cleaning process with lighter cleaning products; (iii) cleaning the surface with a strong cleaner; and (iv) immersion in acid solution. This test aims to identify the applicability of the artificial stone in places such as a kitchen, bathroom or office tops .According to 2O3) and wustite (FeO). Furthermore, iron-based minerals like quartz (SiO2), calcite (CaCO3) and graphite (Cn) are also present.The results of the mineralogical analysis in p ≤ 0.05), with subsequent Tukey test average results. Analyzing the results obtained in As an important parameter, the vibrated density average data were treated with ANOVA considering a CRD performed with 95% confidence level  and a mixture of 70 wt% epoxy and 30 wt% cashew nut shell oil (CNLS) (ASPB7030), were used.Based on the ternary diagram developed in the experimental numerical modeling grid simplex, the composition that displayed the highest vibrated density, and thus the highest packing, was chosen to produce the stones. These data were authenticated using ANOVA and the Tukey test.Both artificial stones presented satisfactory physical indices, with ASPB100 presenting lower porosity and water absorption, making it possible to be applied in humid environments such as kitchens and bathrooms.ASPB7030 and ASPB100 displayed 31.0 ± 2.5 MPa and 25.0 ±1.5 MPa mechanical strength in three-point bending, thus classifying them as stones of very high strength (suitable to be applied as coating) and corroborating the porosity results.Amsler wear tests indicated that ASPB100 with 0.84 mm ± 0.01 thickness reduction can be applied in floors of high traffic areas, while ASPB7030 with 2.09 mm ± 0.27 thickness reduction can be used for medium traffic ones.SEM micrographs proved the physical index and mechanical property results, displaying the optimal load–matrix interaction for both developed stones. ASPB7030 had particles detached from the fractured region, which could be associated with its lower content of epoxy resin. As such, the agglutination of particles in ASPB7030 was not as efficient as in ASPB100.The resistance to staining indicated that both stones are not easily stained, except for the red staining agent, which remained in ASPB7030 even after all cleaning steps.Both artificial stones produced are an economically viable and sustainable alternatives. ASPB7030 stands out for having two wastes (CNSL and blast furnace dust waste) that do not need any prior treatment, which would mean a cost saving. Both demonstrate excellent physical and mechanical properties for civil construction applications.


SMO and GLI1 genes in the hedgehog pathway in malignant mesothelioma specimens. Further study on the expression and prognosis of SMO and GLI1 in malignant mesothelioma tissues and the relationship between the two and the molecular mechanisms of mesothelioma immunity and to further investigate the prognostic value of mesothelioma expression.  To investigate the value of SMO and GLI1 proteins and mRNA in biopsy specimens and plasma cavity effusion specimens from malignant mesothelioma (n = 130) and benign mesothelial tissues (n = 50) and to analyze the clinicopathological significance and survival risk factors of SMO and GLI1 protein expression in mesothelioma. The mechanisms of mesothelioma cell expression and immune cell infiltration were investigated using bioinformatics methods.  Immunohistochemistry and RT-qPCR were applied to detect the expression of SMO and GLI1 in mesothelioma tissues detected high concordance between the diagnostic results of mesothelioma biopsy specimens and plasma cavity effusion specimens. The expression levels of SMO and GLI1 protein and mRNA in mesothelioma tissues were higher than those in benign mesothelioma tissues. The expression levels of SMO and GLI1 protein were correlated with the age, site, and asbestos exposure history of patients with mesothelioma. The expression levels of SMO and GLI1 protein were correlated with the expressions of ki67 and p53 (P < 0.05). SMO and GLI1 gene expression levels were negatively correlated with good prognosis in mesothelioma patients (P < 0.05). Cox proportional risk model indicated that protein expressions of invasion, lymph node metastasis, distant metastasis, staging, and genes were independent prognostic factors of mesothelioma. The GEPIA database showed the overall survival rate and the disease-free survival rate of mesothelioma patients in the high SMO and GLI1 expression groups; the UALCAN database analysis showed lower SMO expression levels in mesothelioma patients with more pronounced TP53 mutations (P = 0.001); GLI1 gene expression levels were strongly correlated with lymph node metastasis in mesothelioma patients (P = 0.009). Timer database analysis showed that the mechanism of immune cell infiltration was closely related to SMO and GLI1 expression. The degree of immune cell infiltration was strongly correlated with the prognosis of mesothelioma patients (P < 0.05). SMO and GLI1 proteins were higher than those of normal mesothelial tissues, and the mRNA expression levels also changed in the same direction. SMO and GLI1 gene expressions in mesothelioma were negatively correlated with age, site of occurrence, and history of asbestos exposure. Positive expression of SMO and GLI1 was negatively correlated with patient survival. The Cox proportional risk model showed that gender, history of asbestos exposure, site of occurrence, SMO, and GLI1 were independent prognostic factors for mesothelioma. The mechanism of immune cell infiltration in mesothelioma is closely related to the gene expression of both and the survival prognosis of mesothelioma patients. The expression levels of both  Malignant mesothelioma is a highly aggressive serous cavity malignancy. It is mainly seen in patients over 60 years of age and is more common in men , 2. MesoSMO genes belonging to class F G protein-coupled receptors, and GLI1 is a direct transcriptional activator downstream of the Hh signaling pathway. The binding of Hh ligands to the transmembrane receptor fragmentation protein-1 (PTCH-1), which has an inhibitory effect on SMO release, causes upregulation of activated SMO downstream of the GLI zinc finger to activate the Hh pathway and promotes fibroblast migration, invasion, and skin wound healing. According to Kaushal Jyoti et al. and Nastase et al.'s research, siRNA transfection tests showed that SMO promotes cell migration by acting upstream of the PI3K pathway [SMO and GLI1 suggests that -catenin activates Hh signaling in turn [GLI1 increases the motility and invasiveness of pancreatic ductal adenocarcinoma cells [The hedgehog (Hh) signaling pathway is one of the important signaling pathways in vivo that regulate organ growth and development and controls tissues in vivo during embryonic development, maintaining their normal structure and function  The Shh  pathway , 12. SMO in turn , 14. Thrma cells , 16. SomPathological specimens from all enrolled patients were collected to obtain information about the patients' general condition, personal hygiene habits, underlying diseases, and type of work. The Ethics Committee  gave its approval for the study. 130 individuals with plasma cavity illness were chosen after screening to receive treatment at Cangzhou People's Hospital between January 2017 and December 2021. The pathological diagnosis complied with the clinical diagnosis and treatment recommendations for malignant mesothelioma in terms of diagnostic criteria. The following were the criteria for inclusion: informed agreement was obtained from patients and their families, and patients were followed up; the age range was 18 to 80; the histology type was patients with a definite pathological diagnosis of thoracoabdominal mesothelioma . The official pathology report identified 4 specimens as having mesothelioma following the discovery of D2-40, WT1, and other markers. The following were the exclusion requirements: patients without genetic disorders in their family history medical records; patients with insufficient pathology data and lost to follow-up; (iii) patients with specimens contaminated due to improper handling of specimens in the experiment .SMO and GLI1 proteins in paraffin tissues. Retrospective analysis of the recruited patients was performed utilizing a diagnostic test evaluation approach. Diagnostic tests were administered to patients who met the inclusion and exclusion requirements, and the test findings were documented. Cases were renumbered using a double-blind approach after those who did not match the inclusion criteria were removed from the research. Four senior pathologists performed pathophysiological diagnoses on each case as part of a new round of diagnostic tests. Then, using immunohistochemical staining for SMO and GLI1, all cases were evaluated. Sensitivity and specificity of the newly developed combination diagnostic approach were recorded to determine the effectiveness of the diagnostic tests.Immunohistochemistry was used to identify the expression of μm, dried overnight at 56-60°C, and removed to cool to room temperature. Antigenic thermorepair preconditioning (EDTA) reagents are used. Then, conventional tissue paraffin sectioning, dewaxing solution, gradient alcohol hydration dewaxing, a special dyeing tank filled with 200 ml of EDTA buffer with pH 8.0, and a boiling water bath were used to put in dewaxed and hydrated slices and boiled for 20 minutes, kept warm for 10 minutes, cooled to room temperature (15-25°C), and washed with water; 3% H2O2 incubated at room temperature (15-25°C) for 10 minutes, PBS solution soaked; remove the PBS solution, add antibody reagent dropwise on the tissue paraffin section, so that the reagent covers the edge of the tissue, incubate in a constant temperature incubator at 37°C for 60 min, remove the reagent on the tissue, immerse the PBS solution for 3 min, and repeat 3 times; remove PBS solution, add anti-mouse/rabbit IgG peroxidase polymer dropwise, incubate for 30 min at room temperature (15-25°C), remove reagent, PBS solution immersion; add DAB chromogenic solution dropwise and incubate for 10 min at room temperature (15-25°C); hematoxylin counterstained for 10 sec, dehydrated, transparent with xylene, and sealed.Use no less than 60 ml of naturally settled serum cavity fluid over a period of 0.5 hours, according to the modified procedure for paraffin sectioning of plasma cavity fluid. Six centrifuge tubes containing the serum luminal fluid samples were spun three times at room temperature (15–25°C) (depending on the size of the centrifuged precipitated cells). The first cycle of centrifugation takes place at 694 × g for 5 minutes, after which the supernatant is spread out and discarded. A few drops of protein, glycerol, and 90% ethanol are added during the second round of centrifugation to revive the precipitate. The precipitate is once more centrifuged for 5 minutes at 694 × g. The second centrifugation and the third centrifugation are identical. After centrifugation, the supernatant was discarded, and the cell blocks were removed, fixed, dehydrated, embedded, and sectioned. Malignant mesothelioma tissue and benign mesothelial tissue samples were collected, neutral formalin was fixed for 12-24 hours, and wax blocks were prepared by dehydration and paraffin embedding. The tissue sections are adhered to the slides at a thickness of 5 SMO (ab124964) against rabbit was obtained from Abcam Inc. The dilution used for immunohistochemistry (paraffin-embedded sections) was 1 : 1000, and the positive control was gastric cancer tissue. GLI1, a rabbit polyclonal antibody , was obtained from Abcam, and the positive control was intestinal cancer tissue. Both antibodies are localized to the cytoplasm. Positive staining is brownish in color. Based on the results of the staining depth assessment, both experts verified the results of any discrepancies, ensuring consistency of the final conclusions.The monoclonal antibody The expression results were graded and counted as follows: the percentages of positive tumor cells were 0 (≤5%), 1 (6%-25%), 2 (26%-50%), 3 (51%-75%), and 4 (>75%). The staining intensity was graded as 0 (no staining), 1(+), 2(++), or 3(++++). Immunohistochemical results were scored according to the percentage of positive tumor cells multiplied by the staining intensity. The final immunostaining score for each section was 0-12, with a score of 0-4 being a low expression, while a score of 5-12 was a high expression.Total RNA was extracted from the cells with a Trizol kit, and the concentration of each group of RNA was measured with a nucleic acid protein assay (repeated three times to take the average), and the RNA was reverse transcribed to cDNA according to the hair transcription system and conditions in the following table, and the system was as follows .The primers were synthesized by TianyiBiotech with the following sequences .ul of RNA with a UV spectrophotometer, and the OD260/280 values were between 1.8 and 2.0. After calculation, the concentration of our extracted RNA was between 0.38-0.96 mg/ml. And the reaction system was as follows   was developed by Peking University and applied to integrate and analyze cancer expression profile data. This interactive web server contains 9,736 tumor samples and 8,587 normal samples of 33 malignant tumors from TCGA and GTEx databases with RNA sequencing expression data to analyze the correlation between candidate genes and the survival and prognosis of colon cancer patients through survival curves. The Kaplan-Meier method was used to analyze the relationship between SMO and GLI1 genes and the prognosis of mesothelioma patients and to draw survival curves. UALCAN  is an online website for analyzing and mining the TCGA database, built on PERL-CGI, javascript, and CSS. It can analyze whether a gene is associated with cancer and paracancer, pathological grade, prognosis, and other factors in TCGA database samples. In this study, we used UALCAN software to analyze the malignant mesothelioma and benign mesothelial tissue data in the TCGA database. The relationship between immune cells and mesothelioma survival prognosis, mesothelioma with associated immune cell infiltration, and SMO and GLI1 gene expression was based on the Timer database online website (http://http://timer.cistrome.org/).GEPIA . Correlation tests were performed using Spearman's rank correlation analysis; survival curves were plotted using GrapHD software. Multifactorial survival risk models established by Cox proportional regression risk analysis were statistically significant (P < 0.05).Statistical analysis was performed using SPSS 23.0 (IBM) statistical software. The correlation between SMO and GLI1 protein expression levels and mesothelioma clinicopathology was measured by correlation analysis, and the correlation between SMO and GLI1 were expressed in the cytoplasm. Statistical analysis showed that the positive expression of SMO in benign mesothelial tissue and malignant mesothelioma was 14.00% (7/50) and 81.54% (106/130) of biopsy specimens, respectively, with statistically significant differences  and 76.15% (99/130), respectively. The differences were statistically significant   and 73.08% (95/130) in benign mesothelial tissue and malignant mesothelioma, respectively  and 66.15% (86/130), respectively, with statistically significant differences  with statistically significant differences . It is suggested that SMO may have a role in the production of GLI1.After comparing the diagnosis of malignant mesothelioma in plasma cavity fluid specimens with that of biopsy specimens, we found a high level of agreement, and a comprehensive analysis of this new diagnostic method has a high clinical application. The results of Spearman's correlation analysis in 130 patients with malignant mesothelioma showed a positive correlation between The amplification curve is a curve describing the dynamic process of RT-qPCR, which is normally S-shaped. During the course of the PCR experiment, the fluorescence signal intensifies as the target gene continues to amplify. The fluorescence amplification curve is divided into three phases, and only during the exponential amplification phase of the fluorescence signal, a logarithmic relationship exists between the logarithmic value of the PCR product volume and the starting template volume, allowing for quantitative analysis. The Ct value indicates the number of cycles in each reaction well when the fluorescence signal reaches a set threshold. It has been confirmed that the higher the starting copy number of the template, the smaller the Ct value, demonstrating that there is also a linear relationship between the logarithm of the starting copy number of the template and the Ct value of this template. The mean Ct value was calculated for all samples tested by replicate wells and used to quantify the target genes contained in the samples.SMO and GLI1) mesothelioma internal reference GAPDH, △CtN represents the relative value of the expression of the target gene in benign mesothelioma tissues and the expression of the internal reference, △CtN = Ct benign mesothelioma target gene (SMO and GLI1) benign. The expression of the target gene in malignant mesothelioma tissues was higher than that in benign mesothelioma tissues if △CtCa < △CtN, and lower than that in benign mesothelioma tissues if △CtCa > △CtN. After statistical analysis, △CtCa and △CtN both conformed to normal distribution. △CtCa < △CtN indicated that the expression of SMO and GLI1 in mesothelioma tissues was higher than that in benign mesothelioma tissues; △CtCa > △CtN indicated that the expression in mesothelioma tissues was lower than that in benign mesothelioma tissues, and P < 0.05 was statistically significant.In this experiment, △CtCa represents the relative value of the expression of the target gene in malignant mesothelioma tissues and the expression of the internal reference, △CtCa = Ct mesothelioma target gene Ct  in malignant mesothelioma tissue is higher than that in benign mesothelial tissue; when X < 2, ΔΔCt < 1, indicating that the expression of target gene  in malignant mesothelioma tissue is the expression in malignant mesothelioma tissues was lower than that in benign mesothelial tissues.In this experiment, the expression of ploidy of mesothelioma tissues compared with benign mesothelial tissues was meaningful when it was greater than or equal to 2 fold and not meaningful when it was less than 2 fold. The relative expression of mesothelioma tissue compared to benign mesothelial tissue was expressed by △△Ct, i.e., △△Ct = △CtA − △CtB = (Ct mesothelioma target gene − Ct mesothelioma endogene XX) − . Using SMO was highly expressed in 28 mesothelioma tissues, with upregulation of more than 2 fold in 24 of the highly expressed samples, no significance in 4 cases, and lower expression than benign mesothelioma tissues in 2 cases .RT-qPCR was applied to detect SMO and GLI1 protein expression and clinicopathological data showed that the expression levels in patients with malignant mesothelioma were negatively correlated with age, history of exposure to asbestos chemicals, mesothelioma site, Ki67, and P53 expression (P < 0.05) and not significantly correlated with patient gender (P > 0.05). High protein expression cases were mostly seen in middle-aged and elderly people . SMO and GLI1 gene expression levels, tumor site, patient's gender, and history of asbestos exposure were all risk factors for survival time in mesothelioma patients (P < 0.05), and the findings were consistent with greater significance for the prognostic value of mesothelioma. Hotta and Fujimoto and Keshava et al. showed that poor prognosis of malignant mesothelioma is usually associated with immune T-lymphocyte infiltration [κB pathway to promote the expansion and differentiation of T cells. Immune evasion of malignant cells is closely associated with high CD70 expression, and overall survival of patients with CD70-expressing tumor cells is significantly reduced [SMO and GLI1 expression, and that CD4+ T cells, neutrophils, and macrophages have an important impact on mesothelioma development and prognosis. We also found that immune cells are closely associated with the copy number immune mechanism of mesothelioma, which contributes to the prognostic assessment and targeted therapy of mesothelioma.Malignant mesothelioma has few treatment options due to its dysfunctional mutant state, and only about 12% of patients with malignant mesothelioma survive longer than 3 years, and its development may be closely related to occupational or environmental asbestos exposure , which il family . bFGF anl family . In addicription , 21. In  related . In thissurvival –25. Our ltration , 27, whi reduced , 29. Tum reduced , 31. OurSMO and GLI1 genes are differentially expressed in malignant mesothelioma and benign mesothelioma tissues, which have more important guiding significance for the diagnosis and prognostic mechanism of mesothelioma and also open up new ideas for gene targeting and personalized immunotherapy of mesothelioma.At present, biopsy specimens are mostly used clinically to diagnose mesothelioma, because mesothelioma cells are indistinguishable from degenerated or proliferating mesothelioma cells, and most patients with malignant mesothelioma effusion in the thorax and abdomen are already in advanced stages at the time of diagnosis, and cytological sections have disadvantages such as superimposed cell obstruction, high false-positive rate, and easy desquamation. We have improved the method by separating the plasma cavity fluid from the pericardium in paraffin sections, and the resulting cells are flattened, the sections are clearly stained, and the diagnostic results are accurate and easy to distinguish, providing an important basis for an accurate cytologic diagnosis of malignant mesothelioma. At present, the definite diagnosis of mesothelioma is mostly obtained by tumor resection or puncture specimens, and pathological specimens are not easy to obtain and harmful to patients; specimens of pleural and peritoneal fluid are easy to obtain and less harmful to patients, and the extracts can alleviate patients' clinical symptoms, which has obvious superiority. In our experiments, we also compared the results of biopsy specimens and effusion specimens, and the accuracy of the two detection methods was consistent, so the use of plasma cavity fluid specimens for the diagnosis of mesothelioma is a relatively novel method. However, the diagnostic accuracy may be slightly lower because of the low cell content in plasma cavity fluid, poor heterogeneity of exfoliated cells, and possible differences in cells in the fluid at different times, in which the accuracy of the location of the extracted fluid may affect the diagnostic accuracy of plasma cavity fluid specimens, but plasma cavity fluid specimens are superior to puncture biopsy, noninvasive, easy to operate, easy to obtain for clinical sampling requirements, and less painful for patients. Moreover, the prevalence of malignant mesothelioma has increased year by year in recent years due to industrialization and environmental changes, with increasing exposure to asbestos chemicals as risk factors, so plasma cavity fluid specimens can be used as an important tool for screening patients with mesothelioma and are of great importance for the clinical diagnostic value of malignant mesothelioma. In summary, SMO and GLI1 expression prognosis and immune cell infiltration mechanisms in malignant mesothelioma tissues by immunohistochemistry, RT-qPCR, and bioinformatics. SMO and GLI1 genes were correlated with gender, age, tumor site, history of asbestos exposure, and staging of mesothelioma patients. Expression levels of both were associated with poor prognosis of mesothelioma and infiltration of immune cells such as CD4+T. This study evaluated the factors affecting the development of malignant mesothelioma, proposed a new idea of SMO, GLI1, and immune cells as possible indicators of personalized immunotherapy and prognosis, and provided an experimental basis for finding new gene targets for mesothelioma.In conclusion, this study analyzed the relationship between


Acquired hemophilia A (AHA) is a rare disease characterized by a prolonged activated partial thromboplastin time (aPTT) and the production of coagulation factor VIII inhibitors. We encountered two cases of AHA in the perioperative period of pancreatoduodenectomy (PD).Case 1: A 76-year-old woman with intraductal papillary mucinous carcinoma developed acute cholecystitis 5 days before PD. Despite immediate improvement in her acute cholecystitis with biliary drainage and antibiotics, her aPTT level was prolonged (55.9 s). PD was performed as scheduled. On postoperative day (POD) 2, she developed intra-abdominal hemorrhaging that required reoperation. However, intra-abdominal bleeding and concomitant anemia persisted after reoperation. On POD 13, she was diagnosed with AHA based on the detection of an inhibitor of coagulation factor VIII. Despite hemostatic and immunosuppressive treatment, including massive blood transfusion, her general condition gradually worsened due to continuous bleeding and secondary infections. She ultimately died of multiple organ failure on POD 71. Case 2: An 82-year-old man received PD for distal cholangiocarcinoma. On POD 3, a small amount of blood via abdominal drainage was observed. On POD 4, his aPTT level was prolonged (61.5 s). On POD 8, subcutaneous hemorrhaging of the median wound was observed, and corticosteroids were administered under suspicion of AHA on POD 9. On POD 15, an inhibitor of FVIII was detected, and he was diagnosed with AHA. On POD 17, the aPTT level had normalized, and an inhibitor of FVIII was undetectable. On POD 41, he was discharged without any serious hemorrhagic events.AHA may be more frequent than previously reported. When unexplained prolonged aPTT or bleeding symptoms are observed, it is important to keep AHA in mind during the perioperative period of invasive surgery. Acquired hemophilia A (AHA) is a rare autoimmune disease characterized by an unjustified prolonged activated partial thromboplastin time (aPTT) and abnormal acute bleeding symptoms with no personal or family history of coagulopathy—due to the production of inhibitor of coagulation factor VIII (FVIII)—with a normal prothrombin time (PT) . The incWe encountered two cases of AHA in just 2 years and herein report those cases that developed during the perioperative period of pancreatoduodenectomy (PD), following markedly different clinical courses.A 76-year-old woman was scheduled for pancreatoduodenectomy (PD) for intraductal papillary mucinous carcinoma of the pancreas head. The aPTT level at the time of the initial visit was 26.8 s . She developed acute cholecystitis 5 days prior to elective surgery because of biliary congestion caused by the tumor. Acute cholecystitis was immediately improved with biliary drainage and the administration of antibacterial agents. However, her aPTT level was prolonged to 55.9 s after cholecystitis, whereas the prothrombin time (PT) was within the normal range . Prolonged aPTT was considered due to infection or bacteremia, and she had no clinical bleeding tendency. PD was performed as scheduled without further investigation. She received a transfusion of 4 units of red blood cells (RBCs) for intraoperative blood loss  due to prolonged oozing from the operative field. On postoperative day (POD) 1, the amylase level of her abdominal drainage fluid was 45 U/L (upper limit of the serum amylase level at the institute: 125 U/L). On POD 2, she had intra-abdominal hemorrhaging, and contrast-enhanced computed tomography (CE-CT) showed extravasation from the hepatic falciform ligament artery . No increase in hematogenous drainage or progression of anemia was observed. On POD 8, subcutaneous hemorrhaging of the median wound was observed. On POD 9, the FVIII activity was found to have decreased to 5%, and immunosuppressive treatment with prednisolone at a dose of 40 mg/day (1 mg/kg/day) was started based on the suspicion of AHA ; and immunosuppressive therapy  for eradication of the inhibitor . To prevIn Case 1, the patient supposedly developed AHA at the time of acute cholecystitis before PD, as evidenced by the prolonged aPTT following cholecystitis. Retrospectively, possible causes of the severe clinical course of Case 1 include: PD being carried out as planned without consideration of the prolonged aPTT; the addition of second surgical invasion; the delayed diagnosis and commencement of treatment for AHA (duration from confirmation of the prolonged aPTT to the commencement of AHA treatment: 11 days); and secondary infection during prolonged treatment period due to uncontrollable hemorrhaging. PD should have been postponed, and the cause of prolonged aPTT should have been investigated in detail for Case 1. In Case 2, the patient developed AHA in the early postoperative period after PD. We suspected AHA in this patient due to minor hemorrhaging and prolonged aPTT, based on our experience with Case 1, and initiated early treatment for AHA (duration from confirmation of the prolonged aPTT to the commencement of AHA treatment: 4 days). As a result, hemorrhaging was controlled, and adverse events from steroids were minimal. Thus, the duration from the onset to the initiation of treatment may be crucial. If malignancies and inflammation from surgery or infection can serve as triggers for AHA development, oncological surgeons may encounter AHA more frequently than previously reported. Keeping AHA in mind during the perioperative period is thus important.AHA may be more common than previously reported. When unexplained prolonged aPTT or abnormal bleeding symptoms are detected, we should consult specialists in hematology as early as possible. The early diagnosis and management of AHA may help to reduce mortality.


Intracranial epidermoid cysts are benign slow-growing ectodermal inclusions that account for less than 1% of all intracranial tumors. We retrospectively reviewed 36 such cases to evaluate the demographic characteristics, clinical manifestations, anatomical distribution, surgical management, and treatment outcome of these tumors. Additionally, we sought to identify the relationship between median or paramedian cistern tumor localization and clinical parameters, such as recurrence risk, hospitalization duration, and postoperative complication rates. The most frequently observed neurological symptoms were transient headaches (77.8%), dizziness (36.1%), CN VII palsy (19.4%), CN VIII hearing difficulty (19.4%) and cerebellar signs (19.4%). The most common surgical approaches included retrosigmoid (36.1%), subfrontal (19.4%) and telovelar (19.4%) approaches; gross total resection was feasible in 83.3% of cases. The postoperative complication rate was 38.9%. Tumors were more frequently found in the paramedian cisterns (47.2%), followed by the median line cisterns (41.6%). Multivariate analysis revealed that postoperative hydrocephalus and age < 40 years were prognostic factors for tumor recurrence. Median-like tumor location was a risk factor for the presence of symptomatic hydrocephalus both preoperatively and postoperatively, increasing the likelihood of protracted hospitalization (> 10 days). Despite their benign histopathological nature, these tumors have an important clinical resonance, with a high rate of postoperative complications and a degree of recurrence amplified by younger age and hydrocephalus. Although called cysts, these “pearly” tumors are solid and are encased by a collagenous capsule that can infiltrate along CSF -filled spaces3. Tumor growth is driven by the division of the stratified squamous epithelium lining its cavity, meaning that the cyst contents are largely composed of acellular keratin debris and cholesterol inclusions. According to the literature, liquefaction of the cyst contents is associated with infection or loss of vascularity4. Approximately 90% of intracranial epidermoid cysts are located in the intradural compartment, although they can also occur extradurally in the intradiploic space of the frontal, parietal, temporal, and occipital bones7. Epidermoid cysts are most commonly found in the cerebellopontine angle , the parasellar region (30%), and the fourth ventricle (5–18%) and less commonly in the middle cranial fossa, diploe or spinal canal9. The role of arachnoid compartments and structures cannot be neglected. The limitations of epidermoid formation into a specific cistern are demonstrated by the similar aspects of the epidermoid cyst at least for a period of time. Once the epidermoid cyst grows, the membranes and other arachnoidian structures, along with the neurovascular structures, will guide the further expansion of the tumor, and as a consequence, extension in different subarachnoid compartments is not an exception10.Epidermoid cysts are benign congenital inclusion cysts that in rare cases may occur intracranially, accounting for approximately 0.3–1.8% of primary intracranial tumors11. The disease course is classic for an extended asymptomatic period, until the tumor capsules begin to impinge on a cranial nerve or eloquent brain tissue, resulting in a rapidly evolving deficit. Magnetic resonance imaging (MRI) is the investigation of choice, and diffusion-weighted imaging (DWI) is the most helpful sequence for differential diagnosis and follow-up12.Despite their slow growth, these tumors have a strong propensity to adhere to critical neurovascular structures and cause impingement secondary to the mass effect, causing significant morbidity and neurologic impairment13. Gross total resection is critical to minimize the risk of recurrence14, as in some studies, incomplete removal of the capsule was associated with recurrence rates as high as 93%15.Surgical resection is at the forefront of patient management, especially because there is currently no concrete chemotherapy or targeted therapy available for this tumor typeThe main aim of the present study was to evaluate the demographic characteristics, clinical manifestations, surgical management and clinical outcomes of patients with intracranial epidermoid tumors. To our knowledge, this is the first study to report such information in a Romanian population. Additionally, we sought to identify the relationship between median or paramedian cistern tumor localization and clinical parameters, such as recurrence risk, hospitalization duration, and postoperative complication rates.All methods were carried out in accordance with the current neurosurgical guidelines available. An observational retrospective study was performed on 36 patients admitted to the Department of Neurosurgery, Cluj-Napoca Country Emergency Hospital during the period 2012–2021. Patients enrolled in this study had a pathologically confirmed diagnosis of an intracranial epidermoid cyst that was surgically resected. All adult patients as well as the guardians of pediatric patients gave their informed consent for both the intervention and the participation within this study. Regarding Figs. Patients were then stratified based on the localization of the tumor to the median, paramedian, or outside cisterns. Median cisterns included the fourth ventricle, cisterna magna, interpeduncular, pericallosal, suprasellar, chiasmatic, quadrigeminal, and cerebellomedullary cisterns. Paramedian cisterns were defined as the cerebellopontine, ambiens, carotid and sylvian cisterns.Further inclusion criteria included patients who presented at the recommended periodic check-ups and who had a postoperative follow-up of at least 6 months. As this was an observational study, the protocol was exempted from approval by the Ethics Committee of the “Iuliu Hatieganu” University of Medicine and Pharmacy.Microscopic resection was performed on all patients. Gross total resection (GTR) was considered if the lesion was completely removed along with its capsule and no residual tumor was seen on 3-month postoperative MRI (DWI sequences); it was termed subtotal resection (STR) if the entire tumor was removed but part of the capsule was left behind or if only some part of the tumor and capsule was excised. The amount of tumor removed was ascertained by verifying operative notes and follow‑up imaging. Brain CT was performed within a day of surgery in all patients post-excision to look for any bleeding in the tumor bed and any other surgery‑related complications. All patients were followed‑up post‑surgery in the neurosurgery outpatient service at our institute.In accordance with the clinic's protocol, after the primary surgical intervention, the patients were evaluated by MRI imaging at 3 months, 6 months, 1 year and then yearly postoperatively. Tumor recurrence was assessed by DWI sequences that demonstrated diffusion restriction at the time of recurrence in the area of tumor resection and the cisterns in the immediate vicinity. If the MRI imaging controls showed tumor recurrence and the patient was clinically symptomatic, they were subjected to surgical reintervention. The exception was the asymptomatic patients who were monitored periodically with MRI imaging at 6-month intervals, some of whom became candidates for surgical intervention at the time of symptomatology appearance. The other cases did not translate into a need for resurgery as the patients continued to be asymptomatic. In the case of patients undergoing surgical reintervention, postoperative scarring distorted the normal anatomy and obliterated the dissection planes. Recurrent epidermoid cysts become more adherent to neighboring critical neurovascular structures, thus making further resection a high-risk procedure. The outcome of patients was characterized by a worsening of preoperative neurological deficits. After discharge, follow-up was performed according to the clinic's protocol.All the data from the study were analyzed using IBM SPSS Statistics 25 and illustrated using Microsoft Office Excel/Word 2013. Qualitative variables were written as counts or percentages and were tested using Fisher’s exact tests. Z tests with Bonferroni correction were used to further detail the results obtained in the contingency tables.This retrospective study involving human participants was in accordance with the ethical standards of the institutional and national research committee and with the 1964 Helsinki Declaration and its later amendments or comparable ethical standards. The Iuliu Hatieganu University of Medicine and Pharmacy, Cluj-Napoca, Research Ethics Committee has confirmed that no ethical approval is needed.The study group included 36 patients aged between 1 and 73 years with a median age of 40.4. In our series, epidermoid cysts showed a slight male predilection . The average period of hospitalization was 11.64 days (between 1 and 2 weeks), with a median of 10 days. Most patients’ cysts were located in the infratentorial region (47.2%) with a predilection for the cerebellopontine angle (38.9%). According to the anatomical distribution of epidermoid cysts, in our study, 38.9% of cases were located at the level of the cerebellopontine angle, 19.4% of cases were located at the level of the IVth ventricle, 13.9% of cases were located at the temporal region, 11.1% of cases were located at the level of the frontal lobe, 8.3% of cases were located at the level of the sellar and parasellar region, and 8.3% of cases were located in other areas, such as the orbital area, the parietal region or the pontine cistern. The Glasgow Coma Scale (GCS) at admission was 15 points in 34 patients and 14 points in 2 patients. Most patients reported an insidious onset of symptoms (77.8%).The most frequently observed neurological symptoms are presented in Fig. Hydrocephalus was present in 16.7% and 19.4% of patients pre- and postoperatively, respectively. In a small subset of patients (16.7%), hydrocephalus failed pharmacologic management and required placement of a shunt, while one patient experienced transient hydrocephalus that responded to conservative management. In all patients with postoperative communicating hydrocephalus, a lumbar drain was placed for approximately 4–5 days to drain any fibrosis-inducing substances or blood clots and promote new CSF formation. If no resolution of hydrocephalus was seen, permanent CSF diversion procedures were considered. As all 6 patients had communicating hydrocephalus, a ventriculoperitoneal shunt was proposed in all cases. Of the 83.3% of patients who benefited from total resection, 5 patients developed postoperative hydrocephalus. Of the 16.7% of patients who benefited from subtotal resection, 2 patients developed postoperative hydrocephalus. In our series, we had 6 patients with hydrocephalus at presentation. Two patients presented with triventricular hydrocephalus and obstruction seen on imaging suggestive of noncommunicating hydrocephalus (obstructive mechanism). Four patients presented with tetraventricular hydrocephalus and no obvious obstruction seen on imaging suggestive of communicating hydrocephalus. In cases with communicating hydrocephalus, the proposed mechanism involves chemical meningitis, subsequent inflammation and a granulomatous reaction in the arachnoid villi with impaired CSF absorption. However, subclinical spillage of cyst contents can occur with no evident manifestation of meningism. Additionally, intricate mechanisms with both obstructive and reduced absorption are possible. Gross total resection was performed in all six cases. Of these six patients, four continued to have persistent hydrocephalus after surgery, and two patients had resolved hydrocephalus. In our series, we included a total of seven patients with postoperative hydrocephalus. Apart from the four cases discussed above, three de novo hydrocephalus cases were observed with no obstructive component.The overall postoperative complication rate was 38.9% and included persistent hydrocephalus (6 cases), aseptic chemical meningitis (2 cases), CN VII and VIII paresis (1 case), pseudomeningocele (1 case), dysarthria (1 case), acute subdural hematoma and seizure (1 case), transient hydrocephalus and subdural hygroma (1 case) and pulmonary embolus (1 case). In our study, 8 of the patients had a duration of symptoms between 1 and 4 years; the overall postoperative complication rate in this subset of patients was 25% (2 cases) and included aseptic chemical meningitis (1 case) and pulmonary embolus (1 case). Ten of the patients had a duration of symptoms under 1 month; the overall postoperative complication rate in this subset of patients was 70% (7 cases) and included persistent hydrocephalus (4 cases), pseudomeningocele (1 case), dysarthria (1 case), acute subdural hematoma and seizure (1 case). Precipitation of symptoms (under 1 month) was associated with a higher rate of postoperative complications (p = 0.026); no association was found between long-standing symptomatic patients (more than 1 year) and the rate of postoperative complications (p = 0.44).In 30 cases, gross total resection was feasible, while in the remaining patients, only subtotal resection was achieved. The most commonly used surgical approaches included retrosigmoid (36.1%), subfrontal (19.4%), telovelar (19.4%) or other 25.1%)  approaches. In 88.9% of cases, an improvement in symptoms was observed with an average Glasgow Outcome Scale (GOS) value of 4.61. In our study, gross total resection was feasible in 30 cases (83.3%)  over the last 10 years, epidermoid cysts represent 0.85% (36 out of 4233) of operated brain tumors. Despite their benign nature, intracranial epidermoid tumors can be challenging to manage due to their tendency to adhere to critical neurovascular structures, forcing conservative management. Failure to remove the entire proliferative capsule of the tumor augments recurrence rates sevenfold (to 21%), as was reported by Shear et al. in a 691-patient meta-analysis22 where epidermoid cysts generally become symptomatic during the fourth decade of life. Symptoms are driven by the compression of local structures and are based on tumor location—in our cohort, chronic intractable headache, dizziness, and cranial nerve palsies were the most common signs. The median symptom duration was 90 days, with minimum and maximum values of 4 days and 4 years, respectively. Stratifying patients into younger  and older  cohorts revealed differences in acute versus chronic symptom presentation. In younger patients, symptom onset was acute in 38.46% of cases, in which the symptoms lasted less than one month and all patients showed signs of increased ICP  at admission. In older patients, the onset of symptoms was acute in only 13.04% of cases, in which the symptoms lasted less than five months and were caused by mass effect, increased ICP, or both.In our study, the median age at presentation was 40.4 years with a 1.2:1 predilection to affect male versus female patients, which is on par with other large case series reported in the literature26. However, in our study, trigeminal neuralgia and hearing impairment were present in 5.6% and 19.4% of patients, respectively, in whom the symptom onset was insidious (more than one month).A substantial part of the literature links trigeminal neuralgia and hearing impairment with more acute symptom onset23. Hydrocephalus was reported in 16.7% of our patients at admission; half of them had a sudden onset of symptoms , while the other half reported a gradual onset over approximately one month. Among patients with hospitalized hydrocephalus, in 66.66% of cases, the hydrocephalus persisted postoperatively  with the need for CSF diversion procedures in the form of a ventriculoperitoneal shunt in all 4 cases. Additionally, there were three more new cases of tetraventricular hydrocephalus in the postoperative setting, requiring shunting in two cases.Rapid symptom onset was also correlated with the presence of hydrocephalus27, these lesions are the third most common lesions in the cerebellopontine angle after meningiomas and acoustic neurinomas. In our study, we found a statistically significant association (P = 0.006) between infratentorial tumor location and the incidence of postoperative complications (78.6%), which was strikingly higher than that for supratentorial tumors (7.1%). This is consistent with similar studies that evaluated the complications of tumors following epidermoid surgery in the posterior fossa28.Intracranial epidermoid lesions are most commonly located in the infratentorial region (47.2%), with a predilection for the cerebellopontine angle (38.9%). According to other studies19. This sequela arises secondary to the spilling of irritant material from the tumor into the subarachnoid space—incomplete resection of the capsule can also contribute to this incidence2. The incidence of meningitis can be reduced with radical resection29. This complication was seen in two patients (8 and 11 years of age) after subtotal resection of the tumor, which improved following lumbar drainage and treatment with dexamethasone for 7 days after surgery. In these two cases, the tumor was located in the cerebellopontine/cerebellomedullary cisterns and in the pontine cistern, causing CN deficits.After transient CN palsies, aseptic meningitis is the second most common postoperative complication30. In our cohort, we used this approach when the tumor was localized to the cerebellopontine, ambient or cerebellomedullary cisterns—lateral suboccipital (retrosigmoid) craniotomy was performed with patients placed in the sitting position. For tumors located in the fourth ventricle, quadrigeminal cistern and cisterna magna, we performed a median suboccipital craniotomy with patients placed in the sitting position. In the current series, we documented one case of pulmonary embolus arising secondary to the use of the sitting position. In cases where median suboccipital craniotomy was chosen, two patients developed subdural hygroma and pseudomeningocele, which were successfully managed by lumbar drainage.Given that the most common location of the tumor is the cerebellopontine angle, the retrosigmoid approach is an important operative approach for epidermoid tumors31, we nonetheless encountered one case of transient postoperative seizures and acute subdural hematoma after using the subfrontal route to remove an epidermoid tumor from the chiasmatic cistern. The frontal paramedian approach was used for tumors located in the pericallosal cistern and subarachnoid space, while the subtemporal approach was used to reach tumors located in the ambiens and interpeduncular cisterns.In 19.4% of cases, a pterional approach or a more limited frontolateral approach was used to reach tumors in the chiasmatic, suprasellar and carotid cisterns. Although postoperative seizures are more common after tumor removal via the subtemporal approach19. Furthermore, a pooled analysis of four case series (n = 71) found no statistically significant differences in transient or permanent complication rates between gross total and subtotal resection. While the resection margin should be carefully balanced against precipitating iatrogenic neurologic deficits, when subtotal resection is not feasible, it can still portend complication rates that are on par with gross total resection32. Preoperative capsular enhancement, diameter > 4.5 cm, calcification, and multicompartmental distribution have previously been identified as predictors for tumor recurrence13. In the case of subtotal resection, capsule fragments adherent to neurovascular structures should be devitalized to reduce the risk of recurrence33. In our study, gross total resection was feasible in 30 cases (83.3%). Tumor recurrence was observed in 22.2% of patients and was reported in both subtotal and gross total resection cases, with a median time to recurrence of 7.5 years. In the recurrent cases, we did not find any instances of malignant transformation.As a general neuro-oncological theme, gross total resection offers definitive treatment and drastically mitigates tumor recurrence risk. This concept is especially relevant for intracranial epidermoid tumors, where subtotal resection yields overall recurrence rates of up to 22%34. To appraise the connection between anatomical localization and the incidence of pre- and postoperative hydrocephalus in our cohort, we classified and evaluated the tumor distribution by median and paramedian cisterns. All cysts growing from the interpeduncular cistern, pericallosal cistern, suprasellar cistern, chiasmatic cistern, quadrigeminal cistern, 4th ventricle, cisterna magna and cerebellomedullary cistern were classified as median line cysts. Cysts growing from the cerebellopontine cistern, ambient cistern, carotid cistern and Sylvian cistern were classified as paramedian cysts18. In our study, tumor distribution among the median and paramedian cisterns was relatively close ; we also found a positive correlation between tumors located in the median cisterns and postoperative hydrocephalus . Midline tumors localized to the fourth ventricle were most likely to present with preoperative hydrocephalus that required postoperative shunting, but there are studies that describe fourth ventricle epidermoid tumors without hydrocephalus39 or report the disappearance of symptoms after surgery44.The most common permanent postoperative complication in the literature, as well as in our cohort, is hydrocephalus that required shuntingp = 0.030), influencing the patient's outcome (p = 0.024). Despite adequate resection, epidermoid cysts have a predilection to recur because of remnant microscopic disease that is often difficult to remove. Those remnants can serve as a nidus for regrowth via continued desquamation. Fortunately, these cysts do not repopulate with exponential growth but rather have a linear pattern of desquamation that may require patients to have a second surgery later in life when they are symptomatic again. However, this continued desquamation can be responsible for sustained inflammation and subsequent hydrocephalus. In such cases with gross total resection but active microscopic remnants, the appearance of hydrocephalus can precede a recurrence45.The proposed mechanism in all cases of postoperative communicating hydrocephalus was impaired CSF absorption via chemical meningitis. In cases where the tumor capsule was firmly adhered to surrounding structures, we did not perform dissection or coagulation of this part of the capsule. Chemical meningitis can occur by spillage of the cyst contents during the operation; such cases are usually transient and self-limiting and can be managed successfully with steroids. In our opinion, hydrocephalus is a prognosticator of recurrence (p = 0.032), which is expected given the higher pre- and postoperative symptom burden in these patients. Approximately half of the patients who were admitted for longer than 10 days also had, on average, at least one postoperative complication. Precipitation of symptoms (under 1 month) was associated with a higher rate of postoperative complications (p = 0.026); no association was found between long-standing symptomatic patients and the rate of postoperative complications (p = 0.44). There was no statistically significant association between tumor location and sex (p = 1.000), patient age (p = 0.068), acute or chronic symptom onset (p = 0.734), or symptom duration (p = 0.856). However, there was a correlation between age ≥ 40 years and paramedian tumor localization (82.4% versus 46.7%), and vice versa—age < 40 years and median tumor localization (53.3% versus 17.6%). This disparity might be explained by the higher incidence of obstructive hydrocephalus in median tumors, where smaller tumor volumes are required to precipitate acute symptoms secondary to impeding CSF outflow. In contrast, paramedian tumors have a longer latent period of growth, where compressed and/or atrophied normal adjacent brain tissue can accommodate larger tumor volumes without the presence of critical symptoms38.Midline tumor location was also associated with a protracted hospitalization course exceeding 10 days . Although the extent of resection is a well-established prognostic factor for recurrence, this criterion did not reach statistical significance in our study (p = 0.109), most likely due to the small sample size. Rather, the incidence of postoperative hydrocephalus was strongly associated with recurrence —this was especially relevant in patients < 40 years of age, in whom the recurrence interval was lower than that in patients ≥ 40 years of age . In our study, recurrence rates in patients with subtotal resection were approximately 50% within a follow-up period of five years, which is consistent with previously reported studies43.Multivariate analysis suggested that median versus paramedian localization had no influence on recurrence rates Despite their benign biology, primary intracranial epidermoid tumors are challenging clinical entities due to their recurrence statistics, lack of systemic treatment options, and high rates of postoperative complications even in patients with more sparing subtotal resection.To our knowledge, this is the first study in the literature to evaluate the relationship between epidermoid cysts and median versus paramedian localization. For median epidermoid cysts discovered accidentally or with minor symptoms in younger patients, major attention should be given due to the increased risk of developing hydrocephalus, which leads to a higher degree of tumor recurrence in less than 5 years after surgery. For these patients, from our point of view, both the neurologist and the neurosurgeon who are following the patient should reevaluate the patient as often as possible to raise the issue of surgical treatment as soon as possible.