Development and comparison of two 3D printed scaffolds of biosilica from marine sponges for bone tissue engineering

Development and comparison of two 3D printed scaffolds of biosilica from marine sponges for bone tissue engineering | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Development and comparison of two 3D printed scaffolds of biosilica from marine sponges for bone tissue engineering Giovanna do Espirito Santo, Amanda de Souza, Gustavo Oliva Amaral, and 8 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4914115/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract This study compared the physicochemical characteristics and biological effects of two 3D printed biosilica (BS) scaffolds (grid and gyroid). Methods included Scanning Electron Microscopy (SEM), Micro-Computed Tomography (Micro-CT), Mass Loss and pH Assessment, Fourier-Transform Infrared Spectroscopy (FTIR), and Energy-Dispersive X-ray Spectroscopy (EDS). The mechanical evaluation involved a Compression Test, and in vitro tests used cell adhesion assays with osteoblastic (MC3T3-E1) and fibroblastic (L929) cell lines. SEM showed BS spicules in both models on day 0, with signs of degradation along the experimental periods of immersion, forming a homogeneous network with the interaction with alginate. Micro-CT revealed rough surfaces in both models, with the gyroid model being more homogeneous and porous, with larger pores compared to the grid model. The gyroid model demonstrated higher values in the compression test and a decrease in pH on the first day and no differences for both models on days 3, 7, and 14. The mass loss was higher in the gyroid model by day 21. FTIR tests showed characteristic peaks for ALG and BS. EDS detected silica (Si), chlorine (Cl), calcium (Ca), carbon (C), and oxygen (O). In cell adhesion assays, both models supported adhesion and proliferation of L929 and MC3T3-E1 cells, with the gyroid model showing better cell elongation and morphology. Overall, the gyroid model demonstrated superior physicochemical properties, greater mechanical strength, and enhanced biological performance compared to the grid model, making it more promising for tissue engineering applications. Biosilica bone healing marine sponges scaffolds 3D printing. Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 INTRODUCTION The incidence of fractures has significantly increased in the last years and represents a public health issue around the world and a serious economic burden, especially in osteoporosis people or in traumatic fractures with great extension [1] . Specifically, in this situation, the process of healing may be impaired, leading to a delay in the process of consolidation or even in the occurrence of non-consolidated fractures [2]. In this context, surgical procedures are the treatment of choice, having the aim of fixating the fractures and/or implanting biomaterial bone grafts for bone healing [3], [4]. Biomaterial bone grafts have been considered one of the preferred alternatives for bone tissue replacement due to their potential for stimulating tissue growth and bone consolidation [5], [6]. To date, numerous biomaterials for bone regeneration have been investigated, including synthetic and natural biomaterials [7]. From the class of natural biomaterial, biocompounds extracted from marine sponges have been demonstrating a remarkable potential to be used in tissue engineering [8]–[11]. One of the main components of marine sponges is biosilica (BS) (glassy amorphous silica- SiO 2 ), which has emerged as a promising raw material for bone grafts [12]. BS makes part of the inorganic skeleton of marine sponges and it is formed by an enzymatic and silicatein-mediated reaction [13]. Some authors have extracted BS from sponges and demonstrated, through in vitro studies, evidence of the osteogenic potential of BS and its ability to stimulate mineralization, upregulate the expression of genes related to bone cell differentiation, and to increase cell proliferation [14]. Gabbai-Armelin et al. (2018) demonstrated through a cytotoxicity assay that BS had a positive influence on MC3T3-E1 cell viability and qRT-PCR showed that this material stimulated Runx2 and BMP4 gene expressions, indicating a potential use of BS to be used as bone for tissue engineering applications. Moreover, the structure and morphology of the bone graft scaffold are crucial factors that need to be carefully considered to effectively promoting healing. Commonly, bone grafts are used in the shape of scaffolds, which present adequate porosity with interconnected pores and mechanical properties [15] . In this context, additive manufacturing, also known as 3D printing, is a technology that has been widely used for the rapid prototyping and manufacturing scaffolds, allowing reaching all the characteristics previously cited [16], [17]. 3D printed scaffolds offer many advantages over other manufacturing techniques, such as the potential patient-specific graft fabrication, facilitating tailored design. Moreover, they allow precise control over porosity, shape, and size, resulting in structures characterized by interconnected pores [18]. All of this planned structure promotes optimized in vitro and in vivo cell growth and proliferation and tissue healing after an injury. Different 3D scaffolds with different structures can be manufactured such as the grid model, which is one of the most common models of scaffolds for bone tissue engineering, with superimposed layers, which create an environment capable of promoting cellular infiltration and proliferation [19]. Another model of 3D printed scaffolds widely used is the gyroid model, which consists of parallel and perpendicular wavy filaments that exhibit a well-distributed arrangement of pores during the deposition of lines and layers [20]. Therefore, the comparative study of both models having BS as the base material can contribute to the understanding of which format is more appropriate for the construction of scaffolds for stimulating bone repair. The hypothesis is that the models of grid and gyroid scaffolds made of BS from sponge marine would exhibit particular properties arising from their geometries capable of stimulating cell proliferation and adhesion in a different way. In this context, this investigation aimed to manufacture 2 different models of 3D-printed scaffolds made with BS from marine sponges, grid, and gyroid, and to study their physicochemical and mechanical characteristics and their biological effects through in vitro tests. MATERIALS AND METHODS BS Extraction BS was extracted from the marine sponge species Dragmacidon reticulatum collected on the site of Praia Grande, São Sebastião, São Paulo, Brazil. Samples were washed with distilled water to remove any unwanted material from the primary collection, then ground and immersed in 5% (v/v) sodium hypochlorite (NaClO) until the organic matter was degraded (Weaver et al. (2003) ) . After this stage, samples were washed with distilled water to remove NaClO and nitric acid, and sulfuric acid (1:4) was added to dissolve the residual organic part. After 24 hours, the BS particles were decantated and distilled water was added until it reached a pH > 6 (Weaver, 2003). Finally, the BS powder obtained was dried in an oven at 37 °C and sieved to produce particles around 106 μm in size. BS powder was then stored in a Falcon tube and kept under vacuum. Printing ink protocol The ink ratio for the 3D printing was set at 70:30, with 70% BS and 30% sodium alginate (ALG). For this proposal, 9.333 g of BS and 4 g of ALG were weighed. BS was weighed and homogenized with 50 mL of distilled water in a Falcon tube in a vortex to avoid future clogging of the printing needle due to the BS particles and stirring for 1 min. The mixture was transferred to a beaker under a magnetic stirrer and heated with another 50 mL of distilled water until it reached 65-75 °C. When the temperature was reached, ALG was added slowly and then homogenized under the same temperature range for 1 hour. After this period, a homogeneous hydrogel was formed and the printing ink was submitted to a primary crosslinker with 1% m/v calcium chloride (CaCl 2 ), stirred, and finally stored under refrigeration. 3D Printing Protocol for BS scaffolds For manufacturing the 3D printed scaffold, a computational model was developed using TinkerCAD software, specifying a scaffold diameter of 20 mm and a thickness of 3 mm. Subsequently, the model was imported into a Cura Ultimaker 5.8 software, for the image slicing, and the grid and gyroid infill patterns were selected, as displayed in Figure 1A and 1B, respectively. Both infill patterns were configured with a filament distance of 1.5 mm, a printing speed of 10 mm/s, and a flow rate of 10 %. Afterwards, the ink was loaded into a 5 mL syringe and inserted into the extruder of the 3D printer (Educational Starter, 3D Biotechnologies Solutions, Campinas, Brazil), with the scaffolds being printed layer by layer. Subsequently, the printed structures underwent a secondary cross-linking process using CaCl 2 and were then immersed for 25 minutes. Then, they were briefly rinsed with distilled water, frozen for subsequent freeze-drying for 1 hour, dried in an oven, and finally subjected to tertiary cross-linking under UV (403 nm) light for 10 minutes on each side of the scaffolds. CHARACTERIZATION OF SCAFFOLDS Scanning Electron Microscopy (SEM) Scanning electron microscopy (SEM- JEOL, model JSM-6610LV) was used for analyzing the morphology of the produced scaffolds. The scaffolds were evaluated without incubation and after 1 and 21 days of incubation in PBS. For the analysis, samples were placed on conductive tape and covered with a thin layer of another (20 mm) using a sputter coater (Balzers model SDS 050). Micro–Computed Tomographic Analysis (Micro-CT) For three-dimensional observation of the models and assessment of pore size after the 3D printing of the scaffold micro-CT images were collected. Imaging was carried out using a Sky Scan 1172 X-ray microtomograph (Bruker, Belgium) with the following parameters: 80 kV, 124 μA, and a 0.5 mm thick aluminum filter to attenuate beam hardening. The exposure period was set to 590 ms with a rotation step of 0.2º, resulting in a cross-sectional pixel size of 9.91 µm. The scaffolds were photographed, and each sample generated 220 photos. From them, 108 pictures per sample were chosen for further investigation. This resulted in binary pictures (BIN), which were used to identify the size and distribution of pores inside the scaffold. To properly estimate pore sizes, the chosen BIN images were analyzed using CT Analyzer (Bruker micro-CT, version 1.14.4.1) and CTvox (Bruker micro-CT, version 3.0) software. Compression Test For the mechanical characteristics of the scaffolds, the maximum tensile stress was measured with the Brazilian disc test . The analysis was carried out in triplicate and the results were obtained using the equation 1: The stress applied to the material is represented by σ, F max is the highest force that the material can bear before breaking, D is the diameter and t is the thickness of the specimen. Mass Loss and pH Assessment For the mass loss test, the produced scaffolds were individually weighed to determine their beginning mass before being divided into Falcon tubes, according to the experimental time periods of 1, 3, 7, and 14 days. They were then immersed in phosphate-buffered saline (10 mM, pH 7.4) and incubated in a 37°C oven. Following each time period, the scaffolds were removed, dried in an oven at 37 °C, and weighed to establish their ultimate mass. The leftover PBS was measured with a pH meter using the same technique. This test was performed using five duplicates, and mean values and standard deviations were used to calculate the results. Fourier-Transform Infrared Spectroscopy (FTIR) To elucidate the chemical bonds present in the produced scaffolds, the FTIR technique was conducted (Thermo Nicolet Nexus 4000, USA). Spectra were acquired in the range of 400 – 4000 cm -1 with a resolution of 2 cm -1 . Energy-Dispersive X-ray Spectroscopy (EDS) The relative quantification of atomic elements present in the scaffolds was determined using a Shimadzu equipment, model EDX-720. The samples were immersed in SBF solution for 0, 1, 3, 7, 14, and 21 days. They were then removed and dried in an oven at 37 ºC until completely dry. For this analysis, the samples passed through an X-ray tube with a Rh anode operating at 5–50 kV and 1–1,000 microA. In vitro culture The biological response of the BS scaffolds was assessed by culturing osteoblast cells (MC3T3-E1) and murine fibroblast cells (L929), following ISO standard 10993-5:2009 guidelines. These cell types were cultured in bottles using α-MEM and Dulbecco's modified Eagle's medium (DMEM) supplemented with 10 % fetal bovine serum (FBS) and 1% antibiotic-antimycotic solution at 37 ºC in a humidified atmosphere of 5% CO 2 for the respective cells. They were maintained at subconfluent densities and passaged weekly until use. Cell adhesion assay The MC3T3-E1 (osteoblasts) and L929 (murine fibroblasts) cell lines were seeded (1×10 6 cells/mL) on the surface of the scaffolds pre-moistened with the culture media, followed by a 3-hour incubation time (5% CO2, 37 ºC and 95% humidity). Cell adhesion was observed by confocal microscopy (SP8 AOBS Tandem Scanner, LEICA) at 1, 3, 7, and 14 days after seeding. Scaffolds were subjected to a three-step washing process with a PBS solution to remove the cells that were not firmly adhered onto the surface of the scaffolds. They were then immersed in a 4 % perfluoroalkoxyalkanes (PFA) solution for cell fixation of the ones adhered onto the surface of the samples. Subsequently, cells were stained with Phalloidin Alexa Fluor®488 for identifying the presence of actin filaments, and DAPI® to analyze the nuclear DNA. Statistical Analysis The distribution of variables was tested using Shapiro-Wilk's normality test. Parametric variables, when comparing groups, underwent a two-way analysis of variance (ANOVA). For non-parametric variables, the analysis involved Welch's T-test. The statistical software used was GraphPad Prism version 8.0, and a significance level of p ≤ 0.05 was adopted, followed by the Bonferroni post-hoc test. RESULTS SEM Analysis Figure 2 demonstrates the SEM micrographies of the grid and gyroid samples, before and after incubation. Figures 2A and 2B demonstrated that at day 0 (without incubation) BS spicules were observed for both grid and gyroid models. Moreover, both samples presented pores distributed around the samples. On day 1 after immersion, for both models, the spicules were still visible but partially incorporated into the ALG matrix, while some agglomerations were present (Figure 2C and 2D). After 21 days of incubation, the integrity of BS spicules was no longer seen for both samples, showing a significant degradation of the material, forming a homogeneous net with the ALG particles. MicroCT Figure 3 presents the micro-CT images for both grid and gyroid models. The 3D images show that for grid scaffolds a rough surface with an irregular edge was observed compared to the gyroid model, which was more homogeneous (Figures 3A and 3C, respectively). Figures 3B and 3D represent the BIN images, demonstrating the pores in both models, with an apparent higher porosity observed in the gyroid model (highlighted by the black coloring). Furthermore, using the same technique, it was possible to quantify the pore size of the models, with values of 57.45 ± 0.38 μm and 64.76 ± 1.35 μm being observed for grid and gyroid models, respectively. Compression test Table 1 shows the average values obtained from the mechanical compression test. The values expressed in this analysis showed that the values of maximum compression capacity were 4.9 ± 0.360 N and 11.71 ± 1.212 N, for grid and gyroid models, respectively, with a statistical difference. In addition, the values equivalent to how much load each of the models can withstand before rupture were also presented, with the gyroid model showing a greater capacity to withstand loads, 431.05 ± 78.281 kPa, when compared to the grid model which showed lower values, 109.26 ± 174.528 kPa, with a statistical difference between the two models. Table 1 . Values of the mechanical compression test for the 3D printed scaffolds . Statistically significant differences are indicated by (*) in Fmax and σmax (Two-way ANOVA, p < 0.05). 3D Printed scaffolds Models F max (N) σ max (kPa) grid 4.9 ± 0.360 109.26 ± 174.528 gyroid 11.71 ± 1.212 (*) 431.05 ± 78.281 (*) pH Evaluation Figure 4 shows that both models showed a decrease in pH values during the experimental periods. On day 1, the pH values were 6.024 ± 0.02 for the grid model and 5.674 ± 0.17 for the gyroid model. On days 3 and 7, both models exhibited similar behavior, although there was a statistically significant difference over the experimental time. On day 3, the grid model had a pH of 5.89 ± 0.06, while the gyroid model had 5.688 ± 0.10. On day 7, the pH of the grid model decreased to 5.492 ± 0.10, and that of the gyroid model was 5.522 ± 0.02. On day 14, these values decreased even further, to 5.396 ± 0.08 and 5.522 ± 0.026, respectively. Mass Loss Both grid and gyroid models showed progressive degradation during the experimental period. On day 1, 98.72 % ± 1.11 was presented for the grid model and 97.20 % ± 1.14 for the gyroid model. On day 3, the grid model remained more stable with 97.14 % ± 1.14, while the gyroid model showed 89.07 % ± 3.27. On day 7, the grid model showed 93.95 % ± 1.12 of its initial mass, while the gyroid model decreased to 86.73 % ± 2.41. On day 14, the grid model showed 93 % ± 1.22 of its initial mass, while the gyroid model showed 84 % ± 2.42. Statistically significant differences were observed between the values found for both models on days 3, 7, and 14. FTIR Analysis The chemical bonds present in the chemical composition of the scaffolds after the manufacturing, cross-linking, and drying stages were described and depicted in Figure 6 and Table 2. Four characteristic peaks of ALG present in the scaffolds were observed, including a stretching vibration of O-H at the 3421 cm -1 . Additionally, there were vibrations of asymmetric and symmetric stretching of C=O present at 1628 cm -1 . The final two peaks corresponded to C-O-H and C-O-C in the 1404 cm -1 and 1022 cm -1 , respectively. Meanwhile, the peaks evidencing the silicon group corresponded to the stretching vibration of the Si-O-Si group at the 1096 cm -1 band. There was also a bending vibration of the Si-O group at the 789 cm -1 band, and finally, an out-of-plane bending vibration corresponding to the 1096 cm -1 band. Table 2 . Band obtained through FTIR analysis of scaffolds. Wavenumber (cm -1 ) Functional group 3421 O-H stretching vibration 1628 C=O asymmetrical and symmetric stretching vibrations 1404 C-O-H 1022 C-O-C 1096 Si-O-Si stretching 789 Si-O bending vibrations 486 Si-O out-of-plane bending vibrations EDS Analysis The relative amounts of the elements carbon (C), oxygen (O), sodium (Na), silicon (Si), phosphorus (P), chlorine (Cl), calcium (Ca), potassium (K), and magnesium (Mg) presented in the samples were measured and are presented in Figure 8. It was observed that on day 0 (without incubation in SBF solution), the gyroid model exhibited elements C, O, and Si, similarly to the grid model, which also showed the presence of Cl and Ca. On Day 1, there was a decrease in C and O but an increase in the other elements (Si, Cl, and Ca). The grid model also showed a significant increase in the same elements. Starting from day 14, the gyroid model showed fewer elements than the grid model, especially in the Si element. There were statistical differences between the groups on the following days and elements: on day 0 for the C element; on day 1 for the Cl element; on day 3 for the C and Ca elements; on day 14 for the C, O, Si, and Cl elements; and on day 21 for the C and Cl elements. On day 7, there was no statistical difference. IN VITRO CULTURE Cell adhesion assay Figure 8 presents the results obtained from confocal microscopy, illustrating the adhesion and proliferation behavior of L929 cells within the grid and gyroid models over experimental periods of 1, 3, 7, and 14 days. Initially, on day 1, both scaffold models displayed limited cell adhesion, with round-shaped cells appearing more scattered. By day 3, a notable difference could be observed, as the number of cells remarkably increased in both models. Moreover, especially in the gyroid model, adherent cells began to spread out across the scaffolds. Continuing to day 7, cell number increased in both models, indicating cell proliferation. In addition, at this time period, changes in cell shape and cytoskeletal rearrangements were more evident, with no visible difference concerning cell spreading in grid and gyroid models. However, on day 14, the cells returned to their initial round morphology on the grid model scaffolds, while they remained elongated, still displaying the characteristic stretched fibroblastic morphology on the gyroid-shaped scaffolds. Figure 9 shows the confocal images obtained from the cell adhesion assay on the grid and gyroid scaffolds with the MC3T3-E1 cells at experimental times of 1, 3, 7, and 14 days. The image reveals that on day 1, the gyroid model exhibited greater cell adhesion compared to the grid model. By day 3, this trend continued, with the gyroid model showing a more extensive cell distribution and spreading. On day 7, cell numbers started to decrease and this process continued until day 14. At this later time point, the grid model had distinguishable fewer cells than the gyroid model, indicating that the gyroid structure provided a more favorable environment for MC3T3-E1 cell adhesion and growth. DISCUSSION This study aimed to manufacture 3D printed scaffolds in 2 different models, namely the grid and gyroid models, with BS extracted from the marine sponge Dragmacidon reticulatum and to study their physicochemical and mechanical characteristics and the biological effects in in vitro tests. SEM and micro-CT analysis demonstrated the morphology of the BS spicules and the interconnected pores for both scaffolds. In addition, the same technique made it possible to quantify the pore sizes in the models, showing that the grid model showed smaller sizes than the gyroid model. A decrease in pH values was observed for both scaffolds up to 14 days post-incubation and the degradation rate of the gyroid model was higher. FTIR and EDS demonstrated characteristic peaks of the alginate and BS (such as O-H, C=O and C, O and Si), and higher values in the compression test were observed for the gyroid model. Moreover, the in vitro studies demonstrated that both scaffolds were able of supporting cell integration for both scaffolds, but with a greater cell adhesion for the gyroid model. The use of BS from marine sponges for manufacturing scaffolds for bone tissue engineering proposals has been considered a goldmine [10], [22]–[27]. Many authors state that BS presents biocompatibility, similarity with the natural extracellular matrix, tunable chemistry [28], and lower production costs compared to other synthetic materials [10]. Moreover, in the present study, 3D-printed BS scaffolds were manufactured and compared. In the SEM analysis, similar findings were seen for both models, with the clear presence of BS spicules, presenting degradation after incubation. Also, micro-CT analysis demonstrated that the gyroid model presented a more homogenous surface, with a higher size of pores. A significant reduction in pH values was obtained for both scaffolds after 14 days of immersion, with higher values found for the gyroid model. Also, Gabbai-Armelin et al. (2019)observed a decrease in the values found for pH of BS samples during 14 days of immersion, and [29] also observed that after the incubation, the 3D printed BS scaffolds presented a significant decrease in pH and mass loss over time after incubation. It is also suggested that this decrease in pH is related to the degradation of sodium alginate, which is composed of a carboxylate group which binds to other ions and molecules forming hydrogen bonds [30]. Furthermore, an intense mass degradation was observed mainly in the gyroid model, reaching around 70% of the initial mass on day 14, which behavior was not observed in the grid model, suggesting that the different scaffolds present different stabilities. The rate of biomaterial degradation is a very important variable for the success of the bone graft due to the need of space liberation into the fracture site of the for newly formed bone tissue ingrowth [31]. Taking together, these data indicated that the behavior of degradation of the gyroid model may culminate in a biological advantage, with an accelerated dissolution of ions from the scaffold and a faster liberation of space, stimulating a higher formation of tissue ingrowth. Higher mechanical properties were observed for the gyroid model, showing greater resistance to maximum force loads, possibly due to its wavy geometry, which provided a higher interconnectivity between the stronger filaments, leading to a higher resistance, compared to the grid model. It is known that the grid structure is the simplest structure used for bone scaffolds. It is constituted by layers, with uniform pore distribution and possibly, the stress concentrations at the intersection nodes of the model grid negatively influencing its mechanical performance [17], [32]. Conversely, for the gyroid model, the structural design such as pore size, shape, and porosity, can be controlled by adjusting the parameter to simulate the porous structure of natural bone. Therefore, this model may be more suitable for constructing bone scaffolds. The findings of the present work corroborate those of Guo et al. (2023) demonstrated that in the compression test, the gyroid model showed higher compression strength than the Grid structure, which was attributed to the continuous curved structure which alleviated stress concentration and had a more uniform stress bearing. Knowledge of the average pore size of a scaffold has a positive correlation with the porosity of the scaffold and, for bone tissue engineering applications, it is a very important characteristic, for supporting cell attachment and proliferation, determining the success of the bone graft [34], [35]. In the present study, the gyroid model had a larger pore size compared to the grid model, which may indicate a more suitable scaffold structure to promote greater bone cell proliferation and tissue growth [36]. Also, Diao et al. (2017), stated that their scaffolds composed of Beta-Tricalcium Phosphate (β-TCP) and with a pore size of up to 100 um showed better results in the critical-sized calvarial defect rat model repair. The FTIR analysis demonstrated both models presented similar compositions, with the characteristic peaks of BS, comprising Si-O-Si stretching, Si-O bending vibrations, and out-of-plane Si-O bending vibrations [14], [38]. Similarly, the characteristic peaks of sodium alginate were found, including O-H stretching vibrations, asymmetric and symmetric C=O stretching vibrations, and C-O-H and C-O-C functional groups [39]–[41]. The relative amounts of elements in the EDS analysis, which was carried out on scaffolds submerged in SBF solution, differed between the groups. On day 0, the gyroid model had the elements C, O, and Si. In contrast, the lattice model included Cl and Ca, both of which may be the result of cross-linking. Thus, it is suggested that the grid model may have more residual material during washing after cross-linking compared to the gyroid model. On subsequent days, Si was more present in the grid model than in the gyroid model. It is worth noting that the elements may interact with the SBF solution and influence their deposition on the scaffolds, as well as intensify their dilution over the experimental periods. It can also indicate that even though extraction is carried out with steps to ensure that all elements other than silicon are degraded and removed, there may still be residues that can be observed in the EDS analysis. The in vitro cell adhesion assay demonstrated that the gyroid-shaped scaffolds model presented a remarkable increase in the number of fibroblast cells compared to the grid model and cells with a stretched fibroblastic morphology. For osteoblasts, the same results were found, with a higher number of cells being observed in the gyroid model, indicating that the gyroid structure provided a more favorable environment for cell ingrowth. Guo et al. (2023) found, through in vitro experiments, a higher number of cells on the gyroid scaffold when compared to the grid porous scaffold model, with better cell adhesion and proliferation also in the gyroid scaffold. These can be explained by the gyroid structural scaffold which presents an improved pore connectivity and permeability, which is more appropriate for supporting cell growth [42]–[44]. The optimization of the structure and morphology of 3D-printed scaffolds for bone tissue engineering are in high demand. In the present study, grid and gyroid models made with marine BS had their morphologies and in vitro effects compared, demonstrating a clear indication of the superiority of the gyroid model. However, further studies involving more detailed in vitro experiments and pre-clinical works remain to be performed to continue the investigation of the gyroid-shaped scaffolds manufactured with BS. CONCLUSION Finally, both scaffolds were created effectively using the 3D printing technique, with regard to the optimization of process parameters, microstructure, mechanical characteristics, and biocompatibility. In addition, the gyroid structural porous bone scaffolds demonstrated higher mechanical strength and better cellular responses than the standard grid structural scaffolds. The present study demonstrates that co-design of material, structure, and technology can result in comprehensive good performance of polymerfor bone scaffolds, as well as the promise of 3D-printed gyroid scaffolds in bone tissue engineering. DECLARATIONS Funding : This study was funded by São Paulo Research Foundation (FAPESP) grant number 2022/04433-8. F.V.S also acknowledge FAPESP for the fellowship (grant number 2022/05316-5) Availability of Data and Material : Datasets created and/or analyzed during the current investigation are accessible from the corresponding author upon reasonable request. Conflicts of Interest/Competing Interests : The authors have no conflict of interest. REFERENCES B. Bouyer, F. Leroy, J. Rudant, A. Weill, and J. 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Barikani, “Synthesis and characterization of calcium alginate nanoparticles, sodium homopolymannuronate salt and its calcium nanoparticles,” Sci. Iran. , vol. 19, no. 6, pp. 2023–2028, 2012, doi: 10.1016/j.scient.2012.10.005. G. Lawrie et al. , “Interactions between alginate and chitosan biopolymers characterized using FTIR and XPS,” Biomacromolecules , vol. 8, no. 8, pp. 2533–2541, 2007, doi: 10.1021/bm070014y. W. P. Voo, B. B. Lee, A. Idris, A. Islam, B. T. Tey, and E. S. Chan, “Production of ultra-high concentration calcium alginate beads with prolonged dissolution profile,” RSC Adv. , vol. 5, no. 46, pp. 36687–36695, 2015, doi: 10.1039/c5ra03862f. J. Ma et al. , “3D Printing of Strontium Silicate Microcylinder-Containing Multicellular Biomaterial Inks for Vascularized Skin Regeneration,” Adv. Healthc. Mater. , vol. 10, no. 16, pp. 1–14, 2021, doi: 10.1002/adhm.202100523. A. Myakinin et al. , “In vitro evaluation of electrochemically bioactivated Ti6Al4V 3D porous scaffolds,” Mater. Sci. Eng. C , vol. 121, no. October 2020, 2021, doi: 10.1016/j.msec.2021.111870. R. Noroozi; et al. , “In vitro static and dynamic cell culture study of novel bone scaffolds based on 3D-printed PLA and cell-laden alginate hydrogel,” Mater. Today Proc. , vol. 22, no. 1, pp. 16–20, 2019, [Online]. Available: https://doi.org/10.1080/10584587.2018.1514889%0Ahttps://doi.org/10.1016/j.matpr. 2019.12.188%0Ahttps://doi.org/10.1016/j.matpr.2019.09.090%0Ahttps://doi.org/10.1080/14484846.2018.1432089. Additional Declarations No competing interests reported. Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4914115","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":350759476,"identity":"284ce732-b089-43d7-864e-5a25c1acf4ba","order_by":0,"name":"Giovanna do Espirito 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1","display":"","copyAsset":false,"role":"figure","size":1696162,"visible":true,"origin":"","legend":"\u003cp\u003eScaffold filling models: (A) grid model and (B) gyroid model\u003c/p\u003e","description":"","filename":"FIGURE1.png","url":"https://assets-eu.researchsquare.com/files/rs-4914115/v1/77cb0ae15b401b9cdf3a9616.png"},{"id":64482189,"identity":"0a23107d-d44f-4240-bc4f-91b1b60cad0d","added_by":"auto","created_at":"2024-09-13 16:38:45","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":2701916,"visible":true,"origin":"","legend":"\u003cp\u003eSEM images of the surface for the grid and gyroid models (2500 × magnification). (A, B) – Images representing the models without incubation in PBS solution; (C, D) – After 1 day incubated in PBS solution; (E, F) – After 21 days incubated in PBS solution. The scale bar represents 10 µm (* yellow indicates BS spicules and à yellow indicates agglomerations incorporated into the ALG network).\u003c/p\u003e","description":"","filename":"FIGURE2.png","url":"https://assets-eu.researchsquare.com/files/rs-4914115/v1/04f92543fdb1056a2b0d3028.png"},{"id":64481390,"identity":"0fbb2571-125e-4cea-a982-b6d188aa4e3b","added_by":"auto","created_at":"2024-09-13 16:30:45","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":3257938,"visible":true,"origin":"","legend":"\u003cp\u003eMicro-CT of the scaffolds of the grid and gyroid models. (A) – 3D image of the grid model surface; (B) – BIN images of the grid model; (C) – 3D image of the gyroid model surface; (D) - BIN image of the gyroid model.\u003c/p\u003e","description":"","filename":"FIGURE3.png","url":"https://assets-eu.researchsquare.com/files/rs-4914115/v1/b33830701215d2f6f7e8c905.png"},{"id":64482685,"identity":"f31727d0-89aa-4001-9cd9-80a30b9a50e5","added_by":"auto","created_at":"2024-09-13 16:46:45","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":112747,"visible":true,"origin":"","legend":"\u003cp\u003epH Values of grid and gyroid models as a function of time period in phosphate-buffered saline. Statistically significant differences (*) were observed on days 1 and 3. (Two-way ANOVA, p \u0026lt; 0.05).\u003c/p\u003e","description":"","filename":"FIGURE4.png","url":"https://assets-eu.researchsquare.com/files/rs-4914115/v1/519e3c962705a2f289190a83.png"},{"id":64481383,"identity":"bcde4274-9bd4-4ed8-bd67-454c7e01235f","added_by":"auto","created_at":"2024-09-13 16:30:45","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":107087,"visible":true,"origin":"","legend":"\u003cp\u003eMass loss, in percentage, for grid and gyroid models as a function of time period in phosphate-buffered saline. Statistically significant differences are indicated by (*) on days 3, 7, and 14 (Two-way ANOVA, p \u0026lt; 0.05).\u003c/p\u003e","description":"","filename":"FIGURE5.png","url":"https://assets-eu.researchsquare.com/files/rs-4914115/v1/929cd99094f3cc2c2e8bd877.png"},{"id":64482193,"identity":"66cb7e7d-4dd8-4a21-8534-2a8cb117ae93","added_by":"auto","created_at":"2024-09-13 16:38:47","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":268192,"visible":true,"origin":"","legend":"\u003cp\u003eBands obtained through FTIR indicate the presence of characteristic functional groups of ALG and BS.\u003c/p\u003e","description":"","filename":"FIGURE6.png","url":"https://assets-eu.researchsquare.com/files/rs-4914115/v1/db068533b6b85fdff9d3bc6a.png"},{"id":64482191,"identity":"a27e3500-74e1-4ab4-a0b2-cd51592ca1eb","added_by":"auto","created_at":"2024-09-13 16:38:45","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":329129,"visible":true,"origin":"","legend":"\u003cp\u003eEDS for identification of chemical elements contained in the grid and gyroid models. (A) – quantification on day 0, without incubation in the SBF solution; (B, C, D, E, F) – with incubations in the PBS solution at times 1, 3, 7, 14, and 21, respectively. * \u0026nbsp;Statistical difference (Two-way ANOVA, p \u0026lt; 0.05)\u003c/p\u003e","description":"","filename":"FIGURE7.png","url":"https://assets-eu.researchsquare.com/files/rs-4914115/v1/4ade81eb3bb4c240b21be529.png"},{"id":64481389,"identity":"0c427a01-002d-4cd3-8f25-b2314dd24f46","added_by":"auto","created_at":"2024-09-13 16:30:45","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":3096689,"visible":true,"origin":"","legend":"\u003cp\u003eGradient of L929 cell adhesion on the grid and gyroid model scaffolds according to experimental times of 1, 3, 7, and 14 days.\u003c/p\u003e","description":"","filename":"FIGURE8.png","url":"https://assets-eu.researchsquare.com/files/rs-4914115/v1/8251af172683dd496d0008f8.png"},{"id":64482192,"identity":"35371ab8-adbf-48fd-b24a-91027ee05f60","added_by":"auto","created_at":"2024-09-13 16:38:45","extension":"png","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":2660620,"visible":true,"origin":"","legend":"\u003cp\u003eThe gradient of MC3T3-E1 cell adhesion on the grid and gyroid model scaffolds according to experimental times of 1, 3, 7, and 14 days.\u003c/p\u003e","description":"","filename":"FIGURE9.png","url":"https://assets-eu.researchsquare.com/files/rs-4914115/v1/03941fd7f316b287bb241f92.png"},{"id":69744296,"identity":"ec122bbe-2ac0-40eb-b072-ce5da62685f4","added_by":"auto","created_at":"2024-11-24 20:16:33","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":19545302,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4914115/v1/087506a1-3514-45de-a7f6-d598751791ca.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Development and comparison of two 3D printed scaffolds of biosilica from marine sponges for bone tissue engineering","fulltext":[{"header":"INTRODUCTION","content":"\u003cp\u003eThe incidence of fractures has significantly increased in the last years and represents a\u0026nbsp;public health issue around the world and a serious economic burden, especially in osteoporosis people or in traumatic fractures with great extension\u0026nbsp;[1]\u003cstrong\u003e.\u0026nbsp;\u003c/strong\u003eSpecifically, in this situation, the process of healing may be\u0026nbsp;impaired, leading to a delay in the process of consolidation or even in the occurrence of non-consolidated fractures\u0026nbsp;[2].\u0026nbsp;In this context, surgical procedures are the treatment of choice, having the aim of fixating the fractures and/or implanting biomaterial bone grafts for bone healing\u0026nbsp;[3], [4].\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eBiomaterial bone grafts have been considered one of the preferred alternatives for bone tissue replacement due to their potential for stimulating tissue growth and bone consolidation\u0026nbsp;[5], [6]. To date, numerous biomaterials for bone regeneration have been investigated, including synthetic and natural biomaterials\u0026nbsp;[7]. From the class of natural biomaterial, biocompounds extracted from marine sponges have been demonstrating a remarkable potential to be used in tissue engineering\u0026nbsp;[8]–[11].\u0026nbsp;One of the main components of marine sponges is\u0026nbsp;biosilica (BS) (glassy amorphous silica- SiO\u003csub\u003e2\u003c/sub\u003e), which has emerged as a promising\u0026nbsp;raw material for bone grafts\u0026nbsp;[12].\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eBS makes part of the inorganic skeleton of marine sponges and it is formed by an enzymatic and silicatein-mediated reaction\u0026nbsp;[13].\u0026nbsp;Some authors have extracted BS from sponges and demonstrated, through \u003cem\u003ein vitro\u0026nbsp;\u003c/em\u003estudies, evidence of the osteogenic potential of BS and its ability to stimulate mineralization, upregulate the expression of genes related to bone cell differentiation, and to increase cell proliferation\u0026nbsp;[14]. Gabbai-Armelin \u003cem\u003eet al.\u003c/em\u003e (2018) demonstrated through a cytotoxicity assay that BS had a positive influence on MC3T3-E1 cell viability and qRT-PCR showed that this material stimulated Runx2 and BMP4 gene expressions, indicating a potential use of BS to be used as bone for tissue engineering applications.\u0026nbsp;Moreover, the structure and morphology of the bone graft scaffold are crucial factors that need to be carefully considered to effectively promoting healing.\u003c/p\u003e\n\u003cp\u003eCommonly, bone grafts are used in the shape of scaffolds, which present adequate porosity with\u0026nbsp;interconnected pores and mechanical properties\u0026nbsp;[15]\u003cstrong\u003e.\u003c/strong\u003e In this context, additive manufacturing, also known as 3D printing, is a technology that has been widely used for the rapid prototyping and manufacturing scaffolds, allowing reaching all the characteristics previously cited\u0026nbsp;[16], [17].\u0026nbsp;3D printed scaffolds offer\u0026nbsp;many advantages over other manufacturing techniques, such as the potential patient-specific graft fabrication, facilitating tailored design. Moreover, they allow precise control over porosity, shape, and size, resulting in structures characterized by interconnected pores\u0026nbsp;[18]. All of this planned structure promotes optimized \u003cem\u003ein vitro\u003c/em\u003e and \u003cem\u003ein vivo\u003c/em\u003e cell growth and proliferation and tissue healing after an injury. Different 3D scaffolds with different structures can be manufactured such as the\u0026nbsp;grid model, which is one of the most common models of scaffolds for bone tissue engineering, with superimposed layers, which create an environment capable of promoting cellular infiltration and proliferation\u0026nbsp;[19]. Another model of 3D printed scaffolds widely used is the gyroid model, which \u0026nbsp;consists of parallel and perpendicular wavy filaments that exhibit a well-distributed arrangement of pores during the deposition of lines and layers\u0026nbsp;[20].\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eTherefore, the comparative study of both models having BS as the base material can contribute to the understanding of which format is more appropriate for the construction of scaffolds for stimulating bone repair. The hypothesis is that the models of grid and gyroid scaffolds made of BS from sponge marine would exhibit particular properties arising from their geometries capable of stimulating cell proliferation and adhesion in a different way. In this context, this investigation aimed to manufacture 2 different models of 3D-printed scaffolds made with BS from marine sponges, grid, and gyroid, and to study their physicochemical and mechanical characteristics and their biological effects through \u003cem\u003ein vitro\u003c/em\u003e tests.\u003c/p\u003e"},{"header":"MATERIALS AND METHODS","content":"\u003cp\u003e\u003cstrong\u003eBS Extraction\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eBS was extracted from the marine sponge species \u003cem\u003eDragmacidon reticulatum\u003c/em\u003e collected on the site of Praia Grande, S\u0026atilde;o Sebasti\u0026atilde;o, S\u0026atilde;o Paulo, Brazil. Samples were washed with distilled water to remove any unwanted material from the primary collection, then ground and immersed in 5% (v/v) sodium hypochlorite (NaClO) until the organic matter was degraded (Weaver \u003cem\u003eet al.\u003c/em\u003e (2003)\u003cstrong\u003e)\u003c/strong\u003e. After this stage, samples were washed with distilled water to remove NaClO and nitric acid, and sulfuric acid (1:4) was added \u0026nbsp;to dissolve the residual organic part. After 24 hours, the BS particles were decantated and distilled water was added until it reached a pH \u0026gt; 6 (Weaver, 2003). Finally, the BS powder obtained was dried in an oven at 37 \u0026deg;C and sieved to produce particles around 106 \u0026mu;m in size. BS powder was then stored in a Falcon tube and kept under vacuum.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePrinting ink protocol\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe ink ratio for the 3D printing was set at 70:30, with 70% BS and 30% sodium alginate (ALG). For this proposal, 9.333 g of BS and 4 g of ALG were weighed. BS was weighed and homogenized with 50 mL of distilled water in a Falcon tube in a vortex to avoid future clogging of the printing needle due to the BS particles and stirring for 1 min. The mixture was transferred to a beaker under a magnetic stirrer and heated with another 50 mL of distilled water until it reached 65-75 \u0026deg;C. When the temperature was reached, ALG was added slowly and then homogenized under the same temperature range for 1 hour. After this period, a homogeneous hydrogel was formed and the printing ink was submitted to a primary crosslinker with 1% m/v calcium chloride (CaCl\u003csub\u003e2\u003c/sub\u003e), stirred, and finally stored under refrigeration.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3D Printing Protocol for BS scaffolds\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFor manufacturing the 3D printed scaffold, a computational model was developed using TinkerCAD software, specifying a scaffold diameter of 20 mm and a thickness of 3 mm. Subsequently, the model was imported into a Cura Ultimaker 5.8 software, for the image slicing, and the grid and gyroid infill patterns were selected, as displayed in Figure 1A and 1B, respectively.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eBoth infill patterns were configured with a filament distance of 1.5 mm, a printing speed of 10 mm/s, and a flow rate of 10 %. Afterwards, the ink was loaded into a 5 mL syringe and inserted into the extruder of the 3D printer (Educational Starter, 3D Biotechnologies Solutions, Campinas, Brazil), with the scaffolds being printed layer by layer. Subsequently, the printed structures underwent a secondary cross-linking process using CaCl\u003csub\u003e2\u003c/sub\u003e and were then immersed for 25 minutes. Then, they were briefly rinsed with distilled water, frozen for subsequent freeze-drying for 1 hour, dried in an oven, and finally subjected to tertiary cross-linking under UV (403 nm) light for 10 minutes on each side of the scaffolds.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCHARACTERIZATION OF SCAFFOLDS\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eScanning Electron Microscopy (SEM)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eScanning electron microscopy (SEM- JEOL, model JSM-6610LV) was used for analyzing the morphology of the produced scaffolds. The scaffolds were evaluated without incubation and after 1 and 21 days of incubation in PBS. For the analysis, samples were placed on conductive tape and covered with a thin layer of another (20 mm) using a sputter coater (Balzers model SDS 050).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMicro\u0026ndash;Computed Tomographic Analysis (Micro-CT)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFor three-dimensional observation of the models and assessment of pore size after the 3D printing of the scaffold \u0026nbsp;micro-CT images were collected. Imaging was carried out using a Sky Scan 1172 X-ray microtomograph (Bruker, Belgium) with the following parameters: 80 kV, 124 \u0026mu;A, and a 0.5 mm thick aluminum filter to attenuate beam hardening. The exposure period was set to 590 ms with a rotation step of 0.2\u0026ordm;, resulting in a cross-sectional pixel size of 9.91 \u0026micro;m. The scaffolds were photographed, and each sample generated 220 photos. From them, 108 pictures per sample were chosen for further investigation. This resulted in binary pictures (BIN), which were used to identify the size and distribution of pores inside the scaffold. To properly estimate pore sizes, the chosen BIN images were analyzed using CT Analyzer (Bruker micro-CT, version 1.14.4.1) and CTvox (Bruker micro-CT, version 3.0) software.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompression Test\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFor the mechanical characteristics of the scaffolds, the maximum tensile stress was measured with the Brazilian disc test . The analysis was carried out in triplicate and the results were obtained using the equation 1:\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cimg src=\"data:image/png;base64,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\"\u003e\u003c/p\u003e\n\u003cp\u003eThe stress applied to the material is represented by \u0026sigma;, F\u003csub\u003emax\u003c/sub\u003e is the highest force that the material can bear before breaking, D is the diameter and t is the thickness of the specimen.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMass Loss and pH Assessment\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFor the mass loss test, the produced scaffolds were individually weighed to determine their beginning mass before being divided into Falcon tubes, according to the experimental time periods of 1, 3, 7, and 14 days. They were then immersed in phosphate-buffered saline (10 mM, pH 7.4) and incubated in a 37\u0026deg;C oven. Following each time period, the scaffolds were removed, dried in an oven at 37 \u0026deg;C, and weighed to establish their ultimate mass. The leftover PBS was measured with a pH meter using the same technique. This test was performed using five duplicates, and mean values and standard deviations were used to calculate the results.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFourier-Transform Infrared Spectroscopy (FTIR)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo elucidate the chemical bonds present in the produced scaffolds, the FTIR technique was conducted (Thermo Nicolet Nexus 4000, USA). Spectra were acquired in the range of 400 \u0026ndash; 4000 cm\u003csup\u003e-1\u003c/sup\u003e with a resolution of 2 cm\u003csup\u003e-1\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEnergy-Dispersive X-ray Spectroscopy (EDS)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe relative quantification of atomic elements present in the scaffolds was determined using a Shimadzu equipment, model EDX-720. \u0026nbsp;The samples were immersed in SBF solution for 0, 1, 3, 7, 14, and 21 days. They were then removed and dried in an oven at 37 \u0026ordm;C until completely dry. For this analysis, the samples passed through an X-ray tube with a Rh anode operating at 5\u0026ndash;50 kV and 1\u0026ndash;1,000 microA.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eIn vitro\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;culture\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe biological response of the BS scaffolds was assessed by culturing osteoblast cells (MC3T3-E1) and murine fibroblast cells (L929), following ISO standard 10993-5:2009 guidelines. These cell types were cultured in bottles using \u0026alpha;-MEM and Dulbecco\u0026apos;s modified Eagle\u0026apos;s medium (DMEM) supplemented with 10 % fetal bovine serum (FBS) and 1% antibiotic-antimycotic solution at 37 \u0026ordm;C \u0026nbsp;in a humidified atmosphere of 5% CO\u003csub\u003e2\u003c/sub\u003e for the respective cells. They were maintained at subconfluent densities and passaged weekly until use.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCell adhesion assay\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe MC3T3-E1 (osteoblasts) and L929 (murine fibroblasts) cell lines were seeded (1\u0026times;10\u003csup\u003e6\u003c/sup\u003e cells/mL) on the surface of the scaffolds pre-moistened with the culture media, followed by a 3-hour incubation time (5% CO2, 37 \u0026ordm;C and 95% humidity).\u003c/p\u003e\n\u003cp\u003eCell adhesion was observed by confocal microscopy (SP8 AOBS Tandem Scanner, LEICA) at 1, 3, 7, and 14 days after seeding. Scaffolds were subjected to a three-step washing process with a PBS solution to remove the cells that were not firmly adhered onto the surface of the scaffolds. They were then immersed in a 4 % perfluoroalkoxyalkanes (PFA) solution for cell fixation of the ones adhered onto the surface of the samples. Subsequently, cells were stained with Phalloidin Alexa Fluor\u0026reg;488 for identifying the presence of actin filaments, and DAPI\u0026reg; to analyze the nuclear DNA.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStatistical Analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe distribution of variables was tested using Shapiro-Wilk\u0026apos;s normality test. Parametric variables, when comparing groups, underwent a two-way analysis of variance (ANOVA). For non-parametric variables, the analysis involved Welch\u0026apos;s T-test. The statistical software used was GraphPad Prism version 8.0, and a significance level of p \u0026le; 0.05 was adopted, followed by the Bonferroni post-hoc test.\u003c/p\u003e"},{"header":"RESULTS","content":"\u003cp\u003e\u003cstrong\u003eSEM Analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFigure 2 demonstrates the SEM micrographies of the grid and gyroid samples, before and after incubation. Figures 2A and 2B demonstrated that at day 0 (without incubation) BS spicules were observed for both grid and gyroid models. Moreover, both samples presented pores distributed around the samples. On day 1 after immersion, for both models, the spicules were still visible but partially incorporated into the ALG matrix, while some agglomerations were present (Figure 2C and 2D). After 21 days of incubation, the integrity of BS spicules was no longer seen for both samples, showing a significant degradation of the material, forming a homogeneous net with the ALG particles.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMicroCT\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFigure 3 presents the micro-CT images for both grid and gyroid models. The 3D images show that for grid scaffolds a rough surface with an irregular edge was observed compared to the gyroid model, which was more homogeneous (Figures 3A and 3C, respectively). Figures 3B and 3D represent the BIN images, demonstrating the pores in both models, with an apparent higher porosity observed in the gyroid model (highlighted by the black coloring). Furthermore, using the same technique, it was possible to quantify the pore size of the models, with values of 57.45 \u0026plusmn; 0.38 \u0026mu;m and 64.76 \u0026plusmn; 1.35 \u0026mu;m being observed for grid and gyroid models, respectively.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompression test\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTable 1 shows the average values obtained from the mechanical compression test. The values expressed in this analysis showed that the values of maximum compression capacity were 4.9 \u0026plusmn; 0.360 N and 11.71 \u0026plusmn; 1.212 N, for grid and gyroid models, respectively, with a statistical difference. In addition, the values equivalent to how much load each of the models can withstand before rupture were also presented, with the gyroid model showing a greater capacity to withstand loads, 431.05 \u0026plusmn; 78.281 kPa, when compared to the grid model which showed lower values, 109.26 \u0026plusmn; 174.528 kPa, with a statistical difference between the two models.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 1\u003c/strong\u003e. Values of the mechanical compression test for the 3D printed scaffolds\u003cem\u003e.\u0026nbsp;\u003c/em\u003eStatistically significant differences are indicated by (*) in Fmax and \u0026sigma;max (Two-way ANOVA, p \u0026lt; 0.05).\u003c/p\u003e\n\u003cdiv align=\"Left\"\u003e\n \u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" width=\"414\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd width=\"26.81159420289855%\"\u003e\n \u003cp\u003e\u003cstrong\u003e3D Printed scaffolds Models\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"34.54106280193237%\"\u003e\n \u003cp\u003e\u003cstrong\u003eF\u003csub\u003emax\u003c/sub\u003e (N)\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"38.64734299516908%\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026sigma;\u003csub\u003emax\u003c/sub\u003e (kPa)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"26.81159420289855%\"\u003e\n \u003cp\u003e\u003cstrong\u003egrid\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"34.54106280193237%\"\u003e\n \u003cp\u003e4.9 \u0026plusmn; 0.360\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"38.64734299516908%\"\u003e\n \u003cp\u003e109.26 \u0026plusmn; 174.528\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"26.81159420289855%\"\u003e\n \u003cp\u003e\u003cstrong\u003egyroid\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"34.54106280193237%\"\u003e\n \u003cp\u003e11.71 \u0026plusmn; 1.212 (*)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"38.64734299516908%\"\u003e\n \u003cp\u003e431.05 \u0026plusmn; 78.281 (*)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n\u003c/div\u003e\n\u003cp\u003e\u003cstrong\u003epH Evaluation\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFigure 4 shows that both models showed a decrease in pH values during the experimental periods. On day 1, the pH values were 6.024 \u0026plusmn; 0.02 for the grid model and 5.674 \u0026plusmn; 0.17 for the gyroid model. On days 3 and 7, both models exhibited similar behavior, although there was a statistically significant difference over the experimental time. On day 3, the grid model had a pH of 5.89 \u0026plusmn; 0.06, while the gyroid model had 5.688 \u0026plusmn; 0.10. On day 7, the pH of the grid model decreased to 5.492 \u0026plusmn; 0.10, and that of the gyroid model was 5.522 \u0026plusmn; 0.02. On day 14, these values decreased even further, to 5.396 \u0026plusmn; 0.08 and 5.522 \u0026plusmn; 0.026, respectively.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMass Loss\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eBoth grid and gyroid models showed progressive degradation during the experimental period. On day 1, 98.72 % \u0026plusmn; 1.11 was presented for the grid model and 97.20 % \u0026plusmn; 1.14 for the gyroid model. On day 3, the grid model remained more stable with 97.14 % \u0026plusmn; 1.14, while the gyroid model showed 89.07 % \u0026plusmn; 3.27. On day 7, the grid model showed 93.95 % \u0026plusmn; 1.12 of its initial mass, while the gyroid model decreased to 86.73 % \u0026plusmn; 2.41. On day 14, the grid model showed 93 % \u0026plusmn; 1.22 of its initial mass, while the gyroid model showed 84 % \u0026plusmn; 2.42. Statistically significant differences were observed between the values found for both models on days 3, 7, and 14. \u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFTIR Analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe chemical bonds present in the chemical composition of the scaffolds after the manufacturing, cross-linking, and drying stages were described and depicted in Figure 6 and Table 2. Four characteristic peaks of ALG present in the scaffolds were observed, including a stretching vibration of O-H at the 3421 cm\u003csup\u003e-1\u003c/sup\u003e. Additionally, there were vibrations of asymmetric and symmetric stretching of C=O present at 1628 cm\u003csup\u003e-1\u003c/sup\u003e. The final two peaks corresponded to C-O-H and C-O-C in the 1404 cm\u003csup\u003e-1\u003c/sup\u003e and 1022 cm\u003csup\u003e-1\u003c/sup\u003e, respectively. Meanwhile, the peaks evidencing the silicon group corresponded to the stretching vibration of the Si-O-Si group at the 1096 cm\u003csup\u003e-1\u003c/sup\u003e band. There was also a bending vibration of the Si-O group at the 789 cm\u003csup\u003e-1\u003c/sup\u003e band, and finally, an out-of-plane bending vibration corresponding to the 1096 cm\u003csup\u003e-1\u003c/sup\u003e band.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 2\u003c/strong\u003e. \u0026nbsp;Band obtained through FTIR analysis of scaffolds.\u003c/p\u003e\n\u003cdiv align=\"Left\"\u003e\n \u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" width=\"61%\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd width=\"42.42424242424242%\"\u003e\n \u003cp\u003e\u003cstrong\u003eWavenumber (cm\u003csup\u003e-1\u003c/sup\u003e)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"57.57575757575758%\"\u003e\n \u003cp\u003e\u003cstrong\u003eFunctional group\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"42.42424242424242%\"\u003e\n \u003cp\u003e3421\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"57.57575757575758%\"\u003e\n \u003cp\u003eO-H stretching vibration\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"42.42424242424242%\"\u003e\n \u003cp\u003e1628\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"57.57575757575758%\"\u003e\n \u003cp\u003eC=O asymmetrical and symmetric stretching vibrations\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"42.42424242424242%\"\u003e\n \u003cp\u003e1404\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"57.57575757575758%\"\u003e\n \u003cp\u003eC-O-H\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"42.42424242424242%\"\u003e\n \u003cp\u003e1022\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"57.57575757575758%\"\u003e\n \u003cp\u003eC-O-C\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"42.42424242424242%\"\u003e\n \u003cp\u003e1096\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"57.57575757575758%\"\u003e\n \u003cp\u003eSi-O-Si stretching\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"42.42424242424242%\"\u003e\n \u003cp\u003e789\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"57.57575757575758%\"\u003e\n \u003cp\u003eSi-O bending vibrations\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"42.42424242424242%\"\u003e\n \u003cp\u003e486\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"57.57575757575758%\"\u003e\n \u003cp\u003eSi-O out-of-plane bending vibrations\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n\u003c/div\u003e\n\u003cp\u003e\u003cstrong\u003eEDS Analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe relative amounts of the elements carbon (C), oxygen (O), sodium (Na), silicon (Si), phosphorus (P), chlorine (Cl), calcium (Ca), potassium (K), and magnesium (Mg) presented in the samples were measured and are presented in Figure 8. It was observed that on day 0 (without incubation in SBF solution), the gyroid model exhibited elements C, O, and Si, similarly to the grid model, which also showed the presence of Cl and Ca. On Day 1, there was a decrease in C and O but an increase in the other elements (Si, Cl, and Ca). The grid model also showed a significant increase in the same elements. Starting from day 14, the gyroid model showed fewer elements than the grid model, especially in the Si element. There were statistical differences between the groups on the following days and elements: on day 0 for the C element; on day 1 for the Cl element; on day 3 for the C and Ca elements; on day 14 for the C, O, Si, and Cl elements; and on day 21 for the C and Cl elements. On day 7, there was no statistical difference.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eIN VITRO\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;CULTURE\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCell adhesion assay\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFigure 8 presents the results obtained from confocal microscopy, illustrating the adhesion and proliferation behavior of L929 cells within the grid and gyroid models over experimental periods of 1, 3, 7, and 14 days. Initially, on day 1, both scaffold models displayed limited cell adhesion, with round-shaped cells appearing more scattered. By day 3, a notable difference could be observed, as the number of cells remarkably increased in both models. Moreover, especially in the gyroid model, adherent cells began to spread out across the scaffolds. Continuing to day 7, cell number increased in both models, indicating cell proliferation. In addition, at this time period, changes in cell shape and cytoskeletal rearrangements were more evident, with no visible difference concerning cell spreading in grid and gyroid models. However, on day 14, the cells returned to their initial round morphology on the grid model scaffolds, while they remained elongated, still displaying the characteristic stretched fibroblastic morphology on the gyroid-shaped scaffolds.\u003c/p\u003e\n\u003cp\u003eFigure 9 shows the confocal images obtained from the cell adhesion assay on the grid and gyroid scaffolds with the MC3T3-E1 cells at experimental times of 1, 3, 7, and 14 days. The image reveals that on day 1, the gyroid model exhibited greater cell adhesion compared to the grid model. By day 3, this trend continued, with the gyroid model showing a more extensive cell distribution and spreading. On day 7, cell numbers started to decrease and this process continued until day 14. At this later time point, the grid model had distinguishable fewer cells than the gyroid model, indicating that the gyroid structure provided a more favorable environment for MC3T3-E1 cell adhesion and growth.\u003c/p\u003e"},{"header":" DISCUSSION ","content":"\u003cp\u003eThis study aimed to manufacture 3D printed scaffolds in 2 different models, namely the grid and gyroid models, with BS extracted from the marine sponge \u003cem\u003eDragmacidon reticulatum\u0026nbsp;\u003c/em\u003eand to study their physicochemical and mechanical characteristics and the biological effects in \u003cem\u003ein vitro\u003c/em\u003e tests. SEM and micro-CT analysis demonstrated the morphology of the BS spicules and the interconnected pores for both scaffolds.\u0026nbsp;In addition, the same technique made it possible to quantify the pore sizes in the models, showing that the grid model showed smaller sizes than the gyroid model.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;A decrease in pH values was observed for both scaffolds up to 14 days post-incubation and the degradation rate of the gyroid model was higher. FTIR and EDS demonstrated characteristic peaks of the alginate and BS (such as O-H, C=O and C, O and Si), and higher values in the compression test were observed for the gyroid model. Moreover, the \u003cem\u003ein vitro\u003c/em\u003e studies demonstrated that both scaffolds were able of supporting cell integration for both scaffolds, but with a\u0026nbsp;greater cell adhesion for the gyroid model.\u003c/p\u003e\n\u003cp\u003eThe use of BS from marine sponges for manufacturing scaffolds for bone tissue engineering proposals \u0026nbsp;has been considered a goldmine\u0026nbsp;[10], [22]–[27]. Many authors state that BS presents biocompatibility, similarity with the natural extracellular matrix, tunable chemistry\u0026nbsp;[28], and lower production costs compared to other synthetic materials\u0026nbsp;[10]. Moreover, in the present study, 3D-printed BS scaffolds were manufactured and compared. In the SEM analysis, similar findings were seen for both models, with the clear presence of BS spicules, presenting degradation after incubation. Also, micro-CT analysis demonstrated that the gyroid model presented a more homogenous surface, with a higher size of pores.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;A significant reduction in pH values was obtained for both scaffolds after 14 days of immersion, with higher values found for the gyroid model. Also,\u0026nbsp;Gabbai-Armelin \u003cem\u003eet al.\u003c/em\u003e (2019)observed a decrease in the values found for pH of BS samples during 14 days of immersion, and\u0026nbsp;[29]\u0026nbsp;also observed that after the incubation, the 3D printed BS scaffolds presented a significant decrease in pH and mass loss over time after incubation.\u0026nbsp;It is also suggested that this decrease in pH is related to the degradation of sodium alginate, which is composed of a carboxylate group which binds to other ions and molecules forming hydrogen bonds\u0026nbsp;[30]. Furthermore, an intense mass degradation was observed mainly in the gyroid model, reaching around 70% of the initial mass on day 14, which behavior was not observed in the grid model, suggesting that the different scaffolds present different stabilities. The rate of biomaterial degradation is a very important variable for the success of the bone graft due to the need of space liberation into the fracture site of the for newly formed bone tissue ingrowth\u0026nbsp;[31]. Taking together, these data indicated that the behavior of degradation of the gyroid model may culminate in a biological advantage, with an accelerated dissolution of ions from the scaffold and a faster liberation of space, stimulating a higher formation of tissue ingrowth. \u0026nbsp; \u0026nbsp;\u003c/p\u003e\n\u003cp\u003eHigher mechanical properties were observed for the gyroid model, showing greater resistance to maximum force loads, possibly due to its wavy geometry, which provided a higher interconnectivity between the stronger filaments, leading to a higher resistance, compared to the grid model. It is known that the grid structure is the simplest structure used for bone scaffolds. It is constituted by layers, with uniform pore distribution and possibly, the stress concentrations at the intersection nodes of the model grid negatively influencing its mechanical performance\u0026nbsp;[17], [32]. Conversely, for the gyroid model, the structural design such as pore size, shape, and porosity, can be controlled by adjusting the parameter to simulate the porous structure of natural bone. Therefore, this model may be more suitable for constructing bone scaffolds. The findings of the present work corroborate those of\u0026nbsp;Guo \u003cem\u003eet al.\u003c/em\u003e (2023)\u0026nbsp;demonstrated that in the compression test, the gyroid model showed higher compression strength than the Grid structure, which \u0026nbsp;was attributed to the continuous curved structure which alleviated stress concentration and had a more uniform stress bearing.\u003c/p\u003e\n\u003cp\u003eKnowledge of the average pore size of a scaffold has a positive correlation with the porosity of the scaffold and, for bone tissue engineering applications, it is a very important characteristic, for supporting cell attachment and proliferation, determining the success of the bone graft\u0026nbsp;[34], [35]. \u0026nbsp;In the present study, the gyroid model had a larger pore size compared to the grid model, which may indicate a more suitable scaffold structure to promote greater bone cell proliferation and tissue growth\u0026nbsp;[36]. Also,\u0026nbsp;Diao \u003cem\u003eet al.\u003c/em\u003e (2017), stated that their scaffolds composed of Beta-Tricalcium Phosphate (β-TCP) and with a pore size of up to 100 um showed better results in the critical-sized calvarial defect rat model repair.\u003c/p\u003e\n\u003cp\u003eThe FTIR analysis demonstrated both models presented similar compositions, with the characteristic peaks of BS, comprising Si-O-Si stretching, Si-O bending vibrations, and out-of-plane Si-O bending vibrations\u0026nbsp;[14], [38]. Similarly, the characteristic peaks of sodium alginate were found, including O-H stretching vibrations, asymmetric and symmetric C=O stretching vibrations, and C-O-H and C-O-C functional groups\u0026nbsp;[39]–[41]. The relative amounts of elements in the EDS analysis, which was carried out on scaffolds submerged in SBF solution, differed between the groups. On day 0, the gyroid model had the elements C, O, and Si. In contrast, the lattice model included Cl and Ca, both of which may be the result of cross-linking. Thus, it is suggested that the grid model may have more residual material during washing after cross-linking compared to the gyroid model. On subsequent days, Si was more present in the grid model than in the gyroid model. It is worth noting that the elements may interact with the SBF solution and influence their deposition on the scaffolds, as well as intensify their dilution over the experimental periods. It can also indicate that even though extraction is carried out with steps to ensure that all elements other than silicon are degraded and removed, there may still be residues that can be observed in the EDS analysis.\u003c/p\u003e\n\u003cp\u003eThe \u003cem\u003ein vitro\u003c/em\u003e cell adhesion assay\u0026nbsp;demonstrated\u0026nbsp;that the gyroid-shaped\u0026nbsp;scaffolds\u0026nbsp;model presented a\u0026nbsp;remarkable increase in the\u0026nbsp;number \u0026nbsp;of fibroblast cells compared to the grid model and cells with a\u0026nbsp;stretched fibroblastic morphology. For osteoblasts, the same results were found, with a higher\u0026nbsp;number of cells being observed in the gyroid model, indicating that the gyroid structure provided a more favorable environment for cell ingrowth.\u0026nbsp;Guo \u003cem\u003eet al.\u003c/em\u003e (2023) found, through \u003cem\u003ein vitro\u003c/em\u003e experiments, a higher number of cells on the gyroid scaffold when compared to the grid porous scaffold model, with better cell adhesion and proliferation also in the gyroid scaffold. These can be explained by the gyroid structural scaffold which presents an improved pore connectivity and permeability, which is more \u0026nbsp;appropriate for supporting cell growth\u0026nbsp;[42]–[44].\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe optimization of the structure and morphology of 3D-printed scaffolds for bone tissue engineering are in high demand. In the present study, grid and gyroid models made with marine BS had their morphologies and \u003cem\u003ein vitro\u003c/em\u003e effects compared, demonstrating a clear indication of the superiority of the gyroid model. However, further studies involving more detailed \u003cem\u003ein vitro\u003c/em\u003e experiments and pre-clinical works remain to be performed to continue the investigation of the gyroid-shaped scaffolds manufactured with BS.\u0026nbsp;\u003c/p\u003e"},{"header":"CONCLUSION","content":"\u003cp\u003eFinally, both scaffolds were created effectively using the 3D printing technique, with regard to the optimization of process parameters, microstructure, mechanical characteristics, and biocompatibility. In addition, the gyroid structural porous bone scaffolds demonstrated higher mechanical strength and better cellular responses than the standard grid structural scaffolds. The present study demonstrates that co-design of material, structure, and technology can result in comprehensive good performance of polymerfor bone scaffolds, as well as the promise of 3D-printed gyroid scaffolds in bone tissue engineering.\u003c/p\u003e"},{"header":"DECLARATIONS ","content":"\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e: This study was funded by São Paulo Research Foundation (FAPESP) grant number 2022/04433-8. F.V.S also acknowledge FAPESP for the fellowship (grant number 2022/05316-5)\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of Data and Material\u003c/strong\u003e: Datasets created and/or analyzed during the current investigation are accessible from the corresponding author upon reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflicts of Interest/Competing Interests\u003c/strong\u003e: The authors have no conflict of interest.\u003c/p\u003e"},{"header":"REFERENCES","content":"\u003col\u003e\n \u003cli\u003eB. Bouyer, F. Leroy, J. Rudant, A. Weill, and J. Coste, \u0026ldquo;Burden of fractures in France: incidence and severity by age, gender, and site in 2016,\u0026rdquo; \u003cem\u003eInt. Orthop.\u003c/em\u003e, vol. 44, no. 5, pp. 947\u0026ndash;955, 2020, doi: 10.1007/s00264-020-04492-2.\u003c/li\u003e\n \u003cli\u003eH. Lv \u003cem\u003eet al.\u003c/em\u003e, \u0026ldquo;A multicenter survey of patients\u0026rsquo; favorite type of nursing care and associated factors in Hebei Province, China,\u0026rdquo; \u003cem\u003ePLoS One\u003c/em\u003e, vol. 17, no. 3 March, pp. 1\u0026ndash;14, 2022, doi: 10.1371/journal.pone.0264169.\u003c/li\u003e\n \u003cli\u003eG. C. Keles, M. Sumer, B. O. Cetinkaya, F. Tutkun, and S. B. Simsek, \u0026ldquo;Effect of Autogenous Cortical Bone Grafting in Conjunction with Guided Tissue Regeneration in the Treatment of Intraosseous Periodontal Defects,\u0026rdquo; \u003cem\u003eEur. J. 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Available: https://doi.org/10.1080/10584587.2018.1514889%0Ahttps://doi.org/10.1016/j.matpr.\u003cbr\u003e2019.12.188%0Ahttps://doi.org/10.1016/j.matpr.2019.09.090%0Ahttps://doi.org/10.1080/14484846.2018.1432089.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Biosilica, bone healing, marine sponges, scaffolds, 3D printing.","lastPublishedDoi":"10.21203/rs.3.rs-4914115/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4914115/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThis study compared the physicochemical characteristics and biological effects of two 3D printed biosilica (BS) scaffolds (grid and gyroid). Methods included Scanning Electron Microscopy (SEM), Micro-Computed Tomography (Micro-CT), Mass Loss and pH Assessment, Fourier-Transform Infrared Spectroscopy (FTIR), and Energy-Dispersive X-ray Spectroscopy (EDS). The mechanical evaluation involved a Compression Test, and \u003cem\u003ein vitro\u003c/em\u003e tests used cell adhesion assays with osteoblastic (MC3T3-E1) and fibroblastic (L929) cell lines. SEM showed BS spicules in both models on day 0, with signs of degradation along the experimental periods of immersion, forming a homogeneous network with the interaction with alginate. Micro-CT revealed rough surfaces in both models, with the gyroid model being more homogeneous and porous, with larger pores compared to the grid model. The gyroid model demonstrated higher values in the compression test and a decrease in pH on the first day and no differences for both models on days 3, 7, and 14. The mass loss was higher in the gyroid model by day 21. FTIR tests showed characteristic peaks for ALG and BS. EDS detected silica (Si), chlorine (Cl), calcium (Ca), carbon (C), and oxygen (O). In cell adhesion assays, both models supported adhesion and proliferation of L929 and MC3T3-E1 cells, with the gyroid model showing better cell elongation and morphology. Overall, the gyroid model demonstrated superior physicochemical properties, greater mechanical strength, and enhanced biological performance compared to the grid model, making it more promising for tissue engineering applications.\u003c/p\u003e","manuscriptTitle":"Development and comparison of two 3D printed scaffolds of biosilica from marine sponges for bone tissue engineering","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-09-13 16:30:40","doi":"10.21203/rs.3.rs-4914115/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"fd546740-cc5e-4a19-b3ec-52b3c7bfa0f6","owner":[],"postedDate":"September 13th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2024-11-24T20:08:14+00:00","versionOfRecord":[],"versionCreatedAt":"2024-09-13 16:30:40","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-4914115","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4914115","identity":"rs-4914115","version":["v1"]},"buildId":"7rjqhiLT3MXkJMwkYKINL","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}