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Obtaining biocompatible ceramic scaffolds of calcium phosphates through ceramic stereolithography

Carolina Duque-Uribe, Valentina López Vargas, Ana Isabel Moreno Florez, Alejandro Pelaez-Vargas, Alex Ossa, Carolina Cárdenas-Ramírez, Sebastián Restrepo-Vélez, Andrés Felipe Vásquez & Claudia Garcia 2025-07-20

Abstract

Ceramic stereolithography scaffolds with designs based on triple periodic minimal surfaces (TPMS) were developed for potential applications in bone tissue regeneration. An acrylic-based resin with calcium phosphate nanoparticles were used. Particles were synthesized via Combustion in solution, resulting in hydroxyapatite and β-TCP phases. Suspensions with 35, 40, and 50 vol% particles, using a 10 wt% of dispersant, were prepared and rheologically characterized to ensure suitable viscosities for printing, and were used to print gyroid scaffolds by DLP technique. The suspension with the highest ceramic load demonstrated the highest viscosity. The green bodies were morphologically and mechanically characterized before and after sintering. Volumetric shrinkage, morphological characteristics by digital and FE-SEM images, and compressive strength were evaluated. Polymeric-ceramic (Hybrid) scaffolds before sintering exhibited better compressive strength than sintered ones. Ceramic scaffolds achieved compressive strength values up to 0.9 MPa, comparable to those of cancellous and cortical bone. The optimal scaffolds (50CPF) were subjected to degradation tests in PBS and were impregnated with ethanolic extract of propolis from Arauca, Colombia, for biological analysis using the L929 cell line. The results indicate that ceramic stereolithography is an effective technique to produce scaffolds with optimal characteristics for potential applications in bone tissue regeneration.

Graphical Abstract

1 Introduction

Bone trauma and diseases are very common around the world and often require partial repairs or complete replacements, which are complex and expensive treatments [1]. One solution is the use of autografts, but this method can compromise the function of healthy organs or tissues, has a long production and acquisition cycle, and may produce grafting material that is insufficient or inappropriate size and shape [2, 3]. Consequently, there is a significant focus on developing novel treatments and techniques for bone tissue regeneration, including the use of materials to produce implantable devices with properties similar to bone in terms of mechanical strength and biocompatibility [4, 5].

Implantable devices, known as scaffolds, need a porous structure with adequate size and pore interconnection to provide cell migration, vascularization, oxygen and nutrient flow, and residue elimination [5]. Additionally, scaffolds must support loads, a primary function of bone tissue. Geometries based on triple periodic minimal surfaces (TPMS) have been studied to satisfy these requirements, as their designs ensure interconnected porosity and controlled pore size. The curvature of TPMS structures influences cellular performance and fluid permeability significantly [1, 6]. One of the best known TPMS structures is the gyroid, which separates spaces into two opposing and congruent labyrinths of passages [7]. This structure can provide good mechanical resistance and an elastic modulus comparable to natural cancellous bone. Cells seeded in gyroid structures show better cell viability compared to other TPMS geometries, as their design allows greater cell penetration and better flow of the medium through their porosities [1, 8].

Geometries such as TPMS are difficult to produce using conventional manufacturing techniques. Additive manufacturing or 3D printing is a strategic tool to create these complex shapes [9]. Stereolithography (SLA) and digital light processing (DLP) stand out among 3D printing techniques for their ability to efficiently produce high-precision pieces [6], making them and attractive candidate for the creation of scaffolds for bone regeneration. These systems use a photocurable resin that polymerizes layer by layer upon light exposure. The DLP system ensures simultaneous light irradiation across the desired cross-section, reducing processing time compared to SLA, which uses a laser [5].

In the SLA and DLP processes, the CAD file of the prototype undergoes a slicing procedure to create layers. This information is sent to the machine, which projects each horizontal section onto the liquid resin using masks. The machine’s mobile platform starts at the bottom of the Z-axis, submerged in the vat containing the liquid resin, leaving a thin layer of material between the bottom of the vat and the platform. Upon exposure to UV light, this layer solidifies, forming the desired shape and thickness. The platform then rises with the solidified layer, allowing the resin to refill the vat, and submerges back to repeat the process until all sections of the object are solidified [10].

In these techniques, the photocurable resin can be modified by adding ceramic particles to produce hybrid (polymer/ceramic) or fully ceramic pieces. In the latter case, the particles are surrounded by the crosslinked polymer during printing. Once printed, the green body is subjected to thermal treatment to remove the resin and sinter the ceramic [11, 12]. High solid loads must be used to avoid crack formation during thermal processing, with a careful viscosity control to ensure self-leveling during printing. Nanometric ceramic particles improve the mechanical properties of the final piece but present challenges in preparing the mixture due to their high specific surface area and greater tendency to agglomerate [12]. In the ceramic SLA process, several essential steps are necessary to obtain fully ceramic pieces. These steps include preparing the photopolymerizable resin-ceramic suspension, printing the piece, debinding the resin, and sintering the green ceramic body. The objective is to achieve a high density of ceramic particles to produce pieces with good dimensional and structural integrity and to ensure proper sintering once the resin debinding process is complete [13].

Materials used to create scaffolds for bone repairs include a wide range of ceramics, polymers, and composite materials [14]. Depending on their ability to stimulate bone tissue, materials are classified as bioinert or bioactive. Bioactive materials include natural polymers like collagen, ceramics like calcium phosphates or bioactive glasses [15]. Calcium phosphates have shown great potential due to their biocompatibility, performance, degradability [16], mechanical stability, and composition, which is very similar to bone tissue, favoring cell adhesion, proliferation and promoting mineralization [17].

Scaffolds, as implantable devices, have demonstrated their efficacy in bone regeneration processes. The addition of natural compounds has been studied to confer properties that some scaffold materials by themselves could not provide, such as antibacterial activity or enhanced biocompatibility and bioactivity [18]. Propolis, a resinous substance produced by bees, has been used for treating many diseases and surgical repairs due to its biological activity [19]. These are produced by bees from various waxes, gums, pollen, and sap found in plants surrounding the hives and used by bees to protect them from the cold and some plagues [20]. The flora around the hive influences in the propolis properties, which vary geographically [21]. Some propolis have been described as potential antioxidants with antibacterial activity [22, 23] and may be considered anti-inflammatory and tissue restorers, promoting wound healing and serving as good alternatives for tissue regeneration [19, 24, 25].

In this work, a composite of acrylic resin with calcium phosphate particles, using a dispersant, was developed for implementation in ceramic SLA printing of gyroid TPMS ceramic scaffolds for potential biomedical applications. The resin was characterized, and the rheological behavior of the resin-calcium phosphate mixture with different ceramic load percentages was studied to ensure favorable printing conditions. High-quality gyroid geometries were obtained. These scaffolds were subjected to a controlled thermal treatment to remove the resin and achieve ceramic sintering. The resulting ceramic scaffolds were morphologically and mechanically characterized. The scaffolds with the percentage of ceramic load that presented the best mechanical behavior were selected for degradation tests and impregnation with propolis extracts from Arauca, Colombia, to evaluate cytotoxicity and proliferation of the L929 cell line on these samples.

2 Materials and methods

2.1 Resin and calcium phosphate particles

For the fabrication of scaffolds via vat photopolymerization, a commercial, pigment-free Portux Print 3D Model resin (New Stetic) was used. This resin was previously analyzed and characterized in a previous work [10] using Differential Scanning Calorimetry (DSC) and thermogravimetry (TG) with a TA Instruments Discovery 550 with air atmosphere and a heating ramp of 10 °C/min up to 1000 °C. Fourier Transform Infrared Spectroscopy (FTIR) was performed using a Shimadzu IRTracer-100 with an accessory to measure transmittance before and after curing. The FTIR spectra were acquired in a spectral range of 500–4000 cm−1.

The calcium phosphate particles used in the resin-ceramic suspension for printing were synthesized using the combustion in solution method, as detailed in a previous study [26]. Calcium nitrate tetrahydrate (Ca(NO3)2·4H2O, Merck) and ammonium hydrogen phosphate ((NH4)2HPO4, Alfa Aesar) were used as precursor raw materials for calcium and phosphorus, respectively. Glycine (C2H5NO2, Merck) was used as fuel, and nitric acid (HNO3) as a catalyst. Unlike the procedure in [26], the ashes were not thermally treated and were used directly for mixing and printing. The ashes were manually ground for 5 min and then subjected to a deagglomeration process in a ball mill (Retsch PM 100) for 15 min at 250 rpm with 5-min intervals and reverse rotation. The ashes were evaluated using X-ray diffraction with a double circle multipurpose Xpert-Pro PANanalytical Radiation Cu-Ka (λ = 0.15406 nm) diffractometer in a range of diffraction of 10–50° (2θ) as is detailed in [26].

Particle size was measured by taking images with a field emission scanning electron microscope (FESEM) and analyzing them using ImageJ software to statistically count 200 particles. For image acquisition, the samples were fixed on graphite tape, given a thin gold (Au) coating using a DENTON VACUUM Desk IV, and analyzed with a Dual Beam Field Emission Scanning Electron Microscope (Beam FIB-FESEM, Thermo Fisher Scientific, model Socios 2 LoVac).

2.2 Suspension of calcium phosphate particles in resin

Suspensions with concentrations of 35 (35CPF), 40 (40CPF), and 50 (50CPF) vol% of calcium phosphate particles (ashes) were prepared. The mixing process was conducted in two stages using a ball mill (Retsch PM 100) over a total period of 6 h. In the first stage, the commercial Portux Print 3D Model resin (New Stetic) was mixed with a dispersant, Disperbyk-167 (Altana), with 10 wt% of the ceramic load. This stage lasted 2 h at 250 rpm, with 30 s pauses every 5 min and reverse rotation. In the second stage, the ceramic powder was added at the required concentration for each batch, and the mixture was returned to the mill under the same conditions for an additional 4 h to achieve a homogeneous mixture. The resulting suspensions were rheologically characterized using shear rate vs. viscosity curves on a Bohlin Visco88BV viscometer (Bohlin Instruments).

The 50CPF suspension was also subjected to a thermogravimetric analysis (TG) and DSC using a TA Instruments Discovery 550 with air atmosphere and a heating ramp of 10 °C/min from room temperature to 1200 °C. This analysis was performed to determine the thermal behavior of the suspension with the highest ceramic load. For this purpose, a specimen with a diameter of 4 mm and a height of 1.5 mm, was printed on the same machine used for printing the scaffolds and used the same printing parameters.

2.3 Fabrication of the scaffolds

In this work, the Asiga PICO2GD printer, which operates using DLP methodology with a light source wavelength of 385 nm, was used. The printing parameters and slicing were configured using Asiga Composer software, with a layer thickness of 0.05 mm, an exposure time of 68 s for the initial layers, and 15 s for the subsequent layers. The selected TPMS geometry for the scaffolds was a gyroid, as shown in Fig. 1a. These scaffolds had an external cylindrical shape with a diameter of 10 mm, a height of 8 mm, and a porosity of 80.5 vol%. The porosity was measured using images of the different kind of samples, the ImageJ software, and a CAD model image to calculate the material and pore volumes. It is important to note that the porosity referred to in this section is macroscopic, resulting from the TPMS geometry.

Fig. 1 CAD Model Image and Thermal Treatment Curve. a Top and side view of the CAD model of the gyroid geometry, b Thermal treatment curve to which the 35CPF, 40CPF, and 50CPF samples were subjected

The interconnected pores of the TPMS structures can be described using trigonometric functions. These were modeled using MathMod® software in a previous study [27]. Initially, a unit cell of the TPMS was created and replicated in three dimensions, three times along the XY axes and twice along the Z-axis, resulting in surfaces consisting of a total of 18-unit cells.

2.4 Post-processing of green ceramic bodies

Once the samples were printed, they were cleaned for 3 min in isopropyl alcohol and were subjected to a post-curing process with an Anycubic Wash & Cure Plus machine (Anycubic, China) at a wavelength of 405 nm for 50 min. Subsequently, the samples were subjected to thermal treatment in an SA-1700X furnace (Sentro Tech Corp, USA) for debinding and subsequent sintering of the ceramic material. The thermal treatment curve used is shown in Fig. 1b and the ramps information is shown in Table 1. The heating of the furnace, where the samples were located, started at room temperature (25 °C). The maximum temperature reached was 1300 °C. The temperature was raised gradually with pauses of about 1 h every 100 °C to ensure the complete and gradual degradation of the resin. The temperature was maintained for 2 h at 800 °C to ensure the phase change of the calcium phosphates. There were six ramps numbered from 1 to 6 in Fig. 1b. Once the samples were sintered after 6 h at 1300 °C, the cooling was performed slowly following the oven conditions until reaching room temperature (25 °C).

Table 1 Heat treatment curve ramps

The sintered ceramic bodies were characterized by X-ray diffraction using a double-circle Xpert-Pro PANalytical diffractometer with Cu-Kα radiation (λ = 0.15406 nm) in a diffraction range of 10–50° (2θ).

2.5 Scaffolds properties

The calcium phosphate scaffolds produced via DLP were characterized before and after the thermal treatment for resin debinding and sintering. Field emission scanning electron microscopy (FESEM) was used for imaging. The samples were fixed on graphite tape, given a thin gold (Au) coating using a DETON VACUUM desk IV machine and analyzed with a dual beam field emission scanning electron microscope (Beam FIB-FESEM, Thermo Fisher Scientific, model Socios 2 LoVac).

To calculate the volumetric shrinkage percentage during thermal treatment, the external dimensions (diameter and height) of both kind of samples (before and after sintering) were measured with an analog caliper (Discover Tools, China). The theoretical volume of the geometry was also measured using the CAD file in ImageJ software, as well as the wall thicknesses of the CAD model, the printed scaffolds, and the scaffolds after sintering.

2.5.1 Mechanical tests

Mechanical compression strength tests were conducted on the scaffolds before and after sintering using a universal testing machine (Instron 3366, Instron, MA, USA) with a constant displacement rate of 1 mm/s. Measurements were taken on five scaffolds from each concentration under identical conditions. The Young’s modulus of the samples was obtained from the slope of the linear section of the stress-strain curve for each test, while the maximum compressive strength of the samples was measured as the maximum stress value reached by each test sample.

2.5.2 Degradation test

An in vitro degradation test was made in accordance with ASTM F 1635-16 to study the weight loss of 50CPF scaffolds post-sintering over a 23-day period. The scaffolds were immersed in phosphate-buffered saline (PBS) solution (pH 7.4, Sigma-Aldrich, USA). Measurements were taken on days 1, 5, 8, 12, 15, and 23. Each sample was individually immersed in 20 ml of PBS within sealed polyethylene containers and incubated at 37 °C with 5% CO2 (Thermo Scientific, USA). On each evaluation day, the pH of the PBS was measured using a PH700 pH meter (Apera, USA). Samples were then removed from the PBS, rinsed with distilled water, and dried at 30 °C for 15 min in an oven (Binder, Germany). Once dried, the samples were weighed using a balance (Adam Equipment, UK). The mass loss was calculated using Eq. 1. Each measurement was performed in triplicate to ensure accuracy and reliability.

Where W0 is the initial dry weight of the sample and Wt is the dry weight of the sample after the selected time.

2.5.3 Impregnation of Sintered Scaffolds with Propolis

The printed and sintered 50CPF scaffolds were impregnated with ethanolic extracts of propolis from Arauca, Colombia, at a concentration of 0.7 mg/mL, obtained from a previous study [23]. Initially, 20 µL of the ethanolic propolis extract was deposited on the surface of each scaffold. The scaffolds were then placed in a positive pressure chamber (Wiropress, BEGO, Germany) for 5 min at 2.4 Bar. The absorption of the extract by the scaffolds was confirmed by observing a color change in the scaffolds and by checking the moisture of a napkin placed under the samples. This process was repeated until a total volume of 80 µL was impregnated into each scaffold.

2.5.4 Biological assays

To evaluate bioactivity, two groups of samples were used: scaffolds composed of 50 vol% of calcium phosphate impregnated with an ethanolic extract of propolis (50CPF + P) and unimpregnated scaffolds (50CPF). Both types of samples were sterilized with UV light before experimentation. A cytotoxicity assay using MTT was conducted in 24-well plates, with each well containing one scaffold of each type. Onto each scaffold, 50,000 cells from the L929 cell line (NCTC clone 929 [L cell, L-929, derivative of Strain L], ATCC Nr. CCL-1) were seeded using DMEM medium supplemented with 10% v/v fetal bovine serum (Gibco, USA). A control group with the same number of cells seeded without any scaffold was maintained to ensure proper cell growth. Measurements were taken at 24 and 96 h, as well as at 6 days. On each measurement day, the medium was removed, the wells and samples were washed with Ringer’s lactate to eliminate dead cells, the sample were moved to a different well, and MTT solution (1 mg/mL in DMEM medium) was added. The plates were then incubated for 2 h. After incubation, the absorbance at 570 nm of each well containing 50CPF and 50CPF + P scaffolds was measured using a spectrophotometer (Multiscan Go, Zeiss, Germany). Comparisons were made between the two scaffold types. All experiments and measurements were performed in triplicate.

2.6 Statistical analysis

The results were expressed as mean ± standard deviation (SD). Statistical analysis was performed using Minitab Statistical Software (Minitab Inc.). To assess the significance of differences between experimental groups, a multivariate analysis of variance (ANOVA) was conducted. A p-value of less than 0.05 was considered statistically significant. Graphs were generated using Wolfram Mathematica (Wolfram) and Origin (OriginLab Corporation) software.

3 Results and discussion

3.1 Characterization of ceramic powder

Figure 2a shows the histogram of particles and agglomerates size distribution (PSD) for the calcium phosphate (ashes) used in this study. The data indicates that 32.5% of the particles range in size from 20 to 40 nm, and 90% are smaller than 100 nm. Figure 2b highlights the presence of aggregates ~150 nm in size, composed of nanoparticles with granular or spherical morphology. Controlling the size of particles and aggregates in DLP printing is crucial, as their dimensions must be smaller than the layer thickness to avoid compromising the vertical resolution of the printed geometry [13]. In this study, most particles are smaller than the used layer thickness of 0.05 mm, ensuring that the printing resolution will not be affected.

Fig. 2 Characterization of Calcium Phosphate Particles. a Resulting histogram from particle size measurements taken with ImageJ software and analyzed by Origin software of the particle size distribution of the calcium phosphate (ashes) used in the resin-ceramic mixture for DLP printing. b Example of FESEM image used for particle and aggregate size measurements

The X-ray diffraction (XRD) pattern of the ashes, are reported in a previous work [26]. This reveals the formation of two main phases of calcium phosphate: hydroxyapatite (HAP), indexed with the JCPDS pattern (010740566), and the β-phase of tricalcium phosphate (β-TCP), corresponding to JCPDS (01-070-2065). Minor phases of calcium pyrophosphate were also identified.

3.2 Characterization of resin and suspension

The thermal behavior and molecular composition of the resin, both before and after curing, have been detailed in a previous study  [10]. In that work the resin was identified as an acrylic type, exhibiting a typical thermogravimetric behavior. Furthermore, it was observed that post-printing analysis showed no significant alterations or changes in functional groups of the polymer [10]. TG also presented in [10], reveals significant mass losses between 300 °C and 470 °C.

Figure 3a presents the TG and DSC curves for a 50CPF sample. The TG curve shows mass loss similar to that of the resin without ceramic loading, as reported in  [10], with significant losses between 300 °C and 470 °C. Beyond this range, the residual weight corresponds to the ceramic particles, indicating no alteration in the resin upon contact with calcium phosphate particles. Thus, no differences are observed in the resin delamination process, regardless of ceramic loading.

Fig. 3 Suspension Characterization. a Thermogravimetric analysis and differential scanning calorimetry of the 50CaP specimen. b Shear rate vs. viscosity curves of the mixtures with different ceramic loading percentages

The DSC analysis highlights three regions marked with dashed boxes. An exothermic peak near 110 °C, shown in the green box, is attributed to polymerization reactions that continue to occur in the solid resin; this same result has been presented by different authors with polymeric resins of acrylic nature [28,29,30]. There is an exothermic region near to 300 °C and 400 °C, corresponding to the degradation reaction of the polymeric chains of the resin [30, 31], reflecting the most substantial mass loss in the sample. This behavior has also been corroborated by Yong Zeng et al. [32]. The purple box highlights an exothermic peak after 650 °C, indicating ongoing polymer decomposition and suggesting residual carbon residues in the sample, despite minimal mass losses observed in the TG [31, 33].

Figure 3b presents the shear rate versus viscosity curves for mixtures with different ceramic loading percentages. All mixtures exhibit non-Newtonian, shear-thinning rheological behavior, attributed to microstructure breakdown under increased shear rates, resulting from particle-particle and particle-resin monomer interactions [34]. This behavior is well-documented in the literature [12, 17, 35,36,37].

For SLA printing, resins with viscosities up to 5 Pa·s are typically suitable [38]. However, for resin-ceramic suspensions, viscosity must be relatively low to allow self-leveling in the vat during printing without interference from particulate materials. Therefore, loaded resins should have viscosities below 3 Pa·s [13]. Additionally, maximizing solids content is crucial for proper sintering without excessive shrinkage, which could lead to defects. However, high solid loading should not result in inappropriate viscosity or alter the suspension’s shear behavior, making the use of dispersants essential [39]. For the mixtures studied, viscosities do not exceed 1.2 Pa·s at shear rates over 400 s−1, with the 50CPF mixture showing the highest viscosity, still below the reported values for resin-ceramic suspensions for SLA. The use of BYK dispersant was essential for achieving mixtures with good flowability and particle stability. This effectiveness may be due to the phosphate group anchoring of BYK, which facilitates its combination with calcium phosphate particles, while its end chain has an affinity for non-aqueous media [34], creating a barrier layer on the particle surface that generates sufficient repulsion force between particles [12].

The viscosities obtained for the three mixtures are successful, as resin viscosity increases exponentially with ceramic content, an expected outcome that does not affect the printing process. Studies incorporating ceramic loadings exceeding 40 vol% often use micrometer-sized particles due to the large surface area per unit volume of nanoparticles, making high nanoparticle content resins complex to prepare, with few studies achieving this [34, 40]. The use of nanostructured calcium phosphate powders in this study yields promising results for ceramic suspensions with suitable viscosities for 3D SLA printing.

3.3 Geometry, contraction, and phases analysis of scaffolds

The scaffolds produced through DLP printing of the resin-ceramic suspension, followed by post-curing and sintering treatments, are shown in Fig. 4a–f. Cylindrical scaffolds with Gyroid geometry, measuring 10 × 8 mm (diameter x height) and 80.5 vol% porosity, were successfully printed. Macroscopically, no morphological differences were observed between scaffolds with varying particle percentages and the original CAD model (Fig. 1a). The scaffolds exhibited interconnected porosity, with the porosity percentage remaining consistent after sintering. Before sintering (Fig. 4a–c), the scaffolds appeared opaque gray due to the high particle content, which had not yet undergone thermal treatment. The surface was smooth and free of prominent defects, with no pore blockages typical of over-curing processes, indicating appropriate printing parameters were used. After sintering (Fig. 4d–f), volumetric contraction was evident due to the thermal treatment, which resulted in the complete removal of the resin. Contraction values for each type of scaffold are presented in Table 2. Thinning of the scaffold walls was also observed before and after sintering, with thickness values provided in Table 2. The greatest contraction occurred in the 35CPF samples, as expected, due to their lowest particle content and highest resin percentage. Despite these contractions, the inherent Gyroid TPMS geometry was preserved without appreciable deformations and without changes in the design porosity percentage.

Fig. 4 Digital photographs of the scaffolds obtained by DLP printing with Gyroid geometry. Before the sintering process: a 35CPF, b 40CPF, c 50CPF, and after the sintering process: d 35CPF, e 40CPF, f 50CPF, and g X-ray diffraction (XRD) pattern of calcium phosphate Scaffolds after thermal treatment

Table 2 Volumetric contraction values, maximum compressive strength, Young’s Modulus, and Wall Thickness for scaffolds with Gyroid geometry, before and after sintering, at different ceramic loading concentrations

Figure 4g shows the XRD pattern of the material after thermal and sintering treatment of the samples once they were printed, showing that the particles present the same main phases as the ashes, that the crystallization of the ashes was enhanced, and that the pyrophosphate phase disappeared. This suggests that the resin removal process and subsequent sintering lead to improved crystallinity and the structural characteristics of the desired crystalline phases in the final product.

3.4 Microscopic analysis of scaffolds

Figure 5 presents FESEM images of scaffolds with Gyroid geometry at different ceramic loading concentrations, both before and after sintering. Before the sintering (Fig. 5a–f), no significant differences were observed among the three types of samples when compared to the CAD model (Fig. 1a). The scaffold walls displayed a high content of ceramic particles, with visible layers typical of the printing process, while the resin, serving as a binder, was not discernible. Wall thicknesses for the samples before and after sintering are indicated with yellow arrows.

Fig. 5 SEM and FESEM images of Gyroid scaffolds before sintering: a, d 35CPF, b, e 40CPF, and c, f 50CPF. After sintering: g, j 35CPF, h, k 40CPF, and i, l 50CPF

Before sintering, no significant differences in wall thicknesses were observed across samples, as measurement uncertainties caused the values to overlap. However, there was a tendency for 35CPF and 40CPF samples to have thicker walls, likely due to increased curing during the printing process; fewer particles result in less light attenuation and therefore longer exposure times. Conversely, the 50CPF samples had thinner walls because the increased particle content attenuated the light more, resulting in insufficient exposure time for adequate curing.

After sintering (Fig. 5g–l), some cracks were evident due to volumetric contractions, indicated by red arrows. The 35CPF scaffolds exhibited more cracks due to their lower particle content and higher resin percentage, leading to greater contractions. In contrast, the 40CPF and 50CPF scaffolds had almost imperceptible cracks, indicating proper thermal treatment. The temperature curve used included 1-h pauses every 100 °C, ensuring gradual temperature increases during debinding to prevent excessive stress. The sintering temperature of 1300 °C was suitable for densifying the scaffolds, as shown in (Fig. 5j, k, l), where sintered necks are visible, and smaller and fewer pores are present with increasing ceramic loading.

3.5 Mechanical characterization of scaffolds

Figure 6 presents the stress vs. strain curves from compression mechanical testing of Gyroid-shaped scaffolds before and after sintering. The maximum compressive strength and Young’s modulus for each composition type are listed in Table 2. Statistical comparison between scaffolds before and after sintering yielded p values less than 0.05 for all three concentration types, indicating significant differences.

Fig. 6 Stress-strain vs. deformation Curves for Gyroid-shaped scaffolds: a Before sintering and b After sintering

Scaffolds fabricated from the polymer-ceramic combination before sintering exhibited higher compressive strength and Young’s modulus, suggesting that the ceramic particles were well adhered to the resin, enhancing the rigidity of the samples. The printed and post-cured composite materials displayed low defect levels. The fact that the mechanical strength of the 35CPF sample before sintering is slightly higher, may indicate a greater presence of defects in the 40CPF and 50CPF samples as the amount of calcium phosphate particles and agglomerates increases, which cause minor decreases for the samples with higher percentage of ceramics for the values of compressive mechanical strengths. However, the values presented do not show significant differences between particle percentages of particles. Similar results were obtained in a previous study with lower loading percentages [10]. The increased mechanical strength of the 35CPF samples may also be attributed to better curing during the printing process and thicker walls, as previously mentioned.

The maximum compressive strength of the hybrid bodies falls within the range of human cancellous bone (0.6–15 MPa) [41], suggesting they could be considered alternatives for bone repairs requiring high resolution, good finishes, and resistance to mechanical loads without accelerated degradation. The Young’s modulus indicates the elastic properties of the material, and the values for the hybrid bodies are higher than those for fully ceramic ones, directly related to the presence of resin.

After sintering, fully ceramic scaffolds exhibited lower compressive strength due to cracks resulting from contraction during resin elimination, the small volume of material in this TPMS geometry, and the characteristic fragility of ceramic material. As expected, higher ceramic content resulted in better mechanical strength. Both compressive strength and elastic modulus for the three concentration types align with those reported for cancellous and cortical bone, making these scaffolds promising for medical applications in bone repairs with versatility [17, 42]. In ceramic bodies, a lower Young’s modulus implies lower resistance for the same deformations, as they are much more fragile. Among the ceramic scaffolds, the 50CaP type exhibited the best mechanical compression behavior, making this geometry the selected choice for impregnation with ethanolic propolis extracts and subsequent biological assays.

It is important to note that analyzing the mechanical response of ceramic materials can be challenging due to the variability in results caused by the presence of cracks and voids within the materials. Nevertheless, we utilize the mechanical responses of the scaffolds to compare the various materials manufactured, both prior to and following the sintering process, finding significant differences.

3.6 In vitro degradation

The in vitro degradation behavior of the 50CPF scaffolds after sintering was studied by immersing them in PBS solution for 1, 5, 8, 12, 15, and 23 days. The results are presented in Fig. 7. For the case where the slope is positive, they are considered as mass losses, and when they are negative, they are considered as mass gains or increases. Throughout the test, the scaffolds maintained their shape with no evident deformations. The pH of the PBS in which each sample was paced remained constant and close to the initial pH of PBS (7.4), indicating the material’s stability over time. In the initial days, a chemical interaction between the scaffolds and the medium led to the deposition of salts on the surface of the samples [43], presenting fluctuations in these initial days of immersion. However, as shown in Fig. 7, the dissolution of these salts occurred rapidly due to the high interaction with the medium facilitated by the scaffold’s roughness, resulting in significant mass loss by day 8. From day 12 to day 23, a mass gain was observed, attributed to the interaction of calcium phosphates with the medium and their bioactive properties, causing apathitic phases to deposit on the scaffold surfaces. This observation is consistent with previous studies confirming the high bioactivity of calcium phosphates, which leads to the formation of apathitic phases on scaffold surfaces [10, 44]. The mass gains increase considerably between days 12 and 15, indicating that the greatest deposition of apathitic phases occurs on these days. On day 23 the increase is not significant respect to the previous days; this means that the stability in the chemical interactions of the material with the surrounding medium is being reached.

Fig. 7 Loss and weight gain values of 50CPF scaffolds after sintering as a function of soaking time in PBS

3.7 Biological assay of propolis-impregnated scaffolds

Upon impregnation, the scaffolds exhibited a shift to brown tones, indicating successful incorporation of propolis into the ceramic matrix without compromising the structural integrity or dimensions of the 50CPF scaffolds. This change in coloration is characteristic of propolis presence. To assess cell viability, the MTT assay was conducted for each scaffold type at three different time points, with control cells remaining viable throughout. Figure 8 illustrates the cell proliferation and cytotoxicity results for each scaffold type, measured in terms of absorbance. The MTT assay relies on cellular oxidoreductase enzymes to reduce MTT dye to insoluble Formazan, producing a purple coloration whose absorbance correlates with the number of live cells [45]. Higher absorbance values thus indicate greater cell viability.

Fig. 8 Cell proliferation of L929 cell line on scaffolds with 50% w/v calcium phosphate (50CPF + P) and without impregnation (50CPF) of ethanolic propolis extract from Arauca, Colombia at 24 h, 96 h, and 6 days

Both propolis-impregnated scaffolds (50CPF + P) and non-impregnated scaffolds (50CPF) supported cell growth. This suggests that the geometry of the printed ceramic scaffolds and the chemical composition of the hydroxyapatite and β-tricalcium phosphate phases facilitate cell adhesion and proliferation of L929 cells. The Gyroid TPMS geometry enhances cell growth due to its interconnected porosity, which is achieved through mathematical functions inherent to the structure and the porosity developed during the sintering process. These results align with previous studies indicating that calcium phosphate or bioactive glass scaffolds promote cell proliferation [46, 47]. The Gyroid TPMS geometry also aids in nutrient transport and waste elimination, effectively mimicking natural bone microstructure [48]. This geometry promotes vascularization, accelerates regeneration [49], and provides surface area for cell-material interaction [49, 50].

Absorbance values for 50CPF + P over the three measurement days were higher than those for 50CPF, indicating that the ethanolic extract of propolis enhances cell growth without exhibiting cytotoxic effects at the used concentration. Propolis contains a variety of organic and inorganic compounds, such as polyphenols, terpenoids, steroids, amino acids, flavonoids, and phenolic acids (e.g., gallic acid, caffeic acid) [25, 51, 52]. These compounds confer antioxidant, anti-inflammatory, anti-allergic, antibacterial, and cell proliferation-promoting properties to propolis [25]. Propolis has shown efficacy in bone and cartilage regeneration, partly due to active components like caffeic acid, and has demonstrated positive effects on connective cells such as fibroblasts, preventing apoptosis [52]. It also enhances collagen production in L929 cells during the proliferation phase [19, 53]. Therefore, propolis is beneficial for tissue regeneration. The use of propolis-impregnated 50CPF scaffolds supports cell growth and tissue regeneration, making them a promising candidate for bone and cartilage repair applications [24].

4 Conclusion

In this study, we successfully fabricated scaffolds with gyroid TPMS geometry using the DLP technique and suspensions of commercial acrylic resin with 35, 40, and 50 vol% calcium phosphate powders. These nanostructured powders, primarily composed of HAP and β-TCP, were synthesized via the combustion in solution method. All suspensions exhibited suitable viscosities for printing, aided by the optimal addition of 10% w/w BYK dispersant.

Thermal treatment effectively removed the resin and sintered the green bodies, producing dense ceramic scaffolds that retained their designed geometry despite expected volumetric shrinkage. FESEM images confirmed smooth surfaces and high structural fidelity. Although some cracking occurred, successful sintering was evident with neck formation between particles.

Before sintering, polymer-ceramic scaffolds showed higher compressive strength and Young’s modulus, suggesting their suitability for load-bearing applications. After sintering, the fully ceramic scaffolds had lower mechanical strength but retained structural integrity.

The 50CPF scaffolds demonstrated bioactivity in a 23-day degradation test, showing salt dissolution at 8 days and apatite formation from day 12, indicating potential for bone regeneration. Additionally, these scaffolds were successfully impregnated with propolis extract without affecting their structure. Cytotoxicity tests confirmed biocompatibility, and enhanced cell proliferation was observed in the propolis-treated scaffolds.

Overall, this work highlights the potential of ceramic SLA for producing complex, high-resolution scaffolds using nanostructured calcium phosphate powders, and supports their application in tissue engineering—especially when combined with bioactive agents like propolis.

Reference: Omitted

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