Production of hydroxyapatite coating on 3D printed PLA parts by powder bed annealing

Production of hydroxyapatite coating on 3D printed PLA parts by powder bed annealing | 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 Production of hydroxyapatite coating on 3D printed PLA parts by powder bed annealing Felipe dos Anjos Rodrigues Campos, Thiago de Oliveira Santos, and 7 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6483704/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 The development of effective and biocompatible coatings for polymeric implants is crucial for advancing orthopedic solutions. This study investigates the feasibility of employing powder bed annealing to deposit hydroxyapatite (HA) coatings on 3D-printed polylactic acid (PLA) parts. The proposed method provides a cost-effective and scalable alternative to conventional coating techniques. The experimental process involved immersing PLA parts in a submicrometric ceramic powder bed followed by thermal treatment to induce adhesion and diffusion of HA particles into the polymer surface. The results demonstrated that the powder bed annealing process successfully generated a uniform HA particulate coating, significantly enhancing the surface roughness, wettability, and hydrophilicity of the PLA substrate. Mechanical characterization revealed an increase in flexural strength and surface microhardness, while maintaining impact resistance. However, a slight reduction in ductility was observed. Biocompatibility tests confirmed that the coated samples supported cell adhesion and proliferation, suggesting their potential for promoting osseointegration in biomedical applications. Compared to existing methods, powder bed annealing allows for the direct integration of bioactive coatings onto polymeric implants without requiring complex post-processing. Additionally, the combination of PLA’s biodegradability with HA’s osteoinductive properties suggests promising applications for resorbable implants in bone regeneration. This study contributes to the ongoing innovation in bioactive coatings, offering a practical pathway to accessible and personalized orthopedic implants. Hydroxyapatite coating Powder bed annealing 3D printing Polylactic acid (PLA) Orthopedic implants Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Figure 10 Figure 11 Figure 12 Figure 13 Figure 14 Figure 15 Figure 16 Figure 17 Figure 18 Figure 19 Figure 20 Figure 21 Figure 22 1. INTRODUCTION With the development of technology and medicine, together with the increase in the quality of life of the population in several countries, there has been a growing increase in the life expectancy of its inhabitants [ 1 ], which has resulted in an aging of the world population [ 2 ]. This creates new challenges related to old age, obesity and a sedentary lifestyle, such as osteoarthritis, which is characterized as a degeneration of the articular cartilage and simultaneous proliferation of bone tissue, cartilage and connective tissue [ 4 ], and causes progressive symptoms of pain, limitations of joint movement, stiffness and muscle weakness [ 5 ]. This situation has been accompanied by an increasing number of orthopedic problems and a greater demand for the use of implants. Therefore, it is extremely important to conduct research to develop technologies that enable the manufacturing of implants in a more financially accessible way and that use more practical manufacturing methods that offer greater capacity for customization according to the patient's characteristics. In this sense, many studies have been carried out on the feasibility of using polymers [ 3 , 6 ], such as the successful implantation of a rib implant made of biocompatible Nylon produced by additive manufacturing [ 7 ]. Among the benefits of using biocompatible polymers, in addition to their much more affordable price than titanium, the most common material used in implants, it is also worth mentioning that some have satisfactory mechanical properties [ 8 ] and an elasticity modulus closer to that of bone tissue, which prevents the effect of bone density loss due to an uneven distribution of load between the implant and bone, known as stress shielding [ 9 ] Another major difference of some polymers, such as polylactic acid (PLA), is their resorbable characteristic, with their degradation occurring through hydrolysis and enzymatic activities [ 10 ], which can be very useful for the production of small implants that can break down over time and be absorbed by the body while the bone tissue around them regenerates and takes their place. Even though there is already a good variety of polymers with good biocompatibility, such as PLA, PCL and Nylon 12, there are studies showing that the compatibility presented by polymers can still be improved using methods for surface modification of the part, since the roughness of the implant has an influence on bone differentiation [ 11 ], and that the use of calcium compounds and titanium dioxide in the form of a coating stimulates the regeneration of bone tissue [ 12 , 13 ]. With this interest in the use of polymers in the manufacture of implants, a manufacturing process that ended up benefiting and standing out was additive manufacturing, such as the Fused Filament Fabrication (FFF), which allows the rapid manufacture of polymeric parts with a high level of customization. However, this technology has as its main limitation its high anisotropy, due to layer-by-layer deposition, resulting in mechanical properties dependent on adhesion between layers in the direction perpendicular to the plane of the deposited layers. Thus, a possible method to solve this problem is to perform an annealing process, to coalesce the layers or even fuse them, making the part more homogeneous and reducing anisotropy. Another possible benefit of annealing is the change in the organizational state of the molecules in semicrystalline polymers, such as PLA. In these cases, their permanence at temperatures above the glass transition temperature favors an increase in crystallinity, which can improve their mechanical properties [ 14 ]. Many works have attempted to improve 3D printed parts mechanical properties by annealing [ 15 – 18 ], and although the results were very promising with increases in mechanical properties up to 100%, the process could deform the geometry of the parts [ 66 ]. However, it has been found that immersing the parts in powder and compacting it could form a mold that would maintain the geometry during annealing and/or remelting [ 19 ]. As explained in other works [ 20 ], the remelting might cause particles from the powder to adhere in the surface, but they could be easily cleaned after by sanding. With this phenomenon in mind, it was hypothesized in this work that this particle adherence could be used to generate a coating in an innovative, simple and inexpensive way, by immersing the 3D printed PLA part in a powder bed and conducing the annealing and/or remelting process. This could in theory be used not only as a new method for depositing calcium phosphate and titanium dioxide compounds in the surface of PLA, but also as a method to improve the 3D printed parts mechanical properties, potentially setting up a new way to manufacture orthopedic implants. Thus, this work aimed to use the powder bed annealing process to generate a ceramic particulate coating on PLA parts manufactured by the FFF process. The project consisted of heating and remelting the part immersed in a tray of submicrometric ceramic particulates and then analyzing the influence of the thermal process parameters in the mechanical resistance of the parts and in the formation of the calcium phosphate and titanium dioxide coating, both materials with recognized capabilities of inducing osseointegration in orthopedic implants [ 64 , 65 ] and widely used for coating commercial implants. The innovative nature of the research stands out, because although annealing in particulate matrix has already been reported in the literature for improving surface roughness and mechanical properties, the possibility of generating a biocompatible coating had not yet been explored. These studies hereby presented may result in reduced manufacturing costs for customized parts for orthopedic implants, contributing to the general health of the population. 2. MATERIALS & METHODS 2.1. PRODUCTION AND PREPARATION OF TEST SPECIMENS Several pre-tests were performed to verify the possibilities and suitable conditions for generating a coating through the powder bed annealing process, in which a significant change in the physical and mechanical appearance of the samples was observed, through visual inspection and flexural tests. For instance, it was found that annealing below 170°C yielded no visible coating, while above 190°C the sample would suffer significant geometrical deformation after annealing. Besides, as both calcium phosphate and titanium dioxide coatings favor osteointegration of implants as mentioned above, it would be desirable to verify in cytologic tests if there was a better component for the proposed coating method. However, it was also verified that annealing in pure titanium dioxide powder led to severe porosity in the samples, while there was no such disadvantage when using calcium phosphate. For this reason, titanium dioxide could only be tested when mixed with calcium phosphate in these pre-tests. Thus, as defined in preliminary studies, test samples were prepared in accordance with ASTM D6272 [ 21 ], since it was desired to compare the behavior of the test specimens in four-point bending, impact, water contact angle, surface roughness, microhardness and layer thickness tests. As the sample geometry could be used for all tests, it facilitated production and guaranteed that there were always extra test specimens available. In addition, the main advantage of choosing the same geometry was to ensure that during the annealing process there would be no major differences in temperature distribution and heating rate. All samples were produced one at a time on a Creality Ender 3 v1®, to guarantee that the thermal history of each sample would be the same prior to annealing. The test specimens were produced with a nozzle temperature of 210°C, table temperature of 60°C, 100% infill, printing speed of 50 mm/s, layer height of 0.2 mm and diameter nozzle of 1 mm. The adopted test specimen had a parallelepiped shape of 127 mm in length, 12.7 mm in width and 3.2 mm in thickness, as shown in Fig. 1, and they were printed with the largest surface in contact with the printing table. The powder bed annealing process was performed using a Britania BFE10V electric oven and the particulate used was pure calcium phosphate (CaPO) and a mixture of 50% calcium phosphate with 50% titanium dioxide (CaPO + TiO 2 ). Eight test specimens were produced for each condition according to the design of experiment presented in Table 1 . Four test specimens were annealed at a time, all of which were positioned inside a single tray and placed at the furnace, as shown in Fig. 2. This procedure was adopted because if two trays were used at once, there could be differences in the heat and temperature distribution on the samples. The annealing temperature was controlled by a thermocouple perforating the tray, with its tip positioned between the samples. For the annealing process, the oven was turned on and regulated on its analogic thermostat to a temperature 10°C above the maximum temperature of the condition being tested. Then the tray was inserted and the temperature inside the tray was monitored as it increased slowly towards the maximum temperature of the condition. It took about 30 minutes to reach 170°C and 40 minutes to reach 190°C, because it takes time for the heat to penetrate the tray and disseminate through the powder by thermal conduction. When the temperature indicated by thermocouple reached the maximum temperature of the experiment, the oven was either turned off and opened immediately for the conditions with 0 minutes at maximum temperature, or the oven was regulated on its thermostat to the maximum temperature of the experiment (170°C or 190°C) and kept turned on for 5 minutes and then turned off and opened. In both cases, the tray was taken off the oven and allowed to cool down naturally until it decreased to 40°C, in a room with temperature of 25°C, which took about 50 minutes. Then the tray was opened, and the samples were retrieved from below the powder and washed smoothly in water, dried in compressed air and stored in plastic containers filled with silica to prevent moisture, until they would be used for the subsequent mechanical and biological experiments. The procedure is patent pending, with code BR 10 2025 000986-2 registered in Brazilian National Institute of Intelectual Protection (INPI). Table 1 Experimental design of the full factorial type 2 3 for powder bed annealing tests. Condition Composition Maximum temperature (°C) Time at maximum temperature (min) A CaPO 190 5 B CaPO 190 0 C CaPO 170 5 D CaPO 170 0 E CaPO + TiO2 190 5 F CaPO + TiO2 190 0 G CaPO + TiO2 170 5 H CaPO + TiO2 170 0 After the test specimens were manufactured, four of each condition were separated for the contact angle measurement, roughness and flexural tests, while the other four of each condition were sectioned in smaller samples for impact tests, layer thickness analysis, microhardness and in-vitro biocompatibility evaluation. First the nondestructive tests such as water contact angle measurements were conducted in the first four samples, and then these same specimens were used for four-point flexural essays, with surface roughness measurements being carried in the fractured parts from the later essay. For the impact tests, layer thickness analysis and microhardness, the other four test specimens were sectioned using an angle grinder equipped with cutting disc. The parts that would be taken to SEM for analysis of layer thickness and chemical composition were embedded in resin, sanded to a 2000-mesh sandpaper, polished and received a 10 nm gold coating on a Leica EM SCD050 metallizer. These samples prepared for analysis in the SEM were the same ones used in the microhardness tests, with all analyses being carried out on the cross section of the sectioned samples. The samples for biocompatibility tests were cut by a hole saw into small disks of approximately 9.5 mm diameter to be fit in a culture plate. 2.2. MATERIALS All the samples were 3D printed with natural PLA by Filamentos 3D Brasil, and its characteristics are provided in Table 2 according to the manufacturer website (F3D, 2024). PLA is a material with recognized effectiveness in composite materials for orthopedic applications [ 22 ], being the most used polymer in composite interference screws [ 23 , 24 ] and in research with resorbable osseointegrated implants [ 25 ]. Neijhoft et al. [ 26 ] found that if there is sufficient contact time between filament and nozzle, which can be achieved through small layer heights, the FFF method presents inherent sterility for PLA. Besides, in accordance with other studies [ 27 , 28 ], it has been shown that autoclaving, the preferred method for sterilizing surgical materials in hospitals, is also able to sterilize 3D printed PLA efficiently and with low distortion for higher size parts [ 29 ]. These characteristics further highlights the adequateness of PLA as a material for implants and tissue engineering. Table 2 Physical and mechanical properties of Premium Natural PLA from Filamentos 3D Brasil [ 30 ]. Physical Properties Value Unit ASTM Standard Density 1.24 +/- 0.05 g/cm³ D792 Fluidity 7–9 g/10 min − (190°C − 2.16 Kg) D1238 Relative Viscosity 4 g/dL Chloroform 30° D5225 Melting point 165–180 °C D3418 Glass Transition Temperature 55–60 °C D3418 Mechanical Properties Value Unit ASTM Standard Tensile Strength 51 MPa D638 Elongation at Break 3,3 % D882 Impact Resistance (IZOD) 118 J/m D256 Heat Deflection Temperature 55–60 °C (0.45 MPa) E2092 The calcium phosphate was obtained from a food additive supplier, since this material is widely used as anti-humectant, while the titanium dioxide was obtained from online stores, since it is the world’s most used pigment load for white paints. These powders, both of which resemble white fine particulates as thin as wheat flour, were analyzed by X-ray diffraction (XRD) to verify which were their crystalline structures, on a Shimadzu XRD6000 difractometer, with 2°/min scanning, 0.02° resolution and 2θ angle of 5 to 60° for calcium phosphate and 20 to 70° for titanium dioxide. The particles were also investigated by SEM (Scanning Electron Microscopy) and EDS (Energy Dispersive Spectroscopy) on a Tescan VEGA 3 and Zeiss EVO MA10 microscopes equipped with Oxford INCAx-act probe, to measure the size of particles and their chemical composition in weight, respectively. 2.3. WATER CONTACT ANGLE MEASUREMENT The water contact angle tests were performed using a 5 µL chromatographic syringe with a fixed needle and a support that ensured the slow and controlled approach of the drop to the surface of the specimen. The drops were 5 µL and three drops were deposited in different positions on the specimens while the procedure was filmed so that it would be possible to later measure the contact angle. This procedure was performed according to Han et al. [ 31 ], following the general guidelines of ASTM D7334 [ 32 ]. The angles were measured using ImageJ software, and the images were taken from the filming approximately 10 seconds after the drop was deposited. The angles were observed as exemplified in Fig. 3 , for the two internal angles at the left and right edges, registering the average for each contact angle measurement. Four measurements were carried in a sample of each condition, for calculation of average and standard deviation. 2.4. ROUGHNESS TEST For roughness measurements the parameters Ra, Rq and Rz were observed using a portable digital Mitutoyo SJ 201roughness meter with resolutions of 0.4 µm, 0.1 µm, 0.05 µm and 0.01 µm for a measurement range of 350 µm, 100 µm, 50 µm and 10 µm, respectively. Three specimens were tested per condition following the general recommendations of ISO 4287 [ 33 ] and ISO 4288 [ 34 ] standards, with three measurements being performed in different positions for each specimen. In addition, a cutoff of 2.5 mm was used, since according to the standard, this value is the most suitable for surfaces with Ra from 2 to 10 µm, resulting in an evaluation length of 12.5 mm. 2.5. FLEXURAL TEST For these bending tests, four specimens of each condition were evaluated using an equipment developed at the Laboratory of Education and Research in Machining (LEPU) in previous works, which follows the requirements of ASTM D6272 (ASTM) standard. The flexural testing machine had 4 parallel load cells with a resolution of 0.05 g and a capacity of 50 kg each, totaling a maximum load of 200 kg. The load cells, with an accuracy of ± 0.1% of the maximum load according to the manufacturer (resulting in ± 200 g for this equipment), were connected to HX711 modules linked to an Arduino Uno for amplification and signal conversion. With the results of the bending tests, data on maximum flexural strength (MFS), modulus of elasticity (E) and flexural elongation (Ɛ) were obtained. To calculate the modulus of elasticity, which has different possibilities according to the standard, the method used considers the origin at 0 and the maximum load point, as can be seen in the example in Fig. 4, which shows the graphs for specimens of conditions B and G, with the points used to calculate the elongation being those highlighted by black dots. 2.6. IMPACT TEST The Izod impact tests were performed according to ASTM D4508 (ASTM) to observe the difference in energy absorbed by the samples. The specimens used were sectioned pieces with length, height and width of 19 mm, 12.7 mm and 3.2 mm respectively. In this test, four specimens were tested per condition, and the pendulum was released at an angle of 0° in relation to the horizontal, reaching a speed of 5.6 m/s when hitting the sample with an energy of 3.8 J. The pendulum used had a length of 0.778 m (distance from the tip to the center of rotation), with a total mass of 0.963 kg and a center of mass at 0.159 m from the center of rotation. The impact testing machine used was adapted from the work of Tsuruta [ 63 ], and the measurement of the energy absorption by the sample was performed by an encoder-type sensor fixed to the pendulum shaft, which measured the maximum angle reached by the pendulum. The encoder consisted of a 220 mm disk with 360 holes 1 mm wide and spaced 1 mm apart, positioned on the edge of the disk together with an LM393 module, fixed to the pendulum support, which was responsible for reading the movement. 2.7. COATING LAYER THICKNESS AND CHEMICAL ANALYSIS For layer thickness analysis, four samples of each condition were embedded in a disk resin, sanded up to 2000 grit sandpaper, polished with 10 µm chromium oxide particles, metallized with a 10 nm gold coating and taken to the same SEM microscopes for BSE (backscattered electron imaging) and EDS analysis. One photo of each of the four samples in the same disk was taken in BSE mode to analyze the cross-section of the original specimen, making possible to measure the layer thickness using the ImageJ software as shown in Fig. 5 , and calculating the average of the layer thickness in 3 different regions of each image. 2.8. MICROHARDNESS ANALYSIS After examination in the SEM, the samples underwent microhardness testing. To prevent interference, microhardness tests were conducted away from SEM-analyzed areas, as prolonged exposure to the electron beam can degrade the surface. The tests followed ASTM E384 standards, utilizing an HV0.2 scale (1.961 N load), with a 15-second load application and 10x magnification. Measurements were taken across the sample’s cross-section, starting at the surface and extending toward the center, with approximately 300 µm spacing between indentations. Each major face of two samples from every annealing condition (A to H) was tested, resulting in four measurements per condition. This approach aimed to assess hardness variations from the coating to the substrate's inner regions. 2.9. BIOCOMPATIBILITY TESTS AND SEM ANALYSIS Biocompatibility of the disks cut from samples was evaluated by cytotoxicity assay, using the Alamar blue assay, in which MC3T3-E1 cells, (ECACC 99072810), which are pre-osteblast derived from mouse calvaria, were cultured in α-MEM 10% FBS medium, after reaching 80% confluence, were plated at a seeding density of 1x10 4 cells on the surface of each disk placed on the bottom of 48 well plate. The plates were incubated overnight at 37°C in a humidified atmosphere with 5% CO2 to allow the cells to adhere to the disks. As a positive control, cells attached to the bottom of the plate, without disks and as negative control, cells were cultivated with medium containing DMSO 5%. After 24 hour, 7 days or 14 days, 20 µL of PBS containing resazurin (7-Hydroxy-3H-phenoxazin-3-one-10-oxide sodium salt) (Sigma-Aldrich, São Paulo, Brazil) at a concentration of 0.3µM was added to each well. The plate was incubated for 4 hours protected from light and then the supernatant was transfered to an appropriate plate for fluorescence Reading, which was measured using a VICTOR NivoTM Plate Reader, at 560 nm excitation and 600 nm emission. For analysis, the absorbance of the samples containing only α-MEM 10% FBS medium (no cells) were subtracted from the samples. The statistical analysis was carried by two-way ANOVA with Tukey's post-test (for multiple comparisons) considering a 95% confidence interval for proliferation analysis with the individual viability after 1, 7 and 14 days. After 14 days, the wells were washed 3 times in Phosphate Buffered Saline (PBS) 1X and then fixed in 3.2% formaldehyde for 1 hour at room temperature. They were rinsed once more in PBS 1X and gradually dehydrated from ethyl alcohol diluted in distilled water, in concentrations rising from 50–100% in 10% increments, for 10 minutes in each, and finally stored in absolute alcohol at 4˚C for 1 day. Then they were dried in vacuum for 12 hours and metallized with a 10 nm gold coating to be analyzed by SEM. 3. RESULTS 3.1. POWDER MATERIAL CHARACTERIZATION The XRD spectrum of calcium phosphate can be seen in Fig. 6 , in which the peaks at 25.9°; 31.74°; 32.16°; and 32.88° are indicative of the hydroxyapatite (HA) crystal structure according to Hu et al. (2020), differentiating it from other structures such as TCP (tri-calcium phosphate) and BCP (bi-calcium phosphate), commonly obtained in calcium phosphate synthesis reactions. In calcium phosphate with the HA structure, with molecular formula Ca 5 (PO 4 ) 3 OH, the OH groups appear trapped in the crystal structure, and several authors (Zhang, 2013; Sossa et al., 2018; de Melo Costa et al., 2009) have already shown that its lower solubility and greater stability in the body make it the preferred structure for implant coating. Regarding TiO 2 , as shown by El-Sherbiny et al. [ 35 ] and Ijadpanah-Saravy et al. [ 36 ], the peaks at 27.44°; 36.1°; 41.28°; and 56.66° are typical of the rutile structure and differentiate it from anatase and brookite, the other possible crystalline forms. Rutile is in fact the most stable in crystals larger than 35 nm [ 37 ] and is the most commonly obtained structure in the industrial production processes of titanium dioxide, with its biological activity being better than that of the amorphous form obtained in some electrochemical processes [ 38 ]. The narrow bands indicate high crystallinity of the material used, in addition to uniformity of crystal size The shape and size of the Ca 5 (PO 4 ) 3 OH particles can be seen in Fig. 7. The crystals are predominantly rod-shaped with a thickness of around 200 nm. Despite the high surface area, the calcium phosphate powder appears to be less adherent than titanium dioxide, being easier to handle and to clean, so it is possible that the formation of large agglomerates reduces the surface energy of this material. The chemical composition of this calcium phosphate deposited on carbon tape and coated in gold is shown in Fig. 8, which also shows a mass content of Ca of around 10% and around 29% of C. This is because the C content and part of the O content comes from the carbon tape substrate, since the EDS rays comes from regions up to 2 µm deep in the sample, and for this analysis the powder was thinly spread on the tape. For the same reason, although the Ca 5 (PO 4 ) 3 OH from hydroxyapatite structure leads to an expected mass percentage in the proportion of 40% : 19% : 41% for Ca : P : O, the lower mass of Ca and P in comparison with O comes from the oxygen present in the polymeric carbon tape. The size and shape of the TiO 2 particulates can be seen in Fig. 9 . It is noticeable the approximately spherical or ellipsoidal shape of these particulates, which agglomerate to form larger grains, as also observed by Hu et al. (2014). Due to the submicrometric scale (with particulates of about 400 nm), the material has a high surface/volume ratio, and therefore adheres easily to the substrate, even when not annealed. Their chemical composition can be seen in Fig. 10, where a variation in the mass percentages of the elements, including carbon, can be noted. In this case, the C content comes from the polymeric substrate and may be higher or lower depending on the thickness of the TiO 2 layer at each point. 3.2. WATER CONTACT ANGLE The water contact angle is shown in Fig. 11 for each annealing condition, for non-annealed samples and for the coated surfaces of the femoral and acetabular components of a commercial hip implant, demonstrated in Fig. 12 . In all cases, except for specimens E and F, the contact angles were smaller than the non-annealed state and much smaller than 90°, which indicates an improvement in surface hydrophilicity [ 39 ]. In fact, together with a high roughness, the contact angles of less than 30° obtained can lead to a superwetting surface as defined by [ 40 ], which is a good predictor for the biocompatibility of the material [ 41 ]. Also, there were annealing conditions in which the wettability of the 3D printed PLA samples was even better than that obtained for the commercial hip implant, highlighting the potential of this new biomaterial as an inductor for cell adhesion and differentiation [ 42 , 43 ]. Analysis of variance (ANOVA) was performed to assess how the annealing conditions affect the water contact angle, from which the most significant parameters were composition (p = 0,000000), time in maximum temperature (p = 0,000009) and the combination of them (p = 0,000001). Calcium phosphate is a common anti-humectant in the food industry due to its affinity with water, preventing the food from absorbing moisture. Therefore, it is possibly for this reason that it presents greater hydrophilicity. Titanium dioxide covered the surface irregularly, leading to smaller wetting angles on average and greater variability in the results, as in conditions E and F. Regarding temperature, it may not have been significant because at both 170°C and 190°C the polymer assumes a similar level of viscosity. The time at maximum temperature may have been more significant because it provided more time for the diffusion of particulates on the surface, whereas in the case of titanium dioxide, this led to a more irregular surface observed visually and by SEM (Fig. 13a and Fig. 13b). In these figures, the white/gray lines to the left of each layer are just flaws resulted from the embedding, sanding and polishing processes. 3.3. ROUGHNESS TEST The roughness parameters Ra, Rz and Rq were measured and are shown in Fig. 14 , from which there is notable difference between the values when changing the chemical composition of the powder bed. According to ANOVA, the chemical composition was in fact significant for a confidence level of 95% for Ra (p = 0,000000), Rq (p = 0,000446) and Rz (p = 0,000000). This greater roughness can also be visually perceived in the test specimens annealed in the mixture of titanium dioxide and calcium phosphate, with the surface of the test specimen having a very rough finish as if the molten PLA was being absorbed by the powder bed. Figure 15 shows two samples annealed in calcium phosphate (type C and D) and two in titanium dioxide with calcium phosphate (type E and H), illustrating the observed results. Comparatively, the roughness values for conditions A to D are close to those observed for the femoral component of the same commercial hip implant of Fig. 12 a, measured in another previous work [ 44 ] with Ra ranging from 4.7 µm to 5.8 µm and Rq from 6 µm to 7.6 µm. This indicate that the surface of the annealed 3D printed PLA parts bear similarity with that of biomaterials already validated for commercial use, which is also a good indicator of the potential of the obtained surfaces. 3.4. FLEXURAL TESTS The results of maximum flexural strength (MFS), modulus of elasticity (E) and flexural elongation (Ɛ) are shown in Fig. 16 . It shows that powder bed annealing resulted in a decrease in elongation as well as an increase in flexural strength and modulus of elasticity. Considering that the coating is very thin in comparison to the bulk material, the mechanical properties will depend almost entirely on the characteristics of the bulk PLA. As the process reaches temperatures above 160°C for which PLA already starts to melt, the changes observed in MFS, E and Ɛ could come from an alteration in the PLA chemical composition, or in its porosity, or in its crystallinity, as explained in other works [ 67 ]. Although neither of these properties were measured in this work, previous works have shown that that the most important factors the justify increases in mechanical resistance are the modification of pores geometry [ 69 ] and polymer crystallization [ 68 ]. For elongation and maximum flexural strength, the influence of composition (p = 0,011332 and p = 0,002948, respectively) and time at maximum temperature (p = 0,001278 and p = 0,015807, respectively) were significant with a 95% confidence level. This can be explained because more crystalline parts tend to be more rigid and have greater mechanical resistance, if they have not become brittle [ 45 ]. This occurs because in the amorphous state the macromolecules are randomly entangled, with a greater possibility of movement when subjected to stresses, while in the crystalline state the macromolecules are rigidly packed, with a lower capacity to stretch and deform [ 46 ]. Above the glass transition (Tg), thermal energy generates the vibration of the macromolecules, which have greater freedom to rotate and move, with a natural tendency to crystallize if there is enough time, given that the crystalline state is the state with the lowest free energy [ 46 ]. Thus, the thermal history of printing and annealing influences the final mechanical properties of the test specimens, explaining the observed variations. Moreover, the particulate alters the regularity and presence of surface pores, which affect the nucleation and propagation of cracks, as in the case of Fig. 15 . Thus, due to the stress concentration, composition was also a preponderant factor for the fracture of the samples. These results show that the powder bed annealing process was able not only to create the coating on the PLA surface, but also to improve its resistance, both in comparison to the non-annealed PLA and to PLA flexural resistance values from the literature, which vary from about 60 MPa to 100 Mpa [ 47 , 48 ]. This further supports the attractiveness of this new manufacturing process for PLA coatings, because besides it being simpler and cheaper in comparison to cold gas shooting, plasma spray and other coating processes [ 49 , 50 ], it can even improve the substrate mechanical properties. It is also important to highlight that as can be seen from Fig. 15 , even after the fracture of the samples during the bending tests, there was no delamination of the coating from the bulk material. Although no direct measurement of the adhesion of the coating was carried out, this aspect serves as an indirect evaluation of coating integrity as exemplified in ASTM B571 [ 70 ], since there was no visible failure in any of the specimens. 3.5. IMPACT TESTS For the impact tests, the graph from Fig. 17 shows that there is no explicit trend in the results as a function of the input parameters, which is reinforced by no significance from any factors in ANOVA. Comparing the results obtained with values from other studies [ 51 ], which presented results close to 50 J/m for parts produced from PLA by 3D printing, it is observed that powder bed annealing did not impair the impact resistance of the substrate. Again, given the relatively thin coating, the results of the impact test might be influenced primarily by the bulk properties of the PLA, leading to similar impact energy values across all specimens. This indicates that the material conserves its toughness after the surface modification process, enabling it for use in the production of biomaterials. 3.6. COATING LAYER THICKNESS AND CHEMICAL ANALYSIS The results of the coating layer thickness measured in the SEM are presented in Fig. 18 and it shows that pure calcium phosphate composition (p = 0,000019) and a longer time at maximum temperature (p = 0,000000) ensured better layer thickness results, whereas both parameters together with the combination (p = 0,023406) of time in maximum temperature and maximum temperature were significant for a 95% of confidence level. A possible explanation for the influence of the particulate is the fact that, although it cannot be perceived from the images, for calcium phosphate the particulate is visually less adhered when handled, which may make it easier for the particulate to diffuse into the molten polymer more homogeneously, forming a new composite material from the mixture of the particulate and PLA. Likewise, a longer time at maximum temperature allows more diffusion to occur. It is recommended that the coating layer formed should not be too small, since it can be absorbed by the bone tissue very quickly in the first few weeks after implantation, but it should also not be too thick that could make the surface fragile. Thus, an ideal thickness is considered by some authors [ 52 ] to be between 30 µm and 80 µm, while others have found it to be between 70 µm to 90 µm [ 53 ]. Therefore, it can be observed that the results obtained in this study could be satisfactory for biomaterial fabrication, since most of the results are between 30 µm and 50 µm. Regarding the chemical composition, it can be inferred that the coating is a composite of calcium phosphate grains interspersed with PLA for samples A to D. In Fig. 19a the BSE image shows that the aligned denser white grains have infiltrated the gray PLA substrate (to the right) during annealing, and the composition maps of Fig. 19b show that there is a well-defined region where the calcium phosphate was deposited, and in this region, there is also carbon from the PLA substrate. The same is true for samples E to H, for which the coating is a composite of calcium phosphate and titanium dioxide grains interspersed with PLA, as shown in Fig. 19c and Fig. 19d. Regarding the applicability to implants, it is expected that the composite coating might have an even better behavior than only pure calcium phosphate coating or pure PLA because, as explained in the work of Bernardo et al. [ 22 ], the PLA component may offer structural support for cell attachment and growth, while the HA phase releases calcium and phosphate ions that both promote osteoinduction and increase the low pH created by PLA degradation in lactic acid. Besides, the use of composite parts of PLA and calcium phosphate [ 54 , 55 ] and PLA and titanium dioxide [ 56 ] have already shown biocompatibility characteristics, validating this new coating method by power bed annealing as an interesting manufacturing method for biomaterials, especially in the case of interference screws, for which this composite mix has been shown to have good results in vitro [ 57 , 58 ] and in vivo [ 59 ] and is now widely used in commercial products [ 25 ]. 3.7. MICROHARDNESS ANALYSIS The microhardness was measured in the cross section of the samples, starting on the coating and moving towards the bulk, with each indentation spaced about 300 µm from the other. As can be seen from the plots in Fig. 20, there is not much difference of the microhardness on the sample section, with major differences only being observed very close the surface. The significance analysis showed that the input parameters had no influence on the microhardness in the cross section. Regarding the large variation in hardness observed in the coating region, the most likely explanation for it is the fact that it borders the embedding resin, which is against the recommendations from ASTM E384 [ 60 ]. Therefore, it can be inferred that there is no significant change of the mechanical properties of the substrate at microscale, indicating that this coating process does not damage the substrate’s resistance. 3.8. BIOCOMPATIBILITY TEST AND SEM ANALYSIS Cytotoxicity tests were performed only in 3 of the 8 production conditions of the coated samples, also comparing them with pure PLA and negative and positive control materials, as shown in Fig. 21 . Although in the first day the samples showed worse cell viability, after 14 days all discs showed greater cell growth than in the positive control, with the calcium phosphate and titanium dioxide coating of sample H performing better. Still, not even for the first day the material can be considered cytotoxic, since the cell viability was above the limit of 70% [ 61 ]. This indicates that the material, in addition to being non-toxic to cells, inducing possible adhesion and proliferation on the surface of the samples, which is an excellent indication that these biomaterials could become an option for new models of orthopedic implants in the future. Interestingly, in Fig. 22a the MC3T3 cell can be seen well attached on the calcium phosphate grains, with its cell process spreading on the surface of sample B. Similarly, other cells can also be seen in the surface of sample B in Fig. 22b, which has a very different aspect from pure PLA surface, for which an isolated cell can be seen in Fig. 22c. In this case, even after 14 days, although the cell attached to the surface, its ovoid morphology suggests it did not differentiate into a mature cell [ 62 ] with the typical spreading process, exactly because this sample lacked the calcium phosphate and/or titanium dioxide that promote the osteogenic stimuli. In contrast, Fig. 22d shows even more osteocytes spread over the surface from sample H, which presented the best cell viability after 14 days. 3.9. COMPARISON AGAINST OTHER COATING TECHNOLOGIES FOR POLYMERIC IMPLANTS As discussed, the integration of PLA and HA into implants is well stablished in the literature [ 71 , 73 , 88 ], given the resorbable nature of the former and the osseointegration properties of the later. Therefore, the proposed coating method should be compared to other technologies capable of combining these components for the same goal. This could be achieved either by methods that create a HA coating on PLA or that mix HA into PLA matrix. Considering the first, there have been studies with plasma spray for coating polymers such as PEEK (poly-ether-ether-ketone) [ 72 ]. However, considering the high temperatures reached in the plasma arc of about 10 4 K [ 76 ], it would most probably not be suitable for coating PLA, which has a melting point of about 160°C compared to about 340°C for PEEK. Besides, other works [ 74 ] have shown that plasma spray deposited HA may present low adhesion with polymer, with failure of the coating under load before the failure of the substrate. Additionally, plasma spray high temperatures might cause HA to transform into α or β tricalcium phosphate, calcium oxide and/or tetracalcium phosphate, impairing not only the crystallinity [ 77 ] and chemical composition of the coating, but also its effectiveness for osseointegration [ 75 ]. Flame spray process is a variation that also could potentially deposit ceramic coatings [ 78 ] but would suffer from the same drawbacks as plasma spray for PLA as the substrate material. Interestingly, some authors have already been able to deposit titanium dioxide onto low melting temperature polymers such as UHMWPE [ 79 ] using vacuum plasma spray, but no other work was found with such feature for PLA. Also, all plasma spray techniques require expensive tooling such as a plasma torch, powder feeder and power supplies with capacity of 25 to 150 kW [ 76 ], besides also requiring expensive materials sometimes [ 80 ], such as pure nitrogen, argon or helium that act as carrier gases and stabilize the plasma arc. Although not less expensive due to the necessary costly equipment [ 81 ], a potential alternative would be the use of CGS (cold gas shooting), in which micrometric particles accelerated to supersonic speeds in a high-speed gas stream impact and adhere to a substrate, creating a distinct coating [ 75 ]. However, almost 90% of the kinetic energy of the particles is converted into heat, and although there have not been found works with CGS onto polymers, it is likely that it would face the same problems related to excessive heat damaging the substrate. Moreover, it is important to highlight that CGS and all thermal spray are line of sight techniques, and coating the totality of a complex geometry would be more time consuming, even with a robotic arm. In comparison, the powder bed annealing method hereby proposed can create a uniform coating around all the part, even for more complex geometries. Also, it is a much more inexpensive and scalable method, because it only requires an electrical oven (about US $ 30) and a metallic tray as tooling (about US $ 5), and only about 100 g of HA powder (bought as additive for food industry for about US $ 4/kg) and 16 g of PLA (natural PLA bought at US $ 20/kg) to produce four samples at each condition. This make the proposed technology especially relevant for developing countries where access to expensive biomedical products is still a health issue. The only drawback of the powder bed annealing process is that it is not able to generate a pure HA and/or HA + TiO 2 coating in comparison to other techniques. However, the biocompatibility tests showed that phenomenon does not pose a problem, since the coating is still not cytotoxic and leads to cell differentiation. Regarding other coating techniques, as explained in other works [ 82 , 83 ], despite the lower temperature of sputtering process, it does not create coatings of more than 3 µm, while sol-gel would be limited to 10 µm and may also require deleterious heating of the part to solidify the coating [ 84 ]. The powder bed annealing, besides producing coatings of up to 50 µm in thickness, applies heat with the part immersed in powder, which avoids heat deflection and maintains the geometry. Electrodeposition methods [ 85 ], on the other hand, cannot be applied to polymers, which are natural electrical insulators. Finally, in relation to process the mix HA into the PLA matrix, two main processes can be highlighted. The first is injection molding, which has been used successfully to incorporate HA into PLA uniformly [ 86 ]. Similarly, these authors also found that annealing the part could enhance the impact resistance as happened for most samples produced by powder bed annealing. However, mixing even small quantities of HA into the whole substrate has been found to decrease the impact [ 87 ] and tensile resistance [ 92 ] in comparison to pure PLA. Here lies another advantage of powder bed annealing, since except for conditions A and H, all others yield samples with average impact resistance above that of natural PLA used in the tests. The second process to incorporate HA into PLA is additive manufacturing, with several examples of composite filaments being produced for later use in FFF process. However, it has been found that concentrations as low as 10% of HA dispersed into PLA could be prejudicial to its mechanical properties, with its tensile resistance dropping from 50 MPa to 30 MPa [ 90 ]. This phenomenon was not observed in the flexural tests carried out with powder bed annealed parts, where the resistance was the same or higher than for natural PLA, while also achieving very low values of water contact angle (mostly less than 30°). This level of hydrophilicity did not happen in other works [ 91 ] that used PLA filament with 12% of HA for which water contact angles stayed at 66°, indicating once more the advantage of the powder bed annealing process as it conserves the toughness and resistance of the PLA substrate while presenting the improved biocompatible properties of HA at the surface. Furthermore, incorporating HA to PLA filaments in a uniform dispersion is still a challenge [ 93 ] since simple mechanical stirring or high-shear mixing may not suffice, while for other additive manufacturing methods such as SLS, that is also not easy due to a small sintering window and flowability of the heterogeneous powder [ 94 ]. It is also important to highlight that conserving a PLA core would be advantageous over creating pure HA parts, due to the inherent low fracture toughness of pure HA manufactured by SLS [ 89 ]. 4. CONCLUSIONS This study demonstrated the viability of using powder bed annealing as an innovative method for producing hydroxyapatite coatings on 3D-printed PLA parts. The process successfully generated a ceramic particulate layer with favorable characteristics for biomedical applications, particularly in orthopedic implants. The results showed that the coating exhibited good thickness control, improved surface roughness, and enhanced hydrophilicity, which are crucial factors for promoting osseointegration. Mechanical tests indicated that powder bed annealing enhanced the flexural strength of the PLA parts while maintaining impact resistance and microhardness in the substrate. Although ductility decreased, the mechanical improvements suggest that this method can contribute to stronger and more reliable implant materials. Additionally, biocompatibility tests confirmed that the hydroxyapatite-coated surfaces supported cell adhesion and proliferation, further validating the potential of this technique for biomedical applications. Compared to conventional coating methods like plasma spraying or chemical deposition, powder bed annealing could offer a cost-effective and scalable alternative. The ability to integrate hydroxyapatite coatings directly onto polymeric implants without complex post-processing steps highlights the practicality of this approach. Furthermore, the combination of PLA’s biodegradability with hydroxyapatite’s osteoinductive properties suggests promising applications in resorbable implants. Overall, this research contributes to the ongoing development of personalized, bioactive implants by optimizing a simple yet effective coating technique. Future work should explore long-term in vivo performance, refine processing parameters for improved adhesion and uniformity, and investigate alternative bioceramic compositions for tailored implant applications. The findings suggest that powder bed annealing could play a crucial role in advancing polymer-based implant technologies, offering a practical pathway to more accessible and efficient orthopedic solutions. Declarations Author Contribution F.A.R.C. and T.O.S. wrote the main manuscript text. K.F.A. and J.V.R.A. prepared Figures 1–3 and conducted the main experiments. L.C.P. and F.C.R.S. performed data analysis and contributed to the preparation of Figures 4–7. L.S.C.F. assisted with literature review and manuscript editing. L.R.R.S. conceived the study, supervised the project, and finalized the manuscript. Á.R.M. contributed to experimental design and provided critical revisions. All authors reviewed and approved the final manuscript. ACKNOWLEDGMENTS The authors would like to thank the support of the Brazilian Association of Engineering and Mechanical Sciences (ABCM), the Brazilian National Council for Scientific and Technological Development (CNPq), Coordination for the Improvement of Higher Education Personnel (CAPES), Minas Gerais State Research Support Foundation (FAPEMIG), Laboratory of Education and Research in Machining of Federal University of Uberlandia (LEPU-UFU), Laboratory of Tribology and Materials (LTM-UFU), Laboratory of Nanobiotechnology (NANOS-UFU), Faculty of Mechanical Engineering (FEMEC-UFU) and the Postgraduate Program in Mechanical Engineering (PPGEM-UFU). References Growing at a slower pace, world population is expected to reach 9.7 billion in 2050 and could peak at nearly 11 billion around 2100 | UN DESA | United Nations Department of Economic and Social Affairs. https://www.un.org/development/desa/en/news/population/world-population-prospects-2019.html. 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C., Gilchrist, F., & Miller, C. (2020). Characterization of a composite polylactic acid-hydroxyapatite 3D-printing filament for bone-regeneration. Biomedical physics & engineering express , 6 (2), 025007. Wu, C. S., Wang, S. S., Wu, D. Y., & Shih, W. L. (2021). Novel composite 3D-printed filament made from fish scale-derived hydroxyapatite, eggshell and polylactic acid via a fused fabrication approach. Additive Manufacturing , 46 , 102169. Marzuki, A. P., Mohd Salleh, F., Rosli, M. N. S., Tharazi, I., Abdullah, A. H., & Abdul Halim, N. H. (2022). Rheological, mechanical and physical properties of poly-lactic acid (PLA)/hydroxyapatites (HA) composites prepared by an injection moulding process. Journal of Mechanical Engineering (JMechE) , 19 (2), 17-40. Omigbodun, F. T., Oladapo, B. I., & Osa-uwagboe, N. (2024). Exploring the frontier of Polylactic Acid/Hydroxyapatite composites in bone regeneration and their revolutionary biomedical applications–A review. Journal of Reinforced Plastics and Composites , 07316844241278045. Schappo, H., Giry, K., Salmoria, G., Damia, C., & Hotza, D. (2023). Polymer/calcium phosphate biocomposites manufactured by selective laser sintering: an overview. Progress in Additive Manufacturing , 8 (2), 285-301. Additional Declarations No competing interests reported. Supplementary Files graphicalabstract.pptx 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. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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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-6483704","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":452789408,"identity":"d94d9b4b-a04d-44f7-a791-4792d7efaa5b","order_by":0,"name":"Felipe dos Anjos Rodrigues Campos","email":"","orcid":"","institution":"Federal University of Uberlândia","correspondingAuthor":false,"prefix":"","firstName":"Felipe","middleName":"dos Anjos Rodrigues","lastName":"Campos","suffix":""},{"id":452789410,"identity":"1a857059-fa12-40e2-b602-17739e719ea0","order_by":1,"name":"Thiago de Oliveira Santos","email":"","orcid":"","institution":"Federal University of Uberlândia","correspondingAuthor":false,"prefix":"","firstName":"Thiago","middleName":"de Oliveira","lastName":"Santos","suffix":""},{"id":452789411,"identity":"2ed1e9d8-5dde-4e2c-a512-97349525a89b","order_by":2,"name":"Kauã Ferreira de Almeida","email":"","orcid":"","institution":"Federal University of Uberlândia","correspondingAuthor":false,"prefix":"","firstName":"Kauã","middleName":"Ferreira","lastName":"de Almeida","suffix":""},{"id":452789412,"identity":"9631e290-0102-4f03-88a5-2bf72b395431","order_by":3,"name":"João Victor Rezende Amaro","email":"","orcid":"","institution":"Federal University of Uberlândia","correspondingAuthor":false,"prefix":"","firstName":"João","middleName":"Victor Rezende","lastName":"Amaro","suffix":""},{"id":452789415,"identity":"529d3e64-fda1-4ef0-b29c-0f982a54ddaa","order_by":4,"name":"Lucas Correia Peres","email":"","orcid":"","institution":"Federal University of Uberlândia","correspondingAuthor":false,"prefix":"","firstName":"Lucas","middleName":"Correia","lastName":"Peres","suffix":""},{"id":452789416,"identity":"c011f24a-56b3-4712-8d1a-44aa7058cad7","order_by":5,"name":"Felipe Chagas Rodrigues de Sousa","email":"","orcid":"","institution":"Federal University of Uberlândia","correspondingAuthor":false,"prefix":"","firstName":"Felipe","middleName":"Chagas Rodrigues","lastName":"de Sousa","suffix":""},{"id":452789418,"identity":"eb3c1550-0d55-4b3f-a56c-5718c2e90f8b","order_by":6,"name":"Letícia de Souza Castro-Filice","email":"","orcid":"","institution":"Federal University of Uberlândia","correspondingAuthor":false,"prefix":"","firstName":"Letícia","middleName":"de Souza","lastName":"Castro-Filice","suffix":""},{"id":452789421,"identity":"011da3e8-9f5d-4c67-a0ff-25dda5c9a8e5","order_by":7,"name":"Leonardo Rosa Ribeiro da Silva","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAmUlEQVRIiWNgGAWjYBACPghlw8DewMBGnBaosjQGngMkajlMihb25mOfK/6cT+yRSGB7XEGUFp5jyTPPtt0GaWE3PEOUFokcY8bGhtuJ+4G2SDYQpUX+jTFjw59zYIcRqUWCB6iF7QApWnjSkhkb25KNe3gethsSpYWf/fBhoMPsZHvYk489JEoLEmAkVcMoGAWjYBSMApwAAKz8K7b7JlwoAAAAAElFTkSuQmCC","orcid":"","institution":"Federal University of Uberlândia","correspondingAuthor":true,"prefix":"","firstName":"Leonardo","middleName":"Rosa Ribeiro da","lastName":"Silva","suffix":""},{"id":452789422,"identity":"3d88ddaa-b435-4657-849b-59a736c0f6f2","order_by":8,"name":"Álisson Rocha Machado","email":"","orcid":"","institution":"Pontifícia Universidade Católica do Paraná","correspondingAuthor":false,"prefix":"","firstName":"Álisson","middleName":"Rocha","lastName":"Machado","suffix":""}],"badges":[],"createdAt":"2025-04-19 09:08:09","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6483704/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6483704/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":82268688,"identity":"a98d06f5-4d03-4e51-b53a-ba105705fc77","added_by":"auto","created_at":"2025-05-08 13:48:27","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":378294,"visible":true,"origin":"","legend":"\u003cp\u003e(a) sample geometry. (b) Samples as printed. (c) Coated samples.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-6483704/v1/d293c3c4cd6a94b59ee46a03.png"},{"id":82268763,"identity":"1922b2f5-2223-4f5d-bf55-d35ade56dd17","added_by":"auto","created_at":"2025-05-08 13:48:30","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":334990,"visible":true,"origin":"","legend":"\u003cp\u003e(a) Representation of PLA samples positioned in the metallic tray (thermocouple indicated by yellow arrow). (b) Representation of tray with samples positioned in the electric oven. Although the powder is not shown in this representation, it is filling the tray to the top, where a plate covers the tray.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-6483704/v1/7ac0a67db9de00b9907bbed5.png"},{"id":82268758,"identity":"760ebc81-ba61-43de-b16b-ff640dce13a2","added_by":"auto","created_at":"2025-05-08 13:48:30","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":40791,"visible":true,"origin":"","legend":"\u003cp\u003eMeasurement of contact angle in accordance with ASTM D7334 [32].\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-6483704/v1/17596f09c46b64788dd99e2f.png"},{"id":82270546,"identity":"92d072ec-5d3b-4a45-9a03-b72ab65c42ae","added_by":"auto","created_at":"2025-05-08 14:04:30","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":49391,"visible":true,"origin":"","legend":"\u003cp\u003eStress-strain curve with points used for calculating the modulus of elasticity highlighted.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-6483704/v1/25667eb4581d4eaee8e626b9.png"},{"id":82268691,"identity":"565a57bf-8265-4cc9-910f-338680be432f","added_by":"auto","created_at":"2025-05-08 13:48:27","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":395557,"visible":true,"origin":"","legend":"\u003cp\u003eImage illustrating the procedure for measuring the thickness of the layer formed by the adhesion of particulate matter to a type A sample.\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-6483704/v1/29ae1a07c995c51e82688a16.png"},{"id":82269968,"identity":"5d331d25-d0e5-470e-a3d8-ad82bdb1bf42","added_by":"auto","created_at":"2025-05-08 13:56:28","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":55485,"visible":true,"origin":"","legend":"\u003cp\u003eX-ray diffraction for calcium phosphate and titanium dioxide with angle 2θ from 5 to 70°.\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-6483704/v1/318f90fe7b1b9b931924d9dc.png"},{"id":82268695,"identity":"eb10b062-3b57-41fe-bba2-70e72f50f83a","added_by":"auto","created_at":"2025-05-08 13:48:27","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":524794,"visible":true,"origin":"","legend":"\u003cp\u003eShape and size of Ca\u003csub\u003e5\u003c/sub\u003e(PO\u003csub\u003e4\u003c/sub\u003e)\u003csub\u003e3\u003c/sub\u003eOH particles at (a) 30,000x and (b) 10,000x magnifications.\u003c/p\u003e","description":"","filename":"7.png","url":"https://assets-eu.researchsquare.com/files/rs-6483704/v1/725547cdab5546d5aa7872ca.png"},{"id":82271898,"identity":"14b233af-42c7-4c6e-b390-345801a89ca0","added_by":"auto","created_at":"2025-05-08 14:20:30","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":295867,"visible":true,"origin":"","legend":"\u003cp\u003eChemical composition of calcium phosphate particles deposited on carbon tape and coated with Au.\u003c/p\u003e","description":"","filename":"8.png","url":"https://assets-eu.researchsquare.com/files/rs-6483704/v1/095d4b232d8b4a40e9cbf476.png"},{"id":82268710,"identity":"f10d4205-cc25-41bf-8fe1-1bd412a66f76","added_by":"auto","created_at":"2025-05-08 13:48:28","extension":"png","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":882094,"visible":true,"origin":"","legend":"\u003cp\u003eShape and size of TiO\u003csub\u003e2\u003c/sub\u003e crystals that cluster into larger structures.\u003c/p\u003e","description":"","filename":"9.png","url":"https://assets-eu.researchsquare.com/files/rs-6483704/v1/13c78215ef5e9e5cfc1ad546.png"},{"id":82270533,"identity":"c0a3063b-e4c5-4e0c-b031-996458cee0f2","added_by":"auto","created_at":"2025-05-08 14:04:28","extension":"png","order_by":10,"title":"Figure 10","display":"","copyAsset":false,"role":"figure","size":251616,"visible":true,"origin":"","legend":"\u003cp\u003eChemical composition at different points of the TiO\u003csub\u003e2\u003c/sub\u003e coating on polyamide substrate (Campos et al., 2021), with region highlighted for magnification in Fig. 9.\u003c/p\u003e","description":"","filename":"10.png","url":"https://assets-eu.researchsquare.com/files/rs-6483704/v1/6f0cf7ab2655fe175bad4c0d.png"},{"id":82269964,"identity":"909dea13-e243-4358-a7dc-17cf26270951","added_by":"auto","created_at":"2025-05-08 13:56:27","extension":"png","order_by":11,"title":"Figure 11","display":"","copyAsset":false,"role":"figure","size":31072,"visible":true,"origin":"","legend":"\u003cp\u003eWetting angles for each condition and comparison to surface of commercial hip implant.\u003c/p\u003e","description":"","filename":"11.png","url":"https://assets-eu.researchsquare.com/files/rs-6483704/v1/6550811e53c2aec49c6eaa1a.png"},{"id":82268714,"identity":"c5be9c47-b0a5-4f97-88b5-d1b676d47492","added_by":"auto","created_at":"2025-05-08 13:48:28","extension":"png","order_by":12,"title":"Figure 12","display":"","copyAsset":false,"role":"figure","size":753709,"visible":true,"origin":"","legend":"\u003cp\u003eCoated surfaces of (a) femoral and (b) acetabular components of commercial hip implant.\u003c/p\u003e","description":"","filename":"12.png","url":"https://assets-eu.researchsquare.com/files/rs-6483704/v1/8f7794490fd4aa45612caa43.png"},{"id":82268699,"identity":"de8f187c-5d00-4a51-a6ef-125a2c368598","added_by":"auto","created_at":"2025-05-08 13:48:28","extension":"png","order_by":13,"title":"Figure 13","display":"","copyAsset":false,"role":"figure","size":456156,"visible":true,"origin":"","legend":"\u003cp\u003eImage obtained by SEM of the coating layer of a test specimen annealed in a) calcium phosphate and b) in a mixture of calcium phosphate and titanium dioxide.\u003c/p\u003e","description":"","filename":"13.png","url":"https://assets-eu.researchsquare.com/files/rs-6483704/v1/a3475a0b2adc098c015d8aa9.png"},{"id":82268748,"identity":"e1a1a594-943d-4489-b1b0-1ba4ff8e486a","added_by":"auto","created_at":"2025-05-08 13:48:29","extension":"png","order_by":14,"title":"Figure 14","display":"","copyAsset":false,"role":"figure","size":41229,"visible":true,"origin":"","legend":"\u003cp\u003eSurface roughness for all the conditions analyzed.\u003c/p\u003e","description":"","filename":"14.png","url":"https://assets-eu.researchsquare.com/files/rs-6483704/v1/c0bd86f691a9d380b989d680.png"},{"id":82270537,"identity":"e4fcf03e-0432-4067-b8c2-deea80bc64c5","added_by":"auto","created_at":"2025-05-08 14:04:28","extension":"png","order_by":15,"title":"Figure 15","display":"","copyAsset":false,"role":"figure","size":395625,"visible":true,"origin":"","legend":"\u003cp\u003ePhotograph of the surface of test specimens used in roughness tests (fractured pieces of the bending test). 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17","display":"","copyAsset":false,"role":"figure","size":25391,"visible":true,"origin":"","legend":"\u003cp\u003eAbsorbed energy data for all the conditions analyzed.\u003c/p\u003e","description":"","filename":"17.png","url":"https://assets-eu.researchsquare.com/files/rs-6483704/v1/fc4206d93e35db9dd3181daa.png"},{"id":82270541,"identity":"44811a68-54c6-4b6d-b518-a0d5f5fb6076","added_by":"auto","created_at":"2025-05-08 14:04:29","extension":"png","order_by":18,"title":"Figure 18","display":"","copyAsset":false,"role":"figure","size":31997,"visible":true,"origin":"","legend":"\u003cp\u003eLayer thickness of particulate coatings measured in the SEM, for all the annealing condition.\u003c/p\u003e","description":"","filename":"18.png","url":"https://assets-eu.researchsquare.com/files/rs-6483704/v1/b523da247831df31d333519a.png"},{"id":82268689,"identity":"4c46048b-a6e8-4943-8244-b450350200ce","added_by":"auto","created_at":"2025-05-08 13:48:27","extension":"png","order_by":19,"title":"Figure 19","display":"","copyAsset":false,"role":"figure","size":1499862,"visible":true,"origin":"","legend":"\u003cp\u003eSEM images for sample D in BSE mode (a) and EDS composition map (b), and for sample E in BSE mode (c) and EDS composition map (d).\u003c/p\u003e","description":"","filename":"19.png","url":"https://assets-eu.researchsquare.com/files/rs-6483704/v1/19dce34bffc84d910e161245.png"},{"id":82270007,"identity":"1b61d99c-bb44-42f2-b3d7-954b6e4e3a9c","added_by":"auto","created_at":"2025-05-08 13:56:31","extension":"png","order_by":20,"title":"Figure 20","display":"","copyAsset":false,"role":"figure","size":84392,"visible":true,"origin":"","legend":"\u003cp\u003eMicrohardness variation in the cross section of the sample for conditions (a) A to D and (b) E to H.\u003c/p\u003e","description":"","filename":"20.png","url":"https://assets-eu.researchsquare.com/files/rs-6483704/v1/ecd68e9a89681c75cd86f337.png"},{"id":82269972,"identity":"e8e66fad-d87f-4775-91e3-67f738f18821","added_by":"auto","created_at":"2025-05-08 13:56:28","extension":"png","order_by":21,"title":"Figure 21","display":"","copyAsset":false,"role":"figure","size":93631,"visible":true,"origin":"","legend":"\u003cp\u003eCell viability after 1, 7 and 14 days for different samples.\u003c/p\u003e","description":"","filename":"21.png","url":"https://assets-eu.researchsquare.com/files/rs-6483704/v1/d9a597b31114256737060083.png"},{"id":82268772,"identity":"60619b4c-e14f-450e-92d1-d17f3f87f35f","added_by":"auto","created_at":"2025-05-08 13:48:30","extension":"png","order_by":22,"title":"Figure 22","display":"","copyAsset":false,"role":"figure","size":596589,"visible":true,"origin":"","legend":"\u003cp\u003eSEM images of cell adhered in sample B (a), cells spread in the calcium phosphate grains of surface from sample B (b), adhered and not differentiated cell in pure PLA sample (c) and cells spread in the calcium phosphate grains of surface from sample D (d).\u003c/p\u003e","description":"","filename":"22.png","url":"https://assets-eu.researchsquare.com/files/rs-6483704/v1/b6bae0658436b38858603cec.png"},{"id":83363174,"identity":"f511b494-1950-43e2-a6bf-086efcdb5383","added_by":"auto","created_at":"2025-05-23 17:46:37","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":8722732,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6483704/v1/219569a7-0daa-4489-8ead-51efed097e24.pdf"},{"id":82268706,"identity":"e16784bd-b6fa-4b29-af19-f975d41e76c7","added_by":"auto","created_at":"2025-05-08 13:48:28","extension":"pptx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":3897484,"visible":true,"origin":"","legend":"","description":"","filename":"graphicalabstract.pptx","url":"https://assets-eu.researchsquare.com/files/rs-6483704/v1/3a2f0b485b4f5ad2d240710c.pptx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Production of hydroxyapatite coating on 3D printed PLA parts by powder bed annealing","fulltext":[{"header":"1. INTRODUCTION","content":"\u003cp\u003eWith the development of technology and medicine, together with the increase in the quality of life of the population in several countries, there has been a growing increase in the life expectancy of its inhabitants [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e], which has resulted in an aging of the world population [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. This creates new challenges related to old age, obesity and a sedentary lifestyle, such as osteoarthritis, which is characterized as a degeneration of the articular cartilage and simultaneous proliferation of bone tissue, cartilage and connective tissue [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e], and causes progressive symptoms of pain, limitations of joint movement, stiffness and muscle weakness [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThis situation has been accompanied by an increasing number of orthopedic problems and a greater demand for the use of implants. Therefore, it is extremely important to conduct research to develop technologies that enable the manufacturing of implants in a more financially accessible way and that use more practical manufacturing methods that offer greater capacity for customization according to the patient's characteristics. In this sense, many studies have been carried out on the feasibility of using polymers [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e], such as the successful implantation of a rib implant made of biocompatible Nylon produced by additive manufacturing [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Among the benefits of using biocompatible polymers, in addition to their much more affordable price than titanium, the most common material used in implants, it is also worth mentioning that some have satisfactory mechanical properties [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e] and an elasticity modulus closer to that of bone tissue, which prevents the effect of bone density loss due to an uneven distribution of load between the implant and bone, known as \u003cem\u003estress shielding\u003c/em\u003e [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e] Another major difference of some polymers, such as polylactic acid (PLA), is their resorbable characteristic, with their degradation occurring through hydrolysis and enzymatic activities [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e], which can be very useful for the production of small implants that can break down over time and be absorbed by the body while the bone tissue around them regenerates and takes their place.\u003c/p\u003e \u003cp\u003eEven though there is already a good variety of polymers with good biocompatibility, such as PLA, PCL and Nylon 12, there are studies showing that the compatibility presented by polymers can still be improved using methods for surface modification of the part, since the roughness of the implant has an influence on bone differentiation [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e], and that the use of calcium compounds and titanium dioxide in the form of a coating stimulates the regeneration of bone tissue [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eWith this interest in the use of polymers in the manufacture of implants, a manufacturing process that ended up benefiting and standing out was additive manufacturing, such as the \u003cem\u003eFused Filament Fabrication\u003c/em\u003e (FFF), which allows the rapid manufacture of polymeric parts with a high level of customization. However, this technology has as its main limitation its high anisotropy, due to layer-by-layer deposition, resulting in mechanical properties dependent on adhesion between layers in the direction perpendicular to the plane of the deposited layers. Thus, a possible method to solve this problem is to perform an annealing process, to coalesce the layers or even fuse them, making the part more homogeneous and reducing anisotropy. Another possible benefit of annealing is the change in the organizational state of the molecules in semicrystalline polymers, such as PLA. In these cases, their permanence at temperatures above the glass transition temperature favors an increase in crystallinity, which can improve their mechanical properties [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eMany works have attempted to improve 3D printed parts mechanical properties by annealing [\u003cspan additionalcitationids=\"CR16 CR17\" citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e], and although the results were very promising with increases in mechanical properties up to 100%, the process could deform the geometry of the parts [\u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e66\u003c/span\u003e]. However, it has been found that immersing the parts in powder and compacting it could form a mold that would maintain the geometry during annealing and/or remelting [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. As explained in other works [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e], the remelting might cause particles from the powder to adhere in the surface, but they could be easily cleaned after by sanding. With this phenomenon in mind, it was hypothesized in this work that this particle adherence could be used to generate a coating in an innovative, simple and inexpensive way, by immersing the 3D printed PLA part in a powder bed and conducing the annealing and/or remelting process. This could in theory be used not only as a new method for depositing calcium phosphate and titanium dioxide compounds in the surface of PLA, but also as a method to improve the 3D printed parts mechanical properties, potentially setting up a new way to manufacture orthopedic implants.\u003c/p\u003e \u003cp\u003eThus, this work aimed to use the powder bed annealing process to generate a ceramic particulate coating on PLA parts manufactured by the FFF process. The project consisted of heating and remelting the part immersed in a tray of submicrometric ceramic particulates and then analyzing the influence of the thermal process parameters in the mechanical resistance of the parts and in the formation of the calcium phosphate and titanium dioxide coating, both materials with recognized capabilities of inducing osseointegration in orthopedic implants [\u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e64\u003c/span\u003e, \u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e65\u003c/span\u003e] and widely used for coating commercial implants. The innovative nature of the research stands out, because although annealing in particulate matrix has already been reported in the literature for improving surface roughness and mechanical properties, the possibility of generating a biocompatible coating had not yet been explored. These studies hereby presented may result in reduced manufacturing costs for customized parts for orthopedic implants, contributing to the general health of the population.\u003c/p\u003e"},{"header":"2. MATERIALS \u0026 METHODS","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\n \u003ch2\u003e2.1. PRODUCTION AND PREPARATION OF TEST SPECIMENS\u003c/h2\u003e\n \u003cp\u003eSeveral pre-tests were performed to verify the possibilities and suitable conditions for generating a coating through the powder bed annealing process, in which a significant change in the physical and mechanical appearance of the samples was observed, through visual inspection and flexural tests. For instance, it was found that annealing below 170\u0026deg;C yielded no visible coating, while above 190\u0026deg;C the sample would suffer significant geometrical deformation after annealing. Besides, as both calcium phosphate and titanium dioxide coatings favor osteointegration of implants as mentioned above, it would be desirable to verify in cytologic tests if there was a better component for the proposed coating method. However, it was also verified that annealing in pure titanium dioxide powder led to severe porosity in the samples, while there was no such disadvantage when using calcium phosphate. For this reason, titanium dioxide could only be tested when mixed with calcium phosphate in these pre-tests.\u003c/p\u003e\n \u003cp\u003eThus, as defined in preliminary studies, test samples were prepared in accordance with ASTM D6272 [\u003cspan class=\"CitationRef\"\u003e21\u003c/span\u003e], since it was desired to compare the behavior of the test specimens in four-point bending, impact, water contact angle, surface roughness, microhardness and layer thickness tests. As the sample geometry could be used for all tests, it facilitated production and guaranteed that there were always extra test specimens available. In addition, the main advantage of choosing the same geometry was to ensure that during the annealing process there would be no major differences in temperature distribution and heating rate.\u003c/p\u003e\n \u003cp\u003eAll samples were produced one at a time on a Creality Ender 3 v1\u0026reg;, to guarantee that the thermal history of each sample would be the same prior to annealing. The test specimens were produced with a nozzle temperature of 210\u0026deg;C, table temperature of 60\u0026deg;C, 100% infill, printing speed of 50 mm/s, layer height of 0.2 mm and diameter nozzle of 1 mm. The adopted test specimen had a parallelepiped shape of 127 mm in length, 12.7 mm in width and 3.2 mm in thickness, as shown in Fig.\u0026nbsp;1, and they were printed with the largest surface in contact with the printing table.\u003c/p\u003e\n \u003cp\u003eThe powder bed annealing process was performed using a Britania BFE10V electric oven and the particulate used was pure calcium phosphate (CaPO) and a mixture of 50% calcium phosphate with 50% titanium dioxide (CaPO\u0026thinsp;+\u0026thinsp;TiO\u003csub\u003e2\u003c/sub\u003e). Eight test specimens were produced for each condition according to the design of experiment presented in Table \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e. Four test specimens were annealed at a time, all of which were positioned inside a single tray and placed at the furnace, as shown in Fig.\u0026nbsp;2. This procedure was adopted because if two trays were used at once, there could be differences in the heat and temperature distribution on the samples. The annealing temperature was controlled by a thermocouple perforating the tray, with its tip positioned between the samples.\u003c/p\u003e\n \u003cp\u003eFor the annealing process, the oven was turned on and regulated on its analogic thermostat to a temperature 10\u0026deg;C above the maximum temperature of the condition being tested. Then the tray was inserted and the temperature inside the tray was monitored as it increased slowly towards the maximum temperature of the condition. It took about 30 minutes to reach 170\u0026deg;C and 40 minutes to reach 190\u0026deg;C, because it takes time for the heat to penetrate the tray and disseminate through the powder by thermal conduction. When the temperature indicated by thermocouple reached the maximum temperature of the experiment, the oven was either turned off and opened immediately for the conditions with 0 minutes at maximum temperature, or the oven was regulated on its thermostat to the maximum temperature of the experiment (170\u0026deg;C or 190\u0026deg;C) and kept turned on for 5 minutes and then turned off and opened. In both cases, the tray was taken off the oven and allowed to cool down naturally until it decreased to 40\u0026deg;C, in a room with temperature of 25\u0026deg;C, which took about 50 minutes. Then the tray was opened, and the samples were retrieved from below the powder and washed smoothly in water, dried in compressed air and stored in plastic containers filled with silica to prevent moisture, until they would be used for the subsequent mechanical and biological experiments. The procedure is patent pending, with code BR 10 2025 000986-2 registered in Brazilian National Institute of Intelectual Protection (INPI).\u003c/p\u003e\n \u003cp\u003e\u003c/p\u003e\u0026nbsp;\u003ctable id=\"Tab1\" border=\"1\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cp\u003eExperimental design of the full factorial type 2\u003csup\u003e3\u003c/sup\u003e for powder bed annealing tests.\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eCondition\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eComposition\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eMaximum temperature (\u0026deg;C)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eTime at maximum temperature (min)\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eA\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eCaPO\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e190\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e5\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eB\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eCaPO\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e190\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eC\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eCaPO\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e170\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e5\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eD\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eCaPO\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e170\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eE\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eCaPO\u0026thinsp;+\u0026thinsp;TiO2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e190\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e5\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eF\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eCaPO\u0026thinsp;+\u0026thinsp;TiO2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e190\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eG\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eCaPO\u0026thinsp;+\u0026thinsp;TiO2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e170\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e5\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eH\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eCaPO\u0026thinsp;+\u0026thinsp;TiO2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e170\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n \u003cp\u003e\u003c/p\u003e\n \u003cp\u003eAfter the test specimens were manufactured, four of each condition were separated for the contact angle measurement, roughness and flexural tests, while the other four of each condition were sectioned in smaller samples for impact tests, layer thickness analysis, microhardness and in-vitro biocompatibility evaluation. First the nondestructive tests such as water contact angle measurements were conducted in the first four samples, and then these same specimens were used for four-point flexural essays, with surface roughness measurements being carried in the fractured parts from the later essay. For the impact tests, layer thickness analysis and microhardness, the other four test specimens were sectioned using an angle grinder equipped with cutting disc. The parts that would be taken to SEM for analysis of layer thickness and chemical composition were embedded in resin, sanded to a 2000-mesh sandpaper, polished and received a 10 nm gold coating on a Leica EM SCD050 metallizer. These samples prepared for analysis in the SEM were the same ones used in the microhardness tests, with all analyses being carried out on the cross section of the sectioned samples. The samples for biocompatibility tests were cut by a hole saw into small disks of approximately 9.5 mm diameter to be fit in a culture plate.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec4\" class=\"Section2\"\u003e\n \u003ch2\u003e2.2. MATERIALS\u003c/h2\u003e\n \u003cp\u003eAll the samples were 3D printed with natural PLA by Filamentos 3D Brasil, and its characteristics are provided in Table \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e according to the manufacturer website (F3D, 2024). PLA is a material with recognized effectiveness in composite materials for orthopedic applications [\u003cspan class=\"CitationRef\"\u003e22\u003c/span\u003e], being the most used polymer in composite interference screws [\u003cspan class=\"CitationRef\"\u003e23\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e24\u003c/span\u003e] and in research with resorbable osseointegrated implants [\u003cspan class=\"CitationRef\"\u003e25\u003c/span\u003e]. Neijhoft et al. [\u003cspan class=\"CitationRef\"\u003e26\u003c/span\u003e] found that if there is sufficient contact time between filament and nozzle, which can be achieved through small layer heights, the FFF method presents inherent sterility for PLA. Besides, in accordance with other studies [\u003cspan class=\"CitationRef\"\u003e27\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e28\u003c/span\u003e], it has been shown that autoclaving, the preferred method for sterilizing surgical materials in hospitals, is also able to sterilize 3D printed PLA efficiently and with low distortion for higher size parts [\u003cspan class=\"CitationRef\"\u003e29\u003c/span\u003e]. These characteristics further highlights the adequateness of PLA as a material for implants and tissue engineering.\u003c/p\u003e\n \u003cp\u003e\u003c/p\u003e\u0026nbsp;\u003ctable id=\"Tab2\" border=\"1\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cp\u003ePhysical and mechanical properties of Premium Natural PLA from Filamentos 3D Brasil [\u003cspan class=\"CitationRef\"\u003e30\u003c/span\u003e].\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003ePhysical Properties\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eValue\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eUnit\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eASTM Standard\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eDensity\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.24 +/- 0.05\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eg/cm\u0026sup3;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eD792\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eFluidity\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e7\u0026ndash;9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eg/10 min \u0026minus;\u0026thinsp;(190\u0026deg;C \u0026minus;\u0026thinsp;2.16 Kg)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eD1238\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eRelative Viscosity\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eg/dL Chloroform 30\u0026deg;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eD5225\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eMelting point\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e165\u0026ndash;180\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u0026deg;C\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eD3418\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eGlass Transition Temperature\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e55\u0026ndash;60\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u0026deg;C\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eD3418\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eMechanical Properties\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eValue\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eUnit\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eASTM Standard\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eTensile Strength\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e51\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eMPa\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eD638\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eElongation at Break\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3,3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eD882\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eImpact Resistance (IZOD)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e118\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eJ/m\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eD256\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eHeat Deflection Temperature\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e55\u0026ndash;60\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u0026deg;C (0.45 MPa)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eE2092\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n \u003cp\u003e\u003c/p\u003e\n \u003cp\u003eThe calcium phosphate was obtained from a food additive supplier, since this material is widely used as anti-humectant, while the titanium dioxide was obtained from online stores, since it is the world\u0026rsquo;s most used pigment load for white paints. These powders, both of which resemble white fine particulates as thin as wheat flour, were analyzed by X-ray diffraction (XRD) to verify which were their crystalline structures, on a Shimadzu XRD6000 difractometer, with 2\u0026deg;/min scanning, 0.02\u0026deg; resolution and 2\u0026theta; angle of 5 to 60\u0026deg; for calcium phosphate and 20 to 70\u0026deg; for titanium dioxide. The particles were also investigated by SEM (Scanning Electron Microscopy) and EDS (Energy Dispersive Spectroscopy) on a Tescan VEGA 3 and Zeiss EVO MA10 microscopes equipped with Oxford INCAx-act probe, to measure the size of particles and their chemical composition in weight, respectively.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec5\" class=\"Section2\"\u003e\n \u003ch2\u003e2.3. WATER CONTACT ANGLE MEASUREMENT\u003c/h2\u003e\n \u003cp\u003eThe water contact angle tests were performed using a 5 \u0026micro;L chromatographic syringe with a fixed needle and a support that ensured the slow and controlled approach of the drop to the surface of the specimen. The drops were 5 \u0026micro;L and three drops were deposited in different positions on the specimens while the procedure was filmed so that it would be possible to later measure the contact angle. This procedure was performed according to Han et al. [\u003cspan class=\"CitationRef\"\u003e31\u003c/span\u003e], following the general guidelines of ASTM D7334 [\u003cspan class=\"CitationRef\"\u003e32\u003c/span\u003e]. The angles were measured using ImageJ software, and the images were taken from the filming approximately 10 seconds after the drop was deposited. The angles were observed as exemplified in Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e, for the two internal angles at the left and right edges, registering the average for each contact angle measurement. Four measurements were carried in a sample of each condition, for calculation of average and standard deviation.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec6\" class=\"Section2\"\u003e\n \u003ch2\u003e2.4. ROUGHNESS TEST\u003c/h2\u003e\n \u003cp\u003eFor roughness measurements the parameters Ra, Rq and Rz were observed using a portable digital Mitutoyo SJ 201roughness meter with resolutions of 0.4 \u0026micro;m, 0.1 \u0026micro;m, 0.05 \u0026micro;m and 0.01 \u0026micro;m for a measurement range of 350 \u0026micro;m, 100 \u0026micro;m, 50 \u0026micro;m and 10 \u0026micro;m, respectively. Three specimens were tested per condition following the general recommendations of ISO 4287 [\u003cspan class=\"CitationRef\"\u003e33\u003c/span\u003e] and ISO 4288 [\u003cspan class=\"CitationRef\"\u003e34\u003c/span\u003e] standards, with three measurements being performed in different positions for each specimen. In addition, a cutoff of 2.5 mm was used, since according to the standard, this value is the most suitable for surfaces with Ra from 2 to 10 \u0026micro;m, resulting in an evaluation length of 12.5 mm.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec7\" class=\"Section2\"\u003e\n \u003ch2\u003e2.5. FLEXURAL TEST\u003c/h2\u003e\n \u003cp\u003eFor these bending tests, four specimens of each condition were evaluated using an equipment developed at the Laboratory of Education and Research in Machining (LEPU) in previous works, which follows the requirements of ASTM D6272 (ASTM) standard. The flexural testing machine had 4 parallel load cells with a resolution of 0.05 g and a capacity of 50 kg each, totaling a maximum load of 200 kg. The load cells, with an accuracy of \u0026plusmn;\u0026thinsp;0.1% of the maximum load according to the manufacturer (resulting in \u0026plusmn;\u0026thinsp;200 g for this equipment), were connected to HX711 modules linked to an Arduino Uno for amplification and signal conversion. With the results of the bending tests, data on maximum flexural strength (MFS), modulus of elasticity (E) and flexural elongation (Ɛ) were obtained. To calculate the modulus of elasticity, which has different possibilities according to the standard, the method used considers the origin at 0 and the maximum load point, as can be seen in the example in Fig. 4, which shows the graphs for specimens of conditions B and G, with the points used to calculate the elongation being those highlighted by black dots.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\n \u003ch2\u003e2.6. IMPACT TEST\u003c/h2\u003e\n \u003cp\u003eThe Izod impact tests were performed according to ASTM D4508 (ASTM) to observe the difference in energy absorbed by the samples. The specimens used were sectioned pieces with length, height and width of 19 mm, 12.7 mm and 3.2 mm respectively. In this test, four specimens were tested per condition, and the pendulum was released at an angle of 0\u0026deg; in relation to the horizontal, reaching a speed of 5.6 m/s when hitting the sample with an energy of 3.8 J. The pendulum used had a length of 0.778 m (distance from the tip to the center of rotation), with a total mass of 0.963 kg and a center of mass at 0.159 m from the center of rotation. The impact testing machine used was adapted from the work of Tsuruta [\u003cspan class=\"CitationRef\"\u003e63\u003c/span\u003e], and the measurement of the energy absorption by the sample was performed by an encoder-type sensor fixed to the pendulum shaft, which measured the maximum angle reached by the pendulum. The encoder consisted of a 220 mm disk with 360 holes 1 mm wide and spaced 1 mm apart, positioned on the edge of the disk together with an LM393 module, fixed to the pendulum support, which was responsible for reading the movement.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec9\" class=\"Section2\"\u003e\n \u003ch2\u003e2.7. COATING LAYER THICKNESS AND CHEMICAL ANALYSIS\u003c/h2\u003e\n \u003cp\u003eFor layer thickness analysis, four samples of each condition were embedded in a disk resin, sanded up to 2000 grit sandpaper, polished with 10 \u0026micro;m chromium oxide particles, metallized with a 10 nm gold coating and taken to the same SEM microscopes for BSE (backscattered electron imaging) and EDS analysis. One photo of each of the four samples in the same disk was taken in BSE mode to analyze the cross-section of the original specimen, making possible to measure the layer thickness using the ImageJ software as shown in Fig. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003e, and calculating the average of the layer thickness in 3 different regions of each image.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e\n \u003ch2\u003e2.8. MICROHARDNESS ANALYSIS\u003c/h2\u003e\n \u003cp\u003eAfter examination in the SEM, the samples underwent microhardness testing. To prevent interference, microhardness tests were conducted away from SEM-analyzed areas, as prolonged exposure to the electron beam can degrade the surface. The tests followed ASTM E384 standards, utilizing an HV0.2 scale (1.961 N load), with a 15-second load application and 10x magnification.\u003c/p\u003e\n \u003cp\u003eMeasurements were taken across the sample\u0026rsquo;s cross-section, starting at the surface and extending toward the center, with approximately 300 \u0026micro;m spacing between indentations. Each major face of two samples from every annealing condition (A to H) was tested, resulting in four measurements per condition. This approach aimed to assess hardness variations from the coating to the substrate\u0026apos;s inner regions.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\n \u003ch2\u003e2.9. BIOCOMPATIBILITY TESTS AND SEM ANALYSIS\u003c/h2\u003e\n \u003cp\u003eBiocompatibility of the disks cut from samples was evaluated by cytotoxicity assay, using the Alamar blue assay, in which MC3T3-E1 cells, (ECACC 99072810), which are pre-osteblast derived from mouse calvaria, were cultured in \u0026alpha;-MEM 10% FBS medium, after reaching 80% confluence, were plated at a seeding density of 1x10\u003csup\u003e4\u003c/sup\u003e cells on the surface of each disk placed on the bottom of 48 well plate. The plates were incubated overnight at 37\u0026deg;C in a humidified atmosphere with 5% CO2 to allow the cells to adhere to the disks. As a positive control, cells attached to the bottom of the plate, without disks and as negative control, cells were cultivated with medium containing DMSO 5%. After 24 hour, 7 days or 14 days, 20 \u0026micro;L of PBS containing resazurin (7-Hydroxy-3H-phenoxazin-3-one-10-oxide sodium salt) (Sigma-Aldrich, S\u0026atilde;o Paulo, Brazil) at a concentration of 0.3\u0026micro;M was added to each well. The plate was incubated for 4 hours protected from light and then the supernatant was transfered to an appropriate plate for fluorescence Reading, which was measured using a VICTOR NivoTM Plate Reader, at 560 nm excitation and 600 nm emission. For analysis, the absorbance of the samples containing only \u0026alpha;-MEM 10% FBS medium (no cells) were subtracted from the samples. The statistical analysis was carried by two-way ANOVA with Tukey\u0026apos;s post-test (for multiple comparisons) considering a 95% confidence interval for proliferation analysis with the individual viability after 1, 7 and 14 days.\u003c/p\u003e\n \u003cp\u003eAfter 14 days, the wells were washed 3 times in Phosphate Buffered Saline (PBS) 1X and then fixed in 3.2% formaldehyde for 1 hour at room temperature. They were rinsed once more in PBS 1X and gradually dehydrated from ethyl alcohol diluted in distilled water, in concentrations rising from 50\u0026ndash;100% in 10% increments, for 10 minutes in each, and finally stored in absolute alcohol at 4˚C for 1 day. Then they were dried in vacuum for 12 hours and metallized with a 10 nm gold coating to be analyzed by SEM.\u003c/p\u003e\n\u003c/div\u003e"},{"header":"3. RESULTS","content":"\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\n \u003ch2\u003e3.1. POWDER MATERIAL CHARACTERIZATION\u003c/h2\u003e\n \u003cp\u003eThe XRD spectrum of calcium phosphate can be seen in Fig. \u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003e, in which the peaks at 25.9\u0026deg;; 31.74\u0026deg;; 32.16\u0026deg;; and 32.88\u0026deg; are indicative of the hydroxyapatite (HA) crystal structure according to Hu et al. (2020), differentiating it from other structures such as TCP (tri-calcium phosphate) and BCP (bi-calcium phosphate), commonly obtained in calcium phosphate synthesis reactions. In calcium phosphate with the HA structure, with molecular formula Ca\u003csub\u003e5\u003c/sub\u003e(PO\u003csub\u003e4\u003c/sub\u003e)\u003csub\u003e3\u003c/sub\u003eOH, the OH groups appear trapped in the crystal structure, and several authors (Zhang, 2013; Sossa et al., 2018; de Melo Costa et al., 2009) have already shown that its lower solubility and greater stability in the body make it the preferred structure for implant coating. Regarding TiO\u003csub\u003e2\u003c/sub\u003e, as shown by El-Sherbiny et al. [\u003cspan class=\"CitationRef\"\u003e35\u003c/span\u003e] and Ijadpanah-Saravy et al. [\u003cspan class=\"CitationRef\"\u003e36\u003c/span\u003e], the peaks at 27.44\u0026deg;; 36.1\u0026deg;; 41.28\u0026deg;; and 56.66\u0026deg; are typical of the rutile structure and differentiate it from anatase and brookite, the other possible crystalline forms. Rutile is in fact the most stable in crystals larger than 35 nm [\u003cspan class=\"CitationRef\"\u003e37\u003c/span\u003e] and is the most commonly obtained structure in the industrial production processes of titanium dioxide, with its biological activity being better than that of the amorphous form obtained in some electrochemical processes [\u003cspan class=\"CitationRef\"\u003e38\u003c/span\u003e]. The narrow bands indicate high crystallinity of the material used, in addition to uniformity of crystal size\u003c/p\u003e\n \u003cp\u003eThe shape and size of the Ca\u003csub\u003e5\u003c/sub\u003e(PO\u003csub\u003e4\u003c/sub\u003e)\u003csub\u003e3\u003c/sub\u003eOH particles can be seen in Fig.\u0026nbsp;7. The crystals are predominantly rod-shaped with a thickness of around 200 nm. Despite the high surface area, the calcium phosphate powder appears to be less adherent than titanium dioxide, being easier to handle and to clean, so it is possible that the formation of large agglomerates reduces the surface energy of this material. The chemical composition of this calcium phosphate deposited on carbon tape and coated in gold is shown in Fig.\u0026nbsp;8, which also shows a mass content of Ca of around 10% and around 29% of C. This is because the C content and part of the O content comes from the carbon tape substrate, since the EDS rays comes from regions up to 2 \u0026micro;m deep in the sample, and for this analysis the powder was thinly spread on the tape. For the same reason, although the Ca\u003csub\u003e5\u003c/sub\u003e(PO\u003csub\u003e4\u003c/sub\u003e)\u003csub\u003e3\u003c/sub\u003eOH from hydroxyapatite structure leads to an expected mass percentage in the proportion of 40% : 19% : 41% for Ca : P : O, the lower mass of Ca and P in comparison with O comes from the oxygen present in the polymeric carbon tape.\u003c/p\u003e\n \u003cp\u003eThe size and shape of the TiO\u003csub\u003e2\u003c/sub\u003e particulates can be seen in Fig. \u003cspan class=\"InternalRef\"\u003e9\u003c/span\u003e. It is noticeable the approximately spherical or ellipsoidal shape of these particulates, which agglomerate to form larger grains, as also observed by Hu et al. (2014). Due to the submicrometric scale (with particulates of about 400 nm), the material has a high surface/volume ratio, and therefore adheres easily to the substrate, even when not annealed. Their chemical composition can be seen in Fig.\u0026nbsp;10, where a variation in the mass percentages of the elements, including carbon, can be noted. In this case, the C content comes from the polymeric substrate and may be higher or lower depending on the thickness of the TiO\u003csub\u003e2\u003c/sub\u003e layer at each point.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\n \u003ch2\u003e3.2. WATER CONTACT ANGLE\u003c/h2\u003e\n \u003cp\u003eThe water contact angle is shown in Fig. 11 for each annealing condition, for non-annealed samples and for the coated surfaces of the femoral and acetabular components of a commercial hip implant, demonstrated in Fig. \u003cspan class=\"InternalRef\"\u003e12\u003c/span\u003e. In all cases, except for specimens E and F, the contact angles were smaller than the non-annealed state and much smaller than 90\u0026deg;, which indicates an improvement in surface hydrophilicity [\u003cspan class=\"CitationRef\"\u003e39\u003c/span\u003e]. In fact, together with a high roughness, the contact angles of less than 30\u0026deg; obtained can lead to a superwetting surface as defined by [\u003cspan class=\"CitationRef\"\u003e40\u003c/span\u003e], which is a good predictor for the biocompatibility of the material [\u003cspan class=\"CitationRef\"\u003e41\u003c/span\u003e]. Also, there were annealing conditions in which the wettability of the 3D printed PLA samples was even better than that obtained for the commercial hip implant, highlighting the potential of this new biomaterial as an inductor for cell adhesion and differentiation [\u003cspan class=\"CitationRef\"\u003e42\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e43\u003c/span\u003e].\u003c/p\u003e\n \u003cp\u003eAnalysis of variance (ANOVA) was performed to assess how the annealing conditions affect the water contact angle, from which the most significant parameters were composition (p\u0026thinsp;=\u0026thinsp;0,000000), time in maximum temperature (p\u0026thinsp;=\u0026thinsp;0,000009) and the combination of them (p\u0026thinsp;=\u0026thinsp;0,000001). Calcium phosphate is a common anti-humectant in the food industry due to its affinity with water, preventing the food from absorbing moisture. Therefore, it is possibly for this reason that it presents greater hydrophilicity. Titanium dioxide covered the surface irregularly, leading to smaller wetting angles on average and greater variability in the results, as in conditions E and F. Regarding temperature, it may not have been significant because at both 170\u0026deg;C and 190\u0026deg;C the polymer assumes a similar level of viscosity. The time at maximum temperature may have been more significant because it provided more time for the diffusion of particulates on the surface, whereas in the case of titanium dioxide, this led to a more irregular surface observed visually and by SEM (Fig. 13a and Fig. 13b). In these figures, the white/gray lines to the left of each layer are just flaws resulted from the embedding, sanding and polishing processes.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e\n \u003ch2\u003e3.3. ROUGHNESS TEST\u003c/h2\u003e\n \u003cp\u003eThe roughness parameters Ra, Rz and Rq were measured and are shown in Fig. \u003cspan class=\"InternalRef\"\u003e14\u003c/span\u003e, from which there is notable difference between the values when changing the chemical composition of the powder bed. According to ANOVA, the chemical composition was in fact significant for a confidence level of 95% for Ra (p\u0026thinsp;=\u0026thinsp;0,000000), Rq (p\u0026thinsp;=\u0026thinsp;0,000446) and Rz (p\u0026thinsp;=\u0026thinsp;0,000000). This greater roughness can also be visually perceived in the test specimens annealed in the mixture of titanium dioxide and calcium phosphate, with the surface of the test specimen having a very rough finish as if the molten PLA was being absorbed by the powder bed. Figure \u003cspan class=\"InternalRef\"\u003e15\u003c/span\u003e shows two samples annealed in calcium phosphate (type C and D) and two in titanium dioxide with calcium phosphate (type E and H), illustrating the observed results. Comparatively, the roughness values for conditions A to D are close to those observed for the femoral component of the same commercial hip implant of Fig. \u003cspan class=\"InternalRef\"\u003e12\u003c/span\u003ea, measured in another previous work [\u003cspan class=\"CitationRef\"\u003e44\u003c/span\u003e] with Ra ranging from 4.7 \u0026micro;m to 5.8 \u0026micro;m and Rq from 6 \u0026micro;m to 7.6 \u0026micro;m. This indicate that the surface of the annealed 3D printed PLA parts bear similarity with that of biomaterials already validated for commercial use, which is also a good indicator of the potential of the obtained surfaces.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e\n \u003ch2\u003e3.4. FLEXURAL TESTS\u003c/h2\u003e\n \u003cp\u003eThe results of maximum flexural strength (MFS), modulus of elasticity (E) and flexural elongation (Ɛ) are shown in Fig. \u003cspan class=\"InternalRef\"\u003e16\u003c/span\u003e. It shows that powder bed annealing resulted in a decrease in elongation as well as an increase in flexural strength and modulus of elasticity. Considering that the coating is very thin in comparison to the bulk material, the mechanical properties will depend almost entirely on the characteristics of the bulk PLA. As the process reaches temperatures above 160\u0026deg;C for which PLA already starts to melt, the changes observed in MFS, E and Ɛ could come from an alteration in the PLA chemical composition, or in its porosity, or in its crystallinity, as explained in other works [\u003cspan class=\"CitationRef\"\u003e67\u003c/span\u003e]. Although neither of these properties were measured in this work, previous works have shown that that the most important factors the justify increases in mechanical resistance are the modification of pores geometry [\u003cspan class=\"CitationRef\"\u003e69\u003c/span\u003e] and polymer crystallization [\u003cspan class=\"CitationRef\"\u003e68\u003c/span\u003e]. For elongation and maximum flexural strength, the influence of composition (p\u0026thinsp;=\u0026thinsp;0,011332 and p\u0026thinsp;=\u0026thinsp;0,002948, respectively) and time at maximum temperature (p\u0026thinsp;=\u0026thinsp;0,001278 and p\u0026thinsp;=\u0026thinsp;0,015807, respectively) were significant with a 95% confidence level. This can be explained because more crystalline parts tend to be more rigid and have greater mechanical resistance, if they have not become brittle [\u003cspan class=\"CitationRef\"\u003e45\u003c/span\u003e]. This occurs because in the amorphous state the macromolecules are randomly entangled, with a greater possibility of movement when subjected to stresses, while in the crystalline state the macromolecules are rigidly packed, with a lower capacity to stretch and deform [\u003cspan class=\"CitationRef\"\u003e46\u003c/span\u003e]. Above the glass transition (Tg), thermal energy generates the vibration of the macromolecules, which have greater freedom to rotate and move, with a natural tendency to crystallize if there is enough time, given that the crystalline state is the state with the lowest free energy [\u003cspan class=\"CitationRef\"\u003e46\u003c/span\u003e]. Thus, the thermal history of printing and annealing influences the final mechanical properties of the test specimens, explaining the observed variations. Moreover, the particulate alters the regularity and presence of surface pores, which affect the nucleation and propagation of cracks, as in the case of Fig. \u003cspan class=\"InternalRef\"\u003e15\u003c/span\u003e. Thus, due to the stress concentration, composition was also a preponderant factor for the fracture of the samples.\u003c/p\u003e\n \u003cp\u003eThese results show that the powder bed annealing process was able not only to create the coating on the PLA surface, but also to improve its resistance, both in comparison to the non-annealed PLA and to PLA flexural resistance values from the literature, which vary from about 60 MPa to 100 Mpa [\u003cspan class=\"CitationRef\"\u003e47\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e48\u003c/span\u003e]. This further supports the attractiveness of this new manufacturing process for PLA coatings, because besides it being simpler and cheaper in comparison to cold gas shooting, plasma spray and other coating processes [\u003cspan class=\"CitationRef\"\u003e49\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e50\u003c/span\u003e], it can even improve the substrate mechanical properties. It is also important to highlight that as can be seen from Fig. \u003cspan class=\"InternalRef\"\u003e15\u003c/span\u003e, even after the fracture of the samples during the bending tests, there was no delamination of the coating from the bulk material. Although no direct measurement of the adhesion of the coating was carried out, this aspect serves as an indirect evaluation of coating integrity as exemplified in ASTM B571 [\u003cspan class=\"CitationRef\"\u003e70\u003c/span\u003e], since there was no visible failure in any of the specimens.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec17\" class=\"Section2\"\u003e\n \u003ch2\u003e3.5. IMPACT TESTS\u003c/h2\u003e\n \u003cp\u003eFor the impact tests, the graph from Fig. \u003cspan class=\"InternalRef\"\u003e17\u003c/span\u003e shows that there is no explicit trend in the results as a function of the input parameters, which is reinforced by no significance from any factors in ANOVA. Comparing the results obtained with values from other studies [\u003cspan class=\"CitationRef\"\u003e51\u003c/span\u003e], which presented results close to 50 J/m for parts produced from PLA by 3D printing, it is observed that powder bed annealing did not impair the impact resistance of the substrate. Again, given the relatively thin coating, the results of the impact test might be influenced primarily by the bulk properties of the PLA, leading to similar impact energy values across all specimens. This indicates that the material conserves its toughness after the surface modification process, enabling it for use in the production of biomaterials.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec18\" class=\"Section2\"\u003e\n \u003ch2\u003e3.6. COATING LAYER THICKNESS AND CHEMICAL ANALYSIS\u003c/h2\u003e\n \u003cp\u003eThe results of the coating layer thickness measured in the SEM are presented in Fig. \u003cspan class=\"InternalRef\"\u003e18\u003c/span\u003e and it shows that pure calcium phosphate composition (p\u0026thinsp;=\u0026thinsp;0,000019) and a longer time at maximum temperature (p\u0026thinsp;=\u0026thinsp;0,000000) ensured better layer thickness results, whereas both parameters together with the combination (p\u0026thinsp;=\u0026thinsp;0,023406) of time in maximum temperature and maximum temperature were significant for a 95% of confidence level. A possible explanation for the influence of the particulate is the fact that, although it cannot be perceived from the images, for calcium phosphate the particulate is visually less adhered when handled, which may make it easier for the particulate to diffuse into the molten polymer more homogeneously, forming a new composite material from the mixture of the particulate and PLA. Likewise, a longer time at maximum temperature allows more diffusion to occur. It is recommended that the coating layer formed should not be too small, since it can be absorbed by the bone tissue very quickly in the first few weeks after implantation, but it should also not be too thick that could make the surface fragile. Thus, an ideal thickness is considered by some authors [\u003cspan class=\"CitationRef\"\u003e52\u003c/span\u003e] to be between 30 \u0026micro;m and 80 \u0026micro;m, while others have found it to be between 70 \u0026micro;m to 90 \u0026micro;m [\u003cspan class=\"CitationRef\"\u003e53\u003c/span\u003e]. Therefore, it can be observed that the results obtained in this study could be satisfactory for biomaterial fabrication, since most of the results are between 30 \u0026micro;m and 50 \u0026micro;m.\u003c/p\u003e\n \u003cp\u003eRegarding the chemical composition, it can be inferred that the coating is a composite of calcium phosphate grains interspersed with PLA for samples A to D. In Fig.\u0026nbsp;19a the BSE image shows that the aligned denser white grains have infiltrated the gray PLA substrate (to the right) during annealing, and the composition maps of Fig.\u0026nbsp;19b show that there is a well-defined region where the calcium phosphate was deposited, and in this region, there is also carbon from the PLA substrate. The same is true for samples E to H, for which the coating is a composite of calcium phosphate and titanium dioxide grains interspersed with PLA, as shown in Fig.\u0026nbsp;19c and Fig.\u0026nbsp;19d. Regarding the applicability to implants, it is expected that the composite coating might have an even better behavior than only pure calcium phosphate coating or pure PLA because, as explained in the work of Bernardo et al. [\u003cspan class=\"CitationRef\"\u003e22\u003c/span\u003e], the PLA component may offer structural support for cell attachment and growth, while the HA phase releases calcium and phosphate ions that both promote osteoinduction and increase the low pH created by PLA degradation in lactic acid. Besides, the use of composite parts of PLA and calcium phosphate [\u003cspan class=\"CitationRef\"\u003e54\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e55\u003c/span\u003e] and PLA and titanium dioxide [\u003cspan class=\"CitationRef\"\u003e56\u003c/span\u003e] have already shown biocompatibility characteristics, validating this new coating method by power bed annealing as an interesting manufacturing method for biomaterials, especially in the case of interference screws, for which this composite mix has been shown to have good results in vitro [\u003cspan class=\"CitationRef\"\u003e57\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e58\u003c/span\u003e] and in vivo [\u003cspan class=\"CitationRef\"\u003e59\u003c/span\u003e] and is now widely used in commercial products [\u003cspan class=\"CitationRef\"\u003e25\u003c/span\u003e].\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec19\" class=\"Section2\"\u003e\n \u003ch2\u003e3.7. MICROHARDNESS ANALYSIS\u003c/h2\u003e\n \u003cp\u003eThe microhardness was measured in the cross section of the samples, starting on the coating and moving towards the bulk, with each indentation spaced about 300 \u0026micro;m from the other. As can be seen from the plots in Fig.\u0026nbsp;20, there is not much difference of the microhardness on the sample section, with major differences only being observed very close the surface. The significance analysis showed that the input parameters had no influence on the microhardness in the cross section. Regarding the large variation in hardness observed in the coating region, the most likely explanation for it is the fact that it borders the embedding resin, which is against the recommendations from ASTM E384 [\u003cspan class=\"CitationRef\"\u003e60\u003c/span\u003e]. Therefore, it can be inferred that there is no significant change of the mechanical properties of the substrate at microscale, indicating that this coating process does not damage the substrate\u0026rsquo;s resistance.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec20\" class=\"Section2\"\u003e\n \u003ch2\u003e3.8. BIOCOMPATIBILITY TEST AND SEM ANALYSIS\u003c/h2\u003e\n \u003cp\u003eCytotoxicity tests were performed only in 3 of the 8 production conditions of the coated samples, also comparing them with pure PLA and negative and positive control materials, as shown in Fig. \u003cspan class=\"InternalRef\"\u003e21\u003c/span\u003e. Although in the first day the samples showed worse cell viability, after 14 days all discs showed greater cell growth than in the positive control, with the calcium phosphate and titanium dioxide coating of sample H performing better. Still, not even for the first day the material can be considered cytotoxic, since the cell viability was above the limit of 70% [\u003cspan class=\"CitationRef\"\u003e61\u003c/span\u003e]. This indicates that the material, in addition to being non-toxic to cells, inducing possible adhesion and proliferation on the surface of the samples, which is an excellent indication that these biomaterials could become an option for new models of orthopedic implants in the future.\u003c/p\u003e\n \u003cp\u003eInterestingly, in Fig.\u0026nbsp;22a the MC3T3 cell can be seen well attached on the calcium phosphate grains, with its cell process spreading on the surface of sample B. Similarly, other cells can also be seen in the surface of sample B in Fig.\u0026nbsp;22b, which has a very different aspect from pure PLA surface, for which an isolated cell can be seen in Fig.\u0026nbsp;22c. In this case, even after 14 days, although the cell attached to the surface, its ovoid morphology suggests it did not differentiate into a mature cell [\u003cspan class=\"CitationRef\"\u003e62\u003c/span\u003e] with the typical spreading process, exactly because this sample lacked the calcium phosphate and/or titanium dioxide that promote the osteogenic stimuli. In contrast, Fig. 22d shows even more osteocytes spread over the surface from sample H, which presented the best cell viability after 14 days.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec21\" class=\"Section2\"\u003e\n \u003ch2\u003e3.9. COMPARISON AGAINST OTHER COATING TECHNOLOGIES FOR POLYMERIC IMPLANTS\u003c/h2\u003e\n \u003cp\u003eAs discussed, the integration of PLA and HA into implants is well stablished in the literature [\u003cspan class=\"CitationRef\"\u003e71\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e73\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e88\u003c/span\u003e], given the resorbable nature of the former and the osseointegration properties of the later. Therefore, the proposed coating method should be compared to other technologies capable of combining these components for the same goal. This could be achieved either by methods that create a HA coating on PLA or that mix HA into PLA matrix. Considering the first, there have been studies with plasma spray for coating polymers such as PEEK (poly-ether-ether-ketone) [\u003cspan class=\"CitationRef\"\u003e72\u003c/span\u003e]. However, considering the high temperatures reached in the plasma arc of about 10\u003csup\u003e4\u003c/sup\u003e K [\u003cspan class=\"CitationRef\"\u003e76\u003c/span\u003e], it would most probably not be suitable for coating PLA, which has a melting point of about 160\u0026deg;C compared to about 340\u0026deg;C for PEEK. Besides, other works [\u003cspan class=\"CitationRef\"\u003e74\u003c/span\u003e] have shown that plasma spray deposited HA may present low adhesion with polymer, with failure of the coating under load before the failure of the substrate. Additionally, plasma spray high temperatures might cause HA to transform into \u0026alpha; or \u0026beta; tricalcium phosphate, calcium oxide and/or tetracalcium phosphate, impairing not only the crystallinity [\u003cspan class=\"CitationRef\"\u003e77\u003c/span\u003e] and chemical composition of the coating, but also its effectiveness for osseointegration [\u003cspan class=\"CitationRef\"\u003e75\u003c/span\u003e]. Flame spray process is a variation that also could potentially deposit ceramic coatings [\u003cspan class=\"CitationRef\"\u003e78\u003c/span\u003e] but would suffer from the same drawbacks as plasma spray for PLA as the substrate material. Interestingly, some authors have already been able to deposit titanium dioxide onto low melting temperature polymers such as UHMWPE [\u003cspan class=\"CitationRef\"\u003e79\u003c/span\u003e] using vacuum plasma spray, but no other work was found with such feature for PLA. Also, all plasma spray techniques require expensive tooling such as a plasma torch, powder feeder and power supplies with capacity of 25 to 150 kW [\u003cspan class=\"CitationRef\"\u003e76\u003c/span\u003e], besides also requiring expensive materials sometimes [\u003cspan class=\"CitationRef\"\u003e80\u003c/span\u003e], such as pure nitrogen, argon or helium that act as carrier gases and stabilize the plasma arc.\u003c/p\u003e\n \u003cp\u003eAlthough not less expensive due to the necessary costly equipment [\u003cspan class=\"CitationRef\"\u003e81\u003c/span\u003e], a potential alternative would be the use of CGS (cold gas shooting), in which micrometric particles accelerated to supersonic speeds in a high-speed gas stream impact and adhere to a substrate, creating a distinct coating [\u003cspan class=\"CitationRef\"\u003e75\u003c/span\u003e]. However, almost 90% of the kinetic energy of the particles is converted into heat, and although there have not been found works with CGS onto polymers, it is likely that it would face the same problems related to excessive heat damaging the substrate. Moreover, it is important to highlight that CGS and all thermal spray are line of sight techniques, and coating the totality of a complex geometry would be more time consuming, even with a robotic arm. In comparison, the powder bed annealing method hereby proposed can create a uniform coating around all the part, even for more complex geometries. Also, it is a much more inexpensive and scalable method, because it only requires an electrical oven (about US\u003cspan\u003e$\u003c/span\u003e30) and a metallic tray as tooling (about US\u003cspan\u003e$\u003c/span\u003e5), and only about 100 g of HA powder (bought as additive for food industry for about US\u003cspan\u003e$\u003c/span\u003e4/kg) and 16 g of PLA (natural PLA bought at US\u003cspan\u003e$\u003c/span\u003e20/kg) to produce four samples at each condition. This make the proposed technology especially relevant for developing countries where access to expensive biomedical products is still a health issue. The only drawback of the powder bed annealing process is that it is not able to generate a pure HA and/or HA\u0026thinsp;+\u0026thinsp;TiO\u003csub\u003e2\u003c/sub\u003e coating in comparison to other techniques. However, the biocompatibility tests showed that phenomenon does not pose a problem, since the coating is still not cytotoxic and leads to cell differentiation. Regarding other coating techniques, as explained in other works [\u003cspan class=\"CitationRef\"\u003e82\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e83\u003c/span\u003e], despite the lower temperature of sputtering process, it does not create coatings of more than 3 \u0026micro;m, while sol-gel would be limited to 10 \u0026micro;m and may also require deleterious heating of the part to solidify the coating [\u003cspan class=\"CitationRef\"\u003e84\u003c/span\u003e]. The powder bed annealing, besides producing coatings of up to 50 \u0026micro;m in thickness, applies heat with the part immersed in powder, which avoids heat deflection and maintains the geometry. Electrodeposition methods [\u003cspan class=\"CitationRef\"\u003e85\u003c/span\u003e], on the other hand, cannot be applied to polymers, which are natural electrical insulators.\u003c/p\u003e\n \u003cp\u003eFinally, in relation to process the mix HA into the PLA matrix, two main processes can be highlighted. The first is injection molding, which has been used successfully to incorporate HA into PLA uniformly [\u003cspan class=\"CitationRef\"\u003e86\u003c/span\u003e]. Similarly, these authors also found that annealing the part could enhance the impact resistance as happened for most samples produced by powder bed annealing. However, mixing even small quantities of HA into the whole substrate has been found to decrease the impact [\u003cspan class=\"CitationRef\"\u003e87\u003c/span\u003e] and tensile resistance [\u003cspan class=\"CitationRef\"\u003e92\u003c/span\u003e] in comparison to pure PLA. Here lies another advantage of powder bed annealing, since except for conditions A and H, all others yield samples with average impact resistance above that of natural PLA used in the tests. The second process to incorporate HA into PLA is additive manufacturing, with several examples of composite filaments being produced for later use in FFF process. However, it has been found that concentrations as low as 10% of HA dispersed into PLA could be prejudicial to its mechanical properties, with its tensile resistance dropping from 50 MPa to 30 MPa [\u003cspan class=\"CitationRef\"\u003e90\u003c/span\u003e]. This phenomenon was not observed in the flexural tests carried out with powder bed annealed parts, where the resistance was the same or higher than for natural PLA, while also achieving very low values of water contact angle (mostly less than 30\u0026deg;). This level of hydrophilicity did not happen in other works [\u003cspan class=\"CitationRef\"\u003e91\u003c/span\u003e] that used PLA filament with 12% of HA for which water contact angles stayed at 66\u0026deg;, indicating once more the advantage of the powder bed annealing process as it conserves the toughness and resistance of the PLA substrate while presenting the improved biocompatible properties of HA at the surface. Furthermore, incorporating HA to PLA filaments in a uniform dispersion is still a challenge [\u003cspan class=\"CitationRef\"\u003e93\u003c/span\u003e] since simple mechanical stirring or high-shear mixing may not suffice, while for other additive manufacturing methods such as SLS, that is also not easy due to a small sintering window and flowability of the heterogeneous powder [\u003cspan class=\"CitationRef\"\u003e94\u003c/span\u003e]. It is also important to highlight that conserving a PLA core would be advantageous over creating pure HA parts, due to the inherent low fracture toughness of pure HA manufactured by SLS [\u003cspan class=\"CitationRef\"\u003e89\u003c/span\u003e].\u003c/p\u003e\n\u003c/div\u003e"},{"header":"4. CONCLUSIONS","content":"\u003cp\u003eThis study demonstrated the viability of using powder bed annealing as an innovative method for producing hydroxyapatite coatings on 3D-printed PLA parts. The process successfully generated a ceramic particulate layer with favorable characteristics for biomedical applications, particularly in orthopedic implants. The results showed that the coating exhibited good thickness control, improved surface roughness, and enhanced hydrophilicity, which are crucial factors for promoting osseointegration.\u003c/p\u003e \u003cp\u003eMechanical tests indicated that powder bed annealing enhanced the flexural strength of the PLA parts while maintaining impact resistance and microhardness in the substrate. Although ductility decreased, the mechanical improvements suggest that this method can contribute to stronger and more reliable implant materials. Additionally, biocompatibility tests confirmed that the hydroxyapatite-coated surfaces supported cell adhesion and proliferation, further validating the potential of this technique for biomedical applications.\u003c/p\u003e \u003cp\u003eCompared to conventional coating methods like plasma spraying or chemical deposition, powder bed annealing could offer a cost-effective and scalable alternative. The ability to integrate hydroxyapatite coatings directly onto polymeric implants without complex post-processing steps highlights the practicality of this approach. Furthermore, the combination of PLA\u0026rsquo;s biodegradability with hydroxyapatite\u0026rsquo;s osteoinductive properties suggests promising applications in resorbable implants.\u003c/p\u003e \u003cp\u003eOverall, this research contributes to the ongoing development of personalized, bioactive implants by optimizing a simple yet effective coating technique. Future work should explore long-term in vivo performance, refine processing parameters for improved adhesion and uniformity, and investigate alternative bioceramic compositions for tailored implant applications. The findings suggest that powder bed annealing could play a crucial role in advancing polymer-based implant technologies, offering a practical pathway to more accessible and efficient orthopedic solutions.\u003c/p\u003e"},{"header":"Declarations","content":"\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eF.A.R.C. and T.O.S. wrote the main manuscript text. K.F.A. and J.V.R.A. prepared Figures 1\u0026ndash;3 and conducted the main experiments. L.C.P. and F.C.R.S. performed data analysis and contributed to the preparation of Figures 4\u0026ndash;7. L.S.C.F. assisted with literature review and manuscript editing. L.R.R.S. conceived the study, supervised the project, and finalized the manuscript. \u0026Aacute;.R.M. contributed to experimental design and provided critical revisions. All authors reviewed and approved the final manuscript.\u003c/p\u003e\u003ch2\u003eACKNOWLEDGMENTS\u003c/h2\u003e \u003cp\u003eThe authors would like to thank the support of the Brazilian Association of Engineering and Mechanical Sciences (ABCM), the Brazilian National Council for Scientific and Technological Development (CNPq), Coordination for the Improvement of Higher Education Personnel (CAPES), Minas Gerais State Research Support Foundation (FAPEMIG), Laboratory of Education and Research in Machining of Federal University of Uberlandia (LEPU-UFU), Laboratory of Tribology and Materials (LTM-UFU), Laboratory of Nanobiotechnology (NANOS-UFU), Faculty of Mechanical Engineering (FEMEC-UFU) and the Postgraduate Program in Mechanical Engineering (PPGEM-UFU).\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eGrowing at a slower pace, world population is expected to reach 9.7 billion in 2050 and could peak at nearly 11 billion around 2100 | UN DESA | United Nations Department of Economic and Social Affairs. https://www.un.org/development/desa/en/news/population/world-population-prospects-2019.html. 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Polymer/calcium phosphate biocomposites manufactured by selective laser sintering: an overview. \u003cem\u003eProgress in Additive Manufacturing\u003c/em\u003e, \u003cem\u003e8\u003c/em\u003e(2), 285-301.\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":"Hydroxyapatite coating, Powder bed annealing, 3D printing, Polylactic acid (PLA), Orthopedic implants","lastPublishedDoi":"10.21203/rs.3.rs-6483704/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6483704/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThe development of effective and biocompatible coatings for polymeric implants is crucial for advancing orthopedic solutions. This study investigates the feasibility of employing powder bed annealing to deposit hydroxyapatite (HA) coatings on 3D-printed polylactic acid (PLA) parts. The proposed method provides a cost-effective and scalable alternative to conventional coating techniques. The experimental process involved immersing PLA parts in a submicrometric ceramic powder bed followed by thermal treatment to induce adhesion and diffusion of HA particles into the polymer surface. The results demonstrated that the powder bed annealing process successfully generated a uniform HA particulate coating, significantly enhancing the surface roughness, wettability, and hydrophilicity of the PLA substrate. Mechanical characterization revealed an increase in flexural strength and surface microhardness, while maintaining impact resistance. However, a slight reduction in ductility was observed. Biocompatibility tests confirmed that the coated samples supported cell adhesion and proliferation, suggesting their potential for promoting osseointegration in biomedical applications. Compared to existing methods, powder bed annealing allows for the direct integration of bioactive coatings onto polymeric implants without requiring complex post-processing. Additionally, the combination of PLA’s biodegradability with HA’s osteoinductive properties suggests promising applications for resorbable implants in bone regeneration. This study contributes to the ongoing innovation in bioactive coatings, offering a practical pathway to accessible and personalized orthopedic implants.\u003c/p\u003e","manuscriptTitle":"Production of hydroxyapatite coating on 3D printed PLA parts by powder bed annealing","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-05-08 13:48:21","doi":"10.21203/rs.3.rs-6483704/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":"cc7faf7d-6032-4406-98fb-3142904eb239","owner":[],"postedDate":"May 8th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-05-23T17:38:23+00:00","versionOfRecord":[],"versionCreatedAt":"2025-05-08 13:48:21","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-6483704","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6483704","identity":"rs-6483704","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}