Engineering 3D-printed PLA/PBS biocomposites based on tricalcium phosphate and bone powder for bone tissue applications

Engineering 3D-printed PLA/PBS biocomposites based on tricalcium phosphate and bone powder for bone tissue applications | 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 Engineering 3D-printed PLA/PBS biocomposites based on tricalcium phosphate and bone powder for bone tissue applications Luana Caroline Lima, Davi Caetano, Leonardo Pinto, Luiz Pessan, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8722217/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 5 You are reading this latest preprint version Abstract The global burden of bone fractures is projected to double by 2050, and this trend is driven by an aging population, higher incidence of car accidents, cancer-related bone damage, and osteoporosis, which compromises bone integrity. Addressing this growing challenge, bone tissue engineering (BTE) emerges as a promising strategy using polymeric scaffolds. These scaffolds must be biocompatible, bioresorbable, mechanically stable, and capable of promoting cell adhesion, proliferation, and differentiation. Therefore, this study focuses on developing biocomposite scaffolds based on a PLA/PBS (75/25 wt%) blend, incorporating biofillers—tricalcium phosphate (TCP) and bone powder (BP)—to enhance BTE. The composites were produced via melt blending and evaluated through rheological and thermal characterizations. The results indicated thermal degradation in the polymer matrix upon filler incorporation, evidenced by reduced viscosities and thermal stability. Despite this, 3D printed scaffolds demonstrated reproducibility and mechanical properties suitable for bone repair. Interestingly, increasing filler concentrations (10 and 20 wt%) did not enhance mechanical performance but did not compromise the elastic modulus compared to the pure blend and pure PLA. The cytotoxicity assay showed that the scaffolds are non-cytotoxic. These results collectively suggest that the developed scaffolds may serve as promising options for bone tissue engineering. Poly (lactic acid) Poly (butylene succinate) bone tissue engineering additive manufacturing biocomposite Figures Figure 1 Figure 2 Figure 3 Figure 4 1. INTRODUCTION The projected doubling of patients at risk of hip fracture by 2050, a trend not limited to the global scale but also affecting Brazil with a surge from 50,000 to 200,000 annual cases, underscores the urgency of our research on new bone tissue engineering strategies 1 , 2 . Population aging, car accidents, neoplasia, and the increasing prevalence of osteoporosis are the primary drivers of this surge. These conditions render bones more susceptible to fractures under minimal stress, necessitating the development of new procedures and materials for repairing bone tissue defects 1 . Tissue engineering, a dynamic and interdisciplinary field that combines biology and engineering principles, offers a promising solution to the fractures that are a hallmark of osteoporosis. It repairs damaged tissues and can potentially substitute the function of a failing organ 2 , 3 . Bone Tissue Engineering (BTE), a sub-area of regenerative medicine, aims to develop biocompatible, bioresorbable, and non-toxic scaffolds. These scaffolds, used as grafts to treat bone fractures, must mimic the characteristics of the tissue they replace. These characteristics include physical, chemical, and mechanical properties, as well as the capacity for vascularization and promoting cell adhesion, proliferation, and differentiation. This practical solution, which ensures superior cell adhesion, proliferation, and differentiation compared to traditional bone grafting techniques, is a significant advancement in the field 4 , 5 . Among the various polymers available for the manufacture of scaffolds, poly (lactic acid) (PLA) and poly (butylene succinate) (PBS) stand out for their desirable and adaptable properties for biomedical applications 6 – 9 . PLA, derived from lactic acid, is a biocompatible and bioabsorbable polymer presenting high mechanical properties. Due to these characteristics, PLA has been widely used in medical applications, such as scaffolds for cell cultures, controlled drug release systems, and bone/skin tissue regeneration. However, it has the disadvantage of a long biodegradation time, hydrophobicity, and no bioactive properties 6 , 9 . PBS is another biopolymer that has stood out in biomedicine due to its biocompatible, bioabsorbable, and biodegradable properties, as well as its good flexibility and high degree of crystallinity. Gigli et al. 10 reviewed the recent applications of PBS as a biomaterial and how its physical, mechanical, and biodegradation properties can be tuned to specific applications via copolymerization, melt blending, and surface modifications. When PLA/PBS blends are developed, PBS enhances some of PLA's properties, such as increasing flexibility, acting as a plasticizer, and reducing biodegradation time 7 , 10 , 11 . Zhang et al. 12 prepared melt-compounded PLA/PBS formulations, varying the composition by 10 wt%, and found improvements in the mechanical properties of PLA/PBS blend components depending on the chosen formulation. When PLA is the major polymeric component in the matrix, PBS acts as a dispersed component, and the blend presents an increase in elongation at break and a reduction in tensile strength and modulus of elasticity. On the other hand, when PLA is the dispersed component, the mixture is enriched by PLA particles, which improves PBS's mechanical properties. Still, more than these characteristics are needed to consolidate their use as biomaterials in BTE since they cannot promote sufficient osteoconductive properties and bone mineralization. Therefore, a myriad of strategies to improve polymer bioactivity have been investigated, including the addition of functional groups via grafting to enhance wettability or the incorporation of functional fillers similar to native bone composition, such as Hydroxyapatite (HA), β-tricalcium phosphate (TCP), and bioglasses 4 , 13 – 15 . TCP belongs to the tricalcium phosphate family. It has received considerable attention as a potential material for bone grafts due to its excellent osteoconductivity and biocompatibility in living tissues 16 , 17 . Compared to hydroxyapatite, TCP has a lower Ca/P ratio, allowing for rapid biodegradation and absorption by the body. Once incorporated into the human body, TCP dissolves and releases Ca 2+ and P 5+ ions, which help precipitate bone-like apatite, which interacts with the host's bones 16 , 18 . TCP is one of the most widely reported bioactive fillers used in BTE biomaterials, and it has also been used in composites with poly (ε-caprolactone) for long bone defect implantation, which, after almost 2 years, achieves weight-bearing properties 5 . Another type of bioceramic that has gained attention in biomedical applications and research is bovine bone matrix, typically in the form of bone powder (BP). BP is a highly processed allograft derivative that is highly compatible and easily incorporated into a polymer matrix 19 – 21 . BP provides cells with structural support and biochemical benefits, promoting efficient tissue remodeling. Additionally, BP contains extracellular matrix (ECM) proteins, such as collagen I, and several growth factors, including vascular endothelial growth factor (VEGF), transforming growth factor-β1 (TGF-β1), and bone morphogenic proteins (BMPs), among others. These components enhance osteogenic differentiation and vascularization, and stimulate bone formation and remodeling 22 , 23 . These characteristics provide support and osteoconduction structure, in addition to having a high content of calcium and phosphorus, which are essential elements for the new formation of bone tissue. For this reason, bovine bone can be used to prepare xenogeneic grafts from cortical or medullary bone substitutes 23 , 24 . Lee et al. 21 investigated the application of bone matrix obtained from demineralized and decellularized bovine bone. This material successfully promoted the mineralization of primary osteoblasts cultured in vitro and improved new bone formation in calvarial defects in mice. While PLA/TCP composites are already well-known in BTE, our research presents multiple unique features that set them apart from previous studies. We used a PLA/PBS (75/25 wt%) blend as the matrix, a formulation that is significantly less studied than pure PLA or traditional PLA-based copolymers. Moreover, we examined two biofillers—tricalcium phosphate (TCP) and bone powder (BP)—in this PLA/PBS blend, using the TCP-filled system as a comparative. Consequently, our findings offer new perspectives on how these fillers affect the composite's thermal transitions, melt rheology, and mechanical behavior. Together, the study provides an extensive evaluation of a previously overlooked polymer–biofiller combination and demonstrates its suitability for scaffolds used in bone repair. The integration of reproducible 3D printing capability, mechanical strength, and non-cytotoxic properties highlights the potential for these materials in practical applications, whereas the established compromises among filler type, concentration, and performance provide a useful foundation for future enhancements 2. EXPERIMENTAL 2.1. MATERIALS The poly (lactic acid) (PLA), supplied by NatureWorks under the trade name Ingeo™ Biopolymer 2003D, has a specific mass of 1.24 g/cm 3 and a melt fluid index (MFI) of 6.0 g/10 min (210 ºC, 2.16 kg). The poly(butylene succinate), supplied by TUNHE under the trade name TH803, has a specific mass of 1.20–1.28 g/cm 3 and an MFI of 25 g/10 min (210 ºC, 2.16 kg). β-tricalcium phosphate (TCP), with a chemical formula of Ca 3 (PO 4 ) 2 , purity greater than 90%, and a particle size distribution (d 50 ) of 6 ± 2 µm, was purchased from Fluidinova and sintered at 1000°C for 1 hour in a laboratory muffle furnace before use. BP used in this work was manufactured in our laboratories and presented a particle size distribution (d 50 ) of 50 ± 10 µm. 2.2 METHODS 2.2.1 Fabrication of Bone Powder (BP) A bovine femur bone was obtained from a local butcher, and a scalpel was used to remove any remaining fat and meat. The bone was then cleaned at approximately 100°C for 2 hours in tap water containing 260g of NaCl, followed by 1 hour in deionized water. Subsequently, the cleaned bone was dried in an oven at 100°C. Once dried, the bone was ground using a knife mill, and the resulting powder was collected and stored for further use. The bone powder was then cryo-milled and separated using a 400-mesh analytical sieve and then cleaned for two hours in a solution of acetone and Milli-Q water (1:1) under agitation. The solution was filtered, and the collected powder was dried at 100°C and then lyophilized. The preparation of the BP was adapted from Lee et al. 21 and a summary of the preparation steps is presented in Scheme 1 . 2.2.2 Characterization of Bone Powder (BP) 2.2.2.1 Particle size distribution A laser diffraction particle size analyzer (Cilas, model 1190L) was used to determine the distribution and average size of the biofiller particles. The samples were prepared as a suspension (sample + water + 1% dispersant (Darvan)) with a solid content of 10% by mass and subjected to ultrasonic agitation (probe ultrasonic, model VCX 500, Sonics) for 15 minutes to ensure complete deagglomeration of the particles. 2.2.2.2 X-ray diffraction and fluorescence The structural components of the fillers were studied using X-ray diffraction with a Siemens D5005 instrument equipped with a Cu K α source (Cu K α , λ = 1.5418 Å). The scan was conducted from 20° to 90° with a step size of 0.02° and a counting time of 1 second. Subsequently, the spectra were analyzed using the DIFFRAC.EVA (Bruker) software was used to determine the phases and structures of the biofillers. X-ray fluorescence was used to qualitatively verify the presence of elements in the composition of BP and TCP. The instrument was a Shimadzu EDX-720 Energy Dispersive X-ray Fluorescence Spectrometer, with a Rh anode operating between 5–50 kV and 1–1,000 microns. 2.2.2.3 Inductively Coupled Plasma Optical Emission Spectroscopy (ICP-OES) and Fourier Transform Infrared Spectroscopy (FTIR) ICP-OES analysis was used to determine the calcium and phosphorus contents of TCP and BP. The samples were digested in HNO 3 (1 M for 10 min), and the assay was performed using Thermo Scientific's ICP-OES iCAP 6500 Duo equipment. Quantification was then determined using the Ca 317.93 nm and P 177.49 nm wavelengths. FTIR was used to identify the organic and inorganic components of BP and TCP. The analysis was conducted using a Nicolet 6700 spectrometer from Thermo Scientific, operating in attenuated total reflection mode. The spectra were acquired in the 4000 − 500 cm − 1 range, 32 scans, and a resolution of 2 cm − 1 . 2.2.2 Development and characterization of PLA/PBS blend and biocomposites PLA, PBS, and PLA/PBS (75/25 wt%) blends, as well as PLA/PBS/TCP and PLA/PBS/BP biocomposites with TCP or BP contents of 10 and 20 wt%, were produced using an internal mixer coupled to a torque rheometer. A Haake torque rheometer, model Rheomix 600p, with counter-rotational and semi-intermeshing roller rotors, was used at 185°C. The mixing time was 5 minutes at a rotation speed of 60 rpm, after which the mixtures were collected. All subsequent analyses were conducted on these samples, including the 3D printing process. The PLA/PBS blend was named the BLD, and the composites have the matrix's name plus the first letter of the filler used and its percentage; for example, BLD20T is the composite obtained from the BLD blend with 20 wt% of TCP, and BLD20B is the composite obtained from the BLD blend with 20 wt% of BP. The rheological characterization of the BLD and the BLD biocomposites was evaluated using a controlled-stress rheometer (AR G2, TA Instruments) with a 25 mm parallel plate geometry, a 1 mm gap, and an inert nitrogen atmosphere. The tests were assessed at 185°C with a shear rate ranging from 0.01 to 100 s − 1 . Differential Scanning Calorimetry (DSC) was used to evaluate the glass transition temperature (T g ), crystalline melting temperature (T m ), and degree of crystallinity (X c ) of the PLA/PBS blend and its variation with the addition of biofillers. A TA Instruments model QS100 was used, and the samples were heated from 30°C to 200°C at a heating rate of 10°C/min. T g was obtained from the second heating cycle. The X c was calculated using the following equation: $$\:{X}_{c}\left(\%\right)=\:\frac{{\varDelta\:H}_{m}}{\:{\varDelta\:H}_{m\:}^{o}(1-N)}\:\times\:\:100$$ 1 Where N is the mass fraction of the fillers and/or PBS in the blend or composite, ∆Hm is the melting enthalpy, and ∆Hmº is the melting enthalpy considering a 100% crystalline PLA (93.7 J/g) and PBS (110.3 J/g) 25,26 . The thermal stability of the BLD and biocomposites was evaluated using thermogravimetric analysis (TGA). A TA Instruments TGA Q50 was used with a heating rate of 20°C/min, from room temperature to 800°C, under a nitrogen atmosphere. TGA was also used to characterize the fillers. The blend morphology and filler distribution in the developed compositions were evaluated using scanning electron microscopy (SEM) with a TESCAN MIRA FEG SEM, operated at 5 kV. Cross-sections of the samples were prepared by cryogenic fracturing in liquid nitrogen. Before SEM analysis, a thin layer of gold was deposited on the scaffolds using a Balzers SCD 004 sputter coater equipment. Subsequently, Energy Dispersive Spectroscopy (EDS) mapping, operated at 10 kV, was performed to analyze the dispersion and distribution of calcium and phosphorus elements in the polymeric matrix. 2.2.3 Additive manufacturing of PLA, PBS, and biocomposites PLA, PBS, BLD, and biocomposites were printed using the 3D Biotechnology Solutions (3DBS) Genesis II model. This printer has a mechanical microextrusion mechanism, which is suitable for printing polymers in pellet or powder form. The printing criteria for scaffolds were determined after several tests, ensuring the best results were achieved using a print speed of 5 mm/s, a bed temperature of 80°C, a nozzle size of 600 µm, and a temperature of 185°C. The materials used in printing were obtained after processing in the internal mixer, crushed into small granules, and dried under vacuum at 40°C for 96 hours. They were then fed directly into the metallic syringe for 3D printing the scaffolds. Individual samples were cylindrical, 8 mm in diameter, comprised of 6 layers of 500 µm, 400 µm between filaments (pore size), and a deposition pattern of 0–90◦. The drawing was sliced by the PrusaSlicer software and printed with the Repetier-Host software, which allows control of the 3DBS printer. 2.2.4 Scaffolds characterization 2.2.4.1. Mechanical analysis Following ASTM D 695 − 15, uniaxial compression tests were conducted to evaluate the mechanical properties of the scaffolds. The tests were performed using an Instron universal testing machine, model 5569, equipped with a 500N load cell and a crosshead speed of 1.3 mm/min. Five specimens were tested for each composition. Data is presented as mean ± standard deviation and analyzed using one-way analysis of variance (ANOVA) followed by Tukey’s post hoc test 2.2.4.2. Scanning electron microscopy (SEM) SEM evaluated the scaffolds' architecture using the TESCAN MIRA FEG SEM, operated at 5 keV. Samples were fractured in cryogenic conditions and gold-coated. Two samples were analyzed via SEM: the 3D-printed scaffolds and a cross-section of the samples produced through compression molding. 2.2.4.3 In vitro Biodegradation The in vitro degradation test was conducted by placing PLA, PBS, BLD, and biocomposite samples in a phosphate buffer solution at 37°C, and their weights were measured at 7, 21, and 35 days to determine mass variation. 2.2.4.4 Cell viability The osteoblast proliferation in the scaffolds was performed using pre-osteoblastic mouse cells MC3T3-1. The cells were cultured in a medium containing 89% v/v α-MEM (Gibco) supplemented with 10% fetal bovine serum (FBS, Vitrocell) and 1% antibiotic-antimycotic (Vitrocell) in an incubator (Series II 3110, Thermo Fisher Scientific) at 37°C and containing 5% CO 2 . Before the biological tests, the scaffolds were sterilized by immersion in 70% alcohol under UV light for 15 minutes on both sides. After sterilization, the samples were rinsed with phosphate buffer solution and incubated with 500 µL of culture medium for 24 hours in an incubator at 37 ºC. After this time, the samples were transferred to a new 48-well culture plate, and 500 µL of an osteoblastic cell solution with 50,000 cells was added to each well. The medium was changed every 2–3 days. A working solution was prepared for cell proliferation assay by diluting resazurin solution (#R7017, Millipore-Sigma) in a culture medium (1:9 dilution). After 1 and 7 days, the samples were transferred to a new plate, the medium was removed, and 500 µL of the working solution was added to each well. The plate was then incubated at 37 ºC for 4 h in the dark. Subsequently, 100 µL of the solution from all samples was transferred to a 96-well plate (clear), and the absorbance (560 nm/590 nm) was measured using a microplate reader (SpectraMax®M5). Three samples in 2 independent experiments per composition were analyzed, including negative controls containing a cell-free resazurin solution and an autoclaved resazurin solution as a positive control for estimating cell viability. After 1 day of cell culture on the scaffolds, cell adhesion and proliferation were analyzed by SEM. The scaffolds were transferred to a new plate, washed with phosphate buffer solution, and immersed in 1 mL of 2.5% glutaraldehyde solution for 30 minutes. The samples were then washed with a phosphate buffer solution and dehydrated using a series of ethanol concentrations (50%, 70%, 90%, and 100%). Finally, the scaffolds were dried, coated with a thin layer of gold, and analyzed using a SEM (TESCAN MIRA FEG) at an acceleration voltage of 5 keV. 3. RESULTS AND DISCUSSION 3.1 Fillers characterization TCP and BP are drawing significant attention as promising biofillers for bone tissue engineering due to their biocompatibility, osteoconductivity, and ability to promote bone regeneration. TCP, a synthetic ceramic commonly produced by wet chemistry, is known for its excellent bioactivity and supports osteoblast attachment and proliferation. BP, obtained from natural bone sources, is inexpensive, easy to produce, and can mimic human bone structure, providing an ideal scaffold for cellular growth and mineralization. Residual organic components, such as collagen, also contribute to the biological signaling required for bone repair 23 , 24 . Therefore, this section aims to characterize these biofillers before their incorporation into PLA/PBS blends. Figure 1 presents an overview of the morphological characterizations performed on BP and TCP. XRD diffractograms are shown in Fig. 1 (a), and it is observed that the TCP shows three prominent peaks at approximately 2θ = 26.72°C, 29.23°C, and 30.62°C; these peaks are attributed to crystalline β-TCP. Similarly, the XRD pattern of BP displayed multiple diffraction peaks characteristic of hydroxyapatite at 2θ = 25.8°, 31.9°, 39.7°, 45.5°, and 47.7° 21 . FTIR-ATR (Fig. 1 b-c) was used to verify the presence of functional groups in BP's structure and investigate their chemical composition after the purification processes and from a commercial TCP. The FTIR-ATR spectrum of TCP (Fig. 1 b), reveals the presence of three peaks referring to the phosphate group (PO 4 −3 ) at 1020 cm − 1 . Bands at 588 and 554 cm − 1 are attributed to the bending modes of [PO] 27 and peaks at 561 and 607 cm − 1 represent PO 4 −3 in β-TCP 28 . On the other hand, FTIR-ATR BP spectrum (Fig. 1 c) shows the presence of phosphate (PO 4 −3 ), hydroxyl (OH-), and carbonate (CO 3 −2 ) groups. An intense broadband at 1092 cm − 1 is associated with the asymmetric stretching of the phosphate group (PO 4 −3 ). The low-intensity peaks at 1460 cm − 1 and 1520 cm − 1 indicate the asymmetric v3 stretching of the carbonate groups (CO 3 −2 ); the appearance of such a group may be related to the decomposition of CO 2 present in the environment, decomposition reaction /carbonate formation. A broad peak around 3417 cm − 1 is evident in the spectrum, demonstrating the presence of adsorbed water, attributed to the ν3 and ν1 stretching modes of water molecules. Furthermore, the band at 3497 cm − 1 indicates the stretching vibration characteristic of the O–H group of HA. Similar bands were identified in previous studies by other authors, corroborating the results of this study 29 , 30 . Table 1 presents the X-ray Fluorescence (XRF) and ICP-OES analysis. The results revealed the composition of the BP sample, highlighting the presence and concentrations of the oligo-elements Zn, Fe, and Si. Surprisingly, TCP also presented some oligo-elements, such as Sr, K, and Si. The XRF is not a quantitative assay; therefore, the concentrations are not accurate. We therefore performed ICP-OES and determined the Ca/P concentration for each component. Due to its bovine nature, BP showed a lower Ca/P ratio than humans (1.67) 31 . The values in Table 1 differ from those reported in the literature; this discrepancy may be attributed to impurity fractions in the sample. Maintaining CaP moieties in the bovine bone grafts may enhance their osteogenic potential 32 . Table 1 Results of TCP and BP composition obtained through characterization by XRF and Ca/P ratio determined by ICP-OES Samples Concentration of elements (%) Ca/P ratio Ca P Si K S Sr Zn Fe Cl TCP 71.90 24.70 1.40 0.70 - 0.03 - - 1.20 1.55 BP 76.20 21.60 1.20 0.40 0.17 0.16 0.05 0.03 - 1.34 3.2 Biocomposites characterization The characterization of PLA, PBS, BLD, and composites with mass contents of 10 and 20 wt% TCP or BP is presented in Fig. 2 . The blends were developed with a PLA/PBS fraction (75/25 wt%) due to a good balance between processability and mechanical properties, and because higher PBS contents would lead to instability during printing due to differences in viscosity and the appropriate processing temperatures of the components 33 . The mixture was performed using an internal mixer coupled to a torque rheometer. As expected, each sample shows an initial peak in the torque curve, which results from feeding raw materials at RT, which are immediately subjected to shear and thermal exchange (Fig. 2 a). Torque decreases due to the melting of PLA and PBS, and a continued reduction after 2–3 min might indicate polymer thermodegradation. The curve for pure PLA gradually decreases as the processing time increases, with complete melting occurring 3 minutes after the start and at the end of 5 minutes; it has an equilibrium torque of 3.5 N.m. Similarly, PBS melts completely after approximately 2 minutes from the beginning of mixing. It has an equilibrium torque of 1.1 min, lower than PLA's, due to its lower melting temperature and higher MFI. The BLD shows an equilibrium torque of 1.6 N.m and complete melting 3 minutes after the start of the mixing process. Compared to pure PLA and pure PBS, it is possible to observe a decrease in the equilibrium peak, and torque showed an intermediate value, as expected. Finally, when analyzing the blends biocomposites with 10 and 20 wt% of TCP or BP, there was an increase in the equilibrium torque compared to the BLD (1.6 N.m for BLD and 1.9 for BLD10T). This result shows that TCP acts as a filler, increasing the mixture's viscosity during the melting process. On the other hand, the composite with 10 wt% of BP maintained the blend's equilibrium torque at 1.6 N.m. The biocomposites with 20 wt% TCP or BP showed an increased equilibrium torque compared to the BDL (2.2 for BLD20T and 2.0 for BLD20BP), suggesting that the fillers act by restraining polymeric chain mobility, reducing it, and causing a slight increase in the final torque. Rheological characterization was performed in steady-state flow, and the viscosity curves versus shear rate for PLA, PBS, BLD, and biocomposites are presented in Fig. 2 b. PLA showed the highest viscosity across all shear rates tested, and PBS, as expected, is among the lowest due to its lower molecular weight, with a 4 times lower MFI than PLA. The BLD presented an intermediate behavior between these two compositions, and it was expected that, due to the higher volume fraction, the BLD would be closer to the PLA curve; however, the low viscosity PBS fraction might act as a lubricant and reduce the chain entanglement during the test. Comparing the biocomposites with TCP and BP, it was observed that the TCP-containing blends exhibited higher viscosity. A similar trend was observed in Fig. 2 a; these curves are as close to pure PLA's. Despite this, viscosity was reduced as a function of shear rate, and these compositions started to deviate from linearity (Newtonian plateau) at lower shear rates (4–5 s − 1 ) compared to pure PLA. This behavior can be attributed to thermodegradation, which reduces molecular weight and widens the molecular weight distribution, making the lower molecular weight chains more easily deformed at lower shear rates. Similarly, the compositions with BP exhibited even lower viscosities than pure PBS, highlighting extensive thermodegradation induced by the addition of BP. Materials that have calcium in their composition can react with polymer chains, as reported by 14,34 . The compositions' thermostability was assessed by thermogravimetric analysis (Fig. 1 c, Table 2 ). PBS has the highest thermal stability, with decomposition starting at 359.5 ± 2.1 ºC, while PLA starts at 343.3 ± 1.2 ºC. Surprisingly, the BLD presented an even lower value, 331.0 ± 1.0 ºC. A possible explanation for this behavior is that the PLA/PBS blend is immiscible (DSC Curves are in Supplementary File - Figure S1 ), and the PBS might act as a plasticizer, reducing its thermal stability. Hassan et al. 35 observed a similar trend. In these compositions, the test was carried out to 800 ºC, and the obtained residue was around 1 % For the biocomposites, the reduction in viscosity observed in rheological tests and the decrease in thermal stability (T onset ) can be related to thermally induced chain scission processes catalyzed by ionic species originating from the fillers. In the TCP-containing composites, thermal degradation is primarily associated with the hydrophilicity and high surface area of TCP particles, which facilitate hydrolysis of ester linkages in PLA during melt processing 36 . This hydrolytic process promotes chain cleavage, resulting in a reduction in molecular weight, which is consistent with the lowered onset degradation temperatures. However, the TCP particles, which are well distributed, increased the viscosity at the Newtonian plateau. In contrast, BP biocomposites exhibited more pronounced degradation effects. Since the BP is retained in its mineral phase, higher concentrations of calcium and trace transition metals, such as Zn, Fe, and Sr, were observed (Table 1 ). As previously reported, the presence of these in polyesters at melt-processing temperatures may lead to severe degradation of the polymeric structure via catalyzed unzipping depolymerization, resulting in a sharp reduction in the thermal and mechanical properties 37 . It is also possible that the BP have structural water and introduced enough water to promote hydrolytic degradation of PLA during processing. This synergistic effect accounts for the substantially lower viscosity of the BP biocomposites compared to both the BLD and the TCP biocomposites. Altogether, these findings indicate that while both fillers influence polymer degradation, the mechanism in TCP composites is predominantly hydrolytic. In contrast, in BP composites, it is catalytic in nature, driven by metal-ion–mediated oxidative. Lastly, the blends with 10 and 20 wt% of TCP presented residues of 9.5 ± 0.4 and 19.5 ± 0.2, respectively. These results are consistent with the addition of 10 and 20 wt% TCP. For blends with BP, the results were slightly lower because pure BP, when heated to 800 ºC, yields a ~ 72.5% residue (Fig. 1 d). The approximately 27.5% mass loss for BP at 800°C is consistent with the presence of significant non-ceramic fractions, including residual organic content (such as collagen, proteins, and fats) and structural water. This multi-stage decomposition is well-described in the literature; for example, a recent TGA characterizations of bovine bone show an initial 10% mass loss related to moisture removal below 200°C, followed by ~ 22% loss in the 200–400°C range due to combustion of organic components, with carbonate and remnant organic groups decomposing above 400°C 38 .The TCP was sintered at 1000°C for 1 hour, and therefore, did not exhibit mass variation within this temperature range. It is essential to acknowledge that the BP used in our study is not purely ceramic (hydroxyapatite or TCP) but rather contains a substantial organic component. This explains why the BLD/BP biocomposite residue values (BLD10B: 6.3%, BLD20B: 13.8%) are lower than the nominal BP addition (10 wt% and 20 wt%), as only the inorganic part survives calcination to 800°C, while the organic content is lost during heating 38 , 39 . Figure 2 d-f presents micrographs of PLA, PBS, and BLD surface fractures, respectively. PLA exhibits a classical fragile fracture characterized by low deformation surfaces, whereas PBS shows slight deformation during fracture due to its glass transition occurring in the range of -15 to -30 ºC. Morphological analysis of the BLD revealed a drop-in-matrix morphology, with PBS forming discrete domains dispersed within the continuous PLA phase (Supplementary Figure S2 ). This morphology has direct implications for the distribution of inorganic fillers (BP and TCP), as we have not used pre-mixing or compatibilization; filler localization is predominantly governed by thermodynamic affinity, surface energy, and melt processing behavior. PBS, with its lower polarity and melting temperature, provides a more favorable interfacial environment for the fillers, particularly during processing at 185°C, leading to preferential filler migration from PLA into PBS-rich regions and at the PLA/PBS interfaces 40 . However, the particle size of the fillers further influences their spatial distribution. The larger BP particles (~ 50 µm) exceed the size of PBS droplets (1–5 µm), promoting their localization at the interface or partial dispersion within the PLA matrix, while the TCP particles (~ 6 µm) can more easily disperse within the PBS domains. Such selective filler distribution creates a heterogeneous microstructure that strongly affects interfacial stress transfer. EDS mapping was performed on blends containing TCP and BP, and this analysis aimed to evaluate their dispersion in the samples (Fig. 2 g). The TCP was better distributed, independent of its concentration, and, because it has a lower particle size, only small yellow (phosphorus) and red (calcium) pixels were observed during EDS mapping. On the contrary, despite cryo-milling, BP showed more irregular, larger agglomerates, with an average particle size of 50–55 µm. Table 2 TGA results Samples T onset (ºC) Residue (%) PLA 343.3 ± 1.2 1.2 ± 0.1 PBS 359.5 ± 2.1 1.1 ± 0.3 BLD 335.0 ± 2.5 0.2 ± 0.1 BLD10T 328.6 ± 8.1 9.5 ± 0.4 BLD10B 345.5 ± 3.5 6.3 ± 0.4 BLD20T 319.5 ± 4.9 19.7 ± 0.2 BLD20B 339.5 ± 2.1 13.8 ± 0.4 3.3 Scaffolds characterization Pure PLA, PBS, BLD, and BLD composite scaffolds were printed at 185°C and are shown in Fig. 3 a. All the scaffolds presented shape and size close to the designed geometry with a pore size of 400 µm, layer height of 500 µm, and layer width of 600 µm, as shown in Fig. 3 b-h and Table 3 , which presents the SEM results of the printed scaffolds. The micrographs of the scaffolds show that the materials are suitable for 3D printing, resulting in a controllable, reproducible structure. The scaffolds showed average pore sizes, layer heights, and layer widths between 430–522 µm, 464–544 µm, and 523–577 µm, respectively. The addition of BP to the BLD blend results in scaffold filaments with a rougher appearance, as seen in the SEM images in Figs. 3 g and 3 h, especially in scaffolds containing 20 wt% BP. In this case, cavities on the surface of the filaments and voids within the filaments are observed, likely due to volatiles that form below 185°C, as shown in the TGA analysis in Fig. 1 d. It should be noted that the scaffold's surface topography plays an important role in implant and cell responses. Macro-roughness increases implant fixation in natural tissues, while micro-roughness can, for example, stimulate osteoblast growth for differentiation and increase mineralization when compared to cells grown on a smooth surface 41 , 42 . Compression tests were used to assess the mechanical properties of the scaffolds, enabling the determination of their elastic moduli (Table 3 ). All scaffolds presented similar elastic moduli within the standard deviation, averaging 62 and 77 MPa, with no significant distinction between groups (p > 0.05). The structure of human trabecular bone is highly porous and, therefore, exhibits apparent modulus values ranging from 10 to 3,000 MPa, and these values are highly influenced by the apparent density of the bone (the mass of bone tissue divided by the total volume of the sample) 43 . Although incorporating BP and TCP led to polymeric degradation, as indicated by TGA and rheometer curves, and the formation of voids within the printed filaments (BLD20B), the extent of these was insufficient to reduce the elastic modulus compared to pure PLA and BLD. SEM corroborates (Fig. 3 h and 3 h’) these interpretations, showing that scaffolds containing higher BP loadings (20 wt%) exhibit rougher filament surfaces and internal voids, likely arising from filler aggregation and the release of volatiles during processing. These features suggest suboptimal interfacial adhesion and localized defects, which can explain the absence of significant improvements in the mechanical modulus despite the addition of reinforcing fillers. Table 3 Morphological characteristics of the scaffolds and the elastic modulus in mean ± SD Sample Layer height ( \(\:\mu\:\) m) Layer width ( \(\:\mu\:\) m) Poro size ( \(\:\mu\:\) m) Elastic modulus (MPa) PLA 491 ± 11 523 ± 15 505 ± 48 65 ± 13 PBS 544 ± 51 550 ± 61 522 ± 19 62 ± 10 BLD 505 ± 41 533 ± 30 421 ± 25 77 ± 11 BLD10T 526 ± 18 568 ± 26 447 ± 26 64 ± 10 BLD10B 464 ± 20 556 ± 37 444 ± 15 71 ± 5 BLD20T 512 ± 14 538 ± 24 430 ± 52 67 ± 8 BLD20B 509 ± 15 577 ± 54 481 ± 36 66 ± 11 The degradation of the scaffolds was analyzed as a function of time of immersion in phosphate-buffered saline by measuring scaffold weight (Fig. 4 a). The scaffolds of PBS lost 1.5 wt% of their initial mass in 35 days. This was expected due to its higher degradation rate, as demonstrated in other studies 44 , 45 . On the other hand, the mass loss of the PLA, BLD, and BLD matrix composites was negligible. Lyyra et. al observed a similar result 45 when evaluating the PLA/PBS blends profile with 25 and 50 wt% PBS. Despite the presence of the PBS component in the BLD blends and composites, the scaffolds continue to exhibit a degree of hydrolytic degradation similar to that of pure PLA, possibly because few PBS particles are exposed on the surface. However, as suggested by Wang et. al 44 when the sample's surface degrades and more PBS particles are exposed, water has a greater chance of penetrating the spaces between the PBS particles and the PLA matrix, and consequently, the hydrolytic degradation of the PLA matrix is greatly accelerated. The effect of the biofillers on cellular response was evaluated using a cell viability assay at 1 and 7 days. After 1 day of culture of MC3T3-1 pre-osteoblastic mouse cells on the scaffolds, no difference was observed in the percentage of resazurin reduction between all the scaffolds. The same behavior was observed after 7 days of cell culture, except for the PBS scaffold, which showed the lowest percentage reduction. Osteoblast cell adhesion was assessed after 1 day of cell culture on the scaffolds using scanning electron microscopy. Figure 4 c-i show the positive effect of the fillers on cell adhesion, especially on the BLD20T scaffold. This scaffold showed greater cell spreading and extended filopodia growth than the other samples. Overall, scaffolds made from BLD, TCP, and BP fillers are cytocompatible and can be further tested for their therapeutic properties in bone tissue engineering. It is worth noting that the presence of these fillers in these scaffolds can increase the proliferation of mesenchymal stem cells, calcium deposition, and the expression of osteogenic-related genes, such as RUNX2, OCN, and OPN, as demonstrated in another study 21 . 4. CONCLUSIONS This study aimed to develop composite scaffolds from the PLA/PBS blend (75/25 wt%) and TCP and BP biofillers for bone tissue regeneration. The composites were produced by melt blending and showed that the addition of TCP and BP led to thermal degradation reactions of the polymer matrix, as suggested by the lower viscosities and thermal stability observed in the rheological and thermal characterization. Despite the signs of degradation, the 3D printed scaffolds produced were reproducible and had suitable mechanical properties for bone tissue replacement. Increasing the concentration of fillers (10 and 20 wt%) did not improve the mechanical properties of the blend, but nor did it reduce the elastic modulus when compared to pure BLD and pure PLA. The scaffolds developed with BLD did not show any variation in degradation in phosphate-buffered saline within the time frame of the analysis. Finally, the cytotoxicity evaluation with mouse pre-osteoblastic cells showed that the scaffolds were not cytotoxic. Future research should explore these structures' long-term in vitro and in vivo behavior, focusing on bioresorption time and their osseointegration and osteoinduction. Declarations ACKNOWLEDGEMENTS This study was financed in part by the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - Brasil (CAPES) - Finance Code 001, Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) grant number 406258/2022-8, and Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) (grant numbers 2019/27415-2, 2022/03157-7, 2022/16119-6). The authors thank the Laboratory of Structural Characterization (LCE/DEMa/UFSCar) for access to SEM-EDS facilities. The authors would like to thank Cromex® for donating PBS matrix. Author contributions Investigation, data collection, original draft writing, review, and editing were performed by L.C.G.L, D.F.C, L.A.P, and E.H.B. Methodology, data curation, and editing of the table and figure were performed by L.A.P, D.F.C and E.H.B. Supervision, and editing of the final draft were performed by E.H.B. All authors have read and agreed to the published version of the manuscript. Conflicts of interest The authors declare that they have no conflict of interest. Data and code availability Not applicable. Supplementary information Not applicable. Ethical approval Not applicable. References Aliotta, L.; Seggiani, M.; Lazzeri, A.; Gigante, V.; Cinelli, P. A Brief Review of Poly (Butylene Succinate) (PBS) and Its Main Copolymers: Synthesis, Blends, Composites, Biodegradability, and Applications. Polymers (Basel) 2022 , 14 (4), 844. https://doi.org/10.3390/polym14040844. Sing, C.; Lin, T.; Bartholomew, S.; Bell, J. S.; Bennett, C.; Beyene, K.; Bosco‐Levy, P.; Bradbury, B. 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FigureS1.tif FigureS2BLD.tif Cite Share Download PDF Status: Under Review Version 1 posted Reviewers agreed at journal 16 Feb, 2026 Reviewers invited by journal 02 Feb, 2026 Editor invited by journal 01 Feb, 2026 Editor assigned by journal 29 Jan, 2026 First submitted to journal 28 Jan, 2026 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. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. 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-8722217","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":584058987,"identity":"3441b97c-3aaa-40d8-958b-10be9de106d0","order_by":0,"name":"Luana Caroline Lima","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Luana","middleName":"Caroline","lastName":"Lima","suffix":""},{"id":584058988,"identity":"e2773520-e418-4123-8b48-8e57c9762c6e","order_by":1,"name":"Davi Caetano","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Davi","middleName":"","lastName":"Caetano","suffix":""},{"id":584058989,"identity":"6a2f5040-400e-47c4-83e6-9cecee217e50","order_by":2,"name":"Leonardo Pinto","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Leonardo","middleName":"","lastName":"Pinto","suffix":""},{"id":584058990,"identity":"85fc86be-bae6-48e9-87f0-18b746234010","order_by":3,"name":"Luiz Pessan","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Luiz","middleName":"","lastName":"Pessan","suffix":""},{"id":584058991,"identity":"4e428d1e-b1a0-4ac6-815c-1300c2a3b0d6","order_by":4,"name":"Eduardo Henrique Backes","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA00lEQVRIiWNgGAWjYDACCR4gcYCBgR/ESSggRYtkA0iLASlaDA6AeMRo4ZfuPfjhxxkbe+PzqxM/PDBgkOcXO4Bfi+Scc8mSPTfSErfdeLtZAugww5mzE/BrMbiRYyDN8OFwgtmNsxtAWhIMbhPQYn8jx/g3w4f/9sYzzm7+QZQWA4kcM2mGGwcYN/D3biPOFokbeWmWPWeSE2fc4N1mkWAgQdgv/DNyD9/4cczOnr//7OabPyps5PmlCWhBsg+sUoJY5WD7DpCiehSMglEwCkYSAADGKEg5PB3PNgAAAABJRU5ErkJggg==","orcid":"https://orcid.org/0000-0003-2604-686X","institution":"Universidade Federal de São Carlos","correspondingAuthor":true,"prefix":"","firstName":"Eduardo","middleName":"Henrique","lastName":"Backes","suffix":""}],"badges":[],"createdAt":"2026-01-28 14:51:35","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-8722217/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-8722217/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":101789225,"identity":"7d6bc088-0b50-41e9-af0f-c0e70f7f402d","added_by":"auto","created_at":"2026-02-03 15:56:31","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":304098,"visible":true,"origin":"","legend":"\u003cp\u003eResults of the physical-chemical and morphological characterizations performed on TCP and BP: a) XRD, b - c) FTIR-ATR, d) TGA, and e-f) SEM.\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-8722217/v1/2c33a7d86102b479fd88f052.png"},{"id":101789236,"identity":"ae49762d-2c4e-4620-afcb-174a763e8fda","added_by":"auto","created_at":"2026-02-03 15:56:35","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":1085996,"visible":true,"origin":"","legend":"\u003cp\u003eCharacterization of PLA, PBS, BLD, and composites: a) Torque versus time; b) viscosity versus shear rate; c) TGA curves. Cross-sectional SEM: d) PLA, e) PBS, f) BLD; and g) EDS mapping of the composites, (calcium (Ca) in red and phosphorus (P) in green).\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-8722217/v1/b0727d87b59f28dbcb75726e.png"},{"id":101881018,"identity":"d8438e6c-4c39-416c-9be7-8583d40a386c","added_by":"auto","created_at":"2026-02-04 15:09:01","extension":"jpeg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":192209,"visible":true,"origin":"","legend":"\u003cp\u003eImages of printed scaffolds and top and cross-sectional SEM micrographs of the scaffolds: b, b’) PLA; c, c’) PBS; d, d’) BLD; e, e’) BLD10T; f, f’) BLD20T; g, g’) BLD10B e h, h’) BLD20B.\u003c/p\u003e","description":"","filename":"floatimage4.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-8722217/v1/d9076ba4733fd4743e000c31.jpeg"},{"id":101880969,"identity":"d47ff650-0193-4837-9c74-003077ecbba2","added_by":"auto","created_at":"2026-02-04 15:08:28","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":742991,"visible":true,"origin":"","legend":"\u003cp\u003ea)\u003cstrong\u003e \u003c/strong\u003eWeight of the scaffolds; b) Cytotoxicity evaluation after 1 and 7 days, and SEM images of the osteoblasts adhered to the scaffold surface after 1 day of cell culture: c) PLA; d) PBS; e) BLD; f) BLD10T; g) BLD10B; h) BLD20T and i) BLD20B.\u003c/p\u003e","description":"","filename":"floatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-8722217/v1/9e58f90eeb64fe4e669ecede.png"},{"id":102295036,"identity":"ce9b4b3e-8362-4d7e-9949-7e4b6e465029","added_by":"auto","created_at":"2026-02-10 10:07:48","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3473397,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8722217/v1/3b18795c-00b0-444e-98ba-e7eb322becb3.pdf"},{"id":101789227,"identity":"8f624c14-1e68-4b92-9911-d240c8997a27","added_by":"auto","created_at":"2026-02-03 15:56:33","extension":"png","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":210211,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eScheme 1: \u003c/strong\u003eProcedural experiment from BP preparation to scaffolds.\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-8722217/v1/fd516cd77d11b1385b09c8a7.png"},{"id":101789223,"identity":"eb9c7f69-19a2-490c-986e-b486f3569e46","added_by":"auto","created_at":"2026-02-03 15:56:31","extension":"tif","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":1756226,"visible":true,"origin":"","legend":"","description":"","filename":"FigureS1.tif","url":"https://assets-eu.researchsquare.com/files/rs-8722217/v1/dfb52b213de7d985d220836f.tif"},{"id":101789140,"identity":"c5a55275-a924-4755-b904-6b978d1c9cbe","added_by":"auto","created_at":"2026-02-03 15:56:20","extension":"tif","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":10656944,"visible":true,"origin":"","legend":"","description":"","filename":"FigureS2BLD.tif","url":"https://assets-eu.researchsquare.com/files/rs-8722217/v1/2e285646515c92e3d8e4f152.tif"}],"financialInterests":"","formattedTitle":"Engineering 3D-printed PLA/PBS biocomposites based on tricalcium phosphate and bone powder for bone tissue applications","fulltext":[{"header":"1. INTRODUCTION","content":"\u003cp\u003eThe projected doubling of patients at risk of hip fracture by 2050, a trend not limited to the global scale but also affecting Brazil with a surge from 50,000 to 200,000 annual cases, underscores the urgency of our research on new bone tissue engineering strategies \u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e,\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e. Population aging, car accidents, neoplasia, and the increasing prevalence of osteoporosis are the primary drivers of this surge. These conditions render bones more susceptible to fractures under minimal stress, necessitating the development of new procedures and materials for repairing bone tissue defects \u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eTissue engineering, a dynamic and interdisciplinary field that combines biology and engineering principles, offers a promising solution to the fractures that are a hallmark of osteoporosis. It repairs damaged tissues and can potentially substitute the function of a failing organ \u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e,\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e. Bone Tissue Engineering (BTE), a sub-area of regenerative medicine, aims to develop biocompatible, bioresorbable, and non-toxic scaffolds. These scaffolds, used as grafts to treat bone fractures, must mimic the characteristics of the tissue they replace. These characteristics include physical, chemical, and mechanical properties, as well as the capacity for vascularization and promoting cell adhesion, proliferation, and differentiation. This practical solution, which ensures superior cell adhesion, proliferation, and differentiation compared to traditional bone grafting techniques, is a significant advancement in the field \u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e,\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eAmong the various polymers available for the manufacture of scaffolds, poly (lactic acid) (PLA) and poly (butylene succinate) (PBS) stand out for their desirable and adaptable properties for biomedical applications \u003csup\u003e\u003cspan additionalcitationids=\"CR7 CR8\" citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e. PLA, derived from lactic acid, is a biocompatible and bioabsorbable polymer presenting high mechanical properties. Due to these characteristics, PLA has been widely used in medical applications, such as scaffolds for cell cultures, controlled drug release systems, and bone/skin tissue regeneration. However, it has the disadvantage of a long biodegradation time, hydrophobicity, and no bioactive properties \u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e,\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e. PBS is another biopolymer that has stood out in biomedicine due to its biocompatible, bioabsorbable, and biodegradable properties, as well as its good flexibility and high degree of crystallinity. Gigli \u003cem\u003eet al.\u003c/em\u003e\u003csup\u003e10\u003c/sup\u003e reviewed the recent applications of PBS as a biomaterial and how its physical, mechanical, and biodegradation properties can be tuned to specific applications via copolymerization, melt blending, and surface modifications.\u003c/p\u003e \u003cp\u003eWhen PLA/PBS blends are developed, PBS enhances some of PLA's properties, such as increasing flexibility, acting as a plasticizer, and reducing biodegradation time \u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e,\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e,\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e. Zhang \u003cem\u003eet al.\u003c/em\u003e\u003csup\u003e12\u003c/sup\u003e prepared melt-compounded PLA/PBS formulations, varying the composition by 10 wt%, and found improvements in the mechanical properties of PLA/PBS blend components depending on the chosen formulation. When PLA is the major polymeric component in the matrix, PBS acts as a dispersed component, and the blend presents an increase in elongation at break and a reduction in tensile strength and modulus of elasticity. On the other hand, when PLA is the dispersed component, the mixture is enriched by PLA particles, which improves PBS's mechanical properties. Still, more than these characteristics are needed to consolidate their use as biomaterials in BTE since they cannot promote sufficient osteoconductive properties and bone mineralization.\u003c/p\u003e \u003cp\u003eTherefore, a myriad of strategies to improve polymer bioactivity have been investigated, including the addition of functional groups via grafting to enhance wettability or the incorporation of functional fillers similar to native bone composition, such as Hydroxyapatite (HA), β-tricalcium phosphate (TCP), and bioglasses \u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e,\u003cspan additionalcitationids=\"CR14\" citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e. TCP belongs to the tricalcium phosphate family. It has received considerable attention as a potential material for bone grafts due to its excellent osteoconductivity and biocompatibility in living tissues \u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e,\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e. Compared to hydroxyapatite, TCP has a lower Ca/P ratio, allowing for rapid biodegradation and absorption by the body. Once incorporated into the human body, TCP dissolves and releases Ca\u003csup\u003e2+\u003c/sup\u003e and P\u003csup\u003e5+\u003c/sup\u003e ions, which help precipitate bone-like apatite, which interacts with the host's bones \u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e,\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e. TCP is one of the most widely reported bioactive fillers used in BTE biomaterials, and it has also been used in composites with poly (ε-caprolactone) for long bone defect implantation, which, after almost 2 years, achieves weight-bearing properties \u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eAnother type of bioceramic that has gained attention in biomedical applications and research is bovine bone matrix, typically in the form of bone powder (BP). BP is a highly processed allograft derivative that is highly compatible and easily incorporated into a polymer matrix\u003csup\u003e\u003cspan additionalcitationids=\"CR20\" citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e. BP provides cells with structural support and biochemical benefits, promoting efficient tissue remodeling. Additionally, BP contains extracellular matrix (ECM) proteins, such as collagen I, and several growth factors, including vascular endothelial growth factor (VEGF), transforming growth factor-β1 (TGF-β1), and bone morphogenic proteins (BMPs), among others. These components enhance osteogenic differentiation and vascularization, and stimulate bone formation and remodeling \u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e,\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e. These characteristics provide support and osteoconduction structure, in addition to having a high content of calcium and phosphorus, which are essential elements for the new formation of bone tissue. For this reason, bovine bone can be used to prepare xenogeneic grafts from cortical or medullary bone substitutes \u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e,\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eLee \u003cem\u003eet al.\u003c/em\u003e \u003csup\u003e21\u003c/sup\u003e investigated the application of bone matrix obtained from demineralized and decellularized bovine bone. This material successfully promoted the mineralization of primary osteoblasts cultured in vitro and improved new bone formation in calvarial defects in mice.\u003c/p\u003e \u003cp\u003eWhile PLA/TCP composites are already well-known in BTE, our research presents multiple unique features that set them apart from previous studies. We used a PLA/PBS (75/25 wt%) blend as the matrix, a formulation that is significantly less studied than pure PLA or traditional PLA-based copolymers. Moreover, we examined two biofillers\u0026mdash;tricalcium phosphate (TCP) and bone powder (BP)\u0026mdash;in this PLA/PBS blend, using the TCP-filled system as a comparative. Consequently, our findings offer new perspectives on how these fillers affect the composite's thermal transitions, melt rheology, and mechanical behavior. Together, the study provides an extensive evaluation of a previously overlooked polymer\u0026ndash;biofiller combination and demonstrates its suitability for scaffolds used in bone repair. The integration of reproducible 3D printing capability, mechanical strength, and non-cytotoxic properties highlights the potential for these materials in practical applications, whereas the established compromises among filler type, concentration, and performance provide a useful foundation for future enhancements\u003c/p\u003e"},{"header":"2. EXPERIMENTAL","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1. MATERIALS\u003c/h2\u003e \u003cp\u003eThe poly (lactic acid) (PLA), supplied by NatureWorks under the trade name Ingeo\u0026trade; Biopolymer 2003D, has a specific mass of 1.24 g/cm\u003csup\u003e3\u003c/sup\u003e and a melt fluid index (MFI) of 6.0 g/10 min (210 \u0026ordm;C, 2.16 kg). The poly(butylene succinate), supplied by TUNHE under the trade name TH803, has a specific mass of 1.20\u0026ndash;1.28 g/cm\u003csup\u003e3\u003c/sup\u003e and an MFI of 25 g/10 min (210 \u0026ordm;C, 2.16 kg). β-tricalcium phosphate (TCP), with a chemical formula of Ca\u003csub\u003e3\u003c/sub\u003e(PO\u003csub\u003e4\u003c/sub\u003e)\u003csub\u003e2\u003c/sub\u003e, purity greater than 90%, and a particle size distribution (d\u003csub\u003e50\u003c/sub\u003e) of 6\u0026thinsp;\u0026plusmn;\u0026thinsp;2 \u0026micro;m, was purchased from Fluidinova and sintered at 1000\u0026deg;C for 1 hour in a laboratory muffle furnace before use. BP used in this work was manufactured in our laboratories and presented a particle size distribution (d\u003csub\u003e50\u003c/sub\u003e) of 50\u0026thinsp;\u0026plusmn;\u0026thinsp;10 \u0026micro;m.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2 METHODS\u003c/h2\u003e \u003cdiv id=\"Sec5\" class=\"Section3\"\u003e \u003ch2\u003e2.2.1 Fabrication of Bone Powder (BP)\u003c/h2\u003e \u003cp\u003eA bovine femur bone was obtained from a local butcher, and a scalpel was used to remove any remaining fat and meat. The bone was then cleaned at approximately 100\u0026deg;C for 2 hours in tap water containing 260g of NaCl, followed by 1 hour in deionized water. Subsequently, the cleaned bone was dried in an oven at 100\u0026deg;C. Once dried, the bone was ground using a knife mill, and the resulting powder was collected and stored for further use. The bone powder was then cryo-milled and separated using a 400-mesh analytical sieve and then cleaned for two hours in a solution of acetone and Milli-Q water (1:1) under agitation. The solution was filtered, and the collected powder was dried at 100\u0026deg;C and then lyophilized. The preparation of the BP was adapted from Lee \u003cem\u003eet al.\u003c/em\u003e \u003csup\u003e21\u003c/sup\u003e and a summary of the preparation steps is presented in Scheme \u003cspan refid=\"Sch1\" class=\"InternalRef\"\u003e1\u003c/span\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section3\"\u003e \u003ch2\u003e2.2.2 Characterization of Bone Powder (BP)\u003c/h2\u003e \u003cdiv id=\"Sec7\" class=\"Section4\"\u003e \u003ch2\u003e2.2.2.1 Particle size distribution\u003c/h2\u003e \u003cp\u003eA laser diffraction particle size analyzer (Cilas, model 1190L) was used to determine the distribution and average size of the biofiller particles. The samples were prepared as a suspension (sample\u0026thinsp;+\u0026thinsp;water\u0026thinsp;+\u0026thinsp;1% dispersant (Darvan)) with a solid content of 10% by mass and subjected to ultrasonic agitation (probe ultrasonic, model VCX 500, Sonics) for 15 minutes to ensure complete deagglomeration of the particles.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section4\"\u003e \u003ch2\u003e2.2.2.2 X-ray diffraction and fluorescence\u003c/h2\u003e \u003cp\u003eThe structural components of the fillers were studied using X-ray diffraction with a Siemens D5005 instrument equipped with a Cu K\u003csub\u003eα\u003c/sub\u003e source (Cu K\u003csub\u003eα\u003c/sub\u003e, λ\u0026thinsp;=\u0026thinsp;1.5418 \u0026Aring;). The scan was conducted from 20\u0026deg; to 90\u0026deg; with a step size of 0.02\u0026deg; and a counting time of 1 second. Subsequently, the spectra were analyzed using the DIFFRAC.EVA (Bruker) software was used to determine the phases and structures of the biofillers.\u003c/p\u003e \u003cp\u003eX-ray fluorescence was used to qualitatively verify the presence of elements in the composition of BP and TCP. The instrument was a Shimadzu EDX-720 Energy Dispersive X-ray Fluorescence Spectrometer, with a Rh anode operating between 5\u0026ndash;50 kV and 1\u0026ndash;1,000 microns.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section4\"\u003e \u003ch2\u003e2.2.2.3 Inductively Coupled Plasma Optical Emission Spectroscopy (ICP-OES) and Fourier Transform Infrared Spectroscopy (FTIR)\u003c/h2\u003e \u003cp\u003eICP-OES analysis was used to determine the calcium and phosphorus contents of TCP and BP. The samples were digested in HNO\u003csub\u003e3\u003c/sub\u003e (1 M for 10 min), and the assay was performed using Thermo Scientific's ICP-OES iCAP 6500 Duo equipment. Quantification was then determined using the Ca 317.93 nm and P 177.49 nm wavelengths.\u003c/p\u003e \u003cp\u003eFTIR was used to identify the organic and inorganic components of BP and TCP. The analysis was conducted using a Nicolet 6700 spectrometer from Thermo Scientific, operating in attenuated total reflection mode. The spectra were acquired in the 4000\u0026thinsp;\u0026minus;\u0026thinsp;500 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e range, 32 scans, and a resolution of 2 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section3\"\u003e \u003ch2\u003e2.2.2 Development and characterization of PLA/PBS blend and biocomposites\u003c/h2\u003e \u003cp\u003ePLA, PBS, and PLA/PBS (75/25 wt%) blends, as well as PLA/PBS/TCP and PLA/PBS/BP biocomposites with TCP or BP contents of 10 and 20 wt%, were produced using an internal mixer coupled to a torque rheometer. A Haake torque rheometer, model Rheomix 600p, with counter-rotational and semi-intermeshing roller rotors, was used at 185\u0026deg;C. The mixing time was 5 minutes at a rotation speed of 60 rpm, after which the mixtures were collected. All subsequent analyses were conducted on these samples, including the 3D printing process. The PLA/PBS blend was named the BLD, and the composites have the matrix's name plus the first letter of the filler used and its percentage; for example, BLD20T is the composite obtained from the BLD blend with 20 wt% of TCP, and BLD20B is the composite obtained from the BLD blend with 20 wt% of BP.\u003c/p\u003e \u003cp\u003eThe rheological characterization of the BLD and the BLD biocomposites was evaluated using a controlled-stress rheometer (AR G2, TA Instruments) with a 25 mm parallel plate geometry, a 1 mm gap, and an inert nitrogen atmosphere. The tests were assessed at 185\u0026deg;C with a shear rate ranging from 0.01 to 100 s\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eDifferential Scanning Calorimetry (DSC) was used to evaluate the glass transition temperature (T\u003csub\u003eg\u003c/sub\u003e), crystalline melting temperature (T\u003csub\u003em\u003c/sub\u003e), and degree of crystallinity (X\u003csub\u003ec\u003c/sub\u003e) of the PLA/PBS blend and its variation with the addition of biofillers. A TA Instruments model QS100 was used, and the samples were heated from 30\u0026deg;C to 200\u0026deg;C at a heating rate of 10\u0026deg;C/min. T\u003csub\u003eg\u003c/sub\u003e was obtained from the second heating cycle. The X\u003csub\u003ec\u003c/sub\u003e was calculated using the following equation:\u003cdiv id=\"Equ1\" class=\"Equation\"\u003e\u003cdiv format=\"TEX\" class=\"mathdisplay\" id=\"FileID_Equ1\" name=\"EquationSource\"\u003e\n$$\\:{X}_{c}\\left(\\%\\right)=\\:\\frac{{\\varDelta\\:H}_{m}}{\\:{\\varDelta\\:H}_{m\\:}^{o}(1-N)}\\:\\times\\:\\:100$$\u003c/div\u003e\u003cdiv class=\"EquationNumber\"\u003e1\u003c/div\u003e\u003c/div\u003e\u003c/p\u003e \u003cp\u003eWhere N is the mass fraction of the fillers and/or PBS in the blend or composite, ∆Hm is the melting enthalpy, and ∆Hm\u0026ordm; is the melting enthalpy considering a 100% crystalline PLA (93.7 J/g) and PBS (110.3 J/g)\u003csup\u003e25,26\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eThe thermal stability of the BLD and biocomposites was evaluated using thermogravimetric analysis (TGA). A TA Instruments TGA Q50 was used with a heating rate of 20\u0026deg;C/min, from room temperature to 800\u0026deg;C, under a nitrogen atmosphere. TGA was also used to characterize the fillers.\u003c/p\u003e \u003cp\u003eThe blend morphology and filler distribution in the developed compositions were evaluated using scanning electron microscopy (SEM) with a TESCAN MIRA FEG SEM, operated at 5 kV. Cross-sections of the samples were prepared by cryogenic fracturing in liquid nitrogen. Before SEM analysis, a thin layer of gold was deposited on the scaffolds using a Balzers SCD 004 sputter coater equipment. Subsequently, Energy Dispersive Spectroscopy (EDS) mapping, operated at 10 kV, was performed to analyze the dispersion and distribution of calcium and phosphorus elements in the polymeric matrix.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section3\"\u003e \u003ch2\u003e2.2.3 Additive manufacturing of PLA, PBS, and biocomposites\u003c/h2\u003e \u003cp\u003ePLA, PBS, BLD, and biocomposites were printed using the 3D Biotechnology Solutions (3DBS) Genesis II model. This printer has a mechanical microextrusion mechanism, which is suitable for printing polymers in pellet or powder form. The printing criteria for scaffolds were determined after several tests, ensuring the best results were achieved using a print speed of 5 mm/s, a bed temperature of 80\u0026deg;C, a nozzle size of 600 \u0026micro;m, and a temperature of 185\u0026deg;C. The materials used in printing were obtained after processing in the internal mixer, crushed into small granules, and dried under vacuum at 40\u0026deg;C for 96 hours. They were then fed directly into the metallic syringe for 3D printing the scaffolds. Individual samples were cylindrical, 8 mm in diameter, comprised of 6 layers of 500 \u0026micro;m, 400 \u0026micro;m between filaments (pore size), and a deposition pattern of 0\u0026ndash;90◦. The drawing was sliced by the PrusaSlicer software and printed with the Repetier-Host software, which allows control of the 3DBS printer.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section3\"\u003e \u003ch2\u003e2.2.4 Scaffolds characterization\u003c/h2\u003e \u003cdiv id=\"Sec13\" class=\"Section4\"\u003e \u003ch2\u003e2.2.4.1. Mechanical analysis\u003c/h2\u003e \u003cp\u003eFollowing ASTM D 695\u0026thinsp;\u0026minus;\u0026thinsp;15, uniaxial compression tests were conducted to evaluate the mechanical properties of the scaffolds. The tests were performed using an Instron universal testing machine, model 5569, equipped with a 500N load cell and a crosshead speed of 1.3 mm/min. Five specimens were tested for each composition. Data is presented as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation and analyzed using one-way analysis of variance (ANOVA) followed by Tukey\u0026rsquo;s post hoc test\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section4\"\u003e \u003ch2\u003e2.2.4.2. Scanning electron microscopy (SEM)\u003c/h2\u003e \u003cp\u003eSEM evaluated the scaffolds' architecture using the TESCAN MIRA FEG SEM, operated at 5 keV. Samples were fractured in cryogenic conditions and gold-coated. Two samples were analyzed via SEM: the 3D-printed scaffolds and a cross-section of the samples produced through compression molding.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section4\"\u003e \u003ch2\u003e2.2.4.3 \u003cem\u003eIn vitro\u003c/em\u003e Biodegradation\u003c/h2\u003e \u003cp\u003eThe in vitro degradation test was conducted by placing PLA, PBS, BLD, and biocomposite samples in a phosphate buffer solution at 37\u0026deg;C, and their weights were measured at 7, 21, and 35 days to determine mass variation.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section4\"\u003e \u003ch2\u003e2.2.4.4 Cell viability\u003c/h2\u003e \u003cp\u003eThe osteoblast proliferation in the scaffolds was performed using pre-osteoblastic mouse cells MC3T3-1. The cells were cultured in a medium containing 89% v/v α-MEM (Gibco) supplemented with 10% fetal bovine serum (FBS, Vitrocell) and 1% antibiotic-antimycotic (Vitrocell) in an incubator (Series II 3110, Thermo Fisher Scientific) at 37\u0026deg;C and containing 5% CO\u003csub\u003e2\u003c/sub\u003e. Before the biological tests, the scaffolds were sterilized by immersion in 70% alcohol under UV light for 15 minutes on both sides. After sterilization, the samples were rinsed with phosphate buffer solution and incubated with 500 \u0026micro;L of culture medium for 24 hours in an incubator at 37 \u0026ordm;C. After this time, the samples were transferred to a new 48-well culture plate, and 500 \u0026micro;L of an osteoblastic cell solution with 50,000 cells was added to each well. The medium was changed every 2\u0026ndash;3 days. A working solution was prepared for cell proliferation assay by diluting resazurin solution (#R7017, Millipore-Sigma) in a culture medium (1:9 dilution). After 1 and 7 days, the samples were transferred to a new plate, the medium was removed, and 500 \u0026micro;L of the working solution was added to each well. The plate was then incubated at 37 \u0026ordm;C for 4 h in the dark. Subsequently, 100 \u0026micro;L of the solution from all samples was transferred to a 96-well plate (clear), and the absorbance (560 nm/590 nm) was measured using a microplate reader (SpectraMax\u0026reg;M5). Three samples in 2 independent experiments per composition were analyzed, including negative controls containing a cell-free resazurin solution and an autoclaved resazurin solution as a positive control for estimating cell viability. After 1 day of cell culture on the scaffolds, cell adhesion and proliferation were analyzed by SEM. The scaffolds were transferred to a new plate, washed with phosphate buffer solution, and immersed in 1 mL of 2.5% glutaraldehyde solution for 30 minutes. The samples were then washed with a phosphate buffer solution and dehydrated using a series of ethanol concentrations (50%, 70%, 90%, and 100%). Finally, the scaffolds were dried, coated with a thin layer of gold, and analyzed using a SEM (TESCAN MIRA FEG) at an acceleration voltage of 5 keV.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003c/div\u003e"},{"header":"3. RESULTS AND DISCUSSION","content":"\u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003e3.1 Fillers characterization\u003c/h2\u003e \u003cp\u003eTCP and BP are drawing significant attention as promising biofillers for bone tissue engineering due to their biocompatibility, osteoconductivity, and ability to promote bone regeneration. TCP, a synthetic ceramic commonly produced by wet chemistry, is known for its excellent bioactivity and supports osteoblast attachment and proliferation. BP, obtained from natural bone sources, is inexpensive, easy to produce, and can mimic human bone structure, providing an ideal scaffold for cellular growth and mineralization. Residual organic components, such as collagen, also contribute to the biological signaling required for bone repair \u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e,\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e. Therefore, this section aims to characterize these biofillers before their incorporation into PLA/PBS blends.\u003c/p\u003e \u003cp\u003eFigure \u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e presents an overview of the morphological characterizations performed on BP and TCP. XRD diffractograms are shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e(a), and it is observed that the TCP shows three prominent peaks at approximately 2θ\u0026thinsp;=\u0026thinsp;26.72\u0026deg;C, 29.23\u0026deg;C, and 30.62\u0026deg;C; these peaks are attributed to crystalline β-TCP. Similarly, the XRD pattern of BP displayed multiple diffraction peaks characteristic of hydroxyapatite at 2θ\u0026thinsp;=\u0026thinsp;25.8\u0026deg;, 31.9\u0026deg;, 39.7\u0026deg;, 45.5\u0026deg;, and 47.7\u0026deg; \u003csup\u003e21\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eFTIR-ATR (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eb-c) was used to verify the presence of functional groups in BP's structure and investigate their chemical composition after the purification processes and from a commercial TCP. The FTIR-ATR spectrum of TCP (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eb), reveals the presence of three peaks referring to the phosphate group (PO\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e\u0026minus;3\u003c/sup\u003e) at 1020 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e. Bands at 588 and 554 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e are attributed to the bending modes of [PO]\u003csup\u003e\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e and peaks at 561 and 607 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e represent PO\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e\u0026minus;3\u003c/sup\u003e in β-TCP \u003csup\u003e\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eOn the other hand, FTIR-ATR BP spectrum (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ec) shows the presence of phosphate (PO\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e\u0026minus;3\u003c/sup\u003e), hydroxyl (OH-), and carbonate (CO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e\u0026minus;2\u003c/sup\u003e) groups. An intense broadband at 1092 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e is associated with the asymmetric stretching of the phosphate group (PO\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e\u0026minus;3\u003c/sup\u003e). The low-intensity peaks at 1460 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e and 1520 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e indicate the asymmetric v3 stretching of the carbonate groups (CO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e\u0026minus;2\u003c/sup\u003e); the appearance of such a group may be related to the decomposition of CO\u003csub\u003e2\u003c/sub\u003e present in the environment, decomposition reaction /carbonate formation. A broad peak around 3417 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e is evident in the spectrum, demonstrating the presence of adsorbed water, attributed to the ν3 and ν1 stretching modes of water molecules. Furthermore, the band at 3497 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e indicates the stretching vibration characteristic of the O\u0026ndash;H group of HA. Similar bands were identified in previous studies by other authors, corroborating the results of this study \u003csup\u003e\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e,\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTable\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e presents the X-ray Fluorescence (XRF) and ICP-OES analysis. The results revealed the composition of the BP sample, highlighting the presence and concentrations of the oligo-elements Zn, Fe, and Si. Surprisingly, TCP also presented some oligo-elements, such as Sr, K, and Si. The XRF is not a quantitative assay; therefore, the concentrations are not accurate. We therefore performed ICP-OES and determined the Ca/P concentration for each component. Due to its bovine nature, BP showed a lower Ca/P ratio than humans (1.67) \u003csup\u003e\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003e. The values in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e differ from those reported in the literature; this discrepancy may be attributed to impurity fractions in the sample. Maintaining CaP moieties in the bovine bone grafts may enhance their osteogenic potential \u003csup\u003e\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eResults of TCP and BP composition obtained through characterization by XRF and Ca/P ratio determined by ICP-OES\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"11\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c9\" colnum=\"9\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c10\" colnum=\"10\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c11\" colnum=\"11\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eSamples\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"9\" nameend=\"c10\" namest=\"c2\"\u003e \u003cp\u003eConcentration of elements (%)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c11\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eCa/P ratio\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCa\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eP\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eSi\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eK\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eS\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003eSr\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c8\"\u003e \u003cp\u003eZn\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c9\"\u003e \u003cp\u003eFe\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c10\"\u003e \u003cp\u003eCl\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTCP\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e71.90\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e24.70\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e1.40\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.70\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e0.03\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e1.20\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c11\"\u003e \u003cp\u003e1.55\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eBP\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e76.20\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e21.60\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e1.20\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.40\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.17\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e0.16\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e0.05\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e0.03\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c11\"\u003e \u003cp\u003e1.34\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003e3.2 Biocomposites characterization\u003c/h2\u003e \u003cp\u003eThe characterization of PLA, PBS, BLD, and composites with mass contents of 10 and 20 wt% TCP or BP is presented in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e. The blends were developed with a PLA/PBS fraction (75/25 wt%) due to a good balance between processability and mechanical properties, and because higher PBS contents would lead to instability during printing due to differences in viscosity and the appropriate processing temperatures of the components \u003csup\u003e\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eThe mixture was performed using an internal mixer coupled to a torque rheometer. As expected, each sample shows an initial peak in the torque curve, which results from feeding raw materials at RT, which are immediately subjected to shear and thermal exchange (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea). Torque decreases due to the melting of PLA and PBS, and a continued reduction after 2\u0026ndash;3 min might indicate polymer thermodegradation. The curve for pure PLA gradually decreases as the processing time increases, with complete melting occurring 3 minutes after the start and at the end of 5 minutes; it has an equilibrium torque of 3.5 N.m. Similarly, PBS melts completely after approximately 2 minutes from the beginning of mixing. It has an equilibrium torque of 1.1 min, lower than PLA's, due to its lower melting temperature and higher MFI.\u003c/p\u003e \u003cp\u003eThe BLD shows an equilibrium torque of 1.6 N.m and complete melting 3 minutes after the start of the mixing process. Compared to pure PLA and pure PBS, it is possible to observe a decrease in the equilibrium peak, and torque showed an intermediate value, as expected. Finally, when analyzing the blends biocomposites with 10 and 20 wt% of TCP or BP, there was an increase in the equilibrium torque compared to the BLD (1.6 N.m for BLD and 1.9 for BLD10T). This result shows that TCP acts as a filler, increasing the mixture's viscosity during the melting process. On the other hand, the composite with 10 wt% of BP maintained the blend's equilibrium torque at 1.6 N.m. The biocomposites with 20 wt% TCP or BP showed an increased equilibrium torque compared to the BDL (2.2 for BLD20T and 2.0 for BLD20BP), suggesting that the fillers act by restraining polymeric chain mobility, reducing it, and causing a slight increase in the final torque.\u003c/p\u003e \u003cp\u003eRheological characterization was performed in steady-state flow, and the viscosity curves versus shear rate for PLA, PBS, BLD, and biocomposites are presented in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb. PLA showed the highest viscosity across all shear rates tested, and PBS, as expected, is among the lowest due to its lower molecular weight, with a 4 times lower MFI than PLA. The BLD presented an intermediate behavior between these two compositions, and it was expected that, due to the higher volume fraction, the BLD would be closer to the PLA curve; however, the low viscosity PBS fraction might act as a lubricant and reduce the chain entanglement during the test.\u003c/p\u003e \u003cp\u003eComparing the biocomposites with TCP and BP, it was observed that the TCP-containing blends exhibited higher viscosity. A similar trend was observed in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea; these curves are as close to pure PLA's. Despite this, viscosity was reduced as a function of shear rate, and these compositions started to deviate from linearity (Newtonian plateau) at lower shear rates (4\u0026ndash;5 s\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) compared to pure PLA. This behavior can be attributed to thermodegradation, which reduces molecular weight and widens the molecular weight distribution, making the lower molecular weight chains more easily deformed at lower shear rates. Similarly, the compositions with BP exhibited even lower viscosities than pure PBS, highlighting extensive thermodegradation induced by the addition of BP. Materials that have calcium in their composition can react with polymer chains, as reported by \u003csup\u003e14,34\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eThe compositions' thermostability was assessed by thermogravimetric analysis (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ec, Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). PBS has the highest thermal stability, with decomposition starting at 359.5\u0026thinsp;\u0026plusmn;\u0026thinsp;2.1 \u0026ordm;C, while PLA starts at 343.3\u0026thinsp;\u0026plusmn;\u0026thinsp;1.2 \u0026ordm;C. Surprisingly, the BLD presented an even lower value, 331.0\u0026thinsp;\u0026plusmn;\u0026thinsp;1.0 \u0026ordm;C. A possible explanation for this behavior is that the PLA/PBS blend is immiscible (DSC Curves are in Supplementary File - Figure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e), and the PBS might act as a plasticizer, reducing its thermal stability. Hassan et al. \u003csup\u003e35\u003c/sup\u003e observed a similar trend. In these compositions, the test was carried out to 800 \u0026ordm;C, and the obtained residue was around 1 %\u003c/p\u003e \u003cp\u003eFor the biocomposites, the reduction in viscosity observed in rheological tests and the decrease in thermal stability (T\u003csub\u003eonset\u003c/sub\u003e) can be related to thermally induced chain scission processes catalyzed by ionic species originating from the fillers. In the TCP-containing composites, thermal degradation is primarily associated with the hydrophilicity and high surface area of TCP particles, which facilitate hydrolysis of ester linkages in PLA during melt processing \u003csup\u003e\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u003c/sup\u003e. This hydrolytic process promotes chain cleavage, resulting in a reduction in molecular weight, which is consistent with the lowered onset degradation temperatures. However, the TCP particles, which are well distributed, increased the viscosity at the Newtonian plateau.\u003c/p\u003e \u003cp\u003eIn contrast, BP biocomposites exhibited more pronounced degradation effects. Since the BP is retained in its mineral phase, higher concentrations of calcium and trace transition metals, such as Zn, Fe, and Sr, were observed (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). As previously reported, the presence of these in polyesters at melt-processing temperatures may lead to severe degradation of the polymeric structure via catalyzed unzipping depolymerization, resulting in a sharp reduction in the thermal and mechanical properties \u003csup\u003e\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u003c/sup\u003e. It is also possible that the BP have structural water and introduced enough water to promote hydrolytic degradation of PLA during processing. This synergistic effect accounts for the substantially lower viscosity of the BP biocomposites compared to both the BLD and the TCP biocomposites. Altogether, these findings indicate that while both fillers influence polymer degradation, the mechanism in TCP composites is predominantly hydrolytic. In contrast, in BP composites, it is catalytic in nature, driven by metal-ion\u0026ndash;mediated oxidative.\u003c/p\u003e \u003cp\u003eLastly, the blends with 10 and 20 wt% of TCP presented residues of 9.5\u0026thinsp;\u0026plusmn;\u0026thinsp;0.4 and 19.5\u0026thinsp;\u0026plusmn;\u0026thinsp;0.2, respectively. These results are consistent with the addition of 10 and 20 wt% TCP. For blends with BP, the results were slightly lower because pure BP, when heated to 800 \u0026ordm;C, yields a\u0026thinsp;~\u0026thinsp;72.5% residue (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ed).\u003c/p\u003e \u003cp\u003eThe approximately 27.5% mass loss for BP at 800\u0026deg;C is consistent with the presence of significant non-ceramic fractions, including residual organic content (such as collagen, proteins, and fats) and structural water. This multi-stage decomposition is well-described in the literature; for example, a recent TGA characterizations of bovine bone show an initial 10% mass loss related to moisture removal below 200\u0026deg;C, followed by ~\u0026thinsp;22% loss in the 200\u0026ndash;400\u0026deg;C range due to combustion of organic components, with carbonate and remnant organic groups decomposing above 400\u0026deg;C \u003csup\u003e38\u003c/sup\u003e.The TCP was sintered at 1000\u0026deg;C for 1 hour, and therefore, did not exhibit mass variation within this temperature range.\u003c/p\u003e \u003cp\u003eIt is essential to acknowledge that the BP used in our study is not purely ceramic (hydroxyapatite or TCP) but rather contains a substantial organic component. This explains why the BLD/BP biocomposite residue values (BLD10B: 6.3%, BLD20B: 13.8%) are lower than the nominal BP addition (10 wt% and 20 wt%), as only the inorganic part survives calcination to 800\u0026deg;C, while the organic content is lost during heating \u003csup\u003e\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e,\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eFigure \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ed-f presents micrographs of PLA, PBS, and BLD surface fractures, respectively. PLA exhibits a classical fragile fracture characterized by low deformation surfaces, whereas PBS shows slight deformation during fracture due to its glass transition occurring in the range of -15 to -30 \u0026ordm;C.\u003c/p\u003e \u003cp\u003eMorphological analysis of the BLD revealed a drop-in-matrix morphology, with PBS forming discrete domains dispersed within the continuous PLA phase (Supplementary Figure \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e). This morphology has direct implications for the distribution of inorganic fillers (BP and TCP), as we have not used pre-mixing or compatibilization; filler localization is predominantly governed by thermodynamic affinity, surface energy, and melt processing behavior. PBS, with its lower polarity and melting temperature, provides a more favorable interfacial environment for the fillers, particularly during processing at 185\u0026deg;C, leading to preferential filler migration from PLA into PBS-rich regions and at the PLA/PBS interfaces \u003csup\u003e\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e\u003c/sup\u003e. However, the particle size of the fillers further influences their spatial distribution. The larger BP particles (~\u0026thinsp;50 \u0026micro;m) exceed the size of PBS droplets (1\u0026ndash;5 \u0026micro;m), promoting their localization at the interface or partial dispersion within the PLA matrix, while the TCP particles (~\u0026thinsp;6 \u0026micro;m) can more easily disperse within the PBS domains. Such selective filler distribution creates a heterogeneous microstructure that strongly affects interfacial stress transfer.\u003c/p\u003e \u003cp\u003eEDS mapping was performed on blends containing TCP and BP, and this analysis aimed to evaluate their dispersion in the samples (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eg). The TCP was better distributed, independent of its concentration, and, because it has a lower particle size, only small yellow (phosphorus) and red (calcium) pixels were observed during EDS mapping. On the contrary, despite cryo-milling, BP showed more irregular, larger agglomerates, with an average particle size of 50\u0026ndash;55 \u0026micro;m.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eTGA results\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"3\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSamples\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eT\u003csub\u003eonset\u003c/sub\u003e (\u0026ordm;C)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eResidue (%)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePLA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e343.3\u0026thinsp;\u0026plusmn;\u0026thinsp;1.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e1.2\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePBS\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e359.5\u0026thinsp;\u0026plusmn;\u0026thinsp;2.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e1.1\u0026thinsp;\u0026plusmn;\u0026thinsp;0.3\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eBLD\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e335.0\u0026thinsp;\u0026plusmn;\u0026thinsp;2.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e0.2\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eBLD10T\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e328.6\u0026thinsp;\u0026plusmn;\u0026thinsp;8.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e9.5\u0026thinsp;\u0026plusmn;\u0026thinsp;0.4\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eBLD10B\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e345.5\u0026thinsp;\u0026plusmn;\u0026thinsp;3.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e6.3\u0026thinsp;\u0026plusmn;\u0026thinsp;0.4\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eBLD20T\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e319.5\u0026thinsp;\u0026plusmn;\u0026thinsp;4.9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e19.7\u0026thinsp;\u0026plusmn;\u0026thinsp;0.2\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eBLD20B\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e339.5\u0026thinsp;\u0026plusmn;\u0026thinsp;2.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e13.8\u0026thinsp;\u0026plusmn;\u0026thinsp;0.4\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003e3.3 Scaffolds characterization\u003c/h2\u003e \u003cp\u003ePure PLA, PBS, BLD, and BLD composite scaffolds were printed at 185\u0026deg;C and are shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea. All the scaffolds presented shape and size close to the designed geometry with a pore size of 400 \u0026micro;m, layer height of 500 \u0026micro;m, and layer width of 600 \u0026micro;m, as shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eb-h and Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e, which presents the SEM results of the printed scaffolds. The micrographs of the scaffolds show that the materials are suitable for 3D printing, resulting in a controllable, reproducible structure. The scaffolds showed average pore sizes, layer heights, and layer widths between 430\u0026ndash;522 \u0026micro;m, 464\u0026ndash;544 \u0026micro;m, and 523\u0026ndash;577 \u0026micro;m, respectively.\u003c/p\u003e \u003cp\u003eThe addition of BP to the BLD blend results in scaffold filaments with a rougher appearance, as seen in the SEM images in Figs.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eg and \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eh, especially in scaffolds containing 20 wt% BP. In this case, cavities on the surface of the filaments and voids within the filaments are observed, likely due to volatiles that form below 185\u0026deg;C, as shown in the TGA analysis in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ed. It should be noted that the scaffold's surface topography plays an important role in implant and cell responses. Macro-roughness increases implant fixation in natural tissues, while micro-roughness can, for example, stimulate osteoblast growth for differentiation and increase mineralization when compared to cells grown on a smooth surface \u003csup\u003e\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e,\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eCompression tests were used to assess the mechanical properties of the scaffolds, enabling the determination of their elastic moduli (Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). All scaffolds presented similar elastic moduli within the standard deviation, averaging 62 and 77 MPa, with no significant distinction between groups (p\u0026thinsp;\u0026gt;\u0026thinsp;0.05).\u003c/p\u003e \u003cp\u003eThe structure of human trabecular bone is highly porous and, therefore, exhibits apparent modulus values ranging from 10 to 3,000 MPa, and these values are highly influenced by the apparent density of the bone (the mass of bone tissue divided by the total volume of the sample) \u003csup\u003e\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e\u003c/sup\u003e. Although incorporating BP and TCP led to polymeric degradation, as indicated by TGA and rheometer curves, and the formation of voids within the printed filaments (BLD20B), the extent of these was insufficient to reduce the elastic modulus compared to pure PLA and BLD. SEM corroborates (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eh and \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eh\u0026rsquo;) these interpretations, showing that \u003cem\u003escaffolds\u003c/em\u003e containing higher BP loadings (20 wt%) exhibit rougher filament surfaces and internal voids, likely arising from filler aggregation and the release of volatiles during processing. These features suggest suboptimal interfacial adhesion and localized defects, which can explain the absence of significant improvements in the mechanical modulus despite the addition of reinforcing fillers.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab3\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eMorphological characteristics of the scaffolds and the elastic modulus in mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"5\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSample\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eLayer height\u003c/p\u003e \u003cp\u003e(\u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\mu\\:\\)\u003c/span\u003e\u003c/span\u003em)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eLayer width\u003c/p\u003e \u003cp\u003e(\u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\mu\\:\\)\u003c/span\u003e\u003c/span\u003em)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003ePoro size\u003c/p\u003e \u003cp\u003e(\u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\mu\\:\\)\u003c/span\u003e\u003c/span\u003em)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eElastic modulus (MPa)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePLA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e491\u0026thinsp;\u0026plusmn;\u0026thinsp;11\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e523\u0026thinsp;\u0026plusmn;\u0026thinsp;15\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e505\u0026thinsp;\u0026plusmn;\u0026thinsp;48\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e65\u0026thinsp;\u0026plusmn;\u0026thinsp;13\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePBS\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e544\u0026thinsp;\u0026plusmn;\u0026thinsp;51\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e550\u0026thinsp;\u0026plusmn;\u0026thinsp;61\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e522\u0026thinsp;\u0026plusmn;\u0026thinsp;19\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e62\u0026thinsp;\u0026plusmn;\u0026thinsp;10\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eBLD\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e505\u0026thinsp;\u0026plusmn;\u0026thinsp;41\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e533\u0026thinsp;\u0026plusmn;\u0026thinsp;30\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e421\u0026thinsp;\u0026plusmn;\u0026thinsp;25\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e77\u0026thinsp;\u0026plusmn;\u0026thinsp;11\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eBLD10T\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e526\u0026thinsp;\u0026plusmn;\u0026thinsp;18\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e568\u0026thinsp;\u0026plusmn;\u0026thinsp;26\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e447\u0026thinsp;\u0026plusmn;\u0026thinsp;26\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e64\u0026thinsp;\u0026plusmn;\u0026thinsp;10\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eBLD10B\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e464\u0026thinsp;\u0026plusmn;\u0026thinsp;20\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e556\u0026thinsp;\u0026plusmn;\u0026thinsp;37\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e444\u0026thinsp;\u0026plusmn;\u0026thinsp;15\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e71\u0026thinsp;\u0026plusmn;\u0026thinsp;5\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eBLD20T\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e512\u0026thinsp;\u0026plusmn;\u0026thinsp;14\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e538\u0026thinsp;\u0026plusmn;\u0026thinsp;24\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e430\u0026thinsp;\u0026plusmn;\u0026thinsp;52\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e67\u0026thinsp;\u0026plusmn;\u0026thinsp;8\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eBLD20B\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e509\u0026thinsp;\u0026plusmn;\u0026thinsp;15\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e577\u0026thinsp;\u0026plusmn;\u0026thinsp;54\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e481\u0026thinsp;\u0026plusmn;\u0026thinsp;36\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e66\u0026thinsp;\u0026plusmn;\u0026thinsp;11\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eThe degradation of the scaffolds was analyzed as a function of time of immersion in phosphate-buffered saline by measuring scaffold weight (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea). The scaffolds of PBS lost 1.5 wt% of their initial mass in 35 days. This was expected due to its higher degradation rate, as demonstrated in other studies \u003csup\u003e\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e,\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e\u003c/sup\u003e. On the other hand, the mass loss of the PLA, BLD, and BLD matrix composites was negligible. Lyyra et. al observed a similar result \u003csup\u003e\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e\u003c/sup\u003e when evaluating the PLA/PBS blends profile with 25 and 50 wt% PBS. Despite the presence of the PBS component in the BLD blends and composites, the scaffolds continue to exhibit a degree of hydrolytic degradation similar to that of pure PLA, possibly because few PBS particles are exposed on the surface. However, as suggested by Wang et. al \u003csup\u003e44\u003c/sup\u003e when the sample's surface degrades and more PBS particles are exposed, water has a greater chance of penetrating the spaces between the PBS particles and the PLA matrix, and consequently, the hydrolytic degradation of the PLA matrix is greatly accelerated.\u003c/p\u003e \u003cp\u003eThe effect of the biofillers on cellular response was evaluated using a cell viability assay at 1 and 7 days. After 1 day of culture of MC3T3-1 pre-osteoblastic mouse cells on the scaffolds, no difference was observed in the percentage of resazurin reduction between all the scaffolds. The same behavior was observed after 7 days of cell culture, except for the PBS scaffold, which showed the lowest percentage reduction. Osteoblast cell adhesion was assessed after 1 day of cell culture on the scaffolds using scanning electron microscopy. Figure\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ec-i show the positive effect of the fillers on cell adhesion, especially on the BLD20T scaffold. This scaffold showed greater cell spreading and extended filopodia growth than the other samples. Overall, scaffolds made from BLD, TCP, and BP fillers are cytocompatible and can be further tested for their therapeutic properties in bone tissue engineering. It is worth noting that the presence of these fillers in these scaffolds can increase the proliferation of mesenchymal stem cells, calcium deposition, and the expression of osteogenic-related genes, such as RUNX2, OCN, and OPN, as demonstrated in another study \u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"4. CONCLUSIONS","content":"\u003cp\u003eThis study aimed to develop composite scaffolds from the PLA/PBS blend (75/25 wt%) and TCP and BP biofillers for bone tissue regeneration. The composites were produced by melt blending and showed that the addition of TCP and BP led to thermal degradation reactions of the polymer matrix, as suggested by the lower viscosities and thermal stability observed in the rheological and thermal characterization. Despite the signs of degradation, the 3D printed scaffolds produced were reproducible and had suitable mechanical properties for bone tissue replacement. Increasing the concentration of fillers (10 and 20 wt%) did not improve the mechanical properties of the blend, but nor did it reduce the elastic modulus when compared to pure BLD and pure PLA. The scaffolds developed with BLD did not show any variation in degradation in phosphate-buffered saline within the time frame of the analysis. Finally, the cytotoxicity evaluation with mouse pre-osteoblastic cells showed that the scaffolds were not cytotoxic. Future research should explore these structures' long-term in vitro and in vivo behavior, focusing on bioresorption time and their osseointegration and osteoinduction.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eACKNOWLEDGEMENTS\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was financed in part by the Coordena\u0026ccedil;\u0026atilde;o de Aperfei\u0026ccedil;oamento de Pessoal de N\u0026iacute;vel Superior - Brasil (CAPES) - Finance Code 001, Conselho Nacional de Desenvolvimento Cient\u0026iacute;fico e Tecnol\u0026oacute;gico (CNPq) grant number 406258/2022-8, and Funda\u0026ccedil;\u0026atilde;o de Amparo \u0026agrave; Pesquisa do Estado de S\u0026atilde;o Paulo (FAPESP) (grant numbers 2019/27415-2, 2022/03157-7, 2022/16119-6). The authors thank the Laboratory of Structural Characterization (LCE/DEMa/UFSCar) for access to SEM-EDS facilities. \u0026nbsp;The authors would like to thank Cromex\u0026reg; for donating PBS matrix.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eInvestigation, data collection, original draft writing, review, and editing were performed by L.C.G.L, D.F.C, L.A.P, and E.H.B. Methodology, data curation, and editing of the table and figure were performed by L.A.P, D.F.C and E.H.B. Supervision, and editing of the final draft were performed by E.H.B. All authors have read and agreed to the published version of the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflicts of interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no conflict of interest.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData and code availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSupplementary information\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthical approval\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eAliotta, L.; Seggiani, M.; Lazzeri, A.; Gigante, V.; Cinelli, P. 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Hydrolytic Degradation of Polylactide/Polybutylene Succinate Blends with Bioactive Glass. \u003cem\u003eMater Today Commun\u003c/em\u003e \u003cstrong\u003e2023\u003c/strong\u003e, \u003cem\u003e37\u003c/em\u003e, 107242. https://doi.org/10.1016/j.mtcomm.2023.107242.\u003c/li\u003e\n\u003c/ol\u003e"},{"header":"Scheme","content":"\u003cp\u003eScheme 1 is available in the Supplementary Files section.\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":true,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"journal-of-polymer-research","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"jpol","sideBox":"Learn more about [Journal of Polymer Research](https://www.springer.com/journal/10965)","snPcode":"10965","submissionUrl":"https://www.editorialmanager.com/jpol/","title":"Journal of Polymer Research","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Poly (lactic acid), Poly (butylene succinate), bone tissue engineering, additive manufacturing, biocomposite","lastPublishedDoi":"10.21203/rs.3.rs-8722217/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8722217/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThe global burden of bone fractures is projected to double by 2050, and this trend is driven by an aging population, higher incidence of car accidents, cancer-related bone damage, and osteoporosis, which compromises bone integrity. Addressing this growing challenge, bone tissue engineering (BTE) emerges as a promising strategy using polymeric scaffolds. These scaffolds must be biocompatible, bioresorbable, mechanically stable, and capable of promoting cell adhesion, proliferation, and differentiation. Therefore, this study focuses on developing biocomposite scaffolds based on a PLA/PBS (75/25 wt%) blend, incorporating biofillers\u0026mdash;tricalcium phosphate (TCP) and bone powder (BP)\u0026mdash;to enhance BTE. The composites were produced via melt blending and evaluated through rheological and thermal characterizations. The results indicated thermal degradation in the polymer matrix upon filler incorporation, evidenced by reduced viscosities and thermal stability. Despite this, 3D printed scaffolds demonstrated reproducibility and mechanical properties suitable for bone repair. Interestingly, increasing filler concentrations (10 and 20 wt%) did not enhance mechanical performance but did not compromise the elastic modulus compared to the pure blend and pure PLA. The cytotoxicity assay showed that the scaffolds are non-cytotoxic. These results collectively suggest that the developed scaffolds may serve as promising options for bone tissue engineering.\u003c/p\u003e","manuscriptTitle":"Engineering 3D-printed PLA/PBS biocomposites based on tricalcium phosphate and bone powder for bone tissue applications","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-02-03 15:55:10","doi":"10.21203/rs.3.rs-8722217/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"reviewerAgreed","content":"","date":"2026-02-16T14:02:19+00:00","index":0,"fulltext":""},{"type":"reviewersInvited","content":"","date":"2026-02-02T06:22:25+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"Journal of Polymer Research","date":"2026-02-01T20:07:48+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-01-29T11:40:01+00:00","index":"","fulltext":""},{"type":"submitted","content":"Journal of Polymer Research","date":"2026-01-28T09:50:36+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"journal-of-polymer-research","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"jpol","sideBox":"Learn more about [Journal of Polymer Research](https://www.springer.com/journal/10965)","snPcode":"10965","submissionUrl":"https://www.editorialmanager.com/jpol/","title":"Journal of Polymer Research","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"6704224e-a886-4a0b-a5ca-b77dec43edd8","owner":[],"postedDate":"February 3rd, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2026-03-31T08:01:48+00:00","versionOfRecord":[],"versionCreatedAt":"2026-02-03 15:55:10","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8722217","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8722217","identity":"rs-8722217","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}