TCP Doped with Metal Ions Reinforced with Tetragonal and Cubic Zirconia

Ceramic biocomposites based on bioactive tricalcium phosphate doped with metal ions are a strategy for obtaining good biomimetics for human bone composition. Manufacturing with PMMA porogen also induces bone-like porosity morphology. The poor strength of tricalcium phosphate can be overcomed by designing ceramic composites reinforced with tetragonal and cubic zirconia. In this work, five different bioceramic composites were manufactured without and with induced porosity and their physical, mechanical, microstructural, and biological properties were studied. With the addition of tetragonal and cubic zirconia, an improvement in strength of 22% and 55%, respectively, was obtained, corresponding to up to 20.7 MPa. PMMA was suitable for adding porosity, up to 30%, with interconnectivity while an excellent hOB cellular viability was achieved for all biocomposites.


Introduction
The average life expectancy has increased, causing the aging of the world population.With this, health complications related to joints and bone tissue, such as osteoporosis and osteoarthritis, can arise [1,2].Therefore, finding alternatives that provide quality of life to the patient is essential.Inert bioceramics (e.g., zirconia, ZrO 2 ), do not form biochemical bonds with surrounding tissues, i.e., do not react with the body [3].Physical, chemical, and mechanical properties such as high compressive strength, wear and corrosion resistance, hardness, elastic modulus similar to that of steel, high fracture toughness, and stability in a physiological environment, make ZrO 2 a material of the highest interest for the manufacture of orthopedic and dental prostheses [2,4].
At room temperature and in pure form, ZrO 2 has a stable monoclinic crystalline structure (m-ZrO 2 ) up to 1170 • C. With increasing temperature, it becomes metastable: tetragonal (t-ZrO 2 ) stable up to 2370 • C and above this temperature, cubic (c-ZrO 2 ) [4][5][6].The m-ZrO 2 is not suitable for high temperature applications due to the volume expansion associated with the transformation of t-ZrO 2 to m-ZrO 2 [7,8], known as martensitic transformation [5,9].In this transformation there is an increase in volume of approximately 4.5% during cooling [6,8] which is detrimental to the mechanical behavior of ZrO 2 because the stress induced during this transformation leads to the formation of cracks [5].In this sense, it can be concluded that the oral environment is a strongly predisposing factor for uncontrolled martensitic transformation [6].
Stabilizers such as yttrium oxide (Y 2 O 3 ) can be added to ZrO 2 to inhibit the transformation of the t-phase to m-phase.Briefly, this addition results in the part of the Zr +4 atoms being replaced by Y +3 atoms, stabilizing the polymorphic modifications of ZrO 2 when subjected to the sintering process.This avoids the volume variations caused by phase transformations [6,10] that form yttrium-stabilized zirconia (YSZ), where 3YSZ is mostly tetragonal and 8YSZ is mostly the cubic crystalline phase [11].However, several studies For successful applications, bone structures must mimic the porosity of native bone and allow it to grow through the interconnectivity of the structure [31].Polymethylmethacrylate (PMMA) has been considered for its particular characteristics, such as mechanical strength, moldability to fill complex defects, low cost, having approval by the FDA and with clean and easy thermal elimination at high temperatures [32][33][34].
The aim of this work was to produce dense ceramic biocomposites and PMMA induced porous ceramic biocomposites, both compositions based in TCP, doped with metal ions of magnesium (Mg 2+ ), manganese (Mn 2+ ), zinc (Zn 2+ ), and iron (Fe 3+ ).The two types of manufactured samples were reinforced with 10 wt% and 20 wt% of tetragonal zirconia (3YSZ) and cubic zirconia (8YSZ).A detailed evaluation of their physical, mechanical, microstructural, and biological properties was performed.
To each of the compositions with YSZ, 3YSZ and 8YSZ were added according to the corresponding mass fraction.Then, 15 g of the doped or substituted TCP (sTCP) was added according to the mass fraction, and 15 g of YSZ balls and 30 g of isopropyl alcohol were mixed in a high energy ball mill [35].The milling, drying, and sieving processes occurred under the previous conditions.The particle size distribution (PSD) of the powders of the reference material (sTCP, which give rise to the material designated 10T) and the other compositions with the mixture between 80% of sTCP, 20% of cubic zirconia (8T2cZ), and 20% of tetragonal zirconia (8T2tZ) are shown in Figure 1.To each of the compositions with YSZ, 3YSZ and 8YSZ were added according corresponding mass fraction.Then, 15 g of the doped or substituted TCP (sTCP added according to the mass fraction, and 15 g of YSZ balls and 30 g of isopropyl a were mixed in a high energy ball mill [35].The milling, drying, and sieving pro occurred under the previous conditions.The particle size distribution (PSD) of the ders of the reference material (sTCP, which give rise to the material designated 10 the other compositions with the mixture between 80% of sTCP, 20% of cubic zi (8T2cZ), and 20% of tetragonal zirconia (8T2tZ) are shown in Figure 1.The PSD of 10T powders is bimodal: with a major peak at 10 µm and another peak at 1 µm.Both compositions have zirconia (8T2cZ and 8T2tZ) present, instead with two small minor peaks of 0.5 and 1.8 microns, respectively.
To induce porosity, PMMA spheres were added to each of the five composit the mass fraction 60:40 (each of the five compositions: PMMA, respectively).In or obtain a homogeneous mixture, the mixture was placed in the Fritsch mill for 20 s rpm.
Cylindric pellets of bioceramic composites with mass of ~0.65 g, ~13 mm dia and 3 mm thickness were made using a universal electromechanical testing machin madzu, AGS-X, Kyoto, Japan) with a 13 mm diameter stainless steel matrix in whic axial pressure of 50 MPa was applied for 10 s.Then, the samples were sintered in a temperature of 1300 °C for 120 min with a heating rate of 5 °C/min.

Microstructural and Mechanical Characterization
To confirm if the doping reaction occurred, the XRD test was performed in the biocomposites 10T, 8T2tZ, and 8T2cZ, since 8T2tZ and 8T2cZ have the highest conc tion of each ZrO2 studied and 10T the same concentration of each ion without the ad of 3YSZ and 8YSZ.An X-ray diffractometer (DMAX III/C, Rigaku, Tokyo, Japan) w The PSD of 10T powders is bimodal: with a major peak at 10 µm and another minor peak at 1 µm.Both compositions have zirconia (8T2cZ and 8T2tZ) present, instead of one, with two small minor peaks of 0.5 and 1.8 microns, respectively.
To induce porosity, PMMA spheres were added to each of the five compositions in the mass fraction 60:40 (each of the five compositions: PMMA, respectively).In order to obtain a homogeneous mixture, the mixture was placed in the Fritsch mill for 20 s at 500 rpm.
Cylindric pellets of bioceramic composites with mass of ~0.65 g, ~13 mm diameter and 3 mm thickness were made using a universal electromechanical testing machine (Shimadzu, AGS-X, Kyoto, Japan) with a 13 mm diameter stainless steel matrix in which uniaxial pressure of 50 MPa was applied for 10 s.Then, the samples were sintered in air at a temperature of 1300 • C for 120 min with a heating rate of 5 • C/min.

Microstructural and Mechanical Characterization
To confirm if the doping reaction occurred, the XRD test was performed in the dense biocomposites 10T, 8T2tZ, and 8T2cZ, since 8T2tZ and 8T2cZ have the highest concentration of each ZrO 2 studied and 10T the same concentration of each ion without the addition of 3YSZ and 8YSZ.An X-ray diffractometer (DMAX III/C, Rigaku, Tokyo, Japan) with the Bragg-Brentano (θ/2θ) horizontal geometry was used.The X-ray tube of copper (wavelength of 1.5405 Å) operated at 40 kV at 30 mA using CuKα radiation.The intensity of diffracted radiation as function of the 2θ diffraction angle was obtained between 5 • and 90 • .The diffractograms obtained were compared with the theoretical cards available in the ICDD database of the MDI/JADE, version 6 analysis software.The contents of each crystalline phase of the compositions in %vol were quantified through Rietveld refinements using the FullProf software, version May2021 [36].Scanning electron microscopy (SEM) (Hitachi S-2700, Tokyo, Japan), was performed for microstructure imaging by applying the SE mode at an accelerating voltage of 20 kV.Chemical analysis was performed using SEM with energy dispersive X-ray probe (EDX, Brucker Quantax 400, Elk Grove Village, IL, USA).This analysis was carried out using the average of three different areas, where the gold peaks were not considered and all other peaks corresponding to Ca, P, and O and also the metals such as Zr, Mg, Mn, Fe, and Zn were quantified.
Fourier transform infrared spectroscopy (FTIR) was performed using a Nicolet TM iS 10 FTIR spectrometer (Thermo Scientific Inc., Waltham, MA, USA).Infrared spectra were recorded in the range of 525-4000 cm −1 at ambient temperature and with a resolution of 4 cm −1 (32 scans).
Apparent porosity and bulk density were measured according to ASTM C20-00 [37].In this procedure three different weights, in grams, were calculated: dry sample weight (D), saturated weight (W), after boiling for 2 h and resting 12 h entirely covered with water, and suspended immersed in water weight (S).Considering the density of the water equal to 1 g/cm 3 , the apparent porosity, P, in %, expressing the relationship of the volume of the open pores in the specimen to its exterior volume, is calculated using Equation ( 1): The bulk density, BD, in g/cm 3 , is the quotient of its dry weight divided by the exterior volume, including pores, and is calculated by Equation ( 2): The diametral compression test was conducted with a universal electromechanical testing machine (Shimadzu, AGS-X, Japan) with a load cell of 10,000 N [38] and a displacement rate of 0.5 mm/min [39].The tensile strength, σ x , was determined by Equation ( 3) where F corresponds to the maximum force applied, d, the diameter, and e, the thickness of the cylindric sample: In Figure 2, the illustrations of dense and porous ceramic samples are observed after the fracture occurs during the diametral compression test.
diffracted radiation as function of the 2θ diffraction angle was obtained between 5 90°.The diffractograms obtained were compared with the theoretical cards availa the ICDD database of the MDI/JADE, version 6 analysis software.The contents of crystalline phase of the compositions in %vol were quantified through Rietveld r ments using the FullProf software, version May2021 [36].Scanning electron micro (SEM) (Hitachi S-2700, Tokyo, Japan), was performed for microstructure imaging b plying the SE mode at an accelerating voltage of 20 kV.Chemical analysis was perfo using SEM with energy dispersive X-ray probe (EDX, Brucker Quantax 400, Elk G Village, IL, USA).This analysis was carried out using the average of three different where the gold peaks were not considered and all other peaks corresponding to Ca, P O and also the metals such as Zr, Mg, Mn, Fe, and Zn were quantified.
Fourier transform infrared spectroscopy (FTIR) was performed using a Nicole 10 FTIR spectrometer (Thermo Scientific Inc., Waltham, MA, USA).Infrared spectra recorded in the range of 525-4000 cm −1 at ambient temperature and with a resolutio cm −1 (32 scans).
Apparent porosity and bulk density were measured according to ASTM C20-00 In this procedure three different weights, in grams, were calculated: dry sample w (D), saturated weight (W), after boiling for 2 h and resting 12 h entirely covered wit ter, and suspended immersed in water weight (S).Considering the density of the equal to 1 g/cm 3 , the apparent porosity, P, in %, expressing the relationship of the vo of the open pores in the specimen to its exterior volume, is calculated using Equatio The bulk density, BD, in g/cm 3 , is the quotient of its dry weight divided by the rior volume, including pores, and is calculated by Equation ( 2): The diametral compression test was conducted with a universal electromech testing machine (Shimadzu, AGS-X, Japan) with a load cell of 10,000 N [38] and a disp ment rate of 0.5 mm/min [39].The tensile strength, σx, was determined by Equatio where F corresponds to the maximum force applied, d, the diameter, and e, the thic of the cylindric sample: In Figure 2, the illustrations of dense and porous ceramic samples are observed the fracture occurs during the diametral compression test.
Discs similar to those used in the mechanical characterization (Figure 2) of ~13 mm in diameter and 3 mm in thickness were broken into six identical parts, and before starting the biological tests, were sterilized by ultraviolet irradiation (UV) for 1 h [1].The cells were seeded at a density of 15,000, 10,000, and 2500 cells/well in three 48-well plates for 24 h at 37 • C.Then, the medium was removed, and the cells were incubated with 10% of material in relation to the well area [41] and 300 µL of DMEM-F12 in all wells.After 1, 3, and 7 days of incubation, the medium and the material were removed and the hOB were incubated with 220 µL resazurin 10% (v/v) [40,42].Cell viability was determined by measuring resorufin fluorescence at λ ex = 545 nm and λ em = 590 nm [40,42].The plate corresponding to day 1 of incubation with material contained a density of 15,000 cells/well, day 3, 10,000 cells/well, and day 7 plate, 2500 cells/well.Negative control (K − ) cells were incubated only with culture medium and positive control (K + ) cells were incubated with bleach.
The cellular attachment to porous biocomposites was assessed by SEM analysis in BSE 3D mode (Backscattered electrons) with an acceleration voltage of 20 kV.The cells were fixed with 500 µL of glutaraldehyde at 2.5% (v/v) for 1 h.After, the samples were dehydrated with increasing ethanol concentrations (50, 70, and 99%) for 5 min each, frozen at −80 • C for 1 h and freeze-dried for 3 h.Then, the samples were coated with gold using a turbomolecular pumped coater.

Microstructural Properties
Representative SEM micrographs of the fracture surfaces of the sintered ceramic composites are shown in Figure 3.In all cases there are mixed fracture surfaces, that is, transgranular (examples illustrated by letter "T" in Figure 3) and intergranular fractures (examples illustrated by letter "I" in Figure 3).Regarding porosity, it is evident that an interconnected porosity network exists even in dense biocomposites.In addition to perfectly spherical pores, that is, pores caused by PMMA spheres, more elongated pores are also observed.
The macropores caused by the PMMA spheres, visible in the image at 200× magnification, present an average size of 100 µm, between 120-150 µm and between 110-140 µm for the 10T, 8T2tZ, and 8T2cZ biocomposites, respectively.In this way, bone growth and cell colonization have good conditions to occur.
XRD assays were performed to confirm the presence of ZrO 2 crystalline phases in the dense biocomposites 8T2tZ and 8T2cZ.Figure 4 shows the X-ray diffraction spectra of the analyzed biocomposites.In the 10T biocomposites, in addition to other small, identified peaks, four main peaks of higher intensity were identified for 2θ = 27 • , 32 • , 34 • , and 53 • that correspond to β-TCP and are coincident with their theoretical card.The predominant structure is β-TCP (~78%vol according to Table 2).Also, in 10T samples, HA is the second most present phase, and α-TCP, with a very low value, can be neglected.The existence of α-TCP was not expected, because of the doping of TCP with 10% MgO.According to the literature [24], with the addition of Mg 2+ to TCP, the transformation temperature of β-TCP to α-TCP is expected to increase.This is also promoted by the relative high temperature of sintering.In biocomposites 8T2tZ and 8T2cZ, four main peaks were identified for 2θ = 30.2• , 31.1 • , 50.4 • , and 59.7 • corresponding to t-ZrO 2 , β-TCP, t-ZrO 2 , and also t-ZrO 2 , respectively, for 8T2tZ; in 8T2cZ main peaks for 2θ = 30 • , 31.1 • , 50.1 • and 59.7 • were identified, corresponding to c-ZrO 2 , β-TCP, c-ZrO 2 , and also c-ZrO 2 , respectively.For both cases, the peaks coincide with the respective theoretical cards.The macropores caused by the PMMA spheres, visible in the image at 200× magnification, present an average size of 100 µm, between 120-150 µm and between 110-140 µm for the 10T, 8T2tZ, and 8T2cZ biocomposites, respectively.In this way, bone growth and cell colonization have good conditions to occur.
XRD assays were performed to confirm the presence of ZrO2 crystalline phases in the dense biocomposites 8T2tZ and 8T2cZ.Figure 4 shows the X-ray diffraction spectra of the analyzed biocomposites.In the 10T biocomposites, in addition to other small, identified peaks, four main peaks of higher intensity were identified for 2θ = 27°, 32°, 34°, and 53° that correspond to β-TCP and are coincident with their theoretical card.The predominant structure is β-TCP (~78%vol according to Table 2).Also, in 10T samples, HA is the second most present phase, and α-TCP, with a very low value, can be neglected.The existence of α-TCP was not expected, because of the doping of TCP with 10% MgO.According to the literature [24], with the addition of Mg 2+ to TCP, the transformation temperature of β-TCP to α-TCP is expected to increase.This is also promoted by the relative high temperature of sintering.In biocomposites 8T2tZ and 8T2cZ, four main peaks were identified for 2θ = 30.2°,31.1°,50.4°, and 59.7° corresponding to t-ZrO2, β-TCP, t-ZrO2, and also t-ZrO2, respectively, for 8T2tZ; in 8T2cZ main peaks for 2θ = 30°, 31.1°,50.1° and 59.7° were identified, corresponding to c-ZrO2, β-TCP, c-ZrO2, and also c-ZrO2, respectively.For both cases, the peaks coincide with the respective theoretical cards.The main phases of the biocomposites analyzed were quantified by Rietveld refinement, as shown on Table 2.According to Table 2, the predominant phase is β-TCP, presenting values of 77.60, 71.09, and 77.50%vol for biocomposites 10T, 8T2tZ, and 8T2cZ, respectively.HA was only detected in 8T2tZ and 10T, with values of 3.05 and 21.69%vol, respectively.In biocomposites with 8YSZ, mostly this phase corresponds to ZrO2, c-ZrO2, The main phases of the biocomposites analyzed were quantified by Rietveld refi ment, as shown on Table 2.According to Table 2, the predominant phase is β-TCP, p senting values of 77.60, 71.09, and 77.50%vol for biocomposites 10T, 8T2tZ, and 8T2 ), and α-TCP, respectively.The main phases of the biocomposites analyzed were quantified by Rietveld refinement, as shown on Table 2.According to Table 2, the predominant phase is β-TCP, presenting values of 77.60, 71.09, and 77.50%vol for biocomposites 10T, 8T2tZ, and 8T2cZ, respectively.HA was only detected in 8T2tZ and 10T, with values of 3.05 and 21.69%vol, respectively.In biocomposites with 8YSZ, mostly this phase corresponds to ZrO 2 , c-ZrO 2 , whose value is 21.02%vol, and a small percentage of t-ZrO 2 , 1.48%vol, which indicates that during the manufacturing process the c-ZrO 2 was converted into t-ZrO 2, probably due to high temperatures.In the biocomposites with 3YSZ, only t-ZrO 2, was quantified, 25.86%vol.
No metal oxides were detected, including MgO, which indicates that during the calcination process, the Ca 2+ ions were replaced by the metal ions of Mg 2+ , Mn 2+ , Zn 2+ , and Fe 3+ into sites of the crystalline structure of β-TCP.
In order to understand the presence of several peaks corresponding to HA, the XRD was carried out for sTCP after the calcination process and compared with the XRD after the sintering process (Figure S1).The main peaks correspond, in both spectra, to the TCP beta phase.The presence of TCP alpha crystalline phase peaks is unclear.In the calcined sTCP sample, the peaks for 2θ = 29 • , 31.8 • , 32.2 • , 32.9 • , 34.0 • , 39.2 • , 46.7 • , 49.5 • , and 63.0 • correspond to the hydroxyapatite phase.In the sintered phase at 1300 • C, the number of HA peaks decreases, essentially leaving the relevant peaks at 31.8 • , 32.2 • , and 32.9 • .
The formation of hydroxyapatite in this case most likely occurs through what in the literature is called mechanochemical synthesis [43,44].These mechanochemical reactions resulted in the formation of a defective phase of calcium-deficient HA, which, when calcined at up to 720 • C, leads to the formation of HA and β-TCP [43].The principle of this dynamic synthesis during grinding is related to the energy during grinding (the impact that the mill balls have on the powdered grains).In other words, a reaction and an interdiffusion mechanism are promoted between different grains or with an intimate chemical reaction of the molecules between them [44].In this sense, similar to the present work, after 5 h of mechanical activation, Yeong et al. [43] obtained a 2θ of 31.8 • as the most prominent peak, corresponding to the crystalline plane of HA (211).
In fact, the growth of the HA phase, which is directly related to the improvement of biocompatibility and osteoinduction, is important in bone reconstruction [1,18,45].
From the elementary chemical analysis (EDX), Table 3, the presence of the elements Ca, P, Zr, and the remaining added metal ions were verified.
The elements Ca and P come from the chemical formula of TCP, (Ca 3 (PO 4 ) 2 ), and the Zr from the doping with the two types (tetragonal and cubic) of ZrO 2 used in the composition of the ceramic biocomposites.The Ca/P ratio, in wt%, of 2.68, 3.53, and 3.37, and in mol% of the 2.07, 2.73, and 2.60 for the ceramic biocomposites 10T, 8T2tZ, and 8T2cZ, respectively is slightly high when compared to the works reported in the literature, namely, Wu et al. [46], which refers to bone minerals with Ca/P ratio between 1.37 and 1.87 mol%.With the addition of the 3YSZ and 8YSZ, this Ca/P ratio, tends to increase, with its highest value being in ceramic biocomposites with 20% of 3YSZ.
Figure 5 shows the characteristic FTIR spectra of the biocomposites.From the FTIR analysis, the characteristic covalent bonds of the different ionic groups are observed.The ceramic compound TCP is formed by Ca 2+ and PO 3− ions.The peak at 943.19 cm −1 and 972.12 cm −1 is related to the presence of pure β-TCP.Thus, the bands between the range 900-1200 cm −1 represent the stretching mode of the PO4 −3 group [47,48].The results show the characteristic peaks of the covalent bonds present in PO4 −3 .Hydroxyapatite (HA) has the OH − ion.The characteristic peaks at 630 cm −1 and 3571 cm −1 were attributed to the stretching mode of the hydroxyl group (OH − ) [47]; however, these peaks were not clearly detected or were too small compared to others.The band at 465-627 cm −1 and the band at 900-1000 cm −1 were expected due to Zr-O, which indicates the formation of cubic ZrO2 and tetragonal ZrO2 crystalline phases, respectively [49,50].In this sense, it is believed that the peaks with the greater width of the two samples with zirconia (~600 cm −1 ), in comparison with the sharp peaks of the 10T composition, and the more pronounced drop (~820 cm −1 ), are due to the presence of Zr-O bonds, thus being almost undetectable due to their low content and the presence of high PO4 3− peaks in this region.From the FTIR analysis, the characteristic covalent bonds of the different ionic groups are observed.The ceramic compound TCP is formed by Ca 2+ and PO 3− ions.The peak at 943.19 cm −1 and 972.12 cm −1 is related to the presence of pure β-TCP.Thus, the bands between the range 900-1200 cm −1 represent the stretching mode of the PO 4 −3 group [47,48].The results show the characteristic peaks of the covalent bonds present in PO 4 −3 .Hydroxyapatite (HA) has the OH − ion.The characteristic peaks at 630 cm −1 and 3571 cm −1 were attributed to the stretching mode of the hydroxyl group (OH − ) [47]; however, these peaks were not clearly detected or were too small compared to others.The band at 465-627 cm −1 and the band at 900-1000 cm −1 were expected due to Zr-O, which indicates the formation of cubic ZrO 2 and tetragonal ZrO 2 crystalline phases, respectively [49,50].In this sense, it is believed that the peaks with the greater width of the two samples with zirconia (~600 cm −1 ), in comparison with the sharp peaks of the 10T composition, and the more pronounced drop (~820 cm −1 ), are due to the presence of Zr-O bonds, thus being almost undetectable due to their low content and the presence of high PO 4 3− peaks in this region.

Mechanical Properties
The apparent porosity of the 10T biocomposite was 12.9%.The porosity increased to 24.6% and 21.9% for the dense biocomposites reinforced with 3YSZ, i.e., 9T1tZ and 8T2tZ, respectively.The similar samples manufactured with PMMA show higher porosity, namely, 26.2%, 29.0%, and 29.3% for 10T, 9T1tZ, and 8T2tZ, respectively.The dense biocomposites of 9T1cZ and 8T2cZ showed lower apparent porosity with reinforcement of 10 wt% of 8YSZ (14.9%) and similar porosity for 20 wt% (20.7%).The samples manufactured with PMMA present similar porosity of 28.9% and 27.6%, for 10 wt% and 20 wt%, respectively.In both manufacturing conditions, the addition of 3YSZ or 8YSZ increased the apparent porosity in relation to the standard, 10T, see Figures 2, 3 and S2.
The bulk density of the sTCP doped with metal ions (reference material, 10T) presented a value of 2.80 g/cm 3 .The density increased for 3.19 g/cm 3 and 3.36 g/cm 3 and for 3.18 g/cm 3 and 3.35 g/cm 3 for the dense biocomposites reinforced with 3YSZ and 8YSZ, respectively.The samples manufactured with PMMA, with 10 wt% and 20 wt% of 3YSZ, present a density of 2.30 g/cm 3 and 2.62 g/cm 3 , respectively.While the porous samples reinforced with 10 wt% and 20 wt% of 8YSZ, show 2.73 g/cm 3 and 2.60 g/cm 3 , respectively.The addition of 3YSZ and 8YSZ in dense biocomposites increased the density related to initial doped sTCP.However, in the samples manufactured with PMMA the density decreased or presented similar values (Figures 3 and S3).
These results are in agreement with the literature [1,18,31,45].There, it was proven that, with the addition of 10 and 20% ZrO 2 , the apparent porosity increased while the bulk density decreased, and this is what happened with the porous biocomposites analyzed in this work.With the addition of the 3YSZ and 8YSZ, porosity and bulk density increased, which can be explained by the effect of the sintering temperature (1300 • C).Considering the particle size distribution of the mixture up to 10 µm, with both zirconias up to 2 µm, the sintering conditions by pressure-less, unassisted sintering where, in general, densification is accompanied by (an undesirable) grain coarsening, the success of avoiding the grain growth is related to the control of the competition between densification and grain growth.That is extremely difficult because the driving forces for both are proportional to the reciprocal grain size and hence comparable in magnitude [51].Thus, for the composition, particle size, and single step sintering in air the temperature used is not sufficient to promote the grain boundary atomic migration of zirconia.In these conditions, only the approximation of zirconia particles promotes the formation of zirconia agglomerates and partial grain growing with poor densification and superior porosity [52].Nonetheless, the addition of ions like Mg 2+ promotes the densification of materials [18,45].Another explanation for the apparent porosity results in the use of the biocomposites manufactured with PMMA (40 wt%).Lee et al. [31] showed that, with different percentages of PMMA, starting with 40%, the porosity tends to increase gradually, and even with 40% the value is already quite considerable.
The diametrical compression tests revealed that the dense biocomposites with 3YSZ and 8YSZ presented mechanical strength values between 12.85 and 16.40 MPa and between 13.36 and 20.74 MPa, respectively (Table 4).
These results revealed that, in comparison with sTCP doped (13.4 MPa), the dense biocomposites with 20 wt% of 3YSZ and 8YSZ are higher.The samples 8T2cZ, are those that have higher mechanical strength, with a value of 20.7 MPa (55% higher than reference doped sTCP).As expected in induced porous biocomposites, the mechanical strength values were greatly decreased.From the initial 1.28 MPa (TCP doped) the addition of the 20 wt% of ZrO 2 decreases to 0.1 and 0.14 MPa for 3YSZ and 8YSZ, respectively (Figure S4).The addition of ZrO 2 to the ions doped with sTCP creates a ceramic microstructure in which the sTCP base matrix is reinforced with micro-and nanoparticles of zirconia (3YSZ and 8YSZ) which is due to its superior mechanical resistance (strength, toughness, and hardness).Furthermore, it is evident that the superior mechanical properties of the cubic phase (8YSZ) contribute more effectively to the increase in resistance than the tetragonal phase (3YSZ) [53].
Similar to previous studies, biocomposites with reinforcement of ZrO 2 have higher mechanical strength than "pure" biocomposites, i.e., biocomposites without the presence of ZrO 2 [1,54,55].In this study the most resistant biocomposites have 20% of 3YSZ or 8YSZ.On the other hand, these results can also be explained by the addition of Mg 2+ , that replaces the Ca 2+ ions in its sites (doping effect), which have already been shown to increase the mechanical strength of the biocomposites [45].Regarding biocomposites manufactured with PMMA, low mechanical strength was already reported by Lee et al. [31].

Biological Properties
The biocompatibility of dense ceramic biocomposites, both with and without porosity induced by PMMA reinforcement with 3YSZ and 8YSZ, was evaluated using a resazurin assay, with the corresponding results displayed in Figure 6.hOB were chosen as the cell model due to their pivotal role in bone matrix production and remodeling [56], a crucial aspect of the osseointegration process.Notably, the data obtained from the resazurin assay, as shown in Figure 6A,B, demonstrated that even after 7 days of incubation, the hOB cells remained highly metabolically active when in contact with both dense and porous biocomposites.
Similar to previous studies, biocomposites with reinforcement of ZrO2 have hig mechanical strength than "pure" biocomposites, i.e., biocomposites without the prese of ZrO2 [1,54,55].In this study the most resistant biocomposites have 20% of 3YSZ or 8Y On the other hand, these results can also be explained by the addition of Mg 2+ , that places the Ca 2+ ions in its sites (doping effect), which have already been shown to incre the mechanical strength of the biocomposites [45].Regarding biocomposites manu tured with PMMA, low mechanical strength was already reported by Lee et al. [31].

Biological Properties
The biocompatibility of dense ceramic biocomposites, both with and without po ity induced by PMMA reinforcement with 3YSZ and 8YSZ, was evaluated using a re urin assay, with the corresponding results displayed in Figure 6.hOB were chosen as cell model due to their pivotal role in bone matrix production and remodeling [56], a cial aspect of the osseointegration process.Notably, the data obtained from the resazu assay, as shown in Figure 6A,B, demonstrated that even after 7 days of incubation, hOB cells remained highly metabolically active when in contact with both dense and rous biocomposites.In fact, their cell viability consistently exceeded the 70% threshold, indicating str biocompatibility.It is worth noting that the porous biocomposites were made f PMMA, a structure porosity that, according to the literature [57], could potentially hin biocompatibility and bioactivity.However, the results of this study are consistent w previous research investigating the in vitro cytotoxicity of biocomposites containin TCP and ZrO2 [1,45,58].Also, as in other reported studies [57,58], the use of PMMA du manufacturing did not leave residues that could harm the cytotoxicity results.
As in previous studies [45], no relevant differences were detected in cell viability proliferation between the pure TCP composition and the TCP compositions doped w combinations of the four metal ions, while the bioceramic composites of TCP with zirco did not show high cell viability.This cell viability can be attributed to the range of m In fact, their cell viability consistently exceeded the 70% threshold, indicating strong biocompatibility.It is worth noting that the porous biocomposites were made from PMMA, a structure porosity that, according to the literature [57], could potentially hinder biocompatibility and bioactivity.However, the results of this study are consistent with previous research investigating the in vitro cytotoxicity of biocomposites containing β-TCP and ZrO 2 [1,45,58].Also, as in other reported studies [57,58], the use of PMMA during manufacturing did not leave residues that could harm the cytotoxicity results.
As in previous studies [45], no relevant differences were detected in cell viability and proliferation between the pure TCP composition and the TCP compositions doped with combinations of the four metal ions, while the bioceramic composites of TCP with zirconia did not show high cell viability.This cell viability can be attributed to the range of molar concentrations of ions incorporated into the crystalline structure of β-TCP, which closely mimics the composition of natural human bone [14,45,59].
SEM was used to visualize cell attachment and growth on the PMMA-induced porous biocomposites.Figure 7 shows the results at 3000× and 5000× magnification after 3 days of incubation.In addition, SEM images of the biocomposites without hOB cells, magnified to 1200×, are included for comparison.These images clearly show that hOB cells not only adhered to the surface but also infiltrated the interior of the biocomposites over time.This observation is consistent with previous research using β-TCP [60].These results may be associated with the porosity present in the materials, whose macropores have an ideal average size between 100-200 µm for cell growth, and according to the literature [61][62][63] this directly influences cell proliferation, promoting cell adhesion and growth.

Figure 3 .
Figure 3. SEM characteristic fracture surfaces of the ceramic biocomposites.Dense biocomposites with a magnification of 1000× and 5000× and porous biocomposites with a magnification of 100× and 200×.Examples of transgranular and intergranular fracture surfaces are illustrated by letters "T" and "I", respectively.

Figure 3 .
Figure 3. SEM characteristic fracture surfaces of the ceramic biocomposites.Dense biocomposites with a magnification of 1000× and 5000× and porous biocomposites with a magnification of 100× and 200×.Examples of transgranular and intergranular fracture surfaces are illustrated by letters "T" and "I", respectively.Biomimetics 2023, 8, x FOR PEER REVIEW 8 of 17

Figure 7 .
Figure 7. SEM images of cellular attachment to porous biocomposites after 3 days and without cellular attachment, with magnifications of 3000×, 5000× and 1200×, respectively.

Table 1 .
Nomenclature and composition of the materials studied in molar fraction (x i ), volumetric fraction (v i ) and mass fraction (w i ).

Table 1 .
Nomenclature and composition of the materials studied in molar fraction (xi), volu fraction (vi) and mass fraction (wi).