Calcium phosphate-based bioceramics are highly valued in biomedical engineering for their remarkable biocompatibility and ability to promote bone regeneration. Hydroxyapatite (HAp) and zinc-doped β-tricalcium phosphate (xZn-TCP) have both been the subject of extensive research as biomaterials. However, the performance of composites combining these two materials still needs to be optimized. In this study, the HAp- and xZn-TCP-based composites, where x=3mol% and 5mol%, were carried out by mixing in water the two powders previously synthesized through a classical coprecipitation process. The stabilized slurries were slip cast on a plaster support to prepare thin pellets to be sintered. The study focused on two series of Zn-doped HAp/TCP composites containing HAp as the main phase (90 and 80wt%) and xZn-TCP as the main phase (90 and 80wt%). Structural analyses confirmed the presence of β-TCP with a rhombohedral structure and HA with a hexagonal structure. Zinc doping significantly improved sinterability, yielding relative densities of up to 99% at 1200°C, particularly for the lower Zn content. Furthermore, zinc stabilized the β-TCP phase, resulting in an increase in the β-TCP→α-TCP transition temperature to approximately 1420°C. The mechanical study highlighted a positive relationship between increased sample density and optimization of material properties. Indeed, the composite made up of 80% (3mol% Zn-TCP) and 20% HAp exhibited superior performance, showing the highest value of Young's modulus – 135GPa and the Vickers hardness – 5.4GPa. Along with these mechanical properties, cytotoxic tests performed by means of MTT assay on HEK293 cells showed that all compositions supported cell viability above 100%, thus indicating the absence of any cytotoxic effects. In addition to that, the ability to stimulate cell proliferation is another important property of these materials. Thanks to these unique properties, the materials under discussion have excellent prospects for usage in clinical practice, namely, as dental implants, spinal cages, and bone defect repair sites.
Las biocerámicas a base de fosfato de calcio son muy valoradas en la ingeniería biomédica por su notable biocompatibilidad y su capacidad para promover la regeneración ósea. Tanto la hidroxiapatita (HAp) como el β-fosfato tricálcico dopado con zinc (xZn-TCP) han sido objeto de numerosas investigaciones como biomateriales. Sin embargo, el rendimiento de los compuestos que combinan estos dos materiales aún requiere optimización. En este estudio, se prepararon compuestos a base de HAp y xZn-TCP, con x = 3% y 5% molar, respectivamente, mediante la mezcla en agua de los dos polvos previamente sintetizados a través de un proceso clásico de coprecipitación. Las suspensiones estabilizadas se vertieron sobre un soporte de yeso para obtener pastillas delgadas que posteriormente se sinterizaron. El estudio se centró en dos series de compuestos de HAp/TCP dopados con zinc, que contenían HAp como fase principal (90% y 80% en peso) y xZn-TCP como fase principal (90% y 80% en peso). Los análisis estructurales confirmaron la presencia de β-TCP con estructura romboédrica y HA con estructura hexagonal. El dopaje con zinc mejoró significativamente la sinterabilidad, obteniendo densidades relativas de hasta el 99% a 1.200 ̊C, especialmente con un menor contenido de Zn. Además, el zinc estabilizó la fase β-TCP, lo que resultó en un aumento de la temperatura de transición β-TCP → α-TCP a aproximadamente 1.420 ̊C. El estudio mecánico destacó una relación positiva entre el aumento de la densidad de la muestra y la optimización de las propiedades del material. De hecho, el compuesto formado por un 80% (3% molar de Zn-TCP) y un 20% de HAp mostró rendimiento superior, con el mayor valor de módulo de Young (135 GPa) y dureza Vickers (5,4 GPa). Además de estas propiedades mecánicas, las pruebas de citotoxicidad realizadas mediante el ensayo MTT en células HEK293 demostraron que todas las composiciones mantenían una viabilidad celular superior al 100%, lo que indica la ausencia de efectos citotóxicos. Asimismo, la capacidad de estimular la proliferación celular es otra propiedad importante de estos materiales. Gracias a estas propiedades únicas, los materiales en cuestión presentan excelentes perspectivas de uso en la práctica clínica, concretamente como implantes dentales, jaulas espinales y materiales para la reparación de defectos óseos.
Bone defects are a common and complex problem in clinical orthopedics; they are often caused by infections, complex fractures, tumors, or congenital anomalies. Since these defects rarely heal on their own, bone grafts or bone coatings are generally required to restore the function of damaged bone tissue. Ideal bone substitute materials must be biocompatible, mechanically strong, and capable of promoting bone regeneration by supporting osteogenesis and integrating well with surrounding tissues. Various biomaterials have been studied for this purpose, including ceramics [1], polymers [2], and composites [3], and advanced manufacturing techniques such as 3D bioprinting have been developed [4]. Increasing importance is being placed on the development of biomaterials capable of effectively promoting bone healing and improving clinical outcomes. Calcium phosphate-based bioceramics, particularly hydroxyapatite (HAp:Ca10(PO4)6(OH)2) and β-tricalcium phosphate (β-TCP:Ca3(PO4)2), have attracted significant interest due to their chemical similarity to the mineral phase of natural bone [5–7]. Both HAp and β-TCP are composed primarily of calcium and phosphate ions, essential components of bone tissue, and offer advantages such as low cost, ease of synthesis, safety, bioactivity, and osteoconductivity, which promote bone regeneration. HAp has long been used as a coating material in dental and orthopaedic implants and finds applications in bone [8–10], skin, cartilage, vascular and other tissues [11]. Its excellent biocompatibility makes HAp one of the most suitable ceramics for artificial bone tissue engineering. While HAp remains highly valued, β-TCP is attracting growing interest due to its potential to improve biological and mechanical performance β-TCP is widely employed in bone grafting for orthopaedic, dental, and maxillofacial procedures due to its osteoconductivity and favorable resorption behavior [12,13]. It has been used as a bone substitute for over 25 years [14]. However, β-TCP ceramics face challenges such as insufficient mechanical strength for load-bearing applications, rapid degradation that may hinder bone healing, and difficulties in densification at high temperatures due to a phase transition from β-TCP to α-TCP occurring near 1125°C, which involves cell expansion and can degrade material properties [14]. To overcome these drawbacks, research has been conducted on the following two approaches. The first approach involves ion substitution within the β-TCP crystal lattice. For example, when Ca2+ ions are replaced with other divalent metal ions such as Mg2+, Sr2+, or Zn2+, it has been found that this significantly influences the structural stability of the β-phase [13–22]. For example, previous research on zinc doped β-TCP has shown that the unit cell volume of the doped TCP is reduced because of the smaller radius of Zn2+ (0.074nm) compared with Ca2+(0.100nm), thus stabilizing the TCP structure [22]. This stabilizing effect shifts the phase transition of β-TCP to α-TCP to a temperature above 1200°C, thus creating a wider sintering window by avoiding the undesirable volume expansion of the α-phase [13,23,24]. Herein, zinc doped β-TCP plays a key role in the acceleration of mineral deposition and stem cell osteogenic differentiation. This factor, combined with its powerful antibacterial activity, should lead to a synergetic improvement in the bone formation capacity of the material. Moreover, adequate levels of zinc are absolutely necessary for the process of bone mineralization, development, growth, and maintenance. The biological significance of this element is highlighted by the observation that a lack of zinc results in various developmental abnormalities, including dwarfism. With Zn added to the material, the latter will not only act as a scaffold material but also deliver a valuable micronutrient that prevents growth restrictions [25]. Therefore, Cuneyt Tas et al. [16] confirmed that In vitro culture tests performed by mouse osteoblast-like cells showed that the β-TCP doped with Zn showed the highest cell viability. The second approach involves developing biphasic systems, traditionally composed of HAp and β-TCP. These composites aim to establish a biological equilibrium based on the high stability of HAp and the rapid resorbability of β-TCP [26–28]. Previous work on these biphasic compositions has shown that as the proportion of β-TCP increases, the porosity of HAp/β-TCP composites decreases [29–34], but few scientific publications have addressed the synergistic effects of combining an HAp phase with a pre-stabilized Zn-doped β-TCP phase. Meanwhile, Atsuo Ito et al. [35] found that the osteoblastic cells MC3T3-E1 grown in vitro and the alkaline phosphatase activity of stromal cells in rats are substantially promoted by Zn-TCP/HAp composites besides a substantial 51% increase in bone formation. Thus, little attention has been paid to structural, mechanical and in some cases biological characterization of both HAp and xZn-TCP system. Thus, the novelty of this work lies in the optimization of the HAp/xZn-TCP ratio in two different series of composites. Unlike conventional studies on biphasic calcium phosphates, in which a single phase is dominant, our study considers both HAp-rich and xZn-TCP-rich compositions. This article investigated the synthesis of zinc-doped tricalcium phosphates via precipitation, followed by the fabrication of biphasic composites by casting a mixture of the suspensions of the two powders. Two distinct series of composite materials (HAp-rich and xZn-TCP-rich) were developed to assess the role of the matrix material on the overall performance of the composite. The main aim of the current research is to determine the ideal ratio of the HAp/xZn-TCP composites that should exhibit the required characteristics in terms of structure, mechanical and biological activity for application purposes. Through determining the proper ratios composites, this research intends to establish adequate composite materials that will be suitable for use in orthopedics and dentistry.
Materials and methodsCalcium phosphate powder synthesisHydroxyapatite (HAp) and zinc-doped tricalcium phosphate (xZn-TCP) powders are synthesized via aqueous precipitation technique [30–33]. This synthesis takes place in a double-walled reactor connected to a thermocryostat (Julabo) to maintain a constant temperature of about 31°C throughout the process. Mixing is achieved with a stirring device (Heidolph RZR 2102 Control) fitted with a Teflon-coated blade. The pH is carefully controlled and adjusted via a pH controller linked to a dosing pump (Black Stone BL 7916) and a pH probe. During synthesis, an aqueous calcium nitrate solution, Ca(NO3)2·4H2O (purity >98%, Honeywell, Germany), is added to the reactor, along with zinc nitrate Zn(NO3)2 (8.0–10.2%, Alfa Aesar, Germany) for the zinc-doped samples. Simultaneously, an aqueous solution of diammonium hydrogen phosphate (NH4)2HPO4 (98.0–102.0%, Carlo Erba, France), is introduced at a flow rate of 10mL/min using a peristaltic pump. The used amounts of reactants for each synthesized product are gathered in Table 1. The reaction mixture's pH is maintained at 6.7 for xZn-TCP by automatic addition of ammonia solution (NH4OH) controlled by the system. After the full addition of the phosphate solution, the mixture is stirred at the synthesis temperature of about 31°C for 20h to mature. The product is then filtered using a Buchner funnel and washed with distilled water to remove by-products, especially ammonium nitrate. Finally, the powder is dried at 70°C for 24h, followed by a calcination at 850°C. For HAp synthesis, a similar method is used, but with an excess of ammonia (9mL NH4OH per 30.96mL water), at 70°C and pH 10.2, followed by calcination at 900°C.
The reactions for HAp and xZn-TCP synthesis are the following respectively:
Following the calcination, powders are wet ball milled for 4h (90g powder with 200g water) in high-density polyethylene (HDPE) jars loaded with 1.320kg of yttria-stabilized zirconia balls of 10, 15, and 20mm diameter. The resulting suspensions are filtered through a Büchner funnel and dried at 70°C for 24h. Particle size distribution is measured by laser granulometry (Malvern Mastersizer 3000), and the specific surface area is determined by BET analysis using a Micromeritics Flowsorb III after degassing the powders under vacuum at 250°C for 20h to remove adsorbed moisture.
Composites processing and characterizationThe composites 90%HAp–10%(xZn-TCP), 80%HAp–20%(xZn-TCP), 10%HAp–90%(xZn-TCP), and 20%HAp–80%(Zn-TCP) are prepared as aqueous suspensions using 65g of the respective HAp and xZn-TCP powders, 34mL of distilled water, and 1.31g of dispersant (Darvan C, R.T. Vanderbilt Co). The mixtures are ground for 4h in a jar containing yttria-stabilized zirconia beads, then poured on plaster supports. After 2h, the formed pellets are removed and dried at 70°C for 24h. The structural characteristics of the green pellets are examined by Fourier-transform infrared spectroscopy (FTIR) using a Jasco 4600 instrument over the range 4000–400cm−1, and by X-ray diffraction (XRD) with a PANalytical PW3040/60 X’Pert PRO diffractometer equipped with a Cu Kα source (λ=1.5406Å). For dilatometric measurements, cylindrical pellets (1cm thick, 5mm diameter) are formed by uniaxial pressing at 4 tons for 2min. The apparatus is a Netzsch DIL 402C horizontal dilatometer with a resistive furnace. The thermal cycle involves heating at 5°C/min to 1100°C with a 3-h hold, then heating to a maximum of 1500°C, followed by a controlled cooling rate of 5°C/min to room temperature.
Sintering was conducted on slip cast pellets in an electric furnace (Nabertherm LHT 08/17 1750°C) with a heating rate of 3°C/min up to temperatures between 1100°C and 1300°C, held for 3h, followed by cooling at 40°C. The relative densities of sintered pellets are measured by the Archimedes method in deionized water at room temperature, using an analytical balance with 0.1mg resolution. Three samples per composition and temperature are tested to obtain average density values. Microstructure observations are made on polished and thermally etched pellet surfaces using scanning electron microscopy (SEM, JEOL-JCM6000). Polishing is performed with SiC paper and diamond pastes down to 0.5–3μm, and thermal etching is done in the Nabertherm furnace. Mechanical properties, including Young's modulus (E), are evaluated by measuring the velocities of transverse and longitudinal ultrasonic waves propagating through the samples. The modulus values are calculated using the appropriate wave velocity equations.
Here, ρ represents the density, VT is the velocity of the transverse wave, and VL is the velocity of the longitudinal wave. Furthermore, the Vickers hardness (Hv) was determined using a microhardness tester (FM-7) through hardness distribution measurements. A load of 9.8N (noted as F) was applied for 10s to induce 10 separated indentation locations. The Hv values were calculated using the following formula [36,37]:
where d is the diagonal of the indentation. Three samples of specified composition and sintering temperature were tested.
The prepared samples were tested for cytotoxicity on HEK293 cells with an MTT assay. Cells were grown in DMEM with 10% FBS at 37°C in a 5% CO2 atmosphere. Material extracts were obtained by incubating the samples in culture medium for 24h. The cells were then exposed to the extracts for 24h. After incubation, MTT solution was added, and the formed formalin crystals were dissolved in DMSO. The absorbance was measured at 570nm, and cell viability was calculated relative to the control.
Results and discussionCharacterization of synthesized powders
The FTIR spectra of HAp powders, shown in Fig. 1, exhibit the absorption bands characteristic of hydroxyapatite. The bands observed at ∼1081cm−1, ∼1030cm−1, ∼1009cm−1, ∼956cm−1, ∼601cm−1, ∼570cm−1, and ∼474cm−1 correspond to P–O stretching vibrations. Additionally, hydroxide ion (OH−) bands appear around ∼631cm−1 and ∼3562cm−1. These results are in good agreement with those reported by Leung et al. [38]. Fig. 2 shows the FTIR spectra of un- and doped TCP powder with zinc at concentrations of 3mol% and 5mol%. All the infrared bands correspond to β-TCP, with no sign of secondary phases such as calcium pyrophosphate (CPP), which would appear at 720cm−1 and 1200cm−1. Most of the bands are attributed to the phosphate group (PO43−) of TCP: the bands at 942 and 957cm−1 arise from the symmetric stretching of PO43− ions, while those at 1075 and 1091cm−1 are related to asymmetric stretching [39]. Minor variations in FTIR spectra were observed upon substituting Ca2+ with 3–5mol% Zn2+ in β-TCP. This limited structural disruption is consistent in one hand to the lower substituted Zn content in the other hand to the smaller ionic radius of Zn2+ (0.74Å) compared to Ca2+ (1.00Å), which suggests a well-tolerated but restricted lattice contraction. Meanwhile, the interaction between metal cations and phosphate ions in TCP materials plays an important role in controlling their structural and functional properties [18,40,41]. Cations such as calcium and zinc participate in ion exchange and electrostatic interaction with the phosphate group. This interaction may slightly affect the structural stability of TCP materials [27] and may lead to changes in the absorption bands of the phosphate group.
The XRD diffractogram of HAp calcined at 1000°C, shown in Fig. 3, reveals that all peaks detected correspond exclusively to the HAp phase, with no indication of other possible compounds such as β-TCP or CaO. Fig. 4 presents the XRD diffractograms of undoped and Zn-doped β-TCP samples, calcined at 1000°C. The diffraction patterns indicate that most of the observed peaks are attributed to β-TCP, with the most intense reflection (0210) located at 2θ≈31°, exhibiting a relative intensity of 100%. No significant peaks corresponding to calcium pyrophosphate (CPP), whose most intense diffraction peak (202) appears at 2θ=28.9°, are observed. Nevertheless, the presence of low-intensity peaks, particularly at 2θ≈31.7°, indicates the presence of a trace amount of HAp as a secondary phase. Rietveld refinements performed using the Jana2020 program confirm that β-TCP remains the predominant phase. A slight shift in peak positions is noted in the Zn-doped samples compared to the undoped β-TCP. The Rietveld refinement results, including lattice parameters and theoretical densities for undoped and zinc-doped Zn-TCP and HAp, are summarized in Table 2. An increase in the Zn2+ content leads to a reduction in the lattice parameters [18,39]. This lattice contraction is attributed to the smaller ionic radius of Zn2+ (0.74Å) compared to Ca2+ (1.00Å) resulting in shorter chemical bonds. Such contraction is also linked to increased thermal stability of β-TCP and a delay in the allotropic transformation from β-TCP to α-TCP, as confirmed by several studies [41,42]. According to Vahabzadeh et al., Kannan et al. and Nishikawa et al. [23,40,41], substitution by ions smaller than calcium reduces the crystal lattice volume and stabilizes the β-TCP phase [43]. The shifts observed in the X-ray diffraction peaks and the changes in the crystal lattice parameters confirm the successful incorporation of Zn2+ dopants into the crystal structure of TCP, as expected. Increasing Zn2+ content causes a reduction in lattice parameters [18,40]. This lattice contraction is attributed to the smaller ionic radius of Zn2+ (0.74Å) compared to Ca2+ (1.00Å), which results in shorter chemical bonds. Such contraction is also linked to increased thermal stability of β-TCP and a delay in the allotropic transformation of β-TCP to α-TCP, as confirmed by several studies [41,42]. According to Vahabzadeh et al., Kannan et al. and Nishikawa et al. [23,40,41], substitution with ions smaller than calcium reduces the crystal lattice volume and stabilizes the β-TCP phase [43]. The shifts observed in the X-ray diffraction peaks and the changes in the crystal lattice parameters confirm the successful incorporation of Zn2+ dopants into the crystal structure of TCP, as expected.
The particle size distribution and specific surface area (BET) of the HAp and xZn-TCP powders after grinding are presented in Table 3. The data show that zinc doping results in a notable reduction in particle size and a significant increase in specific surface area. This behavior suggests that the presence of zinc modifies the physicochemical characteristics of the powders, possibly by inhibiting grain growth or enhancing brittleness during grinding, thus promoting finer particles with greater surface area. In summary, substituting calcium with zinc in β-TCP leads to a gradual reduction in particle and grain size. This phenomenon is explained by the fact that Zn2+ ions, whose ionic radius is 0.74Å (smaller than that of Ca2+ ions, which is 0.99Å), cause the crystal lattice to contract (Kannan et al., 2009) [40]. Furthermore, when zinc is at the grain boundaries, it creates the solute drag effect, which inhibits grain growth during heating (Roy et al., 2013) [44]. Moreover, zinc also stabilizes the β-TCP structure, preventing the transition to the larger α-TCP at high temperatures. These effects result in a finer structure, a higher specific surface area and a finer microstructure as the zinc concentration increases (Kai et al., 2014; Xue et al., 2008) [45,46].
Powder sinteringDensificationThe relative density of the sintered specimens was established by comparing the experimental density to the theoretical value summarized in Table 4. While the theoretical density was determined based on the specific sample stoichiometry, the experimental density was measured via the Archimedes displacement method.
Calculated theoretical densities of samples.
| TCP | 3mol% Zn-TCP | 5mol% Zn-TCP | HAp | |||
|---|---|---|---|---|---|---|
| d | 3.067 | 3.065 | 3.062 | 3.150 | ||
| 90%TCP–10%HAp | 90%(3%ZnTCP)–10%HAp | 90%(5%ZnTCP)–10%HAp | 80%TCP–20%HAp | 80%(3%ZnTCP)–20%HAp | 80%(5%ZnTCP)–20%HAp | |
| d | 3.075 | 3.074 | 3.071 | 3.084 | 3.082 | 3.080 |
| 90%TCP–10%HAp | 10%(3%ZnTCP)–90%HAp | 10%(5%ZnTCP)–90%HAp | 20%TCP–80%HAp | 20%(3%ZnTCP)–80%HAp | 20%(5%ZnTCP)–80%HAp | |
| d | 3.142 | 3.142 | 3.141 | 3.133 | 3.133 | 3.132 |
Fig. 5 shows the variation in the relative density of the different samples as a function of sintering temperature, ranging from 1100 to 1300°C. For the HAp sample, the maximum relative density (96%) was reached at 1200°C and decreased slightly at 1300°C. The relative density of the pure β-TCP sample decreases continuously from 94% to 86% as the temperature rises, which is not the case for the zinc-doped samples. These exhibit a higher level of densification, starting at 95–96% at 1100°C, increasing to 97–98% at 1200°C and decreasing slightly at 1300°C. Conversely, β-TCP reached its maximum densification at 1100°C, followed by a decline. This pattern indicates that zinc doping improves both the thermal stability and the densification behavior of β-TCP. It should be noted that at a zinc concentration of x=3mol% by weight and a sintering temperature of 1200°C, the relative density is close to 98% of the theoretical density of β-TCP. At higher temperatures, the reduction in density of undoped β-TCP is likely due to the grain coarsening leading to increase in porosity. Furthermore, previous studies have shown that the incorporation of zinc into the apatite structure disrupts the crystal lattice of the apatite powders, thereby promoting better densification [41,42].
Figs. 6 and 7 show the relative density values of the various composites (%HAp:%(xZn-TCP)) sintered at temperatures ranging from 1100 to 1300°C. For composites with a higher HAp content, such as (90%HAp:10%(xZn-TCP) and (80%HAp:20%(xZn-TCP)), the addition of zinc-doped TCP to HAp slightly reduces the relative density. Conversely, for composites richer in TCP, such as (90%(xZn-TCP):10%HAp) and (80%(xZn-TCP):20%HAp), the inclusion of HAp leads to an increase in relative density. This indicates that HAp may help to stabilize the microstructure and improve densification in TCP-dominant composites. The highest relative density, reaching 99% of the theoretical value, is observed at 1200°C for the 80% (3mol% Zn-TCP):20%HAp composite. While it is known that lattice expansion generally leads to lower densities, the densities in this study were calculated relative densities, which depend highly on the microstructure. Although there is a trend for some lattice contraction when increasing the amount of zinc, it is found that there is a decrease in relative density for Zn doping level equal or superior to 5mol%, plausibly related to the optimum required amount of dopant was surpassed. Thus, with higher Zn doping levels, microstructural factors, such as porosity and inhomogeneity, dominate, leading to reduced density. In summary, zinc doping improves the densification and thermal stability of β-TCP, particularly at moderate doping levels, while the HAp/TCP ratio in the composites significantly influences the final relative density and microstructural stability of the sintered materials [41,42]. Figs. 8 and 9 present the X-ray diffractograms of crushed pellets from both composite series sintered at 1200°C, composed of varying percentages of HAp and Zn-TCP. The diffractograms reveal no phases other than HAp and β-TCP, indicating the coexistence of these two phases without the formation of secondary phases. This suggests strong chemical compatibility between HAp and Zn-TCP after sintering. Furthermore, the absence of peaks corresponding to the α-TCP phase confirms that the β→α allotropic transformation was suppressed, emphasizing the stabilizing effect of zinc on the TCP structure. This observation aligns with the findings of the subsequent dilatometric study.
The microstructure evolution of the HAp sample versus sintering temperature is presented in Fig. 10. At 1100°C, the material remains 81% dense with numerous 1μm intergranular pores (Fig. 10a). When the temperature increased to 1200°C a reduction in porosity and slight grain coarsening is noted (Fig. 10b), resulting in a peak relative density of 96%. However, by 1300°C, the intergranular pores vanish and are replaced by significant grain coarsening, which accounts for the minor decrease in relative density to 94% (Fig. 10c). The data in Table 5 confirms this trend, showing that the grain size of the sintered HAp increases as the temperature rises, which is a clear indication that heat treatment plays a significant role in enhancing grain growth. Comparable results were reported in previous research, such as the study conducted by Trzaskowska et al. [10].
The microstructure of the sintered xZn-TCP specimens strictly reflects their densification behavior, as shown in Fig. 11. For the Zn-free TCP sample, the decline in densification is attributed to increased porosity as the sintering temperature rises correlated to the β→α phase transition. In contrast, the Zn-doped TCP samples exhibit high densification rates, peaking at 99% for the 3mol% Zn composition at 1200°C; at this point, open porosity is eliminated and only slight grain coarsening occurs. Other samples, specifically those sintered at 1100°C and 1300°C or containing 5mol% Zn, maintain some surface pores, resulting in densification ratios slightly below the 99% maximum. These findings are supported by the grain size measurements for each specimen, which are detailed in Table 5. This indicates that zinc not only enhances densification but also inhibits excessive grain growth. These findings align with those reported by many researchers, particularly Nishikawa et al. show that Zn acts as an effective dopant in β-TCP [16,23,42,43].
Fig. 12 presents the surface microstructures of the 90%HAp:10%(xZn-TCP) and 80%HAp:20%(xZn-TCP) composites, which correlate closely with their measured relative densities. According to Table 6, grain size in these composites increases with both the TCP content (regardless of zinc doping) and the sintering temperature, suggesting that TCP plays a significant role in promoting grain growth during sintering. Similarly, the microstructures of the (90%(xZn-TCP):10%HAp) and (80%(xZn-TCP):20%HAp) composites shown in Fig. 13 are consistent with their measured densities. Table 6 and 7 indicates that grain size decreases as the HAp proportion in the TCP matrix increases, whether the TCP is undoped or zinc-doped. Conversely, grain size increases with rising sintering temperature. Overall, these results highlight the interplay between zinc doping, HAp/TCP ratio, and sintering temperature in controlling grain growth and densification in these composite materials [18,24,41,47,48].
Grain sizes (μm) of 90%HAp:10%(xZn-TCP) and 80%HAp:20%(xZn-TCP) pellets after sintering.
| Sintering temperature | 90%HAp10%TCP | 90%HAp:10%(3mol% Zn-TCP) | 90%HAp:10%(5mol% Zn-TCP) | 80%HAp:20%TCP | 80%HAp:20%(3mol% Zn-TCP) | 80%HAp:20%(5mol%Zn-TCP) |
|---|---|---|---|---|---|---|
| 1100°C | 1.05 | 1.13 | 1.21 | 1.27 | 1.29 | 1.36 |
| 1200°C | 1.11 | 1.25 | 1.32 | 1.36 | 1.42 | 1.76 |
| 1300°C | 5.73 | 6.16 | 6.77 | 7.06 | 8.67 | 9.26 |
Grain size (μm) of (90%(xZn-TCP)–10%HAp) and (80%(xZn-TCP)–20%HAp) pellets after sintering.
| 90%TCP–10%HAp | 90%(3mol% Zn-TCP)–10%HAp | 90%(5mol% Zn-TCP)–10%HAp | 80%TCP–20%HAp | 80%(3mol% Zn-TCP)–20%HAp | 80%(5mol% Zn-TCP)–20%HAp | |
|---|---|---|---|---|---|---|
| 1100°C | 2.88 | 2.02 | 1.89 | 2.45 | 1.93 | 1.50 |
| 1200°C | 3.30 | 2.31 | 2.09 | 2.69 | 2.17 | 1.92 |
| 1300°C | 18.30 | 15.80 | 10.06 | 16.7 | 11.40 | 8.76 |
Fig. 14 presents the thermal cycle, which involves heating at 5°C/min to 1100°C, holding for 3h at 1100°C, then further heating to a maximum of 1500°C, followed by controlled cooling to room temperature. Dilatometric analysis shown in Fig. 15 indicates that the initial allotropic transition of un-doped TCP from the β to α phase occurs at 1210°C. When TCP is doped with zinc at x=3mol%, this phase transition temperature was delayed to 1390°C with a rise of 180°C. For x=5mol%, a slight reduction in the transition temperature to 1385°C is observed [13,49]. The dilatometric analysis of the %HAp:%(xZn-TCP) composites is presented in Fig. 16. The results demonstrate that the allotropic transformation (β→α) was suppressed, a phenomenon credited to the zinc doping within the composite structure, which effectively inhibits this phase transition. This suggests that Zn doping stabilizes the structure and restricts the expected allotropic transformation during dilatometric testing. Fig. 17 and Table 8 display the dilatometric results for the composites (%(xZn-TCP)–%HAp), demonstrating that combining β-TCP with HAp further delays the β-TCP allotropic transformation, consistent with literature findings [50]. Specifically, the composite with 80%(3mol% Zn TCP) doped with and 20% HAp exhibits a maximum transition temperature of 1420°C, achieving an increase of 205°C. Thus, as the substitution of zinc into the TCPlattice increased, it served as a potent thermal stabilizer, effectively increasing the temperature of the β→α phase transformation from the usual range of 1125–1150°C to above 1300°C as indicated by Kai et al. [46]. By favoring the occupation of the Ca(5) sites, the smaller Zn2+ ions (0.74Å) induce a unit cell contraction that energetically stabilizes the β-structure, effectively delaying the transformation into the more reactive and larger-grained α-phase [13,45,50,51]. This improved thermal stability is also important during the sintering process, allowing the formation of high-density ceramics with a refined microstructure of high surface area, which would otherwise be compromised by the fast grain growth and volume expansion resulting from the α-phase transformation [52].
Mechanical propertiesVickers hardness
Hardness is an important property that measures a material's resistance to deformation, specifically its ability to withstand surface indentation. As illustrated in Fig. 18, the hardness values depend on both the zinc content (x=0mol%; 3mol%, and 5mol%) and the sintering temperature (1100, 1200, and 1300°C). For undoped β-TCP sintered at 1100°C, the hardness is 3.8±0.2GPa, which aligns closely with the values reported by Somers et al. [53] and Boilet et al. [54]. When the zinc substitution level is x=3mol%, hardness increases with temperature, reaching a peak of 4.7±0.2GPa at 1200°C. Fig. 19 shows how the Vickers hardness of the composites (%HAp:%(xZn-TCP)) varies with sintering temperature between 1100 and 1300°C. Among the composites with higher TCP content, such as (90%(xZn-TCP)–10%HAp) and (80%(xZn-TCP)–20%HAp), the highest hardness is observed for the (80%(3mol% Zn-TCP):20%HAp) composite, reaching 5.4±0.2GPa at 1200°C. For composites richer in HAp, specifically (90%HAp:10%(xZn-TCP)) and (80%HAp:20%(xZn-TCP)), the maximum hardness is found in the (90%HAp:10%(TCP-xZn)) composite, with a value of 4.6±0.2GPa at 1200°C. The incorporation of zinc into the β-TCP lattice shows a marked improvement in its mechanical hardness, which is primarily driven by microstructural refinement and the improvement of densification. As Zn2+ ions substitute for Ca2+, the inhibition of grain growth, as related to the Hall-Petch effect increases the density of grain boundaries which act as barriers to dislocation movement, thereby raising the material's resistance to deformation [40,44,51]. Additionally, the introduction of zinc stabilizes the compound in the β-phase, preventing the transformation into the α-phase, which is known for its porosity. This leads to a higher fractional density of the compound when subjected to temperatures of up to 1300°C, which is directly related to the compound's hardness [45]. This is also related to the principle of solid solution strengthening, where the lattice strain of the smaller zinc ion also plays a part. However, it should be noted that the compound's hardness tends to reach a peak at moderate levels of substitution (0.25–1.0wt%) before the saturation of the lattice [52].
Fig. 20 depicts the variation of Young's modulus for sintered pellets across the temperature range of 1100–1300°C. It clearly demonstrates the positive impact of zinc doping on Young's modulus, with all zinc-containing compositions exhibiting higher values than undoped β-TCP, which has a modulus of 101GPa at 1100°C, consistent with values reported by Somers et al. [53] and Boilet et al. [54]. The zinc-doped TCP samples show greater Young's modulus values compared to the undoped ones, reaching a maximum of 129GPa at 1200°C for x=3mol%. HAp samples reached 92GPa at 1200°C, indicating that Zn dopants significantly enhance the mechanical properties of β-TCP [45]. Fig. 21 illustrates the change in Young's modulus of the composites (%HAp–%(xZn-TCP)) as a function of sintering temperature between 1100 and 1300°C. For the composites with higher TCP content, such as (90%(xZn-TCP)–10%HAp) and (80%(xZn-TCP)–20%HAp), the highest Young's modulus is observed for the (80%(3mol% ZnTCP)–20%HAp) composite, reaching 134GPa at 1200°C. Similarly, for composites richer in HAp, namely (90%HAp–10%(TCP-xZn) and (80%HAp–20%(xZn-TCP), the maximum modulus is also found in the (80%(3mol%TCP)-20%HAp) composite, with a value of 134GPa at 1200°C. Moreover, the hardness and Young's modulus values obtained in this study are consistent with those reported in the literature [55–60], with the highest values recorded for x=3mol% in the (80%(3mol% Zn-TCP)–20%HA) composite, which corresponds to the lowest porosity observed for this composition.
Biocompatibility test
The biocompatibility test of the sintered samples was performed on CCK-8 cell viability assay on a human embryonic kidney cell line (HEK293) to assess the cytocompatibility of HAp–xZnTCP composites. Fig. 22 demonstrate that the vast majority of samples exhibit viability levels markedly exceeding 100%, frequently reaching 150–200%, confirming not only the non-cytotoxic nature of the biomaterials but also a significant proliferative effect: the tested materials actively stimulate the growth and proliferation of HEK293 cells, well beyond the baseline survival level observed in the control group. This trend is consistent regardless of the HAp/TCP ratio, with both the 90/10 and 80/20 blends maintaining excellent biocompatibility and showing that all compositions show a good cytotoxicity. Furthermore, the progressive increase in cell viability within each compositional group as a function of Zn doping level highlights the beneficial effect of the dopant: doped samples, particularly those with the 3mol% retain very high viability levels, confirming the complete cellular tolerance to zinc at the concentrations tested and its well-established role in stimulating cell proliferation and osteogenic differentiation. Thus, these data provide robust evidence of the cytocompatibility and bioactive character of the HAp/Zn-TCP composites, which not only exhibit no toxicity toward HEK293 cells but also actively promote their growths which present a key advantage for their prospective application in bone tissue engineering and repair.
Discussion
The notable increase in Vickers hardness and Young's modulus observed in zinc-doped samples, especially at a substitution level of x=3mol%, can be attributed to enhanced densification and refined microstructure. At 1200°C, the 80%(3wt.%Zn-TCP):20%HAp composite showed the highest values, with hardness around 5.4GPa and Young's modulus reaching up to 135GPa. The addition of zinc improves the sintering behavior of β-TCP, leading to decreased porosity and a finer microstructure. This aligns with the findings of Kai et al., who reported that ZnO promotes densification and delays the β to α phase transition, thereby enhancing mechanical strength [15]. Additionally, Esfahani et al. [61] demonstrated that substituting Ca2+ with smaller Zn2+ ions cause lattice contraction, which restricts grain growth. Similarly, Nishikawa et al. [41] showed that zinc effectively modifies grain boundaries and inhibits grain coarsening during high-temperature sintering. Compositional differences also significantly influence mechanical performance. Composites with a high proportion of zinc-doped TCP (80–90%) and lower HAp content (10–20%) exhibited superior mechanical properties compared to HAp-rich composites. The 80%(3mol% Zn-TCP):20%HAp formulation delivered the best mechanical response at 1200°C, likely due to the synergistic effect of improved sinterability and a stable microstructure. In contrast, HAp-rich composites (90%HAp:10%(xZn-TCP)) showed slightly lower hardness (∼4.6GPa) and modulus values (113GPa), which can be explained by HAp's lower sintering activity and its tendency to promote microstructural coarsening when present in excess. Previous studies byBoilet et al., Moslim et al., and Zamora et al. support these observations, noting that while HAp contributes to phase stabilization, excessive HAp content can hinder densification and compromise mechanical integrity [54,62,63]. Therefore, the optimized 80%(3mol% Zn-TCP):20%HAp composition offers the best balance of structural and mechanical properties, primarily due to the enhanced thermal stability and densification imparted by zinc incorporation. Moreover, the cytocompatibility and biological activity of HAp/Zn-TCP composites was verified. It was found that there was no toxic effect on the HEK293 cell line at all and, on the contrary, the growth of these cells was effectively stimulated. This would confirm their potential for advanced applications in bone tissue engineering and skeletal repair.
ConclusionThe present paper can be summarized by the following eight key points:
- 1.
The HAp and xZn-TCP powders were prepared through aqueous precipitation and successfully densified into bio-composite ceramics by a novel method of solid-state suspension consolidation technique.
- 2.
This study has been conducted on two different systems, namely HAp rich and xZn-TCP rich material composition with various concentrations of 0, 3.0, and 5.0mol% Zn content.
- 3.
The results showed the existence of both rhombohedral β-TCP and hexagonal HAp phases coexisting in the bio-composites.
- 4.
The phase stability of TCP was greatly improved upon doping with zinc, as it delayed the undesirableβ to α phase transition to above 1390°C, hence extending the sintering range.
- 5.
At 1200°C, the composites could achieve 98% relative densities while the undoped β-TCP attained only 94% density.
- 6.
The microstructure analysis indicated that the zinc doping restricted the grain growth giving a more refined structure, thus improving their mechanical properties.
- 7.
The optimal composite system consists of 80%(3mol% Zn-TCP)/20%HAp with a maximum mechanical strength of about 5.4GPa Vickers hardness, and 135GPa Young's modulus ready for orthopedic load-bearing applications that could serve as effective, next-generation bone tissue engineering scaffolds.
- 8.
All compositions exhibited cell viability levels exceeding 100%, indicating a total lack of cytotoxicity and a significant stimulatory effect on Hek293 cell proliferation highest performance were observed with 80%(3mol% Zn-TCP)/20%HAp composite.
Zohra Sghaier: Investigation; Methodology; Writing – original draft; Data curation; Formal analysis.
Florian Jean: Data curation; Investigation; Formal analysis.
Marie Lasgorceix: Investigation; Methodology; Formal analysis; Writing – review & editing.
Mustapha Hidouri: Investigation; Methodology; Writing – review & editing.
Anne Leriche: Conceptualization; Investigation; Methodology; Project administration, Writing – review & editing.
FundingThis study did not receive any dedicated financial support from public, commercial, or nonprofit funding organizations.
Conflict of interestsThe authors confirm that they have no known financial conflicts or personal relationships that could have influenced the research or the findings presented in this paper.
The authors are grateful to the Biotechnology Center of Sfax (CBS), Tunisia, for granting access to the facility for cytotoxicity analysis. The authors also acknowledge the invaluable contribution of Professor Sami Aifa for his assistance in the biological evaluation.
