Fabrication of boron-containing apatite ceramics via reaction sintering route and their response of lymphocytes for adoptive immunotherapy

Abstract

Conventional cancer treatments have several challenges, such as side effects and physical decline. Recently, adoptive immunotherapy, which has few side effects, has attracted attention, but the use of expensive drugs has become an issue. In addressing this problem, boron-containing apatite (Ca_9.5+0.5x{(PO₄)_6-x(BO₃)_x}{(BO₂)_1-xO_x} (0 ≤ x ≤ 1); BAp) ceramics, which are referred to as “immunoceramics,” have been developed to actively engage the immune system and enhance the activation of CD3+CD8+ cells corresponding to killer T cells. The BAp ceramics have been fabricated via ultrasonic spray pyrolysis (USSP); however, a deviation from the stoichiometric composition occurred when x in the chemical formula was small. Therefore, in this study, the synthesis method was changed from USSP to “reaction sintering”. The material properties of the fabricated BAp ceramics confirmed the synthesis of the BAp phase. In particular, the BAp ceramics fabricated by reaction sintering have a composition closer to stoichiometry than those synthesized by USSP. Furthermore, when immune cells derived from mouse spleen were cultured on BAp ceramics, the immune cells cultured on the BAp ceramics fabricated by reaction sintering remarkably increased the percentage of CD3+CD4+ cells corresponding to helper T cells and CD3+CD8+ cells compared with those of BAp ceramics fabricated by USSP. When the BAp ceramics were used to culture the immune cells again, the percentage of CD3+CD4+ cells and CD3+CD8+ cells could be increased as in the first culture. In addition, when BAp ceramics were reused for immune cell culture, they increased the percentage of CD3+CD4+ cells and CD3+CD8+ cells as in the first culture. The BAp ceramics may be expected as a novel culture substrate for adoptive immunotherapy.

Graphical abstract

1. Introduction

According to statistics conducted in 2019, the World Health Organization estimated that cancer would be one of the leading causes of death. Furthermore, the number of cancer cases worldwide is expected to reach 28.4 million in 2040, accounting for a 47 % increase from 2020 [1]. The three main treatment modalities for cancer are surgery, chemotherapy, and radiation therapy. However, these treatments have harmful effects on the body, including patient’s weakness after surgery and other side effects. Consequently, cancer treatment using the patient’s own immune cells, which is known as immunotherapy, has received considerable attention.

This immunotherapy is a natural way to treat cancer by using the body’s own immune system. Among various types of immunotherapy, adoptive immunotherapy is a common approach. In this method, lymphocytes, which play an important role in the immune system, are collected from the patient, activated by coculturing with cytokines and other agents outside the body, and then returned to the patient to treat the disease [2,3]. Rosenberg et al. showed that naturally occurring tumor-reactive lymphocytes could induce complete regression in patients with melanoma, indicating that adoptive immunotherapy might be used in the treatment of common epithelial cancers [4]. However, the cytokines and other agents required for lymphocyte activation are costly and not covered by health insurance, thereby posing financial challenges [5]. Moreover, the expanded lymphocytes have limited tumor-antigen specificity, and they must be cultured with large amounts of IL-2, which remains a major technical challenge. Therefore, alternative strategies that can activate immune cells in a more efficient and cost-effective manner are highly desirable.

Other substances, such as lectins, are also known to activate immune cells [6]. In particular, borono-lectin has killer properties, and it can be applied in cancer immunotherapy [7,8]. Saito et al. reported that multivalency of glycan-binding sites is essential for lymphocyte activation. These sites can bind to glycans on the lymphocyte surface, transmitting activation signals into the cell, thereby inducing activation [9]. However, lectins are plant-derived proteins that exhibit toxic effects. As mentioned, expensive antibody drugs and lectins are often used to activate immune cells in immunotherapy. Given these factors, the number of clinical applications of immunotherapy is limited.

Therefore, this study aimed to develop an immune-activating material that is inexpensive and easy to handle. We focused on phenylboronic acid–containing polymers, in which the BO₂ group in the phenylboronic acid moiety interacts with glycan regions on immune cells to induce activation. This interaction can be mediated by the formation of B(OH)₃ through the dissociation of the BO₂ group, followed by the binding of the resulting OH groups to cell surface glycans. Boronic acid–containing polymers showed lymphocyte activation similar to that of lectins with regard to their ability to promote lymphocyte proliferation [10].

Inspired by these findings, we focused on boron-containing apatite (Ca_9.5+0.5x{(PO₄)_6-x(BO₃)_x}{(BO₂)_1-xO_x} (0 ≤ x ≤ 1); BAp), a material in which boron-derived functional groups are incorporated into hydroxyapatite (Ca₁₀(PO₄)₆(OH)₂; HAp), which is a compound known for its high biocompatibility [11]. BAp contains BO₂ groups within its crystal structure, which can exhibit immunostimulatory effects similar to those of lectins. The amount of BO₂ groups increases as the value of x in the general formula decreases. BAp was fabricated using ultrasonic spray pyrolysis, and BAp ceramics were successfully fabricated with controlled x values. USSP involves spraying an aqueous solution with a predetermined composition to generate fine droplets, which are then introduced into the heated zone of an electric furnace. This process induces the rapid evaporation of the solvent, thermal decomposition of the precipitated metal salts, and solid-state reactions, resulting in the formation of the desired powder. Furthermore, when mouse-derived immune cells were cultured on the fabricated BAp ceramics, the proportion of CD3+CD4+ cells corresponding to helper T cells and CD3+CD8+ cells corresponding to killer T cells increased. These results indicate that BAp, which contains BO₂ groups incorporated as glycan recognition sites, has immune cell activation properties [12].

However, stoichiometric BAp could not be obtained under conditions with a high content of BO₂ groups (i.e., lower x values) because of the high vapor pressure of boron. As described previously, BO₂ groups play an important role in immune cell activation, and increasing their content is crucial.

In this study, a novel fabrication process for BAp ceramics was established using reactive sintering with hydroxyapatite (HAp), calcium oxide (CaO), and boric acid (H₃BO₃) as starting materials. The material properties and immune cell responsiveness of the resulting BAp ceramics were also evaluated. Furthermore, considering their application as a culture substrate for adoptive immunotherapy, the persistence of immune activation was examined by reusing BAp ceramics that had been previously used for cell culture.

2. Materials and methods

2.1. Preparation and characterization of calcined BAp powders

Calcined BAp powder was synthesized as follows: HAp (Taihei Chemical Co., Japan), CaO, and H₃BO₃ (FUJIFILM Wako Pure Chemical Corp., Osaka, Japan) were mixed to obtain the precursor powder (Table 1). The samples were designated as BAp–x, and three compositions corresponding to x = 0.3, 0.4, and 0.5 in the general formula of BAp were prepared. The CaO used in this study was prepared by calcining calcium carbonate (CaCO₃; FUJIFILM Wako Pure Chemical Corp., Osaka, Japan) at 1,000 °C for 3 h (heating rate: 10 °C/min). Approximately 1.2 g of the mixed powder was uniaxially pressed at 50 MPa using a mold with a diameter of 17.5 mm to form a compact. Then, the green compact was heated at 400 °C for 1 h (heating rate: 10 °C/min). The heated compact was ground using a mortar to obtain the calcined BAp powder. The calcined BAp powder was evaluated as follows: The crystalline phases of the obtained powder were identified by X-ray diffraction (XRD) using CuKα radiation (Ultima IV, Rigaku, Japan). The crystalline phases were identified by comparing their XRD patterns with the reference pattern for HAp (No. 09–0432) as provided by the International Centre for Diffraction Data (ICDD). Fourier transform infrared (FT-IR) spectroscopy was performed in the range of 400–4000 cm⁻¹ using the KBr pellet method (IR Prestige-21, Shimadzu, Japan). For comparison, HAp-100 powder (Taihei Chemical Co., Japan) was also characterized.

Table 1.

General characteristics of BAp ceramics.

Synthesis method	Sample	Starting composition / mass %			Bulk density / g・cm-3	Relative density / %	Latice constaint / nm		Ca/P molar ratio		B content / mol %
		HAp	CaO	H3BO3			a(b)-axis	c-axis	Determined	Expected	Determined	Expected
Reaction sintering	BAp-0.3	93.1	0.821	6.03	2.24	72.2	0.942	0.691	1.74	1.69	5.70	6.12
	BAp-0.4	91.9	2.02	6.06	2.13	70.1	0.904	0.692	1.76	1.73	6.30	6.13
	BAp-0.5	90.7	3.22	6.09	2.49	88.7	0.900	0.691	1.80	1.77	6.38	6.15
USSP	BAp-0.4	-	-	-	2.39	72.4	0.938	0.689	1.73	1.73	5.00	6.13
	BAp-0.5	-	-	-	2.68	91.8	0.937	0.692	1.76	1.77	5.58	6.15

2.2. Fabrication and characterization of BAp ceramics

Approximately 0.8 g of calcined BAp powder was uniaxially pressed at 100 MPa using a mold with a diameter of 17.5 mm to fabricate a compact and then sintered at 1,200 °C for 5 h (heating rate: 10 °C/min) to obtain BAp ceramics. As a comparative material, approximately 1 g of HAp-100 powder (Taihei Chemical Industrial Co., Ltd., Japan) was uniaxially pressed at 50 MPa using a mold with a diameter of 20.0 mm to form a compact and then sintered at 1,200 °C for 5 h (heating rate: 10 °C/min) to obtain HAp ceramics.

The resulting ceramics were characterized by XRD and FT-IR spectroscopy. The surface morphologies of individual ceramics were observed using a scanning electron microscope (JSM-6390LA, JEOL, Japan) at an accelerating voltage of 10 kV. The relative density of the HAp and BAp ceramics was calculated by dividing the bulk density of the sintered bodies by the theoretical density of HAp. The bulk density of the ceramics was determined by dividing the mass of the test specimen by its volume. The diameter and thickness of the test specimen were measured using calipers. In addition, the relative density of BAp was calculated by dividing the bulk density by the true density, which was measured using a pycnometer. The true density was used because BAp contains some α-TCP phases. The true density of HAp was assumed to be the theoretical value of 3.16 g/cm³.

The Ca/P molar ratio and B content of the ceramics were determined by inductively coupled plasma atomic emission spectroscopy (ICP-AES; SPS7800, Hitachi High-Tech, Japan). The B content is calculated as follows:

$$\mathrm{B}\text{contents}[\text{mol}\%]=\frac{\mathrm{B}\left[\text{mol}\right]}{\text{Ca}\left[\text{mol}\right]+P\left[\text{mol}\right]+B\left[\text{mol}\right]}\times 100.(2-2-1)$$

The lattice parameters of the BAp ceramics were determined by XRD (Ultima IV, Rigaku, Japan). Silicon powder (NIST SRM 640d) was used as an internal standard. After measurement, the obtained lattice parameters were compared with those of HAp using the ICDD database.

In evaluating the dissolution behavior of the ceramics, 0.1 g of crushed ceramic powder was immersed in 10 cm³ of 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) buffer (pH 7.3). The samples were incubated in a thermostatic shaking bath set at 37 °C (THERMOSTATIC SHAKING BATH T-3S, Thomas Scientific Instruments Co., Ltd., Japan).

In investigating the ion release behavior of the ceramics, 0.1 g of crushed ceramic powder was immersed in 10 cm³ of HEPES buffer (pH 7.3). The suspensions were incubated at 37 °C in a thermostatic shaking bath (T-3S, Thomas Scientific Instruments Co., Ltd., Japan). On day 1, the samples were centrifuged, and the supernatant was collected for analysis. The remaining solid was re-immersed in fresh HEPES buffer, and incubation was continued. This cycle was repeated on days 1, 3, 5, 7, 10, 14, 21, and 28. The concentration of calcium (Ca), phosphorus (P), and boron (B) in the collected supernatants was measured by ICP-AES to evaluate the release profile of various ions from the ceramics.

The BAp ceramic specimens for cell culture were polished, cleaned by ultrasound, and sterilized by dry heating at 160 °C for 90 min before use. The surface roughness of the ceramic specimens was measured using a surface roughness tester (SURFTEST SV-3100, Mitutoyo Corp., Japan) based on the arithmetic average roughness (R_a), which is a representative parameter of surface roughness.

2.3. Mice and cell culture

All animal experiments were conducted in accordance with the protocols approved by the Animal Care and Use Committee of the School of Science and Technology, Meiji University (approval number: MUIACUC2025–10). Immune cells were isolated from the spleens of 7- to 12-week-old female C57BL/6 N mice. The spleens were harvested, and immune cells were separated using a nylon mesh sheet with a pore size of 67 µm. Red blood cells were removed by washing with an ammonium chloride potassium (ACK) solution and phosphate-buffered saline (PBS). The ACK lysing buffer was prepared using ammonium chloride (NH₄Cl; Wako Pure Chemical Industries Osaka, Japan), potassium bicarbonate (KHCO₃; Wako Pure Chemical Industries, Osaka, Japan), and disodium ethylenediaminetetraacetate dihydrate (EDTA-2Na·2H₂O; Wako Pure Chemical Industries, Osaka, Japan). The isolated immune cells were cultured in an RPMI-1640 medium supplemented with 10 % fetal bovine serum, 100 U/cm³ penicillin, 100 µg/cm³ streptomycin, and 50 mmol/dm³ 2-mercaptoethanol. After preparing the immune cell suspension to a concentration of 1.0 × 10⁶ cells/cm³, the cells were directly seeded onto BAp and HAp ceramics with polystyrene plates used as control materials. The cultures were incubated at 37 °C in a humidified atmosphere containing 5 % CO₂ for 1 day, followed by flow cytometry. In addition, Transwell inserts were used to culture spleen cells in the presence of ceramics under noncontact conditions. 1.0 cm³ suspension of spleen cells at a density of 1.0 × 10⁶ cells/cm³ was seeded onto 12-well polystyrene plates, and individual ceramic specimens were placed on the Transwell insert. Subsequently, 0.8 cm³ of medium was added, and the cultures were incubated at 37 °C in a humidified 5 % CO₂ atmosphere for 1 day. The ceramics used in this study were prepared by uniaxially pressing 0.3 g of calcined BAp powder using a mold with a diameter of 10.0 mm and then sintering at 1,200 °C for 5 h (heating rate: 10 °C/min).

2.4. Flow cytometry

The collected immune cells were washed two times with PBS and resuspended in 1 mL of PBS. The cell suspension was treated with 1 µL of fluorescent reactive dye (LIVE/DEAD Fixable Aqua Dead Cell Stain Kit, Invitrogen) and incubated at 4 °C for 30 min. The reaction was stopped by washing with PBS. Then, the cells were washed two times with 2 mL of fluorescence-activated cell sorting (FACS) buffer and resuspended in 1 mL of FACS buffer. The suspension was incubated with 120 µL of antibody mixture containing 10 µL each of anti-CD3, anti-CD4, and anti-CD8 antibodies. CD3 is a surface marker for T cells, while CD4 and CD8 are markers for helper and killer T cell subsets, respectively. After incubation at 4 °C for 15 min, the cells were washed with 2 mL of FACS buffer. Data were analyzed using Attune Cytometric Software (Applied Biosystems). Gating was set using unstimulated control samples, and voltages were determined on the basis of unstained samples. The proportions of CD3+CD4+ cells and CD3+CD8+ cells were calculated within each sample based on the total lymphocyte population in the spleen, which was defined as 100 %.

2.5. Morphological observation of cells

The morphology of cells after 1 day of culture was observed by using a scanning electron microscope. For scanning electron microscopy (SEM) analysis, cells were washed three times with PBS and fixed with 1 mL of 10 % glutaraldehyde (Wako Pure Chemical Industries, Japan) at 4 °C. After fixation, the cells were washed three times with PBS and three times with sterile water, followed by freeze drying prior to SEM observation.

2.6. Evaluation of the sustainability of immune activation function

The following experiments were performed to verify whether the used BAp ceramics could be reused. After cell culture, the ceramic specimens were analyzed by XRD for crystalline phase identification and by ICP-AES for elemental analysis to assess the changes in material properties. Furthermore, the ceramics were resterilized by dry heating and used to culture mouse spleen-derived immune cells under the same conditions. Flow cytometry was conducted to evaluate the immune-activating effect of the reused BAp ceramics.

2.7. Statistics analysis

Data are presented as mean ± standard deviation (SD). Statistical analyses were performed using Welch’s t-test. Statistical significance was defined as *p < 0.05 and **p < 0.01.

3. Results

3.1. Characterization of calcined BAp powders

The XRD patterns of the calcined BAp powders (x = 0.3–0.5) and HAp powder are shown in Fig. 1(A). All samples exhibited a single crystalline phase of HAp. Based on the FT-IR spectra shown in Fig. 1(B), the absorption bands corresponding to the PO₄ groups were detected at 1100–960, 600, and 560 cm⁻¹, whereas the absorption bands corresponding to the OH groups were observed at 3600–3000 cm⁻¹ in all samples.

Fig. 1.

XRD and IR analyses of BAp calcined powders and BAp ceramics.

(A) XRD patterns of calcined BAp and HAp powders.

(B) FT-IR spectra of calcined BAp and HAp powders.

(C) XRD patterns of HAp and BAp ceramics.

(D) FT-IR spectra of HAp and BAp ceramics.

3.2. Characterization of HAp and BAp ceramics

The XRD patterns of the resulting HAp and BAp ceramics are shown in Fig. 1(C). All fabricated specimens exhibited characteristic XRD patterns of apatite. However, small amounts of α-tricalcium phosphate (α-TCP) phase were detected in BAp-0.3 and BAp-0.4 specimens. Furthermore, the diffraction intensity of α-TCP increased with the decrease of the value of x in the general formula of BAp. Ceramics with lower x values produced larger amounts of α-TCP. Based on the FT-IR spectra shown in Fig. 1(D), the absorption bands of the PO₄ groups were detected around 1100–1000 cm⁻¹ and 600–573 cm⁻¹ in all samples. In addition, for BAp-0.3, 0.4, and 0.5, the absorption bands corresponding to the BO₂ groups (2000 and 1950 cm⁻¹) and BO₃ groups (1300–1200 cm⁻¹ and 800–750 cm⁻¹) were observed.

Fig. 2(A) shows the SEM images of the HAp and BAp ceramics before polishing. SEM observations confirmed pore elimination and grain growth leading to densification. Table 1 shows the relative density, lattice constant, and chemical composition determined by ICP-AES of the BAp ceramics. The true densities of BAp-0.3, BAp-0.4, and BAp-0.5, which were measured by using the pycnometer method, were 3.11, 3.05, and 2.81 g/cm³, respectively. Correspondingly, the relative density of BAp-0.5 was 89 %, indicating a relatively dense structure, whereas that of BAp-0.3 and BAp-0.4 was 72 % and 70 %, respectively. Compared with stoichiometric HAp, the lattice constant of the BAp ceramics decreased along the a-axis and increased along the c-axis. Furthermore, the chemical composition of the synthesized BAp-0.3 to 0.5 ceramics was closer to the theoretical value of BAp than those synthesized by USSP.

Fig. 2.

SEM images and solubility test results of BAp ceramics.

(A) SEM images of HAp and BAp ceramics (Scale bar: 5μm (HAp), 1μm (BAp)).

(B) Release profiles of calcium ions from BAp ceramics.

(C) Release profiles of phosphate ions from BAp ceramics.

(D) Release profiles of boron ions from BAp ceramics.

The results of the solubility test on ceramics are shown in Fig. 2(A–C). Among the ceramic samples, Ca and P were continuously released, whereas B reached its release limit by day 7. The increase in phosphorus release can be attributed to the enhanced liberation of phosphate ions resulting from the increased fraction of the secondary phase, α-TCP. In contrast, for calcium, the synthesized BAp-0.3–0.5 samples showed an increasing Ca/P molar ratio with increasing x value, leading to the highest calcium ion release being observed in the BAp-0.5 sample. In addition, the amount of Ca released from BAp ceramics was greater than that from HAp.

3.3. Cellular responses of mouse spleen cells to HAp and BAp ceramics

Fig. 3(A and C) show the percentages of CD3+CD4+ cells and CD3+CD8+ cells cultured on HAp and BAp-0.3–0.5 ceramic specimens, respectively, based on the results of flow cytometry. The figures also include the percentages of CD3+CD4+ cells and CD3+CD8+ cells cultured on BAp ceramics fabricated by USSP (Fig. 3(B and D)). These results were redrawn using the data reported in reference [12]. Representative cytograms obtained from the flow cytometric analysis are shown in Figs. S-1 (control) and S-2 (BAp-0.4). In addition, Fig. S-3 presents the cell viability after 1 day of culture, calculated based on the Live/Dead results obtained by flow cytometry. For both cells, their percentage increased when BAp-0.3–0.5 ceramics were used as the culture substrate compared with the control and HAp groups. Furthermore, BAp ceramics fabricated by reactive sintering remarkably enhanced the percentages of CD3+CD4+ cells and CD3+CD8+ cells compared with those prepared by USSP.

Fig. 3.

Flow cytometric analysis of immune cells cultured on BAp ceramics and morphological observation of T cells.

(A) Percentage of CD3+CD4+ cells on the culture substrate prepared by reaction sintering.

(B) Percentage of CD3+CD4+ cells on the culture substrate prepared by ultrasonic spray pyrolysis.

(C) Percentage of CD3+CD8+ cells on the culture substrate prepared by reaction sintering.

(D) Percentage of CD3+CD8+ cells on the culture substrate prepared by ultrasonic spray pyrolysis.

(E) SEM images of spleen cells cultured on BAp ceramics (Scale bar: 10 μm (× 1000), 1 μm (× 15,000)).

(F) Percentage of CD3+CD4+ cells in the indirect culture of BAp.

(G) Percentage of CD3+CD8+ cells in the indirect culture of BAp.

In evaluating the effect of the culture substrate on cell morphology, T cells cultured on each ceramic were observed by SEM. The results are shown in Fig. 3(E). On all BAp ceramics, adherent T cells were observed, with notable morphological changes and pseudopodia formation. Considering that the surface roughness of all ceramics used for culture was adjusted to below 0.1 µm, the influence of surface topography can be considered negligible.

3.4. Effect of liquidity factors on HAp and BAp ceramics

In examining the effects of liquidity factors eluted from the ceramics specimens on the percentages of immune cells, an indirect coculture experiment was conducted using a Transwell insert. Fig. 3(F and G) shows the percentage of CD3+CD4+ cells and CD3+CD8+ cells after indirect coculture with HAp and BAp ceramics. No significant differences in the percentage of CD3+CD4+ cells and CD3+CD8+ cells were found among the samples. These findings indicate that eluted factors such as ions released from the ceramics did not affect either CD3+CD4+ cells or CD3+CD8+ cells.

3.5. Sustainability of the immune activation function of BAp ceramics

After the culture of immune cells, the ceramic specimens were analyzed by XRD and ICP-AES to identify the crystalline phases and to determine the elemental composition, respectively. Fig. 4(A) shows the XRD patterns of the ceramics after culture. On all specimens, HAp was identified as the main crystalline phase. Apart from HAp, the α-TCP phase was partially identified in BAp-0.3 and BAp-0.4. These crystalline phases were consistent with those observed before cell culture.

Fig. 4.

Material properties and immunoresponsiveness of BAp ceramics after adherent culture with immune cells.

(A) XRD pattern of HAp and BAp ceramics after cultivation.

(B) Percentage of CD3+CD4+ cells on the culture substrate.

(C) Percentage of CD3+CD8+ cells on the culture substrate.

Table S1 presents the results of elemental analysis of the BAp ceramics after cell culture using ICP-AES. Despite a decrease in the Ca/P molar ratio, the overall chemical composition of the BAp ceramics remained largely unchanged after culture.

Furthermore, the BAp ceramics that had been previously used for cell culture were reused to culture immune cells for 1 day. Fig. 4(B and C) shows the percentages of CD3+CD4+ cells and CD3+CD8+ cells after the second culture. On all BAp ceramics, the percentages of CD3+CD4+ cells and CD3+CD8+ cells were significantly higher than those of the control and HAp groups, showing similar levels to those observed in the initial culture.

4. Discussion

Adoptive immunotherapy is a cancer treatment that involves isolating immune cells from the patient, expanding and activating them through in vitro culture, and then reinfusing them into the patient. This approach has attracted attention because of its minimal side effects, as it uses the patient’s own immune cells. However, this treatment modality is limited by the high cost of reagents used for immune cell activation. In addressing this issue, we have focused on developing cost-effective and easy-to-handle immunoactive materials and have fabricated BAp ceramics using USSP [12]. This process has easy composition control of synthetic powders because the raw powder is prepared from solution [[13], [14], [15]]. Therefore, USSP is suitable for synthesizing materials with complex compositions such as BAp. We have also fabricated BAp ceramics using USSP and evaluated their immune cell responsiveness. Consequently, BAp-0.4 with a high content of BO₂ group (low x value) had the highest immunoactive effect. However, BAp synthesized by USSP could not synthesize stoichiometric BAp under a high BO₂ group content (low x value). Given the high vapor pressure of boron, during USSP, HAp precipitates before BO₂⁻ or BO₃³⁻ groups can be incorporated, leading to the volatilization of boron. As a result, the final B-containing powder exhibits a lower boron content than the theoretical stoichiometric composition.

In this study, the synthesis method for BAp ceramics was reevaluated, and solid-state reaction sintering was used with HAp, H₃BO₃, and CaO as starting materials. BAp ceramics with compositions corresponding to x = 0.3–0.5 in the general formula were fabricated. Subsequently, their material properties were characterized, and the immune cell responses were evaluated under in vitro conditions. XRD analysis of the calcined BAp powders confirmed a single-phase HAp, although their crystallinity was low. The FT-IR spectra showed no absorption bands attributable to borate groups. These results can be explained by the low crystallinity of the apatite at the calcined powder stage and the absence of borate group substitution within the apatite crystal structure. On the contrary, XRD analysis of BAp ceramics, which were fabricated by molding and sintering the calcined powders, confirmed a highly crystalline HAp as the main phase. FT-IR spectra revealed peaks corresponding to the BO₂ and BO₃ groups. These FT-IR results are similar to the absorption spectra of BAp and HAp synthesized by Ito et al. [11]. Considering the FT-IR and XRD data, the ceramics fabricated in this study can be identified as BAp.

In BAp-0.3 and BAp-0.4 samples, the α-TCP phase slightly formed, with the peak intensity of α-TCP increasing as the x value decreased. This trend can be attributed to the low Ca/P ratio in the starting composition of ceramics with smaller x values, which leads to calcium lattice vacancies within the apatite crystal structure, thereby destabilizing the structure. Consequently, the apatite decomposes more easily, resulting in the greater formation of α-TCP, which is a phase with a lower Ca/P ratio than HAp.

By contrast, BAp-0.5, which has a higher x value and higher Ca/P ratio, is less prone to calcium lattice vacancies within the apatite crystal structure. This characteristic results in a more stable crystal structure and makes apatite decomposition less probable. In the general formula of BAp (Ca_9.5+0.5x{(PO₄)_6-x(BO₃)_x}{(BO₂)_1-xO_x} (0≦x≦1)), the {(PO₄)_6-x(BO₃)_x} site corresponds to the a(b)-axis, whereas the {(BO₂)_1-xO_x} site corresponds to the c-axis [11,16]. Compared with the theoretical lattice constants of HAp, the a(b)-axis lattice constants of BAp ceramics contracted, and the a(b)-axis lattice constants decreased as the x value increased (indicating a higher content of BO₃ groups). This result is consistent with previous reports, that is, the P–O bond distance in BAp is shorter than that in HAp [16]. Regarding the c-axis lattice constant, no significant changes were observed, which is consistent with prior studies.

Based on the results of relative density measurements, the relative density of HAp and BAp-0.5 was 96 % and 89 %, respectively, indicating relatively dense structures. By contrast, the relative density of BAp-0.3 and BAp-0.4 was 72 % and 70 %, respectively. This decrease in density is due to the decomposition of the apatite phase into α-TCP, which inhibited densification. In addition, the formed α-TCP may have segregated near grain boundaries, thereby exerting a pinning effect that suppressed grain growth in the apatite phase.

The results of ICP-AES analysis indicate that reactive sintering successfully produced BAp ceramics with a composition close to the stoichiometric target. By contrast, BAp ceramics fabricated via USSP faced challenges in achieving stoichiometric composition under a higher BO₂ content (lower x values). This limitation is due to the high vapor pressure of boron, causing boron to volatilize during USSP before the incorporation of the BO₂ and BO₃ groups into the precipitating HAp. Consequently, the content of boron in the obtained powder is lower than the intended stoichiometric composition.

Flow cytometry following the direct seeding of murine spleen cells onto the fabricated specimens revealed that BAp ceramics fabricated via reactive sintering remarkably increased the proportion of CD3+CD4+ cells compared with the control and HAp groups. A similar trend was also observed in CD3+CD8+ cells. By contrast, when spleen cells were cultured under noncontact conditions using Transwell inserts, no significant changes were observed in either CD3+CD4+ cells or CD3+CD8+ cells populations. These findings indicate that soluble factors, such as ions released from the ceramics upon dissolution, did not directly influence the spleen cells. Collectively, these results indicate that direct physical contact between the ceramics and spleen cells induces immunostimulatory effects via cell–material interactions, thereby increasing the proportion of CD3+CD4+ cells and CD3+CD8+ cells. These observations are consistent with our previous findings [12].

BAp ceramics fabricated via reactive sintering remarkably increased the proportion of CD3+CD4+ cells and CD3+CD8+ cells compared with those prepared using USSP. ICP-AES analysis revealed that the BAp ceramics fabricated by reactive sintering exhibited a higher boron content and a composition closer to the theoretical stoichiometry than those fabricated by USSP. Therefore, the content of BO₂, which may contribute to immune cell activation, was also higher, which may account for the enhanced immunostimulatory effects observed.

Furthermore, SEM observations of T cells cultured on each sample revealed that the T cells on the BAp ceramics extended pseudopodia and adhered to the surface. Although immune cells are generally nonadherent as they are suspension cells, the stimuli from the material surface induced cell adhesion and morphological changes [17]. These findings are consistent with our previous reports [12,18]. In addition, because the amount of α-TCP contained in the BAp ceramics is extremely small, its contribution to the observed immune cell responses may be considered negligible.

As shown in Fig. 3(F and G), no significant differences in the proportion of immune cell populations were observed among the materials when only the soluble factors released from the ceramics, such as calcium (Ca), phosphorus (P), and boron (B), were evaluated. These results indicate that the immunostimulatory effects of the BAp-based immunoceramics are primarily dependent on direct cell–material contact.

After the initial immune cell culture, the ceramics were subjected to XRD analysis for phase identification and ICP-AES for elemental composition. Subsequently, a second round of immune cell culture was conducted using the same ceramics. The XRD and ICP-AES results revealed minimal changes in the crystalline phases and composition before and after cell culture. A slight decrease in the Ca/P molar ratio was observed, which may be due to the dissolution of α-TCP and calcium components present in the ceramics. Flow cytometry analysis of the cells cultured in the second round showed results comparable to those obtained in the initial culture. These findings indicate that the BAp ceramics developed in this study are durable enough to support repeated use as a culture substrate. Compared with cytokines and other immunostimulatory proteins, which are often expensive, ceramics provide a cost-effective alternative. Furthermore, the demonstrated reusability of the ceramics indicates their potential to be highly cost-effective in clinical applications.

In this study, T cell stimulation was limited to 24 h to evaluate the initial immune response induced by direct contact with BAp ceramics. Although conventional T cell culture protocols typically employ 3–7 days of stimulation to assess sustained proliferation and activation, the present experimental design was specifically focused on the early-stage interactions between the material and immune cells. Future studies incorporating longer culture periods will be necessary to comprehensively evaluate long-term functional changes in T cells and the persistence of immune responses.

5. Conclusions

The BAp ceramics were successfully fabricated by reactive sintering using HAp, CaCO₃, and H₃BO₃ as starting materials, and the following three findings were obtained.

• 1)Compared with BAp ceramics prepared by USSP, those fabricated by reactive sintering had a composition closer to the theoretical stoichiometry of BAp.
• 2)BAp ceramics prepared by the reactive sintering significantly enhanced the proportions of CD3+CD4+ cells and CD3+CD8+ cells compared with the control group, and these proportions were consistently higher than those observed for BAp ceramics prepared by the USSP method.
• 3)Furthermore, the BAp ceramics used for cell culture were shown to be reusable for subsequent immune cell culture after sterilization.

Based on these findings, BAp ceramics fabricated via reactive sintering were shown to increase the proportions of CD3+CD4+ cells and CD3+CD8+ cells, suggesting their potential as biomaterials for adoptive immunotherapy in the future.

CRediT authorship contribution statement

Y. Oshima: Writing – original draft, Validation, Formal analysis, Data curation. D. Nakagawa: Validation, Methodology, Data curation. S. Nagai: Writing – review & editing, Supervision, Resources, Methodology, Conceptualization. M. Aizawa: Writing – review & editing, Supervision, Project administration, Funding acquisition, Conceptualization.

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

The author is an Editorial Board Member/Editor-in-Chief/Associate Editor/Guest Editor for [Journal name] and was not involved in the editorial review or the decision to publish this article.

Data availability

Data will be made available on request.