Introduction

Loss of bone tissue can result from severe trauma or tumor formation, and bone regeneration is a time-consuming process. Autologous bone grafting offers optimal biocompatibility; however, the available volume is limited [1]. Allogeneic bones can also be used but involve risks of infection and immune responses. Metals, organic materials, and ceramics are examples of biomaterials used to fill bone defects. Bone biomaterials should be biocompatible and bone-replaceable. In this regard, bioceramics are widely used owing to their biocompatibility and mechanical strength. Calcium phosphate as a bioceramic has been garnered great interest because of its high biocompatibility [2]. Calcium phosphate features many phases, each with a different solubility. Examples include monocalcium phosphate anhydrous, dicalcium phosphate (DCPD), octacalcium phosphate (OCP), hydroxyapatite (HAp) and β - TCP [3]. In particular, OCP undergoes a phase transition to HAp after implantation [4] and can be used as a biomaterial with higher performance when used in combination with collagen [5]. A clinical case of tibial segmental bone loss reconstruction using an autograft and-TCP has been reported [6]. b-TCP, one of the many commercially available calcium phosphate-based bone substitutes, is resorbed by osteoclasts due to its high solubility in acidic conditions [7]. Moreover, b-TCP can be easily obtained by mixing calcium hydroxide and DCPD well and by sintering at approximately 1000 ℃.

The bone is structurally optimized and consists of cortical and cancellous bone segments. Therefore, a three-dimensional 3D-designable bone graft material that mimics these characteristics is desirable [8]. The 3D models of shapes drawn via CAD can be freely shaped by polymer melting or photopolymerization using polyethylene glycol diacrylate. He et al. reviewed methods such as fused deposition modeling (FDM) and stereolithography (SLA) in which 3D printing is likened to the reverse process of cutting a potato with a knife [9]. Other methods reviewed include powder–liquid 3D printing, selective laser sintering, and digital light processing (LCD) [10]. The LCD method usually irradiates ultraviolet rays two-dimensionally from the panel attached to the bottom and reacts with the photocuring resin. Reviews on bioceramics using 3D printing have primarily focused on porous calcium phosphate and bioglass [11]. Elhattab et al. reported FDM-based 3D printing of TCP and polylactic acid composites [12]. In addition, Ye et al. reported FDM-based 3D printing of TCP and polyhydroxyalkanoate composites [13], and Wang et al. reported a calcium phosphate bioceramic for fabricating bone tissue engineering scaffolds using a digital light processing 3D printer [14]. Navarrete-Segado et al. used an LCD 3D printer to produce apatite implants by dispersing apatite in a resin to form a porous apatite model. They reported that the dispersibility of HAp influenced the quality of ceramic molding [15].

In this study, we have constructed a new system that is 3D prints from a mixture of DCPD and Ca(OH)2 with polyethylene glycol diacrylate and dispersants and transforms into β-TCP upon sintering.

Materials and methods

Materials

DCPD (Lot: WTL6929), calcium hydroxide (Lot: WTH3013), and Polyethylene Glycol 200 (Lot: TPJ0504) were purchased from Fujifilm Wako Pure Chemical Corporation (Japan). Water-washable resin(SKU: SK01W), whose main component was polyethylene glycol diacrylate, was purchased from Felidentia Capital Limited (Japan).

Methods

Preparation method of β -TCP model

DCPD and calcium hydroxide were mixed at a Ca/P ratio of 1.50 for a total of 50.0 g. Subsequently, 25 ml of PEG and 75 ml of the water washable resin were mixed with 50.0 g of the prepared calcium phosphate mixture. The prepared slurry was stirred at 500 RPM for 60 min in a planetary ball mill (P6 Classic Line, Fritsch Japan Co., Ltd., Japan) using an agate jar and 10 stones with a diameter of 1.0 cm. A Sonic Mini (Phrozen Technology, Taiwan) was used as the 3D printer. The mixture was poured into the resin vat and printed. The prepared gyroid model with 10.0-mm diameter and 20.0-mm height was designed in Fusion 360 (Autodesk®, USA), as shown in Fig. 1.

Fig. 1
figure 1

The prepared STL model of gyroid model with 10.0-mm diameter and 20.0-mm height

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The printed model was then transferred to the normal printing phase and vertically printed using a circle as the base. The sample after photopolymerization and before sintering is called (AMR)-150. Subsequently, the support material was carefully removed using scissors and tweezers. Each AMR-150 was sintered in a furnace at 1000 °C with SRF650 (Morita Co., Japan). The sintered sample is referred to as AST-150. A disk-type AST-150 with a diameter of 7 mm was prepared for biocompatible cell culture.

Characterization of prepared samples

XRD patterns

X-ray diffraction (XRD) patterns were obtained using a diffractometer (PANalytical X’Pert PRO MPD diffractometer (Malvern PANalytical Co.). The measurements were performed under CuKα radiation (40 mA and 45 Kv), with 0.001° 2θ steps from 3.00° to 60.0°.

FT-IR spectra

Infrared spectra were obtained using a Fourier-transform infrared (FTIR) spectrometer (FT/IR-4200, JASCO Co., Tokyo, Japan) equipped with a diamond type ATR accessory. The FTIR spectra of prepared samples were measured in the range 400–4000 cm− 1 at a resolution of 1.0 cm− 1. The obtained spectra were collected and averaged 64 times.

SEM

The surfaces of the prepared samples were observed using field-emission scanning electron microscopy (SEM; TM4000PlusII, Hitachi, Japan). Prior to observation, the samples were covered with Au–Pd to improve conductivity.

Compression test

The compression test was performed using a cylinder with the circular surface at the bottom. The compression crosshead speed was 2 mm/min. Compression curves were drawn using the compression testing machine EZ test EZ-LX 5 kN and the attached computer.

Cell culture

Commercially available iliac bone marrow-derived mesenchymal stem cell(MSC)s (donors aged 41 years; gender: male Lot No 413042 Lonza, Walkersville, MD, USA) were seeded at 10,000 cells/cm2 on β-TCP in a 24-well dish and α-minimal essential medium with L-glutamine and phenol red (α-MEM: Fujifilm Wako, Japan) with 10% fetal bovine bone (FBS: Hyclone, Logan, USA) and 1% Penicillin-Streptomycin-Amphotericin B Suspension (life Technologies, Waltham, MA, USA) under 37 °C and 5% CO2 for 3 days.

Immunofluorescence staining (evaluation of cytoskeleton and structure): Evaluation of the adhesion of MSCs seeded onto the prepared samples.

Actin and nuclear staining were performed. After 3 days of culture, the cells were washed with phosphate-buffered saline (PBS), fixed with 4% paraformaldehyde (Fujifilm Wako), and stained with phalloidin 488 (Invitrogen, Thermo Fisher, USA) for 1 h at ambient temperature. Washed again with PBS. Nuclei were stained with a mixture of 1ul DAPI (DOJINDO Fujifilm Wako, Japan) and 1ul PBS for 10 min and washed with PBS. Observations were performed using a fluorescence microscope. (OLYMPUS, IX71, Japan).

Results and discussion

Figure 2 shows photographs of a 3D-printed green body and a sintered body. The surface of the printed sample is smooth, and the layer lines often observed in 3D printing are not visible to the naked eye. This polymer had no defects on its surface and no crystal precipitation are present on its surface.

Fig. 2
figure 2

Photo images of prepared green body and sintered samples; left: AMR-150 (a), right: AST-150 (b)

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The size of the sintered body decreased owing to sintering shrinkage. The horizontal and vertical shrinkage were 15.2 ± 1.5% and 34.2 ± 0.2%, respectively. To create a sample with the desired size that matches the bone-defective part, it was necessary to create a larger-sized green body, taking shrinkage into consideration. It has been suggested that the solid filling rate and the organic matter content of the gyroid structure also affect the shrinkage rate. However, further studies are required to elucidate these effects. The surface of the sintered body was rougher than that of the green body owing to the precipitation of crystals. To evaluate these phenomena in detail, we used SEM to observe the surface details.

Figure 3 shows the SEM image of the prepared sample. Figure 3 (a) and (b) show a smooth surface with few precipitated crystals. The SEM image of the green body suggests that it was moderately dispersed in the polymer. Plate-like crystals can be observed in Fig. 3(c) and (d). Degreasing at temperatures above 650 °C suggests degreasing of the polymeric binders. AST-150 is composed of only precipitated crystals. In particular, the plate crystals are intricately intertwined.

Fig. 3
figure 3

SEM image of prepared models; (a) AMR-150 × 30, (b) AMR-150 × 1000, (c) AST-150 × 30, and (d) AST150 × 1000

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Figure 4 shows the XRD patterns of the prepared samples. AMR 150 exhibits a unique peak at 11.69°, which is assigned to DCPD. Only a high-strength peak is observed at 11.69°, indicating that the crystal habit of the precipitation is controlled. In the sintered AST150 sample, it was assigned as β -TCP. Pronounced peaks were observed at 17.03°, 31.05°, and 34.4°. These results indicate that the pure β -TCP was synthesized with 3D modeling in sintered phase [16, 17].

Fig. 4
figure 4

Effects of sintering on XRD patterns of prepared samples: (a) AMR-150 and (b) AST-150

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Figure 5 illustrates the FT-IR spectra of the prepared samples and raw materials. The spectrum of AMR-150 is strongly influenced by PEG200 [18] and the resin. Specifically, peaks attributed to PEG are observed at 1110, 1437, 1564, and 2880 cm− 1, and that of the photopolymerized resin is observed at 1731 cm− 1. The spectral waveform of β -TCP is observed in AST-150 [19].

Fig. 5
figure 5

Effects of sintering on FT-IR spectra of prepared samples: (a)AMR-150 and (b) AST-150

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The results of the XRD pattern and FT-IR spectrum revealed that the fired sample featured a single β -TCP phase. The β- TCP is a crystal with high biocompatibility and proven clinical applications. β -TCP induces new bone formation and has bone resorption properties. Thus, it can be used not only for implants in the mandible but also for orthopedic purposes.

The mechanical strength of the synthesized sample is an important factor when practical applications. Figure 6 shows the results of the compression tests. The compression test was conducted by placing the sample cylinder vertically. The maximum compressive strength for the specimen with a diameter of 8.5 mm was 79.5 ± 9.4 kPa (SD). This compressive strength is such that it does not collapse even when held in the hand and is not damaged even when grabbed with tweezers. It has been suggested that this material is strong enough to withstand surgery. Moreover, a stronger structure can be created by varying the blending ratio of the photopolymerized resin. The solid–pore ratio was 0.5. It was assumed that the strength could be controlled by adjusting this value. Sous et al. reported the mechanical strength of β - TCP with a pore ratio of 65% [20]. Moreover, Ryan et al. reported the mechanical strength of β– TCP scaffolds for tissue engineering [16]. The gyroid structure of calcium phosphate was found to be suitable for biomaterials [21]. Considering these reports, structural comparisons of printed patterns are essential. Although the pores in the structure are crucial for vascularization to attain maximum depth, that is, to improve metabolic efficiency, it is important to consider the balance with strength by adjusting the porosity.

Fig. 6
figure 6

Compression test of prepared AST-150 samples. #1,2,3 presents the trajectory of each compression

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Figure 7 shows the MSC nuclear and actin staining. The cells attach to the surface of the sample and stretch actin. This indicates that cells do not die due to attachment of MSC​. Several papers have reported on the combination of β-TCP and MSC. Liu and Lun reported on β- TCP composites in orthopedics with MSC cells [22]. Moreover, Seebach et al. reported on endothelial progenitor cells and MSC seeded onto β-TCP granules to enhance vascularization and bone healing in a critical-sized bone defect in rats [23]; they also investigated the relationship between β-TCP and MSCs, with their results indicating acceptable coexistence [24].

Fig. 7
figure 7

MSC nuclear staining and actin staining on prepared samples

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In this study, we demonstrate that synthesis can be performed using the effects of sintering and report a one-pot sintering method. The inclusion of various ceramic crystal phases in resins and methods that utilize the reactions caused by sintering will lead to the development of various productive technologies. For example, apatite can be synthesized by setting the calcium-to-phosphorus ratio to 1.67. In particular, this ratio is important for mixing the resin and starting materials. Thus, the compressive strength can be improved by manipulating this ratio. This pilot study validates the feasibility of synthesizing the desired crystalline phase by introducing a variety of starting materials. The contribution of this study as a bioceramic synthesis method can be enhanced by synthesizing various biocompatible ceramics and conducting cell culture and implantation tests. In this study, an inexpensive LCD printer with a low resolution (1080:1920) was used. Moreover, the layer resolution of the green body was low. The screen resolution offers the possibility of reproducing more details in the model. The results clearly indicate that the selection of equipment is an important aspect, depending on the balance between the resolution of the 3D printer screen and the desired model (Table 1).

Table 1 Printing properties of LCD 3d printer
Full size table

Additive manufacturing, or bioprinting, for biological applications is a field that is expected to develop in the future [25,26,27]. A wide range of research is being conducted, from 3D printed living cells to implants that mimic hard tissue, such as the one in this study [28, 29]. Create a computer-aided design of the missing 3D model using an MRI or CT scanner [30]. Four-dimensional printing, which is a further development of these technologies, is attracting attention [31]. It is a technology that generates dynamically controlled architectures and has potential for use in hard and soft tissue implants. This indicates that it can also be applied as a drug delivery system by encapsulating drugs in bioink or bioceramic [32, 33]. In this study, we were able to demonstrate the basic considerations for constructing a system in which drugs are encapsulated in bioceramics in future research.

Conclusion

A 3D printing model consisting of a combination of DCPD, Ca (OH)2, a photopolymerizable monomer, and PEG was synthesized, and a 3D printing slurry solution consisting of calcium phosphate was also prepared. The 3D print method was successfully demonstrated, resulting in dispersed ceramic samples. Upon calcination, DCPD and Ca(OH)2 were observed to undergo a phase transition to a β-TCP phase. In conclusion, we report a method to economically and freely synthesize and mold porous β-TCP.