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We have previously developed a novel chelate-setting β-tricalcium phosphate (β-TCP) cement with non-fragmentation property in vivo. This novel cement has been set on the basis of chelate-setting mechanism of inositol phosphate (IP6). In this study, β-TCP powders were synthesized by mechanochemical method, and the as-prepared powders were heated at 600-1300°C for 1 h. Some properties of the resulting powders were examined. The crystalline phase of the resulting powders in the range of 600-1100°C was of β-TCP single phase. In the cases at 1200°C and 1300°C, the resulting powders were composed of β-TCP and α-TCP. Median particle sizes of the resulting powders increased with heating temperature from 5.35 μm up to 47.7 μm. Dissolution rate of Ca2+ ions from the β-TCP powders was measured by Japanese Industrial Standard T 0330-3. When the heating temperature was at 700°C, the Ca2+ ions solubility was highest among examined ones. The β-TCP powder heated at 700°C for 1 h will be expected as the starting powder for paste-like artificial bone filler with excellent bioresorbability.

Additive printing, commonly known as additive manufacturing, presents transformative potential over conventional scaffold manufacturing methods in the repair of musculoskeletal tissues. With its ability to create complex geometries and microarchitectures that mimic tissue complexity, additive printing can offer various innovative methods for successful patient-specific tissue engineering applications. Here, we review the development in additive printing of the musculoskeletal tissues, in particular, bone and tendon tissue engineering. Promising technologies including (1) light-assisted-based, (2) nozzle-based, and (3) printer-based system, and materials which are involved in additive printing of bone and tendon tissues, will be presented. Lastly, a discussion of the future direction in this field is also provided.

A novel chelate-setting β-tricalcium phosphate (β-TCP) cement with anti-washout properties have been fabricated previously. This cement has been set on the basis of chelating ability of inositol phosphate (IP6). In this study, the ball-milling and surface-modification conditions of starting β-TCP cement powders were optimized in terms of bioresorbability. Starting powders were prepared by simultaneously ball-milling at 300 rpm for 3 h with 1 mm diameter ZrO2 beads and surface-modifying with 40 cm3 of 3000 ppm IP6 solution. The resulting starting powder was consisted of β-TCP single phase, and had high specific surface area of 48.3 m2∙g-1. Cement pastes were prepared by mixing the starting powder and the aqueous solution composed of 2.5 mass% sodium hydrogen phosphate, 1.5 mass% citric acid and 1.0 mass% sodium alginate at a powder/liquid ratio of 1/0.90 [g∙cm-3] for 2 min. After setting in pure water for 72 h, compressive strength of the cement specimens was higher than that of human cancellous bone. Dissolution rate of Ca2+ ions was measured by according to Japanese Industrial Standard T 0330-3. The results of Ca2+ ions dissolution rate test demonstrated that the cement specimens derived from the above starting powder were the highest dissolution rate among examined ones. This cement would be expected as bone fillers with high bioresobability.

Hydroxyapatite (HAp) is one of components of bone and teeth, and has an osteoconductivity. Thus, the HAp has been used as biomaterials for bone graftings. We have succeeded in development of the novel chelate-setting calcium-phosphate cement (CPC) using pure HAp particles surface-modified with inositol phosphate (IP6). While, biological apatite presented in bone and teeth of mammals contains various ions: Na+, K+, Mg2+, Cl-, F- and CO32-, in addition to Ca2+ and PO43- ions. In this work, in order to create the chelate-setting CPC with enhanced osteoconductivity, the above-mentioned biological apatite powder (hereafter, bone HAp), instead of pure HAp, was used as a starting powder for fabrication of the chelate-setting cement. The biocompatibility of the resulting chelate-setting bone HAp cement (hereafter, IP6-bone HAp cement) was examined using a rabbit’s tibia model. When the living reaction to hard tissue was histologically examined after 4 weeks implantation, we could observe that newly-formed bone directly bonded to the surface of the specimen. The newly-formed bone was also present around the cement specimen. The amounts of newly-formed bone around IP6-bone HAp cement was about 1.5 times those around IP6-pure HAp cement without bone minerals. The above findings demonstrate that the present IP6-bone HAp cements are one of the promising candidates as novel CPC with enhanced osteoconductivity.

Novel bioresorbable calcium-phosphate cement (CPC) with anti-washout property was developed by adding thermally cross-linked gelatin particles as pore generator into a CPC. The CPC was composed of α-tricalcium phosphate (α-TCP) and surface-modified hydroxyapatite (HAp) with inositol phosphate as a chelating agent (IP6-HAp). The bioresorbable CPC hybridized with gelatin particles was successfully fabricated by mixing the aqueous sodium chondroitin sulfate solution including Na2HPO4 and the pre-mixed powders composed of α-TCP (72 mass%), IP6-HAp (18 mass%), and the gelatin particles (10 mass%). The hybridized CPC paste showed initial setting time (IST) of 5 minutes and exhibited anti-washout property. Compressive strength after setting for 24 h reached to 4.2 MPa. An in vivo preliminary study using pig’s tibia model demonstrated that the hybridized CPC could be easily injected and set promptly without washout. In addition, no fragmentation in the specimens was observed after 8 weeks implantation. Moreover, a histological observation (Villanueva bone stain) revealed that almost 80% of the hybridized CPC specimens were resorbed and that immature bones were formed inside the specimens.

We have previously developed biodegradable β-tricalcium phosphate (β-TCP) cement based on the chelate-setting mechanism of inositol phosphate (IP6). The β-TCP cement powder for the cement fabrication was prepared via a novel powder preparation process, in which the starting β-TCP powders were prepared by simultaneous ball-milling and surface-modification in the IP6 solution. In the present study, the novel powder preparation process was applied to an α-TCP powder, and effect of milling time and beads size for ball-milling on the material properties of the α-TCP powders was investigated. The α-TCP powder ball-milled in 1000 ppm IP6 solution for 4 h with 2 mm-diameter beads was composed of single phase α-TCP with the smallest particle size of 2.2 µm. Dissolution of 4 h-milled α-TCP powder was approximately twice higher than that of starting α-TCP powder before ball-milling. The α-TCP powder with high dissolution property prepared via the novel powder preparation process is potential candidate for fabrication of the chelate-setting cement.

We have previously synthesized silver-containing hydroxyapatite (Ag-HAp) powders by an ultrasonic spray-pyrolysis (USSP) technique. On the other hand, we have successfully fabricated novel calcium-phosphate cements (CPCs) composed of mainly β-tricalcium phosphate (β-TCP) phase with anti-washout property (hereafter, β-TCP cement), which was set on the basis of chelate-bonding ability of inositol phosphate (IP6). In this study, we developed novel CPCs with both anti-bacterial and anti-washout properties by adding the Ag-HAp powder into the above β-TCP cements, and examined their anti-bacterial property and cytotoxicity. The Ag-HAp powders with Ag contents of 0, 2, and 5 mol% as a nominal composition were synthesized by an USSP technique. The raw powder for β-TCP cement was prepared by ball-milling the commercially-available β-TCP powder in the IP6 solution. The Ag-HAp/β-TCP powders were prepared by mixing Ag-HAp powder and β-TCP cement powder at a ratio of 25:75 in mass. The Ag-HAp/β-TCP cement was fabricated by mixing the above-mentioned Ag-HAp/β-TCP powder and 2.5 mass% Na2HPO4 solution at a powder/liquid ratio of 1/0.3 [g/cm3]. The anti-bacterial property of resulting cements was evaluated using Staphylococcus aureus by biofilm formation test. The Ag-HAp/β-TCP cements containing 2 and 5 mol% Ag showed strong anti-bacterial property among examined specimens. Furthermore, the cytotoxicity of Ag+ ion eluted from these cements was also examined using osteoblastic MC3T3-E1 cells and Transwell® kit. The relative cell viability cultured on each Ag-containing cement specimen was over 80 %, compared with the control (polystyrene plate). These results demonstrate that the present Ag-HAp/β-TCP cements containing 2 mol% Ag are promising one of the candidates as CPCs with both anti-bacterial property and biocompatibility.

Nanometer scale Ca-deficient hydroxyapatite (nanoapatite) is a potential candidate as artificial bone substitute materials owing to its similarity to the bone with respect to composition, morphology and osteoclastic degradation or adsorbent materials for blood purification therapy to remove pathogenic substances. The initial biodegradation behaviors, the initial cell-material interaction and the protein adsorption properties of nanoapatite must depend on the microstructure. The purpose of this study is the preparation of nanoapatite particles and their structural characterization by using X-ray diffraction (XRD) and solid-state NMR spectroscopy. The nanoapatite particles were prepared by precipitation processing method, and the effects of magnesium ions on the precipitation of calcium phosphate were examined, because Mg ions are well-known to play a role of inhibition of crystal growth. The addition of Mg ions led to the precipitation of nanometer scale Ca-deficient apatite crystals having 1.33-1.63 of the molar ratio (Mg+Ca)/P. NMR analyses showed that the microstructure of Mg•HAp particles can be explained by a crystalline HAp core covered with a thin amorphous hydrated calcium phosphate layer.

n our previous study, silicon-containing hydroxyapatite (Si-HAp) powder was prepared via an aqueous precipitation reaction. The Si-HAp powders were synthesized with desired Si contents (0, 0.4, 0.8, 1.6, and 2.4 mass%) as a nominal composition. Another previous study in our group demonstrated surface-modification of HAp powder with inositol phosphate (IP6) enhanced the compressive strength of apatite cement. Thus, to fabricate the cements with higher bioactivity, the above Si-HAp powders were surface-modified with IP6 (IP6-Si-HAp). The IP6-Si-HAp cements with various Si contents were fabricated by mixing with pure water at the powder/liquid ratio of 1/0.4 [w/v]. In order to clarify biocompatibility of the IP6-Si-HAP cements in the present work, MC3T3-E1 cells as a model of osteoblast were seeded on the cement specimens. As for the numbers of cells cultured on the IP6-Si-HAp cements, the substitution of lower levels of Si into HAp lattice did not greatly influence the cell proliferation. However, the substitution of Si amount over 0.8 mass% enhanced the cell proliferation. Especially, the IP6-Si-HAp cement with the Si content of 2.4 mass% showed excellent cell proliferation among examined specimens. Therefore, to fabricate the cements with higher bioactivity, it is necessary to control the amount of Si in the IP6-Si-HAp cements. The usage of these IP6-Si-HAp cements may make it possible to fabricate the cements with higher bioactivity, compare to conventional pure HAp cements.

Cancers frequently metastasize to bone, where it leads to secondary tumor growth, and osteolytic bone degradation. Bone metastases are often associated with fractures and severe pain resulting in decreased quality of life. Accordingly, effective therapies to inhibit the development or progression of bone metastases will have important clinical benefits. Bone cement, one of the powerful tools as bone substitutes, is used to fill the resection voids. The aim of this study was to develop a local drug delivery system using HAp cement as a carrier of chemotherapeutic agents. In the present study, we have fabricated chelate-setting apatite cements (IP6-HAp cements) using HAp particles surface-modified with inositol hexaphosphate (IP6) and evaluated their anti-tumor effect. Human osteosarcoma (HOS) cultured on IP6-HAp cements (over 3000 ppm IP6) resulted in inhibition of cell growth. DNA microarray analysis indicated changes in the expression of apoptosis-related genes on IP6-HAp cement surface-modified with 5000 ppm IP6 compared with HAp cement, suggesting activation of apoptosis machinery by IP6-HAp cement. To clarify the mechanism of anti-tumor effect of IP6-HAp cement, the properties of cement were investigated. The release kinetics of IP6 from IP6-HAp cement showed that the level of released IP6 was insufficient to induce anti-tumor activity. These results led us to consider that locally high concentration of IP6 which was released from cement acts on the cells directly as anti-tumor agent and induces the apoptosis. Consequently, IP6-HAp cement might gain the anti-tumor effect and act as a carrier for local drug delivery system.

We have developed novel calcium-phosphate cements (CPCs) based on the chelate-setting mechanism of inositol phosphate (IP6) using hydroxyapatite (HAp), β-tricalcium phosphate (β-TCP) and α-TCP as starting materials. These cements (IP6-HAp, IP6-β-TCP and IP6-α-TCP cements) have different bioresorbability due to the chemical composition of starting materials. In the present study, biocompatibility and bioresorbability of the above three cements and commercially available cement (Biopex®-R) was histologically evaluated in vivo using rabbit model for 4, 8, and 24 weeks, in addition to their dissolution in vitro. The dissolution of these cements increased in the order of IP6-HAp, IP6-β-TCP and IP6-α-TCP cements. The newly-formed bones were directly in contact with both the IP6-HAp and Biopex®-R cement specimens. As for the IP6-β-TCP and IP6-α-TCP cements, newly-formed bones were formed time-dependently slightly apart from the cement specimens. Resorption rate for Biopex®-R, IP6-HAp, IP6-β-TCP, and IP6-α-TCP cements after 24 weeks implantation were of 7.2, 5.0, 13.7, and 16.2%, respectively, compared to original cements. The present chelate-setting cements with different bioresorbability are promising candidates for application as the novel CPCs.

We have previously developed hydroxyapatite (HAp) cement based on the chelate-setting mechanism of sodium inositol hexaphosphate (IP6), in which HAp powder was prepared by surface-modification with IP6 after ball-milling of the HAp powder (conventional process). Meanwhile, we have recently established novel powder preparation process (modified process). In the present study, the adsorption behavior of IP6 on the surface of HAp at both the processes was circumstantially examined to clarify the chelating mechanism of IP6. The adsorbed amount of IP6 increased with the IP6 concentration in both the processes; however, the adsorbed amount of IP6 at the modified process was lower than that at the conventional process. X-ray photoelectron spectroscopic study revealed that the IP6 adsorbed on the surface of HAp powders. The degree in dispersion of the HAp particles at the modified process was higher than that at conventional process. Furthermore, the elution of IP6 from the powders prepared at the novel process was lower than that of the powders at the conventional process.

We prepared silver-containing hydroxyapatite (Ag-HAp) powders by an ultrasonic spray-pyrolysis (USSP) technique and evaluated their antibacterial activity.　　Ag-HAp powders with Ag contents of 0, 1, 2, 5, and 20 mol% as nominal composition were prepared by the above USSP technique. The droplets of starting solutions were dried at 300 ºC and then pyrolyzed at 850 ºC to prepare the Ag-HAp powders. Ag-HAp powders showed a HAp single phase and were composed of spherical particles with a diameter of 0.5 - 3.0 μm. To evaluate the antibacterial activity of resulting powders, colony count method was performed using Staphylococcus aureus. Ag-HAp powders with the contents of Ag more than 5 mol% showed good antibacterial activity.

We have developed novel hydroxyapatite (HAp) cement, “chelate-setting HAp cement” on the basis of chelate bonding of inositol hexaphosphate (IP6). In order to create the cement with enhanced bioactivity, we used a silicon-substituted hydroxyapatite (Si-HAp) as a starting material instead of pure HAp powder. The Si-HAp powders were prepared via an aqueous precipitation reaction and surface-modified with IP6 (IP6-Si-HAp). The Si-HAp were synthesized with desired Si contents (0, 0.4, 0.8, 1.6, and 2.4 mass%). Regardless of the amount of Si, the XRD patterns and FT-IR spectra of the powders were similar to each other. Additionally, chemical composition of Si-HAp powders were the almost same as the nominal one. The substitution of Si resulted in a decrease in the a-axis and increase in the c-axis of unit cell of HAp. While, the compressive strength of IP6-Si-HAp cements was higher than that of Si-HAp cements. These results suggest that surface-modification of any powders with IP6 is effective for enhancement of mechanical property. Comparison of mechanical property between HAp and Si-HAp cement specimens revealed that the substitution of lower levels of Si into0 HAp lattice did not greatly influence compressive strength. However, the substitution of high dose of Si (over 2.4 mass%) reduced the compressive strength. Therefore, to fabricate the chelate-setting cements with enhanced bioactivity, it is necessary to control the amount of Si.