Kohei Nagata

Histological evaluations of apatite-fiber scaffold cultured with mesenchymal stem cells by implantation at rat subcutaneous tissue

BACKGROUND There is a strong impetus for the development of alternative treatments for bone disease that avoid the complications associated with autografts and allografts. To address this, we previously developed porous apatite-fiber scaffolds (AFSs) which have three-dimensional interconnected pores, and constructed tissue-engineered bone by culturing rat bone marrow cells (RBMCs) using AFSs in a radial-flow bioreactor (RFB). OBJECTIVE To generate additional baseline data for the development of tissue-engineered bone constructed for clinical application using a RFB, we cultured RBMCs using AFSs under static conditions (hereafter, RBMC AFS culture), and monitored RBMC growth and differentiation characteristics in vitro, and two weeks after subcutaneous inoculation into recipient rats. METHODS RBMCs were seeded to AFSs and growth, differentiation and calcification were monitored in vitro and in vivo by histological evaluation using hematoxylin eosin, alkaline phosphatase and alizarin red S stains. RESULTS RBMCs in/on AFSs proliferated and differentiated normally in vitro and in vivo, and calcification was evident two weeks after subcutaneous AFS culture implantation. Histological assays revealed that AFSs and RBMC AFS cultures were biocompatible, and did not induce inflammation or immunological rejection in vivo. CONCLUSIONS These findings suggest that AFSs are a conducive microenvironment for bone regeneration and are well tolerated in vivo. The results provide valuable baseline data for the design of implant studies using tissue-engineered bone constructed by RFB.

Development of Bioresorbable Calcium-Phosphate Cements Hybridized with Gelatin Particles and their In Vivo Evaluation Using Pig’s Tibia Model

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.

Inhibitory Effects of Zoledronic Acid-Loaded Bioresorbable Carrier on Osteosarcoma

Calcium phosphate cements have attracted much attention as a drug carrier for local administration of bisphosphonates which are widely used to treat bone diseases such as osteoporosis and bone tumors. In the present study, to design the novel drug delivery carrier for bone metastasis without undesirable side effects, zoledronic acid (ZOL) loaded-bioresorbable β-tricalcium phosphate (β-TCP) cement was fabricated on the basis of chelate-setting mechanism of inositol phosphate (IP6) using ZOL loaded-β-TCP powders. In order to examine the minimally effective concentration of ZOL on osteosarcoma with no effect on osteoblast viability, cells were treated with ZOL. At a concentration over 10 μM, ZOL significantly inhibited the proliferation of osteosarcoma, whereas osteoblasts proliferated normally. On the other hand, the ZOL showed concentration-dependent adsorption to IP6/β-TCP powders through chemisorption. Based on these results, we have fabricated ZOL loaded-IP6/β-TCP cements and evaluated the anti-tumor effect on osteosarcoma. We found that ZOL loaded-IP6/β-TCP cements have an inhibitory effect on osteosarcoma and induced apoptotic like-cell death. These results suggest that ZOL loaded-IP6/β-TCP cements are promising materials to develop the local treatment for bone metastasis.

Effects of Adding Polysaccharides and Citric Acid into Sodium Dihydrogen Phosphate Mixing Solution on the Material Properties of Gelatin-Hybridized Calcium-Phosphate Cement

We have succeeded in improving the material properties of a chelate-setting calcium-phosphate cement (CPC), which is composed of hydroxyapatite (HAp) the surface of which has been modified with inositol hexaphosphate (IP6) by adding α-tricalcium phosphate (α-TCP) powder. In order to create a novel chelate-setting CPC with sufficient bioresorbability, gelatin particles were added into the IP6-HAp/α-TCP cement system to modify the material properties. The effects of adding polysaccharides (chitosan, chondroitin sulfate, and sodium alginate) into the sodium dihydrogen phosphate mixing solution on the material properties of the gelatin-hybridized CPC were evaluated. The results of mechanical testing revealed that chondroitin sulfate would be the most suitable for fabricating the hybridized CPC with higher compressive strength. Moreover, further addition of an appropriate amount of citric acid could improve the anti-washout capability of the cement paste. In summary, a gelatin-hybridized IP6-HAp/α-TCP cement system prepared with a mixing solution containing chondroitin sulfate and citric acid is expected to be a beneficial CPC, with sufficient bioresorbability and material properties.

Preparation and Characterization of β-Tricalcium Phosphate Powders with High Solubility for Chelate-Setting Calcium-Phosphate Cements

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.

Fabrication and characterization of chelate-setting β-tricalcium phosphate cements with enhanced bioresorbability

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.

In Vivo Evaluation of Chelate-Setting Cement Fabricated from Hydroxyapatite Including Bone Minerals Using a Rabbit’s Tibia Model

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.