Kyushu University Academic Staff Educational and Research Activities Database
List of Papers
Anada Takahisa Last modified date:2021.07.15

Associate Professor / Department of Soft Materials Chemistry / Institute for Materials Chemistry and Engineering


Papers
1. R. Anjum, S. Nishimura, S. Kobayashi, K. Nishida, T. Anada, M. Tanaka, Protein Stabilization Effect of Zwitterionic Osmolyte-bearing Polymer, Chemistry Letters, in press, 2021.07.
2. R. Anjum, K. Nishida, H. Matsumoto, D. Murakami, S. Kobayashi, T. Anada, M. Tanaka, Attachment and Growth of Fibroblast Cells on Poly(2-methoxyethyl acrylate) Analog Polymers as Coating Materials., Coatings, 11, 4, 461-477, 2021.04.
3. M Oikawa, H Masumoto, N Shiraishi, Y Orii, T Anada, O Suzuki, K Sasaki, Effect of surface modification of Ti-6Al-4V alloy by electron resonance plasma oxidation., Dent Mater J, https://doi.org/10.4012/dmj.2020-051, 40, 228-234, 2021.01.
4. K Baba, Y Shiwaku, R Hamai, Y Mori, T Anada, K Tsuchiya, I Oizumi, N Miyatake, E Itoi, O Suzuki., Chemical stability-sensitive osteoconductive performance of octacalcium phosphate bone substitute in an ovariectomized rat tibia defect. , ACS Appl Bio Mater, https://doi.org/10.1021/acsabm.9b01091, 3, 1444-1458, 2020.02.
5. Tsuyoshi Kurobane, Yukari Shiwaku, Takahisa Anada, Ryo Hamai, Kaori Tsuchiya, Kazuyoshi Baba, Masahiro Iikubo, Tetsu Takahashi, Osamu Suzuki, Angiogenesis involvement by octacalcium phosphate-gelatin composite-driven bone regeneration in rat calvaria critical-sized defect, Acta Biomaterialia, 10.1016/j.actbio.2019.02.021, 88, 514-526, 2019.04, Effect of octacalcium phosphate/gelatin composite (OCP/Gel) on angiogenesis was studied by its implantation in rat calvaria critical-sized defect in relation to bone regeneration for 2 and 4 weeks. The implantation of OCP/Gel disks was analyzed by radiomorphometry using a radiopaque material perfusion (Microfil®) method and histomorphometry by hematoxylin and eosin-staining before and after the decalcification. Effect of the OCP dose in the range up to 4 mg per well on the capillary-like tube formation by human umbilical vein endothelial cells (HUVECs) was also examined in a transwell cell culture. The results showed that the blood vessels formation by OCP/Gel group was significantly higher at 2 weeks than other groups but decreased at 4 weeks during the advancement of new bone formation. The capillary-like tube formation was highest in an OCP dose of 1 mg per well while other OCP doses above or below 1 mg did not show such a stimulatory effect. The results established both in vivo and in vitro confirmed that OCP has a positive effect on angiogenesis during bone regeneration in a suitable dose ranges, suggesting that the angiogenesis stimulated by OCP could be involved in the OCP/Gel-enhanced bone regeneration. Statement of significance: We have reported that octacalcium phosphate (OCP) materials display stimulatory capacities on the bone tissue-related cells. However, the effect of OCP on the angiogenesis and its relation to the OCP-enhanced bone regeneration is unknown. This study confirmed the capacity of OCP on angiogenesis before increasing the new bone mass after the implantation of a composite of OCP and gelatin (OCP/Gel). The blood vessels formation took place associated with the area beginning of the new bone formation, which finally decreased together with development of bone formation. Because OCP was ascertained stimulating the capillary-like tube formation in HUVEC culture with a certain OCP dose, the present study is the first report showing the capacity of OCP on angiogenesis during the OCP/Gel-enhanced bone regeneration..
6. Tomoya Sato, Takahisa Anada, Ryo Hamai, Yukari Shiwaku, Kaori Tsuchiya, Susumu Sakai, Kazuyoshi Baba, Keiichi Sasaki, Osamu Suzuki, Culture of hybrid spheroids composed of calcium phosphate materials and mesenchymal stem cells on an oxygen-permeable culture device to predict in vivo bone forming capability, Acta Biomaterialia, 10.1016/j.actbio.2019.03.001, 88, 477-490, 2019.04, Three-dimensional (3-D) cell culture can better mimic physiological conditions in which cells interact with adjacent cells and the extracellular matrix than monolayer culture. We have developed a 3-D cell culture device, the Oxy chip, which can be used to generate and supply oxygen to cell spheroids to prevent hypoxia. Here, we used the Oxy chip to generate hybrid spheroids comprising calcium phosphate (CaP) particles (hydroxyapatite (HA), β-tricalcium phosphate (β-TCP) or octacalcium phosphate (OCP)) and mesenchymal stem cells (MSCs, C3H10T1/2 cells or D1 cells) that can be used to analyze cell differentiation mechanisms. We showed that the 3-D cell-cell and cell-material interactions and oxygenation offered by the Oxy chip promoted osteoblastic differentiation of MSCs. We also used histomorphometric analysis of hematoxylin and eosin staining, quality analyses by μCT and collagen orientation observation with picrosirius red staining in bone regeneration following implantation of three CaPs in a critical-sized defect in mouse calvaria. The in vivo bone formation capacity of the three tested CaP materials was OCP ≥ β-TCP > HA: the newly formed bone by OCP had a structure relatively close to that of the calvaria intact bone. When MSCs were 3-D cultured with the CaP materials using the Oxy chip, the in vitro osteogenic capacity of these materials was highly similar to trends observed in vivo. The in vitro alkaline phosphatase activity of D1 cells had the highest correlation with in vivo bone volume (R = 0.900). Chemical and FTIR spectroscopic analyses confirmed that differentiation of D1 cells could be associated with amorphous calcium phosphate (ACP) precipitation concomitant with OCP hydrolysis. Taken together, hybrid spheroid cultures using the Oxy chip can be used to screen and predict bone forming potential of bone substitute materials. Statement of significance: An oxygen permeable-culture chip (Oxy chip) can be used to induce formation of cell spheroids by mesenchymal stem cells (MSCs). Use of the Oxy chip avoids hypoxia in the spheroid core and enhances MSC osteoblastic differentiation relative to conventional spheroid culture methods. The present study showed that the Oxy chip mimics the in vivo environment associated with bone formation and can be used to generate hybrid spheroids consisting of calcium phosphates and MSCs that are useful for analyzing cell differentiation mechanisms. Bone formation analysis following implantation of calcium phosphate materials in mouse calvaria defects showed positive correlation with the in vitro results. We propose that hybrid spheroids cultured on the Oxy chip can be used to screen and predict the bone forming potential of bone substitute materials..
7. Takahisa Anada, Chi Chun Pan, Alexander M. Stahl, Satomi Mori, Junji Fukuda, Osamu Suzuki, Yunzhi Yang, Vascularized bone-mimetic hydrogel constructs by 3D bioprinting to promote osteogenesis and angiogenesis, International journal of molecular sciences, 10.3390/ijms20051096, 20, 5, 2019.03, Bone is a highly vascularized tissue with a unique and complex structure. Long bone consists of a peripheral cortical shell containing a network of channels for vascular penetration and an inner highly vascularized bone marrow space. Bioprinting is a powerful tool to enable rapid and precise spatial patterning of cells and biomaterials. Here we developed a two-step digital light processing technique to fabricate a bone-mimetic 3D hydrogel construct based on octacalcium phosphate (OCP), spheroids of human umbilical vein endothelial cells (HUVEC), and gelatin methacrylate (GelMA) hydrogels. The bone-mimetic 3D hydrogel construct was designed to consist of a peripheral OCP-containing GelMA ring to mimic the cortical shell, and a central GelMA ring containing HUVEC spheroids to mimic the bone marrow space. We further demonstrate that OCP, which is evenly embedded in the GelMA, stimulates the osteoblastic differentiation of mesenchymal stem cells. We refined the design of a spheroid culture device to facilitate the rapid formation of a large number of HUVEC spheroids, which were embedded into different concentrations of GelMA hydrogels. It is shown that the concentration of GelMA modulates the extent of formation of the capillary-like structures originating from the HUVEC spheroids. This cell-loaded hydrogel-based bone construct with a biomimetic dual ring structure can be potentially used for bone tissue engineering..
8. Naru Shiraishi, Risa Ishiko-Uzuka, Kenta Takahashi, Toru Ogawa, Takahisa Anada, Osamu Suzuki, Takashi Goto, Keiichi Sasaki, Effect of functionally-Graded Calcium titanate film, prepared by metal-organic chemical vapor deposition, on titanium implant, Applied Sciences (Switzerland), 10.3390/app9010172, 9, 1, 2019.01, Calcium Titanate (CaTiO
3
) has been introduced as an attractive biomaterial for the enhancement of calcium phosphate deposition in vivo and in vitro. We hypothesized that CaTiO
3
directly coated on titanium (Ti) by metal-organic chemical vapor deposition (MOCVD) could be a suitable candidate for biocompatible coatings for medical devices, particularly dental implants. To prove this hypothesis, surface characterization, cell culture, and animal study were completed in this study. The result of this study showed that CTO 800, a CaTiO
3
film prepared by heating at 800 °C, had a high hydrophilic surface. Mouse bone marrow stromal ST-2 cells cultured on substrates and CTO 800 exhibited cell differentiation, represented by alkaline phosphatase activity, compared with cells cultured on non-coated Ti and CTO 700 (a CaTiO
3
film prepared by heating at 700 °C). The push-in test value of CTO 800, a parameter that indicates the degree of osseointegration, was significantly higher than that of Ti. Calcium titanate coated on Ti by MOCVD has the potential to accelerate the process of osseointegration; thus, our results support the use of CaTiO
3
coating for biocompatible biomaterial for medical applications, particularly dental implants..
9. Hiroaki Ayabe, Takahisa Anada, Takuo Kamoya, Tomoya Sato, Masaki Kimura, Emi Yoshizawa, Shunyuu Kikuchi, Yasuharu Ueno, Keisuke Sekine, J. Gray Camp, Barbara Treutlein, Autumn Ferguson, Osamu Suzuki, Takanori Takebe, Hideki Taniguchi, Optimal Hypoxia Regulates Human iPSC-Derived Liver Bud Differentiation through Intercellular TGFB Signaling, Stem Cell Reports, 10.1016/j.stemcr.2018.06.015, 11, 2, 306-316, 2018.08, Timely controlled oxygen (O2) delivery is crucial for the developing liver. However, the influence of O2 on intercellular communication during hepatogenesis is unclear. Using a human induced pluripotent stem cell-derived liver bud (hiPSC-LB) model, we found hypoxia induced with an O2-permeable plate promoted hepatic differentiation accompanied by TGFB1 and TGFB3 suppression. Conversely, extensive hypoxia generated with an O2 -non-permeable plate elevated TGFBs and cholangiocyte marker expression. Single-cell RNA sequencing revealed that TGFB1 and TGFB3 are primarily expressed in the human liver mesenchyme and endothelium similar to in the hiPSC-LBs. Stromal cell-specific RNA interferences indicated the importance of TGFB signaling for hepatocytic differentiation in hiPSC-LB. Consistently, during mouse liver development, the Hif1a-mediated developmental hypoxic response is positively correlated with TGFB1 expression. These data provide insights into the mechanism that hypoxia-stimulated signals in mesenchyme and endothelium, likely through TGFB1, promote hepatoblast differentiation prior to fetal circulation establishment. To delineate the influence of O2 on liver development, human induced pluripotent stem cell-derived liver buds were used as a fetal liver model. This model provides insight into hypoxic modulation of mesenchyme-derived TGFB signals that promote hepatoblast differentiation in developing liver buds prior to blood circulation..
10. Sei Tsutsui, Takahisa Anada, Yukari Shiwaku, Kaori Tsuchiya, Hajime Yamazaki, Osamu Suzuki, Surface reactivity of octacalcium phosphate-derived fluoride-containing apatite in the presence of polyols and fluoride, Journal of Biomedical Materials Research - Part B Applied Biomaterials, 10.1002/jbm.b.34026, 106, 6, 2235-2244, 2018.08, The present study was designed to characterize co-precipitated fluoridated apatitic materials from octacalcium phosphate (OCP) precursor and to investigate their surface reactions with polyols including glycerol in the presence of fluoride ions. Laboratory-synthesized fluoridated apatite crystals (LS-FA) were obtained in a solution containing fluoride (F) from 25 to 500 ppm. LS-FAs and commercially available fluoroapatite (FA) and hydroxyapatite (HA) were characterized by physical techniques, such as X-ray diffraction. LS-FA obtained in the presence of 100 ppmF (100 ppm-LS-FA) had an apatitic structure, but its solubility was close to HA in a culture medium (α-MEM) despite the fact it contains over 3 wt % of F. 100 ppm-LS-FA, FA, and HA were then subjected to the human serum albumin (HSA) adsorption test at pH 7.4 (in a 150 mM Tris–HCl buffer) and the dissolution and re-mineralization experiments in the presence of xylitol, D-sorbitol, or glycerol, and F under acidic and neutral conditions. Adsorption affinity of HSA was estimated as highest for FA and lowest for LS-FA. LS-FA, FA, and HA were immersed in a lactic acid solution with the polyols and/or F ion-containing solution up to 200 ppm to analyze the dissolution behavior. LS-FA had the highest dissolution tendency in the conditions examined. Glycerol enhanced the dissolution of phosphate from apatite crystals in particular from LS-FA. The results suggest that the apatite crystals, obtained through the hydrolysis of OCP in the presence of F, provide a more reactive surface than FA or HA under physiological environments..
11. Ryosuke Iwama, Takahisa Anada, Yukari Shiwaku, Kaori Tsuchiya, Tetsu Takahashi, Osamu Suzuki, Osteogenic cellular activity around onlaid octacalcium phosphate-gelatin composite onto rat calvaria, Journal of Biomedical Materials Research - Part A, 10.1002/jbm.a.36335, 106, 5, 1322-1333, 2018.05, The activity and the distribution of osteogenic cells around octacalcium phosphate (OCP) granules combined with gelatin matrix (OCP/Gel) onlaid on rat calvaria were analyzed histomorphometrically and immunohistochemically during vertical bone augmentation under mechanical or nonmechanical environment until 8 weeks. OCP/Gel disk was placed in subperiosteal pocket on the calvaria with or without polytetrafluoroethylene (PTFE) support. The latter is a nonmechanical stress model to alleviate the mechanical stress from the subcutaneous tissues. Onlay grafts of gelatin (Gel) sponge disk and OCP granules were also carried out for the comparison purpose. When bone augmentation was evaluated in first area from bone surface (area until 150 μm high from bone surface) and second area above the newly formed bone (area until 150 µm high from the first area), bone formation was enhanced most in first area followed by second area of OCP/Gel with PTFE. The appearance of tartrate-resistant acid phosphatase (TRAP)-positive osteoclast-like cells was suppressed more in the newly formed tissue with PTFE than those without PTFE with an emphasis of the presence of gelatin. Although Runx2 positive-cells were accumulated more in both OCP/Gel with and without PTFE, osteocalcin-positive cells were abundant in OCP/Gel with PTFE than that without PTFE, suggesting that nonmechanical stress condition is more suitable for osteoblast differentiation. The appearance of receptor activator of nuclear factor (NF)-kappaB ligand (RANKL)-positive cells was restrained in OCP/Gel and Gel with PTFE while osteoprotegerin-positive cells were most accumulated in OCP/Gel without PTFE. The results suggest that OCP/Gel composite under the mechanical stress-alleviated condition can enhance bone augmentation through balanced osteoblastic and osteoclastic cellular activities..
12. Yuko Sai, Yukari Shiwaku, Takahisa Anada, Kaori Tsuchiya, Tetsu Takahashi, Osamu Suzuki, Capacity of octacalcium phosphate to promote osteoblastic differentiation toward osteocytes in vitro, Acta Biomaterialia, 10.1016/j.actbio.2018.01.026, 69, 362-371, 2018.03, Octacalcium phosphate (OCP) has been shown to act as a nucleus for initial bone deposition and enhancing the early stages of osteoblastic differentiation. However, the effect on differentiation at the late stage into osteocytes has not been elucidated. The present study was designed to investigate whether OCP can promote the differentiation lineage from osteoblasts to late osteocytes using a clonal cell line IDG-SW3 compared to commercially available sintered β-tricalcium phosphate (β-TCP) and hydroxyapatite (HA) in a transwell cell culture. Special attention was paid to detect the progress of OCP hydrolysis associated with ionic dissolution products from this material. OCP induced the appearance of an alkaline phosphatase (ALP) peak in the IDG-SW3 cells compared to β-TCP and HA and increased SOST/sclerostin and FGF23 gene expression after 35 days of incubation. Analyses by X-ray diffraction, curve fitting of Fourier transform infrared spectra, and acid phosphate inclusion of the materials showed that OCP tended to hydrolyze to an apatitic structure during the incubation. Since the hydrolysis enhanced inorganic phosphate ion (Pi) release from OCP in the media, IDG-SW3 cells were further incubated in the conditioned media with an increased concentration of Pi in the presence or absence of phosphonoformic acid (PFA), which is an inhibitor of Pi transport within the cells. An increase in Pi concentration up to 1.5 mM raised ALP activity, while its positive effect was eliminated in the presence of 0.1 to 0.5 mM PFA. Calcium ions did not show such an effect. These results indicate the stimulatory capacity of OCP on osteoblastic differentiation toward osteocytes. Statement of Significance: Octacalcium phosphate (OCP) has been shown to have a superior osteoconductivity due to its capacity to enhance initial stage of osteoblast differentiation. However, the effect of OCP on the late osteoblastic differentiation into osteocyte is unknown. This study showed the capacity associated with the structural change of OCP. The data show that OCP released inorganic phosphate (Pi) ions while the hydrolysis advanced if soaked in the media, determined by chemical and physical analyses, and enhanced osteocytes differentiation of IDG-SW3 cells more than hydroxyapatite (HA) and β-tricalcium phosphate (β-TCP). Conditioned elevated Pi-containing media in the absence of OCP enhanced the osteocyte differentiation in the range of the concentration induced by OCP, the effect of which was cancelled by the inhibitor of Pi-transporters..
13. Sei Tsutsui, Takahisa Anada, Yukari Shiwaku, Atsushi Yamagishi, Osamu Suzuki, Effect of polyols on phosphorus-containing calcium fluoride deposition on hydroxyapatite surfaces, Journal of Hard Tissue Biology, 10.2485/jhtb.27.34, 27, 1, 34-38, 2018.01, The present study investigated how polyols (sugar alcohols) affect the formation of calcium fluoride (CaF2) in the presence of fluoride on synthetic hydroxyapatite (HA) as a model of tooth enamel. HA plates were immersed in sugar alcohol solutions containing 10-60 (w/w)% xylitol, D-sorbitol, or glycerol and 1000 ppm fluoride at room temperature to analyze CaF2 formation on the surfaces. Only glycerol enhanced fluoride incorporation significantly and induced a nanoscale spherical deposition on the HA surface composed of calcium, fluoride, and a small amount of phosphorous according to energy-dispersive X-ray spectroscopic analysis. Overall results suggest that glycerol is capable of dissolving HA surfaces and enhancing phosphorous-containing CaF2 deposition, which can potentially prevent dental caries through the enhanced remineralization of enamel surfaces. Thus, fluoride may be involved in controlling the entire process of CaF2 deposition, dissolution, and remineralization of enamel..
14. Risa Ishiko-Uzuka, Takahisa Anada, Kazuhito Kobayashi, Tadashi Kawai, Yuji Tanuma, Keiichi Sasaki, Osamu Suzuki, Oriented bone regenerative capacity of octacalcium phosphate/gelatin composites obtained through two-step crystal preparation method, Journal of Biomedical Materials Research - Part B Applied Biomaterials, 10.1002/jbm.b.33640, 105, 5, 1029-1039, 2017.07, The present study was designed to investigate whether composite of coprecipitating octacalcium phosphate and gelatin (C-OCP/Gel) has an effect in repairing critical-sized defect of rat calvaria with oriented regenerative bone if implanted. The materials were prepared through two steps to disperse homogenous and well-elongated OCP toward long axis of the crystals in gelatin (Gel) matrix with the distinct concentration 17–44 wt %: OCP precipitates recovered from the coprecipitated with Gel molecules in aqueous solution (referred to as C-OCP hereafter) were mixed again in fresh aqueous Gel solution with various mixing ratio to form C-OCP/Gel for implantation. C-OCP/Gel disks with 9 mm diameter and 1 mm thickness after the dehydrothermal treatment was implanted in 9 mm diameter rat calvaria critical-sized defect. The histology, the histomorphometry in the regenerated bone and the quantitative analysis of the orientation of collagen with picrosirius red staining were carried out. It was found that C-OCP/Gel is capable of not only inducing sufficiently regenerative bone over 80% of the defect coupled with practically complete material biodegradation but also forming oriented bone significantly in relation to the amount of C-OCP in Gel matrix until 12 weeks after the implantation..
15. Taisuke Masuda, Hisataka Maruyama, Fumihito Arai, Takahisa Anada, Kaori Tsuchiya, Toshio Fukuda, Osamu Suzuki, Application of an indicator-immobilized-gel-sheet for measuring the pH surrounding a calcium phosphate-based biomaterial, Journal of Biomaterials Applications, 10.1177/0885328217699108, 31, 9, 1296-1304, 2017.04, The present study was designed to investigate the local microenvironment of octacalcium phosphate in a granule form upon biomolecules adsorption utilizing an indicator-immobilized-gel-sheet for measuring pH. We previously showed that octacalcium phosphate enhances bone regeneration during its progressive hydrolysis into hydroxyapatite if implanted in bone defects. The gel-sheet was made from a photocrosslinkable prepolymer solution, which can easily immobilize a pH indicator (bromothymol blue; BTB) in the hydrogel. The indicator-immobilized-gel-sheet was mounted on a biochip which was made of polydimethylsiloxane (PDMS) with a flow channel. The pH value was calculated by detecting the color changes in the gel-sheet and displayed as the pH distribution. After pre-adsorption of bovine albumin, β-lactoglobuline or cytochrome C onto octacalcium phosphate granules, the granules with the gel-sheet were further incubated in Tris-HCl buffer solution in the absence or presence of fluoride, known as an accelerator of octacalcium phosphate hydrolysis. pH values of the gel-sheet surrounding octacalcium phosphate granules showed a decrease from pH 7.4 to 6.6 in relation to the proteins adsorbed. Overall, the proposed pH-sensitive gel can be used to detect the pH around octacalcium phosphate granules with a high spatial resolution..
16. Shinpei Chiba, Takahisa Anada, Kentaro Suzuki, Keisuke Saito, Yukari Shiwaku, Naohisa Miyatake, Kazuyoshi Baba, Hideki Imaizumi, Masami Hosaka, Eiji Itoi, Osamu Suzuki, Effect of resorption rate and osteoconductivity of biodegradable calcium phosphate materials on the acquisition of natural bone strength in the repaired bone, Journal of Biomedical Materials Research - Part A, 10.1002/jbm.a.35828, 104, 11, 2833-2842, 2016.11, The purpose of this study was to compare the biodegradation rate and quality of regenerated bone among four materials. A short time period of 8 weeks was chosen to examine early bone healing. The rod-shaped implants of commercially available two β-tricalcium phosphate (β-TCP) ceramics with porosity 60% and 71–80%, respectively, laboratory prepared octacalcium phosphate/gelatin composite (OCP/Gel), which has been proven to have a highly osteoconductive and biodegradable property in rat calvarial defect, and gelatin sponge (Gelatin) were implanted in rabbit tibia defect of 6 mm diameter and 7 mm depth for 2, 4 and 8 weeks. Analyses by μCT, histomorphometry and push-in test were carried out to evaluate the extent of the tissue regeneration and the material biodegradation in the long bone. OCP/Gel and Gelatin were completely resorbed but only OCP/Gel induced cortical bone bridge until 8 weeks that has strength compatible to that of the natural bone. β-TCP (71%-80%) and β-TCP (60%) were not completely resorbed and never induced the amount of new bone formation beyond that by OCP/Gel. The results indicate that the new bone having enough strength could be regenerated if the material shows not only higher biodegradation rate but also higher osteoconductivity..
17. Takuo Kamoya, Takahisa Anada, Yukari Shiwaku, Teruko Takano-Yamamoto, Osamu Suzuki, An oxygen-permeable spheroid culture chip (Oxy chip) promotes osteoblastic differentiation of mesenchymal stem cells, Sensors and Actuators, B: Chemical, 10.1016/j.snb.2016.03.107, 232, 75-83, 2016.09, Mesenchymal stem cells (MSCs) are being clinically used for bone and cartilage regeneration. However, the preparation of MSCs for implantation is still costly and time consuming, and controlling the differentiation of stem cells remains a challenge. Although much attention has been paid to three-dimensional cultures in the fields of tissue engineering and regenerative medicine, adequate oxygen supply remains a challenge for growing thicker and larger cellular constructs. To solve this problem, we have developed an oxygen-permeable spheroid culture device (Oxy chip) that enables direct oxygen supply to the cells. The aim of this study was to examine the effect of a three-dimensional culture and oxygenation to the cells on the differentiation of mouse MSC strain D1 cells. Our data demonstrated that MSCs grown in the Oxy chip differentiated into osteoblasts more quickly and efficiently than those grown in the conventional non-oxygen permeable chip and monolayer culture. DNA array and energy metabolism analyses revealed that the Oxy chip facilitated osteoblastic differentiation and aerobic glycolysis, rather than chondrogenic differentiation and anaerobic glycolysis. Together, we revealed for the first time that the oxygenation by the Oxy chip was effective on the osteoblastic differentiation and survival of three-dimensional cultured MSCs. This chip is useful for preparing differentiated cells and controlling the direction of differentiation of MSCs. Moreover, this approach may be useful for transitioning spheroid cultures as a therapy in regenerative medicine..
18. Takahisa Anada, Tomoya Sato, Takuo Kamoya, Yukari Shiwaku, Kaori Tsuchiya, Teruko Takano-Yamamoto, Keiichi Sasaki, Osamu Suzuki, Evaluation of bioactivity of octacalcium phosphate using osteoblastic cell aggregates on a spheroid culture device, Regenerative Therapy, 10.1016/j.reth.2016.02.004, 3, 58-62, 2016.03.
19. Susumu Sakai, Takahisa Anada, Kaori Tsuchiya, Hajime Yamazaki, Henry C. Margolis, Osamu Suzuki, Comparative study on the resorbability and dissolution behavior of octacalcium phosphate, β-tricalcium phosphate, and hydroxyapatite under physiological conditions, Dental Materials Journal, 10.4012/dmj.2015-255, 35, 2, 216-224, 2016.03, The dissolution behaviors of octacalcium phosphate (OCP), β-tricalcium phosphate (β-TCP), and hydroxyapatite (HA) were compared by implanting the materials in rat subcutaneous pouches for 8 weeks using a filter chamber or immersing them in simulated body fluid (SBF) or Tris-HCl buffer for 2 weeks at pH 7.4 and 37oC. X-ray diffraction, Fourier transform infrared spectroscopy, scanning electron microscopy, and chemical analysis were conducted on these materials. Degree of supersaturation (DS) in the two solutions immersed with each calcium phosphate material was calculated from their chemical compositions. The results showed that OCP partially converted to apatitic crystals, while β-TCP and HA remained unchanged after the implantation. The DS of the SBF solution remained slightly supersaturated with respect to OCP and β-TCP, but slightly undersaturated in the Tris-HCl buffer. These findings suggest that previously reported OCP and β-TCP biodegradation could be induced through cell-mediated osteoclastic resorption rather than a simple dissolution process..
20. Mayu Tadaki, Takahisa Anada, Yukari Shiwaku, Takashi Nakamura, Masanori Nakamura, Masaru Kojima, Tatsuo Arai, Satoshi Fukumoto, Osamu Suzuki, A 3D culture model study monitoring differentiation of dental epithelial cells into ameloblast-like cells, RSC Advances, 10.1039/c6ra04570g, 6, 67, 62109-62118, 2016.01.
21. Keisuke Saito, Takahisa Anada, Yukari Shiwaku, Shinpei Chiba, Naohisa Miyatake, Kentaro Suzuki, Kaori Tsuchiya, Osamu Suzuki, Dose-dependent enhancement of octacalcium phosphate biodegradation with a gelatin matrix during bone regeneration in a rabbit tibial defect model, RSC Advances, 10.1039/c6ra07602e, 6, 69, 64165-64174, 2016.01.
22. Bunichi Hirayama, Takahisa Anada, Yukari Shiwaku, Naohisa Miyatake, Kaori Tsuchiya, Masanori Nakamura, Tetsu Takahashi, Osamu Suzuki, Immune cell response and subsequent bone formation induced by implantation of octacalcium phosphate in a rat tibia defect, RSC Advances, 10.1039/c6ra10834b, 6, 62, 57475-57484, 2016.01.
23. Akiko Henmi, Hiroshi Okata, Takahisa Anada, Mariko Yoshinari, Yasuto Mikami, Osamu Suzuki, Yasuyuki Sasano, Bone matrix calcification during embryonic and postembryonic rat calvarial development assessed by SEM–EDX spectroscopy, XRD, and FTIR spectroscopy, Journal of Bone and Mineral Metabolism, 10.1007/s00774-014-0647-x, 34, 1, 41-50, 2016.01, Bone mineral is constituted of biological hydroxyapatite crystals. In developing bone, the mineral crystal matures and the Ca/P ratio increases. However, how an increase in the Ca/P ratio is involved in maturation of the crystal is not known. The relationships among organic components and mineral changes are also unclear. The study was designed to investigate the process of calcification during rat calvarial bone development. Calcification was evaluated by analyzing the atomic distribution and concentration of Ca, P, and C with scanning electron microscopy (SEM)–energy-dispersive X-ray (EDX) spectroscopy and changes in the crystal structure with X-ray diffraction (XRD) and Fourier transform infrared (FTIR) spectroscopy. Histological analysis showed that rat calvarial bone formation started around embryonic day 16. The areas of Ca and P expanded, matching the region of the developing bone matrix, whereas the area of C became localized around bone. X-ray diffraction and FTIR analysis showed that the amorphous-like structure of the minerals at embryonic day 16 gradually transformed into poorly crystalline hydroxyapatite, whereas the proportion of mineral to protein increased until postnatal week 6. FTIR analysis also showed that crystallization of hydroxyapatite started around embryonic day 20, by which time SEM–EDX spectroscopy showed that the Ca/P ratio had increased and the C/Ca and C/P ratios had decreased significantly. The study suggests that the Ca/P molar ratio increases and the proportion of organic components such as proteins of the bone matrix decreases during the early stage of calcification, whereas crystal maturation continues throughout embryonic and postembryonic bone development..
24. Kosei Endo, Takahisa Anada, Masumi Yamada, Minoru Seki, Keiichi Sasaki, Osamu Suzuki, Enhancement of osteoblastic differentiation in alginate gel beads with bioactive octacalcium phosphate particles, Biomedical Materials (Bristol), 10.1088/1748-6041/10/6/065019, 10, 6, 2015.12.
25. Naofumi Kanda, Takahisa Anada, Takuto Handa, Kazuhito Kobayashi, Yushi Ezoe, Tetsu Takahashi, Osamu Suzuki, Orthotopic Osteogenecity Enhanced by a Porous Gelatin Sponge in a Critical-Sized Rat Calvaria Defect, Macromolecular Bioscience, 10.1002/mabi.201500191, 15, 12, 1647-1655, 2015.12.
26. Xiaogu Wang, Tetsuo Suzawa, Tomohiko Miyauchi, Baohong Zhao, Rika Yasuhara, Takahisa Anada, Masanori Nakamura, Osamu Suzuki, Ryutaro Kamijo, Synthetic octacalcium phosphate-enhanced reparative dentine formation via induction of odontoblast differentiation, Journal of Tissue Engineering and Regenerative Medicine, 10.1002/term.1669, 9, 11, 1310-1320, 2015.11, Synthetic octacalcium phosphate (OCP) has been suggested to be a useful biomaterial for the regeneration of hard tissues, including bone. However, it remains unknown whether OCP induces dentine formation by dental pulp. We investigated biomineralization of dental pulp exposed to synthetic OCP in vitro and in vivo. When dental pulp was exposed directly to OCP, rapid formation of reparative dentine (RD) was induced and expression of dentine sialoprotein synthesis was observed in dental pulp adjacent to newly synthesized RD. OCP inhibited the proliferation of rat pulp cells and also promoted their odontoblastic differentiation in vitro, as alkaline phosphatase activity, mineralization of pulp cells and the expression level of dentine sialophosphoprotein were enhanced. Direct contact between OCP and pulp cells is required for OCP to exhibit its effects in vitro. The expression level of Runx2, a transcription factor whose downregulation is closely related to odontoblast differentiation, was downregulated in pulp cells cultured with OCP. Structural changes of OCP during culture were determined by Fourier transform infrared spectroscopy. OCP tended to be converted to carbonate hydroxyapatite after incubation with or without pulp cells, which may be analogous to biological apatite crystals. Taken together, our data suggest that synthetic OCP supports RD formation by dental pulp and downregulation of Runx2 may be involved in that stimulatory activity. Furthermore, OCP-apatite conversion is involved in this stimulatory capacity of OCP..
27. Tatsuya Miyazaki, Satoshi Miyauchi, Takahisa Anada, Akira Tawada, Osamu Suzuki, Chondroitin Sulfate-E Binds to Both Osteoactivin and Integrin αvβ3 and Inhibits Osteoclast Differentiation, Journal of Cellular Biochemistry, 10.1002/jcb.25175, 116, 10, 2247-2257, 2015.10, Integrins and their ligands have been suggested to be associated with osteoclast-mediated bone resorption. The present study was designed to investigate whether chondroitin sulfate E (CS-E), which is one of the sulfated glycosaminoglycans (GAGs), is involved in osteoactivin (OA) activity, and osteoclast differentiation. The binding affinity of sulfated GAGs to integrin and its ligand was measured using biotin-labeled CS-E, and the osteoclast differentiation was evaluated by tartrate-resistant acid phosphatase staining and a pit formation assay. CS-E as well as CS-B, synthetic chondroitin polysulfate, and heparin inhibited osteoclast differentiation of bone marrow-derived macrophages. Pre-coating of OA to synthetic calcium phosphate-coated plates enhanced the osteoclastic differentiation of RAW264 cells, and addition of a neutralizing antibody to OA inhibited its differentiation. CS-E bound not only to OA, fibronectin, and vitronectin, but also to its receptor integrin αVβ3, and inhibited the direct binding of OA to integrin αVβ3. Furthermore, CS-E blocked the binding of OA to cells and inhibited OA-induced osteoclastic differentiation. On the other hand, heparinase treatment of RAW264 cells inhibited osteoclastic differentiation. Since binding of OA to the cells was inhibited by the presence of heparan sulfate or heparinase treatment of cells, heparan sulfate proteoglycan (HSPG) was also considered to be an OA receptor. Taken together, the present results suggest that CS-E is capable of inhibiting OA-induced osteoclast differentiation by blocking the interaction of OA to integrin αVβ3 and HSPG. J. Cell. Biochem. 116: 2247-2257, 2015..
28. Yoshiaki Itoigawa, Osamu Suzuki, Hirotaka Sano, Takahisa Anada, Takuto Handa, Taku Hatta, Yoshiyuki Kuwahara, Atsushi Takahashi, Yushi Ezoe, Kazuo Kaneko, Eiji Itoi, The role of an octacalcium phosphate in the re-formation of infraspinatus tendon insertion, Journal of Shoulder and Elbow Surgery, 10.1016/j.jse.2015.01.011, 24, 7, e175-e184, 2015.07, Background: To improve the success rate of rotator cuff repair, we investigated whether octacalcium phosphate (OCP) with gelatin (Gel) vehicle had a positive effect on tendon-to-bone healing. Methods: We assessed the histologic characteristics of the tendon-to-bone healing using the rabbit rotator cuff repair model. We divided the shoulders into 3 groups: control (without OCP/Gel composite), OCP/Gel composite (OCP+group), and Gel alone without OCP (Gel group) to evaluate the effectiveness of gelatin. Results: Both the number of newly formed tendon fibers and the Sharpey fibers at the repair site increased in the OCP+group compared with those in the other 2 groups on hematoxylin-eosin staining (P<.05). On immunohistochemical evaluation, both the bone and the fibers in the OCP+group demonstrated that type I collagen was picked up, whereas the newly formed tendon fibers and Sharpey fibers revealed type III collagen. Conclusion: Treatment with OCP made collagen fibers and the Sharpey fibers, constituted by type I and type III collagens, increase at the tendon-to-bone insertion. It might be beneficial for the healing of rotator cuff tendon to bone..
29. Masakazu Yamada, Takahisa Anada, Taisuke Masuda, Teruko Takano-Yamamoto, Osamu Suzuki, Effect of mechanical stress on differentiation of mouse mesenchymal stem cells seeded into an octacalcium phosphate-gelatin scaffold, Sensors and Actuators, B: Chemical, 10.1016/j.snb.2015.05.073, 220, 125-130, 2015.06, We report the extent of mechanical stress on a bioactive three-dimensional scaffold that can induce differentiation of seeded osteoblastic cells in vitro. Mouse mesenchymal stem D1 cells were seeded on a porous spongy scaffold consists of octacalcium phosphate (OCP) and gelatin (Gel) composite, which was previously proven to be a sufficient bone-replaceable material in vivo. The cellular activity was determined after loading of uni-axial compression by deforming a 5 mm ring-shaped, cell-seeded OCP/Gel composite in the range of 1-3 mm (corresponding to 20-60% of the material thickness) using a cyclic mechanical loading device designed for this study. The extent of the deformation did not affect the proliferation. However, enhancement of differentiation marker genes, such as osteopontin and osteoclacin, occurred after the composite was deformed by just 20%. These results suggest that osteoblastic differentiation of D1 cells is promoted if suitable mechanical stress is provided, which indicates biological relevance of the bone regenerative properties of this material under mechanical stress that were shown in a previous in vivo study..
30. Kenta Takahashi, Naru Shiraishi, Risa Ishiko-Uzuka, Takahisa Anada, Osamu Suzuki, Hiroshi Masumoto, Keiichi Sasaki, Biomechanical evaluation of Ti-Nb-Sn alloy implants with a low young’s modulus, International journal of molecular sciences, 10.3390/ijms16035779, 16, 3, 5779-5788, 2015.03, Dental implants are widely used and are a predictable treatment in various edentulous cases. Occlusal overload may be causally related to implant bone loss and a loss of integration. Stress concentrations may be diminished using a mechanobiologically integrated implant with bone tissue. The purpose of this study was to investigate the biomechanical behavior, biocompatibility and bioactivity of a Ti-Nb-Sn alloy as a dental implant material. It was compared with cpTi. Cell proliferation and alkaline phosphatase (ALP) activity were quantified. To assess the degree of osseointegration, a push-in test was carried out. Cell proliferation and ALP activity in the cells grown on prepared surfaces were similar for the Ti-Nb-Sn alloy and for cpTi in all the experiments. A comparison between the Ti-Nb-Sn alloy implant and the cpTi implant revealed that no significant difference was apparent for the push-in test values. These results suggest that implants fabricated using Ti-Nb-Sn have a similar biological potential as cpTi and are capable of excellent osseointegration..
31. Yushi Ezoe, Takahisa Anada, Hajime Yamazaki, Takuto Handa, Kazuhito Kobayashi, Tetsu Takahashi, Osamu Suzuki, Characterization of partially hydrolyzed OCP crystals deposited in a gelatin matrix as a scaffold for bone tissue engineering, Journal of Nanoparticle Research, 10.1007/s11051-015-2864-1, 17, 3, 2015.01, The present study was designed to investigate how hydrolysis of octacalcium phosphate (OCP) into hydroxyapatite is affected by the presence of gelatin (Gel) molecules and how osteoblastic cells respond to the resultant OCP hydrolyzate/Gel composites as the hydrolysis advances. OCP was prepared from a solution containing calcium and phosphate ions and Gel molecules, having a composition to produce a 40 wt% OCP as a final co-precipitate as the OCP/Gel. The precipitate was further incubated up to 40 h to advance the hydrolysis of OCP. These precipitates were processed to mold OCP/Gel sponges through lyophilization and dehydrothermal treatment. Chemical analysis, X-ray diffraction, Fourier transform infrared spectroscopy, transmission electron microscopy, and selected area electron diffraction revealed that the hydrolysis of OCP/Gel composite in hot water advanced in a time-dependent manner and faster than hydrolysis of OCP alone. The effect of Gel on the OCP hydrolysis was further examined in the presence of distinct concentrations of Gel molecules in hot water, showing that the Gel enhanced the hydrolysis as the concentration increased. Proliferation and differentiation of mouse bone marrow stromal ST-2 cells on the hydrolyzed OCP/Gel composites were compatible with Gel sponge alone after 21 days of culture, suggesting that these composites could be a candidate as a scaffold in bone tissue engineering..
32. Naoko Sato, Toshie Kuwana, Miou Yamamoto, Hanako Suenaga, Takahisa Anada, Shigeto Koyama, Osamu Suzuki, Keiichi Sasaki, Bone response to immediate loading through titanium implants with different surface roughness in rats, Odontology, 10.1007/s10266-013-0107-4, 102, 2, 249-258, 2014.01, Because of its high predictability of success, implant therapy is a reliable treatment for replacement of missing teeth. The concept of immediate implant loading has been widely accepted in terms of early esthetic and functional recovery. However, there is little biological evidence to support this concept. The objective of this study was to examine the interactive effects of mechanical loading and surface roughness of immediately loaded titanium implants on bone formation in rats. Screw-shaped anodized titanium implants were either untreated (smooth) or acid-etched. Two implants were inserted parallel to each other in the tibiae of rats, and a closed coil spring (2.0 N) was immediately applied. Trabecular and cortical bone around both implants was analyzed using microtomographic images, and a removal torque test was performed at weeks 1, 2, and 4. Immediate loading of acid-etched implants resulted in significant decreases in bone mineral density, contact surface area, and cortical bone thickness. These effects were not observed after immediate loading of smooth implants. Conversely, loading did not influence acid-etched implant fixation; however, smooth implant fixation at week 1 was significantly reduced. These results imply that surface roughness regulates bone response to mechanical stress and that immediate loading might not inhibit osseointegration for smooth and rough implants in the late healing stages..
33. Kenji Hoshi, Harumi Kawaki, Ichiro Takahashi, Nobuo Takeshita, Masahiro Seiryu, Sakhr A. Murshid, Taisuke Masuda, Takahisa Anada, Ryushi Kato, Hideki Kitaura, Osamu Suzuki, Teruko Takano-Yamamoto, Compressive force-produced CCN2 induces osteocyte apoptosis through ERK1/2 pathway, Journal of Bone and Mineral Research, 10.1002/jbmr.2115, 29, 5, 1244-1257, 2014.01, Osteocytes produce various factors that mediate the onset of bone formation and resorption and play roles in maintaining bone homeostasis and remodeling in response to mechanical stimuli. One such factor, CCN2, is thought to play a significant role in osteocyte responses to mechanical stimuli, but its function in osteocytes is not well understood. Here, we showed that CCN2 induces apoptosis in osteocytes under compressive force loading. Compressive force increased CCN2 gene expression and production, and induced apoptosis in osteocytes. Application of exogenous CCN2 protein induced apoptosis, and a neutralizing CCN2 antibody blocked loading-induced apoptosis. We further examined how CCN2 induces loaded osteocyte apoptosis. In loaded osteocytes, extracellular signal-regulated kinase 1/2 (ERK1/2) was activated, and an ERK1/2 inhibitor blocked loading-induced apoptosis. Furthermore, application of exogenous CCN2 protein caused ERK1/2 activation, and the neutralizing CCN2 antibody inhibited loading-induced ERK1/2 activation. Therefore, this study demonstrated for the first time to our knowledge that enhanced production of CCN2 in osteocytes under compressive force loading induces apoptosis through activation of ERK1/2 pathway..
34. Kentaro Suzuki, Takahisa Anada, Tatsuya Miyazaki, Naohisa Miyatake, Yoshitomo Honda, Koshi N. Kishimoto, Masami Hosaka, Hideki Imaizumi, Eiji Itoi, Osamu Suzuki, Effect of addition of hyaluronic acids on the osteoconductivity and biodegradability of synthetic octacalcium phosphate, Acta Biomaterialia, 10.1016/j.actbio.2013.09.005, 10, 1, 531-543, 2014.01, The present study was designed to investigate whether three sodium hyaluronic acid (HyA) medical products, Artz®, Suvenyl® and a chemically modified derivative of sodium HyA Synvisc®, can be used as suitable vehicles for an osteoconductive octacalcium phosphate (OCP). OCP granules (300-500 μm diameter) were mixed with these sodium HyAs with molecular weights of 90 × 104 (Artz®), 190 × 104 (Suvenyl®) and 600 × 104 (Synvisc®) (referred to as HyA90, HyA190 and HyA600, respectively). OCP-HyA composites were injected using a syringe into a polytetrafluoroethylene ring, placed on the subperiosteal region of mouse calvaria for 3 and 6 weeks, and then bone formation was assessed by histomorphometry. The capacity of the HyAs for osteoclast formation from RAW264 cells with RANKL was examined by TRAP staining in vitro. Bone formation was enhanced by the OCP composites with HyA90 and HyA600, compared to OCP alone, through enhanced osteoclastic resorption of OCP. HyA90 and HyA600 facilitated in vitro osteoclast formation. The results suggest that the osteoconductive property of OCP was accelerated by the HyAs-associated osteoclastic resorption of OCP, and therefore that HyA/OCP composites are attractive bone substitutes which are injectable and bioactive materials..
35. Osamu Suzuki, Takahisa Anada, Taisuke Masuda, Effect of mechanical stress on bone formation by osteoconductive octacalcium phosphate bone substitute materials, BME = Bio medical engineering / henshu, Nihon ME Gakkai, 10.11239/jsmbe.52.SY-43, 52, SY-43, 2014.01, It is known that bone remodeling process advances under the control of mechanical stress in the body. Synthetic biomaterials, such as hydroxyapatite, are widely used to repair various bone defects. Octacalcium phosphate (OCP) and the related materials are bioactive and bioresorbable materials (Suzuki O. Jpn Dent Sci Rev 49: 58, 2013). The OCP materials show an osteoconductivity if implanted in various bone tissues. However, the resorption of the materials is enhanced under load bearing conditions, which is restored again if the assumed mechanical stress is alleviated. The effect of the mechanical stress on the biomaterial implantation condition was simulated using a culture device, which utilizes deformation of PDMS membrane deformation (Masuda T et al. J Biotechnol 133: 231, 2008), by analyzing bone tissue related cells, including osteoblasts. It is likely that the bone regeneration with the biomaterials could be controlled depending on the extent of the mechanical stress..
36. Ryuhei Nishikawa, Takahisa Anada, Risa Ishiko-Uzuka, Osamu Suzuki, Osteoblastic differentiation of stromal ST-2 cells from octacalcium phosphate exposure via p38 signaling pathway, Dental Materials Journal, 10.4012/dmj.2013-226, 33, 2, 242-251, 2014.01, The present study investigated the role of mitogen-activated protein kinase (MAPK) signaling in osteoblastic differentiation of stromal ST-2 cells induced by synthetic octacalcium phosphate (OCP) incubation. Since our previous studies revealed that OCP consumes calcium ions in media during conversion to hydroxyapatite, the effect of the ions on ST-2 cell differentiation with or without OCP crystals was analyzed. The effect of presence or absence of MAPK inhibitors was also analyzed. OCP increased alkaline phosphatase (ALP) activity and the mRNA expression of differentiation markers via the p38 signaling pathway. The PD98059 MAPK inhibitor increased ALP activity and differentiation marker genes in cells cultured in OCP-coated wells. Reduction of calcium ions in the medium by EGTA increased the ALP activity without OCP in the presence of phosphate ion concentrations up to 7.5 mM. OCP may enhance osteoblastic differentiation through the p38 signaling pathway via the reduction of calcium ions induced by its physicochemical property..
37. Kazuhito Kobayashi, Takahisa Anada, Takuto Handa, Naofumi Kanda, Mariko Yoshinari, Tetsu Takahashi, Osamu Suzuki, Osteoconductive property of a mechanical mixture of octacalcium phosphate and amorphous calcium phosphate, ACS Applied Materials and Interfaces, 10.1021/am5067139, 6, 24, 22602-22611, 2014.01, The present study was designed to investigate the extent of osteoconductive property of a mechanical mixture of octacalcium phosphate (OCP) and amorphous calcium phosphate (ACP). OCP was mixed with ACP in granules that had a diameter of 300 and 500 μm, respectively, and at 25, 50, or 75 wt %. The physicochemical characteristics and the osteoconductive properties of the mixtures were compared with OCP alone or ACP alone through implantation into rat critical-sized calvaria defects for up to 12 weeks and simulated body fluid (SBF) immersion for 2 weeks. The mixtures of OCP and ACP, in particular the OCP 25 wt % and ACP 75 wt % (O25A75), had higher radiopacity compared to ACP and OCP alone. O25A75 induced greater enhancement of bone regeneration than ACP alone at 8 weeks and that than OCP alone at 12 weeks. X-ray diffraction and Fourier transform infrared (FTIR) analyses of the retrieved mixtures showed that ACP, OCP, and O25A75 tended to convert to hydroxyapatite (HA) after the implantation, while the structure of OCP remains without complete conversion after SBF immersion. Analyses by FTIR curve fitting of the solids and the degree of supersaturation of the SBF supported the observation that the existence of ACP enhances the kinetics of the conversion. Scanning electron microscopy found that the surface of O25A75 had distinct characteristics with OCP and ACP after SBF immersion. The results suggest that the extent of the osteoconduction of OCP could be controlled by the copresence of ACP most probably through the prevailing dissolution-precipitation of the surface of ACP crystals to form HA..
38. Kei Ichiro Miura, Takahisa Anada, Yoshitomo Honda, Yukari Shiwaku, Tadashi Kawai, Seishi Echigo, Tetsu Takahashi, Osamu Suzuki, Characterization and bioactivity of nano-submicro octacalcium phosphate/gelatin composite, Applied Surface Science, 10.1016/j.apsusc.2013.05.086, 282, 138-145, 2013.10, The present study was designed to investigate the physicochemical and bioactive properties of a nano-submicro sized octacalcium phosphate (OCP)-dispersed gelatin (Gel) composite (nano-submicro OCP/Gel) used as a bone substitute material in various bone defects. Well-grown, synthesized OCP was mechanically ground from 100 to 300 μm-sieved granules to particles that were approximately 500 nm in size. Then, 50 wt% of the nano-submicro OCP was mixed with porcine skin-derived acid extracted gelatin. The mixture was molded and lyophilized and then subjected to dehydrothermal crosslinking. X-ray diffraction (XRD) and Fourier transform infrared (FTIR) spectroscopy showed that the structure of OCP was retained even after mechanical grinding to a nano-submicro scale level as well as inclusion in the Gel matrix. The bioactivity of nano-submicro OCP/Gel was examined by immersing the composite in simulated body fluid (SBF) for 7 days and by implanting it in rat critical-sized calvaria defects for 8 weeks. The nano-submicro OCP tended to convert to low crystalline hydroxyapatite (HA) in SBF as assessed by XRD. The nano-submicro OCP/Gel exhibited osteoconductivity in vivo, yielding new bone formation that was closely associated with the implanted composite. These results suggest that the nano-submicro OCP/Gel composite exhibits similar osteoconductivity as observed in other OCP-based materials previously reported and could be used as a bone substitute material for repairing various defects in bone..
39. Yoshitomo Honda, Takahisa Anada, Shinji Morimoto, Osamu Suzuki, Labile Zn ions on octacalcium phosphate-derived Zn-containing hydroxyapatite surfaces, Applied Surface Science, 10.1016/j.apsusc.2013.02.040, 273, 343-348, 2013.05, We previously synthesized and characterized zinc-containing octacalcium phosphate (OCP) and its hydrolyzed Ca-deficient hydroxyapatite (HA). In the present report, we attempted to define the state of Zn in the OCP-derived Zn-calcium phosphates (CaPs) in relation to the presence of specific amino acids. Zn-containing OCPs were prepared in solutions that included Zn ions up to a concentration of 3.5 mM, and their hydrolyzates [hydrolyzed (hy)-Zn-CaP] were obtained in hot water. The materials were characterized by x-ray diffraction and scanning electron microscopy. The concentration of Ca and Zn ions at room temperature was determined by analyzing the supernatant after incubating the materials in α-minimal essential medium (α-MEM) and HEPES buffer including cysteine, histidine, lysine, aspartic acid, and glutamic acid. Zn ions were more dissolved in α-MEM than HEPES buffer in the absence of amino acids. The inclusion of the amino acids enhanced Zn dissolution by several hundred fold, even in HEPES buffer. Among the amino acids, both cysteine and histidine enhanced the release of Zn. The effect was particularly remarkable with cysteine even in the presence of the other amino acids tested. These results indicate that Zn ions are present as a surface labile pool, which tends to be preferentially desorbed by cysteine, a ubiquitous molecule present in serum..
40. Isao Shibuya, Kentaro Yoshimura, Yoichi Miyamoto, Atsushi Yamada, Masamichi Takami, Tetsuo Suzawa, Dai Suzuki, Noriharu Ikumi, Fumitaka Hiura, Takahisa Anada, Osamu Suzuki, Ryutaro Kamijo, Octacalcium phosphate suppresses chondrogenic differentiation of ATDC5 cells, Cell and tissue research, 10.1007/s00441-012-1548-8, 352, 2, 401-412, 2013.05, Implantation of octacalcium phosphate (OCP), a hydroxyapatite precursor, has been reported to induce chondrogenesis in vivo. In this study, we examined the effects of OCP on the chondrogenic differentiation of mouse chondroblastic ATDC5 cells in vitro. Contrary to our expectation, chondrogenic differentiation of ATDC5 cells evaluated by the mRNA expression of Col2a1, Acan and Col10a1 was suppressed by OCP. Among Sox9, Sox5 and Sox6, essential transcription factors for chondrogenesis, the expression of Sox6 mRNA was markedly lowered by OCP. Whereas ATDC5 cells dissolved OCP to liberate calcium and inorganic phosphorus, increased calcium or phosphate in the medium had little effect on the differentiation of these cells. Direct contact of ATDC5 cells with OCP was required to suppress the expression of Col2a1 and Sox6 mRNAs, whereas the introduction of Sox6 short interfering RNA lowered the expression of Col2a1 mRNA. On the other hand, the forced expression of Sox6 protein partially but significantly, restored the expression of Col2a1 mRNA suppressed by OCP. These results indicate that OCP suppresses the chondrogenic differentiation of ATDC5 cells, at least in part, at the Sox6 transcription level..
41. Takahisa Anada, Osamu Suzuki, Size regulation of chondrocyte spheroids using a PDMS-based cell culture chip, Journal of Robotics and Mechatronics, 10.20965/jrm.2013.p0644, 25, 4, 644-649, 2013.01, Cartilage self-repair is limited due to a lack of blood supply and the low mitosis rate of chondrocytes. A tissue engineering approach using cells and biomaterials has the potential to treat cartilage injury. Threedimensional cellular aggregates are an excellent model for mimicking condensation and chondrogenic differentiation in vitro. We developed a technique for constructing spheroids utilizing a polydimethylsiloxane (PDMS)-based culture chip. The objective of this study is to determine how the initial cell density on a culture chip affects the chondrogenic ATDC5 cell differentiation. We demonstrate how culture chips having arrays of multicavities are able to generate high numbers of uniform spheroids rapidly and simultaneously with narrow size distribution. Spheroids are collected easily and noninvasively. Higher cell seeding density on the culture chip enhances chondrogenic cell differentiation. These results suggest the usefulness of this chip in engineering 3D cellular constructs with high functionality for tissue engineering..
42. Y. Shiwaku, Takahisa Anada, H. Yamazaki, Y. Honda, S. Morimoto, K. Sasaki, O. Suzuki, Structural, morphological and surface characteristics of two types of octacalcium phosphate-derived fluoride-containing apatitic calcium phosphates, Acta Biomaterialia, 10.1016/j.actbio.2012.07.041, 8, 12, 4417-4425, 2012.12, Octacalcium phosphate (OCP) has been reported to stimulate bone regeneration during hydrolysis into hydroxyapatite (HA). The present study was designed to characterize structural, morphological and surface properties of fluoride-containing apatitic calcium phosphates (CaP) obtained through OCP hydrolysis or direct precipitation of OCP in the presence of 12-230 ppm of fluoride (F). The products were characterized by chemical analysis, X-ray diffraction (XRD), field emission scanning electron microscopy (FE-SEM), transmission electron microscopy (TEM), selected area electron diffraction (SAED) and Fourier transform infrared spectroscopy (FTIR) as well as measurements of surface area, solubility, osteoblastic activities and bovine serum albumin (BSA) adsorption. XRD analysis re-confirmed that both preparations yielded more apatitic CaP with a higher concentration of F. However, the co-precipitated products (CF-CaP) maintained the properties of OCP, in particular the solubility, whereas the hydrolysis products (HF-CaP) had the characteristics of fluoridated apatite. The crystals of plate-like OCP were changed to the crystals of rod-like CF-CaP and small irregular HF-CaP with the advance of the hydrolysis. The SAED analysis detected both OCP and apatite crystals even in the most hydrolyzed CF-CaP. Mouse bone marrow stromal ST-2 cells grew better on CF-CaP compared with HF-CaP. BSA adsorption was inhibited on HF-CaP more than on CF-CaP. These results show that OCP produces physicochemically distinct apatitic fluoridated CaP during hydrolysis, regarding the structure, the crystal morphology and the protein adsorption, depending on the fluoride introduction route, which provides biologically interesting material..
43. Takahisa Anada, Junji Fukuda, Yuko Sai, Osamu Suzuki, An oxygen-permeable spheroid culture system for the prevention of central hypoxia and necrosis of spheroids, Biomaterials, 10.1016/j.biomaterials.2012.08.040, 33, 33, 8430-8441, 2012.11.
44. Naru Shiraishi, Rong Tu, Risa Uzuka, Takahisa Anada, Takayuki Narushima, Takashi Goto, Mitsuo Niinomi, Keiichi Sasaki, Osamu Suzuki, Biomechanical evaluation of amorphous calcium phosphate coated TNTZ implants prepared using a radiofrequency magnetron sputtering system, Materials Transactions, 10.2320/matertrans.M2012078, 53, 7, 1343-1348, 2012.10, Ti29Nb13Ta4.6Zr alloy (TNTZ), a new β-type Ti alloy, has excellent advantages as a biomaterial, such as low Young's modulus and cytotoxicity, and the absence of allergens. However, it is unclear whether TNTZ can achieve sufficient osseointegration for it to be used as a dental implant. The effectiveness of surface modification of TNTZ implants by radiofrequency (RF) magnetron sputtering is also unclear. We investigated the biomechanical behaviors of TNTZ implants in vivo, using cylindrical implants of four types: pure Ti, TNTZ, and pure Ti and TNTZ coated with amorphous calcium phosphate (ACP). The implants were inserted in rat femurs, and the femurs were subjected to biomechanical analyses after various time intervals. The results suggest that TNTZ implants can achieve osseointegration similar to that of pure Ti, and that surface modification with ACP by RF magnetron sputtering improves osseointegration, especially in the later stages of healing..
45. Taisuke Masuda, Natsuki Takei, Takuma Nakano, Takahisa Anada, Osamu Suzuki, Fumihito Arai, A microfabricated platform to form three-dimensional toroidal multicellular aggregate, Biomedical Microdevices, 10.1007/s10544-012-9713-0, 14, 6, 1085-1093, 2012.09, Techniques that allow cells to self-assemble into three-dimensional (3D) spheroid microtissues provide powerful in vitro models that are becoming increasingly popular in fields such as stem cell research, tissue engineering, and cancer biology. Appropriate simulation of the 3D environment in which tissues normally develop and function is crucial for the engineering of in vitro models that can be used for the formation of complex tissues. We have developed a unique multicellular aggregate formation platform that utilizes a maskless gray-scale photolithography. The cellular aggregate formed using this platform has a toroidal-like geometry and includes a micro lumen that facilitates the supply of oxygen and growth factors and the expulsion of waste products. As a result, this platform was capable of rapidly producing hundreds of multicellular aggregates at a time, and of regulating the diameter of aggregates with complex design. These toroidal multicellular aggregates can grow as long-term culture. In addition, the micro lumen can be used as a continuous channel and for the insertion of a vascular system or a nerve system into the assembled tissue. These platform characteristics highlight its potential to be used in a wide variety of applications, e.g. as a bioactuator, as a micro-machine component or in drug screening and tissue engineering..
46. Yuji Tanuma, Takahisa Anada, Yoshitomo Honda, Tadashi Kawai, Shinji Kamakura, Seishi Echigo, Osamu Suzuki, Granule size-dependent bone regenerative capacity of octacalcium phosphate in collagen matrix, Tissue Engineering - Part A, 10.1089/ten.tea.2011.0349, 18, 5-6, 546-557, 2012.03, The present study was designed to determine whether the osteoconductivity of octacalcium phosphate-collagen (OCP/Col) composite can be improved by controlling the granule size of OCP. The granules of synthetic OCP, with diameters in the range of 53 to 300, 300 to 500, and 500 to 1000μm, were used as an inorganic source of composite materials mixed with atelo-Col. After vacuum dehydrothemal treatment, OCP/Col disks were implanted into critical-sized calvaria defects in Wistar rats for 4, 8, and 12 weeks and examined radiographically, histologically, histomorphometrically, and histochemically. The materials were characterized according to mercury intrusion porosimetry and scanning electron microscopy. X-ray diffraction was performed before and after implantation. The dissolution of OCP crystals in a Col matrix was determined by immersing OCP/Col disks in a culture medium. OCP/Col had a constant pore size (∼30μm) regardless of OCP granule size. OCP in the Col matrix tended to convert to hydroxyapatite (HA) during the implantation. OCP/Col with the smallest granules of OCP enhances both bone regeneration and biodegradation the most through tartrate-resistant acid phosphatase (TRAP)-positive osteoclastic cellular resorption of OCP granules. The smallest OCP granules in the Col matrix showed the highest dissolution and had the greatest potential to form HA. The results indicated that the size of the included OCP granules can controll the osteoconductivity of OCP/Col. The overall results suggest that the physicochemical property of OCP crystals is a factor that determines the bone regenerative capacity of OCP/Col in critical-sized calvaria large bone defects in rats..
47. T. Handa, Takahisa Anada, Y. Honda, H. Yamazaki, K. Kobayashi, N. Kanda, S. Kamakura, S. Echigo, O. Suzuki, The effect of an octacalcium phosphate co-precipitated gelatin composite on the repair of critical-sized rat calvarial defects, Acta Biomaterialia, 10.1016/j.actbio.2011.12.002, 8, 3, 1190-1200, 2012.03, This study was designed to investigate the extent to which an octacalcium phosphate/gelatin (OCP/Gel) composite can repair rat calvarial critical-sized defects (CSD). OCP crystals were grown with various concentrations of gelatin molecules and the OCP/Gel composites were characterized by chemical analysis, X-ray diffraction (XRD), Fourier transform infrared (FTIR) spectroscopy, transmission electron microscopy (TEM), selected area electron diffraction (SAED) and mercury intrusion porosimetry. The OCP/Gel composite disks received vacuum dehydrothermal treatment, were implanted in Wistar rat calvarial CSD for 4, 8 and 16 weeks, and then subjected to radiologic, histologic, histomorphometric and histochemical assessment. The attachment of mouse bone marrow stromal ST-2 cells on the disks of the OCP/Gel composites was also examined after 1 day of incubation. OCP/Gel composites containing 24 wt.%, 31 wt.% and 40 wt.% of OCP and with approximate pore sizes of 10-500 μm were obtained. Plate-like crystals were observed closely associated with the Gel matrices. TEM, XRD, FTIR and SAED confirmed that the plate-like crystals were identical to those of the OCP phase, but contained a small amount of sphere-like amorphous material adjacent to the OCP crystals. The OCP (40 wt.%)/Gel composite repaired 71% of the CSD in conjunction with material degradation by osteoclastic cells, which reduced the percentage of the remaining implant to less than 3% within 16 weeks. Of the seeded ST-2 cells, 60-70% were able to migrate and attach to the OCP/Gel composites after 1 day of incubation, regardless of the OCP content. These results indicate that an OCP/Gel composite can repair rat calvarial CSD very efficiently and has favorable biodegradation characteristics. Therefore, it is hypothesized that host osteoblastic cells can easily migrate into an OCP/Gel composite..
48. Shinji Morimoto, Takahisa Anada, Yoshitomo Honda, Osamu Suzuki, Comparative study on in vitro biocompatibility of synthetic octacalcium phosphate and calcium phosphate ceramics used clinically, Biomedical Materials (Bristol), 10.1088/1748-6041/7/4/045020, 7, 4, 2012.01, The present study was designed to investigate the extent to which calcium phosphate bone substitute materials, including osteoconductive octacalcium phosphate (OCP), display cytotoxic and inflammatory responses based on their dissolution in vitro. Hydroxyapatite (HA) and β-tricalcium phosphate (β-TCP) ceramics, which are clinically used, as well as dicalcium phosphate dihydrate (DCPD) and synthesized OCP were compared. The materials were well characterized by chemical analysis, x-ray diffraction and Fourier transform infrared spectroscopy. Calcium and phosphate ion concentrations and the pH of culture media after immersion of the materials were determined. The colony forming rate of Chinese hamster lung fibroblasts was estimated with extraction of the materials. Proliferation of bone marrow stromal ST-2 cells and inflammatory cytokine TNF-α production by THP-1 cells grown on the material-coated plates were examined. The materials had characteristics that corresponded to those reported. DCPD was shown to dissolve the most in the culture media, with a marked increase in phosphate ion concentration and a reduction in pH. ST-2 cells proliferated well on the materials, with the exception of DCPD, which markedly inhibited cellular growth. The colony forming capacity was the lowest on DCPD, while that of the other calcium phosphates was not altered. In contrast, TNF-α was not detected even in cells grown on DCPD, suggesting that calcium phosphate materials are essentially non-inflammatory, while the solubility of the materials can affect osteoblastic and fibroblastic cellular attachment. These results indicate that OCP is biocompatible, which is similar to the materials used clinically, such as HA. Therefore, OCP could be clinically used as a biocompatible bone substitute material..
49. Hirofumi Kaneko, Junichi Kamiie, Hirotaka Kawakami, Takahisa Anada, Yoshitomo Honda, Naru Shiraishi, Shinji Kamakura, Tetsuya Terasaki, Hidetoshi Shimauchi, Osamu Suzuki, Proteome analysis of rat serum proteins adsorbed onto synthetic octacalcium phosphate crystals, Analytical Biochemistry, 10.1016/j.ab.2011.07.022, 418, 2, 276-285, 2011.11, The present study was designed to determine which proteins are selectively adsorbed onto two bone substitute materials, octacalcium phosphate (OCP) and hydroxyapatite (HA) crystals, from rat serum by proteome analysis. Ground crystals of synthetic OCP and commercially available sintered HA, with the same surface area, were incubated in rat serum proteins at 37 °C for 24 h. The proteins from the crystals extracted with guanidine-HCl-EDTA were listed on the basis of the results of liquid chromatography tandem mass spectrometry (LC/MS/MS). A total of 138 proteins were detected from OCP; 103 proteins were detected from HA. Forty-eight proteins were from both crystals. A quantitative analysis of the proteins detected was performed for the extracted two bone formation-related proteins apolipoprotein E (Apo E), a protein known to promote osteoblast differentiation, and complement 3 (C3). HA adsorbed C3 (3.98 ± 0.03 fmol/μg protein) more than OCP (1.81 ± 0.07 fmol/μg protein) did, while OCP adsorbed Apo E (2.42 ± 0.03 fmol/μg protein) more than HA (1.21 ± 0.01 fmol/μg protein) did even after deleting the high-abundance proteins, such as albumin. The results demonstrated that OCP exhibits a similar property but distinct capacity with HA in adsorbing bone formation-related proteins from the serum constituents..
50. Ryo Akatsuka, Keiichi Sasaki, Mohammed Saeed Sepasy Zahmaty, Miyoko Noji, Takahisa Anada, Osamu Suzuki, Tsunemoto Kuriyagawa, Characteristics of hydroxyapatite film formed on human enamel with the powder jet deposition technique, Journal of Biomedical Materials Research - Part B Applied Biomaterials, 10.1002/jbm.b.31781, 98 B, 2, 210-216, 2011.08, This study aimed to create hydroxyapatite (HAp) film by powder jet deposition with manipulating the blasting nozzle above human enamel and to examine the microstructural and mechanical properties of the HAp film and the bonding strength at the interface between the HAp film and the enamel substrate. HAp particles calcinated at 1200°C with an average size of 4.7 μm were used. The HAp particles were mixed with carrier gas (N 2) to form an aerosol flow and was accelerated and blasted from the nozzle onto the enamel substrate at room temperature and atmospheric pressure. To evaluate the microstructure, scanning electron microscope (SEM) images of the surface and cross section of the HAp films and a three-dimensional profile of the HAp films were observed. To evaluate the mechanical properties, the micro-Vickers hardness and the bonding strength of the HAp films to the enamel substrate were measured. The deposition area of the HAp film was over 3 Ã - 4 mm. The average and maximum thickness were about 30 and 40 μm, respectively. No significant difference was observed between the hardness of the HAp film and the enamel (p > 0.05). The bonding strength of the HAp film was the same as the bonding strength between composite resin and enamel. Compared with previous reports, wider and thicker HAp film was created on the enamel substrate successfully. The HAp film, which has same hardness with enamel and same bonding strength to the enamel with composite resin, would be a candidate as dental restorative materials.
51. Sonthaya Numthuam, Takahiro Kakegawa, Takahisa Anada, Ali Khademhosseini, Hiroaki Suzuki, Junji Fukuda, Synergistic effects of micro/nano modifications on electrodes for microfluidic electrochemical ELISA, Sensors and Actuators, B: Chemical, 10.1016/j.snb.2011.02.010, 156, 2, 637-644, 2011.08, Microfluidic electrochemical sensing has been considered to be highly efficient. However, we showed, by using numerical simulations in this study, that a planar electrode formed on the bottom of a microchannel is exposed to only a small fraction of analytes in amperometric detection. We also showed that three-dimensional (3D) micropillar electrodes significantly improve the detection current. The practical performance was evaluated using 3D micropillar electrodes fabricated by photolithography. The output current increased as the diameters of the micropillars decreased, as predicted by the simulations. It is noteworthy that the current enhancements obtained with the 3D electrodes were larger than those expected from an increase in the surface area. Further increase in current was achieved by electrical deposition of nanoporous gold-black onto the surface of the 3D electrode: when a 3D electrode with micropillars 30 μm in diameter was used, the output current was approximately 20 times that obtained with a 2D electrode without modification. The applicability of the micropillar electrodes was demonstrated in electrochemical enzyme-linked immunosorbent assay (ELISA) of bone metabolic marker proteins. Although an increase in the surface area of the electrode leads to more noise in general, there is no significant difference in the signal-to-noise ratio between the modified 3D electrode and the 2D electrode without modification in the ELISA experiments. This nanoporous micropillar electrode could potentially be a useful component for the development of on-site diagnosis systems..
52. Tadashi Kawai, Keiko Matsui, Shinya Iibuchi, Takahisa Anada, Yoshitomo Honda, Kazuo Sasaki, Shinji Kamakura, Osamu Suzuki, Seishi Echigo, Reconstruction of Critical-Sized Bone Defect in Dog Skull by Octacalcium Phosphate Combined with Collagen, Clinical Implant Dentistry and Related Research, 10.1111/j.1708-8208.2009.00192.x, 13, 2, 112-123, 2011.06, The present study was designed to investigate whether synthetic octacalcium phosphate (OCP) combined with collagen (OCP/collagen) can repair a critical-sized defect in dog skull. OCP/collagen has been shown to biodegrade and to tend to be replaced by newly formed bone if implanted in rat calvaria defects. Materials and Methods: An OCP/collagen disk was prepared from pepsin-digested atelocollagen isolated from porcine dermis and synthetic OCP. Two critical-sized defects (20mm in diameter) were made in a dog skull. Ten disks of OCP/collagen or collagen (control) were implanted in the bone defects and resected with surrounding tissues at 3, 6, or 12 months after the implantation. The specimens were analyzed radiographically, crystallographically, histologically, and histomorphometrically. Results: X-ray diffraction and FTIR analyses showed that OCP tended to convert to a poorly crystallized hydroxyapatite, similar to that of biological apatite, by 3 months. Radiographic and histologic analyses showed that the implantation of OCP/collagen disks initiated new bone formation in the defects at 3 months after implantation. However, there was no promotion of bone formation by control collagen disks even with prolonged implantation up to 12 months. Histomorphometric analysis revealed that the percentage of newly formed bone in the defect implanted with OCP/collagen increased significantly, from 30.91±6.65 at 3 months to 51.22±5.99 at 12 months, although the value tended to reach a plateau at 6 months (44.49±3.34). On the other hand, the percentage of remaining OCP was estimated at approximately 10% at 3 months and remained nearly unchanged thereafter. Conclusion: The results suggest that bone regeneration of a critical-sized bone defect of dog calvaria by OCP/collagen can be enhanced for 3 to 6 months and that OCP/collagen holds potential as a bone substitute material..
53. Yoshitomo Honda, Takahisa Anada, Shinji Morimoto, Yukari Shiwaku, Osamu Suzuki, Effect of Zn2+ on the physicochemical characteristics of octacalcium phosphate and its hydrolysis into apatitic phases, Crystal Growth and Design, 10.1021/cg1009835, 11, 5, 1462-1468, 2011.05, Zinc (Zn) ions and OCP crystals have both been reported to be stimulants for bone formation. The present study was designed to investigate how Zn ion affects the precipitation of octacalcium phosphate (OCP) and its hydrolysis process into apatitic phases. Zn-containing octacalcium phosphate preparations (Zn-CaPs) and their hydrolysates (hydrolyzed Zn-CaPs) containing various amounts of Zn (0.06-0.72 mmol/g) were obtained either through a co-precipitation method or by enhancing the OCP hydrolysis in hot water. All of the Zn-CaPs obtained had Ca/P molar ratios similar to that of stoichiometric OCP (133), ranging from 1.23 to 1.37. Zn-CaPs also exhibited similar dissolution behavior to that of original OCP if incubated in a physiological solution (in the 0.1 M HEPES buffer, pH = 7.4, 37 °C). X-ray diffraction analysis of the hydrolyzed samples showed that original OCP converted to the crystalline hydroxyapatite with a specific peak at 2θ = 10.8°, while Zn-CaPs containing Zn above the 0.18 mmol/g retained the amorphous-like structure even after a greater than 25 h hydrolysis period. These results indicate that Zn ions retarded the hydrolysis process of OCP toward apatite, resulting in enhanced formation of the amorphous-like structure..
54. Tatsuya Miyazaki, Satoshi Miyauchi, Takahisa Anada, Hideki Imaizumi, Osamu Suzuki, Evaluation of osteoclastic resorption activity using calcium phosphate coating combined with labeled polyanion, Analytical Biochemistry, 10.1016/j.ab.2010.11.014, 410, 1, 7-12, 2011.03, Osteoclasts are involved in bone resorption, and its activation is considered one of the causes of osteoporosis. The pit assay is the principal method for evaluating osteoclast function by measuring hydroxyapatite resorption in vitro. However, the pit assay requires time and trained techniques, including the pit image analysis, and there is no other easy method for evaluating bone resorption. In this study, we developed a novel approach to quantify the bone resorption activity using a calcium phosphate (CaP) coating labeled with fluorescent polyanion. Fluoresceinamine-labeled chondroitin polysulfate or Hoechst 33258-labeled deoxyribonucleic acid was used for CaP labeling. When macrophage cell line RAW264 was cultured on the labeled CaP under the stimulation with the receptor activator of the NF-κB ligand (RANKL), RAW264 cells differentiated into osteoclastic cells and the fluorescence intensity of the culture supernatant and pit area increased in a time- and dose-dependent manner. Furthermore, drugs for osteoporosis treatment, such as pamidronate and β-estradiol, inhibited fluorescein release by the cells stimulated with RANKL. A positive correlation between the fluorescence intensity and pit area was observed (r = 0.917). These results indicated that this new method using fluorescent polyanion-labeled CaP is a standardized useful assay system for the evaluation of bone resorption activity..
55. Tadashi Kawai, Takahisa Anada, Taisuke Masuda, Yoshitomo Honda, Yuhiro Sakai, Yukio Kato, Shinji Kamakura, Seishi Echigo, Osamu Suzuki, The effect of synthetic octacalcium phosphate in a collagen scaffold on the osteogenicity of mesenchymal stem cells, European Cells and Materials, 10.22203/eCM.v022a10, 22, 124-136, 2011.01, Although the efficacy of the in vivo osteogenic capabilities of synthetic octacalcium phosphate (OCP) crystal implantation can be explained through its stimulatory capacity for the differentiation of the host osteoblastic cell lineage, direct evidence that OCP supports bone regeneration by osteogenic cells in vivo has not been shown. Mesenchymal stem cells (MSCs) isolated from 4-week-old male Wistar rat long bones were pre-incubated in osteogenic or maintenance medium in the presence or absence of basic fibroblast growth factor (bFGF). OCP/Collagen (OCP/Col) or collagen disks were seeded with MSCs that had been pre-incubated in osteogenic medium containing bFGF, which exhibited the highest differentiation induction, and then incubated for an additional day. The disks were implanted in critical-sized calvaria defects of 12-week-old male Wistar rats and the specimens were analysed radiographically, histologically, histomorphometrically, and by micro-computed tomography (CT) imaging at 4 and 8 weeks after the implantation. The OCP/Col·MSCs group rapidly induced more bone regeneration, even within 4 weeks, compared to the OCP/Col group without MSCs. The bone mineral density of the OCP/Col·MSCs group was also greater than the OCP/Col group. The Col·MSCs group did not exhibit prominent osteogenicity. These results indicate that OCP crystals in a collagen matrix efficiently promote exogenously introduced osteogenic cells to initiate bone regeneration if the cells are pre-treated in a suitable differentiation condition..
56. Tatsuya Miyazaki, Satoshi Miyauchi, Akira Tawada, Takahisa Anada, Osamu Suzuki, Effect of chondroitin sulfate-E on the osteoclastic differentiation of RAW264 cells, dental materials journal, 10.4012/dmj.2009-132, 29, 4, 403-410, 2010.12, The present study was designed to investigate whether chondroitin sulfate (CS)-E, a CS structural isomer variant, alter the differentiation of macrophage cell line RAW264 cells to osteoclast-like cells. CS-B, CS-E, low molecular weight CS-E, synthetic chondroitin polysulfate (CPS) and heparin significantly inhibited the formation of tartrate-resistant acid phosphatase-positive multinuclear cells and pit formation on calcium phosphate (CaP)-coated plates. CS-E pre-coated on the CaP plate also inhibited pit formation. Digestion of CS on the cell surface by chondroitinase showed no effect on the osteoclastic differentiation of RAW264 cells whereas inhibitory effect on the differentiation of osteoblastic cell line MC3T3-E1. On the other hand, exogenously added fluoresceinlabeled CS-E directory bound to fibronectin and RAW264 cells. These results suggest that CS-E structure on the surface of osteoblasts or bone matrix binds to cell adhesion molecule such as integrin on the pre-osteoclastic cells and inhibits the differentiation into osteoclasts. CS-E may have a potential in treating bone defect if combined with CaP materials..
57. Taisuke Masuda, Tadashi Kawai, Takahisa Anada, Shinji Kamakura, Osamu Suzuki, Quality of regenerated bone enhanced by implantation of octacalcium phosphate-collagen composite, Tissue Engineering - Part C: Methods, 10.1089/ten.tec.2009.0212, 16, 3, 471-478, 2010.06, The present study was designed to investigate whether mechanical testing in conjunction with microcomputed tomography (μCT) analysis can be used to evaluate the quality of regenerated bone enhanced by the implantation of a composite composed of granular octacalcium phosphate and collagen (OCP/Col) matrix. Previous studies confirmed that the granules of OCP alone or OCP in Col matrix tend to mature into bone-like hydroxyapatite and enhance bone regeneration coupled with its own biodegradation, if implanted in various bony sites. OCP/Col was implanted in rat calvaria critical-sized bone defect for 4 to 12 weeks for microindentation, mechanical testing, μCT imaging, and histological examinations. The microindentation testing of the regenerated bone revealed a progressive increase of the Vickers hardness showing the highest value in 12 weeks. The Vickers hardness was in good agreement with both the parameters of μCT and the mechanical property; the quality of regenerated bone increased progressively with the implantation periods. The regenerated bone exhibited a mature bone-like matrix structure with osteocytes histologically. The quality of the regenerated bone was compatible to that of normal control calvaria bone regarding the mechanical properties. The results indicate that microindentation testing can be used to evaluate the quality of regenerated bone in the initial regeneration and the development of bone enhanced by OCP granules within Col matrix. It appears that the implantation of OCP/Col could be a model study to determine the quality of the regenerated bone..
58. Yusuke Orii, Hiroshi Masumoto, Yoshitomo Honda, Takahisa Anada, Takashi Goto, Keiichi Sasaki, Osamu Suzuki, Enhancement of octacalcium phosphate deposition on a titanium surface activated by electron cyclotron resonance plasma oxidation, Journal of Biomedical Materials Research - Part B Applied Biomaterials, 10.1002/jbm.b.31605, 93, 2, 476-483, 2010.05, The present study was designed to investigate whether the formation of octacalcium phosphate (OCP) is accelerated on titanium (Ti) surface by an electron cyclotron resonance (ECR) plasma oxidation at various pressures and temperatures. X-ray diffraction (XRD) of Ti-oxidized substrates showed that the rutile TiO2 phase on its surfaces appeared at 300°C and was crystallized when the oxidation temperature increased up to 600°C. The thickness of TiO2 film on the substrates increased progressively as the temperature increased. The oxidized Ti surfaces were soaked in calcium and phosphate solutions supersaturated with respect to both hydroxyapatite (HA) and OCP but slightly supersaturated with dicalcium phosphate dihydrate (DCPD). OCP crystals with a blade-like morphology were deposited as the primary crystalline phase on Ti substrates, while DCPD was included as a minor constituent. The amount of OCP deposition was maximized under 0.015 Pa in 300°C. On the other hand, the oxidation temperature did not show a significant effect on the deposit in the range examined. The phase conversion from OCP to HA, determined by XRD, was demonstrated to occur even at 1 day and to advance until 7 days by immersing the Ti substrate with the deposit in simulated body fluid at 37°C. The present results suggest that ECR plasma oxidation could be used to improve a Ti surface regarding its bioactivity due to the enhancement of osteoconductive OCP deposition..
59. Takahisa Anada, Taisuke Masuda, Yoshitomo Honda, Junji Fukuda, Fumihito Arai, Toshio Fukuda, Osamu Suzuki, Three-dimensional cell culture device utilizing thin membrane deformation by decompression, Sensors and Actuators, B: Chemical, 10.1016/j.snb.2010.01.065, 147, 1, 376-379, 2010.05, We have developed a novel multicellular aggregate (spheroid) culture device that utilizes thin polydimethylsiloxane membrane deformation by decompression. The device was capable of producing thousands of spheroids rapidly at a time and regulating the diameter of spheroids with narrow size distribution. The spheroids were easily retrieved from the device noninvasively. These characteristics of the device suggest that it has the potential for use in a wide variety of applications, such as drug screening and tissue engineering..
60. Yoshihisa Murakami, Yoshitomo Honda, Takahisa Anada, Hidetoshi Shimauchi, Osamu Suzuki, Comparative study on bone regeneration by synthetic octacalcium phosphate with various granule sizes, Acta Biomaterialia, 10.1016/j.actbio.2009.10.023, 6, 4, 1542-1548, 2010.04, The present study was designed to investigate whether the granule size of synthetic octacalcium phosphate (OCP) and the resultant intergranular spaces between the granules formed by the filling affect its osteoconductive and biodegradable characteristics in a mouse calvaria critical-sized defect up to 10 weeks after implantation. Mercury intrusion porosimetry showed that OCP granules having distinct diameter sizes ranging from 53 to 300 (S-OCP), 300 to 500 (I-OCP) and 500 to 1000 μm (L-OCP) produced distinct intergranular spaces between OCP granules ranging from 28.8 to 176.6 μm. The dissolution rate of OCP, estimated by the phosphate concentration in the culture medium, was the highest in S-OCP, followed by I-OCP and L-OCP, while the specific surface area of OCP decreased. Histological and histomorphometric analyses showed that bone formation around the implanted granules increased significantly with increasing granule size coupled with activating the appearance of TRAP- and cathepsin K-positive osteoclastic cells. The rate of new bone formation formed with L-OCP was two times higher than that formed with S-OCP at 10 weeks after implantation. The results indicated that the osteoconductive and biodegradable properties of OCP can be augmented by increasing the granule size, most probably by thus providing enough spaces between the granules, suggesting that the intergranular spaces formed by the granules may work similarly to pores, as reported in porous ceramic materials. It seems likely that the enhancement of bone formation by OCP is accompanied by simultaneous activation of osteoclastic resorption of OCP..
61. Naru Shiraishi, Takahisa Anada, Yoshitomo Honda, Taisuke Masuda, Keiichi Sasaki, Osamu Suzuki, Preparation and characterization of porous alginate scaffolds containing various amounts of octacalcium phosphate (OCP) crystals, Journal of Materials Science: Materials in Medicine, 10.1007/s10856-009-3911-1, 21, 3, 907-914, 2010.03, The present study was designed to investigate whether the amount of octacalcium phosphate (OCP) affects the characteristics of alginate (Alg)/OCP scaffolds regarding the pore formation and its distribution, and the thermodynamic stability from OCP to hydroxyapatite (HA) in an in vitro physiological environment. Alg/OCP composites with weight ratios of 100/0, 75/25, 50/50, and 25/75 were prepared through mixing the ground synthesized OCP crystals with an Alg solution and applying lyophilization. Analysis of X-ray diffraction, Fourier transform infrared (FTIR) spectroscopy, and mercury intrusion porosimetry verified that the crystalline phase of OCP and the porosities were retained regardless of the OCP amount. On the other hand, the elastic modulus, determined by mechanical testing, and, interestingly, the pore size increased with increasing the OCP amount. The immersion of the composites in a simulated body fluid up to 14 days revealed that OCP in Alg matrices tends to convert to HA with enhancing the calcium consumption depending on the OCP amount. The results indicated that the inclusion of OCP crystals in the Alg matrix by the mixing process controls the character of the pore distribution in Alg/OCP composites while maintaining the transitory nature of OCP..
62. Yukari Shiwaku, Yoshitomo Honda, Takahisa Anada, Shinji Morimoto, Taisuke Masuda, Keiichi Sasaki, Osamu Suzuki, Analysis of physicochemical properties of octacalcium phosphate prepared by hydrolysis and co-precipitation with fluoride ions, Journal of the Ceramic Society of Japan, 10.2109/jcersj2.118.402, 118, 1378, 402-405, 2010.01, The present study was designed to determine the physicochemical properties of octacalcium phosphate (OCP) and its products obtained by different preparations in the presence of fluoride ions (F-) through hydrolysis of the synthesized OCP and coprecipitation. X-ray diffraction and chemical analyses of the products showed that OCP tended to be hydrolyzed into an apatitic structure in both preparations, while the OCP structure remained when hydrolyzed in alkaline pH and obtained by coprecipitation. The solubility decreased with increasing the F- content in the hydrolyzed products but almost corresponded to that of the original OCP in the co-precipitation products. The results confirmed that the different preparation produces OCP hydrolyzates having distinct physicochemical properties that preserve the characteristics of OCP to some extent. Analysis of cytochrome c (used as a model protein of growth factors) adsorption onto the crystals indicated that the hydrolysis groups had the highest capacity to adsorb..
63. Aritsune Matsui, Takahisa Anada, Taisuke Masuda, Yoshitomo Honda, Naohisa Miyatake, Tadashi Kawai, Shinji Kamakura, Seishi Echigo, Osamu Suzuki, Mechanical stress-related calvaria bone augmentation by onlayed octacalcium phosphate-collagen implant, Tissue Engineering - Part A, 10.1089/ten.tea.2009.0284, 16, 1, 139-151, 2010.01, Previous studies have suggested that the biodegradability of octacalcium phosphate-collagen (OCP/Col) composite by osteoclasts is accelerated in association with mechanical stress suffered by the host tissue around the implant. The present study was designed to investigate whether alleviation of mechanical stress restores the bone regenerative properties of OCP/Col, as previously shown in nonload-bearing sites. OCP/Col discs supported with a polytetrafluoroethylene (PTFE) ring, which has a higher modulus than OCP/Col, were implanted in a rat subperiosteal pocket for up to 12 weeks. The structural features of the implant and biological responses were analyzed by X-ray diffraction, Fourier transform infrared spectroscopy, histomorphometry, histochemistry, and tissue mRNA expression around the implants. The effect of compression was analyzed using mouse stromal ST-2 cells by deforming the cell-seeded OCP/Col discs in vitro with or without a PTFE ring. The results clearly indicated the restoration of bone formation by the alleviation of mechanical stress and the upregulation of osteoblast-related genes, such as osterix on the other hand, the implantation of OCP/Col on calvaria or in an in vitro test without PTFE support resulted in the upregulation of osteoclast-related genes, such as tartrate-resistant acid phosphatase (TRAP) and cathepsin K, in the tissues or receptor activator of the nuclear factor-κB ligand (RANKL) in ST-2 cells. The results confirmed that calvaria augmentation is enhanced by implanting OCP/Col if suitable conditions regarding mechanical stress are provided..
64. Y. Suzukfi, S. Kamakura, Y. Honda, Takahisa Anada, K. Hatori, K. Sasaki, O. Suzuki, Appositional bone formation by OCP-Collagen composite, Journal of Dental Research, 10.1177/0022034509351378, 88, 12, 1107-1112, 2009.12, Synthetic octacalcium phosphate (OCP) has been shown to enhance bone formation and to biodegrade if implanted into bone defects. Here, we hypothesized that an OCP-atelocollagen complex (OCP/Col) is biodegradable and can induce bone formation in a thickness-dependent manner when implanted into the calvaria. OCP/Col disks (diameter, 9 mm; thickness, 1 or 3 mm) were implanted into a subperiosteal pocket in the calvaria of 12-week-old Wistar rats for 4, 8, and 12 weeks and subsequent bone formation was monitored. X-ray diffraction analysis and Fourier transform infrared spectroscopy showed that OCP in the OCP/Col implants was converted into a carbonate-rich apatite after 4 weeks. Although thinner disks tended to be replaced by new bone, thicker disks were progressively resorbed by osteoclast-like cells until 12 weeks, possibly via the increased mechanical load in the subperiosteal pocket. Therefore, OCP/Col can increase appositional intramembranous bone formation if the appropriate size of the implant is applied..
65. Masamichi Takami, Ayako Mochizuki, Atsushi Yamada, Keita Tachi, Baohong Zhao, Yoichi Miyamoto, Takahisa Anada, Yoshitomo Honda, Tomio Inoue, Masanori Nakamura, Osamu Suzuki, Ryutaro Kamijo, Osteoclast differentiation induced by synthetic octacalcium phosphate through receptor activator of NF-κB ligand expression in osteoblasts, Tissue Engineering - Part A, 10.1089/ten.tea.2009.0065, 15, 12, 3991-4000, 2009.12, Synthetic octacalcium phosphate (OCP) has a potential to enhance new bone formation and exhibits biodegradable characteristics when implanted in experimentally created bone defects. The precise mechanisms of OCP biodegradation remain unclear, though histological observations have revealed that bone-resorbing osteoclasts appear and resorb implanted OCP. To investigate how osteoclasts develop around implanted OCP, we examined osteoclast differentiation using OCP crystals in vitro. Coculturing of mouse bone marrow cells and osteoblasts in OCP-coated cell culture plates induced osteoclast differentiation, whereas that did not occur without coating. Further, addition of bone morphogenetic protein-2 significantly increased the number of osteoclasts in the OCP-coated wells. In the presence of OCP, osteoblasts expressed receptor activator of NF-κB ligand (RANKL), an osteoclast differentiation factor. In addition, when half of each culture well was coated with OCP, osteoclasts were formed in both coated and noncoated areas, suggesting that soluble factors mediate osteoclast differentiation induced by OCP. Also, calcium levels in culture medium were significantly decreased in the presence of OCP, while experimental reduction of calcium from 8.0 to 5.0 mg/dL significantly induced RANKL mRNA expression. These results suggest that OCP itself decreases calcium levels around implanted OCP, which induces osteoclast differentiation through RANKL expression by osteoblasts..
66. Takeshi Fuji, Takahisa Anada, Yoshitomo Honda, Yukari Shiwaku, Hiroko Koike, Shinji Kamakura, Keiichi Sasaki, Osamu Suzuki, Octacalcium phosphate-precipitated alginate scaffold for bone regeneration, Tissue Engineering - Part A, 10.1089/ten.tea.2009.0048, 15, 11, 3525-3535, 2009.11, The present study was designed to investigate whether octacalcium phosphate (OCP)-precipitated alginate (Alg) promotes osteoblastic cell proliferation and bone regeneration in comparison with Alg itself. Alg, known to lack mammal cell attachment capability, was used as the matrix to test the distribution effect of OCP in a three-dimensional environment. A series of Alg/OCP scaffolds with different pore sizes was prepared by centrifuging Alg gels precipitated by OCP crystals. The scaffolds had a bimodal distribution of pores (ultrafine pores: approximately 100nm; relatively large pores: from 6.0 to 51.7 μm) and over 86% porosity. The osteoconductive capability of Alg/OCP was determined by examining mouse bone marrow stromal ST-2 cell proliferation after 3 days in vitro and bone regeneration in mouse calvaria critical-sized defect after 21 days. The analyses showed that ST-2 cell proliferation and bone regeneration increased with an increase in the pore size and reached the highest level in the 51.7 μm pore scaffold. The results suggest that OCP-precipitated Alg provides a better scaffold for osteoblasts to attach and proliferate in a three-dimensional environment and promotes bone regeneration, indicating that OCP is a candidate material to modify the surface of non-cell-interactive polymeric scaffolds, such as Alg, into an osteogenic condition..
67. Ichiro Takahashi, Taisuke Masuda, Kumiko Kohsaka, Fumie Terao, Takahisa Anada, Yasuyuki Sasano, Teruko Takano-Yamamoto, Osamu Suzuki, Molecular mechanisms of mechanical stress response during chondrogenesis, Journal of Biomechanical Science and Engineering, 10.1299/jbse.4.307, 4, 3, 307-317, 2009.09, Muscloskeletal tissues, bone, cartilage, muscles and tendons regulate and support the body's actions and are differentiated from mesenchymal stem cells. These organs generate and/or respond to mechanical stress, which is inevitable in daily life. Among these tissues, cartilages play roles in articular function in joints where shear stress is loaded in combination with cyclical or intermittent compressive force by joint action, and provides a template for bone growth under compressive stress directed along the long axis of long bones. Along with soluble factors, such as cytokines and growth factors, mechanical stress has been recognized as one of the epigenetic factors that regulates the gene expression of various types of cells. Thus, the molecular mechanisms of the mechano-sensing, mechano-transduction, and mechano-response of cells have become a focus of connective tissue biology. Here, we will discuss the mechanisms through which differentiated chondrocytes and mesenchymal cells that are differentiating into chondrocytes respond to various types of mechanical stress by altering their phenotype and how these phenotypic changes are molecularly regulated. We will focus on the roles of cell-extracellular matrix interactions through integrins and downstream signaling pathways involving mitogen-activated protein kinases..
68. Takahisa Anada, Yoichi Takeda, Yoshitomo Honda, Kazuo Sakurai, Osamu Suzuki, Synthesis of calcium phosphate-binding liposome for drug delivery, Bioorganic and Medicinal Chemistry Letters, 10.1016/j.bmcl.2009.05.117, 19, 15, 4148-4150, 2009.08, Metastatic bone disease is often associated with bone pain, pathologic fractures, and nerve compression syndromes. Effective therapies to inhibit the progression of bone metastases would have important clinical benefits. Therefore, we developed a novel calcium phosphate-binding liposome for a bone-targeting drug delivery system. We synthesized a novel amphipathic molecule bearing a bisphosphonate (BP) head group to recognize and bind to hydroxyapatite (HA). We demonstrated that the liposomes having BP moieties show high affinity for HA. Doxorubicin-loaded liposomes adsorbed on the surface of HA significantly reduce the number of viable human osteosarcoma MG63 cells. This shows that the liposomes can be excellent carriers for anticancer drugs because they specifically target bone tissue. This calcium phosphate-binding liposome system could be used with many drugs for bone-related diseases such as osteoporosis, rheumatoid arthritis, and multiple myeloma..
69. Yoshitomo Honda, Takahisa Anada, Shinji Kamakura, Shinji Morimoto, Tsunemoto Kuriyagawa, Osamu Suzuki, The effect of microstructure of octacalcium phosphate on the bone regenerative property, Tissue Engineering - Part A, 10.1089/ten.tea.2008.0300, 15, 8, 1965-1973, 2009.08, The present study was designed to investigate whether the microstructure of synthetic octacalcium phosphate (OCP) affects its intrinsic bone regenerative properties as a scaffold and its conversion process into hydroxyapatite (HA). Our previous studies indicated that an agregate of OCP crystals, consisting of randomly oriented plate-like crystals, are capable of enhancing both osteoblastic cell differentiation in vitro and bone regeneration. While the transformation of OCP into HA has been considered in relation to the stimulatory capacity of OCP in bone regeneration, little is known about the effect of the microstructure of OCP granules on these capabilities. Two types of OCP granules, with identical diameters (300-500μm) but composed of crystals with distinct crystal dimensions (4.0 and 26.6μm length), were prepared (hereafter referred to as fine OCP granules [F-OCP] and coarse OCP granules [C-OCP], respectively). The intergranule distances and the porosity, including the intergranule spaces, were 108.5μm and 93.7% for F-OCP, and 67.5μm and 95.7% for C-OCP, as estimated by mercury intrusion. The OCP granules were implanted in mouse critical-sized calvarial defects for up to 14 days. Histological examination demonstrated that osteoblastic cells aligned on the surface of F-OCP at day 7 and formed new bone around the granules up to day 14. On the other hand, cells around C-OCP were sparse at day 7, and resulted in only slight bone formation around the granules at day 14. X-ray diffraction showed that both OCP granules tended to be converted to an apatite structure with similar conversion velocity by the implantation. Adhesion of mouse bone marrow stromal ST-2 cells was markedly inhibited on C-OCP compared to F-OCP in vitro. These results suggested that the microstructure consisting of plate-like crystals of OCP controls cell adhesion on the crystal surfaces and their resultant bone regenerative properties as well as the physicochemical effect associated with the transitory nature of OCP previously reported..
70. Naohisa Miyatake, Koshi N. Kishimoto, Takahisa Anada, Hideki Imaizumi, Eiji Itoi, Osamu Suzuki, Effect of partial hydrolysis of octacalcium phosphate on its osteoconductive characteristics, Biomaterials, 10.1016/j.biomaterials.2008.10.058, 30, 6, 1005-1014, 2009.02, The present study was designed to investigate whether the stoichiometry of octacalcium phosphate OCP affects its osteoconductive and immune response characteristics in rat bone marrow. Those characteristics of synthetic, well-grown OCP but with a non-stoichiometric composition were compared with those of a slightly hydrolyzed OCP (low crystalline OCP: LC-OCP), the fully hydrolyzed apatitic product of OCP or biodegradable β-tricalcium phosphate (β-TCP) ceramic, by their implantation in rat tibia for 56 days. The physicochemical aspect of implants and biological responses were analyzed by X-ray diffraction, histomorphometry, immunohistochemistry and expression of mRNA around the implants. The remarkable findings were that: (1) the highest bone formation rate was obtained for β-TCP whereas the lowest for LC-OCP at Day 14; (2) the rates were reversed and reached the highest for LC-OCP until Day 56; (3) the early expression of ostoeoclast markers TRAP and cathepsin-K was suppressed with LC-OCP; (4) the expression of inflammatory markers IL-β1 and TNF-α was suppressed with LC-OCP. The results confirmed that the partially hydrolyzed OCP with Ca/P molar ratio 1.37 (LC-OCP) enhances bone formation most, suppressing early osteoclast activity and reducing inflammation..
71. Tadashi Kawai, Takahisa Anada, Yoshitomo Honda, Shinji Kamakura, Keiko Matsui, Aritsune Matsui, Kazuo Sasaki, Shinji Morimoto, Seishi Echigo, Osamu Suzuki, Synthetic octacalcium phosphate augments bone regeneration correlated with its content in collagen scaffold, Tissue Engineering - Part A, 10.1089/ten.tea.2008.0141, 15, 1, 23-32, 2009.01, Previous studies have shown that synthetic octacalcium phosphate (OCP) facilitates in vitro osteoblastic cell differentiation in an OCP dose-dependent manner and that a complex of OCP and collagen (OCP/collagen) enhances critical-sized rat calvaria defects more than OCP alone. The present study was designed to investigate whether the bone regenerative properties of OCP/collagen are augmented in an OCP dose-dependent manner, thereby establishing a suitable composition of this composite as a bone substitute material. OCP/collagens with a wide range of mixing ratios from 23:77 to 83:17, including the previously examined composition (77:23), were prepared by blending granules of OCP with atelocollagen and molded into a disk as an implant. A critical-sized defect was made in rat calvaria, and each disk was implanted into the defect for 4 or 12 weeks and then examined radiographically, histologically, and histomorphometrically. Mouse bone marrow-derived stromal ST-2 cells were cultured in dishes pre-coated with OCP/collagen or OCP alone with different OCP contents to determine the capacity of cell attachment and proliferation up to 14 days. Histological and radiographic examinations showed that newly formed bone was observed in relation to OCP granules within the collagen matrix. Histomorphometric analysis confirmed that increasing the amount of OCP in collagen matrices resulted in progressive enhancement of bone regeneration and that the ratio 83:17 generated the maximum repair level of approximately 64% of the defect at 12 weeks. OCP/collagen promoted the proliferation and attachment of ST-2 cells more than OCP alone regardless of OCP content. Fourier transform infrared spectroscopy analysis of the coatings after the incubation indicated that OCP tended to convert to apatite regardless of the presence of collagen. The present study demonstrated that the osteoconductive characteristics of OCP/collagen can be displayed in an OCP dose-dependent manner. The results suggest that collagen promotes the proliferation and attachment of host osteoblastic cells on OCP/collagen composite implants..
72. Osamu Suzuki, Tazunu Kikawa, Takahisa Anada, Shinji Kamakura, Hideki Imaizumi, Intramembranous bone reaction to synthetic ocp during hydroxyapatite deposition onto the OCP template, 8th World Biomaterials Congress 2008, WBC 2008 8th World Biomaterials Congress 2008, 1, 2008.12.
73. Tatsuya Miyazaki, Satoshi Miyauchi, Akira Tawada, Takahisa Anada, Satoshi Matsuzaka, Osamu Suzuki, Oversulfated chondroitin sulfate-E binds to BMP-4 and enhances osteoblast differentiation, Journal of cellular physiology, 10.1002/jcp.21557, 217, 3, 769-777, 2008.12, Small leucine-rich proteoglycans, such as biglycan, and their side chain sulfated glycosaminoglycans (GAGs), have been suggested to be involved in bone formation and mineralization processes. The present study was designed to investigate whether chondroitin sulfate (CS), one of the GAG, and its oversulfated structures coupled with bone morphogenetic protein-4 (BMP-4) alter the differentiation and subsequent mineralization of MC3T3-E1 osteoblastic cells. CS-E, one of the oversulfated CS structure, enhanced cell growth, alkaline phosphatase (ALP) activity, collagen deposition, and mineralization whereas heparin enhanced only ALP activity and mineralization. As well as CS-E, CS-H, and CPS also enhanced the mineralization of the cells. CS-E enhanced the mineralization of the cells by interacting with protein in the conditioned medium. CS-E induced mineralization was significantly inhibited by an antibody against BMP-4. The addition of exogenous BMP-4 further increased the capacity of CS-E to enhance mineralization. Fluorescence correlation spectroscopy method using fluoresceinamine-labeled GAG revealed that the oversulfated GAGs have a high affinity for BMP-4. The disaccharide analysis of the cells indicated that MC3T3-E1 cells are capable of producing oversulfated structures of CS by themselves. The lack of CS from the cells after chondroitinase treatment resulted in the inhibition of mineralization. These results in the present study indicate that oversulfated CS, which possesses 4,6-disulfates in N-acetyl-galactosamine, binds to BMP-4 and promotes osteoblast differentiation and subsequent mineralization..
74. Takahisa Anada, Takashi Kumagai, Yoshitomo Honda, Taisuke Masuda, Ryutaro Kamijo, Shinji Kamakura, Nobuhito Yoshihara, Tsunemoto Kuriyagawa, Hidetoshi Shimauchi, Osamu Suzuki, Dose-dependent osteogenic effect of octacalcium phosphate on mouse bone marrow stromal cells, Tissue Engineering - Part A., 10.1089/ten.tea.2007.0339, 14, 6, 965-978, 2008.06, Octacalcium phosphate (OCP) has been advocated to be a precursor of biological apatite crystals in bones and teeth. Our previous studies showed that synthetic OCP stimulates bone regeneration, followed by the progressive conversion of OCP into hydroxyapatite (HA), when implanted in bone defects. However, the precise mechanism to induce the osteogenic phenotype in osteoblasts by OCP has not been identified. The present study was designed to investigate whether the physicochemical aspect, specific to and derived from the structural properties of OCP, influences the function of an osteoblastic cell line, mouse bone marrow stromal ST-2 cells. Different amounts of synthetic OCP and synthetic sintered ceramic HA were coated onto 48-well tissue culture plates. The amounts of OCP and HA were controlled to strengthen their intrinsic physicochemical properties, in which the milieu around the crystals will be modified during the culture. The roughness of the OCP coatings was independent of the amount of coating. Chemical analyses of the supernatants of the OCP coatings revealed that the concentration of Ca2+ decreased with increasing amounts of OCP, while the concentration of inorganic phosphate increased markedly, most probably through OCP-apatite conversion. ST-2 cells were cultured on the OCP or HA coatings up to day 21. The OCP coating caused a significant decrease in cell attachment and in the initial stage of proliferation, dependent upon the amount of coating. On the other hand, OCP enhanced the expression of osteogenic markers, including type I collagen, alkaline phosphatase, and osterix. However, HA did not alter the expression of these markers in ST-2 cells cultured on different amounts of HA coating. These results demonstrated that OCP is capable of inducing the differentiation of stromal cells into osteoblastic cells, especially differentiation into early stage osteoblastic cells, prior to reaching the stage of mature osteoblastic cell lineage..
75. Taisuke Masuda, Ichiro Takahashi, Takahisa Anada, Fumihito Arai, Toshio Fukuda, Teruko Takano-Yamamoto, Osamu Suzuki, Development of a cell culture system loading cyclic mechanical strain to chondrogenic cells, Journal of Biotechnology, 10.1016/j.jbiotec.2007.08.007, 133, 2, 231-238, 2008.01, Mechanical stimulation is considered to be one of the major epigenetic factors regulating the metabolism, proliferation, survival and differentiation of cells in the skeletal tissues. It is generally accepted that the cytoskeleton can undergo remodeling in response to mechanical stimuli such as tensile strain or fluid flow. Mechanically induced cell deformation is one of the possible mechanotransduction pathways by which chondrocytes sense and respond to changes in their mechanical environment. Mechanical strain has a variety of effects on the structure and function of their cells in the skeletal tissues, such as chondrocytes, osteoblasts and fibroblasts. However, little is known about the effect of the quality and quantity of mechanical strain and the timing of mechanical loading on the differentiation of these cells. The present study was designed to investigate the effect of the deformation of chondrogenic cells, and cyclic compression using a newly developed culture device, by analyzing mechanobiological response to the differentiating chondrocytes. Cyclic compression between 0 and 22% strains, at 23 μHz was loaded on chondrogenic cell line ATDC5 by seeding in a mass mode on PDMS membrane, assuming direct transfer of cyclic deformation from the membrane to the cells at the same frequency. The compressive strain, induced within the membrane, was characterized based on the analysis of the finite element modeling (FEM). The results showed that the tensile strain inhibits the chondrogenic differentiation of ATDC5 cells, whereas the compressive strain enhances the chondrogenic differentiation, suggesting that the differentiation of the chondrogenic cells could be controlled by the amount and the mode of strain. In conclusion, we have developed a unique strain loading culture system to analyze the effect of various types of mechanical stimulation on various cellular activities..
76. Jusaku Minari, Takanori Kubo, Hideki Ohba, Naohiko Shimada, Yoich Takeda, Ryouji Karinaga, Takahisa Anada, Kazuya Koumoto, Takeshi Kawazu, Takeshi Nagasaki, Seiji Shinkai, Kazuo Sakurai, Delivery of antisense oligonucleotides to nuclear telomere RNA by use of a complex between polysaccharide and polynucleotide, Bulletin of the Chemical Society of Japan, 10.1246/bcsj.80.1091, 80, 6, 1091-1098, 2007.12, Telomerase, which is highly activated in neoplastic cells, can be a target for antisense therapy, and for that purpose, antisense oligonucleotides (AS ODNs) have to be effectively delivered into cellular nucleus where the target telomerase is present. The present work shows a new strategy to deliver AS ODNs to nucleus by use of a novel complex made from a natural polysaccharide schizophyllan (SPG) and AS ODNs. Nuclear transport is strictly regulated by the nuclear pore size and the related proteins. If the molecular weight of SPG is decreased, the SPG/AS ODN complex should be easily transported, although the stability of the complex decreases with a decrease in the molecular weight. We optimized the molecular weight of SPG to be 25 K. Furthermore, we attached importing-β (a nuclear transport protein) to the side chain of SPG by use of a streptavidin-biotin interaction. When this complex was added to Jurkat cells, the telomerase activity was more suppressed than the naked dose, indicating that the importin-β in the complex induced the nuclear transport of the complexed AS ODN and the AS ODN inhibited the telomerase. The present work shows a new methodology for nuclear anti-sense therapy that should be important in future anti-cancer therapies..
77. Shinji Kamakura, Kazuo Sasaki, Takahiro Homma, Yoshitomo Honda, Takahisa Anada, Seishi Echigo, Osamu Suzuki, The primacy of octacalcium phosphate collagen composites in bone regeneration, Journal of Biomedical Materials Research - Part A, 10.1002/jbm.a.31332, 83, 3, 725-733, 2007.11, We have engineered a scaffold constructed of synthetic octacalcium phosphate (OCP) and porcine collagen sponge (OCP/Col), and reported that OCP/Col drastically enhanced bone regeneration. In this study, we investigated whether OCP/Col would enhance bone regeneration more than β-tricalcium phosphate (β-TCP) collagen composite (β-TCP/Col) or hydroxyapatite (HA) collagen composite (HA/Col). Discs of OCP/Col, β-TCP/Col, or HA/Col were implanted into critical-sized defects in rat crania and fixed at 4 or 12 weeks after implantation. The newly formed bone and the remaining granules of implants in the defect were determined by histomorphometrical analysis, and radiographic and histological examinations were performed. Statistical analysis showed that the newly formed bone by the implantation of OCP/Col was significantly more than that of β-TCP/Col or HA/Col. In contrast, the remaining granules in OCP/Col were significantly lower than those in β-TCP/Col or HA/Col. Bone regeneration by OCP/Col was based on secured calcified collagen and bone nucleation by OCP, whereas bone regeneration by β-TCP/Col or HA/Col was initiated by poorly calcified collagen and osteoconductivity by β-TCP or HA. This study showed that the implantation of OCP/Col in a rat cranial defect enhanced more bone regeneration than β-TCP/Col and HA/Col..
78. Shinji Kamakura, Kazuo Sasaki, Yoshitomo Honda, Takahisa Anada, Keiko Matsui, Seishi Echigo, Osamu Suzuki, Dehydrothermal treatment of collagen influences on bone regeneration by octacalcium phosphate (OCP) collagen composites, Journal of Tissue Engineering and Regenerative Medicine, 10.1002/term.58, 1, 6, 450-456, 2007.09, We have engineered a scaffold constructed of synthetic octacalcium phosphate (OCP) and collagen composites (OCP-collagen) and report that OCP-collagen significantly enhanced bone regeneration more than the implantation of OCP. We hypothesized that the dehydrothermal treatment (DHT) during the fabrication of OCP-collagen might influence bone regeneration by OCP-collagen. To examine this hypothesis, bone regeneration by the implantation of OCP-collagen with DHT [OCP/Col(+)] was compared with that by OCP-collagen without DHT [OCP/Col(-)]. It was confirmed that both OCP/Col(+) and OCP/Col(-) contained the characteristics of OCP structure in X-ray diffraction. Before implantation, calcium deposition derived from OCP was observed within the collagen of both OCP/Col(+) and OCP/Col(-) by undecalcified histological sections. OCP/Col(+) or OCP/Col(-) was implanted into the critical-sized defects in rat crania. Radiographic and histological examination was performed and the percentage of newly formed bone (n-Bone%) in the defect was determined by a histomorphometrical analysis. N-Bone% treated with OCP/Col(+) was significantly higher than that with OCP/Col(-) at 4 and 12 weeks after implantation, because fast degradation of the implanted collagen of OCP/Col(-) elicited disappearance of the scaffold for bone regeneration. The stiffness of the calcified collagen in OCP-collagen would be more important than the existence of calcified collagen to enhance the bone regeneration by OCP-collagen composites. The present study suggests that the dehydrothermal treatment would influence effective bone regeneration by OCP-collagen..
79. Naohiko Shimada, Cevayir Coban, Yoichi Takeda, Masami Mizu, Jusaku Minari, Takahisa Anada, Yuichi Torii, Seiji Shinkai, Shizuo Akira, Ken J. Ishii, Kazuo Sakurai, A polysaccharide carrier to effectively deliver native phosphodiester CpG DNA to antigen-presenting cells, Bioconjugate Chemistry, 10.1021/bc0700178, 18, 4, 1280-1286, 2007.07, Oligodeoxynucleotides containing unmethylated CpG sequences (CpG DNAs) activate the vertebrate innate immune system via toll-like receptor 9 (TLR-9). Although CpG DNA is a promising immunotherapeutic agent, its short circulation time in biological fluids due to nuclease is the major drawback. This paper proposes that a natural polysaccharide called schizophyllan (SPG) can be used as an effective CpG DNA carrier because SPG can complex with CpG DNA and the resultant complex shows the nuclease resistance of the bound DNA. In order to increase cellular uptake in vitro, we chemically attached spermine, cholesterol, arginine octamer, or RGD peptide to SPG. The complexes made of the chemically modified SPG and CpG DNA having a phosphorothioate (PS) or phosphodiester (PO) backbone led to increased secretion of cytokines of about 4- to 15-fold, compared with the uncomplexed dose. Furthermore, when PO CpG DNA was complexed with unmodified SPG, the IL-12 level increased by almost 3- to 11-fold compared with the naked dose. The PO CpG DNA/unmodified SPG complex data suggested that unmodified SPG might effectively deliver PO in vivo due to the electrically neutral nature of unmodified SPG. When the complexed CpG DNAs were injected intraperitoneally, a large amount of IL-12 production was observed compared with the uncomplexed material. Both in vivo and vitro assays indicated that the SPG complex may be of use for CpG DNA therapy..
80. Shinji Kamakura, Kazuo Sasaki, Yoshitomo Honda, Takahisa Anada, Osamu Suzuki, Octacalcium phosphate combined with collagen orthotopically enhances bone regeneration, Journal of Biomedical Materials Research - Part B Applied Biomaterials, 10.1002/jbm.b.30531, 79, 2, 210-217, 2006.11, Octacalcium phosphate (OCP) is resorbable bone regenerative material, but its brittleness makes it difficult to maintain its shape without restraint. We have engineered a scaffold constructed of synthetic OCP and porcine collagen sponge (OCP/Collagen) and investigated whether OCP/Collagen composite could improve bone regeneration. To examine this hypothesis, bone regeneration by the implantation of OCP/Collagen was compared with those by OCP and collagen. Radiographic and histological examination was performed and the percentage of newly formed bone (n-Bone%) in the defect was determined by a histomorphometrical analysis. OCP/Collagen, OCP, or collagen was implanted into the critical-sized defects in rat crania and fixed at 2, 4, or 8 weeks after implantation. OCP/Collagen improved the handling performance than the granules of OCP, and synergistically enhanced the bone regeneration beyond expectation, which were composed of bone nucleation by OCP and cell infiltration by collagen. Histomorphometrical analysis showed that n-Bone % ± standard error treated with OCP/Collagen (48.4 ± 5.14) was significantly higher than those with OCP (27.6 ± 4.04) or collagen (27.4 ± 5.69) in week 8. The present study suggests that the combination OCP with collagen elicited the synergistic effect for bone regeneration..
81. Yoshitomo Honda, Takahisa Anada, Shinji Kamakura, Masanori Nakamura, Shunji Sugawara, Osamu Suzuki, Elevated extracellular calcium stimulates secretion of bone morphogenetic protein 2 by a macrophage cell line, Biochemical and Biophysical Research Communications, 10.1016/j.bbrc.2006.05.013, 345, 3, 1155-1160, 2006.07, It has been suggested that macrophages and multinucleated giant cells are responsible for phagocytosis of resorbable calcium phosphate (CaP) compounds implanted in bone defects. However, function of macrophages around the CaP, if continuously exposed to various concentration of extracellular calcium ions ([Ca2+]o), is still unknown. The present study showed that when resorbable octacalcium phosphate was implanted in mouse calvaria, macrophage-like cells were observed around the implant during bone formation. Then, experiments were designed to investigate whether secretion of bone morphogenetic protein 2 (BMP-2) is enhanced by [Ca2+]o in a macrophage cell line (J774A.1) in vitro. The mRNA expression and the secretion of BMP-2 in J774A.1 cells were significantly increased when incubated in the medium with [Ca2+]o up to 14 mM. The promotion of mRNA expression was maintained even when incubated with a small amount of minute CaP crystals. The present results suggest that [Ca2+]o above physiological concentration may stimulate macrophages to induce osteogenic cytokine, such as BMP-2, for bone formation by osteoblast..
82. Takahisa Anada, Naoko Okada, Jusaku Minari, Ryouji Karinaga, Masami Mizu, Kazuya Koumoto, Seiji Shinkai, Kazuo Sakurai, CpG DNA/zymosan complex to enhance cytokine secretion owing to the cocktail effect, Bioorganic and Medicinal Chemistry Letters, 10.1016/j.bmcl.2005.11.070, 16, 5, 1301-1304, 2006.03, Zymosan, classified among β-(1→3)-d-glucans, is produced from the cell wall of yeast and well known to induce proinflammatory cytokines when ingested by immune cells. We found that zymosan forms a complex with immunostimulatory CpG DNA, where both zymosan and CpG DNA can induce cytokine secretion according to the different mechanisms (i.e., recognized by different receptors). The complex activated macrophages and induced cytokine secretion, more efficiently than separate administration of zymosan or CpG DNA. Microscopic observation showed that this increment of the cytokine secretion can be explained by the fact that zymosan and zymosan/CpG DNA complex are up-taken more than naked CpG DNA. Additionally, existence of two different immunostimulants in the same cells may enhance the immunoresponse. This report presents a new strategy to construct a delivering vehicle for CpG DNA and to enhance its activity with the 'cocktail effect' of the two immunostimulants..
83. Ryouji Karinaga, Takahisa Anada, Jusaku Minari, Masami Mizu, Kazuya Koumoto, Junji Fukuda, Kohji Nakazawa, Teruaki Hasegawa, Munenori Numata, Seiji Shinkai, Kazuo Sakurai, Galactose-PEG dual conjugation of β-(1→3)-D-glucan schizophyllan for antisense oligonucleotides delivery to enhance the cellular uptake, Biomaterials, 10.1016/j.biomaterials.2005.08.023, 27, 8, 1626-1635, 2006.03, Antisense oligonucleotides (AS ODNs) are applied to silence a particular gene, and this approach is one of the potential gene therapies. However, naked oligonucleotides are easy to be degraded or absorbed in biological condition. Therefore, we need a carrier to deliver AS ODNs. This paper presents galactose moieties that were conjugated to the side chain of SPG to enhance cellular ingestion through endocytosis mediated by asialoglycoprotein receptor specifically located on parenchymal liver cells. We introduced galactose with two types of chemical bonds; amide and amine, and the amine connection showed lower ingestion and more toxicity than the amide one. Since PEG was known to induce endocytosis escape, we combined PEG and galactose aiming to provide both cellular up-take and subsequent endocytosis escape. We designed lactose or galactose moieties to attach to the end of the PEG chain that connects to the SPG side chain. When the PEG had the molecular weight of 5000-6000, the antisense effect reached the maximum. We believe that this new type of galactose and PEG dual conjugation broaden the horizon in antisense delivery..
84. Kazuya Koumoto, Masami Mizu, Takahisa Anada, Takeshi Nagasaki, Seiji Shinkai, Kazuo Sakurai, Cholesterol-appended β-(1→3)-D-glucan schizophyllan for antisense oligonucleotides delivery to enhance the cellular uptake, Bulletin of the Chemical Society of Japan, 10.1246/bcsj.78.1821, 78, 10, 1821-1830, 2005.12, Schizophyllan is a natural β-(1→3)-D-glucan existing as a triple helix in water and as a single chain in dimethyl sulfoxide (DMSO), respectively. As we already reported, when a homo-polynucleotide is added to a schizophyllan solution, the single chain of schizophyllan forms a complex with the polynucleotide. One of the potential applications of this novel complex is an antisense-oligonucleotide (AS ODN) carrier. The present paper describes a modification technique that enables us to introduce a cholesterol group only to the side chain of schizophyllan. We prepared four cholesterol-appended schizophyllans with different modification levels. Using these compounds, we made complexes and carried out an in vitro antisense assay, administrating a phosphorothioate AS ODN to the several cell lines to depress their c-myb mRNA. When we used 2.2-2.3 mol% modified schizophyllan as the carrier, the antisense effect was most enhanced among others. The addition of β-cyclodextrin improved the complexation ability as well as the up-take for highly modified samples. Furthermore, the cytotoxicity for these modified schizophyllan samples was negligibly as small as the natural (unmodified) schizophyllan. The present work has thus clarified that schizophyllan can act as a new potential candidate for AS ODN carriers..
85. Takahisa Anada, Ryouji Karinaga, Kazuya Koumoto, Masami Mizu, Takeshi Nagasaki, Yoshio Kato, Kazunari Taira, Seiji Shinkai, Kazuo Sakurai, Linear double-stranded DNA that mimics an infective tail of virus genome to enhance transfection, Journal of Controlled Release, 10.1016/j.jconrel.2005.08.019, 108, 2-3, 529-539, 2005.11, Our previous work showed that a natural β-(1 → 3)-d-glucan schizophyllan (SPG) can form a stable complex with single-stranded oligonucleotides (ssODNs). When protein transduction peptides were attached to SPG and this modified SPG was complexed with ssODNs, the resultant complex could induce cellular transfection of the bound ODNs, without producing serious cytotoxicity. However, no technique was available to transfect double-stranded DNAs (dsDNA) or plasmid DNA using SPG. This paper presents a new approach to transfect dsDNA, showing preparation and transfection efficiency for a minimal-size gene having a loop-shaped poly(dA)80 on both ends. This poly(dA) loops of dsDNA can form a complex with SPG. An siRNA-coding dsDNA with the poly(dA) loop was complexed with Tat-attached SPG to silence luciferase expression. When LTR-Luc-HeLa cells that can express luciferase under the control of the LTR promoter were exposed to this complex, the expression of luciferase was suppressed (i.e., RNAi effect was enhanced). Cytotoxicity studies showed that the Tat-SPG complex induced much less cell death compared to polyethylenimine, indicating that the proposed method caused less harm than the conventional method. The Tat-SPG/poly(dA) looped dsDNA complex had a structure similar to the viral genome in that the dsDNA ends were able to induce transfection and protection. The present work identifies the SPG and poly(dA) looped minimum-sized gene combination as a candidate for a non-toxic gene delivery system..
86. Ryouji Karinaga, Kazuya Koumoto, Masami Mizu, Takahisa Anada, Seiji Shinkai, Kazuo Sakurai, PEG-appended β-(1→3)-D-glucan schizophyllan to deliver antisense-oligonucleotides with avoiding lysosomal degradation, Biomaterials, 10.1016/j.biomaterials.2004.11.031, 26, 23, 4866-4873, 2005.08, Schizophyllan is a natural β-(1→3)-d-glucan existing as a triple helix in water and as a single chain in dimethylsulfoxide (DMSO). As we already reported, when a homo-polynucleotide [e.g., poly(dA) or poly(C)] is added to the schizophyllan/DMSO solution and subsequently DMSO is exchanged for water, the single chain of schizophyllan forms a complex with the polynucleotide. One of the potential applications for this novel complex is an antisense- oligonucleotide (AS ODN) carrier. The present paper describes a modification technique that enabled us to introduce PEG only to the side chain of schizophyllan. This technique consisted of periodate oxidation of the glucose side chain and subsequent reaction between methoxypolyethylene glycol amine and the formyl terminate, followed by reduction with NaBH4. Subsequently, we made a complex from PEG-appended schizophyllan and an AS ODN sequence, and carried out an in vitro antisense assay, administrating the AS ODN complex to depress A375 c-myb mRNA of A375 melanoma cell lines. The PEG-SPG/AS ODN complex showed more enhanced antisnese effect than naked AS ODN dose, i.e., the same level as that of RGD-appended SPG. Here, the RGD system has been shown one on the most effective AS ODN carrier (Science 261 (1993) 1004-1012). When we added nigericin to the assay system, the antisense effect was not affected in the PEG-SPG system, on the other hand, it was almost eliminated in the RGD system. Nigericin is well known to interrupt transport from endosome to lysosome. Therefore, the difference between the PEG and RGD complexes indicates that, in the PEG system, AS ODN was able to escape from lysosomal degradation. The present work has thus proposed a new strategy to delivery AS ODN using schizophyllan as a new carrier..
87. Ryouji Karinaga, Kazuya Koumoto, Jusaku Minari, Masami Mizu, Takahisa Anada, Kazuo Sakurai, Seiji Shinkai, Competition between polysaccharide/polynucleotide complexation vs., polynucleotide hybridization; salt concentration dependence of the reaction direction, e-Journal of Surface Science and Nanotechnology, 10.1380/ejssnt.2005.38, 3, 38-45, 2005.02, Schizophyllan is a natural β-(1→3)-D-glucan existing as a triple helix in water and as a single chain in dimethylsulfoxide (DMSO), respectively. As we already reported, when a homo-polynucleotide [ex. poly (A) or poly(dA)] was added to the schizophyllan/DMSO solution and subsequently DMSO was exchanged for water, the single chain of schizophyllan formed a complex with the polynucleotide. In this paper, using an adenine-thymine (or uracil) double strand as a model system, we explored whether s-SPG can unzip the polynucleotide duplexes, examining the competition between the schizophyllan/polynucleotide complexation and the polynucleotide hybridization with circular dichroism and fluorescence spectroscopy at various NaCl concentrations. The unzipping reaction happened for the hetero-duplexes (i.e., RNA-DNA duplexes) at low salt concentrations, on the other hand, it was not observed for the homo-duplexes at all the salt concentrations. When we compared dissociation temperatures, the unzipping reaction of the homo-duplexes was expected at low salt concentrations. This discrepancy between the experimental results and the expectation, as well as the different behavior between the hetero- and homo- duplexes, can be ascribed to the difference in the activation energy of intermediate states..
88. Takahisa Anada, Kazuo Sakurai, Seiji Shinkai, A new polynucleotide-polysaccharide complex and its application to functional oligonucleotide delivery, Trends in Glycoscience and Glycotechnology, 10.4052/tigg.17.49, 17, 94, 49-57, 2005.01, Schizophyllan (SPG) is a polysaccharide that belongs to the β-(1-3) glucan family and adopts a triple-helixical conformation in water. When SPG dissolves in dimethyl sulfoxide (DMSO), the triple helix is dissociated to three random coils. When water is added to the DMSO solution (renaturation), the single chain of SPG (s-SPG) collapses owing to both hydrophobic interaction and hydrogen bonding formation, and eventually aggregation takes place with increasing the water content. When this renaturation process is carried out in a mixture containing s-SPG and a single-stranded polynucleotide, a macro-molecular complex is formed, consisting of two s-SPG chains and one polynucleotide chain. This novel complexation was examined with circular dichroism, UV spectroscopy, and gel electrophoresis. We applied to this complex to deliver functional oligonucleotides such as antisense DNA and CpG motifs. The biological functions of these oligonucleotides were extremely enhanced owing to the complexation..
89. Ryouji Karinaga, Masami Mizu, Kazuya Koumoto, Takahisa Anada, Seiji Shinkai, Taro Kimura, Kazuo Sakurai, First observation by fluorescence polarization of complexation between mRNA and the natural polysaccharide schizophyllan, Chemistry and Biodiversity, 10.1002/cbdv.200490054, 1, 4, 634-639, 2004.12, Schizophyllan is a natural β-(1 → 3)-D-glucan that exists as a triple helix in H2O and as a single chain in dimethylsulfoxide (DMSO) or basic solution (pH >13). As we have already reported, when a homopolynucleotide (e.g., poly(dA), poly(A), or poly(C)) is added to a schizophyllan/DMSO solution, and, subsequently, DMSO is exchanged for H 2O, the single chain of schizophyllan forms a complex with the polynucleotide. Since eukaryotic mRNAs have poly(A) tails, we hypothesized that schizophyllan can bind to mRNA by interacting with this tail. However, we have not yet observed complexation between schizophyllan and mRNA after exchanging DMSO for H2O. In this report, we show that the complexation can be accelerated when the solution pHis changed from 13 to 7-8 in the presence of schizophyllan and polynucleotides. By this approach, we found that schizophyllan forms a complex with a yeast mRNA..
90. Kazuya Koumoto, Ryouji Karinaga, Masami Mizu, Takahisa Anada, Kazuo Sakurai, Toyoki Kunitake, Seiji Shinkai, Removal of the side-chain glucose groups from schizophyllan improves the thermal stability of the polycytidylic acid complexes under the physiological conditions, Biopolymers, 10.1002/bip.20148, 75, 5, 403-411, 2004.12, Thermal stabilization of the complex between polycytidylic acid [poly(C)] and the modified schizophyllan (SPG) whose hydrophilic side-chain glucose groups are selectively removed utilizing mild Smith-degradation has been investigated. With the decrease in the side-chain glucose groups of schizophyllan, the complex with poly(C) can be considerably stabilized compared with unmodified SPG; for example, the Tm value after the removal of the side-chain glucose groups from 33.3 (unmodified) to 1.0 is enhanced by 14°C. In addition, the thermal stabilization effect is even operative under the physiological conditions ([NaCl] = 0.15 mol dm-3). This effect is exerted owing to the construction of the hydrophobic atmosphere around the complex. Although schizophyllan lost the side-chain glucose groups, it still kept the protection effect of the bound poly(C) chain against RNaseA-mediated hydrolysis as observed for unmodified schizophyllan. The assessment of the cytotoxicity for A375:human malignant melanoma, and HL60:human promyelocytic leukemia revealed that the modified schizophyllan scarcely increases the cytotoxicity. These results indicate that the present modification for schizophyllan is of great significance in a viewpoint to develop the practical gene carriers operative even under the physiological conditions..
91. Takahisa Anada, Hideshi Matsunaga, Ryouji Karinaga, Kazuya Koumoto, Masami Mizu, Koji Nakano, Seiji Shinkai, Kazuo Sakurai, Proposal of new modification technique for linear double-stranded DNAs using the polysaccharide schizopyllan, Bioorganic and Medicinal Chemistry Letters, 10.1016/j.bmcl.2004.08.043, 14, 22, 5655-5659, 2004.11, A poly(dA) tailed double-stranded DNA and its polysaccharide complexes are reported. A natural polysaccharide schizophyllan (SPG) has been known to form a stable complex with poly(dA). We attached a poly(dA) 80 tail to the both ends of a linear double-stranded DNA, which had been prepared from a plasmid DNA vector. The poly(dA) tailed DNA verified to form complex with SPG by gel electrophoresis and atomic force microscopy (AFM). AFM images indicated that the complexes exhibit a dumbbell-like architecture, that is, quite similar to that of adenovirus genome. The complex demonstrated excellent exonuclease resistance, probably because of the protection effect by SPG complexation..
92. Masami Mizu, Kazuya Koumoto, Takahisa Anada, Takahiro Matsumoto, Munenori Numata, Seiji Shinkai, Takeshi Nagasaki, Kazuo Sakurai, A polysaccharide carrier for immunostimulatory CpG DNAs to enhance cytokine secretion, Journal of the American Chemical Society, 10.1021/ja031978+, 126, 27, 8372-8373, 2004.07, A β-(1 → 3)-D-glucan schizophyllan (SPG) forms a stoichiometric complex with some polynucleotides. This communication describes our attempt to apply the SPG complex to deliver CpG DNA to endosomes to enhance cytokine secretion. To increase cellular uptake, we introduced spermine, arginine-glycine-aspartic acid tripeptide, octaarginine, or cholesterol to the SPG side chain. The chemically modified SPG showed essentially no cytotoxicity. When CpG DNA complex made therefrom was exposed to macrophages, dramatic enhancement in the cytokine secretion was observed. It increased 5-10 times from the naked dose and 100 times from the background. This performance promises that SPG can be an excellent carrier for CpG DNA..
93. Masami Mizu, Kazuya Koumoto, Takahisa Anada, Ryouji Karinaga, Taro Kimura, Takeshi Nagasaki, Seiji Shinkai, Kazuo Sakurai, Enhancement of the antisense effect of polysaccharide-polynucleotide complexes by preventing the antisense oligonucleotide from binding to proteins in the culture medium, Bulletin of the Chemical Society of Japan, 10.1246/bcsj.77.1101, 77, 6, 1101-1110, 2004.06, Schizophyllan is a β-(1 → 3)-D-glucan existing as a triple helix in water and as a single chain in dimethyl sulfoxide (DMSO), respectively. As we already reported, when a homo-phosphodiester-polynucleotide is added to the schizophyllan/DMSO solution and, subsequently, DMSO is exchanged for water, the single chain of schizophyllan (s-SPG) forms a complex with the polynucleotide. In this paper, we report that phosphorothioate oligonucleotides can form a complex with s-SPG in the same manner as phosphodiester oligonucleotides. We carried out an in vitro antisense assay combining melanoma cell lines and a phosphorothioate antisense oligonucleotide (AS ODN) to depress c-myb mRNA. We found that the AS ODN bound to the complex reduces cell growth more efficiently than that of naked AS ODN by preventing the AS ODN from binding to albumin in the culture medium and being hydrolyzed..
94. Masami Mizu, Kazuya Koumoto, Takahisa Anada, Kazuo Sakurai, Seiji Shinkai, Antisense oligonucleotides bound in the polysaccharide complex and the enhanced antisense effect due to the low hydrolysis, Biomaterials, 10.1016/j.biomaterials.2003.11.008, 25, 15, 3117-3123, 2004.01, Schizophyllan is a β-(1→3)-D-glucan and can form a novel complex with some single-chains of DNAs. As the preceding paper revealed, the polynucleotide bound in the complex is more stable to nuclease-mediated hydrolysis than the polynucleotide itself (i.e., naked polynucleotide). This paper examined possibility to apply this complex to an antisense DNA carrier, using an in vitro (cell-free) transcription/translation assay. In this assay, we used a plasmid DNA coding a green fluorescence protein (GFP) and an antisense DNA designed to hybridize the ribosome-binding site in the GFP-coded mRNA. When the antisense DNA was administered as the complex, a lower GFP expression efficiency (or higher antisense effect) is observed over naked DNA. This is because the antisense DNA in the complex is protected from the attack of deoxyribonuclease. When exonuclease I, which specifically hydrolyzes single DNA chains, was present in the GEP assay system, the antisense effect was not changed for the complex while being weakened in the naked antisense DNA system. These results imply that the exonuclease I cannot hydrolyze the antisense DNA in the complex, while it can hydrolyze naked DNA to reduce its antisense effect..
95. Takahiro Matsumoto, Munenori Numata, Takahisa Anada, Masami Mizu, Kazuya Koumoto, Kazuo Sakurai, Takeshi Nagasaki, Seiji Shinkai, Chemically modified polysaccharide schizophyllan for antisense oligonucleotides delivery to enhance the cellular uptake efficiency, Biochimica et Biophysica Acta - General Subjects, 10.1016/j.bbagen.2003.10.019, 1670, 2, 91-104, 2004.01, Schizophyllan is a natural β-(1→3)-D-glucan existing as a triple helix in water and as a single chain in dimethylsulfoxide (DMSO), respectively. As we already reported, when some homo-phosphodiester polynucleotide (for example, poly(dA) or poly(C)) is added to the schizophyllan/DMSO solution and subsequently DMSO is exchanged for water, the single chain of schizophyllan forms a complex with the polynucleotide. Furthermore, we have already demonstrated that one of the potential applications of this novel complex is an antisense-oligonucleotide (AS ODN) carrier. This work describes a versatile and universal modification technique which enables us to introduce various functional groups only to the side chain of schizophyllan. This technique consists of periodate oxidation of the glucose side chain (it does not react with the main chain because of the absence of the 1,2-diol group in β-(1→3)-glucan) and subsequent introduction of the functional groups into the formyl terminate. In the present work, the introduced functional groups were spermine, octa-arginine (R8), arginine-glycine-aspartic acid tripeptide (RGD) and some amino or α-amino acid compounds. Using these compounds, we made the complexes and carried out an in vitro antisense assay for them, administrating a phosphorothioate AS ODN to the melanoma A375 or leukemia HL-60 cell lines to depress their c-myb mRNA. When we used the R8 or RGD modified schizophyllan as the antisense carrier, the antisense effect was most enhanced among others. Their superiority can be ascribed to enhancement of endocytosis due to these functional peptides. Furthermore, the cytotoxicity for these two modified schizophyllans was negligibly as small as the natural (unmodified) schizophyllan. One of the peculiar features of our system is that the complex (i.e., carrier+AS ODN) is charged negatively in total, which is different from the conventional systems. The present work has thus clarified that schizophyllan can act as a new potential candidate for AS ODN carriers..
96. Takahisa Anada, Kazuya Koumoto, Masami Mizu, Ryouji Karinaga, Kazuo Sakurai, Seiji Shinkai, Microanalysis of Nucleic Acids Using the Limulus G Test, Analytical Sciences, 10.2116/analsci.19.1567, 19, 12, 1567-1568, 2003.12, The limulus G test has been used as a quantitative analysis of (1→3)-β-D-glucans, including schizophyllan (SPG) and curdlan. The present work extended the limulus G test to detect polynucleotide/SPG complexes. The complex showed an extremely sensitive response to the test, compared with SPG itself. The minimum concentration of the complex to show the response is almost 10-times as small as that of SPG itself, indicating the possibility to detect (1→3)-β-D-glucans or/and polynucleotides on the pico gram/ml scale..
97. Masaharu Murata, Wataru Kaku, Takahisa Anada, Yoshikuni Sato, Takeshi Kano, Mizuo Maeda, Yoshiki Katayama, Novel DNA/polymer conjugate for intelligent antisense reagent with improved nuclease resistance, Bioorganic and Medicinal Chemistry Letters, 10.1016/j.bmcl.2003.08.062, 13, 22, 3967-3970, 2003.11, Antisense technology provides an effective strategy to inhibit synthesis of the gene product. We prepared a novel antisense reagent comprised of oligodeoxynucleotides (ODN) and a thermo responsive polymer, poly(N-isopropylacrylamide) (PNIPAAm). The conjugate inhibited gene expression in a dose-dependent manner. The ODN-PNIPAAm conjugate demonstrated excellent resistance to S1 nuclease. In particular, PNIPAAm-modified antisense ODN at the 3′,5′-ends of the ODN provided complete resistance against nuclease at 37°C, which is above the phase transition temperature of the PNIPAAm side chain. These characteristics of the conjugate suggest it may have potential for use in a new gene delivery system as part of an antisense strategy..
98. Masaharu Murata, Wataru Kaku, Takahisa Anada, Yoshikuni Sato, Mizuo Maeda, Yoshiki Katayama, Temperature-dependent Regulation of Antisense Activity Using a DNA/poly(N-isopropylacrylamide) Conjugate, Chemistry Letters, 10.1246/cl.2003.986, 32, 11, 986-987, 2003.11, We prepared a novel antisense reagent comprising of oligodeoxynucleotides (ODNs) and a thermo-responsive polymer, poly(N-isopropylacrylamide) (PNIPAAm). The conjugate demonstrated stimuli-responsive regulation of gene expression via conformational change of the polymer chain..
99. Masaharu Murata, Wataru Kaku, Takahisa Anada, Nobuaki Soh, Yoshiki Katayama, Mizuo Maeda, Thermo responsive DNA/polymer conjugate for intelligent antisense strategy, Chemistry Letters, 10.1246/cl.2003.266, 32, 3, 266-267, 2003.03, The conjugate between 20-mer antisense oligodeoxynucleotides (ODN) and thermo-responsive polymer, poly(N-isopropylacrylamide) (PNIPAAm), was prepared. Hybridization reaction between the conjugate and their target RNA was modulated by conformational change of polymer moiety depending on temperature condition..
100. Takahisa Anada, Masako Ogawa, Hisashi Yokomizo, Yoshihisa Ozaki, Tohru Takarada, Yoshiki Katayama, Mizuo Maeda, Oligodeoxynucleotide-modified capillary for electrophoretic separation of single-stranded DNAs with a single-base difference, Analytical Sciences, 10.2116/analsci.19.73, 19, 1, 73-77, 2003.01, We describe here a method of affinity capillary electrophoresis in which oligodeoxynucleotide (ODN) was immobilized onto the inner surface of the capillary. The immobilized ODN functioned successfully as an affinity ligand for sequence-based DNA separation. Six- or 12-mer ODN with a sequence complementary to one of the c-K-ras gene was used as an immobilized ligand. When the 12-mer ODN was used, the detection peak for the complementary ODN disappeared selectively, while the single-base mutant was detected as usual. In contrast, when the 6-mer ODN was used as the affinity ligand with a mixture of the complementary ODN and its single-base mutant, it was possible to detect both as completely separate peaks. That is, the separation mode was dependent on the base number of the immobilized ODN used as an affinity ligand..
101. Takahisa Anada, Takumi Arisawa, Yoshihisa Ozaki, Tohru Takarada, Yoshiki Katayama, Mizuo Maeda, The separation of oligodeoxynucleotides having a single-base difference by affinity capillary electrophoresis using oligodeoxynucleotide-polyacrylamide conjugate, Electrophoresis, 10.1002/1522-2683(200207)23:14<2267::AID-ELPS2267>3.0.CO;2-I, 23, 14, 2267-2273, 2002.07, We have developed an affinity capillary electrophoresis for gene mutation assay. We show a method for separating a mixture of oligodeoxynucleotides having single-base difference using oligodeoxynucleotide-polyacrylamide conjugate. Because the conjugate behaved similarly to nonionic polyacrylamide in terms of migration rate, it can be used as a pseudoimmobilized affinity ligand in a polyacrylamide-coated capillary. Oligodeoxynucleotide having an oncogene sequence and a version with one base substituted were completely separated by this method. The magnesium-ion concentration was found to be a keyfactor in achieving efficient separation of oligodeoxynucleotides with the same chain length..
102. Tatsuya Morozumi, Takahisa Anada, Hiroshi Nakamura, New fluorescent "off-on" behavior of 9-anthryl aromatic amides through controlling the twisted intramolecular charge transfer relaxation process by complexation with metal ions, Journal of Physical Chemistry B, 105, 15, 2923-2931, 2001.04, New fluorophores based on linear polyether N,N′-[oxybis(3-oxapentamethylenoxy)-2-phenyl]bis(9-anthracenecarbonamide) (3) and its analogues (2 and 4) have been synthesized, and their complexation properties with various alkali-metal and alkaline-earth-metal ions were investigated by fluorescence, UV, and 1H NMR spectroscopies. In the absence of metal ion, 2-4 showed almost no fluorescence emission (fluorescence quantum yield φ = 0.0003, fluorescence "off state) since twisted intramolecular charge transfer (TICT) occurred through the amide bond. However, 2-4 demonstrated a significant increase in fluorescence intensity around 430 nm upon complexation with alkaline-earth-metal ions. In the case of 4·Ca2+, a large enhancement effect on the fluorescence quantum yield (φ = 0.014, [Ca2+]/[4] = 5, φ4-ca2+free4 -42, fluorescence "on" state) was observed. This "off-on" fluorescence characteristic was originated from the cooperative strong binding mode between the carbonyl group and the ethylenoxy moiety for alkaline-earth-metal ions, resulting in effective inhibition of photoinduced TICT relaxation. Fluorophores 2-4 formed a 1:1 complex (the order of the complex formation constants was Ca2+ > Sr2+ ≈ Ba2+) and showed no considerable spectral changes upon complexation with alkali-metal ions and Mg2+. The 1H NMR study on 2-4 and their complexes indicated that free 2-4 formed a helical structure. After complexation, the conformational change of 2 from a helical structure to a semicircular structure was observed. The pseudocyclic form was supported as the complex structure of 3, whereas the large conformational change of 4 was not observed after the addition of metal ions..