Papers by Author: Mamoru Aizawa

Abstract: We previously showed enhanced osteoblast differentiation by culturing cells in apatite-fiber scaffold (AFS). The well-developed a-surface of the apatite fibers provides favorable structural features and chemical interactions with the cells, and the scaffold appears to affect cell differentiation. AFS was used here to study its utility for soft-tissue engineering. An embryonal carcinoma cell line, P19.CL6, was cultured in AFS, and the expression and phosphorylation of a gap-junction protein, connexin 43 (Cx43), during cell proliferation and differentiation was examined. We show that treatment with dimethyl sulfoxide appears to induce a change in the isoform composition of Cx43 under the control condition, but not in AFS. We also show that serum starvation induces the phosphorylation of Ser 368 of Cx43 only in functionally mature cells.

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

Abstract: Hydroxyapatite (HAp) is one of components of bone and teeth, and has an osteoconductivity. Thus, the HAp has been used as biomaterials for bone graftings. We have succeeded in development of the novel chelate-setting calcium-phosphate cement (CPC) using pure HAp particles surface-modified with inositol phosphate (IP6). While, biological apatite presented in bone and teeth of mammals contains various ions: Na⁺, K⁺, Mg²⁺, Cl^-, F^- and CO₃^2-, in addition to Ca²⁺ and PO₄^3- ions. In this work, in order to create the chelate-setting CPC with enhanced osteoconductivity, the above-mentioned biological apatite powder (hereafter, bone HAp), instead of pure HAp, was used as a starting powder for fabrication of the chelate-setting cement. The biocompatibility of the resulting chelate-setting bone HAp cement (hereafter, IP6-bone HAp cement) was examined using a rabbit’s tibia model. When the living reaction to hard tissue was histologically examined after 4 weeks implantation, we could observe that newly-formed bone directly bonded to the surface of the specimen. The newly-formed bone was also present around the cement specimen. The amounts of newly-formed bone around IP6-bone HAp cement was about 1.5 times those around IP6-pure HAp cement without bone minerals. The above findings demonstrate that the present IP6-bone HAp cements are one of the promising candidates as novel CPC with enhanced osteoconductivity.

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

Abstract: We have successfully developed the apatite-fiber scaffold (AFS) with enhanced mechanical porosity for tissue engineering of bone and liver via two routes: i) use of two type of carbon beads with diameter of ~150 μm and ~20 μm and following ii) uniaxial pressing of the green compacts. Our Aim is to add vascular formation ability into the above AFS in order to maintain the regenerated tissues for a long time. In the present study, the AFSs with various porosities (68±2.4, 85±1.5, 89±0.6, 92±1.0%) were fabricated, and then loaded with vascular endothelial growth factor (VEGF). Drug release from VEGF-loaded AFSs with various porosities was examined by immersing them into phosphate buffer. The AFSs with the highest porosity (92%) could be released with the most VEGF among examined AFSs. In addition, we carried out preliminary study for the compatibility of vascular endothelial cells, M1 cells established by Matsuura et al. to the VEGF-loaded AFS (porosity: 92%), in order to account for the vascular formation into the pore of the AFS. The numbers of M1 cells cultured in/on the VEGF-loaded AFS were about 1.5 times that of VEGF-free AFS over a period of cell culture. These results demonstrate that the VEGF-loaded AFS with enhanced mechanical property have a good compatibility to the M1 cells as a model of vascular endothelial cells.

Abstract: We have successfully developed porous apatite-fiber scaffolds (AFSs) which have three-dimensional (3D) inter-connected pores; subsequently, we have clarified that the AFSs have an excellent bioactivity on the basis of both in vitro and in vivo evaluations. In addition, we have reconstructed the tissue-engineered bone with 3D structure through 3D-cell culture of mesenchymal stem cells derived from rat bone marrow (RBMC) using the AFS settled into the radial-flow bioreactor (RFB), and examined effect of flow rate of medium in the RFB on the differentiation of osteoblasts in tissue-engineered bone. Aim in the present work is to establish of the optimal conditions of flow rate in this construction method of 3D tissue-engineered bone. The flow rates were set to 0.4, 1.3, 6.3, 11.5 and 16.5 cm³min^-1; tissue-engineered bones cultured by the individual flow rates are defined as bones#1~#5. The level of differentiation of osteoblasts in all the bones#1~#5 was examined by determining the content of two kinds of differentiation maker into osteoblast, alkaline phosphatase (ALP) for initial/middle stage and osteocalcin (OC) for late stage. The ALP activity normalized for DNA content of bone#3 showed the highest value among all of them. Moreover, the OC amount normalized for DNA content of bone#3 also indicated the highest among all the examined samples. These results demonstarate that the flow rate of 6.3 cm³min^-1 may promote the differentiation into osteoblast. In conclusion, we determined that this flow rate was the optimal conditions for the bone regeneratrion in RFB.

Abstract: The aim of the present investigation was to examine Si release from the silicon-containing apatite fiber scaffold (Si-AFS) and the biocompatibility of the Si-AFS. We have successfully synthesized silicon-containing apatite fibers (Si-AF) by a homogenous precipitation method. Three-dimensional Si-AFS were fabricated using these Si-AFs. The concentrations of Si in the starting solution were 0 (AF) and 0.8 (0.8Si-AF) mass%. The 0.8Si-AFS1000 were fabricated by firing Si-AF slurry compacts (carbon/Si-AF [w/ ratio: 10/1) at 1300 °C for 5 h. Solubility experiments were carried out in 0.05 mol/dm³ Tris-HCl buffer solutions at pH 7.30 using 0.8Si-AFS1000 (porosity: ~98%), together with Si-free AFS1000 (~98%) for 21 days. The Ca²⁺, PO₄^3- and SiO₄^4- concentrations in the solution were determined by inductively-coupled plasma atomic emission spectrometry (ICP-AES). The biocompatibility of the Si-AFS was examined in vitro using osteoblastic cell, MC3T3-E1 for 21 days. The results of the ICP-AES analysis indicated that the amount of SiO₄^4- ions released from 0.8Si-AFS1000 rapidly increased at 1 day, and then the released SiO₄^4- ions remained constant over a period for 21 days. The cells seeded on/in the 0.8Si-AFS1000 well-proliferated as compared to those on/in the AFS1000. Consequently, we can conclude that the 0.8Si-AFS offers as a potential novel scaffold material, creating a three-dimensional cell culture environment.

Abstract: Heart disease is the second most common cause of mortality in Japan. Most cases of late stage heart failure can only be effectively treated by a heart transplant. Cardiac tissue engineering is emerging both as a new approach for improving the treatment of heart failure and for developing new cardiac drugs. Apatite-fiber scaffold (AFS) was originally designed as a substitute material for bone. AFS contains two sizes of pores and is appropriate for the three dimensional proliferation and differentiation of osteoblasts. To establish engineered heart tissue, a pluripotent embryonal carcinoma cell line, P19.CL6, was cultured in AFS. P19.CL6 cells seeded into AFS proliferated well. Generally, cardiac differentiation of P19.CL6 cells is induced by treating suspension-cultured cells with dimethyl sulfoxide (DMSO), after which the cells form spheroids. However, our results showed that P19.CL6 cells cultured in AFS differentiated into myocytes without forming spheroidal aggregates, and could be cultured for at least one month. Thus, we conclude that AFS is a good candidate as a scaffold for cardiac tissue engineering.

Abstract: We have developed a chelate-setting apatite cement. Synthesized hydroxyapatite (HAp) powders surface-modified with inositol hexaphosphate (IP6-HAp powder) were set by chelate-bonding with inositol hexaphosphate (IP6). Our aim is to fabricate IP6-HAp cement with anti-bacterial activity by adding lactoferrin (LF). It is known that LF has both anti-bacterial and osteoinductive activity. Gram-positive Staphylococcus aureus and Gram-negative Escherichia coli were used to examine the effect of LF on biofilm formation and localization of living and dead cells. In addition, the cell viability of MC3T3-E1 osteoblastic cells was determined. Our results show that the anti-bacterial activity of LF is not due to a bactericidal effect but to the inhibition of bacterial adhesion to surfaces. Furthermore, LF cement did not affect cell proliferation. Thus, LF cement is a candidate for bifunctional biomaterials having both anti-bacterial and osteo-conductive activity.

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