Michiyo Honda

Enhanced early osteogenic differentiation by silicon-substituted hydroxyapatite ceramics fabricated via ultrasonic spray pyrolysis route

The influence of silicon-substituted hydroxyapatite (Si-HAp) on osteogenic differentiation was assessed by biological analysis. Si-HAp was prepared by ultrasonic spray pyrolysis (USSP) technique using various amounts of Si (0, 0.8, and 1.6 mass%). Chemical analysis revealed that Si was incorporated into the hydroxyapatite (HAp) lattice with no other crystalline phase and which caused the change of crystal structure. Biological analyses showed that the Si contents affected the cell proliferation and morphology, suggesting that there is an optimal Si content for cell culture. As for differentiation, alkaline phosphatase activity and osteocalcin production of Si-HAp were higher than those of HAp. Gene expression profiles also revealed that substitution of Si (0.8 mass%) up-regulated the expression levels of osteocalcin and especially Runx2, a master gene for osteoblast development. These results suggest that incorporating Si into the HAp lattice may enhance the bioactivity, particularly during early osteoblast development.

In vitro and in vivo antimicrobial properties of silver-containing hydroxyapatite prepared via ultrasonic spray pyrolysis route

Hydroxyapatite (HAp), with its high biocompatibility and osteoconductivity, readily absorbs proteins, amino acids and other substances, which in turn favor the adsorption and colonization of bacteria. To prevent bacterial growth and biofilm formation on HAp discs, silver-containing (1-20 mol%) HAp (Ag-HAp) powders were synthesized using an ultrasonic spray pyrolysis (USSP) technique. The X-ray diffraction (XRD) peaks were very broad, indicating low crystallinity, and this induced the release of Ag(+) ions from Ag-HAp powders. In addition, a gradual increase in Ca(2+) ion release was observed. These results suggest that dissolution of Ca(2+) ion in Ag-HAp triggered the release of Ag(+) ions. The antimicrobial efficacy of Ag-HAp disc was tested against Staphylococcus aureus. Samples with Ag contents of more than 5 mol% were found to be highly effective against bacterial colonization and biofilm formation in vitro. In vivo antibacterial tests using bioluminescent strains also showed reductions in the viability of bacteria with Ag-HAp (5 mol%) discs. Biocompatibility tests using a modified Transwell® insert method showed that Ag-HAp (5 mol%) discs have negative effects on osteoblast proliferation. These results indicate that Ag-HAp (5 mol%) has effective antibacterial activity and good biocompatibility both in vitro and in vivo together with good biocompatibility, thus confirming its utility as a bactericidal material.

Specific knockdown of m-calpain blocks myogenesis with cDNA deduced from the corresponding RNAi

Fusion of mononuclear myoblast to multinucleated myotubes is crucial for myogenesis. Both mu- and m-calpain are ubiquitously expressed in most cells and are particularly abundant in muscle cells. Knockout of calpain-1 (catalytic subunit of mu-calpain) induced moderate platelet dysaggregation, preserving the normal development and growth, although knockout of calpain-2 (m-calpain) is lethal in mice. Therefore, there should be muscle-specific function of m-calpain per se. Previous methods lack direct evidence for the involvement of m-calpain, because the specific inhibitor to m-calpain has not been developed yet and the inhibition was less potent. Here, we show that screened RNA interference (RNAi) specifically blocked the m-calpain expression by 95% at both the protein and the activity levels. After transfection of adenovirus vector-mediated cDNA corresponding to the RNAi-induced short hairpin RNA, m-calpain in C(2)C(12) myoblasts was knocked down with no compensatory overexpression of mu-calpain or calpain-3. The specific knockdown strongly inhibited the fusion to multinucleated myotubes. In addition, the knockdown modestly blocked ubiquitous effects, including cell migration, cell spreading, and alignment of central stress fiberlike structures. These results may indicate that m-calpain requiring millimolar Ca(2+) level for the full activation plays specific roles in myogenesis, independent of mu-calpain, and leave us challenging problems in the future.

Antibacterial polyetheretherketone implants immobilized with silver ions based on chelate‐bonding ability of inositol phosphate: Processing, material characterization, cytotoxicity, and antibacterial properties

We developed a novel antibacterial implant by forming a hydroxyapatite (HAp) film on polyetheretherketone (PEEK) substrate, and then immobilizing silver ions (Ag(+) ) on the HAp film based on the chelate-bonding ability of inositol phosphate (IP6). First, the PEEK surface was modified by immersion into concentrated sulfuric acid for 10 min. HAp film was formed on the acid-treated PEEK via the soft-solution process using simulated body fluid (SBF), urea, and urease. After HAp coating, specimens were immersed into IP6 solution, and followed by immersion into silver nitrite solution at concentrations of 0, 0.5, 1, 5 or 10 mM. Ag(+) ions were immobilized on the resulting HAp film due to the chelate-bonding ability of IP6. On cell-culture tests under indirect conditions by Transwell, MC3T3-E1 cells on the specimens derived from the 0.5 and 1 mM Ag(+) solutions showed high relative growth when compared with controls. Furthermore, on evaluation of antibacterial activity in halo test, elution of Ag(+) ions from Ag(+) -immobilized HAp film inhibited bacterial growth. Therefore, the above-mentioned results demonstrated that specimens had both biocompatibility and strong antibacterial activity. The present coating therefore provides bone bonding ability to the implant surface and prevents the formation of biofilms in the early postoperative period.

High Molecular Weight Lectin Isolated from the Mucus of the Giant African Snail Achatina fulica

To understand better the host defense mechanisms of mollusks against pathogens, we examined the anti-microbial activity of mucus from the giant African snail Achatina fulica. Hemagglutination activity of the mucus secreted by the integument of snails inoculated with Escherichia coli was observed to increase and to cause hemagglutination of rabbit red blood cells. Purification of the snail mucus lectin by sequential column chromatography revealed that the relative molecular mass of the lectin was 350 kDa. The hemagglutination activity of the lectin was Ca2+-dependent and was inhibited by galactose. Growth arrest tests showed that the lectin did not inhibit bacterial growth, but did induce agglutination of gram-positive and gram-negative bacteria. Tissue distribution analyses using a polyclonal antibody revealed that the lectin was expressed in the tissues of the mantle collar. The lectin isolated from the mucus of the snail appeared to contribute to its innate immunity.

Topographical analyses of proliferation and differentiation of osteoblasts in micro- and macropores of apatite-fiber scaffold

A variety of calcium phosphates have been used for bone tissue-engineering applications. We developed porous hydroxyapatite (HAp) ceramics by firing green compacts consisting of spherical carbon beads and HAp fiber. The apatite-fiber scaffold (AFS) forms a three-dimensional network of fibers with two different pore sizes (micro- and macropores). In this study, we investigated cell distribution and fine cell structure in AFS by confocal laser scanning microscopy. Osteoblastic cells were permeated homogenously throughout the scaffold under static culture conditions and grew three-dimensionally in macropores of AFS. Cells penetrated into micropores when they were capable of cell-cell formations. Cell proliferation and differentiation were also evaluated by biochemical and molecular biological approaches. The expression levels of early-phase osteogenic genes in AFS increased immediately, and those of middle-phase genes were maintained during the 2-week study period. Furthermore, the expression of late-phase markers increased gradually during the incubation period. These data indicate that macropores provide sufficient space for cell growth and proliferation and that micropores facilitate cell differentiation via cell-cell networks. This study provides evidence for the effectiveness of three-dimensional culture systems comprising AFS, which mimics the microenvironment of bone cells.

Fabrication of novel biodegradable α-tricalcium phosphate cement set by chelating capability of inositol phosphate and its biocompatibility

Biodegradable α-tricalcium phosphate (α-TCP) cement based on the chelate-setting mechanism of inositol phosphate (IP6) was developed. This paper examined the effect of the milling time of α-TCP powder on the material properties of the cement. In addition, biocompatibility of the result cement in vitro using osteoblasts and in vivo using rabbit models will be studied as well. The α-TCP powders were ballmilled using ZrO2 beads in pure water for various durations up to 270 minutes, with a single-phase α-TCP obtained at ballmilling for 120 minutes. The resulting cement was mostly composed of α-TCP phase, and the compressive strength of the cement was 8.5±1.1MPa, which suggested that the cements set with keeping the crystallite phase of starting cement powder. Thecell-culture test indicated that the resulting cementswere biocompatiblematerials. In vivo studies showed that the newly formed bones increased with milling time at a slight distance fromthe cement specimens and grewmature at 24weeks, and the surface of the cementwas resorbed by tartrate-resistant acid phosphatase-(TRAP-)positive osteoclast-like cells until 24weeks of implantation. The present α-TCP cement is promising for application as a novel paste-like artificial bone with biodegradability and osteoconductivity.

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

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

Preparation of Spherical Zn-Substituted Tricalcium Phosphate Powder by Ultrasonic Spray-Pyrolysis Technique and Its Characterization

Zinc, an essential trace element, has a stimulatory effect on bone formation. To enhance the osteoconduction of implant materials such as hydroxyapatite and tricalcium phosphate TCP, zinc was incorporated into them. In this study, we have synthesized spherical zinc-substituted TCP Zn-TCP powders using ultrasonic spray-pyrolysis technique, fabricated Zn-TCP ceramics, and characterized their powder properties. Four kinds of Zn-TCPs with various amounts of zinc content were prepared, to examine the influence of zinc addition to the crystal structure of TCP and to compare the different Zn-TCPs with each other. Zn-TCP ceramics were fabricated from the resulting powders under different sintering temperatures. Zn-TCP samples were analyzed and led to the following results: the resulting powders were composed of α- and β-TCP phases, whereas the fabricated ceramics mainly consisted of β-TCP phase. The determination of zinc content in the resulting powders and fabricated ceramics showed a maximum content of ~12 mol%. The resulting powders consisted of spherical particles with diameters <2 μm. The lattice constants of ceramics did not show a significant change of the a-axis, b-axis, and c-axis. The bulk density of Zn-TCPs showed their maximum at 1100°C, while the bulk density of Zn-TCP0 was greatest at 1000°C.

A new system to evaluate the influence of immunosuppressive drugs on pancreatic islets using epigenetic analysis in a 3-dimensional culture.

Objective The present study aimed to establish a new method to evaluate the influence of immunosuppressive drugs on pancreatic islets in short-term in vitro cultures using epigenetic analysis in a 3-dimensional culture. Methods For this purpose, we selected (a) a 3-dimensional culture system utilizing thermoreversible gelation polymer, (b) pancreatic duodenal homeobox-1 (pdx-1)-Venus transgenic pigs expressing the green fluorescent protein, (c) FK506 as an immunosuppressive drug of the evaluation, and (d) the bisulfite sequencing technique to evaluate the methylation levels of pdx-1 and insulin genes. Each isolated pancreatic islet was cultured with several doses of FK506. The viability of the each islet was evaluated by analyzing the emission of Venus in real time and by propidium iodide staining. Epigenetic analysis was performed at several time points. Results Each single pancreatic islet was stably cultured for 30 days in this system. At day 30 in culture, we observed that insulin DNA methylation levels in the group that received a high dose of FK506 dramatically increased, although there was no change in pdx-1 DNA methylation level and configuration of the islets. Conclusions Our system may be useful to determine immunosuppressive drugs that are specifically suitable for islet transplantation.