Cytochrome P450 CYP71BE5 in grapevine (Vitis vinifera) catalyzes the formation of the spicy aroma compound (-)-rotundone.

(-)-Rotundone is a potent odorant molecule with a characteristic spicy aroma existing in various plants including grapevines (Vitis vinifera). It is considered to be a significant compound in wines and grapes because of its low sensory threshold and aroma properties. (-)-Rotundone was first identified in red wine made from the grape cultivar Syrah and here we report the identification of VvSTO2 as a α-guaiene 2-oxidase which can transform α-guaiene to (-)-rotundone in the grape cultivar Syrah. It is a cytochrome P450 (CYP) enzyme belonging to the CYP 71BE subfamily, which overlaps with the very large CYP71D family and, to the best of our knowledge, this is the first functional characterization of an enzyme from this family. VvSTO2 was expressed at a higher level in the Syrah grape exocarp (skin) in accord with the localization of (-)-rotundone accumulation in grape berries. α-Guaiene was also detected in the Syrah grape exocarp at an extremely high concentration. These findings suggest that (-)-rotundone accumulation is regulated by the VvSTO2 expression along with the availability of α-guaiene as a precursor. VvSTO2 expression during grape maturation was considerably higher in Syrah grape exocarp compared to Merlot grape exocarp, consistent with the patterns of α-guaiene and (-)-rotundone accumulation. On the basis of these findings, we propose that VvSTO2 may be a key enzyme in the biosynthesis of (-)-rotundone in grapevines by acting as a α-guaiene 2-oxidase.

Size and Surface Properties of Functionalized Organosilica Particles Impact Cell-Particle Interactions Including Mitochondrial Activity.

Understanding of the interactions between macrophages and multifunctional nanoparticles is important for development of novel macrophage-based immunotherapies. Here, we investigated the effects of fluorescent thiol-organosilica particle size and surface properties on cell-particle interactions, including mitochondrial activity, using the mouse macrophage cell line J774A.1. Three different sizes of thiol-organosilica particles (150, 400, and 680 nm in diameter) containing fluorescein (OS/F150, OS/F400, and OS/F680) and particles surface functionalized with polyethylenimine (PEI) (OS/F150PEI, OS/F400PEI, and OS/F680PEI) were prepared. Flow cytometric analysis, time-lapse imaging, and single-cell analysis of particle uptake and mitochondrial activity of J774A.1 cells demonstrated variations in uptake and kinetics depending on the particle size and surface as well as on each individual cell. Cells treated with OS/F150 and OS/F150PEI showed higher uptake and mitochondrial activity than those treated with other particles. The interaction between endosomes and mitochondria was observed using 3D fluorescent imaging and was characterized by the involvement of iron transport into mitochondria by iron-containing proteins adsorbed on the particle surface. Scanning electron microscopy of the cells treated with the particles revealed alterations in cell membrane morphology, depending on particle size and surface. We performed correlative light and electron microscopy combined with time-lapse and 3D imaging to develop an integrated correlation analysis of particle uptake, mitochondrial activity, and cell membrane morphology in single macrophages. These cell-specific characteristics of macrophages against functional particles and their evaluation methods are crucial for understanding the immunological functions of individual macrophages and developing novel immunotherapies.

Effects of Au States in Thiol-Organosilica Nanoparticles on Enzyme-like Activity for X-ray Sensitizer Application: Focus on Reactive Oxygen Species Generation in Radiotherapy.

In radiotherapy, the use of Au nanoparticles (Au NPs) has been proposed to enhance cell damage by X-ray irradiation. Although the role of Au in radiotherapy is not fully understood, the catalytic activity of Au has been actively studied in the industrial field. Moreover, owing to their enzyme-like activity and high biocompatibility in vitro and in vivo, Au NPs present significant potential for biological applications. In this study, we incorporated different Au states both on the surface and embedded in thiol-organosilica (thiol-OS/Au series) to investigate the efficiency of anticancer cell activity of Au in radiotherapy. The thiol-OS/Au series comprised different Au(I)/Au(0) ratios and Au NPs, and different sizes of Au NPs were embedded in thiol-OS/Au. These thiol-OS/Au series samples were evaluated for enzyme-like activities in reactive oxygen species (ROS) generation by X-ray irradiation. Thiol-OS/Au embedded with small Au NPs (AC600/thiol-OS/Au) exhibited peroxidase (POD)-like activity under acidic conditions. This POD-like activity improved ROS generation and cytotoxicity under X-ray irradiation. Furthermore, AC600/thiol-OS/Au exhibited catalase (CAT)-like activity under basic conditions and showed no cytotoxicity toward nonirradiated cells. These results revealed the efficiency of functionalizing with small Au NPs that possess pH-controlled POD- and CAT-like activity as a radiosensitizer. We compared the suitability of using Au with different states to obtain the thiol-OS/Au series samples for application as radiosensitizers. The findings of this study will aid the design of efficacious strategies for the Au nanostructure-based radiotherapy of cancer cells.

Size effect of fluorescent thiol-organosilica particles on their distribution in the mouse spleen.

We investigated the distribution of intravenously administered thiol-organosilica particle (thiol-OS) in the spleen to evaluate their size effect in mice. A single administration of particles of thiol-OS containing rhodamine B (Rh) (90, 280, 340, 450, 630, 1110, 1670, and 3030 nm in diameter) was performed. After 24 h, we conducted a combination analysis using histological studies by fluorescent microscopy and quantitative inductively coupled plasma optical emission spectrometry (ICP-OES), which revealed no clear correlation between the particle size and spleen uptake of particle weight and number per tissue weight, and the injection dose. Moreover, Rh with 450 nm diameter (Rh450) showed the highest uptake, and Rh with 340 nm diameter (Rh340) showed the lowest uptake. Histologically, large fluorescent areas in the marginal zone (MZ) and red pulp (RP) of the spleen were observed for all particle sizes, but less in the follicle of white pulp. Using combination analysis using the particle weights of ICP-OES and the fluorescent area, we compared the distributions of each particle in each region. Rh450 had the largest accumulated weight in the MZ and RP. Particles larger than Rh450 showed negative correlations between their sizes and accumulated weight in the MZ and RP. Simultaneous dual administration of particles using Rhs and thiol-OS containing fluorescein (90 nm in diameter) showed the size-dependent difference in cellular distribution and intracellular localization. Immunohistochemical staining against macrophage markers, CD169, and F4/80 showed various colocalization patterns with macrophages that uptook particles, indicating differences in particle uptake in each macrophage may have novel significance.

Preparation of Fetal Bovine Serum-Copper Phosphate Hybrid Particles under Cell Culture Conditions for Cancer Cell Treatment.

Fetal bovine serum (FBS) particles, which mainly consist of bovine serum albumin, have the potential for biological and medical applications as drug carriers. The coacervation of albumin is a common technique for preparing albumin-based particles. The replacement of salt with novel metal salts such as Cu is an affordable way to embed the metal ion in the albumin-based particles. Further, increased Cu distribution is prevalent in many cancers. Here, we prepared adhesive cell-like FBS-copper phosphate hybrid particles [FBS-Cu3(PO4)2], which exhibited toxicity toward cancer cells, with a narrow size distribution under cell culture conditions for preventing tumor progression. FBS-Cu3(PO4)2 showed peroxidase-like activity. In addition, FBS-Cu3(PO4)2 was successfully loaded with rhodamine B and conjugated with fluorescein isothiocyanate as models of drugs by coincubation. Thus, we designed a simple preparation method for optimizing FBS-Cu3(PO4)2 synthesis under cell culture conditions. FBS-Cu3(PO4)2 has significant potential as an efficient reactive oxygen species generator and drug-delivery agent against cancer cells. Furthermore, the RhoB-loaded FBS-Cu3(PO4)2 successfully interacted with 4T1 mouse mammary tumor cells and were confirmed to exhibit toxicity.

Surface Functionalization of Organosilica Nanoparticles With Au Nanoparticles Inhibits Cell Proliferation and Induces Cell Death in 4T1 Mouse Mammary Tumor Cells for DNA and Mitochondrial-Synergized Damage in Radiotherapy.

Radiotherapy is one of the most effective cancer treatments. Au nanoparticles (NPs) are one of the most used X-ray sensitizing materials however the effective small sub-nm size of Au NPs used for X-ray sensitizers is disadvantageous for cellular uptake. Here, we propose the surface functionalization of organosilica NPs (OS) with Au NPs (OS/Au), which combined the 100 nm size of OS and the sub-nm size of Au NPs, and synthesized effective Au materials as an X-ray sensitizer. The X-ray sensitizing potential for 4T1 mouse mammary tumor cells was revealed using a multifaceted evaluation combined with a fluorescence microscopic cell imaging assay. The number of polyethyleneimine (PEI)-modified OS (OS/PEI) and OS/Au (OS/Au/PEI) uptake per 4T1 mouse mammary tumor cell was the same; however, 4T1 cells treated with OS/Au/PEI exhibited significant inhibition of cell proliferation and increases in cell death by X-ray irradiation at 8Gy. The non-apoptotic death of OS/Au/PEI-treated 4T1 cells was increased by DNA and mitochondrial-synergized damage increase and showed potential applications in radiotherapy.

Analysis of cell-nanoparticle interactions and imaging of in vitro labeled cells showing barcorded endosomes using fluorescent thiol-organosilica nanoparticles surface-functionalized with polyethyleneimine.

Biomedical imaging using cell labeling is an important technique to visualize cell dynamics in the body. To label cells, thiol-organosilica nanoparticles (thiol-OS) containing fluorescein (thiol-OS/Flu) and rhodamine B (thiol-OS/Rho) were surface-functionalized with polyethyleneimine (PEI) (OS/Flu-PEI and OS/Rho-PEI) with 4 molecular weights (MWs). We hypothesized PEI structures such as brush, bent brush, bent lie-down, and coiled types on the surface depending on MWs based on dynamic light scattering and thermal gravimetric analyses. The labeling efficacy of OS/Flu-PEIs was dependent on the PEI MW and the cell type. A dual-particle administration study using thiol-OS and OS-PEIs revealed differential endosomal sorting of the particles depending on the surface of the NPs. The endosomes in the labeled cells using OS/Flu-PEI and thiol-OS/Rho revealed various patterns of fluorescence termed barcoded endosomes. The cells labeled with OS-PEI in vitro were administrated to mice intraperitoneally after in situ labeling of peritoneal cells using thiol-OS/Rho. The in vitro labeled cells were detected and identified in cell aggregates in vivo seamlessly. The labeled cells with barcoded endosomes were also identified in cell aggregates. Biomedical imaging of in vitro OS-PEI-labeled cells combined with in situ labeled cells showed high potential for observation of cell dynamics.

Development of Non-Porous Silica Nanoparticles towards Cancer Photo-Theranostics.

Nanoparticles have demonstrated several advantages for biomedical applications, including for the development of multifunctional agents as innovative medicine. Silica nanoparticles hold a special position among the various types of functional nanoparticles, due to their unique structural and functional properties. The recent development of silica nanoparticles has led to a new trend in light-based nanomedicines. The application of light provides many advantages for in vivo imaging and therapy of certain diseases, including cancer. Mesoporous and non-porous silica nanoparticles have high potential for light-based nanomedicine. Each silica nanoparticle has a unique structure, which incorporates various functions to utilize optical properties. Such advantages enable silica nanoparticles to perform powerful and advanced optical imaging, from the in vivo level to the nano and micro levels, using not only visible light but also near-infrared light. Furthermore, applications such as photodynamic therapy, in which a lesion site is specifically irradiated with light to treat it, have also been advancing. Silica nanoparticles have shown the potential to play important roles in the integration of light-based diagnostics and therapeutics, termed "photo-theranostics". Here, we review the recent development and progress of non-porous silica nanoparticles toward cancer "photo-theranostics".

Protein corona components of polyethylene glycol-conjugated organosilica nanoparticles modulates macrophage uptake.

Fluorescent organosilica nanoparticles (FNP) conjugated with polyethylene glycol (PEG) of variant molecular weight (2 K, 12 K, 20 K, and 30 K) were prepared to investigate their cellular uptake by murine-derived macrophages. In a medium with FBS, the cellular uptake of FNP-PEGs was decreased as compared to a medium without FBS, indicating that protein corona on FNP-PEGs reduced cellular uptake. Bovine serum albumin (BSA) and hemoglobin (Hb) were detected as the most abundant components on all FNP-PEGs. Pre-coating of FNP-PEGs with BSA and Hb reduced the macrophage uptake in a medium without FBS, suggesting that these components might strengthen the stealth function of PEGs. Furthermore, there was more reduction in uptake of BSA- and Hb-coated FNP-PEGs from a medium with FBS than without FBS. BSA and Hb could be the stealth enhancement protein of FNP-PEGs in vitro.

In vivo evaluation of composites of PLGA and apatite with two different levels of crystallinity.

The cortical bone response towards poly(lactide-co-glycolide) (70/30) (PLGA) (70/30)/apatite complex scaffolds with different levels of crystallinity was investigated. Apatite with different levels of crystallinity, Ca-deficient hydroxyapatite (CDHA), which has a low crystallinity, and a mixture of carbonated hydroxyapatite (CHA) and CDHA, which has a higher crystallinity, were prepared from an aqueous mixture of Ca-EDTA complex, H(2)O(2), H(3)PO(4), and NH(4)OH. Two porous PLGA(70/30)/apatite composite scaffolds, composite scaffold A (containing low crystallinity CDHA) and composite scaffold B (containing the higher crystallinity CHA/CDHA mixture), were prepared. Afterwards, pure porous PLGA and the two composite scaffolds were implanted into the cortical bone of rabbit tibiae for 12 weeks. High-resolution microfocus X-ray computed tomography and histological examinations revealed a better bone response for composite scaffold A compared with PLGA and composite scaffold B. For composite scaffold A, the original bone defect was almost filled with new bone. Quantitative analysis revealed that composite scaffold A produced a significantly greater amount of new bone. The present study demonstrated that the level of apatite crystallinity influences bone response. A PLGA/apatite porous composite with a low level of apatite crystallinity shows promise as a bone substitute or scaffold material for bone tissue engineering.

Snake venom rhodocytin induces plasma extravasation via toxin-mediated interactions between platelets and mast cells.

Venomous snakebites can induce local tissue damage, including necrosis of soft tissues, haemorrhage, blistering and local swelling associated with plasma extravasation, which can lead to lethal complications such as hypovolemic shock. However, the details of the underlying mechanisms remain unknown. In this study, we showed that intradermal treatment of mice with venom rhodocytin from the Malayan viper Calloselasma rhodostoma induced plasma extravasation, dependent on C-type lectin-like receptor 2 (CLEC-2) on platelets. Rhodocytin-induced plasma extravasation also relied on mast cells and histamine. In vitro co-culture of rhodocytin-activated platelets with mast cells induced histamine release from mast cells in an ATP/P2X7-dependent manner. Consistent with this, blockade or deficiency of P2X7 in mast cells suppressed rhodocytin-induced plasma extravasation in the skin. Together, these findings indicate that rhodocytin induces plasma extravasation by triggering platelet activation via CLEC-2, followed by activation of mast cells and histamine release via the ATP/P2X7 pathway. These results reveal a previously unrecognized mechanism by which snake venom increases vascular permeability via complex venom toxin-mediated interactions between platelets and mast cells.

Clinicopathological features of gastric cancer detected by endoscopy as part of annual health checkup.

BACKGROUND AND AIM: Upper gastrointestinal endoscopy is generally accepted as the gold standard for the clinical evaluation of gastric cancer (GC). However, the efficacy of endoscopic screening for asymptomatic GC remains controversial. The present study is designed to clarify the efficacy of endoscopic screening for the detection of early GC by investigating the clinicopathological features. METHODS: A total of 17 522 patients who had underwent endoscopic screening as a part of their annual health checkup at the Seirei Center for Health Promotion and Preventive Medicine between April 2002 and March 2006 were enrolled in this study. We investigated the clinicopathological findings of GC detected by endoscopy. Furthermore, in accordance with the screening interval at our center, patients with GC were categorized into two groups: group A, patients with repeated endoscopic screening within the last 2 years, and group B, patients without endoscopic screening within the last 2 years. RESULTS: Thirty-nine GC (mean age of patients: 62.2 +/- 8.0 years, 36 males and three females) were detected in total (0.22%). The proportion of early GC was 87.2%. Notable differences between groups A and B were not found in the rate of early GC (P = 0.6342). However, eight of 27 cases (29.6%) in group A were treated by endoscopic resection, but none in group B (P = 0.0344). In six of 26 cases (23.1%) in group A, the recorded images from the previous endoscopic examination indicated some macroscopic abnormalities at the same location, suggesting GC or premalignant lesions. CONCLUSION: Endoscopic screening is useful for detecting GC at the early stages, and repeated examinations at short-time intervals contribute to the detection of resectable lesions by endoscopy. Further studies are needed to decrease the false negative rate of endoscopic screening.

Cobalt hematoporphyrin inhibits CLEC-2-podoplanin interaction, tumor metastasis, and arterial/venous thrombosis in mice.

The platelet activation receptor C-type lectin-like receptor 2 (CLEC-2) interacts with podoplanin on the surface of certain types of tumor cells, and this interaction facilitates tumor metastasis. CLEC-2 is also involved in thrombus formation and its stabilization. Because CLEC-2-depleted mice are protected from experimental lung metastasis and thrombus formation and do not show increased bleeding time, CLEC-2 may serve as a good target for antimetastatic or antithrombotic drugs. We screened 6770 compounds for their capability to inhibit CLEC-2-podoplanin binding using an enzyme-linked immunosorbent assay. In the first screening round, 63 compounds were identified and further evaluated by flow cytometry using CLEC-2-expressing cells. We identified protoporphyrin IX (H2-PP) as the most potent inhibitor and modified its hematoporphyrin moiety to be complexed with cobalt (cobalt hematoporphyrin [Co-HP]), which resulted in an inhibitory potency much stronger than that of H2-PP. Surface plasmon resonance analysis and molecular docking study showed that Co-HP binds directly to CLEC-2 at N120, N210, and K211, previously unknown podoplanin-binding sites; this binding was confirmed by analysis of CLEC-2 mutants with alterations in N120 and/or K211. Co-HP at a concentration of 1.53 µM inhibited platelet aggregation mediated through CLEC-2, but not that mediated through other receptors. IV administration of Co-HP to mice significantly inhibited hematogenous metastasis of podoplanin-expressing B16F10 cells to the lung as well as in vivo arterial and venous thrombosis, without a significant increase in tail-bleeding time. Thus, Co-HP may be a promising molecule for antimetastatic and antiplatelet treatment that does not cause bleeding tendency.

Strong chiroptical activity from achiral gold nanorods assembled with proteins.

The side-by-side assembly of gold nanorods and proteins, particularly human serum albumin, exhibits a distinct chiroptical activity in a wide range of visible and near-infrared wavelengths corresponding to the surface plasmon resonance. The anisotropy factor was the highest in a colloidal solution system and was able to be inverted by an external environment.

Characterization and Bone Response of Carbonate-Containing Apatite-Coated Titanium Implants Using an Aqueous Spray Coating.

We performed thin carbonate-containing apatite (CA) coating on titanium (Ti) by an aqueous spray coating (ASC) method that consisted of a Ca-CO3-PO4 complex. Two different CA coatings were produced by two different spray amounts and were heat-treated after spraying. We evaluated three-dimensional structures, adhesiveness to Ti, and durability of the CA film. In addition, we performed immersion experiments in simulated body fluid (SBF), and bone responses were evaluated after implantation into a femoral bone defect in rats. The bonding ability of ASC-coated implant into the bone was examined by push-in tests. Unique network structures with small particles were identified on CA coatings. Although heat treatment produced no significant difference in surface morphology, scratch tests revealed that heat treatment improved the adhesion of CA coatings to Ti. Crystal formation progressed on CA-coated specimens, and the sample placement direction influenced crystal formation and growth in SBF immersion. Animal implantation experiments revealed significantly greater bone-to-implant contact ratio and bone mass in both cortical and bone marrow, respectively, four weeks after implantation. Push-in tests suggested that the bonding of the CA coating to Ti is clinically acceptable. Therefore, we conclude that CA coating to Ti by the ASC method would be possible for clinical applications, including dentistry.

Growth and maturation of small hepatocytes.

Proliferation of adult rat hepatocytes is observed in serum-free Dulbecco's modified Eagle's medium (DMEM) supplemented with 10 mmol/L nicotinamide and 10 ng/mL epidermal growth factor (EGF). The proliferating cells are mainly mononucleate and form small cell colonies surrounded by mature hepatocytes. Although these cells in focal colonies have a less-differentiated appearance, immunocytochemically and ultrastructurally they possess hepatic characteristics. The size of small hepatocytes is one-third to half that of mature hepatocytes. Therefore, we call the cells forming a colony, small hepatocytes. The small hepatocytes can be subcultured for several passages. Furthermore, the cells are rich in the supernatant following 50 g centrifugation for 1 min after collagenase liver perfusion. When the cells are cultured in DMEM supplemented with 10% foetal bovine serum, 10 mmol/L nicotinamide, 1 mmol/L ascorbic acid 2-phosphate, 10 ng/mL EGF and 1% dimethyl sulphoxide, each small hepatocyte can clonally proliferate for more than 3 months. A small hepatocyte divides to form a colony and the number of cells reaches more than 100 within 20 days. With time in culture, cells with a large cytoplasm appear within a colony. They have many mitochondria and large peroxisomes with crystalline nucleoids and are typical, mature hepatocytes. Immunoreactivity to connexin 32 and well-developed bile canaliculus structures are often observed in the cell-cell borders. Thus, we suggest that small hepatocytes may be considered to be 'committed progenitor cells' that can further differentiate into mature hepatocytes.

Behaviors of MC3T3-E1 cells on carbonated apatite films, with a characteristic network structure, fabricated on a titanium plate by aqueous spray coating.

Four carbonated apatite films having average thicknesses of 1.3-0.11µm, proportions of network sizes above 10µm of 41-68%, and average border heights of the characteristic network structure of 0.98-0.29µm were fabricated on a titanium plate by aqueous spray coating. These carbonated apatite films after heat treatment showed good mineralization ability in Hanks' balanced salt solution. Assessment of initial cell attachment and calcination on these films and on the Ti plate using osteoblastic MC3T3-E1 indicated that the carbonated apatite film heat treated at 600°C, whose film thickness, proportion of network sizes above 10µm, and border height were 0.11µm, 61%, and 0.31µm, respectively, was most preferred by osteoblastic cells. Field emission scanning electron microscopic observation of the cells attached to the films showed that the wide network and low border height of the network structure on the carbonated apatite film play an important role in the development of the filopodia of the osteoblastic cells.

Application of carbonated apatite coating on a Ti substrate by aqueous spray method.

The fabrication and characterization of a carbonate-containing apatite film deposited on a Ti plate via an aqueous spray method is described. The mist of the spray solution emitted from a perpendicularly oriented airbrush was made to strike a warmed Ti substrate. The thicknesses of the sprayed film and those heat-treated at 400 °C-700 °C under Ar gas flow were in the range 1.21-1.40 µm. The results of elemental analyses and Fourier transform infrared spectroscopy of the powders that were mechanically collected from the surface of the sprayed film suggest that the film was Ca(10)(PO4)6(CO3) · 2CO2 · 3H2O. The presence of the carbonate ion and the lattice CO2 molecule was confirmed via the aforementioned analyses; the finding was also consistent with the X-ray diffraction patterns of the films and the chemical identity of the sprayed and heat-treated films that were measured using X-ray photoelectron spectroscopy. The sprayed film comprises a characteristic network structure, which contains round particles within the networks, as was observed by field-emission scanning electron microscopy. A scratch test indicated that the shear stress of the sprayed film (21 MPa) significantly improved to 40 and >133 MPa after heat-treatment at 600 °C and 700 °C, respectively, under Ar gas flow for 10 min.

The effect of composition of calcium phosphate composite scaffolds on the formation of tooth tissue from human dental pulp stem cells.

Different approaches towards making 3-dimensional (3-D) bioengineered tooth for future replacement therapy have been developed including scaffold-based tooth regeneration. However, selection of optimal scaffold for future clinical application remains a challenge. In the present study, we tested biocompatibility of four different types of 3-D scaffolds for tooth-tissue regeneration, including a pure poly(lactide-co-glycolide) (PLGA) (70/30, mol/mol) scaffold and three types of calcium phosphate contained composites scaffolds that were 50 wt% of PLGA combined with 50 wt% of hydroxyapatite (HA), tricalcium phosphate (TCP) or calcium carbonate hydroxyapatite (CDHA) respectively. These scaffolds were fabricated by the particle leaching in combination with phase separation technology. Surface modification of these scaffolds was further performed by an ammonia plasma treatment and anchorage of collagen technology. Effect of composition of the composite scaffolds on proliferation of human dental pulp stem cells (DPSCs) was accessed using in vitro MTT assay and in vivo BrdU labeling. Differentiation capability of the DPSCs in the scaffolds was analyzed by measurement of the levels of calcified tissue formation and ALP activity. Our results showed that while the calcium phosphate contained compound is able to support regeneration of tooth tissue effectively, the PLGA/TCP scaffold is more appropriate for the proliferation and differentiation of DPSCs. Furthermore, seeding of dissociated 4-dpn rat tooth bud cells on the PLGA/TCP scaffold generated dentin- and pulp-like tissues. Our results demonstrate that the PLGA/TCP scaffold is superior to the other three scaffolds for tooth-tissue regeneration, especially for dentin formation.

Crystallinity control of apatite through Ca-EDTA complexes and porous composites with PLGA.

Apatite compounds with different levels of crystallinity were prepared using Ca-EDTA complexes. Ca-deficient hydroxyapatite (CDHA) with low crystallinity was synthesized by ultrasonic stirring of a mixture of Ca-EDTA complex, phosphoric acid, and ammonium hydroxide in hydrogen peroxide aqueous solution. Mixtures of carbonate hydroxyapatite (HA) and CDHA with higher crystallinity were also prepared from a solution involving the same complex. The porous composites with lower or higher crystallinity apatite with a copolymer of poly(L-lactide-co-glycilide)(70/30) (PLGA(70/30)) were fabricated by a solution-casting/particles leaching method. The apatites and porous composites were characterized, and it was found that the degradation of composites of apatite with a low level of crystallinity was fastest in phosphate-bufferd saline (PBS) solution compared with other apatite composites with higher levels of crystallinity; however, the rate was smaller than that of PLGA alone. Plasma treatment influenced the degradation of composites in PBS and apatite precipitation in simulated body fluid (SBF). Hydroxyapatite deposition on the PLGA composite with the low crystallinity occurred six times faster than that on PLGA alone after immersion in SBF. The incorporation of apatite into the PLGA matrix did not cause any adverse effects on cell attachment in an assay employing human gingival fibroblasts. This study suggested that the current apatite and PLGA porous composite will be a promising scaffold material for tissue engineering.