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The synthesis of the saccharide β-D-fructopyranosyl-(2→6)-D-glucopyranose, which was isolated from Super Ohtaka^®, has recently been reported. During the synthesis of this saccharide, the formation of two novel saccharides from D-glucose and D-fructose was observed. The present study aimed to confirm the structures of the two disaccharides synthesized from D-glucose and D-fructose by thermal treatment. Furthermore, various properties of the saccharides were investigated. Both saccharides were isolated from the reaction mixture by carbon-Celite column chromatography and an HPLC system and were determined to be novel sucrose-isomers, β-D-fructopyranosyl-(2↔1)-β-D-glucopyranoside (1) and β-D-fructofuranosyl-(2↔1)-β-D-glucopyranoside (2), by MALDI-TOF MS and NMR analyses. Both saccharides showed low digestibility in vitro, and the sweetness of saccharide 2 was 0.45 times that of sucrose.

Ideal biomaterials for bone tissue engineering should have the capability to guide the osteogenic differentiation of mesenchymal stem cells and, at the same time, to stimulate angiogenesis of endothelia cells. In this study it was found that three Ca-Mg-Si-containing bioceramics (bredigite Ca7MgSi4O16, akermanite Ca2MgSi2O7 and diopside CaMgSi2O6) had osteogenic and angiogenic potential. The effects of three silicate ceramics on the osteogenic differentiation of human bone marrow mesenchymal stem cells (hBMSCs) and the angiogenesis of human aortic endothelial cells (HAECs) were explored in comparison with β-tricalcium phosphate (β-TCP) bioceramics. The proliferation, alkaline phosphatase (ALPase) activity and bone-related gene expression (COL1, ALPase, OP, BSP and OC) of hBMSCs were significantly enhanced upon stimulation with ionic extracts of these silicate bioceramics. In addition, the results showed that extracts from the three silicate bioceramics also stimulated HAEC proliferation and in vitro angiogenesis with improved NO synthesis and angiogenic gene expression (KDR, FGFR1, ACVRL1 and NOS3). Among the three silicate ceramics bredigite showed the highest osteogenic and angiogenic potential and with the highest extract Si (possibly Si(OH)3O(-)) concentration, while diopside had the lowest osteogenic and angiogenic potential with the lowest extract Si concentration. Furthermore, it was found that the concentration of Si ions in extracts of the three silicate bioceramics was obviously higher than that of β-TCP ceramics, indicating an important role of Si ions in stimulating cell proliferation, osteogenic differentiation and angiogenesis. The results suggest that the silicate-based akermanite and bredigite ceramics might be good scaffold biomaterials for bone tissue engineering applications due to their distinctive dual functions of osteogenesis/angiogenesis stimulation.

Stem cells are a promising cell source for regenerative medicine. Stem cell differentiation must be regulated for applications in regenerative medicine. Stem cells are surrounded by extracellular matrix (ECM) in vivo. The ECM is composed of many types of proteins and glycosaminoglycans that assemble into a complex structure. The assembly of ECM molecules influences stem cell differentiation through orchestrated intracellular signaling activated by many ECM molecules. Therefore, it is important to understand the comprehensive role of the ECM in stem cell differentiation as well as the functions of the individual ECM molecules. Decellularized ECM is a useful in vitro model for studying the comprehensive roles of ECM because it retains a native-like structure and composition. Decellularized ECM can be obtained from in vivo tissue ECM or ECM fabricated by cells cultured in vitro. It is important to select the correct decellularized ECM because each type has different properties. In this review, tissue-derived and cell-derived decellularized ECMs are compared as in vitro ECM models to examine the comprehensive roles of the ECM in stem cell differentiation. We also summarize recent studies using decellularized ECM to determine the comprehensive roles of the ECM in stem cell differentiation.

The capacity to induce rapid vascular ingrowth during new bone formation is an important feature of biomaterials that are to be used for bone regeneration. Akermanite, a Ca-, Mg- and Si-containing bioceramic, has been demonstrated to be osteoinductive and to promote bone repair. This study further demonstrates the ability of akermanite to promote angiogenesis and investigates the mechanism of this behavior. The akermanite ion extract predominantly caused Si-ion-stimulated proliferation of human aortic endothelial cells. The Si ion in the extract was the most important component for the effect and the most effective concentration was found to be 0.6–2 μg ml−1. In this range of Si ion concentration, the stimulating effect of the ceramic ion extract was demonstrated by the morphology of cells at the primary, interim and late stages during in vitro angiogenesis using ECMatrix™. The akermanite ion extract up-regulated the expression of genes encoding the receptors of proangiogenic cytokines and also increased the expression level of genes encoding the proangiogenic downstream cytokines, such as nitric oxide synthase and nitric oxide synthesis. Akermanite implanted in rabbit femoral condyle model promoted neovascularization after 8 and 16 weeks of implantation, which further confirmed its stimulation effect on angiogenesis in vivo. These results indicate that akermanite ceramic, an appropriate Si ion concentration source, could induce angiogenesis through increasing gene expression of proangiogenic cytokine receptors and up-regulated downstream signaling. To our knowledge, akermanite ceramic is the first Si-containing ceramic demonstrated to be capable of inducing angiogenesis during bone regeneration.

A fermented beverage of plant extract was prepared from about 50 kinds of fruits and vegetables. Natural fermentation was conducted by lactic acid bacteria (Leuconostoc spp.) and yeast (Zygosaccharomyces spp. and Pichia spp.). Eighteen kinds of oligosaccharides were isolated from this beverage, and their structures were confirmed by methylation analysis, MALDI-TOF-MS and NMR measurements. In these saccharides, eight novel oligosaccharides were found to be constructed by di- and trisaccharides with the fructosyl residue of pyranose form, and other trisaccharides with fructosyl residues of sucrose bond with the β-D-galactose and β-D-glucose. The characteristics of one of novel saccharide, O-β-D-fructopyranosyl-(2→6)-D-glucopyranose (Fp2-6G) were investigated, and it was shown to be non-cariogenicity and have low digestibility. Furthermore, the saccharide was selectively used by beneficial bacteria, Bifidobacterium adolescentis and B. longum, but was not used by unfavorable bacteria, Clostridium perfringens, Escherichia coli and Enterococcus faecalis that produce mutagenic substances.

This review focuses on the NMR methods for the oligosaccharides containing fructopyranoside that were previously isolated from the fermented beverage of an extract from 50 kinds of fruits and vegetables. The ^1H and ^C-NMR signals of each saccharide were assigned using 2D-NMR including COSY, HSQC, HSQC-TOCSY, CH_2-selected HSQC-TOCSY, and CT (constant time)-HMBC. The fructose in pyranosyl form showed different ^C chemical shifts from those of furanosyl form. Further confirmation of the pyranosyl form could be obtained from the HMBC correlation peak between C-2 and H-6 of fructose residue (Fru), whereas the C-2 of Fru in furanosyl form chould give the HMBC correlation peak between H-5 of Fru. Problems encountered were signal overlapping of protons and low peak separation. The key correlation peak between C-2 and H-6 of Fru was overlapped by the correlation peak indicating a glycosidic linkage between the C-2 of Fru and the H-6 of the glucose residue (Glc, or Glc’). These were solved using HSQC and CT-HMBC spectra rather than HMQC and conventional HMBC spectra, which have an inherent broad-line-shape in the carbon dimension.

Fermented beverage of plant extract was prepared from 55 kinds of fruits and vegetables. Natural fermentation was conducted by lactic acid bacteria (Leuconostoc spp.) and yeast (Zygosaccharomyces spp. and Pichia spp.). We have previously found that the fermented beverage contained the novel saccharide, O-β-D-fructopyranosyl-(2→6)-D-glucopyranose, which is produced by the fermentation process. The characteristics of O-β-fructopyranosyl-(2→6)-D-glucopyranose were investigated. The saccharide showed 0.2 times the sweetness of sucrose, non-cariogenicity and low digestibility. Furthermore, the unfavorable bacteria, Clostridium perfringens, Escherichia coil and Enterococcus faecalis that produce mutagenic substances did not use the saccharide. Therefore, it was thought that saccharide 1 could be a new material for foods. 植物エキス発酵飲料は約50種類の植物を原料として，ショ糖の浸透圧を利用してエキスを抽出し，おもに酵母(Zygosaccharomyces spp. and Pichia spp.)，乳酸菌(Leuconostoc spp.)の発酵によって製造される。この飲料からO-β-D-fructopyranosyl-(2→6)-D-glucopyranose(糖 1)が見出され，発酵によって生成される。糖1は，ショ糖と比較し0.2倍の甘さであり，酸に対する安定性はショ糖とほぼ同等であった。また，低う蝕性，難消化性糖であることが認められ，さらに，腸内細菌による資化性を調査したところ，この糖質はクロストリディウム属菌などに利用されず，ビフィドバクテリウム属菌によってのみ資化された。