Authorship：Lead author   Language：English   Publishing type：Research paper (scientific journal)  

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Authorship：Lead author   Language：English   Publishing type：Research paper (scientific journal)  

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Language：English   Publishing type：Research paper (scientific journal)  

Although vital pulp therapy should be performed by promoting the wound-healing capacity of dental pulp, existing pulp-capping materials were not developed with a focus on the pulpal repair process. In previous investigations of wound healing in dental pulp, we found that organic dentin matrix components (DMCs) were degraded by matrix metalloproteinase-20, and DMC degradation products containing protein S100A7 (S100A7) and protein S100A8 (S100A8) promoted the pulpal wound-healing process. However, the direct use of recombinant proteins as pulp-capping materials may cause clinical problems or lead to high medical costs. Thus, we hypothesized that functional peptides derived from recombinant proteins could solve the problems associated with direct use of such proteins. In this study, we identified functional peptides derived from the protein S100 family and investigated their effects on dental pulp tissue. We first performed amino acid sequence alignments of protein S100 family members from several mammalian sources, then identified candidate peptides. Next, we used a peptide array method that involved human dental pulp stem cells (hDPSCs) to evaluate the mineralization-inducing ability of each peptide. Our results supported the selection of 4 candidate functional peptides derived from proteins S100A8 and S100A9. Direct pulp-capping experiments in a rat model demonstrated that 1 S100A8-derived peptide induced greater tertiary dentin formation compared with the other peptides. To investigate the mechanism underlying this induction effect, we performed liquid chromatography–tandem mass spectrometry analysis using hDPSCs and the S100A8-derived peptide; the results suggested that this peptide promotes tertiary dentin formation by inhibiting inflammatory responses. In addition, this peptide was located in a hairpin region on the surface of S100A8 and could function by direct interaction with other molecules. In summary, this study demonstrated that a S100A8-derived functional peptide promoted wound healing in dental pulp; our findings provide insights for the development of next-generation biological vital pulp therapies.

DOI： 10.1177/00220345221135766

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DOI： 10.1038/s41598-023-27412-x

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER SCI LTD  

During wound regeneration, both cell adhesion and adhesion-inhibitory functions must be controlled in parallel. We developed a membrane with dual surfaces by merging the properties of carboxymethyl cellulose (CMC) and collagen using vitrification. A rigid membrane was formed by vitrification of a bi-layered CMC and collagen hydrogel without using cross-linking reagents, thus providing dual functions, strong cell adhesion-inhibition with the CMC layer, and cell adhesion with the collagen layer. We referred to this bi-layered CMC-collagen vitrigel membrane as "Bi-C-CVM" and optimized the process and materials. The introduction of the CMC layer conferred a "tough but stably wet" property to Bi-C-CVM. This enables Bi-C-CVM to cover wet tissue and make the membrane non-detachable while preventing tissue adhesion on the other side. The bi-layered vitrification procedure can expand the customizability of collagen vitrigel devices for wider medical applications.

DOI： 10.1016/j.carbpol.2022.119223

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Authorship：Lead author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：Expert Opinion on Biological Therapy  

Background: Several studies demonstrated the therapeutic potential of mesenchymal stem cell–derived exosomes (MSC-exs) based on their anti-inflammatory properties. The objective was to determine the therapeutic effects of MSC-exs on aortic aneurysms (AAs) caused by atherosclerosis. Research design and methods: Apolipoprotein E knockout mice with AAs induced by angiotensin II were injected with MSC-exs or saline as a control. The change in the diameter of the aorta was measured. The expression of AA-related proteins and the histology of the aortic wall were investigated at 1 week after treatment. MicroRNA and protein profiles of MSC-exs were examined. Results: MSC-exs significantly attenuated AA progression (2.04 ± 0.20 mm in the saline group and 1.34 ± 0.13 mm in the MSC-ex group, P = 0.004). In the MSC-ex group, the expression of IL-1β, TNF-α and MCP-1 decreased, and expression of IGF-1 and TIMP-2 increased. MSC-ex induced the M2 phenotype in macrophages and suppressed the destruction of the elastic lamellae in the aortic wall. MSC-exs contained high levels of 10 microRNAs that inhibit AA formation and 13 proteins that inhibit inflammation and promote extracellular matrix synthesis. Conclusions: MSC-ex might be a novel alternative therapeutic tool for treatment of existing AAs.

DOI： 10.1080/14712598.2022.2005575

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Authorship：Lead author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：ACS Omega  

Water-insoluble cationic poly(vinyl alcohol) (PVA) films were fabricated using a mixed aqueous solution of PVA and poly([2-(methacryloyloxy)ethyl]trimethylammonium chloride (METAC)-co-methacrylic acid (MAAc)-co-5-methacrylamido-1,2-benzoxaborole (MAAmBO)) copolymer (3D). The surface of the PVA film is typically negatively charged, and simple fabrication methods for water-insoluble PVA films with cationic surface charges are required to expand their application fields. METAC, which has a permanent positive charge owing to the presence of a quaternary ammonium cation, was selected as the cationic unit. The MAAc and MAAmBO units were used as two types of cross-linking structures for the thermal cross-linking of the hydroxy and carboxy groups of the MAAc unit (covalent bonding) as well as the diol and benzoxaborole groups of the MAAmBO unit (dynamic covalent bonding). The films were thermally cross-linked at 135 °C for 4 h without the addition of materials. After immersion in surplus water at 80 °C for 3 h, the cross-linked PVA/3D films retained almost 100% of their weights. The ζ-potential of the water-insoluble PVA/3D film was 9.4 ± 0.8 mV. The PVA/3D film was strongly dyed using anionic acid red 1 (AR1) because of its positively charged surface. Interestingly, it could also be slightly dyed using cationic methylene blue (MB) and became transparent (original state) after immersion in water for 2 days. These results suggested that positive and negative charges coexisted in the PVA/3D film, and the surface properties were positively inclined. Moreover, the degree of hemolysis of the PVA/3D films was similar to that of the negative control, which showed high blood compatibility. To our knowledge, this is the first report on the fabrication of water-insoluble cationic PVA films using two types of cross-linking structures containing carboxy and benzoxaborole groups. The cross-linked PVA films were analyzed using Fourier transform infrared (FT-IR) spectroscopy, differential scanning calorimetry (DSC), and contact angle (CA) and ζ-potential measurement, as well as by determining the mechanical properties, adsorption of charged molecules, and biocompatibility. These readily fabricated water-insoluble PVA films with positive charges can show potential applications in sensors, adsorption systems, and antimicrobial materials.

DOI： 10.1021/acsomega.1c02013

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For decades, researchers have investigated the ideal material for clinical use in the cardiovascular field. Several substitute materials are used clinically, but each has drawbacks. Recently we developed biodegradable and elastic poly(ε-caprolactone-co-D,L-lactide) (P(CL-DLLA)) copolymers by adjusting the CL/DLLA composition, and evaluated the long-term efficacy and outcomes of these copolymers when used for right ventricular outflow tract (RVOT) replacement. This P(CL-DLLA) material was processed into a circular patch and used to replace a surgical defect in the RVOT of adult rats. Control rats were implanted with expanded polytetrafluoroethylene (ePTFE). Histologic evaluation was performed at 8, 24, and 48 weeks post-surgery. All animals survived the surgery with no aneurysm formation or thrombus. In all periods, ePTFE demonstrated fibrous tissue. In contrast, at 8 weeks P(CL-DLLA) showed infiltration of macrophages and fibroblast-like cells into the remaining material. At 24 weeks, P(CL-DLLA) was absorbed completely, and muscle-like tissue was present with positive staining for α-sarcomeric actinin and cardiac troponin T (cTnT). At 48 weeks, the cTnT-positive area had increased. The biodegradable and elastic P(CL-DLLA) induced cardiac regeneration throughout the 48-week study period. Future application of this material as a cardiovascular scaffold seems promising. Statement of significance: Biomaterials for reconstruction of tissue deficiencies in cardiovascular surgery require having suitable mechanical properties for cardiac tissue and biodegradation resulting in native tissue growth. Several biodegradable polymers such as poly-ε-caprolactone (PCL) and polylactic acid (PLA) have excellent biocompatibility and already been widely used clinically. In general, PCL and PLA are quite mechanically rigid. Meanwhile, significant elasticity is required in the high-pressure environment of the heart while the material is being replaced by new tissue. The present study provides a novel four-armed crosslinked poly(ε-caprolactone-co-D,L-lactide) (i.e., P(CL-DLLA)) material for cardiac patch, which was demonstrated properties including tissue-compatible, super-elastic nature, that made it suitable for long-term, in vivo RVOT repair. This super-elastic biomaterial could be useful for reconstruction of various muscular tissues deficiencies.

DOI： 10.1016/j.actbio.2021.01.022

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Springer Science and Business Media Deutschland GmbH  

We previously reported the effectiveness of autologous mesenchymal stromal cells (MSCs) for the treatment of aortic aneurysm (AA), mediated mainly by these cells’ anti-inflammatory properties. In this study, we investigate whether the therapeutic effects of allogeneic MSCs on AA are the same as those of autologous MSCs. To examine the immune response to allogeneic MSCs, C57BL/6 lymphocytes were co-cultured with BALB/c MSCs for 5 days in vitro. Apolipoprotein E-deficient C57BL/6 mice with AA induced by angiotensin II were randomly divided into three groups defined by the following intravenous injections: (i) 0.2 ml of saline (n = 10, group S) as a control, (ii) 1 × 106 autologous MSCs (isolated from C57BL/6, n = 10, group Au) and (iii) 1 × 106 allogeneic MSCs (isolated from BALB/c, n = 10, group Al). Two weeks after injection, aortic diameters were measured, along with enzymatic activities of MMP-2 and MMP-9 and cytokine concentrations in AAs. Neither allogenic (BALB/c) MSCs nor autologous (C57BL/6) MSCs accelerated the proliferation of lymphocytes obtained from C57BL/6. Compared with group S, groups Au and Al had significantly shorter aortic diameters (group S vs Au vs Al
2.29 vs 1.40 vs 1.36 mm, respectively, p &lt
 0.01), reduced MMP-2 and MMP-9 activities, downregulated IL-6 and MCP-1 and upregulated expression of IGF-1 and TIMP-2. There were no differences in these results between groups Au and Al. Thus, our study suggests that treatment with allogeneic MSCs improves chronic inflammation and reduced aortic dilatation. These effects were equivalent to those of autologous MSCs in established mouse models of AA.

DOI： 10.1007/s00441-020-03295-6

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：MDPI AG  

Vital pulp therapy is an important endodontic treatment. Strategies using growth factors and biological molecules are effective in developing pulp capping materials based on wound healing by the dentin-pulp complex. Our group developed biodegradable viscoelastic polymer materials for tissue-engineered medical devices. The polymer contents help overcome the poor fracture toughness of hydroxyapatite (HAp)-facilitated osteogenic differentiation of pulp cells. However, the composition of this novel polymer remained unclear. This study evaluated a novel polymer composite, P(CL-co-DLLA) and HAp, as a direct pulp capping carrier for biological molecules. The biocompatibility of the novel polymer composite was evaluated by determining the cytotoxicity and proliferation of human dental stem cells in vitro. The novel polymer composite with BMP-2, which reportedly induced tertiary dentin, was tested as a direct pulp capping material in a rat model. Cytotoxicity and proliferation assays revealed that the biocompatibility of the novel polymer composite was similar to that of the control. The novel polymer composite with BMP-2-induced tertiary dentin, similar to hydraulic calcium-silicate cement, in the direct pulp capping model. The BMP-2 composite upregulated wound healing-related gene expression compared to the novel polymer composite alone. Therefore, we suggest that novel polymer composites could be effective carriers for pulp capping.

DOI： 10.3390/polym12040937

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DOI： 10.1007/s11748-019-01195-3

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Authorship：Lead author   Language：English   Publishing type：Research paper (scientific journal)  

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Authorship：Lead author   Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.1155/2019/9104680

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OBJECTIVE: The pathogenesis of aortic aneurysm (AA) is associated with chronic inflammation in the aortic wall with increased levels of matrix metalloproteinases (MMPs). Clarithromycin (CAM) has been reported to suppresses MMP activity. In this study, we investigated whether CAM could prevent the formation and rupture of AA. METHODS: Male apolipoprotein E-deficient mice (28-30 weeks of age) were infused with angiotensin II for 28 days. CAM (100 mg/kg/d) or saline (as a control) was administered orally to the mice every day (CAM group, n = 13; control group, n = 13). After the administration period, the aortic diameter, elastin content, macrophage infiltration, MMP levels, and levels of inflammatory cytokines, including nuclear factor κB (NF-κB), were measured. RESULTS: The aortic diameter was significantly suppressed in the CAM group (P < .001). No rupture death was observed in the CAM group in contrast to five deaths (38%) in the control group (P < .01). CAM significantly suppressed the degradation of aortic elastin (56.3% vs 16.5%; P < .001) and decreased the infiltration of inflammatory macrophages (0.05 vs 0.16; P < .01). Compared with the controls, the enzymatic activity of MMP-2 and MMP-9 was significantly reduced in the CAM group (MMP-2, 0.15 vs 0.56 [P < .01]; MMP-9, 0.12 vs 0.60 [P < .01]), and the levels of interleukin 1β (346.6 vs 1066.0; P < .05), interleukin 6 (128.4 vs 346.2; P < .05), and phosphorylation of NF-κB were also decreased (0.3 vs 2.0; P < .01). CONCLUSIONS: CAM suppressed the progression and rupture of AA through the suppression of inflammatory macrophage infiltration, a reduction in MMP-2 and MMP-9 activity, and the inhibition of elastin degradation associated with the suppression of NF-κB phosphorylation.

DOI： 10.1016/j.jvs.2017.12.047

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Authorship：Lead author   Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.3390/polym10090947

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Nagoya University  

Recently, the effects of stem cell supernatants or exosomes, such as skin wounds, have attracted attention. However, the effects of the induced pluripotent stem (iPS) cell-derived exosomes (iPS-Exos) have not been investigated in detail. Here, we investigated the effects of iPS-Exos on skin wound healing using an animal model. We isolated iPS-Exos from the iPS cell culture media. Control exosomes were isolated from unused iPS cell culture media (M-Exos). We first observed the morphologic characteristics of the isolated exosomes and examined the expression of surface antigens. The effects of these exosomes on the migratory response and proliferation of fibroblasts were analyzed as well. Additionally, using a diabetic ulcer model, the effects of iPS-Exos and M-Exos on skin wound healing were investigated. Transmission electron microscope analysis demonstrated that the size of iPS-Exos (120 ± 25 nm) was significantly larger than that of M-Exos (≤ 100 nm). Flow cytometry analyses showed that iPS-Exos were positive for CD9, CD63, and CD81, whereas they were negative for HLA-ABC and -DR expression. The migratory ability of fibroblasts cocultured with iPS-Exos was shown to be higher than that of the cells cocultured with M-Exos, as demonstrated using scratch assay. Skin wound healing model results showed that the administration of iPS-Exos results in a faster wound closure compared with that observed in the M-Exo group. In conclusion, the results obtained here indicate that iPS-Exos may promote the migration of fibroblasts in vitro and in vivo, suggesting the possibility of using iPS-Exos for the treatment of diabetic ulcer.

DOI： 10.18999/nagjms.80.2.141

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER SCI LTD  

Background aims. We have confirmed that aortic aneurysm (AA) can be regressed by the administration of bone marrow derived mesenchymal stromal cells (BM-MSCs). We investigated the kinetics of signaling pathways in AA following treatment with BM-MSCs. Methods. Angiotensin II-infused apolipoprotein E-deficient mice were treated by intravenous injection of 1 x 10(6) BM-MSCs in 0.2 mL saline (BM-MSCs group, n = 5) or 0.2 mL saline (saline group, n = 5). Mice were sacrificed 2 weeks after injection and subjected to measurements of the incidence of AA and levels of phosphorylated proteins. Levels of proteins in conditioned media of BM-MSCs were also measured. Results. The incidence of AA in the BM-MSCs group was reduced (BM-MSC 40% versus saline 100%, P &lt; 0.05). Levels of pNF-kB and pSTAT1 were reduced (pNF-kB: 0.28 versus 0.45 unit/mL, P &lt; 0.05, pSTAT1: 0.16 versus 0.34, P &lt; 0.05), whereas levels of pAkt and pSmad3 were elevated (pAkt: 0.13 versus 0.07, P &lt; 0.01, pSmad3: 1.07 versus 0.47, P &lt; 0.05) in the BM-MSCs group. The levels of pNF-kB, pAkt, and pSmad3 were correlated with aortic diameters. Trophic factors including IGFPB-3, NRF, Activin A and PDGF-AA were secreted from BM-MSCs (IGFBP-3: 35.2 pg/mL, NRF: 3.1 pg/mL, Activin A: 3.1 pg/mL, PDGF-AA: 0.45 pg/mL). Conclusions. Our findings suggested that the therapeutic mechanism of BM-MSC mediated AA regression could contribute to regulation of the NF-kB, Smad3 and Akt signaling pathways. In addition, paracrine actions by factors including NRF, IGFBP-3, Activin A and PDGF-AA might have affected these signaling pathways.

DOI： 10.1016/j.jcyt.2017.07.010

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Language：Japanese   Publishing type：Research paper (scientific journal)   Publisher：(NPO)日本心臓血管外科学会  

Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER SCI LTD  

The aim of this study was to isolate mesenchymal stem cells (MSCs) from feline peripheral blood (fPB-MSCs) and to characterise the cells' in vitro properties. The mononuclear cell fractions were isolated from venous blood of cats by density gradient centrifugation and cultured on plastic dishes under various culture conditions to isolate MSCs. When these cells were cultured with 5% autologous plasma (AP) and 10% foetal bovine serum (FBS), adherent spindle shaped fibroblast-like cells (fPB-MSCs) were obtained from 15/22 (68%) cats. These cells were isolated only from medium containing both AP and FBS. The morphology of these MSCs was similar to those isolated from other species and from other feline tissues. fPB-MSCs expanded steadily up to 5-6 passages, but had increased population doubling time during passaging and almost all cells stopped proliferation at passages 7-9. These cells expressed CD44 and CD90, and were mostly negative for major histocompatibility class II and CD4. The cells could be induced to differentiate into adipogenic, osteogenic and chondrogenic cell lineages. These findings indicate that fPB-MSCs can be generated but appear to require specific culture conditions. (C) 2016 Elsevier Ltd. All rights reserved.

DOI： 10.1016/j.tvjl.2016.08.009

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Language：Japanese   Publishing type：Research paper (scientific journal)   Publisher：(NPO)日本心臓血管外科学会  

Language：English   Publishing type：Research paper (scientific journal)   Publisher：MARY ANN LIEBERT, INC  

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：LIPPINCOTT WILLIAMS & WILKINS  

Background: Impaired wound healing as one of the complications arising from diabetes mellitus is a serious clinical issue. Recently, various cell therapies have been reported for promotion of wound healing. Skin-derived precursor cells (SKPs) are multipotent adult stem cells with the tendency to differentiate into neurons. We investigated the potency of promoting diabetic wound healing by the application of SKPs.
Methods: Skin-derived precursor cells isolated from diabetic murine skin were cultured in sphere formation medium. At passage 2, they were suspended in phosphate-buffered saline (PBS), and applied topically to full-thickness excisional cutaneous wounds in diabetic mice. Application of PBS served as controls (n = 21 for each group; n = 42 total).
Time to closure and percentage closure were calculated by morphometry. Wounds were harvested at 10 and 28 days and then processed, sectioned, and stained (CD31, alpha-smooth muscle actin, and neurofilament heavy chain) to quantify vascularity and neurofilaments.
Results: Wounds treated with SKPs demonstrated a significantly decreased time to closure (18.63 days) compared with PBS-control wounds (21.72 days, P &gt; 0.01), and a significant improvement in percentage closure at 7, 10, 14, and 18 days compared with PBS-control wounds (P &gt; 0.01). Histological analysis showed that the Capillary Score (the number of vessels/mm(2)) was significantly higher in SKP-treated wounds at day 10 but not at day 28. Nerve Density (the number of neurofilaments/mm(2)) had increased significantly in SKP-treated wounds at day 28 compared with control group. Some applied SKPs were stained by neurofilament heavy chain, which demonstrates that SKPs directly differentiated into neurons.
Conclusions: Skin-derived precursor cells promoted diabetic wound healings through vasculogenesis at the early stage of wound healing. Skin-derived precursor cells are a possible therapeutic tool for diabetic impaired wound healing.

DOI： 10.1097/SAP.0000000000000342

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DOI： 10.4252/wjsc.v6.i3.278

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：OXFORD UNIV PRESS INC  

An aortic aneurysm (AA) is caused by atherosclerosis with chronic inflammation. Mesenchymal stem cells (MSCs) have potential anti-inflammatory properties. In this study, we examined whether an already-formed AA can be treated by intravenous injection of bone marrow-derived (BM)-MSCs in a mouse model.
AA was induced in apolipoprotein E-deficient mice by angiotensin II-infusion for 28 days through sub-cutaneous osmotic mini-pumps. After that, 1 x 10(6) BM-MSCs (in 0.2 ml saline) or 0.2 ml saline as a control was injected via the tail vein. Mice were sacrificed at 2 (saline group n = 10, BM-MSC group n = 10), 4 (saline group n = 6, BM-MSC group n = 7) or 8 weeks (saline group n = 5, BM-MSC group n = 6) after injection. The aortic tissues of each group were dissected. Aortic diameter, elastin content, matrix metalloproteinase (MMP)-2 and -9 enzymatic activity and cytokine concentrations were measured, as was macrophage infiltration, which was also evaluated histologically.
The incidence of AA in the BM-MSC group was reduced at 2 weeks (BM-MSC 40% vs saline 100%, P &lt; 0.05), and aortic diameter was reduced at 2 and 4 weeks (2 weeks: 1.40 vs 2.29 mm, P &lt; 0.001; 4 weeks: 1.73 vs 2.32 mm, P &lt; 0.05). The enzymatic activities of MMP-2 and -9 were reduced in the BM-MSC group at 2 weeks (active-MMP-2: 0.28 vs 0.45 unit/ml, P &lt; 0.05; active-MMP-9: 0.16 vs 0.34 unit/ml, P &lt; 0.05). Inflammatory cytokines were down-regulated in the BM-MSC group (interleukin-6: 2 weeks: 1475.6 vs 3399.5 pg/ml, P &lt; 0.05; 4 weeks: 2184.7 vs 3712.8 pg/ml, P &lt; 0.05 and monocyte chemotactic protein-1: 2 weeks: 208.0 vs 352.7 pg/ml, P &lt; 0.05) and insulin-like growth factor (IGF)-1 and tissue inhibitor of metalloproteinase (TIMP)-2 were up-regulated in the BM-MSC group at 2 weeks (IGF-1: 4.7 vs 2.0 ng/ml, P &lt; 0.05; TIMP-2: 9.5 vs 4.0 ng/ml, P &lt; 0.001). BM-MSC injection inhibited infiltration of M1 macrophages and preserved the construction of elastin.
Our results suggest that BM-MSCs might be an effective treatment for AA. Further investigation is necessary to optimize the injected dosage and the frequency of BM-MSCs to prevent a transient effect.

DOI： 10.1093/ejcts/ezu018

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Language：Japanese   Publishing type：Research paper (scientific journal)   Publisher：(NPO)日本心臓血管外科学会  

Language：English   Publishing type：Research paper (scientific journal)   Publisher：BIOMED CENTRAL LTD  

Background: Mesenchymal stem cells (MSCs) are known to be capable of suppressing inflammatory responses. We previously reported that intra-abdominal implantation of bone marrow-derived MSCs (BM-MSCs) sheet by laparotomy attenuated angiotensin II (AngII)-induced aortic aneurysm (AA) growth in apolipoprotein E-deficient (apoE(-/-)) mice through anti-inflammation effects. However, cell delivery by laparotomy is invasive; we here demonstrated the effects of multiple intravenous administrations of BM-MSCs on AngII-induced AA formation.
Methods: BM-MSCs were isolated from femurs and tibiae of male apoE(-/-) mice. Experimental AA was induced by AngII infusion for 28 days in apoE(-/-) mice. Mice received weekly intravenous administration of BM-MSCs (n=12) or saline (n=10). After 4 weeks, AA formation incidence, aortic diameter, macrophage accumulation, matrix metalloproteinase (MMP)' activity, elastin content, and cytokines were evaluated.
Results: AngII induced AA formation in 100% of the mice in the saline group and 50% in the BM-MSCs treatment group (P &lt; 0.05). A significant decrease of aortic diameter was observed in the BM-MSCs treatment group at ascending and infrarenal levels, which was associated with decreased macrophage infiltration and suppressed activities of MMP-2 and MMP-9 in aortic tissues, as well as a preservation of elastin content of aortic tissues. In addition, interleukin (IL)-1 beta, IL-6, and monocyte chemotactic protein-1 significantly decreased while insulin-like growth factor-1 and tissue inhibitor of metalloproteinases-2 increased in the aortic tissues of BM-MSCs treatment group.
Conclusions: Multiple intravenous administrations of BM-MSCs attenuated the development of AngII-induced AA in apoE(-/-) mice and may become a promising alternative therapeutic strategy for AA progression.

DOI： 10.1186/1479-5876-11-175

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OBJECTIVESArterial graft spasm occasionally causes circulatory collapse immediately following coronary artery bypass graft. The aim of this study is to evaluate the efficacy of our developed materials, which were composed of milrinone (phosphodiesterase III inhibitor) or diltiazem (calcium-channel blocker), with nano-scaled fibre made of biodegradable polymer to prevent arterial spasm.METHODSMilrinone- or diltiazem-releasing biodegradable nano-scaled fibres were fabricated by an electrospinning procedure. In vivo milrinone- or diltiazem-releasing tests were performed to confirm the sustained release of the drugs. An in vivo arterial spasm model was established by subcutaneous injection of noradrenalin around the rat femoral artery. Rats were randomly divided into four groups as follows: those that received 5 mg of milrinone-releasing biodegradable nano-scaled fibre (group M, n = 14)
5 mg of diltiazem-releasing biodegradable nano-scaled fibre (group D, n = 12)
or those that received fibre without drugs (as a control
group C, n = 14) implanted into the rat femoral artery. In the fourth group, sham operation was performed (group S, n = 10). One day after the implantation, noradrenalin was injected in all groups. The femoral arterial blood flow was measured continuously before and after noradrenalin injection. The maximum blood flow before noradrenalin injection and minimum blood flow after noradrenalin injection were measured.RESULTSIn vivo drug-releasing test revealed that milrinone-releasing biodegradable nano-scaled fibre released 78% of milrinone and diltiazem-releasing biodegradable nano-scaled fibre released 50% diltiazem on the first day. The ratios of rat femoral artery blood flow after/before noradrenalin injection in groups M (0.74 ± 0.16) and D (0.72 ± 0.05) were significantly higher than those of groups C (0.54 ± 0.09) and S (0.55 ± 0.16) (P &lt
0.05).CONCLUSIONNoradrenalin-induced rat femoral artery spasm was inhibited by the implantation of milrinone-releasing biodegradable nano-scaled fibre or diltiazem-releasing biodegradable nano-scaled fibre. These results suggested that our materials might be effective for the prevention of arterial graft spasm after coronary artery bypass graft. © 2013 The Author. Published by Oxford University Press on behalf of the European Association for Cardio-Thoracic Surgery. All rights reserved.

DOI： 10.1093/icvts/ivt092

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CiNii Research

Language：English   Publishing type：Research paper (scientific journal)   Publisher：SPRINGER JAPAN KK  

The concept of tissue engineered small-caliber vascular grafts (TE-SCVGs) is theoretically ideal. In this study, we evaluated the long-term (more than 1 year) course of TE-SCVGs using a rat carotid arterial replacement model. We fabricated a TE-SCVG scaffold (0.7 mm in diameter) with electrospun nano-scale fibers. Poly-epsilon-caprolactone was used as a biodegradable polymer. These artificial vessels were then used in carotid arterial replacement performed on Sprague-Dawley rats. The implanted grafts were removed at an early phase (1, 2, 6 weeks), middle phase (12, 24 weeks), and late phase (48, 72 weeks) after implantation. Twenty-nine patent grafts from among the 40 implanted grafts (patency 72.5 %) could be evaluated. No aneurysm formation was observed during the follow-up period. Endothelial cells positive for immunostaining with von Willebrand factor were found to be already attached to the inner surface of the TE-SCVGs in the early phase. The percentage of smooth muscle cell specific marker (a-smooth muscle actin and calponin with fluorescent immunostaining) positive cells, which seemed to be mesenchymal cells in the graft wall, increased with time, while, in contrast, the scaffold material decreased. Even after 72 weeks, however, although the scaffold material had degraded, it had not disappeared completely. These results show that the novel TE-SCVGs we developed were still functioning in the rat carotid arterial circulation after more than 1 year. However, further investigations will be required with regard to regeneration of the SMC layer and the complete degradation of graft materials.

DOI： 10.1007/s10047-012-0652-6

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：WILEY-BLACKWELL  

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Language：Japanese   Publishing type：Research paper (scientific journal)   Publisher：一般社団法人日本外科学会  

CiNii Research

Authorship：Lead author   Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.1111/j.1601-0825.2011.01871.x

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER SCIENCE INC  

Background. Both rapid endothelialization and the prevention of intimal hyperplasia are essential to improve the patency of small-caliber vascular grafts (SCVGs). Using the peptide array based screening system, we identified the peptide CAG (cysteine-alanine-glycine), which has a high affinity for endothelial cells and a low adhesive property for smooth muscle cells (SMCs). In this article, we report an in vivo analysis of novel vascular grafts that were constructed with a biodegradable polymer (poly-epsilon-caprolactone [PCL]) containing CAG peptide.
Methods. The novel SCVG, which measured 0.7 mm in diameter and 7 mm in length, was fabricated using the electrospinning technique. Carotid arterial replacement was performed on Sprague-Dawley rats using SCVGs with (group CAG) or without CAG (group C). Histologic and biochemical assessments were performed at 1, 2, and 6 weeks after implantation.
Results. The ratio of endothelialization was significantly higher in group CAG compared with group C (CAG versus C, 64.4 +/- 20.0% versus 42.1 +/- 8.9% at 1 week; p = 0.017; 98.2 +/- 2.3% versus 72.7 +/- 12.9% at 2 weeks; p = 0.001; and 97.4 +/- 4.6% versus 76.7 +/- 5.4% at 6 weeks; p &lt; 0.001). Additionally, Western blot analysis showed that the level of endothelial nitric oxide synthase (eNOS) at 1 week in group CAG was significantly higher than that in group C (CAG versus C, 1.20 +/- 0.37 versus 0.34 +/- 0.16; p = 0.013), and that alpha-smooth muscle actin (ASMA) at 6 weeks in group CAG was significantly lower than that in group C (CAG versus C, 0.89 +/- 0.06 versus 1.25 +/- 0.22; p = 0.04).
Conclusions. The graft with CAG promoted rapid endothelialization and the potential for inhibition of intimal hyperplasia. (Ann Thorac Surg 2012;93:156-63) (C) 2012 by The Society of Thoracic Surgeons

DOI： 10.1016/j.athoracsur.2011.07.055

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：MOSBY-ELSEVIER  

01,Objective:Aortic aneurysm (AA) is associated with loss of elastin and structural integrity, accompanied by increased matrix metalloproteinase (MMP) expression. These processes are supported by inflammatory macrophages, with mediators such as tumor necrosis factor-alpha (TNF-alpha). Mesenchymal stem cells (MSCs) contribute to aortic remodeling. Therefore, to clarify whether MSCs might be useful for AA cell therapy, we examined the effect of MSCs on vascular smooth muscle cells (SMCs) and macrophages in vitro, on aortic tissue ex vivo, and on aorta in vivo.
Methods: Murine macrophages and SMCs were cultured, with or without bone marrow-derived murine MSCs, for 96 hours in vitro. Gene expression of MMPs and TNF-alpha from macrophages and that of elastin from SMCs were measured. The murine aortic tissues were cultured with or without MSCs for up to 14 days, followed by measurement of MMP enzyme activity and elastin content. The in vivo aneurysm model used apolipoprotein E-deficient (apoE(-/-)) male mice receiving angiotensin (Ang II) infusion for 28 days. MSCs were implanted by laparotomy to the abdominal aortic adventitial surface from the superior mesenteric artery origin to the left renal artery. Age-matched apoE(-/-) mice with or without Ang II infusion were used for control groups. At the end point, aortic diameter, elastin content, MMPs' activity, and cytokines expressed, including interleukin-6 (IL-6), monocyte chemotactic protein-1 (MCP-1), TNF-alpha, insulin-like growth factor-1 (IGF-1), and tissue inhibitor of metalloproteinases-1 (TIMP-1) were quantified.
Results: MSCs suppressed MMP-2 with or without MSCs (2.59 vs 3.94, P &lt; .05), MMP-9 (5.83 vs 9.70, P &lt; .05), and TNF-alpha (2.79 vs 3.38, P &lt; .05) expression in macrophages, and promoted elastin expression in SMCs (19.35 vs 3.23, P &lt; .05) in vitro. MSCs also decreased active MMP-2 activity (0.310 vs 0.0609 U/mu L, P &lt; .05) and preserved elastin content (68.05 vs 40.29 mu g/mg, P &lt; .05) ex vivo. AA development was site-specifically inhibited (0.73 vs 1.04 mm aortic diameter, P &lt; .05) and elastin content was preserved (46.9 vs 25.6 mu g/mg, P &lt; .05) at 4 weeks. Downregulation of MMPs and IL-6, MCP-1, and TNF-alpha, and upregulation of IGF-1 and TIMP-1 were demonstrated with MSC implantation in vivo.
Conclusions: MSC implantation inhibits Ang II-induced AA development in apoE(-/-) mice through elastin preservation in the aortic wall and is associated with attenuated levels of MMTs and inflammatory cytokines. (J Vase Surg 2011;54:1743-52.)

DOI： 10.1016/j.jvs.2011.06.109

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：SPRINGER  

Tissue engineering is an effective approach for the treatment of bone defects. Statins have been demonstrated to promote osteoblastic differentiation of bone marrow-derived stromal cells (BMSCs). Electrospun biodegradable fibers have also shown applicability to drug delivery in the form of bone tissue engineered scaffolds with nano- to microscale topography and high porosity similar to the natural extracellular matrix (ECM). The aim of this study was to investigate the feasibility of a simvastatin-releasing, biodegradable, nano- to microscale fiber scaffold (SRBFS) for bone tissue engineering with BMSCs. Simvastatin was released from SRBFS slowly. BMSCs were observed to spread actively and rigidly adhere to SRBFS. BMSCs on SRBFS showed an increase in alkaline phosphatase activity 2 weeks after cell culture. Furthermore, osteoclastogenesis was suppressed by SRBFS in vitro. The new bone formation and mineralization in the SRBFS group were significantly better than in the biodegradable fiber scaffold (BFS) without simvastatin 12 weeks after implantation of the cell-scaffold construct into an ectopic site on the murine back. These results suggest that SRBFS promoted osteoblastic differentiation of BMSCs in vitro and in vivo, and demonstrate feasibility as a bone engineering scaffold.

DOI： 10.1007/s10439-011-0327-0

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Language：English   Publishing type：Part of collection (book)   Publisher：CRC Press  

Nanotechnology will play a great role in the field of cardiovascular disease in the near future. It will be a very useful tool in drug delivery approaches to treat cardiovascular disease. Nanomedicine (medical treatment using nanotechnology) can be considered a refinement of molecular medicine and integrates advances in genomics and proteomics to facilitate the development of personalized medicine. Nanomedicine for cardiovascular disease has focused on materials at super-molecular levels to deliver increased therapeutic efficacy and diminish adverse effects, which have ultimately resulted in their clinical application. All the aforementioned developments have revolutionized the application of nanotechnologies and have led to improved understanding of the possibilities for drug delivery to diseased tissues and organs. In this chapter we introduce a novel drug delivery system (DDS) using biodegradable nano-scaled fiber for use in cardiovascular medicine. Occasionally, systemic administration of the drug for cardiovascular diseases not only fails to work sufficiently, but also causes adverse side effects due to the limited effective threshold of the drug. Therefore, these medicines may need to be released locally and gradually. Our newly developed DDS applied biodegradable polymer mixed with a drug used for surgical treatment of cardiovascular diseases, to achieve sustained release. The mechanisms of sustained release of a drug are quite simple
the drug is released with diffusion, hydrolysis of the biodegradable polymer and/or degradation of polymer by enzymatic or phagocytotic effects in vivo. An electrospinning procedure is used to fabricate the nanofiber configurations for these materials. In this chapter, we review the DDS scaffold using electrospinning nanofiber technology, and also highlight our recent results using our new DDS with biodegradable nano-scaled fiber for cardiovascular diseases to prevent anastomotic stricture and treat aortic aneurysm.

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER SCI LTD  

The pathogenesis of aortic aneurysm (AA) is characterized by degradation of extracellular matrix with Increased matrix metalloproteinases (MMPs) and inflammatory reaction Doxycycline (DOXY) has been reported to control the extension of AA by regulation of MMP However systemic administration may cause adverse side effects In this study we demonstrated the possibility of local administration of DOXY controlled-release biodegradable fiber (DCRBF) for AA in mice DCRBF was fabricated by biodegradable polymer (polylactic acid PLA) mixed with DOXY using an electrospinning technique DCRBF was cocultured with SMCs macrophages and aortic tissue and placed on an abdominal aortic aneurysm which induced apolipoprotein E-deficient mice We evaluated gene and protein expression of proteases elastin and inflammatory markers In the presence of DCRBF MMP-12 was significantly decreased TGF-beta 1 and Lox were significantly increased in SMC gene expression MMP-9 and -12 significantly decreased gene expression of macrophages The DCRBF preserved elastin content and decreased MMP-2 and -9 in aortic tissue In addition IGF-1 and TIMP-1 were significantly increased and IL-6 and TNF-alpha were significantly decreased with DCRBF in vivo In conclusion our results suggested that local administration of DCRBF may become a promising alternative therapeutic strategy for AA (C) 2010 Elsevier Ltd All rights reserved

DOI： 10.1016/j.biomaterials.2010.08.069

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：KARGER  

Background: Bone marrow-derived mesenchymal stem cells (MSCs) can differentiate into various types of cell, and the extracellular matrix (ECM) is acknowledged to be important for the regulation of cell functions. In this study, we demonstrated the effects of ECMs on the differentiation of human bone marrow-derived MSCs into a smooth muscle cell (SMC) lineage. Methods: Human MSCs (hMSCs) were cultured on dishes coated with 3 types of ECM including laminin (LM), collagen type IV (Col-IV) and fibronectin for 7 days, and simultaneously cultured on a noncoated dish as a control. Cell numbers of these cultured hMSCs were counted, and their expression of SMC-specific genes and proteins was evaluated. hMSCs were then seeded on LM-coated biodegradable sheets and implanted into rat subcutaneous space. After 2 weeks of implantation, these tissues were evaluated. Results: The number of hMSCs was significantly increased by culture on Col-IV-coated dishes. The expression of SMC-specific genes and proteins (alpha-smooth muscle actin, ASMA; h1-calponin, CALP) in hMSC was significantly upregulated from culture on LM-coated dishes. LM-coated sheets showed
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a significantly increased expression of ASMA and CALP protein in vivo. Moreover, a fully differentiated marker (SM2) was expressed in the in vivo implanted hMSCs in the course of 2 weeks on the LM-coated sheet. Conclusion: These results suggest that the signal transduction of the cell-matrix interaction for the differentiation of hMSCs into SMCs was activated when cultured with LM. LM-coated materials may thus be useful for cardiovascular tissue engineering. Copyright (C) 2009 S. Karger AG, Basel

DOI： 10.1159/000260061

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Language：English   Publishing type：Research paper (international conference proceedings)  

Pulmonary venous obstruction (PVO) after correction of total anomalous pulmonary venous connection (TAPVC) frequently occurs due to intimal-hyperplasia and the required re-operation. We have developed a novel sustained-release drug delivery system, using Tacrolimuseluting biodegradable nano-fiber (TEBN). It consists of nano-scale fiber composed of biodegradable polymer and Tacrolimus. This study evaluated the effects of TEBN for prevention of venous anastomotic stricture in a rat model to apply to PVO operation. Tacrolimus was incorporated into poly (L-lactide-co-glycolide). The venous stricture model was made by rat inferior vena cava anastomosis. The IVC anastomosis was covered with TEBN with 1.0 wt% Tacrolimus (n=12) or without TEBN as a control (n=12), and evaluated histologically at 1, 2, and 4 weeks after operation. The ratio of intimal area was significantly reduced in the TEBN group compared with the control group (ratio
1 week: 0.43 ± 0.26 vs. 0.07 ± 0.04, P=0.04, 2 weeks: 0.39 ± 0.19 vs. 0.05 ± 0.02, P=0.01, 4 weeks: 0.31±0.15 vs. 0.09 ± 0.04, P=0.03, control vs. TEBN, respectively). Histological findings showed endothelialization along the inner surface of the vein even in TEBN. The TEBN reduced intimal hyperplasia and preserved endothelialization even in a venous stricture. These results suggested that this strategy might be useful for prevention of recurrent PVO after TAPVC correction. © 2009 Published by European Association for Cardio-Thoracic Surgery. All rights reserved.

DOI： 10.1510/icvts.2008.192831

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：MOSBY-ELSEVIER  

Objective: We developed a novel sustained drug-eluting device using tacrolimus-eluting biodegradable nanofiber to prevent anastomotic stricture and evaluated the effects in a rat abdominal aortic anastomosis model.
Methods: In vitro and in vivo tacrolimus release tests for tacrolimus-eluting biodegradable nanofiber were performed to confirm its sustained release. To verify the prevention of anastomotic stricture, tacrolimus-eluting biodegradable nanofiber was placed around the end-to-end anastomosis of abdominal aorta in rats. Five rats were allocated to the following 5 groups: ( 1) control without tacrolimus-eluting biodegradable nanofiber, ( 2) 5 mg of nanofiber only ( 0 wt% of tacrolimus), ( 3) 5 mg of tacrolimus-eluting biodegradable nanofiber containing 0.04 wt% of tacrolimus, ( 4) 5 mg of tacrolimus-eluting biodegradable nanofiber containing 0.1 wt% of tacrolimus, and ( 5) 5 mg of tacrolimus-eluting biodegradable nanofiber containing 1.0 wt% of tacrolimus. Morphometric and histologic analyses including immunohistochemistry were performed in each of the groups 2 weeks after the operation.
Results: The tacrolimus-eluting biodegradable nanofiber gradually released tacrolimus for at least 1 month in vitro and in vivo. The ratio of intimal area was significantly reduced in the 1.0 wt% tacrolimus-eluting biodegradable nanofiber group compared with the other groups (0.26, 0.24, 0.25, 0.21, and 0.08 in control, 0 wt%, 0.04 wt%, 0.1 wt%, and 1.0 wt%, respectively, P &lt;. 05). The cells, which constitute intimal hyperplasia, were positive for smooth muscle actin and SMemb, and factor VIII revealed that endothelial cells covered the surface of the aortic lumen even in the 1.0 wt% tacrolimus-eluting biodegradable nanofiber group in immunohistochemistry.
Conclusion: Tacrolimus-eluting biodegradable nanofiber reduced neointimal hyperplasia and preserved endothelialization. This device may be useful in the prevention of anastomotic stricture.

DOI： 10.1016/j.jtcvs.2008.11.017

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：SPRINGER  

Bone-marrow-derived mesenchymal stem cells (MSCs) can differentiate into a variety of cell types including smooth muscle cells (SMCs). We have attempted to demonstrate that, following treatment with transforming growth factor-beta 1 (TGF-beta 1) and ascorbic acid (AA), human bone-marrow-derived MSCs differentiate into the SMC lineage for use in tissue engineering. Quantitative polymerase chain reaction for SMC-specific gene (alpha smooth muscle actin, h1-calponin, and SM22 alpha) expression was performed on MSCs, which were cultured with various concentrations of TGF-beta 1 or AA. TGF-beta 1 had a tendency to up-regulate the expression of SMC-specific genes in a dose-dependent manner. The expression of SM22 alpha was significantly up-regulated by 30 mu M AA. We also investigated the additive effect of TGF-beta 1 and AA for differentiation into SMCs and compared this effect with that of other factors including platelet-derived growth factor BB (PDGF-BB). In addition to SMC-specific gene expression, SMC-specific proteins increased by two to four times when TGF-beta 1 and AA were used together compared with their administration alone. PDGF did not increase the expression of SMC-specific markers. MSCs cultured with TGF-beta 1 and AA did not differentiate into osteoblasts and adipocytes. These results suggest that a combination of TGF-beta 1 and AA is useful for the differentiation of MSCs into SMCs for use in tissue engineering.

DOI： 10.1007/s00441-008-0654-0

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Language：English   Publishing type：Research paper, summary (international conference)   Publisher：LIPPINCOTT WILLIAMS & WILKINS  

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Language：English   Publishing type：Research paper, summary (international conference)   Publisher：LIPPINCOTT WILLIAMS & WILKINS  

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Language：Japanese   Publisher：(NPO)日本心臓血管外科学会  

Language：English   Publishing type：Rapid communication, short report, research note, etc. (scientific journal)  

OBJECTIVES:<br />
The effect of growth differentiation factor 5 and bone morphogenetic protein 2 on human periodontal ligament-derived cells was investigated with special reference to tendo/ligamentogenesis-related markers.<br />
MATERIALS AND METHODS:<br />
Effects of each factor were analyzed by quantitative PCR for scleraxis and tenomodulin and by western blotting for scleraxis. After exposure to those factors, STRO-1-positive and STRO-1-negative fractions of human periodontal ligament tissues were isolated with an immunomagnetic cell sorting system, and the expression of scleraxis in each fraction was analyzed by western blotting. Non-separated crude cells were used as a control.<br />
RESULTS:<br />
Growth differentiation factor 5 and bone morphogenetic protein 2 did not increase alkaline phosphatase activity in crude periodontal ligament-derived cells. Growth differentiation factor 5, but not bone morphogenetic protein 2, increased the expression of scleraxis in crude, STRO-1-positive and STRO-1-negative periodontal ligament-derived cells. The expression of scleraxis in STRO-1-positive periodontal ligament-derived cells was significantly less compared to that in crude P2 and STRO-1-negative periodontal li

Language：Japanese   Publisher：(NPO)日本心臓血管外科学会  

Language：Japanese   Publisher：(一社)日本人工臓器学会  

Language：English  

Background and Objective: Although periodontal ligament-derived cells are expected to be a useful source of cells for periodontal tissue engineering, the characteristic changes of primary cultured cells have not been well studied. Therefore, the aim of this study was to investigate the characteristics of periodontal ligament-derived cells and their changes during passage. Material and Methods: Human periodontal ligament tissue was obtained from extracted third molars. Cells were subcultured until passage 6 and the cell characteristics from early to late passages were evaluated using immunofluorescence microscopy, alkaline phosphatase activity analyses, reverse transcription-polymerase chain reaction and quantitative real-time polymerase chain reaction. To examine the function of periodontal ligament-derived cells further, cells were transplanted into the renal subcapsule of an immunocompromised rat. Results: Immunofluorescence results showed relatively uniform expression of MSX-2 and osteonectin from passage 1 until passage 6. The STRO-1-positive fraction was 33.5% at passage 0, which was reduced to 14.7% at passage 3. Cultured cells at passage 1 expressed mRNA for collagen type I, collagen type XII, Runx2, alkaline phosphatase, osteonectin, osteopontin, scleraxis, tenomodulin, Msx2, GDF5 and GDF7 genes, but not for bone sialoprotein. The level of mRNA expression from tenomodulin and collagen type XII genes decreased after passage 3. Alkaline phosphatase activity decreased in cells at later passages. Osteogenic induction of periodontal ligament-derived cells resulted in a down-regulation of the tenomodulin gene. Transplanted cells from both early and late passages produced dense collagen fiber bundles without calcified tissue. Conclusion: Cultured periodontal ligament-derived cells were a morphologically homogeneous population, although expression of STRO-1 was limited in primary culture. Cultured cells showed de-differentiation during passage for both osteogenesis- and tendo/ligamentogenesis-related genes. © 2008 Blackwell Munksgaard.

DOI： 10.1111/j.1600-0765.2008.01137.x

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