Development of novel waxy bone haemostatic agents composed of biodegradable polymers with osteogenic-enhancing peptides in rabbit models.

OBJECTIVES: The use of bone wax (BW) is controversial for sternal haemostasis because it increases the risk of wound infection and inhibits bone healing. We developed new waxy bone haemostatic agents made from biodegradable polymers containing peptides and evaluated them using rabbit models. METHODS: We designed 2 types of waxy bone haemostatic agents: peptide wax (PW) and non-peptide wax (NPW), which used poly(ε-caprolactone)-based biodegradable polymers with or without an osteogenesis-enhancing peptide, respectively. Rabbits were randomly divided into 4 groups based on treatment with BW, NPW, PW or no treatment. In a tibial defect model, the bleeding amount was measured and bone healing was evaluated by micro-computed tomography over 16 weeks. Bone healing in a median sternotomy model was assessed for 2 weeks using X-ray, micro-computed tomography, histological examination and flexural strength testing. RESULTS: The textures of PW and NPW (n = 12 each) were similar to that of BW and achieved a comparable degree of haemostasis. The crevice area of the sternal fracture line in the BW group was significantly larger than that in other groups (n = 10 each). The PW group demonstrated the strongest sternal flexural strength (n = 10), with complete tibial healing at 16 weeks. No groups exhibited wound infection, including osteomyelitis. CONCLUSIONS: Waxy biodegradable haemostatic agents showed satisfactory results in haemostasis and bone healing in rabbit models and may be an effective alternative to BW.

A review of current status of cell-based therapies for aortic aneurysms.

An aortic aneurysm (AA) is defined as focal aortic dilation that occurs mainly with older age and with chronic inflammation associated with atherosclerosis. The aneurysmal wall is a complex inflammatory environment characterized by endothelial dysfunction, macrophage activation, vascular smooth muscle cell (VSMC) apoptosis, and the production of proinflammatory molecules and matrix metalloproteases (MMPs) secreted by infiltrated inflammatory cells such as macrophages, T and B cells, dendritic cells, neutrophils, mast cells, and natural killer cells. To date, a considerable number of studies have been conducted on stem cell research, and growing evidence indicates that inflammation and tissue repair can be controlled through the functions of stem/progenitor cells. This review summarizes current cell-based therapies for AA, involving mesenchymal stem cells, VSMCs, multilineage-differentiating stress-enduring cells, and anti-inflammatory M2 macrophages. These cells produce beneficial outcomes in AA treatment by modulating the inflammatory environment, including decreasing the activity of proinflammatory molecules and MMPs, increasing anti-inflammatory molecules, modulating VSMC phenotypes, and preserving elastin. This article also describes detailed studies on pathophysiological mechanisms and the current progress of clinical trials.

Administration of anti-inflammatory M2 macrophages suppresses progression of angiotensin II-induced aortic aneurysm in mice.

Aortic aneurysm (AA) is a vascular disorder characterized pathologically by inflammatory cell invasion and extracellular matrix (ECM) degradation. It is known that regulation of the balance between pro-inflammatory M1 macrophages (M1Ms) and anti-inflammatory M2 macrophages (M2Ms) plays a pivotal role in AA stabilization. We investigated the effects of M2M administration in an apolipoprotein E-deficient (apoE-/-) mouse model in which AA was induced by angiotensin II (ATII) infusion. Mice received intraperitoneal administration of 1 million M2Ms 4 weeks after ATII infusion. Compared with a control group that was administered saline, the M2M group exhibited reduced AA expansion; decreased expression levels of interleukin (IL)-1ß, IL-6, tumor necrosis factor-α (TNF-α), and monocyte chemoattractant protein-1 (MCP-1); and a lower M1M/M2M ratio. Moreover, the M2M group exhibited upregulation of anti-inflammatory factors, including IL-4 and IL-10. PKH26-labeled M2Ms accounted for 6.5% of cells in the aneurysmal site and co-expressed CD206. Taken together, intraperitoneal administration of M2Ms inhibited AA expansion by reducing the inflammatory reaction via regulating the M1M/M2M ratio. This study shows that M2M administration might be useful for the treatment of AA.

Bi-layered carboxymethyl cellulose-collagen vitrigel dual-surface adhesion-prevention membrane.

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.

Alternative therapeutic strategy for existing aortic aneurysms using mesenchymal stem cell-derived exosomes.

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-1ß, 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.

Fabrication of Cationic Poly(vinyl alcohol) Films Cross-Linked Using Copolymers Containing Quaternary Ammonium Cations, Benzoxaborole, and Carboxy Groups.

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.

Long term efficacy and fate of a right ventricular outflow tract replacement using an elastomeric cardiac patch consisting of caprolactone and D,L-lactide copolymers.

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.

Therapeutic effect of allogeneic bone marrow-derived mesenchymal stromal cells on aortic aneurysms.

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 < 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.

Performance of a Biodegradable Composite with Hydroxyapatite as a Scaffold in Pulp Tissue Repair.

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.

Hydrogen water alleviates obliterative airway disease in mice.

OBJECTIVE: Bronchiolitis obliterans syndrome arising from chronic airway inflammation is a leading cause of death following lung transplantation. Several studies have suggested that inhaled hydrogen can protect lung grafts from ischemia-reperfusion injury via anti-inflammatory and -oxidative mechanisms. We investigated whether molecular hydrogen-saturated water can preserve lung allograft function in a heterotopic tracheal allograft mouse model of obliterative airway disease METHODS: Obliterative airway disease was induced by heterotopically transplanting tracheal allografts from BALB/c donor mice into C57BL/6 recipient mice, which were subsequently administered hydrogen water (10 ppm) or tap water (control group) (n = 6 each) daily without any immunosuppressive treatment. Histological and immunohistochemical analyses were performed on days 7, 14, and 21. RESULTS: Hydrogen water decreased airway occlusion on day 14. No significant histological differences were observed on days 7 or 21. The cluster of differentiation 4/cluster of differentiation 3 ratio in tracheal allografts on day 14 was higher in the hydrogen water group than in control mice. Enzyme-linked immunosorbent assay performed on day 7 revealed that hydrogen water reduced the level of the pro-inflammatory cytokine interleukin-6 and increased that of forkhead box P3 transcription factor, suggesting an enhancement of regulatory T cell activity. CONCLUSIONS: Hydrogen water suppressed the development of mid-term obliterative airway disease in a mouse tracheal allograft model via anti-oxidant and -inflammatory mechanisms and through the activation of Tregs. Thus, hydrogen water is a potential treatment strategy for BOS that can improve the outcome of lung transplant patients.

Montelukast, a Cysteinyl Leukotriene Receptor 1 Antagonist, Induces M2 Macrophage Polarization and Inhibits Murine Aortic Aneurysm Formation.

BACKGROUND: The pathogenesis of abdominal aortic aneurysm (AAA) is characterized by atherosclerosis with chronic inflammation in the aortic wall. Montelukast is a selective cys-LT 1 receptor antagonist that can suppress atherosclerotic diseases. We evaluated the in vitro properties of montelukast and its in vivo activities in an angiotensin II-infused apolipoprotein E-deficient (apoE-/-) AAA mouse model. METHODS: The mouse monocyte/macrophage cell line J774A.1 was used in vitro. M1 macrophages were treated with montelukast, and gene expressions of inflammatory cytokines were measured. Macrophages were cultured with montelukast, then gene expressions of arginase-1 and IL (interleukin)-10 were assessed by quantitative polymerase chain reaction, arginase-1 was measured by fluorescence-activated cell sorting, and IL-10 concentration was analyzed by enzyme-linked immunosorbent assay. In vivo, one group (Mont, n=7) received oral montelukast (10 mg/kg/day) for 28 days, and the other group (Saline, n=7) was given normal Saline as a control for the same period. Aortic diameters, activities of matrix metalloproteinases (MMPs), cytokine concentrations, and the number of M2 macrophages were analyzed. RESULTS: Relative to control, montelukast significantly suppressed gene expressions of MMP-2, MMP-9, and IL-1ß, induced gene expressions of arginase-1 and IL-10, enhanced the expression of the arginase-1 cell surface protein, and increased the protein concentration of IL-10. In vivo, montelukast significantly decreased aortic expansion (Saline vs Mont; 2.44 ± 0.15 mm vs 1.59 ± 0.20 mm, P<.01), reduced MMP-2 activity (Saline vs Mont; 1240 µM vs 755 µM, P<.05), and induced infiltration of M2 macrophages (Saline vs Mont; 7.51 % vs 14.7 %, P<.05). CONCLUSION: Montelukast induces M2 macrophage polarization and prevents AAA formation in apoE-/- mice.

The oral administration of clarithromycin prevents the progression and rupture of aortic aneurysm.

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 1ß (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.

Evaluation of the Bactericidal and Fungicidal Activities of Poly([2-(methacryloyloxy)ethyl]trimethyl Ammonium Chloride)(Poly (METAC))-Based Materials.

Poly([2-(methacryloyloxy)ethyl]trimethyl ammonium chloride) (METAC) and the gels were prepared and evaluated for their bactericidal and fungicidal activities. The antimicrobial properties of poly(METAC) were tested against Escherichia coli (E. coli), Bacillus subtilis (B. subtilis), Saccharomyces cerevisiae (Sa. cerevisiae), methicillin-susceptible Staphylococcus aureus (MSSA), methicillin-resistant Staphylococcus aureus (MRSA), Pseudomonas aeruginosa (P. aeruginosa), and Candida albicans (C. albicans). Moreover, the structural forms of the linear and cross-linked poly(METAC) were investigated for their influences on bacterial aggregation, precipitation, and cell-death. To our knowledge, this is the first report on the comparison of the antimicrobial properties of poly(METAC) and poly(METAC)-gels. The bactericidal and fungicidal activities were evaluated by determining minimum inhibitory concentrations (MICs), UV⁻Vis spectroscopy, and fluorescence and confocal microscopies. The MICs were found to be 123 (MSSA), 123 (MRSA), 123 (P. aeruginosa), 370 (E. coli), 123 (B. subtilis), 370 (C. albicans), and 370 µg/mL (Sa. cerevisiae), as determined by broth dilution, and 370 (MSSA), 370 (MRSA), 370 (P. aeruginosa), 3300 (E. coli), 370 (B. subtilis), 1100 (C. albicans), and >10,000 µg/mL (Sa. cerevisiae), as determined by paper disc diffusion (on solid medium). The poly(METAC)-gels achieved rapid adsorption/precipitation of bacteria via the cationic surface charge. Thus, these poly(METAC)-based polymers can potentially be used as antibacterial materials.

Bone marrow-derived mesenchymal stromal cells regress aortic aneurysm via the NF-kB, Smad3 and Akt signaling pathways.

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 × 106 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 <0.05). Levels of pNF-kB and pSTAT1 were reduced (pNF-kB: 0.28 versus 0.45 unit/mL, P <0.05, pSTAT1: 0.16 versus 0.34, P <0.05), whereas levels of pAkt and pSmad3 were elevated (pAkt: 0.13 versus 0.07, P <0.01, pSmad3: 1.07 versus 0.47, P <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.

Isolation and characterisation of peripheral blood-derived feline mesenchymal stem cells.

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.

Skin-derived precursor cells promote wound healing in diabetic mice.

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, α-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 < 0.01), and a significant improvement in percentage closure at 7, 10, 14, and 18 days compared with PBS-control wounds (P < 0.01). Histological analysis showed that the Capillary Score (the number of vessels/mm2) was significantly higher in SKP-treated wounds at day 10 but not at day 28. Nerve Density (the number of neurofilaments/mm2) 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.

Mesenchymal stem cells for treatment of aortic aneurysms.

An aortic aneurysm (AA) is a silent but life-threatening disease that involves rupture. It occurs mainly in aging and severe atherosclerotic damage of the aortic wall. Even though surgical intervention is effective to prevent rupture, surgery for the thoracic and thoraco-abdominal aorta is an invasive procedure with high mortality and morbidity. Therefore, an alternative strategy for treatment of AA is required. Recently, the molecular pathology of AA has been clarified. AA is caused by an imbalance between the synthesis and degradation of extracellular matrices in the aortic wall. Chronic inflammation enhances the degradation of matrices directly and indirectly, making control of the chronic inflammation crucial for aneurysmal development. Meanwhile, mesenchymal stem cells (MSCs) are known to be obtained from an adult population and to differentiate into various types of cells. In addition, MSCs have not only the potential anti-inflammatory and immunosuppressive properties but also can be recruited into damaged tissue. MSCs have been widely used as a source for cell therapy to treat various diseases involving graft-versus-host disease, stroke, myocardial infarction, and chronic inflammatory disease such as Crohn's disease clinically. Therefore, administration of MSCs might be available to treat AA using anti-inflammatory and immnosuppressive properties. This review provides a summary of several studies on "Cell Therapy for Aortic Aneurysm" including our recent data, and we also discuss the possibility of this kind of treatment.

Therapeutic potential of bone marrow-derived mesenchymal stem cells in formed aortic aneurysms of a mouse model.

OBJECTIVES: 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. METHODS: AA was induced in apolipoprotein E-deficient mice by angiotensin II-infusion for 28 days through sub-cutaneous osmotic mini-pumps. After that, 1 × 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. RESULTS: The incidence of AA in the BM-MSC group was reduced at 2 weeks (BM-MSC 40% vs saline 100%, P < 0.05), and aortic diameter was reduced at 2 and 4 weeks (2 weeks: 1.40 vs 2.29 mm, P < 0.001; 4 weeks: 1.73 vs 2.32 mm, P < 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 < 0.05; active-MMP-9: 0.16 vs 0.34 unit/ml, P < 0.05). Inflammatory cytokines were down-regulated in the BM-MSC group (interleukin-6: 2 weeks: 1475.6 vs 3399.5 pg/ml, P < 0.05; 4 weeks: 2184.7 vs 3712.8 pg/ml, P < 0.05 and monocyte chemotactic protein-1: 2 weeks: 208.0 vs 352.7 pg/ml, P < 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 < 0.05; TIMP-2: 9.5 vs 4.0 ng/ml, P < 0.001). BM-MSC injection inhibited infiltration of M1 macrophages and preserved the construction of elastin. CONCLUSIONS: 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.

Intravenous administration of mesenchymal stem cells prevents angiotensin II-induced aortic aneurysm formation in apolipoprotein E-deficient mouse.

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 < 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)-1ß, 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.

Prevention of arterial graft spasm in rats using a vasodilator-eluting biodegradable nano-scaled fibre.

OBJECTIVES: Arterial 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. METHODS: Milrinone- 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. RESULTS: In 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 < 0.05). CONCLUSION: Noradrenalin-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.