MATERIALS AND METHODS: Human adipose-derived stem cells at passage 4 were evaluated by flow cytometry to examine the expression of surface markers. These adipose-derived stem cells were tested for adipogenic and osteogenic differentiation capacity. Ribonucleic acid was extracted from the cells for quantitative polymerase chain reaction analysis to determine the expression levels of chondrogenic genes after chondrogenic induction.
RESULTS: Human adipose-derived stem cells were strongly positive for the mesenchymal markers CD90, CD73, CD44, CD9, and histocompatibility antigen and successfully differentiated into adipogenic and osteogenic lineages. The human adipose-derived stem cells aggregated and formed a dense matrix after chondrogenic induction. The expression of chondrogenic genes (collagen type II, aggrecan core protein, collagen type XI, COMP, and ELASTIN) was significantly higher after the first week of induction. However, a significantly elevated expression of collagen type X was observed after three weeks of chondrogenic induction.
CONCLUSION: Human adipose-derived stem cells retain stem cell characteristics after expansion in culture to passage 4 and serve as a feasible source of cells for cartilage regeneration. Chondrogenesis in human adipose-derived stem cells was most prominent after one week of chondrogenic induction.
MATERIALS AND RESULTS: Thirty-five paraffin-embedded ameloblastoma cases, ameloblastoma-derived cell lines (AM-1), and primary cultures of ameloblastoma stromal fibroblasts (ASF) were used. Immunohistochemistry, MTT assay, Western blotting, and RT-PCR were performed on these samples. Parenchyma-stromal CCN2 overexpression correlated significantly with fibrous-type stroma, but not with myxoid-type stroma, suggesting a role of CCN2 in fibrosis (P < 0.05). Recombinant CCN2 induction of enhanced ASF proliferation in AM-1 medium supports this view. Conversely, BMP4 and TGF-β were expressed in myxoid-type fibroblasts, but little expression was found in parenchyma. RANKL-positive and CD68-positive stromal cell populations were significantly greater in myxoid-type tumor areas than in fibrous-type tumor areas, while a higher Ki-67 labeling index was recorded in ameloblastoma with fibrous-type stroma. These data suggest that stromal properties influence bone resorption-related activities and growth rates, respectively.
CONCLUSIONS: These results suggest that the effects of secreted growth factors are governed by ameloblastoma parenchyma-stromal interactions. CCN2 promotes fibrogenesis independent of TGF-β signaling. Absence of CCN2 expression is associated with a phenotypic switch to a myxoid-type microenvironment that is conducive for TGF-β/BMP4 signaling to promote osteoclastogenesis.
MATERIALS AND METHOD: Eighty-seven paraffin-embedded ameloblastoma cases (20 unicystic, 47 solid/multicystic, 3 desmoplastic and 17 recurrent) were subjected to immunohistochemistry for expression of cortactin, N-WASP, WIP, Src kinase and F-actin, and findings correlated with clinicopathological parameters.
RESULTS: Invadopodia proteins (except Src kinase) and F-actin were widely detected in ameloblastoma (cortactin: n = 73/87, 83.9%; N-WASP: n = 59/87; 67.8%; WIP: n = 77/87; 88.5%; and F-actin: n = 87/87, 100%). Protein localization was mainly cytoplasmic and/or membranous, and occasionally nuclear for F-actin. Cortactin, which functions as an actin-scaffolding protein, demonstrated significantly higher expression levels within ameloblastoma tumoral epithelium than in stroma (P < 0.05). N-WASP, which coordinates actin polymerization and invadopodia-mediated extracellular matrix degradation, was overexpressed in the solid/multicystic subtype (P < 0.05). WIP, an upstream regulator of N-WASP, and F-actin were significantly upregulated along the tumour invasive front compared to tumour centres (P < 0.05). Except for males with cortactin overexpression, other clinical parameters (age, ethnicity and anatomical site) showed no significant correlations.
CONCLUSIONS: Present results suggest that local invasiveness of ameloblastoma is dependent upon the migratory potential of its tumour cells as defined by their distribution of cortactin, N-WASP and WIP in correlation with F-actin cytoskeletal dynamics.
OBJECTIVES: This study has aimed to establish optimum conditions to generate and characterize MSC from human umbilical cord (UC-MSC).
MATERIALS AND METHODS: To optimize MSC population growth, basic fibroblast growth factor (bFGF) was utilized in culture media. Effects of bFGF on expansion kinetics, cell cycle, survival of UC-MSC, cytokine secretion, expression of early stem-cell markers and immunomodulation were investigated.
RESULTS: bFGF supplementation profoundly enhanced UC-MSC proliferation by reducing population doubling time without altering immunophenotype and immunomodulatory function of UC-MSC. However, cell cycle studies revealed that bFGF drove the cells into the cell cycle, as a higher proportion of cells resided in S phase and progressed into M phase. Consistent with this, bFGF was shown to promote expression of cyclin D proteins and their relevant kinases to drive UC-MSC to transverse cell cycle check points, thus, committing the cells to DNA synthesis. Furthermore, supplementation with bFGF changed the cytokine profiles of the cells and reduced their apoptotic level.
CONCLUSION: Our study showed that bFGF supplementation of UC-MSC culture enhanced the cells' growth kinetics without compromising their nature.
METHODS: MSCs and Oh-LAAO were isolated and characterized by standard methodologies. The effects of the experimental therapies were evaluated in C57/BL6 mice. The animal study groups consisted of full-thickness uninfected and MRSA-infected wound models which received Oh-LAAO, MSCs, or both. Oh-LAAO was administered directly on the wound while MSCs were delivered via intradermal injections. The animals were housed individually with wound measurements taken on days 0, 3, and 7. Histological analyses and bacterial enumeration were performed on wound biopsies to determine the efficacy of each treatment.
RESULTS: Immunophenotyping and differentiation assays conducted on isolated MSCs indicated expression of standard cell surface markers and plasticity which corresponds to published data. Characterization of Oh-LAAO by proteomics, enzymatic, and antibacterial assays confirmed the identity, purity, and functionality of the enzyme prior to use in our subsequent studies. Individual treatments with MSCs and Oh-LAAO in the infected model resulted in reduction of MRSA load by one order of magnitude to the approximate range of 6 log10 colony-forming units (CFU) compared to untreated controls (7.3 log10 CFU). Similar wound healing and improvements in histological parameters were observed between the two groups. Co-administration of MSCs and Oh-LAAO reduced bacterial burden by approximately two orders of magnitude to 5.1 log10 CFU. Wound closure measurements and histology analysis of biopsies obtained from the combinational therapy group indicated significant enhancement in the wound healing process compared to all other groups.
CONCLUSIONS: We demonstrated that co-administration of MSCs and Oh-LAAO into a mouse model of MRSA-infected wounds exhibited a synergistic antibacterial effect which significantly reduced the bacterial count and accelerated the wound healing process.