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Generating mesenchymal stem-like cells (MSLCs) from induced pluripotent stem cells (iPSCs) can be a practical method for obtaining the sufficient cells for autologous tissue engineering. Single-cell culturing in specific medium and non-feeder cells is an alternative and promising strategy to overcome problems of embryo culture; however, little is known about how different culture media affect the proliferation and differentiation of MSLCs. We first derived MSLCs from iPSCs with non-integrating episomal plasmid vectors (hereafter 409B2 cells) using three different cell culture media, including single-cell culture medium in feeder-free condition: mTeSR1, DEF-CS500, or StemFit AK02N. The morphology of all MSLCs was completely altered to a fibroblastic morphology after four passages. Surface antigens CD29, CD44, CD73, CD90, but not CD34 and CD45, were expressed in all passages. RUNX2 was expressed in MSLCs cultured in all three feeder-free media, while SOX9 and PPARγ were expressed in MSLCs cultured in only DEF-CS500. MSLCs derived from DEF-CS500, which is a single-cell culture medium, grew at a slightly faster rate than those cultured in other media and expressed early-stage genes for tri-lineage differentiation. Taken together, these findings provide valuable information for generating MSLCs using single-cell culture methods. [ABSTRACT FROM AUTHOR]

We evaluated the effect of porous alpha-tricalcium phosphate (α-TCP) with immobilized basic fibroblast growth factor (bFGF) on periodontal regeneration in a canine model of 2-wall periodontal defects. Identical bone defects were made in the canine mandible; six defects in each animal were filled with porous α-TCP with bFGF bound via heparin (bFGF group), and the remaining defects were filled with unmodified porous α-TCP (control group). Micro-computed tomography and histological evaluation were performed at 2, 4, and 8 weeks post-implantation. The bone mineral content of the bFGF group was higher than that of the control group at 2 and 4 weeks (p < 0.05). Histological evaluation at 2 weeks post-implantation revealed degradation of the porous α-TCP, and bone had formed on the surface of α-TCP particles in the bFGF group. Some of these collagen fibers connected the newly formed cementum with the alveolar bone, revealing the formation of new periodontal ligaments with Sharpey’s fibers. At 8 weeks, continuous cortical bone with a Haversian structure covered the top of the bone defects in the bFGF group. These findings indicate that porous α-TCP with immobilized bFGF could promote periodontal regeneration at the early regeneration phase in a canine model of 2-wall periodontal defects. [ABSTRACT FROM AUTHOR]

This study investigated cell survival and gene expression under various compressive stress conditions mimicking orthodontic force by using a newly developed in vitro model of human periodontal ligament-like tissue (HPdLLT). The HPdLLT was developed by three-dimensional culturing of human periodontal ligament fibroblasts in a porous poly- l-lactide matrix with threefold increased culture media permeability due to hydrophilic modification. In vitro HPdLLTs in experimental groups were subjected to 5, 15, 25 and 35 g/cm compressive stress for 1, 3, 7 or 14 days; controls were cultured over the same periods without compressive stress. Cell morphology and cell apoptosis in the experimental and control groups were investigated using scanning electron microscopy and caspase-3/7 detection. Real-time polymerase chain reaction was performed for seven osteogenic and osteoclastic genes. Similar extracellular matrix and spindle-shaped cells were observed inside or on the surface of in vitro HPdLLTs, with no relation to compressive stress duration or intensity. Similar caspase-3/7 activity indicating comparable apoptosis levels was observed in all samples. Receptor activator of nuclear factor kappa-B ligand and bone morphogenetic protein 2 genes showed characteristic 'double-peak' expression at 15 and 35 g/cm on day 14, and alkaline phosphatase and periodontal ligament-associated protein 1 expression peaked at 5 g/cm on day 14; other genes also showed time-dependent and load-dependent expression patterns. The in vitro HPdLLT model system effectively mimicked the reaction and gene expression of the human periodontal ligament in response to orthodontic force. This work provides new information on the effects of compressive stress on human periodontal ligament tissue. [ABSTRACT FROM AUTHOR]

We compared the osteoblastic differentiation abilities of dedifferentiated fat cells (DFATs) and human bone marrow mesenchymal stem cells (hMSCs) as a cell source for bone regeneration therapies. In addition, the utility of DFATs in bone tissue engineering in vitro was assessed by an alpha-tricalcium phosphate (α-TCP)/collagen sponge (CS). Human DFATs were isolated from the submandibular of a patient by ceiling culture. DFATs and hMSCs at passage 3 were cultured in control medium or osteogenic medium (OM) for 14 days. Runx2 gene expression, alkaline phosphatase (ALP) activity, as well as osteocalcin (OCN) and calcium contents were analyzed to evaluate the osteoblastic differentiation ability of both cell types. DFATs seeded in a α-TCP/CS and cultured in OM for 14 days were analyzed by scanning electron microscopy (SEM) and histologically. Compared with hMSCs, DFATs cultured in OM generally underwent superior osteoblastogenesis by higher Runx2 gene expression at all days tested, as well as higher ALP activity at day 3 and 7, OCN expression at day 14, and calcium content at day 7. In SEM analyses, DFATs seeded in a α-TCP/CS were well spread and covered the α-TCP/CS by day 7. In addition, numerous spherical deposits were found to almost completely cover the α-TCP/CS on day 14. Von Kossa staining showed that DFATs differentiated into osteoblasts in the α-TCP/CS and formed cultured bone by deposition of a mineralized extracellular matrix. The combined use of DFATs and an α-TCP/CS may be an attractive option for bone tissue engineering. [ABSTRACT FROM AUTHOR]

Objectives: The purpose of this study was to evaluate and compare the osteoblastic differentiation ability of dedifferentiated fat (DFAT) cells and adipose stem cells (ASCs) from the buccal fat pad (BFP). Materials and methods: We isolated human DFAT cells and ASCs from the BFP of a patient who underwent oral and maxillofacial surgery and then analyzed their cell surface antigens by flow cytometry. Then, the cells were cultured in osteogenic medium for 14 days. Measurement of bone-specific alkaline phosphatase (BAP), osteocalcin (OCN), and calcium deposition and alizarin red staining were performed to evaluate the osteoblastic differentiation ability of both cell types. Results: ASCs and DFAT cells were positive for CD90 and CD105 and negative for CD11b, CD34, and CD45. BAP (days 3 and 7), OCN (day 14), and calcium deposition (days 7 and 14) within DFAT cell cultures were significantly higher than those in ASC cultures. The alizarin red-stained area in DFAT cell cultures, which indicates mineralized matrix deposition, was stained more strongly than that in ASC cultures. Conclusions: The cell surface antigens of ASCs and DFAT cells tend to be similar. Furthermore, the osteoblastic differentiation ability of human DFAT cells is higher than that of ASCs from the BFP. Clinical relevance: Isolation of DFAT cells from the BFP has an esthetic advantage because the BFP can be obtained via the oral cavity without injury to the external body surface. Therefore, we consider that DFAT cells from the BFP are an ideal cell source for bone tissue engineering. [ABSTRACT FROM AUTHOR]

Mature adipocyte-derived dedifferentiated fat (DFAT) cells rapidly differentiate into osteoblasts under three-dimensional culture conditions. However, it has not been demonstrated that DFAT cells can differentiate into osteoblasts in a rigid scaffold consisting of titanium fiber mesh (TFM). We examined the proliferation and osteogenic differentiation ability of DFAT cells using TFM as a scaffold. DFAT cells derived from rabbit subcutaneous fat were seeded into TFM and cultured in osteogenic medium containing dexamethasone, l-ascorbic acid 2-phosphate and β-glycerophosphate for 14 days. In scanning electron microscopy (SEM) analysis, well-spread cells covered the titanium fibers on day 3, and appeared to increase in number from day 3 to 7. Numerous globular accretions were found and almost completely covered the fibers on day 14. Cell proliferation, as measured by DNA content in the TFM, was significantly higher on day 7 compared with that of day 1. Osteocalcin and calcium content in the TFM were significantly higher on day 14 compared to those of days 1, 3, and 7, indicating DFAT cells differentiated into osteoblasts. We theorize that globular accretions observed in SEM analysis may be calcified matrix resulting from osteocalcin secreted by osteoblasts binding calcium contained in fetal bovine serum. In this study, we demonstrated that DFAT cells differentiate into osteoblasts and deposit mineralized matrices in TFM. Therefore, the combination of DFAT cells and TFM may be an attractive option for bone tissue engineering. [ABSTRACT FROM AUTHOR]