In the present study, three-dimensional (3D) porous poly(lactic acid) (PLA) scaffold was fabricated by using progen and freeze-dry method and then fibronectin was immobilized after NaOH treatment on 3D porous scaffold. The biological effect of fibronectin immobilization on PLA scaffold was evaluated by the measurement of initial attachment of MC3T3-E1 osteoblast-like cells and by the morphological observation of attached cells. The number of attached cells onto FN-PLA scaffolds were significantly greater than on PLA scaffolds after 90 min culture (p<0.05). The attached cells were flattened and fastened on FN-PLA scaffold surface and the presence of lamelipodia and flopodia was recognized. Moreover the attached cell could be observed not only the surface of 3D porous PLA scaffold but also the inside of the scaffold by fibronectin immobilization. On the other hand, cells were rounded shape on PLA scaffold surface and there are no formation of lamelipodia and flopodia. The attached cells were not seen in the inside area of PLA scaffold.In conclusion, it revealed that fibronectin immobilization of 3D porous PLA scaffold was effective for biological activities of osteoblast-like cells.

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Orthodontic therapy applies physical stimulation to the compression-side periodontium, which produces inflammatory cytokines and receptor activators of nuclear factor kappa-B ligand (RANKL). With these proteins, osteoclast precursors such as monocytes and macrophages migrate from vessels into the periodontium and then differentiate into osteoclasts, causing bone resorption. Phosphatidylinositol 3-kinase (PI3-kinase) regulates this differentiation into osteoclasts and bone resorption. PDK1(3-phosphoinositide-dependent protein kinase 1) and Akt (protein kinase B) exist downstream of PI3-kinase. Therefore, in order to clarify the involvement of Akt in the differentiation into osteoclasts, we tested the effects of Akt Inhibitor V, RANKL, and the PDK inhibitor, OSU-03012 on the differentiation of RAW264.7 cells into osteoclasts. Both Akt Inhibitor V and OSU-03012 inhibited the differentiation into osteoclasts of RAW264.7 cells stimulated with RANKL. RANKL-induced phosphorylation of Akt (Thr³⁰⁸) was inhibited in RAW264.7 cells by OSU-03012, while RANKL-induced phosphorylation of Akt (Ser⁴⁷³) was not. These results suggest that Akt (Thr³⁰⁸, but not Ser⁴⁷³) exist upstream of the RANKL stimulation in the differentiation cascade of RAW264.7 cells into osteoclasts.

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Apoptosis of cardiomyocytes plays an important role in the development of cardiovascular diseases (CVD). Numerous of studies have investigated the relationship between periodontal and CVD, particularly myocardial infarction (MI) and stroke. Pathological sympathetic overactivation and vagal withdrawal are thought to reduce the survival rate after acute MI (AMI) and chronic heart failure. Recent studies also suggested that acetylcholine (ACh), a parasympathetic nerve system neurotransmitter, on the gap junction component connexin43 (Cx43) using H9c2 cells. We tested whether exogenous of ACh improved the H₂O₂-induced death of H9c2 cell. In this study, H₂O₂-induced H9c2 cardiomyoblasts death was not were improved by ACh (10, 100, 1000 μM). These results suggest that the ACh is not involved in the H₂O₂-induced death of H9c2 cell.

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The Embryo Stem Cell Test (EST) using mouse ES cell differentiation is an in vitro embryotoxicity screening test protocol. This EST protocol requires a 10-day or longer culture because the differentiation of contracting myocardial cells. Therefore, we developed a novel assessment method using alkaline phosphatase (ALP) activity as an endpoint, and compared the results obtained with the EST and our methods regarding four kinds of dental monomer. Bis-GMA was found to be weakly embryotoxic by the EST method, but non-embryotoxic by our method using ALP activity. No difference was found in the results for the other monomers by these methods. In the future, more data should be collected to establish our method.

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Porcine atelocollagen has traditionally been used as a scaffold for cell culture in the field of regenerative medicine. Marine collagen, rather than collagen derived from mammals has recently attracted attention. However, the collagen of fish (e.g., salmon) that inhabits cold oceans has already been demonstrated to be unsuitable as a three-dimensional scaffold for long-term culture at 37°C. In the present study, we examined whether type I collagen, extracted from the scales of tilapia inhabiting tropical and subtropical areas, can be used as a scaffold for ES cells. As a result, it had no effect on ES cell differentiation as compared with conventional porcine collagen. In a previous study, salmon collagen could not withstand long-term culture. Thus, the collagen obtained from tilapia, featuring a high denaturation temperature (35-37°C), is useful as a scaffold for ES cell differentiation.

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Tissue regeneration is the important issue in the field of operative dentistry. We demonstrated in series of studies that human bone marrow mesenchymal stem cells (hMSCs) can differentiate into odontblasts under the certain conditions (Izawa S et al., 2009). Mesenchymal stem cells can differentiate into multiple types of cells derived from mesenchyme (Liechty KW et al., 2000). At the same time odontblasts are primarily derived from mesenchyme; thus, we expected that mesenchymal stem cells can differentiate into odontblasts.The results in series of our study indicated that TNFα is necessary for hMSCs to differentiate into odontblasts and suggest that TNFα activates potential totipotency of hMSCs and the activation can lead to the regeneration of target cells or tissues.

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The influence of the reproduction and generating on the man of a biomaterial is still unknown. Spielmann et al. developed the embryonic stem cell test (EST), which is an in vitro embryotoxicity test method that can be used to estimate the risk of embryotoxicity of chemical substances relatively quickly compared to the conventional methods that involve animal experiments. The EST has been evaluated in a validation study by the European Centre for the Validation of Alternative Methods: ECVAM in which two other in vitro embryotoxicity tests (micromass test, whole embryo culture test) were validated against a set of test chemicals characterized by high levels of in vivo embryotoxicity data in laboratory animals and humans. In a validation study in Europe, the EST was found to be reproducible, demonstrating an overall accuracy of 80% and 100% correct prediction of strong embryotoxicity. In EST original protocol, there are some points involving the chemical characteristics of biomaterials that could be improved. Near future, we will release the 4-dimensional experimental model into which it was made to development.

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