Imiquimod is a TLR7/8 agonist that has anti-cancer therapeutic efficacy in the treatment of pre-cancerous skin lesions and certain non-melanoma skin cancers. To test our hypothesis that imiquimod enhances DNA repair as a mechanism for its anticancer activity, the nucleotide excision repair genes were studied in bone marrow derived cells. Imiquimod enhanced the expression of XPA and other DNA repair genes (qPCR analysis), and resulted in an increased nuclear localization of the DNA repair enzyme XPA. This was dependent on MyD88, as bone marrow derived cells from MyD88−/− mice did not increase XPA gene expression, and did not enhance the survival of MyD88−/− derived bone marrow derived cells after UVB exposure as was observed in bone marrow derived cells from MyD88+/+ mice. Imiquimod also enhanced DNA repair of UVL irradiated gene expression constructs, and accelerated the resolution of cyclobutane pyrimidine dimers (CBPD) after UVL exposures in P388 and XS52. Lastly, topical treatment of mouse skin with 5% imiquimod cream prior to UVL irradiation resulted in a decrease in the number of cyclobutane pyridimine dimer positive, APC that were found in local lymph nodes 24 hours after UVL irradiation in both wild type and IL-12 gene targeted mice. In total, these data support the idea that TLR7 agonists such as imiquimod enhance DNA repair in bone marrow derived cells. This property is likely to be an important mechanism for its anti-cancer effects, because it protects cutaneous APC from the deleterious effects of UVL.

A low concentration of boron (B) accelerates the proliferation and differentiation of mammalian osteoblasts. The aim of this study was to investigate the effects of 0.1 mM of B on the membrane function of osteoblastic cells in vitro. Genes involved in cell activity were investigated using gene expression microarray analyses. The Ca influx and efflux were evaluated to demonstrate the activation of L-type Ca channel for the Ca influx, and that of Na/K-ATPase for the Ca efflux. A real-time PCR analysis revealed that the messenger RNA (mRNA) expression of four mineralization-related genes was clearly increased after 3 days of culture with a B-supplemented culture medium. Using microarray analyses, five genes involved in cell proliferation and differentiation were upregulated compared to the control group. Regarding the Ca influx, in the nifedipine-pretreated group, the relative fluorescence intensity for 1 min after adding B solution did not increase compared with that for 1 min before addition. In the control group, the relative fluorescence intensity was significantly increased compared with the experimental group (P < 0.05). Regarding the Ca efflux, in the experimental group cultured in 0.1 mM of B-supplemented medium, the relative fluorescence intensity for 10 min after ouabain treatment revealed a significantly lower slope value compared with the control group (P < 0.01). This is the first study to demonstrate the acceleration of Ca flux by B supplementation in osteoblastic cells. Cell membrane stability is related to the mechanism by which a very low concentration of B promotes the proliferation and differentiation of mammalian osteoblastic cells in vitro.

A hypoxic condition is known to contribute to pluripotency. In the present article, the effects of transcription factors were first assessed regarding the proliferation and differentiation of human induced pluripotent stem (iPS) cells under hypoxic conditions using cell morphology and real-time polymerase chain reaction (RT-PCR). Morphology evaluations and RT-PCR revealed that the colony formation was promoted and the expression of pluripotent markers was increased under hypoxic conditions. In addition, the function of hypoxia inducible factors (HIFs) in human iPS cells under hypoxic conditions was evaluated in relation to the morphology and the expression of pluripotency markers by siRNA and RT-PCR. The HIF-2α silencing group showed a reduction in the colony size of human iPS cells and a statistically significant reduction in the expression of undifferentiation markers compared to the control group. Furthermore, the expression of HIF-2α was decreased when signal transducer and activator of transcription 3 (STAT3) was suppressed by its inhibitor, Stattic or S31 201. The inhibition using Stattic did not produce colony formation. The expression of pluripotent markers was also decreased using Stattic or S31 201. This study indicates that the HIF-2α expression in human iPS cells was activated under hypoxic conditions, similarly to that in murine iPS cells, and that HIF-2α among HIFs is the most effective compound for maintaining the pluripotency of human iPS cells. Furthermore, the STAT3 signal pathway regulates the expression of HIF-2α.

Fish collagen peptides (FCP) derived from the skin, bones and scales are commercially used as a functional food or dietary supplement for hypertension and diabetes. However, there is limited evidence on the effects of FCP on the osteoblast function in contrast to evidence of the effects on wound healing, diabetes and bone regeneration, which have been obtained from animal studies. In this narrative review, we expound on the availability of FCP by basic research using osteoblasts. Low-concentration FCP upregulates the expression of osteoblast proliferation, differentiation and collagen modifying enzyme-related genes. Furthermore, it could accelerate matrix mineralization. FCP may have potential utility as a biomaterial to improve collagen quality and promote mineralization through the mitogen-activated protein kinase and Smad cascades. However, there are few clinical studies on bone regeneration in human subjects. It is desirable to be applied clinically through clinical study as soon as possible, based on the results from basic research.

Tilapia type I atelocollagen (TAC) is a strong candidate for clinical application as its biological scaffold due to a high degeneration temperature and biologically safe properties. The aim of this study was to confirm the biological effects of TAC in vitro on osteoblastic cells, simulating its clinical application. The proliferation and differentiation of typical preosteoblasts, MC3T3-E1 cells, were investigated using a microarray analysis, staining assay for mineralization, and real-time PCR analysis of the expression of mineralization-related genes. The mRNA expression of 10 genes involved in proliferation and differentiation increased after 3-day culture on an TAC gel, with an average balanced score ratio exceeding 1.5 compared to the control. After two weeks of culture, all three experimental groups showed stronger alkaline phosphatase staining than after one week. The genes expression of alkaline phosphatase, osteocalcin, and bone sialoprotein increased under the experimental conditions. The gene expression of osteopontin did not increase, and no statistical differences were noted among the three experimental groups. The present and previous findings suggest that TAC is not only a suitable alternative to collagen products originating from mammals but also a novel biomaterial with cell differentiation ability for regenerative medicine.