New wrinkling substrate assay reveals traction force fields of leader and follower cells undergoing collective migration.

Physical forces play crucial roles in coordinating collective migration of epithelial cells, but details of such force-related phenomena remain unclear partly due to the lack of robust methodologies to probe the underlying force fields. Here we develop a method for fabricating silicone substrates that detect cellular traction forces with a high sensitivity. Specifically, a silicone elastomer is exposed to oxygen plasma under heating. Removal of the heat shrinks the substrate so as to reduce its critical buckling strain in a spatially uniform manner. Thus, even small cellular traction forces can be visualized as micro-wrinkles that are reversibly emerged on the substrate in a direction orthogonal to the applied forces. Using this technique, we show that so-called leader cells in MDCK-II cell clusters exert significant magnitudes of traction forces distinct from those of follower cells. We reveal that the direction of traction forces is highly correlated with the long axis of the local, individual cells within clusters. These results suggest that the force fields in collective migration of MDCK-II cells are predominantly determined locally at individual cell scale rather than globally at the whole cell cluster scale.

Melatonin­induced miR­181c­5p enhances osteogenic differentiation and mineralization of human jawbone­derived osteoblastic cells.

Our previous study revealed that treatment with a combination of fibroblast growth factor­2 and melatonin (MEL) synergistically augmented osteogenic activity and mineralization of MC3T3­E1 mouse preosteoblast cells. Thus, the objective of the present study was to assess the effect of MEL on osteogenetic characteristics in human osteoblastic cells. Human jawbone­derived osteoblastic (hOB) cells were isolated from mandibular bone fragments. RUNX family transcription factor 2 (Runx2) expression, alkaline phosphatase (ALP) enzyme activity and the mineralization ability of hOB cells in the presence of MEL were evaluated. Microarray analysis was also performed to assess the expression of MEL­induced microRNAs (miRNAs/miRs) in hOB cells. Treatment with MEL significantly enhanced Runx2 expression, ALP activity and mineralization staining. However, this effect was significantly reduced following transforming growth factor­ß1 treatment. In total, 124 miRNAs were differentially expressed in MEL­treated hOB cells, compared with untreated cells. Of the upregulated miRNAs, miR­181c­5p exhibited the largest fold change. Runx2 mRNA expression and mineralization staining in the presence of MEL were significantly reduced following transfection with a miR­181c­5p inhibitor. In addition, transfection with miR-181c-5p mimics significantly increased Runx2 expression and mineralization staining. These results suggested that MEL­induced miR­181c­5p was involved in osteogenic differentiation and mineralization of hOB cells. Using TargetScan, a putative miR­181c­5p binding site was identified in the Notch2 gene. Moreover, Notch2 mRNA and protein expression levels in hOB cells were significantly reduced following transfection with miR­181c­5p mimics, confirming Notch2 as a target gene for miR­181c­5p. Notch2 siRNA knockdown significantly increased Runx2 expression and mineralization staining, which suggested that Notch2 may negatively regulate osteogenic differentiation of hOB cells by downregulating Runx2. In conclusion, MEL­induced expression of miR­181c­5p enhanced osteogenic differentiation and calcification of hOB cells.

High HPV16 E6 viral load in the oral cavity is associated with an increased number of bacteria: A preliminary study.

In a previous study, the present research group reported that males had a significantly higher prevalence of human papillomavirus (HPV)16 than females in oral rinse samples. The objective of the present study was to examine the relationship between HPV16 viral load and clinical factors, including remaining teeth, denture use and numbers of oral bacteria. A total of 124 patients (48 males and 76 females; mean age, 61.6 years; age range, 20-97 years) who visited the Department of Oral and Maxillofacial Reconstructive Surgery of Hiroshima University Hospital (Hiroshima, Japan) between November 2016 and August 2017 were analyzed. None of the patients had evidence of oral cancer or pre-malignant lesions, including epithelial dysplasia and leukoplakia. Quantitative polymerase chain reaction (qPCR) analysis was employed to examine the number of HPV16 viral copies. Furthermore, the number of oral bacteria was determined using the dielectrophoretic impedance measurement method. HPV16 was below the limit of detection in qPCR findings for samples obtained from 30 of the 124 subjects, thus the association of HPV16 viral copy number with clinical parameters was examined in the remaining 94 patients. The average number of HPV16 E6 DNA copies was 1.65±3.47 copies/cell (range, 0.07-25.3 copies/cell) and was significantly higher in subjects with a high oral bacteria count [≥106.5 colony forming unit (CFU)/ml] than in those with a low count (<106.5 CFU/ml) (0.79±0.98 vs. 2.06±4.11 copies/cell; P=0.030). The present results indicated that HPV16 viral load may be related to an increased bacterial number in the oral cavity. Further investigations are required to clarify the correlation between oral HPV load and oral hygiene status.

Microcontact Peeling: A Cell Micropatterning Technique for Circumventing Direct Adsorption of Proteins to Hydrophobic PDMS.

Microcontact printing (µCPr) is one of the most popular techniques used for cell micropatterning. In conventional µCPr, a polydimethylsiloxane (PDMS) stamp with microfeatures is used to adsorb extracellular matrix (ECM) proteins onto the featured surface and transfer them onto particular areas of a cell culture substrate. However, some types of functional proteins other than ECM have been reported to denature upon direct adsorption to hydrophobic PDMS. Here we describe a detailed protocol of an alternative technique--microcontact peeling (µCPe)--that allows for cell micropatterning while circumventing the step of adsorbing proteins to bare PDMS. This technique employs microfeatured materials with a relatively high surface energy such as copper, instead of using a microfeatured PDMS stamp, to peel off a cell-adhesive layer present on the surface of substrates. Consequently, cell-nonadhesive substrates are exposed at the specific surface that undergoes the physical contact with the microfeatured material. Thus, although µCPe and µCPr are apparently similar, the former does not comprise a process of transferring biomolecules through hydrophobic PDMS. © 2017 by John Wiley & Sons, Inc.

Microcontact peeling as a new method for cell micropatterning.

Micropatterning is becoming a powerful tool for studying morphogenetic and differentiation processes of cells. Here we describe a new micropatterning technique, which we refer to as microcontact peeling. Polydimethylsiloxane (PDMS) substrates were treated with oxygen plasma, and the resulting hydrophilic layer of the surface was locally peeled off through direct contact with a peeling stamp made of aluminum, copper, or silicon. A hydrophobic layer of PDMS could be selectively exposed only at the places of the physical contact as revealed by water contact angle measurements and angle-resolved X-ray photoelectron spectroscopy, which thus enabled successful micropatterning of cells with micro-featured peeling stamps. This new micropatterning technique needs no procedure for directly adsorbing proteins to bare PDMS in contrast to conventional techniques using a microcontact printing stamp. Given the several unique characteristics, the present technique based on the peel-off of inorganic materials may become a useful option for performing cell micropatterning.