Gabriele Ruth Anisah Froemming

Orbital fluid shear stress promotes osteoblast metabolism, proliferation and alkaline phosphates activity in vitro.

Prolonged disuse of the musculoskeletal system is associated with reduced mechanical loading and lack of anabolic stimulus. As a form of mechanical signal, the multidirectional orbital fluid shear stress transmits anabolic signal to bone forming cells in promoting cell differentiation, metabolism and proliferation. Signals are channeled through the cytoskeleton framework, directly modifying gene and protein expression. For that reason, we aimed to study the organization of Normal Human Osteoblast (NHOst) cytoskeleton with regards to orbital fluid shear (OFS) stress. Of special interest were the consequences of cytoskeletal reorganization on NHOst metabolism, proliferation, and osteogenic functional markers. Cells stimulated at 250 RPM in a shaking incubator resulted in the rearrangement of actin and tubulin fibers after 72 h. Orbital shear stress increased NHOst mitochondrial metabolism and proliferation, simultaneously preventing apoptosis. The ratio of RANKL/OPG was reduced, suggesting that orbital shear stress has the potential to inhibit osteoclastogenesis and osteoclast activity. Increase in ALP activity and OCN protein production suggests that stimulation retained osteoblast function. Shear stress possibly generated through actin seemed to hold an anabolic response as osteoblast metabolism and functional markers were enhanced. We hypothesize that by applying orbital shear stress with suitable magnitude and duration as a non-drug anabolic treatment can help improve bone regeneration in prolonged disuse cases.

Short-term moderate hypothermia stimulates alkaline phosphatase activity and osteocalcin expression in osteoblasts by upregulating Runx2 and osterix in vitro.

Exposure of Normal Human Osteoblast cells (NHOst) to a period of hypothermia may interrupt their cellular functions, lead to changes in bone matrix and disrupt the balance between bone formation and resorption, resulting in bone loss or delayed fracture healing. To investigate this possibility, we exposed NHOst cells to moderate (35 °C) and severe (27 °C) hypothermia for 1, 12, 24 and 72 h. The effects of hypothermia with respect to cell cytoskeleton organization, metabolic activity and the expression of cold shock chaperone proteins, osteoblast transcription factors and functional markers, were examined. Our findings showed that prolonged moderate hypothermia retained the polymerization of the cytoskeletal components. NHOst cell metabolism was affected differently according to hypothermia severity. The osteoblast transcription factors Runx2 and osterix were necessary for the transcription and translation of bone matrix proteins, where alkaline phosphatase (Alp) activity and osteocalcin (OCN) bone protein were over expressed under hypothermic conditions. Consequently, bone mineralization was stimulated after exposure to moderate hypothermia for 1 week, indicating bone function was not impaired. The cold shock chaperone protein Rbm3 was significantly upregulated (p<0.001) during the cellular stress adaption under hypothermic conditions. We suggest that Rbm3 has a dual function: one as a chaperone protein that stabilizes mRNA transcripts and a second one in enhancing the transcription of Alp and Ocn genes. Our studies demonstrated that hypothermia permitted the in vitro maturation of NHOst cells probably through an osterix-dependent pathway. For that reason, we suggest that moderate hypothermia can be clinically applied to counteract heat production at the fracture site that delays fracture healing.

Human Papilloma Virus 18 Detection in Oral Squamous Cell Carcinoma and Potentially Malignant Lesions Using Saliva Samples

BACKGROUND Oral cancer has become one of the most prevalent cancers worldwide and human Papillomavirus is one of the risk factors for developing oral cancer. For this study HPV18 was chosen as it is one of the high risk HPV types and may lead to carcinogenesis. However, prevalence of HPV18 infection in Oral Squamous Cell Carcinoma in Malaysia remains unclear. OBJECTIVE This study aimed to investigate the viral load of HPV18 DNA in OSCC and potentially malignant lesions using saliva samples. MATERIALS AND METHODS Genomic DNAs of thirty saliva samples of normal subjects and thirty saliva samples compromised of 16 samples from potentially malignant lesions and 14 of OSCC patients were amplified for HPV18 DNA using a nested polymerase chain reaction analysis. All PCR products were then analyzed using the Bioanalyzer to confirm presence of HPV18 DNA. RESULT From thirty patients examined, only one of 30 (3.3%) cases was found to be positive for HPV18 in this study. CONCLUSION The finding of this study revealed that there is a low viral detection of HPV18 in Malaysian OSCC by using saliva samples, suggesting that prevalence of HPV18 may not be important in this group of Malaysian OSCC.

Delta- and gamma-tocotrienol isomers are potent in inhibiting inflammation and endothelial activation in stimulated human endothelial cells

Background Tocotrienols (TCTs) are more potent antioxidants than α-tocopherol (TOC). However, the effectiveness and mechanism of the action of TCT isomers as anti-atherosclerotic agents in stimulated human endothelial cells under inflammatory conditions are not well established. Aims 1) To compare the effects of different TCT isomers on inflammation, endothelial activation, and endothelial nitric oxide synthase (eNOS). 2) To identify the two most potent TCT isomers in stimulated human endothelial cells. 3) To investigate the effects of TCT isomers on NFκB activation, and protein and gene expression levels in stimulated human endothelial cells. Methods Human umbilical vein endothelial cells were incubated with various concentrations of TCT isomers or α-TOC (0.3–10 µM), together with lipopolysaccharides for 16 h. Supernatant cells were collected and measured for protein and gene expression of cytokines (interleukin-6, or IL-6; tumor necrosis factor-alpha, or TNF-α), adhesion molecules (intercellular cell adhesion molecule-1, or ICAM-1; vascular cell adhesion molecule-1, or VCAM-1; and e-selectin), eNOS, and NFκB. Results δ-TCT is the most potent TCT isomer in the inhibition of IL-6, ICAM-1, VCAM-1, and NFκB, and it is the second potent in inhibiting e-selectin and eNOS. γ-TCT isomer is the most potent isomer in inhibiting e-selectin and eNOS, and it is the second most potent in inhibiting is IL-6, VCAM-1, and NFκB. For ICAM-1 protein expression, the most potent is δ-TCT followed by α-TCT. α- and β-TCT inhibit IL-6 at the highest concentration (10 µM) but enhance IL-6 at lower concentrations. γ-TCT markedly increases eNOS expression by 8–11-fold at higher concentrations (5–10 µM) but exhibits neutral effects at lower concentrations. Conclusion δ- and γ-TCT are the two most potent TCT isomers in terms of the inhibition of inflammation and endothelial activation whilst enhancing eNOS, possibly mediated via the NFκB pathway. Hence, there is a great potential for TCT isomers as anti-atherosclerotic agents.

Morphological and phenotypical characteristics of human osteoblasts after short-term space mission.

Morphological and phenotypical signs of cultured readaptation osteoblasts were studied after a short-term space mission. The ultrastructure and phenotype of human osteoblasts after Soyuz TMA-11 space flight (2007) were evaluated by scanning electron microscopy, laser confocal microscopy, and ELISA. The morphofunctional changes in cell cultures persisted after 12 passages. Osteoblasts retained the drastic changes in their shape and size, contour deformation, disorganization of the microtubular network, redistribution of organelles and specialized structures of the plasmalemma in comparison with the ground control cells. On the other hand, the expression of osteoprotegerin and osteocalcin (bone metabolism markers) increased; the expression of bone resorption markers ICAM-1 and IL-6 also increased, while the expression of VCAM-1 decreased. Hence, space flight led to the development of persistent shifts in cultured osteoblasts indicating injuries to the cytoskeleton and the phenotype changes, indicating modulation of bone metabolism biomarkers.

Employing different types of phytoestrogens improve bone mineralization in bisphenol A stimulated osteoblast

Aims: Phytoestrogens and xenoestrogens act as agonists/antagonists in bone formation and differentiation. Strong bones are depending of the ability of osteoblasts to form new tissue and to mineralize the newly formed tissue. Dysfunctional or loss of mineralization leads to weak bone and increased fracture risk. In this study, we reported the effect of different types of phytoestrogens (daidzein, genistein and equol) on mineralization in hFOB 1.19 cells stimulated with bisphenol A (BPA). Main methods: Cell mineralization capacity of phytoestrogens was investigated by evaluating calcium, phosphate content and alkaline phosphatase activity. Bone related markers, osteocalcin and osteonectin, responsible in maintaining mineralization were also measured. Key findings: BPA is significantly interfering with bone mineralization in hFOB 1.19 cells. However, the enhanced mineralization efficacy of daidzein and genistein (particularly at a dose of 5 and 40 &mgr;g/mL, respectively) was evidenced by increasing calcium and phosphate content, higher ALP activity, compared to the untreated BPA group. The quantitative analyses were confirmed through morphological findings. Osteocalcin and osteonectin levels were increased in phytoestrogens‐treated cells. These findings revealed the potential effect of phytoestrogens in reverting the demineralization process due to BPA exposure in hFOB 1.19 cells. Significance: We found that osteoblast differentiation and mineralization were maintained following treatment with phytoestrogens under BPA exposure.