Browsing by Author "Windsor, Lester Jack"

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    Kalirin : novel role in osteocyte function
    (2013) Wayakanon, Kornchanok; Bruzzaniti, Angela; Windsor, Lester Jack; Chu, Tien-Min Gabriel; Cho, Sopanis D.; Cook, Norman Blaine, 1954-
    Communication between bone cells is important for the maintenance of bone mass. Although osteocytes are deeply embedded within the mineralized matrix, they are essential for the regulation of osteoblast and osteoclast functions. However, the intracellular proteins that control the morphology and function of osteocytes, and their ability to communicate with other bone cells are still unknown. Kalirin is a novel multi-domain GTP exchange factor (GEF) protein that activates the RhoGTPases. Recently, we found that 14 week old female Kalirin knockout (Kal-KO) mice exhibit a 45% decrease in trabecular bone density and have significantly lower cortical area, perimeter, thickness and polar cross-sectional moment of inertia (-12.6%, -7.2%, -7.6% and -21.9%, respectively) than WT mice. Kalirin was found to be expressed in osteoclasts and osteoblasts but its expression and function in osteocytes is currently unclear. We examined the role of Kalirin on the morphology and function of osteocytes. Primary osteocytes were isolated by sequential collagenase digestions from long bones (femurs and tibias) of 10-week old WT and Kal-KO mice. Immunofluorescent staining revealed Kalirin was localized to the perinuclear region of primary osteocytes and MLO-Y4 cells, and was detected along the cytoplasmic processes of primary osteocytes. We also examined primary osteocytes isolated from the long bones of Kal-KO and WT mice for changes in the length and number of cytoplasmic processes. Kal-KO osteocytes were found to express significantly fewer cytoplasmic processes per cell (3.3±0.21) than WT osteocytes (4.7±0.3). In addition, the cytoplasmic processes of Kal-KO osteocytes were shorter (79.5±4.6 µm) than those observed for WT osteocytes (85.4±3.6 µm) (p <0.01). Quantitative PCR revealed the expression of mRNA for the three major Kalirin isoforms (Kal-7, Kal-9, Kal-12) in primary osteocytes and in MLO-Y4 cells. Moreover, the mRNA levels of osteoprotegerin (OPG) and SOST, which are important for controlling osteoclast differentiation and Wnt signaling leading to bone formation, respectively, were reduced in Kal-KO osteocytes. Next, the role of Kalirin in osteocyte morphology and function was further examined. Treatment of MLO-Y4 cells for 5 days with nerve growth factor, which is known to activate Kalirin in neurons, or over-expression of the Ser-Thr kinase domain of Kal-12, promoted cytoplasmic process elongation and upregulated phosphorylated ERK and RhoA levels. Together, these results suggest that Kalirin controls osteocyte morphology and function in part by regulating cytoskeletal remodeling and the activity of ERK and RhoA. Furthermore, Kalirin may control the bone remodeling cycle by regulating osteocyte signaling to osteoclasts and osteoblasts.
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    Synthesis of multifunctional chlorhexidine-doped thin films for titanium-based implant materials
    (Elsevier, 2020-12) Oliveira Matos, Adaias; Bandeira de Almeida, Amanda; Beline, Thamara; Tonon, Caroline C.; Casarin, Renato Corrêa Viana; Windsor, Lester Jack; Duarte, Simone; Nociti, Francisco Humberto, Jr.; Rangel, Elidiane Cipriano; Gregory, Richard L.; Barão, Valentim Adelino Ricardo; Cariology, Operative Dentistry and Dental Public Health, School of Dentistry
    Our goal was to create bio-functional chlorhexidine (CHX)-doped thin films on commercially pure titanium (cpTi) discs using the glow discharge plasma approach. Different plasma deposition times (50, 35 and 20 min) were used to create bio-functional surfaces based on silicon films with CHX that were compared to the control groups [no CHX and bulk cpTi surface (machined)]. Physico-chemical and biological characterizations included: 1. Morphology, roughness, elemental chemical composition, film thickness, contact angle and surface free energy; 2. CHX-release rate; 3. Antibacterial effect on Streptococcus sanguinis biofilms at 24, 48 and 72 h; 4. Cytotoxicity and metabolic activity using fibroblasts cell culture (NIH-F3T3 cells) at 1, 2, 3 and 4 days; 5. Protein expression by NIH-F3T3 cells at 1, 2, 3 and 4 days; and 6. Co-culture assay of fibroblasts cells and S. sanguinis to assess live and dead cells on the confocal laser scanning microscopy, mitochondrial activity (XTT), membrane leakage (LDH release), and metabolic activity (WST-1 assay) at 1, 2 and 3 days of co-incubation. Data analysis showed that silicon films, with or without CHX coated cpTi discs, increased surface wettability and free energy (p < 0.05) without affecting surface roughness. CHX release was maintained over a 22-day period and resulted in a significant inhibition of biofilm growth (p < 0.05) at 48 and 72 h of biofilm formation for 50 min and 20 min of plasma deposition time groups, respectively. In general, CHX treatment did not significantly affect NIH-F3T3 cell viability (p > 0.05), whereas cell metabolism (MTT assay) was affected by CHX, with the 35 min of plasma deposition time group displaying the lowest values as compared to bulk cpTi (p < 0.05). Moreover, data analysis showed that films, with or without CHX, significantly affected the expression profile of inflammatory cytokines, including IL-4, IL-6, IL-17, IFN-y and TNF-α by NIH-F3T3 cells (p < 0.05). Co-culture demonstrated that CHX-doped film did not affect the metabolic activity, cytotoxicity and viability of fibroblasts cells (p > 0.05). Altogether, the findings of the current study support the conclusion that silicon films added with CHX can be successfully created on titanium discs and have the potential to affect bacterial growth and inflammatory markers without affecting cell viability/proliferation rates.