Browsing by Author "SONG, F."

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    Heterogeneity of Human Gingival Fibroblasts in Tobaccostimulated Collagen Degradation
    (Office of the Vice Chancellor for Research, 2010-04-09) ZHANG, W.; FANG, M.; SONG, F.; WINDSOR, L. J.
    Matrix metalloproteinases (MMPs) are a large family of zinc-dependent endopeptidases and their activity is modulated by tissue inhibitors of metalloproteinases (TIMPs). Smoking is a risk factor for periodontal disease. Cigarette smoke condensate (CSC) is the particulate matter of cigarette smoke. Human gingival fibroblasts (HGFs) are one of major cellular components in periodontal tissue. CSC can increase collagen degradation of HGFs by enhancing and altering the localization of MMPs. Previous clinical studies also showed that some smoking people even with very high dental plaque index still had good periodontal status and did not develop periodontal disease. Objectives: The aim of this study was to investigate the heterogeneity of HGFs to CSC-stimulated collagen degradation and to start examining its mechanisms. Methods: Eleven HGF cell lines were established from healthy gingival tissue from patients undergoing crown-lengthening surgery. HGFs were seeded as single colony (75,000 cells/well) in 6-well Type I collagen coated plates and exposed to 100 µg/ml CSC (Murty Pharmaceuticals, Lexington, KY) diluted in serum-free media with/without a MMPs inhibitor (GM6001, 100 nM, Chemicon, Temecla, CA) for 3 days. HGFs were seeded with serum free media alone as controls. The mRNA levels of multiple MMPs/TIMPs were measured by reverse transcriptionpolymerase chain reaction. Results: CSC increased collagen degradation in 7 HGF cell lines (CSC-susceptible HGFs), but not in 4 HGF cell lines (CSC-unsusceptible HGFs). GM6001 inhibited CSC-stimulated collagen degradation in all of CSC-susceptible HGFs. The mRNA levels of MMP-1, MMP-2, MMP-3, MMP-14, TIMP-1, and TIMP-2 increased 2.5, 1.3, 3.9, 2.0, 1.6, and 1.3 fold, respectively, in the CSC-susceptible HGFs. However, expression of MMPs/TIMPs basically didn’t change in the CSC-unsusceptible HGFs, except for MMP-3 which increased 1.4 fold. Conclusions: Heterogeneity of HGFs existed in regard to the CSC-stimulated collagen degradation and the altered expression of the MMPs/TIMPs may be responsible for this heterogeneity. This project was supported by the IUPUI Tobacco Cessation and Biobehavioral Center.
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    INTERACTIONS OF HUMAN ORAL CELLS WITH ORAL BACTERIAL
    (Office of the Vice Chancellor for Research, 2012-04-13) HEEKE, K.S.; GREGORY, R.L.; SONG, F.; WINDSOR, L.J.
    Introduction: Streptococcus mutans is the main etiological cause of den-tal caries, and it has been shown that individuals who smoke have increased dental caries. S. mutans is known to bind to or interact with MG63 osteo-blasts. However, very little is known about the effects of tobacco directly on these bacteria on their ability to affect human pulp MG63 osteoblasts. We are hypothesizing that tobacco upregulates the expression of pro-inflammatory cytokines and MMPs to increase the pathogenic potential of S. mutans. The objective of this research project is to investigate the effects that nicotine, cigarette smoke condensate (CSC), and dissolvable smokeless tobacco (DST)-extract treated bacterial cells have on humanMG63 osteo-blasts, in respect to their release of pro-inflammatory and anti-inflammatory cytokines, as well as MMP expression. In addition, the effects of the S. mutans cells will be examined for the ability to affect MG63 osteoblast growth. The long-term goal is to develop treatment modalities to reduce the effects of smoking on dental caries. Materials and Methods: S. mutans UA159 was incubated in Tryptic Soy Broth (TSB), with the following concentrations: 2 mg/mL nicotine, 0.125 mg/mL CSC, 100 uL/3 mL DST-extract, and a 0 mg/mL control group. The cultures were grown in the presence of the tobacco products for 8 h at 37oC in 5% CO2, and centrifuged to isolate cells and supernatants. The cells were washed and heat-killed for 1 h at 60oC. Human MG63 osteoblasts were iso-lated from extracted teeth, and cell passages 3-8 will be used. The tobacco-treated S. mutans cells and supernatants will be incubated with the osteo-blasts in culture plates for 72 h and cytokine expression evaluated by re-verse transcriptase polymerase chain reaction. Results: The protein concentration of each tobacco-treated sample was found. The undiluted concentrations of the nicotine- and CSC-treated cells were slightly lower and the DST-treated cells was slightly higher than the control cells. The undiluted nicotine (p<0.05) and DST-treated supernatants were higher than the control, while the CSC supernatant protein concentra-tion was lower. From our previous studies, it was found that nicotine in-creases bacteriocin production of S. mutans, so we might hypothesize that nicotine induces bacteriocin secretion, thus increasing dental caries.
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    Matrix Metalloproteinases Expression during Limb Regeneration
    (Office of the Vice Chancellor for Research, 2010-04-09) SANTOSH, N.; MAHMOUDI, B. S.; WINDSOR, L. J.; STOCUM, D. L.; SONG, F.
    Axolotl (regeneration-competent) is one of the unique vertebrates which can regenerate missing organs such as limbs, jaws, spinal cord, and tail anytime during their life cycle. There also exists a recessive mutant of axolotl which has a phenotype called short toes (s/s, regenerationdeficient). The s/s mutant can regenerate its tail and spinal cord but cannot maintain the growth of the limb blastema, which results in the failure of limb regeneration. Remodeling of extracellular matrix (ECM) during early blastema formation, also known as histolysis, leads to the release of stem cells and activation of various growth factors. Therefore, histolysis is considered to be a crucial step in regenerating the exact replica of missing limbs in axolotls. Matrix metalloproteinases (MMPs) are zinc dependent endopeptidase that have been suggested to play roles in histolysis. However, it still remains unclear if histolysis is different in limb regeneration between regeneration competent and deficient animals. In this study, we analyzed the expression patterns of MMPs and the tissue inhibitors of the MMPs (TIMPs) in axolotl and s/s utilizing MMP arrays (RayBiotech, Inc., Norcross, GA), zymography and western blots. The cut-off limbs of the axolotls and s/s were used as controls. The animals were allowed to regenerate and the blastema was collected at three stages: epidermis closure (EC), dedifferentiation (DD), and early bud (EB). The total proteins were extracted from all the samples. 20 μg of protein was used to perform MMP arrays according to manufacturer’s protocol. They detected MMP-1, -2, -3, -8, -9, -10, and -13, as well as TIMP-1, -2 and -4 in the controls, EC, DD and EB samples from axolotl and s/s. Gelatin zymograghy with 20 μg of protein confirmed that MMP-2 and -9 were expressed at all the same time points in the axolotl and s/s samples. The expression patterns of MMP-9 were similar in the axolotl and s/s until the DD stage. While later in the EB stage, the axolotl showed a decrease in MMP-9 expression and s/s showed increased expression. Western blots were performed with 40 μg of protein using MMP-2 and -9 antibodies, and confirmed the zymography results. These results suggested that the expression patterns of the MMPs, especially MMP-9, are different in regeneration competent and deficient animals. One of the keys for a healthy blastema formation, which can multiply and later repattern into the missing limb, might be the release of the critical amount of MMP at the right time. This study was supported by an IUSD start-up grant to F. Song and a grant from W. M. Keck Foundation to D. L. Stocum.