2008 Research Forum Poster Session Abstracts

Abstracts for presentations at the 94th AAP Annual Meeting in Seattle

Basic Research
Clinical Research

The Research Forum provides a platform for clinical and basic research to be presented by those in the field of periodontics. The eight poster finalists below were on display in the Annual Meeting Exhibit Hall, and the following researchers won cash prizes:

Clinical Impact Award: Megumi Inomata, DDS
Clinical Science: Richard A Reinhardt, DDS, PhD
Basic Science: Tomoyuki Kawase, DDS, PhD

Our thanks to The Natural Dentist for sponsoring the Research Forum.

BASIC RESEARCH ABSTRACTS

Elastin in Gingival Connective Tissue Is Involved in Epithelial Keratinization

Category: Basic Research
Primary Author: Kuo Yuan, DDS, M.S. Institute of Oral Medicine, College of Medicine, National Cheng Kung University
Secondary Authors: Po-Chen Hsieh, Institute of Oral Medicine, College of Medicine, National Cheng Kung University; Chia-Wen Chang, Institute of Basic Medicine, College of Medicine, National Cheng Kung University
Financial Support: None
Background: Keratinized epithelium exclusively expresses cytokeratin-1 and -10, while non-keratinized epithelium expresses cytokeratin-4 and -13. The underlying connective tissue can determine the phenotypes of oral epithelia. One of the most discernable differences between keratinized and non-keratinized gingiva connective tissues is the quantity of elastin they contain: it is abundant elastin in the latter but scarce in the former. Whether elastin modulates the cytokeratin expression of oral epithelial cells is unknown.
Methods: Ten gingival specimens containing both keratinized and non-keratinized portions were obtained from crown-lengthening procedures. Four were processed for immunohistochemical (IHC) stainings for elastin and cytokeratins. Keratinized and non-keratinized portions of six gingiva were separated and then transferred to a organ culture system. Purified proteins of elastin and elastase were added separately, and then the gingiva were cultured for 14 days and processed for the same IHC staining. Cell cultures of keratinized and non-keratinized gingiva fibroblasts were established and tested for elastin expression. Oral mucosa equivalents were engineered by embedding different fibroblasts in fibrin gels with oral keratinocytes on the top. The equivalents were cultured for 2 weeks and collected for cytokeratin analysis using IHC.
Results: The results of IHC for keratinized and non-keratinized gingiva were consistent with previous findings. The results of immunostaining and immunoblotting showed that only non-keratinized gingival fibroblasts from cell culture expressed elastin. Both the native and engineered keratinized gingiva changed the phenotypes and expressed cytokeratin-4 and -13 when treated with exogenous elastin. On the contrary, the native and engineered non-keratinized gingiva expressed cytokeratin-1 and -10 when exogenous elastase was used to eradicate inherent elastin. However, the switch in cytokeratin expression was not complete: the expression of specific cytokeratins were not as exclusive as in the native gingiva without treatments of elastin or elastase.
Conclusion: Our study provided evidence that the elastin in the connective tissue of non-keratinized gingiva is important in determining the non-keratinized phenotypes of overlaying epithelium. The treatment of elastase on non-keratinized gingiva switched specific cytokeratin expressions. However, elastin may not be the only determining factor in the differentiating process. With more understanding of the molecular mechanisms in the keratinization of gingival epithelia, non-surgical or less invasive techniques may be developed and clinically applied to replace conventional mucogingival surgery.

Arginine-specific gingipain A from Porphyromonas gingivalis induces Weibel-Palade body exocytosis and enhanced activation of vascular endothelial cells through protease-activated receptors

Category: Basic Research
Primary Author: Megumi Inomata, DDS, Department of Oral Disease Research, National Insitute for Longevity Science, National Center for Gerontology
Secondary Authors: Takeshi Into, Department of Oral Disease Research, National Institute for Longevity Science, National Center for Gerontology; Yuichi Ishihara, Department of Periodontology, School of Dentistry, Aichi gakuin University; Kenji Matsushita, Department of Oral Disease Research, National Insitute for Longevity Science, National Center for Gerontology; Toshihide Noguchi, Department of Periodontology, School of Dentistry, Aichi gakuin University
Financial Support: None
Background: Porphyromonas gingivalis is a principal periodontopathic pathogen. P. gingivalis has a number of virulence factors, including LPS and fimbriae, and the most notable factors are secretory proteases termed gingipains. P. gingivalis infection is thought to cause proinflammatory effects on periodontal tissue, in which gingipain-induced cytokine production and adhesion molecule expression in vascular endothelial cells may play important roles. In this study, we investigated how gingipains exert such proinflammatory effects on vascular endothelial cells.
Methods: P. gingivalis ATCC33277 whole cells and their LPS were obtained from commercial sources. Gingipain (arginine-specific gingipain A; RgpA) was prepared from released vesicles from P. gingivalis HG66 culture. The amount of IL-8 released from human umbilical vein endothelial cells (HUVECs) was determined by ELISA. The expression level of IL-8 mRNA was determined by quantitative RT-PCR. Quantification of Weibel-Palade body (WPB) exocytosis was performed by measuring the amount of von Willebrand factor (VWF), in HUVEC culture media by ELISA. The ‘WPB exocytosis (%)’ was calculated by taking the value of VWF release by the calcium ionophore A23187 as 100%. RNA interference HUVECs were transfected with short interfering RNA (siRNA) for protease-activated receptor (PAR)1, PAR2, PAR3 or angiopoietin-2 (Ang-2) using Lipofectin reagent.
Results: RgpA increased the responsiveness of vascular endothelial cells to P. gingivalis LPS or P. gingivalis whole cells to induce enhanced IL-8 production through PARs and phospholipase C (PLC)γ, a common mediator of PAR signaling. We investigated whether RgpA-induced enhanced cell activation was mediated through exocytosis of the endothelial cell-specific granule WPB because WPB contains vasoactive substances. RgpA rapidly activated PAR- and PLCγ-dependent WPB exocytosis. In addition, we found that Ang-2, a substance of WPB, had a capability to enhance IL-8 production by P. gingivalis LPS.
Conclusion: A novel role for gingipain in induction of enhanced proinflammatory events through the PAR-PLCγ mediated WPB exocytosis in vascular endothelial cells. Ang-2, a substance of WPB, was found to mediate the enhanced cell responses. Our results also imply the synergism of Toll-like receptors (TLRs) and PARs because proinflammatory responces by TLR agonists of P. gingivalis, including LPS, was upregulated by the PAR activator gingipain. Thus, the presence of gingipain at the site of P. gingivalis infection may affect responsiveness of the vascular endothelium to TLR-stimulating virulence factors of P. gingivalis through such mechanisms.

Osteoprotegerin is induced during differentiation of monocytes

Category: Basic Research
Primary Author: Yasuhiro Notohara, DDS, Osaka Dental University
Secondary Authors: Yutaka Nagano, Department of Internal Medicine, Osaka Dental University; Naochika Domae, Department of Internal Medicine, Osaka Dental University; Masatoshi Ueda, Department of Periodontology, Osaka Dental University
Financial Support: None
Background: To prevent and treat osteoporosis including alveolar bone resorption caused by periodontitis, it is important to understand the changes in expression of molecules during differentiation of osteoclasts from their precursor cells, since osteoclasts play central roles in bone resorption. Considering that osteoclasts originate from monocytes/macrophages, we conducted experiments to see changes in mRNA expression of osteoprotegerin (OPG), RANK, and RANKL during differentiation of THP-1 cells, a human monocytic leukemia cell line, into macrophages.
Methods: THP-1 cells were cultured using RPMI 1640 supplemented with 10% fetal calf serum in a 5% CO2 environment at 37oC. The cells were induced to differentiate into macrophage-like cells using phorbol 12-myristate 13-acetate (PMA). Total RNA from the cells was extracted by the single-step guanidinium-isothiocyanate method using a commercial reagent (TRYZOL). The concentration and integrity of extracted RNA were analyzed by Bradford method using SmartSpec 3000. Then, total RNA was reverse-transcribed by Quiagen one-step PCR kit, and the cDNA was amplified by PCR. PCR products were separated by agarose gel electrophoresis, and band intensities were quantified digitally using Image software (VersaDoc). Real-time PCR was performed using StepOnePlus system (Applied Biosystems).
Results: THP-1 cells in suspension culture became adhesive and attached to culture dishes within 24h exposure to PMA. Before PMA treatment, OPG mRNA was not identified at all. However, the mRNA was clearly induced after the treatment with PMA. The induction was maximal when the cells were incubated with 200nM PMA for 48h. In contrast, THP-1 cells expressed RANK and RANKL mRNA before the stimulation with PMA, and their expression did not change after the PMA treatment.
Conclusion: We have demonstrated that OPG, a secretory protein and a competitive inhibitor of RANK binding to RANKL, was induced when THP-1 monocytes differentiated into Bmacrophages. It has been reported that the binding of receptor activator of NF ligand (RANKL) to RANK expressed on the surface of osteoclast precursor cells is one of the most important steps in osteoclast differentiation. OPG competitively inhibits this binding. Therefore, this molecule has been considered as an important regulator of osteoclast differentiation, which was conformed when marked osteoporosis was observed in mice that are deficient in OPG. In summary, in contrast to RANK and RANKL, OPG was induced during macrophage differentiation of monocytes, which may contribute to the regulation of the differentiation of osteoclasts.

Evidence to support the clinical application of cultured human periodontal sheets: Evaluation of its bone-forming potential

Category: Basic Research
Primary Author: Tomoyuki Kawase, DDS, PhD, Division of Oral Bioengineering, Niigata University
Secondary Authors: Kazuhiro Okuda, Division of Periodontology, Institute of Medicine & Dentistry, Niigata University; Hiroyuki Kogami, Division of Oral Bioengineering, Institute of Medicine & Dentistry, Niigata University; Masaki Nagata, Division of Oral and Maxillofacial Surgery, Institute of Medicine & Dentistry, Niigata University; Koh Nakata, Bioscience Medical Research Center, Niigata University Hospital; Hiromasa Yoshie, Division of Periodontology, Institute of Medicine & Dentistry, Niigata University
Financial Support: This study was supported in part by a Grant-in-Aid for Scientific Research from the Japan Society for the Promotion of Science and by Practical　Application　Research from the Japan Science and Technology Agency (Tokyo, Japan).
Background: Our recent clinical studies have demonstrated that periodontal regenerative therapy, using cultured autologous human periosteal sheets (hCP) in combination with autologous platelet-rich plasma (PRP) and porous hydroxylapatite (HAp) particles, is potentially important for stimulating alveolar bone formation at specific sites. However, more needs to be known about how hCP-based therapy functions in vivo. To improve our understanding, we have now studied hCP-based bone formation both in in vitro and in animal implantation studies.
Methods: Under approval of our ethical committee, human periosteal segments were aseptically dissected from alveolar bone of informed consent volunteers and cultured with Medium 199 containing 10% FBS and antibiotic agents in 100-mm dishes. Alkaline phosphatase (ALP) activity was examined by staining with a commercial kit. For cytokine assays, hCP was incubated in 1% FBS-containing medium for 2 days, and supernatant was directly added to the commercial cytokine antibody array. Spots were visualized with a cooled CCD camera and quantified with a software. After being cultured for 30-40 days in the absence or presence of the osteoblastic (OB) inducing agent (incl. dexamethasone), hCP was implanted into the dorsal subcutaneous tissue of nude mice or extracted for proteome analysis. Implanted hCP was picked out and subjected to histological examination.
Results: Periosteal cells migrated out from tissue segments within 4-8 days after plating, and grew more rapidly with longer culture times. Although ALP activity did not increase with culture time under basal conditions, this important bioactivity (and concomitant biomineralization) was gradually up-regulated with OB induction. The cytokine array demonstrated that OB induction strikingly down-regulated IL-6 and thrombopoietin, but substantially up-regulated IL-8, IL-13, IGF-I and IGFBP-2. In proteome analysis, HSP27 appeared to be markedly up-regulated in differentiated hCP. When differentiated hCP was implanted alone (without any scaffolds), osteoid tissue was formed within 2 weeks. Implantation of immature hCP was notably less potent.
Conclusion: These findings suggest that hCP has high potential for inducing ectopic bone formation without the necessity of adding ceramic or similar tissue scaffolds, and that hCP function as a living drug delivery system to influence cells near the implantation site through production of several important cytokines. In our parallel study, furthermore, hCP combined with porous HAp blocks appeared to facilitate osteoclast formation and resorption of the ceramics. Therefore, it is expected that hCP not only directly provides bone formation system, but also efficiently induces bone resorption system in the presence of porous HAp. In conclusion, hCP by itself could serve as an “osteoinductive biomaterial”, and addition of appropriate scaffolds would be better system for bone regenerative therapy. This study provides evidence supporting our clinical application of autologous hCP with porous HAp particles.

CLINICAL RESEARCH ABSTRACTS

Effect of platelet-rich plasma in combination with demineralized freeze-dried bone allograft for the treatment of vertical osseous defects.

Category: Clinical Research
Primary Author: Matteo Piemontese, DDS, DMD, Institute of Dental Science, Division of Periodontology and Oral Pathology
Secondary Authors: Simone Domenico Aspriello, Institute Of Dental Science, Division of Periodontology and Oral Pathology; Antonio Zizzi, Dipartimento Di Patologia Molecolare E Terapie Innovative, Institute of Istologyguendalina Lucarini dipartimento Di Patologia Molecolare E Terapie Innovative, Institute of Istologycorrado Rubini dipartimento Di Neuroscienze, Institute of Harmed Anatomyluigi Ferrante institute of Microbiology and Biomedical Science
Financial Support: None
Background: The aim of the present randomized, double-masked clinical trial was to compare platelet-rich plasma (PRP) combined with a demineralized freeze-dried bone allograft (DFDBA) against DFDBA mixed with a saline solution in the treatment of human intrabony defects.
Methods: Sixty interproximal intrabony osseous defects in 60 healthy, non-smoking subjects diagnosed with chronic periodontitis were treated in this study. Thirty subjects each were randomly assigned to either test group (PRP and DFDBA) or control group (DFDBA and saline). Clinical and radiographic measurements were made at baseline and at the 12-month evaluation.
Results: When compared to baseline, the 12-month results indicated that, while both treatment modalities resulted in significant changes in all clinical parameters (gingival index, bleeding on probing, probing depth, clinical attachment level, gingival recession; P<0.001) and radiographyc examinations (hard tissue fill, bone depth reduction; P<0.001), the test group exhibited statistically significant changes compared to the control sites in probing depth reduction: 4.3±1.7mm versus 2.6±2.2mm (P<0.05); clinical attachment gain 3.5±2.1mm versus 2.3±2.4mm (P<0.001).
Conclusion: Treatment with a combination of PRP and DFDBA compared to DFDBA with saline has led to a significantly more favorable clinical improvement in intrabony periodontal defects. No statistically significant differences were observed in hard tissue response between the two treatment groups confirming no effect of PRP on hard tissue fill or any gain in new hard tissue formation.

Longitudinal GCF Biomarkers Associated with Ongoing Clinical Attachment Loss

Category: Clinical Research
Primary Author: Richard A Reinhardt, DDS, PhD, University of Nebraska Medical Center College of Dentistry
Secondary Authors: Richard A. Reinhardt, University of Nebraska Medical Center College of Dentistry; Julie A. Stoner, University of Oklahoma Health Sciences Center; Lorne M. Golub, Stony Brook University School of Dental Medicine; Hsi-Ming Lee, Stony Brook University School of Dental Medicine; Jeffrey B. Payne, University of Nebraska Medical Center College of Dentistry
Financial Support: Supported by NIDCR/NIH grant (R01DE012872)
Background: Previously-reported clinical outcomes from a double-blind placebo-controlled randomized 2-year clinical trial of postmenopausal osteopenic patients with moderate-severe periodontitis undergoing periodontal maintenance therapy (PMT) showed 2.5% of sites with progressive clinical attachment level (CAL) loss. The purpose of this follow-up analysis was to determine how gingival crevicular fluid (GCF) biomarkers of inflammation (IL-1β) and bone resorption (ICTP) were associated with CAL.
Methods: One hundred twenty-eight subjects in the clinical trial contributed pooled 10-second GCF samples at baseline, 1 and 2 years from the same 2 sites probing 5 mm or more. Interleukin (IL)-1β and pyridinoline cross-linked carboxyl-terminal telopeptide of type I collagen (ICTP) were measured with immunoassays. Subjects were randomized to receive subantimicrobial dose doxycycline (SDD, 20 mg BID; n = 64) or placebo (n = 64). GCF biomarkers were modeled as categorical variables of reduction, constant or increased levels at 1 and 2 years vs. baseline based on tertiles; CAL (Florida probe) was categorized based on improvement, no change or progression (1.5 mm or more) at 1 and 2 years. Generalized estimating equations were used with adjustments for baseline CAL, randomized treatment group, smoking status, study center, and assay batch.
Results: 97% of sites were stable or improved over 2 years, yet some subjects had multiple progressing sites. Placebo subjects with IL-1β increases had a concurrent 55% increase in the odds of more progressive periodontitis (OR = 1.55, 95% CI: 1.10 to 2.20, p = 0.01) versus subjects with IL-1β reductions. The SDD group did not show this association, and had a 19% (p = 0.03) reduction in the odds of more progressive periodontitis versus placebo. Among all subjects, those with increases in IL-1β during the first year of PMT had 67% increased odds of more progressive periodontitis after 2 years (OR = 1.67, 95% CI: 1.13 to 2.46, p = 0.01) versus subjects with IL-1β reductions. ICTP levels were not significantly associated with CAL changes.
Conclusion: Postmenopausal osteopenic patients with moderate-severe periodontitis who are compliant with 3-4 month PMT, yet have persistently high levels of GCF IL-1β, are more likely to demonstrate concurrent or future progression of their disease as measured by clinical attachment loss. SDD appears to inhibit this association and results in less progressive disease. While ICTP levels were not significantly associated with CAL changes, further analysis from this trial indicated a marginal association between increased baseline GCF ICTP and future alveolar bone height loss. GCF IL-1β may serve as a predictive measure of the effectiveness of PMT. Supported by NIDCR/NIH grant (R01DE012872).

The Effects of Smoking on the Survival of Smooth and Rough Surface Dental Implants

Category: Clinical Research
Primary Author: Ayman A Balshe, DDS, Mayo Clinic
Secondary Authors: Steven E. Eckert, Mayo Clinic; Sreenivas Koka, Mayo Clinic; Daniel A. Assad, Mayo Clinic; Amy L. Weaver, Mayo Clinic
Financial Support: None
Background: Smoking has been described as a risk factor that may influence the survival of dental implants. Most of the studies that demonstrated smoking as a risk factor for failure of dental implants have generally used early generation smooth surface implants. The purpose of this study is to compare the long-term survival rates of smooth and rough surface dental implants among smokers and non-smokers.
Methods: A retrospective chart review was conducted for two time periods: January 1, 1991, through December 31, 1996, during which smooth surface implants were utilized, and January 1, 2001, through December 31, 2005, during which rough surface implants were utilized. This review included all implants placed and restored in one institution during the two time frames. Data were specifically collected relative to patient age, gender, smoking status, implant diameter, implant length, and anatomic location of implants. Associations of patient/implants characteristics with implant survival were evaluated using marginal Cox proportional hazards models and summarized with hazard ratios (HR) and corresponding 95% confidence intervals (CI).
Results: Among the rough surface implants, smoking was not identified as significantly associated with implant failure (HR=0.8; 95% CI=0.3-2.2; p=0.72). In contrast, smoking was associated with implant failure among the group with smooth surface implants (HR=3.3; 95% CI 2.8-6.2; p < 0.001). Implant anatomic location was not associated with implant survival among patients with rough surface implants (p=0.52) and among non-smokers with smooth surface implants (p=0.16). However, anatomic location affected the implant survival among smokers with smooth surface implants (p=0.004).
Conclusion: Smoking was identified as a risk factor for implant failure of smooth surface implants only. Among the smokers who received smooth surface implants, an association was identified between implant failure and location of the implant placement. No association was identified between implant failure and location among the smokers who received rough surface implants

Mucosal Wound Healing, Sex Hormones and Inflammation

Category: Clinical Research
Primary Author: Bahareh Zyaei, DDS, University of Illinois at Chicago, Department of Periodontics
Secondary Authors: Phillip T Marucha, University of Illinois, Department of Periodontics; Christopher G Engeland, University of Illinois, Department of Periodontics
Financial Support: None
Background: Most wound healing studies have examined dermal wounds and reported a female advantage in healing rates. Recently our laboratory demonstrated women heal mucosal wounds more slowly than men (Engeland et al., Arch Surg, 141:1193-7, 2006) and with greater inflammation. The objective of this study is to determine the role of sex hormones in mucosal wound healing. We hypothesized sex hormones play a role in wound healing, possibly through their modulating effects on inflammation in different subgroups.
Methods: This study involved 384 volunteers (198 men, 186 women) between the ages 18-43, and 93 older subjects between the ages of 50-88 (60 female and 33 male). A 3.5 mm diameter wound was created on the oral hard palate and videographed at 24h intervals for 7 days. Tissue biopsy samples were obtained from a second longitudinal wound at 6h or 24h post-wounding and pro-inflammatory mediators were measured by rt-PCR. Plasma levels of testosterone, progesterone and estradiol were determined by EIA. Wound healing rates measured in different groups: younger and older men, naturally cycling young women, versus women on OCPs, naturally cycling women in the follicular phase, versus the luteal phase, young women on OCP, peri versus post-menopausal women with and without HRT.
Results: Young men with higher testosterone levels exhibited significantly delayed wound closure on days 5-7 compared to individuals with lower testosterone levels. Similarly, naturally cycling young females with higher testosterone levels exhibited significantly delayed wound closure on days 4-7 compared to individuals with lower testosterone levels. Older females not on HRT, with higher testosterone levels exhibited significantly faster wound closure compared to individuals with lower testosterone levels. In this study, progesterone and estradiol did not exhibit a relationship to wound healing. Also no significant relationship found between the levels of sex hormones and inflammatory mediators or HPA axis hormones.
Conclusion: This study found repeatedly that wound healing was related to testosterone, possibly accounting for the previously reported observed gender effects. Importantly, a sexual dichotomy in how these hormones impact upon inflammation may be a critical factor in healing. Interestingly, naturally cycling women exhibited delayed healing with higher testosterone levels similar to young men. This however was irrespective to the phase of the menstrual cycle. This study showed that healing in older post- menopausal women on HRT is faster than post menopausal women not on HRT suggesting beneficial effects of HRT on wound healing. Our data also suggests that testosterone only relates to wound healing in people who are not taking exogenous synthetic hormones in the form of oral contraceptives or hormonal replacement therapy.