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2010 AAP Research Forum Poster Session Abstracts

Abstracts for finalists in the Research Forum Poster competition during the 96th AAP Annual Meeting in Honolulu.

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 poster finalists below were on display in the Annual Meeting Exhibit Hall Sunday, October 31, through Tuesday, November 2, and the following researchers won cash prizes:

Clinical Impact Award: Gustavo Garlet
Clinical Science: Christina Park
Basic Science: Azusa Yamada

BASIC RESEARCH ABSTRACTS

Comparison of Different Tissue-derived Cell Sheets for Periodontal Regeneration in a Canine 1-wall Defect Model

Primary Author: Yuka Tsumanuma, Tokyo Medical and Dental University, Tokyo, Japan
Secondary Authors: Drs. Takanori Iwata, Kaoru Washio, Toshiyuki Yoshida, Masayuki Yamato, Isao Ishikawa, Teruo Okano, Tokyo Women's Medical University, Tokyo, Japan, Drs. Azusa Yamada, Yuichi Izumi, Tokyo Medical and Dental University, Tokyo, Japan
Financial Support: None
Background: Our previous study indicated that the transplantation of 3-layered periodontal ligament cell (PDLC) sheets combined with β-tricalcium phosphate (β-TCP) regenerated true periodontal tissue in a canine 3-wall defect model. Other studies suggested that both bone marrow derived mesenchymal stromal cells (BMMSC) and alveolar periosteal cells (PSC) also enhance periodontal regeneration. In this study, we compared the potential of PDLC, BMMSC, and PSC using a canine 1-wall defect model.
Methods: All experimental protocols were approved by the animal welfare committee of Tokyo Women’s Medical University. BMMSC, PDLC, and PSC were obtained from 4 beagle dogs, respectively. Cells were cultured on temperature-responsive dishes with osteoinductive medium containing 50 μg/ml ascorbic acid, 10 mM β-glycerophosphate, and 10 nM dexamethasone for 5 days. Periodontal defects were surgically created at the mandibular mesial and distal of P3 and mesial of M1 bilaterally. Total 6 defects were created in each dog. Different tissue derived cell sheets were fabricated and transplanted to the denuded root surface of 1-wall-defects (5 mm x 5 mm x 5 mm) autologously. Bone defects were filled with β-TCP. β-TCP alone was transplanted in the control group. The dogs were sacrificed, dissected and fixed 8 weeks after the transplantation for histological analysis. Following parameters were evaluated. (i) Newly-formed cementum thickness (µm): the length of cementum regenerated in the direction vertical to denuded root surface. The length were measured every 500 µm on the newly formed cementum and the mean was calculated. (ii) Vertical ligament regeneration rate (%): the length of newly formed PDL-like fibers attached to denuded root surface at an angle of more than 45° divided by the length of denuded root surface. (iii) Bone regeneration rate (%): the distance between the bottom of bone defect and coronal extension of new alveolar bone by defect height. (iv) Apical extension of the junctional epithelium (mm): the distance between the apical extension of junctional epithelium and cement-enamel junction.
Results: Newly-formed cementum thickness was significantly higher in PDLC group (p < 0.05). The means ± SD of PDLC, BMMSC, PSC, and control group were 14.37 ± 4.38, 7.80 ± 2.67, 3.99 ± 2.64, and 6.81 ± 3.35 µm, respectively. The vertical ligament regeneration rate of PDLC group (23.02%) was significantly higher compared to PSC group (8.14%) (p < 0.05), but not to those of BMMSC group (18.43%) and control group (13.98%). No significant differences were observed in the bone regeneration rate within all the groups. Means of alveolar bone regeneration in all groups were approximately 70~80%. The apical extension of junctional epithelium exhibited no remarkable differences within all the groups.
Conclusions: These results suggest that there are significant differences between cells derived from different tissues in the regeneration of PDL and cementum. PDLC may be a suitable cell source for periodontal regeneration. Histometric analysis (group means ± SD)

*p < 0.05 compared to PDLC group; Post-hoc test
BMMSC, PDLC, PSC group (n = 4)
Control group (n = 6)

One-wall infrabony defect in dog. Left: One wall defects were surgically created (5 mm × 5 mm × 5 mm) and cell sheets (arrow head) were transplanted to the denuded root surface. Right: Bone defects were filled with β-TCP.

Photomicrographs of sites implanted with PDLC sheet (left) and BMMSC sheet (right).
(Bar, 1 mm; Azan staining)

High magnification of Fig.2. Thick newly formed cementum is observed only in the PDLC group.
(Bar, 100 µm; Azan staining)

Transplantation of Embryonic Stem Cells Enhance Regeneration of Periodontal Furcation Defects in a Porcine Model

Primary Author: Kuo Yuan, National Cheng Kung University, Tainan City, Taiwan
Secondary Authors: Dr. Chia-Wen W. Hsu, Tainan Municipal Hospital, Tainan City, Taiwan, Dr. Shih-Chung C. Liao, National Cheng Kung University, Tainan City, Taiwan, Dr. Jenn-Rong R. Yang, Council of Agriculture, Tainan, Taiwan
Financial Support: None
Background: Stem cell-based therapy is promising to regenerate lost tissues. Among different stem cells, embryonic stem cells (ESCs) are pluripotent that can differentiate to almost any kind of cell in the body. Their self-renewal ability is much stronger than adult stem cells and provides a more unlimited transplantation source. Although various techniques are available to regenerate periodontal furcation defects, none of them is completely satisfactory. We aimed to investigate whether ESCs transplantation could enhance regeneration of furcation defects in a porcine model.
Methods: Experimental periodontitis were created in the buccal furcations of bilateral mandibular first molars of four 5-month-old male minipigs under general anesthesia. Bony lesion with 4 mm width, 5 mm length and 3 mm horizontal depth were generated using a low speed bur (Figure 1). Temporary filling material was applied into the lesions to cause inflammation. After 4 weeks, the lesions were surgically debrided and implanted with collagen membrane alone (control site) or collagen membrane overlaid with 1×106 porcine ESCs expressing green fluorescent protein (GFP) (Figure 2). The cultured pESCs were regularly tested for their undifferentiated state before the surgery (Figure 3). Following 3 months of healing, the pigs were euthanized. The mandibles were dissected and assessed for clinical parameters including probing depth and attachment level (Figure 4). The fluorescence levels of the mandibles were measured using an in vivo fluorescence imaging system (IVIS) (Figure 5) to detect the persistence and quantity of transplanted cells. The treated molars with adjacent tissues as well as part of the lungs, livers and maxillary bone were obtained and processed for immunohistochemical (IHC) staining.
Results: No obvious teratoma or rejection was seen in all the examined specimens. The test group had significantly shallower probing depth and attachment level than the control group (Figure 4). The IVIS data demonstrated fluorescence level in the test group was about 2 fold of that in the control group. The IHC staining for GFP provided evidence that transplanted pESCs differentiated to new periodontal attachments in the test sites. Surprisingly, GFP(+) cementocytes were also detectable in the reparative cementum of the control sites (Figure 6).
Conclusions: The transplantation of ESCs leads to formation of new periodontal ligament and cementum, which, in turn, leads to formation of a new attachment. There is no significantly adverse effect. Homing of the ESCs to wound healing sites was suspected. This study demonstrates the feasibility of using ESCs to enhance the regeneration in the periodontal furcation defects. More studies are required to assess the efficacy and safety of this potential treatment.

Figure1. Experimental periodontitis was created on the buccal furcation of mandibular first molar.

Figure 2. H&E and SEM photomicrographs for pESCs.

Figure 3. Embryonic stem cell markers (rodamine) were regularly checked in our pESC clones which express GFP.
Graph

Figure 4. Comparison of probing depths and attachment levels between test and control groups.

Figure 5. Comparison of the fluorescence levels between test and control sites using an in vivo fluorescence imaging system (IVIS).

Figure 6. H&E and Immunohistochemical staining for GFP in test and control furcation areas.

Role of Intercellular Interactions between Mast Cells and Gingival Fibroblasts in Mediating Inflammation

Primary Author: Reza Termei, University of Toronto, Toronto, ON, Canada
Secondary Authors: Drs. Carol Laschinger, Christopher A. McCulloch, University of Toronto, Toronto, ON, Canada
Financial Support: None
Background: Host inflammatory response plays a critical role in the pathogenesis of periodontitis. Chronic periodontitis advances through bursts in activity and tissue destruction followed by remission and repair. The mechanisms that control these episodes of acute exacerbation in periodontitis are not fully understood. Interleukin-8 (IL-8) is a pro-inflammatory cytokine that promotes neutrophils chemotaxis into the gingival crevice. It is increased in periodontal lesions and tends to decrease following treatment. IL-8 levels in the gingival crevice can reflect the activity of periodontal destruction. Our objective was to study the role of interactions between residents of human gingival tissue mast cells and fibroblasts in regulating IL-8 expression and neutrophil chemotaxis.
Methods: Human gingival fibroblasts (HGF) were co-cultured with human mast cells (HMC-1) in 2:1 ratio. After 8 hours of co-culture, the concentration of IL-8 was measured by ELISA. Co-cultures were treated with the gap junction inhibitor β-glycyrrhetinic acid (BGA) or with the calcium release agonists thapsigargin and ionomycin. To study the effect of direct cell-cell contact, mast cells were grown on Cyclopore® membranes to physically separate them from HGFs while still allowing for small molecules and mediators to pass through. Chemotaxis analysis was done using mouse neutrophils in response to the co-culture conditioned medium. Also, spatial relationships between mast cells and gingival fibroblasts were examined by light and confocal laser microscopy using fluorescein molecules of different sizes: Dextran (MW= 10-70 kDa) and Calcein-AM (MW< 1kDa). Student t-test was used for statistical analysis.
Results: HMC co-cultured with HGF significantly increased IL-8 secretion compared to the control groups(p<0.001). This effect was blocked by the gap junction inhibitor, BGA (p<0.05) or when HMCs were separated from HGFs by membranes (p<0.01). Elevation of intracellular calcium caused a 15-fold increase in IL-8 levels from co-culture samples(p<0.001). Neutrophils chemotaxis was significantly enhanced in response to the co-culture conditioned medium (p<0.001). This effect was efficiently inhibited when co-cultures were treated with BGA (p<0.001). Microscopic analysis showed that HMCs adhered to HGFs. Dye transfer studies with confocal laser microscopy confirmed the presence of cell-cell communication between HMCs and HGFs however, only small molecules (MW<1000 Da) were able to transfer from mast cells into the fibroblasts.
Conclusions: Mast cells adhere to gingival fibroblasts via gap junctions and promote IL-8 secretion and enhanced neutrophil chemotaxis. This may then lead to perpetuation of the inflammatory response and tissue destruction. Calcium plays an important role in this phenomenon. Our study is the first to show the potential role of direct cellular interactions between mast cells and gingival fibroblasts in mediating inflammation within the periodontium.

IL-8 level was increased significantly in co-culture media
Graph

Separating HMCs from having direct contact with HGFs and use of gap junction inhibitor (BGA) significantly dropped the IL-8 level in the co-culture media. Treating fibroblasts with mast cell sonicate did not cause any increase in IL-8 level.
Graph

Mast cells-Fibroblasts co-culture media enhanced neutrophil chemotaxis. This effect was significantly blocked with the gap junction inhibitor (BGA).

Light microscopic view of HGF alone (A) and in co-culture with mast cells after 24 hours and following rinsing with PBS (B)

Gene Expression Profiles from Jaw and Alveolar Bones are Uniquely Different from Profiles of Trunk and Limb Bones

Primary Author: Tomohiro Taguchi, Showa University Dental Hospital, Tokyo, Japan
Secondary Authors: Drs. Junichi Watahiki, Tomoki Nampo, Akiko Enomoto, Miki Ono, Koutaro Maki, Showa University Dental Hospital, Tokyo, Japan, Drs. Gou Yamamoto, Tarou Irie, Tetsuhiko Tachikawa, Showa University School of Dentistry, Tokyo, Japan
Financial Support: None
Background: Developmentally, most bones of the trunk and limb, including the iliac bone, are formed by endochondral ossification, while the jaw and alveolar bones are formed by intramembranous ossification. Using bone with a different ossification pattern from those of the jaw and alveolar bones as a graft material for jawbone reconstruction is a matter of concern. Jaw and alveolar bones both differentiate from neural crest cells, while the aforementioned bones derive from mesoblasts. In this study, we investigated differences in gene expressions from the jaw bone (mandible and maxilla) and from bones of the trunk and limb (ilium and thigh bone). The calvarium is unique in that it arises from two embryonic tissue origins, namely the neural crest for frontal formation and the mesoderm for parietal formation. Thus, we transplanted mandible, maxilla, ilium and thigh bone grafts into two different types of calvarial regional defects to assess bone regeneration.
Methods: Gene expressions from the above tissues were evaluated by means of a DNA microarray and real-time PCR. Preliminary bone regeneration data from the calvarial defects was evaluated by real-time PCR, micro-computed tomography and histological analyses.
Results: Our data indicate that there is a close homology between ilium and thigh bone whole gene expressions. However, gene expression profiles from the jaw bone were different from those of the ilium and thigh bone. Specifically, P0, P75, Musashi-1, Nestin, Sox-10 and Slug were expressed more strongly in jaw bone as compared with profiles from the ilium and thigh bone. As expected, histological analysis showed differing bone formation processes between grafts of the jaw bone and of the extremities.
Conclusions: Jaw bone exhibits a unique gene expression profile that is associated with neural crest cells and has a positive influence on bone regeneration when used as a graft material. Thus, it is recommended to fully investigate the biological and mechanical properties of jaw bone as an alternative graft material for jaw bone reconstructive surgery.

Effect of Bone Configuration on Stress Distribution Around Screw-type Parallel and Tapered Implants

Primary Author: Deborah Termeie, University of California, Los Angeles, Los Angeles, CA
Secondary Authors: Drs. Perry R. Klokkevold, Angelo A. Caputo, University of California, Los Angeles, Los Angeles, CA
Financial Support: None
Background: The long-term clinical success of a dental implant is dependent upon acquiring and maintaining sufficient osseointegration and bone support to resist forces of occlusion. The purpose of this study was to investigate the effects of bone configuration on stress distribution around screw-type dental implants using photoelastic models.
Methods: Twelve composite photoelastic models were assembled using two different resins to stimulate trabecular and cortical bone. Half of the models had standard dimensions for ridge width and the other half had narrower buccal-lingual dimensions. Internal connection implants (13mm length) with a standard (4mm), wide (5mm), or narrow (3.3mm) diameter were embedded in photoelastic models. Half of the implants were tapered and half were straight. Full gold crowns in the shape and dimension of a mandibular first molar were fabricated and attached to the implants. Vertical (0 degrees) and angled (20 degrees to implant axis) loads of 15 pounds and 30 pounds were applied to specific points on the crown by means of a calibrated load cell.
Results: Narrow diameter implants generated higher stress especially in narrow ridges. Wide diameter implants produced the least stress in all ridges. Models with wide diameter implants loaded axially had a more symmetrical distribution of the stress compared to standard and narrow diameter implants. Vertically (axial) loaded models had the highest stress in the crest and apical areas. A more asymmetrical stress distribution pattern developed with angled (non-axial) loads, which also generated greater stress at the apex.
Conclusions: Implant diameter and ridge width had considerable influence on stress distribution. Narrow diameter implants produced more stress than wide diameter implants in all ridge widths.

Dimensions of bone in the narrow ridge model
Table

Dimensions of bone in the regular ridge model Table

Model Description
Table

Regular ridge with a standard (4 mm) diameter implant
Diagram

Narrow ridge with a standard (4 mm) diameter implant

Sequential Histology and Immunohistochemistry Analysis of Cementum Regeneration in Vivo Study

Primary Author: Yong-tae Kim, Yonsei University, College of Dentistry, Seoul, Republic of Korea
Secondary Authors: Drs. Jung Chul Park, Seong-Ho Choi, Kyoo-Sung Cho, Chang-sung Kim, Yonsei University, College of Dentistry, Seoul, Republic of Korea
Financial Support: This research was supported by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science and Technology (KRF-2008-313-E00587)
Background: Many studies reported that periodontal repair and regeneration were originated by periodontal ligament tissues. Isolation of PDLSC from periodontal ligament tissues and cryopreservation technique were also studied. Periodontal regeneration has been the ultimate goal of periodontal treatment. It has been known that human periodontal ligament stem cell(hPDLSC) is appropriate source for periodontal regeneration. The purpose of this study is to investigate the proliferation and differentiation of hPDLSC with passage of time for periodontal tissue engineering.
Methods: hPDLSCs were harvested from patients’(n=3) extracted premolars(n=12) due to orthodontic treatment. Cells were isolated from obtained periodontal tissues. Cell cultures were conducted under the defined condition. Immunohistochemical staining, fluorescence activated cell sorter(FACS) were used to identify characteristics of stem cells. hPDLSCs were transplanted into immunocompromised mice(n=5) to assess the potential for new cementum and periodontal tissue formation. Histologic and immunohistochemical studies were conducted and analyzed after 3 days, 7 days, 2 weeks, 4 weeks, 8 weeks.
Results: hPDLSCs had mesenchymal stem cell-makers such as STRO-1, CD-146, CD-44 and CD-90. Under defined culture condition, hPDLSCs were differentiated to cementoblasts. hPDLSCs, when transplanted into immunocompromised mice using a hydroxyapatite/tricalcium phosphate(HA/TCP) carrier, showed the potential to induce new cementum and collagen fiber formation. At 7 days, PDLSCs were scattered and increased among carrier particles. At 2 weeks, collagen fibers were embedded to HA/TCP carriers and then cememtum were mineralized around carriers at 4 weeks. Fibers had been more and thicker, and cementum had matured and mineralized with the passing of time.
Conclusions: This study showed that periodontal ligament tissues obtained after extraction due to orthodontic treatment contained stem cells, and these cells had potential to induce cementum and collagen fibers in vivo study. Tissue engineering using these cells, which could be achieved easily without ethical issues, might be alternative for clinical approach for reconstruction of periodontal tissue.

Expression of WNT Related Genes in Human Periodontal Ligament Cells during Osteogenic Differentiation

Primary Author: Azusa Yamada, Tokyo Medical and Dental University, Tokyo, Japan
Secondary Authors: Dr. Yuichi Izumi, Tokyo Medical and Dental University, Tokyo, Japan, Drs. Takanori Iwata, Masayuki Yamato, Isao Ishikawa, Teruo Okano, Tokyo Women's Medical University, Tokyo, Japan
Financial Support: None
Background: Periodontal ligament (PDL) is a connective tissue that exists between cementum and alveolar bone socket, and considered to contain mesenchymal stromal cells. When PDL cells are cultured with osteoinductive medium (OIM), alkaline phosphatase (ALP) activity and osteogenic gene expressions are enhanced and PDL cells form mineralized nodules after the long-term cultivation. The aim of this study was to investigate the involvement of Wnt signaling during osteogenic differentiation in human PDL cells.
Methods: Following an approved institutional review board protocol from Tokyo Women's Medical University, human PDL tissues were obtained from 3 donors, which were extracted due to impaction. Donors had given written informed concent. To investigate the multipotency of hPDL cells, colony forming assay (CFA) and differentiation assay (osteogenesis and adipogenesis) were performed. hPDL cells were cultured in OIM, which contained 50 μg/ml ascorbic acid, 10 mM β-glycerophosphate, and 10 nM dexamethasone for 5 days, and ALP activity of cells was measured. To analyze the Wnt-related genes during osteoblastic differentiation, hPDL cells were cultured with or without OIM for 4, 7, and 14 days and mRNA of each sample was collected. RT2 Profiler PCR Array (Human WNT Signaling Pathway; SABiosciences) was performed. The mRNA expression levels of interested genes were quantitatively analyzed by real-time PCR (ABI Prism 7300 Sequence detection system, Applied Biosystems) using the sequence specific primers. Primers used were as follows; β-actin (4326315E), SFRP3 (Hs00173503_m1), and SFRP4 (Hs00180066_m1). The samples were analyzed in triplicate. The mRNA expression levels relative to β-actin were determined.
Results: hPDL cells used in this study showed highly replicative activity in CFA assay, and were confirmed to form mineralized nodules in osteogenesis study and lipid droplets in adipogenesis study,respectively. These data showed that hPDL cells possessed mesenchymal stromal cell-like properties. hPDL cells grown in OIM for 5 days showed 4-folds ALP activity compared to that of cells grown in normal medium (p < 0.05). From the results of PCR array analysis, CT values of Wnt1, 3A, 6, 7A, 8A, 10A were greater than 35 ,therefore considered not to have been expressed. When cells were cultured in OIM, the expression of secreted Frizzled-related protein 3 (SFRP3) was up-regulated, on the other hand, that of SFRP4 was down-regulated in all samples. TaqMan PCR assay also confirm the gene expressions, and time course studies revealed the expression of SFRP3 was gradually increased, whereas that of SFRP4 was decreased in time course for 2 weeks.
Conclusions: These results suggest that Wnt signaling, especially SFRPs, involves in the osteogenic differentiation of hPDL cells. SFRPs, which are extracellular antagonist of Wnt signaling, may be differentially regulated during this process and can be useful as markers of osteogenic differentiation of hPDL cells.

Table. Alterations in Gene Expression by Cultured Human Periodontal Ligament Cells
Table

6 genes showed both statistically significant differential expression and OIM/- ratio greater than ± 2 in response to osteoinductive medium.
Graph

Figure 1. Effect of OIM on ALP levels of hPDL cells from 3 donors. hPDL cells grown in OIM showed high ALP activity compared to that of cells grown in normal medium.
Graph

Figure 2. Cycle threshold distribution for experimental and control group. Histogram showing the mean cycle threshold (CT) distribution for experimental and control groups. The mean values were determined from 3 replicate microarray plates. Genes with CT values of greater than 35 (absent calls) were considered to lie outside the detection threshold of the system.
Graphs

Figure 3. Effect of OIM on ALP levels of hPDL cells from 3 donors.

Correlation of Bone Mineral Density and Bone Volume in Human Jaw Bones Using Micro Computed Tomography

Primary Author: Yoon Jeong Kim, Loma Linda University, Loma Linda, CA
Secondary Authors: Drs. Pedram Fakheri, Elham Javadi, Elisa Sin, Loma Linda University, Loma Linda, CA
Financial Support: None
Background: Function of dental implants depends upon load transmission at the bone-to-implant interface. The higher failure rates of implants were attributed to poor bone quality. Various methods have been developed to evaluate the bone quality, type and architecture of bone. Two existing methods of analysis would be considered the gold standards of bone density measure are histological/histomorphometric analysis and micro computed tomography (micro-CT). There has not been any study available regarding the correlation between bone mineral density and morphometric bone volume evaluated using micro-CT. accordingly, this study was designed to study the bone density measures of human alveolar jaw bones evaluated using micro-CT bone mineral density and morphometric bone volume analysis
Methods: Human cadavers were screened and a total of 16 edentulous bone specimen blocks, 9 maxillary and 7 mandibular blocks from various regions of the jaws were selected. Each specimen block provides a minimum dimension of alveolar bone quantity accommodating a 4.0 mm in diameter and 10 mm in length dental implant. A radiopaque indicator in 2mm diameter was inserted into each block to locate the dental implant position, a region of interest.Each bone block was scanned using a micro-CT machine with 10 micron voxel size (VivaCT 40, Scanco USA). Consecutive images representing 1.5 mm thick buccolingual slice immediately mesial to the indicator and immediately distal to the indicator were selected for analysis. A region of interest, 4mm X10mm a rectangular area parallel to the indicator on each image was analyzed to measure the mean bone mineral density expressed in mg Hydroxyapatite (HA)/cm3. The total volume, bone volume and the relative bone volume within the volume of interest were also measured.
Results: A wide range of the bone mineral density was observed from 48 to 840 mg HA/cm3. The relative bone volume varies as well; the minimum 2.4 % and the maximum 68.9 % were noted (Figure 1, 2). The significantly high linear correlation was observed between the bone mineral density and the relative bone volume (Pearson’s correlation coefficient r = o.99. p<0.5). (Figure 3)
Conclusions: The study investigated the two aspects of bone quality using micro-CT. The high correlation between the bone mineral density and the bone volume suggest that the bone mineral density can be utilized to evaluate the quality of the jaw bones. The bone mineral density measure in jaw bones, the non- invasive method will enable clinicians to evaluate the pre-implant sites adequately and plan surgical and/or prosthetic treatment accordingly since there is significant variation in the jaw bone density found in the study.

Figure 1. Three dimensional photo of micro-CT scan of a low density bone block.

Figure 2. Three dimensional photo of micro-CT scan of a high density bone block
Graph

Figure 3. Relationship between the relative bone volume and bone mineral density

CLINICAL RESEARCH ABSTRACTS

The Effect of Splinted and Non-splinted Prostheses on Crestal Bone Loss around Posterior Dental Implants: A Retrospective Study

Primary Author: Jaffer Y. Kermalli, University of Toronto, Toronto, ON, Canada
Secondary Authors: Drs. Douglas A. Deporter, Ernest Lam, University of Toronto, Toronto, ON, Canada
Financial Support: None
Background: The purpose of this project was to examine the effect of splinting adjacent dental implants placed in posterior sites. Implant survival and success was assessed clinically and radiographically, the latter by measuring crestal bone levels. In addition, a comparison of two intraoral radiographic techniques to assess crestal bone levels were compared.
Methods: Patient records from a database were searched according to an inclusion criteria of posterior jaw site dental implants placed in the Oral Reconstruction Center of the Faculty of Dentistry, University of Toronto by graduate or continuing education students. Implants had to have been restored for a minimum of 1 year and have a periapical (PA) and vertical bitewing radiograph (vBW) taken at the most recent recall. Charts were reviewed and radiographs were digitalized and measured using SigmaScan Pro V4. Descriptive statistics, ANOVA, Kaplan Meier survival curve, and multiple regression analysis was performed with SAS V9.1.
Results: Of the 995 implants that qualified, 799 had both PA and vBW radiographs taken at least 1 year after prosthesis insertion. These implants were located in 345 patients (59.1% Female, 40.9% Male) with a mean age of 59.0 years (range 29.5-83.7 years). Three different implant designs with various dimensions (sintered porous surface (SPS), threaded with dual acid-etched surface (THR-DAA), threaded with SLA surface (THR-SLA)) were included. SPS implant length was shorter than the threaded implants(P<0.0001). There was no effect of implant length or width on crestal bone loss. The implants in this study were placed in the posterior maxilla (45.93%) and mandible (54.07%); 51.2% were restored with splinted prostheses and 48.8% with non-splinted prostheses. Mean crestal bone loss for each type of implant was significantly different than for the other implant types (P<0.0001). SPS had less bone loss than THR-DAA and THR-SLA. PA views showed approximately 0.10 mm (Range 0.07-0.40 mm) less crestal bone loss than vBW'S but more thread distortion (P<0.01). The data from the three different implant systems showed a consistent trend for splinted prostheses to have more crestal bone loss (0.30 mm; range 0.16-0.38 mm). The data included 49 failed implants with an overall failure rate of 4.9%. No THR-SLA implants failed, while 21 (10.9%) SPS implants and 28 (4.3%) THR-DAA implants failed. There was a significant difference in the failure rate of SPS and THR-DAA (P<0.0005) and in the time to fail (P<0.0036). SPS implants were more likely to fail several years after loading in contrast to THR-DAA implants which were more likely to fail to osseointegrate in the first 9 months.
Conclusions: Splinted implant prostheses led to more crestal bone loss than non-splinted ones, however the difference (0.30mm) may not be clinically significant. Successful SPS implants showed significantly less crestal bone loss than threaded implants. vBW radiographs show more crestal bone loss (0.10mm) and less thread distortion.

Distribution of implants in study
Table

*2 implants were observed to be failing and subsequently failed during the data collection phase of the study.

Distribution of Crestal Bone Loss
Table

Splinted vs. Non-Splinted Crestal Bone Loss
Table

Tooth Loss and Patient Compliance in Periodontally Treated Patients in an Institutional Practice

Primary Author: Marianne Ong, National Dental Centre Singapore, Singapore, Singapore
Secondary Authors: Drs. Mervyn Ng, Chu Guan Koh, National Dental Centre Singapore, Singapore, Singapore, Dr. Lum Peng Lim, National University of Singapore, Singapore, Singapore, Dr. Yiong Huak Chan, Yong Loo Lin School of Medicine, National University Health System, Singapore, Singapore
Financial Support: None
Background: Supportive periodontal therapy(SPT) is important for the maintenance of oral health in periodontally treated patients. Use of true endpoints (e.g., tooth loss) versus surrogate endpoints (e.g., probing depths) to evaluate the effectiveness of periodontal therapy may be more tangible to patients in understanding the benefits of treatment.The aim of this study was to investigate incidence of tooth loss during active periodontal therapy(APT) & SPT in periodontal patients treated at the National Dental Centre Singapore(NDCS) & to compare them to a group of patients who did not have SPT after APT.
Methods: Periodontal patients treated prior to December 1997 were identified from an electronic database. Records were retrieved and reviewed by a single examiner who had not treated them. The longitudinal retrospective study comprised of all compliers(AC): regular compliers(RC)+irregular compliers(IC), on SPT (classification by Novaes et al 1996). These were chronic periodontitis patients treated & maintained for at least 7 years by periodontists at NDCS. The clinical cross-sectional study comprised of patients who failed to attend SPT after APT, i.e., non-compliers(NC). 273 out of 478 reviewed patient folders for AC and 207 out of 1318 reviewed patient folders for NC met the inclusion criteria. Only 39 of the 207 patients agreed to participate in a single recall visit assessment. Relevant data collected from folders for AC and from both folders & the single clinical examination for NC were entered into an Excel spreadsheet. All analyses were performed using SPSS17.0 with significance set at p<0.05. Differences in tooth loss between groups were assessed using paramatric tests if normality and homogenity assumptions were satisfied otherwise non-parametric Mann Whitney U test was applied. Logistic regression was performed to determine predictors for tooth loss.
Results: Findings of this study are summarised in Tables 1 to 3. During APT, AC(N=273) & NC(N=39) lost a mean of 1.3 teeth/patient and 1.1 teeth/patient respectively, with multi-rooted teeth having a 2-fold odds of being extracted compared to single-rooted teeth. During SPT (mean period 10.7 years), AC lost a mean of 0.9 teeth/patient with an annual tooth loss due to periodontitis of 0.03 teeth/patient/year. During discontinuation of SPT (mean period 9.6 years), NC lost a mean of 2.7 teeth/patient with an annual tooth loss due to periodontitis of 0.22 teeth/patient/year. This was a 7-fold higher odds of losing teeth than those in the SPT group (p<0.05). Logistic regression analysis only showed age >/= 60 was a highly significant predictor of tooth loss during SPT (p=0.015), with adjusted odds ratio of 2.1 (95%CI:1.14-3.4) for AC.
Conclusions: Within the limitations of this study, SPT following APT led to minimal tooth loss, especially due to periodontitis, for a period of 10 years after APT in this group of patients. Patients who completed APT without SPT tended to lose more teeth compared to patients who completed APT with SPT.

Table 1: Demographics
Table

RC - regular compliers, IC - irregular compliers, AC - all compliers (RC+IC), NC - non-compliers

Table 2: Duration, number of teeth present & lost during APT
Table

APT - active periodontal therapy. No significant differences between groups (p>0.05)

Table 3: Duration, number of teeth present & lost during SPT for compliers & discontinuation of SPT for non-compliers
Table

SPT - supportive periodontal therapy, *statistically significant difference between groups (p<0.05)

Periodontal Regeneration with and without Limited Orthodontics for the Treatment of Advanced Osseous Lesions

Primary Author: Shigeki Ogihara, Ogihara Dental Clinic, Tokyo, Japan
Secondary Author: Dr. Hom-Lay Wang, University of Michigan, Ann Arbor, MI
Financial Support: None
Background: Limited orthodontics has been shown to correct some infrabony defects. Studies have demonstrated the efficacy of using enamel matrix derivative (EMD) with demineralized freeze-dried bone allograft (DFDBA) to treat intraosseous defects. To our knowledge, no study has examined the limited orthodontic treatment together with EMD/DFDBA in their ability of treating advanced periodontal disease. Therefore, the purpose of this study was to compare the clinical efficacy of limited orthodontics combined with EMD/DFDBA or EMD/DFDBA alone for the treatment of 2- or 3-wall infrabony lesions.
Methods: A total of 47 consecutive patients with 50 2-, 3-wall or combination infrabony defects of ≥6mm were reported. Defects were first treated with EMD and DFDBA (i.e. 0.25cc of DFDBA containing 0.3 ml of EMD). Four weeks after surgery, orthodontic extrusive force was applied utilizing fixed appliances to enable extrusion and uprighting/concurrent extrusion. Defects were randomly assigned to the below 2 treatment groups: Ortho/ EMD/DFDBA {OED} (n=26) and EMD/DFDBA {ED} (n=24) alone. Re-entry surgeries were performed at 6 months after initial surgery and open probing attachment level (OPAL) recorded. Clinical attachment level (CAL) and probing depth (PD) were obtained 1 year after initial surgery to prevent the influence of orthodontic fixed appliance. Data were then statistically analyzed using paired t test and Wilcoxon matched-pairs signed ranks tests.
Results: Both treatment groups showed a significant improvement from baseline. However, no significant difference was found between two groups except 2-wall defect sites. Relative improvements at 2-wall defect sites were in CAL for 45±5% (ED) and 50±8% (OED) (p=0.043) and in OPAL for 42±11% (ED) and 51±7% (OED)(P=0.067).
Conclusions: Results obtained from this study indicated that both treatments were effective for the treatment of 2- or 3-wall infrabony defects. However, the limited orthodontics provided additional benefit to EMD/DFDBA in managing 2-wall infrabony defects.

Chronic Gingivitis as the Control Group for Case/Control Studies of Periodontitis Genetics: A Paradigm Change Based in Case/Control Definition Reappraisal and Supported by Classic SNP Analysis

Primary Author: Gustavo P. Garlet, School of Dentistry of Bauru, Bauru, Sao Paulo, Brazil
Secondary Authors: Drs. Ana Paula F. Trombone, Joao S. Silva, São Paulo University, Sao Paulo, Brazil, Drs. Gabriela Gennaro, Walter Martins, Ana Paula Camapanelli, School of Dentistry of Bauru, Bauru, Sao Paulo, Brazil, Dr. Cristina R. Cardoso, Federal University of Triângulo Mineiro, Minas Gerais, Brazil, Drs. Ariadne Letra, Renato Menezes, Alexandre R. Vieira, University of Pittsburgh, Pittsburgh, PA
Financial Support: None
Background: Scientific literature regarding chronic periodontitis (CP) pathogenesis includes thousands of studies focused in the possible contribution of genetic factors to CP outcome; usually case/control (C/C) studies evaluating single nucleotide gene polymorphism (SNPs). However, these results are usually highly inconsistent and controversial. A case-control study is designed to determine if an exposure is associated with an outcome. However, a control group comprising periodontally healthy (H) subjects, not exposed to the risk factor (i.e. periodontopathogens), does not allow the determination of ‘susceptible’ or ‘resistant’ phenotypes. In this scenario, a critical reappraisal in C/C definitions might lead to a paradigm change in PD genetics studies.
Methods: Therefore, we propose that chronic gingivitis (CG) patients (exposed to the risk factor, presenting periodontal tissue inflammation, but not bone loss), could represent a genetically resistant group opposing the susceptible group of CP, and tested this hypothesis in C (N=190), CP (N=178) and CG (N=153) groups through the FLRP analysis of classic SNPs IL1B-3954, IL6-174,TNFA-308 and IL10-592.
Results: Our results demonstrate that when CP and H groups were compared, IL1B-3954 T allele (p=0.063, OR=0.66) and IL10-592 A allele (p=0.03, OR=0.68) presented a significant but weak association, while TNFA-308 A allele (p=0.29, OR=0.77) and IL6-174 C allele (p=0.45, OR=0.85) frequency was similar in both groups. However, when CG was set up as a “resistant” control versus the CP susceptible group, IL1B-3954 T allele (p=0.0075, OR=0.53), IL10-592 A allele (p=0.0006, OR=0.50) and TNFA-308 A allele (p=0.018, OR=0.51) degree of association with resistant/susceptible phenotypes were dramatically increased, but IL6-174 C allele (p=0.19, OR=0.76) frequency remained statistically similar between these groups. Similar results were obtained when the genotypes were analyzed: IL1B-3954CC (HvCP: p=0.016, OR=0.58; CGvsCP: p=0.0011, OR=2.20); IL10-592AA (H vs CP: p=0.01, OR=2.3; CG vs CP: p<0.001, OR=0.28); TNFA-308AA (HvsCP: p=0.18, OR=0.72; CGvsCP: p=0.005, OR=2.20); IL6-174CC (HvsCP: p=0.34, OR=0.80; CGvsCP: p=0.09, OR=1.45), when the risk/protective effect of the SNPs analyzed was significantly more evident in the CG vs CP comparison. These increases in statistical significance degree are possibly due to the analysis of extreme resistant and susceptible phenotypes, which is not possible with a H group including subjects genetically prone to develop periodontitis, but able to control the etiological bacterial factor. It is very important to mention that bacterial challenge may differ in CP and CG groups, and this co-variate must be considered in future studies.
Conclusions: Our results demonstrate that a reappraisal of case/control study design may impact futures studies of periodontitis genetics, and that the inclusion of chronic gingivitis as the resistant phenotype control group may contribute to unravel the genetic basis of PD.

Contribution of Local and Systemic Inflammatory Responses in Non-controlled Type II Diabetic Patients with Periodontal Disease

Primary Author: Ruben F. Mesia, University of Florida, Gainesville, FL
Secondary Authors: Drs. Shannon M. Wallet, Ikramuddin Aukhil, Michael J. Clare-Salzler, Luciana M. Shaddox, University of Florida, Gainesville, FL
Financial Support: None
Background: Numerous studies have shown a correlation between chronic inflammatory periodontal disease and diabetes, in which both of them influence the progression and response to treatment of the other. However, the mechanisms behind the association of these two diseases are not yet elucidated. The objective of this study was to evaluate whether the local and/or systemic inflammatory responses in non-controlled Type II diabetic patients with periodontal disease were more robust than those of non-diabetic patients with periodontal disease.
Methods: Gingival crevicular fluid (GCF) and blood samples were collected from 20 patients with chronic periodontal disease (10 uncontrolled type II diabetes-DBT; 10 non-diabetic-NDBT). GCF samples were collected from a diseased site (pocket depth-PD≥5mm and attachment loss–AL≥2mm) and a healthy site (no AL, no bleeding on probing-BoP). Blood samples were stimulated with ultra-pure TLR-2 and TLR-4 agonists for 24h. Twenty-two cyto/chemokines were quantified in GCF and culture supernatants using Luminex.
Results: DBT patients showed higher unstimulated levels of IL-8, TNFα, IL-10, MIP1α and MIP1β; higher stimulated levels of IL-6, IL-8, IL-10, MIP1α and MIP1β; and lower stimulated and unstimulated levels of GM-CSF than NDBT (p<0.05). DBT patients also showed higher levels of MIP1β in GCF from healthy sites (p<0.001) than NDBT, while NDBT showed higher levels of IL-7 in GCF from diseased sites.
Conclusions: Significantly higher systemic pro-inflammatory response in DBT patients could explain the increased susceptibility to periodontal disease in these patients. Conversely, this increase in mediators could be a reflection of the negative effect periodontal disease has on diabetic metabolic control. In addition, the lower levels of certain defensive cytokines in DBT patients upon stimulation with TLRs could indicate an impaired immune response in this patient population.

Comparative Microbiological Analyses of Biofilms in Patients with Prosthetic Joint Infections and Periodontitis

Primary Author: Volker Clar, Medical University Graz, Graz, Austria
Secondary Authors: Drs. Alexander Heschl, Michael Haas, Reinhard Windhager, Heimo Clar, Gernot Wimmer, Medical University Graz, Graz, Austria, Dr. Rutger Persson, Medical University Bern, Bern, Switzerland
Financial Support: None
Background: Biofilms play an important role in infections of the prosthetic joint as well as periodontitis. The aim of the present pilot study was to compare the biofilm of the subgingival plaque of periodontitis infected teeth with the biofilm of prosthetic joint infections (PJI).
Methods: Biofilm were taken from 10 patients with PJI and chronic periodontitis. Microbial samples were obtained using paper-points from the subgingival tooth surface and the explanted orthopaedic implants. Each paper-point-sample was sent in separated tubes to microbiological test (checkerboard + RT-PCR). The results of both locations were compared.
Results: Bacteria found in biofilms of periodontal lesions were similar to the biofilms in PJI of the same patient in 7 cases. Positive detected bacteria implicated A.actinomycetemcomitans (Aa), F.nucleatum (Fn), P.aeruginosa, P.gingivalis (Pg), P.intermedia (Pi), Tann.forsythia (Tf). In three patients no correlation could be found.
Conclusions: Bacteria from periodontal lesions could be disseminated and settle on artificial surfaces of prosthetic joints. Further data are needed to evaluate the interaction between prosthetic joint infections (PJI) and periodontitis.

Factors Influencing the Periodontal Referral Process

Primary Author: Christina Park, University of Kentucky, Lexington, KY
Secondary Authors: Drs. Mohanad Al-Sabbagh, Adam Branscum, Ershal Harrison, Mark V. Thomas, University of Kentucky, Lexington, KY
Financial Support: None
Background: In North America, most periodontal specialists see patients via the referral process from general dental practitioners (GP). GPs assess patient’s periodontal status and make decisions either to treat or refer to a periodontist. Ideally, referrals should be a smooth transfer of care of patients from one practitioner to another. In reality, this process is complicated by many factors. Studies indicate that a substantial number of patients diagnosed with periodontal disease remain untreated. There is little information available regarding the referral process. The purpose of this study is to identify factors that GPs consider most important in influencing the referral process. The findings will be compared and contrasted with periodontists’ responses in order to gain perspectives from both groups involved in the referral process.
Methods: An online cross-sectional survey of GPs (n=533) and periodontists (n=533) practicing in the southeastern region of U.S. was conducted using a questionnaire. Online address lists were obtained from the American Dental Association and the American Academy of Periodontology. Subjects were asked to rate the importance of each of 16 identified factors’ influence on the referral process on a 5 point LIkert scale (with 1=not important to 5=most important). Demographic data of the subjects were also collected for the purpose of comparing the characteristics of the two study groups. The results were analyzed to rank-order the factors by importance for each study group and to identify statistically significant differences in responses between the two groups.
Results: “Specialist’s clinical skill” was identified as the most important factor influencing referral by the GPs (86%), while periodontists (72.5%) rated “previous positive experience between GP and periodontist” as the most important factor. General dentists rated the following as most important (In descending order of importance): clinical skill (4.84), previous positive experience (4.57), communication (4.52), likelihood of good patient-specialist rapport (4.50), and similar practice philosophies (4.20). The periodontists’ top five factors were: previous positive experience (4.66), communication (4.40), likelihood of good patient-periodontist rapport (4.29), similar practice philosophies (4.25), and specialist returns patients back to GP within a reasonable time period (4.19). Factors that showed statistically significant differences (p value <0.05) in the responses between the two groups were clinical skill, social interaction between GP and periodontist, periodontist’s board certification, and reciprocity of referrals to GP. (Table 1)
Conclusions: The clinical skill of the periodontist was identified by the GPs as the primary factor influencing the GP’s referral decision. Periodontists perceived “previous positive experience between the GP and periodontist” as the most influential factor. Further studies are required to gain more insight into the referral process.

Table 1: Ranking Importance of Factors: GPs vs. Periodontists Table

Note: *indicates statistically significant differences. Ratings on a scale of 1(=not important) to 5(=most important).

Aggregatibacter Actinomycetemcomitans is Associated with an Increased Risk for Adverse Pregnancy

Primary Author: Emi Hirano, Niigata University School of Medical and Dental Sciences, Niigata, Japan
Secondary Authors: Drs. Noriko Sugita, Akira Kikuchi, Yasuko Shimada, Jun Sasahara, Ruriko Iwanaga, Kenichi Tanaka, Hiromasa Yoshie, Niigata University School of Medical and Dental Sciences, Niigata, Japan
Financial Support: None
Background: Periodontitis has been considered a systemic exposure implicated in a higher risk of adverse pregnancy. The aim of this study was to determine whether maternal periodontitis and oral microbial burden were associated with the development of adverse pregnancy.
Methods: The sample was composed of systemic healthy 130 women who had giving birth at the Department of Gynecology of Niigata University Medical and Dental Hospital. Maternal demographic and medical data (body mass index, urine protein, preeclampsia, pregnancy induced hypertension) were collected from medical records. Preeclampsia was defined as blood pressure >140/90 mm Hg and > or =1+ proteinuria after 20 weeks of gestation. The clinical periodontal parameters, the levels of Aggregatibacter actinomycetemcomitans (A.a.), Porphyromonas gingivalis (P.g.), Prevotella intermedia (P.i.) in subgingival plaque collected from 4 teeth per participant with the sites of the deepest probing depths, and the maternal immunoglobulin G (IgG) responses in serum to these bacteria were measured within 5 days after labor. We used the Mann-Whitney U test, and χ2 test for statistical analyses.
Results: There were 38 women with periodontitis. Positive correlations were found between the numbers of bacteria with the serum IgG antibody levels to these bacteria. There was no association between adverse pregnancy and presence of periodontitis. The number of A.a. was associated with an increased risk for preeclampsia (P=0.0103), pregnancy induced hypertension (P=0.0169), urine protein (P=0.0084), PTB (P=0.0254), and maternal obese before pregnancy (P=0.0062). The maternal clinical measurements of periodontitis were correlated positively with the number of P.g. and P.i. (the mean probing pocket depth; r=0.313, P=0.0003 and r=0.345, P<0.0001, the mean clinical attachment level; r=0.285, P=0.0010 and r=0.344, P<0.0001, percentages of the sites with bleeding on probing; r=0.388, P<0.0001 and r=0.263, P=0.0024, and the mean gingival index; r=0.319, P=0.0002 and r=0.185, P=0.00352, respectively), although, no statistically significant association was found between these pathogens and maternal obstetric parameters.
Conclusions: The present study shows that maternal infection for A.a during pregnancy may be associated with an increased risk for the adverse pregnancy.A.a has been implicated in the pathogenesis of various infectious diseases, such as endocarditis and meningitis, as well as in several types of periodontitis. A.a produces a leukotoxin family and cytolethal distending toxin (CDt). The combined actions of leukotoxin and Cdt may provide a certain efficient virulence mechanism for adverse pregnancy. While, this increased number of A.a may not be inducing factor of advised pregnancy but a passive result. Further studies are needed to evaluate the specific involvement of A.a in the pathogenesis of adverse pregnancy outcome.

Obstetric and periodontal characteristics with periodontopathic bacteria.
Table

Associations between periodontopathic bacteira and preterm birth, preeclampsia and pregnancy induced hypertension were analysed with Mann-Whitney U test. Correlation between periodontopathic bacteira and mean CAL or BOP (%) were analysed with chi square test. * Statistically significant (P<0.05).

Graph