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2011 Research Forum Poster Session Abstracts

Abstracts for finalists in the Research Forum Poster competition during the 97th AAP Annual Meeting in Miami Beach.

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, and the following researchers won cash prizes:

Clinical Impact Award: Kazuhiro Okuda
Clinical Research: Rodrigo López
Basic Research: Po-Chun Chang

Abstracts can also be accessed using the AAP's Abstract Viewer.

BASIC RESEARCH ABSTRACTS

Effect of DMP1 on Periodontal Ligament Stem Cells

Primary Author: Sangeetha Chandrasekaran, University of Illinois at Chicago, Chicago, IL
Financial Assistance: None
Background: Periodontitis and the resultant inflammation can lead to destruction of connective tissue attachment to the tooth, resorption of alveolar bone and ultimately tooth loss. The periodontal ligament has necessary stem cells that has the ability to regenerate lost periodontal tissues. Many natural and synthetic materials have been tried to achieve periodontal regeneration but the results remain variable and unpredictable.
During periodontal ligament formation, periodontal progenitor cells penetrate disintegrated hertwigs epithelial root sheath and contact with root dentin to form periodontium. Clinically, direct contact of the conditioned root surfaces with periodontal cells seems to be a pre-requisite for periodontal regeneration. Previous studies have demonstrated that dentin molecules might function as regulatory signals for various mesenchymal and inflammatory cells to enhance healing potential of periodontal tissues. One such non-collagenous protein in dentin is Dentin Matrix Protein 1 (DMP1). The purpose of this project is to study the biologic effects of DMP1 on human periodontal ligament stem cells In Vitro and assess the advantages of using DMP1 as a biological mediator to enhance the regenerative potential of the periodontium.
Methods: hPDLSC cells were a kind gift from Dr. Shi (USC, School of Dentistry). Briefly PDL was isolated from extracted teeth and pooled as single cell suspensions. Stem cells were identified by bromodeoxyuridine incorporation for 24 hours. hPDLSC were cultured in the presence or absence of rDMP1. Quantitative PCR reactions were performed to analyze the expression of several genes involved in periodontal regeneration. hPDLSC’s were plated on glass cover slips, treated with rDMP1and processed for immunocytochemical analysis. After incubation with primary antibodies for DMP1 and phospho-ERK, the cells were incubated with the appropriate secondary antibodies and examined by confocal microscopy. Western blot analysis of rDMP1 treated and control samples were done using pERK and ERK antibodies. Alkaline phosphatase and VonKossa staining were performed to characterize the differentiation of hPDLSCs into osteoblasts. Field emission scanning electron microscopic analysis of the treated and control cell cultures were also performed.
Results: Treatment with rDMP1 resulted in the upregulation of genes like Matrix metalloproteinase-2, Alkaline phosphatase, Osteoprotegerin and transforming growth factor β1. Activation of Erk MAP kinase signaling pathway and translocation of pERK from the cytoplasm to the nucleus was observed. Overall, DMP1 treated cells showed increased expression of alkaline phosphatase, increased matrix and mineralized nodule formation when compared to untreated controls.
Conclusion: DMP1 can orchestrate a coordinated expression of genes and phenotypic changes in hPDLSCs by activation of ERK signaling pathway which may provide a valuable strategy for tissue engineering approaches in periodontal regeneration.

Differential Expression of Mesenchymal Stem Cells and Periodontal Ligament Fibroblasts in Diabetic Conditions: An in Vitro Investigation

Primary Author: Po-Chun Chang, National University of Singapore, Singapore
Financial Assistance: Supported by NUS/MOE R-222-000-034-133 and R-222-000-041-112 (Singapore)
Background: Diabetes is known to influence periodontal health according to the hyperglycemic status and extensive tissue glycation. However, there is still lack of suitable model to investigate the cellular behvaior of periodontal ligament fibroblasts (PLFs) and mesenchymal stem cells (MSCs) in diabetic periodontitis. Thus, in the present study we fabricated an artificial matrix to simulate glycation under diabetes and investigated the gene expression and viability of PLFs and MSCs in vitro.
Methods: Glycation of extracellular matrix was fabricated by incubating type I collagen with glucose-6-phosphate (G6P), and matrix cross-link was characterized by fluorescence and under confocal microscopy. We simulated several in vitro clinically-relevant conditions by adding the lipopolysaccharide (LPS) of P. gingivalis, adjusting the concentration of glucose in the culture medium, glycating collagen matrix, or combinations. The viability of human PLFs and MSCs was examined by MTT and LDH assays at day 1 and 4. Gene expression of the cellular receptors, including toll-like receptor 2 and 4 (TLR2, TLR4) and receptor for advanced glycation end product (RAGE), inflammatory signalling, including initiator (NF-kB) and late response gene (HMGB1), oxidative stressor gene (heme oxygenase-1, HO-1), attachment (integrin-α2), and type I and IV collagen, were evaluated by realtime PCR at 6 and 24 hours.
Results: The intensity of autofluorescence increased in G6P treated collagen matrix, indicating the enhancement of crosslinking and resistance to degradation. The fiber density and orientation did not significantly change after G6P treatment. Reduction of mitogenesis and increasing apoptotsis were noted in PLFs and MSCs seeding on glycated matrix or under LPS treatment. However, high glucose content appeared to favor the viability of PLFs but inhibit MSCs. Furthermore, glucose concentration could only down-regulate integrin-α2 without significant alteration of other gene expression of MSCs and PLFs. Both matrix glycation and LPS treatment promoted expression of TLR and RAGE-mediated inflammation and increase oxidative stress, whereas the levels of type I and IV collagen were reduced. However, although not significantly altered the level of cellular receptors, with the presence of RAGE or LPS, high glucose can further interfere the matrix synthesis and up-regulate the oxidative stressor.
Conclusion: Our artificial matrix successfully simulated the properties of glycated matrix in diabetes. PLFs and MSCs behaved differently under high glucose content culture condition, whereas the concentration of glucose did not significant affect expression of inflammatory signaling genes unless the presence of matrix glycation or LPS. The coincidence of TLR and RAGE up-regulation under LPS treatment and/or matrix glycation implied the existence of cross-talk between diabetes and periodontitis. Supported by NUS/MOE R-222-000-034-133 and R-222-000-041-112 (Singapore).

Freeze-Dried Platelet-Rich Plasma (PRP)-Coated Polyglactin Mesh: A Promising Alternative for Wound Care and Periodontal Regenerative Therapy

Primary Author: Tomoyuki Kawase, Institute of Medicine and Dentistry, Niigata University, Niigata, Japan
Financial Assistance: None
Background: In clinical periodontal studies, we have demonstrated that topical application of autologous platelet-rich plasma (PRP) at the site of a periodontal bone defect contributes to regeneration of periodontal tissues. However, there are two major challenges with this approach for regeneration. First, autologous PRP requires immediate preparation prior to clinical application, and second, its liquid form reduces handling efficiency. To expand the clinical applicability and practical availability, the autogeneic source of PRP should be expanded to an allogeneic source and its liquid form should be converted to a solid or semi-solid form. We have developed a wound-dressing coated with freeze-dried PRP and tested its biological effectiveness using both cultured epidermal fibroblasts and a skin wound model produced in genetically bred diabetic mice.
Methods: PRP was prepared from blood of young healthy volunteers and coated onto polyglactin mesh (Fig. 1A). PRP-coated mesh was then freeze-dried and stored at 4°C for at least 4 weeks until used (Fig. 1B, C: 4 months). Growth factors contained in the PRP-coated mesh and frozen PRP, which were derived from the same blood sample, were measured by a commercial cytokine-antibody array. Mouse fibroblasts were isolated from dorsal epidermal tissues, passaged 3-5 times, and utilized in the in vitro experiments. Cells were treated with PRP-coated mesh through polyethylene terephthalate membrane for 3 days in a low-serum medium (Fig. 2A), and their mitogenic activity was evaluated by the ability to reduce tetrazolium salt. On the back of genetically bred diabetic mice (5-week old, female), full-thickness skin defects (10 x 10mm, 2 defects per mouse) were prepared, dressed with PRP-coated mesh, and covered by polyurethane film. Wound-healing was evaluated macroscopically and microscopically.
Results: In PRP-coated mesh, CD41-positive platelets were well trapped (Fig 1D vs. E). Growth factors in PRP-coated mesh were almost equally detected in the frozen PRP preparation (Table 1). In the in vitro study, PRP-coated mesh formed a fibrin clot (Fig. 2B vs. C) and significantly stimulated cell proliferation (Fig. 2D). In the in vivo study, PRP-coated mesh increased the number of cells positive for α-smooth muscle actin, presumably myofibroblasts, and collagen deposition (Fig. 3). At 20 days, macroscopic observations demonstrated that wound almost healed in PRP-coated mesh (Fig. 4B vs. D). Microscopic observations demonstrated that PRP-coated mesh increased angiogenesis and facilitated formation of granulation tissue and re-epithelialization (Fig 5).
Conclusion: These findings suggest that our PRP-coated mesh could maintain the bioactivity of growth factors in PRP at sufficient levels for a month or longer a 4°C. Therefore, this PRP-coated mesh could be clinically used as an alternative for freshly prepared autologous PRP and could conveniently be utilized in periodontal regenerative therapy.

Immunohistoflourescence and MRNA Expression of Bone Markers during Early Healing around Dental Implants. A Novel Approach in Miniature Pigs.

Primary Author: Yung Kyun Kim, University of Connecticut, Farmington, CT
Financial Assistance: Institut Straumann AG: supporter of this study
Background: To better understand the effect of implant surface modification on bone, more information is needed on the early stages of bone healing around dental implants in vivo. Quantitative reverse transcription polymerase chain reaction (QRT-PCR) is a method that can be utilized to evaluate the early stages of peri-implant wound healing at molecular level by analyzing the level of gene transcription. In addition, the level of protein expression can be analyzed by immunohistoflourescence (IHF).
A pilot study was performed to evaluate the expression of the osteogenic genes/biomarkers, and the quantity/quality of bone in contact with the implant during the early healing around titanium SLA, titanium SLActive, and titanium zirconium SLActive (Roxolid) implant surfaces in the miniature pigs. Main objective of this pilot study was to optimize the research methodology of studying the early bone healing at the molecular level with QRT-PCR and IHF in the miniature pigs.
Methods: Total of 36 bone chamber implants were implanted in the mandible of 6 mini pigs. Each hemi mandible received three randomly allocated bone chamber implants on both sides in a split-mouth design. Animals were sacrifice at 3 days and 2 weeks of healing period. One hemi mandible was foreseen for histological and histomorphometrical analysis. The other hemi mandible was foreseen for IHF analysis. The presence of B-catenin, Runx2, Osteopontin(Opn), and Osteocalcin(Ocn) were measured by IHF. RNA was isolated from two animals at 2 weeks; Osterix(Osx), Opn and Ocn mRNA expression levels were evaluated for osteoblasts differentiation by QRT-PCR.
Results: No significant histological difference was recorded between the different implant types at each sacrifice time point. Histomorphometrical analysis showed that there was no significant difference between the groups concerning the bone area in total area in bone chambers at 3 days and 2 weeks. QRT-PCR at 2 weeks showed that SLActive have the highest Opn and Ocn mRNA expression and the lowest Osx expression. Two fold higher expression of Osx was reported on SLA and Roxolid compared to SLActive. At 3 days, none of the biomarkers were detected by IHF in this miniature pig model. At 2 weeks, Ocn was only detectable biomarker which was expressed highest by SLActive followed by Roxolid and SLA; however, there was no statistically significant difference between the groups in the level of Ocn expression.
Conclusion: Within the limitations of this initial study, it might be concluded that the combination of QRT-PCR and IHF is a valuable technique to evaluate the early stages of wound healing around endosseous implants. These preliminary data suggest earlier expression of Ocn with SLActive compared to SLA and Roxolid. Thus SLActive may entail enhanced osteoconductive properties when compared to the other surfaces tested in this experiment. However, further experimental studies of higher power are needed in order to validate the research methods and to confirm these preliminary findings.

Figure 1. Gene expression was measured for Osterix (green), Osteopontin (purple), and Osteocalcin (blue) at 2 weeks – mean values and standard error. Gapdh was used for endogenous control. SLActive demonstrated lowest level of Osterix, but highest level of Osteopontin & Osteocalcin. However, due to small sample number and large difference between two animals, no statistical significance level of gene expression was found between SLActive, SLA, and Roxolid.

Figure 2. Representative immunohistoflourescence views at 3 days. (left) DAPI (4',6-diamidino-2-phenylindole) + Cy5 (Cyanine 5); DAPI’s blue emission illustrates DNA and outlines the chamber. (middle) Cy5; red emission illustrates Osteocalcin antigen reactivity, which outlines native bone and not detected inside the chamber. (right) Negative control shows there was no background staining.

Figure 3. Representative immunohistoflourescence views of Roxolid implant at 2 weeks. (left) DAPI + Cy5; combination of blue (DAPI) and red (Cy5) clearly demarcate the bone chamber. (middle) Cy5; a pronounced Osteocalcin antigen reactivity (red) was observed inside the bone chamber. (right) Negative control.

Figure 4. Quantification of Osteocalcin expression inside the bone chambers detected by immunohistoflourescence at 2 weeks - Osteocalcin / total area [%], mean value and standard error. There was no statistical difference between SLActive, SLA, and Roxolid.

Small Interfering RNA-Mediated Silencing of Toll-like Receptors 2 and 4 Modify Interleukin (IL)-6 and IL-8 Production by Human Gingival and Periodontal Ligament Fibroblasts

Primary Author: Ana Carolina Morandini, University of São Paulo Bauru School of Dentistry, Bauru, SP, Brazil
Financial Assistance: None
Background: Increasing interest has been directed to the role of the innate immune system in inflammatory diseases, including periodontitis. In essence, several pathogen associated molecular patterns (PAMPs) from bacteria can interact with pattern recognition receptors (PRRs) such as Toll-like receptors (TLRs) and have been found to be important for the innate immune system. The innate host response to lipopolysaccharide (LPS) from Porphyromonas gingivalis is unusual in that different studies have reported that it can be an agonist for TLR2 as well as an antagonist or agonist for TLR4. Besides inflammatory cells, fibroblasts express TLRs and the osteotropic cytokine Interleukin (IL)-6 as well as the chemokine IL-8 are expressed by human gingival fibroblasts (HGF) and human periodontal ligament fibroblasts (HPLF). The present study investigated whether signaling through TLR2 or TLR4 can affect the production of IL-6 and IL-8 in HGF and HPLF from the same volunteer donors.
Methods: Fibroblasts were isolated and cultured from gingival and periodontal ligament of systemically healthy volunteers. Cells were seeded in 96-well plates at 8.5 x 103cells/well in Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 10% fetal bovine serum (FBS). After overnight attachment, the fibroblasts were transfected with a scrambled siRNA, TLR2 or TLR4 siRNA. Twenty-four hours after transfection, the media were changed and DMEM with 10% FBS was added with or without the addition of P. gingivalis LPS (1µg/mL), E. coli LPS (1µg/mL), Pam2CSK4 (TLR2/6 agonist, 50 ng/mL), or Pam3CSK4 (TLR2/1 agonist, 500 ng/mL). Six hours after being challenged, the supernatant was collected and the levels of IL-6 and IL-8 were analyzed by enzyme-linked immunosorbent assay (ELISA). Quantitative real-time PCR analysis using the TaqMan Gene Expression PCR Master Mix Kit and specific primers was performed to confirm the TLR2 and TLR4 Knock-down.
Results: The silencing of TLR2 and TLR4 was approximately 75%. Knock-down of TLR2 by siRNA resulted in significantly decreased IL-6 and IL-8 production in response to P. gingivalis LPS as well as to Pam2CSK4 and Pam3CSK4 both in HGF and HPLF. Interestingly, the silencing of TLR4 also resulted in lower levels of IL-6, but not of IL-8 for the cells stimulated with Pam2CSK4 and Pam3CSK4. When challenged by E.coli LPS, only HGF showed a significantly decrease in IL-6 and IL-8 levels under TLR2 and TLR4 knock-down and the reduction was more pronounced with TLR4 silencing.
Conclusion: Using two synthetic ligands acting specifically at TLR2 and siRNA knock-down of TLR2 and TLR4, it was demostrated the important role of TLR2 in the production of IL-6 and IL-8 in HGF and HPLF. These data suggests that signaling through the innate immune system by the most numerous resident non-professional immune cells in periodontium controls the production of important cytokines that modulate the inflammatory microenvironment.

Effect of TLR4 silencing in HGF and HPLF as assessed by mRNA analyses of TLR4. The mRNA levels were assessed by quantitative real-time PCR with TaqMan assays and normalized against RPL13A (reference gene). Cells were seeded in 96-well plates at 8.5 x 103cells/well in Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 10% fetal bovine serum (FBS). After overnight attachment, fibroblasts were transfected with a scrambled siRNA or TLR4 siRNA. Twenty-four hours after transfection, the media were changed and DMEM with 10% FBS was added with or without the addition of P. gingivalis LPS (1µg/mL), E. coli LPS (1µg/mL), Pam2CSK4 (TLR2/6 agonist, 50 ng/mL), or Pam3CSK4 (TLR2/1 agonist, 500 ng/mL). The values represent mean ± SEM from 4 wells/group. Asterisk denote statistical difference compared with SCR siRNA (*p < 0.01).

Effect of TLR2 and TLR4 silencing in IL-6 production by HGF. Cells were seeded in 96-well plates at 8.5 x 103cells/well in Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 10% fetal bovine serum (FBS). After overnight attachment, fibroblasts were transfected with a scrambled siRNA, TLR2 or TLR4 siRNA. Twenty-four hours after transfection, the media were changed and DMEM with 10% FBS was added with or without the addition of P. gingivalis LPS (1µg/mL), E. coli LPS (1µg/mL), Pam2CSK4 (TLR2/6 agonist, 50 ng/mL), or Pam3CSK4 (TLR2/1 agonist, 500 ng/mL). The values represent mean ± SEM from 4 wells/group. Asterisk denote statistical difference compared with SCR siRNA (*p < 0.001).

Effect of TLR2 and TLR4 silencing in IL-6 production by HPLF. Cells were seeded in 96-well plates at 8.5 x 103cells/well in Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 10% fetal bovine serum (FBS). After overnight attachment, fibroblasts were transfected with a scrambled siRNA, TLR2 or TLR4 siRNA. Twenty-four hours after transfection, the media were changed and DMEM with 10% FBS was added with or without the addition of P. gingivalis LPS (1µg/mL), E. coli LPS (1µg/mL), Pam2CSK4 (TLR2/6 agonist, 50 ng/mL), or Pam3CSK4 (TLR2/1 agonist, 500 ng/mL). The values represent mean ± SEM from 4 wells/group. Asterisk denote statistical difference compared with SCR siRNA (*p < 0.001).

Effect of TLR2 and TLR4 silencing in IL-8 production by HGF. Cells were seeded in 96-well plates at 8.5 x 103cells/well in Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 10% fetal bovine serum (FBS). After overnight attachment, fibroblasts were transfected with a scrambled siRNA, TLR2 or TLR4 siRNA. Twenty-four hours after transfection, the media were changed and DMEM with 10% FBS was added with or without the addition of P. gingivalis LPS (1µg/mL), E. coli LPS (1µg/mL), Pam2CSK4 (TLR2/6 agonist, 50 ng/mL), or Pam3CSK4 (TLR2/1 agonist, 500 ng/mL). The values represent mean ± SEM from 4 wells/group. Asterisk denote statistical difference compared with SCR siRNA (*p < 0.001).

Effect of TLR2 and TLR4 silencing in IL-8 production by HPLF. Cells were seeded in 96-well plates at 8.5 x 103cells/well in Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 10% fetal bovine serum (FBS). After overnight attachment, fibroblasts were transfected with a scrambled siRNA, TLR2 or TLR4 siRNA. Twenty-four hours after transfection, the media were changed and DMEM with 10% FBS was added with or without the addition of P. gingivalis LPS (1µg/mL), E. coli LPS (1µg/mL), Pam2CSK4 (TLR2/6 agonist, 50 ng/mL), or Pam3CSK4 (TLR2/1 agonist, 500 ng/mL). The values represent mean ± SEM from 4 wells/group. Asterisk denote statistical difference compared with SCR siRNA (*p < 0.01).

Localization of Porphyromonas Gingivalis and Tannerella Forsythia in Gingival and Subgingival Granulation Tissues Using Novel Monoclonal Antibodies

Primary Author: Amodini Rajakaruna Gamaralalage, Tokyo Medical and Dental University, Bunkyo-ku, Tokyo, Japan
Financial Assistance: None
Background: In the last few decades several molecular techniques have been developed to detect periodontopathic bacteria such as PCR, whole-genomic probes and DNA microarray. Despite accuracy and specificity in these methods, it is not possible to locate periodontopathic bacteria in tissue specimens while preserving the tissue architecture. Immunohistochemistry (IHC) is a methodology that allows location of antigen-antibody reactions while observing cell morphology and tissue architecture. However IHC has challenging requirements such as necessity of specific antibodies and preservation of bacteria during tissue processing. Therefore the objectives of this study were to develop specific monoclonal antibodies for Porphyromonas gingivalis and Tannerella forsythia and to locate these bacteria in formalin fixed and paraffin embedded diseased gingival/subgingival granulation tissues.
Methods: BALB/c mice were immunized with whole bacterial cell lysate of P. gingivalis ATCC 53978 and T. forsythia ATCC 43037 respectively. Hybridoma cell lines producing anti- P. gingivalis and anti- T. forsythia antibodies were checked for specificity with ELISA and with IHC using formalin-fixed, paraffin-embedded rat liver sections infected with each bacterial species. The specificity and cross reactivity of monoclonal antibodies were confirmed with IHC using rat liver sections infected with pathogenic and commensal bacteria commonly present in human body.
Gingival & subgingival granulation tissues were collected during periodontal surgery from 60 patients with chronic periodontitis who have undergone initial periodontal therapy. Tissues were immediately fixed in 10% buffered neutral formalin and paraffin embedded. IHC was done to investigate the localization of P. gingivalis and T. forsythia in granulation tissues and their existences were also confirmed by real time detection.
Results: Anti-P. gingivalis and anti-T. forsythia gave positive reactions only with P. gingivalis and T. forsythia infected rat liver sections respectively and did not cross react with other bacterial species.
Out of 60 patients P. gingivalis was detected extracellularly in 35(58%) and intracellularly in 13(27%). T. forsythia was detected extracellularly among 45(75%) patients and intracellularly in 23(38%) patients. Out of the cases with extracellular positivity, intracellular positivity was detected in 31 % and 51% for P. gingivalis and T. forsythia respectively.
Conclusion: The produced monoclonal antibodies could be used as specific tools to locate Porphyromonas gingivalis and Tannerella forsythia in human tissues. These bacteria may exist intra-cellularly in gingival/ subgingival granulation tissues in patients with chronic periodontitis.

Potential of a Novel PDGF-BB / VEGF-A PLGA Microspheres Scaffold for Hard Tissue Regeneration

Primary Author: Vinicius Rodrigues, Harvard University, Boston, MA
Financial Assistance: None
Background: Bone regeneration has been widely done in treating bone loss, especially in the periodontal practice. Shortcomings of it have been linked to limited blood supply, while good site vascularity has been shown to enhance bone graft results. Angiogenesis may, therefore, improve the results of such treatment, if promoted on that site. To stimulate vessel sprouting, growth factors (GF) have been tested, with potential results found in pre-clinical models. Among these results, some of the most promising come with recombinant human vascular endothelial growth factor-A 165 (VEGF), especially when used with recombinant human platelet-derived growth factor-BB (PDGF) in a sustained delivery system. In many studies these GF have also been implicated in enhanced osteogenesis. Thus, we hypothesize that sustained local delivery of PDGF and VEGF may improve bone regeneration.
Methods: 25 μL of rhPDGF-BB (300 ng/μL - Gem 21S®), rhVEGF-A165 (1.3 ng/μL), rhBMP-2 (2 ng/μL - Infuse®) (positive control) or saline (negative control) were injected SC over one the Parietal bones of mice (4-6 week-old, male, C57Bl/6 wild-type), 3x/day for 5 days (n=5). Calcein was injected IP to identify new bone. 3 days after the last calvarial injection μCT was taken and histological sections were prepared. Next, this protocol of injections was performed with VEGF, PDGF, PDGF+VEGF and saline over a 2 mm critical size defect created on the Parietal bone of mice (n=4-6). On a 3rd experiment, a similar defect was treated as above, but also with a mix of bone grafts [DFDBA (RegenerOss)+FDBA (27 / 3 mg)] (n=4-6). Finally, a new set of mice had defects covered by a scaffold of FDBA and poly(lactic-co-glycolic acid) (PLGA) microspheres incorporating VEGF (0.1 μg), PDGF (0.15 μg), VEGF+PDGF (0.1 / 0.15 μg) or saline (n=4-6). 28 days after the surgeries μCT were done and samples prepared for histology.
Results: The results of the injections over bone show that PDGF, but not VEGF, induced bone growth, modestly. On the defects (with bone grafts or not), PDGF induced significant bone formation, while VEGF had a mild effect. When combined, VEGF appeared to not add to, or slightly blunt, the effects of PDGF. Finally, the PLGA/FDBA scaffold, as a local sustained delivery system of GF, caused great inflammation, with few signs of bone formation in all groups. Still, the PDGF+VEGF group had statistically more bone volume on the scaffold area than the other groups and the original scaffolds. This possibly was due a reduced FDBA resorption with bone formation. Also, histology seems to indicate that this group had more vascularity than the controls.
Conclusion: In conclusion, taking in account the limitations of this study, our data shows that PDGF is osteogenic; suggests that PDGF+VEGF may improve bone formation in the context of defects, but intermittent delivery of PDGF alone appears to be better for that; and indicates that PLGA, possibly, is not ideal for bone regeneration.

Fig. 1 - Calvarial injections region of interest (yellow box on the right Parietal bone). Image correspond to μCT of a positive control sample (injected with rhBMP-2). Red lines correspond to the areas of the histological sections.

Fig. 2 – Histological sections of Parietal bones on the region of interest - Fluorescence Microscopy of Control Groups. Top: rhBMP-2 (Positive Control); Bottom: Saline (Negative Control)

Fig. 3 - Histological sections of Parietal bones on the region of interest - Fluorescence microscopy of test groups. Top: rhPDGF-BB; Bottom: rhVEGF-A165.

Fig. 4 - Micro-CT of mice calvariae - Calvarial injections over critical size defects. Left: Saline injections (control); Right: rhPDGF-BB.

Fig. 5 – New bone formation and mineralized bone graft particles on the calvarial defect experiment groups with bone grafts and growth factors injections. Bars represent mean area (in mm3) of mineralized tissue measured by μCT.

Fig. 6 – Change in bone volume above the calvaria induced by scaffolds with growth factors or saline. Bars represent mean amount of fold change in each group.

Effect of Systemic Parathyroid Hormone (1-34), Bone Morphogenetic Protein-2, and Demineralized Bone Matrix on Local Bone Formation in the Rat Critical-Size Calvarial Defect Model

Primary Author: Brian Stancoven, US Army Advanced Education Program in Periodontics, Ft. Gordon, GA
Financial Assistance: None
Background: Identifying osteoconductive/inductive biomaterials and understanding biological processes associated with their application is a present focus of periodontal research. The purpose of this study was to investigate local bone formation following application of recombinant human bone morphogenetic protein-2 (rhBMP-2) in an absorbable collagen sponge (ACS) carrier, human demineralized bone matrix (hDBM), and systemic parathyroid hormone (1-34) [PTH] alone, or in the above combinations, using an established discriminating preclinical model.
Methods: Routine, critical-size (ø8-mm), through-and-through calvarial osteotomy defects were created in 160 adult male Sprague-Dawley rats using a trephine diamond bur and sterile saline irrigation under general anesthesia. Experimental defects were randomized to receive, rhBMP-2(0.1 mg/mL)/ACS or hDBM (35 mg). Control defects received ACS (soak-loaded with saline) and sham-surgery controls. A custom titanium mesh was fitted over the defects to prevent soft tissue collapse. Eighty animals (20/group) were randomized to receive daily injections of PTH (15 µg/kg SQ). Surgical staples were used to evert the wound margins to ensure wound closure for primary intention healing. Radiographic and histometric analysis were performed following 4- and 8-week healing intervals.
Results: The multivariable analysis showed that rhBMP-2/ACS supports substantially increased bone formation compared with hDBM (p<0.01), ACS and sham-surgery controls (p<0.01) throughout the 4- and 8-week healing intervals. ACS alone significantly enhanced local bone formation compared with sham-surgery control and hDBM group (p<0.01). Notably, hDBM treated defects showed significantly less bone formation than the sham-surgery control (p<0.01). In general, systemically administered PTH, alone or in combination with the other groups, had no significant effect on local bone formation (p=0.70).
Conclusion: Systemic PTH exerts no discernable effects on local bone formation. rhBMP-2/ACS has a significant potential to support bone formation. hDBM elicits no apparent osteoconductive or osteoinductive properties and might be considered osteo-obstructive.

Surgery

4 Weeks

8 Weeks

4 Weeks, + PTH

8 Weeks, + PTH

Histometric Defect Closure

CLINICAL RESEARCH ABSTRACTS

The Effects of a Static Magnet Field on the Osseointegration Process Following Immediate Implant Placement into Fresh Extraction Sockets: A Randomized Controlled Clinical Trial

Primary Author: Seyed Hossein Bassir, Tehran University of Medical Sciences, Tehran, Islamic Republic of Iran
Financial Assistance: None
Background: The traditional implant installation protocol leads to almost 1 year of reduced life quality which may affect the decision for dental implants rehabilitation. Overall treatment length could be optimized by shortening the healing period following implant installation . Electromagnetic fields have demonstrated promising results in shortening the osseointegration period in both in vitro and in vivo studies. However, there is still a lack of clinical studies on the effects of electromagnetic fields on the implant osseointegration.
The aim of this study was to evaluate the effects of static magnet field on the osseointegration process of implants immediately placed into fresh extraction sockets. This was accomplished by determining the changes in implant stability and marginal bone levels during the 3-month healing period.
Methods: In this randomized-controlled clinical trial, 20 patients who were in need of 20 fixed implant-supported single crowns in the anterior segment of maxilla were included. After tooth extraction, screw type Straumann SLA oral implants (Straumann AG, Waldenburg, Switzerland) were placed immediately into extraction socket. Then, according to the randomization table, ten implants were covered with magnetic abutment (test group) and ten implants were covered with normal healing abutment (control group). Implant stability was assessed by means of radio frequency analysis (RFA) at implant placement and after 1,2 and 3 months. The crestal bone level was recorded using digital subtraction radiography technique 1, 2 and 3 months after implant placement. The normal distribution of the data was tested (Kolmogorov–Smirnov test) and statistical analysis for determination of differences in marginal bone levels were performed by Wilcoxon Signed Ranks test. Comparisons of ISQ values between groups were performed using Paired T-tests. A significance level of α = 0.05 was used for all comparisons.
Results: The RFA measurements showed a statistically significant higher stability for implants in test group than that of control group after one month (P=0.04). The ISQ values of both groups increased during the 3 months. However, there was no statistically significant difference between both after 3 months (P=0.12). After two months, test group had less crestal bone loss (0.30±0.10) compared to control (0.39±0.16) group (P=0.03). At month 3, both test (0.39±0.20) and control (0.35±0.11) groups had closely similar bone levels (P=0.12).
Conclusion: Static magnetic field generated by magnetic abutments could shorten the implant healing period probably by affecting the osseointegration process at initial stage of bone healing.

Clinical Evaluation of Guided Bone Regeneration in Horizontally Challenged Edentulous Ridges during Implant Placement Surgery

Primary Author: Jia-Hui Fu, University of Michigan, Ann Arbor, MI
Financial Assistance: Zimmer Dental Inc.: Sponsor
Background: Loss of a tooth results in a significant yet unavoidable horizontal resorption of the buccal bone. This poses as a challenge to clinicians because the long-term success of a dental implant used in the rehabilitation of edentulous ridges hinges on its prosthodontically driven three-dimensional position in bone. Regenerative techniques, such as the Sandwich Bone Augmentation (SBA), were thus proposed to augment the ridge horizontally for implant placement. This study aims to investigate the effect of a pericardium barrier membrane on SBA for the management of buccal dehiscence defects around implants.
Methods: Twenty-six healthy patients, each with a single buccal implant dehiscence defect, were randomly assigned into two groups: bone augmentation with pericardium membrane group (test) and bone augmentation with no membrane group (control). Both groups received an inner and outer layer of mineralized human cancellous and cortical allograft. Defects in the test group were subsequently covered with a pericardium barrier membrane. Clinical parameters including flap thickness, horizontal ridge width, defect height (distance between smooth-rough junction to the base of the defect), defect width and horizontal defect depth (at crest, 2mm, 4mm and 6mm below crest), were measured at implant placement and 6 months re-entry, using a reference stent. A bone core biopsy of the regenerated bone was obtained using a trephine at the re-entry surgery. Cone beam computed tomography (CBCT) scans were taken at baseline, implant placement and 6 months post surgery. CBCT, micro-computed tomography (microCT) and histological analyses were performed to assess the thickness and quality of buccal bone regenerated.
Results: All baseline characteristics, such as age, gender, flap thickness, bone width at crest and 3mm below crest, initial defect depth, defect height and width were not significantly different between the 2 groups. All implants placed were successfully osseointegrated at 6 months. Significant buccal bone resorption was found in the control group at 2mm, 4mm and 6mm below the bone crest (p<0.05). There were no significant differences in the percentage of defect height and width changes between the 2 groups (p>0.05). Results from the CBCT analyses demonstrated significant differences in the relative horizontal bone width changes on the buccal surface of the implant, with the control group having a greater magnitude of change. MicroCT assessment revealed no significant differences in bone volume density and extent of mineralization between the 2 groups. The test group showed significantly less radiographic vertical bone loss at 6 months after surgery. Histological examination showed active woven bone formation and maturation in both groups.
Conclusion: The use of SBA with a pericardium membrane can predictably regenerate mature buccal bone on implant dehiscence defects. Addition of a barrier membrane prevented significant buccal bone resorption in the osseointegration phase of dental implants.

Control group
a) Baseline defect after flap reflection (buccal view)
b) Baseline defect after flap reflection (occlusal view)
c) Implant placement (buccal view)
d) Implant placement (occlusal view)
e) Placement of mineralized human cancellous allograft on the buccal implant dehiscence defect (buccal view)
f) Placement of mineralized human cortical allograft (buccal view)
g) Placement of mineralized human cortical allograft (occlusal view)

Test group
a) Baseline defect (buccal view)
b) Baseline defect (occlusal view)
c) Baseline defect after flap reflection (buccal view)
d) Baseline defect after flap reflection (occlusal view)
e) Implant placement (buccal view)
f) Implant placement (occlusal view)

Test group
g) Placement of mineralized human cancellous allograft on the buccal implant dehiscence defect (buccal view)
h) Placement of mineralized human cancellous allograft on the buccal implant dehiscence defect (occlusal view)
i) Placement of mineralized human cortical allograft (buccal view)
j) Placement of mineralized human cortical allograft (occlusal view)
k) Placement of pericardium membrane over the bone allografts (buccal view)
l) Placement of pericardium membrane over the bone allografts (occlusal view)

a) Clinical re-entry at control site 6 months post implant placement (buccal view)
b) Clinical re-entry at control site 6 months post implant placement (occlusal view)
c) Clinical re-entry at test site 6 months post implant placement (buccal view)
d) Clinical re-entry at test site 6 months post implant placement (occlusal view)
e) Histological view showing bone formation around bone allograft particles at 6 months
f) Magnified histological view

CBCT analysis
Bone width at control site at
a) Baseline
b) Implant placement
c) 6 months post surgery
Bone width at test site at
d) Baseline
e) Implant placement
f) 6 months post surgery

Radiographic assessment
Vertical bone level at control site at
a) Baseline
b) Implant placement
c) 6 months post implant placement
d) 12 months post implant placement
Vertical bone level at test site at
e) Baseline
f) Implant placement
g) 6 months post implant placement
h) 12 months post implant placement

Interleukin-6 Gene Promoter Methylation in Rheumatoid Arthritis and Periodontitis

Primary Author: Kohei Ishida, Niigata University, Niigata, Japan
Financial Assistance: None
Background: Methylation status of the cytokine genes may play a key role in the pathogenesis of inflammatory diseases such as rheumatoid arthritis (RA) and periodontitis. Interleukin-6 (IL-6) is one of the most potent cytokines that play a crucial role in the pathogenesis of both diseases. Therefore, this study was undertaken to evaluate the DNA methylation status of the IL-6 promoter region in subjects with RA and periodontitis and healthy controls.
Methods: After periodontal examination, genomic DNA was isolated from peripheral blood mononuclear cells obtained from 30 patients with RA, 30 patients with periodontitis and 30 healthy controls. Genomic DNA was bisulfite-modified and subjected to the polymerase chain reaction (PCR) with three pairs of oligonucleotide forward and reverse primers. DNA methylation status of individual CpG motifs in the IL-6 promoter region was determined with the direct sequencing of the PCR products. Serum levels of IL-6 were also determined with an enzyme-linked immunosorbent assay (ELISA).
Results: A total of 19 CpG motifs were identified in the promoter region (-1200 bp to +27 bp ) of the IL-6 gene. The methylation levels of individual CpG motifs were comparable among the three subject groups, except at -74 bp and +19 bp. The CpG motif at -74 bp was significantly less methylated in the patients with RA and periodontitis, compared to the controls. In contrast, +19 CpG motifs in the patients were more methylated than those in the controls (P = 0.0002 for -74 bp, and P = 0.0005 for +19 bp, respectively). Of three CpG sites, serum levels of IL-6 were found to be significantly different between individuals with unmethylation and partially methylation at -74 bp (P = 0.04), which was obtained by ELISA.
Conclusion: These results suggest that methylation status of CpG motifs in the IL-6 promoter region may affect serum IL-6 levels, thus implicating a role in the pathogenesis of RA and periodontitis.

Validation of Self-Reported Measures for Periodontitis –Type 1 Diabetes Mellitus Patients with Disease Duration of 50 Years or Longer

Primary Author: Nolan Laborde, Harvard University, Boston, MA
Financial Assistance: None
Background: The aim of this study was to validate a set of eight questions developed by the Centers for Disease Control (CDC) for use in the Behavioral Risk Factor Surveillance System and the National Health and Nutrition Examination Survey (NHANES) for self-reported periodontal disease. These questions were administered to a group of insulin dependent diabetic patients with disease duration of 50 years or longer as part of the Joslin 50 Year Medalist Study.
Methods: The study involved an analysis of 150 insulin-dependent patients participating in the 50 Year Medalist Study (Medalists). These patients completed the self-reported periodontal disease questionnaire followed by a clinical examination consisting of complete full mouth periodontal examinations including periodontal pocket depth, clinical attachment level, plaque index, gingival index, caries and missing teeth evaluation. Statistical analyses were performed using Analysis of Variance (ANOVA) for measurement data followed by multiple comparisons using post hoc analysis (LSD and Sheffe test). Chi-Square tests were performed for categorical data. Significance was determined at α=0.05. Cases of no/mild, moderate, or severe periodontitis were defined according to the CDC/AAP classification. Logistic regression modeling was performed to predict the prevalence of periodontal disease.
Results: Based on the AAP/CDC disease category, moderate to severe periodontitis was detected in 62.6% (94) of the Medalists. Age (≥ 65 yrs old) was found strongly related with the severity of periodontal disease (p<0.001) while there was no significant difference between males and females with respect to the periodontal disease. Analysis of the questionnaire demonstrated that 66% (33/50) of patients with no disease/mild periodontitis accurately predicted their periodontal condition while in the moderate to severe group, 81.8% (63/77) of patients precisely predicted their periodontal condition. Among the 8 questions, questions about health of gums and previous periodontal treatment (scaling and root planing) were strong predictors in addition to age.
Conclusion: The results of this study demonstrate that this self-reported questionnaire can successfully predict the periodontal condition in this patient population. Periodontal disease occurs more frequently and in greater severity in patients with diabetes. This survey could be a useful screening tool for physicians as a basis for dental referral, which in turn can help patients with increased risk for oral and systemic inflammation.

Polymorphisms in Pattern Recognition Receptors in Periodontitis

Primary Author: Rodrigo Lopez, Aarhus University, Aarhus C, Denmark
Financial Assistance: None
Background: Despite intensive research into the role of immune responses in subjects with periodontitis, little is known about early responses of the innate immune system. Pattern recognition receptors (PRR) function as sensors of microbial structures and mediate the initial immune response against bacteria. Several genetic polymorphisms of the PRR system have been associated with functions that are associated with various immunoinflammatory disorders, and PRR polymorphisms may also be associated with periodontitis. We therefore aimed at investigating the association between selected polymorphisms for PRR and periodontitis.
Methods: Study population: The study group included 87 cases with Clinical Attachment Level (CAL) ≥ 3 mm in at least two teeth and 73 controls who did not fulfill these criteria. Ethical approval for the study was obtained from the local Ethics Committee, and informed written consent was obtained from each participant.
A trained and periodontist conducted all periodontal examinations, which comprised recordings of CAL, probing pocket depth, bleeding on probing, and supragingival deposits at six sites per tooth. Cases presented with more supragingival deposits than controls, more pocketing and bleeding on probing.
Genotyping of polymorphisms: A sample of 10 ml blood was obtained from each participant by venepuncture using BD Vacutainer tubes with anticoagulant. All samples were centrifuged for 10 min at 800 g to obtain buffy coats. Genomic DNA was extracted from thawed cell pellets on a Maxwell-16 robot (Promega Corporation, WI, USA) and was subsequently genotyped using two in-house multiplex single nucleotide polymorphism (SNP) assays covering a total of 45 SNPs in the genes encoding TLR1-10, DDX58 (RIG-I), IFIH1 (MDA5), NOD1 (CARD4), and NOD2 (CARD15). Allele-specific primers were labeled in an allele-specific primer extension (ASPE) reaction, using polymerase chain reaction-amplified SNP-sites as their target sequences. The labeled ASPE-primers were subsequently hybridized to MicroPlex-xTAG beadsets for detection and counting on the Luminex flow-cytometer platform (Luminex Corporation, Austin, TX, USA).
Assessment of Hardy-Weinberg equilibrium was performed using the software Haploview v4.2 (Broad Institute, Massachusetts Institute of Technology, Cambridge, USA). P-values for association tests were based on Fisher’s exact test, exact logistic regression analyses, and the Cochran Armitage Trend test and were considered significant at P < 0.05.
Results: Results: Nonconformance to Hardy-Weinberg equilibrium was tested for all investigated SNPs in the healthy control group, but none were found. At the nominal level, TLR1-rs5743611 (p=0.01), TLR7-rs3853839 (p=0.02), and TLR8-rs3764879 (p=0.01) showed significant associations with periodontitis.
Conclusion: Selected mutations in genes encoding for pattern recognition receptors may play a role in the occurrence of periodontitis.

Immunohistochemical Evaluation of Labial Salivary Glands in Xerostomic Patients

Primary Author: Yat Ho Ma, US Army Advanced Education Program in Periodontics, Ft. Gordon, GA
Financial Assistance: None
Background: Xerostomia, the subjective complaint of dry mouth, affects a substantial proportion of the population, and can have serious adverse effects on the quality of life. It can arise for several reasons, particularly insufficient production of saliva that in turn can result from several underlying causes, such as chronic use of xerostomic medications, or a disease affecting gland function. In particular, Sjögren's Syndrome, an autoimmune disease that affects salivary gland function, is an important cause of xerostomia due to hyposalivation. Previous studies in an animal model for Sjögren's showed that salivary gland dysfunction was associated strongly with expression of cell proliferation markers (Ki-67 and PCNA). However, similar human studies are limited, and the relationship between marker expression, Sjögren's, and xerostomia has not been examined. Our objective was to determine the expression levels of proliferation markers in the labial salivary gland (LSG) of xerostomic patients.
Methods: Archived LSG biopsies from 15 xerostomic and 4 non-xerostomic subjects were retrieved. H&E staining and TUNEL (apoptotic assay) of all biopsies were performed at the GHSU core laboratory. In addition, immunohistochemistry using antibodies for Ki-67 and PCNA were performed to detect cell proliferation. Focus scores were obtained from the H&E histology while the percentages of cells positive for TUNEL, Ki-67 and PCNA were determined by counting 1000 cells and converted into apoptotic and proliferation indices. The biopsies were categorized into four groups: subjects with Sjögren’s Syndrome (SS), xerostomia subjects with positive serology (ANA titer, Anti-SSA/SSB, or RF), xerostomia subjects without serology, and non xerostomic normal. ANOVA analyses were performed on apoptotic and proliferation indices between the four groups. Statistically significant differences between treatment groups were determined with P < 0.01.
Results: Sections of LSG epithelial cells showed similar modest levels of apoptosis and Ki-67 positive staining from xerostomic patients and normal control subjects (Figures 1 and 2). However, the proportion of PCNA positive cells in xerostomic patient samples was significantly higher than that of the non-xerostomic group (Figure 3).
Conclusion: Xerostomic subjects uniformly show high levels of PCNA expression in LSG biopsies compared to non-xerostomic subjects regardless of focus score or serology. Since PCNA is also a marker for DNA repair, elevated staining indicates that an increase in DNA damage occurs in the xerostomic LSG. The activation of DNA repair may cause the reduction in gland function. Proliferation and apoptosis of salivary epithelial cells do not appear to play major roles in salivary dysfunction of xerostomic and Sjögren’s Syndrome subjects. Screening LSG biopsies for PCNA expression might provide an objective tool for the evaluation of glandular abnormalities in xerostomic patients, regardless of their Sjögren's Syndrome status.

Figure 1: The percentage of TUNEL positive cells (apoptosis) between subject groups. Subject groups were statistically non-significantly different (P>0.20).

Figure 2: The percentage of Ki-67 positive cells (proliferation) between subject groups. Subject groups were statistically non-significantly different (P>0.60).

Figure 3: The percentage of PCNA positive cells (proliferation and DNA repair) between subject groups. Subject groups were statistically significantly different (P<0.001).

Tissue Engineered Cultured Periosteal Sheet Application to Periodontal Regeneration: Three Year Results

Primary Author: Kazuhiro Okuda, Niigata University, Niigata, Japan
Financial Assistance: None
Background: One year results of autologous grafting of human cultured periosteal (hCP) sheets in combination with platelet-rich plasma (PRP) and hydroxyapatite (HA) granules in the treatment of infrabony periodontal defects have shown favorable clinical results (Yamamiya and Okuda, et al. Journal of Periodontology 2008;79:811-818). The present report addresses the three year clinical, radiographic and histological results of subjects treated with a combination of hCP sheets, PRP and HA granules.
Methods: Twenty-two patients exhibiting at least one infrabony periodontal defect with a probing depth (PD) ≥6 mm, clinical attachment level (CAL) ≥6 mm and radiographic presence of an infrabony osseous defect (IBD) ≥3 mm were included in this investigation. The periosteum tissue samples were dissected aseptically from each patient, and were incubated for approximately six weeks. Prior to placement of hCP sheets, the osseous defects were thoroughly debrided and filled with HA granules in combination with PRP (Figure 1). Standardized clinical (Figure 2) and radiographic (Figure 3) measurements were determined during follow-up examinations at one, two and three years.
Results: At baseline, the mean PD was 7.6±1.4 mm, CAL was 8.0±1.4 mm, and IBD was 4.6±1.0 mm. One year after treatment the mean PD, CAL and IBD was 4.8±1.4, 2.9±0.5 and 1.4±1.1 mm, respectively. At the three-year time period, mean CAL was 4.7±1.3 mm, PD was 3.0±0.2 mm, and IBD was 0.5±0.9 mm. Statistically significant clinical and radiographic improvements had occurred between baseline and the one- and three-year time periods (p <0.01)(Figure 4). There were significant gains in the IBD at one- and three-years when compared to baseline and at three-years when compared to two years (p <0.01) (Figure 4). Histological results demonstrated that alkaline phosphatase activity in the hCP sheets was consistent with von Kossa-positive mineral deposits (Figure 5). In addition, the hCP sheets were positive for CD73, CD90 and CD105 antigens, whereas negative for CD19, CD34, and CD45 antigens (Figure 6), which supported evidence for marker genes of bone marrow-derived mesenchymal stem cells.
Conclusion: The treatment with a combination of hCP sheets, PRP, and HA were found to be stable and clinically maintaining after three years. The long-term osseous structure re-growth as an indicator of true regeneration following re-establishment of a functional attachment, as proposed by Heijl et al. 1997, is also supported by the results of the present study. These findings reported here further strengthen our belief the hCP sheets function as a living drug delivery system to favorably influence cellular functions and serves as a seed for ectopic bone formation near the implantation site through production of important growth factors related to periodontal regeneration.

Figure 1. Representative clinical case of a site surgically treated with hCP sheets in combination with a PRP and HA granule mixture

A) Pretreatment view of mandibular right lateral incisor with clinical attachment Level (CAL) of 8 mm and probing depth (PD) of 8 mm on the mesial aspect.
B) Reflection of a surgical flap and debridement of the area revealed the two- and three-walled osseous defect.
C) Grafting with the PRP + HA granules into the intrabony osseous defect.
D) Intraoperative facial view of the surgical site following placement of the hCP sheet covering the PRP + HA granules.

Figure 2. Clinical results at 1, 2 and 3 years of a site surgically treated with hCP sheets in combination with a PRP and HA granule mixture.

1Y) 1 year post-treatment view of the surgical site with CAL of 5 mm and PD of 2 mm.
2Y) 2 years post-treatment view of the surgical site with CAL of 5 mm and PD of 2 mm.
3Y) 3 years post-treatment view of the surgical site with CAL of 5 mm and PD of 3 mm.

Figure 3. Radiographic results at baseline, one, two and three years, of a site surgically treated with hCP sheets in combination with a PRP and HA granule mixture.

Baseline) Pretreatment standardized radiograph suggests an intrabony defect on the mesial surface of the mandibular right lateral incisor.
1Y) Radiopaque fill of the mesial osseous defect at 1 year post-surgery.
2Y) Radiopaque fill of the mesial osseous defect at 2 years post-surgery.
3Y) Radiopaque fill of the mesial osseous defect at 3 years post-surgery.

Figure 4. Change in mean (±SD) clinical and radiographic measurements from baseline to the 1- and 3-year time periods (n=22, p <0.01).

Figure 5. In vitro observation of the alkaline phosphatase (ALP) activity and mineralization of hCP sheets.

ALP activity: ALP activity of a hCP sheet cultured in growth medium for 35 days.
Mineral deposits: Dark-brown areas represent von Kossa-positive mineral deposits. The hCP sheet was cultured in growth medium for 60 days.

Figure 6. Surface markers associated with the hCP sheet.

The hCP sheets were positive for CD73, CD90 and CD105 antigens, whereas negative for CD19, CD34, and CD45 antigens.

The Influence of Biofilm in Gingival Overgrowth in Renal Transplant Recipients Receiving Tacrolimus or Ciclosporin-A. A 12 Months Prospective Study.

Primary Author: Ricardo Sekiguchi, University of São Paulo, São Paulo, Brazil
Financial Assistance: None
Background: OBJECTIVE: The aim of this study was to determine the influence of biofilm in gingival overgrowth (GO) in renal transplant recipients receiving tacrolimus (Tcr) or cyclosporine-A (CsA) in the absence of calcium channel blockers after 365 days of follow up.
Methods: Methods: Sixty-seven subjects (33 Tcr/34 CsA) were evaluated before, 30 days, 90 days, 180 days and 365 days after kidney transplant surgery. Demographic, periodontal parameters, Plaque Index (PI) and GO score were recorded for all subjects. In every examination the same data were collected and all the subjects received oral hygiene instructions and professional prophylaxis. Clinical assessment was performed by a single trained and calibrated examiner (ICC=0.927) who was blinded to the patient's drug regime. GO was analyzed by visual examination of the buccal and lingual papillae of the six most anterior teeth of the upper and lower arch. A score ranging from 0 to 5 was attributed to each papilla according to the quantity of GO both in the horizontal and vertical axes. Patients showing a score ≥30 were classified as presenting clinically significant GO (CSGO). Included in the study were subjects of both genders, older than 18 years, who presented no clinical or radiographic signs of periodontitis. Smokers, diabetics, patients presenting some type of gingival overgrowth during the pre-transplant period and taking any of the following drugs were excluded: nifedipine, diltiazem, verapamil, phenytoin, sodium valproate, azithromycin. The individuals were divided in two groups according to their gingival overgrowth. Group CSGO had gingival overgrowth >30 and group NCSGO had no gingival overgrowth or GO <30.
Results: At 365 days post-transplant, clinically significant GO was observed in 20.8% of all individuals. When comparing the groups CSGO and NCSGO in regard to PI it was noted a statistically significant difference at 90 days(p<0.001), 180 days (p<0.001) and 365 days (p<0.001) (see table 1). The clinical inflammatory assessment was made through bleeding on probing (BoP) and had similar results as shown in table 2.
Conclusion: Within the limits of this prospective study, it can be concluded that the group that developed clinically significant GO always presented higher PI and BoP index compared to the group without GO but it was only statistically significant after 90 days, 180 days and 365 days of the beginning of immunosuppressive therapy. Thefore, Plaque Index could have positive association in the incidence of clinically significant GO.