International Poster Journal of Dentistry and Oral Medicine, Supplement Osteology/2019

Introduction: Vestibuloplasty is a frequently performed surgical procedure to create, restore or even increase the soft tissue sealing around dental restorations if possible with keratinized mucosa. Avascular porcine collagen matrices reveal comparable clinical result as free gingival grafts in the context of tissue regeneration around dental implants. The process of graft vascularization presenting the basic requirement for local healing and colonization of collagen grafts is still incompletely understood. Material and Methods: In 10 patients vestibuloplasty was performed during implant uncovering using collagen matrices (Mucograft®). Tissue perfusion of the collagen matrices was measured using laser- doppler- spectrophotometer (Oxygen- to- See, Lea Medizintechnik, Gießen, Germany) intraoperatively and on postoperative days 2, 5, 7, 14, 30 and 90. Graft perfusion expressed by oxygen saturation [SO2%], relative amount of hemoglobin [rHb], blood flow and velocity [AU] was detected and compared the surrounding mucosa. In another 10 patients vestibuloplasty was performed with free gingival grafts (FGG) as control. On postoperative day 14 biopsies were taken from the matrices and analysed via Rt-PCR- analysis for expression of angiogentic mediators (Angiopoetin1/2, VEGF, Dll4, Notch, Tie2) and via immunhistochemistry for CD31- expression. Results: Blood flow and velocity significantly increased until postoperative day five and approximated to perfusion values of the surrounded mucosa already during the following days. Likewise, measured matrix oxygen saturation also significantly increased within the first five postoperative days whereas hemoglobin content did not show any differences during the investigated period. Collagen matrix reperfusion was comparable to vascularization of free gingival grafts in a control group. Two weeks after vestibuloplasty angiogenesis and vessel maturation are still progressing Dll4 and Tie2 are highly expressed in the matrices. Interestingly, CD31- expression did not differ between FGG and collagen matrices on day 14. Conclusion: Mucograft perfusion mainly progresses within the first postoperative week with only minimal further detectable alterations until day 90. Therefore, vessel maturation and capillary- network formation progressed from day 14 onwards without detectable alterations in matrix tissue perfusion. Keywords: collagen matrix, angiogenensis, perfusion

There is a gap in the literature regarding RNA extraction and human alveolar bone gene expression in type 2 diabetes.The overall objective of this novel study is to investigate the gene expression from alveolar bone in health and diabetes.There are two broad objectives;1To improve RNA extraction protocol from human alveolar bone and improve RIN (RNA integrity values) 2 Analyze gene expression profiles in health and diabetes RNA sequencing.Methods-Healthy and Type 2 diabetic subjects undergoing periodontal surgeries were screened and consented.The subjects were undergoing procedures like osseous surgery,tori removal,crown lengthening,extraction and alveoplasty were selected. A total of 43 subjects were recruited until now for the study (25 healthy and 18 diabetes).At the surgery appointment, alveolar bone was harvested and immediately placed in a vial containing 500ul of Trizol and immediately frozen in liquid nitrogen.Multiple vials were obtained if there was more bone available.Samples were processed within two months of collection.During the RNA extraction stage, bone samples were homogenized with a bone crusher in liquid nitrogen to uniformly break larger pieces of bone and then were processed in a bullet blender with metal beads in a cold room.The homogenized sample was extracted and processed by Qiagen mini columns.RNA was then analyzed on nano-drop spectrophotometer for quality and quantity and a bio-analyzer to obtain RNA integrity number(RIN).Samples that had greater than 3.5 RIN were then processed by a rRNA depletion method to further purify the samples and were submitted for RNA sequencing for differential gene expression.Results-Extracting stable and high quality RNA from human bone samples is technique and patient dependent.Our data showed that maintaining low or below freezing temperatures of the bone samples are key to good RIN values that may influence techniques like RT-PCR and RNA sequencing. Until now 25 healthy and 18 diabetics subjects were recruited.Our results show that a total of 10 healthy and 8 diabetic samples had RIN Values of 3.5 or more and were submitted for RNA sequencing for differential gene expression.The mean age, HbA1c% and RIN values of healthy vs diabetic subjects is;age (55.3 ± 17.5 vs 63.9 ± 8.7 years), HbA1c% ( 5.6 ± 0.29 vs 7.3 ± 2.4) and RIN ( 4.94 ± 1.41 vs 4.43 ± 1.54) respectively. Sequencing analysis shows that overall gene expression was downregulated in diabetes samples. Conclusions-Understanding alveolar gene expression in diabetes is very important its molecular and clinical effects on periodontal regeneration or dental implants are relatively unknown.Our results here showed that our protocol for RNA extraction from human bone samples results in improved RIN values. Higher RIN values (> 6) are preferred for sensitive gene expression techniques like RT-PCR and RNA sequencing, however methods like rRNA depletion techniques are available to purify and amplify the RNA samples. Data from RNA sequencing shows that overall gene expression and specific pathways for inflammation and bone regulation were down regulated in diabetic subjects compared to healthy controls. Keywords: bone, diabetes, genes

Guided virtual surgery (GVS) has as premise a better accuracy for dental implants placement. However, the reproducibility of the implant planned position by means of surgical guides is still under investigation. This study had as objective to assess the angular and the linear (point of entry and apical extremity) deviations of single-tooth dental implants placed by two different techniques: GVS with stereolithographic guide and conventional surgery (CS) with handmade guide. This split-mouth randomized clinical trial was approved by the Research Ethical Committee involving Human Beings of the Federal University of Santa Catarina (CEPSH - UFSC; Protocol no. 1,658,040/2016) and was registered on the Brazilian Register of Clinical Trials (register number: RBR-2h745w). Twelve patients with bilateral homologous single-tooth missing in posterior mandible were selected (n=24). After scan appliance manufacturing and cone beam computed-tomography (CBCT) by dual scanning technique, cases were virtually planned and forwarded to a prototyping center to stereolitographic surgical guide manufacturing (CAD/CAM). The conventional surgical guides were obtained by scan appliances' preparation. During the surgical procedure, patients were randomized on GVS or CS by a coin toss. Each side of the mandible received one implant by a different technique. After one week, patients underwent a new CBCT scan and the images were superimposed to evaluate the differences of implants positioning between planned and performed. Student's t-test was applied at a significance level of 5% (p=0.05). Angular deviations of GVS implants showed 2.2±1.1 degrees of discrepancy while those placed by CS presented a discrepancy of 3.5±1.6 degrees, and this difference was statistically significant (p-value=0.032). For the variables coronal and apical distances (deviations), however, the null hypothesis was accepted (p-value>0.05), that is, the means between the groups of GVS and CS were not different statistically. It can be concluded that single-tooth implant placement by GVS is more accurate, at least for the angular deviation, when compared to CS with a surgical guide made by hand. Considering the linear deviations (cervical extremity and apical end), the difference between both groups cannot be demonstrated in this study. More randomized clinical trials with larger samples are needed to confirm such trends and to provide solid evidence on the subject. Keywords: dental implants, computer-assisted surgery, oral surgery, cone-beam computed tomography, single-tooth dental implant

Objectives: This pilot study aimed at investigating the safety and feasibility of pre-augmentation soft tissue expansion (STE) as a potential approach in facilitating bone augmentation surgery, by enhancing soft tissue quantity and quality. Methods: Self-inflating osmotic soft tissue expanders of different sizes (expander final volume range: 240 - 1300 mm3) were implanted subperiosteally in four patients requiring vertical and/or horizontal bone augmentation, and left in situ for 20-60 days. Guided bone regeneration was carried out after STE completion & expander removal. In all cases, bone augmentation was performed using particulate autogenous bone, mixed with xenograft. In case of vertical bone augmentation, the bone graft was covered with titanium reinforced PTFE high-density membrane, while collagen membrane was used in horizontal bone augmentation. In patients that completed the study, dental implants were placed 6 months post-augmentation. CBCT scans were taken before placement of soft tissue expanders and 4-6 months following bone augmentation procedures. Vertical & horizontal bone gains were calculated on CBCT scans by subtracting bone height or width "before augmentation" from bone height or width "before placement of dental implants"at landmark sites. For volumetric analysis, optical scanning of pre-and post-expansion cast models fabricated was done through an optic scanner. Afterwards, superimposition of the optically scanned pre- and post-expansion models was done for each patient through two CAD software applications, to calculate the soft tissue volume increase. Results: Four patients (3 females, 1 male, mean age= 53.6 ±7.1 years) were included in this pilot investigation. Expanders were placed at 5 surgical sites (in one case, 2 expanders were placed in the same patient). During STE, healing was uneventful in 2 patients (3 surgical sites) while STE failed in 2 patients (2 surgical sites). One patient dropped out after failed STE; therefore, only 3 patients completed the scheduled treatment. Horizontal bone gain averaged 3 mm in 2 successfully expanded sites while one site had a vertical bone gain of 8 mm. All 3 patients received dental implants in the augmented areas (total= 7 implants). All implants were successfully osseointegrated. Regarding volumetric analysis (2 patients, 3 surgical sites), soft tissue volume increase (STVI) was 259.4 mm3for the 0.24 ml expander, 436.1 mm3for the 0.7 ml cylinder expander, and 755.9 mm3 for the 1.3 ml expander (mean STVI of the 3 successful sites= 483.8 ± 251.7 mm3). Thus, STVI corresponded only to the 0.24 ml expander, while this increase was almost half of the final expander volume for the 0.7 ml and 1.3 ml expanders, probably due to higher pressure distribution to the underlying bone surface with bigger final volume expanders. Nonetheless, the surplus amount of soft tissues through STE still allowed for tension-free primary flap closure without the need for deep periosteal and/or vertical releasing incisions during bone augmentation. Conclusions: Soft tissue expansion (STE) might be a beneficial pre-augmentation approach, especially in the vertical dimension. The ideal area for this specific application would be the posterior mandible with the presence of alveolar mucosa. However, the presented technique still requires improvement before being applied routinely in everyday dental practice. Keywords: guided bone regeneration, soft tissue expanders, soft tissue expansion, soft tissue management

Objectives: Amelogenins contribute to the early resolution of inflammation in periodontal lesions, which suggests their anti-inflammatory effect. Therefore, the main objective of this study was to compare surgical treatment of periodontal infrabony defects with and without the adjunct of an enamel matrix derivative (EMD) in terms of acute-phase responses, in systemically healthy patients. The primary outcome measure was the change of C Reactive Protein (CRP) at 24 hours after the surgery. Methods: The study was a randomized single-blind clinical trial with 2 treatment arms: surgical therapy with the adjunctive application of EMD and surgical therapy without the EMD, respectively. The study type was monocentric. The follow-up was 6 months. The patients were eligible if they were in a good health (ASA I) and without previous periodontal surgery done. Patients who reported current smoking over 20 cigarettes per day or pipe or cigar were excluded. Thirty-eight periodontitis patients with intrabony defects of ≥ 4 mm in vertical depth were randomly allocated to one of the two treatment arms. For randomization purposes, each patient received a code that corresponded to an assignment of the groups and was placed in a sealed envelope that was opened only at the time of the treatment. Clinical parameters were measured at baseline and at 6 months control. Following measurements were taken: periodontal pocket depth (PPD), clinical attachment level (CAL), recession. Systemic parameters were taken at baseline and at day 1, day 7 and day 180 after periodontal surgery. Blood sample analysis included lipid profile, CRP, fibrinogen, D-dimer, cystatin, glucose levels. Statistical analysis was performed with multivariate analysis of covariance and the level of significance was set at 0.05. Covariates included in the model were age, smoking and body mass index. Results: A total of 121 subjects were screened, 38 included and completed the study. A total number of 38 patients completed the study. There was similar sex distribution among participants, while the test group patients were younger (p0.05). All smokers belonged to the test group (n=2). The CRP values changed in both groups after periodontal surgical treatment. In the test group, the CRP increased from baseline values of 2.10 mg/L (95% CI: 0.56, 3.62) to 3.10 mg/L (95% CI: 1.57, 4.62) at day 1. In the control group, the CRP increased from 1.84 mg/L (95% CI: 0.44, 3.24) at baseline to 4.36 mg/L (95% CI: 2.99, 5.78) at day 1 (p0.05), resulting in a higher CRP values for the control group at day 1 when compared to test group (p=0.004). The CRP values decreased gradually in both groups, and this decrease was more pronounced for the control group, when compared to its day 1 values (p0.0001 vs. day 1). Fibrinogen levels increased at day 1 and decreased afterwards for both groups. In the test group, the baseline values of 295.63 mg/dL (95% CI: 260.46, 330.78) changed to 327.79 mg/dL (95% CI: 292.62, 362.96) at day 1. In the control group, the values of 275.47 mg/dL (95% CI: 246.33, 304.62) at baseline increased to 317.68 mg/dL (95% CI: 288.54,346.82) at day 1 (p0.0001 vs. baseline). Surgical treatment proved efficacious in both groups, since it resulted in reduction of all measured clinical parameters at 6 months (p0.01 vs. baseline). However, CAL gain was higher in the test group: 4.26 ± 2.182 mm versus 3.26 ± 2.207 mm in the control group. Conclusion: To the best of the authors knowledge, this is the first study assessing the systemic effect of regenerative surgery of periodontal infrabony defects. Our data show that the adjunction of EMD was associated with lower increase of CRP and fibrinogen in test group at day 1 after periodontal surgery, suggesting a possible systemic anti-inflammatory effect. This local and systemic effect of EMD can be of interest when surgically treating periodontitis patients with systemic comorbidities. More research is needed to gain further insight into present findings. The study was registered in ClinicalTrials.gov: NCT03590093. Keywords: periodontitis, enamel matrix derivative, EMD, C-reactive protein, CRP

Objectives: Whereas stationary stability of implants has been postulated for decades, recent studies suggested a phenomenon termed implant migration. This describes a change in position of implants as a reaction to applied forces. The present study aims at employing image registration of in-vivo micro CT scans from different time points, and to assess (i) if migration of continuously loaded implants is possible and (ii) migration correlates with the force magnitude. Material & Methods: Two customized machined implants were placed in the dorsal portion of caudal vertebrae in n=61 rats, and exposed to standardized forces (0.5N, 1.0N, 1.5N) applied through a flat nickel titanium contraction spring, or no forces (control). Micro CT scans were performed at 0, 1, 2, 4, 6, and 8 weeks after surgery. The baseline image was registered with the forthcoming scans. Implant migration was measured as the Euclidean distance between implant tips. Bone remodelling was assessed between the baseline and the forthcoming scans. Results: The findings confirmed a positional change of the implants at 2 and 8 weeks of healing, and a linear association between applied force and velocity of movement (anterior implant: X2=12.12, Df=3, p=0.007, posterior implant: X2=20.35, Df=3, p 0.001). Bone apposition was observed around the implants and accompanied by formation of load-bearing trabeculae and a general cortical thickening close and also distant to the implants. Conclusion: The present analysis confirmed that implants can migrate in bone. The applied forces seemed to stimulate bone thickening, which could explain why implants migrate without affecting stability. Keywords: implant migration, animal study, bone to implant interface

Objective: Studies demonstrated immediate implant sites commonly have ridge dimensional alterations accompanied with post-op mucosal recession that compromise esthetic outcomes. The use of an autogenous tissue graft or an acellular dermal matrix was proposed to compensate these changes. However, the results of studies were equivocal. This randomized controlled trial was aimed to evaluate the impact of immediate implant combined with soft tissue augmentation on preserving tissue contour. Methods: This study had three groups: immediate implant (IM), immediate implant combined with the subepithelial connective tissue graft (IMCT), and immediate implant combined with the acellular dermal matrix (IMAD). The included patients had a single unrestorable tooth in the esthetic zone (maxillary incisors, canines and premolars) with adjacent teeth. The surgical sites should have acceptable buccal gingival level, interproximal bone level (recession or bone loss≤ 2mm) and mostly intact buccal plate. All implants were immediately placed following extraction and xenografts were placed to fill in any bony defects. Each implant was randomly assigned to one group (the size of the graft was standardized). A healing abutment was placed and allowed to heal for six months before the permanent crown delivery. Smokers (≤10 cigarettes per day) were included. The primary outcome was ridge dimensional alteration while the secondary outcomes were changes of peri-implant mucosal level, mucosal thickness, bone level and bone dimension. Based on the power calculation, 15 patients in each group was planned to be recruited. Ridge dimensional changes were measured by computer-assisted scanned images of dental models. The images of periapical radiographs and cone beam computed tomography were used to measure bone levels and dimensions. All clinical measurements were performed by a single examiner using a stent with reference marks at four visits (baseline, 3, 6, 12 months following the surgery). Results: To date, fifteen patients completed the 12-month visit and nineteen patients completed the 6-month visit. Only 6-month results of these nineteen patients (n=9, 6, 4 in the IM, IMCT, and IMAD groups) were reported. The reduction of buccal horizontal ridge dimension (BHRD) or buccal horizontal bone dimension (BHBD) did not seem to be different between groups (mean BHRD: 0.65±0.66, 0.47±0.95, 0.12±1.16mm; mean BHBD: 0.42±1.21, 0.05±1.69, 0.78±0.71mm in the IM, IMCT, and IMAD respectively). The mean changes of peri-implant mucosal levels were within 1mm (mean mid-buccal mucosal recession: -0.33±1.03, 0.50±0.77, 0.16±1.55 mm; mean mesiobuccal mucosal recession: 0.61±0.65, 0.33±0.52, 0.75±0.65mm; mean distobuccal mucosal recession: 0.44±0.81, 0.33±0.61, 0.63±1.60mm in IM, IMCT and IMAD respectively). Soft tissue augmentation appeared to increase peri-implant mucosal thickness (mean mucosal thickness gain: 0.18±0.15, 0.78±0.34, 1.27±0.83mm in IM, IMCT and IMAD respectively). Radiographic alveolar bone levels appeared to be stable (mean marginal bone loss at the mesial site:0.99±1.34, 0.07±0.16, 0.75±1.07mm; at the distal site: 0.73±1.15, 0.02±0.04, 1.23±1.13mm in the IM, IMCT and IMAD respectively). No further statistical analysis was performed since the study was not finished. Conclusions: The preliminary results demonstrated soft tissue augmentation increased tissue thickness but did not appear to have significant impact on preserving peri-implant mucosal level, bone level, bone dimension and ridge dimension. No conclusive statement could be made until the study is finished. Keywords: alveolar ridge, bone resorption, dental implants, esthetics, dental

Objectives: The aim of this study was to histomorphometrically evaluate the effects of the association of platelet rich fibrin (PRF) with a xenograft, in critical bone defects in rabbits' calvaria. Materials and methods: Twelve New Zealand adult male rabbits were distributed in two groups, according to the graft material that was used: Control (CG) - Bio-Oss xenograft (n=6); and Test (TG) - Bio-Oss xenograft associated to PRF (n=6). In all animals two bilateral bone defects with twelve millimeters in diameter were created, but just one defect received the Bio-Gide collagen membrane coverage. After eight weeks the animals were sacrificed and a histomorphometric analysis was done. PRF Obtention: Following Choukroun's original protocol (Dohan et al., 2006), blood samples were centrifuged for ten minutes in a 3,000 rpm spin, by the Intra-Spin L-PRF centrifuge (Intra-lock, USA). The PRF was cut in small pieces before being associated with the xenograft for TG. Surgical Procedure: A full thickness flap was performed, two bone defects were created (with a trephin bur) and the grafts placed inside the defect. After the membrane coverage (just in one side), the flaps were sutured with nylon thread. Histomorphometric Analysis: After histological processing, images were captured using an optical microscope coupled to a digital camera, and reproduced on a computer screen using the Infinity Analyze software (Lumenera, Canada). Histomorphometric analysis was performed by evaluating the following tissues: Non Vital Mineralized Tissue (NVMT), Vital Mineralized Tissue (VMT) and Non Mineralized Tissue (NMT). Statistical Analysis: The intra and intergroup statistical comparison was done by Kuskal-Wallis test, using a p value of 0.05. Results: The histomorphometric results showed, for the side with membrane coverage, no statistical significant difference between CG and TG for all parameters. On the other hand, for the side without membrane coverage, a statistical significant difference between CG and TG was observed for VMT and NMT. Concerning the side with membrane coverage, TG and CG had 17.88 ± 7.57% and 13.50 ± 0.09% for NVMT, respectively (p=0.2971). For VMT, TG and CG had 13.88 ± 5.68% and 13.29 ± 0.16%, respectively (p=0.5211). For NMT, TG and CG had 68.24 ± 5.05% and 73.21 ± 0.12%, respectively (p=0.0776). Regarding the side without membrane coverage, TG and CG had 16.59 ± 3.54% and 13.09 ± 0.06% for NVMT, respectively (p=0.0542). For VMT, TG and CG had 9.52 ± 0.60% and 6.56 ± 0.08%, respectively (p=0.0039). For NMT, TG and CG had 73.74 ± 3.48% and 80.34 ± 0.05%, respectively (p=0.0038). Conclusions: In this experimental model: 1) Platelet rich fibrin does not result in higher levels of bone formation when a guided bone regeneration technique (i.e. with membrane coverage) is used. 2) The usage of the collagen membrane has a synergistic effect on bone healing when associated with a xenograft. 3) Platelet rich fibrin may increase the level of newly formed bone only in bone grafting procedures using xenograft without collagen membrane coverage. Keywords: platelet rich fibrin, bone regeneration

Objectives: To determine the effect of alveolar ridge preservation (ARP) for molar sites without primary flap closure. Materials and Methods: Three groups were established: the sockets grafted with deproteinized bovine bone mineral containing 10% collagen (DBBM-C) and covered by a native bilayer collagen membrane (CM) (test group 1), the sockets grafted with DBBM-C only (test group 2), and the sockets healed naturally (control group). Primary flap closure was not attempted. Cone-beam computed tomography scans were obtained immediately after ARP and 4 months later. A biopsy was performed at the time of implant surgery. The change of marginal bone level was measured. Results: There was significantly less horizontal resorption in the test group 1 than the control group at 1 mm (-1.02±0.88 [mean±SD] vs. -4.44±3.71 mm) and 3 mm (-0.31±1.51 vs. -2.27±1.15 mm) levels below the crest, and significantly less vertical reduction in the midcrestal area in test group 1 than in test group 2 (-0.25±0.95 vs. -1.15±1.63 mm) (p0.05). There were no significant differences between the test groups in clinical and histomorphometric measurements. All groups exhibited stable marginal bone level after one year loading. Conclusion: ARP without primary flap closure in molar areas was effective in minimizing ridge resorption and favored implant treatment. Keywords: bone regeneration, bone substitute, randomized controlled trial, tooth extraction, wound healing

Success of bone grafts depends on their ability to induce favorable cellular responses. Accordingly, bone regeneration is thought to depend on the ability of macrophages to undergo transition from M1 to M2 phenotype. Macrophages from grade C periodontitis subjects are hyper-inflammatory, nonetheless it is unknown whether these cells undergo efficient M1-M2 transition. The aim of this study is to evaluate the ability of macrophages from grade C periodontitis to undergo phenotypic transitions. In brief, macrophages from generalized grade C periodontitis (n=10), generalized grade B periodontitis (n=10) and periodontally healthy (n=10) subjects will be exposed to M1- and M2-polarizing stimuli and in vitro kinetics of macrophage phenotype switch will be assessed using flow cytometry analysis of the cells and multiplex assays of conditioned media. Data will be analyzed by one-way ANOVA followed by post-hoc Tukey's test (α = 0.05). Preliminary experiments were conducted to determine ideal experimental conditions for peripheral blood mononuclear cell (PBMC) isolation, CD14+ isolation, differentiation of PMBCs into macrophages and polarization of M0 macrophages into M1 and M2 macrophages. CD14+ negative magnetic isolation and an adherence protocol were necessary to obtain predictable macrophage differentiation. Ideal methods for PBMC isolation, PBMC differentiation into macrophages, and M0 macrophage polarization into M1 and M2 macrophages were established. Keywords: periodontitis, macrophages, inflammation, healing

Objectives: To examine the influence of demineralization process using 4 different concentrations and reaction times of HCl acid on physicochemical properties, BMP-2 releasing and degradation rate at 30 days of human tooth matrix. Materials and methods: Tooth particles with size 500-1,000 µm were divided to 5 groups: 0M/0min, 0.5M/10min, 0.5M/20min, 1M/10min and 1M/20min. Chemical compositions were analyzed by XRD and XRF. Surface morphology was observed by SEM and BET analysis. Bradford protein assay was used to quantify total protein and human ELISA kit was used for BMP-2 quantification. Degradation rate was assessed by using 50 mM Tris-HCl solution for 30 days. Results: Increasing reaction time led to more collagen exposed, larger dentinal tubules, less crystallinity, and less calcium-phosphate percentage but increased Ca/P ratio. The highest total protein and BMP-2 concentration were found in 1M/20min group compared to 0M/0min (p=0.000). Increasing HCl concentration led to more degradation rate. Conclusions: The reaction time of HCl had effect on tooth particles in aspect of surface morphology, crystallinity, element components, Ca/P ratio, quantity of BMP-2 releasing while increasing concentration led to more degradation. Keywords: bone graft, tooth, demineralization, hydrochloric acid, BMP-2, degradation

Objectives: Additive manufacture techniques are revolutionizing the concept of biomaterials production and treatment personalization. The objective of this pilot study was to evaluate the viability and cellular migration of preosteoblasts on scaffolds made with a 3D-printable polyurethane-based material. In addition, the potential use of a platelet extract (PE) gel as a carrier of growth factors, like PDGF-AB and IGF-I, was evaluated to enhance cellular proliferation at imbibed scaffolds. Methods: For the 3D-printable material production, polyurethane was heated and stirred at high speed and hyaluronic acid, C6H10O3 (HEMA) and C22H21O2P (TPO) were added to the vial. The scaffolds were manufactured using a Miicraft 125 3D printer (Miicraft®, Taiwan). The PE was obtained after a first centrifugation of human whole blood (BioIVT®, USA) at 1,000g for 2min and 15s and a second centrifugation of the supernatant at 1,000g for 5min. The pellet was isolated and later resuspended in platelet poor plasma. For the PE-gel preparation, the PE was left on cold bath sonification for 5min and then 1/10 volume of 10x PBS was added. Then, PE solution was diluted with VitroGel™ 3D (TheWell Bioscience Inc.®, USA) at a 5-fold. Scaffolds were imbibed on PE-gel while in a cold sonification bath. Cellular viability was evaluated with preosteoblasts (MC3T3-E1) after different scaffold detoxication protocols (boiling, ultraviolet light exposure, dimethyl sulfoxide submersion and vacuum chamber use) using CellTiter-Glo® (Promega®, USA). The migration assay was evaluated using imbibed or non-imbibed scaffolds placed under an 8 µm-pore cell-culture insert where MC3T3-E1 were seeded. Cell counting was performed after 24, 48 and 72h using CCK-8® (Dojindo®, Japan). The concentration of PDGF-AB and IGF-I released by imbibed scaffolds were measured using DuoSet® ELISA (R&D Systems®, USA) at 5, 15 and 30min and 1, 3, 6, 9 and 24h. PDGF-AB concentration was also assessed after addition of PAR1 to PE. Results: The results indicated that the 3D-printed polyurethane-based scaffolds are biocompatible and good carriers for the PE-gel. The best post-processing method for detoxification of the material prior to cell culture was the use of a vacuum chamber with the scaffold submerged at hot deionized water, achieving results close to positive control after 24h (7,547 RLU against 7,750 RLU, respectively). Detoxification with overnight ultraviolet light exposure showed better results when compared to boiling and dimethyl sulfoxide submersion, however, results were much lower than positive control (63 RLU, 74.5 RLU and 93 RLU against 6,424 RLU, 11073 RLU and 15,005 RLU at 24, 48 and 72h). At the migration assay, a higher number of cells could be counted at 24, 48 and 72h on the scaffolds imbibed with PE-gel when compared to those without PE-gel, with values of 49,214, 100,857 and 101,428 cells for the first and 38,857, 83,000 and 83,142 cells for the later, respectively. Regarding the concentration of PDGF-AB and IGF-I released by the scaffold imbibed with PE-gel at the cell culture medium, a reduction in concentration was noted over time. No difference was notice concerning the time the scaffold remained submerged on PE-gel during cold sonification bath or if a different scaffold design was used. However, the addition of PAR1 to PE was related to an increase at the concentration of PDGF-AB over time, showing gradual release of the growth factor to the medium. Conclusions: The results from this pilot study suggest that 3D printed polyurethane-based scaffolds are viable constructs for cell culture migration after a post-processing detoxification. The best protocol is the use of a vacuum chamber with the scaffold submerged at hot deionized water. Boiling, overnight ultraviolet light exposure and dimethyl sulfoxide submersion do not appear to be effective methods for that purpose. The use of PE-gel seems to increase cell counting, favoring cell proliferation. However, our results showed that some modifications still can be done to optimize the PDGF-AB and IFG-I stability. For that, the addition of PAR1 seems to be necessary to optimize platelet activation. These results will guide the methodology design of our future research aiming the clinical applications of 3D-printing for bone regeneration, such as the production of biodegradable scaffolds, membranes and fixation devices. Keywords: 3D-print, polyurethane, digital light processing, scaffold, membrane

Objective: Periodontal and peri-implant diseases are similar and can result in the destruction of supporting tissues. Although clinical indexes and radiological examinations are the standardized method of diagnosis, the literature is concerned with the diagnostic potential of gingival crevicular fluid (GCF) and peri-implant sulcus fluid (PISF). This cross-sectional study aimed to evaluate the effect of periodontal healthy and diseased conditions on the volume of GCF and PISF. Methods: Our study included GCF and PISF collected from 40 patients who have at least 3 dental implant [26 periodontally healthy (H), 27 gingivitis (G), 26 periodontitis (P); 26 healthy implants (HI), 27 peri-implant mucositis (PM), and 27 peri-implantitis (PI)]. The inclusion criteria for healthy teeth and implants were absence of bone loss around the teeth or implant and bleeding on probing (BOP)(-); for peri-implantitis, radiologic bone loss (RBL)>1 mm around the implant and BOP(+); for peri-implant mucositis RBL1 mm around the implant and BOP(+); for gingivitis with no RBL and attachment loss, and BOP(+); for periodontitis attachment loss and BOP(+). In the periodontal examination of the patients, the periodontal pocket depth (PPD), gingival index (GI), plaque index (PI), and clinical attachment level (CAL) were measured by a calibrated periodontist. GCF and PISF samples were collected and measured in a PERIOTRON 8000 device and the results were converted into volume by third party software. The periodontal parameters and GCF and PISF volumes were compared between groups using SPSS statistical analysis software v.24. Results: Although we found a statistically significant difference between the G and P, PM and PI, H and P, and HI and PI groups (p0.005), no statistically significant difference was found between the HI and PM, and H and G groups (p>0.005). In addition, there was a correlation between GCF volume and CAL and RBL in the PI and P groups. Conclusions: Despite the limitations of our study, it can be said that GCF volume increases in the presence of periodontal and peri-implant disease especially in patients with periodontitis and peri-implantitis, and that PISF and GCF can be used in addition to radiological examinations during the differential diagnosis of peri-implant diseases. Keywords: periodontal disease, peri-implant disease, gingival crevicular fluid, peri-implant sulcus fluid

Objectives: If considering the morphological, physical and chemical similarity of dentine and bone, it gives us biological ground for using dentine blocks for local alveolar ridge augmentation in cases with a small amount of atrophy prior to implant placement. The aim of the study is to prove both clinically and histologically whether the use of dentine blocks for local alveolar ridge augmentation is a valuable treatment modality. Methods: A single cohort of fifteen patients, both male and female was selected. The age range was from 21 to 60. Inclusion criteria were: non-functional vital teeth, which were to be extracted; local bone atrophy with less than 5 mm ridge width. Clinical procedure. After donor-tooth extraction, followed separation of clinical crown, periodontal ligament and root cement. All procedures were performed with irrigation. Then the blocks were adapted to the recipient site and fixed with titanium screws. Minimum healing period before reopening was 4 months. At second surgery stage, biopsy with trephine was collected, following implant placement in this site. Radiological evaluation was done at baseline and four months after transplantation. Results: Fifteen patients had 17 implants placed in newly regenerated bone. After 8 to 10 weeks all implants were loaded with final restorations. Morphological data shows incorporation of bone structures and dentine blocks. Conclusions: T-bone concept showed promising clinical and histological results in terms of alveolar ridge augmentation for consecutive implant placement, and crestal bone stability around implants after loading in one year observation period. However, with limitations of the study further clinical evaluation and long-term observations are needed before recommending it as a definitive treatment modality. Keywords: dentineblocks autologous GBR

Objective: The objective of this investigation was to compare the periodontal condition of liver transplant candidates (LTCs) with healthy controls. Methods: A complete periodontal examination was performed on fifty liver transplant candidates (LTC group) and fifty patients without liver disease (control group). Demographic data, systemic health and information related to liver diseasewere collected using a structured questionnaire. Full-mouth complete periodontal examination of six sites per tooth was performed: gingival recession (GR), probing depth (PD), attachment loss (AL), bleeding on probing (BOP), and visible plaque index (VPI). The groups were compared in regard to periodontal clinical variables. Results: LCT demonstrated greater prevalence of periodontitis than healthy controls (P 0.001). In addition, they had greater mean percentage of sites with AL ≥3 mm (P = 0.008) and AL ≥5 mm (P = 0.023), greater mean AL (P = 0.003), greater mean gingival recession (P 0.001), and more missing teeth than in the control group (P = 0.02). Conclusion: LTCs exhibited increased prevalence, extent, and severity of periodontitis when compared with control patients. Keywords: end-stage liver disease, liver cirrhosis, liver transplantation, liver, periodontal diseases

Objective: To evaluate osteogenesis and angiogenesis coupling in different bone materials (Autograft, xenograft and synthetic) used around dental implants, we design this study to compare osteogenesis and angiogenesis coupling in 3 types of bone graft for dental implants. Implant restorations are an important every day treatment to replace missing teeth in the general dental practice. An understanding of implant design and placement for optimum clinical results is common knowledge for dental practitioners. Materials and Methods: Twelve healthy adult male dogs are randomly assigned into three Groups. Under general anesthesia the area corresponding to the 2nd to 6th sternebra is exposed. A 6 mm depth hole in the each sternebra was created, using a 10 mm diameter trephine drill. After performing 2 mm further drilling at the end of the hole using ICX surgical kit, a 3.45 mm * 6.5 mm ICX dental implant (Medentis Medical GmbH) is inserted within each hole. The gap around the implants is filled with synthetic bone material (OSTEON™, South Korea) in Group I, xenogenic bone material (cerabone, GmbH, Germany) in Group II and autogenous bone particles in Group III. To evaluate the effect of time on the bone healing and implant osseointegration every 2 months 4 dogs will be sacrificed (at 2, 4 and 6 months). We evaluated osseointegration process surrounding dental implant by osteogenesis and angiogenesis. As osseointegration composes of angiogenesis and osteogenesis processes, osteogenesis and angiogenesis expresses genes such as ALP, BMP7, RUNX, OCN and TIE1, Bfgf, ANG2, VEGFA, VEGFR, CD34 respectively, which these genes express in around of dental implant in 12 implants (4 implants from each group; divided into 2, 4, and 6 months). This evaluation of gene expression and proteins have performed by Real time PCR and ELISA test, respectively Results: Osteon as synthetic bone material revealed osteogenesis process by high gene expression ALP, RUNX2 and OCN during 2 months. While, cerabone as xenogen bone material showed these genes expression after 4 months. BMP-7 as a osteogenic gene has same level of expression by both of bone materials after 2 months. In angiogenesis process, two genes of ANG2 and VEGFA have expression by osteon in 4 months while these genes start expressing by cerabone after 6 months. Osteon can start TIE1 gene expression after 2 months and continue until 4 months while this gene has expression by cerabone after 6 months. CD34 and Bfgf genes have same levels of expression by both of bone materials in 2,4 and 6 months. All of data are based on real time gene expression analysis and for RUNX2 as osteogenic gene and ANG2 as angiogenic gene also immunoassay. Conclusion: Based on these preliminary in vivo results which related to osteogenesis and angiogenesis coupling, we can conclude that osteon as synthetic bone material which is osteoconductive quickly provoke osseointegration in implant place in compare with cerabone as xenogenic bone material. This bone material can produce appropriate osseointegration around dental implant. Keywords: bone graft material, osseointegration, osteogenesis, angiogenesis

Objective: Rheumatoid Arthritis is an inflammatory disease causing degenerative changes in joints including the temporomandibular joint. Currently, structural imaging is used to detect pathologic alterations in the TMJ. Molecular imagining techniques offer an alternative that might demonstrate inflammatory changes in the joint prior to the structural breakdown. To address this possibility we compared PET/CT scans of the TMJ using FDG and NaF taken in cohorts of RA and healthy control subjects. Method: 18 patients diagnosed with RA (mean age 55±12.1years: 4 females and 14 males) were included in the test group. Eighteen age-sex- and race-matched healthy control subjects were selected from the CAMONA clinical trial. PET/CT images were acquired 180-minute post-intravenous administration of FDG and NaF. OsirX® software was used for image analysis. For FDG analysis, regions of interest (ROIs) were manually assigned per anatomical boundaries using a closed polygon tool. The first ROI of the mask was assigned on the trans-axial CT slice of the joint up to two to three slices inferiorly. The ROI followed the anatomy of TMJ. Averaged SUVmean and SUVmax were used to semi-quantify FDG and NaF uptake in the joint. For NaF, A3-D ball tool of 1.5 cm was used to assign ROIs with the head of the mandibular condyle located at the center including the osseous compartment of the joint extending inferiorly to the neck of the condyle. The average SUVmean of the right and the left TMJ was determined. For normalization, a Target to Background Ratio (TBR) was calculated for each subject by dividing the average SUVmean by SUVmean uptake in superior vean cava. The student's t-test and regression analysis were used. The severity of RA was assessed by determining the serum C reactive protein level (DAS-28 CRP) and erythrocyte sedimentation rate (DAS-28 ESR) for each subject. Results/conclusions: The FDG average SUVmean of RA patients was 1.18±0.47 compared to 1.09±0.27 in healthy controls (p=0.48). FDG TBRmean for the test group was (1.21±0.33) compared to 0.91±0.2 in controls,(p=0.003.) No correlation was found between FDG uptake and DAS28-CRP or DAS28-ESR. The NaF average SUVmean was significantly higher in RA patients than healthy control subjects (2.4±0.8 versus 1.9±0.4, p=0.02). TMJ TBRmean was also higher in RA patients relative to healthy controls (4.2±2.1 versus 2.7±0.9, p=0.01) A significant positive correlation was found between TBRmean and DAS28-CRP (r=0.49, p=0.03), while there was not a significant correlation with DAS28-ESR (r=0.37, p=0.12). Our results illustrate the potential value of using PET/CT for evaluation of TMJ disorders and other inflammatory conditions within the oral cavity. Since this approach yields structural images along with data that can be quantified it provides more information than conventional structural imaging techniques. The RA patients appeared to have significantly higher metabolic activity in the TMJ relative to the healthy control subjects. The degree of NaF uptake in the TMJ correlated with biochemical parameters of RA disease severity based on DAS28-CRP and DAS28-ESR scores. Thus, the results suggest that NaF uptake does have the capacity to detect inflammatory changes prior to overt structural breakdown of the joint. Keywords: PET scan, rheumatoid arthritis, temporomandibular joint