Isolation and characterization of human periodontal ligament stem cells under the terms of use in clinical application: A pilot study.

Background: The aim of the present study is to determine the possibility of isolation and characterization of the human periodontal ligament stem cells (hPDLSCs) using limited harvested periodontal ligament (PDL) tissue of only one patient's wisdom teeth (2-4 teeth) under the more compatible terms of use in clinical application without using the fetal bovine serum (FBS). Materials and Methods: In this pilot study, hPDLSCs were isolated from the impacted third molar, and tissue was scraped from the roots of the impacted third molar of 10 volunteers to enzymatically digest using collagenase. The cells were sub-cultured. The samples of the first seven patients and half of the eighth patient's sample were cultured in alpha modified of Eagle's medium (α-MEM) (-FBS) medium and the other part of the eighth patient's sample was cultured with prior medium supplemented with +FBS 15% as a control of the cultivation protocol. While for the past two patients (9th and 10th the α-MEM medium was supplemented with L-Glutamine, anti/anti 2X, and 20% knock-out serum replacement (KSR). Two more nutritious supplements (N2 and B27) were added to the medium of the tenth sample. Flow-cytometric analysis for the mesenchymal stem cell surface markers CD105, CD45, CD90, and CD73 was performed. Subsequent polymerase chain reaction was undertaken on three samples cultured with two growth media. Results: Cultivation failed in some of the samples because of the lack of cell adhesion to the culturing dish bottom (floating cells), but it was successful for the 9th and 10th patients, which were cultured in the α-MEM serum supplemented with KSR 20%. Flow cytometry analysis was positive for CD105, CD90, and CD73 and negative for CD45. The PDL stem cells (PDLSCs) expressed CD105, CD45, and CD90 but were poor for CD73. Conclusion: According to the limited number of sample tests in this study, isolation and characterization of PDLSCs from collected PDL tissue of one patient's wisdom teeth (2-4) may be possible by the proper setup in synthetic FBS-free serum.

The effect of Vitamin E supplementation on treatment of chronic periodontitis.

BACKGROUND: The purpose of this study was to evaluate the effect of Vitamin E supplements on chronic periodontitis based on the clinical parameters of pocket depth and clinical attachment level and total antioxidant capacity (TAC) of saliva. MATERIALS AND METHODS: In this clinical trial, 16 patients with chronic periodontitis were selected and divided into two groups. The indices of pocket depth and attachment loss for 6 teeth per person were measured with a periodontal probe. A total of 41 teeth in the control group and 42 teeth in the case group were examined. Then, 2 ml nonstimulated saliva was collected from each patient. All patients were treated with scaling and root planing (SRP). The case group consumed 200 IU supplementary Vitamin E daily for up to 2 months. After 2 months, clinical indices were re-measured and 2 ml nonstimulated saliva was collected. The TAC of saliva samples was measured by using Zellbio's TAC Kit. Data were analyzed by the SPSS software and were evaluated in each group between the first session and 2 months later with paired t-test. The differences between the two groups were evaluated through the independent t-test (α ≤ 0.05). RESULTS: Independent t-test showed that mean change in TAC (P = 0.14) and pocket depth changes (P = 0.33) was not significant between two groups 2 months after SRP, but mean attachment loss changes in the case group was significantly less than the control group (P = 0.03). CONCLUSION: The results of this study indicate that Vitamin E supplementation with SRP can reduce the inflammatory process of periodontitis and improve periodontal clinical indices and decrease the amount of attachment loss.

The efficacy of metal artifact reduction (MAR) algorithm in cone-beam computed tomography on the diagnostic accuracy of fenestration and dehiscence around dental implants.

BACKGROUND: The aim of the present study was to investigate the impact of metal artifact reduction (MAR) algorithm of cone-beam computed tomography (CBCT) on the diagnostic accuracy of fenestration and dehiscence around dental implants. METHODS: Twenty-three dental implants were placed adjacent to the dehiscence and 23 adjacent to the fenestration defects on bovine bone blocks. The blocks were scanned with CBCT unit in two modes, with and without MAR algorithm. The area under the receiver operator characteristic (ROC) curves (Az value), specificity, sensitivity, positive predictive value, negative predictive value, and accuracy were determined for all modes. RESULTS: For both defects, the Az values were higher in off MAR condition. The values of sensitivity, positive predictive value, negative predictive value, and accuracy, were higher in off MAR condition for both defects. However, the specificity in both defects in the two modes was equal. CONCLUSION: The MAR algorithm in CBCT unit may not be helpful in increasing the diagnostic accuracy of fenestration and dehiscence around dental implants.

Retro MTA and tricalcium phosphate/retro MTA for guided tissue regeneration of periodontal dehiscence defects in a dog model: a pilot study.

OBJECTIVES: Retro MTA is a fast setting Calcium silicate cement used in endodontic regeneration procedures in recent years. Beta-tricalcium phosphate (ß-TCP) is another common biomaterial used for bone augmentation procedures. The present pilot study was undertaken to evaluate and compare the efficacy of Retro MTA and a mixture of Retro MTA / ß-TCP for periodontal tissue regeneration. MATERIALS AND METHODS: In 4 beagle dogs, periodontal dehiscence type defects were created. In each side, one dehiscence defect was left empty as a control site and three treatment modalities were randomly applied for the others: Retro MTA covered with a collagen membrane, Retro MTA + ß-TCP covered with a membrane and covering the defect with a membrane without any bone augmentation. After 8 weeks Animals were sacrificed and Histomorphometric and histologic analysis were conducted. RESULTS: Histologic analysis showed more cementum formation for both Retro MTA+ ß-TCP (3.74 ± 0.34 mm) and Retro MTA group (3.24 ± 0.56 mm) compared to control group 1 (1. 15 ± 0.45 mm) and control group 2 (0.78 ± 0.65 mm). Formation of newly formed bone and cementum in the experimental groups were significantly higher as compared to the control groups (P < 0.0001). CONCLUSIONS: Retro MTA or Retro MTA+ ß-TCP covered with a collagen membrane resulted in regeneration of periodontal tissues. However, Retro MTA+ ß-TCP showed tendency towards better results than the use of Retro MTA alone.

The Efficacy of Green Tea Chewing Gum on Gingival Inflammation.

STATEMENT OF THE PROBLEM: According to previous studies, the components of green tea extracts can inhibit the growth of a wide range of gram-pos-itive and -negative bacterial species and might be useful in controlling oral infections. PURPOSE: The aim of this study was to determine the effect of green tea chewing gum on the rate of plaque and gingival inflammation in subjects with gingivitis. MATERIALS AND METHOD: In this double-blind randomize controlled clinical trial, 45 patients with generalized marginal gingivitis were selected and divided into two groups of green tea (23) and placebo (22) chewing gum. The patients chewed two gums for 15 minutes daily for three weeks. Sulcus bleeding index (SBI) and approximal plaque index (API) were studied at the baseline, 7 and 21 days later. Saliva sampling was conducted before and after 21 days for evaluation of IL-1ß. The results were analyzed and compared by using repeated measures ANOVA, paired t test, and independent two-sample t test (α=0.05). RESULT: The results showed that chewing gum significantly affected the SBI and API (p< 0.001). Paired t test showed that the two groups were significantly different regarding the mean changes of SBI and API at different periods of 1-7, 1-21, and 7-21 (p< 0.001). Concerning IL-1ß, the repeated measures ANOVA revealed that the effect of chewing gum was significant (p<0.001). Moreover, paired t-test represented no significant difference between the mean changes of IL-1ß within 1-21 day (p= 0.086). CONCLUSION: The green tea chewing gum improved the SBI and API and effectively reduced the level of IL-1ß.

Serum, saliva, and GCF concentration of RANKL and osteoprotegerin in smokers versus nonsmokers with chronic periodontitis.

BACKGROUND: The role of host response in periodontitis pathogenesis is confirmed, and it is well established that immune response plays a major role in the alveolar bone destruction. In the investigation of these responses, the role of receptor activator of the nuclear factor-kB ligand (RANKL)-osteoprotegerin (OPG) system is the most promising. Smoking can affect the RANKL-OPG system in a manner that will further enhance bone loss in periodontitis. The aim of this study is to assess the serum, saliva, and gingival crevicular fluid (GCF) concentration of RANKL and OPG in smoker versus nonsmoker untreated chronic periodontitis (CP) patients. MATERIALS AND METHODS: Thirty-nine subjects were included in the present cross-sectional study: 29 systemically healthy CP male patients (15 smokers, 14 nonsmokers) and 10 systemically and periodontally healthy nonsmoker male subjects. Serum, GCF, and whole saliva samples were obtained from the subjects. The enzyme-linked immunosorbent assay (ELISA) kits were used for assaying the concentrations of RANKL and OPG in the samples. The one-way analysis of variance (ANOVA) test and the least significant difference (LSD) post hoc test were utilized to compare differences between the groups. RESULTS: RANKL and OPG concentrations in saliva, serum, and GCF did not show any significant difference among all groups (P > 0.05). Salivary RANKL/OPG ratios were significantly higher in the nonsmoker CP group than in the healthy control group (P > 0.05) but they were not statistically significant among smoker periodontitis patients. CONCLUSIONS: The salivary RANKL/OPG ratio was higher in nonsmokers with periodontitis in comparison with smoker periodontitis patients.

Evaluation of the effects of two different bone resorption inhibitors on osteoclast numbers and activity: An animal study.

BACKGROUND: The aim of this study was to evaluate the effects of bone resorption inhibitors, doxycycline (DOX) and erythromycin (EM), on osseous wound healing in rat alveolar socket. MATERIALS AND METHODS: In this randomized controlled trial, 45 8-10-week-old male Wistar rats had their maxillary right molar extracted. They were divided into three groups of 15. In Group 1 normal saline, Group 2 DOX, and Group 3 EM were administered at the doses of 5 ml/kg/day, 5 mg/kg/day, and 2 mg/kg/day, respectively, for 7 consecutive days. The rats were sacrificed 7, 14, and 21 days after surgery. Real-time polymerase chain reaction was employed to evaluate the mRNA expression of receptor activator of nuclear factor κB ligand (RANKL) and osteoprotegerin (OPG) and immunohistochemical staining for tartrate-resistant acid phosphatase (TRAP) to determine osteoclasts. The data were analyzed by one-way analysis of variance followed by Tukey's post hoc test using SPSS version 20. Significant level was set at 0.05. RESULTS: The results showed that when drug-treated groups compared to control groups, RANKL gene expression significantly decreased, TRAP+ cells decreased on day 7. The RANKL/OPG ratios in the first two weeks in the test groups were significantly lower than the control group. There was no significant difference in the studied indices between DOX and EM groups. CONCLUSION: Following administration of DOX and EM, the number of osteoclasts and RANKL/OPG ratio decreased suggesting their anti-osteoclastogenesis activity. These two drugs have no advantage over each other in increasing the bone formation.

A histomorphometric study of the effect of doxycycline and erythromycin on bone formation in dental alveolar socket of rat.

BACKGROUND: The aim of the present study was to evaluate whether subantimicrobial doses of doxycycline (DOX) and erythromycin (EM) used for the treatment of peri-implant osteolysis due to their anti-osteoclastogenesis can interfere with the osseous wound healing process in rat alveolar socket. MATERIALS AND METHODS: Forty-five male Wistar rats had their first maxillary right molar extracted and were divided into three groups. DOX and EM at the doses of 5 mg/kg/day orally (p.o.) and 2 mg/kg/day intraperitoneally (i.p.) were administered respectively to two separate groups for 7 days after operation. In the control group the animals received normal saline (5 ml/kg). Five rats were sacrificed at 7, 14 and 21 days post-extraction in each study group. A histomorphometric analysis was used to evaluate new bone formation inside the alveolar socket. Significant level was set at 0.05. RESULTS: The findings showed that the percentage of new bone formation (NBF) enhanced significantly on days 7 and 14. There was no significant difference in the NBF between DOX and EM groups. CONCLUSION: Short-term treatment with both DOX and EM enhanced new bone formation without any advances in favor of each drug.

Socket preservation using demineralized freezed dried bone allograft with and without plasma rich in growth factor: A canine study.

BACKGROUND: The accelerating effect of plasma rich in growth factors (PRGFs) in the healing of extraction sockets has been demonstrated by some studies. The aim of the present study was to histologically and histomorphometrically evaluate whether bone formation would increase by the combined use of PRGF and demineralized freeze-dried bone allograft (DFDBA). MATERIALS AND METHODS: In four female dogs, the distal root of the second, third and fourth lower premolars were extracted bilaterally and the mesial roots were preserved. The extraction sockets were randomly divided into DFDBA + PRGF, DFDBA + saline or control groups. Two dogs were sacrificed after 2 weeks and two dogs were sacrificed after 6 weeks. The extraction sockets were evaluated from both histological and histomorphometrical aspects. The data were analyzed by Mann-Whitney followed by Kruskal-Wallis tests using the Statistical Package for the Social Sciences version 20 (SPSS Inc., Chicago, IL, USA). Significant levels were set at 0.05. RESULTS: The least decrease in socket height was observed in the DFDBA + PRGF group (0.73 ± 0.42 mm). The least decrease in the coronal portion was observed in the DFDBA + PRGF group (1.38 ± 1.35 mm²). The least decrease in the middle surface was observed in the DFDBA group (0.61 ± 0.80 mm²). The least decrease in the apical portion was observed in the DFDBA group (0.34 ± 0.39 mm²). CONCLUSION: The present study showed better socket preservation subsequent to the application of DFDBA and PRGF combination in comparison with the two other groups. However, the difference was not statistically significant.

Expression levels of th-2 and th-17 characteristic genes in healthy tissue versus periodontitis.

OBJECTIVE: The T helper 17 (Th17) cells have been suggested to have osteoclast activating effects while T helper 2 (Th2) cells are considered to have an osteoprotective role in periodontitis. This study was to compare the markers of Th17 cells (RORC2 and IL-17 genes) with that of Th2 cells (IL-4 and GATA-3 genes) between healthy and periodontitis tissues. MATERIALS AND METHODS: The samples were obtained from patients with periodontitis and healthy tissues. The mRNA expression levels of IL-17, RORC2, IL-4 and GATA-3 were measured in both groups using quantitative RT-PCR. The results were compared using SPSS 11.0. Correlation coefficient was analyzed by Spearman's rho test. Mann-Whitney was used to measure the difference between IL-17 and IL-4 as well as RORC2 and GATA-3. RESULTS: In periodontal lesions, the expression levels of all markers were significantly higher than the healthy tissue (p≤ 0.001). The results showed a significant increase in the number of markers of Th17 (RORC2 and IL-17 genes) compared to markers of Th2 (GATA-3, IL-4) in patients with periodontitis compared to controls (p≤0.002). A positive correlation between IL-17 and RORC2 (p≤0.05) and between IL-4 and GATA-3 (p≤0.001) was found. CONCLUSION: The results show that expression levels of IL-4, GATA-3, IL-17 and RORC2 increase significantly in periodontal lesions compared with the controls. In periodontal lesions, IL-17 levels are significantly greater than IL-4, which plays a protective role against alveolar bone loss.

Comparison of bone regeneration using three demineralized freeze-dried bone allografts: A histological and histomorphometric study in rabbit calvaria.

BACKGROUND: It has been stated that the bone allografts from different tissue banks may lead to various amount of bone induction, so the aim of this study was to evaluate bone regeneration of three demineralized allografts both histologically and histomorphometrically in rabbits calvaria bone defects. MATERIALS AND METHODS: In this double-blind randomized experimental animal study, 32 critical size defects (11-mm diameter) in the calvaria of 16 male New Zealand white rabbits were randomly filled with three demineralized freeze-dried bone allografts (DBM, CENOBONE, DEMBONE), while the nongrafted defect was regarded as control group. After 6 and 12 weeks of healing, the experimental animals were euthanized for specimen preparation. After histological evaluation, histomorphometric analysis was performed to quantify new bone formation and remained graft particles. The data were analyzed by one-way ANOVA with Tukey's ad-hoc test and t-test. (P < 0.05 was considered to be statistically significant). RESULTS: Mean percentage of bone formation increased between two healing time, but it was not statistically significant in all groups except DBM which the bone formation significantly decreased (P = 0.04). There were not statistically significant differences between three allografts in remained particles and bone formation in both healing times and they could not induce significantly more bone formation than control group. CONCLUSION: Both test and control groups resulted in successful new bone formation. No difference was noted in bone formation and remained particles between three commercial bone allografts. Further studies in this issue may be needed.

Histological and histomorphometric analysis of animal experimental dehiscence defect treated with three bio absorbable GTR collagen membrane.

BACKGROUND: Guided tissue regeneration (GTR) allows mesenchymal cells to repopulate the defects. However, there is limited information regarding the efficacy of different membranes. The present study was designed to histologically and histomorphometrically compare three collagen membranes in regenerative treatment of dehiscence defects in dogs. MATERIALS AND METHODS: This 8 weeks experimental animal study comprised 4 healthy dogs. 5 × 5 mm periodontal dehiscences were created in each side of the mandible (4 dehiscences in each side of dogs' mandible). In each side, one dehiscence defect was left uncovered as a control site and three other sites were randomly covered with different collagen membranes (Biogide (BG), Biomend (BM), and Cytoplast (CYT)). Histomorphometric and histologic analysis were conducted at 4 and 8 weeks. Data were analyzed using ANOVA, Mann-Withney, Kruskal-Wallis and Fisher 's exact tests (α = 0.05). RESULTS: According to histomorphometric analysis there was a significant difference between treatment and control groups regarding the bone formation and the distance between the reference point and apical end of junctional epithelium (DJE) (P < 0.05). At 4 weeks, the maximum amount of bone thickness and height was observed in BG and CYT respectively, and this maximum rate was seen with the use of BG at 8 weeks. It was shown that DJE reached its highest rate in BM and CYT at 4 and 8 weeks, respectively. Organized PDL was formed in treatment groups. CONCLUSION: The membrane-treated groups had a statistically significant increase in bone formation and connective tissue attachment compared to control groups. However, there are some differences among experimental groups, which should be considered in GTR treatments.

Comparative Evaluation of IFNγ, IL4 and IL17 Cytokines in Healthy Gingiva and Moderate to Advanced Chronic Periodontitis.

BACKGROUND: Considering the role of T cells in the pathogenesis of periodontitis, the purpose of this study was to compare the amount of IFNγ, IL4 and IL17 in advanced periodontal lesions with healthy gum areas to determine each TH1, TH2 and TH17 cells activity in comparison with each other and finally, to compare the value and the role of humoral, cell mediate and autoimmune responses. METHODS: In this descriptive analytical study, those with moderate to advanced periodontitis, having pocket with 4 to 7 mm in depth, were randomly selected. After preparing the healthy and affected sample tissues, the amount of the specific antibody in I IFNγ, IL4 and IL17 cytokines were measured using ELISA method and were compared between the two groups. The findings were analyzed using the software and descriptive statistical methods and Pearson correlation statistical analysis (α = 0.05). RESULTS: This study was performed on 37 patients with moderate to severe periodontitis and 22 healthy individuals without any periodontal disease. IL4 and IFNγ levels in the patients with chronic periodontitis compared to those of healthy gingival samples showed a significant reduction (P > 0.05) whereas the amount of IL17 in tissue samples of chronic periodontitis compared to healthy gums had a significant increase (P < 0.05). CONCLUSION: It appears that in the periodontitis pathogenesis, as well as TH1 and TH2 responses, IL17 causes the host immunological response to the periodontal pathogenesis.