Miroslav Andric

(a) To quantify the relative gene expression of matrix metalloproteinases (MMP) -2, -9, their tissue inhibitor (TIMP) -2, receptor activator of nuclear factor kappa-B ligand (RANKL), and osteoprotegerin (OPG) in human apical periodontitis (AP) and to associate it with levels of these molecules in healthy dental pulps. (b) To assess differences in gene expression among AP lesions stratified by clinical, radiographic, and histopathological features. (c) Finally, a potential correlation of investigated molecules was explored in AP samples. The study cohort comprised 80 AP lesions obtained from 80 adult volunteers during apicoectomy procedures. The healthy control cohort consisted of 50 dental pulp samples from intact teeth extracted for orthodontic purposes, collected from 50 voluntary donors. Relative gene expression of MMP-2, MMP-9, TIMP-2, RANKL, and OPG was assessed in all tissue samples using reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR). Statistical analyses included the Chi-square test, Student’s t-test, Mann–Whitney U test, and Spearman correlation. Higher gene expression levels of RANKL, OPG, MMP-2, and MMP-9 were observed in the study compared to the healthy cohort (p=0.048, p=0.002, p=0.048, and p=0.012, respectively). Higher relative expression of the MMP2 gene was observed in smaller lesions compared to large lesions (p=0.021). Significant positive correlations were observed between the investigated mediators. Concurrent overexpression of the studied mediators, along with their positive correlations in AP lesions, indicates their coordinated role in disease progression and alveolar bone resorption.

The primary objective of this study was to examine the potential association between glutathione S-transferases (GSTT1/GSTM1) deletion polymorphisms and the development of apical periodontitis (AP) in a population of patients at two university centers: the Faculty of Medicine at the University of Banja Luka in Bosnia and Herzegovina and the School of Dental Medicine at the University of Belgrade in Serbia. The study involved 200 patients with AP in the experimental and 250 healthy individuals without AP in the control group. As a source of genomic DNA, sterile buccal swabs were taken from each patient. Genotyping of GSTM1 and GSTT1 deletion polymorphisms was conducted using multiplex Polymerase Chain Reaction (PCR). The risk of AP development with regard to the genotypes was evaluated based on odds ratios (ORs) and 95% confidence intervals (CIs) that were calculated via unconditional logistic regression. There were significant differences in demographic characteristics between the investigated groups (p = 0.446, p = 0.154, respectively). GSTM1 and GSTT1 deletions were associated with a 3.05-fold and 5.69-fold risk (OR = 3.05, 95% CI = 2.07-4.49, OR = 5.69, 95% CI = 3.66-8.86, p < 0.001, p < 0.001, respectively) for the AP development. The co-occurrence of both deletions posed a significantly higher risk for AP development (OR = 52.76. 95% CI = 18.20-152.94, P < 0.001). The carriers of null GSTT, null GSTM, and double null GSTT/GSTM genotypes are more susceptible to AP development in the populations examined at the two centers.

In regenerative bone tissue medicine, combining artificial bone substitutes with progenitor cells is a prospective approach. Surface modification via cold atmospheric plasma (CAP) enhances biomaterial–cell interactions, which are crucial for successful bone regeneration. Using a rabbit calvarial critical-size defect model, we assessed the use of CAP-pretreated beta-tricalcium phosphate (β-TCP), alone or with periodontal ligament stem cells (PDLSCs), for bone regeneration. Histological and histomorphometric analyses at two and four weeks revealed significantly improved bone regeneration and reduced inflammation in the CAP-treated β-TCP with PDLSCs compared to β-TCP alone. Immunohistochemical analysis also showed an increase in the bone healing markers, including bone morphogenic proteins 2 and 4, runt-related transcription factor 2, collagen-1, and osteonectin, after two and four weeks in the CAP-treated β-TCP implants with PDLSC. This in vivo study demonstrates for the first time the superior bone regenerative capacity of CAP-pretreated β-TCP seeded with PDLSCs, highlighting the therapeutic potential of this combined approach in osteoregeneration.

Background: Despite numerous studies indicating a high prevalence of herpesviruses in both apical and marginal periodontitis samples, their exact role in the pathogenesis of a periodontal disease is still unclear. Objective: This umbrella review aimed to summarize data on herpesviruses detection in marginal periodontitis (MP) and apical periodontitis of endodontic origin (APEO) samples. Methods: The study protocol has been drafted a priori and registered to the International Prospective Register of Systematic Reviews (PROSPERO) (CRD42020215922). The literature search was conducted using the following electronic databases: Clarivate Analytics’ Web of Science, Scopus, PubMed and Cochrane Database of Systematic Reviews, from inception to October 2020, with no language restrictions. Systematic reviews with or without meta-analysis that evaluated the association between the occurrence of herpesviruses and different forms of periodontal diseases were included. Other types of studies, including narrative reviews, were excluded. Two reviewers independently performed a literature search, data extraction, and quality assessment of included studies. Any disagreements or doubts were resolved by a third reviewer. The quality of the reviews was assessed using the AMSTAR 2 tool (A measurement tool to assess systematic reviews). Results: Six systematic reviews were included in the current review. One was graded as high quality, another one was graded as moderate quality, whereas the other four were graded as critically low-quality reviews. The presence of herpesviruses in subgingival samples was associated with an increased risk of MP, supported by the corresponding meta-analyses. Although the association was strong (OR > 3.0), the confidence intervals were wide, heterogeneity was significant, and studies were of small sample size. In addition, publication bias was detected. Contrary, data from systematic reviews that assessed APEO and herpesviruses did not show any significant associations. Conclusions: Low-quality studies with high uncertainty suggest a strong association between herpesviruses and MP, but not with APEO. Keywords marginal periodontitis periapical periodontitis of endodontic origin herpesviruses herpes simplex virus Epstein-Barr virus human cytomegalovirus systematic review

Objectives The aim of this systematic review was to critically analyze available data on gene polymorphisms in odontogenic keratocysts and ameloblastomas, including their possible relationship with clinical and histological features of these lesions. Materials and Methods A comprehensive search of Web of Science Scopus, PubMed, Cochrane Central Register of Controlled Trials and EMBASE was conducted using relevant key terms and supplemented by a gray literature search. Quality assessment of included studies was performed using criteria from the Strengthening the Reporting of Genetic Association (STREGA) statement. Results Ten studies were included in the final review. Survivin ‐31G/C, interleukin IL‐1α ‐889 C/T, p53 codon 72 G/C, tumor necrosis factor TNF‐α (−308G>A) and its receptor TNF‐R1 (36A>G), glioma‐associated oncogene homolog 1 rs2228224 and matrix metalloproteinase 2 rs243865 gene polymorphisms were reported to be associated with odontogenic keratocysts. For ameloblastomas, p53 ‐ codon 72 G/C, X‐ray repair cross‐complementing protein 1 ‐ codons 194 and 399 and matrix metalloproteinase 9 rs3918242 gene polymorphisms were identified as risk factors. It wasn’t possible to establish a relationship between specific polymorphisms and clinical and histological features of investigated lesions. Conclusions Several gene polymorphisms might be considered as a risk factor for the development of these lesions. Future studies should investigate whether these polymorphisms might be used to identify patients with increased risk of recurrence or aggressive disease.

Aim: To investigate the possible association between TNFα (−308 G/A) and IL‐1β (−511 C/T) single nucleotide polymorphisms (SNPs) and GSTT and GSTM deletion polymorphisms and risk of apical periodontitis (AP) development, and determine the association of different genotypes with the presence of herpesviral infection in AP.Methodology: The study included 120 periapical lesions and 200 control samples. Gene polymorphism analysis was performed using either polymerase chain reaction (PCR) or PCR/ restriction fragment length polymorphism (RFLP). Relative gene expression of TNF‐α and IL‐1β was analysed using reverse transcriptase – real‐time PCR. The presence of Epstein–Barr virus (EBV) and human cytomegalovirus (HCMV) was assessed by nested PCR. Chi‐square and Fisher’s exact tests and logistic regression analyses were done for polymorphisms, whilst Mann–Whitney U‐test was performed for the expression analysis. The expected frequency of variants was analysed by the Hardy–Weinberg equilibrium test.Results: TNF‐α (−308 G/A) SNP increased AP susceptibility for heterozygous (odds ratio (OR) = 1.72, 95% confidence interval (CI) = 1.06–2.80, P = 0.027) and homozygous (OR = 8.55, 95% CI = 1.77–41.36, P < 0.001) carriers of the variant A allele. On the other hand, IL‐1β (−511 C/T) polymorphism exerted a protective effect both in heterozygotes (OR = 0.540, 95% CI = 0.332–0.880, P = 0.013) and homozygotes (OR = 0.114, 95% CI = 0.026–0.501, P < 0.001). In addition, GSTM1 and GSTT1 null genotypes separately, as well as concomitantly, were associated with an increased risk for AP development (P < 0.001). The null GSTT1 genotype increased approximately twice the risk of Epstein–Barr infection (EBV) in AP (OR = 2.17, 95% CI = 1–4.71, P = 0.048), whilst TNF‐α SNP decreased it, both in heterozygotes (OR = 0.20, 95% CI = 0.08–0.48, P < 0.001) and AA homozygotes (OR = 0.07, 95% CI = 0.01–0.37, P = 0.001).Conclusions: GSTM and GSTT deletion polymorphisms, as well as TNFα (−308 G/A) SNP, are associated with increased risk, whereas IL‐1β (−511 C/T) polymorphism decreases the risk of AP development. GSTT and TNFα polymorphisms also appear to modulate the risk of EBV infection in Serbian patients with AP.

Objectives: This study aimed to investigate whether Epstein-Barr virus (EBV) positive periapical lesions exhibited higher mRNA levels of Notch signalling molecules (Notch2 and Jagged1), bone resorption regulators (receptor activator of nuclear factor kappa-β ligand (RANKL) and osteoprotegerin (OPG)), and proinflammatory cytokines (tumour necrosis factor-alpha (TNF-α), interleukin-1 beta (IL-1β) and IL-6) compared to EBV negative lesions. Additionally, the potential correlation between investigated molecules in periapical lesions was analyzed. Materials and methods: Sixty-four apical periodontitis lesions were obtained subsequent to standard apicoectomy procedure. The presence of EBV was determined using nested PCR. Based on the presence of EBV all periapical lesions were divided into two groups, 29 EBV positive and 35 EBV negative lesions. A reverse transcriptase real-time PCR was used to determine mRNA levels of Notch2, Jagged1, RANKL, OPG, TNF-α, IL-1β and IL-6. Results: Significantly higher mRNA levels of Notch2, Jagged1, RANKL and IL-1β were observed in EBV positive compared to EBV negative lesions. Significant positive correlation was present between Notch2 and Jagged1, Jagged1 and RANKL, and IL-β and TNF-α in EBV positive periapical lesions. Conclusions: Notch signalling pathway may be involved in alveolar bone resorption in apical periodontitis lesions infected by EBV.

Aim: To investigate whether apical periodontitis lesions infected by Epstein–Barr virus (EBV) exhibit higher levels of oxidative stress biomarkers [8‐hydroxydeoxyguanosine (8‐OHdG) and oxidized glutathione (GSSG)] and bone resorption regulators [receptor activator of nuclear factor (NF‐κB) ligand (RANKL) and osteoprotegerin (OPG)] compared to EBV‐negative periapical lesions and healthy pulp tissues.Methodology: The experimental group consisted of 30 EBV‐positive and 30 EBV‐negative periapical lesions collected in conjunction with apicoectomy. The pulp tissues of 20 impacted third molars were used as healthy controls. The qualitative and quantitative analysis of EBV was performed by nested and real‐time polymerase chain reaction (PCR), respectively. The levels of RANKL and OPG were analysed by reverse transcriptase real‐time PCR. The levels of 8‐OHdG and GSSG were determined by enzyme‐linked immunosorbent assay (ELISA). Mann–Whitney U‐test and Spearman's correlation were used for statistical analysis.Results: The levels of RANKL, OPG, 8‐OHdG and GSSG were significantly higher in apical periodontitis lesions compared to healthy pulp controls (P = 0.001, P < 0.001, P < 0.001 and P < 0.05, respectively). RANKL and OPG mRNA expression was significantly higher in EBV‐positive compared to EBV‐negative periapical lesions (P < 0.05). There was no significant correlation between EBV copy numbers and levels of RANKL, OPG, 8OH‐dG and GSSG in apical periodontitis.Conclusion: Levels of bone resorption regulators and oxidative stress biomarkers were increased in apical periodontitis compared to healthy pulp tissues. EBV‐positive periapical lesions exhibited higher levels of RANKL and OPG compared to EBV‐negative periapical lesions. EBV may contribute to progression of apical periodontitis via enhanced production of bone resorption regulators.

Background/Aim. The use of autogenous bone grafts for augmentation of the resorbed alveolar ridge is still considered the gold standard in implant dentistry. The aim of this study was to analyze the resorption rate of autogenous bone block grafts from the retromolar region placed in the frontal segment of the upper jaw unprotected by barrier membranes, to assess the stability of implants placed into the grafted bone, as well as to monitor its changes during the healing period. Methods. The study included 18 patients with a total of 20 grafted sites. The residual alveolar ridge was measured before and after the augmentation and prior to implant placement. All implants were restored with provisional crowns within 48 hours after the placement. Implant stability was assessed using resonance frequency analysis. Results. The average period from ridge augmentation to reentry was 5.4 months (range 4?6 months). At reentry the healed alveolar ridge had a mean width of 6.1 ? 1.27 mm. The mean calculated width gain was 3.04 ? 1.22 mm. The overall surface resorption of block grafts was 0.68 ? 0.69 mm (18.85%). At the time of implant placement the mean value of implant stability quotient (ISQ) was 71.25 ? 5.77. The lowest ISQ values were noted after three weeks of healing, followed by a gradual increase until week 12. After 12 weeks implants showed significantly higher ISQ values compared to primary stability (p < 0.05 Wilcoxon signed ranks test). During the 3-years followup period no cases of implant loss were recorded. Conclusion. Despite a significant resorption of bone grafts, it was possible to place implants in all the cases and to use the immediate loading protocol without affecting implant survival rate.