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OBJECTIVE: The objective of the study was to determine the concentration of matrix metalloproteinase 9 (MMP-9) and changes in the presence of periodontopathogens in the gingival crevicular fluid before and after tooth preparation with the subgingival and equigingival finish line position. PATIENTS AND METHODS: The clinical prospective study included 20 subjects with an indication for upper canine preparation, with the subgingival (group 1) and equigingival finish line (group 2). Samples were taken in four observation intervals: 5 minutes before (control samples), as well as 15 minutes, 24 and 72 hours after tooth preparation (experimental samples). Measurement of MMP-9 was done using Enzyme-linked Immunosorbent Assay (ELISA). The presence of bacteria in the gingival fluid was proven by the Polymerase chain reaction (PCR) analysis. RESULTS: The MMP-9 values did not differ statistically significantly between the groups (p=0.524). The MMP-9 values showed a statistically significant difference in the given observation period (p<0.001) with a significant linear increase in values (p<0.001). A significant quadratic trend recorded a decrease in the MMP-9 values 15 minutes after preparation, and an increase 24 hours after preparation, without a significant difference in the interaction between groups (p=0.392). After preparation, a significant difference in the presence of periodontopathogens was confirmed, i.e., a decrease in the presence of Prevotella intermedia (p=0.025) and Tannerella forsythia (p=0.016) in group 1, and an increase in the presence of Aggregatibacter actinomycetemcomitans in both groups (p=0.029, p=0.026). CONCLUSIONS: The study is a good basis for determining the influence of tooth preparation on gingival inflammation, with therapeutic (choice of preparation technique) and preventive significance regarding the protection of the periodontal tissue from possible iatrogenic damage. [ABSTRACT FROM AUTHOR]

Abstract: Objective: A clinical prospective study was designed to evaluate microbiological, cytomorphometric and clinical efficacy of low‐level laser therapy (LLLT) as an adjunct to periodontal therapy in the treatment of chronic periodontitis. Methods: Sixty subjects were included and randomly assigned into 2 groups: SRP (scaling root planning) group (n = 30) and LLLT + SRP group (n = 30). Clinical parameters were measured before intervention, after the fifth treatment, and after a month. All subjects received oral hygiene instructions and full‐mouth conservative periodontal treatment (removal of dental plaque followed by SRP). Afterwards, in group II, Kavo LLLT (980 nm, 0.2 W, 6 J/cm2) was applied. Subgingival samples were collected at baseline and after the fifth treatment to quantify Aggregatibacter actinomycetemcomitans, Prevotella intermedia, Porphyromonas gingivalis, Tannerella forsythensis and Treponema denticola by polymerase chain reaction (PCR). Gingival swabs were taken, and direct smears were prepared on slides for cytomorphometric analysis. Results: Evaluation using clinical parameters showed better results in LLLT group. A statistically significant decrease in the prevalence of bacteria after treatment in LLLT group was observed for the following: T. forsythensis and T. denticola (P < .001), P. gingivalis (P < .01), A. actinomycetemcomitans and P. intermedia (P < .05). The values of nuclear area, perimeter and Ferret's diameter were significantly lower in both studied groups after treatment, but statistical significance was higher in LLLT group (P < .001) than in the SRP therapy group (P < .05). Conclusion: Low‐level laser therapy as an adjunct to periodontal therapy demonstrates short‐term additional bacteriological, cytological and clinical benefits. [ABSTRACT FROM AUTHOR]

Introduction/Objective. The objective of the study was to investigate whether there are differences in therapeutic effect between initial treatments of chronic periodontitis [scaling and root planning (SRP)] alone and SRP in conjunction with injectable platelet-rich fibrin (I-PRF) application, comparing clinical parameters after three months. Methods. Twenty-four patients with chronic periodontitis who had at least two sites with probing pocket depth (PPD) ≥ 5 mm on contralateral side participated in the study. Using a split-mouth design, the patients were treated with SRP + I-PRF (study group) or SRP only (control group). The clinical parameters, clinical attachment level (CAL), gingival margin level (GML), PPD, bleeding on probing, and plaque index, were recorded on both sides. Results. Compared to baseline, both treatment modalities demonstrated an improvement in investigated clinical parameters. The mean value of CAL was reduced from 1.97 ± 0.75 (0.25–3.31) to 1.07 ± 0.44 (0.12–1.78) in the study group, whereas it decreased from 1.81 ± 0.66 (0.42–2.96) to 1.48 ± 0.55 (0.22–2.30) in the control group. Similarly, the corresponding values for GML and PPD showed statistically significant difference between the groups (p = 0.040 and p = 0.006, respectively). Conclusion. Regardless the limited number of patients in the study, initial periodontal therapy in conjunction with injectable platelet-rich fibrin proved to display significant improvement in all clinical parameters compared to initial periodontal therapy alone.

Background/Aim: Current approaches in therapy of head and neck cancers are surgery, radiotherapy and chemotherapy. However, recurrence, development of multidrug resistance, side effects, and high costs of therapy are significant problems which point to the need for more efficient and less toxic drugs and interventions.

Introduction/Objective. The purpose of this study was to compare polypropylene and silk suture materials in terms of bacterial adherence and clinical features including the impact on soft tissue healing. Methods. Ten healthy patients were included in this study. Unilateral upper and lower wisdom teeth were extracted at the same time and wounds were sutured with different threads (one monofilament – polypropylene – and one multifilament – silk suture). Stitches were removed seven days postoperatively. Real-time polymerase chain reaction was used to analyze bacterial adherence. Intraoperative handling and ease of removal were assessed with the help of Visual Analogue Scale. Landry healing index was used for evaluation of soft tissue healing. Results. Significantly more pronounced bacterial adherence was found on silk compared to polypropylene sutures (p = 0.005). Superior intraoperative handling properties were registered suturing with polypropylene compared to silk (p = 0.005). Soft tissue healing was significantly better around polypropylene sutures, both on the third and the seventh postoperative day (p = 0.016). Patient discomfort was slightly higher for polypropylene sutures, but without statistical significance. Conclusion. Polypropylene suture material showed significantly lower bacterial adherence and superior clinical features compared to silk, including better soft tissue healing. [Project of the Serbian Ministry of Education, Science and Technological Development, Grant no. 175075]

Introduction. Periodontal diseases are associated with the presence of elevated levels of bacteria within the gingival crevice. Objective. The aim of this study was to evaluate a total amount of bacteria in subgingival plaque samples in patients with a periodontal disease. Methods. A quantitative evaluation of total bacteria amount using quantitative real-time polymerase chain reaction (qRT-PCR) was performed on 20 samples of patients with ulceronecrotic periodontitis and on 10 samples of healthy subjects. The estimation of total bacterial amount was based on gene copy number for 16S rRNA that was determined by comparing to Ct values / gene copy number of the standard curve. Results. A statistically significant difference between average gene copy number of total bacteria in periodontal patients (2.55.107) and healthy control (2.37.106) was found (p=0.01). Also, a trend of higher numbers of the gene copy in deeper periodontal lesions (>7 mm) was confirmed by a positive value of coefficient of correlation (r=0.073). Conclusion. The quantitative estimation of total bacteria based on gene copy number could be an important additional tool in diagnosing periodontitis. [Projekat Ministarstva nauke Republike Srbije, br. 175075]

Introduction Caries is one of the most significant and widespread oral diseases. It has been confirmed that dental plaque, i.e. microorganisms in it, are the most important factor in the development of dental caries. Caries profunda represents deep carious lesion from where bacterial toxins may affect pulp through dentinal tubules. The aim of this study is to assess the efficacy of indirect pulp capping based on microbiological findings of bacteria present in deep carious lesions before and after the treatment. Material and Methods The clinical study included 29 patients of both genders, aged 16 to 40 and 45 permanent teeth with deep caries lesions. The first microbiological sample was taken after cavity preparation and removal of soft dentin from the bottom of the cavity. The second sample was taken after the removal of temporary filling and calcium hydroxide paste 60 days after the indirect pulp capping treatment. The collected samples were stored in special sterile micro tubes (Eppendorf) and kept at the temperature of -80°C until microbiological analysis was performed. Samples were tested for the presence of the following microorganisms: Porphyromonas gingivalis, Aggregatibacter actinomycetemcomitans and Enterococcus faecalis using the multiplex polymerase chain reaction (PCR) method. Results The results showed that prior to the treatment of deep carious lesions the most common species was E. faecalis (80% of samples), followed by A. actinomycetemcomitans (32% of samples), while the least common was P. gingivalis (16% of samples). After the treatment with products based on calcium hydroxide, E. faecalis was registered in 18% of samples, A. actinomycetemcomitans in 16% of samples and P. gingivalis was not registered in any sample. Conclusion The most common bacterial species in teeth with deep caries lesions was E. faecalis, whereas A. actinomycetemcomitans and P. gingivalis were found in lower number of samples.

Introduction Deep caries is a reversible process where caries lesion has affected bigger part of dentin and only thin layer of softened dentin that separates lesion from the pulp is remained. The objective of this study was to identify and determine serotypes of Aggregatibacter actinomycetemcomitans in teeth with deep caries lesions at the beginning of their treatment. Material and methods Clinical research included 29 patients of both genders, aged 16 to 40 and 45 permanent teeth with diagnosed deep caries lesions based on medical history, clinical and radiographic examination. After cavity preparation and removal of softened dentin, microbiological swab was taken from the bottom of the cavity. Swabs were disposed in special sterile micro tubes and stored at the temperature of -80oC until serotyping was done (determination of serotypes of A. actinomycetemcomitans bacterium). Results In one of the 3 samples two serotypes of A. actinomycetemcomitans (b and c) were identified which is relatively rare finding, while in the second and third sample serotypes (a) and serotype (b) was identified, respectively. Conclusion In the three samples the 3 serotypes were found (a, b and c) and one of the samples was carrying even two different serotypes, which is a rare phenomenon. For more serious epidemiological study of A. Actinomycetemcomitans serotypes at the population level incomparably larger starting material is necessary, at least few hundred of samples.

Introduction. Periodontitis is a destructive inflammatory disease of the tooth-supporting tissues, primarily caused by Gram-negative microorganisms. Thus, the primary objective of cause-related initial periodontal therapy is disruption and removal of the subgingival biofilm. Objective. The aim of this study was to evaluate the clinical and microbiological effects of the initial therapy in patients diagnosed with chronic periodontitis. Methods. Forty patients with chronic periodontitis were included in the study. As a part of the clinical assessment undertaken prior to the initial therapy, as well as one month and three months post-therapy, plaque index, gingival index, papilla bleeding index, probing pocket depth and clinical attachment level were recorded. Microbiological testing was performed prior to the initial therapy and three months after therapy. Polymerase chain reaction assays were used to determine the presence of Porphyromonas gingivalis, Tannerella forsythensis, Prevotella intermedia and Aggregatibacter actinomycetemcomitans. Results. All clinical parameters were significantly reduced after therapy. The prevalence of Aggregatibacter actinomycetemcomitans was reduced by 22.5%, which was a statistically significant decrease compared to the baseline. The prevalence of Porphyromonas gingivalis, Tannerella forsythensis and Prevotella intermedia tended to decrease after therapy; however, the difference did not reach statistical significance. Conclusion. The results of the present study demonstrated the beneficial effects of the initial periodontal therapy on both the clinical and microbiological parameters. [Projekat Ministarstva nauke Republike Srbije, br. 175075]

Introduction. Pathogenesis and some characteristics of periodontitis cannot be fully explained by bacterial etiology alone. Herpes viruses may bridge the gap between clinical characteristics and molecular understanding of periodontal destruction. Objective. The aim of this study was to investigate the prevalence of herpes simplex virus type 1 (HSV-1) in gingival crevicular fluid (GCF) of healthy and damaged periodontium in Serbian population and to explore potential correlation between the presence of this virus and the level of periodontal destruction. Methods. Samples were collected from gingival sulcus/periodontal pockets by sterile paper points and the presence of viral DNA in gingival crevicular fluid was assessed by PCR. Results. There was no statistically significant difference in HSV-1 in presence between periodontitis patients (PG=38.9%) and healthy controls (HC=32.3%), (Chi-square test, with Yates’ correction p=0.7574). However, HSV-1 positive patients showed significantly higher values of parameters of periodontal destruction (PPD=7.11±2.52, CAL=5.46±2.34) than periodontitis patients without HSV-1 in gingival crevicular fluid (PPD=4.70±1.79, CAL=3.39±2.65) (p values respectively, p=0.002 and p=0.023, Independent Samples T-Test). HSV-1 occurred more often in deeper (PPD≥6 mm) (69.2%) than in shallow pockets (3 mm

Introduction. Clinically acceptable antiseptic should possess organolithic-mineralolithic properties and antimicrobial efficacy, and should be non-toxic. Objective. The aim of the paper was to assess the presence of genomes of the most common microorganisms (Porphyromonas gingivalis, Agregatibacter actinomycetemcomitans, Tanerella forsythensis, Prevotella intermedia, Treponema denticola and Enterococcus faecalis) in infected tooth root canals before and after rinsing with solution of doxycycline, citric acid and detergent Tween-80 (MTAD) in patients with clinically diagnosed primary apex periodontitis. Methods. The content of primarily infected canals before and after using the MTAD solution was used as a biological material in which the presence of microorganisms DNA was proved. For the detection of bacterial genome the multiplex PCR technique was applied. Results. The percentage of positive samples before canal treatment was 100%. In infected root canals E. faecalis was most dominant (37%). In a relatively high percentage we detected P. intermedia (25%), A. actinomycetemcomitans (20%), T. denticola (17%), T. forsythensis (15%) and P. gingivalis (10%). After rinsing the canal system using MTAD solution, there was a statistically significant decrease in E. faecalis (12%), P. intermedia (0%), T. forsythensis (0%) and P. gingivalis (0%). The presence of other bacteria was also diminished but not statistically significantly. Conclusion. With the application of multiplex PCR technique which provided a simultaneous amplification of various genomic sequences, using several pairs of primers, the most dominant in infected root canals were E. faecalis. P. intermedia, A. actinomycetemcomitans, T. denticola, T. forsythensis and P. gingivalis. After mechanic treatment and irrigation of root canals with MTAD solution, P. intermedia, P. gingivalis and T. forsythensis were not found. The presence of E. faecalis, A. actinomycetemcomitans and T. denticola was diminished, however, not statistically significantly.

Background: Genetic studies of salivary gland neoplasms were mainly focused on chromosomal changes, and some specific patterns of chromosome translocations have been described. However, molecular alterations, in particular the role of HER-2/H-ras/c-myc signalling cascade in pleomorphic adenoma pathogenesis (PA), are less well characterized. In addition, data on single nucleotide polymorphisms (SNPs) as potential susceptibility factors for PA development are also quite scarce. Methods: Mutational analyses were performed by means of real-time PCR (HER-2 and c-myc amplification analysis), PCR-SSCP and sequencing (H-ras point mutation detection). Polymorphisms analysis was performed by PCR-RFLP (survivin and MMP-9 genes). Results: Amplification of HER-2 and c-myc has been found in 13% and 9% of PA cases respectively. Point mutations in H-ras codons 12/13 have been detected in 17% of PAs. No correlation could be established between these alterations and clinical characteristics of PAs, whereas they might play a role in a subset of malignant salivary gland tumours. As for survivin -31 G/C polymorphism, C allele carriers had a 4-fold decrease of the risk of developing PA (p=0.05). Carriers of the variant allele T of the -1562C/T SNP in MMP-9 gene had a 4-fold increase of the risk of developing PA (p

Background: Head and neck squamous cell carcinoma, including oral cancer, is the sixth most common cancer worldwide. Despite advances in surgery and treatment, the 5-year survival rate has not improved significantly. Therefore, reliable molecular markers for oral cancer progression are badly needed. Methods: We conducted a copy number analysis to estimate amplification status of c-myc, cycD1 and EGFR oncogenes, mutational PCR-SSCP analysis to determine activation of H-ras oncogene and inactivation of TP53 tumour suppressor gene and methylation specific PCR analysis to evaluate hypermethylation of p16 and MGMT genes. Results: c-myc oncogene was amplified in 56.7%, cycDI in 20% and EGFR in 16.7% of Oral Squamous Cell Carcinoma (OSCC) cases while H-ras was activated in 33.3% of samples. Amplification of c-myc was significantly associated with the tumour grade 2. Interestingly, EGFR and H-ras alterations were mutually exclusive. p16 and MGMT were inactivated by hypermethylation in 30% and 13.3% of cases. Co-alteration of cycDI and p16 were not observed in any of the analyzed samples. TP53 was inactivated in 56.7% of samples and was significantly associated with progression of OSCC, grade 2 and stage 2. Moreover, TP53 and c-myc oncogene were simultaneously altered in grade 2 OSCC. Conclusions: The most promising marker of OSCC progression remains the TP53 tumour suppressor, which is the most frequently mutated gene in oral cancers. Since there is synergism between TP53 and c-myc, it seems that co-alteration of these two genes could be also a good marker of OSCC progression from grade1 to grade 2 tumours.

Tumor necrosis factor (TNF) which exerts its effects through two different receptors known as TNF-R1 and TNF-R2, is a major proinflammatory cytokine involved in the pathogenesis of different types of tumors. We have investigated whether polymorphisms in TNF-alpha (-308G>A), TNF receptor 1 (36A>G) and TNF receptor 2 (676T>G) genes modulate the susceptibility for oral squamous cell carcinomas (OSCCs) and basal cell carcinomas (BCCs) of the skin, two frequent types of head and neck cancers. Genotyping was done on 50 OSCC patients, 50 BCC patients and 60 healthy individuals, using PCR/RFLP. A significant difference in genotype and allele frequencies was found between patients and controls for the TNF-R2 polymorphism, in both OSCC and BCC. There was no statistically significant difference between patients and controls for TNF-alpha and TNF-R1 polymorphisms. Carriers of G allele had an approximately 2.5- and 5-fold higher risk for OSCC and BCC, respectively, in the Serbian population.

Myelodysplastic syndromes (MDS) are rare in children and only a few studies have analyzed their molecular mechanisms. The NPM1 gene encodes for nucleophosmin (NPM) which regulates hematopoiesis. Mutations in exon 12 of the NPM1 cause the nucleophosmin cytoplasmic dislocation and disrupt its functions. We have analyzed mutations of the NPM1 gene in archival bone marrow samples from 17 children with MDS and detected, in one patient, transition C to T in codon 293. To the best of our knowledge, this is the first analysis of NPM1 mutations in childhood MDS and the very first missense mutation of the NPM1 gene reported so far.

Dentin provides a protective enclosure for genomic and mitochondrial DNA. In the present study, DNA was obtained from pulverized or ground teeth. The quality of the DNA extracted from the teeth of 70 unrelated individuals was tested in the context of assessing the allelic and genotypic frequencies of autosomal loci D19S216, D20S502 and D20S842, and calculating a number of parameters of population genetics and forensic interest. This study illustrates that teeth can be a convenient tissue to extract DNA from large numbers of individuals for population genetic studies as well as for forensic case work.

Oral squamous cell carcinomas (OSCCs) are associated with poor prognosis, and despite advances in therapy approaches, no major improvement in survival has been achieved in the recent years. Efforts are now directed toward finding new biological markers that could predict tumor behavior more accurately. OSCCs, as the majority of malignant tumors, arise from progressive accumulation of genetic and epigenetic lesions, transforming normal cells into malignant. In this paper, an analysis of current studies directed to understanding the underlying mechanisms of OSCC pathogenesis was presented. The emphasis was put on mutational analysis of cancer genes, as well as on the role of viral infections and methylation processes in OSCC. Finally, an overview of studies that tried to determine the possibility for developing OSCC was given.

Introduction/Aim. Gingivitis is a common occurrence in children and may well be thought as a risk factor for the appearance and progression of the diseases of parodontal tissues. It is thus necessary to react in a timely and adequate fashion to prevent the disease to become serious and produce parodontopathy. The aim of the study was to establish the efficacy of hyaluronic acid in the treatment of chronic gingivitis in children. Methods. The study enrolled 130 children with permanent dentition. All of the examinees were divided into three groups: group I - 50 patients with chronic gingivitis in which only the basic treatment was applied; group II - 50 patients with chronic gingivitis in which hyaluronic acid was applied in addition to basic treatment; group III - 30 examinees with healthy gingiva (control group). Assessment of oral hygiene and status of the gingiva and parodontium was done using the appropriate indexes before and after the treatment. Inflammation of the gingiva was monitored by way of cytomorphometric studies. Results. The pretreatment values of the plaque index (PI) were high: in the group I PI was 1.94; in the group II PI was 1.68. After the treatment, the PI value was reduced to null in both groups (PI = 0). In the group III PI was 0.17. The bleeding index (BI) in the group I was 2.02 before and 0.32 after the treatment; the BI value in the group II was 1.74 before and 0.16 after the treatment. In the group III BI was 0. In the group I, the Community Periodontal Index of Treatment Needs (CPITN) was 1.66 before and 0.32 after the treatment; in the group II, the CPITN value was 1.5 before and 0.24 after the treatment. In the group III, the CPITN value was 0. In the group I, the size of the nuclei of the stratified squamous epithelium of the gingiva was reduced, although not so much as the nuclear size in the group II of examinees. Conclusion. Basic treatment is able to successfully treat chronic gingivitis in children. The use of hyaluronic acid together with the basic treatment can markedly improve the treatment effect.

Background/Aim. Periodontitis is a chronic inflammatory disease of periodontal tissues with consequential is bone loss as a result of host immunological reactions caused by periopathogens. The aim of the study was to investigate if there is a correlation between clinical parameters and the presence of two most aggressive periopathogens (Aggregatibacter actinomycetemcomitans - Aa and Porphyromonas gingivalis - Pg) in patients with progressive periodontal lesions. Methods. A total of 34 systemic healthy people, 23 to 70 years old, were included in the study. The patients were clinically and radiologically examined, and after that, the representative pocket with greatest pocket depth was chosen and the sample was collected from that place. The measured clinic parameters were: gingival index, index of gingival bleeding, pocket depth and plaque indices. The multiplex Polymerase Chain Reaction (PCR) method was used for detection of periopathogens. After obtaining results, appropriate statistical tests were used to correlate the clinical and microbiological results. Results. Aa and Pg were detected in the same percentage of samples. Aa and Pg were detected in 35.29% samples alone, and in 29.41% both were detected. The values of measured clinical parameters did not show a statistical significance between the groups. In analysis of correlations among clinical parameters inside the groups, a statistical significance was found only between gingival and plaque index in the group with Aa. Conclusion. Clinical course of periodontitis in the developed stage does not differ in relation to the presence of different periopathogens as the major inductors of immunologically guided destructive processes.

Introduction. Short tandem repeats and more specifically microsatellites represent a powerful tool in forensic medicine. In the past years, they have been extensively used in human identification and paternity testing. Objective The aim of the present study was to analyze two microsatellite markers in the Serbian population, i.e. to determine the number of alleles and the relevant forensic parameters. Methods. DNA was isolated from teeth samples using standard proteinase K digestion and phenol/chloroform alcohol extraction. PCR products were analyzed on polyacrilamide gels and visualized by AgNO3 staining. Forensic parameters were calculated using the Cervus software. Results. The loci D18S70 and D20S116 were analyzed on a sample of 70 unrelated, healthy adult individuals from Serbia. The number of alleles was determined and Hardy Weinberg equilibrium was confirmed for both loci. D18S70 and D20S116 demonstrated 6 and 8 alleles, respectively. The power of discrimination (PD) and the power of exclusion (PE) for the tested STR loci, D18S70 and D20S116 were 0.92 (PD), 0.41 (PE) and 0.95 (PD), 0.480 (PE), respectively. Conclusion. According to the presented data, D18S70 and D20S116 are most informative markers. Based on allelic frequencies and statistical parameters for forensic testing, it may be suggested that these two microsatellites represent useful markers for individual identification and parentage analysis in the Serbian population.

Introduction. According to clinical and epidemiological studies, ovarian cancer ranks fifth in cancer deaths among women. The causes of ovarian cancer remain largely unknown but various factors may increase the risk of developing it, such as age, family history of cancer, childbearing status etc. This cancer results from a succession of genetic alterations involving oncogenes and tumour suppressor genes, which have a critical role in normal cell growth regulation. Mutations and/or overexpression of three oncogenes, c-erbB-2, c-Myc and K-ras, and of the tumour suppressor gene p53, have been frequently observed in a sporadic ovarian cancer. Objective. The aim of the present study was to analyze c-Myc and c-erbB-2 oncogene alterations, specifically amplification, as one of main mechanisms of their activation in ovarian cancers and to establish a possible association with the pathogenic process. Methods. DNA was isolated from 15 samples of malignant and 5 benign ovarian tumours, using proteinase K digestion, followed by phenol-chloroform isoamyl extraction and ethanol precipitation. C-Myc and c-erbB-2 amplification were detected by differential PCR. The level of gene copy increase was measured using the Scion image software. Results. The amplification of both c-Myc and c-erbB-2 was detected in 26.7% of ovarian epithelial carcinoma specimens. Only one tumour specimen concomitantly showed increased gene copy number for both studied genes. Interestingly, besides amplification, gene deletion was also detected (26.7% for c-erbB-2). Most of the ovarian carcinomas with alterations in c-Myc and c-erbB-2 belonged to advanced FIGO stages. Conclusion. The amplification of c-Myc and c-erbB-2 oncogenes in ovarian epithelial carcinomas is most probably a late event in the pathogenesis conferring these tumours a more aggressive biological behaviour. Similarly, gene deletions point to genomic instability in epithelial carcinomas in higher clinical stages as the result of clonal evolution and selection.

INTRODUCTION Periodontitis is an inflammatory disease of the supporting tissues of teeth and is a major cause of tooth loss in adults. The onset and progression of periodontal disease is attributed to the presence of elevated levels of a consortium of pathogenic bacteria. Gram negative bacteria, mainly strict anaerobes, play the major role. OBJECTIVE The present study aimed to assess the presence of the main types of microorganisms involved in the aetiopathogenesis of periodontal disease: Porphyromonas gingivalis, Aggregatibacter actinomycetemcomitans, Eikenella corrodens, Treponema denticola, Tanerella forsythia and Prevotella intermedia in different samples collected from the oral cavity of 90 patients diagnosed with periodontitis. METHOD Bacterial DNA detection was performed in diverse biological materials, namely in dental plaque, gingival tissue and saliva, by means of multiplex PCR, a technique that allows simultaneous identification of two different bacterial genomes. RESULTS In the dental plaque of the periodontitis patients, Treponema denticola dominated. In the gingival tissue, Tannerella forsythia and Treponema denticola were the microbiota most frequently detected, whilst in saliva Treponema denticola and Eikenella corrodens were found with the highest percentage. CONCLUSION The identification of microorganisms by multiplex PCR is specific and sensitive. Rapid and precise assessment of different types of periodontopathogens is extremely important for early detection of the infection and consequently for the prevention and treatment of periodontal disease. In everyday clinical practice, for routine bacterial evaluation in patients with periodontal disease, the dental plaque is the most suitable biological material, because it is the richest in periodontal bacteria.

Background/Aim. Graves-Basedow disease is a common multifactorial genetic syndrome, which is determined by several genes and environmental factors. The aim of the present study was to investigate the presence of risk factors for developing Graves-Basedow disease between the groups of individuals with and without Graves-Basedow disease, and to compare the presence of risk factors between the affected individuals with or without positive family history for Graves- Basedow disease. Methods. This cross-sectional study was conducted in Ćuprija (central Serbia) during a period from December 2001 to April 2002. The case group comprised 132 individuals diagnosed with Graves-Basedow disease. The control group comprised 130 subjects without any of endocrine diseases. All participants were interviewed at the Medical Center Ćuprija using structural questionnaire. Data were collected on basic demographic characteristics, exposure to various chemical and physical agents, stress, smoking and family history of Graves-Basedow disease. In statistical analysis chi-square test was used. Results. The individuals with Graves-Basedow disease were statistically significantly older (above 50) (p = 0.020), exposed to stress (p = 0.024) and to physical agents (p = 0.031), and had significantly (p = 0.000) more relatives with Graves-Basedow disease than those without the disease. Among the affected individuals with positive family history of Graves-Basedow disease the number of women was significantly higher (p = 0.000), than the affected individuals without positive family history of Graves- Basedow disease. Conclusion. In our study, as in many other, gender, age, positive family history of Graves-Basedow disease and exposure to physical agents were identified as potential risk factors for the increased incidence of Graves- Basedow disease. Different risk factors are probably responsible for developing Graves-Basedow disease among the affected individuals with or without positive family history of Graves-Basedow disease. .

Aim: The aim of this study was to analyze the presence of the anti-apoptotic protein bcl-2 in oral squamous cell carcinoma and determine its potential role in the development and progression of this type of tumor. Materials and methods: The expression of bcl-2 was determined in 28 paraffin blocks of oral squamous cell carcinoma using the immunohistochemical method. The percentage of the immuno-reactive cells in positively stained tumor regions was determined using the microscopic analysis and Ozaria software. Results: Positive immunohistochemical test was observed in 19 out of 28 samples (68%) as follows: in 11 samples there was a low (+), in four a moderate (++) and in the last four a high percentage (+++) of stained cells. In the group of patients at the low stage of the disease (T2), 50% of tumor samples showed bcl-2 protein expression whereas in the higher stages (T3 and T4) of positively stained samples, this percentage was 67%. There was a trend of an increasing number of cells with positive bcl-2 staining in the tumors of higher clinical stages but not the level of bcl-2 protein expression. Conclusion: Both parameters, the presence of bcl-2 staining and the percentage of cells with bcl-2 immunoexpression, may act as additional prognostic parameters that indicate an increased proliferative tumor potential.

Although myc genes are an important family of oncogenes involved in human carcinogenesis, data concerning its role in head and neck tumors are quite limited. Moreover, information regarding precancerous lesions is almost inexisting. In order to better understand mechanisms of progression of premalignant lesions into malignant and the possible role of c-myc amplification, the status of this oncogene was analyzed in two types of pathological changes known to possess malignant transformation potential oral leukoplakia and chronic laryngitis. In the first type of lesions a high percentage of c-myc activation (33%) was observed pointing to a probable role of myc in leukoplakia malignant transformation, while in the other type of lesion there were no signs of myc amplification.

Liver phospholipid fatty acid composition depends on the dietary lipid intake and the efficiency of hepatic enzymatic activity. Our study aimed to simultaneously investigate the liver phospholipid fatty acid composition in response to chronic linseed, palm, or sunflower oil diets. We used adult female C57/BL6 mice and randomly divided them into control and three groups treated with 25 % dietary oils. Prior to treatment, we analyzed the fatty acid profiles in dietary oils and hepatocytes and, after 100 days, the fatty acid composition in the liver using gas-liquid chromatography. Linseed oil treatment elevated alpha-linolenic, eicosapentaenoic, and docosapentaenoic acids and reduced arachidonic and docosatetraenoic acids, consequently lowering the n-6/n-3 ratio. Palm oil treatment increased linoleic acid and decreased docosahexaenoic acid, contributing to an elevated n-6/n-3 ratio. Sunflower oil treatment elevated total monounsaturated fatty acids by increasing palmitoleic, oleic, and vaccenic acids. The estimated activity of Δ9 desaturase was significantly elevated in the sunflower oil group, while Δ5 desaturase was the highest, and Δ6 desaturase was the lowest after the linseed oil diet. Our findings demonstrate that chronic consumption of linseed, palm, or sunflower oil alters the distribution of liver phospholipid fatty acids differently. Sunflower oil diet elevated total monounsaturated fatty acids, proposing potential benefits for liver tissue health. Considering these outcomes, a substantial recommendation emerges to elevate linseed oil intake, recognized as the principal ALA source, thereby aiding in reducing the n-6/n-3 ratio. Moreover, modifying dietary habits to incorporate specific vegetable oils in daily consumption could substantially enhance overall health., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Despite advancements in treatment, the squamous cell carcinoma (OSCC) patient survival rate remains stagnant. Conventional therapies have limited effectiveness, necessitating novel agents. Our study aims to synthesize and characterize amorphous calcium phosphate nanoparticles (nACPs), assess their potential cytotoxic effects on premalignant and malignant OSCC cells, and investigate possible mechanisms of action. The morphological features of nACP were investigated by field emission scanning coupled with energy dispersive spectroscopy (EDS), Fourier transform infrared spectroscopy (FTIR), and particle size distribution (PSD). Then, we examined the effect of nACPs on nanoparticle uptake, cell adhesion, viability, invasion ability, cell cycle, and gene expression. nACP uptake was dose-dependent, induced limited selectivity in cytotoxicity between healthy and malignant cells, and affected cellular adhesion and invasion. Early apoptosis was the predominant type of cell death. The nACP effect on viability was verified by alterations in the genes associated with apoptosis and proliferation. A high concentration of nACP was shown to arrest the cell cycle progression in the G0/G1 phase of both malignant and premalignant cells. This type of nACP justifies the development of a strategy for its potential use as an anti-cancer agent and/or anti-cancer active carrier for various drugs in oral cancer treatments.

Objectives: In order to ensure improved and accelerated bone regeneration, nano-hydroxyapatite scaffolds are often enriched with different bioactive components to further accelerate and improve bone healing. In this review, we critically examined whether the enrichment of nHAp/polymer scaffolds with growth factors, hormones, polypeptides, microRNAs and exosomes improved new bone formation in vivo., Materials and Methods: Out of 2989 articles obtained from the literature search, 106 papers were read in full, and only 12 articles met the inclusion criteria for this review., Results: Several bioactive components were reported to stimulate accelerated bone regeneration in a variety of bone defect models, showing better results than bone grafting with nHAp scaffolds alone., Conclusions: The results indicated that composite materials based on nHAp are excellent candidates as bone substitutes, while nHAp scaffold enrichment further accelerates bone regeneration. The standardization of animal models should be provided in order to clearly define the most significant parameters of in vivo studies. Only in this way can the adequate comparison of findings from different in vivo studies be possible, further advancing our knowledge on bone regeneration and enabling its translation to clinical settings.

Type 2 diabetes mellitus (T2DM) is associated with functional deterioration of the salivary gland and dental pulp, related to oxidative stress. The aim was to integrate experimental and bioinformatic findings to analyze the cellular mechanism of melatonin (MEL) action in the human parotid gland and dental pulp in diabetes. Human parotid gland tissue was obtained from 16 non-diabetic and 16 diabetic participants, as well as human dental pulp from 15 non-diabetic and 15 diabetic participants. In human non-diabetic and diabetic parotid gland cells (hPGCs) as well as in dental pulp cells (hDPCs), cultured in hyper- and normoglycemic conditions, glial cell line-derived neurotrophic factor (GDNF), MEL, inducible nitric oxide synthase (iNOS) protein expression, and superoxide dismutase (SOD) activity were measured by enzyme-linked immunosorbent assay (ELISA) and spectrophotometrically. Bioinformatic analysis was performed using ShinyGO (v.0.75) application. Diabetic participants had increased GDNF and decreased MEL in parotid ( p < 0.01) and dental pulp ( p < 0.05) tissues, associated with increased iNOS and SOD activity. Normoglycemic hDPCs and non-diabetic hPGCs treated with 0.1 mM MEL had increased GDNF ( p < 0.05), while hyperglycemic hDPCs treated with 1 mM MEL showed a decrease in up-regulated GDNF ( p < 0.05). Enrichment analyses showed interference with stress and ATF/CREB signaling. MEL induced the stress-protective mechanism in hyperglycemic hDPCs and diabetic hPGCs, suggesting MEL could be beneficial for diabetes-associated disturbances in oral tissues.

We appraised the relationship between the biological and the chronological age and estimated the rate of biological aging in HIV-infected patients. Two independent biomarkers, the relative telomere length and iron metabolism parameters, were analyzed in younger (<35) and older (>50) HIV-infected and uninfected patients (control group). In our control group, telomeres of younger patients were significantly longer than telomeres of older ones. However, in HIV-infected participants, the difference in the length of telomeres was lost. By combining the length of telomeres with serum iron, ferritin, and transferrin iron-binding capacity, a new formula for determination of the aging process was developed. The life expectancy of the healthy population was related to their biological age, and HIV-infected patients were biologically older. The effect of antiretroviral HIV drug therapies varied with respect to the biological aging process. IMPORTANCE This article is focused on the dynamics of human aging. Moreover, its interdisciplinary approach is applicable to various systems that are aging., Competing Interests: The authors declare no conflict of interest.

Due to the unique functional properties of shape memory alloys (SMAs) and current scientific interest in Cu-containing biomaterials, a continuously cast Cu-Al-Ni alloy in the form of rods has been investigated as a potential candidate for biomedical application. Additionally, the fact that Cu- complexes have an antitumour effect served as a cornerstone to develop more efficient drugs based on trace element complexes. In line with that, our study aimed to analyse the basic properties of the Cu-Al-Ni alloy, along with its anticancer properties. The detailed chemical analysis of the Cu-Al-Ni alloy was performed using XRF and SEM/EDX analyses. Furthermore, a microstructural and structure investigation was carried out, combined with hardness measurements using the static Vickers method. Observations have shown that the Cu-Al-Ni microstructure is homogeneous, with the presence of typical martensitic laths. XRD analysis confirmed the presence of two phases, β' (monoclinic) and γ' (orthorhombic). The viability of osteosarcoma cells in contact with the Cu-Al-Ni alloy was evaluated using epifluorescence microscopy, while their morphology and attachment pattern were observed and analysed using a high-resolution SEM microscope. Biocompatibility testing showed that the Cu-Al-Ni alloy exerted a considerable antineoplastic effect.

High elution and diffusion of 2-hydroxylethyl methacrylate (HEMA) and camphorquinone (CQ) through dentinal tubules may induce pulp injury and postoperative sensitivity. We aimed to investigate the melatonin protective effect in HEMA- and CQ-treated human dental pulp cells (hDPCs) as well as its relevance in a mechanism for postoperative sensitivity in diabetic patients. hDPCs were exposed to HEMA (5 mM) and/or CQ (1 mM) in the absence and presence of melatonin (MEL) (0.1 mM and 1 mM). Heme oxygenase-1 (HMOX1), NADPH oxidase-4 (NOX4), BCL-2-associated X-protein (BAX), B-cell lymphoma-2 (BCL-2) and caspase-3 (CASP3) gene expression levels, and superoxide dismutase (SOD) activity were measured in hDPCs while inducible nitric oxide synthase (iNOS) and melatonin protein expression were measured in human dental pulp as well, by RT-PCR, by ELISA, and spectrophotometrically. Bioinformatic analyses were performed by using the ShinyGO (v.0.75) application. Type 2 diabetic patients showed a higher incidence of postoperative sensitivity and lower melatonin and higher iNOS content in dental pulp tissue compared with non-diabetic patients. Melatonin, when co-added in hDPC culture, reverses HEMA and CQ cytotoxic effects via anti-apoptotic and anti-inflammatory/antioxidant iNOS-related effects. Enrichment analyses showed that genes/proteins, altered by HEMA and CQ and normalized by melatonin, are the most prominently overrepresented in type 2 diabetes mellitus pathways and that they share subcellular localization in different oligomeric protein complexes consisting of anti- and pro-apoptotic regulators. This is the first evidence of the ability of melatonin to counteract iNOS-mediated inflammatory and stress effects in HEMA- and CQ-treated hDPCs, which could be of significance for the modulation of presently observed immediate postoperative sensitivity after composite restoration in type 2 diabetic patients.

Nickel-titanium alloys used in dentistry have a variety of mechanical, chemical, and biofunctional properties that are dependent on the manufacturing process. The aim of this study was to compare the mechanical and biofunctional performances of a nickel-titanium alloy produced by the continuous casting method (NiTi-2) with commercial nitinol (NiTi-1) manufactured by the classical process, i.e., from remelting in a vacuum furnace with electro-resistive heating and final casting into ingots. The chemical composition of the tested samples was analyzed using an energy dispersive X-ray analysis (EDX) and X-ray fluorescence (XRF). Electron backscatter diffraction (EBSD) quantitative microstructural analysis was performed to determine phase distribution in the samples. As part of the mechanical properties, the hardness on the surface of samples was measured with the static Vickers method. The release of metal ions (Ni, Ti) in artificial saliva (pH 6.5) and lactic acid (pH 2.3) was measured using a static immersion test. Finally, the resulting corrosion layer was revealed by means of a scanning electron microscope (SEM), which allows the detection and direct measurement of the formatted oxide layer thickness. To assess the biocompatibility of the tested nickel-titanium alloy samples, an MTT test of fibroblast cellular proliferation on direct contact with the samples was performed. The obtained data were analyzed with the IBM SPSS Statistics v22 software. EDX and XRF analyses showed a higher presence of Ni in the NiTi-2 sample. The EBSD analysis detected an additional NiTi 2 -cubic phase in the NiTi-2 microstructure. Additionally, in the NiTi-2 higher hardness was measured. An immersion test performed in artificial saliva after 7 days did not induce significant ion release in either group of samples (NiTi-1 and NiTi-2). The acidic environment significantly increased the release of toxic ions in both types of samples. However, Ni ion release was two times lower, and Ti ion release was three times lower from NiTi-2 than from NiTi-1. Comparison of the cells' mitochondrial activity between the NiTi-1 and NiTi-2 groups did not show a statistically significant difference. In conclusion, we obtained an alloy of small diameter with an appropriate microstructure and better response compared to classic NiTi material. Thus, it appears from the present study that the continuous cast technology offers new possibilities for the production of NiTi material for usage in dentistry.

Chronic periodontitis is a common complication in diabetes. The aim of this study was to evaluate some clinical and microbiological parameters in controlled and uncontrolled type 2 diabetes mellitus (type 2 DM) patients compared to non-diabetic (NDM) individuals, as well as to assess the effect of non-surgical periodontal therapy on these parameters. The study was performed in 61 type 2 DM patients with periodontitis (group 1A: 29 patients having achieved good metabolic control, HbA1c <7%; group 1B: 32 patients with poor metabolic control, HbA1c ≥7%), and 31 NDM individuals suffering from periodontitis. Periodontal indices (plaque index, PI; gingival index, GI; probing pocket depth, PPD; and clinical attachment level, CAL) were measured and subgingival plaque samples were analyzed using polymerase chain reaction prior to treatment initiation and 3 months post-treatment. The results recorded on the majority of measured parameters indicated that differences in treatment success achieved in the three treatment groups were not statistically significant (∆PI p=0.646; ∆GI p=0.303; and ∆CAL p=0.233). Likewise, comparison of the effectiveness in microorganism reduction revealed no significant differences between DM groups and NDM patients. Therefore, study results supported the hypothesis that periodontal therapy outcome was unaffected by the level of glycemic control in patients with diabetes.

Background: Components of the metabolic syndrome (MetS) play an important role in the accelerated aging process. Relative telomere length (RTL) is a marker of biological aging. The aim of our study was to determine RTL and its possible association with MetS and the components of MetS in HIV-infected patients treated with cART., Methods: We included 24 HIV-infected men, all Caucasians, with successful cART (<50 HIV-RNA copies/mL) and on stable cART for at least 24 months. The presence of MetS and its components was determined by the criteria prescribed by the International Diabetes Federation. RTL was determined by Real-Time PCR and ΔΔCt method. We performed a multiple linear regression modeling on log-transformed RTL (dependant variable) to evaluate which components of the metabolic syndrome as well as cART duration and cART type, had an impact on RTL., Results: Eleven (45.8%) patients had and 13 (54.2%) had not MetS. All patients, had an undetectable viral RNA and a relatively good immune status. The mean RTL was 0.62 ± 0.15 and 0.95 ± 0.36 in patients with and without MetS, respectively (p = 0.01). Multiple linear regression model showed no significant association between duration of cART, cART type and RTL (p = 0.2165, p = 0.8628, respectively). The same analysis showed that an increase in number of MetS components was associated with shorter telomere length (β = -0.4982, p = 0.042)., Conclusions: We showed for the first time association between RTL shortening in HIV-infected men with metabolic syndrome. Furthermore, our study also indicated that an increment of metabolic syndrome components is strongly associated with shorter telomere length., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Compromised dentition has been suggested to pose a significant risk factor for dementia. It was mainly investigated through insufficient tooth number, disregarding contact between opposing teeth (dental occlusion). The ɛ4 allele of apolipoprotein (APOE4) is the primary genetic marker for the late onset of Alzheimer's disease (AD). However, APOE4 and dental occlusion have not yet been investigated as possible associated risk factors for AD. The study was aimed to examine the impact of dental status and different APOE gene variants on AD occurrence. Secondly, sociodemographic variables were investigated as factors potentially associated with AD. The case-control study included two groups: 116 patients with AD (according to the NINDS-ADRDA criteria) and 63 controls (Mini-Mental State Examination scores ≥24). The analysis of APOE gene polymorphism was conducted through PCR reaction. Dental examination included recording of number of teeth, presence of fixed or removable dentures, and number of functional tooth units (FTU). Regression analysis was used to investigate the joint effect of the clinical and genetic variables on AD. Results showed that patients with AD were more often carriers of ɛ3/ɛ4 genotype and ɛ4 allele, had lower number of teeth and FTU, and were less likely to be married, live in home, and had less chronic diseases, compared to the controls. Regression analysis showed that presence of APOE4 allele and the number of total FTU remained associated with AD, even when adjusted for age, sex, and level of education. In conclusion, deficient dental occlusion and presence of APOE4 may independently increase risk for AD.

Aim: To characterize stem cells originating from different dental tissues (apical papilla [SCAP], dental follicle [DFSC], and pulp [DPSC]) and test the capacity of Raman microspectroscopy to distinguish between the three dental stem cell types., Methods: SCAP, DFSC, and DPSC cultures were generated from three immature wisdom teeth originating from three patients. Cell stemness was confirmed by inducing neuro-, osteo-, chondro-, and adipo-differentiaton and by mesenchymal marker expression analysis by flow-cytometry and real-time polymerase chain reaction. Cellular components were then evaluated by Raman microspectroscopy., Results: We found differences between SCAP, DFSC, and DPSC Raman spectra. The ratio between proteins and nucleic acids (748/770), a parameter for discriminating more differentiated from less differentiated cells, showed significant differences between the three cell types. All cells also displayed a fingerprint region in the 600-700 cm-1 range, and characteristic lipid peaks at positions 1440 cm-1 and 1650 cm-1., Conclusion: Although different dental stem cells exhibited similar Raman spectra, the method enabled us to make subtle distinction between them.

The importance of oral microflora composition in HIV-infected patients is well recognized. However, no studies so far have dealt with age-related changes in periodontal pathogens occurrence in HIV+ individuals. The aim of the present study was to assess and compare temporal changes of bacteria frequency in younger (≤35 years) and older (≥50 years) HIV-infected and non-infected individuals. Bacterial DNA was isolated from buccal swabs of 30 younger and 30 older subjects in both HIV+ and HIV- groups. By means of PCR the following microorganisms were detected: Aggregatibacter actinomycetemcomitans, Eikenella corrodens, Peptostreptococcus micros, Porphyromonas gingivalis, Prevotella intermedia, Tannerella forsythia and Treponema denticola. Oral and periodontal examinations were performed in all subjects. The prevalence of microorganisms was significantly higher in HIV+ patients compared to controls, and their distribution showed a notable shift. The decreasing incidence in HIV- subjects was: Pi>Pm>Pg>Aa>Ec>Tf>Td whilst in HIV+ it was: Pi>Pm>Ec>Pg>Tf>Aa>Td. Oral manifestations of HIV infection were more frequent in older compared to younger patients. All measured values of clinical periodontal parameters were significantly higher in older compared to younger HIV+ patients. Ageing in HIV+ subjects is accompanied with a substantial increase and rearrangements of periodontal microflora, potentially aggravating oral and systemic health.