Salivary Diagnosis of Dental Caries: A Systematic Review.

The activity of dental caries, combined with its multifactorial etiology, alters salivary molecule composition. The present systematic review was developed to answer the following question: "Are salivary biomarkers reliable for diagnosis of dental caries?". Following the "Preferred Reporting Item for Systematic Reviews and Meta-analysis" (PRISMA) guidelines, the review was conducted using multiple database research (Medline, Web of Science, and Scopus). Studies performed on healthy subjects with and without dental caries and providing detailed information concerning the clinical diagnosis of caries (Decayed, Missing, Filled Teeth-DMFT and International Caries Detection and Assessment System-ICDAS criteria) were included. The quality assessment was performed following a modified version of the Joanna Briggs Institute Prevalence Critical Appraisal Checklist. The protocol was registered in the International Prospective Register of Systematic Reviews (PROSPERO, ID: CRD42022304505). Sixteen papers were included in the review. All studies reported statistically significant differences in the concentration of salivary molecules between subjects with and without caries (p < 0.05). Proteins were the most investigated molecules, in particular alpha-amylase and mucins. Some studies present a risk of bias, such as identifying confounding factors and clearly defining the source population. Nevertheless, the 16 papers were judged to be of moderate to high quality. There is evidence that some salivary compounds studied in this review could play an important diagnostic role for dental caries, such as salivary mucins, glycoproteins (sCD14), interleukins (IL-2RA, 4,-13), urease, carbonic anhydrase VI, and urea.

The Association between Salivary Metabolites and Gingival Bleeding Score in Healthy Subjects: A Pilot Study.

Periodontal diseases, including gingivitis and periodontitis, are among the most prevalent diseases in humans. Gingivitis is the mildest form of periodontal disease, characterized by inflammation of the gingiva caused by the accumulation of dental plaque. Salivary diagnostics are becoming increasingly popular due to the variation in saliva composition in response to pathological processes. We used a metabolomics approach to investigate whether a specific saliva metabolic composition could indicate preclinical stage of gingivitis. 1H-NMR spectroscopy was used to obtain the salivary metabolite profiles of 20 healthy subjects. Univariate/multivariate statistical analysis evaluated the whole saliva metabolite composition, and the Full-Mouth Bleeding Score (FMBS) was employed as a classification parameter. Identifying a signature of specific salivary metabolites could distinguish the subjects with high FMBS scores but still within the normal range. This set of metabolites may be due to the enzymatic activities of oral bacteria and be associated with the early stages of gingival inflammation. Although this analysis is to be considered exploratory, it seems feasible to establish an FMBS threshold that distinguishes between the absence and presence of early inflammatory alterations at the salivary level.

Human Serum and Salivary Metabolomes: Diversity and Closeness.

Saliva, which contains molecular information that may reflect an individual's health status, has become a valuable tool for discovering biomarkers of oral and general diseases. Due to the high vascularization of the salivary glands, there is a molecular exchange between blood and saliva. However, the composition of saliva is complex and influenced by multiple factors. This study aimed to investigate the possible relationships between the salivary and serum metabolomes to gain a comprehensive view of the metabolic phenotype under physiological conditions. Using 1H-NMR spectroscopy, we obtained the serum metabolite profiles of 20 healthy young individuals and compared them with the metabolomes of parotid, submandibular/sublingual, and whole-saliva samples collected concurrently from the same individuals using multivariate and univariate statistical analysis. Our results show that serum is more concentrated and less variable for most of the shared metabolites than the three saliva types. While we found moderate to strong correlations between serum and saliva concentrations of specific metabolites, saliva is not simply an ultrafiltrate of blood. The intense oral metabolism prevents very strong correlations between serum and salivary concentrations. This study contributes to a better understanding of salivary metabolic composition, which is crucial for utilizing saliva in laboratory diagnostics.

Sample optimization for saliva 1H-NMR metabolic profiling.

Nuclear Magnetic Resonance (NMR) based metabolomic analysis of whole saliva has provided potential diagnostic biomarkers for numerous human diseases contributing to a better understanding of their mechanisms. However, a comprehensive interpretation of the significance of metabolites in whole, parotid, and submandibular/sublingual saliva subtypes is still missing. Precision and reproducibility of sample preparation is an essential step. Here, we present a simple and efficient protocol for saliva 1H-NMR metabolic profiling. This procedure has been specifically designed and optimized for the identification and quantification of low concentration metabolites (as low as 1.1 µM) and is suitable for all the saliva subtypes.

Salivary Cytokines as Biomarkers for Oral Squamous Cell Carcinoma: A Systematic Review.

The prognosis of patients with oral squamous carcinoma (OSCC) largely depends on the stage at diagnosis, the 5-year survival rate being approximately 30% for advanced tumors. Early diagnosis, including the detection of lesions at risk for malignant transformation, is crucial for limiting the need for extensive surgery and for improving disease-free survival. Saliva has gained popularity as a readily available source of biomarkers (including cytokines) useful for diagnosing specific oral and systemic conditions. Particularly, the close interaction between oral dysplastic/neoplastic cells and saliva makes such fluid an ideal candidate for the development of non-invasive and highly accurate diagnostic tests. The present review has been designed to answer the question: "Is there evidence to support the role of specific salivary cytokines in the diagnosis of OSCC?" We retrieved 27 observational studies satisfying the inclusion and exclusion criteria. Among the most frequent cytokines investigated as candidates for OSCC biomarkers, IL-6, IL-8, TNF-α are present at higher concentration in the saliva of OSCC patients than in healthy controls and may therefore serve as basis for the development of rapid tests for early diagnosis of oral cancer.

Salivary MicroRNA for Diagnosis of Cancer and Systemic Diseases: A Systematic Review.

: Background: The aberrant expression of microRNAs (miRNAs) has been associated with several diseases, including cancer, inflammatory, and autoimmune conditions. Interest in salivary miRNAs as non-invasive tools for the diagnosis of malignancies and systemic diseases is rapidly increasing. The present systematic review was developed for answering the question: "Are salivary microRNAs reliable biomarkers for diagnosis of cancer and systemic diseases?" METHODS: The application of inclusion and exclusion criteria led to the selection of 11 papers. Critical appraisals and quality assessments of the selected studies were performed through the National Institute of Health "Study Quality Assessment Tool" and the classification of the Oxford Center for Evidence-Based Medicine. RESULTS: Seven studies reported statistically significant correlations between one or more salivary miRNAs and the investigated disease. The critical analysis allowed us to classify only two studies (18.2%) as having "good" quality, the rest being scored as "intermediate" (8; 73%) and "poor" (1; 9%). Evidence exists that salivary miR-940 and miR-3679-5p are reliable markers for pancreatic cancer and that miR140-5p and miR301a are promising molecules for the salivary diagnosis of gastric cancer. CONCLUSIONS: Further studies, possibly avoiding the risk of bias highlighted here, are necessary to consolidate these findings and to identify new reliable salivary biomarkers.

Auto-Fluorescence and Histopathologic Evaluation of Medication-Related Osteonecrosis of the Jaws: Perspectives for Treatment.

Auto-fluorescence (AF) of healthy bone tissue has recently been described. Loss of AF (LAF) has, on the contrary, been reported in necrotic bone. Further, the use of LAF as a possible guidance to distinguish viable from necrotic bone during surgical treatment of osteonecrosis has been proposed. The aim of this study is to detail 8 patients of medication-related osteonecrosis of the jaws treated through an AF-guided surgical resection. The authors also provide the histopathologic description of hypo-fluorescent and hyper-fluorescent bone in each patient. After removal of necrotic bone block, Er:YAG laser was used for vaporizing further necrotic bone, up to the detection of strongly hyper-fluorescent bone. Samples of hyper-fluorescent bone were collected around areas of necrosis. Histopathologic evaluation revealed viable bone tissue in all hyper-fluorescent specimens. On the basis of these data, AF-guided surgical resection could be effective in highlighting surgical margins of necrotic bone tissue and it might have some utility in a range of applications of bone surgery.

Erbium Yttrium-Aluminum-Garnet Laser Versus Traditional Bur in the Extraction of Impacted Mandibular Third Molars: Analysis of Intra- and Postoperative Differences.

INTRODUCTION: Different osteotomy techniques have been proposed in order to improve postoperative course of impacted third molar extraction. The aim is to evaluate the possible advantages achieved with erbium yttrium-aluminum-garnet (Er:YAG) laser osteotomy compared with traditional burs. MATERIALS AND METHODS: Seventy-six extractions were randomly classified into 2 groups according to osteotomy instrument: group 1 (G1)-Er:YAG laser: 35 patients; group 2 (G2)-traditional bur: 41 patients. Intraoperative parameters: total time, stitches number, and patient compliance. Postoperative: pain, health-related quality of life (HR-QoL), need for analgesics, edema, trismus, intra- and extraoral hematoma, and postoperative complications. RESULTS: Mean time for G1 resulted 1069.4 seconds; for G2 1913.5 seconds (P < 0.0001). Mean number of stitches (P = 0.773) and patient compliance (P = 0.063) were not statistically different. Regarding pain, mean visual analog scale (VAS), and numeric rating scale (NRS) scores were lower in G1 than in G2. Statistically significant differences were highlighted at days 0, 1, and 3 with VAS scale and at days 0, 1, 3, and 7 with NRS scale. The HR-QoL scores resulted lower in G1 than in G2 (P < 0.0001). Mean facial swelling and trismus resulted statistically lower in G1 than in G2 at day 2 (P < 0.0001). Trismus resulted statistically lower in G1 than in G2 at days 2 (P < 0.0001) and 7 (P = 0.004). Two patients (5.71%) of subcutaneous emphysema was recorded in G1 and 2 patients (4.88%) of lip paresthesia in G2. CONCLUSION: Data confirm that the use of Er:YAG laser for osteotomy may achieve several advantages both technical and biological.

Clinical Differences in Autofluorescence Between Viable and Nonvital Bone: A Case Report With Histopathologic Evaluation Performed on Medication-Related Osteonecrosis of the Jaws.

Medication-related osteonecrosis of the jaws (MRONJ) is an adverse side effect of several drug therapies, including bisphosphonates (BPs). Osteonecrosis of the jaw specifically related to BP therapy is usually referred to using the acronym BRONJ. However, no consensus has yet been reached regarding the most appropriate management of BRONJ. The greatest success rates have been recorded with surgical removal of necrotic bone. In particular, erbium:yttrium-aluminum-garnet (Er:YAG) laser-assisted surgery has shown significantly better results than conventional surgical approaches. According to a position paper reported by the American Association of Oral and Maxillofacial Surgeons in 2007, the identification of necrotic bone margins during osteonecrosis removal can be very difficult. In 2015, a review of treatment perspectives for MRONJ reported that both surgical debridement and resection cannot be standardized owing to the lack of guidance to define the necrotic margins. Recently, the use of autofluorescence (AF) of the bone as a possible suitable guide to visualize necrotic bone during surgical debridement or resection was proposed. It seems that vital bone could be highlighted by its very strong AF. In contrast, necrotic bone loses AF and, thus, appears much darker. The molecular sources of the phenomenon of AF are the specific amino acids of the collagen molecules that show AF when irradiated by ultraviolet or blue light. The use of AF as an intraoperative diagnostic tool is entirely new in the management of MRONJ, although it has been used for several years in other fields (eg, intervertebral disc surgery). The aim of the present report was to describe a case of mandibular BRONJ treated with a new surgical approach performed with an Er:YAG laser and guided by AF. The histopathologic evaluation of the removed hypofluorescent bone block and hyperfluorescent surrounding bone has also been reported in detail.

Medication-Related Osteonecrosis of the Jaw Around Dental Implants: Implant Surgery-Triggered or Implant Presence-Triggered Osteonecrosis?

INTRODUCTION: Dentoalveolar surgery including tooth extractions and dental implants placement is considered the major risk factor for developing medication-related osteonecrosis of the jaw (MRONJ).In this study, a patient series of MRONJ around dental implants were carefully analyzed to describe the findings and to assess the possible risk factors. METHODS: Fifteen patients with peri-implant bone osteonecrosis were selected out of a group of 250 patients (6%). Patients were divided into 2 groups according to the temporal relationship. Group 1 (G1)-necrosis immediately after implant placement (from 2 to 10 months) and defined as "implant surgery-triggered" MRONJ. Group 2-necrosis distant (from 1 to 15 years) from implant placement and defined as "implant presence-triggered" MRONJ. Epidemiological and pharmacological variables were recorded as well as specific data about osteonecrosis and dental implants. RESULTS: G1 included 6 patients: 5 (83.4%) treated with oral bisphosphonates (BPs) for osteoporosis and 1 (16.6%) with intravenous BPs for breast cancer. Mean duration of BP therapy (BPT) was 83.7 months. G2 included 9 patients: 8 patients (88.89%) treated with intravenous BPs for malignant disease and 1 (11.11%) with oral BPs for osteoporosis. CONCLUSIONS: Data confirms that not only surgical insertion of dental implants is a potential risk factor for the development of osteonecrosis but also the presence itself of the implant into the bone can be associated with this disease. Therefore, it is necessary to inform of the increased risk for MRONJ also the patients who have already osteointegrated implants and are going to start the BPT.The risk is lower for patients receiving oral BPs but it exists and seems to be higher if the implant is located in the posterior areas, if the duration of BPT is more than 3 years and if the patient is under corticosteroid therapy.

Different laser wavelengths comparison in the second-stage implant surgery: an ex vivo study.

The implant surgery consists of two distinct techniques, the transmucosal, also known as "one-stage" and the "two-stage" technique. Lasers represent a possible aid in implant dentistry, especially in the two-stage technique and its main characteristics are represented by a decreased trauma to bone and soft tissues, a reduction of pain as well as a reduction of the risk of postoperative infections. The aim of this study was to analyze in an animal model the thermal elevation induced by four different laser wavelengths (diode, Nd:YAG, Er:YAG, KTP) during the implant uncovering. Four pig jaws were used to carry out this study. Five implants were placed in each anatomical specimen for a total of 20 fixtures. Four wavelengths (532, 810, 1,064 and 2,940 nm) were used to uncover the implants. Two thermocouples were used to measure temperature changes during laser irradiation at bone level, peri-implant tissues and on the fixture surface The thermocouples were connected with two probes of 1.5 mm in diameter, in order to simultaneously recording two temperature variations. Surface temperature was also checked during all procedures with a thermal camera (Thermovision A 800, Flyr Systems, Stockolm, Sweden) connected to a PC. The mean temperatures of each specimen (five fixtures) were calculated (TM1, mean temperature at the beginning; TM2, mean peak temperature). Furthermore, a record of the temperature at 1 min after the end of the surgical procedure was taken (mean: TM3). All the recorded values were statistically evaluated by one-way analysis of variance (ANOVA). The thermocouples recorded a lower increase in temperature for Er:YAG and KTP laser; Nd:YAG and diode laser produced similar increases characterized by higher values. The thermo-camera pointed out the lower increase for Er:YAG and higher for diode laser. KTP laser resulted faster in uncovering implants and diode laser was the one that needed more time. This ex vivo study showed that laser utilization with the recommended parameters gives no risks of dangerous thermal elevation to the tissues and implants.

Tooth extractions in high-risk patients under bisphosphonate therapy and previously affected with osteonecrosis of the jaws: surgical protocol supported by low-level laser therapy.

Trauma during dental surgery is a predisposing factor for medication-related osteonecrosis of the jaws (MRONJ). There are no specific guidelines for the management of dental extractions in patients under bisphosphonate therapy (BPT). The authors proposed in 2013 a successful protocol for tooth extractions in patients under BPT supported by Nd:YAG low-level laser therapy (LLLT). The aim of this study was to validate the safety and efficacy of this protocol reporting the data related to its application in a particular category of patients under BPT at high risk for MRONJ and who were previously affected with MRONJ. Eighty-two tooth extractions were performed in 36 patients previously affected with MRONJ. Antibiotic treatment was administered 3 days before and 2 weeks after tooth extractions. Patients were additionally treated with Nd:YAG LLLT, 5 applications of 1 minute each. Patients were evaluated 3 days and once a week for 2 months after the extractions and every time they received LLLT. In a total of 82 extractions, minimal bone exposure was observed in 2 cases, treated with Er:YAG laser vaporization and then completely healed. The data confirmed that laser biostimulation is a reliable technique that can be considered in the surgical protocol for patients under BPT.

Odontogenic myxofibroma: a concise review of the literature with emphasis on the surgical approach.

PURPOSE: The aim of this work is to report a review of the literature concerning epidemiology, clinical and radiographic features as well as treatment of odontogenic myxofibroma (MF). METHODS: The PubMed database was searched using the following keywords: "odontogenic myxofibroma", "odontogenic fibromyxoma", "myxofibroma of the jaw" and "fibromyxoma of the jaw". RESULTS: Fifteen articles reporting the experience with 24 patients were identified. Male/female ratio was 1:1.4 and the average age 29.5 years. The most frequent location was the mandible. In 66.7% of the cases the radiographic appearance was a multilocular radiolucency. Swelling was observed in 13 patients (92.86%), varying degrees of pain in 5 (35.71%) and paresthesia in only one patient (7.14%). Six out of 24 patients (26.09%) were treated with radical surgery and 17 out of 24 (73.91%) with a conservative approach. In two out of 21 cases (9.52%) a recurrence was reported. CONCLUSIONS: MF is an extremely rare tumour and no agreement exist on the causes of its development. According to the present review, the choice of treatment should depend on variables such as localization, presence of a primary or of a recurrent lesion, age, general medical conditions and aesthetic needs of the patient.

Laser welded versus resistance spot welded bone implants: analysis of the thermal increase and strength.

INTRODUCTION: The first aim of this "ex vivo split mouth" study was to compare the thermal elevation during the welding process of titanium bars to titanium implants inserted in pig jaws by a thermal camera and two thermocouples. The second aim was to compare the strength of the joints by a traction test with a dynamometer. MATERIALS AND METHODS: Six pigs' jaws were used and three implants were placed on each side of them for a total of 36 fixtures. Twelve bars were connected to the abutments (each bar on three implants) by using, on one side, laser welding and, on the other, resistance spot welding. Temperature variations were recorded by thermocouples and by thermal camera while the strength of the welded joint was analyzed by a traction test. RESULTS: For increasing temperature, means were 36.83 and 37.06, standard deviations 1.234 and 1.187, and P value 0.5763 (not significant). For traction test, means were 195.5 and 159.4, standard deviations 2.00 and 2.254, and P value 0.0001 (very significant). CONCLUSION: Laser welding was demonstrated to be able to connect titanium implant abutments without the risk of thermal increase into the bone and with good results in terms of mechanical strength.

Is the use of Pentoxifylline and Tocopherol effective in the treatment of Osteoradionecrosis of the jaws or for the treatment of medicationosteonecrosis of the jaw? An overview.

PURPOSE: The aim of the present study was to determine the methodological quality of systematic reviews that evaluated the effectiveness of pentoxifylline and tocopherol (PENTO) in the treatment of osteoradionecrosis of the jaw (ORNJ) and medication-related osteonecrosis of the jaw (MRONJ). METHODS: Searches were performed in Databases including PubMed, Scopus, LILACS, DARE, Cochrane Library, and SIGLE through OpenGrey until March 2024, were evaluated by two independent reviewers to answer the following question: Is the use of PENTO protocol effective in the treatment of ORNJ or for the treatment of MRONJ? RESULTS: A total of 256 articles were initially identified; however, following the use of appropriate inclusion and exclusion criteria, five systematic reviews were identified for detailed analysis. The final study sample comprised 588 patients: 397 patients with ORN and 197 patients with MRONJ who were treated with PENTO. The total recovery of individuals who used the PENTO protocol was 62,2 % for ORN and 100 % for MRONJ, with a follow-up period of 1 month to 10 years. The methodological quality of the studies was assessed using the AMSTAR 2 tool, in which four were of low quality and 1 moderate quality. CONCLUSION: The treatment of ORN and MRONJ with pentoxifylline and tocopherol has shown good results in the studies presented, with a partial or total reduction in bone exposure. However, the low quality of the relevant reports highlights the need for primary and secondary studies with better methodological rigor to reduce bias and provide reassurance for this treatment option.

Laser-assisted surgery with different wavelengths: a preliminary ex vivo study on thermal increase and histological evaluation.

Since the introduction of laser in clinical practice, different wavelengths have been used for oral surgery on the basis of the different characteristics and affinities of each one. The aim of this study was a comparison of different laser wavelengths in relation to both thermal increase and "histological quality" in a model of soft tissue surgery procedures. Thermal evaluation was realized, during laser-assisted surgery excision performed on a bovine tongue, by a thermal camera device to evaluate thermal increase on the surface of the sample and with four thermocouples to evaluate thermal increase on the depth of the specimen; temperature was recorded before starting surgical procedure and at the peak of every excision. The quality of excision, in terms of tissue damage and regularity, was realized by two blind examiners on the basis of established criteria. The highest superficial thermal increase was recorded for Superpulse 5-W CO2 laser, the lowest one for Er:YAG laser. The highest in depth thermal increase was recorded for 5 W Diode laser, the lowest one for Er:YAG laser. The best quality of incision was obtained with a 3-W CO2 laser and 3-W diode laser; epithelial, stromal, and vascular damages were evaluated with different degrees for all the used wavelengths with the best result, in terms of "tissue respect," for Er:YAG laser. In all the surgical procedures performed, thermal increase was evaluated until the end of the procedure; at remaining tissue level, thermal decrease was evaluable in the few seconds after surgery. The Er:YAG laser was the device with a lower influence on thermal increase; CO2 and diode lasers revealed a good histological quality. Further studies may be necessary to test the reliability of laser devices for the excision of all the types of specimens needing histological evaluation and diagnosis.

Clinicopathological evaluation of 164 dental follicles and dentigerous cysts with emphasis on the presence of odontogenic epithelium in the connective tissue. The hypothesis of "focal ameloblastoma".

OBJECTIVES: Some ameloblastomas presumably originate from odontogenic epithelium within the connective tissue of dental follicles and dentigerous cysts. Therefore, it would seem reasonable to discuss as whether odontogenic epithelium proliferations, frankly displaying ameloblastomatous features ("focal ameloblastoma"), should be considered as an "early" ameloblastoma. STUDY DESIGN: Histopathological reports from 164 dental follicles and dentigerous cysts from the Department of Oral and Maxillofacial Surgery/Oral Pathology of the VU Free University medical center in Amsterdam, The Netherlands, were reviewed. Histopathological slides from 39 cases reporting the presence of odontogenic epithelium within the connective tissue were re-evaluated in order to assess the possible presence of focal ameloblastomas. RESULTS: Focal ameloblastomas were detected in one dental follicle and in two dentigerous cysts. During a follow-up period of 6, 8 and 22 years, respectively, no clinical signs of (recurrent) ameloblastoma have occurred in these patients. CONCLUSIONS: Focal ameloblastoma possibly represents the early stage of ameloblastoma development.

Case series of 589 tooth extractions in patients under bisphosphonates therapy. Proposal of a clinical protocol supported by Nd:YAG low-level laser therapy.

OBJECTIVE: Trauma during dental surgery is a predisposing factor for bisphosphonates (BP)-related osteonecrosis of the jaws (BRONJ). However, about 40% of cases of BRONJ are not related to dental invasive procedures, being probably associated to endodontic or periodontal infections. Extraction of non-treatable teeth is considered a reliable choice, to improve symptoms and to reduce the risk of BRONJ. Here we report our experience of tooth extractions in patients under oral or intravenous BP therapy. STUDY DESIGN: Two-hundred and seventeen patients (38 males, 179 females; mean age 68.72 ± 11.26 years, range 30 to 83 years) under BP therapy received 589 tooth extractions at the Unit of Oral Medicine, Pathology and Laser-assisted Surgery of the University of Parma, Italy, between June 2006 and December 2010. Ninety five patients were under BP therapy for oncological disease (multiple myeloma (MM): 23; bone metastases (BM): 72) and 122 patients for non oncological diseases: 119 osteoporosis (OP), 2 rheumatoid arthritis (RA) and 1 Paget's disease (PD). The mean duration of BP was of 35 months. Antibiotic treatment was administered three days before and 2 weeks after tooth extractions. Patients were additionally treated with low level laser therapy (LLLT) through Nd:YAG laser (1064 nm--power 1.25 W; frequency 15 Hz; fibre diameter: 320 µm), 5 application of 1 minute each. Patients were evaluated 3 days and once a week for 2 months after the extractions and every time they received LLLT. Mean follow-up was 15 months (ranging from 4 to 31 months). RESULTS: In a total of 589 extractions (285 mandibular, 304 maxillary) performed, a minimal bone exposure was observed in 5 cases, treated with Er:YAG laser vaporization and than healed. CONCLUSIONS: Our experience supports the hypothesis that the association of antibiotic treatment and LLLT can be effective in preventing ONJ after tooth extractions in patients under BPT.

Ultra-high performance liquid chromatography high-resolution mass spectrometry for metabolomic analysis of dental calculus from Duke Alessandro Farnese and Maria D'Aviz.

Dental calculus is a valuable resource for the reconstruction of dietary habits and oral microbiome of past populations. In 2020 the remains of Duke Alessandro Farnese and his wife Maria D'Aviz were exhumed to get novel insights into the causes of death. This study aimed to investigate the dental calculus metabolome of the noble couple by untargeted metabolomics. The pulverized samples were decalcified in a water-formic acid mixture, extracted using methanol/acetonitrile and analyzed by ultra-high performance liquid chromatography coupled to high-resolution mass spectrometry (UHPLC-HRMS) using a reversed-phase separation followed by electrospray ionization and full scan in positive and negative ion mode. Waters Synapt-G2-Si High-Definition hybrid quadrupole time-of-flight mass spectrometer was used. Significant features were then identified using MSE acquisition mode, recording information on exact mass precursor and fragment ions within the same run. This approach, together with data pre-treatment and multivariate statistical analysis allowed for the identification of compounds able to differentiate between the investigated samples. More than 200 metabolites were identified, being fatty acids, alcohols, aldehydes, phosphatidylcholines, phosphatidylglycerols, ceramides and phosphatidylserines the most abundant classes. Metabolites deriving from food, bacteria and fungi were also determined, providing information on the habits and oral health status of the couple.