Combined loss of expression of involucrin and cytokeratin 13 is associated with poor prognosis in squamous cell carcinoma of mobile tongue.

BACKGROUND: This study aimed to evaluate the prognostic significance of expression levels of involucrin (IVL), cytokeratin (CK)-10 and -13 at different intratumor sites (tumor center and invading area) of oral tongue squamous cell carcinoma (OTSCC). METHODS: IVL, CK13 and CK10 expression levels were examined in a multicenter cohort of 146 OTSCCs using immunohistochemistry. External mRNA datasets were used for expression analysis and/or to validate survival associations. RESULTS: External transcriptomic datasets showed downregulation of IVL and KRT13 in oral malignancies including OTSCC as compared to normal controls. The combined loss of IVL and CK13 expression at the invading core but not at the center core was significantly associated with poor differentiation and reduced 5-year overall survival. Multivariate Cox analysis confirmed the loss of CK13 and IVL expression to be an independent prognostic factor. Transcriptomic dataset corroborated immunohistochemistry results. CONCLUSIONS: Combined expression levlels of IVL and CK13 might be useful as prognostic biomarkers in OTSCC.

Correlations between the Peptide Hormone Ghrelin and Proinflammatory Cytokines in Experimental Periodontitis Models of Female Rats at Different Stages of the Life Cycle.

OBJECTIVE: The aim of this study was to examine the correlations between the levels of ghrelin and inflammatory and bone metabolism markers in rats with periodontitis. DESIGN: Thirty female Wistar rats (6 trial rats and 4 control rats in each group) were divided into pubertal, adult and postmenopausal groups. Periodontitis was induced by ligatures. On the 21 st day, blood was collected and all rats were then sacrificed. The levels of osteocalcin, osteoprotegerin, alkaline phosphatase, tumour necrosis factor alpha (TNF-α), interleukin 1 beta (IL-1ß), acylated ghrelin, total ghrelin and soluble receptor activator of nuclear factor-kB ligands in the blood samples were measured using enzyme-linked immunosorbent assays. The jaws were decalcified in a Tris-EDTA solution and embedded in paraffin and 4-5 µm sections were cut for IL-ß, TNF -α and ghrelin staining. RESULTS: Significantly higher serum alkaline phosphatase levels were detected in the trial rats in the pubertal group than in the control rats (p = 0.033). In the postmenopausal group, ghrelin levels positively correlated with interleukin 1 beta levels (r = 0.708, p < 0.05). Among all trial rats, the postmenopausal group exhibited significantly higher levels of acylated ghrelin than the other groups (p = 0.001). Significantly higher osteoprotegerin levels were observed in the control rats than in the trial rats in the postmenopausal group (p = 0.012). Inflammation scores were significantly higher in adult trial rats than in controls (p = 0.024); significantly higher TNF-α levels were detected in postmenopausal experimental rats than in the adult experimental group (p = 0.025). CONCLUSIONS: We concluded that total ghrelin levels in serum only correlated with IL-ß levels in postmenopausal rats.

The landscape of genetic alterations in ameloblastomas relates to clinical features.

Ameloblastoma is a mostly benign, but locally invasive odontogenic tumor eliciting frequent relapses and significant morbidity. Recently, mutually exclusive mutations in BRAF and SMO were identified causing constitutive activation of MAPK and hedgehog signaling pathways. To explore further such clinically relevant genotype-phenotype correlations, we here comprehensively analyzed a large series of ameloblastomas (98 paraffin block of 76 patients) with respect to genomic alterations, clinical presentation, and histological features collected from the archives of three different pathology centers in France, Germany, and Turkey. In good agreement with previously published data, we observed BRAF mutations almost exclusively in mandibular tumors, SMO mutations predominantly in maxillary tumors, and single mutations in EGFR, KRAS, and NRAS. KRAS, NRAS, PIK3CA, PTEN, CDKN2A, FGFR, and CTNNB1 mutations co-occurred in the background of either BRAF or SMO mutations. Strikingly, multiple mutations were exclusively observed in European patients, in solid ameloblastomas and were associated with a very high risk for recurrence. In contrast, tumors with a single BRAF mutation revealed a lower risk for relapse. We here establish a comprehensive landscape of mutations in the MAPK and hedgehog signaling pathways relating to clinical features of ameloblastoma. Our data suggest that ameloblastomas harboring single BRAF mutations are excellent candidates for neo-adjuvant therapies with combined BRAF/MEK inhibitors and that the risk of recurrence maybe stratified based on the mutational spectrum.

The genomic road to invasion-examining the similarities and differences in the genomes of associated oral pre-cancer and cancer samples.

BACKGROUND: It is frequently assumed that pre-invasive lesions are simpler precursors of cancer and will contain a limited subset of the genomic changes seen in their associated invasive disease. Driver mutations are thought to occur early, but it is not known how many of these are present in pre-invasive lesions. These assumptions need to be tested with the increasing focus on both personalised cancer treatments and early detection methodologies. METHODS: We examined genomic copy number changes in 256 pre-invasive and invasive samples from 69 oral cancer patients. Forty-eight samples from 16 patients were further examined using exome sequencing. RESULTS: Evidence of a shared ancestor of both dysplasia and carcinoma was seen in all but one patient. One-third of dysplasias showed independent copy number events. The remainder had a copy number pattern that was similar to or simpler than that of the carcinoma. All dysplasias examined contained somatic mutations absent in the related carcinoma. Previously observed copy number changes and TP53 mutations were very frequently observed, and almost always shared between dysplasia and carcinoma. Other gene changes were more sporadic. Pathway analysis confirmed that each patient's disease developed in a different way. Examining the numbers of shared mutations and the rate of accumulation of mutations showed evidence that all samples contain a population of sub-clones, with little evidence of selective advantage of a subset of these. CONCLUSIONS: These findings suggest that most of the genomic changes driving oral cancer occur in the pre-cancerous state by way of gradual random accumulation rather than a dramatic single event.

Unusual Presentation of an Adenocarcinoma of the Lung Metastasizing to the Mandible, Including Molecular Analysis and a Review of the Literature.

Lung cancer is the most frequent cause of cancer-related death worldwide. Metastases of non-small cell lung carcinoma to the oral and maxillofacial region are rare. Thus, the diagnosis of a metastatic lesion in the oral cavity is challenging to the clinician and to the pathologist. This report presents a case of a 72-year-old man with metastatic lung adenocarcinoma located in the posterior mandibular region. Next-generation sequencing analysis showed no important mutations in the relevant genes except in the TP53 tumor suppressor gene.

Management of a Recurrent Pyogenic Granuloma of the Hard Palate with Diode Laser: A Case Report.

INTRODUCTION: Pyogenic granuloma (PG) is a prevalent inflammatory hyperplasia of skin and oral mucosa which is often caused by constant low-grade local irritation, traumatic injury or hormonal factors. In many cases, gingival irritation and inflammation due to poor oral hygiene are precipitating factors. Oral PG occurs predominantly on the gingiva, but it is also encountered on the lips, tongue, buccal mucosa and rarely on the hard palate. Although surgical excision is the first choice of treatment, many other treatment modalities could be counted such as cryosurgery, sodium tetradecyl sulfate sclerotherapy, intralesional steroids, flash lamp pulsed dye laser, neodymium-doped yttrium aluminium garnet (Nd:YAG) laser, carbon dioxide (CO2) laser, erbium-doped yttrium aluminum garnet (Er:YAG) lasers and diode laser have been suggested. After surgical excision recurrence occurs up to 16% of these lesions. It is believed that recurrence ensues as a result of incomplete excision, failure to eliminate etiologic factors or repeated trauma. CASE REPORT: A 50-year-old female was referred to the Department of Oral Surgery, Gazi University, School of Dentistry, complaining of a swelling and growth on the right side of the hard palate for four months. Patient reported a similar growth in the same area about two years earlier, which had turned out to be a PG by histopathology. The treatment plan included surgical excision of the lesion using diode laser. RESULTS: The patient reported no pain after the surgery. She was discharged with a prescription of chlorhexidine mouthwash and necessary post-operative instructions. At 7 days follow up visit, immediate recurrence of the lesion was observed, and it was excised by diode laser with 2 mm margins at its clinical periphery, to a depth up to the periosteum, by the same operator. No recurrence or scarring was observed in 14 months follow-up. CONCLUSION: Although diode laser is a secure and efficient technique for the treatment of intraoral PG, in order to minimize its recurrence, the lesion should be excised with a wider margin down to the periosteum or to the causing agent. Also due to its high recurrence rate, long-term follow-up is recommended.

Solitary plasmacytoma of the mandible: report of two cases.

Plasma cell neoplasms (plasmacytoma) are discrete, solitary masses of lymphoid neoplastic proliferations of B cells. Plasmacytomas comprise three groups: multiple myeloma, solitary plasmacytoma (SP) and extramedullary plasmacytoma. SP originates as a clone of transformed malignant plasma cells in the bone marrow. SP of the jaw is a rare condition; therefore diagnosis is quite difficult and often results in misdiagnosis. MM is a lymphoproliferative disease the prognosis of which is worse than SP. SP can progress to MM in a few months to years after diagnosis. In this regard, early diagnosis of the disease is of utmost importance. This article presents two cases of SP diagnosed in the mandible and documented with clinical, radiographic and histological findings.

Osteocalcin and osteonectin expression after double application of platelet-rich plasma in rabbits.

PURPOSE: Platelet-rich plasma (PRP) is a novel method for transferring autogenous growth factors to the wound area. The aim of this study was to evaluate the efficacy of double-application of PRP (DA-PRP) on bone healing in rabbit cranial defects by examining osteonectin (ON) and osteocalcin (OC) expression. MATERIALS AND METHODS: Twenty-eight rabbits, each with two surgically prepared calvarial bone defects, were included in this study and divided into six groups: The defects (N=56) were treated with either a single-application of PRP (SA-PRP) (n=10), a combination of SA-PRP and betatricalciumphosphate (SA-PRP+ß-TCP) (n=10), only DAPRP (n=8), both DA-PRP and beta-tricalciumphosphate (DA-PRP+ß-TCP) (n=8), only beta-tricalciumphosphate (ß-TCP) (n=10), or controls (n=10). The animals were sacrificed at 30th day postoperatively and samples were immunohistochemically examined for ON and OC expressions. RESULTS: It was determined that DA-PRP did not significantly improve the ON and OC percentages achieved by SA-PRP or the controls. The three groups treated with ß-TCP showed a higher percentage of ON than those treated without ß-TCP (p<0.05). The ß-TCP treated groups and SA-PRP group demonstrated higher OC percentage than DA-PRP and control groups (p<0.05). CONCLUSION: The present findings suggest that DAPRP did not have a significant effect on the healing of non-critical size rabbit cranial bone defects.

The clonal relationships between pre-cancer and cancer revealed by ultra-deep sequencing.

The study of the relationships between pre-cancer and cancer and identification of early driver mutations is becoming increasingly important as the value of molecular markers of early disease and personalised drug targets is recognized, especially now the extent of clonal heterogeneity in fully invasive disease is being realized. It has been assumed that pre-cancerous lesions exhibit a fairly passive progression to invasive disease; the degree to which they, too, are heterogeneous is unknown. We performed ultra-deep sequencing of thousands of selected mutations, together with copy number analysis, from multiple, matched pre-invasive lesions, primary tumours and metastases from five patients with oral cancer, some with multiple primary tumours presenting either synchronously or metachronously, totalling 75 samples. This allowed the clonal relationships between the samples to be observed for each patient. We expose for the first time the unexpected variety and complexity of the relationships between this group of oral dysplasias and their associated carcinomas and, ultimately, the diversity of processes by which tumours are initiated, spread and metastasize. Instead of a series of genomic precursors of their adjacent invasive disease, we have shown dysplasia to be a distinct dynamic entity, refuting the belief that pre-cancer and invasive tumours with a close spatial relationship always have linearly related genomes. We show that oral pre-cancer exhibits considerable subclonal heterogeneity in its own right, that mutational changes in pre-cancer do not predict the onset of invasion, and that the genomic pathway to invasion is neither unified nor predictable. Sequence data from this study have been deposited in the European Nucleotide Archive, Accession No. PRJEB6588.

A novel genomic signature reclassifies an oral cancer subtype.

Verrucous carcinoma of the oral cavity (OVC) is considered a subtype of classical oral squamous cell carcinoma (OSCC). Diagnosis is problematic, and additional biomarkers are needed to better stratify patients. To investigate their molecular signature, we performed low-coverage copy number (CN) sequencing on 57 OVC and exome and RNA sequencing on a subset of these and compared the data to the same OSCC parameters. CN results showed that OVC lacked any of the classical OSCC patterns such as gain of 3q and loss of 3p and demonstrated considerably fewer genomic rearrangements compared to the OSCC cohort. OVC and OSCC samples could be clearly differentiated. Exome sequencing showed that OVC samples lacked mutations in genes commonly associated with OSCC (TP53, NOTCH1, NOTCH2, CDKN2A and FAT1). RNA sequencing identified genes that were differentially expressed between the groups. In silico functional analysis showed that the mutated and differentially expressed genes in OVC samples were involved in cell adhesion and keratinocyte proliferation, while those in the OSCC cohort were enriched for cell death and apoptosis pathways. This is the largest and most detailed genomic and transcriptomic analysis yet performed on this tumour type, which, as an example of non-metastatic cancer, may shed light on the nature of metastases. These three independent investigations consistently show substantial differences between the cohorts. Taken together, they lead to the conclusion that OVC is not a subtype of OSCC, but should be classified as a distinct entity.

P16(INK 4a) and Ki-67 expression in human papilloma virus-related head and neck mucosal lesions.

Human papilloma virus (HPV) is postulated as a risk factor in the etiology of some specific mucosal pathologies in the head and neck regions. Despite the frequent use of p16(INK4a) as a surrogate marker for HPV-infection, there is still controversy with respect to its reliability. This study has been undertaken to assess the potential role of p16(INK 4a) and Ki-67 expression in HPV-related lesions. The study was conducted on 71 specimens of oral, tonsillar and laryngeal lesions which comprised 25 dysplasia and 46 papilloma specimens. Specimens were immunohistochemically stained for p16(INK4A) and Ki-67 proteins. HPV DNA was determined by one step multiplex polymerase chain reaction. HPV DNA was detected in 33.8% of all lesions. Tonsil and larynx lesions showed significant differences with oral lesions for HPV positivity (p < 0.001). p16(INK 4a) over-expression was seen in 56.5% of papilloma and 60% of dysplasia specimens. HPV status showed a positive correlation with p16(INK 4a) expression in tonsillar dysplasias (p < 0.001). p16(INK 4a) expression may have a value as a marker in high risk HPV induced dysplasias, but not in low risk infected lesions. The proliferation index is not related to HPV-induced lesions and may be evaluated as an independent marker in head and neck premalignant lesions.

Oral mucosal lesions: a retrospective review of one institution's 13-year experience.

BACKGROUND/AIM: To investigate the relative frequency of biopsied nonneoplastic oral mucosal lesions in Ankara, Turkey. MATERIALS AND METHODS: Biopsy records of a single center from 2000-2012 were retrospectively collected. Diagnosis was recorded and evaluated with respect to patient demographics (age, sex) and location of the lesion. RESULTS: Of 11,980 biopsies, 1732 (14.5%) were mucosal nonneoplastic lesions. Hyperplastic lesions (n= 1000, 57.7%) with fibroepithelial hyperplasia in 30.9% of patients were the most common type of oral nonneoplastic lesions. The mean age of patients differed with respect to type of mucosal lesion, tending to be lower in patients with reactive lesions. Dermatoses showed a female predominance. CONCLUSION: Our ,findings revealed that hyperplastic lesions were the most common among nonneoplastic oral mucosa lesions. Geographic and ethnic.differences of patients with various types of oral mucosal lesions require further investigation.

p16INK 4a and Ki-67 expression in human papilloma virus-related head and neck mucosal lesions / Expresión de p16INK 4a y Ki-67 en las lesiones de cabeza y cuello relacionados con el virus del papilloma humano

Human papilloma virus (HPV) is postulated as a risk factor in the etiology of some specific mucosal pathologies in the head and neck regions. Despite the frequent use of p16INK4a as a surrogate marker for HPV-infection, there is still controversy with respect to its reliability. This study has been undertaken to assess the potential role of p16INK 4a and Ki-67 expression in HPV-related lesions. The study was conducted on 71 specimens of oral, tonsillar and laryngeal lesions which comprised 25 dysplasia and 46 papilloma specimens. Specimens were immunohistochemically stained for p16INK4A and Ki-67 proteins. HPV DNA was determined by one step multiplex polymerase chain reaction. HPV DNA was detected in 33.8% of all lesions. Tonsil and larynx lesions showed significant differences with oral lesions for HPV positivity (p<0.001). p16INK 4a over-expression was seen in 56.5% of papilloma and 60% of dysplasia specimens. HPV status showed a positive correlation with p16INK 4a expression in tonsillar dysplasias (p<0.001). p16INK 4a expression may have a value as a marker in high risk HPV induced dysplasias, but not in low risk infected lesions. The proliferation index is not related to HPV-induced lesions and may be evaluated as an independent marker in head and neck premalignant lesions.

El virus del papiloma humano (VPH) se postula como un factor de riesgo en la etiología de algunas patologías de la mucosa, específicas en las regiones de cabeza y cuello. A pesar de usar con frecuencia el p16INK4A como un marcador sustituto para la infección por VPH, todavía existe controversia con respecto a su fiabilidad. Este estudio se ha llevado a cabo para evaluar el papel potencial de la expresión de p16INK 4a y de Ki-67 en las lesiones relacionadas con el VPH. El estudio se realizó en 71 muestras de lesiones orales, tonsilares y laríngeas que comprendían 25 displasias y 46 especímenes de papiloma. Los especímenes fueron teñidos inmunohistoquímicamente para p16INK4a y Ki-67. El ADN del VPH se determinó mediante una PCR multiplex de un paso. ADN del VPH se detectó en el 33,8% de todas las lesiones. Las lesiones de la amígdala y laringe mostraron diferencias significativas con lesiones orales para la positividad de VPH (p <0,001). Sobre-expresión de p16INK 4a se observó en 56,5% de las muestras de papiloma y 60% de las muestras de displasia. El estatus del VPH mostró una correlación positiva con la expresión de p16INK4a en displasias tonsilares (p <0,001). La expresión de p16INK4a puede tener valor como marcador en las displasias inducidas por VPH de alto riesgo, pero no en las lesiones infectadas de bajo riesgo. El índice de proliferación no está relacionado con las lesiones inducidas por VPH y puede ser evaluado como un marcador independiente en las lesiones premalignas de la cabeza y del cuello.

Peripheral osteoma of the mandible: a case report.

Osteomas are benign tumors which are composed of mature compact or cancellous bone. They can be either peripheral, central or extraskeletal. The peripheral osteoma arises from surface of the bone (periosteal) whereas the central osteoma arises from the bone medullary (endosteal) and the extra-skeletal soft tissue osteoma usually develops within the muscle. Osteomas are most commonly found in the skull and facial bones. Multiple osteomas may be associated with Gardner's Syndrome. These lesions are usually painless and recurrence is uncommon after local excision. In this case report clinical, radiographic findings and treatment of a 24-year-old male patient with peripheral osteoma in the anterior mandible are presented.

Comparison of methods for the extraction of DNA from formalin-fixed, paraffin-embedded archival tissues.

AIM: Discussing a protocol involving xylene-ethanol deparaffinization on slides followed by a kit-based extraction that allows for the extraction of high quality DNA from FFPE tissues. METHODS: DNA was extracted from the FFPE tissues of 16 randomly selected blocks. Methods involving deparaffinization on slides or tubes, enzyme digestion overnight or for 72 hours and isolation using phenol chloroform method or a silica-based commercial kit were compared in terms of yields, concentrations and the amplifiability. RESULTS: The highest yield of DNA was produced from the samples that were deparaffinized on slides, digested for 72 hours and isolated with a commercial kit. Samples isolated with the phenol-chloroform method produced DNA of lower purity than the samples that were purified with kit. The samples isolated with the commercial kit resulted in better PCR amplification. CONCLUSION: Silica-based commercial kits and deparaffinized on slides should be considered for DNA extraction from FFPE.

Investigation of p16(INK4a) as a prognostic biomarker in oral epithelial dysplasia.

BACKGROUND: Human papilloma virus is a risk factor for oropharyngeal cancer. Evidence for a similar aetiological role in the development of oral dysplasia or its transformation to oral cancer is not as clear. Meta-analyses estimate the prevalence of high-risk human papilloma virus (HPV) serotypes to be three times higher in pre-malignant lesions and cancer than in normal oral mucosa. However, this does not imply a causal relationship. Conflicting results are reported from the few studies examining the prognostic significance of HPV positivity in the development of oral cancer. We aimed to examine the ability of p16(INK4a) protein expression, a surrogate marker of HPV infection, to predict malignant progression in a large cohort of oral dysplasia patients. METHODS: One hundred forty eight oral dysplasia cases underwent immunohistochemical analysis using a monoclonal antibody against p16(INK4a) . Clinical factors were also collated on each case. Slides were double scored independently by two trained observers. Univariate analyses using both logistic and Cox regression models were performed. RESULTS: Thirty nine of 148 cases progressed to cancer. Ten of 148 cases (7%) were p16(INK4a) positive. High grade of dysplasia (P = 0.0002) and lesion morphology (P = 0.03) were found to be prognostic of malignant progression. p16(INK4a) score was not prognostic in this cohort (P = 0.29). This did not change with a time to event analysis (P = 0.24). CONCLUSION: Few studies have assessed the aetiological role of HPV in cancer development from dysplastic lesions. Our study, using one of the largest cohorts of oral dysplasia, demonstrated a low rate of p16(INK4a) positivity and was unable to confirm a prognostic ability for this biomarker.

Recurrent unicystic mural type ameloblastoma in a 9-year-old boy, 8 years follow-up.

Unicystic ameloblastoma is not a rare odontogenic tumor in the pediatric population. A significant care should be given to unicystic ameloblastoma if it has mural invasions due to its local aggressiveness, high recurrence rates and radical management options as in conventional ameloblastoma. Fine needle aspiration (FNA) cytology is a rapid, non-traumatic diagnostic method that provides a required attention prior to surgery. We present an excisionsl biopsy proved FNA diagnosed mural type unicystic ameloblastoma in a 9-year-old child recurred as a solid ameloblastoma after 8 years. When distinctive features of ameloblastoma are known, an accurate diagnosis can be made by FNA cytology, in combination with clinicoradiological findings. This method gives benefit to the patients especially the younger ones both for the pre-operative surgical planning and the post-operative follow-up.

Primary oral myeloid sarcoma: Report of a case.

Myeloid sarcoma is defined as a tumor mass of immature myeloid cells that may be observed in a variety of locations including bone, skin, lymph nodes and soft tissues. However, oral involvement of myeloid sarcoma is extremely rare. These tumors are considered as specific lesions of acute myeloid leukemia. We present a case of a myeloid sarcoma of the upper vestibular gingiva in a 29-year-old woman who has no hematologic disease history. Multiple metastases were found in floor of the nasal cavity, left breast, and left lacrimal gland 12 months after primary diagnosis.

Histologic and histomorphometric assessment of eggshell-derived bone graft substitutes on bone healing in rats.

OBJECTIVE: The objective of this study was to histologically and histomorphometrically evaluate the efficacy of the new formulations of eggshell-derived calcium carbonate in rats. STUDY DESIGN: The study was conducted on 30 adult male rats. Four standardized and circular intrabony defects were created in the both maxilla and mandibula of each animal. Three different graft materials were prepared as follows: 1) Material A: Eggshell-derived calcium carbonate combined with carrageenan gel, 2) Material B: Eggshell-derived calcium carbonate combined with xanthan gum gel, and 3) Material C: Eggshell-derived calcium carbonate powder. The right mandibular defect sites were grafted with Material A in all animals, and defects on the left were grafted with Material B. Defects on the right side of maxilla were received Material C in all animals, and all left maxillary defects were remained untreated as controls. The animals were sacrificed either postoperatively on the 15th day, postoperatively on the 30th day or postoperatively on the 45th day. Histomorphometric measurements were made of the areas of newly formed bone, necrotic bone, fibrous tissue and residual graft material. RESULTS: Material A exhibited the highest level of osteoid formation followed by Material B and Material C on the 45th day. In terms of osteoid formation, statistically significant differences were observed between graft materials and controls at 45th day compared to 15th and 30th day (p<0.05). CONCLUSIONS: Eggshell-derived graft substitutes in both gel and powder forms are biocompatible materials which may have the potential to enhance the new bone formation. Key words:Bone graft material, bone defects, eggshell, histopathological evaluation, rat.

Effects of low-dose doxycycline and bisphosphonate clodronate on alveolar bone loss and gingival levels of matrix metalloproteinase-9 and interleukin-1ß in rats with diabetes: a histomorphometric and immunohistochemical study.

BACKGROUND: Bisphosphonates (BPs) and low-dose doxycycline (LDD) have been shown to inhibit bone resorption and to improve the levels of proinflammatory mediators and destructive enzymes in gingival tissues, respectively. The purpose of this study is to evaluate the effect of mono and combined BP clodronate and LDD therapies in reducing gingival levels of matrix metalloproteinase-9 (MMP-9), interleukin-1ß (IL-1ß), and alveolar bone loss in rats with diabetes. METHODS: Fifty adult Wistar rats were divided into five study groups as follows: 1) group 1 = diabetes control; 2) group 2 = diabetes + periodontitis; 3) group 3 = diabetes + periodontitis + LDD; 4) group 4 = diabetes + periodontitis + clodronate; and 5) group 5 = diabetes + periodontitis + LDD + clodronate. LDD and clodronate were given as a single agent or as combination therapy during the 7 days of the post-experimental periodontitis period. On day 7, the rats were sacrificed, the mobility of the tooth was recorded, and block biopsies were removed. The gingival tissues were analyzed histologically and immunohistochemically for expression of MMP-9 and IL-1ß. Alveolar bone loss was evaluated morphometrically under a light microscope. Data analysis was performed statistically by Kruskal-Wallis and post hoc Tukey and Spearman correlation tests. RESULTS: Alveolar bone loss was significantly greater in groups 2 through 5 than group 1 (P <0.05) but was not significantly different among groups 2 through 5 (P >0.05). Animals with periodontitis (group 2) expressed significantly higher levels of MMP-9 and IL-1ß compared with those without periodontitis (group 1) (P <0.05). MMP-9 expression was significantly lower in group 3 than groups 1, 2, and 5 (P <0.05). IL-1ß expression was significantly lower in the groups 1, 3, 4, and 5 than 2 (P <0.01) but was not significantly different among groups 1, 3, 4, and 5. Positive correlations were found between alveolar bone loss and density of inflammation (ρ = 0.319, P = 0.021) and between MMP-9 and IL-1ß (ρ = 0.418, P = 0.002), respectively. CONCLUSION: Our findings suggest that ligature-induced periodontitis in animals with diabetes results in significantly higher levels of MMP-9 and IL-1ß expression in gingiva. The use of mono and combined clodronate and LDD administrations may significantly reduce levels of MMP-9 and IL-1ß expression. However, drug administration did not affect alveolar bone levels during the study period.