Primary Oral Mixed Neuroendocrine-Non-neuroendocrine Neoplasm (MiNEN): A Rare Case Report and Review of the Literature.

Mixed neuroendocrine-non-neuroendocrine neoplasms (MiNENs) are rare tumors recently characterized by the presence of both neuroendocrine and non-neuroendocrine components within the same tumor tissue. Although MiNEN found their place in the WHO classification for various organs, this composite tumor in the head and neck region remains exceptionally rare. We present a case of primary oral MiNEN in a 64-year-old male located on the left side of lower gingiva. Biopsy raised suspicion of neuroendocrine carcinoma (NEC) and the patient underwent partial mandibulectomy. The resected specimen showed two distinct components of NEC and squamous cell carcinoma (SCC) with the confirmation of immunohistochemical markers. There has been no sign of recurrence nor metastasis 6 years after the surgery. In addition, we have conducted a review of published cases with potential relevance to this entity, resulting in five cases. The diverse terminology reinforces the need for a standardized classification system of oral/head and neck MiNENs.

Immunoexpression of SIRT1, 6, and 7 in oral leukoplakia and oral squamous cell carcinoma.

Sirtuins (SIRTs) are a family of proteins involved in the metabolic process responsible for extending the lifespan. The role of SIRT1, 6, and 7 in oral squamous cell carcinoma (OSCC) and oral leukoplakia (OLP), one of its precursors, is still elusive. In this study, 82 OLP and 77 OSCC were immunohistochemically examined for SIRT1, 6, and 7. Stained sections were thoroughly scanned and evaluated using a digital image analysis program. The SIRT1, 6, and 7 expressions were detected in the nuclei of epithelial and carcinoma cells in various degrees. Afterward, any correlations among SIRTs, including associations with clinicopathological features and the Kaplan-Meier curves were analyzed. OSCC demonstrated significantly higher SIRT1 expression than OLP, while non-dysplastic lesions showed significantly higher SIRT6 expression than other lesions. A strong correlation was observed between SIRT6 and 7 in OLP, SIRT1 and 6 in in OSCC and in SIRT6 and 7 when all lesion types were considered. There were no significant differences between SIRTs reactivity and the clinical features in OLP. For OSCC, SIRT1 and 6 was found to be directly associated with site of the lesion, while SIRT7 showed a direct relationship between gender, stromal lymphocytic infiltration, and depth of the invasion. OSCC with high SIRT7 expression revealed a slightly lower survival probability, although not statistically significant (p = 0.1019). Our findings suggest that SIRT1, 6, and 7 may play correlated and diverse roles in the development and advancement of OSCC.

A case of giant dental calculus in a patient with centronuclear myopathy.

INTRODUCTION: Centronuclear myopathy is a hereditary congenital muscle disease. It is characterized by generalized muscle hypotonia from early childhood, elongated cacial appearance, mandibular undergroth, and dental malposition. In this report, we discuss the clinical course and management of a patient with centronuclearmyopathy, who developed a giant dental calculus in the floor of the mouth and underwent surgical excision. CASE REPORT: A 37-year-old Japanese man was referred to our hospital, and reported a swelling in the floor of the mouth. The patient affects centronuclear myopathy and has generalized muscle weakness. CT images showed a high-density area in the floor of the mouth measuring 35 × 28 × 20 mm. The lesion was clinically diagnosed as giant dental calculus, and surgically removed. CONCLUSION: We have experienced a case of giant dental calculus in a patient with centronuclear myopathy. In dental treatment, we must consider generalized muscle weakness.

Condylar Resorption Following Compressive Mechanical Stress in Rabbit Model - Association of Matrix Metalloproteinases.

BACKGROUND/AIM: Idiopathic condylar resorption (ICR) is a morphological change of the condylar head that occurs following orthodontic treatment or orthognathic surgery. This complication is serious, as it can cause relapse after mandible treatment. The aim of this experimental study was to evaluate the mechanism of influence of condylar resorption on compressive mechanical stress in temporomandibular joint following a change in occlusal position by mandible advancement. MATERIALS AND METHODS: An osteotomy procedure at the midline of mandible was performed in 15 rabbits, with the left side moved forward by 3.5 mm. Advancement of the left side of the mandible resulted in compressive mechanical stress on condylar head on the left side. Samples were subjected to micro-computed tomography, histological staining and immunohistochemistry. RESULTS: The area and depth of anterior condylar resorption at two weeks were significantly different as compared to those at one week (p<0.05). TRAP staining confirmed the significantly largest number of TRAP-positive cells after two weeks (p=0.02), compared to one week. MMP-3 and MMP-13 immunostaining of the anterior condylar head at two weeks revealed high levels of both proteins from the surface to the deep layer of cartilage. CONCLUSION: Compressive mechanical stress following mandible advancement results in load on the anterior surface of the condylar head, which leads to bone resorption there, and induces MMP-3 and MMP-13 related to degradation of condylar head cartilage.

Pulmonary pleomorphic carcinoma metastasizing to the anterior mandibular gingiva: A case report and literature review.

INTRODUCTION AND IMPORTANCE: Pulmonary pleomorphic carcinoma (PPC) is a rare subtype of non-small cell lung cancer. No standard therapy has been established for advanced disease, which results in poor prognosis. Some cases of PPC metastasizing to mandibular gingiva have been reported, and the involved site in these reports is all unilateral mandible. CASE PRESENTATION: We report a case of PPC metastasizing to the anterior mandibular gingiva in a 68-year-old man. The patient was referred to our hospital with tumor bleeding and difficulty with intake. One month before, he had been diagnosed as PPC. The size of oral tumor was 28 × 25 mm, and we performed surgical resection. Although there was no recurrence of oral lesion, he died of systemic metastases after 3 months since the surgery. CLINICAL DISCUSSION: The prognosis of patients with metastatic tumor in oral region is poor. Radical treatment for oral lesion is often difficult due to the existence of other metastasis or the refractory, in particular cases with high grade malignancies such as PPC. On the other hand, because of the development of cancer treatment and the arrival of super-aging society, the number of patients with metastatic tumor in oral region has been expected to increase in future. CONCLUSION: PPC metastasizing to the gingiva of mandibular symphysis is extremely rare. If there are possibilities to improve the prognosis or quality of life, radical or palliative treatment for metastatic tumor in oral region should be performed.

Biodegradation behaviors of magnesium(Mg)-based alloy nails in autologous bone grafts: In vivo study in rabbit skulls.

OBJECTIVE: In this study, autologous bone grafts using bone-fixing nails made of magnesium-zinc-calcium ternary alloys were performed using rabbit skulls. MATERIAL AND METHODS: Two types of nails for bone fixation were prepared: 2.5 mm width, 3 mm length and 2.5 mm width, 2 mm length. A disk-shaped bone with a diameter of 5 mm was resected from the parietal bone and fixed with a 3 mm long nail. As a control group, a 2 mm long nail was driven into the existing bone. The rabbits were sacrificed at 1, 4, 12, and 24 weeks after surgery. The resected samples were observed with micro X-ray CT, and embedded in methyl methacrylate to prepare non-decalcified specimens. The in vivo localization of elements was examined using energy-dispersive X-ray spectroscopy (EDS). RESULTS: Micro X-ray CT images of samples showed volume reduction due to degradation in both the bone graft and control groups. No significant difference in the amount of degradation between the two groups was observed, however characteristic degradation processes were observed in each group. The samples stained with alizarin red S showed amorphous areas around the nails, which were considered as corrosion products and contacted directly with the newly formed bones. EDS analysis showed that corrosion products were mainly composed of magnesium and oxygen at an early stage, while calcium and phosphorus were detected on the surface layer during the long-term observation. CONCLUSIONS: The degradation speed of the magnesium alloy nails varied depending on the shapes of the nails and surrounding tissue conditions. A calcium phosphate layer was formed on the surface of magnesium alloy nails, suggesting that the degradation rate of the nail was slow.

Comparison of different diagnostic imaging techniques for the detection of bone invasion in oral cancers.

OBJECTIVES: To evaluate the ability of different imaging modalities to accurately detect bone invasion in oral squamous cell carcinomas. PATIENTS AND METHODS: Patients with oral squamous cell carcinoma, who were scheduled for mandibulectomy or maxillectomy, underwent clinical evaluation using five preoperative imaging diagnosis methods-contrast-enhanced MRI, CT, 99mTc scintigraphy (Tc scan), FDG-PET CT (PET/CT), and panoramic radiography. The sensitivity and specificity of each modality in detecting bone invasion were calculated by comparing the findings on the images with postoperative histopathological findings. In a subgroup of patients, we further assessed the ability of MRI and CT to detect the accurate extent of bone invasion, including the height, width, and depth in patients with pathological mandibular invasion. RESULTS: Overall, 50 patients were enrolled in this study, and nine patients with pathological mandibular invasion were included in our subgroup analysis. MRI was found to be the most useful method in detecting bone invasion, showing the highest sensitivity (88.9%) and negative predictive values (92.3%). CT (87.5% specificity and 77.8% sensitivity) was more specific than MRI, though less sensitive. Combined PET/CT was more sensitive (83.3%) and less specific (71.9%) than CT. Tc scan had high sensitivity (88.9%); however, the specificity was relatively low (71.9%). CONCLUSION: MRI was the most useful method in detecting bone invasion. A negative MRI result definitively excludes bone marrow invasion. In patients with positive MRI findings, a negative CT may be useful in ruling out bone marrow invasion.

In vivo behaviors of highly flexible paper consisting of ultralong hydroxyapatite nanowires.

This study was conducted in order to investigate biological compatibility of a thin and flexible hydroxyapatite (HAP) paper which consists of ultralong hydroxyapatite nanowires. Circular-shaped cranial bone defects with a diameter of 8.8 mm were prepared to expose the dura maters in Wistar rats. The similar-sized, circular-shaped HAP paper was placed at the bottom of the bone defects. After 2, 4, and 8 weeks, the rats were sacrificed, and the experimental sections were examined by micro-CT scanning and histological observation. The HAP paper covered with fibrous tissues showed no inflammatory cell infiltration, and their thicknesses decreased over time. Tartrate-resistant acid phosphatase-positive osteoclast-like cells were induced around the edges of the HAP paper along with the exfoliation of the HAP paper. The newly-formed bones were observed in the bone-defected areas, either with a direct contact with the HAP paper or through thin fibrous tissues. The HAP paper-induced osteoblast differentiation was confirmed since the alkaline phosphatase activities were detected on the surfaces of the HAP paper. These results indicated that the HAP paper may induce osteogenesis without causing any harmful effects. The highly flexible HAP paper can contribute to further development of bone regenerative therapy.

Osteogenic response under the periosteum by magnesium implantation in rat tibia.

This study was designed to examine osteoconductive effects of Mg in rats tibia. The animals were sacrificed after 1, 2, and 8 weeks. The elemental analysis was performed using SEM/EDX at week 1. Following X-ray micrography at weeks 2 and 8, samples were embedded in paraffin. The expression of osteocalcin was observed by immunohistochemical staining. The element concentrations of fibrous capsules around the specimens were also measured by ICP-MS. The concentrations of Ca and P on the surface of the Mg specimen increased in SEM/EDX. The tissue specimen showed new bone formation on the bone surface near the implanted area. The concentrations of Mg, Ca, and P were high in the fibrous capsules surrounding Mg. Implantation induced differentiation of osteoblasts, and this process was considered to be associated with new bone formation. Induction of cell differentiation may be influenced by corrosion products in addition to corroding magnesium.

In vivo behavior of untreated and compressed concentrated growth factors as biomaterials in rabbits.

To characterize concentrated growth factors (CGFs) in vivo, we examined the degradation of implanted CGF in rabbits. Untreated CGF (U-CGF) and compressed CGF (C-CGF) were subcutaneously implanted into the dorsum. Histological analyses showed that the U-CGF and C-CGF induced very few inflammatory cells and that the U-CGF and C-CGF were subsequently degraded with dendritic invasion of granulation tissue. The C-CGF histopathologically remained for longer term than the U-CGF. Aggregated CD31+ and RAM11+ cells appeared in and around the implanted CGF. The number of macrophages and blood vessels in the CGF-implanted groups was greater than that in the sham group. There were more blood vessels in the U-CGF group than that in the C-CGF and sham group. We showed that CGF was degraded by macrophages in 4 weeks and enhanced angiogenesis with dendritically branching new capillaries. Therefore, the U-CGF and C-CGF can be clinically applied as a biomaterial inducing angiogenesis.

Florid Cemento-Osseous Dysplasia-Associated Simple Bone Cyst Showing Marked Irregular Border and High Apparent Diffusion Coefficient Value.

A simple bone cyst (SBC) is an intrabone cavity without epithelial lining, which occasionally occur with fibrous-osseous lesions. The imaging finding of the scalloped border and conserving the lamina dura, periodontal ligament spaces, or follicular spaces are considered radiographically pathognomonic of SBC. SBC has been reported to occur with fibrous-osseous lesions, including cemento-osseous dysplasia (COD). Computed tomography (CT) imaging findings are sparse, and there are no reports of magnetic resonance imaging (MRI) findings, including apparent diffusion coefficient (ADC) value calculated by diffusion-weighted MRI for the florid COD-associated SBC. We report a case of a 39-year-old woman who was referred to our hospital because a panoramic radiograph of her right mandible showed asymptomatic radiolucency in the apical molar area. CT images showed a low-density lesion in the periapical areas of the right mandible, which accompanied a well-defined, high-density lesion, and a mixed low/high-density lesion with an unusual marked irregular border in the left mandible. The MRI showed cyst-like extremely hyperintense signals on fat-suppressed T2-weighted images at the right mandibular low-density and the left mandibular mixed low/high-density areas. ADC map showed high values (over 2.5 × 10-3 mm2/s) at the cystic areas in the bilateral mandible. We performed incisional biopsies of the bilateral mandibular lesions and resections of the bilateral maxillary lesions. Surgical and histopathological findings established a diagnosis of florid COD in the bilateral mandible and maxilla, simultaneously complicated by multiple SBCs in the bilateral mandible. The ADC value of cystic component is useful for the diagnosis, if the COD-associated SBC exhibits solitary, nonspecific, or rare imaging findings.

Influence of oestrogen deficiency and excessive mechanical stress on condylar head of mandible.

OBJECTIVE: We assessed the aetiology of idiopathic condylar resorption by examining the effects of oestrogen and compressive mechanical stress under a low systemic oestrogen condition in temporomandibular joints (TMJ) caused by an ovariectomy. MATERIALS AND METHODS: Female rabbits were divided into non-ovariectomy (non-OVX) and ovariectomy (OVX) groups. A cortical osteotomy was performed with a custom device that was increased in length by 0.25 mm every 12 hr for 1 week after the operation, during which the TMJs in the rabbits received compressive mechanical stress. Samples from both groups were examined with micro-computed tomography and histological staining. RESULTS: Area and depth of bone resorption were both greater in the OVX group. Furthermore, a significantly earlier and greater prevalence of sub-condylar bone resorption was noted in that group, while cells positive for tartrate-resistant acid phosphatase were increased in the OVX group. CONCLUSIONS: The present findings suggest that oestrogen induced a much greater amount of bone resorption on the anterior surface of the condylar head at an earlier stage in the TMJs of the present model rabbits. Thereafter, restoration of TMJ function appeared to occur in a normal manner.

Immunohistochemical assessment of growth factor signaling molecules: MAPK, Akt, and STAT3 pathways in oral epithelial precursor lesions and squamous cell carcinoma.

Several growth factors and their receptors, such as epidermal growth factor receptor, have been studied as prognostic biomarkers for many epithelial malignancies. The signal transduction cascade of those receptors includes RAS/RAF/ERK, PI3K/Akt/mTOR, and STAT3 pathways. The aim of this study was to investigate the expression levels of several key proteins of those pathways in patients with oral squamous cell carcinoma (OSCC) and oral epithelial precursor lesions (OEPLs), and to correlate the expressions of these proteins with clinicopathologic features and prognosis. Fifteen leukoplakia (LP), 15 low-grade epithelial dysplasia, 15 high-grade epithelial dysplasia (HD), and 132 OSCC specimens were immunohistochemically examined for KRAS, HRAS, NRAS, BRAF, pERK1/2, pAkt, pmTOR, and pSTAT3 expression. Immunoreactivity for these molecules predominantly occurred in regions OEPL basal to prickle layers and in most OSCC cells. KRAS and NRAS expression was significantly lower in OSCC than in OEPLs, while pAkt and pmTOR showed higher expression in OSCC than in OEPLs. pERK1/2 expression was significantly higher in HD than in LP. In OSCC, KRAS and NRAS immunoreactivity was significantly higher in advanced age and male gender. In addition, higher immunoreactivity was shown in pERK1/2 in female gender and advanced TNM stage, pAkt in advanced T classification and cases without postoperative metastasis, pmTOR in advanced mode of invasion, and pSTAT3 in invasion depth. Correlations between these markers and clinicopathological variables were also noted. MAPK, Akt, and STAT3 pathways might play diverse roles in oral carcinogenesis.

Immunohistochemical assessment of Eph/ephrin expression in oral squamous cell carcinoma and precursor lesions.

To evaluate erythropoietin-producing hepatocellular carcinoma (Eph)/Eph receptor-interaction protein (ephrin) expression in oral squamous cell carcinoma (OSCC) and oral epithelial precursor lesions (OEPLs), EphA2, EphB4, and ephrinB2 were examined and compared with microvessel density (MVD) and lymphatic vessel density (LVD). Samples from 73 OSCC and 43 OEPLs patients were immunohistochemically analyzed with antibodies against EphA2, EphB4, ephrinB2, CD34, and D2-40. Results were compared with clinicopathological findings. Immunohistochemical reactivity for EphA2, EphB4, and ephrinB2 was detected in epithelial cells and some stromal vascular cells in OEPLs and OSCC, proportionately with the level of malignancy. The number of blood vessel endothelial cells stained with CD34 and lymphatic vessel endothelial cells stained with D2-40 was increased in OEPLs and OSCC. In OSCC, ephrinB2 and EphB4 exhibited significant correlation with recurrence and invasion depth, respectively. MVD was significantly lower in slight lymphocytic reaction than in prominent stromal reaction. Association was found between LVD and T classification, postoperative metastasis, survival, mode of invasion, and invasion depth. Expression of EphA2, EphB4, ephrinB2, MVD, and LVD might be associated with malignant potential of the oral epithelium. Angiogenesis and lymphangiogenesis appear to be related to progression of potentially malignant oral lesions.

Immunohistochemical assessment of chromatin licensing and DNA replication factor 1, geminin, and γ-H2A.X in oral epithelial precursor lesions and squamous cell carcinoma.

BACKGROUND: Carcinogenesis occurs when the cell cycle is compromised. Chromatin licensing and DNA replication factor 1, geminin, and γ-H2A histone family member X are expressed in cells in G1 phase, S/G2 /M phases, and apoptosis, respectively, and these three markers may be useful for histological evaluation of malignant lesions. Here, we aimed to identify cell cycle phases and apoptosis using immunohistochemistry in oral epithelial precursor lesions and oral squamous cell carcinoma. METHODS: Chromatin licensing and DNA replication factor 1, geminin, and γ-H2A histone family member X expression levels were immunohistochemically examined in tissue specimens from 55 patients with oral epithelial precursor lesions and 50 patients with oral squamous cell carcinoma. Associations of clinicopathological variables with marker expression were assessed. RESULTS: Chromatin licensing and DNA replication factor 1 was expressed in the prickle cell layer of oral epithelial precursor lesions and many carcinoma cells of oral squamous cell carcinoma. Geminin reactivity was widely distributed in high-grade dysplasia and oral squamous cell carcinoma rather than low-grade or no dysplastic cases. γ-H2A histone family member X was expressed in the superficial layer of oral epithelial precursor lesions and scattered carcinoma cells of oral squamous cell carcinoma. In oral squamous cell carcinoma, lower geminin expression was observed in recurrent cases. Geminin and γ-H2A histone family member X were associated with the degree of differentiation and mode of invasion. CONCLUSION: Chromatin licensing and DNA replication factor 1, geminin, and γ-H2A histone family member X expression levels were correlated with oral carcinogenesis; these markers were associated with clinicopathological behaviors in oral squamous cell carcinoma.

In vitro and in vivo analysis of the biodegradable behavior of a magnesium alloy for biomedical applications.

The present study was designed to investigate the biodegradation behavior of Mg alloy plates in the maxillofacial region. For in vitro analysis, the plates were immersed in saline solution and simulated body fluid. For in vivo, the plates were implanted into the tibia, head, back, abdominal cavity, and femur and assessed at 1, 2, and 4 weeks after implantation. After implantation, the plate volumes and the formed insoluble salt were measured via micro-computed tomography. SEM/EDX analysis of the insoluble salt and histological analysis of the surrounding tissues were performed. The volume loss of plates in the in vitro groups was higher than that in the in vivo groups. The volume loss was fastest in the abdomen, followed by the head, back, tibia, and femur. There were no statistically significant differences in the insoluble salt volume of the all implanted sites. The corrosion of the Mg alloy will be affected to the surrounding tissue responses. The material for the plate should be selected based on the characteristic that Mg alloys are decomposed relatively easily in the maxillofacial region.

Expression of immunoregulatory molecules PD-L1 and PD-1 in oral cancer and precancerous lesions: A cohort study of Japanese patients.

OBJECTIVE: An association of the programmed cell death-1 (PD-1) and its ligand PD-L1 with various types of malignant tumors has been established. This study aimed to investigate the role of the PD-L1/PD-1 pathway in oral squamous cell carcinoma (OSCC) and oral epithelial precursor lesions (OEPL). MATERIALS AND METHODS: We examined 106 OSCC and 79 OEPL specimens for PD-L1 and PD-1 expression by immunohistochemistry. The results were compared with clinicopathological features of OSCC patients. RESULTS: In OSCC and OEPL specimens, PD-L1 expression was detected predominantly in epithelial or carcinoma cells, whereas PD-1 expression was found mainly in infiltrating or stromal lymphocytes. Seventy-two OSCC (67.9%) and 21 OEPL (26.6%) specimens were positive for PD-L1, and 73 OSCC (68.9%) and 23 OEPL (29.2%) specimens were positive for PD-1. PD-L1 and PD-1 expression levels were significantly different between OEPL and OSCC specimens (P < 0.001). There were significant positive correlations between PD-L1 and PD-1 expression in OEPL and OSCC specimens (P < 0.001). PD-L1 and PD-1 immunoreactivity was significantly associated with tumor size (P < 0.05). PD-L1 and PD-1 immunoreactivity in cases with advanced TNM staging was significantly higher than that in low staging cases (P < 0.01). There were significant correlations between PD-L1 and PD-1 expression in OSCC specimens and pathological variables such as stromal lymphocytic reaction (P < 0.05) and invasion depth (P < 0.01). CONCLUSION: PD-L1 and PD-1 immunohistochemical status may be related to carcinogenesis, tumor progression, and prognosis in oral epithelial lesions. Agents targeting PD-1 and PD-L1 might be useful for OSCC treatment.

Peripheral clear cell variant of calcifying epithelial odontogenic tumor devoid of calcification.

A clear cell variant of calcifying epithelial odontogenic tumor (CCEOT) affecting an extraosseous site is described. A 60-year-old male patient presented with gingival swelling on the lingual side of the anterior mandible. The results of biopsy suggested clear cell odontogenic carcinoma, and marginal resection of the mandible was performed. The resected specimen was composed of eosinophilic and clear cells with deposits of amyloid-like material. The clear cells exhibited granules that were positive for PAS. There was no calcification in the resected lesion. Based on these features, the conclusive diagnosis was peripheral CCEOT without calcification. No signs of recurrence were evident after 3 years of follow-up.

Roles of Aryl Hydrocarbon Receptor in Aromatase-Dependent Cell Proliferation in Human Osteoblasts.

Aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor and its expression is influenced by environmental compounds, such as 3-methylcholanthrene (3-MC) and ß-naphthoflavone (ß-NF). AhR and its downstream genes, such as CYP1A1, are considered to play a pivotal role in xenobiotic responses. AhR signaling has also been proposed to mediate osteogenesis in experimental animals, but its details have remained unclear. Therefore, in this study, we examined the possible roles of AhR in human bone. Immunohistochemical analysis revealed that AhR was detected in both osteoblasts and osteoclasts. We then screened AhR-target genes using a microarray analysis in human osteoblastic hFOB cells. Results of microarray and subsequent PCR analysis did reveal that estrogen metabolizing and synthesizing enzymes, such as CYP1B1 and aromatase, were increased by 3-MC in hFOB and osteosarcoma cell line, MG-63. The subsequent antibody cytokine analysis also demonstrated that interleukin-1ß and -6 expression was increased by 3-MC and ß-NF in hFOB cells and these interleukins were well known to induce aromatase. We then examined the cell proliferation rate of hFOB and MG-63 cells co-treated with 3-MC and testosterone as an aromatase substrate. The status of cell proliferation in both hFOB and MG-63 cells was stimulated by 3-MC and testosterone treatment, which was also inhibited by an estrogen blocker, aromatase inhibitor, or AhR antagonist. These findings indicated that AhR could regulate estrogen synthesis and metabolism in bone tissues through cytokine/aromatase signaling.