Deep-learning algorithm to detect fibrosing interstitial lung disease on chest radiographs.

BACKGROUND: Antifibrotic therapies are available to treat chronic fibrosing interstitial lung diseases (CF-ILDs), including idiopathic pulmonary fibrosis. Early use of these treatments is recommended to slow deterioration of respiratory function and to prevent acute exacerbation. However, identifying patients in the early stages of CF-ILD using chest radiographs is challenging. In this study, we developed and tested a deep-learning algorithm to detect CF-ILD using chest radiograph images. METHOD: From the image archive of Sapporo Medical University Hospital, 653 chest radiographs from 263 patients with CF-ILDs and 506 from 506 patients without CF-ILD were identified; 921 were used for deep learning and 238 were used for algorithm testing. The algorithm was designed to output a numerical score ranging from 0 to 1, representing the probability of CF-ILD. Using the testing dataset, the algorithm's capability to identify CF-ILD was compared with that of doctors. A second dataset, in which CF-ILD was confirmed using computed tomography images, was used to further evaluate the algorithm's performance. RESULTS: The area under the receiver operating characteristic curve, which indicates the algorithm's detection capability, was 0.979. Using a score cut-off of 0.267, the sensitivity and specificity of detection were 0.896 and 1.000, respectively. These data showed that the algorithm's performance was noninferior to that of doctors, including pulmonologists and radiologists; performance was verified using the second dataset. CONCLUSIONS: We developed a deep-learning algorithm to detect CF-ILDs using chest radiograph images. The algorithm's detection capability was noninferior to that of doctors.

[Breast and Advanced Ovarian Double Cancer with a BRCA2 Pathogenic Variant-A Case Report].

A 49-year-old woman presented with abdominal pain. A computed tomography scan revealed a left breast mass with suspected peritoneal dissemination. She had no notable family history of the disease. Following a detailed examination, she was diagnosed with ovarian cancer(Stage ⅢC)and left breast cancer(Stage Ⅰ). After confirming the diagnosis of ovarian cancer with a laparoscopic biopsy, neoadjuvant chemotherapy was scheduled. Due to its efficiency in reducing tumor burden, debulking surgery was also performed. While receiving adjuvant chemotherapy for ovarian cancer, concomitant anastrozole was administered for breast cancer. A pathogenic variant of BRCA2 was subsequently identified. Once adjuvant chemotherapy for ovarian cancer had been completed, a left mastectomy and sentinel lymph node biopsy were performed as management for breast cancer. The patient will continue treatment with anastrozole for breast cancer and olaparib for ovarian cancer and will be followed up appropriately.

Cytoplasmic expression of maspin predicts unfavourable prognosis in patients with squamous cell carcinoma of the lung.

AIMS: Maspin is known to be a tumour suppressor protein, and its prognostic significance in patients with several types of cancer, including lung squamous cell carcinoma (SCC), has been reported. However, its prognostic impact on lung SCC has been controversial. We explored the prognostic value of maspin expression with particular reference to its subcellular localization in patients with lung SCC. METHODS AND RESULTS: Paraffin-embedded tissue samples from 101 curatively resected patients with lung SCC were analysed immunohistochemically using an antibody for maspin. Maspin positivity was defined as strong expression in only the cytoplasm and observed in 25 patients (24.6%). It correlated significantly with the presence of lymph node metastasis (P = 0.006) and higher pathological stage (P = 0.003). The patients were followed-up for 2-119 months (median: 50 months), and the maspin-positive group had shorter disease-free survival (DFS) and disease-specific survival (DSS) by log-rank test (P = 0.002, P = 0.016, respectively). Multivariate analysis revealed that the status of maspin was the only independent prognostic factor for DFS and DSS (P = 0.017, P = 0.047, respectively). CONCLUSIONS: Cytoplasmic expression of maspin could be an independent unfavourable prognostic indicator in patients with lung SCC.

Cytoplasmic maspin expression is a predictor of poor prognosis in patients with lung adenocarcinoma measuring <3 cm.

AIMS: Maspin is known to be a tumour suppressor protein, and few studies focused upon its prognostic significance in patients with small-size lung adenocarcinoma have been reported; however, its clinical significance remains controversial. We explored the prognostic value of maspin with particular reference to its subcellular localization in patients with resected lung adenocarcinoma measuring <3 cm. METHODS AND RESULTS: Immunohistochemical analyses were performed on resected 110 specimens of lung adenocarcinoma measuring <3 cm. Maspin positivity was defined as strong expression in only the cytoplasm and was observed in 27 patients (24.5%). It correlated significantly with the presence of lymph node metastasis (P = 0.009) and micropapillary component (P < 0.001). The patients were followed-up for 6-88 months (median: 71 months), and the maspin-positive group had shorter disease-free survival (DFS) and overall survival (OS) by log-rank test (P < 0.001, P < 0.001, respectively). Using Cox's multivariate analysis, the status of maspin was an independent prognostic factor for DFS and OS (P = 0.004, P = 0.022, respectively), as well as lymph node metastasis. CONCLUSIONS: Cytoplasmic maspin expression could be an independent poor prognostic indicator of patients with lung adenocarcinoma measuring <3 cm.

TGF-ß in dentin matrix extract induces osteoclastogenesis in vitro.

Previously, we have demonstrated that the extracellular matrix from dentin affects osteoclastic activity in co-culture between osteoclast and osteoblast-rich fraction from mouse marrow cells. In the present study, we aimed to investigate the mechanisms of dentin matrix extract-induced osteoclastogenesis in mouse bone marrow macrophages (BMMs). Dentin proteins were extracted from bovine incisor root dentin using 0.6 M HCl. BMMs were cultured in α-MEM containing macrophage colony-stimulating factor/receptor activator of nuclear factor kappa-B ligand in the presence or absence of dentin matrix extract. Tartrate-resistant acid phosphatase (TRAP)-positive cell number, total TRAP activity, and the mRNA levels of osteoclast-related genes, assayed by real-time RT-PCR, were determined as markers of osteoclastogenesis. A neutralizing antibody against transforming growth factor-ß1 (TGF-ß1), SB431542, a TGF-ß receptor inhibitor, and ELISA were used to determine the role of TGF-ß1. We observed increases in TRAP-positive cell number, TRAP activity, and the mRNA levels of osteoclast-related genes of BMMs cultured with dentin extract. The use of a neutralizing antibody against TGF-ß1 or SB431542 inhibited the inductive effect of dentin extract, suggesting TGF-ß1 involvement. The addition of exogenous TGF-ß1, but not bone morphogenic protein-2, also increased osteoclastogenesis, corresponding to the ELISA determination of TGF-ß1 in the dentin extract. In conclusion, our results indicate that proteins from dentin matrix have an inductive effect in osteoclastogenesis, which is mediated, in part, by TGF-ß1.

A fluorescence spotlight on the clockwork development and metabolism of bone.

Biological phenomena that exhibit periodic activity are often referred as biorhythms or biological clocks. Among these, circadian rhythms, cyclic patterns reflecting a 24-h cycle, are the most obvious in many physiological activities including bone growth and metabolism. In the late 1990s, several clock genes were isolated and their primary structures and functions were identified. The feedback loop model of transcriptional factors was proposed to work as a circadian core oscillator not only in the suprachiasmatic nuclei of the anterior hypothalamus, which is recognized as the mammalian central clock, but also in various peripheral tissues including cartilage and bone. Looking back to embryonic development, the fundamental architecture of skeletal patterning is regulated by ultradian clocks that are defined as biorhythms that cycle more than once every 24 h. As post-genomic approaches, transcriptome analysis by micro-array and bioimaging assays to detect luminescent and fluorescent signals have been exploited to uncover a more comprehensive set of genes and spatio-temporal regulation of the clockwork machinery in animal models. In this review paper, we provide an overview of topics related to these molecular clocks in skeletal biology and medicine, and discuss how fluorescence imaging approaches can contribute to widening our views of this realm of biomedical science.

The effect of chewing exercise in preschool children on maximum bite force and masticatory performance.

BACKGROUND: Mastication is a developing function affected by various factors. There is a need for further research on methods of promoting masticatory function in young children. AIM: The aim of this study was to evaluate the effects of gum chewing exercise on the maximum bite force (MBF) and the masticatory performance of preschool children. DESIGN: The study population included 98 preschool children age 4-6years. MBF was measured by Occlusal Force-Meter(®) , and masticatory performance values were evaluated by using the colour-changeable chewing gum. The examinations were performed four times with an interval of 4weeks. An exercise group of 70 subjects was instructed to chew the exercise gum twice daily for 5min during a 4-week period. The chewing gum used for this study was specially developed with the physical property of maintaining hardness during chewing. A control group of 28 subjects was instructed not to chew any gum during the study period. RESULTS: No significant differences were found between the exercise group and the control group in MBF and a* values at the start of the study. After 4weeks of chewing exercise, MBF and a* values were significantly increased in the exercise group compared with those of the control group. These increases were maintained for 4weeks after exercise had finished. CONCLUSIONS: Gum chewing exercise is effective to increase MBF and a* values of preschool children and the effects are maintained after exercise completion.

Objective assessment of internal stress in children during dental treatment by analysis of autonomic nervous activity.

AIMS: The aim of this study was to assess the internal stress of children during dental treatment based on autonomic nerve activity and facial muscle activity. METHODS: We recorded the electrocardiogram of children during the treatment of composite resin restoration and analysed autonomic nerve activity by means of power spectral analysis of heart rate variability. Simultaneously, electromyography (EMG) activity of the corrugator muscle was recorded in children during dental treatment, and the relationship between sympathetic nerve activity and corrugator EMG activity was analysed. RESULTS: In all subjects, the mean sympathetic nerve activity was significantly higher during oral examination and after treatment compared with pre-treatment. Depending on the sympathetic nerve responses to the other treatment procedures, the subjects could be classified into two groups: the stress group and the nonstress group. Sympathetic nerve activity was significantly higher during infiltration anaesthesia and cavity preparation compared with pre-treatment activity in the stress group, whereas it was consistently lower than the pre-treatment levels during most treatment procedures in the nonstress group. The mean amplitudes of the averaged corrugator muscle EMG during dental treatment did not differ between the stress and nonstress groups. CONCLUSION: The present results suggest that the measurement of autonomic nervous activity, especially sympathetic nervous activity, is quite useful in assessing the internal stress of children, even when no expressed sign of unease are present during dental treatment.

Wear of resin composites and primary enamel and their applicability to full crown restoration of primary molars.

PURPOSE: To determine whether resin composites are appropriate for full crown restoration of primary molars by evaluating their wear characteristics. Specifically, the wear properties of resin composite specimens and the opposing enamel surfaces were characterized by means of impacting-sliding wear testing. METHODS: Three types of light-cured resin composites (Estelite Sigma quick, Litefill IIP, and Metafil C), one type of chemical-cured resin composite (Clearfil FII), and a hybrid composite (Estenia C&B) were tested in this study. The enamel sample was used as the control. The hemispherically prepared specimens were subjected to impacting-sliding wear testing against the flattened enamel of primary molars. The worn surfaces were examined by laser scanning microscopy and scanning electron microscopy. The volumetric loss was estimated by using micro-CT images. The areas of worn enamel surfaces were measured by 3D color laser microscopy. On most of the worn enamel surfaces, cracks appeared. Scatter plot analyses between their width and depth were carried out. Data for each specimen were statistically analyzed by multiple comparisons among the means of treatment by Bonferroni's method (P< 0.01). RESULTS: Clearfil showed significantly higher surface area wear, volumetric loss, and worn enamel surface area than did the other resin composites and the control enamel (P< 0.01). There was no significant difference among the worn surface areas ofEstelite, Litefill, Metafil, and Estenia (P< 0.01). The control enamel showed significantly lower worn surface area than did the resin composites (P< 0.01). There was no significant difference in volumetric loss and worn enamel surface areas among Estelite, Litefill, Metafil, Estenia, and the control enamel (P< 0.01). Cracks larger than that on the control enamel were seen on the worn enamel surface opposing Estenia.

Comparison between robot-assisted thoracoscopic surgery and video-assisted thoracoscopic surgery for mediastinal and hilar lymph node dissection in lung cancer surgery.

OBJECTIVES: Lymph node dissection (LND) with robot-assisted thoracoscopic surgery (RATS) in lung cancer surgery has not been fully evaluated. The aim of this study was to compare LND surgical results between video-assisted thoracoscopic surgery (VATS) and RATS. METHODS: We retrospectively compared perioperative parameters, including the incidence of LND-associated complications (chylothorax, recurrent and/or phrenic nerve paralysis and bronchopleural fistula), lymph node (LN) counts and postoperative locoregional recurrence, among 390 patients with primary lung cancer who underwent lobectomy and mediastinal LND by RATS (n = 104) or VATS (n = 286) at our institution. RESULTS: The median total dissected LN numbers significantly differed between the RATS and the VATS groups (RATS: 18, VATS: 15; P < 0.001). They also significantly differed in right upper zone and hilar (#2R + #4R + #10L) (RATS: 12, VATS: 10; P = 0.002), left lower paratracheal and hilar (#4L + #10L) (RATS: 4, VATS: 3; P = 0.019), aortopulmonary zone (#5 + #6) (RATS: 3, VATS: 2; P = 0.001) and interlobar and lobar (#11 + #12) LNs (RATS: 7, VATS: 6; P = 0.041). The groups did not significantly differ in overall nodal upstaging (P = 0.64), total blood loss (P = 0.69) or incidence of LND-associated complications (P = 0.77). CONCLUSIONS: In this comparison, it was suggested that more LNs could be dissected using RATS than VATS, especially in bilateral superior mediastinum and hilar regions. Accumulation of more cases and longer observation periods are needed to verify whether RATS can provide the acceptable quality of LND and local control of lung cancer.

A rare case of combined thymoma and a multilocular thymic cyst discovered due to chest pain.

BACKGROUND: A thymoma with chest pain and multilocular thymic cysts (MTCs) is very rare. CASE PRESENTATION: A 49-year-old man presented to another hospital complaining of an anterior chest pain. Chest computed tomography (CT) showed an anterior mediastinal tumor 60 × 30 × 55 mm in size. The boundary with the pericardium or left brachiocephalic vein seemed to be partially unclear while enhanced by the contrast medium, and so the tumor could have invaded them. No definitive diagnosis of myasthenia gravis was made although the serum anti-acetylcholine receptor antibody count was high. We performed an extended thymectomy with combined partial resection of left brachiocephalic vein, left upper lobe, and left phrenic nerve. He was discharged with no chest pain and no complications post-surgery. The tumor was pathologically type B2 thymoma with hemorrhage necrosis and MTCs, and we diagnosed Masaoka stage II because of no histological infiltration to the organs. CONCLUSIONS: We speculated that hemorrhagic necrosis due to infarction in tumor caused the inflammation to spread to the surrounding organs, which was related to the chest pain and the development of MTCs.

Three-Dimensional Comparison in Palatal Forms Between Modified Presurgical Nasoalveolar Molding Plate and Hotz's Plate Applied to the Infants With Unilateral Cleft Lip and Palate.

The presurgical nasoalveolar molding plate appliance with stent (PNAM) extended from the palatal molding plate; to correct the nostril shape of infants with cleft lip and palate is well known. The PNAM appliance is based on the finding that a high degree of plasticity is maintained in the cartilage of infants during the first 6 weeks after birth. However, on the current PNAM protocol described by Grayson et al. the nasal stent is supposed to be an adjunct to the palatal molding plate after reducing the severity of the alveolar cleft width. We have used the modified Hotz's plate from the setup model and built up the nasal stent even before reducing the severity of the alveolar deformity. In this study we assess the effects of the modified Hotz's plate and the modified PNAM appliance for the alveolar and palatal form. The lateral deviation of the incisal point, the width of the palatal cleft, and the degree of curvature of the palatal vault were first evaluated on plaster models. The PNAM group is smaller on the lateral deviation of the incisal point than the modified Hotz's group. The decreased average width of the palatal cleft and curvature of the palate, was almost the same in both the modified Hotz's and PNAM groups. In comparison with the modified Hotz's plate, the modified PNAM appliance also improves the molding of the alveolar segments and reduces cleft width.

Transformation from adenocarcinoma to squamous cell carcinoma associated with long-term administration of EGFR-TKIs.

Although patients with non-small cell lung cancer exhibiting EGFR mutations generally respond to tyrosine kinase inhibitors (TKIs), the majority of patients acquire resistance ~1 year after treatment. EGFR T790M mutations, MET or HER2 amplifications and phenotypic transformations contribute to the mechanism of EGFR-TKI resistance. The transformation of small cell lung cancer frequently occurs, although few convert to squamous cell carcinoma associated with the administration of EGFR-TKIs. The current study reports a case of EGFR-mutated adenocarcinoma of the lung that transitioned to squamous cell carcinoma in association with long-term EGFR-TKIs administration.

Comparison of medium-term survival outcomes between robot-assisted thoracoscopic surgery and video-assisted thoracoscopic surgery in treating primary lung cancer.

OBJECTIVES: Robot-assisted thoracoscopic surgery (RATS) for primary lung cancer has been spreading rapidly in Japan. While RATS has various technical advantages over video-assisted thoracoscopic surgery (VATS), the quality of surgery from an oncologic viewpoint must be maintained, and the evaluation of medium- to long-term survival outcomes is momentous. METHODS: This study included 299 patients with primary lung cancer (VATS, n = 246; RATS, n = 53) who underwent lobectomy and mediastinal lymph node dissection at our hospital. We reviewed and compared perioperative factors, the number of dissected lymph nodes, and postoperative recurrence between the VATS and RATS groups. We also compared the postoperative survival rates among 98 patients (49 patients in each group) whose background factors were adjusted by propensity score matching (PSM). RESULTS: After PSM, a significant difference was found in the total operative time between the two groups (p < 0.01); however, no difference was found between the RATS console time and VATS total operative time (p = 0.68). Postoperative recurrence was not significantly different between the groups (VATS, 14%; RATS, 6%; p = 0.18). The 3-year recurrence-free survival rate was not significantly different between the groups (VATS, 91.7%; RATS, 91.2%; p = 0.21). CONCLUSIONS: RATS had promising survival results, which were similar to those of VATS even after PSM. Further case series and longer observation periods are needed to determine what technical benefits of RATS may affect the prognosis of lung cancer.

Absolute transmitted light plethysmography for assessment of dental pulp vitality through quantification of pulp chamber hematocrit by a three-layer model.

After confirming that the gingival circulation had little effect on transmitted light plethysmography measurement in the upper central incisor in both in vivo experiments and numerical Monte Carlo simulation studies, a three-layer model comprising of a pulp chamber sandwiched between two dentin layers has been introduced to quantify the pulp chamber hematocrit (Hctp) from the measured optical density. Two-flux theory was utilized to derive a mathematical equation for transmitted intensity in terms of tooth dimensions, Hctp, and light-source wavelength. Each layer was assumed homogeneous so as to represent its optical properties by the bulk absorption and scattering constants. The mean error between the Hctp estimate based on the three-layer-model equation and the Hctp actual in the extracted model tooth was -0.00115 with standard deviation (SD) of 0.00733 at 522 nm wavelength, while for 810 nm +0.09157 and 0.02493. The Hctp estimate of the upper central incisor in 10 young volunteers at 522 nm using the three-layer model ranged from 0.002 to 0.061 with the mean of 0.032. The Hctp change reflects blood volume shift in the pulp microcirculation to possibly indicate dental pulp vitality.

Initial Results of Robotic Surgery for Primary Lung Cancer: Feasibility, Safety and Learning Curve.

BACKGROUND: At the end of 2016, robot-assisted thoracoscopic surgery (RATS) was still not covered by Japanese national health insurance. Therefore, few institutions in Japan perform RATS and even fewer have reported procedures as they occurred earlier. So, we decided to focus on the initial results of RATS for primary lung cancer. METHODS: We retrospectively reviewed 44 patients who underwent RATS for primary lung cancer from January 2011 to August 2016. After mastering the initial procedure, we introduced a completely portal robotic pulmonary resection procedure using a carbon dioxide insufflation system. Cases were divided into 2 groups: the early period (20 cases) and the later period (24 cases). RESULTS: There was no case of conversion to video-assisted thoracoscopic surgery or thoracotomy. In the 44 cases of primary lung cancer, median operating time was 239.5 min, console time was 179 min, blood loss was 10 mL, drainage period was 2 days, morbidity of Grade 2 or more (Clavien-Dindo classification) was 18.2%, morbidity of Grade 3 or more was only 4.6%, and there was no 30-day mortality. Median operating and console times were significantly shorter in the later period (215 min and 159.5 min, respectively) than in the initial period (300.5 min and 228 min, respectively). Median blood loss was significantly lower in the later period (5 mL) than in the initial period (50 mL). Five-year overall and disease-free survival rates were 100% and 88.9%, respectively. CONCLUSION: RATS for primary lung cancer is feasible and safe, has a faster learning curve, and provides satisfactory. Studies with longer follow-ups and larger numbers of cases are necessary.

Effect of turpentine-induced fever during the enamel formation of rat incisor.

OBJECTIVE: Some epidemiological studies have indicated that diseases resulting in prolonged and sustained fever, such as exanthemata, respiratory infections and otitis media in infantile period or childhood are likely to have a marked deleterious effect on enamel formation, but the relationship between fever and enamel defects is unknown. The purpose of the present study was to induce a persistent high fever and examine the effects on the developing tooth enamel. METHODS: Twenty male Wistar rats weighting 140+/-10 g were used in this study. For the experimental group, a dose of 2.3 ml/kg steam-distilled turpentine was subcutaneously injected into both hind limbs five times at 12h intervals. Control rats received 2.3 ml/kg of sterile saline into the same injection site. The rectal temperatures of animals were measured at the febrile period. After constant periods, the animals were sacrificed, and the mandibular incisors were examined by contact microradiography (CMR) and histological observation. RESULTS: The febrile state lasted for 57 h and the average temperature rose 1.51 degrees C higher than that of the control group. The ground sections, semi-thin and ultra-thin sections of mandibular incisors were prepared and the enamel was observed. The microradiographs showed a radiolucent line along with the incremental line in the enamel. Moreover, microscopic examination indicated disorientation of enamel prism and crystal-free area within this radiolucent lesion. CONCLUSIONS: Persistent high fever pattern was established firmly by the turpentine injections and the process of enamel formation was influenced by the febrile period.

[Inspection by infection control team of the University Hospital, Faculty of Dentistry, Tokyo Medical and Dental University].

Factors affecting infection are the existence of infectious microorganisms, sensitivity of hosts, number of microorganisms, and infectious routes. Efforts to prevent infection focus on not allowing these factors to reach the threshold level. Inspection by an infection control team (ICT) of a hospital is one countermeasure for preventing nosocomial infection. We summarize here the problems for complete prevention of nosocomial infection based on the results of inspection by our ICT, so that staff working in the hospital can recognize the importance of preventing nosocomial infection. The following were commonly observed problems in our clinics found by the ICT : (1) incomplete practice of standard precautions and/or isolation precautions, (2) noncompliance with guidelines for the prevention of cross-infection, and (3) inappropriate management of medical rejectamenta. Infection control can be accomplished by strictly observing the standard precautions and isolation precautions. The ICT inspection round in the hospital could be an effective metaff working in the hod to clarify and overcome the problems involved in infection.

Prognostic Significance of Solid and Micropapillary Components in Invasive Lung Adenocarcinomas Measuring ≤3 cm.

BACKGROUND/AIM: We aimed to analyze the clinical impact of solid and micropapillary components in a series of Japanese patients resected for ≤3 cm lung adenocarcinoma. PATIENTS AND METHODS: A total of 115 patients with ≤3 cm lung adenocarcinomas were reviewed and classified according to the American Thoracic Society and the European Respiratory Society classification. The presence of solid (S+) or micropapillary component (MP+) was defined when the component constituted ≥1% of the entire tumor. The impact of these components on disease-free (DFS) and disease-specific (DSS) survival was analyzed. RESULTS: Thirty (26.1%) cases with S+ and 27 (23.5%) with MP+ were identified, and multivariate analysis indicated that S+ status significantly reduced the duration of DFS and DSS. In 86 patients of acinar- and papillary-predominant subgroups, S+ and/or MP+ had the most significant effect on DFS and DSS by multivariate analysis. CONCLUSION: S+ and/or MP+ status predict worse prognosis in patients with acinar- and papillary-predominant lung adenocarcinoma.

Effects of retinoic acid on the differentiation of chondrogenic progenitor cells, ATDC5.

Chondrocyte differentiation is a fundamental process during endochondral ossification. Retinoic acid (RA) has been shown to regulate this process, however, the mechanisms underlying RA regulation of chondrogenesis are not clearly understood. Chondroprogenitor cells, ATDC5 have been shown to be a useful in vitro model for examining the multiple step differentiation of chondrocytes. The present study investigated the mechanisms underlying RA regulation of chondrogenesis using ATDC5 cell culture. In this study, we show that RA suppresses the cell growth, cartilage nodule formation, accumulation of proteoglycan, alkaline phosphatase (ALPase) activity and mineralization and that RA dose dependently upregulates the levels of type X collagen and matrix metalloproteinase-13 (MMP-13) mRNA which are marker proteins of hypertrophic chondrocytes, in ATDC5 cells. The addition of protein synthesis inhibitor, cycloheximide (CHX), partially inhibits the induction of type X collagen and MMP-13 mRNA by RA. In this system, RA upregulates the mRNA level of Runx2/Cbfa1 (type II), a positive regulator for mineralization, and downregulates the mRNA of Indian hedgehog (Ihh), parathyroid hormone related protein (PTHrP), negative regulators for terminal differentiation. However, RA downregulates ALPase, bone gla protein (BGP) mRNAs and mineralization. These data indicate that RA stimulates cartilage differentiation, however, cell condensation and cartilage nodule formation may be candidates of primary importance in the terminal differentiation of chondrocytes.