Spheroid culture enhances osteogenic potential of periodontal ligament mesenchymal stem cells.

OBJECTIVE AND BACKGROUND: Human periodontal ligament mesenchymal stem cells (hPDLMSCs) are reported to be responsible for homeostasis and regeneration of periodontal tissue. Although hPDLMSCs are commonly cultured in monolayers, monolayer cultures have been reported as inferior to 3-dimensional cultures such as spheroids, which are spherical clusters of cells formed by self-assembly. The aim of this study was to examine the osteogenic phenotype of spheroids of hPDLMSCs, compared with monolayer cultures of hPDLMSC, in vitro and in vivo. MATERIAL AND METHODS: Spheroids were formed using microwell chips that were tagged with polyethylene glycol. Mesenchymal stem cell (MSC) markers in hPDLMSC spheroids were examined by flow cytometer. Real-time polymerase chain reaction analysis was examined to measure the expressions of stemness markers and osteogenesis-related genes in monolayer and spheroid-cultured hPDLMSCs. Immunofluorescence analysis was performed to confirm protein expressions of stemness markers in PDLMSC spheroids. Nodule formation assay, alkaline phosphatase (ALP) activity assay and transplantation assay in a mouse calvarial defect model were performed to confirm the osteogenic potential of hPDLMSC spheroids. To elucidate the mechanism of spheroid culture enhanced osteogenesis in hPDLMSCs with osteoinductive medium (OIM), a small interfering RNA (siRNA) assay targeted with secreted frizzled-related protein 3 (SFRP3) was examined. The levels of SFRP3 expression in monolayer and spheroid-cultured hPDLMSCs with OIM were measured by real-time polymerase chain reaction and western blotting analysis. ALP gene expression and ALP activity were examined in SFRP3-deficient hPDLMSC spheroids. RESULTS: The hPDLMSC spheroids expressed MSC markers, which were similar to hPDLMSCs grown in monolayer cultures. Intriguingly, the protein and mRNA expressions of transcription factors that regulate "stemness" were significantly increased in hPDLMSC spheroids, compared with hPDLMSCs in monolayer cultures. Nodule formation by hPDLMSCs was significantly increased in spheroid cultures grown with OIM, compared with monolayer-cultured hPDLMSCs. ALP activity and expression of osteogenesis-related genes were also significantly enhanced in hPDLMSC spheroids, compared with monolayer cultures. Treatment with hPDLMSC spheroids significantly enhanced new bone formation in a murine calvarial defect model, compared with hPDLMSCs in monolayer culture. Finally, to elucidate mechanisms by which spheroid culture enhances ALP activation in hPDLMSCs grown with OIM, an siRNA assay was used to manipulate expression of SFRP3, a Wnt signaling antagonist. Knockdown of SFRP3 suppressed ALP gene expression in hPDLMSCs grown in OIM; further, it suppressed ALP activity in spheroid culture. These data suggest that the enhancement of osteogenic potential in hPDLMSC spheroids is regulated through SFRP3-mediated ALP activation. CONCLUSION: Spheroid cultures of hPDLMSCs may be a novel and useful tool in regenerative medicine.

Gingival epithelial cells support osteoclastogenesis by producing receptor activator of nuclear factor kappa B ligand via protein kinase A signaling.

BACKGROUND AND OBJECTIVE: Periodontal disease is dental plaque-induced inflammatory disease of the periodontal tissues that results in bone loss in the affected teeth. During bone resorption, receptor activator of nuclear factor kappa B ligand (RANKL) is an essential factor that regulates osteoclastogenesis. Recently, we found that gingival epithelial cells (GECs) in periodontal tissue produce RANKL, the expression of which is regulated by tumor necrosis factor-α and protein kinase A signaling. In this study, we asked whether RANKL-producing GECs induce bone marrow macrophages (BMMs) to form osteoclasts in a co-culture system. MATERIAL AND METHODS: Ca9-22 GECs and osteoclast precursor BMMs were co-cultured with or without the protein kinase A signaling activator forskolin or inhibitor H89 to examine whether the RANKL-producing GECs could be induced to form osteoclasts, as determined using a pit formation assay. RESULTS: Osteoclasts formed spontaneously in co-cultures of Ca9-22 cells and BMMs, even in the absence of RANKL. The cells were cultured on bone slices for 14 d, at which time resorption pits were observed. Forskolin treatment significantly increased osteoclast numbers in these co-cultures, but forskolin alone did not induce osteoclast formation by BMMs. CONCLUSION: GECs producing RANKL are able to support osteoclastogenesis in an in vitro co-culture system using GECs and BMMs, in a process promoted by forskolin.

Ammonium hydrogen tartronate at 240 and 20 K.

The low-temperature structure determination of the title compound, alternatively called ammonium hydrogen hydroxypropanedioate, NH(4)(+) x C(3)H(3)O(5)(-), has revealed that the H atom involved in a very short asymmetric O--H...O hydrogen bond [O...O = 2.448 (2) A at 240 K and 2.4393 (10) A at 20 K] is disordered.

Novel, potent, and selective phosphodiesterase 5 inhibitors: synthesis and biological activities of a series of 4-aryl-1-isoquinolinone derivatives.

A novel class of potent and selective phosphodiesterase 5 (PDE5) inhibitors, 4-aryl-1-isoquinolinone derivatives, which have been designed by the comparison of the structure of cGMP and a previously reported 1-arylnaphthalene lignan, was disclosed. Among these compounds, methyl 2-(4-aminophenyl)-1,2-dihydro-1-oxo-7-(2-pyridinylmethoxy)-4-(3,4,5-trimethoxyphenyl)-3-isoquinoline carboxylate dihydrochloride (36a) exhibited potent PDE5 inhibitory activity (IC(50) = 1.0 nM) with high isozyme selectivities (IC(50) ratio: PDE1/PDE5 = 1300, PDE2/PDE5 > 10 000, PDE3/PDE5 > 10 000, PDE4/PDE5 = 4700, PDE6/PDE5 = 28). Compound 36a also showed the most potent relaxant effect on isolated rabbit corpus cavernosum (EC(30) = 7.9 nM). Compound 63 (T-1032), the sulfate form of 36a, was selected for further biological and pharmacological evaluation of erectile dysfunction.

An efficient synthesis of the anti-asthmatic agent T-440: a selective N-alkylation of 2-pyridone.

6,7-Diethoxy-1-[1-(2-methoxyethyl)-2-oxo-1,2-dihydropyridin- 4-yl]naphthalene-2,3-dimethanol [T-440, (1)] is a potential anti-asthmatic agent based on selective phosphodiesterase 4 inhibition. It was necessary for the further evaluation of 1 to develop an efficient synthetic route for 1, especially the construction of the 1-(2-methoxyethyl)-2-pyridone moiety. We examined an N-selective alkylation of pyridone derivative (2) in basic media. 2-Methoxyethylation of 2 with 2-methoxyethyl iodide utilizing LiH as the base gave predominantly an N-alkyl pyridone derivative (3a) in 82% yield (N/O-alkylation=92/8), which is compatible with an ab initio calculation of transition-state structures for the methylation of 2-pyridone. Single crystallization of a crude mixture of 3a and 4a furnished pure 3a, which is a key synthetic intermediate of 1.

Novel selective PDE IV inhibitors as antiasthmatic agents. Synthesis and biological activities of a series of 1-aryl-2,3-bis(hydroxymethyl)naphthalene lignans.

A series of 1-aryl-2,3-bis(hydroxymethyl)naphthalene lignans have been synthesized and evaluated for their ability to selectively inhibit PDE IV isolated from guinea pig. Replacement of the 1-phenyl ring by a pyridone ring led to marked improvement of their selectivity for PDE IV over PDE III. The compounds that were most potent and selective involved those bearing an N-alkylpyridone ring at C-1. These compounds also showed potent antispasmogenic activity without causing significant changes in heart rate in the guinea pig. The most potent compound was 6,7-diethoxy-2, 3-bis(hydroxymethyl)-1-[1-(2-methoxyethyl)-2-oxo-pyrid-4-yl]nap hth alene (17f), ED50 values of histamine-induced and antigen-induced bronchoconstriction in the guinea pig being 0.08 and 2.3 mg/kg iv, respectively. This compound was chosen as a candidate for further pharmacological evaluation.

[Gingival metastasis of large-cell lung cancer that produced G-CSF].

A 68-year-old man was referred to our hospital for further examination of a gingival mass. Chest radiographs and magnetic resonance imaging disclosed a bulky mass originating in the upper portion of the left lung, in contact with a chronic empyema lesion that first occurred after resection for pulmonary tuberculosis. Examination of a specimen obtained by percutaneous needle biopsy of the mass led to the diagnosis of large-cell carcinoma. Laboratory findings on admission showed marked leukocytosis (48,100/microliter) without evidence of severe a bacterial infection. The level of G-CSF in serum was abnormally high (246 pg/ml, normal value: < 30 pg/ml). Chemotherapy with vindesine, ifosfamide, and cisplatin resulted in shrinkage of the gingival mass, and a decrease in the G-CSF level to 66 pg/ml. Immunohistochemical staining with an anti-G-CSF monoclonal antibody to the primary lung tumor and the gingival mass obtained at autopsy was positive for cytoplasmic G-CSF.

[Six cases of primary pulmonary cryptococcosis].

Six patients with asymptomatic primary pulmonary Cryptococcosis are reported. In all of the patients, the disease was detected by annual chest X-ray during mass screening for lung cancer or during follow-up for pulmonary tuberculosis or gastric cancer. The chest X-ray findings consisted of a solitary pulmonary nodule in 4 patients and multiple pulmonary nodules in 2. Only one patient who could not be histologically diagnosed by bronchofiberscopy underwent surgical resection. However, the other 5 patients were histologically diagnosed by transbronchial biopsy with bronchofiberscopy. They were treated with oral antifungal agents, namely flucytosine (5-FC) and/or fluconazole, with marked improvement of chest X-ray findings. These results indicate that transbronchial biopsy with bronchofiberscopy and oral administration of antifungal agents instead of initial surgical resection are useful in the diagnosis and treatment of primary pulmonary cryptococcosis.

Outcome of patients with lung cancer detected via mass screening as compared to those presenting with symptoms.

We performed lung cancer resection in 721 patients between 1980 and 1989. Cancers were detected via mass screening programs by annual chest X-ray examination in the majority of cases. We evaluated the surgical results in patients with tumors detected by mass screening and compared them to those in whom the malignancy was detected by symptoms. Lesions in the mass screened group were T1 to T2 tumors in 90% of the cases, and NO in 73%. Stage I disease accounted for 65.3% in the mass screened group. The overall 5-year survival rate was 56.2% in the mass screened group, which was significantly better than the 25.3% for the symptom group (P less than 0.001). The surgical results in the lung cancer cases detected by the mass screening program had better results than the cases who presented with symptoms.

Dental findings of patients who underwent liver transplantation.

Cyclosporin-induced gingival enlargement was presented. Enlargement was noted at several sites in the mouth of five patients taking Cyclosporin following liver transplantation. Papillary gingival enlargement was observed to affect the entire maxilla and mandible. Enlargement was particularly marked in the interdental papillary region on the labial side of the anterior teeth of the maxilla. Discoloration of the teeth was observed. Radiographic findings were unremarkable. Oral management of these patients is the prevention of dental caries and infection originating in the area of gingival enlargement. It is important to begin proper brushing as early as possible for suppressing progression of gingival enlargement. For prevention of dental caries, prophylaxis is more important in the case of liver transplantation than with normal patients. It should be stressed to the patients or their parents that periodic examination, topical application of fluoride and pit and fissure sealants are of special importance.

[Dental treatment under general anesthesia of a patient with pulmonary atresia with intact ventricular septum].

A case of pulmonary atresia with intact ventricular septum in a 4-year-4-month boy, whose dental treatment was performed under general anesthesia, was reported. For the general anesthesia, the medication Digoxin was stopped 24 hours before the treatment. For the prevention of bacterial endocarditis, medication consisting on antibiotics of the penicillin series was administered 24 hours before the treatment. The anesthesia was administered with the modified N.L.A. method with Ketamine and Diazepam. The dental treatment went smoothly, and was finished within 1-hour-15 mins from the start. Anesthesia was administered for 1-hour-45 mins. The prognosis was good, and the patient was discharged the next day. It should be noted that as for performing dental treatment under anesthesia or not using anesthesia, there are some different opinions. We think that it must be discussed from the point of view of the effect of the dental treatment on the patient versus the danger of the anesthesia. In this case, we found that, under general anesthesia, stress of the patient was reduced, and the dental treatment was able to be performed smoothly and safely.

[The efficacy of X-ray signs for differential diagnosis of small lung cancer and tuberculoma. Committee for Lung Cancer Mass Screening, JATA].

In order to evaluate the efficacy of X-ray signs for the differential diagnosis of small lung cancer from tuberculoma, a cooperative study was carried out. X-ray films of 64 cases (lung cancer 35, tuberculoma 29) were read by 11 experienced chest physicians independently. The positivity of various X-ray signs were assessed respectively and obtained data were analysed with ROC analysis method. "Ill defined contour", "unevenness of density", "paleness" were proved to be relatively useful as a diagnostic tool, but "notch", "pleural indentation" were not useful in differentiating lung cancer from tuberculoma. It was also noted that great interindividual variations existed on the judgements of X-ray signs among chest specialists, and the conquest of which may be a crucial key for the universal validity of these signs.

Cytologic and histologic correlation in primary lung cancer. A study of 154 cases with resectable tumors.

The accuracy of cytologic diagnosis and typing was examined in 154 patients, 113 males and 41 females, who underwent radical surgery during the past six years. There were 42 central and 112 peripheral lesions: 6 adenocarcinomas and 28 squamous-cell carcinomas were centrally located and 69 adenocarcinomas and 27 squamous-cell carcinomas were peripherally located. Repetition of sputum sampling at least three times was preferred, especially in central lesions, which were detected in 57% to 64% of the cases by either three-day-pooled or aerosol-induced specimens. Peripheral lesions required brushing to enhance the accuracy. The overall typing accuracy was 64.3%, ranging from 83.6% in squamous-cell carcinoma to 25.0% in large-cell carcinoma. Cytologic positivity correlated well with the finding of tumors more than 3 cm in diameter. Adenocarcinoma and squamous-cell carcinoma showed no significant difference in frequency of regional lymph nodal metastases. The value of judging the accuracy of cytologic diagnosis and typing on the histologic evaluation of the entire resected lesion, rather than on biopsy specimens, is emphasized.