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.

Msx2 Prevents Stratified Squamous Epithelium Formation in the Enamel Organ.

Tooth enamel is manufactured by the inner enamel epithelium of the multilayered enamel organ. Msx2 loss-of-function mutation in a mouse model causes an abnormal accumulation of epithelial cells in the enamel organ, but the underlying mechanism by which Msx2 regulates amelogenesis is poorly understood. We therefore performed detailed histological and molecular analyses of Msx2 null mice. Msx2 null ameloblasts and stratum intermedium (SI) cells differentiated normally in the early stages of amelogenesis. However, during subsequent developmental stages, the outer enamel epithelium (OEE) became highly proliferative and transformed into a keratinized stratified squamous epithelium that ectopically expressed stratified squamous epithelium markers, including Heat shock protein 25, Loricrin, and Keratin 10. Moreover, expression of hair follicle-specific keratin genes such as Keratin 26 and Keratin 73 was upregulated in the enamel organ of Msx2 mutants. With the accumulation of keratin in the stellate reticulum (SR) region and subsequent odontogenic cyst formation, SI cells gradually lost the ability to differentiate, and the expression of Sox2 and Notch1 was downregulated, leading to ameloblast depolarization. As a consequence, the organization of the Msx2 mutant enamel organ became disturbed and enamel failed to form in the normal location. Instead, there was ectopic mineralization that likely occurred within the SR. In summary, we show that during amelogenesis, Msx2 executes a bipartite function, repressing the transformation of OEE into a keratinized stratified squamous epithelium while simultaneously promoting the development of a properly differentiated enamel organ competent for enamel formation.

Osteopontin Is Essential for Type I Collagen Secretion in Reparative Dentin.

Osteopontin (OPN) is a highly phosphorylated glycoprotein that is a prominent component of the mineralized extracellular matrix of bone. The secretion of OPN by immunocompetent cells plays a role in the differentiation of odontoblast-like cells during pulpal healing following tooth transplantation. This study aimed to clarify the role of OPN during reparative dentinogenesis. A groove-shaped cavity was prepared on the mesial surface of the upper first molars of wild-type (WT) and Opn knockout (KO) mice, and the samples were collected at intervals of 1 to 14 d. The demineralized sections were processed for immunohistochemistry for Ki67, nestin, OPN, dentin sialoprotein (DSP), integrin αvß3, and type I collagen; in situ hybridization for Opn, col1a1, and dentin sialophosphoprotein (Dspp); and apoptosis assay. For the loss and gain of function experiments, an in vitro culture assay for evaluating dentin-pulp complex regeneration was performed. On day 1 in WT mice, odontoblasts beneath the affected dentin lost nestin immunoreactivity. On day 3, the expression of Opn was recognized at the mesial dental pulp, and OPN was deposited along the predentin-dentin border. Nestin-positive newly differentiated odontoblast-like cells expressed both Dspp and col1a1 and showed positive immunoreactivity for integrin αvß3, DSP, and type I collagen. Until day 14, reparative dentin formation continued next to the preexisting dentin at the mesial coronal pulp. In contrast, there was no reparative dentin in the Opn KO mice where nestin- and DSP-positive newly differentiated odontoblast-like cells lacked immunoreaction for type I collagen. The in vitro organ culture demonstrated that the administration of recombinant OPN rescued the type I collagen secretion by odontoblast-like cells in the Opn KO mice. The results suggested that the deposition of OPN at the calcification front is essential for the type I collagen secretion by newly differentiated odontoblast-like cells to form reparative dentin during pulpal healing following cavity preparation.

Evc regulates a symmetrical response to Shh signaling in molar development.

Tooth morphogenesis involves patterning through the activity of epithelial signaling centers that, among other molecules, secrete Sonic hedgehog (Shh). While it is known that Shh responding cells need intact primary cilia for signal transduction, the roles of individual cilia components for tooth morphogenesis are poorly understood. The clinical features of individuals with Ellis-van Creveld syndrome include various dental anomalies, and we show here that absence of the cilial protein Evc in mice causes various hypo- and hyperplasia defects during molar development. During first molar development, the response to Shh signaling is progressively lost in Evc-deficient embryos and, unexpectedly, the response consistently disappears in a buccal to lingual direction. The important role of Evc for establishing the buccal-lingual axis of the developing first molar is also supported by a displaced activity of the Wnt pathway in Evc mutants. The observed growth abnormalities eventually manifest in first molar microdontia, disruption of molar segmentation and symmetry, root fusions, and delayed differentiation. Analysis of our data indicates that both spatially and temporally disrupted activities of the Shh pathway are the primary cause for the variable dental anomalies seen in patients with Ellis-van Creveld syndrome or Weyers acrodental dysostosis.

Sonic hedgehog signaling is important in tooth root development.

Hertwig's epithelial root sheath (HERS) is important for tooth root formation, but the molecular basis for the signaling of root development remains uncertain. We hypothesized that Sonic hedgehog (Shh) signaling is involved in the HERS function, because it mediates epithelial-mesenchymal interactions during embryonic odontogenesis. We examined the gene expression patterns of Shh signaling in murine developing molar roots. Shh and Patched2 transcripts were identified in the HERS, whereas Patched1, Smoothened, and Gli1 were expressed in the proliferative dental mesenchyme in addition to the HERS. To confirm whether Shh signaling physiologically functions in vivo, we analyzed mesenchymal dysplasia (mes) mice carrying an abnormal C-terminus of the PATCHED1 protein. In the mutant, cell proliferation was repressed around the HERS at 1 wk. Moreover, the molar eruption was disturbed, and all roots were shorter than those in control littermates at 4 wks. These results indicate that Shh signaling is important in tooth root development. Abbreviations used: BrdU, 5-bromo-2'-deoxyuridine; HERS, Hertwig's epithelial root sheath; NFI-C/CTF, nuclear factor Ic/CAAT box transcription factor; PCNA, proliferating cell nuclear antigen; Ptc, patched; Shh, sonic hedgehog; Smo, smoothened.

[The clinical evaluation of MTD false-positive & false-negative results].

The usefulness of MTD (Amplified Mycobacterium Tuberculosis Direct Test) for a rapid diagnosis of tuberculosis was evaluated. A total of 400 clinical samples obtained from July, 1995 to June, 1997 were tested by MTD, direct microscopy and culture. The results of MTD and smear/culture were coincident in 387 out of 400 samples. Eight samples (2%) were MTD false-positive (i.e. they were MTD positive but smear and culture negative), and 5 (1.25%) were MTD false-negative (i.e. MTD negative but smear and/or culture positive). Despite a careful review of the clinical data of those patients whose samples showed discrepant results, the reasons of discrepancy were not clear in 2 (0.5%) of the 8 false positives and 3 (0.75%) of the 5 false negatives. In the other cases, the MTD false positives may be accounted for the presence of previous M. tuberculosis infection, the influence of anti-tuberculous medication and so on, and the MTD false negatives are most likely due to the presence of inhibitors (blood, for example) or to the small number of organisms in the specimens. It can be concluded that adequate samples should be obtained, and that MTD should be repeated in case of discrepant results.

[A study of virulence factors produced by MRSA strains isolated from blood samples].

Toxic shock syndrome toxin-1 (TSST-1) and enterotoxins are important virulence factors produced by Staphylococcus aureus. It is reported that these toxins are associated with septic shock and toxic shock syndrome. We investigated the toxin production and coagulase types of 701 MRSA strains isolated in Sasebo City General Hospital between 1994 and 1996 TSST-1 or/and enterotoxins were detected in 67% of all MRSA strains, and those were detected in 88% of MRSA strains isolated from blood samples. 45% of all MRSA strains produced both TSST-1 and enterotoxin C, and 70% of MRSA strains obtained from blood produced those toxins. Frequency of TSST-1 or/and enterotoxin production by MRSA strains isolated from blood samples was significantly higher than that by MRSA strains isolated from urine and pharynx (p < 0.05), and frequency of both TSST-1 and enterotoxin C production by MRSA isolates from blood was significantly higher than that by MRSA strains isolated from pharyngeal sample (p < 0.05). This study indicated that investigation of virulence factors produced by MRSA might give the useful information on prevention and treatment of MRSA infection.

Right ventricular impairment in patients with chronic respiratory failure on home oxygen therapy--non-invasive assessment using a new Doppler index.

We evaluated the usefulness of the newly defined Doppler index combining systolic and diastolic myocardial performance, in assessing right-heart dysfunction in 29 patients with chronic respiratory failure caused by old tuberculosis who were on 24-h home oxygen therapy. We measured tricuspid inflow velocity, right-ventricular outflow velocity, late/early diastolic peak velocities (A/E), the ratio between pre-ejection period and ejection time (PEP/ET), and the new index of systolic and diastolic myocardial performance (SDMP) calculated as (isovolumetric contraction time + isovolumetric relaxation time)/ejection time. The calculated A/E, PEP/ET and SDMP in our patients were significantly higher than those in age-matched healthy subjects (n = 37, mean age 67 +/- 8 years). There was no overlap in the SDMP index between healthy subjects and patients and the index was not influenced by heart rate. Our results suggest that SDMP index is a better marker than A/E and PEP/ET for the assessment of right-ventricular impairment.

[Angioscopic findings of the peripheral pulmonary arteries in patients with chronic pulmonary diseases].

Angioscopic observation of the peripheral pulmonary arteries was performed in 14 patients with chronic lung diseases (male 9/female 5, age 70.8 +/- 8.3 years, pulmonary tuberculosis sequelae 6, chronic bronchitis 4, chronic pulmonary emphysema 3, pneumoconiosis 1). The blood flow was stopped by the inflated balloon of a 7F guiding catheter, and then the peripheral pulmonary arteries (inner diameter 1.0-4.5 mm) were observed with a fiberoptic catheter (outer diameter 0.7 mm). In 10 out of 14 patients abnormal findings were obtained, which included redness, erosion, ulceration of the vascular wall, thrombus, fibrous tissue, and occlusion of the lumen. The incidence of abnormal findings tended to be higher in chronic bronchitis and pulmonary tuberculosis sequelae than in chronic pulmonary emphysema. These results suggest that 1) various changes including "vasculitis" and thrombus exist in the peripheral pulmonary arteries of chronic lung diseases, and 2) that angioscopy may provide the detailed information about both the vascular wall and the lumen which cannot be obtained by pulmonary angiography.

[Extravascular thermal volume in pulmonary emphysema].

The extravascular thermal volume of the lung (ETVL) was measured by the heat-sodium double indicator dilution technique in 10 patients with chronic pulmonary emphysema. ETVL was 4.99 +/- 1.23 ml/kg, and correlated significantly with TLC(r = 0.747), %VC(r = 0.664), PaO2(r = 0.779), AaDO2(r = -0.796), shunt ratio(r = -0.7), and total pulmonary vascular resistance(r = 0.698). ETVL tended to decrease with the progression of the severity of emphysema as assessed by computed tomography. These results suggest that 1) the extravascular thermal volume decreases in chronic pulmonary emphysema parallel with an increase in the severity of the complaint, and that 2) the decrease of extravascular thermal volume may reflect a decrease in the size of the pulmonary capillary bed. It is concluded that the measurement of extravascular thermal volume may be useful for the evaluation of the severity of pulmonary emphysema.

[Pulmonary hemodynamics and home oxygen therapy in patients with chronic respiratory failure].

The authors examined pulmonary hemodynamics with respect to underlying diseases, severity and type of chronic respiratory failure, and the incidence and effect of home oxygen therapy (HOT) in 155 patients with chronic lung diseases (old pulmonary tuberculosis (OTB) 45, chronic pulmonary emphysema (CPE) 54, chronic bronchitis (CBR) 42 and fibrosing lung disease (FLD) 14). They underwent right heart catheterization during a stable period, while breathing room air. The arterial PO2 ranged from 64.3 +/- 9.7 Torr (CBR) to 69.9 +/- 10.0 Torr (CPE), and the mean pulmonary arterial pressure ranged from 17.3 +/- 4.6 mmHg (CPE) to 20.6 +/- 5.4 mmHg (OTB). The incidence of pulmonary hypertension (PH, PA mean greater than or equal to 20 mmHg) was 53.3% in OTB, 40% in CBR, 35.7% in FLD, 23.8% in CPE, 69% in respiratory failure, 40% in quasi-respiratory failure, and 2.1% in non-respiratory failure. The percentage of patients who received HOT was 84.5% in respiratory failure and 54.1% in quasi-respiratory failure. Comparing Type I with Type II chronic respiratory failure, the incidence of PH was lower in the former than the latter (38.3% vs 80.6%), whereas HOT was applied to an equal percentage of patients (67.4%) in both groups. The effect of HOT was evaluated in 11 patients with chronic respiratory failure. The mean pulmonary arterial pressure was 22.7 +/- 4.7 mmHg before HOT, and decreased to 20.7 +/- 5.6 mmHg after 24.5 +/- 10.1 months of HOT. Although this difference was not significant statistically, this result suggests the desirable effect of HOT on pulmonary hemodynamics.

[A cases of isoniazid-induced lupus].

A case of isoniazid (INH)-induced lupus occurring in a 62-year-old man is presented. He visited our hospital in May 1986 and a cavitary lesion was found in the right upper lobe on a chest roentgenogram. He had no previous history of treatment with antituberculotic agents. Though acid-fast bacilli were not found in his sputum, pulmonary tuberculosis was strongly suspected and INH, rifampicin and ethanbutol were administered. Four days after starting the treatment, minimal left pleural effusion was seen on chest X-ray film. Three months later he began to complain polyarthralgia in his digital joints. In a pleural effusion many lymphocytes were found; and the antinuclear antibody (ANA), the anti-extractable nuclear antigens (ENA) antibody, and the RNase resistant anti-ENA antibody were positive, and their titres were 20x, 1000x and 1000x, respectively, and the immune complex (IC) was 16.0 micrograms/ml (LT5). In blood serum, the ANA test the anti-ENA antibody and the RNase resistant anti-ENA antibody were positive with titres 40x, 640x and 640x respectively; and the IC was 14.0 micrograms/ml, and the RA test was positive. The improvement of clinical findings and disappearance of auto-antibodies seen after stopping INH confirmed the diagnosis as INH-induced lupus.

Ciprofloxacin therapy in cystic fibrosis.

There is great need for an oral agent that could be used to treat pulmonary exacerbations in patients with cystic fibrosis. In this study, the use of oral ciprofloxacin as sole therapy was evaluated in 18 patients with 39 infectious episodes; 13 episodes were classified as severe, 19 were classified as moderate, and seven were classified as mild. Patients ranged in age from eight to 36 years (mean, 23 years). Dosage varied according to severity of disease, body size, and the susceptibility of the Pseudomonas isolate to ciprofloxacin; the dose ranged from 750 to 2,250 mg daily (mean, 1,800 mg). Ten patients received one course of ciprofloxacin, and eight received repeated courses. The overall clinical response rate was 82 percent. There was a response to the initial treatment course in 96 percent of the patients. Those in whom therapy failed had been re-treated with ciprofloxacin and were severely ill. Failure to respond correlated poorly with pretreatment minimal inhibitory concentration (MIC) values (0.6 microgram/ml for failures versus 0.4 microgram/ml for responses). Pseudomonas could not be eradicated from the sputum of any of the patients, although there was a marked reduction in purulence and bacterial counts. In general, patients who did not require re-treatment for three months would again have susceptible organisms. When organisms became resistant to ciprofloxacin (MIC greater than 2 micrograms/ml), they showed no concomitant new aminoglycoside or beta-lactam resistance. No serious toxicity occurred in any of the 39 episodes of treatment. In seven patients treated with combination therapy (tobramycin or azlocillin), the infecting organisms were reduced in number, but eradication of Pseudomonas generally could not be achieved. Increases in MIC occurred during combination therapy. Ciprofloxacin is a major advance in the treatment of bronchopulmonary infection in patients with cystic fibrosis.

The effect of the combination of erythromycin with new beta-lactam antibiotics against gram-negative aerobic respiratory pathogens.

The effect of erythromycin on the in vitro activity of cefotaxime, ceftizoxime, cefoperazone, moxalactam, ceftazidime, cefmenoxime, aztreonam, imipenem, piperacillin, and gentamicin against 89 bacteria isolated from sputum and tracheal aspirates of patients admitted to intensive care units was evaluated. There were 30 Pseudomonas aeruginosa, 27 Klebsiella pneumoniae, 9 Enterobacter cloacae, 5 Escherichia coli, 4 Enterobacter aerogenes, 3 each of Klebsiella oxytoca, Pseudomonas maltophilia, Serratia marcescens, 2 each of Morganella morganii, Acinetobacter anitratus; 1 Proteus mirabilis. All isolates were resistant to erythromycin. Organisms were screened for synergy by fixed erythromycin concentration at 1, 8 and 16 micrograms/ml. There were 16 isolates that showed a greater than four-fold difference in MICs. These strains were analyzed by the checkerboard broth method. No antagonism was seen for any drug combination with erythromycin. One Enterobacter cloacae showed synergy of ceftizoxime and erythromycin, and 1 E. cloacae showed synergy with cefotaxime. Addition was found for 2 P. aeruginosa, 1 each E. aerogenes, E. coli, P. mirabilis, K. pneumoniae and S. marcescens. At concentrations of erythromycin achievable in blood or pulmonary tissue, the activity of newer beta-lactams and gentamicin is not altered by erythromycin.