Not single but periodic injections of synovial mesenchymal stem cells maintain viable cells in knees and inhibit osteoarthritis progression in rats.

OBJECTIVE: We investigated the effects of single or repetitive intra-articular injections of synovial mesenchymal stem cells (MSCs) on a rat osteoarthritis (OA) model, and elucidated the behaviors and underlying mechanisms of the stem cells after the injection. DESIGN: One week after the transection of the anterior cruciate ligament (ACL) of wild type Lewis rats, one million synovial MSCs were injected into the knee joint every week. Cartilage degeneration was evaluated with safranin-o staining after the first injection. To analyze cell kinetics or MSC properties, luciferase, LacZ, and GFP expressing synovial MSCs were used. To confirm the role of MSCs, species-specific microarray and PCR analyses were performed using human synovial MSCs. RESULTS: Histological analysis for femoral and tibial cartilage showed that a single injection was ineffective but weekly injections had significant chondroprotective effects for 12 weeks. Histological and flow-cytometric analyses of LacZ and GFP expressing synovial MSCs revealed that injected MSCs migrated mainly into the synovium and most of them retained their undifferentiated MSC properties though the migrated cells rapidly decreased. In vivo imaging analysis revealed that MSCs maintained in knees while weekly injection. Species-specific microarray and PCR analyses showed that the human mRNAs on day 1 for 21 genes increased over 50-fold, and increased the expressions of PRG-4, BMP-2, and BMP-6 genes encoding chondroprotective proteins, and TSG-6 encoding an anti-inflammatory one. CONCLUSION: Not single but periodic injections of synovial MSCs maintained viable cells without losing their MSC properties in knees and inhibited osteoarthritis (OA) progression by secretion of trophic factors.

Monoiodoacetic acid induces arthritis and synovitis in rats in a dose- and time-dependent manner: proposed model-specific scoring systems.

OBJECTIVE: In a rat monoiodoacetic acid (MIA)-induced arthritis model, the amount of MIA commonly used was too high, resulting in rapid bone destruction. We examined the effect of MIA concentrations on articular cartilage and infrapatellar fat pad (IFP). We also established an original system for "macroscopic cartilage and bone score" and "IFP inflammation score" specific to the rat MIA-induced arthritis model. DESIGN: Male Wistar rats received a single intra-articular injection of MIA in the knee. The amount of MIA was 0.1, 0.2, 0.5, and 1 mg respectively. Articular cartilage was evaluated at 2-12 weeks. IFP was also observed at 3-14 days. RESULTS: Macroscopically, low MIA doses induced punctate depressions on the cartilage surface, and cartilage erosion proceeded slowly over 12 weeks, while higher MIA doses already induced cartilage erosion at 2 weeks, followed by bone destruction. MIA macroscopic cartilage and bone score, OARSI histological score, and Mankin score increased in a dose- and time-dependent manner. The IFP inflammation score peaked at 5 days in low dose groups, then decreased, while in high dose groups, the IFP score continued to increase over 14 days due to IFP fibrosis. CONCLUSIONS: Punctate depressions, cartilage erosion, and bone destruction were observed in the MIA-induced arthritis model. The macroscopic cartilage and bone scoring enabled the quantification of cartilage degeneration and demonstrated that MIA-induced arthritis progressed in a dose- and time-dependent manner. IFP inflammation scores revealed that 0.2 mg MIA induced reversible synovitis, while 1 mg MIA induced fibrosis of the IFP body.

IL-1ß-induced matrix metalloproteinase-3 regulates cell proliferation in rat dental pulp cells.

OBJECTIVE: We previously reported that matrix metalloproteinase-3(MMP-3) accelerates wound healing following dental pulp injury. In this study, we tested the hypothesis that induction of MMP-3 activity by interleukin-1ß would promote proliferation and apoptosis of dental pulp cells. MATERIALS AND METHODS: Dental pulp cells were isolated from rat incisors and subjected to interleukin-1ß. Matrix metalloproteinase-3 mRNA and protein expression were assessed using reverse transcription-polymerase chain reaction and Western blotting, respectively. Matrix metalloproteinase-3 activity was measured using fluorescence. Dental pulp cell proliferation and apoptosis were determined using enzyme-linked immunosorbent assays (ELISA) for BrdU and DNA fragmentation, respectively. siRNA was used to reduce MMP-3 transcripts in these cells. RESULTS: Treatment with interleukin-1ß increased MMP-3 mRNA and protein levels as well as its activity in dental pulp cells. Cell proliferation was also markedly increased, with no changes in apoptosis observed. Treatment with siRNA against MMP-3 potently suppressed this interleukin-1ß-induced increase in MMP-3 expression and activity, and also suppressed cell proliferation but unexpectedly increased apoptosis in these cells (P < 0.05). This siRNA-mediated increase in apoptosis could be reversed with exogenous MMP-3 stimulation (P < 0.05). CONCLUSIONS: Interleukin-1ß induces MMP-3-regulated cell proliferation and suppresses apoptosis in dental pulp cells.

Cytokines induce MMP-3-regulated proliferation of embryonic stem cell-derived odontoblast-like cells.

OBJECTIVES: Matrix metalloproteinase (MMP)-3 expression increases after pulpectomy and accelerates angiogenesis in rat dental pulp by an uncharacterised mechanism. Odontoblasts, a major component of dental pulp, could represent a therapeutic target. We investigated whether MMP-3 activity is induced by cytokines and/or is associated with cell proliferation and apoptosis in embryonic stem cell-derived odontoblast-like cells. MATERIALS AND METHODS: We used reverse transcriptase polymerase chain reaction, western blotting, an MMP-3 activity assay, a BrdU-cell proliferation enzyme-linked immunosorbent assay and DNA fragmentation analysis to evaluate siRNA-mediated downregulation of MMP-3 expression and activity, and any changes in the proliferative and apoptotic responses associated with this reduced expression. RESULTS: Pro-inflammatory cytokines (interleukin-1ß, tumour necrosis factor-α and interferon-γ, at relatively low concentrations) induced MMP-3 mRNA and protein expression, and increased MMP-3 activity and cell proliferation, but not apoptosis. MMP-3 silencing produced a potent and significant suppression of cytokine-induced MMP-3 expression and activity, decreased cell proliferation and increased apoptosis. These effects were rescued by application of exogenous MMP-3. CONCLUSIONS: Our results suggest that pro-inflammatory cytokines induce MMP-3-regulated cell proliferation and anti-apoptosis effects in odontoblast-like cells derived from embryonic stem cells, in addition to their well-documented destructive role in inflammation.

Mouse ES cells have a potential to differentiate into odontoblast-like cells using hanging drop method.

OBJECTIVES: We examined whether mouse embryonic stem (ES) cells can differentiate into odontoblast-like cells without epithelial-mesenchymal interaction. MATERIALS AND METHODS: Cells were cultured by the 'hanging drop' method using a collagen type-I scaffold (CS) combined with bone morphogenetic protein (BMP)-4 (CS/BMP-4). Expression of odontoblast-related mRNA and protein, and cell proliferation were performed by reverse transcription-polymerase chain reaction (RT-PCR), immunofluorescence staining and WST-1 assay, respectively. RESULTS: Cells potently expressed odontoblast-related cell marker mRNAs following induction of odontoblastic differentiation. Dentin sialophosphoprotein, a marker of mature odontoblasts, was strongly expressed in differentiated ES cells. The cells also acquired an odontoblast-like functional phenotype, as evidenced by the appearance of alkaline phosphatase activity and calcification. The cell-surface expression of α2, α6, αV and αVß3 integrin proteins was rapidly upregulated in differentiated cells. Finally, anti-α2 integrin antibody suppressed the expression of odontoblastic markers in cells grown using this culture system, suggesting that α2 integrin expression in ES cells triggers their differentiation into odontoblast-like cells. CONCLUSIONS: Mouse ES cells cultured by the 'hanging drop' method are able to differentiate into cells with odontoblast-specific physiological functions and cell-surface integrin protein expression.

Dual roles for NF-kappaB activation in osteoblastic cells by serum deprivation: osteoblastic apoptosis and cell-cycle arrest.

To clarify the mechanisms of osteoblastic cell death, we examined whether serum deprivation would cause activation of the apoptotic signal cascade and arrest of the cell cycle in mouse osteoblastic MC3T3-E1 cells. Serum withdrawal from osteoblastic cell cultures resulted in growth arrest and cell-cycle arrest at G0/G1, which actions were accompanied by transient and potent activation of NF-kappaB, caspase-8, caspase-2, caspase-3, and caspase-9 in this order. Apoptosis, but not necrosis, in serum-deprived cells could be detected by FACS using Annexin-V/propidium iodine double staining. Serum deprivation also resulted in transient activation of the 20S proteasome, which is an important component for regulation of the cell cycle by the ubiquitin-proteasome system. The 20S proteasome inhibitor (PSI) but not NF-kappaB inhibitor SN50 suppressed the activation of proteasomes in serum-deprived cells. Although caspase inhibitors could not prevent the G0/G1 arrest in the serum-deprived cells, SN50 and the 20S proteasome inhibitor could block it. Since SN50, 20S proteasome inhibitor and caspase inhibitor could rescue cells from serum deprivation-induced apoptosis, the pathway for NF-kappaB/caspase activation is independent of the NF-kappaB/cell-cycle pathway, and the events downstream of the NF-kappaB/caspase-9 cascade lead to apoptosis. Taken together, our present results identify a novel role for NF-kappaB in cell-cycle and apoptosis regulation and underscore the significance of each independent signal cascade in serum-deprived osteoblastic cells.

Evaluation of duodenal papillary function by ERCP-CT and comparison with endoscopic measurement of pressure in the papillary sphincter zone.

Imaging of the main pancreatic duct on ERCP-CT (computerized tomography performed immediately after endoscopic retrograde cholangiopancreatography) was evaluated in 192 patients. Favorable images were obtained in elderly patients, as well as in those with main pancreatic duct dilation, chronic pancreatitis, papillary disease, and choledocholithiasis. In addition, we used a microtransducer inserted through a duodenoscope to measure papillary sphincter zone and main pancreatic duct pressure. We then compared the endoscopic results with the images achieved on ERCP-CT. The patients with favorable images showed higher pancreatic and papillary basal pressure and a faster wave cycle than those with poor images. In addition, significant differences in pancreatic duct pressure and basal pressure were seen. Peak papillary pressure showed no consistent correlations with imaging. Patients with favorable images exhibited an increase in the frequency of irregular papillary sphincter zone waves. We conclude from these findings that good images on ERCP-CT reflect a slow flow of pancreatic juice, which is closely associated with papillary stenosis.

Ultrastructural identification of light microscopic giant mitochondria in alcoholic liver disease.

Ultrastructural identification of light microscopic giant mitochondria was performed on the same specimens for light and electron microscopic observations. The liver tissue specimens were fixed in OsO4, embedded in epoxy resin, cut 4 microns thick and stained with polychrome. At the beginning of the study a light microscopic observation was made, and a microphotograph was taken. The identification of light microscopic giant mitochondria by conventional microscopy was identified by the occupation rate in liver cells, the negative findings of stainability and the morphological consistency (round, cigar-shaped and granular). The specimens were subsequently embedded again in epoxy resin and cut into ultrathin sections of 400 A. A transillumination electron microscope was used for the observation, and ultrastructural images of light microscopic giant mitochondria revealed that they were crystalloid bodies with a crystalline latticelike structure. The occupation rates within liver cells and the morphological shapes of the crystalloid bodies corresponded with those of light microscopic giant mitochondria. The light microscopic giant mitochondria obviously had different features from those of electron microscopic giant mitochondria and Mallory bodies (Yokoo's type II), although Mallory bodies showed the same staining properties as light microscopic giant mitochondria.

[The usefulness of diving reflex--the effect on PSVT].

We attempted to use the diving reflex to treat PSVT in five patients, in whom carotid-sinus massage had failed to terminate, PSVT. In three cases PSVT converted to normal sinus rhythm. The patient was seated comfortably, and a pan of ice and water (4 degrees C) was placed on the table in front of the patient. He then took a deep breath and, without expiration, submerged his face in the cold ice and water for about 30 seconds. In case 1, a 68-year-old male, PSVT stopped after facial immersion of 11 seconds. Subsequently, sinus arrest with junctional or ventricular contractions continued for 7 seconds followed by conversion to the sinus rhythm. The duration of facial immersion was 18 seconds. In case 2, a 51-year-old male, PSVT converted to the sinus rhythm in the same way. The duration of facial immersion was 23 seconds. Case 3 was a 27-year-old female with WPW syndrome. PSVT converted to the sinus rhythm after a 6.6 seconds facial immersion. Case 4 and 5 were respectively 72 and 73-year-old males, whose attacks did not respond to the diving reflex for the reason that they could not maintain breath-holding for a sufficient time to activate the reflex adequately. We postulated that apnea might effect the termination of PSVT in case 1 and 2 because relatively long duration of about 20 seconds was necessary to convert PSVT to the sinus rhythm. On the other hand, in case 3, cold water stimulation on the face might play an important role because PSVT converted to the sinus rhythm immediately.(ABSTRACT TRUNCATED AT 250 WORDS)

[A case of spontaneous pneumomediastinum in a trombonist].

A case of spontaneous mediastinal emphysema in a 21-year-old female trombonist was reported. During light work, she experienced pain in her neck which later radiated into her chest. She had neither causal disease nor episode of straining at the onset of her work. On admission, physical examination revealed subcutaneous emphysema over the upper part of the chest and neck, and mediastinal crunch on auscultation (Hamman's sign). Roentgenograms revealed the presence of a considerable amount of air in the mediastinum and this extended upward through the mediastinum into the soft tissue of the neck bilaterally. The chest and neck CT yielded clearer information concerning the location and degree of mediastinal emphysema. She was treated with bed rest and recovered completely within five days. Spontaneous mediastinal emphysema without causal disease or apparent precipitating episode is infrequently recognized. In our case, though the trombonist had no apparent straining episode, the causative factor can be assumed to be the tenderness of the alveoli originating from frequent over-inflations of the lungs and high intra-alveolar pressures of about 150 cmH2O during trombone performance, which may result in alveolar rupture under normal intralveolar pressures.

Measurement of portal blood flow by an ultrasonic "duplex" system composed of a pulsed Doppler flowmeter and a linear-type real time B mode electroscanner.

The portal vein hemodynamics of patients with various liver diseases were investigated by means of a duplex system consisting of a linear electroscanner and a pulsed Doppler flowmeter. In cases of chronic liver disease, the cross-sectional area of the portal vein trunk became greater as the liver injury proceeded, while the maximum velocity tended to decrease. However, blood flow volume was no different between the controls and the chronic liver disease group. On the other hand, the splenic venous flow volume tended to increase as the liver injury advanced, suggesting that the increase in splenic venous flow volume is closely related to the formation of esophageal varices. This method permits non-invasive observation of changes during the course of various types of liver disease.