Purified Human Dental Pulp Stem Cells Promote Osteogenic Regeneration.

Human dental pulp stem/progenitor cells (hDPSCs) are attractive candidates for regenerative therapy because they can be easily expanded to generate colony-forming unit-fibroblasts (CFU-Fs) on plastic and the large cell numbers required for transplantation. However, isolation based on adherence to plastic inevitably changes the surface marker expression and biological properties of the cells. Consequently, little is currently known about the original phenotypes of tissue precursor cells that give rise to plastic-adherent CFU-Fs. To better understand the in vivo functions and translational therapeutic potential of hDPSCs and other stem cells, selective cell markers must be identified in the progenitor cells. Here, we identified a dental pulp tissue-specific cell population based on the expression profiles of 2 cell-surface markers LNGFR (CD271) and THY-1 (CD90). Prospectively isolated, dental pulp-derived LNGFR(Low+)THY-1(High+) cells represent a highly enriched population of clonogenic cells--notably, the isolated cells exhibited long-term proliferation and multilineage differentiation potential in vitro. The cells also expressed known mesenchymal cell markers and promoted new bone formation to heal critical-size calvarial defects in vivo. These findings suggest that LNGFR(Low+)THY-1(High+) dental pulp-derived cells provide an excellent source of material for bone regenerative strategies.

Differential cellular localization of antioxidant enzymes in the trigeminal ganglion.

Because of its high oxygen demands, neural tissue is predisposed to oxidative stress. Here, our aim was to clarify the cellular localization of antioxidant enzymes in the trigeminal ganglion. We found that the transcriptional factor Sox10 is localized exclusively in satellite glial cells (SGCs) in the adult trigeminal ganglion. The use of transgenic mice that express the fluorescent protein Venus under the Sox10 promoter enabled us to distinguish between neurons and SGCs. Although both superoxide dismutases 1 and 2 were present in the neurons, only superoxide dismutase 1 was identified in SGCs. The enzymes relevant to hydrogen peroxide degradation displayed differential cellular localization, such that neurons were endowed with glutathione peroxidase 1 and thioredoxin 2, and catalase and thioredoxin 2 were present in SGCs. Our immunohistochemical finding showed that only SGCs were labeled by the oxidative damage marker 8-hydroxy-2'-deoxyguanosine, which indicates that the antioxidant systems of SGCs were less potent. The transient receptor potential vanilloid subfamily member 1 (TRPV1), the capsaicin receptor, is implicated in inflammatory hyperalgesia, and we demonstrated that topical capsaicin application causes short-lasting mechanical hyperalgesia in the face. Our cell-based assay revealed that TRPV1 agonist stimulation in the presence of TRPV1 overexpression caused reactive oxygen species-mediated caspase-3 activation. Moreover, capsaicin induced the cellular demise of primary TRPV1-positive trigeminal ganglion neurons in a dose-dependent manner, and this effect was inhibited by a free radical scavenger and a pancaspase inhibitor. This study delineates the localization of antioxidative stress-related enzymes in the trigeminal ganglion and reveals the importance of the pivotal role of reactive oxygen species in the TRPV1-mediated caspase-dependent cell death of trigeminal ganglion neurons. Therapeutic measures for antioxidative stress should be taken to prevent damage to trigeminal primary sensory neurons in inflammatory pain disorders.

Repeated longitudinal in vivo imaging of neuro-glio-vascular unit at the peripheral boundary of ischemia in mouse cerebral cortex.

Understanding the cellular events evoked at the peripheral boundary of cerebral ischemia is critical for therapeutic outcome against the insult of cerebral ischemia. The present study reports a repeated longitudinal imaging for cellular-scale changes of neuro-glia-vascular unit at the boundary of cerebral ischemia in mouse cerebral cortex in vivo. Two-photon microscopy was used to trace the longitudinal changes of cortical microvasculature and astroglia following permanent middle cerebral artery occlusion (MCAO). We found that sulforhodamine 101 (SR101), a previously-known marker of astroglia, provide a bright signal in the vessels soon after the intraperitoneal injection, and that intensity was sufficient to detect the microvasculature up to a depth of 0.8 mm. After 5-8 h from the injection of SR101, cortical astroglia was also imaged up to a depth of 0.4 mm. After 1 day from MCAO, some microvessels showed a closure of the lumen space in the occluded MCA territory, leading to a restructuring of microvascular networks up to 7 days after MCAO. At the regions of the distorted microvasculature, an increase in the number of cells labeled with SR101 was detected, which was found as due to labeled neurons. Immunohistochemical results further showed that ischemia provokes neuronal uptake of SR101, which delineate a boundary between dying and surviving cells at the peripheral zone of ischemia in vivo. Finally, reproducibility of the MCAO model was evaluated with magnetic resonance imaging (MRI) in a different animal group, which showed the consistent infarct volume at the MCA territory over the subjects.

Postoperative nerve conduction changes after open and endoscopic carpal tunnel release.

OBJECTIVE: To assess the improvement of motor distal latency (MDL), sensory nerve conduction velocity (SCV) of the median nerve and the amplitudes of compound muscle action potential (CMAP) and sensory nerve action potential (SNAP) in patients with idiopathic carpal tunnel syndrome subjected to surgical treatment according to the open carpal tunnel release method and the endoscopic carpal tunnel release. METHODS: Sixty-six hands of sixty-six patients were divided into two groups: the ECTR group and the OCTR group. The patients were evaluated preoperatively, and at 1, 3, 6, and 12 months postoperatively. RESULTS: Although no statistically significant difference of the recovery of MDL and the amplitude of CMAP and SNAP was detected between the two groups at any time point during follow-up, one patient in the ECTR group in whom the operation had been converted to OCTR, showed delay of MDL and decrease in the amplitude of CMAP. CONCLUSIONS: There is a risk of nerve damage in patients undergoing ECTR. Although statistical analysis suggests that nerve conduction improves by about the same degree 12 months after ECTR or OCTR, slightly faster improvement after OCTR cannot be excluded.

Anionic sites in articular cartilage revealed by polyethyleneimine staining.

Articular cartilage is a unique tissue that contains neither blood vessels nor nerves, and that performs mechanical loading during joint movement. These properties are endowed by abundant glycosaminoglycans (GAGs), which are capable of retaining water-soluble substances. The GAGs attach to core proteins and form proteoglycans. Although many studies have focused on proteoglycans and collagen fibrils in cartilage, little is known about the nature of the negative charge of GAGs. Recently, we investigated this subject using a cationic dye, polyethyleneimine (PEI), with several different techniques such as pre-embedding, post-embedding, and quick-freezing and deep-etching methods. In addition, we investigated whether the anionic charge is altered at low pH, using PEI and cationic colloidal gold (CCG) labeling. The shapes of PEI-positive structures revealed by the pre-embedding method varied at different pHs. Three-dimensional analysis using the quick-freezing and deep-etching method demonstrated that meshwork structures composed of fine filaments were decorated with tiny PEI granules. Additionally, the meshwork structure was broken down after chondroitinase ABC digestion. These data indicate that the large PEI deposits observed in pre-embedding preparations are, at least in part, artificial images, and that the meshwork structure consists of chondroitin sulfate-retaining anionic sites. Low pH conditions changed PEI or CCG labeling patterns, showing that negative charges of GAGs in articular cartilage are altered under environmental pH conditions. These findings demonstrate that binding capacities of anionic sites to water-soluble or ionic substances are greatly affected by pH alterations without actually decreasing the number of anionic sites. Therefore, to understand cartilage dynamics and the pathogenesis of joint diseases in greater detail, alterations of anionic charge during mechanical loading or under pathological conditions should be examined in future studies.

Ultrastructural study of upper surface layer in rat articular cartilage by "in vivo cryotechnique" combined with various treatments.

The ultrastructures of the upper surface layer of rat articular cartilage were studied with our "in vivo cryotechnique" followed by freeze-substitution method for scanning electron microscopy (SEM) or transmission electron microscopy (TEM). Rat hip or knee articular cartilage was quickly frozen by the in vivo cryotechnique with liquid isopentane-propane cryogen (-193 degrees C), and surface areas of some frozen specimens were freeze-fractured with a scalpel in liquid nitrogen. They were freeze-substituted and freeze-dried, ion-sputtered, and then observed in SEM. Other frozen specimens were routinely freeze-substituted and embedded in epoxy resin for TEM. Many globular structures were detected in the thick upper surface layer that had not been revealed by the conventional fixation methods. Their sizes were reduced by Triton X-100 treatment, and their localization was also detected in synovial fluid, as revealed by SEM. Such globular lipid-like structures in the upper surface layer of hip or knee articular cartilage might contribute to joint lubrication.

Nucleocapsid formation and/or subsequent conformational change of rabies virus nucleoprotein (N) is a prerequisite step for acquiring the phosphatase-sensitive epitope of monoclonal antibody 5-2-26.

We investigated the antigenic maturation of rabies virus N protein, for which we used some conformational epitope-specific monoclonal antibodies (MAbs) and an MAb (5-2-26) against a phosphorylation-dependent linear epitope. Infected cells were lysed with a deoxycholate-free lysis buffer and separated by ultracentrifugation into the soluble top and the nucleocapsid fractions. None of the study MAbs recognized N proteins in the top fraction, whereas nucleocapsid-associated N proteins were recognized by all of the MAbs. Immunoprecipitation with polyclonal anti-N antibodies coprecipitated the P proteins from the top fraction, indicating that soluble N proteins are mostly associated with the P protein. The N proteins dissociated from both the N-P complex and nucleocapsids were recognized by none of the study MAbs, whereas the MAb 5-2-6 recognized the SDS-denatured N proteins of the nucleocapsid but not of the top fraction. In addition, the phosphorylation-deficient mutant N proteins were shown to be similarly accumulated as the wild-type N proteins into the viral inclusion bodies, defined as the virus-specific structures composed of viral nucleocapsids, that are produced in the cytoplasm of the infected cells. Based on these results, we believe that newly synthesized N proteins are not immediately phosphorylated at serine-389 (a common phosphorylation site) but are first associated with the P protein. After being used for encapsidation of the viral RNA, the N proteins undergo conformational changes, whereby epitopes for the conformation-specific MAbs are formed and become phosphorylated at serine-389.

Longitudinal Median Nerve Conduction Studies After Endoscopic Carpal Tunnel Release.

Forty hands of 36 patients who had undergone endoscopic carpal tunnel release (ECTR), utilising Chow's two-portal technique after being diagnosed with idiopathic carpal tunnel syndrome, were subjected to longitudinal median nerve conduction studies. The distal motor latency (DML) was examined pre-operatively on all the hands, which were re-examined at the post-operative 1st, 3rd, 6th and 12th months. Rapid improvement of DML was observed post-operatively in the first three months. These improvements patterns are not much different from those after open carpal tunnel release (OCTR) reported in the literatures. We consider that the data reported herein can be used as standards of DML course after ECTR.

Utilization of a partially threaded Kirschner wire in the treatment of femoral neck fractures.

OBJECTIVE: This report introduces a new type of pin for treatment of femoral neck fractures. DESIGN: Retrospective analysis. METHODS: The pin used in this study has a tapered thread in its middle portion to facilitate fixation in the lateral femoral cortex, as well as a simple tip that allows percutaneous pinning without displacement of the reduced position. Between 1978 and 1993, we used our pins on 218 hips in 214 patients, all age sixty years or more. We were able to observe postoperative osteosynthesis in 176 hips (172 patients) during a mean follow-up period of thirty-two months; the incidence of osteosynthesis and local postoperative complications was also charted. RESULTS: Osteosynthesis was obtained in 153 hips (87 percent); for groups based on Garden's classification, results were as follows: twenty-four of twenty-four hips in type 1, fifteen of sixteen in type 2, sixty-three of seventy-one in type 3, and fifty-one of sixty-five in type 4. Some local postoperative complications were observed, including second fractures at the pin insertion point (three hips), pin penetration of the femoral head (twenty-four hips), and pin slippage (three hips). CONCLUSION: This pin is effective in the treatment of femoral neck fractures and has the added benefits of being minimally invasive and low in cost.

Studies on the rabies virus RNA polymerase: 2. Possible relationships between the two forms of the non-catalytic subunit (P protein).

We investigated the relationship between the two forms of rabies virus P protein, a non-catalytic subunit of rabies virus RNA polymerase. The two displayed different electrophoretic mobilities as 37- and 40-kDa polypeptides, hence termed as p37 and p40, respectively. Double labeling experiments with [3H]leucine and [32P]orthophosphate demonstrated that p40 was much more phosphorylated than p37. Treatment of the virion proteins with alkaline phosphatase eliminated only p40, and not 37-kDa polypeptide. The p37 was a major product of the P gene, and was accumulated in the infected cell and incorporated into the virion. On the other hand, p40 was apparently detected only in the virion, and little detected in the cells. Treatment of infected cells with okadaic acid, however, resulted in significant accumulation of p40 in the cell, suggesting that p40 was continuously produced in the cell but dephosphorylated quickly. We detected both 37- and 40-kDa products in P cDNA-transfected animal cells, while only a 37-kDa product was produced in Escherichia coli. Incubation of 37-kDa products from E. coli with the lysates of animal cells in vitro resulted in the production of a 40-kDa product, which was also shown to be suppressed by the heparin. From these results, it is suggested that p40 is produced by the hyperphosphorylation of a 37-kDa polypeptide, which depends on certain heparin-sensitive cellular enzyme(s) and occurs even in the absence of the other viral gene products, and that p40 is reverted quickly to p37 in the infected cells, probably being dependent on some virus-induced factor(s).

Nontraumatic bilateral first rib fractures.

The authors report the case of a 21-year old woman who presented bilateral spontaneous fractures of the first ribs, in the posterior portion on the right side, in the anterior portion on the left side. The pathogenesis of spontaneous fractures of the first rib is discussed.

A histochemical study of anionic sites in the intermediate layer of rat femoral cartilage using polyethyleneimine at different pH levels.

Anionic sites in the intermediate layer of young rat hyaline cartilages were examined using a cationic dye, polyethyleneimine (PEI), at different pH levels. Femoral heads were resected and fixed in 2.5% glutaraldehyde and treated with 0.5% PEI at pH 7.4, pH 2.5 or pH 1.0. Some cartilage samples were first digested with chondroitinase ABC or hyaluronidase. The PEI deposits at pH 7.4 appeared to be irregular shapes. Their sizes seemed to be larger than those at pH 2.5 or pH 1.0. The PEI deposits were also found on the surface of collagen fibrils at both pH 7.4 and pH 2.5 even after the chondroitinase ABC digestion, but were not found at pH 1.0. Moreover, they disappeared after hyaluronidase digestion. Accordingly, it is suggested that PEI-positive structures varied depending on pH levels. In addition, hyaluronan may be localized near collagen fibrils, but most sulphated proteoglycans may not.

Proteoglycans in articular cartilage revealed with a quick freezing and deep etching method.

OBJECTIVES: To clarify the three dimensional ultrastructure of proteoglycans, and their relationship with other matrix components in articular cartilage. METHODS: Specimens from rat femoral heads were examined using three techniques: (1) Histochemical staining with cationic polyethyleneimine (PEI), using a pre-embedding or a postembedding method. Some tissues were pretreated with chondroitinase ABC or hyaluronidase. (2) Quick freezing and deep etching (QF-DE). Some specimens were fixed with paraformaldehyde and washed in buffer solution before quick freezing; others were frozen directly. (3) Ultrathin sections were studied after conventional preparation. RESULTS: Proteoglycans were observed as aggregated clumps with PEI staining by the pre-embedding method, but as fine filaments by the postembedding method. They were lost with enzyme digestion; this was also demonstrated by the QF-DE method. The ultrastructure was well preserved by the QF-DE method when fixation and washing procedures were included, but not without these procedures. A fine mesh-like structure was connected to the cell membrane in the pericellular matrix. Filamentous structures suggestive of aggrecans were observed among collagen fibrils. They had side chains, approximately 50 nm in length, which branched from the central filaments at intervals of 10-20 nm, and were occasionally linked to other structures. Many thin filaments were also attached to the collagen fibrils. CONCLUSIONS: The QF-DE method incorporating paraformaldehyde fixation and buffer washing procedures revealed three dimensional, extended structures suggestive of proteoglycans.

Radiographic factors predicting non-union of displaced intracapsular femoral neck fractures.

After 82 patients underwent osteosynthesis with multiple pinning for a displaced intracapsular femoral neck fracture, 58 fractures were united, and 24 were not. These 82 patients were included in a discriminant analysis. Union and non-union groups were the dependent variables. Eleven presumably predictive factors measured on X-ray films obtained from these patients before and immediately after operation were taken as independent variables. Three radiographic factors were selected by a stepwise method according to the effect they had on union. These were: the distance of medial displacement between fragments before reduction; the varus angle after reduction; and the distance of medial displacement between fragments after reduction. The distance of medial displacement between fragments before reduction proved the most important factor in making a choice between osteosynthesis and primary arthroplasty, and it was revealed that osteosynthesis could not be expected to provide union if the distance was 20 mm or more. It was also confirmed that union was promoted, if the cranial fragment was reduced to a valgus position and the medial cortical bones were aligned as much as possible at the time of osteosynthesis.

Efficacy of ivermectin against live mites and eggs of Sarcoptes scabiei in pigs.

Sows infested with Sarcoptes scabiei var. suis (ssvs) were treated with 75, 150 and 300 micrograms/kg of ivermectin by a single subcutaneous injection at the neck region. Compared to the numbers of mites and eggs just before injection, those on post treatment weeks (PTW) 1, 2 and 4 showed significant decreases. Especially at 300 micrograms/kg, the counts showed almost all mites and eggs were eradicated on PTW 1, manifesting ivermectin to possess potential effect on ssve without apparent abnormal side effect. Potential mitocide effect of ivermectin on ssvs was revealed.

Spontaneous and induced sister chromatid exchanges in lymphocytes of healthy persons.

Spontaneous and mitomycin C-induced sister chromatid exchanges (SCEs) in lymphocytes were analyzed in 46 healthy persons. Our results suggest that the frequency of spontaneous SCE in lymphocytes is useful for evaluation of the biological effects of environmental mutagens, and that the induced-SCE test with a mutagen and lymphocytes may be used to detect persons with a high sensitivity to the mutagen.

Effects of smoking on spontaneous and induced sister chromatid exchanges in lymphocytes.

Spontaneous and mitomycin C-induced sister chromatid exchanges (SCEs) in lymphocytes were analyzed in 24 non-smokers and 24 sex- and age-matched smokers. Mean spontaneous SCE frequency for non-smokers was 9.8 SCEs/cell, and that for smokers was 11.5 SCEs/cell. The difference was statistically significant (P less than 0.001 by t-test). These results suggest that spontaneous SCE frequency in lymphocytes is useful for evaluation of biological effects of environmental mutagens. However, we could not find any effects of smoking on the sensitivities of lymphocytes to mitomycin C in vitro. The effects of mutagens on humans may be independent of one another.

Lead exposure among 3-year-old children and their mothers living in a pottery-producing area.

The present study's aim was to compare the levels of lead exposure between 3-year-old children and their mothers in a pottery-producing area, and to assess the levels of lead exposure for 3-year-old children of the parents engaged in pottery-manufacturing work. For these purposes urinary lead, coproporphyrin, and delta-aminolevulinic acid were determined for the children and their mothers, living in a pottery-producing area and in a control area. The mean urinary lead was 9.3 to 10.8 micrograms/1 for the mothers in the pottery-producing area and 10.6 micrograms/1 for the children in the control area. In contrast, among the children in the pottery-producing area, those whose parents had been engaged in the pottery production had a much higher mean urinary lead of 13.6 to 15.8 micrograms/1. These results suggest that parental work related to lead contributes appreciably to lead exposure for children.

Home lead-work as a potential source of lead exposure for children.

Health examinations for lead poisoning were made on 62 family members from 15 families of homes carrying on lead work, such as quench-hardening in a molten lead bath and type-printing, as work at home. The most interesting findings concern the occurrence of cases with an unduly high lead absorption among children, but not among adult family members other than home lead-workers. The home environments of the children with an unduly high lead absorption represented contamination with housedust high in lead contents. The ingestion of the contaminated housedust by hand-to-mouth is probably responsible for the excessive lead exposure of the affected children. The results of the present study suggest that contamination of housedust with lead due to home lead-work constitutes a possible hazardous source of lead exposure for children.