Extracellular Vesicles of Stem Cells to Prevent BRONJ.

Extracellular vesicles (EVs), several tens to hundreds of nanometers in size, are vesicles secreted by cells for intercellular communication. EVs released from mesenchymal stem cells (MSC-EVs) have the potential to treat multiple diseases. This study aimed to determine the effects of MSC-EVs on bisphosphonate-related osteonecrosis of the jaw (BRONJ), whose pathogenesis and treatment are not yet established. To this end, zoledronic acid (ZOL) was administered to bone marrow cells and fibroblasts in vitro. In vivo, a BRONJ model was produced by administering ZOL to rats and extracting teeth. Each MSC-EV-treated and nontreated group was compared histologically and molecularly. In vitro, the nontreated group showed an increased number of ß-galactosidase-positive cells and expression of senescence-associated genes p21, pRB and senescence-related inflammatory cytokines. Conversely, MSC-EV administration decreased the number of senescent cells and expression levels of p21, pRB and inflammatory cytokines. In vivo, in the nontreated group, the socket was partially uncovered by the oral epithelium, leaving an exposed bone. Conversely, in the MSC-EV-treated group, the socket was healed. Besides, in the nontreated group, ß-galactosidase-positive cells existed in the socket and colocalized with the CD90 and periostin-positive cells. However, there were few ß-galactosidase-positive cells in the MSC-EV-treated group. Furthermore, gene expression of stem cell markers Bmi1 and Hmga2 and the vascular endothelial marker VEGF was significantly increased in the MSC-EV-treated group, compared with that in the nontreated group. These results indicate that MSC-EVs prevent ZOL-induced senescence in stem cells, osteoblasts, and fibroblasts and reduce inflammatory cytokines. Furthermore, administration of MSC-EVs prevented senescence of cells involved in wound healing and the spread of chronic inflammation around senescent cells, thereby promoting angiogenesis and bone regeneration and preventing BRONJ.

Identification of the Novel Tooth-Specific Transcription Factor AmeloD.

Basic-helix-loop-helix (bHLH) transcription factors play an important role in various organs' development; however, a tooth-specific bHLH factor has not been reported. In this study, we identified a novel tooth-specific bHLH transcription factor, which we named AmeloD, by screening a tooth germ complementary DNA (cDNA) library using a yeast 2-hybrid system. AmeloD was mapped onto the mouse chromosome 1q32. Phylogenetic analysis showed that AmeloD belongs to the achaete-scute complex-like ( ASCL) gene family and is a homologue of ASCL5. AmeloD was uniquely expressed in the inner enamel epithelium (IEE), but its expression was suppressed after IEE cell differentiation into ameloblasts. Furthermore, AmeloD expression showed an inverse expression pattern with the epithelial cell-specific cell-cell adhesion molecule E-cadherin in the dental epithelium. Overexpression of AmeloD in dental epithelial cell line CLDE cells resulted in E-cadherin suppression. We found that AmeloD bound to E-box cis-regulatory elements in the proximal promoter region of the E-cadherin gene. These results reveal that AmeloD functions as a suppressor of E-cadherin transcription in IEE cells. Our study demonstrated that AmeloD is a novel tooth-specific bHLH transcription factor that may regulate tooth development through the suppression of E-cadherin in IEE cells.

Restricted localization of thrombospondin-2 protein during mouse embryogenesis: a comparison to thrombospondin-1.

Thrombospondin-1 and -2 (TSP1 and TSP2) are multifunctional, multimodular extracellular matrix proteins encoded by separate genes. We compared the distributions of TSP1 and TSP2 in mouse embryos (day 10 and later) by immunohistochemistry. TSP1 was detected on day 10 in the heart and intestinal epithelium, on day 11 in megakaryocytes, and on day 14 in the lung. TSP2 was not detected until day 14, with strongest staining in mesenchymal condensation that gives rise to cartilage and bone. The distribution of TSP2 was different from but overlapped with the distribution of TSP1. TSP1 was found in cartilage proper with diminished staining around chondrocytes undergoing differentiation and hypertrophy, whereas TSP2 was restricted to the matrix surrounding chondrocytes of the growth zone cartilage. TSP2 and TSP1 were both expressed in centers of intramembranous ossification that form the skull bones, in reticular dermis, on the apical surface of nasal epithelium, in skeletal muscle, and in the sheath surrounding vibrissae. Areas of exclusive staining for TSP2 included the perichondrium surrounding the cartilage of the nasal cavities, developing bone of the lower mandible, and adrenal gland. The distinct localizations of TSP1 and TSP2 indicate that the two proteins have specific functions during mouse embryogenesis.

Nuclei of origin of monoaminergic, peptidergic, and cholinergic afferents to the cat trigeminal motor nucleus: a double-labeling study with cholera-toxin as a retrograde tracer.

The aim of the present study was to determine the brainstem afferents and the location of neurons giving rise to monoaminergic, cholinergic, and peptidergic inputs to the cat trigeminal motor nucleus (TMN). This was done in colchicine treated animals by using a very sensitive double immunostaining technique with unconjugated cholera-toxin B subunit (CT) as a retrograde tracer. After CT injections in the TMN, retrogradely labeled neurons were most frequently seen bilaterally in the nuclei reticularis parvicellularis and dorsalis of the medulla oblongata, the alaminar spinal trigeminal nucleus (magnocellular division), and the adjacent pontine juxtatrigeminal region and in the ipsilateral mesencephalic trigeminal nucleus. We further observed that inputs to the TMN arise from the medial medullary reticular formation (the nuclei retricularis magnocellularis and gigantocellularis), the principal bilateral sensory trigeminal nucleus, and the dorsolateral pontine tegmentum. In addition, the present study demonstrated that the TMN received 1) serotonergic afferents, mainly from the nuclei raphe obscurus, pallidus, and dorsalis; 2) catecholaminergic afferent projections originating exclusively in the dorsolateral pontine tegmentum, including the Kölliker-Fuse, parabrachialis lateralis, and locus subcoeruleus nuclei; further, that 3) methionin-enkephalin-like inputs were located principally in the medial medullary reticular formation (nuclei reticularis magnocellularis and gigantocellularis and nucleus paragigantocellularis lateralis), in the caudal raphe nuclei (Rpa and Rob) and the dorsolateral pontine tegmentum; 4) substance P-like immunoreactive neurons projecting to the TMN were present in the caudal raphe and Edinger-Westphal nuclei; and 5) cholinergic afferents originated in the whole extent of the nuclei reticularis parvicellularis and dorsalis including an area located ventral to the nucleus of the solitary tract at the level of the obex. In the light of these anatomical data, the present report discusses the possible physiological involvement of TMN inputs in the generation of the trigeminal jaw-closer muscular atonia occurring during the periods of paradoxical sleep in the cat.

Photochemical grafting of alpha-propylsulphate-poly(ethylene oxide) on polyurethane surfaces and enhanced antithrombogenic potential.

The aim of this study is the grafting of photoreactive alpha-propylsulphate-poly(ethylene oxide) (PEO-SO3), one end of which is capped with an azidophenyl group, on polyurethane (PU) surfaces via a photochemical technique. The anti-Factor Xa activity and the platelet adhesion characteristics of the modified PU surface were evaluated by a chromogenic assay method and by a flow-controlled chamber method, respectively. X-ray photoelectron spectroscopy analysis showed that PEO-SO3 was covalently grafted on the PU surface. The grafted surface showed anti-Factor Xa activity in the presence of antithrombin III, and significantly reduced platelet adhesion characteristics as compared with those of the unmodified PU surface. These results suggest that the grafting of PEO-SO3 improves the antithrombogenicity of PU surfaces.

Temperature-dependent modulation of blood platelet movement and morphology on poly(N-isopropylacrylamide)-grafted surfaces.

Poly(N-isopropylacrylamide) (PIPAAm) exhibits a reversible, temperature-dependent soluble/insoluble transition at its lower critical solution temperature (LCST) of 32 degrees C in aqueous media. The temperature-responsive PIPAAm was grafted onto tissue culture polystyrene (TCPS) dish surfaces by electron beam irradiation. Blood platelet behaviors on PIPAAm-grafted surface were examined by computerized image analysis and scanning electron microscopy. Platelet behaviors on this surface were dramatically dependent upon temperature, but those on poly(ethylene glycol)(PEG)-grafted or polystyrene remained unchanged. Below the 32 degrees C (LCST), platelets on PIPAAm-grafted surfaces retained a rounded shape and an oscillating vibratory microbrownian motion for extended times, similarly to those on PEG-grafted surfaces. Above the LCST, platelets readily adhered, spread and developed characteristic pseudopodia on PIPAAm-grafted surface similarly to those on TCPS. An ATP synthesis inhibitor failed to hinder prevention of platelet adhesion onto PIPAAm-grafted surface (below the LCST) suggesting that the preventive mechanism is ATP-independent similarly to that of PEG-grafted surfaces. These results correlate platelet surface activation state with the hydration and structure of polymer surfaces, and demonstrate the ability to modulate such reactions by a small temperature change in situ.

Cell-free formation of disulfide-bonded multimer from isolated plasma fibronectin in the presence of a low concentration of SH reagent under a physiological condition.

Purified plasma fibronectin in Tris-buffered saline aggregated on incubation at 37 degrees C in the presence of dithiothreitol without the presence of cells. On SDS polyacrylamide gel electrophoresis without reduction, dimeric fibronectin showed a 460 kDa band, while the protein band of aggregated fibronectin remained at the top of the running gel. The aggregate was a disulfide-bonded multimer, since both the dimeric and the multimeric fibronectins migrated as 230 kDa polypeptides after reduction. The multimer formation required SH reagent and proceeded faster with higher SH concentration, suggesting the occurrence of a disulfide exchange reaction during the aggregation. Since dimeric fibronectin with carboxymethylated sulfhydryl groups also formed multimers under the same condition, the free sulfhydryl groups of dimeric fibronectin may not be involved in the multimer formation, suggesting involvement of disulfide exchange from intramolecular bonds to intermolecular bonds. The multimerization was not influenced by Na+, Ca2+, or EDTA, while urea-treated fibronectin required a higher concentration of dithiothreitol for multimer formation. Fibronectin partially degraded by m-calpain did not form multimers. The multimeric fibronectin retained heparin-binding and cell attachment activities, but had lost gelatin-binding activity. Involvement of the terminal regions containing type I and type II repeats was suggested in the interaction of pFN leading to the multimerization.

Surgical treatment of aortic dissection: application of Ivalon sponge to the dissected lumen.

With canine disease models for aortic dissection, we performed comparative evaluations of several surgical procedures for the management of this dissection. From these experimental procedures, we developed a new operative technique, the Ivalon sponge occlusion method, designed to promote thrombus formation and to mesh (organize) effectively with the tissue ingrowth of the peripheral dilated, dissected aortic lumen. The details of the technique are described. It has been used successfully in 12 patients with DeBakey type I and type III aortic dissection. Based on our experimental and clinical evaluations, although the results are preliminary, we believe this operative technique is a simple, effective approach for the management of extended aortic dissection.

Contribution of calcium ion sequestration by polyoxyethylated nonionic surfactants to the enhanced colonic absorption of p-aminobenzoic acid.

Enhanced absorption of p-aminobenzoic acid (PABA) from the colon by polyoxyethylated nonionic surfactants was investigated using an in situ perfusion technique. The order of their absorption-enhancing effect was as follows: polyoxyethylene lauryl ether greater than polyoxyethylene sorbitan fatty acid esters approximately equal to polyoxyethylene fatty acid esters. The coexistence of calcium chloride in the perfusing solution caused a partial reverse in this enhancement. The calcium ion sequestration capacity of the surfactants was correlated with their ability to enhance colonic absorption of PABA. The findings suggest that calcium ion sequestration by the surfactants contributes to their enhancement of the colonic absorption of PABA.

Apical leakage of obturated canals prepared by Er:YAG laser.

The present study evaluates the degree of apical leakage in vitro after root canal preparation using Er:YAG laser irradiation followed by obturation. Twenty-four single-rooted teeth were divided into 2 groups of 12. One group served as a control and these root canals were conventionally prepared up to a #50K file. The other group was prepared by Er:YAG laser irradiation at parameters of 2 Hz and 170 to 230 mJ/pulse. After obturation the teeth were immersed in a vacuum flask containing 0.6% rhodamine for 48 h, longitudinally bisected, and observed by stereoscopy and scanning electron microscopy. The degree of apical leakage from an apical stop was measured and statistical analysis was performed. The degree of apical leakage from the teeth prepared by laser was not significantly less than that from control teeth (p > 0.01). Morphological findings showed that contact between the root canal walls and obturated materials was hermetic in both groups, but canal walls prepared by laser were rough and irregular. These results show that root canal preparation by laser does not affect apical leakage after obturation compared with leakage in canals prepared using the conventional method.

Compressed allograft chips for acetabular reconstruction in revision hip arthroplasty.

We report the results of 24 acetabular reconstructions in which cemented polyethylene cups and tamped corticocancellous allografts were used for severe acetabular bone deficiency. Eleven hips had type-II (cavitary) bone deficiency and 13 had type-III (combined) defects. At a mean follow-up of 5.8 years, two components had migrated more than 5 mm and had accompanying radiolucent zones of more than 2 mm width. A radiolucency 5 mm wide was also seen in zone III of an acetabular implant which had not migrated. None of the patients had required revision because of loosening or infection.

Technical evaluation of oxygen transfer rates of fish gills and artificial gills.

An artificial gill, which takes oxygen from water, would enhance the ability of people to function under water for extended periods. Increasing oxygen transfer rate, however, would be essential to the realization of compact, commercially viable equipment. Fish have evolved a variety of techniques to enable them to breathe under water, and their mechanisms must be clarified before compact, high-performance artificial gills can be developed. A model of the secondary lamellae of fish gills, through which oxygen is taken up from water to the blood, was devised, and its structure and oxygen transfer rate were evaluated by computer simulation analysis for carp and dogfish. Oxygen transfer rates were also found for an outside-water-flow artificial gill using a hollow fiber membrane at various fiber packing ratios. The biologic membrane is rate-determining for oxygen transfer through the secondary lamellae. Blood and water side boundary film resistances are small for fish because the blood and water channels are very narrow and numerous. When the fiber packing ratio of the artificial gill is raised, the oxygen transfer rate increases because of lower water side boundary film resistance. An optimum fiber packing ratio should be selected so that there is no major increase in pressure drop and no channeling occurs.

Theoretical comparison of filtration by the renal glomerulus and artificial membranes.

Improvement in filtration performance of artificial membranes will be possible if their structure mimics the renal glomerulus. Blood filtration with glomerular capillary and artificial membranes was, therefore, modeled to clarify the effects of their structure on filtration rates. Filtration rates were obtained by dividing membrane modules axially into a number of sections and using a calculus of finite differences. The modules were assumed to be composed of straight hollow fibers arranged in parallel, with a membrane surface area of 1.5 m2. The mean transmembrane pressure (TMP) was assumed to be too low for a protein gel layer to form on the membrane surface. A decrease in the inner diameter of membrane hollow fibers led to an increase in filtration rate because of an increased film mass transfer coefficient. A decrease in hollow fiber length also produced an increase in filtration rate because of decreased axial TMP drop. The glomerular capillary has a higher filtration rate than artificial membranes because of the low TMP drop and the low osmotic pressure at the membrane surface. Decreasing both the inner diameter and the length of the hollow fibers is effective in increasing the filtration rate at constant TMP.

Zeta potential of hollow fiber dialysis membranes and its effects on hydrogen phosphate ion permeability.

To clarify ion transport, dialysis membranes are evaluated in terms of zeta potential calculated by the Helmholtz-Smoluchowski equation from data on streaming potential delta E and pressure drop delta P, depending upon the operating conditions at which the values are measured. The objective of the current study is to design an improved method for measurement of delta E and delta P of hollow fiber dialysis membranes and to clarify the diffusive permeability of hydrogen phosphate ion. A polytetrafluoroethylene cylindrical cell with an inside diameter of 14 mm and a height of 10 mm was packed with 2,000-3,000 pieces of hollow fibers, and glass filters were set on either side of the cell. Deaerated water purified by ion exchange and reverse osmosis with an electric conductivity of approximately 150 microS/m was caused to flow in the hollows at 293 K to determine delta E and delta P. A good linear relationship between delta E and delta P and the reproducibility of the data was obtained and is shown in Figures 5 and 6, demonstrating the utility of the improved method to measure delta E and delta P, and the validity of the Helmholtz-Smoluchowski equation to calculate zeta potential from data on delta E and delta P. Hydrogen phosphate ion permeability increased with zeta potential for the membranes at about the same rate as pure water permeability. This indicates that hydrogen phosphate ion permeability depends upon the charge and internal structure of dialysis membranes.

A novel method for the continuous measurement of endotoxin concentration.

Conventional limulus amebocyte lysate tests involved procedures to prevent contamination by atmospheric endotoxins. To address this problem, the authors have proposed a technique in which the sampling, reagent mixing, and reaction steps are carried out consecutively in a single tube. Since reagents do not come in contact with the atmosphere, the new technique promises stable determination of the concentration of endotoxins in dialysate fluid. An aqueous solution of endotoxin simulating dialysate fluid was sampled in a silicone rubber tube from a sterile infusion bag, then mixed with an indicator in the same tube. After reaction at 310 K, measurements were made of light absorbance at 405 nm and its linearity with endotoxin concentration was determined. Results showed a high degree of linearity (correlation coefficient of not less than 0.99) at endotoxin concentrations of 0-15 pg/ml. The time for the reaction was shortened to 12 min, in which case the response time was 15 min. It is suggested that this new test for determining endotoxin concentration using limulus amebocyte lysate reagent, in which all three steps--sampling, mixing, and reaction--proceed continuously in a single tube, offers higher reliability, greater ease of operation, and shorter response time than conventional tests.

An artificial gill system for oxygen uptake from water using perfluorooctylbromide.

If the oxygen dissolved in seawater could be used for breathing, human beings could spend more time under water, greatly increasing their mobility. As yet, however, sufficient oxygen to enable us to live in the sea for long periods is not available. An artificial gill system has been developed for oxygen uptake from water to deoxygenated air using perfluorooctylbromide (PFOB), which has high oxygen solubility. It was found that oxygen was transferred rapidly from water to PFOB when water flowed outside the hollow fibers and PFOB flowed inside. Oxygen transfer through the membrane from PFOB to air was found to be the rate determining step. Use of PFOB gave a stable supply of oxygen from water to deoxygenated air over long periods. It was found that PFOB acts as a storage medium for oxygen.

New polymer alloy dialysis membranes with varying permeabilities and sievings.

The superstructure of polymer alloys that is responsible for their function is best controlled by varying the polymers, polymer blend ratio, solvents, temperature, or all four. Dialysis membranes made of polymer alloys can have varying pure water and solute permeabilities, pore sizes, pore size distributions, and mechanical strengths when wet. Resistance to heat and mass transfer can also vary. The optimal superstructure design of a polymer alloy dialysis membrane enables it to remove efficiently newly identified uremic toxins with higher molecular weights from patients on hemodialysis. The authors prepared new hollow fiber dialysis membranes using a polyethersulfone/polyacrylate (PEPA) polymer alloy, and evaluated pure water and solute permeabilities and reflection coefficients that are dependent upon the superstructure of the PEPA membranes. The new PEPA membranes are asymmetric, with skin layers on either side. The values for pure water permeability and overall mass transfer coefficients for urea, creatinine, and vitamin B12 are quite adequate for hemodialysis. The reflection coefficients for substances with molecular weights greater than 3,000 are strongly dependent upon the conditions under which the membranes were prepared. The values for cytochrome C permeability ranged from 0.42 to 0.71 microns/s.

Surface roughness of cellulose hollow fiber dialysis membranes and platelet adhesion.

A great deal of research has been conducted focusing on membrane materials with reference to their blood compatibility, but blood compatibility is influenced both by the material used in membranes and their structure, and by the flow conditions at the membrane surface. Accordingly, the relationship between membrane surface roughness and hemocompatibility has been evaluated using five types of membranes of differing surface roughness by evaluating the inner surfaces of the hollow fibers by atomic force microscopy (AFM) and by measuring platelet adhesion ratios using bovine blood. The yield stress, which equates to flow characteristics, was also evaluated using a glycerol suspension of polymethylmethacrylate (PMMA), a Bingham fluid. It was found that membranes having rough surfaces had high platelet adhesion ratios and poor hemocompatibility, whereas those with smoother surfaces had lower platelet adhesion ratios and better hemocompatibility. Measurement of the yield stresses for these membranes revealed higher values for those with rough surfaces, and lower values for those with smoother polyethylene glycol (PEG) grafted surfaces. This suggests that flow conditions at the membrane surface differ according to its surface roughness, and that this difference in flow conditions also influences hemocompatibility.

Comparative blood compatibility of polyether vs polycarbonate urethanes by epifluorescent video microscopy.

The segmented polyether urethanes (PEUs) have been used in implantable medical devices due to excellent mechanical properties, acceptable blood compatibility, and good biostability. However, recent studies demonstrate that the polyether soft segment of PEU is susceptible to oxidative degradation in vivo due to scission of the polyether group. Recently, polycarbonate urethanes (PCUs) having no ether linkage in the soft segment have been developed, and show improved stability against oxidative degradation over PEUs. The current study evaluates blood compatibility of these PCUs in comparison with PEUs using epifluorescent video microscopy (EVM) combined with a parallel plate flow cell. The authors selected two PCUs, Corethane 80A (Corvita Corporation, Miami, FL) and PCU(1560), and two PEUs, Pellethene 2363-80AE (Dow Chemical Japan, Tokyo, Japan) and Tecoflex EG80A (Thermedics, Inc., Woburn, MA), all of which have similar hard segment compositions (MDI or HMDI:1,4-butanediol(BD)) and the same hardness of 80A. The EVM measured the amount of platelet coverage on the surfaces using human whole blood perfused at a wall shear rate of 100/sec for 20 min. Complement activation (C3a) also was measured. Both PEUs, especially Pellethane, showed significantly higher platelet adhesion than the PCUs (p < 0.05). There were no significant differences in platelet adhesion between the two PCUs. As for C3a measurements, Tecoflex showed higher complement activation than the others. Based on these results, it is recommended that PEUs should be replaced by ether free PCUs for use in implantable blood contacting devices such as artificial hearts and pacemaker lead insulators.

A new amphiphilic block co-polymer with improved elastomeric properties for application in various medical devices.

The authors have demonstrated that an amphiphilic block co-polymer composed of 2-hydroxyethyl methacrylate (HEMA) and styrene (HEMA-st) showed excellent blood compatibility in in vitro, ex vivo, and in vivo experiments. The poor elastomeric properties of HEMA-st, however, have been an obstacle to its wider application in medical devices. To improve the mechanical properties of HEMA-st, the authors have developed a new amphiphilic block co-polymer composed of HEMA and octylstyrene (HEMA-oct). The size and morphology of the microdomain structures of HEMA-oct observed by transmission electron microscopy were similar to those of HEMA-st. Kink resistance tests showed improved elastomeric properties of HEMA-oct over HEMA-st. The blood compatibility of HEMA-oct was evaluated using an in vitro flow cell system combined with an epifluorescent video microscope, in which real time platelet adhesion and activation in whole blood can be observed and quantified, and ex vivo rabbit A-A shunt experiments. HEMA-st and a polyurethane (Pellethane 2363-80AE) were used for comparison. In a flow cell system, both HEMA-st and HEMA-oct showed minimal platelet coverage on the surfaces and less platelet activation as measured by beta-thromboglobulin (beta-TG), whereas Pellethane showed a considerable amount of platelet coverage with high beta-TG production. A-A shunt occlusion times were 309 +/- 31.2 min for HEMA-st, 251 +/- 47.7 min for HEMA-oct, and 30 +/- 3.4 min for Pellethane.(ABSTRACT TRUNCATED AT 250 WORDS)