A novel purification method for multipotential skeletal stem cells

At least some cells within bone marrow stromal populations are multipotential (i.e., differentiate in vitro into osteoblasts, chondrocytes, and adipocytes) and thus designated skeletal stem cells (SSCs) or mesenchymal stem cells (MSCs) amongst other names. Recently, a subpopulation of stromal cells, notably osteoblasts or their progenitors, has been identified as a definitive regulatory component of the hematopoietic stem cell (HSC) niche. Thus, the development of methods for purifying not only SSCs but cells comprising the HSC niche is of interest. Here, we report a method for purifying a novel bone marrow‐derived population with a high frequency of osteoprogenitors and high expression levels of osteoblast differentiation markers (highly purified osteoprogenitors (HipOPs)) as well as markers of the bone niche for HSCs. In vivo transplantation experiments demonstrated that donor HipOPs differentiated into not only osteoblasts, osteocytes and cells around sinusoids but also hematopoietic cells. Thus, HipOPs represent a novel population for simultaneous reconstruction of bone and bone marrow microenvironments. J. Cell. Biochem. 108: 368–377, 2009. © 2009 Wiley‐Liss, Inc.     

Assay of succinate dehydrogenase activity by the tetrazolium method: evaluation of an improved technique in skeletal muscle fractions

An improved spectrophotometric method for measuring succinate dehydrogenase (EC 1.3.99.1) activity with the use of 2-(p-iodophenyl)-3-(p-nitrophenyl)-5-phenyltetrazolium chloride (INT) is described. The procedure has been evaluated in mitochondrial fractions and homogenates of frog skeletal muscle. For mitochondrial suspensions, extraction of formazan with alcohol was found to be superior to extraction with ethyl acetate. For homogenates, complete extraction of formazan required sequential treatment with alcohol and ethyl acetate; the generally employed procedure of extracting once with ethyl acetate alone led to serious underestimation of the amount of formazan in the tissue. Observations of mitochondrial suspension incubated with various concentrations of INT led to the selection of 0.8 mM INT for optimal results. Higher concentrations, although commonly used, can exert undesirable inhibitory effects on succinate dehydrogenase activity, especially at low concentrations of mitochondria and after longer periods of incubation. The problem of instability of succinate dehydrogenase was solved by the addition of buffer at pH 7.5.     

A novel sensitive immunoassay method based on the Invader technique

A novel and sensitive immunoassay method has been developed in which the conventional sandwich immunoassay and the highly sensitive DNA detection method, the Invader method, are combined. The signal amplification function of the latter method has been successfully used to enhance the sensitivity of the sandwich immunoassay. The new assay method may be called the Immuno-Invader assay. The assay format involves three important steps: (1) a target antigen is captured and flagged with a biotin-conjugated detection antibody by the sandwich method, (2) streptavidin and a biotin-conjugated oligonucleotide are added to form a complex with the detection antibody, and (3) the oligonucleotide in the complex is detected using the Invader method. The method was applied to the assay of human tumor necrosis factor-α (hTNF-α). Detection limits obtained were 0.1 pg/ml hTNF-α when a luminescent europium chelate was used with a time-resolved measurement mode, and 0.8 pg/ml when fluorescein was used with a normal prompt fluorescence measurement mode. On the other hand, the detection limit of a commercially available hTNF-α enzyme-linked immunosorbent assay that uses horseradish peroxidase was 3.5 pg/ml. These results demonstrate the feasibility and potential of the new assay method for highly sensitive immunoassay.     

A novel method for the isolation of calsequestrin from porcine skeletal muscle sarcoplasmic reticulum

Bovine heart submitochondrial particles depleted of F1 by treatment with urea ("F1-depleted particles') were incubated with soluble F1-ATPase. The binding of F1 to the particles and the concomitant conferral of oligomycin sensitivity on the ATPase activity required the presence of cations in the incubation medium. NH4+, K+, Rb+, Na+ and Li+ promoted reconstitution maximally at 40-74 mM, guanidinium+ and Tris+ at 20-30 mM, and Ca2+ and Mg2+ at 3-5 mM. The particles exhibited a negative zeta-potential, as determined by microelectrophoresis, and this was neutralized by mono- and divalent cations in the same concentration range as that needed to promote F1 binding and reconstitution of oligomycin-sensitive ATPase. It is concluded that the cations act by neutralizing negative charges on the membrane surface, mainly negatively charged phospholipids. These results are discussed in relation to earlier findings reported in the literature with F1-depleted thylakoid membranes and with submitochondrial particles depleted of both F1 and the coupling proteins F6 and oligomycin sensitivity-conferring protein.     

A novel voltage clamp technique for mapping ionic currents from cultured skeletal myotubes.

The biophysical properties and cellular distribution of ion channels largely determine the input/output relationships of electrically excitable cells. A variety of patch pipette voltage clamp techniques are available to characterize ionic currents. However, when used by themselves, such techniques are not well suited to the task of mapping low-density channel distributions. We describe here a new voltage clamp method (the whole cell loose patch (WCLP) method) that combines whole-cell recording through a tight-seal pipette with focal extracellular stimulation through a loose-seal pipette. By moving the stimulation pipette across the cell surface and using a stationary whole-cell pipette to record the evoked patch currents, this method should be suitable for mapping channel distributions, even on large cells possessing low channel densities. When we applied this method to the study of currents in cultured chick myotubes, we found that the cell cable properties and the series resistance of the recording pipette caused significant filtering of the membrane currents, and that the filter characteristics depended in part upon the distance between the stimulating and recording pipettes. We describe here how we determined the filter impulse response for each loose-seal pipette placement and subsequently recovered accurate estimates of patch membrane current through deconvolution.     

Annexin A1: novel roles in skeletal muscle biology

Annexin A1 (ANXA1, lipocortin-1) is the first characterized member of the annexin superfamily of proteins, so called since their main property is to bind (i.e., to annex) to cellular membranes in a Ca(2+) -dependent manner. ANXA1 has been involved in a broad range of molecular and cellular processes, including anti-inflammatory signalling, kinase activities in signal transduction, maintenance of cytoskeleton and extracellular matrix integrity, tissue growth, apoptosis, and differentiation. New insights show that endogenous ANXA1 positively modulates myoblast cell differentiation by promoting migration of satellite cells and, consequently, skeletal muscle differentiation. This suggests that ANXA1 may contribute to the regeneration of skeletal muscle tissue and may have therapeutic implications with respect to the development of ANXA1 mimetics.     

美皮康在骨科外固定架患者护理中的应用

目的:探讨美皮康应用于外固定架针孔换药方式的临床效果。方法:将骨科50例四肢骨折行外固定架固定的患者随机分成实验组24例和对照组26例。实验组以美皮康有边型敷料换药,对照组以临床常用的络合碘纱布条进行换药,比较两组每天更换敷料频次,外固定针孔及周围皮肤红肿、渗出情况,护士更换污染床单被套频次及护士满意度。结果:实验组每天更换敷料频次少于对照组(P<0.05),外固定针孔及周围皮肤红肿、渗出等情况,实验组均优于对照组(P<0.05),实验组中护士更换污染床单被套频次明显少于对照组(P<0.05),护士满意度高于对照组(P<0.05)。结论:美皮康对保护骨科外固定针孔的治疗效果优于传统络合碘纱布条换药,对预防外固定针道感染有效,同时减轻护士维持床单位整洁、减少床单被套的更换频次,提高护士的满意度。     

A method for the use of Zenker-formol fixation and the periodic acid Schiff staining technique in light microscopic radioautography

Summary Although providing superior histological preservation, Zenker-formol fixation has not been utilized in radioautographic experiments, since some component of the fixative (presumably mercury) desensitizes the emulsion and thus prevents the appearance of a radioautographic reaction. Attempts to identify and eliminate the effects of this desensitizing agent led to experiments with Zenker-formol fixed tissues from rats injected with ³H-leucine or ³H-thymidine. Radioautographs of such tissues contained a brown-black precipitate and occasionally a heavy background fog, but no normal radioautographic reaction. Moreover, when the radioautographs were exposed to light, the emulsion was transparent over the tissues, indicating that a tissue-bound component had completely desensitized the emulsion. The two components of Zenker's fluid-mercuric chloride and potassium dichromate-plus the mercurous chloride which forms as a precipitate in the course of fixation, were then tested for their individual effects on the radioautographic emulsion. It was found that mercuric chloride was primarily responsible for the desensitization of the emulsion. Merourous chloride, which formed the dark precipitate in radioautographs, produced the background fog. While potassium dichromate caused some desensitization of the emulsion, in normal histological processing where tissues had a lengthy post-fixation wash, this substance was for the most part washed out.A number of attempts were made to remove the mercury by treating the Zenker-fixed tissues with solutions of iodine, sodium thiosulfate and cysteine. It was found that the most intense radioautographic reaction was obtained with the following procedure: prior to embedding, tissue blocks were treated with 0.5% iodine solution in 70% alcohol for 17 hours. Optionally, sections of this material could be given a second treatment with 0.5% alcoholic iodine for 10 minutes. Although simple iodination eliminated the mercurous chloride precipitate and the background fog, there was still no radioautographic reaction. However, subsequent treatment of sections of this iodinated material in 5% aqueous sodium thiosulfate for 5 minutes yielded an adequate radioautographic reaction. Therefore, the simultaneous removal of both mercurous chloride precipitate and tissue bound mercury required iodination followed by sodium thiosulfate. Using this procedure, a method was described for preparing tissues for radioautography using Zenker-formol fixation and the periodic acid-Schiff staining technique.This work was supported by a grant from the Medical Research Council of Canada to Dr. C. P. Leblond.     

A novel calcium current in dysgenic skeletal muscle

The whole-cell patch-clamp technique was used to study voltage-dependent calcium currents in primary cultures of myotubes and in freshly dissociated skeletal muscle from normal and dysgenic mice. In addition to the transient, dihydropyridine (DHP)-insensitive calcium current previously described, a maintained DHP-sensitive calcium current was found in dysgenic skeletal muscle. This current, here termed ICa-dys, is largest in acutely dissociated fetal or neonatal dysgenic muscle and also in dysgenic myotubes grown on a substrate of killed fibroblasts. In dysgenic myotubes grown on untreated plastic culture dishes, ICa-dys is usually so small that it cannot be detected. In addition, ICa-dys is apparently absent from normal skeletal muscle. From a holding potential of -80 mV. ICa-dys becomes apparent for test pulses to approximately -20 mV and peaks at approximately +20 mV. The current activates rapidly (rise time approximately 5 ms at 20 degrees C) and with 10 mM Ca as charge carrier inactivates little or not at all during a 200-ms test pulse. Thus, ICa-dys activates much faster than the slowly activating calcium current of normal skeletal muscle and does not display Ca-dependent inactivation like the cardiac L-type calcium current. Substituting Ba for Ca as the charge carrier doubles the size of ICa-dys without altering its kinetics. ICa-dys is approximately 75% blocked by 100 nM (+)-PN 200-110 and is increased about threefold by 500 nM racemic Bay K 8644. The very high sensitivity of ICa-dys to these DHP compounds distinguishes it from neuronal L-type calcium current and from the calcium currents of normal skeletal muscle. ICa-dys may represent a calcium channel that is normally not expressed in skeletal muscle, or a mutated form of the skeletal muscle slow calcium channel.     

A novel potassium channel in skeletal muscle mitochondria

In this work we provide evidence for the potential presence of a potassium channel in skeletal muscle mitochondria. In isolated rat skeletal muscle mitochondria, Ca(2+) was able to depolarize the mitochondrial inner membrane and stimulate respiration in a strictly potassium-dependent manner. These potassium-specific effects of Ca(2+) were completely abolished by 200 nM charybdotoxin or 50 nM iberiotoxin, which are well-known inhibitors of large conductance, calcium-activated potassium channels (BK(Ca) channel). Furthermore, NS1619, a BK(Ca)-channel opener, mimicked the potassium-specific effects of calcium on respiration and mitochondrial membrane potential. In agreement with these functional data, light and electron microscopy, planar lipid bilayer reconstruction and immunological studies identified the BK(Ca) channel to be preferentially located in the inner mitochondrial membrane of rat skeletal muscle fibers. We propose that activation of mitochondrial K(+) transport by opening of the BK(Ca) channel may be important for myoprotection since the channel opener NS1619 protected the myoblast cell line C2C12 against oxidative injury.     

A novel in vitro three-dimensional skeletal muscle model

A novel three-dimensional (3D) skeletal muscle model composed of C2C12 mouse myoblasts is described. This model was generated by cultivating myoblasts in suspension using the rotary cell culture system (RCCS), a unique culture environment. Single-cell suspensions of myoblasts were seeded at 5 × 10⁵/ml in growth medium without exogenous support structures or substrates. Cell aggregation occurred in both RCCS and suspension control (SC) conditions within 12 h but occurred more rapidly in the SC at all time intervals examined. RCCS-cultured myoblasts fused and differentiated into a 3D construct without serum deprivation or alterations. Syncitia were quantified at 3 and 6+ d in stained thin sections. A significantly greater number of syncitia was found at 6+ d in the RCCS cultures compared to the SC. The majority of syncitia were localized to the periphery of the cell constructs for all treatments. The expression of sarcomeric myosin heavy chain (MHC) was localized at or near the periphery of the 3D construct. The majority of MHC was associated with the large cells (syncitia) of the 6+-d aggregates. These results show, for the first time, that myoblasts form syncitia and express MHC in the presence of growth factors and without the use of exogenous supports or substrates. This model test system is useful for investigating initial cell binding, myoblast fusion and syncitia formation, and differentiation processes.     

Successful replantation in the face of absent venous drainage: an experimental study

Inadequate venous outflow is the factor most responsible in digital replantation failure and is a common cause of tissue loss in general. An experimental replantation model utilizing the rabbit ear was used to study the extreme example of venous congestion: absent venous drainage in the replanted part. Results of this study support the use of single arterial inflow along with an efferent AV fistula for outflow in the management of replants with absent venous drainage. Potential indications for the use of an efferent arteriovenous fistula in digital revascularization include the following: (1) the finding of distal veins too small to reanastomose, as is often the case in children and at distal levels in adults; (2) preferential destruction of distal venous structures, as commonly seen in degloving or other dorsal injuries; and (3) in the management of postreplant venous thrombosis.