Introduction Erectile dysfunction (ED) remains a major complication after radical prostatectomy. The use of adipose tissue-derived stem cells (ADSC) has shown promising results for the treatment of ED. However, the mechanisms of action for stem cell therapy remain controversial, with increasing evidence pointing to paracrine pathways. Aim To determine the effects and to identify the mechanism of action of ADSC and ADSC-derived lysate in a rat model of cavernous nerve (CN) crush injury. Methods Thirty-two male Sprague-Dawley rats were randomly divided into four equal groups: one group underwent sham operation, while three groups underwent bilateral CN crush. Crush-injury groups were treated at the time of injury with intracavernous injection of ADSC, lysate, or vehicle only (injured controls). Erectile function was assessed by cavernous nerve electrostimulation at 4 weeks. Penile tissue was collected for histology. Main Outcome Measures Intracavernous pressure increase upon CN stimulation; neuronal nitric oxide synthase (nNOS) content in the dorsal penile nerve; smooth muscle content, collagen content, and number of apoptotic cells in the corpus cavernosum. Results Both ADSC and lysate treatments resulted in significant recovery of erectile function, as compared to vehicle treatment. nNOS content was preserved in both the ADSC and lysate group, with significantly higher expression compared to vehicle-treated animals. There was significantly less fibrosis and a significant preservation of smooth muscle content in the ADSC and lysate groups compared to injured controls. The observed functional improvement after lysate injection supports the hypothesis that ADSC act through release of intracellular preformed substances or by active secretion of certain biomolecules. The underlying mechanism of recovery appears to involve neuron preservation and cytoprotection by inhibition of apoptosis. Conclusions Penile injection of both ADSC and ADSC-derived lysate can improve recovery of erectile function in a rat model of neurogenic erectile dysfunction.
In-cell NMR 1-3 provides information about how the crowded environment in cells, where the concentration of macromolecules reaches hundreds of grams per liter, 4 affects protein structure and dynamics. Several successes, including target protein overexpression in Escherichia coli 1,5-9 and injection of isotope-enriched protein into Xenopus laevis oocytes, 10,11 have been reported, but in-cell NMR remains in its infancy, and several potential problems need to be addressed. One problem is protein leakage from the cell during the experiment. 12-14 When this occurs, sharp signals from the protein molecules in the less viscous media mask the broader signals from the protein molecules in the more viscous cytosol. Here we examine two proteins. The intrinsically disordered protein, α-synuclein (αSN, ∼14 kDa), does not leak and is observed by in-cell NMR. The globular protein, chymotrypsin inhibitor 2 (CI2, ∼7 kDa), 15 leaks, and the remaining intracellular CI2 is not detectable. We show that the difference in detectability between αSN and CI2 is consistent with a differential dynamical response to macromolecular crowding. Figure 1A shows the 15 N-1 H HSQC spectrum of an in-cell NMR experiment on αSN. The spectrum is consistent with that from previous studies. 9,16 Figure 1B shows the spectrum from the supernatant collected immediately after sample preparation. Only metabolite signals 17 are observed. Figure 1C shows the spectrum from the supernatant recovered after the in-cell NMR experiment. Again, only metabolites are observed. The data demonstrate that the αSN spectrum in panel A comes from αSN in the cell. We have obtained similar results with the intrinsically disordered protein FlgM. 8 We performed the same experiments with CI2 expressing cells. In contrast to αSN, all three spectra are nearly identical ( Figure 1E-G) (and typical of a CI2 spectrum 18 in dilute solution). These data suggest that CI2 leaks from the cells. SDS-PAGE confirms that ∼20% of the CI2 is lost from cells. Figure 1D), proving that encapsulated cells can provide useful in-cell spectra. NIH Public AccessWe repeated the experiment with CI2-expressing cells. No CI2 signal was observed even though we increased the sensitivity by accumulating the data for a longer time compared to the other samples ( Figure 1H). However, a typical CI2 spectrum was recovered after dissolving the encapsulates with EDTA (data not shown). These observations suggest that the signal from the intracellular CI2, which we know is present in detectable amounts, is too broad to observe. We reasoned that the broadening arises from an alteration in the dynamics of CI2, either from binding a larger species in cells or from the higher viscosity of E. coli cytoplasm, which can be 10-11 times that of water. 21,22Why would the intrinsically disordered proteins αSN and FlgM react differently compared with a globular protein CI2 to the increased viscosity in cells such that we detect αSN and FlgM, but not CI2? The ability to detect a protein by high-resolution NMR depends on its dy...
Background-Effective treatment for stress urinary incontinence (SUI) is lacking. This study investigates whether transplantation of adipose tissue-derived stem cells (ADSCs) can treat SUI in a rat model.
Tissue engineered vascular grafts (TEVGs) are beginning to achieve clinical success and hold promise as a source of grafting material when donor grafts are unsuitable or unavailable. Significant technological advances have generated small-diameter TEVGs that are mechanically stable and promote functional remodeling by regenerating host cells. However, developing a biocompatible blood-contacting surface remains a major challenge. The TEVG luminal surface must avoid negative inflammatory responses and thrombogenesis immediately upon implantation and promote endothelialization. The surface has therefore become a primary focus for research and development efforts. The current state of TEVGs is herein reviewed with an emphasis on the blood-contacting surface. General vascular physiology and developmental challenges and strategies are briefly described, followed by an overview of the materials currently employed in TEVGs. The use of biodegradable materials and stem cells requires careful control of graft composition, degradation behavior, and cell recruitment ability to ensure that a physiologically relevant vessel structure is ultimately achieved. The establishment of a stable monolayer of endothelial cells and the quiescence of smooth muscle cells are critical to the maintenance of patency. Several strategies to modify blood-contacting surfaces to resist thrombosis and control cellular recruitment are reviewed, including coatings of biomimetic peptides and heparin.
Thermal oxidation, which serves as a low-cost, effective and relatively simple/facile method, was used to modify a micro-structured titanium surface in ambient atmosphere at 450 °C for different time periods to improve in vitro and in vivo bioactivity. The surface morphology, crystallinity of the surface layers, chemical composition and chemical states were evaluated by field-emission scanning electron microscopy (FESEM), X-ray diffraction (XRD), and X-ray photoelectron spectroscopy (XPS). Cell behaviours including cell adhesion, attachment, proliferation, and osteogenic differentiation were observed in vitro study. The ability of the titanium surface to promote osseointegration was evaluated in an in vivo animal model. Surface thermal oxidation on titanium implants maintained the microstructure and, thus, both slightly changed the nanoscale structure of titanium and enhanced the crystallinity of the titanium surface layer. Cells cultured on the three oxidized titanium surfaces grew well and exhibited better osteogenic activity than did the control samples. The in vivo bone-implant contact also showed enhanced osseointegration after several hours of oxidization. This heat-treated titanium enhanced the osteogenic differentiation activity of rBMMSCs and improved osseointegration in vivo, suggesting that surface thermal oxidation could potentially be used in clinical applications to improve bone-implant integration.
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