Improved Models for the Porous Surface with Passive Control

A computational investigation of afterbody flow using a passive control method is conducted. The passive control method consists of a porous surface placed over a plenum. The purpose of the passive control method is to exploit the adverse pressure gradient present in afterbody flow in an attempt to reduce boundary layer separation and afterbody drag. Four different porous wall models are used to model the transpiration velocity in the region of passive control. A three-dimensional, time-dependent, Reynolds-averaged, simplified Navier-Stokes solver, PAB3D, is used to simulate afterbody flow with and without passive control. Three afterbody configurations with boat-tail angles of 10, 20, and 30 deg. are used to obtain two-dimensional solutions with a freestream Mach number of 0.6 and nozzle pressure ratio of 6. The region of passive control was initially placed from 20-60% of the nozzle length. The effect of the porous placement and porous extent is also studied. Baseline (no porosity) two-dimensional solutions are qualitatively similar to experimental data but under-predict the magnitude of the pressure recovery. Results for the subsonic solutions show losses in the pressure recovery for some cases with passive control. Three-dimensional effects are also investigated and seen to be very significant. Three-dimensional baseline solutions, for both sub- and super-critical freestream Mach numbers, compare very favorably with the experimental data in comparison to the two-dimensional solution. Future work is required to examine three-dimensional afterbody flows with passive porosity

Smooth Muscle-Like Cells Generated from Human Mesenchymal Stromal Cells Display Marker Gene Expression and Electrophysiological Competence Comparable to Bladder Smooth Muscle Cells

The use of mesenchymal stromal cells (MSCs) differentiated toward a smooth muscle cell (SMC) phenotype may provide an alternative for investigators interested in regenerating urinary tract organs such as the bladder where autologous smooth muscle cells cannot be used or are unavailable. In this study we measured the effects of good manufacturing practice (GMP)-compliant expansion followed by myogenic differentiation of human MSCs on the expression of a range of contractile (from early to late) myogenic markers in relation to the electrophysiological parameters to assess the functional role of the differentiated MSCs and found that differentiation of MSCs associated with electrophysiological competence comparable to bladder SMCs. Within 1-2 weeks of myogenic differentiation, differentiating MSCs significantly expressed alpha smooth muscle actin (αSMA; ACTA2), transgelin (TAGLN), calponin (CNN1), and smooth muscle myosin heavy chain (SM-MHC; MYH11) according to qRT-PCR and/or immunofluorescence and Western blot. Voltage-gated Na+ current levels also increased within the same time period following myogenic differentiation. In contrast to undifferentiated MSCs, differentiated MSCs and bladder SMCs exhibited elevated cytosolic Ca2+ transients in response to K+-induced depolarization and contracted in response to K+ indicating functional maturation of differentiated MSCs. Depolarization was suppressed by Cd2+, an inhibitor of voltage-gated Ca2+-channels. The expression of Na+-channels was pharmacologically identified as the Nav1.4 subtype, while the K+ and Ca2+ ion channels were identified by gene expression of KCNMA1, CACNA1C and CACNA1H which encode for the large conductance Ca2+-activated K+ channel BKCa channels, Cav1.2 L-type Ca2+ channels and Cav3.2 T-type Ca2+ channels, respectively. This protocol may be used to differentiate adult MSCs into smooth muscle-like cells with an intermediate-to-late SMC contractile phenotype exhibiting voltage-gated ion channel activity comparable to bladder SMCs which may be important for urological regenerative medicine applications

Levels of intracellular Ca²⁺.

<p>Ca²⁺ imaging of (A) bladder SMCs and (B) differentiated MSCs (d7). K⁺-induced depolarization increased the intracellular Ca²⁺ content (black trace). Depolarization in the presence of 50 μM Cd²⁺ prevented the Ca²⁺ increase (dashed trace). (C) In undifferentiated MSCs (expanded in GMP expansion medium) no transient increase in cytosolic Ca²⁺ was observed in response to K+ induced depolarization. Arrow indicates time point in which 15 mM K⁺ was added to the bath solution.</p

Expression levels of αSMA <i>ex vivo</i>, after MSCs expansion and myogenic differentiation <i>in vitro</i>.

<p>(A) Mononuclear cells were directly isolated from bone marrow <i>ex vivo</i> and assessed for expression of αSMA by flow cytometry. Viable cells were gated (SSC/FSC), CD45⁺ cells were excluded, and expression of αSMA was recorded in the cytoplasm of the CD45^-CD271⁺ fraction of the mononuclear cells. The histogram represents αSMA in the cytoplasm of CD271⁺ cells (solid line) compared to the unstained controls (dotted line). (B) MSCs were expanded in GMP-compliant expansion medium and then assessed for expression of αSMA in the cytoplasm of the cells. Viable cells were gated (SSC/FSC) and αSMA⁺ cells were recorded (solid line). The dotted histogram represents the unstained controls. The broad profile of the histogram indicates that a large portion of MSCs express αSMA after expansion and prior to induction of differentiation (27% of MSCs were positive, MFI of 38). (C) After expansion in GMP-compliant expansion medium MSCs were differentiated for 14 days. Then expression of αSMA in the cytoplasm of differentiating MSCs was explored (solid line). The histogram indicates that more cells contain αSMA after differentiation (27% of MSCs were positive, MFI of 42, right panel). The dotted lines represent unstained controls.</p

Levels of voltage-activated Na⁺ currents and Na⁺ channels.

<p>(A) Voltage-activated Na⁺ currents were elicited by a voltage step from -120 mV to +20 mV. MSCs: trace from undifferentiated MSCs in control medium (undiff MSC-FBS) and GMP expansion medium (MSC-GMP); d7: trace from MSCs after 7 days in myogenic differentiation medium; SMC: trace from primary bladder SMCs. Capacitive transient is blanked for better visualization. (B) Na⁺ current density for undifferentiated MSCs in control medium (MSC FBS) and GMP medium (MSC GMP), as well as MSCs differentiated for 7, 14 or 21 days and SMCs, respectively. <i>n</i> = 10–20. * p<0.05. Error bars indicate SEM. (C) Effect of TTX on Na⁺ currents in undifferentiated MSCs (here: MSC in control medium). Superposition of Na⁺ currents elicited at +20mV. Na⁺ channels could be blocked by the specific Na⁺ channel inhibitor TTX. (D) Effect of TTX on Na⁺ currents in MSCs that were differentiated for 7 days. Superposition of Na⁺ currents elicited at +20mV. TTX blocks the current concentration-dependently.</p

Expression of contractile SMC-specific proteins analyzed by immunofluorescence.

<p>MSCs were expanded in GMP expansion medium until they were 70% confluent and at passage 2 treated with control medium or SMC differentiation medium for 14 days, fixed and then analyzed by immunofluorescence for expression of αSMA, transgelin, calponin and SM-MHC. Primary human bladder smooth muscle cells (HBdSMC) served as the positive control. Nuclei were stained with DAPI. Magnification 20x. Representative of <i>n</i> = 3.</p

Blockage of voltage-gated Na⁺ channel subtypes.

<p>(A) Application of 100nM ranolazine reduced the peak amplitude of voltage-activated Na⁺ channels [(<i>n</i> = 3 for undifferentiated MSCs (cultured in GMP expansion medium), <i>n</i> = 4 for SMCs, <i>n</i> = 5 for MSCs that were differentiated for 5–10 days and <i>n</i> = 7 for MSCs differentiated for 13–21 days (Diff MSC)] compared to the respective control (= 1.0, not shown). (B) Application of pro-toxin II inhibited voltage-gated Na⁺ channels in SMCs. Superposition of single current traces obtained in control (bold), at 2nM (dashed) and 100nM (dotted) from one donor, respectively. (C) Summary plot of current inhibition by pro-toxin II. Data obtained from n = 4 experiments. * p<0.05. Error bars indicate SEM.</p

Determination of osteogenic and adipogenic differentiation.

<p>MSCs were cultured in control medium, SMC differentiation medium, osteogenic differentiation medium or adipogenic differentiation medium as indicated for 14 days. HbdSMCs were cultured in SMC medium (smooth muscle cell growth medium 2). Osteogenic differentiation was assessed by Von Kossa staining (A-D) and adipogenic differentiation by Oil Red O staining (E-H). Magnification 20x. Representative of <i>n</i> = 3.</p

Expression levels of potassium and L- and T-type calcium ion channels.

<p>(A) Bladder SMCs were compared to (B) MSCs treated with SMC differentiation medium for 7 days for expression of ion channels. Transcript levels of <i>KCNMA1</i> (potassium ion channel), <i>CACNA1C</i> (Ca_v1.2 L-type Ca²⁺ channel) and <i>CACNA1H</i> (Ca_v3.2 T-type Ca²⁺ channel) were measured by qRT-PCR and expressed as target/reference ratio relative to <i>GAPDH</i> and <i>PPIA</i>. <i>n</i> = 3. Error bars indicate SEM.</p

Expression levels of contractile SMC-specific genes.

<p>MSCs were expanded in GMP expansion medium until they were 70% confluent and at passage 2 treated with control medium (CM) or SMC differentiation medium (5 ng/mL human TGF-β1, 5 ng/mL human PDGF-AB and 30 μM ascorbic acid) for 0, 3, 7, 14, 21 and 28 days. Differentiation was analyzed by qRT-PCR. Transcript levels were calculated relative to <i>GAPDH</i> and <i>PPIA</i>. (A) Data was calculated relative to CM for that time point. * p<0.05 compared to CM at the respective day of differentiation. (B) Data was calculated relative to day 0, the starting point of differentiation. * p<0.05 compared to CM at the respective day of differentiation; ^† p<0.05 compared to day 0. <i>n</i> = 6–8. Error bars indicate SEM.</p