A homeostatic role of nucleus-actin filament coupling in the regulation of cellular traction forces in fibroblasts.

Cellular traction forces are contractile forces that depend on the material/substrate stiffness and play essential roles in sensing mechanical environments and regulating cell morphology and function. Traction forces are primarily generated by the actin cytoskeleton and transmitted to the substrate through focal adhesions. The cell nucleus is also believed to be involved in the regulation of this type of force; however, the role of the nucleus in cellular traction forces remains unclear. In this study, we explored the effects of nucleus-actin filament coupling on cellular traction forces in human dermal fibroblasts cultured on substrates with varying stiffness (5, 15, and 48 kPa). To investigate these effects, we transfected the cells with a dominant-negative Klarsicht/ANC-1/Syne homology (DN-KASH) protein that was designed to displace endogenous linker proteins and disrupt nucleus-actin cytoskeleton connections. The force that exists between the cytoskeleton and the nucleus (nuclear tension) was also evaluated with a fluorescence resonance energy transfer (FRET)-based tension sensor. We observed a biphasic change in cellular traction forces with a peak at 15 kPa, regardless of DN-KASH expression, that was inversely correlated with the nuclear tension. In addition, the relative magnitude and distribution of traction forces in nontreated wild-type cells were similar across different stiffness conditions, while DN-KASH-transfected cells exhibited a different distribution pattern that was impacted by the substrate stiffness. These results suggest that the nucleus-actin filament coupling play a homeostatic role by maintaining the relative magnitude of cellular traction forces in fibroblasts under different stiffness conditions.

Construction of a corresponding empirical model to bridge thermal properties and synthesis of thermoresponsive poloxamines.

The thermoresponsive properties of poloxamine (tetra-branch PEO-PPO block copolymer) hydrogels are related to several variables. Of particular interest to this study were the molecular weight of the polymer, the molar ratio between PEO and PPO blocks, and the concentration of the aqueous solution. Accurately controlling the thermoresponsive behaviors of the polymer is critical to the application of such materials; therefore, the structure-property relationship of tetra-branch PEO-PPO block copolymer was studied by synthesis via anionic ring-opening polymerization (AROP). The structure-property relationships were studied by measuring the thermoresponsive behavior via differential scanning calorimetry (DSC) and developing an empirical model which statistically fit the collected data. This empirical model was then used for designing poloxamines that have critical micellization temperatures (CMT) between room temperature and physiological temperature. The model was validated with three polymers that targeted a CMT of 308 K (35°C). The empirical model showed great success in guiding the synthesis of poloxamines showing a temperature difference of less than 3 K between the predicted and the observed CMTs. This study showed a great potential of using an empirical model to set synthesis parameters to control the properties of the polymer products.

Enzyme-induced hypoxia leads to inflammation in urothelial cells in vitro.

PURPOSE: To determine the contributions of different durations of hypoxia to NLRP3 inflammasome activation in urothelial cells and how ischemic changes in bladder tissues is an important chemical que that leads to pathological changes seen in BOO. METHODS: A rat urothelial cell line (MYP3) was exposed to either a short duration (2 h) or long duration (6 h) of enzyme-induced hypoxia. Following exposure to a short duration of hypoxia, NO and ATP concentrations were measured from supernatant media and caspase-1 levels were measured from cell lysates. In a separate experiment, cells were fixed following hypoxia exposure and immunostained for HIF-1α stabilization. RESULTS: Although short exposure of low oxygen conditions resulted in a hypoxic response in MYP3 cells, as indicated by HIF-1α stabilization and increased NO activity, NLRP3 inflammasome activation was not observed as caspase-1 activity remained unchanged. However, exposure of MYP3 cells to a longer duration of hypoxia resulted in an increase in intracellular caspase-1 activity. Furthermore, treatment with antioxidant (GSH) or TXNIP inhibitor (verapamil) attenuated the hypoxia-induced increase in caspase-1 levels indicating that hypoxia primarily drives inflammation through a ROS-mediated TXNIP/NLRP3 pathway. CONCLUSION: We conclude that hypoxia induced bladder damage requires a duration that is more likely related to elevated storage pressures/hypoxia, seen in later stages of BOO, as compared to shorter duration pressure elevation/hypoxia that is encountered in normal micturition cycles or early in the BOO pathology where storage pressures are still normal.

A Novel Bio-Adhesive Mesh System for Medical Implant Applications: In Vivo Assessment in a Rabbit Model.

Injectable surgical sealants and adhesives, such as biologically derived fibrin gels and synthetic hydrogels, are widely used in medical products. While such products adequately adhere to blood proteins and tissue amines, they have poor adhesion with polymer biomaterials used in medical implants. To address these shortcomings, we developed a novel bio-adhesive mesh system utilizing the combined application of two patented technologies: a bifunctional poloxamine hydrogel adhesive and a surface modification technique that provides a poly-glycidyl methacrylate (PGMA) layer grafted with human serum albumin (HSA) to form a highly adhesive protein surface on polymer biomaterials. Our initial in vitro tests confirmed significantly improved adhesive strength for PGMA/HSA grafted polypropylene mesh fixed with the hydrogel adhesive compared to unmodified mesh. Toward the development of our bio-adhesive mesh system for abdominal hernia repair, we evaluated its surgical utility and in vivo performance in a rabbit model with retromuscular repair mimicking the totally extra-peritoneal surgical technique used in humans. We assessed mesh slippage/contraction using gross assessment and imaging, mesh fixation using tensile mechanical testing, and biocompatibility using histology. Compared to polypropylene mesh fixed with fibrin sealant, our bio-adhesive mesh system exhibited superior fixation without the gross bunching or distortion that was observed in the majority (80%) of the fibrin-fixed polypropylene mesh. This was evidenced by tissue integration within the bio-adhesive mesh pores after 42 days of implantation and adhesive strength sufficient to withstand the physiological forces expected in hernia repair applications. These results support the combined use of PGMA/HSA grafted polypropylene and bifunctional poloxamine hydrogel adhesive for medical implant applications.

BOO induces fibrosis and EMT in urothelial cells which can be recapitulated in vitro through elevated storage and voiding pressure cycles.

PURPOSE: To determine the unique contributions from elevated voiding and storage pressures in the development of fibrosis and the epithelial-to-mesenchymal transition (EMT) in urothelial cells, and how progressive BOO pressure cycling is an important mechanical cue leading to these pathological changes. MATERIALS AND METHODS: Urothelial cells isolated from control, SHAM, 2 (acute)- or 6 (chronic)-week BOO rats treated with an inflammasome inhibitor or no drug. Total RNA was isolated and RT-PCR was conducted with custom primers for pro-fibrotic and EMT genes. In separate experiments, a rat urothelial cell line was exposed to cyclic pressure regimes characteristic of acute and chronic BOO in the presence or absence of an inflammasome inhibitor. Following exposure, RT-PCR was conducted, collagen content was determined and intracellular caspase-1 activity was measured. RESULTS: Urothelial cells isolated from acute and chronic BOO rat models demonstrated expression of pro-fibrotic and EMT genes. Similarly, MYP3 rat urothelial cells subjected to pressure cycling regimes that reflect intravesical pressures in the acute or chronic BOO bladder also demonstrated increased expression of pro-fibrotic and EMT genes, along with elevated soluble collagen. Treatment with inflammasome inhibitors reduced expression of pro-fibrotic genes in the rat model and pressure cycling model but had a limited effect on EMT. CONCLUSION: These results indicate that acute and chronic BOO pressure cycling are essential in the initiation and progression of fibrosis in the bladder via the NLRP3 inflammasome, but also provide new evidence that there is also an alternative NLRP3-independent pathway leading to EMT and fibrosis.

Evaluation of Poly (Carbonate-Urethane) Urea (PCUU) Scaffolds for Urinary Bladder Tissue Engineering.

Although the previous success of bladder tissue engineering demonstrated the feasibility of this technology, most polyester based scaffolds used in previous studies possess inadequate mechanical properties for organs that exhibit large deformation. The present study explored the use of various biodegradable elastomers as scaffolds for bladder tissue engineering and poly (carbonate-urethane) urea (PCUU) scaffolds mimicked urinary bladder mechanics more closely than polyglycerol sebacate-polycaprolactone (PGS-PCL) and poly (ether-urethane) urea (PEUU). The PCUU scaffolds also showed cyto-compatibility as well as increased porosity with increasing stretch indicating its ability to aid in infiltration of smooth muscle cells. Moreover, a bladder outlet obstruction (BOO) rat model was used to test the safety and efficacy of the PCUU scaffolds in treating a voiding dysfunction. Bladder augmentation with PCUU scaffolds led to enhanced survival of the rats and an increase in the bladder capacity and voiding volume over a 3 week period, indicating that the high-pressure bladder symptom common to BOO was alleviated. The histological analysis of the explanted scaffold demonstrated smooth muscle cell and connective tissue infiltration. The knowledge gained in the present study should contribute towards future improvement of bladder tissue engineering technology to ultimately aide in the treatment of bladder disorders.

Surface modification of polypropylene surgical meshes for improving adhesion with poloxamine hydrogel adhesive.

Tissue adhesive has notable clinical benefits in hernia repair fixation. A novel poloxamine tissue adhesive was previously shown to successfully bond collagen tissue with adequate adhesive strength. In application related to attachment of polypropylene (PP) mesh, the adhesive strength between the mesh and poloxamine hydrogel adhesive is limited by the hydrophobicity of PP monofilaments and lack of covalent bond formation. The purpose of this study was to compare two different surface modifications [bovine serum albumin (BSA) adsorption and poly-glycidyl methacrylate/human serum albumin (PGMA/HSA) grafting] of PP mesh for improving the adhesive strength between poloxamine hydrogel adhesive and PP mesh. The PGMA/HSA surface modification significantly improved the adhesive strength for meshes attached with poloxamine hydrogel tissue adhesive compared with unmodified meshes and meshes modified by BSA adsorption. An area of 1 cm2 adhesive provided for a maximum adhesive strength of 65-70 kPa for meshes modified by PGMA/HSA, 4-13 kPa for meshes modified by BSA, and 22-45 kPa for unmodified meshes. Optical microscopy and infrared spectroscopy (FTIR) confirmed the improved adhesive strength was achieved through mechanical interlock of the hydrogel tissue adhesive into the PP mesh pores and chemical bonding of the albumin after successful PGMA/HSA grafting onto the PP monofilaments. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater 107B: 1047-1055, 2019.

Elevated hydrostatic pressure stimulates ATP release which mediates activation of the NLRP3 inflammasome via P2X4 in rat urothelial cells.

Partial bladder outlet obstruction (pBOO) is a prevalent urological condition commonly accompanied by increased intravesical pressure, inflammation, and fibrosis. Studies have demonstrated that pBOO results in increased NLRP3 inflammasome and caspase-1 activation and that ATP is released from urothelial cells in response to elevated pressure. In the present study, we investigated the role of elevated pressure in triggering caspase-1 activation via purinergic receptors activation in urothelial cells. Rat urothelial cell line, MYP3 cells, was subjected to hydrostatic pressures of 15 cmH2O for 60 min, or 40 cmH2O for 1 min to simulate elevated storage and voiding pressure conditions, respectively. ATP concentration in the supernatant media and intracellular caspase-1 activity in cell lysates were measured. Pressure experiments were repeated in the presence of antagonists for purinergic receptors to determine the mechanism for pressure-induced caspase-1 activation. Exposure of MYP3 cells to both pressure conditions resulted in an increase in extracellular ATP levels and intracellular caspase-1 activity. Treatment with P2X7 antagonist led to a decrease in pressure-induced ATP release by MYP3 cells, while P2X4 antagonist had no effect but both antagonists inhibited pressure-induced caspase-1 activation. Moreover, when MYP3 cells were treated with extracellular ATP (500 µM), P2X4 antagonist inhibited ATP-induced caspase-1 activation, but not P2X7 antagonist. We concluded that pressure-induced extracellular ATP in urothelial cells is amplified by P2X7 receptor activation and ATP-induced-ATP release. The amplified ATP signal then activates P2X4 receptors, which mediate activation of the caspase-1 inflammatory response.

Nanometer-sized extracellular matrix coating on polymer-based scaffold for tissue engineering applications.

Surface modification can play a crucial role in enhancing cell adhesion to synthetic polymer-based scaffolds in tissue engineering applications. Here, we report a novel approach for layer-by-layer (LbL) fabrication of nanometer-size fibronectin and gelatin (FN-G) layers on electrospun fibrous poly(carbonate urethane)urea (PCUU) scaffolds. Alternate immersions into the solutions of fibronectin and gelatin provided thickness-controlled FN-G nano-layers (PCUU(FN-G) ) which maintained the scaffold's 3D structure and width of fibrous bundle of PCUU as evidenced by scanning electron miscroscopy. The PCUU(FN-G) scaffold improved cell adhesion and proliferation of bladder smooth muscles (BSMCs) when compared to uncoated PCUU. The high affinity of PCUU(FN-G) for cells was further demonstrated by migration of adherent BSMCs from culture plates to the scaffold. Moreover, the culture of UROtsa cells, human urothelium-derived cell line, on PCUU(FN-G) resulted in an 11-15 µm thick multilayered cell structure with cell-to-cell contacts although many UROtsa cells died without forming cell connections on PCUU. Together these results indicate that this approach will aid in advancing the technology for engineering bladder tissues in vitro. Because FN-G nano-layers formation is based on nonspecific physical adsorption of fibronectin onto polymer and its subsequent interactions with gelatin, this technique may be applicable to other polymer-based scaffold systems for various tissue engineering/regenerative medicine applications.

Mechanical characterization of a bifunctional Tetronic hydrogel adhesive for soft tissues.

Although a number of tissue adhesives and sealants for surgical use are currently available, attaining a useful balance in high strength, high compliance, and low swelling has proven difficult. Recent studies have demonstrated that a four-arm poly(propylene oxide)-poly(ethylene oxide) block copolymer, Tetronic, can be chemically modified to form a hydrogel tissue adhesive (Cho et al., Acta Biomater 2012;8:2223-2232; Barrett et al., Adv Health Mater 2012;1-11; Balakrishnan, Evaluating mechanical performance of hydrogel-based adhesives for soft tissue applications. Clemson University, All Theses, Paper 1574: Tiger Prints; 2013). Building on the success of these studies, this study explored bifunctionalization of Tetronic with acrylates for chemical crosslinking of the hydrogel and N-hydroxysuccinimide (NHS) for reaction with tissue amines. The adhesive bond strengths of various uni and bifunctional Tetronic blends (T1107 ACR: T1107 ACR/NHS) determined by lap shear testing ranged between 8 and 74 kPa, with the 75:25 (T1107 ACR: T1107 ACR/NHS) blend displaying the highest value. These results indicated that addition of NHS led to improvement of tissue bond strength over acrylation alone. Furthermore, ex vivo pressure tests using the rat bladder demonstrated that the bifunctional Tetronic adhesive exhibited high compliance and maintained pressures under hundreds of filling and emptying cycles. Together, the results of this study provided evidence that the bifunctional Tetronic adhesive with a proper blend ratio may be used to achieve an accurate balance in bulk and tissue bond strengths, as well as the compliance and durability for soft tissue such as the bladder.

Tetronic(®)-based composite hydrogel scaffolds seeded with rat bladder smooth muscle cells for urinary bladder tissue engineering applications.

Natural hydrogels such as collagen offer desirable properties for tissue engineering, including cell adhesion sites, but their low mechanical strength is not suitable for bladder tissue regeneration. In contrast, synthetic hydrogels such as poly (ethylene glycol) allow tuning of mechanical properties, but do not elicit protein adsorption or cell adhesion. For this reason, we explored the use of composite hydrogel blends composed of Tetronic (BASF) 1107-acrylate (T1107A) in combination with extracellular matrix moieties collagen and hyaluronic acid seeded with bladder smooth muscle cells (BSMC). This composite hydrogel supported BSMC growth and distribution throughout the construct. When compared to the control (acellular) hydrogels, mechanical properties (peak stress, peak strain, and elastic modulus) of the cellular hydrogels were significantly greater. When compared to the 7-day time point after BSMC seeding, results of mechanical testing at the 14-day time point indicated a significant increase in both ultimate tensile stress (4.1-11.6 kPa) and elastic modulus (11.8-42.7 kPa) in cellular hydrogels. The time-dependent improvement in stiffness and strength of the cellular constructs can be attributed to the continuous collagen deposition and reconstruction by BSMC seeded in the matrix. The composite hydrogel provided a biocompatible scaffold for BSMC to thrive and strengthen the matrix; further, this trend could lead to strengthening the construct to match the mechanical properties of the bladder.

Clinical applications of surgical adhesives and sealants.

In the United States and Europe, the number of topical adhesives, surgical sealants, and hemostats approved for use in the surgical setting is ever expanding although no single device fills all medical and surgical needs to replace sutures. As more surgical procedures are performed through laparoscopic and robotic approaches, these devices are becoming more important, and current research is focused on solving the limitations of conventional wound treatments. This review article discusses clinical applications of various biologically derived and synthetic products that are currently available to surgeons and those that are in development.

Examining the role of mechanosensitive ion channels in pressure mechanotransduction in rat bladder urothelial cells.

Until recently, the bladder urothelium had been thought of only as a physical barrier between urine and underlying bladder tissue. Recent studies, however, have demonstrated that the urothelium is sensitive to mechanical stimuli and responds by releasing signaling molecules (NO, ATP). This study sought to investigate the role of select ion channels in urothelial cell (UC) pressure mechanotransduction. Using a custom-made pressure chamber, rat bladder UCs cultured on tissue culture plastic dishes were exposed to sustained hydrostatic pressure (5-20 cmH(2)O) for up to 30 min. When compared to the control, UCs exposed to 10 cmH(2)O (5 min), and 15 cmH(2)O (5 and 15 min), exhibited a significant (p < 0.05) increase in ATP release. In the absence of extracellular calcium, ATP release due to hydrostatic pressure was attenuated. Blocking the L-type voltage-gated channel with nifedipine during pressure exposure did not affect ATP release. However, blocking TRP channels, stretch-activated channels (SACs), and the epithelial sodium channel (ENaC) with ruthenium red, gadolinium chloride, and amiloride, respectively, all abolished hydrostatic pressure-evoked ATP release. These results have provided evidence for the first time that cultured UCs are sensitive to hydrostatic pressure in the physiologically relevant range. The results of this study also provide evidence that one or multiple mechanosensitive ion channels play a role in the mechanotransduction of hydrostatic pressure, which supports the view that not only tissue stretch or tension, but also pressure is an important parameter for mechanosensing of bladder fullness.

Effect of sustained hydrostatic pressure on rat bladder smooth muscle cell function.

OBJECTIVES: To test a hypothesis that bladder smooth muscle cells (BSMCs) shift their phenotype from contractile to synthetic in response to elevated hydrostatic pressure. Although mechanical stimuli are needed for development of the bladder, the exact mechanisms for this process are poorly understood. METHODS: Rat BSMCs were exposed to 7.5 cm H(2)O of hydrostatic pressure in custom-made columns to a maximum of 48 hours. After exposure to pressure, the smooth muscle cells were fixed, stained, and imaged to quantify cell morphology and proliferation. Additionally, Western blotting was used to quantify extracellular signal-regulated kinase (ERK(1/2)) activation as well as phenotype marker proteins, alpha-smooth muscle actin, and SM-22. RESULTS: Compared with the control, BSMCs exposed to hydrostatic pressure exhibited a more spread morphology after 4 hours and the expression of activated ERK(1/2) was a maximum of two-fold at 1.5-3 hours. Moreover, cell density of BSMCs exposed to hydrostatic pressure exhibited an increase after 48 hours when compared with their respective controls. In contrast, alpha-smooth muscle actin and SM-22 expression was similar in the control and in cells exposed to hydrostatic pressure for 48 hours. CONCLUSIONS: The morphologic and proliferative changes of BSMC in response to hydrostatic pressure possibly indicate a phenotypic shift from contractile to synthetic. Moreover, the activation of ERK(1/2) intracellular signaling pathway may represent a potential mechanism for the pressure-induced BSMC proliferation. The comparable levels of contractile proteins observed in both control and pressure group BSMCs suggest that not all the phenotype markers are regulated concomitantly by a single stimulus.

Increased synthetic phenotype behavior of smooth muscle cells in response to in vitro balloon angioplasty injury model.

Restenosis remains a common problem following balloon angioplasty, and it has been speculated that changes in the mechanical environment due to endovascular interventions are correlated with shifts in smooth muscle cell (SMC) phenotype. In order to study SMC response to forces similar to those exerted during balloon angioplasty, an in vitro concurrent shear and tensile forces simulator has been developed. After 24 hr of exposure to cyclic tension (5%) and shear (0.1-0.5 dynes/cm(2)) following simulated angioplasty injury (12% stretch), rat aortic SMCs exhibited significant synthetic behavior. These responses included increased cell proliferation, apoptosis, and cell hypertrophy compared to cells exposed to strain alone. While all SMCs exposed to dynamic stimuli (strain, strain+balloon injury, strain+balloon injury+shear) demonstrated a decrease in contractile protein expression, the injury group also exhibited significantly greater expression of the synthetic marker vimentin. These in vitro findings agree with in vivo events following balloon angioplasty and present a refined dynamic model to be implemented for better understanding of SMC activation and prevention of responses through pharmacological treatment.

Layer by layer three-dimensional tissue epitaxy by cell-laden hydrogel droplets.

The ability to bioengineer three-dimensional (3D) tissues is a potentially powerful approach to treat diverse diseases such as cancer, loss of tissue function, or organ failure. Traditional tissue engineering methods, however, face challenges in fabricating 3D tissue constructs that resemble the native tissue microvasculature and microarchitectures. We have developed a bioprinter that can be used to print 3D patches of smooth muscle cells (5 mm x 5 mm x 81 microm) encapsulated within collagen. Current inkjet printing systems suffer from loss of cell viability and clogging. To overcome these limitations, we developed a system that uses mechanical valves to print high viscosity hydrogel precursors containing cells. The bioprinting platform that we developed enables (i) printing of multilayered 3D cell-laden hydrogel structures (16.2 microm thick per layer) with controlled spatial resolution (proximal axis: 18.0 +/- 7.0 microm and distal axis: 0.5 +/- 4.9 microm), (ii) high-throughput droplet generation (1 s per layer, 160 droplets/s), (iii) cell seeding uniformity (26 +/- 2 cells/mm(2) at 1 million cells/mL, 122 +/- 20 cells/mm(2) at 5 million cells/mL, and 216 +/- 38 cells/mm(2) at 10 million cells/mL), and (iv) long-term viability in culture (>90%, 14 days). This platform to print 3D tissue constructs may be beneficial for regenerative medicine applications by enabling the fabrication of printed replacement tissues.

An exploratory pathways analysis of temporal changes induced by spinal cord injury in the rat bladder wall: insights on remodeling and inflammation.

BACKGROUND: Spinal cord injuries (SCI) can lead to severe bladder pathologies associated with inflammation, fibrosis, and increased susceptibility to urinary tract infections. We sought to characterize the complex pathways of remodeling, inflammation, and infection in the urinary bladder at the level of the transcriptome in a rat model of SCI, using pathways analysis bioinformatics. METHODOLOGY/PRINCIPAL FINDINGS: Experimental data were obtained from the study of Nagatomi et al. (Biochem Biophys Res Commun 334: 1159). In this study, bladders from rats subjected to surgical SCI were obtained at 3, 7 or 25 days post-surgery, and Affymetrix GeneChip Rat Genome U34A arrays were used for cRNA hybridizations. In the present study, Ingenuity Pathways Analysis (Ingenuity Systems, www.ingenuity.com) of differentially expressed genes was performed. Analysis of focus genes in networks, functional analysis, and canonical pathway analysis reinforced our previous findings related to the presence of up-regulated genes involved in tissue remodeling, such as lysyl oxidase, tropoelastin, TGF-beta1, and IGF-1. This analysis also highlighted a central role for inflammation and infection, evidenced by networks containing genes such as CD74, S100A9, and THY1. CONCLUSIONS/SIGNIFICANCE: Our findings suggest that tissue remodeling, infection, inflammation, and tissue damage/dysfunction all play a role in the urinary bladder, in the complex response to SCI.

Role of cytoskeletal components in stress-relaxation behavior of adherent vascular smooth muscle cells.

A number of recent studies have demonstrated the effectiveness of atomic force microscopy (AFM) for characterization of cellular stress-relaxation behavior. However, this technique's recent development creates considerable need for exploration of appropriate mechanical models for analysis of the resultant data and of the roles of various cytoskeletal components responsible for governing stress-relaxation behavior. The viscoelastic properties of vascular smooth muscle cells (VSMCs) are of particular interest due to their role in the development of vascular diseases, including atherosclerosis and restenosis. Various cytoskeletal agents, including cytochalasin D, jasplakinolide, paclitaxel, and nocodazole, were used to alter the cytoskeletal architecture of the VSMCs. Stress-relaxation experiments were performed on the VSMCs using AFM. The quasilinear viscoelastic (QLV) reduced-relaxation function, as well as a simple power-law model, and the standard linear solid (SLS) model, were fitted to the resultant stress-relaxation data. Actin depolymerization via cytochalasin D resulted in significant increases in both rate of relaxation and percentage of relaxation; actin stabilization via jasplakinolide did not affect stress-relaxation behavior. Microtubule depolymerization via nocodazole resulted in nonsignificant increases in rate and percentage of relaxation, while microtubule stabilization via paclitaxel caused significant decreases in both rate and percentage of relaxation. Both the QLV reduced-relaxation function and the power-law model provided excellent fits to the data (R(2)=0.98), while the SLS model was less adequate (R(2)=0.91). Data from the current study indicate the important role of not only actin, but also microtubules, in governing VSMC viscoelastic behavior. Excellent fits to the data show potential for future use of both the QLV reduced-relaxation function and power-law models in conjunction with AFM stress-relaxation experiments.

Diabetes-induced alternations in biomechanical properties of urinary bladder wall in rats.

OBJECTIVES: To determine whether diabetes mellitus and the associated changes in bladder function will trigger bladder wall tissue remodeling and concomitant alterations in the mechanical properties. We investigated the time course of changes in function and mechanical properties of diabetic and diuretic rat bladders using both in vivo and in vitro techniques METHODS: Cystometry was performed at 2, 4, and 8 weeks on female Sprague-Dawley rats that had received either a single injection of streptozotocin (65 mg/kg intraperitoneally) or 5% sucrose in drinking water for the duration of the experiments. At each point, the biaxial mechanical properties of 10 x 10-mm tissue specimens obtained from the posterior part of bladder wall were quantified. The changes in overall tissue compliance and mechanical anisotropy as a function of time were examined RESULTS: Both diabetic and diuretic conditions led to increases in bladder weight, bladder capacity, and in vivo compliance compared with the controls at all points tested. Under biaxial loading, all bladder wall tissues exhibited a nonlinear stress-strain relationship and mechanical anisotropy, with greater tissue compliance in the circumferential direction than in the longitudinal direction. Although the compliance of the bladder wall increased progressively and synchronously in both diabetic and diuretic bladders for < or = 4 weeks, only the diabetic bladders continued to increase the compliance for < or = 8 weeks (diabetic 0.64 +/- 0.04 vs diuretic 0.48 +/- 0.05, P = .03) CONCLUSIONS: The results of our study have shown that diuresis mainly contributes to the "early" changes of mechanical properties of the bladder, with diabetes inducing additional "late" changes of mechanical properties of the rat bladders after 4 weeks.

Effect of sustained tension on bladder smooth muscle cells in three-dimensional culture.

Previous studies demonstrated that bladder cells respond to changes in their mechanical environments by exhibiting alterations in cellular functions, such as hypertrophy or fibrosis. In the present study, we hypothesize that changes in smooth muscle cell (SMC) behavior triggered by mechanical stimuli may represent a phenotypic shift between contractile and synthetic phenotypes. Using a custom-made device, rat bladder SMCs were cultured in three-dimensional (3-D) collagen gels and exposed to sustained tension. When compared to no-tension controls, SMCs exposed to tension exhibited significantly (p < 0.05) higher expression of alpha-smooth muscle actin (alpha-SMA), while cell population density was similar in both groups. In addition, both mean and median aspect ratios of SMCs in 3-D collagen constructs exposed to tension were significantly (p < 0.05) greater than those of cells cultured under no externally applied tension, indicating that there are more elongated, spindle-shaped cells in the tension group. These SMCs in 3-D cultures exposed to tension also exhibited cellular alignment along the direction of applied tension. Since contractile SMCs are known to exhibit greater expression of phenotypic marker proteins as well as a more elongated morphology, we concluded that sustained tension on cells is an important mechanical stimulus for maintenance of the contractile phenotype of bladder SMCs in vitro.