应用脂肪干细胞构建组织工程化小口径血管平滑肌层的实验研究

目的探索利用脂肪干细胞在生物反应器内构建组织工程血管平滑肌层的可行性。方法用抽吸的脂肪获取脂肪干细胞,在生长因子TGF-β1和BMP4作用下诱导成平滑肌细胞,然后将诱导的平滑肌细胞接种于PGA上,将细胞-材料复合物置于生物反应器内进行培养,在模拟胚胎发育血流动力学的刺激下(搏动频率:75次/分;扩展量<5%),构建小口径的血管平滑肌组织。培养8周后,取材行组织学和生物力学检测并与正常血管对比。结果脂肪干细胞在TGF-β1和BMP4的诱导下,细胞呈现平滑肌细胞特有的"波峰-波谷"样生长特点,并表达平滑肌细胞的特异性标记物α-SMA、SM22α、calponin和SM-MHC;反应器内培养8周后,构建的管状组织胶原分泌旺盛,具有一定的力学强度和弹性。结论利用脂肪干细胞可在体外生物反应器内构建组织工程化小口径血管平滑肌层,为临床上小血管病变的修复提供了一种新的可能的途径。     

Effects of Pulsatile Bioreactor Culture on Vascular Smooth Muscle Cells Seeded on Electrospun Poly (lactide‐co‐ε‐caprolactone) Scaffold

Electrospun nanofibrous scaffolds have several advantages, such as an extremely high surface‐to‐volume ratio, tunable porosity, and malleability to conform over a wide variety of sizes and shapes. However, there are limitations to culturing the cells on the scaffold, including the inability of the cells to infiltrate because of the scaffold's nano‐sized pores. To overcome the limitations, we developed a controlled pulsatile bioreactor that produces static and dynamic flow, which improves transfer of such nutrients and oxygen, and a tubular‐shaped vascular graft using cell matrix engineering. Electrospun scaffolds were seeded with smooth muscle cells (SMCs), cultured under dynamic or static conditions for 14 days, and analyzed. Mechanical examination revealed higher burst strength in the vascular grafts cultured under dynamic conditions than under static conditions. Also, immunohistology stain for alpa smooth muscle actin showed the difference of SMC distribution and existence on the scaffold between the static and dynamic culture conditions. The higher proliferation rate of SMCs in dynamic culture rather than static culture could be explained by the design of the bioreactor which mimics the physical environment such as media flow and pressure through the lumen of the construct. This supports regulation of collagen and leads to a significant increase in tensile strength of the engineered tissues. These results showed that the SMCs/electrospinning poly (lactide‐co‐ε‐caprolactone) scaffold constructs formed tubular‐shaped vascular grafts and could be useful in vascular tissue engineering.     

The effect of a cyclic uniaxial strain on urinary bladder cells

Purpose

Pre-conditioning of a cell seeded construct may improve the functional outcome of a tissue engineered construct for augmentation cystoplasty. The precise effects of mechanical stimulation on urinary bladder cells in vitro are not clear. In this study we investigate the effect of a cyclic uniaxial strain culture on urinary bladder cells which were seeded on a type I collagen scaffold.

Methods

Isolated porcine smooth muscle cells or urothelial cells were seeded on a type I collagen scaffolds and cultured under static and dynamic conditions. A uniform cyclic uniaxial strain was applied to the seeded scaffold using a Bose Electroforce Bio-Dynamic bioreactor. Cell proliferation rate and phenotype were investigated, including SEM analysis, RT-PCR and immunohistochemistry for α-Smooth muscle actin, calponin-1, desmin and RCK103 expression to determine the effects of mechanical stimulation on both cell types.

Results

Dynamic stimulation of smooth muscle cell seeded constructs resulted in cell alignment and enhanced proliferation rate. Additionally, expression of α-Smooth muscle actin and calponin-1 was increased suggesting differentiation of smooth muscle cells to a more mature phenotype.

Conclusions

Mechanical stimuli did not enhance the proliferation and differentiation of urothelial cells. Mechanical stimulation, i.e., preconditioning may improve the functional in vivo outcome of smooth muscle cell seeded constructs for flexible organs such as the bladder.

     

Chronic high pressure potentiates the antiproliferative effect and abolishes contractile phenotypic changes caused by endothelial cells in cocultured smooth muscle cells

High in vitro pressures have been reported to alter smooth muscle cell (SMC) and endothelial cell (EC) phenotype, while endothelial cells (ECs) can influence the proliferation, phenotype, and contractile features of smooth muscle cells (SMC) in coculture systems. However, little is known about the in vitro effects of pressure on EC/SMC cocultures. We therefore sought to compare SMC proliferation in independent and EC coculture under ambient and high pressure, and identify changes in the contractile phenotype of SMCs by measuring levels of the L-type Ca(2+) channel a(1) subunit (dihydropyridine-DHP receptor) which is critical for Ca(2+) transients, differentiation and contractility in SMC. METHODS: Rat aortic SMCs in independent culture (SMC/0) and coculture with ECs (SMC/EC) were maintained in 5% CO(2) under either atmospheric or high pressure (130 mmHg). SMC were counted at 0, 1, 3, and 5 days and compared to initial cell counts of day 0 before the exposure to experimental conditions. DHP receptor levels were quantitated by Western blotting (three similar studies). RESULTS: ECs suppressed SMC proliferation on day 1 of coculture in both atmospheric and high pressure (20% inhibition vs independent culture, P < or = 0.05). By day 3, cocultured SMC under atmospheric pressure displayed no EC-mediated inhibition, and at day 5, atmospheric cocultured SMCs revealed statistically significant enhanced proliferation as compared with SMCs in independent cultures. However, cocultured SMCs exposed to 130 mmHg pressure displayed sustained sensitivity to EC growth inhibition at both days 3 and 5 of the experiment. Coculture decreased SMC DHP-receptor levels under atmospheric pressure. However, this effect was abolished in cocultures exposed to high pressure. CONCLUSIONS: High pressure substantially alters the regulatory influence of EC on SMC proliferation and contractile potential. This pressure/coculture model should increase our understanding of cellular interaction in hypertensive vasculopathy.     

应用生物反应器构建组织工程化血管的实验研究

目的探讨利用生物反应器体外构建具有完整结构的组织工程化血管的可行性。方法从6月龄毕格犬颈总动脉获取平滑肌细胞,颈外静脉获取内皮细胞,经体外培养扩增。然后,先将平滑肌细胞接种于聚羟基乙酸(PGA)上,形成细胞—材料复合物,将该复合物置于生物反应器内培养。模拟成年哺乳动物循环系统的参数,予以动态力学刺激(搏动频率:75次/分;扩张量<5%)。8周后,在其上接种内皮细胞,对照组不接种内皮细胞,培养5d后取材检测。结果组织学染色显示,平滑肌纤维成分排列较规则,有层次感,内皮细胞完整;对照组未见内皮细胞层结构。结论利用生物反应器可在体外构建具有完整结构的组织工程化血管。     

Time-dependent modulation of alignment and differentiation of smooth muscle cells seeded on a porous substrate undergoing cyclic mechanical strain

The orientation of cellular alignment in smooth muscle tissue engineering is directly related to optimal movement of engineered tissue when it is transplanted in vivo. Cyclic mechanical strain has been applied to modulate the alignment, proliferation, and differentiation of smooth muscle cells. This study was conducted to investigate the effects of cyclic mechanical strain on primary cultured myofibroblasts seeded onto three-dimensional polymeric scaffolds, and to determine the optimal mechanical treatment time required to produce artificial smooth muscle. The cells were primary cultured from rabbit esophageal smooth muscle layer, and a self-designed stretching chamber was used to modulate the cells on porous polyurethane (PU) scaffolds with 10% strain at a frequency of 1 Hz. The applied cyclic strain induced cellular alignment. In particular, cellular alignment perpendicular to the direction of strain was generated in the condition strained over 18 h. In terms of proliferation, the strained groups differed significantly from the statically cultured group, but no difference was observed between groups that were subjected to straining for different lengths of time. Quantitative analysis of alpha-smooth muscle actin (SMA) showed that differentiation was significantly promoted at 18 h of strain. Penetration of primary cultured cells into the pores of PU scaffolds was shown after cyclic strain application, especially in 18 and 24 h of strain. Consequently, it is expected that myofibroblast/scaffold hybrids, cyclically strained in the defined time course, could be practically applied to organize functional smooth muscle tissues having consistent cell alignment and up-regulated SMA.     

小口径组织工程血管体外构建的研究

目的 探讨间充质干细胞体外构建组织工程血管的可行性.方法 将体外培养扩增的犬骨髓间充质干细胞(MSCs)定向分化为平滑肌样细胞和内皮样细胞,接种于ε-己内酯/L-丙交酯(PCLA)支架上,将其置于生物反应器内,在搏动性力学(100±20/55±20)mm Hg(1 mm Hg=0.133 kPa)刺激条件下培养.3 d后行血管组织学检测.结果 血管支架拉伸强度6.1 MPa;骨髓间充质干细胞成功定向分化为平滑肌样细胞和内皮样细胞;血管腔内表面完全为细胞覆盖,表面的细胞沿液体流动的方向分布;种植的部分细胞已经渗透入血管壁内.结论 骨髓间充质干细胞可作为种子细胞,与PCLA支架在生物反应器内构建组织工程血管.     

Influence of topography of an endovascular stent material on smooth muscle cell response

The phenotypic shift of smooth muscle cells (SMCs) from contractile to synthetic phenotype after endovascular stenting has been deemed to be the predominant cause of restenosis. Although substrate topography has been shown to affect SMC response for a variety of polymers, effect of topography on phenotype of SMCs has not been well studied for endovascular stent materials. Thus, the objective of the present study was to evaluate SMC phenotype, manifested in morphology, proliferation, and contractile marker smooth muscle α-actin expression, as a function of 316L stainless steel topography. Results of this study showed that the cells grown on micro-grooved surface (groove depth: 13 μm) were significantly more elongated than those on the electropolished surface. Ascertained by repeated proliferation studies, cells grown on micro-grooved surface demonstrated a significantly lower proliferation rate relative to the electropolished surface. An enhanced expression of smooth muscle α-actin grown on micro-grooved surface was also found near confluence. Furthermore, cells on electropolished surface demonstrated a substantial loss of smooth muscle α-actin between days 1 and 4. Therefore, given the favorable SMC response reported in this study, our findings suggest that a micro-grooved topography might prove beneficial for endovascular stent applications.     

The contractile wall of the caput epididymidis in men affected by congenital or postinflammatory obstructive azoospermia

The transport and storage of spermatozoa in the epididymis depend on the contractile activity of its tubular wall. It is not known what differences exist in the contractile wall of the human epididymis in cases of obstructive azoospermia. The contractile wall in the tubules of the caput epididymidis was analyzed by light microscopy and transmission electron microscopy in 10 azoospermic men, 5 with a bilateral congenital absence of vas deferens (CBAVD) and 5 with a bilateral postinflammatory congestive obstruction of the epididymis. Five specimens from the same region of the caput epididymidis, obtained from fertile men who had undergone an orchidectomy because of testicular cancer, served as controls. No differences were observed between congenital and congestive obstructions. The contractile wall in caput tubules proximal to the obstructed level was strongly thickened when compared with controls (62.98 +/- 5.84 micro; 80.82 +/- 7.72 micro vs 19.59 +/- 2.23 micro, respectively, for congestive and congenital obstructions vs controls; P <.0001 vs controls), and the spindle-shaped myoid cells, which formed the contractile wall in normal cases, were replaced by large smooth muscle cells (SMCs) that showed features of coexisting contractile and secretory functions. The former included crowded cytoplasmic bundles of thin myofilaments (5-6 nm in diameter) converging to a large number of dense bodies, numerous micropinocytotic vesicles of the plasma membrane, and a continuous cell basement membrane. The presence of a developed rough endoplasmic reticulum and a Golgi complex, associated with the accumulation of thick layers of pericellular basement membrane-like material and ground substance, was indicative of a secretory phenotype of SMCs. The increased mechanical forces on the epididymal wall upstream from the obstruction might eventually activate the differentiation of myoid cells into SMCs, leading to an altered physiology of the contractile wall that could have possible clinical relevance in the case of microsurgical epididymovasostomy.     

Sustained orbital shear stress stimulates smooth muscle cell proliferation via the extracellular signal-regulated protein kinase 1/2 pathway

OBJECTIVE: Nonlaminar shear stress stimulates smooth muscle cell (SMC) proliferation and migration in vivo, especially after an endothelial-denuding injury. To determine whether sustained shear stress directly stimulates SMC proliferation in vitro, the effect of orbital shear stress on SMC proliferation, phenotype, and extracellular signal-regulated protein kinase 1/2 (ERK1/2) phosphorylation was examined. METHODS: Bovine SMCs were exposed to orbital shear stress (210 rpm) for up to 10 days, with and without the ERK1/2 upstream pathway inhibitor PD98059 (10 microM) or the p38 pathway inhibitor SB203580 (10 microM). Proliferation was directly counted and assessed with proliferation cell nuclear antigen. Western blotting was used to assess activation of SMC ERK1/2 and SMC phenotype markers. RESULTS: SMCs exposed to sustained orbital shear stress (10 days) had 75% increased proliferation after 10 days compared with static conditions. Expression of markers of the contractile phenotype (alpha-actin, calponin) was decreased, and markers of the synthetic phenotype (vimentin, beta-actin) were increased. ERK1/2 was phosphorylated in the presence of orbital shear stress, and orbital shear-stress-stimulated SMC proliferation was inhibited in the presence of PD98059 but sustained in the presence of SB203580. Orbital shear-stress-induced changes in SMC phenotype were also inhibited in the presence of PD98059. CONCLUSION: Orbital shear stress directly stimulates SMC proliferation in long-term culture in vitro and is mediated, at least partially, by the ERK1/2 pathway. The ERK1/2 pathway may also mediate the orbital shear-stress-stimulated switch from SMC contractile to synthetic phenotype. These results suggest that shear-stress-stimulated SMC proliferation after vascular injury is mediated by a pathway amenable to pharmacologic manipulation.     

Endothelial cell activation of the smooth muscle cell phosphoinositide 3-kinase/Akt pathway promotes differentiation

OBJECTIVE: Interactions between endothelial cells (ECs) and smooth muscle cells (SMCs) are fundamental in diverse cardiovascular processes such as arteriogenesis, atherosclerosis, and restenosis. We aimed to determine the intracellular signaling mechanisms by which ECs promote a differentiated SMC phenotype. METHODS: Bovine thoracic aorta ECs and SMCs were isolated and cultured. For co-culture studies, ECs were grown to confluence on one side of a semi-permeable Cyclopore membrane. SMCs were then plated on the opposite side of the membrane and cultured for 24 to 48 hours. For adenovirus experiments, SMCs were infected prior to plating opposite ECs. For conditioned media studies, SMCs cultured alone on plastic were treated with media harvested from EC/SMC in co-culture. SMC phenotype was assayed by microscopy and measurement of two-dimensional area, or by western blotting for contractile protein markers of differentiation. Akt activation was measured by western blotting for phospho-Serine 473. RESULTS: Although SMCs cultured alone exhibit a dedifferentiated synthetic phenotype, we report that bilayer co-culture with ECs induced a differentiated SMC phenotype as measured by morphology and cell area and expression of protein markers of differentiation, including contractile proteins and the cyclin-dependent kinase inhibitor p27 kip . The EC/SMC bilayer co-culture resulted in activation of the SMC protein kinase Akt, with no effect on total Akt expression. Similarly, conditioned media from co-cultured EC/SMC promoted rapid Akt phosphorylation and subsequent expression of differentiation protein markers in SMCs cultured alone. Adenoviral overexpression of constitutively active Akt in SMCs cultured alone mimicked the ability of ECs to induce SMC differentiation. Notably, inhibition of phosphoinositide 3 (PI 3)-kinase activity with wortmannin or adenoviral overexpression of a dominant-negative Akt prevented the EC-mediated effect on SMC morphology and differentiation protein marker expression. CONCLUSIONS: ECs direct SMCs towards a differentiated phenotype through activation of the SMC PI 3-kinase/Akt pathway. CLINICAL RELEVANCE: Interactions between endothelial cells (ECs) and smooth muscle cells (SMCs) are fundamental in diverse cardiovascular processes such as arteriogenesis, collateral blood vessel development, atherosclerosis, and restenosis. Alterations in SMC phenotype occur in each of these processes. Endothelial denudation has been suggested to contribute to the SMC proliferative response to vessel injury by angioplasty or other catheterization procedures. We have employed a co-culture approach to dissect the molecular signals that are dependent on the spatial relationship between ECs and SMCs, and have identified the importance of the PI3K/Akt pathway in EC-induced SMC differentiation. This pathway may suggest targets for therapeutic interventions for intimal hyperplasia and restenosis.     

Hyperplastic vascular smooth muscle cells of the intrarenal arteries in angiotensin II type 1a receptor null mutant mice

BACKGROUND: Angiotensin II (Ang II), which contracts vascular smooth muscle cells (VSMCs), has been reported to regulate VSMC growth. Recently formed transgenic mice without angiotensinogen or Ang II receptors showed vascular alterations. However, it is still unclear how their VSMCs alter. We explored the role of Ang II via the Ang II type 1a receptor (AT1a) in VSMCs in vivo using AT1a null mutant mice. METHODS: We analyzed the ultrastructure of the intrarenal arteries in AT1a null mutant mice that were homozygous for a targeted disruption of AT1a receptor gene using light and electron microscopy. RESULTS: The structural changes of the intrarenal arteries in AT1a null mutant mice showed the wall thickening, which in the interlobar, arcuate, and proximal interlobular arteries consisted of two additional populations of VSMCs, on the luminal and abluminal sides of the media. The luminal overpopulation of smooth muscle cells (SMCs) was arranged in a longitudinal direction separated by increased interposed elastic laminae. The abluminal overpopulation of SMCs ran in circumferential directions separated from the main population. The cytological structure of VSMCs in AT1a null mutant mice was smaller in size, contained more organelles for protein synthesis and secretion than in control mice, and had poorly developed contractile apparatus. CONCLUSIONS: The lack of AT1a signaling causes structural abnormalities in the renal vascular system and transforms the phenotype of VSMCs into cell proliferation, induces the escape of VSMCs from the circular mechanical integrity, and results in increased synthesis of extracellular matrices.     

Changes in smooth muscle cell phenotype and contractile function following ischemia-reperfusion injury in the rat urinary bladder

Twenty-eight adult male Sprague-Dawley rats were divided into four groups: Group 1 received 1 hour (h) of bilateral ischemia alone. Groups 2 and 3 received 1 h ischemia followed by 1 and 4 h of reperfusion (I-R), respectively. Group 4 consisted of age-matched control rats. Bladder strips were studied using electrical field stimulation (EFS) and KCl stimulation. Maximal contractile responses were recorded and analyzed. Temporal patterns of changes in phenotypic (non-contractile and contractile) expression of bladder smooth muscle cells were investigated using electron microscopy. The mean ratio of non-contractile to contractile phenotype (nc/c) of smooth muscle cells (SMCs) in the control group was 0.169. In the ischemia alone group, the ratio was 0.991. In the 1 h I-R group, the ratio 0.865 whereas in 4 h I-R group the ratio 1.601. The contractile responses to EFS and KCl showed decreased responses in all groups. These results clearly demonstrated that the ratio of nc/c increased significantly in the ischemia group and further increased significantly in both I-R groups. The contractile responses decreased in all ischemic groups although the magnitude of the contractile changes did not correspond in the change of phenotype ratio.     

Development of a novel vascular simulator and injury model to evaluate smooth muscle cell response following balloon angioplasty

Following balloon angioplasty, denudation of endothelial cells exposes vascular smooth muscle cells (SMCs) to normally unseen shear forces from blood flow. In vivo studies investigate the response to angioplasty injury, but limited studies have been performed using in vitro systems. In order to study SMC response in vitro, a concurrent shear and tensile forces simulator has been developed to provide clinically significant levels of strain and shear stresses in addition to simulating forces similar to those during balloon angioplasty. In this acute study (8 hr), rat aortic SMCs demonstrated significant cell proliferation following applied increased tensile forces of angioplasty injury and shear exposure when compared to lower levels of tensile exposure similar to a normal physiological level, with an average 75% increase in the number of cells of the injury group compared to the normal dynamic group. SMCs exposed to balloon angioplasty injury and concurrent shear and tensile mechanical forces demonstrated decreased expression of the contractile phenotypic marker smooth muscle alpha-actin. These findings demonstrate the efficacy of the developed model for in vitro angioplasty and the simulated mechanical environment to the cells. This provides an in vitro model to isolate the effects of concurrent mechanical forces and could also potentially act as a preliminary step toward use in pharmaceutical research for reduction or prevention of SMC proliferation due to altered mechanical forces during endovascular procedures.     

Growth of bone marrow stromal cells on small intestinal submucosa: an alternative cell source for tissue engineered bladder

OBJECTIVE: To assess the potential use of bone marrow stromal cell (BMSC)-seeded biodegradable scaffold for bladder regeneration in a canine model, by characterizing BMSCs and comparing them to bladder smooth muscle cells (SMCs) by immunohistochemistry, growth capability, and contractility. MATERIALS AND METHODS: Bone marrow was taken by direct needle aspiration from the femurs of five beagle dogs for the in vitro study. Mononuclear cells were isolated by Ficoll-Paque density gradient centrifugation and cultivated in medium 199 with 10% fetal bovine serum. BMSCs were characterized by cell proliferation, in vitro contractility, immunohistochemical analysis, and the growth pattern on small intestinal submucosa (SIS) scaffolds compared to bladder SMC cultures from the same dogs. Another six dogs had a hemicystectomy and bladder augmentation with BMSC-seeded (two), bladder cells including urothelial cells plus SMC-seeded SIS (two) and unseeded SIS scaffolds (two). The six dogs were followed for 10 weeks after augmentation. RESULTS: In vitro BMSCs had a significant contractile response to calcium-ionophore, with a mean (sem) 36 (2)%, relative contraction (P < 0.01), which was similar to bladder SMCs but markedly different from fibroblasts. BMSCs also expressed alpha-smooth muscle actin by immunohistochemical staining and Western blotting, but did not express desmin or myosin. In vivo, both BMSC-seeded and bladder cell-seeded SIS grafts had solid smooth-muscle bundle formation throughout the graft. CONCLUSIONS: BMSCs had a similar cell proliferation, histological appearance and contractile phenotype as primary cultured bladder SMCs. SIS supported three-dimensional growth of BMSCs in vitro, and BMSC-seeded SIS scaffold promoted bladder regeneration in a canine model. BMSCs may serve as an alternative cell source in urological tissue engineering.     

Morphologic and mechanical characteristics of engineered bovine arteries

OBJECTIVE: The ideal small-caliber arterial graft remains elusive despite several decades of intense research. A novel approach to the development of small-caliber arterial prostheses with a biomimetic system for in vitro vessel culture has recently been described. In this study we examined the effects of culture time and tissue culture scaffolding on engineered vessel morphology and function and found that these parameters greatly influence the function of engineered vessels. METHODS: This report describes the effects of culture time and scaffold type on vessel morphology, cellular differentiation, and vessel mechanical characteristics. Engineered vessels were cultured from bovine aortic smooth muscle cells (SMCs) and endothelial cells that were seeded onto biodegradable polymer scaffolds and cultured under physiologically pulsatile conditions. Engineered vessels were subjected to histologic, ultrastructural, immunocytochemical, and mechanical analyses. RESULTS: Vessel morphology and mechanical characteristics improved as time in culture increased to 8 weeks. SMCs in the engineered vessel wall were organized into a highly lamellar structure, with cells separated by alternating layers of collagen fibrils. Polymer scaffold remnants were present in vessels cultured for 8 weeks, and SMCs that were in proximity to polymer remnants exhibited a dedifferentiated phenotype. CONCLUSIONS: These findings aid in the systematic understanding of the effects of in vitro parameters on engineered vessels and will be useful for the translation of vessel culture techniques to human cells for the development of autologous human vascular grafts.     

Bladder smooth muscle cell phenotypic changes and implication of expression of contractile proteins (especially caldesmon) in rats after partial outlet obstruction

BACKGROUND: The purpose of the present study was to investigate morphological changes in bladder smooth muscle of rats with partial outlet obstruction. We investigated smooth muscle cell phenotypic changes and implication of synthetic phenotype in contractility decrease and bladder compliance after bladder outlet obstruction. METHODS: Partial bladder outlet obstruction was introduced in female rats. Bladder were removed at 1, 3, 6, 10 and 20 weeks after the obstruction. Temporal pattern of changes in bladder mass, light microscopic pathogenesis and phenotypic expression of the bladder smooth muscle cells in the electron micrographs were investigated. Expression of contractile protein was also investigated by the immunoblotting method. RESULTS: Marked increase in bladder mass with marked thickening of smooth muscle layer was observed at 1 week after obstruction. The ratio of myocytes exhibiting contractile and synthetic phenotypes was almost constant until 6 weeks after the obstruction, but thereafter, synthetic phenotypes gradually increased and the ratio (synthetic/contractile phenotype) was 1.5-fold at 20 weeks after the obstruction. Caldesmon was most markedly expressed after the obstruction among contractile proteins examined by the immunoblotting method. CONCLUSION: Phenotypic changes were confirmed in bladder smooth muscle, and the decrease of the ratio of contractile phenotype was observed after long-term obstruction of the bladder outlet. Among the contractile proteins in the bladder smooth muscle cell, caldesmon was considered a reliable marker for predicting the pathogenetic conditions of the bladder.     

Phenotypic changes in human bladder smooth muscle cell

Tissue specimens were resected from 15 patients (age 7 to 82, average 52.2 years old) with bladder diseases; i.e., 2 with neurogenic bladder, 6 recipients of kidney transplantation with defunctionalized bladder, 3 with benign prostatic hypertrophy, 3 with bladder cancer and 1 with vesicoureteral reflux. We investigated the phenotypic expression of the bladder smooth muscle cells with bladder diseases. The ratio of non-contractile to contractile phenotypes (nc/c ratio) showed a rising tendency with aging. The increase of nc/c ratio was especially notable with neurogenic bladder. Phenotypic expression was observed in human bladder smooth muscle cells as reported in vascular smooth muscle cells. Several bladder diseases cause a conversion of contractile smooth muscle cell phenotype from contractile type to non-contractile type, and this modulation of smooth muscle cell phenotype may play an important role in detrusor function.     

Fabrication of compliant hybrid grafts supported with elastomeric meshes

We devised tubular hybrid medial tissues with mechanical properties similar to those of native arteries, which were composed of bovine smooth muscle cells (SMCs) and type I collagen with minimal reinforcement with knitted fabric meshes made of synthetic elastomers. Three hybrid medial tissue models that incorporated segmented polyester (mesh A) or polyurethane-nylon (mesh B) meshes were designed: the inner, sandwich, and wrapping models. Hybrid medial tissues were prepared by pouring a cold mixed solution of SMCs and collagen into a tubular glass mold consisting of an inner mandrel and an outer sheath and subsequent thermal gelation, followed by further culture for 7 days. For the inner model, the mandrel was wrapped with a mesh. For the sandwich model, a cylindrically shaped mesh was incorporated into a space between the mandrel and the sheath. The wrapping model was prepared by wrapping a 7-day-incubated nonmesh gel with a mesh. The inner diameter was 3 mm, irrespective of the model, and the length was 2.5-4.0 cm, depending on the model. The intraluminal pressure-external diameter relationship showed that nonmesh and inner models had a very low burst strength below 50 mmHg, while the sandwich model ruptured at around 110-120 mmHg; no rupturing below 240 mmHg was observed for the wrapping model, regardless of the type of mesh used. Compliance values of wrapping and sandwich models were close to those of native arteries. Pressure-dependent distensibility characteristics similar to native arteries were observed for a mesh A wrapping model, whereas a mesh B wrapping model expanded almost linearly as intraluminal pressure increased, which appeared to be due to elasticity of the incorporated mesh. Thus, design criteria for hybrid vascular grafts with appropriate biomechanical matching with host arteries were established. Such hybrid grafts may be mechanically adapted in an arterial system.