Tissue-engineered vascular grafts composed of marine collagen and PLGA fibers using pulsatile perfusion bioreactors

Novel tubular scaffolds of marine source collagen and PLGA fibers were fabricated by freeze drying and electrospinning processes for vascular grafts. The hybrid scaffolds, composed of a porous collagen matrix and a fibrous PLGA layer, had an average pore size of 150+/-50 microm. The electrospun fibrous PLGA layer on the surface of a porous tubular collagen scaffold improved the mechanical strength of the collagen scaffolds in both the dry and wet states. Smooth muscle cells (SMCs)- and endothelial cells (ECs)-cultured collagen/PLGA scaffolds exhibited mechanical properties similar to collagen/PLGA scaffolds unseeded with cells, even after culturing for 23 days. The effect of a mechanical stimulation on the proliferation and phenotype of SMCs and ECs, cultured on collagen/PLGA scaffolds, was evaluated. The pulsatile perfusion system enhanced the SMCs and ECs proliferation. In addition, a significant cell alignment in a direction radial to the distending direction was observed in tissues exposed to radial distention, which is similar to the phenomenon of native vessel tissues in vivo. On the other hand, cells in tissues engineered in the static condition were randomly aligned. Immunochemical analyses showed that the expressions of SM alpha-actin, SM myosin heavy chain, EC von Willebrand factor, and EC nitric oxide were upregulated in tissues engineered under a mechano-active condition, compared to vessel tissues engineered in the static condition. These results indicated that the co-culturing of SMCs and ECs, using collagen/PLGA hybrid scaffolds under a pulsatile perfusion system, leads to the enhancement of vascular EC development, as well as the retention of the differentiated cell phenotype.     

Formation of corporal tissue architecture in vivo using human cavernosal muscle and endothelial cells seeded on collagen matrices

We explored the feasibility of developing corporal tissue, consisting of human cavernosal smooth muscle and endothelial cells in vivo, using three-dimensional acellular collagen matrices, which are similar in architecture to native corpora. Acellular collagen matrices were derived from processed donor rabbit corpora, using cell lysis techniques. Human corpus cavernosal muscle and endothelial cells were seeded on the acellular matrices. A total of 80 matrices, 20 without cells and 60 with cells, were implanted subcutaneously in athymic mice. An additional 36 matrices seeded with cells were maintained in culture for up to 4 weeks. Hydroxyproline quantification, Western blot analysis, RT-PCR, and scanning electron microscopy of the matrices, with and without cells, were performed at various time points. Animals were killed 3 days and 1, 2, 3, 4, 6, and 8 weeks after implantation. Immunocytochemical and histological analyses were performed to confirm the muscle and endothelial phenotype. Organ bath studies were performed in order to determine the degree of tissue contraction. Western blot analysis detected alpha-actin, myosin, and tropomyosin proteins from human corporal smooth muscle cells. Expression of muscarinic acetylcholine receptor (mAChR) subtype m4 mRNA was demonstrated by RT-PCR from corporal muscle cells before and 8 weeks after seeding. The implanted matrices showed neovascularity into the sinusoidal spaces by 1 week after implantation. Increasing organization of smooth muscle and endothelial cells lining the sinusoidal walls was observed at 2 weeks and continued with time. The matrices were covered with the appropriate cell architecture 4 weeks after implantation. The matrices showed a stable collagen concentration over 8 weeks, as determined by hydroxyproline quantification. Immunocytochemical studies using alpha-actin and factor VIII antibodies confirmed the presence of corporal smooth muscle and endothelial cells, both in vitro and in vivo, at all time points. There was no evidence of cellular organization in the control matrices. Organ bath studies showed that the cell-seeded corporal tissue matrices responded to electrical field stimulation, whereas the unseeded implants failed to respond. This study demonstrates that human cavernosal smooth muscle and endothelial cells seeded on three-dimensional acellular collagen matrices derived from donor corpora are able to form well-vascularized corporal tissues in vivo.     

The performance of poly-epsilon-caprolactone scaffolds in a rabbit femur model with and without autologous stromal cells and BMP4

The ability of a cellular construct to guide and promote tissue repair strongly relies on three components, namely, cell, scaffold and growth factors. We aimed to investigate the osteopromotive properties of cellular constructs composed of poly-epsilon-caprolactone (PCL) and rabbit bone marrow stromal cells (BMSCs), or BMSCs engineered to express bone morphogenetic protein 4 (BMP4). Highly porous biodegradable PCL scaffolds were obtained via phase inversion/salt leaching technique. BMSCs and transfected BMSCs were seeded within the scaffolds by using an alternate flow perfusion system and implanted into non-critical size defects in New Zealand rabbit femurs. In vivo biocompatibility, osteogenic and angiogenic effects induced by the presence of scaffolds were assessed by histology and histomorphometry of the femurs, retrieved 4 and 8 weeks after surgery. PCL without cells showed scarce bone formation at the scaffold-bone interface (29% bone/implant contact and 62% fibrous tissue/implant contact) and scarce PCL resorption (16%). Conversely, PCL seeded with autologous BMSCs stimulated new tissue formation into the macropores of the implant (20%) and neo-tissue vascularization. Finally, the BMP4-expressing BMSCs strongly favoured osteoinductivity of cellular constructs, as demonstrated by a more extensive bone/scaffold contact.     

Engineering of vaginal tissue in vivo

Congenital vaginal anomalies and cloacal malformations may require extensive surgical reconstruction. Surgical challenges are often encountered because of the limited amounts of native tissue available. We investigated the feasibility of using vaginal epithelial and smooth muscle cells for the engineering of vaginal tissues in vivo. Vaginal epithelial and smooth muscle cells of female rabbits were grown, expanded in culture, and characterized immunocytochemically. Vaginal epithelial and smooth muscle cells were seeded on polyglycolic acid (PGA) scaffolds at 10 x 10(6) and 20 x 10(6) cells/cm(3), respectively. The cell-seeded scaffolds were subcutaneously implanted into nude mice. The animals were killed 1, 4, and 6 weeks after implantation. Immunocytochemical and histochemical analyses were performed with pancytokeratins AE1/AE3 and with smooth muscle-specific alpha-actin antibodies to confirm the reconstituted tissue phenotype. Western blot analyses and electrical field stimulation studies were also performed to further characterize the tissue-engineered constructs. Vaginal epithelial cells were serially identified with anti-pancytokeratins AE1/AE3 at all culture stages. Smooth muscle cells in culture stained positively with alpha-smooth muscle actin antibodies. One week after implantation in vivo, the retrieved polymer scaffolds demonstrated multilayered tissue strips of both cell types, and penetrating native vasculature was also noted. Increased organization of the smooth muscle and epithelial tissue was evident by 4 weeks. There was no evidence of tissue formation in the controls. Immunocytochemical analyses using anti-pancytokeratins confirmed the presence of vaginal epithelial cells in each of the constructs. Anti-alpha-actin smooth muscle antibodies also confirmed the presence of multilayered smooth muscle fibers and tissue at each time point. Western blot analyses of the scaffolds confirmed the expression of cytokeratin and smooth muscle actin proteins when compared with controls. The contractile properties of the tissue-engineered vaginal constructs in response to electrical field stimulation were similar to those of normal vaginal tissue. Vaginal epithelial and smooth muscle cells can be easily cultured and expanded in vitro. Cell-seeded polymer scaffolds are able to form vascularized vaginal tissue in vivo that have phenotypic and functional properties similar to those of normal vaginal tissues. This is the first demonstration in tissue engineering wherein vaginal epithelial and smooth muscle cells are reconstituted in vivo into vaginal tissue. This technology may be pursued further experimentally in order to achieve the engineering of vaginal tissues for clinical applications.     

Engineering multi-layered skeletal muscle tissue by using 3D microgrooved collagen scaffolds

Preparation of three-dimensional (3D) micropatterned porous scaffolds remains a great challenge for engineering of highly organized tissues such as skeletal muscle tissue and cardiac tissue. Two-dimensional (2D) micropatterned surfaces with periodic features (several nanometers to less than 100 μm) are commonly used to guide the alignment of muscle myoblasts and myotubes and lead to formation of pre-patterned cell sheets. However, cell sheets from 2D patterned surfaces have limited thickness, and harvesting the cell sheets for implantation is inconvenient and can lead to less alignment of myotubes. 3D micropatterned scaffolds can promote cell alignment and muscle tissue formation. In this study, we developed a novel type of 3D porous collagen scaffolds with concave microgrooves that mimic muscle basement membrane to engineer skeletal muscle tissue. Highly aligned and multi-layered muscle bundle tissues were engineered by controlling the size of microgrooves and cell seeding concentration. Myoblasts in the engineered muscle tissue were well-aligned and had high expression of myosin heavy chain and synthesis of muscle extracellular matrix. The microgrooved collagen scaffolds could be used to engineer organized multi-layered muscle tissue for implantation to repair/restore the function of diseased tissues or be used to investigate the cell–cell interaction in 3D microscale topography.     

聚左旋乳酸/壳聚糖纳米纤维三维多孔支架复合骨髓间充质干细胞修复兔骨缺损**△

背景：骨髓间充质干细胞具有向多种间质细胞谱系分化的能力,且支架材料的性能对骨缺损的修复有重要影响。 目的：观察聚左旋乳酸/壳聚糖纳米纤维三维多孔支架复合骨髓间充质干细胞治疗骨缺损。 方法：对骨缺损模型兔分别采用空白植入、髂后上棘自体松质骨移植、聚左旋乳酸/壳聚糖纳米纤维多孔支架移植和复合了骨髓间充质干细胞的聚左旋乳酸/壳聚糖纳米纤维多孔支架移植修复缺损部位。 结果与结论：至移植12周,移植复合了骨髓间充质干细胞的聚左旋乳酸/壳聚糖纳米纤维多孔支架的实验兔的缺损处有骨组织生成,支架材料降解,已完成缺损修复,其修复情况接近松质骨组;髂后上棘自体松质骨移植的实验兔的缺损修复完好,新形成的骨组织较规则;只植入聚左旋乳酸/壳聚糖纳米纤维多孔支架的实验兔有少量骨组织形成,材料部分降解;空白植入的实验兔缺损处无新生骨组织生成,主要由纤维结缔组织填充。说明新型的生物支架材料聚左旋乳酸/壳聚糖纳米纤维三维多孔支架与来源于新西兰大白兔的骨髓间充质干细胞复合培养后,植入同种异体兔股骨髁缺损处,使骨缺损的修复速度加快,表现为较好的体内诱导成骨的作用。     

Ectopic bone regeneration by human bone marrow mononucleated cells, undifferentiated and osteogenically differentiated bone marrow mesenchymal stem cells in beta-tricalcium phosphate scaffolds

Tissue engineering approaches using the combination of porous ceramics and bone marrow mesenchymal stem cells (BMSCs) represent a promising bone substitute for repairing large bone defects. Nevertheless, optimal conditions for constructing tissue-engineered bone have yet to be determined. It remains unclear if transplantation of predifferentiated BMSCs is superior to undifferentiated BMSCs or freshly isolated bone marrow mononucleated cells (BMNCs) in terms of new bone formation in vivo. The aim of this study was to investigate the effect of in vitro osteogenic differentiation (β-glycerophosphate, dexamethasone, and l-ascorbic acid) of human BMSCs on the capability to form tissue-engineered bone in unloaded conditions after subcutaneous implantation in nude mice. After isolation from human bone marrow aspirates, BMNCs were divided into three parts: one part was seeded onto porous beta-tricalcium phosphate ceramics immediately and transplanted in a heterotopic nude mice model; two parts were expanded in vitro to passage 2 before cell seeding and in vivo transplantation, either under osteogenic conditions or not. Animals were sacrificed for micro-CT and histological evaluation at 4, 8, 12, 16, and 20 weeks postimplantation. The results showed that BMSCs differentiated into osteo-progenitor cells after induction, as evidenced by the altered cell morphology and elevated alkaline phosphatase activity and calcium deposition, but their clonogenicity, proliferating rate, and seeding efficacy were not significantly affected by osteogenic differentiation, compared with undifferentiated cells. Extensive new bone formed in the pores of all the scaffolds seeded with predifferentiated BMSCs at 4 weeks after implantation, and maintained for 20 weeks. On the contrary, scaffolds containing undifferentiated BMSCs revealed limited bone formation only in 1 out of 6 cases at 8 weeks, and maintained for 4 weeks. For scaffolds with BMNCs, woven bone was observed sporadically only in one case at 8 weeks. Overall, this study suggests that ectopic osteogenesis of cell/scaffold composites is more dependent on the in vitro expansion condition, and osteo-differentiated BMSCs hold the highest potential concerning in vivo bone regeneration.     

Increased smooth muscle actin expression from bone marrow stromal cells under retinoic acid treatment: an attempt for autologous blood vessel tissue engineering

Vascular replacement in vital organs is sometimes necessary for human life for example because of atherosclerosis. Blood vessel tissue engineering is applied for autologous transplantations to avoid graft rejections. Stem cells are used for blood vessel tissue engineering because they are the origin of smooth muscle cells, endothelial cells and fibroblasts. This paper shows that bone marrow stromal cells (BMSCs) can be induced to differentiate into the early stage of smooth muscle cells by using 0.01 microM retinoic acid. The differentiation of BMSCs to smooth muscle cells was detected by the expression of smooth muscle alpha actin (SM alpha-actin), the earliest smooth muscle cell marker. The SM alpha-actin marker expression was demonstrated using indirect immunofluorescence technique and Western blot analysis. The induction of BMSC to form early stages of smooth muscle cells in this study is appropriate for blood vessel tissue engineering because the early stage smooth muscle cells may be stimulated to develop vascular walls with endothelial cells using a co-culture system.     

重组hTGF-β1基因转染的BMSCs在体内成软骨能力的初步研究

目的重组hTGF-β1腺病毒(adeno-hTGF-β1)转染的BMSCs在体内成软骨能力的初步研究,探讨其作为组织工程化软骨的种子细胞的可行性。方法重组adeno-hTGF-β1转染猪BMSCs,酶联免疫吸附定量检测(ELISA法)重组腺病毒转染hTGF-β1蛋白的表达。然后消化收集重组腺病毒转染后的BMSCs,均匀接种于圆盘状PGA支架上,对照组转染adeno-LacZ,然后植入裸鼠背部皮下,在植入后第3周取材,分别行大体观察、组织学、II型胶原免疫组化和蛋白聚糖含量检测对形成组织进行评价。结果 ELISA结果显示adeno-hTGF-β1转染的BMSCs,其hTGF-β1表达量是转染adeno-lacZ 的BMSCs 2.65倍( P0.05)。植入裸鼠体内后3周取材,实验组HE染色观察可见有软骨形成,但较不均匀,并被纤维组织分割,形成的软骨组织区域可见软骨细胞包埋在软骨陷窝内;对照组可见仅有少量软骨形成,被大量的纤维组织和未降解的PGA支架包裹,实验组和对照组形成软骨占总组织百分比,分别为(41.83±4.64)%和(17.50±2.85)%,P0.05。Safranin’O染色显示,实验组形成的软骨组织区域都有被染成桔红色蛋白多糖类基质分泌,着色比对照组更深。实验组形成的软骨组织区域有大量棕黄色的II型胶原颗粒,而对照组仅有少量的棕黄色的II型胶原颗粒,实验组形成的软骨组织中的蛋白聚糖含量多于正常猪耳软骨。结论重组hTGF-β1腺病毒转染BMSCs作为种子细胞,在裸鼠体内能促使软骨组织形成,从而为hTGF-β1基因转染的BMSCs在软骨组织工程应用中奠定了基础。     

Mechano-active tissue engineering of vascular smooth muscle using pulsatile perfusion bioreactors and elastic PLCL scaffolds

Blood vessels are subjected in vivo to mechanical forces in a form of radial distention, encompassing cyclic mechanical strain due to the pulsatile nature of blood flow. Vascular smooth muscle (VSM) tissues engineered in vitro with a conventional tissue engineering technique may not be functional, because vascular smooth muscle cells (VSMCs) cultured in vitro typically revert from a contractile phenotype to a synthetic phenotype. In this study, we hypothesized that pulsatile strain and shear stress stimulate VSM tissue development and induce VSMCs to retain the differentiated phenotype in VSM engineering in vitro. To test the hypothesis, rabbit aortic smooth muscle cells (SMCs) were seeded onto rubber-like elastic, three-dimensional PLCL [poly(lactide-co-caprolactone), 50:50] scaffolds and subjected to pulsatile strain and shear stress by culturing them in pulsatile perfusion bioreactors for up to 8 weeks. As control experiments, VSMCs were cultured on PLCL scaffolds statically. The pulsatile strain and shear stress enhanced the VSMCs proliferation and collagen production. In addition, a significant cell alignment in a direction radial to the distending direction was observed in VSM tissues exposed to radial distention, which is similar to that of native VSM tissues in vivo, whereas VSMs in VSM tissues engineered in the static condition randomly aligned. Importantly, the expression of SM alpha-actin, a differentiated phenotype of SMCs, was upregulated by 2.5-fold in VSM tissues engineered under the mechano-active condition, compared to VSM tissues engineered in the static condition. This study demonstrates that tissue engineering of VSM tissues in vitro by using pulsatile perfusion bioreactors and elastic PLCL scaffolds leads to the enhancement of tissue development and the retention of differentiated cell phenotype.     

Three-dimensional engineered bone from bone marrow stromal cells and their autogenous extracellular matrix

Most bone tissue-engineering research uses porous three-dimensional (3D) scaffolds for cell seeding. In this work, scaffold-less 3D bone-like tissues were engineered from rat bone marrow stromal cells (BMSCs) and their autogenous extracellular matrix (ECM). The BMSCs were cultured on a 2D substrate in medium that induced osteogenic differentiation. After reaching confluence and producing a sufficient amount of their own ECM, the cells contracted their tissue monolayer around two constraint points, forming scaffold-less cylindrical engineered bone-like constructs (EBCs). The EBCs exhibited alizarin red staining for mineralization and alkaline phosphatase activity and contained type I collagen. The EBCs developed a periosteum characterized by fibroblasts and unmineralized collagen on the periphery of the construct. Tensile tests revealed that the EBCs in culture had a tangent modulus of 7.5 +/- 0.5 MPa at 7 days post-3D construct formation and 29 +/- 9 MPa at 6 weeks after construct formation. Implantation of the EBCs into rats 7 days after construct formation resulted in further bone development and vascularization. Tissue explants collected at 4 weeks contained all three cell types found in native bone: osteoblasts, osteocytes, and osteoclasts. The resulting engineered tissues are the first 3D bone tissues developed without the use of exogenous scaffolding.     

不同来源骨髓间充质干细胞的体内成软骨

背景：骨髓间充质干细胞经体外诱导后可修复软骨缺损,但目前采用的种子细胞多来源于自体或同种异体。 目的：观察同种异体及异种来源的骨髓间充质干细胞诱导成软骨后修复喉软骨缺损的效果。 方法：分别取人胚胎骨髓间充质干细胞和刚出生兔骨髓间充质干细胞的第3代细胞种植于聚乳酸-羟基乙酸共聚物生物支架上,并加入转化生长因子β1和软骨形态发生蛋白诱导成软骨细胞。将两种细胞体系植入新西兰白兔体内,并于植入后4,8周取材行大体、组织学观察。 结果与结论：植入后4,8周人胚胎骨髓间充质干细胞和兔骨髓间充质干细胞均有新生组织填充,经组织学观察大部分为软骨细胞,分泌软骨细胞基质糖胺聚糖和Ⅱ型胶原,且两种细胞支架复合物所生成的软骨细胞数大致相同,并无明显的免疫排斥反应。提示异种来源的骨髓间充质干细胞复合聚乳酸-羟基乙酸共聚物在转化生长因子β1和软骨形态发生蛋白联合诱导下所得的组织工程化软骨,与同种来源的骨髓间充质干细胞所获得的组织工程化软骨修复喉软骨缺损具有可比性。     

Analysis of the dynamics of bone formation, effect of cell seeding density, and potential of allogeneic cells in cell-based bone tissue engineering in goats

After decades of research, relatively little is known about the role of bone marrow stromal cells (BMSCs) for bone tissue engineering. Although homogeneous cell seeding is regarded optimal, cell survival in large constructs is unlikely, except for the very periphery. Also no minimal and optimal BMSC densities have been identified. An interesting development is the use of allogeneic BMSCs. These have not yet been compared directly to autologous BMSCs. Culture-expanded BMSCs of 10 Dutch milk goats were cryopreserved and peroperatively seeded on 7 mm cubic scaffolds of 65% porous biphasic calcium phosphate (BCP). A range of BMSC densities (per cm3 scaffold) were prepared of 8E2 (= 8 x 10(2)), 8E3, 8E4, 8E5, 8E6 (considered the standard), and 1.6E7. Each goat received a control without cells, the six densities, and an 8E6 allogeneic BMSCs construct intramuscularly. After 3, 5, and 7 weeks, fluorochrome markers were administrated. At 9 weeks, implants were retrieved. The BCP scaffolds appeared to be autoinductive as the controls (without BMSCs) showed some bone. Early bone formation (before 3 weeks) appeared only at the peripheral 2mm of the BMSC-seeded constructs; the later 5- and 9-week labels were found more centrally, suggesting bone migration to the center. There was a minimum of 8E4 and optimum of 8E6 BMSCs/cm3. Allogeneic cells yielded comparable new bone.     

人骨形成蛋白7修饰骨髓间充质干细胞复合仿生型支架材料的体内成骨研究

目的 应用携带人骨形成蛋白7(hBMP7)基因的兔骨髓间充质干细胞(BMSC)与仿生型生物玻璃-胶原-透明质酸-磷脂酰丝氨酸(BG-COL-HYA-PS)支架材料复合培养,植入兔桡骨缺损模型中观察其在体内成骨的能力.方法 携带hBMP7基因或增强型绿色荧光蛋白(EGFP)基因的2型重组腺相关病毒(rAAV2)载体在体外分别转染兔BMSC,再将转染后和未转染的兔BMSC分别与BG-COL-HYA-PS支架材料复合培养7 d后植入3组兔桡骨缺损模型,每组6只兔.各组在分别术后8周、12周通过大体标本观察、影像学、组织学等方法观察骨缺损的修复情况.以正常兔桡骨为对照组(n=3),术后12周比较各组骨缺损修复组织生物力学差异.结果 rAAV2-hBMP7转染的兔BMSC与BG-COL-HYA-PS复合支架材料有良好的生物相容性,植入兔桡骨缺损模型内表现出明确的成骨能力和骨修复能力,而形成的新骨最大压力载荷值低于正常桡骨组织[(188.46±12.24)N比(203.25±19.29)N,P＜0.05].结论 用hBMP7修饰BMSC复合仿生型BG-COL-HYA-PS支架材料构建的组织工程骨具有较强的骨修复能力,但形成的新生骨组织与正常骨组织比较仍然有早期生物力学方面的不足.     

Chondrogenic derivatives of embryonic stem cells seeded into 3D polycaprolactone scaffolds generated cartilage tissue in vivo

In spite of recent scientific advances, treatment and repair of cartilage using tissue engineering techniques remains challenging. The major constraint is the limited proliferative capacity of mature autologous chondrocytes used in the tissue engineering approach. This problem can be addressed by using stem cells, which can self-renew with greater proliferative potential. Cartilage tissue engineering using adult mesenchymal stem cells derived from bone marrows has met with limited success. In this study we explored cartilage tissue generation from embryonic stem cells (ESCs). ESCs were induced to differentiate into chondroprogenitors, capable of proliferating and subsequently differentiating into cartilage-producing cells. The chondrogenic cells expressed chondrocyte-specific markers and deposited extracellular matrix proteins, proteoglycans. ESC-derived chondrogenic cells and polycaprolactone scaffolds seeded with these cells implanted in mice (129 SvImJ) generated cartilage tissue in vivo. Postimplant analysis of the retrieved tissues demonstrated cartilage-like tissue formation in 3-4 weeks. The cells of retrieved tissues also expressed the chondrocyte-specific marker collagen II. These findings suggest that ESCs can be used for tissue engineering and cultivation of cartilage tissues.     

Immunohistochemical studies on expression of human vascular smooth muscle myosin heavy chain isoforms in normal mammary glands, benign mammary disorders and mammary carcinomas

The expression of myosin in normal and diseased mammary glands of 199 Japanese women was evaluated immuno-histochemically by the avidin-biotin peroxldase complex method using antibodies to three human smooth muscle myosin heavy chain isoforms derived from the vascular smooth muscle: myosin SM1 is expressed consistently from fetal stage to adulthood, myosin SM2 appears only in well-differentiated smooth muscle after birth, and myosin SMemb is more abundant in embryonic aortas. SM1 was expressed in myoepithelial cells of normal mammary glands and fibrocystic diseases and in myoepithelial-like tumor cells In the basal layer of fibroadenomas and phyllodes tumors. SM2 was expressed only In the myoeplthelial cells of mammary glands in breastfeeding women. SMemb was expressed more Intensely In the cytoplasm of luminal epithelial cells in larger fibroadenomas ( P <0.01), or In the cytoplasm of carcinoma cells In lnvaslve ductal carcinomas with metastasized lymph nodes ( P <0.001) and In those of higher histological grade ( P <0.0001). Multivariate logistic analysis showed a significant correlation only between the expression of SMemb and histological grade ( P <0.0001), which is a prognostic factor of mammary carcinomas. These findings suggested the possible prognostic value of SMemb.     

Contractile protein isoforms of single and cultured smooth muscle cells from guinea pig ileum.

Single smooth muscle cells isolated from guinea pig ileum using collagenase and papain produce contractile response to muscarinic agents, while the cultured cells do not. Using fluo-3/AM and a confocal laser scanning fluorescence microscope, it was observed that carbachol, a muscarinic agent, caused an increase in the intracellular Ca2+ of both single and cultured cells. SDS-PAGE and Western Blot analyses revealed the expression of myosin heavy chain isoforms of SM1 (204 kDa) and SM2 (200 kDa) in single smooth muscle cells, and non muscle isoform (196 kDa) of myosin heavy chain only in the cultured cells. With respect to actin isoforms, alpha-actin was predominant in single cells and beta-actin was major in the cultured cells. Two types of tropomyosin monomer, 39 kDa and 41 kDa, were detected in single cells, while the 41 kDa monomer was lost in cultured cells. These differences in contractile protein profiles between single and cultured cells were collaborated with the observation of cells using immunofluorescence microscope with responsible antibodies to isoforms of myosin heavy chain, actin and tropomyosin. These results suggest that the loss of contractility in cultured smooth muscle cells is profoundly related to changes in contractile protein profiles from smooth muscle type to non muscle type.