Tissue reorganization in response to mechanical load increases functionality

In the rapidly growing field of tissue engineering, the functional properties of tissue substitutes are recognized as being of the utmost importance. The present study was designed to evaluate the effects of static mechanical forces on the functionality of the produced tissue constructs. Living tissue sheets reconstructed by the self-assembly approach from human cells, without the addition of synthetic material or extracellular matrix (ECM), were subjected to mechanical load to induce cell and ECM alignment. In addition, the effects of alignment on the function of substitutes reconstructed from these living tissue sheets were evaluated. Our results show that tissue constructs made from living tissue sheets, in which fibroblasts and ECM were aligned, presented higher mechanical resistance. This was assessed by the modulus of elasticity and ultimate strength as compared with tissue constructs in which components were randomly oriented. Moreover, tissue-engineered vascular media made from a prealigned living tissue sheet, produced with smooth muscle cells, possessed greater contractile capacity compared with those produced from living tissue sheets that were not prealigned. These results show that the mechanical force generated by cells during tissue organization is an asset for tissue component alignment. Therefore, this work demonstrates a means to improve the functionality (mechanical and vasocontractile properties) of tissues reconstructed by tissue engineering by taking advantage of the biomechanical forces generated by cells under static strain.     

Acellular Blood Vessels Combined Human Hair Follicle Mesenchymal Stem Cells for Engineering of Functional Arterial Grafts

Tissue-engineered vessels offer options for autologous vascular grafts in cardiovascular repair and regeneration. The experiments aimed to construct functional arterial grafts by combining human hair follicle mesenchymal stem cells (HF-MSCs) with acellular umbilical arteries. We isolated mesenchymal stem cells from human hair follicles. Under appropriate culture conditions, these cells displayed CD44, CD90 and CD105, and exhibited the potential for differentiation to adipocytes, osteoblasts and chondrocytes. Very promisingly, HF-MSCs expressed the vascular smooth muscle specific markers in the presence of transforming growth factor-β. We created acellular arterial scaffolds by digesting human umbilical arteries with trypsin and sodium dodecyl sulfate. These acellular arterial scaffolds retained major components of the extracellular matrix. The mechanical properties of these acellular arterial scaffolds were very similar to those of native blood vessels. We then seeded HF-MSCs into acellular arterial scaffolds and found that they still expressed vascular smooth muscle specific markers. The arterial grafts derived from HF-MSCs demonstrated vasoreactivity in response to humoral constrictors. We constructed arterial grafts that are very close to native blood vessels in their structures and physiological functions. These properties suggest that these arterial grafts could be used as small diameter arterial grafts for cardiovascular repair and regeneration.     

C1q binding to human vascular smooth muscle cells mediates immune complex deposition and superoxide generation

Evidence was obtained for the binding of Clq to the membrane of cultured vascular smooth muscle cells derived from human umbilical cord veins. Clq was fixed to the cell membrane at 4°C, whereas it was ingested into the cytoplasm, as a cytoplasmic inclusion, when tested at 37°C. The addition of Clq in advance inhibited the subsequent binding of Clq. Neither fibronectin nor laminin was detected on the cell membrane. Aggregated IgG bound to vascular smooth muscle cells in the case of preincubation with Clq at 4°C, whereas aggregated IgG did not bind to the cells in the absence of Clq. The addition of Clq molecules to the cells in suspension enhanced Superoxide generation by vascular smooth muscle cells. There was no effect of Clq on Superoxide generation by the cells in monolayer. These results suggest that Clq binds on the membrane of vascular smooth muscle cells via its specific receptor that mediates immune complex binding to the cells and Superoxide generation. These properties elucidate the mechanisms by which circulating immune complexes deposit in the vascular wall, and subsequent degradation of tissue components surrounding vascular smooth muscle cells occurs through oxidative burst of the cells.     

人脐动脉平滑肌细胞培养及转染TFPI基因的实验研究

目的 探讨组织因子途径抑制因子（TFPI）基因对人脐动脉血管平滑肌细胞生长的影响,为TFPI基因用于血管再狭窄的治疗提供理论依据和实验基础.方法 从人脐动脉分离平滑肌细胞,通过免疫组化方法进行细胞鉴定;用不同剂量pIRES-TFPI基因（分别为1,2,3 μg/mL）转染血管平滑肌细胞,采用RT-PCR测定细胞内TFPI表达以优化基因转染条件;通过MTT法测定TFPI基因对人脐动脉血管平滑肌细胞生长的影响.结果 分离得到的血管平滑肌细胞的纯度高于90%;3个剂量的基因转染后,细胞内TFPI基因的表达水平无明显差异.采用2 μg/mL转染剂量时,TFPI基因转染后第5天,脐动脉血管平滑肌的生长受到明显抑制.结论 通过基因转染的方式将TFPI基因导入细胞对人脐动脉平滑肌的增殖具有抑制作用.     

Production of the third and fourth component of complement (C3, C4) by smooth muscle cells.

Production of the third and fourth components of complement (C3, C4) by smooth muscle cells was investigated by using normal human aortic smooth muscle cells (AoSMC), human smooth muscle cell line (G402) and vascular smooth muscle cells obtained from human umbilical cord vein (UVSMC). AoSMC spontaneously produced both C3 and C4 at 15 ng/10(6) cells/72 hr and 22 ng/10(6) cells/72 hr, respectively, and both were enhanced by interferon-gamma (IFN-gamma). Although phorbol 12-myristate 13-acetate (PMA) and tumour necrosis factor-alpha (TNF-alpha) enhanced C3 production, C4 production was reduced by these agents. On the other hand, G402 produced C4 but not C3 in a dose-dependent manner when cultured with IFN-gamma. UVSMC produced only a small amount of C3 and C4 compared with AoSMC or G402. C3 and C4 produced by AoSMC were confirmed to be identical with their human serum counterparts as determined by sodium dodecyl sulphate-polyacrylamide gel electrophoresis and measurement of haemolytic activity. Northern blotting analysis showed that the expression of mRNA of C3 and C4 was enhanced by TNF-alpha and IFN-gamma, respectively, in AoSMC. Our findings suggest the importance of smooth muscle cells as a source of components of complement in vascular diseases including vasculitis.     

Vascular smooth muscle cells on hyaluronic acid: culture and mechanical characterization of an engineered vascular construct

Esterified hyaluronic acid (HYAFF) is routinely used for clinical tissue-engineering applications such as skin and cartilage. The material is degraded by neotissue formation and degradation products are highly biocompatible. In the present article we investigate the possibility to culture vascular smooth muscle cells on this biodegradable material for the generation of tubular constructs to be used for vascular tissue engineering. We have evaluated cell attachment and growth, and the possibility to obtain a three-dimensional tubular shape culture from flat HYAFF sheets. We also evaluated the mechanical properties of the cell constructs, using a specific testing protocol, and compared them with the properties of segments of porcine coronary artery. Morphology and viability tests demonstrated that vascular cells, either from porcine or human origin, adhere and grow on nonwoven meshes of HYAFF, and that precoating of the material with fibronectin or collagen had a modest effect on cell growth and extracellular matrix production. Cell growth reached a maximum 7 days after seeding. Simple wrapping of flat sheets of nonwoven meshes containing vascular cells around a cylindrical mandrel, and culture under static conditions for 14 days, yielded tubular constructs suitable for mechanical tests. Despite cell colonization, constructs showed lower mechanical resistance as compared with porcine coronary arteries. The material used and the technique developed result in highly cellularized tubular constructs. Whether the mechanical properties may be improved by dynamic culture conditions is worthy of investigation.     

Development of the human umbilical vein scaffold for cardiovascular tissue engineering applications

Biologic function and the mechanical performance of vascular grafting materials are important predictors of graft patency. As such, "functional" materials that improve biologic integration and function have become increasingly sought after. An important alternative to synthetic materials is the use of biomaterials derived from ex vivo tissues that retain significant biologic and mechanical function. Unfortunately, inconsistent mechanical properties that result from tedious, time consuming, manual dissection methods have reduced the potential usefulness of many of these materials. We describe the preparation of the human umbilical vein (HUV) for use as an acellular, three-dimensional, vascular scaffold using a novel, automated dissection methodology. The goal of this investigation was to determine the effectiveness of the autodissection methodology to yield an ex vivo biomaterial with improved uniformity and reduced variance. Mechanical properties, including burst pressure, compliance, uniaxial tension testing, and suture holding capacity, were assessed to determine the suitability of the HUV scaffold for vascular tissue engineering applications. The automated methodology results in a tubular scaffold with significantly reduced sample to sample variation, requiring significantly less time to excise the vein from the umbilical cord than manual dissection methods. Short-term analysis of the interactions between primary human vascular smooth muscle cells and fibroblasts HUV scaffold have shown an excellent potential for cellular integration by native cellular remodeling processes. Our work has shown that the HUV scaffold is mechanically sound, uniform, and maintains its biphasic stress-strain relationship throughout tissue processing. By maintaining the mechanical properties of the native blood vessels, in concert with promising cellular interactions, the HUV scaffold may lead to improved grafts for vascular reconstructive surgeries.     

Human vascular smooth muscle in culture. Growth and ultrastructure.

Mixed primary cultures of endothelial and smooth muscle cells were obtained from human umbilical cord vessels after prolonged collagenase digestion of their luminal surfaces. Morphologically homogeneous populations of vascular smooth muscle were then selectively isolated and subcultured for up to 16 weeks. Ultrastructurally, cultured cells were characterized by the presence of bundles of myofilaments with dense bodies similar to native umbilical vessel smooth muscle. Mature cultures developed a distinctive topographical organization consisting of bands of parallel cells and intertwined, multilayered areas. Elaborate intercellular attachments formed along contiguous cell surfaces. Large amounts of extracellular material accumulated, including amorphous substance, elastic fiber microfibrils, and 250- to 300-A,faintly-banded fibrils. In older cultures, focal proliferation, extracellular material secretion and cellular degeneration produced nodular protrusions somewhat resembling atherosclerotic lesions in vivo. Endothelium and smooth muscle cultured from this readily available human source may provide useful comparative material for pathophysiologic studies of vascular disease.     

Spatial and temporal pattern of smooth muscle cell differentiation during development of the vascular system in the mouse embryo

The initial phase of smooth muscle differentiation in the vascular system of the mouse embryo was observed immunohistochemically with monoclonal antibody against -smooth muscle actin. Few smooth muscle cells were detected in the vascular system of the 9.5-day embryo, where only the dorsal aorta and umbilical artery showed signs of smooth muscle differentiation. In the 10.5-day embryo, smooth muscle cells were observed in the dorsal aorta, ventral aorta, omphalomesenteric artery and vein, umbilical artery and vein, internal carotid artery, aortic arches III and IV, and subclavian artery. The extent of smooth muscle differentiation varied among these vessels and among regions of a vessel. At 11.5 days of gestation, smooth muscle cells appeared in the basilar artery, vertebral artery, aortic arches VI, intersomitic artery, ductus venosus, and caudal artery. Smooth muscle cells were absent from the venous system characteristic of the embryo at the stages examined. Alpha-smooth muscle actin-positive cells were also observed in allantoic mesoderm in the placenta at 9.5 days, when the umbilical vessels were not surrounded by smooth muscle cells. Vascular smooth muscle cells appear to arise independently from mesenchyme at multiple sites in the vascular system.     

In vitro and in vivo studies of ePTFE vascular grafts treated with P15 peptide

The purpose of this study is to evaluate the effectiveness of P15 cell-binding peptide treated ePTFE vascular grafts in vitro and in vivo. The P15 peptide was covalently immobilized onto ePTFE vascular grafts by an atmospheric plasma coating method. In vitro cell growth properties were studied using primary human umbilical vein endothelial cells (HUVECs) and primary human umbilical artery smooth muscle cells (HUASMCs). X-ray photoelectron spectroscopy and amino-acid analysis were used to analyze the surface characteristics of the peptide treated and untreated grafts. The cell growth study showed that the P15 peptide effectively promoted the adhesion and proliferation of endothelial cells. 700% more endothelial cells were proliferated on the P15-treated ePTFE grafts compared to the untreated ePTFE controls. In contrast, the P15 peptide was significantly less effective for promoting the adhesion and proliferation of smooth muscle cells than endothelial cells; only about 100% more smooth muscle cells proliferated on the P15-treated samples compared to the untreated control samples. The sheep model was used in the in vivo study. The amount of neointimal hyperplasia present at the arterial and venous sides of the anastomosis and the degree of endothelialization on the luminal surface of the grafts were assessed. Four P15-treated grafts and two control grafts were implanted as arteriovenous grafts between the femoral artery and vein or the carotid artery and jugular vein in two sheep (n = 6). The in vivo study showed that the thickness of the neointimal hyperplasia of untreated grafts was 3-times thicker than that of P15-treated grafts (P < 0.05) at the venous side of the anastomosis. P15-treated grafts also had a higher degree of endothelialization on the graft lumen.     

Genetic modification of smooth muscle cells to control phenotype and function in vascular tissue engineering

Rat smooth muscle cells (SMCs) stably transfected with the gene for the phenotype regulating protein cyclic guanosine monophosphate-dependent protein kinase (PKG) were used as a cell source in the preparation of three-dimensional (3D) collagen type I vascular constructs. PKG-transfected cells expressed severalfold higher levels of the contractile protein smooth muscle alpha-actin (SMA), relative to untransfected SMCs, both in monolayer culture and in 3D gels. The proliferation rate of PKG-transfected cells was lower than that of untransfected cells in both culture geometries. Three-dimensional collagen constructs made with PKG-transfected cells compacted to a similar degree as those made with untransfected cells, and this compaction could be augmented by biochemical stimulation with platelet-derived growth factor BB (PDGF) or transforming growth factor beta(1) (TGF). Application of cyclic mechanical strain to tubular collagen gels seeded with PKG-transfected cells resulted in a higher degree of gel compaction and circumferential matrix alignment, relative to statically grown controls, but cell proliferation and SMA expression were not affected. These results show that genetic modification of SMCs can be used as a tool to control cell function in vascular tissue engineering, and that the function of such cells can be further modulated by application of biochemical and mechanical stimulation.     

Functional characterization of human coronary artery smooth muscle cells under cyclic mechanical strain in a degradable polyurethane scaffold

There are few synthetic elastomeric biomaterials that simultaneously provide the required biological conditioning and the ability to translate biomechanical stimuli to vascular smooth muscle cells (VSMCs). Biomechanical stresses are important physiological elements that regulate VSMC function, and polyurethane elastomers are a class of materials capable of facilitating the translation of stress induced biomechanics. In this study, human coronary artery smooth muscle cells (hCASMCs), which were seeded into a porous degradable polar/hydrophobic/ionic (D-PHI) polyurethane scaffold, were subjected to uniaxial cyclic mechanical strain (CMS) over a span of four weeks using a customized bioreactor. The distribution, proliferation and contractile protein expression of hCASMCs in the scaffold were then analyzed and compared to those grown under static conditions. Four weeks of CMS, applied to the elastomeric scaffold, resulted in statistically greater DNA mass, more cell area coverage and a better distribution of cells deeper within the scaffold construct. Furthermore, CMS samples demonstrated improved tensile mechanical properties following four weeks of culture, suggesting the generation of more extracellular matrix within the polyurethane constructs. The expression of smooth muscle α-actin, calponin and smooth muscle myosin heavy chain and the absence of Ki-67+ cells in both static and CMS cultures, throughout the 4 weeks, suggest that hCASMCs retained their contractile character on these biomaterials. The study highlights the importance of implementing physiologically-relevant biomechanical stimuli in the development of synthetic elastomeric tissue engineering scaffolds.     

An Expanded Population of CD34+ Cells from Frozen Banked Umbilical Cord Blood Demonstrate Tissue Repair Mechanisms of Mesenchymal Stromal Cells and Circulating Angiogenic Cells in an Ischemic Hind Limb Model

Peripheral vascular disease affects ~20 % of the population over 50 years of age and is a complication of type 2 diabetes. Cell therapy studies revealed that cells from older or diabetic donors have a reduced capacity to induce tissue repair compared to healthy and younger cells. This fact greatly impedes the use of autologous cells for treatment. Umbilical cord blood CD34+ cells are a source of angiogenic cells but unlike bone marrow CD34+ angiogenic cells, achieving clinically significant cell numbers has been difficult without in vitro expansion. We report here that culturing CD34+/CD45+ blood cells from frozen umbilical cord blood units in a medium supplemented with FGF4, SCF and FLT3-ligand produced a population of cells that remain CD34+/CD45+ but have an increased capacity for tissue healing. The cultured CD34+ cells were compared directly to non-cultured CD34+ cells in a mouse model of ischemia. Cultured CD34+ cells demonstrated strong paracrine signaling as well as the capacity to differentiate into endothelial cells, smooth muscle and striated muscle. We observed an improvement in blood flow and a significant reduction in foot necrosis. A second study was completed to assess the safety of the cells. No adverse effects were associated with the injection of the cultured cells. Our method described here for culturing umbilical cord blood cells resulted in cells with a strong paracrine effect that induces substantial tissue repair in a murine model of hind limb ischemia and evidence of engraftment and differentiation of the cultured cells into new vasculature and muscle.     

New stent surface materials: The impact of polymer-dependent interactions of human endothelial cells,smooth muscle cells,and platelets

Despite the development of new coronary stent technologies, in-stent restenosis and stent thrombosis are still clinically relevant. Interactions of blood and tissue cells with the implanted material may represent an important cause of these side effects. We hypothesize material-dependent interaction of blood and tissue cells. The aim of this study is accordingly to investigate the impact of vascular endothelial cells, smooth muscle cells and platelets with various biodegradable polymers to identify a stent coating or platform material that demonstrates excellent endothelial-cell-supportive and non-thrombogenic properties. Human umbilical venous endothelial cells, human coronary arterial endothelial cells and human coronary arterial smooth muscle cells were cultivated on the surfaces of two established biostable polymers used for drug-eluting stents, namely poly(ethylene-co-vinylacetate) (PEVA) and poly(butyl methacrylate) (PBMA). We compared these polymers to new biodegradable polyesters poly(l-lactide) (PLLA), poly(3-hydroxybutyrate) (P(3HB)), poly(4-hydroxybutyrate) (P(4HB)) and a polymeric blend of PLLA/P(4HB) in a ratio of 78/22% (w/w). Biocompatibility tests were performed under static and dynamic conditions. Measurement of cell proliferation, viability, glycocalix width, eNOS and PECAM-1 mRNA expression revealed strong material dependency among the six polymer samples investigated. Only the polymeric blend of PLLA/P(4HB) achieved excellent endothelial markers of biocompatibility. Data show that PLLA and P(4HB) tend to a more thrombotic response, whereas the polymer blend is characterized by a lower thrombotic potential. These data demonstrate material-dependent endothelialization, smooth muscle cell growth and thrombogenicity. Although polymers such as PEVA and PBMA are already commonly used for vascular implants, they did not sufficiently meet the criteria for biocompatibility. The investigated biodegradable polymeric blend PLLA/P(4HB) evidently represents a promising material for vascular stents and stent coatings.     

In vitro and in vivo studies of ePTFE vascular grafts treated with P15 peptide

The purpose of this study is to evaluate the effectiveness of P15 cell-binding peptide treated ePTFE vascular grafts in vitro and in vivo. The P15 peptide was covalently immobilized onto ePTFE vascular grafts by an atmospheric plasma coating method. In vitro cell growth properties were studied using primary human umbilical vein endothelial cells (HUVECs) and primary human umbilical artery smooth muscle cells (HUASMCs). X-ray photoelectron spectroscopy and aminoacid analysis were used to analyze the surface characteristics of the peptide treated and untreated grafts. The cell growth study showed that the P15 peptide effectively promoted the adhesion and proliferation of endothelial cells. 700% more endothelial cells were proliferated on the P15-treated ePTFE grafts compared to the untreated ePTFE controls. In contrast, the P15 peptide was significantly less effective for promoting the adhesion and proliferation of smooth muscle cells than endothelial cells; only about 100% more smooth muscle cells proliferated on the P15-treated samples compared to the untreated control samples. The sheep model was used in the in vivo study. The amount of neointimal hyperplasia present at the arterial and venous sides of the anastomosis and the degree of endothelialization on the luminal surface of the grafts were assessed. Four P15-treated grafts and two control grafts were implanted as arteriovenous grafts between the femoral artery and vein or the carotid artery and jugular vein in two sheep (n = 6). The in vivo study showed that the thickness of the neointimal hyperplasia of untreated grafts was 3-times thicker than that of P15-treated grafts (P < 0.05) at the venous side of the anastomosis. P15-treated grafts also had a higher degree of endothelialization on the graft lumen.     

Vascular smooth muscle cells for use in vascular tissue engineering obtained by endothelial-to-mesenchymal transdifferentiation (EnMT) on collagen matrices

The discovery of the endothelial progenitor cell (EPC) has led to an intensive research effort into progenitor cell-based tissue engineering of (small-diameter) blood vessels. Herein, EPC are differentiated to vascular endothelial cells and serve as the inner lining of bioartificial vessels. As yet, a reliable source of vascular smooth muscle progenitor cells has not been identified. Currently, smooth muscle cells (SMC) are obtained from vascular tissue biopsies and introduce new vascular pathologies to the patient. However, since SMC are mesenchymal cells, endothelial-to-mesenchymal transdifferentiation (EnMT) may be a novel source of SMC. Here we describe the differentiation of smooth muscle-like cells through EnMT. Human umbilical cord endothelial cells (HUVEC) were cultured either under conditions favoring endothelial cell growth or under conditions favoring mesenchymal differentiation (TGF-beta and PDGF-BB). Expression of smooth muscle protein 22alpha and alpha-smooth muscle actin was induced in HUVEC cultured in mesenchymal differentiation media, whereas hardly any expression of these markers was found on genuine HUVEC. Transdifferentiated endothelial cells lost the ability to prevent thrombin formation in an in vitro coagulation assay, had increased migratory capacity towards PDGF-BB and gained contractile behavior similar to genuine vascular smooth muscle cells. Furthermore, we showed that EnMT could be induced in three-dimensional (3D) collagen sponges. In conclusion, we show that HUVEC can efficiently transdifferentiate into smooth muscle-like cells through endothelial-to-mesenchymal transdifferentiation. Therefore, EnMT might be used in future progenitor cell-based vascular tissue engineering approaches to obtain vascular smooth muscle cells, and circumvent a number of limitations encountered in current vascular tissue engineering strategies.     

Immunohistochemical localisation of tissue plasminogen activator and urokinase in the vessel wall.

Immunoreactive plasminogen activators were studied in tissue sections using a peroxidase method and monospecific antibodies to tissue plasminogen activator produced by a melanoma. Tissue plasminogen activator reactivity was found in skin melanomas and in endothelial and smooth muscle cells of arteries and veins. Vessels of the umbilical cord showed higher reactivity than peripheral vessels. Only faint antiurokinase reactivity was found. By means of the fibrin slide technique, fibrinolytic activity could be shown in peripheral vessel walls but not in the umbilical cord, which suggests that immunoreactivity of tissue plasminogen activator bound to an inhibitor can also be demonstrated. This method may be a useful tool in further studies of tissue plasminogen activator in physiological as well as pathological processes.     

T-cell intravascular lymphomatosis (angiotropic large cell lymphoma): association with Epstein–Barr viral infection

Amniotic fluid bacterial infection is an occasional cause of second trimester septic abortion. We describe an autolysis-related histological artifact, umbilical cord ‘pseudo-vasculitis’, which can erroneously implicate amniotic bacterial infection in fetal death. Clinicopathological features of 13 second trimester fetal deaths with umbilical cord pseudo-vasculitis are reported. In four cases (31%), an incorrect pathological diagnosis of umbilical vasculitis had initially been rendered. Umbilical cords from five cases of pseudo-vasculitis and one comparison fetus (18-week septic abortion with true umbilical vasculitis), were studied with chloroacetate esterase and with immunohistochemical staining for myeloperoxidase, muscle-specific actin (HHF35) and smooth muscle actin. Histologically, umbilical pseudo-vasculitis exhibited numerous small, rounded, degenerating cells with irregular, multilobed nuclei (closely resembling neutrophils) located within the umbilical vessel wall. Immunohistochemical studies demonstrated that all cells resembling neutrophils were of smooth muscle origin. Moderate to severe fetal autolysis was present in all cases of umbilical pseudo-vasculitis, suggesting that this finding represents autolysis of umbilical vascular smooth muscle secondary to post mortem fetal retention. Vascular smooth muscle in other fetal and placental locations did not demonstrate the finding, suggesting that this striking degenerative artifact of smooth muscle is restricted to the umbilical cord.     

In vitro analysis of extracellular matrix production by porcine glomerular mesangial and vascular smooth muscle cells.

Proliferation potential and extracellular matrix production were compared in cultured porcine glomerular mesangial cells and arterial and venous smooth muscle cells. Mesangial and arterial smooth muscle cells proliferated more rapidly than venous smooth muscle cells. In immunofluorescence studies, mesangial and arterial smooth muscle cells stained strongly for collagen types I, III, and V; venous smooth muscles showed weaker staining for collagens III and V. Total collagen synthesis in cultured mesangial and arterial smooth muscle cells was lower than in venous smooth muscle cells. Electrophoretic analysis showed type I collagen predominated in all cell types, although levels were highest in mesangial and arterial smooth muscle cells. Collagen V (alpha 3) occurred only in venous smooth muscle cells. Mesangial and arterial smooth muscle cells showed cellbound fibronectin and laminin, which also were secreted into the medium. Venous smooth muscle cells secreted fibronectin, but all laminin was cell bound. The findings suggest a strong similarity between mesangial and arterial smooth muscle cells.     

Hydrostatic pressure independently increases elastin and collagen co-expression in small-diameter engineered arterial constructs

Prior studies have demonstrated that smooth muscle cell (SMC) proliferation, migration, and extracellular matrix production increase with hydrostatic pressure in vitro. We have engineered highly compliant small-diameter arterial constructs by culturing primary adult baboon arterial SMCs under pulsatile perfusion on tubular, porous, elastomeric scaffolds composed of poly(glycerol sebacate) (PGS). This study investigates the effect of hydrostatic pressure on the biological and mechanical properties of PGS-based engineered arterial constructs. Pressure was raised using a downstream needle valve during perfusion while preserving flow rate and pulsatility, and constructs were evaluated by pressure-diameter testing and biochemical assays for collagen and elastin. Pressurized constructs contained half as much insoluble elastin as baboon common carotid arteries but were significantly less compliant, while constructs cultured at low hydrostatic pressure contained one-third as much insoluble elastin as baboon carotids and were similar in compliance. Hydrostatic pressure significantly increased construct burst pressure, collagen and insoluble elastin content, and soluble elastin concentration in culture medium. All arteries and constructs exhibited elastic recovery during pressure cycling. Hydrostatic pressure did not appear to affect radial distribution of SMCs, collagens I and III, and elastin. These results provide insights into the control of engineered smooth muscle tissue properties using hydrostatic pressure.