Human amniotic membrane for guided bone regeneration of calvarial defects in mice.

Due to its biological properties, human amniotic membrane (hAM) is widely studied in the field of tissue engineering and regenerative medicine. hAM is already very attractive for wound healing and it may be helpful as a support for bone regeneration. However, few studies assessed its potential for guided bone regeneration (GBR). The purpose of the present study was to assess the potential of the hAM as a membrane for GBR. In vitro, cell viability in fresh and cryopreserved hAM was assessed. In vivo, we evaluated the impact of fresh versus cryopreserved hAM, using both the epithelial or the mesenchymal layer facing the defect, on bone regeneration in a critical calvarial bone defect in mice. Then, the efficacy of cryopreserved hAM associated with a bone substitute was compared to a collagen membrane currently used for GBR. In vitro, no statistical difference was observed between the conditions concerning cell viability. Without graft material, cryopreserved hAM induced more bone formation when the mesenchymal layer covered the defect compared to the defect left empty. When associated with a bone substitute, such improved bone repair was not observed. These preliminary results suggest that cryopreserved hAM has a limited potential for GBR.

Layer-by-layer bioassembly of cellularized polylactic acid porous membranes for bone tissue engineering.

The conventional tissue engineering is based on seeding of macroporous scaffold on its surface ("top-down" approach). The main limitation is poor cell viability in the middle of the scaffold due to poor diffusion of oxygen and nutrients and insufficient vascularization. Layer-by-Layer (LBL) bioassembly is based on "bottom-up" approach, which considers assembly of small cellularized blocks. The aim of this work was to evaluate proliferation and differentiation of human bone marrow stromal cells (HBMSCs) and endothelial progenitor cells (EPCs) in two and three dimensions (2D, 3D) using a LBL assembly of polylactic acid (PLA) scaffolds fabricated by 3D printing. 2D experiments have shown maintain of cell viability on PLA, especially when a co-cuture system was used, as well as adequate morphology of seeded cells. Early osteoblastic and endothelial differentiations were observed and cell proliferation was increased after 7 days of culture. In 3D, cell migration was observed between layers of LBL constructs, as well as an osteoblastic differentiation. These results indicate that LBL assembly of PLA layers could be suitable for BTE, in order to promote homogenous cell distribution inside the scaffold and gene expression specific to the cells implanted in the case of co-culture system.

Altered nanofeature size dictates stem cell differentiation.

The differentiation of stem cells can be modulated by physical factors such as the micro- and nano-topography of the extracellular matrix. One important goal in stem cell research is to understand the concept that directs differentiation into a specific cell lineage in the nanoscale environment. Here, we demonstrate that such paths exist by controlling only the micro- and nano-topography of polymer surfaces. Altering the depth (on a nanometric scale) of micro-patterned surface structures allowed increased adhesion of human mesenchymal stem cells (hMSCs) with specific differentiation into osteoblasts, in the absence of osteogenic medium. Small (10 nm) depth patterns promoted cell adhesion without noticeable differentiation, whereas larger depth patterns (100 nm) elicited a collective cell organization, which induced selective differentiation into osteoblast-like cells. This latter response was dictated by stress through focal-adhesion-induced reorganization of F-actin filaments. The results have significant implications for understanding the architectural effects of the in vivo microenvironment and also for the therapeutic use of stem cells.

uPA and MMP-2 were involved in self-assembled network formation in a two dimensional co-culture model of bone marrow stromal cells and endothelial cells.

Two dimensional (2D) co-cultures of human bone marrow stromal cells (HBMSCs) and human umbilical vein endothelial cells (HUVECs) stimulate osteoblastic differentiation of HBMSCs, induce the formation of self-assembled network and cell interactions between the two cell types involving many vascular molecules. Because of their strong activities on angiogenesis and tissue remodeling, urokinase plasminogen activator (uPA), plasminogen activator inhibitor-1 (PAI-1), matrix metalloproteinase-2 (MMP-2) as well tissue inhibitors of matrix metalloproteinase-2 (TIMP-2) were investigated in this 2D co-culture model. We found that the expression of uPA, MMP-2 in the co-cultured cells was significantly higher than those in mono-cultured cells. In opposite, PAI-1, expressed only by HUVECs is not regulated in the co-culture. Inhibition assays confirm that uPA played a critical role in the formation of self-assembled network as neutralization of uPA disturbed this network. In the same context, inhibition of MMP-2 prevented the formation of self-assembled network, while the inhibition of uPA abolished the over expression and the activity of MMP-2. This upregulation could initiate the uPA expression and proteolysis processes through the MMP-2 activity, and may contribute to endothelial cell migration and the formation of this self-assembled network observed in these 2D co-cultured cells.

Composition and properties of silver-containing calcium carbonate-calcium phosphate bone cement.

The introduction of silver, either in the liquid phase (as silver nitrate solution: Ag(L)) or in the solid phase (as silver phosphate salt: Ag(S)) of calcium carbonate-calcium phosphate (CaCO3-CaP) bone cement, its influence on the composition of the set cement (C-Ag(L) and C-Ag(S) cements with a Ca/Ag atomic ratio equal to 10.3) and its biological properties were investigated. The fine characterisation of the chemical setting of silver-doped and reference cements was performed using FTIR spectroscopy. We showed that the formation of apatite was enhanced from the first hours of maturation of C-Ag(L) cement in comparison with the reference cement, whereas a longer period of maturation (about 10 h) was required to observe this increase for C-Ag(S) cement, although in both cases, silver was present in the set cements mainly as silver phosphate. The role of silver nitrate on the setting chemical reaction is discussed and a chemical scheme is proposed. Antibacterial activity tests (S. aureus and S. epidermidis) and in vitro cytotoxicity tests (human bone marrow stromal cells (HBMSC)) showed that silver-loaded CaCO3-CaP cements had antibacterial properties (anti-adhesion and anti-biofilm formation) without a toxic effect on HBMSC cells, making C-Ag(S) cement a promising candidate for the prevention of bone implant-associated infections.

Analysis of collagen expression during chondrogenic induction of human bone marrow mesenchymal stem cells.

Adult mesenchymal stem cells (MSCs) are currently being investigated as an alternative to chondrocytes for repairing cartilage defects. As several collagen types participate in the formation of cartilage-specific extracellular matrix, we have investigated their gene expression levels during MSC chondrogenic induction. Bone marrow MSCs were cultured in pellet in the presence of BMP-2 and TGF-ß3 for 24 days. After addition of FGF-2, at the fourth passage during MSC expansion, there was an enhancing effect on specific cartilage gene expression when compared to that without FGF-2 at day 12 in pellet culture. A switch in expression from the pre-chondrogenic type IIA form to the cartilage-specific type IIB form of the collagen type II gene was observed at day 24. A short-term addition of FGF-2 followed by a treatment with BMP-2/TGF-ß3 appears sufficient to accelerate chondrogenesis with a particular effect on the main cartilage collagens.

Development and characterization of a PLGA-HA composite material to fabricate 3D-printed scaffolds for bone tissue engineering.

Additive manufacturing is a rising field in bone tissue engineering. Additive fabrication offers reproducibility, high precision and rapid manufacture of custom patient-specific scaffolds. The development of appropriate composite materials for biomedical applications is critical to reach clinical application of these novel biomaterials. In this work, medical grade poly(lactic-co-glycolic) acid (PLGA) was mixed with hydroxyapatite nanoparticles (nHA) to fabricate 3D porous scaffolds by Fused Deposition Modeling. We have first confirmed that the composite material could be printed in a reproductive manner. Physical characterization demonstrated a low degradation of the material during manufacturing steps and an expected loading and homogeneous distribution of nHA. In vitro biodegradation of the scaffolds showed modifications of morphological and physicochemical properties over time. The composite scaffolds were biocompatible and high cell viability was observed in vitro, as well as a maintain of cell proliferation. As expected, the addition of nHA displayed a positive impact on osteodifferentiation in vitro. Furthermore, a limited inflammatory reaction was observed after subcutaneous implantation of the materials in the rat. Overall, this study suggests that this composite material is suitable for bone tissue engineering applications.

Role of neural-cadherin in early osteoblastic differentiation of human bone marrow stromal cells cocultured with human umbilical vein endothelial cells.

In our previous studies, roles of gap junction and vascular endothelial growth factor in the cross-talking of human bone marrow stromal cells (HBMSCs) and human umbilical vein endothelial cells (HUVECs) have been extensively studied. The present study focused on the investigation of the roles of neural (N)-cadherin in early differentiation of HBMSCs in direct-contact cocultures with HUVECs for 24 and 48 h. Quantitative real-time polymerase chain reaction, immunofluorescence, Western blot, as well as functional studies were applied to perform the studies at both protein and gene levels. Results showed that cocultured cells expressed much higher N-cadherin than monocultured cells after 24 and 48 h of culture. We observed that N-cadherin concentrated in the membrane of cocultured HBMSCs (co-HBMSCs) while distributed within the cytoplasm of monocultured HBMSCs, which indicated that the cell-cell adhesion was improved between cocultured cells. In addition, more beta-catenin was found to translocate into the cocultured cells nuclei and more T cell factor-1 (TCF-1) were detected in cocultured cells than in the monocultured cells. Moreover, mRNA levels of early osteoblastic markers including alkaline phosphatase (ALP) and type I collagen (Col-I) of co-HBMSCs were significantly upregulated, whereas neutralization of N-cadherin led to a downregulation of ALP and Col-I in both of the HBMSCs and co-HBMSCs compared with untreated cells. Taking our findings together it can be concluded that cocultures of HBMSCs with HUVECs increased N-cadherin expression and improved cell-cell adhesion. Whether this applies only to osteoprogenitor cells or to all the cell types in the culture will need to be determined by further studies. Subsequently, signaling transduction might be induced with the participation of beta-catenin and TCF-1. With the N-cadherin-mediated cell-cell adhesion and signaling transductions, the early osteoblastic differentiation of co-HBMSCs was significantly upregulated.

In vitro comparison of three percutaneous atrial septal defect closure devices for endothelialisation and haemocompatibility.

BACKGROUND: Percutaneous device closure of atrial septal defect (ASD) is the gold-standard treatment, but several delayed complications may occur as a result of incomplete device endothelialisation. AIMS: In this in vitro study, we compared three ASD closure devices [Nit-Occlud® ASD-R (device 1); Hyperion™ ASDO (device 2); and Amplatzer™ Septal Occluder (device 3)] in terms of the endothelialisation process, using human endothelial progenitors cells (EPCs), and haemocompatibility. METHODS: EPCs from umbilical cord blood were extracted, cultured and characterised. Device samples were seeded with 100,000 cells/cm2. EPC adhesion was investigated at 3 and 24hours, and EPC proliferation was monitored, which allowed longitudinal follow-up (days 1-12). Haemocompatibility of device samples was assessed using a complement C3a assay and platelet and coagulation activation. RESULTS: With regard to EPC adhesion and proliferation, no statistically significant differences were found between the three devices. We observed for each device a significant time-dependent EPC proliferation, appearing at day 8 for devices 2 and 3 and day 10 for device 1. No complement or platelet activation occurred within 15minutes of contact with devices. However, there was minimal activation of coagulation for the three devices. CONCLUSIONS: In this in vitro study we showed that, despite the three ASD occluders having different device designs and coatings, adhesion and proliferation of human endothelial cells was similar for all devices. This should be further confirmed by similar studies including shear stress forces and anti-thrombotic treatments.

Role of vascular endothelial growth factor in the communication between human osteoprogenitors and endothelial cells.

Proper bone remodeling requires an active process of angiogenesis which in turn supplies the necessary growth factors and stem cells. This tissue cooperation suggests a cross-talk between osteoblasts and endothelial cells. This work aims to identify the role of paracrine communication through vascular endothelial growth factor (VEGF) in co-culture between osteoblastic and endothelial cells. Through a well defined direct contact co-culture model between human osteoprogenitors (HOPs) and human umbilical vein endothelial cells (HUVECs), we observed that HUVECs were able to migrate along HOPs, inducing the formation of specific tubular-like structures. VEGF(165) gene expression was detected in the HOPs, was up-regulated in the co-cultured HOPs and both Flt-1 and KDR gene expression increased in co-cultured HUVECs. However, the cell rearrangement observed in co-culture was promoted by a combination of soluble chemoattractive factors and not by VEGF(165) alone. Despite having no observable effect on endothelial cell tubular-like formation, VEGF appeared to have a crucial role in osteoblastic differentiation since the inhibition of its receptors reduced the co-culture-stimulated osteoblastic phenotype. This co-culture system appears to enhance both primary angiogenesis events and osteoblastic differentiation, thus allowing for the development of new strategies in vascularized bone tissue engineering.

A Unique Triculture Model to Study Osteoblasts, Osteoclasts, and Endothelial Cells.

IMPACT STATEMENT: In this article, we first developed a new medium to culture together primary human osteoblastic, osteoclastic, and endothelial cells (ECs) chosen to represent the three major bone cell tissues. Indeed, no study has been conducted on primary human cells and on the phenotype/activity retention of these three primary human cell types. Thus, we established an original triculture model with osteoblastic, osteoclastic, and ECs, where not only both cell phenotype and cell activity were maintained but also cell culture homeostasis. These promising results will permit further investigations to create in vitro conditions to mimic the bone microenvironment and analyze cell interactions in ex vivo studies.

Layer-by-layer bioassembly of poly(lactic) acid membranes loaded with coculture of HBMSCs and EPCs improves vascularization in vivo.

Layer-by-layer (LBL) BioAssembly method was developed to enhance the control of cell distribution within 3D scaffolds for tissue engineering applications. The objective of this study was to evaluate in vivo the development of blood vessels within LBL bioassembled membranes seeded with human primary cells, and to compare it to cellularized massive scaffolds. Poly(lactic) acid (PLA) membranes fabricated by fused deposition modeling were seeded with monocultures of human bone marrow stromal cells or with cocultures of these cells and endothelial progenitor cells. Then, four cellularized membranes were assembled in LBL constructs. Early osteoblastic and endothelial cell differentiation markers, alkaline phosphatase, and von Willebrand's factor, were expressed in all layers of assemblies in homogenous manner. The same kind of LBL assemblies as well as cellularized massive scaffolds was implanted subcutaneously in mice. Human cells were observed in all scaffolds seeded with cells, but not in the inner parts of massive scaffolds. There were significantly more blood vessels observed in LBL bioassemblies seeded with cocultures compared to all other samples. LBL bioassembly of PLA membranes seeded with a coculture of human cells is an efficient method to obtain homogenous cell distribution and blood vessel formation within the entire volume of a 3D composite scaffold.

Unusual transduction response of progenitor-derived and mature endothelial cells exposed to laminar pulsatile shear stress.

Progenitor-derived endothelial cells (PDECs) isolated from human umbilical cord blood generate a great hope in the fields of vascular tissue engineering. Endothelial cells subjected to shear stress convert mechanical stimuli into intracellular signals that affect cellular functions. It is essential to ensure that PDECs are able to sense shear stress as mature endothelial cells from human saphenous veins (HSVECs) do with mitogen-activated protein (MAP) kinase and nuclear factor (NF)-kappaB signal transduction pathways. HSVECs and PDECs were seeded on glass slides coated with gelatin and exposed to 12 dyn/cm2 in a parallel-plate flow chamber. In both cell types, shear stress activated extracellular signal-related kinase (ERK)1/2 with a rapid time course (maximum 5 min) followed by a reduced phosphorylation, and p38 pathway. c-Jun N-terminal protein kinase (JNK) phosphorylation is observed only in PDECs. With respect to NF-kappaB translocation to the nucleus, the NF-kappaB pathway is not activated by flow in HSVECs and PDECs although interleukin-1alpha (IL-1alpha) activates this pathway in both cell types. In our experimental conditions, shear stress does not modify the nuclear translocation of NF-kappaB in HSVECs after IL-1alpha stimulation. It can be stated that PDECs are shear stress sensitive and capable of signal transduction as mature HSVECs are, despite the unusual transduction response of both cell types.

Influence of External Beam Radiotherapy on the Properties of Polymethyl Methacrylate-Versus Silicone-Induced Membranes in a Bilateral Segmental Bone Defect in Rats.

INTRODUCTION: Standard care for malignant tumors arising next to a bone structure is surgical removal with safety margins, followed by external beam radiotherapy (EBRT). Complete tumor removal can result in large bone defects. A two-step bone reconstruction technique using the induced membrane (IM) technique has proven its efficacy to bridge gap nonunion. During the first step, a spacer is placed in the bone gap. The spacer then is removed and the IM around it is filled with autologous cancellous bone graft. However, the feasibility of this technique with the addition of adjuvant EBRT between the two reconstruction steps has not yet been studied. Polymethyl methacrylate (PMMA) used to be the standard spacer material for the first step. Silicone spacers could replace them owing to their good behavior when submitted to EBRT and their easier removal from the surgical site during the second step. The aim of this study was to evaluate the influence of EBRT on the histological and biochemical properties of IM induced using PMMA or silicone as spacer. MATERIALS AND METHODS: The analyses were performed on PMMA- or silicone-IM with and without EBRT in a 6-mm bilateral femoral defect in 32 rats. Thickness and vessel content were measured in both groups. Bone morphogenetic protein 2 (BMP2) and vascular endothelial growth factor (VEGF) content in lysates of the crushed membranes were measured by enzyme immunoassay. Finally, alkaline phosphatase activity was analyzed in human bone marrow stromal cell cultures in contact with the same lysates. RESULTS: EBRT did not change the histological structure of the cellular internal layer or the fibrous outer layer. The nature of the spacer only influenced IM thickness, PMMA-IM with external radiotherapy being significantly thicker. EBRT decreased the vascular density of IM but was less effective on VEGF/BMP2 production. In vitro, IM could have an osteoinductive potential on human bone marrow stem cells. CONCLUSION: EBRT did not modify the histological properties of IMs but decreased their vascular density. VEGF and BMP2 production within IMs was not affected by EBRT. Silicone spacers are able to induce membranes with similar histological characteristics to PMMA-IM.

Influence of the three-dimensional culture of human bone marrow mesenchymal stromal cells within a macroporous polysaccharides scaffold on Pannexin 1 and Pannexin 3.

Because cell interactions play a fundamental role for cell differentiation, we investigated the expression of Pannexin 1 and Pannexin 3 in human bone marrow mesenchymal stromal cells (HBMSCs) in a three-dimensional (3D) microenvironment provided by a polysaccharide-based macroporous scaffold. The pannexin (Panx) family consists of three members, Panx1, Panx2, and Panx3. The roles of Panx large-pore ion and metabolite channels are recognized in many physiological and pathophysiological scenarios, but the role of these proteins in human physiological processes is still under investigation. Our study demonstrates that HBMSCs cultured within 3D scaffolds have induced Panx1 and Panx3 expression, compared with two-dimensional culture and that the Panx3 gene expression profile correlates with those of bone markers on mesenchymal stromal cells culture into the 3D scaffold. We showed that Panx1 is involved in the HBMSCs 3D cell-cell organization, as acting on the size of cellular aggregates, demonstrated by the use of Probenecid and the mimetic peptide 10panx1 as specific inhibitors. Inhibition of Panx3 using siRNA strategy shows to reduce the expression of osteocalcin as osteoblast-specific marker by HBMSCs cultured in 3D conditions, suggesting a role of this Panx in osteogenesis. Moreover, we evaluated Panx1 and Panx3 expression within the cellularized scaffolds upon subcutaneous implantation in NOG (NOD/Shi-scid/IL-2Rγnull ) mice, where we could observe a more intense expression in the constructs than in the surrounding tissues in vivo. This study provides new insights on the expression of pannexins in HBMSCs on a 3D microenvironment during the osteogenic differentiation, in vitro and in vivo.

Colorectal wall regeneration resulting from the association of chitosan hydrogel and stromal vascular fraction from adipose tissue.

Chitosan hydrogel and adipose derived stem cells (ADCS) have been reported as the optimal partnership for colorectal tissue engineering. In that field, the aim of the current experiment was to assess the interest of seeding ADSC on chitosan hydrogel patches in an in vivo comparative study and on a tube intended replace a colonic segment in an in vivo feasibility study. In the comparative study, a 2 × 3 cm colonic wall defect was performed in 20 swine and repaired by suturing a chitosan hydrogel patch: acellular matrix (group A, n = 10) versus matrix seeded with autologous stromal vascular fraction (SVF) (group B, n = 10). In the feasibility study, a circular colonic section was performed and a 2-cm-length chitosan hydrogel tube (seeded with autologous SVF) was implanted between the two edges of the colon in 3 pigs. Graft areas were explanted at 8 weeks for examinations. Endpoints were technical feasibility, fibrosis ratio, and smooth muscle layer regeneration. A complete coverage of the patched area was observed with an ad integrum regeneration of the colonic wall including smooth muscle cells layer around a thin fibrosis scare. Fibrosis ratio was significantly lower group B: 13% versus 55% (p = 0.013). Segmental colonic replacement appeared accurate. Our data confirmed in a large animal model the healing effect of chitosan on colorectal tissue. The very low rate of the fibrosis ratio in the cellularized group emphasizes inflammatory control effect of the chitosan hydrogel and SVF association. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 106A: 460-467, 2018.

Characterization of printed PLA scaffolds for bone tissue engineering.

Autografts remain the gold standard for orthopedic transplantations. However, to overcome its limitations, bone tissue engineering proposes new strategies. This includes the development of new biomaterials such as synthetic polymers, to serve as scaffold for tissue production. The objective of this present study was to produce poly(lactic) acid (PLA) scaffolds of different pore size using fused deposition modeling (FDM) technique and to evaluate their physicochemical and biological properties. Structural, chemical, mechanical, and biological characterizations were performed. We successfully fabricated scaffolds of three different pore sizes. However, the pore dimensions were slightly smaller than expected. We found that the 3D printing process induced decreases in both, PLA molecular weight and degradation temperatures, but did not change the semicrystalline structure of the polymer. We did not observe any effect of pore size on the mechanical properties of produced scaffolds. After the sterilization by γ irradiation, scaffolds did not exhibit any cytotoxicity towards human bone marrow stromal cells (HBMSC). Finally, after three and seven days of culture, HBMSC showed high viability and homogenous distribution irrespective of pore size. Thus, these results suggest that FDM technology is a fast and reproducible technique that can be used to fabricate tridimensional custom-made scaffolds for tissue engineering. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 106A: 887-894, 2018.

Decellularized and matured esophageal scaffold for circumferential esophagus replacement: Proof of concept in a pig model.

Surgical resection of the esophagus requires sacrificing a long portion of it. Its replacement by the demanding gastric pull-up or colonic interposition techniques may be avoided by using short biologic scaffolds composed of decellularized matrix (DM). The aim of this study was to prepare, characterize, and assess the in vivo remodeling of DM and its clinical impact in a preclinical model. A dynamic chemical and enzymatic decellularization protocol of porcine esophagus was set up and optimized. The resulting DM was mechanically and biologically characterized by DNA quantification, histology, and histomorphometry techniques. Then, in vitro and in vivo tests were performed, such as DM recellularization with human or porcine adipose-derived stem cells, or porcine stromal vascular fraction, and maturation in rat omentum. Finally, the DM, matured or not, was implanted as a 5-cm-long esophagus substitute in an esophagectomized pig model. The developed protocol for esophageal DM fulfilled previously established criteria of decellularization and resulted in a scaffold that maintained important biologic components and an ultrastructure consistent with a basement membrane complex. In vivo implantation was compatible with life without major clinical complications. The DM's scaffold in vitro characteristics and in vivo implantation showed a pattern of constructive remodeling mimicking major native esophageal characteristics.

Gene response in endothelial cells cultured on engineered surfaces is regulated by shear stress.

In vitro endothelialization of small-diameter synthetic vascular prostheses confluently lined with cultured autologous endothelial cells (ECs) before implantation has been shown to increase their patency. Many authors have studied the effects of shear stress on EC gene response seeded on various substrates showing different gene expression profiles according to cell type, flow times, or shear type with different molecular biology techniques, but few studies have reported any EC gene response to shear stress when cells are seeded on vascular grafts. The purpose of this in vitro study was to investigate whether ECs were able to transduce shear stress at the level of the nucleus. Human saphenous vein ECs were seeded on glass slides coated with gelatin or fibrin glue or on 6-mm fibrin-glue-coated grafts. Then cells were exposed to 12 dyn/cm(2) for 4 h and ribonucleic acid (RNA) were extracted. The relative messenger RNA (mRNA) expression was studied using real-time quantitative polymerase chain reaction for the following mRNAs: von Willebrand Factor, tissue-plasminogen activator, CD31, vascular endothelial (VE)-cadherin, beta(1) integrin, and vascular endothelial growth factor receptor type 2. From parallel flow chambers, results have shown similar EC gene response on gelatin and fibrin glue under laminar shear stress with downregulation of prothrombotic genes, as well as upregulation of nonthrombotic genes and upregulation of adhesion molecules such as VE-cadherin, but some discrepancies are noted, with a downregulation of CD31 and kinase insert domain receptor (KDR) for the former, without significant variation for the latter. In comparison, results show upregulation of tissue type plasminogen activator gene and downregulation of KDR, VE-cadherin, and beta(1) integrin genes in ECs lining grafts. To conclude, the major finding of our study is to show that human saphenous vein ECs seeded on fibrin glue (in planar flow chambers or in tubular grafts) can be regulated using shear stress via gene expression changes in a nonthrombotic way.

Signal transduction and procoagulant state of human cord blood--progenitor-derived endothelial cells after interleukin-1alpha stimulation.

Isolation of endothelial progenitors from human umbilical cord blood generated great hope in vascular tissue engineering. However, before clinical use, progenitor derived endothelial cells (PDECs) have to be compared with mature endothelial cells (ECs). The aim of this study was to explore the behavior of PDECs exposed to a proinflammatory cytokine (interleukin-1alpha; IL-1alpha) according to the mitogen-activated protein (MAP) kinase and nuclear factor (NF)-kappaB signal transduction pathways as well as procoagulant activity (PCA). CD34(+) mononuclear cells were isolated using magnetic beads, cultured, and compared with human saphenous vein ECs (HSVECs). PDECs express endothelial markers: CD31, VE-cadherin, von Willebrand factor, KDR, and incorporate acetylated low-density lipoprotein (Dil-Ac-LDL). IL-1alpha similarly activates c-Jun N-terminal protein kinase (JNK) and p38 pathways in HSVECs and PDECs, whereas extracellular signal-related kinase (ERK)1/2 phosphorylation is lower in PDECs than in HSVECs. Low ERK1/2 phosphorylation in PDECs was specific to IL-1alpha as vascular endothelial growth factor (VEGF) similarly stimulated ERK1/2 pathway. With respect to inhibitor of NF-kappa B (Ikappa B) degradation, NF-kappa B translocation and phosphorylation, the NF-kappa B pathway is comparable in HSVECs and PDECs after stimulation. PCA and tissue factor level induced by IL-1alpha are lower in PDECs than in HSVECs. Thus, our data show that PDECs display the characteristics of functional mature ECs under IL-1alpha stimulation. However, we observed significant differences between PDECs and HSVECs related to both ERK1/2 pathway activation and tissue factor production.