Construction Of Blood Vessels Research Articles

Targeting KDM1A in Neuroblastoma with NCL-1 Induces a Less Aggressive Phenotype and Suppresses Angiogenesis.

Background: The KDM1A histone demethylase regulates the cellular balance between proliferation and differentiation, and is often deregulated in human cancers including the childhood tumor neuroblastoma. We previously showed that KDM1A is strongly expressed in undifferentiated neuroblastomas and correlates with poor patient prognosis, suggesting a possible clinical benefit from targeting KDM1A. Methods: Here, we tested the efficacy of NCL-1, a small molecule specifically inhibiting KDM1A in preclinical models for neuroblastoma. Results: NCL-1 mimicked the effects of siRNA-mediated KDM1A knockdown and effectively inhibited KDM1A activity in four neuroblastoma cell lines and a patient-representative cell model. KDM1A inhibition shifted the aggressive tumor cell phenotypes towards less aggressive phenotypes. The proliferation and cell viability was reduced, accompanied by the induction of markers of neuronal differentiation. Interventional NCL-1 treatment of nude mice harboring established neuroblastoma xenograft tumors reduced tumor growth and inhibited cell proliferation. Reduced vessel density and defects in blood vessel construction also resulted, and NCL-1 inhibited the growth and tube formation of HUVEC-C cells in vitro. Conclusions: Inhibiting KDM1A could attack aggressive neuroblastomas two-fold, by re-directing tumor cells toward a less aggressive, slower-growing phenotype and by preventing or reducing the vascular support of large tumors.

Significantly Positive Impact of Nonsteroidal Anti-inflammatory Drugs Combined With Osmoprotectant (Osmolytes) in Cancer Treatment.

Osmoprotectant osmolyteand nonsteroidal anti-inflammatory drug (NSAID) coadministration can work synergistically in cancer chemotherapy since most tumors are inflammatory and cancer cells experience osmotic stress. NSAIDs have been shown to inhibit cyclooxygenase (COX) enzymes, which in turn reduces prostaglandin synthesis and prevents inflammation. They also encourage cell death to prevent tumor growth and its spread to other tissues and prevent the construction of new blood vessels, which contributes to the growth of cancer. Taurine belongs to the class of osmolytes since it has been shown to stabilize macromolecular structures and maintain cellular osmotic balance when combined with betaine and glycine. When these drugs are taken together, as opposed to separately, the effectiveness of cancer treatment is increased by increasing cancer cell death and suppressing tumor growth. Notable therapeutic benefits include the reduction of local inflammatory milieu by NSAIDs, which promotes tumor development, and the protection of surviving, normal cells and tissues from treatment-induced damage caused by cancer. By enhancing this synergy, side-effect risk can be decreased and treatment outcomes improved in terms of quality. Put another way, peptides can increase the therapeutic index of NSAIDs in cancer patients by preventing cell damage, which may lessen the gastrointestinal (GI), cardiovascular (CV), and renal side effects of the drug. However, there are drawbacks because using NSAIDs for an extended period of time is linked to serious side effects that call for strict supervision. More research is required because the usefulness and significance of osmolytes in cancer therapy are still very unclear, if not fragmented. In addition, people who live in places with limited resources may find it difficult to afford the possible expenditures associated with osmolytes and selective cyclooxygenase-2 (COX-2) inhibitors. Only the molecular mechanisms of the two drugs' interactions, the appropriate dosages for combination therapy, and clinical trials to validate the efficacy and safety of this dosage should be the focus of future research. The request is inviting because it presents hope for an extremely successful antiviral strategy; nevertheless, in order to implement this approach successfully, it is likely to be necessary to create affordable formulations and scalable solutions that do not necessitate excessive treatment regimen individualization. Due to their complementary capacities to demonstrate anti-inflammatory and cytoprotective effects, Akta and5-aminosalicylic acid (5-ASA) administration may thus represent a significant advancement in the treatment of cancer.

Review on the Method of Generating Vascular Cells from iPSCs and Its Application

Induced Pluripotent Stem Cells (iPSCs) embryonic-like stem cells are reprogrammed from skin or adult somatic cells. iPSC-derived cardiovascular cells have been widely used in many cardiovascular disease models and drug discovery. This paper reviews and summarizes the method of generating three different types of iPSC-derived cardiovascular cells, including iPSC-derived Endothelial Cells (ECs), Cardiomyocytes (CMs), and Vascular Smooth Muscle Cells (vSMCs), and the application of these iPSC-derived cardiovascular cells. The diseases discussed in this paper are pulmonary arterial hypertension (PAH) and critical limb ischemia (CLI). Additionally, construction of tissue-engineered blood vessels (TEBVs) for vascular grafting has also been discussed. To conclude, iPSCs, as a promising technique, is used in disease modeling, drug screening, tissue engineering, and the discovery of specific cardiovascular therapies for various patients.

Electrical Stimulation Promotes the Vascularization and Functionalization of an Engineered Biomimetic Human Cardiac Tissue.

The formation of multiscale vascular networks is essential for the in vitro construction of large-scale biomimetic cardiac tissues/organs. Although a variety of bioprinting processes have been developed to achieve the construction of mesoscale and large-scale blood vessels, the formation of microvascular networks still mainly depends on the self-assembly behavior of endothelial cells (ECs), which is inefficient and demanding without appropriate stimulus. To address this problem, the elongation and connection of endothelial cells in engineered cardiac tissue (ECT) are sought to promote by electrical stimulation (ES) to achieve vascularization. As proof of the concept, bio-inks are composed of GelMA/fibrin hydrogel, human pluripotent stem cells induced cardiomyocytes (iPSC-CM), and human umbilical vein endothelial cells (HUVEC) are used for the bioprinting of ECTs. It is demonstrated that electrical stimulation significantly promotes the elongation, migration, and interconnection of HUVECs in ECT and increases the expression of related genes. Moreover, ES also enhances the secretion of signal factors interacting between CMs and HUVECs. It seems that the HUVECs further strengthen the contractility of cardiac tissue. Taken together, electrical stimulation promotes vascularization and CMs functionalization in ECT, which has important application potential in the fabrication of vascularized ECT and its clinical transplantation.

Bioprinting Technologies and Bioinks for Vascular Model Establishment.

Clinically, large diameter artery defects (diameter larger than 6 mm) can be substituted by unbiodegradable polymers, such as polytetrafluoroethylene. There are many problems in the construction of small diameter blood vessels (diameter between 1 and 3 mm) and microvessels (diameter less than 1 mm), especially in the establishment of complex vascular models with multi-scale branched networks. Throughout history, the vascularization strategies have been divided into three major groups, including self-generated capillaries from implantation, pre-constructed vascular channels, and three-dimensional (3D) printed cell-laden hydrogels. The first group is based on the spontaneous angiogenesis behaviour of cells in the host tissues, which also lays the foundation of capillary angiogenesis in tissue engineering scaffolds. The second group is to vascularize the polymeric vessels (or scaffolds) with endothelial cells. It is hoped that the pre-constructed vessels can be connected with the vascular networks of host tissues with rapid blood perfusion. With the development of bioprinting technologies, various fabrication methods have been achieved to build hierarchical vascular networks with high-precision 3D control. In this review, the latest advances in 3D bioprinting of vascularized tissues/organs are discussed, including new printing techniques and researches on bioinks for promoting angiogenesis, especially coaxial printing, freeform reversible embedded in suspended hydrogel printing, and acoustic assisted printing technologies, and freeform reversible embedded in suspended hydrogel (flash) technology.

3D bioprinting of multi-layered segments of a vessel-like structure with ECM and novel derived bioink.

3D-Bioprinting leads to the realization of tridimensional customized constructs to reproduce the biological structural complexity. The new technological challenge focuses on obtaining a 3D structure with several distinct layers to replicate the hierarchical organization of natural tissues. This work aims to reproduce large blood vessel substitutes compliant with the original tissue, combining the advantages of the 3D bioprinting, decellularization, and accounting for the presence of different cells. The decellularization process was performed on porcine aortas. Various decellularization protocols were tested and evaluated through DNA extraction, quantification, and amplification by PCR to define the adequate one. The decellularized extracellular matrix (dECM), lyophilized and solubilized, was combined with gelatin, alginate, and cells to obtain a novel bioink. Several solutions were tested, tuning the percentage of the components to obtain the adequate structural properties. The geometrical model of the large blood vessel constructs was designed with SolidWorks, and the construct slicing was done using the HeartWare software, which allowed generating the G-Code. The final constructs were 3D bioprinted with the Inkredible + using dual print heads. The composition of the bioink was tuned so that it could withstand the printing of a segment of a tubular construct up to 10 mm and reproduce the multicellular complexity. Among the several compositions tested, the suspension resulting from 8% w/v gelatin, 7% w/v alginate, and 3% w/v dECM, and cells successfully produced the designed structures. With this bioink, it was possible to print structures made up of 20 layers. The dimensions of the printed structures were consistent with the designed ones. We were able to avoid the double bioink overlap in the thickness, despite the increase in the number of layers during the printing process. The optimization of the parameters allowed the production of structures with a height of 20 layers corresponding to 9 mm. Theoretical and real structures were very close. The differences were 14% in height, 20% internal diameter, and 9% thickness. By tailoring the printing parameters and the amount of dECM, adequate mechanical properties could be met. In this study, we developed an innovative printable bioink able to finely reproduce the native complex structure of the large blood vessel.

Cell Adhesion Strength Indicates the Antithrombogenicity of Poly(2-methoxyethyl acrylate) (PMEA): Potential Candidate for Artificial Small-Diameter Blood Vessel

Poly(2-methoxyethyl acrylate) (PMEA) is a US FDA-approved biocompatible polymer, although there is insufficient work on human umbilical vein endothelial cells (HUVECs) and platelet interaction analysis on PMEA-analogous polymers. In this study, we extensively investigated HUVEC–polymer and platelet–polymer interaction behavior by measuring the adhesion strength using single-cell force spectroscopy. Furthermore, the hydration layer of the polymer interface was observed using frequency-modulation atomic force microscopy. We found that endothelial cells can attach and spread on the PMEA surface with strong adhesion strength compared to other analogous polymers. We found that the hydration layers on the PMEA-analogous polymers were closely related to their weak platelet adhesion behavior. Based on our results, it can be concluded that PMEA is a promising candidate for the construction of artificial small-diameter blood vessels owing to the presence of IW and a hydration layer on the interface.

Tissue Engineering Approaches to Uncover Therapeutic Targets for Endothelial Dysfunction in Pathological Microenvironments.

Endothelial cell dysfunction plays a central role in many pathologies, rendering it crucial to understand the underlying mechanism for potential therapeutics. Tissue engineering offers opportunities for in vitro studies of endothelial dysfunction in pathological mimicry environments. Here, we begin by analyzing hydrogel biomaterials as a platform for understanding the roles of the extracellular matrix and hypoxia in vascular formation. We next examine how three-dimensional bioprinting has been applied to recapitulate healthy and diseased tissue constructs in a highly controllable and patient-specific manner. Similarly, studies have utilized organs-on-a-chip technology to understand endothelial dysfunction’s contribution to pathologies in tissue-specific cellular components under well-controlled physicochemical cues. Finally, we consider studies using the in vitro construction of multicellular blood vessels, termed tissue-engineered blood vessels, and the spontaneous assembly of microvascular networks in organoids to delineate pathological endothelial dysfunction.

Poly(2-Methoxyethyl Acrylate) (PMEA)-Coated Anti-Platelet Adhesive Surfaces to Mimic Native Blood Vessels through HUVECs Attachment, Migration, and Monolayer Formation

Confluent monolayers of human umbilical vein endothelial cells (HUVECs) on a poly(2-methoxyethyl acrylate) (PMEA) antithrombogenic surface play a major role in mimicking the inner surface of native blood vessels. In this study, we extensively investigated the behavior of cell–polymer and cell–cell interactions by measuring adhesion strength using single-cell force spectroscopy. In addition, the attachment and migration of HUVECs on PMEA-analogous substrates were detected, and the migration rate was estimated. Moreover, the bilateral migration of HUVECs between two adjacent surfaces was observed. Furthermore, the outer surface of HUVEC was examined using frequency-modulation atomic force microscopy (FM-AFM). Hydration was found to be an indication of a healthy glycocalyx layer. The results were compared with the hydration states of individual PMEA-analogous polymers to understand the adhesion mechanism between the cells and substrates in the interface region. HUVECs could attach and spread on the PMEA surface with stronger adhesion strength than self-adhesion strength, and migration occurred over the surface of analogue polymers. We confirmed that platelets could not adhere to HUVEC monolayers cultured on the PMEA surface. FM-AFM images revealed a hydration layer on the HUVEC surfaces, indicating the presence of components of the glycocalyx layer in the presence of intermediate water. Our findings show that PMEA can mimic original blood vessels through an antithrombogenic HUVEC monolayer and is thus suitable for the construction of artificial small-diameter blood vessels.

Gelatin/sodium alginate hydrogel-coated decellularized porcine coronary artery to construct bilayer tissue engineered blood vessels

Cardiovascular diseases and vascular trauma can be commonly found in the population. Scholars worldwide hope to develop small-diameter vascular grafts that can replace autologous vessels for clinical use. Decellularized blood vessels can retain the original morphology, structure, and physical properties of blood vessels, which is conducive to cell growth, proliferation, and differentiation. In this study, porcine coronary arteries (PCAs) were decellularized to prepare decellularized porcine coronary artery (DPCA), and bilayer hybrid scaffolds were prepared by coating gelatin and sodium alginate mixed hydrogel of seven different proportions and combined with mouse fibroblasts (L929 cells) to study the construction of tissue engineering vessels in vitro. The obtained bilayer hybrid scaffolds were 3–7 cm in length, 5 mm in external diameter, and 1 mm in average wall thickness. All seven bilayer hybrid scaffolds showed good biocompatibility after cell inoculation. Compared with 2D culture, cells on 3D scaffolds grew relatively slowly in the first 4 days, and the number of cells proliferated rapidly at 7 days. In the same culture days, different concentrations of hydrogel also had an impact on cell proliferation. With the increase of hydrogel content, cells on the 3D scaffold formed cell colonies faster. The results showed that the scaffold had good biocompatibility and could meet the needs of artificial blood vessel construction.

Development of a 3D Printed Lung Model Made of Synthetic Materials for Simulation.

Considering the complexity of lung structures and the difficulty of thoracoscopic surgery, simulation-based training is of paramount importance for junior surgeons. Here, we aim to design a high-fidelity lung model through utilizing the three-dimensional (3D) printing technology combined with synthetic materials to mimic the real human lung. The 3D printed lung model was manufactured based on the computed tomography images of a randomly selected male patient. Synthetic materials were used for the construction of lung parenchyma, blood vessels, and bronchi. Then, the model was assessed in terms of its visual, tactile, and operational features by participants (the senior surgeons, junior surgeons, and medical students), who were asked to complete the specially designed survey-questionnaires. A 3D printed model of the right lung made of synthetic materials was successfully fabricated. Thirty subjects participated in our study (10 senior surgeons, 10 junior surgeons, and 10 medical students). The average visual evaluation scores for senior surgeons, junior surgeons, and medical students were 3.97 ± 0.61, 4.56 ± 0.58, 4.76 ± 0.49, respectively. The average tactile evaluation scores were 3.40 ± 0.50, 4.13 ± 0.68, 4.00 ± 0.64, respectively. The average operation evaluation scores were 3.33 ± 0.83, 3.93 ± 0.66, 4.03 ± 0.66, respectively. Significant lower scores were obtained in the group of the senior surgeons compared with the other two groups. A high level of fidelity was exhibited in our 3D printed lung model and it could be applied as a promising simulator for the surgical training in the future.

Hypoxia - as a Possible Regulator of the Activity of Epicardial Mesothelial Cells After Myocardial Infarction.

Aim To study the effect of hypoxia on the activity of epithelial-mesenchymal transition (EMT) in epicardial cells, which provides formation of a specialized microenvironment.Material and methods This study used a model of experimental myocardial infarction created by ligation of the anterior descendent coronary artery. The activity of epicardial cells after a hypoxic exposure was studied with the hypoxia marker, pimonidazole, bromodeoxyuridine, immunofluorescent staining of heart cryosections, and in vitro mesothelial cell culture.Results The undamaged heart maintained the quiescent condition of mesothelial cells and low levels of their proliferation, extracellular matrix protein production, and of the EMT activity. Acute ischemic injury induced moderate hypoxia in the epicardial/subepicardial region. This caused a global rearrangement of this region due to the initiation of EMT in cells, changes in the cell composition, and accumulation of extracellular matrix proteins. We found that the initiation of EMT in mesothelial cells may result in the formation of smooth muscle cell precursors, fibroblasts, and a population of Sca-1+ cardiac progenitor cells, which may both participate in construction of new blood vessels and serve as a mesenchymal link for the paracrine support of microenvironmental cells. In in vitro experiments, we showed that 72‑h hypoxia facilitated activation of EMT regulatory genes, induced dissembling of intercellular contacts, cell uncoupling, and increased cell plasticity.Conclusion The epicardium of an adult heart serves as a "reparative reserve" that can be reactivated by a hypoxic exposure. This creates a basis for an approach to influence the epicardium to modulate its activity for regulating reparative processes.

A case of simultaneous laparoscopic surgery for double cancer comprising multiple early gastric cancer and advanced sigmoid colon cancer after revascularization

BackgroundTraditionally, the surgery for simultaneous double cancer of the stomach and colon required a large incision to the upper and lower region of the abdomen. In this case, an artificial blood vessel was located under the skin after revascularization. Considering ischemia due to graft compression by incision retractor during laparotomy, this was difficult to do. This is a report on laparoscopic surgery for simultaneous double cancer of the stomach and colon after revascularization.Case presentationA 69-year-old man had early gastric cancer and advanced sigmoid colon cancer. He had suffered from thromboangitis obliterans and has undergone revascularization many times due to poor blood flow in his lower limbs. He had had some artificial blood vessels inserted under the skin, confirmed by blood vessel construction image by preoperative computed tomography (CT). There was a bypass vessel from the left axillary artery to the left femoral artery under the skin of the left thoracoabdominal. In addition, there were two bypass vessels from the left external iliac artery to the right femoral artery under the skin of the lower abdomen. One of the two bypasses was occluded. In the blood flow to the intestinal tract, the inferior mesenteric artery was already occluded. Peripheral blood flow in the common iliac artery depended on blood flow from the artificial blood vessel, and blood flow from the internal iliac artery to the rectum was poor. Laparoscopic Hartmann’s operation was performed for Stage II B (UICC 8th Edition) sigmoid colon cancer. Because the blood flow in the intestinal tract on the anal side was poor, we thought that anastomosis was at a high risk for leakage. Laparoscopic total gastrectomy was also performed simultaneously for two Stage I (UICC 8th edition) gastric cancers in the cardia and body. The location of the port site and stoma was carefully determined preoperatively to prevent damage and infection to the artificial blood vessels. Minimal invasive surgery was performed using laparoscopic surgery.ConclusionsLaparoscopic surgery with small incisions is useful for patients with double cancer who need an approach to the upper and lower abdomen. Furthermore, laparoscopic surgery has less interference on graft in patients with artificial blood vessels under the skin by intraperitoneal approach.

The Combination of Tissue-Engineered Blood Vessel Constructs and Parallel Flow Chamber Provides a Potential Alternative to In Vivo Drug Testing Models.

Cardiovascular disease is a major cause of death globally. This has led to significant efforts to develop new anti-thrombotic therapies or re-purpose existing drugs to treat cardiovascular diseases. Due to difficulties of obtaining healthy human blood vessel tissues to recreate in vivo conditions, pre-clinical testing of these drugs currently requires significant use of animal experimentation, however, the successful translation of drugs from animal tests to use in humans is poor. Developing humanised drug test models that better replicate the human vasculature will help to develop anti-thrombotic therapies more rapidly. Tissue-engineered human blood vessel (TEBV) models were fabricated with biomimetic matrix and cellular components. The pro- and anti-aggregatory properties of both intact and FeCl3-injured TEBVs were assessed under physiological flow conditions using a modified parallel-plate flow chamber. These were perfused with fluorescently labelled human platelets and endothelial progenitor cells (EPCs), and their responses were monitored in real-time using fluorescent imaging. An endothelium-free TEBV exhibited the capacity to trigger platelet activation and aggregation in a shear stress-dependent manner, similar to the responses observed in vivo. Ketamine is commonly used as an anaesthetic in current in vivo models, but this drug significantly inhibited platelet aggregation on the injured TEBV. Atorvastatin was also shown to enhance EPC attachment on the injured TEBV. The TEBV, when perfused with human blood or blood components under physiological conditions, provides a powerful alternative to current in vivo drug testing models to assess their effects on thrombus formation and EPC recruitment.

Poly(acrylic acid) as an adhesion promoter for UV-assisted thermoplastic bonding: Application for the in vitro construction of human blood vessels

In this study, we introduced a novel adhesion bonding method for fabricating thermoplastic microdevices using poly(acrylic acid) (PAA) as a UV-assisted adhesion promoter. The bonding mechanism was based on the covalent cross-links between poly(methyl methacrylate) (PMMA) and PAA via the free radicals in their carbon backbone generated under UV irradiation. The water contact angle and Fourier-transformed infrared (FTIR) analysis were performed to analyze the surface characteristics of the PAA-coated PMMA. PMMAs were bonded under UV treatment for 60 s with the highest bond strength of around 1.18 MPa. The PMMA microdevice was leak-proof for over 200 h. Besides, clog-free PMMA microdevices with various-sizes microchannels were performed to demonstrate such a high applicable bonding method for microdevice fabrication. Moreover, PMMAs were bonded with other thermoplastics with a bond strength of around 0.5 MPa. Notably, collagen was easily coated inside the PMMA microchannels via electrostatic interaction between PAA and collagen which is beneficial for on-device cell culture. As a result, a layered co-culture model of smooth muscle cells (SMCs) and human umbilical vein endothelial cells (HUVECs) was realized inside simple straight microchannels mimicking human blood vessel wall. Therefore, the introduced bonding method could pave the way for fabricating microdevice for cell-related applications.

Bioprinting small diameter blood vessel constructs with an endothelial and smooth muscle cell bilayer in a single step

Bioengineered artificial blood vessels have been a major area of interest over the last decade. Of particular interest are small diameter vessels, as surgical options are currently limited. This study aimed to fabricate a small diameter, heterogeneous bilayer blood vessel-like construct in a single step with gelatin methacryloyl (GelMA) bioink using a 3D micro-extrusion bioprinter on a solid platform. GelMA was supplemented with Hyaluronic acid (HA), glycerol and gelatin to form a GelMA bioink with good printability, mechanical strength, and biocompatibility. Two separate concentrations of GelMA bioink with unique pore sizes were selected to fabricate a heterogeneous bilayer. A higher concentration of GelMA bioink (6% w/v GelMA, 2% gelatin, 0.3% w/v HA, 10% v/v glycerol) was used to load human umbilical vein endothelial cells (HUVECs) and form an inner, endothelial tissue layer. A lower concentration of GelMA bioink (4% w/v GelMA, 4% gelatin, 0.3% w/v HA, 10% v/v glycerol) was used to load smooth muscle cells (SMCs) and form an outer, muscular tissue layer. Bioprinted blood vessel-like grafts were then assessed for mechanical properties with Instron mechanical testing, and suture-ability, and for biological properties including viability, proliferation, and histological analysis. The resulting 20 mm long, 4.0 mm diameter lumen heterogeneous bilayer blood vessel-like construct closely mimics a native blood vessel and maintains high cell viability and proliferation. Our results represent a novel strategy for small diameter blood vessel biofabrication.

Nanotechnology in the Future Treatment of Diabetic Wounds.

Diabetic wounds have a large and increasing burden on the healthcare of the UK. Currently, none of the standard treatment options for the treatment of diabetic wounds specifically target the physiological processes behind their enhanced severity. This review evaluated recent studies in the field of nanotechnology concerned with treating diabetic wounds. The studies had each developed novel therapeutics involving nanomedicines that sought to either enhance angiogenesis, the construction of new blood vessels, or increase collagen production, as well as limit the augmented inflammation, in wounds in diabetic rat or mice models. The investigations tended to either target specific anti-inflammatory or pro-proliferative receptors on endogenous cells, or transport growth factors to the wound. Previous studies have shown the beneficial effects of growth factors on healing, but they are easily broken down. By transporting them in nanoscaffolds and liposomes, it has been shown that the longevity of growth factors can be enhanced. Gold nanoparticle matrices have also been shown to have a beneficial effect on healing, by both conveying proliferative factors and independently triggering angiogenesis and collagen production. The most impressive results in the review were achieved by nanomedicines involving multiple growth factors, hence, the review will highlight the beneficial factors to wound healing and suggest a composite therapy to be trialled in the future. The review will evaluate each set of papers using similar nanomedicines and highlight the challenges of transferring this therapy to the clinic.

Silencing of RBP‑JK promotes the differentiation of bone marrow mesenchymal stem cells into vascular endothelial cells.

Bone marrow mesenchymal stem cells (BM-MSCs) are important for postnatal angiogenesis and are suitable for use in construction of blood vessels by tissue engineering. The present study aimed to investigate the influence of recombination signal binding protein for immunoglobulin kappa J region (RBP-JK) on the differentiation of BM-MSCs into vascular endothelial cells, and to assess the underlying mechanisms. BM-MSCs were isolated and identified by flow cytometry. Lentiviral vectors encoding RBP-JK shRNA (shRBPJK) were constructed to knockdown RBP-JK expression and endothelial differentiation of BM-MSCs was induced. The experimental groups were treated with: empty lentiviral vector (vector group), growth factors (bFGF and VEGF; induced group), shRBPJK (shRBPJK group), and growth factors + shRBPJK (induced + shRBPJK group). The expression of endothelial markers, vascular endothelial growth factor receptor 2 (Flk-1), and von Willebrand factor (vWF) were detected by immunofluorescence. Additionally, in vitro blood vessel formation and phagocytosis were assessed using acetylated LDL, Dil complex and the underlying molecular mechanisms evaluated by western blotting. BM-MSCs were separated and transduced with shRBPJK to reduce RBP-JK expression. Compared with the vector group, the expression of the endothelial cell markers, Flk-1 and vWF, in vitro tubule formation, and phagocytosis ability increased, while the expression levels of p-AKT/AKT and p-NF-κB/NF-κB were significantly decreased (P<0.05) in the induced, shRBPJK, and induced + shRBPJK groups. Compared with the induced group, the expression of Flk-1 and vWF, the number of tubules, and phagocytosis were higher in the induced + shRBPJK group, while the expression levels of p-AKT/AKT and p-NF-κB/NF-κB were lower (P<0.05). Collectively, the present data indicated that silencing of RBP-JK promotes the differentiation of MSCs into vascular endothelial cells, and this process is likely regulated by AKT/NF-κB signaling.

In vitro construction of artificial blood vessels using spider silk as a supporting matrix

Recent research has vigorously pursued the development of tissue-engineered vascular grafts (TEVs). One of the striking points of the early phase is the instability of the artificial blood vessels and lack of vascular resistance, which is supposed to be a consequence of unstable scaffolds. Therefore, alternative biological approaches are necessary to improve the physical properties of the artificial vessel walls.We developed blood vessel-like constructs based on native spider silk as a scaffold. C2C12 and ST1.6R cells were seeded on the surface of scaffolds and cultivated under pulsatile flow (max. ~135 mmHg and min. ~90 mmHg) in a bioreactor. Constructed grafts were compared to human blood vessels and cell seeded scaffolds cultured in the absence of bioreactor. Mechanical properties, morphological structures and expression of marker genes were assessed by strength and strain experiments, SEM, histological staining, immunohistology, Western blotting and quantitative real-time PCR. The results indicate that the constructed vessel resembles native blood vessels in morphological structure as well as in function and expression of biomarkers. Spider silk scaffolds seem to provide an optimal and stable basis for vessel constructs.

Fabrication of engineered tubular tissue for small blood vessels via three-dimensional cellular assembly and organization ex vivo

Although there is a great need for suitable vascular replacements in clinical practice, much progress needs to be made toward the development of a fully functional tissue-engineered construct. We propose a fabrication method of engineered tubular tissue for small blood vessels via a layer-by-layer cellular assembly technique using mouse smooth muscle cells, the construction of a poly-(l-lactide-co-ε-caprolactone) (PLCL) scaffold, and integration in a microfluidic perfusion culture system. The cylindrical PLCL scaffold is incised, expanded, and its surface is laminated with the cell layers. The construct confirms into tubular structures due to residual stress imposed by the cylindrical PLCL scaffold. The perfusion culture system allows simulation of static, perfusion (laminar flow), and perfusion with pulsatile pressure (Pulsatile flow) conditions in which mimicking the in vivo environments. The aim of this evaluation was to determine whether fabricated tubular tissue models developed their mechanical properties. The cellular response to hemodynamic stimulus imposed by the dynamic culture system is monitored through expression analysis of fibrillin-1 and fibrillin-2, elastin and smooth muscle myosin heavy chains isoforms transcription factors, which play an important role in tissue elastogenesis. Among the available materials for small blood vessel construction, these cellular hybrid vascular scaffolds hold much potential due to controllability of the mechanical properties of synthetic polymers and biocompatibility of integrated cellular components.