Polymeric nanofiber leveraged co-delivery of anti-stromal PAK1 inhibitor and paclitaxel enhances therapeutic effects in stroma-rich 3D spheroid models.

The role of tumor stroma in solid tumors has been widely recognized in cancer progression, metastasis and chemoresistance. Cancer-associated fibroblasts (CAFs) play a crucial role in matrix remodeling and promoting cancer cell stemness and resistance via reciprocal crosstalk. Residual tumor tissue after surgical removal as well as unresectable tumors face therapeutic challenges to achieve curable outcome. In this study, we propose to develop a dual delivery approach by combining p21-activated kinase 1 (PAK1) inhibitor (FRAX597) to inhibit tumor stroma and chemotherapeutic agent paclitaxel (PTX) to kill cancer cells using electrospun nanofibers. First, the role of the PAK1 pathway was established in CAF differentiation, migration and contraction using relevant in vitro models. Second, polycaprolactone polymer-based nanofibers were fabricated using a uniaxial electrospinning technique to incorporate FRAX597 and/or PTX, which showed a uniform texture and a prolonged release of both drugs for 16 days. To test nanofibers, stroma-rich 3D heterospheroid models were set up which showed high resistance to PTX nanofibers compared to stroma-free homospheroids. Interestingly, nanofibers containing PTX and FRAX597 showed strong anti-tumor effects on heterospheroids by reducing the growth and viability by > 90 % compared to either of single drug-loaded nanofibers. These effects were reflected by reduced intra-spheroidal expression levels of collagen 1 and α-smooth muscle actin (α-SMA). Overall, this study provides a new therapeutic strategy to inhibit the tumor stroma using PAK1 inhibitor and thereby enhance the efficacy of chemotherapy using nanofibers as a local delivery system for unresectable or residual tumor. Use of 3D models to evaluate nanofibers highlights these models as advanced in vitro tools to study the effect of controlled release local drug delivery systems before animal studies.

In Vitro and In Vivo Degradation of Photo-Crosslinked Poly(Trimethylene Carbonate-co-ε-Caprolactone) Networks.

Three-armed poly(trimethylene carbonate) (PTMC) and poly(trimethylene carbonate-co-Ɛ-caprolactone) (P(TMC-co-ε-CL)) macromers with molecular weights of approximately 30 kg mol-1 are synthesized by ring-opening polymerization and subsequent functionalization with methacrylic anhydride. Networks are then prepared by photo-crosslinking. To investigate the in vitro and in vivo degradation properties of these photo-crosslinked networks and assess the effect of ε-caprolactone content on the degradation properties, PTMC networks, and copolymer networks with two different TMC:ε-CL ratios are prepared. PTMC networks degraded slowly, via an enzymatic surface erosion process, both in vitro and in vivo. Networks prepared from P(TMC-co-ε-CL) macromers with a 74:26 ratio are found to degrade slowly as well, via a surface erosion process, albeit at a higher rate compared to PTMC networks. Increasing the ε-CL content to a ratio of 52:48, resulted in a faster degradation. These networks lost their mechanical properties much sooner than the other networks. Thus, PTMC and P(TMC-co-ε-CL) networks are interesting networks for tissue engineering purposes and the exact degradation properties can be tuned by varying the TMC:ε-CL ratio, providing researchers with a tool to obtain copolymer networks with the desired degradation rate depending on the intended application.

Hybrid Networks of Hyaluronic Acid and Poly(trimethylene carbonate) for Tissue Regeneration.

To improve the mechanical performance of hyaluronic acid (HA)-based hydrogels, we prepared novel hybrid hydrogels consisting of hydrophilic HA and hydrophobic poly(trimethylene carbonate) (PTMC). Both polymers were functionalized with methacrylic anhydride, yielding HAMA and PTMC-tMA. Hybrid networks with different ratios of PTMC-tMA:HAMA were prepared by photo-cross-linking, using DMSO pH 2.7 as a common solvent for both macromers. The hybrid networks had high gel contents. The hydrophilicity of the networks increased with increasing HAMA content. The networks consisted of the intended amounts of both macromers. The suture retention strength and compression modulus of the networks increased with increasing PTMC-tMA content. While the 100% HAMA network could not be sutured, the 50:50 PTMC-tMA:HAMA network had a suture retention strength of 5.3 N/mm. This is comparable to that of natural vascular tissues. Also the compression modulus (867 kPa) was significantly higher than that of the 100% HAMA network (13 kPa). Moreover, the networks were compatible with human mesenchymal stem cells. In conclusion, these resilient PTMC-tMA:HAMA networks are promising new biomaterials for tissue regeneration.

3D Lung-on-Chip Model Based on Biomimetically Microcurved Culture Membranes.

A comparatively straightforward approach to accomplish more physiological realism in organ-on-a-chip (OoC) models is through substrate geometry. There is increasing evidence that the strongly, microscale curved surfaces that epithelial or endothelial cells experience when lining small body lumens, such as the alveoli or blood vessels, impact their behavior. However, the most commonly used cell culture substrates for modeling of these human tissue barriers in OoCs, ion track-etched porous membranes, provide only flat surfaces. Here, we propose a more realistic culture environment for alveolar cells based on biomimetically microcurved track-etched membranes. They recreate the mainly spherical geometry of the cells' native microenvironment. In this feasibility study, the membranes were given the shape of hexagonally arrayed hemispherical microwells by an innovative combination of three-dimensional (3D) microfilm (thermo)forming and ion track technology. Integrated in microfluidic chips, they separated a top from a bottom cell culture chamber. The microcurved membranes were seeded by infusion with primary human alveolar epithelial cells. Despite the pronounced topology, the cells fully lined the alveoli-like microwell structures on the membranes' top side. The confluent curved epithelial cell monolayers could be cultured successfully at the air-liquid interface for 14 days. Similarly, the top and bottom sides of the microcurved membranes were seeded with cells from the Calu-3 lung epithelial cell line and human lung microvascular endothelial cells, respectively. Thereby, the latter lined the interalveolar septum-like interspace between the microwells in a network-type fashion, as in the natural counterpart. The coculture was maintained for 11 days. The presented 3D lung-on-a-chip model might set the stage for other (micro)anatomically inspired membrane-based OoCs in the future.

Development of an In Vitro Airway Epithelial-Endothelial Cell Culture Model on a Flexible Porous Poly(Trimethylene Carbonate) Membrane Based on Calu-3 Airway Epithelial Cells and Lung Microvascular Endothelial Cells.

Due to the continuing high impact of lung diseases on society and the emergence of new respiratory viruses, such as SARS-CoV-2, there is a great need for in vitro lung models that more accurately recapitulate the in vivo situation than current models based on lung epithelial cell cultures on stiff membranes. Therefore, we developed an in vitro airway epithelial-endothelial cell culture model based on Calu-3 human lung epithelial cells and human lung microvascular endothelial cells (LMVECs), cultured on opposite sides of flexible porous poly(trimethylene carbonate) (PTMC) membranes. Calu-3 cells, cultured for two weeks at an air-liquid interface (ALI), showed good expression of the tight junction (TJ) protein Zonula Occludens 1 (ZO-1). LMVECs cultured submerged for three weeks were CD31-positive, but the expression was diffuse and not localized at the cell membrane. Barrier functions of the Calu-3 cell cultures and the co-cultures with LMVECs were good, as determined by electrical resistance measurements and fluorescein isothiocyanate-dextran (FITC-dextran) permeability assays. Importantly, the Calu-3/LMVEC co-cultures showed better cell viability and barrier function than mono-cultures. Moreover, there was no evidence for epithelial- and endothelial-to-mesenchymal transition (EMT and EndoMT, respectively) based on staining for the mesenchymal markers vimentin and α-SMA, respectively. These results indicate the potential of this new airway epithelial-endothelial model for lung research. In addition, since the PTMC membrane is flexible, the model can be expanded by introducing cyclic stretch for enabling mechanical stimulation of the cells. Furthermore, the model can form the basis for biomimetic airway epithelial-endothelial and alveolar-endothelial models with primary lung epithelial cells.

Development of Porous and Flexible PTMC Membranes for In Vitro Organ Models Fabricated by Evaporation-Induced Phase Separation.

Polymeric membranes are widely applied in biomedical applications, including in vitro organ models. In such models, they are mostly used as supports on which cells are cultured to create functional tissue units of the desired organ. To this end, the membrane properties, e.g., morphology and porosity, should match the tissue properties. Organ models of dynamic (barrier) tissues, e.g., lung, require flexible, elastic and porous membranes. Thus, membranes based on poly (dimethyl siloxane) (PDMS) are often applied, which are flexible and elastic. However, PDMS has low cell adhesive properties and displays small molecule ad- and absorption. Furthermore, the introduction of porosity in these membranes requires elaborate methods. In this work, we aim to develop porous membranes for organ models based on poly(trimethylene carbonate) (PTMC): a flexible polymer with good cell adhesive properties which has been used for tissue engineering scaffolds, but not in in vitro organ models. For developing these membranes, we applied evaporation-induced phase separation (EIPS), a new method in this field based on solvent evaporation initiating phase separation, followed by membrane photo-crosslinking. We optimised various processing variables for obtaining form-stable PTMC membranes with average pore sizes between 5 to 8 µm and water permeance in the microfiltration range (17,000-41,000 L/m2/h/bar). Importantly, the membranes are flexible and are suitable for implementation in in vitro organ models.

In vitro modelling of alveolar repair at the air-liquid interface using alveolar epithelial cells derived from human induced pluripotent stem cells.

Research on acute and chronic lung diseases would greatly benefit from reproducible availability of alveolar epithelial cells (AEC). Primary alveolar epithelial cells can be derived from human lung tissue but the quality of these cells is highly donor dependent. Here, we demonstrated that culture of EpCAM+ cells derived from human induced pluripotent stem cells (hiPSC) at the physiological air-liquid interface (ALI) resulted in type 2 AEC-like cells (iAEC2) with alveolar characteristics. iAEC2 cells expressed native AEC2 markers (surfactant proteins and LPCAT-1) and contained lamellar bodies. ALI-iAEC2 were used to study alveolar repair over a period of 2 weeks following mechanical wounding of the cultures and the responses were compared with those obtained using primary AEC2 (pAEC2) isolated from resected lung tissue. Addition of the Wnt/ß-catenin activator CHIR99021 reduced wound closure in the iAEC2 cultures but not pAEC2 cultures. This was accompanied by decreased surfactant protein expression and accumulation of podoplanin-positive cells at the wound edge. These results demonstrated the feasibility of studying alveolar repair using hiPSC-AEC2 cultured at the ALI and indicated that this model can be used in the future to study modulation of alveolar repair by (pharmaceutical) compounds.

Synthesis and characterization of rGO-graft-poly(trimethylene carbonate) for nerve regeneration conduits.

Graphene-graft-polymer has been used to improve the compatibility between graphene and a polymer matrix, and to further enhance electrical, mechanical and biological properties of polymer/graphene composites. In this study, poly(trimethylene carbonate) (PTMC) was successfully grafted onto graphene surface via 'grafting from' method. Reduced graphene oxide (rGO) initiator was synthesized by azido ethanol reaction with graphene oxide (GO) at high temperature. This resulted in thermal reduction of the GO and stable hydroxyl groups on the graphene surface. Subsequently, rGO initiator was used for the ring-opening polymerization of TMC monomer. rGO-graft-PTMC composites with PTMC molecular weights of 430, 480, 2150 and 7030 g mol-1 were successfully synthesized using different amounts of TMC. Single layer graphene nanosheets remained after graft polymerization by this method. rGO-graft-PTMC dispersions in chloroform were stable. The rGO-graft-PTMC composites with PTMC molecular weights of 430-7030 g mol-1 had electrical conductivities ranging from 0.2 to 0.016 s cm-1. To investigate the biocompatibility of rGO-graft-PTMC, PTMC-based films containing rGO-graft-PTMC were prepared and used in cell culturing experiments. The composite films showed good biocompatibility with PC12 neuronal cells. It is concluded that rGO-graft-PTMC composite is a promising material for the preparation of nerve regeneration conduits.

Fabrication of poly (trimethylene carbonate)/reduced graphene oxide-graft-poly (trimethylene carbonate) composite scaffolds for nerve regeneration.

One of the key challenges for neural tissue engineering is to exploit functional materials to guide and support nerve regeneration. Currently, reduced graphene oxide (rGO), which is well-known for its unique electrical and mechanical properties, has been incorporated into biocompatible polymers to manufacture functional scaffolds for nerve tissue engineering. However, rGO has poor dispersity in polymer matrix, which limits its further application. Here, we replaced rGO with rGO-graft-PTMC. The rGO-graft-PTMC was firstly prepared by grafting trimethylene carbonate (TMC) oligomers onto rGO. Subsequently, PTMC/rGO-graft-PTMC composite fibrous mats were fabricated by electrospinning of a dispersion of PTMC and rGO-graft-PTMC. The loading of rGO-graft-PTMC could reach up to 6 wt% relative to PTMC. Scanning electron microscopy images showed that the morphologies and average diameters of PTMC/rGO-graft-PTMC composite fibrous mats were affected by the content of rGO-graft-PTMC. Additionally, the incorporation of rGO-graft-PTMC resulted in enhanced thermal stability and hydrophobicity of PTMC fibers. Biological results demonstrated that PC12 cells showed higher cell viability on PTMC/rGO-graft-PTMC fibers of 2.4, 4.0 and 6.0 wt% rGO-graft-PTMC compared to pure PTMC fibers. These results suggest that PTMC/rGO-graft-PTMC composite fibrous structures hold great potential for neural tissue engineering.

Surface curvature in triply-periodic minimal surface architectures as a distinct design parameter in preparing advanced tissue engineering scaffolds.

Reproduction of the anatomical structures and functions of tissues using cells and designed 3D scaffolds is an ongoing challenge. For this, scaffolds with appropriate biomorphic surfaces promoting cell attachment, proliferation and differentiation are needed. In this study, eight triply-periodic minimal surface (TPMS)-based scaffolds were designed using specific trigonometric equations, providing the same porosity and the same number of unit cells, while presenting different surface curvatures. The scaffolds were fabricated by stereolithography using a photocurable resin based on the biocompatible, biodegradable and rubber-like material, poly(trimethylene carbonate) (PTMC). A numerical approach was developed to calculate the surface curvature distributions of the TPMS architectures. Moreover, the scaffolds were characterized by scanning electron microscopy, micro-computed tomography and water permeability measurements. These original scaffold architectures will be helpful to decipher the biofunctional role of the surface curvature of scaffolds intended for tissue engineering applications.

Differentiation of adipose stem cells seeded towards annulus fibrosus cells on a designed poly(trimethylene carbonate) scaffold prepared by stereolithography.

Cell-based therapies could potentially restore the biomechanical function and enhance the self-repair capacity of annulus fibrosus (AF) tissue. However, choosing a suitable cell source and scaffold design are still key challenges. In this study, we assessed the in vitro ability of human adipose stem cells (hASCs), an easily available cell source to produce AF-like matrix in novel AF-mimetic designed scaffolds based on poly(trimethylene carbonate) and built by stereolithography. To facilitate efficient differentiation of hASCs towards AF tissue, we tested different culture medium compositions and cell seeding techniques. This is the first study to report that medium supplementation with transforming growth factor (TGF)-ß3 is essential to support AF differentiation of hASCs while TGF-ß1 has negligible effect after 21 days of culture. Fibrin gel seeding resulted in superior cell distribution, proliferation and AF-like matrix production of hASCs compared to direct and micromass seeding under TGF-ß3 stimulation. Not only the production of sulphated glycosaminoglycans (sGAG) and collagen was significantly upregulated, but the formed collagen was also oriented and aligned into bundles within the designed pore channels. The differentiated hASCs seeded with fibrin gel were also found to have a comparable sGAG:collagen ratio and gene expression profile as native AF cells demonstrating the high potential of this strategy in AF repair. Copyright © 2016 John Wiley & Sons, Ltd.

Biomaterials for the Treatment of Alzheimer's Disease.

Alzheimer's disease (AD) as a progressive and fatal neurodegenerative disease represents a huge unmet need for treatment. The low efficacy of current treatment methods is not only due to low drug potency but also due to the presence of various obstacles in the delivery routes. One of the main barriers is the blood-brain barrier. The increasing prevalence of AD and the low efficacy of current therapies have increased the amount of research on unraveling of disease pathways and development of treatment strategies. One of the interesting areas for the latter subject is biomaterials and their applications. This interest originates from the fact that biomaterials are very useful for the delivery of therapeutic agents, such as drugs, proteins, and/or cells, in order to treat diseases and regenerate tissues. Recently, manufacturing of nano-sized delivery systems has increased the efficacy and delivery potential of biomaterials. In this article, we review the latest developments with regard to the use of biomaterials for the treatment of AD, including nanoparticles and liposomes for delivery of therapeutic compounds and scaffolds for cell delivery strategies.

Regeneration of the lung: Lung stem cells and the development of lung mimicking devices.

Inspired by the increasing burden of lung associated diseases in society and an growing demand to accommodate patients, great efforts by the scientific community produce an increasing stream of data that are focused on delineating the basic principles of lung development and growth, as well as understanding the biomechanical properties to build artificial lung devices. In addition, the continuing efforts to better define the disease origin, progression and pathology by basic scientists and clinicians contributes to insights in the basic principles of lung biology. However, the use of different model systems, experimental approaches and readout systems may generate somewhat conflicting or contradictory results. In an effort to summarize the latest developments in the lung epithelial stem cell biology, we provide an overview of the current status of the field. We first describe the different stem cells, or progenitor cells, residing in the homeostatic lung. Next, we focus on the plasticity of the different cell types upon several injury-induced activation or repair models, and highlight the regenerative capacity of lung cells. Lastly, we summarize the generation of lung mimics, such as air-liquid interface cultures, organoids and lung on a chip, that are required to test emerging hypotheses. Moreover, the increasing collaboration between distinct specializations will contribute to the eventual development of an artificial lung device capable of assisting reduced lung function and capacity in human patients.

Efficient generation of smooth muscle cells from adipose-derived stromal cells by 3D mechanical stimulation can substitute the use of growth factors in vascular tissue engineering.

Occluding artery disease causes a high demand for bioartificial replacement vessels. We investigated the combined use of biodegradable and creep-free poly (1,3-trimethylene carbonate) (PTMC) with smooth muscle cells (SMC) derived by biochemical or mechanical stimulation of adipose tissue-derived stromal cells (ASC) to engineer bioartificial arteries. Biochemical induction of cultured ASC to SMC was done with TGF-ß1 for 7d. Phenotype and function were assessed by qRT-PCR, immunodetection and collagen contraction assays. The influence of mechanical stimulation on non-differentiated and pre-differentiated ASC, loaded in porous tubular PTMC scaffolds, was assessed after culturing under pulsatile flow for 14d. Assays included qRT-PCR, production of extracellular matrix and scanning electron microscopy. ASC adhesion and TGF-ß1-driven differentiation to contractile SMC on PTMC did not differ from tissue culture polystyrene controls. Mesenchymal and SMC markers were increased compared to controls. Interestingly, pre-differentiated ASC had only marginal higher contractility than controls. Moreover, in 3D PTMC scaffolds, mechanical stimulation yielded well-aligned ASC-derived SMC which deposited ECM. Under the same conditions, pre-differentiated ASC-derived SMC maintained their SMC phenotype. Our results show that mechanical stimulation can replace TGF-ß1 pre-stimulation to generate SMC from ASC and that pre-differentiated ASC keep their SMC phenotype with increased expression of SMC markers.

Biodegradable elastomers for biomedical applications and regenerative medicine.

Synthetic biodegradable polymers are of great value for the preparation of implants that are required to reside only temporarily in the body. The use of biodegradable polymers obviates the need for a second surgery to remove the implant, which is the case when a nondegradable implant is used. After implantation in the body, biomedical devices may be subjected to degradation and erosion. Understanding the mechanisms of these processes is essential for the development of biomedical devices or implants with a specific function, for example, scaffolds for tissue-engineering applications. For the engineering and regeneration of soft tissues (e.g., blood vessels, cardiac muscle and peripheral nerves), biodegradable polymers are needed that are flexible and elastic. The scaffolds prepared from these polymers should have tuneable degradation properties and should perform well under long-term cyclic deformation conditions. The required polymers, which are either physically or chemically crosslinked biodegradable elastomers, are reviewed in this article.

Flexible and elastic scaffolds for cartilage tissue engineering prepared by stereolithography using poly(trimethylene carbonate)-based resins.

The aim of this study is to investigate the applicability of flexible and elastic poly(trimethylene carbonate) (PTMC) structures prepared by stereolithography as scaffolds for cartilage tissue engineering. A three-armed methacrylated PTMC macromer with a molecular weight of 3100 g mol(-1) is used to build designed scaffolds with a pore diameter of 350 ± 12 µm and a porosity of 54.0 ± 2.2%. Upon seeding of bovine chondrocytes in the scaffolds, the cells adhere and spread on the PTMC surface. After culturing for 6 weeks, also cells with a round morphology are present, indicative of the differentiated chondrocyte phenotype. Sulphated glycosaminoglycans and fibrillar collagens are deposited by the cells. During culturing for 6 weeks, the compression moduli of the constructs increases 50% to approximately 100 kPa.

Fluorescently labeled branched polymers and thermal responsive nanoparticles for live cell imaging.

Branched poly(methoxy-PEG acrylate) and thermally responsive poly(methoxy-PEG acrylate)-block-poly(N-isopropylacrylamide) are synthesized by RAFT polymerization. After reduction, these polymers are fluorescently labeled by reacting the free thiol groups with N-(5-fluoresceinyl)maleimide. As shown by DLS, the labeled copolymer poly(methoxy-PEG acrylate)-block-poly(N-isopropylacrylamide) forms nanoparticles at body temperature (37 °C) due to the presence of the thermosensitive poly(N-isopropylacrylamide). These materials were used as bioprobes for imaging HUVECs in vitro and chick embryo CAM in vivo. Both labeled polymer and nanoparticles are biocompatible and can be used as efficient fluorescent bioprobes.

Evaluation of tubular poly(trimethylene carbonate) tissue engineering scaffolds in a circulating pulsatile flow system.

Tubular scaffolds (internal diameter approximately 3 mm and wall thickness approximately 0.8 mm) with a porosity of approximately 83% and an average pore size of 116 µm were prepared from flexible poly(trimethylene carbonate) (PTMC) polymer by dip-coating and particulate leaching methods. PTMC is a flexible and biocompatible polymer that crosslinks upon irradiation; porous network structures were obtained by irradiating the specimens in vacuum at 25 kGy before leaching soluble salt particles. To assess the suitability of these scaffolds in dynamic cell culturing for cardiovascular tissue engineering, the scaffolds were coated with a thin (0.1 to 0.2 mm) non-porous PTMC layer and its performance was evaluated in a closed pulsatile flow system (PFS). For this, the PFS was operated at physiological conditions at liquid flows of 1.56 ml/s with pressures varying from 80-120 mmHg at a frequency of 70 pulsations per minute. The mechanical properties of these coated porous PTMC scaffolds were not significantly different than non-coated scaffolds. Typical tensile strengths in the radial direction were 0.15 MPa, initial stiffness values were close to 1.4 MPa. Their creep resistance in cyclic deformation experiments was excellent. In the pulsatile flow setup, the distention rates of these flexible and elastic scaffolds were approximately 0.10% per mmHg, which is comparable to that of a porcine carotid artery (0.11% per mmHg). The compliance and stiffness index values were close to those of natural arteries.?In long-term deformation studies, where the scaffolds were subjected to physiological pulsatile pressures for one week, the morphology and mechanical properties of the PTMC scaffolds did not change. This suggests their suitability for application in a dynamic cell culture bioreactor.

Dynamic culturing of smooth muscle cells in tubular poly(trimethylene carbonate) scaffolds for vascular tissue engineering.

Porous, tubular, flexible, and elastic poly(trimethylene carbonate) (PTMC) scaffolds (length 8 cm and inner diameter 3 mm) for vascular tissue engineering were prepared by means of a dip-coating and particulate leaching procedure. Using NaCl as porogen, scaffolds with an average pore size of 110 µm and a porosity of 85% were obtained. Before leaching the salt, the structures were made creep-resistant by means of crosslinking at 25 kGy gamma irradiation. To increase the efficiency of cell seeding, the scaffolds were provided with a microporous outer layer of 0.2 mm with an average pore size of 28 µm and a porosity of 65% (total wall thickness 1 mm). Human smooth muscle cells (SMCs) were seeded in these scaffolds with an efficiency of 43%, as determined after 24 h cell adhesion. SMCs were cultured in the scaffolds up to 14 days under stationary conditions or under pulsatile flow conditions in a bioreactor (pressure 70-130 mmHg, 69 pulsations/min, and average wall shear rate 320 s(-1)). Although SMCs proliferated under both conditions, cell numbers were three to five times higher in case of dynamic culturing. This was qualitatively confirmed by means of histology. Also, in terms of mechanical properties, the dynamically cultured constructs performed better than the statically cultured constructs. After culturing for 14 days, the maximum tensile strengths of the constructs, determined in the radial direction, had increased from 0.16 MPa (unseeded scaffold) to 0.48 MPa (dynamic culturing) and 0.38 MPa (static culturing). The results of this study indicate that a potentially useful medial layer for tissue-engineered vascular grafts can be prepared by dynamic culturing of human SMCs seeded in porous tubular poly(trimethylene carbonate) scaffolds.

A microfluidic wound-healing assay for quantifying endothelial cell migration.

Endothelial migration is an important process in the formation of blood vessels and the repair of damaged tissue. To study this process in the laboratory, versatile and reliable migration assays are essential. The purpose of this study was to investigate whether the microfluidic version of the conventional wound-healing assay is a useful research tool for vascular science. Endothelial cells were seeded in a 500-mum-wide microfluidic channel. After overnight incubation, cells had formed a viable and confluent monolayer. Then, a wound was generated in this monolayer by flushing the channel with three parallel fluid streams, of which the middle one contained the protease trypsin. By analyzing the closing of the wound over time, endothelial cell migration could be measured. Although the migration rate was two times lower in the microfluidic assay than in the conventional assay, an identical 1.5-times increase in migration rate was found in both assays when vascular endothelial growth factor (VEGF(165)) was added. In the microfluidic wound-healing assay, a stable gradient of VEGF(165) could be generated at the wound edge. This led to a two-times increase in migration rate compared with the untreated control. Finally, when a shear stress of 1.3 Pa was applied to the wound, the migration rate increased 1.8 times. In conclusion, the microfluidic assay is a solid alternative for the conventional wound-healing assay when endothelial cell migration is measured. Moreover, it offers unique advantages, such as gradient generation and application of shear stress.