A textile-reinforced composite vascular graft that modulates macrophage polarization and enhances endothelial cell migration, adhesion and proliferation in vitro.

At the present time, there is no successful off-the-shelf small-caliber vascular graft (<6 mm) for the repair or bypass of the coronary or carotid arteries. In this study, we engineer a textile-reinforced hydrogel vascular graft. The textile fibers are circularly knitted into a flexible yet robust conduit to serve as the backbone of the composite vascular graft and provide the primary mechanical support. It is embedded in the hydrogel matrix which seals the open structure of the knitted reinforcement and mediates cellular response toward a faster reendothelialization. The mechanical properties of the composite vascular graft, including bursting strength, suture retention strength and radial compliance, significantly surpass the requirement for the vascular graft application and can be adjusted by altering the structure of the textile reinforcement. The addition of hydrogel matrix, on the other hand, improves the survival, adhesion and proliferation of endothelial cells in vitro. The composite vascular graft also enhances macrophage activation and upregulates M1 and M2 related gene expression, which further improves the endothelial cell migration that might favor the reendothelialization of the vascular graft. Taken together, the textile-reinforced hydrogel shows it potential to be a promising scaffold material to fabricate a tissue engineered vascular graft.

Same-Single-Cell Analysis of Pacemaker-Specific Markers in Human Induced Pluripotent Stem Cell-Derived Cardiomyocyte Subtypes Classified by Electrophysiology.

Insights into the expression of pacemaker-specific markers in human induced pluripotent stem cell (hiPSC)-derived cardiomyocyte subtypes can facilitate the enrichment and track differentiation and maturation of hiPSC-derived pacemaker-like cardiomyocytes. To date, no study has directly assessed gene expression in each pacemaker-, atria-, and ventricular-like cardiomyocyte subtype derived from hiPSCs since currently the subtypes of these immature cardiomyocytes can only be identified by action potential profiles. Traditional acquisition of action potentials using patch-clamp recordings renders the cells unviable for subsequent analysis. We circumvented these issues by acquiring the action potential profile of a single cell optically followed by assessment of protein expression through immunostaining in that same cell. Our same-single-cell analysis for the first time revealed expression of proposed pacemaker-specific markers-hyperpolarization-activated cyclic nucleotide-modulated (HCN)4 channel and Islet (Isl)1-at the protein level in all three hiPSC-derived cardiomyocyte subtypes. HCN4 expression was found to be higher in pacemaker-like hiPSC-derived cardiomyocytes than atrial- and ventricular-like subtypes but its downregulation over time in all subtypes diminished the differences. Isl1 expression in pacemaker-like hiPSC-derived cardiomyocytes was initially not statistically different than the contractile subtypes but did become statistically higher than ventricular-like cells with time. Our observations suggest that although HCN4 and Isl1 are differentially expressed in hiPSC-derived pacemaker-like relative to ventricular-like cardiomyocytes, these markers alone are insufficient in identifying hiPSC-derived pacemaker-like cardiomyocytes. Stem Cells 2016;34:2670-2680.

Development of Optical Nanosensors for Detection of Potassium Ions and Assessment of Their Biocompatibility with Corneal Epithelial Cells.

Imbalance of potassium-ion levels in the body can lead to physiological dysfunctions, which can adversely impact cardiovascular, neurological, and ocular health. Thus, quantitative measurement of potassium ions in a biological system is crucial for personal health monitoring. Nanomaterials can be used to aid in disease diagnosis and monitoring therapies. Optical detection technologies along with molecular probes emitting within the near-infrared (NIR) spectral range are advantageous for biological measurements due to minimal interference from light scattering and autofluorescence within this spectral window. Herein, we report the development of NIR fluorescent nanosensors, which can quantitatively detect potassium ions under biologically relevant conditions. The optical nanosensors were developed by using photoluminescent single-walled carbon nanotubes (SWCNTs) encapsulated in polymers that contain potassium chelating moieties. The nanosensors, polystyrene sulfonate [PSS-SWCNTs, nanosensor 1 (NS1)] or polystyrene-co-polystyrene sulfonate [PS-co-PSS-SWCNTs, nanosensor 2 (NS2)], exhibited dose-dependent optical responses to potassium ion level. The nanosensors demonstrated their biocompatibility via the evaluation of cellular viability, proliferation assays, and expression of cytokeratin 12 in corneal epithelial cells (CEpiCs). Interestingly, the nanosensors' optical characteristics and their responses toward CEpiCs were influenced by encapsulating polymers. NS2 exhibited a 10 times higher fluorescence intensity along with a higher signal-to-noise ratio as compared to NS1. NS2 showed an optical response to potassium ion level in solution within 5 min of addition and a limit of detection of 0.39 mM. Thus, NS2 was used for detailed investigations including potassium ion level detection in serum. NS2 showed a consistent response to potassium ions at the lower millimolar range in serum. These results on optical sensing along with biocompatibility show a great potential for nanotube sensors in biomedical research.

Application of Noggin-Coated Electrospun Scaffold in Corneal Wound Healing.

Purpose: The objective of this study is to develop and characterize electrospun corneal bandage infused with Noggin protein and evaluate its therapeutic potential in the treatment of superficial nonhealing corneal ulceration. Methods: Electrospun nanofibrous scaffolds were created with different blend ratios of polycaprolactone and gelatin and coated with different concentrations of Noggin protein. Morphologic, mechanical, degradation, and surface chemistry of the developed scaffold was assessed. Biocompatibility of the developed scaffold with corneal epithelial cells was evaluated by looking at cell viability, proliferation, and immunostaining. In vitro wound healing in the presence of Noggin-coated scaffold was evaluated by measuring wound closure rate after scratch. Results: Uniform nanofibrous scaffolds coated with Noggin were constructed through optimization of electrospinning parameters and demonstrated mechanical properties better than or similar to commercially available contact lenses used in corneal wound healing. In the presence of Noggin-coated scaffold, corneal epithelial cells showed higher proliferation and wound-healing rate. Conclusions: This Noggin-coated electrospun scaffold represents a step toward, expanding treatment options for patients with indolent corneal ulcers. Translational Relevance: In this study, the feasibility of Noggin-coated electrospun scaffold as a therapeutic for indolent corneal ulcer was evaluated. This study also provides a better perspective for understanding electrospun scaffolds as a tunable platform to infuse topical therapeutics and use as a corneal bandage.

Heparin Affinity-Based IL-4 Delivery to Modulate Macrophage Phenotype and Endothelial Cell Activity In Vitro.

Macrophages play a pivotal role in wound healing and tissue regeneration, as they are rapidly recruited to the site of injury or implanted foreign material. Depending on their interaction with the material, macrophages can develop different phenotypes, with the M1 pro-inflammatory and M2 pro-regenerative phenotypes being highly involved in tissue regeneration. M2 macrophages mitigate inflammation and promote tissue regeneration and extracellular matrix remodeling. In this study, we engineered a gelatin-heparin-methacrylate (GelMA-HepMA) hydrogel that gradually releases interleukin-4 (IL-4), a cytokine that modulates macrophages to adopt the M2 phenotype. Methacrylation of heparin improved the retention of both heparin and IL-4 within the hydrogel. The GelMA-HepMA hydrogel and IL-4 synergistically downregulated M1 gene expression and upregulated M2 gene expression in macrophages within 48 h of in vitro cell culture. However, the M2-like macrophage phenotype induced by the GelMA-HepMA-IL-4 hydrogel did not necessarily further improve endothelial cell proliferation and migration in vitro.

Comparison of NIH 3T3 Cellular Adhesion on Fibrous Scaffolds Constructed from Natural and Synthetic Polymers.

Polymer scaffolds are increasingly ubiquitous in the field of tissue engineering in improving the repair and regeneration of damaged tissue. Natural polymers exhibit better cellular adhesion and proliferation than biodegradable synthetics but exhibit inferior mechanical properties, among other disadvantages. Synthetic polymers are highly tunable but lack key binding motifs that are present in natural polymers. Using collagen and poly(lactic acid) (PLA) as models for natural and synthetic polymers, respectively, an evaluation of the cellular response of embryonic mouse fibroblasts (NIH 3T3 line) to the different polymer types was conducted. The samples were analyzed using LIVE/DEAD™, alamarBlue™, and phalloidin staining to compare cell proliferation on, interaction with, and adhesion to the scaffolds. The results indicated that NIH3T3 cells prefer collagen-based scaffolds. PLA samples had adhesion at the initial seeding but failed to sustain long-term adhesion, indicating an unsuitable microenvironment. Structural differences between collagen and PLA are responsible for this difference. Incorporating cellular binding mechanisms (i.e., peptide motifs) utilized by natural polymers into biodegradable synthetics offers a promising direction for biomaterials to become biomimetic by combining the advantages of synthetic and natural polymers while minimizing their disadvantages.

Development of a Pneumatic-Driven Fiber-Shaped Robot Scaffold for Use as a Complex 3D Dynamic Culture System.

Cells can sense and respond to different kinds of continuous mechanical strain in the human body. Mechanical stimulation needs to be included within the in vitro culture system to better mimic the existing complexity of in vivo biological systems. Existing commercial dynamic culture systems are generally two-dimensional (2D) which fail to mimic the three-dimensional (3D) native microenvironment. In this study, a pneumatically driven fiber robot has been developed as a platform for 3D dynamic cell culture. The fiber robot can generate tunable contractions upon stimulation. The surface of the fiber robot is formed by a braiding structure, which provides promising surface contact and adequate space for cell culture. An in-house dynamic stimulation using the fiber robot was set up to maintain NIH3T3 cells in a controlled environment. The biocompatibility of the developed dynamic culture systems was analyzed using LIVE/DEAD™ and alamarBlue™ assays. The results showed that the dynamic culture system was able to support cell proliferation with minimal cytotoxicity similar to static cultures. However, we observed a decrease in cell viability in the case of a high strain rate in dynamic cultures. Differences in cell arrangement and proliferation were observed between braided sleeves made of different materials (nylon and ultra-high molecular weight polyethylene). In summary, a simple and cost-effective 3D dynamic culture system has been proposed, which can be easily implemented to study complex biological phenomena in vitro.

Serum immune checkpoint profiling identifies soluble CD40 as a biomarker for pancreatic cancer.

Pancreatic ductal adenocarcinoma (PDAC) responds poorly to systemic treatment, including new immunotherapeutic approaches. Biomarkers are urgently needed for early disease detection, patient stratification for treatment, and response prediction. The role of soluble CD40 (sCD40) is unknown in PDAC. In this study, we performed a quantitative multiplex analysis of 17 immune checkpoint proteins in serum samples from patients with various stages of PDAC in a discovery study (n = 107) and analyzed sCD40 by ELISA in a validation study (n = 317). Youden's J statistic was used for diagnostic cut-off optimization. A Cox proportional hazards regression model was applied in an empiric approach for prognostic threshold optimization. Kaplan-Meier estimator and multivariable Cox regression analyses were used for survival analysis. sCD40 was significantly increased in the serum of patients with PDAC compared to healthy cohorts and patients with IPMN. In the validation cohort, the area under the receiver operating characteristic (ROC) c-statistic was 0.8, and combining sCD40 with CA19-9 yielded a c-statistic of 0.95. sCD40 levels were independent of the tumor stage. However, patients who received neoadjuvant chemotherapy had significantly lower sCD40 levels than those who underwent upfront surgery. Patients with a sCD40 level above the empirical threshold of 0.83 ng/ml had a significantly reduced overall survival with a hazard ratio of 1.4. This observation was pronounced in patients after neoadjuvant chemotherapy. Collectively, soluble CD40 may be considered as both a diagnostic and prognostic non-invasive biomarker in PDAC.

In Vitro Biocompatibility and Degradation Analysis of Mass-Produced Collagen Fibers.

Automation and mass-production are two of the many limitations in the tissue engineering industry. Textile fabrication methods such as electrospinning are used extensively in this field because of the resemblance of the extracellular matrix to the fiber structure. However, electrospinning has many limitations, including the ability to mass-produce, automate, and reproduce products. For this reason, this study evaluates the potential use of a traditional textile method such as spinning. Apart from mass production, these methods are also easy, efficient, and cost-effective. This study uses bovine-derived collagen fibers to create yarns using the traditional ring spinning method. The collagen yarns are proven to be biocompatible. Enzymatic biodegradability was also confirmed for its potential use in vivo. The results of this study prove the safety and efficacy of the material and the fabrication method. The material encourages higher cell proliferation and migration compared to tissue culture-treated plastic plates. The process is not only simple but is also streamlined and replicable, resulting in standardized products that can be reproduced.

Effects of sterilization methods on gelatin methacryloyl hydrogel properties and macrophage gene expressionin vitro.

To assure the long-term safety and functional performance after implantation, it is of critical importance to completely sterilize a biomaterial implant. Ineffective sterilization can cause severe inflammation and infection at the implant site, leading to detrimental events of morbidity and even mortality. Macrophages are pivotal players in the inflammatory and foreign body response after implanting a biomaterial in the body. However, the relationship between the sterilization procedure and macrophage response has not been established. In this study, three commonly used sterilization methods, including autoclaving, ethylene oxide gas and ethanol treatment, were used to sterilize a gelatin methacryloyl hydrogel. The impacts of different sterilization methods on the structure and physical properties of the hydrogel were compared. Macrophage responses to the sterilized hydrogel were analyzed based on their morphology, viability andin vitrogene expression. It was found that the sterilization methods only marginally altered the hydrogel morphology, swelling behavior and elastic modulus, but significantly impacted macrophage gene expression within 48 h and over 7 din vitro. Therefore, when selecting sterilization methods for GelMA hydrogel, not only the sterility and hydrogel properties, such as material destruction and degradation caused by temperature and moisture, should be taken into consideration, but also the cellular responses to the sterilized material which could be substantially different.

Electrospun Carbon Nanotube-Based Scaffolds Exhibit High Conductivity and Cytocompatibility for Tissue Engineering Applications.

Carbon nanotubes (CNTs) are known for their excellent conductive properties. Here, we present two novel methods, "sandwich" (sCNT) and dual deposition (DD CNT), for incorporating CNTs into electrospun polycaprolactone (PCL) and gelatin scaffolds to increase their conductance. Based on CNT percentage, the DD CNT scaffolds contain significantly higher quantities of CNTs than the sCNT scaffolds. The inclusion of CNTs increased the electrical conductance of scaffolds from 0.0 ± 0.00 kS (non-CNT) to 0.54 ± 0.10 kS (sCNT) and 5.22 ± 0.49 kS (DD CNT) when measured parallel to CNT arrays and to 0.25 ± 0.003 kS (sCNT) and 2.85 ± 1.12 (DD CNT) when measured orthogonally to CNT arrays. The inclusion of CNTs increased fiber diameter and pore size, promoting cellular migration into the scaffolds. CNT inclusion also decreased the degradation rate and increased hydrophobicity of scaffolds. Additionally, CNT inclusion increased Young's modulus and failure load of scaffolds, increasing their mechanical robustness. Murine fibroblasts were maintained on the scaffolds for 30 days, demonstrating high cytocompatibility. The increased conductivity and high cytocompatibility of the CNT-incorporated scaffolds make them appropriate candidates for future use in cardiac and neural tissue engineering.

Induced Pluripotent Stem Cell-Derived Corneal Cells: Current Status and Application.

Deficiency and dysfunction of corneal cells leads to the blindness observed in corneal diseases such as limbal stem cell deficiency (LSCD) and bullous keratopathy. Regenerative cell therapies and engineered corneal tissue are promising treatments for these diseases [1]. However, these treatments are not yet clinically feasible due to inadequate cell sources. The discovery of induced pluripotent stem cells (iPSCs) by Shinya Yamanaka has provided a multitude of opportunities in research because iPSCs can be generated from somatic cells, thus providing an autologous and unlimited source for corneal cells. Compared to other stem cell sources such as mesenchymal and embryonic, iPSCs have advantages in differentiation potential and ethical concerns, respectively. Efforts have been made to use iPSCs to model corneal disorders and diseases, drug testing [2], and regenerative medicine [1]. Autologous treatments based on iPSCs can be exorbitantly expensive and time-consuming, but development of stem cell banks with human leukocyte antigen (HLA)- homozygous cell lines can provide cost- and time-efficient allogeneic alternatives. In this review, we discuss the early development of the cornea because protocols differentiating iPSCs toward corneal lineages rely heavily upon recapitulating this development. Differentiation of iPSCs toward corneal cell phenotypes have been analyzed with an emphasis on feeder-free, xeno-free, and well-defined protocols, which have clinical relevance. The application, challenges, and potential of iPSCs in corneal research are also discussed with a focus on hurdles that prevent clinical translation.

Plasma-Induced Diallyldimethylammonium Chloride Antibacterial Hernia Mesh.

A hernia is a pathological condition caused by a defect or opening in the muscle wall, which leads to organs pushing through the opening or defect. Hernia recurrence, seroma, persistent pain, tissue adhesions, and wound infection are common complications following hernia repair surgery. Infection after hernia mesh implantation is the third major complication leading to hernia recurrence. In order to reduce the incidence of late infections, we developed a polypropylene mesh with antibacterial properties. In this study, knitted polypropylene meshes were exposed to radio-frequency plasma to activate their surfaces. The antibacterial monomer diallyldimethylammonium chloride (DADMAC) was then grafted onto the mesh surface using pentaerythritol tetraacrylate as the cross-linker since it is able to engage all four functional groups to form a high-density cross-linked network. The subsequent antibacterial performance showed a 2.9 log reduction toward Staphylococcus aureus and a 0.9 log reduction for Escherichia coli.

Neoadjuvant chemotherapy drives intratumoral T cells toward a proinflammatory profile in pancreatic cancer.

BACKGROUNDPancreatic ductal adenocarcinoma (PDAC) has a dismal prognosis. At diagnosis, only 20% of patients with PDAC are eligible for primary resection. Neoadjuvant chemotherapy can enable surgical resection in 30%-40% of patients with locally advanced and borderline resectable PDAC. The effects of neoadjuvant chemotherapy on the cytokine production of tumor-infiltrating T cells are unknown in PDAC.METHODSWe performed multiplex immunofluorescence to investigate T cell infiltration in 91 patients with PDAC. Using flow cytometry, we analyzed tumor and matched blood samples from 71 patients with PDAC and determined the frequencies of T cell subsets and their cytokine profiles. Both cohorts included patients who underwent primary resection and patients who received neoadjuvant chemotherapy followed by surgical resection.RESULTSIn human PDAC, T cells were particularly enriched within the tumor stroma. Neoadjuvant chemotherapy markedly enhanced T cell density within the ductal area of the tumor. Whereas infiltration of cytotoxic CD8+ T cells was unaffected by neoadjuvant chemotherapy, the frequency of conventional CD4+ T cells was increased, and the proportion of Tregs was reduced in the pancreatic tumor microenvironment after neoadjuvant treatment. Moreover, neoadjuvant chemotherapy increased the production of proinflammatory cytokines by tumor-infiltrating T cells, with enhanced TNF-α and IL-2 and reduced IL-4 and IL-10 expression.CONCLUSIONNeoadjuvant chemotherapy drives intratumoral T cells toward a proinflammatory profile. Combinational treatment strategies incorporating immunotherapy in neoadjuvant regimens may unleash more effective antitumor responses and improve prognosis of pancreatic cancer.FUNDINGThis work was supported by the Jung Foundation for Science and Research, the Monika Kutzner Foundation, the German Research Foundation (SE2980/5-1), the German Cancer Consortium, and the Faculty of Medicine Carl Gustav Carus, Technische Universität Dresden.

LAG-3-Expressing Tumor-Infiltrating T Cells Are Associated with Reduced Disease-Free Survival in Pancreatic Cancer.

T cells are the predominant immune cell population in the pancreatic tumor microenvironment. High CD8+ and Th1-polarized CD4+ T cell infiltration is associated with prolonged survival in human pancreatic ductal adenocarcinoma (PDAC). However, the expression pattern of co-stimulatory and inhibitory receptors by PDAC-infiltrating T cells and their prognostic significance are not well defined. In this study, we employed multiplex immunofluorescence to investigate the intratumoral expression of the co-stimulatory receptor inducible T-cell co-stimulator (ICOS), the inhibitory receptors lymphocyte-activation gene 3 (LAG-3), programmed death 1 (PD-1), and V-domain immunoglobulin suppressor of T cell activation (VISTA) by tumor-infiltrating T cells (CD3) in a cohort of 69 patients with resected PDAC. T cells were enriched particularly within the stromal area and were highly heterogeneous across tumors. Further, T cells were associated with prolonged disease-free survival (DFS). However, LAG-3 expression by PDAC-infiltrating T cells was correlated with reduced DFS. Our study highlights the biological importance of LAG-3 expression by tumor-infiltrating T cells. LAG-3+ T cells may represent a novel prognostic marker and a particularly attractive target for immunotherapeutic strategies in PDAC.

Electrospun Scaffolds and Induced Pluripotent Stem Cell-Derived Cardiomyocytes for Cardiac Tissue Engineering Applications.

Tissue engineering (TE) combines cells, scaffolds, and growth factors to assemble functional tissues for repair or replacement of tissues and organs. Cardiac TE is focused on developing cardiac cells, tissues, and structures-most notably the heart. This review presents the requirements, challenges, and research surrounding electrospun scaffolds and induced pluripotent stem cell (iPSC)-derived cardiomyocytes (CMs) towards applications to TE hearts. Electrospinning is an attractive fabrication method for cardiac TE scaffolds because it produces fibers that demonstrate the optimal potential for mimicking the complex structure of the cardiac extracellular matrix (ECM). iPSCs theoretically offer the capacity to generate limitless numbers of CMs for use in TE hearts, however these iPSC-CMs are electrophysiologically, morphologically, mechanically, and metabolically immature compared to adult CMs. This presents a functional limitation to their use in cardiac TE, and research aiming to address this limitation is presented in this review.

Biochemical and biomechanical properties of the pacemaking sinoatrial node extracellular matrix are distinct from contractile left ventricular matrix.

Extracellular matrix plays a role in differentiation and phenotype development of its resident cells. Although cardiac extracellular matrix from the contractile tissues has been studied and utilized in tissue engineering, extracellular matrix properties of the pacemaking sinoatrial node are largely unknown. In this study, the biomechanical properties and biochemical composition and distribution of extracellular matrix in the sinoatrial node were investigated relative to the left ventricle. Extracellular matrix of the sinoatrial node was found to be overall stiffer than that of the left ventricle and highly heterogeneous with interstitial regions composed of predominantly fibrillar collagens and rich in elastin. The extracellular matrix protein distribution suggests that resident pacemaking cardiomyocytes are enclosed in fibrillar collagens that can withstand greater tensile strength while the surrounding elastin-rich regions may undergo deformation to reduce the mechanical strain in these cells. Moreover, basement membrane-associated adhesion proteins that are ligands for integrins were of low abundance in the sinoatrial node, which may decrease force transduction in the pacemaking cardiomyocytes. In contrast to extracellular matrix of the left ventricle, extracellular matrix of the sinoatrial node may reduce mechanical strain and force transduction in pacemaking cardiomyocytes. These findings provide the criteria for a suitable matrix scaffold for engineering biopacemakers.

Effect of fiber orientation of collagen-based electrospun meshes on human fibroblasts for ligament tissue engineering applications.

Within the past two decades polylactic-co-glycolic acid (PLGA) has gained considerable attention as a biocompatible and biodegradable polymer that is suitable for tissue engineering and regenerative medicine. In this present study, we have investigated the potential of PLGA, collagen I (ColI), and polyurethane (PU) scaffolds for ligament tissue regeneration. Two different ratios of PLGA (50:50 and 85:15) were used to determine the effects on mechanical tensile properties and cell adhesion. The Young's modulus, tensile stress at yield, and ultimate tensile strain of PLGA(50:50)-ColI-PU scaffolds demonstrated similar tensile properties to that of ligaments found in the knee. Whereas, scaffolds composed of PLGA(85:15)-ColI-PU had lower tensile properties than that of ligaments. Furthermore, we investigated the effect of fiber orientation on mechanical properties and our results indicate that aligned fiber scaffolds demonstrate higher tensile properties than scaffolds with random fiber orientation. Also, human fibroblasts attached and proliferated with no need for additional surface modifications to the presented electrospun scaffolds in both categories. Collectively, our investigation demonstrates the effectiveness of electrospun PLGA scaffolds as a suitable candidate for regenerative medicine, capable of being manipulated and combined with other polymers to create three-dimensional microenvironments with adjustable tensile properties to mimic native tissues.

Microenvironment influences vascular differentiation of murine cardiovascular progenitor cells.

We examined the effects of the microenvironment on vascular differentiation of murine cardiovascular progenitor cells (CPCs). We isolated CPCs and seeded them in culture exposed to the various extracellular matrix (ECM) proteins in both two-dimensional (2D) and 3D culture systems. To better understand the contribution of the microenvironment to vascular differentiation, we analyzed endothelial and smooth muscle cell differentiation at both day 7 and day 14. We found that laminin and vitronectin enhanced vascular endothelial cell differentiation while fibronectin enhanced vascular smooth muscle cell differentiation. We also observed that the effects of the 3D electrospun scaffolds were delayed and not noticeable until the later time point (day 14), which may be due to the amount of time necessary for the cells to migrate to the interior of the scaffold. The study characterized the contributions of both ECM proteins and the addition of a 3D culture system to continued vascular differentiation. Additionally, we demonstrated the capability bioengineer a CPC-derived vascular graft.

Hyaluronan-Based Three-Dimensional Microenvironment Potently Induces Cardiovascular Progenitor Cell Populations.

The relationship between stem cell niches in vivo and their surrounding microenvironment is still relatively unknown. Recent advances have indicated that extrinsic factors within the cardiovascular progenitor cell niche influence maintenance of a multipotent state as well as drive cell-fate decisions. We have previously shown the direct effects of extracellular matrix (ECM) proteins and have now investigated the effects of dimension on the induction of a cardiovascular progenitor cell (CPC) population. We have shown here that the three-dimensionality of a hyaluronan-based hydrogel greatly induces a CPC population, as marked by Flk-1. We have compared the effects of a 3D microenvironment to those of conventional 2D cell culture practices and have found that the 3D microenvironment potently induces a progenitor cell state.