Electrospun Tubular Scaffolds Research Articles

Polycaprolactone/Testicular Extracellular Matrix/Graphene Oxide-Based Electrospun Tubular Scaffolds for Reproductive Medicine: Biomimetic Architecture of Seminiferous Tubules.

Numerous scaffolds are developed in the field of testicular bioengineering. However, effectively replicating the spatial characteristics of native tissue, poses a challenge in maintaining the requisite cellular arrangement essential for spermatogenesis. In order to mimic the structural properties of seminiferous tubules, the objective is to fabricate a biocompatible tubular scaffold. Following the decellularization process of the testicular tissue, validation of cellular remnants' elimination from the specimens is conducted using 4',6-diamidino-2-phenylindole staining, hematoxylin and eosin staining, and DNA content analysis. The presence of extracellular matrix (ECM) components is confirmed through Alcian blue, Orcein, and Masson's trichrome staining techniques. The electrospinning technique is employed to synthesize the scaffolds using polycaprolactone (PCL), extracted ECM, and varying concentrations of graphene oxide (GO) (0.5%, 1%, and 2%). Subsequently, comprehensive evaluations are performed to assess the properties of the synthetic scaffolds. These evaluations encompass Fourier-transform infrared spectroscopy, scanning electron microscopy imaging, scaffold degradation testing, mechanical behavior analysis, methylthiazolyldiphenyl-tetrazolium bromide assay, and in vivo biocompatibility assessment. The PCL/decellularized extracellular matrix with 0.5% GO formulation exhibits superior fiber morphology and enhanced mechanical properties, and outperforms other groups in terms of in vitro biocompatibility. Consequently, these scaffolds present a viable option for implementation in "in vitro spermatogenesis" procedures, holding promise for future sperm production from spermatogonial cells.

Luminal endothelialization of small caliber silk tubular graft for vascular constructs engineering.

The constantly increasing incidence of coronary artery disease worldwide makes necessary to set advanced therapies and tools such as tissue engineered vessel grafts (TEVGs) to surpass the autologous grafts [(i.e., mammary and internal thoracic arteries, saphenous vein (SV)] currently employed in coronary artery and vascular surgery. To this aim, in vitro cellularization of artificial tubular scaffolds still holds a good potential to overcome the unresolved problem of vessel conduits availability and the issues resulting from thrombosis, intima hyperplasia and matrix remodeling, occurring in autologous grafts especially with small caliber (<6 mm). The employment of silk-based tubular scaffolds has been proposed as a promising approach to engineer small caliber cellularized vascular constructs. The advantage of the silk material is the excellent manufacturability and the easiness of fiber deposition, mechanical properties, low immunogenicity and the extremely high in vivo biocompatibility. In the present work, we propose a method to optimize coverage of the luminal surface of silk electrospun tubular scaffold with endothelial cells. Our strategy is based on seeding endothelial cells (ECs) on the luminal surface of the scaffolds using a low-speed rolling. We show that this procedure allows the formation of a nearly complete EC monolayer suitable for flow-dependent studies and vascular maturation, as a step toward derivation of complete vascular constructs for transplantation and disease modeling.

Engineered Full Thickness Electrospun Scaffold for Esophageal Tissue Regeneration: From In Vitro to In Vivo Approach.

Acquired congenital esophageal malformations, such as malignant esophageal cancer, require esophagectomy resulting in full thickness resection, which cannot be left untreated. The proposed approach is a polymeric full-thickness scaffold engineered with mesenchymal stem cells (MSCs) to promote and speed up the regeneration process, ensuring adequate support and esophageal tissue reconstruction and avoiding the use of autologous conduits. Copolymers poly-L-lactide-co-poly-ε-caprolactone (PLA-PCL) 70:30 and 85:15 ratio were chosen to prepare electrospun tubular scaffolds. Electrospinning apparatus equipped with two different types of tubular mandrels: cylindrical (∅ 10 mm) and asymmetrical (∅ 10 mm and ∅ 8 mm) were used. Tubular scaffolds underwent morphological, mechanical (uniaxial tensile stress) and biological (MTT and Dapi staining) characterization. Asymmetric tubular geometry resulted in the best properties and was selected for in vivo surgical implantation. Anesthetized pigs underwent full thickness circumferential resection of the mid-lower thoracic esophagus, followed by implantation of the asymmetric scaffold. Preliminary in vivo results demonstrated that detached stitch suture achieved better results in terms of animal welfare and scaffold integration; thus, it is to be preferred to continuous suture.

BFGF and SDF-1α Improve In Vivo Performance of VEGF-Incorporating Small-Diameter Vascular Grafts.

Tissue-engineered vascular grafts are widely tested as a promising substitute for both arterial bypass and replacement surgery. We previously demonstrated that incorporation of VEGF into electrospun tubular scaffolds from poly(3-hydroxybutyrate-co-3-hydroxyvalerate)/poly(ε-caprolactone) enhances formation of an endothelial cell monolayer. However, an overdose of VEGF can induce tumor-like vasculature; thereby, other bioactive factors are needed to support VEGF-driven endothelialization and successful recruitment of smooth muscle cells. Utilizing emulsion electrospinning, we fabricated one-layer vascular grafts with either VEGF, bFGF, or SDF-1α, and two-layer vascular grafts with VEGF incorporated into the inner layer and bFGF and SDF-1α incorporated into the outer layer with the following structural evaluation, tensile testing, and in vivo testing using a rat abdominal aorta replacement model. The latter graft prototype showed higher primary patency rate. We found that the two-layer structure improved surface topography and mechanical properties of the grafts. Further, the combination of bFGF, SDF-1α, and VEGF improved endothelialization compared with VEGF alone, while bFGF induced a rapid formation of a smooth muscle cell layer. Taken together, these findings show that the two-layer structure and incorporation of bFGF and SDF-1α into the vascular grafts in combination with VEGF provide a higher primary patency and therefore improved in vivo performance.

Gelatin-polytrimethylene carbonate blend based electrospun tubular construct as a potential vascular biomaterial.

The present work details the fabrication of electrospun tubular scaffolds based on the biocompatible and unexploited blend of gelatin and polytrimethylene carbonate (PTMC) as a media (middle layer of blood vessel) equivalent for blood vessel regeneration. An attempt to resemble the media stimulated the selection of gelatin as a matrix (substitution for collagen) with the inclusion of the biodegradable elastomer PTMC (substitution for elastin). -The work highlights the variation of electrospinning parameters and its assiduous selection based on fiber diameter distribution and pore size distribution to obtain smooth microfibers and micropores which is reported for the first time for this blend. Electrospun conduits of gelatin-PTMC blend had fibers sized 6-8 μm and pores sized ~100-150 μm. Young's modulus of 0.40 ± 0.045 MPa was observed, resembling the tunica media of the native artery (~0.5 MPa). An evaluation of the surface properties, topography, and mechanical properties validated its physical requirements for inclusion in a vascular graft. Preliminary biological tests confirmed its minimal in-vitro toxicity and in-vivo biocompatibility. MTT assay (indirect) elucidated cell viability above 70% with scaffold extract, considered to be non-toxic according to the EN ISO-10993-5/12 protocol. The in-vivo subcutaneous implantation in rat showed a marked reduction in macrophages within 15 days revealing its biocompatibility and its possibility for host integration. This comprehensive study presents for the first time the potential of microporous electrospun gelatin and PTMC blend based tubular construct as a potential biomaterial for vascular tissue engineering. The proposed media equivalent included in a bilayer or trilayer polymeric construct can be a promising off-shelf vascular graft.

Multiscale constitutive model with progressive recruitment for nanofibrous scaffolds

Biomedical applications need tailor-made scaffolds that exhibit biomimetic mechanical properties. In this context, electrospinning has emerged as a technique with promising features for their production. However, the electrospun scaffolds mechanical behavior as a function of the microstructure and nanofiber properties is still poorly understood. Besides, multiscale constitutive modeling appears as a powerful design tool, not only able to characterize electrospun structures, but also to determine the fiber properties and scaffold microstructure that would achieve the objective response. With focus in this last aspect, we developed a multiscale constitutive model for nanofibrous structures that takes into account the material constitutive properties, scaffold microstructure, and nanofiber progressive recruitment. A statistical approach of the nanofibers tortuosity with a modified Gaussian distribution was adopted, which allowed for reproducing the scaffolds macroscopic nonlinear mechanical behavior. It was observed that such behavior arises even if the nanofibers response is considered as mechanically linear. Experimental data from pressure vs. diameter inflation tests of electrospun tubular scaffolds was used to validate the model. In addition, the influence of the microstructural parameters upon the macroscopic constitutive behavior was studied. Finally, the model parameters were adjusted to obtain a vascular graft able to reproduce the mechanical response of a target natural tissue. The current study presents a step towards understanding, characterizing, and optimizing the mechanical properties of nanofibrous biomaterials.

Embryonic Mesenchymal Multipotent Cell Differentiation on Electrospun Biodegradable Poly(ester amide) Scaffolds for Model Vascular Tissue Fabrication.

Vascular differentiation of stem cells and matrix component production on electrospun tubular scaffolds is desirable to engineer blood vessels. The mouse embryonic multipotent mesenchymal progenitor cell line (10T1/2) provides an excellent tool for tissue engineering since it shares similar differentiation characteristics with mesenchymal stem cells. Although 10T1/2 cells have been widely studied in the context of skeletal tissue engineering, their differentiation to smooth muscle lineage is less known. In this study, we fabricated tubular electrospun poly(ester amide) (PEA) fibers from L-phenylalanine-derived biodegradable biomaterials and investigated cell-scaffold interactions as well as their differentiation into vascular smooth muscle cell and subsequent elastin expression. PEA scaffolds fabricated under different collector speeds did not have an impact on the fiber directionality/orientation. 10T1/2 cytocompatibility and proliferation studies showed that PEA fibres were not cytotoxic and were able to support proliferation for 14days. Furthermore, cells were observed infiltrating the fibrous scaffolds despite the small pore sizes (~ 5µm). Vascular differentiation studiesof 10T1/2 cells using qPCR, Western blot, and immunostaining showed a TGFβ1-induced upregulation of vascular smooth muscle cell (VSMC)-specific markers smooth muscle alpha-actin (SM-α-actin) and smooth muscle myosin heavy chain (SM-MHC). Differentiated 10T1/2 cells produced both elastin and fibrillin-1 suggesting the potential of fibrous PEA scaffolds to fabricate model vascular tissues.

Electrospun Tubular Scaffold for Stenting Application: A Proof of Concept

Bioresorbable Stents (BRS) offer enormous potential, however they must accomplish some specific requirements such as: (I) their manufacturing process must be precise; (II) degradation should have minimal toxicity; (III) their degradation rate should match the recovery rate of vascular tissue; (IV) ideally, they should induce rapid endothelialization to restore the functions of vascular tissue, but at the same time reduce the risk of restenosis; and (V) their mechanical behavior should comply with the medical requirements. With the recent inclusion of polymeric materials and additive manufacturing technologies, a new idea makes its way, Electrospun Tubular Scaffolds (ETS) for stenting process. Unlike current stents, ETS can present some advances (I) Longitudinal flexibility to help the placement of the stent, (II) Material matrix that mimic body tissue to help the rapid endothelization. The present work aims to study the feasibility of this new idea by analyzing how the electrospinning (ES) parameters affect the ETS properties. ETS were fabricated with a Rotational Electrospinning Machine. Poly (ε-caprolactone) (PCL) and Acetone were chosen as biopolymer and non-toxic solvent respectively. Electrospinning Voltage (Ev), Flow Rate (Fr), Polymer Concentration (PCL%), and Target Volume (Tv) were analyzed on the final ETS properties, namely, ETS Thickness (TK), ETS Maxim Radial Expansion (Mø), ETS Recoil Ratio (Rø). ETS thickness were measured by micrometer. Finally ETS radial behavior was studied by Radial Tests. Target Volume proved to be one of the most important parameter to obtain different ETS thickness, while Flow Rate and PCL Concentration has been the most important parameters to obtain a uniform ETS both longitudinally as radially. Regarding the radial behavior, ETS has proved their dependence of the fiber diameter obtained during the ES process. ETS have been able to expand radially a maximum of 67% with an average recoil ratio of 20% which left the ETS expand at 47%. These results allow us to conclude that ETS for stenting applications could be a possible solution for the current BRS problems.

Gelatin — Oxidized carboxymethyl cellulose blend based tubular electrospun scaffold for vascular tissue engineering

The present work deals with the fabrication of electrospun tubular scaffold based on in-situ crosslinked blend of gelatin − oxidized carboxymethyl cellulose (OCMC) for vascular tissue engineering. The flow behavior and spinability of the hydrogel despite the in-situ crosslinked gelatin chains evaluated by Raman spectroscopic studies and rheological studies was utilized for electrospinning. The study highlights the tunable pore size and fiber diameter of the nanofibers with the manipulation of electrospinning parameters. With a future perspective of vascular tissue engineering, the electrospinning parameters yielding smooth bead free fibers and maximum magnitude in pore size and fiber diameter as well their homogenous distribution were selected for the fabrication of tubular constructs which is rarely reported. The surface and mechanical properties were evaluated to validate its properties to the native vessel. Biocompatibility was studied in vitro with BALB/c 3T3 cells and in vivo after subcutaneous implantation in rats. MTT assay confirmed its no-toxicity and no abnormal foreign body reaction were observed by 7 and 15days after implantation. Crosslinking with biocompatible crosslinker OCMC has rendered insolubility to gelatin yet making it spinable for electrospinning to fabricate porous, nanofibrous vascular biomaterial.

Development of an �off-the-shelf� implantable and absorbable electrospun polycaprolactone-chitosan (PCL-CS) device to regenerate damaged esophageal tissue

Event Abstract Back to Event Development of an “off-the-shelf” implantable and absorbable electrospun polycaprolactone-chitosan (PCL-CS) device to regenerate damaged esophageal tissue Derek C. Dashti1, 2, Jed Johnson3 and Aline M. Betancourt4, 5 1 Tulane University, Bioinnovation (IGERT) Program, United States 2 D&P Bioinnovations, LLC, United States 3 Nanofiber Solutions, Inc., United States 4 Tulane University, Department of Medicine, Tulane Center for Stem Cell Research & Regenerative Medicine, United States 5 Commence Bio, Inc., United States Introduction: A severely impaired esophagus requires a largely invasive standard of care surgery known as an esophagectomy, whereby the surgeon removes the affected tissue of the esophagus and splices the stomach or intestine on to the remaining tissue [1]. This results in high morbidity rates and a low quality of life [2]-[5]. Our solution for esophageal resection, is to develop a decellularized synthetic electrospun tubular scaffold from absorbable polymers such as polycaprolactone (PCL) and chitosan (CS) loaded with established therapeutic factors from anti-inflammatory allogeneic bone marrow derived human mesenchymal stem cells (MSC2s) [6] as an “off-the shelf” implantable device that can robustly regenerate a damaged portion of an esophagus. Materials and Methods: 1) Electrospinning: Prepare a concentration solution of CS:PCL at 10% (w/v). Dispense the solution onto a rotating stainless steel rod through a 20 gauge blunt needle. Sterilize the tubular scaffold in 70% ethanol for 30 minutes. Wash 3X with sterile deionized water and freeze-dry. 2) Cell Culture of MSCs on PCL-CS: Seed the PCL-CS scaffold with MSCs at 600,000/cm2 in a sterile polypropylene cell culture tube. Culture MSCs on the PCL-CS scaffold for 2-3 days in the polypropylene tube within a humidified incubator at 37oC and 5% CO2. 3) Freeze/thaw Decellularization: Freeze the MSC seeded PCL-CS construct in deionized water at -80oC overnight. Afterwards, thaw the construct at room temperature and immediately place the construct in liquid nitrogen for 10 minutes followed by an immediate immersion in a 37oC water bath for 10 minutes. Repeat 2X. 4) Scanning Electron Microscopy (SEM): Fix samples with 10% formalin and then dehydrate in a graded ethanol (EtOH) series. Mount on SEM and sputter coat. 5) DAPI Staining: Fix samples with 10% formalin and incubate in a 30 uM DAPI solution for 10 minutes at room temperature. Wash samples with phosphate buffer saline (PBS) and record fluorescent images. Results and Discussion: An effective improved approach with the device is to utilize the MSC2’s potent and predictive anti-inflammatory therapeutic factors to provide robust wound healing of a damaged esophagus. In order to obtain the necessary secreted factors on the PCL-CS scaffold, first MSCs need to viably adhere and grow on the material [Figure 1]. The PCL-CS MSC2 seeded construct is decellularized to develop an implantable “off-the-shelf” absorbable device for potential use by thoracic surgeons to repair a damaged esophagus. To avoid residual detergents that are traditionally used to decellularize tissues, a freeze/thaw decellularization strategy is suggested and assessed here [Figure 2]. Overall the freeze/thaw method established is properly decellularizing the PCL-CS construct. Conclusion: MSCs can be viably seeded on a tubular PCL-CS scaffold and subsequently this construct can be decellularized. Electrospinning the PCL-CS tube allows for a specific biomechanical design to not only allow MSC2s to properly adhere, but potentially allow native esophageal precursor cells to infiltrate the scaffold and robustly regenerate the esophagus [7],[8]. The freeze/thaw decellularization method provides an ideal translational medical device that can be packaged in saline solution for up to 1 year. This research initiates a path in developing a synthetic construct to be used by a thoracic surgeon as a one time use device implant to robustly regenerate an impaired esophagus. Funding was provided by the National Institutes of Health 1P20RR20152-01, 1R43AR061902, Department of Defense OC073102 Concept Award, National Science Foundation IGERT Award 1144646. Research support from the Tulane Cancer Center and the Center for Stem Cell Research and Regenerative Medicine, and Histogen, Inc.

Constitutive modeling of an electrospun tubular scaffold used for vascular tissue engineering.

In this study, we sought to model the mechanical behavior of an electrospun tubular scaffold previously reported for vascular tissue engineering with hyperelastic constitutive equations. Specifically, the scaffolds were made by wrapping electrospun polycaprolactone membranes that contain aligned fibers around a mandrel in such a way that they have microstructure similar to the native arterial media. The biaxial stress-stretch data of the scaffolds made of moderately or highly aligned fibers with three different off-axis fiber angles α (30°, 45°, and 60°) were fit by a phenomenological Fung model and a series of structurally motivated models considering fiber directions and fiber angle distributions. In particular, two forms of fiber strain energy in the structurally motivated model for a linear and a nonlinear fiber stress-strain relation, respectively, were tested. An isotropic neo-Hookean strain energy function was also added to the structurally motivated models to examine its contribution. The two forms of fiber strain energy did not result in significantly different goodness of fit for most groups of the scaffolds. The absence of the neo-Hookean term in the structurally motivated model led to obvious nonlinear stress-stretch fits at a greater axial stretch, especially when fitting data from the scaffolds with a small α. Of the models considered, the Fung model had the overall best fitting results; its applications are limited because of its phenomenological nature. Although a structurally motivated model using the nonlinear fiber stress-strain relation with the neo-Hookean term provided fits comparably as good as the Fung model, the values of its model parameters exhibited large within-group variations. Prescribing the dispersion of fiber orientation in the structurally motivated model, however, reduced the variations without compromising the fits and was thus considered to be the best structurally motivated model for the scaffolds. It appeared that the structurally motivated models could be further improved for fitting the mechanical behavior of the electrospun scaffold; fiber interactions are suggested to be considered in future models.

Electrospun Tubular Scaffold with Circumferentially Aligned Nanofibers for Regulating Smooth Muscle Cell Growth

Simulation for the smooth muscle layer of blood vessel plays a key role in tubular tissue engineering. However, fabrication of biocompatible tube with defined inner nano/micro-structure remains a challenge. Here, we show that a biocompatible polymer tube from poly(l-lactide) (PLLA) and polydimethylsiloxane (PDMS) can be prepared by using electrospinning technique, with assistance of rotating collector and parallel auxiliary electrode. The tube has circumferentially aligned PLLA fibers in the inner surface for cell growth regulation and has a PDMS coating for better compressive property. MTT assay showed the composite PLLA/PDMS tube was suitable for various cells growth. In vitro smooth muscle cells (SMCs) cultured in the tube showed that the aligned PLLA fibers could induce SMCs' orientation, and different expression of α-SMA and OPN genes were observed on the aligned and random PLLA fibers, respectively. The successful fabrication of composite PLLA/PDMS tubular scaffold for regulating smooth muscle cells outgrowth has important implications for tissue-engineered blood vessels.

Aspirin-loaded electrospun poly(ε-caprolactone) tubular scaffolds: potential small-diameter vascular grafts for thrombosis prevention

Thrombosis is the main cause of failure of small-diameter synthetic vascular grafts when used for by-pass procedures. The development of bioresorbable vascular scaffolds with localized and sustained intra-luminal antithrombotic drug release could be considered a desirable improvement towards a valuable solution for this relevant clinical need. For this aim, we present the fabrication and characterization of aspirin-loaded electrospun poly(ε-caprolactone) tubular scaffolds as a vascular drug-delivery graft. Three different drug concentrations were considered (i.e., 1, 5 or 10 % w/w). Although a fibrous structure was clearly observed for all the collected scaffolds, aspirin content was directly implied in the final microstructure leading to a bimodal fiber diameter distribution and fused fibers at crossing-points (5 or 10 % w/w). Mechanical response highlighted a direct relationship for modulus and stress at break with the aspirin content, while the elongation at break was not remarkably different for the investigated cases. The temporal drug release was strongly dependent from the amount of loaded aspirin, reaching a steady state release after about 50 h. Finally, the adhesion assay confirmed the capability of the electrospun scaffolds to reduce platelet adhesion/aggregation onto aspirin loaded polymeric fibers. Aspirin-loaded electrospun tubular scaffold could represent a feasible candidate to develop a novel bioresorbable drug-releasing graft for small-diameter vessel replacements.

Electrospun tubular scaffolds: On the effectiveness of blending poly(ε‐caprolactone) with poly(3‐hydroxybutyrate‐co‐3‐hydroxyvalerate)

Tissue engineering can effectively contribute to the development of novel vascular prostheses aimed to overcome the well-known drawbacks of small-diameter grafts. To date, poly(ε-caprolactone) (PCL), a bioresorbable synthetic poly(α-hydroxyester), is considered one of the most promising materials for vascular tissue engineering. In this work, the potential advantage of intimate blending soft PCL and hard poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV), a polymer of microbial origin, has been evaluated. Nonwoven mats and small-diameter tubular scaffolds of PCL, PHBV, and PCL/PHBV were fabricated by means of electrospinning technique. Mechanical properties and suture retention strength were investigated according to the international standard for cardiovascular implants. Biological tests demonstrated that both PCL-based scaffolds supported survival and growth of rat cerebral endothelial cells in a short time. The fiber alignment of the electrospun tubular scaffolds contributed to a more rapid and homogeneous cell colonization of the luminal surface.

Construction and characterization of an electrospun tubular scaffold for small-diameter tissue-engineered vascular grafts: A scaffold membrane approach

Based on a postulate that the microstructure of a scaffold can influence that of the resulting tissue and hence its mechanical behavior, we fabricated a small-diameter tubular scaffold (∼3mm inner diameter) that has a microstructure similar to the arterial media using a scaffold membrane approach. Scaffold membranes that contain randomly oriented, moderately aligned, or highly aligned fibers were fabricated by collecting electrospun poly([epsilon]-caprolactone) fibers on a grounded rotating drum at three different drum rotation speeds (250, 1000, and 1500rpm). Membranes of each type were wrapped around a small-diameter mandrel to form the tubular scaffolds. Particularly, the tubular scaffolds with three different off-axis fiber angles (30, 45, and 60 degree) were formed using membranes that contain aligned fibers. These scaffolds were subjected to biaxial mechanical testing to examine the effects of fiber directions as well as the distribution of fiber orientations on their mechanical properties. The circumferential elastic modulus of the tubular scaffold was closely related to the fiber directions; the larger the off-axis fiber angle the greater the circumferential elastic modulus. The distribution of fiber orientations, on the other hand, manifested itself in the mechanical behavior via the Poisson effect. Similar to cell sheet-based vascular tissue engineering, tubular cell-seeded constructs were prepared by wrapping cell-seeded scaffold membranes, alleviating the difficulty associated with cell seeding in electrospun scaffolds. Histology of the construct illustrated that cells were aligned to the fiber directions in the construct, demonstrating the potential to control the microstructure of tissue-engineered vascular grafts using the electrospun scaffold membrane.

Hierarchically Designed Electrospun Tubular Scaffolds for Cardiovascular Applications

Hierarchically designed tubular scaffolds with bi-layer and multi-layer structures are expected to mimic native vessels in its structural geometry. A new approach for the fabrication of hierarchically designed tubular scaffold with suitable morphology was introduced through electrospinning technique. Among these scaffolds, bi-layer scaffold had a single inner and outer layer whereas multilayer scaffold had more number of inner layers. The inner layer/layers of the scaffolds were made up of aligned poly (lactic acid) (PLA) fibers for EC adhesion where as outer layers were composed of random fibers of poly (caprolactone) (PCL) and PLA providing larger pores for SMC penetration. The fabricated scaffolds were characterized by FTIR spectroscopy and Differential Thermal Analysis (DTA) and examined by evaluating cellular interactions. Human Umbilical Vein Endothelial Cells (HUVECs) seeded on aligned PLA fibers showed enhanced cellular orientation and cytoskeletal organization. In addition, the PCL-PLA composite random fibers supported SMC adhesion and proliferation sufficiently. The functionality of the endothelial cells grown on the PLA-aligned scaffold was also found to be satisfactory. Lining the constructs with a luminal monolayer of well-organized ECs along with homogenously distributed SMCs surrounding them might result in vascular conduits suitable for in vivo applications. Since this hierarchically designed tubular scaffold closely mimics the morphology of native vessel, this could be a better candidate for vascular tissue engineering.

Dynamic culture conditions to generate silk-based tissue-engineered vascular grafts

Tissue engineering is an alternative approach for the preparation of small-diameter (<6 mm) vascular grafts due to the potential to control thrombosis, anastomotic cellular hyperplasia and matrix production. This control also requires the maintenance of graft patency in vivo, appropriate mechanical properties and the formation of a functional endothelium. As a first step in generating such tissue-engineered vascular grafts (TEVGs), our objective was to develop a tissue-engineered construct that mimicked the structure of blood vessels using tubular electrospun silk fibroin scaffolds (ESFSs) with suitable mechanical properties. Human coronary artery smooth muscle cells (HCASMCs) and human aortic endothelial cells (HAECs) were sequentially seeded onto the luminal surface of the tubular scaffolds and cultivated under physiological pulsatile flow. The results demonstrated that TEVGs under dynamic flow conditions had better outcome than static culture controls in terms of cell proliferation and alignment, ECM production and cell phenotype based on transcript and protein level assessments. The metabolic activity of HCASMCs present in the TEGs indicated the advantage of dynamic flow over static culture in effective nutrient and oxygen distribution to the cells. A matrigel coating as a basement membrane mimic for ECM significantly improved endothelium coverage and retention under physiological shear forces. The results demonstrate the successful integration of vascular cells into silk electrospun tubular scaffolds as a step toward the development of a TEVG similar to native vessels in terms of vascular cell outcomes and mechanical properties.

Silk-based electrospun tubular scaffolds for tissue-engineered vascular grafts

Electrospinning was used to fabricate non-woven nanofibrous tubular scaffolds from Bombyx mori silk fibroin using an all aqueous process. Cell studies and mechanical characterization tests were performed on the electrospun silk tubes to assess the viability of their usage in bioengineering small-diameter vascular grafts. Human endothelial cells and smooth muscle cells were successfully cultured on the electrospun silk. Mechanical characterization tests demonstrated burst strength sufficient to withstand arterial pressures and tensile properties comparable to native vessels. These cellular and mechanics outcomes demonstrate potential utility of these electrospun silk scaffolds for small-diameter vascular grafts.

Functionally graded electrospun scaffolds with tunable mechanical properties for vascular tissue regeneration

Electrospun tubular scaffolds (4 mm inner diameter) based on bio-artificial blends of polyglyconate (Maxon®) and proteins such as gelatin and elastin having a spatially designed multilayer structure were prepared for use as vascular tissue scaffolds. Scanning electron microscopy analysis of scaffolds showed a random nanofibrous morphology with fiber diameter in the range of 200–400 nm for protein-blended Maxon, which mimics the nanoscale dimensions of collagen (50–500 nm). The scaffolds have a well interconnected pore structure and porosity up to 82%, with protein blending and multi-layering in contrast to electrospun Maxon® scaffolds (67%). Fourier-transform infrared spectroscopy, x-ray diffraction and differential scanning calorimetry results confirmed the blended composition and crystallinity of fibers. Uniaxial tensile testing revealed a strength of 14.46 ± 0.42 MPa and a modulus of 15.44 ± 2.53 MPa with a failure strain of 322.5 ± 10% for a pure Maxon® scaffold. The blending of polyglyconate with biopolymers decreased the tensile properties in general, with an exception of the tensile modulus (48.38 ± 2 MPa) of gelatin/Maxon mesh, which was higher than that of the pure Maxon® scaffold. Trilayered tubular scaffolds of gelatin/elastin, gelatin/elastin/Maxon and gelatin/Maxon (GE-GEM-GM) that mimic the complex trilayer matrix structure of natural artery have been prepared by sequential electrospinning. Tensile testing under dry conditions revealed a tensile strength of 2.71 ± 0.2 MPa and a modulus of 20.4 ± 3 MPa with a failure strain of 140 ± 10%. However, GE-GEM-GM scaffolds tested under wet conditions after soaking in a phosphate buffered saline medium at 37 °C for 24 h exhibited mechanical properties (2.5 MPa tensile strength and 9 MPa tensile modulus) comparable to those of native femoral artery.

Engineering of Vascular Grafts With Genetically Modified Bone Marrow Mesenchymal Stem Cells on Poly (Propylene Carbonate) Graft

Bone marrow mesenchymal stem cells (MSCs) have demonstrated their pluripotency to differentiate into different cell lineages and may be an alternative cell source for vascular tissue engineering. The objective of this study is to create small diameter vessels by seeding and culture of genetically modified MSCs onto a synthetic polymer scaffold produced by an electrospinning technique. A tubular scaffold (2 mm in diameter) with a microstructure of nonwoven fibers was produced by electrospinning of poly (propylene carbonate) (PPC). Rat MSCs obtained from bone marrow were expanded in culture and modified with vasculoprotective gene endothelial nitric oxide synthase (eNOS) or marker gene green fluorescent protein (GFP). These MSCs were seeded onto the electrospun fibrous grafts (internal diameter = 2 mm), and cultured in 5% CO(2) at 37 degrees C. The growth of MSCs in the scaffold was analyzed with scanning electron microscopy (SEM) and hematoxylin and eosin (H&E) staining. The gene transfer and transgenic gene expression were examined with fluorescence-activated cell sorting (FACS), immunochemical staining, reverse transcriptase-polymerase chain reaction (RT-PCR), and western blot. The production of nitric oxide (NO) by the engineered vessels was measured with an NO detection kit. Our data showed that the seeded cells integrated with the microfibers of the scaffold to form a three-dimensional cellular network, indicating a favorable interaction between this synthetic PPC scaffold with MSCs. High transduction efficiency was obtained with the use of concentrated retrovirus in the gene transfection of MSCs. The eNOS gene transcripts and protein were detected in the grafts seeded with eNOS-modified MSCs by RT-PCR and immunochemical staining. The amount of NO produced by grafts seeded with eNOS-modified MSCs was comparable to that produced by native blood vessels, and it was significantly higher than that in the grafts seeded with nonmodified MSCs. In summary, the vascular graft produced by culture of eNOS gene-modified MSCs onto the electrospun tubular scaffolds shows promising results in terms of function. The use of MSCs and therapeutic genes in tissue engineering of blood vessels could be helpful in improving vessel regeneration and patency.