Jed Johnson

Tissue-Engineered Small Diameter Arterial Vascular Grafts from Cell-Free Nanofiber PCL/Chitosan Scaffolds in a Sheep Model

Tissue engineered vascular grafts (TEVGs) have the potential to overcome the issues faced by existing small diameter prosthetic grafts by providing a biodegradable scaffold where the patient’s own cells can engraft and form functional neotissue. However, applying classical approaches to create arterial TEVGs using slow degrading materials with supraphysiological mechanical properties, typically results in limited host cell infiltration, poor remodeling, stenosis, and calcification. The purpose of this study is to evaluate the feasibility of novel small diameter arterial TEVGs created using fast degrading material. A 1.0mm and 5.0mm diameter TEVGs were fabricated with electrospun polycaprolactone (PCL) and chitosan (CS) blend nanofibers. The 1.0mm TEVGs were implanted in mice (n = 3) as an unseeded infrarenal abdominal aorta interposition conduit., The 5.0mm TEVGs were implanted in sheep (n = 6) as an unseeded carotid artery (CA) interposition conduit. Mice were followed with ultrasound and sacrificed at 6 months. All 1.0mm TEVGs remained patent without evidence of thrombosis or aneurysm formation. Based on small animal outcomes, sheep were followed with ultrasound and sacrificed at 6 months for histological and mechanical analysis. There was no aneurysm formation or calcification in the TEVGs. 4 out of 6 grafts (67%) were patent. After 6 months in vivo, 9.1 ± 5.4% remained of the original scaffold. Histological analysis of patent grafts demonstrated deposition of extracellular matrix constituents including elastin and collagen production, as well as endothelialization and organized contractile smooth muscle cells, similar to that of native CA. The mechanical properties of TEVGs were comparable to native CA. There was a significant positive correlation between TEVG wall thickness and CD68+ macrophage infiltration into the scaffold (R2 = 0.95, p = 0.001). The fast degradation of CS in our novel TEVG promoted excellent cellular infiltration and neotissue formation without calcification or aneurysm. Modulating host macrophage infiltration into the scaffold is a key to reducing excessive neotissue formation and stenosis.

Evaluation of Changes in Morphology and Function of Human Induced Pluripotent Stem Cell Derived Cardiomyocytes (HiPSC-CMs) Cultured on an Aligned-Nanofiber Cardiac Patch

Introduction Dilated cardiomyopathy is a major cause of progressive heart failure. Utilization of stem cell therapy offers a potential means of regenerating viable cardiac tissue. However, a major obstacle to stem cell therapy is the delivery and survival of implanted stem cells in the ischemic heart. To address this issue, we have developed a biomimetic aligned nanofibrous cardiac patch and characterized the alignment and function of human inducible pluripotent stem cell derived cardiomyocytes (hiPSC-CMs) cultured on this cardiac patch. This hiPSC-CMs seeded patch was compared with hiPSC-CMs cultured on standard flat cell culture plates. Methods hiPSC-CMs were cultured on; 1) a highly aligned polylactide-co-glycolide (PLGA) nanofiber scaffold (~50 microns thick) and 2) on a standard flat culture plate. Scanning electron microscopy (SEM) was used to determine alignment of PLGA nanofibers and orientation of the cells on the respective surfaces. Analysis of gap junctions (Connexin-43) was performed by confocal imaging in both the groups. Calcium cycling and patch-clamp technique were performed to measure calcium transients and electrical coupling properties of cardiomyocytes. Results SEM demonstrated >90% alignment of the nanofibers in the patch which is similar to the extracellular matrix of decellularized rat myocardium. Confocal imaging of the cardiomyocytes demonstrated symmetrical alignment in the same direction on the aligned nanofiber patch in sharp contrast to the random appearance of cardiomyocytes cultured on a tissue culture plate. The hiPSC-CMs cultured on aligned nanofiber cardiac patches showed more efficient calcium cycling compared with cells cultured on standard flat surface culture plates. Quantification of mRNA with qRT-PCR confirmed that these cardiomyocytes expressed α-actinin, troponin-T and connexin-43 in-vitro. Conclusions Overall, our results demonstrated changes in morphology and function of human induced pluripotent derived cardiomyocytes cultured in an anisotropic environment created by an aligned nanofiber patch. In this environment, these cells better approximate normal cardiac tissue compared with cells cultured on flat surface and can serve as the basis for bioengineering of an implantable cardiac patch.

Preclinical study of patient-specific cell-free nanofiber tissue-engineered vascular grafts using 3-dimensional printing in a sheep model

Background: Tissue‐engineered vascular grafts (TEVGs) offer potential to overcome limitations of current approaches for reconstruction in congenital heart disease by providing biodegradable scaffolds on which autologous cells proliferate and provide physiologic functionality. However, current TEVGs do not address the diverse anatomic requirements of individual patients. This study explores the feasibility of creating patient‐specific TEVGs by combining 3‐dimensional (3D) printing and electrospinning technology. Methods: An electrospinning mandrel was 3D‐printed after computer‐aided design based on preoperative imaging of the ovine thoracic inferior vena cava (IVC). TEVG scaffolds were then electrospun around the 3D‐printed mandrel. Six patient‐specific TEVGs were implanted as cell‐free IVC interposition conduits in a sheep model and explanted after 6 months for histologic, biochemical, and biomechanical evaluation. Results: All sheep survived without complications, and all grafts were patent without aneurysm formation or ectopic calcification. Serial angiography revealed significant decreases in TEVG pressure gradients between 3 and 6 months as the grafts remodeled. At explant, the nanofiber scaffold was nearly completely resorbed and the TEVG showed similar mechanical properties to that of native IVC. Histological analysis demonstrated an organized smooth muscle cell layer, extracellular matrix deposition, and endothelialization. No significant difference in elastin and collagen content between the TEVG and native IVC was identified. There was a significant positive correlation between wall thickness and CD68+ macrophage infiltration into the TEVG. Conclusions: Creation of patient‐specific nanofiber TEVGs by combining electrospinning and 3D printing is a feasible technology as future clinical option. Further preclinical studies involving more complex anatomical shapes are warranted.

Development of Novel, Bioresorbable, Small-Diameter Electrospun Vascular Grafts

This study proposes a production method capable of producing vascular grafts from fully synthetic, resorbable polymers that both meet basic minimum mechanical requirements for potential vascular grafts, and have a compliance similar to that of the intended vasculature being replaced. All of the electrospun vascular grafts in this work meet the minimum mechanical requirements for compliance, burst pressure, and suture retention strength, and could be potential candidates for off-the-shelf tissue engineered vascular grafts. Each polymer investigated in this paper has FDA approval for medical use and has been shown to be successful in various tissue engineering applications. Only recently has an electrospun small-diameter graft been fabricated with compliance and burst pressure greater than that of the human saphenous vein. We show a significant advancement in burst pressure, compliance, and suture retention strength in the novel electrospun grafts presented in this work which demonstrates the potential use of these tissue engineered vascular grafts for coronary artery bypass graft and other smalldiameter graft indications.

Full-thickness oesophageal regeneration in pig using a polyurethane mucosal cell seeded graft

Malignant oesophageal pathology typically requires resection of a portion of oesophagus. The aim of this study was to investigate attachment and growth of swine oesophageal mucosal cells on electrospun synthetic nanofibre matrices of varying chemistries and to determine whether a mucosal‐seeded graft, in a swine animal model, could induce regeneration. Swine mucosal oesophageal cells were isolated and seeded them onto five different matrix materials. Matrix samples were cultured for up to 14 days, after which matrices were analysed for cell attachment. Attachment varied for each of the matrix materials tested, with the most rigid showing the lowest levels of attachment. Importantly, sections of these matrices illustrated that multiple layers of mucosal cells formed, mimicking endogenous oesophageal structure. A tdTomato reporter line (mucosaltdt cells) was created to enable cell tracking. As polyurethane matrix was found optimal through in vitro testing, a graft was prepared using mucosaltdt cells, along with an unseeded control, and implanted into swine for determination of oesophageal regeneration. Mucosal seeded polyurethane grafts initiated full thickness regeneration of the oesophagus, including epithelial, submucosal, and skeletal muscle layers which were highly vascularized. Interestingly, an unseeded graft showed similar regeneration, indicating that the role of cells in the process of oesophageal regeneration is still unclear. The electrospun polyurethane matrix does appear suitable for multilayered cellular attachment and growth of oesophageal mucosal cells, and implantation of polyurethane grafts initiated full thickness regeneration of the oesophagus, indicating potential for oesophageal reconstruction in humans. Copyright

Virtual surgical planning, flow simulation, and 3-dimensional electrospinning of patient-specific grafts to optimize Fontan hemodynamics

Background: Despite advances in the Fontan procedure, there is an unmet clinical need for patient‐specific graft designs that are optimized for variations in patient anatomy. The objective of this study is to design and produce patient‐specific Fontan geometries, with the goal of improving hepatic flow distribution (HFD) and reducing power loss (Ploss), and manufacturing these designs by electrospinning. Methods: Cardiac magnetic resonance imaging data from patients who previously underwent a Fontan procedure (n = 2) was used to create 3‐dimensional models of their native Fontan geometry using standard image segmentation and geometry reconstruction software. For each patient, alternative designs were explored in silico, including tube‐shaped and bifurcated conduits, and their performance in terms of Ploss and HFD probed by computational fluid dynamic (CFD) simulations. The best‐performing options were then fabricated using electrospinning. Results: CFD simulations showed that the bifurcated conduit improved HFD between the left and right pulmonary arteries, whereas both types of conduits reduced Ploss. In vitro testing with a flow‐loop chamber supported the CFD results. The proposed designs were then successfully electrospun into tissue‐engineered vascular grafts. Conclusions: Our unique virtual cardiac surgery approach has the potential to improve the quality of surgery by manufacturing patient‐specific designs before surgery, that are also optimized with balanced HFD and minimal Ploss, based on refinement of commercially available options for image segmentation, computer‐aided design, and flow simulations.

Endoscopic management of tissue-engineered tracheal graft stenosis in an ovine model

To evaluate the safety and efficacy of bronchoscopic interventions in the management of tissue‐engineered tracheal graft (TETG) stenosis.

Quantification of tissue-engineered trachea performance with computational fluid dynamics: Computational Fluid Dynamics for Ovine TETG

Current techniques for airway characterization include endoscopic or radiographic measurements that produce static, two‐dimensional descriptions. As pathology can be multilevel, irregularly shaped, and dynamic, minimal luminal area (MLA) may not provide the most comprehensive description or diagnostic metric. Our aim was to examine the utilization of computational fluid dynamics (CFD) for the purpose of defining airway stenosis using an ovine model of tissue‐engineered tracheal graft (TETG) implantation.