Despite significant advances in the fabrication of bioengineered scaffolds for tissue engineering, delivery of nutrients in complex engineered human tissues remains a challenge. By taking advantage of the similarities in the vascular structure of plant and animal tissues, we developed decellularized plant tissue as a prevascularized scaffold for tissue engineering applications. Perfusion-based decellularization was modified for different plant species, providing different geometries of scaffolding. After decellularization, plant scaffolds remained patent and able to transport microparticles. Plant scaffolds were recellularized with human endothelial cells that colonized the inner surfaces of plant vasculature. Human mesenchymal stem cells and human pluripotent stem cell derived cardiomyocytes adhered to the outer surfaces of plant scaffolds. Cardiomyocytes demonstrated contractile function and calcium handling capabilities over the course of 21 days. These data demonstrate the potential of decellularized plants as scaffolds for tissue engineering, which could ultimately provide a cost-efficient, “green” technology for regenerating large volume vascularized tissue mass.
Large-scale musculoskeletal wounds, such as those seen in trauma injuries, present poor functional healing prognoses. In severe trauma, when the native tissue architecture is destroyed or lost, the regenerative capacity of skeletal muscle is diminished by scar formation. Here we demonstrate that a scaffold system composed of fibrin microthreads can provide an efficient delivery system for cell-based therapies and improve regeneration of a large defect in the tibialis anterior of the mouse. Cell-loaded fibrin microthread bundles implanted into a skeletal muscle resection reduced the overall fibroplasia-associated deposition of collagen in the wound bed and promoted in-growth of new muscle tissue. When fibrin microthreads were seeded with adult human cells, implanted cells contributed to the nascent host tissue architecture by forming skeletal muscle fibers, connective tissue, and PAX7-positive cells. Stable engraftment was observed at 10 weeks postimplant and was accompanied by reduced levels of collagen deposition. Taken together, these data support the design and development of a platform for microthread-based delivery of autologous cells that, when coupled to an in vitro cellular reprogramming process, has the potential to improve healing outcomes in large skeletal muscle wounds.
There is a critical need to engineer a neotrachea because currently there are no long-term treatments for tracheal stenoses affecting large portions of the airway. In this work, a modular tracheal tissue replacement strategy was developed. High-cell density, scaffold-free human mesenchymal stem cell-derived cartilaginous rings and tubes were successfully generated through employment of custom designed culture wells and a ring-to-tube assembly system. Furthermore, incorporation of transforming growth factor-β1-delivering gelatin microspheres into the engineered tissues enhanced chondrogenesis with regard to tissue size and matrix production and distribution in the ring- and tube-shaped constructs, as well as luminal rigidity of the tubes. Importantly, all engineered tissues had similar or improved biomechanical properties compared to rat tracheas, which suggests they could be transplanted in a small animal model for airway defects. The modular, bottom up approach used to grow stem cell-based cartilaginous tubes in this report is a promising platform to engineer complex organs (e.g., trachea), with control over tissue size and geometry, and has the potential to be used to generate autologous tissue implants for human clinical applications.
The goal of this study was to develop a system to rapidly generate engineered tissue constructs from aggregated cells and cell-derived extracellular matrix (ECM) to enable evaluation of cell-derived tissue structure and function. Rat aortic smooth muscle cells seeded into annular agarose wells (2, 4 or 6 mm inside diameter) aggregated and formed thick tissue rings within 2 weeks of static culture (0.76 mm at 8 days; 0.94 mm at 14 days). Overall, cells appeared healthy and surrounded by ECM comprised of glycosoaminoglycans and collagen, although signs of necrosis were observed near the centers of the thickest rings. Tissue ring strength and stiffness values were superior to those reported for engineered tissue constructs cultured for comparable times. The strength (100–500 kPa) and modulus (0.5–2 MPa) of tissue rings increased with ring size and decreased with culture duration. Finally, tissue rings cultured for 7 days on silicone mandrels fused to form tubular constructs. Ring margins were visible after 7 days, but tubes were cohesive and mechanically stable, and histological examination confirmed fusion between ring subunits. This unique system provides a versatile new tool for optimization and functional assessment of cell-derived tissue, and a new approach to creating tissue-engineered vascular grafts.
SummaryThere is an urgent need for an efficient approach to obtain a large-scale and renewable source of functional human vascular smooth muscle cells (VSMCs) to establish robust, patient-specific tissue model systems for studying the pathogenesis of vascular disease, and for developing novel therapeutic interventions. Here, we have derived a large quantity of highly enriched functional VSMCs from human induced pluripotent stem cells (hiPSC-VSMCs). Furthermore, we have engineered 3D tissue rings from hiPSC-VSMCs using a facile one-step cellular self-assembly approach. The tissue rings are mechanically robust and can be used for vascular tissue engineering and disease modeling of supravalvular aortic stenosis syndrome. Our method may serve as a model system, extendable to study other vascular proliferative diseases for drug screening. Thus, this report describes an exciting platform technology with broad utility for manufacturing cell-based tissues and materials for various biomedical applications.
Over 6.5million people annually in the United States suffer chronic wounds; many will become infected with antibiotic-resistant bacteria. Treatments used to prevent and fight infection are toxic and may hinder wound healing. AMPs are broad-spectrum antimicrobials that also promote healing; however, their instability and toxicity are major challenges. To overcome treatment gaps, we functionalized collagen scaffolds with chimeric antimicrobial peptides (AMPs) with collagen binding domains to create antimicrobial and non-cytotoxic scaffolds that may promote healing. This is the first report of CBD-mediated delivery of AMPs onto collagen scaffolds that demonstrates no cytotoxicity toward fibroblasts. This study also suggests that retention of antimicrobial activity is CBD-dependent, which provides foundations for fundamental studies of CBD-AMP mechanisms and clinical explorations.
Each year, hundreds of thousands of patients undergo coronary artery bypass surgery in the United States.1 Approximately one third of these patients do not have suitable autologous donor vessels due to disease progression or previous harvest. The aim of vascular tissue engineering is to develop a suitable alternative source for these bypass grafts. In addition, engineered vascular tissue may prove valuable as living vascular models to study cardiovascular diseases. Several promising approaches to engineering blood vessels have been explored, with many recent studies focusing on development and analysis of cell-based methods. [2][3][4][5] Herein, we present a method to rapidly self-assemble cells into 3Dtissue rings that can be used in vitro to model vascular tissues.To do this, suspensions of smooth muscle cells are seeded into round-bottomed annular agarose wells. The non-adhesive properties of the agarose allow the cells to settle, aggregate and contract around a post at the center of the well to form a cohesive tissue ring. 6,7 These rings can be cultured for several days prior to harvesting for mechanical, physiological, biochemical, or histological analysis. We have shown that these cell-derived tissue rings yield at 100-500 kPa ultimate tensile strength 8 which exceeds the value reported for other tissue engineered vascular constructs cultured for similar durations (<30 kPa). 9,10 Our results demonstrate that robust cell-derived vascular tissue ring generation can be achieved within a short time period, and offers the opportunity for direct and quantitative assessment of the contributions of cells and cell-derived matrix (CDM) to vascular tissue structure and function. Video LinkThe video component of this article can be found at https://www.jove.com/video/3366/ Protocol Cell seeding mold fabricationBegin by milling a 1/2" thick piece of polycarbonate to create 15, round-bottomed, annular wells with a center post diameter of 2 mm. The milled channels are 6 mm deep and 3.75 mm wide. Clean and dry the polycarbonate mold to remove any plastic debris from the milling process.Mix polydimethylsiloxane (PDMS) at a 10:1 ratio (w/w) of base to curing agent, degas to remove all air bubbles, and pour onto the polycarbonate mold. Degas again to remove any remaining bubbles, and cure in the oven at 60°C for 4 hours.Once cured, carefully remove the PDMS template by slowly peeling it away from the polycarbonate, wash with soap and water, and autoclave. Also autoclave a solution of two percent agarose (w/v) dissolved in Dulbecco's modified Eagle medium (DMEM).Place the PDMS template on a level surface and fill with molten agarose by first pipetting agarose into each of the center post molds, and then pipetting into the space around it. Allow the agarose to solidify (approximately 15 minutes), then invert the mold to release the agarose from the PDMS. Cut excess agarose from around each of the wells, and place the agarose wells into 12-well plates. Cell culture and ring seedingAdd cell culture media (DMEM with 10% ...
Myocardial infarction results in the loss of irreplaceable contractile elements. Necrotic tissue is removed by macrophages and replaced with granulation tissue, which is eventually transformed into a collagenous scar. During this process the infarcted wall thins, the left ventricular (LV) chamber dilates, and there is interstitial fibrosis and cardiomyocyte hypertrophy in the noninfarcted region of the ventricle. These changes are associated with cardiac dysfunction and progression to heart failure. Based on the observation that late reperfusion (too late to salvage myocardium) both increases the rate of wound healing and attenuates ventricular dilation, 1 we have proposed that identifying growth factors involved in infarct repair could provide additional targets to prevent ventricular dysfunction. 2 We previously studied baseline variables of repair in a murine model of myocardial infarction induced by permanent coronary artery ligation. We found that fibroblast and endothelial cell proliferation peak at 4 days, that vascular density decreases as the infarct heals (although the total vessel area remains constant), and infarct scars contract to ϳ50% of the original infarct size. 2These baseline measurements provide a means to understand the factors that govern the process of infarct repair and for investigating therapeutic molecules that promote healing.Fibroblast growth factor-2 (FGF2, bFGF) has long been known to stimulate proliferation of cultured mesenchymal
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