Creation of Laryngeal Grafts from Primary Human Cells and Decellularized Laryngeal Scaffolds.

Current reconstruction methods of the laryngotracheal segment fail to replace the complex functions of the human larynx. Bioengineering approaches to reconstruction have been limited by the complex tissue compartmentation of the larynx. We attempted to overcome this limitation by bioengineering laryngeal grafts from decellularized canine laryngeal scaffolds recellularized with human primary cells under one uniform culture medium condition. First, we developed laryngeal scaffolds which were generated by detergent perfusion-decellularization over 9 days and preserved their glycosaminoglycan content and biomechanical properties of a native larynx. After subcutaneous implantations in rats for 14 days, the scaffolds did not elicit a CD3 lymphocyte response. We then developed a uniform culture medium that strengthened the endothelial barrier over 5 days after an initial growth phase. Simultaneously, this culture medium supported airway epithelial cell and skeletal myoblast growth while maintaining their full differentiation and maturation potential. We then applied the uniform culture medium composition to whole laryngeal scaffolds seeded with endothelial cells from both carotid arteries and external jugular veins and generated reendothelialized arterial and venous vascular beds. Under the same culture medium, we bioengineered epithelial monolayers onto laryngeal mucosa and repopulated intrinsic laryngeal muscle. We were then able to demonstrate early muscle formation in an intramuscular transplantation model in immunodeficient mice. We supported formation of three humanized laryngeal tissue compartments under one uniform culture condition, possibly a key factor in developing complex, multicellular, ready-to-transplant tissue grafts. Impact Statement For patients undergoing laryngectomy, no reconstruction methods are available to restore the complex functions of the human larynx. The first promising preclinical results have been achieved with the use of biological scaffolds fabricated from decellularized tissue. However, the complexity of laryngeal tissue composition remains a hurdle to create functional viable grafts, since previously each cell type requires tailored culture conditions. In this study, we report the de novo formation of three humanized laryngeal tissue compartments under one uniform culture condition, a possible keystone in creating vital composite tissue grafts for laryngeal regeneration.

Two Methods for Decellularization of Plant Tissues for Tissue Engineering Applications.

The autologous, synthetic, and animal-derived grafts currently used as scaffolds for tissue replacement have limitations due to low availability, poor biocompatibility, and cost. Plant tissues have favorable characteristics that make them uniquely suited for use as scaffolds, such as high surface area, excellent water transport and retention, interconnected porosity, preexisting vascular networks, and a wide range of mechanical properties. Two successful methods of plant decellularization for tissue engineering applications are described here. The first method is based on detergent baths to remove cellular matter, which is similar to previously established methods used to clear mammalian tissues. The second is a detergent-free method adapted from a protocol that isolates leaf vasculature and involves the use of a heated bleach and salt bath to clear the leaves and stems. Both methods yield scaffolds with comparable mechanical properties and low cellular metabolic impact, thus allowing the user to select the protocol which better suits their intended application.

Optical Method to Quantify Mechanical Contraction and Calcium Transients of Human Pluripotent Stem Cell-Derived Cardiomyocytes.

Differentiation of human pluripotent stem cells into cardiomyocytes (hPS-CMs) holds promise for myocardial regeneration therapies, drug discovery, and models of cardiac disease. Potential cardiotoxicities may affect hPS-CM mechanical contraction independent of calcium signaling. Herein, a method using an image capture system is described to measure hPS-CM contractility and intracellular calcium concurrently, with high spatial and temporal resolution. The image capture system rapidly alternates between brightfield and epifluorescent illumination of contracting cells. Mechanical contraction is quantified by a speckle tracking algorithm applied to brightfield image pairs, whereas calcium transients are measured by a fluorescent calcium reporter. This technique captured changes in contractile strain, calcium transients, and beat frequency of hPS-CMs over 21 days in culture, as well as acute responses to isoproterenol and Cytochalasin D. The technique described above can be applied without the need to alter the culture platform, allowing for determination of hPS-CM behavior over weeks in culture for drug discovery and myocardial regeneration applications.

Creation of a Bioengineered Skin Flap Scaffold with a Perfusable Vascular Pedicle.

Full-thickness skin loss is a challenging problem due to limited reconstructive options, demanding 75 million surgical procedures annually in the United States. Autologous skin grafting is the gold standard treatment, but results in donor-site morbidity and poor aesthetics. Numerous skin substitutes are available on the market to date, however, none truly functions as full-thickness skin due to lack of a vascular network. The creation of an autologous full-thickness skin analogue with a vascular pedicle would result in a paradigm shift in the management of wounds and in reconstruction of full-thickness skin defects. To create a clinically relevant foundation, we generated an acellular skin flap scaffold (SFS) with a perfusable vascular pedicle of clinically relevant size by perfusion decellularization of porcine fasciocutaneous flaps. We then analyzed the yielded SFS for mechanical properties, biocompatibility, and regenerative potential in vitro and in vivo. Furthermore, we assessed the immunological response using an in vivo model. Finally, we recellularized the vascular compartment of an SFS and reconnected it to a recipient's blood supply to test for perfusability. Perfusion decellularization removed all cellular components with preservation of native extracellular matrix composition and architecture. Biaxial testing revealed preserved mechanical properties. Immunologic response and biocompatibility assessed via implantation and compared with native xenogenic skin and commercially available dermal substitutes revealed rapid neovascularization and complete tissue integration. Composition of infiltrating immune cells showed no evidence of allorejection and resembled the inflammatory phase of wound healing. Implantation into full-thickness skin defects demonstrated good tissue integration and skin regeneration without cicatrization. We have developed a protocol for the generation of an SFS of clinically relevant size, containing a vascular pedicle, which can be utilized for perfusion decellularization and, ultimately, anastomosis to the recipient vascular system after precellularization. The observed favorable immunological response and good tissue integration indicate the substantial regenerative potential of this platform.

Biofunctionalized Plants as Diverse Biomaterials for Human Cell Culture.

The commercial success of tissue engineering products requires efficacy, cost effectiveness, and the possibility of scaleup. Advances in tissue engineering require increased sophistication in the design of biomaterials, often challenging the current manufacturing techniques. Interestingly, several of the properties that are desirable for biomaterial design are embodied in the structure and function of plants. This study demonstrates that decellularized plant tissues can be used as adaptable scaffolds for culture of human cells. With simple biofunctionalization technique, it is possible to enable adhesion of human cells on a diverse set of plant tissues. The elevated hydrophilicity and excellent water transport abilities of plant tissues allow cell expansion over prolonged periods of culture. Moreover, cells are able to conform to the microstructure of the plant frameworks, resulting in cell alignment and pattern registration. In conclusion, the current study shows that it is feasible to use plant tissues as an alternative feedstock of scaffolds for mammalian cells.

Crossing kingdoms: Using decellularized plants as perfusable tissue engineering scaffolds.

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.

Design of a Fibrin Microthread-Based Composite Layer for Use in a Cardiac Patch.

The ability to modulate the mechanical properties, and cell alignment within a cardiac patch without hindering cell functionality may have significant impact on developing therapies for treating myocardial infarctions. We developed fibrin-based composite layers comprising aligned microthreads distributed uniformly throughout a hydrogel. Increasing the microthread volume fraction (∼5%, 11% and 22%) significantly increased the moduli of the scaffolds (20.6 ± 8.1, 46.4 ± 23.0, and 97.5 ± 49.3 kPa, respectively), p < 0.05. Analyses of cell-mediated contractile strains and frequencies showed no significant differences among composite layers and fibrin hydrogel controls, suggesting that microthread-based composite layers exhibit similar active functional properties. Cell orientation in composite layers suggests an increase in nuclear alignment within 100 µm of fibrin microthreads and suggests that microthreads influence the alignment in adjacent areas. In this study, we developed composite layers with tunable, mechanical patch properties that improve cell alignment and support cell functionality.

Bioengineering Human Myocardium on Native Extracellular Matrix.

RATIONALE: More than 25 million individuals have heart failure worldwide, with ≈4000 patients currently awaiting heart transplantation in the United States. Donor organ shortage and allograft rejection remain major limitations with only ≈2500 hearts transplanted each year. As a theoretical alternative to allotransplantation, patient-derived bioartificial myocardium could provide functional support and ultimately impact the treatment of heart failure. OBJECTIVE: The objective of this study is to translate previous work to human scale and clinically relevant cells for the bioengineering of functional myocardial tissue based on the combination of human cardiac matrix and human induced pluripotent stem cell-derived cardiomyocytes. METHODS AND RESULTS: To provide a clinically relevant tissue scaffold, we translated perfusion-decellularization to human scale and obtained biocompatible human acellular cardiac scaffolds with preserved extracellular matrix composition, architecture, and perfusable coronary vasculature. We then repopulated this native human cardiac matrix with cardiomyocytes derived from nontransgenic human induced pluripotent stem cells and generated tissues of increasing 3-dimensional complexity. We maintained such cardiac tissue constructs in culture for 120 days to demonstrate definitive sarcomeric structure, cell and matrix deformation, contractile force, and electrical conduction. To show that functional myocardial tissue of human scale can be built on this platform, we then partially recellularized human whole-heart scaffolds with human induced pluripotent stem cell-derived cardiomyocytes. Under biomimetic culture, the seeded constructs developed force-generating human myocardial tissue and showed electrical conductivity, left ventricular pressure development, and metabolic function. CONCLUSIONS: Native cardiac extracellular matrix scaffolds maintain matrix components and structure to support the seeding and engraftment of human induced pluripotent stem cell-derived cardiomyocytes and enable the bioengineering of functional human myocardial-like tissue of multiple complexities.

Beta 1 integrin binding plays a role in the constant traction force generation in response to varying stiffness for cells grown on mature cardiac extracellular matrix.

We have previously reported a unique response of traction force generation for cells grown on mature cardiac ECM, where traction force was constant over a range of stiffnesses. In this study we sought to further investigate the role of the complex mixture of ECM on this response and assess the potential mechanism behind it. Using traction force microscopy, we measured cellular traction forces and stresses for mesenchymal stem cells (MSCs) grown on polyacrylamide gels at a range of stiffnesses (9, 25, or 48 kPa) containing either adult rat heart ECM, different singular ECM proteins including collagen I, fibronectin, and laminin, or ECM mimics comprised of varying amounts of collagen I, fibronectin, and laminin. We also measured the expression of integrins on these different substrates as well as probed for ß1 integrin binding. There was no significant change in traction force generation for cells grown on the adult ECM, as previously reported, whereas cells grown on singular ECM protein substrates had increased traction force generation with an increase in substrate stiffness. Cells grown on ECM mimics containing collagen I, fibronectin and laminin were found to be reminiscent of the traction forces generated by cells grown on native ECM. Integrin expression generally increased with increasing stiffness except for the ß1 integrin, potentially implicating it as playing a role in the response to adult cardiac ECM. We inhibited binding through the ß1 integrin on cells grown on the adult ECM and found that the inhibition of ß1 binding led to a return to the typical response of increasing traction force generation with increasing stiffness. Our data demonstrates that cells grown on the mature cardiac ECM are able to circumvent typical stiffness related cellular behaviors, likely through ß1 integrin binding to the complex composition.

Mesenchymal stem cells ability to generate traction stress in response to substrate stiffness is modulated by the changing extracellular matrix composition of the heart during development.

In this study we present a novel method for studying cellular traction force generation and mechanotransduction in the context of cardiac development. Rat hearts from three distinct stage of development (fetal, neonatal and adult) were isolated, decellularized and characterized via mechanical testing and protein compositional analysis. Stiffness increased ~2-fold between fetal and neonatal time points but not between neonatal and adult. Composition of structural extracellular matrix (ECM) proteins was significantly different between all three developmental ages. ECM that was solubilized via pepsin digestion was cross-linked into polyacrylamide gels of varying stiffness and traction force microscopy was used to assess the ability of mesenchymal stem cells (MSCs) to generate traction stress against the substrates. The response to increasing stiffness was significantly different depending on the developmental age of the ECM. An investigation into early cardiac differentiation of MSCs demonstrated a dependence of the level of expression of early cardiac transcription factors on the composition of the complex ECM. In summary, this study found that complex ECM composition plays an important role in modulating a cell's ability to generate traction stress against a substrate, which is a significant component of mechanotransductive signaling.