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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 ...

Worcester Polytechnic Institute

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.

Bioengineering Human Myocardium on Native Extracellular Matrix.

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.

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

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.

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

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.

Joshua R. Gershlak

Department of Biomedical Engineering

Worcester Polytechnic Institute

Two Methods for Decellularization of Plant Tissues for Tissue Engineering Applications

Joshua R. Gershlak

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Department of Biomedical Engineering

Worcester Polytechnic Institute

Two Methods for Decellularization of Plant Tissues for Tissue Engineering Applications

Department of Biomedical Engineering