A subcutaneous pancreatic islet transplantation platform using a clinically applicable, biodegradable Vicryl mesh scaffold - an experimental study.

Pancreatic islet transplantation into the liver is an effective treatment for type 1 diabetes but has some critical limitations. The subcutaneous site is a potential alternative transplant site, requiring minimally invasive procedures and allowing frequent graft monitoring; however, hypoxia is a major drawback. Our previous study without scaffolding demonstrated post-transplant graft aggregation in the subcutaneous site, which theoretically exacerbates lethal intra-graft hypoxia. In this study, we introduce a clinically applicable subcutaneous islet transplantation platform using a biodegradable Vicryl mesh scaffold to prevent aggregation in a diabetic rat model. Islets were sandwiched between layers of clinically proven Vicryl mesh within thrombin-fibrin gel. In vitro, the mesh prevented islet aggregation and intra-islet hypoxia, which significantly improved islet viability. In vivo rat syngeneic islet transplantations into a prevascularized subcutaneous pocket demonstrated that the mesh significantly enhanced engraftment, as measured by assays for graft survival and function. Histological examination at 6 weeks showed well-vascularized grafts sandwiched in a flat shape between the mesh layers. The biodegradable mesh was fully absorbed by three months, which alleviated chronic foreign body reaction and fibrosis, and supported long-term graft maintenance. This simple graft shape modification approach is an effective and clinically applicable strategy for improved subcutaneous islet transplantation.

Optimizing Temperature and Oxygen Supports Long-term Culture of Human Islets.

Islet transplantation is a promising treatment for type-1 diabetes; however, donor shortage is a concern. Even when a pancreas is available, low islet yield limits the success of transplantation. Islet culture enables pooling of multiple low-yield isolations into an effective islet mass, but isolated islets rapidly deteriorate under conventional culture conditions. Oxygen (O2) depletion in the islet core, which leads to central necrosis and volume loss, is one of the major reasons for this deterioration. METHODS: To promote long-term culture of human islets in PIM-R medium (used for islet research), we adjusted temperature (12°C, 22°C, and 37°C) and O2 concentration (21% and 50%). We simulated the O2 distribution in islets based on islet O2 consumption rate and dissolved O2 in the medium. We determined the optimal conditions for O2 distribution and volume maintenance in a 2-week culture and assessed viability and insulin secretion compared to noncultured islets. In vivo islet engraftment was assessed by transplantation into diabetic nonobese diabetic-severe combined immunodeficiency mouse kidneys. We validated our results using CMRL 1066 medium (used for clinical islet transplantation). RESULTS: Simulation revealed that 12°C of 50% O2 PIM-R culture supplied O2 effectively into the islet core. This condition maintained islet volume at greater than 90% for 2 weeks. There were no significant differences in viability and function in vitro or diabetic reversal rate in vivo between 2-week cultured and noncultured islets. Similar results were obtained using CMRL 1066. CONCLUSIONS: By optimizing temperature and O2 concentration, we cultured human islets for 2 weeks with minimal loss of volume and function.

Mathematical modeling of oxygen release from hyperbarically loaded polymers.

Polymer-based scaffolds are used extensively in the field of regenerative medicine. These biomaterials may induce therapeutic responses through modulating a wound microenvironment with or without the addition of cells. It has long been known that oxygen is a crucial component of the microenvironment that influences cellular and physiological processes such as metabolism, proliferation, differentiation, matrix deposition, phagocytic killing, and wound healing. Consequently, several studies have investigated the potential for using oxygen-eluting biomaterials to regulate the oxygen tension within a wound microenvironment and to tune the regenerative response. We recently demonstrated that hyperbarically loaded polymers could be used as oxygen delivery devices for biomedical uses. To further develop this strategy, it is important to quantitatively characterize the spatiotemporal oxygen diffusion profile from scaffolds. Here, we use analytical and numerical solutions to describe the profiles of oxygen diffusion from hyperbarically loaded polymers as a function of different scaffold geometries, material compositions, and ambient temperatures. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 2018 © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 35: e2751, 2019.

Oxygen transporter for the hypoxic transplantation site.

Cell transplantation is a promising treatment for complementing lost function by replacing new cells with a desired function, e.g. pancreatic islet transplantation for diabetics. To prevent cell obliteration, oxygen supply is critical after transplantation, especially until the graft is sufficiently re-vascularized. To supply oxygen during this period, we developed a chemical-/electrical-free implantable oxygen transporter that delivers oxygen to the hypoxic graft site from ambient air by diffusion potential. This device is simply structured using a biocompatible silicone-based body that holds islets, connected to a tube that opens outside the body. In computational simulations, the oxygen transporter increased the oxygen level to >120 mmHg within grafts; in contrast, a control device that did not transport oxygen showed <6.5 mmHg. In vitro experiments demonstrated similar results. To test the effectiveness of the oxygen transporter in vivo, we transplanted pancreatic islets, which are susceptible to hypoxia, subcutaneously into diabetic rats. Islets transplanted using the oxygen transporter showed improved graft viability and cellular function over the control device. These results indicate that our oxygen transporter, which is safe and easily fabricated, effectively supplies oxygen locally. Such a device would be suitable for multiple clinical applications, including cell transplantations that require changing a hypoxic microenvironment into an oxygen-rich site.

Characterization of a novel bioreactor system for 3D cellular mechanobiology studies.

In vitro engineering systems can be powerful tools for studying tissue development in response to biophysical stimuli as well as for evaluating the functionality of engineered tissue grafts. It has been challenging, however, to develop systems that adequately integrate the application of biomimetic mechanical strain to engineered tissue with the ability to assess functional outcomes in real time. The aim of this study was to design a bioreactor system capable of real-time conditioning (dynamic, uniaxial strain, and electrical stimulation) of centimeter-long 3D tissue engineered constructs simultaneously with the capacity to monitor local strains. The system addresses key limitations of uniform sample loading and real-time imaging capabilities. Our system features an electrospun fibrin scaffold, which exhibits physiologically relevant stiffness and uniaxial alignment that facilitates cell adhesion, alignment, and proliferation. We have demonstrated the capacity for directly incorporating human adipose-derived stromal/stem cells into the fibers during the electrospinning process and subsequent culture of the cell-seeded constructs in the bioreactor. The bioreactor facilitates accurate pre-straining of the 3D constructs as well as the application of dynamic and static uniaxial strains while monitoring bulk construct tensions. The incorporation of fluorescent nanoparticles throughout the scaffolds enables in situ monitoring of local strain fields using fluorescent digital image correlation techniques, since the bioreactor is imaging compatible, and allows the assessment of local sample stiffness and stresses when coupled with force sensor measurements. In addition, the system is capable of measuring the electromechanical coupling of skeletal muscle explants by applying an electrical stimulus and simultaneously measuring the force of contraction. The packaging of these technologies, biomaterials, and analytical methods into a single bioreactor system has produced a powerful tool that will enable improved engineering of functional 3D ligaments, tendons, and skeletal muscles. Biotechnol. Bioeng. 2016;113: 1825-1837. © 2016 Wiley Periodicals, Inc.

Oxygen delivery from hyperbarically loaded microtanks extends cell viability in anoxic environments.

Oxygen diffusion limitations within nascent tissue engineered (TE) grafts lead to the development of hypoxic regions, cell death, and graft failure. Previous efforts have been made to deliver oxygen within TE scaffolds, including peroxide-doping, perfluorocarbons, and hyperbaric oxygen therapy, to mitigate these effects and help maintain post transplantation cell viability, but these have suffered from significant drawbacks. Here we present a novel approach utilizing polymeric hollow-core microspheres that can be hyperbarically loaded with oxygen and subsequently provide prolonged oxygen delivery. These oxygen carriers are termed, microtanks. With an interest in orthopedic applications, we combined microtanks within polycaprolactone to form solid phase constructs with oxygen delivery capabilities. The mathematical laws governing oxygen delivery from microtank-loaded constructs are developed along with empirical validation. Constructs achieved periods of oxygen delivery out to 6 days, which was shown to prolong the survival of human adipose derived stem cells (hASCs) and human umbilical vein endothelial cells (HUVECs) as well as to enhance their cellular morphology under anoxic conditions. The results of this study suggest the microtank approach may be a feasible means of maintaining cell viability in TE scaffolds during the critical period of vascularization in vivo.

Engineering anatomically shaped vascularized bone grafts with hASCs and 3D-printed PCL scaffolds.

The treatment of large craniomaxillofacial bone defects is clinically challenging due to the limited availability of transplantable autologous bone grafts and the complex geometry of the bones. The ability to regenerate new bone tissues that faithfully replicate the anatomy would revolutionize treatment options. Advances in the field of bone tissue engineering over the past few decades offer promising new treatment alternatives using biocompatible scaffold materials and autologous cells. This approach combined with recent advances in three-dimensional (3D) printing technologies may soon allow the generation of large, bioartificial bone grafts with custom, patient-specific architecture. In this study, we use a custom-built 3D printer to develop anatomically shaped polycaprolactone (PCL) scaffolds with varying internal porosities. These scaffolds are assessed for their ability to support induction of human adipose-derived stem cells (hASCs) to form vasculature and bone, two essential components of functional bone tissue. The development of functional tissues is assessed in vitro and in vivo. Finally, we demonstrate the ability to print large mandibular and maxillary bone scaffolds that replicate fine details extracted from patient's computed tomography scans. The findings of this study illustrate the capabilities and potential of 3D printed scaffolds to be used for engineering autologous, anatomically shaped, vascularized bone grafts.