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.

BackgroundType 1 diabetes is an autoimmune disease that destroys insulin-producing beta cells in the pancreas. Pancreatic islet transplantation could be an effective treatment option for type 1 diabetes once several issues are resolved, including donor shortage, prevention of islet necrosis and loss in pre- and post-transplantation, and optimization of immunosuppression. This study seeks to determine the cause of necrotic loss of isolated islets to improve transplant efficiency.MethodologyThe oxygen tension inside isolated human islets of different sizes was simulated under varying oxygen environments using a computational in silico model. In vitro human islet viability was also assessed after culturing in different oxygen conditions. Correlation between simulation data and experimentally measured islet viability was examined. Using these in vitro viability data of human islets, the effect of islet diameter and oxygen tension of the culture environment on islet viability was also analyzed using a logistic regression model.Principal findingsComputational simulation clearly revealed the oxygen gradient inside the islet structure. We found that oxygen tension in the islet core was greatly lower (hypoxic) than that on the islet surface due to the oxygen consumption by the cells. The hypoxic core was expanded in the larger islets or in lower oxygen cultures. These findings were consistent with results from in vitro islet viability assays that measured central necrosis in the islet core, indicating that hypoxia is one of the major causes of central necrosis. The logistic regression analysis revealed a negative effect of large islet and low oxygen culture on islet survival.Conclusions/SignificanceHypoxic core conditions, induced by the oxygen gradient inside islets, contribute to the development of central necrosis of human isolated islets. Supplying sufficient oxygen during culture could be an effective and reasonable method to maintain isolated islets viable.

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.

Adipose-derived stem/stromal cell (ASC)-based tissue engineered muscle grafts could provide an effective alternative therapy to autografts – which are limited by their availability – for the regeneration of damaged muscle. However, the current myogenic potential of ASCs is limited by their low differentiation efficiency into myoblasts. The aim of this study was to enhance the myogenic response of human ASCs to biochemical cues by providing biophysical stimuli (11% cyclic uniaxial strain, 0.5 Hz, 1 h/day) to mimic the cues present in the native muscle microenvironment. ASCs elongated and fused upon induction with myogenic induction medium alone. Yet, their myogenic characteristics were significantly enhanced with the addition of biophysical stimulation; the nuclei per cell increased approximately 4.5-fold by day 21 in dynamic compared to static conditions (23.3 ± 7.3 vs. 5.2 ± 1.6, respectively), they aligned at almost 45° to the direction of strain, and exhibited significantly higher expression of myogenic proteins (desmin, myoD and myosin heavy chain). These results demonstrate that mimicking the biophysical cues inherent to the native muscle microenvironment in monolayer ASC cultures significantly improves their differentiation along the myogenic lineage.

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.

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The ability to physically manipulate specific cells is critical for the fields of biomedicine, synthetic biology, and living materials. Ultrasound has the ability to manipulate cells with high spatiotemporal precision via acoustic radiation force (ARF). However, because most cells have similar acoustic properties, this capability is disconnected from cellular genetic programs. Here, we show that gas vesicles (GVs)—a unique class of gas-filled protein nanostructures—can serve as genetically encodable actuators for selective acoustic manipulation. Because of their lower density and higher compressibility relative to water, GVs experience strong ARF with opposite polarity to most other materials. When expressed inside cells, GVs invert the cells’ acoustic contrast and amplify the magnitude of their ARF, allowing the cells to be selectively manipulated with sound waves based on their genotype. GVs provide a direct link between gene expression and acoustomechanical actuation, opening a paradigm for selective cellular control in a broad range of contexts.