Development of an automated biomaterial platform to study mosquito feeding behavior.

Mosquitoes carry a number of deadly pathogens that are transmitted while feeding on blood through the skin, and studying mosquito feeding behavior could elucidate countermeasures to mitigate biting. Although this type of research has existed for decades, there has yet to be a compelling example of a controlled environment to test the impact of multiple variables on mosquito feeding behavior. In this study, we leveraged uniformly bioprinted vascularized skin mimics to create a mosquito feeding platform with independently tunable feeding sites. Our platform allows us to observe mosquito feeding behavior and collect video data for 30-45 min. We maximized throughput by developing a highly accurate computer vision model (mean average precision: 92.5%) that automatically processes videos and increases measurement objectivity. This model enables assessment of critical factors such as feeding and activity around feeding sites, and we used it to evaluate the repellent effect of DEET and oil of lemon eucalyptus-based repellents. We validated that both repellents effectively repel mosquitoes in laboratory settings (0% feeding in experimental groups, 13.8% feeding in control group, p < 0.0001), suggesting our platform's use as a repellent screening assay in the future. The platform is scalable, compact, and reduces dependence on vertebrate hosts in mosquito research.

Perfusable cell-laden matrices to guide patterning of vascularization in vivo.

The survival and function of transplanted tissue engineered constructs and organs require a functional vascular network. In the body, blood vessels are organized into distinct patterns that enable optimal nutrient delivery and oxygen exchange. Mimicking these same patterns in engineered tissue matrices is a critical challenge for cell and tissue transplantation. Here, we leverage bioprinting to assemble endothelial cells in to organized networks of large (>100 µm) diameter blood vessel grafts to enable spatial control of vessel formation in vivo. Acellular PEG/GelMA matrices with perfusable channels were bioprinted and laminar flow was confirmed within patterned channels, beneficial for channel endothelialization and consistent wall shear stress for endothelial maturation. Next, human umbilical vein endothelial cells (HUVECs) were seeded within the patterned channel and maintained under perfusion culture for multiple days, leading to cell-cell coordination within the construct in vitro. HUVEC and human mesenchymal stromal cells (hMSCs) were additionally added to bulk matrix to further stimulate anastomosis of our bioprinted vascular grafts in vivo. Among multiple candidate matrix designs, the greatest degree of biomaterial vascularization in vivo was seen within matrices fabricated with HUVECs and hMSCs encapsulated within the bulk matrix and HUVECs lining the walls of the patterned channels, dubbed design M-C_E. For this lead design, vasculature was detected within the endothelialized, perfusable matrix channels as early as two weeks and αSMA+ CD31+ vessels greater than 100 µm in diameter had formed by eight weeks, resulting in durable and mature vasculature. Notably, vascularization occurred within the endothelialized, bioprinted channels of the matrix, demonstrating the ability of bioprinted perfusable structures to guide vascularization patterns in vivo. The ability to influence vascular patterning in vivo can contribute to the future development of vascularized tissues and organs.

Comprehensive dynamic and kinematic analysis of the rodent hindlimb during over ground walking.

The rat hindlimb is a frequently utilized pre-clinical model system to evaluate injuries and pathologies impacting the hindlimbs. These studies have demonstrated the translational potential of this model but have typically focused on the force generating capacity of target muscles as the primary evaluative outcome. Historically, human studies investigating extremity injuries and pathologies have utilized biomechanical analysis to better understand the impact of injury and extent of recovery. In this study, we expand that full biomechanical workup to a rat model in order to characterize the spatiotemporal parameters, ground reaction forces, 3-D joint kinematics, 3-D joint kinetics, and energetics of gait in healthy rats. We report data on each of these metrics that meets or exceeds the standards set by the current literature and are the first to report on all these metrics in a single set of animals. The methodology and findings presented in this study have significant implications for the development and clinical application of the improved regenerative therapeutics and rehabilitative therapies required for durable and complete functional recovery from extremity traumas, as well as other musculoskeletal pathologies.

Projection-based stereolithography for direct 3D printing of heterogeneous ultrasound phantoms.

Modern ultrasound (US) imaging is increasing its clinical impact, particularly with the introduction of US-based quantitative imaging biomarkers. Continued development and validation of such novel imaging approaches requires imaging phantoms that recapitulate the underlying anatomy and pathology of interest. However, current US phantom designs are generally too simplistic to emulate the structure and variability of the human body. Therefore, there is a need to create a platform that is capable of generating well-characterized phantoms that can mimic the basic anatomical, functional, and mechanical properties of native tissues and pathologies. Using a 3D-printing technique based on stereolithography, we fabricated US phantoms using soft materials in a single fabrication session, without the need for material casting or back-filling. With this technique, we induced variable levels of stable US backscatter in our printed materials in anatomically relevant 3D patterns. Additionally, we controlled phantom stiffness from 7 to >120 kPa at the voxel level to generate isotropic and anisotropic phantoms for elasticity imaging. Lastly, we demonstrated the fabrication of channels with diameters as small as 60 micrometers and with complex geometry (e.g., tortuosity) capable of supporting blood-mimicking fluid flow. Collectively, these results show that projection-based stereolithography allows for customizable fabrication of complex US phantoms.

Generation of model tissues with dendritic vascular networks via sacrificial laser-sintered carbohydrate templates.

Sacrificial templates for patterning perfusable vascular networks in engineered tissues have been constrained in architectural complexity, owing to the limitations of extrusion-based 3D printing techniques. Here, we show that cell-laden hydrogels can be patterned with algorithmically generated dendritic vessel networks and other complex hierarchical networks by using sacrificial templates made from laser-sintered carbohydrate powders. We quantified and modulated gradients of cell proliferation and cell metabolism emerging in response to fluid convection through these networks and to diffusion of oxygen and metabolites out of them. We also show scalable strategies for the fabrication, perfusion culture and volumetric analysis of large tissue-like constructs with complex and heterogeneous internal vascular architectures. Perfusable dendritic networks in cell-laden hydrogels may help sustain thick and densely cellularized engineered tissues, and assist interrogations of the interplay between mass transport and tissue function.

Analysis and Modeling of Rat Gait Biomechanical Deficits in Response to Volumetric Muscle Loss Injury.

There is currently a substantial volume of research underway to develop more effective approaches for the regeneration of functional muscle tissue as treatment for volumetric muscle loss (VML) injury, but few studies have evaluated the relationship between injury and the biomechanics required for normal function. To address this knowledge gap, the goal of this study was to develop a novel method to quantify the changes in gait of rats with tibialis anterior (TA) VML injuries. This method should be sensitive enough to identify biomechanical and kinematic changes in response to injury as well as during recovery. Control rats and rats with surgically-created VML injuries were affixed with motion capture markers on the bony landmarks of the back and hindlimb and were recorded walking on a treadmill both prior to and post-surgery. Data collected from the motion capture system was exported for post-hoc analysis in OpenSim and Matlab. In vivo force testing indicated that the VML injury was associated with a significant deficit in force generation ability. Analysis of joint kinematics showed significant differences at all three post-surgical timepoints and gait cycle phase shifting, indicating augmented gait biomechanics in response to VML injury. In conclusion, this method identifies and quantifies key differences in the gait biomechanics and joint kinematics of rats with VML injuries and allows for analysis of the response to injury and recovery. The comprehensive nature of this method opens the door for future studies into dynamics and musculoskeletal control of injured gait that can inform the development of regenerative technologies focused on the functional metrics that are most relevant to recovery from VML injury.