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ABSTRACT: The parents of broiler (meat) chickens (ie, broiler breeders) are food-restricted until sexual maturity, ensuring good health and reproduction, but resulting in hunger. We investigated whether diets with added insoluble fiber promote satiety and reduce behavioral, motivational, and physiological signs of hunger. Ninety-six broiler breeders were fed 1 of 4 feed treatments (n = 24 per diet) from 6 to 12 wk of age: 1) a commercial diet fed to the recommended ration (R) or 2) ad libitum (AL), the same diet as R but mixed with oat hulls at 3) 20% (OH20%) or 4) 40% (OH40%). The R, OH20% and OH40% diets were approximately iso-energetic and resulted in mean 12 wk of age weights within 2.5% of each other (1.21 kg), while AL birds weighed 221% as much (2.67kg). At 12 wk of age, agouti-related protein (AGRP) expression, was, on average, more than 12 times lower in AL birds (P < 0.001) but did not differ between the fiber diet treatments and R. Pro-opiomelanocortin (POMC) expression, was, on average, over 1.5 times higher in AL birds, but was not statistically significantly affected by feed treatments (P = 0.33). In their home pens, AL birds stood/sat more, foraged less and fed more in total (P < 0.001) and OH40% birds spent longer feeding than R (P = 0.001). Motivation to forage tested by willingness to walk through water to access an area of wood shavings (without food) was not significantly affected by diet (P = 0.33). However, restricted birds were willing to cross in only 7.3% to 12.5% of tests. Mostly birds stayed on the start platform, where AL birds sat more than other treatments and OH40% birds reduced walking relative to R birds (P = 0.016). Across the behavioral and physiological measurements there was a dichotomy of effects in response to approximately iso-energetic diets differing in fiber. There were some potentially beneficial behavioral effects related to reduced foraging and walking. However, there was no evidence that these diets significantly improved physiological measures of satiety of broiler breeders.

OBJECTIVES/GOALS: To determine if decellularized costal cartilage (DCC), which could theoretically be obtained “off the shelf,” would provide similar results to autologous cartilage grafts previously studied in this lab, thereby widening the application of our novel nipple engineering approach to all patients undergoing nipple reconstruction. METHODS/STUDY POPULATION: PLA scaffolds (diameter: 1.0 cm, height: 1.0 cm) were printed using a PRUSA 3D printer and sterilized. Lamb costal cartilage was minced (1 mm3) or zested (

Purpose: While tissue engineering offers the promise of revolutionary innovation, scalable three-dimensional tissue culture is limited by the diffusion of nutrients and oxygen making media perfusion obligatory. Unfortunately, the cost of bioreactors for large construct tissue culture can be prohibitive, with a typical perfusion chamber costing several thousand dollars, and even small petri-dish-sized devices costing hundreds of dollars each. We have developed a low-cost perfusion setup that seals collagen-based perfusable cellular constructs within a sterile PDMS well between coverslips, allowing for repeated live-imaging of perfused 3D engineered tissues. Herein we describe fabrication of this novel system and validate its utility. Methods: Molds and frames were designed on 3D-modeling software (Fusion 360) and printed on a Prusa i3 MK3S 3D printer in poly(lactic acid) (PLA). Molds were filled with poly(dimethyl siloxane) (PDMS), which was cured to form chambers, bubble traps, mason jar lid chambers, and media reservoir lid adapters. In total, the tissue culture chamber device, mason jar lid inset, media reservoir lid, and bubble trap require 4, 1, 2, and 4 unique printed components, respectively. Results: Each perfusion chamber can be assembled for under 8 USD per device and reused repeatedly. The current model has a tissue chamber custom-built with 18x10x4 (LxWxH)mm3 dimensions, but this chamber can be readily customized to experiment-specific dimensions. These devices allow cellular hydrogel constructs to be maintained in a sterile environment after assembly, perfused at varying rates to expose cells to different levels of shear stress, and the cells can be intermittently imaged with light, fluorescent or confocal microscopy - an unparalleled benefit for monitoring of experiments and collection of timepoint imaging data. The perfusion circuit consists of autoclavable glass and PDMS components, including a bubble trap, a crucial component of the circuit for preventing air bubbles that can damage cells and block microchannels, and a lid adapter, which allows 50 mL conical tubes to serve as self-oxygenating media reservoirs. Media changes can be performed via peristaltic pump perfusion or with syringe-based cell culture techniques for static culture. Constructs have been perfused within standard incubators for up to 14 days demonstrating normal cell viability without contamination or evidence of infection, with longer perfusion culture intervals (>1 month) currently being tested. Conclusion: The increasing accessibility of 3D-modeling and 3D-printing has enabled rapid prototyping of devices to address the problems that we face as surgeon-scientists. We have developed a low-cost tissue-engineering perfusion circuit that facilitates 3D-tissue-culture while allowing for repeated live-imaging as the cultured tissue develops. The instructions for our setup can be utilized to replicate our devices in other labs, and these designs can be readily customized to meet the needs of specific experimental aims.

Background Hemorrhage remains a major cause of death around the world. Eighty percent of trauma patients in India do not receive medical care within the first hour. The etiology of these poor outcomes is multifactorial. We describe findings from the first Stop the Bleed (StB) course recently offered to a group of medical providers in southern India.Methods A cross-sectional survey of 101 participants who attended StB trainings in India was performed. Pre-training and post-training questionnaires were collected from each participant. In total, 88 healthcare providers’ responses were analyzed. Three bleeding control skills were presented: wound compression, wound packing, and tourniquet application.Results Among participants, only 23.9% had received prior bleeding control training. Participants who reported feeling ‘extremely confident’ responding to an emergency medical situation rose from 68.2% prior to StB training to 94.3% post-training. Regarding hemorrhage control abilities, 37.5% felt extremely confident before the training, compared with 95.5% after the training. For wound packing and tourniquet application, 44.3% and 53.4%, respectively, felt extremely confident pre-training, followed by 97.7% for both skills post-training. Importantly, 90.9% of StB trainees felt comfortable teaching newly acquired hemorrhage control skills. A significant majority of participants said that confidence in their wound packing and tourniquet skills would improve with more realistic mannequins.Conclusion To our knowledge, this is the first StB training in India. Disparities in access to care, long transport times, and insufficient numbers of prehospital personnel contribute to its significant trauma burden. Dissemination of these critical life-saving skills into this region and the resulting civilian interventions will increase the number of trauma patients who survive long enough to reach a trauma center. Additionally, considerations should be given to translating the course into local languages to increase program reach.Level of Evidence Level IV.

ABSTRACT IMPACT: This project uses rapid prototyping, 3D printing, and cell seeding to solve the most common post-operative complications that arise from the current methods of urethral elongation during phalloplasty. OBJECTIVES/GOALS: Post-op fistula and stricture formation occur in up to 50% of phalloplasty patients. These complications arise from the mismatch between skin and uroepithelium, or from scarring secondary to ischemia. Here we describe the fabrication of a novel vascularized urothelial flap for phalloplasty that contains discrete urothelial and vascular channels. METHODS/STUDY POPULATION: A custom designed 3D negative mold, with a urethral channel and a vascular inlet and outlet channel was prototyped in Adobe Fusion 360 and printed on a Prusa i3 MK3S printer in PLA. A 2mm diameter pluronic sacrificial macrofiber was used to connect the channels to form a vascular loop, and 1% type-I collagen was extruded over the mold. After solidifying, the scaffold was demolded and seeded with grade I urothelial carcinoma (SW780 cells, at 5-10 x 106 cells/mL) in the urethral channel, and adenovirus-infected E4 endothelial cells (at 3x106 cells/mL) in the vascular channel. The scaffolds were cultured up to 14 days and then fixed for histologic analysis. RESULTS/ANTICIPATED RESULTS: Collagen scaffolds were fabricated reliably using the custom 3D negative molds. After both seven and fourteen days of culture, the urothelial channel contained a robust, stable urothelial monolayer lining throughout the channel. By 14 days urothelial multilayer formation was seen, providing definitive evidence of a more mature urothelial layer. Grooves within the collagen allowed for nests of urothelial cells to develop, leading to increased multilayer formation. In addition, the vascular channels supported a healthy endothelial lining at both seven and fourteen days. There were no significant histological differences between constructs seeded with 5x106 urothelial cells/mL and 107 cells/mL. We anticipate that multilayer formation will increase with time, and that constructs will survive beyond 28 days. DISCUSSION/SIGNIFICANCE OF FINDINGS: We have developed a novel strategy to engineer vascularized urethral tissue. These constructs can be kept for at least 14 days and form stable monolayers and multilayers consistent with native urothelial architecture. Using 3D printing and autologous cell seeding promises to create patient-specific vascularized urethral flaps for phalloplasty.