OBJECTIVES/SPECIFIC AIMS: This study’s aims are to optimize the isolation and growth of chondrocytes from pig auricular cartilage; to identify the ideal seeding conditions onto 3D printed auricular bioscaffolds to maximize chondrocyte growth; and to investigate what quantity and types of host tissue can grow on the bioscaffold. Primary outcomes will include comparisons between different seeding conditions in various objective measures of bioscaffold growth and survival as listed in the methods section. Secondary outcomes will include continued optimization of bioscaffolds to minimize extrusion rates and maximize morphologic and histologic similarity to human auricular cartilage. METHODS/STUDY POPULATION: For chondrocyte-seeded scaffolds, cartilage will be collected from freshly harvested porcine auricular tissue and digested in type II collagenase. Chondrocytes derived from the harvest will be seeded into auricular PCL scaffolds using a type I collagen/hyaluronic acid composite gel, which has been previously shown to support chondrogenesis. For scaffolds containing cartilage, punch biopsies will be collected and embedded in specific areas of the scaffold previously shown to experience excessive stress/strain compared to the rest of the construct. From there, five of each chondrocyte-seeded bioscaffolds, chondrocyte-unseeded bioscaffolds, and cartilage-containing bioscaffolds will be implanted into athymic rats. Total follow up will be for six months, with outcomes as measured by clinical assessments, morphologic measurements, radiological imaging, histological analysis, biomechanical evaluation, and photodocumentation. Once these measures are obtained, we will work closely with Dr. Myra Kim, an adjunct professor with the Biostatistics Department, to appropriately analyze differences between the models. RESULTS/ANTICIPATED RESULTS: We believe that while all scaffolds (chondrocyte-seeded, chondrocyte-unseeded, and cartilage-containing) will be structurally sound, the chondrocyte-seeded scaffolds and cartilage-containing scaffolds will exhibit improved soft tissue coverage and have lower exposure and fracture rates. Additionally, between the two, we posit that there will not be appreciable differences histologically, radiologically, or morphologically. DISCUSSION/SIGNIFICANCE OF IMPACT: Auricular reconstruction is a geometrically complex and technically challenging problem. Reconstruction hinges on the physical characteristics of the deformity, patient preferences, and reconstructive materials available. The current gold standard for auricular reconstruction uses autologous rib cartilage as foundational support for overlying soft tissue and these techniques involve freehand carving of the cartilage, requiring high levels of technical skill. Harvesting the materials for this procedure is invasive, and the outcomes of the surgery are largely variable and sometimes undesirable. As alternatives, implantable scaffolds including those made from high density porous polyethylene (commercially referred to as MedPor) have been investigated. However, many of these have proven inadequate due to factors including infection, extrusion, and morphologic and biomechanical dissimilarity from native tissue. 3D printing represents an exciting new avenue through which to address many of these difficulties. Our group has previously demonstrated the successful design, production, and implantation of 3D-printed models: in auricular reconstruction, we have demonstrated the successful creation and implementation of a 3D printed ear scaffold into an athymic rodent model. We now turn our attention to optimization of seeding of our ear scaffold with chondrocytes derived from porcine auricular cartilage or with cartilage punch biopsies, all while maintaining emphasis on regulatory feasibility. With success in this arena, we will be able to provide a much less invasive and technically challenging alternative to the current gold standard, create patient-specific bioscaffolds which are more form fitting and individualized, and provide children with ear malformations better alternatives and treatments for their conditions.