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1267 - Combination Of Direct Write 3D Printed Scaffolds With Bioprinted Chondrocytes


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Presented at

ORS 2019 Annual Meeting





INTRODUCTION: 3D printing and bioprinting are potential game changers in tissue engineering both for in vitro modelling and for biological implants. Direct write is a melt electrospinning writing method whereby the polymer (polycaprolactone; PCL) is melted through a charged spinneret and the liquid PCL is attracted to a collector plate which is moved to produce fibers which are deposited in defined geometries. Bioprinted chondrocytes in a collagen hydrogel were printed on top of the PCL scaffold and grown in vitro for 1 month. The combination of these techniques allows for better control over the material properties and higher fidelity of the in vitro matured print with the initial design than a bioprint alone. METHODS: Direct write PCL scaffolds (10 μm fiber diameter) were printed (RegenHu 3D Discovery) with 70 μm, 60 μm, 50 μm, 40 μm, 30 μm, 40 μm, 50 μm, 60 μm, 70 μm mesh size (Fig. 1A) resulting in scaffolds that were ~140 μm thick. Scaffolds were sterilized with sterile 70% ethanol for 1h then dried in a laminar flow cabinet. Scaffolds were coated with fibronectin (BD Bioscience, 5 μg/cm2) for 30 min then dried. Rabbit articular chondrocytes (3 donors) were isolated in accordance with IACUC guidelines of Baylor College of Medicine from the femoral head, and seeded on synoviocyte-derived extracellular matrix coated T175 flasks as previously described1. At the end of primary culture, chondrocytes are trypsinized, counted and resuspended to give a cell concentration of 50 x 106 cells/ml in defined chondrogenic media1. Chondrocytes are then mixed with collagen (Lifeink® 200; Advanced Biomatrix) at a 2:5 ratio by 50 passes through a luer lock connector between two syringes. Bubbles were removed from the mixture by centrifugation of the syringe (1000 RCF, 5 min). The syringe was then loaded and the cells printed onto the PCL scaffold in 3 layers with 100 % infill (r3bEL mini, Se3d). Composite constructs were grown in complete media (DMEM-LG supplemented with 10% FBS and 1% penicillin/streptomycin) for 5 days before exchange with defined chondrogenic media (DMEM-HG supplemented with 1% insulin, transferrin, selenium + premix; 130 mM ascorbate-2-phosphate, 2mM glutamax, 1% sodium pyruvate, 1% MEM non-essential amino acids, 100 nM dexamethasone, 1.25 μg/ml fungizone, 1% penicillin/streptomycin) and growth for a further month, all at 37°C, 5% O2, 5%CO2. At the end of culture, constructs were washed with Tyrodes and incubated with Calcein-AM (Invitrogen) for 30 minutes in Tyrodes solution at room temperature. Following this, constructs were washed in Tyrodes then placed on ice before being imaged by confocal microscopy (Nikon A1-Rs). RESULTS SECTION: At the end of primary culture, over 190 million chondrocytes are typically achievable from a single femoral head (range 1.9x108 – 4.9x108). Initial experiments, where constructs were immediately grown in chondrogenic media, saw very poor cell viability; this was altered for this study to have a brief, 5-day, recovery period in complete media before exchange with the defined chondrogenic media resulting in significant improvements in cell viability. Chondrocytes migrated out of the collagen hydrogel both into the PCL scaffold and onto non-tissue culture treated culture plate. Chondrocytes that remained in the construct were viable at 1 month post-seeding with varying morphology (Fig. 3). Studies on biochemical, and histological outcomes revealed similar glycosminoglycan per cell but with non-homogenous distribution in printed constructs. DISCUSSION: Tissue engineering of cartilage constructs for both in vitro models and, eventually, biological replacement tissue still faces significant challenges. Pore size has been reported to modulate cell differentiation/migration. A composite product, with a defined pore size for boney ingrowth, whilst supporting chondrogenesis, could address limitations in cartilage repair. Direct write printing has the ability to create defined pore size constructs. Bioprinting can be used to deposit cells in defined geometries but, because hydrogel/cell inks must exist within a ”Goldilocks” range being neither too stiff to print but stiff enough to retain their structure during cross-linking, 3-dimensional resolution is limited. The combination of these two technologies has the potential to address the limitations of each: shape fidelity, resolution and mechanical strength for bioprinting, and chondrogenesis and bioactivity for direct write. SIGNIFICANCE/CLINICAL RELEVANCE: Controlled scaffold pore size with fine fiber diameter, possible using direct write printing, provides a thin, biocompatible surface to bioprint chondrocytes. These techniques are combined here and demonstrate feasibility of this method as a composite system for the precise control of 3D organized constructs. REFERENCES: [1] Kean T.J. and Dennis J.E., 2015 PLoS One; [2] Wagner, T and Lipinski, H., 2013 Journal of Open Research Software; [3] Kean T.J. 2019 https://3dprint.nih.gov/discover/3DPX-010438 ; [4] Zheng C. and Levenston M.E., 2015 Eur Cell Mater; [5] Kean T.J., Mera H., Whitney G.A., MacKay D.L., Awadallah A., Fernandes R.J., Dennis J.E., 2016 Connect Tissue Res.


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© Copyright 2020 Morressier GmbH.
All rights reserved.