3D-printed Polyurethane-based Scaffolds for Bone Regeneration

Objectives: Additive manufacture techniques are revolutionizing the concept of biomaterials production and treatment personalization. The objective of this pilot study was to evaluate the viability and cellular migration of preosteoblasts on scaffolds made with a 3D-printable polyurethane-based material. In addition, the potential use of a platelet extract (PE) gel as a carrier of growth factors, like PDGF-AB and IGF-I, was evaluated to enhance cellular proliferation at imbibed scaffolds. Methods: For the 3D-printable material production, polyurethane was heated and stirred at high speed and hyaluronic acid, C6H10O3 (HEMA) and C22H21O2P (TPO) were added to the vial. The scaffolds were manufactured using a Miicraft 125 3D printer (Miicraft®, Taiwan). The PE was obtained after a first centrifugation of human whole blood (BioIVT®, USA) at 1,000g for 2min and 15s and a second centrifugation of the supernatant at 1,000g for 5min. The pellet was isolated and later resuspended in platelet poor plasma. For the PE-gel preparation, the PE was left on cold bath sonification for 5min and then 1/10 volume of 10x PBS was added. Then, PE solution was diluted with VitroGel™ 3D (TheWell Bioscience Inc.®, USA) at a 5-fold. Scaffolds were imbibed on PE-gel while in a cold sonification bath. Cellular viability was evaluated with preosteoblasts (MC3T3-E1) after different scaffold detoxication protocols (boiling, ultraviolet light exposure, dimethyl sulfoxide submersion and vacuum chamber use) using CellTiter-Glo® (Promega®, USA). The migration assay was evaluated using imbibed or non-imbibed scaffolds placed under an 8 µm-pore cell-culture insert where MC3T3-E1 were seeded. Cell counting was performed after 24, 48 and 72h using CCK-8® (Dojindo®, Japan). The concentration of PDGF-AB and IGF-I released by imbibed scaffolds were measured using DuoSet® ELISA (R&D Systems®, USA) at 5, 15 and 30min and 1, 3, 6, 9 and 24h. PDGF-AB concentration was also assessed after addition of PAR1 to PE. Results: The results indicated that the 3D-printed polyurethane-based scaffolds are biocompatible and good carriers for the PE-gel. The best post-processing method for detoxification of the material prior to cell culture was the use of a vacuum chamber with the scaffold submerged at hot deionized water, achieving results close to positive control after 24h (7,547 RLU against 7,750 RLU, respectively). Detoxification with overnight ultraviolet light exposure showed better results when compared to boiling and dimethyl sulfoxide submersion, however, results were much lower than positive control (63 RLU, 74.5 RLU and 93 RLU against 6,424 RLU, 11073 RLU and 15,005 RLU at 24, 48 and 72h). At the migration assay, a higher number of cells could be counted at 24, 48 and 72h on the scaffolds imbibed with PE-gel when compared to those without PE-gel, with values of 49,214, 100,857 and 101,428 cells for the first and 38,857, 83,000 and 83,142 cells for the later, respectively. Regarding the concentration of PDGF-AB and IGF-I released by the scaffold imbibed with PE-gel at the cell culture medium, a reduction in concentration was noted over time. No difference was notice concerning the time the scaffold remained submerged on PE-gel during cold sonification bath or if a different scaffold design was used. However, the addition of PAR1 to PE was related to an increase at the concentration of PDGF-AB over time, showing gradual release of the growth factor to the medium. Conclusions: The results from this pilot study suggest that 3D printed polyurethane-based scaffolds are viable constructs for cell culture migration after a post-processing detoxification. The best protocol is the use of a vacuum chamber with the scaffold submerged at hot deionized water. Boiling, overnight ultraviolet light exposure and dimethyl sulfoxide submersion do not appear to be effective methods for that purpose. The use of PE-gel seems to increase cell counting, favoring cell proliferation. However, our results showed that some modifications still can be done to optimize the PDGF-AB and IFG-I stability. For that, the addition of PAR1 seems to be necessary to optimize platelet activation. These results will guide the methodology design of our future research aiming the clinical applications of 3D-printing for bone regeneration, such as the production of biodegradable scaffolds, membranes and fixation devices. Keywords: 3D-print, polyurethane, digital light processing, scaffold, membrane