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Biocompatibility and mechanical/physical properties of 3D printed, milled, and conventionally processed denture base materialsUlmer, Mallory; Biomedical Sciences (Augusta University, 2019-12)According to the American College of Prosthodontists, over 36 million people in the USA are edentulous with a 2:1 predilection for geriatric patients1. Each year, an estimated 15% of edentulous Americans will seek denture treatment1. Conventional dentures require multiple visits and lab processing time. 3D printing technology offers the potential to reduce the number of appointments and speed up the time until patient rehabilitation. However, the newly FDA-certified 3D printer denture resins, featuring secretive and proprietary formulae, lack studies concerning their biocompatibility/safety and mechanical strength. This study aims to investigate the biocompatibility and physical properties of one such 3D printer resin, NextDent® Base (Vertex, Soesterberg, The Netherlands), and compare it to pre-existing conventional polymethyl methacrylate (PMMA) denture base (Lucitone 199, Dentsply Sirona, York, Pennsylvania) and milled PMMA denture base (IvoBase CAD®, Ivoclar Vivadent AG, Schaan, Liechtenstein). The cytotoxicity was examined using of 12 discs: conventional PMMA, milled PMMA, as-printed 3D printer resin, post-cured 3D printer resin, and Teflon controls. An MTT assay using human periodontal ligament (900L) cells was employed, and specimens were aged for 1, 3, 7, 10, and 14 days. After day 7, there were no statistically significant differences among the groups, excluding the Teflon control, which showed significantly less cell viability on day 14. Bars of conventional PMMA, milled PMMA, as-printed 3D printer resin, and post-cured 3D printer resin were subjected to a 3-point bend test to examine flexural strength and moduli differences. The mean flexural strength was 63.8 ± 3.06, 82.6 ± 1.9, 5.1 ± 0.4, and 22.1 ± 6.4 MPa, respectively, while the flexural moduli were 1757.3 ± 109.5, 2226.7 ± 76.3, 110.3 ± 20.3, and 537.0 ± 210.6 MPa, respectively. The flexural strength and modulus were significantly different among all groups. Weibull analyses for conventional PMMA, milled PMMA, as-printed 3D printer resin, and post-cured 3D printer resin revealed a Weibull modulus of 23.5, 42.8, 16.6, and 3.7, respectively, and a characteristic strength of 65.2, 83.5, 5.3, and 24.5 MPa, respectively. The characteristic strength was significantly different among all groups as well. The Weibull modulus was significantly different between all groups, except for conventional vs. as-printed, which were not significantly different. In summary, milled PMMA featured significantly greater mechanical properties. Both 3D printed groups proved to be very weak, with the as-printed group being the weakest of all. The differences between the as-printed and post-cured groups highlight the importance of properly post-curing the resin. While the biocompatibility results showed promise, the mechanical testing results were disappointing. Unfortunately, the findings suggest that 3D-printed denture base resin is not yet ready for clinical use.
Dendritic Cell Derived Exosomes Loaded with Immunoregulatory Cargo Reprogram local Immune Responses and Inhibit Degenerative Bone Disease In vivoElashiry, Mahmoud; Biomedical Sciences (Augusta University, 2020-12)Background: Histopathological study of periodontitis (PD) lesions at sites of bone loss reveals infiltration with dendritic cells (DC) CD4+ T cell clusters and other inflammatory cells. DCs can direct bone protective T-regulatory cell (Tregs) responses, or bone destructive T-helper 17 (Th17). The use of exosomes (EXO), natural nanoparticles released by DCs and other cells, are under intense scrutiny in clinical trials for autoimmune diseases and cancer, but no studies to date have harnessed DC-derived EXO to regulate alveolar bone loss in PD. Aim: To determine the ability of custom DC-derived EXO to reprogram immune cell functions of recipient DCs and T cells and mitigate inflammatory bone loss in mice. Methods: Murine bone marrow derived donor DC subtypes, including immune regulatory DCs (regDC), immature DCs (iDC) and immune stimulatory (stimDC) DCs were the source of purified DC EXO. Reg DC EXO were actively loaded with TGFB1/IL10 using ultrasonication. Preliminary in vitro studies of EXO cargo, stability and resistance of cytokine cargo to proteolysis, as well as immune functions and osteoclastogenesis was investigated. The following DC EXO subtypes were then tested in vivo in six groups of mice, in the ligature induced PD model: Group 1, no ligature, Groups 2, 3, 4, 5 and 6, 8 ligature plus gingival injection of, respectively, PBS, regDC EXO, iDC EXO, stimDC EXO and free TGFB1/IL10. Biodistribution and in vivo uptake of EXO by gingival recipient DCs and T cells were tracked. The ability of DC EXO to modulate gingival recipient DC and CD4 T cells and cytokine expression was confirmed. TRAP staining of histological sections measured osteoclast number, while bone loss volume was measured in 3-D by micro-CT. Results: Injected EXO showed a high affinity for gingival site of inflammatory bone loss. RegDC EXO containing TGFb/IL-10 cargo, protected cargo against proteolytic degradation and were taken up by recipient DCs and T cells in vivo, promoting Tregs, while inhibiting Th17 recruitment and inhibiting bone loss. In contrast, EXO subtypes lacking TGFb/IL-10 or free TGFB/IL-10 did not shift the Treg-Th17 balance and did not inhibit bone loss. Mechanistically, a key role for TGFb1 in induction of Tregs by regDC EXO was found using blocking antibodies to TGFb and/or IL-10. T.E.M. analysis revealed TGFb1 localized in the EXO lumen and in the transmembrane domain, which sustained signaling in recipient DCs. Blocking experiments revealed that sustainable prolonged TGFb1 signaling required initial interaction between regDCs EXO and TGFBR1 complex on acceptor cells, followed by internalization of regDC EXO with TGFB1-TGFBR1 complex for sustained SMAD2/3 phosphorylation. Conclusion: This is the first study to demonstrate the efficacy of DCs exosomes for inhibition of experimental bone loss and the cellular immune mechanisms involved. This provides the basis for a future novel immunotherapeutic strategy for PD in humans.