Abstract
Dental caries destroys the enamel and dentine of the tooth and as infection progresses can lead to pulpal inflammation, infection, and severe pain. An inflamed pulp is characterised by a high amount of cytokines including tumour necrosis factor (TNF)-a, interleukin (IL)-1a, IL-1b, IL-4, IL-6, IL-8, IL-17, and IL-23 resulting in a complex cellular immune response and uncontrolled inflammation. Subsequently, making it difficult for the pulp to heal and restore normal function. Pulp tissue has an innate capacity to heal if inflammation is mild, but in cases of severe inflammation the pulp inevitably becomes non-vital. Controlling the intensity of inflammation would enable the pulp to repair. Chronic inflammatory diseases such as psoriasis, inflammatory bowel diseases and rheumatoid arthritis are characterised by a dysregulated immune response with relatively high cytokine levels. Whilst IL-23 inhibitors have been successfully used in the treatment of these chronic inflammatory diseases, this approach is yet to be attempted in cases of pulp inflammation. This thesis aimed to investigate the ability of an IL-23 inhibitor to function as a dental pulp immunotherapeutic medicament in vitro and in vivo.
Ethical approval for the in vitro study was obtained from the University of Otago Human Ethics Committee (Health) and consultation with Māori has been undertaken through the Ngāi Tahu Research Consultation Committee. In vitro, human dental pulp cells (HDPCs) were isolated and cultured from both non-carious and carious teeth (n = 5, each) using the explant technique. Expression of vimentin was assessed using immunocytochemistry. Cell growth curve, colony forming unit and mineralisation assays were evaluated in both non-carious and carious HDPCs at passage 4. Gene expression of Toll-like receptors (TLR)-2, TLR-4,TLR-9, TNF-a, IL-1b, IL-6, IL-8, IL-17A, IL-17R, IL-23A, nuclear factor- kappa B (NF-kB1), mitogen activated protein kinase (MAPK1), dentine matrix protein (DMP)-1, dentine sialophosphoprotein (DSPP), sex determining region Y-box 2 (SOX2), MKi67 (marker of proliferation) and mesenchymal (vimentin, CD44 and CD105) genes were determined for non- carious and carious HDPCs using quantitative real-time reverse-transcription polymerase chain reaction. Further, we also assessed the gene expression of TLR-2, TLR-4, TLR-9, TNF-a, IL- 1b, IL-6, IL-8, IL-17A, IL-17R, IL-23A, NF-kB1, MAPK1, DMP-1, DSPP, MKi67 and SOX- 2 for non-carious and carious pulp tissue (n = 3, each). This group provided baseline data and served as a reference to observe the trend in the gene expression between primary cell culture (non-carious (nc) HDPCs and carious (c) HDPCs) findings and pulp tissue (non-carious and carious). Furthermore, HDPCs were seeded in a range of concentrations for the IL-23 inhibitor to evaluate cell viability using the MTS assay.
For in vivo studies, ethics was obtained through the Animal Ethics Committee, University of Otago. A Wistar rat pulp exposure model was used (n = 12, ~350 gms). Mechanically exposed pulps were capped with IL-23 inhibitor (n = 6) while controls received sterile saline (n = 6). After 1-, 15- and 30-days mandibular jaws were removed and prepared for histological analysis. The effect of IL-23 inhibitor on pulpal inflammation and healing in comparison to other therapies (MTA, Odontopasteâ) and untreated control in Wistar rat was also investigated.
Findings from the in vitro studies showed that both non-carious and carious HDPCs were successfully isolated and cultured from non-carious and carious human dental pulp tissue. Both HDPC types at passage 4 demonstrated a typical spindle morphology with positive vimentin expression. No statistical difference was observed in the growth characteristics and ability to differentiate into a mineralising phenotype between both non-carious and carious HDPCs. Statistically significant differences were found in colony forming efficiency between the two cell populations (p < 0.05). Significantly higher levels of TLR-2, TLR-4, TLR-9, TNF- a, IL-6, IL-8, IL-17R, IL-23A, NF-kB1, MAPK1, DMP-1, DSPP, SOX2, CD44 and vimentin were detected in carious HDPC cultures compared with ncHDPCs cultures (p < 0.05). We also found higher levels of TLR-2 and TLR-4, IL-6, IL-8, IL-17A, IL-23A, NF-kB1 and MKi67 in inflamed pulp tissue compared with non-carious pulp tissue (p < 0.05). IL-23 inhibitor (5 μg/mL) treatment enhanced the proliferation of carious HDPCs (p < 0.05).
Histological analysis of rat tissue at day one demonstrated inflammation and no mineralised tissue formation in the control and IL-23 inhibitor groups. On day 15, inflammation was observed in both the groups, but the presence of mineralised tissue was only observed in the IL-23 inhibitor treatment group. On day 30, the IL-23 inhibitor group demonstrated the presence of mineralised tissue within the viable pulp with no evidence of inflammation while the control group exhibited inflammation and mineralised tissue. Furthermore, qPCR results demonstrated that inflammatory genes (IL-6 and IL-23A) were expressed at a lower level in the IL-23 inhibitor group compared to Odontopasteâ and MTA groups. Both DMP-1 and BSP genes were expressed at a higher level in IL-23 inhibitor and MTA groups.
In summary, the aforementioned results demonstrate that IL-23 blockade induced the proliferation of carious HDPCs with an inflammatory phenotype, and in a rat pulp exposure model treatment promoted mineralisation. Clinically, IL-23 inhibition has the potential to modulate inflammation and promote mineralisation.