Periodontal disease is globally prevalent and ultimately leads to the destruction of alveolar bone and tooth loss. Tissue engineering approaches seeking to regenerate alveolar bone have begun to explore the use of stem-cell laden hydrogels for minimally invasive surgery within small and spatially complex maxilla-oral defects. However, the oral environment is unique when compared to other sites for bone regeneration in the body, with the presence of periodontal tissues and oral bacteria in the local environment potentially affecting oral healthcare therapies. Herein, we have designed and fabricated a facile 3D in vitro model of the bone-dentine interface to elucidate the effect of the dentine surface and oral bacteria on human mesenchymal stem cells (hMSCs) encapsulated in a hydrogel matrix. The model is fabricated via 3D bioprinting, as it provides high levels of automation and scalable additive-manufacture of 3D constructs with spatio-temporal control over biomaterial deposition. Before incorporating hMSCs into the model, the properties of the bioink and methodology for preparation of the dentine disc were investigated. Thereafter, the viability, spatial distribution, and proliferative capacity of hMSCs within the bioprinted constructs were characterised, with hMSCs further found to interact with the dentine surface whilst remaining anchored within the bioink. The model is then used to investigate the effect of a dentine substrate on bone tissue engineering within the bioink. Assays developed to assess hMSC fate revealed that dentine substrate induces osteogenic differentiation within the bioink. Analysis of the engineered tissues revealed that extracellular matrix production was primarily occurring within the bioink at the hydrogel-media interface, i.e, distal to the bone-dentine interface, and the resulting mineral phase was confirmed as being biologically derived hydroxyapatite. These results suggest that endogenous tissues affect hMSC-based bone tissue engineering and are important to consider in preclinical investigations of periodontal regenerative therapies. Finally, Fusobacterium periodonticum was incorporated to evaluate the effect of oral bacteria in the model. Initially, 2D cocultures were established to understand morphological and viability changes for both hMSCs and F. periodonticum. Next, multiple methods were employed for the fabrication of 3D culture models that included F. periodonticum. The most suitable approach for inoculation of F. periodonticum in 3D was then used in coculture with bioprinted hMSCs. This 3D coculture model was then characterized with respect to cell morphologies and viabilities to explore the effect of infection in a 3D environment, establishing a robust model for investigation of oral healthcare therapies.