Establishment of a three-dimensional in vitro peri-implant bone-mucosa composite model

Background: Peri-implant health depends on the complex interactions between the dental implant, surrounding soft/hard tissues and the oral microbial environment. However, existing 2D and monoculture models fail to replicate this complexity, limiting their clinical relevance. Therefore, this study aimed to develop a clinically relevant 3D in vitro model that integrates oral soft tissue, hard tissue and a titanium implant in a 3D setup to accurately replicate the peri-implant environment. In addition, the model was designed to integrate bacterial biofilms, in order to mimic peri-implant infections. Methods: As a hard tissue component, osteoblast-covered HA/TCP scaffold structures were developed and merged with peri-implant mucosa, resulting in a 3D in vitro peri-implant bone-mucosa composite model. The composite model was then cultivated for 2, 7 and 14 days. At each time point, histological analysis, live/dead staining and collagen immunofluorescence staining were performed to assess its structural integrity, osteoblast viability and bone ECM characteristics. To demonstrate proof-of concept for suitability in simulating implant infection, an oral multispecies biofilm was integrated on top of the implant in the peri-implant bone-mucosa model. Results: Cell viability and osteoblastic phenotype were maintained throughout the study period. Microscopic and histological analyses confirmed a homogenous structure, with a stratified epithelium overlying collagen-embedded human gingival fibroblasts closely connected to the underlying scaffold structure interspersed with bone cells. Combined with a living multi-species biofilm, this model represents all essential components observed in a peri-implant infection. Conclusions: By combining oral soft tissue, hard tissue and a titanium implant in a 3D setup, this model represents the first and most complex model for evaluating innovative implant materials and novel treatment strategies as well as studying the progression of peri-implant diseases. Incorporating different biofilms could enhance the model's clinical relevance, enabling the study of pro-inflammatory responses to bacterial infections in a setting that includes both soft and hard tissue.