Modeling periodontal host-microbe interactions using vascularized gingival connective tissue equivalents

Abstract

Gingival connective tissue and its vasculature play a crucial role in the host's immune response against the periodontal microbiome and serve as a bridge between the oral and systemic environments. However, there is a lack of representative models that mimic the complex features of vascularized gingival connective tissue and its interaction with the periodontal microbiome, hindering our understanding of periodontal health and disease. Towards this pursuit, we present the characterization of vascularized gingival connective tissue equivalents (CTEs) as a model to study the interactions between oral biofilm colonizers and gingival tissues in healthy and diseased states. Whole-mount immunolabelling and label-free confocal reflectance microscopy of human fibrin-based matrix embedded with gingival fibroblasts and microvascular endothelial cells demonstrated the generation of bi-cellular vascularized gingival CTEs. Next, we investigated the response of the vascularized gingival CTEs to early, intermediate, and late oral biofilm colonizers. Despite colonization, the early colonizers did not elicit any significant change in the production of the cytokines and chemokines by the CTEs representative of the commensal and homeostatic state. In contrast, intermediate and late colonizers representing a transition to a diseased state exhibited connective tissue and vascular invasion, and elicited a differential immune response accompanied by increased monocyte migration. The culture supernatants produced by the vascularized gingival CTEs in response to early and intermediate colonizers polarized macrophages towards an immunomodulatory M2-like phenotype which activates and protects the host, while the late colonizers polarized towards a pro-inflammatory M1-like phenotype. Lastly, in silico analysis showed a high strength of associations between the proteins and transcripts investigated with periodontitis and vascular diseases. In conclusion, the vascularized gingival CTEs provide a biomimetic in vitro platform to study host-microbiome interactions and innate immune response in periodontal health and diseased states, which potentially paves the way toward the development and assessment of novel periodontal therapeutics.

Microphysiological Modeling of Gingival Tissues and Host‐Material Interactions Using Gingiva‐on‐Chip

“…Further, inclusion of endothelial cells would enable the recapitulation of microvasculature and incorporation of immune cells. [ 51 ] Similarly, for the ulcer‐on‐chip, inclusion of keratinocytes and a wound can enable closer recapitulation of oral ulcers and its application for wound healing studies. These improvisations could then pave way to study the impact of the microbiome and external agents on the innate immune response of the gingival tissues and its modulation using oral‐care products, as well as of effective transmucosal drug and nutraceutical delivery.…”

Biofabrication Strategies for Oral Soft Tissue Regeneration

“…These cell‐derived reticular ECM fibers are interspersed between the amorphous native fibrin matrix and organized around the vasculature. [ 123 ]…”

“…Incorporating vasculature within gingival tissue constructs provides vital functionality with the prospects of enabling rapid inosculation and integration with host vasculature, oxygenation, nutrient transport, removal of metabolic waste, and host innate and adaptive defense. [ 125 ] Tissue constructs with microvascular networks have been fabricated using 3D culture and self‐assembly of endothelial and mural cells, [ 123,126–127 ] 3D bioprinting, [ 128–130 ] and microfluidic organ‐on‐chip systems. [ 131–132 ] Some key considerations for incorporating vasculature within the gingival constructs include the source of endothelial cells, ranging from microvascular origin or large vessels.…”

“…[ 131–132 ] Some key considerations for incorporating vasculature within the gingival constructs include the source of endothelial cells, ranging from microvascular origin or large vessels. [ 123,127,130,132–133 ] Additionally, incorporating mesenchymal stromal or mural cells is essential for vascular maturation and function. [ 127 ] The source of stromal cells and their ratio to endothelial cells can play a crucial role in angiogenic events, vascular maturation, and barrier function.…”

“…Integrating blood vessels into tissue equivalents remains an ongoing challenge, demanding optimization of cell culture conditions, biomaterial selection, fabrication methodologies, and comprehensive clinical investigations. [ 138 ] Makkar et al., [ 123 ] recently demonstrated the fabrication of vascularized gingival connective tissue equivalents using primary gingival fibroblasts and endothelial cells of microvascular origin embedded within a fibrin‐based matrix (Figure 6A–C).…”