Investigating Periodontal Mechanobiology at Cellular and Tissue Levels Using Novel In Vitro Bioengineering Techniques

Abstract

The periodontal ligament (PDL) is a specialized connective tissue, that anchors teeth to alveolar bone lining the tooth sockets. PDL is crucial for transmitting mechanical loads from teeth to bone during oral functions like mastication. Several mechanical and biochemical regulators like Rho/ROCK signaling and inflammatory cytokines, intersect to regulate periodontal cellular and tissue behavior during health and disease. Hence, research like the current study, that explores the effects of mechanical loads and inflammatory mediators is key to develop clinical interventions for periodontal applications like regeneration and facilitating efficient orthodontic tooth movement. However, the in vivo complexity and inaccessibility of human PDL has necessitated the use of in vitro models for PDL research. In the present study, aspects of periodontal mechanobiology in both 2- and 3-dimensions have been studied, by employing latest bioengineering advancements to develop multiple novel in vitro PDL models. In chapter 2 of this thesis, I investigate the effects of inflammatory cytokine tumor necrosis factor-alpha (TNF-α) with and without the involvement of the Rho/ROCK signaling pathway on single cells using a novel reference free traction force microscopy technique called as ‘Black dots’. This chapter also demonstrates the presence of the myofibroblast phenotype with alpha smooth muscle actin stress fibers and details myofibroblast properties and their regulation by TNF-α. These crucial findings highlight the link between TNF-α induced alterations in cells’ mechanical and morphometric properties, such as actin cytoskeleton, contractile force and cell area, with functional outcomes like cell differentiation. Understanding cellular mechanisms at the 3-dimensional (3D) level is necessary to gain insights on cell-cell and cell-matrix cross-talks that are closer to in vivo reality. In Chapter 3, I have developed a novel 3D in vitro PDL model that reproduces certain key in vivo periodontal design features and can be potentially used to test drugs, signaling molecules & various biological agents on the PDL. This model is also equipped to apply tensile loads with built-in magnets, thus providing a platform to test tensile loading effects on PDL as during mastication or orthodontic tooth movement. The expected effects of tensile loading on cell number, collagen remodeling, cytoskeletal changes and osteogenic gene expression are also demonstrated using this model. In Chapter 4, using the suspended tissue open microfluidic patterning (STOMP) system, I have spatially patterned tissue constructs with embedded entheses made up of PDL and osteoblastic cells, as an in vitro cellular representation of the in vivo PDL-alveolar bone enthesis. This model provides a potentially valuable platform to investigate cell biomechanics, mechanotransduction and tissue behaviour at the junction of disparate cell types. Overall, the current research project showcases three new in vitro models that can enable new discoveries in the field of periodontics and orthodontics.