Bioreactor for investigating the effect of controlled mechanical stimulations on periodontal ligament 3D constructs: design, developement and validation tests

Periodontal ligament (PDL), connecting alveolar bone and tooth root, is crucial for mechanical stabilization and mastication-related forces absorption. The capacities deteriorate if PDL is affected by periodontitis, which is ranked as the sixth most prevalent disease worldwide, affecting approximately 10% of adults. It is characterized by the progressive degeneration of periodontium, eventually leading to the detachment of teeth from their alveolar cavity. In the field of periodontal research, studies have demonstrated that PDL cells possess the ability to regenerate periodontal tissue when exposed to specific physiological stimuli (e.g., tension, compression, and shear stress) in an ex vivo environment. Indeed, tissue engineering could represent a promising strategy for treating periodontitis; however, due to the PDL complexity, any regeneration strategy should entail studying specific signals that guide the formation and remodeling of the PDL. In this context, the aim of this thesis was to design, develop, and validate gripping systems (GSs) for applying both compression and shear stress to a biomimetic three-layered construct, with an existing uniaxial stretch bioreactor. Each GS, designed in SolidWorks (Dassault Systèmes), consists in one mobile frame coupled with a crankshaft and one fixed frame. Each frame is composed of: 1) a sample holder, made of PDMS (Sylgard 184, Dow Corning, 10:1), which might house up to three constructs at the same time; 2) a rigid part to be coupled with the existing bioreactor, responsible of the transmission of compression or shear stress to the constructs through the PDMS. The sample holders were designed for facilitating the insertion and removal of the constructs. For supporting the geometry design and optimization, computational simulations were conducted using static analysis in SolidWorks. In particular, four different domains were modeled with different mechanical properties, a displacement of 0.2 mm was applied to the mobile frame while the other frame was constrained. For the compression GSs, the deformation transferred to the construct was found to be 70% of the imposed value, uniformly distributed across the sections of the constructs. Concerning the shear stress GSs, values between 1.2 kPa and 1.3 kPa were uniformly obtained on the transversal section of the constructs. As explanatory, the compression GS was manufactured and coupled with a stepper motor, several images were collected, and the strain value was calculated. The results were then compared with the strain values obtained with computational approach. The proposed bioreactor introduces an in vitro controlled 3D dynamic culture environment, allowing the application of various physical stimuli by changing the GS. The computational modelling demonstrated that the GSs allow obtaining uniform stimulations of the construct. The in-house tests proved the ease of assembly and use of the GSs. Moreover, since the proposed GSs are built from modular components and offer tunable stimulation, they are versatile and suitable for several tissue engineering applications. Further characterization and validation tests are ongoing on both the compression and shear stress GS, with the final aim of producing an advanced platform for in vitro investigating the effects of different physical stimuli (tension, compression and shear stress) on 3D constructs for periodontal ligament engineering applications.