Disco intervertebral “on-a-chip”: estabelecimento de um modelo in vitro revolucionário para o estudo da lombalgia

The IVDisCHIP specific objectives (SO) are to: SO1: Identify the best biomaterials and formulations for developing artificial ECM to biofabricate IVD-on-a-chip models (WP1). Optimize biomaterials based on gellan gum, gelatin, silk fibroin and others to develop photo-crosslinkable hydrogels and formulations suitable for 3D printing of on-a-chip models. The hydrogels should: 1) enable to create stiffness gradients, 2) be printable, 3) support cell viability and differentiation, and 4) be biocompatible and biodegradable, while some of them should also 5) be immunomodulatory and 6) be non- or anti-angiogenic. Fully characterize the hydrogels regarding structure, mechanical properties, injectability, gelation time, swelling behavior, degradation, and cytocompatibility. SO2: Design the chips and optimize the bioinks and 3D printing conditions for mechanical biomimicry (gradients) and IVD tissue interfaces (WP2). Investigate different designs for IVD-on-a-chip in vitro models, focusing on replicating native stiffness gradients, regional microstructure, and cell morphology and distribution. Optimize bioink development and digital light processing 3D printing conditions to obtain an accurate and reproducible in vitro IVD-on-a-chip model. Thoroughly characterize bioinks and 3D structures in terms of physicochemical and biological properties. SO3: Develop and validate a bioprinted healthy IVD-on-a-chip tissue models (WP3). Create a healthy model using the photo-crosslinked hydrogels and 3D structures from WP1-2, replicating the native stiffness gradient (AF>TZ>NP), regional microstructure (modular design with separate channels and side channels), and regional cell morphology and distribution of IVD (incorporating human-derived healthy NP and AF cells). Targeted control of vascularization in the AF zone using non- or anti-angiogenic materials or molecules, and inflammation in the NP zone using immunomodulatory materials. Optimize fluid perfusion and culture conditions to simulate physiological conditions (normoxia and hypoxia). These models provide a physiologically relevant platform for effectively evaluating cell- or biomaterial-based tissue engineered strategies. SO4: Develop and validate bioprinted inflammatory and degenerative IVD-on-a-chip models in a personalized manner (WP4)(using patients own cells). Create physiologically relevant representations of both the initial (immune) and later (degenerative) stages of the disease. Incorporate inflammatory mediators in the healthy model directly through the NP channel or by the NP side channel to simulate an early stage of degeneration (immune model), while controlling vascularization. Incorporate human degenerated NP and AF cells at lower densities compared to the healthy model in the degenerative model, while altering the stiffness gradient (NP stiffness closer to AF) to mimic the later stage of IDD. Induce vascularization in the AF zone by perfusion of angiogenic molecules either directly through the AF channel or by the AF side channel. Optimize fluid perfusion and culture conditions to simulate pathological conditions. Test anti-inflammatory drugs (immune and degenerative models) and anti-angiogenic molecules (degenerative model) as proof-of-concept studies. These models facilitate drugs testing at reducing inflammation and vascularization levels, while also enabling the study of key processes involved in IDD initiation and establishment and identification of potential therapeutic targets.