# In Vitro Synovial Membrane 3D Model Developed by Volumetric Extrusion Bioprinting
## 4 Discussion  
Synovial tissue plays a fundamental role in the immuno-inflammatory processes characteristic of OA and RA, the most common rheumatic diseases affecting joints [21]. Therefore, understanding the mechanisms that regulate physiological and pathological synovium functions is crucial to shed light on the etiology and development of those diseases, and that might be helpful in discovering more effective therapies to minimize or counteract their progression. Recently, the use of synovial biopsies to investigate pathological mechanisms has been increasingly applied [2], but reliability and culture condition issues limit their use. Recreating an in vitro synovial tissue 3D model would allow the evaluation of the critical pathways in a more biomimetic context and, in the meantime, represent a suitable alternative to 2D in vitro models for drug testing. Our study represents a proof of concept (PoC) towards the possibility of fabricating through volumetric extrusion bioprinting a 3D engineered construct composed of GelMA embedded with a human synovial cell line. The construct could represent the first step towards the development of an in vitro synovial model. To the best of our knowledge, a 3D bioprinted in vitro model of synovial tissue has no precedent in the literature. Previous in vitro attempts at recapitulating the synovium have been limited and primarily based on synovial fluid or synovial fibroblast monoculture or co-culture within 2D and 3D systems or tissue explants [6; 7]. However, those models did not mimic the synovial structure accurately. More advanced applications, such as organoids or multicompartment organ-on-chip [6; 7; 8; 9], significantly improved the in vitro model's relevance. However, they still need to enable the fabrication of a relevant-size, three-dimensional engineered synovium. Only one study, by [PERSON] et al. [22], used 3D bioprinting technology by mixing RA-patients derived synoviocytes (MH/A) and vascular endothelial cells in a gelatin/alginate bioink. Nonetheless, their aim was to create a specific disease model to mimic the pathological characteristics of RA pannus vascular tissue [23]. Interestingly, the authors have selected a grid structure design, as in our study. A grid structure favors cell well-being, guarantees nutrient transport, and promotes a new extracellular matrix (ECM) deposition. For these reasons the grid structure design is a well-established model used to bioprint and biofabricate a wide range of engineered tissues characterized by different internal microarchitectures (i.e., adipose tissue, liver, and neural tissue) [24; 25; 26]. The use of hydrogels in bioprinting has recently emerged as a significant area of research. Hydrogels are the ideal materials for cell encapsulation due to their hydrated state that, on the other hand, provides challenges for generating high-fidelity constructs. Due to its rheological and mechanical features, we utilized GelMA, a widely diffused hydrogel biomaterial for 3D bioprinting. The high versatility of GelMA makes it suitable for recapitulating the soft tissue's natural environment by enhancing stimuli responsiveness and integrating complex biochemical/biomechanical signals [27]. GelMA presents, anyway, some drawbacks in printability due to its high temperature-dependent rheological properties that negatively impact the bioink processability, printing accuracy, and fidelity. To overcome these limitations, it is common to blend GelMA with different polymers or increase its concentration (above 15%). However, these approaches may be both detrimental to cell viability [28; 29]. In this study, we selected a 10% GelMA concentration, which allows better biocompatibility but may present printability issues causing an up-to-twofold increase of the extruded filament size in comparison with the nozzle one or impaired stacking behavior due to discontinuities in the deposited filaments [29; 30; 31; 32].