# In Vitro Synovial Membrane 3D Model Developed by Volumetric Extrusion Bioprinting
## 4 Discussion
Therefore, we selected an Additive Manufacturing technology termed volumetric extrusion that allowed us to exert a finer control over GelMA material flow and printability. Furthermore, we implemented a dual gelation/crosslinking approach based on accurate thermal control of the printhead and the substrate, and layer-by-layer light irradiation, greatly improving material shape retention and stacking. As a result, we were able to fabricate relevant-sized 3D structures with microarchitectural features closely matching CAD design, with an average deviation of less than 1% in fiber diameter and pore size. Atthe same time, cell viability and functionality were proven not to be negatively affected by the bioprinting process in the selected conditions. However, volumetric extrusion also provides some limitations. In fact, cylindrical nozzles are preferred to conical ones to control the flow rate. This, combined with the need for longer needles to print inside a multi-well plate format, led to some restrictions regarding minimum fiber diameter. As observed during preliminary bioprinting tests, lower-size needles (200 um inner diameter) resulted in low cell viability and critical failures in material deposition. Cell aggregates likely formed within the nozzle, leading to inhomogeneous material extrusion and frequent clogging phenomena. It would be challenging to fabricate structures with finer internal fibers. For this purpose, it is crucial to provide optimal conditions tailored for the specific bioink formulation. This is achieved not only by fine-tuning the bioprinting parameters but also by selecting the most suitable dispensing technology and process conditions. The human synovial cell K4 IM line that we used was embedded in the biomaterial and 3D bioprinted without detrimental effect on viability, showing that the bioprinted hydrogel provides a proper microenvironment for cell survival. We already demonstrated that this cell line might be a useful tool to test the effects of different biological compounds used in the treatment of OA [33] and to evaluate different cellular pathways in OA studies [34]. However, although the use of cell lines presents many advantages (i.e., easy to manipulate in culture; no ethical concerns associated with the use of animal and human tissue; reproducibility of results also obtained from different laboratories), it represents one of the major limitations of this study. Further research will be performed by using primary human cells, expecting to include also the macrophage component. [PERSON] et al. compared human synovioocytes isolated from OA patients at two different passages (Passage 1 versus Passage 5) [35]. They showed that synovial macrophages are present at Passage 1, but not at Passage 5. Although the K4 IM cell line has a fibroblast-like phenotype without the macrophage component, in our study, it was a tool to prepare a synovial membrane 3D model in vitro suitable for further development to study different cellular pathways mechanisms, as well as the effect of pharmacological drugs in OA and other pathologies in which synovial membrane is involved. Further studies will be needed based on our PoC to mimic more accurately the structure and cellular composition of the anatomical synovium, possibly based on the coordinated bioprinting of multiple materials and cell populations or on the integration of bioprinting and electrospinning/writing technologies to provide zonal differentiation through construct microarchitecture, as successfully proven by other studies [36; 37].