| Rheumatoid Arthritis(RA)is a chronic autoimmune disease with joint erosion.Pannus is a typical pathological product of RA,which mainly includes hyperplastic synovial tissue,new blood vessels and cellulose.Since the disease cannot be completely cured at present,most RA patients need long-term dependence on drugs to relieve symptoms.However,at this stage,drugs have the problems of large side effects and high inefficiency.Hence,the development and exploration of high-efficiency and low-toxic drugs is still a research hotspot in the treatment of RA,but the current RA model(ordinary monolayer cell culture and animal model)is no longer sufficient for the rapid development of high-throughput drug screening.In recent years,organoid models have been gradually applied to drug screening,which provides a new reference for the screening platform of RA treatment drugs.In this study,we propose to construct RA pannus pathological tissues in vitro through 3D bioprinting technology to explore the role of this model in drug screening.This thesis first used M1 type macrophages,synovial fibroblasts(MH7A)and vascular endothelial cells(EA.hy 926)as the research objects to explore the cell communication within RA pannus.First,we need to induce Monocytes from Human Peripheral Blood(THP-1)and identify its characteristic.We detected chemokines CCL2,Interleukin(IL)-1β,TNF-α and chemokine receptor CCR7 gene expression by RT-PCR,analyzed the expression of CD80 in M1 macrophages by flow cytometry,and measured IL-1β and IL-8 secreted protein content by ELISA.We finally chose200 ng/ml Phorbol-12-myristate-13-acetate(PMA)-induced THP-1 as the M1 macrophage model.The serum-free supernatant of THP-1 derived M1 macrophages was collected to simulate the inflammatory environment.It mixed with RPMI-1640 complete medium at the ratio of 1:1 to stimulate MH7 A for 24 h.After RT-PCR detection,we found that the expression of IL-1β,IL-8 and VEGF gene of MH7 A is significantly increased.In addition,we explored the effect of THP-1 derived M1 macrophage serum-free supernatant on the proliferation of MH7 A and EA.hy 926.The results showed that the proliferation of MH7 A cells was not affected by the serum-free inflammatory environment of THP-1 derived M1 macrophages,while the proliferation of EA.hy 926 was significantly inhibited,suggesting that there is information exchange between cells.The above results lay the foundation for the composition of a cell co-culture model in the next step.The three-dimensional co-culture model has a closer cell composition and behavior of human physiology than the traditional two-dimensional planar culture model,exhibiting similar responses to in vivo pharmacological effects.Therefore,it can be used as a drug screening model to evaluate the efficacy and safety of drugs in vitro.In this study,we provided a 3D cell co-culture environment.The bio-ink was composed of MH7 A,EA.hy 926 and gelatin/alginate hydrogel,and a 3D space required for cell growth was constructed using bioprinting technology.During the7-day culture process,we observed the cell viability in the 3D scaffold,and the cell proliferation rate was steadily increasing.In addition,we evaluated the interaction between cells in the 3D printed scaffold.We found that as time increases,the cells would assemble and the size of the sphere increased.When it comes to 3D pannus model,we used TNF-α to stimulate MH7 A and EA.hy 926 in advance to simulate the pathological state of RA pannus tissue.Through ELISA detection,we found that the expression of angiopoietin(ANG)proteins in the pannus model increased over time.However,when we tested the first-choice RA drug methotrexate in this model,we found that the angiogenic protein content has decreased.In summary,the results of this paper indicated that there was information exchange between cells in pannus,and the cell co-culture model better mimiced the pathological structure in vivo.We further used 3D cell bioprinting technology to provide a three-dimensional pathological environment for cell co-cultivation,which could be more accurate to construct RA pannus tissue in vitro.This 3D co-cultured pannus model with biological characteristics may provide an effective platform for the research and development of RA therapeutics. |
