| Cartilage is one of the most important connective tissues in the human body and its main function in human joints is to lubricate and prevent wearing.Arthritis is one of the most common chronic complications in the skeletal system,and also one of the diseases with a high incidence in the world.Patients with arthritis are often accompanied with pain,swelling,stiffness,and other symptoms,leading to the mobility inconvenience and sometimes even paralysis,which seriously affects the quality of life of patients.Considering these aspects,this paper will demonstrate the fabrication of scaffolding systems based on poly(lactic-co-glycolic acid)(PLGA),and gelatin(Gel)as the porogen,as raw materials using the microfluidic technology.Further,these highly porous microspheres are harbored with chondrocytes and osteoblasts/endothelial cell co-culture in vitro towards constructing the cartilage globular microstructures and osteogenesis-endothelial globular microstructures.In this context,two kinds of composite spherical microstructures are then fixed with hydrogels,respectively to construct a joint model in which the upper layer is cartilage tissue and the lower layer is osteogenic/endothelial tissue.Then,the pathological changes induced by the lipopolysaccharide(LPS)were used to construct the arthritis model.Finally,the drug screening is conducted to explore the medicinal value of the drug and provided a feasible scheme for the treatment of arthritis.Initially,the PLGA-based large porous microspheres are fabricated using a microfluidic technique and characterized.A T-shaped microchannel was constructed from a polyvinyl chloride(PVC)pipe design,with a 2 % PLGA solution,a 6 % Gel solution,and a 2 % polyvinyl alcohol(PVA)solution.Using the principle of microfluidic droplet shearing,the particle size distribution of the large porous microspheres was between 350-700 μm and 10-60 μm,with uniform particle size,large pore size and excellent morphology.The results of Fourier infrared spectroscopy,X-ray diffraction and thermogravimetric analysis,compared to the raw materials showed that,the functional groups,crystal types and glass transition temperatures of macroporous polymer microspheres,respectively,have no significant changes.After 6weeks of incubation for degradation in vitro,the weight loss rate analysis showed less than 90 %.Further,the cells are co-cultured with microspheres to construct spherical microtissues in vitro and the bioefficacy is demonstrated.The effect of microsphere scaffold suspension on the cell growth is investigated.The results showed that microsphere scaffold would affect neither the morphology of cells,nor the proliferation and growth of cells.The experimental results showing the proliferation,adhesion and growth condition on microspheres displayed that with the extension of incubation time,total growing on microsphere support cell proliferation,and growth in excellent condition.Using the laser confocal microscope,static cultivation and dynamic observation after the distribution of cells on the bracket of microspheres,we found that the dynamic culture is more conducive to cell proliferation in the porousmicrospheres scaffolds.After 7 days of dynamic culture,the cells could basically highly populated in the large porous microsphere scaffold and the cells could extend and grow into the interior of the scaffold.Hematoxylin-eosin(HE)staining and protein immunohistochemical staining results showed that the microtissues constructed in vitro could express cartilage specific proteins,indicating the initial success of microtissue construction.In summary,dynamic co-culture can be used to construct chondroglobular and osteoblastic microtissues in vitro.Then,a model of arthritis was constructed in vitro and the bioefficacy studies are performed.The microtissues constructed in vitro were immobilized by hydrogels.It can be seen from the scanning electron microscopy that hydrogels have porous structures that facilitate the exchange of nutrients,metabolic wastes and gases.After the hydrogels are mixed with the microtissues constructed in vitro,the microtissues with osteoblasts/endothelial cells in the lower layer and chondrocytes in the upper layer could be cross-linked through blue light irradiation constructing the microtissues.The tissue structure can be constructed to simulate the joint model,and then the pathological changes can be induced by LPS to construct the arthritis model.CCK-8method is used to investigate the effects of different concentrations of LPS on cell activity.The experimental results showed that LPS concentration of 1 μg/mL had little effect on cell activity and that LPS concentration of 10 μg/mL had little effect on cell activity.After investigating the effects of different concentrations of LPS on the constructed microtissues in different periods,the contents of interleukin-1(IL-1β)and tumor necrosis factor-α(TNF-α)in the microtissue-supernatant are determined by kit.The experimental results showed that the contents of IL-1β and TNF-α in the microtissue-supernatant are the highest after 48 h of dipping in LPS with a concentration of 0.5 μg/mL.Therefore,the optimal concentration of LPS that causes joint lesions is 0.5 μg/mL at the optimal action time of 48 h.Finally,the effects of curcumin on the content of IL-1β and TNF-α in the supernatant of the arthritis model were studied.CCK-8 method was used to detect the toxicity of curcumin at different concentrations on cells.The results of cytotoxicity showed that curcumin with a concentration of 10 μg/mL for 48 h.From the changes in cell morphology and the relative proliferation rate of cells,it could be seen that curcumin of 10 μg/mL had been toxic to cells.When the concentration of curcumin was 8 μg/mL,the cell morphology is not changed and cell growth is not affected.Therefore,8 μg/m L curcumin is used as the optimal concentration.With confocal laser scanning microscope 8 μg/mL of curcumin on joint model preprocessing,after 4h of curcumin be swallowed up by the cell.The experimental results showed that curcumin pretreatment could reduce the levels of IL-1β and TNF-α in the tissues of the arthritis model induced by LPS,and inhibiting the release of pro-inflammatory factors was a protective mechanism for the articular cartilage,which further proved that the joint model constructed in this paper could be used for drug screening studies.In summary,PLGA-based large porous degradable microspheres could be successfully prepared based on the microfluidic technology.Large porous microspheres have good biocompatibility.C518 cells and MC3T3-E1 cells could adhere,grow,and proliferate on the large porous microspheres,which can constructosteoblastic microtissues and chondrospherical microtissues.After the composite hydrogel having been solidified,a joint model with osteoblast/endothelial tissue in the lower layer and cartilage tissue in the upper layer could be constructed.Moreover,the model induced by LPS to construct an arthritis model for curcumin drug screening is successful.It could provide a feasible scheme for constructing a new drug screening model for arthritis. |
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