ObjectiveTo develop an anti-inflammatory poly (lactic-co-glycolic acid) (PLGA) scaffold by loading xanthohumol, and investigate its anti-inflammatory and cartilage regeneration effects in goats. Methods The PLGA porous scaffolds were prepared by pore-causing agent leaching method, and then placed in xanthohumol solution for 24 hours to prepare xanthohumol-PLGA scaffolds (hereinafter referred to as drug-loaded scaffolds). The PLGA scaffolds and drug-loaded scaffolds were taken for general observation, the pore diameter of the scaffolds was measured by scanning electron microscope, the porosity was calculated by the drainage method, and the loading of xanthohumol on the scaffolds was verified by Fourier transform infrared (FTIR) spectrometer. Then the two scaffolds were co-cultured with RAW264.7 macrophages induced by lipopolysaccharide for 24 hours, and the expressions of inflammatory factors [interleukin 1β (IL-1β) and tumor necrosis factor α (TNF-α)] were detected by RT-PCR and Western blot to evaluate the anti-inflammatory properties in vitro of two scaffolds. Bone marrow mesenchymal stem cells (BMSCs) was obtained from bone marrow of a 6-month-old female healthy goat, cultured by adherent method, and passaged in vitro. The second passage cells were seeded on two scaffolds to construct BMSCs-scaffolds, and the cytocompatibility of scaffolds was observed by live/dead cell staining and cell counting kit 8 (CCK-8) assay. The BMSCs-scaffolds were cultured in vitro for 6 weeks, aiming to verify its feasibility of generating cartilage in vitro by gross observation, histological staining, collagen type Ⅱ immunohistochemical staining, and biochemical analysis. Finally, the two kinds of BMSCs-scaffolds cultured in vitro for 6 weeks were implanted into the goat subcutaneously, respectively. After 4 weeks, gross observation, histological staining, collagen type Ⅱ immunohistochemical staining, biochemical analysis, and RT-PCR were performed to comprehensively evaluate the anti-inflammatory effect in vivo and promotion of cartilage regeneration of the drug-loaded scaffolds. Results The prepared drug-loaded scaffold had a white porous structure with abundant, continuous, and uniform pore structures. Compared with the PLGA scaffold, there was no significant difference in pore size and porosity (P>0.05). FTIR spectrometer analysis showed that xanthohumol was successfully loaded to PLGA scaffolds. The in vitro results demonstrated that the gene and protein expressions of inflammatory cytokines (IL-1β and TNF-α) in drug-loaded scaffold significantly decreased than those in PLGA scaffold (P<0.05). With the prolongation of culture, the number of live cells increased significantly, and there was no significant difference between the two scaffolds (P>0.05). The in vitro cartilage regeneration test indicated that the BMSCs-drug-loaded scaffolds displayed smooth and translucent appearance with yellow color after 6 weeks in vitro culture, and could basically maintained its original shape. The histological and immunohistochemical stainings revealed that the scaffolds displayed typical lacunar structure and cartilage-specific extracellular matrix. In addition, quantitative data revealed that the contents of glycosaminoglycan (GAG) and collagen type Ⅱ were not significantly different from BMSCs-PLGA scaffolds (P>0.05). The evaluation of cartilage regeneration in vivo showed that the BMSCs-drug-loaded scaffolds basically maintained their pre-implantation shape and size at 4 weeks after implantation in goat, while the BMSCs-PLGA scaffolds were severely deformed. The BMSCs-drug-loaded scaffolds had typical cartilage lacuna structure and cartilage specific extracellular matrix, and no obvious inflammatory cells infiltration; while the BMSCs-PLGA scaffolds had a messy fibrous structure, showing obvious inflammatory response. The contents of cartilage-specific GAG and collagen type Ⅱ in BMSCs-drug-loaded scaffolds were significantly higher than those in BMSCs-PLGA scaffolds (P<0.05); the relative gene expressions of IL-1β and TNF-α were significantly lower than those in BMSCs-PLGA scaffolds (P<0.05). ConclusionThe drug-loaded scaffolds have suitable pore size, porosity, cytocompatibility, and good anti-inflammatory properties, and can promote cartilage regeneration after implantation with BMSCs in goats.
Objective To prepare a novel hyaluronic acid methacrylate (HAMA) hydrogel microspheres loaded polyhedral oligomeric silsesquioxane-diclofenac sodium (POSS-DS) patricles, then investigate its physicochemical characteristics and in vitro and in vivo biological properties. Methods Using sulfhydryl POSS (POSS-SH) as a nano-construction platform, polyethylene glycol and DS were chemically linked through the “click chemistry” method to construct functional nanoparticle POSS-DS. The composition was analyzed by nuclear magnetic resonance spectroscopy and the morphology was characterized by transmission electron microscopy. In order to achieve drug sustained release, POSS-DS was encapsulated in HAMA, and hybrid hydrogel microspheres were prepared by microfluidic technology, namely HAMA@POSS-DS. The morphology of the hybrid hydrogel microspheres was characterized by optical microscope and scanning electron microscope. The in vitro degradation and drug release efficiency were observed. Cell counting kit 8 (CCK-8) and live/dead staining were used to detect the effect on chondrocyte proliferation. Moreover, a chondrocyte inflammation model was constructed and cultured with HAMA@POSS-DS. The relevant inflammatory indicators, including collagen type Ⅱ, aggrecan (AGG), matrix metalloproteinase 13 (MMP-13), recombinant A disintegrin and metalloproteinase with thrombospondin 5 (Adamts5), and recombinant tachykinin precursor 1 (TAC1) were detected by immunofluorescence staining and real-time fluorescence quantitative PCR, with normal cultured chondrocytes and the chondrocyte inflammation model without treatment as control group and blank group respectively to further evaluate their anti-inflammatory activity. Finally, by constructing a rat model of knee osteoarthritis, the effectiveness of HAMA@POSS-DS on osteoarthritis was evaluated by X-ray film and Micro-CT examination. Results The overall particle size of POSS-DS nanoparticles was uniform with a diameter of about 100 nm. HAMA@POSS-DS hydrogel microspheres were opaque spheres with a diameter of about 100 μm and a spherical porous structure. The degradation period was 9 weeks, during which the loaded POSS-DS nanoparticles were slowly released. CCK-8 and live/dead staining showed no obvious cytotoxicity at HAMA@POSS-DS, and POSS-DS released by HAMA@POSS-DS significantly promoted cell proliferation (P<0.05). In the chondrocyte anti-inflammatory experiment, the relative expression of collagen type Ⅱ mRNA in HAMA@POSS-DS group was significantly higher than that in control group and blank group (P<0.05). The relative expression level of AGG mRNA was significantly higher than that of blank group (P<0.05). The relative expressions of MMP-13, Adamts5, and TAC1 mRNA in HAMA@POSS-DS group were significantly lower than those in blank group (P<0.05). In vivo experiments showed that the joint space width decreased after operation in rats with osteoarthritis, but HAMA@POSS-DS delayed the process of joint space narrowing and significantly improved the periarticular osteophytosis (P<0.05). Conclusion HAMA@POSS-DS can effectively regulate the local inflammatory microenvironment and significantly promote chondrocyte proliferation, which is conducive to promoting cartilage regeneration and repair in osteoarthritis.