RESUMO
The aim of this article is to study how andrographolide-releasing collagen scaffolds influence rabbit articular chondrocytes in maintaining their specific phenotype under inflammatory environment. Physical blending combined with vacuum freeze-drying method was utilized to prepare the andrographolide-releasing collagen scaffold. The characteristics of scaffold including its surface morphology and porosity were detected with environmental scanning electron microscope (ESEM) and a density instrument. Then, the release of andrographolide from prepared scaffolds was measured by UV-visible spectroscopy. Rabbit chondrocytes were isolated and cultured and seeded on andrographolide-releasing collagen scaffolds. Following culture with normal medium for 3 d, seeded chondrocytes were cultured with medium containing interleukin-1 beta (IL-1β) to stimulate inflammation for 7 d. The proliferation, morphology and gene transcription of tested chondrocytes were detected with Alamar Blue assay, fluorescein diacetate (FDA) staining and reverse-transcriptase quantitative polymerase chain reaction (RT-qPCR) test respectively. The results showed that the collagen scaffolds prepared by vacuum freeze-dry possess a high porosity close to 96%, and well-interconnected chambers around (120.7±17.8) μm. The andrographolide-releasing collagen scaffold continuously released andrographolide to the PBS solution within 15 d, and collagen scaffolds containing 2.22% andrographolide significantly inhibit the proliferation of chondrocytes. Compared with collagen scaffolds, 0.44% andrographolide-containing collagen scaffolds facilitate chondrocytes to keep specific normal morphologies following 7 d IL-1β induction. The results obtained by RT-qPCR confirmed this effect by enhancing the transcription of tissue inhibitor of metalloproteinase-1 ( ), collagen II ( ), aggrecan ( ) and the ratio of / collagen I( ), meanwhile, reversing the promoted transcription of matrix metalloproteinase-1 ( ) and matrix metalloproteinase-13 ( ). In conclusion, our research reveals that andrographolide-releasing (0.44%) collagen scaffolds enhance the ability of chondrocytes to maintain their specific morphologies by up-regulating the transcription of genes like , and , while down-regulating the transcription of genes like and which are bad for phenotypic maintenance under IL-1β simulated inflammatory environment. These results implied the potential use of andrographolide-releasing collagen scaffold in osteoarthritic cartilage repair.
RESUMO
PURPOSE: To confirm the adhesion and matrix formation of chondrocytes which were cultured on chitosan beads and to elucidate the difference between the porous chitosan beads and non-porous chitsan beads as scaffold for chondrocytes. MATERIALS AND METHODS: Chondrocytes isolated from rabbit articular cartilage were cultured in vitro on porous and non-porous chitosan bead for 2 weeks. Histochemical (H&E stain, Toluidin blue stain) and scanning electromicroscopic approaches were used to compare the differences between two groups. RESULTS: In both groups, adhesion and proliferation of chondrocytes were observed on scanning electron microscopy. which were more active in the porous chitosan bead group. On histochemical staining with toluidine blue, the porous chitosan bead group showed stronger metachromasia than that of the non-porous chitosan bead. CONCLUSION: It is concluded that both chitosan beads could work as an effective scaffold for culturing chondrocytes, and that porous chitosan bead may be a better scaffold than non-porous chitosan bead because of cavities in former bead.