Methods: The decellularization was achieved using a developed closed sonication treatment system for 10 hrs, and continued with a washing process for 5 days. For the control, a simple immersion treatment was set as a benchmark to compare the decellularization efficiency. Histological and biochemical assays were conducted to investigate the cell removal and retention of the vital extracellular matrix. Surface ultrastructure of the prepared scaffolds was evaluated using scanning electron microscope at 5,000× magnification viewed from cross and longitudinal sections. In addition, the biomechanical properties were investigated through ball indentation testing to study the stiffness, residual forces and compression characteristics. Statistical significance between the samples was determined with p-value =0.05.
Results: Histological and biochemical assays confirmed the elimination of antigenic cellular components with the retention of the vital extracellular matrix within the sonicated scaffolds. However, there was a significant removal of sulfated glycosaminoglycans. The surface histoarchitecture portrayed the preserved collagen fibril orientation and arrangement. However, there were minor disruptions on the structure, with few empty micropores formed which represented cell lacunae. The biomechanical properties of bioscaffolds showed the retention of viscoelastic behavior of the scaffolds which mimic native tissues. After immersion treatment, those scaffolds had poor results compared to the sonicated scaffolds due to the inefficiency of the treatment.
Conclusion: In conclusion, this study reported that the closed sonication treatment system had high capabilities to prepare ideal bioscaffolds with excellent removal of cellular components, and retained extracellular matrix and biomechanical properties.
MATERIALS AND METHODS: Chondrocyte/agarose constructs were treated with varying concentrations of TNFα (0.1-100 ng/ml) and cultured at 5 and 21 % oxygen tension for 48 h. In separate experiments, constructs were subjected to dynamic compression (15 %) and treated with TNFα (10 ng/ml) and/or L-NIO (1 mM) at 5 and 21 % oxygen tension using an ex vivo bioreactor for 48 h. Markers for catabolic activity (NO, PGE2) and tissue remodelling (GAG, MMPs) were quantified by biochemical assay. ADAMTS-5 and MMP-13 expression were examined by real-time qPCR. 2-way ANOVA and a post hoc Bonferroni-corrected t test were used to analyse data.
RESULTS: TNFα dose-dependently increased NO, PGE2 and MMP activity (all p
METHODS: We established an in vitro OA culture model by exposing human chondrocytes to proinflammatory cytokine and interleukin (IL)-1β for 36 hours prior to treatment with the herbals: C. quadrangularis, W. somnifera, and the combination of the two herbals. Cell viability, toxicity, and gene expression of OA modifying agents were examined. In addition, expression of survivin, which is crucial for cell growth, was analyzed. In vivo work on osteotomized rats studied the bone and cartilage regenerative effects of C. quadrangularis, W. somnifera, and the combination therapy.
RESULTS: Exposure of chondrocytes to IL-1β induced significant toxicity and cell death. However, herbal treatment alleviated IL-1β induced cell toxicity and upregulated cell growth and proliferation. C. quadrangularis inhibited gene expression of cytokines and matrix metalloproteinases, known to aggravate cartilage and bone destruction, and augmented expression of survivin by inhibiting p38 MAPK. Interestingly, osteotomized rats treated with C. quadrangularis drastically enhanced alkaline phosphatase and cartilage tissue formation as compared to untreated, W. somnifera only, or the combination of both herbals.
CONCLUSION: Our findings demonstrate for the first time the signaling mechanisms regulated by C. quadrangularis and W. somnifera in OA and osteogenesis. We suggest that the chondroprotective effects and regenerative ability of these herbals are via the upregulation of survivin that exerts inhibitory effects on the p38 MAPK signaling pathway. These findings thus validate C. quadrangularis as a potential therapeutic for rheumatic disorders.