Background and Object: Articular cartilage is known as an avascular tissue, which has per se anti-angiogenic factors. Several molecules in cartilage extracellular matrix (ECM) are reported to exhibit antiangiogenic activity, some of which already went through clinical trial for harmaceutical purpose such as chondromodulin-I and type XVIII-derived endostatin. We have found previously that the natural cartilage ECM could be a promising treatment with anti-angiogenic activity. However, wide application of drug necessitates various formula especially in soluble phase. We fabricated evenly distributed water suspension-form biomaterial using the cartilage ECM that could be easily deliverable into the body. In this study, we evaluated whether a water-soluble form of the porcine cartilage-derived ECM biomaterial (PCP-ws, porcine cartilage powder – water suspension) still possesses antiangiogenic property.
Materials and Methods: The slices of cartilage tissues obtained from 6-weeks-old porcine knees were lyophilized and comminuted into a particulate form, which was then decellularized by hypotonic buffer and DNase-I treatments. The decellularized ECM powder was solubilized by pepsin/HCl. The toxicity of the PCP-ws was evaluated on mouse fibroblast cell line (L929), rabbit chondrocytes and human umbilical vein endothelial cells (HUVEC) by MTT assay. The effect of PCP-ws on the function of endothelial cells was evaluated including cell adhesion, migration, proliferation and tube formation ability were performed.
Results: In this study, it is demonstrated that the porcine cartilage powder (PCP) was successfully decellularized and made to a suspension form (PCP-ws) for easily deliverable biomaterial of anti-angiogenic therapy. The viability of L929 and chondrocyte were slightly decreased. In contrast, the viability of HUVEC was severely decreased by PCP-ws in a dose dependent. PCP-ws was prove to be an excellent substrates for inhibiting endothelial cell adhesion, migration and proliferation, particularly for suppression formation of tube-like structure in vitro. In the tube formation assay, the formation of tube network of HUVECs was significantly inhibited by PCP-ws in a dose-dependent manner. Remarkably, the experimental group treated with 5 mg/ml of PCP-ws showed almost no tube-like structure. The length of tube structure decreased approximately by 17 folds, when compared with the untreated group.
Discussion and Conclusion: In this study, it is demonstrated that the porcine cartilage powder (PCP) was successfully decellularized and made to a water suspension form (PCPws) for easily deliverable biomaterial of anti-angiogenic therapy. PCP-ws was prove to be an excellent substrates for inhibiting endothelial cell adhesion, migration and proliferation, particularly for suppression formation of tube-like structure in vitro. Supposedly, PCP-ws have a variety of bioactive factors such as chondromodulin-I, endostatin and sulfated glycosaminoglycan side chains that are known to exist in the articular cartilage. Therefore, these results suggest that PCP-ws has an inhibitory effect on angiogenesis in vitro and could be a useful biomaterial for many applications.