Inhibition Of Vascularized Cartilage Regeneration By Loaded Curcumin Scaffold

Autologous chondrocytes are important seed cells for cartilage regeneration.Due to their limited sources,autologous chondrocytes cannot be popularized.Bone marrow stem cells are known for their strong proliferative ability and multi-directional differentiation.At present,ectopic ossification of cartilage constructed by bone marrow stem cells in subcutaneous environment is an internationally recognized problem.It is generally believed that ectopic ossification is mainly related to vascularization.Therefore,the key to solve this problem might be vascularization inhibition.Curcumin has anti-angiogenesis effect.By inhibiting vascular endothelial growth factor and its corresponding receptors,curcumin significantly inhibits the angiogenesis of solid tumor in Ehrlich ascites cancer cells in tumor-bearing mice,indicating that it is indeed an angiogenesis inhibitor.Objective The aims of this study were to 1)investigate the regeneration of chondrocytes in the presence of curcumin in-vivo and clarify how to regulate curcumin loading materials to inhibit angiogenesis without affecting the cartilage regeneration activities of stem cells,2)optimize the curcumin release from loaded materials and curcumin concentrations in order to inhibit the vascularization without affecting stem cell cartilage regeneration,3)determine whether the chondrocytes loaded with curcumin could be used to construct chondrocytes in-vitro.Methods The curcumin loaded materials were first divided into 2 groups(serum-free medium and PBS)and treated at 3 different time-point stages.For the PBS group,the absorbance rate was measured at the beginning of each stage to detect the curcumin concentration standard line.For the three different time points(24 h,48 h,and72 h),tests for concentrations of curcumin solution,cell scratch test,cell cycle,and apoptosis tests were carried.The outcome cells of each stage(of serum-free medium group)were then implanted subcutaneously into nude mice.The materials were then harvested after 3,6,and 12 weeks and tests like HE,S/O staining,CD31 immunohistochemical,CD31 immunofluorescence staining were performed.Gelatin and chondrocytes were set up as control group(PC group)and mixture of gelatin/curcumin and chondrocytes as experiment group(Exp group).After that,The above constructs were implanted subcutaneously into the nude mice.The samples were then harvested 3,6 weeks after implantation for the evaluation of cartilage formation.Results The results of cell scratch test showed that the apoptosis of vascular endothelial cells began at the curcumin concentration of 30 um while bone marrow stem cells apoptosis began at 40 um.Therefore,the curcumin concentration of 30 um can be considered as appropriate since it can inhibit the growth of vascular endothelial cells without affecting the proliferation of bone marrow stem cells.Conclusion With the appropriate concentration,curcumin loaded scaffolds can inhibit the vascular endothelial cells without any obvious effects on bone marrow stem cells proliferation.Such loaded scaffolds can clearly inhibit vascularization in the subcutaneous environment.Therefore,they could be successfully used in the construction and tissue engineering of cartilages in subcutaneous animal models.That gives curcumin loaded scaffolds a great potential application prospect.