Study on the biological characteristics of human nucleus pulposus decellularized extracellular matrix scaffolds with different degrees of degeneration

English Keywords:Nucleus pulposus  Degrees of degeneration  Nucleus pulposus extracellular matrix removal  Tissue engineering

English Abstract:

  【Abstract】 Objectives: To explore the biological characteristics of human nucleus pulposus decellularized extracellular matrix(NP-dECM) scaffolds with different degrees of degeneration(Ⅱ, Ⅲ, Ⅳ), and to select the NP-dECM scaffold with the best biological characteristics. Methods: Nucleus pulposus mesenchymal stem cells(NPMSCs) were isolated by collagenase type Ⅱ digestion method. Cell adhesion, cell morphology and growth of NPMSCs were observed under microscope. The cell surface markers CD73, CD90, CD105, CD34, CD45, HLA-DR were detected by flow cytometry, and the differentiation ability of NPMSCs was observed by adipogenic, osteogenic and chondrogenic induction, so as to confirm that they were the seed cells needed for this study. Human NP-dECM scaffolds with different degrees of degeneration were prepared by physical, stain remover and nuclease(group Ⅱ-D, Ⅲ-D and Ⅳ-D). The decellular effect of NP-dECM scaffold was evaluated by visual observation, quantitative and qualitative detection of DNA and extracellular matrix components, and SEM. The cytocompatibility of human NP-dECM scaffolds with different degrees of degeneration was evaluated using CCK-8 method and living/dead cells in vitro. Results: Human NPMSCs isolated and cultured from the nucleus pulposus tissue grew adherent, and the cells were short spindle shaped and spindle-shaped. The third passage of human NPMSCs expressed high levels of surface markers such as CD73, CD90 and CD105, and low levels of surface markers such as CD34, CD45 and HLA-DR. It met the criteria of mesenchymal stem cells. Human NP-dECM scaffolds with three degrees of degeneration remained intact, and the scaffolds of each group were loose and porous under electron microscope. HE staining, DAPI staining and DNA determination showed that the NP-dECM scaffold effectively removed cell components in the three degrees of degeneration. The quantitative and qualitative detection results of GAG and HYP showed that ECM was well retained, and the GAG retention was in the order of group Ⅱ-D > group Ⅲ-D > group Ⅳ-D from high to low. There was no statistical significance in HYP content between groups Ⅱ-D and Ⅲ-D, which was higher than that in group Ⅳ-D. The results of CCK-8 and live/dead cell staining showed that groups Ⅱ-D, Ⅲ-D and Ⅳ-D showed good cytocompatibility. Conclusions: The Ⅱ degree degeneration of human nucleus pulposus tissue is the best choice for the preparation of human NP-dECM scaffold.