［INTRODUCTION］Our research has focused on acquiring highly functional nucleus pulposus (NP) cells for the development of regenerative medicine products aimed at treating intervertebral disc degeneration. Previously, we reported that culturing medullary nucleus cells on laminin-coated dishes enhances the expression of Tie2 and type II collagen (Col2). In this study, we aim to reveal a more detailed analysis of the underlying mechanism.
［METHODS］Human NP tissues (nucleus pulposus tissues) were collected from patients undergoing surgery after obtaining approval from the authors’ Institutional Ethics Review Board (17R173) and informed consent from the patients. The samples were collected under three conditions: (1) immediately after tissue culture, (2) after one week of tissue culture on two types of dishes – non-coated (group N) and laminin-coated (MAX iMatrix-511, 0.5 μL/mL) (group L) – with culture conditions of 20% fetal bovine serum (FBS), 37°C, 5% O2, 5% CO2, and 3 x 104 cells/5 mL, and (3) continued culture on group N and group L for an additional week. We compared the acquired cells in terms of Tie2 expression and evaluated intracellular extracellular matrix (ECM) products, specifically type II collagen (Col2), using flow cytometry. Additionally, we assessed PI3K/p85 and ER stress/JNK levels through Western blotting. For statistical analysis, we considered p < 0.05 as statistically significant, and the results are presented as mean ± standard deviation.
［RESULTS］Tie2 expression significantly increased in group L at both 1 and 2 weeks after tissue culture. We observed enhanced expression of PI3K/p85 in group L, as confirmed by western blot analysis.
［DISCUSSION］Our findings suggest that the use of laminin-coated dishes during the culture of medullary nucleus cells primarily affects proliferation signals. Ongoing RNA-seq analysis for five of the samples used in this study will provide a more comprehensive understanding of our results. In the future, we intend to further investigate the interaction between integrin receptors and laminin in medullary nucleus cells.