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Long-term load duration induces N-cadherin down-regulation and loss of cell phenotype of nucleus pulposus cells in a disc bioreactor culture.

ABSTRACT: Long-term exposure to a mechanical load causes degenerative changes in the disc nucleus pulposus (NP) tissue. A previous study demonstrated that N-cadherin (N-CDH)-mediated signalling can preserve the NP cell phenotype. However, N-CDH expression and the resulting phenotype alteration in NP cells under mechanical compression remain unclear. The present study investigated the effects of the compressive duration on N-CDH expression and on the phenotype of NP cells in an ex vivo disc organ culture. Porcine discs were organ cultured in a self-developed mechanically active bioreactor for 7 days. The discs were subjected to different dynamic compression durations (1 and 8 h at a magnitude of 0.4 MPa and frequency of 1.0 Hz) once per day. Discs that were not compressed were used as controls. The results showed that long-term compression duration (8 h) significantly down-regulated the expression of N-CDH and NP-specific molecule markers (Brachyury, Laminin, Glypican-3 and Keratin 19), attenuated Alcian Blue staining intensity, decreased glycosaminoglycan (GAG) and hydroxyproline (HYP) contents and decreased matrix macromolecule (aggrecan and collagen II) expression compared with the short-term compression duration (1 h). Taken together, these findings demonstrate that long-term load duration can induce N-CDH down-regulation, loss of normal cell phenotype and result in attenuation of NP-related matrix synthesis in NP cells.

SUBMITTER: Li P

PROVIDER: S-EPMC5408662 | biostudies-literature | 2017 Apr

REPOSITORIES: biostudies-literature

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Long-term load duration induces N-cadherin down-regulation and loss of cell phenotype of nucleus pulposus cells in a disc bioreactor culture.

Li Pei P Zhang Ruijie R Wang Liyuan L Gan Yibo Y Xu Yuan Y Song Lei L Luo Lei L Zhao Chen C Zhang Chengmin C Ouyang Bin B Tu Bing B Zhou Qiang Q

Bioscience reports 20170428 2

Long-term exposure to a mechanical load causes degenerative changes in the disc nucleus pulposus (NP) tissue. A previous study demonstrated that N-cadherin (N-CDH)-mediated signalling can preserve the NP cell phenotype. However, N-CDH expression and the resulting phenotype alteration in NP cells under mechanical compression remain unclear. The present study investigated the effects of the compressive duration on N-CDH expression and on the phenotype of NP cells in an <i>ex vivo</i> disc organ cu ...[more]

PMID: 28351894

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Project description:BackgroundThe development of mechanically active culture systems helps increase the understanding of the role of mechanical stress in intervertebral disc (IVD) degeneration. Motion segment cultures allow for preservation of the native IVD structure, and adjacent vertebral bodies facilitate the application and control of mechanical loads. The purpose of this study was to establish loading and organ culture methods for rabbit IVD motion segments to study the effect of static load on the whole disc organ.MethodsIVD motion segments were harvested from rabbit lumbar spines and cultured in no-loading 6-well plates (control conditions) or custom-made apparatuses under a constant, compressive load (3 kg, 0.5 MPa) for up to 14 days. Tissue integrity, matrix synthesis, and the matrix gene expression profile were assessed after 3, 7, and 14 days of culturing and compared with those of fresh tissues.ResultsThe results showed that ex vivo culturing of motion segments preserved tissue integrity under no-loading conditions for 14 days whereas the static load gradually destroyed the morphology after 3 days. Proteoglycan contents were decreased under both conditions, with a more obvious decrease under static load, and proteoglycan gene expression was also downregulated. However, under static load, immunohistochemical staining intensity and collagen Type II alpha 1 (COL2A1) gene expression were significantly enhanced (61.54 ± 5.91, P = 0.035) and upregulated (1.195 ± 0.040, P = 0.000), respectively, compared with those in the controls (P < 0.05). In contrast, under constant compression, these trends were reversed. Our initial results indicated that short-term static load stimulated the synthesis of collagen Type II alpha 1; however, sustained constant compression led to progressive degeneration and specifically to a decreased proteoglycan content.ConclusionsA loading and organ culture system for ex vivo rabbit IVD motion segments was developed. Using this system, we were able to study the effects of mechanical stimulation on the biology of IVDs, as well as the pathomechanics of IVD degeneration.

Project description:STUDY DESIGN:Profiling proteins expressed in the nucleus pulposus of fetal intervertebral disc (IVD). PURPOSE:To evaluate the molecular complexity of fetal IVDs not exposed to mechanical, traumatic, inflammatory, or infective insults to generate improved knowledge on disc homeostasis. OVERVIEW OF LITERATURE:Low back pain is the most common musculoskeletal disorder, causing a significant reduction in the quality of life, and degenerative disc disorders mainly contribute to the increasing socioeconomic burden. Despite extensive research, the causative pathomechanisms behind degenerative disc disorders are poorly understood. Precise molecular studies on the intricate biological processes involved in maintaining normal disc homeostasis are needed. METHODS:IVDs of nine fetal specimens obtained from medical abortions were used to dissect out the annulus fibrosus and nucleus pulposus under sterile operating conditions. Dissected tissues were transferred to sterile Cryovials and snap frozen in liquid nitrogen before transporting to the research laboratory for protein extraction and further liquid chromatography tandem mass spectrometry (LC-MS/ MS) analysis. Collected data were further analyzed using Gene Functional Classification Tool in DAVID and STRING databases. RESULTS:A total of 1,316 proteins were identified through LC-MS/MS analysis of nine fetal IVD tissues. Approximately 247 proteins present in at least four fetal discs were subjected to further bioinformatic analysis. The following 10 clusters of proteins were identified: collagens, ribosomal proteins, small leucine-rich proteins, matrilin and thrombospondin, annexins, protein disulfide isomerase family proteins and peroxiredoxins, tubulins, histones, hemoglobin, and prolyl 4-hydroxylase family proteins. CONCLUSIONS:This study provides fundamental information on the proteome networks involved in the growth and development of healthy fetal discs in humans. Systematic cataloging of proteins involved in various structural and regulatory processes has been performed. Proteins expressed most abundantly (collagen type XIV alpha 1 chain, biglycan, matrilin 1, and thrombospondin 1) in their respective clusters also elucidate the possibility of utilizing these proteins for potential regenerative therapies.

Dataset Information

Long-term load duration induces N-cadherin down-regulation and loss of cell phenotype of nucleus pulposus cells in a disc bioreactor culture.

Publications

Long-term load duration induces N-cadherin down-regulation and loss of cell phenotype of nucleus pulposus cells in a disc bioreactor culture.

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