Static compression down-regulates N-cadherin expression and facilitates loss of cell phenotype of nucleus pulposus cells in a disc perfusion culture

Mechanical compression often induces degenerative changes of disc nucleus pulposus (NP) tissue. It has been indicated that N-cadherin (N-CDH)-mediated signaling helps to preserve the NP cell phenotype. However, N-CDH expression and the resulting NP-specific phenotype alteration under the static compression and dynamic compression remain unclear. To study the effects of static compression and dynamic compression on N-CDH expression and NP-specific phenotype in an in vitro disc organ culture. Porcine discs were organ cultured in a self-developed mechanically active bioreactor for 7 days and subjected to static or dynamic compression (0.4 MPa for 2 h once per day). The noncompressed discs were used as controls. Compared with the dynamic compression, static compression significantly down-regulated the expression of N-CDH and NP-specific markers (laminin, brachyury, and keratin 19); decreased the Alcian Blue staining intensity, glycosaminoglycan and hydroxyproline contents; and declined the matrix macromolecule (aggrecan and collagen II) expression. Compared with the dynamic compression, static compression causes N-CDH down-regulation, loss of NP-specific phenotype, and the resulting decrease in NP matrix synthesis.

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Dynamic Compression Promotes the Matrix Synthesis of Nucleus Pulposus Cells Through Up-Regulating N-CDH Expression in a Perfusion Bioreactor Culture

Cellular Physiology and Biochemistry ◽

10.1159/000488616 ◽

2018 ◽

Vol 46 (2) ◽

pp. 482-491 ◽

Cited By ~ 4

Author(s):

Yichun Xu ◽

Hui Yao ◽

Pei Li ◽

Wenbin Xu ◽

Junbin Zhang ◽

...

Keyword(s):

Nucleus Pulposus ◽

Dynamic Compression ◽

Akt Pathway ◽

Bioreactor Culture ◽

Nucleus Pulposus Cells ◽

Matrix Synthesis ◽

Static Compression ◽

Matrix Deposition ◽

The Matrix

Background/Aims: An adequate matrix production of nucleus pulposus (NP) cells is an important tissue engineering-based strategy to regenerate degenerative discs. Here, we mainly aimed to investigate the effects and mechanism of mechanical compression (i.e., static compression vs. dynamic compression) on the matrix synthesis of three-dimensional (3D) cultured NP cells in vitro. Methods: Rat NP cells seeded on small intestinal submucosa (SIS) cryogel scaffolds were cultured in the chambers of a self-developed, mechanically active bioreactor for 10 days. Meanwhile, the NP cells were subjected to compression (static compression or dynamic compression at a 10% scaffold deformation) for 6 hours once per day. Unloaded NP cells were used as controls. The cellular phenotype and matrix biosynthesis of NP cells were investigated by real-time PCR and Western blotting assays. Lentivirus-mediated N-cadherin (N-CDH) knockdown and an inhibitor, LY294002, were used to further investigate the role of N-CDH and the PI3K/Akt pathway in this process. Results: Dynamic compression better maintained the expression of cell-specific markers (keratin-19, FOXF1 and PAX1) and matrix macromolecules (aggrecan and collagen II), as well as N-CDH expression and the activity of the PI3K/Akt pathway, in the 3D-cultured NP cells compared with those expression levels and activity in the cells grown under static compression. Further analysis showed that the N-CDH knockdown significantly down-regulated the expression of NP cell-specific markers and matrix macromolecules and inhibited the activation of the PI3K/Akt pathway under dynamic compression. However, inhibition of the PI3K/Akt pathway had no effects on N-CDH expression but down-regulated the expression of NP cell-specific markers and matrix macromolecules under dynamic compression. Conclusion: Dynamic compression increases the matrix synthesis of 3D-cultured NP cells compared with that of the cells under static compression, and the N-CDH-PI3K/Akt pathway is involved in this regulatory process. This study provides a promising strategy to promote the matrix deposition of tissue-engineered NP tissue in vitro prior to clinical transplantation.

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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

Bioscience Reports ◽

10.1042/bsr20160582 ◽

2017 ◽

Vol 37 (2) ◽

Cited By ~ 8

Author(s):

Pei Li ◽

Ruijie Zhang ◽

Liyuan Wang ◽

Yibo Gan ◽

Yuan Xu ◽

...

Keyword(s):

Nucleus Pulposus ◽

Mechanical Load ◽

Dynamic Compression ◽

Blue Staining ◽

Cell Phenotype ◽

Alcian Blue Staining ◽

Bioreactor Culture ◽

Down Regulation ◽

Load Duration

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.

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Engineered Peptide-Functionalized Hydrogels Modulate the RNA Transcriptome of Human Nucleus Pulposus Cells In Vitro

10.1101/2021.03.05.434094 ◽

2021 ◽

Author(s):

Marcos N. Barcellona ◽

Julie E. Speer ◽

Liufang Jing ◽

Munish C. Gupta ◽

Jacob M. Buchowski ◽

...

Keyword(s):

Nucleus Pulposus ◽

Intracellular Signaling ◽

Drug Targets ◽

A Priori ◽

Enrichment Analysis ◽

Gene Set Enrichment Analysis ◽

Cell Phenotype ◽

Nucleus Pulposus Cells ◽

Catabolic State

AbstractDegeneration and aging of the nucleus pulposus (NP) of the intervertebral disc (IVD) is accompanied by alterations in NP cell phenotype marked by a shift towards a fibroblast-like, catabolic state. We have recently demonstrated an ability to manipulate the phenotype of human adult degenerative NP cells through 2D culture upon poly(ethylene glycol) (PEG) based hydrogels dually functionalized with integrin- and syndecan-binding laminin-mimetic peptides (LMPs). In the present study, we sought to understand the transcriptomic changes elicited through NP cell interactions with the LMP-functionalized hydrogel system (LMP gel) by examining targets of interest a priori and by conducting unbiased analysis to identify novel mechanosensitive targets. The results of gene specific analysis demonstrated that the LMP gel promoted adult degenerative NP cells to upregulate 148 genes including several NP markers (e.g. NOG and ITGA6) and downregulate 277 genes, namely several known fibroblastic markers. Additionally, 13 genes associated with G protein-coupled receptors, many of which are known drug targets, were identified as differentially regulated following culture upon the gel. Furthermore, through gene set enrichment analysis we identified over 700 pathways enriched amongst the up- and downregulated genes including pathways related to cell differentiation, notochord morphogenesis, and intracellular signaling. Together these findings demonstrate the global mechanobiological effects induced by the LMP gel and confirm the ability of this substrate to modulate NP cell phenotype.

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Cell Phenotype and Matrix Regeneration of Human Nucleus Pulposus Cells of Different Ages in a Laminin-Rich Pseudo-3D Culture System in Vitro

Global Spine Journal ◽

10.1055/s-0034-1376648 ◽

2014 ◽

Vol 4 (1_suppl) ◽

pp. s-0034-1376648-s-0034-1376648

Author(s):

X. Tang ◽

L. Jing ◽

L. A. Setton ◽

W. J. Richardson ◽

R. D. Fitch ◽

...

Keyword(s):

Nucleus Pulposus ◽

Culture System ◽

3D Culture ◽

Cell Phenotype ◽

Nucleus Pulposus Cells ◽

Different Ages ◽

3D Culture System

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Η χρήση του πεπτιδίου Ec της ισόμορφης IGF-1Ec στην επισκευή χόνδρινων βλαβών

10.12681/eadd/43322 ◽

2018 ◽

Author(s):

Νικόλαος Αρμακόλας

Keyword(s):

Polymerase Chain Reaction ◽

Trypan Blue ◽

Quantitative Polymerase Chain Reaction ◽

Blue Staining ◽

Alcian Blue Staining ◽

Chain Reaction ◽

Polymerase Chain ◽

Tgf Β1 ◽

Invasion Assays

Το πεπτίδιο Ec (PEc) του IGF-1Ec (IGF-1Ec) επάγει την κινητοποίηση των ανθρωπίνων μεσεγχυματικών βλαστικών κυττάρων (hMSC) και ενεργοποιεί την εξωκυτταρική κινάση 1 και 2 (ERK 1/2) διαφόρων κυττάρων. Σκοπός της παρούσας μελέτης ήταν η διερεύνηση της επιδρασης του PEc στην κινητοποίηση και τη διαφοροποίηση των hMSCs, καθώς και η δυνατότητα εφαρμογής του σε συνδυασμό με τον TGF-β1 (TGF-β1) στην επιδιόρθωση του αρθρικού χόνδρου. Τα αποτελέσματα της εξωγενούς χορήγησης του ΡΕc και του ΤGF-β1, ξεχωριστά και σε συνδυασμό, σε hMSCs εκτιμήθηκαν χρησιμοποιώντας trypan blue assay, reverse transcription-quantitative polymerase chain reaction, western blot analysis, Alcian blue staining, wound healing assays και migration/invasion assays. Προσδιορίστηκε ότι το PEc εμπλέκεται στη διαδικασία διαφοροποίησης των hMSCs προς υαλώδη χόνδρο. Η χορήγηση PEc ή / και TGF-β1 σε hMSCs έδειξε συγκρίσιμη εναπόθεση χονδρικής θεμέλειας ουσίας. Ακόμα, η χορήγηση του ΡΕc σε συνδυασμό με τον ΤGF-β1 συσχετίστηκε με μια σημαντική αύξηση στην κινητοποίηση των hMSC σε σύγκριση με την χορήγηση μόνο του TGF-β1 ή του ΡEc (Ρ <0,05). Επομένως, το ΡΕc φαίνεται να διευκολύνει in vitro την κινητοποίηση των hMSC και την διαφοροποίηση τους προς χονδροκύτταρα, ενισχύοντας το ρόλο του ΤGF-β1.

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Ligustrazine Prevents Intervertebral Disc Degeneration via Suppression of Aberrant TGFβ Activation in Nucleus Pulposus Cells

BioMed Research International ◽

10.1155/2019/5601734 ◽

2019 ◽

Vol 2019 ◽

pp. 1-9

Author(s):

Shufen Liu ◽

Yuhao Cheng ◽

Yuqi Tan ◽

Jingcheng Dong ◽

Qin Bian

Keyword(s):

Growth Factor ◽

Mouse Model ◽

Intervertebral Disc ◽

Nucleus Pulposus ◽

Disc Degeneration ◽

Intervertebral Disc Degeneration ◽

Ex Vivo ◽

Tissue Growth ◽

Nucleus Pulposus Cells

Objectives. Aberrant transforming growth factor β (TGFβ) activation is detrimental to both nucleus pulposus (NP) cells and cartilage endplates (CEPs), which can lead to intervertebral disc degeneration (IDD). Ligustrazine (LIG) reduces the expression of inflammatory factors and TGFβ1 in hypertrophic CEP to prevent IDD. In this study, we investigate the effects of LIG on NP cells and the TGFβ signaling. Design. LIG was injected to the lumbar spinal instability (LSI) mouse model. The effect of LIG was evaluated by intervertebral disc (IVD) score in the LSI mouse model. The expression of activated TGFβ was examined using immunostaining with pSmad2/3 antibody. The upright posture (UP) rat model was also treated and evaluated in the same manner to assess the effect of LIG. In ex vivo study, IVDs from four-week old mice were isolated and treated with 10−5, 10−6, and 10−7 M of LIG. We used western blot to detect activated TGFβ expression. TGFβ-treated human nucleus pulposus cells (HNPCs) were cotreated with optimized dose of LIG in vitro. Immunofluorescence staining was performed to determine pSmad2/3, connective tissue growth factor (CCN2), and aggrecan (ACAN) expression levels. Results. IVD score and the percentage of pSmad2/3+ NP cells were low in LIG-treated LSI mice in comparison with LSI mice, but close to the levels in the Sham group. Similarly, LIG reduced the overexpression of TGFβ1 in NP cells. The inhibitory effect of LIG was dose dependent. A dose of 10−5 M LIG not only strongly attenuated Smad2/3 phosphorylation in TGFβ-treated IVD ex vivo but also suppressed pSmad2/3, CCN2, and ACAN expression in TGFβ-treated NP cells in vitro. Conclusions. LIG prevents IDD via suppression of TGFβ overactivation in NP cells.

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Differentiation of Human Wharton's Jelly Cells Toward Nucleus Pulposus-Like Cells After Coculture with Nucleus Pulposus Cells In Vitro

Tissue Engineering Part A ◽

10.1089/ten.tea.2011.0186 ◽

2012 ◽

Vol 18 (1-2) ◽

pp. 167-175 ◽

Cited By ~ 20

Author(s):

Dike Ruan ◽

Yan Zhang ◽

DeLi Wang ◽

Chao Zhang ◽

JianHong Wu ◽

...

Keyword(s):

Nucleus Pulposus ◽

Wharton’S Jelly ◽

Nucleus Pulposus Cells ◽

Wharton's Jelly ◽

Wharton’S Jelly Cells

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Duhuo Jisheng Decoction inhibits SDF-1-induced inflammation and matrix degradation in human degenerative nucleus pulposus cells in vitro through the CXCR4/NF-κB pathway

Acta Pharmacologica Sinica ◽

10.1038/aps.2018.36 ◽

2018 ◽

Vol 39 (6) ◽

pp. 912-922 ◽

Cited By ~ 8

Author(s):

Zong-chao Liu ◽

Zhen-long Wang ◽

Chen-yi Huang ◽

Zhi-jiang Fu ◽

Yong Liu ◽

...

Keyword(s):

Nucleus Pulposus ◽

Matrix Degradation ◽

Nucleus Pulposus Cells

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The response of nucleus pulposus cell senescence to static and dynamic compressions in a disc organ culture

Bioscience Reports ◽

10.1042/bsr20180064 ◽

2018 ◽

Vol 38 (2) ◽

Cited By ~ 1

Author(s):

Jianmin Shi ◽

Lianglong Pang ◽

Shouguo Jiao

Keyword(s):

Organ Culture ◽

Nucleus Pulposus ◽

Dynamic Compression ◽

Nucleus Pulposus Cell ◽

Cell Senescence ◽

Degenerative Changes ◽

Mechanical Stimuli ◽

Static Compression

Mechanical stimuli obviously affect disc nucleus pulposus (NP) biology. Previous studies have indicated that static compression exhibits detrimental effects on disc biology compared with dynamic compression. To study disc NP cell senescence under static compression and dynamic compression in a disc organ culture, porcine discs were cultured and subjected to compression (static compression: 0.4 MPa for 4 h once per day; dynamic compression: 0.4 MPa at a frequency of 1.0 Hz for 4 h once per day) for 7 days using a self-developed mechanically active bioreactor. The non-compressed discs were used as controls. Compared with the dynamic compression, static compression significantly promoted disc NP cell senescence, reflected by the increased senescence-associated β-galactosidase (SA-β-Gal) activity, senescence-associated heterochromatic foci (SAHF) formation and senescence markers expression, and the decreased telomerase (TE) activity and NP matrix biosynthesis. Static compression accelerates disc NP cell senescence compared with the dynamic compression in a disc organ culture. The present study provides that acceleration of NP cell senescence may be involved in previously reported static compression-mediated disc NP degenerative changes.

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