The Stimulatory Effect of Notochordal Cell-Conditioned Medium in a Nucleus Pulposus Explant Culture

Vries, Stefan A.H. de; Doeselaar, Marina van; Meij, Björn P.; Tryfonidou, Marianna A.; Ito, Keita

doi:10.1089/ten.tea.2015.0121

Cited by 26 publications

(27 citation statements)

References 37 publications

(51 reference statements)

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“…This open mechanobiology question is critically important because NCs are considered signaling centers that orchestrate IVD growth and maintenance and a source of NP progenitor cells. [30][31][32][33] NCs and their soluble factors may also prevent painful IVD degeneration by stimulating GAG production in mature sNPCs and mesenchymal stem cells, 32,[34][35][36][37] by preventing apoptosis, and/or by inhibiting neurite cell growth and angiogenesis. 36,[38][39][40] This work used a mouse whole IVD organ culture model and determined that static hyperosmotic loading was capable of inducing a transition of NCs to sNPCs and identified important osmoregulatory and mechanotransduction proteins involved in this process (Fig.…”

Section: Discussionmentioning

confidence: 99%

Hyperosmolarity induces notochordal cell differentiation with aquaporin3 upregulation and reduced N‐cadherin expression

Palacio-Mancheno

Evashwick‐Rogler

Laudier

et al. 2017

Journal Orthopaedic Research

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The nucleus pulposus (NP) of intervertebral discs (IVD) undergoes dramatic changes with aging including loss of its gelatinous structure and large, vacuolated notochordal cells (NCs) in favor of a matrix-rich structure populated by small NP cells (sNPCs). NP maturation also involves a loading-pattern shift from pressurization to matrix deformations, and these events are thought to predispose to degeneration. Little is known of the triggering events and cellular alterations involved with NP maturation, which remains a fundamental open spinal mechanobiology question. A mouse IVD organ culture model was used to test the hypotheses that hyperosmotic overloading will induce NP maturation with transition of NCs to sNPCs while also increasing matrix accumulation and altering osmoregulatory and mechanotransductive proteins. Results indicated that static hyperosmolarity, as might occur during growth, caused maturation of NCs to sNPCs and involved a cellular differentiation process since known NC markers (cytokeratin-8, -19, and sonic hedgehog) persisted without increased cell apoptosis. Osmosensitive channels Aquaporin 3 (Aqp3) and transient receptor potential vanilloid-4 (TRPV4) expression were both modified with altered osmolarity, but increased Aqp3 with hyperosmolarity was associated with NC to sNPC differentiation. NC to sNPC differentiation was accompanied by a shift in cellular mechanotransduction proteins with decreased N-cadherin adhesions and increased Connexin 43 connexons. We conclude that hyperosmotic overloading can promote NC differentiation into sNPCs. This study identified osmolarity as a triggering mechanism for notochordal cell differentiation with associated shifts in osmoregulatory and mechanotransductive proteins that are likely to play important roles in intervertebral disc aging. © 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 36:788-798, 2018.

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Section: Discussionmentioning

confidence: 99%

Hyperosmolarity induces notochordal cell differentiation with aquaporin3 upregulation and reduced N‐cadherin expression

Palacio-Mancheno

Evashwick‐Rogler

Laudier

et al. 2017

Journal Orthopaedic Research

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“…The bovine NP culture potentially represents a space-efficient and relatively inexpensive approach to screen for molecular therapeutics that can be used in human medicine. Stable bovine and human NP cultures were used for investigating the effects of ECM anabolics such as LinkN, notochordal cell-conditioned medium [52] and bone morphogenic protein 7 (BMP-7) [53]. However, the stable bovine NP culture exhibits neither an inflammatory phenotype nor ECM degradation.…”

Section: Discussionmentioning

confidence: 99%

An Inflammatory Nucleus Pulposus Tissue Culture Model to Test Molecular Regenerative Therapies: Validation with Epigallocatechin 3-Gallate

Krupková

Hlavna

Tahmasseb

et al. 2016

IJMS

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Organ cultures are practical tools to investigate regenerative strategies for the intervertebral disc. However, most existing organ culture systems induce severe tissue degradation with only limited representation of the in vivo processes. The objective of this study was to develop a space- and cost-efficient tissue culture model, which represents degenerative processes of the nucleus pulposus (NP). Intact bovine NPs were cultured in a previously developed system using Dyneema jackets. Degenerative changes in the NP tissue were induced either by the direct injection of chondroitinase ABC (1–20 U/mL) or by the diffusion of interleukin-1 beta (IL-1β) and tumor necrosis factor alpha (TNF-α) (both 100 ng/mL) from the culture media. Extracellular matrix composition (collagens, proteoglycans, water, and DNA) and the expression of inflammatory and catabolic genes were analyzed. The anti-inflammatory and anti-catabolic compound epigallocatechin 3-gallate (EGCG, 10 µM) was employed to assess the relevance of the degenerative NP model. Although a single injection of chondroitinase ABC reduced the proteoglycan content in the NPs, it did not activate cellular responses. On the other hand, IL-1β and TNF-α significantly increased the mRNA expression of inflammatory mediators IL-6, IL-8, inducible nitric oxide synthase (iNOS), prostaglandin-endoperoxide synthase 2 (PTGS2) and matrix metalloproteinases (MMP1, MMP3, and MMP13). The cytokine-induced gene expression in the NPs was ameliorated with EGCG. This study provides a proof of concept that inflammatory NP cultures, with appropriate containment, can be useful for the discovery and evaluation of molecular therapeutic strategies against early degenerative disc disease.

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“…There is compelling evidence to suggest that NC cells are the early progenitors for adult NP cells and that the disappearance of cells with the recognizable NC‐like morphology (ie, large, rounded and vacuolated cells) and the onset of DIVD are associated chronologically, which may imply causation . As such, NC cells are thought to have substantial therapeutic application and may promote a healthy phenotype in adult NP cells of several species and NP tissue explants …”

Section: Introductionmentioning

confidence: 99%

“…2,3 As such, NC cells are thought to have substantial therapeutic application and may promote a healthy phenotype in adult NP cells of several species [4][5][6] and NP tissue explants. 7 The NP matrix consists of an irregular mesh of type II collagen and elastin fibers with abundant and highly hydrophilic proteoglycans, most significantly aggrecan. [8][9][10][11][12] DIVD commonly results in a change from type II to type I collagen and a decrease in proteoglycan content leading to dehydration of the NP, 13 an increase in expression of matrix degrading enzymes, [14][15][16][17] an upregulation of inflammatory cytokines [18][19][20] and an increase in cell senescence and apoptosis.…”

Section: Introductionmentioning

confidence: 99%

“…However, current culture systems for NC cells, such as the gold standard alginate bead culture, do not allow for proliferation of NC cell populations. 5,6,[32][33][34][35][36] Thus even in commonly used animals where NC cells are retained throughout the majority of lifespan, such as porcine, 7,34,37 canine 24,36 and rat, 38,39 multiple disc levels or multiple animals are required to pool sufficient cells for experimentation. This represents a significant limitation both in current studies investigating NC cell phenotype and function, as well as for future potential clinical translation of human cells.…”

Section: Introductionmentioning

confidence: 99%

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An optimized culture system for notochordal cell expansion with retention of phenotype

et al. 2018

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Background Notochordal (NC) cells display therapeutic potential in treating degeneration of the intervertebral disc. However, research on their phenotype and function is limited by low‐cell yields and a lack of appropriate methodology for cell expansion. Utilizing porcine cells, this study aimed to develop an optimized culture system which allows expansion of NC cell populations with retention of phenotype. Methods Post‐natal porcine and foetal human nucleus pulposus tissue was compared histologically and expression of known NC cell marker genes by porcine NC cells was analyzed. Porcine NC cells were isolated from six‐week post‐natal discs and cultured in vitro under varied conditions: (1) DMEM vs αMEM; (2) laminin‐521, fibronectin, gelatin and uncoated tissue culture‐treated polystyrene (TCP); (3) 2% O 2 vs normoxia; (4) αMEM (300 mOsm/L) vs αMEM (400 mOsm/L); (5) surface stiffness of 0.5 and 4 kPa and standard TCP. Adherence, proliferation, morphology and expression of NC cell markers were assessed over a 14‐day culture period. Results Native porcine nucleus pulposus tissue demonstrated similar morphology to human foetal tissue and porcine NC cells expressed known notochordal markers (CD24, KRT8, KRT18, KRT19, and T). Use of αMEM media and laminin‐521‐coated surfaces showed the greatest cell adherence, proliferation and retention of NC cell morphology and phenotype. Proliferation of NC cell populations was further enhanced in hypoxia (2%) and phenotypic retention was improved on 0.5 kPa culture surfaces. Discussion Our model has demonstrated an optimized system in which NC cell populations may be expanded while retaining a notochordal phenotype. Application of this optimized culture system will enable NC cell expansion for detailed phenotypic and functional study, a major advantage over current culture methods described in the literature. Furthermore, the similarities identified between porcine and human NC cells suggest this system will be applicable in human NC cell culture for investigation of their therapeutic potential.

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The Stimulatory Effect of Notochordal Cell-Conditioned Medium in a Nucleus Pulposus Explant Culture

Cited by 26 publications

References 37 publications

Hyperosmolarity induces notochordal cell differentiation with aquaporin3 upregulation and reduced N‐cadherin expression

Hyperosmolarity induces notochordal cell differentiation with aquaporin3 upregulation and reduced N‐cadherin expression

An Inflammatory Nucleus Pulposus Tissue Culture Model to Test Molecular Regenerative Therapies: Validation with Epigallocatechin 3-Gallate

An optimized culture system for notochordal cell expansion with retention of phenotype

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