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Research Papers

The Role Of Extracellular Matrix Elasticity and Composition In Regulating the Nucleus Pulposus Cell Phenotype in the Intervertebral Disc: A Narrative Review

[+] Author and Article Information
Priscilla Y. Hwang

Department of Biomedical Engineering,
Duke University,
136 Hudson Hall, Box 90281,
Durham, NC 27708
e-mail: ph17@duke.edu

Jun Chen

Department of Orthopaedic Surgery,
Duke University,
375 Medical Science Research Building, Box 3093,
Durham, NC 27710
e-mail: junchen@duke.edu

Liufang Jing

Department of Biomedical Engineering,
Duke University,
136 Hudson Hall, Box 90281,
Durham, NC 27708
e-mail: lfjing@duke.edu

Brenton D. Hoffman

Department of Biomedical Engineering,
Duke University,
136 Hudson Hall, Box 90281,
Durham, NC 27708
e-mail: brenton.hoffman@duke.edu

Lori A. Setton

Department of Biomedical Engineering and Orthopaedic Surgery,
Duke University,
136 Hudson Hall, Box 90281,
Durham, NC 27708
e-mail: setton@duke.edu

1Corresponding author.

Contributed by the Bioengineering Division of ASME for publication in the JOURNAL OF BIOMECHANICAL ENGINEERING. Manuscript received October 3, 2013; final manuscript received December 13, 2013; accepted manuscript posted December 26, 2013; published online February 5, 2014. Editor: Beth Winkelstein.

J Biomech Eng 136(2), 021010 (Feb 05, 2014) (9 pages) Paper No: BIO-13-1460; doi: 10.1115/1.4026360 History: Received October 03, 2013; Revised December 13, 2013; Accepted December 26, 2013

Intervertebral disc (IVD) disorders are a major contributor to disability and societal health care costs. Nucleus pulposus (NP) cells of the IVD exhibit changes in both phenotype and morphology with aging-related IVD degeneration that may impact the onset and progression of IVD pathology. Studies have demonstrated that immature NP cell interactions with their extracellular matrix (ECM) may be key regulators of cellular phenotype, metabolism and morphology. The objective of this article is to review our recent experience with studies of NP cell-ECM interactions that reveal how ECM cues can be manipulated to promote an immature NP cell phenotype and morphology. Findings demonstrate the importance of a soft (<700 Pa), laminin-containing ECM in regulating healthy, immature NP cells. Knowledge of NP cell-ECM interactions can be used for development of tissue engineering or cell delivery strategies to treat IVD-related disorders.

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References

Figures

Grahic Jump Location
Fig. 1

The intervertebral disc is situated between vertebral bodies in the spinal column, and acts to support loads, provide flexibility, and dissipate energy in the spine. The disc is comprised of distinct anatomic zones: the anulus fibrosus (AF), nucleus pulposus (NP), and cartilage endplates. The AF consists of concentric lamella of highly-aligned collagen fibers, with cells typically aligned along the fiber direction. The NP is a gelatinous, highly-hydrated tissue, with cells typically exhibiting rounded, unaligned morphologies. Staining is safranin O and fast green. Images of specific cell morphology in each region were obtained via light microscopy.

Grahic Jump Location
Fig. 2

Porcine NP cells preferentially attach and spread upon laminin-containing substrates. (a) Fraction of adherent cells remaining attached to ECM substrates following application of centrifugal detachment force. Higher numbers of NP cells resist detachment when adherent to laminin ligands (isoforms LM-332, LM-511, LM-111), as compared to collagen and fibronectin ECM ligands ((b) and (c)) NP cell spreading and NP cell shape dynamics on ECM substrates. NP cells on laminin isoforms LM-332 and LM-511 spread rapidly and to a greater extent as compared to other matrix substrates NP cells on laminin isoforms. Additionally, NP cells lost their original shape factor as the cells spread on laminin isoforms (error bars omitted for clarity, significant effects of substrate and time were detected via two-way ANOVA, p < 0.05; substrates not labeled with same letter were statistically different. (LM = laminin, FN = fibronectin, BSA = bovine serum albumin, CM = cultured media) Specific methods described in detail and image adapted from Gilchrist et al. 2011 [83].

Grahic Jump Location
Fig. 3

Soft laminin-containing substrates promote immature NP cells to form multicell clusters, while retaining cell dimensions and rounded morphology. Actin immunostaining of immature porcine NP cell behavior on BME-functionalized polyacrylamide gel (BME-PAAm) (100 and 290 Pa), “soft” BME (300 Pa), and “stiff” BME (2900 Pa) substrates after 7 days of culture (green = actin (phalloidin), red = cell nuclei (propidium iodide), bar = 100 μm). Specific methods and image adapted from Gilchrist et al. 2011 [70].

Grahic Jump Location
Fig. 4

Changes in immature porcine NP cell morphology on substrates. (a) Immature porcine NP cells spread out on stiff BME but maintain rounded morphology on soft BME. (b) Immature porcine NP cells have significantly decreased cell velocity on soft BME upon formation of cell cluster. On stiff BME, NP cells continue to send out lamellipodia and filopodia as if sensing the underlying substrate. (c) Immature porcine NP cells transfected with GFP-actin display distinct actin fibers as the cell spreads and attaches to the underlying stiff BME substrate. On soft BME, NP cells remain rounded and do not have any actin stress fiber formation. Methods for substrate development were adapted from Gilchrist et al. 2011 [70]. Imaging and analysis performed using the Olympus VivaView Fluorescent Incubator Microscope and Metamorph Software, in collaboration with the Duke Light Microscopy Core Facility.

Grahic Jump Location
Fig. 5

Matrix production and changes in gene expression in immature porcine NP cells cultured upon various substrates (a) Matrix production in immature porcine NP cells on soft BME substrates is significantly higher (*p < 0.05, One-way ANOVA, with Tukey's post hoc analysis) than matrix production in NP cells on all other substrates. (b) Gene expression was calculated relative to values for 18 s mRNA and normalized by values for stiff BME. mRNA values for NP-specific and NP-matrix-related markers were higher in immature porcine NP cells on soft BME substrates compared to all other substrates Methods for biochemical assays are adapted from Gilchrist et al. 2011 [70], and methods for gene expression are adapted from Tang et al. 2012 [34]

Grahic Jump Location
Fig. 6

Treatment of immature porcine NP cells with Rho GTPase inhibitors, ROCK (Y27632) and Rac1 (NSC23766). (a) Immature porcine NP cells are unable to form cell clusters on soft BME substrates after treatment with ROCK inhibitor but not Rac1 inhibitor (green = phalloidin, red = propidium iodide, bar = 50 μm). (b) Decreased matrix production in NP cells after 4-day treatment with ROCK inhibitor on soft BME substrates (*p < 0.01, **p < 0.05, Two-way ANOVA with Tukey's post hoc analysis); matrix production is unaffected by ROCK inhibitor in NP cells on all other substrates. (c) Gene expression was calculated relative to values for 18 s mRNA and normalized by values for stiff BME. mRNA values for NP-specific and NP-matrix-related markers are decreased in immature porcine NP cells after 4-day treatment with ROCK inhibitor on soft BME substrates. Methods for biochemical assays are adapted from Gilchrist et al. 2011 [70], and methods for gene expression are adapted from Tang et al. 2012 [34].

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