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Research Papers

Dynamic Hydrostatic Pressure Regulates Nucleus Pulposus Phenotypic Expression and Metabolism in a Cell Density-Dependent Manner

[+] Author and Article Information
Bhranti S. Shah

Department of Orthopedic Surgery,
Columbia University,
New York, NY 10032

Nadeen O. Chahine

Department of Orthopedic Surgery,
Columbia University,
650 West 168th Street, 14-1408E,
New York, NY 10032;
Department of Biomedical Engineering,
Columbia University,
New York, NY 10032
e-mail: noc7@columbia.edu

1Corresponding author.

Manuscript received August 14, 2017; final manuscript received December 12, 2017; published online January 12, 2018. Editor: Beth A. Winkelstein.

J Biomech Eng 140(2), 021003 (Jan 12, 2018) (10 pages) Paper No: BIO-17-1360; doi: 10.1115/1.4038758 History: Received August 14, 2017; Revised December 12, 2017

Dynamic hydrostatic pressure (HP) loading can modulate nucleus pulposus (NP) cell metabolism, extracellular matrix (ECM) composition, and induce transformation of notochordal NP cells into mature phenotype. However, the effects of varying cell density and dynamic HP magnitude on NP phenotype and metabolism are unknown. This study examined the effects of physiological magnitudes of HP loading applied to bovine NP cells encapsulated within three-dimensional (3D) alginate beads. Study 1: seeding density (1 M/mL versus 4 M/mL) was evaluated in unloaded and loaded (0.1 MPa, 0.1 Hz) conditions. Study 2: loading magnitude (0, 0.1, and 0.6 MPa) applied at 0.1 Hz to 1 M/mL for 7 days was evaluated. Study 1: 4 M/mL cell density had significantly lower adenosine triphosphate (ATP), glycosaminoglycan (GAG) and collagen content, and increased lactate dehydrogenase (LDH). HP loading significantly increased ATP levels, and expression of aggrecan, collagen I, keratin-19, and N-cadherin in HP loaded versus unloaded groups. Study 2: aggrecan expression increased in a dose dependent manner with HP magnitude, whereas N-cadherin and keratin-19 expression were greatest in low HP loading compared to unloaded. Overall, the findings of the current study indicate that cell seeding density within a 3D construct is a critical variable influencing the mechanobiological response of NP cells to HP loading. NP mechanobiology and phenotypic expression was also found to be dependent on the magnitude of HP loading. These findings suggest that HP loading and culture conditions of NP cells may require complex optimization for engineering an NP replacement tissue.

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Figures

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Fig. 1

Schematic and the components of the hydrostatic pressure bioreactor system. The system (a) consists of a bioreactor chamber connected to the servo stimulator placed within an incubator (Adapted from Reinwald et al. [23]) (b). The chamber can hold a standard tissue culture plate (c). To apply HP to the samples within the bioreactor chamber, compressed incubator air is pumped into the bioreactor chamber via an air heater. The stimulation regime is applied via a growthworks control box and laptop accompanied by growthworks software.

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Fig. 2

Cell viability and cytotoxicity of NP cells seeded at either 1 M/mL or 4 M/mL in 3D microtissues. (a) DNA content was similar between the control (0 MPa) and HP loaded (0.1 MPa) samples of the same group, however high (4 M/mL) cell density group had significantly higher DNA content than the low (1 M/mL) cell density group. (b) Metabolism was assessed by measuring ATP levels and was normalized to total DNA content. ATP levels were significantly different between the low (1 M/mL) and high (4 M/mL) groups; however, only HP loaded low (1 M/mL) celldensity group promoted higher ATP levels in comparison to the control (0 MPa) group. (c) Cytotoxicity was assessed by measuring LDH levels and was normalized to total DNA content. LDH release levels were significantly elevated in HP-treated groups compared to the unloaded groups. Overall, high (4 M/mL) cell density group significantly promoted higher amount of LDH release in comparison to the low (1 M/mL) cell density group. Results are presented as mean ± SD (n = 3). *Control versus HP loaded; #Control 1 M/mL versus control 4 M/mL or HP 1M/mL versus 4 M/mL, p < 0.05.

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Fig. 3

Gene expression for NP cell phenotypic markers. Quantitative RT-PCR was performed to assess fold-changes in gene expression of NP cells in response to varying cell density group and HP treatment. High (4 M/mL) HP-treated cell density group promoted matrix protein, aggrecan (a) but no significant change in collagen II (b) expression. Low (1 M/mL) HP-treated cell density group promoted an increasing trend in aggrecan but not collagen II expression. However, collagen I (c) expression increased only in the high (4 M/mL) cell density HP-treated group. Only low (1 M/mL) cell density HP-treated group promoted putative NP makers, keratin-19 (d), N-cadherin (e) but not FOXF1 (f) expression in comparison to the control group. No significant changes were observed in any of the matrix-degrading genes—MMP-2 (g), MMP-3 (h) and MMP-13 (i) in any of the varying cell density group in response to varying HP loading regimens. Results are presented as mean ± SD (n = 3). *Control versus HP loaded; #Control 1 M/mL versus control 4 M/mL or HP 1 M/mL versus 4 M/mL, p < 0.05.

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Fig. 4

HP treatment maintained GAG and Collagen production levels in varying cell density beads. A significant increase in GAG (a) and collagen (b) accumulation was observed in low (1 M/mL) group in comparison to high (4 M/mL) group. However, HP treatment failed to stimulate additional GAG and collagen production in HP-treated samples in comparison to their respective control (unloaded) samples. Results are presented as mean ± SD (n = 3). *Control versus HP loaded; #Control 1 M/mL versus control 4 M/mL or HP 1 M/mL versus 4 M/mL, p < 0.05.

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Fig. 5

Cell viability, metabolic activity and cytotoxicity of NP cells in response to varying HP loading regimens in 3D microtissues. (a) Representative live/dead images of cells within unloaded, low-magnitude (0.1 MPa) and high-magnitude (0.6 MPa) loaded constructs. (b) No difference in the ATP levels were observed between the unloaded (0 MPa) and low-magnitude (0.1 MPa) and high-magnitude (0.6 MPa) loaded samples. (c) High-magnitude (0.6 MPa) HP promoted higher amount of LDH release in comparison to the low-magnitude (0.1 MPa) and unloaded group. Results are presented as mean ± SD (n = 3). *p < 0.05.

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Fig. 6

Gene expression for NP cell phenotypic markers. Quantitative RT-PCR was performed to assess fold-changes in gene expression of NP cells in response to varying HP loading regimens in 3D microtissues. A significant increase in matrix protein, aggrecan (a) but no significant change in Collagen II (b) was observed with increasing HP magnitude treatments. Only high (0.6 MPa) HP treatment promoted Collagen I expression (c). However, only low-magnitude (0.1 MPa) HP treatment promoted Keratin19 (d), and N-cadherin (e) expression in comparison to high-magnitude (0.6 MPa) HP and unloaded group. No significant changes were observed in FOXF1 expression (f) and any of the matrix-degrading genes—MMP-2 (g), MMP-3 (h) and MMP-13 (i) in response to varying HP loading regimens. Results are presented as mean ± SD (n = 3). *p < 0.05.

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Fig. 7

Effect of varying HP loading regimens on GAG and collagen deposition. No significant change in GAG (a) and collagen (b) accumulation was observed in unloaded, low-magnitude (0.1 MPa) and high-magnitude (0.6 MPa) HP treatment. Results are presented as mean ± SD (n = 3). *p < 0.05.

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