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Research Papers

Physiologic Medium Maintains the Homeostasis of Immature Bovine Articular Cartilage Explants in Long-Term Culture

[+] Author and Article Information
Krista M. Durney, Akaljot Singh, Lucie Karbowski, Hyeon Jin Koo, Gerard A. Ateshian

Department of Biomedical Engineering,
Columbia University,
500 West 120th Street,
New York, NY 10027

Danial Sharifi Kia, Tianbai Wang

Department of Mechanical Engineering,
Boston University,
110 Cummington Mall,
Boston, MA 02215

Michael B. Albro

Department of Mechanical Engineering,
Boston University,
110 Cummington Mall,
Boston, MA 02215
e-mail: albro@bu.edu

1Corresponding author.

Manuscript received February 20, 2018; final manuscript received October 18, 2018; published online December 5, 2018. Assoc. Editor: David Corr.

J Biomech Eng 141(2), 021004 (Dec 05, 2018) (12 pages) Paper No: BIO-18-1096; doi: 10.1115/1.4041901 History: Received February 20, 2018; Revised October 18, 2018

The ability to maintain living articular cartilage tissue in long-term culture can serve as a valuable analytical research tool, allowing for direct examination of mechanical or chemical perturbations on tissue behavior. A fundamental challenge for this technique is the recreation of the salient environmental conditions of the synovial joint in culture that are required to maintain native cartilage homeostasis. Interestingly, conventional media formulations used in explanted cartilage tissue culture investigations often consist of levels of metabolic mediators that deviate greatly from their concentrations in synovial fluid (SF). Here, we hypothesize that the utilization of a culture medium consisting of near-physiologic levels of several highly influential metabolic mediators (glucose, amino acids, cortisol, insulin, and ascorbic acid) will maintain the homeostasis of cartilage explants as assessed by their mechanical properties and extracellular matrix (ECM) contents. Results demonstrate that the aforementioned mediators have a strong effect on the mechanical and biochemical stability of skeletally immature bovine cartilage explants. Most notably, (1) in the absence of cortisol, explants exhibit extensive swelling and tissue softening and (2) in the presence of supraphysiologic levels of anabolic mediators (glucose, amino acids, insulin), explants exhibit increased matrix accumulation and tissue stiffening. In contrast, the administration of physiologic levels of these mediators (as present in native SF) greatly improves the stability of live cartilage explants over one month of culture. These results may have broad applicability for articular cartilage and other musculoskeletal tissue research, setting the foundation for important culture formulations required for examinations into tissue behavior.

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Figures

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Fig. 1

Remaining glucose levels in culture medium after 48 h of explant incubation for varying media to tissue volume ratios. Dashed line represents initial supplemented glucose level (1 mg/mL).

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Fig. 2

Mechanical properties and biochemical contents of cartilage explants initially and after 20 days of culture in SF: (a) SR, (b) compressive Young's modulus, (c) GAG content per initial wet weight, and (d) GAG content per final weight wet. *p < 0.05 versus day 0 value.

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Fig. 3

Critical chemical mediator experiment #1: Mechanical properties, biochemical contents, and histology of cartilage explants cultured for 32 days in medium with varying levels of cortisol: (a) SR, (b) compressive Young's modulus, (c) GAG content per initial wet weight, (d) GAG content per final weight wet, (e) collagen content, and (f) cell content. Insulin (1 ng/mL), glucose (1 mg/mL), amino acid (1.0X), and ascorbic acid (20 μg/mL) levels were maintained constant for all groups. Arrow indicates physiologic level of cortisol present in SF. Dashed lines represent day 0 values. *p < 0.05 versus day 0 value. (g) Representative images of cartilage explants initially (day 0) and after 32 days of culture with varying cortisol levels. Scale bar = 1.0 mm. (h) Representative safranin-O stained sections of explants initially and after 32 days of culture with varying cortisol levels. Arrow represents crack formation in tissue center of 0 ng/mL cortisol samples, which is distinct from cartilage vascular canals (* labeled) that are present in other groups and characteristic of immature cartilage tissue [37]. Scale bar = 1.0 mm.

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Fig. 4

Swelling ratio of cartilage explants cultured for 32 days in cortisol-free medium while subjected to devitalization, cortisol supplementation, or specific enzyme inhibitors. *p < 0.05 versus control value.

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Fig. 5

Critical chemical mediator experiment #2: Mechanical properties, biochemical contents, and histology of cartilage explants cultured for 32 days in medium with varying levels of insulin and glucose: (a) SR, (b) compressive Young's modulus, (c) GAG content per initial wet weight, (d) GAG content per final weight wet, (e) collagen content, and (f) cell content. Cortisol (25 ng/mL), amino acid (1.0X), and ascorbic acid (20 μg/mL) levels were maintained constant for all groups. Arrow indicates physiologic levels of insulin and glucose present in SF. Dashed lines represent day 0 values. *p < 0.05 versus day 0 value. p < 0.05 represents significant variation from corresponding 1 ng/mL insulin value. (g) Linear correlation between SR and GAG content. (h) Representative images of cartilage explants initially (day 0) and after 32 days of culture for each media group. Scale bar = 1.0 mm. (i) Representative safranin-O stained sections of explants initially and after 32 days of culture for 6000 ng/mL insulin and 4.5 mg/mL glucose group. Arrow represents crack-induced tissue void in tissue center. Scale bar = 1.0 mm.

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Fig. 6

Critical chemical mediator experiment 3: Mechanical properties, biochemical contents, and viability of cartilage explants cultured for 7, 16, or 32 days in medium with varying levels of amino acids: (a) SR, (b) compressive Young's modulus, (c) GAG content per initial wet weight, and (d) GAG content per final wet weight. Insulin (1 ng/mL), glucose (1 mg/mL), cortisol (25 ng/mL), and ascorbic acid (20 μg/mL) levels were maintained constant for all groups. Arrow indicates physiologic level of amino acids present in SF. 1X amino acid level represents concentrations present in standard DMEM. Dashed lines represent day 0 values. *p < 0.05 versus day 0 value. p < 0.05 versus corresponding day 7 value. (e) Representative viability images (live cells: green, dead cells: red) of cartilage explants after 32 days of culture for 0.25X and 0.03X amino acid groups.

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Fig. 7

Secretion rate of endogenous cartilage growth factor, latent TGF-β1, from cartilage explants cultured in medium of varying levels of amino acids. Secretion rate is normalized to explant volume. Arrow indicates physiologic level of amino acids present in SF *p < 0.05 versus 1.0X value.

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Fig. 8

Mechanical properties and biochemical contents of cartilage explants cultured for 32 days in a corticosteroid-free, physiologic, or supraphysiologic culture medium while subjected to dynamic mechanical loading or free swelling conditions: (a) SR, (b) compressive Young's modulus, and (c) GAG content per initial wet weight. *p < 0.05, p = 0.07 versus corresponding free swelling value.

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