Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2022 Jan-Mar;13(1):19476035221081466.
doi: 10.1177/19476035221081466.

Stiffness- and Bioactive Factor-Mediated Protection of Self-Assembled Cartilage against Macrophage Challenge in a Novel Co-Culture System

Ryan P Donahue  1 , Jarrett M Link  1 , Vijaykumar S Meli  1   2 , Jerry C Hu  1 , Wendy F Liu  1   2   3   4 , Kyriacos A Athanasiou  1
Affiliations

Stiffness- and Bioactive Factor-Mediated Protection of Self-Assembled Cartilage against Macrophage Challenge in a Novel Co-Culture System

Ryan P Donahue et al. Cartilage. 2022 Jan-Mar.

Abstract

Objective: Tissue-engineered cartilage implants must withstand the potential inflammatory and joint loading environment for successful long-term repair of defects. The work's objectives were to develop a novel, direct cartilage-macrophage co-culture system and to characterize interactions between self-assembled neocartilage and differentially stimulated macrophages.

Design: In study 1, it was hypothesized that the proinflammatory response of macrophages would intensify with increasing construct stiffness; it was expected that the neocartilage would display a decrease in mechanical properties after co-culture. In study 2, it was hypothesized that bioactive factors would protect neocartilage properties during macrophage co-culture. Also, it was hypothesized that interleukin 10 (IL-10)-stimulated macrophages would improve neocartilage mechanical properties compared to lipopolysaccharide (LPS)-stimulated macrophages.

Results: As hypothesized, stiffer neocartilage elicited a heightened proinflammatory macrophage response, increasing tumor necrosis factor alpha (TNF-α) secretion by 5.47 times when LPS-stimulated compared to construct-only controls. Interestingly, this response did not adversely affect construct properties for the stiffest neocartilage but did correspond to a significant decrease in aggregate modulus for soft and medium stiffness constructs. In addition, bioactive factor-treated constructs were protected from macrophage challenge compared to chondrogenic medium-treated constructs, but IL-10 did not improve neocartilage properties, although stiff constructs appeared to bolster the anti-inflammatory nature of IL-10-stimulated macrophages. However, co-culture of bioactive factor-treated constructs with LPS-treated macrophages reduced TNF-α secretion by over 4 times compared to macrophage-only controls.

Conclusions: In conclusion, neocartilage stiffness can mediate macrophage behavior, but stiffness and bioactive factors prevent macrophage-induced degradation. Ultimately, this co-culture system could be utilized for additional studies to develop the burgeoning field of cartilage mechano-immunology.

Keywords: cartilage; immunology; macrophage; tissue engineering.

PubMed Disclaimer

Conflict of interest statement

Declaration of Conflicting Interests: The author(s) declared the following potential conflicts of interest with respect to the research, authorship, and/or publication of this article: Kyriacos A. Athanasiou and Jerry C. Hu are scientific consultants for Cartilage Inc.

Figures

Figure 1.
Figure 1.
Stiffness is modulated via C-ABCcomp., and macrophages are adhered and stimulated in a direct co-culture system. Constructs were cultured in either CHG only or with TCL treatment. After 27 days, compressive stiffness was modulated via C-ABCcomp. application. Constructs were assayed for baseline properties (t = 0). The next day, macrophages adhered and were cultured in a 1:1 mix of CHG and MΦM medium, then stimulated with LPS in study 1 and LPS or IL-10 in study 2. Unstimulated macrophage controls, construct-only controls, and macrophage-only controls were also included. After 2 weeks of co-culture, constructs were assayed again (t = 2W). C-ABCcomp. = chondroitinase ABC to modulate compressive stiffness; CHG = chondrogenic medium; TCL = transforming growth factor beta 1/chondroitinase ABCeng./lysyl oxidase-like 2; MΦM = macrophage medium; LPS = lipopolysaccharide; IL-10 = interleukin 10.
Figure 2.
Figure 2.
C-ABCcomp. modulates the compressive stiffness of CHG-treated constructs. (A) GAG/WW increased across soft to stiff groups and decreased as higher C-ABCcomp. concentrations were used to modulate compressive stiffness. (B) Similarly, aggregate modulus also trended higher from soft to stiff, as expected. Statistics: One-way ANOVA with post hoc Tukey’s HSD, α = 0.05, n = 3 per group. C-ABCcomp. = chondroitinase ABC to modulate compressive stiffness; CHG = chondrogenic medium; GAG = glycosaminoglycan; WW = wet weight; ANOVA = analysis of variance; HSD = honestly significant difference.
Figure 3.
Figure 3.
CHG-treated constructs of soft and medium stiffness differ in staining intensity. Following 2 weeks of co-culture, stiff constructs maintain cell morphology and tissue staining intensity in co-culture groups more than the soft and medium groups. Scale bar = 200 µm. CHG = chondrogenic medium; LPS = lipopolysaccharide.
Figure 4.
Figure 4.
Soft and medium stiffness CHG-treated constructs suffer losses in aggregate modulus values despite no increases in TNF-α production. (A) GAG/WW was significantly affected by the ΜΦ factor, with significant decreases within the medium stiffness constructs between the construct-only control and LPS-stimulated co-culture group. (B) Trending with GAG/WW, aggregate modulus significantly decreased with the addition of macrophages (unstimulated or LPS-stimulated) in soft and medium stiffness constructs. (C) Conversely, after 48 hours of co-culture, only the stiff construct group had significant increases in TNF-α secretion between the construct-only control and the LPS-stimulated group. Statistics: Two-way ANOVA with post hoc Tukey’s HSD among groups within a stiffness (dotted lines), α = 0.05, n = 3-6 per group. CHG = chondrogenic medium; TNF-α = tumor necrosis factor alpha; GAG = glycosaminoglycan; WW = wet weight; ΜΦ = macrophage; LPS = lipopolysaccharide; ANOVA = analysis of variance; HSD = honestly significant difference.
Figure 5.
Figure 5.
C-ABCcomp. modulates the compressive stiffness of TCL-treated constructs. (A) GAG/WW does not differ significantly for TCL-treated constructs at t = 0. (B) Aggregate modulus significantly decreases with application of C-ABCcomp. (C) Soft constructs show less intense and H&E and Saf-O staining due to C-ABCcomp. application and only peripheral loss of GAG. Also, some cells are visible near the soft construct edge. Conversely, stiff constructs show intense Saf-O staining at the periphery indicating high GAG content, and cells are not present at the periphery of the construct. Scale bar = 100 µm. Statistics: Student t test, α = 0.05. C-ABCcomp. = chondroitinase ABC to modulate compressive stiffness; TCL = transforming growth factor beta 1/chondroitinase ABCeng./lysyl oxidase-like 2; GAG = glycosaminoglycan; WW = wet weight; H&E = hematoxylin and eosin; Saf-O = Safranin O.
Figure 6.
Figure 6.
Saf-O staining after 2 weeks of macrophage co-culture shows donor-related variation, with diminished staining among LPS-stimulated co-cultures. Both construct-only controls and co-culture groups show variability between donors in both (A) soft and (B) stiff TCL-treated constructs. However, on average, staining is slightly diminished in some LPS-stimulated groups compared to construct-only controls. Scale bar = 100 µm. Saf-O = Safranin O; LPS = lipopolysaccharide; Unstim. = unstimulated; IL-10 = interleukin 10; TCL = transforming growth factor beta 1/chondroitinase ABCeng./lysyl oxidase-like 2.
Figure 7.
Figure 7.
Aggregate modulus values of TCL-treated constructs only decrease in soft construct, LPS-stimulated co-cultures. (A) The construct-only control and macrophage co-cultures in the soft constructs are significantly different in GAG/WW, while the stiff group does not exhibit significant differences between groups. (B) Aggregate modulus also trends downward for the soft group when co-cultured with macrophages, but only the LPS-stimulated co-culture group is significantly different from the construct-only control. Stiff group aggregate moduli were largely unaffected by macrophage treatment. Statistics: Two-way ANOVA with post hoc Tukey’s HSD among groups within a stiffness (dotted lines), α = 0.05, n = 5-6 per group. TCL = transforming growth factor beta 1/chondroitinase ABCeng./lysyl oxidase-like 2; LPS = lipopolysaccharide; GAG = glycosaminoglycan; WW = wet weight; MΦ = macrophage; IL-10 = interleukin 10; ANOVA = analysis of variance; HSD = honestly significant difference.
Figure 8.
Figure 8.
TNF-α secretion of TCL-treated construct co-cultures increases with stiffness, decreases with construct co-culture, and diminishes when macrophages are stimulated toward an anti-inflammatory phenotype. In unstimulated co-culture conditions, TNF-α production increases with stiffness. Similarly, TNF-α secretion also trends higher for stiff construct co-culture compared to the soft construct condition in the LPS-stimulated groups. Interestingly, construct addition significantly decreases the TNF-α production compared to macrophage-only controls in LPS-stimulated groups. For IL-10 stimulation, a decrease in TNF-α levels is seen with increasing construct stiffness. Statistics: One-way ANOVA with post hoc Tukey’s HSD (for each stimulation condition), α = 0.05, n = 5-6 per group. TNF-α = tumor necrosis factor alpha; TCL = transforming growth factor beta 1/chondroitinase ABCeng./lysyl oxidase-like 2; MΦ = macrophage, LPS = lipopolysaccharide; IL-10 = interleukin 10; ANOVA = analysis of variance; HSD = honestly significant difference.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

References

    1. Koh TJ, DiPietro LA. Inflammation and wound healing: the role of the macrophage. Expert Rev Mol Med. 2011;13:e23. - PMC - PubMed
    1. Kennedy A, Fearon U, Veale DJ, Godson C. Macrophages in synovial inflammation. Front Immunol. 2011;2:52. - PMC - PubMed
    1. Chen Y, Jiang W, Yong H, He M, Yang Y, Deng Z, et al.. Macrophages in osteoarthritis: pathophysiology and therapeutics. Am J Transl Res. 2020;12(1):261-8. - PMC - PubMed
    1. Wang KH, Wan R, Chiu LH, Tsai YH, Fang CL, Bowley JF, et al.. Effects of collagen matrix and bioreactor cultivation on cartilage regeneration of a full-thickness critical-size knee joint cartilage defects with subchondral bone damage in a rabbit model. PLoS One. 2018;13(5):e0196779. - PMC - PubMed
    1. Kondo M, Kameishi S, Kim K, Metzler NF, Maak TG, Hutchinson DT, et al.. Safety and efficacy of human juvenile chondrocyte-derived cell sheets for osteochondral defect treatment. NPJ Regen Med. 2021;6(1):65. - PMC - PubMed

Publication types

MeSH terms