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
. 2020 Nov 13:8:590743.
doi: 10.3389/fbioe.2020.590743. eCollection 2020.

Physioxia Stimulates Extracellular Matrix Deposition and Increases Mechanical Properties of Human Chondrocyte-Derived Tissue-Engineered Cartilage

James E Dennis  1 , George Adam Whitney  1 , Jyoti Rai  2 , Russell J Fernandes  2 , Thomas J Kean  1
Affiliations

Physioxia Stimulates Extracellular Matrix Deposition and Increases Mechanical Properties of Human Chondrocyte-Derived Tissue-Engineered Cartilage

James E Dennis et al. Front Bioeng Biotechnol. .

Abstract

Cartilage tissue has been recalcitrant to tissue engineering approaches. In this study, human chondrocytes were formed into self-assembled cartilage sheets, cultured in physiologic (5%) and atmospheric (20%) oxygen conditions and underwent biochemical, histological and biomechanical analysis at 1- and 2-months. The results indicated that sheets formed at physiological oxygen tension were thicker, contained greater amounts of glycosaminoglycans (GAGs) and type II collagen, and had greater compressive and tensile properties than those cultured in atmospheric oxygen. In all cases, cartilage sheets stained throughout for extracellular matrix components. Type II-IX-XI collagen heteropolymer formed in the neo-cartilage and fibrils were stabilized by trivalent pyridinoline cross-links. Collagen cross-links were not significantly affected by oxygen tension but increased with time in culture. Physiological oxygen tension and longer culture periods both served to increase extracellular matrix components. The foremost correlation was found between compressive stiffness and the GAG to collagen ratio.

Keywords: articular cartilage; biomechanical testing; chondrocyte; chondrogenesis; collagen cross linking; hypoxia; tissue-engineered cartilage; type II collagen.

PubMed Disclaimer

Figures

FIGURE 1
FIGURE 1
Biochamber model and setup. A circular biochamber design with a 1.13 cm2 cell seeding area (1) was used with three seeding chambers (3) stacked on top of each other giving a 2.1 ml seeding volume. Stainless steel screws (2) were used to raise the biochambers 1.2 cm allowing media access to the cell sheet through the polyester membrane (7) sandwiched between the bottom 0.2 cm plate and the seeding chambers. Biochambers were assembled and placed in Nalgene containers (6) with a ceramic filter on the lid (4). Defined chondrogenic media was added to the level of the membrane, cells seeded, then media added to 0.2 cm below the top of the topmost seeding chamber.
FIGURE 2
FIGURE 2
Tissue engineered cartilage sample preparation and tensile testing. Panel (A) shows the custom manufactured dog bone punch (A1), the 5 mm punched tissue-engineered human cartilage sheet on the dogbone punch (A2), the resulting dogbone (A3), and the dogbone fixed to the OHP film adapter to insert into the grips of the mechanical testing device. Panel (B) shows sequential images of a piece of tissue-engineered human cartilage being tested in tension to failure. Panel (C) shows the stress strain curve of the cartilage piece with approximate points shown in the graph of the images in (B), with (B2) indicating the yield stress, in this case close to the ultimate yield stress (C′), and the best fit line showing the tensile Young’s modulus.
FIGURE 3
FIGURE 3
Extracellular matrix deposition in tissue-engineered human cartilage sheets. Sheets grown under Physioxia are more easily manipulated than those grown in Atm O2; an example of a large (16 cm2) sheet from a single donor grown in Physioxia or Atm O2 at 7-weeks (A). Cell normalized glycosaminoglycan (GAG/DNA) content of cartilage from samples grown at Physioxia and Atm O2 are shown at 3- and 7-weeks (B). Cell normalized total collagen content of tissue-engineered cartilage when grown in Physioxia and Atm O2 at 3 and 7 weeks is shown (C). Trivalent collagen cross-link density (HP + LP/Collagen) at 3- and 7-weeks under both Physioxia and Atm O2 are shown (D). Note, all measures are shown on a log (base 2) scale. The average of data for each experiment is shown by a symbol with lines connecting an experiment, six donors, eight experiments.
FIGURE 4
FIGURE 4
Collagen heteropolymer crosslink analysis. Collagen in human tissue-engineered cartilage was analyzed by SDS page and western blot. Lane 1 human type II collagen (control); Lane 2 7-week Atm O2 (20%); Lane 3 7-week Physioxia (5%); Lane 4 3-week Atm O2; Lane 5 3-week Physioxia. Panels (A) Coomassie blue stain; (B) Western blot type using II collagen antibody to α1(II) chain in helical region; (C) Western blot using antibody to C-telopeptide of α1(II) chains of type II collagen; (D) Western blot using antibody to N-telopeptide of α1(XI) chains of type IX collagen; (E) Western blot of antibody that recognizes C-telopeptide of the non-collagenous domain of α1(IX) chains of type IX collagen of type IX collagen. (F) Locations of the epitopes in the α1(II) chain or in telopeptide stubs crosslinked to the collagen chains in type II-IX-XI heteropolymer.
FIGURE 5
FIGURE 5
Mechanical testing of tissue-engineered human cartilage sheets. Tissue-engineered human cartilage sheet thickness over time (3- and 7-weeks) in both Physioxia and Atm O2 (A). Compressive stiffness (Equilibrium Modulus) of tissue-engineered human cartilage sheets when grown in Physioxia vs Atm O2 both at 3- and 7-weeks (B). Elastic tensile Modulus of tissue engineered human cartilage sheets in Physioxia and Atm O2 at 3- and 7-weeks (C). Correlation of GAG/Collagen ratio with Equilibrium modulus (D). (A–C) The average of data for each experiment is shown by a symbol with lines connecting a donor/experiment, (A,B) six donors, eight experiments, (C) 4 donors, 4 experiments. In (D) the symbols represent average sheet data regardless of the time or oxygen tension.
FIGURE 6
FIGURE 6
Histological analysis of human tissue-engineered cartilage sheets. Tissue-engineered human cartilage sheets from a single donor are shown (see Supplementary Figures 1,2 for other donors and Supplementary Figure 3 for native immunohistochemistry controls), they were stained for GAG (safranin-O, column A), type I collagen (column B), type II collagen (column C), and type X collagen (column D). Rows 1 and 2 show data from the 3-week time point and rows 3 and 4 show data from week-7. Atm O2 sheets are shown in rows 1 and 3, Physioxic sheets are shown in rows 2 and 4. The scale bar indicates a 1 mm distance.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Adkar S. S., Wu C.-L., Willard V. P., Dicks A., Ettyreddy A., Steward N., et al. (2019). Step-Wise chondrogenesis of human induced pluripotent stem cells and purification via a reporter allele generated by CRISPR-Cas9 Genome editing. Stem Cell. 37 65–76. 10.1002/stem.2931 - DOI - PMC - PubMed
    1. Anderson D. E., Markway B. D., Bond D., McCarthy H. E., Johnstone B. (2016). Responses to altered oxygen tension are distinct between human stem cells of high and low chondrogenic capacity. Stem Cell Res. Ther. 7:154. 10.1186/s13287-016-0419-8 - DOI - PMC - PubMed
    1. Anderson D. E., Markway B. D., Weekes K. J., McCarthy H. E., Johnstone B. (2018). Physioxia promotes the articular chondrocyte-like phenotype in human chondroprogenitor-derived self-organized tissue. Tissue Eng. Part A 24 264–274. 10.1089/ten.TEA.2016.0510 - DOI - PMC - PubMed
    1. Bianchi V. J., Weber J. F., Waldman S. D., Backstein D., Kandel R. A. (2017). Formation of hyaline cartilage tissue by passaged human osteoarthritic chondrocytes. Tissue Eng. Part A 23 156–165. 10.1089/ten.TEA.2016.0262 - DOI - PubMed
    1. Dennis J. E., Bernardi K. G., Kean T. J., Liou N. E., Meyer T. K. (2018). Tissue engineering of a composite trachea construct using autologous rabbit chondrocytes. J. Tissue Eng. Regen. Med. 12 e1383–e1391. 10.1002/term.2523 - DOI - PMC - PubMed

LinkOut - more resources