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. 2022 Dec 23:13:20417314221143484.
doi: 10.1177/20417314221143484. eCollection 2022 Jan-Dec.

In vitro elastic cartilage reconstruction using human auricular perichondrial chondroprogenitor cell-derived micro 3D spheroids

Takayoshi Oba  1   2 , Satoshi Okamoto  1 , Yasuharu Ueno  3 , Megumi Matsuo  1 , Tomomi Tadokoro  1 , Shinji Kobayashi  4 , Kazunori Yasumura  4 , Shintaro Kagimoto  5 , Yutaka Inaba  2 , Hideki Taniguchi  1   3
Affiliations

In vitro elastic cartilage reconstruction using human auricular perichondrial chondroprogenitor cell-derived micro 3D spheroids

Takayoshi Oba et al. J Tissue Eng. .

Abstract

Morphologically stable scaffold-free elastic cartilage tissue is crucial for treating external ear abnormalities. However, establishing adequate mechanical strength is challenging, owing to the difficulty of achieving chondrogenic differentiation in vitro; thus, cartilage reconstruction is a complex task. Auricular perichondrial chondroprogenitor cells exhibit high proliferation potential and can be obtained with minimal invasion. Therefore, these cells are an ideal resource for elastic cartilage reconstruction. In this study, we aimed to develop a novel in vitro scaffold-free method for elastic cartilage reconstruction, using human auricular perichondrial chondroprogenitor cells. Inducing chondrogenesis by using microscopic spheroids similar to auricular hillocks significantly increased the chondrogenic potential. The size and elasticity of the tissue were maintained after craniofacial transplantation in immunodeficient mice, suggesting that the reconstructed tissue was morphologically stable. Our novel tissue reconstruction method may facilitate the development of future treatments for external ear abnormalities.

Keywords: Elastic cartilage; auricular perichondrial chondroprogenitor cells; micro three-dimensional culture; microtia; rotating culture.

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Conflict of interest statement

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Figures

Figure 1.
Figure 1.
Analysis of fetal auricular hillocks and human perichondrial chondroprogenitor cells. (a) Schematic representation of a human embryo at 6 weeks gestation, showing the emerging auricular hillocks. (b) Image of the auricular hillocks of an E11.5 mouse embryo, with a magnified view shown on the right. Dashed line indicates the outline of the hillocks. White arrowheads indicate the first pharyngeal groove. (c) Immunohistochemistry of E11.5 mouse embryonic auricular hillocks. Left panel: type I collagen (COL1) (green), type II collagen (COL2) (red), and diamidino-phenylindole (DAPI) (blue) staining; right panel: SOX9 and DAPI staining. (d) Image of an excised auricular perichondrium (scale: 1 mm). (e) Image of expanded auricular perichondrial chondroprogenitor cells (scale bars: 100 μm). (f) Representative flow cytometry analysis of perichondrial cells to determine CD44, CD90, CD73, and CD105 expression in four patient samples. Left panels: unstained cells (negative control). Data are shown as the mean ± SD.
Figure 2.
Figure 2.
Comparison of 2D, micro 3D, and macro 3D cultures. (a) Image of micro 3D culture with a magnified panel below. Scale bars: 2 mm. (b) Time course images of 2D (top row), micro 3D (middle row), and macro 3D (bottom row) cultures. Scale bars: 100 μm. (c) Diameter of micro 3D and macro 3D spheroids. (d) Gene expression of SOX9 and COLL11A2 against 18S in each culture system on a time course. (e) Secretion level of melanoma inhibitory activity (MIA) and hyaluronic acid (HA) in each culture system on a time course. (f) Merged and magnified immunohistochemical images of 2D (top row), micro 3D (middle row), and macro 3D (bottom row) cultures after staining with type I collagen (COL1) (green), type II collagen (COL2) (red) (left panels), and SOX9 (green) (right panels), along with diamidino-phenylindole (DAPI) (blue). Scale bars: 50 μm. (g) Areas positive for COL1 staining (%) and cells positive for SOX9 staining (%) based on immunohistochemical images of each culture system. Data are shown from eight independent experiments. ns: not significant. *p < 0.01.
Figure 3.
Figure 3.
Maturation of fused micro 3D–cultured spheroids into elastic cartilage via rotating wall vessel (RWV) culture. (a) Schematic representation of micro 3D–cultured spheroids transferred into an RWV. (b) Representative image of tissue cultured in an RWV. Scale bars: 1 mm (c) Secretion level of melanoma inhibitory activity (MIA) and hyaluronic acid (HA) on a time course (n = 8). (d) Gene expression of AGGRECAN and ELASTIN against 18S in passage 2 chondroprogenitor cells, day 3 micro 3D–cultured spheroids and week 4 RWV–cultured cartilage (n = 4). ns: not significant. *p < 0.05. **p < 0.01.
Figure 4.
Figure 4.
Characterization of in vitro human perichondrial chondroprogenitor cell–derived reconstructed elastic cartilage. (a) Images of the frontal and side view. Scale bars: 2 mm. (b) Histological images of in vitro reconstructed elastic (top row), human auricular (middle row), and human costal (bottom row) cartilages stained with HE, Alcian blue (AB), Elastica Van Gieson (EVG), type I collagen (COL1) (green), type II collagen (COL2) (red), and diamidino-phenylindole (DAPI) (blue), each with magnified images below. Elastica Van Gieson (EVG) panels have further magnified panels of the section with dashed lines with white arrowheads indicating elastic fibers. Scale bars: 50 μm.
Figure 5.
Figure 5.
Craniofacial implantation treatments and in vivo analysis. (a) Representative images of the craniofacial area of elastic cartilage–implanted mice (upper row) and sham-treated mice (lower row), with (b) measured forehead thickness. Scale bars: 1 cm. Yellow arrows: Implanted elastic cartilage. Data are shown as the mean ± SD. (c) In vivo images of the implanted elastic cartilage after 2 months of transplantation. (d) Images of the frontal view and side view of the ex vivo elastic cartilage. Scale bars: 2 mm. (e) Diameter of in vitro and ex vivo elastic cartilage. (f) Elastic modulus of human costal hyaline cartilage, human auricular elastic cartilage, in vitro, elastic cartilage reconstruction and ex vivo elastic cartilage reconstruction. (g) Histological images of ex vivo elastic cartilage reconstruction stained with HE, Alcian blue (AB), Elastica Van Gieson (EVG), type I collagen (COL1) (green), type II collagen (COL2) (red), and diamidino-phenylindole (DAPI) (blue), each with magnified images below. Elastica Van Gieson (EVG) panels have further magnified panels of the section with dashed lines with white arrowheads indicating elastic fibers. Scale bars: 50 μm. n = 8 mice for the treated group, and n = 4 mice for the sham-treated group. ns: not significant. *p < 0.01.
Figure 6.
Figure 6.
Protocol for reconstructing elastic cartilage tissue using human auricular perichondrial chondroprogenitor cells.
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References

    1. Luquetti DV, Leoncini E, Mastroiacovo P. Microtia-anotia: a global review of prevalence rates. Birth Defects Res A Clin Mol Teratol 2011; 91: 813–822. - PMC - PubMed
    1. Luquetti DV, Heike CL, Hing AV, et al.. Microtia: epidemiology and genetics. Am J Med Genet A 2012; 158: 124–139. - PMC - PubMed
    1. Mastroiacovo P, Corchia C, Botto LD, et al.. Epidemiology and genetics of microtia-anotia: a registry based study on over one million births. J Med Genet 1995; 32: 453–457. - PMC - PubMed
    1. Bly RA, Bhrany AD, Murakami CS, et al.. Microtia reconstruction. Facial Plast Surg Clin North Am 2016; 24: 577–591. - PMC - PubMed
    1. Griffin MF, O’Toole G, Sabbagh W, et al.. Comparison of the compressive mechanical properties of auricular and costal cartilage from patients with microtia. J Biomech 2020; 103: 109688. - PubMed

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