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. 2020 Nov 11;21(22):8496.
doi: 10.3390/ijms21228496.

Development of a Method for Scaffold-Free Elastic Cartilage Creation

Masahiro Enomura  1 , Soichiro Murata  1   2 , Yuri Terado  1 , Maiko Tanaka  1 , Shinji Kobayashi  3 , Takayoshi Oba  4 , Shintaro Kagimoto  5 , Yuichiro Yabuki  5 , Kenichi Morita  6 , Toshimasa Uemura  6   7 , Jiro Maegawa  5 , Hideki Taniguchi  1   2
Affiliations

Development of a Method for Scaffold-Free Elastic Cartilage Creation

Masahiro Enomura et al. Int J Mol Sci. .

Abstract

Microtia is a congenital aplasia of the auricular cartilage. Conventionally, autologous costal cartilage grafts are collected and shaped for transplantation. However, in this method, excessive invasion occurs due to limitations in the costal cartilage collection. Due to deformation over time after transplantation of the shaped graft, problems with long-term morphological maintenance exist. Additionally, the lack of elasticity with costal cartilage grafts is worth mentioning, as costal cartilage is a type of hyaline cartilage. Medical plastic materials have been transplanted as alternatives to costal cartilage, but transplant rejection and deformation over time are inevitable. It is imperative to create tissues for transplantation using cells of biological origin. Hence, cartilage tissues were developed using a biodegradable scaffold material. However, such materials suffer from transplant rejection and biodegradation, causing the transplanted cartilage tissue to deform due to a lack of elasticity. To address this problem, we established a method for creating elastic cartilage tissue for transplantation with autologous cells without using scaffold materials. Chondrocyte progenitor cells were collected from perichondrial tissue of the ear cartilage. By using a multilayer culture and a three-dimensional rotating suspension culture vessel system, we succeeded in creating scaffold-free elastic cartilage from cartilage progenitor cells.

Keywords: chondrocyte progenitor cells; elastic cartilage; scaffold-free; three-dimensional rotating suspension culture.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Regenerated cartilage culture method. (A) Macro images of human auricular cartilage tissue, along with hematoxylin and eosin (H&E), Alcian blue (AB), elastica–van Gieson (EVG), safranin O, and collagen type I (Col1) and type II (Col2) immunohistological staining. Scale Bars: 200 μm. (B) First, 1.5 × 106 chondrocyte progenitor cells collected from the auricular cartilage tissue were seeded, cultured in 1 day DMEM/F12 FBS, and then cultured in a differentiation induction medium for 6 days. After culturing, 1.5 × 106 chondrocyte progenitor cells were seeded and the same operation was repeated twice on a multilayer sheet. (C) The multilayer sheet was collected with a scraper, placed in a three-dimensional rotating wall vessel (RWV), and allowed to stand overnight. The RWV rotation culture was conducted so that the multilayer sheet floated.
Figure 2
Figure 2
Chondrocyte progenitor cell multilayer culture. (A) Culture images of chondrocyte progenitor cells passaged 5 and 16 times. Scale Bars: 500 μm. (B) Culture images of chondrocyte progenitor cells in multilayer culture numbers 1 to 3. (C) Measurement of melanoma-inhibiting activity (MIA) during subculture and layered culture of chondrocyte progenitor cells. Holm–Sidak multiple comparisons test: * p < 0.01 vs. 2D culture and layer number (n = 7–26). (D) Measurement of hyaluronic acid secretion by layered culture. Holm–Sidak multiple comparisons test: * p < 0.01 vs. layer number (n = 26)
Figure 3
Figure 3
Three-dimensional multilayer cultured elastic cartilage. (A) Multilayer sheets cultured in the RWV at 1 day and 1, 2 and 3 weeks. Scale Bars: 1 cm. (B) RWV quantitative analysis using Holm–Sidak multiple comparisons test: * p < 0.01 vs. RWV culture (n = 20–26). (C) Measurement of MIA secretion in 2D culture and RWV culture for chondrocyte progenitor cells. (n = 7–45). (D) Measurement of shear stress in RWV culture at 1, 2, and 3 weeks. Holm–Sidak multiple comparisons test: * p < 0.01 vs. RWV culture 3w (n = 3).
Figure 4
Figure 4
Transplanted elastic cartilage tissue showing maturation. (A) Macroscopic images, along with hematoxylin and eosin (HE), Alcian blue (AB), elastica–van Gieson (EVG), safranin O, DAPI, collagen type I (Col1), and collagen type II (Col2) staining of elastic cartilage tissue after RWV culture (in vitro), transplanted elastic cartilage tissue (in vivo), and primary cartilage. Scale Bars: 200 μm. (B) Shear stress values of cultured elastic cartilage tissue (in vitro), transplanted elastic cartilage tissue (in vivo), and primary cartilage. Holm–Sidak multiple comparisons test: * p < 0.01 vs in vivo samples (n = 4–41) (C) Appearance scale changes over time up to 8 weeks after transplantation. Holm–Sidak multiple comparisons test: * p < 0.01 vs. 1 and 2 w after transplantation (n = 22–28).
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