Development of a Method for Scaffold-Free Elastic Cartilage Creation

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.

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

“…Exposing tissues to shear stress by using bioreactors has also been reported to be an effective culture method for elastic cartilage reconstruction. 16,32,33 Our results also indicated that this is a promising method, based on the progressive increase in chondrogenesis-related protein secretion levels and the in vitro histological analysis of elastic cartilage cultured via rotating wall vessel. Surprisingly, 3000 micro 3D-cultured spheroids fused together within a day strongly enough to tolerate a speed of 15 rpm (Figure 3(b)).…”

“…The expression of the auricular perichondrial chondroprogenitor markers CD90 and CD44 was detected by flow cytometry. 16,20 CD90+CD44+ auricular perichondrial chondroprogenitor cells comprised 98.2% ± 0.9% of the auricular perichondrial cells. Furthermore, 98.0% ± 2.1% of the auricular perichondrial cells were CD73+CD105+ (Figure 1(f), Supplemental Figure 1(a)).…”

“…These results indicate the benefit of using rotating wall vessel culture to promote cartilage maturation, consistent with previous reports. 16,32,33 After 4 weeks of rotating wall vessel culture, the reconstructed elastic cartilage reached 4.3 mm (3.9-4.5 mm) in diameter (Figures 4(a) and 5(e)). Histological analysis revealed positive Alcian blue, EVG, and type II collagen staining, which was consistent with the results for the human native auricular cartilage.…”

“…In our previous study, we succeeded in developing a method for creating scaffold-free elastic cartilage in vivo. 16 However, the tissue shrank to approximately half its initial size during transplantation. 16 Mesenchymal aggregation, which occurs during cartilage differentiation, may explain this shrinkage.…”

“… 16 However, the tissue shrank to approximately half its initial size during transplantation. 16 Mesenchymal aggregation, which occurs during cartilage differentiation, may explain this shrinkage. 17 , 18 Therefore, a method in which cartilage differentiation is mostly completed in vitro is required.…”