A Novel Scaffold-Free Engineered Cartilage Using Combined Chondrocyte Pellets and Sheets in Chondrogenic Differentiation Medium

Abstract

BACKGROUND: The scaffold-free approach has emerged with a focus on creating cartilage-like tissues using cell pellets, cell spheroids, and cell sheets. However, complete repair of damaged cartilage using these tissues remains an ongoing challenge due to the limitation of thin structure and poor structural integrity. METHOD: In this study, we proposed a novel method to produce scaffold-free cartilage by combining cell pellets and cell sheet technology as chondrocyte pellet-sheet tissues. The chondrocyte sheets acted as a support platform at the top and the bottom of the ten chondrocyte pellets. At day 7, the quality of the tissues cultured in a chondrogenic differentiation medium (CDM) and basal medium was compared using real-time PCR, immunofluorescence staining, proteomics, and atomic force microscopy (AFM). RESULTS: Our method supported the enhancement of tissue thickness. Compared to the control basal medium, the diameter and thickness of the chondrocyte pellet-sheet tissues in CDM were 1.47- and 2.21-fold increase, respectively. The level of mRNA expression and immunostaining of collagen type II were higher in the tissues cultured in CDM, compared to those in basal medium. Using proteomics, transferrin was found in both fresh and cultured CDM. The protein profiles of the tissues in CDM revealed the downregulation of actin and the upregulation of fibromodulin (FMOD), which related to the reorganization of cell shape and the production of cartilage ECM, respectively. Pathway analysis of chondrocyte pelletsheet tissues in CDM also revealed the inhibition of RhoA and the presence of a TGFb signaling pathway with SMAD protein signals. Moreover, Young’s modulus indicating structural integrity of the tissues cultured in CDM (28.25 ± 13.13 kPa) was higher than those in basal medium (4.63 ± 2.25 kPa). CONCLUSION: Combining chondrocyte pellets and sheets in CDM allowed the generation of thick tissues and enhanced structural integrity. The compacted structure of the tissues in CDM might inhibit actin expression via RhoA inhibition. Growth factors in CDM, especially transferrin might be involved in chondrogenic differentiation via TGFb signaling pathway with SMAD protein signals.