Abstract
A major challenge of cell-based therapy for cartilage lesions is the preservation of the chondrogenic phenotype during ex vivo cell cultivation. In this in vitro study, the chondro-inductive capacity of two different hyaline cartilage-conditioned cell culture media on human chondrocytes in 3D spheroids was determined. Media were conditioned by incubation of 200 mg/mL vital or devitalized cartilage matrix in growth media over 35 days. The media were analyzed for the content of soluble procollagen type (Col) II and glycosaminoglycans (GAGs) as well as released TGF-β1, IGF-1 and IGFBP3. Unconditioned medium served as a negative control while the positive medium control was supplemented with TGF-β1 and IGF-1. Spheroid cultures prepared from human chondrocytes were cultivated at 37 °C, 5% CO2 and 21% O2 in the respective media and controls. After 14 and 35 days, the deposition of ECM components was evaluated by histological analysis. Vital cartilage-conditioned medium contained significantly higher levels of Col II and active TGF-β1 compared to medium conditioned with the devitalized cartilage matrix. Despite these differences, the incubation with vital as well as devitalized cartilage conditioned medium led to similar results in terms of deposition of proteoglycans and collagen type II, which was used as an indicator of re-differentiation of human chondrocytes in spheroid cultures. However, high density 3D cell cultivation showed a positive influence on re-differentiation.
Highlights
Defects of hyaline cartilage have recently been identified as the majority of diseases of the musculoskeletal system in both humans and animals [1]
To proof the devitalization by High Hydrostatic Pressure (HHP), the samples of cartilage were digested with collagenase A and the number of living cells was determined by Trypan blue staining
Long-term incubation of hyaline cartilage in defined cell culture medium led to the release of molecules, such as GAGs, procollagen type II and transforming growth factor (TGF-)β1, resulting in the conditioning of culture medium
Summary
Defects of hyaline cartilage have recently been identified as the majority of diseases of the musculoskeletal system in both humans and animals [1]. Defect regeneration leads often to fibrocartilage-like tissue in patients [1]. This replacement tissue is characterized by a high amount of collagen type I and low proteoglycan content and lacks important properties such as resistance to biophysical loading. Some surgical treatment procedures have emerged including arthroscopic debridement and lavage, subchondral bone microfracture, osteochondral transplantation and autologous chondrocyte implantation (ACI). These treatments can lack in restoration of functional properties of the replaced cartilaginous tissue [4]. The loss of the chondrogenic phenotype already after the first passage in vitro is well documented [5,6] and is characterized by morphological changes, a decrease in secretion of cartilage-specific matrix proteins such as collagen II and aggrecan and the up-regulation of mesenchymal stem cell genes [6,7,8,9,10,11,12,13,14]
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