Current treatments for cartilage lesions are often associated with fibrocartilage formation and donor site morbidity. Mechanical and biochemical stimuli play an important role in hyaline cartilage formation. Biocompatible scaffolds capable of transducing mechanical loads and delivering bioactive instructive factors may better support cartilage regeneration. In this study we aimed to test the interplay between mechanical and FGF-18 mediated biochemical signals on the proliferation and differentiation of primary bovine articular chondrocytes embedded in a chondro-conductive Fibrin-Hyaluronan (FB/HA) based hydrogel. Chondrocytes seeded in a Fibrin-HA hydrogel, with or without a chondro-inductive, FGFR3 selective FGF18 variant (FGF-18v) were loaded into a joint-mimicking bioreactor applying controlled, multi-axial movements, simulating the natural movements of articular joints. Samples were evaluated for DNA content, sulphated glycosaminoglycan (sGAG) accumulation, key chondrogenic gene expression markers and histology. Under moderate loading, samples produced particularly significant amounts of sGAG/DNA compared to unloaded controls. Interestingly there was no significant effect of FGF-18v on cartilage gene expression at rest. Following moderate multi-axial loading, FGF-18v upregulated the expression of Aggrecan (ACAN), Cartilage Oligomeric Matrix Protein (COMP), type II collagen (COL2) and Lubricin (PRG4). Moreover, the combination of load and FGF-18v, significantly downregulated Matrix Metalloproteinase-9 (MMP-9) and Matrix Metaloproteinase-13 (MMP-13), two of the most important factors contributing to joint destruction in OA. Biomimetic mechanical signals and FGF-18 may work in concert to support hyaline cartilage regeneration and repair. Statement of significance: Articular cartilage has very limited repair potential and focal cartilage lesions constitute a challenge for current standard clinical procedures. The aim of the present research was to explore novel procedures and constructs, based on biomaterials and biomechanical algorithms that can better mimic joints mechanical and biochemical stimulation to promote regeneration of damaged cartilage. Using a hydrogel-based platform for chondrocyte 3D culture revealed a synergy between mechanical forces and growth factors. Exploring the mechanisms underlying this mechano-biochemical interplay may enhance our understanding of cartilage remodeling and the development of new strategies for cartilage repair and regeneration.
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While the expression of COL1 and 10 showed no significant differences between treatment groups (Fig. 5A and C, respectively), the synergistic action of mechanical loading and 100 ng/mL FGF-18v led to significant COL2 upregulation (Fig. 5B), further supported by IHC analysis (Fig. 6). Furthermore, when looking at unnormalized gene expression data (Fig. S1A), COL2 overall expression was higher than COL1 expression for all groups. Thus, we calculated COL2/COL1 absolute expression ratio (Fig. S1B), confirming a ratio favorable to COL2, suggesting that the chondrocytic phenotype was maintained within the 3D environment in both treated and untreated conditions. This should lead to the production of hyaline cartilaginous tissue in favor of fibrocartilage . In addition, in our system COL10 expression decreased after seeding into the hydrogel (Fig. S1A), further suggesting the preservation of the differentiated state of chondrocytes, not leading to hypertrophy, characteristic of OA cartilage .
This project has received funding from the European Union's Horizon 2020 research and innovation programme under Marie Sklodowska-Curie Grant Agreement No. 642414.
© 2020 Acta Materialia Inc.
- Chondrogenic differentiation
- Fibrin-hyaluronan hydrogel
- Fibroblast growth factor-18
- Multi-axial loading