Abstract
Autologous chondrocyte implantation (ACI) depends on the quality and quantity of implanted cells and is hindered by the fact that chondrocytes cultured for long periods of time undergo dedifferentiation. Here we have developed a reproducible and efficient chondrogenic protocol to redifferentiate chondrocytes isolated from osteoarthritis (OA) patients. We used morphological, histological and immunological analysis together with a RT-PCR detection of collagen I and collagen II gene expression to show that chondrocytes isolated from articular cartilage biopsies of patients and subjected to long-term culture undergo dedifferentiation and that these cells can be redifferentiated following treatment with the chimeric Activin A/BMP2 ligand AB235. Examination of AB235-treated cell pellets in both in vitro and in vivo experiments revealed that redifferentiated chondrocytes synthesized a cartilage-specific extracellular matrix (ECM), primarily consisting of vertically-orientated collagen fibres and cartilage-specific proteoglycans. AB235-treated cell pellets also integrated into the surrounding subcutaneous tissue following transplantation in mice as demonstrated by their dramatic increase in size while non-treated control pellets disintegrated upon transplantation. Thus, our findings describe an effective protocol for the promotion of redifferentiation of autologous chondrocytes obtained from OA patients and the formation of a cartilage-like ECM that can integrate into the surrounding tissue in vivo.
Highlights
Autologous chondrocyte implantation (ACI) depends on the quality and quantity of implanted cells and is hindered by the fact that chondrocytes cultured for long periods of time undergo dedifferentiation
We previously reported the creation of chimeric ligands based on systematic swapping of BMP2 and Activin-A sequences using a strategy termed Random Assembly of Segmental Chimera and Heteromers (RASCH)[10]
Isolated chondrocytes were grown in monolayer culture up to passage 6 (P6) to ensure a complete dedifferentiation
Summary
Autologous chondrocyte implantation (ACI) depends on the quality and quantity of implanted cells and is hindered by the fact that chondrocytes cultured for long periods of time undergo dedifferentiation. This dedifferentiation causes chondrocytes to lose their round shape and become flattened fibroblast-like cells with an increased proliferative capacity and is accompanied by changes in gene expression and surface markers including decreased Col II and aggrecan and increased levels of Col I, Col X and COMP3 This loss of the differentiated chondrocyte phenotype upon culture in vitro represents a major disadvantage of the ACI technique because a decreased ratio of collagen type II/I results in production of an extracellular matrix typical of fibrotic tissue that might compromise cartilage regeneration[4]. Since the success of ACI depends on the number and quality of the cells to be implanted into the chondral lesion, approaches to revert dedifferentiation, called redifferentiation, are being investigated In this respect, some studies have focused on using 3D cultures[5] or growth factors, such as members of the TGF-β superfamily including bone morphogenetic proteins (BMPs)[6,7]. We found that one of these chimeras, AB235, significantly promotes chondrogenic differentiation of adipose-derived stem cells[11]
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