Cartilage Tissue Formation by Passaged Human Chondrocytes is Not Influenced by the Severity of Osteoarthritis from which the Cells are Obtained
Bianchi, Vanessa 1, 2 ; Backstein, David 3 ; Kandel, Rita 1, 2, 4
1. Lunenfeld-Tannenbaum Research Institute, Toronto Canada; 2. Institute of Biomaterials and Biomedical Engineering, University of Toronto.; 3. Department of Orthopaedics, Mt. Sinai Hospital, Toronto Canada; 4. Pathology and Laboratory Medicine, Mt Sinai Hospital, Toronto Canada
Purpose: Biological therapies to repair damaged articular cartilage (AC) are being explored as a better alternative to total joint replacement with a prosthetic. We have shown in previous work that serially passaged human chondrocytes from osteoarthritic (OA) donors can be used to form robust and phenotypically stable AC in vitro. However, it is still unclear whether the severity of OA from which the cells are derived affects the capacity to form new AC. As cartilage is vastly acellular, the isolated cells need to be serially passaged. When passaged they acquire a progenitor-like phenotype similar to that of mesenchymal stromal cells (MSC), characterized by the ability to differentiate into all 3 of the mesenchymal lineages, and expression of MSC surface markers (CD105, CD166, CD73 and CD90). Therefore, we hypothesize that the severity of OA will not affect the ability of serially passaged human chondrocytes to redifferentiate and form AC-like tissue in vitro.
Methods: AC was attained with patient consent and research board approval. Tissue was examined macroscopically for indications of OA prior to dissection and cell isolation. Areas showing minimal osteoarthritic changes (Low OA) and more severe osteoarthritic changes (High OA) were dissected and separated. Representative samples of tissue were fixed in formalin for histological analysis. Chondrocytes were isolated enzymatically and passaged twice (P2) to 9 population doublings in DMEM 20% FBS. P2 cells were seeded onto collagen type 2 coated Millicell inserts (12mm) at 2 x 106 cells per membrane. Cells were cultured for 3 weeks in serum-free redifferentiation media with 10ng/ml TGFß3 as previously established (Bianchi et al. Tiss Eng Part A. 2017). In vitro formed tissues were assessed histologically, biochemically and by immunohistochemistry.
Results: Histological evaluation and grading of native tissue (OARSI Pritzker et al.) revealed that tissues isolated from areas of High OA were significantly different than Low OA. High OA tissue was thinner, had clusters of proliferative chondrocytes and had complete loss of proteoglycan in the proximal region, hallmarks of severe OA. After serial passaging and redifferentiation, no significant differences were observed in the amount of sulphated proteoglycan or collagen accumulated in the in vitro formed tissues. Furthermore, when assessed via immunohistochemistry the in vitro formed tissues from both groups accumulated hyaline cartilage matrix (collagen type 2 and aggrecan). Collagen type X and collagen type 1 were absent in the in vitro formed tissues in both groups.
Conclusions: One of the major concerns with using chondrocytes from OA cartilage for biological therapies is the potential for the diseased phenotype to be maintained. In this study we demonstrated that chondrocytes originating from AC with a high severity of OA were able to form tissues with negligible differences from that of cells isolated from AC with a low severity of OA.