Passaged chondrocytes will form hyaline cartilage-like tissue in vitro independent of whether cells are obtained from early or late osteoarthritic cartilage

Purpose: Biological therapies to repair damaged or diseased articular cartilage (AC) are being explored as an alternative to prosthetic joint replacement. Autologous chondrocytes can easily be obtained from a small biopsy of articular cartilage excised arthroscopically in a minimally invasive procedure, and may be the optimal cell source for a biological therapy. We have shown previously that serially passaged human chondrocytes from low grade osteoarthritic (OA) donors can be used to form robust and phenotypically stable AC in vitro. This may be due in part to the fact that when chondrocytes are serially 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 increased expression of MSC surface markers, CD105, CD166, CD73 and CD90. As studies continue to emerge identifying changes in the chondrocyte phenotype with OA it is important to investigate whether these changes have the potential to influence the suitability of OA chondrocytes for bioengineering applications Therefore we hypothesize that following serial passaging in monolayer culture, chondrocytes will redifferentiate and form hyaline cartilaginous tissues regardless of the severity of OA affecting the tissues from which they were isolated. Methods: AC was obtained with patient consent and Research Ethics Board approval (Mt. Sinai Hospital). Tissue was graded macroscopically for OA changes prior to harvest using the ICRS Clinical Cartilage Injury Evaluation System. Areas of AC showing minimal osteoarthritic changes (Low OA, grade 1) or more severe osteoarthritic changes (High OA, grade of 3 adjacent to eroded areas) were dissected separately. Representative samples of tissue were formalin fixed for histological analysis. Chondrocytes were isolated enzymatically and either used immediately (P0) or passaged twice (P2, approx. 9 doublings) in DMEM supplemented with 20% FBS. P0 and P2 cells were placed onto collagen type 2 coated membranes (MillicellTM 12mm, 2x106 cells). Cells were cultured in these 3D cultures for 3 weeks in serum-free (1% ITS) redifferentiation media supplemented with 10ng/ml TGFβ3. Selected cultures were grown in the presence of (B-glycerophosphate). In vitro formed tissues were assessed histologically, biochemically and by immunohistochemistry. N=3. Results: When assessed histologically, AC from High OA was fibrillated and thinner due to erosion and loss of proteoglycans. Additionally, there were clusters of chondrocytes, hallmarks of more severe OA. Low OA cartilage showed intact cartilage tissue with no chondrocyte cloning. Histological sections were also graded using the OARSI grading system by Pritzker K. P. H. et al. The average grade of Low OA tissue was 1.50 (± 0.86 SD) which was significantly lower (p=0.02) than High OA tissue which had an average grade of 4.34 (± 0.28 SD). Primary chondrocytes obtained from either low or high grade OA cartilage did not form cartilage tissue in vitro. However after passaging, cells from both types of cartilage redifferentiated and formed a continuous layer of cartilage-like tissue. There were no significant differences in the amounts of sulphated proteoglycan (p=0.6) or collagen (p=0.9) accumulated in the tissues formed by P2 cells from either group (Fig 1). Immunohistochemical studies showed that tissues formed by passaged chondrocytes isolated from either High or Low OA cartilage accumulated matrix rich in collagen type 2 and aggrecan, the major extracellular matrix molecules of hyaline AC. No collagen type X or collagen type 1 were detected in these tissues. When these tissues were cultured in the presence of mineralizing inducing media (B-glycerophosphate) the tissue did not calcify. Conclusions: This study demonstrated that the grade of OA did not adversely affect the ability of the isolated chondrocytes to form articular cartilage-like tissue. Interestingly non-passaged cells were not able to form cartilage tissue suggesting that passaging induces changes in the cell phenotype allowing them to acquire chondrogenic potential. This would be in keeping with the study by Bernstein P. et al. that showed that acquisition of progenitor characteristics was not influenced by the grade of OA. Further study is required to determine if these changes are contributing to the ability of OA cells to form cartilage in vitro.