Enzymatic Isolation of Articular Chondrons: Is It Much Different Than That of Chondrocytes?

Abstract

In native articular cartilage, chondrocytes are completely capsulated by a pericellular matrix (PCM), together called the chondron. Due to its unique properties (w.r.t. territorial matrix) and importance in mechanotransduction, the PCM and chondron may be important in regenerative strategies. The current gold standard for the isolation of chondrons from cartilage dates from 1997. Although previous research already showed the low cell yield and the heterogeneity of the isolated populations, their compositions and properties have never been thoroughly characterized. This study aimed to compare enzymatic isolation methods for chondrocytes and chondrons and characterize the isolation efficiency and quality of the PCM. Bovine articular cartilage was digested according to the 5-hour gold standard chondron isolation method (0.3% dispase + 0.2% collagenase II), an overnight chondron isolation (0.15% dispase + 0.1% collagenase II), and an overnight chondrocyte isolation (0.15% collagenase II + 0.01% hyaluronidase). Type VI collagen staining, fluorescence-activated cell sorting (FACS) analysis, specific cell sorting and immunohistochemistry were performed using a type VI collagen staining, to study their isolation efficiency and quality of the PCM. These analyses showed a heterogeneous mixture of chondrocytes and chondrons for all three methods. Although the 5-hour chondron isolation resulted in the highest percentage of chondrons, the cell yield was significantly lower compared to the other isolation methods. FACS, based on the type VI collagen staining, successfully sorted the three identified cell populations. To maximize chondron yield and homogeneity, the overnight chondron enzymatic digestion method should be combined with type VI collagen staining and specific cell sorting.