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

In native articular cartilage, chondrocytes (Chy) are completely capsulated by a pericellular matrix (PCM), together called the chondron (Chn). Due to its unique properties (w.r.t. territorial matrix) and importance in mechanotransduction, the PCM and Chn may be important in regenerative strategies. The current gold standard for the isolation of Chns 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 Chy and Chns and characterizes the isolation efficiency and quality of the PCM. Bovine articular cartilage was digested according to the 5-h (5H) gold standard Chn isolation method (0.3% dispase +0.2% collagenase II), an overnight (ON) Chn isolation (0.15% dispase +0.1% collagenase II), and an ON Chy 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 Chy and Chns for all three methods. Although the 5H Chn isolation resulted in the highest percentage of Chns, 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 Chn yield and homogeneity, the ON Chn enzymatic digestion method should be combined with type VI collagen staining and specific cell sorting. Impact statement Since chondrocytes are highly dependent on their microenvironment for maintaining phenotypic stability, it is hypothesized that using chondrons results in superior outcomes in cartilage tissue engineering. This study reveals the constitution of cell populations obtained after enzymatic digestion of articular cartilage tissue and presents an alternative method to obtain a homogeneous population of chondrons. These data can improve the impact of studies investigating the effect of the pericellular matrix on neocartilage formation.

HR-pQCT for the Evaluation of Muscle Quality and Intramuscular Fat Infiltration in Ageing Skeletal Muscle.

Myosteatosis is the infiltration of fat in skeletal muscle during the onset of sarcopenia. The quantification of intramuscular adipose tissue (IMAT) can be a feasible imaging modality for the clinical assessment of myosteatosis, important for the early identification of sarcopenia patients and timely intervention decisions. There is currently no standardized method or consensus for such an application. The aim of this study was to develop a method for the detection and analysis of IMAT in clinical HR-pQCT images of the distal tibia to evaluate skeletal muscle during the ageing process, validated with animal and clinical experimentation. A pre-clinical model of ovariectomized (OVX) rats with known intramuscular fat infiltration was used, where gastrocnemii were scanned by micro-computed tomography (micro-CT) at an 8.4 µm isotropic voxel size, and the images were analyzed using our modified IMAT analysis protocol. IMAT, muscle density (MD), and muscle volume (MV) were compared with SHAM controls validated with Oil-red-O (ORO) staining. Furthermore, the segmentation and IMAT evaluation method was applied to 30 human subjects at ages from 18 to 81 (mean = 47.3 ± 19.2). Muscle-related parameters were analyzed with functional outcomes. In the animal model, the micro-CT adipose tissue-related parameter of IMAT% segmented at −600 HU to 100 HU was shown to strongly associate with the ORO-positively stained area (r = 0.898, p = 0.002). For the human subjects, at an adjusted threshold of −600 to −20 HU, moderate positive correlations were found between MV and MD (r = 0.642, p < 0.001), and between MV and IMAT volume (r = 0.618, p < 0.01). Moderate negative correlations were detected between MD and IMAT% (r = −0.640, p < 0.001). Strong and moderate associations were found between age and MD (r = −0.763, p < 0.01), and age and IMAT (r = 0.559, p < 0.01). There was also a strong correlation between IMAT% and chair rise time (r = 0.671, p < 0.01). The proposed HR-pQCT evaluation protocol for intramuscular adipose-tissue produced MD and IMAT results that were associated with age and physical performance measures, and were of good predictive value for the progression of myosteatosis or sarcopenia. The protocol was also validated on animal skeletal muscle samples that showed a good representation of histological lipid content with positive correlations, further supporting the clinical application for the rapid evaluation of muscle quality and objective quantification of skeletal muscle at the peripheral for sarcopenia assessment.