Development of Reliable and High – Throughput Human Biomimetic Cartilage and Bone Models to Explore Senescence and Personalized Osteoarthritis Treatment Options

Abstract

To facilitate effective preclinical testing of senescence treatments for osteoarthritis (OA), we have created reliable biomimetic and high-throughput models using aged human joint tissues. Moreover, concerns regarding scalability led to the concurrent development of a high-throughput human in vitro senescence cartilage organoid model. Osteochondral explants and cells for the cartilage organoid model were isolated from patients undergoing joint replacement surgery due to OA. To induce senescence, explants and organoids were subjected to radiation and/or mechanical loading. Samples were harvested; gene expression of relevant senescent and cartilage genes was measured using RT-qPCR, and protein expression was evaluated using histology. A general senescence phenotype was induced by the perturbations, as shown by senescence-associated β-galactosidase staining. In-depth gene expression analysis revealed that hyperphysiological mechanical loading upregulated gene expression of IL8 and SERPINE1, representing aspects of a senescence-associated secretory phenotype (SASP) profile. Irradiation upregulated CDKN1A, encoding p21, and downregulated LMNB1, representing a cell cycle arrest profile with the absence of a SASP response. Combining the two perturbations showed upregulation of CDKN1A, IL8, and SERPINE and downregulation of LMNB1, representing a complementary senescence model. The high-throughput human in vitro cartilage organoid senescence model showed similar effects to the irradiation explant model. In this study, we present a variety of senescence models of human aged chondrocytes that allows for rapid initial screening of anti-senescence compounds in high-throughput, as well as in-depth, characterization of post-mitotic aged chondrocytes prone to OA pathophysiology. This research advances the development of essential therapeutics for OA.

Irradiation and Loading Regime

As seen in Figure 1, 3 days after extraction, explants were placed in sterile PBS and irradiated with 20 Gy (Yxlon, Germany). After 5 days of culture, 65% dynamic unconfined compression was applied using the MACH‐1 (Biomomentum Inc., Laval, Quebec, Canada) apparatus for 4 days. At Day 12, the explants were harvested for IHC and RNA analysis. The human in vitro cartilage organoids were exposed to 10 or 20 Gy ionizing radiation on Day 7 of the culture, and on Day 10, the cartilage organoids were harvested for RNA analysis. In total, 142 osteochondral explants were used from 29 donors, divided into controls (31 osteochondral explants from N = 29 donors), irradiated (16 osteochondral explants from N = 12 donors), hyperphysiological mechanical loaded (20 osteochondral explants from N = 18 donors), and a combination group (42 osteochondral explants from N = 29 donors). Additional details are available in the Supplementary methods, section “Irradiation and loading regime.”

Bone; cartilage; knee; osteoarthritis.