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.

Keywords: bone; cartilage; knee; osteoarthritis.

Figure 1

Experiment timeline of (A), the osteochondral explants; timeline of the irradiation (IR), hyperphysiological mechanical loading (ML), and combination (IR + ML) models. Bone and cartilage were separated for gene expression analysis. Before separation, cross‐sections were taken for histology. (B) The high‐throughput human in vitro cartilage organoids; timeline of the IR and harvest of the human in vitro mini cartilage organoid model. Complete pellets were taken for gene expression analysis and histology. Created with BioRender.com.

Figure 2

Representative images and quantification of SA‐B‐gal staining in (A) Control explant. (B) Hyperphysiological mechanical loading model. (C) Irradiation model. (D) Combination model. (E) Overview image of entire control explant, scale bar is 600 μm. Scale bar is 100 μm in A–D. (F) For the quantification of the staining, four squares (black squares in E) were used per histological section. In total, two histological sections were quantified per condition. N = 1 donor and two histological sections. Abbreviations: Combi = combination model, IR = irradiation model, ML = hyperphysiological mechanical loading model, SD = standard deviation.

Figure 3

Gene expression of the cell cycle arrest profile and SASP genes in (A), the cartilage of the hyperphysiological mechanical loading (17–51 osteochondral explants and N = 12–29 donors) and in the irradiation groups (16–47 osteochondral explants and N = 11–29 donors). (B) The bone cartilage of the hyper physiological mechanical loading (45–51 osteochondral explants and N = 27–29 donors) and in the irradiation groups (45–47 osteochondral explants and N = 28–29 donors). (C) The high‐throughput human neo‐cartilage mini pellet model (19–31 mini pellets and N = 3 donors). Neo‐cartilage organoids = high‐throughput human in vitro cartilage organoids, ML = hyperphysiological mechanical loading model, IR = irradiation model, *p < 0.05, **p < 0.01, ***p < 0.001.