Osteochondral Live Slices as Physiological in-vitro Model of the Human Joint for High-throughput Applications

Abstract

Background: Current advances in musculoskeletal research yielded numerous new insights and therapy options for diseases of the joints but preclinical in-vitro testing is currently limited to self-manufactured 2D and 3D cell cultures only remotely mimicking the in-vivo situation in regards of tissue composition and cell configuration. While in-vivo animal models are a lot more lifelike, they are inherently connected with ethical concerns, translation to the human situation, logistic efforts and high costs. Here we explored the use of native human life slice cultures from explanted tibial plateaus upon their feasibility to serve as a highly lifelike and easy-to-handle model of the human joint which can be obtained in masses from few tissue samples.Methods: Osteochondral slices (1cm x 1cm x 500µm) were prepared with a special microtome from 23 human tibial plateaus and subsequently cultivated in hanging inserts over the course of 7 or 21 days. Short-term cultivated slices were stimulated with either 800 pM/1.2 nM TNF-α or 800 pM TGF-β3. During cultivation, viability of tissue cells was assessed via laser microscopy / resazurin assay. After cultivation, gene expression of cartilage ECM Marker proteins was quantified with RT-PCR. TNF-α stimulated slices and their controls were stained with Safranin-O and analyzed via histomorphometry to quantify tissue proteoglycan content. Results: Tissue cell viability remained >90% over the three weeks. Laser scanning microscopy revealed highly conserved spatial alignment of cells inside the cartilage. Incubation of the slice cultures with TNF-α showed a significant, dose dependent decrease in mRNA expression of cartilage proteins collagen II, cartilage oligomeric matrix protein and aggrecan while incubation with TGF-β3 caused a significant increase of early bone formation proteins collagen I and also cartilage oligomeric matrix protein. Histologically, TNF-α incubation caused a 32% reduction of proteoglycans, detachment of collagen fibers and cell swelling.Conclusion: In summary, native osteochondral slice cultures provide a stable and manipulable, physiological model of human joint bone and cartilage biology. The setup is easy to produce and handle, scalable, and contributes to 3R principles in biomedical research, suggesting itself as a platform for preclinical testing especially regarding high-throughput applications.