Investigating the role of lysyl oxidase in lateral integration of engineered cartilage tissue spheroids

Abstract

Introduction: Degeneration of articular cartilage leading to osteoarthritis is the most common joint disease in the world. Attempts to regenerate defects show inadequate lateral integration. Copper (Cu)-dependent lysyl oxidase-like 2 (LOXL2) is a critical endogenous enzyme for healthy cartilage growth and remodeling. In this study, LOXL2 and Cu were studied for their effect on differentiation of tissue-engineered cartilage spheroids (TS) and cartilage health in inflammatory and nutrient-deprived conditions.

Methods: TS were cultured from human articular chondrocytes (hACs) in both isolated and tissue fusion conditions to assess LOXL2 and Cu impact on lateral integration. Supplementation of LOXL2 and Cu was implemented either once or twice, at the individual TS and/or TS-TS fusion stage, to elucidate the time of greatest effect and/or any synergistic benefits for double dosing. Histological, immunofluorescent, mechanical, and biochemical metrics were used to measure cartilage health, integration, and LOXL2 activity. In parallel, chicken eggshell membrane (ESM) was investigated as a possible supplement for acellular collagen crosslinking given its high natural LOXL2 content. Cu delivery via biomaterial doping of PCL fusion scaffolds was also pursued in parallel experiments. Cu cytotoxicity, printing feasibility, and Cu leeching from scaffolds were all assessed.

Results: LOXL2 and Cu supplementation lead to increased mechanical properties in individual TS. Exposure of TS to inflammatory cytokines depleted glycosaminoglycans (GAGs) but were regained and then some during a period of healing culture, with LOXL2 and Cu displaying a greater increase in GAGs than without. TS-TS integration was induced most by LOXL2 and Cu, then Cu only, then non-treated (NT) conditions, with synergism seen with multiple doses of LOXL2 and Cu. Four weeks of fusion culture had vast benefits in integration compared to two weeks. ESM treated with collagenase to further isolate LOX2 showed some promise in enhancing collagen hydrogel mechanics. Copper sulfate was cytotoxic to hACs over 0.16 mg/mL and Cu was steadily released from 3D-printed PCL fusion constructs over 15 days following a Day 1 burst release.

Conclusion: LOXL2 aids in GAG production and lateral integration even in disease-like conditions. Cu release can be integrated into the established fusion system.