Type II Collagen Scaffolds for Osteochondral Regeneration: Repair Efficacy under Osteoarthritic Condition, the Impact of Microgravity and Cosmic Radiation, and Mechanism Investigations

Abstract

Cartilage, particularly the hyaline cartilage in weight-bearing joints, exhibits minimal self-healing ability due to its avascularity and aneural nature. Once significant discomfort arises, the defect typically permeates the non-sensitive nerve layer of the cartilage and invades the subchondral bone. Therefore, the repair of osteochondral defects (OCDs) is a more prevalent and formidable challenge than the repair of cartilage injuries. Current treatments in clinical practice, such as microfracture, frequently result in the development of fibrocartilage with suboptimal mechanical properties in the long term. Alternative therapies, like autologous chondrocyte implantation, are primarily constrained by the scarcity of donor tissue and the requirement for a second surgery, which may inflict additional therapeutic trauma to the subchondral bone. In short, OCDs are prevalent without effective methods for complete regeneration.

Type II collagen (Col2), an essential element of cartilaginous tissues, presents an excellent choice for creating biomimetic scaffolds. Among the various Col2 scaffolds, we biosynthesized a cartilaginous graft (LhCG) by leveraging the anchorage-independent proliferation of chondrocytes. LhCG can undergo mild decellularization (yielding dLhCG), which produces a cross-linked 3D network of Col2 with small amounts of other cartilage proteins and proteoglycans. Large animal studies have indicated that dLhCG engraftment in traumatic OCDs can induce fine restoration of cartilage and subchondral bone.

The first section of this thesis examined the use of Col2 scaffolds in tissue engineering, emphasizing their significant role in regenerating hyaline cartilage and intervertebral discs. It also explores the diverse applications of Col2 in disease modeling, bone regeneration, and the creation of artificial immune organs.

The second section of this thesis investigated the repair efficiency of Col2 scaffolds in rat osteoarthritic (OA) knee joints. OA, a degenerative joint disease characterized by cartilage deterioration, inflammation, and subchondral bone alterations, presents a significant challenge for OCD regeneration due to the bidirectional relationship between these conditions. Our 90-day post-implantation results demonstrated complete histological healing of critical-sized OCDs, with excellent integration into surrounding tissues. The regenerated tissue exhibited biochemical and biomechanical properties comparable to native tissue. We further validated the Col2 scaffolds' regenerative potential in over-critical-sized OCDs, observing promising histological outcomes 150 days post-implantation. Gene expression analysis revealed a unique profile in the regenerated tissue, with reduced expression of cartilage degradation-associated genes compared to surrounding osteoarthritic tissue, while maintaining expression levels of osteochondral anabolism genes similar to healthy tissue. Transcriptomic and proteomic analyses indicated suppression of the TGF-β-Smad1/5/8 pathway in regenerated OA tissue, consistent with findings in over-critical-sized OCDs.

The third section of this thesis explored the effects of space environments, specifically microgravity and cosmic radiation, on the scaffold's characteristics and reparative efficacy. In May 2023, multiple dLhCG scaffolds were delivered to the China Tiangong Space Station for a 6-month exposure test, both inside (In-dLhCG) and outside (Out-dLhCG) the spacecraft, with corresponding dLhCG scaffolds maintained on the ground (G-dLhCG) as controls. When examined under TEM, dLhCG displayed a structure morphologically and dimensionally similar to native cartilage fibers. Given that dLhCG is an engineered cartilaginous tissue that underwent decellularization without physical destruction or protease digestion, we reasonably infer that its ultrastructure mirrors the microfibrillar arrangement observed in native cartilage. Additionally, we found that dLhCG maintained their macroporous structure, ultrastructure, and immunohistochemistry characteristics after six months of exposure to space conditions. In vivo studies revealed no significant differences in the regenerative capacity among G-, In-, and Out-dLhCG.

The fourth section of this thesis investigated the intricate regeneration process and its underlying mechanisms. We observed that upon implantation into the OCD site, dLhCG undergoes four stages of regeneration: acute inflammatory infiltration, inflammation subsidence with osteochondral cell entry, exclusive occupation by mixed-phenotype osteochondral cells, and development of distinct articular cartilage and subchondral bone phases. The relative osteoblast count peaked at day 60 before declining to day 40 levels by day 80, while chondrocyte numbers steadily increased. dLhCG facilitates osteochondral regeneration by modulating interactions with chondrocytes and osteoblasts in a temporally regulated manner, activating specific signaling pathways at distinct postoperative stages.

This study marked the first effort to examine the repair efficiency of Col2 scaffolds in OA conditions. Additionally, this study is expected to significantly contribute by conducting the first space experiment on cartilage repair scaffolds, laying the foundation for fulfilling the future requirement of repairing articular cartilage in space.

Date of Award	31 Dec 2024
Original language	English
Awarding Institution	City University of Hong Kong
Supervisor	Dongan WANG (Supervisor)