Type II Collagen Scaffold Used as Platforms for Mechanism Study of Hyaline Cartilage Regeneration and Anti-arthritis Drug Evaluation In Vitro

Abstract

Cartilage injuries are escalating worldwide, particularly in the aging society. Maintenance and rejuvenation of articular cartilage with hyaline phenotype remains a challenge as the underlying mechanism hasn’t been completely understood. Due to the native nature, type II collagen-based scaffold (Col2S) provides a suitable environment for cartilage repair in scientific research and clinical use. Although extensive studies have focused on cartilage regeneration of Col2S, the underlying mechanism remains largely unknown. Col2S offers an ideal platform to build in vitro cartilaginous tissue models for mechanistic study and notably pathological conditions modeling. Degenerative joint disorders including rheumatoid arthritis (RA) are the foremost causes of cartilage damage. RA is an inflammatory condition marked by chronic inflammation of synovial joints, contributing to progressive destruction of cartilage. Despite efforts to unearth effective treatment methods for RA, it remains an unmet need in the field. Consequently, there is a surge in efforts to develop standardized and reproducible in vitro models for efficient and cost-effective drug screening.

This thesis commences with an introductory overview of research objectives in the Chapter 1 and further delves into the pathophysiological milieu of joints, as well as current pathological models for RA therapeutics in Chapter 2. The focal point of this thesis lies in the mechanism study in Chapter 3 exploiting chondrocyte membrane nanoaggregates as antagonists for Col2S in which dedifferentiated chondrocytes can be rejuvenated into hyaline cartilaginous phenotype. This study develops an anti-Col2 strategy to explain how Col2 based scaffolds modulate chondrocyte phenotype and highlights the contribution of Col2 in rejuvenation and maintenance of hyaline cartilaginous phenotype. This thesis presents a synovium-cartilage system simulating RA microenvironment in vitro using 3D-bioprinted hydrogel and Col2S in Chapter 4. A biomimetic in vitro RA model was successfully established and their response to anti-arthritis drugs was confirmed, suggesting its potential for high-throughput preclinical drug testing. Details are elucidated as follows.

In the chapter 1, the introduction of this thesis begins with the overview of cartilage repair and inflammatory joint disease. This chapter will highlight the current challenges for maintenance and rejuvenation of articular cartilage with hyaline phenotype, as well as difficulties in modeling joint disease by in vitro studies. Current status of mechanisms studies in hyaline cartilage regeneration and the manufacturing approaches of biomaterials mimicking pathophysiological conditions will be described. Despite potent cartilage restoration of Col2 based scaffolds, the underlying mechanism remains unclear. To date, there have been no documented reports of both synovium and cartilage composite unit of the human joint model other than the individual cartilaginous or synovial tissue model reported previously. This chapter proposes research objectives of this thesis to address these limitations. The thesis aims to (1) Report the state of the art in hyaline cartilage regeneration and in vitro models for anti-arthritis drug evaluation; (2) Explore the mechanism underlying cartilage restoration of Col2-based scaffolds; (3) Establish a physiologically relevant in vitro model fully recapitulating the synovium-cartilage units in RA.

To achieve the proposed objectives, Chapter 2 initially offers a literature review to discuss the features of cartilage and the pathological characteristics of RA, 3D matrices for cartilage repair, 3D printing technique, in vitro pathological models for anti-arthritis drug evaluation, along with RA pharmacologic therapies. In this chapter, the features present in the microenvironment of cartilage and the pathological characteristics of RA are highlighted, with a specific focus on the microstructure, surface charge, and composition of cartilage, as well as the alterations of synovium and cartilage with the progress of RA. The research works on a diverse range of 3D matrices, are retraced to offer insights into recent advances in biomaterials for cartilage treatment applications, with emphasis on Col2S for the treatment of cartilage injuries. After a brief introductory overview of in vitro arthritis models for drug screening, 3D printing technique for the fabrication of in vitro models is discussed. Subsequently, a comprehensive survey of disease-modifying antirheumatic drugs (DMARDs) is provided.

To unveil the mechanism underlying cartilage regeneration of Col2 based scaffolds, a mechanism study in Chapter 3 has been designed and performed using scaffolds made of Col2 as the 3D cell cultural platforms, on some of which nanoaggregates comprising extracts of chondrocyte membrane (CCM) were coated as the antagonist of Col2. As the model cells, dedifferentiated chondrocytes (P5) were respectively seeded into these Col2-based scaffolds with (antCol2S) or without CCM coating. After 6 weeks, in Col2S, the chondrocytes were rejuvenated to regain hyaline phenotype; whereas, this redifferentiation effect was attenuated in antCol2S. Transcriptomic and proteomic profiling indicated that the Wnt/β-catenin signaling pathway, which is opponent to the maintenance of hyaline cartilaginous phenotype, was inhibited in Col2S, but it was contrarily upregulated in antCol2S due to the antagonism and shielding against Col2 by the CCM coating. Specifically, in antCol2S, since the coated CCM nanoaggregates contain the same components as those present on the surface of the seeded chondrocytes, the corresponding ligand sites on Col2 had been pre-occupied and saturated by CCM coating before exposure to the seeded cells. The results indicated that the ligation between Col2 ligands and integrin α5 receptors on the surface of the seeded chondrocytes in antCol2S was antagonized by CCM coating, which facilitates the Wnt/β-catenin signaling toward the loss of hyaline cartilaginous phenotype. This finding reveals the contribution of Col2 for the maintenance and rejuvenation of hyaline cartilaginous phenotype in chondrocytes by inhibition of Wnt/β-catenin signaling pathway via Col2-integrin α5 ligation.

For the establishment of a synovium-cartilage system simulating RA microenvironment in vitro, 3D bioprinting produces layer-by-layer architectures, enabling precisely recapitulating the complicated structures in vivo. The study in Chapter 4 explores the potential of anti-inflammatory drugs using bio-printed inflamed human synovium-cartilage units for evaluation. A biomimetic stratified geometric structure was bio-printed using gelatin-based composite bioink encapsulating fibroblast-like synoviocytes (FLS), pro-inflammatory M1-like macrophages, and anti-inflammatory macrophages for protection to create an in vitro RA model. Inflammatory stimuli were induced in the model by the incorporation of pro-inflammatory M1-like macrophages. In this project, we will focus on the creation of accurate and reliable 3D RA models that would allow us to study the inflammatory joint process in the context of ECM degradation and to investigate the influence of distinct human cell populations such as FLS and macrophages on chondrocytes.

Higher secretion of tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) and elevated gene expression of pro-inflammatory mediators (TNFA and IL6), degradative enzymes (MMP1 and MMP3), angiogenesis-related factors (VEGFA), and type I collagen (COL1A1), as well as the decreased production of type II collagen (COL2A1) and aggrecan (ACAN), validated the establishment of the proposed in vitro RA model. Subsequently, the effectiveness of anti-inflammatory drugs including Celecoxib and Tofacitinib, was validated in the established model. In the inflamed construct, the initial augmented secretion of key pro-inflammatory mediators (TNF-α and IL-6) was downregulated post-drug treatment. Additionally, the expression of fibroblast activation protein (FAP) indicating the activation and invasion of FLS, inducible nitric oxide synthase (iNOS) associated with inflammation, and TUNEL representing apoptosis of chondrocytes, was significantly diminished in the drug-treated group. Overall, the in vitro model simulates inflammation in RA and serves as a potential reliable platform to screen novel anti-inflammatory drugs for RA therapy. The significance of this study lies in that the 3D disease tissue models will fulfill the requirements for a cost-effective and reproducible in vitro model and will aid in bridging the gap between the overly simplified human cellular models and highly intricate animal models for future drug development.

In summary, the sophisticated pathophysiological microenvironment of cartilage poses challenges to the mechanism study of hyaline cartilage regeneration and the establishment of in vitro pathological model for anti-arthritis drug evaluation. To this end, this thesis provides a novel strategy to antagonize Col2 at nano-dimension and insights into the molecular dialogue in the regulation of hyaline articular cartilage regeneration. In addition, an in vitro synovium-cartilage model mimicking RA microenvironment has been successfully established and will be a preclinical drug high-throughput screening platform for RA therapeutics.

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