Osteoarthritis (OA) is a degenerative joint disease. Clinically, it manifests as joint pain, tenderness, stiffness, locking, and sometimes effusion, affecting the mobility and quality of life of OA patients. OA belongs to the ten most disabling diseases in the developed countries. OA affects over 240 million people globally, and the number of OA patients is projected to increase in the next 30 years due to ageing and increasing in world population. The annual cost for each OA patient’s treatment ranges from hundreds to thousands of euros, posing a heavy burden not only on the patient’s budget but also on national, and even international economies. Advanced OA can lead to irreversible joint disability, and there are no disease-modifying OA drugs available. In general, surgical interventions are used to relieve joint pain and improve joint functions.
It is recognized that subchondral bone sclerosis, cartilage erosion, and osteophyte formation are hallmarks of advanced OA. However, it is still poorly known what happens during the very early stages of OA. Moreover, most of the tissue-level studies with human materials have been limited to late-stage OA. Thus, animal models are usually utilized to examine the osteochondral changes in early OA. Though some previous studies have been conducted to monitor the early osteochondral changes in a rabbit model with post-traumatic OA (PTOA), those studies have tended to focus on the changes in either articular cartilage or subchondral bone, or only a few locations or at a single time point. Instead, a relatively comprehensive understanding of osteochondral changes during OA progression would be gained by evaluating multiple tissues in multiple locations at multiple time points. Furthermore, although it is known that the subchondral plate becomes thicker with OA, there is a limited armory of tools available for exploring how this occurs, especially in a three-dimensional (3D) characterization. In fact, calcified cartilage is usually neglected in the 3D analysis of the subchondral structure, and deep learning methods now show potential for 3D calcified cartilage segmentation and analysis. On the other hand, if one wishes to elucidate the tissue-level changes of articular cartilage in early OA, then the metabolic and inflammatory activities of articular cartilage need to be analyzed. Finally, since the strain is a crucial factor for mechanotransduction, the biomechanics of in situ chondrocytes and cartilage matrix should be explored in the early phases of OA.
In this thesis, the early development of OA was investigated using the anterior cruciate ligament transection (ACLT) induced PTOA rabbit model. The earliest changes in osteochondral tissues were studied two weeks after ACLT, and the more progressive changes were studied eight weeks after ACLT. The 3D subchondral morphology was analyzed using a micro-computed tomography (µCT), and a novel pipeline was proposed for automatic calcified cartilage segmentation and thickness analysis from high-resolution µCT images. The thickness, degeneration, proteoglycan content, and collagen orientation of articular cartilage were examined using optical coherence tomography, histology, digital densitometry, and polarized light microscopy, respectively.
As strain is an essential regulator of mechanotransduction, different experimental mechanical strain measurement methods were reviewed. In an attempt to clarify the changes in chondrocytes' deformation behavior, chondrocyte morphology was evaluated with confocal microscopy during the indentation experiment. In addition, the cartilage local strains and chondrocyte viability were assessed from the indentation experiments. Finally, when comparing biomechanical and chondrocyte function, gene expression levels related to cartilage matrix synthesis, and catabolic and inflammatory activities were analyzed using a quantitative polymerase chain reaction technique.
The results showed that the subchondral structure and articular cartilage were altered in a sitespecific manner during the OA progression in the PTOA rabbit model. During the advancement of PTOA, the subchondral bone morphology deteriorated progressively, while the collagen orientation angle in the articular cartilage displayed only minor changes at both time points. The two-week ACLT presented with a greater OARSI score in the femoral condyles and thinner trabeculae in the lateral tibial plateau and groove locations, together with a loss of proteoglycans in the femoral condyles, medial tibial plateau, groove and patella. Eight weeks after ACLT, the OARSI score was greater in the femoral condyles and lateral tibial plateau, while a deterioration of subchondral bone was observed in most of the knees’ locations. Proteoglycan loss was observed in the femoral condyles, groove and patellar cartilage of the ACLT group when compared to the age-matched control (CNTRL) group eight weeks after ACLT. In addition, a novel pipeline was developed and validated; it was confirmed to be reliable for 3D µCT-based calcified cartilage segmentation and thickness analysis. From the proposed pipeline, site-specific calcified cartilage thickness was determined in the CNTRL knees; the patella and tibial plateaus had the thickest and thinnest calcified cartilage respectively. Furthermore, a preliminary histological assessment indicated that ACLT surgery could result in thicker calcified cartilage in both surgical and contralateral knees in a site-specific manner.
During the indentation testing, the chondrocyte surface area did not decrease in the lateral cartilage of the ACLT group as occurred in the CNTRL group. The mRNA expression levels of matrix synthesizing enzymes and compounds including aggrecan, type VI collagen hyaluronan synthases-1 and Dickkopf-related protein 1, and proteins involved in matrix catabolism including matrix metalloproteinase-3 and 13, as well as inflammation factor interleukin-6, were all upregulated in the surgically-operated knees at two weeks’ post-ACLT.
In conclusion, ACLT induced site-specific changes in subchondral bone, articular cartilage and calcified cartilage in the rabbit model. It was observed that subchondral bone became progressively deteriorated during the progression of early OA, while there were only minor changes in the collagen orientation angle of articular cartilage. The tibial plateaus were sensitive to the morphological changes in subchondral bone, while the femoral condyles were sensitive to the composition changes of articular cartilage. ACLT-evoked changes in the animals’ activities, rather than surgery itself induced a sitespecific thicker calcified cartilage. In early OA, the metabolic and inflammatory activities of chondrocytes were increased site-specifically in terms of mRNA levels; the biosynthesis of ACAN occurred in the lateral and groove compartments of surgical knees, contributing to the ability of the altered cartilage to resist mechanical loading.