Osteoarthritis (OA) is a degenerative joint disease mostly occurring in large weightbearing joints such as the knee joint. It is generally characterised by a moderate degree of inflammation and synovitis;​ its other common features are degeneration of the articular cartilage, thickening of the joint capsule, osteophyte formation, meniscal erosion and exposed bone surfaces. Another characteristic feature of OA is angiogenesis, which is the physiological process of forming blood vessels that is vital in wound healing and in the development of many organs. However, angiogenesis is not expected to occur in articular cartilage, which is avascular in nature. This avascularity is maintained by a proper balance between angiogenic and antiangiogenic molecules within the tissue. Nonetheless, during OA this balance becomes impaired which may result in neovascularisation at various sites of the knee joint and possibly in the calcified cartilage. The roles of these angioregulatory molecules in OA pathology, or OA severity and progression, are not well described.

Another characteristic phenomenon from which OA patients suffer the most, is pain. It is assumed that angiogenesis plays a stimulatory role in the growth of nerves in knee joint tissue, as it is believed that nerve growth always follows the course of blood vessels in many other tissues. Some angioregulatory molecules, such as vascular endothelial growth factor-alpha (VEGF-A) and nerve growth factor (NGF), have been reported to stimulate both blood vessel and nerve growth. Therefore, blood vessel and nerve growth may be closely integrated and involved in OA pain. However, evidence of the detailed vascular and nerve networks in the knee joint tissue, specifically in the subchondral bone and the cartilage, is lacking. Also, it is not clear how angioregulatory molecules affect the cartilage phenotype. Therefore, this PhD project aimed to:​ 1) determine the relationship of angioregulatory molecules and OA, 2) visualise the neurovascular network in the knee joint and investigate their contribution to OA pain and finally, 3) investigate the effects of LSKL (an antagonist peptide of the anti-angiogenic molecule thrombospondin 1 [TSP1]) on cartilage and/or chondrocyte phenotypes.

A meniscectomised (MMX) rat model of OA and a human osteoarthritic osteochondral tissue model were evaluated with microscopic computed tomography (micro–CT) scanning, histological safranin-O staining, followed by Mankin scoring, immunohistochemical detection for VEGF-A, TSP1, TSP2, endostatin (EST) and CD31, and gene expression analysis for COL2A1, COL10A1, VEGF-A, THBS1, THBS2 and COL18A1, respectively, to assess the role of angioregulatory molecules in OA severity and progression.

To achieve the second aim, MMX and sham control rats were subjected to von Frey filament testing to assess pain behaviour (distal allodynia). At termination, vessels were perfused with a radio-opaque contrast agent (Microfil) and scanned with micro– CT, followed by image processing, to visualise the blood vessel networks inside and outside the knee bones. Immunofluorescence staining was performed on frozen sections from both rat and human osteochondral tissue to visualise the blood vessels and nerve fibres, followed by image analysing to evaluate vascularity.

Finally, to achieve the third aim, both human cartilage explants and articular chondrocytes in high-density monolayer culture were treated with various concentrations of the thrombospondin 1 inhibitor LSKL (0.1, 1.0, 10, 100, 1000 µM), a scrambled control peptide SLLK (1000 µM) and TGF-β3 (5 ng/mL), for 24 hours (for cartilage explants) and 48 hours (for monolayer chondrocytes), respectively, followed by cell viability assay, cell content determination assay (deoxyribonucleic acid [DNA] quantification assay), glycosaminoglycan (GAG) determination assay and gene expression analysis to assess chondrocyte phenotype and cytokine expression.

In rats, more severe OA was associated with lower levels of expression of TSP1 and EST, suggesting that angiogenesis could further stimulate OA severity and progression. A lack of healthy tissue from human patients meant it was not possible to reproduce these results in a human tissue model. In the second study, there was increased pain and vascularity in the OA-affected knee joint of the hind limbs of MMX rats as measured by von Frey filament testing and immunofluorescence staining, respectively. While micro–CT analysis revealed a significant increase in total vessel volume in the knee joint, Geomagic Wrap® software-based analysis found no change in total vessel volume or in vessel volume inside either the tibia or the femur. Human osteochondral tissue sections also revealed increased vascularity as well as nerve fibres running alongside the blood vessels in the subchondral bone of the medial tibial plateau, and vascularity at or near the osteochondral junction as well as in the calcified cartilage. Moreover, bone marrow lesions (BMLs) were also detected in the subchondral bone. Results for the second aim suggested that subchondral bone may act as a potential source of characteristic OA pain. Results in the third study revealed no significant change in cell viability or in GAG content released by the cells and in the media, compared to that of the control in either the explant or monolayer chondrocyte culture model. However, there was a significant increase in GAG/DNA content in cells treated with 100 µM LSKL, compared to that of cells in the non-treated control. Although gene expression analysis found no significant change in the explant model, monolayer chondrocytes in the LSKL-treated groups exhibited significant upregulation of ACAN, COL2A1, COL10A1 and ADAMTS4. Similar gene expressions were also observed in the cells treated with TGF-β3. The extended gene expression analysis of TGF-β1 responsive genes showed a downregulation of the CDKN1A gene in both LSKL– and TGF-β3–treated cells, while TGF-β1–treated cells also showed a downregulation of JUN. Results of the third study suggested that there might be multiple mechanisms responsible for TGF-β activation such that if one mechanism is blocked, another may be activated to maintain an adequate level of TGF-β in the cellular environment.

Therefore, this PhD project revealed a negative correlation between OA severity and expression of anti-angiogenic molecules:​ the higher the OA severity, the lower the expression of anti-angiogenic molecules. Furthermore, this study also developed a technique to capture detailed vascular networks both inside the bones and around the knee joints. Moreover, it also developed a modified immunofluorescence staining technique that reveals BMLs and neurovascular networks in the subchondral bone as well as in the articular cartilage. The findings of the study also suggest that subchondral bone may play a vital role in the pain mechanism underlying OA. Finally, the findings of this PhD project suggested that multiple mechanisms are involved in maintaining a constant level of active TGF-β in the cellular environment, provided an insight into the role of angioregulatory molecules in OA severity and developed a potential investigative tool to aid research into the roles of angiogenesis, nerve growth and BMLs in OA pain.