Articular cartilage is a viscoelastic, supportive soft tissue serving as an interface between the articulating bones of diarthrodial joints. It distributes the applied load and provides almost frictionless contact, enabling smooth movement. Articular cartilage can be considered as a biphasic material having both solid and interstitial fluid phases. The solid phase has a highly organized collagen type II network that enmeshes negatively charged proteoglycans into the tissue matrix. The charges of proteoglycans form a fixed charged density into the tissue. The interstitial fluid phase is mainly water with dissolved charged macromolecules and proteins. Chondrocytes are the only cell type found in articular cartilage. They are responsible for the cartilage homeostasis, which is regulated by the biomechanical and biochemical environment of chondrocytes.

Osteoarthritis is the most common knee joint disease affecting the cartilage structure and function, and the chondrocytes’ ability to maintain cartilage. The first osteoarthritic changes in cartilage are the increase in water content, the loss of proteoglycans and collagen network fibrillation in the superficial cartilage, followed by weakened biomechanical responses. These alterations affect the biomechanical and biochemical environment of chondrocytes. Alterations from the chondrocyte deformation and morphology change the cartilage turnover, hence accelerating the disease progression. However, it is not known how early after anterior cruciate ligament trauma that chondrocytes deformation is altered and how their vicinity changes. Furthermore, it is unclear what the specific changes in the cartilage tissue collagen network, proteoglycan content and biomechanical response during the disease progression are, and in which knee joint locations the possible changes occur. Cartilage structure is typically evaluated with destructive two-dimensional histological methods, leading to the local evaluation of the sample. More detailed local and global three-dimensional evaluation would be beneficial in cartilage and soft tissue research, and also, in the characterization of pathological changes. This thesis aims to clarify the relationship of articular cartilage tissue and chondrocyte deformation in situ in different knee joint locations shortly after surgically induced anterior cruciate ligament trauma in rabbit knee joints. Additionally, the suitability of a contrast agent-free micro-computed tomography imaging to evaluate three-dimensional articular cartilage collagen network orientation is investigated and if alterations in three-dimensional collagen network can be observed early after anterior cruciate ligament transection of rabbit knee joints.

In this thesis, the fixed charged density reflecting the proteoglycan content of the cell vicinity was evaluated 4 weeks after anterior cruciate ligament surgery and correlated with cell shape using histology. Chondrocyte deformation and morphology were investigated under static loading 2 weeks after the anterior cruciate ligament transection of rabbits. Evaluation was conducted with a custom-made confocal laser scanning microscope system combined with a biomechanical testing device with indentation geometry. The setup enables imaging of living superficial cartilage cells in their natural environment in situ under static loading. Image stacks before and after indentation were acquired and cell morphologic responses to indentation were evaluated. After confocal microscopy, biomechanical stress-relaxation tests were conducted to evaluate the cartilage tissue function. Histological analysis was also used to investigate the depth-wise collagen network orientation and fixed charged density with polarized light microscopy and digital densitometry, respectively. In addition, biomechanical and histological evaluation was performed on the latter surgical group, 8 weeks after the anterior cruciate ligament trauma. 2- and 8-week timepoints were used to evaluate the temporal changes caused by the anterior cruciate ligament transection. All evaluations were conducted on six different knee joint locations:​ the lateral and medial femoral condyle, lateral and medial tibial plateau, femoral groove, and patellar cartilage. Moreover, the aforementioned investigations included three groups:​ healthy control, the group which underwent surgery, and the intact contralateral knee joints. Finally, structure tensor analysis was used to estimate three-dimensional cartilage structure orientation and anisotropy of the micro-computed tomography imaged lateral and medial femoral condyle cartilage acquired from healthy rabbits and rabbits 2 weeks after anterior cruciate ligament transection. The acquired depth-wise configurations were verified with corresponding histology performed with polarized light microscopy.

4 weeks post surgery, the fixed charged density was smaller in the group which underwent surgery compared with the control group in the lateral and medial femoral condyle and patellar cartilage. As fixed charged density in the vicinity of the cells was normalized as such further into the tissue, higher values in the pericellular matrix were observed in the lateral and medial femoral condyle, the medial tibial plateau, and patellar cartilage 4 weeks post surgery as compared with the healthy rabbits. Cell height and width were different in the surgical group compared with the control group in various locations and cartilage depths, with a focus on the lateral femoral condyle and the medial tibial plateau cartilage 4 weeks post operation. At the same timepoint, the cell aspect ratio (height divided by width) correlated negatively with the measured, fixed charged density of the pericellular matrix, and moreover, the cell aspect ratio correlated positively with the normalized, fixed charged density of the pericellular matrix.

2 weeks post surgery, the cell morphology and deformation differed from the healthy controls, with the most vulnerable locations being the lateral and medial femoral condyle and patellar cartilage. At the same time, cartilage structure was different in virtually all analysed sites in the group which underwent surgery when compared with the control group. However, the greatest differences occurred in the medial femoral condyle, lateral tibial plateau, and patellar cartilage. In addition, cartilage biomechanical response, represented by Young’s modulus, was smaller in the medial femoral condyle of the group which underwent surgery when compared with the control group, and in the lateral femoral condyle of the group which was operated on compared with the contralateral group 2 weeks post operation. The greatest temporal changes appeared in the lateral and medial femoral condyle, and the patellar cartilage. Interestingly, the patellar and femoral groove deep cartilage witnessed changes in fixed charged density at the 2-week timepoint, but the changes subsided 8 weeks post operation.

Structure tensor analysis was able to estimate the cartilage tissue orientation and anisotropy of the three-dimensional micro-computed tomography images quantitatively and visually, and the depth-wise evaluation of structure orientation had excellent correlation with the histological evaluation. However, the structure tensor analysis witnessed only minor differences between the healthy and experimental rabbit knee joints 2 weeks post surgery.

Anterior cruciate ligament transection affected the analysed knee joint surfaces differently. The superficial cartilage, especially in the lateral and medial femoral condyle cartilage, were the most vulnerable sites for changes after the transection of the anterior cruciate ligament, in which alterations were present on the cell and the tissue level. In addition, these locations witnessed progressive changes in the cartilage tissue structure and composition. On the other hand, in the femoral groove and patellar cartilage, the fixed charged density was reduced early after surgery, but subsided at later on, suggesting cartilage fixed charged density recovery. Less fixed charged density loss was observed in the cell vicinity than in the surrounding tissue, especially in the superficial and middle cartilage at almost all locations 4 weeks post surgery. At least two explanations could account for this:​ (i) fixed charged density is bound more tightly to the cell surrounding the pericellular matrix, or (ii) fixed charged density turnover has been increased due to early osteoarthritis. The differences in the cell morphology between the group which was operated upon and the healthy group were greatest at the lateral femoral condyle. 2 weeks post surgery, chondrocytes of the lateral and medial femoral condyle went through the greatest changes, which might be due to the reduced fixed charged density and lower equilibrium modulus of the cartilage tissue at these locations. Only slight changes occurred in the collagen network investigated with histology, suggesting that proteoglycan content reduction precedes collagen fibrillation, which might predispose the collagen network to change. This is also supported by the three-dimensional evaluation of the cartilage structure, which did not observe significant differences between the operated and healthy knee joints. Nevertheless, these results highlight the difference between the observations made with the two- and three-dimensional evaluations used in this study. Differences might occur due to the differences of these two methods, or the analysed regions and sizes. On the other hand, the reason for the bleaching of proteoglycans might be due to the possible fibrillated network, from which proteoglycan can escape easily.

To conclude, this study suggests that the first target for rehabilitation and biochemical treatment after anterior cruciate ligament trauma should focus on preventing proteoglycan bleaching from the tissue and maintaining the healthy cartilage turnover especially in the femoral condyle cartilage. Moreover, the structure tensor analysis combined with micro-computed tomography showed potential in cartilage structure evaluation in three-dimensions, even though, it did not find significant differences in the early osteoarthritic rabbit cartilage as compared with healthy cartilage.