tech-nique analogous to dGEMRIC [37, 135]. Similarly as with dGEMRIC, there are several successfulin vitrostudies where the relationship be-tween anionic contrast agent concentration and GAG concentration
in cartilage has been shown [13,37,79,135,163–165,209]. The contrast agent concentration measured with CECT has also been shown to correlate with the cartilage biomechanical properties [9, 13]. How-ever, it has been reported that the diffusion time for contrast agent to reach the equilibrium may well be several hours, which suggests that it cannot be reached in clinical settings [79, 163–165, 199].
According to an earlier study [164], GAG and contrast agent concentrations have correlated already before the equilibrium has been reached. In addition, CECT has enabled the detection of cartilage injuries already after 30–60 minutes of diffusion [87]. These results imply that imaging of diffusion dynamics instead of the situation at equilibrium may enable evaluation of cartilage integrity.
Recently, a new cationic iodinated contrast agent (CA4+) has been introduced to be used in CECT [75] and it has been evaluated in vitro [14, 15, 97] andin vivoin rabbits [170]. Carrying a positive charge of 4 it is effectively attracted by the GAGs and accumulates in higher concentrations in cartilage compared to anionic contrast agents and this makes easier the segmentation of cartilage [14, 15, 75, 170]. CA4+has been shown to provide a better correlation between contrast agent and GAG concentrations than anionic contrast agents [14, 15]. Similar to the situation of CECT with anionic contrast agents, CECT with CA4+has also been reported to correlate with the biomechanical properties of cartilage [97].
The capability of CECT to detect cartilage degeneration has been investigated in vivo in small animal models, and the method has been able to distinguish between healthy and degenerated carti-lage [138, 139, 161]. These animal studies and ex vivo studies on cadaveric knee joints have reported encouraging results showing that CECT provides valuable information on cartilage status even without reaching the diffusion equilibrium [160, 189].
X-ray arthrography has been in clinical use for decades [23, 105, 136, 143]. Contrast agents have been used to visualize cartilage morphology and superficial lesions, and to assess the menisci and ligaments [5, 44, 55, 147, 190]. In contrast to arthrography, a delay between the contrast agent injection and image acquisition is
ap-Contrast enhanced imaging of cartilage
plied in delayed quantitative CT arthrography (dQCTAi.e. in vivo application of CECT). The clinical potential of this technique has been recently investigated for the first time [86]. In that study, the contrast agent concentration in cartilage that was normalized with the concentration in the synovial fluid was higher in an OA knee than in a healthy knee at 30 minutes after intra-articular injection of anionic contrast agent. Since each patient has a different amount of synovial fluid which dilutes the injected contrast agent, it is difficult to decide on the optimal amount and concentration of the contrast agent in order to provide the best possible diagnostic quality.
In addition, the minimum number of acquisitions needed in dQCTA is still unclear. Three acquisitions were proposed by Kokko-nen et al. [86]: one before contrast agent injection (non-contrast image), one after the injection followed by a few minutes of light exercise of the joint to aid distribution of the contrast agent (arthro-graphic image), and one at 30–60 minutes after the injection (delayed image). By subtracting the non-contrast image from the arthro-graphic and delayed images, it is possible to determine the absolute contrast agent concentration in cartilage in these two time points.
However, considering the radiation dose, acquisition of the non-contrast image may not be always acceptable. It is possible that the amount of contrast agent penetrating to cartilage between the acquisitions of the arthrographic and delayed images could serve as an adequate indicator of the state of the cartilage (Figure 3.2) [85].
Figure 3.2:Examples of dQCTA images of human tibiofemoral joint [85]: A) arthrographic image with clearly visible superficial lesions, B) delayed image acquired at 45 min after contrast agent injection, and C) subtraction image which is acquired by subtracting the arthrographic image from the delayed image. The high contrast agent concentration visualized in the figures B and C is indicative of poor cartilage quality.
4 Aims of the present study
In this thesis the diffusion of X-ray contrast agents in cartilage was investigatedin vitroandin vivo. The aim was to assess the effects of cartilage integrity and composition on contrast agent diffusion and to investigate the diagnostic potential of contrast enhanced CT.
The specific aims of this thesis were
1. to determine the tissue-averaged axial diffusion coefficients for four different CT and MRI contrast agents and to compare diffusion through the articular surface with diffusion through deep cartilage.
2. to investigate the ability of microCECT to distinguish sponta-neously healed osteochondral lesions from normal cartilage tissue in equine intercarpal joint.
3. to determine the individual contribution of cross-linking in-duced increases in FCD and steric hindrance to the change in the partition of clinical X-ray contrast agents in cartilage.
4. to compare dGEMRIC and dQCTA with each other, and their association to the arthroscopic findings in human knee joints in vivo.
5 Materials and methods
This thesis consists of four independent studies (I–IV). The materials and methods used in the studies are summarized in Table 5.1. The experimental CECT data in study I was obtained from an earlier study by Silvastet al.[165]. The rest of the data is original.
Table 5.1:Summary of materials and methods used in studies I–IV.
Study Species and tissue Number of samples or patients
Methods
I Bovine cartilage n=6 per group 4 groups
III Bovine cartilage n=6 in group I n=7 in group II
The bovine knees for studies I and III were acquired from a local abattoir (Atria Oyj, Kuopio, Finland). The samples were detached
from upper lateral quadrant of visually intact patellae. In study I, eight adjacent full-thickness (1.3±0.1 mm, mean±SD) cartilage discs (diameter 4 mm) were detached from patellae and randomly divided into four groups according to the contrast agent to be used in CECT imaging [165].
In study II, osteochondral lesions (diameter 6 mm) were sur-gically created in the intercarpal joints of horses under general anesthesia. After 12 months of spontaneous healing, the horses were sacrificed, and osteochondral plugs (diameter 14 mm) were har-vested. The plugs included the repair cartilage and intact adjacent tissue. The procedures were approved by the Utrecht University Animal Experiments Committee.
In study III, cylindrical plugs (diameter 24.5 mm) were drilled from the upper lateral quadrant of bovine patellae and cut into four pieces. Osteochondral samples (diameter 6 mm) were punched from each piece, and divided into two groups according to the contrast agent to be used in CECT imaging. One of the paired samples served as a reference while the other was treated with threose (Sigma Aldrich Co., St. Louis, MO, USA) to induce collagen cross-linking, which resembles the process of natural ageing. The samples were incubated in humidified 5%CO2/95% air atmosphere at 37◦C for seven days.
Study IV involved contrast enhanced CT and MR imaging of human patients (n=11) referred to an arthroscopic surgery of the knee. One patient declined to undergo arthroscopy but completed all imaging studies. One patient was excluded from the analysis due to irregular distribution of contrast agent in the joint. An informed consent was obtained from all patients. The study was approved by the Ethical Committee of the Northern Ostrobothnia Hospital District, Oulu, Finland (No. 33/2010).
The rather low number of samples in the studies was accepted for practical reasons. However, it was ensured that in the in vitro studies the control and experimental samples were paired, and the test groups included independent sample pairs.
Materials and methods