1Department of Orthopaedic Surgery, Nara Medical University, 840, Shijyo-cho, Kashihara, Nara, 634-8522 and 2Institute for Frontier Medical Sciences, Kyoto University, 53 Shogoin Kawahara-cho, Kyoto, 606-8397, Japan
Correspondence to: K. Hattori, Department of Orthopaedic Surgery, Nara Medical University, 840, Shijyo-cho, Kashihara, Nara, 634-8522, Japan. E-mail: hattori{at}naramed-u.ac.jp
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Abstract |
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Methods. Three experimental rabbit models (spontaneous repair model, large cartilage defect model, treatment model) were examined using our ultrasonic evaluation system and a histological grading scale. From resulting wavelet map, the percentage of maximum magnitude was selected as the quantitative index of the ultrasonic evaluation system.
Results. The percentage maximum magnitude in the spontaneous repair model was 61.1%, that in the large defect model was 29.8% and that in the treatment model was 36.3%. There was modest correlation between the percentage maximum magnitude and the histological grading scale (r = -0.594)
Conclusion. Our findings indicate that ultrasound analysis can predict the microstructure of regenerated cartilage.
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Introduction |
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We developed an intra-articular ultrasonic probe and a new evaluation system for articular cartilage in the human knee, and revealed that this system was able to quantitatively evaluate cartilage degeneration [3, 4]. However, it remains to be shown whether this system can accurately evaluate regenerated cartilage. The purpose of this study was to determine the efficacy of our system for evaluating regenerated cartilage. To this end, we quantitatively evaluated regenerated cartilage using experimental rabbit models.
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Materials and methods |
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Experimental models
For the spontaneous repair model, adolescent rabbits were anaesthetized and an anteromedial arthrotomy was performed in one knee. The patella was dislocated laterally and two defects, 3 mm in diameter and 3 mm in depth, penetrating the subchondral bone plate, were created on the patellar groove of the femur. The defects were washed with saline but left without any further treatment. The wound was then closed in layers. As a control, the procedure was not performed in the other knee.
For the large cartilage defect model, adult rabbits were operated on as described above. The articular cartilage was resected with a chisel to make a 5 mm diameter defect down to the subchondral bone. The defects were washed with saline and the wound was then closed in layers.
For the treatment model, The adult rabbits were operated on as described for the large cartilage defect model. A periosteal graft was obtained from the medial side of the proximal end of the tibia, transplanted to the defect with the cambium layer facing up into the joint, and fixed with 40 nylon sutures. The wound was then closed in layers.
The animals were returned to their cages and allowed to move freely without joint immobilization. Two adolescent rabbits were excluded from this series: one died 3 days after the operation and another showed signs of infection 6 days after the operation. The rabbits were killed at 12 weeks with an overdose of phenobarbital sodium salt. The knee joints were opened and dissected free from all soft tissues, and the tibia was removed. The distal femur was cut proximally to the patellofemoral joint and the cartilage samples were taken.
Ultrasonic evaluation
Ultrasonic examination was made in saline using a transducer and pulsar receiver (Panametrics Japan, Tokyo, Japan) (Fig. 1A). The reflex echogram from the cartilage was transformed into a wavelet map using wavelet transformation. For the mother wavelet function, Gabor's function was selected. The maximum magnitude was selected as a quantitative index on the wavelet map (Fig. 1B).
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Histological evaluation and scoring
After ultrasonic evaluation, cartilage samples were fixed in 10% formalin, decalcified in EDTA and embedded in paraffin. Sagittal sections of 5 µm thickness were prepared from the centre of the defect area, and stained with haematoxylin and eosin, toluidine blue O and safranin O. Each section was graded using the histological scale described by Wakitani et al. [5]. The total score was low for cases of good cartilage regeneration.
Statistical analysis
Differences in ultrasonic data were analysed with the non-parametric MannWhitney U-test. Pearson correlations were calculated to determine the association between the ultrasonic data and the histological data. The significance level was set at P < 0.05.
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Results |
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Histological grading scores and correlation between the ultrasonic data and the histological scale
The histological grading score was low for cases of good cartilage regeneration. The score of the spontaneous repair model was 4.6 points (range 27 points). The score of the large cartilage defect model was 10.5 points (range 912 points). The score of the treatment model was 7.2 points (range 411 points). There was modest correlation between the percentage maximum magnitude for the ultrasonic examination and the score on the histological grading scale for the results of all the measurements (Fig. 2B).
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Discussion |
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Several researchers have reported a relationship between the acoustic properties and matrix degeneration of cartilage using enzyme-induced degenerated cartilage samples in vitro. Joiner et al. [6] revealed that matrix degeneration and proteoglycan loss resulted in a decrease in the speed of sound and an increase in the frequency-dependent attenuation. Töyräs et al. [7] investigated enzyme-induced degenerated cartilage using a scanning ultrasound system that enables the generation of A-, B-, C-, D-, F- and M-mode images. These high-resolution images suggested that ultrasound could be used for the evaluation of spatial changes in the cartilage properties. Therefore, ultrasound could provide valuable information concerning structural and material abnormalities in articular cartilage. Our evaluation method using wavelet transformation is highly reproducible and the ultrasonic probe used in the evaluation is so small that it should be useful for the clinical assessment of articular cartilage condition in arthroscopy.
As new cartilage treatment methods come into use, there will be a requirement for non-invasive quantitative evaluation of the regenerated cartilage. A reliable evaluation method, if established, would help answer the important clinical question of whether repair tissue is mainly hyaline cartilage or mainly fibrocartilage. In our study, all hyaline cartilage (control sites) had a higher maximum magnitude than the repaired tissue, and imperfect regenerated cartilage had a reduced maximum magnitude, even if fibrous tissue and fibrocartilage were seen only in the superficial layer of the repaired tissue in the case of the spontaneous repair model. However, we could not judge the borderline of the two types of regenerated tissue by the percentage of maximum magnitude, and further detailed research on our evaluation method is now required.
In conclusion, ultrasonic evaluation using wavelet maps can predict the histological findings of regenerated cartilage using three experimental models. This evaluation system should contribute greatly to orthopaedics and rheumatology, and assist in the progress of cartilage regeneration.
No benefits in any form have been received or will be received from a commercial party related directly or indirectly to the subject of this article.
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References |
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