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Does Low-intensity pulsed ultrasound treatment repair articular cartilage injury? A rabbit model study
- Shan-Wei Yang†1, 2,
- Chien-Lin Kuo†3,
- Shwu Jen Chang†4,
- Po-Chou Chen4,
- Yen Ting Lin4,
- Ioannis Manousakas4 and
- Shyh Ming Kuo4Email author
© Yang et al.; licensee BioMed Central Ltd. 2014
Received: 4 February 2013
Accepted: 6 February 2014
Published: 10 February 2014
Low-intensity pulsed ultrasound (LIPUS) regiment has been used to treat fractures with non-union and to promote bone union in general. The effect of LIPUS on articular cartilage metabolism has been characterized. Yet, the effect of LIPUS to repair articular cartilage injury remains unclear in vivo.
We designed a study to investigate the effect of LIPUS on articular cartilage repairing in a rabbit severe cartilage injury model. Eighteen rabbits were divided into three groups: Sham-operated group, operated group without-LIPUS-treatment, operated group with-LIPUS-treatment (a daily 20-minute treatment for 3 months). Full-thickness cartilage defects were surgically created on the right side distal femoral condyle without intending to penetrate into the subchondral bone, which mimicked severe chondral injury. MR images for experimental joints, morphology grading scale, and histopathological Mankin score were evaluated.
The preliminary results showed that the operated groups with-LIPUS-treatment and without-LIPUS-treatment had significantly higher Mankin score and morphological grading scale compared with the sham-operated group. However, there was no significant difference between the with-LIPUS-treatment and without-LIPUS-treatment groups. Cartilage defects filled with proliferative tissue were observed in the with-LIPUS-treatment group grossly and under MR images, however which presented less up-take under Alcian blue stain. Furthermore, no new deposition of type II collagen or proliferation of chondrocyte was observed over the cartilage defect after LIPUS treatment.
LIPUS has no significant therapeutic potential in treating severe articular cartilage injury in our animal study.
Chondral injuries are common lesions of the knee joint. Variety of causes results to chondral injury, such as trauma, aging degeneration and infection. Currently, many treatments are available for chondral injury, such as physical therapy, lifestyle modification, pharmacological medications with non-steroid anti-inflammatory drugs (NSAID) or glucosamine, and intra-articular injection of Hyaluronan . The very limited capability for self repair and subsequent degeneration of injuried cartilage and other articular tissues often lead to osteoarthritis, which may eventually lead to the need for total knee arthroplasty . However, the better treatments for chondral injury should not only target the symptoms of the patient but also promote biological repair of the destructed articular cartilage tissue.
Ultrasound is a form of mechanical energy that can be transmitted into the biological tissue as high frequency acoustical pressure waves. It was used as a diagnostic and therapeutic tool. The therapeutic ultrasound achieves its biological result such as muscle pain relief and decrease of joint stiffness by increasing the temperature of the tissue, with intensities ranging from 1 to 3 W/cm2. In contrast, the intensities of diagnostic images are of much lower level (0.5 to 50 mW/cm2) without thermogenic and destructive actions. Low-intensity pulsed ultrasound (LIPUS) is a recommended therapy to treat fractures with non-union and promotes bone union clinically [3–8]. Application of high-intensity continuous ultrasound generates considerable heat in living tissues, whereas LIPUS (< 100 mW/cm2) has much lower intensity with non-thermogenic and non-destructive actions.
Previous studies presented the LIPUS enhances the endochondral ossification in the healing process of fractured bone and promote bone formation, possibly by inducing chondrocyte proliferation [4–8]. It also regulated vascular endothelial growth factor (VEGF) expression in early fracture healing phase and subsequent chondrogenesis . Furthermore, some in vitro studies demonstrated the LIPUS may potentially protect cartilage by inhibiting matrix metalloproteinase-13 (MMP-13) mRNA expression, and stimulate chondrocyte proliferation and matrix production in chondrocytes [10–12]. It also has been reported to promote the mRNA expression of type II collagen, type X collagen, aggrecan, and transforming growth factor (TGF)-ß in chondrocytes . In this context, the effect of LIPUS on articular cartilage metabolism has been characterized.
Most animal studies that analyze the histological and biochemical changes in osteoarthritis are anterior cruciate ligament (ACL) transection model in canines or partial meniscus resection model in rabbits or rats, which result to joint instability and induced cartilage degeneration gradually [14–18]. However, the studies had difficulties in controlling the consistency of the cartilage injuries among the animals.
We designed an experimental rabbit model of severe articular cartilage injury to evaluate the effect of cartilage repair. Surgically created defects of full-thickness cartilage were performed to build consistent severe cartilage injuries. The destruction of the full-thickness cartilage could be controlled, and the effect of cartilage repair could be evaluated quantitatively. Following the model, we investigated the effect of LIPUS on cartilage repair.
Materials of animal model and grouping
Eighteen female Japanese white rabbits with post-natal 12 weeks and body weight between 2.0 to 2.5 kilograms were selected into this trial. Two rabbits per cage were housed under a specific pathogen-free condition (controlled temperature of 24 ± 3°C and humidity of 55 ± 15%) and fed the same standard laboratory food ad libitum. All rabbits were allowed to move freely in the cages. Eighteen rabbits were randomly divided into three groups: 1. Sham-operated control group (sham: n = 6); 2. Operated experimental group without LIPUS treatment (without-LIPUS-treatment: n = 6); 3. Operated experimental group with LIPUS treatment (with-LIPUS-treatment: n = 6). The present investigation conforms to the Guide for the Animal Use Protocol of Institutional Animal Care and Use Committee of I-Shou University (IACUC-ISU99010).
Surgical preparation for cartilage defect
The Exogen low-intensity pulsed ultrasound device (Smith & Nephew Inc, Memphis, TN, USA) was used on the operated right knees of each rabbit in the with-LIPUS-treatment group for a daily 20-minute treatment for 3 months after the surgery. The LIPUS device produced a 200 μs burst of 1.5 MHz acoustic sine waves with a pulse repetition frequency of 1 kHz, and provided a peak intensity of 30 mW/cm2. The knees of each rabbit in the Sham and without-LIPUS-treatment groups were not treated with LIPUS. All animals were sacrificed after the 3-months phase for further assessment.
Assessment of cartilage repair
Magnetic resonance image (MRI)
All MRI experiments were performed on a 1.5 T GE Signa HDxt clinical whole body scanner. MR images of right knee joints were taken for all experimental rabbits under sedation with intra-muscular injection anesthesia with Zoletil 50 (1.0 ml/kg) for the sham, with-LIPUS-treatment and without-LIPUS-treatment groups. Proton density fast spin echo (PDFSE) pulse sequence was used to acquire sagittal view MR images and to assess the articular cartilage. Scanning parameters for sagittal PDFSE pulse sequence were 4000 ms TR (Repetition time), 30 ms TE (Echo time), 2 mm slice thickness, 2 NEX (Number of excitation), 16 ETL (Echo train length), and 8 cm × 8 cm FOV (Field of view). Under this sequence, MR images could provide good contrast between intermediate-signal articular cartilage and high-signal joint fluid, allowing clear visualization of the cartilage defects. Variation of cartilage defect among different phases was investigated to evaluate the effect of LIPUS on articular cartilage repair.
Gross morphological evaluation
Morphological changes to the femoral condylar surfaces in the rabbits were assessed before the collection of cartilage samples during operation after sacrifice. The grading scale reported by Yoshiaka  was used, which divided the conditions of cartilage into grade I: intact surface; grade II: minimal fibrillation; grade III: overt fibrillation; and grade IV: erosion. Two independent blinded observers performed evaluation of the gross morphology.
Histopathological evaluation was performed on the sagittal sections of the operated cartilage in the femoral condyle. Knee joint samples were dissected, fixed in 10% formalin for 24 hr, decalcified by Gooding and Stewart’s fluid (equal volume of 10% formalin and 10% formic acid solution), and embedded in paraffin. H&E stain and Alcian blue stain were employed to observe the injuried cartilage layer and to evaluate the effect of cartilage repair.
The modified Mankin scoring system [20, 21] was also used to evaluate cartilage repair histologically. The severity of the cartilage injury lesions was graded on a scale of 0–13, with a combined score of structure (0 – 6 points), matrix staining (0 – 4 points), and cellular abnormalities (0 – 3 points). In this scoring system lower score indicates more healthy cartilage. The histopathological evaluation was performed by two independent blinded observers.
Statistical analysis of data
The data were presented with mean ± SD. The Fisher-exact Test was used to analyze the results of gross morphology. Histological Mankin scores were analyzed using Wilcoxin signed rank test using SPSS (Statistical Package for Social Science; version 10.0; SPSS, Chicago, IL, USA). Statistical significance was set at p < 0.05.
Many animal models have been developed to investigate the pathogenesis of osteoarthritis, such as spontaneous models in aging animals, enzymatically or chemically induced models, and surgical induced models, as well as ACL transection combined partial meniscus resection [14–17]. In above models, cartilage degenerations as osteoarthritis were induced gradually. Most damage of articular cartilage was relatively mild, similar to early or middle stages of osteoarthritis . Besides, they were difficult to control in the consistency of the cartilage injuries among the animals in the models. In our study, we designed an experimental rabbit model with a uniform articular cartilage defect created surgically to establish the consistency in chondral injuries. Even the location and degree of chondral damage were not characterized histologically. The repair tissues on the defect were evaluated qualitatively. In gross observation of joints after sacrifice, persisted defect over the articular surface without proliferation of healthy tissue was observed in the without-LIPUS-treatment group, whereas the proliferative tissue was observed within the margin of defect in the with-LIPUS-treatment group. Significantly increased number of severe morphological grades in the operated knees without LIPUS treatment indicated the articular defects were severe chondral injuries. Besides Mankin score changes between the sham group and the operated groups also confirmed the severity of the chondral injuries that were created surgically. They indicated that a severe chondral injury has been demonstrated to be feasible to build in this study.
Low-intensity pulsed ultrasound (LIPUS) is a pressure or sound wave with the capability to transfer mechanical energy into biological tissues . It stimulates osteogenesis and fracture healing [3–8]. It produces micromechanical stresses in tissues and elicits an increase in nitric oxide production and activation of hypoxia-inducible factor-1α, thereby inducing the expression of vascular endothelial growth factor levels in osteoblasts [3, 22]. The stimulation mechanism of low-intensity ultrasound was suggested to be derived from electrical potentials (piezoelectricity) and not thermal effects. This may lead to the stimulation of angiogenesis, which plays a role in early bone repair and endochondral ossification. Chondrocyte cultures exposed to ultrasound were studied in vitro and showed increased aggrecan mRNA levels and proteoglycan synthesis, suggesting direct ultrasound stimulation of aggrecan expression [11, 23]. An increased rate of endochondral ossification was demonstrated in mouse metatarsal rudiments using low-intensity ultrasound . Hence, the LIPUS up-regulating the expression of extracellular matrix proteins and collagens in chondrocytes was characterized. Moreover, it was suggested by Ebisawa that LIPUS has a potential to enhance chondrogenic differentiation, but not cell proliferation .
Although many in vitro studies reported the effectiveness of LIPUS on articular cartilage metabolism, only few in vivo studies have been presented. Li X et al.  found that LIPUS promoted cartilage repair through the down-regulation of MMP-13 in rabbit knee osteoarthritis with ACL transection model. Naito K et al.  also presented that LIPUS increased type II collagen synthesis via induction of type II collagen mRNA expression and activation of chondrocytes in a rat osteoarthritis with ACL and meniscus transection model. In our study, though filling tissue was observed over the chondral defect after LIPUS treatment grossly and under MR images, the layer was acellular tissue without up-take of Alcian blue histologically. No proliferation of chondrocyte or new deposition of normal extra-cellular matrix was observed. The filling tissue may be necrosis or degeneration of remanent debris. No statistically significant difference of the morphology scale and the Mankin score between the with-LIPUS-treatment and the without-LIPUS-treatment groups was assessed. No therapeutic effect of LIPUS on repair of chondral injury was observed in our study, which was not compatible with previous studies. The reason might be the degree of chondral injuries in our model was more severe than previous studies, which presented fibrillation or chondromalacia as mild osteoarthritis induced gradually. The chondral injuries in our model which presented a full-thickness cartilage defect deep to exposure of subchondral bone were too severe injury to repair by stimulation of LIPUS. In other words, the effect of cartilage repair by LIPUS might be less effective in repairing such severe chondral injury extensively.
We designed a rabbit model of severe chondral injury to investigate the effect of cartilage repair by LIPUS. The results were evaluated using a morphology grading scale, and histopathology. Preliminarily, no significant therapeutic effect of LIPUS was observed in our animal study. On the other hand, the therapeutic effect of LIPUS on human cartilage may be different from rabbit. Besides, the repairing effect may be different in different dosed of LIPUS, which was not test in our study. It needs further study to clarify the effect in the future.
This study was financially supported by a grant from Gang-Shang Armed Forces Hospital, Taiwan (GSAFH 100–02).
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