Abstract
Background:
Appropriate animal models of osteoarthritis (OA) are essential to develop new treatment modalities for OA. A combination of surgical and chemical induction could be appropriate for OA models.
Methods:
Rabbit knee OA models developed by surgical induction (anterior cruciate ligament transection [ACLT]), chemical induction (monosodium iodoacetate [MIA] injection), and a combination of both were compared to assess compositional and structural destruction of the knee joint. Twenty-one New Zealand white rabbits were randomly divided into 3 groups to induce OA (group 1: ACLT, n = 3; group 2: MIA [3, 6, 9 mg] injection, n = 9; group 3: ACLT + MIA [3, 6, 9 mg] injection, n = 9).
Results:
In all groups, the Modified Mankin score was significantly higher in the osteoarthritis-induced knee than in the control. Modified Mankin scores were compared by category. The ACLT group was observed to score high in cartilage structure. In the MIA group, chondrocytes and matrix staining showed higher scores, and the ACLT+MIA group scored higher in all categories for cartilage structure, chondrocytes, matrix staining, and tidemark integrity. The ACLT + 3 mg MIA showed definite OA characteristics such as cartilage surface destruction and degeneration of cartilage layers, and the ACLT + 6 mg MIA and ACLT + 9 mg MIA showed more prominent OA characteristics such as cartilage surface destruction, matrix disorganization, and osteophyte formation.
Conclusion:
The combination of MIA injection and ACLT could be an appropriate method for OA induction in rabbit models.
Keywords: Cartilage, Knee joint, Osteoarthritis, Animal model
Introduction
Osteoarthritis (OA) is the most common joint disease and is characterized by a variety of articular cartilage abnormalities ranging from simple articular cartilage degeneration to extensive damage to the articular cartilage. In severe cases, it results in permanent joint deformation [1]. As life expectancy increases in an aging society, the incidence of OA is rapidly increasing, causing millions of people to suffer. To treat OA, medication or injection of hyaluronic acid or steroid is administered in the early stage, and surgical treatments such as cartilage regeneration are required in the advanced stage [2–4].
To develop new treatment modalities for OA, proper animal models of OA are essential. In animal models, the preclinical process of defining the etiology of the disease and confirming the effectiveness of the treatment plays an important role in the clinical application of the treatment [5]. Each animal model has advantages and limitations in the study of therapeutic agent efficacy [6].
As for OA animal models, various animal species, such as mice, rats, rabbits, and guinea pigs, are used for research and can be divided into spontaneous and induced models [7]. The spontaneous model is composed of naturally occurring genetic models. These models are slow-paced and time-consuming but can be used to understand the pathophysiology of human degenerative OA [8]. The induced models are either surgically or chemically induced. The surgically induced model produces OA by causing instability during surgery, such as anterior cruciate ligament transection (ACLT) and meniscectomy [9, 10]. Chemically-induced OA is induced by injecting chemical drugs, such as monosodium iodoacetate (MIA), papain, and collagenase to alter homeostasis and destroy joint structures [11–13]. The OA induction method and animal species are selected depending on the purpose of the study and in consideration of various conditions, such as time and cost [14, 15].
An animal model that shows cartilage defects or osteoarthritic changes 2 to 3 months after induction may be appropriate for research purposes.
Rabbits, rats, and mice are the most popular models in the laboratory. Rat models that are induced surgically or chemically are well known OA models. However, the joints in rats are too small and become severely damaged after MIA injection. Therefore, they may not be appropriate as OA models. On the other hand, rabbit knee joint size is more appropriate for macroscopic evaluation. However, surgical OA induction of the rabbit knee takes a long time and the degree of joint destruction is variable. Also, MIA induced OA in the rabbit knee is not well known.
In this study, the ACLT and MIA models, which are representative osteoarthritis models, were compared and combined to investigate the subsequent changes in rabbit knee cartilage. The possibility of proper OA model development was evaluated through the combination of surgical induction to alter biomechanical stability and chemical induction to induce degradation of the cartilage matrix.
Materials and methods
Animals
Twenty-one New Zealand white rabbits weighing 3.5 kg (3–4 kg) were studied. All rabbits were maintained under the same environmental conditions. The rabbits were randomly divided into 3 groups to induce OA (group 1: ACLT, n = 3; group 2: MIA [3, 6, 9 mg] injection, n = 9; group 3: ACLT + MIA [3, 6, 9 mg] injection, n = 9). In each group, the contralateral knee, without any treatment, was used as a control (Fig. 1). All rabbits were anesthetized by intramuscular injection of ketamine (0.7 ml/kg) and xylazine (0.2 ml/kg). This study was approved by the Institutional Animal Care and Use Committee (IACUC) of our institute (UJA2019-10A).
Fig. 1.
Design of an experimental animal model for the induction of osteoarthritis of the rabbit knee. The ACLT group was observed for 12 weeks after ACLT surgery on the knee. The MIA group was observed 12 weeks after 3, 6, and 9 mg of MIA injection into the knee. In the ACLT + MIA group, ACLT surgery was performed on the rabbit's knee, followed by 3, 6, and 9 mg of MIA injection 2 weeks later, and observed for 4 weeks (total experimental period of 6 weeks)
Induction of osteoarthritis
ACLT surgery
The rabbits were placed in a supine position. A longitudinal midline skin incision was made from the superior pole of the patella to the level of the right tibial tubercle. The subcutaneous tissue was divided along the line of the skin incision. A medial skin flap was developed to expose the medial border of the patella and patellar tendon. A medial para-patellar capsular incision was then made, and the patella was dislocated laterally to expose the ACL. The ACL was transected using a No. 11 surgical blade (Paragon, Sheffield, UK). The wound was then closed layer-by-layer. The rabbits were sacrificed 12 weeks after the surgery (Fig. 1).
MIA injection
MIA (I2512, Sigma-Aldrich Inc, St. Louis, MO, USA) was dissolved in sterile physiological saline to prepare concentrations of 3, 6, and 9 mg. Intra-articular injection of 300 µl of MIA at each concentration was administered on the right knee of the rabbits. Rabbits were sacrificed 12 weeks after the MIA injection (Fig. 1).
MIA injection after ACLT surgery
The right ACL was resected as described in Sect. 2.2.1. Two weeks after surgery, MIA at each concentration (3, 6, and 9 mg) was injected into the right knee. Rabbits were sacrificed 4 weeks after the MIA injection (Fig. 1).
Histology and scoring
Knee joints, along with the femur and tibia, were extracted from each group and fixed in 10% formalin (Sigma-Aldrich Inc., St. Louis, MO, USA) for 14 days. The tissues were decalcified in formic nitric acid (Duksan, Seoul, Korea) solution for 21 days, dipped and neutralized in 5% sodium sulfate solution (Sigma-Aldrich Inc., Poole, UK), and rinsed with water. The tissues were dehydrated with ethanol, cleaned with xylene, and embedded in paraffin. Specimens were sectioned to a thickness of 4 μm using a microtome (Leica, Wetzlar, Germany), and hematoxylin–eosin staining was applied to observe tissue structures. Alcian blue staining was applied to observe glycosaminoglycans and proteoglycans. For immunohistochemical analysis, collagen type II staining was performed using an EnVision Detection Kit (K5007, Dako, Glostrup, Denmark) and antibodies (ab185430, 1:200, Abcam, Cambridge, MA, USA), and histological sections were visualized using an Olympus microscope BX53 (Olympus, Tokyo, Japan). After the slides were prepared, morphological analysis of the severity of chondral lesions was performed using the modified Mankin score (Table 1) [16].
Table 1.
Modified Mankin score
| Category | Score | |
|---|---|---|
| Cartilage structure | Normal | 0 |
| Surface irregularities | 1 | |
| Pannus and surface | 2 | |
| Clefts to transitional zone |
4 3 |
|
|
Clefts to calcified zone Clefts to radial zone |
5 | |
| Complete disorganization | 6 | |
| Cartilage cells | Normal | 0 |
| Pyknosos, lipid degeneration | 1 | |
| Clusters | 2 | |
| Hypocellularity | 3 | |
| Matrix staining (Alcian blue) | Normal | 0 |
| Slight reduction | 1 | |
| Moderate reduction | 2 | |
| Severe reduction | 3 | |
| No staining | 4 | |
| Tidemark integrity | Intact | 0 |
| Destroyed | 1 |
Statistical analysis
Statistical analyses were performed using GraphPad Prism 5 (GraphPad Software Inc., San Diego, CA, USA). All variables were summarized using standard descriptive statistics such as mean, standard deviation, median, and range.
The Mann-Whitney test was used to analyze OA-induced rabbit knees versus controls in each group. Statistical significance was defined as *** p < 0.001.
Results
ACLT surgery
Macroscopic observation of the OA model produced by ACLT revealed smooth edges and intact cartilage in the femur and tibia of the control (Fig. 2A, upper, 1st and lower, 3th). The cartilage surface was not smooth in the ACLT. Erosion was observed on the surface of the femoral cartilage (Fig. 2B, upper, 1st). Osteophyte development was observed on the sides of the femur and the medial edge of the tibia (Fig. 2 upper, 1st and lower, 3th).
Fig. 2.
Macroscopic observation and histopathological changes of the femoral and tibial cartilage 12 weeks after ACLT. A Control. Articular cartilage structures are normal with well-arranged chondrocytes according to layers (upper, 2nd and lower, 2nd), good staining of Alcian blue (upper, 4th and lower, 4th) and collagen type II (upper, 5th and lower, 5th). B ACLT. Osteophytes are observed (black arrow; upper, 1st and lower, 1st). The density of chondrocytes and staining are lower than those of the control, and fibrillation (arrowhead; upper, 2nd) is observed on the surface of the femoral cartilage. A thin delaminated layer is observed on the articular cartilage of the tibia (lower, 3rd). Reduced Alcian blue staining is observed in the femoral condyle and the surface of the tibia (black star; upper, 4th and lower, 4th). Brown color staining in collagen type II staining is reduced on the cartilage surface (black star; upper, 5th and lower, 5th). Magnification: ×100; Scale bar, 100 μm. Magnification: ×400; Scale bar, 20 μm (green box).
In the histopathological analysis, hematoxylin–eosin staining of the control showed normal cells and cartilage (Fig. 2A, upper, 2nd and lower, 2nd). Alcian blue staining for proteoglycans in the cartilage matrix was shown in blue throughout the cartilage (Fig. 2A, upper, 4th and lower, 4th). The cartilage matrix, assessed by collagen type II immunostaining, was brown throughout the cartilage (Fig. 2A, upper, 5th and lower, 5th).
Hematoxylin–eosin staining of the ACLT showed irregular fibrillation, and the femoral cartilage surface was not smooth (Fig. 2B, upper, 2nd). Slight fibrillation was observed on the tibial cartilage surface (Fig. 2B, lower, 3rd). Compared with the control, the density of chondrocytes was lower and hypertrophy was observed (Fig. 2B, upper, 3rd and lower, 3rd).
Staining of the cartilage matrix with Alcian blue is shown in blue in the femur and tibia. However, its expression was slightly decreased on the cartilage surface (Fig. 2B, upper, 4th and lower, 4th). Cartilage matrix staining with collagen type II was weakened at the cartilage surface of the femur and tibia (Fig. 2B, upper, 5th and lower, 5th). Weak staining with Alcian blue and collagen type II indicated loss of the cartilage matrix.
MIA injection
The cartilage of the control group was normal, and the femur and tibia had smooth and intact cartilage (Fig. 3A, upper, 1st and lower, 1st). Hematoxylin–eosin staining revealed normal cells and cartilage in the control (Fig. 3A, upper, 2nd and lower, 2nd). Alcian blue and collagen type 2 staining were observed throughout the cartilage (Fig. 3, upper, 4th and lower, 4th; Fig. 3A, upper, 5th and lower, 5th).
Fig. 3.
Histopathological changes in the femur and tibial cartilage after 12 weeks of induction using each concentration of MIA. A Control. Chondrocytes and structures are normal and show normal Alcian blue and collagen type II staining. 12 weeks after B 3 mg C 6 mg and D 9 mg MIA injection. The cartilage surface of the MIA group was slightly irregular. A defect is observed in the deep zone of the cartilage (arrowhead; D, upper, 3rd). Overall expression of Alcian blue staining reduced. Collagen type II staining was not expressed throughout the cartilage. Magnification: ×100; Scale bar, 100 μm. Magnification: ×400; Scale bar, 20 μm (green box).
In MIA injection, no significant erosion or defects in cartilage were observed, and no bone tissue formation was observed (Fig. 3B, upper, 1st and lower, 1st; 3D, upper, 1st and lower, 1st). The cartilage surface was slightly irregular; however, no serious damage was observed. Chondrocytes were less dense than those in the controls and were irregularly arranged.
For the 3 mg MIA, hematoxylin–eosin staining showed that the tibial matrix cracked (Fig. 3B, lower, 3rd; black star). Alcian blue staining for cartilage matrix showed significantly decreased expression in the whole cartilage (Fig. 3B, upper, 4th and lower, 4th). Collagen type II staining was not observed throughout the cartilage (Fig. 3B, upper, 5th and lower, 5th).
Next, for the 6 mg MIA, the chondrocytes of the femur showed a disordered arrangement (Fig. 3C, upper, 3rd), in the tibia, chondrocytes without lacuna were present (Fig. 3C, lower, 3rd; black arrow), and the matrix was split (Fig. 3C, lower, 3rd; black star). Alcian blue staining of the matrix showed a slight decrease in expression (Fig. 3C, upper, 4th and lower, 4th). Collagen type II staining was not observed throughout the cartilage (Fig. 3C, upper, 5th and lower, 5th).
In the femur and tibia of the 9 mg MIA, empty lacuna (Fig. 3D, upper, 3rd and lower, 3rd) and large defects in the deep zone were observed (Fig. 3, upper, 3rd and lower, 3rd; arrowhead). Alcian blue staining for cartilage matrix was weakly expressed (Fig. 3, upper, 4th and lower, 4th). Collagen type II staining was not observed in cartilage (Fig. 3, upper, 5th and lower, 5th).
MIA injection after ACLT surgery
The combination model in which MIA was injected into the knee of rabbits who underwent ACLT had various cartilage defects depending on the MIA dose.
On visual observation, the control showed smooth and intact cartilage at the edges of the femur and tibia (Fig. 4, upper, 1st and lower, 1st). Histopathological analysis revealed normal cells and cartilage (Fig. 4A, upper, 2nd and lower, 2nd). Alcian blue and collagen type II staining were expressed throughout the cartilage (Fig. 4, upper, 4th and lower, 4th; 4A, upper, 5th and lower, 5th).
Fig. 4.
Macroscopic observation and histopathological changes in the femur and tibial cartilage according to the MIA concentration in OA induced by ACLT + MIA. A Control. Chondrocytes and surfaces are normal and show normal Alcian blue and collagen type II staining. B ACLT + 3mg MIA. Osteophyte is observed in the macroscopic image (black arrow; lower, 1st). Chondrocyte clusters (black arrow; upper, 3rd) and cartilage surface cracks (black arrow; upper, 2nd) are observed. Expansion of the calcified cartilage and tidemark duplication (dotted line; upper, 3rd). Alcian blue staining is weakly expressed in the femur and tibia (upper, 4th and lower, 4th). Collagen type II is expressed in the middle region (upper, 5th) of the femur. C ACLT + 6 mg MIA. Osteophytes (black arrow; upper, 1st and lower, 1st) and defects on the cartilage surface (arrowhead; upper, 1st and lower, 1st) are observed. The femoral cartilage was lost because of severe detachment. Cracks and fibrosis are observed in the tibia (arrowhead; lower, 2nd). Alcian blue staining is weakly expressed in the femur and tibia (upper, 4th and lower, 4th). Collagen type II staining is not observed in the femur or tibia (upper, 5th and lower, 5th). D ACLT + 9 mg MIA. Osteophytes (black arrow; upper, 1st and lower, 1st) and erosion (arrowhead; upper, 1st and lower, 1st) are observed. Damage to cartilage surface (arrowhead; upper, 2nd). Vascular invasion (black arrow; lower, 3rd). Alcian blue staining is weakly expressed in the femur and tibia (upper, 4th and lower, 4th). Collagen type II staining is not observed in the femur or tibia (upper, 5th and lower, 5th). Magnification: ×100; scale bar: 100 μm. Magnification: ×400; scale bar: 20 μm (green box).
In the ACLT + 3 mg MIA, bone tissue formed on the medial edge of the tibia (Fig. 4B, lower, 1st; black arrow). In the ACLT + 6 mg MIA, the cartilage surfaces of the femur and tibia were not smooth and erosion was observed (Fig. 4C, upper, 1st and lower, 1st; arrowhead). Osteophytes formed on the medial and lateral sides of the femur, and severe osteophytes were observed on the medial edge of the tibia (Fig. 4C, upper, 1st and lower, 1st; black arrow). This group had malformed cartilage with severe bone tissue development. In the ACLT + 9 mg MIA, severe erosion occurred on both the femoral and lateral tibial cartilage surfaces (Fig. 4D, upper, 1st and lower, 1st; arrowhead). Osteophytes were observed all over the rim of the outer femur and tibia (Fig. 4D, upper, 1st and lower, 1st; black arrow).
In the ACLT + 3 mg MIA, hematoxylin–eosin staining revealed cracks on the cartilage surface of the femur (Fig. 4B, upper, 2nd; black arrow) and the chondrocytes cluster (Fig. 4B, upper, 3rd; black arrow). Expansion of calcified cartilage and tidemark duplication were observed (Fig. 4B, upper, 3rd; dotted line). Tibial chondrocytes were disordered and had lost their lacuna (Fig. 4B, lower, 3rd; black arrow). In cartilage matrix staining, the expression of Alcian blue decreased in the entire cartilage of the femur and tibia (Fig. 4B, upper, 4th and lower, 4th). The expression on the cartilage surface of the femur was further reduced (Fig. 4B, upper, 4th; black star). Collagen type II was only expressed in the middle region of the femur (Fig. 4B, upper, 5th; black star).
In the ACLT + 6 mg MIA, hematoxylin–eosin staining was lost in most of the femoral cartilage. In the tibia, the stromal fissure extended to the deep part of the cartilage (Fig. 4C, lower, 2nd; black arrow) and severe fibrillation was observed (Fig. 4C, lower, 2nd; arrowhead). Acian blue and collagen type II were not expressed in the tibia (Fig. 4C, upper, 4th and lower, 4th; 4C, upper, 5th and lower, 5th).
In the ACLT + 9 mg MIA, no chondrocytes were observed in the femur. Complex fibrillation and typical fibrillation occurred in the cartilage (Fig. 4D, upper, 2nd). The tibial cartilage was deeply eroded (Fig. 4D, lower, 2nd) and vascular invasion was observed in the calcified cartilage and tidemark (Fig. 4D, lower, 3rd; black arrow). Alcian blue staining of cartilage matrix was not observed in the femur or tibia. (Fig. 4D, upper, 4th and lower, 4th). Staining of the cartilage matrix by collagen type II showed very weak expression in the femur and tibia (Fig. 4D, upper, 5th and lower, 5th).
Modified Mankin score
In all groups, the Modified Mankin score was significantly higher in the osteoarthritis-induced knee than in the control (Fig. 5). In femur total score, ACLT (3.78 ± 0.19), 3 mg MIA ( 3.67 ± 0.0) and 6 mg MIA (3.67 ± 0.0) showed mild OA. 9mg MIA (6.67 ± 0.33) and ACLT + 3 mg MIA (7.89 ± 0.84) showed severe OA. ACLT + 6 mg MIA (13.78 ± 0.19) and ACLT + 9 mg MIA (13.11 ± 0.38) had the highest scores and very severe osteoarthritis was observed (Figure 5A). In tibia total score, ACLT (3.33 ± 0.33), 3 mg MIA (4.44 ± 0.84), 6 mg MIA (4.33 ± 0.33), 9 mg MIA (4.67 ± 0.33) and ACLT + 3 mg MIA (3.33 ± 0.0) showed mild OA. ACLT + 6 mg MIA (11.67 ± 0.33) and ACLT + 9 mg MIA (11.56 ± 0.38) scored high and severe osteoarthritis was observed (Figure 5B). Modified Mankin scores were compared by category. The ACLT group was observed to score high in cartilage structure. In the MIA group, chondrocytes and matrix staining showed higher scores, and the ACLT+MIA group scored higher in all categories for cartilage structure, chondrocytes, matrix staining, and tidemark integrity (Table 2).
Table 2.
Scores for each category in the modified Mankin score for the femur and tibia
| Femur | Tibia | |||||||||||||||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| Cartilage structure | Cartilage cells | Matrix staining (Alcian blue) |
Tidemark integrity | Cartilage structure | Cartilage cells | Matrix staining (Alcian blue) |
Tidemark integrity | |||||||||||||||||
| ACLT | ||||||||||||||||||||||||
| Control | 0.11 | ± | 0.19 | 0.00 | ± | 0.00 | 0.00 | ± | 0.00 | 0.00 | ± | 0.00 | 0.22 | ± | 0.38 | 0.00 | ± | 0.00 | 0.00 | ± | 0.00 | 0.00 | ± | 0.00 |
| ACLT | 1.56 | ± | 0.19 | 1.00 | ± | 0.00 | 1.22 | ± | 0.19 | 0.00 | ± | 0.00 | 1.33 | ± | 0.00 | 1.11 | ± | 0.19 | 0.89 | ± | 0.19 | 0.00 | ± | 0.00 |
| MIA | ||||||||||||||||||||||||
| Control | 0.11 | ± | 0.19 | 0.00 | ± | 0.00 | 0.00 | ± | 0.00 | 0.00 | ± | 0.00 | 0.11 | ± | 0.19 | 0.00 | ± | 0.00 | 0.00 | ± | 0.00 | 0.00 | ± | 0.00 |
| 3 mg | 0.44 | ± | 0.19 | 1.00 | ± | 0.00 | 2.22 | ± | 0.19 | 0.00 | ± | 0.00 | 0.33 | ± | 0.33 | 2.22 | ± | 0.19 | 1.89 | ± | 0.38 | 0.00 | ± | 0.00 |
| 6 mg | 0.44 | ± | 0.19 | 1.00 | ± | 0.00 | 2.22 | ± | 0.19 | 0.00 | ± | 0.00 | 0.56 | ± | 0.19 | 2.11 | ± | 0.19 | 1.67 | ± | 0.00 | 0.00 | ± | 0.00 |
| 9 mg | 0.67 | ± | 0.00 | 3.00 | ± | 0.00 | 3.00 | ± | 0.33 | 0.00 | ± | 0.00 | 0.56 | ± | 0.19 | 2.22 | ± | 0.38 | 1.89 | ± | 0.38 | 0.00 | ± | 0.00 |
| ACLT + MIA | ||||||||||||||||||||||||
| Control | 0.11 | ± | 0.19 | 0.00 | ± | 0.00 | 0.00 | ± | 0.00 | 0.00 | ± | 0.00 | 0.11 | ± | 0.19 | 0.00 | ± | 0.00 | 0.00 | ± | 0.00 | 0.00 | ± | 0.00 |
| 3 mg | 3.00 | ± | 0.33 | 2.22 | ± | 0.19 | 1.67 | ± | 0.33 | 1.00 | ± | 0.00 | 0.44 | ± | 0.19 | 1.00 | ± | 0.00 | 1.89 | ± | 0.19 | 0.00 | ± | 0.00 |
| 6 mg | 5.78 | ± | 0.19 | 3.00 | ± | 0.00 | 4.00 | ± | 0.00 | 1.00 | ± | 0.00 | 5.00 | ± | 0.00 | 2.78 | ± | 0.19 | 2.89 | ± | 0.19 | 1.00 | ± | 0.00 |
| 9 mg | 5.11 | ± | 0.38 | 3.00 | ± | 0.00 | 4.00 | ± | 0.00 | 1.00 | ± | 0.00 | 5.00 | ± | 0.00 | 2.78 | ± | 0.19 | 2.78 | ± | 0.19 | 1.00 | ± | 0.00 |
Fig. 5.
Modified Mankin score of the femur and tibia in ACLT, MIA, and ACLT + MIA groups. Osteoarthritis severity was assessed on a scale of 0-14 total scores by summing each category score through the modified Mankin scoring system. A Modified Mankin score of the femur. B Modified Mankin score of the tibia. ***p < 0.001 vs control (Mann-Whitney test)
Discussion
Making an appropriate OA animal model is necessary for research on OA pathophysiology and treatment modalities [5]. Popular OA models include ACLT and meniscectomy, which cause mechanical instability [17–20]. However, it is difficult to evaluate OA due to the inconsistency in the degree of OA and long induction time, such as several months, in a rabbit model [21, 22]. In preliminary experiments, 4 and 8 weeks after ACLT were studied but were deemed insufficient, as the characteristics of OA in the knee only become prominent after at least 12 weeks. Furthermore, the changes should be observed microscopically.
Another method of OA induction is MIA intraarticular injection [23, 24]. In the case of rats and mice, the destruction was severe for several days after injection [25]. However, there are few reports on MIA injection into rabbit knees for OA induction [26, 27]. In the preliminary experiment, the rabbit knee was injected with 3 to 100 mg of MIA with diverse densities. Even after three months, the articular surface was not destroyed, but the internal structure and cells were disorganized and degenerated. Only side effects were observed, and there were no definite advantages of a higher MIA density for OA induction. Higher than 30 mg of MIA injection showed some toxic effects on rabbits, such as ulceration of surgical wounds, brown or gray discoloration of the cartilage surface, and sudden death. Therefore, it is necessary to determine the minimal density of MIA required for effective OA induction. As 3 mg of MIA is commonly used in rat models of OA, 6 mg or 9 mg may not be harmful to rabbits, considering the weight and size of the animal.
OA typically presents with surface destruction and internal disorganization in the articular cartilage. Therefore, a combination of MIA intra-articular injection and ACLT could be suggested for effective OA induction, such as surface destruction and cartilage disorganization. This combination method was attempted and good progression of OA after ACLT with MIA injection over four weeks was observed. We determined that if the appropriate result in two or three months could be obtained with 3, 6, and 9 mg of MIA + ACLT, this could be a proper model for OA. At 12 weeks, structural destruction of the articular cartilage surface, reduced staining in the middle of the cartilage layer, and no staining of Alcian blue and collagen type II on the surface of the cartilage layer were observed in the ACLT group. These findings are consistent with those of other studies using surgically induced OA models [28, 29]. For the MIA group at 12 weeks, there was a smooth articular surface and reduced staining for Alcian blue and collagen type II. This indicates reduced proteoglycan content in the cartilage. MIA activates matrix metalloproteinase and suppresses proteoglycan synthesis in the cartilage [11, 30], resulting in cartilage necrosis and arthritis. Among the MIA concentrations, 9 mg showed some structural destruction, but this was in the middle layer of the cartilage and not on the surface. This was completely different from the rat model, as destruction after MIA injection was observed for several days. The ACLT and MIA models showed OA characteristics but were not perfect. The ACLT model showed structural destruction, such as surface destruction of the articular cartilage, and the MIA model showed good matrix degeneration.
Recently, a new aspect of OA has been suggested by The Osteoarthritis Research Society International [31], which is cell stress and extracellular matrix degradation induced by micro-and macro-injury that activates maladaptive repair responses, including pro-inflammatory pathways of innate immunity. This is abnormal metabolism of the cartilage and is followed by cartilage degradation. Therefore, chemical induction, such as MIA injection, could be suggested as the initial event of OA progression in the OA model. However, MIA injection alone was not appropriate for cartilage structural destruction in rabbits in our preliminary study. Therefore, the combination of MIA injection and ACLT could be suggested as a proper model of OA with respect to molecular and anatomical derangement in the cartilage of rabbits. In the ACLT + MIA group, MIA was injected two weeks after ACLT. In a preliminary study, an OA model injected with MIA immediately after ACLT surgery caused problems such as ulcers and delayed wound healing. Therefore, an interval of two weeks was used to ensure surgical wound healing. The ACLT + 3 mg MIA showed definite OA characteristics such as cartilage surface destruction and degeneration of cartilage layers, and the ACLT + 6 mg MIA and ACLT + 9 mg MIA showed more prominent OA characteristics such as cartilage surface destruction, matrix disorganization, and osteophyte formation.
This study had some limitations. The number of animals was too small to draw an appropriate conclusion. Additionally, only the cartilage changes of the femur and tibia were investigated. More research is needed to investigate changes in other sites, such as the meniscus and synovium. However, this model has several advantages over the other mechanical and chemical OA induction models. First, evident OA characteristics can be obtained. The ACLT model showed some cartilage surface destruction, while the other points were not prominent. The MIA injection model showed some cartilage matrix degeneration, but surface destruction was minimal. The combination of MIA and ACLT resulted in total structural destruction, including matrix degradation. Second, the induction time was shorter in the ACLT + MIA group. Other models require at least 12 weeks for OA induction, but 6 weeks was sufficient for OA induction in the combination model. This could encourage researchers to conduct active research on OA, such as new treatment modalities for OA after OA induction. Therefore, the combination of MIA injection and ACLT could be suggested as an appropriate method for OA induction in a rabbit model, with respect to OA induction time and joint destruction characteristics.
Acknowledgements
This study received no funding.
Declarations
Conflict of interest
The authors declare that there is no conflict of interest.
Ethical statement
The animal studies were performed after receiving approval of the Institutional Animal Care and Use Committee (IACUC) (Approval No. UJA2019-10A).
Footnotes
Publisher's Note
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
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