- Research article
- Open Access
Differences in joint morphology between the knee and ankle affect the repair of osteochondral defects in a rabbit model
© The Author(s). 2016
Received: 8 July 2016
Accepted: 20 September 2016
Published: 4 October 2016
Although differences in the results of the bone marrow stimulation technique between the knee and ankle have been reported, a detailed mechanism for those differences has not been clarified. The purpose of this study was to examine whether morphological differences between the knee and ankle joint affect the results of drilling as treatment for osteochondral defects in a rabbit model.
Osteochondral defects were created at the knee and ankle joint in the rabbit. In the knee, osteochondral defects were created at the medial femoral condyle (MFC) and patellar groove (PG). At the ankle, defects were created in the talus at either a covered or uncovered area by the tibial plafond. After creating the osteochondral defect, drilling was performed. At 4, 8, and 12 weeks after surgery, repair of the osteochondral defects were evaluated histologically. The proliferation of rabbit chondrocytes and proteoglycan release of cartilage tissue in response to IL-1β were analyzed in vitro in both joints.
At 8 weeks after surgery, hyaline cartilage repair was observed in defects at the covered area of the talus and the MFC. At 12 weeks, hyaline cartilage with a normal thickness was observed for the defect at the covered area of the talus, but not for the defect at the MFC. At 12 weeks, subchondral bone formation progressed and a normal contour of subchondral bone was observed on CT in the defect at the covered area of the talus. No significant differences in chondrocyte proliferation rate and proteoglycan release were detected between the knee and ankle in vitro.
Our results demonstrate that the covered areas of the talus show early and sufficient osteochondral repair compared to that of the knee and the uncovered areas of the talus. These results suggest that the congruent joint shows better subchondral repair prior to cartilage repair compared to that of the incongruent joint.
Articular cartilage has a limited ability of repair, and untreated lesions of articular cartilage may progress to osteoarthritis (OA) [1, 2]. Many strategies for the treatment of articular cartilage have been developed including bone marrow stimulation techniques [3, 4], osteochondral grafts , and tissue engineering approaches . Articular cartilage consists of sparse chondrocytes and dense extracellular matrix of mainly type 2 collagen and proteoglycans. Articular cartilage lacks blood vessels and nervous innervation, which makes the repair of articular cartilage difficult . The constitution of the cartilage differs between joints in individuals, which may result in differences in the results of treatment and the progression of OA. Several reports have demonstrated constitutional and biochemical differences of articular cartilage between the knee and ankle [7, 8].
For the repair of articular cartilage, bone marrow stimulation techniques have been performed widely. Bone marrow stimulation creates blood clots in the area of the defect through methods such as microfracture or drilling. Results after drilling of the talus have reported good outcome in 80–96 % of cases [9–11]. On the other hand, results after drilling of the medial femoral condyle (MFC) have reported good outcome in 69–80 % of cases [12–14]. As a whole, the results of the talus are better than those of the MFC.
Factors influencing differences in results after bone marrow stimulation between the knee and ankle have not been explored. As suggested in previous reports, biochemical and constitutional differences between the knee and ankle cartilages may affect the capacity of the cartilage to repair after bone marrow stimulation . However, differences in the joint morphology between the knee and ankle should be examined. As for the knee joint, the femoral condyle and the tibia plateau do not have good congruency, while the ankle joint has good congruency as a tenon and mortise structure. This difference in joint morphology may affect the capacity of cartilage repair. OA is induced by cartilage injury, and the incidence of primary OA is different between the knee and ankle. Symptomatic OA with radiographical signs occurs about eight to nine times more frequently in the knee than in the ankle joint [16, 17]. OA induced by cartilage injury may progress due to the degeneration of adjacent cartilage, and catabolic factors, such as inflammatory cytokines and biomechanical stress, in the cartilage around the defect may affect the progression of OA after cartilage injury. There may be differences in the chondrocyte response to inflammatory cytokines and proliferation between the knee and ankle, which may lead to a difference in the progression of OA after cartilage injury between the knee and ankle joint. Understanding the factors that may contribute to differences in the outcome of cartilage repair and incidence of OA between knee and ankle may improve outcome after treatment.
We hypothesize that the repair of articular cartilage in the congruent joint is better than that in the non-congruent joint. The purpose of this study was to examine differences in cartilage repair after drilling between the talus (congruent joint) and knee (non-congruent joint) in a rabbit model. In addition, differences between the knee and ankle as to the proliferation ability of chondrocytes, the production of proteoglycans (patellar groove (PG)), and the reaction of cartilage to IL-1β were also examined in vitro. Together, results may clarify the roles of morphological and biochemical factors in differences in cartilage degeneration between the knee and ankle.
Rabbits were housed in the research facilities for laboratory animal science. The experimental research protocol was reviewed and approved by the Hiroshima University ethical committee.
The osteochondral defects (3.0 mm in diameter and 2.0 mm in depth) were created using a punch. Drilling was performed at four points using a 0.7-mm Kirschner wire (K-wire) as described previously .
Micro-CT and histological evaluation
Pineda histologic score for cartilage repair 
Filling of defect (%)
Reconstruction of osteochondral junction
Significantly reduced staining
Mostly hyaline and fibrocartilage
Some fibrocartilage, but mostly
Non-chondrocytic cells only
The sections at 12 weeks were pretreated with antigen retrieval reagent (Immunoactive, Matsunami Glass Ind., Osaka, Japan) for 1 h followed by 0.3 % H2O2 for 30 min, normal blocking serum for 30 min, and primary antibody against type 2 collagen (dilution, 1:100; anti-hCL(II); Daiichi Fine Chemical, Toyama, Japan), type 1 collagen (dilution, 1:250; Novus Biologicals, USA), and MMP13 (dilution, 1:15; Neo Markers, CA, USA) overnight at 4 °C. The next day, the sections were visualized using the avidin-biotin system (Vectastain Elite ABC Mouse IgG kit, Vector Laboratories, Inc., Burlingame, CA) and 3,3′diaminobenzidine (Peroxidase Substrate Kit, Vector Laboratories, Inc.) according to the manufacturer’s instructions.
To quantitate the immunochemistry results, the number of immune-positive cells in each type of osteochondral defect was counted in a microscopic field (100 μm × 100 μm) under ×40 magnification.
Proliferation ability of chondrocytes
Cartilage tissue was obtained from the distal femur and talus. Chondrocytes were isolated from the cartilage tissues and cultured. The tissues were minced and incubated in trypsin (Tryp LE Express; Life Technologies, Carlsbad, CA) for 15 min at 37 °C, after which the cartilage was treated with Dulbecco’s modified Eagle’s medium (DMEM; Gibco BR, Grand Island, NY, USA) containing 0.2 % collagenase (Sigma, St. Louis, MO) at 37 °C for 4 h. Dissociated cells were cultured in DMEM supplemented with 10 % fetal bovine serum (FBS; Biowhittaker, Walkersville, MD, USA) and 100 U/ml of penicillin-streptomycin. After overnight culture, non-adherent cells were removed, and adherent cells were further incubated in fresh medium. Chondrocytes were seeded in 96-well plates at a density of 5 × 103 cells per well in DMEM medium supplemented with 10 % FBS. At 4 h after cell plating, cell proliferation was assessed using a Cell Counting Kit-8 (Dojindo, Kumamoto, Japan) (4-[3-(4-iodophenyl)-2-(4-nitrophenyl)-2H-5-tetrazolio]-1,3-benzene disulfonate (WST) assay) at day 1 according to the manufacturer’s instruction. The values, corresponding to the number of viable cells, were read at OD 450 mm using a Microplate Reader (BioTek Instrument, Winooski, VT, USA). The cell increase ratios at 2 and 3 days were compared between the femur and talus.
Production ability of proteoglycans and the reaction to IL-1β
The cartilage tissue was obtained from the distal femur and talar domes. Samples are incubated at 37 °C for 72 h in 48-well plates. Each well contained 500 μl of DMEM with 10 % FBS and 1 % penicillin/streptomycin. The cartilage samples were washed three times and cultured at 37 °C for an additional 72 h in 500 μl of serum-free DMEM with IL-1β (1 ng/ml; Peprotech, Rocky Hill, NJ, USA) or without IL-1β. The assay was performed in at least three independent experiments with duplicate wells. The concentration of the released glycosaminoglycan in the cartilage-conditioned medium was determined using the Blyscan Glycosaminoglycan assay kit (Biocolor, UK) according to the manufacturer’s protocol. Proteoglycan release quantity with IL-1β/without IL-1β ratio was compared between the femur and talus.
The Kruskal-Wallis test was used to analyze the histological scoring data and quantitative values of immunochemistry among the groups. If a significant difference was obtained, the Steel-Dwass test was used to perform multiple comparisons between the groups. In vitro data were analyzed using the Mann-Whitney U test to determine significant differences between the femur and talus. A P value of <0.05 was considered significant.
Production of proteoglycans and reaction to IL-1β
Our results demonstrate that osteochondral defects in the covered talus, which has good congruency, exhibit early and good repair at 12 weeks after surgery, with the cartilage at the defect appearing similar to the adjacent cartilage. Immunoreactivity of covered talus demonstrated hyaline cartilage in the defect site. The covered talus showed good repair compared to that of the uncovered talus. This indicates that morphological factors may affect osteochondral repair. The covered talus has high congruency, while the uncovered talus does not. Decreased joint congruency results in increased contact pressure per area . Congruent joints can distribute load and reduce the progress of cartilage defects and subchondral cysts. Thus, our results indicate that cartilage repair of congruent joints (covered talus) is faster and better than that of incongruent joints (uncovered talus).
In addition, the improved lesion repair of congruent joints compared to that of incongruent joints relates to subchondral bone repair. Our results indicate that subchondral bone in the osteochondral defect of the covered talus showed early recovery to the level of adjacent bone, which suggests that good cartilage repair may be required for early subchondral bone repair. There is a dynamic relationship between cartilage and subchondral bone, such that an abnormality in either can lead to the loss of balance of the bone cartilage unit. Damaged subchondral bone cannot support the overlying cartilage . Tidemark advancement and thickening of the subchondral plate induced by remodeling of injured subchondral bone are early signs of OA . Shahgaldi et al. reported that contact pressures of reparative articular surfaces were either higher or lower than normal controls and suggested that these differences lead to thickness variations of the surface of repaired tissue and the presence of an abnormal thickness of subchondral plate . Messner  and Shapiro et al.  postulated that inadequate subsurface support of the subchondral bone bed may be a reason for unsuccessful repair. Thus, the formation of a normal contour of subchondral bone is an important factor for successful osteochondral repair.
Other important factors of osteochondral repair are the characteristics of the cartilage. Several studies have shown differences between the knee and ankle cartilages. Schumacher et al. demonstrated a difference in cell distribution between the knee and ankle cartilages . Horizontal sections of the superficial zone of the ankle contain chondrocytes organized into clusters or chondrons of two to six cells that lie horizontal to the surface. This cell clustering is not observed in knee articular cartilage. In the knee, chondrocytes in the superficial zone exist as single cells or as doublets that are isolated from each other. Chondrocytes in the deep zone of the ankle are observed as either single cells or doublets. Within the knee, 90 % of chondrocytes are present as single cells, whereas only 3.8 % of chondrocytes in the ankle exist as single cells. Chubinskaya et al. and Aurich et al. demonstrated that the GAG content is significantly higher in the ankle than in the knee [28, 29]. The GAG content is reduced in OA cartilage, but this may be due to proteoglycan release from the damaged matrix. In regard to the equilibrium modulus, dynamic stiffness and hydraulic permeability, which define the ability of the extracellular matrix to withstand compressive loads, are higher in the ankle cartilage than in the knee cartilage . Therefore, the ankle cartilage has stiffer cartilage and a greater mean compressive modulus than knee cartilage . The response of ankle chondrocytes to inflammatory molecules is lower than that in the knee because of differences between transport properties within the ankle and knee. Molecules diffuse through avascular cartilage and the rate of diffusion is determined by diffusion and partition coefficients. The diffusion coefficient is similar between the knee and ankle, whereas the partition coefficient is 47 % lower in the ankle than in the knee . Orazizadeh reported a difference of tolerance to biomechanical stress on the cartilage between the knee and ankle. Marked differences in the relative levels of the aggrecan gene mRNA are observed in the response of ankle chondrocytes to 20 min of mechanical stimulation at 0.33 Hz within a sealed pressure chamber compared to the response of knee joint chondrocytes. Ankle chondrocytes are more metabolically active than those of the knee . Deep zone chondrocytes synthesize more PG and collagen than those in the superficial zones in the knee and ankle joints, although there is variation between the different zones within the cartilage [30, 32, 34]. The incidence of OA in the ankle joint is lower than that in the knee joint due to these differences between the knee and ankle cartilages [35, 36]. However, our results showed no significant difference in the proliferation of chondrocytes, the production of PG, or the response to the IL-1β between the knee and ankle cartilages. Thus, our results may reflect differences in joint morphology between the knee and ankle, rather than differences in biochemical properties of cartilage.
There are a few limitations to this study. First, this research did not consider sufficiently the influence of weight bearing. Takahashi et al. reported that loading and unloading in the early phase of cartilage repair has merits and demerits . Yokota et al. reported that loading at a moderate intensity appears to be necessary for cartilage maintenance . In our model, the degree of load varies at each defect site. Therefore, this difference in load on the osteochondral defect site may influence the results of this study. Second, bone marrow cells from the knee and ankle were not evaluated. The talus of the rabbits is very small, and harvesting bone marrow is difficult. Further investigation of differences in bone marrow cells between the knee and ankle is needed. Third, in this study, subchondral bone cysts were not evaluated. Loading compressed cartilage forces its water into subchondral bone that has been drilled, leading to a localized high increased flow and pressure of fluid in the subchondral bone. This results in local osteolysis and development of a subchondral cyst. Subchondral cyst formation is assumed to be caused by the damaged cartilage functioning as a valve . The presence of subchondral bone cysts may affect the results of this study, but obvious subchondral bone cysts were not observed in either histological or radiographical evaluation.
Our results demonstrate that the covered talus shows early and good osteochondral repair compared to the knee and uncovered talus. These results suggest that the congruent joint has advantages over the non-congruent joint for subchondral repair prior to cartilage repair.
This work was supported in part by a grant-in-aid to Prof. Adachi N. for scientific research from the Ministry of Education, Culture, Sports, Science and Technology, Japan (no. 25253089).
Availability of data and materials
The data was all shown in the manuscript.
MM performed the examination using a rat and the analysis on all samples, interpreted the data, wrote the manuscript, and acted as the corresponding author. NA and TN supervised the development of work, helped in the analysis on all samples and data interpretation and manuscript evaluation, and also contributed to the experiment design and concept. TK and RS helped to evaluate and edit the manuscript. MO helped to evaluate and final proof the manuscript. All authors read and approved the final manuscript.
The authors declare that they have no competing interests.
Consent for publication
The experimental research protocol was reviewed and approved by the Hiroshima University ethical committee.
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