The animal experiment was carried out in accordance with relevant guidelines and regulations and was approved by the Medical Ethics Committee of the First Affiliated Hospital of Shenzhen University. Fresh adult bovine knees purchased from the Guangdong Medical Laboratory Animal Center were used in this experiment. The animals were around 18 months old with closed epiphyses. The entire knee joint was harvested with the joint capsule intact and stored at 4°C and delivered to the lab within 1 day. Chondrocyte viability was maintained during a 3 to 6 day period of storage, which has been demonstrated by a previous publication . The experiment was performed 1 day after the animals’ death.
Under sterile conditions, a model of grade-II cartilage degeneration was established by opening the knee joints, exposing the articular cartilage on the femoral condyles, scraping chondral membrane off with scalpel, and abrading the cartilage on both the medial and lateral condyles with a rasp . Grids consisting of six squares of 1 × 1 cm2 area were created on the medial and lateral femoral condyles at the weight-bearing area with a sterile ink marker. Each grid was marked with a number from 0 to 5 (Fig. 1A). 0 represented the control without any bRFE treatment, while the others served as the experiment groups treated with different time durations of bRFE (N=6/group).
Treatment with bRFE
Radiofrequency ablation was generated from the SAPHYRE 60º angle bipolar ablation probe on the Vulcan EAS electrothermal system (Smith and Nephew, Inc., Andover, Massachusetts, USA). The probe was set at bipolar ablation mode at 70 W. The probe was moved in a meandering pattern in contact with the cartilage surface but not applying and pressure; in addition, no fluid flow was used during bRFE treatment. The model was submerged in normal saline in order to simulate the arthroscopic environment. Squares No. 1 to 5 were treated in the aforementioned method for 10, 20, 30, 40, and 50 seconds, respectively. After the treatment, the squares were taken off one by one with a scalpel blade ensuring the removal of full-thickness cartilage containing subchondral bone. Each cartilage square was dissected into three parts: part A, B and C. Part A was used for hematoxylin/eosin (HE) staining, Part B was used for observation of the cartilage contour under scanning electron microscope (SEM). Part C was used for the measurement of glycosaminoglycan (GAG) content in the articular cartilage.
All the Part A’s from the six groups were fixed in 10% neutral buffered formalin (NBF) for 2 days. Then, they were decalcified and paraffin embedded. Sections were cut at 5μm thickness using a microtome, deparaffinized through xylene, and hydrated via ethanol gradient and water. Afterwards, HE staining was performed to reveal the cartilage morphology. The slices were dyed in hematoxylin solution for 5 min, given a 1 min water soak, differentiated with 1% hydrochloric acid ethanol for 30s, given a 15 min water soak, dyed with 0.5% eosin for 3 min, given a distilled water soak, and finally sealed for observation after dehydration.
Part B was fixed in 10% NBF, dehydrated in a graded series of ethanol, dried at critical point, and coated with gold in an Autoconductavac IV (Seevac, Pittsburgh, PA) before their contouring was examined with SEM ( Hitachi S 3000N, Tokyo, Japan) and scored according to the system provided in Table 1 . A higher score indicates a smoother articular cartilage surface.
GAG content was measured using the described dimethylmethylene blue (DMB) method . After freeze-drying for 1 day, the cartilage specimens (Part C) were weighed on an electronic scale to determine their dry weight. Then, they were immersed in papain at 60°C 1 day for enzymolysis. 3 mL DMB was added for every 100μl of the solution. The optical density (OD) value was determined using a UV spectrophotometer. Compared with standard curves, the GAG content of each specimen was calculated using the following formula: GAG content (μg/mg)= GAG content of the specimen/dry weight of the specimen.
Sample results are presented in the text as mean ± standard deviation. The software SPSS 16.0 (version 15.0 for Windows; SPSS Inc., Chicago, IL, USA) was applied for statistical analysis and management. The one-way analysis of variance (ANOVA), SNK-q, and Dunnett’s T3 were applied for comparisons of multi-sample means and heterogeneity of variance. Results were considered significant at a value of P < 0.05.