Ethics statement
The experimental protocol was approved by the Animal Care and Use Committee of Jinan University, Guangzhou, China. All procedures performed in this study involving animals were in accordance with the revised Animals (Scientific Procedures) Act 1986 in the UK and Directive 2010/63/EU in Europe.
Preliminary experiment
In order to investigate the feasibility of this study, including training operations and sample size estimation, two healthy New Zealand white rabbits were performed the preliminary experiment first. Sample size was determined by the calculation of the pre-test results and the relevant literatures [12, 28].
Animal model
Thirty-two healthy New Zealand white rabbits (16 male and 16 female, with a mean age of 8 months and weighting from 2.0 to 3.0 kg) were used for the formal experiment. Rabbits were randomly numbered 1–32, and divided into 5 groups. Numbers 1 to 6 were included in Group A, 7 to 12 in Group B, 13 to 18 in Group C, 19 to 24 in Group D, and numbers 25 to 32 were included in Group E (among the 8 rabbits in Group E, 2 rabbits were randomly selected for each subgroup EA, EB, EC, and ED, which were considered as the blank controls of Group A, B, C, and D, respectively). The left knees of experimental groups (Group A, B, C, D) were served as the experimental side to establish articular cartilage models at different stages of early degeneration with papain, which is a known and validated mediator of inducing PG catabolism [28–31], and the right knees acted as the control side. On the 1st, 4th, and 7th day of the experiment, the twenty-four rabbits of experimental groups were administered an injection of 0.5 ml of 1.6 % papain solution into the joint cavity of the left knee by using a 16-gauge hypodermic needle, and a control injection of the same amount of solution without papain in the right knee. The rabbits were sedated via ear vein injection of Ketamine (10 mg/kg) and Midazolam (1 mg/kg) while being monitored by an anesthesiologist for the period of the surgical procedure. Postoperatively, the rabbits were housed in individual cages without restriction of joint movement.
MRI Protocol
T1ρ and T2-weighted images of the bilateral knee joints were obtained for 32 rabbits by using a clinical 3.0 T MRI scanner (GE discovery MR750; the First Affiliated Hospital of Jinan University, Guangzhou, China) and human 8ch knee array coil. Group A, B, C, and D were scanned in sequence at 1, 2, 3 and 4 weeks post-operation (from the last injection of papain) respectively, and rabbits in the subgroups (EA, EB, EC, and ED) underwent MRI in the same period for comparison. For the duration of the imaging session (approximately 15 mins), each rabbit was under general anesthesia with intravenous Ketamine (8 mg/kg) and Midazolam (0.8 mg/kg), and the protocol included 2 sequences: the first, conventional T2-weighted imaging (T2WI) for morphological evaluation were acquired using a fast spin-echo (FSE) imaging on sagittal view, with the following parameters: repetition time/echo time (TR/TE) = 2000 ms/85 ms, slice thickness/spacing (thn/spa) = 2 mm/0 mm, field of view (FOV) = 12 × 12 cm, acquisition matrix = 352 × 224, NEX = 2; the second MRI pulse sequence used for a series of T1ρ-weighted sagittal images was 3D Magnetization-Prepared Angle-Modulated Partitioned k-Space Spoiled Gradient Echo Snapshots (3D MAPSS) with a spin-lock pulse amplitude of 500 Hz, under the following parameters: time of spin-lock pulse (TSL) = 0, 10, 40, or 80 ms, TR/TE = 7.8 ms/3.7 ms, thn/spa = 12 mm/0 mm, FOV = 12 × 12 cm, matrix = 256 × 128, NEX = 1, scan time = 3 min 33 s, and the B1 of spin-lock radio frequency (RF) was 0.145 Gauss.
Histology
Immediately after imaging, rabbits in each group were euthanized by intravenous pentobarbital (100 mg/kg), and both knees were harvested, formalin-fixed, and decalcified. Tissue blocks of 0.5 cm in thickness were harvested from the medial and lateral femoral condyle cartilage (MFCC and LFCC) on the sagittal plane, and made into paraffin-embedded tissue sections. Morphology of the chondrocytes was observed after hematoxylin and eosin (H&E) staining, and the PG content was determined by Safranin-O and Fast Green staining [12, 32].
T1ρ quantification
Sun Advantage Workstation 4.5 (GE) was used for the post-processing of T1ρ images. With an exponential decay, T1ρ maps were reconstructed on a pixel-by-pixel basis by fitting a linear regression of the image intensity data to the following equation:
$$ \mathbf{S}\ \left(\mathbf{T}\mathbf{S}\mathbf{L}\right) = {\mathbf{S}}_{\mathbf{0}} \times \mathbf{exp}\ \left(-\mathbf{T}\mathbf{S}\mathbf{L}/{\mathbf{T}}_{\mathbf{1}\boldsymbol{\uprho }}\right), $$
where S is the signal intensity on T1ρ images with the given TSL.
On the intermediate layer of the T1ρ sagittal image, three regions of interest (ROIs) were placed from anterior to posterior on the images of MFCC and LFCC for measurement of the T1ρ values. Each ROI was drawn using a circular tool by the radiologist with more than 10 years of the professional experience of MRI, and in order to reduce the influence of partial volume effect on results, the ROIs were not allowed to be set on the adjacent tissue. Average values of the three ROIs were calculated and recorded as the T1ρ values of MFCC and LFCC, which were statistically analyzed.
Image interpretation
The interpretations of images acquired were determined by two radiologists with more than 10 years of experience, which blinded to all experimental information and the final histological diagnosis, and independently readed the T1ρ original and T2-weighted images of the 32 rabbits. Each observer performed the same interpretation twice, with a delay of two weeks, to assess intraobserver agreement. The image interpretation results were determined according to the agreement and disagreement between the two observers as follows: the same results and the final results after discussion.
Statistical analysis
Interobserver and intraobserver reliability of image interpretation was evaluated with the interclass and intraclass correlation coefficient (ICC): poor (< 0.40), fair (0.40 – 0.59), good (0.60 – 0.74), and excellent (0.75 – 1.00). The mean ± standard deviation of measurements were calculated for the T1ρ values of MFCC and LFCC. A normal distribution of our data was shown by using the normality test, and then the differences in the T1ρ values of LFCC and MFCC within experimental groups were compared using one-way analysis of variance (ANOVA). Comparison of the T1ρ values in the control side between Group E and experimental groups was conducted with independent sample T-test. A P value of < 0.05 was considered statistically significant. Statistical analyses were performed with SPSS statistics software package, version 10.0 (SPSS Inc., Chicago, Illinois, USA).