Animal preparation
This laboratory-based animal study used a judgement sampling technique. Forty-eight male Japanese white rabbits were used because of their history of use for histomorphometric analyses of the ACL insertion and to eliminate the influence of female hormones on the ACL [8,9,10,11, 15]. Because the skeletal growth of rabbits is complete at 6 months [16], we set the evaluation period at 24 weeks of age. These rabbits were purchased from Japan SLC, Inc. (Hamamatsu, Japan) and Hamada farm (Miho, Japan). The rabbits were maintained in accordance with the guidelines of the Ethical Committee of Ibaraki Prefectural University of Health Sciences and Ibaraki University, and the National Institutes of Health Guidelines for the Care and Use of Laboratory Animals (NIH Pub. No. 86–23 Rev. 1985). Six animals at each age (1 day, 1, 2, 4, 6, 8, 12, and 24 weeks) were euthanized by over dose intravenous barbiturate injection (200 mg/kg, Somnopentyl®, Kyoritsu Seiyaku Corporation, Tokyo, Japan). Because this study was an evaluation of the development of fibrocartilage layers in ACL tibial insertions, it could not be considered an in vitro experiment. The animal species to be studied was determined based on previous reports [8,9,10,11]. Moreover, it is considered difficult to prepare tissue specimens using animals smaller than rabbits. Therefore, we chose rabbits for this study.
Histomorphological analysis
Knees from the animals were fixed in 10% neutral-buffered formalin for 1 week. After fixation, the specimens from rabbits 2–24 weeks of age were decalcified in 10% ethylenediaminetetraacetic acid (pH 7.4) for 7–12 weeks and then embedded in paraffin. The specimens from rabbits 1 day and 1 weeks of age could be sliced without decalcification. The specimens were sliced at 5-μm thickness in the center of the ACL tibial insertion site. The slices were stained with hematoxylin-eosin, and safranin O to assess the histomorphology and GAG contents [8,9,10,11]. We also used proliferating cell nuclear antigen (PCNA) staining to detect proliferating cells [8,9,10,11] (Fig. 1a), terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate-biotin nick-end labeling (TUNEL) staining to detect apoptotic cells [8,9,10,11] (Fig. 1b), and Sox9 staining to evaluate the developmental differentiation of chondrocytes (Fig. 1c).
PCNA immunostaining was carried out with a Histofine® SAB-PO(M) Kit (Nichirei Biosciences Inc., Tokyo, Japan) according to the manufacturers’ instructions. Briefly, sections were deparaffinized, rinsed in phosphate-buffered saline (PBS) for 5 min, and immersed in 3% hydrogen peroxide (H2O2) in methanol for 10 min to block endogenous peroxidase activity. After rinsing in PBS for 5 min, the sections were blocked in 10% normal rabbit serum at 25 °C for 10 min, and incubated with an anti-PCNA monoclonal antibody (PC-10; Code No. M0879; Dako, Glostrup, Denmark) at 1:100 dilution for 12 h at 4 °C. Antibody Diluent (Code No. S0809; Dako) was used instead of a primary antibody for the negative controls [8,9,10,11].
TUNEL staining was performed using an Apoptag® Plus Peroxidase In Situ Apoptosis Detection Kit (Merck Millipore, Billerica, MA, USA) according to the manufacturers’ instructions. TUNEL-positive nuclei of chondrocytes were stained dark brown, and TUNEL-negative nuclei were stained blue [8,9,10,11].
Sox9 immunohistochemical staining was performed with a Histofine® SAB-PO (R) Kit (Nichirei Biosciences Inc.) and a Rabbit-To-Rabbit Blocking Reagent (ScyTek Laboratories Inc., Logan, UT, USA) according to the manufacturers’ instructions. Deparaffinized sections were rinsed twice with PBS for 3 min each and immersed in 3% H2O2 in methanol for 10 min to block endogenous peroxidase activity. After three rinses in PBS for 5 min each, the sections were blocked with 10% normal goat serum at room temperature for 10 min, incubated with a Rabbit-To-Rabbit Blocking Reagent at room temperature for 30 min, and washed four times in PBS for 5 min each. The sections were then incubated with an anti-Sox9 rabbit polyclonal antibody (Bioworld Technology Inc., Louis Park, MN, USA) at 1:100 dilution for 24 h at 4 °C. The immunoreaction product was developed in diaminobenzidine, and the sections were counterstained with Mayer’s hematoxylin for 30 s. Sox9-positive nuclei were stained dark brown and Sox9-negative nuclei were stained blue.
Histomorphometric analyses were performed using similar methods to those in our previous study [8,9,10,11]. The sections were examined using a BX-51 light microscope (Olympus Optical Co. Ltd., Tokyo, Japan). The GAG areas stained red by safranin O were evaluated in the fibrocartilage layers in the ACL tibial insertion (Fig. 2). In the specimens from rabbits 1 day and 1 and 2 weeks of age, we defined the fibrocartilage layers in the ACL tibial insertion as those having lower-density staining of cartilaginous tissue than articular cartilage by safranin O with round cells, and between the ligament and hyaline cartilage area continuous with articular cartilage. In the specimens from rabbits ≥4 weeks of age, we defined the fibrocartilage layers in the ACL tibial insertion as the cartilaginous tissue with round cells between the ligament and bone [9, 10]. The length of the tidemark in the ACL tibial insertion was measured as the sum-total length that stained with hematoxylin-eosin. The histological ACL length was defined as the distance between the anterior attachment of the femur and the posterior attachment of the tibia [6]. The width of the ACL tibial insertion was defined as the anterior-to-posterior distance of the ACL tibial attachment. Mac Scope software (Mitani Co., Fukii, Japan) was used to determine the total numbers of chondrocytes and the numbers of TUNEL-positive, PCNA-positive, and Sox9-positive chondrocytes in the safranin O-stained GAG areas in the fibrocartilage layers in the ACL tibial insertion. Each red-stained GAG area and tidemark length was divided by the width of the ACL insertion to define the average thickness of the red-stained GAG areas and the percentage of the tidemark length relative to the ACL insertion width, respectively. The TUNEL-, PCNA-, and Sox9-positive rates were calculated based on the total numbers of chondrocytes in the safranin O-stained GAG areas in the fibrocartilage layers.
Statistical analysis
For each parameter, normality of the data was tested using the Shapiro-Wilk normality test. The time-dependent histological changes were evaluated by one-way analysis of variance (ANOVA) when the assumption of normality of all variables for each parameter was accepted. Factors determined to show significant differences by ANOVA were further evaluated by Dunnett’s test. When the assumption of normality failed for all variables in each parameter, the Kruskal-Wallis test and Bonferroni adjustment technique were applied. All parameters were compared with those at 24 weeks. The level of significance was set at 5%. All analyses were performed with IBM SPSS Statistics version 24.0 (IBM Corp., Armonk, NY, USA).
According to a previous study [9], a power calculation was performed with a confidence level of 95% (α = 0.05) and power (1 – β) of 80% using the POWER Procedure in SAS software (SAS Institute, Cary, NC, USA). Calculation of the smallest sample size that produced a significant difference yielded an estimated sample size of five to six specimens per age group. We enrolled six specimens per age group to reduce the number of animals used.
