Experimental animals
Twenty-six skeletally mature female Japanese white rabbits (age: 6 months old, body weight: 2.5–3.0 kg, KITAYAMA LABES, Nagano, Japan) were used for this experiment. The Institute for Experimental Animals, Kanazawa University Advanced Science Research Center approved all experimental protocols, using an animal model. All surgical procedures were performed in accordance with the Guide for the Care and Use of Laboratory Animals published by the United States National Institutes of Health (Bethesda, MD; NIH publication no.86-23, revised 1985).
Induction of experimental OA
Induction of OA was performed as described in previous study [17]. The rabbits were anesthetized by intramuscular injection of ketamine hydrochloride (35mg/kg) and xylazine (5 mg/kg), and intravenous injection of pentobarbital sodium (50 mg/kg). Both knees were shaved and sterilized with iodine. The right knee joint was exposed through a medial parapatellar incision. The patella was dislocated laterally, and the knee was placed in full flexion. The ACL was visualized optimally and transected with a micro-scissor (Fig. 1A). A positive anterior drawing test was performed to confirm complete ligament transection. The joint was irrigated and sutured in a routine fashion. An identical operation was performed in the left knee. Postoperatively, free activity was allowed in the cage without immobilization. Traumatic degeneration was induced as described previously for the ACLT model [18], which is characterized by OA-like damage. The rabbits showed considerable individual variability in OA progression at 4 weeks after ACLT [19]. Therefore, we used matched-pair analysis to examine the chondroprotective effect of ADSC sheets on OA progression in a stricter manner.
Isolation of ADSCs
ADSCs were isolated by a previously reported method [5]. Adipose tissue (approximately 1–2 g) was harvested from the posterior neck regions of the rabbits and washed with phosphate-buffered saline (PBS; Wako Pure Chemical Industries, Osaka, Japan). The tissue was cut into strips over 5 minutes. Collagenase (Wako Pure Chemical Industries) was dissolved in PBS to a concentration of 0.12% in 25 mL and used to digest adipose tissue at 37°C for 45 minutes in a water bath. The mixture was shaken every 15 minutes during the digestion period. Immediately after the reaction was completed, 25 mL of Dulbecco’s modified Eagle’s medium (DMEM; Wako Pure Chemical Industries), was added to neutralize collagenase activity. The resulting solution was filtered and centrifuged at 1300 revolutions per minute (rpm) for 6 minutes at 25°C, and the supernatant was removed. Next, a pellet of cells was seeded at 5 × 104 cells/cm2 in 100 mm tissue culture dishes and cultured in DMEM containing 10% fetal bovine serum (FBS; Nichirei Biosciences, Tokyo, Japan) and 1% Penicillin-Streptomycin Solution (P/S; Wako Pure Chemical Industries) at 37°C in 5% CO2 incubator. After 24 hours, debris was removed by washing with PBS and fresh medium was added. Plastic-adherent spindle-shaped cells became apparent 3–4 days later and were isolated with trypsinization after reaching 80%–90% confluence. More than three passage ADSCs were prepared for the subsequent experiment. Previous reports have confirmed the presence of stemness in ADSCs extracted by this method [6,20].
Fabrication of ADSC sheets
ADSC sheets were fabricated as reported previously [21]. To create cell sheets, ADSCs were seeded in the 100 mm tissue culture dishes by 1 × 106 cells/dish for 7days. The culture medium consisted of DMEM, 10% FBS, 1% P/S, and 50 mM ascorbate-2-phosphate (vitamin C [Vc]). The culture medium was refreshed every 2–3 days in 1 week. The fabricating medium containing ascorbate-2-phosphate was used to enhance the collagen protein secretion by the ADSCs to make the ADSC sheets (Fig. 1B).
Injection of ADSC sheets
Twenty-four rabbits (48 knees) were used for matched-pair analysis. Autologous ADSC sheets were fabricated from the subcutaneous adipose tissue parallel to induction of experimental OA. ADSC sheets were rinsed with PBS three times. Autologous cell death sheets were made for control by liquid nitrogen according to a previous report [22]. At 4 weeks after ACLT, autologous ADSC sheets in 1 mL PBS were injected intra-articularly into the right knee (ADSC sheets group), using 14-gage needles (Fig. 1C). Similarly, autologous cell death sheets were injected into the left knee (control group). Subsequent injections were administered once weekly. The rabbits were sacrificed following the intravenous injection of 6 ml sodium pentobarbital at 6, 8, 10 and 12 weeks after ACLT (6 rabbits in each week), and femoral condyles from both knees were harvested (Fig. 1D).
Macroscopic analysis
To assess cartilage lesions, the femoral condyles were stained with India ink (American MasterTech, CA, USA). A Canon IXY 650 digital camera (Canon, Tokyo, Japan) was used to take macroscopic pictures. Gross findings were classified and scored as described previously [23, 24]. The medial and lateral femoral condyles were scored individually as grade 0, intact articular surface; grade 1, minimal fibrillation; grade 2, overt fibrillation; grade 3, erosion of 0–2 mm; grade 4, erosion of 2–5 mm; and grade 5, erosion of >5 mm. The two scores were summed to obtain a cumulative macroscopic OA score. All evaluations were performed by two blinded researchers and their scores were averaged to obtain an overall score.
Histologic analysis
The dissected distal femurs were fixed in a 4% paraformaldehyde solution after gross morphologic examination, and subsequently decalcified in 4% ethylenediaminetetraacetic acid (EDTA) solution. The specimens were dehydrated with a gradient ethanol series, embedded in paraffin blocks, and cut into 5-μm sections. Safranin O staining was used to assess general morphology and proteoglycans/collagen content in cartilage. Histologic sections were visualized using a fluorescence microscope (Keyence Japan, Osaka, Japan). Ten coronal sections were prepared in the coronal plane through the middle of the femoral condyles, and one section from each sample, which included the most severely degenerated area, was used for each histologic analysis. Two blinded investigators evaluated the severity of cartilage degeneration using the cartilage OA histopathology grading system methodology of the Osteoarthritis Research Society International (OARSI) [25].
Immunohistochemical analysis
The analyses were performed as follows to evaluate cartilage matrix protein catabolic enzymes [26]. After deparaffinization, sections were incubated with 0.3% hydrogen peroxide for 30 minutes, and subsequently treated with hyaluronidase for 60 minutes. Then, they were incubated at room temperature with mouse anti-human MMP-1 monoclonal antibody (1:100; Kyowa Pharma Chemical Co. Toyama, Japan), mouse anti-rabbit MMP-13 monoclonal antibody (1:20; Thermo Fisher Scientific Inc. Waltham, USA), and mouse anti-human ADAMTS-4 monoclonal antibody (1:150; Thermo Fisher Scientific Inc. Waltham, USA) respectively. All antibody dilutions were made in PBS. After an overnight reaction with the primary antibody at 4℃, sections were incubated with labeled polymer-HRP anti-mouse IgG (Dako, Tokyo, Japan) at room temperature for 30 minutes. Signals were visualized with 3, 3’-diaminobenzidine tetrahydrochloride, and nuclei were counterstained with hematoxylin. Six microscopic fields (×100 magnification) relative to the medial, central and lateral regions in cartilage tissue were used to perform a semi-quantitative analysis of immunohistochemistry. A semi-quantitative method that assigns immunohistochemistry values as a percentage of positive cells (MMPs, ADAMTS-4) was provided for a complete assessment of protein expression, with maximum scoring being 100% [27]. Immunohistochemical analysis results were evaluated by two observers blinded to the identity of each sample.
1,10-dioctadecyl-3,3,30,30-tetramethylindocarbocyanine perchlorate (DiI) labeling
A DiI labeling examination was performed to monitor the fate of the ADSCs after the intra-articular injection. Two rabbits (four knees) were used for this examination. Experimental OA was induced and autologous ADSC sheets and autologous ADSCs were fabricated. At 4 weeks after ACLT, autologous ADSC sheets (2.0 × 106 cells) were injected into the right knee, and ADSCs (2.0 × 106 cells) were injected into the left knee. On the day of injection, ADSC sheets and ADSCs were labeled for cell tracking with a fluorescent lipophilic tracer, DiI (Molecular Probes, Eugene, OR, USA) as indicated by the manufacturer. The rabbits were sacrificed at 6 weeks after ACLT, and the femoral condyles were dissected. Fresh frozen sagittal sections containing a central portion of the knee joint were prepared using Kawamoto’s method [28].
Statistical analysis
SPSS ver.23.0 (SPSS, Inc., Chicago, IL, USA) was used to perform statistical analysis. The results are shown as the mean ± standard deviation. A paired t-test was used to perform matched-pair analyses. P < 0.05 were considered significant.