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SUPPLEMENTAL METHODS
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Animals
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All experiments were conducted in accordance with the institutional guidelines
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of our university for the care and use of experimental animals. Seven microminipigs
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(Fujimicra Corporation, Shizuoka, Japan) were used (14). Microminipigs are pigs newly
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developed for experimentation and are smaller than minipigs. They averaged 21.1 months
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old and 18.3 kg. All pigs had free access during the study period to food and water in a
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post-operative care cage (75 cm wide, 70 cm long and 90 cm high). Seven posterior parts
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of intact lateral menisci (LM) were used for analyzing T1rho value, histological
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evaluation, and biochemical content in order to investigate intact menisci. In intact LM,
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anterior parts of LM were not examined, since they might be influenced by the surgical
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procedures. Seven incised MM were examined for analyzing T1rho value and histological
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evaluation. In incised MM, biochemical content was not measured, since incised MM
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were so fragile that we could not cut them into small pieces corresponding to each zone.
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Surgical Procedures
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With the animal under general anesthesia, each knee was approached through a
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medial parapatella arthrotomy. After cutting the medial collateral ligament (MCL) and
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the anterior part of meniscofemoral ligament (Fig. 1a), the knee was maximally flexed
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and externally rotated to expose a whole MM. The middle part of the MM was incised
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radially from rim to peripheral (Fig. 1b). The tear was repaired using 4-0 nylon (Bear
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Medical Corp, Chiba, Japan) by a mattress suture (Fig. 1c). MCL, capsule, and skin were
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closed in layers with absorbable sutures. Pigs were allowed to move freely in their cages
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without any fixation method. Incised MM and intact LM of the same leg were harvested
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four weeks after surgery.
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Histological Examination
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Specimens of LM and MM were fixed separately in 4% paraformaldehyde for
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seven days, then dehydrated with a gradient ethanol series. In order to section finely, the
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specimens were decalcified with 20% EDTA (pH 7.4) at room temperature for 21 days.
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The specimens were embedded in paraffin and sectioned into 5 μm thick slices sagittally
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in order to correspond with sagittal T1rho mapping images. The sections that were
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morphologically close to the T1rho mapping image were stained by hematoxylin and
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eosin (HE), safranin-o/fast green, and Masson trichrome stainings.
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Immunohistochemistry
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The paraffin-embedded sections were deparaffinized and sequentially pretreated
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with proteinase K, hydrogen peroxidase, and horse serum. For type I collagen, sections
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were covered with mouse anti-human type I collagen antibody (1:800 in dilution;
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ab90395, Abcam) at 4°C overnight, then with secondary antibody of biotinylated horse
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anti-mouse IgG (1:200 in dilution; Vector Laboratories, Burlingame, CA) for 30 minutes
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at room temperature. For type II collagen, sections were incubated with rabbit anti-human
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type II collagen antibody (1:1000 in dilution; ab34172, Abcam) at 4°C overnight, then
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with secondary antibody of biotinylated goat anti-rabbit IgG (1:200 in dilution; Vector
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Laboratories) for 30 minutes at room temperature. Immunostaining was detected with the
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Vectastain ABC reagent (Vector Laboratories) followed by diaminobenzidine staining.
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The sections were counterstained with hematoxylin.
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Transmission Electron Microscopy
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In order to examine the morphological difference of cell and extracellular matrix
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among each zone of intact LM, a transmission electron microscope (TEM) was used. The
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specimens of LM were fixed in 2.5% glutaraldehyde in 0.1M phosphate buffered saline
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(PBS) for 2 h. The specimens were washed overnight at 4°C in the same buffer and post-
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fixed with 1% OsO4 buffered with 0.1 M PBS for 2 h. The specimens were then
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dehydrated in a graded series of ethanol and embedded in Epon 812. Ultrathin sections
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(90 nm) were collected on copper grids, double-stained with uranyl acetate and lead
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citrate, and then observed with TEM (H-7100; Hitachi, Tokyo, Japan)
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Measurement of Sulfated Glycosaminoglycan (GAG) and Hydroxyproline (Collagen)
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Concentration
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The posterior part of intact LM was divided into zones in the sagittal direction
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in order to correspond to the T1rho analysis. Two slices of the central and lateral slice
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were examined in each meniscus (Fig. 2A, Fig. 3A). Each meniscus was digested for 24
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h at 60°C in papain buffer (200 mg/ml papain [Sigma-Aldrich, St. Louis, MO]) The GAG
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concentration of supernatant was determined by the Blyscan-assay (Biocolor Ltd, County
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Antrim, UK) according to the manufacturer’s instructions. For hydroxyproline content,
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dissolved sample solution was added to the same volume of 12N concentrated HCl (Wako,
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Osaka, Japan) and hydrolyzed at 120°C for 3 h. Collagen concentration of hydrolyzed
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samples was estimated using the Hydroxyproline Colorimetric Assay Kit (Biovision Corp,
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Milpitas, CA) according to the manufacturer’s instructions.
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