Materials and chemicals
Collagen peptides manufactured by Nitta Gelatin Inc. Osaka, Japan were used; higher molecular weight collagen peptides (HP): fish collagen peptides (average molecular weight 5000–7000 Da), lower molecular weight collagen peptides (LP): fish collagen peptides (average molecular weight < 1000 Da). Dipeptide content including Pro-Hyp and Hyp-Gly was about 0.1 g/kg in the HP and 3 g/kg in the LP, respectively. Pro-Hyp was purchased from BACHEM (Purity > 98%; Bubendorf, Switzerland). The other analytical grade chemicals were purchased from Sigma-Aldrich Japan Inc. (Tokyo, Japan) and FUJIFILM Wako Chemicals U.S.A. Co. (Osaka, Japan).
Chemicals for LC–MS/MS
Methanol (high-performance liquid chromatography grade), ammonium acetate (high-performance liquid chromatography grade), and formic acid (high-performance liquid chromatography grade) were purchased from FUJIFILM Wako Chemicals U.S.A. Co. Ammonium bicarbonate was purchased from Sigma-Aldrich Japan Inc. Pro-Hyp and Hyp-Gly were purchased from Bachem (Bubendort, Germany). Standards prepared for Pro-Hyp and Hyp-Gly were dissolved in water, mixed, and diluted to 1 nmol/L, 10 nmol/L, 100 nmol/L, 1,000 nmol/L, and 10,000 nmol/L with 50 mM ammonium bicarbonate buffer. After filtration with a 0.2-mm regenerated cellulose filter (RC4, Sartorius Japan, Tokyo, Japan), 1 mL of the resulting filtrate was used into the LC–MS/MS system.
Sample preparation for LC–MS/MS
The plasma was deproteinized by adding threefold amounts of 100% (w/v) ethanol. The supernatant was then centrifuged at 13,000 rpm for 10 min at 4 °C. The supernatant was diluted tenfold with 50 mM ammonium bicarbonate buffer. After filtering through a 0.2-mm regenerated cellulose filter, the resulting filtrate was injected into the LC–MS/MS system.
LC–MS/MS analysis
Samples were analyzed by LC–MS/MS. The LC analysis was performed using an ACQITY UPLC H-class Biosystem (Waters, Milford, MA, USA). The MS/MS analysis was performed using a Xevo TQ-XS system (Waters, Milford, MA, USA). Hypersil GOLD PFP column (2.1 × 150 mm, 5 μm, Thermo Fisher Scientific Inc., Tokyo, Japan) was used for separation. Gradient elution was carried out: A buffer with 0.2% (v/v) formic acid and 2 mM ammonium acetate and B buffer with 100% methanol. The gradient profile with the following proportions (v/v) of B buffer was applied (t (min), % B buffer, flow rate): (0 min, 2%, 200 µL/min), (3.50 min, 2%, 200 µL/min), (3.51 min, 95%, 400 µL/min), (7.00 min, 95%, 400 µL/min), (7.10 min, 2%, 200 µL/min) (10 min: time was required to reach initial conditions). The column temperature was maintained at 40 °C. The tandem quadrupole mass spectrometer was used in positive ion electrospray mode. The ion source was operated at 150 °C with a capillary voltage of 1.5 kV. Nitrogen was employed for the desolvation gas at 500 °C. The mode of acquisition was multiple reaction monitoring (MRM) at an argon collision gas pressure of 3.5–3.8 × 10–3 mbar. The list of peptides and the MRM transitions and collision energies for the method are presented below. The data were acquired using MassLynx Software version 4.2 (Waters, Milford, MA, USA).
| MRM (m/z, precursor > product) | Collision energies | |
|---|---|---|
| Pro-Hyp | 229 > 132 | 15 |
| Hyp-Gly | 189 > 86 | 15 |
Animals
Animal experiments were conducted with the approval of the Fukuoka University Animal Experiment Committee (No. 1812097) on December 18, 2018, and study protocols complied with the institution’s animal care guidelines. Eight to 10-week-old C57BL/6 female mice (Japan SLC Inc., Shizuoka, Japan) were used. All procedures were conducted under aseptic conditions, using autoclaves, ethylene oxide gas, 70% ethanol, and povidone-iodine. Mice were anesthetized with pentobarbital (Somnopentyl; KYORITU SEIYAKU, Tokyo, Japan). Hematological analyses, including red blood cell (RBC), and white blood cell (WBC) counts (Celltac-α, NIHON KOHDEN, Tokyo, Japan) were done in the blood drawn from the eyelids using a heparinized 75-µL capillary (Hirschmann Laborgeräte GmbH & CO., Eberstadt, Germany).
Abdominal muscle wall excision model
The model was established; In brief, after removing hair on the abdomen, mice under general anesthesia were set down in a supine position, and a 1.5 cm line alongside the midline in the abdominal skin was marked. After applying iodine to the abdominal area, the skin was resected along the marked line. After separating the skin around the cut line from the abdominal muscle wall with scissors, the fascia at the umbilicus spot was pulled up and a needle with a suture was inserted. The tissue was then lifted with the thread and the spot, where the suture had been inserted, was grasped with round-top tweezers. The tissue was cut away around the circular edge of the tweezers, and the amounts of excised tissue were all set in more than 6 mg. The opened skin was placed together and sutured at upper and lower positions. The abdominal area of the wound was covered with a film dressing (Tegaderm; SUMITOMO 3 M, Tokyo, Japan). After the surgery, a silicon vest was placed on the mice, and the vests were kept on the mice for 1 week to complete surficial skin healing. After 1 week, the vest was removed.
Pro-Hyp administration
Drinking experiment
Different molecular weight collagen peptides HP and LP, and milk-derived Casein (CS) (Sigma-Aldrich Japan, Tokyo, Japan) were dissolved at a concentration of 8% in water and autoclaved. The solutions and water alone (HO) were given to mice without restriction. Each group had 5 or 6 mice and 2 or 3 mice were housed in the same cage with a normal diet. The amount of solution and body weight were monitored every other day during the 21 days of the study. On day 21, blood was obtained for the quantitative analysis of peptides using the LC–MS/MS method. Thereafter, the mice were sacrificed, and the abdominal tissue was harvested and photographed with a digital camera (NEX-C3; SONY Co., Tokyo, Japan) and used for histological examination.
Intrapreneurial administration experiment
Intraperitoneal injections of 500 nmol per 200 µL Pro-Hyp (group PH) or 200 µL saline (group SC) were administered daily. On days 7, 14, and 21, blood was obtained for the general hematological analysis, and the blood on day 21 was used for the quantitative analysis of peptides using the LC–MS/MS method. Thereafter, the mice were sacrificed, and the abdominal tissue was harvested and photographed with a digital camera (NEX-C3; SONY Co., Tokyo, Japan) and used for histological examination. Seven mice were used in each group in different time points. In some analyses, the plasma level of Pro-Hyp after a single i.p. injection of Pro-Hyp was monitored for 7 days (n = 3). Saline injection was used as a control (n = 3).
Histological examination
The tissue was fixed in 5% buffered formaldehyde (pH7.4) for several days. Two pathological tissue samples from the wound were excised and embedded in paraffin blocks using a tissue processor (ASP200S; LEICA Biosystems, Nussloch, Germany). Four-µm-thick tissue sections were cut with a microtome (LEICA RM2235; LEICA Biosystems).
Histological staining
Tissue sections were stained with hematoxylin and eosin (HE) and Masson’s trichrome (MT) stains. For immunohistological examination, the following primary antibodies were used: rat anti-NIMP-R14, a Ly-6G/-6C neutrophil marker (1:400; Hycult Biotech, Netherlands), rat anti-CD31 (1:30; Dianova GmbH, Hamburg, Germany), rat anti-F4/80 (1:200; Abcam plc), rabbit anti-TGF-β1 (1:800; Abcam plc) rabbit anti-αSMA (1:2000; Abcam plc), rabbit anti-Smad3 (1:1000; Gene Tex Inc., CA, USA), rabbit anti-phosphorylated Smad3 (p-Smad3) (1:8000; Rockland Immunochemicals Inc., Limeric, PA), rabbit anti-myoglobin (1:1000; Abcam plc), and rabbit anti-myogenin (1:200; Abcam plc). All were diluted with Antibody Diluent (DAKO Japan Inc., Tokyo, Japan). For the CD31, TGF-β1, myoglobin, and myogenin antibodies, antigens were activated by heating in 0.01 M citrate buffer (pH 6.0) for 5 min. Sections were incubated overnight with the primary antibody at 4 °C. The immunobound antibodies were detected using biotin-conjugated secondary antibodies in the N-Histofine Simple Stain Mouse MAX PO (Rat) detection system (Nichirei Biosciences Inc., Tokyo, Japan) and the EnVision kit (DAKO Japan Inc.), and the sections were developed by 3,3′-diaminobenzidine stain and counterstained with hematoxylin. Stained sections were observed under a microscope (Bio-Zero and BZ-X800; KEYENCE Co.).
Morphometrical analysis for granulation tissue
Total lesion area: Using histological pictures stained by HE and MT stains, the area of granulation tissue developed between the abdominal muscle wall, including fibrosis and regenerative and migrated muscle cells, was morphometrically measured with a computer-assisted-software BZ-II (Keyence).
Fibrosis area: representative lesions in MT stain were taken using 10-time lens in the Bio-Zero (Keyence), and the blue staining collagenous fibers were highlighted, binarized, and measured (VH-analyzer: Keyence, Osaka, Japan), and the percentage of collagen distribution in the granulation tissue was calculated.
Myoblast number: Myogenin-stained granulation tissue was taken using a 10-time lens, and the positive spots in the area of granulation tissue were calculated.
Regenerated myocyte area: Myoglobin-stained granulation tissue was taken using a 10-time lens, and the positive area of regenerative muscles in the granulation area was calculated.
Positive cell number for p-Smad3: Cells with a positive nucleus for p-Smad3 were counted out of the cells in tissue photographed with a 20-time lens. Examined tissues were of the granulation tissue and the stumped muscle (Fig. 7B).
Statistical analysis
Numerical results are presented as the mean ± standard error (SE) or standard deviation (SD). Two groups were compared using a Student’s t-test, and a linear regression analysis was performed to compare the two values. P values less than 0.05 were considered significant. P values less than 0.05 and 0.01 are indicated by the asterisk (*) and (**) in the graphs, respectively.