Collagen-derived dipeptide Pro-Hyp administration accelerates muscle regenerative healing accompanied by less scarring after wounding on the abdominal wall in mice

Collagens act as cellular scaffolds in extracellular matrixes, and their breakdown products may also have important biological functions. We hypothesize that collagen dipeptide Pro-Hyp induces favorable healing activities and examined the effects of Pro-Hyp administered via different routes on wound healing using our novel murine model, in which an advanced fibrosis-prone scar lesion was developed in the abdominal muscle wall under the skin. After excising a part of the abdominal wall, a free-drinking experiment was performed using solutions with casein (CS), high molecular weight collagen peptides (HP), and low molecular weight collagen peptides including Pro-Hyp and Hyp-Gly (LP), in addition to water (HO). On day 21 of the study, when compared to the HO and CS groups, muscle regeneration in the LP group was significantly advanced in the granulation tissue, which was associated with a decrease in fibrosis. To clarify the effects of Pro-Hyp, daily intraperitoneal administration of pure Pro-Hyp was performed. Pro-Hyp administration induced many myogenically differentiated cells, including myogenin-positive myoblasts and myoglobin-positive myocytes, to migrate in the granulation tissue, while scar tissue decreased. These results indicated that Pro-Hyp administration accelerates muscle regenerative healing accompanied by less scarring after wounding on the abdominal wall.

Granulation tissue and muscle regeneration. On day 21, granulation tissue was developed in the excised lesion between the stumped abdominal muscle wall, and histological analysis was performed. The following parameters were analyzed in the cross-sectional lesion area, fibrosis area using MT stain, myogenin-positive myoblast number, and myoglobin-positive regenerative myocyte area. No differences in the lesion area were found among the groups ( Fig. 2A, upper left), however, the fibrosis area was significantly smaller in the LP group than in the CS group ( Fig. 2A, upper right; Fig. 2B). In muscle regenerative responses, myoblast number tended to increase in the LP group (p < 0.1 vs. CS) ( Fig. 2A, lower left), but regenerative muscle area in the granulation tissue was significantly increased in the LP group ( Fig. 2A, lower right), which was followed by the HP group. Representative morphological alterations are shown in Fig. 2B.

Pro-Hyp intraperitoneal injection experiment.
Pro-Hyp appearance in the blood after its i.p. injection. Pro-Hyp appeared at its maximum level in the blood 30 min after a single i.p. injection of Pro-Hyp (500 nmol/200 μL) to the mice and then decreased significantly 1 h after the injection (Fig. S2). After 3 h, the level returned to its untreated level.
Body weight and hematological alterations. After abdominal muscle wall excision in mice, daily i.p. injection of Pro-Hyp (group PH) or saline control (group SC) was continued for 21 days. No changes were found in body weight, WBC, RBC, and platelet in the blood among the groups (Table S1). www.nature.com/scientificreports/ Lesion area and fibrosis. During the study, tissue samples were obtained on days 7, 14, and 21. Granulation tissue was developed in the lesion between the stumped abdominal muscle wall under the skin (Fig. 3A), and using MT stain, the total lesion area of the granulation tissue and fibrosis area in cross-section was measured. Lesion areas in both groups similarly changed, but a temporal expansion was seen on day 14, however, no differences were noted between the groups (Fig. 3B, left). On the other hand, fibrosis increased time-dependently in both groups, and in the SC group, it reached the maximum level early on day 14. However, fibrosis level was significantly lower in group PH than group SC after 14 days of the study (Fig. 3B, right).
Histological characteristics of muscle regeneration. Muscle regeneration after muscle injury may be initiated by inflammation, growth factors, and cytokines. In this study, TGF-β involving pathways were histologically examined in the granulation tissue on day 21. Inflammatory cell infiltrations including neutrophils (data not shown) and macrophages (F4/80) were scarcely found in the granulation tissue in both groups (Fig. 4A), however, p-Smad3 expression increased over time (Fig. 4A). This phenomenon was detected in both the muscle and granulation tissue (Fig. 4B), with more positive cells in the granulation tissue. Thus, p-Smad3 expression in the granulation tissue was examined. Although all examined cells in the tissue were positive for Smad3 in nuclei, the cells positive for p-Smad3 increased significantly in the Pro-Hyp group compared to the control group on day 21 (Fig. 4C).
Fibrosis and muscle regeneration. After abdominal muscle wall excision, granulation tissues were shown to consist of collagen-rich-fibrous elements and muscle regeneration. Among them, representative two different lesions on day 21 from different groups at the muscle-stumped area are shown in Fig. 5A; myogenic regenerative cells migrated from the stumped edge of muscle tissue into the granulation tissue, but when fibrous tissue was formed ahead of the stumped edge, regenerative muscle cell migration was strictly prohibited. We thus analyzed the relationship between fibrosis and myocyte regeneration using mice in both groups on day 21. There is a significant negative correlation between the two parameters ( Fig. 5B).

Figure 1.
The presence of collagen-derived peptides in the blood after consuming the different peptides. Mice with the abdominal surgery were fed a normal diet and consumed just water (HO) or water that contained milkderived casein (CS), high molecular weight collagen peptides (HP), and low molecular weight collagen peptides (LP) for 21 days. After the study was completed, collagen peptides in the plasma were measured using a LC-MS/ MS technique. Higher contents of peptides were detected in the following groups Hyp-Gly, Pro-Hyp, Gly-Pro-Hyp, and Pro-Hyp-Gly. The comparison was done between the levels on day 0 and day 21 in the same group and between the groups on day 21. N = 5 or 6/group, Mean ± SE, # p > 0.1, *p < 0.05. www.nature.com/scientificreports/ Myogenic differentiation in granulation tissues. Figure 6A shows histological alterations in the granulation tissue formed around the stumped muscle tissue on day 14 in the Pro-Hyp group. Fibrosis progressed around the stumped area, but myoglobin-positive muscle regeneration and migration also started from the stumped edge, while myogenin-positive myoblasts were found to scatter near and far from the stumped lesion. αSMA-positive myofibroblasts were densely distributed over the granulation tissue, but not in the muscle-stumped area. CD-31-positive neovessels were densely distributed around the stumped area. To confirm muscle regeneration in the granulation tissue, electron microscopy was performed. Myotubes forming multinuclear cells in an envelope were found (Fig. 6B, left), and elongated myofibers (Fig. 6B, meddle) and large matured muscle fibers (Fig. 6B, right) were also detected in the granulation tissue, but no facia was found.
Pro-Hyp administration and muscle regeneration. The effects of Pro-Hyp on muscle regeneration were analyzed. No statistical differences were found in myoblasts number during the study in groups SC and PH, but the highest level was noted in group PH on day 21 (Fig. 7A, left). Regenerative muscles shown by myoglobin expression significantly increased on day 21 in group PH (Fig. 7A, right). The highlighted images of granulation tissue in the two groups are quite different (Fig. 7B), showing that muscle regeneration is limited in the enormous fibrous lesion detected in group SC, however, regenerated muscle cells spread to all areas of the granulation tissue in group PH. Whereas, no differences in p-Smad3 expression were found between groups PH and SC during the study (data not shown).

Discussion
It has been known that scar lesions are hardly developed in mice. Our established abdominal muscle-wall excision model, develops fibrosis-prone scar lesions under an in situ tensile force on the abdomen. After 21 days of the drinking study, in granulation tissue, group LP had dominant muscle regeneration with lesser fibrosis among the groups. Although the original level of dipeptides, including Pro-Hyp and Hyp-Gly, in the collagen peptides in the group HP was about 1/30 lower than the group LP, the blood levels of Pro-Hyp after the study showed similar levels in both groups. This discrepancy could be due to Hyp-containing peptides' resistance against intracellular www.nature.com/scientificreports/ hydrolysis 24 ; it can be therefore speculated that Pro-Hyp and Hyp-Gly were accumulated in the blood even in the mice that consumed HP peptides during the study. Regarding the relocation of administered peptides to the wound lesion, it has been addressed that when Pro-Hyp and Hyp-Gly are orally administrated, Pro-Hyp alone selectively accumulated in the inflamed lesion 25 . Moreover, our previous results showed that Pro-Hyp alone, but not Hyp-Gly, is greatly generated during the development of the granulation tissue 15 , we thus focused on Pro-Hyp, and afterward, daily i.p. injection of pure Pro-Hyp was next performed using the abdominal mouse model. However, it is still possible that low molecule weight collagen peptides other than Pro-Hyp may have a favorable effect on the abdominal wall wound healing.
In the abdominal muscle wall excision model, fibrosis and muscle regeneration simultaneously took place in the granulation tissue. These two pathological changes are contradicted and thus the healing outcome was impacted. In wound healing, granulation tissue formation with collagen matrix is an essential event to reconstitute the defected tissues and provide an emergent scaffold for cell migration and replication. However, if exceeded, lesions would be overwhelmed by collagen-rich fibrous tissues, leading to scar-prone lesions, which may affect subsequent regenerative healings by preventing cellular activities, including neovascularization and stem cell differentiation and proliferation 1,2 . Such morphological transformation in the granulation tissue was evidenced in this study. These alternative changes may affect clinical outcomes in post-surgery patients by forming an unfunctional hypertrophic scars-like collagenous lesion, not only in the skin but also in other organs.
Scar tissues developed in the model may be due to tensile force on the abdominal muscle wall. It has been shown that mechanical stresses alter not only cellular shape and structure but also function 4,5 . It is well established that the pattern of the developed scar lesions in keloid patients is physically followed by the direction of the tensile force on the skin, forming a distinct shape associated with excess fibrosis 2 . Mechanical stresses stimulate and transduce as an intracellular signal, where there are many possible pathways, such as TGF-β/Smad, integrins, MAPK/G-protein, TNF-α/NF-κB, Wnt/β-catenin, interleukin, and calcium-ion channel, by which gene transcriptions for the development of fibrous lesions could progress 26 .
When the skeletal muscle is damaged, various growth factors for muscle repair are involved, including but not limited to, HGF, FGFs, IGFs, PDGF, and TGF-β family 27 . In the process of muscle regeneration after injury, satellite cells in the muscular fasciae start to proliferate and differentiate into myoblasts, and myoblasts fuse with each other, forming myotubes, and they are furthermore maturated into muscle fibers, i.e., myocytes 11,28 . In our study, these cell types were all detected in the granulation tissue that was shown by using morphological and immunohistochemical techniques. This cellular differentiation may be initiated by the factors from not only traumatic tissue damage but also a tensile force on the muscle tissue 29 . Among many possible factors, we focused www.nature.com/scientificreports/ on the TGF-β/Smad pathway 30 . Although inflammatory cells including macrophages and TGF-β1-possessing cells did not appear in the granulation tissue during the study, p-Smad3 time-dependently increased not only around the stumped muscle tissue but also in the granulation tissue. These results indicate that phosphorylation of Smad3 may occur in the tissues under stimulation of tensile force even in the absence of TGF-β1, and induce progression of fibrosis with collagen deposition, and also activate αSMA expression in myofibroblasts. A randomized double-blind placebo-controlled study using 120 patients with pressure ulcers indicated that collagen-derived peptides, especially hydrolysates accelerate wound healing reactions 17 . Furthermore, in animal experiments, a diet with Pro-Hyp and Hyp-Gly ameliorate skin barrier defects against skin drying using a mouse model for skin barrier dysfunction 31 . Interestingly, in the skin tissue obtained from the treated mice, up-regulated genes were preferably found in the skeletal muscle-related genes 31 , which could be derived from the cutaneous muscle in the mouse skin, but they unfortunately could not elucidate the phenomenon at that moment. In the present study, Pro-Hyp administration accelerated muscle regeneration after excision of a part of the abdominal muscle wall in mice. These findings strongly support that Pro-Hyp may at a certain point be involved in the myogenic differentiation during the muscle regeneration process.
Intracellular mechanisms of Pro-Hyp effects on cell differentiation are still unclear. However, some mechanisms have been reported; Pro-Hyp induces differentiation in osteoblasts 19 , in which Pro-Hyp binds to Foxg1 and increases Runx2 expression. Runx2 and Smad form a transcriptional complex. In a recent study (2021) 20 , it was shown that Pro-Hyp promotes differentiation of tendon cells with modulation of lineage-specific factors and induces chemotactic activity, proliferation, and type I collagen-network organization. In our recent study using primary fibroblasts derived from mouse skin tissue 32 , Pro-Hyp selectively stimulates migration and proliferation of fibroblasts, which express p75 neurotrophin receptor (p75 NTR ) when cultured on collagen gels. Moreover, nerve growth factor and its receptor p75 NTR in myoblasts act for myogenesis and myoprotection when they are damaged 33 . More exact intracellular mechanisms of Pro-Hyp roles especially in myogenic regenerative healing accompanied with reduced fibrosis should be physiologically defined in the future.

Conclusion
The effects of Pro-Hyp were examined using a unique abdominal wound healing mouse model, in which fibrous scarring and myogenic regeneration simultaneously occurred in the granulation tissue formed in the abdominal muscle wall. Pro-Hyp administration, which had no side effects at all, strongly induced migration and differentiation of myogenin-positive myoblasts and myoglobin-positive myocytes in the granulation tissue, but fibrous scar tissue formation was suppressed (Fig. 8). Therefore, it could be effective to administer Pro-Hyp to patients with bedsores, who underwent surgery, or have trauma to activate the proper wound healing process with less scarring. However, additional preclinical studies should be performed.
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  www.nature.com/scientificreports/ 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).
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). 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-assistedsoftware 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. www.nature.com/scientificreports/