Histopathological Study on Collagen in Full-Thickness Wound Healing in Fraser's Dolphins (Lagenodelphis hosei)

Abstract

Fraser's dolphins (Lagenodelphis hosei) possess great healing abilities. Their skin composition can be restored after wounding, including collagen spacing, orientation, and bundle thickness. However, it remains unclear how collagens are involved in the wound-healing process and eventually regain normality in Fraser's dolphins. Learned from the other two scarless healing animals, changes in type III/I collagen composition are believed to modulate the wound healing process and influence the scarring or scarless fate determination in human fetal skin and spiny mouse skin. In the current study, Herovici's, trichrome, and immunofluorescence staining were used on normal and wounded skin samples in Fraser's dolphins. The results suggested that type I collagens were the main type of collagens in the normal skin of Fraser's dolphins, while type III collagens were barely seen. During the wound healing process, type III collagens showed at early wound healing stages, and type I collagen increased in the mature healed wound. In an early healed wound, collagens were organized in a parallel manner, showing a transient hypertrophic-like scar, and eventually restored to normal collagen configuration and adipocyte distribution in the mature healed wound. The remarkable ability to remove excessive collagens merits further investigation to provide new insights into clinical wound management.

Keywords: Fraser’s dolphins; type I collagen; type III collagen; wound healing.

Figure 1

Overview of the epidermis, dermis, and blubber in normal skin of a sub-adult male Fraser’s dolphin (a). The blue color in trichrome staining represented collagen fibrils mainly located in the papillary dermis (PD) and reticular dermis (RD) and less in the blubber layer (B). The cytoplasm in the epidermis (Epi) is stained red, including stratum externum (SE), stratum spinosum (SS), and rete ridges (RR). Magnified figures showed (b) reticular dermis, (c) upper blubber layer, and (d) lower blubber layer. Dermis (Der). Hypodermis (Hy). Scale bars in (a) = 1 mm; in (b–d) = 100 μm.

Figure 2

Differential staining of type I and type III collagens in normal skin of Fraser’s dolphins. (a) In Herovici’s staining, collagen fibers in the papillary dermis, reticular dermis, and blubber of normal skin were stained fuchsia to red, with no blue fiber observed. (b–e): Immunohistochemical staining. (b) Type I collagen signals (brown) existed in the papillary dermis, reticular dermis, and whole blubber layer. (c) Type III collagen signals were barely visible. (d) Negative control of type III collagen. (e) Liver tissue with fibrotic lesions served as a positive control for type III collagen. Scale bars = 100 μm.

Figure 3

Collagen distribution during wound healing in Fraser’s dolphins. The definition of the wound healing stage is based on [21]. Open healing wound, early stage 3 (a–c); nearly closed wound, late stage 3 (d–f); immature healed wound, stage 4 (g–i) were stained with trichrome to compare collagen distribution. The blue color in trichrome staining indicates collagen fibril, and the red color represents cytoplasm in the epidermis and muscle tissue underlying blubber. Scale bars = 1 mm.

Figure 4

Differential staining of type I and type III collagen in stage 3 wound in Fraser’s dolphins. Left panel: low power view of stage 3 wound in Herovici’s staining. Particular regions: (a) The middle layer of granulation tissue; (b) the bottom layer of granulation tissue; (c) the reticular dermis at the wound adjacent are enlarged in the right panel. Right panel: differential staining of type I (red) and type III (blue) collagen with Herovici’s staining and IHC staining (brown). Scale bars = 50 μm.

Figure 5

Differential staining of type I and type III collagens in the healed wound and unwounded skin in Fraser’s dolphins. In Herovici’s staining, the collagen fibers in the reticular dermis of the stage 4 wound (immature healed wound) were stained purple to fuchsia (a), whereas those in the stage 5 wound (mature healed wound) and the unwounded skin were stained fuchsia to red (b,c). Scale bars = 100 μm.

Figure 6

Illustrative diagrams of collagen type III/I ratio in animal skin. (a) Comparison of collagen type III/I ratio in scarring or scarless healing animals. In humans, the collagen type III/I ratio is higher in fetus skin than in adult skin, both in normal skin and during wound healing [25,26]. In mice, the collagen type III/I ratio in spiny mice in the early wound healing stages is higher than in the laboratory mouse [22,24]. (b) Changes in collagen composition during wound healing of stages 3 (healing wound), 4 (immature healed wound), and 5 (mature healed wound). Red fibril, type I collagen; blue fibril, type III collagen, yellow circle, adipocytes. These schematic diagrams, not drawn to scale, represented the epidermis and upper reticular dermis in different animals.