Qualitative study on diabetic cutaneous wound healing with radiation crosslinked bilayer collagen scaffold in rat model

Abstract

Diabetes may leave patients more prone to skin problems, and minor skin conditions can more easily turn into serious damage to the extracellular matrix, which further impairs the skin's mechanical properties and delays wound healing. Therefore, the aim of the work is to develop extracellular matrix substitution to remodel the mechanical properties of diabetic cutaneous wound and thus accelerate diabetic wound healing. A green fabrication approach was used to prepare radiation crosslinked bilayer collagen scaffold from collagen dispersion. The morphological, mechanical and swelling characteristics of radiation crosslinked bilayer collagen scaffold were assessed to be suitable for cutaneous wound remodeling. The feasibility of radiation crosslinked bilayer collagen scaffold was performed on full-skin defect of streptozotocin-induced diabetic rats. The tissue specimens were harvested after 7, 14, and 21 days. Histopathological analysis showed that radiation crosslinked bilayer collagen scaffold has beneficial effects on inducing skin regeneration and remodeling in diabetic rats. In addition, immunohistochemical staining further revealed that the radiation crosslinked bilayer collagen scaffold could not only significantly accelerate the diabetic wound healing, but also promote angiogenesis factor (CD31) production. Vascularization was observed as early as day 7. The work expands the therapeutic ideas for cutaneous wound healing in diabetes.

Figure 1

Schematic illustration of the preparation progress of radiation crosslinked bilayer collagen scaffold. (A) The preparation process of radiation crosslinked bilayer collagen scaffold. (a) Extraction. (b) Mixture. (c) Radiation. (d) Combination. (B) The proposed mechanisms of crosslinking for collagen under radiation. (a) Water radiolysis. (b) Chain-transfer. (c) Cross-linking. (*) Representative segment.

Figure 2

Physico-chemical characterization of radiation crosslinked bilayer collagen scaffold (rcBCS). (A) Scanning electron microscopy observation of the microstructures of silicone membrane (*) with liquid silicone. (B) Scanning electron microscopy observation of cross-section structure of rcBCS. (C) The distribution of pore sizes of rcBCS. (D) Tensile stress at maximum load of rcBCS relative to collagen scaffold (CS). (E) Compressive stress at maximum load of rcBCS relative to CS. Data are expressed as mean ± SD. (F) Swelling ratio of the rcBCS and CS shows high swelling capacity.

Figure 3

Wound healing in vivo in STZ-induced diabetic rat. (A) Schematic illustration of 20 mm diameter and full-thickness wounds created at either side of the dorsal central line. (B) Representative photographs of visual appearance of wound excised on rat on days 0, 3, 7, 14 and 21; (C) Representative traces of wound-bed closure during 21 days. (D) Monitoring of blood glucose level at varied time points. (Treatment = rcBCS group, Control = sham operation group).

Figure 4

Representative images of H&E staining. (A, B, C represent rcBCS group, and D, E, F represent sham operation group; 1 represents panorama image, 2 represents epidermis image, 3 represent dermis image. Blood vessels: black arrow; Inflammatory cell: *; The blue lines indicate the support force of bilayer dermal equivalent; The red lines indicate the skin contraction force; The areas inside the red dashed lines indicate granulation tissue. Bar = 200 μm).

Figure 5

Representative images of the Masson’s trichrome staining on the day 7 and 14 (A, B represent rcBCS group and C, D represent sham operation group; Bar in 1 = 500 μm, bar in 2 = 50 μm).

Figure 6

Representative immunohistochemical staining images of CD31 on (A) dermis and (B) epidermal layer on days 7, 14 and 21 in rcBCS group.

Figure 7

The possible cellular mechanisms involved in diabetic wound healing with treatment of rcBCS. (A) Hemostasis ability due to the high swelling ratio. (B) Facilitating vascularization and providing mechanical support. (C) Antibacterial ability due to silicone membrane. (D) In comparison with impaired healing in diabetic wound (right), the healing ability is improved for the treatment of rcBCS (left). (a) Keratinocyte migration. (b) Fibroblast proliferation and attachment. (c) Collagen deposition. (d) Endothelial cell migration. (e) Inflammation. (f) Neutrophil removed. (E) The temporal sequence of overlapping processes involved in the healing of diabetes wound with and without the treatment of rcBCS. (F) The interaction of rcBCS with cells during diabetes healing processes. (a) Hemostasis. (b) Inflammation. (c) Proliferation. (1) advancing epithelial layer. (2) collagen deposition. (3) increased fibroblast activity. (4) new vessel. (5) degradation of rcBCS. (d) Remodeling. (1) reconstruction of the ECM. (2) superficial scar. (3) rich in blood vessels.