The sustained release of basic fibroblast growth factor accelerates angiogenesis and the engraftment of the inactivated dermis by high hydrostatic pressure

Abstract

We developed a novel skin regeneration therapy combining nevus tissue inactivated by high hydrostatic pressure (HHP) in the reconstruction of the dermis with a cultured epidermal autograft (CEA). The issue with this treatment is the unstable survival of CEA on the inactivated dermis. In this study, we applied collagen/gelatin sponge (CGS), which can sustain the release of basic fibroblast growth factor (bFGF), to the inactivated skin in order to accelerate angiogenesis. Murine skin grafts from C57BL6J/Jcl mice (8 mm in diameter) were prepared, inactivated by HHP and cryopreserved. One month later, the grafts were transplanted subcutaneously onto the back of other mice and covered by CGS impregnated with saline or bFGF. Grafts were harvested after one, two and eight weeks, at which point the engraftment was evaluated through the histology and angiogenesis-related gene expressions were determined by real-time polymerase chain reaction. Histological sections showed that the dermal cellular density and newly formed capillaries in the bFGF group were significantly higher than in the control group. The relative expression of FGF-2, PDGF-A and VEGF-A genes in the bFGF group was significantly higher than in the control group at Week 1. This study suggested that the angiogenesis into grafts was accelerated, which might improve the engraftment of inactivated dermis in combination with the sustained release of bFGF by CGSs.

Fig 1. Micrographs of the inactivated skin grafts by HHP and photographs of grafting procedures.

(A) A micrograph of murine skin before HHP, (B) a micrograph of murine skin just after HHP, (C) a micrograph of murine skin after 4 weeks cryopreservation; the blue broken lines indicate the dermis of grafts, (D) a gross photo of the inactivated graft position, (E) gross photos of the grafts covered by NSS- or bFGF-impregnated (sandwich-technique) CGSs. Magnification: 20x, scale bar: 100 μm.

Fig 2. The infiltration of recipient cells into inactivated dermis after implantation.

(A) Micrographs of HE-stained sections of skin grafts at Weeks 1 and 2 after implantation. The original dermal cell remnants were absorbed, and the epidermis indicated by black arrowheads was detached from the papillary dermis at Week 1. Cells from recipients showed infiltration to the dermis, and the dermal cellular density increased over the time. No inflammation reactions or dermal collagen fiber degeneration was observed. The blue broken lines indicate the dermis of grafts, CGS: collagen gelatin sponge after implantation, SC: subcutaneous layer; magnification: 10x, scale bar: 100 μm. (B) The diagram shows significant differences in the dermal cellular density of the bFGF group between Weeks 1 and 2 (*** p < 0.001); the dermal cellular density of the bFGF group at Week 2 was significantly higher than that in the NSS group (** p < 0.01).

Fig 3. The infiltration of recipient fibroblasts into inactivated dermis after implantation.

(A) Micrographs of anti-vimentin-stained sections differentiating fibroblasts from other cells at Weeks 1 and 2 after implantation. Recipient fibroblasts began to infiltrate the grafts at Week 1 and increased at Week 2. ↙: fibroblast; magnification: 20x, scale bar: 100 μm. (B) A comparison of the dermal fibroblast density of the grafts; a significant increase in the number of fibroblasts in the bFGF group was noted between Weeks 1 and 2 as well as between the bFGF and NSS groups in Week 2 (*** p < 0.001).

Fig 4. The comparison of the angiogenesis-related genes expression at Weeks 1 and 2 after implantation.

The relative expressions of FGF-2, PDGF-A and VEGF-A were strongly up-regulated in the bFGF group at Week 1 (** p < 0.01, *** p < 0.001). The expression of FGF-2 was lower in the bFGF group than in the NSS group (** p < 0.01), while that of PDGF-A was higher in the bFGF group than in the NSS group (* p < 0.05).

Fig 5. Evaluation of newly formed capillaries after implantation.

(A) Micrographs of immunohistochemical staining of anti-CD31 at Weeks 1 and 2. Few capillaries were observed at Week 1 in both groups. The capillaries increased in number and size at Week 2 in both groups. ▲: newly formed capillary; magnification: 20x, scale bar: 100 μm. (B) A comparison of the number of newly formed capillaries in the grafts. The number of capillaries significantly increased from Weeks 1 to 2 in both groups. The number of capillaries in the bFGF group was significantly higher than that in the NSS group at Weeks 1 and 2 (* p < 0.05, ** p < 0.01, *** p < 0.001). (C) A comparison of the newly formed capillaries area in the grafts. The area of the capillaries also significantly increased from Weeks 1 to 2 in both groups. The number of capillaries in the bFGF group was significantly higher than that in the NSS group at Weeks 1 and 2 (* p < 0.05, ** p < 0.01).

Fig 6. The assessment of dermal thickness and area after 8 weeks implantation.

(A) A micrograph of the Azan staining at Week 8. The areas of the grafts are indicated by broken black lines. No signs of infection or necrosis were observed in either group. The yellow broken line indicates the evaluated thickness of the grafts, M: recipients’ muscle; Magnification: 2x, scale bar: 1 mm. (B) A comparison of the thickness of the grafts between the two groups at Week 8. The grafts in the bFGF group were significantly thicker than those in the NSS group (* p < 0.05). (C) A comparison of the area of the grafts between the two groups at Week 8. The dermis of the bFGF group was significantly larger than that in the NSS group (* p < 0.05).