Pilot Study of the Biological Properties and Vascularization of 3D Printed Bilayer Skin Grafts

Abstract

The skin is the largest human organ, and defects in the skin with a diameter greater than 4 cm do not heal without treatment. Allogeneic skin transplantation has been used to allow wound healing, but many grafts do not survive after implantation, due to multiple complications in the procedure. In the present study, the vascularization of three-dimensional (3D) printed full-thickness skin grafts was investigated. Dermal-epithelial grafts were transplanted into a nude mouse model to evaluate integration with the host tissue and the extent of wound healing. To create microvessels in the skin grafts, a bilayer structure consisting of human dermal fibroblasts, keratinocytes, and microvascular endothelial cells was designed and fabricated using an extruded 3D printer. Human dermal fibroblasts and human microvascular endothelial cells were mixed with gelatin-sodium alginate composite hydrogel as the dermis, and human keratinocytes were mixed with gel as the epithelium. Confocal imaging allowed visualization of the location of the cells in the double-layer skin grafts. A full-thickness wound was created on the backs of nude mice and then covered with a double-layer skin graft. Various groups of mice were tested. Animals were euthanized and tissue samples collected after specified time points. Compared with the control group, wound contraction improved by approximately 10%. Histological analysis demonstrated that the new skin had an appearance similar to that of normal skin and with a significant degree of angiogenesis. The results of the immunohistochemical analysis demonstrated that the transplanted cells survived and participated in the healing process.

Keywords: Bilayer skin graft; Gelatin-alginate complex hydrogel; Three-dimensional printing; Vascularization.

Figure 1

The extruded three-dimensional printing equipment consists of control, motion, feed, and nozzle systems. The main body of the equipment was placed on an ultra-clean platform.

Figure 2

(A) Schematic diagram of transplantable printed skin. The top layer consisted of keratinocytes and gel and the bottom layer fibroblasts, microvascular endothelial cells and gel. (B) A macroscopic image of the printed skin graft.

Figure 3

The surgical procedure involved establishing a full-thickness skin wound and transplantation. (A) Marking of the wound incision lines; (B) establishment of a full-thickness skin wound; (C) placement of the printed skin graft; (D) covering by Tegaderm.

Figure 4

CCK-8 cytotoxicity assay used to assess the cytotoxicity of the hydrogel over 24 h, 48 h, and 72 h on NHDFs.

Figure 5

Three-dimensional images of printed cells after live/dead staining. A, B, and C represent cells 1, 4, and 7 days after printing, respectively. Green staining indicates cell survival. Sample dimensions were 1265 μm (length) × 1265 μm (width) × 305 μm (height).

Figure 6

Fluorescent cell tracking in the double skin grafts on the (A) day 1 and (B) day 7 after printing, respectively. Keratinocytes are labeled blue, microvascular endothelial cells red, and fibroblasts green.

Figure 7

Gross observation of the mice after 4 weeks. A represents the printed skin graft, B is the control group without endothelial cells, C is the control group with no cells, and D is the blank group.

Figure 8

H&E staining of (A) the printed skin graft group with three types of cell; (B) the control group without endothelial cells; (C) the acellular control group; and (D) the blank group.

Figure 9

A and C represent the printed skin graft group, at ×100 and ×400, respectively. B and D represent the control group, at ×100 and ×400, respectively. Because there was no significant difference between the three control groups, only one set is displayed. The microvessels marked as brown in A and C can be clearly seen, while visible in B and D only sparsely.

Figure 10

Immunohistochemical staining of CK10. A and C represent the printed skin graft group with HMVECs. B and D represent the control group without HMVECS.