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.

Bioprinting and plastic compression of large pigmented and vascularized human dermo-epidermal skin substitutes by means of a new robotic platform

“…We based our SkinFactory experiments according to the third approach, as it was already successfully proved that endothelial cells spontaneously assembled and formed a vascular plexus in vitro. 14 , 72 , 73 Indeed, many 3D bioprinting protocols also rely on the remarkable self-assembly property of endothelial cells, yet with two variants: (a) the printing of the dermal component (with fibroblasts and endothelial cells) and consecutive printing of the epidermal compartment, followed by direct transplantation with the assembly of the vascular structures occurring in vivo, 74 or (b) a short microvascular development-phase in vitro is interposed between dermal and epidermal printing. 63 For the formation of a vascular network in our substitutes, according to the second variant, we included an apparently long (2 weeks), but, in our opinion, necessary development-phase in vitro.…”

In Vitro Strategies to Vascularize 3D Physiologically Relevant Models

“…In this perspective, large‐scale expansion methods have moved from 2D culture systems, in which cells are expanded by multiplying the number of culture dishes, to bioreactor systems, with the advantage of introducing dynamic culturing conditions, monitoring and controlling of the culture environment, less user‐dependent variability, and higher cost and time efficiency. With the variety of bioreactors and culture methods established nowadays, [ 308 ] protocols for scalable GMP production of PSCs, hiPSC‐derived cells and multipotent SCs, especially MSCs, fundamental during the angiogenesis process, have been successfully developed, [ 309 , 310 , 311 ] although some critical aspects are still debated. For instance, media formulation still represents one of the bottlenecks and an homogenization is required, notably to prevent any unwanted differentiation during the expansion process and to cope with the high costs of the components.…”

Bioengineered Efficacy Models of Skin Disease: Advances in the Last 10 Years

“…An HSE composed of keratinocytes, fibroblasts, and human microvascular endothelial cells (HMVECs) in a gelatin alginate composite hydrogel was used to generate another, vascularized HSE [125]. This HSE was composed of a mixture of fibroblasts, HMVECs, and hydrogel with keratinocytes seeded on top.…”

“…This staining revealed two distinct capillary networks, a blood capillary network, and a lymphatic capillary network. Manual Deposition Immune System MUTZ-LC, NHFs, NHKs [128][129][130] Manual Deposition Immune System NHFs, NHKs, LCs, DCs [131] Manual Deposition Immune System NHFs, NHKs, MUTZ-3-LCs [132] Manual Deposition Immune System NHFs, NHKs, DCs [133] Manual Deposition Immune System NHFs, NHKs, Macrophages [134] Manual Deposition Immune System NHKs, NHFs, Peripheral Blood Mononuclear Cells, CD4 + T cells [135] Skin-On-A-Chip Vasculature HaCaT Cells, HS27 Fibroblasts, HUVECs [94] 3D Bioprinting, Extrusion Nervous System hNSCs [115] Manual Deposition Nervous System hNSCs [116] 3D Bioprinting, Extrusion Nervous System Schwann Cells [117] Manual Deposition Immune System, Nervous System NHKs, NHFs, hiNSCs [118] Manual Deposition Hair Follicle SKPs, Epi-SCs [119] 3D Bioprinting, Extrusion Hair Follicle, Sweat Gland NHKs, NHFs, MSCs [120] Manual Deposition Hair Follicle Dermal Progenitor Cells, Epi-SCs [121] Manual Deposition Vasculature, Hair Follicle DPCs, NHKs, NHFs, HUVECs [107] 3D Bioprinting, Extrusion Vasculature NHKs, NHFs, Pericytes, Endothelial Cells [124] 3D Bioprinting, Extrusion Sweat Gland Epithelial Progenitor Cells [122] Manual Deposition Sebaceous Gland hiPSCs [123] 3D Bioprinting, Extrusion Vasculature NHKs, NHFs, HMVECs [125] 3D Bioprinting, Extrusion Vasculature Adipose-Derived Stem Cells, Endothelial Progenitor Cells [126] Skin-On-A-Chip Vasculature NHKs, NHFs, HUVECs [101] Manual Deposition Lymphatic System, Vasculature NHFs, HUVECs, NHKs, NHDLMECs [127] Manual Deposition Lymphatic System, Vasculature LECs, NHFs [127] 8. Future Directions…”