Early-stage bilayer tissue-engineered skin substitute formed by adult skin progenitor cells produces an improved skin structure in vivo

Abstract

Background In recent years, significant progress has been made in developing highly complex tissue-engineered skin substitutes (TESSs) for wound healing. However, the lack of skin appendages, such as hair follicles and sweat glands, and the time required, are two major limitations that hinder its broad application in the clinic. Therefore, it is necessary to develop a competent TESS in a short time to meet the needs for clinical applications. Methods Adult scalp dermal progenitor cells and epidermal stem cells together with type I collagen as a scaffold material were used to reconstitute bilayer TESSs in vitro. TESSs at 4 different culture times (5, 9, 14, and 21 days) were collected and then grafted onto full-thickness wounds created in the dorsal skin of athymic nude/nude mice. The skin specimens formed from grafted TESSs were collected 4 and 8 weeks later and then evaluated for their structure, cell organization, differentiation status, vascularization, and formation of appendages by histological analysis, immunohistochemistry, and immunofluorescent staining. Results Early-stage bilayer TESSs after transplantation had a better efficiency of grafting. A normal structure of stratified epidermis containing multiple differentiated layers of keratinocytes was formed in all grafts from both early-stage and late-stage TESSs, but higher levels of the proliferation marker Ki-67 and the epidermal progenitor marker p63 were found in the epidermis formed from early-stage TESSs. Interestingly, the transplantation of early-stage TESSs produced a thicker dermis that contained more vimentin- and CD31-positive cells, and importantly, hair follicle formation was only observed in the skin grafted from early-stage TESSs. Finally, early-stage TESSs expressed high levels of p63 but had low expression levels of genes involved in the activation of the apoptotic pathway compared to the late-stage TESSs in vitro. Conclusions Early-stage bilayer TESSs reconstituted from skin progenitor cells contained more competent cells with less activation of the apoptotic pathway and produced a better skin structure, including hair follicles associated with sebaceous glands, after transplantation, which should potentially provide better wound healing when applied in the clinic in the future.

Accelular nanofibrous bilayer scaffold intrapenetrated with polydopamine network and implemented into a full-thickness wound of a white-pig model affects inflammation and healing process

“…To date, of all acellular scaffolds, only human acellular dermis products, such as clinically proven AlloDerm and DermaMatrix appear to be the best option in skin tissue [ 51 , 52 ]. Implantation of acellular scaffolds with cell cultures of fibroblasts and/or keratinocytes is associated with enormous costs and difficult regulations [ 53 ]. Currently, an example of a cell-free extracellular matrix represents Integra, made of Coll and chondroitin-6-sulfate with a silicone backing [ 54 ].…”

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

“…Another hair follicle generation model used human adult scalp dermal progenitor cells and epidermal stem cells to create a bilayered HSE that exhibited hair follicle formation [120]. To form this HSE, an initial acellular layer of collagen was generated, and a second collagen matrix containing dermal progenitor cells was then deposited on top of it.…”

“…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…”

Basic Quality Controls Used in Skin Tissue Engineering

“…Most TESSs are first evaluated ex vivo and, based on these findings, some of these substitutes could prove suitable for further in vivo preclinical evaluation [84,85]. Currently, there is a wide range of methods available to characterize TESSs generated ex vivo and tested in vivo, which include functional analyses, histology (light and electron microscopy), molecular biology and biomechanical testing [86,87].…”