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.

Keywords: Hair follicle regeneration; Skin progenitor cell; Skin regeneration; Tissue-engineered skin; p63.

Fig. 1

TESSs from different time points produce pigmented skin after grafting. a Representative H&E-stained images of TESSs at different time points (bars = 50 μm). b Representative images of the skin in graft areas at 4 weeks after transplantation of different TESSs (bars = 5 mm). The white dashed line indicates the border of the host mouse skin and the pigmented human graft skin area. c Representative image of H&E staining of the pigmented area from b. The black dashed line indicates the boundary between the human skin graft area and the host mouse skin. d, e IF staining of human pan-ck (red, d) and human vimentin (green, e) in the pigmented area from b; DAPI stains the nuclei (blue). The white dashed line indicates the boundary between the human skin graft and the host mouse skin (bars = 50 μm). f, g The average size of pigmented skin areas at 4 and 8 weeks after transplantation. *p < 0.05, **p < 0.01 when two groups were compared as indicated; none of the other comparisons was significantly different (3 mice for each group, n = 3)

Fig. 2

A normal structure of the epidermis is formed from transplantation of different stages of TESSs at 4 and 8 weeks after grafting. a, b Representative images of H&E staining of the epidermis formed at 4 (a) and 8 (b) weeks after grafting. c–f Representative images of IF staining of β-catenin (red, c), human pan-ck (red, d), K10 (red, e), and loricrin (red, f) at 4 weeks; DAPI stains the nuclei (blue); bars = 50 μm

Fig. 3

The epidermis from early-stage TESSs contains more Ki-67- and p63-positive cells. a, b Representative images of Ki-67 IHC staining of the epidermis formed 4 (a) and 8 (b) weeks after grafting different stages of TESSs. c, d Representative images of IF staining (red) of p63 in the epidermis formed 4 (c) and 8 (d) weeks after grafting different stages of TESSs; DAPI stains the nuclei (blue). e, f Quantification of the percentage of Ki-67-positive cells in the basal cell layer by counting the number of Ki-67-positive cells in a total of 200 basal cells with DAPI staining from a for 4 and b for 8 weeks after transplantation. g, h Quantification of the percentage of p63-positive cells in the epidermis formed by counting the number of p63-positive cells in a total of 200 epidermal cells with DAPI staining from c for 4 and d for 8 weeks after transplantation. *p < 0.05, **p < 0.01, ***p < 0.005 when two groups were compared as indicated; none of the other comparisons was significantly different (3 mice for each group, n = 3); bars = 50 μm

Fig. 4

Thicker dermis appears in the skin formed from transplantation of early-stage TESSs. a–d Representative H&E-stained images of the dermis formed from the transplantation of different stages of TESSs at 4 (a) and 8 weeks (c) after transplantation. Blue dashed bars indicate the thickness of the dermis measured, and the averages of dermis thickness (n = 3) at 4 and 8 weeks are shown in b and d, respectively. e, g Representative images of IF staining of vimentin (green) in the dermis with DAPI staining (blue). f, h Corresponding quantification of vimentin-positive cells per high-power field (× 400) in a light microscope from e for 4 and g for 8 weeks after transplantation, respectively. i, j Representative images of Masson’s trichome staining of the dermis formed for collagen fibers (blue) from the transplantation of different stages of TESSs at 4 (i) and at 8 weeks (j) after transplantation. *p < 0.05, **p < 0.01, ***p < 0.005 when two groups were compared as indicated; none of the other comparisons was significantly different (3 mice for each group, n = 3); bars = 50 μm

Fig. 5

More CD31-positive cells and hair follicles are formed in the dermis of early-stage TESSs. a, b PAS staining at 4 (a) and 8 (b) weeks, the red layer shows the location and clarity of the basement membrane zone (yellow arrows). c, d CD31-positive microvessels (black arrows) were widely distributed in newly formed tissues from early-stage TESSs, while the microvessels were scattered in tissues from TESS-14d and TESS-21d. e, f Quantification of CD31-positive microvessels per high-power field (× 400) in a light microscope from c for 4 and d for 8 weeks after transplantation. **p < 0.01, ***p < 0.005 when two groups were compared as indicated; none of the other comparisons was significantly different (3 mice for each group, n = 3); bars = 50 μm. g–k Representative images of hair follicles (H&E staining, Ki-67, pan-ck, p63-vimentin, K10) in the skin formed from transplants TESS-5d at 8 weeks after grafting. The blue arrow in g indicates the dermal papilla, and the green arrow in g indicates a sebaceous gland. Ki-67 staining demonstrates an active hair matrix with highly proliferating cells (brown arrows in h); bars = 50 μm

Fig. 6

Early-stage TESS expresses high levels of p63, but less activation of the apoptotic pathway. a Different stages of TESSs were collected before in vivo grafting for western blot analysis of protein levels of K5, K1, p63, Pro-Caspase-3, and C-Caspase-3. The housekeeping gene GAPDH was used as a loading control. b Quantification of western blot analysis of p63 and C-Caspase-3 relative to the band density of GAPDH. c TESSs as described for a were collected for qRT-PCR analysis of p63, p21, Bax, and Bcl-2. The relative mRNA level of each gene was calculated relative to TESS-5d (expression level as 1) after adjustment by the housekeeping gene 36B4. All experiments were repeated 3 times. *p < 0.05, **p < 0.01, ***p < 0.005 when two groups were compared; none of the other comparisons was significantly different