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

Abstract

Extensive availability of engineered autologous dermo-epidermal skin substitutes (DESS) with functional and structural properties of normal human skin represents a goal for the treatment of large skin defects such as severe burns. Recently, a clinical phase I trial with this type of DESS was successfully completed, which included patients own keratinocytes and fibroblasts. Yet, two important features of natural skin were missing: pigmentation and vascularization. The first has important physiological and psychological implications for the patient, the second impacts survival and quality of the graft. Additionally, accurate reproduction of large amounts of patient's skin in an automated way is essential for upscaling DESS production. Therefore, in the present study, we implemented a new robotic unit (called SkinFactory) for 3D bioprinting of pigmented and pre-vascularized DESS using normal human skin derived fibroblasts, blood- and lymphatic endothelial cells, keratinocytes, and melanocytes. We show the feasibility of our approach by demonstrating the viability of all the cells after printing in vitro, the integrity of the reconstituted capillary network in vivo after transplantation to immunodeficient rats and the anastomosis to the vascular plexus of the host. Our work has to be considered as a proof of concept in view of the implementation of an extended platform, which fully automatize the process of skin substitution: this would be a considerable improvement of the treatment of burn victims and patients with severe skin lesions based on patients own skin derived cells.

Keywords: 3D-Bioprinting; autologous dermo-epidermal skin substitute; collagen plastic compression; pigmentation; tissue engineering; vascularization.

Figure 1.

Schematic illustration of the robotic platform SkinFactory. (a) Loading of the cartridge with collagen type I and human dermal cells. (b) Production of the dermal component by pressure assisted extrusion. (c) Hydrogel plastic compression. (d) Inkjet-based bioprinting of human epidermal cells. (e) Cell culture system. (f) Overview of the turning SkinFactory platform. Station b: pressure assisted extrusion, station c: plastic compression, station d: Inkjet-bioprinting. Ah: cartridge adapter head; Ca: cap; Cf: cell culture flask; Ch: cartridge holder; Cp: compression piston; Crt: cartridge; Cw: compression weights; Fr: insert frame; In: culture insert; Li: culture flask lid; Ma: microvalve actuator; Mv: microvalve; Mx: mixer; Pl: plunger; Ps: o-ring pistons; Pv: pressure valve; St: stage.

Figure 2.

Skin substitute production process. (a) Front view of the SkinFactory. (b) Production of the dermal component by pressure assisted extrusion. (d) Hydrogel plastic compression. (e) Inkjet-based bioprinting of human epidermal cells. (g) Cell culture system. (h) Pre-vascularized pigmented human dermo-epidermal skin substitute of 6 × 6 cm². Abbreviations as for Figure 1. At: aspiration tube; Hg: hydrogel; Ih: insert holder.

Figure 3.

Patterned cell printing resulted in a coherent layer of human keratinocytes and melanocytes. (a) FdA staining of human fibroblasts in a collagen type I hydrogel 7 days after hydrogel production. (b) The programed printing pattern for keratinocyte and melanocyte seeding was visualized by printing of trypan blue on a filter paper. (c–f) Inkjet-based bioprinting of epidermal cells: FdA staining of keratinocytes and melanocytes 1 day (c), 3 days (d), 6 days (e), and 8 days (f) after printing. (g) Immunofluorescence staining of cells 4 days after seeding with antibodies against panCK (keratinocytes, green) and HMB45 (melanocytes, red). Counterstaining of cell nuclei with Hoechst (blue). Scale bars: 100 µm in (a); 2 mm in (c–f).

Figure 4.

Analysis of a pigmented human DESS in vivo. (a) Macroscopic view of the human graft 3 weeks after transplantation. (b) Hematoxylin/eosin staining of sections showing the stratum basale (arrows), stratum spinosum and stratum granulosum (star), and stratum corneum (square). (c) Fontana-Masson melanin staining. Cell nuclei and cytoplasm were counterstained with Hematoxylin/eosin. Melanocyte dendrites projecting to the upper layers of the epidermis (black arrows) and melanosomes shielding keratinocytes nuclei (blue arrows) are visible. (d–j) Immunofluorescence staining using antibodies to: Tyrosinase ((d), green), HMB45 (green)/Lam332 (red) (e), Ki67 (green)/Lam332 (red) (f), CK15 ((g), green), CK5 (green)/Lam332 (red) (h), CK10 (green)/Lam322 (red) (i), CK1 (green)/Lam332 (red) (j). Counterstaining of cell nuclei with Hoechst (blue). (k) Quantification (n = 5) of proliferating cells (Ki67⁺) in DESS and in foreskin as a control. (l) Quantification (n = 5) of melanocytes (HMB45⁺) in DESS and in foreskin as a control. Results are reported as mean ± standard error of the mean (±SEM). Comparison between two groups was performed using the unpaired Student’s t test. Differences were considered significant with a p < 0.05. Scale bars: 100 µm.

Figure 5.

SkinFactory production of pre-vascularized human DESS in vitro. (a) CD31 immunofluorescence whole-mount staining showing the formation of capillaries from HDMECs in collagen type I hydrogels upon 3 weeks and (b) 4 weeks of culture after SkinFactory production. (c) CD31/CD90 double staining and confocal imaging with volume reconstruction of HDMECs-derived capillaries upon 3 weeks of culture in a collagen type I hydrogel displaying the presence of mural cells (CD90 positive) around endothelial cells (CD31 positive). (d–f) CD31 (red)/Lyve1 (green) immunofluorescence staining of HDMECs (BECs and LECs) raised in collagen hydrogels for 3 weeks under three different conditions: uncompressed (d), compressed upon HDMECs inclusion (e), or compressed 2 weeks after inclusion (f). Counterstaining of cell nuclei with Hoechst (blue). In all three cases lymphatic (yellow CD31/lyve1 double positive) and blood capillaries (red CD31 single positive) were visible. Scale bars are 100 µm.

Figure 6.

In vivo investigation of bio-printed pigmented and pre-vascularized human DESS using the SkinFactory. (a) Human capillaries (CD31, red, arrow heads) are present in the human neo-dermis, which is highlighted by human dermal fibroblasts (CD90, green, arrows), 1 and 2 weeks after transplantation (upper panels). Human Lyve1 (green) and human CD31 expression (red) demonstrate the presence of both lymphatic (arrow heads) and blood capillaries (arrows) 1 and 2 weeks after transplantation (lower panels). (b) Hematoxylin/eosin staining (upper panel): stratum corneum (white star), stratum spinosum (black star), pigmented clusters of melanocytes (black arrow heads), unpigmented clusters of basal keratinocytes (white arrow heads), melanocyte dendrites (blue arrow), and melanosome supranuclear caps in keratinocytes (black arrows). (b′) Four-color immunofluorescence staining demonstrating the presence and the localization of the four included cell types: fibroblasts (CD90, blue), endothelial cells (CD31, white), Keratinocytes (CK1, red), and melanocytes (HMB45, green). The white dotted line indicates the dermo-epidermal border. (c) Additional histological and immunofluorescence analysis. First row: Hematoxylin/eosin staining showing the formation of multi-layered epidermis, basal, and cornified layers are indicated by arrowheads and stars, respectively. Second row: CK10 (green)/Lam332 (red) immunofluorescence double staining with arrowheads indicating the basal layer. Third row: Ki67 (green)/Lam332 (red) double staining with arrowheads indicating the proliferative cells. Fourth row: HMB45 (green)/Lam332 (red) double staining with the arrowheads indicating the melanocytes. Counterstaining of cell nuclei with Hoechst (blue). (d) Quantification (n = 5) of proliferative basal cells (Ki67⁺) in DESS 3 weeks after transplantation and comparison to foreskin. (e) Quantification (n = 5) of melanocytes (HMB45⁺) in the basal layer of DESS 3 weeks upon transplantation compared to foreskin. Results are reported as mean ± standard error of the mean (±SEM). Comparison between two groups was performed using the unpaired Student’s t test. Differences were considered significant with a p < 0.05. (f) Macroscopic view of a DESS 3 week after transplantation. Scale bars are 200 µm.