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. 2020 Mar;26(5-6):227-238.
doi: 10.1089/ten.TEA.2019.0201. Epub 2019 Dec 3.

Three Dimensional Bioprinting of a Vascularized and Perfusable Skin Graft Using Human Keratinocytes, Fibroblasts, Pericytes, and Endothelial Cells

Tânia Baltazar  1 , Jonathan Merola  2 , Carolina Catarino  3   4 , Catherine B Xie  1 , Nancy C Kirkiles-Smith  1 , Vivian Lee  5 , Stephanie Hotta  6 , Guohao Dai  5 , Xiaowei Xu  7 , Frederico C Ferreira  8 , W Mark Saltzman  9 , Jordan S Pober  1 , Pankaj Karande  3   4
Affiliations

Three Dimensional Bioprinting of a Vascularized and Perfusable Skin Graft Using Human Keratinocytes, Fibroblasts, Pericytes, and Endothelial Cells

Tânia Baltazar et al. Tissue Eng Part A. 2020 Mar.

Abstract

Multilayered skin substitutes comprising allogeneic cells have been tested for the treatment of nonhealing cutaneous ulcers. However, such nonnative skin grafts fail to permanently engraft because they lack dermal vascular networks important for integration with the host tissue. In this study, we describe the fabrication of an implantable multilayered vascularized bioengineered skin graft using 3D bioprinting. The graft is formed using one bioink containing human foreskin dermal fibroblasts (FBs), human endothelial cells (ECs) derived from cord blood human endothelial colony-forming cells (HECFCs), and human placental pericytes (PCs) suspended in rat tail type I collagen to form a dermis followed by printing with a second bioink containing human foreskin keratinocytes (KCs) to form an epidermis. In vitro, KCs replicate and mature to form a multilayered barrier, while the ECs and PCs self-assemble into interconnected microvascular networks. The PCs in the dermal bioink associate with EC-lined vascular structures and appear to improve KC maturation. When these 3D printed grafts are implanted on the dorsum of immunodeficient mice, the human EC-lined structures inosculate with mouse microvessels arising from the wound bed and become perfused within 4 weeks after implantation. The presence of PCs in the printed dermis enhances the invasion of the graft by host microvessels and the formation of an epidermal rete. Impact Statement Three Dimensional printing can be used to generate multilayered vascularized human skin grafts that can potentially overcome the limitations of graft survival observed in current avascular skin substitutes. Inclusion of human pericytes in the dermal bioink appears to improve both dermal and epidermal maturation.

Keywords: bioprinting; microvasculature; regenerative medicine; skin; tissue engineering.

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Conflict of interest statement

No competing financial interests exist.

Figures

FIG. 1.
FIG. 1.
Immunocharacterization and phenotyping of cultured human dermal FBs, placental PCs, and ECFC-derived ECs. (A) Dermal FBs express α-smooth muscle actin, PDGFR-α, PDGFR-β, and CD90 but not NG2, as confirmed by immunofluorescence microscopy. Placental PCs stained positive for α-smooth muscle actin, PDGFR-β, NG2, and CD90, but not PDGFR-α. (B) Flow cytometry analysis of dermal FBs confirmed the expression of PDGFR-α, PDGFR-β, and CD90. Furthermore, these cells lack expression of NG2, CD31, and CD45. PCs showed positive expression of PDGFR-β, NG2, and CD90, but lack expression of PDGFR-α, CD31, and CD45, as confirmed by flow cytometry. (C) Human ECFC-derived ECs stained positive for VE-cadherin by immunofluorescence microscopy and showed positive expression of CD31, but not CD45 by flow cytometry analysis. Specific staining by flow cytometry is shown in green; isotype-matched control staining is shown in red. Similar results were observed in three independent isolations from different donors. Scale bar: 100 μm. FB, fibroblast; PC, pericyte; ECFC, endothelial colony-forming cell. Color images are available online.
FIG. 2.
FIG. 2.
Optimization, maturation, and characterization of dermal/epidermal compartments of bioprinted skin constructs in vitro. (A) Evaluation of the effect of nebulized NaHCO3 as a collagen crosslinking agent on distribution of dermal FBs and differentiation of KCs in 3D bioprinted constructs. Top images are shown as maximum projections of z-stack epifluorescence images of printed constructs at day 3. FBs and KCs were fluorescently labeled with CellTracker™ Red CMPTX and CellTracker Green CMFDA dyes, respectively. Bottom images show H&E staining of bioprinted constructs 20 days after printing demonstrating heterogeneous distribution of FBs when collagen layers were nebulized. Scale bar: 100 μm. (B) Schematic showing layer-by-layer bioprinting of human skin equivalents. (C) Representative images of H&E and immunofluorescence staining of human foreskin and bioprinted constructs 30 days after in vitro maturation. Printed grafts show positive expression of filaggrin, cytokeratin 14, cytokeratin 10, and collagen type IV, similar to human foreskin. Cell nuclei were stained with DAPI (blue). Scale bar: 100 μm. KC, keratinocyte. Color images are available online.
FIG. 3.
FIG. 3.
Evaluation of culture conditions to allow endothelial network assembly in bioprinted dermal constructs in vitro. (A) Timeline of culture conditions tested: RFP-expressing ECs cocultured with dermal FBs in bioprinted dermal constructs cultured in skin medium for 4 days (protocol A); or EGM2 for 4 days and subsequently in skin medium until day 37 of in vitro culture (protocol B). (B) Live cell fluorescence microscopy of bioprinted dermal constructs cultured with protocol A, showing absence of EC network formation, whereas protocol B promoted self-assembly and long-term maintenance of EC networks. Scale bar: 100 μm. (C) 3D reconstruction of self-assembled endothelial networks in skin medium in vitro at day 10. Scale bar: 50 μm. Grids define the 3D space. (D) Printed samples were followed for 50 days to evaluate long-term endothelial network stability and viability in skin medium. Live cells were stained with calcein (green) and nuclei with Hoechst (blue). (E) Live imaging of cocultures of RFP-expressing ECs, Cy5-dermal FBs, and GFP-expressing PCs, 7 days after printing. Scale bar: 100 μm. Color images are available online.
FIG. 4.
FIG. 4.
Characterization of 3D bioprinted vascularized skin equivalents before engraftment. (A) Timeline of 2-stage protocol to fabricate human vascularized skin equivalents. First, bioprinting of a vascularized dermal compartment cultured in EGM2 for 4 days and, second, bioprinting of the epidermal compartment and culture in skin medium. At day 8, bioprinted constructs were characterized or implanted onto an immunodeficient mouse model. (B) Representative images of H&E and immunofluorescence staining of human adult skin and bioprinted constructs at the time of engraftment. Bioprinted skin grafts containing ECs show human CD31+ vessel-like structures, whereas bioprinted grafts without human ECs do not. Bioprinted skin grafts showed positive Ki67 and CK14 expression in the epidermal basal layer. Skin grafts containing human PCs showed increased expression of laminin 5 and CK10+ suprabasal terminally differentiated KCs. Cell nuclei were stained with DAPI (blue). Scale bar: 50 μm. Color images are available online.
FIG. 5.
FIG. 5.
Characterization of 3D bioprinted skin grafts at the time of engraftment and 2 weeks postengraftment onto immunodeficient mice. (A) Photographs of bioprinted grafts with and without incorporation of ECs and PCs at the time of engraftment. (B) Representative images of H&E and immunofluorescence staining of bioprinted constructs 2 weeks postengraftment. H&E staining shows a higher degree of hemorrhage in nonvascularized bioprinted skin grafts compared to grafts containing ECs. UEA-1 staining shows the presence of human EC-lined vessels. Formation of rete ridges was observed particularly in vascularized bioprinted grafts containing PCs. Scale bar: 50 μm. Color images are available online.
FIG. 6.
FIG. 6.
Characterization of 3D bioprinted skin equivalents 4 weeks postengraftment. (A) Immunohistochemical staining of human involucrin, at the wound edge, showing that epidermis of 3D bioprinted vascularized skin is of human origin. Scale bar: 50 μm. (B) Representative H&E images of bioprinted grafts showing the extent of contraction. Bioprinted grafts without ECs and PCs were significantly smaller with a larger area occupied by the mouse skin. The border between bioprinted graft and mouse skin was identified by thin epidermis, presence of hair follicles and deep dermis with adipose tissue. Scale bar: 1 mm. (C) Substitutes formulated with human ECs contained vascular structures 4 weeks postengraftment. Presence of mouse microvessels and infiltration of mouse macrophages were assessed by staining with GSL-B4 and F4/80 antibody, respectively. To demonstrate that human EC-lined vessels were perfused, fluorescent UEA-1 was injected 30 min before explant. Arrows point to perfused human-lined vessels. Vascularized bioprinted grafts displayed formation of rete ridge-like structures, particularly in bioprinted grafts containing PCs. Scale bar: 100 μm. Color images are available online.
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