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. 2022 Dec 27;13(1):55. doi: 10.3390/biom13010055

Table 2.

Structural advances in 3D bioprinting of skin.

Classifications Materials Cell Components (Origin) Printing Methods Printability (Fidelity) and Scalability Biological Assessment of Printed Constructs Advantages (+)
Challenges (−)		 Ref.
Hair GelMA and HAMA NHDFs (human dermis), HaCaT cells (human epidermis), and HFDPCs (human scalp) Extrusion Triple-layered micron-sized lattice structure
  • -

    Live/dead assay

  • -

    IF staining on hair-inducing properties and skin morphology

 + A papillary layer was recapitulated by 3D printing.
+ Enhanced epidermis-dermis interaction supported spontaneous hair pore development.
− Long-term observation is required for full hair shaft development.		 [23]
GelMA and rhCol3 HaCaT cells and HDFs (human skin) Extrusion Filament fusion test at millimeter scales
  • -

    Live/dead assay

  • -

    CCK-8 assay on proliferation

  • -

    rtPCR on cytoskeletons and ECM production

  • -

    IHC analysis on epithelialization and in vivo wound healing properties

 + rhCol3 enhanced the growth of HaCaT cells and HDFs.
+ Enhanced wound healing and hair follicle development on in vivo rat model.
− higher cell spreading and population in dermal layers are required.		 [78]
Col KCs (human neonatal foreskin dermis), HUVECs (human umbilical cord), and HFDPCs (human scalp) Extrusion Micropillar mold 500 μm in diameter and 4 mm in length
  • -

    IF staining and IHC on HFU development and vasculature both in vitro and in vivo

 + The HFU-developed and vascularized dermal constructs were fabricated.
+ The skin engraftment allows for human hair growth in nude mice.
− Reproducibility on hair shaft protrusion should be confirmed.		 [79]
Vascularization PGA, and xeno-free dermal and epidermal bioink HECs (umbilical cord blood), FBs (human dermis), PCs (human placentas), and KCs (human epidermis) Extrusion NA
  • -

    Flow cytometry on cell phenotypes

  • -

    IF staining and IHC analysis on tissue structures

 + A mature stratified epidermis with rete ridge-like structures was developed.
+ The developed microvessels prevented graft necrosis and induced perfusion with host microvessels.
+ A xeno-free approach to complex tissue engineering was achieved.
− Further studies are required on the efficacy of the xeno-free strategy and the degree of wound bed contraction.		 [80]
GelMA, SCS, and DA BMSCs, HUVECs, NHDFs, and HaCaT cells (from human origin) Extrusion Lattice-structured constructs
  • -

    Hoechst staining on the viability

  • -

    IF staining on angiogenesis

  • -

    ARS staining on osteogenesis

  • -

    Cell scratch assay on wound healing

 + Micro-vascularization as tubelike structures with endothelial cell marker expression were confirmed.
+ Enhanced in vitro skin wound healing activity and maintained multipotency of BMSCs.
− Further biological evaluations are required.		 [81]
GelMA, HA-NB, and LAP HFBs and HUVECs (from human origin) DLP Cylinder with submicron lattices
  • -

    Live/dead assay

  • -

    TIANamp Genomic DNA Kit on cell proliferation

  • -

    IF staining on cell tracking, migration, and adhesion

  • -

    IF and IHC analysis on inflammatory cell infiltration, wound healing, and angiogenic markers expression.

 + The DLP enabled interconnected microchannel formation that facilitates cellular behaviors.
+ Efficient neovascularization was achieved by mimicking the physiological structure of native skin.
+ Induced instant defense function and dermal regeneration with skin appendages in large animals.
− In-depth studies on underlying mechanisms in the hair follicle and blood vessel regeneration are required.		 [82]
Rat tail Col I HFBs, HUVECs, HECFCs, PCs, and HKCs (from human origin) Extrusion NA
  • -

    IF staining and IHC analysis on skin structure, epithelialization, ECM production, and vascularization.

 + In vitro, HKCs formed a multilayered barrier, while the HUVECs and PCs self-assemble into interconnected microvascular networks.
+ Transplantable skin grafts composed of an irrigational microvascular system were developed.
− Harvesting plenty of healthy cells from the patients are required.		 [83]
Full thickness Gel, glycerol, and HA KCs, dark melanocytes, HDFs, HFDPCs, HDMECs, and preadipocytes (from human origin) Extrusion 2.5 × 2.5 cm triple-layered patch with micron-sized lattices
  • -

    Picrosirius red staining for Col fiber

  • -

    IHC analysis on structural maturation

 + Epidermis-dermis-hypodermis triple−layered skin mimetics were 3D bioprinted.
+ Matured normal and basket weave Col was observed.
− Immune responses in the large animal models should be elucidated.		 [84]
GelMA and Alg HDFs (human dermis), HUVECs (human umbilical cord), HKCs Extrusion Micron-sized lattice
  • -

    Live/dead assay

  • -

    IF staining on cell morphology and proliferation

  • -

    ELISA on ECM synthesis and migration

 + A 3D full-thickness skin model composed of epidermis-dermis with vasculature was fabricated.
+ Controlled matrix stiffness regulated pro−Col1α1 and MMP-1 expression.
+ Repeated HKCs seeding and Gel coating support epidermal differentiation.
− Epidermal markers should be further elucidated.		 [85]
Alg, Gel, and DCEL FBs (human dermis) and KCs (human epidermis) Extrusion Micron-sized highly fine lattice-structured cylinder
  • -

    MTT assay on cytotoxicity

  • -

    Live/dead assay

  • -

    IF staining on Col and keratin expression.

 + The incorporated DCEL can induce the uniform distribution of cellulose fibers within bioinks.
+ The distinct epidermal-dermal histological features were visualized with specific marker expressions.
− Further biological assessments should be conducted.		 [86]
Col HDFs (human neonatal dermis), HKCs (human neonatal epidermis), and MCs (human darkly pigmented neonatal epidermis) Extrusion 2 × 2 cm stratified constructs
  • -

    IHC analysis on skin structures and differentiation

 + KC formed the stratum corneum and freckle-like pigmentations were developed by MCs at the dermal-epidermal junction.
+ First developed engineered ephelides in biomimetic skin.
−In−depth studies for melanin production and pigmentation should be conducted.		 [87]
Gel, Col I, elastin, fibrinogen, laminin, and entactin HDF (neonatal human dermis) and HKCs (neonatal human epidermis) Extrusion Directly 3D printed on a well plate.
  • -

    H&E staining on epidermal differentiation

  • -

    IHC analysis on the tight junction,—ECM proteins, and proliferation markers.

  • -

    MTT assay on the viability

  • -

    OCT on tissue morphology

 + Four primary layers of the epidermis were developed.
+ Stratum granulosum formed f-TKD shape allowing homeostasis by tight junction barrier.
− Need to apply iPSCs to obtain consistent and reproducible KCs sources.		 [88]

Abbreviations: ARS, Alizarin red S; BMSC, bone marrow-derived stem cells; DA, dextran aldehyde; DCEL, diethylaminoethyl cellulose; DLP, digital light processing; GelMA, Gel methacryloyl; HAMA, hyaluronic acid (HA) methacryloyl; HA-NB, NB-linked HA; HDMECs, human dermal microvascular endothelial cells, HECFCs human endothelial colony-forming cells; HECs, human endothelial cells; HFDPCs, hair follicle dermal papilla cells; HFU, hair follicle unit; HKCs, human KCs; IHC, immunohistochemical; iPSCs, induced pluripotent stem cells; LAP, lithium phenyl-2,4,6-trimethylbenzoylphosphinate; MCs, melanocytes; MMP, matrix metalloproteinase; NB, N-(2-aminoethyl)-4-(4-(hydroxymethyl)-2-methoxy-5-nitrosophenoxy) butanamide; NHEKs, normal human epithelial KCs; OCT, optical coherence tomography; PCs, placental pericytes; rhCol, recombinant human Col; SCS, succinylated chitosan; and SKPs, skin-derived precursors.