Biomolecules, Vol. 13, Pages 55: Advances and Innovations of 3D Bioprinting Skin

HairGelMA and HAMANHDFs (human dermis), HaCaT cells (human epidermis), and HFDPCs (human scalp)ExtrusionTriple-layered micron-sized lattice structure-

Live/dead assay

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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 rhCol3HaCaT cells and HDFs (human skin)ExtrusionFilament fusion test at millimeter scales-

Live/dead assay

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CCK-8 assay on proliferation

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rtPCR on cytoskeletons and ECM production

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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]ColKCs (human neonatal foreskin dermis), HUVECs (human umbilical cord), and HFDPCs (human scalp)ExtrusionMicropillar 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]VascularizationPGA, and xeno-free dermal and epidermal bioinkHECs (umbilical cord blood), FBs (human dermis), PCs (human placentas), and KCs (human epidermis)ExtrusionNA-

Flow cytometry on cell phenotypes

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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 DABMSCs, HUVECs, NHDFs, and HaCaT cells (from human origin)ExtrusionLattice-structured constructs-

Hoechst staining on the viability

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IF staining on angiogenesis

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ARS staining on osteogenesis

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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 LAPHFBs and HUVECs (from human origin)DLPCylinder with submicron lattices-

Live/dead assay

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TIANamp Genomic DNA Kit on cell proliferation

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IF staining on cell tracking, migration, and adhesion

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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 IHFBs, HUVECs, HECFCs, PCs, and HKCs (from human origin)ExtrusionNA-

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 thicknessGel, glycerol, and HAKCs, dark melanocytes, HDFs, HFDPCs, HDMECs, and preadipocytes (from human origin)Extrusion2.5 × 2.5 cm triple-layered patch with micron-sized lattices-

Picrosirius red staining for Col fiber

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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 AlgHDFs (human dermis), HUVECs (human umbilical cord), HKCsExtrusionMicron-sized lattice-

Live/dead assay

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IF staining on cell morphology and proliferation

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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 DCELFBs (human dermis) and KCs (human epidermis)ExtrusionMicron-sized highly fine lattice-structured cylinder-

MTT assay on cytotoxicity

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Live/dead assay

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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]ColHDFs (human neonatal dermis), HKCs (human neonatal epidermis), and MCs (human darkly pigmented neonatal epidermis)Extrusion2 × 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 entactinHDF (neonatal human dermis) and HKCs (neonatal human epidermis)ExtrusionDirectly 3D printed on a well plate.-

H&E staining on epidermal differentiation

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

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MTT assay on the viability

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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]

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