Design and Bioprinting of Decellularized Extracellular Matrix-Based Bioinks for Skin Tissue Engineering

Figures & data

Figure 1. Design, preparation and bioprinting of dECM-based bioinks for skin tissue engineering.

Several sources can be used to obtain dECM, which is extracted and processed via a combination of chemical, physical and/or biological methods, followed by sterilization and quality control steps. For (bio)ink design, the dECM can be combined with cells and used as either a single material or combined with other biomaterials that impart specific properties at rheological, biomechanical and/or biological levels. In the context of skin bioprinting, extrusion- and inkjet-based bioprinting have been used to create acellular and cell-laden constructs as in vitro skin models and grafts for wound healing.

dECM: Decellularized extracellular matrix; ECM: Extracellular matrix.

Table 1. Examples of processing methodologies applied for the design of dECM-based bioinks for skin bioprinting.

Tissue	Origin	Pretreatment	Decellularization protocol	Disinfection or sterilization	Post-processing	Processing time (h)	Ref.
Skin	Porcine	Subcutaneous fat, connective tissue, epidermis mechanically remove + frozen (-80°C) + thawed + chopped	0.25% trypsin (6 h) + DI rinse (15 min, 3X) + 70% ethanol (10 h) + 3% H2O2 (15 min) + DI rinse (15 min, 2X) + 1% TX-100 in 0.26% EDTA/0.69% Tris (24 h) + DI rinse (15 min, 2X)	n/d	Frozen + freeze-dried + cryogenic milled + filtered (100 μm pore)	>140	[11,75]
Skin	Porcine	Chopped into small pieces (1 × 1 cm^2)	0.25 wt% trypsin and 1 mM EDTA (37°C, 6 h) + 1% v/v TX-100 and 25 mM EDTA (24 h) + PBS rinse (14 h) + DNase (30 U/ml) and MgCl2 (10 mM) (37°C, 24 h) + PBS rinse (48 h) + DI rinse (15 min)	0.1% PAA in 4% EtOH (2 h) + PBS rinse	Freeze-dried	>118	[13,76]
Skin	Porcine	Chopped into small pieces (1 × 1 cm^2)	0.25 wt% trypsin and 1 mM EDTA (6 h, 37°C) + fat tissues physically removed + 1 wt% TX-100 and 25 mM EDTA (24 h) + PBS rinse (14 h) + DNase (30 U/ml) and MgCl2 (10 mM) (37°C, 24 h) + PBS rinse (48 h) + DI rinse (15 min)	0.1% PAA in 4% EtOH (2 h) + PBS rinse (15 min, 2X) + DI rinse (15 min, 2X)	Freeze-dried	>118	[10]
Skin	Porcine	Subcutaneous fat, connective tissue, and epidermis mechanically removed	2% NaOH and 2% TX-100 (12 h, 4°C) + PBS rinse	n/d	n/d	>12 h	[77]
Skin	Porcine	n/d	Proprietary (T&R Biofab, deCelluid, Siheung, Korea)	n/d	n/d	n/d	[78]
Skin	Porcine	Epidermis and subcutaneous tissue removed	Hypertonic solution (24 h) + saline rinse + frozen (-80°C, 24 h) + 0.25% Trypsin (6 h, 37°C) + 1% TX-100, 25 mM EDTA + PBS rinse (3X)	n/d	Lyophilized Freeze-dried (72 h)	n/d	[39]
Adipose	Human	Centrifugation + PBS rinse	0.5% SDS (12 h, 4X) + isopropanol (12 h, 4X) + PBS rinse	0.1% PAA in 4% EtOH (4 h) + PBS and DI rinse	Freeze-dried + frozen with liquid N2 + pulverized with mortar and pestle	>100	[47,62]
Adipose	Human	PBS rinse (3X)	Freeze-thaw (-80°C,1 h/37°C, 40 min, 3X) + centrifuged (7000 g, 3 min, RT) + oil removal + 0.25% trypsin/0.1% EDTA (37°C,120 rpm/min, ON) + PBS rinse (30 min, 3X) + 99.9% isopropanol (12 h, 4X) + PBS rinse (30 min, 3X) + 0.25% trypsin/0.1% EDTA (4 h) + PBS rinse (30 min, 3X) + 1000 U of nuclease and type II lipase (ON) + PBS rinse (30 min, 3X) + 99.9% isopropanol (6 h) + PBS rinse (30 min, 3X)	n/d	Freeze-dried (48 h)	>91	[79]
Adipose	Porcine	Cut in cubes 3 mm	0.5% w/v SDS (48 h) + 0.1% v/v TX-100 (5 min) + isopropanol	0.1% PAA in 4% EtOH (4 h) + DI (72 h)	Frozen (-80°C) + freeze-dried (48 h)	∼172	[78,80]
Esophagus (mucosal)	Porcine	Cut into pieces 1–2 mm thick + PBS wash	1% TX-100 (72 h) + 1% SDS (48 h) + DNase (37°C, 72 h) + PBS rinse (72 h)	0.1% peracetic acid in 4% EtOH (2 h) + autoclaved DI rinse	Freeze-dried	>266	[81]
Small intestine submucosa	Porcine	Jejunum DI rinse + fat removed + cut into pieces (∼10 cm in length) + tunica serosa and muscularis layers removed + PBS rinse	0.1% (w/v) peracetic acid + PBS and DI rinse	n/d	Freeze-dried + cryogenic grinded	n/d	[82]
Small intestine	Porcine	Cut longitudinally to obtain tissue sheets + DI rinse	0.1% TX-100 (8 h) + 3 DI rinses (15 min each) + 2% SDC (4 h) + PBS rinse (1 h) + 1 M NaCl (1 h)	0.1% PAA in 4% EtOH (3 h) + sterile water rinse (1 h) + sterile water rinse (24 h)	Freeze-dried + milled	>42.75	[83]
Small intestine submucosa	Porcine	Porcine jejunum mechanical dissociated	Enzyme digestion + detergent	n/d	Freeze-dried + grinding with cryogenic tissue-lyser (5 min)	n/d	[9]
Small intestine submucosa	Porcine	Tap water rinse + removal of tunica serosa, muscularis externa and tunica mucosa	NaCl and H2O + PBS and autoclaved type II H2O rinse	n/d	Dried in laminar flow hood (RT) + freeze-milled	n/d	[84]
Placenta	Human	n/d	1X PBS with heparin and pen/strep (3X) + PBS with heparin and pen/strep (2 h, 3 ml min-1) + 0.1% SDS with pen/strep (3d, 3 ml/min) + 0.01% SDS with pen/strep (3d, 3 ml/min) + 0.001% SDS with pen/strep (3d, 3 ml min-1) + Tris-HCl solution containing DNase/RNase (24 h) + 1% TX-100 in PBS (24 h) + PBS (3d)	n/d	n/d	>338	[85]
Placenta	Human	Cut into fragments + DI rinse	0.3%/0.5% SDS/TX-100 (30 min) + PBS rinse (5d)	n/d	n/d	>120.5	[14]

dECM: Decellularized extracellular matrix; DI: Distilled water; EtOH: Ethanol; n/d: Not defined; PAA: Peracetic acid; ON: Overnight; PBS: Phosphate-buffered saline; PMSF: Phenylmethylsulfonyl fluoride; SDC: Sodium deoxycholate.

Figure 2. Design and bioprinting of dECM bioinks for skin tissue engineering.

(A) Quantification of the major ECM components (collagen, GAG, and elastin) after the decellularization process. (B) Representative images of 3D cell-printed in vitro skin equivalents using type I collagen (C-HSE) and skin-derived bioink (S-HSE). Scale bar: 2 mm. (C) Morphological changes occurring in the diverse types of skin grafts during 28 days after the surgery. (D) Bioprinted skin model recreating diabetic hallmarks by the inclusion of perfusable and vascularized hypodermal compartment and augmented diabetic features.

C-HSE: Human skin equivalent using collagen bioink; dECM: Decellularized extracellular matrix; ECM: Extracellular matrix; GAG: Glycosaminoglycans; IS: Inguinal split-thickness skin grafts; PCL: Polycaprolactone; PDA: 3D-printed dermal analogues; PL: Pelnac Dermal Substitute; ROS: Reactive oxygen species; S-HSE: Human skin equivalent using skin-derived extracellular matrix bioink.

Modified with permission from [10,125,126,145].

Figure 3. Design and bioprinting of multimaterial dECM-containing bioinks for skin tissue engineering.

(A) Manipulation of the bioprinted dECM patch (i) and its ability to fit to the wound bed (ii). (B) Effect of gelatin incorporation on the printability of dECM inks and comparison to Pluronic F 127 ink (scale bar: 10 mm). (C) Schematic illustration of the design, bioprinting and implantation of ADSCs-laden ECM–GelMA–HAMA constructs. (D) Macroscopic images of the wound size after treatment with Alg/gel and 5%ECM-Alg/gel 3D printed scaffolds during 21 days upon implantation. (E) Schematic illustration of the process of fabrication of the skin model of hypertrophic scar.

ADSC: Adipose-derived stem cells; Alg: Alginate; dECM: Decellularized extracellular matrix; ECM: Extracellular matrix; GelMA: Gelatin-methacryloyl; HAMA: Methacrylated hyaluronic acid; PCA: Preformed cellular aggregates.

Modified with permission from [7,14,79,161,162].

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