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3D bioprinting of functional human skin: production and in vivo analysis

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Biofabrication 9 (2017) 015006 doi:10.1088/1758-5090/9/1/015006

PAPER

3D bioprinting of functional human skin: production and in vivoanalysis

NievesCubo1,5,MartaGarcia1,2,3,5, Juan F del Cañizo4, DiegoVelasco1,3 and Jose L Jorcano1,2

1 Department of Bioengineering andAerospace Engineering, UniversidadCarlos III deMadrid (UC3M), Spain2 Division of Epithelial Biomedicine, CIEMAT-CIBERER,Madrid, Spain3 Instituto de Investigación Sanitaria de la Fundación JiménezDíaz,Madrid, Spain4 Department of Surgery, UniversidadComplutense deMadrid, ExperimentalMedicine and Surgery,Hospital General Universitario

GregorioMarañón,Madrid, Spain5 These authors contributed equally.

E-mail: [email protected] and [email protected]

Keywords: 3Dbioprinting, skin bioprinting, artificial skin, skin equivalents, skin tissue engineering, 3D skin culture, fibrin hydrogel

Supplementarymaterial for this article is available online

AbstractSignificant progress has beenmade over the past 25 years in the development of in vitro-engineeredsubstitutes thatmimic human skin, either to be used as grafts for the replacement of lost skin, or forthe establishment of in vitro human skinmodels. In this sense, laboratory-grown skin substitutescontaining dermal and epidermal components offer a promising approach to skin engineering. Inparticular, a humanplasma-based bilayered skin generated by our group, has been applied successfullyto treat burns as well as traumatic and surgical wounds in a large number of patients in Spain. Thereare some aspects requiring improvements in the production process of this skin; for example, therelatively long time (three weeks)needed to produce the surface required to cover an extensive burn ora large wound, and the necessity to automatize and standardize a process currently performedmanually. 3Dbioprinting has emerged as a flexible tool in regenerativemedicine and it provides aplatform to address these challenges. In the present study, we have used this technique to print ahuman bilayered skin using bioinks containing human plasma aswell as primary human fibroblastsand keratinocytes that were obtained from skin biopsies.Wewere able to generate 100 cm2, a standardP100 tissue culture plate, of printed skin in less than 35min (including the 30min required forfibringelation).We have analysed the structure and function of the printed skin using histological andimmunohistochemicalmethods, both in 3D in vitro cultures and after long-term transplantation toimmunodeficientmice. In both cases, the generated skinwas very similar to human skin and,furthermore, it was indistinguishable frombilayered dermo-epidermal equivalents, handmade in ourlaboratories. These results demonstrate that 3Dbioprinting is a suitable technology to generatebioengineered skin for therapeutical and industrial applications in an automatizedmanner.

1. Introduction

Skin injuries caused by burns, chronic ulcers fromdifferent etiology, infections, cancer surgery, and othergenetic and somatic diseases require effective treat-ment to prevent morbidity or mortality. The WorldHealth Organization estimates that nearly 11 millionburn injuries per year worldwide require medicalattention, with approximately 265 000 leading to death[1]. To restore the function of the skin after damageand to facilitate wound-healing, autologous grafts

(autografts) obtained from own-patients donor sitesare commonly used to repair the skin, while avoidingimmune-rejection. Unfortunately, the availability ofautografts for wound coverage is insufficient whendealing with large and/or severe wounds [2–4]. As aresult, several approaches have been explored for skinreplacement therapy, such as cultured autologousepithelial autografts (for a review see [5]), but theirresults are far from ideal, since they are limited by theirfragility and the difficulty of handling, unpredictabletake rate and sensitivity to mechanical shearing forces

RECEIVED

27 July 2016

REVISED

11 September 2016

ACCEPTED FOR PUBLICATION

14 September 2016

PUBLISHED

5December 2016

© 2016 IOPPublishing Ltd

for at least twomonths post grafting [6–8]. In responseto these limitations, new approaches for skin engineer-ing have been tested and developed in recent years.These advances have led to the development of moresophisticated laboratory-grown skin substitutes whichcontain dermal and epidermal components that inter-act dynamically with each other during in vitromaturation and also after transplantation [9–12]. Inparticular, fibrinogen (and its derivative fibrin) is ablood component that has been used extensively as astromal substitute to construct human skin since it hasthe advantages of low price, availability, and goodtolerance to cells; in addition, if required, it can beproduced as an autologous scaffold [13–15]. In thiscontext, a human plasma-derived bilayered (includingdermis and epidermis) skin model was generated byour group and applied successfully to treat burns andtraumatic and surgical wounds [16, 17].

The limitations of the current process: high pro-duction costs, the need for specialized personnel, andthe time required for production of a surface of ther-apeutically useful skin (3–4 weeks to generate 1 m2),combined with a foreseen higher demand for artificialskin, have all led to an increasing need to develop newmethods that offer automation, standardization, andreduction in time and production costs [7, 18, 19].Three-dimensional (3D) bioprinting, has emerged as aflexible tool in regenerative medicine and provides aplatform to address these needs. 3D bioprinting opensup the possibility of constructing artificial tissues ororgans, either autologous or allogeneic, by printingcells, soluble factors and biomaterials in a desired pat-tern with the help of high-precision Cartesian robots[20–23]. A variety of biomaterials have been widelystudied as scaffolds for bioprinting in tissue engineer-ing [24, 25] examples include hydrogels [e.g. 26, 27]which aremostly used for the generation of soft tissuessuch as skin (for a review, see [28]), and polymers[e.g. 29] and ceramics, which are frequently used forthe generation of hard tissues such as bone (e.g.[30, 31]). Additionally, many efforts have been madein the field of biomaterials to design multifunctionalscaffolds, which could be used in the future for the 3Dprinting of advanced tissue engineered constructs e.g.[32], see for reviews [33, 34]. A very common strategyinvolves printing layers of hydrogel matrix precursorsand a posteriori crosslinking in order to form a scaffoldthat provides structural support to the cells and otherextracellular components embedded within it [35–37]. After an in vitro culture period of time, whichdepends on the tissue generated and is required for tis-sue fusion, remodelling and maturation, the printedtissue or organ construct can potentially be applied toreplace the function of the damaged tissue.

Very recently, a comprehensive review about skin3Dbioprinting was published [38]. As discussed in thisreview, there are two main strategies concerning theuse of skin bioprinting for wound treatment. The first

strategy is in situ bioprinting. Using this technique,fibrin-collagen layers containing either amniotic fluid-derived stem cells [39] or human fibroblasts (hFBs)and keratinocytes (hKCs) [40] were printed on full-thickness wounds of nude mice. Although the resultsof these experiments were promising, further optim-ization is still required before application to humanpatients [38].

In the second strategy, two different bioprintingapproaches have recently been explored for the in vitroproduction and in vitro and in vivo analysis of skinconstructs containing dermal and epidermal compo-nents. In the first approach [41, 42], the authors used afree-form fabrication (FFF) technique to deposit avariable number of layers of crosslinked collagen andcollagen containing either hFBs or hKCs. In the sec-ond approach [43, 44], laser assisted bioprinting(LaBP) was used to deposit alternating layers, com-posed of 20 sublayers each, containing immortal mur-ine fibroblasts and immortal hKCs (NIH-3T3 andHaCaT cell lines, respectively) embedded in a collagenmatrix. These approaches aremainly aimed at demon-strating the feasibility of generating artificial skin bybioprinting. However, in our opinion (see also [38] foradditional comments), they present several draw-backs: (1) In general, they do not use human primaryfibroblasts and keratinocytes simultaneously. Theused cells might be less sensitive to the stresses of thebioprinting process, and their proliferation and differ-entiation characteristics are far different from those ofthe cells contained in human native skin. (2) The prin-ted layered structures are not reminiscent of normalskin and the skin constructs produced did not possessthe structural quality of the normal human skin.Moreover, the time required in these multi-layereddeposition methods is currently far from allowing theeffective generation of the relatively large skin surfacesneeded inwound treatment.

Based on our previous experience producing skinequivalents for transplantation to human patients, wedecided to follow the second strategy. In this study wehave used a FFF 3D bioprinting technique to engineera human plasma-derived bilayered skin using hFBsand hKCs obtained from skin biopsies. The printedhuman skin was analysed both in 3D in vitro culturesand in vivo upon grafting to immunodeficient athymicmice (skin-humanized mice), using histological andimmunohistological methods [45–47]. Our resultsshowed that the printed skin had structural and func-tional characteristics as well as appearance and con-sistency similar to those of normal human skin, andskin equivalents produced manually in our group. Wealso demonstrated the capacity of our process toreproducibly print large areas of human skin, usefulfor the treatment of diverse cutaneous pathologiessuch as burns, ulcers and surgical wounds.

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2.Materials andmethods

2.1. Bioprinter design and set-upThe model used in this study was the open sourcePrintrbot (original), modified to deposit cell-ladenhydrogels (figure 1(A)). Most of the structural partswere generated by a normal 3D printer in acryloni-trile-butadiene-styrene (ABS).

An external module (extrusion module) com-posed of two electric stepper motors (NEMA17) andfour sterile disposable plastic syringes (5 or 20 ml)wasdesigned to contain and extrude the hydrogel pre-cursors and the cell suspensions. The plastic syringesand the sterile connecting tubes were replacedbetween experiments to avoid contamination. Thecontent of each syringe will be described in section 2.3.Three of these tubes ((a)–(c) in figure 1(B)) convergedat the head into a trifurcated connector where theircontents were mixed; then, the mixture went througha luer 1.2×40 mm extrusion needle without a bevel,acting as a‘nozzle’ (needle 1 in figure 1(B)). The flow ofeach syringe (3–6ml min−1)was directly proportionalto its volume content in order to obtain a constanthomogeneous mixture in the extrusion needle. Typi-cally, the flow in the extrusion needle was 12 mlmin−1. The fourth tube ((d) in figure 1(B))was directlyconnected to an independent extrusion needle (needle2 infigure 1(B)) and had aflowof 4mlmin−1.

The x–y plane contained a heated surface in orderto maintain the temperature at 37 °C (figure 1(A)).The printer and the extrusionmodule were placed in acell culture laminar flow hood; all the parts were ster-ilizedwithUV light in the hood.

The printer firmware was installed in the micro-controller (ATmega2560) of a RepRap Arduino Mega

Pololu Shield (RAMPS), and it manages the mechan-ical sensors and actuators as well as the thermal con-trol of the heated bed. The selected firmware wasMarlin, also open source, because it is able to controlmore than one extruder. Deposition trajectories weregenerated and sent to the RAMPS using Repetierv0.53. This programme transformed the geometricdata into paths or spatial coordinates to be followed bythe printer head. It also controlled which of the dis-pensers should be active and the operative proceduretime. A script in C++ was developed to establish thevolume of liquid to be deposited.

Liquids were pumped by a module composed offour separated dispensers, as previously described.Each of them had its own syringe. As the RAMPS hadonly two available ports to connect extruders, to con-trol three of the syringes we designed a system similarto a syringe pump but with a reduction system, basedon timing belts and pulleys, which allowed to movethree different syringes with only one stepmotor (sup-plementary figure 1). Each syringe driving screw hadits own angular speed (wi) determined by the corresp-onding reduction coefficient which depended on therelation between the teeth number (ni) of its pulleyand that of themain pulley (n). These coefficients werecalculated to obtain a constant stoiquiometry of thethree components in the final mixture at the nozzle.The fourth syringe was moved independently by thesecond stepmotor.

The motors, NEMA17, presented a step angle of1.8° and a holding torque of 4.8 kg cm, and were con-trolled by a DRV8825 driver that provides six differentstep resolutions (full-step, half-step, 1/4-step, 1/8-step, 1/16-step, and 1/32-step). Thefinal resolution ofthe deposition depended on this parameter and it

Figure 1. (A)Bioprinter setup and components. Human plasma, hFBs, calcium chloride (CaCl2) and hKCs, respectively, are pumpedthrough four sterile tubes from the extrusionmodule E to the headA. The extruder needles B deposit the components on the printingplastic dish (P100, Corning 100×20mm). The heated bedCmaintains the temperature at 37 °C.The system is controlled by thecontrol unitD, which is composed of anArduinowith RAMPS 1.4 and LCD interface. (B)Picture of the head showing the three tubes(a)–(c) carrying the components of the dermal compartment (hFBs, human plasma andCaCl2), converging to the trifurcatedconnector which is itself connected to needle 1 and the fourth tube, carrying the hKCs, connected directly to needle 2.

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could be easily tuned with jumpers. All these drivers,sensors and actuators were connected to the RAMPS1.4 shield, which was mounted over an Arduino Mega2560 board that contained a microcontrollerATmega2560.

2.2. Primary hKCs and hFBs culturehFBs and hKCs obtained from skin biopsies of healthydonors were obtained from the collections of biologi-cal samples of human origin; these samples areregistered in the ‘Registro Nacional de Biobancos paraInvestigación Biomédica del Instituto de Salud CarlosIII’. hKCs were cultured following previouslydescribed methods [48] as modified by our laboratory[15, 49]. The growing media for hKCs was a 3:1mixture of Dulbecco’s Modified Eagle Medium(DMEM) (GIBCO-BRL) and HAM’S F12 (GIBCO-BRL) (hKC medium) containing 10% of fetal bovineserum (FBS), 0.1 nM choleric toxin, 2 nM T3, 5 mgml−1 insulin, 0.4 mg ml−1 hydrocortisone and 10 ngml−1 EGF (Sigma, St Louis, MO). hFBs were culturedin Dulbecco’s modified Eagle’s medium (DMEM,BiochromKG) containing 10%FBS.

2.3. Preparation andprinting offibrin-based skinSkin substitutes formed by two layers, representing thedermis (lower layer) and the epidermis (upper layer),were generated following the method developed in[15, 17]. The lower layer was a plasma-derived fibrinmatrix populated with hFBs and the upper layer wasformed by hKCs, seeded on the top of the fibrinscaffold. Fresh frozen human plasma was provided bya local blood bank (Banco de Sangre del CentroComunitario de Transfusión del Principado de Astur-ias (CCST) Spain) and was obtained according to thestandards of the American Association of Blood Banks[50]. The fibrin matrix was prepared as previously

described in [15, 17]with somemodifications requiredfor the bioprinting process, as described below.

Figure 2 describes the bioprinting process andin vitro and in vivo experiments. To generate a dermalsubstitute, 7×104 cultured hFBs were resuspendedin 4ml of DMEMand loaded in the first syringe. In thesecond syringe, a volume of human plasma containing30 mg of fibrinogen (typically 13 ml) was mixed with200 μl of tranexamic acid (antifibrinolytic agentAmchafibrin, Fides- Ecopharma). Finally, in a thirdsyringe 2.3 ml of CaCl2 (prepared at 1% w/v in saline,(NaCl 0.9% w/v)) was loaded. The function of CaCl2is to induce the coagulation of the plasma fibrinogeninto a fibrin hydrogel. The total volume of the threesyringes was adjusted to 25 ml by adding saline to thethird syringe. After this, the syringes were put into theextrusion module of the bioprinter and their contentwas mixed (as described in section 2.1) and depositedon a P100 tissue culture plate (Corning100×20mm).

Printed dermal substitutes were left in a cell cul-ture incubator (at 37 °C in 5%CO2) for thirty minutesto allow them to polymerize, and then 10 ml of hKCsmedium, containing 6×106 hKCs per P100 platewere loaded into the fourth syringe of the extrusionsystem and deposited over the dermal equivalent.hKCs were allowed to attach and spread overnight in acell culture incubator. This number was established inorder to generate a confluent hKCs monolayer at thismoment. Immediately after the overnight incubation,the printed skin equivalents were transplanted on tothe backs of immunodeficientmice (see section 2.5).

Alternatively, skin substitutes were printed ontranswell inserts. The components were placed onpolycarbonate transwell inserts (1μmpore) in a 6-wellculture plate (Corning Costar Corp., Cambridge, MA,4.15 cm2) for the 3D in vitro assays (see section 2.5). In

Figure 2. Scheme of the bioprinting process. The extrusionmodule contained four syringes, loadedwith hFBs (a), plasma (b), CaCl2(c) and hKCs (d), respectively. The contents of the syringes (a)–(c)were continuously pumped out at the appropriate speed,mixed asthey arrived at the head, extruded through the needle and deposited on the corresponding plate type (P100 or transwell), following thetrajectories dictated by the control unit. Thismixture was allowed to polymerize for 30min at 37 °C to form afibroblast-containingfibrin hydrogel, which became the dermal compartment of the skin equivalent. Immediately after this polymerization step, the hKCssuspension contained in syringe (d)was similarly deposited on top of this hydrogel to form a confluentmonolayer. (A)Equivalentsprinted on transwell inserts were allowed to differentiate at the air–liquid surface for 17 d and then analysed. (B)Equivalents printedon P100 plates were grafted on to the backs of immunodeficientmice for eight weeks and then analysed.

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this case, the deposition volume and the trajectorieswere adjusted to the geometry of these plates. After thehKCs attaching and spreading step, hKCs wereallowed to differentiate at the air–liquid interface andformed amultilayered skin as explained in section 2.5.

2.4. ImmunodeficientmiceImmunodeficient athymic nude mice were purchasedfrom IFFA-Credo-Charles River (St. Aulin-les-Elbeuf,France) and kept and used at the CIEMAT AnimalFacility (Spanish registration number 28079-21A)under sterile conditions. The animals were housed inindividually ventilated type II cages, a maximum offour mice to each cage with 25 air changes per hourand 10 KGy gamma irradiated soft wood pellets asbedding. Experiments were carried out according toEuropean and Spanish laws and regulations.

2.5. In vivo and in vitromaturation anddifferentiation of printed skin equivalentsAs explained in section 2.3, the printing process wasdesigned to produce a fibroblast-containing fibrinhydrogel covered with a monolayer of hKCs. In vivoand in vitro assays were performed to analyse theviability of these constructs and their capacity togenerate a terminally differentiated skin.

For the in vivo assays, once hKCs were attached tothe fibrin surface, the cultured equivalents weremanually detached from the P100 plate and grafted onto the backs of immunodeficient mice. Four femalemice were aseptically cleansed and grafted as pre-viously described [17]. Full thickness circular woundsof 12 mm diameter were produced by means of apunch on the dorsum of each mouse. Then, circularsamples of the same diameter were obtained by thesame punch from the printed skin substitutes, placedon the generated wounds and covered by the skin, pre-viously removed from these mice, devitalized by threecycles of freezing and thawing. The devitalized skinwas kept in place with the help of sutures. The graftswere analysed eight weeks after grafting took place.

For the in vitro assays, skin constructs deposited ontranswells were allowed to differentiate at the air–liquid interface for 17 d at 37 °C in a CO2 incubator indifferentiating medium (hKCs medium containing0.5% FBS and 50 μm of ascorbic acid). The mediumwas changed every three days [51–53].

2.6.Histology and immunostainingFour-mm biopsies of human skin regenerated eitherin vitro or in vivo were collected with the help of apunch. For histological analysis, samples were fixed in3.7% buffered formaldehyde, and embedded in paraf-fin. 3 μm cross-sections were dewaxed, rehydratedand stainedwith hematoxylin-eosin (H/E).

For immunohistochemistry experiments, thebiopsies were frozen and 5 μm cryosections were ana-lysed using primary specific antibodies against well-

known skin markers: anti human-vimentin (V9, Bio-Genex, San Ramon, CA to distinguish hFBs), anti-keratin 5 (polyclonal AF138, BabCO, Berkeley, CA; tolabel hKCs of the proliferative basal layer), antikeratin10 (monoclonal AE2, ICN Biomedicals, Cleveland,OH; to label suprabasal hKCs), and antihuman filag-grin (polyclonal AF-62, BabCO) to label the epidermalgranular layer. To determine the formation of the der-moepidermal basal membrane, a specific antibodyagainst human-collagen VII (Clone LH7.2, Sigma)wasused. To detect blood vessel formation, biopsies fromhuman skin regenerated on nude mice were labelledwith anti-SMA (Smooth Muscle Actin, C6198, Sigma,St. Louis, USA). Samples were coverslipped usingMowiol (Hoechst, Somerville, NJ) mounting mediacontaining 46-diamidino-2-phenyl indole (DAPI,ROCHE, Germany, 20 μg ml−1) for nucleivisualization.

2.7. Analysis of epidermal cells viabilityTo determine if the printing process compromised theviability of the epidermal cells, freshly trypsinizedhKCs (as a control) and hKCs, pumped through theextrusion system after trypsinization, were subjectedto colony-forming assay as described else-where [54, 55].

In brief, approximately 500 hKCs were placed intoeach well of a six multi-well plate containing a feederlayer of lethally irradiated 3T3 cells. Three wells wereseeded with control hKCs and the other three wereseeded with extruded hKCs. After nine days, cultureswere stained with fluorescent Rhodamine B (R-6626,Sigma St Louis, USA) to estimate the number of epi-dermal colonies using an inverted fluorescencemicro-scope. The number of colonies was calculated bycounting ten fields per well.

3. Results

3.1. Analysis of printed human skin differentiatedin vitroBioengineered equivalents deposited into transwellswere allowed to differentiate at the air–liquid interfacefor 17 d. As shown in the histological staining(figure 3(B)), printed equivalents generated a tissuewith a structure similar to that obtained differentiatinghandmade skin equivalents (figure 3(A)) and alsosimilar to normal human skin (figure 4(C)). A well-formed, orthokeratotic stratum corneum was presentindicating terminal differentiation. The dermal com-partment contained well spread hFBs in the fibrinmatrix. To analyse the nature of this differentiationmore carefully, immunofluorescent analysis was car-ried out. Expression of keratin K10 was detected insuprabasal cells (figure 3(C)), where this intracellularstructural protein is specifically synthesized in normalskin. To visualize the state and persistence of the hFBs,after this relatively prolonged culture time, we stained

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cryosections with a specific antibody against humanvimentin, a cytoskeletal protein characteristic of thiskind of cell. This demonstrated the proper growth andspreading of the hFBs comprising the dermal com-partment of the bioprinted skin (figure 3(D)). The lackof strateum corneum in figures 3(C) and (D) is due tothe cryosectioning method which frequently removesthese structures.

3.2. Analysis of printed human skin differentiatedin vivoTo study if bioprinted skin substitutes had the capacityto differentiate in vivo, we grafted them on to the backof immunodeficient athymic mice. These grafts wereperformed orthotopically, so that the printed equiva-lents were placed on the wound beds generated on theback of the immunodeficient mice, as described in[17, 49] (see also section 2.5). Between four and sixweeks after grafting, the devitalized mouse skin, usedas a biologic bandage, fell off and the grafted humanskin became visible (figure 4(A)). It exhibited acharacteristic wrinkled, thick and whitish aspect, verysimilar to the appearance of native human skin andclearly different from the surrounding thin and

pinkish mouse skin. Histological analysis demon-strated that the regenerated human skin presented astructure very similar to that of normal human skin(compare figure 4(B) with figure 4(C)). All the stratacharacteristic of normal skin, stratum basale, stratumspinosum, stratum granulosum and a well-developedstratum corneum are easily identified in theprinted skin.

A more detailed analysis of the original printedskin was performed by immunofluorescence usingwell-established skin markers. Accordinlgy, the basalproliferative stratum was clearly revealed by labellingwith an antibody recognizing keratin K5 (figure 5(A),green staining). The correct formation of the dermo-epidermal junction of the skin was confirmed by label-ling with an antibody against human collagen VII(figure 4(B) green staining), the protein forming theanchoring fibrils that bind together epidermis anddermis. This structure is very important for themechanical stability of the skin; its lack leads to severeblistering due to the separation of the two compart-ments of the tissue, observed in patients suffering fromdysthrophic epidermolysis bullosa [56]. We considerthe presence of the basal lamina and the stratum

Figure 3. In vitro 3Dhuman skin equivalents obtained after 17 d of differentiation at the air–liquid interface. (A) ‘Handmade’ skinequivalent following our previous protocol. (B)–(D)Printed skin equivalents. (A), (B)Histological analysis offixed samples, usinghematoxilyn-eosin. (C), (D) Immunostaining of frozen samples using an anti-K10 antibody ((C), green immunofluorescence) and ananti-human vimentin antibody ((D), red immunofluorescence). Blue colour in (C) and (D) denotesDAPI staining of the nuclei. EpandDe in (A)–(D)denote the epidermal and the dermal compartments, respectively. Thewhite dotted line indicates the dermo-epidermal junction (basalmembrane). Scale bar: 100μm.

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corneum as clear indications of proper and completedifferentiation of the grafted printed skin. As in nor-malmature skin, there was a strong suprabasal expres-sion of keratin K10 (figure 5(C), red staining) and ofthe late differentiation marker filaggrin, characteristicof the granular layer (figure 5(D), green staining). Alsorete ridges (a hallmark of mature human skin, notfound inmouse skin)were detected in some regions ofthe grafted printed skin (asterisks infigure 5(D)).

In addition, immunostaining with a specific anti-body against human vimentin, showed the persistenceof hFBs exclusively in the dermal compartment of theregenerated skin (figure 5(B), red staining). Anotherimportant parameter to assess in the regeneration pro-cess is the vascularization of the grafted human skin,which allows oxygenation and nutrition of the new tis-sue and therefore, its long-term persistence. As shownby the red staining and arrows in figure 5(D), bloodvessels (SMA+, red)were detected in the dermis of theprinted skin upon in vivo regeneration.

3.3. hKCs survival analysishKCs are more delicate and difficult to keep in culturethan fibroblasts. In particular, it is well- establishedthat hKCs differentiate terminally when cultured insuspension [57]. Therefore, it is necessary to analysethe viability of these cells after going through thebioprinting process. To do this, we performed colony-forming efficiency assays as described in materials andmethods (section 2.7). We found (figure 6) that the

number and the size of the colonies were very similar,both before and after the cells underwent the bioprint-ing process (the same number of cells were seeded inboth cases).

4.Discussion

This work shows the automation and in vitro produc-tion of printed human skin containing dermal andepidermal components, with structural and functionalproperties similar to those of skin constructed byconventional manual procedures developed pre-viously in our group, and also to those of in vivohuman skin. Due to the relatively simple structure ofskin tissue and the strong clinical relevance ofmethodsfacilitating the treatment of wounds, production ofskin tissue containing dermal and epidermal compo-nents by bioprinting is currently an area of activedevelopment. To our knowledge, two main in vitrodeposition approaches containing in vitro and in vivoanalysis have been published [41–44]. The firstapproach, which was based on a layer by layerdeposition technique, demonstrated the feasibility ofthe multi-layered deposition of fibroblasts and kerati-nocytes in a collagen scaffold. The dermal compart-ment contained eight acellular collagen layers,interspersed with three fibroblast-containing collagenlayers. On top of this, two keratinocytes-containingcollagen layers were deposited. This requires that, afterdeposition, the fibroblasts migrate to generate a

Figure 4.Histological analysis (8weeks postgrafting) of bioprinted human skin grafted to immunodeficientmice. (A)Visualappearance of the grafted human skin. The dotted linemarks the boundary between human andmouse skin. (B)H/E staining of theregenerated human skin. (C)H/E staining of normal human skin. Thewhite dotted line in (B) and (C) indicates the dermo-epidermaljunction (basalmembrane, BM). Ep andDe in (B) and (C)denote the epidermal and the dermal compartments, respectively. Scale bar:100μm.

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homogeneous distribution throughout the collagenmatrix, as can be observed in a normal dermis.

Apart from this, it is difficult to evaluate the qualityof the 3D skin obtained, since the authors do not showthe localization of any of the several well-known skindifferentiation markers. In addition, the histology ofthe skin tissue does not show proper stratification andterminal differentiation as compared to human skin.As the authors recognize, this can be, at least in part,due to the use of an immortalized keratinocyte cell line(HaCaT cells) instead of primary hKCs. Alternatively,it could be a consequence of the anomalous severeshrinking and compaction of the dermal compart-ment reported by the authors during the air–liquidinterface culture, the step in which keratinocyte strati-fication and differentiation to form an epidermis takesplace. Finally, based on the data provided by theauthors, the estimated printing speed allows thedeposition of 1 cm2 skin per hour which is a very slowprocess considering the large surfaces needed for clin-ical or commercial applications.

The second approach used a LaBP technique toarrange immortal human HaCaT keratinocytes andmouse NIH-3T3 fibroblasts. In this technique, 20 col-lagen sublayers containing fibroblasts were printedonto a sheet of Matriderm® and subsequently 20 col-lagen sublayers containing keratinocytes were printedon top of it. A clear problem of thismethod is again the

use of immortalized keratinocyte and fibroblast celllines; in particular the NIH-3T3 murine fibroblaststhat are very different from hFBs.Moreover, the histo-logical and immunohistochemical data presented bythe authors indicated relevant differences when com-paring their printed skin with normal human skin: (1)From the histological point of view, it is apparent thatin the histological sections of the in vivo implants usingthe dorsal skin full chambers [44], the human epi-dermis, although apparently well differentiated, asindicated by the presence of a stratum corneum, hadan abnormal thickness: it is thinner than the mouseepidermis while it is known that human skin is thickerthan mouse skin, in particular in this type of graftingexperiment [17, 49]. (2) From the immunohistochem-ical point of view, K14 is a well-established marker ofepidermal basal cells as shown by the own authors innormal mouse skin. However, this marker is found inall the epidermal layers of both in vivo and in vitro dif-ferentiated printed skin. Similarly, Ki67, which is amarker of cell proliferation that should be mostlyrestricted to basal cells, was found evenly distributedthrough the whole epidermis of in vitro cultures. Inagreement with the authors, we think that the timeused in these experiments, probably due to the limita-tions imposed by the dorsal skin full chambermethod,is a too short time to obtain a fully differentiated epi-dermis. It has been reported that HaCaT cells need

Figure 5. Immunohistochemical analysis (eight weeks postgrafting) of bioprinted human skin grafted to immunodeficientmice usingantibodies against skinmarkers. (A)KeratinK5 detection (green immunofluorescence). (B)CollagenVII (green line between dermisand epidermis) and vimentin (the red colour in the dermal compartment)detection. (C)HumankeratinK10 detection (red suprabasalstaining: notice that the basal layer is negative). (D) Filaggrin (green staining in the stratum granulosum) and SMA (red staining)detection. Arrows point to some of the capillaries present in the dermal compartment. Asterisksmark rete ridges. Nuclei were stainedwithDAPI (blue). Thewhite dotted line in panels (A) and (C), indicates the epidermal-dermal boundary. Inside the images: Ep—epidermal compartment, De—dermal compartment. Scale bar: 100μm.

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between three and six weeks to develop a fully differ-entiated epidermis upon in vivo transplantation to theback of nude mice [58, 59]. The delocalized K14 andKi67 staining observed in these experiments is also ahallmark of the early stages of HaCaT 3D differentia-tion [58, 59]. Finally, although it is difficult to make anaccurate estimation from the data provided by theauthors, a caveat concerning this technology is whatwould be the time required to produce a skin surfaceof clinical or commercial interest (50–100 cm2).

From a practical point of view, our method is sim-pler and quicker than the two described methodssince, instead of printing a high number of cellular andacellular layers to form the dermis and epidermis, wedeposit simultaneously all the elements (hFBs, humanplasma and CaCl2) required to form the dermis and,on top of this, the hKCs required to form a confluentlayer of epidermal cells. These constructs give rise to afully differentiated human skin upon in vitro or in vivodifferentiation.

To overcome the limitations present in the twoapproaches discussed above and, therefore, to fullyvalidate the bioprinting method for the production ofa tissue with structural and functional properties,similar to normal human skin, the use of human pri-mary fibroblasts and keratinocytes is necessary.According to this, we used primary human cells toge-ther with a fibrin-based dermal matrix previouslydeveloped by our group for the production of largeskin surfaces, useful in the treatment of severe andextensive burns, wounds with loss of substance andskin fragility diseases [16, 17]. Based on the foregoing,we developed an extrusion bioprinting method thatdid not harm these biological components, in

particular, hKCs, which are known to terminally dif-ferentiate when they are kept in suspension [57].

We did not observe any contraction or shrinkingof thefibrin hydrogels as reported in [42]with collagenhydrogels. Initially, we analysed the printed skinequivalent upon in vitro differentiation. The resultsshowed that the generated skin had correct archi-tecture (figure 3(B), H/E staining) and differentiation(figures 3(C) and (D), staining with dermal and epi-dermal markers) as well as persistence, homogeneousdistribution and spreading of the hFBs.

Secondly, the structure and functionality of theprinted human skin was further analysed on skin-humanized mice. This model recapitulates faithfullythe characteristics of the skin from which human cellswere obtained (donor’s skin) and overcomes the timelimitations imposed by the dorsal skin full chamberused in [44]. Our laboratory has extensive experienceusing it to model diverse cutaneous diseases [45] andprocesses [60, 61]. To our knowledge, it constitutes thebest system to perform long-term experiments withhuman skin in an in vivo scenario.We usedH/E stain-ing (figure 4) and several differentiation markers suchas keratin 5 (a marker of proliferative basal keratino-cytes), human vimentin (a marker of hFBs), human-collagen type VII (a marker of dermoepidermal basalmembrane), keratin 10 (a marker of suprabasal differ-entiated keratinocytes) and human-filaggrin (amarkerof keratinocyte terminal differentiation) (figure 5).Thesemarkers labelled the same cells and structures inthe bioprinted skin as they do in human normal skin,including the presence of a well-developed stratumcorneum and a basal membrane. In addition, it isimportant to highlight the in vivo formation of rete

Figure 6.Colony-forming efficiency assay.Microscopic appearance (phase contrast) of the hKC colonies grown in the presence of afeeder layer of lethally irradiated 3T3 cells; before (A) and after (B), passing them through the printing system. (C)Number ofkeratinocyte colonies permicroscopic field before (left) and after (right). Scale bar: 200μm.

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ridges, a hallmark of mature human skin, not found inmouse skin and so far, not reported with printedhuman skin.

Neoangiogenesis formation is one of themost cru-cial events for successful skin grafting to take place.Obviously, this is particularly relevant if one considersthe clinical applications of bioengineered skin equiva-lents. As shown in figure 5(D), and also reported in[44], small blood vessels were found in the grafted bio-printed equivalents beneath the epidermis. They aresimilar to the capillary network found in the papillarydermis of normal skin, which is critical for nourishingthe avascular epidermis. These blood vessels seem togrow in from the depth of the wound bed into the der-mal compartment. This is also observed in patientswith grafted skin and it is attributed to endothelialgrowth factors, and other soluble factors, largely pro-duced by keratinocytes and fibroblasts; for review see[62]. This finding represents additional evidence of thefunctionality of our skin regenerated fromhuman bio-printed equivalents.

We consider that the two approaches discussedabove [41–44], are clearly relevant as they describemulti-layered deposition techniques for skin bioprint-ing. Together with our work, they demonstrate thatdifferent bioprinters and bioprinting technologies canbe potentially used to produce human skin. Clearly,the design of appropriate bioinks is a critical step forthe production of printed skin with structure andfunctionality increasingly similar to normal humanskin. Based on our previous experience of treatingpatients with skin equivalents, we have developed asimple, flexible and robust method to produce humanskin which is useful in the clinic (e.g. for treatment ofskin wounds) and in industry (e.g. for drug screening).Our approach allows the deposition of 100 cm2 ofhuman skin in less than 35 min, which is much fasterthan approaches reported in the literature [41–44].

In addition, other bioprinting approaches use col-lagen as a 3D matrix, a biomaterial commonly used inthe production of dermo-epidermal equivalents that,in our opinion, presents twomain disadvantages whencompared to the human plasma-based dermal scaf-folds used in this work. Firstly, human plasma, unlikeanimal renatured collagen, provides a more suitable3D scaffold to promote migration, proliferation anddifferentiation of the cells in the wound bed[15, 17, 63]. Secondly, human plasma-based scaffoldsallow efficient production of collagen by hFBs and theconcomitant remodelling of the scaffold to generate adermal extracellular matrix similar to that found innormal human skin [15, 17].

5. Conclusions

We have developed a simple and robust bioprintingmethod and bioinks that allow the production ofhuman bilayer skin, using human plasma and primary

hFBs and hKCs. Based on careful histological andimmunohistochemical in vitro and in vivo analysis, wedemonstrated that the printed skin was very similar tonormal human skin and indistinguishable frombilayered dermo-epidermal equivalents, previouslyproduced manually in our laboratory and successfullyused in the clinic. This method allows the productionof human skin in amounts and times appropriate forits clinical and commercial use. The method alsoopens up the possibility of producing skin equivalentsin an automatized and standardized manner, whichshould lead to a reduction in the cost of the productand an improvement in the production line, therebyovercoming some of the problems presented by thecurrentmanual productionmethod.

Acknowledgments

We kindly thank Angélica del Corral, GuillermoVizcaíno, Miguel Iñigo from UC3M and BlancaDuarte, Almudena Holguin, Nuria Illera and LuisaRetamosa from CIEMAT and IIS-FJD for theirexcellent technical assistance; and Jesus Martinez(CIEMAT) for animal care.We are also indebted to DrAlvaro Meana, Eva Garcia (from CCST), Dr FernandoLarcher (from CIEMAT and IIS-FJD) and Dr MarcelaDel Rio (from UC3M and IIS-FJD) for helpful discus-sions and providing materials required for this work.We also thank Lucia Gullon and Alfredo Brisac fromBioDan Group for collaboration and support in thisproject, in particular in the development of thebioprinter. This work was partially supported bygrants DPI2014-61887-EXP and DPI2015-68088-Pfrom the Spanish Ministerio de Economía y Competi-tividad. The authors declare no conflict of interest.

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